Monographs

Formulated Preparations:
General~onographs
2014
FORMULATED PREPARATIONS:
GENERAL MONOGRAPHS
Pharmaceutical Preparations *****
** **
(Ph Eur monograph 2619) ***
~E~ ____________________________________________
INTRODUCTION
This monograph is intended to be a reference source of
standards in the European Pharmacopoeia on active
substances, excipients and dosage forms, which are to be
applied in the manufacture/preparation of pharmaceuticals,
but not a guide on how to manufacture as there is specific
guidance available covering methods of manufacture and
associated controls.
It do es not cover investigational medicinal products, but
competent authorities may refer to pharmacopoeial standards
when authorising clinical trials using investigational medicinal
products.
DEFINITION
Pharmaceutical preparations are medicinal products generally
consisting of active substances that may be combined with
excipients, formulated into a dosage form suitable for the
intended use, where necessary after reconstitution, presented
in a suitable and appropriately labelled container.
Pharmaceutical preparations may be licensed by the
competent authority, or unlicensed and made to the specific
needs of patients according to legislation. There are 2
categories of unlicensed pharmaceutical preparations:
-- extemporaneous preparations, i.e. pharmaceutical
preparations individually prepared for a specific patient or
patient group, supplied after preparation;
-- stock preparations, i.e. pharmaceutical preparations
prepared in advance and stored until a request for a
supply is received.
In addition to this monograph, pharmaceutical preparations
also comply with the General Notices and with the relevant
general chapters of the Pharmacopoeia. General chapters are
normally given for information and become mandatory when
referred to in a general or specific monograph, unless such
reference is made in a way that indica tes that it is not the
intention to make the text referred to mandatory but rather
to cite it for information.
Where relevant, pharmaceutical preparations also comply
with the dosage form monographs (e.g. Capsules (0016) ,
Tablets (0478) and general monographs relating to
pharmaceutical preparations (e.g. Allergen products (1063),
Herbal teas (1435), Homoeopathic preparations (1038),
Immunosera for human use, animal (0084), Immunosera for
veterinary use (0030) , M onoclonal antibodies for human use
(2031), Radiopharmaceutical preparations (0125), Vaccines for
human use (0153), Vaccines for veterinary use (0062) .
Where pharmaceutical preparations are
manufactured/prepared using material s of human or animal
origin, the general requirements of general chapters 5.1.7.
Viral safety, 5.2.6. Evaluation of safety of veterinary vaccines
and immunosera and 5.2.8. Minimising ¡he risk of transmitting
animal spongiform encephalophathy agents via human and
veterinary medicinal products apply, where appropriate.
General Monographs IV-37
ETHICAL CONSIDERATIONS AND GUIDANCE IN
THE PREPARATION OF UNLICENSED
PHARMACEUTICAL PREPARATIONS
The underlying principie of legislation for pharmaceutical
preparations is that, subject to specific exemptions, no
pharmaceutical preparation may be placed on the market
without an appropriate marketing authorisation.
The exemptions from the formal licensing requirement allow
the supply of unlicensed products to meet the special needs
of individual patients. However, when deciding to use an
unlicensed preparation all health professionals involved
(e.g. the prescribing practitioners and/or the preparing
pharmacists) have, within their area of responsibilities, a duty
of care to the patient receiving the pharmaceutical
preparation.
In considering the preparation of an unlicensed
pharmaceutical preparation, a suitable level of risk assessment
is undertaken.
The risk assessment identifies:
-- the criticality of different parameters (e.g. quality of active
substances, excipients and containers; design of the
preparation process; extent and significance of testing;
stability of the preparation) to the quality of the
preparation; and
-- the risk that the preparation may present to a particular
patient group.
Based on the risk assessment, the person responsible for the
preparation must ensure, with a suitable level of assurance,
that the pharmaceutical preparation is, throughout its
shelf-life, of an appropriate quality and suitable and fit for its
purpose. For stock preparations, srorage conditions and
shelf-life have to be justified on the basis of, for example,
analytical data or professional judgement, which may be
based on literature references.
PRODUCTION
Manufacture/preparation must take place within the
framework of a suitable quality system and be compliant with
the standards relevant to the type of product being made.
Licensed products must comply with the requirements of
their licence. For unlicensed products a risk assessment as
outlined in the section 'Ethical considerations and guidance
in the preparation of unlicensed pharmaceutical preparations'
is of special importance, as these products are not previously
assessed by the competent authority.
Fonnulation
During pharmaceutical development or prior ro
manufacture/preparation, suitable ingredients, processes, tests
and specifications are identified and justified in order to
ensure the suitability of the product for the intended
purpose . This includes consideration of the properties
required in order to identify whether specific ingredient
properties or process steps are critical to the required quality
of the pharmaceutical preparation.
Active substances and excipients
Active substances and excipients used in the formulation of
pharmaceutical preparations comply with the requirements of
the relevant general monographs, e.g. Substances for
pharmaceutical use (2034), Essential oils (2098), Extracts
(0765), Herbal drugs (1433), Herbal drug preparations (1434),
Herbal drugs for homoeopathic preparations (2045), Mother
tinctures for homoeopathic preparations (2029), Methods of
preparation of homoeopathic stocks and potentisation (2371),
Products of fermentation (1468), Products with risk of
transmitting agents of animal spongzform encephalopathies (1483),
 IV-38 General Monographs
Products of recombinant DNA technology (0784), Vegetable fatty
oz1s (J 579).
In addition, where specific monographs exist, the quality of
the active substances and excipients used complies with the
corresponding monographs.
Where no specific monographs exist, the required quality
must be defined, taking into account the intended use and
the involved risk.
When physicochemical characteristics of active substances
and functionality-related characteristics (FRCs) of excipients
(e.g. particJe-size distribution, viscosity, polymorphism) are
critical in relation to their role in the manufacturing process
and quality attributes of the pharmaceutical preparation, they
must be identified and controlled.
D etailed information on FRCs is given in general chapter
5.15. Functionality-related characteristics of excipients.
Microbiological quality
The formulation of the pharmaceutical preparation and its
container must ensure that the microbiological quality is
suitable for the intended use.
During development, it shall be demonstrated that the
antimicrobial activity of the preparation as such or, if
necessary, with the addition of a suitable preservative or
preservatives, or by the selection of an appropriate container,
provides adequate protection from adverse effects that may
arise from microbial contamination or proliferation during
the storage and use of the preparation. A suitable test
method together with criteria for evaluating the preservative
properties of the formulation are provided in general chapter
5.1.3. Efficacy of antimicrobial preservation.
If preparations do not have adequate antimicrobial efficacy
and do not contain antimicrobial preservatives they are
supplied in single-dose containers, or in multidose containers
that prevent microbial contamination of the contents after
opening.
In the manufacture/preparation of non-sterile pharmaceutical
preparations, suitable measures are taken to ensure their
microbial quality; recommendations on this aspect are
provided in general chapters 5.1.4. Microbiological quality of
non-sterile pharmaceutical preparations and substances for
pharmaceutical use and 5.1.8. Microbiological quality of herbal
medicinal products for oral use.
Sterile preparations are manufactured/prepared using
materials and methods designed to ensure sterility and to
avoid the introduction of contaminants and the growth of
micro-organisms; recommendations on this aspect are
provided in general chapter 5.1. 1. Methods of preparation of
sterile products.
Containers
A suitable container is selected. Consideration is given to the
intended use of the preparation, the properties of the
container, the required shelf-life, and product/container
incompatibilities. Where applicable, containers for
pharmaceutical preparations comply with the requirements
for containers (3.2 and subsections) and materials used for
the manufacture of containers (3. 1 and subsections).
Stability
Stability requirements of pharmaceutical preparations are
dependent on their intended use and on the desired storage
time.
Where applicable, the probabilíty and criticality of possible
degradation products of the active substance(s) and/or
reaction products of the active substance(s) with an excipient
and/or the immediate container must be assessed. Depending
2014
on the result of this assessment, limits of degradation and/or
reaction products are set and monitored in the
pharmaceutical preparation. Licensed products require a
stability exercise.
Methods used for the purpose of stability testing for all
relevant characteristics of the preparation are validated as
stability indicating, Le. the methods allow the quantification
of the relevant degradation products and physical
characteristic changes.
TESTS
Relevant tests to apply in order to ensure the appropriate
quality of a particular dosage form are described in the
specific dosage form monographs.
Where it is not practical, for unlicensed pharmaceutical
preparations, to carry out the tests (e.g. batch size, time
restraints), other suitable methods are implemented to ensure
that the appropriate quality is achieved in accordance with
the risk assessment carried out and any local guidance or
legal requirements.
Stock preparations are normally tested to a greater extent
than extemporaneous preparations.
The following tests are applicable to many preparations and
are therefore listed here.
Appearance
The appearance (e.g. size, shape and colour) of the
pharmaceutical preparation is controlled.
Identity and purity tests
Where applicable, the following tests are carried out on the
pharmaceutical preparation:
- identification of the active substance(s);
- identification of specific excipientes), such as
preservatives;
- purity tests (e.g. investigation of degradation products,
residual solvents (2.4.24) or other related impurities,
sterility (2. 6.1));
- safety tests (e.g. safety tests for biological products) .
Uniformity (2.9.40 or 2.9.5/2.9.6)
Pharmaceutical preparations presented in single-dos e units
comply with the testes) as prescribed in the relevant specific
dosage form monograph. If justified and authorised, general
chapter 2.9.40 can be applicable only at the time of release.
Special uniformity requirements apply in the following cases:
- for herbal drugs and herbal drug preparations, compliance
with general chapter 2.9.40 is not required;
- for homoeopathic preparations, the provisions of general
chapters 2.9.6 and 2.9.40 are normally not appropriate,
however in certain circumstances compliance with these
chapters may be required by the competent authority;
- for single- and multivitamin and trace-element
preparations, compliance with general chapters 2.9.6 and
2.9.40 (content uniformity only) is not required;
- in justified and authorised circumstances, for other
preparations, compliance with general chapters 2.9.6 and
2.9.40 may not be required by the competent authority.
Reference standards
Reference standards may be needed at various stages for
quality control of pharmaceutical preparations. They are
established and monitored taking due account of general
chapter 5.12. Reference standards.
ASSAY
Unless otherwise justified and authorised, contents of active
substances and specific excipients such as preservatives are
2014 General Monographs IV-39

determined in pharmaceutical preparations. Limits must be

defined and justified.
Suitable and validated methods are used. If assay methods
prescribed in the respective active substance monographs are
used, it must be demonstrated that they are not affected by
the presence of the excipients and/or by the formulation.
Reference standards
See Tests .
LABELLING AND STORAGE
The relevant labelling requirements given in the general
dosage form monographs apply. In addition, relevant
European Union or other applicable regulations apply.
GLOSSARY
Formulation
The designing of an appropriate formula (inc1uding materials,
processes, etc.) that will ensure that the patient receives the
suitable pharmaceutical preparation in an appropriate form
that has the required quality and that will be stable and
effective for the required length of time.
Licensed pharmaceutical preparation
A medicinal product that has been granted a marketing
authorisation by a competent authority. Synonym: authorised
pharmaceutical preparation.
Manufacture
AH operations of purchase of materials and products,
Production, Quality Control, release, storage, distribution of
medicinal products and the related controls.
Preparation (of an unlicensed pharmaceutical
preparation)
The 'manufacture' of unlicensed pharmaceutical preparations
by or at the request of pharmacies or other healthcare
establishments (the term 'preparation' is used instead of
'manufacture' in order c1early 10 distinguish it from the
industrial manufacture of Iicensed pharmaceutical
preparations).
ReconstÍtution
Manipulation to enable the use or application of a medicinal
product with a marketing authorisation in accordance with
the instructions given in the summary of product
characteristics or the patient information leafiet.
Ftisk assessrnent
The identification of hazards and the analysis and evaluation
of risks associated with exposure 10 those hazards.
Unlicensed pharmaceutical preparation
A medicinal product that is exempt from the need of having
a marketing authorisation issued by a competent authority
but is made for specific patients' needs according 10
legislation.
_ _ __ _ __ _ _ __ _ _ _ __ _ _ _ _ _ _ Ph Eur
Monographs

Herbal Drugs, Herbal Drug

Preparations and Herbal
Medicinal Products
 2014
Herbal Drugs *****
** **
(Ph Eur monograph 1433) ***
Herbal Drugs comply with the requirements of the European
Phannacopoeia. These requirements are reproduced below.
~E~ ____________________________________________
DEFINITION
Herbal drugs are mainly whole, fragrnented, or broken
plants, parts of plants, algae, fungi or lichen, in an
unprocessed sta te, usually in dried form but sometimes fresh.
Certain exuda tes that have not been subjected to a specific
treatment are also considered to be herbal drugs . Herbal
drugs are precisely defined by the botanical scientific name
according to the binominal system (genus, species, variety
and author).
Whole describes a herbal drug that has not been reduced in
size and is presented, dried or undried, as harvested;
for example: dog rose, bitter fennel or sweet fennel, Roman
chamomile ftower.
Fragmented describes a herbal drug that has been reduced in
size after harvesting to permit ease of handling, drying andJor
packaging; for example: cinchona bark, rhubarb, passion
ftower.
Broken describes a herbal drug in which the more-fragile
parts of the plant have broken during drying, packaging or
transportation; for example : belladonna leaf, matricaria
ftower, hop strobile.
Cut describes a herbal drug that has been reduced in size,
other than by powdering, to the extent that the macroscopic
description in the monograph of the herbal drug can no
longer be applied. When a herbal drug is cut for a specific
purpose that results in the cut herbal drug being
homogeneous, for example when cut for herbal teas, it is a
herbal drug preparation. Certain cut herbal drugs processed
in this way may be the subject of an individual monograph.
A herbal drug that complies with its monograph and is
subsequently cut for extraction shall comply in its cut form,
except for its macroscopic description, with the monograph
for that herbal drug, unless otherwise justified.
The term herbal drug is synonymous with the term herbal
substance used in European Community legislation on herbal
medicinal products.
PRODUCTION
Herbal drugs are obtained from cultivated or wild plants.
Suitable collection, cultivation, harvesting, drying,
fragrnentation and storage conditions are essential to
guarantee the quality of herbal drugs.
Herbal drugs are, as far as possible, free from impurities such
as soil, dust, dirt and other contaminants such as fungal,
insect and other animal contaminations. They are not rotten.
If a decontaminating treatment has been used, it is necessary
to demonstrate that the constituents of the plant are not
affected and that no harmful residues remain. The use of
ethylene oxide is prohibited for the decontamination of
herbal drugs.
IDENTIFICATION
Herbal drugs are identified using their macroscopic and
microscopic descriptions and any further tests that may be
required (for example, thin-Iayer chromatography).
TESTS
Foreign matter (2.8.2)
Carry out a test for foreign matter, unless otherwise
prescribed or justified and authorised. The content of foreign
General Monographs IV-43
matter is not more than 2 per cent 111/117, unless otherwise
prescribed or justified and authorised. An appropriate specific
test may apply to herbal drugs liable to be adulterated.
It may not be possible to perform the test for foreign matter
on a herbal drug that is cut, as described under Definition,
for either a specific purpose or for extraction. Under these
circumstances' the cut material is presumed to comply with
the test for foreign matter providing that the herbal drug
prior to cutting was compliant with this test.
Loss on drying (2.2.32)
Carry out a test for loss on drying, unless otherwise
prescribed or justified and authorised.
Water (2.2.13)
A determination of water may be carried out instead of a test
for los s on drying for herbal drugs with a high essential-oil
contento
Pesticides (2.8.13)
Herbal drugs comply with the requirements for pesticide
residues. The requirements take into account the nature of
the plant, where necessary the preparation in which the plant
might be used, and where available the knowledge of the
complete record of treatment of the batch of the plant.
Heavy metals (2.4.27)
Unless otherwise stated in an individual monograph or unless
otherwise justified and authorised:
-- cad117iwn: maximum 1.0 ppm;
-- lead: maximum 5.0 ppm;
-- 117ercUly: maximum 0.1 ppm.
Where necessary, limits for other heavy metals may be
required.
Where necessary herbal drugs comply with other tests, such
as the followiI).g, for example.
Total ash (2.4.16).
Ash insoluble in hydrochloric acid (2.8.1).
Extractable matter.
Swelling index (2.8. 4).
Bitterness vaIue (2.8.1 S).
Aftatoxin B¡ (2.8.18)
Where necessary, limits for aftatoxins may be required.
Ochratoxin A (2.8.22)
Where necessary, a limit for ochratoxin A may be required.
Radioactive contamination
In sorne specific circumstances, th e risk of radioactive
contamination is to be considered.
MicrobiaI contamination
Where a herbal drug is used whole, cut or powdered as an
ingredient in a medicinal product, the microbial
contamination is controlled (Microbiological quality of herbal
medicinal products for oral use (5.1.8) or Microbiological quality
of non-sterile phannaceutical preparations and substances for
phannaceutical use (5.1.4) (for example, for cutaneous use» .
ASSAY
Unless otherwise prescribed or justified and authorised,
herbal drugs are assayed by an appropriate method.
STORAGE
Protected from light.
____________________________________________
~E~
 IV-44 General Monographs
Processed Herbal Drugs
DEFINITION
Processed Herbal Drugs are obtained by subjecting Herbal
Drugs to traditional processing methods.
Processed Herbal Drugs are defined precisely by the
botanical scientific name according to the binomial system
(genus, species, subspecies, variety, and author) and plant
parto Monographs for Processed Herbal Drugs may refer to
the relevant monograph for the unprocessed material where
the binomial name is given.
PRODUCTION
Processed Herbal Drugs are obtained by subjecting Herbal
Drugs to specific types of processing according to traditional
processing methods. These traditional processing methods
have the potential to alter the physical characteristics and/or
chemical constituents of a Herbal Drug. Traditional
processing methods may require the addition of processing
aids to the herbal drug, for example, honey, vinegar, wine,
mil k and salto The additional processing aids used should be
of a suitable quality or of pharmacopoeial quality where a
monograph exists. The method of traditional processing is
provided under the Production section in individual
monographs.
IDENTIFICATION
Processed Herbal Drugs are identified using their
macroscopical and, where appropriate, microscopical
descriptions and any further tests that may be required.
TESTS
A test for foreign matter, Appendix XI D, is carried out,
unless otherwise prescribed in the individual monographs.
A specific appropriate test may be prescribed to detect
potential contaminants in processed herbal drugs.
If appropriate, the Processed Herbal Drugs comply with
other tests, for example, total ash, Appendix XI J, Method II,
ash insoluble in hydrochloric acid, Appendix XI K, Method II,
extractable matter, swelling index, Appendix XI C and bittemess
value, Appendix XI N.
The test for loss on drying, Appendix IX D, is carried out on
Processed Herbal Drugs, unless otherwise prescribed in the
individual monographs. A determinatíon of water by distillation,
Appendix IX C, Method II, is carried out for Processed
Herbal Drugs with a high essential oil contento
Processed Herbal Drugs comply with the requirements for
pesticide residues, Appendix XI L. The requirements take into
account the nature of the Processed Herbal Drugs, where
necessary the preparation in which the plant might be used,
and where available, the knowledge of the complete record of
treatrnent of the batch of the Processed Herbal Drugs during
cultivation, harvesting and processing. The content of
pesticide residues may be determined by the method
described in the annex to the general method.
The risk of contamination of Processed Herbal Drugs by
heavy metal s must be considered. In an individual
monograph either a generallimit for heavy metals or specific
limits for individual heavy metal may be required.
Where necessary limits for specific toxins, for example
afiatoxins or ochratoxins, may be applied.
Where processing is carried out to remove or limit specific
constituents from the herbal drug a suitable limit test should
be carried out.
In sorne specific circumstances, the risk of radioactive
contamination is to be considered.
2014
ASSAY
Unless otherwise justified and authorised Processed Herbal
Drugs are assayed by an appropriate method.
***
Herbal Drug Preparations * *
** **
(Ph Bur monograph 1434) ***
Herbal Drug Preparations comply with the requirements of the
Buropean Pharmacopoeia. These requirements are reproduced
below.
~E~ ____________________________________________
DEFINITION
Herbal drug preparations are homogeneous products
obtained by subjecting herbal drugs to treatrnents such as
extraction, distillation, expression, fractionation, purification,
concentration or fermentation.
Herbal drug preparations inc1ude, for example, extracts,
essential oils, expressed juices, processed exuda tes, and
herbal drugs that have been subjected to size reduction for
specific applications, for example herbal drugs cut for herbal
teas or powdered for encapsulation.
Herbal teas comply with the monograph Herbal teas (1435).
NOTE: the term comminuted used in European Community
legislation on herbal medicinal products describes a herbal
drug that has been either cut or powdered.
The term herbal drug preparation is synonymous with the term
herbal preparation used in European Community legislation
on herbal medicinal products.
____________________________________________ ~E~
***
Essential Oils
*** ***
(Ph Bur monograph 2098) ***
Essential Oils comply with the requirements of the Buropean
Pharmacopoeia. These requirements are reproduced below.
~E~ ____________________________________________
The statements in this monograph are intended to be read in
conjunction with individual monographs on essential oils in the
European Pharmacopoeia. Application of the monograph to other
essential oils may be decided by the competent authority.
DEFINITION
Odorous product, usually of complex composition, obtained
from a botanically defined plant raw material by steam
distillation, dry distillation, or a suitable mechanical process
without heating. Essential oils are usually separated from the
aqueous phase by a physical process that does not
significantly affect their composition.
Essential oils may be subjected to a suitable subsequent
treatment. Thus an essential oil may be commercially known
as being deterpenated, desesquiterpenated, rectified or
'x'-free.
- A deterpenated essential oil is an essential oil from which
monoterpene hydrocarbons have been removed, partially
or totally.
- A deterpenated and desesquiterpenated essential 011 is an
essential oil from which mono- and sesquiterpene
hydrocarbons have been removed, partially or totally.
2014 General Monographs IV-45

- A rectified essencial oil is an essential oil that has been Chromatographic profile
subjected to fractional distillation to remove certain Gas chromatography (2.2.28): use the normalisation
constituents or modify the contento procedure.
- An 'x'-free essencial oil is an essential oil that has been In addition to the system suitability test given in the specific
subjected to partial or complete removal of one or more monograph, it is necessary to check the suitability of the
constituents. chromatographic system using the following test, which is to
PRODUCTION be carried out periodically within the framework of
Depending on the monograph, the plant raw material may be performance qualification.
fresh, wilted, dried, whole, broken or ground. The chromatogram shown in Figure 2098.-1 is given as an
Steam distillation The essential oil is produced by the example.
passage of steam through the plant raw material in a suitable Reference solution essential oil CRS. If necessary, the reference
apparatus. The steam may be introduced from an extemal solution can be diluted with heptane R .
source or generated by boiling water below the raw material Column:
or by boiling water in which the raw material is immersed. -- material: fused silica;
The steam and oil vapours are condensed. The water and - size: 1 = 60 m, 0 = 0.25 mm;
essential oil are separated by decantation. - stationary phase: macrogol 20 000 R (0.25 ¡lm).
Dry distillation The essential oil is produced by high- Cam'er gas heliwn for chromatography R.
temperature heating of stems or barks in a suitable apparatus Flow rate 1.5 mUmin.
without the addition of water or steam.
Split ratio 1:500. The split ratio/injection volume can be
Mechanical process The essential oil, usually known as adjusted in order to fit the specific equipment used, provided
'cold-pressed', is produced by a mechanical process without that the column load stays the same.
any heating. It is mainly applied to Citrus fruit and involves
Temperature:
expression of the oil from the pericarp and subsequent
separation by physical means. Time Temperature
(min) (OC)
In certain cases, a suitable antioxidant may be added to the
essential oil. Column 0·15 70

15· 100 70 -t 240

CHARACTERS
The appearance and the odour of the essential oil is 100 - JOS 240
determined. Injection port 250
IDENTIFICATION Detector 270
Essential oils are identified by their gas chromatographic
profile, or failing this, by any other test that may be required Detection F lame ionisation.
(for example, a test by thin-layer chromatography) . Injection 1 ¡lL.
TESTS ldentification of components Use the chromatogram supplied
GENERAL TESTS with essential oil CRS.
The essential oil complies with the prescribed limits for the System suitabllity Reference solution:
following tests. - resolution: minimum 1.5 between the peaks due to linalol
Relative density (2.2.5) and linalyl aceta te;
- signal-to-noise ratio: minimum 100 for the peak due to
Refractive index (2.2.6)
decanal;
Optical rotation (2.2.7) - limits: the percentage content of each of the 9 components
Fatty oils and resinified essential oils (2.8.7) is within the limits stated on the leaflet provided with
SUPPLEMENTARY TESTS essential oil CRS.
If necessary, the essential oil complies with the prescribed STORAGE
limits for the following tests. In a well-filled, airtight container, protected from light.
Freezing point (2.2.18)
LABELLING
Acid value (2.5.1) The label states:
Peroxide value (2.5.5) - the scientific name of the plant raw material used;
Foreign esters (2.8.6) - where applicable, the type and/or the chemotype of the
essential oil;
Residue on evaporation (2.8.9) - where applicable, the method of production;
Water (2.8.5) -- where applicable, the name and concentration of any
Solubility in alcohol (2.8.10) added antioxidant;
- where applicable, additional processing steps that are not
F alsification
specified under Definition.
If appropriate, a test for one or more falsifications may be
___________________________________________ PhE~

carried out by thin-layer chromatography (2.2.27), by gas

chromatography (2.2.28) using a chiral colurnn if necessary,
or by any other suitable method.
IV-46 General Monographs
2 5 6
3
-t-- .,
I ~ ti, ¡ I
O 40
1. a-pinene 3. hexanol
2. cineole 4. decanal
Figure 2098.-1. - Chromatogram for the test for chromatographic profile of essential oi/s
EXTRACTS
(Ph Eur monograph 0765)
Extracts comply with the requirements of the European
Pharmacopoeia. These requirements are reproduced below.
~ Ew ____________________________________________
DEFINITlON
Extraets are preparations of liquid (liquid extraets and
tinetures), semi-solid (soft extraets and oleoresins) or solid
(dry extraets) eonsisteney, obtained from herbal drugs or
animal matter, whieh are usually in a dry state.
Where medicinal produets are manufaetured using extraets of
animal origin, the requirements of ehapter 5.1.7. Viral safety
apply.
Different types of extraet may be distinguished. Standardised
extraets are adjusted within an aeeeptable toleranee to a
given eontent of eonstituents with known therapeutie aetivity;
standardisation is aehieved by adjustrnent of the extraet with
inert material or by blending batehes of extraets. Quantified
extraets are adjusted to a defined range of eonstituents;
adjustrnents are made by blending batehes of extraets. Other
extraets are essentially defined by their production proeess
(state of the herbal drug or animal matter to be extracted,
solvent, extraetion conditions) and their specifieations.
2014
9
i I I I I i , ¡ , i i
50 60 70 80 90 min
5.linalol 7. [3-caryophyllene 9. benzyl salicylate
6_ linalyl acetate 8. eugenol
PRODUCTlON
Extraets are prepared by suitable methods using ethanol or
other suitable solvents. Different batches of the herbal drug
or animal matter may be blended prior to extraetion.
The herbal drug or animal matter to be extracted may
undergo a preliminary treatment, for example, inactivation of
enzymes, grinding or defatting. In addition, unwanted matter
may be removed after extraetion.
Herbal drugs, animal matter and organic solvents used for
the preparation of extracts comply with any relevant
monograph of the Pharmacopoeia. For soft and dry extracts
where the organic solvent is removed by evaporation,
recovered or recycIed solvent may be used, provided that the
reeovery proeedures are controlled and monitored to ensure
that solvents meet appropriate standards before re-use or
admixture with other approved materials. Water used for the
preparation of extracts is of a suitable quality. Exeept for the
test for bacterial endotoxins, water complying with the
section on Purified water in bulk in the monograph on
Purified water (0008) is suitable. Potable water may be
suitable if it complies with a defined specifieation that alIows
the eonsistent production of a suitable extracto
Where applicable, concentration to the intended eonsistency
is earried out using sUÍtable methods, usually under redueed
pressure and at a temperature at which deterioratíon of the
constituents ís redueed to a minimum. Essentíal oíls that
2014
have been separated during proeessing may be resto red to the
extraets at an appropriate stage in the manufaeturing proeess.
Suitable exeipients may be added at various stages of the
manufaeturing proeess, for example to improve teehnologieal
qualities sueh as homogeneiry or eonsisteney. Suitable
stabilisers and antimicrobial preservatives may also be added.
Extraetion with a given solvent leads to rypieal proportions of
eharaeterised eonstituents in the extraetable matter; during
produetion of standardised and quantified extraets,
purifieation proeedures may be applied that inerease these
proportions with respeet to the expeeted values; sueh extraets
are referred to as 'refined'.
IDENTIFICATION
Extraets are identified using a suitable method.
TESTS
Where applieable, as a result of analysis of the herbal drug or
animal matter used for produetion and in view of the
produetion proeess, tests for mierobiologieal qualiry (5.1.4),
heavy metal s, afiatoxins and pestieide residues (2.8.13) in the
extraets may be neeessary.
ASSAY
Wherever possible, extraets are assayed by a suitable method.
LABELLING
The label states:
- the herbal drug or animal matter used;
- whether the extraet is liquid, soft or dry, or whether it is a
tineture;
- for standardised extraets, the eontent of eonstituents with
known therapeutie aetiviry;
- for quantified extraets, the eontent of eonstituents
(markers) used for quantifieation;
- the ratio of the starting material to the genuine extraet
(extraet without exeipients) (DER);
- the solvent or solvents used for extraetion;
- where applieable, that a fresh herbal drug or fresh animal
matter has been used;
- where applieable, that the extraet is 'refined';
- the name and amount of any exeipient used including
stabilisers and antimicrobial preservatives;
- where applieable, the pereentage of dry residue.
LIQUID EXTRACTS
DEFINITION
Liquid extraets are liquid preparations of whieh, in general,
1 part by mass or volume is equivalent to 1 part by mass of the
dried herbal drug or animal matter. These preparations are
adjusted, if neeessary, so that they satisfY the requirements
for eontent of solvent, and, where applieable, for
eonstituents.
PRODUCTION
Liquid extracts are prepared by using ethanol of a suitable
eoneentration or water to extraet the herbal drug or animal
matter, or by dissolving a soft or dry extraet (whieh has been
produeed using the same strength of extraetion solvent as is
used in preparing the liquid extraet by direet extraetion) of
the herbal drug or animal matter in either ethanol of a
suitable eoneentration or water. Liquid extraets may be
filtered, if neeessary.
A slight sediment may form on standing, whieh is aeeeptable
as long as the eomposition of the liquid extraet is not
ehanged signifieantly.
General Monographs IV-47
TESTS
Relative density (2.2.5)
Where applieable, the liquid extraet eomplies with the limits
preseribed in the monograph.
Ethanol (2.9.10)
For a1coholie liquid extraets, carry out the determination of
ethanol eontent. The ethanol eontent eomplies with that
preseribed.
Methanol and 2-propanol (2.9. 11)
Maximum 0.05 per eent V/V of methanol and maximum
0.05 per eent V/V of 2-propanol for a1coholie liquid extraets,
unless otherwise preseribed.
Dry residue (2.8. 16)
Where applieable, the liquid extraet eomplies with the limits
preseribed in the monograph, eorreeted if necessary, taking
into account any exeipient used.
STORAGE
Proteeted from light.
LABELLING
The label sta tes in addition to the requirements listed aboye:
- where applicable, the ethanol eontent in per eent V/V in
the final extraet.
TINCTURES
DEFINITION
Tinetures are liquid preparations that are usually obtained
using either 1 part of herbal drug or animal matter and
10 parts of extraction solvent, or 1 part of herbal drug or
animal matter and 5 parts of extraetion solvent.
PRODUCTION
Tinetures are prepared by maeeration or pereolation (outline
methodology is given below) using only ethanol of a suitable
concentration for extraction of the herbal drug or animal
matter, or by dissolving a soft or dry extract (whieh has been
produced using the same strength of extraetion solvent as is
used in preparing the tincture by direct extraction) of the
herbal drug or animal matter in ethanol of a suitable
concentration. Tinctures are filtered, if neeessary.
Tinetures are usually c1ear. A slight sediment may form on
standing, whieh is aeceptable as long as the composition of
the tincture is not changed significantly.
Production by maceration
Unless otherwise prescribed, reduce the herbal drug or
animal matter to be extracted to pieces of suitable size, mix
thoroughly with the preseribed extraction solvent and allow
to stand in a c10sed container for an appropriate time.
The residue is separated from the extraetion solvent and, if
neeessary, pressed out. In the latter case, the 2 liquids
obtained are eombined.
Production by percolation
If neeessary, reduce the herbal drug or animal matter to be
extraeted to pie ces of suitable size. Mix thoroughly with a
portion of the preseribed extraction solvent and allow to
stand for an appropriate time. Transfer to a pereolator and
allow the pereolate to fiow at room temperature slowly
making sure that the herbal drug or animal matter to be
extraeted is always eovered with the remaining extraction
solventoThe residue may be pressed out and the expressed
liquid combined with the pereolate.
 IV-48 General Monographs 2014

TESTS los s on drying with a different limit or a test on water is

Relative density (2.2.5) prescribed in the monograph.
Where applicable, the tincture complies with the limits TESTS
prescribed in the monograph. Water (2.2.13)
Ethanol (2. 9.10) Where applicable, the dry extract complies with the limits
The ethanol content complies with that prescribed. prescribed in the monograph.
Methanol and 2-propanol (2.9.11) Loss on drying (2.8.17)
Maximum 0.05 per cent V/V of methanol and maximum Where applicable, the dry extract complies with the limits
0.05 per cent V/V of 2-propanol, unless otherwise prescribed. prescribed in the monograph.
Dry residue (2.8. 16) Solvents
Where applicable, the tincture complies with the limits Residual solvents are controlled as described in chapter 5.4,
prescribed in the monograph, corrected if necessary, taking unless otherwise prescribed 01' justitied and authorised.
into account any excipient used.
STORAGE
STORAGE In an airtight container, protected from light.
Protected from light. _ _________________________________________ PhE~

LABELLING
The label sta tes in addition ro the requirements listed aboye:
- for tinctures other than standardised and quantified
tinctures, the ratio of starting material ro extraction liquid
or of starting material ro final tincture;
- the ethanol content in per cent V/V in the final tincture.
SOFT EXTRACTS
DEFINITlON
Soft extracts are semi-solid preparations obtained by
evaporation or partial evaporation of the solvent used for
extraction.
TESTS
Dry residue (2.8.16)
The soft extract complies with the limits prescribed in the
monograph.
Solvents
Residual solvents are controlled as described in chapter 5.4,
unless otherwise prescribed or justitied and authorised.
STORAGE
Protected from light.
OLEORESINS
DEFINITlON
Oleoresins are semi-solid extracts composed of a resin in
solution in an essential andlor fatty oil and are obtained by
evaporation of the solventes) used for their production.
This monograph applies to oleoresins produced by extraction
and not to natural oleoresins.
TESTS
Water (2.2.13)
The oleoresin complies with the limits prescribed in the
monograph.
Solvents
Residual solvents are controlled as described in chapter 5.4,
unless otherwise prescribed or justified and authorised.
STORAGE
In an airtight container, protected from light.
DRY EXTRACTS
DEFINITlON
Dry extracts are solid preparations obtained by evaporation
of the solvent used for their production. Dry extracts have a
los s on drying of not greater than 5 per cent m/m, unless a
Tinctures 01 the British Pharmacopoeia
In addition to the requirements for TincUlres of the European
Pharmacopoeia (stated under Extracts), the follozoing statements
apply to those tinctures that are the subject of an individual
mOl1ograph in the British Phannacopoeia.
DEFINITlON
Certain preparations of the British Pharmacopoeia entitled
Tinctures do not conform strictly ro the definition of the
European Pharmacopoeia and consequently application of
sorne of the aboye requirements is inappropriate.
Any necessary exceptions are stated in the relevant individual
monographs.
Herbal Teas ***
*** ***
(Ph Eur monograph 1435) ***
Herbal Teas comply zoith the requirements of the European
Pharmacopoeia. These requimnents are repl'Oduced be/ozo.
Ph Eur ___________________________________________
DEFINITlON
Herbal teas consist exclusively of one or more herbal drugs
intended for oral aqueous preparations by means of
decoction, infusion or maceration. The preparation is
prepared immediately before use.
Herbal teas are usually supplied in bulk form or in bags for
single use.
The herbal drugs used comply with the appropriate
individual European Pharmacopoeia monographs or in their
absence with the general monograph Herbal dnlgs (1433) .
IDENTIFICATlON
The identity of herbal drugs present in herbal teas is checked
by suitable methods such as botanical examinations andlor
chromarographic protiles.
TESTS
Recommendations on the microbiological quality of herbal
teas (5.1.8.) take into account the prescribed preparation
method (use of boiling or non-boiling water).
The proportion of herbal drugs present in herbal teas is
checked by appropriate methods.
Herbal teas in bags comply with the following test:
 2014
Uniformity of mass
Determine the individual and the average mas s of the
contents of 20 randomly chosen units as follows: weigh a
single full bag of herbal tea, open it without losing any
fragments. Empty it completely using a brush. Weigh the
empty bag and calcula te the mass of the contents by
subtraction. Repeat the operation on the 19 remaining bags
and calculate the average mass of the contents of the
20 units. Unless otherwise justified, not more than 2 of the
20 individual mas ses deviate from the average mass by more
than the percentage deviation shown in the table below and
none deviates by more than rwice that percentage.
Average mass Percentage deviation
less than 1.5 g 15 per cent
1.5 g to 2.0 g included 10 per cent
more than 2.0 g 7.5 per cent
STORAGE
Protected from light.
____________________________________________ ~E~
***
Instant Herbal Teas
*** ***
(Ph Eu1' monogmph 2620) ***
~E~ ____________________________________________
DEFINITION
Instant herbal teas consist of I or more herbal drug
preparations (primarily extracts with or without added
essential oils), and are intended for the preparation of an oral
solution immediately before use.
Instant herbal teas may also contain, in addition to herbal
drug preparations, suitable excipients such as maltodextrin
and added ftavourings.
Instant herbal teas are presented as a powder or granules and
are usually supplied in bulk form or in sachets.
The herbal drug preparations used comply with the
appropriate individual European Pharmacopoeia monographs
or, in the absence of such individual monographs, with the
general monograph Herbal drug prepamnons (1434) and with
other appropriate general monographs, for example Extraets
(0765) or Essential oils (2098).
IDENTIFICATION
The identity of herbal drug preparations present in instant
herbal teas is checked by suitable methods.
TESTS
General chapter 5.1.8 contains recommendations on the
microbiological quality of extract-containing herbal medicinal
products such as instant herbal teas.
The proportion of herbal drug preparations present in instant
herbal teas is checked by suitable methods.
Instant herbal teas in sachets comply with the following test.
Uniformity of mass
Determine the individual and the average mass of the
contents of 20 randomly chosen units as follows: weigh a
single full sachet of instant herbal tea, open it without losing
any fragments. Empty it completely using a brush. Weigh the
empty sachet and calculate the mass of the contents by
subtraction. Rep eat the operation on the 19 remaining
Acanthopanax Bark IV-49
sachets and calculate the average mass of the contents of the
20 units . Unless otherwise justified, not more than 2 of the
individual masses deviate from the average mas s by more
than the percentage deviation shown in the table below and
none deviates by more than twice that percentage.
Average mass Percentage deviation
less than 1.5 g 15 per cent
1.5 g to 2.0 g included 10 per cen t
more than 2.0 g 7.5 per cent
STORAGE
Protected from light.
____________________________________________ ~E~
***
Acanthopanax Bark * *
*
******
(Ph Eu1' monograph 2432)
~E~ ____________________________________________
DEFINITION
Dried root bark of Eleuthe1'Oeoccus graeilistylus (W.W.Sm.)
S.Y.Hu var. nodijlorus (Dunn) H .Ohashi (Aeanthopanax
gmcilistylus W.W.Sm.) collected in summer and autumn.
IDENTIFICATION
A. The bark occurs in irregular quills, 5-15 cm long,
0.4-1.4 cm in diameter, about 2 mm thick. The outer surface
is greyish-brown, with slightly twisted longitudinal wrinkles
and transverse lenticel-like scars. The inner surface is pale
yellow or greyish-yellow, with fine longitudinal striations.
The texture is light, fragile, easily broken. The fracture is
irregular, greyish-white.
B. Microscopic examination (2.8.23). The powder is greyish-
white. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
characters: cluster crystals of calcium oxalate, 8-64 11m in
diameter, sometimes included in crystal cells arranged in
rows; cork cells, rectangular or polygonal, thin-walled,
sometimes walls of cork cells of older barks unevenly
thickened, slightly pitted; fragments of secretory canals
containing colourless or pale yellow secretions. Examine
under a microscope using a 50 per cent V/V solution of
glyce1'Ol R. The powder shows abundant starch granules,
simple, polygonal or subspherical, 2-8 ~lm in diameter, or
compound with 2-10 components.
C. Examine the chromatogram obtained in the test for
Periploea sepium.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
TESTS
Periploca sepium
Thin-Iayer chromatography (2.2.27).
Test solution To 0.3 g of the powdered herbal drug (355)
(2.9.12) add 3 mL of methanol R, heat in a water-bath at
60 oC for 1 min and filter.
Reference solution Dissolve 5 mg of thymol R and 8 mg of
borneol R in 5 mL of methanol R.
IV-50 Agnus Castus Fruit 2014

Top oC the plate Sorne of the fruits may retain a stalk, about 1 mm long.
- -- - --
A transverse section of the fruit shows 4 locules, each
containing an elongated seed.
Thyrnol : an orange zone
B. Reduce 10 a powder (355) (2.9.12). Examine under a
--- --- microscope using chloral hydrate solution R. The powder
Borneol: a brown zone shows the foIlowing diagnostic characters: fragments of the
outer epidermis of the calyx composed of polygonal ceIls
A broad pink zone
denseIy covered with short, bent or undulate, uni-, bi- or
ReCerence solution Test solution tri-ceIlular uniseriate covering trichomes; ceIls of the epicarp
with thick waIls and weIl-marked, large pits; isolated
glandular trichomes with a uniceIlular stalk and a uni- or
multi-ceIlular head; layers of parenchyma from the outer part
Plate TLC silica gel plate R (5-40 ~lm) [or TLC silica gel
of the mesocarp, sorne containing brown pigment, others
plate R (2-10 J..lm)).
extending into septa; fragments from the inner part of the
Mobile phase ethyl acetate R, methylene chloride R (2:98 V/V) . mesocarp composed of thin-waIled, pitted, sclerenchymatous
Application 20 J..lL [or 1 ~lL) as bands of 10 mm [or 8 mm) . ceIls and of typical isodiametric sclerous ceIls with very thick,
Development Over a path of 10 cm [or 6 cm). deeply grooved waIls and a narrow, steIlate lumen; smaIl
Drying In airo brown ceIls of the endocarp; fragments of the testa
containing areas of fairly large, thin-waIled lignified ceIls with
Detection Treat with anisaldehyde solution R , heat at 105 oC
reticulate bands of thickening; numerous fragments of the
for 5 min and examine in daylight.
endosperm composed of thin-waIled parenchymatous ceIls
Results The chromatogram obtained with the test solution containing aleurone grains and oil droplets.
shows no intense coloured zones aboye the zone due to
C. Thin-Iayer chromatography (2. 2.27) .
borneol in the chromatogram obtained with the reference
solution. Test solution T o l.0 g of the powdered drug (355) (2.9. 12)
add 10 mL of methanol R. Heat in a water-bath at 60 oC for
Acanthopanax giraldii
10 mino AIlow to cool and filter.
The outer surface of the root bark must not be covered with
scaly covering trichomes. R eference solution Dissolve 0.5 mg of aucubin R and 1 mg of
agnuside R in methanol R and dilute to 1.0 mL with the same
Loss on drying (2.2.32) solvento
Maximum 12.0 per cent, determined on l.000 g of the
powdered herbal drug (355) (2.9. 12) by drying in an oven at Plate TLC silica gel F254 plate R (5-40 J..lm) [or TLC szlica gel
105 oC for 2 h. F 254 plate R (2-10 J..lm)).
Mobile phase water R, methanol R, ethyl acetate R
Total ash (2.4.16)
(8: 15:77 V/V/V).
Maximum 12.0 per cent.
Application 10 J..lL [or 8 J..lL) as bands.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cenl. Development Over a path of 8 cm [or 5 cm) .
Drying In air.
Extractable matter
Minimum 16.0 per cenl. Detection Spray withformic acid R and heat at 120 oC for
10 min; examine in daylight.
To 2.00 g of the powdered herbal drug (250) (2.9.12) add a
mixture of 8 g of water R and 12 g of ethanol (96 per cent) R Results See below the sequence of zones present in the
and aIlow to macerate for 2 h, shaking frequently. Filter, chromatograms obtained with the reference solution and the
evaporate the filtrate to dryness on a water-bath in vacuo and test solution. Furthermore, other zones may be present in the
dry in an oven at 100-105 oC for 2 h. The residue weighs a chromatogram obtained with the test solution.
mínimum of 320 mg.
_ __ _ _ _ _ __ _ __ _ _ _ _ __ _ ____ ~Ew
Top of the plate

- -- ---
Agnuside: a blue zone A blue zone (agnuside)
Agnus Castus Fruit ***
*** *** - -- - --
(Ph Bur monograph 2147) *** Aucubin: a blue zone A blue zon e (aucubin)
~Ew ____ _ __ __ _ __ __ _ __ __ __ ___
Reference solution Test solution
DEFINITlON
Whole, ripe, dried fruit of Vitex agnus-castus L.
TESTS
Content
Foreign matter (2.8.2)
Minimum 0.08 per cent of casticin (C19H 1SOS; M r 374.3) Maximum 3.0 per cenl.
(dried drug).
Other species of Vitex, in particular Vitex negundo
IDENTIFICATlON No fruit of other species with a much greater diameter is
A. Agnus casrus fruit is oval or almost globular, with a presento
diameter of up to 5 mm. The persistent calyx is greenish-
Total ash (2.4.16)
grey, finely pubescent, ends in 4-5 short teeth and envelops
Maximum 5.0 per cent.
2/3 to 3/4 of the surface of the fruil. The blackish-brown
fruit consists of a pericarp that becomes progressively
sclerous up to the endocarp. The style scar is often visible.
2014 Agnus Castus Fruit IV-51

o 2 4 6 8 10 min

l. penduletin 2. casticin

Figure 2147.'1. - Chromatogram for the assay of casticin in Agnus castus frui!: test solution

Loss on drying (2.2.32) Time Mobile phase A Mobile phase B

Maximum 10.0 per eent, determined on 1.000 g of the (min) (percent VM (percent VM
powdered drug (355) (2.9. 12) by drying in an oven at 0-13 50 ~35 50 ~ 65
105 oC for 2 h. 13 - 18 35 ~ O 65 ~ 100
ASSAY 18 - 23 O ~ 50 lOO ~ 50
Liquid ehromatography (2.2.29).
Test solution Extraet 1.000 g of the powdered drug (355) Flow rate 1.0 mUmin.
(2. 9. 12) with 40 mL of methanol R for 2 min using a Detection Speetrophotometer at 348 nm.
suitable-speed homogeniser. Colleet the supernatant liquid Injection 10 flL.
and filter into a 250 mL fiask. Repeat the extraetion with a System suitability Test solution:
further 40 mL of methanol R, eolleeting the supernatant - resolution: minimum 1.5 between the peaks due to
liquid and filtering as before. Rinse the residue earefully with penduletin and eastiein (see Figure 2147.-1 ).
a small quantity of methanol R. Combine the methanol
Calculate the pereentage eontent of eastiein using the
extraets and rinsings and evaporate to dryness in vaeuo in a
following expression:
water-bath at not more than 30 oC. With the aid of
ultrasound, dissolve the residue obtained in methanol R and
dilute to 20 .0 mL with the same solvento Filter the solution
through a membrane filter (nominal pore size 0.45 flm).
Dilute 1.0 mL to 10.0 mL with methanol R.
Referenee solution Dissolve 100.0 mg of agnus eastusfruit area of the peak due to eastiein in the ehroma1Ogram
standardised dry extraet CRS in 20.0 mL of methanol R with obtained with the test solution;
the aid of ultrasound for 20 min, then dilute 10 25.0 mL with area of the peak due 10 easticin in the ehroma1Ogram
the same solvento Filter the solution through a membrane obtained with the referenee solution;
filter (nominal pore size 0.45 flm). mas s of the drug used to prepare the test solution, in
Column: grams;
- size: 1 = 0.125 m, 0 = 3.0 mm; mas s of agnus eastus fruit standardised dry extraet CRS
- stationary phase: octadecylsilyl siliea gel for ehromatography R used 10 prepare the referenee solution, in grams;
(3 flm); PI pereentage eontent of eastiein in agnus castus fruit
- temperature: 25 oc. standardised dry extract CRS.
Mobile phase: __________________________________________ ~Ew

- mobile phase A: 5.88 gIL solution of phosphoric acid R;

- mobile phase B: methanol R;
 IV-52 Agrimony 2014

Agrimony *****
** *
(Ph Eur monograph 1587) *** * A
~Eif ____________________________________________

DEFINITION
Dried flowering tops of Agn:monia eupatoria L.
Ab
Content
Minimum 2.0 per cent ofta=ins, expressed as pyrogalIol Aa---t\'~n

(C 6H 6 0 3 i M r 126.1) (dried drug).

IDENTIFICATION
A. The stem is green or, more usualIy, reddish, cylindrical
and infrequently branched. It is covered with long, erect or
tangled hairs . The leaves are compound imparipennate with
3 or 6 opposite pairs of leaflets, with 2 or 3 smaller leaflets
between. The leaflets are deeply dentate to serrate, dark
green on the upper surface, greyish and densely tomentose
on the lower face. The flowers are small and form a terminal
spike. They are pentamerous and borne in the axils of hairy
bracts, the calyces closely surrounded by numerous terminal
hooked spires, which occur on the rim of the hairy
receptacle. The petals are free, yelIow and deciduous. Fruit-
bearing obconical receptacles, with deep furrows and hooked
bristles, are usually present at the base of the inflorescence.
B. Reduce to a powder (355) (2.9.12). The powder is
yelIowish-green or grey. Examine under a microscope using
ehloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1587.-1) : numerous straight or
bent, unicellular, long, thick-walled (about 500 ¡.tm) covering Figure 1587.-1.- Illustration for identification test B of
trichomes [Ab, Ca, F], finely warty, and sometimes spirally powdered herbal drug of agrimony
marked, often fragmented (F)i fragments of the epidermis of
the stems [A] with stomata [Aa], covering trichomes (Ab)
and glandular trichomes [Ac]i fragments of upper leaf the plate to dry in air for 30 min and examine in ultraviolet
epidermis in surface view [C] with straight walIs bearing light at 365 nm.
covering trichomes (Ca), accompanied by palisade Results See below the sequence of zones present in the
parenchyma [Cb], with sorne of the celIs containing calcium chromatograms obtained with the reference solution and the
oxalate prisms [Ce]; fragments of lower leaf epidermis in test solution.
surface view m with sinuous walIs and abundant stomata
Ua], mostly anomocytic (2.8.3) but occasionally anisocytic,
Top of the plate
and glandular trichomes Ub] i ovoid to subspherical polIen
grains, with 3 pores and a smooth exine [D]i glandular An orange fluorescent zone may
trichomes with a multicellular, uniseriate stalk and a be present (quercitroside)
unicellular to quadricellular head [B, Jb] i fragments of the lsoquercitroside: an orange An orange fluorescent zone
fluorescent zone (isoquercitroside)
stems [H] with groups of fibres [Ha] and parenchymatous
cells, sorne of which contain cluster crystals of calcium An orange fluorescent zone
(hyperoside)
oxalate [Hb]i small spiral vessels from the leaflets [G]i
Rutin: an orange fluorescent zone An orange fluorescent zone (rutin)
fragments of large, spiral or bordered-pitted ves seIs from the
Reference solution Test solution
stem [E] .
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 2.0 g of the powdered drug (355) (2. 9.12)
TESTS
add 20 mL of methanol R. Heat with shaking at 40 oC for
Loss on drying (2.2.32)
10 mino Filter.
Maximum 10.0 per cent, determined on l.000 g of the
Referenee solution Dissolve 1.0 mg of isoquercitroside R and powdered drug (355) (2.9.12) by drying in an oven at
1.0 mg of rutin R in 2 mL of methanol R. 105 oC for 2 h.
Plate TLC siliea gel plate R.
Total ash (2.4.16)
Mobile phase anhydrous formie acid R, water R , ethyl aeetate R Maximum 10.0 per cent.
(10:10:80 VIV/V).
ASSAY
Application 10 ¡.tL as bands.
Carry out the determination of tannins in herbal drugs
Development Over a path of 12 cm. (2.8.14). Use l.000 g ofpowdered drug (180) (2. 9.12).
Drying At 100-105 oc. ____________________________________________ ~Eif

Detection Spray the still-warm plate with a 10 giL solution

of diphenylborie acid aminoethyl ester R in methanol R and then
with a 50 gIL solution of maerogol 400 R in methanol Ri allow
 2014 Aloes IV-53

Alchemilla *** Dlying At 100-105 oC for 5 mino

*** ***
(Ph Eu/' monograph 1387) ***
nE~ ____________________________________________
DEFINITION
Whole or cut, dried, flowering, aerial parts of Alehemilla
vulgaris L. sensu latiore.
Content
Minimum 6.0 per cent of tannins, expressed as pyrogallol
(C 6 H 6 0 3 ; M r 126.1) (dried drug).
IDENTIFICATION
A. The greyish-green, partly brownish-green, radical leaves
which are the main part of the drug are reniform or slightly
semicircular with a diameter generally up to 8 cm, seldom up
to 11 cm and have 7 to 9, or 11 lobes and a long petiole.
The smaller, cauline leaves, which have a pair of large
stipules at the base, have 5-9 lobes and a shorter petiole or
they are sessile. The leaves are densely pubescent especially
Detection Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R. Subsequently spray with a
50 gIL solution of macrogol 400 R in methanol R . Allow to dry
in air for about 30 mino Examine in ultraviolet light at
365 nm.
Results See below the sequence of the zones present in the
chromatograms obtained with the referenee solution and the
test solution. Furthermore, other fluoreseent zones may be
present in the chromatograrn obtained with the test solution.
Top of the plate
2 red fluorescent zones
(chlorophyll)
Caffeic acid: a light blue florescent 1 or 2 intense light blue fluo rescent
zone zones
One or several in tense green or
greenish·yellow fluorescent zones
----- - --

on the lower surface and have a coarsely serrated margino Chlorogenic acid: a light blue An in tense yellow or orange
Young lea ves are folded with a whitish-silvery pubescence; fluorescent zone fluorescent zone
older leaves are slightly pubescent and have a finely meshed ----- ---
venation, prominent on the lower surface. The greyish-green
Reference solution Test solution
or yellowish-green petiole is pubescent, about 1 mm in
diameter, with an adaxial groove. The apetalous flowers are
yellowish-green or light green and about 3 mm in diameter. TESTS
The calyx is double with 4 small segments of the epicalyx Loss on drying (2.2.32)
altemating with 4 larger sepals, subacute or triangular. They Maximum 10.0 per eent, determined on 1.000 g of
are 4 short stamens and a single carpel with a cap ita te powdered drug (355) (2.9.12) by drying in an oven at
stigma. The greyish-green or yellowish-green srem is 105 oC for 2 h .
pubescent, more or less longitudinally wrinkled and hollow.
Total ash (2.4.16)
B. Reduce to a powder (355) (2.9.12). The powder is
Maximum 12.0 per cenr.
greyish-green. Examine under a microscope using ehloral
hydrate solution R. The powder shows the following diagnostic ASSAY
characters: unicellular, narrow trichomes up to 1 mm long Carry out the determination of tannins in herbal drugs
partly tortuous, acuminate, and bluntly pointed at the apex, (2.8.14). Use 0.50 g ofthe powdered drug (355) (2.9.12).
with thick lignified walls, somewhat enlarged and pitted at ____________________________________________ PhE~

the base; fragments of leaves with 2 layers of palisade

parenchyma, the upper layer of which is 2-3 times longer
than the lower layer and with spongy parenchyma, containing
scattered cluster crystals of ealcium oxalate, up to 25 ~lm in Barbados Aloes ***
diameter; leaf fragments in surface view with sinuous or wavy *** ***
epidermal eells, the anticlinal walls unevenly thickened and Cura~ao Aloes ***
beaded, anomoeytie stomata (2.8.3); groups of vascular tissue (Ph Eur monograph 0257)
and lignified fibres from the petioles and stems, the ves seis
Preparation
spirally thiekened or with bordered pits; oecasional thin-
Standardised Aloes Dry Extraet
walled conieal trichomes, about 300 ¡.lm long; thin-walled
nE~ ____________________________________________
parenchyma containing cluster crystals of calcium oxalate;
spherical pollen grains, about 15 ~lm in diameter, with DEFINITION
3 distinet pores and a granular exine; occasional fragments of Concentrated and dried juiee of the leaves of Aloe barbadensis
the ovary wall with cells eontaining a single crystal of calcium Miller.
oxalare.
Content
C. Thin-layer chromatography (2.2.27). Minimum 28.0 per eent ofhydroxyanthracene derivatives,
Test solution To 0.5 g of the powdered drug (355) (2.9.12) expressed as barbaloin (C21H2Z09; M r 418.4) (dried drug).
add 5 mL of methanol R and heat in a water-bath at 70 oC
CHARACTERS
under a reflux condenser for 5 mino Cool and filter.
Appearance
Reference solution Dissolve 1.0 mg of caffeic acid R and
Dark brown masses, slightly shiny or opaque with a
1.0 mg of chlorogenic acid R in 10 mL of methanol R.
conchoidal fracture, or brown powder.
Plate TLC silica gel plate R.
Solubility
Mobile phase anhydrous formic acid R, water R, ethyl aceta te R Partly soluble in boiling water, soluble in hot ethanol
(8:8:84 V/V/V). (96 per cent).
Applieation 20 ~lL of the test solution and 10 ¡.lL of the
reference solution, as bands.
D evelopment Over a path of 10 cm.
 IV-54 Cape Aloes
IDENTIFICATION
A. Thin-layer chromatography (2.2.2 7) .
Test solution To 0.25 g of the powdered drug add 20 mL of
methanol R and heat to boiling in a water-bath. Shake for a
few minutes and decant the solution. Store at about 4 oC
and use within 24 h.
Reference solution Dissolve 25 mg of barbaloin R in
methanol R and dilute to 10 mL with the same solvento
Plate TLC silica gel G plate R.
M obile phase water R, methanol R, echyl acetate R
(13: 17:100 V/V/ V) .
Application 10 fiL, as bands of 20 mm by maximum 3 mm.
Develop117ent Over a path of 10 cm.
Drying In air.
Detection A Spray with a 100 gIL solution of potassium
hydroxide R in methanol R and examine in ultraviolet light at
365 nm .
Results A The chromatogram obtained with the test solution
shows in the central pan a yellow fluorescent zone
(barbaloin) similar in position to the zone due to barbaloin in
the chromatogram obtained with the reference solution and
in the lower pan a light blue fluorescent zone (aloesine).
Detection B Heat at 110 oC for 5 mino
Results B In the chromatogram obtained with the test
solution, a violet fluorescent zone appears just below the zone
due to barbaloin.
B. Shake 1 g of the powdered drug with 100 mL of boiling
water R . Cool, add 1 g of talc R and filter. To 10 mL of the
filtra te add 0.25 g of disodiu117 tecraborate R and heat to
dissolve. Pour 2 mL of this solution into 20 mL of water R .
Yellowish-green fluorescence appears which is panicularly
m arked in ultraviolet light at 365 nm.
C. To 5 mL of the filtrate obtained in identification test B
add 1 mL of freshly prepared bromine water R. A brownish-
yellow precipita te is formed and the supernatant liquid is
violet.
TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered drug by drying in an oven at 105 oc.
Total ash (2.4.16)
Maximum 2. 0 per cent.
ASSAY
Carry out the assay protected !ro117 briglu light.
Introduce 0.3 00 g ofpowdered drug (180) (2.9.12) into a
250 mL conical flask. Moisten with 2 mL of 117ethanol R, add
5 mL of water R warmed to about 60 cC, mix, then add a
further 75 mL of water R at about 60 cC and shake for
30 mino Cool, filter into a volumetric flask, rinse the conical
flask and filter with 20 mL of water R, add the rinsings to the
volumetric flask and dilute to 1000.0 mL with water R.
Transfer 10.0 mL of this solution to a 100 mL round-
bottomed flask containing 1 mL of a 600 gIL solution of
fem'c chloride R and 6 mL of hydrochlon'c acid R. Heat in a
water-bath under a reflux condenser for 4 h, with the water
level aboye that of the liquid in the flask. Allow to cool,
transfer the solution to a separating funnel, rinse the flask
successively with 4 mL of water R, 4 mL of 1 M sodiu117
hydroxide and 4 mL of water R and add the rinsings to the
separating funnel. Shake the contents of the separating funnel
with 3 quantities, each of 20 mL, of echer R. Wash the
combined ether layers with 2 quantities, each of 10 mL, of
2014
water R. Discard the washings and dilute the organic phase to
100.0 mL with ether R. Evaporate 20.0 mL of the solution
carefully to dryness on a water-bath and dissolve the residue
in 10.0 mL of a 5 gIL solution of 117agnesiu117 acerate R in
methanol R . Measure the absorbance (2.2.25) at 512 nm
using methanol R as the compensation liquido
Calculate the percentage content of hydroxyanthracene
derivatives, as barbaloin, from the following expression:
A x 19.6
m
i.e. taking the specific absorbance of barbaloin to be 255.
A = absorbance at 512 nm,
m = mass of the substance to be examined, in grams.
STORAGE
In an airtight container.
_ _ _ _ _ __ _ __ _ __ _ __ _ _ __ _ _ Ph Eur
Cape Aloes
(Ph Eur 111onograph 0258)
Preparation
Standardised Aloes Dry Extract
nE~ _______________________
DEFINITION
Concentrated and dried juice of the leaves of various species
of Aloe, mainly Aloe ferox Miller and its hybrids.
Content
Minimum 18.0 per cent of hydroxyanthracene derivatives,
expressed as barbaloin (C 21 H 22 0 9 ; M,. 418.4) (dried drug) .
CHARACTERS
Appearance
Dark brown masses tinged with green and having a shiny
conchoidal fracture, or greenish-brown powder.
Solubility
Partly soluble in boiling water, soluble in hot ethanol
(96 per cent).
IDENTIFICATION
A. Examine the chromatograms obtained in the test for
Barbados aloes.
Results The chromatogram obtained with the test solution
shows in the central pan a yellow fluorescent zone
(barbaloin) similar in position to the zone due to barbaloin in
the chromatogram obtained with the reference solution and
in the lower part 2 yellow fluorescent zones (aloinosides A
and B) and 1 blue fluorescent zone (aloesine).
B. Shake 1 g ofthe powdered drug with 100 mL ofboiling
water R. Cool, add 1 g of talc R and filter. To 10 mL of the
filtrate add 0.25 g of disodiu117 tetraborace R and heat to
dissolve. Pour 2 mL of the solution into 20 mL of water R.
A yellowish-green fluorescence appears which is particularly
marked in ultraviolet light at 365 nm.
C . To 5 mL of the filtra te obtained in identification test B
add 1 mL of freshly prepared bromine water R. A yellow
precipitate is formed. The supernatant liquid is not violeto
TESTS
Barbados aloes
Thin-layer chromatography (2.2.27).
 2014 Aloes Preparations IV-55

*****
Test solutwn To 0.25 g of the powdered drug add 20 mL of
methanol R and heat ro boiling in a water-bath. Shake for a
Standardised Aloes Dry Extraet
** **
few minutes and decant the solution. Srore at about 4 oC (Ph Eur monograph 0259) ***
and use within 24 h. ~E~ ____________________________________________
Rejerence solution Dissolve 25 mg of barbaloin R in
methanol R and dilute to 10 mL with the same solvento DEFINITION
Standardised dry extract prepared from Barbados aloes or
Plate TLC silica gel G plate R.
Cape aloes, or a mixture of both.
Mobile phase water R, methanol R, ethyl aceta te R
(13:17:100 VIV/V).
Conteot
19.0 per cent to 21.0 per cent of hydroxyanthracene
Application 10 ¡,tL, as bands of 20 mm by maximum 3 mm. derivatives, expressed as barbaloin (C 21 R 22 0 9 ; M r 418.4)
Development Over a path of 10 cm. adjusted, if necessary (dried extract) .
Drying In air. PRODUCTION
Detection Spray with a 100 gIL solution of potassium The extract is produced from the herbal drug by a suitable
hydroxide R in methanol R . Reat at 110 oC for 5 min and procedure using boiling water.
examine in ultraviolet light at 365 nm.
CHARACTERS
Results The chromarogram obtained with the test solution
Appearaoce
shows no violet fluorescent zone just below the zone due to
Brown or yellowish-brown powder.
barbaloin.
Solubility
Loss on drying (2.2.32)
Sparingly soluble in boiling water.
Maximum 10.0 per cent, determined on 1.000 g of the
powdered drug by drying in an oven at 105 oC . IDENTIFICATION
Total ash (2.4.16) A. Thin-layer chromarography (2.2.27).
Maximum 2.0 per cent. Test solution To 0.25 g of the extract to be examined add
20 mL of methanol R and heat ro boiling in a water-bath.
ASSAY
Shake for a few minutes and decant the solution. Store at
Carry out the assay protected jrom bright light.
about 4 oC and use within 24 h.
Introduce 0.400 g ofpowdered drug (180) (2.9.12) into a
Rejerence solution Dissolve 25 mg of barbaloin R in
250 mL conical fiask. Moisten with 2 mL of methanol R, add
methanol R and dilute ro 10 mL with the same solvento
5 mL of water R warmed ro about 60 oC, mix, then add a
further 75 mL of water R at about 60 oC and shake for Plate TLC silica gel G plate R.
30 mino Cool, filter into a volumetric fiask, rinse the conical Mobile phase water R, methanol R, ethy l aceta te R
fiask and filter with 20 mL of water R, add the rinsings ro the (13:17 :100 VIV/V).
volumetric fiask and dilute to 1000.0 mL with water R. Application 10).lL as bands of 20 mm by not more than
Transfer 10.0 mL ofthis solution ro a 100 mL round- 3 mm.
bottomed fiask containing 1 mL of a 600 giL solution of Development Over a path of 10 cm.
jemc chloride R and 6 mL of hydrochloric acid R. Reat in a
Drying In air.
water-bath under a refiux condenser for 4 h, with the water
level aboye that of the liquid in the fiask. AlIow to cool, Detection Spray with a 100 gIL solution of potassium
transfer the solution to a separating funnel, rinse the fiask hydroxide R in methanol R and examine in ultraviolet light at
successively with 4 mL of water R, 4 mL of 1 M sodium 365 nm.
hydroxide and 4 mL of water R and add the rinsings ro the Results The chromatogram obtained with the test solution
separating funne!. Shake the contents of the separating funnel shows, in the central part, a zone of yellow fiuorescence
with 3 quantities, each of 20 mL, of ether R. Wash the (barbaloin) similar in position to the zone due to barbaloin in
combined ether layers with 2 quantities, each of 10 mL, of the chromatogram obtained with the reference solution and
water R. Discard the washings and dilute the organic phase to in the lower part, a zone of light blue fiuorescence (aloesine) .
100.0 mL with ether R . Evaporate 20.0 mL of the solution In the lower part of the chromatogram obtained with the test
carefully to dryness on a water-bath and dissolve the residue solution 2 zones of yellow fiuorescence (aloinosides A and B)
in 10.0 mL of a 5 gIL solution of magnesium acetate R in (Cape aloes) and a zone of violet fiuorescence just below the
methanol R . Measure the absorbance (2.2.25) at 512 nm zone due to barbaloin (Barbados aloes) may be presento
using methanol R as the compensation liquido B. Shake 1 g with 100 mL ofboiling water R. Cool, add 1 g
Calculate the percentage content of barbaloin from the of talc R and filter. To 10 mL of the filtra te add 0.25 g of
following expression: disodium tetraborate R and heat to dissolve. Pour 2 mL of this
solution into 20 mL of water R. A yellowish-green
fiuorescence appears which is particularly marked in
A x 19.6
ultraviolet light at 365 nm.
m
TESTS
Loss 00 drying (2.8.17)
i.e . taking the specific absorbance of hydroxyanthracene
derivatives, as barbaloin, ro be 255. Maximum 4.0 per cent mimo
A = absorbance at 512 nm, Total ash (2.4.16)
m = mass of the substance ro be examined in grams. Maximum 2.0 per cent.

STORAGE
In an airtight container.
____________________________________________ ~E~
 IV-56 Anethum Graveolens Sowa Fruit
ASSAY
Carry out the assay protected fro111 brighr light.
Introduce 0.400 g into a 250 mL conical fiask. Moisten with
2 mL of methanol R, add 5 mL of water R warmed to about
60 oC, mix, add a further 75 mL of water R at about 60 oC
and shake for 30 min o Cool, filter into a volumetric flask,
rinse the conical flask and the filter with 20 mL of warer R,
add the rinsings to the volurnetric fiask and dilute to
1000.0 mL with warer R. Transfer 10.0 mL of this solution
ro a 100 mL round-bottomed flask containing 1 mL of a
600 giL solution of fen·jc chloride R and 6 mL of hydrochlorie
acid R. Heat in a water-bath under a reflux condenser for
4 h, with the water level aboye that of the liquid in the fiask.
AlIow to cool, transfer the solution to a separating funnel,
rinse the fiask successively with 4 mL of warer R, 4 mL of
1 M sodium hydroxide and 4 mL of water R, and add the
rinsings to the separating funnel. Shake the contents of the
separating funnel with 3 quantities, each of 20 mL, of
echer R. Wash the combined ether layers with 2 quantities,
each of 10 mL, of water R. Diseard the washings and dilute
the organic layer to 100.0 mL with erher R. Evaporate
20.0 mL earefully ro dryness on a water-bath and dissolve
the residue in 10.0 mL of a 5 gIL solution of magnesium
acetare R in methanol R . Measure the absorbanee (2.2.25) at
51 2 nm using methanol R as the compensation liquid.
Calculate the pereentage content of hydroxyanthracene
derivatives, expressed as barbaloin, using the fo llowing
expression:
A x 19.6
m CONFIRMATION
i.e. taking the speeific absorbance of barbaloin ro be 255.
A absorbance at 512 nm;
m = mass of the substance to be examined, in grams.
____________________________________________ PhEw
Anethum Graveolens Sowa Fruit
DEFINITION
Anethum Graveolens Sowa Fruit is the dried ripe fruit of
Anerhum graveolens L. Sowa Group.
Content
Ir eontains not less than 3.0% v/w of essential oil ealculated
with reference ro the anhydrous drug.
IDENTIFICATION
A. The dried fruits usually occur as separate mericarps,
pedicels normally absent; broadly oval, highly compressed
dorsally, about 4 mm long, 2 ro 3 mm broad, with 5 dorsal
ridges, each meriearp exhibiting 3 pale brown dorsal ridges,
the two lateral ridges elongated into eharacteristic
membranous wings; surface glabrous; remnants of the
stylopod at the apex; commissural surface fiat, often with
attached, paler brown carpophore; vittae visible as two
darker, arc-shaped, longitudinal bands.
B. Reduce ro a powder (355). The powder is pale brown.
Examine under a microscope using ehloral hydrate solution.
The powder contains numerous fragments of the epicarp,
with cuticular striations and infrequent anomoeytie stomata;
parquetry layer of endoearp in surface view; endosperm of
oval to rectangular thick-walled cells eontaining oil globules
and aleurone grains with embedded mierorosette erystals of
calcium oxalate; fragments of yellowish-brown septate vittae;
2014
parenchyma of mesocarp consisting of elongated, lignified,
reticulately thickened cells; sclereids of mesocarp thick-walled
with few pits.
C. Carry out the method for thin-layer chromarography,
Appendix III A, using the following solutions.
(1) Add 10mL of methanol (70%) to 0.5 g ofthe powdered
drug (355), mix and place in an ultrasonie bath for
30 minutes. Filter (a 0.45-flm PTFE is suitable) into a
10 mL volumetrie fiask and dilute ro 10 mL with
methanol (70%) .
(2) 0.05% w/v each of carvone and dillapiole in
methanol (70%).
CHROMATOGRAPHIC CONDITIONS
(a) Use high-performance silica gel 60 F 254 plates (Merek
silica gel 60 F 254 HPTLC plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 10 flL of each solution as 6 mm bands.
(d) Develop the plate to 8 cm.
(e) After removal of the plate, dry in air, spray with vanillin
reagent, heat the plate at 110 0 until the coloured bands
appear and examine in daylight.
MOB/LE PHASE
2 volumes of acetic acid, 10 volumes of ethyl aceta te and
88 volumes of roluene.
SYSTEM SUITABILITY
The test is not valid unless the ehromatogram obtained with
solution (3) shows two clearly separated bands.
The ehromarogram obtained with solution (1) shows a
purple band eorresponding in position and colour to the
band due to carvone in the chromarogram obtained with
solution (2); a brown band corresponding in position and
colour ro the band due to dillapiole and other bands as
shown in the table. Other bands may be presento
Top of the plate
A faint brown band
A brown band (dillapiole) A brown band (di llapiole)
A purple band (earvone) A purple band (earvo ne) A purple band (earvone)
A grey band
Solution (1) Solution (2) Solution (3)
TESTS
Apiole
Carry out the method for gas chromarography,
Appendix II! B, using the following solutions.
(1) Use the oil retained in the Assay of Essential oj].
(2) 1.0% w/v of apiole in roluene.
(3) 0.05 % v/v of {J-myrcene and 0.8% v/v of limonene in
roluene.
CHROMATOGRAPHIC CONDITIONS
(a) Use a fused silica eapillary colurnn (30 m x 0.53 mm)
bonded with a 1 flm film thickness, polyethylene glycol 20,000
(DB-Wax is suitable).
(b) Use helium as the carrier gas at l.5 mL per minute.
(e) Use the gradient conditions described in the table.
 2014 Angelica Archangelica Root IV-57

(d) Use a ftame ionisation detector maintained at a ASSAY

temperarure of 260°. Essential oil
(e) Inject 1 ¡.¡L of each solution at a temperature of 250°. Carry out the method for Essential Oils in Herbal Drugs,
Appendix XI E using 18 g of freshly prepared powdered drug
(1400) with 250 mL of water as the distillation liquido Distil
Time Temperature Comment at arate of 2 to 3 mL per minute for 2 hours using 0.50 mL
(min) of toluene in the graduated tube. Measure the quantity of
0-+5 60 isothermal
essential oil distilled and use in the tests for Apiole and
Chromatographic profile.
5 -+ 68 60 ...... 250 linear gradient

68 -+ 75 250 isothermal

SYSTEM SUITABILITY Angelica Archangelica Root *****

The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peaks due
to ~-myrcene and limonene, is at least 4.5.
In the chromatogram obtained with solution (3), the
substances elute in the following order: ~-myrcene and
limonene.
CONFIRMATION
In the chromatogram obtained with solution (1), there is no
peak corresponding to the peak due to apiole obtained with
solution (2).
Water
Not more than 10.0% v/w, Appendix IX C, method II using
30 g ofpowdered drug (1400).
Total Ash
Not more than 8.0%, Appendix XI J, Method lI.
Chromatographic profile
Carry out the method for gas chromatography,
Appendix III B, using the following solutions.
(1) Use the oil retained in the Assay of Essential oil.
(2) 0.4% w/v each of limonene, dihydrocarvone, carvone and
dz7lapiole in toluene.
(3) 0.05% v/v of {J-myrcene and 0.8% v/v of limonene in
toluene
CHROMATOGRAPHIC CONDITIONS
The chromatographic procedure described under the test for
Apiole may be used.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peaks due
to ~-myrcene and limonene is at least 4.5.
In the chromatogram obtained with solution (2), the
substances elute in the following order: limonene,
dihydrocarvone isomer 1, dihydrocarvone isomer 2, carvone
and dillapiole.
In the chromatogram obtained with solution (3), the
substances elute in the following order: ~-myrcene and
limonene.
Calculate the content of limonene, dihydrocarvone, carvone
and dillapiole by normalisation. Disregard the peak due to
toluene.
Limits:
-- limonene: 15.0 to 28.0%,
-- sum of dihydrocarvone isomers 1 and 2: 5.0 to 30.0%,
-- carvone: 20.0 to 45.0%,
-- dillapiole: 15.0 to 35.0%.
Disregard any peak with an area less than 0.025 times the
area of the peak due to carvone in the chromatogram
obtained with solution (2).
** **
(Ph Eur monograph 1857) ***
~E~ ____________________________________________
DEFINITION
Whole or cut, carefully dried rhizome and root of Angelica
archangelica L. (syn. A. officinalis Hoffm.).
Content
Minimum 2.0 mL/kg of essential oil (dried drug).
CHARACTERS
Bitter taste.
IDENTIFICATION
A. The rhizome is greyish-brown or reddish-brown, with
transversely annulated thickenings. The base bears greyish-
brown or reddish-brown, cylindrical, longitudinally furrowed,
occasionally branched roots often with incompletely
encircling, transverse ridges. The apex sometimes shows
remnants of stem and leaf bases. The fracture is uneven.
The transversely cut surface shows a greyish-white, spongy,
distinctly radiate bark, in which the secretory channels are
visible as brown spots, and a bright yellow or greyish-yellow
wood which, in the rhizome, surrounds the greyish or
brownish-white pith.
B. Microscopic examination (2.8.23). The powder is
brownish-white. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1857.-1): fragrnents of cork consisting of
severallayers of thin-walled, greyish-brown or reddish-brown
cells, in surface view [C) or in transverse section [E]; large,
yellowish-brown secretory channels, whole or fragrnented, in
transverse section [A] or in longitudinal section [F];
fragrnents of medullary rays, 2 or 4 cells wide [G]; fragrnents
of xylem [B] consisting of lignified vessels with reticulate
thickening [Ba] occurring singly or in small groups, and
unlignified parenchyma in which sorne of the cells associated
with the vessels are collenchymatously thickened. Examine
under a microscope using a 50 per cent V/V solution of
glycerol R. The powder shows numerous, simple starch
granules 2-4 ~lm in diameter, free or included in parenchyma
cells [D].
C. Examine the chromatograms obtained in the test for other
species of Angelica, Levisticum and Ligusticum described in the
European Pharrnacopoeia.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint ftuorescent zones may
be present in the chromatogram obtained with the test
solution.
 IV-58 Angelica Archangelica Root 2014

Top of the plate

(Z)·Ligustilide: a bluish·white
f1uorescent zone
--- ---
Osthole: a blue f1uorescent zone A blue f1uorescent zone

Imperatorin: a whitish f1uorescent A whitish f1uorescent zone

zone
A blue f1uorescent zone

--- ---
3 blue f1uorescent zones

Reference solution Test solution

Results B See below the sequence of zones present in the

chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint quenching zones may
be present in the chromatogram obtained with the test
solution.

Top of the plate

(Z}-Ligustilide: a blue f1uorescent

zone
--- ---
Osthole: a quenching zone A quenching zone

Imperatorin: a quenching zone A quenching zone

A quenching zone

--- ---
Figure 1857.·1. - Illustration for identification test B of
Several quenching zones
powdered herbal drug of angelica root
Reference solution Test solution

Loss on drying (2.2.32)

TESTS Maximum 10.0 per cent, determined on 1.000 g of the
Other species of Angelica, Levisticum and Ligusticum powdered herbal drug (355) (2.9.12) by drying in an oven at
described in the European Phannacopoeia 105 oC for 2 h.
Thin-layer chromatography (2.2.27). Total ash (2.4.16)
Test solution To 1 g of the freshly powdered herbal drug Maximum 10.0 per cent.
(355) (2.9.12) add 4 mL of heptane R, close and sonicate for
Ash insoluble in hydrochloric acid (2.8.1)
5 mino Centrifuge the mixture and use the supernatant.
Maximum 2.0 per cent.
Referenee solutwn Dissolve 1 mg of imperatonn R, 1 mg of
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of ASSAY
methanol R. Carry out the determination of essential oils in herbal drugs
(2.8. 12). Reduce the herbal drug to a powder (500) (2.9.12)
Plate TLC siliea gel F 254 plate R (2-10 Jlm).
and immediately use 40.0 g for the determination. Use a 2 L
Mobile phase glacial acetic acid R, ethyl aceta te R, toluene R round-bottomed fiask, 10 drops of liquid paraffin R, 500 mL
(1:10:90 VIVIJ;''). of water R as distillation liquid and 0.50 mL of xylene R in
Application 4 JlL as bands of 8 mm. the graduated tube. Distil at arate of 2-3 mUmin for 4 h.
Development Over a path of 6 cm. _ _ __ __ _ __ __ __ _ _ _ __ _ _ _ _ Ph Eur
Drying In air.
Deteetion A Examine in ultraviolet light at 365 nm.
Results A The chromatogram obtained with the test solution
shows no zone at the position of (Z)-ligustilide in the
chromatogram obtained with the reference solution.
Detection B Examine in ultraviolet light at 254 nm.
Results B The chromatogram obtained with the test solution
shows no zone at or just below the position of (Z)-ligustilide
in the chromatogram obtained with the reference solution.
Foreign matter (2.8.2)
Maximum 5 per cent of leaf bases and stem bases, maximum
5 per cent of discoloured pie ces and maximum 1 per cent of
other foreign matter.
 2014 Angelica Dahurica Root IV-59

Angelica Dahurica Root *** Results B See below the sequence of zones present in the
*** *** chromatograms obtained with the reference solution and the
(Ph Bur monograph 2556) *** test solution. Furthermore, other faint quenching zones may
PhE~ ______________________________________________ be present in the chromatogram obtained with the test
solution.
DEFINITION
Dried, whole or fragmented root, with roodets removed, of
Top of the plate
Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. &
Sayo collected in summer or autumn. (Z)-Ligustilide: a blue fluorescent A faint quenchi ng zone
zone
Content --- - --
Minimum 0.08 per cent of imperatorin (C 16H 140 4; Mr
270.3) (dried drug) . A quenching zone

IDENTIFICATION Osthole: a quenching zone

A. The non-fragmented drug consists of conical roots, about Imperatorin: a quenching zone A quenching zone (imperatorin)
10-25 cm long and 1.5-2.5 cm in diameter. The root crown,
more or less quadrangular, is obtuse and shows stem scars on
--- - --

prominences. It tapers to the tip oThe outer surface is

brownish-grey or yellowish-brown and clearly striated
Reference solution Test solution
longitudinally, showing scars of the secondary roots and
lenticel-like transverse protuberances, sorne of them arranged
in 4 longitudinal rows. The texture is compact, hard and Results C See below the sequence of zones present in the
heavy. The fracture, white or whitish grey and mealy, is chromatograms obtained with the reference solution and the
marked with concentric striations. The cambium occurs as a test solution. Furthermore, other faint zones may be present
brown ringo Very many brown dots, corresponding to a in the chromatogram obtained with the test solution.
transverse section of the secretory canals, are visible in the
cortical parto Top of the plate
E. Microscopic examination (2.8.23). The powder is
2 prominent reddish zones
yellowish-white. Examine under a microscope using chloral
hydrate solution R . The powder shows the following diagnostic (Z)-Ligustilide: a grey zone
characters: reticulate lignified ves seis, free or in groups of 2 --- ---
or 3 and accompanied by ligneous parenchyma cells with fine
cellulose walls; numerous fragments of parenchyma with A faint blue zone
ovoid cells; a few orange cork fragments, consisting of several Osthole : a violet zone
layers of superimposed cells; secretory canals, usually broken,
Imperatorin : a grey zone A yellow and violet double·zone
with yellow or pale brown contents and oil droplets . Examine
under a microscope using a 50 per cent V/V solution of --- - - -
glycerol R. The powder shows very many starch granules
A prominent violet zone
varying in size from 5 to 25 ¡.¡m; sorne are simple and
rounded, others consist of 2-8 elements, but most are Ayellow zone
polyhedral, either due to compound granules breaking up or Reference solution Test solution
to compression in the cells.
C . Examine the chromatograms obtained in the test for other
officinal species of Angelica, L evisticum and Ligusticum. TESTS
Results ASee below the sequence of zones present in the Other officinal species of Angelica, Levisticum and
chromatograms obtained with the reference solution and the Ligusticum
test solution. Furthermore, other faint fiuorescent zones may Thin-layer chromatography (2.2. 27).
be present in the chromatogram obtained with the test Test solution To 1 g of the powdered herbal drug (355)
solution. (2.9.12) add 4 mL of heptane R, close and sonicate for 5 min.
Centrifuge the mixture and use the supernatant.
Reference solution Dissolve 1 mg of imperatorin R, 1 mg of
Top of the plate
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of
(Z)-Ligustilide: a bluish·white A bluish-white f1uorescent zone methanol R.
f1uorescent zone
Plate TLC silica gel F 254 plate R (2-10 ¡.¡m).
- -- - --
Mobile phase glacial acetic acid R , ethyl acetate R, toluene R
A whitish f1uorescent zone (1 : 10:90 VIV/V).
Osthole: a blue f1uorescent zone A blue f1uorescent zone Application 4 ¡.¡L as bands of 8 mm.
Imperatorin : a whitish f1uorescent A whitish fluorescent zone Development Over a path of 6 cm.
zone (imperatorin) Drying In airo
--- --- Detection A Examine in ultraviolet light at 365 nm.
Results A The chromatogram obtained with the test solution
Reference solution Test solution shows no intense blue fiuorescent zone below the position of
imperatorin in the chromatogram obtained with the reference
solution.
 IV-60 Angelica Pubescens Root 2014

Detection B Examine in ultraviolet Iight at 254 nm.
Results B The chromatogram obtained with the test solution
shows no blue fiuorescent zone corresponding to the zone
due to (Z)-ligustilide in the chromatogram obtained with the
reference solution; the chromatogram obtained with the test
solution shows no quenching zone at the position of osthole
or below the position of imperatorin in the chromatogram
obtained with the reference solution.
Detection C Treat with a 10 per cent V/V solution of sulfuric
acid R in methanol R, heat at 100 oC for 5 min and examine
in daylight.
Results C The chromatogram obtained with the test solution
shows no violet zone corresponding to the zone due to
osthole in the chromatogram obtained with the reference
solution.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
System suitability Reference solution eb):
-- resolution: minimum 1.5 between the peaks due to
imperatorin and phellopterin.
Calculate the percentage content of imperatorin using the
following expression:
Al area of the peak due to imperatorin in the
chromatogram obtained with the test solution;
A2 area of the peak due to imperatorin in the
chromatogram obtained with reference solution (a);
mi mas s of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mass of imperatorin CRS used to prepare reference
solution (a), in grams;
p percentage content of imperatorin in
imperatorin CRS.
____________________________________________ ~Ew

Total ash (2.4. 16)

Maximum 6.0 per cent.
Ash insoluble in hydroch1oric acid (2.8.1)
Maximum 1.5 per cent. Angelica Pubescens Root *****
ASSAY
** **
Liquid chromatography (2.2.29).
(Ph Eur monograph 2557) ***
PhEw ____________________________________________
Test solution Disperse 0.400 g of the powdered herbal drug
(355) (2.9.12) in 45 mL of methanol R and sonicate for 1 h . DEFINITION
Cool and dilute to 50.0 mL with methanol R . Filter through a Dried root, without rootlets, of Angelica pubescens Maxim.
membrane filter (nominal pore size 0.45 ¡.¡m). f. biserrata R.H.Shan et C .Q .Yuan, collected in early spring
before sprouting, or in the end of autumn when stem and
Reference solution (a) Dissolve 5.0 mg of imperatorin CRS in
methanol R and dilute to 50.0 mL with the same solvent. lea ves wither.
Dilute 1.0 mL of the solution to 10.0 mL with methanol R. Content
Reference solution (b) Disperse 80 mg of Angelica dahurica Minimum 0.50 per cent of osthole (CIsHI 603; M r 244.3)
root HRS in 9 mL of methanol R and sonicate for 1 h . Cool (dried drug) .
and dilute to 10 mL with methanol R. Filter through a IDENTIFICATION
membrane filter (nominal pore size 0.45 )lm). A. The taproot is more or less cylindrical, branching rapidly
Precolumn: into 2-3 or more principal roots at the lower part; the whole
-- size: 1 = 4 mm, 0 = 4.0 mm; is about 5-30 cm long. The root crown is enlarged, with
-- stationary phase: octadecylsilyl silica gel for chromatography R transverse, annulated wrinkles and mea sures about
(5 ¡.¡m). 0.5-4.5 cm in diameter; it shows the remains of stems, leaves
Column: or buds. The greyish-brown or dark brown outer surface is
-- size: 1 = 0.125 m, 0 = 4.0 mm; longitudinally wrinkled and shows slightly prominent rootlet
-- stanonary phase: octadecylsilyl si/ica gel for chromatography R scars and transverse lenticel-like protuberances. The fracture
(5 )lm). shows greyish-yellow bark, with abundant brown dots due to
Mobile phase: secretory canal s; the cambium ring is brown and the wood is
mobile phase A: water R; greyish-yellow or yellowish-brown.
mobile phase B: acetonitrile R1; B. Microscopic examination (2.8.23). The powder is
Time
yellowish-brown or brown. Examine under a microscope
Mobile phase A Mobile phase B
(min) (per ce nI V!JI) (per cenl V!JI)
using chloral hydrate solution R. The powder shows the
0·15 45 55 following diagnostic characters: fragments of Iignified vessels
up to 90 )lm in diameter with spiral or reticulate thickenings,
15 - 33 45 .... 5 55 .... 95 free or in groups of 2 or 3; fragments of ph10em parenchyma
33·35 5 95 with fine, sinuous fusiform cells, about 7-38 ~lm in diameter,
with slightIy thickened walls and fine, oblique criss-cross
Flow rate 1.0 mUmin. striations; orange-brown cork fragments, consisting of several
Detection Spectrophotometer at 210 nm. layers of superimposed, somewhat polyhedral cells in surface
1njection 2O )lL. view; secretory canals, usually broken, with yellow or pale
Identification of peaks Use the chromatogram supplied with brown contents and droplets of essential oi!. Examine under
a microscope using a 50 per cent V/V solution of glycerol R.
Angelica dahurica root HRS and the chromatogram obtained
with reference solution (b) to identify the peak due to The powder shows numerous small, rounded or ovo id,
phellopterin. simple starch granules, about 10 ¡.¡m in size, with a
punctiform hilum that is visible on the largest granules; a few
Relative retention With reference to imperatorin (retention starch granules consisting of 2-1 O components are also
time = about 5 min): phellopterin = about 1.1. presento
2014 Angelica Pubescens Root IV-61

C . Examine the chromatograms obtained in the test for other TESTS

officinal species of Angelica, Levisticum and Ligusticum. Other officinal species of Angelica, Levisticum and
R esults ASee below the sequence of zones present in the Ligusticum
chromatograms obtained with the reference solution and the Thin-layer chromatography (2.2.27).
test solution. Furthermore, other faint fiuorescent zones may Test solution To 1 g of the powdered herbal drug (355)
be present in the chromatogram obtained with the test (2.9.12) add 4 mL of heptane R, close and sonicate for 5 mino
solution. Centrifuge the mixture and use the supernatant.
Reference solution Dissolve 1 mg of imperatonn R, 1 mg of
Top of the plate (Z) -ligustilide R and 1 mg of osthole R in 10 mL of
methanol R.
(Z)·Ligustilide: a bluish-white A bluish-white fluorescent zone
fluorescent zone Plate TLC silica gel F 254 plate R (2-10 llm).
--- --- Mobtle phase glacial acetic acid R, ethyl acetate R, toluene R
(1:10:90 VIV/V).
A very faint whitish zone
Application 4 llL as bands of 8 mm.
Osthole: a blue fluorescent zone A prominent blue fluorescent zone
(osthole) Development Over a path of 6 cm.
Imperatorin: a whitish fluorescent A whitish fluorescent zone (may Drying In air.
zone be missing) Detection A Examine in ultraviolet Iight at 365 nm.
- -- ---
Results A The chromatogram obtained with the test solution
A blue fluorescent zone shows no intense whitish fiuorescent zone directly aboye the
position of osthole and no blue fiuorescent zone just below
3 blue fluorescent zones
the position of imperatorin in the chromatogram obtained
Reference solution Test solution with the reference solution.
D etection B Examine in ultraviolet light at 254 nm.
R esults B See below the sequence of zones present in the Results B The chromatogram obtained with the test solution
chromatograms obtained with the reference solution and the shows no blue fiuorescent zone corresponding to the zone
test solution. Furthermore, other faint quenching zones may due to (Z)-ligustilide in the chromatogram obtained with the
be present in the chromatogram obtained with the test reference solution.
solution. Detection C Treat with a 10 per cent V/V solution of sulfunc
acid R in methanol R, heat at 100 oC for 5 min and examine
in daylight.
Top of the plate
Results C The chromatogram obtained with the test solution
(Z)·Ligustilide: a blue fluorescent A faint quenching zone shows no zone corresponding to the zone due to
zone
(Z)-ligustilide in the chromatogram obtained with the
--- ---
reference solution.
Osthole: a quenching zone A quenching zone (osthole) Loss on drying (2.2.32)
A blue fluorescen t zone Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
Imperatorin: a quenching zone A quenching zone (may be missing)
105 cc for 2 h .
- - - ---
Total ash (2.4.16)
2 or 3 quenching zones Maximum 8.0 per cent.
Reference solution Test solution Ash insoluble in hydroch1oric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
Results C See below the sequen ce of zones present in the
Liquid chromatography (2.2.29).
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present Test solution Disperse 0.500 g of the powdered herbal drug
in the chromatogram obtained with the test solution. (355) (2.9.12) in 18 mL of methanol R and sonicate for
30 mino Cool and dilute to 20.0 mL with methanol R.
Mix and filter. Dilute 5.0 mL of the filtra te to 20.0 mL with
Top of the plate methanol R.
A prominent reddish zone Reference solution (a) Dissolve 5.0 mg of osthole CRS in
methanol R and dilute to 100.0 mL with the same solvent.
(Z)-Ligustilide: a grey zone
Reference solution (b) Disperse 0.250 g of Angelica pubescens
--- ---
root HRS in 9 mL of methanol R and sonicate for 30 mino
Osthole: a viole t zone A violet zone (osthole) Cool and dilute to 10.0 mL with methanol R . Mix and filter.
Dilute 5.0 mL of the filtrate to 20.0 mL with methanol R.
Imperatorin: a grey zone A violet zone (may be missing)
Column:
--- --- - size: 1 = 0.125 m, 0 = 2.0 mm;
A prominent violet zone
- stationary phase: octadecylsilyl silica gel for chromatography R
(4 ~lm).
A yellow zone
Mobile phase water R, acetonitnle R (40:60 V/V).
Reference solution Test solution Flow rate 0.23 mUmin.
IV-62 Angelica Root
Detection Spectrophotometer at 322 nm.
Injection 1O ~.
Retention time Osthole = about 8 mino
System suitability Reference solution (b):
-- resolution: minimum 1.5 between the peak due to osthole
and peak 2; use the chromatogram supplied with Angelica
pubescens root HRS to identify peak 2.
Calculate the percentage content of osthole using the
following expression:
Al x m2 x p x 0.8
A 2 x mI
area of the peak due to osthole in the
chromatogram obtained with the test solution;
area of the peak due to osthole in the
chromatogram obtained with reference solution (a);
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of osthole CRS used to prepare reference
solution (a), in grams;
p percentage content of osthole in osthole CRS.
____________________________________________ PhEw
Angelica Sinensis Root
DEFINITION
Angelica Sinensis Root is the dried whole root of Angelica
sinensis (Oliv.) Diels . (Angelica polymorpha Maxim. var.
sinensis Oliv.). The dried root consists of the top (uppermost
part), main body and smalllateral roots (tails).
It is collected in late autumn, removed from rootlets and
dried.
It contains not less than 0.1 % of Z-ligustilide (ClzHI40Z),
calculated with reference to the dried material.
IDENTIFICATION
A. The whole root is yellowish-brown to brown, up to 25 cm
long, irregularly cylindrical with 3 to 5 or more branch roots
arising from the lower end. The upper part is 1.5 to 5 cm in
diameter, annulated on the surface and rounded at the apex
which may show purple or yellowish-green remains of stems
and leaves; the surface of the remainder of the main root is
strongly longitudinally wrinkled and has pale, transverse
lenticels; the branch roots are 0.3 to 1 cm in diameter in the
upper part, twisted and tapering towards the base, the outer
surface is strongly striated and has few rootlet scars.
B. Reduce to a powder (355). The powder is pale yellowish
to buff. Examine under a microscope using chloral hydrate
solution. The powder shows brown fragments of cork
composed of thin-walled cells; abundant thin-walled
parenchyma from the secondary cortex, phloem and
medullary rays, sorne of the phloem cells fusiform with
slightly thickened walls; lignified ves seis in groups of 2 or 3
associated with smalI celIed and pitted xylem parenchyma;
the vessels are up to 80 ~lm in diameter and have reticulate
or scalariform thickening. Examine under a microscope using
50% v/v of glycerol. The powder shows smalI groups of single
starch granules, spherical to ovoid, up to about 8 11m in
diameter.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
2014
(1) Add 4 mL of heptane to 1.0 g of the powdered drug, mix
with the aid of ultrasound for 5 minutes and filter (use a
0.22 11m membrane filter).
(2) 0.1 % w/v of lino/eic acid in methanol.
(3) 0.1% w/v ofjerulic acid in methanol.
(4) 0.1 % w/v of Z-ligustilide CRS in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a silica gel F 254 precoated plate (Merck silica gel 60
F Z54 HPTLC plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 10 I1L of solution (1) and 5 ~lL of solutions (2) to
(4), as bands.
(d) Develop the plate to 15 cm.
(e) Remove the plate, alIow to dry in a stream of warm air
for 5 minutes or until the solvents are completely removed.
Examine under ultraviolet light (254 nm). Spray the plate with
methanolic sulfuric acid (5%), heat at 105° for 3 minutes and
examine in daylight.
MOBILE PHASE
1 vol ume of jormic acid, 10 volumes of ethyl acetare and
90 volumes of toluene.
SYSTEM SUITABILITY
When examined under ultraviolet light (254 nm) the violet
band with an Rf value of approximately 0.7 the
chromatogram obtained with solution (4) corresponds in
colour and position to that in the chromatogram obtained
with solution (1). A band with an Rf value of approximately
0.23 in the chromatogram obtained with solution (3)
corresponds in position to a band in the ehromatogram
obtained with solution (1). Other bands may be present in
the ehromatogram obtained with solution (1).
CONFIRMATION
When sprayed with methanolic sulfuric acid (5%) the
ehromatogram obtained with solution (1) shows three spots
with similar Rf values to the spots in the chromatograms
obtained with solutions (2), (3) and (4). Other spots may be
present in the ehromatogram obtained with solution (1).
TESTS
Lovage root (Levisticum officinale)
Carry out the method for gas chromatography,
Appendix !II B, using the following solutions.
(1) Extraet approximately 20 g of the eoarsely powdered
drug in a 500 mL round-bottomed flask by hydrodistilIation
for 2 to 3 hours in 200 mL of water, eolleeting the distillate
in suitable glassware. Extraet the oily drops on top of the
distilIate with 5 mL of toluene.
(2) 0.2% w/v ea eh of couman'n and eugenol in toluene.
(3) 0.1 % w/v of Z-ligustilide CRS in acetonitrile.
(4) 0.1 % w/v of benzyl alcohol in toluene.
(5) 0.1 % w/v of (-)-cG1-vone in toluene.
(6) 0.1 % w/v of octanoic acid in toluene.
(7) 0.1 % w/v of 3-propylidenephthalide in toluene.
CHROMATOGRAPHIC CONDITIONS
(a) Use a fused siliea eapillary eolumn (50 m x 0.32 mm)
bonded with a film (1.05 11m) of 5% phenyl/95%
dimethylpolysiloxane (HP 5 is suitable).
(b) Use helium as the earrier gas at 1 mL per minute.
(e) Use an oven maintained at an initial temperature of 40°
increasing linearly to 220° at arate of 5° per minute, then
maintained at 220°.
 2014
(d) Use a split injection system having a split ratio of 20: 1
maintained at 250°.
(e) Use a flame ionisation detector maintained at a
temperature of 250°.
(f) Inject 1 pL of each solution.
(g) Record the chromatograms for a sufficient length of time
to elute all the peaks in the chromatogram obtained with
solution (1) (55 minutes may be suitable).
SYSTEM SUITABILITY
The test is not val id unless, in the chromatogram obtained
with solution (2) , the resolution between coumarin (eluting
at approximately 34 minutes) and eugenol (eluting at
approximately 37 minutes) is at least 3.0.
CONFIRMATION
In the chromatogram obtained with solution (1):
- there are no peaks corresponding to the principal peaks in
the chromatograms obtained with solutions (4), (5), (6)
and (7);
- there is a peak corresponding to the principal peak in the
chromatogram obtained with solution (3).
Loss on drying
When dried at 100° to 1050 for 2 hours, loses not more than
12.0% of its weight. Use 1 g.
Total ash
Not more than 7.0%, Appendix XI J, Method 11.
Acid-insoluble ash
Not more than 2.0%, Appendix XI K.
Ethanol-soluble extractive
Not less than 45%, Appendix XI B1.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Finely powder not less than 5.0 g of the drug being
examined. Transfer 0.5 g of the powder into a 25 mL
volumetric flask and add 20 mL of methanol, place in an
ultrasonic bath (maintained at a low temperature by adding
ice to the bath) for 100 minutes, equilibrate to ambient
temperature and dilute to volume with methanol. Centrifuge
the solution at 5000 rpm for 5 minutes or until a c!ear
supernatant is obtained. FiIter through a 0.45-~lm fiIter.
(2) 0.025 % w/v of Z-ligusti/ide CRS in acetonitrile.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
with octadecylsi/yl silica gel for chromatography (5 ~lm) (Hypersil
ODS is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a flow rate of 1.0 mL per minute.
(d) Use a detection wavelength of 350 nm .
(e) Inject 10 pL of each solution.
MOBILE PHASE
A mixture of 8 volumes of water and 12 volumes of
acetonitrl1e.
SYSTEM SUITABILITY
Inject solution (2) not less than five times. The test is not
valid unless the relative standard deviation of the peak areas
of the Z-ligustilide peak is not more than 3.0%, the relative
standard deviation of the retention times of the Z-ligustilide
peak is not more than 3.0%. The column efficiency,
dete=ined on the Z-ligustilide peak, is not less than 5000
Angelica Sinensis Root IV-63
theoretical plates. The symmetry factor, dete=ined on the
Z-ligustilide peak, is not more than 1.3.
Inject solution (1). The test is not va lid unless the reso/ution
factor between the Z-ligustilide peak and the closest peak
(relative retention about 0.9 with respect to Z-ligustilide) is
not less than 1.5.
DETERMINATION OF CONTENT
U sing the retention time and the peak area from the
chromatograms obtained with solution (2), locate and
integrate the peak due to Z-ligustilide in the chromatogram
obtained with solution (1) .
Calculate the content of Z-ligustilide in the sample using the
dec!ared content of Z-ligustilide (C12H¡402) in
Z-ligustilide CRS and the following expression:
A, ¡lb V, 100
- x - x - x p x -- -
A, V, m, IOO-d
A¡ Area of the peak due to Z-ligustilide in the
chromatogram obtained with solution (1).
A2 Area of the peak due to Z-ligustilide in the
chromatogram obtained with solution (2).
m¡ Weight of the drug being examined in mg.
m2 Weight of Z-ligustílide CRS in mg.
V¡ Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content of C12H¡ 40 2 in Z-ligustilide CRS.
d Percentage loss on drying of the herbal drug being
examined.
STORAGE
Angelica Sinensis Root should be protected from moisture.
Processed Angelica Sinensis Root *****
* *
(Angelica Sinensis Root, Ph Eur monograph 2558) **** *
_ _ _ _ __ _ _ _ _ _ __ _ __ __ _ ____
~Eif
DEFINITION
Smoke-dried, whole or fragmented root, with rootlets
removed, of Angelica sinensis (Oliv.) Diels collected in late
autumn.
Content
Minimum 0.050 per cent of trans-ferulic acid (C lO H lO 0 4 ; M r
194.2) (dried drug) .
IDENTIFICATION
A. Taproot branching rapidly into lOor more conical
principal roots; the whole is about 15-25 cm long.
The annulated root crown is about 1.5-4 cm in diameter;
its blunt, rounded tip shows the yellowish-green remains of
stems and petioles of leaves. The outer surface is light
brownish-yellow 01' dark brown, lumpy, irregularly striated
longitudinally and shows scars of secondary roots and
transversallenticel-like markings. The branching roots have a
thick upper part (0.3-1 cm in diameter) and a thin lower
part. They are frequently twisted and show few scars of
secondary roots. The texture is friable. The fracture,
yellowish-white or yellowish-brown, shows a thick bark with
sorne c!efts and numerous brown dots due to secretory
canals . The cambium occurs as a yellowish-brown ringo
The wood is light coloured.
The fragmented roots occur as long strips about 1.5-2 mm
thick, 1.5-4 cm wide at the root crown and 10-15 cm long.
 IV-64 Angelica Sinensis Root
B. Microscopic examination (2.8.23). The powder is
yellowish-white. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: reticulate or scalariform lignified ves seis up to
80 ¡.1m in diameter, free or in groups of 2 or 3 and
accompanied by ligneous parenchyma cells with thick walls;
numerous fragments of parenchyma with ovo id cells; orange
cork fragments, consisting of several layers of superimposed
cells, more or les s rectangular in surface view; very small
calcium oxalate prisms, visible in polarised light, in the cork;
rare secretory canals, usually broken, with orange-yellow
contents, up to 170 ¡.tm in diameter. Examine under a
microscope using a 50 per cent V/V solution of glycerol R: the
powder shows small (less than 10 ¡.tm), simple, rounded or
ovoid starch granules, usually included in parenchyma cells.
e. Examine the chromatograms obtained in the test for other
officinal species of Angelica, L evisticum and L igusticum.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint ftuorescent zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
(Z}-Ligustilide: a bluish·white A prominent bluish·white
fluorescent zone fluorescent zone ((Z)·ligusti lide)
---
Osthole: a blue fluorescent zone
Imperatorin: a whitish fluorescent
zone
---
Referenee solution Test solution
R esults B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint quenching zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
(Z)·Ligustilide: a blue fluorescent A prominent blue flu orescent zone
((Z)·ligustilide)
zone
A faint quenching zone
- --
Osthole: a quenching zone A faint quenching zone
Imperatorin: a quenching zone
---
Referenee solution Test solution
TESTS
Other officina1 species of Angelica, Levisticum and
Ligusticum
Thin-layer chromatography (2.2.27) .
Test solution To 1 g of the powdered herbal drug (355)
(2.9.12) add 4 mL of heptane R, close and sonicate for 5 mino
Centrifuge and use the supernatant.
2014
Reference solution Dissolve 1 mg of (Z)-ligustilide R, 1 mg of
imperarorin R and 1 mg of osthole R in 10 mL of methanol R .
Plate TLC silica gel F Z54 plate R (2-10 ¡.tm).
Mobile phase Glacial acetic acid R, ethyl acetate R, roluene R
( 1:10:90 VIV/V) .
Application 4 ¡.tL as bands of 8 mm.
Development Over a path of 6 cm.
Drying In airo
Detection A Examine in ultraviolet light at 365 nm.
R esults A The chromatogram obtained with the test solution
shows no intense blue ftuorescent zone at or below the
position of osthole in the chromatogram obtained with the
reference solution.
Detection B Examine in ultraviolet light at 254 nm.
R esults B The chromatogram obtained with the test solution
shows no quenching zone at or below the position of
imperatorin in the chromatogram obtained with the reference
solution.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (35 5) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
---
Maximum 2.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Disperse 0.200 g of the powdered herbal drug
- --
(355) (2.9. 12) in 20.0 mL of a 70 per cent VIV solution of
methanol R in a conical ftask, stopper tightly and weigh. Heat
under a reftux condenser for 30 min, cool and weigh again.
Compensate the loss of solvent with a 70 per cent V/V
solution of methanol R, mix well and allow to stand. Filter the
supernatant through a membrane filter (nominal pore size
0.45 ~lm); use the filtrate .
R eference solurion (a) In a brown-glass volumetric ftask,
dissolve 10.0 mg of ferulic acid CRS in a 70 per cent VIV
solution of methanol R and dilute to 100.0 mL with the same
solvento
R eference solution (b) In order to prepare cis-ferulic acid
in situ, introduce 2 mL of reference solution (a) into a
transparent vial and expose to ultraviolet light at 254 nm for
about 60 mino
Colunm:
---
- size: 1 = 0.150 m, 0 = 2.0 mm;
stationmy phase: octadecylsilyl süica gel for chromarography R
(4 ¡.tm);
temperarure: 35 0e.
--- Mobile phase aceronitrile R , 0.085 per cent VIV solution of
phosphol1·C acid R (17 :83 V/V).
Flow rate 0.23 mUmin.
Detection Spectrophotometer at 316 nm.
Injection 1O ~lL.
Retention time trans-ferulic acid = about 13 mini czS-ferulic
acid = about 14 mino
Sysrem suitability Reference solution (b):
- resolution: minimum 1.3 between the peaks due to
trans-ferulic acid and cis-ferulic acid.
Calculate the percentage content of trans-ferulic acid using
the following expression:
 2014 Aniseed IV-65

carpophore and the pedicel [Ab], accompanied by ves seIs

with spiral or annular thickening [Aa, F] .

A¡ area ofthe peak due to trans-ferulic acid in the

chromatogram obtained with the test solution;
A2 area of the peak due to trans-ferulic acid in the
chromatogram obtained with reference solution (a);
m¡ mass of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mas s of ferulic acid CRS used to prepare reference
solution (a), in grams;
p percentage content of trans-ferulic acid in f erulic
acid CRS.
____________________________________________ ~E~

Aniseed ***
*** ***
Anise ***
(Ph Eur monog¡·aph 0262)
When Powdered Aniseed is prescribed or demanded,
material complying with the requirements below, with the
exception of Identification test A and the test for Foreign
matter, shall be dispensed or supplied.
~E~ ____________________________________________

DEFINITION
Whole, dry cremocarp of Pimpinella anisum L.
Content
Minimum 20 mUkg of essential oil (anhydrous drug).
CHARACTERS
Reminiscent odour of anethole.
The fruit is a cremocarp and generally entire; a small
fragment of the thin, rigid, slightly curved pedicel is
frequently attached.
IDENTIFICATION
A. The cremocarp is ovoid or pyriform and slightly
compressed laterally, yellowish-green or greenish-grey,
3-5 mm long and up to 3 mm wide, surmounted by a
stylopod with 2 short, reflexed stylar points. The mericarps
are attached by their tops to the carpophore with aplane
commissural surface and a convex dorsal surface, the latter
being covered with short, warty trichomes visible using a
lens; each mericarp shows 5 primary ridges, running
longirudinally, comprising 3 dorsal ridges and 2 lateral ridges,
non-prominent, and lighter in colour.
B. Microscopic examination (2.8.23) . The powder is
greenish-yellow or brownish-green. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters (Figure 0262 .-1):
fragments of epicarp in surface view [D] with a striated
cuticle, occasional anomocytic stomata (2.8.3) [Da], bases of
covering trichomes [Dc] and whole covering trichomes [Db],
mostly unicellular, sometimes curved, with a blunt apex and
a warty cuticle; isolated fragments of covering trichomes [E];
fragments [H] of numerous narrow, branched vittae [Ha],
often accompanied by elongated cells of the commissural
surface [Hb]; fragments of testa [B] consisting of a layer of
brown, polyhedral, thin-walled cells; fragments of endosperm
[G] containing oil droplets [Ga], aleurone grains and small
cluster crystals of ca1cium oxalate [Gb]; oblong sclereids from
the mesocarp [C] or the commissural surface of the fruit;
bundles of short sclerenchymatous fibres [A] from the
Figure 0262.-1. - Illustration for identification test B of
powdered herbal drug of aniseed
e. Thin-layer chromatography (2.2.27).
Test solution Shake 0.10 g of the powdered herbal drug
(1400) (2.9.12) with 2 mL of methylene chloride R for 15 mino
Filter and carefully evaporate the filtrate to dryness on a
water-bath at 60 0e. Dissolve the residue in 0.5 mL of
toluene R .
Reference solution Dissolve 3 ¡.¡L of anethole R and 40 ¡.¡L of
olive oil R in 1 mL of toluene R .
Plate TLC silica gel GF254 plate R.
Mobile phase toluene R .
Application 2 ¡.¡L and 3 ¡.¡L of the test solution, then 1 ~lL,
2 ~lLand 3 ¡.tL of the reference solution, at 2 cm intervals.
Developmem Over a path of 10 cm.
Drying In air.
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms show a quenching zone
(anethole) in the central part against a light background.
Detection B Spray with a freshly prepared 200 gIL solution
of phosphomolybdic acid R in ethanol (96 per cent) R, using
10 mL for a 200 mm square plate, and heat at 120 oC for
5 mino
Results B The spots due to anethole appear blue against a
yellow background. In the chromatogram obtained with 2 ~lL
of the test solution, the spot due to anethole is intermediate
in size between the cortesponding spots in the
chromatograms obtained with 1 ¡.tL and 3 ¡.tL of the reference
solution. The chromatograms obtained with the test solution
IV-66 Star Anise
show in the lower third a blue spot (triglycerides) similar in
position to the spot in the lower third of the chromatograms
obtained with the reference solution (triglycerides of olive
oil).
TESTS
Water (2.2.13)
Maximurn 70 mUkg, determined on 20.0 g of the powdered
herbal drug.
Total ash (2.4.16)
Maximurn 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.5 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8. 12). Use 10.0 g ofthe herbal drug reduced to a coarse
powder immediately before the determination, a 250 mL
round-bottomed fiask, and 100 mL of water R as the
distillation liquido Place 0.50 mL of xylene R in the graduated
tube. Distil at arate of 2.5-3.5 mUmin for 2 h.
____________________________________________ PhEw
Star Anise *****
*
(Ph Eur monograph 1153) ******
Preparation
Concentrated Anise Water
~Ew ____________________________________________
DEFINITION
Dried composite fruit of Illicium verum Hooker fil.
Content:
-- minimum 70 mUkg of essential oil (anhydrous drug),
-- minimum 86.0 per cent of trans-anethole in the essential
oil.
CHARACTERS
The fruit carpels are brown.
Odour of anethole.
IDENTIFICATION
A. The fruit generally consists of 8 developed, one-seeded
follides, each 12-22 mm long and 6-12 mm high, radially
arranged around a shon, central, blunt-ending colurnella.
In some fruits 1 or 2 follides may be missing, but their
position is dearly visible. Each follide is boat-shaped or boot-
shaped, with a greyish-brown dorsal surface showing rough
markings and lateral surfaces bearing scars from the
neighbouring follides. One or more follides are split open
along the ventral suture, exposing a single, lenticular, shiny,
reddish-brown seed about 8 mm in diameter. The markings
on the dorsal surface are not visible from the ventral surface.
Some of the follides (1 -3) may be imperfectly developed.
Isolated follides, pedicels and seeds may be presento
B. Reduce to a powder (355) (2.9.12) . The powder is
reddish-brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: brown epicarpal cells, polygonal in surface view,
with a strongly striated cutide and occasional anomocytic
stomata (2.8.3); fragments of the endocarp with long
palisade-like cells; fragments of the mesocarp with large
parenchymatous cells, vessels, oil-containing cells and groups
of stone cells; fragments of the seed testa with palisade-like,
sderified, strongly pitted, yellow cells up to 200 flm long;
2014
fragments of the columella and the fruit stalk with strongly
and irregularly thickened, star-shaped stone cells about
400 fAm long and 150 ~lm wide; rhomboidal or rectangular
crystals of ca1ciurn oxalate.
C. Examine the chromatograms obtained in test B for Illiciwn
anisatum ( = 1. religiosum) and certain other Illicium spp.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other weaker zones may be
present in the chromatogram obtained with the test solution.
Top of the plate
--- ---
Caffeic acid: a Iight blue
f1uorescent zone
Quercitrin: a brownish·yellow
fluorescent zone
A brownish-yellow f1uorescent
zone
--- ---
A greenish f1uorescent zone
Hyperoside: a brownish-yellow A brownish-yellow f1uorescent
f1uorescent zone zone
Chlorogenic acid: a light blue
f1uorescent zone
A green f1uorescent zone
Rutin: a brownish-yellow A brownish·yellow f1uorescent
f1uorescent zone zone
Reference solution Test solution
TESTS
Illicium anisatum (= 1. religiosum) and certain other
Illicium spp.
A. Adulteration with Illicium anisatum or certain other Illicium
spp. is indicated by the presence of fruits mainly consisting of
more than 8 follides; fruits either smaller than 2.5 cm or
greater than 3.5 cm; follides with the suture edged with a
thickening extending to the neighbouring follic1e, or with
dorsal markings visible from the ventral surface; follides
somewhat undulate and ending in a fine beak or a small,
ventrally tumed hook; follides with a profile fitting into a
rectangle; pedicels more than 5 cm long; seedless fruits; seeds
either very flat or almost spherical.
B. Thin-layer chromatography (2.2.27).
Test solution To 2.0 g ofthe powdered drug (355) (2.9.12)
add 10 mL of methanol R and heat under a reflux condenser
in a water-bath at 60 oC for 5 mino AlIOW to cool and filter.
Reference solution Dissolve 1 mg of caffeic acid R , 1 mg of
chlorogenic acid R, 2.5 mg of quercitrin R, 2.5 mg of rutin R
and 2.5 mg of hyperoside R in 10 mL of methanol R.
Plate TLC silica gel plate R (2- 10 fAm) .
Mobile phase anhydrous fonnic acid R, glacial acetic acid R,
water R, ethyl acetate R (11: 11 :26: 100 V/V/V/V) .
Application 5 fAL as bands.
Development Over a path of 6 cm.
Drying In a current of warm air.
Detection Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R; after 30 min,
examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the test solution
shows no brownish-yellow fluorescem zone at or aboye the
2014 Star Anise Oil IV-67

position of the zone due to quercitrin in the chromatogram 0.05 per cent of the area of the principal peak in the
obtained with the reference solution. No yellow ftuorescent chromatogram obtained with the test solution.
zone is seen at or aboye the position of the zone due to _ __ _ _ _ __ _ _ _ _ _ _ _ __ _ _ _ _ _ Ph Eur
caffeic acid in the chromatogram obtained with the reference
solution. No brownish-yellow ftuorescent zone is seen directly
aboye the zone due to hyperoside in the chromatogram
obtained with the reference solution.
Star Anise Oil ** *
*** ***
Water (2.2.13)
Maximum 100 mUkg, determined by distillation on 20.0 g
ofthe powdered drug (355) (2.9.12).
(Ph Eur monograph 2108) ***
ffiE~ _ __ _ __ _ __ _ _ _ _ _ _ _ _ _ __ __
Total ash (2.4.16)
Maximum 4.0 per cent. DEFINITlON
Essential oil obtained by steam distillation from the dry ripe
ASSAY
fruits of Illicium verum Hook. fi!.
Essential oH
Carry out the determination of essential oils in herbal drugs CHARACTERS
(2.8.12) . Use a 250 mL round-bottomed ftask and 100 mL Appearance
of water R as the distillation liquido Immediately before the Clear, colourless or pale yellow liquido
determination, reduce 50.0 g of the drug to a coarse powder
IDENTIFICATlON
(1400) (2.9. 12) and mix. Further reduce about 10.0 g ofthis
First identification B.
mixture to a finer powder (710) (2.9.12) . Use 2.50 g of the
powder for the determination. Introduce 0.50 mL of xylene R Second identification A.
into the graduated tube. Distil at arate of 2-3 mUmin for A. Thin-Iayer chromatography (2.2.27) .
2 h. Test solution Dissolve 1 g of the substance to be examined
trans- Anethole in toluene R and dilute to 10 mL with the same solvent.
Gas chromatography (2.2.28): use the normalisation Reference solution Dissolve 10 IlL of linalol R , 30 [lL of
procedure. anisaldehyde R and 200 IlL of anethole R and in toluene R and
Test solution Dilute the mixture of essential oi! and xylene R di!ute to 15 mL with the same solvent. Dilute 1 mL of this
obtained in the assay of essential oil to 5.0 mL with xylene R solution to 5 mL with toluene R.
by rinsing the apparatus. Plate TLC silica gel F 254 plate R .
Reference solution To 1.0 mL of xylene R add 20 IlL of Mobile phase ethyl acetate R, toluene R (7:93 V/V) .
estragole R, 20 mg of a-terpineol R and 60 IlL of anethole R. Application 5 IlL as bands of 10 mm (for normal
Column: TLC plates) or 2 IlL as bands of 10 mm (for fine partic1e
- material: fused si!ica; TLC plates).
- size: 1 = 30 m, 0 = 0.25 mm; Development Over a path of 15 cm (for normal TLC plates)
- stationary phase: macrogol 20 000 R. or over a patb of 6 cm (for fine partic1e size plates) .
Carrier gas helium for chromatography R . Drying In airo
Flow rate l.0 mUmin. Detection A Examine in ultraviolet light at 254 nm.
Split ratio 1:1OO. Results ASee below the sequence of zones present in the
Temperature: chromatograms obtained with the reference solution and the
Time Temperature test solution. Furtbermore, other zones may be present in the
(min) (oC) chromatogram obtained with the test solution.
Column 0-5 60

5·80 60 -7 210 Top of the plate

80 - 95 210 A quenching zone, partly separated
Injection port 200
Anethole: a quenching zone A very strong quenching zone
Detector 220 (anethole)
--- ---
Detection Flame ionisation.
Anisaldehyde: a quenching zone A quenching zone (anisaldehyde)
Injection 1 1lL.
Elution order Order indicated in the preparation of the --- ---
reference solution. Reference solution Test solution
System suitability Reference solution:
- resolution: minimum 5 between tbe peaks due to estragole
and ex-terpineol. Detection B Spray with methyl 4-acetylbenzoate reagent R and
heat at 100-105 oC for 10 min; examine the still hot plate in
Use the retention times from the chromatogram obtained
daylight within 10 mino
with the reference solution locate tbe components of the
reference solution in the chromatogram obtained with the Results B See below the sequence of zones present in the
test solution. chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
Calculate the percentage content of trans-anethole . Disregard
chromatogram obtained with the test solution.
any peak due to the solvent or with an area less than
IV-68 Star Anise Oil 2014

Top of the plate Fatty oils and resinified essential oils (2.8.7)
It complies with the test for fatty oils and resinified essential
A violet·brown zone, not fully
separated oils.
Anethole: a brown zone A very strong brown zone Chromatographic profile
(anethole) Gas chromatography (2.2.28): use the normalisation
- -- --- procedure.
Anisaldehyde: a yellow zone A yellow zone (anisaldehyde) Test solution Dissolve 200 IlL of the substance to be
examined in 1.0 mL of hexane R.
--- - -- Rejerence solution To 1.0 mL of hexane R, add 20 !-1L of
Linalol: a grey zone A grey zone (linalol)
linalol R, 20 IlL of estragole R, 20 IlL of (J.-terpineol R, 60 IlL
Reference solution Test solution of anethole R and 30 IlL of anisaldehyde R.
Column:
- material: fused silica,
B. Examine the chromatograms obtained in the test for - size: l = 30 m, 0 = 0.25 mm,
chromatographic profile. - stationary phase: macrogol 20 000 R (film thickness
Results The characteristic peaks in the chromatogram 0.25 11m).
obtained with the test solution are similar in retention time to Canier gas helium jor chromatography R.
those in the chromatogram obtained with the reference
Flow rate 1.0 mUmin.
solution.
Split ratio 1:1OO.
TESTS Temperature:
Relative density (2.2.5)
0.979 to 0.985. Time Temperature
(min) ('C)
Refractive index (2.2.6) Column 0·5 60
1.553 to 1.556.
5·80 60 ~ 210
Freezing point (2.2.18)
15 oC to 19 oc. 80·95 210

Fenchone Injection port 200

Gas chromatography (2.2.28) as described in the test for
chromatographic profile with the following modifications.
Test solution Dissolve 400 IlL of the substance to be
examined in 2.0 mL of hexane R .
Rejerence solution (a) Dilute 10 IlL ofjenchone R to 1.2 g
with hexane R.
Rejerence solution (b) Dilute 100 IlL of reference solution (a)
to 100 mL with hexane R .
System suitability Reference solution (b):
- signal-to-noise ratio: minimum 10 for the principal peak.
Limit:
- jenchone: maximum 0.01 per cent.
Pseudoisoeugenyl 2-methylbutyrate
Gas chromatography (2.2.28) as described in the test for
chromatographic pro file with the following modifications.
Test solution The substance to be examined.
Rejerence solution (a) Dilute 10 mg of the test solution to
1.000 g with hexane R . Dilute 0.5 mL of this solution to
100 mL with hexane R.
Rejerence solution (b) Pseuaoisoeugenyl 2-methylbutyrate jor
peak identification CRS.
System suitability:
- the chromatogram obtained with reference solution (b) is
similar to the chromatogram provided with
pseudoisoeugenyl 2-methylbutyrate jor peak
identification CRS.
- signal-to-noise ratio: minimum 10 for the principal peak in
the chromatogram obtained with reference solution (a).
Limit Locate the peak due to pseudoisoeugenyl
2-methylbutyrate by comparison with the chromatogram
provided with pseudoisoeugenyl 2-methylbutyrate jor peak
identification CRS.
- pseudoisoeugenyI2-methylbutyrate: maximum 0.01 per cent.
Detector 220
Detection Flame ionisation.
1njection 0.2 1lL.
Elution order Order indicated in the composition of the
reference solution; record the reteinion times of these
substances.
System suitability Reference solution:
- resolution: minimum 1.5 between the peaks due to
estragole and a-terpineol.
Using the retention times determined from the
chromatogram obtained with the reference solution, locate
the components of the reference solution in the
chromatogram obtained with the test solution and locate
cis-anethole and foeniculin using the chromatogram shown in
Figure 2108.-1 (disregard any peak due to hexane) .
Determine the percentage content of these components.
The percentages are within the following ranges:
- linalol: 0.2 per cent to 2.5 per cent,
- estragole: 0.5 per cent to 6.0 per cent,
- (J.-terpineol: maximum 0.3 per cent,
- cis-anethole: 0.1 per cent to 0.5 per cent,
- trans-anethole: 86 per cent to 93 per cent,
- anisaldehyde: 0.1 per cent to 0.5 per cent,
- joeniculin: 0.1 per cent to 3.0 per cent.
STORAGE
At a temperature not exceeding 25 oc.
_ __ _ _ __ _ _ _ __ __ _ _ __ _ __ _ Ph Eur
 2014 Anise Oil IV-69

2 5

4
6
3

~ J, 1.tIl 1 Itt 1 , , ,
J aJ LJJ J

I , , , I ' , I I I ' , , I ' I ' I ' J I I I I I I J , I I I I I

o 10 20 30 40 50 60 70 80 min

1. linalol 3. a-terpineol 5. trans-anethole 7. foeniculin

2. estragole 4. cis-anethole 6. anisaldehyde

Figure 2108.-1. - Chromatogram for the test for chromatographic profile of star anise oi!

*****
Application 5 llL as bands of 10 mm (for normal
Anise Oil
** ** TLC plates) or 2 llL as bands of 10 mm (for fine partic1e
Aniseed Oil *** size plates).
(Ph Bur monograph 0804) Development Over a path of 15 cm (for normal TLC plates)
or over a path of 6 cm (for fine partic1e size plates) .
Preparation
Concentrated Anise Water Drying In airo
nEw ____________________________________________ Deteetion A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of zones present in the
DEFINITION
chromatograms obtained with the reference solution and the
Essential oil obtained by steam distillation from the dry ripe
test solution. Furthermore, other zones may be present in the
fruits of Pimpinella anisum L.
chromatogram obtained with the test solution.
CHARACTERS
Appearance
Top of the plate
Clear, colourless or pale yellow liquido
Anethole: a quenching zone A very strong quenching zone
IDENTIFICATION (anethole)
First identification B. ---- ---
Second identification A.
A quenching zone
A. Thin-Iayer chromatography (2.2.27).
Anisaldehyde: a quenching zone A quenching zone (anisaldehyde)
Test solution Dissolve 1 g of the substance to be examined
in toluene R and dilute to 10 mL with the same solvent. --- ---
Reference solution Dissolve 10 llL of linalol R, 30 llL of Reference solution Test solution
anisaldehyde R and 200 llL of anethole R in toluene R and
dilute to 15 mL with the same solvent. Dilute 1 mL of this
solution to 5 mL with toluene R. Detection B Spray with methyl 4-acetylbenzoate reagent R and
Plate TLC si/ica gel F 254 plate R . heat at 100-105 oC for 10 min; examine the still hot plate in
Mobz7e phase ethyl acetate R , toluene R (7:93 V/V) . daylight within 5 mino
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
 IV- 7 O Anise Oil 2014

test solution. Furthermore, other zones may be present in the -- joeniculin: maximum 0.01 per cent.
chromatogram obtained with the test solution. Fatty oils and resinified essential oils (2.8.7)
It complies with the test for fatty oils and resinified essential
Top oC tbe plate
oils.
Chromatographic profile
A violet·brown zone (monoterpene
hydrocarbons) (solvent front) Gas chromatography (2.2.28): use the normalisation
Anethole: a brown zone A very strong brown zone procedure.
(anethole). distinctly separated Test solution Dissolve 200 ¡¡L of the substance to be
--- --- examined in 1.0 mL of hexane R.
A grey zone Rejerence solution To 1.0 mL of hexane R, add 20 ¡¡L of
Anisaldehyde: a yellow zone
linalol R, 20 ¡,tL of estragole R, 20 ¡¡L of CJ.-terpineol R, 60 ¡¡L
A yellow zone (anisaldehyde)
of anethole R and 30 ¡,tL of anisaldehyde R.
- -- --- Column:
Linalol: a grey zone A grey zone (linalol) -- material: fused silica,
-- size: 1 = 30 m, 0 = 0.25 mm,
A grey zone
-- stationary phase: macrogol 20 000 R (film thickness
Reference solution Test solution 0.25 ¡¡m).
Carrier gas helium jor chromatography R.
Flow rate 1.0 mUmin.
B. Examine the chromatograms obtained in the test for
chromatographic profile. Split ratio 1: 1OO.
Results The characteristic peaks in the chromatogram Temperature:
obtained with the test solution are similar in retention time to Time Temperature
those in the chromatogram obtained with the reference (min) (OC)
solution. Column 0-5 60

TESTS 5 - 80 60 ~ 210
Relative density (2.2.5) 80 - 95 210
0.980 to 0.990.
Injection port 200
Refractive index (2.2.6)
Detector 220
1.552 to 1.561.
Freezing point (2.2.18) Detection Flame ionisation.
15 oC to 19 oC. Injection 0.2 ¡¡L.
Fenchone Elution order Order indicated in the composition of the
Gas chromatography (2.2.28) as described in the test for reference solution. Record the retention times of these
chromatographic profile with the following modifications. substances.
Test solution Dissolve 400 ¡¡L of the substance to be System suitability Reference solution:
examined in 2.0 mL of hexane R. -- resolution: minimum 1.5 between the peaks due to
Rejerence solution (a) Dilute 10 ¡¡L ofjenchone R to 1.2 g estragole and IX-terpineol.
with hexane R. Using the retention times determined from the
Rejerence solution (b) Dilute 100 ¡¡L of reference solution (a) chromatogram obtained with the reference solution, locate
to 100 mL with hexane R. the components of the reference solution in the
System suitability Reference solution (b): chromatogram obtained with the test solution and locate
-- signal-to-noise ratio: minimum 10 for the principal peak. cis-anethole and pseudoisoeugenyl 2-methylbutyrate using the
Limit: chromatogram shown in Figure 0804.-1 (disregard any peak
-- jenchone: maximum 0.01 per cent. due to hexane).
Foeniculin Determine the percentage content of these components.
Gas chromatography (2.2.28) as described in the test for The percentages are within the following ranges:
chromatographic profile with the following modifications. -- linalol: maximum 1.5 per cent,
-- estragole: 0.5 per cent to 5.0 per cent,
Test solution The substance to be examined. -- rx-terpineol: maximum 1.2 per cent,
Rejerence solution (a) Dilute 10 mg of the test solution to -- cis-anethole: 0.1 per cent to 0.4 per cent,
1.000 g with hexane R. Dilute 0.5 mL of this solution to -- trans-anethole: 87 per cent to 94 per cent,
100 mL with hexane R. -- anisaldehyde: 0.1 per cent to 1.4 per cent,
Rejerence solution (b) Foeniculin jor peak identification CRS. -- pseudoisoeugenyI2-methylbutyrate: 0.3 per cent to
System suitability: 2.0 per cent.
-- the chromatogram obtained with reference solution (b) is STORAGE
similar to the chromatogram provided with joeniculin jor At a temperature not exceeding 25 oC.
peak identification CRS, ____________________________________________ PhE~

-- signal-w-noise ratio: minimum 10 for the principal peak in

the chromatogram obtained with reference solution (a).
Limit Locate the peak due to foeniculin by comparison with
the chromatogram provided with joeniculin jor peak
identification CRS.
2014 Arnica Flower IV- 71

2
6
3 7

I
I

I
l. I
, ,
I

I
J 1.
, , ,
ilL
I
U, , I
.1
I
, ,
¡ .1
,
JI
, I
,
~
, , I I
o 10 20 30 40 50 60 70 80 min

1. linalol 3. a-terpineol 5. trans-anethole 7. pseudoisoeugenyl 2·rnethylbutyrate

2. estragole 4. cis-anethole 6. anisaldehyde

Figure 0804.-l. - Chromatogram for the test for chromatographic profile of anise oi!

Concentrated Anise Water Arnica Flower ***

* *
DEFINITION (Ph Eur monograph 1391) ******
*
Anise Oil or Star Anise Oil 20mL
Preparation
Ethanol (90 per cent) 700 mL
Arnica Tincture
Water Sufficient to produce
~E~ ____________________________________________
1000 mL
Extemporaneous preparation DEFINITION
The following directions apply. Whole or partially broken, dried fiower-heads of Amica
Dissolve the Anise Oil or Star Anise Oil in the Ethanol montana L.
(90 per cent) and add gradually, with vigorous shaking after Content
each addition, sufficient Water to produce 1000 mL. Mínimum 0.40 per cent m/m of total sesquiterpene lactones,
Add 50 g of previously sterilised Purified Tale, or other expressed as dihydrohelenalín tiglate (dried drug).
suitable filtering aid, allow to stand for a few hours, shaking CHARACTERS
occasionally, and filter. Aromatic odour.
The water complies with ¡he requiremenls stated under Aromatic The capitulum, when spread out, is about 20 mm in
Waters and with the following requiremenls. diameter and about 15 mm deep, and has a pedunde 2-3 cm
TESTS long. The involucre consists of 18-24 elongated lanceolate
Ethanol content bracts, with acute apices, arranged in 1-2 rows: the bracts,
60 to 64% v/v, Appendix VIII F . about 8-10 mm long, are green with yellowish-green external
Weight per mL hairs visible under a lens. The receptad e, about 6 mm in
0.898 to 0.908 g, Appendix V G. diameter, is convex, alveolate and covered with hairs.
Its periphery bears about 20 ligulate fiorets 20-30 mm long;
the disc bears a greater number of tubular fiorets about
15 mm long. The ovary, 4-8 mm long, is crowned by a
pappus of whitish bristles 4-8 mm long. Sorne brown
achenes, crowned or not by a pappus, may be presento
IDENTIFICATION
A. The involucre consists of elongated oval bracts with acute
apices; the margin is ciliated. The ligulate fioret has a
 IV-72 Arnica Flower 2014

A~.~~........
reduced calyx crowned by fine, shiny, whitish bristIes,
bearing small coarse trichomes. The orange-yellow corolla %
bears 7-10 parallel veins and ends in 3 small lobes.
The stamens, with free anthers, are incompletely developed. Ab

B~gn,
The narrow, brown ovary bears a stigma divided into 2
branches curving outwards. The tubular f10ret is
actinomorphic. The ovary and the calyx are similar to those
of the ligulate f1oret. The short corolIa has 5 reflexed
triangular lobes; the 5 fertile stamens are fused at the
anthers . ~'
B. Microscopic examination (2.8.23). Separate the capitulum

O'·
into its different parts . Examine under a microscope using
chloral hydrate solution R . The powder shows the folIowing .,
H
diagnostic characters (Figure 1391.-1): the epidermis es ofthe
A
bracts of the involucre [L, M, 0, Q] have stomata [Lb, Oa, V
Qa] and trichomes, more abundant on the outer (abaxial) F
surface. There are several different types of trichomes:
uniseriate multicellular covering trichomes, varying in length
from 50-500 ¡.tm, particularIy abundant on the margins of the
bract, whole [La] or fragmented [P]; secretory trichomes with
uni- or biseriate multicellular stalks and with multicelIular,
globular heads, about 300 ¡.tm long, abundant on the outer
surface of the bract [Qb]; secretory trichomes with
multicelIular stalks and with multicelIular, globular heads,
about 80 ¡.tm long, abundant on the inner surface of the
bract, in surface view [Ob] or in side view [Ma].
The epidermis of the ligulate coroIla [C, G, H, TI consists of
lobed or elongated celIs covered by a striated cuticle [Ga], a
few sto mata and trichomes of different types: covering
Ha
trichomes, with very sharp ends, whose length may exceed
500 ¡.tm, consisting of 1-3 proximal, thick-waIled ceIls and
2-4 distal, thin-walIed celIs [C, Hb]; secretory trichomes with AN
biseriate multicelIular heads in surface view [Gb] or in side ~
view ITa]; secretory trichomes with multicellular stalks and
multicellular globular heads [K] . The ligule ends in rounded
papilIose ceIls [Ha]. Fragments ofthe epidermis of the ovary
[A, B, D] are covered with trichomes of 2 types: secretory
trichomes with short stalks and multicellular globular heads,
in surface view [Aa] or in side view [Da]; twinned covering
trichomes usuaIly consisting of 2 longitudinalIy united ceIls,
with common pitted waIls, in surface view [Ab] or in side
view [Ba]; their ends are sharp and sometimes bifid.
The epidermis es of the calyx consist of elongated ceIls
bearing short, unicellular, covering trichomes pointing
towards the upper end of the bristIe [E]. The polIen grains
have a diameter of about 30 ¡.tm, are rounded, with a spiny
exine, and have 3 germinal pores [F, N].
C. Examine the chromatograms obtained in the test for
Calendula officinalis L. - Heterotheca inuloides Cass.
Results The chromatogram obtained with the test solution
shows, in the middle, a f1uorescent blue zone corresponding
to the zone due to chlorogenic acid in the chromatogram
obtained with the reference solution; it shows, aboye this
zone, 3 f1uorescent yeIlowish-brown or orange-yeIlow zones,
and aboye these 3 zones a f1uorescent greenish-yelIow zone
due to astragalin; the zone located below the astragalin zone
is due to isoquercitroside; the zone located just below this
zone is due to luteolin-7 -glucoside; it also shows a f1uorescent 3~
greenish-blue zone below the zone due to caffeic acid in the
chromatogram obtained with the reference solution.
Figure 1391.·1. - Illustration for identification test B of
TESTS powdered herbal drug of arnica flower
Foreign matter (2.8.2)
Maximum 5.0 per cent. Test solution To 2.00 g of the powdered herbal drug (710)
(2.9.12) add 10 mL of methanol R . Heat in a water-bath at
Calendula officinalis L. - Heterotheca inuloides Cass
60 oC for 5 min with shaking. Cool and filter .
Thin-Iayer chromatography (2.2.27).
 2014 Arnica Preparations IV-73

Reference solution Dissolve 2.0 mg of caffeic acid R , 2.0 mg -- stationary phase: octadecylsilyl szlica gel for chromatography R
of chlorogenic acid R and 5.0 mg of rutin R in methanol R and (4 ¡.Lm).
dilute to 30 mL with the same solvent. Mobile phase:
Plate TLC silica gel plate R. -- mobile phase A: water R;
Mobile phase anhydrous formic acid R, water R, methyl ethyl -- mobile phase B: methanol R;
ketone R, ethyl acetate R (10:10:30:50 V!V!V/V) . Time Mobile phase A Mobile phase B
Application 15 ¡.LL as bands. (min) (per cent VM (per cent VM
0-3 62 38
Development Over a path of 15 cm.
Drying In air for a few minutes. 3·20 62 -7 55 38 ...., 45

Deteetion Spray with a 10 gIL solution of diphenylboric acid 20·30 55 45

aminoethyl ester R in methanol R, and then with a 50 gIL 30 - 55 55...., 45 45 ...., 55
solution of macrogol400 R in methanol Ri heat at 100-105 oC
55 - 57 45...., O 55...., 100
for 5 min, allow to dry in air and examine in ultraviolet light
at 365 nm. 57 - 70 O 100
Results The chromatogram obtained with the reference 70 - 90 62 38
solution shows in the lower part an orange-yellow fluorescent
zone due to rutin, in the middle part a fluorescent zone due Flow rate 1.2 mUmin.
to chlorogenic acid and in the upper part a light bluish DeteetÍon Spectrophotometer at 225 nm.
fluorescent zone due to caffeic acidi the chromatogram Injection 20 ¡.LL loop injec1Or.
obtained with the test solution does not show a fluorescent
Calculate the percentage content of total sesquiterpene
orange-yellow zone corresponding to the zone due 10 rutin in
lac1Ones, expressed as dihydrohelenalin tiglate, using the
the chromatogram obtained with the reference solution, nor
following expression:
does it show a zone below this.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
SLS X e x v x 1.187 x 100
Ss x m x 1000
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
area of all peaks due 10 sesquiterpene lactones
Total ash (2.4.16)
appearing after the santonin peak in the
Maximum 10.0 per cent.
chromatogram obtained with the test solution;
ASSAY Ss area of the peak due 10 santonin in the
Liquid chromatography (2.2.29) . chroma1Ogram obtained with the test solution;
Internal standard solution Dissolve immediately before use m mass of the herbal drug to be examined, in
0.010 g of santonin CRS, accurately weighed, in 10.0 mL of gramsi
methanol R. C concentration of santonin in the intemal
Test solution Introduce 1.00 g of the powdered herbal drug standard solution used for the test solution, in
(355) (2.9.12) into a 250 mL round-bonomed flask, add milligrams per millilitrei
50 mL of a mixture of equal volumes of methanol R and v volume of the intemal standard solution used for
water R and heat under a reflux condenser in a water-bath at the test solution, in millilitresi
50-60 oC for 30 min, shaking frequently. Allow 10 cool and 1.187 peak correlation factor between dihydrohelenalin
filter through a paper filter. Add the paper filter, cut into tiglate and santonin.
pieces, 10 the residue in the round-bottomed flask, add ____________________________________________ ~Ew

50 mL of a mixture of equal volumes of methanol R and

water R and heat under a reflux condenser in a water-bath at
50-60 cC for 30 min, shaking frequently. Repeat this
procedure twice. To the combined filtrates add 3.00 mL of
Arnica Tincture ***
the intemal standard solution and evaporate 10 18 mL under
reduced pressure. Rinse the round-bottomed flask with *** ***
water R and dilute, with the washings, 10 20.0 mL. Transfer
(Ph Bur monograph 1809) ***
~Ew ____________________________________________
the solution 10 a chromatography column about 0.15 m long
and about 30 mm in intemal diameter containing 15 g of DEFINITION
kieselguhr for chromatography R . Allow 10 stand for 20 mino Tincture produced from Arnicafiower (1391).
Elute with 200 mL of a mixture of equal volumes of ethyl
Content
acetate R and methylene chloride R . Evaporate the eluate 10
Minimum 0.04 per cent of sesquiterpene lactones expressed
dryness in a 250 mL round-bottomed flask. Dissolve the
as dihydrohelenalin tiglate (C2oH2605i M r 346.42).
residue in 10 .0 mL of methanol R and add 10.0 mL of
water R. Add 7.0 g of neutral aluminium oxide R, shake for PRODUCTION
120 s, centrifuge at 5000 g for 10 min and filter through a The tincture is produced from the herbal drug by a suitable
paper filter. Evaporate 10.0 mL of the filtra te to dryness . procedure using 10 parts of ethanol (60-70 per cent V/V) for
Dissolve the residue in 3.0 mL of a mixture of equal volumes 1 part of drug.
of methanol R and water R and filter.
CHARACTERS
Column: Appearance
-- size: 1 = 0.12 m, 0 =4 mm; Yellowish-brown liquid.
IV- 7 4 Arnica Preparations 2014

IDENTIFICAnON equal volumes of methanol R and water R and filter.

Examine the chromatograms obtained in the test for Evaporate the filtrate 10 dryness. Dissolve the residue in
Calendula officinalis - Heterotheca inuloides. 2.0 mL of a mixture of 20 volumes of water R and
Chromatogram obtained with the test solution: 80 volumes of methanol R and filter through a membrane
- in the middle, a fluorescent blue zone corresponding 10 filter (nominal pore size 0.45 ¡.tm) .
the zone due 10 chlorogenic acid in the chromatogram Reference solution Dissolve 0.02 g of methyl
obtained with the reference solution; 4-hydroxybenzoate R and 0.02 g of ethyl 4-hydroxybenzoate R
- aboye this zone, 3 fluorescent yellowish-brown to orange- in methanol R and dilute to 10.0 mL with the same solvento
yellow zones, and aboye these 3 zones a fluorescent Column:
greenish-yellow zone corresponding 10 astragalin; the zone - size: 1 = O.1 2 m, 0 = 4 mm;
located below the astragalin zone corresponds to - stationary phase: end-capped octadecylsilyl siliea gel for
isoquercitrin; the zone located just below this zone chromatography R (5 ¡.tm);
corresponds to luteolin-7-glucoside; - temperature: 20 oc.
- a flu orescent greenish-blue zone below the zone due 10 Mobile phase:
caffeic acid in the chromatogram obtained with the - mobile phase A: water R;
reference solution. - mobile phase B: methanol R;
TESTS Time Mobile phase A Mobile phase B
Calen dula officinalis - Heterotheca inuloides (min) (percent VI!') (per cent VI!')
Thin-Iayer chromatography (2.2.27). 0·3 62 38
Test solution The tincture 10 be examined. 3·20 62 ~ 55 38 ~45

Reference solution Dissolve 2.0 mg of caffeic acid R, 2.0 mg 20·30 55 45

of chlorogenic acid R and 5.0 mg of rutin R in methanol R and
dilute 10 30.0 mL with the same solvento 30·55 55 ~ 45 45 ~ 55

Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel
plate R (2-1 0 ¡.tm)).
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (10: 10:30:50 VIVIV/V).
Application 30 ¡.tL [or 8 ¡.tL) as bands.
Development Over a path of 15 cm [or 8 cm) .
Drying At 80-105 oc.
Detection Spray the plate whilst still hot with a 10 gIL
solution of diphenylboric acid aminoethyl ester R in methanol R
and then with a 50 gIL solution of macrogol 400 R in
methanol R; heat 5 min at 100-105 oC, allow the plate 10 dry
in air and examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower part an orange-yellow fluorescent
zone (rutin), in the middle part a fluorescent zone due to
chlorogenic acid and in the upper part a light bluish
fluorescent zone (caffeic acid). The chromatogram obtained
with the test solution do es not show any fluorescent orange-
yellow zone corresponding to rutin in the chromatogram
obtained with the reference solution and no zone below the
zone corresponding 10 rutin.
Ethanol (2.9. 10)
The final ethanol concentration is not less than 90 per cent
of that of the initial extraction solvento
Methanol and 2-propanol (2.9.11)
Maximum 0.05 per cent VIV of methanol and maximum
0.05 per cent VIV of 2-propanol.
Dry residue (2.8.16)
Minimum 1.7 per cent.
Flow rate 1.2 mUmin.
Detector Spectrophotometer at 225 nm.
Injection 20 ¡.tL.
Relative retention With reference to santonin
(retention time = about 9.5 min):
butyl 4-hydroxybenzoate = about 4.6.
System suitabz1ity Reference solution:
- resolution: minimum 5 between the peaks due to methyl
4-hydroxybenzoate and ethyl 4-hydroxybenzoate.
Calculate the percentage of lactone sesquiterpenes, expressed
as dihydrohelenalin tiglate, using the following expression:
F¡ x e x v x 1.187
F2 x m x 10
area of all peaks appearing between the peaks due
to santonin and butyl 4-hydroxybenzoate in the
chromatogram obtained with the test solution;
area of the peak due 10 santonin in the
chromatogram obtained with the test solution;
m mass of the tincture 10 be examined, in grams;
C concentration of santonin in the intemal standard
solution used to prepare the test solution, in
milligrams per millilitre;
v volume of the internal standard solution used 10
prepare the test solution, in millilitres;
1.187 peak correlation factor between dihydrohelenalin
tiglate and santonin.
__________________________________________ ~Ew

ASSAY
Liquid chromatography (2.2.29).
Internal standard solution Dissolve immediately before use
0.010 g accurately weighed of santonin CRS and 0.02 g of
butyl 4-hydroxybenzoate R in 10.O mL of methanol R.
Test solution In a round-bottomed flask introduce 5.00 g of
the tincture 10 be examined, add 2.00 mL of the internal
standard solution and 3 g of anhydrous aluminium oxide R,
shake for 120 s and filter through a filter paper. Rinse the
round-bottomed flask and filter with 5 mL of a mixture of
 2014 Artichoke Leaf IV-75

Artichoke Leaf ***

*** ***
(Ph Eur monograph 1866) ***
Preparation
Artichoke Leaf Dry Extract
~Eif ____________________________________________

DEFINITlON
Whole or cut, dried leaf of Cynara cardunculus L. (syn. C.
scolymus L.).
Content
Minimum 0.8 per cent of chlorogenic acid (C16HlS09; M r
354.3) (dried drug) .
IDENTIFICATlON
A. The entire leaf may be up to 70 cm long and 30 cm wide.
The lamina is deeply lobed in the upper part to within
1-2 cm of the petiole on either side, in the lower part the leaf
becomes pinnate; all the segments have markedly dentate
margins and taper at the apex. Spines are absent. The upper
surface of the lamina is green with a fine covering of whitish
hairs, the lower surface is pale green or white and densely
tomentose with long, tangled hairs. The petiole and main
veins are flat on the upper surface, prominently raised and
longitudinally ridged on the lower surface, with conspicuous
hairs on both surfaces.
E. Reduce to a powder (1000) (2.9.12) . The powder is
greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1866.-1): fragments ofthe epidermis es of I 40 flm I

the lamina, in surface view; the upper epidermis [F] is

composed of cells with straight or slightly sinuous walls [Fa], Figure 1866.-1. - Illustration for identification test B of
accompanied by palisade parenchyma [Fb]; the lower powdered herbal drug of artichoke leaf
epidermis [C] is composed of more sinuous-walled cells;
abundant anomocytic stomata ( 2.8.3) on both surfaces [D]
and multicellular, uniseriate covering trichomes in felted Results See below the sequence of fluorescent zones present
masses, the majority fragmented [Ca] with a short stalk in the chromatograms obtained with the reference solution
composed of several cells and a very long, narrow and and the test solution. Furthermore, other fluorescent zones
frequently curled terminal cell, others consisting of 4-6 may be present in the chromatogram obtained with the test
cylindrical cells; very occasional glandular trichomes with a solution.
short stalk and a uniseriate or biseriate head, in surface view
[E] or in transverse section [Ea]; abundant fragments of
Top of the pI ate
covering trichomes [G]; fragments of the lamina, in
transverse section [E]; abundant fragments of vascular tissue A light blue fluorescent zone
from the petiole and veins [A]. --- ---
C. Thin-layer chromatography (2.2.27).
Luteolin·7·glucoside: a yellow or A yellow or orange fluorescent
Test solution To 2.0 g ofthe powdered drug (1000) (2.9.12) orange fluorescent zone zone (luteolin-7·glucoside)
add 20 mL of ethanol (60 per cent V/Ji? R. Allow to stand for Chlorogenic acid: a light blue A light blue fluorescent zone
2 h with occasional stirring. Filter. fluorescent zone (chlorogenic acid)
Reference solution Dissolve 5 mg of luteolin-7-g1ucoside R and --- ---

5 mg of chlorogenic acid CRS in methanol R and dilute to Reference solution Test solution
10 mL with the same solvento
Plate TLC silica gel plate R (5-40 j1m) [or TLC siliea gel
plate R (2- 10 j1m)]. TESTS
Mobile phase anhydrous formic acid R, glacial acetic acid R, Total ash (2.4.16)
water R, ethyl acetate R (11 :11 :27:100 V/V/V/V) . Maximum 20.0 per cent.
Application 10 j1L [or 2 j1L] as bands of 10 mm [or 8 mm] . Loss on drying (2.2.32)
Development Over a path of 13 cm [or 6 cm]. Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug (710) (2.9.12) by drying in an oven at
Drying In airo
105 oC for 2 h .
Detection Heat at 100 oC for 5 min; spray the warm plate
with a 10 gIL solution of diphenylboric acid aminoethyl ester R ASSAY
in methanol R followed by a 50 giL solution of macrogol Liquid chromatography (2.2.29).
400 R in methanol R; examine in ultraviolet light at 365 nm. Test solution To 0.500 g ofthe powdered drug (1000)
(2.9.12) add 50.0 mL of methanol R and heat under a reflux
condenser on a water-bath at 70 oC for 1 h. Centrifuge and
 IV- 76 Artichoke Leaf 2014

o 5 10 15 20 25 30 min

1. chlorogenic acid 2. unknown substance

Figure 1866.-2. - Chromatogram for the assay of artichoke leaf: test solution

transfer the supematant to a 200 mL volumetric fiask. System suitability Test solution:
Repeat the pro ce dure and dilute to 200.0 mL with water R . - the chromatogram obtained is similar to the
Referenee solution Dissolve 5.0 mg of ehlorogenie acid CRS in chromatogram shown in Figure 1866.-2;
50.0 mL of methanol R . Transfer 5.0 mL of this solution to a - resolution: mínimum 2.0 between the peak due to
volumetric fiask, add 5 mL of methanol R and dilute to chlorogenic acid and the subsequent peak (peak 2).
20.0 mL with water R. Calculate the percentage content of chlorogenic acid using
Column: the following expression:
- size: 1 = 0.25 m, 0 = 4.6 mm;
- stationary phase: oetadeeylsilyl siliea gel for ehromatography R
(5 ¡.¡m);
- temperature: 40 oc.
Mobile phase: area of the peak due to chlorogenic acid in the
- mobile phase A: phosphorie aeid R, water R (0.5:99.5 V/V); chromatogram obtained with the test solution;
- mobile phase B: phosphorie acid R, aeetonitrile R area of the peak due to chlorogenic acid in the
(0.5:99.5 V/V); chromatogram obtained with the reference solution;
Time MobiJe phase A MobiJe phase B mI mass of the drug to be examined in the test
(min) (per cent VM (per cent V/JI) solution, in grams;
0-1 92 8 m2 mass of ehlorogenie acid CRS in the reference
1 - 20 92 -7 75 8 -7 25 solution, in grams;
p percentage content of chlorogenic acid in chlorogenic
20 - 33 75 25
acid CRS.
33 - 35 75 --) O 25 --) 100 _ __ __ _ _ _ _ __ __ _ __ _ __ _ __ _ Ph f ur

Flow rate 1.2 mUmin.

Deteetion Spectrophotometer at 330 nm.
InJeetion 25 ¡.¡L.
 2014 Artichoke Leaf Preparations IV - 77

Test solution Dissolve 30.0 mg of the extraet to be examined

Artichoke Leaf Dry Extract in the solvent mixture and dilute to 25.0 mL with the solvent
(Ph Eur monograph 2389) mixture.
~Ew ____________________________________________ Reference solution (a) Dissolve 5.0 mg of chlorogenic
acid CRS in 50.0 mL of methanol R. Transfer 5.0 mL of this
DEFINITION solution to a volumetrie ftask, add 5 mL of methanol R and
Dry extraet produeed from Artichoke lea! (1866) . dilute to 20.0 mL with water R.
Content Reference solution (b) Dissolve 30 mg of the artichoke leaf dry
Minimum 0.6 per eent of ehlorogenie aeid (CI6HIS09; M r extract HRS in the solvent mixture and dilute to 25 .0 mL
354.3) (dried extraet). with the solvent mixture.
PRODUCTION Column:
The extraet is produeed from the herbal drug by a suitable -- size: l = 0.25 m, 0 = 4.6 mm;
proeedure using water of minimum 80 oC. -- stationary phase: octadecylsilyl silica gel for chromatography R
(5 ¡.tm);
CHARACTERS -- temperature: 40 oC .
Appearance
Mobz1e phase:
Light brown or brown, amorphous powder.
-- mobz1e phase A: phosphoric acid R, water R (0.5 :99.5 V/V);
IDENTIFICATION -- mobile phase B: phosphoric acid R, acetonitrile R
Thin-Iayer ehromatography (2.2.27). (0.5:99 .5 V/V);
Test solution Dissolve 1.0 g of the extraet to be examined in Time Mobile phase A Mobile phase B
10 mL of ethanol (60 per cent V/V) R. Sonieate for 5 min and (min) (pereent VM (pereent VM
filter. 0- 1 92 8
Reference solution Dissolve 5 mg of luteolin-7-glucoside R and 1 - 20 92 -7 75 8 -7 25
5 mg of chlorogenic acid R in 10 mL of methanol R.
20 - 33 75 25
Plate TLC silica gel plate R (5-40 ¡.un) [or TLC silica gel
plate R (2-10 ¡.tm)]. 33 - 35 75 -7 O 25 -7 100

Mobz1e phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (11:11 :27:100 VIVIV/V).
Application 10 ¡.tL [or 2 ¡.tL] as bands of 10 mm [or 8 mm].
Development Over a path of 13 cm [or 6 cm].
Drying In airo
Detection Heat at 100 oC for 5 mini spray the warm plate
with a 10 gIL solution of diphenylboric acid aminoethyl ester R
in methanol R followed by a 50 gIL solution of macrogol
400 R in methanol R; examine in ultraviolet light at 365 nm.
Results See below the sequenee of ftuoreseent zones present
in the ehromatograms obtained with the referenee solution
and the test solution. Furthermore, other ftuoreseent zones
may be present in the ehromatogram obtained with the test
solution.
Flow rate 1.2 mUmin.
Detection Speetrophotometer at 330 nm.
Injection 25 ¡.tL.
System suitabz1ity Referenee solution (b) :
-- peak-to-valley ratio: minimum 2.5, where Hp = height
aboye the baseline of the peak irnmediately after the peak
due to ehlorogenie aeid and H v = height aboye the
baseline of the lowest point of the curve separating this
peak from the peak due to ehlorogenie aeid;
-- the ehromatogram obtained is similar to the
ehromatogram supplied with the artichoke leaf dry extract
HRS.
Calculate the pereentage eontent of ehlorogenie aeid using
the following expression:

Top of the plate Al X m2 X p X 0.125

A light blue f1uorescent zone A 2 x mI
----- -----
Al area of the peak due to ehlorogenie aeid in the
Luteolin-7-glucosíde: a yellow or A yellow or orange f1uorescent zone
orange f1uorescent zone (luteolín-7-glucosíde) ehromatogram obtained with the test solution;
A2 area of the peak due to ehlorogenie aeid in the
ehromatogram obtained with referenee solution (a);
Chlorogeníc acíd : a light blue A light blue fluorescent zone
f1uorescent zone (chlorogeníc acíd) mI mass of the extraet to be examined used to prepare
----- ----- the test solution, in milligrams;
m2 mas s of chlorogenic acid CRS used to prepare
Referenee solution Test solution referenee solution (a), in milligrams;
p pereentage eontent of ehlorogenie aeid in chlorogenic
acid CRS.
TESTS
____________________________________________ ~E~

Loss on drying (2.8_17)

Maximum 6.0 per eent.
Total ash (2.4.16)
Maximum 30.0 per eent.
ASSAY
Liquid ehromatography (2.2.29).
Solvent mixture methanol R, water R (30:70 V/V).
IV-78 Ash Leaf 2014

Ash Leaf *****

** **
(Ph Bu/" monograph 1600) ***
~E~ ____________________________________________

DEFINITION
Dried leaf of Fraxinus excelsior L. or Fraxinus angustijolia Vahl
(syn. Fraxinus oxyphylla M. Bieb) or of hybrids of these 2
species or of a mixture.
Content
Minimum 2.5 per cent of total hydroxycinnamic acid
derivatives, expressed as chlorogenic acid (CI6HI809; M r
354.3) (dried drug).
IDENTIFICATION
A. The leaf consists of leaflets that are sometimes detached
and separated from the rachis . The leaflet is about 6 cm long
and 3 cm wide. Each leaflet is subsessile or shortly petiolate,
oblong, lanceolate, somewhat unequal at the base, acuminate
at the apex, with fine, acute teeth on the margins; the upper
surface is d ark green and the lower surface is greyish-green.

Ga_ G
The midrib and secondary veins are whitish and prominent
on the lower surface.
B. Microscopic examination (2.8. 23) . The powder is greyish-
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1600.-1 ): fragments ofthe upper
epidermis of the lamina in surface view [Bl, with sorne of the
25 ~m
cells showing cuticular striations, accompanied by underlying I-----i
Gb
palisade parenchyma [Ba); fragments of the lower epidermis
in surface view [A) consisting of cells covered by fine
cuticular striations [Aa), numerous anomocytic stomata Figure 1600.-1. - Illustration for identification test B of
(2.8.3) [Ab) and rare peltate glandular trichomes with a powdered herbal drug of ash leaf
unicellular stalk and a glandular head composed of radiating
cells [Ac) ; fragments of lamina in transverse section [F] with
2 layers of palisade parenchyma [Fa], spongy parenchyma Reference solution Dissolve 5 mg of rutin R and 5 mg of
[Fb) and, occasionally, glandular trichomes embedded in the chlorogenic acid R in 10 mL of methanol R.
epidermis [Fc); occasional multicellular, uniseriate, conical
Plate TLC silica gel plate R (5-40 J1IIl) [or TLC silica gel
covering trichomes composed of cells with thick striated
plate R (2-10 11m)) .
walls, either on an epidermis [C] or fragmented [D);
fragments of vascular tissue from the leaflets [E) composed of Mobile phase anhydrous formic acid R, water R, ethyl acetate R
spiral ves seis [Ea), short fibres [Eb) and sometimes palisade (10:10:80 VIV/V).
parenchyma [Ec); fragments of vascular tissue from the veins Application 10 llL [or 4 llL) as bands of 10 mm [or 8 mm) .
[G) composed of fibres [Ga), sometimes accompanied by Development Over a path of 10 cm [or 6 cm] .
cells with thick, pitted walls from the medullary rays [Gb] . Drying In airo
C. Examine the chromatograms obtained in the test for Detection Heat at 100 oC for 3 min; treat the still-warm
Fraxinus omus. plate with a 10 gIL solution of diphenylboric acid aminoethyl
Results See below the sequence of zones present in the ester R in methanol R; dry in air; treat with a 50 gIL solution
chromatograms obtained with the reference solution and the of macrogol 400 R in methanol R; dry in air; examine in
test solution. The intensity of the zones present in the ultraviolet light at 365 nm.
chromatogram obtained with the test solution may vary
depending on the presence of F. excelsior, F. angustifolia, their
hybrids or their concentration in a mixture. Furthermore,
Top of the plate
other fluorescent zones may be present in the chromatogram
obtained with the test solution. - -- - - -
TESTS A light blue flu orescent zone
Foreign matter (2.8.2) (acteoside)
Maximum 3.0 per cent of stems and maximum 2.0 per cent Chlorogenic acid: a light blue A light blue fluorescent zone may
fluorescent zone be present (chlorogenic acid)
of other foreign matrer.
--- - --
Fraxinus ornus
Thin-Iayer chromatography (2.2.27) . A light blue fluorescent zone

Test solution To 1 g of the powdered herb al drug (355) Rutin : an orange fluorescent zone An orange flu orescent zone (rutin)
(2.9.12) add 20 mL of methanol R . Stir with a magnetic Reference solution Test solution
stirrer for 10 mino Filter.
2014
Results The chromatogram obtained with the test solution
does not show any intense light blue fluorescent zones in the
upper third of the chromatogram.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 12.0 per cent.
ASSAY
Test solution (a) To 0.300 g of the powdered herbal drug
(355) (2.9.12) add 95 mL of ethanol (50 per eent V/v,¡ R. Boil
in a water-bath under a reflux condenser for 30 mino Allow
to cool and filter. Rinse the filter with 5 mL of ethanol
(50 per cent V/v,¡ R. Combine the filtrate and the rinsings in
a volumetric flask and dilute to 100.0 mL with ethanol
(50 per cent V/v,¡ R.
Test solution (b) To 1.0 mL of test solution (a) in a test
tube, add 2 mL of 0.5 M hydrochloric acid, 2 mL of a
solution prepared by dissolving 10 g of sodium nitrite R and
10 g of sodium molybdate R in 100 mL of water R, then add
2 mL of dilute sodium hydroxide solution R and dilute to
10.0 mL with water R; mix.
Imrnediately measure the absorbance (2.2.25) oftest solution
(b) at 525 nm, using as compensation liquid a solution
prepared as follows: mix 1.0 mL of test solution (a), 2 mL of
0.5 M hydrochloric acid, 2 mL of dilute sodium hydroxide
solution R and dilute to 10.0 mL with water R.
Calculate the percentage content of total hydroxycinnamic
acid derivatives, expressed as chlorogenic acid, using the
following expression:
A x 5.3
m Plate TLC siliea gel F254 plate R (2-10 flm).
taking the specific absorbance of chlorogenic acid to be 188.
A absorban ce at 525 nm;
m = mass of the herbal drug to be examined, in grams.
____________________________________________ ~Ew
Astragalus Mongholicus Root
(Ph Eur monograph 2435)
Ph Eur __________________________________________
DEFINITION
Whole, dried root of Astragalus mongholicus varomongholieus
(Syn. Astragalus membranaeeus Bunge varo mongholieus
(Bunge) P.I<. Rsiao) and Astragalus mongholieus varo dahurieus
(De.) Podlech (Syn. Astragalus membranaeeus Bunge), freed
from rootlets and rootstock, collected from spring to autumn.
Content
Minimum 0.040 per cent of astragaloside IV (C41R68014; M r
785) (dried drug).
IDENTIFICATION
A. Cylindrical, often with branches, upper part relatively
thick, 30-90 cm long and 1-3.5 cm in diameter. Extemally
pale brownish-yellow or pale brown, with irregular,
longitudinal wrinkles or furrows. Texture hard and tenacious;
uneasily broken, fracture highly fibrous and weakly
(cultivated origin) or strongly starchy (wild origin), bark
yellowish-white, wood pale yellow, with radiate striations and
Astragalus Mongholicus Root IV-79
fissures; the central region is dark brown and in older roots
may be broken down to form a hollow surrounded by
fragments of disintegrating tissue.
B. Reduce to a powder (355) (2.9.12). The powder is
yellowish-white. Examine under a microscope using ehloral
hydrate solution R. The powder shows the following diagnostic
characters: fibres, in bundles or scattered, 8-30 flm in
diameter, thick-walled with longitudinal fissures on the
surface, the primary walls often separated from the secondary
walls, both ends often broken or tassel-like, or slightly
truncated; colourless or orange ves seis with closely arranged
bordered pits; cork fragments consisting of severallayers,
often accompanied by collenchymatous phelloderm; stone
cells occasionally visible, rounded, oblong or irregular,
slightly thick-walled. Examine under a microscope using a
50 per cent V/V solution of glyeerol R: the powder shows
small, rounded or ovoid starch granules, usually simple or
sometimes 2- or 3-compound, about 5 flm in diameter.
e. Thin-layer chromatography (2.2.27).
Test solution Reat 3 g ofthe powdered drug (355) (2.9.12)
with 50 mL of methanol R for 50 min under reflux and then
filter. Evaporate the filtrate under reduced pressure to
dryness and take up the residue in 1 mL of water R . Apply
the solution to a 6 mL solid phase extraction column
containing octadecylsilyl siliea gel for ehromatography R
previously conditioned with 3 mL of methanol R and then
with 3 mL of water R. Wash the column with 15 mL of
water R followed by 15 mL of a 30 per cent VIV solution of
methanol R. Discard the washings. Elute with 20 mL of
methanol R and collect the eluate. Evaporate the eluate under
reduced pressure to dryness and take up the residue with
2 mL of methanol R.
Referenee solution Dissolve 10.0 mg of daidzin R and 5.0 mg
of daidzein R in 5.0 mL of methanol R .
Mobile phase water R, methanol R, ethyl aeetate R
(10:13.5:100 VIV/V).
Application 3 flL as bands of 8 mm.
Development Over a path of 7 cm.
Drying In air.
Detection A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top oC the plate
A blue fluorescent zone
Daidzein: a quenching zone
A quenching zone
----- -----
A quenching zone
Daidzin: a quenching zone A quenching zone
----- -----
ReCerence solution Test solution
Detection B Treat with anisaldehyde solution R. Reat at
100 oC for 3 mino Examine in ultraviolet light at 366 nm.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
 IV-80 Astragalus Mongholicus Root 2014

test solution. Furthermore, other faint zones may be present Mobile phase:
in the chromatogram obtained with the test solution. -- mobile phase A: water R;
-- mobile phase B : aeetonitrile R;

Top oC the plate Time Mobile phase A Mobile phase B

(min) (pereent VM (per cent VM
A violet zone 0-5 90 10
Daidzein: a pale blue zone 5 - 10 90 -> 80 10 -> 20
A violet zone 10 - 20 80 -> 75 20 -> 25

- -- --- 20 - 30 75 -> 67 25 -> 33

A violet zone 30 - 40 67 -> 65 33 -> 35

Daidzin: a pale blue zone A brown zone 40 - 50 65 -> 40 35 -> 60

5 brown zones 50 - 55 40 60

--- - -- Flow rate 0.5 mUmin .

Referenee solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent.
Loss on drying (2.2. 32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9. 12) by drying in an oven at
105 oC for 3 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Weigh 4.0 g of the powdered drug (355)
(2.9. 12) into a Soxhlet type extractor and add 40 mL of
methanol R . Macerate ovemight. Add again 40 mL of
methanol R . Heat under a reflux condenser for 4 h. Evaporate
to dryness. Dissolve the residue in 10 mL of water R, heating
slightly if necessary. Shake with 4 quantities, each of 40 mL,
of butanol R saturated with water R . Combine the butanol
extracts and wash with 2 quantities, each of 40 mL, of
ammonia R . Discard the arnmonia layers and evaporate the
butanol layers to dryness. Dissolve the residue in 5 mL of
water R and cool. Apply the solution to a solid phase
extraction column containing 1 g of oetadeeylsilyl siliea gel for
ehromatography R previously washed with 5 mL of methanol R
and 5 mL of water R . Wash the column with 20 mL of
water R and 20 mL of ethanol (25 per eent V/V) R. Elute with
25 mL of ethanol (70 per eent V/V) R . Evaporate the eluate to
dryness. Dissolve the residue in 5.0 mL of methanol R .
R eferenee solution (a) Dissolve 10.0 mg of astragaloside
Deteenon Evaporative light-scattering detector; the following
settings have been found to be suitable; if the detector has
different setting parameters, adjust the detector settings so as
to comply with the system suitability criterion :
-- earrier gas: air;
-- fiow rate: 1.5 mUmin;
-- evaporator temperature: 50 oC .
Injeetion 20 ¡¡L of the test solution and reference solutions
(b), (c), (d) and (e).
R elative reten non With reference to ginsenoside Rb1
(retention time = about 33.6 min):
astragaloside IV = about 1.05.
System suitability:
-- resolunon: minimum 4.0 between tlle peaks due to
astragaloside IV and ginsenoside Rb1 in the
chromatogram obtained with reference solution (e).
Establish a calibration curve with the logarithm of the
concentration (mg/mL) of reference solutions (b), (c) and (d)
(corrected by the dec1ared percentage content of astragaloside
IV CRS) as the abscissa and the logarithm of the
corresponding peak area as the ordinate. Calculate the
percentage content of astragaloside IV using the following
expression:
lOA x 0.5
m
A logarithm of the concentration corresponding to the
astragaloside IV peak in the chromatogram obtained
with the test solution, determined from the calibration
curve;
m m ass of the drug to be examined used to prepare the
test solution, in grams.
____________________________________________ ~E~

IV CRS in methanol R and dilute to 10.0 mL with the same

solvent.
R eferenee solutions (b), (e), (d) Dilute reference solution (a)
to obtain 3 reference solutions of astragaloside IV, the
concentrations of which span the expected value in the test
solution.
R eferenee solution (e) Dissolve 5.0 mg of ginsenoside Rbl R in
5 mL of methanol R and dilute to 10.0 mL with reference
solution (a) .
Column:
-- size: 1 = 0.25 m, 0 = 3.2 mm;
-- stationary phase: oCladeeylsilyl siliea gel for ehromatography R
(3 ¡¡m);
-- temperature: 25 oc.
 2014 Atractylodes Rhizome, Largehead IV-81

*** Top of the plate

Atractylodes Lancea Rhizome * *
** ** j3-Caryophyllene: a pink zone A pink or violet zone
(Ph Eur monograph 2559) ***
nE~ ____________________________________________ An orange zone may be presen t

- -- - --
DEFINITION
Dried, whole or fragrnented rhizome of Atractylodes lancea An intense greyish·green zone
(Thunb.) De. (syn. Atractylodes chinensis (Bunge) Koidz.) A very faint violet zone
with the roots removed, collected in spring and autumn.
- -- ---
Content
Minimum 14 mUkg of essential oil (anhydrous drug). Bornyl aceta te : a brown zone A violet zone

IDENTIFICATION Several violet zones

A. The whole rhizome is curved, irregular, nodular and
cylindrical, 3-10 cm long and 1-3 cm in diameter.
The external surface is transversally wrinkled, dark greyish-
brown or yellowish-brown; it shows numerous rounded
protuberances and large circular stem scars and smaller roOl
scars.
The fragrnented rhizome occurs in slices with a highly
variable diameter (1-4 cm) and a thickness of about 0.5 cm.
The external surface is wrinkled, dark greyish-brown or
yellowish-brown and shows numerous scars. The transverse
section is pale yellow or brownish-yellow, consisting of
fibrous tissues scattered with particularly abundant orange oil
cavities appearing as dots.
B. Microscopic examination (2.8.23) . The powder is
brownish-yellow. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: fragments of orange cork, with polyhedral cells,
often accompanied by sub rectangular or ovo id sclereids with
very thick channelled walls from the phelloderrn; isolated,
ovoid or subrectangular sclereids with very thick, channelled
walls and a narrow lumen, variable in shape (20-80 )..lm in
diameter); fragrnents of parenchyma with polyhedral or
subrectangular cells containing small needle-shaped crystals
of caJcium oxalate (5-30 )..lm) clearIy visible in polarised Iight;
fragrnents of fibres in bundles, with heavily thickened and
slightly pitted walls (40 ~lm in diameter) and a narrow
lumen, very often associated with xylem ves seIs; fragrnents of
short, reticulate or pitted ves seIs, usually included in
parenchyma with thin-walled cells; fragrnents of oil glands
Reference solution Test solution
Application 5)..lL [or 3 )..lL] as bands of 10 mm [or 6 mm].
Development In an unsaturated tank, over a path of 10 cm
[or 6 cm].
Drying In airo
Detection Treat with anisaldehyde solution R and heat at
105-110 oC for 5-10 min; examine in daylight.
Results The chromatogram obtained with the test solution
shows an intense greyish-green zone in the middle third.
In the case of a substitution by Atractylodes macrocephala, no
intense greyish-green zone is present in the middle third.
Water (2.2.13)
Maximum 100 mUkg, deterrnined on 20.0 g of the
powdered herbal drug (355) (2.9.12).
Total ash (2.4.16)
Maxirnum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 15.0 g offreshly powdered herbal drug (710)
(2.9.12), a 500 mL round-bottomed fiask, 200 mL of water R
as the distillation liquid and 0.50 mL of xylene R in the
graduated tube. Distil at arate of 2-3 mUmin for 2 h.
____________________________________________ nE~

with thin-walled cells and granular orange-brown contents

and orange oil droplets. Examine under a microscope,
without heating, using glycerol R: the powder shows pieces of
inulin, free or included in parenchyma cells.
e. Examine the chromatograms obtained in the test for
Atractylodes macrocephala.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furtherrnore, other faint zones may be present
in the chromatogram obtained with the test solution.
TESTS
Atractylodes macrocephala
Thin-layer chromatography (2.2.27).
Test solution Introduce 0.5 g of the powdered herbal drug
(355) (2.9.12) into a centrifuge tube, add 2 mL of
methanol R and stopper the tube. Sonicate at 25 oC for
15 min and centrifuge.
R eference solution Dissolve 10 mg of {J-caryophyllene R and
10 mg of bomyl acetate R in 5 mL of methanol R.
Plate TLC silica gel plate R (5-40 )..lm) [or TLC silica gel
plate R (2-10 ~lm)].
Mobile phase ethyl acetate R, heptane R (5:95 VIV).
***
Largehead Atractylodes Rhizome
*** ***
(Ph Eur monograph 2560) ***
nE~ ____________________________________________
DEFINITION
Dried, whole or fragrnented rhizome of Atractylodes
macrocephala Koidz. with the roots removed, collected in
winter when the lower leaves of the plant turn yeIlow and the
upper leaves become fragile.
Content
Minimum 9 mUkg of essential oil (anhydrous drug).
IDENTIFICATION
A. The whole rhizome is irregularly shaped, 3-13 cm long
and 1.5-7 cm in diameter. ExternaIly yeIlowish-grey or dark
brown, with small knob-like protrusions, interrupted
longitudinal wrinkles and grooves.
The fragrnented rhizome occurs in slices with a highly
variable diameter (1 -7 cm) and a thickness of about 0.5 cm.
The external surface is wrinkled or grooved, more or less
IV-82 Azadirachta Indica Leaf
dark yellowish-brown with numerous root scars.
The transverse section is pale yellow, consisting of tissues
with wide spaces between them and scattered with many
orange oil cavities appearing as dots that are particularly
abundant in the external tissues.
The fracture is hard and fibrous.
B. Microscopic examination (2.8. 23). The powder is
brownish-yellow. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: fragments of orange cork, with polyhedral cells;
fragments of parenchyma with polyhedral or subrectangular
cells, many of which contain small needle-shaped crystals of
ca1ciurn oxalate (1 0-32 ~m) c1early visible in polarised light;
sc1ereids, isolated or in small groups, with very thick,
channelled walls, variable in shape (35-65 ~lm in diameter);
fragments of fibres, isolated or in bundles, with moderately
thickened and slightly pitted walls (40 ~m in diameter);
fragments of short, reticulate or pitted vessels, usually
inc1uded in parenchyma with thin-walled cells; fragments of
oil glands with thin-walled cells and granular orange-brown
contents. Examine under a microscope, without heating,
using glycerol R; the powder shows numerous pieces of inulin,
free or inc1uded in parenchyma cells.
C. Examine the chromatograms obtained in the test for
Atraccylodes lancea.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top oC the plate
p·Caryophyllene : a pink zone A pink or violet zone
An orange zone
---
A very faint violet zone
---
Bornyl aceta te: a brown zone
A very faint viole! zone
Several fain! viole! zones
Reference solution Tes! solution
D. To 0.5 g of the powdered herbal drug (355) (2.9. 12) add
5 mL of ethanol (96 per cent) R, heat in a water-bath at 60 oC
for 2 min and filter. To 1 mL of the filtrate add 0.25 mL of
a solution freshly prepared as follows: dissolve 5 mg of
vanillin R in 0.5 mL of ethanol (96 per cene) R, to this
solution add 0.5 mL of water R and 3 mL of hydrochloric
acid R. Shake immediately; a red or reddish-purple colour
develops and persists.
TESTS
Atractylodes lancea
Thin-layer chromatography (2.2.27).
Test solution Introduce 0.5 g of the powdered herbal drug
(355) (2.9.12) into a centrifuge tube, add 2 mL of
111ethanol R and stopper the tube. Sonicate at 25 oC for
15 min and centrifuge.
Reference solution Dissolve 10 mg of p-caryophyllene R and
10 mg of bornyl acetate R in 5 mL of 111elhanol R.
Plate TLC silica gel place R (5-40 J1m) [or TLC si/ica gel
plate R (2-10 ~m ) ) .
2014
Mobile phase elhyl acetale R, heptane R (5:95 V/V).
Applicalion 5 ~L [or 3 ~L) as bands of 10 mm [or 6 mm).
Develop111ent In an unsaturated tank, over a path of 10 cm
[or 6 cm).
Drying In airo
Detection Treat with anisaldehyde solution R and heat at
105-110 oC for 5-10 min; examine in daylight.
Results The chromatogram obtained with the test solution
shows no greyish-green zone in the middle third, aboye the
very faint violet zone.
Water (2.2.13)
Maximum 100 mUkg, determined on 20.0 g ofthe
powdered herbal drug (710) (2.9.12).
Total ash (2.4.16)
Maximurn 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 15.0 g offreshly powdered herbal drug (710)
(2.9.12), a 500 mL round-bottomed ftask, 200 mL of water R
as the distillation liquid and 0.50 mL of xylene R in the
graduated tube. Distil at arate of 2-3 mUmin for 2 h.
____________________________________________ PhE~
Azadirachta Indica Leaf
Nimba Leaf
DEFINITION
Azadirachta Indica Leaf is the dried leaf of Azadirachta indica
A. Juss.
--- It contains not les s than 1.0% of tetranortriterpinoids,
expressed as salannin, ca1culated with reference to the dried
drug.
---
IDENTIFICAnON
A. Leaftets thin and fragile, ovate to lanceolate, 3 to 10 cm
long and 1 to 2.5 cm wide, curved with a serrate rnargin;
base markedly asyrnmetrical, apex acuminate and terminating
in a fine point; upper surface dark brownish-green, lower
surface paler with distinct midrib and lateral veins running to
the margins; both surfaces glabrous. Fragments of the rachis
may be present; these are pale brown, slender, up to about
10 cm long, cylindrical with faint longitudinal striations and
bearing alternating pairs of scars where the leaftets were
attached.
B. Reduce to a powder (355) . The powder is green. Examine
under a microscope using chloral hydrate solution. The powder
shows fragments of the epidermis composed of thin-walled
tangentially elongated cells with abundant ano111ocylic
sto mata, Appendix XI H; abundant fragments of single
layered palisade and thin-walled parenchymatous cells of the
spongy mesophyll present, sorne with associated ves seis; sorne
fragments display rosette crystals of ca1cium oxalate ofien in
rows.
C. Carry out the method for thin-layer chro111atography,
Appendix III A, using the following solutions.
(1) Add 30 rnL of 111elhanol to approximately 5 g of
powdered herbal drug, mix thoroughly by hand and with the
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for
5 minutes and collect the c1ear supernatant liquid. Repeat the
extraction twice, combine the supernatant liquid and dilute
 2014 Azadirachta Indica Leaf IV -83

10 100 mL with methanol. Filter approximately 30 mL of the extraction twice, combine the supematant liquid and dilute
solution through a 0.45-¡.tm filter and use the filtrate. to 100 mL with methanol. Filter approximately 30 mL of the
(2) 0.025% w/v each of azadirachtin, salannin CRS and solution through a 0.45-¡.tm filter and use the filtra te.
f3-sitosterol in methanol. (2) 0.0025% w/v of salannin CRS and 0.001 % w/v of
CHROMATOGRAPHIC CONDITIONS azadirachtin-A CRS in methanol.
(a) Use a silica gel 60 or high-performance sz7ica gel 60 CHROMATOGRAPHIC CONDlTIONS
precoated plate [Merck silica gel 60 HPTLC plates are (a) Use a stainless steel column (15 cm x 2.1 mm) packed
suitable]. with octadecylsilyl silica gel for chromatography (5 ¡.tm)
(b) Use the mobile phase as described below. (Spherisorb ODSl is suitable).
(c) Apply as bands 5 ¡.tL of each solution. (b) Use gradient elution and the mobile phase described
(d) Develop the plate to 15 cm [or 7 cm]. below.
(e) After removal of the plate, spray with vanillin reagent, heat (c) Use a fiow rate of 0.5 mL per minute.
the plate at 100° for 3 minutes and examine in daylight. (d) Use a column temperature of 30°.
MOBILE PHASE (e) Use a detection wavelength of217 nm.
3 volumes of hexane and 7 volumes of ethyl acetate. (f) Inject 10 ¡.tL of each solution.
When the chromatograms are recorded under the prescribed
SYSTEM SUITABILITY
conditions the retention time of the peak due to azadirachtin-
The test is not valid unless the chroma1Ogram obtained with A is about 15 minutes and the retention time of the peak due
solution (2) shows three cIearly separated spots. to salannin is about 22 minutes.
CONFIRMATION MOBILE PHASE
In the chromatogram obtained with solution (1), a black Mobile phase A 0.1 volume of trifiuroacetic acid and
band with an Rf value of approximately 0.15 corresponding 100 volumes of water.
in position to a brown band in the chromatogram for
Mobile phase B 0.1 volume of trifiuroacetic acid and
solution (2) is obtained. A black band with an Rf value of
100 volumes of acetonitrile.
approximately 0.3 corresponding in position to the indigo
band in the chromatogram for solution (2) is obtained for
salannin. An indigo band with an Rf value of approximately Time Mobile phase A Mobile phase B Comment
0.6 corresponding in position to a purple band in the (Minutes) (%v/v) (%v/v)
chromatogram for solution (2) is obtained for P-sitosterol. 0-10 90--+70 10--+30 linear grad ient

10-25 70--+30 30--+ 70 linear gradient

Top ofthe plate 25-40 30 70 isocratic

40-45 30--+90 70--+ 10 linear gradient

45-50 90 10 re-equilibration
Purple band ~-sitosterol : a purple
band

SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
Salannin: an ¡ndigo
with solution (2):
Black band
band the symmetry factor of the peak due to azadirachtin-A is at
most 1.2;
Black band Azadirachtin: a brown
band the symmetry factor of the peak due to salannin is at most 1.4.
Solution (1) Solution (2) DETERMINATION OF CONTENT
Calculate the total content of tetranortriterpinoids, expressed
as salannin, from the sum of the areas of the peaks eluting
TESTS from three minutes before to three minutes after the
Foreign matter retention time of salannin and from the dec1ared content of
Not more than 2%, Appendix XI D. salannin in salannin CRS using the following expression:
Loss on drying
When dried for 2 hours at 105°, loses not more than 10.0% Al m 2 VI 100
of its weight. Use 1 g. --x--x--xpx
Ash
A 2 V 2 mi 100 - d
Not more than 10.0%, Appendix XI J, method ll.
Water-soluble extractive combined areas of the peaks in the chromatogram
Not less than 20.0%, Appendix XI B2. obtained with solution (1) with retention times from
three minutes before 10 three minutes after the
ASSAY
retention time of peak due 10 salannin in the
Carry out the method for liquid chromatography, chromatogram obtained with solution (2),
Appendix III D, using the following solutions. area of the peak due to salannin in the chromatogram
(1) Add 30 mL of methanol to approximately 5 g of obtained with solution (2),
powdered herbal drug, mix thoroughly by hand and with the mi weight of the drug being examined in mg,
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for m2 weight of salannin CRS in mg,
5 minutes and collect the cIear supematant liquido Repeat the VI dilution volume of solution (1),
 IV-84 Bacopa Monnieri
dilution volume of solution (2),
percentage content of salannin in salannin CRS,
percentage loss on drying of the herbal drug being
examined.
STORAGE
Azadirachta Indica Leaf should be protected from moisture.
Bacopa Monnieri
DEFINITION
Bacopa Monnieri is the dried aerial parts of Bacopa monnieri
(L.) Wettst.
It contains not less than 1.0% w/w of bacopa saponins,
expressed as bacopaside n (C47H760S), calculated with
reference to the dried drug.
IDENTIFICATION
A. Pie ces of herb, consisting mainly of stem and leaf; buff or
greenish brown, angular stems, 1 to 2 mm in diameter and
10 to 30 cm long, nodes prominent, often showing sprouting
rootlets and with numerous ascending branches; greenish
leaves sessile or short petioled, fleshy, glabrous on the upper
surface, simple, opposite, decussate, 0.6 to 2.5 cm long and
3 to S mm wide, reniform, spathulate or oblanceolate,
margin entire or, rarely, dentate. If present, flowers are
axillary and solitary, on pedunc1es usually longer than the
leaves; corolla up to 1 cm long, five lobed, oblong, obtuse;
fruit capsule ovoid-acuminate or slightly beaked at the apex,
glabrous, up to 5 mm long.
B. Reduce to a powder (355) . The powder is greenish or
yellowish-brown. Examine under a microscope using chloral
hydrate solution. The powder shows fragments of the
epidermis, with a thin striated cutic1e, multicellular glandular
trichomes, anomocytic stomata; numerous xylem ves seIs with
reticulate thickening. Examine under a microscope using
50% v/v of glycerol in water. Starch granules are present,
usually simple, round or ovo id, 4 to 14 ¡.tm in diameter,
without a visible hilum.
C. Carry out the method for thin-layer chromatography,
Appendix nI A, using the following solutions.
(1) Add 30 mL of methanol (70%) to 5.0 g of the powdered
herbal drug, heat on a water-bath under reflux for
30 minutes, cool, centrifuge at 3000 rpm for 5 minutes and
decant the supernatant liquid. Repeat the extraction
procedure with a further rwo 30-mL quantities of methanol
(70%). Combine the supernatant liquid, dilute to 100 mL
with methanol (70%) and filter (0.45 !-lm PTFE is suitable).
(2) 0.05% w/v of bacopaside JI CRS in methanol (70%) .
(3) 0.05% w/v each of bacopaside 1 CRS and bacopaside
JI CRS in methanol (70%).
CHROMATOGRAPHIC CONDITIONS
(a) Use silica gel 60 precoated plates or high-performance
silica gel 60 (Merck silica gel 60 plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 20 !-lL [or 10 !-lL] of each solution, as bands.
(d) Develop the plate to 15 cm [or S cm].
(e) After removal of the plate, dip in anisaldehyde solution Rl,
heat in an oven at 105° for 5 minutes and examine in
daylight.
MOBILE PHASE
10 volumes of 1% v/v of formic acid, 20 volumes of methanol
and 70 volumes of ethyl acetate.
2014
SYSTEM SurT ABILITY
The test is not valid unless the chromatogram obtained with
solution (3) shows rwo c1early separated bands.
CONFIRMATION
The chromatogram obtained with solution (1) shows a dark
band with an Rf value of approximately 0.4 corresponding to
the band obtained with bacopaside JI in the chromatogram
obtained with solution (3), a lighter band with an Rf value of
approximately 0.3 corresponding to the band obtained with
bacopaside 1 in the chromatogram obtained with solution (3) .
Bands with Rf values of approximately 0.2 and O.S are also
presento Other bands may be present in solution (1).
Top of theJ!.late
unknown: dark band
bacopaside 11: dark band bacopaside 11: dark band bacopaside 11: dark band
bacopaside 1: light band bacopaside 1: tight band
unknown: Jight band
Solution (1) Solution (2) Solution (3)
TESTS
Foreign matter
Not more than 1.0%, Appendix XI D.
Loss on drying
Not more than 11.0%, Appendix IX D . Use 1 g.
Ash
Not more than 13.0%, Appendix XI J, Method n .
Water-soluble extractive
Not les s than 15.0%, Appendix XI B2.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions .
(1) Reduce to a powder (355). To 5.0 g of the powder, add
30 mL of methanol (70%) and heat under reflux for
30 minutes. Allow to cool, centrifuge and collect the
supernatant liquido Repeat the extraction rwice with rwo
further 30-mL quantities of methanol (70%). Combine the
three supernatant liquids, dilute to 100 mL with methanol
(70%) and filter (0.45 ¡.tm PTFE is suitable).
(2) 0.05% w/v of bacopaside 11 CRS in methanol (70%).
(3) 0.05% w/v of bacoside A CRS in methanol (70%).
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with end-capped octadecylsilyl silica gel for chromatography
(5 ¡.tm) (Phenomenex Luna C1S is suitable) .
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a flow rate of 1.0 mL per minute.
(d) Use a column temperature of 30°.
(e) Use a detection wavelength of 205 nm.
(t) Inject 20 !-lL of each solution.
 2014 Bearberry Leaf IV-S5

(g) Record the chromatograms for 75 minutes .
MOBILE PHASE
315 volumes of acetonitrile and 685 volumes of 0.71 % w/v
anhydrous sodium sulfate, previously adjusted to pH 2.3 with
sulfuric acid.
When the chromatograms are recorded under the prescribed
conditions the retention time of bacopaside Ir is about
36 minutes. The retention times relative 10 bacopaside Ir are:
bacoside A3 , about 0.9; bacopaside X, about 1.2;
bacopasaponin C, about 1.3; bacopaside 1, about 1.4.
SYSTEM SUIT ABILITY
The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peaks due
10 bacoside A3 and bacopaside Ir is at least 1.5 and the
resolution factor between the peaks due 10 bacopaside X and
bacopasaponin C is at least 2.4.
DETERMINATION OF CONTENT
Calculate the total content of bacopa saponins (bacoside A3 ,
bacopaside II, bacopaside X, bacopasaponin C and
bacopaside 1), expressed as bacopaside II from the
chromatograms obtained, and using the deelared content of
bacopaside II in bacopaside 11 CRS.
pericyele [D]; fragments of the vascular system [F] consisting
ofpitted vessels [Fa] and fibres [Fb] accompanied by rows of
ceIls containing prisms of calcium oxalate [Fc]; oil droplets
are present in the parenchymatous ceIls; occasional fragments
of conical, uniceIlular covering trichomes [C].
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of a mixture of equal volumes of
methanol R and water R, and heat under a refiux condenser
for 10 mino Filter whilst hoto Wash the fiask and the filter
with a mixture of equal volumes of methanol R and water R
and dilute to 5 mL with the same mixture of solvents.
Reference solution Dissolve 50 mg of arbutin R and 25 mg of
gallic acid R in methanol R and dilute 10 20.0 mL with the
same solvent.
Plate TLC silica gel plate R (5-40 ¡lm) [or TLC silica gel
plate R (2-10 ¡lm)].
Mobile phase anhydrous formic acid R, water R, ethyl aceta te R
(6 :6:88 VIV/V).
Application 10 ¡lL [or 2 ¡lL] as bands of 15 mm [or 8 mm] .
Development Over a path of 15 cm [or 6 cm] .
Drying At 105-110 oC until the mobile phase has
evaporated.
Detection Treat with a 10 gIL solution of
dichloroquinonechlorimide R in methanol R, then treat with a

Bearberry Leaf *** 20 gIL solution of anhydrous sodium carbonate R.

*** *** Results See below the sequence of zones present in the
Uva Ursi *** chromatograms obtained with the reference solution and the
(Ph Eur monograph 1054) test solution. Furthermore, other blue or brown zones may
PhEur _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ __ _ __ be present in the chromatogram obtained with the test
solution.
DEFINITION
Whole or fragmented, dried leaf of Arctostaphylos uva-ursi
(L.) Spreng. Top of the plate

Content
Minimum 7.0 per cent of anhydrous arbutin (C 12 H 1ó 0 7 ; M r
272 .3) (dried drug).
Callic acid: a brownish zone A brownish zone
IDENTIFlCATION
A. The leaf, shiny and dark green on the adaxial surface,
--- ---

Iighter on the abaxial surface, is generaIly 7-30 mm long and

5-12 mm wide. The entire leaf is obovate with smooth
A brown zone
margins, somewhat refiexed downwards, narrowing at the
base into a short petiole. The leaf is obtuse or retuse at its --- ---
apex. The lamina is thick and coriaceous. The venation, Arbutin: a blue zone An intense blue zone (arbutin)
pinnate and finely reticulate, is elearIy visible on both
surfaces. The adaxial surface is marked with sunken veinlets, Reference solution Test solution
giving it a characteristic grainy appearance. Only the young
leaf has ciliated margins. Old leaves are glabrous.
TESTS
B. Microscopic examination (2.8.23). The powder is green,
greenish-grey or yeIlowish-green. Examine under a
Foreign matter (2.8.2)
microscope using chloral hydrate solution R . The powder Maximum 5 per cent of stems and maximum 3 per cent of
shows the foIlowing diagnostic characters (Figure 1054.-1) : other foreign matter.
fragments of adaxial epidertnis in surface view [A] showing Leaves of different colour
thick and irregularIy pitted polygonal ceIls [Aa] usuaIly Maximum 10 per cent, determined in the same mauner as
accompanied by palisade parenchyma [Ab); fragments of foreign matter (2.8.2) .
adaxial epidermis in transverse section [G], showing straight- Loss on drying (2.2.32)
waIled ceIls [Ga] covered by a thick smooth cutiele [Gb], and Maximum 10.0 per cent, detertnined on 1.000 g of the
accompanied by palisade parenchyma [Gc] consisting of 3 or powdered herbal drug (355) (2.9.12) by drying in an oven at
4 layers of ceIls of unequal lengths, sorne of which contain 105 oC for 2 h.
numerous prisms of calcium oxalate [Gd]; fragments of
Total ash (2.4.16)
abaxial epidertnis, in surface view [B, E] , showing
Maximum 5.0 per cent.
anomocytic stomata (2.8.3) [Ba] surrounded by 5-11
subsidiary ceIls, scars of hair bases [Ea], and accompanied by ASSAY
spongy parenchyrna [Eb]; groups of Iignified fibres frorn the Liquid chromatography (2.2.29).
 IV-86 Belladonna Leaf
Figure 1054.-1. - Illustration for identification test B of
powdered herbal drug of bearberry leaf
Test solution In a 100 mL flask with a ground-glass neck,
place 0.800 g ofthe powdered herbal drug (250) (2.9.12).
Add 20 mL of water R and heat under a reflux condenser on
a water-bath for 30 min. Allow to cool and filter the liquid
through a plug of absorbent corton. Add the absorbent
cotton to the residue in the 100 mL flask and extract with
20 mL of water R under a reflux condenser on a water-bath
for 30 mino Allow to cool and filter through a paper filter.
Combine the filtrates and dilute to 50.0 mL with water R.
Filter through a paper filter. Discard the first 10 mL of the
filtrate .
Reference solutlon (a) Dissolve 50.0 mg of arbutin CRS in
the mobile phase and dilute to 50.0 mL with the mobile
phase.
Ref erence solution (b) Dissolve 2.5 mg of hydroquinone R in
the mobile phase and dilute to 10.0 mL with the mobile
phase. To 5.0 mL of the solution, add 2.5 mL of reference
solution (a) and dilute to 10.0 mL with the mobile phase.
Column:
-- size: 1 = 0.25 m, 0 = 4 mm;
-- stationary phase: base-deactivated oaadecylsilyl silica gel for
chromatography R (5 ¡lm).
M obile phase methanol R, water R (10 :90 V/V).
Flow rate 1.2 mUmin.
Detection Spectrophotometer at 280 nm.
Injection 20 ¡.¡L.
System suitability Reference solution (b):
-- resolution: minimum 4.0 between the peaks due to arbutin
and hydroquinone.
Calculate the percentage content of arbutin using the
following expression:
2014
are a of the peak due to arbutin in the
chromatogram obtained with the test solution;
area of the peak due to arbutin in the
chromatogram obtained with reference solution (a);
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mass of arbutin CRS used to prepare reference
solution (a), in grams;
p percentage content of arbutin in arbutin CRS.
_____ __ _ _ __ __ _ _ _ _ _ _ __ ___ PhEur
Belladonna Leaf ***
** **
Belladonna Herb **** *
(Ph Eur monograph 0221)
Preparations
Prepared Belladonna
Standardised Belladonna Leaf Dry Extract
Belladonna Tincture
When Belladonna Herb, Belladonna Leaf or Powdered
Belladonna Herb is prescribed, Prepared Belladonna shall be
supplied.
nEw _ _ _ _ _ __ _ __ __ _ __ __ _ _ ___
~
DEFINITION
Dried leaf or dried leaf and flowering, and occasionally fruit-
bearing, tops of Atropa belladonna L.
Content
Minimum 0.30 per cent of total alkaloids, expressed as
hyoscyamine (C 17H 23N0 3 ; M r 289.4) (dried drug) .
The alkaloids consist mainly of hyoscyamine together with
small quantities of hyoscine (scopolamine).
CHARACTERS
Slightly nauseous odour.
IDENTIFICATION
A. The leaves are green or brownish-green, slightly darker on
the upper surface, often crumpled and rolled and partly
matted together in the drug. The leaf is petiolate and the
lamina is acute and decurrent. The margin is entire.
The flowering stems are flattened and bear at each node a
pair of leaves unequal in size, in the axils of which occur
singly the flowers or occasionally fruits. The flowers have a
gamosepalous calyx and campanulate corolla. The drug may
contain fruits, as globular berries, green or brownish-black
and surrounded by the persistent calyx with widely spread
lobes.
B. Microscopic examination (2.8.23). The powder is dark
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 0221.-1) : fragments of the lamina showing
sinuous-walled epidermal cells with striated cutiele [A, C]
and pan of the underlying palisade parenchyma [Aa]
associated with the upper epidermis [A]; numerous stomata
[Ca] more frequent on the lower epidermis [C] , anisocytic
and also sorne anomocytic (2.8.3); multicellular, uniseriate
covering trichomes with a smooth cutiele [F] , glandular
trichomes with unicellular heads and multicellular, uniseriate
stalks [D] or with multicellular heads and unicellular stalks
[B]; parenchyma cells ineluding rounded cells, sorne of which
 2014
contain microsphenoidal crystals of calcium oxalate [E];
a=ularly and spirally thickened vessels [K] . The powdered
drug may also show: fibres and reticulately thickened ves seis
from the stems; subspherical pollen grains, 40-50 ¡.tm in
diameter, with 3 germinal pores, 3 furrows and an extensively
pitted exine [H]; fragments of the corolla with a papillose
epidermis m or bearing numerous covering or glandular
trichomes of the types previously described [L]; fragments of
the brownish-yellow testa consisting of irregularly sclerified
cells [G] .
Figure 0221.-1. - Illustration for identification test B of
powdered herbal drug of belladonna leaf
C. Shake 1 g ofthe powdered herbal drug (180) (2. 9.12)
with 10 mL of 0.05 M sulfuric acid for 2 mino Filter and add
to the filtrate 1 mL of concentrated ammonia R and 5 mL of
water R. Shake cautiously with 15 mL of ether R, avoiding
formation of an emulsion. Separate the ether layer and dry
over anhydrous sodium sulfate R . Filter and evaporate the ether
in a porcelain dish. Add 0.5 mL of fuming nitric acid R and
evaporate to dryness on a water-bath. Add 10 mL of
acetone R and, dropwise, a 30 giL solution of potassium
hydroxide R in ethanol (96 per cent) R . A deep violet colour
develops.
D. Examine the chromatograms obtained in the
chromatography test.
Results The principal zones in the chromatograms obtained
with the test solution are similar in position, colour and size
to the principal zones in the chromatograms obtained with
the same volume of the reference solution.
TESTS
Chromatography
Thin-Iayer chromatography (2.2.27) .
Test solution To 0.6 g of the powdered herbal drug (180)
(2.9. 12) add 15 mL of 0.05 M sulfuric acid, shake for 15 min
Belladonna Leaf IV-S?
and filter. Wash the filter with 0.05 M sulfuric acid until
20 mL of filtrate is obtained. To the filtrate add 1 mL of
concentrated ammonia R and shake with 2 quantities, each of
10 mL, of peroxide-free ether R. If necessary, separate by
centrifugation. Dry the combined ether layers over anhydrous
sodium sulfate R, filter and evaporate to dryness on a water-
bath. Dissolve the residue in 0.5 mL of methanol R.
Reference solution Dissolve 50 rng of hyoscyamine sulfate R in
9 mL of methanol R. Dissolve 15 mg of hyoscine
hydrobromide R in 10 mL of methanol R . Mix 1.8 mL of the
hyoscine hydrobromide solution and 8 mL of the
hyoscyamine sulfate solution.
Plate TLC silica gel G plate R .
Mobile phase concentrated ammonia R, water R, acetone R
(3:7:90 VIV/V) .
Application 1O ~IL and 20 ¡.tL, as bands of 20 mm by 3 mm,
leaving 1 cm between the bands.
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 min; allow to cool.
Detection A Spray with potassium iodobismuthate soludon R2,
using about 10 mL for a plate 200 mm square, until the
orange or brown zones become visible against a yellow
background.
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the
lower third, hyoscine in the upper third of the
chromatograms) and colour to the bands in the
chromatograms obtained with the reference solution.
The zones in the chromatograms obtained with the test
solution are at least equal in size to the corresponding zones
in the chromatogram obtained with the same volume of the
reference solution. Faint secondary zones may appear,
particularly in the middle of the chromatogram obtained with
20 ¡.tL of the test solution or near the starting point in the
chromatogram obtained with 1O ~IL of the test solution.
Detection B Spray with sodium nitrite solution R until the
coating is transparent; examine after 15 mino
Results B The zones due to hyoscyamine in the
chromatograms obtained with the reference solution and the
test solution change from brown to reddish-brown but not to
greyish-blue (atropine) and any secondary zones disappear.
Foreign matter (2.8.2)
Maximum 3 per cent of stems with a diameter greater than
5 mm.
Total ash (2.4.16)
Maximum 16.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
a) Determine the loss on drying (2.2.32) on 2.000 g of the
powdered herbal drug (180) (2.9.12), by drying in an oven at
105 oC.
b) Moisten 10.00 g ofthe powdered herbal drug (180)
(2. 9.12) with a mixture of 5 mL of ammonia R, 10 rnL of
ethanol (96 per cent) R and 30 mL of peroxide-free ether R and
mix thoroughly. Transfer the mixrure to a suitable percolator,
if necessary with the aid of the extracting mixture. Allow to
macerate for 4 h and percolate with a mixture of 1 volume of
chloroform R and 3 volumes of peroxide-free ether R until the
alkaloids are completely extracted. Evaporate to dryness a
few millilitres of the liquid fiowing from the percolator,
dissolve the residue in 0.25 M sulfuric acid and verify the
absence of alkaloids using potassium tetraiodomercurate
 IV-88 Prepared Belladonna
solution R. Concentrate the percolate 10 about 50 mL by
distilling on a water-bath and transfer it to a separating
funnel, rinsing with peroxide-free ether R. Add a quantity of
peroxide-free ether R equal to at least 2.1 times the volume of
the percolate 10 produce a liquid of a density well below that
of water. Shake the solution with no fewer than 3 quantities,
each of 20 mL, of 0.25 M sulfuric acid, separate the 2 layers
by centrifugation if necessary and transfer the acid layers to a
2nd separating funnel. Make the acid layer alkaline with
ammonia R and shake with 3 quantities, each of 30 mL, of
chloroform R. Combine the chloroform layers, add 4 g of
anhydrous sodium sulfate R and allow 10 stand for 30 min with
occasional shaking. Decant the chloroform and wash the
sodium sulfate with 3 quantities, each of 10 mL, of
chloroform R . Add the washings 10 the chloroform extract,
evaporate 10 dryness on a water-bath and heat in an oven at
100-105 oC for 15 mino Dissolve the residue in a few
millilitres of chloroform R, add 20.0 mL of 0.01 M sulfuric acid
and remove the chloroform by evaporation on a water-bath.
Titrate the excess of acid with 0.02 M sodium hydroxide using
methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, using the following expression:
57.88 x (20 - n)
(100 - d) x m
d loss on drying, as a percentage;
n volume of 0.02 M sodium hydroxide, in millilitres;
m mass of the powdered herbal drug, in grams.
_ _ _ _ _ _ __ _ __ __ _ _ _ _ _ __ _ _ PhEur
Prepared Belladonna ***
*** ***
Prepared Belladouna Herb ***
(Ph Eur monograph 0222)
PhE~ _ _ _ _ __ __ _ _ _ _ _ _ __ _ _ _ _ __
DEFINITION
Belladonna leafpowder (180) (2.9.12) adjusted, ifnecessary,
by adding powdered lactose or belladouna leaf powder with a
lower alkaloidal content.
Content
0.28 per cent 10 0.32 per cent of total alkaloids, expressed as
hyoscyamine (Mr 289 .4) (dried drug) .
CHARACTERS
Slightly nauseous odour.
IDENTIFICATION
A. The powder is dark green. Examine under a microscope,
using chloral hydrate solution R. The powder shows the
following diagnostic characters: fragments of leaf lamina
showing sinuous-walled epidermal cells, a striated cuticle and
numerous s10mata predominantly present on the lower
epidermis (anisocytic and also sorne anomocytic) (2.8.3);
multicellular uniseriate covering trichomes with smooth
cuticle, glandular trichomes with unicellular heads and
multicellular, uniseriate stalks or with multicellular heads and
unicellular stalks; parenchyma cells including rounded cells
containing microsphenoidal crystals of calciurn oxalate;
aunular and spirally thickened vessels . The powdered drug
may also show the following: fibres and reticulately thickened
ves seis from the stems; subspherical pollen grains, 40-50 ¡.¡m
in diameter, with 3 germinal pores, 3 furrows and an
2014
extensively pined exine; fragments of the corolla, with a
papillose epidermis or bearing numerous covering or
glandular trichomes of the types previously described;
brownish-yellow seed fragments containing irregularly
sclerified and pitted cells of the testa. Examined in glycerol
(85 per cent) R, the powder may be seen 10 contain lactase
crystals.
B. Shake 1 g with 10 mL of 0.05 M sulfuric acid for 2 mino
Filter and add 10 the filtrate 1 mL of concentrated ammonia R
and 5 mL of water R . Shake cautiously with 15 mL of
ether R, avoiding formation of an emulsion. Separate the
ether layer and dry over anhydrous sodium sulfate R. Filter and
evaporate the ether in a porcelain dish. Add 0.5 mL of
fuming nitric acid R and evaporate to dryness on a water-bath.
Add 10 mL of acetone R and, dropwise, a 30 gIL solution of
potassium hydroxide R in ethanol (96 per cent) R . A deep violet
colour develops.
C. Examine the chromatograms obtained in the test
Chroma1Ography.
Results The principal zones in the chromatograms obtained
with the test solution are similar in position, colour and size
to the principal zones in the chromatogram obtained with the
same volume of the reference solution.
TESTS
Chromatography
Thin-Iayer chromatography (2.2.27).
Test solution To 0.6 g of the drug to be examined add
15 mL of 0.05 M sulfuric acid, shake for 15 min and filter.
Wash the filter with 0.05 M sulfuric acid until 20 mL of
filtrate is obtained. To the filtrate add 1 mL of concentrated
ammonia R and shake with 2 quantities, each of 10 mL, of
peroxide-free ether R. If necessary, separate by centrifugation.
Dry the combined ether layers over anhydrous sodium
sulfate R, filter, and evaporate to dryness on a water-bath.
Dissolve the residue in 0.5 mL of methanol R .
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in
9 mL of methanol R. Dissolve 15 mg of hyoscine
hydrobromide R in 10 mL of methanol R . Mix 1.8 mL of the
hyoscine hydrobromide solution and 8 mL of the
hyoscyamine sulfate solution.
Plate TLC silica gel G plate R.
Mobile phase concentrated ammonia R, water R, acetone R
(3:7:90 VIV/V).
Application 10 ¡.¡L and 20 ¡.¡L of each solution, as bands of
20 mm by 3 mm, leaving 1 cm between each bando
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 min; allow 10 cool.
Detection A Spray with potassium iodobismuthate solution R2,
using about 10 mL for a plate 200 mm square, until orange
or brown zones become visible against a yellow background.
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the
lower third, hyoscine in the upper third) and colour to those
in the chroma1Ograms obtained with the reference solution;
the zones in the chromatograms obtained with the test
solution are at least equal in size 10 the corresponding zones
in the chroma1Ogram obtained with the same volume of the
reference solution; faint secondary zones may appear,
particularly in the middle of the chromatogram obtained with
20 ¡.tL of the test solution or near the point of application in
the chroma1Ogram obtained with 10 ¡.¡L of the test solution.
Detection B Spray with sodium nitrite solution R until the
coating is transparent and examine after 15 mino
 2014
Results B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
reference solution change from brown to reddish-brown but
not to greyish-blue (atropine), and any secondary zones
disappear.
Loss on drying (2.2.32)
Maximum 5.0 per cent, determined on l.000 g by drying in
an oven at 105 oC.
Total ash (2.4.16)
Maximum 16.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
a) Determine the loss on drying (2.2.32) on 2.000 g by
drying in an oven at 105 oc.
b) Moisten 10.00 g with a mixture of 5 mL of ammonia R,
10 mL of ethanol (96 per cent) R and 30 mL of perox¡de-free
ether R and mix thoroughly. Transfer the mixture to a
suitable percolator, if necessary with the aid of the extracting
mixture. Allow to macerate for 4 h and percolate with a
mixture of 1 volume of chloroform R and 3 volumes of
peroxide-free ether R until the alkaloids are completely
extracted. Evaporate to dryness a few millilitres of the liquid
fiowing from the percolator, dissolve the residue in 0.25 M
sulfuric acid and verify the absence of alkaloids using
potassium tetraiodomercurate solution R. Concentrate the
percolate to about 50 mL by distilling on a water-bath and
transfer it to a separating funnel, rinsing with peroxide-free
ether R. Add a quantity of peroxide-free ether R equal to at
least 2.1 times the volume of the percolate to produce a
liquid of a density well below that of water. Shake the
solution with no fewer than 3 quantities, each of 20 mL, of
0.25 M sulfuric acid, separate the 2 layers by centrifugation if
necessary and transfer the acid layers to a 2 nd separating
funnel. Make the acid layer alkaline with ammonia R and
shake with 3 quantities, each of 30 mL, of chloroforn¡ R .
Combine the chloroform layers, add 4 g of anhydrous sodium
sulfate R and allow to stand for 30 min with occasional
shaking. Decant the chloroform and wash the sodium sulfate
with 3 quantities, each of 10 mL, of chloroform R. Add the
washings to the chloroform extract, evaporate to dryness on a
water-bath and heat in an oven at 100-105 oC for 15 mino
Dissolve the residue in a few millilitres of chloroform R, add
20.0 mL of 0.01 M sulfun'c acid and remove the chloroform
by evaporation on a water-bath. Titrate the excess of acid
with 0.02 M sodium hydroxide using methyl red mixed
solution R as indicator.
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, using the following express ion:
57.88 x (20 - n)
(100 - d) x m
d loss on drying as a percentage;
n volume of 0.02 M sodium hydroxide used, in millilitres;
m mass of drug used, in grams.
STORAGE
In an airtight container.
_ __ _ _ __ __ _ _ _ _ __ _ _ _ __ __ Ph Eur
Belladonna Preparations IV-89
Standardised Belladonna Leaf Dry *****
** **
Extract ***
(Ph Eur monograph 1294)
~E~ _ _ _ _ __ _ __ _ _ _ _ _ _ _ __ _ _ __
DEFINITION
Standardised dry extract obtained from Belladonna leaf
(0221).
Content
0.95 per cent to l.05 per cent of total alkaloids, expressed as
hyoscyamine (C 17 H 23N0 3 ; M r 289.4) (dried extract) .
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (70 per cent V/V) .
CHARACTERS
Appearance
Brown or greenish, hygroscopic powder.
IDENTIFICATION
A. Thin-Iayer chromatography (2.2. 27).
Test solution To 1 g of the extract to be exarnined add
5.0 mL of methanol R. Shake for 2 min and filter.
Reference solution Dissolve 1.0 mg of chIorogenic acid R and
2.5 mg of rutin R in 10 mL of methanol R.
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (10:10:30:50 VIVIV/V).
Application20 fLL as bands.
Development Over a path of 15 cm.
Drying At 100-105 oC .
Detection Spray the warm plate with a 10 gIL solution of
diphenylbon'c acid aminoethyl ester R in methanol R, then spray
with a 50 gIL solution of macrogol 400 R in methanol R; allow
to dry in air for 30 min and examine in ultraviolet light at
365 nm.
Results The chromatograms obtained with the reference
solution and the test solution show in the central part a hght
blue fiuorescent zone (chlorogenic acid) and in the lower part
a yellowish-brown fiuorescent zone (rutin); furthermore, the
chromatogram obtained with the test solution shows a httle
aboye the start a yellowish-brown fiuorescent zone and
directly aboye that a yellow fiuorescent zone, and a yellow or
yellowish-brown fiuorescent zone between the zone due to
rutin and the zone due to chlorogenic acid. Further zones
may be present.
B. Examine the chromatograms obtained in the test for
atropine.
R esults The principal zones in the chromatogram obtained
with the test solution are similar in position and colour to the
principal zones in the chromatogram obtained with the
reference solution.
TESTS
Atropine
Thin-Iayer chromatography (2.2.27).
Test solution To 0.20 g of the extract to be examined add
10.0 mL of 0.05 M sulfuric acid, shake for 2 min and filter.
Add l.0 mL of concentrated ammonia R and shake with
2 quantities, each of 10 mL, of peroxide-free ether R.
If necessary, separate by centrifugation. Dry the combined
ether layers over about 2 g of anhydrous sodium sulfate R, filter
and evaporate to dryness on a water-bath. Dissolve the
residue in 0.5 mL of methanol R.
 IV-90 Belladonna Preparations
R eferenee solution Dissolve 50 mg of hyoseyamine sulfate R in
9 mL of methanol R . Dissolve 15 mg of hyoseine
hydrobromide R in 10 mL of methanol R . Mix 1.8 mL of the
hyoscine hydrobromide solution and 8 mL of the
h yoscyamine sulfate solution.
Plare TLC siliea gel plate R.
M obile phase coneentrated ammonia R, water R, aeetone R
(3: 7:90 VIV/V) .
Applieation 20 ¡.tL as bands.
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 min; allow to coo!.
Deteetion A Spray with potassium iodobismuthate solution R2,
until orange or brown zones become visible against a yellow
background.
Results A The zones in the chromatogram obtained with the
test solution are similar in position (hyoscyamine in the lower
third, hyoscine in the upper third) and colour to those in the
chromatogram obtained with the reference solution. Other
faint zones may be present in the chromatogram obtained
with the test solution.
D eteetion B Spray with sodium nitrite solution R until the
coating is transparent and examine after 15 mino
R esults B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
reference solution change from orange or brown to reddish-
brown but not to greyish-blue (atropine) .
Loss on drying (2.8.17)
Maximum 5.0 per cent.
Microbial contamination
TAMC: acceptance criterion 10 4 CFU/g (2.6.12).
TYMC: acceptance criterion 10 2 CFU/g (2.6.12).
Absence of Eseheriehia eoli (2.6.13).
Absence of Salmonella (2.6.13) .
ASSAY
At each extraction stage it is necessary to check that the
alkaloids have been completely extracted. If the extraction is
into the organic phase this is done by evaporating to dryness
a few millilitres of the last organic layer, dissolving the
residue in 0.25 M sulfurie acid and verifying the absence of
alkaloids using potassium tetraiodomereurate solution R . If the
extraction is into the acid aqueous phase, this is done by
taking a few millilitres of the last acid aqueous phase and
verifying the absence of alkaloids using potassium
retraiodomereurate solution R .
Disperse 3.00 g in a mixture of 5 mL of ammonia R and
15 mL of water R. Shake with no fewer than 3 quantities,
each of 40 mL, of a mixture of 1 volume of methylene
ehloride R and 3 volumes of peroxide-free ether R until the
alkaloids are completely extracted. Concentrate the combined
organic layers to about 50 mL by distilling on a water-bath
and transfer the resulting liquid to a separating funnel,
rinsing with peroxide-free ether R . Add a quantity of peroxide-
free ether R equal to at least 2.1 times the volume of the
liquid to produce a layer having a density well below that of
water. Shake the resulting solution with no fewer than
3 quantities, each of 20 mL, of 0.25 M sulfuric acid until the
alkaloids are completely extracted. Separate the layers by
centrifugation, if necessary, and transfer the acid layers to a
2 nd separating funne!. Make the combined acid layers
alkaline with ammonia R and shake with no fewer than
3 quantities, each of 30 mL, of methylene ehloride R until the
alkal oids are completely extracted. Combine the organic
layers, add 4 g of anhydrous sodium sulfate R and allow to
2014
stand for 30 min with occasional shaking. Decant the
methylene chloride and wash the sodium sulfate with
3 quantities, each of 10 mL, of methylene ehloride R . Combine
the organic extracts and evaporate to dryness on a water-
bath. Reat the residue in an oven at 100-105 cC for 15 min o
Dissolve the residue in a few millilitres of methylene ehloride R ,
evaporate to dryness on a water-bath and again heat the
residue in an oven at 100-105 oC for 15 min o Dissolve the
residue in a few millilitres of methy lene ehloride R, add
20.0 mL of 0.01 M sulfurie acid and remove the methylene
chloride by evaporation on a water-bath. Titrate the excess of
acid with 0.02 M sodium hydroxide using methyl red mixed
solution R as indicator.
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, using the following expression:
57.88 x (20 - n)
100 x m
n volume of 0.02 M sodium hydroxide used, in millilitres;
m mas s of drug used, in grams.
____________________________________________ ~E~
Belladonna Tincture ***
*** ***
(S tandardised B elladonna Leaf Tincture, ***
Ph Eur monograph 1812)
Ph Eur ____________________________________________
DEFINITION
Tincture produced from B elladonna leaf (0221).
Content
0.027 per cent to 0.033 per cent of total alkaloids, calculated
as hyoscyamine (C 17H 23N0 3 ; M r 289.4) . The alkaloids
consist mainly of hyoscyamine together with small quantities
of hyoscine.
PRODUCTION
The tincture is produced from 1 part of the powdered drug
(355) (2.9.12) and 10 parts of ethanol (70 per cent V/V) b y a
suitable procedure.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
T est solution Evaporate to dryness 10.0 mL of the tincture
to be examined in a water-bath at 40 oC under reduced
pressure. Dissolve the residue in 1.0 mL of methanol R .
R eferenee solution Dissolve 1.0 mg of ehlorogenie acid R and
2.5 mg of rutin R in 10 mL of methanol R .
Plate TLC siliea gel plate R .
Mobile phase anhydrous formie acid R , water R , methyl ethyl
ketone R, ethyl acetare R (10: 10:30:50 VIVIV/V) .
Application 40 ¡.tL as b ands.
Development Over a path of 15 cm.
Drying At 100- 105 oc.
Detection Spray the warm plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R;
subsequently spray the plate with a 50 gIL solution of
macrogol 400 R in methanol R ; allow the plate to dry in air for
30 min and examine in ultraviolet light at 365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
 2014 Belladonna Preparations IV-91

test solution. Furthermore, other fluorescent zones may be Detection B Spray with sodium nitrite solution R until the
present in the chromatogram obtained with the test solution. plate is transparento Examine after 15 mino
Results B The zones due to hyoscyamine in the
Top of the plate
chromatograms obtained with the test solution and the
reference solution change from brownish-orange to reddish-
--- - - - brown but not to greyish-blue (atropine) and any secondary
Chlorogenic acid: a light blue A light blue f1uorescent zone zones disappear.
f1uorescent zone (chlorogenic acid) Ethanol (2.9.10)
A yellow or yellowish-brown 64 per cent V/V to 69 per cent V/V.
f1uorescent zone
--- - - - ASSAY
Rutin: a yellowish-brown A bluish-grey f1uorescent zone
Evaporate 50.0 g of the tincture to be examined to a volume
f1uorescent zone of about 10 mL. Transfer quantitatively to a separating
A yellow f1uorescent zone funnel, with the minimum volume of alcohol (70 per cent
V/V) R. Add 5 mL of ammonia R and 15 mL of water R.
A yellowish-brown f1uorescent
zone
Shake with not fewer than 3 quantities each of 40 mL of a
Reference soIution Test solution
mixture of 1 volume of methylene chloride R and 3 volumes of
peroxide-free ether R, carefully to avoid emulsion, until the
alkaloids are completely extracted. Combine the organic
B. Examine the chromatograms obtained in the test for layers and concentrate the solution to a volume of about
atropine, detection A. 50 mL by distilling on a water-bath. Transfer the resulting
R esults ASee below the sequence of zones present in the solution quantitatively to a separating funnel, rinsing with
chromatograms obtained with the reference solution and the peroxide-free ether R. Add a quantity of peroxide-free ether R
test solution. Faint secondary zones may appear, particularly equal to at least 2.1 times the volume of the solution to
in the middle of the chromatogram obtained with 40 ¡.tI- of produce a layer having a density well below that of water.
the test solution or near the point of application in the Shake the resulting solution with not fewer than 3 quantities
chromatogram obtained with 20 ~IL of the test solution. each of 20 mL of 0.25 M sulfuric aeid until the alkaloids are
completely extracted. Separate the layers by centrifugation if
necessary and transfer the layers to a separating funnel. Make
Top of the plate the combined layers alkaline with ammonia R and shake with
Hyoscine: a brownish·orange zone A brownish-orange zone (hyoscine)
not fewer than 3 quantities each of 30 mL of methylene
ehloride R until the alkaloids are completely extracted.
--- --- Combine the organic layers, add 4 g of anhydrous sodium
Faint secondary zones sulfate R and allow to stand for 30 min with occasional
shaking. Decant the methylene chloride and filter. Wash the
--- - - - sodium sulfate with 3 quantities each of 10 mL of methylene
Hyoscyamine : a brownish·orange A brownish-orange zone ehloride R . Combine the organic extracts, evaporate to
zone (hyoscyamine) dryness on a water-bath. Heat the residue in an oven at
Faint secondary zones 100-105 oC for 15 mino Dissolve the residue in a few
Reference solution Test solution millilitres of methylene chloride R, evaporate to dryness on a
water-bath and heat the residue in an oven at 100-105 oC for
15 min again. Dissolve the residue in a few millilitres of
TESTS methylene ehloride R . Add 20.0 mL of 0.01 M sulfuric aeid and
Atropine remove the methylene chloride by evaporation on a water-
Thin-Iayer chromatography (2.2.27). bath. Titrate the excess of acid with 0.02 M sodium hydroxide
using methyl red mixed solution R as indicator.
Test solution To 15.0 mL of the tincture to be examined
add 15 mL of 0.05 M sulfurie acid. Filter. Add 1 mL of Calculate the percentage content of total alkaloids, expressed
coneentrated ammonia R to the filtrate and shake with as hyoscyamine, using the following expression:
2 quantities, each of 10 mL, of peroxide-free ether R. Separate
by centrifugation if necessary. Dry the combined ether layers 57.88 x (20 - n)
over anhydrous sodium sulfate R . Filter and evaporate to 100 x m
dryness on a water-bath . Dissolve the residue in 0.5 mL of
methanol R. n volume of 0.02 M sodium hydroxide used, in millilitres,
R eferenee solution Dissolve 50 mg of hyoseyamine sulfate R in m mass of drug used, in grams.
9 mL of methanol R. Dissolve 15 mg of hyoscine ____________________________________________ ~Em

hydrobromide R in 10 mL of methanol R . Mix 1.8 mL of the

hyoscine hydrobromide solution and 8 mL of the
hyoscyamine sulfate solution.
Plate TLC siliea gel plate R.
Mobile phase eoncentrated ammonia R, water R, acetone R
(3:7:90 VIV/V).
Applieation 20 ~L and 40 ~L of each solution, as bands.
Development Over a path of 10 cm.
Drying At 100-105 cC for 15 mino
Detection A Spray with potassium iodobismuthate solution R2.
 IV-92 Siam Benzoin 2014

Siam Benzoin *** Mobzle phase glacial aeetie acid R, di-isopropyl ether R,
*** *** hexane R (10:40:60 VIV/V) .
(Ph Eur monograph 2158) *** Application 10 ¡.¡L as bands.
Prepararlon Development Over a path of 12 cm.
Siam Benzoin Tincture Drying In air.
____________________________________________
Detection Examine in ultraviolet light at 254 nm.
~E~

DEFINITION Results The chromatogram obtained with the test solution

Resin obtained by incising the trunk of Styrax tonkinensis
(Pierre) Craib ex Hartwich.
Content
45.0 per cent to 55.0 per cent of total acids, calculated as
benzoic acid (C 7 H 6 0 2 ; M r 122.1) (dried drug).
CHARACTERS
Characteristic odour of vanillin.
IDENTIFICATION
A. Siam benzoin occurs as opaque, granular, rounded or
ovoid masses (tears), varying in size from a few millimeters
up to 3 cm, separated or sometimes agglomerated together
by a reddish-brown, transparent resino Individual tears are
yellowish-white to reddish externally with a waxy, whitish
fracture which becomes reddish on exposure to airo
B. Examine the chromatograms obtained in test B for Styrax
benzoin.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint fluorescent zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
-----
Methyl cinnamate: a very
prominent quenching zone
Benzoic acid: a quenching zone A quenching zone (benzoic acid)
shows no zone in the same position as the zone due to
cinnamic acid in the chromatogram obtained with the
reference solution.
Matter insoluble in ethanol
Maximum 5 per cent.
To 2 g of the powdered drug add 25 mL of ethanol
(90 per cent V/ J-) R. Boil until almost completely dissolved.
Filter through a previously tared sintered-glass filter (16)
(2.1.2) and wash with 3 quantities, each of 5 mL, of boiling
ethanol (90 per cent V/ J-) R. Heat the glass filter and its
contents in an oven at 100-105 oC for 2 h . Weigh after
cooling.
Loss on drying (2.2.32)
Maximum 5.0 per cent, determined on 2.00 g of the coarsely
powdered drug by drying in vacuo for 4 h.
Total ash (2.4.16)
Maxirnum 2.0 per cent.
ASSAY
Place 0.750 g of the finely powdered drug in a 250 mL
borosilicate glass flask and add 15.0 mL of 0.5 M alcoholic
potassium hydroxide. Boil under a reflux condenser on a
water-bath for 30 mino Allow to cool and rinse the condenser
with 20 mL of ethanol (96 per cent) R. Titrate the excess of
potassium hydroxide with 0.5 M hydrochlorie acid. Determine
----- the end-point potentiometrically (2.2.20). Carry out a blank
titration.
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to
61.05 mg ofbenzoic acid (C7H 60 Z).
____________________________________________ ~E~

Cinnamic acid: a prominent

quenching zone
- - - -- ---
A quenching zone
Siam Benzoin Tincture ***
A very prominent quenching zone
** **
Vanillin: a quenching zone A quenching zone (vanillin) (Ph Eur monograph 2157 **** *
~E~ ____________________________________________
Series of unresolved zones
includinl! a quenching zone
DEFINITION
Reference solution Test solution
Tincture produced from Siam benzoin (2158).
Content
TESTS Minimum 5.0 per cent m/m of total acids, calculated as
Styrax benzoin benzoic acid (C 7 H 6 0 2 ; M r 122.1 ).
A. To 0.2 g of the finely powdered drug add 10 mL of PRODUCTION
ethanol (96 per een!) R. Shake vigorously until almost The tincture is produced from 1 part of the drug and 5 parts
completely dissolved and filter. Place 5 mL of the filtrate in a of ethanol (75 per cent V/V to 96 per cent V/V) by a suitable
test-tube and add 0.5 mL of a 50 gIL solution of jerrie procedure.
ehloride R in ethanol (96 per een!) R. A green colour is
produced. No yellow colour is produced. CHARACTERS
Appearance
B. Thin-Iayer chromatography (2.2.27).
Orange-yellow liquido
Test solu!Íon Sonicate 0.2 g of the finely powdered drug in
It has a characteristic odour of vanillin.
5 mL of ethanol (96 per een!) R and filter. Collect the filtrate .
Rejerenee solution Dissolve 20 mg of benzoie acid R, 10 mg of IDENTIFICATION
trans-cinnamie acid R, 4 mg of vanillin R and 20 mg of methyl A. Place 10 mL in a test tube; add 0.5 mL of a 50 gIL
einnamate R in 10 mL of ethanol (96 per eent) R. solution of jerrie ehloride R in ethanol (96 per eent) R. A green
colour is produced.
Plate TLC siliea gel F 254 plate R.
2014 Sumatra Benzoin IV-93

B. Examine the chromatograms obtained in the test for

Sumatra Benzoin ***
Sumatra benzoin tincture. *** ***
R esults See below the sequence of the zones present in the (Ph Eur monograph 1814) ***
chromatograms obtained with the reference solution and the Benzoin
test solution. Furthermore, other faint fiuorescent zones may Preparations
be present in the chromatogram obtained with the test Benzoin Inhalation
solution.
Compound Benzoin Tincture
Sumatra Benzoin Tincture
Top of t he plate PhE~ ______________________________________________

- - - --- DEFINITION
Methyl cinnamate: a very Resin obtained by incising the trunk of Styrax benzoin
prominent quenching zone Dryander.
Benzoic acid : a quenching zone A quenching zone (benzoic acid)
Content
Cinnamic acid: a prominent 25.0 per cent to 50.0 per cent of total acids, ca1culated as
quenching zone
benzoic acid (C7H60Z; M r 122.1) (dried drug).
- -- ---
IDENTIFICATION
A quenching zone
A. Sumatra benzoin occurs as creamy white, rounded to
A very prominent quenching zone ovoid tears, which may be embedded in a dull greyish-brown
A quenching zone (vanillin)
or reddish-brown matrix. It is hard and brittle and the
Vanillin: a quenching zone
fractured surface is dull and uneven.
Series of unresolved zones
ineludin~ a Quenchin!! zone
B. Examine the chromatograms obtained in test B for Styrax
Reference solution Test solution tonkinensis.
Results See below the sequence of quenching zones present
in the chromatograms obtained with the reference solution
TESTS and the test solution. Furthermore, other faint quenching
Sumatra benzoin tincture zones may be present in the chromatogram obtained with the
Thin-Iayer chromatography (2.2.27). test solution.
Test solution The tincture to be examined.
R eference solution Dissolve 20 mg of benzoic acid R, 10 mg of Top of the plate
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl A very intense dark zone
cinnamate R in 20 mL of ethanol of the same concentration
- -- ---
as that used for the production of the tincture.
Plate TLC silica gel F254 plate R (5-40 ¡¡m) [or TLC sitica gel Methyl cinnamate: a very intense A dark zone
F 254 plate R (2-10 ¡¡m)]. dark zone
Benzoic acid: a dark zone A very weak dark zone (benzoic
Mobile phase glacial acetic acid R , di-isopropyl ether R, acid)
hexane R (10:40:60 VIVIV). Cinnamic acid: an intense dark A very intense dark zone (cinnamic
Application 20 ¡¡L [or 8 ¡¡L] as bands. zone acid)

Development Over a path of 12 cm [or 6 cm] . --- ---

Drying In air. A dark zone

Detection Examine in ultraviolet light at 254 nm. A very intense dark zone
R esults The chromatogram obtained with the test solution A dark zone
does not show any zone in the same position as the zones
Vanillin: a dark zone A very weak dark zone (vanillin)
due to cinnamic acid and methyl cinnamate in the
chromatogram obtained with the reference solution. Series of unresolved zones
ineludin!! 2 dark zones
Ethanol (2.9.10)
Reference solution Test solution
95 per cent to 105 per cent of the content stated on the
labe!'
ASSAY TESTS
Place 3.50 g in a 250 mL borosilicate glass fiask and add Darnmargum
15.0 mL of 0.5 M alcoholic potassium hydroxide. Boil under a Thin-Iayer chromatography (2.2.27).
refiux condenser on a water-bath for 30 mino Allow to cool
Test solution Dissolve 0.2 g of the drug to be examined with
and rinse the condenser with 20 mL of ethanol
gentle heating in 10 mL of ethanol (90 per cene Vl f.? R and
(96 per cene) R. Titrate the excess of potassium hydroxide
centrifuge.
with 1 M hydrochloric acid, determining the end-point
potentiometrically (2.2.20) . Carry out a blank titration. Plate TLC aluminium oxide G plate R .
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to Mobite phase Light petroleum R4, ethe/" R (40:60 VIV).
61.05 mg ofbenzoic acid (C7H 60Z). Application 5 ¡¡L.
______________________________________________ PhE~
Developmene Over a path of 10 cm.
Drying In airo
IV-94 Benzoin Preparations 2014

*****
Detection Spray with anisaldehyde solution R and heat at
Sumatra Benzoin Tincture
100-105 oC for 5 mino ** **
Results The chroma1Ogram obtained does not show any (Ph Bur monograph 1813) ***
prominent spot with an RF between 0.4 and 1.0. ~E~ ____________________________________________

Styrax tonkinensis DEFINITION

A. To 0.2 g ofthe finely powdered drug add 10 mL of Tincture produced from Sumatra benzoin (1814) .
ethanol (96 per cent) R. Shake vigorously until almost
completely dissolved and filter. Place 5 mL of the filtrate in a Content
test-tube and add 0.5 mL of a 50 gIL solution of jerric Minimum 4.0 per cent m/m of total acids, calculated as
chloride R in ethanol (96 per cent) R. A yellowish, slightly benzoic acid (C 7 H 6 0 2 ; M r 122.1).
green colour is produced. PRODUCTION
B. Thin-Iayer chromatography (2.2.27). The tincture is produced from I part of the drug and 5 parts
Test solution Sonicate 0.2 g of the finely powdered drug in of ethanol (75 per cent VIV 10 96 per cent V/V) by a suitable
5 mL of ethanol (96 per cent) R and filter. Collect the filtrate. procedure.
Rejerence solution Dissolve 20 mg of benzoic acid R, 10 mg of CHARACTERS
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl Appearance
cinnamate R in 10 mL of ethanol (96 per cent) R. Orange-yellow liquido
Plate TLC silica gel F254 plate R (5-40 ¡.tm) [or TLC silica gel IDENTIFICATION
F254 plate R (2-10 ¡.tm)]. Examine the chromatograms obtained in the test for Siam
Mobile phase glacial acetic acid R, di-isopropyl ether R, benzoin tincture.
hexane R (10:40:60 VIV/V). Results See below the sequence of quenching zones present
Application 10 ¡.tL [or 2 ¡.tL] as bands. in the chromatograms obtained with the reference solution
Development Over a path of 12 cm [or 5 cm]. and the test solution. Furthermore, other faint quenching
Drying In air. zones may be present in the chroma1Ogram obtained with the
test solution.
Detection Examine in ultraviolet light at 254 nm.
Results The chromatogram obtained with the test solution
shows 2 faint zones in the same positions as the dark zones Top of the pI.te
due 10 benzoic acid and vanillin in the chroma1Ogram A very intense dark zane
obtained with the reference solution.
----- -----
Matter insoluble in ethanol
MethyI cinnamate: • very intense A d.rk zane
Maxirnum 20.0 per cent. d.rk zane
To 2.0 g of the powdered drug add 25 mL of ethanol Benzaic acid: • d.rk zane A very weak d.rk zane (benzaic .cid)
(90 per cent V/rJ R. Boil until almost completely dissolved.
Cinnamic .cid: .n intense d.rk zane A very in tense d.rk zane (cinn.mic
Filter through a tared sintered-glass filter (16) (2.1.2) and .cid)
wash with 3 quantities, each of 5 mL, of boiling ethanol ----- -----
(90 per cent V/rJ R . Heat the glass filter and its contents in
A dark zane
an oven at lOO-lOS oC for 2 h. Allow 10 cool and weigh.
A very intense d.rk zone
Loss on d.rying (2.2.32)
Maxirnum 5.0 per cent, determined on 2.000 g of the A d.rk zane
coarsely powdered drug by drying in vacuo for 4 h. Vanillin : a dark zane A very weak dark zane (v.nillin)
Total ash (2.4.16) Series af unresalved d.rk zanes
Maxirnum 2.0 per cent.
Reference soIution Test soIution
ASSAY
Place 0.750 g ofthe finely powdered drug in a 250 mL
borosilicate glass fiask and add 15.0 mL of 0.5 M alcoholic TESTS
potassium hydroxide. Boil under a refiux condenser on a Siam benzoin tincture
water-bath for 30 minoAllow to cool and rinse the condenser Thin-layer chromatography (2.2.27) .
with 20 mL of ethanol (96 per cent) R. Titrate the excess of
Test solution The tincture 10 be examined.
potassium hydroxide with 0.5 M hydrochlorie aeid,
determining the end-point potentiometrically (2.2.20). Carry Rejerenee solution Dissolve 20 mg of benzoie acid R, 10 rng of
out a blank titration. trans-cinnamic aeid R, 4 mg of vanillin R and 20 mg of methyl
einnamate R in 20 mL of ethanol of the same concentration
I mL of 0.5 M aleoholie potassium hydroxide is equivalent 10
as that used for the production of the tincture.
61.05 mg ofbenzoic acid (C 7 H 6 0 2 ).
____________________________________________ ~E~
Plate TLC siliea gel F254 plate R (5-40 ¡.tm) [or TLC siliea gel
F254 plate R (2-10 ¡.tm)] .
Mobz1e phase glacial acetie acid R, di-isopropyl ether R,
hexane R (10:40:60 VIV/V).
Application 20 ¡.tL [or 8 ¡.tL] as bands.
Development Over a path of 12 cm [or 6 cm] .
Drying In airo
Deteetion Examine in ultraviolet light at 254 nm.
2014 Benzoin Preparations IV-95

Results The chromatogram obtained with the test solution

does not show zones due to benzoic acid and vanillin that are
Benzoin Inhalation
more intense than the corresponding zones in the Benzoin Inha1ation Vapour
chromatogram obtained with the reference solution. DEFINITION
Ethanol (2.9. 10) Benzoin Inhalation is an inhalation vapour, solution.
95 per cent to 105 per cent of the content stated on the Sumatra Benzoin crushed 100 g
labe!' Prepared storax of 50 g
commerce
ASSAY
Ethanol (96 per cent) Sufficient to produce 1000 mL
Place 3.50 g in a 250 mL borosilicate glass fiask and add
15.0 mL of 0.5 M alcoho/ic potassium hydroxide. Boil under a In making Benzoin Inhalation, Ethanol (96 per cent) may be
refiux condenser on a water-bath for 30 mino Allow to cool replaced by Industrial Methylated Spirit 1 .
and rinse the condenser with 20 mL of ethanol Extemporaneous preparation
(96 per cent) R. Titrate the excess of potassium hydroxide The following directions apply.
with 1 M hydrochlon'c acid, determining the end-point Macerate the crushed Sumatra Benzoin and the prepared
potentiometrically (2.2.20). Carry out a blank titration. storax with 750 mL of Ethanol (96 per cent) for 24 hours.
1 mL of 0.5 M a/coho/ic potassium hydroxide is equivalent to Filter and pass sufficient Ethanol (96 per cent) through the
61.05 mg ofbenzoic acid (C7Hó02) ' filter to produce 1000 mL.
____________________________________________ ~Ew

The inhalation complies with lhe requirements stated under

Preparations for Inhalation and with the following requirements.
Content of total balsamic acids
Not less than 3.0% w/v, ca1culated as cinnamic acid,
Compound Benzoin Tincture C9 H s 0 2'
Friars' Balsam Total solids
0
9.0 to 12.0% w/v when determined by drying at 105 for
DEFINITION 4 hours, Appendix XI A. Use 2 mL.
Barbados Aloes or Cape Aloes 20 g
Prepared storax of commerce 100 g ASSAY
Sumatra Benzoin crushed 100 g Boil 10 mL with 25 mL of O.5M ethanolic potassium hydroxide
Ethanol (90 per cent) Sufficient to produce under a refiux condenser for 1 hour. Evaporate the ethanol,
1000 mL disperse the residue in 50 mL of hot water, cool, add 80 mL
of water and 1.5 g of magnesium sulfate dissolved in 50 mL of
Extemporaneous preparation water. Mix thoroughly and allow to stand for 10 minutes.
The following directions apply. Filter, wash the residue on the filter with 20 mL of water,
Macerate the Barbados Aloes or Cape Aloes, the prepared acidify the combined filtrate and washings with hydrochloric
storax and the Sumatra Benzoin with 800 mL of Ethanol acid and extract with four 40-rnL quantities of ether. Discard
(90 per cent) in a c10sed vessel for not less than 2 days, the aqueous solution, combine the ether extracts and extract
shaking occasionally, filter and pass sufficient Ethanol with successive quantities of 20,20, 10, 10 and 10 rnL of
(90 per cent) through the filter to produce 1000 mL. sodium hydrogen carbonate solution, washing each aqueous
The tinclure comp/ies with the requirements for Tinctures stated extract with the same 20 mL of ether. Discard the ether
under Extracts and wilh the following requirements. layers, carefully acidify the combined aqueous extracts with
hydrochloric acid and extract with successive quantities of 30,
Content oftotal balsamic acids
20, 20 and 10 mL of chlorofonn, filtering each extract through
Not less than 4.5% w/v, ca1culated as cinnamic acid,
anhydrous sodium sulfate supported on absorbent cotton. Distil
C 9 H s 02'
the chloroform from the combined filtra tes until 10 mL
TESTS remains and remove the remainder in a current of airo
Ethano1 content Dissolve the residue, with the aid of gentle heat, in 10 mL of
70 to 76% v/v, Appendix VIII F, Method IlI. ethanol (96%), previously neutralised to phenol red solution,
Dry residue cool and titrate with O.lM sodium hydroxide VS using phenol
15 to 19% w/v. red solution as indicator. Each mL of 0.1 M sodium hydroxide
VS is equivalent to 14.82 mg of total balsamic acids,
Relative density
ca1culated as cinnamic acid, C 9 H s 02'
0.880 to 0.910, Appendix V G.
ASSAY 1 Statutory regulations governing the use oi Industrial Methylated Spirit must

Carry out the Assay described under Benzoin Inhalation be observed.

using 10 mL of the tincture. Each mL of O.lM sodium
hydroxide VS is equivalent to 14.82 mg of total balsamic
acids, ca1culated as cinnamic acid, C 9 H s 02'
 IV-96 Berberís Arístata
Berberis Aristata
DEFINITION
Berberis Aristata is the dried, cut stem of Berberis aristata
De.
It contains not less than 1.4% ofberberine (C2oHI9NOs),
ca1culated with reference to the dried material.
IDENTIFICATION
A. The cut pieces of stem are subcylindrical, often branched
and somewhat swollen at the nodes, ftom about 15-20 mm
diameter and varying in length. The bark is soft, about
4-8 mm thick, with a yellowish brown outer surface, finely
wrinkled longitudinally or deeply furrowed, peeling off in
places and exposing the inner dark yellow wood. Fracture
short in the region of the bark, hard and fibrous in the wood.
B. Reduce to a powder. The powder is yellowish brown.
Examine under a microscope using chloral hydrate solution.
The powder contains numerous fragments of xylem, the
vessels reticulately and spirally thickened, sorne tracheids;
thick-walled, short, spindle-shaped, lignified, yellowish fibres
of the phloem and xylem with a wide lumen; stone cells
elongated with thick, pitted walls, sorne containing a single
ca1cium oxalate crystal, normally present in groups;
parenchyma cells of the medullary rays, sorne with yellow-
brown contents, single prism crystals of ca1ciurn oxalate, or
simple starch granules; cork cells yellowish-brown, thin-
walled; numerous scattered starch grains and calcium oxalate
crystals.
e. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Add 4 mL of methanol (80%) to 250 mg of the powdered
herbal drug (180) in a centrifuge tube. Mix with the aid of
ultrasound for 10 minutes. Centrifuge at 3000 rpm for
5 minutes and collect the clear supernatant. Repeat the
extraction twice with a further two 2-mL portions of methanol
(80%). Combine the supernatants and dilute to 20 mL with
methanol (80%).
(2) 0.04% w/v each of berberine chloride BPCRS and palmatine
chloride in methanol (80%) .
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F 254.
(b) Use the mobile phase as described below.
(e) Apply 20 ¡.IL of each solution as 6 mm bands.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (254 nm).
MOBILE PHASE
10 volurnes of anhydrous formic acid, 10 volurnes of water and
80 volumes of ethyl acetate.
SYSTEM SUITABILlTY
The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated bands .
CONFIRMATION
The chromatogram obtained with solution (1) shows a
principal yellow band eorresponding in eolour and position to
the band obtained for berberine chloride in solution (2), a
yellow band corresponding in colour and position to the
band obtained for palmatine in solution (2) and several other
bands as shown in the table . Other bands may be presento
2014
Top of the plate
Yellow band (berberine chloride) Yellow band (berberine chloride)
Faint yellow band
Yellow band (palmatine) Yellow band (palmatine)
Purple band
Solution (1) Solution (2)
TESTS
D- Tetrahydropabnatine
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) To 0.5 g of powdered sample, add 400 mL of a mixture
of equal volumes of acetonitrile and 0.1 % v/v orthophosphoric
acid. Mix with the aid of ultrasound for 40 minutes and
allow to cool. Dilute to 500 mL with the mobile phase and
filter through a 0.45-¡.Im filter.
(2) 0.01 % w/v each of palmatine chloride and berberine
chloride BPCRS in the mobile phase.
(3) 0.01 % w/v of D-tetrahydropalmatine hydrochloride in the
mobile phase.
CHROMATO GRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm x 4.6 mm) paeked
with end-capped octadecylsilyl silica gel for chromatography
(5 ¡.1m) (Phenomenex Luna C18 is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(e) Use a fiow rate of l.2 mL per minute.
(d) Use an ambient eolumn temperature.
(e) Use a deteetion wavelength of 235 nm.
(f) Injeet 10 ¡.IL of eaeh solution.
When the chromatograms are recorded under the preseribed
eonditions the retention times relative to palmatine (retention
time = about 8 minutes) are berberine ehloride = about l.1;
D-tetrahydropalmitine = about 1.6.
MOBILE PHASE
27 volurnes of acetonitrile and 73 volumes of a 1.36% w/v
solution of potassium dihydrogen orthophosphate.
SYSTEM SUITABILlTY
The test is not valid unless, in the chromatogram obtained
with solution (2), the resolutionfactor between the peaks due
to palmatine and berberine chloride is at least 2.0.
CONFIRMATION
In the ehromatogram obtained with solution (1), there are no
peaks eorresponding to the peak due to D-tetrahydropalmitine
in the ehromatogram obtained with solution (3).
Loss on drying
When dried for 2 hours at 105°, loses not more than 10.0%
of its weight, Appendix IX D . Use 1 g.
Total Ash
Not more than 3.0%, Appendix XI J, Method n.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D , using the following solutions.
 2014
(1) To 0.5 g of powdered sample, add 400 mL of a mixture
of equal volumes of aeetonitnle and 0.1% v/v orthophosphoric
acid. Mix with the aid of ultrasound for 40 minutes and
allow to cool. Dilute to 500 mL with the mobile phase and
filter through a 0.45-¡.¡m filter.
(2) 0.01 % w/v each of palmatine ehlonde and berberine
ehlonde BPCRS in the mobile phase.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under the test for
D-tetrahydropalmatine may be used.
MOBILE PHASE
27 volumes of aeetonitn"le and 73 volumes of a 1.36% w/v
solution of potassium dihydrogen orthophosphate.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (2), the resolution factor between the peaks due
to palmatine and berberine chIoride is at least 2.0.
DETERMINATION OF CONTENT
Using the retention time and the peak area from the
chromatogram obtained with solution (2), locate and
integrate the peak due to berberine chloride in the
chromatogram obtained with solution (1). Calculate the
content of berberine in the sample using the dec1ared content
of berberine in berbenne ehlonde BPCRS and the following
expression:
Al ID, VI 100
-x-x-xpx - --
A2 V, mr 100-d
Al area of the peak due to berberine in the
chromatogram obtained with solution (1),
A2 area of the peak due to berberine in the
chromatogram obtained with solution (2),
mi weight of the drug being examined in mg,
m2 weight of berbenne ehlonde BPCRS in mg,
VI dilution volume ofsolution (1) in mL,
V2 dilution volume of solution (2) in mL,
p percentage content of berberine in berbenne
ehlonde BPCRS,
d percentage loss on drying of the herbal drug being
examined.
STORAGE
Berberis Aristata should be protected from moisture.
Dried Bilberry ***
*** ***
(Dned Bilbeny Fruit, Ph Eur monograph 1588) ***
~Ew ____________________________________________
DEFINITION
Dried ripe fruit of Vaecinium myrtillus L.
Content
Minimum 1.0 per cent of ta=ins, expressed as pyrogallol
(C 6 H 6 0 3 ; M r 126.1 ) (dried drug).
CHARACTERS
Sweet and slightly astringent taste.
IDENTIFICATION
A. Dried bilberry is a dark blue, subglobular, shrunken berry
about 5 mm in diameter, with a scar at the lower end and
Bilberry IV-97
surmounted by the persistent calyx, which appears as a
circular fold and the remains of the style . The deep violet,
fteshy mesocarp contains numerous small, brown, ovoid
seeds.
B. Reduce to a powder (355) (2.9.12). The powder is violet-
brown. Examine under a microscope using ehloral hydrate
solutíon R. The powder shows: violet-pink sc1ereids from the
endocarp and the mesocarp, usually aggregated, with thick,
cha=elled walls; reddish-brown fragments of the epicarp
consisting of polygonal cells with moderately thickened walls;
brownish-yellow fragments of the outer seed testa made up of
elongated cells with U-shaped thickened walls; c1usters and
prisms crystals of various size of calcium oxalate.
C. Thin-Iayer chromatography (2.2.27)
Test solution To 2 g of the powdered drug (355) (2.9.12)
add 20 mL of methanol R. Shake for 15 min and filter.
Referenee solution Dissolve 5 mg of ehrysanthemín R in
10 mL of methanol R.
Plate TLC siliea gel plate R .
Mobile phase anhydrous formie aeid R, water R, butanol R
(16:19:65 VIV/V).
Applieation 10 ¡.¡L, as bands.
Development Over a path of 10 cm.
Dlying In air.
Deteetion Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference and test
solutions .
Top of the plate
A violet-red zone of low intensity
Chrysanthemin: a violet-red zone A principal violet·red zone
A compacl set of other principal
zones:
- a violet·red zone
- several violet-blue zones
Reference solution Test solution
TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug by drying in an oven at 105 oC for 2 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14) . Use 1.500 g ofthe powdered drug (355) (2.9.12).
____________________________________________ Ph Eur
IV-98 Bilberry 2014

*** Loss on drying (2.2.32)

Fresh Bilberry
**
(Fresh Bzlberry Fruit, Ph Eur monograph 1602)
Preparation
Fresh Bilberry Fruit Dry Extract, Refined and Standardised
~Ew __________________________________________
DEFINITION
Fresh or frozen, ripe fruit of Vaccinium myrtil/us L.
Content
Minimum 0.30 per cent of anthocyanins, expressed as
cyanidin 3-0-glucoside chloride (chrysanthemin,
C21H21CIOll; M r 484.8) (dried drug) .
CHARACTERS
Sweet and slighdy astringent taste.
IDENTIFICATION
A. The fresh fruit is a blackish-blue globular berry about
5 mm in diameter. Its lower end shows asear or, rarely, a
fragment of the pedicel. The upper end is ftattened and
surmounted by the remains of the persistent style and of the
calyx, which appears as a circular fold. The violet, fteshy
mesocarp includes 4 to 5 locules containing numerous small,
brown, ovoid seeds.
B. The crushed fresh fruit is violet-red. Examine under a
microscope using chloral hydrate solution R. It shows violet-
pink sclereids from the endocarp and the mesocarp, usually
aggregated, with thick, channelled walls; reddish-brown
* *
**
80.0 per cent to 90.0 per cent, determined on 5.000 g of the
*** freshly crushed drug by drying in an oven at 105 oc.
ASSAY
Crush 50 g extemporaneously. To about 5.00 g of the
crushed, accurately weighed drug, add 95 mL of methanol R .
Stir mechanically for 30 mino Filter into a 100.0 mL
volumetric ftask. Rinse the filter and dilute to 100.0 mL with
methanol R . Prepare a 50-fold dilution of this solution in a
0.1 per cent VIV solution of hydrochloric acid R in methanol R.
Measure the absorbance (2.2.25) of the solution at 528 nm,
using a 0.1 per cent VIV solution of hydrochloric acid R in
methanol R as the compensation liquid.
Calculate the percentage content of anthocyanins, expressed
as cyanidin 3-0-glucoside chloride, using the following
expression:
A X 5000
718 x m
718 specific absorbance of cyanidin 3-0-glucoside
chloride at 528 nm
A absorbance at 528 nm
m mass of the substance to be examined in grams.
STORAGE
When frozen, store at or below - 18 oc.
__________________________________________ ~Ew

fragments of the epicarp consisting of polygonal cells with

moderately thickened walls; brownish-yellow fragments of the
outer layer of the testa composed of elongated cells with
U-shaped thickened walls; cluster crystals of calcium oxalate .
***
Refined and Standardised Fresh
C. Thin-Iayer chromatography (2.2.27) .
*** ***
Test solution To 5 g of the freshly crushed drug, add 20 mL Bilberry Fruit Dry Extraet ***
of methanol R . Stir for 15 min and filter.
(Ph Eur monograph 2394)
Reference solution Dissolve 5 mg of chrysanthemin R in ~ __________________________________________
Ew

10 mL of methanol R.
Plate TLC silica gel plate R. DEFINITION
Refined and standardised dry extract produced from Bilberry
Mobile phase anhydrous formic acid R, water R, butanol R
jruit, fresh (I 602) .
(16:19:65 VIV/V) .
Application 10 ¡.¡L, as bands. Content
32.4 per cent to 39.6 per cent of anthocyanins, expressed as
Development Over a path of 10 cm.
cyanidin 3-0-glucoside chloride [chrysanthemin
Drying In airo (C 21 H 21 CIO ll ; M r 484 .8)J (dried extract).
Detection Examine in daylight.
PRODUCTION
R esults See below the sequence of the zones present in the The extract is produced from the herbal drug by a suitable
chromatograms obtained with the reference solution and the procedure using ethanol (96 per cent V/V) or methanol
test solution. (minimum 60 per cent V/V). Refinement may be performed
by ion-exchange chromatography.
Top of the plate CHARACTERS
A violet·red zone Appearance
Dark reddish-violet, amorphous, hygroscopic powder.
Chrysanthemin: a violet·red zone A principal violet·red zone
IDENTIFICATION
A compact set of other principal
First identification B.
zones:
- a violet·red zone Second identification A.
A. Thin-Iayer chromatography (2. 2.27) .
- several violet-blue zones
Test solution Dissolve 0.10 g of the extract to be examined
Reference solution Test solution
in 25 mL of methanol R. Stir for 15 min and filter.
Reference solution Dissolve 2 mg of chrysanthemin R and
2 mg of myrtillin R in 5 mL of methanol R .
TESTS
Plate TLC plate coated with cel/ulose for chromatography R
Total ash (2.4.16)
Maximum 0.6 per cent. (5-40 ¡.¡m) [or TLC plate coated with cel/ulose for
chromatography R (2-10 ¡.¡m)J.
2014 Bilberry Fruit Preparations IV-99

Mobile phase: mobile phase A: hydrochloric acid R, acetic Top oC the plate
acid R, water R (3: 15:82 V/V/V);
--- - --
mobile phase B: water R, acetic acid R (40:60 V/V).
A violet·red zone
Application 10 ~L [or 2 ~L] as bands of 10 mm [or 6 mm].
Development A Over a path of 10 cm [or 6 cm] with mobile Chrysanthemin: a violet·red zone A violet·red zone (chrysanthemin)
phase A.
Myrtillin: a violet·red zone A violet·red zone (myrtillin)
Drying A In warm airo
Development B Over a path of 10 cm [or 6 cm] with mobile
--- ---
phase B. ReCerence solution Test solution
Drying B In air.
Detection Examine in daylight.
B. Liquid chromatography (2.2.29) as described in the test
R esults See below the sequence of zones present in the for total anthocyanidins.
chromatograms obtained with the reference solution and the
The characteristic anthocyanin peaks (peaks 1-8, 10-15 and
test solution. Furthermore, other faint zones may be present
17) in the chromatogram obtained with the test solution are
in the chromatogram obtained with the test solution.
similar in their retention times to those in the chromatogram
obtained with reference solution (b).

4
3 5

7
8

15

6 12

11 13
17
10

~A
14
16 18 1J10
L
r------r t:. D. ¡:" i l l 'D. D. m • D. De :¿ffi" D. D.
~A Vt.::D.
Ll<:.\.1I!i;
J. D.
1
\.L> 'IlS:i>
JI,

O 5 10 15 20 25 30 35 40 45 50 55 min

1. delphinidin 3·().galactoside chloride 11. petunidin 3·Q·arabinoside chloride

2. myrtillin (delphinidin 3·().glucoside chloride) 12. peonidin 3·().glucoside chloride

3. cyanidin 3·Q·galactoside chloride 13. malvidin 3·Q·galactoside chloride

4, delphinidin 3·Q·arabinoside chloride 14. peonidin 3·().arabinoside chloride

5, chrysanthemin (cyanidin 3·().glucoside chloride) 15, malvidin 3·().glucoside chloride

6. petunidin 3·Q·galactoside chloride 16. cyanidin chloride

7. cyanidin 3·().arabinoside chloride 17. malvidin 3·Q·arabinoside chloride

8. petunidin 3·().glucoside chloride 18. petunidin chloride

9. delphinidin chloride 19, peonidin chloride

10. peonidin 3·Q·galactoside chloride 20. malvidin chloride

Figure 2394.-1. - Chromatogram for the assay of refined and standardised fresh bilberry fruit dry extraet
IV-100 Birch Leaf 2014

TESTS Al sum of the areas of the peaks d ue to the

Loss on drying (2.8.1 7) anthocyanidins (peaks 9, 16, 18-20) in the
Maximum 4.5 per cent. chromatogram obtained with the test solution;
Total ash (2.4.16) A2 area of the peak due to cyanidin chloride (peak 16) in
MaxÍInum 2.0 per cent. the chromatogram obtained with reference solution
(a);
T ota! anthocyanidins mi mas s of the extract to be examined used to prepare
Liquid chromatography (2.2.29). Maintain the solutions at the test solution, in grams;
4 oC .
m2 mass of eyanidin ehloride CRS used to prepare
Solvem mixture hydroehlorie acid R, methanol R (2:98 V/V). reference solution (a), in grams;
Test solution Dissolve 0.1250 g ofthe extract to be p percentage content of cyanidin chloride in eyanidin
examined in the solvent mixture and dilute to 25.0 mL with ehloride CRS.
the solvent mixture. Dilute 5.0 mL of this solution to Limits Not more than 1.0 per cent of total anthocyanidins,
20.0 mL with dllute phosphorie acid R. expressed as cyanidin ch1oride.
Reference solution (a) Dissolve 10.0 mg of eyanidin
ASSAY
ehloride CRS in the solvent mixture and dilute to 25.0 mL
Liquid chromatography (2.2.29) as described in the test for
with the solvent mixture. Dilute 2.0 mL of this solution to
total anthocyanidins with the following modification.
100.0 mL with dilute phosphorie acid R.
Injeetion Test solution and reference solution (b).
Referenee solution (b) Dissolve 0.1250 g of bilberry dry
extraet CRS in the solvent mixture and dilute to 25 .0 mL Calculate the percentage content of total anthocyanins,
with the solvent mixture. Dilute 5.0 mL of this solution to expressed as cyanidin 3-0-glucoside chloride, using the
20.0 mL with dilute phosphorie acid R. following expression:
Column:
-- size: 1 = 0.250 m, 0 = 4.6 mm;
-- stationary phase: octadeeylsilyl slliea gel for ehromatography R
(5 ~lm);
-- temperature: 30 oc. sum of the areas of the peaks due to the anthocyanins
Moblle phase: (peaks 1-8, 10-15 and 17) in the chromatogram
-- mobile phase A: anhydrous formie acid R, water R obtained with the test solution;
(8.5:9 1.5 V/V); area of the peak due to cyanidin 3-0-glucoside
-- mobile phase B: anhydrous formie ae¡d R, aeewnitrile R, chloride (peak 5) in the chromatogram obtained with
methanol R, water R, (8.5:22.5:22.5:41.5 VIVIV/V); reference solution (b);
mi mass of the extract to be examined used to prepare
Time Mobile phase A Mobile phase B
the test solution, in grams;
(min) (per eent VM (pereent VM
mas s of bilberry dry extract CRS used to prepare
0-35 93 ~ 75 7 ~ 25
reference solution (b), in grams;
35 - 45 75 ~35 25 ~ 65 p percentage content of cyanidin 3-0-glucoside chloride
45 - 46 35 ~ O 65 ~ 100 in bilberry dry extraet CRS.
____________________________________________ ~E~

46 - 50 O 100

Flow rate 1.0 mUmin.

Deteetion Spectrophotometer at 535 nm.
Injection 1O ~lL.
Idemifieation of peaks Use the chromatogram supplied with
bilberry dry extract CRS and the chromatograms obtained with
reference solutions (a) and (b) to identify the peaks due to
the anthocyanins and the anthocyanidins.
Retention times The retention times and the elution order of
the peaks are similar to those shown in the chromatogram
(Figure 2394.-1 ).
System suitability Reference solution (b):
-- peak-to-valley ratio: minimum 2.0, where H p = height
aboye the baseline of the peak due to cyanidin 3-0-
galactoside (peak 3) and H v = height aboye the baseline
of the lowest point of the curve separating this peak from
the peak due to delphinidin 3-0-arabinoside (peak 4).
Calculate the percentage content of total anthocyanidins,
expressed as cyanidin chloride, using the following
expression:
Al x m2 x 100 x p
mI x A2 x 1250
Birch Leaf *****
* *
(Ph Bur monograph 1174) *****
~E~ ____________________________________________
DEFINITION
Whole or fragmented, dried leaves of Bentla pendula Roth
and/or Betula pubeseens Ehrh. as well as hybrids of both
species.
Content: minimum 1.5 per cent of flavonoids, expressed as
hyperoside (C21H20012; M r 464.4) (dried drug).
IDENTIFICATION
A. The leaves of both species are dark green on the adaxial
surface and lighter greenish-grey on the abaxial surface; they
show a characteristic dense reticulate venation. The veins are
light brown or almost white.
The leaves of B. pendula are glabrous and show c10sely
spaced glandular pits on both surfaces. The leaves of B.
pendula are 3-7 cm long and 2-5 cm wide; the petiole is long
and the doubly dentate lamina is triangular or rhomboid and
broadly cuneate or trunca te at the base. The angle on each
side is unrounded or slight1y rounded, and the apex is long
and acuminate.
 2014
The leaves of B . pubescens show few glandular trichomes and
are slightly pubescent on both surfaces. The abaxial surface
shows small bundles of yellowish-grey trichomes at the
branch points of the veins. The leaves of B . pubescens are
slightly smaller, oval or rhomboid and more rounded. They
are more roughly and more regularly dentate. The apex is
neither long nor acuminate.
Figure 1174.-1. - Illustration for identification test B of
powdered herbal drug of birch leaf
B. Microscopic examination (2.8.23). The powder is
greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1174.-1): numerous fragments ofthe
lamina, in surface view, with sttaight-walled, adaxial
epidermal cells accompanied by underlying palisade
parenchyma [E] and cells of the abaxial epidermis
surrounding anomocytic stomata (2.8.3) [G]; large, free,
glandular trichomes usually measuring 100-120 J..lm [D];
fragments of the lamina in transverse section [B], showing
glandular trichomes on the epidermis es [Ba], heterogeneous,
asymmetrical mesophyll containing cluster crystals [Bb] and
prisms [Be] of calcium oxalate; fragments of spongy
parenchyma [A] accompanied by crystal sheaths [Aa] and
cells containing cluster crystals of calcium oxalate [Ab];
fragments of vessels and sclerenchyrna fibres [C] . If B.
pubescens is present, the powder also contains unicellular
covering trichomes with very thick walls, about 80-600 J..lm
long, usually 100-200 ~lm, numerous on the margin of the
lamina [F] or on the epidermis es, in surface view [H].
C. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R and shake. Heat on a
water-bath at 60 oC for 5 mino Cool and filter the solution.
Birch Leaf IV-10 1
Reference solution Dissolve 1 mg of chlorogenic acid R, 1 mg
of caffeic acid R, 2.5 mg of hyperoside R and 2.5 mg of rutin R
in 10 mL of methanol R.
Plate TLC si/ica gel plate R.
Mobzle phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethy! acetate R (10:10:30:50 VIVIV/V) .
Application 10 J..lL as bands.
Development Over a path of 10 cm.
Drying In a current of warm airo
Detection Treat with a 10 giL solution of dipheny!boric acid
aminoethyl ester R in methanol R; subsequently treat with a
50 gIL solution of macrogo! 400 R in methanol R; allow to dry
in air for 30 min and examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows 3 zones in its lower half: in increasing order
of R p , a yellowish-brown fiuorescent zone (rutin), a light blue
fiuorescent zone (chlorogenic acid) and a yellowish-brown
fiuorescent zone (hyperoside), and in irs upper third, a light
blue fiuorescent zone (caffeic acid). The chromatogram
obtained with the test solurion shows 3 zones similar in
position and fiuorescence to the zones due to rutin,
chlorogenic acid and hyperoside in the chromatogram
obtained with the reference solution. The zone due to rutin is
very faint and the zone due ro hyperoside is intense. Ir also
shows other yellowish-brown faint fiuorescent zones berween
the zones due to caffeic acid and chlorogenic acid in the
chromatogram obtained with the reference solution. Near the
solvent front, the red ftuorescent zone due to chlorophylls is
visible. In the chromatogram obtained with the test solution,
berween this zone and the zone due ro caffeic acid in the
chromatogram obrained with the reference solution, there is a
brownish-yellow zone due to quercetin.
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of fragments of female catkins and
maxirnum 3 per cent of other foreign manero
Loss on drying (2.2.32)
Maxirnum 10.0 per cent, derermined on l.000 g of
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maxirnum 6.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed fiask introduce
0.200 g of the powdered herbal drug (355) (2.9.12) , 1 mL of
a 5 gIL solution of hexamethy!enetetramine R, 20 mL of
acetone R and 2 mL of hydrochloric acid R1 . Boil the mixture
under a refiux condenser for 30 mino Filter the liquid
through a plug of absorbent conon into a 100 mL fiask.
Add the absorbent corton to the residue in the round-
bottomed fiask and extraer with 2 quantiries, each of 20 mL,
of acetone R, each time boiling under a reftux condenser for
10 mino Allow to cool to room temperature, filter the liquid
through a plug of absorbent corton then through a filter
paper into the volumetric fiask, and dilute to 100.0 mL with
acetone R by rinsing the fiask and filter. Introduce 20.0 mL of
the solution into a separating funnel, add 20 mL of water R
and extract the mixture with 1 quanrity of 15 mL and then
3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acerate extracts in a separating funnel, wash with
2 quantities, each of 50 mL, of water R, and filter the extract
over 10 g of anhydrous sodium sulfate R into a 50 mL
volumetric fiask and dilure to 50.0 mL with ethy! acetate R.
 IV-102 Bistort Rhizome 2014

Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R .
Compensation liquid Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetie
acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after
30 min, by comparison with the compensation liquid at
425 nm.
Calculate the percentage content of fiavonoids, expressed as
hyperoside, using the following expression:
A x 1.25
m
Referenee solution Dissolve 5 mg of fructose R and 5 mg of
eateehin R in 5 mL of methanol R.
Plate TLC siliea gel plate R (2-10 ¡lm).
Mobile phase water R, anhydrous fomúc acid R, ethyl acetate R
(5:10:85 VIV/V) .
Application 2 ¡lL as bands.
Development Over a path of 7 cm.
Drying In airo
Detection Spray with anisaldehyde solution R and heat at
100-105 oC for 5 min; examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.

i.e. taking the specific absorbance of hyperoside to be 500.

A absorbance at 425 nm; Top oí the plate
m = mas s ofthe herbal drug to be examined, in grams. Catechin: a brown zone A brown zone (catechin)
____________________________________________ Phf~

--- -----
A brown zone

A violet zone

Bistort Rhizome ***

*** ***
A brown zone

An orange zone
(Ph Eur monograph 2384) ***
~f~ ____________________________________________ --- -----

DEFINITION Fructose: a green zone A green zone (fructose)

Whole or fragmented, dried rhizome of Persicaria bistorta (L.) Reference solution Test solution
Samp. (syn. Polygonum bistorta L.) without adventitious roots.
Content
Minimum 3.0 per cent oftannins, expressed as pyrogallol TESTS
(C 6H 6 0 3; M r 126.1) (dried drug) . Paris polyphylla or Paris quadrifolia
Examine the powdered drug (355) (2.9. 12) under a
CHARACTERS
microscope using chloral hydrate solution R. The presence of
The whole rhizome is up to 13 cm long and 2.5 cm in
raphides of calcium oxalate, free or in bundles, indica tes
diameter. The remnants of the roots are not longer than
adulteration by the rhizome of Paris polyphylla Smith var.
1 cm and are about 1 mm in diameter.
yunnanensis (Franch.) Hand.-Mazz or Pans polyphy lla Smith
IDENfIFICATION var. chinensis (Franch.) Hara or París quadrifolia L.
A. The whole rhizorne, reddish-brown or blackish-brown, is Loss on drying (2.2.32)
thick, twisted, and tumed back on itself. Its outer surface Maximurn 12.0 per cent, determined on 1.000 g ofthe
shows transverse striations and blackish spots. It is fiattened powdered drug (355) (2. 9. 12) by drying in an oven at
and somewhat depressed on the upper surface, convex on the 105 De.
lower surface. It shows adventitious root scars on the surface.
The fracture, pinkish-beige, shows an elliptical zone of Total ash (2.4.16)
whitish pits corresponding to the vessels. The drug may also Maximurn 9.0 per cent.
be obtained as more-or-less cylindrical fragments about Ash insoluble in hydrochloric acid (2.8.1)
0.3 cm in diameter and up to 1 cm long, with a reddish- Maximurn 1.0 per cent.
brown outer surface, marked by adventitious root scars and a ASSAY
pinkish-beige fracture.
Carry out the determination of tannins in herbal drugs
B. Reduce to a powder (355) (2.9.12). The powder is (2.8.14) . Use 1.000 g ofthe powdered drug (180) (2.9. 12).
reddish-brown. Examine under a microscope using ehloral ____________________________________________ Ph fur
hydrate solution R . The powder shows the following diagnostic
characters: fragments of parenchyma; very nurnerous calciurn
oxalate cluster crystals either free or inside parenchyma cells;
a few reticulate lignified vessels; rare cork fragments.
Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows rounded to ovoid
starch granules, simple, about 10 ¡lm in diameter.
e. Thin-layer chromatography (2.2.27).
Test solution To 1.0 g ofthe powdered drug (355) (2.9. 12)
add 10 mL of a mixture of equal volumes of water R and
methanol R, heat on a water-bath at about 65 oC for 30 min
and filter.
 2014 Black Cohosh IV-103

Black Cohosh *** present in the chromatograms obtained with reference

** ** solution (a) and the test solution.
(Ph E ur monogmph 2069) **** *
~E~ ____________________________________________
Top of the plate
DEFlNITION
Dried, whole or fragmented rhizome and root of Actaea
- -- - - - ---
mcemosa L. (syn. Cimicijuga mcemosa (L.) Nutt.).
Content
Minimum 1.0 per cent of triterpene glycosides, expressed as Actein: a brown zone Actein: a brown zone A brown zone (actein)
monoammonium glycyrrhizate (C 42 H ó5 NO, ó; M r 840) (dried
23-Epi-26- A brown zone
drug) . deoxyactein : a brown (23-epi-26-deoxyactein )
zone
IDENTIFICATION
A. W'hole drug The rhizome is dark brown, hard,
subcylindrical and somewhat motted; l.5-2.5 cm in diameter --- --- ---
and 2-15 cm long; it shows numerous cJosely arranged,
upright or curved branches each terminating in the remains
of a bud or in a circular, cup-shaped scar. The fracture is A violet zone A violet zone
horny, the transverse section shows a thin outer bark A violet zone A violet zone
surrounding a ring of numerous pale, narrow wedges of
vascular tissue alternating with darker medullary rays and a A brown zone A brown zone
large central pith. Roots attached to the lower surface of the
rhizome are usually broken off, leaving circular scars.
Reference Reference
The roots are dark brown, 1-3 mm in diameter, brittle, solution (a) solution (b)
Test solution
nearly cylindrical or obtusely quadrangular and longírudinally
wrinkled; the fracture is shon; the transverse section shows a
wide outer bark, a dark brown cylinder, in which the central TESTS
regio n is composed of 3-6 lighter wedges of vascular tissue Loss on drying (2.2.32)
united at the centre and separated by broad, non-lignified Maximum 12 per cent, determined on l.000 g of the
medullary rays. powdered herbal drug (355) (2.9.12) by drying in an oven at
Fmgmented drug More or Jess angular, irregular pieces of 105 oC for 2 h.
the rhizome and cylindrical pie ces of the roots. The hard, Foreign matter (2.8.2)
horny rhizome fragments usually show a dark brown surface Maximum 5 per cent.
corresponding to the outer surface and several frequently Total ash (2.4.16)
striated, light brown surfaces corresponding to the section. Maximum 10 per cent.
The dark brown, more or less cylindrical root fragments are
wrinkled longitudinally. The lighter coloured transverse Ash insoluble in hydrochloric acid (2.8.1)
section shows a distinct cambium line separating a thick Maximum 5 per cent.
outer bark from a central regíon composed of 3-6 wedges of Substitution by Címícifuga americana Michx., C.
vascular tissue united at the centre and separated by broad joetida L., C. dahurica (Turcz.) Maxim. or C.
medullary rays. heracleifolia Kom
B. Microscopic examination (2.8.23). The powder is light Thin-layer chromatography (2.2.27).
brown. Examine under a microscope using chloral hydrate Test solution To 0.50 g of the powdered herbal drug (355)
solution R. The powder shows the following diagnostic (2.9.12) add 10 mL of ethanol (50 per cent V/V) R and shake
characters: numerous fragments of thin-walled parenchyma; well. Sonicate for 10 min and centrifuge. Use the
groups of small, lignified vessels with cJosely-arranged supernatant.
bordered pits or, less frequently, with reticulate thickening; Reference solution (a) To 0.50 g of Actaea racemosa HRS add
lignified thin-walled fibres and xylem parenchyma; fragments 10 mL of ethanol (50 per cent V/Y] R and shake well.
of brown, suberised cells with moderately thickened walls. Sonicate for 10 min and centrifuge. Use the supernatant.
Examine under a microscope using a mixture of equal Reference solution (b) Dissolve 2 mg of actein R in
volumes of glycerol R and water R. The powder shows methanol R and dilute to 10 mL with the same solvento
abundant starch granules, spherical or polygonal, simple or 2
Plate TLC si/iea gel F 254 plate R (2-10 ¡.¡m).
or 3 (so me times up to 6) compound; individual granules are
3-15 ¡.¡m in diameter with a central, slit-shaped hilum. Mobile phase anhydrous formic acid R, ethyl fonnale R,
toluene R (20:30:50 VIV/V).
C . Examine the chromatograms obtained in the test for
substitution by Cimicijuga americana Michx., C. foetida L., C. Applieation 2 ¡.¡L as bands of 8 mm (see Table 2069.-1).
dahurica (Turcz.) Maxim. or C. heracleijolia Kom. Development Over a path of 6 cm.
Results B Use the chromatograms supplied with Actaea Drying In air.
racemosa HRS and the chromatogram obtained with reference System suitability Reference solution (b):
solution (a) ro identify the bands corresponding to A. -- the RF value of the zone due to actein is between 0.35
racemosa. and 0.40 (detection B).
See below the sequence of zones present in the Deteetion A Examine in ultraviolet light at 254 nm.
chromatograms obtained with reference solutions (a) and (b) Results A The chromatogram obtained with the test solution
and the test solution. Funhermore, other faint zones may be does not show any quenching zones more intense than those
 IV-104 Black Cohosh 2014

Table 2069.-1.- Application scheme

Traek 1 2 3 4 5 6 7

Application 2 2
2 2 2 2
volume (¡JL)

Referenee Referenee Referenee Referenee Test

Solution Test solution Blank
solution (a) solution (b) solution (a) solution (b) solution

After development. the plate is cut along traek 4 (blank). Traeks 1-3 are used for deteetion of a substitution by C. americana, C. foetida, C. dahurica or
C. heracleifolia (deteetion A), traeks 5-7 for identifieation e (deteetion B).

Table 2069.-2.- Application scheme

Traek 1 2 3 4 5 6 7 8 9

Applieation
20 2 2 20 20 2 2 20
volume (¡JL)

Referenee Referenee Referenee Referenee Referenee Reference

Solution Test solution Blank Test solution
solution (a) solution (b) solution (e) solution (al solution (bl solution (e)

After development and examination for detection of C. americana (deteetion A), the plate is cut along traek 5 (blank). Traeks 1-4 are used for deteetion of
adulteration with C. foetida (deteetion Bl, traeks 6-9 for detection of adulteration with C. heracleifolia and/ or C. dahurica (deteetion e).

in the ehromatogram obtained with referenee solution (a) Top of the plate
between R p value 0.2 and Rp value 0.3 5.
Detection B Treat with a 10 per eent V/V solution of sulfuric
- -- ---
acid R in methanol R; heat at 100 oC for 5 mini allow to eool
to room temperarure and examine in daylight. A weak zone A weak zone

Adulteration with Cimicifuga americana Michx., C. 2 weak zones 2 weak zones

foetida L., C. dahurica (Turcz.) Maxim. andlor C. A weak zone A weak zone
heracleifolia Kom
Thin-Iayer ehromatography (2.2.27) as deseribed in the test
for substitution by Cimicijuga americana Michx., C. foetida L., - -- _ _ _A DARK ZONE

C. dahurica (Turez.) Maxim. or C. heracleijolia Kom., with
the following modifieations .
Reference solution (c) Dissolve 2 mg of cimijugin R in
methanol R and dilute to 10 mL with the same solvento
Application 2 IlL of referenee solutions (b) and (e), 20 ¡lL of
the test solution and referenee solution (a), as bands of
8 mm (se e Table 2069.-2).
System suitability Referenee solution eb) :
- the R p value of the zone due to aetein is between 0.35
and 0.40 (deteetions B and C).
Detection A Examine in ultraviolet light at 254 nm.
Results A Absenee of more than 10 per eent of C.
amencana.
Compare the ehromatogram supplied with Actaea racemosa
HRS for C. americana and the ehromatograms obtained with
the test solution and referenee solution (a) .
The ehromatogram obtained with the test solution do es not
show any quenehing zone at Rp value 0.3 (zone presented in
eapitals in the ehromatogram of C. americana, see below).
The presenee of this zone in the ehromatogram obtained
with the test solution indicates adulteration with C. amen'cana
at a level greater than 10 per eent.
A weak zone A weak zone
A dark zone A dark zone
A dark zone A dark zone
Reference solution (a) C. americana (lO per cen!)
Detection B Dissolve 4.5 g of boric acid R in 150 mL of
anhydrous ethanol R (solution A); dissolve 5 g of oxalic acid R
in 50 mL of anhydrous ethanol R (solution B); combine
solutions A and B and mix well; treat the plate with this
freshly prepared solution and heat at 120 oC for 5 mini
examine in ultraviolet light at 365 nm.
Results B Absenee of more than 5 per eent of C. foetida .
Compare the ehromatogram supplied with Actaea racemosa
HRS for C. foetida and the ehromatograms obtained with the
test solution and referenee solutions (a), (b) and (e).
The ehromatogram obtained with the test solution does not
show any intense fiuoreseent zone between R p value 0.03
and R p value 0.06 or at the same position as the bright
fiuoreseent zone in the ehromatogram obtained with
referenee solution (e) (zones presented in eapitals in the
ehromatogram of C. foetida, see below). The presenee of 1 or
both zones in the ehromatogram obtained with the test
solution indica tes adulteration with C. foetida at a level
greater than 5 per eent.
 2014 Black Cohosh IV - 105

Top of tbe plate Referenee solution (e) Dilute 2.0 mL of reference solution (a)
--- --- --- --- to 10.0 mL with methanol R.
Referenee solution (d) Dilute 1.0 mL of reference solution (a)
Actein: a weak Actein: a weak A weak whitish
whitish zone whitish zone zone (actein) to 20.0 mL with methanol R.
- -- - -- --- - - - Reference solution (e) Dissolve 500 mg of Actaea racemosa dry
A bluish zone extract for system suitability HRS in methanol R and dilute to
A bluish zone
10.0 mL with the same solvent; sonicate and filter through a
membrane filter (nominal pore size 0.45 ~tm).
Cimifugin: A BRIGHT Column:
a bright FLUORESCENT
ZONE (CIMIFUGINI
- size: 1 = 0.25 m, 0 = 4.6 mm;
fluorescent zone
A brownish zone - stationary phase: octadecylsilyl siliCa gel for chromatography R
A brownish zone
(5 ¡lm).
A bluish zone A bluish zone
Mobile phase:
A FLUORESCENT - mobile phase A: 0.1 per cent V/V solution of anhydrous
ZONE
fonnic acid R in water R;
- mobile phase B: 0 .1 per cent V/V solution of anhydrous
Reference Reference Refe rence C. foetida (5 per fonnic acid R in a mixture of equal volumes of
solution (a) solutíon (b) solutioo (e) ceot)
acetonitrile R and methanol R;
Tíme Mobile phase A Mobile phase B
Detection C Dissolve 8 g of antimony triehloride R in 200 mL
(mín) (per een! VM (pereent VM
of methylene chloride R; treat with this solution and heat at
0·40 50 -> 20 50 -> 80
120 oC for 10 min; examine in ultraviolet light at 365 nm.
40 - 41 20 -> 5 80 -> 95
Results C Absence of more than 5 per cent of C. heracleifolia
and/or C. dahurica. 41 - 44 5 95
Compare the chromatogram supplied with Actaea racemosa
Flow rate 1.0 mUmin.
HRS for C. heracleifolia and C. dahunca and the
chromatograms obtained with the test solution and reference Detection Evaporative light-scattering detector; the following
solutions (a) and (b). The chromatogram obtained with the settings have been found to be suitable; if the detector has
test solution does not show any bright fiuorescent zone just different setting parameters, adjust the detector settings so as
aboye the zone due to actein (zone presented in capitals in to comply with the system suitability criterion for the signal-
the chromatogram of C. heracleifolia or C. dahurica, see to-noise ratio:
be1ow). The presence of this zone in the chromatogram - carrier gas: nitrogen R;
obtained with the test solution indicates adulteration with - flow rate: 0.8 mUmin;
C. heracleifolia andlor C. dahurica at a level greater than - evaporator temperature: 100 oC;
5 per cent. - nebuliser temperature: 60oc.
Injection 1O ~tL.
Top of the plate Identification of peaks Use the chromatogram supplied with
Actaea racemosa dry extraet for system suitability HRS and the
- - - - -- - - -
chromatogram obtained with reference solution (e) to identify
A BRIGHT FLlIORESCENT the peaks to be quantified.
ZONE
Actein: a weak Actein: a weak A weak brownish zone
System suitability:
brownish zone brownish zone (actein) - signal-to-noise ratio: minimum 4.0 for the peak due to
--- - -- --- monoammonium glycyrrhizate in the chromatogram
A brownish zone A brownish zone
obtained with reference solution (d);
- peak-to-valley ratio: minimum 3, where H p = height aboye
A bluish zone A bluish zo ne the baseline of peak 4 and H v = height aboye the baseline
of the lowest point of the curve separating this peak from
peak 5 in the chromatogram obtained with reference
C. herac/eifolia
(5 per ceot) aod/ or solution (e).
Refereoce solutíon (a) Refereoce solutíoo (b)
C. dilhurica (5 per
cent)
Establish a calibration curve with the logarithm to base 10 of
the concentration (in milligrams per millilirre) of reference
solutions (a), (b), (c) and (d) (corrected by the assigned
ASSAY percentage content of monoammonium glycyrrhizate in
Liquid chromatography (2.2.29). Aetaea raeemosa for assay CRS) as the abscissa and the
Test solution Introduce 4.00 g of the powdered herbal drug logarithm to base 10 of the corresponding peak are a as the
(355) (2.9.12) into a 200 mL screw-cap bottle. Add 50.0 mL ordinate.
of a mixture of equal volumes of methanol R and water R. Calculate the percentage content of each peak using the
Sonicate for 45 min and shake for 15 min. Filter through a following expression:
membrane filter (nominal pore size 0.45 ¡lm) .
Reference solution (a) Dissolve 10.0 mg of Actaea racemosa
for assay CRS (containing monoarnmonium glycyrrhizate) in m
methanol R with the aid of ultrasound and dilute to 10.0 mL
with the same solvento A logarithm to base 10 of the concentration of each peak
Reference solution (b) Dilute 5.0 mL of reference solution (a) in the chromatogram obtained with the test solution,
to 10.0 mL with methanol R. determined from the calibration curve;
IV-106 Black Currant
m = mass of the herbal drug to be examined used to
prepare the test solution, in grams.
Calculate the percentage content of triterpene glycosides by
taking the sum of the percentage contents of peaks 1 to 12.
____________________________________________ ~E~
Black Currant
Preparation
Black Currant Syrup
DEFINITION
Black Currant consists of the fresh ripe fruits of Ribes nigrum
L., together with their pedicels and rachides.
CHARACTERISTICS
Odour, strong and characteristic.
Macroseopieal Berries: globose, ranging in diameter from
about 7 ro 15 mm, occurring in pendulous racemes; epicarp
shiny black externally, enclosing a yellowish green translucent
pulp containing numerous fiattened ovoid seeds, about
2.5 mm long, 1.25 mm wide and 1 mm thick; berry crowned
with withered remains of five-cleft calyx; pedicels thin, up ro
about 10 mm long, attached to a rachis of variable length.
Mieroseopieal Epicarp: glands yellow, disc-shaped, roughly
circular or broadly ellipucal, varying in diameter from about
140 to 240 ¡.tm, each consisting of a single layer of cells
attached in the centre to the epicarp by means of a short,
multiseriate stalk. Calyx: trichomes unicellular, blunt-ended
with thin, crooked walls, about 10 to 14 ¡.tm wide and
averaging about 350 ¡.tm in length. Seed: testa with pigment
layer composed of small cells with horseshoe-shaped wall
thickenings as seen in cross section, each cell containing one
or rwo prismatic crystals of calcium oxalate; endosperm cells
with irregularly thickened walls.
Black Currant Syrup
DEFINITION
Black Currant Syrup is prepared either from the c1arified
juice of Black Currant or from concentrated black currant
juice of commerce. It contains a suitable antioxidant.
Permitted food grade colours may be added.
PRODUCTION
It is prepared by dissolving 700 g of Sucrose either in
560 mL of clarified juice, previously diluted with Water to a
weight per mL of 1.045 g, or in 560 mL of a solution of the
same weight per mL prepared from the concentrated juice of
commerce and Water, and adding ro this solution sufficient
Benzoic Acid ro give a final concentration of not more than
800 ppm, or sufficient Sodium Metabisulfite or other suitable
sulfite to give a final concentration of not more than
350 ppm of sulfur dioxide.
The syrup complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of ascorbic acid 1, C 6H s0 6
Not less than 0.055% w/w.
TESTS
Sulfur dioxide
Not more than 350 ppm, Appendix IX B.
2014
Weight per rnL
1.27 to 1.30 g, Appendix V G.
ASSAY
Mix 5 g with 25 mL of a freshly prepared 20% w/v solution
of metaphosphorie aeid, add 20 mL of aeewne and dilute to
100 mL with water. To four 3 mL quantities of this solution
add 0.4, 0.5, 0.6 and 0.7 mL, respectively, of double-strength
standard 2,6-diehlorophenolindophenol solution, mix well by
agitation with a fine stream of carbon dioxide, add 3 mL of
ehloroform, agitate for a further 15 seconds, examine the
soluuons against a white background and select the rwo that
are on either side of the end point (that is, one colourless
and one pink). Prepare a further six solutions as directed
aboye, but adding to the first an amount of dye solution
equal to that added to the selected colourless solution,
successively increasing this volume by 0.02 mL increments in
the second to the fifth solutions and adding to the sixth
solution a volume equal to that added to the selected pink
solution. Select the solution exhibiting the faintest pink
colour. Each mL of double-strength standard
2,6-diehlorophenolindophenol solution added to this solution is
equivalent to 0.200 mg of C 6H s 0 6 •
STORAGE
Black Currant Syrup should be kept in a well-filled container
and protected from Iight.
B1ack Currant Syrup contains, in 10 mL, about 7.5 mg of
ascorbic acid.
lThe requiremenl for Comem of ascorbic acid does nOI apply when Black
Curram Syrup is used as a fiavouring agem for pharmaceutical purposes.
Blackcurrant Leaf ***
*** ***
(Ph Eur monograph 2528) ***
~E~ ____________________________________________
DEFINITION
Dried leaf of Ribes nigrum L.
Content
Minimum 1.0 per cent of fiavonoids, expressed as
isoquercitroside (C21H20012; M r 464.4) (dried drug) .
IDENTIFICATION
A. The leaf is simple. The lamina may be up ro 10 cm long
and 12 cm wide and shows 3 (rarely 5) rounded triangular
lobes, dentate or crenate on the margins, with the median
lobe being the largest. The light-brown midrib and secondary
veins are very visible on the lower surface, and form a
characteristic nerwork through numerous anastomoses.
The rigid, light-brown petiole shows a very distinct gutter on
the upper part and its length is equal to half the length of the
lamina.
B. Microscopic examination (2.8.23). The powder is
brownish-green. Examine under a microscope using ehloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 2528.-1): curved, unicellular covering
trichomes, with moderately thickened, slightly verrucose walls
[D] ; orange-yellow, globular or ovoid glandular trichomes,
lacking a visible stalk, with a multicellular head up to 200 ¡.tm
in diameter, in surface view [A]; fragments of the lower
epidermis, in surface view [B], composed of cells with
irregularly thickened walls [Ba], numerous anomocytic
sto mata (2.8.3) [Bb] accompanied by spongy parenchyma
[Bc]; fragments, in surface view [C] or in transverse section
 2014 Blackcurran t Leaf IV-1 07

[E], of the upper epidennis [Ca, Ea], accompanied by Top of the plate
palisade parenchyma [Cb, Eb]; cluster crystals of ca1ciurn
--- ---
oxalate up to 30 ¡.1m in diameter, isolated [F] or included in
parenchymatous cells [Bd, Ce, Ec]. A green zone

Isoquercitroside: an orange zone An orange zone (mainly

isoquercitroside)
A Iight blue zo ne

- -- ---

Rutin: an orange·yellow zone An orange·yellow zone (rutin)

Referenee soIution Test soIution

Loss on drying (2.2.32)

Maximum 10.0 per cent, detennined on 1.000 g of rhe
powdered herbal drug (355) (2.9.12) by drying in an oven ar
105 oC for 2 h.
Total ash (2.4.16)
Maximum 12.0 per cent.
ASSAY
Liquid chromatography (2.2. 29).
Test solution Disperse 0.200 g of rhe powdered herbal drug
(355) (2.9.12) in 10 mL of an 80 per cent V/V solution of
methanol R. Heat under a reflux condenser in a water-bath at
60 oC for 30 mino Sonicate for 15 minoAllow to cool, dilute
to 20.0 mL with an 80 per cent V/V solurion of methanol R.
Filter through a membrane filter (nominal pore size
0.45 ¡.1m).
Reference solution (a) Dissolve 5.0 mg of isoquereitroside CRS
in an 80 per cent V/V solution of methanol R and dilute to
100.0 mL with the same solution.
Referenee solution (b) Dissolve 5.0 mg of rutin R in
Figure 2528.-1. - Illustration for identification test B of methanol R and dilure to 100.0 mL wirh the same solvento
powdered herbal drug of blackcurrant leaf Referenee solution (e) Dilute 10.0 mL of reference solution
(a) to 20.0 mL with reference solution (b).
C. Thin-layer chromatography (2.2.27). Referenee solution (d) Dilure 1.0 mL of reference solurion (a)
Test solution To 1 g of the powdered herbal drug (3 55) to 20.0 mL wirh an 80 per cent V/V solution of methanol R.
(2.9.12) add 10 mL of methanol R. Heat in a water-bath at Column:
60 oC for 10 min with occasional stirring. Allow to coo!. - size: 1 = 0.25 m, 0 = 4 mm;
Filter. - stationary phase: end-eapped oetadeeylsi/yl si/iea gel for
ehromatography R (5 ¡.1m);
Referenee solution Dissolve 5 mg of isoquercitroside R and
5 mg of rutin R in 10 mL of methanol R.
- temperature: 30 oc.
Mobile phase:
Plate TLC siliea gel plate R (5-40 ~lm) [or TLC si/iea gel
- mobile phase A: 0.05 per cent V/V solurion of trifiuoroaeetie
plate R (2-10 ¡.1m)].
acid R;
Mobile phase anhydrous formie aeid R, water R, ethyl aeetate R - mobile phase B: aeetonitrile R;
(10:10:80 V/V/V).
Time Mobile phase A Mobile phase B
Applieation 10 ¡.IL [or 5 ¡.IL] as bands of 10 mm [or 8 mm]. (min) (per een! VM (pereen! VM
Development Over a path of 10 cm [or 6 cm]. 0·45 97 ..... 60 3 ..... 40
Drying At 100-105 oC for 10 mino
Flow rate 1.0 mUmin.
Deteetion Treat with a 10 giL solution of diphenylborie aeid
Deteetion Spectrophotometer at 350 nm.
aminoethyl ester R in methanol R, then with a 50 gIL solution
of maerogol 400 R in methanol R; allow to dry in air for about Injeetion 10 ¡.IL.
30 min, then examine in ultraviolet light at 365 nm. Identifieation of peaks Use the chromatogram obtained with
R esults See below the sequence of zones present in the reference solution (a) to identify the peak due to
chromatograms obtained with the reference solution and the isoquercitroside and the chromatogram obtained with
test solution. Furthermore, other fluorescent zones may be reference solution (b) to identify the peak due to rutin.
present in the chromatogram obtained wirh the test solution. Retention time Rutin = abour 28 min;
TESTS isoquercitroside = abour 29 mino
Foreign matter (2.8.2) System suitability Reference solurion (e):
Maximurn 3 per cent. - resolution: minimum 3.0 between the peaks due to rutin
and isoquercitroside.
IV-108 Black Horehound 2014

Disregard limit: the area of the principal peak in the

chromatogram obtained with reference solution (d).
Calculate the percentage content of total fiavonoids,
expressed as isoquercitroside, using the following expression:

sum of the areas of the peak due to rutin and all

peaks eluting after the peak due to rutin in the
chromatogram obtained with the test solution;
area of the peak due to isoquercitroside in the
chromatogram obtained with reference solution (a);
mI mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of isoquercitroside CRS used to prepare
reference solution (a), in grams;
p percentage content of isoquercitroside in
isoquercitroside CRS.
____________________________________________ ~Ew

Black Horehound ***

*** ***
(Ph Eur monograph 1858) ***
~Ew ____________________________________________

DEFINITION
Dried fiowering tops of Ballota nigra L.
Content
Minimum 1.5 per cent of total ortho-dihydroxycinnamic acid
derivatives, expressed as acteoside (C29H 3601 5; M r 625)
(dried drug).
IDENTIFICATION
A. The stems are conspicuously 4-angled, longitudinally
striated, dark green or reddish-brown and more or less
pubescent. The leaves are greyish-green, petiolate, the lamina
ovate or orbicular, 2-4 cm wide, the margin irregularly
crenate, and cuneate or cordate at the base; both surfaces are
covered with abundant whitish hairs; the venation is pinnate,
prominent on the lower surface, slightly depressed on the
upper. The fiowers are sessile or very shortly pedicellate, the
calyx is infundibuliform, densely pubescent, with 10
prominent ribs and 5 subequal, broadly ovate teeth;
the corolla, with a tube slightly shorter than the calyx tube, is
purple and bilabiate, the upper lip pubescent on the outer
surface and the lower lip with 3 lobes, the middle of which is
notched.
B. Microscopic examination (2.8.23). The powder is greyish-
green and slightly fiocculent . Examine under a microscope
using chloral hydrate solution R . The powder shows the
following diagnostic characters (Figure 1858.-1 ): numerous
long, uniseriate, multicellular covering trichomes consisting of
4 or more cells, thickened and swollen at the junctions, with
slightly lignified and pitted walls, free [C] or on an epidermis
in transverse section [Ea]; fewer glandular trichomes, usually
on epidermises, in transverse section [E, F, G]: sorne with a
unicellular or multicellular stalk and a globose, uni- or
bicellular h ead [Galo others with a unicellular stalk and a
multicellular head in surface view [Ac] or in transverse
section [Eb], others with a unicellular stalk and an 8-celled
head of lamiaceous type, in surface view [Ad] or in transverse
section [Fa]; fragments of the adaxialleaf epidermis [B] with
cells with sinuous walls, accompanied by cells of the palisade
Figure 1858.-1 . - Illustration for identification test B of
powdered herbal drug of black horehound
parenchyma, most containing fine, needle-shaped crystals
[Ea]; fragments of the abaxialleaf epidermis [A] bearing
numerous sto mata, the majority anomocytic ( 2.8.3) [Aa] but
sorne diacytic [Ab] ; fragments of the epidermis of the corolla
composed of polygonal cells, th ose of the ¡nner epidermis of
the lips papillose [H] and those of the inner epidermis of the
tube bearing uni- or bicellular covering trichomes in a stellate
arrangement [K] ; pollen grains subspherical with 3 pores and
a smooth exine [D]; fragments from the stem (G) with
groups of collenchymatous cells [Gb] and lignified ves seis,
with annular or spiral thickenings m.
C. Thin-layer chromatography (2.2.27).
Test solution T o 2 g of the p owdered herbal drug (35 5)
(2.9.12) add 100 mL of methanol R. Heat on a water-bath
under a refiux condenser for 30 mino Allow to cool. Filter.
Evaporate the filtrate under reduced pressure until a volume
of about 10 mL is obtained.
R eference solution Dissolve 1 mg of chlorogenic acid R and
2.5 mg of rutin R in 10 mL of methanol R.
Plate TLC silica gel plate R .
Mobile phase anhydrous fonnic acid R, glacial acetic acid R,
water R, ethyl acetate R (7 .5:7.5:18:67 VIVIV/V).
Application 20 ~lL as bands.
Development Over a path of 15 cm.
Drying In air.
Detection spray with a solution containing 10 gIL of
diphenylbonc acid aminoethyl ester R and 5 O gIL of macrogol
400 R in methanol R ; allow to dry in a current of warm air;
examine in ultraviolet light at 365 nm after 30 mino
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
 2014 Bogbean Leaf IV- 109

test solution. Furthermore, other tluorescent zones may be

Bogbean Leaf ***
present in the chromatogram obtained with the test solution. *** ***
(Ph Eur monograph 1605) ***
~E~ ____________________________________________
Top of the plate

A reddish fluorescent zone DEFINITION

Dried, entire or fragmen ted leaf of M enyanthes mloliara L.
A faint yellow fluorescent zone
CHARACTERS
A light blue fluorescent zane
(caffeoylmalic acid) Very bitter and persistant taste.
A greenish·blue f1uorescent zane IDENTIFICATION
(acteoside)
A. The leaf is long-petiolated, trifoliate, with long sheaths
A yellawish·brown f1uorescent
zone (Iuteolin 7·lactate)
from the basej the petiole is up to 5 mm in diameter and
Chlarogenic acid: a Iight blue
strongly striated 10ngitudinalIy. The lamina is divided into
f1uorescent zone equal leatlets, sessile, obovate up to 10 cm long and up to
A greenish-blue fluorescent zane 5 cm wide, with an entire, occasionalIy sinuous margin with
(forsythoside B) brownish or reddish hydathodes and a spathulate basej it is
Rutin: an orange·yellow 2 greenish-blue f1uorescent zones glabrous, dark green on the upper surface and paler green on
f1uorescent zone (arenariaside) the lower surface, with a wide, whitish, finely striated
A yellow f1uorescent zone (Iuteolin prominent midrib.
7·lactate glucoside).
A faint greenish·blue f1uorescent
B. Reduce to a powder (355) (2.9.12). The powder is
zone (ballotetroside). yellowish-green. Examine under a microscope using chloral
Reference solution Test solution hydrate solution R. The powder shows fragments of upper
epidermis with polyhedral cells and thin wavy wallsj
fragments of lower epidermis with sinuous wallsj anomocytic
TESTS stomata (2.8.3), on both surfaces, with the subsidiary cells
Loss on drying (2.2.32) showing radiating striationsj epidermal celIs from the veins
Maximum 12.0 per cent, determined on 1.000 g of the straight walIed and papilIosej fragments of mesophylI
powdered herbal drug (355) (2.9.12) by drying in an oven at parenchyma with large intercellular spaces (aerenchyma)j
105 oC for 2 h. irregular celIs with rare scJereidsj fragments of spiral or
a=ular vessels.
Total ash (2.4.16)
Maximum 13.0 per cent. C. Thin-Iayer chromatography (2.2.27) .
Test solution To 1.0 g of the powdered drug (355) (2.9.12)
ASSAY
add 10 mL of methanol R. Heat, with stirring, in a water-bath
Stock solution Place 1.000 g of the powdered drug (3 55)
at 60 oC for 5 min. AlIow to cool and filter. Evaporate to
(2.9. 12) in a tlask. Add 90 mL of ethanol (50 per cent V/V) R.
dryness under reduced pressure in a water-bath at 60 oC.
Heat under a retlux condenser on a water-bath for 30 min.
Dissolve the residue in 2.0 mL of methanol R.
Allow to cool and filter, collecting the filtrate in a 100 mL
volumetric tlask. Rinse the tlask and the filter with 10 mL of Reference solution Dissolve 5 mg of loganin R in 15 mL of
ethanol (50 per cent V/V) R. Add the rinsings to the filtrate methanol R .
and dilute to 100.0 mL with ethanol (50 per cent V/V) R. Plate TLC silica gel plate R.
Test solution Into a 10 mL volumetric tlask, introduce Mobile phase water R, methanol R, ethyl acetare R
successively, with shaking after each addition, 1.0 mL of the (8:15:77 V/V/V).
stock solution, 2 mL of 0.5 M hydrochloric acid, 2 mL of a Application 30 ¡.¡L, as bands.
solution containing 100 gIL of sodium nitrite R and 100 gIL of Development Over a path of 15 cm.
sodium molybdate R, and 2 mL of dilute sodium hydroxide
Drying In air.
solution R, and dilute to 10.0 mL with water R .
Detection Spray with vanillin reagent R. H eat in an oven at
Compensation liquid Into a 10 mL volumetric tlask,
100-105 oC for 10 mino Examine in daylight.
introduce 1.0 mL of the stock solution, 2 mL of 0.5 M
hydrochlO11c acid and 2 mL of dilute sodium hydroxide R esults See below the sequence of the zones present in the
solution R, and dilute to 10.0 mL with water R. chromatograms obtained with the reference and test
solutions. Furthermore, other zones are present in the
Measure immediateJy the absorbance (2.2.25) of the test
chromatogram obtained with the test solution.
solution at 525 nm, by comparison with the compensation
liquido
Calculate the percentage content of total ortho- Top of the plate
dihydroxycinnamic acid derivatives, expressed as acteoside, A violet zone
using the folIowing expression:
An intense blue zone

A x 1000 Loganine: a greyish·violet zone A violet to greyish·violet zone

185 x m A grey to greyish·blue zone

A brownish zone
i.e. taking the specific absorbance of acteoside to be 185.
A absorbance at 525 nmj Reference solution Test solution
m = mas s of the substance to be examined, in grams.
____________________________________________ PhE~
IV-110 Boldo Leaf 2014

TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g of the
powdered drug (355) (2. 9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
Bitterness value (2.8.15)
Minimum 3000.
____________________________________________ ~Ew

Boldo Leaf ***

*** ***
(Ph Bur monograph 1396) ***
Preparation
Boldo Leaf Dry Extract
~ Ew _____________________________________________

DEFINITION
Whole or fragmented dried leaf of Peumus boldus Molina.
Content
Minimum 0.1 per cent of total aIkaloids, expressed as
boldine (C1 9H 21N04; M r 327.4) (anhydrous drug).
CHARACTERS
Characteristic odour, especially when rubbed.
A. Fragment of the lamina, in G. Fragment of the lamina, in
IDENTIFICATION surface view, showing the upper transverse section, showing
epidermis (Aa), hypodermis with the upper epidermis (Ga),
A. The leaf is oval or elliptical usually 5 cm long with a short thickened and beaded walls (Ab), hypodermis (Gb), palisade
petiole, an obtuse or slightly emarginate or mucronate apex and palisade parenchyma (Ac) parenchyma (Gc) and spongy
and an equal and rounded base; the margin is entire and parenchyma (Gd) containing oil
cells (Ge)
slightly undulate and the thickened edges are more or less
B and C. Lower epidermis with H. Spongy parenchyma containing
revolute. The lamina is greyish-green, thick, tough and stomata surrounded by 4·7 fine needle·shaped crystals and oil
brittle. The upper surface is rough with numerous prominenf subsidiary cells cells (Ha)
small protuberances and a depressed venation. The lower D. Unicellular covering trichome, J. Vascular tissue with fibres
solitary
surface is finely pubescent, with the protuberances less welJ-
E and F. Unicellular covering
marked, and a prominent, pinnate venation. trichomes, stellate c1ustered
B. Reduce to a powder (355) (2.9.12). The powder is Figure 1396.-1. - Illuslration of powdered herbal drug of boldo
greyish-green. Examine under a microscope using ehloral leaf (see Idenlification B)
hydrate solurion R. The powder shows fragments of the upper
epidermis and underlying hypodermis with straight or slightly Applieation 40 JlL [or 6 JlL] of the test solution and 20 J.LL
sinuous thickened and beaded walls, those of the lower [or 2 JlL] of the reference solution, as bands of 15 mm [or
epidermis with numerous stomata surrounded by 4-7 8 mm].
subsidiary cells; solitary, bifurcated or stellate cJustered Development Over a path of 15 cm [or 6 cm].
unicellular covering trichomes with more or les s thickened
Drying In airo
and lignified walls; fragments of the lamina showing a two-
layered palisade; debris of the spongy mesophyll incJuding Deteetion Spray with potassium iodobismuthate solution R2, dry
numerous large, rounded oil cells and parenchyma containing for 5 min in air and spray with sodium nitrite solution R;
fine needle-shaped crystals; thick-walled fibres and lignified, examine in daylight after 30 mino
pitted parenchymatous cells associated with vascular tissue Results See below the sequence of zones present in the
fram the veins. chromatograms obtained with the reference solution and the
C. Thin-layer chromatography (2.2.27). test solution.
Test solution Mix l.5 g of the powdered drug (355) (2.9.12) TESTS
and 5 mL of methanol R and sonÍcate for 10 min o Filter the Essential oil (2.8.12)
supernatant through a 3 cm x 0.5 cm column of eellulose for Maximum 40 mUkg (anhydrous drug).
ehromatography Rl. Use the first 1 mL ofthe eluate as the Use 10.0 g of the freshly fragmented drug, a 1000 mL flask
test solution. and 300 mL of water R as the distillation liquido Distil at a
Referenee solution Dissolve 2 mg of boldine R and 10 mg of rate of 2-3 mUmin for 3 h.
hyoscine hydrobromide R in 5 mL of methanol R . Foreign matter (2.8.2)
Plate TLC siliea gel plate R (5-40 Jlm) [or TLC silica gel Maxirnum 4 per cent of twigs and maximum 2 per cent of
plate R (2-10 Jlm)]. other foreign matter.
Mobile phase diethylamine R, methanol R, toluene R
(10:10:80 VIV/V).
 2014 Boldo Leaf Preparations IV-111

Top of the plate Calculate the pereentage eontent of total alkaloids expressed
as boldine using the following expression:
--- ---
A yellowish·brown zone
(LA!) x m2 x p
Hyoscine: a pale brown zone A 2 x mi x 100
Ayellow zone
m¡ mass of the drug to be examined, in grams
A brown zone
mz mass of boldíne CRS in the referenee solution, in
A brown zone grams
--- --- LA¡ sum of the areas of the peaks due to the 6 alkaloids
identified in the ehromatogram obtained with the
Boldine: a brown zone A brown zone (boldine) test solution
Several zones Az of the peak due to boldine in the ehromatogram
obtained with the referenee solution area;
Reference solution Test solution
p percentage eontent of boldine in boldine CRS
_ __ __ _ _ _ __ _ _ _ __ _ _ _ _ __ _ ~Ew

Water (2.2.13)
Maximum 100 mUkg, determined by distillation of 20.0 g of
the powdered drug (355) (2.9.12).
Total ash (2.4.16)
Maximum 13.0 per cent.
ASSAY
AIkaloids
liquid chromatography (2.2.29).
Test solution To 1.000 g of the powdered drug (355)
(2.9.12) add 50 mL of dilute hydroehlorie acid R. Shake in a
water-bath at 80 oC for 30 mino Filter, take up the residue
with 50 mL of dilute hydroehlorie acid R and shake in a water-
bath at 80 oC for 30 mino Filter and repeat the operation
once on the residue obtained. Filter. Combine the cooled
filtrates and shake with 100 mL of a mixture of equal
volumes of ethyl aeetate R and hexane R. Discard the organic
layer. Adjust the aqueous layer to pH 9.5 with dilute
ammonia R1 . Shake successively with 100 mL, 50 mL and
50 mL of methylene ehloride R. Combine the lower layers and
evaporate to dryness under redueed pressure. Dissolve the
residue in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Referenee solution Dissolve 12 mg of boldine CRS in the
mobile phase and dilute to 100.0 mL with the mobile phase.
Dilute 1.0 mL of this solution to 10.0 mL with the mobile
phase.
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: oetadecylsilyl siliea gel for ehromalOgraphy R
(5 ~m).
Solution A Mix 0.2 mL of diethylamine R and 99.8 mL of
aeelOnitrile R .
Solution B Mix 0.2 mL of diethylamine R and 99 .8 mL of
water R and adjust to pH 3 with anhydrous formie acid R.
Mobile phase Solution A, solution B (16:84 V/V) .
Flow rate 1.5 mUmin.
Deteetion Speetrophotometer at 304 nm.
Injeetion 20 ~L.
Relative retention With reference to boldine
(retention time = about 6 min): isoboldine = about 0.9;
isoeorydine N-oxide = about 1.8; laurotetanine = about 2.2;
isocorydine = about 2.8; N-methyIlaurotetanine = about 3.2.
Additional peaks may be presento
System suitability Test solution:
- resolution: minimum 1 between the peaks due to
isoboldine and boldine.
Boldo Leaf Dry Extraet *****
** **
(Ph Eur monograph 1816) ***
~Ew _ __ _ _ _ __ _ _ _ __ _ _ _ _ __ _ __
DEFINITION
Extraet produeed from Boldo leaf (1396).
Content:
- for aqueous extracts: minimum 0.5 per eent of total
alkaloids, expressed as boldine (C19H21N04; M r 327 .4)
(dried extraet);
- for hydroalcoholie extraets: minimum 1.0 per eent of total
alkaloids, expressed as boldine (C19H21N04; M r 327.4)
(dried extraet).
PRODUCTION
The extraet is produced from the herbal drug by a suitable
proeedure using either hot water at not less than 65 oC or a
hydroalcoholie solvent equivalent in strength to ethanol
(45-75 per eent V/V).
CHARACTERS
Appearance
Brown or greenish-brown, hygroscopie powder.
IDENTIFICATION
Thin-layer ehromatography (2.2.27).
Test solution To 0.5 g of the extract to be examined add
1 mL of hydroehloric acid R and 20 mL of water R. Sonieate
for 10 min o Transfer the liquid to a separating funnel and
make alkaline with 2 mL of dilute ammonia R1. Shake with
2 quantities, eaeh of 20 mL, of methylene ehloride R.
Evaporate the eombined organie layers to dryness. Dissolve
the residue in 1 mL of methanol R.
Reference solution Dissolve 2 mg of boldine R and 10 mg of
hyoscine hydrobromide R in 5 mL of methanol R.
Plate TLC siliea gel plate R (5-40 ~m) [or TLC silica gel
plate R (2-10 ~m) ] .
Mobile phase diethylamine R, methanol R, lOluene R
( 10 :10:80 V/V/V).
Application 20 ~L [or 3 ~), as bands of 15 mm [or 8 mm) .
Development Over a path of 15 em [or 6 em).
Drying In air.
Deteetion Spray with potassium iodobismuthate solution R2,
allow to dry in air for 5 min and spray with sodium nitrite
solutíon R; examine in daylight after 30 mino
 IV-112 Buckwheat Herb
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
---
A yellowish·brown zone
An orange·yellow zone
Hyoscine: a pale brown zone
An orange zone
An orange zone
---
Boldine: a brown zone A brown zone (boldine)
Several orange zones
Reference solution Test solution
ASSAY
Liquid chromatography (2.2.29).
Test solution To 1.000 g of the extract to be examined add
50 mL of dilute hydrochloric acid R and sonicate for 10 mino
Transfer to a separating funnel and wash with 10 mL of a
mixture of equal volumes of ethyl acetate R and hexane R.
Adjust the aqueous phase to pH 9.5 with dilute ammonia R1.
After cooling, shake successively with 100 mL, 50 mL, and a
further 50 mL of methylene chloride R, taking care not to form
an emulsiono Evaporate the combined lower layers to dryness
under reduced pressure. Dissolve the residue in the mobile
phase and transfer the solution to a volumetric flask. Rinse
and dilute to 10.0 mL with the mobile phase.
Reference solution Dissolve 12.0 mg of boldine CRS in the
mobile phase and dilute to 100.0 mL with the mobile phase.
Column:
-- size: 1 = 0.25 m, 0 = 4 .6 mm;
-- stau'onary phase: octadecylsi1yl silica gel for chromatography R
(5 ¡.¡m).
Solution A Mix 0.2 mL of diethylamine R with 99.8 mL of
acetonitrile R .
Solution B Mix 0.2 mL of diethylamine R with 99.8 mL of
water R and adjust to pH 3 with anhydrous formic acid R.
Mobile phase Solution A, solution B (16:84 V/V) .
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 304 nm.
Inject:ion 20 ¡.¡L.
Relative retention With reference to boldine
(retention time = about 6 min): isoboldine = about 0.9;
isocorydine N-oxide = about 1.8; laurotetanine = about 2.2;
isocorydine = about 2.8; N-methyIlaurotetanine = about 3.2.
Additional peaks may be presento
System suitability Test solution:
-- resolution: minimum 1.0 between the peaks due to
isoboldine and boldine.
Calculate the percentage content of total alkaloids, expressed
as boldine, using the foIlowing expression:
(¿Al) x m2 x p
A 2 x mI x 10
2014
sum of the areas of the peaks due to the 6 alkaloids
identified in the chromatogram obtained with the
test solution
area of the peak due to boldine in the
chromatogram obtained with the reference solution
m¡ mass of the extract to be examined used to prepare
the test solution, in grams
--- mas s of boldine CRS used to prepare the reference
solution, in grams
p percentage content of boldine in boldine CRS.
______ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ ~E~
Buckwheat Herb ***
*** ***
---
(Ph Eur monograph 2184) ***
PhEuf _ _ _ _ __ __ _ _ _ _ _ __ __ _ _ _ __
DEFINITION
Whole or fragmented aerial parts of Fagopyrum esculentum
Moench, coIlected in the early flowering period prior to
fruiting and dried immediately.
Content
Minimum 3.0 per cent ofrutin (C 2 7 H 3 0 0¡ 6; M r 611) (dried
drug) .
IDENTIFICAnON
A. The stem is cylindrical, hollow, finely ridged
longitudinally, about 2-6 mm in diameter, brownish-green or
reddish, with few branches and thickened at the internodes;
the leaves are arranged spirally and have membranous,
sheathing stipules; the surface is glabrous except in the region
of the stipu1es, where short, white hairs may occur.
The leaves are dark green, paler on the lower surface, up to
7 cm wide and 11 cm long, saggitate or corda te, almost
pentagonal with 2 widely rounded lobes; the lower lea ves are
petiolate, the upper leaves sessile or amplexicaul; the lamina
is glabrous and the margin finely sinuate and fringed with
minute, reddish-brown projections; similar projections occur
on the veins on the upper surface. The inflorescence is a
cymose panicle, the individual flowers 1-2 mm long and
6 mm in diameter with 5 free, white or reddish petals.
B. Microscopic examination (2.8.23) . The powder is dark
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 2184.-1) : fragments ofthe epidermis of
the stem, in surface view [D] , composed of elongated ceIls
showing striations on the outer walls [Da] and anomocytic
sto mata (2.8.3) [Db]; fragments ofthe upper epidermis of
the lamina, in surface view [B], consisting of polygonal ceIls
covered by a striated cuticle [Ba] and anomocytic stomata
[Bb], often accompanied by palisade parenchyma [Be];
fragments of the epidermis of the leaf margins [A] and of the
epidermis covering the veins, often showing ovoid or rounded
papilla-like projections, often reddish, with thickened and
striated walls; fragments of the lower epidermis of the lamina
[C] with thin-walled polygonal ceIls, numerous stomata [Ca]
and rare glandular trichomes with a biseriate stalk and a
globular head usuaIly composed of 8 cells [Cb] ; fragments of
mesophyll [F] with narrow, annular or spiral vessels [Fa] and
of spongy parenchyma, numerous ceIls of which contain
cluster crystals of calcium oxalate, varying in di ame ter
(25-100 ¡.¡m) [Fb], smaller prismatic crystals of calcium
oxalate [Fe], occurring scattered in the mesophyll and also in
the parenchyma of the stem; fragments of lignified tissue [H]
 20 14 Buckwheat Herb IV - 113

with bordered-pitted [Ha], reticulate or annular [Hb] vessels Top of the plate
and thin-walled, pitted fibres [He]; occasional fragments of
2 red zones
the corolla with a papillose epidennis [E] ; spherical or ovoid
pollen grains, about 50 ~lm in diameter, with a pitted exine 1-2 light blue zones
and 3 furrows [G]. - -- ---

An orange zone

An orange zone

Hyperoside : an orange zone 2 blue zones

--- ---
Rutin: an orange-yellow zone An orange-yellow zone (rutin)

Reference solution Test solution

TESTS
Loss 00 drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximurn 15.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution To 0.500 g of the powdered herbal drug (355)
(2.9.12), add 30 mL of an 80 per cent VIV solution of
methanol R. Heat the mixture under a reflux condenser in a
water-bath at 60 oC for 30 min, then extract the mixture in
an ultrasonic bath for 15 mino Allow to cool, dilute to
50.0 mL with an 80 per cent VIV solution of methanol R and
filter.
Reference so/ution (a) Dissolve 25.0 mg of rutoside
trihydrate CRS in an 80 per cent V/V solution of methanol R
and dilute to 50.0 mL with the same solvento
Figure 2184.-l. - Illustration for identification test B of Reference solution (b) Dissolve 20.0 mg of troxerutin R and
powdered herbal drug of buckwheat herb 5.0 mg of quercitrin R in an 80 per cent VIV solution of
methanol R and dilute to 50.0 mL with the same solvento
C . Thin-layer chromatography (2.2.27). Colwnn:
Test solution To 0.5 g of the powdered herbal drug (355) - size: l = 0.125 m, 0 = 4 mm;
(2.9.12) add 5.0 mL of 111ethanol R and heat in a water-bath - stationary phase: octadecylsilyl silica gel fol' chromatography R
at 60 oC under a reflux condenser for 10 mino Cool and (5 ¡.tm);
filter. - temperature: 30 oc.
Reference solution Dissolve 10 mg of hyperoside R and 10 mg Mobile phase:
of rutin R in 10 mL of methanol R. - mobile phase A : mix 50 volumes of acetonitrile R and
Plate TLC silica gel plate R (5-40 ~lm) [or TLC si/iea gel 950 volumes of water R adjusted to pH 2 with phosphoric
plate R (2-10 ¡.tm)]. acid R;
- mobile phase B: mix 95 volumes of water R adjusted to
Mobile phase anhydrous fonnic acid R, water R, ethy/ acetate R pH 2 with phosphon'c acid R and 905 volumes of
(10:10:80 VIV/V).
acetonitrile R;
Application 20 ¡.tL [or 5 ¡.tL] as bands of 15 mm [or 8 mm].
Time Mobile phase A Mobile phase B
Development Over a path of 10 cm [or 6 cm]. (min) (percent VM (percent VM
Dlying At 100-105 oc. 0 ·6 94 6
Detect¡on Treat with a 10 giL solution of diphenylboric acid 6·16.5 94485 6415
aminoethyl ester R in methanol R, subsequently treat with a
16.5 - 22 85476 15 424
50 gIL solution of macrogol 400 R in methanol R; allow to dry
in air for about 30 min and examine in ultraviolet light at 22·25 76459 24441
365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the Plow rate 1.0 mUmin.
test solution. Furthermore, other f1uorescent zones may be Detection Spectrophotometer at 350 nm.
present in the chromatogram obtained with the test solution. Injection 10 ¡.tL.
System suitability Reference solution (b):
IV-114 Greater Burnet Root 2014

-- elution order: order indicated in the composition of
reference solution (b), when the chromatogram is
recorded in the prescribed conditions;
-- resolution: minimum 3 between the peaks due to
troxerutin and quercitrin.
U sing the retention times determined from the
chromatogram obtained with reference solution (a), locate
the peak due to rutin in the chromatogram obtained with the
test solution.
Calculate the percentage content of rutin using the following
expression:
area of the peak due to rutin in the chromatogram
obtained with the test solution;
area of the peak due to rutin in the chromatogram
obtained with reference solution (a);
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of rutoside trihydrate CRS used to prepare
reference solution (a), in grams;
p percentage content of rutin in rutoside
trihydrate CRS.
____________________________________________
Sorne starch granules are found in the parenchyma cells or in
cells of the medullary rays.
C. Thin-layer chromatography (2.2.27).
Test solution To 2.0 g of the powdered drug (355) (2.9.12)
add 50 mL of water R and boil under a refiux condenser for
30 min. Cool the solution and centrifuge for 10 mino Shake
the supernatant with 2 quantities, each of 15 mL, of
di-isopropyl ether R saturated with hydroehlorie acid R . Combine
the ether layers. Evaporate to dryness and dissolve the
residue in 1.0 mL of methanol R. Filter through a
polypropylene syringe filter (nominal pore size 0.45 ¡.tm) .
Rejerenee solution Dissolve 5 mg of gallie acid R and 20 mg
of resorcinol R in 20 mL of methanol R.
Plate TLC siliea gel F 254 plate R (5-40 ¡.tm) [or TLC siliea gel
F254 plate R (2-10 ¡.tm)).
Mobile phase anhydrous jormie acid R, ethyl aeetate R,
toluene R (10:30:60 V/V/V).
Applieation 10 ¡.tL [or 4 ¡.tL) as bands.
Development Over a path of 10 cm [or 6 cm).
Drying In airo
Deteetion A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of quenching zones
present in the chromatograms obtained with the reference
solution and the test solution. Furthermore, other faint
Ph E~
quenching zones may be present in the chromatogram
obtained with the test solution.

Top of the plate

Greater Burnet Root *** A quenehing zone

*** *** --- ---
(Sanguisorba Root, Ph Eur monograph 2385) ***
~E~ ____________________________________________ Resorcinol: a quenehing zone

DEFINITION A quenehing zone

Whole or fragmented, dried underground parts of --- ---
Sanguisorba officinalis L. without rootlets.
Gallie acid: a quenehing zone A quenehing zone (gallie acid)
Content
Minimum 5.0 per cent of tannins, expressed as pyrogallol A quenehing zone
(C 6 H 6 0 3 ; M r 126.1) (dried drug). A quenehing zone
CHARACTERS Reference solution Test solution
The adventitious roots are about 5-25 cm long and up to
2 cm in diameter.
Deteetion B Spray with a 10 gIL solution of jerrie ehloride R
IDENTIFICATION in anhydrous ethanol R and heat at 100-105 oC for 15 min;
A. The whole drug consists of the rhizome, often ramified, examine in daylight.
thick, short, fusiform or cylindrical and the adventitious roots
Results B See below the sequence of the zones present in
whose surface is reddish-brown or blackish-brown, with
the chromatograms obtained with the reference solution and
longitudinal striations, sometimes with transverse fissures,
the test solution. Furthermore, other faint zones may be
and showing rootlet scars.
present in the chromatogram obtained with the test solution.
It may also be found as more or les s cylindrical fragments up
to 2 cm long or elliptical or irregular discs. The fracture is
light-coloured and very fibrous. Top of the plate
B. Reduce to a powder (355) (2.9.12). The powder is light --- -----
yellowish-brown. Examine under a microscope using ehloral
Resorcinol: a brown zone
hydrate solution R. The powder shows the following diagnostic
characters: numerous, whole or fragmented phloem fibres, A blaekish·blue zone
usually isolated, narrow, sometimes more than 500 ¡.tm long
and often rough-walled; calcium oxalate cluster crystals, free
--- -----

or inside parenchyma cells; a few reticulate lignified vessels; Gallic acid: a blaekish-blue zone A blaekish-blue zone (gallie acid)
rare cork fragments. Examine under a microscope using a A blaekish-blue zone
50 per cent V/V solution of glyeerol R. The powder shows
rounded or ovoid starch granules, single or in groups of 2-4; Reference solution Test solution
the diameter of a component granule may reach 30 ¡.tm.
 2014 Butcher's Broom IV-lIS

TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.16)
Maximum 10.0 per cent.
Ash insoluble in hydrochloric acid (2.8. 1) A
Maximum 2.0 per cent.
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14). Use 0.500 g ofthe powdered drug (180) (2.9.12).
____________________________________________ ~Em

Butcher's Broom ***

*** ***
***
(J:.
(Ph Eur monograph 1847)
~Em ____________________________________________
:: L
...
DEFINITION

M\~/
Dried, whole or fragmented underground parts of Ruseus
aeuleatus L.
Content 100~m
Minimum 1.0 per cent of total sapogenins, expressed as
ruscogenins [mixture of neoruscogenin (C27H4004; M r
428.6) and ruscogenin (Cz7H4Z04; M r 430.6)] (dried drug). Figure 1847.-1.- Illustration for identification test B of
IDENTIFICATION powdered herbal drug ofbutcher's broom
A. The rhizome consists of yellowish, branched, articulated,
somewhat knotty pieces, cylindrical or subconical, about
5-10 cm long and about 5 mm thick. The surface is marked evaporate to dryness. Dissolve the residue in 5 mL of
with thin annulations about 1-3 mm wide, separated from methanol R.
one another; rounded scars of the aerial stems are present on Referenee solution Dissolve 1 mg of ruseogenins CRS and
the upper surface. On the lower surface numerous roots, or 1 mg of stigmasterol R in merhanol R and dilute to 5 mL with
their scars, OCCUf; the roots are about 2 mm in diameter and the same solvento
similar in colour to the rhizome. The outer layer is easily Plate TLC siliea gel plate R (5-40 ~lm) [or TLC siliea gel
detached, revealing a yellowish-white, very hard central plate R (2-10 ~m) ].
cylinder.
Mobile phase methanol R, methylene ehloride R (7:93 V /V).
B. Reduce to a powder (355) (2.9.12). The powder is
Applieation 1O ~L [or 4 ~Ll as bands.
yellowish. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic Development Over a path of 15 cm [or 6 cm].
characters (Figure 1847. -1): groups of sclereids of the Drying In air.
rhizome, with variously-shaped cells, rounded, elongated or Deteetion Spray with vanillin reagent R , dry in an oven at
rectangular; the walls are moderately thickened and distinctly 100-105 oC for 1 min and examine in daylight.
beaded, with large, rounded or oval pits [F, G, L, P, Q] ; Results See below the sequence of zones present in the
fragments of the endodermis composed of a single layer of chromatograms obtained with the reference solution and the
irregularly thickened cells [K] ; groups of rounded test solution. Furthermore, other weak zones may be present
parenchymatous cells, thickened at the corners, with small, in the chromatogram obtained with the test solution.
triangular intercellular spaces [D, E, N]; thin-walled
parenchyma m with sorne cells containing raphides of
calcium oxalate [C]; groups [H] of thick-walled tibres [Ha] Top of the plate
and small ves seis, up to about 50 ~m in diameter, the walls Several zones of various colours
showing numerous small, slit-shaped pits [A, Hb]; rare
fragments of dermal tissue of the root [B] ; raphides of Stigmasterol : a violet zone A violet zone
calcium oxalate, isolated [M]. --- ---
C. Thin-layer chromatography (2.2.27).
A violet zone
Test solution Introduce 1.0 g of the powdered drug (355)
Ruscogenins : a yellow zone A yellow zone (ruscogenins)
(2.9.12) and 50 mL of dilute hydroehlorie acid R into a
100 mL ftask with a ground-glass neck. Heat on a water-bath Several zones of various colours
under a reftux condenser for 40 mino Allow to cool and
--- ---
extract the unfiltered mixture with 3 quantities, each of
25 mL, of methylene ehloride R. Combine the organic Reference solution Test solution
solutions and dry over anhydrous sodium sulfate R . Filter and
 IV-116 Calendula Flower 2014

TESTS
Foreign matter (2.8.2)
Maximum 5 per cent.
Loss on drying (2.2.32) area of the peak due to ruscogenin in the
Maximum 12.0 per cent, determined on 1.000 g of the chromatogram obtained with the test solution;
powdered drug (355) (2.9.12) by drying in an oven at area of the peak due to ruscogenin in the
105 oC for 2 h. chromatogram obtained with the reference solution;
Total ash (2.4.16) are a of the peak due to neoruscogenin in the
Maximum 12.0 per cent. chromatogram obtained with the test solution;
area of the peak due to neoruscogenin in the
Ash insoluble in hydrochloric acid (2.8.1) chromatogram obtained with the reference solution;
Maximum 5.0 per cent. mas s of the herbal drug to be examined used to
ASSAY prepare the test solution, in grams;
Liquid chromatography (2.2.29). mas s of ruscogenins CRS used to prepare the reference
Test solution To 2.000 g of the powdered drug (355) solution, in grams;
(2.9.12) add 60 mL of anhydrous ethanol R, 15 mL of water R PI percentage content of ruscogenin in ruscogenins CRS;
and 0.2 g of potassium hydroxide R. Extract on a water-bath P2 percentage content of neoruscogenin in
under a reflux condenser for 4 h. AlIow to cool and filter into ruscogenins CRS.
a 100 mL volumetric flask. Rinse the extraction flask and the _ _ _ __ _ __ _ __ _ __ __ __ _ __ _ Ph Eur
residue in the filter with 3 quantities, each of 10 mL, of
anhydrous elhanol R and add the rinsings to the volumetric
flask. Dilute to 100.0 mL with anhydrous ethanol R. Introduce
25.0 mL of this solution into a round-bottomed flask fitted
to a rotary evaporator and evaporate to dryness. Dissolve the Calendula Flower
residue in 10 mL of butanol R and add 3 mL of hydrochloric
(Ph Bur monograph 1297)
acid R1 and 8 mL of water R. Heat on a water-bath under a
~ Ew ____ _ __ _ _ _____ __ _ __ _ _ _ __
reflux condenser for 1 h . AlIow to cool and transfer to a
100 mL volumetric flask. Rinse the round-bottomed flask DEFINITION
with 3 quantities, each of 20 mL, of methanol R. Add the Whole or cut, dried, and fully opened flowers that have been
rinsings to the volumetric flask and dilute to 100.0 mL with detached from the receptacle of the cultivated, double-
methanol R . flowered varieties of Calendula officinalis L.
Reference solution Dissolve 5.0 mg of ruscogenins CRS in Content
100 mL of methanol R. Minimum 0.4 per cent of flavonoids, expressed as hyperoside
Column: (C2IH20012; M r 464.4) (dried drug).
-- size: l = 0.25 m, 0 = 4.6 mm;
-- stationary phase: octadecylsilyl silica gel for chromatography R IDENTIFICATION
(5 ¡.tm) . A. The ligulate florets consist of a yellow or orange-yellow
ligule, about 3-5 mm wide and about 7 mm in the middle
Mobile phase:
part, with a 3-toothed apex and a hairy, partly sickle-shaped,
-- mobile phase A: water R;
yellowish-brown or orange-brown tube with a projecting style
mobile phase B: acetonitrile Rl;
and a bifid stigma occasionally with a partly bent yellowish-
Time Mobile phase A Mobile phase B brown or orange-brown ovary. The tubular florets, about
(min) (percent VM (percent VM 5 mm long, are present and consist of the yellow, orange-red
0·25 40 60 or reddish-violet 5-lobed corolla and the yellowish-brown or
25·27 40 -) O 60 -) 100 orange-brown tube, hairy in its lower part, mostly with a
partly bent yellowish-brown or orange-brown ovary.
27·37 O 100
B. Reduce to a powder (355) (2.9.12) . The powder is
Flow rate 1.2 mUmin. yellowish-brown. Examine under a microscope using chloral
Detection Spectrophotometer at 203 nm . hydrate solution R. The powder shows the following diagnostic
characters (Figure 1297.-1) : fragments of epidermis es of the
Injection 20 pL.
corolla [C, F, K] containing light yellow oil droplets, sorne
Identification of peaks Use the chromatogram supplied with with fairly large anomocytic stomata (2.8.3) [Fa, Ka];
ruscogenins CRS and the chromatogram obtained with the covering trichomes biseriate, multicellular and conical [G],
reference solution to identify the peaks due to neoruscogenin usually fragmented, and glandular trichomes with a
and ruscogenin. multicellular stalk [E], very abundant on the base of the
Relative retention With reference to neoruscogenin corolla [D]; fragments of parenchyma of the corolla [B]
(retention time = about 16 min): ruscogenin = about 1.2. containing prisms and very small cluster crystals of calcium
Syslem suitabililY Reference solution: oxalate [Ba, Da] and small vessels [Bb]; spherical pollen
-- resolution: minimum 1.5 between the peaks due to grains up to about 40 pm in diameter with a sharply spiny
neoruscogenin and ruscogenin. exine and 3 germinal pores [A, TI; occasional fragments of
Calculate the percentage content of sapogenins, expressed as the stigmas with short, bulbous papillae [H].
ruscogenins (neoruscogenin and ruscogenin), using the
following expression:
2014
A B\~Ba
~
Q in the chromatogram obtained with the test solution.
~
Figure 1297.-1.-Illustration for identification test B of
powdered herbal drug of calen dula flower
e. Thin-Iayer chromatography (2.2.21) .
Test solution Mix 1.0 g ofthe powdered drug (500) (2. 9.12)
and 10 mL of methanol R and heat on a water-bath under a
refiux condenser for 10 mino Cool and filter.
Reference solution Dissolve 1.0 mg of caffeic acid R, 1.0 mg
of chlorogenic acid R and 2.5 mg of nttin R in 10 mL of
methanol R .
Plate TLC silica gel plate R .
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
(10 :10:80 V/V/V) .
Application 20 ~L of the test solution and 1O ~L of the
reference solution, as bands.
Development Over a path of 10 cm.
Drying At 100-105 ce.
D etection Spray the still-warm plate with a 10 giL solution
of dipheny lboric acid aminoethyl ester R in methanol R and then
spray with a 50 gIL solution of macrogol 400 R in methanol R;
allow to dry in air for 30 min and examine in ultraviolet light
at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower part a yellowish-brown
ftuorescent zone (rutin), in the middle part a light bluish
ftuorescent zone (chlorogenic acid) and in the upper part a
light bluish ftuorescent zone (caffeic acid).
The chromatogram obtained with the test solution shows a
yellowish-brown ftuorescent zone corresponding in position
to the zone due to rutin in the chromatogram obtained with
the reference solution, below and directly aboye it, it shows a
yellowish-green ftuorescent zone and a light bluish
ftuorescent zone corresponding to the zone due to
chlorogenic acid in the chromatogram obtained with the
reference solution, a yellowish-green ftuorescent zone aboye it
Calendula Flower IV -117
and a light bluish ftuorescent zone shortly below the zone
due to caffeic acid in the chromatogram obtained with the
reference solution. Furthermore, other zones may be present
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of bracts and maximum 2 per cent of
other foreign matter.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered drug (500) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
ASSAY
Stock solution Into a 100 mL round-bottomed ftask
introduce 0.800 g ofthe powdered drug (500) (2.9. 12),
1 mL of a 5 gIL solution of hexamethylenetetramine R, 7 mL
of hydrochloric acid R1 and 20 mL of acetone R . Boil the
mixture under a reftux condenser for 30 mino Filter the
liquid through a plug of absorbent cotton into a 100 mL
volumetric ftask. Add the absorbent cotton to the residue in
the round-bottomed ftask and extract with 2 quantities, each
of 20 mL, of acetone R, each time boiling under a reftux
condenser for 10 min oAllow to cool to room temperature,
filter the liquid through a plug of absorbent cotton, then filter
the combined acetone solution through a filter-paper into the
volumetric ftask, and dilute to 100.0 mL with acetone R by
rinsing the ftask and filter. Introduce 20. O mL of this solution
into a separating funnel, add 20 mL of water R and extract
the mixture with 1 quantity of 15 mL and then with
3 quantities, each of 10 mL, of ethyl acetate R . Combine the
ethyl acetate extracts in a separating funnel, rinse with
2 quantities, each of 50 mL, of water R, filter the extract over
10 g of anhydrous sodium sulfate R into a 50 mL volumetric
ftask and dilute to 50.0 mL with ethyl acerate R.
Test solution To 10.0 mL ofthe stock solution add 1 mL of
aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial aceuc acid R in methanol R.
Compensation liquid Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent V/V solution of glacial acetic
acid R in methanol R .
Measure the absorbance (2.2.25) of the test solution after
30 min, by comparison with the compensation liquid at
425 nm.
Calculate the percentage content of ftavonoids, expressed as
hyperoside, using the following expression:
A x 1.25
m
i.e. taking the specific absorbance of hyperoside to be 500 .
A absorbance at 425 nm
m = mass of the drug to be examined, in grams.
____________________________________________
PhE~
 IV-118 Capsicum 2014

Capsicum
(Ph Eur monograph 1859)
Preparations
Refined and Quantified Capsicum Oleoresin
Standardised Capsicum Tincture
~E~ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ __ __

DEFINITlON
Dried ripe fruits of Capsieum annuum L. var. minimum
(Miller) Heiser and small-fruited varieties of Capsieum
fruteseens L.
Content
Minimum 0.4 per cent of total capsaicinoids, expressed as
capsaicin (ClsHz7N03; M r 305.4) (dried drug).
CHARACTERS
Extremely pungent taste.
IDENTIFICATlON
A. The fruit is yellowish-orange or reddish-brown, oblong
conical with an obtuse apex, about 1-3 cm long and up ro
1 cm in diameter at the widest part, occasionally attached to
a 5-toothed inferior calyx and a straight peduncle. Pericarp
somewhat shrivelled, glabrous, enclosing about 10-20 fiat,
reniform seeds 3-4 mm long, either loose or attached to a
KQLa
25 ~m
reddish dissepiment.
B. Reduce to a powder (355) (2.9.12). The powder is Figure 1859.-1.- Illustration for identifícation test B of
orange. Examine under a microscope using ehloral hydrate powdered herbal drug of capsicum
solution R . The powder shows the following diagnostic
characters (Figure 1859.-1): fragments ofthe epicarp, in
surface view, with cells ofien arranged in rows of 5 ro 7 [E], Deteetion Spray with a 5 gIL solution of
thick-walled when close to the peduncle [B] and with a diehloroquinoneehlorimide R in methanol R, and expose ro
cuticle uniformly striated [A]; fragments of the pericarp, in ammonia vapour until blue zones appear; examine in
transverse section [D], showing the epicarp covered by a daylight.
thick cuticle [Da] and parenchymatous cells frequently
Results See below the sequence of zones present in the
containing droplets of red oil, occasionally containing
chromarograms obtained with the reference solution and the
microsphenoidal crystals of calcium oxalate [Db]; fragments
test solution. Furthermore, other zones may be present in the
of endocarp [C] with characteristic island groups of
chromatogram obtained with the test solution.
sclerenchymarous cells [Ca], the groups being separated by
thin-walled parenchymatous cells [Cb]; fragments of the
seeds having an episperm composed of large, greenish-yellow, Top of the plate
sinuous-walled sclereids with thin outer walls and strongly
and unevenly thickened radial and inner walls which are
--- ---

conspicuously pitted [G]; endosperm parenchymatous cells Capsaicin: a blue zone A blue zone (capsaicin)
with drops of oil and aleurone grains, 3-6 ¡.tm in diameter
Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin)
[H]; occasional fragments from the calyx having an outer
epidermis with anisocytic stomata (2.8.3) m,
an inner --- -----
epidermis with no stomata and many glandular trichomes Reference solution Test solution
with uniseriate stalks and multicellular heads [N], and a
mesophyll [L] with many idioblasts containing prisms of
calcium oxalate [La] or microsphenoidal crystals of calcium TESTS
oxalate [Lb]; prisms [K] or clusters [M] of calcium oxalate,
Nonivamide
isolated; annularly and spirally thickened vessels [F] .
Liquid chromatography (2.2.29).
C. Thin-Iayer chromatography (2.2.27).
Test solution To 2.5 g ofthe powdered drug (500) (2.9_12)
Test solution To 0.50 g of the powdered drug (500) (2.9.12) add 100 mL of methanol R . AlIow to macerate for 30 mino
add 5.0 mL of ether R, shake for 5 min and filter. Place in an ultrasonic bath for 15 mino Filter into a 100 mL
Referenee solution Dissolve 2 mg of eapsaicin R and 2 mg of volumetric fiask, rinse the fiask and filter with methanol R,
dihydroeapsaicin R in 5.0 mL of ether R . then dilute ro 100.0 mL with the same solvento
Plate TLC oetadeeylsilyl siliea gel plate R . Referenee solution Dissolve 20.0 mg of eapsaicin CRS and
Mobile phase water R , methanol R (20 :80 V/V). 4.0 mg of nonivamide CRS in methanol R and dilme to
Applieation20 ¡.tL as bands. 100.0 mL with the same solvento
Column:
Development Over a path of 12 cm.
-- size: 1 = 0.25 m, 0 = 4.6 mm;
Drying In air. -- stationary phase: phenylsilyl siliea gel for ehromatography R
(5 ¡.tm);
2014 Capsicum Oleoresin IV-119

- temperature: 30 uC.
Refined and Standardised ***
Mobile phase Mix 40 volumes of acetonitrile R and *** ***
60 volumes of a 1 gIL solution of phosphoric acid R. Capsicum Oleoresin ***
Flow rate 1.0 mIJmin. (Ph Bur monograph 2336)
___________________________________________
Detection Spectrophotometer at 225 nm. ~E~

1njection 1O ¡.lL. DEFINITION

Blution order Nordihydrocapsaicin, nonivamide, capsaicin,
dihydrocapsaicin.
System suitability Reference solution:
- resolution: minimum 1.5 between the peaks due to
noruvamide and capsaicin.
Calculate the percentage content of nonivamide using the
following expression:
FI area of the peak corresponding to nonivamide in the
chromatogram obtained with the test solution;
Fz area ofthe peak corresponding to nonivamide in the
chromatogram obtained with the reference solution;
mi mas s of the drug to be examined, in grams;
mz mass of nonivamide CRS used to prepare the
reference solution, in grams;
PI percentage content of nonivamide in nonivamide CRS.
Limit:
- nonivamide: maximum 5.0 per cent of the total
capsaicinoid contento
Foreign matter (2.8.2)
Fruits of C. annuum L. var. longum (Sendtn.) are absent.
Loss on drying (2.2.32)
Maximum 11.0 per cent, deterrnined on 1.000 g of the
powdered drug (500) (2.9. 12) by drying in an oven at
105 DC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
nonivamide.
Calculate the percentage content of total capsaicinoids,
expressed as capsaicin, using the following expression:
Refined and standardised oleoresin produced from Capsicum
(1859).
Content
12.0 per cent to 18.0 per cent m/m of total capsaicinoids,
expressed as capsaicin (C 1s H 27N0 3; M r 305.4) .
PRODUCTION
The oleoresin is produced from the herbal drug by an
appropriate procedure, using ethanol (minimum
90 per cent V/V).
CHARACTERS
Appearance
Red or brown mobile extracto
IDENTIFICATION
Thin-layer chromatography (2.2.27) .
Test solution Dissolve 50 mg of the oleoresin to be examined
in 5 mL of ether R.
Reference solution Dissolve 2 mg of capsaicin R and 2 mg of
dihydrocapsaicin R in 5 mL of ether R.
Plate TLC octadecylsilyl silica gel plate R (5-40 ¡.lm) [or
TLC octadecylsilyl silica gel plate R (2-10 ¡.lm) J.
Mobile phase water R, methanol R (20:80 V/V) .
Application 20 ~IL [or 2 ¡.lLJ as bands of 15 mm [or 8 mm] .
Development Over a path of 12 cm [or 6 cm] .
Drying In airo
Detection Treat with a 0.25 gIL solution of
dichloroquinonechlorimide R in ethyl acerate R, expose to
ammonia vapour until blue zones appear. Examine in
daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate

----- ---

(F3 + F5 + F6) x m4 x P2 Capsaicin: a blue zone A blue zone (capsaicin)

F 4 Xm3
Dihydrocapsaicin: a blue zone A fainl blue zone (dihydrocapsaicin)

----- -----
are a of the peak corresponding to capsaicin in the
chromatogram obtained with the test solution; Reference soIution Test soIution
area of the peak corresponding to capsaicin in the
chromatogram obtained with the reference solution;
area of the peak corresponding to dihydrocapsaicin in TESTS
the chromatogram obtained with the test solution; Nonivamide
area of the peak corresponding to Liquid chromatography (2.2.29).
nordihydrocapsaicin in the chromatogram obtained Test solution Dissolve 0.300 g of the oleoresin to be
with the test solution; examined in 60 mL of methanol R and dilute to 100.0 mL
mas s of the drug to be examined, in grams; with the same solvento
mass of capsaicin CRS used to prepare the reference Reference solution Dissolve 20.0 mg of capsaicin CRS and
solution, in grams; 4.0 mg of nonivamide CRS in 100.0 mL of methanol R.
pz percentage content of capsaicin in capsaicin CRS.
Column:
___________________________________________ Ph Eur - size: 1 = 0.25 m, 0 = 4.6 mm;
- stationary phase: phenylsilyl silica gel for chromatography R
(5 ¡.lm);
- temperature: 30 oC .
IV-120 Capsieum Soft Extraet, Standardised
Mobz1e phase acetonitrile R1, 1 gIL solution of phosphoric
acid R (40:60 V/V).
Flow rate 1.0 mUmin.
Detection Speetrophotometer at 225 nm .
Injection 10).lL.
Run time 1.2 times the retention time of dihydroeapsaiein.
Elution order Nordihydroeapsaicin, nonivamide, capsaiein,
dihydroeapsaiein.
R elative retention With reference to eapsaiein
(retention time = about 19 min):
n ordihydroeapsaicin = about 0.9; nonivamide = about 0.95;
dihydroeapsaicin = about 1.3.
System suitability Referenee solution:
- resolution: minimum 1.5 between the peaks due to
nonivamide and eapsaicin.
Calculate the pereentage eontent of nonivamide with
reference to the total eapsaieinoid eontent, using the
following expression:
Al x m2 x PI X 100
A 2 x mI x e
area of the peak due to nonivamide in the
ehromatogram obtained with the test solution;
area of the peak due to nonivarnide in the
ehromatogram obtained with the referenee solution;
mass of the oleoresin to be examined used to
prepare the test solution, in grams;
mass of nonivamide CRS used to prepare the
referenee solution, in grams;
PI pereentage eontent of nonivamide in
nonivamide CRS;
C pereentage eontent of total capsaieinoids, as
deterrnined in the assay.
Limit:
- nonivamide: maximum 5.0 per eent ofthe total
eapsaieinoid eontent.
Water (2.5.12)
Maximum 8.0 per eent, determined on 5.00 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
n onivamide.
Calculate the pereentage eontent of total eapsaieinoids,
expressed as eapsaicin, using the following expression:
(A 3 + As + A6) x m3 x P2
A4 x mI
area of the peak due to capsaicin in the
ehromatogram obtained with the test solution;
area of the peak due to capsaiein in the
ehromatogram obtained with the referenee solution;
area of the peak due to dihydrocapsaiein in the
ehromatogram obtained with the test solution;
area of the peak due to nordihydroeapsaicin in the
ehromatogram obtained with the test solution;
mass of the oleoresin to be examined used to
prepare the test solution, in grams;
mass of capsaicin CRS used to prepare the referenee
solution, in grams;
pereentage eontent of capsaicin in capsaicin CRS.
____________________________________________ ~E~
2014
*****
Capsicum Soft Extract,
** **
Standardised ***
(Ph Eur monograph 2529)
___________________________________________
~E~
DEFINITION
Standardised soft extraet produeed from Capsicum (1859) .
Content
2.0 per eent to 2.4 per eent of total eapsaicinoids, expressed
as eapsaiein (CIsH 27N0 3; M r 305.4).
PRODUCTION
The extraet is produeed from the herbal drug by a suitable
procedure using ethanol (80 per eent V/V).
The eontent of total eapsaieinoids in the extraet is
determined and adjusted, if neeessary, to the value specified
by adding a suitable inen excipient, for example liquid
glucose.
CHARACTERS
Appearanee: reddish-brown, glutinous manero
IDENTIFICATION
Thin-layer ehromatography (2.2.27).
Test solution To 0.25 g ofthe extraet to be examined add
10 mL of a mixture of water R and propanol R (40:60 V/V).
Shake for 5 mino Filter, if necessary.
R eference solution Dissolve 2 mg of capsaicin R and 1 mg of
dihydrocapsaicin R in 5 mL of methanol R .
Plate TLC octadecylsilyl sz1ica gel plate R (5-40 ).1m) [or
TLC octadecylsilyl sz1ica gel plate R (2-10 ).1m)].
Mobz1e phase water R, methanol R (20:80 V/V).
Application 20).lL [or 2 ).IL] as bands of 15 mm [or 8 mm] .
Development Over a path of 12 cm [or 6 cm].
D rying In air.
D etection Treat with a 0.25 gIL solution of
dichloroquinonechlorimide R in ethyl acetate R , expose to
ammonia vapour until blue zones appear. Examine in
daylight.
R esults See below the sequenee of zones present in the
chromatograms obtained with the referenee solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
----- -----
Capsaicin: a blue zone A blue zone (capsaicin)
Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin)
----- -----
Reference solution Test solution
TESTS
Nonivamide
Liquid ehromatography (2.2.29).
Test solution Stir the extraet to be exarnined until
homogeneous, heating, if neeessary, to not more than 60 oc.
Disperse 0.350 g of the homogeneous extraet in 35 mL of a
mixture of water R and propanol R (40 :60 V/V). Shake for
30 min and dilute to 50.0 mL with propanol R. Dilute
25 .0 mL of the solution to 50.0 mL with the mobile phase
 2014
and filter through a membrane filter (nominal pore size
0.45 ¡.un).
Reference solurion Dissolve 8.0 mg of nonivamide CRS in the
mobile phase and dilute to 100.0 mL with the mobile phase
(solution A). Dissolve 8.0 mg of capsaicin CRS in a mixture
of 5.0 mL of solution A and 45 mL of the mobile phase .
Dilute to 100.0 mL with the mobile phase.
Column:
-- size: l = 0.25 m, 0 = 4.6 mm;
-- stationary phase: phenylsilyl silica gel for chromatography R
(5 ~m);
-- temperature: 30 oc.
Mobile phase acetonitrile R1, 1 giL solution of phosphoric
aád R (40:60 V/V).
Flow rate 1.0 mUmin.
Detection speetrophotometer at 225 nm.
Injection 1O ~L.
Run time 1.2 times the retention time of dihydroeapsaiein.
Elution order nordihydroeapsaiein, nonivamide, eapsaiein,
dihydroeapsaiein.
Relative retention With referenee to eapsaiein (retention
time = about 19 min): nordihydroeapsaiein = about 0.9;
nonivamide = about 0.95; dihydroeapsaicin = about 1.3.
System suitability Referenee solution:
-- resolution: minimum 1.5 between the peaks due to
nonivamide and eapsaiein.
Calculate the pereentage eontent of nonivamide with
referenee to the total eapsaicinoid eontent, using the
following expression:
Al x mz x PI x 5
A z x mi x e Yellowish-orange or reddish-orange liquido
Al area of the peak due to nonivamide in the
ehromatogram obtained with the test solution;
Az area of the peak due to nonivamide in the
ehromatogram obtained with the referenee solution;
mi mass of the extraet to be examined used to prepare
the test solution, in grams;
mz mas s of nonivamide CRS used to prepare the
referenee solution, in grams;
PI pereentage eontent of nonivamide in nonivamide CRS;
C pereentage eontent of total eapsaicinoids, as
determined in the assay.
Limit:
-- nonivamide: maximum 5.0 per eent of the total
eapsaicinoid eontent.
Dry residue (2.8.16)
Minimum 70.0 per eent m/m, determined on 2.00 g.
ASSAY
Liquid ehromatography (2.2.29) as deseribed in the test for
nonivamide.
Calculate the pereentage eontent of total eapsaicinoids,
expressed as eapsaiein, using the following expression:
(A3 + As + A6) x m3 x pz
A4 x mi
A3 area of the peak due to eapsaiein in the
ehromatogram obtained with the test solution;
A4 area of the peak due to eapsaiein in the
ehromatogram obtained with the referenee solution;
Capsicum Preparations IV-121
As are a of the peak due to dihydroeapsaiein in the
ehromatogram obtained with the test solution;
A6 area of the peak due to nordihydroeapsaiein in the
ehromatogram obtained with the test solution;
mi mas s of the extraet to be examined used to prepare
the test solution, in grams;
m3 mass of capsaicin CRS used to prepare the referenee
solution, in grams;
p2 pereentage eontent of eapsaiein in capsaicin CRS.
____________________________________________ PhE~
Standardised Capsicum Tincture *****
** **
(Ph Eur monograph 2337) ***
____________________________________________
~E~
DEFINITION
Standardised tineture produeed from Capsicum (1859) or
Refined and quantijied capsicum oleoresin (2336).
Content
90 per eent to 110 per eent of the nominal eontent of total
eapsaieinoids, expressed as eapsaiein (CI8Hz7N03; M r
305.4), stated on the label, which is between 0.020 per eent
m/m and 0.060 per eent mimo
PRODUCTION
The tincture is produeed from the herbal drug or oleoresin
and ethanol (70 per eent V/V to 85 per eent V/V) by an
appropriate proeedure.
CHARACTERS
Appearance
IDENTIFICATION
Thin-layer ehromatography (2.2.27).
Test solution Shake 10 mL of the tineture to be examined
with 10 mL of hexane R . Allow to separate and use the lower
layer.
Reference solution Dissolve 1 mg of capsaicin R and 1 mg of
dihydrocapsaicin R in 5 mL of ether R.
Plate TLC octadecylsilyl silica gel plate R (5-40 ~m) [or
TLC octadecylsilyl silica gel plate R (2-10 ¡¡m)].
Mobile phase water R, methanol R (20:80 V/V).
Application 20 ~L [or 2 ¡¡L] as bands of 15 mm [or 8 mm].
Development Over a path of 12 cm [or 6 cm] .
Drying In airo
Detection Spray with a 0.25 gIL solution of
dichloroquinonechlorimide R in ethyl acetate R, expose to
ammonia vapour until blue zones appear, then examine in
daylight.
Results See beIow the sequenee of zones present in the
ehromatograms obtained with the referenee solution and the
test solution. Furthermore, other zones may be present in the
ehromatogram obtained with the test solution.
 IV-122 Caraway 2014

Top of the plate area of the peak due to capsaicin in the

- -- - -- chromatogram obtained with the test solution;
area of the peak due to capsaicin in the
Capsaicin: a blue zone A blue zone (capsaicin) chromatogram obtained with the reference solution;
Dihydrocapsaicin: a blue zone A faint blue zone (dihydrocapsaicin) area of the peak due to dihydrocapsaicin in the
chromatogram obtained with the test solution;
--- --- area of the peak due to nordihydrocapsaicin in the
Reference solution Test solution chromatogram obtained with the test solution;
mass of the tincture to be examined, in grams;
mass of capsaicin CRS in the reference solution, in
TESTS grams;
Nonivamide percentage content of capsaicin in capsaicin CRS.
P2
Liquid chromatography (2.2.29).
_ _ _ _ __ __ _ __ _ __ _ __ ______ ~ Ew

Test solution Dilute 50.0 g of the tincture to be examined to

100.0 mL with methanol R.
Reference solution Dissolve 20.0 mg of capsaicin CRS and
4.0 mg of nonivamide CRS in 100.0 mL of methanol R.
Column: Caraway *****
- size: 1 = 0.25 m, 0 = 4.6 mm; ** **
- stationary phase: phenylsilyl silica gel for chromatography R (Caraway Fruit, Ph Eur monograph 1080) ***
(5 ¡.tm); When Powdered Caraway is prescribed or demanded,
- tempera tu re: 30 oC. material complying with the appropriate requirements below
Mobüe phase acetonitrile R, 1 giL solution of phosphoric acid and containing not less than 2.5% v/w (25 mUkg) of
R (40:60 V/V) . essential oil shall be dispensed or supplied.
PhEw _ _ _ _ _ _ _ _ __ _ __ __ __ __ __ _
Flow rate l.0 mUmin.
Detection Spectrophotometer at 225 nro. DEFINITION
Injection 10 ¡.tL. Whole, dry mericarp of Carum carvi L.
Elution order Nordihydrocapsaicin, nonivamide, capsaicin, Content
dihydrocapsaicin. Minimum 30 mUkg of essential oil (anhydrous drug).
System suitabüity Reference solution: CHARACTERS
- resolution: minimum 1.5 between the peaks due 10 Odour reminiscent of carvone.
nonivamide and capsaicin.
IDENTIFICATION
Calculate the percentage content of nonivamide using the A. The fruit is a cremocarp of almost cylindrical shape. It is
following expression: generally 3-6.5 mm long and 1-l.5 mm wide. The mericarps,
usually free, are greyish-brown or brown, glabrous, mostly
sickle-shaped, with both ends sharply terminated. Each bears
5 prominent narrow ridges. When cut transversely the pro file
shows an almost regular pentagon and 4 vittae on the dorsal
FI area of the peak due to nonivamide in the surface and 2 on the commissural surface may be seen with a
chromatogram obtained with the test solution; lens.
F2 area of the peak due to nonivamide in the B. Reduce to a powder (355) (2.9.12). The powder is
chromatogram obtained with the reference solution; yellowish-brown. Examine under a microscope using chloral
mi mass of the tincture to be examined, in grams; hydrate solution R. The powder shows the following diagnostic
m2 mass of nonivamide CRS in the reference solution, in characters: fragments of the secretory cells composed of
grams; yellowish-brown or brown, thin-walled, polygonal secretory
PI percentage content of nonivamide in nonivamide CRS. cells, frequently associated with a layer of thin-walled,
Limit: transversely elongated cells, 8-12 ~lm wide; fragments of the
- nonivamide: maximum 5.0 per cent ofthe total epicarp with thick-walled cells and occasional anomocytic
capsaicinoid contento stomata (2.8.3); numerous endosperm fragments containing
Ethanol (2.9.10) aleurone grains, droplets of fatty oil and microcrystals of
95 per cent to 105 per cent of the content stated on the calcium oxalate in rosette formation; spiral ves seIs
labe!' accompanied by sclerenchymatous fibres; rarely sorne fibre
bundles from the carpophore; groups of rectangular to sub-
Methanol and 2-propanol (2.9.11)
rectangular sclereids from the mesocarp with moderately
Maximum 0.05 per cent V/V of methanol and maximum thickened and pitted walls may be presento
0.05 per cent VIV of 2-propano!.
e. Thin-layer chromatography (2.2.27) .
ASSAY Test solution Shake 0.5 g of the powdered drug (710)
Liquid chromatography (2.2.29) as described in the test for (2.9.12) with 5.0 mL of ethyl acetate R for 2-3 mino Filter
nonivamide. over 2 g of anhydrous sodium sulfate R .
Calculate the percentage content of total capsaicinoids, Reference solution Dissolve 2 ¡.tL of carvone R and 5 ¡.tL of
expressed as capsaicin, using the following expression: olive oü R in 1.0 mL of ethyl acetate R.
Plate TLC silica gel plate R.
(F3 + Fs + F6) x m4 x P2
Mobüe phase ethyl acetate R, toluene R (5:95 V/V).
F 4 x m3
 2014 Caraway Oil IV-123

Application 20 ~lL of the test solution and 10 llL of the Drying In airo
reference solution, as bands. Detection A Examine in ultraviolet light at 254 nm.
Development Over a path of 10 cm. R esults ASee below the sequence of zones present in the
Drying In airo chromatograms obtained with the reference solution and the
Detection A Examine in ultraviolet light at 254 nm. test solution. Furthermore, other zones may be present in the
R esults A The chromatograms obtained with the test
chromatogram obtained with the test solution.
solution and with the reference solution show a quenching
zone (carvone) in the central part against a light background. Top of the plate
Detection B Spray with anisaldehyde solution R and, while --- ---
observing, heat at 100-105 oC for 2-4 minó examine in
daylight. Carvone: a quenching zone A quenching zone (carvone)

R esults B The zones due to carvone are dark orange-brown; --- ---
the chromatogram obtained with the test solution shows Reference solution Test solution
aboye the zone due to carvone a violet zone similar in
position and colour to the zone due to triglycerides of olive
oil in the chromatogram obtained with the reference solution; Detection B Spray with anisaldehyde soluticm R and heat at
the chromatogram obtained with the test solution shows 100-105 oC for 5-10 minoExamine immediately in daylight.
close to the solvent front a weak violet zone due to terpene Results B See below the sequence of zones present in the
hydrocarbons and in the lower part sorne weak, mostly violet- chromatograms obtained with the reference solution and the
greyish and brownish zones. test solution. Furthermore, several zones of weak intensity are
TESTS present, particularly in the lower third, in the chromatogram
Water (2. 2.13) obtained with the test solution.
Maximum 100 mI.Jkg, determined on 10.0 g ofpowdered
drug.
Top of the plate
Total ash (2.4.16)
A reddish·violet zone
Maximum 7.0 per cent.
ASSAY - -- ---
Carry out the determination of essential oils in herbal drugs A reddish·violet zone
(2.8. 12) . Use 10.0 g of drug reduced to a powder (710)
Carvone: a red to orange·brown An intense red to orange·brown
(2.9.12) irnmediately before the determination, a 500 mL zone zone (carvone)
round-bottomed flask, 200 mL of water R as the distillation --- - --
liquid, and 0.50 mL of xylene R in the graduated tube. Distil
Carveol : a reddish·violet zone A reddish·violet zone (carveo!)
at arate of 2-3 mI.Jmin for 90 mino
____________________________________________ ~Eif
A violet·blue zone

Reference solution Test solution

B. Examine the chromatograms obtained in the test for

Caraway Oil chromatographic profile.
(Ph Eur monograph 1817) Results The characteristic peaks in the chromatogram
~Eif ____________________________________________ obtained with the test solution are similar in retention time to
those in the chromatogram obtained with the reference
DEFINITION solution.
Oil obtained by steam distillation from the dry fruits of
TESTS
Carum carvi L.
Relative density (2.2.5)
CHARACTERS 0.904 to 0.920 .
Appearance Refractive index (2.2.6)
Clear, colourless or yellow liquido 1.484 to 1.490.
IDENTIFICATION Optical rotation (2.2.7)
First identification B. + 650
to + 81 0 •
Second identification A. Acid value (2.5. 1)
A. Thin-layer chromatography (2.2.27). Maximum 1.0, determined on 5.00 g.
Test solutwn Dissolve 40 llL of the substance to be Chromatographic pro file
examined in 1.0 mL of toluene R. Gas chromatography (2. 2.28): use the normalisation
R eference solution Dissolve 10 llL of carvone R and 5 llL of procedure.
carveol R in 1.0 mL of toluene R. Test solution Dissolve 0.200 g of the substance to be
Plate TLC silica gel F 254 plate R (5-40 llm) [or TLC silica gel examined in heptane R and dilute to 10.0 mL with the same
plate R (2-10 llm)] . solvento
Mobile phase ethyl acetate R, toluene R (5:95 V/V). Reference solutwn (a) Dissolve 5 llL of {J-myrcene R , 80 llL
Application 10 llL [or 2 llL] as bands. of limonene R, 5 llL of dihydrocarvone R, 100 llL of carvone R
and 5 llL of carveol R in heptane R and dilute to 10.0 mL
D evelopment Over a path of 10 cm [or 5 cm].
with the same solvento
IV-124 Cardamom Fruit 2014

Reference solution (b) Dissolve 10 ~lL of carvone R in Temperature:

heptane R and dilute to 10 mL with the same solvent. Dilute Time Temperature
0.1 mL of this solution to 10 mL with heptane R. (min) (oC)
Column: Column 0-80 50 ~ 170
- material: fused silica,
Injection port 230
- size: 1 = 30 m, 0 = 0.53 mm,
- stationary phase: macrogol 20000 R, (film thickness 1 ¡lm). Detector 230

Carrier gas helium for chromatography R.

Detection Flame ionisation.
Flow rate 1.5 mUmin.
Injection 1 ¡lL.
Split ratio 1:50.
System suitability Reference solution:
Temperature: - resolution: minimum 2.4 between the peaks due to
Time Temperature (- )-carvone (1 sr peak) and carvone Rl (2 nd peak).
(min) (oC) Calculate the percentage content of the (- )-carvone from the
Column 0·5 60 following expression:
5·68 60 ~ 250
Al
68 -75 250 ....,---....,-- x 100
Al +Az
Injection port 250
Detector 260 Al = area of the peak due to (- )-carvone,
Az = area of the peak due to carvone Rl.
Detection Flame ionisation.
Limit:
Injection 1.0 ¡lL. - (- )-carvone: maximum 1 per cent.
Elution order Order indicated in the composition of
STORAGE
reference solution (a). Record the retention times of these
At a temperature not exceeding 25 oC.
substances.
____________________________________________ ~Ew

System suitability Reference solution (a):

- resolution: minimum 4.5 between the peaks due to
p-myrcene and Iimonene.
Using the retention times determined from the
chromatogram obtained with the reference solution, locate
the components of the reference solution in the
chromatogram obtained with the test solution.
Limits:
- p-myrcene: 0.1 per cent to 1.0 per cent,
- limonene: 30.0 per cent to 45.0 per cent,
- trans-dihydrocarvone: maximum 2.5 per cent,
- carvone: 50.0 per cent to 65.0 per cent,
- trans-carveol: maximum 2.5 per cent.
- disregard limit: the area of the peak in the chromatogram
obtained with reference solution (b).
Chira! purity
Gas chromatography (2.2.28).
Test solution Dissolve 20 mg of the substance to be
examined in heptane R and dilute to 10.0 mL with the same
solvent.
R eference solution Dissolve 10 mg of (- )-carvone R and
10 mg of carvone Rl in heptane R and dilute to 10.0 mL with
the same solvent.
Column:
- material: fused silica,
- size: 1 = 30 m, 0 = 0.25 mm,
- stationary phase: modified p-cyclodextrine for chiral
chromatography RI (film thickness 0.25 ¡lm) .
Carrier gas helium for chromatography R.
Flow rate 2.0 mUmin.
Split ratio 1:30.
Cardamom Fruit
In making preparations of Cardamom, only the seed is used.
The seed is removed from the fruit, immediately powdered
or bruised and used immediately in making the preparation.
Cardamom seed, after removal from the fruit, should not be
stored.
DEFINITION
Cardamom Fruit consists of the dried, nearly ripe fruit of
Elettaria cardamomum Maton var. minuscula Burkill.
CHARACTERISTICS
Odour and taste of the seeds, strongly aromatic.
Macroscopical Fruit: a trilocular inferior capsule, up to about
2 cm long, ovoid or oblong, dull green to pale buff, plump or
slightly shrunken, obtusely triangular in cross section, nearly
smooth or longitudinally striated. Seeds in each loculus in
two rows, forming an adherent mass attached to the axile
placenta. Seed: pale to dark reddish brown, about 4 mm long
and 3 mm broad, irregularly angular, marked with six to
eight transverse wrinkles, with a longitudinal channel
containing the raphe, each seed enveloped by a colourless,
membranous aril. Transversely cut surface of seed showing a
brown testa, white starchy perisperm, grooved on one side,
yellowish endosperm and a paler embryo.
Microscopical Seed: aril composed of flattened, thin-walled,
parenchymatous cells. Testa composed of the following
layers: (i) outer epidermis of thick-walled, narrow, axially
elongated cells; (ii) a layer of collapsed parenchyma subjacent
to the outer epidermis; (iii) a single layer (two or three layers
near the raphe) of large, thin-walled, rectangular cells
containing volatile oil; (iv) two or three layers of parenchyma;
(v) layers of thin-walled, flattened cells; (vi) distinctive
sclerenchymatous layer of c10sely packed brown, thick-walled
cells, each with a bowl-shaped cavity in the upper part
containing a warty silica body; (vii) inner layer consisting of
flattened cells. Perisperm: cells thin-walled, packed with
 2014 Carrageenan IV-125

numerous starch granules up ro 6 11m in diameter and, in a

small cavity, one to seven prisms of calcium oxalate about
Compound Cardamom Tincture
10 ro 30 11m long. Endosperm parenchymatous, thin-walled, DEFINITION
with a granular hyaline mass of protein in each cel!. Embryo: Cardamom Oil 0.450 mL
cells small, containing aleurone grains . Caraway Oil 0.400 mL
TESTS Cinnamon Oil 0.225 mL
Foreign matter Cochineal, in moderately coarse 7g
Of the fruit, not more than 1.0%; of the separated seeds, not powder
more than 3.0%, Appendix XI D. Glycerol 50 mL
Ethanol (60 per cent) Sufficient ro produce
Vo1atile oil
1000 mL
In the seeds, not less than 4.0% v/w, Appendix XI E,
Method 1. Use 20 g of the unground seeds and distil for Extemporaneous preparation
5 hours. The following directions apply.
Acid-inso1uble ash Moisten the Cochineal with a sufficient quantity of Ethanol
Ofthe seeds, not more than 3.5%, Appendix XI K. (60 per cent) and prepare 900 mL of tincture by percolation,
Ash Appendix XI F. Add the Cardamom Oil, the Caraway Oil,
Of the seeds, not more than 6.0%, Appendix XI J. the Cinnamon Oil and the Glycerol and sufficient Ethanol
(60 per cent) ro produce 1000 mL; mix. Filter, if necessary.
The tincture complies with the requirements for Tinctures stated
Cardamom Oil under Extracts and with the following requirements.
Preparations TESTS
Aromatic Cardamom Tincture Ethanol content
Compound Cardamom Tincrure 52 to 57% v/v, Appendix VIII F, Method III .
DEFlNITION Glycerol
Cardamom Oil is obtained by distillation from crushed 4.5 ro 5.5 % v/v when determined by the following method.
Cardamom Fruit. Dilute 20 mL ro 100 mL with water. To 20 mL of this
CHARACTERISTICS solution add 100 mL of water and 1 g of activated charcoal
A c1ear, colourless or pale yellow liquid, visibly free from and boil under a refiux condenser for 15 minutes. Filter and
water; odour, that of Cardamom Fruit. wash the filter and charcoal with sufficient water ro produce
150 mL. Add 0.25 mL of bl'Omocresol purple solution and
TESTS neutralise with O.lM sodium hydroxide or 0.05M sulfuric acid to
Ester value the blue colour of the indicaror. Add 1.4 g of sodium periodate
90 ro 156, Appendix XC. and allow ro stand for 15 minutes. Add 3 mL of propane-l)2-
Optical rotation diol, shake and allow ro stand for 5 minutes. Add 0.2 5 mL of
0
+ 20 ro +40°, Appendix V F . bl'Omocresol purple solution and titrate with 0.1M sodium
Refractive index hydroxick VS to the same blue colour. Each mL of
1.461 ro 1.467, Appendix V E. O.l M sodium hydroxick VS is equivalent ro 9.210 mg of
glycero!. Calculate the percentage v/v of glycerol, taking its
Solubility in ethanol
weight per mL to be 1.260 g.
Soluble, at 20°, in 6 volumes of ethanol (70%),
Appendix X M. Relative density
0.925 to 0.937, Appendix V G.
Weight per mL
0.917 to 0.940 g, Appendix V G.
STORAGE
Cardamom Oil should be kept in a well-fiIled container and
Carrageenan ***
*** ***
protected from light.
(Ph Eur monograph 2138) ***
__________________________________________
Aromatic Cardamom Tincture
PhE~

DEFINITION DEFINITION
Cardamom Oil 3 mL Carrageenans are polysaccharides extracted from different
Caraway Oil 10 mL Rhodophyceae with boiling water or aqueous alkali solutions.
Cinnamon Oil 10mL Carrageenan is separated by alcohol precipitation, potassium
Clove Oil 10 mL chloride precipitation, gel pressing, drum drying or freezing.
Strong Ginger Tincrure 60mL The alcohol used during separation and purification is
Ethanol (90 per cent) Sufficient to produce generally 2-propano!. The main components are potassium,
1000 mL sodium, calcium or magnesium salts of the sulfate esters of
D-galacrose and 3,6-anhydro-D-galactose copolymers. They
The tincture complies with the requirements for Tinctures stated exist in different proportions depending on the biological
under Extracts and with the following requirements. origin of the polyrner.
TESTS The prevalent copolymers are designated as K-, [- and
Ethanol content A-carrageenan.
84 to 87% v/v, Appendix VIII F, Method III.
Relative density
0.825 to 0.845, Appendix V G.
IV-126 Cascara 2014

CHARACTERS Total ash (2.4.16)

Appearance
Yellowish, brownish, or white or almost white powder.
Solubility
Soluble in water giving a viscous or colloidal solution,
insoluble in organic solvents.
IDENTIFICATION
A. Prepare a 20 gIL dispersion and heat in a water-bath at
80 oC (Solution A). Allow to cool; it becomes more viscous
upon cooling and may form a gel.
To 10 mL of solution A, while still hot, add 4 drops of a
100 gIL solution of potassium chloride R, mix and allow to
cool. A 'brittle' gel indicates a carrageenan of a
predominantly K-type; an 'elastic' gel indicates a
predominantly t-type; if the solution do es not forrn a gel, the
carrageenan is of a predominantly A-type.
B. Dilute 1 volurne of solution A with about 4 volumes of
water R and add 2-3 drops of a 0.5 giL solution of methylene
blue R in ethanol (96 per cent) R. A blue precipitate is formed.
e. Infrared absorption spectrophotometry (2.2.24).
Preparation Prepare a 2 gIL solution of the substance to be
examined and cast films (5 ~m thick when dry) on a suitable
non-sticking surface.
Carrageenan has strong, broad absorption bands, typical of
all polysaccharides, in the 1000-1100 cm _ 1 region.
Absorption maxima are 1065 cm- I and 1020 cm- I for
Maximum 40.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximurn 2.0 per cent.
LABELLING
The labe! sta tes the type of carrageenan.
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics lhal are
recognised as being relevant control parameters for one or more
functions of the substance when used as an excipient (see chapter
5.15). This section is a non-mandatory part of the monograph
and it is not necessary to verify the characteristics lO demonstrate
compliance. Control of these characteristics can however contribute
lO the quality of a medicinal product by improving the consistency
of the manufacturing process and the performance of the medicinal
product during use. W'here control methods are cited, they are
recognised as being suitable for the purpose, but other methods can
also be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for carrageenan used
as viscosity-increasing agent.
Gel formation
See Identification A.
Apparent viscosity
See Tests.
____________________________________________ ~ Ew

gelling and non-gelling types, respectively. Other

characteristic absorption bands and their intensities relative to
the absorbance at 1050 cm- I are shown in Table 2138.-1.

Cascara ***
Table 2138.-1. - Characteristic absorption bands for
carrageenan identification by infrared absorption
*** ***
spectrophotometry
(Ph Eur monograph 0105) ***
Preparation
Absorbance relative to the
Wave-
absorbance at 1050 cm- I
Standardised Cascara Dry Extract
number Molecular structure
(cm- I ) When Powdered Cascara is prescribed or demanded,
K l A material complying with the requirements below with the
1220 -1260 Ester sulfate 0.7 - 1.2 1.2- 1.6 1.4-2.0 exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
3,6-anhydro·D· ,; 0.2 ____________________________________________
928-933 0.3-0.6 0.2-0.4 ~Ew

galactose

840-850 Galactose-4-sul fa te 0.3-0.5 0.2-0.4 DEFINITION

Dried, whole or fragmented bark of Rhamnus purshiana De.
825-830 Galactose-2-sulfate 0.2-0.4
(syn. Frangula purshiana (De.) A.Gray).
810-820 Galactose-6-sulfate 0.1- 0.3 Content
3,6-anhydro-D· Minimum 8.0 per cent of hydroxyanthracene glycosides of
800 - 805 ,; 0.2 0.2-0.4
galactose-2-sulfate which minimum 60 per cent consists of cascarosides, both
expressed as cascaroside A (C27H 32014; M r 580.5) (dried
drug).
TESTS
IDENTIFICATION
Apparent viscosity (2.2.10)
A. The bark occurs in slightly channelled or nearly fiat
Minimum 5 mPa·s. Heat a 15 gIL dispersion (dried
substance) at 80 oC for at least 15 min to dissolve. pie ces, usually 1-5 mm in thickness, usually varying greatly in
length and width. The outer surface is grey or dark greyish-
Compensate for any loss of water by evaporation, allow to
cool to 75 oC and carry out the test at this temperature. brown and shows occasionallenticels that are orientated
transversally. It is usually more or less completely covered by
Heavy metals (2.4. 8) a whitish coat of lichens, epiphytic moss and foliaceous
Maximum 20 ppm. liverwort. The inner surface is yellow or reddish-brown or
Dissolve 2.0 g in 30 mL of water R and shake for 2 min. almost black with fine longitudinal striations; it turns red
AlIow to stand and separate the aqueous layer. 12 mL of the when treated with alkali. The yellow fracture is short and
solution complies with test A. Prepare the reference solution granular in the outer part and somewhat fibrous in the inner
using lead standard solution (1 ppm Pb) R. parto
Loss on drying (2.2.32) B. Microscopic examination (2.8.23) . The powder is
Maximum 12.0 per cent, deterrnined on 1.000 g by drying in yellowish-brown. Examine under a microscope using chloral
an oven at 105 De. hydrate solution R. The powder shows the following diagnostic
 2014
characters (Figure 0105.-1): bundles [A] ofpardy lignified
ph10em fibres [Aa], accompanied by crystal sheaths
containing prisms of calcium oxalate [Ab] and sometimes
including medullary rays [Ac]; isolated sclereids [G] or
groups of sclereids [B] accompanied by crystal sheaths [Ba];
isolated cluster crystals [C] or prisms [E] of calcium oxalate;
parenchymatous cells [F, H] containing a yellow substance
that becomes deep red when treated with alkali, sometimes
accompanied by cells containing cluster crystals of calcium
oxalate [Ha] ; cork cells, in surface view [D] or in transverse
section m, associated with parenchyma, sorne cells of which
contain cluster crystals of calcium oxalate ITa]; frequendy
epiphytes [K] , which may be liverworts, entire or in
fragments, having a lamina 1 cell thick without a midrib and
composed of isodiametric cells, or 1eaves of mosses, having a
lamina 1 cell thick composed of elongated cells and
possessing a midrib several cells thick.
H solution shows, in the central part, a reddish-brown zone due
Figure 0105 .-1. - Illustration for identification test B of
powdered herbal drug of cascara
C. Examine the chromarograms obtained in test A for Other
species of Rhamnus; anthrones.
R esults The chromatogram obtained with the test solution
shows several reddish-brown zones with different intensities:
there are 4 faint zones, 3 being situated at about the mid-
point of the chromatogram and 1 in the lower third and
there is a strong zone in the upper third of the
chromarogram. Examine in ultraviolet light at 365 nm.
The chromarogram obtained with the test solution shows
several zones with the same fiuorescence, situated aboye and
particularly below (cascarosides) that due to barbaloin in the
chromarogram obtained with the reference solution.
D . Heat 0.2 g of the powdered herbal drug (180) (2.9.12)
with 50 mL of water R on a water-bath for 15 mino Allow ro
Cascara IV-127
cool and filter. To 10 mL of the filtrate add 20 mL of
hydrochloric acid R1 and heat on a water-bath for 15 min o
Allow to cool, transfer to a separating funnel and shake with
3 quantities, each of 20 mL, of ether R. Reserve the aqueous
layer (solution A). Combine the 3 ether extracts and shake
with 10 mL of dilute ammonia R2. The aqueous layer
becomes reddish-violet. Transfer solution A ro a small fiask,
add 5 g of fem'c ehlonde R and heat on a water-bath for
30 mino Allow to cool, transfer ro a separating funnel and
shake with 15 mL of ether R. Wash the ether layer with
10 mL of water R , discard the aqueous layer and shake the
ether layer with 5 mL of dilute ammonia R2. A red colour
develops in the aqueous layer.
TESTS
Other species of Rharnnus; anthrones
Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g ofthe powdered herbal drug (180)
(2. 9.12) add 5 mL of ethanol (70 per cent VIV) R and heat to
boiling. Cool and centrifuge. Decant the supernatant
irnmediately and use within 30 min o
Reference solution Dissolve 20 mg of barbaloin R in ethanol
(70 per cent VIV] R and dilute ro 10 mL with the same
solvent.
Plates TLC sz7ica gel plate R (2 plates).
Mobile phase water R , methanol R, ethyl acetate R
(13: 17: 100 VIVIV).
A. Application: 10 ¡.tL as bands.
Development Over a path of 10 cm.
Drying In air for 5 min o
Detection Spray with about 10 mL of a 50 gIL solution of
potassium hydroxide R in ethanol (50 per cent V/V] R and heat
at 100-105 oC for 15 min; examine immediately after
heating.
R esults The chromatogram obtained with the reference
to barbaloin; examine in ultraviolet light at 365 nm; the zone
due to barbaloin shows intense yellowish-brown fiuorescence;
in the chromatogram with the test solution, no zone with
orange-brown fiuorescence is seen between the zone due ro
barbaloin and the zones due to cascarosides.
B. Application: 10 ¡.tL of the test solution, as a bando
Development Over a path of 10 cm.
Drying In air for not more than 5 mino
Detection Spray irnmediately with a 5 gIL solution of
nitrotetrazolium blue R in methanol R and examine
immediately.
R esults No violet or greyish-blue zones appear.
Foreign rnatter (2.8.2)
Maximum 1 per cent.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (180) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Carry out the assay in 24 h, protectedfrom bright light.
Stir 1.00 g of the powdered herbal drug (180) (2.9. 12) into
100 mL of boiling water R and continue boiling and stirring
for 5 mino Allow ro cool, dilute ro 100.0 mL with water R,
shake, filter and discard the first 20 mL of filtrate . Transfer
10.0 mL of the filtrate to a separating funnel, add 0.1 mL of
IV-128 Cascara Preparations
1 M hydrochlO1ic acid and shake with 2 quantities, each of
20 mL, of a mixture of 1 volume of ether R and 3 volumes of
hexane R . Wash the combined organic extracts with 5 mL of
water R , discard the organic layer and retum the rinsings to
the aqueous layer. Shake the combined aqueous layers with
4 quantities, each of 30 mL, of ethyl acetate R freshly
saturated with water R (to 150 mL of ethyl acetate R add
15 mL of water R, shake for 3 min and allow to stand) on
each occasion allowing separation to take place until the
organic layer is c1ear. Combine the ethyl acetate extracts.
Use the aqueous layer for the assay for cascarosides and the
organic layer for the assay for hydroxyanthracene glycosides
other than cascarosides.
Hydroxyanthracene glycosides other than cascarosides
Transfer the organic layer to a suitable flask and remove the
solvent by distillation, evaporating almost to dryness .
Dissolve the residue in 0.3-0.5 mL of methanol R and transfer
to a volumetric flask, rinsing the 1St flask with warm water R
and adding the rinsings to the methanolic solution. Allow to
cool and dilute to 50.0 mL with water R . Transfer 20.0 mL
of this solution to a 100 mL round-bouomed flask with a
ground-glass neck and containing 2 g of ferric chloride R and
12 mL of hydrochloric acid R. Anach a reflux condenser and
place the flask in a water-bath so that the level of the water is
aboye that of the liquid in the flask and heat for 4 h. Allow
to cool, transfer the solution to a separating funnel and rinse
the flask successively with 3-4 mL of 1 M sodiwn hydroxide
and 3-4 mL of water R, adding the rinsings to the separating
funnel. Shake the contents of the separating funnel with
3 quantities, each of 30 mL, of a mixture of 1 volume of
ether R and 3 volumes of hexane R. Wash the combined
organic layers with 2 quantities, each of 10 mL, of water R
and discard the rinsings. Dilute the organic layer to
100.0 mL with the mixture of ether and hexane. Take
20. 0 mL, evaporate carefully to dryness on a water-bath and
dissolve the residue in 10.0 mL of a 5 gIL solution of
magnesium aceta te R in methanol R . Measure the absorbance
(2. 2.25) at 440 nm and 515 nm using methanol R as the
compensation liquido If the ratio of the absorbance at
515 nm to that at 440 nm is less than 2.4, the assay is
invalido
Calculate the percentage content of hydroxyanthracene
glycosides other than cascarosides, expressed as cascaroside
A, using the following expression:
A x 6.95
m
i.e. taking the specific absorbance to be 180.
A = absorbance at 515 nm;
m = m ass of the substance to be examined, in grams.
Cascarosides
Dilute the aqueous layer to 50.0 mL with water R . Treat
20.0 mL of this solution as described aboye in the assay of
hydroxyanthracene glycosides other than cascarosides.
Measure the absorbance (2.2.25) of the test solution at
440 nm and 515 nm. If the ratio of the absorbance at
515 nm to that at 440 nm is less than 2.7, the assay is
invalido
Calculate the percentage content of cascarosides, expressed
as cascaroside A, using the following expression:
A x 6.95
m
2014
i.e. taking the specific absorbance to be 180.
A absorbance at 515 nm;
m = mass of the substance to be examined, in grams.
____________________________________________ ~Em
Standardised Cascara Dry Extract ***
*** ***
(Ph Eur monograph 1844) ***
Preparation
Cascara Tablets
Ph Eur ____________________________________________
DEFINITION
Standardised dry extract obtained from Cascara ( 0105).
Content
90 per cent to 110 per cent of the nominal content of
hydroxyanthracene glycosides, expressed as cascaroside A
(C 27H 32 0 14 ; MI' 580.5), stated on the label; minimum
60 per cent of the hydroxyanthracene glycosides are
cascarosides, expressed as cascaroside A. The nominal
content of hydroxyanthracene glycosides is within the range
8.0 per cent to 25.0 per cent m/m (dried extract).
PRODUCTION
The extract is produced from the herbal drug by an
appropriate procedure using either boiling water or a
hydroalcoholic solvent at least equivalent in strength to
ethanol (60 per cent V/V).
CHARACTERS
Appearance
Brown, free-flowing powder.
IDENTIFICATION
Thin-Iayer chromatography (2.2. 27).
Test solution To 0.2 g of the extract to be examined add
5 mL of ethanol (70 per cene V/V) R and heat to boiling. Cool
and centrifuge. Decant the supematant solution immediately
and use within 30 mino
R eference solution Dissolve 20 mg of barbaloin R and 2 mg of
emodin R in ethanol (70 per cene V/V) R and dilute to 10 mL
with the same solvent.
Plate TLC silica gel plate R (5-40 ~lm) [or TLC silica gel
plate R (2-10 ~m) ].
Mobile phase water R, methanol R , ethyl acetate R
(13:17:100 VIV/V).
Application 10 ¡.¡L [or 2 ~lL] as bands.
Developmene Over a path of 10 cm [or 6 cm].
Dlying In air for 5 min.
Detection Spray with a 50 gIL solution of potassium
hydroxide R in ethanol (50 per cene V/V) R and heat to
100-105 oC for 15 minó examine in ultraviolet light at
365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
2014 Cascara Preparations IV-129

Top of the plate
Emodin: a red f1uorescent zone
---
Barbaloin: a yellowish·brown
f1uorescent zone
A faint red fluorescent zone
---
A yellowish·brown fluorescent zone
A blue fluorescent zone
water-bath and dissolve the residue in 10.0 mL of a 5 gIL
solution of magnesium acetate R in methanol R . Measure the
absorbance (2.2.25) at 440 nm and 515 nm, using
methanol R as the compensation liquido If the ratio of the
absorbance at 515 nm to that at 440 nm is les s than 2.4, the
assay is invalido
Calculate the percentage content of hydroxyanthracene
glycosides other than cascarosides, expressed as cascaroside
A, using the following expression:

---
An intense yellowish·brown
fluorescent zone
3 yellowish·brown fluorescent zones
Reference solution Test solution
TESTS
Loss on drying (2.8.17)
Maximum 5.0 per cent.
ASSAY
Carry out the assay within 24 h, protected jrom bright light.
To 0.500 g of the extract to be examined add 80 mL of
ethanol (70 per cent V/V) R. Shake, and allow to stand in the
dark for at least 8 h. Dilute to 100.0 mL with ethanol
(70 per cent V/V) R. Shake and filter, discarding the first
20 mL of filtrate. Transfer 10.0 mL of the filtrate to a
separating funnel, add 0.1 mL of 1 M hydrochlonc acid and
shake with 2 quantities, each of 20 mL, of a mixture of
1 volume of ether R and 3 volumes of hexane R. Wash the
combined organic extracts with 5 mL of water R. Discard the
organic layer and return the rinsings to the hydroalcoholic
layer. Shake with 4 quantities, each of 30 mL, of ethyl
acetate R freshly saturated with water R (prepared as follows :
to 150 mL of ethyl acetate R add 15 mL of water R, shake for
3 min and allow to stand), on each occasion allowing the
---
A x 6.95
m
i.e. taking the specific absorbance to be 180.
A = absorbance at 515 nm;
m = mass of the substance to be examined, in grams .
Cascarosides
Dilute the aqueous layer to 50.0 mL with water R. Treat
20.0 mL of this solurion as described aboye in the assay of
hydroxyanthracene glycosides other than cascarosides.
Measure the absorbance (2.2.25) at 440 nm and 515 nm.
If the ratio of the absorbance at 515 nm to that at 440 nm is
les s than 2.7, the assay is invalido
Calculate the percentage content of cascarosides, expressed
as cascaroside A, using the following express ion:
A x 6.95
m
i.e. taking the specific absorbance to be 180.
A = absorbance at 515 nm;
m = mass of the substance to be examined, in grams.
LABELLING
The labe! sta tes the nominal content of hydroxyanthracene
glycosides, expressed as cascaroside A.
____ _ _ _ _ _ _ __ __ __ _ _ ______ ~E~

layers to separate until the organic layer is c1ear. Combine

the ethyl acetate extracts. Use the aqueous layer for the assay
of cascarosides and the organic layer for the assay of
hydroxyanthracene glycosides other than cascarosides.
Hydroxyanthracene glycosides other than cascarosides
Transfer the organic layer to a round-bottomed ftask and
remove the solvent by distillation, evaporating almost to
dryness. Dissolve the residue in 0.5 mL of methanol R, add
10 mL of water R at 40 oC and transfer to a 50 mL
volumetric ftask, rinsing the round-bottomed ftask with
water R at 40 oC and adding the rinsings to the
hydromethanolic solution. Allow to cool and dilute to
50.0 mL with water R. Transfer 20.0 mL of the solution to a
100 mL round-bottomed ftask with a ground-glass neck
containing 2 g of jeme chlonde R and 12 mL of hydrochloric
acid R. Attach a reftux condenser and place the ftask in a
water-bath so that the level of the water is aboye that of the
liquid in the ftask and heat for 4 h. Allow to cool, transfer
the solution to a separating funnel and rinse the ftask
successively with 4 mL of 1 M sodium hydroxide and 4 mL of
water R, adding the rinsings to the separating funnel. Shake
the contents of the separating funnel with 3 quantities, each
of 30 mL, of a mixture of 1 volume of ether R and 3 volumes
of hexane R. Wash the combined organic layers with
2 quantities, each of 10 mL, of water R and discard the
rinsings. Dilute the organic layer to 100.0 mL with a mixture
of 1 volume of ether R and 3 volumes of hexane R. Take
20.0 mL of the solution, evaporate carefully to dryness on a
Cascara Tablets
DEFINITION
Cascara Tablets contain Standardised Cascara Dry Extract.
They are coated.
The tablets comply with the requirements stated under Tablets and
with the jollowing requirements.
Content of total hydroxyanthracene derivatives
17.0 to 23 .0 mg, of which not les s than 60% consists of
cascarosides, both expressed as cascaroside A.
IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Boil a quantity of the powdered tablets containing the
equivalent of 32 mg of total hydroxyanthracene derivatives
with 5 mL of 70% v/v of ethanol, cool and centrifuge. Decant
the supernatant liquid immediately and use within
30 minutes .
(2) Dissolve 20 mg of barbaloin and 2 mg of emodin in
70% v/v of ethanol and dilute to 10 mL with the same
solvent.
IV-130 Cascara Preparations
CHROMATOGRAPHIC CONDITIONS
(a) Use siliea gel F254 precoated plates or high-performance
siliea gel F 254 (Merck siliea gel F254 HPTLC plates are
suitab1e) .
(b) Use the mobile phase as described below.
(c) Apply 10 ¡lL [or 2 )lL] of each solution, as bands.
(d) Develop the plate to 10 cm [or 6 cm].
(e) After removal of the plate, dry in air, spray with a 5% w/v
solution of potassium hydroxide in 50% v/v ethanol, heat at
0
100 to 105 for 15 minutes and examine under ultraviolet
light (365 nm).
MOBILE PHASE
13 volurnes of water, 17 volurnes of methanol and
100 volumes of ethyl aeetate.
CONFIRMATION
The chromatogram obtained with solution (1) show
yellowish-brown fiuorescent bands with Rf values of between
0.2 and 0.25, an intense yellowish-brown fiuorescent band
with an Rf value of about 0.3, a blue fiuorescent band with
an Rf value of about 0.6, a yellowish-brown fiuorescent band
with an Rf value of about 0.7 corresponding in colour and
position to the band obtained with barbaloin in solution (2)
and a faint reddish fiuorescent band with an Rf value of
about 0.9 corresponding in position to emodin in
solution (2) .
Top of the plate
A faint red fluorescent band Emodin a red fluorescent band
A yellow-brown fluorescent band Barbaloin: a yellow-brown
fluorescent band
A blue fluorescent band
An intense yellow-brown
fluorescent band
3 yellow-brown fluorescent bands
Solution (1) Solution (2)
TESTS
Disintegration
Comply with the requirements stated under Tablets but for
sugar-coated tablets the maximum time is 120 minutes.
ASSAY
Cany out the assay within 24 hours, proteeted from bright light.
Add 80 mL of 70% v/v ethanol to a quantity of the powdered
tablets containing 75 mg of total hydroxyanthracene
derivatives. Shake and allow to stand in the dark for at least
8 hours. Dilute to 100.0 mL with 70% v/v of ethanol. Shake
and filter, discarding the first 20 mL of filtrate. Transfer
10.0 mL of the filtrate to a separating funnel, add 0.1 mL of
1M hydroehlorie acid and shake with 2-quantities, each of
20 mL, of a mixture of 1 volume of ether and 3 volumes of
hexane. Wash the combined organic extracts with 5 mL of
water. Discard the organic layer and return the rinsings to the
hydroalcoholic layer. Shake with 4-quantities, each of 30 mL,
of ethyl aceta te freshly saturated with water prepared by
shaking 150 rnL of ethyl acetate with 15 mL of water for
2014
3 minutes and allowing to stand until the layers have
separated and the organic layer is c1ear. Combine the ethyl
acetate extracts and use the aqueous layer for the assay of
cascarosides and the organic layer for the assay of
hydroxyanthracene glycosides other than cascarosides.
Hydroxyanthracene glycosides other than cascarosides
Transfer the organic layer to a round-bottomed fiask and
remove the solvent by distillation, evaporating almost to
dryness. Dissolve the residue in 0.5 mL of methanol, add
10 mL of water at 40° and transfer to a 50 mL volumetric
fiask, rinsing the round-bottomed fiask with water at 40° and
adding the rinsings to the hydromethanolic solution. Allow to
cool and dilute to 50.0 mL with water. Transfer 20.0 rnL of
the solution to a 100 mL round-bottomed fiask with a
ground-glass neck containing 2 g of iron (m) chloride
hexahydrate and 12 mL of 7M hy drochloric acid. Attach a
refiux condenser and place the fiask in a water-bath so that
the level of the water is aboye that of the liquid in the fiask
and heat for 4 hours. Allow to cool, transfer the solution to a
separating funnel and rinse the fiask successively with 4 mL
of 1M sodium hydroxide and 4 rnL of water, adding the
rinsings to the separating funne!. Shake the contents of the
separating funnel with 3-quantities, each of 30 mL, of a
mixture of 1 volume of ether and 3 volurnes of hexane. Wash
the combined organic layers with 2-quantities, each of
10 mL, of water and discard the rinsings. Dilute the organic
layer to 100.0 mL with a mixture of 1 volurne of ether and
3 volurnes of hexane. Take 20.0 mL of the solution,
evaporate carefully to dryness on a water-bath and dissolve
the residue in 10.0 mL of a 0.5% w/v solution of magnesium
aceta te in methanol. Measure the absorbance of the resulting
solution at 440 nm and at 515 nm, Appendix 11 B, using
methanol in the reference cel!. The assay is not valid if the
ratio of the absorbance at 515 nm to that at 440 nm is les s
than 2.4.
Calculate the percentage content of hydroxyanthracene
glycosides other than cascarosides, expressed as cascaroside
A, using the following expression:
A X 6.95
m
i.e. taking the specific absorbance to be 180.
A = absorbance at 515 nm;
m = weight of the substance being examined, in grams.
Cascarosides
To the aqueous solution reserved from the preliminary
extraction add sufficient water to produce 50.0 mL. Carry
out the Assay for hydroxyanthracene gycosides other than
cascarosides, beginning at the words, 'Transfer 20 mL ... '.
Measure the absorbance of the resulting solution at 440 nm
and at 515 nm, Appendix II B, using methanol in the
reference cel!. The assay is not valid if the ratio of the
absorbance at 515 nm to that at 440 nm is les s than 2.7.
Calculate the percentage content of cascarosides, expressed
as cascaroside A, using the following expression:
A x 6.95
m
i.e. taking the specific absorbance to be 180.
A absorbance at 515 nm;
m = weight of the substance being examined, in grams.
2014 Cassia Oil IV-131

LABELLING Optical rotation (2.2.7)

The label states the nominal content of hydroxyanthtacene _ l Oto + 1°.
glycosides, expressed as cascarosides A. Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution The oil to be examined.
Cassia Oil *** Reference solution Dissolve 100 JlL of trans-cinnamic
*** *** aldehyde R, 10 JlL of cinnamyl acetate R, 10 JlL of eugenol R,
(Ph Eur monograph 1496) *** 10 JlL of trans-2-methoxycinnamaldehyde R and 20 mg of
8007-80-5 coumarin R in 1 mL of acetone R.
~E~ ____________________________________________ Column:
- material: fused silica;
DEFINITION - size: l = 60 m, 0 = about 0.25 mm;
Essential oil obtained by steam distillation of the leaves and - stationary phase: bonded macrogol 20 000 R .
young branches of Cinnamomum cassia Blume (e. aromaticum
Carrier gas helium for chromatography R.
Nees) .
Flow rate 1.5 mIJmin.
CHARACTERS
Split ratio 1: 1OO.
Appearance
Temperature:
Clear, mobile, yellow or reddish-brown liquido
Characteristic odour reminiscent of cinnamic aldehyde. Time Temperature
(min) (oC)
IDENTIFICATION Co!umn 0·10 60
First identijication B.
10 ·75 60 -7190
Second identijication A.
75· 160 190
A. Thin-layer chromatography (2.2.27) .
Injection port 200
Test solution Dissolve 0.5 mL of the oil to be examined in
acetone R and dilute to 10 mL with the same solvento Detector 240
Reference solution Dissolve 50 JlL of trans-cinnamic
Detection Flame ionisation.
aldehyde R, 10 JlL of eugenol R and 50 mg of coumarin R in
acetone R and dilute to 10 mL with the same solvento Injection 0.2 JlL.
Plate TLC silica gel plate R. Elution arder Order indicated in the composition of the
reference solution, depending on the operating conditions
Mobzle phase methanol R, toluene R (10 :90 V/V).
and the state of the column, coumarin may elute before or
Application 10 JlL as bands. after trans-2-methoxycinnamaldehyde; record the retention
Development Over a path of 15 cm. times of these substances.
Drying In air. System suitability Reference solution:
Detection A Examine in ultraviolet light at 365 nm. - resolution: minimum 1.5 between the peaks due to
Results A The zone of blue fiuorescence in the trans-2-methoxycinnamaldehyde and coumarin.
chtomatogram obtained with the test solution is similar in Identijication of components Using the retention times
position and colour to the zone in the chtomatogram determined from the chromatogram obtained with the
obtained with the reference solution (coumarin) . reference solution, locate the components of the reference
Detection B Spray with anisaldehyde solution R; examine in solution in the chtomatogram obtained with the test solution.
daylight while heating at 100-105 oC for 5-10 mino Determine the percentage content of each of these
Results B The chromatogram obtained with the reference components. The percentages are within the following
solution shows in its upper part a violet zone (eugenol) and ranges:
above this zone a greenish-blue zone (trans-cinnamic - trans-cinnamic aldehyde: 70 per cent to 90 per cent;
aldehyde). The chtomatogram obtained with the test solution - cinnamyl acetate: 1.0 per cent to 6.0 per cent;
shows a zone similar in position and colour to the zone due - eugenol: maximum 0.5 per cent;
to trans-cinnamic aldehyde in the chromatogram obtained - trans-2-methoxycinnamaldehyde: 3.0 per cent to
with the reference solution and may show a very faint zone 15 per cent;
due to eugenol. Other faint zones are presento - coumarin: 1.5 per cent to 4.0 per cent.
B. Examine the chtomatograms obtained in the test for STORAGE
chtomatographic profile. Protected from heat.
Results The principal peaks in the chromatogram obtained ____________________________________________ ~E~

with the test solution are similar in retention time to those in

the chtomatogram obtained with the reference solution.
Eugenol may be absent from the chromatogram obtained
with the test solution.
TESTS
Re1ative density (2.2.5)
1.052 to 1.070.
Refractive index (2.2.6)
1.600 to 1.614.
 IV-132 Greater Celandine 2014

Greater Celandine ***

*** ***
(Ph Eur monograph 1861) ***
~Eif ____________________________________________

DEFINITION
Dried, whole or cut aerial parts of Chelidonium majus L.
collected during flowering.
Content
Minimum 0.6 per cent of total alkaloids, expressed as
chelidonine (C20H19NOs; M r 353.4) (dried drug).
IDENTIFICATION
A. The stems are rounded, ribbed, yellowish or greenish-
brown, somewhat pubescent, about 3-7 mm in diameter,
hollow and mostly collapsed. The leaves are thin, irregularly
pinnate, the leaflets ovate to oblong with coarsely dentate
margins, the terminalleaflet often 3-lobed; the adaxial
surface is bluish-green and glabrous, the abaxial surface paler
and pubescent, especially on the veins. The flowers have 2
deeply concavo-convex sepals, readily removed, and 4 yellow,
broadly ova te, spreading petals about 8-10 mm long;
the stamens are numerous, yellow, and a short style arises
from a superior ovary; long, capsular, immature fruits are
rarely presento
B. Microscopic examination (2.8.23) . The powder is dark
greyish-green or brownish-green. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters (Figure 1861.-1):
numerous fragments of upper epidermis, composed of cells
Figure 1861.-1. - Illustration for identification test B of
with sinuous walls in surface view [B], accompanied by
powdered herbal drug of greater celandine
underlying palisade parenchyma [Ba]; numerous fragments of
lower epidermis in surface view [A, E] bearing anomocytic
stomata (2.8.3) [Aa] and bases of covering trichomes [Ab],
sometimes accompanied by underlying spongy parenchyma Results See below the sequence of zones present in the
[Ea]; long, uniseriate, multicellular covering trichomes, chromatograms obtained with the reference solution and the
usually fragmented, with thin-walled cells, sometimes test solution. Furthermore, other weaker zones may be
collapsed [G]; vascular tissue from the leaves and stems present in the chromatogram obtained with the test solution.
consisting of pitted and spirally thickened vessels [D]; groups
of fibres [C]; articulated latex rubes with yellowish-brown Top of the plate
contents [F]; occasional fragments of the corolla [H]
consisting of thin-walled cells containing numerous pale --- -----

yellow droplets of oil [Ha]; spherical pollen grains about Methyl red: a red zone A brown zone
30-40 J.lm in diameter with 3 pores and a finely pitted
exine m. A brown zone

C . Thin-Iayer chromatography (2.2.27) . Papaverine: a greyish·brown zone A greyish·brown zone

Test solution To 0.4 g ofthe powdered herbal drug (710) - -- -----

(2.9.12) add 50 mL of dilute acetic acid R. Boil in a water-

bath under a reflux condenser for 30 min. Cool and filter.
To the filtra te add concentrated ammonia R until a strong 2 brown zones
alkaline reaction is produced. Shake with 30 mL of methylene Reference solution Test solution
chloride R . Dry the organic layer over anhydrous sodium
sulfate R, filter and evaporate in vacuo to dryness. Dissolve
the residue in 1.0 mL of methanol R. TESTS
Reference solution Dissolve 2 mg of methyl red R and 2 mg of Foreign matter (2.8.2)
papaverine hydrochloride R in 10 mL of ethanol (96 per cent) R. Maximum 10.0 per cent.
Plate TLC silica gel plate R. Loss on drying (2.2.32)
Mobile phase anhydrous formic acid R, water R, propanol R Maximum 10.0 per cent, determined on 1.000 g ofthe
(1:9:90 V/VIV) . powdered herbal drug (355) (2.9.12) by drying in an oven at
Application 10 J.lL as bands. 105 oC for 2 h .
Development Over a path of 10 cm. Total ash (2.4.16)
Drying In air. Maximum 13.0 per cent.
Detection Spray with potassium iodobismuthate solution R and ASSAY
dry in air; spray with sodium nitrite solution R and allow to dry Test solution To 0.750 g ofthe powdered herbal drug (710)
in air; examine in daylight. (2.9.12) add 200 mL of dilute acetic acid R and heat on a
2014
water-bath for 30 min, shaking frequently. Cool and dilute to
250 .0 mL with dilute aeetie acid R. Filter. Discard the first
20 mL of the filtrate . To 30.0 mL of the filtrate add 6.0 mL
of eoneentrated ammonia R and 100.0 mL of methylene
ehloride R. Shake for 30 mino Separate the organic layer,
place 50.0 mL in a 100 mL round-bonomed fiask and
evaporate 10 dryness in vaeuo at a temperature not exceeding
40 ce. Dissolve the residue in about 2-3 mL of ethanol
(96 per eent) R, warming slightly. Transfer the solution to a
25 mL volumetric fiask by rinsing the round-bottomed fiask
with dilute sulfurie acid R and dilute to 25.0 mL with the
same solvento To 5.0 mL of the solution add 5.0 mL of a
10 gIL solution of ehromotropie acid, sodium salt R in sulfurie
acid R in a 25 mL volumetric fiask, stopper the fiask and mix
carefully. Dilute to 25.0 mL with sulfurie acid R and stopper
the fiask.
Compensation liquid Prepare at the same time and in the
same manner as for the test solution: place in a 25 mL
volumetric fiask 5.0 mL of dilute sulfuric acid R and 5.0 mL
of a 10 gIL solution of ehromotropie aeid, sodium salt R in
sulfurie acid R, stopper the flask and mix carefully. Dilute 10
25.0 mL with sulfuric acid R and stopper the flask.
Place both solutions on a water-bath for 10 mino Cool to
about 20 oC and dilute if necessary to 25.0 mL with sulfurie
acid R. Measure the absorbance (2.2.25) of the test solution
at 570 nm by comparison with the compensation liquid.
Calculate the percentage content of total alkaloids, expressed
as chelidonine, using the following expression:
A x 2.23
m
i.e . taking the specific absorbance of chelidonine 10 be 933 .
A absorbance at 570 nm;
m = mass of the herbal drug to be examined, in grams.
_ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Centaury ***
*** ***
(Ph Eur monograph 1301) ***
~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __
DEFINITION
Whole or fragmented dried flowering aerial parts of
Centaurium erythraea Rafn s. 1. including C. maJus (H. et L.)
Zeltner and C. suffruticosum (Griseb.) Ronn. (syn.: Erythraea
eentaurium Persoon; C. umbellatum Gilibert; C. minus Gars.).
CHARACTERS
Bitter taste.
IDENTIFICATION
A. The hollow cylindrical, light green to dark brown stem has
longitudinal ridges, and is branched only in its upper parto
The sessile leaves are entire, decussately arranged, and have
an ovate to lanceolate lamina, up 10 about 3 cm long. Both
surfaces are glabrous and green to brownish-green.
The inflorescence is diaxially branched. The tubular calyx is
green and has 5 lanceolate, acuminate teeth. The corolla
consists of a whitish tube divided into 5 elongated lanceo late
pink to reddish lobes, about 5-8 mm long. 5 stamens are
present attached to the top of the corolla tube. The ovary is
superior and has a short style, a broad bifid stigma and
numerous ovules. Cylindrical capsules, about 7-10 mm long,
Centaury IV-133
with small brown markedly rough seeds are frequently
presento
B. Reduce 10 a powder (355) (2.9.12). The powder is
greenish-yellow or brownish. Examine under a microscope,
using ehloral hydrate solution R. The powder shows the
following diagnostic characters: fragments from the stem with
lignified groups offibres associated with narrow ves seis,
tracheidal ves seis occasional ves seis with spiral thickening; .
pitted parenchyma of the pith and medullary rays; fragments
of leaf lamina with sinuous epidermal cells and striated
cuticle, especially over the margins and surrounding the
stomata; numerous stomata, mainly anisocytic (2.8.3);
fragments of the palisade mesophyll, each cell containing a
single prism crystal or, les s frequently, a cluster crystal of
calcium oxalate; fragments of calyx and corolla, those of the
calyx with straight-walled epidermal cells, those of the inner
epidermis of the corolla with obtuse papillae and radially
striated cuticle; parts of the endothecium with reticulate or
ridge-shaped wall thickenings; triangularly rounded or
elliptical, yellow pollen grains, about 30 J.lm in diameter, with
a distinctly pitted exine and 3 germinal pores; fragments of
the wall of the fruit capsule composed of crossed layers of
fusiform cells; oil droplets from the seeds, fragments of the
epidermis of the testa showing large, brown reticulations and
a pitted surface.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the powdered drug (355) (2.9.12)
add 25 mL of methanol R, shake for 15 min and filter.
Evaporate the filtrate to dryness under reduced pressure and
at a temperature not exceeding 50 oC. Take up the residue
with small quantities of methanol R so as 10 obtain 5 mL of
solution, which may contain a sedimento
Referenee solution Dissolve 1 mg of rutin R and 1 mg of
swertiamarin R in methanol R and dilute to 1 mL with the
same solvent.
Plate TLC siliea gel F254 plate R (5-40 J.lm) [or TLC siliea gel
F254 plate R (2-10 J.lm)].
Mobile phase water R, anhydrous formie aeid R, ethyl
formate R (4:8:88 V/V/V).
Applieation 10 J.lL [or 5 j.tL] as bands.
Development In an unsaturated tank over a path of 12 cm
[or 6 cm].
Drying In air.
Deteetion A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other less intense quenching
zones may be present in the chromatogram obtained with the
test solution.
Top of the plate
--- ---
--- - --
Swertiamarin : a quenching zone A prominent quenching zone
(swertiamarin)
Rutin: a quenching zone
Reference solution Test solution
Deteetion B Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 mino Examine in daylight.
 IV-134 Centella 2014

Results B See below the sequence of the zones present in B. Reduce the drug to a powder (355) (2.9.12). The powder
the chromatograms obtained with the reference solution and is greenish-grey. Examine under a microscope using ehloral
the test solution. Furthermore, other les s intense coloured hydrate solution R. The powder shows the foIlowing diagnostic
zones may be present in the chromatogram obtained with the characters: numerous fragments of leaf epidermis with
test solution. polygonal ceIls having an irregularly striated cutiele, and
paracytic stomata (2.8.3) that are more numerous in the
lower epidermis; fragments of petiole epidermis with
Top of the plate
elongated ceIls; uniseriate, long, ftexuous uniceIlular covering
--- --- trichomes, occasionaIly multiceIlular; young leaves; spiral
ves seis; resiniferous canals; caIcium oxalate prisms and
--- ---
macIes up to 40 /lm in diameter; bundles of narrow septate
Swertiamarin: a brown zone A brown zone (swertiamarin) fibres from the stem; fragments of the fruit: layers of wide
Rutin: a yellow zone ceIls in a parquetry arrangement, annular vessels,
parenchyma ceIls containing simple or compound starch
A brownish·grey zone granules.
A yellow zone C. Thin-Iayer chromatography (2.2.27).
Test solution To 5.0 g of the powdered drug (355) (2.9.12)
add 50 mL of ethanol (30 per eent V/V] R; heat to boiling
A grey zone
under a reftux condenser and centrifuge.
Reference solution Test solution Referenee solution Dissolve 5 mg of asiaticoside R in
methanol R and dilute to 10 mL with the same solvent.
Plate TLC siliea gel G plate R .
TESTS
Mobile phase aeetie aeid R, formie acid R, water R, ethyl
Foreign matter (2.8.2)
aeetate R (11:11 :27 :100 VIVIV/V) .
Maximum 3 per cent.
Applieation 10 /lL, as bands.
Bitterness value (2.8.15)
Minimum 2000. Development Over a path of 15 cm.
Loss on drying (2.2.32) Drying In airo
Maximum 10.0 per cent, determined on 1.000 g of Deteetion Spray with anisaldehyde solution R and heat at
powdered drug (355) (2.9.12) by drying in an oven at 100-105 oC; examine in daylight.
105 oC for 2 h. Results The chromatograms obtained with the reference
Total ash (2.4.16) solution and the test solution show in the lower third a
Maximum 6.0 per cent. greenish-blue zone (asiaticoside) . The chromatogram
____________________________________________ ~Ew
obtained with the test solution shows also below this zone a
violet zone (madecassoside); near the solvent front it shows a
light blue zone (asiatic acid) and just below a pinkish-violet
zone (madecassic acid); in the lower half it shows brown,
grey and brownish-green zones between the point of
Centella *** application and the zone due to madecassoside, and other
*** *** brownish-yeIlow or Iight yeIlow zones aboye the zone due to
(Ph Eur monograph 1498) *** asiaticoside.
Ph Eur ____________________________________________
TESTS
DEFINITION Foreign matter (2.8.2)
Dried, fragmented aerial parts of Centella asiatica (L.) Urbano Maximum 7 per cent, of which maximum 5 per cent of
underground organs and maximum 2 per cent of other
Content
foreign matter.
Minimum 6.0 per cent of total triterpenoid derivatives,
expressed as asiaticoside (C4sH78019; M r 959 .15) (dried Loss on drying (2.2.32)
drug) . Maximum 10.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
CHARACTERS 105 oC for 2 h.
The leaves are very variable in size; the petiole is usuaIly
5-10, sometimes 15, times longer than the lamina, which is Total ash (2.4.16)
10-40 mm long and 20-40 mm, sometimes up to 70 mm, Maximum 12.0 per cent.
wide. ASSAY
IDENTIFICATION Liquid chromatography (2.2.29).
A. The leaves are alternate, sometimes grouped together at Test solution Place 5 .0 g of the powdered drug (355)
the nodes, reniform or orbicular or oblong-eIliptic and have (2.9.12) in a ceIlulose fingerstaIl in a continuous extraction
palmate nervation, usuaIly with 7 veins, and a crenate apparatus (Soxhlet type). Add 100 mL of methanol R and
margino Young lea ves show a few trichomes on the lower heat for 8 h. Cool and dilute the extract to 100.0 mL with
surface while adult leaves are glabrous . The inftorescence, if methanol R . Filter through a 0.45 /lm filter. Dilute 2.0 mL of
present, is a single umbel which usuaIly consists of 3 ftowers, the filtrate to 20.0 mL with methanol R.
rarely 2 or 4; the ftowers are very smaIl (about 2 mm) Reference solution Dissolve 20.0 mg of asiaticoside R in
pentamerous and have an inferior ovary; the fruit, a methanol R, if necessary using sonication, and dilute to
brownish-grey, orbicular cremocarp, up to 5 mm long, is very
ftattened lateraIly and has 7-9 prominent curved ridges.
2014 Chamomile Flowers IV-135

20.0 mL with the same solvent. Dilute 2.0 mL of this B area of the peak due to madecassoside in the
solution to 100.0 mL with methanol R. chromatogram obtained with the test solution;
Column: C area of the peak due to madecassic acid in the
- size: 1 = 0.25 m, 0 = 4 mm; chromatogram obtained with the test solution;
- stationa¡y phase: octadecylsilyl si/iea gel for chromatography R D area of the peak due to asiatic acid in the
(5 ~lm). chromatogram obtained with the test solution;
Phase mobi/e: mean response factor of asiaticoside.
- mobíle phase A: acetonitrile for chromatography R; _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ __ _ _ _ _ Ph Eur
- mobile phase B: dilute 3 mL of phosphon'c acid R to
1000 mL with water R;

Time Mobile phase A Mobile phase B

Chamomile Flowers ***
(min) (per cen! V/JI) (per cen! V/JI) ** **
0-65 22 78 (Roman Chamomile Flower, Ph Eur monograph 0380) **** *
45 _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ __ __
55
~E~

65 - 66

66 - 76 95 5 DEFINITION
76 - 85 22 78
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 200 run.
Injection 20 ¡lL.
Relative retention With reference to the solvent:
madecassoside = about 5.8; asiaticoside = about 8.1;
madecassic acid = about 17.6; Asiatic acid = about 21. 7.
Calculate the response factor RF of asiaticoside using the
following expression:
Al X VI X 100
mI x HPLCp
area of the peak due to asiaticoside in the
chromatogram obtained with the reference
solution;
volurne of the reference solution, in rnillilitres
rnass of asiaticoside in the reference solution, in
rnilligrarns;
HPLCp purity determined for asiaticoside.
Calculate the mean response factor (RF) for asiaticoside
using the following expression:
N paleae have epidermal cells in longitudinal rows, sclerified at
¿RFi
i=l
N a long, bent, terminal cell about 20 ¡lm wide. The corolla of
N the ligulate flowers consists of papillary cells with cuticular
sum of response factors of asiaticoside for the
L: RFi chromatograms obtained with the reference
i= l
solution;
N number of injections of reference solution
(N = 4, at least).
Calculate the percentage content of total triterpenoid
derivatives, expressed as asiaticoside, using the following
expression:
V [A + (E x 1.017) + (C x 0.526) + (D x 0.509)]
m RF
V volurne of the test solution, in millilitres;
m mass of the substance to be examined in the test
solution, in milligrams;
A area of the peak due to asiaticoside in the
chromatogram obtained with the test solution;
Dried flower-heads of the cultivated double variety of
Chamaemelum nobíle (L.) All. (Anthemis nobilis L.) .
Content
Mínimum 7 mUkg of essential oil (dried drug).
CHARACTERS
The flower-heads are white or yellowish-grey, composed of
solitary hemispherical capitula, made up of a solid conical
receptacle bearing the florets, each subtended by a
transparent small palea.
Strong and characteristic odour.
IDENTIFICATION
A. The capitula have a diameter of 8-20 mm; the receptacle
is solid; the base of the receptacle is surrounded by an
involucre consisting of 2-3 rows of compact and imbricated
bracts with scarious margíns. Most florets are ligulate, but a
few pale yellow tubular florets occur in the central regíon.
Ligulate florets are white, dull, lanceolate and reflexed with a
dark brown, inferior ovary, a filiform style and a bifid stigma;
tubular florets have a five-toothed corolla tube, 5
syngenesious, epipetalous stamens and a gynoeciurn similar
to that of the ligulate florets.
B. Separate the capitulum into its different parts . Examine
under a microscope using chloral hydrate solution R. All parts
of the flower-heads are covered with numerous small yellow
glistening glandular trichomes. The involucral bracts and
the base and they are covered with conical trichomes, about
500 ¡.un long, each composed of 3-4 very short base cells and
striations. The ovaries of both kinds of florets have at their
base a sclerous ring consisting of a single row of cells.
The receptacle and the ovaries contain small clusters of
calciurn oxalate. The pollen grains have a di ame ter of about
35 ¡lm and are rounded or triangular with 3 germinal pores
and a spiny exine.
C. Thin-layer chromatography (2.2.27) .
Test solution To 0.5 g of the powdered drug (710) (2.9.12)
add 10 mL of methanol R and heat with shaking in a water-
bath at 60 oC for 5 min oAlIow to cool and filter.
Reference solution Dissolve 2.5 mg of apigenin R and 2.5 mg
of apigenin 7-glucoside R in 10 mL of methanol R.
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, water R, butanol R
(17:17:66 V/V/V).
Application 10 ¡lL, as bands.
 IV-136 Cinchona Bark 2014

Development Over a path of 10 cm. Content

Drying At 100-105 oC for 5 mino Minimum 6.5 per cent of total alkaloids, of which
Detection Spray the warm plate with a 10 gIL solution of 30 per cent to 60 per cent consists of quinine-type alkaloids
diphenylboric acid aminoethyl ester R in methanol R, using about (dried drug).
10 mL for a plate 200 mm square; subsequently spray with CHARACTERS
the same volume of a 50 gIL solution of macrogol 400 R in Intense bitter, somewhat astringent taste.
methanol R; allow to stand for about 30 min and examine in
IDENTIFICATlON
ultraviolet light at 365 nm.
A. The stem and branch bark is supplied in quilled or curved
Results The chromatogram obtained with the reference pieces 2-6 mm thick. The outer surface is dull brownish-grey
solution shows in the upper third a yellowish-green or grey and frequently bears lichens; it is usually rough,
fiuorescent zone (apigenin) and in the middle third a marked with transverse fissures and longitudinally furrowed
yellowish fluorescent zone (apigenin 7-glucoside) . or wrinkled; exfoliation of the outer surface occurs in sorne
The chromatogram obtained with the test solution shows a varieties. The inner surface is striated and deep reddish-
yellowish-green fluorescent zone and a yellowish fiuorescent brown; the fracture is short in the outer part and fibrous in
zone similar in position and fiuorescence to the zones due to the inner parto
apigenin and apigenin 7-glucoside in the chromatogram
B. Reduce to a powder (355) (2.9.12). The powder is
obtained with the reference solution; aboye the apigenin
reddish-brown. Examine under a microscope using ehloral
7-glucoside zone there is a brownish fluorescent zone
hydrate solution R. The powder shows the following diagnostic
(Iuteolin); immediately below the apigenin 7-glucoside zone
characters (Figure 0174.-1): thin-walled cork cells filled with
there is a light brownish fiuorescent zone (apiin);
reddish-brown contents, in surface view [K] and transverse
immediately below the apiin zone there is a bright blue
section [B]; yellow, spindle-shaped striated phloem fibres up
fiuorescent zone and below this zone a bright blue
to 90 ~Lm in diameter and up to 1300 ¡lm in length, very
fiuorescent zone; other faint zones may be present.
thick-walled with an uneven lumen and with conspicuous,
TESTS funnel-shaped pits, whole [A] or fragmented [F, TI;
Diameter of the flower-heads parenchymatous idioblasts filled with microprisms of calcium
Maximum 3 per cent of fiower-heads have a diameter smaller oxalate [E, G]; clusters of thin-walled phloem parenchyma
than 8 mm. cells [L] accompanied by medullary rays in tangential section
Deteriorated flower-heads [D] . Examine under a microscope using a 50 per cent VIV
Brown or darkened fiower-heads are absent. solution of glycerol R. The powder shows a few starch
granules 6-10 ¡lm in diameter, mostly simple but occasionally
Loss on drying (2.2.32)
with 2 or 3 components, free [B] or included in
Maximum 11.0 per cent, determined on 1.000 g ofthe
parenchymatous cells [C].
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 8.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12) . Use 20.0 g of whole drug, a 500 mL round-
bottomed fiask, 250 mL of water R as the distillation liquid
and 0.50 mL of xylene R in the graduated tube. Distil at a
rate of 3-3.5 mUmin for 3 h.
_ _________________________________________ PhEw

Cinchona Bark ****

*** *
Cinchona; Red Cinchona Bark *** *
(Ph Eur monograph 0174)
Preparation
Cinchona Liquid Extract, Standardised
When Powdered Cinchona is prescribed or demanded,
material complying with the requirements below with the
exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
Ph Eur __________________________________________

DEFINITlON
Whole or cut, dried bark of Cinchona pubescens Vahl
(Cinchona succirubra Pav.), of Cinchona calisaya Wedd., of
Cinchona ledgeriana Moens ex Trimen, or of their varieties or
hybrids. Figure 0174.-1.- Illustration for identification test B of
powdered herbal drug of cinchona bark
 2014 Cinchona Bark IV-137

C. Thin-layer chromatography (2.2.27) . 7 mL of dilute hydrochloric acid R. Heat in a water-bath for

Test solution To 0.10 g ofthe powdered drug (180) (2.9.12) 30 min, allow to cool and add 25 mL of methylene chloride R,
in a test -tube add 0.1 mL of concentrated ammonia R and 50 mL of ether R and 5 mL of a 200 gIL solution of sodium
5 mL of methylene chloride R. Shake vigorously occasionally hydroxide R . Shake the mixture repeatedly for 30 min, add
during 30 min and filter. Evaporate the filtrate to dryness on 3 g of powdered tragacanth R and shake until the mixture
a water-bath and dissolve the residue in 1 mL of anhydrous becomes clear. Filter through a plug of absorbent cotton and
ethanol R. rinse the fiask and the cotton with 5 quantities, each of
Reference solution Dissolve 17.5 mg of quinine R, 2.5 mg of
20 mL, of a mixture of 1 volume of methylene chloride R and
quinidine R, 10 mg of cinchonine R and 10 mg of
2 volumes of ether R. Combine the filtrate and washings,
evaporate to dryness and dissolve the residue in 10.0 mL of
cinchonidine R in 5 mL of anhydrous ethanol R.
anhydrous ethanol R. Evaporate 5.0 mL of this solution to
Plate TLC silica gel plate R.
dryness, dissolve the residue in 0.1 M hydrochloric acid and
Mobile phase diethylamine R, ethyl acetate R, toluene R dilute to 1000.0 mL with the same acid.
(10:20:70 VIV/V). Reference solutions Dissolve separately 30.0 mg of quinine R
Application 10 i-!L as bands. and 30.0 mg of cinchonine R in 0.1 M hydrochloric acid and
Development Twice over a path of 15 cm. dilute each solution to 1000.0 mL with the same acid.
Drying At 100-105 oC, then allow to cool. Measure the absorbances (2.2.25) of the 3 solutions at
Detection A Spray with anhydrous fOr111ic acid R and allow to 316 nm and 348 nm using 0.1 M hydrochloric acid as the
dry in air; examine in ultraviolet light at 365 nm. compensation liquido
Results ASee below the sequence of zones present in the Calculate the percentage content of alkaloids using the
chromatograms obtained with the reference solution and the following equations:
test solution. Furthermore, other fiuorescent zones are
present in the chromatogram obtained with the test solution. [A 316 x A348c] - [A 316c x A 348 ] 100 2
x= x x
[A 316q x A 348c ] - [A 316c x A348q] m 1000
Top of the plate
--- --- [A316 x A348q] - [A316q x A348] 100 2
y= X X --
Quinidine: a distinct blue A distinct blue fluorescent zone [A 31 6c x A348q]- [A316q x A348c] m 1000
fluorescent zone (quinidine)
--- ---
m mas s of the drug used, in grams;
Quinine: a distinct blue fluorescent A distinct blue fluorescent zone x percentage content of quinine-type alkaloids;
zone (auinine)
percentage content of cinchonine-type alkaloids;
Reference solution Test solution
absorbance of the test solution at 316 nm;
absorbance of the test solution at 348 nm;
Detection B Spray with iodoplatinate reagent R. absorbance of the reference solution containing
Results B See below the sequence of zones present in the cinchonine at 316 nm, corrected to a
concentration of 1 mg/1000 mI;
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones are present in the absorbance of the reference solution containing
chromatogram obtained with the test solution. quinine at 316 nm, corrected to a concentration of
1 mg/1000 mI;
absorbance of the reference solution containing
Top of the plate cinchonine at 348 nm, corrected to a
--- --- concentration of 1 mg/1000 mi;
absorbance of the reference solution containing
Cinchonine: a violet zone that A violet zone that becomes
becomes violet-grey violet-grey (cinchonine)
quinine at 348 nm, corrected to a concentration of
Quinidine: a violet zone that A violet zone that becomes
1 mg/1000 mI.
becomes violet-grey violet-grey (quinidine) Calculate the content of total alkaloids (x + y), and calculate
Cinchonidine: an intense dark An intense dark blue zone the relative content of quinine-type alkaloids using the
blue zone (cinchonidine)
---
following expression:
---

Quinine: a violet zone that A violet zone that becomes 100x

becomes violet-¡<rey violet-¡<rev (auinine)
Reference solution Test solution x+y

___ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ __ _ _ _ PhE~

TESTS
Total ash (2.4.16)
Maximum 6.0 per cent.
Loss 00 dryiog (2.2.32)
Maximum 10 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
ASSAY
Test solution In a 250 mL conical fiask mix 1.000 g of the
powdered drug (180) (2.9.12) with 10 mL of water R and
IV-138 Cinchona Preparations 2014

*****
Top of the plate
Standardised Cinchona Liquid
** ** --- ---
Extract *** Cinchonine: a violet·grey zone A violet-grey zone (cinchonine)
(Ph Eur monograph 1818)
PhEur _ __ _ _ _ __ _ _ _ __ _ _ __ __ _ __ Quinidine: a violet·grey zone A violet·grey zone (quinidine)
Cinchonidine: an intense dark blue An intense dark blue zone
DEFINITION zone (cinchonidine)
Liquid extraet produeed from Cinehona bark (0174). --- ---
Content Quinine: a violet·grey zone A violet·grey zone (quinine)
Minimum 4.0 per eent and maximum 5.0 per eent of total
Reference solution Test solution
alkaloids, of whieh 30 per eent to 60 per eent are alkaloids of
the quinine type (C2oH24N20 2; M r 324.4).
PRODUCTION TESTS
Standardised einchona liquid extraet is produeed from the Ethanol (2.9.10)
herbal drug by an appropriate proeedure using: 95 per eent to 105 per eent of the eontent stated on the
- ethanol (30 per eent VIV to 90 per eent V/V), oró labe!.
- a mixture of diluted hydrochlorie aeid, ethanol Methanol and 2-propanol (2.9.11)
(96 per eent V/V), glycerol, water (1 :2:5:20 V/V). Maximum 0.05 per cent VIV of methanol and maximum
CHARACTERS 0.05 per eent VIV of 2-propano!.
Appearance Dry residue (2.8.16)
Brownish-red liquido Minimum 12.0 per eent for glyeerol-free standardised
It has a bitter, astringent taste. einehona liquid extraet and minimum 30.0 per eent for
glyeerol-eontaining standardised cinchona extract, determÍned
IDENTIFICATION
on 2.0 g.
Thin-Iayer chromatography (2.2.27) .
Test solution Dilute 1 mL of the extraet to be examined in ASSAY
1 mL of anhydrous ethanol R. Test solution In a 250 mL conical fiask, mix about 1.000 g
of the extraet to be examined with 10 mL of water R and
R ef erenee solution Dissolve 2.5 mg of quinidine R, 10 mg of
7 mL of dilute hydroehlorie acid R. Heat in a water-bath for
cinehonidine R, 10 mg of cinehonine R and 17.5 mg of
30 min, allow to eool and add 25 mL of methylene ehloride R,
quinine R in 5 mL of anhydrous ethanol R.
50 mL of ether R and 5 mL of a 200 giL solution of sodium
Plate TLC siliea gel plate R (5-40 J.lm) [or TLC si/iea gel hydroxide R. Shake the mixture frequently for 30 min, add
plate R (2-10 J.lm)]. 3 g of powdered tragaeanth R and shake Ulltil the mixture
Mobile phase diethylamine R, ethyl aeetate R, toluene R becomes clear. Filter through a plug of absorbent eotton,
(10 :20:70 VIV/V) . rinse the fiask and the cotton with 5 quantities, eaeh of
Applieation 10 J.lL [or 2 J.lL] as bands. 20 mL, of a mixture of 1 volume of methylene ehloride R and
D evelopment Twiee over a path of 15 cm [or 6 cm]. 2 volumes of ether R. Combine the filtrate and washings,
evaporate to dryness and dissolve the residue in 10.0 mL of
Drying At 100-105 oC then allow to eoo!.
ethanol (96 per eent) R. Evaporate 5.0 mL of this solution to
Detection A Spray with a 50 giL solution of anhydrous formie dryness, dissolve the residue in 0.1 M hydroehlorie acid and
acid R and allow to dry in airó examine in ultraviolet light at dilute to 1000.0 mL with the same aeid.
365 nm.
Referenee solution (a) Dissolve 30.0 mg of cinehonine R in
Results ASee below the sequenee of the zones present in 0. 1 M hydroehloric aeid and dilute to 1000.0 mL with the
the ehromatograms obtained with the reference solution and same aeid.
the test solution. Furthermore, other fiuoreseent zones may
Referenee solution (b) Dissolve 30.0 mg of quinine R in
be present in the chromatogram obtained with the test
0.1 M hydroehlorie acid and diJute to 1000.0 mL with the
solution.
same acid.
Measure the absorbances (2.2.25) of the 3 solutions at
Top of the plate 316 nm and 348 nm, using 0.1 M hydroehlorie acid as the
--- --- eompensation liquid.
Quinidine : a distinct blue f1uorescent A distinct blue f1uorescent zone Calculate the pereentage content of alkaloids from the
zone (quinidine) following equations:
- -- ---

Quinine: a distinct blue fluorescent A distinct blue fluorescent zone

zone _lquinin~
Reference solution Test solution

Detection B Spray with iodoplatinate reagent R .

R esults B See below the sequenee of the zones present in
the ehromatograms obtained with the reference solution and mass of the liquid extraet to be examÍned in grams;
the test solution. Furthermore, other zones may be present in pereentage eontent of quinine-type alkaloids;
the ehromatogram obtained with the test solution. pereentage content of cinehonine-type alkaloids;
absorbanee of the test solution at 316 nm;
absorbanee of the test solution at 348 nm;
 2014 Cinnamon IV-139

Ala absorbance ofreference solution (a) at 316 nm,

corrected to a concentration of 1 mg!1000 mL;
Alb absorbance ofreference solution (b) at 316 nm,
corrected to a concentration of 1 mg!1000 mL;
A 2a absorbance of reference solution (a) at 348 nm,
corrected to a concentration of 1 mg!1000 mL;
A 2b absorbance of reference solution (b) at 348 nm,
corrected to a concentration of 1 mg!1000 mL.
Calculate the content of total alkaloids (nI + n2), and the
relative content of quinine-type alkaloids, from the following
expression:

LABELLING
The label states the solvent composition used for the
production.
____________________________________________ ~Ew

Cinnamon
Cinnamon Bark; Ceylon Cinnamon
(Ph Eur monograph 0387)
Preparation
Cinnamon Tincture J
When Powdered Cinnamon is prescribed or demanded,
material complying with the requirements beJow with the
Figure 0387.-1. - Illustratian far identificatian test B af
exception of Identification test A and containing not less than
pawdered herbal drug af cinnaman
1.0% v/w (10 mUkg) of essential oil shall be dispensed or
supplied.
~Ew ____________________________________________ C. Thin-layer chromatography (2.2.27).
Test solution Shake 0.1 g ofthe powdered drug (500)
DEFINITION (2.9.12) with 2 mL of methylene ehloride R for 15 min o Filter
Dried bark, freed from the outer cork and the underlying and evaporate the filtrate carefully almost to dryness on a
parenchyma, of the shoots grown on cut stock of water-bath. Dissolve the residue in 0.4 mL of toluene R.
Cinnamomum verum lPres!.
Referenee solution Dissolve 50 ¡.tL of cinnamie aldehyde R and
Content 10 ¡.tL of eugenol R in toluene R and dilute to 10 mL with the
Minimum 12 mUkg of essential oi!. same solvento
CHARACTERS Plate TLC si/iea gel GF254 plate R.
Characteristic, aromatic odour. Mobile phase methylene ehlonde R.
IDENTIFICATION Applieation 10 ¡.tL as bands of 20 mm by 3 mm.
A. The bark is about 0.2-0.8 mm thick and occurs in closely Development Over a path of 10 cm.
packed compound quills made up of single or double quills. Drying In air.
The outer surface is smooth, yellowish-brown with faint scars
Deteetion A Examine in ultraviolet light at 254 nm and
marking the position of leaves and axillary buds and has fine,
mark the quenching lones, then examine in ultraviolet light
whitish and wavy longitudinal striations. The inner surface is
at 365 nm and mark the fiuorescent lones.
slightly darker and longitudinally striated. The fracture is
short and fibrous . Results A Examined in ultraviolet light at 254 nm, the
chromatograms obtained with the test solution and the
B. Microscopic examination (2.8.23). The powder is
reference solution show a quenching lone due to
yellowish or reddish-brown. Examine under a microscope
cinnamaldehyde in the median part and, just aboye it, a
using ehloral hydrate solution R. The powder shows the
weaker quenching lone due to eugenol; examined in
following diagnostic characters (Figure 0387.-1): rounded
ultraviolet light at 365 nm, the chromatogram obtained with
scJereids with pitted, cha=elled and moderately thickened
the test solution shows a fiuorescent light blue lone due to
walls, single [E, F] or in groups [C); numerous colourless,
o-methoxycinnamaldehyde just below the lone due to
single fibres, often whole [A], or fragmented [D], with a
cinnamaldehyde.
narrow lumen, thickened, lignifted walls and few pits; small
acicular crystals of ca1cium oxalate in parenchymatous cells Deteetian B Spray with phloroglucinol solution R.
m; very numerous oil droplets [B] . Cork fragments [G] are Results B The lone due to cinnamaldehyde is yellowish-
absent or very rare. Examine under a microscope using a brown and the lone due to o-methoxyci=amaldehyde is
50 per cent VIV solution of glyeerol R . The powder shows violeto
abundant starch granules [H].
 IV-140 Ceylon Cinnamon Bark Gil 2014

TESTS Refractive index (2.2.6)

Total ash (2.4.16) 1.572 to 1.591.
Maximum 6.0 per cent. Optical rotation (2.2. 7)
ASSAY _ 2° to + 1°.
Carry out the determinarion of essenrial oils in herbal drugs Chromatographic pro file
(2.8.12). Use 20.0 g of drug reduced to a powder (710) Gas chromatography (2.2.28): use the normalisation
(2.9.12) immediately before the determination, a 500 mL procedure.
fiask, 200 mL of 0.1 M hydrochloric acid as the disrillation Test solution The essential oil to be examined.
liquid, and 0.50 mL of xylene R in the graduated tube. Distil
Reference solution Dissolve 10 ¡.tL of cineole R, 10 ¡.tL of
at arate of 2.5-3.5 mUmin for 3 h.
linalol R, 10 ¡.tL of ~-caryophyllene R, 10 ¡.tL of safrole R,
____________________________________________ ~E~

100 ¡.tL of trans-cinnamic aldehyde R, 10 ¡.tL of eugenol R,

20 mg of coumarin R, 10 ¡.tL of trans-2-
methoxycinnamaldehyde R and 10 ¡.tL of benzyl benzoate R in
1 mL of acetone R.

Ceylon Cinnamon Bark Oil *** Column:

** ** -- material: fused silica;
Cinnamon Oil ***** -- size: 1 = 60 m, 0 = 0.25 mm;
(Ph Eur monograph 1501) -- stationary phase: bonded macrogol 20 000 R.
Preparation Cam'er gas helium for chromatography R.
Concentrated Cinnamon Water Flow rate 1.5 mUmin.
~E~ ____________________________________________ Split ratio 1:1 OO.
DEFlNlTlON Temperature:
Essential oil obtained by steam distillation of the bark of the Time Temperature
(min) (OC)
shoots of Cinnamomum verum J.Presl.
Column 0 · 10 60
CHARACTERS
10·75 60 -) 190
Appearance
Clear, mobile, light yellow liquid becoming reddish over 75 - 200 190
time. Injection port 200
Characterisric odour reminiscent of cinnamic aldehyde.
Detector 240
IDENTIFICATlON
First identification B. Detectíon F1ame ionisation.
Second identification A. Injection 0.2 ¡.tL.
A. Thin-Iayer chromatography (2.2.27) . Elution order arder indicated in the composition of the
reference solution; depending on the operating conditions
Test solution Dissolve 1 mL of the essential oil to be
and the state of the column, coumarin may elute before or
examined in acetone R and dilute to 10 mL with the same
solvent. after trans-2-methoxycinnamaldehyde; record the retention
times of these substances .
Reference solution Dissolve 50 ¡.tL of trans-cinnamic
System suitability Reference solution:
aldehyde R, 10 ¡.tL of eugenol R, 10 ¡.tL of linalol R and 10 ¡.tL
-- resolution: minimum 1.5 between the peaks due to linalol
of ~-caryophyllene R in ethanol (96 per cent) R and dilute to
and p-caryophyllene.
10 mL with the same solvento
Plate TLC silica gel plate R . Identificatían of components Using the retention times
determined from the chromatogram obtained with the
Mobile phase methanol R, toluene R (10:90 V/V). reference solution, locate the components of the reference
Application 10 ¡.tL as bands. solution in the chromatogram obtained with the test solution.
Development Over a path of 15 cm. Determine the percentage content of each of these
Drying In air. components. The percentages are within the following
Detection Spray with anisaldehyde solution R; heat at ranges:
100-105 oC for 5-10 min and examine in daylight. -- cineole: maximum 3.0 per cent;
-- linalol: 1.0 per cent to 6.0 per cent;
Results The zones in the chromatogram obtained with the
-- ~-caryophyllene: 1.0 per cent to 4.0 per cent;
test solution are similar in position and colour to those in the
-- safrole: maximum 0.5 per cent;
chromatogram obtained with the reference solution.
-- trans-cinnamic aldehyde: 55 per cent to 75 per cent;
B. Examine the chromatograms obtained in the test for -- eugenol: maximum 7.5 per cent;
chromatographic profile. -- coumarin: maximum 0.5 per cent;
Results The principal peaks in the chromatogram obtained -- trans-2-methoxycinnamaldehyde: 0.1 per cent to
with the test solution are similar in retention time to those in 1. O per cent;
the chromatogram obtained with the reference solution. -- benzyl benzoate: maximum 1.0 per cent.
Safrole, coumarin and cineole may be absent from the
STORAGE
chromatogram obtained with the test solution.
Protected ftom heat.
TESTS ____________________________________________ PhE~

Re1ative density (2.2.5)

1.000 to 1.030.
2014 Ceylon Cinnamon Leaf Oil IV-141

Top oí tbe plate

Cinnamon Tincture
1 blue zone (terpenhydrocarbons)
(Ph Eur monograph 1819)
----- -----
~Ew ____________________________________________
Eugenol: a blue zone A blue zone (eugenol)
DEFINITION
Trans-cinnamic aldehyde: a blue A blue zone (trans-dnnamic
Tincture produced from Cinnamon (0387). zone aldehyde)
PRODUCTION Trans-2-methoxycinnamaldehyde: A weak orange-brown zone
an orange-brown zone (the colour (trans-2-methoxycinnamaldehyde)
The tincture is produced from 1 part of the drug and 5 parts fades away)
of ethanol (70 per cent V/V) by an appropriate procedure. ----- -----

CHARACTERS 2 or 3 blue zones above the line of

Appearance application
Clear, brownish-red liquid, with a characteristic odour. Reference solution Test solution

IDENTIFICATION
Thin-layer chromatography (2.2.27). TESTS
Test solution Place 10 mL of the tincture to be examined, Ethanol (2.9.10)
10 mL of saturated sodium ehloride solution R and 5 mL of 64 per cent VIV to 70 per cent VIV.
toluene R in a ground glass-stoppered tube. Shake for 2 min
Methanol and 2-propanol (2.9.11)
and centrifuge for 10 mino Use the organic layer. Maximum 0.05 per cent V/V of methanol and maximum
Referenee solution Dissolve 5 ¡lL of eugenol R, 0.05 per cent V/V of 2-propanol.
25 ¡lL of trans-cinnamie aldehyde R and 5 ¡lL of
Dry residue (2.8.16)
trans-2-methoxycinnamaldehyde R in toluene R and dilute to
Minimum 1.5 per cent m/m, determined on 5.0 g.
10 mL with the same solvento
____________________________________________ PhEw
Plate TLC siliea gel G plate R.
Mobile phase methylene ehloride R.
Applieation 20 ¡lL, as bands.
Development Over a path of 10 cm.
Drying In airo
Concentrated Cinnamon Water
Deteetion A Examine in ultraviolet light at 365 nm. DEFINITION
Cinnamon Oil 20 mL
Results ASee below the sequence of the zones present in
Ethanol (90 per cent) 600 mL
the chromatograms obtained with the reference solution and
Water Sufficient to produce 1000 mL
the test solution.
Extemporaneous preparation
The following directions apply.
Top oí the plate
Dissolve the Cinnamon Oil in the Ethanol (90 per cent) and
----- ----- add gradually, with vigorous shaking after each addition,
Trans·2·methoxycinnamaldehyde: a A light blue fluorescent zone sufficient Water to produce 1000 mL. Add 50 g of previously
light blue fluorescent zone (trans-2- methoxycinnamaldehyde) sterilised Purified Talc, or other suitable filtering aid, allow to
----- ----- stand for a few hours, shaking occasionally, and filter.
A greenish fluorescent zone (above The water eomplies with the requirements stated under Aromatic
the line of application) Waters and with the following requirements.
TESTS
Reference solution Test solution
Ethanol content
52 to 56% v/v, Appendix VIII F.
Detection B Spray with a 200 gIL solution of Weight per mL
phosphomolybdie acid R in ethanol R. Examine in daylight 0.914 to 0.922 g, Appendix V G.
while heating at 100-105 oC for 5-10 mino
Results B See below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones may be present in
Ceylon Cinnamon Leaf Oil ****
the chromatogram obtained with the test solution. *** *
(Ph Eur monograph 1608) *** *
~Ew ____________________________________________

DEFINITION
Oil obtained by steam distillation of the leaves of
Cinnamomum verum J,S. Presl.
CHARACTERS
Appearance
Clear, mobile, reddish-brown or dark brown liquido
Characteristic odour reminiscent of eugenol.
 IV-142 Cítronella Oíl 20 14

IDENTIFICATION Temperature:
First identification B. Time Temperature
Second identification A. (min) (oC)

A. Thin-Iayer chromarography (2.2.27). Column 0·10 45

10·78 45 -7 180
Test solution Dilute 1 mL of the substance ro be examined 78·88 180
in acetone R and dilute to 10 mL with the same solvento lnjection port 200
R eference solution Dilute about 50 ¡.¡L of trans-cinnamic
Detector 240
aldehyde R , 10 flL of eugenol R , 10 ¡.tL of linalol R and 10 ¡.tL
of ~-caryophyllene R in alcohol R and dilute ro 10 mL with the Detection Flame ionisation.
same solvento
Injection 0.2 ¡.tL.
Plate TLC siliea gel place R .
Elution order The order indicated in the composition of the
Mobile phase methanol R , toluene R (10:90 V/V). reference solution. Record the retention times of these
Application 10 flL, as bands . substances.
D evelopment Over a path of 15 cm. System suitability Reference solution:
D rying In airo - resolution: minimum of 1.5 between the peaks due ro
Deteetion Spray with anisaldehyde solution R . Examine in day linalol and ~- caryophyllene .
light while heating at 100-105 oC for 5-10 mino Using the retention times determined from the
R esults The zones in the chromatogram obtained with the chromarogram obtained with the reference solution, locate
test solution are similar in position and colour to those in the the components of the reference solution in the
chromatogram obtained with the reference solution. chromatogram obtained with the test solution.
The zone due to trans-cinnamic aldehyde may be very faint Determine the percentage content of these components.
or absent. The percentages are within the following ranges:
B. Examine the chromatogram obtained in the test for - eineole: maximum 1.0 per cent,
chromatographic pro file. - linalol: 1.5 per cent ro 3.5 per cent,
R esults The characteristic peaks in the chromarogram - ~-earyophyllene: 1.5 per cent ro 7.0 per cent,
obtained with the test solution are similar in retention time ro - safrole: maximum 3.0 per cent,
those in the chromarogram obtained with the reference - trans-cinnamic aldehyde: maximum 3.0 per cent,
solution. The peaks corresponding to cineole, safrole, - cinnamyl acetate: maximum 2.0 per cent,
trans-cinnamic aldehyde, cinnamyl acetate and coumarin may - eugenol: 70 per cent to 85 per cent,
be absent in the chromatogram obtained with the test - eouman·n: maximum 1.0 per cent.
solution. STORAGE
TESTS Protected from heat.
Relative density (2.2.5) __________________________________________ ~Ew

1.030 to 1.059.
Refractive index (2.2.6)
1.527 to 1.540.
Optical rotation (2.2.7) Citronella Oi! *****
** *
- 2.5° to + 2.0°.
(Ph Eur monograph 1609) ****
Chromatographic pro file ~Ew ___________________________________________
Gas chromatography (2.2.28): use the normalisation
procedure. DEFINITION
Test solution The substance to be examined. Oil obtained by steam distillation from the fresh or partially
dried aerial parts of Cymbopogon winterianus Jowitt.
R eference solution Dissolve 10 ¡.tL of cineole R, 10 ¡.tL of
linalol R, 10 flL of ~-earyophyllene R , 10 ¡.tL of safrole R, CHARACTERS
10 ¡.tL of trans-cinnamic aldehyde R, 10 ¡.tL of cinnamyl Appearance
acetate R, 100 ¡.tL of eugenol R and 10 mg of coumarin R in Pale yellow or brown-yellow liquid.
1 mL of acetone R. Very strong odour of citronellal.
Column:
IDENTIFICATION
- material: fused silica,
First identification B.
- size: 1 = 60 m, 0 = 0.25 mm,
- stationary phase: macrogol 20 000 R. Second identification A.
Carrier gas helium for chromatography R. A. Thin-layer chromatography (2.2.27).
Flow rate 1.5 mUmin. Test solution Dilute 0.1 g of citronella oil in 10.0 mL of
alcohol R .
Split ratio 1/100.
Reference solution Dilute 20 f!L of citronellal R in 10.0 mL of
alcohol R.
Plate TLC silica gel plate R.
M obile phase ethyl acetate R, toluene R (10:90 V/V) .
Applieation 5 ¡.tL, as bands.
Development Over a path of 15 cm.
Drying In airo
2014 Clematis Armandii Stem IV-143

Detection Spray with anisaldehyde solution R and heat at System suitabllity Reference solution:
100-105 oC for 10 mino Examine in ultraviolet light at -- reso!ution: minimum of 1.2 between the peaks due to
365 nm. geranyl acetate and citroneIlol.
Result See below the sequence of the zones present in the Using the retention times determined from the
chtomatograms obtained with the reference and test chromatogram obtained with the reference solution, locate
solutions. Furthermore, other zones are present in the the components of the reference solution in the
chroma1Ogram obtained with the test solution. chtomatogram obtained with the test solution.
Determine the percentage content of each of these
components.
Top of the plate
The percentages are within the foIlowing values:
Citronellal: a violet zone A zone similar in colour to the
citronellal zone
-- limonene: 1.0 per cent 10 5.0 per cent,
An orange zone (citronellol·geraniol)
-- citronella!: 30.0 per cent to 45.0 per cent,
-- citronelly! acetate: 2.0 per cent ro 4.0 per cent,
Reference solution Test solution -- neral: maximum 2.0 per cent,
-- gerania!: maximum 2.0 per cent,
-- gerany! acetate: 3.0 per cent to 8.0 per cent,
B. Examine the chromatograms obtained in the test for -- citronellol: 9.0 per cent to 15.0 per cent,
chromatographic profile. -- geranio!: 20.0 per cent to 25 .0 per cent.
Results The characteristic peaks in the chtomatogram ____________________________________________ ~E~

obtained with the test solution are similar in retention time to

those in the chromatogram obtained with the reference
solution. Neral and geranial may be absent in the
Clematis Armandii Stem ****
***
chtoma1Ogram obtained with the test solution.
*
TESTS (Ph Eur monograph 2463) *** *
Relative density (2.2.5) ~E~ ____________________________________________
0.881 to 0.895.
DEFINITION
Refracdve index (2.2.6)
Whole or fragmented, dried stem of Clematis annandii
1.463 to 1.475.
Franch., with cork removed, coIlected in spring or autumn.
Opdcal rotadon (2.2.7)
Content
- 4° to + 1.5°.
Minimum 0.30 per cent of oleanolic acid (C30H4S03; M r
Chromatographic profile 456.7) (dried drug).
Gas chromatography (2.2.28): use the normalisation
procedure. IDENTIFICATION
A. The whole stem is long and cylindrical, slightly twisted on
Test solution The substance to be examined.
itself, about 1-6.5 cm in diameter. It shows nodes, usuaIly
Reference solution Dilute 25 J..lL of limonene R, 100 J..lL of swoIlen, with leaf and branch scars. The outer surface is
citronellal R, 25 J..lL of citronellyl acetate R, 25 J..lL of citral R, brownish-yeIlow or duIl brownish-yeIlow, showing
25 J..lL of geranyl acetate R, 25 J..lL of citronello! R and 100 J..lL longitudinal gro oves and striations corresponding to the ends
of geranio! R in 5 mL of hexane R. of the meduIlary rays. Rare cork remnants are easily removed
Co!umn: as longitudinal strips. The texture is hard. The fracture is
-- materia!: fused silica, difficuIt.
-- size: l = 60 m, 0 = 0.25 mm, The fragmented stem occurs in thick slices, about 2-5 mm
-- stationary phase: macrogol20 000 R (0.2 J..lm) . thick, with uneven margins; most of the transverse section
Cam'er gas helium for chromatography R. consists of the pale yeIlow or slightly brownish-yeIlow wood
Flow rate 1.0 mlJmin. and shows numerous radial striations and cracks
Split ratio 1: 1OO. corresponding to the meduIlary rays; the ves seis are cIearly
visible in transverse section. The pale yeIlow or whitish pith,
Temperature:
sometimes replaced by a hoIlow, is reduced .
Time Temperature
B. Microscopic examination (2.8.23) . The powder is
(min) (OC)
brownish-yeIlow. Examine under a microscope using chloral
Column O" 2 80
hydrate solution R. The powder shows the foIlowing diagnostic
2" 26 80 ~ 150 characters: very numerous fragments of ves seis, up to
26" 42 150 ~ 185 250 J..lm in diameter, with pitted waIls, isolated or associated
with elongated tracheids about 15-25 J..lm in diameter with
42" 49 185 ~ 250
lignified, thickened and pitted waIls; fibres 25-30 ~lm in
Injection port 260 diameter, with narrow lumen and thick and partly, slightly
Detector 260 pitted waIls; parenchymatous ceIls of the secondary phloem
and outer parts of the meduIlary rays, thin-waIled, from the
Detection Flame ionisation. secondary xylem, inner parts of the meduIlary rays and pith,
Injection 1 J..lL of the reference solution, 0.2 J..lL ofthe test with slightly thickened, pitted and lignified ceIl waIls; sub-
solution. rectangular or fusiform scIereids, about 100 J..lffi long and
35 J..lm wide, with thick and pitted waIls; rare orange-brown
E!ution order The order indicated in the composition of the
cork fragments. Examine under a microscope using a
reference solution. Record the retention times of these
50 per cent V/V solution of glycerol R. The powder shows
substances.
rare starch granules, simple or 2-3 compound, spherical or
 IV-144 Clove 2014

ova te, individual granules up to 17 ¡.tm in diameter, with a residue in 10.0 mL of methanol R, shake and filter through a
punctiform or slit-shaped hilum. membrane filter (nominal pore size 0.45 ¡.tm).
C. Thin-layer chromatography (2.2.27). Referenee solution (a) Dissolve 10.0 mg of oleanolie acid CRS
Test solurion To 1 g of the powdered herbal drug (1400) in methanol R and dilute to 20.0 mL with the same solvento
(2.9.12) add 5 mL of methanol R and heat on a water-bath at Referenee solution (b) Dissolve 5.0 mg of ursolic aeid R in
60 oC for 5 min. Filter. reference solution (a) and dilute to 10.0 mL with the same
Reference solution Dissolve 4 mg of hederagenin R and 4 mg solution.
of oleanolic acid R in 10 mL of merhanol R. Column:
Plate TLC silica gel F 254 plate R (5-40 ¡.tm) [or TLC si/iea gel -- siz e: l == 0.25 m, 0 == 4.6 mm;
F Z54 plate R (2-10 ¡.tm)). -- stationary phase: end-capped oetadeeylsilyl si/iea gel for
ehromatography R (5 ¡.tm);
MoblZe phase aeelÍe aeid R, aeetone R, toluene R
-- temperature: 30 oc.
(2 :8:32 VIV/V).
Mobi/e phase 0.4 per cent VIV solution of aeetie aeid R,
Applieation 40 ¡.tL [or 10 ¡.tL) as bands of 10 mm [or
methanol R (15:85 V/V).
8 mm).
Flow rate 1.0 mUmin.
Development Over a path of 13 cm [or 6 cm).
Deteetion Spectrophotometer at 210 nm.
Drying In air.
Injeetion 20 ¡.tL.
DereclÍon Treat with vanillin reagent R, heat at 100 oC for
5 min and examine in daylight. Run time 1.2 times the retention time of ursolic acid.
R esults See below the sequence of zones present in the Retention time Oleanolic acid == about 21 min;
chromatograms obtained with the reference solution and the ursolic acid == about 22 mino
test solution. Furthermore, other mainly grey zones may be System suitability Reference solution (b):
present in the chromatogram obtained with the test solution. -- resolution: minimum 1.3 between the peaks due to
oleanolic acid and ursolic acid.
Calculate the percentage content of oleanolic acid using the
Top of the plate
following expression:

--- ---
A bluish·violet zone

Oleanolic acid: a reddish·violet A reddish·violet zone area of the peak due to oleanolic acid in the
zone chromatogram obtained with the test solution;
A weak light blue or grey zone area of the peak due to oleanolic acid in the
--- --- chromatogram obtained with reference solution (a);
mass of the herbal drug to be examined used to
Hederagenin: a greenish·brown prepare the test solution, in grams;
zone
mass of oleanolic aeid CRS used to prepare reference
An orange zone
solution (a), in grams;
Reference solution Test solution p percentage content of oleanolic acid in oleanolie
acid CRS.
_ _ _ _ _ _ __ _ _ _ _ ________________ ~E~

TESTS
Aristolochia manshuriensis Kom. and other species of
Aristolochia
Clove ***
*** ***
Examine the powdered herbal drug (355) (2.9.12) under a
microscope using ehloral hydrate solution R; no cluster crystals
are visible. (Ph Eur monograph 0376) ***
When Powdered Clove is prescribed or demanded, material
Aristolochic acids (2.8.21, Method A)
complying with the requirements below with the exception of
It complies with the test.
Identification test A and the test for Foreign matter and
Loss on drying (2.2.32) containing not less than 12.0% v/w (120 mUkg) of essential
Maximum 12.0 per cent, determined on 1.000 g of the oil shall be dispensed or supplied.
powdered herbal drug (1400) (2.9.12) by drying in an oven ~E~ ____________________________________________
at 105 oC for 2 h.
Total ash (2.4.16) DEFINITION
Maximum 3 .0 per cent. Whole ftower buds of Syzygium aromaticum (L.) Merr.
et L.M.Perry (syn. Eugenia earyophyllus (Spreng.) Bullock et
ASSAY S.G.Harrison) dried until they become reddish-brown.
Liquid chromatography (2.2.29).
Content
Test solution Disperse 1.00 g of the powdered herbal drug Minimum 150 mUkg of essential oi!.
(355) (2.9.12) in methanol R, add 3 mL of 6 M hydroehlorie
acid R and dilute to 30.0 mL with methanol R. Shake for 2 h. CHARACTERS
Filter, add to the filtrate 10 mL of water R by rinsing the Characteristic, aromatic odour.
ftask and the filter, and extract with 3 quantities, each of IDENTIFICATION
30 mL, of methylene ehloride R. Combine the methylene A. The ftower bud is reddish-brown and consists of a
chloride extracts and evaporate to dryness. Dissolve the quadrangular stalked portion, the hypanthium, 10-12 mm
 2014
long and 2-3 mm in diameter, surmounted by 4 divergent
lobes of sepals which surround a globular head 4-6 mm in
di ame ter. A bilocular ovary containing numerous ovules is
situated in the upper part of the hypanthium. The head is
globular and dome-shaped, composed of 4 imbricated petals
that enclose numerous incurved stamens and a short, erect
style with a nectary disc at the base. The hypanthium exudes
essential oil when indented with the finger-nail.
B. Reduce to a powder (355) (2.9.12). The powder is dark
brown and has the odour and taste of the unground drug.
Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters:
fragments of the hypanthium showing the epidermis and
underlying parenchyma containing large oil glands; short
fibres occurring singly or in small groups, with thickened,
lignified walls and few pits; abundant fragments of
parenchyma containing cluster crystals of ca1cium oxalate;
numerous triangular pollen grains about 15 ¡.tm in diameter
with 3 pores in the angles. Starch granules are absent.
C. Thin-Iayer chromatography (2.2.27).
Test solution Shake 0.1 g ofthe powdered drug (500)
(2.9.12) with 2 mL of methylene chloride R for 15 mino Filter
and carefully evaporate the filtra te to dryness on a water-
bath. Dissolve the residue in 2 mL of toluene R.
Reference solution Dissolve 20 ¡.tL of eugenol R in 2 mL of
toluene R.
Plate TLC silica gel GF254 plate R.
Mobile phase toluene R.
Application 10 ¡.tL of the reference solution and 20 ¡.tL of
the test solution, as bands of 20 mm by 3 mm.
Development Twice, in an unsaturated tank over a path of
10 cm; allow the plate to stand for 5 min between the 2
developments.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm and
mark the quenching zones.
Results A In the chromatogram obtained with the test
solution there is in the median part a quenching zone due to
eugenol similar in position 10 the quenching zone in the
chromatogram obtained with the reference solution and there
may be a weak quenching zone due to acetyleugenol just
below the zone due to eugenol.
Detection B Spray with anisaldehyde solution R using 10 mL
for a plate 200 mm square and heat at 100-105 oC for
5-10 min. Examine in daylight.
R esults B The zones due to eugenol in the chromatograms
obtained with the test and reference solutions are strong
brownish-violet and the zone due to acetyleugenol in the
chromatogram obtained with the test solution is faint violet-
blue. In the chromatogram obtained with the test solution
there are other coloured zones, particularly a faint red zone
in the lower part and a reddish-violet zone due to
caryophyllene in the upper parto
TESTS
Foreign matter (2.8.2)
Maximum 6 per cent of peduncles, petioles and fruits,
maximum 2 per cent of deteriorated cloves and maximum
0.5 per cent of other foreign matter.
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use a 250 mL f1ask, 100 mL of water R as the
Clove Oil IV-145
distillation liquid and 0.50 mL of xylene R in the graduated
tube. Grind 5.0 g of the drug with 5.0 g of diatomaceous
earth R to form a fine, homogeneous powder and proceed
immediately with the determination using 4.0 g of the
mixture. Distil at arate of 2.5-3 .5 mUmin for 2 h.
____________________________________________ ~Ew
***
Clove Oi!
*** ***
(Ph Eur monograph 1091) ***
PhEw ____________________________________________
DEFINITION
Essential oil obtained by steam distillation from the dried
f10wer buds of Syzygium aromaticum (L.) Merr. et L.M.Perry
(syn. Eugenia caryophyllus (Spreng.) Bullock et S.G.Harrison).
CHARACTERS
Appearance
Clear, yellow liquid, which becomes brown when exposed to
airo
Solubility
Miscible with methylene chloride, with toluene and with fatty
oils.
IDENTIFICATION
First identification B.
Second identification A.
A. Thin-layer chromatography (2.2.27) .
Test solution Dissolve 20 ¡.tL of the substance to be
examined in 2.0 mL of toluene R .
Reference solution Dissolve 15 ¡.tL of eugenol R and 15 ¡.tL of
acetyleugenol R in 2.0 mL of toluene R.
Plate TLC silica gel F 254 plate R .
Mobile phase toluene R.
Application 20 ¡.tL of the test solution and 15 ¡.tL of the
reference solution, as bands.
Development Twice in an unsaturated tank over a path of
10 cm; allow to stand for 5 min between the 2 developments.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm and
mark the quenching zones.
Results A The chromatogram obtained with the test solution
shows in the middle part a quenching zone (eugenol) that is
similar in position to the quenching zone in the
chroma1Ogram obtained with the reference solution; just
below, there is a weak quenching zone (acetyleugenol) that is
similar in position to the zone of acetyleugenol in the
chromatogram obtained with the reference solution.
Detection B Spray with anisaldehyde solution R and examine
in daylight while heating at 100-105 oC for 5-10 mino
Results B The zone due to eugenol in the chromatograms
obtained with the test and reference solutions is strong
brownish-violet and the zone due 10 acetyleugenol in the
chromatogram obtained with the test solution is faint violet-
blue; in the chromatogram obtained with the test solution
there are other coloured zones, particularly a faint red zone
in the lower part and a reddish-violet zone (~-caryophyllene)
in the upper parto
B. Examine the chromatograms obtained in the test for
chromatographic profile.
Results The 3 principal peaks in chromatogram obtained
with the test solution are similar in retention time to the
IV-146 Coix Seed 2014

*****
3 principal peaks in the chromatogram obtained with the
Coix Seed
reference solution. * *
TESTS (Ph Bur monograph 2454) *****
____________________________________________
Relative density (2.2.5)
~E~

1.030 to 1.063. DEFINITION

Refractive index (2.2.6) Dried, ripe, caryopsis, freed from the shell, of Coix laeryma-
1.528 to 1.537. jobi L. subsp. ma-yuen (Rom. Caill.) T.Koyama.
Optical rotation (2.2.7) Content
_2° to O°. Minimum 0.50 per cent of triolein (Cs7HI 040 6; Me 885)
Fatty oils and resinified essential oils (2.8.7) (dried drug).
It complies with the test. IDENTIFICATION
Solubility in alcohol (2.8.10) A. The white or pale yellow caryopsis freed from the shell is
1.0 mL is soluble in 2.0 mL and more of ethanol (70 per eent roughly ovoid or elongated-elliptical, about 4-8 mm long and
V/V) R. 3-6 mm wide. The dorsal surface is rounded, milky white
Chromatographic pro file and smooth; the ventral surface shows a deep longitudinal
Gas chromatography (2.2.28): use the normalisation furrow; yellowish-brown remnants of the membranous fioral
procedure. parts may be presento One end is obtusely rounded, the other
end is relatively fiat and slightly dented with an indistinct,
Test solution Dissolve 0.2 g of the substance to be examined
pale brown hilum.
in 10 g of hexane R.
B. Microscopic examination (2.8.23). The powder is light
Referenee solution Dissolve 7 mg of {J-earyophyllene R, 80 mg
grey or light brown. Examine under a microscope using
of eugenol R and 4 mg of aeeTyleugenol R in 10 g of hexane R.
ehloral hydrate solution R. The powder shows the following
Column: diagnostic characters: fragments of endosperm with polygonal
-- material: fused silica; cells arranged in a network; fragments of epicarp with
-- size: 1 = 60 m, 0 = about 0.25 mm; elongated, slightly sinuous cells; cells of the middle layer of
-- stationary phase: maerogol 20 000 R. the pericarp are yellowish-brown, irregularly tube-like, slightly
Canier gas helium for ehromatography R. curved and are irregularly crossed. Examine under a
Flow rate 1.5 mUmin. microscope using a 50 per cent V/V solution of glyeerol R.
Split ratio 1:1OO. The powder shows very numerous starch granules, simple or
2-3 compound, sphericaJ or slightly polyhedral, 3-20 ¡.tm in
Temperature:
diameter, with a stellate, Y-shaped, cJeft-like or point-like
Time Temperature hilum.
(min) (OC)
C. Thin-layer chromatography (2.2.27) .
Column 0·8 60
Test solution To 1 g ofthe powdered herbal drug (710)
8 · 48 60 --+ 180
(2. 9.12) add 10 mL of light petroleum R1 and sonicate for
48·53 180 30 min o Filter and reduce in vaeuo to l mL.
Injection por! 270 Reference solution Dissolve 2 mg of oleie acid R and 2 mg of
triolein R in methanol R and dilute to 1 mL with the same
Detector 270
solvento
Detection Flame ionisation. Place TLC octadeeylsilyl si/iea gel plate R (5-40 ¡.tm) [or
Injeetion 1.0 ¡.tL. TLC octadeeylsilyl siliea gel plate R (2-10 ¡.tm)].
Blution order Order indicated in the composition of the Mobile phase methylene ehloride R, glacial aeetie aeid R,
reference solution. Record the retention times of these acetone R (20:40:50 VIV/V).
sub stances. Applieation 10 ¡.tL [or 2 ¡.tL] as bands of 10 mm [or 8 mm] .
System suitabi/iTy Reference solution: Development Over a path of 7 cm.
-- resolution: minimum 1.5 between the peaks due to eugenol Drying In airo
and acetyleugenol;
Deteetion Treat with a 100 gIL solution of phosphomolybdie
-- number of theoretieal plates: minimum 30 000, ca1culated acid R in ethanol (96 per eent) R, heat at 120 oC for about
for the peak due to ~-caryophyllene at 110 oC.
3 min and examine in daylight.
Identifieation of components Using the retention times
Results See below the sequence of zones present in the
determined from the chromatogram obtained with the
chromatograms obtained with the reference solution and the
reference solution, locate the components of the reference
test solution. Furthermore, other faint zones may be present
solution on the chromatogram obtained with the test
in the chromatogram obtained with the test solution.
solution.
Determine the percentage content of each of these
components. The limits are within the following ranges:
-- {J-earyophyllene: 5.0 per cent to 14.0 per cent;
-- eugenol: 75.0 per cent to 88 .0 per cent;
-- aeeTyleugenol: 4.0 per cent to 15.0 per cent.
STORAGE
Protected from heat.
____________________________________________ ~E~
2014 Cola IV-147

Top of the plate solution (b); use the chromatogram supplied with coix seed
HRS to identify peak 2;
-- signal-to-noise ratio: minimum 30 for the peak due to
triolein in the chromatogram obtained with reference
A purple zone
solution (a).
Establish a calibration curve with the logarithm of the mass
Oleic acid: a purple zone A purple zone (oleic acid)
of triolein (in milligrams) per 50 mL of reference solutions
- -- --- (c), (d), (e), (f), (g) and (h) (corrected by the assigned
percentage content of triolein CRS) as the abscissa and the
A faint purple zone
logarithm of the corresponding peak area as the ordinate.
A purple zone
Calculate the percentage content of triolein using the
A purple zone following expression:
- -- ---
Triolein : a purple zone A purple zone (triolein)

Reference solution Test solution
TESTS
Loss on drying (2.2. 32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (710) (2.9. 12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 3.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution To 0.600 g of the powdered herbal drug (355)
(2.9.12) add 50 mL of the mobile phase and stir with a
magnetic stirrer for 2 h. Sonicate for 30 min. AlIow to cool,
dilute to 50.0 mL with the mobile phase and filter.
Referenee solution (a) Dissolve 10.0 mg of triolein CRS in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Referenee solution (b) To 0.600 g of coix seed HRS add
50 mL of the mobile phase and stir with a magnetic stirrer
for 2 h. Sonicate for 30 min. AlIow to cool, dilute to
50.0 mL with the mobile phase and filter.
Referenee solutions (e), (d), (e), (f), (g), (h) Dilute reference
solution (a) to obtain 6 reference solutions of triolein, the
concentrations of which span the expected value in the test
solution.
Column:
-- size: 1 = 0.25 m, 0 = 4.6 mm;
-- stationary phase: end-eapped oetadecylsz1yl siliea gel for
ehromatography R (5 !lm).
Mobz1e phase methylene chloride R, acetonitrile R (35:65 V/V).
Flow rate 2.0 mUmin.
Detection Evaporative light-scattering detector; the following
settings have been found to be suitable; if the detector has
different setting parameters, adjust the detector settings so as
to comply with the system suitability criterion for signal-to-
noise ratio:
-- eamá gas: nitrogen R;
-- fiow rate: 0.8 mUmin;
-- evaporator temperature: 100 0e.
Injection 10 J.LL.
Run time 35 min.
Retention time Triolein = about 18 min.
System suitability:
resolution: minimum 1.5 between the peak due to triolein
and peak 2 in the chromatogram obtained with reference
A logarithm of the mass of triolein in the test solution,
determined from the calibration curve and the area of
the corresponding peak in the chromatogram obtained
with the test solution;
m mas s of the herbal drug to be examined used to
prepare the test solution, in grams.
____________________________________________ PhEm
Cola ****
*** *
(Ph Bur monograph 1504) ****
n Em ____________________________________________
DEFINITION
Whole or fragmented dried seeds, freed from the testa, of
Cola nitida (Vent.) Schott et Endl. (e. vera K. Schum.) and
its varieties, as well as of Cola acuminata (P. Beauv.) Schott
et Endl. (Stereulia aeuminata P. Beauv.).
Content
Minimum 1.5 per cent of caffeine (Mr 194.2) (dried drug).
IDENTIFICATION
A. The kernels have an oblong, somewhat obruse, sub-
tetragonal shape, with deformations resulting from mutual
pressure inside the fruit; they vary in size and mas s, ranging
from 5-15 g; the outside is hard, smooth and very dark
brown, the inside is more reddish-brown. In C. nitida and its
varieties, the kernels are divided in 2 parts, almost plano-
convex, corresponding to the cotyledons and usually
occurring separated in the commercial drug; the cotyledons
are 3-4 cm long, 2-2.5 cm wide and 1-2 cm thick. In e.
acuminata, the cotyledons are smaller and divided into 4-6
irregular parts.
B. Reduce to a powder (355) (2.9.12). The powder is
reddish-brown. Examine under a microscope using a
50 per cent V/V solution of glycerol R. The powder shows the
following diagnostic characters: numerous ovoid or reniform
starch granules, 5-25 flm in size, with concentric striations
and a stellate, slightly eccentric hilum; fragments of cotyledon
tissue showing large, thick-walled, reddish polygonal cells
filled with starch granules; occasional fragments of the
external epidermis of the cotyledons.
e. Thin-layer chromatography (2.2.27).
Test solurion To 1.0 g of the powdered drug (355) (2.9.12)
add 5 mL of ethanol (60 per cent V/Ji) R. Shake mechanically
at 40 oC for 30 min and filter.
 IV-148 Colophony 2014

Reference solution (a) Dissolve 25 mg of caffeine R in 10 mL
of ethanol (60 per cent V/V) R.
Reference solution (b) Dissolve 50 mg of theobromine R in
10 mL of the mobile phase. Filter.
Plate TLC silica gel F 254 plate R.
Mobzle phase water R, methanol R, ethyl acetate R
(l0:13:77 V/V/V).
Application 20 ~L, as bands.
Development Over a path of 10 cm.
Drying In air for 5 min.
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatogram obtained with the test solution
shows 2 principal quenching zones which are similar in
position ro the zones in the chromatograms obtained with
reference solutions (a) and (b).
-- resolution: minimum 2.5 between the peaks due ro caffeine
and theobromine. If necessary, adjust the volume of
water R in the mobile phase.
Calculate the caffeine content using the following expression:
A¡ =area of the peak due ro caffeine in the chromatogram
obtained with the test solution,
A 2 = area of the peak due ro caffeine in the chromatogram
obtained with the reference solution,
m¡ = mass of the drug to be examined in the test solution, in
grams,
m2 = mass of caffeine CRS in the reference solution, in grams.
____________________________________________ ~Em

Detection B Spray with a mixture of equal volumes of

ethanol (96 per cene) R and hydrochloric acid R and then with a
solution prepared immediately before use by dissolving 1 g of
iodine R and 1 g of potassium iodide R in 100 mL of ethanol
Colophony ***
*** ***
(96 per cent) R.
Results B The chromatogram obtained with the test solution
shows a reddish-brown principal zone similar in position and
(Ph Bur monograph 1862) ***
colour to the zone in the chromatogram obtained with Preparation
reference solution (a). Flexible Collodion
~Em ____________________________________________
TESTS
Loss on drying (2.2.32) DEFINITION
Maximum 12.0 per cent, determined on 2.00 g ofthe Residue remaining after distillation of the volatile oil from the
powdered drug (355) (2.9.12) by drying in an oven at oleoresin obtained from various species of Pinus.
105 cC for 2 h. IDENTIFICATION
Total ash (2.4.16) A. Translucent, pale yellow to brownish-yellow, angular,
Maximum 9.0 per cent. irregularly-shaped, brittle, glassy pie ces of different sizes the
ASSAY surfaces of which bear conchoidal markings.
Liquid chromatography (2.2.29). B. Thin-layer chromarography (2.2.27).
Test solution To 1.00 g (mI) of the powdered drug (355) Test solution Dissolve 1 g in 10 mL of methanol R by gently
(2.9.12), add 50 mL of methanol R. Heat under a refiux warming.
condenser on a water-bath for 30 min. Allow ro cool and Reference solution Dissolve 10 mg of thymol R and 10 mg of
filter. Rinse the filter with 10 mL of methanol R. Take up the linalol R in 10 mL of methanol R.
residue with 50 mL of methanol R . Proceed as before. Plate TLC silica gel plate R.
Combine the filtra tes and the washings in a 200.0 mL
Mobzle phase methylene chloride R.
volumetric fiask and dilute to 200.0 mL with methanol R.
Transfer 20.0 mL of this solution into a round-bottomed Application 1O ~L, as bands.
fiask and evaporate ro dryness under reduced pressure. Take Development Over a path of 15 cm.
up the residue with the mobile phase, transfer ro a 50.0 mL Drying In airo
volumetric fiask and dilute to 50.0 mL with the mobile Detection Spray with anisaldehyde solution R and heat at
phase. 100-105 oC for 10 minó examine in daylight.
Reference solution In a 100.0 mL volumetric fiask, dissolve
30.0 mg (m2) of caffeine CRS and 15.0 mg of theobromine R
in the mobile phase and dilute to 100.0 mL with the mobile Top of the plate
phase. Transfer 10.0 mL ofthis solution to a 100.0 mL
A purple band
volumetric fiask and dilute to 100.0 mL with the mobile
phase. A purple band
Column:
-- size: 1 = 0.25 m, 0 = 4.6 mm; --- ---
-- stationary phase: octadecylsilyl szlica gel for chromatography R 2 purple bands
(5 11m).
Mobzle phase methanol R, water R (25:75 V/V). Thyrnol: an orange band

Flow rate 1 mUmin. --- ---

Detection Spectrophotometer at 272 nm.
Linalol: a purple band Sequen ce of narrow purple bands
Injection The chosen volume of each solution; loop injecror.
System suitability Reference solution: Purple extended baseline band

Reference solution Test solution

 2014 Coriander IV-149

Results See below the sequence of the zones present in the

chromatograms obtained with the reference solution and the
test solution. Furthermore, other coloured zones are present
in the chromatogram obtained with the test solution.
TESTS
Acid value (2.5.1)
145 to 180, determined on 1.0 g.
Total ash (2.4.16)
Maximum 0.2 per cent.
STORAGE
Do not reduce to a powder.
_ _ _ _ _ _ _ _ _ _ _ __ __ _ __ _ _ _ _ PhEur

Coriander ***
*** ***
(Ph Eur monograph 1304) ***
When Powdered Coriander is prescribed or demanded,
material complying with the appropriate requirements below
but containing not less than 0.2% v/w of essential oil shall be
dispensed or supplied. 01?

~J 2~m
~E~ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ __ __

DEFINITION
Dried cremocarp of Coriandrum sativum L.
Content
Minimum 3 mUkg of essential oil (dried drug).
IDENTIFICATION
A. The fruit is brown or light brown, more or less spherical,
about 1.5-5 mm in diameter, or oval and 2-6 mm long.
It consists of the entire cremocarp, with the mericarps usually
tightly connected. The fruit is glabrous and has 10 wavy,
slightly raised primary ridges and 8 straight, more prominent
secondary ridges. The mericarps are concave on the intemal
surface. The stylopod crowns the apex and a small fragment
of the pedicel may be presento
B. Microscopic examination (2.8.23). The powder is brown.
Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters
(Figure 1304.-1) : numerous oil droplets [B]; fragments of
endosperm [A] with small, thick-walled, regular cells
containing microrosettes [Aa] and microcrystals of calcium
oxalate and oil droplets [Ab]; fragments of endocarp, in
surface view [C, TI or in transverse section [H], with very
narrow cells having a parquetry arrangement [Ca, Ha] and
usually associated with a layer of thin-walled [Cb, Hb] or
thicker-walled Ua] rectangular sc1ereids of the mesocarp;
fragments from the sc1erenchymatous layer of the mesocarp
[G] with short, strongly thickened, pitted, fusiform cells
occurring in layers with the cells of adjacent layers
approximately at right angles to one another; fragments of
parenchyma of the mesocarp in transverse section [E] with
small cells with slightly thickened walls [Ea], the remains of
secretory canals [Eb] and sc1ereids [Ec]; fragments of
epicarp, in surface view [F], with thin-walled polyhedral cells,
sorne ofwhich contain small prisms of calcium oxalate [Fa];
rare fragments of secretory canals with brown cells, in surface
view [D]; occasional fragments ofvascular bundles [K] .
K
~Ja
Figure 1304.-1. - Illustration for identification test B of
powdered herbal drug of coriander
C. Thin-layer chromatography (2.2.27) .
Test solution Shake 0.5 g of the freshly powdered herbal
drug (355) (2.9.12) with 5 mL of hexane R for 2-3 min and
filter over 2 g of anhydrous sodium sulfate R.
Reference solution Dissolve 15 ¡.tL of linalol R and 25 ¡.tL of
olive oil R in 5 mL of hexane R immediately before use.
Plate TLC silica gel plate R .
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 20 ¡.tL of the test solution and 10 ¡.tL of the
reference solution, as bands.
Development Twice over a path of 10 cm.
Drying In air.
Detection Spray with anisaldehyde solution R and examine in
daylight while heating at 100-105 oC for 5-10 mino
Results The chromatogram obtained with the reference
solution shows in the lower half a violet or greyish-violet zone
(linalol) and in the upper half a bluish-violet zone
(triglycerides); the chromatogram obtained with the test
solution shows zones similar in position and colour to the
zones in the chromatogram obtained with the reference
solution; several violet-grey or brownish zones, inc1uding the
zone due to geraniol, are shown between the point of
application and the zone due to linalol in the chromatogram
obtained with the reference solution; several faint violet-grey
zones may also be shown between the zone due to
triglycerides and that due to linalol in the chromatogram
obtained with the reference solution.
TESTS
Foreign matter (2.8.2)
It complies with the test. None of the cremocarps show
perforations due to insects.
IV-ISO Coriander Oil 2014

Loss on drying (2.2.32) Results The characteristic peaks in the chromatogram

Maximum 10.0 per cent, determined on l.000 g ofthe obtained with the test solution are similar in retention time to
powdered herbal drug (355) (2.9. 12) by drying in an oven at those in the chromatogram obtained with the reference
105 oC for 2 h . solution.
Total ash (2.4.16) TESTS
Maximum 8.0 per cent. Relative density (2.2.5)
ASSAY 0.860 to 0.880.
Carry out the determination of essential oils in herbal drugs Refractive index (2.2.6)
(2.8.12). Use a 500 mL round-bottomed flask, 200 mL of l.462 to l.470.
water R as the distillation liquid and 0.5 mL of xylene R in Optical rotation (2.2.7)
the graduated tube. Reduce the drug to a coarse powder and + 7° to + 13°.
imrnediately use 30.0 g for the determination. Distil at arate
Acid value (2.5.1)
of 2-3 mUmin for 2 h.
Maxirnum 3.0, determined on 5.00 g of the substance to be
____________________________________________ PhEw
examined.
Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
procedure.
Coriander Oil ****
**
Test solution The substance to be examined.
(Ph Eur monograph 1820) **** ** Reference solution (a) Dissolve 10 IlL of a-pinene R, 10 IlL of
~Ew ____________________________________________
limonene R, 1O ¡.tL of y-terpinene R, 10 ¡.tL of p-cymene R,
10 mg of camphor R, 20 IlL of linalol R, 10 IlL of
DEFINITION a-terpineol R, 10 ¡.tL of geranyl acetate R and 10 IlL of geraniol R
Essential oil obtained by steam distillation from the fruits of in 1 mL of hexane R.
Coriandrum sativum L. Reference solution (b) Dissolve 5 IlL of geraniol R in hexane R
CHARACTERS and dilute to 10 mL with the same solvento
Appearance Column:
Clear, colourless or pale yellow liquido -- material: fused silica,
-- size: 1 = 60 m, 0 = 0.25 mm,
Characteristic spicy odour.
-- stationary phase: macrogol 20 000 R (film thickness
IDENTIFICATION 0.25 11m).
First identification B. Carrier gas helium for chromatography R.
Second identification A. Flow rate 1 mUmin.
A. Examine by thin-layer chromatography (2.2.27). Split ratio 1:65.
Test solution Dissolve 10 IlL of the substance to be Temperature:
examined in 1.0 mL of toluene R.
Time Temperature
Reference solution Dissolve 10 IlL linalol R and 2 IlL of (mio) (oC)
geranyl acetate R in l.0 mL of toluene R. Colurnn 0·10 60
Plate TLC si/ica gel plate R. 10·75 60 --) 190
Mobile phase ethyl acetate R, toluene R (5:95 VIV). 75· 120 190
Injection port 220
Application 1O ~IL as bands.
Detector 240
Development Over a path of 10 cm.
Drying In airo Detection Flame ionisation.
Detection Spray with anisaldehyde solurion R and heat at In.fection 0.2 1lL.
100-105 oC for 10-15 mino Examine immediately in daylight.
Elution order Order indicated in the composition of
Results See below the sequence of the zones present in the reference solution (a). Record the retention times of these
chromatograms obtained with the reference solution and the substances.
test solution.
System suitability Reference solution (a):
-- resolution: minimum 1.5 between the peaks due to linalol
Top of the plate and camphor.
--- U sing the retention times determined from the
---
chromatogram obtained with reference solution (a), locate
Geranyl acetate: a violet·blue zone A violet·blue zone (geranyl acetate) the components of reference solution (a) in the
--- ----- chromatogram obtained with the test solution.
Determine the percentage content of each of these
Linalol: an intense violet zone An intense violet zone (linalo!)
components. The percentages are within the following
A violet·blue zone (geranio!) ranges:
Reference solutioo Test solutioo -- a-pinene: 3.0 per cent to 7.0 per cent,
-- limonene: l.5 per cent to 5.0 per cent,
-- )'-terpinene: 1.5 per cent to 8.0 per cent,
B. Examine the chromatograms obtained in the test for -- p-cymene: 0.5 per cent to 4.0 per cent,
chromatographic profile. -- camphor: 3.0 per cent to 6.0 per cent,
2014 Couch Grass Rhizome IV -151

*****
-- linalol: 65 .0 per cent to 78.0 per cent,
Couch Grass Rhizome
-- rx-terpineol: 0.1 per cent to 1.5 per cent, ** **
-- geranyl acetate: 0.5 per cent to 4.0 per cent, (Ph Bur monograph 1306) ***
-- geraniol: 0.5 per cent to 3.0 per cent, ~E~ ____________________________________________
-- disregard limit: area of the peak in the chromatogram
obtained with reference solution (b) (0.05 per cent). DEFINITION
Chiral purity Whole or cut, washed and dried rhizome of Agropyron repens
(L.) P.Beauv. (Blymus repens (L.) Gould); the adventitious
Gas chromatography (2.2.28).
roots are removed.
Test solution Dissolve 0.02 g of the substance to be
examined in pentane R and dilute to 10 mL with the same IDENTIFICATION
solvento A. The shiny yellowish, light brown or yellowish-brown
Reference solution Dissolve 10 ¡tL of linalol R and 5 mg of pieces ofthe rhizome are 2-3 mm thick and longitudinally
borneol R in pentane R and dilute to 10 mL with the same furrowed. At the nodes are the remains of very thin, more or
solvento less branched roots and whitish or brownish scale-like leaves;
the intemodes, up to 6 cm long, are furrowed and hollow
Column:
inside. The transverse section of the no des shows a yellowish
-- material: fused silica,
medulla.
-- size: 1 = 25 m, 0 = 0.25 mm,
-- stationary phase: modified fJ-cyclodextrin for chiral B. Microscopic examination (2.8.23). The powder is whitish-
chromatography R (film thickness 0.25 ¡tm). yellow. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
Carrier gas helium for chromatography R.
characters (Figure 1306.-1): fragments of the epidermis in
Flow rate 1.3 mUmin. surface view [A) covered with a thick cuticle and composed
Split ratio 1:30. of rectangular and elongated, thick-walled cells with pitted,
Temperature: slightly wavy walls, which usually alternate with small, thin-
walled, rounded or almost square twin cells; fragments in
Time Temperature
(min) (oC) transverse section [B) showing the epidermis [Ea) associated
Column 0·65 50 ~ 180 with thick-walled cells of the hypodermis; fragments in
transverse section [F] consisting of endodermic cells with
Injection port 230 U-shaped thickening ofthe walls [Fa) accompanied by
Detector 230 pericyc1ic fibres [Fb); numerous fragments of moderately
thickened fibres [C); groups of vessels [D, G) with slit-
Detection Flame ionisation. shaped pits [Da) or with spiral and annular thickening [Ga),
Injection 1 ¡tL. accompanied by fibres [Db, Gb); numerous fragments of the
System suitability Reference solution:
-- resolution: minimum 5.5 between the peaks due to
(R)-linalol (1 st peak) and (S)-linalol (2nd peak) and
minimum 2.9 between the peaks due to (S)-linalol and
borneol (3 rd peak).
Limit Calculate the percentage content of (R)-linalol from
the expression:

AR
A s+ A R x 100

As = area of the peak due to (S)-linalol,

A R = area of the peak due to (R)-linalol.
-- (R)-linalol: maximum 14 per cent.
STORAGE
At a temperature not exceeding 25 oC.
____________________________________________ ~E~

Gb

Figure 1306.-1. - Illustration for identification test B of

powdered herbal drug of couch grass rhizome
IV-152 Dandelion Herb with Root 2014

cortical parenchyma and the pith with slightly thickened and (2.8.3) [Ca, Ea); elongated, multicellular covering trichomes
pitted cells [E). with constrictions, which are more or less abundant
depending on the variety or sub-variety [B, D); fragments of
TESTS
the upper (E) epidermis usually accompanied by underlying
Cynodon dactylon, Imperata cylindrical
palisade parenchyma [Eb) and fragments of the lower (C)
Examine under a microscope using iodine solution RJ.
epidermis accompanied by underlying spongy parenchyma
No blue starch grains are visible.
[Cb); lignified, spirally or annularly thickened vessels;
Foreign matter (2.8.2) fragments of ftower-stem epidermis with stomata and rigid-
Maximum 15 per cent of blackish-grey pieces of rhizome in walled, elongated cells [A); pollen grains with a pitted exine
the cut herbal drug. UJ. Examine under a microscope using glyeerol R.
Water-soluble extractive The powder shows angular, irregular inulin fragments, free or
Minimum 25 per cent. included in the parenchyma cells.
To 5.0 g ofthe powdered herbal drug (355) (2.9.12) add
200 mL of boiling water R. Allow to stand for 10 min,
shaking occasionally. Allow to cool, dilute to 200.0 mL with
water R and filter. Evaporate 20.0 mL of the filtrate to
dryness on a water-bath. Dry the residue in an oven at
100-105 oC . The residue weighs a minimum of 0.125 g.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4. 16)
Maximum 5.0 per cent.
Ash insoluble in hydrochIoric acid (2.8.1)
Maximum 1.5 per cent.
____________________________________________ ~Ew

Dandelion Herb with Root ***

*** ***
(Ph Bur monograph 1851) ***
~ Ew ____________________________________________

DEFINITION
Mixture of whole or fragmented, dried aerial and
underground parts of Taraxaewn officinale F.H. Wigg.
CHARACTERS
Bitter taste.
IDENTIFICATION
A. The underground parts consist of dark brown or blackish
fragments 2-3 cm long, deeply wrinkled longirudinally on the Figure 1851.-1 - Illustration for identification test B of
outer surface . The thickened crown shows many scars left by powdered herbal drug of dandelion herb with root
the rosette of leaves. The fracture is short. A transverse
section shows a greyish-white or brownish cortex containing C. Thin-Iayer chromatography (2.2.27) .
concentric layers of brownish laticiferous ves seis and a Test sohaian To 2.0 g of the powdered herbal drug (355)
porous, pale yellow, non-radia te wood. Leaf fragments are (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
green, glabrous or densely pilose. They are crumpled and 60 oC or sonicate for 10 mino Cool and filter.
usually show a clearly visible midrib on the i=er surface. Referenee solution Dissolve 2 mg of ehlorogenie aeid R and
The lamina, with deeply dentate margins, is crumpled. 2 mg of rutin R in methanol R and dilute to 20 mL with the
The solitary ftower heads, on hollow stems, consist of an same solvent.
involucre of green, foliaceous bracts surrounding the yellow
Plate TLC siliea gel plate R (5-40 pm) [or TLC siliea gel
ftorets, all of which are ligulate; a few achenes bearing a
plate R (2-10 ~Lm»).
white, silky, outspread pappus may be presento
Mobile phase anhydrous formie acid R , water R, ethyl aeetate R
B. Microscopic examination (2.8.23) . The powder is
(10 :10:80 VIV/V).
yellowish-brown. Examine under a microscope using ehloral
hydrate solutian R. The powder shows the following diagnostic
Applieation 20 pL [or 5 pL) as bands of 10 mm [or 8 mm).
characters (Figure 1851.-1): fragments of cork [G) with Developmenc Over a path of 12 cm [or 7 cm).
ftattened, thin-walled cells; reticulate lignified vessels [H) Drying In airo
from the roots; fragments of parenchyma containing Deteetion Heat at 100 oC for 5 min; spray with or dip
branched laticiferous vessels [F); fragments of leaves, in briefty into a 10 giL solution of dipheny lborie acid aminoethyl
surface view, showing upper [E) and lower [C] epidermises ester R in methanol R and dry at 100 oC for 5 min; spray with
consisting of interlocking lobed cells and anomocytic sto mata or dip briefty into a 50 gIL solution of maerogal 400 R in
 2014 Dandelion Herb with Root IV-153

methanol R; heat at 100 oC for 5 min and examine in hydrate solution R. The powder shows the foIlowing diagnostic
ultraviolet Iight at 365 nm. characters (Figure 1852.-1): fragments of brown or reddish-
R esults See below the sequence of zones present in the brown cork, in surface view [G] and transverse section [C]
chromatograms obtained with the reference solution and the with fiattened, thin-waIled ceIls [Ca] , sometimes
test solution. Furthermore, other fa int zones may be present accompanied by parenchyma [Cb] ; reticulate lignified vessels
in the chromatogram obtained with the test solution. [E, J, M]; fragments of parenchyma [A, D, K, L] , sorne
containing branched laticiferous ves seIs, in longitudinal
section [Ka] and transverse section [Da]; granular contents
Top of the plate
of laticiferous vessels [B, R] . Examine under a microscope
A faint red zone using glycerol R. The powder shows numerous irregular,
angular inulin fragments, free [F] or incIuded in the
A faint yellow zone parenchyma ceIls [La] .
--- - --
Chlorogenic acid: a blue zone 2 li ght blue zones

- -- - --
Rutin : a yellowish·brown zone

A light blue zone

Reference solution Test solution

TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 17.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 5.0 per cent.
Extractable matter
Minimum 30.0 per cent.
To 2.000 g ofthe powdered herbal drug (250) (2.9. 12) add
40 g of water R . Stir for 1 h and filter. Evaporate 10 g of the
filtrate to dryness on a water-bath and dry in an oven at
100-105 oC for 2 h. The residue weighs a minimum of
0.15 g.
Bitterness value (2.8. 15)
Minimum 100.
____________________________________________ PhE~
Figure 1852.-1.- Illus!ration for identification tes! B of
powdered herbal drug of dandelion roo!

C. Thin-Iayer chromatography (2.2. 27) .

Dandelion Root
** **
(Ph Eur monograph 1852)
Ph Eur ___________________________________________
DEFINITION
Whole or cut, dried underground parts of Taraxacum
officinale F.H.Wigg.
CHARACTERS
Bitter taste .
IDENTIFICATION
A. The dark brown or blackish taproot shows Iittle branching
and is deeply wrinkled 10ngitudinaIly on the outer surface.
The thickened crown shows many scars left by the rosette of
leaves . The fracture is short. A transverse section shows a
greyish-white or brownish cortex containing concentric layers
of brownish laticiferous vessels and a porous, pale yeIlow,
non-radiate wood.
B. Reduce 10 a powder (35 5) (2.9.12). The powder is
yeIlowish-brown. Examine under a microscope using chloral
***** Test solution To 2.0 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R . R eat in a water-bath at
*** 60 oC or sonicate for 10 mino Cool and filter.
R eference solution Dissolve 2 mg of chlorogenic acid R and
2 mg of rutin R in methanol R and dilute to 20 mL with the
same solvent.
Plate TLC silica gel F254 plate R (5-40 ~m) [or TLC silica gel
F254 plate R (2-10 ~m)].
Mobile phase anhydrous formic acid R , water R, ethyl acerate R
(10:10 :80 VIVIV) .
Application 20 ~LL [or 5 ~LL] as bands of 10 mm [or 8 mm] .
Development Over a path of 12 cm [or 7 cm].
Drying In air.
Detection Reat at 100 oC for 5 min; spray with or dip
briefiy into a 10 giL solution of diphenylboric acid aminoethyl
ester R in methanol R and dry at 100 oC for 5 min; spray with
or dip briefiy into a 50 giL solution of macrogol 400 R in
methanol R; heat at 100 oC for 5 min and examine in
ultraviolet light at 365 nm.
 IV- 154 Devil's Claw 2014

Results See below the sequence of zones present in the B. Reduce to a powder (355) (2.9.12). The powder is
chromatograms obtained with the reference solution and the brownish-yellow. Examine under a microscope using chloral
test solution. Furthermore, other faint zones may be present hydrate solution R . The powder shows the following diagnostic
in the chromatogram obtained with the test solution. characters (Figure 1095.-1 ): fragments of cork consisting of
yellowish-brown, thin-walled cells, in surface view [B] and in
transverse section [C]; fragments of cortical parenchyma
Top of the plate
consisting of large, thin-walled cells [E, K, N, P], sometimes
A Iight blue zone containing reddish-brown granular inclusions and isolated
--- yellow droplets (P); fragments of reticulately thickened or
---
pitted ves seis [D, F, G, M] and fragments of lignified
Chlorogenic acid: a blue zone A blue zone (chlorogenic acid) parenchyma [L], sometimes associated with vessels, from the
--- --- central cylinder; prism crystals [A] and rare small needles of
calcium oxalate in the parenchyma. The powder may also
Rutin : a yellowish-brown zone show rectangular or polygonal sclereids with dark reddish-
Reference solution Test solution brown contents [H, TI. With a solution of phloroglucinol in
hydrochloric acid, the parenchyma tums green.

TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
Extractable matter
Minimum 20.0 per cent.
To 2.000 g of the powdered herbal drug (250) (2.9.12) add
40 g of water R. Stir for 1 h and filter. Evaporate 10 g of the
filtrate to dryness on a water-bath and dry in an oven at
100-105 oC for 2 h. The residue weighs a minimum of
0.10 g.
Bitterness value (2.8.15)
Minimum 100.
____________________________________________ ~E~

~
K

Devil's Claw *****

** ** ~
Harpagophytum *** N
~ \ ,

(Devil's Claw Root, Ph Bur monograph 1095) P

Preparation
Devil's Claw Dry Extract
~E~ ____________________________________________ Figure 1095.-1.- Illustration (or identi(ication test B o(
powdered herbal drug o( devil's claw root
DEFINITION
Cut and dried, tuberous secondary roots of Harpagophytum
procumbens De. and/or Harpagophytum zeyheri Decne. C. Thin-Iayer chromatography (2.2.27).
Content Test solution Heat 1.0 g of the powdered drug (355)
Minimum 1.2 per cent of harpagoside (C 24 H 30 0¡¡; M r (2.9.12) with 10 mL of methanol R on a water-bath at 60 oC
494.5) (dried drug). for 10 min. Filter and reduce the filtrate to about 2 mL
under reduced pressure at a temperature not exceeding
CHARACTERS 40 oC.
The root is greyish-brown or dark brown.
Reference solution Dissolve 1 mg of harpagoside R and 2.5 mg
IDENTIFICATION of fructose R in 1 mL of methanol R.
A. It consists of thick, fan-shaped or rounded slices or of Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel
roughly crushed discs. The darker outer surface is traversed plate R (2-10 ¡.tm)] .
by tortuous longitudinal wrinkles. The paler cut surface
Mobile phase water R , methanol R , ethyl acetate R
shows a dark cambial zone and xylem bundles distinctly (8:15:77 VIV/ V) .
aligned in radial rows. The central cylinder shows fine
concentric striations. Seen under a lens, the cut surface Application 20 ¡.tL [or 5 ¡.tL] as bands.
presents yellow or brownish-red granules. Development Over a path of 10 cm [or 7.5 cm] .
 2014 D evil's Claw Preparations IV-155

Dlying In a current of warrn air. - stationary phase: octadecylsilyl silica gel for chromatography R
Detection A Examine in ultraviolet light at 254 nm. (5 ¡.tm).
R esults ASee below the sequence of zones present in the Mobile phase methanol R, water R (50:50 V/V).
chromatograms obtained with the reference solution and the Flow rate 1.5 mUmin.
test solution; the chromatogram obtained with the test Detection Speetrophotometer at 278 nm.
solution shows other distinct zones, mainly above the zone In..jection 10 ¡.tL.
due to harpagoside. Furtherrnore, other faint zones may be
Run time 3 times the retention time of harpagoside .
present in the chromatogram obtained with the test solution.
R etention time Harpagoside = about 7 mino

Top oC the plate

Calculate the percentage eontent of harpagoside using the
following express ion:
--- ---
Harpagoside : a quenching zone A quenching zone: harpagoside

- - -
ReCerence solution Test solution
Detection B Spray with a 10 gIL solution of phloroglucinol R
in ethanol (96 p er cent) R and then with hydrochloric acid R;
heat at 80 cC for 5-10 min and examine in daylight.
R esults B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution; the chromatogram obtained with the test
solution also shows several yellow or brown zones above the
---
area of the peak due to harpagoside in the
ehromatogram obtained with the test solution;
area of the peak due to harpagoside in the
ehromatogram obtained with the referenee solution;
mass of the drug to be examined used to prepare the
test solution, in grams;
mas s of harpagoside CRS in the referenee solution, in
grarns.
_ _ __ ___________________________________ ~E~

zone due to harpagoside. Furthermore, other faint zones may

be present in the chromatogram obtained with the test
solution.

Devil's Claw Dry Extract ***

Top oC the plate
*** ***
--- --- (Ph Bur monograph 1871) ***
~E~ __________________________________________
Harpagoside: a green zone A green zone (harpagoside)

- - - --- DEFINITION
Dry extraet obtained from Devil's claw root (1095).
Ayellow zone
Content
A light green zone Minimum l.5 per eent ofharpagoside (Cz4H30011; M r
Fructose: a yellowish-grey zone A yellowish-grey zone may be 494.5) (dried extraet).
present (Eructose)
PRODUCTION
A brown zone
The extraet is produeed fro m the herbal drug by an
ReCerence solution Test solution appropriate proeedure using either water or a hydroalcoholie
solvent that is at most equivalent in strength to ethanol
(95 per cent V/V).
TESTS
Starch CHARACTERS
Examine the powdered drug (355) (2.9. 12) under a Appearance
microscope using water R. Add iodine solution R1 . N o blue Light brown powder.
colour develops. IDENTIFICATION
Loss on drying (2.2.32) Thin-layer ehromatography (2.2.27).
Maximum 12.0 per cent, deterrnined on l.000 g of the Test solution T o 1.0 g of the extraet to be examined add
powdered drug (35 5) (2.9.12) by drying in an oven at 10 mL of methanol R and heat in a water-bath at 60 oC for
105 oC. 10 mino Coo! and filter.
Total ash (2.4.16) R eference solution Dissolve l.0 mg of harpagoside R and
Maximum 10.0 per cent. 2.5 mg offructose R in l.0 rnL of methanol R .
ASSAY Plate TLC silica gel plate R .
Liquid ehromatography (2.2.29). Mobile phase water R , methanol R, ethyl acetate R
Test solution To 0.500 g of the powdered drug (355) (8:15:77 VIV/V).
(2.9.12) add 100.0 mL of methanol R. Shake for 4 h and Application 20 ¡.tL as bands.
filter through a membrane filter (nominal pore size 0.45 ¡.un). Development Over a path of 10 cm.
R eference solution Dissolve the eontents of a vial of Drying In a eurrent of warrn air.
harpagoside CRS in methanol R and dilute to 10.0 mL with Detection Spray with a 10 gIL solution of phloroglucinol R in
the same solvent. ethanol (96 per cent) R and then with hydrochloric acid R ; heat
Column: at 80 oC for 5-10 min and examine in daylight.
- size: l = 0.10 m, 0 = 4.0 mm;
IV-156 Digitalis Leaf 2014

Results See below the sequence of zones present in the

Digitalis Leaf ***
chromatograms obtained with the reference solution and the ** **
test solution. Furthermore, other faint zones may be present (Ph Eur monograph 011 7) *****
in the chromatogram obtained with the test solution. When Powdered Digitalis is prescribed or demanded,
material complying with the requirements below with the
Top of the plate exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
--- - -- ~E~ _ _ __ _ _ __ _ _ __ _ _ _ _ _ __ _ _ __
Harpagoside: a green zooe A greeo zone (harpagoside)
DEFINITION
--- --- Dried leaf of Digitalis purpurea L.
Ayellow zone
Content
A light greeo zooe Minimum 0.3 per cent of cardenolic glycosides, expressed as
Fructose: a yellowish·grey zooe Ayellowish·grey zooe may be present digitoxin (Mr 765) (dried drug).
(fructose)
CHARACTERS
A brown zooe
Faint but characteristic odour.
Reference solution Test solution The whole leaf is about 10-40 cm long and 4-15 cm wide.
The lamina is ovate lanceo late or broadly ovate. The winged
petiole is from 1/4 as long as to equal in length to the
ASSAY lamina.
Liquid chromatography (2.2.29) .
IDENTIFICATION
Test solution Introduce 0.350 g of the extract to be
A. The leaf is brittle and often occurs broken. The upper
examined into a 100 mL volumetric fiask, add 90 mL of
surface is green and the lower surface is greyish-green.
methanol R and sonicate for 20 mino Cool to room
The apex is subacute and the margin is irregularly crenate,
temperature, dilute to 100.0 mL with methanol R and filter
dentate or serrate. The base is decurrent. The venation is
through a membrane filter (nominal pore size 0.2 ¡.tm).
pinnate, the lateral veins being prominent especially on the
Reference solution Dissolve the contents of 1 vial of lower surface, leaving the midrib at about 45° and
harpagoslde CRS in methanol R and dilute to 10.0 mL with anastomosing near the margin; a veinlet termina tes in each
the same solvento tooth of the margin and the lower veins run down the winged
Column: petiole. The upper surface is rugose and pubescent; the lower
- size: l = 0.10 m, 0 = 4.0 mm; surface shows a network of raised veinlets and is densely
- stationary phase: octadecylsilyl silica gel for chromatography R pubescent.
(5 ¡.tm). B. Microscopic examination (2.8.23) . Examine under a
Mobile phase methanol R, water R (50:50 V/V). microscope using chloral hydrate solution R. The powder
Flow rate 1.5 mUmin. shows the following diagnostic characters (Figure 0117.-1):
Detection Spectrophotometer at 278 nro. fragments of the upper epidermis, in surface view [K, L],
1njection 1O ¡.tL. with cells with a smooth cutic1e and antic1inal walls that are
slightly thickened, are straight or slightly sinuous, and may
Run time 3 times the retention time of harpagoside. show slight beading and pitting [La] and sometimes scars of
Retention time Harpagoside = about 7 min. covering trichomes [Ka], accompanied by underIying palisade
Calculate the percentage content of harpagoside using the parenchyma [Lb]; fragments of the lower epidermis, in
following expression: surface view [G], with markedly sinuous cells and
anomocytic stomata ( 2.8.3) [Ga]; trichomes are of 2 types:
a) uniseriate covering trichomes with blunt apex, usually
consisting of 3-5 cells [H,n, often with 1 or more collapsed
cells Ua], walls mostly finely warty or faintly striated;
b) glandular rrichomes usually with a unicellular [C, D],
Al area of the peak due to harpagoside in the sometimes a multicellular, uniseriate [A, B, E] stalk and a
chromatogram obtained with the test solution; unicellular head [A, B, C, E] or bicellular head, in side view
A2 area of the peak due to harpagoside in the [D] and in surface view [F] or exceptionally a tetracellular
chromatogram obtained with the reference solution; head.
mi mass of the extract to be examined used to prepare
C. Thin-Iayer chromatography (2.2.27).
the test solution, in grams;
m2 mass of harpagoside contained in 1 vial of Test solution To 1.0 g of the powdered herbal drug (180)
harpagoside CRS, in grams. (2.9.12) add a mixture of 20 mL of ethanol (50 per cent
_ _ _ _ _ __ _ _ _ _ _ __ __ _ _ _ __ __ ~E~
V/V) R and 10 mL of lead acetate solution R. Boil for 2 min,
allow to cool and centrifuge. Shake the supematant solution
with 2 quantities, each of 15 mL, of chloroform R; separate
the 2 layers by centrifugation if necessary. Dry the
chloroform layers over anhydrous sodium sulfate R and filter.
Evaporate 10 mL of the solution to dryness on a water-bath
and dissolve the residue in 1 mL of a mixture of equal
volumes of chloroform R and methanol R.
Reference solutwn Dissolve 5 mg of purpureaglycoside A CRS,
2 mg of purpureaglycoside B CRS, 5 mg of digitoxin R and
2014
..: J
~
:
. •.'.•.. Ja
H ;::::
~~i/;
~.:' $
: :) :.
Ka
Figure 0117.-1. - Illustration for identification test B of
powdered herbal drug of digitalis leaf
2 mg of gitoxin R in a mixture of equal volumes of
chlorofonn R and methanol R, then dilute to 10 mL with the
same mixture of solvents.
Plate TLC silica gel G plate R .
Mobile phase water R, methanol R, ethyl acetate R
(7.5:10:75 VIV/V).
Application 20 IlL as bands of 2 cm by 0.3 cm.
Development Over a path of 10 cm.
Drying Until the solvents have evaporated.
Detection Treat with a mixture of 2 volumes of a 10 gIL
solution of chloramine R and 8 volumes of a 250 gIL solution
of trichloroacetic acid R in ethanol (96 per cent) R, then heat at
100-105 oC for 10 min; examine in ultraviolet light at
365 nm.
Results The chromatogram obtained with the reference
solution shows a zone of light blue fluorescence in the lower
part of the chromatogram, due to purpureaglycoside B, and,
just aboye it, a zone of brownish-yelIow fluorescence due to
purpureaglycoside A; a zone of light blue fluorescence, due to
gitoxin, appears in the middle of the chromatogram and
aboye it a zone of brownish-yelIow fluorescence, due to
digitoxin; the zones in the chromatogram obtained with the
test solution are similar in position, colour and size to the
zones in the chromatogram obtained with the reference
solution. Other zones of fluorescence may also appear in the
chromatogram obtained with the test solution.
D. Evaporate 5 mL of the chloroformic solution obtained in
identification test C to dryness on a water-bath. To the
residue add 2 mL of dinitrobenzoic acid solution R and 1 mL
of 1 M sodium hydroxide. A reddish-violet colour develops
within 5 mino
Digitalis Leaf IV-157
E. Evaporate 5 mL of the chloroformic solution obtained in
identification test C to dryness on a water-bath. To the
residue add 3 mL of xanthydrol solution R and heat on a
water-bath for 3 mino A red colour develops.
TESTS
Digitalis lanata Ehrh
The presence of leaves with few or no trichomes and with
paralIel venation or the presence of celIs of the abaxial
epidermis with beaded anticlinal walIs and of celIs of the
adaxial epidermis with numerous stomata indicates
adulteration by Digitalis lanata Ehrh.
Loss on drying (2.2.32)
Maximum 6.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 5.0 per cent.
ASSAY
Shake 0.250 g of the powdered herbal drug (180) (2.9.12)
with 50.0 mL of water R for 1 h . Add 5.0 mL of a 150 gIL
solution of lead acetate R, shake, and after a few minutes add
7.5 mL of a 40 gIL solution of disodium hydrogen phosphate R.
Filter through a pleated paper filter. Heat 50 .0 mL of the
filtrate with 5 mL ofhydrochloric acid (150 gIL HCI) under
a reflux condenser on a water-bath for 1 h. Transfer to a
separating funnel, rinse the flask with 2 quantities, each of
5 mL, of water R and shake with 3 quantities, each of
25 mL, of chlorofonn R. Dry the combined chloroform layers
over anhydrous sodium sulfate R and dilute to 100.0 mL with
chloroform R. Evaporate 40.0 mL of the chloroformic solution
to dryness, dissolve the residue in 7 mL of ethanol
(50 per cent V/V) R, add 2 mL of dinitrobenz oic acid solution R
and 1 mL of 1 M sodium hydroxide. At the same time prepare
a reference solution as follows. Dissolve 50.0 mg of
digitoxin CRS in ethanol (96 per cent) R and dilute to 50.0 mL
with the same solvent. Dilute 5.0 mL of the solution to
50 .0 mL with ethanol (96 per cent) R. To 5.0 mL ofthe
resulting solution add 25 mL of water R and 3 mL of
hydrochloric acid (150 giL HCl). Heat the solution under a
reflux condenser on a water-bath for 1 h and complete the
preparation as described aboye. Measure the absorbance
(2.2.25) of the 2 solutions at 540 nm several times during the
first 12 min until the maximum is reached, using as the
compensation liquid a mixture of 7 mL of ethanol (50 per cent
V/V) R, 2 mL of dinitrobenzoic acid solution R and 1 mL of
1 M sodium hydroxide.
From the absorbances measured and the concentrations of
the solutions, calculate the content of cardenolic glycosides,
expressed as digitoxin.
STORAGE
Protected from moisture.
____________________________________________ PhE~
 IV-158 Dill Oíl
Dill Oi!
DEFINITION
DiIl Oil is obtained by distillation from the dried ripe fruits of
Anethum graveolens L.
CHARACTERISTICS
A clear, colourless or pale yellow liquid, visibly free from
water; odour, characteristic of the crushed fruit.
TESTS
Optical rotation
+70° to + 80°, Appendix V F .
Refractive index
l.481 to l.492, Appendix V E.
Solubility in ethanol
Soluble, at 20°, in 1 volume or more of ethanol (90%) and in
10 volumes or more of ethanol (80%), Appendix X M.
Weight per mL
0.895 to 0.910 g, Appendix V G.
Content of carvone
43.0 to 63.0% w/w when determined by the following
method. To 1.5 g in a glass-stoppered tube (approximately
150 mm x 25 mm) add 10 mL of a solution prepared in the
following manner. Dissolve 7.0 g of hydroxylamine
hydrochloride in 90 mL of ethanol (90%), warming gently if
necessary, add l.6 mL of dimethyl yellow solution and
sufficient 1M potassium hydroxide in ethanol (90%) to produce
apure yellow colour and dilute to 100 mL with ethanol
(90%). Titrate with 1M potassium hydroxide in ethanol (90%)
VS until the red colour changes to yelJow. Place the tube in a
water bath at 75° to 80° and, at 5-minute intervals, neutralise
with 1M potassium hydroxide in ethanol (90%) VS; after
40 minutes complete the titration to the full yellow colour of
the indicator. This procedure gives an approximate value for
the carvone content of the oi!. Repeat the procedure, using as
the colour standard for the end point of the titration the
titrated liquid of the first determination with the addition of
0.5 mL of 1M potassium hydroxide in ethanol (90%) VS.
Calculate the content of carvone from the second
determination. Each mL of 1M potassium hydroxide in ethanol
(90%) VS is equivalent to 15l.4 mg of carvone, C lO H 140.
STORAGE
DiJl Oil should be kept in a weJl-filled container and
protected from light. It darkens in colour on storage.
D09 Rose ****
**
(Ph Bur monograph 1510) **** **
PhE~ ____________________________________________
DEFINITION
Rose hips made up by the receptac1e and the remains of the
dried sepals of Rosa canina L., R. pendulina L. and other Rosa
species, with the achenes removed.
Content
Minimum 0.3 per cent of ascorbic acid (C 6 H s0 6 ; M r 176.1)
(dried drug).
IDENTIFICATION
A. It consists of fragments of the fteshy, hollow, urceolate
receptacle, bearing the remains of the reduced sepals, light
pink or orange-pink, the convex outer surface shiny and
2014
strongly wrinkled; bearing on its lighter inner surface
abundant bristle-like hairs .
B. Reduce to a powder (355) (2.9. 12). The powder is
orange-yellow. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: numerous fragments of receptacle, the outer
epidermis with orange-yellow contents and a thick cuticle,
the inner epidermis composed of thin-walled cells containing
cluster crystals and occasional prisms of calcium oxalate;
scattered lignified cells, isodiametric, with thickened and
pitted walls forming the trichome bases; abundant unicellar
trichomes, up to 2 mm long and 30-45 ¡.tm thick, tapering
towards each end, walls heavily thickened and with a waxy
cuticle which may show fissures in a spiral arrangement;
numerous oily orange-yellow globules.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 5 g of the powdered drug (355) (2.9. 12)
add 25 mL of ethanol (96 per cent) R, shake for 30 min and
filter.
Reference solution Dissolve 10 mg of ascorbic acid R in
5.0 mL of ethanol (60 per cent VIV) R.
Plate TLC silica gel F Z54 plate R.
Mobile phase acetone R, glacial acetic acid R, methanol R,
toluene R (5:5:20:70 VIVIV/V).
Application 20 ¡.tL of the test solution and 2 ¡.tL of the
reference solution.
Development Over a path of 15 cm.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatogram obtained with the test solution
shows a quenching zone similar in position to the principal
zone in the chromatogram obtained with the reference
solution.
Detection B Spray with a 0.2 gIL solution of
dichlorophenolindophenol, sodium salt R in ethanol
(96 per cent) R. Examine in daylight.
Results B The chromatogram obtained with the test solution
shows a white zone on a pink background (ascorbic acid)
similar in position and colour to the principal zone in the
chromatogram obtained with the reference solution.
The chromatogram also shows an intense orange-yeJlow zone
near the solvent front and a yellow zone in the upper third
(carotenoids) .
TESTS
Foreign matter (2.8.2)
Maximum 1 per cent.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oc.
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Test solurion In a round-bottomed ftask, weigh 0.500 g of
the freshly powdered drug (710) (2.9.12). Add a solution of
l.0 g of oxalic acid R in 50.0 mL of methanol R. Boil under a
reftux condenser for 10 min, and cool in iced water until the
temperature reaches 15-20 oc. Filter. Transfer 2.0 mL of the
filtrate to a 50 mL conical ftask. Add successively, with
gentle shaking after each addition, 2.0 mL of
dichlorophenolindophenol standard solution R and then, exactly
60 s later, 0.5 mL of a 100 gIL solution of thiourea R in
 2014
ethanol (50 per cene VIV) R and 0.7 mL of
dinitrophenylhydrazine-sulfuric acid solution R . Heat under a
refiux condenser at 50 oC for 75 min, and place immediately
in iced water for 5 min. Add dropwise 5.0 mL of a mixture
of 12 mL of water R and 50 mL of sulfurie aeid R, taking care
to carry out the addition over a period of minimum 90 s and
maximum 120 s while maintaining vigorous stirring in iced
water. Allow to stand for 30 min at room temperature and
measure the absorbance (2.2.25) at 520 nm using solution A
as compensation liquido
Solurion A Treat 2.0 mL of the filtrate obtained during the
preparation of the test solution as described but adding the
dinitrophenylhydrazine-sulfuric acid solurion R just before the
absorbance is measured.
R eferenee solution Dissolve 40.0 mg of ascorbie acid R in a
freshly prepared 20 gIL solution of oxalie acid R in
methanol R and dilute to 100.0 mL with the same solvento
Dilute 5.0 mL ofthis solution to 100.0 mL with a freshly
prepared 20 gIL solution of oxalie acid R in methanol R . Treat
2.0 mL of the solution as described aboye for the filtrate
obtained during the preparation of the test solution. Measure
the absorbance (2.2.25) at 520 nm using solution B as the
compensation liquido
Solution B Treat 2.0 mL of the reference solution as
described aboye for solution A.
Calculate the percentage content of ascorbic acid from the
following expression:
Al absorbance of the test solution;
Az absorbance of the reference solution;
mI m ass of the substance to be examined, in grams;
mz mass of ascorbic acid used, in grams.
____________________________________________
Drynaria Rhizome ***
** **
**** *
(Ph Eur monograph 2563)
~Em ____________________________________________
DEFlNITION
Dried rhizome of Drynaria fortunei (Kunze) J. Sm. The
ramenta m ay be removed.
Content
Minimum 0.5 per cent of naringin (C z7 H 3 2 0 14; M r 580 .5)
(dried drug).
IDENfIFICATION
A. Long, fiattened, slat-shaped rhizome, often curved and
branched, 5-15 cm long and 1-1.5 cm thick. The surface is
either completely covered in scaly, dark brown hairs (rhizome
with ramenta) or glabrous with dark brown dots (rhizome
without ramenta). The upper surface and both sides show
circular frond scars, rarely the frond bases. The lower surface
shows scars or the remains of fibrous roots. The texture is
light, fragile, easily broken. The section is reddish-brown;
the steles form a ring of small yellow dots.
B. Microscopic examination (2.8.23). The powder is reddish-
brown. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
characters: numerous parenchyma fragments, consisting of
polyhedral cells with slightly and regularly thickened and
Drynaria Rhizome IV-159
pitted walls; scalariform lignified vessels of variable diameter
up to 60 ¡.1m; fragments of scaly hairs forming a tissue
consisting of many reddish-brown cells forming expansions
on the margins (rhizome with ramenta).
C. Thin-layer chromatography (2.2.27).
Test solution To 0.5 g of the powdered h erbal drug (355)
(2.9. 12) add 5 mL of methanol R and sonicate for 10 mino
Cool, centrifuge and use the supernatant.
Referenee solution Dissolve 1 mg of naringin R and 1 mg of
hyperoside R in 2 mL of methanol R.
Plate TLC siliea gel plate R (2-10 ¡.1m).
Mobile phase aeetie acid R, anhydrous formie acid R , water R,
ethy l aeetate R (11 : 11 :26: 100 VIVIV/V).
Applieation10 ¡.IL as bands of 8 mm.
Development Over a path of 6 cm.
Drying In air.
Detection Treat with aluminium ehloride reagent R; examine
in ultraviolet light at 365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones m ay be present
in the chromatogram obtained with the test solution.
Top of the plate
--- - --
Hyperaside: a yellaw zane
Naringin: a bluish-white zane A bluish-white zane (naringin)
A bluish-white zane
- -- - --
~Em
Reference solution Test solution
TESTS
Loss on drying (2.2.32)
Maximum 13.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Disperse 0.100 g of the powdered herbal drug
(355) (2.9.12) in a 50 per cent VIV solution of methanol R
and dilute to 10.0 mL with the same solvento Weigh,
sonicate for 45 min. Allow to cool, weigh and compensate
the loss of solvent with a 50 per cent V/V solution of
methanol R, shake well. Filter through a membrane filter
(nominal pore size 0.45 ¡.1m).
Referenee solution (a) Dissolve 10.0 mg of naringin CRS in
methanol R and dilute to 20.0 mL with the same solvento
R eferenee solution (b) Dissolve 5.0 mg of neohesperidin R in
reference solution (a) and dilute to 10.0 mL with reference
solution (a) .
Referenee solution (e) Dilute 1.0 mL of reference solution (a)
to 10.0 mL with methanol R .
 IV-160 Echinacea 2014

Column: 60 11m in diameter); abundant sclereids oeeuring singly or,

- size: 1 = 0.15 m, 0 = 4.6 mm; more usuaIly, in groups of 2 to 10, mostly elongated to
- stationary phase: oetadeeylsilyl si/iea gel for ehromatography R rectangular, (up to about 150 ~Lm in length and 40 11m wide),
(5 11m). with intereelIular spaees filIed with phytomelanin deposit;
Mobile phase aeetonitnle R, 0.4 per eent V/V solution of fragments of oleoresin canal (80-150 11m in diameter) with
aeetie acid R (18:82 V/V) . yelIowish-orange to reddish-brown eontent; groups of
Flow rate 1.0 mUmin. squarish to rectangular eelIs, about 30-45 11m from the outer
layers of the roots; abundant fine-walIed pitted parenehyma
D eteetion Speetrophotometer at 283 nm. with sphaerocrystalIine masses of inulin.
Injeetion 20 ~lL of the test solution and referenee solutions
C. Examine the chromatograms obtained in the test for
(b) and (e).
E chinacea purpurea.
Run time T wiee the retention time of naringin.
Results See below the sequenee of zones present in the
Retention time Naringin = about 9 min; ehromatograms obtained with the referenee solution and the
neohesperidin = about 12 mino test solution. Furthermore, other faint dark blue ftuoreseent
System suitability Referenee solution (b): zones may be present between the zones of echinaeoside and
- resolution: minimum 5.0 between the peaks due to eynarin in the chromatogram obtained with the test solution .
naringin and neohesperidin.
Calculate the pereentage eontent of naringin using the Top of the plate
folIowing expression:
Caffeic acid: a strong blue
fluarescent zane

Cynarin: a strang greenish A greenish fluarescent zane

fluorescent zane (cynarin)
Al are a of the peak due to naringin in the
ehromatogram obtained with the test solution;
A2 area of the peak due to naringin in the
ehromatogram obtained with referenee solution (e); Echinacoside : a strong greenish A strang greenish fluarescent zane
mI mass of the herbal drug to be examined used to fluarescent zane (echinacoside)
prepare the test solution, in grams;
m2 mass of naringin CRS used to prepare referenee
Reference solution Test solution
solution (a), in grams;
p pereentage eontent of naringin in na1ingin CRS.
_ _ __ _ __ __ _ _ _ __ _ __ _ _ __ _ PhEur D. Examine the ehromatograms obtained in the assay.
R esults The ehromatogram obtained with the test solution
shows 1 major peak due to eehinaeoside and a minor peak
due to eynarin. Peaks due to eaffeie aeid, eaftarie acid and
ehlorogenie aeid are minor peaks or may be absent.
Echinacea Angustifolia Root *****
* * Figure 1821.-1. - Chromawgram f or the assay of eehinaeoside in
(Narrow-leaved Conefiower R oot, **** * narrow-leaved conefiower root
Ph Eur monograph 1821) TESTS
~E~ _ _ _ __ _ _ _ _ __ __ __ _ __ _ __ _ Foreign matter (2.8.2)
DEFINITlON Maximum 3 per cent.
Dried, whole or cut underground parts of Eehinaeea Echinacea purpurea
angustifolia (D .C.). Thin layer chromatography (2. 2.27).
Content Test solution To 1.0 g of the powdered drug (355) (2.9.12)
Minimum 0.5 per eent of eehinaeoside (C3sH460 20; M r add 10 mL of methanol R , treat in an ultrasonie bath for
786.5) (dried drug). 5 min. Centrifuge and use the supematant solution.
R eference solution Dissolve 1 mg of eehinacoside R, 1 mg of
IDENTIFICATlON
cynarin R and 0.5 mg of caffeic acid R in 5.0 mL of
First identification A , B, C.
methanol R .
Seeond identifieation A, B, D.
Plate TLC si/ica gel F2 54 plate R (5-40 11m) [or TLC siliea gel
A. The root erown is up to about 30 mm in diameter and F 254 plate R (2-1 0 11m)] .
shows only a few stem bases. The roots are not very
Mobile phase anhydrous fonnie acid R , water R, methyl ethyl
numerous, up to about 15 mm in diameter, eylindrieal or
ketone R, ethy l acetate R (3:3 :9:15 V/V/V/V).
slightly tapering and sometimes spiralIy twisted, the outer
surfaee is pale brown to yeIlowish-brown. The fracture is Application 25 I1L [or 5 I1L] of the test solution and 10 I1L
short, dark brown with a radiate strueture. [or 2 I1L] of the referenee solution as bands.
B. Reduce to a powder (355) (2.9. 12) . The powder is Development Over a path of 15 cm [or 5 cm] .
greyish-brown. Examine under a mieroseope using ehloral Dly ing In a stream of eold air for about 10 min folIowed by
hydrate solution R . The powder shows the folIowing diagnostie 2 min at 100-105 oc.
eharaeters: narrow lignified fibres (up to about 800 11m in Detection Treat the hot plate using a 5 gIL solution of
length and 50 ~lm in diameter) joined together in long diphenylboric aeid aminoethyl ester R in ethyl aeetate R; examine
bundles surrounded by phytomelanin deposits; lignified in ultraviolet light at 365 nm after 30 min o
retieulately or sealariformly thiekened vessels (up to about
 2014 Echinacea IV -161

2
6
3

I I I I I I I I I
2 4 6 8 10 12 14 16 18 min

1. caftaric acid 3. caffeic acíd 5. echinacoside

2. chlorogenic acid 4. cynarin 6. cíchoric acíd

Figure 1821.-1. - Chromalogram for lhe assay of echinacoside in narrow-leaved coneflower rool

Results The chromatogram obtained with the test solution Mobile phase:
shows no greenish ftuorescent zone just below the zone due - mobile phase A: phosphon'c acid R, water R (1 :999 V/V) ;
to caffeic acid in the chromatogram obtained with the - mobile phase B: acetonitnle R;
reference solution and no greenish ftuorescent zone below the Time Mobile phase A Mobile phase B
zone due to cynarin in the chromatogram obtained with the (min) (per cent V/12 (per cent V/12
reference solurion. In the chromatogram obtained with the O 90 10
test solution no zones apart from faint dark blue ftuorescent
0-13 90 ~ 78 10 ~ 22
zones are visible between the zones due to echinacoside and
cynarin. 13 - 14 78 ~ 60 22 ~40

Loss on drying (2.2.32) 14 - 14.5 60 40

Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 9.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution In a 100 mL volumetric ftask place 0.500 g of
powdered drug (355) (2.9.12) and add 80 mL of ethanol
(70 per cent V/Ji) R. Treat in an ultrasonic bath for 15 min
and dilure to 100.0 mL with ethanol (70 per cent VIV) R.
Mix the suspension and aIlow to stand for a few minutes so
that visible solids settle. Filter a suitable proportion of the
solution through a membrane filter (nominal pore size
0.45 J..lm) before injection.
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
and 10.0 mg of caffeic acid R in ethanol (70 per cent VIV) R,
sonicate for 15 min and dilure to 10.0 mL with the same
solvento Dilure 4.0 mL of this solution to 100.0 mL with
ethanol (70 per cent V/Ji) R.
Column:
- size: 1 = 0.25 m, 0 = 4 .6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 J..lm);
- temperature: 35 oc.
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 330 nm.
Injection 1O ~lL.
Relative retention With reference to chlorogenic acid:
caftaric acid = abour 0.8; caffeic acid = about 1.5;
cynarin = about 1.6; echinacoside = abolit 1.7;
cichoric acid = about 2.3.
System suitability Reference solution:
- resolution: minimlim 10 berween the peaks due to caffeic
acid and chlorogenic acid.
Locate the peaks due to caffeic acid and chlorogenic acid
using the chromatogram obtained with the reference solution.
Locate the peaks due to echinacoside and cynarin using the
chromatogram in Figure 1821 .-1.
Calculate the percentage content of echinacoside from the
foIlowing expression:
Al X C2 X 100 x 2.221
A 2 x Cl
area of the peak dlie to echinacoside in the
chromatogram obtained with the test solution;
area of the peak due to chlorogenic acid in the
chromatogram obtained with the reference
solution;
IV-162 Echinacea
concentration of the test solution, in milligrams
per millilitre;
concentration of chlorogenic acid in the reference
solution, in milligrams per millilitre;
2.221 peak correlation factor between chlorogenic acid
and echinacoside.
STORAGE
Store uncomminuted.
____________________________________________ ~E~
Echinacea Pallida Root ***
*** ***
(Pale Coneflower Rom, Ph Eur monograph 1822) ***
~E~ ____________________________________________
DEFINITION
Dried, whole or cut underground parts of Eehinaeea pallida
Nutt.
Content
Minimum 0.2 per cent of echinacoside (C35H46020;Mr
786.5) (dried drug).
IDENTIFICATION
A. The rhizome and roots are 4-20 mm in diameter,
cylindrical and sometimes spirally twisted, longitudinally
wrinkled or deeply furrowed; the outer surface is reddish-
brown to greyish-brown.
B. Reduce to a powder (355) (2.9.12). The powder is
greyish-brown to light yellow. Examine under a microscope
using ehloral hydrate solution R. The powder shows the
following diagnostic characters: short lignified fibres
(100-300 Jlm in length, up to about 80 Jlm in diameter)
occurring singly or joined together in long bundles,
sometimes with phytomelanin deposits; lignified reticulately
or scalariforrnly thickened vessels (up to about 70 Jlm in
diameter); abundant sclereids, occurring singly or in small
groups of less than 10, varying considerably in shape from
rounded to rectangular or irregular, sometimes much
elongated and fibre-like and measuring up to 400 Jlm in
length; aH the sclereids have associated black, phytomelanin
deposits; fragments of oleoresin canals (up to 240 Jlm in
diameter) with yellowish-orange content; groups of squarish
to rectangular cells of the outer layers (about 40 x 80 Jlm);
abundant thin-walled pitted parenchyma with
sphaerocrystalline mas ses of inulin.
C. Examine the chromatograms obtained in the test for other
Eehinaeea species and Panhenium integrifolium.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. The chromatogram obtained with the test
solution may also show a weak zone close to the solvent
front.
D. Examine the chromatograms obtained in the assay.
Results The major peak in the chromatogram obtained with
the test solution is due to echinacoside. Peaks due to caftaric
acid, caffeic acid, cynarin, chlorogenic acid and cichoric acid
are minor peaks or may be absent.
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent.
Other Echinacea species and Parthenium integrifolium
Thin-Iayer chromatography (2.2.21).
2014
Top of the plate
A greenish-brown to brown zone
A yellow zone
A violet zone
,8Sitosterol: a violet lo pink zone A violet to pink zone (,8silosterol)
N-lsobutyldodecatetraenamide: a
greyish-blue zone
A dark grey-blue zone
Reference solution Test solution
Test solUlion To 1.0 g of the powdered drug (355) (2.9.12)
add 10 mL of methylene ehloride R and sonicate for 5 mino
Centrifuge and use the supernatant solution.
Reference solution Dissolve 1 mg of fJ-sitosterol R and a
volume of N-isobutyldodeeatetraenamide solution R
corresponding to 1 mg of N-isobutyldodecatetraenamide R in
methanol R and dilute to 5.0 mL with the same solvent.
Plate TLC siliea gel F254 plate R (5-40 Jlm ) [or TLC si/iea gel
F254 plate R (2-10 Jlm)].
Mobile phase anhydrous formie aeid R, eyelohexane R, ethyl
acetate R, toluene R (0.9:3:6:24 VIVIV/V).
Applieation 25 JlL [or 5 JlL] of the test solution and 10 J.lL
[or 4 JlL] of the reference solution as bands.
Development Over a path of 15 cm [or 5 cm].
Drying In a stream of cold air for about 10 mino
Deteetion Treat the plate using anisaldehyde solurion R and
heat at 105 oC for 3 min; examine in daylight.
Results The chromatogram obtained with the test solution
shows no greyish-blue zone at the position of
N-isobutyldodecatetraenamide in the chromatogram obtained
with the reference solution, and no blue zone at the position
of the violet zone due to f3-sitosterol in the chromatogram
obtained with the reference solution.
Loss on drying (2.2.32)
Maximum 12.0 per cent, deterrnined on 1.000 g of the
powdered drug (355) (2.9.1 2) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution In a 100 mL volumetric ftask place 0.500 g of
the powdered drug (355) (2.9.12) and add 80 mL of ethanol
(70 per eent VIV) R. Sonicate for 15 min and dilute to
100.0 mL with ethanol (70 per eent V/V? R . Mix the
suspension and allow to stand for a few minutes to aHow
visible solids to settle. Filter a suitable proportion of the
solution through a membrane filter (nominal pore size
0.45 Jlm) before injection.
Referenee solution Dissolve 10.0 mg of ehlorogenic acid CRS
and 10.0 mg of eaffeie acid R in ethanol (70 per cent V/V? R,
sonicate for 15 min and dilute to 10.0 mL with the same
2014 Echinacea IV-163

2 3 4

I I I I I I I I I I
2 4 6 8 10 12 14 16 18 min

1. eaftaric acid 3. eaffeie acid 5. eehinaeaside

2. ehlarogenic add 4. eynarin 6. deharie acid

Figure 1822.-1. - Chromatogram for the assay of echinacoside in pale coneflower root

solvent. Dilute 4.0 mL ofthis solution to 100.0 mL with area of the peak due to echinacoside in the
ethanol (70 per cent V/V) R. chromatogram obtained with the test solution;
eOlu111n: area of the peak due to chlorogenic acid in the
-- size: 1 == 0.25 m, 0 == 4.6 mm; chromatogram obtained with the reference
-- stationary phase: octadecylsilyl silica gel for chro111atography R solution;
(5 J..lm); el concentration of the test solution, in milligrams
-- te111perature: 35 oC. per millilitre;
Mobile phase: concentration of chlorogenic acid in the reference
-- 1110bile phase A: phosphoric acid R, water R (1 :999 V/V); solution, in milligrams per millilitre;
-- 1110bile phase B: acetonitrile R; 2.221 peak correlation factor between chlorogenic acid
and echinacoside.
Time Mobile phase A Mobile phase B
(min) (per cent V(lI) (per cent V(lI) STORAGE
O 90 10 Uncomminuted.
0·13 90 ~ 78 10 ~22 _ _ _ __ __ _ _ __ __ _ _ _ _ __ ____ Ph fUf

13·14 78 ~ 60 22 ~40

14·20 60 40

Flow rate 1.5 mUmin. Echinacea Purpurea Herb ***

Detection Spectrophotometer at 330 nm. *** ***
Injection 10 J..lL.
(Purple eonefiower Herb, Ph Eur 111onograph 1823) ***
~E~ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ __ _ _ ___
Relative retention With reference to chlorogenic acid
(retention time == about 7 min): caftaric acid == about 0.8; DEFINITION
caffeic acid == about 1.5; cynarin == about 1.6; Dried, whole or cut flowering aerial parts of Echinacea
echinacoside == about 1.7; cichoric acid == about 2.3. purpurea (L.) Moench.
Syste111 suitability Reference solution: Content
-- resolution: minimum 10 between the peaks due 10 caffeic Minimum 0.1 per cent for the sum of caftaric acid
acid and chlorogenic acid. (C 13H IZ 0 9 ; M r 312.2) and cichoric acid (C2zHlS012; M r
Locate the peaks due to caffeic acid and chlorogenic acid 474.3) (dried drug).
using the chromatogram obtained with the reference solution. IDENTIFICATION
Locate the peaks due to echinacoside, caftaric acid and First identification A, B, e.
cichoric acid using the chromatogram in Figure 1822.-1.
Second identification A, B, D.
Calculate the percentage content of echinacoside using the
A. The herbaceous perennial plant is 60-150 cm, rarely up to
following expression:
180 cm high. The stem is green to red, upright and slightly
branched. The leaves are alternate, ovate to ovate-Ianceolate,
Al X C2 X 100 x 2.221 irregularly serrate, rugose on both surfaces, dark green with
A2 XCI prominent light green veins; the lamina is thick and shiny.
IV-164 Echinacea 2014

The involucral bracts of the large capitulum are arranged in Top of the plate
2 or 3 rows. The solid receptacle is slightly convexo Each of
An intense red f1uorescent zone
the outer violet ligulate fiorets (4-6 cm) and of the inner
violet-pink tubular fiorets is attached to a reddish acute and Caffeic acid: a strong blue A blue f1uorescent zone
coriaceous bract, which overtops the tubular fiorets . f1uorescent zone
The calyx is reduced to a very short crown, one of the sepals --- ---
is up to 1 mm long.
B. Reduce to a powder (355) (2.9.12). The powder is green. A blue f1uorescent zone
Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters: Chlorogenic acid: a strong blue
f1uorescent zone
whitish-green groups of fibres, 150-200 Jlm in length,
A faint yellow-orange f1uorescent
10-15 ~lm in diameter, sometimes with black deposits; zone
fragments of leaves in surface view showing anomocytic or --- ---
anisocytic sto mata (2.8.3) (about 35-40 Jlm in length);
uniseriate covering trichomes or fragments thereof consisting
mainly of 3 or 4 thick-walled cells of which the apical cell is Referenee solution Test solution
markedly longer than the others; fragments of leaves with
rosette-like arranged epidermal cells around the base of the
covering trichomes; uniseriate glandular trichomes composed
of very thin-walled cells; pitted parenchymatous cells from
Test solution In a 100 mL volumetric fiask place 0.500 g of
the powdered drug (355) (2.9.12) and add 80 mL of ethanol
the pith of the stem as well as pitted elongated cells from the
(70 per cent V/V) R. Sonicate for 15 min and dilute to
mesocarp of the achenes; fragments of parenchyma from the
100.0 mL with ethanol (70 per cent V/V) R. Mix the
seeds with oil droplets; fragments of the epidermis of ligulate
suspension and allow to stand for a few minutes to allow
fiorets composed of red to violet papillous cells; spheroidal
visible solids to settle.
pollen grains, 30-40 Jlm in diameter, with a spiny exine.
C. Thin-Iayer chromatography (2.2.27) .
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
and 10.0 mg of cafJeic acid R in ethanol (70 per cent V/V) R,
Test solution To 1.0 g of the powdered drug (355) (2.9.12) sonicate for 15 min and dilute to 10.0 mL with the same
add 10 mL of methanol R and sonicate for 5 mino Centrifuge solvento Dilute 4.0 mL ofthis solution to 100.0 mL with
and use the supernatant solution. ethanol (70 per cent V/V) R.
Reference solution Dissolve 0.5 mg of cafJeic acid R and Column:
0.5 mg of chlorogenic acid R in 5.0 mL of methanol R. - size: l = 0 .25 m, 0 = 4 .6 mm;
Plate TLC silica gel plate R (5-40 ~lm) [or TLC silica gel - stationary phase: octadecylsilyl silica gel for chromacography R
plate R (2-1 0 Jlm)]. (5 Jlm);
Mobile phase anhydrous formic acid R, water R, methyl ethyl - temperature: 35 oC .
kecone R, ethyl acetate R (3:3:9:15 VIVIV/V). Mobile phase:
Application 25 ~IL [or 5 JlL] of the test solution and 10 JlL - mobile phase A : phosphoric acid R, water R (1:999 V/V);
[or 2 ~IL] of the reference solution, as bands. - mobile phase B: acetonitnle R;
Development Over a path of 15 cm [or 5 cm]. Time Mobile phase A Mobile phase B
Drying In a stream of cold air for about 10 min, then at (min) (per eent V!JI) (pereent VM
100 oC for 2 mino O 90 10

Detection Spray the still-warm plate with a 5 gIL solution of 0·13 90 ~ 78 10 ~22

diphenylboric acid aminoethyl ester R in ethyl acetate R; after 13·14 78 ~ 60 22 ~40

30 min, examine in ultraviolet light at 365 nm.
14 - 20 60 40
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the Flow rate 1.5 mUmin.
test solution. Furthermore, other faint blue fiuorescent zones Detection Spectrophotometer at 330 nm.
may be present in the chromatogram obtained with the test
Injection 10 JlL.
solution.
Relative retention With reference to chlorogenic acid
D. Examine the chromatograms obtained in the assay.
(retention time := about 7 min): caftaric acid := about 0.8;
The principal peak in the chromatogram obtained with the
caffeic acid := about 1.5; cynarin = about 1.6;
test solution is due to cichoric acid and a smaller peak is due
echinacoside = about 1.7; cichoric acid := about 2.3.
to caftaric acid. Peaks due to caffeic acid and chlorogenic
acid are minor or may be absent. System suitability Reference solution:
- resolution: minimum 5 between the peaks due to caffeic
TESTS acid and chlorogenic acid.
Loss on drying (2.2.32)
Locate the peaks due to caffeic acid and chlorogenic acid
Maximum 10.0 per cent, determined on 1.000 g ofthe
using the chromatogram obtained with the reference solution.
powdered drug (355) (2. 9.12) by drying in an oven at Locate the peaks due to caftaric acid and cichoric acid using
105 oC for 2 h.
the chromatogram in Figure 1823.-1.
Total ash (2.4. 16) Calculate the percentage content of caftaric acid using the
Maximum 12.0 per cent. following expression:
ASSAY
Liquid chromatography (2.2.29).
2014 Echinacea IV-165

A
2

I I I I I I

2 4 6 8 10 12 14 16 18 min

1. caftaric acid 2. chlorogenic acid 3. cichoric acid

Figure 1823.-1. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower herb

Al X O2 X 100 x 0.881
A2 x 0 1 Echinacea Purpurea Root
(Purple Conefiower Root, Ph Eur monograph 1824)
Calculate the percentage content of cichoric acid using the ~E~ ____________________________________________
following expression:
DEFINITION
Dried, whole or cut underground parts of Eehinaeea purpurea
A3 X O2 X 100 x 0.695 (L.) Moench.
A2 x 0 1
Content
Minimum 0.5 per cent for the sum of caftaric acid
area of the peak due to caftaric acid in the (C 13H 12 0 9 ; M r 312.2) and cichoric acid (C22Hl S01 2; M r
chromatogram obtained with the test solution; 474.3) (dried drug).
area of the peak due 10 chlorogenic acid in the
IDENTIFICATION
chromatogram obtained with the reference
solution;
First identifieation A, B, e, E.
area of the peak due 10 cichoric acid in the Second identifieation A, B, D, E.
chromatogram obtained with the test solution; A. The rhizome is up to 15 cm long, branched, reddish-
concentration of the test solution, in milligrams brown 10 dark brown on the surface and carries many stem
per millilitre; bases; the inside is fibrous and white. The numerous roo18
concentration of chlorogenic acid in the reference are spirally twisted, light to dark brown and show a fine cross
solution, in milligrams per millilitre; structuring on the surface.
0.695 peak correlation factor based upon the liquid B. Reduce 10 a powder (355) (2.9.12). The powder is light
chromatography response observed; yellow to pinkish-beige. Examine under a microscope using
0.881 peak correlation factor between caftaric acid and ehloral hydrate solution R. The powder shows the following
chlorogenic acid. diagnostic characters: numerous light-brown spindle-shaped
STORAGE fibres that are joined together in long bundles without black
deposits; rare sclereids from the rhizomes and roots, usually
Uncomminuted.
occuring singly, those from the rhizomes being isodiametric,
____________________________________________ ~E~

about 60 /lm in diameter, with black deposits, those from the

roots being 50-120 /lm in length with no black deposits;
secretory cavities up 10 180 /lm in diameter with yellow oil
droplets; squarish to rectangular cells of the outer layers,
sorne with reddish walls; bordered-pined ves seis from the
rhizome, 30-40 /lm in diameter.
C . Examine the chromatogram obtained in the test for other
Eehinaeea species and Parthenium integrifolium.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
IV-166 Echinacea 2014

test solution. Furthermore, faint greenish fluorescent zones TESTS

may be present just below the zone situated in the middle of Other Echinacea species and Parthenium integrifolium
the chromatogram obtained with the test solution. Thin-layer chromatography (2.2.27) .
Test solution To 1.0 g ofthe powdered drug (355) (2.9. 12)
Top oí the plate add 10 mL of methanol R and sonicate for 5 mino Centrifuge
and use the supernatant soIution.
Caffeic acid: a strong blue A strong blue fluorescent zone
fluorescent zone Reference solution DissoIve 1 mg of echinacoside R, 1 mg of
cynarin R and 0.5 mg of caffeic acid R in 5.0 mL of
methanol R .
--- --- Plate TLC silica gel plate R (5-40 !lm) [or TLC silica gel
Cynarin : a strong greenish plate R (2-10 !lm)].
fIuorescent zone
Mobile phase anhydrous formic acid R, water R, methyl ethyl
A blue fIuorescent zone
ketone R, ethyl aceta te R (3:3:9: 15 V/V/V/V).
--- --- Application lO!lL [or 5 !lL] of the test solution and 5 !lL
Echinacoside: a strong greenish [or 2 !lL] of the reference soIution, as bands.
fluorescent zone Development Over a path of 10 cm [or 5 cm].
Drying In a stream of coId air for about 10 min, then at
Reference solution Test solution 105 oC for 2 mino
Detection Spray the stilI-warm pIate with a 5 gIL solution of
diphenylboric acid aminoethyl ester R in ethyl acetate R; after
D. Examine the chromatograms obtained in the assay. 30 min, examine in ultraviolet light at 365 nm.
The principal peak in the chromatogram obtained with the
Results The chromatogram obtained with the test solution
test solution is due to cichoric acid and a smaller peak is due
to caftaric acid. Peaks due to caffeic acid and chlorogenic shows no greenish fluorescent zone corresponding to the
acid are minor or may be absent. zone due to echinacoside in the chromatogram obtained with
the reference solution, and no greenish fluorescent zone
E. Thin-layer chromatography (2.2.27).
corresponding to the zone due to cynarin in the
Test solution To 1.0 g of the powdered drug (355) (2.9.12) chromatogram obtained with the reference solution. No other
add 10 mL of methylene chloride R and sonicate for 5 mino zones apart from very faint dark blue fluorescent zones are
Centrifuge and use the supernatant solution. seen in the lower half of the chromatogram of the test
Reference solution Dissolve 1 mg of fJ-sitosterol R and a solution.
volume of N-isobutyldodecatetraenamide solution R Foreign matter (2.8.2)
corresponding to 1 mg of N-isobutyldodecatetraenamide R in Maximum 3 per cent.
5.0 mL of methanol R.
Loss on drying (2.2.32)
Plate TLC silica gel plate R (5-40 !lm) [or TLC silica gel
Maximum 10.0 per cent, determined on 1.000 g of the
plate R (2-10 !lm)]. powdered drug (355) (2.9.12) by drying in an oven at
Mobile phase anhydrous formic acid R, cyclohexane R, ethyl 105 oC for 2 h.
acetate R, toluene R (0.9:3:6:24 V/V/V/V).
Total ash (2.4.16)
Application 25!lL [or 5 !lL], as bands. Maximum 9.0 per cent.
Development Over a path ofabout 15 cm [or 5 cm]. Ash insoluble in hydrochloric acid (2.8.1)
Drying In a stream of cold air for about 10 mino Maximum 2.0 per cent.
Detection Dip the pIate into anisaldehyde solution R for 1 s ASSAY
and heat at 100-105 oC for 3 min; examine in daylight.
Liquid chromatography (2.2.29).
Results See beIow the sequence of zones present in the
Test solution In a 100 mL volumetric flask place 0.500 g of
chromatograms obtained with the reference soIution and the
the powdered drug (355) (2.9.12) and add 80 mL of ethanol
test soIution. Furthermore, other faint zones may be present
(70 per cent V/V; R. Sonicate for 15 min and dilute to
in the chromatogram obtained with the test soIution.
100.0 mL with ethanol (70 per cent V/V; R . Mix the
suspension and allow to stand for a few minutes to allow
Top oí the plate visible solids to settle.
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
and 10.0 mg of caffeic acid R in ethanol (70 per cent V/V; R ,
--- --- sonicate for 15 min and dilute to 10.0 mL with the same
A bluish-violet zone solvent. Dilute 4.0 mL of this solution to 100.0 mL with
ethanol (70 per cent V/V; R.
j}-Sitosterol: a violet or pink zone A violet or pink zone (j}-sitosterol)
Column:
N-Isobutyldodecatetraenamide: a A greyish-blue zone - size: l = 0.25 m, 0 = 4.6 mm;
greyish-blue zone (N-isobutyldodecatetraenamide) - stationary phase: octadecylsilyl silica gel for chromatography R
--- --- (5 !lm);
A dark greyish-blue zone - temperature: 35 oc.
Mobile phase:
Reference solution Test solution
- mobile phase A: phosphoric acid R , water R (1:999 V/V);
- mobile phase B: acetonitrile R ;
2014 Eclipta Prostrata IV-167

2
A
L,'-'-'-'I'-'-'-'--'I-'-'-''-TI-'-'--'-'I-'--'-'-'I-''-'-'-II--'-'-'-~I'-'-'-'--'I-'-'--'-'I-'--'-'--1
2 4 6 8 10 12 14 16 18 min

1. caftaric acid 2. chlorogenic acid 3. cichoric acid

Figure 1824.-1. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower root

Time Mobile phase A Mobile phase B area of the peak due to chlorogenic acid in the
(min) (per cent V/JI) (per cent V/JI) chromatogram obtained with the reference
O 90 10 solution;
0·13 90 ..... 78 10 ..... 22 area of the peak due to cichoric acid in the
chromatogram obtained with the test solution;
13" 14 78 ..... 60 22 ..... 40
concentration of the dried drug in the test
14·20 60 40 solution, in milligrams per millilitre;
concentration of cWorogenic acid in the reference
Flow rate 1.5 mUmin. solution, in milligrams per millilitre;
Detection Spectrophotometer at 330 nm. 0.695 peak correlation factor based upon the liquid
Injection 10)lL. chromatography response observed;
Relative retention With reference to chlorogenic acid 0.881 peak correlation factor between caftaric acid and
(retention time = about 7 min): caftaric acid = about 0.8; cWorogenic acid.
caffeic acid = about 1.5; cynarin = about 1.6; STORAGE
echinacoside = about 1.7; cichoric acid = about 2.3. Uncornminuted.
System suitability Reference solution: ___________________________________________ ~Ew

-- resolution: minimum 5 between the peaks due to caffeic

acid and chlorogenic acid.
Locate the peaks due to caffeic acid and chlorogenic acid
using the chromatogram obtained with the reference solution.
Locate the peaks due to caftaric acid and cichoric acid using Eclipta Prostrata Whole Plant
the chromatogram in Figure 1824.-1. DEFINITlON
Calculate the percentage content of caftaric acid using the Eclipta Prostrata Whole Plant is the dried whole plant, either
following expression: entire or fragmented, of Eclipta prostrata (L.) L.
It contains not les s than 0.04% of wedelolactone
Al X C2 X 100 x 0.881 (C 16H IO 0 7), calculated with reference to the dried material.
A 2 x C1 IDENTIFICATlON
A. Stems cylindrical, four sided or fiattened, 2 to 5 mm in
Calculate the percentage content of cichoric acid using the diameter, greyish, with appressed, whitish hairs pointing
following expression: towards the tip, longitudinally striated, occasionally
branching and nodes distinct. Leaves dark green, sessile or
A3 X C2 X 100 x 0.695 subsessile, opposite, lanceo late, 2 to 8.5 cm long and 1 to
A2 x C1 2.5 cm wide with an entire or slightly dentate margin and
appressed trichomes on both surfaces. Flowerheads 2 to
Al area of the peak due to caftaric acid in the 6 mm in diameter, greenish-brown, solitary or in pairs on
chromatogram obtained with the test solution; unequal axillary peduncles, up to 8 involucral bracts, ovate,
IV-168 Ec1ipta Prostrata 2014

with appressed hairs; ray fiorets spreading, no longer than the Top of the plate
bracts, not toothed; disc fiorets with 4-toothed coralla, A red zene
pappus usually absent or reduced to minute teeth; 5 stamens, A diffuse pale blue zene
filaments epipetalous and anthers united into a tube; pistil A greenish-white zene

bicarpellary, ovary inferior, unilocular with one basal ovule. A pale blue zene
Fruits 2 to 3 mm long, pappi persistent and coroniform;
unfertilised achenes pale yellow, fiattened and smooth;
A blue-white zene (wedelelactene) A blue-white zene (wedelelactene)
fertilised achenes pale to dark brown, 3 to 4 angled, A blue-white zene
tuberculate and bulbous. Root, if present, cylindrical, greyish,
main root up to about 7 mm in diameter, with secondary
branching. Resmarinic acid : A blue-white zene
A blue-white zene
B. Reduce to a powder (355). The powder is greenish Solution (1) Solution (2)
brown. Examine under a microscope using ehloral hydrate
solution. The main diagnostic characters include numerous
free, whole or braken, large covering trichomes, with warty or
spiny walls, up to 700 f.lm long, uniseriate, usually tricellular, TESTS
with a broad basal cell, a long median cell and a short, Loss on drying
0

pointed sub-triangular apical cell; les s frequently, smaller,
unicellular, pointed covering trichomes from the midrib and
stem. Fragments of leaf show sinuous walled epidermal cells,
underlying palisade, anomocytic stomata, cuticular striations
and covering trichomes on both surfaces. Stem fragments
with unicellular and multicellular trichomes, epidermis of
elongated cells, or in mature stem, poorly developed
rectangular cork cells; secondary cortex of parenchyma with
numerous air-spaces, pericyclic fibres thick-walled, lignified,
simple pitted; secretory canals may be visible; xylem vessels
usually simple pitted or spirally thickened, xylem parenchyma
lignified and pitted. Fragments of root, if present, show
poorly developed cork, consisting of 3-5 rows of thin-walled
elongated cells, a secondary cortex of parenchyma, with
scattered stone cells and fibres either singly or in groups,
xylem ves seIs and tracheids, and fibres with peg-like
projections. Pollen grains with spiny exine and 3 pores.
C. Carry out the method for thin-Iayer chromatography,
Appendix III A, using the following solutions.
(1) Reduce to a powder (355). To 2.0 g ofpowdered sample
add 40 mL of methanol. Mix with the aid of ultrasound for
2 hours at 50° with occasional shaking and allow to cool.
Dilute to 50 mL with methanol and filter.
(2) 0.05% w/v each of wedelolactone EPCRS and rosmarinic
acid in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F254 (Merck silica gel
HPTLC plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 10 f.lL of each solution as 8 mm bands.
(d) Develop the plate to 6 cm.
0
(e) After removal ofthe plate, dry in air, heat at 100 for
5 minutes, treat the plate whilst still hot with a 0.5% v/v
solution of diphenylboric acid aminoethyl ester in ethyl acetate
and examine under ultraviolet light (365 nm) .
MOBILE PHASE
1 volume of anhydrous formic acid, 6 volumes of acetone and
11 volumes of toluene.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated spots.
CONFIRMATION
The chromatogram obtained with solution (1) shows a
fiuorescent band corresponding to wedelolactone and several
other fiuorescent bands as shown in the table. Other
fiuorescent bands may be presento
When dried at 100 to 105° for 2 hours, loses not more than
11.0% of its weight. Use 1 g.
Total Ash
Not more than 22.0%, Appendix XI], Method n.
Acid-inso1uble Ash
Not more than 11.0%, Appendix XI K.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Reduce to a powder (355). To 2.0 g ofpowdered sample
add 40 mL of methanol. Mix with the aid of ultrasound for
2 hours at 50° with occasional shaking and allow to cool.
Dilute to 50 mL with methanol and filter.
(2) 0.005% w/v each of wedelolactone EPCRS and coumestrol
in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
with octadecylsilyl si/ica gel for chromatography (5~lm) (Waters
Symmetry C18 is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a fiow rate of 1.0 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 249 nm.
(f) Inject 10 f.lL of each solution.
MOBILE PHA SE
24 volumes of acetonitrile and 76 volumes of a 0.2% v/v
solution of orthophosphoric acid.
SYSTEM SUITABILITY
The assay is not valid unless, in the chromatogram obtained
with solution (2):
the symmetry factor of the peak due to wedelolactone is less
than 1.2;
the symmetry factor of the peak due to coumestrol is less than
1.1.
DETERMINATIO N OF CONTENT
Calculate the content of wedelolactone in the sample using
the declared content of wedelolactone (C 16H IO 0 7) in
wedelolactone EPCRS and the following expression:
2014 Elder Flower IV-169

A, m2 V, 100
-x-x-xpx
A2 V2 m i 100-d

area of the peak due to wedelolactone in the

chromatogram obtained with solution (1);
area of the peak due to wedelolactone in the
chromatogram obtained with solution (2);
weight of the herbal drug being examined in mg; Ca
weight of wedelolactone EPCRS in mg;
dilution volume of solution (1) in mL; Cb -':\"\AlW~IV
dilution volume of solution (2) in mL; Ce
percentage content of wedelolactone
(C 16H lO 0 7) in wedelolactone EPCRS;
d percentage loss on drying of the herbal drug being
examined.

Elder Flower
(Ph Eur monograph 121 7)
~E~ ____________________________________________

DEFINITION
Dried flowers of Sambueus nigra L.
Content
Minimum 0.80 per cent of flavonoids, expressed as
isoquercitroside (CzI H zoOl Z; M r 464.4) (dried drug).
IDENTIFICATION
A. The flower, about 5 mm in diameter, has 3 small bracts,
visible under a lens, and may have a pedunde.
The 5-toothed calyx is small; the corolla is light yellow, with
5 broadly oval petals fused at their bases into a tube.
The filaments of the 5 yellow stamens alternate with the
petals. The corolla is often isolated or artached to the
starnens, to which it is fused at the base. The ovary is inferior
and it bears a short style with 3 obtuse stigmata.
B. Microscopic examination (2.8.23). The powder is
greenish-yellow. Examine under a microscope using ehloral
hydrate solution R . The powder shows the following diagnostic
characters (Figure 1217.-1): numerous spherical, sometimes
ellipsoidal, pollen grains about 30 11m in diameter, with
3 germinal pores and very finely pitted exine [G]; cells of the
lower epidermis of the sepals often containing oil globules
and covered by a striated cutide in surface view [A]; rare
fragments of the rim of the sepals showing unicellular
marginal teeth, in transverse section [E] ; petal fragments with
numerous small globules of essential oil [H]; fragments of
upper epidermis of the sepals [B] or petals [F], in surface
view, with slightly and irregularly thickened walls [Ba, Fa],
anomocytic stomata ( 2.8.3) [Bb, Fb] and a striated cutide;
mesophyll cells of petals and sepals with idioblasts containing
numerous microsphenoid crystals of ca1cium oxalate [Bc];
fragments of anthers in transverse section [C] and in surface
view [D], showing the outer layer [Ca] and the cells of the
fibrous layer [Cb, Cc, D].
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R and sonicate for 10 mino
Centrifuge for 5 mino
Figure 1217.-1. - Illustration for identification B ofpowdered
herbal drug of elder flow er
R eferenee solution Dissolve 1 mg of caffeie acid R, 1 mg of
ehlorogenie acid R, 2.5 mg of hyperoside R and 2.5 mg of
rutin R in 10 mL of methanol R.
Plate TLC siliea gel plate R (2-10 11m).
Mobzle phase anhydrous formie acid R, water R , methyl ethyl
ketone R, ethyl acetate R (10:10:30:50 VIVIV/V).
Applieation ~IL as bands of 8 mm.
4
Development Over a path of 6 cm.
Drying In air.
Deteetion Heat the plate for 5 min at 100 cC and treat with
a 1 gIL solution of diphenylborie acid aminoethyl ester R in ethyl
aeetate R , then treat with a 5 gIL solution of maerogol 400 R
in methylene ehloride R ; allow to dry in air for 30 min.
Examine in daylight (results A) and in ultraviolet light at
365 nm (results B).
R esults ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
R esults B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
TESTS
Foreign matter (2.8.2)
Maximum 8 per cent of fragments of coarse pedicels and
other foreign matter and maximum 15 per cent of
discoloured, brown flowers . Carry out the determination on
10 g.
Sambucus ebulus L
Examine the chromatograms obtained in identification C.
 IV-17 O Eleutherococcus 2014

Top oC the plate absorbent cotton into the flask. After cooling, filter the
combined acetone extracts through a filter paper into a
volumetric flask and dilute to 100.0 mL with acetone R by
--- --- rinsing the flask and the filter papero Introduce 20.0 mL of
An orange zone this solution into a separating funnel, add 20 mL of water R
and shake the mixture with 1 quantity of 15 mL and then
Hyperoside : a dark yellow zone 3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acetate extracts in a separating funnel, wash with
2 quantities, each of 50 mL, of water R, and filter the
--- ---
extracts over 10 g of anhydrous sodium sulfate R into a
volumetric flask and dilute to 50.0 mL with ethyl acetate R .
Rutin: a dark yellow zone A dark yellow zone Test solution To 10.0 mL ofthe stock solution add 1 mL of
aluminium chlonde reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R.
ReCerence solution Test solution Compensation liquid Dilute 10.0 mL ofthe stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic
acid R in methanol R.
Top oC the plate After 30 min, measure the absorbance (2.2.25) of the test
solution at 425 nm, by comparison with the compensation
Caffeic acid: a blue fluorescent liquido
zone
An in tense, Iight blue f1uorescent Calculate the percentage content of flavonoids, expressed as
zone isoquercitroside, using the following expression:
2 Iight blue f1uorescent zones
A x 1.25
--- ---
m
An orange f1uorescent zone
Hyperoside: an orange fluorescent
i.e. taking the specific absorbance of isoquercitroside to be
zone 500.
Chlorogenic acid: a Iight blue An intense, light blue f1uorescent A absorbance at 425 nm;
f1uorescent zone zone m = mass of the herbal drug to be examined, in grams.
____________________________________________ ~E~

--- ---

Rutin: an orange fluorescent zone An orange f1uorescent zone

Eleutherococcus
Siberian Ginseng
ReCerence solution Test solution (Ph Eur monograph 1419)
~E~ ____________________________________________

Results B The chromatogram obtained with the test solution DEFINITION

does not show a greenish-white zone aboye the zone due to
caffeic acid in the chromatogram obtained with the reference
solution; in the chromatogram obtained with the test
solution, no green fluorescent zone is seen just below the
orange fluorescent zone due to rutin in the chromatogram
obtained with the reference solution.
Loss 00 drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4. 16)
Maximum 10.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed flask, introduce
0.600 g of the powdered herbal drug (355) (2.9.12), add
1 mL of a 5 gIL solution of hexamethylenetetramine R, 20 mL
of acetone R and 2 mL of hydrochlonc acid R1. Boil the
mixture under a reflux condenser for 30 mino Filter the
mixture through a plug of absorbent cotton into a flask.
Add the absorbent cotton to the residue in the round-
bottomed flask and extract with 2 quantities, each of 20 mL,
of acetone R , ea eh time boiling under a reflux condenser for
10 mino Allow to cool, filter each extract through the plug of
Dried, whole or cut underground organs of Eleutherococcus
senticosus (Rupr. et Maxim.) Maxim.
Content: minimum 0.08 per cent for the sum of
eleutheroside B (Mr 372.4) and eleutheroside E (Mr 742.7).
IDENTIFICATION
A. The rhizome is knotty, of irregular cylindrical shape,
1.5 cm to 4.0 cm in diameter; the surface is rugged,
longitudinally wrinkled and greyish-brown to blackish-brown;
the bark, about 2 mm thick, closely adheres to the xylem;
the heartwood is light brown and the sapwood is pale yellow;
the fracture shows short thin fibres in the bark and is coarsely
fibrous, especially in the internal part of the xylem.
The lower surface bears numerous cylindrical and knotty
roots, 3.5 cm to 15 cm long and 0.3 cm to 1.5 cm in
diameter; with a smooth, greyish-brown to blackish-brown
surface; the bark is about 0.5 mm thick, closely adhering to
the pale yellow xylem; the fracture is slightly fibrous;
in places where the outer layer has been removed, the outer
surface is yellowish-brown.
B. Reduce to a powder (355) (2.9.12). The powder is
yellowish-brown. Examine under a microscope, using eh/oral
hydrate solution R. The powder shows numerous groups of
thick-walled, lignified fibres; fragments of reticulate and
bordered pitted ves seIs with a wide lumen; groups of
 2014 Eleutherococcus IV-171

secretory canals, up to 20 [lm in diameter with brown water-bath at 60 oC for 30 mino Allow to cool and filter
contents; parenchymatous cells containing cluster crystals of through a sintered-glass filter (2.1.2) . Collect the liquid in a
calcium oxalate 10 [lm to 50 [lID in diameter. Examine under 250 mL round-bottomed ftask. Repeat this operation twice,
a microscope, using a 50 per cent V/V solution of glycerol R. using the residue obtained in the filtration step instead of the
The powder shows small starch granules, rounded to slightly powdered drug. Add both fractions of supernatant liquid to
angular in outline, single compounds or with 2 or 3 the 250 mL round-bottomed ftask. Evaporate under reduced
components. pressure until about 10 mL of supernatant liquid is left in the
C. Thin-Iayer chromatography (2.2.27). ftask. Transfer the supernatant liquid quantitatively to a
20.0 mL volumetric ftask and dilute to 20.0 mL with a
Test solution To 1.0 g of the powdered drug (355) (2.9.12)
add 10 mL of alcohol (50 per cent V/V] R and boil under mixture of equal volumes of alcohol R and water R. Filter
through a nylon filter (pore size 0.45 [lm) .
reflux for 1 h . Cool and filter. Evaporate the filtrate to
dryness on a water-bath. Dissolve the residue in 2.5 mL of a Reference solution (a) Dissolve 10 mg offerulic acid R in a
mixture of 5 volumes of water R and 20 volumes of alcohol mixture of equal volumes of methanol R and water R and
(50 per cent V/V] R and filter. dilute to 20.0 mL with the same mixture of solvents.
Reference solution Dissolve 2.0 mg of esculin R and 2.0 mg of Reference solution (b) Dissolve 10 mg of caffeic acid R in a
catalpol R in 20 mL of a mixture of 2 volumes of water R and mixture of equal volumes of methanol R and water R and
8 volumes of alcohol (50 per cent V/V] R. dilute to 20.0 mL with the same mixture of solvents.
Plate TLC silica gel plate R . Reference solution (e) Transfer 1 mL of reference solution (a)
to a 25 mL volumetric flask and dilute to 25.0 mL with a
Mobile phase water R, methanol R, methylene chloride R
(4:30:70 VIV/V). mixture of equal volumes of methanol R and water R . Filter
through a nylon filter (pore size 0.45 ).lm) .
Application 20 [lL, as bands.
Referenee solution (d) Transfer 1 mL of reference solution
Development Over a path of 10 cm.
(a) and 1 mL of reference solution (b) in a mixture of equal
Drying In airo volumes of methanol R and water R and dilute to 25.0 mL
Detection A Examine in ultraviolet light at 365 nm. with the same mixture of solvents. Filter through a nylon
Results A The chromatogram obtained with the reference filter (pore size 0.45 [lm).
solution shows in the upper half a blue ftuorescent zone Precolumn:
(esculin). - size: 1= 4 mm, 0 = 4.6 mm,
Detection B Spray with anisaldehyde solution R and examine - stationary phase: octadecylsilyl silica gel for chromatography R
in daylight while heating at 100-105 oC for 5-10 mino (5 ~lm).
Results B See below the sequence of the zones present in Column:
the chromatograms obtained with the reference solution and - size: l = 0.25 m, 0 = 4.6 mm,
the test solution. Furthermore, other faint zones are present - stationary phase: octadecylsilyl silica gel for chromatography R
in the chromatogram obtained with the test solution. (5 [lm).
Mobile phase:
- mobile phase A: phosphoric acid R, water R (0.5:99.5 V/V),
Top of the plate
- mobile phase B: aeetonitrile for chromatography R,
A brown zone (eleutheroside B)
Time Mobile phase A Mobile phase B
Esculin: a blue fluorescent zone (min) (pereent VM (pereent VM
(marked at 365 nm) O-S 90 10
A reddish-brown zone
(eleutheroside E) 5 - 27 90 -7 80 10 -7 20
--- --- 27 - 30 80 -7 50 20 -7 50

Catalpol: a violet-brown zone 30 - 35 50 50

--- ---
2 brown zones Flow rate 1.0 mUmin.
Reference solution Test solution Detection Spectrophotometer at 220 nm.
Injection 20 [lL of the test solution and reference solutions
(e) and (d) .
TESTS
Foreign matter (2.8.2) Retention time Eleutheroside B = about 10 min;
Maximum 3 per cent. eleutheroside E = about 22 min o
Locate the peaks due to eleutheroside B and eleutheroside E
Loss on drying (2.2.32)
using the UV spectra shown in Figures 1419.-1 and 1419.-2 .
Maximum 10.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at System suitability Reference solution (d):
105 oC for 2 h. - resolution: minimum 15 between the peaks due to caffeic
acid and ferulic acid.
Total ash (2.4.16)
Maximum 8.0 per cent. Calculate the total percentage content of eleutheroside B and
eleutheroside E from the expression:
ASSAY
Liquid chromatography (2.2.29) .
Test solution To 0.500 g of the powdered drug (355)
(AB X e x 0 .73 x 2) + -'.:(A..::E~x__C=--x-=1__.9-=0_x_ 22)
(A R x m) (A R x m)
(2.9. 12) in a 100 mL round-bottomed ftask, add 30 mL of a
mixture of equal volumes of alcohol R and water R. Heat in a
IV-172 Eleutherococcus 2014

AB area of the peak due to eleutheroside B in the lIIjJ

ehromatogram obtained with the test solution,
AE area of the peak due to eleutheroside E in the
ehromatogram obtained with the test solution,
l5III
AR area of the peak due to ferulie acid in the
ehromatogram obtained with referenee solution Ce),
e eoneentration of ferulie aeid in referenee solution Ce),
in micrograms per millilitre,
In mas s of the drug to be examined, in milligrams.

:mi

151111

10

10IIII

lIII

10 o

220 !lO 210 2!0 3110 l2I) 3«1 ., ., 111

Figure 1419.-2. - UV spectrum of eleutheroside E for the assay

of eleutherococcus

_ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ Ph f ur

Figure 1419.-1. - UV spectrum of eleutheroside B for the assay

of eleutherococcus
2014 Ephedra Herb IV-173

Ephedra Herb Top of the plate

--- ---
(Ph Eur monograph 2451)
2-Indanamine: a purple spot
PhEur _ _ _ _ _ __ _ __ _ _ _ _ __ _ __ _ __
A purple spot may be present
DEFINITION
Dried herbaceous stem of Ephedra sinica Stapf, Ephedra --- ---

intermedia Schrenk et C.A.Mey. or Ephedra equisetina Bunge. Ephedrine: a purple spot at the A purple spot (ephedrine) at the
Content border between the middle and border between the middle and
lower thirds lower thirds
Minimum 1.0 per cent of ephedrine (ClOHISNO; M r 165.2)
Reference solution Test solution
(dried drug).
IDENTIFlCATION
A. Thin cylindrical pale green or yellowish-green stems up to TESTS
30 cm long and 1-3 mm in diameter; longitudinally striated Loss on drying (2.2.32)
and slighdy rough; intemodes varying in length between Maximum 10.0 per cent, determined on 1.000 g ofthe
1 cm and 6 cm; opposite and decussate leaves reduced to powdered drug (355) (2.9.12) by drying in an oven at
sheaths surrounding the stem, carrying diminutive laminae 105 oC for 2 h.
1.5-4 mm long with 2 lobes (rarely 3), acutely triangular,
Total ash (2.4. 16)
apex greyish-white, base tubular and reddish-brown or
Maximum 9.0 per cent.
blackish-brown. Fracture slighdy fibrous .
B. Reduce to a powder (355) (2.9. 12) . The powder is ASSAY
greenish-yellow. Examine under a microscope using chloral Liquid chromatography (2.2.29) .
hydrate solution R . The powder shows the following diagnostic Test solution To 0.200 g of the powdered drug (355)
characters: fragments of the epidermis, in surface view, (2. 9.12) add 25.0 mL of methanol R, weigh and sonicate for
composed of rectangular cells and numerous stomata with a 45 min. Allow to cool, weigh and adjust to the original mass
small depression at each end, the guard cells large and with methanol R, shake well and filter. Transfer 1.0 mL of the
broadly elliptical; epidermal fragments, in transverse section, filtrate to a small column (1 cm in diameter) packed with
showing a thick cunde and some of the cells extended to 1.50 g of neutral aluminium oxide R (60-210 ¡¡m) . Elute with
form projections; fibres in groups or single, with thick, a mixture of equal volumes of methanol R and water R .
usually lignified walls; fragments of lignified tissue composed Collect about 9 mL of the eluate, add 0.5 mL of phosphoric
of small, bordered-pitted tracheids, vessels with spiral acid R and dilute to 10.0 mL with a mixture of equal
thickening and groups of sdereids; groups of parenchyma, volumes of methanol R and water R .
sorne with thickened and pitted walls; scattered prism crystals Reference solution (a) Dissolve 10.0 mg of ephedrine
of calcium oxalate. hydrochloride CRS in methanol R and dilute to 100.0 mL with
C. Thin-layer chromatography (2.2.27) . the same solvento Dilute 2.0 mL ofthe solution to 25.0 mL
Test solution To 0.2 g ofthe powdered drug (355) (2.9.12) with the mobile phase.
add 0.5 mL of concentrated ammonia R and 10 mL of Reference solution (b) Dissolve 1 mg of ephedrine
methylene chloride R. Boil in a water-bath under a refiux hydrochloride CRS and 1 mg of terbutaline sulfate CRS in
condenser for 1 h. Allow to cool, filter and evaporate the methanol R and dilute to 10 mL with the same solvento
filtrate to dryness; dissolve the residue in 2 mL of Dilute 2 mL of the solution to 25 mL with the mobile phase.
methanol R . Column:
Reference solution Dissolve 1 mg of ephedrine - size: l = 0.25 m, 0 = 4.6 mm;
hydrochloride CRS and 1 mg of 2-indanamine hydrochloride R - stationary phase: octadecylsilyl silúa gel for chromatography R
in 2 mL of methanol R. (5 ¡¡m).
Plate TLC silica gel plate R (5-40 ¡¡m) [or TLC silica gel Mobt7e phase acetonimle R1, 0.1 per cent V/V solution of
plate R (2-10 ¡¡m)]. phosphoric acid R (15:85 V/V).
Mobile phase concentrated ammonia R, methanol R, methylene Flow rate 2.0 mL/min.
chlonde R (0.5 :5:20 VIV/V). Detection Spectrophotometer at 207 nm.
Application 10 ¡¡L [or 1 ¡¡L] as spots with a diameter of 1njection 1O ¡.¡L.
5 mm [or 2 mm]. Run time 3 times the retention time of ephedrine.
Development Over a path of 10 cm [or 6 cm]. System suitability Reference solution (b):
Drying In airo - resolution: minimum 3.5 between the peaks due to
Detection Spray with a 2 gIL solution of ninhydrin R in terbutaline and ephedrine.
ethanol (96 per cent) R; heat at 110 oC for 10 min and Calculate the percentage content of ephedrine using the
examine irnmediately in daylight. following expression:
Results See below the sequence of spots present in the
chromatograms obtained with the reference solution and the Al x m2 x P x 165 .2
test solution. Furthermore, other faint spots may be present A 2 x ml x 5 x 201.7
in the chromatogram obtained with the test solution.
Al area of the peak due to ephedrine in the
chromatogram obtained with the test solution;
Az area of the peak due to ephedrine in the
chromatogram obtained with reference solution (a);
IV-174 Eucalyptus Leaf
mI mass of the drug to be examined used to prepare the
test solution, in gtamSj
m2 mass of ephedrine hydroehlon'de CRS used to prepare
reference solution (a), in gramSj
p percentage content of ephedrine hydrochloride in
ephedrine hydroehloride CRS.
____________________________________________ ~Ew
Eucalyptus Leaf ***
** **
(Ph Eur monograph 1320) *****
PhEw ____________________________________________
DEFINITION
Whole or cut dried leaves of older branches of Euealyptus
globulus Labill.
Content
Minimum 20 mUkg of essential oil for the whole drug
(anhydrous drug) and minimum 15 mUkg of essential oil for
the cut drug (anhydrous drug) .
CHARACTERS
Aromatic odour of cineole.
IDENTIFICATION
A. The leaves which are mainly greyish-green and relatively
thick are elongated, elliptical and slightly sickle-shaped and
usually up to 25 cm in length, and up to 5 cm in width.
The petiole is twisted, strongly wrinkled and is 2-3 cm, rarely
5 cm, in length. The coriaceous, stiff leaves are entire and
glabrous and have a yellowish-gteen mid ribo Lateral veins
anastomose near the margin to a continuous line.
The margin is even and somewhat thickened. On both
surfaces are minute, irregularly distributed, warty dark brown
spots. Small oil glands may be seen in transmitted light.
B. Reduce to a powder (355) (2. 9.12). The powder is
greyish-gteen. Examine under a microscope, using ehloral
hydrate solution R . The powder shows the following diagnostic
characters: fragments of glabrous lamina with small thick-
walled epidermal cells bearing a thick cuticle, numerous
anomocytic stomata (2.8.3) of more than 80 J.Lm in diameter
and occasionally groups of brown cork cells, 300 J.lffi in
diameter and brownish-black in their centre; fragments of
iso bilateral mesophyll with 2-3 layers of palisade parenchyma
on each side and in the centre severallayers of spongy
mesophyll with elongated cells with the same orientation as
the palisade cells and containing prisms and cluster crystals
of calcium oxalatej fragments of mesophyll containing large
schizogenous oil glands.
C . Thin-Iayer chromatogtaphy (2.2.27).
Test solution Shake 0.5 g of the freshly powdered drug (355)
(2.9.12) with 5 mL of toluene R for 2-3 min and filter over
about 2 g of anhydrous sodium sulfate R.
R eference solution Dissolve 50 J.LL of cineole R in toluene R
and dilute to 5 mL with the same solvent.
Plate TLC siliea gel plate R .
Mobile phase ethyl acetate R, toluene R (10:90 V/V) .
Applieation 1O ~IL, as bands.
D evelopment Over a path of 15 cm.
D rying In airo
Detection Spray with anisaldehyde solution R. Examine in
daylight while heating at 100-105 oC for 5-10 mino
2014
A. Thick-walled epidermal cells and F and G. Epidermis covered by a thick
anomocytic sto mata, in surface view culicle (Fa and Ga), in transverse
section
B. Thick-walled epidermal cells H and l. Palisade parenchyrna (la)
and anornocytic sto mata, with with altached spongy mesophyll (lb)
atlached palisade parenchyma (Ba), containing prisms and cluster crystals
in surface view o( caJcium oxalate
c. Parenchyma cells with cluster K. Ce lis containing prisrns o( caJciurn
crystal of calcium oxalate oxalate
D. Vascular tissue L. Fibres
E. Schizogenous oil gland with
attached palisade parenchyma (Ea)
Figure 1320.-1. - Illustration of powdered herbal drug of
eucalyptus leaf (see Identification B)
Results The chromatogtam obtained with the reference
solution shows in the middle a zone due to cineole.
The main zone in the chromatogtam obtained with the test
solution is similar in position and colour to the zone due to
cineole in the chromatogtam obtained with the reference
solution, it also shows an intense violet zone (hydrocarbons)
near the solvent front and there may also be other fainter
zones.
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of dark and brown leaves, maximum
5 per cent of stems and maximum 2 per cent of other foreign
matter. Cordate or ovate sessile leaves of young branches,
with numerous glands on both sides, visible as points in
transmitted light, are not presentoDetermine by using 30 g
of the drug to be examÍned.
Water (2.2.13)
Maxirnum 100 mUkg, determined on 20.0 g of powdered
drug (355) (2.9.12).
Total ash (2.4.16)
Maxirnum 6.0 per cent.
ASSAY
Carry out the determination of essential oil in herbal drugs
(2.8.12). Use 10.0 g ofthe drug, cut immediately before
2014 Eucalyptus Oil IV-175

detennination, a 500 mL round-bottomed fiask, 200 mL of reference solution (a). Sabinene and camphor may be present
water R and 100 mL of g!ycerol R as the distillation liquid and in the chromatogram obtained with the test solution.
0.5 mL of xylene R in the graduated tube. Distil at arate of TESTS
2-3 mUmin for 2 h .
Relative density (2.2.5)
____________________________________________ PhE~

0.906 to 0.927.
Refractive index (2.2.6)
1.458 to 1.470.
*** Optical rotation (2.2.7)
Eucalyptus Oi!
*** **
*
00 to + 100.
(Ph Eur monograph 0390) *** Solubility in alcohol (2.8.10)
ffiE~ ____________________________________________ It is soluble in 5 volumes of ethanol (70 per cene V/V) R .
DEFINITION Aldehydes
Essential oil obtained by steam distillation and rectification To 10 mL in a ground-glass-stoppered tube 25 mm in
from the fresh leaves or the fresh tenninal branchlets of diameter and 150 mm long, add 5 mL of toluene R and 4 mL
various species of Euca!yptus rich in 1,8-cineole. The species of alcoholic hydroxylamine solurion R . Shake vigorously and
mainly used are Euca!yptus globulus Labill., Eucalyptus titrate irnmediately with 0.5 M potassium hydroxide in alcohol
polybracrea R.T.Baker and Euca!yptus smithii R.T.Baker. (60 per cent VN) until the red colour changes to yellow.
Continue the titration with shaking; the end-point is reached
CHARACTERS when the pure yellow colour of the indicator is permanent in
Appearance the lower layer after shaking vigorously for 2 min and
Colourless or pale yellow liquido allowing separation to take place. The reaction is complete in
Odour about 15 mino Repeat the titration using a further 10 mL of
Reminiscent of 1,8-cineole. the substance to be examined and, as a reference solution for
the end-point, the titrated liquid from the 1st detennination
IDENTIFICATION
to which has been added 0.5 mL of 0.5 M potassium
First ideneificarion B.
hydroxide in alcohol (60 per cent V/V). Not more than
Second identification A. 2.0 mL of 0.5 M potassium hydroxide in alcohol (60 per cent
A. Thin-layer chromatography (2.2.27). V/V) is required in the 2nd titration .
Test solution Dissolve 0.1 g of the oil to be examined in Chromatographic pro file
toluene R and dilute to 10 mL with the same solvent. Gas chromatography (2.2.28): use the nonnalisation
Reference solution Dissolve 20 ¡¡L of a-terpineol R and 50 ¡¡L procedure.
of cineole R in toluene R and dilute to 5 mL with the same Test solution Dissolve 200 ¡¡L of the oil to be examined in
solvento heptane R and dilute to 10.0 mL with the same solvent.
Plate TLC silica gel plate R (5-40 ¡¡m) [or TLC silica gel Reference solution (a) Dissolve 10 ¡¡L of a-pinene R, 5 ¡¡L of
plate R (2-10 ¡¡m)). {3-pinene R , 5 ¡¡L of sabinene R, 5 ¡¡L of a-phellandrene R ,
Mobile phase ethyl acetate R, toluene R (10:90 V/V). 10 ¡¡L of limonene R , 50 fLL of cineole R and 5 mg of
Application 10 fLL [or 2 ¡¡L) as bands of 10 mm [or 6 mm) . camphor R in heptane R and dilute to 10 mL with the same
solvento
Developmene Over a path of 15 cm [or 6 cm).
Reference solution (b) Dissolve 5 ¡¡L of limonene R in
Drying In airo
heptane R and dilute to 50.0 mL with the same solvento
Detection Spray with anisaldehyde solution R and heat at Dilute 0.5 mL of the solution to 5.0 mL with heptane R.
100-105 oC for 5-10 min; examine in daylight.
Column:
Results See below the sequence of zones present in the -- material: fused silica;
chromatograms obtained with the reference solution and the -- size: 1 = 60 m, 0 = about 0.25 mm;
test solution. Furthennore, other faint zones may be present -- stationary phase: macrogol 20 000 R (film thickness
in the chromatogram obtained with the test solution, near the 0.25 ¡¡m).
solvent front and at the level of a-terpineol.
Carrier gas helium for chromatography R.
Flow rate 1.5 mUmin.
Top of the plate
Split ratio 1:50.
- - Tempera rure:
1,8-Cineole: a violet-brown zone An intense violet-brown zone Time Temperature
(1,8-cineole)
(min) (oC)
- - Colurnn 0-5 60
a-Terpineol: a violet-brown zone 5 - 33 60 -t 200
Reference solution Test solution 33 - 38 200

Injection port 220

B. Examine the chromatograms obtained in the test for Detector 220
chromatographic profile.
Results The characteristic peaks due to a-pinene, p-pinene, Detection Flame ionisation.
a-phellandrene, limonene and 1,8-cineole in the Injecrion 1 ¡¡L.
chromatogram obtained with the test solution are similar in
retention time to those in the chromatogram obtained with
 IV-176 Fennel
E/ution order Order indicated in the composition of
reference solution (a). Record the retention times of these
substances.
System suitabüity Reference solution (a):
- resolution: minimum 1.5 between the peaks due to
limonene and cineole.
Identijieation of eomponents Using the retention times
determined from the chromatogram obtained with reference
solution (a), locate the components of reference solution (a)
in the chromatogram obtained with the test solution.
Determine the percentage content of each of these
components. The percentages are within the following
ranges:
- rx-pinene: 0.05 per cent to 10.0 per cent;
- f3-pinene: 0.05 per cent to 1.5 per cent;
- sabinene: maximum 0.3 per cent;
- rx-phellandrene: 0.05 per cent to 1.5 per cent;
- limonene: 0.05 per cent to 15.0 per cent;
- 1,8-cineole: minimum 70.0 per cent;
- eamphor: maximum 0.1 per cent;
- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0. 05 per cent).
STORAGE
At a temperature not exceeding 25 0e.
_ _ _ _ _ __ _ __ __ __ __ _ _ _ _ _ _ Ph Eur
Bitter Fennel
(Ph Eur monograph 0824)
~ E~ _ _ _ __ _ __ _ _ __ __ _ _ _ __ __ __
DEFINITION
Dry cremocarps and mericarps of Foenieulum vulgare MilI.
ssp . vulgare varo vulgare.
Content:
- essentialoil: minimum 40 mUkg (anhydrous drug);
- anethole: minimum 60.0 per cent in the essential oil;
- fenehone: minimum 15.0 per cent in the essential oi!.
CHARACTERS
Bitter fennel is greenish-brown, brown or green.
IDENTIFICATION
A. The fruit of bitter fennel is a cremocarp, of almost
cylindrical shape with a rounded base and a narrower summit
crowned with a large stylopod. It is generally 3-12 mm long
and 3-4 mm wide. The mericarps, usually free, are glabrous.
Each bears 5 prominent, slightly carenated ridges. When cut
transversely, 4 vittae on the dorsal surface and 2 on the
commissural surface may be seen with a lens.
B. Microscopic examination (2.8.23). The powder is greyish-
brown or greyish-yellow. Examine under a microscope using
ehloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 0824.-1 ): yellow fragments of
wide secretory canals, often made up of yellowish-brown-
walled polygonal secretory cells [D, H] ; reticulate
parenchyma of the mesocarp [B]; numerous fibre bundles
[G] from the ridges [Ga] , often accompanied by narrow
spiral vesseIs [Gb] ; very numerous endosperm fragments [F]
containing aleurone grains [Fb] and very small cluster
crystals of ca1cium oxalate [Fa]; sorne fibre bundles from the
carpophore [E]; fragments of the endocarp, in surface view
[A, K], consisting of thin-walled, transversely elongated cells,
2014
2-9 Ilm wide, having a parquetry arrangement, sometimes
accompanied by the inner layer of the mesocarp [Aa];
fragments of the epicarp with stomata accompanied by oil
droplets [C]; very numerous oil droplets UJ.
. . G
.~ :
Fb
J
deJo
o~
O
Figure 0824.-1. - Illustration (or identi(ication test B o(
powdered herbal drug o( bitter (ennel
e. Thin-Iayer chromatography (2.2.21) .
Test solutíon Shake 0.3 g of the freshly powdered herbal
drug (1400) (2. 9.12) with 5.0 mL of methylene ehloride R for
15 mino Filter and careful1y evaporate the filtrate to dryness
on a water-bath at 60 oC . Dissolve the residue in 0.5 mL of
IOluene R .
Reference solution Dissolve 50 IlL of anethole R and 10 IlL of
fenchone R in 5.0 mL of hexane R .
Plate TLC siliea gel GFZ54 plate R.
Mobile phase hexane R, IOluene R (20:80 V/V).
Applicatíon 10 IlL as bands of 20 mm by 3 mm.
Development Over a path of 10 cm.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms show in the central part a
quenching zone due to anethole.
Detection B Treat with sulfuric acid R and heat at 140 oC for
5-10 min until a yellow zone due to fenchone appears in the
lower third of the chromatograms.
Results B Anethole appears as a violet band in the central
part; the chromatogram obtained with the test solution also
shows a reddish-brown zone in its upper third (terpenes).
TESTS
Estragole
Gas chromatography (2.2.28) : use the norma lis atio n
procedure.
Test solution Dilute the mixture of essential oil and xylene R
obtained in the determination of essential oil to 5.0 mL with
xylene R, by rinsing the apparatus.
2014 Bitter-Fennel Fruit Oil IV-177

Reference solution Dissolve 5 mg of estragole R in 0.5 mL of

Bitter-Fennel Fruit Oi! ***
xylene R . *** ***
Column: (Ph Eur monograph 1826) ***
-- size: 1 = 30-60 m, 0 = 0.3 mm; ~Ew ____________________________________________
-- stationary phase: macrogol 20 000 R.
DEFlNITlON
Camá gas nitrogen for chromatography R.
Essential oil obtained by steam distilIation from the ripe fruits
Flow rate 0.40 mUmin. of Foeniculum vulgare MilIer, ssp . vulgare var. vulgare.
Split ratio 1 :200.
Content:
Temperature: -- fenchone: 12.0 per cent to 25.0 per cent,
Time Temperature -- trans-anethole: 55.0 per cent to 75.0 per cent.
(min) (oC)
CHARACTERS
Colurnn 0-4 60
Appearance
4 - 26 60 -t 170 Clear, colourless or pale yelIow liquido
26 - 41 170 Characteristic odour.
Injection port 220 IDENTIFlCATlON
Detector 270 First identification B.
Second identification A.
Detection Flame ionisation. A. Thin-layer chromatography (2.2.27).
Injection 1 ¡.tL. Test solution Dissolve 0.1 mL of the oil to be examined in
Limit: 5 mL of toluene R.
-- estragole: maximum 5.0 per cent in the essential oil Reference solution Dissolve 10 ¡.tL of fenchone R and 80 ¡.tL of
obtained in the assay. anethole R in 5 mL of toluene R.
Foreign matter (2.8.2) Plate TLC silica gel plate R.
Maximum 1.5 per cent of peduncIes and maximum Mobile phase ethyl acetate R, toluene R (5 :95 V/V) .
1.5 per cent of other foreign matter.
Application 10 ¡.tL as bands.
Water (2.2.13) Development Over a path of 15 cm.
Maximum 100 mUkg, determined on 20.0 g ofthe
Drying In airo
powdered herbal drug (710) (2.9.12).
Detection Spray with a freshly prepared 200 gIL solution of
Total ash (2.4.16)
phosphomolybdic acid R in ethanol (96 per cent) R and heat at
Maximum 10.0 per cent.
150 oC for 15 mini examine in daylight.
ASSAY Results See below the sequence of the zones present in the
Essential oil chromatograms obtained with the reference solution and the
Carry out the determination of essential oils in herbal drugs test solution. Furthermore, other zones may be present in the
(2.8.12). Use a 500 mL round-bottomed fIask and 200 mL chromatogram obtained with the test solution.
of water R as the distilIation liquid. Reduce the herbal drug
to a coarse powder (1400) (2.9.12) and immediately use
5.0 g for the determination. Introduce 0.50 mL of xylene R in Top oC the plate
the graduated tube. Distil at arate of 2-3 mUmin for 2 h.
Anethole: a dark blue to dark A dark blue to dark violet zone
Anethole and fenchone violet zone (anethole)
Gas chromatography (2.2.28) as described in the test for
estragole with the folIowing modifications . ----- ---

Reference solution Dissolve 5 mg of fenchone R and 5 mg of Fenchone: a blue or bluish-grey A blue or bluish-grey zone
anethole R in 0.5 mL of xylene R. zone (fenchone)
Elution order The order indicated in the composition of the
----- ---
reference solution; record the retention times of these
substances . ReCerence solution Test solution

STORAGE
Protected from moisture. B. Examine the chromatograms obtained in the test for
___________________________________________ ~Ew chromatographic pro file .
Results The characteristic peaks in the chromatogram
obtained with the test solution are similar in retention time to
those in the chromatogram obtained with the reference
solution.
TESTS
Relative density (2.2.5)
0.961 to 0.975.
Refractive index (2.2. 6)
1.528 to 1.539.
Optical rotation (2.2.7)
+ 10.0°to + 24.0°.
IV-178 Bitter-Fennel Fruit Oil 2014

3 6

5 7
.1 ,1 j I A I A

J' l ' l' 1 j , , r ( , ¡ ' 1 " 1 I , , I ' l ' , , r ., , l' \ I I , , I ' , , I ' , , r ' , , I ' I , I I (' '1'
o
1

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 38 42 min

1. a-pinene 3. fenchone 5. cis·anethole 7. anisaldehyde

2. limonene 4. estragole 6. trans-anethole

Figure 1826_-1. - Chromatogram for the test for chromatographic profile of bitter-fennel fruit oi!

Chromatographic pro file Temperature:

Gas chromatography (2.2.28): use the nonnalisation Time Temperature
procedure. (min) (oC)
Column 0-4 60
Test solution Dissolve 0.20 mL of the oil to be examined in
heptane R and dilute to 10.0 mL with the same solvento 4 - 26 60 -7 170
Rejerence solution Dissolve 20 IlL of a-pinene R, 20 IlL of 26 - 41 170
limonene R, 50 IlL ofjenchone R, 20 IlL of estragole R, 100 IlL
lnjection port 220
of anethole R and 20 IlL of anisaldehyde R in heptane R and
dilute to 10.0 mL with the same solvento Detector 270
Column:
- material: fused silica, Using the retention times detennined from the
- size: 1 = 60 m, 0 = 0.25 mm, chromatogram obtained with the reference solution, locate
- stationary phase: macrogol 20 000 R (film thickness the components of the reference solution on the
0.25 11m). chromatogram obtained with the test solution and locate
Carrier gas helium jor chromatography R. cis-anethole using Figure 1826.-l. (Disregard the peak due to
Flow rate 1 mUmin. heptane).
Split ratio 1:200. Determine the percentage content of each of these
components . The percentages are within the following
Detection Flame ionisation.
ranges:
lr¡jection l. O 1lL. - a-pinene: l.0 per cent to 10.0 per cent,
Elution order Order indicated in the composition of the - limonene: 0.9 per cent to 5.0 per cent,
reference solution. Record the retention times of these - jenchone: 12.0 per cent to 25.0 per cent,
substances. - estragole: maximum 6.0 per cent,
System suitability Reference solution: - cis-anethole: maximum 0.5 per cent,
- resolution: minimum 5.0 between the peaks due to - trans-anethole: 55.0 per cent to 75.0 per cent,
estragole and trans-anethole. - anisaldehyde: maximum 2.0 per cen!.
2014 Bitter-Fennel Herb Oil IV-179

The ratio of ex-pinene content to limonene content is greater Development Over a path of 8 cm [or 6 cm].
than 1.0. Drying In airo
STORAGE Detection Spray with a freshly prepared 200 gIL solution of
At a temperature not exceeding 25 oC. phosphomolybdic acid R in ethanol (96 per cent) R and heat at
____________________________________________ PhE~
150 oC for 15 min; examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Bitter-Fennel Herb Oil ***
*** ***
(Ph Bur monograph 2380) *** Top of the plate
~E~ ____________________________________________
Anethole: a dark blue or dark A dark blue or dark violet zone
violet zone (anethole)
DEFINITION
--- ---
Essential oil obtained by steam distillation of the aerial parts
of Foeniculum vulgare MilI. ssp. vulgare, varo vulgare coIlected Fenchone: a blue or bluish-grey A sometimes faint blue or
during fruiting. zone bluish-grey zone (fenchone)
--- ---
CHARACTERS
Reference solution Test solution
Appearance
Clear, pale or intense yeIlow liquido
Anise-like odour. B. Examine the chromatograms obtained in the test for
IDENTIFICATION chromatographic profile.
First identification B. Results:
Second identification A. -- Spanish type: the characteristic peaks due to ex-pinene,
p-pinene, p-myrcene, o:-pheIlandrene, limonene,
A. Thin-Iayer chromatography (2.2.27).
fenchone, estragole and trans-anethole in the
Test solution Dissolve 0.1 mL of the oil to be examined in chromatogram obtained with the test solution are similar
5 mL of toluene R. in retention time to those in the chromatogram obtained
Reference solution Dissolve 10 ¡.tL of fenchone R and 40 ¡.tL of with reference solution (a);
anethole R in 5 mL of toluene R . -- Tasmanian type: the characteristic peaks due to o:-pinene,
Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel o:-pheIlandrene, limonene, fenchone, estragole and trans-
plate R (2-10 ¡.tm)]. anethole in the chromatogram obtained with the test
Mobile phase ethyl acetate R, toluene R (5:95 V/V) . solution are similar in retention time to those in the
chromatogram obtained with reference solution (a).
Application 10 ¡.tL [or 3 ¡.tL] as bands of 10 mm [or 8 mm].

5 6
3

o 5 10
r :11. 1
15 20 25
í 8

30 35
10 11

40 min

1. a-pinene 4. a-phellandrene 7. estragole 10. anisaldehyde

2. ~-pinene 5. limonene 8. cis-anethole ll. anise ketone

3. ~myrcene 6. fenchone 9. trans-anethole

Figure 2380.-1. - Chromatogram for the test for chromatographic profile of Spanish-type
bitter-fennel herb oil
IV-ISO Bitter-Fennel Herb Oil 2014

T ESTS Cam'er gas helium for chromatography R.

Relative density (2.2_5) : Flow rate 1 mUmin.
- Spanish type: 0_877 to 0.921; Split ratio 1:50.
- Tasmanian type: 0.940 to 0.973.
Temperature:
Refractive index (2.2.6):
- Spanish type: 1.487 to 1.501; Time Temperature
(min) (oC)
- Tasmanian type: 1.512 to 1.538.
Column 0-35 70 ---7 210
Optical rotation (2.2.7):
- Spanish type: + 42° to + 68°; 35 - 42 210
- Tasmanian type + 11 ° to + 35 °. ¡njeelion porl 250
Solubility in alcohol (2.8.10) : Detector 270
- Spanish type: 1 volume is soluble in 2 volumes and more
of ethanol (90 per cent V/V! R; Detection Flame ionisation.
- Tasmanian type: 1 volume is soluble in 10 volumes and 1njection 1 ¡..tL.
more of ethanol (85 per cent V/V! R. Elution order Order indicated in the composition of the
Chromatographic pro file reference solution; record the retention times of these
Gas chromatography (2.2.28): use the normalisation substances _
procedure. System suitability Reference solution (a):
Test solution Dissolve 0.20 mL of the oil to be examined in - resolution: minimum 1.5 between the peaks due to
acetone R and dilute to 10.0 mL with the same solvent. ~-myrcene and cx-phellandrene_

Reference solution (a) Dissolve 20 ¡..tL of IX-pinene R, 10 ¡..tL of Using the chromatogram obtained with the reference
f3-pinene R, 20 ¡..tL of f3-myrcene R, 20 ¡..tL of IX-phellandrene R, solution, locate the relevant components for the type of the
20 ¡..tL of limonene R, 40 ¡..tL of fenchone R, 10 ¡..tL of essential oil to be examined in the chromatogram obtained
estragole R, 40 ¡..tL of anethole R, 10 ¡..tL of anisaldehyde R and with the test solution, and locate cis-anethole using Figures
10 ¡..tL of anise ketone R in acetone R and dilute to 10.0 mL 2380.-1 and 2380.-2.
with the same solvent. Determine the percentage content of each of these
Reference solution (b) Dissolve 5 ¡..tL of anethole R in components.
25 _0 mL of acetone R. Dilute 0.5 mL of this solution to For Spanish-type bitter-fennel herb oil, the percentages are
20 _0 mL with acetone R. within the following ranges:
Column: - IX-pinene: 2 .0 to 8.0 per cent;
- material: fused silica; - f3-pinene: 1.0 to 4.0 per cent;
- siz e: 1 = 60 m, 0 = 0.25 mm; - f3-myrcene: 1.0 to 12.0 per cent;
- stationary phase: macrogol 20 000 R (film thickness - IX-phellandrene: 1.0 to 25.0 per cent;
0_25 ¡..tm). - limonene: 8.0 to 30.0 per cent;
- fenchone: 7. O to 16.O per cent;

23

1I L l.
¡ 6 8 9

O 5 10 15 20 25 30 35 40 min
1. a-pinene 4_ fenehone 7. trans-anethole

2_ a-phellandrene 5. estragole 8. anisaldehyde

3. limonene 6. cis-anethole 9_ anise ketone

Figure 2380.-2. - Chromatogram for the test for chromatographic profile of Tasmanian-type
bitter-fennel herb oil
 2014
- estragole: 2.0 to 7.0 per cent;
- cis-anethole: maximum 0.5 per cent;
- trans-anethole: 15.0 to 40.0 per cent;
- anisaldehyde: maximum 1.0 per cent;
- anise ketone: maximum 0.05 per cent;
- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.025 per cent) .
For Tasmanian-type bitter-fennel herb oil, the percentages
are within the following ranges:
- rx-pinene: 2.0 to 11.0 per cent;
- rx-phellandrene: 1.0 to 8.5 per cent;
- limonene: 1.0 to 6.0 per cent;
- fenchone: 10.0 to 25.0 per cent;
- estragole: 1.5 to 6.0 per cent;
- cis-anethole: maximum 0.5 per cent;
- trans-anethole: 45.0 to 78.0 per cent;
- anisaldehyde: maximum l.0 per cent;
- anise ketone: maximum 0.05 per cent;
- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.025 per cent).
STORAGE
At a temperature not exceeding 25 oC.
LABELLING
The label sta tes that the content is Spanish-type or
Tasmanian-type.
_ _ _ __ _ _ __ _ _ _ _ _ _ _ __ _ _ _ _ PhE~
Sweet Fennel *****
** **
(Ph Bur monograph 0825) ***
~E~ _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ ___
DEFINITION
Dry cremocarps and mericarps of Foeniculum vulgare Mili.
subsp. vulgare varo dulce (Mili.) Batt. & Trab.
Content:
- essential oil: minimum 20 mUkg (anhydrous drug);
- anethole: minimum 80.0 per cent in the essential oil.
CHARACTERS
Sweet fennel is pale green or pale yellowish-brown.
IDENTIFICATION
A. The fruit of sweet fenne! is a cremocarp of almost
cylindrical shape with a rounded base and a narrowed
summit crowned with a large stylopod. It is generally
3-12 mm long and 3-4 mm wide. The mericarps, usually
free, are glabrous . Each bears 5 prominent, slightly carenated
ridges. When cut transversely, 4 vittae on the dorsal surface
and 2 on the commissural surface may be seen with a lens.
B. Microscopic examination (2.8.23). The powder is greyish-
brown or greyish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 0825.-l.) : yellow fragments of
wide secretory canals, often made up of yellowish-brown-
walled polygonal secretory cells [D, H]; reticulate
parenchyma of the mesocarp [B]; numerous fibre bundles
[G] from the ridges [GaJ, often accompanied by narrow
spiral vessels [Gb]; very numerous endosperm fragments [F]
containing aleurone grains [Fb] and very small calcium
oxalate cluster crystals [Fa]; sorne fibre bundles from the
carpophore [E]; fragments of the endocarp, in surface view
Sweet Fenne! IV-1Sl
[K, A], consisting of thin-walled, transversely elongated cells
2-9 ¡¡m wide, having a parquetry arrangement, sometimes
accompanied by the inner layer of the mesocarp [Aa];
fragments of the epicarp with stomata accompanied by oil
droplets [C]; very numerous oil droplets m.
..
.' :o
o
Fb
J
o°00
o
°,0
O
K
Figure 0825.-1. - Illustration for identification test B of
powdered herbal drug of sweet fennel
e. Thin-Iayer chromatography (2.2.27).
Test solution Shake 0.3 g of the freshly powdered herbal
drug (1400) (2.9.12) with 5.0 mL of methylene chloride R for
15 mino Filter and carefully evaporate the filtrate to dryness
on a water-bath at 60 0e. Dissolve the residue in 0.5 mL of
toluene R.
Reference solution Dissolve 60 ¡¡L of anethole R in 5.0 mL of
hexane R.
Plate TLC silica gel GF254 plate R.
Mobile phase hexane R, toluene R (20:80 VIV).
Application 10 ¡¡L as bands of 20 mm by 3 mm.
Development Over a path of 10 cm.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms show in the central pan a
quenching zone due to anethole.
Detection B Spray with sulfuric acid R and heat at 140 oC for
5 mini examine in daylight.
Results B The chromatograms show in the central part a
violet band due to anethole; the chromatogram obtained with
the test solution also shows a reddish-brown zone in the
upper third (terpenes).
TESTS
Estragole and fenchone
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Dilute the mixture of essential oil and xylene R
obtained in the assay of essential oil to 5.0 mL with xylene R,
by rinsing the apparatus.
 IV-182 Fenugreek 2014

Reference solution Dissolve 5 mg of estragole R and 5 mg of long, 2-3 mm wide and 1.5-2 mm thick. The widest surfaces
fenchone R in 0.5 mL of xylene R. are marked by a groove that divides the seed into 2 unequal
Colunm: parts . The smaller part contains the radide; the larger part
- size: 1 = 30-60 m, 0 = 0.3 mm; contains the cotyledons.
- stationary phase: macrogol 20 000 R. B. Reduce ro a powder (355) (2. 9.12). The powder is
Cam'er gas nitrogen for chromatography R. yellowish-brown. Examine under a microscope using chloral
Flow rate 0.40 mUmin. hydrate solution R. The powder shows the following diagnostic
characters: fragments of the testa in sectional view with thick
Split ratio 1:200.
cutide covering lageniform epidermal cells, with an
underlying hypodermis of large cells, narrower at the upper
Time Temperature end and constricted in the middle, with bar-like thickenings
(min) (OC)
of the radial walls; yellowish-brown fragments of the
Colurnn 0·4 60 epidermis in surface view, composed of small, polygonal cells
4·26 60 -> 170 with thickened and pitted walls, frequently associated with
the hypodermal cells, circular in outline with thickened and
26·41 170
dosely beaded walls; fragments of the hypodermis viewed
Injection port 220 from below, composed of polygonal cells whose bar-like
Detector 270 thickenings extend to the upper and lower walls; parenchyma
of the testa with elongated, rectangular cells with slightly
thickened and beaded walls; fragments of endosperm with
Detection Flame ionisation. irregularly thickened, sometimes elongated cells, containing
1njection 1 ¡.¡L. mucilage.
Limits: C. Thin-layer chromatography (2.2.27).
- estragole: maximum 10.0 per cent in the essential oil;
Test solution Place l.0 g of the powdered drug (710)
- fenchone: maximum 7.5 per cent in the essential oi!.
(2.9.12) in a 25 mL conical flask and add 5.0 mL of
Foreign matter (2.8.2) methanol R. Heat in a water-bath at 65 cC for 5 mino Cool
Maximum 1.5 per cent of pedundes and maximum and filter.
1.5 per cent of other foreign matter. Reference solution Dissolve 3.0 mg of tngonelline
Water (2.2.13) hydrochloride R in 1.0 mL of methanol R.
Maximum 80 mUkg, determined on 20.0 g of the powdered Plate TLC silica gel F254 plate R .
herbal drug (710) (2.9.12) .
Mobile phase water R, methanol R (30:70 VIV) .
Total ash (2.4.16) Application 20 ¡.¡L of the test solution and 10 ¡.¡L of the
Maximum 10.0 per cent. reference solution, as bands.
ASSAY Developmem Over a path of 10 cm.
Essential oil (2.8.12) Drying In airo
Use 10.0 g ofthe herbal drug reduced ro a coarse powder
Detection A Examine in ultraviolet light at 254 nm.
(1400) (2. 9.12) immediately before the assay, a 500 mL
round-bottomed flask, 200 mL of water R as the distillation Results A The chromatogram obtained with the test solution
liquid, and 0.50 mL of xylene R in the graduated tube. Distil shows in its lower half a quenching zone similar in position
at arate of 2-3 mUmin for 2 h . and fluorescence to the zone in the chromatogram obtained
with the reference solution.
Anethole
Gas chromatography (2.2.28) as described in the test for Detection B Spray with potassium iodobismuthate solution R2.
estragole and fenchone with the following modification. Results B The chromatogram obtained with the test solution
shows an intense orange-red zone similar in position and
Reference solution Dissolve 5 mg of anethole R in 0.5 mL of
xylene R . colour to the zone in the chromatogram obtained with the
reference solution. It also shows in its upper half, a broad
STORAGE light brownish-yellow zone (triglycerides).
Protected from moisture.
TEST S
----------------------______________________ ~f~
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g ofthe
powdered drug by drying in an oven at 105 oC for 2 h .
Total ash (2.4.16)
Fenugreek Maximum 5.0 per cent.
Swelling index (2.8.4)
(Ph Eur monograph 1323)
Minimum 6, determined on the powdered drug (710)
Ph fUf __________________________________________
(2.9. 12) .
DEFINITION __________________________________________ Ph E~

Dried, ripe seeds of Trigonella foenum-graecum L.

CHARACTERS
Strong characteristic aroma tic odour.
IDENTIFICATION
A. The seed is hard, flattened, brown or reddish-brown and
more or less rhomboidal with rounded edges. It is 3-5 mm
2014
*****
Feverfew
** **
(Ph Bur monograph 1516) ***
~E~ ____________________________________________
DEFINITION
Dried, whole or fragrnented aerial parts of Tanacetum
parthenium (L) Schultz Bip.
Content
Minimum 0.20 per cent ofparthenolide (C1sH 2003; M r
248.3) (dried drug).
CHARACTERS
Camphoraceous odour.
IDENTIFICATION
A. The leafy, more or less branched stem has a diameter of
up to 5 mm; it is almost quadrangular, channelled
longitudinally and slightly pubescent. The lea ves are ova te,
2-5 cm long, sometimes up to 10 cm, yellowish-green,
petiolate and alternate. They are pinnate or bipinnate, deeply
divided into 5-9 segments, each with a coarsely crenate
margin and an obtuse apex. Both surfaces are somewhat
pubescent and the midrib is prominent on the lower surface.
When present, the ftowering heads are 12-22 mm in
diameter with long pedicels; they are elustered into broad
corymbs consisting of 5-30 ftower-heads . The hemispherical
involucre is 6-8 mm wide and consists of many overlapping
bracts, which are rather narrow, obtuse and scarious and
have membranous margins. The central ftowers are yellow,
hermaphrodite, tube-shaped with 5 teeth and have 5 stamens
inserted in the corolla; the filaments of the stamens are
separate from each other but the anthers are fused into a
tube through which passes the style, bearing 2 stigrnatic
branches. The peripheral ftowers are female and have a
white, three-toothed ligule, 2-7 mm long. The fruit is an
achene, 1.2-1.5 mm long, brown when ripe, with 5-10 white
longitudinal ribs. It is glandular and bears a short, crenate,
membranous crown.
B. Reduce to a powder (355) (2.9.12). The powder is
yellowish-green. Examine under a microscope using chloral
hydrate solution R . The powder shows the following diagnostic
characters: numerous large, mulúcellular, uniseriate covering
trichomes consisting of a rhomboidal basal cell, 3-5 smaller,
thick-walled rectangular cells and a very long, fiat, slender
terminal cell, often curved at a right angle to the axis of the
basal cell; glandular trichomes with a short, biseriate, 2-4
celled stalk and a biseriate head of 4 cells around which the
cuúele forms a bladder-like covering; epiderrnal cells with
very sinuous, antielinal walls, a striated cutiele and
anomocyúc stomata (2.8.3); numerous spirally and annularly
thickened ves seis; stratified parenchyma and collenchyma.
Fragrnents of disc ftorets containing pale yellow amorphous
masses and small rosene crystals of calcium oxalate may be
present; spherical pollen grains about 25 Ilm in diameter,
with 3 pores and a spiny exine may be presento
C. Thin-Iayer chromatography (2.2.27).
Test solution To 1 g ofthe powdered drug (355) (2. 9.12)
add 20 mL of methanol R. Heat in a water-bath at 60 oC for
15 mino Allow to cool and filter. Evaporate to dryness under
reduced pressure and dissolve the residue in 2 mL of
methanol R .
R eference solution Dissolve 5 mg of parthenolide R in
methanol R and dilute to 5 mL with the same solvent.
Plate TLC silica gel plate R.
Mobile phase acetone R, toluene R (15 :8 5 V/V).
Application 20 ~LL, as bands.
Development Over a path of 10 cm.
Feverfew IV-183
Drying In air.
Detection Spray with a 5 gIL soluúon of vanillin R in a
mixture of 20 volumes of anhydrous ethanol R and
80 volumes of sulfuric acid R. Examine in daylight after
5 min o
Results The chromatogram obtained with the test solution
shows in its central part a blue principal zone that is similar
in position, colour and size to the principal zone in the
chromatogram obtained with the reference solution, and
somewhat below the principal zone a 2nd blue zone may be
present; 1 or 2 blue zones are also present in its lower third;
other violet zones may be presento
TESTS
Foreign matter (2.8.2)
Maximum 10.0 per cent ofstem with a diameter greater than
5 mm and maximum 2.0 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 10.0 per cent, deterrnined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 12.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Completely reduce about 50 g of the drug to
be examined to a powder (355) (2.9.12). After
homogenisaúon, introduce 1.00 g of the powdered drug into
a ftask and add 40 mL of methanol R. Heat in a water-bath at
60 oC for 10 mino Allow to cool and filter. Rinse the filter
with 15 mL of methanol R. Take up the residue with 40 mL
of methanol R. Repeat the operaúon. Collect the filtra tes and
rinsings and evaporate to dryness under reduced pressure.
Take up the residue with methanol R and dilute to 20.0 mL
with the same solvent. Dilute 10.0 mL of this solution to
50.0 mL with the mobile phase. Filter (0.45 Ilm) .
Reference solution Dissolve 5.0 mg of parthenolide R in
methanol R and dilute to 10.0 mL with the same solvent.
Dilute 2.0 mL of this solution to 50.0 mL with the mobile
phase.
Column:
-- size: ! = 0.25 m, 0 = 4.6 mm;
-- stationary phase: octadecylsily! silica gel for chromatography R
(5 Ilm).
Mobile phase acetonitrile R, water R (40:60 V/V).
Flow rate 1 mUmin.
Detection Spectrophotometer at 220 nm.
Injection 20 ¡.tL.
Retention time Parthenolide = about 11.5 mino
Calculate the percentage content of parthenolide using the
following expression:
area of the peak due to parthenolide in the
chromatogram obtained with the test solution;
area of the peak due to parthenolide in the
chromatogram obtained with the reference solution;
m1 mass of the drug to be examined in the test solution,
in grams;
mass of parthenolide in the reference solution, in
grams.
____________________________________________ Ph Eur
 IV-184 Fig 2014

with numerous greyish, transversely elongated lenticels; when

Fig the outer layers are removed, a dark red layer is exposed.
DEFINITlON The orange-brown or reddish-brown inner surface is smooth
The sun-dried succulent fruit of Ficus canea L. and bears fine longitudinal striations; it becomes red when
CHARACTERISTlCS treated with alkali. The fracture is short, fibrous in the inner
part.
Odour, pleasantly fruity; taste, sweet.
B. Microscopic examination (2.8.23). The powder is
Macroscopical Fruit compound: soft, fleshy, brown or
yellowish or reddish-brown. Examine under a microscope
yellowish brown, sometimes covered with a saccharine
using chloral hydrate solution R. The powder shows the
effiorescence; at the summit a small opening surrounded by
following diagnostic characters (Figure 0025 .-1 ): numerous
scales and at the base a short, stalk-like prolongation; fruit up
phloem fibres, in tangential section [D] or in longitudinal
to about 5 cm in length and breadth, consisting of a hollow
section [K], partiaIly lignified, in groups [Da, Ka] with
receptacle bearing on the inner surface numerous drupelets,
crystal sheaths containing calcium oxalate prisms [Db, Kb],
each containing a S10ne about 1.5 10 2.0 mm long; seed
sometimes including medullary rays [Dc]; reddish-brown
containing endosperm and a curved embryo.
fragments of cork [H]; fragments of phloem parenchyma, in
Microscopical Receptacle: epidermal cells polyhedral, longitudinal section [G] containing calcium oxalate cluster
sto mata raised, trichomes unicellular, thick walled, of varying crystals [A, E] or in tangential section [e) including
length up to about 300 ~m; hypodermis composed of medullary rays [Ca] and cells containing calcium oxalate
rounded polyhedral cells, sorne containing small rosette cluster crystals [Cb]; a few ftagments of collenchyma [F] ;
crystals of calcium oxalate; parenchyma made up of large,
irregular cells, forming the greater part of the receptacle,
isolated calcium oxalate cluster crystals [B] and prisms m.
containing large rosette crystals of calcium oxalate and
interspersed with numerous latex tubes, about 30 10 50 ~m
A
wide, and slender vascular bundles. Pericarp: epicarp
consisting of radially elongated cells with mucilaginous outer
walls; mesocarp of delicate, often disorganised cells; endocarp
of radially elongated sclereids with pitted walls . Endosperm
and embryo: small cells containing aleurone grains and ftxed
oil; starch absent.
Water-soluble extractive
Not less than 60.0% when determined by the following
method. To 25 g, minced, add 500 mL of water, boil under
a reflux condenser for 1 hour, cool and filter. To 20 mL of
the filtrate add 20 g of washed and ignited sand, evaporate 10
dryness in a tared, flat bottomed shallow dish and dry the
0
residue 10 constant weight at 100 Calculate the water-
•

soluble extractive by subtracting the weight of sand from the

weight of the residue obtained.
STORAGE
Figs should be s10red in a dry place.

Frangula Bark ***

*** ***
(Ph Bur monograph 0025) ***
Preparation
Standardised Frangula Bark Dry Extract
When Powdered Frangula Bark is prescribed or demanded, Figure 0025.-1. - Illustration for identification test B of
material complying with the requirements below, with the powdered herbal drug of frangula bark
exception of Identification test A and the test for Foreign
matter, shaIl be dispensed or supplied.
C. Examine the chromatogram obtained in test A for other
Ph Eur _ _ _ _ _ _ __ _ _ _ __ _ _ _ _ _ _ _ __
species of Rhamnus; anthrones in ultraviolet light at 365 nm.
DEFINITlON Results The chromatogram obtained with the test solution
Dried, whole or fragmented bark of the stems and branches shows 2 orange-brown zones (glucofrangulins) in the lower
of Rhamnus frangula L. (Frangula alnus MiIler) . third and 2-4 red zones (ftangulins, not always clearly
Content separated, and aboye them frangula-emodin) in the upper
Minimum 7.0 per cent of glucoftangulins, expressed as third.
glucofrangulin A (C27H 300 14; M r 578.5) (dried drug). D . To about 50 mg of the powdered herbal drug (180)
(2.9. 12) add 25 mL of dilute hydrochloric acid R and heat the
IDENTIFICATlON
mixture on a water-bath for 15 mino Allow to cool, shake
A. The bark occurs in curved, almost flat or rolled fragments
with 20 mL of ether R and discard the aqueous layer. Shake
or in single or double quilled pieces usually 0.5-2 mm thick
the ether layer with 10 mL of dilute ammonia RI .
and variable in length and width. The greyish-brown or dark
The aqueous layer becomes reddish-violet.
brown outer surface is wrinkled 10ngitudinaIly and covered
2014
TESTS
Other species of Rhamnus; anthrones
Thin-1ayer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herba1 drug (180)
(2.9.12) add 5 mL of ethanol (70 per cent V/V) R and heat to
boiling. Coo1 and centrifuge. Decant the supernatant solution
immediate1y and use within 30 mino
Rejerence solution Disso1ve 20 mg of barbaloin R in ethanol
(70 per cent V/V) R and di1ute to 10 mL with the same
solvent.
Plates TLC silica gel plate R (2 plates).
Mobile phase water R , methanol R, ethyl acetate R
(13 : 17: 100 VIV/V).
A. Application: 10 J.lL as bands.
Development Over a path of 10 cm.
Drying In air for 5 mino
Detection Spray with a 50 giL solution of potassium
hydroxide R in ethanol (50 per cent V/V) R, and heat at
100-105 oC for 15 mini examine in ultraviolet 1ight at
365 nm.
Results The chromatogram obtained with the reference
solution shows a brownish-yellow zone due to barbaloin in
the central part; the chromatogram obtained with the test
solution shows no zones of intense yellow ftuorescence and
no zone of orange or reddish ftuorescence similar in position
to the zone due to barbaloin in the chromatogram obtained
with the reference solution.
B. Application: 10 J.lL of the test solution as a bando
Development Over a path of 10 cm.
Drying In air for maximum 5 mino
Detection Spray immediately with a 5 gIL solution of
nitrotetrazolium blue R in methanol R; examine immediately.
R esults No violet or greyish-b1ue zones appear.
Foreign matter (2.8.2)
Maximum 1 per cent.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 6.0 per cent.
ASSAY
Carry out the assay protected jrom bright light.
In a tared, round-bottomed ftask with a ground-glass neck,
weigh 0.250 g of the powdered herbal drug (180) (2.9. 12).
Add 25.0 mL of a 70 per cent V/V solution of methanol R;
mix and weigh. Heat in a water-bath under a reftux
condenser for 15 mino Allow to cool, weigh and adjust to the
original mass with a 70 per cent VIV solution of methanol R.
Filter and transfer 5.0 mL of the filtrate to a separating
funnel. Add 50 mL of water R and 0.1 mL of hydrochloric
acid R. Shake with 5 quantities, each of 20 mL, of light
petroleum R. Allow the layers to separate and transfer the
aqueous layer to a 100 mL volumetric ftask . Combine the
light petroleum layers and wash with 2 quantities, each of
15 mL, of Waler R . Use this water for washing the separating
funnel and add it to the aqueous solution in the volumetric
ftask. Add 5 mL of a 50 giL solution of sodium carbonate R
and dilute to 100.0 mL with water R . Discard the light
petroleum layer. Transfer 40.0 mL of the aqueous solution to
a 200 mL round-bottomed ftask with a ground-glass neck.
Add 20 mL of a 200 gIL solution ofjerric chloride R and heat
Frangula Preparations IV-185
under a reftux condenser for 20 min in a water-bath with the
water level aboye that of the liquid in the ftask. Add 2 mL of
hydrochloric acid R and continue heating for 20 min, shaking
frequently, unti1 the precipitate is disso1ved. Allow to cool,
transfer the mixture to a separating funnel and shake with
3 quantities, each of 25 mL, of ether R , previously used to
rinse the ftask. Combine the ether extracts and wash with
2 quantities, each of 15 mL, of water R. Transfer the ether
1ayer to a volumetric ftask and dilute to 100.0 mL with
ether R. Evaporate 20.0 mL carefully to dryness and dissolve
the residue in 10.0 mL of a 5 giL solution of magnesium
acetate R in methanol R . Measure the absorbance (2.2.25) at
515 nm using methanol R as the compensation liquid.
Calculate the percentage content of glucofrangulins,
expressed as glucofrangulin A, using the following expression:
A X 3.06
m
i.e. taking the specific absorbance of glucofrangulin A to be
204.
A absorbance at 515 nm;
m = mass of the substance to be examined, in grams .
____________________________________________ PhE~
Standardised Frangula Bark Dry
Extract
(Ph Eur monograph 1214)
~E~ ____________________________________________
DEFINITION
Standardised dry extract obtained from Frangula bark (0025).
Content
15.0 per cent to 30.0 per cent of glucofrangulins, expressed
as glucofrangulin A (Cz7H30014; M r 578.5) (dried extract);
the measured content do es not deviate from that stated on
the label by more than ± 10 per cent.
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-90 per cent V/V).
CHARACTERS
Appearance
Yellowish-brown, fine powder.
IDENTIFICATION
A. Thin-Iayer chromatography (2.2.27).
Test solution To 0.05 g of the extract to be examined add
5 mL of ethanol (70 per cent VIV) R and heat to boiling. Cool
and centrifuge. Decant the supernatant solution immediately
and use within 30 mino
R ejerence solution Dissolve 20 mg of barbaloin R in ethanol
(70 per cent V/V) R and dilute to 10 mL with the same
solvent.
Plate TLC silica gel plate R.
Mobile phase water R, methanol R , ethyl acetate R
(13:17:100 VIV/V).
Application1O ~IL as bands.
Development Over a path of 10 cm.
Drying In air for 5 mino
 IV-186 Indian Frankincense
Detection Spray with a 50 gIL solution of potassium
hydroxide R in ethanol (50 per cent V/V) R and heat at
100-105 oC for 15 min; examine irnmediately after heaúng.
R esults The chromatogram obtained with the reference
soluúon shows in the middle third a reddish-brown zone due
to barbaloin. The chromatogram obtained with the test
soluúon shows 2 orange-brown zones (glucofrangulins) in the
lower third and 2-4 red zones (frangulins, not always clearly
separated, and aboye them frangula-emodin) in the upper
third.
B. To about 25 mg add 25 mL of dilute hydrochloric acid R
and heat the mixture on a water-bath for 15 mino Allow to
cool, shake with 20 mL of ether R and discard the aqueous
layer. Shake the ether layer with 10 mL of dilute ammonia R1 .
The aqueous layer becomes reddish-violet.
TESTS
Loss on drying (2.8.17)
Maximum 5.0 per cent.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6. 12) .
TYMC: acceptance criterion 10 2 CFU/g (2.6.12) .
Absence of Bscherichia coti (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Carry out the assay protected from bright tight.
Into a tared round-bonomed flask with a ground-glass neck,
weigh 0.100 g. Add 25.0 mL of a 70 per cent VIV solution
of methanol R, mix and weigh again. Heat the flask in a
water-bath under a refiux condenser at 70 oC for 15 mino
Allow to cool, weigh and adjust to the original mas s with a
70 per cent V/V solution of methanol R. Filter and transfer
5.0 mL of the filtrate to a separaúng funnel. Add 50 mL of
water R and 0.1 mL of hydrochloric acid R. Shake with
5 quantiúes, each of 20 mL, of light petroleum R1. Allow the
layers to separate and transfer the aqueous layer to a 100 mL
volumetric flask. Combine the light petroleum layers and
wash with 2 quantities, each of 15 rnL, of water R . Use this
water for washing the separating funne! and add it to the
aqueous solution in the volumetric flask. Add 5 mL of a
50 gIL solution of sodium carbonate R and dilute to 100.0 mL
with water R. Discard the Iight petroleum layer. Transfer
40.0 mL of the aqueous solution to a 200 mL round-
bottomed fiask with a ground-glass neck. Add 20 mL of a
200 gIL solution of jerric chloride R and heat under a refiux
condenser for 20 min in a water-bath with the water leve!
aboye that of the Iiquid in the fiask. Add 2 mL of hydrochloric
acid R and continue heating for 20 min, shaking frequently,
until the precipitate is dissolved. Allow to cool, transfer the
mixture to a separating funnel and shake with 3 quantities,
each of 25 mL, of ether R, previously used to rinse the flask.
Combine the ether extracts and wash with 2 quantities, each
of 15 mL, of water R. Transfer the ether layer to a volumetric
flask and dilute to 100.0 mL with ether R . Evaporate
20.0 mL carefully to dryness and dissolve the residue in
10.0 mL of a 5 gIL solution of magnesium acetate R in
methanol R. Measure the absorbance (2.2.25) at 515 nm
using methanol R as the compensation Iiquid.
Calculate the percentage content of glucofrangulins,
expressed as glucofrangulin A, using the following expression:
A x 3.06
m
2014
i.e. taking the specific absorbance of glucofrangulin A to be
204, calculated on the basis of the specific absorbance of
barbaloin.
A = absorbance at 515 nm;
m = mass of the preparaúon to be examined, in grams.
LABELLING
The label sta tes the content of glucofrangulins.
____________________________________________ ~E~
Indian Frankincense *****
** **
(Ph Bur monograph 2310) ***
~E~ ____________________________________________
DEFINITION
Air-dried gum-resin exuda te, obtained by incision in the stem
or branches of Boswellia serrata Roxb. ex Colebr.
Content:
-- ll-keto-f3-bosweltie aeid (C30H4604; M r 470.7): minimum
l.0 per cent (dried drug);
-- aeetyl-ll-keto- f3-boswellic acid (C 32 H 4SOS; M r 512.7):
minimum l.0 per cent (dried drug) .
IDENTIFICATION
A. Indian frankincense consists of translucent, roundish or
irregularly shaped, variable size pie ces of up to 3 cm. They
are yellowish or reddish-brown. Their surface is covered with
grey dust. The fracture is dull or slightly glossy.
B. Thin-Iayer chromatography (2.2.27).
Test solurion To 1.0 g of the powdered drug (355) (2.9.12)
add 90 mL of methanol R and sonicate for 10 mino Shake the
mixture vigorously 3 or 4 times during this procedure. Dilute
to 100 mL with methanol R. Centrifuge and use the clear
supernatant solution.
Rejerenee solution Dissolve 2 mg of 11-keto-f3-boswellie acid R
and 2 mg of aeetyl-11-keto-f3-boswellie acid R in 20 mL of
methanol R.
Plate TLC siliea gel F254 plate R (5-40 ¡lm) [or TLC siliea gel
F254 plate R (2-10 ¡lm)].
Mobile phase anhydrous jormie acid R, heptane R, ethyl
aeerate R, toluene R (3: 10:20:80 VIVIV/V).
Applieation 10 ¡lL [or 3 ¡lL] as bands.
Development Over a path of 8 cm [or 6 cm] .
Drying In air.
Deteetion Examine in ultraviolet Iight at 254 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. The zones due to 11-keto-~-boswellic acid and
acetyl-11-keto-~-boswel1ic acid in the test solution are of
approximately equivalent intensity. Furthermore, other weak
quenching zones may be present in the chromatogram
obtained with the test solution.
2014 Fumitory IV-187

Top of the plate area of the peak due to 11-keto-~-bo swellic acid in
the chromatogram obtained with the reference
--- ---
solution;
--- --- m mass of the substance to be examined, in grams;
Acetyl-ll-keto-~-boswellic acid: a A quenching zone mI mas s of 11-kelO-f3-boswellic acid R in the reference
quenching zone (acetyl-ll-keto-~-boswellic acid) solution, in grams;
ll-Keto-¡3-boswellic acid: a A quenching zone PI percentage content of 11 -keto-f3-boswellic acid in
quenching zone (ll·keto·S-boswellic acid) 11-keto-f3-boswellic acid R .
Reference solution Test solution
Calculate the percentage content of acetyl-11-keto-~­
boswellic acid using the following expression:
TESTS
Loss on drying (2.2.32)
Maximum 8.0 per cent, determined on 1.000 g of powdered
drug (355) (2.9.12) by drying in an oven at 105 oC for 3 h.
area of the peak due to acetyl-11-keto-~-boswellic
Total ash (2.4.16) acid in the chromatogram obtained with the test
Maximum 10.0 per cent. solution;
ASSAY area of the peak due to acetyl-ll-keto-~-boswellic
Liquid chromatography (2.2.29). acid in the chromatogram obtained with the
reference solution;
Test solution To 1.0 g of the powdered drug (35 5) (2.9. 12)
mass of the substance to be examined, in grams;
add 90 mL of methanol R and sonicate for 10 mino Shake the
mass of acetyl-ll-keto-f3-boswellic acid R in the
mixture vigorously 3 or 4 times during this procedure. Dilute
reference solution, in grams;
to 100.0 mL with methanol R. Centrifuge for 5 mino Dilute
P2 percentage content of acetyl-1 1 -keto-~-boswellic
1.0 mL of the clear solution to 10.0 mL with a mixture of
acid in acetyl-l1-keto-f3-boswellic acid R.
16 volumes of mobile phase A and 84 volumes of mobile
____________________________________________
phase B.
PhE~

Reference solwion Dissolve 1.0 mg of 11-keto-f3-boswellic

acid R and 1.0 mg of acetyl-ll-keto-f3-boswellic acid R in
20.0 mL of methanol R. Dilute 1.0 mL of this solution to
10.0 mL with a mixture of 16 volumes of mobile phase A
Fumitory *****
and 84 volumes of mobile phase B. ** *
Column: (Ph Eur monograph 1869) ****
-- size: 1 = 0.25 m, 0 = 4.6 mm; ~E~ ____________________________________________
-- stationary phase: octadecylsilyl silica gel for chromatography R
(5 f!m) .
DEFINITION
Whole or fragmented, dried aerial parts of Fumaria officinalis
Mobile phase:
L. harvested in full bloom .
-- mobile phase A: phosphoric acid R, water R (0.1 :99 .9 V/V);
-- mobile phase B: phosphoric acid R, acelOnitrile R Content
(0 .1:99.9 V/V); Minimum 0 .40 per cent of total alkaloids, expressed as
protopine (C2oHI9NOs; M r 353.4) (dried drug).
Time Mobile phase A Mobile phase B
(min) (per cent V!JI) (per cent VI!') IDENTIFICATION
O- 12.5 16 -? 6 84 -? 94 A. The hollow, angular stem is light green or greenish-
12.5 . 13.5 6 -? O 94 -? lOO brown. The leaves are alternate, bipinnatisect with 2 or 3 leaf
segments, the ultimate lobes lanceolate or obovate; they are
13.5 - 28 O lOO greenish-b lue and glabrous on both surfaces. The flowers are
Flow rate 1.0 mUmin. small and occur in loose racemes; each has a short pedicel
and is subtended by a leafY bract; they are pink or purplish-
Detection Spectrophotometer at 250 nm. red, dark purple or brown at the apex; the calyx is short,
Injection 20 f!L. composed of 2 petalloid sepals and the corolla is tubular with
Retemion time 11-keto-~-boswellic acid = about 8 min; 4 petals, the upper petal slightly spurred; there are 6 stamens
acetyl-11 -keto- ~-boswellic acid = about 12 mino united by their filaments into 2 groups of 3. The greenish-
System suitability Reference solution: brown, indehiscent fruits are globular or keel-shaped,
-- resolution: minimum 6.0 between the peaks due to truncated or slightly emargin ate at the apex, and each
11-keto - ~-boswe llic acid and acetyl-11-keto- ~-boswellic contains a small brown seed.
acid. B. Microscopic examination (2.8.23). The powder is green.
Calculate the percentage content of 11-keto-~-boswellic acid Examine under a microscope using chloral hydrate solution R.
using the following expression: The powder shows the following diagnostic characters
(Figure 1869.-1 ): fragments ofthe leaflamina in surface view
with the upper epidermis [D] composed of irregularly
Al x mI x 5 x PI
polygonal cells [Da], sorne of which contain microcrystals of
A2 x m
calcium oxalate [Db] , and underlying palisade parenchyma
[De]; marginal cells at the apex of the lamina elongated to
Al area of the peak due to 11-keto-~-boswellic acid in form blunt papillae [Dd], and with the lower epidermis [A]
the chromatogram obtained with the test solution; composed of cells having wavier walls [Aa] and underlying
spongy parenchyma [Ac]; anomocytic stomata ( 2.8.3) [Ab,
IV-188 Fumitory 2014

De] on both surfaces; groups [G] of lignified fibres [Ga] and test solution. Furthermore, other blue fluorescent zones are
spiral [Gb], reticulate or bordered-pitted [B] ves seIs from the present in the chromatogram obtained with the test solution.
stem; fragments of the epidermis of the petals [F] composed
of polygonal cells with sinuous or wavy anticlinal walls and
Top of the plate
no papillae; spherical pollen grains [E], about 30 ¡.1m in
diameter, with a pitted exine and 6 large pores; fragments of
the fruit with polygonal cells with a thick, warty cuticle, from 4 blue fluorescent zones
the epicarp [H], and sinuous sclereids with thick and
channelled walls, from the endocarp [C]. --- - - -

Quinine: a blue fluorescent zone

A greenish-blue fluorescent zone

--- ---

Reference solutlon Test solution

Detection B Treat with a mixture of potassium iodobismuthate

solution R2, acetic acid R and water R (1 :2: 10 V/V/V) until
orange zones appear against a yellow background.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other les s intense orange zones
are present in the chromatogram obtained with the test
solution.

Top of the plate

Protopine: an orange zone An orange zone (protopine)

2 orange zones

--- - --

Quinine: an orange zone A faint orange zone

--- ---
Reference solution Test solution

Figure 1869.-1. -Illustration for identification test B of
powdered herbal drug offumitory
C . Thin-Iayer chromatography (2.2.27).
Test solution To 2 g of the powdered herbal drug (355)
(2.9.12) add 15 mL of 0. 05 M sulfuric acid and stir for
15 mino Filter. Dilute the filtrate lO 20 mL with 0.05 M
sulfuric acid. Add 1 mL of concentrated ammonia R and 10 mL
of ethyl aceta te R. Stir and centrifuge. Collect the upper
organic layer. Repeat the extraction in the same manner.
Collect the organic layers and dry over anhydrous sodium
sulfate R . Evaporate to dryness under reduced pressure. Take
up the residue with 0.5 mL of methanol R.
Reference solution Dissolve 5 mg of protopine hydrochloride R
and 5 mg of quinine R in 10 mL of methanol R.
Place TLC silica gel place R.
Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
acetone R, toluene R (2:6:40:52 VIVIV/V).
Application 30 ¡.IL as bands.
Development Over a path of 15 cm.
Drying In air.
D etection A Examine in ultraviolet light at 365 nm.
Results ASee below the sequence of zones present in the
chromatogram obtained with the reference solution and the
TESTS
Cadmium (2.4.27)
Maximum 1.5 ppm.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4. 16)
Maximum 15.0 per cent.
ASSAY
To 5.000 g ofthe powdered herbal drug (355) (2.9.12) add
5 mL of dilute ammonia Rl and 50 mL of ethyl acetate R.
Shake for 15 mino Filter. Repeat the procedure in the same
manner and combine the filtrates. Evaporate the filtra tes to
dryness under reduced pressure. Dissolve the residue by
sonication for 10 min in 50 mL of 0.05 M sulfuric acid. Filter.
Dilute the filtrate to 100 mL with 0.05 M sulfuric acid. Adjust
to pH 9-10 with concentrated ammonia R and then add
50 mL of ethyl acetate R. Shake gently. Collect the upper
organic layer, after centrifugation if necessary. Repeat the
procedure in the same manner. Combine the organic layers
and dry over anhydrous sodium sulfate R. Evaporate lO dryness
under reduced pressure. Take up the residue with 100 mL of
anhydrous acetic acid R. Titrate with 0.02 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
1 mL of 0.02 M perchlon'c acid is equivalent to 7.068 mg of
protopine.
 2014 Gentian IV-189

Calculate the percentage content of total alkaloids, expressed Total ash (2.4.16)
as protopine, using the following expression: Maximum 5.0 per cent.
ASSAY
n x 706.8 Liquid chromatography (2.2.29). Carry out the assay as
m quickly as possible.
Internal standard solution Dissolve 20.0 mg of butyl
n volume of 0.02 M perchloric acid used, in millilitres;
parahydroxybenzoate CRS in 100.0 mL of a mixture of equal
m mas s of the herbal drug to be examined, in milligrams. volumes of methanol R and water R.
____________________________________________ PhE~

Test solution To 0.800 g of garlic powder add 20.0 mL of

water R and homogenise the mixture in an ultrasonic bath at
4 oC for 5 mino AlIow to stand at room temperature for
30 min. Then centrifuge for 30 min. Dilute 10.0 mL of the
*** supematant to 25.0 mL with a mixture of 40 volumes of a
Garlic Powder * *
******* 1 per cent VIV solution of anhydrous formic acid R and
60 volumes of methanol R (stock solution). Shake and
(Ph Eur monograph 1216)
~E~ ____________________________________________ centrifuge for 5 min. Place 0.50 mL of the internal standard
solution in a volumetric ftask and dilute to 10.0 mL with the
DEFINITION stock solution.
Bulbs of Allium sativum L., cut, freeze-dried or dried at a Precolumn:
temperature not exceeding 65 oC and powdered. -- size: 1 = 20 mm, 0 = 4 mm,
Content -- stationary phase: silanised octadecylsilyl silica gel for
Minimum 0.45 per cent of allicin (C 6 HlQOS2; M r 162.3) chromatography R (5 J.!ffi).
(dried drug). Column:
CHARACTERS -- size: 1 = 0 .25 m, 0 = 4 mm,
-- stationary phase: silanised octadecylsilyl silica gel for
Appearance
chromatography R (5 J.!m).
Light yellowish powder.
Mobile phase Mix 40 volumes of a 1 per cent V/V solution
IDENTIFICATION of anhydrous formic acid R and 60 volumes of methanol R.
A. Examine under a microscope using chloral hydrate
Flow rate 0.8 mUmin.
solution R. The powder shows the following diagnostic
characters: numerous fragments of parenchyma and groups Detection Spectrophotometer at 254 nm.
of spiral or annular vessels accompanied by thin-walled Injection Loop injector, 1 J.!L of the internal standard
parenchyma. solution and 10 J.!L of the test solution.
B. Thin-layer chromatography (2.2.27). Calculate the percentage of allicin using the following
Test solution To 1.0 g of garlic powder add 5.0 mL of expression:
methanol R, shake for 60 s and filter.
Reference solution Dissolve 5 mg of alanine R in 10 mL of 8 1 x m2 x 22.75
water R and dilute to 20 mL with methanol R . 8 2 x m1
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, propanol R, water R, area of the peak due to allicin (principal peak) in the
anhydrous ethanol R (20:20:20:40 VIVIV/V). chromatogram obtained with the test solution,
Application 20 J.!L of the test solution and 10 J.!L of the
area of the peak due to butyl parahydroxybenzoate in
reference solution, as bands. the chromatogram obtained with the test solution,
mass of the drug to be examined, in grams,
Development Over a path of 10 cm.
mass ofbutyl parahydroxybenzoate in 100.0 mi ofthe
Drying In air. internal standard solution, in grams. 1 mg of
Detection Spray with a 2 gIL solution of ninhydrin R in a butylparahydroxybenzoate corresponds to 8.65 mg of
mixture of 5 volumes of glacial acetic acid R and 95 volumes allicin.
of butanol R and heat at 105-110 oC for 5-10 min; examine ____________________________________________ PhE~

in daylight.
Results The chromatogram obtained with the reference
solution shows a violet zone (alanine) in its central third.
The chromatogram obtained with the test solution shows a ***
violet or brownish-red zone similar in position to that in the Gentian * *
chromatogram obtained with the reference solution and ** **
(Gemian Root, Ph Eur monograph 0392) ***
corresponding to alliin; above and below this zone are other,
generally fainter, violet zones. Preparations
Compound Gentian Infusion
TESTS
Starch Gentian Tincture
Examine the powdered drug under a microscope using When Powdered Gentian is prescribed or demanded,
water R. Add iodine solution R1. No blue colour develops. material complying with the requirements below with the
exception of Identification test A shall be dispensed or
Loss on drying (2.2.32)
supplied.
Maximum 7.0 per cent, determined on 1.000 g ofthe
powdered drug by drying in an oven at 105 oC.
 IV-190 Gentian 2014

~E~ ____________________________________________ Top of the plate

DEFINITION A prominent quenching zone
Dried, fragmented underground organs of Gentiana lutea L.
Phenazone: a quenching zone
CHARACTERS A weak quenching zone
Characteristic odour. (amarogentin)
Strong and persistent bitter taste. --- -----
Gentian root occurs as single or branched subcylindrical --- -----
pieces of various lengths and usually 10-40 mm thick but
Hyperoside : a quenching zone A prominent quenching zone
occasionally up to 80 mm thick at the crown. (~entiopicroside)

IDENTIFICATION Reference solution Test solution

A. The surface is brownish-grey, and the colour of a
transverse section is yellowish or reddish-yellow, but not
reddish-brown. The root is longitudinally wrinkled and bears Detection B Spray with a 100 gIL solution of potassium
occasional rootlet scars. The branches of the rhizome hydroxide R in methanol R and then with a freshly prepared
frequently bear a terminal bud and are always encircled by 2 giL solution offast blue B salt R in a mixture of 50 volumes
closely arranged leaf scars. The rhizome and root are brittle of anhydrous ethanol R and 50 volumes of water R . Examine
when dry and break with a short fracture but they absorb in daylight.
moisture readily to become flexible. The smoothed, Results B See below the sequence of the zones present in
transversely cut surface shows a bark, occupying about one- the chromatograms obtained with the reference solution and
third of the radius, separated by the well-marked cambium the test solution. Furthermore, other zones may be present in
from an indistinctly radiate and mainIy parenchymatous the chromatogram obtained with the test solution.
xylem.
B. Reduce to a powder (355) (2.9.12). The powder is light Top of the plate
brown or yellowish-brown. Examine under a microscope
using chloral hydrate solution R. The powder shows the A prominent dark violet zone
following diagnostic characters: fragments of the subero- A violet-red zone (amarogentin)
phellodermic layer, consisting of thin-walled yellowish-brown
--- -----
cork cells and thick-walled collenchyma (phelloderm);
fragments of cortical and ligneous parenchymatous cells with - -- -----
moderately thickened walls containing droplets of oi! and Hyperoside: a brownish-red zone A weak Iight brown zone
small prisms and minute needles of calcium oxalate; (gentiopicroside)
fragments of lignified ves seis with spiral or reticulate Reference solution Test solution
thickening.
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 1.0 g of the powdered drug (355) (2.9.12) TESTS
add 25 mL of methanol R, shake for 15 min and filter. Other species of Gentiana
Evaporate the filtra te to dryness under reduced pressure, at a Examine the chromatograms obtained in identification test
temperature not exceeding 50 oC . Take up the residue with C, detection B.
small quantities of methanol R so as to obtain 5 mL of a Results The chromatogram obtained with the test solution
solution, which may contain a sedimento does not show violet zones immediately aboye the zone due
Reference solution Dissolve 5 mg of hyperoside R and 5 mg of to amarogentin.
phenazone R in 10 mL of methanol R. Totalash (2.4.16)
Plate TLC silica gel F254 plate R . Maximum 6.0 per cent.
Mobile phase water R, anhydrous formic acid R, ethyl Bitterness value (2.8.15)
formate R (4:8:88 V/VIV). Minimum 10000.
Application 20 /lL as bands. Water-soluble extractive
Development In an unsaturated tank, over a path of 8 cm. Minimum 33 per cent.
Drying In airo To 5.0 g ofpowdered drug (7 10) (2.9. 12) add 200 mL of
Detection A Examine in ultraviolet light at 254 nm. boiling water R . Allow to stand for 10 min, shaking
occasionally. Allow to cool, dilute to 200.0 mL with water R
Results ASee below the sequence of the zones present in
and filter. Evaporate 20.0 mL of the filtrate to dryness on a
the chromatograms obtained with the reference solution and
water-bath. Dry the residue in an oven at 100-105 oc.
the test solution. Furthermore, other zones may be present in
The residue weighs a minimum of 0.165 g.
the chromatogram obtained with the test solution. ____________________________________________ PhE~
2014 Gentian Preparations IV-191

Results ASee below the sequence of the zones present in

Compound Gentian Infusion the chromatograms obtained with the reference solution and
DEFINITION the test solution. Furthermore, other zones may be present in
Concentrated Compound Gentian 100 mI the chromatogram obtained with the test solution.
Infusion
Water Sufficient to produce
Top of the plate
1000 mI
A prominent quenching zone
The infusion complies with the requirements stated under Infusions.
Phenazone : a quenching zone

A weak quenching zone

CONCENTRATED COMPOUND GENTIAN (amarogentin)
INFUSION --- ---

DEFINITION --- ---

Gentian, cut small and bruised 125 g
Hyperoside: a quenching zone A prominent quenching zone
Dried Bitter-orange Peel, cut small 125 g (¡<entiopicroside)
Dried Lemon Peel, cut small 125 g Reference solution Test solution
Ethanol (25 per cent) 1200 mI
Extemporaneous preparation Detection B Spray with a 10 per cent VIV solution of
The following directions apply. potassium hydroxide R in methanol R and then with a freshly
Macerate the Gentian, the Dried Bitter-orange Peel and the prepared 2 giL solution of fast blue B salt R in a mixture of
Dried Lemon Peel in a covered vessel for 48 hours with ethanol R and water R (50:50 V/V). Examine in daylight.
1000 mI of the Ethanol (25 per cent); express the liquido Results B See below the sequence of the zones present in
To the pressed marc add 200 mI of the Ethanol the chromatograms obtained with the reference solution and
(25 per cent), macerate for 24 hours, press and add the the test solution. Furthermore, other zones may be present in
liquid to the product of the first pressing. Allow to stand for the chromatogram obtained with the test solution.
not less than 14 days; filter.
TESTS Top of the plate
Ethanol content
A prominent dark violet zone
20 to 24% v/v, Appendix VIII F.
A violet-red zone (amarogentin)
Total solids
Not less than 9.5% w/v, Appendix XI A. --- ---
--- ---
Hyperoside: a brownish-red zone A weak light brown zone
¡~entiopicroside)
Gentian Tincture *** Test solution
*** ***
Reference solution

(Ph Eur monograph 1870) ***

PhEur _ _ __ _ _ _ _ _ _ _ _ _ __ _ _ __ __ _ TESTS
Ethanol content (2.9.10)
DEFINITION 62 per cent VIV to 67 per cent V/V.
Tincture produced from Gentian root (0392).
Bitterness value (2.8.15)
PRODUCTION Minimum 1000.
The tincture is produced from 1 part of the comminuted Dry residue (2.8.16)
drug and 5 parts of ethanol (70 per cent V/V) by a suitable Minimum 5.0 per cent mlm, determined on 3.00 g.
procedure. _ __ _ _ _ __ _ __ _ __ _ _ _ _ __ _ _ PhEw

CHARACTERS
Appearance
Yellowish-brown or reddish-brown liquido
It has a strong bitter taste.
Acid Gentian Mixture
IDENTIFICATION
Acid Gentian Oral Solution
Thin-Iayer chromatography (2.2.27).
Test solution The tincture to be examined. DEFINITlON
Acid Gentian Mixture is an oral solution containing 10% v/v
Reference solutwn Dissolve 5 mg of phenazone R and 5 mg of
of Concentrated Compound Gentian Infusion and 5% v/v of
hyperoside R in 10 mL of metharwl R.
Dilute Hydrochloric Acid in a suitable vehic1e.
Plare TLC silica gel F 254 plate R.
Extemporaneous preparation
Mobile phase water R, anhydrous formic acid R, ethyl It is recently prepared according ro the following formula.
formate R (4:8:88 VIV/V). Concentrated Compound Gentian 100 mL
Application 20 IlL, as bands. Infusion
Development Over a path of 8 cm, in an unsaturated tank. Dilute Hydrochloric Acid 50 mL
Drying In air. Double-strength Chloroform Water 500 mL
Water Sufficient to produce
Detection A Examine in ultraviolet light at 254 nm.
1000 mL
 IV-192 Gentian Preparations
The mixture complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of hyd.rochloric acid, HCl
0.48 to 0.56% w/v.
ASSAY
To 10 mL add 10 mL of water, adjust the pH to between 5.0
and 6.0 with 2M sodium hydroxide and dilute to 25 mL with
water. Add 75 mL of acetate buffer pH 5. O and titrate with
O.lM silver nitrate VS determining the end point
potentiometrically using a sil ver indicator electrode and a
glass reference electrode and stirring throughout the titration.
Each mL of O.lM silver nitrate VS is equivalent to 3.646 mg
ofHCl.
Alkaline Gentian Mixture
Alkaline Gentian Oral Solution
DEFINITION
Alkaline Gentian Mixture is an oral solution containing
10% v/v of eoncentrated eompound Gentian Infusion and
5% w/v of Sodium Bicarbonate in a suitable vehicle.
Extemporaneous preparation
It is recently prepared according to the following formula.
eoncentrated eompound Gentian 100 mL
Infusion
Sodium Bicarbonate 50 g
Double-strengrh ehloroform Water 500 mL
Water Sufficient to produce
1000 mL
The mixture complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of sodium bicarbonate, NaHC0 3
4.75 to 5.25% w/v.
ASSAY
To 10 mL of the mixture add 100 mL of water and 25 mL
of O.5M hydrochloric acid VS, boil for 10 minutes and titrate
the excess of hydrochloric acid with O.5M sodium hydroxide
VS using 0.5 mL of methyl red solution as indicator. Each mL
of O.5M hydrochloric acid VS is equivalent to 42 .00 mg of
NaHe0 3 ·
Ginger ****
***
(Ph Bur monograph 1522) *** *
Preparation
Strong Ginger Tincture
Ginger may be known in commerce as unbleached ginger.
When Powdered Ginger is prescribed or demanded, material
complying with the appropriate requirements below shall be
dispensed or supplied.
____________________________________________
~Eif
DEFINITION
Dried, whole or cut rhizome of Zing¡'ber officinale Roscoe,
with the cork removed, either completely or from the wide,
fiat surfaces only.
Content
Minimum 15 mUkg of essential oil (anhydrous drug).
2014
CHARACTERS
eharacteristic aroma tic odour.
Spicy and burning taste.
IDENTIFICATION
A. The rhizome is laterally compres sed, bearing short,
fiattened, obovate oblique branches on the upper side, each
sometimes having a depressed scar at the apex; the whole
rhizomes are about 5-10 cm long, 1.5-3 cm or 4 cm wide
and 1-1.5 cm thick, sometimes split longitudinally.
The scraped rhizome with a light-brown externa! surface
shows longitudinal striations and occasionalloose fibres;
the outer surface of the unscraped rhizome varies from pale
to dark brown and is more or less covered with cork that
shows conspicuous, narrow, longitudinal and transverse
ridges; the cork readily exfolia tes from the lateral surfaces but
persists between the branches. The fracture is short and
starchy with projecting fibres. The smoothed transversely cut
surface exhibits a narrow cortex separated by an endodermis
from a much wider stele; it shows numerous, scattered,
fibrovascular bundles and abundant scattered oleoresin cells
with yellow contents. The unscraped rhizome shows, in
addition, an outer layer of dark brown cork.
B. Reduce to a powder (355) (2.9.12). The powder is pale
yellow or brownish. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1522.- 1): groups of large, thin-
walled, septate fibres, with one wa!1 frequently dentate [e, D,
G]; fragrnents [K] containing vessels with reticulate
thickening [Ka] often accompanied by narrow, thin-walled
cells containing brown pigment [Kb] and amyliferous
parenchyma [Kc]; abundant reticulate vessels, fairly large,
isolated [H, L]; abundant thin-walled parenchyma of the
ground tissue U, M], sorne cells containing brown oleoresin
Ua] ; fragrnents of brown cork, usually seen in surface view
[F] but sometimes in transverse section [E]. Examine under
a microscope using a 50 per cent V/V solution of glycerol R.
The powder shows abundant starch granules, simple,
fiattened, oblong or oval or irregular, up to about 50 Ilm long
and 25 Ilm wide, with a small point hilum situated at the
narrower end; sometimes, granules show faint, transverse
striations, and may be free [A], agglomerated [B] or included
in parenchymatous cells (Kc).
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 1.0 g of the powdered drug (710) (2.9.12)
add 5 mL of methanol R . Shake for 15 min and filter.
Reference solution Dissolve 10 IlL of citral R and 10 mg of
resorcinol R in lO mL of methanol R . Prepare the solution
* immediately before use.
Plate TLC silica gel plate R .
Mobile phase hexane R, ether R (40:60 V/V).
Application20 IlL as bands.
Development In an unsaturated tank, over a path of 15 cm.
Drying In air.
Detection Spray with a 10 gIL solution of vanillin R in
sulfuric acid R and examine in daylight while heating at
100-105 oC for 10 mino
Results The chromatogram obtained with the reference
solution shows in the lower half an intense red zone
(resorcinol) and in the upper half 2 violet zones (citral);
the chromatogram obtained with the test solution shows
below the zone due to resorcinol in the chromatogram
obtained with the reference solution 2 intense violet zones
(gingerols) and in the middle, between the zones due to
 2014 Ginkgo Leaf IV-193

The tincture complies with the requirements for Tinctures stated

under EXlracts and with the following requirements.
TESTS
Ethanol content
80 to 88% v/v, Appendix VIII F, Method III.
Dry residue
2 .0 to 3.0% w/v.
Relative density
0.832 10 0.846, Appendix V G.

Weak Ginger Tincture

DEFINITION
o Strong Ginger Tincture 200 mI
Ethanol (90 per cent) Sufficient to produce 1000 mI
l' The linClure complies Wilh the requirements for Tinclures stated
under EXlracls and with the following requirements.
TESTS
Ethanol content
86 10 90% v/v, Appendix VIII F, Method III.
Dry residue
Not less than 0.4% w/v. Use 10 mI.
M Relative density
0.825 10 0.835, Appendix V G.

Figure 1522.-1.- Illustration for identification test B of

powdered herbal drug of ginger

Ginkgo Leaf ***

*** ***
resorcinol and citral in the chromatogram obtained with the (Ph Eur monograph 1828) ***
reference solution, 2 other less intense violet zones
Preparation
(shogaols); other zones may be presento
Ginkgo Leaf Dry Extract, Refined and Quantified
TESTS ~E~ __________________________________________
Water (2.2.13)
Maximum 100 mUkg, determined by distillation on 20.0 g DEFINITION
of the powdered drug (710) (2.9.12). Whole or fragmented, dried leaf of Ginkgo biloba L.

Total ash (2.4.16) Content

Maximum 6.0 per cent. Not less than 0.5 per cent of ftavonoids, expressed as ftavone
glycosides (Mr 757) (dried drug).
ASSAY
Carry out the determination of essential oils in herbal drugs IDENTIFICATION
(2.8.12) . Use 20 .0 g of the freshly, coarsely powdered drug, a A. The leaf is greyish or yellowish-green or yellowish-brown.
1000 mL round-bottomed ftask, 10 drops of liquid paraffin R The upper surface is slightly darker than the lower surface.
or other antifoam, 500 mL of water R as distillation liquid The petioles are about 4-9 cm long. The lamina is about
and 0.5 mL of xylene R in the graduated tube. Distil at arate 4-10 cm wide, fan-shaped, usually bilobate or sometimes
of 2-3 mUmin for 4 h. undivided. Both surfaces are smooth, and the venation
__________________________________________ ~E~
dichotomous, the veins appearing 10 radiate from the base;
they are equally prominent on both surfaces. The distal
margin is incised, irregularly and to different degrees, and
irregularly lobate or emarginate. The lateral margins are
entire and taper 10wards the base.
Strong Ginger Tincture B. Reduce to a powder (355) (2.9.12) . The powder is greyish
Ginger Essence or yellowish-green or yellowish-brown. Examine under a
microscope using chloral hydrate solution R. The powder
DEFINITION shows the following diagnostic characters (Figure 1828.-1) :
Ginger, in moderately coarse powder 500 g irregularly-shaped fragments ofthe lamina [A, B, D, E], with
Ethanol (90 per cent) Sufficient to produce the upper epidermis, in surface view (D) and transverse
1000 mI section (E), consisting of elongated cells with irregularly
Extemporaneous preparation sinuous walls [Da], often accompanied by palisade
The following directions apply. parenchyma [Db], and the lower epidermis, in surface view
Prepare by percolation, Appendix XI F. (A) and transverse section (B) , consisting of small cells, with
a finely striated cutic1e and each cell shortly papillose [Aa],
 IV-194 Ginkgo Leaf 2014

and stomata [Ab) about 60 [.lm, wide, deeply sunken with Top of the plate
6-8 subsidiary cells; fragments of vascular tissue from the
A yellowish-brown fluorescent
petiole and veins [C] with xylem [Ca) and parenchyma, zone
sorne cells containing abundant cluster crystals of calcium A green fluorescent zone
oxalate of various sizes [Cb) .
2 yellowish-brown fluorescent
zones
An intense light blue fluorescent
zone sometimes overlapped by a
greenish-brown fluorescent zone
Chlorogenic acid: a light blue
fluorescent zone
A green fluorescent zone

Rutin: a yellowish-brown 2 yellowish·brown fluorescent

fluorescent zone zones
A green fluorescent zone

A yellowish-brown fluorescent
zone
Reference solution Test solution

eb
25~m
Figure 1828.-1.- Illustratian far identificatian test B af
pawdered herbal drug af ginkga leaf
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 2.0 g ofthe powdered drug (710) (2.9.12)
add 10 mL of methanol R . Heat in a water-bath at 65 oC for
10 mino Shake frequently. Allow to cool to room temperature
and filter.
Reference solution D issolve 1.0 mg of chlorogenic acid R and
3.0 mg of rutin R in 20 mL of methanol R .
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R , glacial acetic acid R,
water R , ethyl acetate R (7.5:7.5:17.5:67.5 V/V/V/V) .
Application 20).lL as bands.
Development Over a path of 17 cm.
Drying At 100-105 oc.
Detection Spray the warm plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R, then with
the same volume of a 50 gIL solution of macrogol 400 R in
methanol R; allow to dry in air for about 30 min and examine
in ultraviolet light at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other weak f1uorescent zones may
be present in the chromatogram obtained with the test
solution.
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems and 2 per cent of other
foreign matter.
Loss on drying (2.2.32)
Maximum 11.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 11 .0 per cent.
ASSAY
Flavonoids
Liquid chromatography (2.2.29) .
Test solution Heat 2.500 g ofthe powdered drug (710)
(2.9.12) in 50 mL of a 60 per cent V/V solution of acetone R
under a reflux condenser for 30 mino Filter and collect the
filtrate. Extract the drug residue a 2 nd time in the same
manner, using 40 mL of a 60 per cent V/V solution of
acetone R and filter. Collect the filtrates and dilute to
100.0 mL with a 60 per cent V/V solution of acetone R .
Evaporate 50.0 mL of the solution to eliminate the acetone
and transfer to a 50 .0 mL vial, rinsing with 30 mL of
methanol R. Add 4.4 mL of hydrochloric acid R1, dilute to
50.0 mL with water R and centrifuge. Place 10 mL ofthe
supernatant in a 10 mL brown-glass vial. Close with a rubber
seal and an aluminium cap and heat on a water-bath for
25 mino Allow to cool to room temperature.
Reference solution Dissolve 10.0 mg of quercetin dihydrate R
in 20 mL of methanol R . Add 15 .0 mL of dilute hydrochloric
acid R and 5 mL of water R and dilute to 50.0 mL with
methanol R.
Column:
- size: 1 = 0.125 m, 0 = 4 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 [.lm);
- temperature: 25 oc.
Mobüe phase:
- mobüe phase A : 0.3 gIL solution of phosphoric acid R
adjusted to pH 2.0;
- mobüe phase B: methanol R;
 2014 Ginkgo Preparations IV-195

Time Mobile phase A MobUe phase B IDENTIFICATION

(min) (percent VM (percent VM Thin-Iayer ehromatography (2.2.27).
0-1 60 40
Test solutwn Dissolve 20.0 mg of the extraet to be examined
1 - 20 60 -> 45 40 -> 55 in 10 mL of a mixture of 2 volumes of water R and
20 - 21 45 -> O 55 -> 100 8 volumes of methanol R.
21- 25
Reference solution Dissolve 1.0 mg of chlorogenic acid R and
100
3.0 mg of rutin R in 20 mL of methanol R .
Flow rate l.0 mUmin. Plate TLC silica gel plate R (5-40 ¡.¡m) or [TLC silica gel
Detection Speetrophorometer at 370 nm. plate R (2-10 11m)] .
Injection 10 ¡lL. Mobile phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (7.5 :7.5: 17.5 :67.5 VIVIV/V) .
Relative retention With referenee ro quereetin
(retention time = about 12.5 min): kaempferol = about 1.4; Application 20 ¡lL [or 5 ¡lL], as bands.
isorhamnetin = about 1.5. Development Over a path of 17 cm [or 6 cm].
System suitabüity: Drying At 100-105 oC.
- resolution: minimum 1.5 between the peaks due to Detection Spray the plate whilst still hot with a 10 gIL
kaempferol and isorhamnetin. solution of diphenylboric acid aminoethyl ester R in methanol R,
Do not take into aeeount peaks eluting before the quereetin then spray with a 50 gIL solution of macrogol 400 R in
peak or after the isorhamnetin peak in the ehromatogram methanol R; allow to dry in air for about 30 min and examine
obtained with the test solution. in ultraviolet light at 365 nm.
Calculate the pereentage eontent of fiavonoids, expressed as Results See below the sequence of zones present in the
fiavone glyeosides, using the following expression: chromatograms obtained with the referenee solution and the
test solution. Furthermore, other, weaker fiuorescent zones
F I x mI x 2.514 x p may be present in the chromarogram obtained with the test
2 x ---:::,-------''- solution.
F2 x m2
ASSAY
sum of the areas of all the eonsidered peaks in the Flavonoids
ehromatogram obtained with the test solution; Liquid chromatography (2.2.29).
area of the peak eorresponding to quereetin in the Test solution Dissolve 0.200 g of the extract to be examined
ehromatogram obtained with the referenee solution; in 20 mL of methanol R. Add 15.0 mL of dilute hydrochloric
mi mass of quereetin used ro prepare the referenee acid R and 5 mL of water R and dilute to 50.0 mL with
solution, in grams; methanol R. Transfer 10.0 mL of this solution into a 10 mL
mass of the drug to be examined used to prepare the brown-glass vial. Close the vial with a tight rubber membrane
test solution, in grams; stopper and secure with an aluminium crimped cap. Heat on
p pereentage eontent of anhydrous quereetin in a water-bath for 25 mino Allow to cool to 20 oC.
quercetin dihydrate R. Reference solution Dissolve 10.0 mg of quercetin
_ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ ~E~ dihydrate CRS in 20 mL of methanol R. Add 15.0 mL of
dilute hydrochloric acid R and 5 mL of water R and dilute to
50.0 mL with methanol R.
Column:
Refined and Quantified Ginkgo *** - size: l = 0.125 m, 0 = 4 mm;
*** *** - stationary phase: octadecylsilyl silica gel for chromatography R
Dry Extraet *** (5 ¡lm);
(Ph Eur monograph 1827) - temperature: 25 oC.
~E~ _ _ _ _ __ _ __ __ _ _ __ _ _ __ _ __ Mobüe phase:
- mobile phase A: 0.3 gIL solution of phosphoric acid R
DEFINITION adjusted to pH 2.0;
Refined and quantified dry extraet produeed from Ginkgo leaf - mobile phase B: methanol R;
(1828).
Content:
Time Mobile phase A Mobile phase B
- fiavonoids, expressed asfiavone glycosides (Mr 756 .7):
(min) (percent VM (percent VM
22.0 per eent to 27.0 per eent (dried extraet);
0- 1 60 40
- bilobalide: 2.6 per eent to 3.2 per eent (dried extraet);
- ginkgolides A, B and C: 2.8 per eent to 3.4 per eent (dried 1- 20 60 -> 45 40 -> 55
extraet); 20 - 21 45 -> O 55 -> 100
- ginkgolic acids: maximum 5 ppm (dried extraet).
21 - 25 O 100
PRODUCTION
The extraet is produeed from the herbal drug by an Flow rate l.0 mUmin.
appropriate proeedure using organie solvents and their Detector Spectrophotometer at 370 nm.
mixtures with water, physieal separation steps as well as other Injection 10 ¡lL.
suitable proeesses.
Relative retention With reference ro quercetin
CHARACTERS (retention time = about 12.5 min): kaempferol = about 1.4;
Appearance isorhamnetin = about l.5.
Bright yellow-brown, powder or friable mass.
 IV-196 Ginkgo Preparations 2014

o 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 min

1. quercetin 2. kaempferol 3. isorhamnetin

Figure 1827.-1. - Chromatogram for the assay offlavonoids in refined and quantified ginkgo dryextract

Top of the plate

FI X mI X 2.514 X P
F2 x m2
A blue fluorescent zone

Several faint coloured zones

FI sum of the areas of aH the peaks from the peak due
--- --- to quercetin 10 the peak due to isorhamnetin in the
A brown fluorescent zone chromatogram obtained with the test solution
Fz area of the peak due to quercetin in the
A green fluorescent zone
chromatogram obtained with the reference solution
An intense light blue fluorescent mi mass of quercetin dihydrate CRS in the reference
zone sometimes overlapped by a
greenish-brown fluorescent zone
solution, in grams
Chlorogenic acid: a light blue m2 mas s of the extract to be examined used 10 prepare
fluorescent zone the test solution, in grams
One or two green fluorescent zones p percentage content of anhydrous quercetin in
Rutin : a yellowish-brown fluorescent One or two yellowish-brown
quercetin dihydrate CRS.
zone fluorescent zones
Terpene lactones
--- --- Liquid chromatography (2.2.29).
Several green and yellowish-brown Test solution Place 0.120 g ofthe extract to be examined in
fluorescent zones
a 25 mL beaker and dissolve it in 10 mL of phosphate buffer
solution pH 5.8 R by stirring. Transfer the solution into a
Reference solution Test solution chromatography column, about 0.15 m long and about
30 mm in internal diameter, containing 15 g of kieselguhr for
chromatography R. Wash the beaker with 2 quantities, each of
System suitability Test solution: 5 mL, of phosphate buffer solution pH 5.8 R and transfer the
- resolution: minimum 1.5 between the peaks due to washings to the chromatography column. AHow to stand for
kaempferol and isorhamnetin. 15 mino Elute with 100 mL of ethyl acetate R. Evaporate the
eluate to dryness at a pressure not exceeding 4 kPa in a
Determine the sum of the areas including aH the peaks from water-bath at 50 oC. The residue of solvent is eliminated by
the peak due to quercetin to the peak due to isorhamnetin in an air-current. Take up the residue in 2.5 mL of the mobile
the chroma1Ogram obtained with the test solution (see Figure phase.
1827.-1).
Reference solution (a) Dissolve 30.0 mg of benzyl alcohol CRS
Calculate the percentage content of flavonoids, expressed as in the mobile phase and dilute 10 100.0 mL with the mobile
flavone glycosides, using the foHowing expression: phase.
2014 Ginkgo Preparations IV-197

Reference solution (b) Place 0.120 g ofthe ginkgo dry extract
for peak identification CRS in a 25 mL beaker and dissolve it
in 10 mL of phosphate buffer solution pH 5.8 R by stirring,
then proceed as described for the test solution.
Column:
- size: 1 = 0.25 m, 0 = 4 mm;
- stationary phase: octylsuyl silica gel for chromatography R
(5 /lm);
- temperature: 25 0e.
Mobtle phase tetrahydrofuran R, methanol R, water R
(10:20 :75 VIV/V).
Flow rate 1.0 mlJmin.
Detection Refractometer maintained at 35 De.
Injection 100 /lL.
Identification of peaks Use the chromatogtam supplied with
ginkgo dry extraet for peak identification CRS and the
chromatogtam obtained with the reference solution (b) to
identify the peaks due to bilobalide and ginkgolides A, B and
e.
System suitabtlity:
- the chromatogtam obtained with reference solution (b) is
similar to the chromatogtam supplied with ginkgo dry
extraet for peak identification CRS.
Calculate the percentage content of bilobalide, using the
following expression:
Ft x ml x P x 0.025 x 1.20
F5 x m2
Calculate the percentage content of the sum of ginkgolides
A, B and C, using the following expression:
GA percentage content of ginkgolide A
GB percentage content of ginkgolide B
Gc percentage content of ginkgolide e.
Ginkgolic acids
Liquid chromatogtaphy (2.2.29).
Test solution Dissolve 0.500 g of the powdered extract to be
examined in 8 mL of methanol R, sonicating if necessary, and
dilute to 10.0 mL with the same solvento Centrifuge if
necessary.
Reference solution Dissolve 10.0 mg of ginkgolic acids CRS in
8 mL of methanol R, sonicating if necessary, and dilute to
10.0 mL with the same solvento Dilute 2.0 mL ofthis
solution to 10.0 mL with methanol R .
Column:
- size: 1 = 0.25 m, 0 = 4 .6 mm;
- stationary phase: oetylsilyl silica gel for chromatography R
(5 /lm);
- temperature: 35 oC.
Mobzle phase:
- mobile phase A: dilute 0.1 mL of trifiuoroacetic acid R to
1000 mL with water R;
- mobile phase B: dilute 0.1 mL of tnfluoroaeetie aeid R to
1000 mL with acetonitrile R;

Calculate the percentage content of ginkgolide A, using the Time Mobile phase A Mobile phase B
following expression: (min) (percent VID (percent VID
0-30 25 -0 10 75 -0 90
F2 x ml x p x 0.025 x 1.22 30 - 35 10 90
F5 x m2 35 - 36 10 -025 90 -0 75
36 - 45 25 75
Calculate the percentage content of ginkgolide B, using the
following expression: Flow rate 1.0 mUmin.
Detection Spectrophotometer at 210 nm.
F3 x ml x p x 0.025 x 1.19
Injection 50 /lL.
F5 x m2
ldentification of components Use the chromatogtam supplied
with ginkgolic acids CRS and the chromatogtam obtained with
Calculate the percentage content of ginkgolide C, using the the test solution to identify the peaks due to ginkgolic acids
following expression: C13, C15 and C17 .
System suitability Reference solution:
F 4 x ml x p x 0.025 x 1.27 - resolution: minimum 2.0 between the peaks due ro
F5 x m2 ginkgolic acids C13 and C15;
- symmetry factor: 0.8 to 2.0 for the peaks due to ginkgolic
area of the peak due to bilobalide in the acids C13, C15 and C17.
chromatogram obtained with the test solution Calculate the content in parts per million of ginkgolic acids
area of the peak due to ginkgolide A in the expressed as ginkgolic acid C 17, using the following
chromatogram obtained with the test solution expression:
area of the peak due to ginkgolide B in the
chromatogram obtained with the test solution
area of the peak due to ginkgolide C in the
Al x m2 x p x 2000
chromatogram obtained with the test solution A 2 x ml
area of the peak due to benzyl alcohol in the
chromatogram obtained with reference solution (a) Al sum of the areas of the peaks due to the ginkgolic
mass of benzyl alcohol CRS in reference solution (a), acids C 13, C 15 and C 17 in the chromatogram
in grams obtained with the test solution
mass of the extract to be examined used to prepare Az area of the peak due to ginkgolic acid Cl7 in the
the test solution, in gtams chromatogram obtained with the reference solution
p percentage content of benzyl alcohol in benzyl mi mass of the extract to be examined used to prepare
alcohol CRS. the test solution, in grams
 IV-198 Ginseng
mass of gz'nkgolic acids CRS used to prepare the
reference solution, in grams
p percentage content of ginkgolic acid C 17 in ginkgolic
acids CRS.
____________________________________________
Ginseng ***
** **
(Ph Eur monograph 1523) *****
~Ew ____________________________________________
DEFINITlON
Whole or cut dried root, designated white ginseng; treated
with steam and then dried, designated red ginseng, of Panax
ginseng C. A. Meyer.
Content
Minimum 0.40 per cent for the sum of ginsenosides Rg1
(C42H72014,2H20; M r 837) and Rb1 (Cs4H92023,3H20; M r
1163) (dried drug) .
IDENTIFICATlON
A. The principal root is fusiform or cylindrical, sometimes
branched, up ro about 20 cm long and 2.5 cm in diameter,
and may be curved or markedly re-curved. The surface is
pale yellow ro cream in white ginseng, brownish-red in red
ginseng and shows longitudinal ridges. Stem scars may be
seen at the crown. The fracture is short. The transversely-cut
surface shows a wide outer zone with scanered orange-red
resin canals and a finely radiate i=er region. The rootlets,
numerous in the lower part of white ginseng, are normally
absent in red ginseng.
B. Reduce to a powder (355) (2.9.12). The powder is light
yellow. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters: abundant fragments of thin-walled
parenchymatous cells and fragments of large secretory canal s
containing yellowish-brown resin, non-lignified tracheids and
partially-lignified ves seis with spiral or reticulate thickening,
isolated or in groups; scanered cluster crystals of calcium
oxalate. Examine under a microscope using a mixture of
equal volumes of g1:ycerol R and water R. The starch granules
are very abundant, simple or 2 or 3 compound, and range
from 1-10 ¡.un in diameter. In red ginseng the starch granules
are often deformed and destroyed by treating with steam, or
may be absent.
C. Thin-layer chromatography (2.2.27;.
Test solution Eoil 1.0 g of the powdered drug (355) (2.9.12)
under a reftux condenser with 10 mL of a 70 per cent V/V
solution of methanol R for 15 min. Filter after cooling and
dilute ro 10.0 mL with methanol R.
Reference solution Dissolve 5.0 mg of aescin R and 5.0 mg of
arbutin R in 1 mL of methanol R.
Plate TLC silica gel plate R (5-40 11m) [or TLC silica gel
plate R (2-10 11m)].
Mobile phase ethyl acetate R, water R, butanol R
(25:50: 100 VIV/V), allow the mixture to separate for 10 mino
Use the upper layer.
Application 20 I1L [or 4 I1L] as bands.
Development Over 10 cm [or 5 cm] in an unsaturated tank.
Drying In air.
Detection Spray with anisaldehyde solution R and heat at
105-110 oC for 5-10 minoExamine in daylight.
2014
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution.
~Ew
Top oí the plate
Arbutin: a brown zone
--- ---
A violet zone (ginsenosides Rg 1
+ Rg2)
A faint violet zone (ginsenoside Rf)
A violet zone (ginsenoside Re)
A violet zone (ginsenoside Rd)
A faint violet zone
--- ----
A violet zone (ginsenoside Re)
Aescin: a grey zone A violet zone (ginsenosides Rb 1
+Rb~
Reíerence solution Test solution
TESTS
Panax quinquefolium
Examine the chromatograms obtained in the assay.
The chromatogram obtained with the test solution shows a
peak due to ginsenoside Rf (se e Figure 1523.-1). In the case
of a substitution by Panax quinquefolium no peak due ro
ginsenoside Rf is presento
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.1Ó)
Mximum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum l.O _per cent.
ASSAY
Liquid chromatography (2.2.29) .
Test solution Reduce about 50 g ro a powder (355) (2.9.12).
Place l.00 g of the powdered drug and 70 mL of a
50 per cent V/V solution of methanol R in a 250 mL round-
bottomed ftask. After adding a few grains of pumice, boil on
a water-bath under a reftux condenser for 1 h. After cooling,
centrifuge and collect the supernatant liquido Treat the
residue as described aboye. Mix the collected liquids and
evaporate to dryness under reduced pressure at a temperature
not exceeding 60 oc. Take up the residue with 20.0 mL of a
mixture of 20 volumes of acetonitrile R and 80 volumes of
water R. Dilute 2.0 mL of the solution to 10.0 mL with a
mixture of 20 volumes of acetonitrile R and 80 volumes of
water R. Filter through a suitable membrane filter (nominal
pore size 0.45 11m) before injection.
Reference solution Dissolve 3.0 mg of ginsenoside Rg1 R,
3.0 mg of ginsenoside Re R, 3.0 mg of ginsenoside Rf R and
3.0 mg of ginsenoside Rb1 R in methanol R and dilute to
10.0 mL with the same solvento
Column:
-- size: l = 0.125 m, el = 4.6 mm;
-- stationary phase: octadecylsi1:yl silica gel for chromatography R
(5 11m);
-- temperature: 35 ce.
2014 Ginseng Dry Extraet IV-199

0.35
4 5
0 .30

0.25 6
0.20
7
0.15
2
0.10

0.05

0 .00

O
LN 5 10 15 20 25 30 35 40 45 min

1. ginsenoside Rgl 3. ginsenoside Rf 5. ginsenoside Re 7. ginsenoside Rd

2. ginsenoside Re 4. ginsenoside Rb 1 6. ginsenoside Rb2

Figure 1523.-1. - Chromatogram for the assay of ginseng: test solution

Mobile phase: mass of the drug to be examined, in grams,

-- mobile phase A: water R adjusted to pH 2 with phosphoric mass of ginsenoside Rb 1 in the referenee solution, in
acid R; milligrams,
mobile phase B: acetonitrile R; mass of ginsenoside Rg1 in the referenee solution, in
milligrams,
Time Mobile phase A Mobile phase B
(min) (per cent V!VJ (per cent V!VJ
pereentage eontent of ginsenoside Rb1 in the reagent,
0·8 80 20 pereentage eontent of ginsenoside Rg1 in the reagent.
____________________________________________ ~Ew

8·40 80 -> 60 20 -t 40

40·45 60 -t 40 40 -t 60

45 · 47 40 -t O 60 -t 100

47·52 100 Ginseng Dry Extract *****

** **
52·55 O -t 80 100 -> 20
(Ph Eur monograph 2356) ***
Flow rate 1.0 mIJmin . ~Ew ____________________________________________

Detection Speetrophotometer at 203 nro. DEFINITION

Equilibration 20 mino
Injection 20 ¡.IL.
Elution order Order indieated in the eomposition of the
referenee solution; record the retention times of these
substanees.
System suitability Referenee solution:
-- resolution: minimum 1.0 between the peaks due to
ginsenoside Rg1 and ginsenoside Re.
Loeate the peaks due to ginsenoside Rb1 and ginsenoside
Rg1 in the ehromatogram obtained with the test solution.
Calculate the pereentage eontent of ginsenosides Rb 1 and
Rg1 using the following expression:
Al x m2 x PI + A z x m3 X pz
A3 X mI X 100 A4 x mI x 100
area of the peak due to ginsenoside Rb 1 in the
ehromatogram obtained with the test solution,
area of the peak due to ginsenoside Rg1 in the
ehromatogram obtained with the test solution,
area ofthe peak due to ginsenoside Rb1 in the
ehromatogram obtained with the referenee solution,
area of the peak due to ginsenoside Rg1 in the
ehromatogram obtained with the referenee solution,
Dry extraet produeed from Ginseng (1523).
Content
Minimum 4.0 per eent of the sum of ginsenosides Rb1, Rb2,
Re, Rd, Re, Rf, Rg1 and Rg2, expressed as ginsenoside Rb1
(C s4H 92 0 23 ; M r 1109) (dried extraet) .
PRODUCnON
The extraet is produeed from the herbal drug by a suitable
proeedure using a hydroalcoholie solvent equivalent in
strength to ethanol (35-90 per eent V/V).
CHARACTERS
Appearance
Light brownish-yellow, hygroseopie powder or briule mass.
IDENTIFICAnON
Thin-layer ehromatography (2.2.27).
Test solution Dissolve 0.15 g of the extraet to be examined
in 10 roL of a 70 per eem V/V solution of methanol R.
Reference solution Dissolve 0.15 g of ginseng dry extract HRS
in 10 mL of a 70 per eent V/V solution of methanol R.
Plate TLC silica gel plate R (5-40 ¡.1m) [or TLC silica gel
plate R (2-10 ¡.1m)].
Mobile phase ethyl acetate R, water R, butanol R
(25 :50:100 VIV/V); allow the phases to separate for 10 min
and use the upper layer.
 IV-200 Ginseng Dry Extraet 2014

Applieation 20 ~L [or 4 ~L) as bands of 10 mm [or 8 mm). water R. Apply 5.0 mL of the solution to be analysed to the
Development Over a path of 10 cm [or 5 cm) in an top of the eartridge. Wash the cartridge with 20 mL of
unsaturated tank. water R followed by 15 mL of a 30 per cent VIV solution of
Drying In airo methanol R. Diseard the eluates after eonfirming that no
ginsenosides are present, otherwise repeat the preparation of
Detection Treat with anisaldehyde solution R and heat at
the solution with another brand of eartridge where no
105-110 cC for 5-10 min; examine in daylight.
ginsenosides are eluted with a 30 per eent V/V solution of
Results See below the sequence of zones present in the methanol R . Elute the cartridge with 20 mL of methanol R;
chromatograms obtained with the reference solution and the colleet the eluate. Under reduced pressure, evaporate the
test solution. Furthermore, other faint zones may be present eluate to dryness. Dissolve the residue in 2.0 mL of
in the chromatograms obtained with the test solution and the methanol R . Filter through a suitable membrane filter
reference solution. (nominal pore size 0.45 ¡.Lm).
Referenee solution (b) Dissolve 3.0 mg of ginsenoside
Top of the pla!e Rb1 CRS in methanol R and dilute to 5.0 mL with the same
solvent.
Reference solution (e) Dissolve 3.0 mg of ginsenoside Rg2 R in
--- - --
methanol R and dilute to 5.0 mL with the same solvent.
A viole! zone (ginsenosides Rgl A viole! zone (ginsenosides Reference solution (d) Dilute 1.0 mL of reference solution
+ Rg2) Rgl + Rg2)
(b) to 2.0 mL with referenee solution (e).
A fain! viole! zone (ginsenoside Rf) A fain! viole! zone (ginsenoside Rf)
Column:
A viole! zone (ginsenoside Re) A viole! zone (ginsenoside Re) - size: 1 = 0.125 m, 0 = 4.6 mm;
- stationary phase: octadeeylsilyl siliea gel for ehromatography R
(5 ¡.Lm);
A viole! zone (ginsenoside Rd) A viole! zone (ginsenoside Rd)
- temperature: 35 oC.
A fain! viole! zone A fain! viole! zone Mobile phase:
A viole! zone (ginsenoside Re) A viole! zone (ginsenoside Re) - mobile phase A: water R adjusted to pH 2 with phosphorie
A fain! violet zone
acid R;
A fain! violet zone
- mobile phase B: acetoninile R1;
--- ---
Time Mobile phase A Mobile phase B
A violet zone (ginsenosides Rbl A viole! zone (ginsenosides (min) (per cent VM (per cen! VM
+ Rb2) Rbl + Rb2)
0·8 80 20
Several unresolved viole! and Several unresolved viole! and
l!reenish zones . greenish zones 8 · 40 80 -7 60 20 -t 40
Reference solution Test solution
40·45 60 -7 40 40 -t 60

TESTS
Loss on drying (2.8.17)
Maximum 7.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Buffer solution Dissolve 3.5 g of disodium hydrogen phosphate
dihydrate R and 7.2 g of potassium dihydrogen phosphate R in
water R and dilute to 1000 mL with the same solvent.
Test solution Dissolve 0.100 g ofthe extract to be examined
in the buffer solution and dilute to 10.0 mL with the buffer
solution. Prepare a ready-to-use sample-preparation cartridge
containing 0.50 g of octadecylsilyl silica gel (45 ~m), using
5 mL of methanol R followed by 20 mL of water R. Apply
5.0 mL of the solution to be analysed to the top of the
cartridge. Wash the cartridge with 20 mL of water R followed
by 15 mL of a 30 per cent VIV solution of methanol R.
Discard the eluates after confirming that no ginsenosides are
present, otherwise repeat the preparation of the solution with
another brand of cartridge where no ginsenosides are eluted
with a 30 per cent VIV solution of methanol R . Elute the
cartridge with 20 mL of methanol R; eollect the eluate. Under
reduced pressure, evaporate the eluate to dryness. Dissolve
the residue in 2.0 mL of methanol R. Filter through a suitable
membrane filter (nominal pore size 0.45 ¡.Lm).
Referenee solution (a) Dissolve 0.100 g of ginseng dry extract
HRS in the buffer solution and dilute to 10.0 mL with the
buffer solution. Prepare a ready-to-use sample-preparation
cartridge eontaining 0.50 g of octadeeylsilyl siliea gel
(45 ¡.Lm), using 5 mL of methanol R followed by 20 mL of
45·47 40 -7 O 60 -7 100
Flow rate 1.0 mUmin.
Detection Speetrophotometer at 203 nm.
Infection 20 ¡.LL.
Elution order Ginsenoside Rg1, ginsenoside Re,
ginsenoside Rf, ginsenoside Rb 1, ginsenoside Rg2,
ginsenoside Re, ginsenoside Rb2, ginsenoside Rd; depending
on the operating eonditions and the state of the eolumn,
ginsenoside Rb1 may elute before or after ginsenoside Rg2.
Identification of peaks Use the chromatogram supplied with
ginseng dry extraet HRS and the ehromatogram obtained with
reference solution (a) to identify the peaks due to
ginsenosides Rg1, Re, Rf, Re, Rb2 and Rd; use the
ehromatogram obtained with referenee solution (b) to
identify the peak due to ginsenoside Rb1; use the
ehromatogram obtained with reference solution (e) to identify
the peak due to ginsenoside Rg2.
Relative retention With referenee to ginsenoside Rb1
(retention time = about 33 min):
ginsenoside Rg1 = about 0.53; ginsenoside Re = about 0.54;
ginsenoside Rf = about 0.88; ginsenoside Rg2 = about 0.98;
ginsenoside Re = about 1.04; ginsenoside Rb2 = about 1.08;
ginsenoside Rd = about 1.17.
System suitability Reference solution (d):
- resolution: minimum 1.5 between the peaks due to
ginsenosides Rg2 and Rb 1.
Calculate the pereentage content of the sum of ginsenosides
Rb1, Rb2, Re, Rd, Re, Rf, Rg1 and Rg2, expressed as
ginsenoside Rb1, using the following expression:
 2014
Al x m2 X p x 0.8
A 2 x mI
sum of the areas of the peaks due to ginsenosides
Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2 in the
chromatogram obtained with the test solution;
area of the peak due to ginsenoside Rb1 in the
chromatogram obtained with reference solution (b);
mass of the extract to be examined used to prepare
mI
the test solution, in grams;
mass of ginsenoside Rb1 CRS used to prepare
reference solution (b), in grams;
p percentage content of ginsenoside Rb1 in gimenoside
Rb1 CRS.
____________________________________________ PhE~
Goldenrod ***
*** ***
(Ph Eur monograph 1892) ***
____________________________________________
~E~
DEFINITION
Whole or cut, dried, fiowering aerial parts of Solidago gigantea
Ait or Solidago canadensis L., their varieties or hybrids and/or
mixtures of these.
Content
Minimum 2.5 per cent of fiavonoids, expressed as hyperoside
(C 21 H 20 0 ' 2; M r 464.4) (dried drug).
IDENTIFICATION
A. The stems are greenish-yellow or greenish-brown, partly
tinted reddish, roundish, more or less conspicuously grooved,
glabrous and smooth in the lower part, slightly or densely
pubescent in the upper parto They are solid with a whitish
pith.
The leaves are green, sessile, lanceolate, with a serrate
margin, 8-12 cm long and about 1-3 cm wide, the upper
surface is green and more or less glabrous, the lower surface
is greyish-green and pubescent, especially on the veins.
The infiorescence consists of a number of unilateral, curved
racemes which together form a pyramidal panicJe at the end
of the stems.
Each capitulum has an involucre composed oE linear-
lanceo late, imbricated yellowish-green bracts, surrounding a
single row of yellow ligulate fiorets about the same length as
the involucre; yellow, radially arranged tubular fiorets, as
long as, or longer, than the ligulate fiorets; a brownish
inferior ovary surmounted by a white pappus of silky hairs.
B. Reduce to a powder (355) (2.9.12) . The powder is
greyish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows pappus bristles and
their fragments, consisting of multiseriate trichomes
composed of elongated cells with the tips free from the
surface and forming pointed projections over the entire
length; fragments of the leaf mesophyll with vascular bundles
accompanied by secretory cells; fragments of the leaf
epidermis with sinuous to wavy-walled cells and stomata of
the anomocytic type (2.8.3); uniseriate covering trichomes
with up to 5 or 6 cells, sorne whip-like with a thicker-walled
Goldenrod IV-20 1
terminal cell; fragments of the style with long, slender
papillae; fragments of the stem with reticulate and spiral
ves seIs; pollen grains, with 3 germinal pores and a spiny
exine; numerous whisk-shaped hairs, a few isolated twin-hairs
from the ovary, absence of multicellular trichomes with a
terminal cell bent at a right angle.
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 0.75 g ofthe powdered drug (355) (2.9.12)
add 5 mL of methanol R and boil in a water-bath under a
refiux condenser for 10 mino Cool and filter.
Reference solution Dissolve 1.0 mg of chlorogenic acid R,
2.5 mg of quercitrin R and 2.5 mg of rutin R in 10 mL of
methanol R.
Plate TLC si/ica gel plate R .
Mobile phase anhydrous formic acid R, water R , methyl ethyl
ketone R, ethyl acetate R (6:6:18:30 VIVIVIV).
Application 20 ilL of the test solution and 1O ~lL of the
reference solution as bands.
Development Over a path of 10 cm.
Drying At 100-105 oc.
Detection Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R. AlIow to stand for
30 mino Examine in ultraviolet light at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
A bluish-green f1uorescent zone
Quercitrin: a yellowish-brown A faint to intense yellowish-brown
f1uorescent zone f1uorescent zone (quercitrin)
A more or less intense yellowish
brown zone
Chlorogenic acid: a Iight blue A Iight blue zone and/ or a yellow
f1uorescent zone f1uorescent zone (chlorogenic
acid)
Rutin: an orange f1uorescent zone A faint to intense yellowish-brown
f1uorescent zonejrutiIli
Refereoce solutioo Test solutioo
TESTS
Foreign matter (2.8.2)
Maxirnum 5 per cent of brownish parts and maximum
2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 10 per cent, determined on 0.500 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 7.0 per cent.
Ash insoluble in hydroch10ric acid (2.8. 1)
Maximum 1.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed fiask, introduce
0.200 g of the powdered drug (250) (2.9.12), add 1 mL of a
5 giL solution of hexamethylenetetramine R, 20 mL of
acetone R and 2 mL of hydrochloric acid R1 . Boil the mixture
under a refiux condenser for 30 mino Filter the liquid
through a small plug of absorbent cotton into a 100 mL
 IV-202 European Goldenrod
flask. Add the absorbent cotton to the residue in the round-
bottomed flask, extract with 2 quantities, each of 20 mL of
acetone R, each time boiling under a reflux condenser for
10 mino Allow to cool. Filter the combined acetone extracts
through a filter paper into a volumetric flask. Rinse the flask
and the filter paper and dilute to 100.0 mL with acetone R.
Introduce 20.0 mL of the solution into a separating funnel,
add 20 mL of water R and shake the mixture with 1 quantity
of 15 mL and then 3 quantities, each of 10 mL, of ethyl
acetate R. Combine the ethyl acetate extracts in a separating
funnel, wash twice with 50 mL of water R and filter the
extracts over 10 g of anhydrous sodium sulfate R into a
volumetric flask. Dilute to 50.0 mL with ethyl acetate R,
rinsing the separating funnel and the sodium sulfate.
Test solution To 10.0 mL ofthe stock solution add 1.0 mL
of aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R.
Compensation solution Dilute 10.0 mL of the stock solution
to 25 .0 mL with a 5 per cent VIV solution of glacial acetic
acid R in methanol R .
Measure the absorbance of the test solution (2.2.25) at
425 nm after 30 min by comparison with the compensation
solution.
Calculate the percentage content of flavonoids, expressed as
hyperoside, from the expression:
A x 1.25
m others are uniseriate, multicellular (up to about 10 cells),
i.e. taking the value of the specific absorbance of hyperoside
to be 500.
A absorbance measured at 425 nm,
In = mass of the drug to be examined, in grams.
____________________________________________ ~ E~
***
European Goldenrod
** *
******
(Ph Eur Inonograph 1893)
~E~ ____________________________________________
DEFINITION
Whole or fragmented, dried, flowering aerial parts of Solidago
virgaurea L.
Content
Minimum 0.5 per cent and maximum 1.5 per cent of
flavonoids, expressed as hyperoside (C21H20012; M r 464.4)
(dried drug).
IDENTIFICATION
A. The stem is cylindrical, striated, the lower part often
reddish-violet, sometimes entirely glabrous or pubescent with
short, bent, apically directed hairs. The basal leaves are
obovate or oblanceolate, with a serrate margin, and taper at
the base into a long, winged petiole; the cauline leaves are
alternate, smaller than the basal lea ves and more elliptical in
outline, with an entire or slightly toothed marginó they are
sessile or with onIy a short petiole. Both surfaces of the leaves
are glabrous or only slightly pubescent with a prominent
reticulate venation on the lower surface. The capitula form a
tightly packed paniele. At the base of the pedicels there are 2
small, linear bracts with scarious margins. The involucre
consists of 2-4 rows of loosely arranged, imbricate bracts,
each bract greenish-yellow with a smooth and shiny inner
surface, the outer surface hairy or glabrous, with a scarious
2014
margino Each capitulum contains 6-12 widely separated
female ray florets, about twice as long as the bracts, and
about 10-30 hermaphrodite, tubular florets. All florets are
yellow. The brown, inferior ovary tapers towards the base
and has a ribbed surface, covered with scattered hairs; it is
surmounted by a whitish pappus composed of smooth or
rough, bristly hairs.
B. Microscopic examination (2.8.23) . The powder is light
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figures 1893.-1 and 1893.-2): fragments ofthe
upper epidermis of the leaf in surface view [B, H, M],
covered by a distinctly striated cutiele, composed of
polygonal cells with straight, beaded, thickened walls [Ba,
Ma], uniseriate, multicellular covering trichomes [Ha] or
rounded, thick-walled, covering trichome scars with a pitted
lumen [Mb], and a few anomocytic stomata ( 2.8.3) [Bb]
sometimes accompanied by underlying palisade parenchyma
[Bc]; fragments of the lower epidermis of the leaf in surface
view [A, K, N] covered by a slightly striated cutiele
composed of cells with sinuous walls in the area of the
lamina [Aa] or with more rigid walls near the veins (N),
numerous anomocytic stomata (2.8.3) [Ab], occasional
glandular trichomes with a unicellular stalk and a unicellular
head [Ka, Na], covering trichomes sorne of which are
pennant-like [Ac, F], uniseriate, multicellular, with 1-3 thin-
walled basal cells [Fa], a flagella-like distal cell [Fb], and an
enlarged, more or les s rounded cell [Fc] between them,
with thick, finely wrinkled walls and a rigid conical distal cell
in side view [E]; rare fragments from the ovary [G] bearing
paired, covering trichomes with a distinctly pitted central wall
and a bifid apex in surface view [Ga] or in side view [Gb];
vascular tissue from the stems [L] composed ofvessels [La]
and groups of fibres [Lb]; fragments of the epidermis of the
petals with a striated cutiele, through which run fine spiral
ves seIs [S], and bearing biseriate glandular trichomes in side
view [P]; spherical pollen grains, with 3 germinal pores and a
spiny exine UJ; abundant pappus hairs and their fragments
[C, D], multiseriate with the marginal cells overlapping
outwards; fragments ofparenchyma [Q], sorne showing cells
containing small, isolated eluster crystals of calcium oxalate
[Qa]; fragments of bracts [R] with a finely striated cutiele,
polygonal cells [Ra], bearing pennant-Iike covering trichomes
[Rb] and whose margin bears uniseriate, multicellular
covering trichomes [Rc] .
2014
Figure 1893.-1. - Illustration for identification test B of
powdered herbal drug of European goldenrod
Ka
Figure 1893.-2. - Illustration for identification test B of
powdered herbal drug of European goldenrod
European Goldenrod IV-203
C. Thin-layer chroma1Ography (2.2.27) as described in the
test for Solidago gigantea Ait. and Solidago canadensis L.
Results See below the sequence of the zones present in the
chroma1Ograms obtained with the reference solution and the
test solution. Furthermore, other ftuorescent zones may be
present in the chromatogram obtained with the test solution.
Top of the plate
A light blue fluorescent zone
Quercitrin: an orange fluorescent
zone
- -- ---
Chlorogenic acid: a light blue A light blue fluorescent zone
fluorescent zone (chlorogenic acid)
Rutin: an orange fluorescent zone An orange fluorescent zone (rutin)
--- - --
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of brown coloured matter and
maximum 5 per cent of other foreign matter.
Solidago gigantea Ait. and Solidago canadensis L
Thin-layer chroma1Ography (2.2.27) .
Test solution To 0.75 g ofthe powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R and heat on a water-bath
under a reftux condenser for 10 mino Cool and filter.
Reference solution Dissolve 1.0 mg of chlorogenic acid R,
2.5 mg of quercitnn R and 2.5 mg of ruán R in 10 mL of
methanol R.
Plate TLC silica gel plate R .
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (6:6:18:30 VIVIV/V) .
Application 20 ¡.¡L as bands .
Development Over a path of 10 cm.
Drying In air.
Detection Treat the plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R and then
with a 50 gIL solution of macrogol 400 R in methanol R.
Examine in ultraviolet light at 365 nm after 30 mino
Results The chroma1Ogram obtained with the test solution
shows no strong orange ftuorescent zone similar in position
10 the zone of quercitrin in the chromatogram obtained with
the reference solution.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9. 12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4. 16)
Maximum 8.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed ftask, place
0.200 g ofthe powdered herbal drug (355) (2.9.12) , add
1 mL of a 5 gIL solution of hexamethylenetetramine R, 20 mL
of acetone R and 2 mL of hydrochlonc acid R1 . Boil the
mixture in a water-bath under a reftux condenser for 30 mino
Filter the liquid through a small plug of absorbent cotton
into a 100 mL ftask. Add the absorbent cotton 10 the residue
in the round-bottomed ftask and extract with 2 quantities,
each of 20 mL, of acetone R, each time boiling under a reftux
 IV-204 Goldenseal Root 2014

condenser for 10 mino Allow to cool. Filter the combined
acetone extracts through filter paper, dilute to 100.0 mL with
acetone R, rinsing the volumetric f1ask and the filter paper
with acetone. Introduce 20.0 mL of the solution into a
suitable separating funnel, add 20 mL of water R and shake
the mixture with 1 quantity of 15 mL and then with
3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acetate extracts in a separating funnel, wash twice with
50 mL of water R and filter the extracts over 10 g of
anhydrous sodium sulfate R into a volumetric f1ask. Dilute to
50.0 mL with ethyl acetate R, rinsing the separating funnel
and the sodium sulfate.
Test solution To 10.0 mL of the stock solution add 1.0 mL
of aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R.
Compensation liquid Dilute 10.0 mL of the stock solution to
25 .0 mL with a 5 per cent VIV solution of glacial acetic
acid R in methanol R.
After 30 min, measure the absorbance (2.2.25) ofthe test
solution at 425 nm by comparison with the compensation
liquido
Calculate the percentage content of f1avonoids, expressed as
hyperoside, using the following expression:
A x 1.25
m
i.e. taking the specific absorbance of hyperoside to be 500.
A measured absorbance at 425 nm;
m = mass of the herbal drug to be examined, in grams.
____________________________________________ PhE~
infrequent groups of thin-walled, pitted fibres [H] , usually
found associated with the vessels; numerous ovo id or
spherical, orange-brown granular masses. Examine under a
microscope using a 50 per cent V/V solution of glycerol R .
The powder shows abundam starch granules [C] , mostly
simple but sometimes compound with up to 4 components;
the granules are small, spherical or ovoid, up to about 10 Jlm
in di ame ter, occasionally with a small, rounded or slit-shaped
hilum.
C. Thin-layer chromatography (2.2.21).
Test solution To 250 mg of powdered drug (180) (2.9.12)
add 4 mL of a mixture of 20 volumes of water R and
80 volumes of methanol R. Sonicate for 10 min and filter.
Wash the residue with 2 quantities, each of 2 mL, of
methanol R . Combine the solutions and dilute to 20 mL with
methanol R .
R eference solution Immediately before use, dissolve 5 mg of
hydrastine hydrochloride R and 5 mg of berberine chloride R in
20 mL of methanol R .
Plate TLC silica gel plate R (5-40 ~lm) [or TLC silica gel
plateR (2-10 Jlm)J.
Mobz1e phase anhydrous fonnic acid R , water R, ethyl acera te R
(10:10:80 VIV/V).
Application 20 JlL [or 2 ¡lLJ as bands.
Development Over a path of 15 cm [or 6 cmJ.
Drying In air.
Detection Examine in ultraviolet light at 365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the

test solution. Furthermore, other f1uorescent zones may be

present in the chromatogram obtained with the test solution.

Top of the plate

Goldenseal Root ***
*** *** --- ---
(Goldenseal Rhizome, Ph Eur monograph 1831) *** Berberine: a bright yellow A bright yellow fl uorescent zone
~E~ ____________________________________________ flu orescent zone (berberine)
Hydrastine: a deep blue A deep blue fluorescent zone
DEFINITION fluorescent zone (hydrastine)
Whole or cut, dried rhizome and root of Hydrastis canadensis --- ---
L. A bright Iight blue fluorescent
Content: zone (hydrastinine)
hydrastine (C 2IH 21 N0 6 ; M r 383.4): minimum A deep blue fluorescent zone
2.5 per cent (dried drug); Reference solution Test solution
berberine (C2oHISN04; M r 336.4): minimum 3.0 per cent
(dried drug).
IDENTIFICATION TESTS
A. The rhizome is tortuous and knotty, about 5 cm long and Loss on drying (2.2.32)
5-10 mm thick. The surface is yellowish or brownish-grey, Maximum 10.0 per cent, determined on 1.000 g of the
irregularly wrinkled, and bears the remains of numerous powdered drug (180) (2.9.12) by drying in an oven at
slender, wiry roots; stem bases and scale leaves occur on the 105 oC for 2 h.
upp er surface. The fracture is short and resinous. Total ash (2.4.16)
The transversely cut surface is yellowish-brown and shows a M aximum 8.0 per cent.
fairly wide bark, a ring of 12-20 widely separated xylem
Ash insoluble in hydrochIoric acid (2.8.1)
bundles and a large, central pith.
M aximum 4.0 per cent.
B. Reduce to a powder (180) (2.9.12). The powder is
greenish-yellow. Examine under a microscope using chloral ASSAY
hydrate solution R . The powder shows the following diagnostic Liquid chromatography (2.2.29).
characters (Figure 1831.-1 ): abundant thin-walled fragrnents Test solution To 1.000 g of the powdered drug (355)
of parenchyma [A, G, KJ; occasional fragrnents of yellowish- (2.9.12) in a 100 mL round-bottomed f1ask, add 50 mL of a
brown cork from the rhizome and roots, in surface view m 1 per cent V/V solution of concentrated ammonia R in ethanol
or in transverse section [FJ; groups of small ves seIs with (96 per cent) R and boil the mixture under a reflux condenser
conspicuous perforations in the oblique end walls [L] and for 30 mino AlIow to cool to room temperature and filter the
with simple or bordered, slit-shaped pits [B, D, EJ; liquid through a plug of absorbent cotton into a f1ask.
2014 Hamamelis Leaf IV-205

Using the retention times determined from the

chromatogram obtained with the reference solution, locate in
the chromatogram obtained with the test solution the
components of the referenee solution.
o o O
Calculate the percentage content of each alkaloid (hydrastine
Ql and berberine) using the following expression:
CP
@¡ e
A
<l>
area of the peak due to hydrastine or berberine in the
chromatogram obtained with the test solution;
area of the peak due to hydrastine or berberine in the
ehromatogram obtained with the reference solution;
F mI mas s of the herbal drug to be examined used to
prepare the test solution, in grams;
mas s of hydrastine hydrochloride or berberine
ehloride used to prepare the referenee solution, in
grams;
p percentage content of hydrastine in hydrastine
hydroehloride CRS or berberine in berberine
ehloride CRS.
H
_ _ __ _ _ _ _ _ __ _ _ __ __ _ _ _ _ _ Ph fUf

50 IJm

~
:=::S~L
,,:,\\ .... ,'
.' ,
"\'"

Figure 1831.-1.- Illustration for identification test B of

".
:... ~'.,.'

powdered herbal drug ofgoldenseal rhizome

Add the plug of absorbent cotton to the residue in the

round-bottomed fiask and repeat the extraetion with a further
2 quantities, eaeh of 30 mL, of a 1 per eent V/V solution of
concentrated ammonia R in ethanol (96 per cent) R, each time
boiling under a refiux condenser for 10 min and filtering
through a plug of absorbent cotton in the same fiask as
previously. Filter the combined filtrates through a filter paper
into a 250 mL round-bottomed fiask, and rinse the fiask and
the filter with 20 mL of a 1 per cent V/V solution of
concentrated ammonia R in ethanol (96 per cent) R. Evaporate
the filtrate to dryness in vacuo in a water-bath at 55 oc.
Dissolve the residue in 50.0 mL of the mobile phase. Dilute
10.0 mL ofthis solution to 50.0 mL with the mobile phase.
Reference solution Immediately before use, dissolve 10.0 mg
of hydrastine hydrochloride CRS and 10.0 mg of berberine
ehloride CRS in methanol R and dilute to 100.0 mL with the
same solvento
Column:
- size: l =: 0.125 m, 0 =: 4 mm;
- stationary phase: end-capped octadeeylsilyl silica gel for
chromatography R (5 ~m).
Mobzle phase Dissolve 9.93 g of potassium dihydrogen
phosphate R in 730 mL of water R, add 270 mL of
acetonitrile R and mix.
Flow rate 1.2 mLlmin.
Detection Speetrophotometer at 235 nm.
Injection 1O ~L.
System suitabzlity Reference solution:
- elution order: order indicated in the eomposition of the
reference solution; record the retention times of these
substances;
- resolution: minimum 1.5 between the peaks due to
hydrastine and berberine .
Hamamelis Leaf ****
** *
(Ph Eur monograph 0909) **** *
~f~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __
DEFINITION
Whole or cut, dried leaf of Hamamelis virginiana L.
Content
Minimum 3 per cent of tannins, expressed as pyrogallol
(C 6 H 6 0 3 ; M r 126.1) (dried drug).
IDENTIFICATION
A. The leaf is green or greenish-brown, often broken,
crumpled and compressed into more or less compaet mas ses.
The lamina is broadly ovate or obovate; the base is oblique
and asymmetric and the apex is acute or, rarely, obtuse.
The margins of the lamina are roughly crenate or dentate.
The venation is pinnate and prominent on the abaxial
surface. Usually, 4-6 pairs of secondary veins are attached to
the main vein, emerging at an acute angle and curving gently
to the marginal points where there are fine veins often at
right angles to the secondary veins.
B. Reduce to a powder (355) (2.9.12). The powder is
brownish-green. Examine under a microscope using ehloral
hydrate solution R . The powder shows the following diagnostic
eharacters (Figure 0909.-1): fragments of adaxial epidermis
n,
with wavy antic1inal walls, in surface view [C, often
accompanied by small, cylindrical cells of the palisade
parenchyma, in surface view ITa], or elongated, in transverse
section [F]; fragments of abaxial epidermis with sto mata
mainly paracytic (2.8.3) , in surface view [B], which may be
aeeompanied by irregular-shaped cells of spongy mesophyll
[K, L]; star-shaped covering trichomes, either entire or
broken [A, D , M], composed of 4-12 unicellular branches
 IV-206 Hawthorn Berries 2014

that are united by their bases, elongated, conical and curved,

usually up to 250 ¡lm long, thick-walled and with a clearly
visible lumen whose contents are often brown; fibres are
lignified and thick-walled, isolated or in groups, and
accompanied by a sheath of prismatic ca1cium oxalate crystals
[N, P]; sclereids, frequently enlarged at 1 or both ends,
150-180 ¡lm long, whole or fragmented [H] ; fragments of
annular or spiral ves seIs [E] ; isolated prisms of ca1cium
oxalate [G].
C. Thin-layer chromatography (2.2.27).
Test solution To 1.0 g ofthe powdered drug (355) (2.9.12)
add 10 mL of ethanol (60 per cent V!V) R, shake for 15 min
and filter.
R eference solution (a) Dissolve 30 mg of tannic acid R in
5 mL of ethanol (60 per cent VIV) R.
R eference solution (b) Dissolve 5 mg of gallic acid R in 5 mL
of ethanol (60 per cent V/V) R .
Plate TLC silica gel G plate R .
Mobile phase anhydrous formic acid R, water R, ethyl
formate R (10:10:80 VIV/V).
Application 10 ¡lL, as bands.
Development Over a path of 10 cm.
Drying At 100-105 oC for 10 min, then allow to coo!.
D etection Spray with ferric chloride solution R2 until bluish-
grey zones (phenolic compounds) appear.
R esults The chromatogram obtained with the test solution
shows in its lower third a principal zone similar in position to
the principal zone in the chromatogram obtained with
reference solution (a) and, in its upper part, a narrow zone
similar in position to the principal zone in the chromatogram Figure 0909.-1.- Illustration for identification test B of
powdered herbal drug of hamamelis leaf
obtained with reference solution (b); the chromatogram
obtained with the test solution shows, in addition, several
slightly coloured zones in the central part.
Content
TESTS Minimum 0.06 per cent of procyanidins, expressed as
Foreign matter (2.8.2) cyanidin chIoride (C 1s H Il CI0 6; Me 322.7) (dried drug).
Maximum 7 per cent of stems and maximum 2 per cent of
other foreign matter, determined on 50 g. IDENTIFICATION
A. The false fruit of C. monogyna is obovate or globular,
Loss on drying (2.2.32)
generally 6-10 mm long and 4-8 mm wide, reddish-brown or
Maximum 10.0 per cent, determined on 2.000 g of
dark red. The surface is pitted or, more rarely, reticulated.
powdered drug (355) (2.9.12) by drying in an oven at
The upper end of the fruit is crowned by the remains of 5
105 oC for 4 h.
refiexed sepals surrounding a small, sunken disc with a
Total ash (2.4. 16) shallow, raised rimo The remains of the style occur in the
Maximum 7.0 per cent. centre of the disc with tufts of stiff, colourless hairs at the
Ash insoluble in hydrochloric acid (2.8.1) base. At the lower end of the fruit is a short length of pedicel
Maximum 2.0 per cent. or, more frequently, a small, pale, circular scar where the
pedicel was attached. The receptacle is fieshy and encloses a
ASSAY
yellowish-brown, ovoid fruit with a hard, thick wall
Carry out the determination of tannins in herbal drugs
containing a single, elongated, pale brown, smooth and shiny
(2.8.14). Use 0.750 g ofthe powdered drug (180) (2.9.12).
seed.
____________________________________________ ~Em

The false fruit of C. laevigata is up to 13 mm long.

It contains 2-3 stony fruits, ventrally fiattened, with short
hairs at the topo Frequently, in the centre of the disc of the
false fruit occur the remains of the 2 styles.
Hawthorn Berries
(Ph Eur monograph 1220)
Ph Eur ____________________________________________

DEFINITION
Dried false fruits of Crataegus monogyna Jacq. (IJndm.) or C.
laevigata (Po ir.) De. (syn. C. oxyacantha L.) or their hybrids
or a mixture of these false fruits.
2014
Figure 1220.-1. - Illustratian far identificatian test B af
pawdered herbal drug af hawtharn berries
B. Microscopic examination (2.8.23). The powder is greyish-
red. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1220.-1 ): covering trichomes [F] from
inside the disc that are long, unicellular, frequently bent,
tapering to a point, with much thickened and lignified walls;
fragments of the red outer layer of the receptacle, in surface
view [GJ; fragments of the inner layers of the receptacle [A],
sorne cells containing cluster crystals [Aa] or prisms [Ab] of
calcium oxalate; occasional fragments IT, K] including groups
of sclereids [Ka] and vascular bundles ITa, Kb] associated
with rows of cells containing prisms of calcium oxalate Ub,
Kc]; fragments of the pericarp [B] consisting of parenchyma
including sorne ceJIs containing cluster crystals of calcium
oxalate [Ba] and groups of sclereids of various sizes with
numerous pits [Bb]; thick-walled sclereids [E, H], sorne
channelled (E), sorne with conspicuously branched channels
(H); a few fragments of the testa [C] having an outer layer
composed of hexagonal, mucilaginous cells [Ca] beneath
which is a yeJIowish-brown pigment layer containing
numerous prisms of calcium oxalate [Cb]; parenchyma ofthe
endosperm and cotyledons consisting of cells containing
aleurone grains and globules of fixed oil [D] .
e. Thin-Iayer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R and heat on a water bath
at 65 oC for 5 min, shaking frequently. Allow to cool to
room temperature and filter. Dilute the filtrate to 10 mL
with methanol R.
Reference solution Dissolve 2 mg of chlorogenic acid R, 2 mg
of caffeic acid R, 5 mg of hyperoside R and 5 mg of rutin R in
20 mL of methanol R.
Plate TLC si/ica gel plate R.
Hawthorn Berries IV-207
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethy l acetate R (10:10:30 :50 VIVIV/V).
Application 30 ¡.¡L of the test solution and 10 ¡.¡L of the
reference solution, as bands.
Development Over a path of 15 cm.
Drying At 100-105 De.
Detection Spray whilst hot with a 10 giL solution of
diphenylboric acid aminoethyl ester R in methanol R;
subsequently spray with a 50 giL solution of macrogol 400 R
in methanol R; allow to dry in air for 30 min and examine in
ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower half, in order of increasing R p
values, a yellowish-brown fiuorescent zone (rutin), a light
blue fiuorescent zone (chlorogenic acid) and a yellowish-
brown fiuorescent zone (hyperoside); in the upper third
appears a light blue fiuorescent zone (caffeic acid).
The chromatogram obtained with the test solution shows 3
zones similar in position and fiuorescence to the zones due to
chlorogenic acid, hyperoside and caffeic acid in the
chromatogram obtained with the reference solution, and 3
weak reddish fiuorescent zones, one corresponding to the
zone due to rutin in the chromatogram obtained with the
reference solution and both of the others located aboye the
zone due to hyperoside; below and aboye the zone due to
caffeic acid sorne light blue zones appear.
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of deteriorated false fruit and
maximum 2 per cent of other foreign matter. It does not
contain false fruits of other Crataegus species (C. nigra Waldst.
et Kit., C. pentagyna Waldst. et Kit. ex Willd. and C. azarolus
L.), which are characterised by the presence of more than 3
hard stones.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
ASSAY
To 2.50 g ofthe powdered herbal drug (355) (2.9.12) add
30 mL of ethanol (70 per cent V/V) R. Heat under a refiux
condenser for 30 min and fiiter. Wash the residue with
10.0 mL of ethanol (70 per cent VIV) R. Add to the filtrate
15 .0 mL of hydrochlon'c acid R1 and 10.0 mL of water R .
Heat under a refiux condenser for 80 mino Allow to cool,
filter and wash the residue with ethanol (70 p er cene V/V) R
until the filtrate is colourless. Dilute the filtrate to 250 .0 mL
with ethanol (70 per cent V/V) R. Evaporate 50.0 mL of this
solution in a round-bottomed fiask to about 3 mL and
transfer to a separating funnel. Rinse the round-bottomed
fiask sequentially with 10 mL and 5 mL of water R and
transfer to the separating funnel. Shake the combined
solution with 3 quantities, each of 15 mL, of butanol R.
Combine the organic layers and dilute to 100.0 mL with
butanol R .
Measure the absorbance (2.2.25) ofthe solution at 555 nm.
Calculate the percentage content of procyanidins, expressed
as cyanidin chloride, using the following express ion:
A x 500
1200 x m
IV-208 Hawthorn Leaf and Flower 2014

i.e. taking the specific absorbance of cyanidin chloride to be lignified sc1erenchymarous fibres with narrow lumina;
1200. numerous spherical to elliptical or triangular pollen grains up
A absorbance at 555 nm; ro 45 flm in diameter, with 3 germinal pores and a faintly
m = mass of the substance ro be examined, in grams. granular exine.
_ _ _ __ _ __ _ __ _ __ _ __ _ _ ____ ~E~ C. Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g ofthe powdered drug (355) (2.9.12)
add 10 mL of methanol R and heat in a water-bath at 65 oC
under a reflux condenser for 5 mino Cool and filter.

Hawthorn Leaf and Flower *** Reference solution Dissolve 1.0 mg of chlorogenic acid R and
*** *** 2.5 mg of hyperoside R in 10 mL of methanol R.
(Ph Bur monograph 1432) *** Plate TLC silica gel plate R .
Preparations Mobile phase anhydrous formic acid R, water R, methyl ethyl
Hawthorn Leaf and Flower Dry Extract ketone R, ethyl acetate R (10: 10:30:50 VIVIV/V) .
Quantified Hawthorn Leaf and Flower Liquid Extract Application 20 flL as bands.
~E~ _ _ _ __ _ __ _ __ _ __ _ _ __ _ _ ___ Development Over a path of 15 cm.
DEFINITlON Drying At 100-105 cC.
Whole or cut, dried flower-bearing branches of Crataegus Detection Spray the still-warm plate with a 10 giL solution
monogyna Jacq. (Lindm.), C. laevigata (Poir.) DC. of diphenylboric acid aminoethyl ester R in methanol R, then
(synonyms: C. oxyacanthoides Thuill.; C. oxyacantha auct.) or spray with a 50 gIL solution of macrogol 400 R in methanol R;
their hybrids or, more rarely, other European Crataegus allow to dry in air for about 30 min and examine in
species inc1uding C. pentagyna Waldst. et Kit. ex Willd., C. ultraviolet light at 365 nm.
nigra Waldst. et Kit. and C. azarolus L. Results See below the sequence of zones present in the
Content chromarograms obtained with rhe reference solution and the
Minimum 1.5 per cent of total flavonoids, expressed as test solution. Furthermore, other fluorescent zones may be
hyperoside (C21H 20012; M r 464.4) (dried drug). present in the chromarogram obtained with the test solution.

IDENTIFICATlON
A. The stems are dark brown, woody, 1-2.5 mm in diameter, Top oC the plate
bearing alterna te, petiolate lea ves with small, often deciduous --- ---
stipules and corymbs of numerous small white flowers.
A yellowish·green f1uorescent zone
The leaves are more or les s deeply lobed with slight1y serrate (vitexin)
or almost entire margins; those of C. laevigata are pinnately
Hyperoside: a yellowish-orange A yellowish·orange f1uorescent
lobed or pinnatifid with 3, 5 or 7 obtuse lobes, those of fluorescent zone zone (hyperoside)
C. monogyna pinnatisect with 3 or 5 acute lobes; the adaxial Chlorogenic acid: a light blue A light blue fluorescent zone
surface is dark green or brownish-green, the abaxial surface is fluorescent zone (chlorogenic acid)
lighrer greyish-green and shows a prominent, dense, A yellowish·green fluorescent zone
(vitexin-2"-rhamnoside)
reticulate venation. The leaves of C. laevigata, C. monogyna
and C. pentagyna are glabrous or bear only isolated --- ---
trichomes, those of C. azarolus and C. nigra are densely ReCerence solution Test solution
pubescent. The flowers have a brownish-green tubular calyx
composed of 5 free, reflexed sepals, a corolla composed of 5
free, yellowish-white or brownish, rounded or broadly ovate TESTS
and shortly unguiculate petals and numerous stamens. Foreign matter (2.8.2)
The ovary is fused to the calyx and consisrs of 1-5 carpels, Maximum 8 per cent of lignified branches with a diameter
each with a long style and containing a single ovule; in C. greater than 2.5 mm and maximum 2 per cent of other
monogyna there is 1 carpel, in C. laevigata 2 or 3, in C. foreign matter.
azarolus 2 or 3, or sometimes on1y 1, in C. pentagyna 5 or,
Loss on drying (2.2.32)
rarely,4 .
Maximum 10.0 per cent, determined on 1.000 g of
B. Reduce ro a powder (355) (2.9.12). The powder is powdered drug (355) (2.9. 12) by drying in an oven at
yellowish-green. Examine under a microscope using chloral 105 oC for 2 h .
hydrate solution R. The powder shows the following diagnostic
Total ash (2.4.16)
characrers: unicellular covering trichomes, usually with a
Maximum 10.0 per cent.
thick wall and wide lumen, almost straight or slight1y curved,
pitted at the base; fragments of leaf epidermis with cells ASSAY
which have sinuous or polygonal anticlinal walls and with Stock solution Into a 200 mL flask introduce 0.400 g of the
large anomocytic stomata (2.8.3) surrounded by 4-7 powdered drug (250) (2.9.12) and 40 mL of ethanol
subsidiary cells; parenchymatous cells of the mesophyll (60 per cent VIV) R . Heat in a water-bath at 60 oC for
containing calcium oxalate c1usters, usually measuring 10 min, shaking frequently. Allow to cool and filter through a
10-20 flm, those associared with the veins containing groups plug of absorbent cotton into a 100 mL volumetric ftask.
of small prism crystals; fragments of petals showing rounded Transfer the absorbent cotton with the drug residue back to
polygonal epidermal cells, strongly papillose, with thick walls, the 200 mL flask, add 40 mL of ethanol (60 per cent V/V) R
the cutic1e of which c1earIy shows wavy striations; fragments and heat again in a water-bath at 60 oC for 10 min, shaking
of anthers showing endothecium wirh an arched and frequent1y. Allow to cool and filter into the same 100 mL
regularIy thickened margin; fragments of stems containing volumetric flask. Rinse the 200 mL flask with a further
collenchymatous cells, bordered pitted ves seis and groups of quamity of ethanol (60 per cent V/V) R, filter and transfer ro
 2014 Hawthorn Preparations IV-209

the same 100 mL volumetric ftask. Dilute to 100.0 mL with Test solution Suspend 0.2 g of the extract to be examined in
ethanol (60 per cent VIV) R and filter. 20 mL of ethanol (70 per cent V/V) R and filter.
Test solution Introduce 5.0 mL of the stock solution into a R eference solution Dissolve 1 mg of chlorogenic acid R , 2.5 mg
round-bottomed ftask and evaporate to dryness under of hyperoside R and 2.5 mg of rutin R in 10 mL of
reduced pressure. Take up the residue with 8 mL of a methanol R.
mixture of 10 volumes of methanol R and 100 volumes of Plate TLC silica gel plate R.
anhydrous acetic acid R and transfer to a 25 mL volumetrie
Mobile phase anhydrous formic acid R, water R, 111ethyl ethyl
ftask. Rinse the round-bottomed ftask with 3 mL of a
ketone R, ethyl acetate R (10:10:30:50 VIVIV/V).
mixture of 10 volumes of methanol R and 100 volumes of
anhydrous acetic acid R and transfer to the same 25 mL Application 20 IlL of the test solution and 1O ~lL of the
volumetric ftask. Add 10.0 mL of a solution eontaining referenee solution, as bands.
25.0 gIL of boric acid R and 20.0 gIL of oxalic acid R in D evelopment Over a path of 15 cm.
anhydrous formic acid R and dilute to 25 .0 mL with anhydrous Drying At 100-105 oc.
acetic acid R . D etection Spray the still-warm plate with a 10 gIL solution
Compensarion l¡'quid Introduce 5.0 mL of the stock solution of diphenylboric acid a111inoethyl ester R in methanol R , then
into a round-bottomed ftask and evaporate to dryness under spray with a 50 gIL solution of 111acrogol 400 R in methanol R;
reduced pressure. Take up the residue with 8 mL of a allow to dry in air for 30 min and examine in ultraviolet light
mixture of 10 volumes of methanol R and 100 volumes of at 365 nm.
anhydrous acetic acid R and transfer to a 25 mL volumetric Results See below sequence of zones present in the
ftask. Rinse the round-bottomed ftask with 3 mL of a chromatograms obtained with the reference solution and the
mixture of 10 volumes of methanol R and 100 volumes of test solution. Furthermore, other ftuorescent zones may be
anhydrous acetic acid R and transfer to the same 25 mL present in the ehromatogram obtained with the test solution.
volumetric ftask. Add 10.0 mL of anhydrous formic acid R and
dilute to 25.0 mL with anhydrous acetic acid R.
After 30 min, measure the absorbance (2.2.25) of the test Top of the plate
solution at 410 nm, by comparison with the compensation
A light yell ow f1uorescent zone
liquid.
Calculate the percentage content of total ftavonoids, Hyperoside: a yellowish-orange A yellowish·orange fluorescent zone
f1uorescent zone {hyperoside}
expressed as hyperoside, using the following expression:
Chlorogenic acid: a light blue A light blue fluorescent zone
A x 1.235 fluorescent zone {chlorogenic acid}
m A yellowish·green fluorescent zone
{vitexin 2"·rhamnoside}
i.e. taking the specific absorbanee of hyperoside to be 405.
A absorbanee at 410 nm; Rutin: a yellowish-orange A yellowish·orange fluorescent zone
f1uorescent zone {rutin}
m = mass of the drug to be examined, in grams.
____________________________________________ PhE~
Reference solution Test solution

TESTS
Loss on drying (2.2.32)
Hawthorn Leaf and Flower Dry ***
** **
**** *
Maximum 6.0 per cent, determined on 0.500 g of the extract
Extract to be examined by drying in an oven at 105 oC for 2 h.
(Ph Bur monograph 1865) ASSAY
~E~ ____________________________________________ Stock solution Dissolve 0.100 g of the extract to be
examined in ethanol (60 per cent VIV) R and dilute to
DEFINITION 100.0 mL with the same solvent.
Dry extract produced from Hawthorn leaf and fiower (1432).
Test solution Introduce 5.0 mL of the stock solution into a
Content: round-bottomed ftask and evaporate to dryness under
-- for aqueous extracts: minimum 2.5 per cent of total reduced pressure. Take up the residue in 8 mL of a mixture
ftavonoids, expressed as hyperoside (C21H20012; M r of 10 volumes of methanol R and 100 volumes of anhydrous
464.4) (dried extract); acetic acid R and transfer to a 25 mL volumetric ftask. Rinse
-- for hydroalcoholic extracts: minimum 6.0 per eent of total the round-bottomed ftask with 3 mL of a mixture of
ftavonoids, expressed as hyperoside (C21H 2001 2; M r 10 volumes of methanol R and 100 volumes of anhydrous
464.4) (dried extract). acetic acid R and transfer to the same 25 mL volumetrie ftask.
PRODUCTION Add 10.0 mL of a solution eontaining 25 .0 gIL of boric
The extraet is produced from the herbal drug by a suitable acid R and 20.0 giL of oxalic acid R in anhydrous formic acid R
proeedure using either water or a hydroalcoholie solvent at and dilute to 25.0 mL with anhydrous acetic acid R.
least equivalent in strength to ethanol (45 per cent V/V) . Compensation liquid Introduce 5.0 mL of the stock solution
into a round-bottomed ftask and evaporate to dryness under
CHARACTERS
reduced pressure . Take up the residue in 8 mL of a mixture
Appearance
of 10 volumes of 111ethanol R and 100 volumes of anhydrous
Light brown or greenish-brown powder.
acetic acid R and transfer to a 25 mL volumetric ftask. Rinse
IDENTIFICATION the round-bottomed ftask with 3 mL of a mixture of
Thin-Iayer ehromatography (2.2.27). 10 volumes of methanol R and 100 volumes of anhydrous
IV-210 Hawthom Leaf Preparations
acetic acid R and transfer to the same 25 mL volumetric flask.
Add 10.0 mL of anhydrousjormic acid R and dilute to
25.0 mL with anhydrous acetic acid R.
After 30 min, measure the absorbance (2.2.25) ofthe test
solution at 4 10 nm, by comparison with the compensation
liquido
Calculate the percentage content of total flavonoids,
expressed as hyperoside, using the following expression:
A x 1.235
m
i.e. taking the specific absorbance of hyperoside to be 405.
A absorbance at 410 nm;
m = mass of the extract to be examined, in grams.
____________________________________________ PhE~
Quantified Hawthorn Leaf and
Flower Liquid Extract
(Ph Eur monograph 1864)
Ph Eur ____________________________________________
DEFINITION
Quantified liquid extract produced from Hawthorn leaj with
fiower (1432).
Content
0.8 per cent to 3.0 per cent of flavonoids, expressed as
hyperoside (C21 H 2001 2; M r 464.4).
PRODUCTION
The extract is produced from the herbal drug and ethanol
(30 per cent V/V to 70 per cent V/V) by an appropriate
procedure.
IDENfIFICATION
Thin-layer chromatography (2.2.27).
Test solution Dilute 1.0 g in methanol R and dilute to 5 mL
with the same solvento Shake and filter.
Rejerence solution Dissolve 1.0 mg of chlorogenic acid R and
2.5 mg of hyperoside R in methanol R and dilute to 10 mL
with the same solvento
Plate TLC silica gel plate R (5-40 flm) [or TLC silica gel
plate R (2-10 ~lm») .
Mobüe phase anhydrous jormic acid R, water R , methyl ethyl
ketone R , ethyl acetate R (10:10:30:50 V/V/V/V) .
Application20 flL [or 5 flL) as bands.
Developmem Over a path of 15 cm [or 6 cm).
D¡ying At 100-105 oc.
Detection Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R. Subsequently spray with a
50 gIL solution of macrogol 400 R in methanol R . Allow the
plate to dry in air for about 30 mino Examine in ultraviolet
light at 365 nm .
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other fluorescent zones may be
present in the chromatogram obtained with the test solution.
2014
Top of the plate
----- -----
A yellowish·green fluorescent zone
Hyperoside: a yellowish-orange A yellowish·orange fluo rescent zone
f1uorescent zone (hyperoside)
Chlorogenic acid: a Iight blue A Iight blue fluorescent zone
fluorescent zone (chlorogenic acid)
A yellowish·green fluorescent zone
----- -----
Referente solution Test solution
TESTS
Ethanol (2.9. 10): 95 per cent V/V to 105 per cent V/V of the
quantity stated on the labe!'
ASSAY
Stock solution Dilute about 0.400 g, accurately weighed, in
ethanol (60 per cent V/V> R and dilute to 100.0 mL with the
same solvento
Test solution Introduce 5.0 mL of the stock solution into a
round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
flask. Rinse the round-bottomed flask with 3 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the 25 mL volumetric
flask. Add 10.0 mL of a solution containing 25.0 gIL of boric
acid R and 20.0 giL of oxalic acid R in anhydrous jormic acid R
and dilute to 25.0 mL with anhydrous acetic acid R .
Compensation liquid Introduce 5.0 mL of the stock solution
into a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
flask. Rinse the round-bottomed flask with 3 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the 25 mL volumetric
flask. Add 10.0 mL of anhydrous jom1Íc acid R and dilute to
25.0 mL with anhydrous acetic acid R .
After 30 min measure the absorbance (2.2.25) of the test
solution at 410 nm.
Calculate the percentage content of total flavonoids,
expressed as hyperoside, from the following expression:
A x 1.235
m
i.e. taking the value of the specific absorbance of hyperoside
to be 405.
A absorbance at 410 nm,
m = mass of the extract to be examined, in grams.
____________________________________________ ~E~
Hop Strobile *****
** *
(Ph Eur monograph 1222) *** *
~E~ ____________________________________________
DEFINITION
Dried, generally whole, female inflorescence of Humulus
lupulus L.
2014
CHARACTERS
Characteristic, aromatic odour.
IDENTIFICATION
A. Hop strobiles are generally isolated and 2-5 cm long,
petiolate, ovoid, made up of many oval, greenish-yellow,
sessile, membranous, overlapping bracts. The external bracts
are ftattened and symmetrical. The internal bracts are longer
and asymmetrical at the base because of a fold generally
encircling an induviate fruit (achene). The ovary or rarely the
fruit, the base of the bracts and especially the induvial fold,
are covered with small orange-yellow glands.
B. Reduce to a powder (355) (2.9.12). The powder is
greenish-yellow. Examine under a microscope using chloraJ
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1222.-1): fragments ofbracts and
bracteoles covered by polygonal, irregular or wavy-walled
epidermal cells [D, L, M]; unicellular, conical, straight or
curved covering trichomes with thin, smooth walls,
fragmented [E, G] or attached to an epidermis [A]; rare
anomocytic sto mata (2.8.3) [K]; glandular trichomes, usually
free, with bicellular biseriate stalks and heads consisting of 8
small cells [H, N], rarely attached to an epidermis [La];
fragments of mesophyll containing small calcium oxalate
cluster crystalsm; many characteristic orange-yellow
glandular trichomes with short, bicellular biseriate stalks,
bearing a part widening into a cup, 150-250 Jlm in diameter,
made up of a hemispherical layer of secretory cells with a
cuticle that has been detached and distended by the
accumulation of oleoresinous secretions, in surface view [B]
or in side view [C]; fragments of elongated sclerenchymatous
cells of the testa with thick walls showing striations and
numerous pits [F].
e daylight.
G To 10.0 g ofthe powdered drug (35 5) (2.9.12) add 300 mL
H~
~
ffi
~
N
100~m
>--t
La
Figure 1222.-1.- Illustration for identification test B of
powdered herbal drug of hop strobile
Hop Strobile IV-211
C. Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the freshly powdered drug (355)
(2.9.12) add 10 mL of a mixture of 3 volumes of water R and
7 volumes of methanol R; shake for 15 min and filter.
Reference solution Dissolve 1.0 mg of Sudan orange R,
2.0 mg of curcumin R and 2.0 mg of
dimethylaminobenzaldehyde R in 20 mL of methanol R.
Plate TLC silica gel FZ54 plate R.
Mobile phase anhydrous acetic acid R, ethyl acetate R,
cyclohexane R (2:38:60 VIV/V).
Application 20 JlL as bands.
Development Over a path of 15 cm.
Drying In air.
Detection A Examine in ultraviolet light at 254 nm.
R esults A The chromatogram obtained with the reference
solution shows 3 quenching zones; in the lower quarter is the
faint zone due to curcumin, somewhat below the middle is
the zone due to dimethylaminobenzaldehyde, and aboye, the
zone due to Sudan orange. The chromatogram obtained with
the test solution shows a number of quenching zones similar
in position to the zones in the chromatogram obtained with
the reference solution: at about the level of the zone due to
curcumin is a faint zone due to xanthohumol, near the level
of the zone due to dimethylaminobenzaldehyde are zones due
to humulones, and near the level of the zone due to Sudan
orange are zones due to lupulones.
Detection B Examine in ultraviolet light at 365 nm.
Results B In the chromatogram obtained with the test
solution the zones due to lupulones show blue ftuorescence,
the zones due to humulones show brown ftuorescence and
the zone due to xanthohumol shows dark brown
ftuorescence.
Detection C Spray with dilute phosphomolybdotungstic
reagent R; expose to ammonia vapour and examine in
Results C In the chromatogram obtained with the test
solution the zones due to humulones and to lupulones are
bluish-grey and the zone due to xanthohumol is greenish-
grey; in the chromatogram obtained with the reference
solution the zones are bluish-grey or brownish-grey.
TESTS
Matter extractable by ethanol (70 per cent VIV)
Minimum 25.0 per cent.
of ethanol (70 per cent V/V? R and heat for 10 min on a
water-bath under a reftux condenser. Allow to cool, filter,
and discard the first 10 mL of the filtra te. Evaporate
30.0 mL of the filtrate to dryness on a water-bath and dry in
an oven at 100-105 oC for 2 h . The residue weighs a
minimum of 0.250 g.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9. 12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 12.0 per cent.
____________________________________________ ~E~
 IV-212 White Horehound 2014

White Horehound *** Application 20 pL [or 5 pL] oftest solutions (a) and (b)
*** *** and 1O ~IL [or 2 pL] of the reference solution, as bands.
(Ph Eur monograph 1835) *** Development Over a path of 10 cm [or 6 cm] .
~ ____________________________________________
Ew

D rying In airo
DEFINITION Detection Spray with a 5 gIL solution of vanillin R in a
Whole or fragmented dried flowering aerial parts of mixture of 20 volumes of ethanol (96 per cent) R and
Mamtbiwn vulgare L. 80 volumes of sulfuric acid R and examine in daylight
irnmediately after heating at 130 oC for 5-10 mino
Content
R esults See below the sequence of the zones present in the
Minimum 0.7 per cent of marrubiin (C2oH2S0 4; M r 332.4)
(dried drug). chromatograms obtained with the reference solution and test
solutions (a) and (b). Further zones in the chromatograms
CHARACTERS obtained with test solutions (a) and (b) may be presento
Bitter taste. The zone due to marrubiin in the chromatogram obtained
IDENTIFICAnON with test solution (a) is more intense than that in the
A. The stems are up to 50 cm long, quadrangular, up to chromatogram obtained with test solution (b) . During
7 mm wide, young stems are densely covered with whitish extraction with hydrochloric acid and methanol, conversion
downy hairs, older stems are greenish-grey and less hairy. of pre-marrubiin to marrubiin takes place which leads to an
The lower leaves are broadly ovate to almost orbicular, upper increase in intensity of the zone.
leaves less broadly ova te, both petiolate; lamina 1.5-4 cm
long, 1-3.5 cm wide, apex sub-acute, base tapering or Top of the plate
somewhat corda te, margin dentate to crenate, petiole up to
Guaiazulene: a A bluish-violet zone A bluish-violet zone
3 cm long; venation pinnate, prominent on the lower surface, reddish-violet zone
distinctly depressed on the upper surface. Both leaf surfaces --- ---
are densely covered with fine, white, woolly hairs, older
leaves having fewer hairs on the dark greyish-green upper A bluish-violet zone A bluish-vioJet zone
surface. The flowers are small, sessile in dense axillary --- ---
clusters. The calyx is 5 mm long, persistent, with 5 long and
Cholesterol: a An intense bluish-violet A bluish-violet zone
5 short, alternating, hooked, recurved fringing spines; throat bluish-violet zone zone (marrubiin) (marrubiin)
of calyx with an internal ring of long silky hairs; corolla A bluish-violet zone A bluish·violet zone
7 mm long, dull white, 4-lobed, upper lobe 2-lipped, lower-
lobe 3-lipped; 4 short stamens; style with bifid stigma. A bluish-violet zone A bluish-violet zone

B. Reduce to a powder (710) (2.9.12). The powder is Reference solution Test solution (a) Test solution (b)
greyish-green. Examine under a microscope under chloral
hydrate solution R . The powder shows the following diagnostic
characters: fragments of leaves with sinuous, polygonal TESTS
epidermal cells, diacytic stomata (2.8.3), more numerous on Loss on drying (2.2.32)
the lower surface and cells of the mesophyll with small Maximum 10.0 per cent, determined on 1.000 g of the
needles and cluster crystals of ca1cium oxalate; covering powdered drug (710) (2.9.12) by drying in an oven at
trichomes very numerous, twisted or coiled, 100-200 pm 105 oC for 2 h.
long, unicellular or multicellular and unseriate with 2-6 cells, Total ash (2.4.16)
enlarged at the joints; stellate trichomes of 2 types, one with Maximum 15.0 per cent.
15-20 branches arising from a short unicellular stalk and the
Ash insoluble in hydrochloric acid (2.8.1)
other with fewer branches arising from a sessile base; 8-celled
Maximum 3.0 per cent.
secretory trichomes of lamiaceous type; glandular trichomes
with 1 or 2 celled stalk and 1 to 4 celled head; the covering ASSAY
trichomes on the inner surface of the calyx are up to Liquid chromatography (2.2.29).
1000 pm long with 2 to 3 cells, strongly thickened at the Test solution Reduce 50 g of the drug to a powder (250)
swollen joint and with the upper cell elongated; pollen grains (2.9.12) and homogenise. To 1.00 g of the powdered drug in
spherical, about 25 pm in diameter with smooth exine; a 50 mL round-bottomed flask add 15 mL of a mixture of
fragments of vascular tissue from the stems and veins. 2 volumes of dilute hydrochloric acid R and 8 volumes of
C. Thin-Iayer chromatography (2.2.27). methanol R . Heat in a water bath at 80 oC under a reflux
Test solution (a) To 1.0 g ofthe powdered drug (710) condenser for 30 mino Allow to cool at room temperature
(2.9.12) add 2 mL of dilute hydrochloric acid R and 8 mL of and filter through a plug of adsorbent cotton into a 25 mL
methanol R. Heat under a reflux condenser for 30 min, cool volumetric flask. Dilute to 25 .0 mL with methanol R by
and filter. rinsing the round-bottomed flask and the filter.
Test solution (b) To 1.0 g of the powdered drug (710) Reference solution Dissolve 2.0 mg of marrubiin R in
(2.9.12) add 10 mL of methanol R . Heat under a reflux 10.0 mL of methanol R.
condenser for 30 min, cool and filter. Column:
R ef erence solution Dissolve 10 mg of cholesterol R and 10 mg -- size: l = 0.25 m, 0 = 4 mm,
of guaiazulene R in 10 mL of methanol R. -- stationary phase: end-capped occadecylsilyl silica gel for
chromatography R (5 pm).
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
Mobile phase:
plate R (2-10 pm)].
-- m obile phase A: acetonitrile R,
Mobile phase methanol R, toluene R (5:95 V/V).
mobile phase B: dilute 0.5 mL of phosphoric acid R to
1000 mL with water R,
 2014 Horsetail IV-213

Time Mobile phase A Mobile phase B surface view [B, C] composed of rectangular cells with wavy
(min) (per cen! V!JI) (per cen! V!JI) walls and paracytic stomata ( 2.8.3) in 2-4 rows, the 2
0->15 40 -> 90 60 -> 10 subsidiary cells are in the same plane as the epidermis, cover
15 -> 20 90 -> 40 10 -> 60 the guard cells and show radial ridges; small silica pilulae are
scattered on the surface of the subsidiary cells and appear
20 -> 25 40 60
more frequent at the margin forming a distinct ring
surrounding the subsidiary cells (C); 2-celled papillae on the
Flow rate 1.5 mUmin.
ridges, less distinct on the main stem [A] but large and
Detection Spectrophotometer at 217 nm. rectangular on the branches, oriented longitudinally [F];
lnjection 20 ¡.¡L. in surface view, the epidermis of the main stems consists of
Locate the peak due to marrubiin by comparison with the elongated cells [G], the epidermis of the secondary branches
chromatogram obtained with the reference solution. shows the 2-celled papillae which resemble pairs of small
Calculate the percentage content of marrubiin from the cells separated by a larger cell [D]; fragments of large-celled
following expression: parenchyma [H] and groups of long unlignified fibres with
narrow lumens; small vessels with spiral or annular
Al x m2 x p x 2.5 thickening [E].
A 2 x mI C. Examine the chromatograms obtained in the test for
Equisetum palustre.
area of the peak due to marrubiin in the Results See below the sequence of zones present in the
chromatogram obtained with the test solution, chromatograms obtained with reference solution (b) and the
area of the peak due ro marrubiin in the test solution. Furthermore, other weak fiuorescent zones may
chromatogram obtained with the reference solution, be present in the chromatogram obtained with the test
mass of the drug to be examined, in milligrams, solution.
mass of malTubiin R, in milligrams,
percentage content of marrubiin in marrubiin R.
Top of the plate
____________________________________________ PhE~

2 red fluorescent zones

Caffeic acid: a greenish-blue

fluorescent zone
2 greenish-blue fluorescent zones
Horsetail ***
*** *** --- - --
(Equisetum Stem, Ph Eur monograph 1825) *** An orange fluorescent zone
~E~ ____________________________________________
Hyperoside: an orange fluorescen!
DEFINITION zone

Whole or cut, dried sterile aerial parts of Equisetum G/'Vense L. 2 greenish-blue fluorescent zones

Content --- ---

Minimum 0.3 per cent of total fiavonoids, expressed as Rutin: an orange fluorescent zone
isoquercitroside (C21H20012; M r 464.4) (dried drug).
Reference solution (b) Test solution
IDENTIFICATION
A. It consists of fragments of grooved main stems, branches
with longitudinal sharp ridges and leaves in whorls, united at TESTS
the base into a sheath, light green or greenish-grey. Foreign matter (2.8.2)
The fragments are rough to the touch, brittle and crunchy Maximum 5 per cent.
when crushed. The main stems are about 1-4.5 mm in
Equisetum palustre
diameter, hollow, jointed at the nodes, which occur at
Thin-layer chromatography (2.2.27) .
intervals of about 1.5-4.5 cm; distinct vertical grooves are
present on the internodes, ranging in number from 4 to 14 Test solution To 1.0 g of the powdered herbal drug (355)
or more. The central hollow is less than 50 per cent but (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
more than 25 per cent of the diameter of the main stem. 60 oC for 10 min with occasional shaking. Allow to coo1.
Verticils of widely spaced and erect branches, usually simple, Filter.
each about 1 mm thick with 3-5 longitudinal, sharp ridges, Reference solution (a) To 100.0 mg of Equisetum palustre
occur at the no des; at the end of each ridge is a protruding, HRS add 10 mL of methanol R. Heat in a water-bath at
distinct collenchymatic bundle under the epidermis. 60 oC for 10 min with occasional shaking. Allow to coo1.
The branches are not hollow. The leaves are small, linear, Filter.
verticillate at each node, concrescent at the base; they form a Reference solution (b) Dissolve 1.0 mg of caffeic acid R,
toothed sheath around the stem with the number of teeth 2.5 mg of hyperoside R and 2.5 mg of rutin R in 20 mL of
corresponding to the number of gro oves on the stem. Each methanol R.
tooth, often brown, is lanceolate-triangular. The lowest Plate TLC silica gel plate R (2-10 ¡.¡m).
interno de of each branch is longer than the sheath of the
Mobile phase anhydrous formic acid R, glacial acetic acid R,
stem to which it belongs.
water R, ethyl acetate R (7.5:7.5:18:67 VIVIV/V).
B. Microscopic examination (2.8.23) . The powder is
greenish-grey. Examine under a microscope using chloral Application5 ¡.¡L as bands of 8 mm.
hydrate solution R. The powder shows the following diagnostic Development Over a path of 6 cm.
characters (Figure 1825.-1): fragments ofthe epidermis in Drying In a current of cold air for 5 mino
IV-214 Iceland Moss
Figure 1825.-1. - Illustration for identification test B of
powdered herbal drug of equisetum stem
D etection Heat at 100 oC for 3 min and treat the stilI-warm
plate with a 10 gIL solution of diphenylboríc acid aminoethyl
ester R in methanol R, then treat with a 50 gIL solution of
macrogol 400 R in methanol R; alIow to dry in a current of
cold air and examine after 10 min in ultraviolet light at
365 run.
System suitability The chromatogram obtained with
reference solution (a) shows 2 greenish fiuorescent zones just
above the line of application.
Results In the chromatogram obtained with the test
solution, any greenish fiuorescent zones just above the line of
application are not more intense than the corresponding
zones (characteristic of E. palustre L.) in the chromatogram
obtained with reference solution (a).
Loss on drying (2.2.32)
Maxim um 10.O per cent, determined on 1. 000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Ash insoluble in hydrochloric acid (2.8. 1)
Minimum 3.0 per cent and maximum 15.0 per cent.
Total ash (2.4.16)
Minimum 12.0 per cent and maximum 27.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed fiask, introduce
0.800 g of the powdered herbal drug (355) (2.9.12) and add
1 mL of a 5 gIL solution of hex amethylenetetramine R, 20 mL
of acetone R and 2 mL of hydrochlon'c acid Rl. Boil the
mixture under a refiux condenser for 30 min. Filter the
liquid through a plug of absorbent cotton into a fiask.
Add the absorbent cotton to the residue in the round-
bottomed fiask and extract with 2 quantities, each of 20 mL,
of acetone R, each time boiling under a refiux condenser for
10 mino Allow to cool and filter each extract through a plug
of absorbent cotton into the fiask. After cooling, filter the
2014
combined acetone eXtracts through a filter paper into a
volumetric fiask and dilme to 100.0 mL with acetone R by
rinsing the fiask and the filter paper. Introduce 20.0 mL of
the solution into a separating funnel, add 20 mL of water R
and shake the mixture with 1 quantity of 15 mL and then
3 quantities, each of 10 mL, of ethyl acetate R . Combine the
ethyl acetate extracts in a separating funnel, wash with
2 quantities, each of 50 mL, of water R, and filter the
extracts over 10 g of anhydrous sodium sulfate R into a
volumetric fiask. Dilute to 50.0 mL with ethyl acetate R.
Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium ehloride reagent R and dilute to 25 .0 mL with a
5 per cent VIV solution of glacial aeetie aeid R in methanol R.
Compensation solution Dilute 10.0 mL of the stock solution
to 25.0 mL with a 5 per cent VIV solution of glacial aeetie
acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after
30 min, by comparison with the compensation solution at
425 nm. Calculate the percentage content of fiavonoids,
expressed as isoquercitroside, using the following expression:
A x 1.25
m
i.e. taking the specific absorbance of isoquercitroside to be
500.
A absorbance at 425 nm;
m = mass of the substance to be examined, in grams.
____________________________________________ ~E~
Iceland Moss ***
*** ***
(Ph Bur monograph 1439) ***
~ E~ ____________________________________________
DEFINITION
Whole or cut, dried thallus of Cetraría islandiea (L) Acharius
s.l.
IDENTIFICATION
A. The thallus, up to 15 cm long, is irregularly dichotomous
and consists of glabrous, groove-shaped or almost fiat, stiff,
brittle bands, 0.3-1.5 cm wide and about 0.5 mm thick,
sometimes serrated with the margin appearing ciliated
(pycnidia). The upper surface is greenish or greenish-brown,
the lower surface is greyish-white or light brownish and
shows whitish, depressed spots (so-called respiratory cavities).
On the apices of the terminallobes, very rarely, there are
brown, discoid apothecia.
B. Reduce to a powder (355) (2.9. 12). The powder is
greyish-brown. Examine under a microscope, using ehloral
hydrate solution R. The powder shows the following diagnostic
characters: numerous fragments of the pseudoparenchyma
consisting of narrow-lumened, thick-walIed hyphae from the
marginal layer and wide-lumened hyphae from the adjacent
layer consisting of loosely entwined hyphae, in which, in the
medullary zone, greenish or brownish alga e cells up to 15 ¡.tm
in diameter, are embedded; occasionally marginal fragments
of the thallus with tube-like or cylindrical spermogonia, up to
about 160 ¡.tm wide and up to about 400 ¡.tm long.
C . To 1.0 g ofthe powdered drug (355) (2.9.12) add 10 mL
of water R and boil for 2-3 mino The greyish-brown solution
forms a gel after cooling which gives a blue colour with iodine
solution R.
2014 Ipecacuanha IV- 215

D. Thin-Iayer chromatography (2.2.27) . Standardised Ipecacuanha Liquid Extract

Test solurion To 1.0 g of the powdered drug (355) (2.9.12) Standardised Ipecacuanha Tincture
add 5 mL of acetone R and heat in a water-bath under a ~E~ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ ___
reflux condenser for 2-3 mino Cool and filter.
DEFINITION
Reference solution Dissolve 5 mg of anethole R and 5 mg of
Fragmented and dried underground organs of Cephaelis
caffeic acid R in 2 mL of acetone R.
ipecacuanha (Brot.) A. Rich., known as Matto Grosso
Plate TLC silica gel plate R (5-40 ¡.1m) [or TLC silica gel ipecacuanha, or of Cephaelis acuminata Karsten, known as
plate R (2-10 ¡.1m)] . Costa Rica ipecacuanha, or of a mixture of both species.
Mobile phase acetone R, methanol R, anhydrous formic acid R, The principal alkaloids are emetine and cephaeline.
toluene R (5:5:10:80 V/V/V/V).
Content
Application 20 ¡.IL [or 4 ¡.IL) of the test solution and 10 ¡.IL Minimum 2.0 per cent of total alkaloids, expressed as
[or 2 ¡.IL] ofthe reference solution, as bands . emetine (C29H40N204; M r 480.7) (dried drug).
Development Over a path of 10 cm [or 6 cm).
CHARACTERS
Drying In airo Slight odour.
Detection Spray with anisaldehyde solution R . Heat at
IDENTIFICATION
100-105 oC for 5-10 min and examine in daylight.
A. C. ipecacuanha. The root occurs as somewhat tortuous
Results See below the sequence of zones present in the pieces, dark reddish-brown or very dark brown, seldom more
chromatograms obtained with the reference solution and the than 15 cm long or 6 mm thick, closely annulated externally,
test solution. Furthermore, other faint zones may be present having rounded ridges completely encircling the root;
in the chromatogram obtained with the test solution. the fracture is short in the bark and splintery in the wood.
The transversely cut surface shows a wide greyish bark and a
Top oí the plate small uniformly dense wood. The rhizome occurs as short
A greyish-blue zone
lengths usually attached 10 roots, cylindrical, up to 2 mm in
diameter, finely wrinkled 10ngitudinal1y and with pith
Anethole : a blue or bluish-violet occupying approximately one-sixth of the whole diameter.
zone
--- --- C. acuminata. The root in general resembles the root of C.
ipecacuanha, but differs in the following particulars: it is often
2 weak greyish-blue zones up to 9 mm thick; the external surface is greyish-brown or
A weak greyish-brown or grey zone reddish-brown with transverse ridges at intervals of usually
1-3 mm, the ridges being about 0.5-1 mm wide, extending
--- --- about half-way round the circumference and fading at the
A greyish-violet zone extremities into the general surface leve!.
Caffeic acid: a greyish-blue zone B. Reduce to a powder (355) (2.9.12). The powder is light
grey or yellowish-brown. Examine under a microscope, using
chloral hydrate solution R . The powder shows the following
Reference solution Test solution diagnostic characters: parenchymatous cells, raphides of
calcium oxalate up 10 80 ¡.1m in length either in bundles or
scattered throughout the powder; fragments of tracheids and
TESTS ves seis usually 10-20 ¡.tm in diameter, with bordered pits;
Foreign matter (2.8.2) larger vessels and sclereids from the rhizome. Examine under
Maximum 5 per cent. a microscope using a 50 per cent V/V solution of glycerol R.
Lead (2.4.27) The powder shows simple or two- 10 eight-compound starch
Maximum 10.0 ppm. granules contained in parenchymatous cells, the simple
granules being up to 15 ¡.1m in diameter in C. ipecacuanha
Loss on drying (2.2.32)
and up 10 22 ¡.1m in diameter in C. acuminata.
Maximum 12.0 per cent, determined on 1.000 g of
powdered drug (355) (2.9.12) by drying in an oven at C. Thin-Iayer chromatography (2.2.27).
105 oC for 2 h. Test solurion To 0.1 g of the powdered drug (I80) (2.9.12)
in a test-tube add 0.05 mL of concentrated ammonia R and
Total ash (2.4.16)
5 mL of ether R and stir the mixture vigorously with a glass
Maximum 3.0 per cent.
rod. AlIow to stand for 30 min and filter.
Swelling value (2.8.4)
Reference solution Dissolve 2.5 mg of emetine
Minimum 4.5, determined on the powdered drug (355) hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
(2.9.12).
methanol R and dilute to 20 mL with the same solvent.
~ _ _ __ _ __ _ _ __ _ _ _ _ _ _ _ _ _ _ Ph Eur
Plate TLC silica gel plate R.
Mobz7e phase concentrated ammonia R, methanol R, ethyl
aceta te R, toluene R (2:15:18 :65 V/V/V/V) .
Application 10 ¡.IL, as bands.
Ipecacuanha Development Over a path of 10 cm.
(lpecacuanha Root, Ph Bur monograph 0094) Drying In airo
Preparations Detection A Spray with a 5 gIL solution of iodine R in
Prepared Ipecacuanha ethanol (96 per cent) R and heat at 60 oC for 10 mino
Examine in daylight.
Ipecacuanha Liquid Extract
 IV-216 Ipecacuanha
ResLúts A The chromatograms obtained with the test
solution and with the reference solution show in the lower
part a yellow zone due to emetine and below a light brown
zone due to cephaeline.
Detection B Examine in ultraviolet light at 365 nm.
Results B The zone due to emetine shows an intense yellow
f1uorescence and that due to cephaeline a light blue
f1uorescence . The chromatogram obtained with the test
solution shows also faint f1uorescent zones.
With C. acuminata the principal zones in the chromatogram
obtained with the test solution are similar in position,
f1uorescence and size to the zones in the chromatogram
obtained with the reference solution.
With C. ipecacuanha the only difference is that the zone due
to cephaeline in the chromatogram obtained with the test
solution is much smaller than the corresponding zone in the
chromatogram obtained with the reference solution.
TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of
powdered drug (180) (2.9. 12) by drying in an oven at
105 oc.
Total ash (2.4.16)
Maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
To 7.5 g of the powdered drug (180) (2.9. 12) in a dry f1ask,
add 100 mL of ether R and shake for 5 mino Add 5 mL of
dilute ammonia R1, shake for 1 h. Add 5 mL of water R and
shake vigorously. Decant the ether layer into a f1ask through
a plug of cotton. Wash the residue in the f1ask with
2 quantities, each of 25 mL, of ether R, decanting each
portion through the same plug of cotton. Combine the ether
solutions and eliminate the ether by distillation. Dissolve the
residue in 2 mL of ethanol (90 per cent V/T-] R, evaporate to
dryness and heat at 100 oC for 5 min. Dissolve the residue in
5 mL of previously neutralised ethanol (90 per cent V /T-] R,
warming on a water-bath. Add 15.0 mL of 0. 1 M hydrochloric
acid and titrate the excess acid with 0.1 M sodium hydroxide
using 0.5 mL of methyl red mixed solution R as indicator.
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg of
total alkaloids, expressed as emetine.
STORAGE
Protected from moisture.
_ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ Ph Eur
Prepared Ipecacuanha *****
** **
(Ph Eur monograph 0093) ***
~E~ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ __
DEFINITION
Ipecacuanha root powder (180) (2.9.12) adjusted, if
necessary, by the addition of powdered lactose or
ipecacuanha root powder with a lower alkaloidal contento
Content
1. 9 per cent to 2.1 per cent of total alkaloids, expressed as
emetine (C29H40N204; M r 480.7) (dried drug).
CHARACTERS
Appearance
Light grey or yellowish-brown powder.
2014
Slight odour.
IDENTIFICATION
A. Examine under a microscope, using chloral hydrate
solution R. The powder shows the following diagnostic
characters: parenchymatous cells, raphides of calcium oxalate
up to 80 ¡.1m in length either in bundles or scattered
throughout the powder; fragments of tracheids and vessels
usually 10-20 ~un in diameter, with bordered pits; larger
vessels and sclereids from the rhizome. Examine under a
microscope using a 50 per cent V/V solution of glycerol R.
The powder shows simple or 2-8-compound starch granules
contained in parenchymatous cells, the simple granules being
up to 15 ~lm in diameter in Cephaelis ipecacuanha and up to
22 ¡.1m in diameter in C. acuminata. Examined in glycerol
(85 per cent) R, it may be seen to contain lactose crystals.
B. Thin-layer chromatography (2.2.27).
Test solution To 0.1 g ofthe drug to be examined in a test-
tube add 0.05 mL of concentrated ammonia R and 5 mL of
ether R and stir the mixture vigorously with a glass rod. Allow
to stand for 30 min and filter.
Reference solution Dissolve 2.5 mg of emetine
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
methanol R and dilute to 20 mL with the same solvento
Plate TLC silica gel plate R.
Mobile phase concentrated ammonia R, methanol R, ethyl
acetate R, toluene R (2:15 :18:65 V/V/V/V) .
Application 1O ~lL, as bands.
Development Over a path of 10 cm.
Drying In airo
Detection A Spray with a 5 gIL solution of iodine R in
ethanol (96 per cent) R; heat at 60 oC for 10 min and examine
in daylight.
Results A The chromatograms obtained with the test
solution and the reference solution show in the lower part a
yellow zone due to emetine and below it a light brown zone
due to cephaeline.
Detectíon B Examine in ultraviolet light at 365 nm.
Results B The zone due to emetine shows an intense yellow
f1uorescence and that due to cephaeline a light blue
f1uorescence . The chromatogram obtained with the test
solution also shows faint fiuorescent zones.
With prepared C. acuminata, the principal zones in the
chromatogram obtained with the test solution are similar in
position, f1uorescence and size to the zones in the
chromatogram obtained with the reference solution.
With prepared C. ipecacuanha , the only difference is that the
zone due to cephaeline in the chromatogram obtained with
the test solution is much smaller than the corresponding zone
in the chromatogram obtained with the reference solution.
TESTS
Loss on drying (2.2.32)
Maximum 5.0 per cent, determined on 1.000 g by drying in
an oven at 105 oC.
Total ash (2.4. 16)
Maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
To 7.5 g in a dry f1ask, add 100 mL of ether R and shake for
5 min. Add 5 mL of dilute ammonia R1, shake for 1 h, add
5 mL of water R and shake vigorously. Decant the ether layer
into a fiask through a plug of cotton. Wash the residue in the
 2014
fiask with 2 quantities, each of 25 mL, of ether R, decanting
each portion through the same plug of cotton. Combine the
ether solutions and elimina te the ether by distillation.
Dissolve the residue in 2 mL of ethanol (90 per cent V/V? R,
evaporate the ethanol to dryness and heat at 100 oC for
5 mino Dissolve the residue in 5 mL of previously neutralised
ethanol (90 per cent V/V) R, warming on a water-bath, add
15.0 mL of 0.1 M hydrochlol1'c acid and titrate the excess acid
with 0.1 M sodium hydroxide using 0.5 mL of methyl red mixed
solution R as indicator.
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg of
total alkaloids, expressed as emetine.
STORAGE
In an airtight container.
____________________________________________
Ipecacuanha Liquid Extract
DEFINITION
Ipecacuanha Liquid Extract is prepared from Ipecacuanha by
a method stated under the general monograph for Extracts.
It contains not less than 1.90% and not more than 2.10% of
total alkaloids, calculated as emetine, C29H40N204.
EXTEMPORANEOUS PREPARATION
Prepare by extracting Ipecacuanha with Ethanol
(80 per cent) according to the following formula and
directions.
Ipecacuanha in fin e powder 1000 g
Ethanol (80 per cent) A sufficient quantity
Exhaust the Ipecacuanha by percolation, Appendix XI F, with
Ethanol (80 per cent), reserving the first 750 mL of the
percolate. Remove the ethanol from the remainder of the
percolate by evaporation under reduced pressure at a
temperature not exceeding 60° and dissolve the residual
extract in the reserved portion. Determine the proportion of
alkaloids in the liquid thus obtained by the Assay described
below. To the remainder of the liquid add sufficient Ethanol
(80%) to produce an Ipecacuanha Liquid Extract containing
2 % w/v of total alkaloids calculated as emetine. Allow to
stand for not less than 24 hours; filter.
The extract complies with the requirements stated unde1' Bx tracts
and with the following requirements.
TESTS
Ethanol content
63 to 69% v/v, Appendix VIII F, Method III .
Relative density
0.910 to 0.960, Appendix V G.
Dry residue
The requirement for Dry residue does not apply to
Ipecacuanha Liquid Extract.
ASSAY
To 5 mL in a separating funnel add 20 mL of water, 5 mL of
1M sulfuric acid and 10 mL of chlorofonn and shake well.
Transfer the chloroform extract to a second separating funnel
containing a mixture of 4 mL of ethanol (96%) and 20 mL of
0.05M sulfuric acid, shake, allow to separate and discard the
chloroform layer. Continue the extraction of the liquid in the
first separating funnel with two further 10 mL quantities of
chloroform, transferring the chIoroform solution each time to
the second separating funnel and washing as before. Transfer
Ipecacuanha Preparations IV-217
the acidic liquid from the second separating funnel to the
first separating funnel, make distinctIy alkaline with
5M ammonia and shake with successive quantities of
chlo1'ofol111 until complete extraction of the alkaloids is effected,
Appendix XI G, washing each chIoroform solution with the
same 10 mL of water contained in a third separating funnel.
Remove the chloroform, add to the residue 2 mL of ethanol
(96%), evaporate to dryness and dry for 5 minutes at 80° in
a current of airo Dissolve the residue in 2 mL of ethanol
(96%), previously neutralised to methyl red solution, add
10 mL of 0.05M sulfuric acid VS and titrate with O.IM sodium
hydroxide VS using methyl red mixed solution as indicator. Each
mL of 0.05M sulfuric acid VS is equivalent to 24.03 mg of
total alkaloids, calculated as emetine, C29H40N204'
~E~
Standardised Ipecacuanha Liquid *****
** **
Extract ***
(Ph Bur monograph 1875)
~E~ ____________________________________________
DEFINITION
Standardised liquid extract produced from Ipecacuanha root
(0094).
Content
1.80 per cent to 2.20 per cent of total alkaloids, calculated as
emetine (C29H40N20 4; M r 480.7).
PRODUCTION
The extract is produced from the herbal drug and ethanol
(60 to 80 per cent V/V) by an appropriate procedure.
CHARACTERS
Appearance
Dark brown liquido
IDENTIFICATION
Thin-Iayer chromatography (2.2.27) .
Test solution Dilute 5.0 mL of the extract to be examined to
50 mL with ethanol (70 per cent V/V? R. To 2.0 mL of this
solution add 2 mL of water R and 0.1 mL of concentrated
ammonia R. Add 10 mL of ether R and shake. Separate the
upper layer, dry it over about 2 g of anhydrous sodium
sulfate R and filter.
R eference solution Dissolve 2.5 mg of emetine
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
methanol R and dilute to 10 mL with the same solvent.
Plme TLC silica gel plme R.
Mobile phase concentrated ammonia R, methanol R, ethyl
acetate R, toluene R (2: 15 :18:65 V/VIV/V).
Application 10 ¡.tL as bands.
Development Over a path of 10 cm.
D¡ying In air.
Detection A Spray with a 5 gIL solution of iodine R in
ethanol (96 per cene) R . Heat at 60 oC for 10 min and allow
to cool for 30 min. Examine in daylight.
Results ASee below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones may be present in
the chromatogram obtained with the test solution.
 IV-218 Ipecacuanha Preparations 2014

*****
Top of the plate
Standardised Ipecacuanha
--- --- ** **
Tincture ***
--- ---
(Ph Bur monograph 1530)
Emetine : a yellow zone A yellow zone (emetine) ~E~ _ _ _ _ __ _ __ _ _ _ ____ __ _ __ _ _ __
Cephaeline : a light brown zone A light brown zone (cephaeline)
DEFINITlON
Reference solution Test solution Tineture produeed from Ipecacuanha root (0094).
Content
D etection B Examine the plate in ultraviolet light at 365 nm. 0.18 per eent (m /m) to 0.22 per eent (ml1n) of total alkaloids,
ealculated as emetine (C29H 40N 204; M r 480.7).
R esults B See below the sequenee of the zones present in
the ehromatograms obtained with the referenee solution and PRODUCTlON
the test solution. Furtherrnore, other faint ftuoreseent zones The tineture is produeed from the herbal drug and ethanol
are present in the ehromatogram obtained with the test (70 per eent V/V) by an appropriate proeedure.
solution.
CHARACTERS
Appearance
Top of the plate Yellowish-brown liquido
--- --- IDENTIFICATlON
--- --- Thin-Iayer ehromatography (2.2. 27).
Test solution To 2.0 mL of the tineture to be examined add
Emetine: an intense yellow An intense yellow fluorescent zone
fluorescent zone (emetine) 2 mL of water R and 0.1 mL of concentrated ammonia R .
A light blue fluorescent zone Add 10 mL of ether R and shake. Separate the ether layer,
(cephaeline) dry it over about 2 g of anhydrous sodium sulfate R and filter.
Reference solution Test solution
R eference solution Dissolve 2.5 mg of emetine
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
methanol R and dilute to 10 mL with the same solvento
With a liquid extraet from Cephaelis acuminata root, the zones
of emetine and eephaeline in the ehromatogram obtained Plate TLC silica gel plate R .
with the test solution are of similar size. Mobile phase concentrated ammonia R, methanol R, ethyl
With a liquid extraet from Cephaelis ipecacuanha root, the acetate R, toluene R (2:15 :18:65 VIVIV/V).
zone of emetine is mueh larger than the zone of eephaeline in Application 10 iJL as bands.
the ehromatogram obtained with the test solution. Development Over a path of 10 cm.
TESTS Drying In airo
Ethanol (2.9. 10) Detection A Spray with a 5 gIL solution of iodine R in
95 per eent to 105 per eent of the quantity stated on the ethanol (96 per cent) R and heat at 60 oC for 10 mino
labe!' Examine in daylight.
ASSAY R esults ASee below the sequenee of zones present in the
Dilute 1.00 g of the extraet to be examined to 10 mL with ehroma1Ograms obtained with the referenee solution and the
ethanol (70 per cent V/V; R and transfer to a ehromatography test solution.
eolumn about 0.2 m long and about 15 mm in intemal
diameter, eontaining 8 g of basic aluminium oxide R, using a
Top of the plate
glass rod. After infiltration into the aluminium oxide layer,
rinse the ftask, glass rod and ·intemal wall ofthe eolumn with --- - --
3 quantities, eaeh of 2 mL, of ethanol (70 per cent V/V; R. --- - --
Elute in portions with 40 mL of ethanol (70 per cent V/V; R .
Emetine: a yellow zone A yellow zone (emetine)
Avoid disturbanee or drying of the surfaee of the aluminium
oxide layer. Colleet the whole of the eluate. Evaporate the Cephaeline: a light brown zone A light brown zone (cephaeline)
eluate on a water-bath to about 10 mL. Allow to eoo!. Reference solution Test solution
Add 10.0 mL of 0.02 M hydrochloric acid and 20 mL of
earbon dioxide-free water R . Titrate the exeess aeid with
0.02 M sodium hydroxide using 0.15 mL of methyl red mixed Detection B Examine the plate in ultraviolet light at 365 nm.
solution R as indieator. R esults B See below the sequenee of zones present in the
Perforrn a blank assay by replacing the extraet 10 be ehromatograms obtained with the referenee solution and the
examined with 10.0 mL of alcohol of the strength stated on test solution. Furtherrnore, other faim ftuoreseent zones are
the labe!' present in the ehroma1Ogram obtained with the test solution.
1 mL of 0.02 M hydrochloric acid is equivalent to 4.807 mg of
total alkaloids, ea1culated as emetine.
_ _ _ __ _ _ __ __ _ _ _ _ _ __ __ ___ ~E~
 2014
Top oí the plate
---
---
Emetine: an intense yellow An intense yellow fluorescent zone
f1uorescent zone (emetine)
A Iight blue fluorescent zone
(cephaeline)
Reference solution Test solution
With a tincture from Cephaelis acuminata root, the zones of
emetine and cephaeline in the chromatogram obtained with
the test solution are similar in size.
With a tincture from Cephaelis ipecacuanha root, the zone of
emetine is much larger than the zone of cephaeline in the
chroma1Ogram obtained with the test solution.
TESTS
Ethanol (2.9.10)
95 per cent to 105 per cent of the quantity stated on the
labe!'
ASSAY
Transfer 10.00 g ofthe tincture to be examined to a
chroma1Ography column about 0.2 m long and about 15 mm
in intemal diameter, filled with 8 g of basic aluminium
oxide R . After infiltration into the aluminium oxide layer rinse
the intemal wall of the column with 3 quantities, each of
2 mL, of ethanol (70 per cent VIV) R. Elute in portions, with
40 mL of ethanol (70 per cent V/V) R . Avoid whirling or
drying of the surface of the aluminiurn oxide layer. Collect
the whole of the eluate. Evaporate the eluate on a water-bath
10 about 10 mL. Allow 10 coo!. Add 10.0 mL of 0.02 M
hydrochloric acid and 20 mL of carbon dioxide-free water R .
Titrate the excess acid with 0.02 M sodium hydroxide using
0.15 mL of methyl red mixed solution R as indica1Or.
Perform a blank assay replacing the tincture to be examined
with 10.0 roL of alcohol of the strength stated on the labe!'
1 mL of 0.02 M hydrochloric acid is equivalent 10 4.807 mg of
total alkaloids, ca1culated as emetine.
_____ _ _ _ _ _ _ _ _ __ _ _ __ _ _ ___
Paediatric Ipecacuanha Emetic Mixture
Paediatric Ipecacuanha Emetic; Paediatric Ipecacuanha
Emetic Oral Solution
DEFINITlON
Paediatric Ipecacuanha Emetic Mixture is an oral solution.
Ipecacuanha Liquid Extract 70 roL
Hydrochloric Acid 2.5 mL
Glycerol 100 mL
Syrup Sufficient to produce 1000 mL
The mixture complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of total alkaloids
0.1210 0.16% w/v, ca1culated as emetine, C29H40N20 4.
IDENTIFICATlON
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Mix 5 mL with 10 mL of 1m su/fun·c acid, shake with two
10 mL quantities of chloroform and diseard the ehloroform.
Add suffieient 5m ammonia to make the aqueous solution
distinetly alkaline 10 ¡itmus paper, extraet with four 10 mL
Isatis Root IV-219
quantities of chloroform, evaporate the eombined extraets to
--- dryness, cool the residue and dissolve it in 0.5 mL of ethanol
(96%).
---
(2) 0.1 % w/v of cephaeline hydrochloride EPCRS in ethanol
(96%).
(3) 0.1% w/v of emetine hydrochloride EPCRS in ethanol
(96%) .
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel G.
(b) Use the mobile phase as deseribed below.
(e) Apply 2 !lL of eaeh solution.
(d) Develop the plate to 15 cm.
0
(e) After removal of the plate, dry it at 105 to 110 for0
30 minutes, allow to cool and spray with dilute potassium
iodobismuthate solution.
MOBILE PHASE
10 volurnes of diethylamine and 90 volurnes of chloroform.
CONFIRMATION
The principal spots in the chroma1Ogram obtained with
solution (1) eorrespond in eolour and position to the spots in
the ehroma1Ograms obtained with solutions (2) and (3).
Disregard any secondary spots.
ASSAY
To 25 mL in a separating funnel add 20 mL of water and
5 mL of 1M su/fun·c acid, shake with three 10 mL quantities
of chloroform and wash eaeh ehloroform extraet with a
mixture of 20 mL of 0.05M su/furic acid and 4 mL of ethanol
(96%) contained in a seeond separating funne!. Transfer the
aeid-ethanol mixture from the seeond separating funnel 10
the first, make the eombined liquids distinetly alkaline 10
litmus paper with 5M ammonia and extract with suecessive
quantities of chloroform until complete extraction of the
alkaloids is etfeeted, Appendix XI G. Wash eaeh ehloroform
extraet with the same 10 mL of water, combine the
chloroform extraets, evapora te the ehloroform, add 2 mL of
ethanol (96%) to the residue, evaporate to dryness and dry
~E~
the residue at 80° in a current of air for 5 minutes. Dissolve
the residue in 2 mL of ethanol (96%) previously neutralised
10 methyl red solution, add 10 mL of O. O1M suifuric acid VS
and titrate the exeess of acid with 0.02M sodium hydroxide VS
using methyl red solution as indicator. Each mL of
O.OIM su/furic acid VS is equivalent to 4.806 mg of
CZ9H40Nz04.
Isatis Root *****
** **
(Ph Eur monograph 2566) ***
~E~ ___ _ _ __ _ __ _ _ _ _ _ _ __ _ _ _ ___
DEFINITlON
Dried root of Isatis tinctoria L. (l. indigotica Fortune)
eolleeted in autumn.
Content
Minimum 1.0 per eent of arginine (C6H14N402; M r 174.2)
(dried drug).
IDENTIFICATlON
A. The root is eylindrical, slightly 1ortuous, 10-20 cm long,
0.5-1 cm in diameter, extemally greyish-yellow or brownish-
yellow, wrinkled longitudinally and lentieellate transversally,
with rootlets or rootlet sears. Root stock slightly expanded,
exhibiting dark green or dark brown petiole bases arranged in
IV-220 Isatis Root
whorls, and dense tubercles. The fracture is yellowish-white,
brown or dark brown in bark and yellow or brown in wood.
B. Microscopic examination (2.8.23). The powder is whitish-
yellow or yellow. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: fragments of cork consisting of 5-8 thin-walled
layers; fragments of xylem with reticulate structure; thin-
walled, rounded parenchyma cells. Examine under a
microscope using a 50 per cent V/V solution of glycerol R .
The powder shows abundant, single or compound (2, 3 or 4)
starch grains. The starch grains, 1.5-3.4 ~m in diameter, with
spot, cleft or V-shaped hilum.
C. Thin-layer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of ethanol (70 per cent VIV) R and
sonicate for 10 mino Centrifuge and use the supematant.
Reference solution Dissolve 4 mg of arginine R and 4 mg of
cysteine hydrochloride R in 1 mL of ethanol (70 per cent
V/V) R.
Plate TLC silica gel F 254 plate R (5-40 ~m) [or TLC silica gel
F254 plate R (2-10 ~)].
Mobile phase anhydrous formic acid R, water R, acetonitrile R
(2:8:30 VIV/V) .
Application 4 ~L as bands of 10 mm [or 8 mm].
Development Over a path of 8.5 cm [or 6 cm] .
Drying In airo
Detection Expose to concentrated ammonia R vapour for
5 min, treat with ninhydrin solution R4, then heat at 120 oC
for 3 mino
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint coloured zones may be
present in the chromatogram obtained with the test solution.
Top of tbe plate
---
---
A prominent brown zone
Cysteine: a brown zone
A brown zone
Arginine: a brown zone A brown zone (arginine)
A faint brown zone
Reference solution Test solution
TESTS
Loss on drying (2.2.32)
Maximum 9.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution To 0.100 g of the powdered herbal drug (355)
(2.9.12) add 20 mL of ethanol (70 per eent V/V) R, sonicate
2014
for 20 min, filter, and evaporate the filtrate to dryness.
Dissolve the residue in ethanol (70 per eent V/V) R and dilute
to 10.0 mL with the same solvento
Referenee solution (a) Dissolve 25.0 mg of arginine CRS in
ethanol (70 per eent V/V) R and dilute to 50.0 mL with the
same solvento
Referenee solution (b) Dissolve 3.0 mg of eysteine
hydroehloride R in 6.0 mL of reference solution (a) and dilute
to 10.0 mL with ethanol (70 per cent V/V) R .
Reference solutwns (e), (d), (e), (j), (g), (h) Dilute reference
solution (a) to obtain 6 reference solutions of arginine, the
concentrations ofwhich span the expected value in the test
solution.
Column:
-- size: 1 = 0.15 m, 0 = 4.6 mm;
-- stationary phase: end-eapped oetadecylsilyl siliea gel for
ehromatography R (3 ~m);
-- temperature: 30 oC.
Mobile phase trifiuoroaeetic acid R, water R (0.2:99.8 V/V).
Flow rate 0.2 mUmin.
Detection Evaporative light-scattering detector; the following
settings have been found to be suitable; if the detector has
different setting parameters, adjust the detector settings so as
to comply with the system suitability criterion for the signal-
to-noise ratio:
-- carrier gas: nitrogen R;
-- pressure: 330 kPa;
-- evaporator temperature: 80 oc.
Injeetion1O ~L.
Run time 25 mino
System suitability:
-- resolution: minimum 1.5 between the peaks due to
cysteine and arginine in the chromatogram obtained with
reference solution (b);
-- signal-to-noise ratio: minimum 50 for the peak due to
arginine in the chromatogram obtained with reference
--- solution (a).
Establish a calibration curve with the logarithm of the
---
concentration (in milligrams per 10 mL) of reference
solutions (c), (d), (e), (f), (g) and (h) (corrected by the
assigned percentage content of arginine CRS) as the abscissa
and the logarithm of the corresponding peak areas as the
ordinate.
Calculate the percentage content of arginine using the
following expression:
lOA
m x 10
A logarithm of the concentration of arginine in the test
solution, determined from the calibration curve;
m mass of the herbal drug to be examined used to
prepare the test solution, in grams .
____ _____________________________________ PhE~
2014
Ispaghula Husk ***
*** ***
(Ph Eur monograph 1334) ***
Preparations
Ispaghula Husk Oral Powder
Ispaghula Husk Granules
Ispaghula Husk Effervescent Granules
~E~~ __________________________________________
DEFINITION
Episperm and collapsed adjacent layers removed from the
seeds of Plantago ovata Forssk. (P. ispaghula Roxb.).
IDENTIFICATION
A. The husk consists of pinkish-beige fragments or flakes up
10 about 2 mm long and 1 mm wide, sorne showing a light
brown spot corresponding to the location of the embryo
before it was removed from the seed.
B. Reduce to a powder (355) (2.9.12) . The powder is pale
yellow. Examine under a microscope using laetie reagent R .
The powder shows the following diagnostic characters:
mainly fragments of the episperm with polygonal cells filled
with mucilage; fragments of the inner layers of the testa with
brownish thin-walled ceUs often associated with the outer
layers of the endosperm. Examine under a microscope using
a 50 per cent VIV solution of glyeerol R . The powder shows
occasional starch granules, single or in groups of 2-4,
measuring 3-25 ¡lm in diameter.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 10 mg ofthe powdered drug (355) (2.9.12)
in a thick-waIled centrifuge tube, add 2 mL of a 230 gIL
solution of trifiuoroacetic aeid R and shake vigorously. Stopper
the test tube and heat at 120 oC for 1 h. Centrifuge the
hydrolysate, transfer the clear supernatant liquid into a
50 mL ftask, add 10 mL of water R and evaporate to dryness
under reduced pressure. Take up the residue in 10 mL of
water R and evaporate again to dryness under reduced
pressure. Take up the residue with 2 mL of methanol R.
Referenee solution (a) Dissolve 10 mg of arabinose R in a
small quantity of water R and dilute to 10 mL with
methanol R.
Reference solutwn (b) Dissolve 10 mg of xylose R in a smaIl
quantity of water R and dilute to 10 mL with methanol R.
Referenee solution (e) Dissolve 10 mg of galactose R in a
small quantity of water R and dilute to 10 mL with
methanol R.
Plate TLC silica gel plate R.
Mobile phase water R, acetonitrile R (15:85 V/V).
Application 10 ¡lL, as bands.
Development Over a path of 15 cm.
Detection Spray with aminohippurie acid reagent R and heat
at 120 oC for 5 min; examine in daylight.
Results The chromatogram obtained with the test solution
shows 2 orange-pink zones (arabinose and xylose) and a
yellow zone (galactose) similar in position and colour 10 the
zones in the chromatograms obtained with the reference
solutions.
TESTS
Foreign matter (2.8.2)
Carry out the determination using 5.0 g.
Ispaghula Preparations IV-221
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 4.0 per cent.
Swelling index (2.8.4)
Minimum 40, determined on 0.1 g of the powdered drug
(355) (2.9.12) .
~ __________________________________________ PhE~
Ispaghula Husk Granules
DEFINITION
Ispaghula Husk Granules contain Ispaghula Husk with or
without suitable excipients.
The granules eomply with the requirements stated under Granules
and with the following requirements.
IDENTIFICATION
A. Powder the granules and examine under a microscope
using laetic reagent. Fragments of the episperm with polygonal
cells filled with mucilage and fragments of the inner layers of
the testa with brownish thin-walled cells often associated with
the outer layers of the endosperm are seen.
B. When mounted in ruthenium red solution, the particles of
the powder are stained red.
TESTS
Swelling index
Not less than 40, Appendix XI C. Use a quantity of the
granules containing 1.0 g of Ispaghula Husk and a 100-mL
ground-glass-stoppered cylinder graduated in 1 mL divisions.
Loss on drying
When dried at 100° to 105°, loses not more than 12 .0% of
its weight. Use 1 g.
Ash
Not more than 5.0%, Appendix XI J, Method II.
STORAGE
Ispaghula Husk Granules should be protected from moisture.
Ispaghula Husk Effervescent Granules
DEFINITION
Ispaghula Husk Effervescent Granules contain Ispaghula
Husk in a suitable, effervescent basis.
The granules eomply with the requirements stated under Granules
and with the following requirements.
TESTS
Disintegration
Carry out the test stated under Effervescent Granules with
the foIlowing modifications. Stir the contents of the beaker
occasionally 10 disperse the mucilage formed; evolution of gas
is complete after 5 minutes.
Swelling index
Not less than 40, Appendix XI C. Use a quantity of the
powdered granules containing 1.0 g of Ispaghula Husk and a
100 mL ground-glass-stoppered cylinder graduated in 1 mL
divisions .
IV-222 Ispaghula Preparations
Ispaghula Husk Oral Powder
DEFINITION
Ispaghula Husk Oral Powder contains Ispaghula Husk with
or without suitable excipients.
The powder eomplies with the requirements stated under Oral
Powders and with the following requirements.
IDENTIFICATION
A. Carry out the method for thin-Iayer ehromatography,
Appendix III A, using the following solutions.
(1) Add 2 mL of a 23% w/v solution of trifluoroaeetie aeid to
a quantity of the powder containing 10 mg of Ispaghula
Husk in a thick-walled centrifuge tube, shake vigorously,
close the tube and heat at 120° for 1 hour. Centrifuge the
hydrolysate, transfer the clear supernatant liquid into a
50 mL flask, add 10 mL of water and evaporate the solution
to dryness under reduced pressure. Take up the residue in
10 mL of water, again evaporate to dryness under reduced
pressure and take up the residue in 2 mL of methanol.
(2) Dissolve 10 mg of arabinose in a small quantity of water
and dilute to 10 mL with methanol.
(3) Dissolve 10 mg of xylose in a small quantity of water and
dilute to 10 mL with methanol.
(4) Dissolve 10 mg of galaetose in a small quantity of water
and dilute to 10 mL with methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel.
(b) Use the mobile phase as described below.
(c) Apply 10 JlL of each solution.
(d) Develop the plate to 15 cm.
(e) Afier removal of the plate, dry in air, spray with
0
aminohippurie acid reagent, heat at 120 for 5 minutes and
examine in daylight.
MOBILE PHASE
15 volumes of water and 85 volumes of aeetonitrile.
CONFIRMATION
The chromatogram obtained with solution (1) shows two
orange-pink zones (arabinose and xylose) and a yellow zone
(galactose) similar in position and colour to the zones in the
chromatograms obtained with solutions (2), (3) and (4).
B. When mounted in ruthenium red solution, the particles of
the powder are stained red.
TESTS
Swelling index
Not less than 40, Appendix XI C. Use a quantity ofthe oral
powder containing 1.0 g of Ispaghula Husk and a 100 mL
ground-glass-stoppered cylinder graduated in 1 mL divisions.
Ash
Not more than 4.0%, Appendix XI J, Method Il. Use a
quantity of the powder containing 1 g of Ispaghula Husk.
STORAGE
Ispaghula Husk Oral Powder should be protected from
moisture.
2014
Ispaghula Seed ***
*** ***
(Ph Eur monograph 1333) ***
~E~ ____________________________________________
DEFINITION
Dried ripe seeds of Plantago ovata Forssk. (P. ispaghula
Roxb.).
IDENTIFICATION
A. Ispaghula seed is pinkish-beige, smooth, boat-shaped and
curved. It is l.5 mm to 3.5 mm long, l.5 mm to 2 mm wide
and 1 mm to l.5 mm thick. The concave surface shows in
the centre a light coloured spot corresponding to the hilum.
The convex surface shows a light brown spot corresponding
to the location of the embryo and takes up about one quarter
of the length of the seed.
B. Reduce to a powder (355) (2.9.12). The powder is pale
brown. Examine under a microscope using lactie reagent R.
The powder shows mainly fragments of the episperm with
polygonal cells filled with mucilage; fragments of the inner
layers of the testa with brownish thin-walled cells ofien
associated with the outer layers of the endosperm; fragments
of the endosperm with cells with thick cellulose walls
containing aleurone grains and oil droplets; a few fragments
of embryo with thin-walled cells. Examine under a
microscope using a 50 per cent V/V solution of glyeerol R .
The powder shows starch granules, single or in groups of
2 to 4 and measuring 3 !lm to 25 !lm in diameter.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 50 mg of the powdered drug (355) (2.9.12)
in a thick-walled centrifuge tube add 2 mL of a 230 giL
solution of trifluoroaeetie aeid R, and shake vigorously.
Stopper the test tube and heat the mixture at 120 oC for 1 h.
Centrifuge the hydrolysate, transfer the clear supernatant
liquid into a 50 mL flask, add 10 mL of water R and
evaporate the solution to dryness under reduced pressure.
Take up the residue in 10 mL of water R and evaporate again
to dryness under reduced pressure. Take up the residue in
2 mL of methanol R .
Reference solution (a) Dissolve 10 mg of arabinose R in a
small quantity of water R and dilute to 10 mL with
methanol R.
Referenee solution (b) Dissolve 10 mg of xylose R in a small
quantity of water R and dilute to 10 mL with methanol R.
Reference solution (e) Dissolve 10 mg of galaetose R in a
small quantity of water R and dilute to 10 mL with
methanol R.
Plate TLC siliea gel plate R
Mobile phase water R, aeetonitrile R (15:85 V/V).
Applieation 10 !lL, as bands.
Development Over a path of 15 cm.
Deteetion Spray with aminohippurie acid reagent R and heat
at 120 oC for 5 mino Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference and the test
solutions.
2014 Ivy Leaf IV-223

Top of the plate side view [A]; cluster crystals of calcium oxalate, about
40 flm in diameter, scauered [C] or occurring throughout the
Xylose: an orange-pink zone An orange-pink zone (xylose)
parenchyma (Ed, Fe) ; groups of lignified fibro-vascular tissue
Arabinose: an orange-pink zone An orange-pink zone (arabinose) from the veins [D].
Galactose: a yellow zone A yellow zone (galactose)
Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Carry out the determination using 10.0 g.
Swelling index (2.8.4)
Minimum 9.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4. 16)
Maximum 4.0 per cent.
____________________________________________ ~Ew

Ivy Leaf
(Ph Eur monograph 2148)
~Ew ____________________________________________

DEFINITION
Whole or cut, dried leaves of Hedera helix L., collected in
spring and summer.
Content
Minimum 3.0 per cent of hederacoside C (Cs9H96026; M r
1221) (dried drug).
Figure 2148.-1. - Illuslration for identification test B of
IDENTIFICATION powdered herbal drug of ivy leaf
A. Whole leaves are coriaceous, 4-10 cm in length and width,
cordate at the base. The lamina is palmately 3-5 lobed, the C . Thin-Iayer chromatography (2.2.27).
lobes more or les s triangular with entire margins . The upper
Test solution Extract 0.50 g ofthe powdered herbal drug
surface is dark green with a paler, radiate venation, the lower
(355) (2.9.12) under a reflux condenser in a water-bath at
surface more greyish-green and the venation is distinctly
60 oC with 5 mL of methanol R for 30 mino Cool and filter.
raised. The petioles are long, cylindrical, about 2 mm in
diameter and grooved longitudinally. Scattered white hairs Referenee solution Dissolve 1.0 mg of hederaeoside C R and
occur on the petioles and on the surfaces of younger lea ves, 1.0 mg of ex-hederin R in 1.0 mL of methanol R.
the older leaves are glabrous. Occasional entire, ova te- Plate TLC si/iea gel place R .
rhombic lO lanceolate leaves 3-8 cm long from the flowering Mobile phase anhydrous fonnie acid R , aeetone R, methanol R,
stems may be presento ethyl aeetate R (4:20:20:30 VIVIVIV) .
B. Microscopic examination (2.8.23). The powder is green. Applieation 20 flL as bands of 15 mm.
Examine under a microscope using ehloral hydrate solution R. Development Over a path of 12 cm.
The powder shows the following diagnostic characters
Drying At 100-105 oc.
(Figure 2148.-1): fragments ofthe upper epidermis, in
surface view [F], showing cells with thickened, rather Deteetion Treat with alcoholie solution of sulfurie acid R, heat
sinuous, finely pitted anticlinal walls [Fa] usually at 110 oC for 10 min and examine in daylight.
accompanied by underlying palisade parenchyma [Fb] Results See below the sequence of zones present in the
including sorne cells containing cluster crystals of ca1cium chromatograms obtained with the reference solution and the
oxalate [Fe]; fragments of the lower epidermis, in surface test solution. Furthermore, other zones may be present in the
view [E], showing cells with sinuous, irregularly thickened chromatogram obtained with the test solution.
and pitted walls [Ea] , slOmata that are mostly anomocytic
[Eb] but occasionally anisocytic (2.8.3), surrounded by cells
including sorne that show faint cuticular striations; the lower
epidermis is accompanied by underlying spongy parenchyma
[Ec] including sorne cells containing cluster crystals of
ca1cium oxalate [Ed]; scauered stellate covering trichomes
may be present, composed of 4-8 branches joined at the base
on a multicellular, biseriate stalk, in surface view [B] or in
IV-224 Java Tea 2014

Top of the plate Injeetion 20 JlL.

A green zone System suitability Reference solution:
-- retention time: hederacoside C = about 20 min;
- -- --- if necessary, adjust the time intervals of the gradient.
(X-Hederin: a purple zone A very faint purple zane Calculate the percentage content of hederacoside C with
«X-hederin)
reference to the dried drug using the following expression:
A broad yellow zone
2-3 purple or green zanes
--- ---

Hederacoside e: a purple zone A purple zone (hederacaside C)

Reference solution Test solution are a of the peak due to hederacoside C in the
chromatogram obtained with the test solution;
area of the peak due to hederacoside C in the
TESTS chromatogram obtained with the reference solution;
Foreign matter (2.8.2) mass of the herbal drug to be examined used to
Maxirnum 10 per cent of discoloured leaves, maximum prepare the test solution, in grams;
10 per cent of stems, and maximum 2 per cent of other mass of ivy leaf tincture HRS used to prepare the
foreign matter. reference solution, in grams;
Loss on drying (2.2.32) p percentage content of hederacoside C in ivy leaf
Maximum 10.0 per cent, determined on 1.000 g of the tineture HRS.
powdered herbal drug (355) (2.9.12) by drying in an oven at ____________________________________________ ~E~

105 oC for 2 h .
Total ash (2.4.16)
Maxirnum 10.0 per cent.
ASSAY Java Tea ****
Liquid chromatography (2.2.29). ** *
(Ph Bur monograph 1229) *****
Solvent mixture water R, methanol R (20:80 V/V).
Ph Eur ________________ ______________________
Test solution To 1.00 g of the powdered herbal drug (355)
(2.9.12) in a 250 mL round-bottomed fiask add 50 mL of DEFINITION
the solvent mixture and heat under a refiux condenser in a Fragmented, dried lea ves and tops of stems of Orthosiphon
water-bath at 80 oC for 1 h. Cool and filter through a plug of stamineus Benth. (O. aristatus Miq.; O. spieatus Bak.).
absorbent cotton into a 100 mL volumetric fiask. The plug
Content
of absorbent cotton together with the residue is again
Minimum 0.05 per cent of sinensetin (C2oH2007; M r 372.4)
extracted with 30 mL of the solvent mixture under refiux for (dried drug).
30 mino Filter and combine the filtra tes. Rinse the round-
bottomed fiask and the plug of absorbent cotton with the IDENTIFICATION
solvent mixture and use the solvent mixture to dilute the A. The leaves are friable, up to 7.5 cm in length and 2.5 cm
contents of the volumetric fiask to exactly 100.0 mL. Filter in width. The petiole is short. The lamina is oval or
through a suitable membrane before use. lanceolate, the apex acuminate and the base cuneate.
Referenee solution Dissolve an amount of ivy leaf tineture The abaxial surface of the lea ves is light greyish-green and
HRS corresponding to 3.0 mg ofhederacoside C in the adaxial surface is dark green or brownish-green.
methanol R and dilute to 5.0 mL with the same solvento The venation is pinnate with few secondary veins. Examined
under a lens (x 10), the secondary veins, after running
Column:
parallel to the midrib, diverge at an acute angle. The margin
-- size: 1 = 0.125 m, 0 = 4 mm;
is irregularly and roughly denta te, sometirnes crenate and the
-- stationary phase: end-eapped octadecylsilyl siliea gel for
abaxial surface is slightly curved. The petioles are thin,
ehromatography R (5 Jlm).
quadrangular, 4-8 mm long and, like the primary venation,
Mobile phase:
usually violet-coloured. Occasionally, infiorescences in
-- mobzle phase A: mix 14 volumes of aeetonimle R with c1usters of bluish-white or violet fiowers, not yet opened, are
88 volumes of water R and adjust to pH 2.0 with
found.
phosphorie aeid R;
-- mobile phase B: phosphorie acid R, aeetonitrile R B. Reduce to a powder (355) (2.9.12). The powder is dark
(0 .2:99.8 V/V); green. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
Time MobiJe phase A MobiJe phase B characters: fragments of epidermis, with cells with sinuous
(m in) (pereent VM (per cent VM outlines, bearing unicellular or bicellular conical covering
O-S 100 O
trichomes and articulated uniseriate trichomes up to 450 ~lm
5-6 100 -> 94 0->6 long, consisting of 3-8 cells with thick pitted walls; capitate
6 - 40 94 -> 60 6 -> 40
trichomes with unicellular or bicellular heads; secretory
trichomes with unicellular stalks and usually tetracellular
40·41 60 -> O 40 -> 100 heads; diacytic stomata (2.8.3), which are more numerous on
41 - 55 O 100 the lower epidermis.

Flow rate 1.5 mUmin.

Deteetion Spectrophotometer at 205 nm.
 2014 Java Tea I V-225

Top of the plate

1 or 2 more or less intense blue or
violet-blue fluoreseent zones
--- ---

Sinensetin : an intense light blue A major blue fluoreseent zone

fluoreseent zone (sinensetin)
--- ---

2 bluish fluoreseent zones

Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems with a diameter greater than
1 mm and maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 1l.0 per cent, detennined on 1.000 g of the
powdered drug (355) (2.9. 12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 12.5 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Heat 2.5 g of the powdered drug (355)
(2.9.12) and 100 mL of methylene chloride R on a water-bath
for 30 min with stirring. Filter. Collect the filtrate and repeat
the operation twice, in the same manner, on the filtration
residue. Combine the filtrates. Evaporate the solvent under
A. Lamina, in transverse section, F. Margin 01 the lamina reduced pressure . Dissolve the residue in 25.0 mL of the
showing a secretory trichome with a G. Secretory trichome mobile phase, using an ultrasonic bath if necessary. Filter the
tetracellular head (Aa) and a capitate
lrichome with a unicellular head (Ab) H. Lower epidermis, in surface view, solution through a nitrocellulose filter with a pore size of
with diacytic sto mata (Ha), capitate
B. Upper epidermis, in surface view, trichome with a unicellular head 0.45 flm .
showing a capitate trichome with a (Hb) and multicellular eovering
bicellular head (Ba) and underIying Reference solution Dissolve 5 mg (m2) of sinensetin R in
trichome (He)
palisade parenchyma (Bb) 80 mL of the mobile phase using an ultrasonic bath if
J. Covering trichomes on the margin
C. and E. Articulated covering of the lamina necessary and dilute to 100.0 mL with the mobile phase.
trichomes (usually only fragments
observed) Column:
D. Lower epidermis, in surlace -- size: 1 = 0.25 m, 0 = 4.6 mm;
view, with diacytic stomata (Da) -- stationary phase: octadecylsilyl szlica gel for chromatography R
and secretory trichome with a
tetracellular head (Db) (5 !lm).
Mobz7e phase tetrahydrofuran R, acetic acid R, water R,
Figure 1229.-1. - Illustration of powdered herbal drug ofJava
methanol R (5:8:42:45 VIVIVIV).
tea (see Identification B)
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 258 nm.
C. Thin-Iayer chromatography (2.2.27) .
Injection 20 ¡.¡L.
Test solution Shake 1 g ofthe powdered drug (710) (2.9.12)
Calculate the percentage content of sinensetin using the
with 10 mL of methanol R in a water-bath at 60 oC for 5 min
following expression:
and filter the cooled solution.
Reference solution Dissolve 1 mg of sinensetin R in methanol R
and dilute to 20 mL with the same solvent.
Plate TLC silica gel plate R.
Mobile phase methanol R , ethyl acetate R, toluene R
(5:40:55 VIVIV) . area of the peak due to sinensetin in the
chromatogram obtained with the test solution;
Application 10 flL as bands.
are a of the peak due to sinensetin in the
Development Over a path of 10 cm. chromatogram obtained with the reference solution;
Drying In air. mass of the drug to be examined, in grams;
Detection Examine in ultraviolet light at 365 nm. mass of sinensetin in the reference solution, in grams.
Results See below the sequence of the zones present in the ______________________________________________ Ph Em

chromatogram obtained with the reference solution and the

test solution. Furthermore, red fiuorescent zones are present
in the lower third and near the solvent front of the
chromatogram obtained with the test solution.
 IV-226 Juniper 2014

obtained with the reference solution, a greyish-violet zone

Juniper (terpinen-4-01) a little below the zone due to cineole in the
(Ph Eur monograph 1532) chromatogram obtained with the reference solution, and just
~E~ ____________________________________________ below that a blue zone; a faint violet zone may be present in
a similar position to the zone due to cineole; further zones
DEFINITION are presento
Dried ripe cone berry of Juniperus communis L.
TESTS
Content Foreign matter (2.8.2)
Minimum 10 mlJkg of essential oil (anhydrous drug). Maximum 5 per cent of unripe or discoloured cone berries
CHARACTERS and maximum 2 per cent of other foreign matter.
Strongly aromatic odour, especially if crushed. Water (2.2.13)
IDENTIFICATION Maximum 120 mUkg, determined on 20.0 g of the crushed
A. The berry-shaped cone is globular, up to 10 mm in drug.
diameter, and violet-brown or blackish-brown, frequently Total ash (2.4.16)
with a bluish bloom. It consists of 3 fteshy scales. The apex Maximum 4.0 per cenr.
has a 3-rayed closed cleft and 3 not very clearly defined ASSAY
projections. A remnant of peduncle is frequently attached at Carry out the determination of essential oils in herbal drugs
the base. The fteshy part is crumbly and brownish. (2.8.12). Use 20.0 g ofthe herbal drug reduced to a coarse
It contains 3 or, more rarely, 2 small, elongated, extremely powder using a suitable mili immediately before the assay, a
hard seeds that have 3 sharp edges and are slightly rounded
500 mL round-bottomed ftask, 200 mL of water R as the
at the back, acuminate at the apex. The seeds are fused with distillation liquid and 0.5 mL of xylene R in the graduated
the fteshy part of the cone berry in the lower part on the
tube. Distil at arate of 3-4 mUmin for 90 mino
outside of their bases. Very large, oval oil glands containing
____________________________________________ Ph Eur
sticky resin lie at the outer surface of the seeds.
B. Microscopic examination (2.8.23) . The powder is brown.
Examine under a microscope using chloral hydrate solutian R .
The powder shows the following diagnostic characters:
fragments of epidermis of the cone berry wall containing cells Juniper Oil ***
with thick, pitted, colourless walls and brown glandular *** ***
content, occasio=ally with anomocytic stomata (2.8.3); (Ph Eur monograph 1832) ***
fragments of the 3-rayed apical cleft of the cone berry with ~E~ ____________________________________________
spaces and epidermal cells interlocked by papillous
outgrowths; fragments of the hypodermis with DEFINITION
collenchymatous thickened cells; fragments of the mesocarp Essential oil obtained by steam distillation from the ripe,
consisting of large thin-walled parenchymatous cells, usually non-fermented berry eones of Juniperus communis L.
rounded, with large intercellular spaces and irregular, large, A suitable antioxidant may be added.
usually scarcely pitted, yellow idioblasts (barrel cells); CHARACTERS
fragments of schizogenous oil cells; fragments of the testa Appearance
with thick-walled, pitted, colourless sclereids containing 1 or Mobile, colourless or yellowish liquid.
several prism crystals of calcium oxalate; fragments of the Characteristic odour.
endosperm and embryonic tissue with thin-walled cells
containing fatty oil and aleurone grains. IDENTIFICATION
C. Thin-Iayer chromatography (2.2.27). First identification R
Second identification A.
Test solution Dilute the oil-xylene mixture obtained in the
assay to 5.0 mL with hexane R. A. Thin-Iayer chromatography (2.2.27) .
Reference solution Dissolve 4.0 mg of guaiazulene R and Test solution Dissolve 0.2 mL of the substance to be
50 ¡.tL of cineole R in 10 mL of hexane R. examined in 5 mL of heptane R.
Plate TLC silica gel plate R. Reference solution Dissolve 20 mg of a-terpineol R and 20 ¡.tL
Mobile phase ethyl acetate R, toluene R (5:95 V/V). of terpinen-4-ol R in 25 mL of heptane R.
Plate TLC silica gel plate R.
Application 20 ¡.tL of the test solution and 10 ¡.tL of the
reference solution, as bands . Mobile phase ethyl acetate R, toluene R (5:95 V/V) .
Development Over a path of 15 cm. Application 20 ¡.tL, as bands.
Drying In airo Development Over a path of 12 cm.
Detection Treat with anisaldehyde solution R, heat at Drying In air.
100-105 oC for 5-10 min and examine in daylight. Detection Treat with anisaldehyde solution R and heat at
Results The chromatogram obtained with the reference 100-105 oC until the zones appear; examine immediately in
solution shows a red zone (guaiazulene) in the upper half and daylight.
a brownish-violet or greyish-violet zone (cineole) in the lower Results See below the sequence of zones present in the
half; the chromatogram obtained with the test solution shows chromatograms obtained with the reference solution and the
a strong violet zone (mono- and sesquiterpenes) similar in test solution.
position to the zone due to guaiazulene in the chromatogram
obtained with the reference solution, a reddish-violet zone a
little aboye the zone due to cineole in the chromatogram
2014 Kelp IV-227

Top of Ihe plale Elution order Order indicated in the composition of the
reference solution. Record the retention times of these
An intense brownish·violet zone
substances.
A brown zone System suitability Reference solution:
A violet·pink zone -- resolution: minimum 1.5 between the peaks due to
sabinene and ~-pinene.
Terpinen4-o1: a brownish·violet A brownish·violet zone
zone (terpinen4·01) U sing the retention times determined from the
A violet zone chromatogram obtained with the reference solution, locate
a·Terpineol: a violet or A violet or brownish·violet zone the components of the reference solution in the
brownish·violet zone (a·terpineol) chromatogram obtained with the test solution.
Reference solution Test solulion Determine the percentage content of the components.
Disregard the peak due to trimethylpentane and peaks
comprising les s than 0.01 per cent ofthe total surface area.
B. Examine the chromatograms obtained in the test for The percentages are within the following ranges:
chromatographic profile. -- rx-pinene: 20 per cent to 50 per cent;
Results The characteristic peaks in the chromatogram -- sabinene: maximum 20 per cent;
obtained with the test solution are similar in retention time to -- f-pinene: 1.0 per cent to 12 per cent;
those in the chromatogram obtained with the reference -- f-myrcene: 1.0 per cent to 35 per cent;
solution. -- rx -phellandrene: maximum 1.0 per cent;
TESTS -- limonene: 2.0 per cent to 12 per cent;
-- terpinen-4-ol: 0.5 per cent to 10 per cent;
Relative density (2.2.5)
-- bomyl acetate: maximum 2.0 per cent;
0.857 to 0.876.
-- f -caryophyllene: maximum 7.0 per cent.
Refractive index (2.2.6)
1.471 to 1.483. STORAGE
Optical rotation (2.2.7)
At a temperature not exceeding 25 oc.
____________________________________________ ~Ew

- ISo to - 0.5°.
Peroxide value (2.5.5)
Maximum 20.

*****
Fatty oils and resinified essential oils (2.8.7)
Kelp
It complies with the test for fatty oils and resinified essential ** **
oils. Bladderwrack; Fucus ***
Chromatographic profile (Ph Eur monograph 1426)
Gas chromatography (2.2.28): use the normalisation ~Ew ____________________________________________
procedure.
DEFINITION
Test solution Dissolve 60 mg of the substance to be
examined in trimethylpentane R and dilute to 5.0 mL with the Fragmented dried thallus of Fucus vesiculosus L. or F. serratus
same solvent. L. or Ascophyllum nodosum Le Jolis.
Reference solution Mix 25 !lL each of rx-pinene R, sabinene R, Content
f-pinene R, f-myrcene R, rx-phellandrene R, limonene R, Minimum 0.03 per cent and maximum 0.2 per cent of total
terpinen-4-ol R, bomyl acetate R and f-caryophyllene R and iodine (A r 126.9) (dried drug) .
dilute to 25 .0 mL with trimethylpentane R. CHARACTERS
Column: Salty and mucilaginous taste.
-- material: fused silica; Unpleasant marine odour.
-- size: 1 = 30 m (a film thickness of 1 pm may be used) to
60 m (a film thickness of 0.2 !lm may be used), IDENTIFICATION
0= 0.25-0.53 mm; A. The drug consists of fragments with a corneous
-- stationary phase: poly(dimethyl) (diphenyl) siloxane R. consistency, blackish-brown to greenish-brown, sometimes
covered with whitish effiorescence. The thallus consists of a
Canier gas helium for chromatography R.
ribbon-like blade, branching dichotomously with prominent
Flow rate 2.0 mUmin. central ribs (pseudoveins). F. vesiculosus typically shows a
Split ratio 1:50. foliose blade with smooth edges and bears occasional ovoid,
Temperature: single or paired, air vesicles. The ends of certain branches are
of ovoid shape and a liule widened. They bear numerous
Time Temperalure
(min) (OC) reproductive organs (conceptacles). F. serraLUS has a foliose
Colurnn 0·1 60
blade with a serrate margin and no vesicles, the branches
bearing conceptacles are less swollen. The thallus of
1· 58 60~230
A. nodosum is irregularly branched, without pseudo-midrib.
Injection port 250 It shows single ovoid air vesicles; the falciform conceptacles
Detector 250
are located at the end of small branches.
B. Reduce to a powder (355) (2.9.12). The powder is
Detection Flame ionisation. greenish-brown. Examine under a microscope using chloral
Injection 0.5 pL. hydrate solution R. The powder shows fragments of surface
tissue with regular isodiametric cells with brown contents,
and fragments of deep tissue with colourless, elongated cells
IV-228 Knotgrass
arranged in long filaments with large mucilaginous spaces
between them. Thick-walled cells in files and in closely
packed groups, from the pseudovein, are sometimes visible.
C . To 1 g of the powdered drug (355) (2.9.12) add 20 roL
of a 2 per cent VIV solution of hydrochloric acid R. Shake
vigorously and filter. Wash the residue with 10 mL of
water R and filter. To the residue add 10 mL of a 200 giL
solution of sodium carbonate R. Shake and centrifuge. Collect
the supernatant liquido Adjust to pH 1.5 using sulfuric acid R.
A white, flocculent precipitate is slowly formed.
TESTS
Arsenic (2.4.27)
Maximurn 90 ppm.
Cadmium (2.4.27)
Maximum 4 ppm.
Lead (2.4.27)
Maximum 5 ppm.
Mercury (2.4.27)
Maximum 0.1 ppm.
Swelling index (2.8. 4)
Minimum 6.
Loss on drying (2.2.32)
Maximum 15.0 per cent, determined on 1.000 g by drying in
an oven at 105 oC, for 2 h .
Total ash (2.4. 16)
Maximum 24 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximurn 3.0 per cent.
ASSAY
Total iodine
To 1.000 g of the powdered drug, in a tall silica crucible,
add 5 mL of water R and 5 g of potassium hydroxide R. Stir
with a magnesium rod. Heat on a water bath. Add 1 g of
potassium carbonate R. Mix, add the tip of the magnesium rod
with the residues of the drug and dry, first on a water-bath
then over an open flameo Incinerate raising the temperature
progressively to not more than 600 oc. Allow to cool.
Add 20 mL of water R and heat gently to boiling, stirring
with a glass rod. Filter the hot mixture through an unpleated
filter, into a conical flask. Rinse the residue with 4 quantities,
each of 20 mL, of hot water R. Rinse the filter and the
crucible with 50 mL of hot water R. Combine the solutions.
Allow ro cool. Neutralise with dilute sulfuric acid R in the
presence of methyl orange solution R. Add 3 mL of dilute
sulfuric acid R and 1 mL of bromine water R . The solution is
yellow. After 5 min add 0.6 mL of a 50 giL solution of
phenol R . The solution is clear. Acidify with 5 roL of
phosphoric acid R and add 0.2 g of potassium iodide R. Allow
to stand for 5 min protected from light. Add 1 mL of starch
solution R and titrate with O. 01 M sodium thiosulfate.
1 mL of O. 01 M sodium thiosulfate is equivalent to 0.2115 mg
ofiodine.
LABELLING
The label sta tes the species of kelp present.
____________________________________________ ffiEm
2014
Knotgrass ******
**
(Ph Eur monograph 1885) *** *
ffiEm ____________________________________________
DEFINITlON
Whole or fragmented, dried flowering aerial parts of
Polygonum aviculare L. s.l.
Content
Minimum 0.30 per cent of flavonoids, expressed as
hyperoside (C21H20012; M r 464.4) (dried drug).
IDENTIFICATlON
A. The stem is 0.5-2 mm thick, branched, with nodes,
cylindrical or slightly angular, and longitudinally striated.
Ir bears sessile or shortly petiolate, glabrous, entire leaves,
which differ widely in shape and size. The sheath-like stipules
(ochrea) are lacerate and silvery. The small, axillary flowers
have 5 greenish-white perianth segments, the tips of which
are often red. The dry, indehiscent fruits are 2-4 mm, brown
or black, triangular, usually punctate or striate .
B. Microscopic examination (2.8.23) . The powder is
greenish-brown. Examine under a microscope using chloral
hydrate solution R . The powder shows the following diagnostic
characters (Figure 1885.-1) : fragments oflower [A] and
upper [D] leaf epidermis es with a striated cuticle and
anisocytic sromata (2.8.3) [Aa, Da]; polygonal cells ofthe
upper epidermis [D] with slightly thickened beaded walls,
often associated with palisade parenchyma [Db]; cells of the
lower epidermis [A], with thin, sinuous walls; fragments of
the margin of the lamina of the leaf with irregular cells m;
fragments of parenchyma [G] with numerous cells containing
cluster crystals of calcium oxalate, sorne of which are very
large [Galo often associated with ves seIs [Gb]; groups of
fibres [B, C] with thick walls [Ba, Cb] from the hypodermis
of the stem associated either with the epidermis [Ca] or with
parenchyma consisting of cells containing cluster crystals of
calcium oxalate [Bb]; fragments of the ochrea [E] with
elongated, thin-walled cells [Ea], along which run very
elongated fibres [Eb]; globular pollen grains with a smooth
exine and 3 germinal pores [H]; occasional brown fragments
of the exocarp composed of cells with thick, sinuous walls
[F] .
C. Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the powdered herbal drug (355)
(2. 9.12) add 10 mL of methanol R . Heat the mixture in a
water-bath under a reflux condenser for 10 mino Cool and
filter.
Reference solution Dissolve 1 mg of caffeic acid R, 1 mg of
chlorogenic acid R and 2.5 mg of hyperoside R in 10 mL of
methanol R.
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (7:7 :14:72 VIVIVIV) .
Application 20 ¡.tL as bands.
Development Over a path of 10 cm.
Drying At 100-105 oC.
Detection Treat with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R; subsequently treat with a
50 gIL solution of macrogol 400 R in methanol R. Allow ro dry
in air for about 30 mino Examine in ultraviolet light at
365 nm.
2014 Knotgrass IV-229

Loss on drying (2.2.32)

Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (710) (2.9.12) by drying in an oven at
105 oC for 2 h.

Total ash (2.4.16)

Maximum 10.0 per cent.

ASSAY
Stock solution In a 100 mL round-bottomed flask, place
0.800 g ofthe powdered herbal drug (355) (2.9.12), and add
1 mL of a 5 gIL solution of hexamethylenetetramine R, 20 mL
of acetone R and 2 mL of hydrochloric acid R1 . Boil the
mixture under a reflux condenser for 30 mino Filter the
liquid through a plug of absorbent cotton into a flask.
Add the absorbent cotton to the residue in the round-
bottomed flask and extract with 2 quantities, each of 20 mL,
of acetone R, each time boiling under a reflux condenser for
10 mino Allow to cool, filter each extract through the plug of
absorbent cotton into the flask. Filter the combined acetone
extracts through a filter paper into a volumetric flask and
dilute to 100.0 mL with acetone R, rinsing the flask and the
filter paper. Introduce 20.0 mL of the solution into a
separating funnel, add 20 mL of water R and shake the
mixture with 1 quantity of 15 mL and then 3 quantities, each
of 10 mL, of ethyl acetate R. Combine the ethyl acetate
extracts in a separating funnel and wash with 2 quantities,
each of 50 mL, of water R. Dry the extracts over 10 g of
anhydrous sodium sulfate R, filter into a 50 mL volumetric
flask and dilute to volume with ethyl acetate R.

Figure 1885.-1. - Illustration for identification test B of
powdered herbal drug of knotgrass
Results See below the sequence of fluorescent zones present
in the chromalOgrams obtained with the reference solution
and the test solution. Furthermore, other fluorescent zones
are present in the chromatogram obtained with the test
solution.
Top of the plate
Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium ch/oride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R.
Compensation liquid Dilute 10.0 mL of the stock solution lO
25.0 mL with a 5 per cent V/V solution of glacial acetic
acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after
30 min by comparison with the compensation liquid at
425 nm. Calculate the percentage content of flavonoids,
calculated as hyperoside, using the following expression:

Caffeic acid: a light blue 1 or 2 blue f1uorescent zones A x 1.25

fluorescent zone (caffeic acid)
--- --- m

1 or 2 yellowish-green f1uorescent
zones
A yellow f1uore scent zone
Hyperoside: a yellowish·brown
f1uoresce nt zone
i.e. taking the specific absorbance of hyperoside to be 500.
A absorbance at 425 nm;
m = mas s of the herbal drug to be examined, in grams.
___ _ _ _ _ _ _ _ __ _ _ _ __ _ _ __ ____ ~E~

A yellowish·brown f1uorescent
zone
Chlorogenic acid: a Iight blue A light blue f1uorescent zone
f1uorescent zone (chlorogenic acid)
--- ---
A yellowish-brown f1uorescent
zone
Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Maximum 2 per cent of roots and maximum 2 per cent of
other foreign matter.
 IV-230 Kudzuvine Root 2014

*****
TESTS
Kudzuvine Root
** ** Foreign matter (2.8.2)
(Ph Eur monograph 2434) *** Maximum 5 per cent.
~Ew ____________________________________________ Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
DEFINITION
powdered herbal drug (355) (2.9.12) by drying in an oven at
Fragmented, dried root of Pueraria lobata (Willd.) Ohwi. 105 0e.
Content
Total ash (2.4.16)
Minimum 6.5 per cent of total isofiavonoids, expressed as
Maximum 7.0 per cent.
puerarin (C J2H 20 0 9 ; M r 416.4) (dried drug), ofwhich
minimum 45 per cent consists of puerarin. Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
IDENTIFICATION
A. Small, square pie ces or thick, rectangular slices, 5-35 cm ASSAY
long and 0.5-1 cm thick. The outer bark is pale brown, with Liquid chromatography (2.2.29).
longitudinal wrinkles and rough; the section is yellowish- Test solution Introduce 0.100 g of the powdered herbal drug
white and shows indistinct striations. The texture is strongly (355) (2.9. 12) into a 250 mL conical fiask, add 50.0 mL of
fibrous. ethanol (30 per eent V/V) R and weigh. Reat under a refiux
B. Microscopic examination (2.8.23) . The powder is pale condenser for 30 mino Allow to coo! and weigh again. Adjust
brown. Examine under a microscope using ehloral hydrate to the initial mas s with ethanol (30 per eent V/V) R, mix well
solution R. The powder shows the following diagnostic and filter.
characters: thick-walled lignified fibres, which occur in Referenee solution Introduce an amount of kudzuvine root dry
groups, surrounded by a calcium oxalate prism sheath; crystal extraet HRS corresponding to 3.0 mg of puerarin into a
cells with thickened walls; rare sclereids, subrounded or 250 mL conica! fiask, add 50.0 mL of ethanol (30 per eent
elliptical, about 50 ¡.tm in diameter; relatively large bordered- V/V) R and weigh. Reat under a refiux condenser for
pitted ves seIs with hexagonal or elliptical pits arranged very 30 mino Allow to coo! and weigh again. Adjust to the initia!
densly. Examine under a microscope using a 50 per cent V/V mass with ethanol (30 per cent V/V) R, mix well and filter.
solution of glyeerol R. The powder shows numerous starch Column 2 columns coupled in series:
granules, simple or 2-20 compound; the starch granules are -- size: 1 == 0.10 m, 0 == 4.6 mm;
spheroidal, semi-rounded or polygonal with a pointed, cleft -- stationary phase: monolithic octadecylsilyl si/iea gel for
or stellate hilum, about 15 ¡.tm in diameter. ehromatography R .
e. Thin-layer chromatography (2.2.27). Mobile phase:
Test solutwn Sonicate 0.5 g of the powdered herbal drug -- mobile phase A: glacial acetic acid R, water R
(355) (2.9. 12) with 5 mL of methanol R, then centrifuge; (0.1:99 .9 V/V);
use the supematant. mobile phase B: aeetonitrile R;
Referenee solutwn Dissolve 5 mg of puerarin R and 5 mg of Time Mobile phase A Mobile phase B
daidzin R in 5 mL of methanol R. (mio) (per cent VM (per ceot VI(/)
Plate TLC si/iea gel F 254 plate R (2-10 ¡.tm). 0·16.5 90 --7 71 10 --7 29

Mobile phase water R, methylene ehloride R, methanol R, ethyl Flow rate 3.0 mUmin.
aeetate R (10:20:22:40 VIVIV/V); use the lower layer.
Deteetion Spectrophotometer at 260 nm.
Applieation 7 ¡.tL as bands of 8 mm.
Infection 10 ¡.tL.
Development Over a path of 6 cm.
Identifieation of peaks Use the chromatogram supp!ied with
Drying In air.
kudzuvine root dry extract HRS and the chromatogram
Deteetion Examine in ultraviolet light at 254 nm. obtained with the reference solution to identify the peaks due
Results See below the sequence of zones present in the to the isofiavonoids (3-hydroxypuerarin, puerarin,
chromatograms obtained with the reference solution and the 3-methoxypuerarin, 6-0 "-D-xy!osylpuerarin and daidzin).
test solution. Furthermore, other zones may be present in the Relative retention With reference to puerarin (retention
chromatogram obtained with the test solution. time == about 3.4 min): 3-hydroxypuerarin = about 0.7;
3-methoxypuerarin == about 1.09; 6-0 "-D-
Top oC the plate
xylosylpuerarin = about 1.15; daidzin == about 1.4.
System suitability Reference so!ution:
A weak quenching zone
-- peak-to-valley ratio: minimum 10, where H p = height
--- --- aboye the baseline of the peak due to 3-methoxypuerarin
and H v == height aboye the baseline of the !owest point of
A quenching zone
the curve separating this peak from the peak due to
Daidzin: a quenching zone A quenching zone puerarin.
Puerarin: a quenching zone A quenching zone Calculate the percentage content of puerarin using the
following expression:
----- ---
At least 5 quenching zones

ReCerence solution Test solution

Al area of the peak due to puerarin in the

chromatogram obtained with the test solution;
 2014
A2 area of the peak due to puerarin in the
chromatogram obtained with the reference solution;
mI mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mz mass of kudzuvine root dry extraet HRS used to
prepare the reference solution, in grams;
p percentage content of puerarin in kudzuvine root dry
extract HRS.
Calculate the percentage content of total isofiavonoids
(3-hydroxypuerarin, puerarin, 3-methoxypuerarin, 6-0 "-D-
xylosylpuerarin and daidzin) using the folIowing expression:
sum of the areas of the peaks due to the
isofiavonoids (3-hydroxypuerarin, puerarin,
3-methoxypuerarin, 6-0"-D-xylosylpuerarin and
daidzin) in the chromatogram obtained with the
test solution;
area of the peak due to puerarin in the
chromatogram obtained with the reference solution;
mas s of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mass of kudzuvine root dry extraet HRS used to
prepare the reference solution, in grams;
p percentage content of puerarin in kudz uvine root dry
extraet HRS.
____________________________________________ ~E~
Thomson Kudzuvine Root ***
**
(Ph Eur monograph 2483) *****
~Ew ____________________________________________
DEFINITION
Whole or fragmented, dried root of Pueraria thomsonii Benth.,
with the outer bark removed.
Content
Minimum 0.4 per cent of total isofiavonoids, expressed as
puerarin (CI 2R 2009; M r 416.4) (dried drug), ofwhich
minimum 55 per cent consists of puerarin.
IDENTIFICATION
A. Cylindrical, subfusiform or semi-cylindrical, 12-15 cm
long and 4-8 cm in diameter, sometimes in 10ngitudinalIy or
obliquely cut thick slices, varying in size. ExtemalIy
yelIowish-white or pale brown. The root is heavy, texture
hard and starchy, a transverse section shows pale brown
concentric rings formed by fibres, a longitudinal section
shows several longitudinal striations formed by fibres.
B. Microscopic examination (2.8.23). The powder is
yelIowish-white. Examine under a microscope using ehloral
hydrate solution R . The powder shows the folIowing diagnostic
characters: thick-walIed lignified fibres, which occur in
groups, surrounded by a calcium oxalate prism sheath; crystal
celIs with thickened walIs; rare sc1ereids, subrounded or
elIiptical, about 50 ¡.tm in diameter; relatively large bordered-
pitted vesseIs with hexagonal or elliptical pits, arranged very
densely. Examine under a microscope using a
50 per cent V/V solution of glyeerol R . The powder shows
numerous starch granules, simple or 2-20 compound;
the starch granules are spheroidal, semi-rounded or polygonal
Thomson Kudzuvine IV-231
with a pointed, cleft or stelIate hilum, about 15 ¡.tm in
diameter.
C. Thin-layer chromatography (2.2.27) .
Test solution Sonicate 0.5 g of the powdered herbal drug
(355) (2.9.12) with 5 mL of methanol R , then centrifuge;
use the supematant.
Referenee solution Dissolve 5 mg of daidz in R and 5 mg of
puerarin R in 5 mL of methanol R .
Plate TLC siliea gel F254 plate R (2-10 ¡.tm).
Mobile phase water R, methylene ehloride R, methanol R, ethyl
aeetate R (10:20:22 :40 V/V/V/V); use the lower layer.
Applieation 7 ¡.tL as bands of 8 mm.
Development Over a path of 6 cm.
Drying In airo
Deteetion Examine in ultraviolet light at 254 nm.
Results See below the sequen ce of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
A weak quenching zone
----- - --
Daidzin: a quenching zone A weak quenching zone
Puerarin: a quenching zone A weak quenching zone
--- - --
Several qu enching zones
Reference solution Test solution
*
*
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g ofthe
powdered herbal drug (355) (2.9. 12) by drying in an oven at
105 oC .
Total ash (2. 4.16)
Maximum 7.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Liquid chromatography (2.2.29) .
Test solution Introduce 1.00 g of the powdered herbal drug
(355) (2.9.12) into a 250 mL conical fiask, add 50.0 mL of
ethanol (30 per eent V/V) R and weigh. Reat under a refiux
condenser for 30 mino AlIow to cool and weigh again. Adjust
to the initial mass with ethanol (30 per eent V/V) R, mix welI
and filter.
Referenee solution Introduce an amount of kudz uvine root dry
extraet HRS corresponding to 3.0 mg of puerarin into a
250 mL conical fiask, add 50.0 mL of ethanol (30 per eent
V/V) R and weigh. Reat under a refiux condenser for
30 minoAlIow to cool and weigh again. Adjust to the initial
mas s with ethanol (30 per eent V/V) R, mix welI and filter.
Column 2 columns coupled in series:
-- siz e: 1 = 0.10 m, 0 = 4.6 mm;
-- stationary phase: monolithie oetadeeylsilyl siliea gel for
ehromatography R.
 IV-232 Lavender Flower
Mobile phase:
-- mob17e phase A: glacial aeetie acid R, water R
(0.1:99.9 V/V);
-- mobile phase B: aeetonitrile R;
Time Mobile phase A Mobile phase B
(min) (percent VM (per cent VIII)
90....., 71 10....., 29
O • 16.5
Flow rate 3.0 mUmin.
Detection Spectrophotometer at 260 nm.
Injection 10 ¡.¡L.
Identifieation of peaks Use the chromatogram supplied with
kudzuvine root dry extract HRS and the chromatogram
obtained with the reference solution to identify the peaks due
to the isoflavonoids (puerarin, 3-methoxypuerarin, 6-0"-D-
xylosylpuerarin and daidzin).
Relative retention With reference to puerarin (retention
time = about 3.4 min): 6-0"-D-
xylosylpuerarin = about 1.15; daidzin = about 1.4.
System suitability Reference solution:
-- peak-to-valley ratio: minimum 10, where H p = height
aboye the baseline of the peak due to 3-methoxypuerarin
and H v = height aboye the baseline of the lowest point of
the curve separating this peak from the peak due to
puerarin.
Calculate the percentage content of puerarin using the
following expression:
Al area of the peak due to puerarin in the
chromatogram obtained with the test solution;
A2 area of the peak due to puerarin in the
chromatogram obtained with the reference solution;
mI mass of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mass of kudzuvine root dry extract HRS used to
prepare the reference solution, in grams;
p percentage content of puerarin in kudzuvine root dry
extraet HRS.
Calculate the percentage content of total isoftavonoids
(puerarin, 6-0"-D-xylosylpuerarin and daidzin) using the
following expression:
sum of the areas of the peaks due to the
isoftavonoids (puerarin, 6-0"-D-xylosylpuerarin and
daidzin) in the chromatogram obtained with the test
solution;
area of the peak due to puerarin in the
chromatogram obtained with the reference solution;
mI mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of kudzuvine root dry extraet HRS used to
prepare the reference solution, in grams;
p percentage content of puerarin in kudzuvine root dry
extract HRS.
----------___________________________________ ~Ew
2014
*****
Lavender Flower
**** **
*
(Ph Eur monograph 1534)
~Ew ____________________________________________
DEFINITION
Dried flower of Lavandula angustifolia MilI. (L. officinalis
Chaix).
Content
Minimum 13 mUkg of essential oil (anhydrous drug).
CHARACTERS
Strongly aromatic odour.
IDENTIFICATION
First identification A, B, D.
Second identification A, B, C.
A. The ftower has a short peduncle and consists of a bluish-
grey tubular calyx divided distally into 4 very short teeth and
a small rounded lobe, a blue bilabial corolla with the upper
lip bifid and the lower lip trilobate and 4 didynamous
stamens with ovoid anthers.
B. Microscopic examination (2.8.23) . The powder is bluish-
grey. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1534.-1): covering trichomes bifurcating at
one or more levels [C, L]; secretory trichomes with short
stalks and 8-celled heads of the Lamiaceae type in side view
[H], in surface view [M]; glandular trichomes with
unicellular [O] or multicellular [K] stalks and unicellular
heads; glandular trichomes with long uneven stalks and
unicellular heads, separated from the stalk by an intermediary
cell with a smooth cuticle, certain trichomes show a crown of
small spheroid protuberances just below the insertion point
of the intermediary cell on the stalk [G]; fragments of
papillose epidermis from the inner surface of the petals, in
surface view m, in side view [P]; fragments of calyx
epidermis with sinuous-walled cells and containing prismatic
crystals of calcium oxalate [Ol; spherical pollen grains which
have a diameter of about 45 ¡.¡m and an exine with 6 slit-like
germinal pores and 6 ribbon-like groins radiating from the
poles [A, D, E, F]; rare fragments of leaf epidermis with
stomata, mostly of the diacytic type (2.8.3) [B]; fragments of
vascular tissue with spiral ves seis included in parenchyma
with sorne cells containing small calcium oxalate cluster
crystals [N] .
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of hexane R, shake for 5 min and filter.
Reference solution Dissolve 10 ¡.¡L of linalol R and 10 ¡.¡L of
linalyl acetate R in 5 mL of hexane R.
Plate TLC silica gel plate R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 10 ¡.¡L as bands.
Development Over a path of 15 cm.
Drying In air.
Detection Spray with anisaldehyde solution R. Heat at
100-105 oC for 5-10 min and examine in daylight.
Results The chromatogram obtained with the reference
solution shows in the lower third a greyish-blue zone (linalol)
and in the middle third a greyish-blue zone (linalyl acetate).
The chromatogram obtained with the test solution shows the
zones due to linalol and linalyl acetate and in the middle,
between these zones, a redish-violet zone
(epoxydihydrocaryophyllene). Further zones are also presento
 2014 Lavender Oil IV-233

Temperature:
Time Temperature
(min) (OC)
Column 0 - 15 70
15 - 70 70 -> 180

Injection port 220

Detector 220

Detection Flame ionisation.

Figure 1534.-1. - Illustration for identification test B of
powdered herbal drug of lavender flower
D. Examine the chromatograms obtained in the test for other
species and varieties of lavender.
Results The 5 principal peaks in the chromatogram obtained
with the reference solution are similar in retention time to the
Injection The same volume of each solution.
Elution order Order indicated in the composition of the
reference solution. Record the retention times of these
substances.
System suitability Reference solution:
- resolution: minimum 1.5 between the peaks due to
limonene and cineole;
- number of theoretical plates: minimum 30 000, calculated
for the peak due to limonene at 110 D e.
U sing the retention times determined from the
chromatogram obtained with the reference solution, locate
the 6 components of the reference solution in the
chromatogram obtained with the test solution. Disregard the
peaks due to hexane and xylene.
Limit:
- camphor: maximum 1 per cent.
Water (2.2.13)
Maximum 100 mlJkg, determined on 20.0 g.
Total ash (2.4.16)
Maximum 9.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 20.0 g of the herbal drug, a 1000 mL round-
bottomed fiask, 500 mL of water R as the distillation liquid
and 0.5 mL of xylene R in the graduated tube. Distil at arate
of 2-3 mlJmin for 2 h.
____________________________________________ Ph E~

corresponding peaks in the chromatogram obtained with the

test solution. Among them are mainly linalol and linalyl
acetate peaks.
TESTS Lavender Oil
Foreign matter (2.8.2)
Maximum 3 per cent of stems and maximum 2 per cent of (Ph Eur monograph 1338)
other foreign matter. ~E~ ____________________________________________

Other species and varieties of lavender DEFINITION

Gas chromatography (2.2.28): use the normalisation
Essential oil obtained by steam distillation from the fiowering
procedure.
tops of Lavandula angustifolia MilI. (Lavandula officinalis
Test solution Dilute 0.2 mL of the essential oil-xylene Chaix).
mixture obtained in the assay to 5 mL with hexane R, add
1 g of anhydrous sodium sulfate R, shake and use the CHARACTERS
supernatant liquido Appearance
Colourless or pale yellow, clear liquido
Reference solution Dissolve 0.1 g of limonene R, 0.2 g of
cineole R, 0.05 g of camphor R, 0.4 g of linaZol R, 0.6 g of Odour
linalyl acetate R and 0.2 g of rx-terpineol R in 100 mL of Complex, reminiscent of linalyl aceta te.
hexane R. IDENTIFICATION
Column: First identification B.
- material: fused silica; Second identification A.
- size: l = 60 m, 0 = 0.25 mm;
A. Thin-Iayer chromatography (2.2.27).
- stationary phase: macrogol 20 000 R.
Test solution Dissolve 20 I-I-L of the oil to be examined in
Carrier gas helium for chromatography R.
1 mL of toluene R.
Flow rate 1.5 mlJmin.
Reference solution Dissolve 10 I-I-L of linalol R, 10 I-I-L of
Split ratio 1:1 OO. cineole R and 10 I-I-L of linalyl acetate R in 1 mL of toluene R.
 IV-234 Lavender Oil 2014

Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel Reference solution (b) Dissolve 5 ~lL of limonene R in
plate R (2-10 ¡.tm)]. heptane R and dilute to 50.0 mL with the same solvent.
Mobile phase ethyl acetate R, toluene R (5:95 V/V) . Dilute 0.5 mL of the solution to 5.0 mL with heptane R.
Application 10 ¡.tL [or 2 ¡.tL] as bands of 10 mm [or 6 mm] . Column:
- material: fused silica;
Development Over a path of 10 cm [or 8 cm].
- size: l = 60 m, 0 = 0.25 mm;
Drying In air. - stationary phase: macrogol 20 000 R (film thickness
Detection Spray with anisaldehyde solution R and heat at 0.25 ¡.tm).
100-105 oC for 5-10 min; examine imrnediately in daylight. Carrier gas helium for chromatography R.
Results See below the sequence of zones present in the Flow rate 1.5 mLlmin.
chromatograms obtained with the reference solution and the
Split ratio 1:50.
test solution. Furthermore, other violet-red or greenish-brown
zones are present in the chromatogram obtained with the test Temperature:
solution aboye the zone of linalyl acetate up to the solvent Time Temperature
front. (min) (oC)
Column 0-15 70

15 - 70 70 -7 180
Top of the plate
Injection port 220
A violet·red or greenish-brown
zone Detector 220
--- ---
Detection Flame ionisation.
Linalyl aceta te : a violet or brown A violet or brown zone (linalyl
zone acetate) 1n/ection 1 ¡.tL.
A violet-red zone Elution arder arder indicated in the composition of
reference solution (a). Record the retention times of these
substances.
--- --- System suitability Reference solution (a):
1.8-Cineole: a violet-brown zone Possibly a weak violet-brown zone - resolution: minimum 1.4 between the peaks due to
(l.8-cineole) terpinen-4-o1 and lavandulyl acetate.
Linalol : a violet or brown zone A violet or brown zone (linalol) U sing the retention times determined from the
A weak yellowish-brown zone chromatogram obtained with reference solution (a), locate
the components of reference solution (a) in the
Several unresolved zones
chromatogram obtained with the test solution.
Reference soIution Test soIution Determine the percentage content of each of these
components. The percentages are within the following
ranges:
B. Examine the chromatograms obtained in the test for - limonene: maximum 1.0 per cent;
chromatographic profile. - 1J 8-cineole: maximum 2.5 per cent;
Results The characteristic peaks in the chromatogram - 3-octanone: 0.1 per cent to 5.0 per cent;
obtained with the test solution are similar in retention time to - camphor: maximum 1.2 per cent;
those in the chromatogram obtained with reference - linalol: 20.0 per cent to 45 .0 per cent;
solution (a). - linalyl acetate: 25.0 per cent to 47 .0 per cent;
TESTS - terpinen-4-ol: 0.1 per cent to 8.0 per cent;
Relative density (2.2.5) - lavandulyl acetate: minimum 0.2 per cent;
0.878 to 0.892. - lavandulol: minimum 0.1 per cent;
- rI.-terpineol: maximum 2.0 per cent;
Refractive index (2.2.6) - disregard limit: the area of the principal peak in the
1.455 to 1.466. chromatogram obtained with reference solution (b)
Optical rotation (2.2. 7) (0.05 per cent).
- 12S to - 6.0°.
Chiral purity
Acid value (2. 5.1) Gas chromatography (2.2.28) .
Maximum 1.0, determined on 5.0 g of the substance to be Test solution Dissolve 0.02 g ofthe oil to be examined in
examined dissolved in 50 mL of the prescribed mixture of pentane R and dilute to 10 mL with the same solvent.
solvents.
Reference solution Dissolve 10 ¡.tL of linalol R (mixture of
Chromatographic pro file (R)-linalol and (S)-linalol), add 5 mg of borneol R and 10 ¡.tL
Gas chromatography (2.2.28): use the normalisation of linalyl aceta te R (mixture of (R)-linalyl acetate and
procedure. (S)-linalyl acetate) in pentane R and dilute to 10 mL with the
Test solution Dissolve 200 ¡.tL of the oil to be examined in same solvent.
heptane R and dilute to 10.0 mL with the same solvent. Column:
R eference solution (a) Dissolve 5 ¡.tL of limonene R, 5 ¡.tL of - material: fused silica;
cineole R, 5 ¡.tL of 3-octanone R, 5 mg of camphor R, 40 ¡.tL of - size: l = 25 m, 0 = 0.25 mm;
linalol R, 50 ¡.tL of linalyl acetate R, 10 ¡.tL of terpinen-4-ol R, - stationary phase: modified ¡J-cyclodextrin for chiral
5 ¡.tL of lavandulyl acetate R, 5 ¡.tL of lavandulol R and 5 mg chromatography R (film thickness 0.25 ¡.tm) .
of rI.-terpineol R in heptane R and dilute to 10 mL with the Carrier gas helium for chromatography R .
same solvent.
 2014 Lavender Oil IV-235

Flow rate 1.3 mUmin. Mobile phase ethyl acetate R, toluene R (5 :95 V/V) .
Split ratio 1 :30. Application 10 flL [or 2 flL], as bands of 10 mm [or 6 mm].
Temperature: Development Over a path of 10 cm [or 8 cm].
Time Temperatnre Drying In air.
(min) ('C) Detection Spray with anisaldehyde solution R and heat at
Column 0-65 50 -> 180 100-105 DC for 5-10 min; examine immediately in daylight.
Injection port 230 Results See below the sequence of zones present in the
Detector 230 chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
Detection Flame ionisation. in the chromatogram obtained with the test solution.
¡njection 1 ¡LL.
Blution order (R)-linalol, (S)-linalol, borneol, (R)-linalyl
Top oC the plate
acetate, (S)-linalyl acetate; depending on the operating
conditions and the state of the column, borneol may elute A pink zone
before or after (S)-linalol. --- - --
System suitability Reference solution:
LinalyI acetate: a violet or brown A faint violet or brown zone may
- resolution: minimum 5.5 between the peaks due to zone be present (linalyl acetate)
(R)-linalol and (S)-linalol; minimum 2.9 between the A pink zone
peaks due to (S)-linalol and borneol; minimum 2.0
between the peaks due to (R)-linalyl acetate and --- - --
(S)-linalyl acetate. Cineole: a violet-brown zone An intense violet-brown zone
(cineole)
Calculate the percentage content of the specified
Linalol: a violet or brown zone An intense violet or brown zone
(S)-enantiomers using the following expression: (linalol)
A greyish or brownish zone
As
A
s+ A R X 100 A faint violet zone

ReCerence solution Test solntion

As area of the peak due to the corresponding
(S) -enantiomer;
area of the peak due to the corresponding B. Examine the chromatograms obtained in the test for
(R)-enantiomer. chromatographic pro file .
Limits: Results The characteristic peaks in the chromatogram
- (S)-linalol: maximum 12 per cent; obtained with the test solution are similar in retention time to
- (S)-linalyl acetate: maximum 1 per cent. the peaks due to limonene, cineole, camphor, linalol, linalyl
aceta te, ex-terpineol and trans-ex-bisabolene in the
STORAGE chromatogram obtained with reference solution (a).
At a temperature not exceeding 25 oC.
_ _ _ __ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ Ph fur
TESTS
Re1ative density (2.2.5)
0.894 to 0.907 .
Refractive index (2.2. 6)
1.461 to 1.468.
Spike Lavender Oil *****
** ** Optical rotation (2.2. 7)
(Ph Bur monograph 2419) *** - 7° to + 2°.
Ph fur _ __ __ __ _ __ _ __ _ _ _ _ _ _ __ _ Acid value (2.5.1)
DEFINITION Maximum 1.5, determined on 5.00 g of the substance to be
examined.
Essential oil obtained by steam distillation of the ftowering
tops of Lavandula latifolia Medik. Solubility in alcohol (2.8.10)
1.0 mL of the substance to be examined is soluble,
CHARACTERS sometimes with opalescence, in 3.0 mL of ethanol (70 pe}' cent
Appearance VIV) R.
Clear, mobile, light yellow or greenish-yellow liquido
Chromatographic pro file
Odour reminiscent of cineole and camphor.
Gas chromatography (2.2.28) : use the normalisation
IDENTIFICATION procedure.
First identification B. Test solution Dissolve 200 flL of the substance to be
Second identification A. examined in heptane R and dilute to 10.0 mL with the same
A. Thin-Iayer chromatography (2.2.27) . solvento
Test solution Dissolve 20 ¡LL of the substance to be Reference solution (a) Dissolve 200 flL of spike lavender
examined in 1 mL of toluene R. oil CRS in heptane R and dilute to 10 .0 mL with the same
solvent.
Reference solution Dissolve 10 ¡LL of cineole R, 10 flL of
linalol R and 10 flL of linalyl acetate R in 1 mL of toluene R. Reference solution (b) Dissolve 5 flL of limonene R in
50.0 mL of heptane R . Dilute 0.5 mL of this solution to
Plate TLC silica gel plate R (5-40 flm) [or TLC silica gel
5.0 mL with heptane R.
plate R (2-10 flm)].
 IV-236 Lemon Balm 2014

Column: IDENTIFICATION
-- material: fused silica; A. The leaves have a petiole of varying length; the lamina is
-- size: 1 = 60 m, 0 = 0.25 mm; broadly ova te, up to about 8 cm long and 5 cm wide, acute
-- stationary phase: macrogol 20 000 R (film thickness at the apex and rounded to corda te at the base; the margins
0.25 ¡lm) . are crenate to dentate. The upper surface is intense green,
Carner gas helium for chromatography R. the lower surface is paler green and shows a conspicuous
Flow rate 1.5 mUmin. midrib and a raised, reticulate venation; scattered hairs occur
on the upper surface and along the veins on the lower
Split ratio 1:50.
surface, which is also finely punctuate.
Temperature:
B. Reduce to a powder (355) (2.9.12). The powder is
Time Temperature greenish. Examine under a microscope using chloral hydrate
(min) (oC) solution R . The powder shows the following diagnostic
Column 0-15 70 characters (Figure 1447.-1): fragments ofthe upper
15 -70 70 - 180 epidermis, in surface view, with sinuous walls [A, B, G],
Injection port 220
sometimes accompanied by palisade parenchyma [Aa] ;
Detector 220 fragments of the lower epidermis [D] with diacytic stomata
(2.8.3) [Db]; short, straight, unicellular, conical covering
Detection Flame ionisation. trichomes with a finely striated cutic1e, free [E] or attached to
1njection 1)lL. an epidermis [Da]; multicellular, uniseriate covering
Identijication of peaks Use the chromatogram supplied with trichomes with pointed ends and thick, warty cutic1es [C];
spike lavender oil CRS and the chromatogram obtained with eight-celled secretory trichomes of lamiaceous type, in surface
reference solution (a) to identify the peaks due to limonene, view [Ga]; secretory trichomes with unicellular to tricellular
cineole, camphor, linalol, linalyl acetate, cHerpineol and stalks and unicellular or, more rarely, bicellular heads, in
trans-(X- bisa bo lene. surface view [Ba] or in transverse section [F].
System suitabztity Reference solution (a):
-- the chromatogram obtained is similar to the
chromatogram supplied with spike lavender oil CRS;
-- resolution: minimum 1.5 between the peaks due to
limonene and cineole.
Determine the percentage content of each of these
components. The percentages are within the following
ranges:
-- limonene: 0.5 per cent to 3.0 per cent;
-- cineole: 16.0 per cent to 39.0 per cent;
-- camphor: 8.0 per cent to 16.0 per cent;
-- linalol: 34.0 per cent to 50.0 per cent;
-- linalyl acetate: maximum 1.6 per cent;
-- rt-terpineol: 0.2 per cent to 2.0 per cent;
-- trans-rt-bisabolene: 0.4 per cent to 2.5 per cent;
-- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0 .05 per cent) .
STORAGE
At a temperature not exceeding 25 oC .
____________________________________________ ~Ew

Lemon Balm ***

*** ***
(Melissa Leal, Ph Bur monograph 1447) ***
Preparation
Lemon Balm Dry Extract
~Ew ____________________________________________ Figure 1447.-1.- Illustration for identification test B of
powdered herbal drug of melissa leaf
DEFINITION
Dried leaf of Melissa officinalis L.
C. Thin-Iayer chromatography (2.2.27).
Content
Test solution Place 2.0 g of the powdered drug (355)
Minimum 1.0 per cent of rosmarinic acid (ClsHlóOS; M r
360.3) (dried drug). (2.9.12) in a 250 mL round-bottomed flask and add 100 mL
of water R. Distil for 1 h using the apparatus for the
CHARACTERS determination of essential oils in herbal drugs (2.8.12) and
Odour reminiscent of lemon. 0.5 mL of xylene R in the graduated tube. After distillation
transfer the organic phase to a 1 mL volumetric flask, rinsing
the graduated tube of the apparatus with the aid of a small
 2014 Lemon Balm Preparations IV-237

portion of xylene R, and dilute to 1.0 mL with the same - stationary phase: octadecylsilyl silica gel for chromatography R
solvent. (5 ¡lm).
Reference solution Dissolve 1.0 ¡lL of citronellal R and Mobile phase:
10.0 ¡lL of citral R (composed of neral and geranial) in - mobile phase A: phosphoric acid R, acetonitrile R, water R
25 mL of xylene R . (1:19:80 VIV/V);
Plate TLC silica gel plate R (5-40 ¡lm) [or TLC silica gel - mobile phase B: phosphon'c acid R, methanol R, acetonitnle R
plate R (2-10 ¡lm)]. (1:40 :59 VIV/V);

Mobile phase ethyl acetate R, hexane R (10:90 V/V). Time Mobile phase A Mobile phase B
(min) (per cent V/JI) (per cent V/JI)
Application 20 ¡lL [or 4 ¡lL] as bands.
0-20 100 --7 55 O --7 45
Development In an unsaturated tank over a path of 15 cm
[or 6 cm]. 20 - 25 55 --7 O 45 --7 100

Drying In air. 25 - 30 O --7 100 100 --7 O

Detection Spray with anisaldehyde solution R and heat at

Flow rate 1.2 mUmin.
100-105 oC for 10-15 min; examine in daylight.
Detection Spectrophotometer at 330 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the Injection 20 ¡lL.
test solution. Furthermore, other zones may be present in the Relative retention With reference to rosmarinic acid
chromatogram obtained with the test solution. (retention time = about 11 min): ferulic acid = about 0.8.
System suitability Reference solution (b):
- resolution: minimum 4.0 between the peaks due to ferulic
Top of the plate
acid and rosmarinic acid.
--- --- Calculate the percentage content of rosmarinic acid using the
Citronellal: a grey or greyish-violet A grey or greyish-violet zone following expression:
zone at the border between the (citronellal) at the border between
upper and middle thirds the upper and middle thirds
A reddish-violet zone
Al x m2 x p x 0.2
A 2 x mI
--- ---
Citral: 2 greyish-violet or 2 greyish-violet or bluish-violet Al area of the peak due to rosmarinic acid in the
bluish-violet zones at the border zones (citral) at the border between
between the middle and lower the middle and lower thirds chromatogram obtained with the test solution;
thirds A2 area of the peak due to rosmarinic acid in the
Reference soIution Test soIution chromatogram obtained with reference solution (a);
mi mass of the drug to be examined used to prepare the
test solution, in grams;
TESTS m2 mas s of rosmarinic acid CRS used to prepare reference
Foreign matter (2.8.2) solution (a), in grams;
Maximum 10 per cent of stems with a diameter greater than p percentage content of rosmarinic acid in rosmarinic
1 mm and maximum 2 per cent of other foreign maner, acid CRS.
determined on 20 g. _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ _ _ _ PhEur
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Lemon Balm Dry Extract ***
Total ash (2. 4.16) *** ***
Maximum 12.0 per cent. (Me/issa Leaf Dry Extract, Ph Eur monograph 2524) ***
ASSAY ~Ew _ __ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Liquid chromatography (2.2.29). DEFINITION
Test solution Use brown-glass flasks. Disperse 0.100 g ofthe Dry extract produced from Melissa leaf (1447).
powdered drug (355) (2.9.12) in 90 mL of ethanol
Content
(50 per cent V/V) R . Boil in a water-bath under a reflux
Minimum 2.0 per cent of rosmarinic acid (CIsHI608; M r
condenser for 30 min, cool, and filter into a 100 mL
360.3) (dried extract).
volumetric flask. Rinse the flask and the filter with 10 mL of
ethanol (50 per cent V/V) R and dilute to 100.0 mL with the PRODUCTION
same solvent. Filter through a 0.45 ¡lm filter. The extract is produced from the herbal drug by a suitable
Reference solution (a) Dissolve 20.0 mg of rosmarinic procedure using either hot water (not less than 70 oC) or a
acid CRS in ethanol (50 per cent V/V) R and dilute to hydroalcoholic solvent that is at most equivalent in strength
100.0 mL with the same solvento Dilute 20.0 mL of this to ethanol (70 per cent V/V).
solution to 100.0 mL with ethanol (50 per cent V/V) R. CHARACTERS
Reference solution (b) Dissolve 5.0 mg offerulic acid R in Appearance
reference solution (a) and dilute to 50.0 mL with the same Brown or greenish-brown, amorphous powder.
solution.
IDENTIFICATION
Column: Thin-layer chromatography (2.2.27) .
- size: l = 0.25 m, 0 = 4.6 mm;
 IV-238 Lemon Peel 2014

Test solution To 0.2 g ofthe extract to be examined add - mobile phase B: phosphoric acid R, methanol R, acetonitrile R
5 mL of methanol R. Sonicate for 5 min and filter. (1:40:59 VIV/V);
Rejerence solution Dissolve 1.0 mg of hyperoside R, 1.0 mg of Time Mobile phase A Mobile phase B
rutin R and 5.0 mg of rosmarinic acid R in 10 mL of (min) (per cent V!I1 (per cent V!I1
methanol R. 0-20 100 -4 55 0-445
Plate TLC silica gel plate R (5-40 ¡.¡m) [or TLC si/ica gel 20 - 25 55 -4 O 45 -4 100
plateR (2-10 ¡.¡m)].
Mobile phase anhydrous jormic acid R, water R, ethyl acetate R Flow rate 1.2 mUmin.
(6:6:90 VIV/V). Detection Spectrophotometer at 330 nm.
Application 10 ¡.¡L [or 2 ¡.¡L] as bands of 15 mm [or 8 mm]. Injection 20 ¡.¡L.
Development Over a path of 8 cm [or 6 cm]. Relative retention With reference to rosmarinic acid
D1ying In air. (retention time = about 11 min): ferulic acid = about 0.8 .
Detection Heat at 100 oC for 5 min, spray the plate whilst System suitability Reference solution (b):
still hot with a 5 gIL solution of diphenylboric acid aminoethyl - resolution: minimum 4 .0 between the peaks due to femlic
ester R in ethyl acetate R, and examine in ultraviolet light at acid and rosmarinic acid.
365 nm. Calculate the percentage content of rosmarinic acid using the
Results See below the sequence of fluorescent zones present following expression:
in the chromatograms obtained with the reference solution
and the test solution. Furthermore, other weaker fluorescent Al x m2 x p x 0.2
zones may be present in the chromatogram obtained with the A 2 x mI
test solution.
Al area of the peak due to rosmarinic acid in the
Top of the plate
chromatogram obtained with the test solution;
A2 area of the peak due to rosmarinic acid in the
chromatogram obtained with reference solution (a);
Rasmarinic acid: a light blue An intense light blue fluarescent mI mas s of the extract to be examined used to prepare
fluarescent zane zane (rasmarinic acid) the test solution, in grams;
A blue fluarescent zone m2 mas s of rosman'nic acid CRS used to prepare reference
- -- --- solution (a), in grams;
p percentage content of rosmarinic acid in rosmarinic
A blue fluarescent zane
acid CRS.
--- ---
PhEur
Hyperaside: an orange ar
greenish-yellaw fluarescent zane
A light blue fluarescent zone

Rutin: an arange ar greenish-yellaw

fluorescent zane Dried Lemon Peel
Reference solution Test solution
DEFINITlON
Dried Lemon Peel is the dried outer part of the pericarp of
TESTS the ripe, or nearly ripe, fmit of Citrus limon (L.) Burm. f.
Loss on drying (2.8.17) CHARACTERISTlCS
Maximum 6.0 per cent. It has the macroscopical and microscopical characters
ASSAY described under Identification tests A and B.
Liquid chromatography (2.2.29). IDENTIFICATlON
Test solution Use brown glass flasks. To 0.200 g of the A. Dried Lemon Peel consists of strips or pieces, showing a
extract to be examined add 50 mL of ethanol (50 per cent marked thickening of the epicarp around the calyx. The outer
V/V) R. Sonicate for 10 min and dilute to 100.0 mL with surface is yellow and somewhat rough from the presence of
ethanol (50 per cent V/V) R . Filter through a membrane filter numerous minute pits, each corresponding to an oil gland;
(nominal pore size 0.45 ¡.¡m). the inner surface with only a small remnant of white, spongy
Rejerence solution (a) Dissolve 20.0 mg of rosmarinic pericarp. Fracture, short.
acid CRS in ethanol (50 per cent V/V) R and dilute to B. Prepare thin cross sections from material softened by
100.0 mL with the same solvento Dilute 20.0 mL of this soaking in water and examine under a microscope using
solution to 100.0 mL with ethanol (50 per cent V/V) R. chloral hydrate solution. The sections show a yellow epidermis
Rejerence solution (b) Dissolve 5 mg ofjerulic acid R in composed of small, thin-walled cells and an underlying
reference solution (a) and dilute to 50 mL with reference parenchyma with numerous large oil glands and scattered
solution (a). small strands of vascular tissue; prismatic crystals of calcium
Column: oxalate occur throughout the parenchyma and are
- size: 1 = 0.25 m, 0 = 4.6 mm; particularly abundant in the layers adjacent to the epidermis.
- stationalY phase: octadecylsi1y1 silica gel jor chromatography R C. Carry out the method for thin-layer chromatography,
(5 ¡.¡m). Appendix III A, using the following solutions.
Mobile phase: (1) Add 1 g of freshly cut peel to 10 mL of methanol and
mobile phase A: phosphoric acid R, acetonitrile R, water R heat in a water-bath at 65° for 5 minutes, shaking frequently.
(1:19:80 VIV/V); Allow to cool and filter.
2014 Lemon Oil IV-239

(2) 0.01 % w/v eaeh of caffeic acid and naringin and Mobile phase ethyl acetate R, toluene R (1 5:85 V/V) .
0.025% w/v of rntin in methanol. Application 10 ¡.tL, as bands.
CHROMATOGRAPHIC CONDITIONS Development Over a path of 15 cm.
(a) Using a TLC silica gel plateo Drying In air.
(b) Use the mobile phase as described below. Detection A Examine in ultraviolet light at 254 nm.
(e) Apply 20 ¡.tL of ea eh solution as bands. Results ASee below the sequenee of the zones present in
(d) Develop the plate to 15 cm. the ehromarograms obtained with the referenee solution and
(e) After removal ofthe plate, dry the plate at 105° and spray the test solution.
the warm plate with a 1% w/v solution of diphenylboric acid
aminoethyl ester in methanol and then with a 5% w/v solution Top of the plate
of polyethylene glycol 400 in methanol. Allow the plate to stand A quenching zone (bergamotin)
for 30 minutes and examine under ultraviolet light (365 nm).
Citral: a quenching zone A quenching zone (citral)
MOB/LE PHASE
A dark blue zone
A mixture of 10 volumes of anhydrousformic acid, 10 volumes (S-geranyloxy-7-methoxycoumarin)
of water, 30 volumes of butan-2-one and 50 volumes of ethyl A light blue fluorescent zone
Citropten: a light blue fluorescent
aceta te. zone (citropten)
CONFIRMATION A quenching zone (psoralen
derivative)
The ehromatogram obtained with solution (1): A quenching zone (biakangelicin)
exhibits a yellowish-brown fiuoreseent zone eorresponding in
Reference solution Test solution
eolour and position ro the zone for rutin in the
ehromarogram obtained with solution (2);
aboye it an intense red fiuoreseent zone and further aboye a Detection B Examine in ultraviolet light at 365 nm.
very weak greenish fiuoreseent zone eorresponding in eolour R esults B See below the sequenee of the zones present in
and position to the zone for naringin in the ehromatogram the ehromarograms obtained with the referenee solution and
obtained with solution (2); the test solution.
other eoloured zones are present in the ehromatogram.
TESTS Top of the plate
Volatile oil A yellow fluorescent zone
Not less than 2.0% v/w (1.7% w/w), Appendix XI E, using (bergamotin)
300 mL of water as the distillation liquid and no xylene in Citral: a quenching zone A quenching zone (citral)
the graduated tube. Use 20 g, soaked in water and maeerated
A bright blue fluorescent zone
in a suitable blender, and distil for 3 hours. (S-geranyloxy-7-methoxycoumarin)
Citropten: a bright blue fluorescent A bright violet-blue fluorescent
zone zone (citropten)
A yellow fluorescent zone
(psoralen derivative)
Lemon Oil ***
*** *** An orange zone (biakangelicin)

(Ph Eur monograph 0620) *** Reference solution Test solution

P reparation
Terpeneless Lemon Oil B. Examine the ehromarograms obtained in the test for
____________________________________________
ehromatographie profile.
ffiE~

DEFINITION R esults The eharaeteristie peaks in the ehromarogram

Essential oil obtained by suitable meehanieal means, without obtained with the test solution are similar in retention time to
the aid of heat, from the fresh peel of Citrns limon (L.) those in the ehromatogram obtained with the referenee
Burman fil. solution.
CHARACTERS TESTS
Appearance Relative density (2.2.5)
Clear, mobile, pale yellow or greenish-yellow liquid. It may 0.850 to 0.858 .
beeome cloudy at low temperatures. Refractive index (2.2.6)
Charaeteristie odour. 1.473 ro 1.476.
IDENTIFICATION Optical rotation (2.2.7)
First identijication B. + 57° to + 70°.
Second identijication A. Absorbance (2.2.25)
Dissolve 0.250 g of the substanee to be examined in
A. Thin-Iayer ehromatography (2.2.27).
alcohol R, mix and dilute ro 100.0 mL with the same solvent.
Test solution Mix 1 mL of the substanee to be examined in Measure the absorbanee over the range 260 nm ro 400 nm.
1 mL of toluene R. If a manual instrument is used, measure the absorbanee at
Reference solution Dissolve 10 mg of citropten R and 50 ¡.tL of 5 nm intervals from 260 nm ro about 12 nm before the
citral R in toluene R and dilute ro 10 mL with the same expeeted absorption maximum, then at 3 nm intervals for 3
solvent. readings and at 1 nm intervals ro about 5 nm beyond the
Plate TLC silica gel GF254 plate R. maximum and finally at 10 nm intervals ro 400 nm. Plot a
 IV-240 Lemon Oil 2014

curve representing the absorption spectrum with the Temperature:

absorbances as ordinates and the wavelengths as abscissae.
Time Temperature
Draw as a baseline the tangent between A and B (Figure (min) (oC)
0620.-1). The absorption maximum C is situated at 315 ± Column 0·6 45
3 mu. From C draw a line perpendicular 10 the axis of 6·21 45 __ 90
abscissae and intersecting AB at D. Deduct the absorbance 21· 39 90 __ 180
corresponding 10 point D from that corresponding to point 39·55 180
C. The value C - D is 0.20 10 0.96 and for Italian-type Inieclion port 220
lemon oil it is not less than 0.45 . Detector 220

Detection Flame ionisation.

Ir
1 .00,-----,.-_----,-_-,--_-,------,_-,-----,
Injection 0.5 ~L of the reference solution and 0.2 ~L of the
test solution.
Elution order Order indicated in the composition of the
I
751---_\~A--ft--'-l\-I+---+-_+-_+______l
reference solution. Record the retention times of these
substances.
O. System suitability Reference solution:

\1\ \1
~ resolution: minimum 1.5 between the peaks due to
~-pinene and sabinene and minimum 1.5 between the

g peaks due to geranial and geranyl acetate.

U sing the retention times determined from the
~ 0.501-----_ ----L~-'__..\+
: _~--\I-~_+__-!-----I chromatogram obtained with the reference solution, locate

~.\\ 1\
the components of the reference solution in the

j I chromatogram obtained with the test solution.

Determine the percentage content of these components.
I \ The percentages are within the following ranges:
0.251---- + --+-- +--1-7+-+---+--1 ~ p-pinene: 7.0 per cent 10 l7.0 per cent,

i\\~
-- sabinene: 1.0 per cent to 3.0 per cent,
-- limonene: 56.0 per cent 10 78.0 per cent,
-- y-terpinene: 6.0 per cent to 12.0 per cent,
-- p-caryophyllene: maximum 0.5 per cent,
o -- neral: 0.3 per cent 10 1.5 per cent,
260 280 300 320 340 360 380 400 ~ a-terpineol: maximum 0.6 per cent,
Wavelength (nm) -- neryl acetate: 0.2 per cent to 0.9 per cent,
-- geranial: 0.5 per cent to 2.3 per cent,
Figure 0620.-1. - Typical spectrum oflemon oi! for the test -- geranyl acetate: 0.1 per cent to 0.8 per cent.
for absorbance
Residue on evaporation (2.8.9)
1.8 per cent 10 3.6 per cent after heating on the water-bath
Fatty oils and resinified essential oils (2.8.7) for 4 h.
It complies with the test for fatty oils and resinified essential
STORAGE
oils.
At a temperature not exceeding 25 oc.
Chromatographic profile
Gas chroma1Ography (2.2.28): use the normalisation LABELLING
procedure. The label sta tes, where applicable, that the contents are
Italian-type lemon oil.
Test solution The substance to be examined.
____________________________________________ ~Em

Reference solution Dissolve 20 ~L of p-pinene R, 1O ~L of

sabinene R, 100 ~L of limonene R, 1O ~L of y-terpinene R,
5 ~L of p-caryophyllene R, 20 ~L of citral R, 5 ~L of
a-terpineol R, 5 ~L of neryl acetate R and 5 ~L of
geranyl acetate R in 1 mL of acetone R. Terpeneless Lemon Oil
Column:
Preparations
-- material: fused silica,
Lemon Spirit
-- size: l = 30 m (a film thickness of 1 ~m may be used) to
60 m (a film thickness of 0.2 ~m may be used), Compound Orange Spirit
o = 0.25-0.53 mm, DEFINITION
-- stationary phase: macrogol 20 000 R. Terpeneless Lemon Oil may be prepared by concentrating
Carrier gas helium for chromatography R . Lemon Oil under reduced pressure until most of the terpenes
Flow rate 1.0 mUmin. have been removed or by solvent partition. It contains not
less than 40% w/w of aldehydes calculated as citral,
Split ratio 1:1 OO.
C lOH I6 0.
CHARACTERISTICS
A clear, colourless or pale yellow liquid, visibly free from
water; odour and taste, those of lemon.
 2014 Lemon Verbena Leaf IV-241

TESTS CHARACTERISTICS
Optical rotation Lemon Syrup has a weight per mL of about 1.33 g.
_5° to + 2°, Appendix V F. The syrup complies with the requirements stated under Oral
Refractive index Liquids and with the following requirements.
1.475 to 1.485, Appendix V E . Content of citric acid monohydrate, C 6 H g 0 7 ,HzO
Solubility in ethanol 2.2 to 2.6% w/v.
Soluble, at 20°, in 1 volume of ethanol (80%), ASSAY
Appendix X M .
Mix 8 g with 100 rnL of water and titrate with O.IM sodium
Weight per mL hydroxide VS using phenolphthalein solution R1 as indicator.
0.880 to 0 .895 g, Appendix V G. Each mL of O.IM sodium hydroxide VS is equivalent to
ASSAY 7.005 mg of C 6H s0 7 ,HzO. Determine the weight per mL,
Appendix V G, and calculate the content of C óH S 0 7 ,H zO,
Carry out the method for determination of aldehydes,
weight in volume.
Appendix X K, using 1 g, omitting the toluene and using a
volume, not less than 7 mL, of alcoholic hydroxylamine solution
that exceeds by 1 to 2 mL the volume of O.5M potassium
hydroxide in ethanol (60%) VS required. Each mL of
O.5M potassium hydroxide in ethanol (60%) VS is equivalent to
Lemon Verbena Leaf ***
76 .73 mg of C iOH 16 0. ** **
(Ph Eur monograph 1834) *****
STORAGE
ffiE~ __________________________________________
Terpeneless Lemon Oil should be kept in a well-filled
container and protected from light. DEFINITION
Whole or fragmented, dried leaves of Aloysia citrodora Paláu
(syn. Aloysia triphylla (L'Hér.) Kuntze; Verbena triphylla
L'Hér.; Lippia citriodora Kunth).
Lemon Spirit Content:
-- acteoside (Cz9H3ó015; Mr 625): minimum 2.5 per cent,
DEFINITION expressed as ferulic acid (dried drug);
Terpeneless Lemon Oil 100 mL
-- essentialoil: minimum 3.0 mUkg for the whole drug and
Ethanol (96 per cent) Sufficient to produce 1000 mL
minimum 2.0 mUkg for the fragmented drug (dried
The spirit complies with the requirements stated under Spirits and drug) .
with the following requirements.
CHARACTERS
Content of aldehydes After grinding, it has a characteristic odour reminiscent of
3.45 to 4.60% w/v, calculated as citral, C lO H 16 0 . lemon.
TESTS IDENTIFICATION
Ethanol content A. The lea ves are simple with short petioles. They are
84 to 88% v/v, Appendix VIII F . narrow, lanceolate, and about 4 times longer than they are
Weight per mL wide. The entire, slightly undulating margins are curled
0.814 to 0.823 g, Appendix V G . towards the upper surface. The upper surface is dark green
and rough to the touch; the lower surface is paler green and
ASSAY
shows a prominent midrib with secondary veins running to
Carry out the method for the determination of aldehydes,
the margins.
Appendix X K, using 10 mL, omitting the toluene and using
a volume, not less than 7 mL, of alcoholic hydroxylamine B. Microscopic examination (2.8.23). The powder is light
solution that exceeds by 1 to 2 mL the volume of green. Examine under a microscope using chloral hydrate
O.5M potassium hydroxide in ethanol (60%) VS required. Each solution R. The powder shows the following diagnostic
mL of O.5M potassium hydroxide in ethanol (60%) VS is characters (Figure 1834.-1): fragments ofthe upper
equivalent to 76.73 mg of C iO H 16 0. epidermis of the lamina in surface view [A, B, H], composed
of polygonal cells with numerous short, unicellular, thick-
walled cystolithic trichomes, each arising from a rosette of
cells at the base and containing calcium concretions (B),
glandular trichomes with a unicellular stalk and a unicellular,
Lemon Syrup globular head of variable size in surface view [Ha] and
transverse section [D, F] ; these fragments are usually
DEFINITION
accompanied by palisade parenchyma [Aa, Hb]; fragments of
Lemon Spirit 5 mL
the lower epidermis of the lamina in surface view [E],
Citric Acid Monohydrate 25 g
covered by a striated cutic1e and composed of cells more
Invert Syrup 100 mL
irregular and somewhat sinuous in outline, with abundant
Syrup Sufficient to produce
anomocytic sto mata (2.8.3) [Ea] and numerous glandular
1000 mL
trichomes in surface view [Eb] and/or their scars [Ec];
Extemporaneous preparation fragments of the lamina in transverse section [G] with 2
The following directions apply. layers of palisade parenchyma [Ga] and spongy parenchyma
Dissolve the Citric Acid Monohydrate in sorne of the Syrup, [Gb]; lignified tissue from the veins [C].
add the Invert Syrup, the Lemon Spirit and sufficient Syrup
to produce 1000 mL and mix.
IV-242 Lemon Verbena Leaf 2014

Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel
plate R (2-10 ¡.tm)].
Mobile phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (11:11:27:100 VIVIV/li').
Application 20 ¡.tL [or 5 ¡.tL] as bands.
Development Over a path of about 12 cm [or 6 cm].
Drying In air.
Detection Spray with anisaldehyde solution R and dry at
100-105 oC for about 10 minó examine in daylight.
Results The chromatogram obtained with the test solution
shows no brownish-grey zone at a posítíon between that of
arbutin and rutin in the chromatogram obtained wíth the
reference solutíon.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (710) (2.9.12) by drying ín an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 13.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.5 per cent.
ASSAY
Acteoside
Liquíd chromatography (2.2.29).
Test solution To 1.00 g of the powdered herbal drug (710)
(2.9.12) add 50.0 mL of the reference solutíon and stir for
2 h wíth a magnetíc stirrer. Centrifuge for 15 min and pass
the supernatant through a membrane filter (nomínal pore size
Figure 1834.-1. - Illustration for identification test B of
powdered herbal drug of lemon verbena leaf 0.45 ¡.tm).
Reference solution Díssolve 10.0 mg of ferulic acid CRS in
ethanol (60 per cent V/V) R and dílute 10 100.0 mL wíth the
C. Examine the chromatograms obtained in the test for
same solvent.
Verbena officinalis.
Precolumn:
Results See below the sequence of zones present in the
- size: l = 0.01 m, 0 = 4.0 mm;
chromatograms obtained with the reference solution and the
- stationary phase: octadecylsilyl silica gel for chromatography R
test solution. Furthermore, other faint zones may be present
(5 ¡.tm) .
in the chromatogram obtained with the test solution. Zones
may be present in the chromatogram obtained with the test Column:
solution below the zone due to rutin in the chromatogram - size: l = 0.25 m, 0 = 4.0 mm;
obtained with the reference solution. - stationary phase: octadecylsilyl silica gel for chromatography R
(5 ¡.tm);
- temperature: 20 oC.
Top of the plate Mobz1e phase:
--- --- - mobile phase A: 0.3 per cent VIV solutíon of phosphoric
acid R;
An intense greyish·green zone
- mobz1e phase B: acetonitrile R;
Arbutin: a blue or brown zone
Time Mobile phase A Mobile phase B
(min) (per cent V!JI) (per cent V!JI)
0·20 93 -) 83 7 -) 17
Rutin: a dark brownish·yellow A blue or violet zone
zone 20·30 83 17
- -- --- 30 · 35 83 -) 75 17 -) 25
Reference solution Test solution 35·40 75 -) 20 25 -) 80
40·45 20 -) 93 80 -) 7
TESTS
Flow rate 1.0 mUmin.
Verbena officinalis
Thin-Iayer chromatography (2.2.27). Detection Spectrophotometer at 330 nm.
Injection 20 ¡.tL.
Test solution To 0.50 g ofthe powdered herbal drug (710)
(2.9. 12) add 5 mL of methanol R. Heat in a water-bath at System suitability Test solutíon:
60 oC for 10 mino Cool and filter. - resolution: minimum 3.5 between the peaks due 10 ferulic
Reference solution Dissolve 10 mg of arbutin R and 10 mg of acíd and acteosíde.
rutin R in methanol R and dilute to 10 mL with the same Calculate the percentage content of acteosíde, expressed as
solvent. ferulic acíd, usíng the followíng expression:
 2014
Al x mz x p x 0.5 x 3.1
Az x mI
area of the peak due to acteoside in the
chromatogram obtained with the test solution;
area of the peak due to ferulic acid in the
chromatogram obtained with the reference solution;
mI mas s of the herbal drug in the test solution, in
grams;
m2 mass of ferulic acid CRS in the reference solution, in
grams;
p for 5 min. AlIow to cool and filter.
percentage content of ferulic acid inferulic
acid CRS;
3.1 correlation factor between acteoside and ferulic
acid.
Essential oil (2.8.12)
Introduce 25.0 g of the freshly crushed herbal drug into a
1000 mL flask and add 500 mL of a 10 gIL solution of
sodium chloride R as the distillation liquido Use 0.50 mL of
xylene R in the graduated tube. Distil at arate of
3.0-3.5 mUmin for 3 h.
____________________________________________ ~E~
Lime Flower
(Ph Eur monograph 0957)
Ph Eur _________________________________________
DEFlNITION
Whole, dried inflorescence of Tilia cordata Miller, of Tilia
platyphyllos Scop., of Tilia x vulgaris Heyne or a mixture of
these .
CHARACTERS
Faint aromatic odour.
Faint, sweet and mucilaginous taste.
IDENTIFlCATION
A. The inflorescence is yellowish-green. The main axis of the
inflorescence bears a linguiform bract, membranous,
yellowish-green, practically glabrous, the central vein of
which is joined for up to about half of its length with the
peduncle. The inflorescence usually consists of 2-7 flowers,
occasionally up to 16. The sepals are detached easily from
the perianth; they are up to 6 mm long, their abaxial surface
is usually glabrous, their adaxial surface and their borders are
strongly pubescent. The 5 spatulate, thin petals are yellowish-
white, up to 8 mm long. They show fine venation and their
borders only are sometimes covered with isolated trichomes.
The numerous stamens are free and usually constitute
5 groups. The superior ovary has a pistil with a somewhat
5-lobate stigma.
B. Separate the inftorescence into its different parts . Examine
under a microscope using chloral hydrate solution R.
The adaxial epidermis of the bract shows cells with straight
or slightly sinuous anticlinal walls; the abaxial epidermis
shows cells with wavy-sinuous anticlinal walls and
anomocytic stomata (2.8.3) . Isolated cells in the mesophyll
contain smalI calcium oxalate cluster crystals.
The parenchyma of the sepals shows, particularly near the
veins, numerous mucilaginous cells and cells containing small
calcium oxalate clusters. The adaxial epidermis of sepals
bears bent, thick-walled covering trichomes, unicelIular or
stelIate with up to 5 cells. The epidermal cells of the petals
show straight anticlinal walls with a striated cuticle without
Lime Flower IV-243
sto mata. The parenchyma of the petals shows small calcium
oxalate clusters and especialIy in its acuminate part
mucilaginous cells. The pollen grains have a diameter of
about 30-40 ¡.¡m and are oval or slightly triangular with
3 germinal pores and a finely granulated exine. The ovary is
glabrous or densely covered with trichomes, often very
twisted, unicellular or stellate with 2-4 branches.
e. Thin-Iayer chromatography (2.2.27).
Test solution Shake 1.0 g of the powdered drug (355)
(2.9.12) with 10 mL of methanol R in a water-bath at 65 oC
Reference solution Dissolve 2.0 mg of caffeic acid R, 5 mg of
hyperoside R and 5 mg of rutin R in 10 mL of methanol R .
Plate TLC silica gel plate R .
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl aceta te R (10:10:30 :50 VIVIV/V).
Application 10 ¡.¡L, as bands.
Development Over a path of 15 cm.
Drying At 100-105 oC.
Detection Spray the warm plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R. Then spray
with a 50 gIL solution of macrogol 400 R in methanol R . Allow
to dry for about 30 min and examine in ultraviolet light at
365 nm.
Results The chromatogram obtained with the reference
solution shows in order of increasing Rp value yellowish-
orange or brownish-orange fluorescent zones due to rutin and
hyperoside and a greenish-blue fluorescent zone due to
caffeic acid. In the chromatogram obtained with the test
solution, the main zone shows brownish-yellow or orange
fluorescence. This zone is situated just aboye the zone due to
hyperoside in the chromatogram obtained with the reference
solution. In daylight, this zone stand s out from the other
zones as the main zone. At the Rp level of rutin there is also
a brownish-yellow fluorescent zone. Below this zone, 2 yellow
fluorescent zones may be presento Between the zones due to
rutin and hyperoside, orange and yellow fluorescent zones are
visible. Between the zones due to hyperoside and caffeic acid,
up to 5 yellow or orange fluorescent zones are presento
Immediately below the zone due to caffeic acid is a a blue
fluorescent zone.
TESTS
Foreign matter (2.8.2)
Maxirnum 2 per cent, determined on 30 g. There are no
inflorescences with a bract bearing at the abaxial face stellate,
five- to eight-rayed trichomes and flowers having an apparent
double corolla by transformation of five stamens into petal-
like staminoids and having a pistil which is not lobular nor
indented. Hexamerous flowers occur only occasionalIy
(Tilia americana L., Tilia tomentosa Moench).
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maxirnum 8.0 per cent.
__________________ _ _____________________ Ph fur
IV-244 Linseed
Linseed
(Ph Eur monograph 0095)
When Powdered Linseed is prescribed or demanded, material
complying with the appropriate requirements below shall be
dispensed or supplied.
nE~ ____________________________________________
DEFINITION
Dried, ripe seeds of Linum usitatissimum L.
IDENTIFICATION
A. The seed has a flattened, elongated ovoid shape. The testa
is dark reddish-brown or yellow, smooth and shiny.
The seeds are 4-6 mm long, 2-3 mm wide and 1.5-2 mm
thick; one end is rounded and the other end forms an
oblique point near which the hilum appears as a slight
depression. When viewed with a lens, the surface of the seed-
coat is seen to be minutely pitted. Inside the testa a narrow,
whitish endosperrn and an embryo composed of 2 large,
flattened, yellowish and oi!y cotyledons are present;
the radic1e points towards the hilum.
B. Microscopic examination (2.8.23) . The powder is greasy
to the touch. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 0095.-1): fragments ofthe outer testa [A,
B] with cells that are polygonal in surface view [Aa] or
narrow in transverse section [Ba], and filled with muci!age
[Bb]; fragments of the collenchymatously thickened sub-
epiderrnallayer, in transverse section [Be], or in surface view
[Ab] with rounded cells with triangular intercellular spaces
Bb
Figure 0095.-1. - Illustration for identification test B of
powdered herbal drug of linseed
2014
often attached to the sc1erenchymatous layer composed of
elongated cells, with thickened and pitted walls [Ca], sorne
with strongly thickened and pitted walls [G]; fragments, in
surface view [C], consisting of the hyaline layer with thin-
walled cells [Cb] often remaining attached to the layer of
elongated sc1ereids and crossing them at approximately right
angles [Ca]; fragments of the inner testa, in surface view [D],
composed of moderately thickened polygonal cells filled with
brown-orange pigment; small polyhedral mas ses of pigment
[H]; numerous fragments ofparenchyma from the testae,
with large, slightly and regularly thickened cells, in surface
view U, L]; parenchyma of the endosperrn [K] and
cotyledons [E] containing aleurone grains and oi! droplets;
very numerous isolated oil droplets [F].
TESTS
Foreign matter (2.8.2)
Maximum 10 per cent of seeds with a dull coat and
maximum 1.5 per cent of other foreign matter.
Swelling index (2.8. 4)
Minimum4.
Cadmium (2.4.27)
Maximum 0.5 ppm.
Loss on drying (2.2.32)
Maximum 8.0 per cent, deterrnined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 5.0 per cent.
____________________________________________ nE~
Liquorice ****
*** *
(Liquorice Root, Ph Eur monograph 0277) *** *
Preparations
Liquorice Dry Extract for Flavouring Purposes
Standardised Liquorice Ethanolic Extract
Liquorice Liquid Extract
When Powdered Liquorice is prescribed or demanded,
material complying with the appropriate requirements below
shall be dispensed or supplied.
n Ew ____________________________________________
DEFINITION
Dried, unpeeled or peeled, whole or cut root and stolons of
Glycyrrhiza glabra L. andlor of Glycyrrhiza inflata Bar. andlor
Glycyrrhiza uralensis Fisch.
Content
Minimum 4.0 per cent of 18~-glycyrrhizic acid (C42H6Z016;
M r 823) (dried drug).
IDENTIFICATION
A. The root has few branches. Its bark is brown or brownish-
grey with longitudinal striations and bears traces of lateral
roots. The cylindrical stolons are 1-2 cm in diameter; their
external appearance is similar to that of the root but there are
occasional small buds. The fracture of the root and the
stolon is granular and fibrous. The cork layer is thin;
the secondary phloem region is thick and light yellow with
radial striations. The yellow xylem cylinder is compact, with
a radiate structure. The sto Ion has a central pith, which is
absent from the root. The external part of the bark is absent
from the peeled root.
 2014
B. Microscopic examination (2.8.23) . The powder is light
yellow or faintly greyish. Examine under a microscope using
chloral hy drate solution R. The powder shows the following
diagnostic characters: fragments of yellow thick-walled fibres,
700-1200 ¡.¡m long and 10-20 ~lm wide with a punctiform
lumen, often accompanied by crystal sheaths containing
prisms of calcium oxalate 10-35 ¡.¡m long and 2-5 ~lm wide.
The walls of the vessels are yellow, 5-10 ¡.¡m thick, lignified
and have numerous bordered pits with a slit-shaped aperture;
fragments of cork consisting of thin-walled cells and isolated
prisms of calcium oxalate occur as well as fragments of
parenchymatous tissue. Fragments of cork are absent from
the peeled rOOL Examine under a microscope using a
mixture of equal volumes of glycerol R and water R.
The powder shows the following diagnostic characters:
simple, round or oval starch granules, 2-20 ¡.¡m in diameter.
C. Thin-layer chromatography (2.2.27) .
Test solution To 0.50 g ofthe powdered herbal drug (ISO)
(2.9.12) in a 50 mL round-bottomed flask add 16.0 mL of
water R and 4.0 mL of hydrochloric acid R1 and heat on a
water-bath under a reflux condenser for 30 mino Cool and
filter. Dry the filter and the round-bottomed flask at 105 oC
for 60 mino Place the filter in the round-bottomed fiask, add
20.0 mL of ether R and heat in a water-bath at 40 oC under
a reflux condenser for 5 mino Cool and filter. Evaporate the
filtrate to dryness. Dissolve the residue in 5.0 mL of ether R.
Referenee solution Dissolve 5.0 mg of glycyrrhetie acid R and
5.0 mg of thymol R in 5.0 mL of ether R.
Plate TLC si/ica gel F 254 plate R .
Mob¡le phase eoneentrated ammonia R, water R, ethanol
(96 per eent) R , ethyl acetate R (1:9:25:65 VIVIVIV) .
Applieation 1O ~lL.
D evelopment Over a path of 15 cm.
Drying In air for 5 mino
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms obtained with the test
solution and the reference solution show in the lower half a
quenching zone due to glycyrrhetic acid.
Detection B Treat with anisaldehyde solution R, and heat at
100-105 oC for 5-10 min; examine in daylight.
Results B The chromatogram obtained with the reference
solution shows in the lower half a violet zone due to
glycyrrhetic acid and in the upper third a red zone due to
thymo!. The chromatogram obtained with the test solution
shows in the lower half a violet zone corresponding to the
zone of glycyrrhetic acid in the chromatogram obtained with
the reference solution and a yellow zone (isoliquiridigenine)
in the upper third under the zone of thymol in the
chromatogram obtained with the reference solution. Further
zones may be presento
TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent for the unpeeled drug and
maximum 6.0 per cent for the peeled drug.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent for the unpeeled drug and maximum
0.5 per cent for the peeled drug.
Ochratoxin A (2.8.22)
Maximum 20 [.lg per kilogram of herbal drug.
Liquorice IV-245
ASSAY
Liquid chromatography (2.2.29).
Test solution Place 1.000 g of the powdered herbal drug
(ISO) (2.9.12) in a 150 mL ground-glass conical flask.
Add 100.0 mL of an S gIL solution of ammonia R and treat
in an ultrasonic bath for 30 mino Centrifuge a part of the
solution and dilute 1.0 mL of the supernatant layer to
5.0 mL with an S gIL solution of ammonia R. Filter through
a membrane filter (nominal pore size 0.45 [.lm); use the
filtrate as the test solution.
Solution A Dissolve 0.130 g of monoammonium
glyeyrrhizate CRS in an S gIL solution of ammonia R and
dilute to 100.0 mL with the same solvento
Referenee solution (a) Dilute 5.0 mL of solution A to
100.0 mL with an S gIL solution of ammonia R.
Reference solution (b) Dilute 10.0 mL of solution A to
100.0 mL with an S gIL solution of ammonia R.
Reference solution (e) Dilute 15.0 mL of solution A to
100.0 mL with an S gIL solution of ammonia R.
Column:
- size: 1 = 0.10 m, 0 = 4 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 ¡.¡m).
Mobile phase glacial aeetic acid R, acetonimle R, water R
(6:30:64 VIVIV).
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 254 nm.
Injection 10 ¡.¡L.
Establish a calibration curve with the mass of
monoammonium glycyrrhizate in the reference solutions, in
grams, as the abscissa and the corresponding peak areas as
the ordinate.
Using the retention times and the peak areas determined
from the chromatograms obtained with the reference
solutions, locate and integrate the peak due to lSp-
glycyrrhizic acid in the chromatogram obtained with the test
solution.
Calculate the percentage content of 18P-glycyrrhizic acid
using the following expression:
5 823
Ax-xBx -
m 840
A mass equivalent of monoammonium glycyrrhizate
in the test solution, determined from the
calibration curve, in grams;
B declared percentage content of monoammonium
glycyrrhizate CRS;
m mass of the herbal drug to be examined used to
prepare the test solution, in grams;
S23 molecular mass of lSP-glycyrrhizic acid;
840 molecular mass of monoammonium glycyrrhizate
(without any water of crystallisation) .
LABELLING
The label states whether the drug is peeled or unpeeled.
_ _ _ _ __ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ PhEur
IV-246 Liquorice Preparations 2014

Liquorice Dry Extract tor *** Ochratoxin A (2.8.22)

*** *** MaxÍmum 80 ~lg per kilogram of extracto
Flavouring Purposes *** The maxÍmum content applies to the pure undiluted extracto
(Ph Eur monograph 2378) Where excipients are added to reduce the strength of the
~ E~ _ _ _ __ __ _ _ _ _ __ _ _ ______________ extract, the maximum content should be reduced
proportionally.
DEFINITION
Dry extract produced from Liquorice root (0277). ASSAY
Liquid chromatography (2.2.29).
Content
Solvent mixture water R, methanol R (20:80 V/V) .
5.0 per cent ro 7.0 per cent of 18~-glycyrrhizic acid
(C42H 6201 6; M r 823) (dried extract). Test solUlion Place 0.200 g of the extract to be examÍned in
a 150 mL ground-glass conical ftask. Add 100.0 mL of the
PRODUCTION solvent mixture and sonicate for 2 mino Filter through a
The extract is produced from the cut herbal drug by a membrane filter (nominal pore size 0.45 ¡.tm).
suitable procedure using water.
R eference solutlon Dissolve 50.0 mg of monoammonium
CHARACTERS glycyrrhiz ate CRS in the solvent mixture and dilute to
Appearance 50.0 mL with the solvent mixture. Dilute 1.0 mL of this
Yellowish-brown or brown powder. solution ro 10.0 mL with the solvent mixture.
Very sweet taste. Column:
-- size: 1 = 0.10 m, 0 = 4.0 mm;
IDENTIFICATION
-- stationary phase: octadecylsilyl silica gel for chromatography R
Thin-layer chromatography (2.2.27).
(5 ¡.tm) .
Solvenr mixture ethyl acetate R, methanol R (50:50 V/V).
Mobile phase glacial acetic acid R, acetonitrile R, water R
Test solUlion To 0.30 g of the extract to be examined add (6:30:64 VIV/V).
30 mL of hydrochloric acid R1 and boil on a water-bath under
Flow rate 1.5 mUmin.
a reftux condenser for 60 mino After cooling, extract the
mixture with 2 quantities, each of 20 mL, of ethyl acetate R . Detection Spectrophotometer at 254 nm.
Combine the organic layers and filter through a filter covered Injection 10 ¡.tL.
with anhydrous sodium sulfate R . Evaporate the filtrate ro Run time 3 times the retention time of 18~-glycyrrhizic acid.
dryness in vacuo and dissolve the residue in 2.0 mL of the Retention time 18~-glycyrrhizic acid = about 9 mino
solvent mixture.
Identijication of peaks Use the chromarogram supplied with
R eference solw ion Dissolve 5.0 mg of glycyrrhetic acid R and monoammonium glycyrrhiz ate CRS and the chromarogram
5.0 mg of thymol R in 5.0 mL of the solvent mixture. obtained with the reference solution ro identify the peaks due
Plate TLC silica gel F 254 plate R (5-40 ¡.tm) [or TLC silica gel ro 18~-glycyrrhizic acid and 18a-glycyrrhizic acid.
F 254 plate R (2-10 ¡.tm)]. Sy stem suitability Reference solution:
Mobile phase concentrated ammonia R, water R, ethanol -- the chromatogram obtained with the reference solution is
(96 per cene) R, ethyl acetare R (1:9:25:65 VIVIV/V) . similar to the ehromatogram supplied with
Application 20 ¡.tL [or 10 ¡.tL] as bands. monoammonium glycyrrhiz ate CRS;
D evelopmenr over a path of 15 cm [or 7 cm] . resolution: minimum 2.0 between the peaks due ro 18~­
glycyrrhizic acid and 18a-glycyrrhizic acid.
Drying In air for 5 mino
Calculate the percentage content of 18~-glycyrrhizic acid,
Detection Spray with anisaldehyde solution R and heat at
using the following expression:
100-105 oC for 5-10 min; examine in daylight.
R esults See below the sequence of zones present in the
A l x m2 x p x 0.979
chromarograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present A 2 x mI x 5
in the chromatogram obtained with the test solution.
area of the peak due to 18~-glycyrrhizic aeid in
the chromatogram obtained with the test
Top of the plate
solution;
area of the peak due to 18~-glycyrrhizic acid in
Thyrnol: a red zone the chromatogram obtained with the referenee
solution;
A yellow zone
mass of the extraet to be examined used ro
----- ---- prepare the test solution, in grams;
- -- - - - -- --
mass of monoammonium glycy rrhizate CRS used
to prepare the reference solution, in grams;
Glycyrrhetic acid : a violet zone A violet zone (glycyrrhetic acid) p pereentage eontent of 18~-glycyrrhizie acid in
monoammonium glycyrrhizate CRS;
0.979 peak correlation faetor between 18~-glycyrrhizic
Reference solution Test solution
aeid and monoammonium glycyrrhizate.
____________________________________________ ~ E~

TESTS
Loss on drying (2.8.17)
Maximum 7.0 per cent.
2014
*****
Standardised Liquoriee Ethanolie
** **
Liquid Extraet
(Ph Eur monograph 1536)
Ph Eur _ __ __ _ _ __ _ __ _ _ _ __ __ _ __
DEFINITION
Standardised ethanolie liquid extract produced from Liquorice
root (0277).
Content
3.0 per eent to 5.0 per cent of 18~-glycyrrhizie acid
(C42H62016; M r 823).
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure for liquid extracts using ethanol (70 per cent V/V).
CHARACTERS
Appearance
Dark brown, clear liquido
1t has a faint characteristic odour and a sweet taste.
IDENTIFICATION
Thin-layer chromatography (2.2.27).
Test solution Place 1.0 g of the extract to be examined in a
50 mL round-bottomed fiask, add 16.0 mL of water R and
4.0 mL of hydroehlorie aeid R1 and heat on a water-bath
under a refiux condenser for 30 minoAllow to cool and filter.
Dry the filter and the round-bottomed fiask at 105 oC for
60 minoTransfer the filter to the round-bottomed fiask, add
20 mL of ether R and heat in a water-bath at 40 oC under a
refiux condenser for 5 mino AIlow to eool and filter.
Evaporate the filtrate to dryness and dissolve the residue in
5.0 mL of ether R.
Referenee solution Dissolve 5.0 mg of glycyrrhetie acid R and
5.0 mg of thymol R in 5 mL of ether R .
Plate TLC siliea gel F 254 plate R.
Mobile phase coneentrated ammonia R, water R, ethanol
(96 per eent) R, ethyl aeetate R (1:9:25:65 VIVIV/V).
Applieation 10 flL as bands.
Development Over a path of 15 cm.
Drying In air for 5 mino
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms obtained with the test
solution and the reference solution show in the lower half a
quenehing zone due to glycyrrhetic aeid.
Deteetion B Treat with anisaldehyde solution R; heat at
100-105 oC for 5-10 min and examine in daylight.
Results B The chromatogram obtained with the referenee
solution shows in the lower half a violet zone (glycyrrhetic
aeid), and in the upper third a red zone (thymol);
the ehromatogram obtained with the test solution shows in
the lower half a violet zone corresponding to glycyrrhetie acid
in the ehromatogram obtained with the reference solution,
and in the upper third, below the zone of thymol in the
chromatogram obtained with the referenee solution, a yeIlow
zone due to isoliquiritigenin; further zones are presento
TESTS
Ethanol (2.9.10)
52 per cent V/V to 65 per cent V/V.
Methano1 and 2-propanol (2.9.11)
Maximum 0.05 per cent V/V of methanol and maxirnum
0.05 per cent V/V of 2-propanol.
Liquorice Preparations IV-247
Ochratoxin A (2.8.22)
Maxirnum 80 Jlg per kilogram of extracto
*** ASSAY
Liquid ehromatography (2.2.29).
Solvent mixture dilute ammonia R1, water R (8:92 V/V).
Test solution Dilute 1.000 g of the extraet to be examined to
100 mL with the solvent mixture and centrifuge. Dilute
2.0 mL ofthe supematant to 10.0 mL with the solvent
mixture.
Solution A Dissolve 0.130 g of monoammonium
glyeyrrhizate CRS in the solvent mixture and dilute to
100.0 mL with the solvent mixture.
Reference solution (a) Dilute 5.0 mL of solution A to
100.0 mL with the solvent mixture.
Referenee solution (b) Dilute 10.0 mL of solution A to
100.0 mL with the solvent mixture.
Referenee solution (e) Dilute 15.0 mL ofsolution A to
100.0 mL with the solvent mixture.
Column:
- size: l = 0.10 m, 0 = 4 mm;
- stationary phase: oetadeeylsilyl siliea gel for ehromatography R
(5 flm).
Mobile phase glacial aeetie acid R, aeetonitrile R, water R
(6:30:64 VIV/V).
Flow rate 1.5 mUmin.
Deteetion Spectrophotometer at 254 nm.
Injeetion 10 flL.
Establish a calibration curve with the mass of
monoammonium glycyrrhizate in the reference solutions, in
grams, as the abscissa and the corresponding peak areas as
the ordinate.
Using the retention times and the peak areas determined
from the ehromatograms obtained with the reference
solutions, locate and integrate the peak due to 18~­
glycyrrhizic acid in the chromatogram obtained with the test
solution.
Calculate the percentage content of 18~-glycyrrhizic aeid
using the following expression:
5 823
Ax-xBx-
m 840
A mass equivalent of monoammonium glyeyrrhizate
in the test solution, determined from the
ealibration curve, in grams;
B declared percentage content of monoammonium
glycyrrhizate CRS;
m mass of the extract to be examined used to prepare
the test solution, in grams;
823 molecular mass of 18~-glyeyrrhizic acid;
840 molecular mas s of monoammonium glycyrrhizate
(without any water of crystaIlisation).
_ _ _ _ _ _ __ _ __ _ __ _ _ _ _ __ _ _ Ph Eur
 IV-248 Liquorice Preparations
Liquoriee Liquid Extraet
DEFINITION
Liquorice Liquid Extract is prepared by extracting Liquorice
with Purified Water and adding sufficient Ethanol
(90 per cent) to give an ethanol content of 18% v/v in the
final extract.
Extemporaneous preparation
The following formula and directions apply.
Liquorice, unpeeled, in coarse powder 1000 g
Purified Water A sufficient quantity
Ethanol (90 per cent) A sufficient quantity
Exhaust the Liquorice with Purified Water by percolation,
Appendix XI F. Boil the percolate for 5 minutes and set
aside for not less than 12 hours. Decant the clear liquid, filter
the remainder, mix the two liquids and evaporate until the
weight per mL of the liquid is 1.198 g, Appendix V G. Add to
this liquid, when cold, one quarter of its volume of Ethanol
(90 per cent). Allow to stand for not less than 4 weeks; filter.
The extract complies with the requirements stated under Extracts
and with the following requirements.
IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Extract 5 mL of the liquid extract with 20 mL of
chloroform. Heat 2.5 mL of the aqueous layer with 30 mL of
O.5M sulfuric acid under a reftux condenser for 1 hour, cool
and extract with two 20 mL quantities of ehloroform. Dry the
combined chloroform extracts over anhydrous sodium sulfate,
filter and evaporate the filtrate to dryness; dissolve the
residue in 4 mL of a mixture of equal volumes of ehloroform
and methanol.
(2) Dissolve 10 mg of glycyrrhetinie acid in 2 mL of a mixture
of equal volumes of ehloroform and methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating siliea gel F254 (Merck 10 x 20 cm
plates are suitable).
(b) Use the mobile phase as described below.
(e) Apply 10 ¡.¡L of solution (1) and 5 ~lL of solution (2).
(d) Develop the plate to 15 cm.
(e) After removal ofthe plate, allow it to dry and examine
under ultraviolet light (254 nm).
MOBILE PHASE
5 volumes of methanol and 95 volumes of ehloroform.
CONFIRMATION
The chromatogram obtained with solution (1) exhibits a dark
spot corresponding to the principal spot in the chromatogram
obtained with solution (2) .
TESTS
Ethanol content
16 to 20% v/v, Appendix VIII F, Method III.
Ammonia
Place 5 mL in a distillation apparatus of about 500 mL
capacity fitted with an efficient splash-head and a delivery
tube that dips below the surface of a mixture of 10 mL of
0.05M sulfuric acid VS and 50 mL of water contained in the
receiver. Add 200 mL of water, a little pumiee powder and
30 mL of a solution prepared by dissolving 1.43 g of
potassium dihydrogen orthophosphate and 9.1 g of dipotassium
hydrogen orthophosphate in 100 mL of water and distil until
about 175 mL of distillate has been collected. Titrate the
excess of acid with O.lM sodium hydroxide VS using methyl red
2014
solution as indicator. Not les s than 5 mL of O.lM sodium
hydroxide VS is required.
Dry residue
40 to 45% w/v, Appendix XIP
Relative density
1.125 to 1.140, Appendix V G.
Ochratoxin A
Not more than 40 ppb, Appendix XI S2.
Liquoriee Root for use in TCM
DEFINITION
Liquorice Root for use in TCM is the dried unpeeled root
and rhizome of Glycyrrhiza uralensis Fisch., and/or Glycyrrhiza
inflata Bat. and/or Glycyrrhiza glabra L.
Ir is collected in spring and autumn, separated from the
rootlets and dried in the sun.
Ir contains not less than 2.0% of glycyrrhizic acid
(C42H62016), calculated with reference to the dried material.
IDENTIFICATION
A. The roo [ has few branches. Irs bark is brownish-grey or
brown with longitudinal striations and bears traces of lateral
roots . The cylindrical stolons are 1-2 cm in diameter; their
external appearance is similar to that of the root but there are
occasional small buds. The fracture of the root and the
stolon is granular and fibrous. The cork layer is thin;
the secondary phloem region is thick and light yellow with
radial striations. The yellow xylem cylinder is compact, with
a radiate structure. The stolon has a central pith, which is
absent from the root.
B. Reduce to a powder. The powder is yellowish-brown.
Examine under a microscope using chloral hydrate solution.
The powder shows abundant fibres occurring in groups, each
group surrounded by a calcium oxalate prism crystal sheath,
the individual fibres with very thick, partially lignified walls
and few small pits; lignified bordered-pitted ves seis, singly or
in small groups and sometimes accompanied by lignified
parenchymatous cells with moderately thickened and pitted
walls, sorne of the individual ves seis are very large, with pit
apertures much elongated and the borders difficult to
discerní prism crystals of calcium oxalate up to about 35 ¡.¡m
long occur scattered and in parenchymatous tissue; fragments
of cork composed of thin-walled, slightly lignified polygonal
cells . Examine under a microscope using 50% w/w glycerol
in water. The powder shows abundant starch granules,
mostly simple, spherical to ovo id, 3 ¡.¡m to 20 ¡.¡m in
diameter.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Add 16 mL of water and 4 mL of hydroehlorie acid Rl to
0.5 g of the powdered drug, heat on a water-bath under
reftux for 30 minutes, cool and filter. Dry the filter paper and
residue in a ftask at 105 0 for 60 minutes, add 20 mL of
ether, heat on a water-bath at 40°C under reftux for
5 minutes, cool and filter. Evaporate the filtrate to dryness
and dissolve the residue in 5 mL of ether.
(2) 0 .1% w/v each of glyeyrrhetie acid and thymol in ether.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating substance siliea gel F254 (Mere k 10 x
20 cm plates are suitable) .
(b) Use the mobile phase as described below.
2014
(c) Apply 10 ~lL of each solution as a bando
(d) Develop the plate to 15 cm.
(e) Remove the plate and aIlow it to dry in air for 5 minutes.
Examine under ultraviolet light (254 nm). Spray the plate with
anisaldehyde solution and heat at 100° to 105° for 10 minutes
and examine in daylight.
MOBILE PHASE
1 volume of concentrated ammonia, 9 volumes of water,
25 volumes of ethanol (96%) and 65 volumes of ethyl acetate.
SYSTEM SUITABILITY
Under ultra-violet light, the chromatogram obtained with
solution (2) shows in the lower half a quenching zone due to
glycyrrhetic acid. When sprayed, the chromatogram obtained
with solution (2) shows in the lower half the violet zone of
glycyrrhetic acid and in the upper third the red zone of
thymol.
CONFIRMATION
The chromatogram obtained with solution (1) shows in the
lower half a violet zone corresponding to the zone of
glycyrrhetic acid in the chromatogram obtained with solution
(2) and a yeIlow zone (isoliquiridigenine) in the upper third
under the zone of thymol in the chromatogram obtained with
solution (2) . Further zones may be presento
TESTS
Total Ash
Not more than 7.0%, Appendix XI J, Method n.
Acid-insoluble ash
Not more than 2.0%, Appendix XI K, Method n.
Loss on drying
When dried for 2 hours at 100° to 105°, loses not more than
12.0%. Use 1 g.
Ochratoxin A
Not more than 20 ppb, Appendix XI S2.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the foIlowing solutions.
(1) Mix 1 g of the powdered drug with 100 mL of 0.8% w /v
of ammonia, place in an ultrasonic bath for 30 minutes,
centrifuge, dilute 1 mL of supernatant solution to 5 mL with
0.8% w/v of ammonia and filter through a 0.45-flm filter.
(2) 0.0065 % w/v of monoammonium glycyrrhizate EPCRS in
0 .8% w/v of ammonia.
(3) 0.013 % w/v of monoammonium glycyrrhizate EPCRS in
0.8% w /v of ammonia.
(4) 0.0195 % w/v of monoammonium glycyrrhizate EPCRS in
0.8 % w/v of ammonia.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (12.5 cm x 4 mm) packed
with octadecylsilyl siliea gel for chromatography (5 flm) (Hypersil
ODS SS is suitable).
(b) Use isocratic elution and the mobile phase described
beIow.
(c) Use a flow rate of 1.5 mL per minute.
(d) Use ambient column temperature.
(e) Use a detection wavelength of 254 nm.
(f) Inject 10 flL of each solution.
MOBILE PHASE
6 volumes of glacial aceuc acid, 30 volumes of acetonitrile and
64 volumes of water.
Liquorice Root IV-249
SYSTEM SUITABILITY
The assay is not valid unless (a) the column efficiency,
determined on the peak due to glycyrrhizic acid in the
chromatogram obtained with solution (2), is at least 30,000
theoretical plates per metre and (b) the symmetry factor of the
peak is not more than 1.3.
DETERMINATION OF CONTENT
Inject solution (4). Adjust the sensitivity of the system so mat
the height of the peaks is at least 50% of the fuIl scale of the
recorder. Inject solutions (2), (3) and (4) and determine the
peak areas . Prepare a calibration curve with the concentration
of the solutions (g per 100 mL) as the abscissa and the
corresponding areas as the ordinate. Inject solution (1).
Using the retention time and the peak area from the
chromatograms obtained with solutions (2), (3) and (4),
locate and integra te the peak due to glycyrrhizic acid in the
chromatogram obtained with solution (1).
Calculate the percentage content of glycyrrhizic acid from the
foIlowing expression:
5 822
Ax-xBx-
m 840
A concentration of monoammonium glycyrrhizate in
solution (1) determined from the calibration curve,
in g per 100 mL,
B decIared percentage content of monoammonium
glycyrrhizate EPCRS,
m weight of the substance being examined in grams,
822 molecular weight of glycyrrhizic acid,
840 molecular weight of monoammonium glycyrrhizate
(without any water of crystaIlisation).
STORAGE
Liquorice Root for use in TCM should be protected from
moisture.
Processed Liquorice Root for use in
TCM
DEFINITION
Processed Liquorice Root for use in TCM is the processed
Liquorice Root for use in TCM.
It contains not less than 2.0% of glycyrrhizic acid
(C42H62016) calculated with reference to the dried material.
PRODUCTION
Processed Liquorice Root for use in TCM is cIeaned,
softened thoroughly, sliced transverseIy or 10ngitudinaIly to
form uniform pieces and dried.
IDENTIFICATION
A. The transversely-cut pieces are 0.5 to 2.5 cm in diameter,
irregularly circular to ovo id, up to about 3 mm thick.
The outer surface is dark reddish-brown and 10ngitudinaIly
wrinkled. The transverse surface is cream to yellow and
shows a thin layer of cork and a pale brown cambium line
separating the radiate phloem region from the distinctly
radiate xylem. In pieces cut from the rhizome there is a
central, whitish pithi in those cut from the roots the radia te
xylem continues to the centre.
The longitudinally-cut pieces have been sliced obliquely and
usuaIly incIude a smaIl portion of the outer surface at the
tapering endsi they are about 6 to 8 cm long, 1 to 1.5 cm
IV-250 Liquorice Root
wide and 3 mm thick. The outer surface is reddish-brown
and longitudinally wrinkled with scattered transverse ridges;
the smooth cut surface is cream to yellow, faintly fibrous and
shows a distinct cambium; on sorne of the pieces the vessel
cavities are visible in the central region.
B. Reduce to a powder. The powder is yellowish-brown.
Examine under a microscope using chloral hydrate solution.
The powder shows abundant fibres occurring in groups, each
group surrounded by a calcium oxalate prism crystal sheath,
the individual fibres have very thick, partially lignified walls
and few small pits; lignified bordered-pitted vessels, singly or
in small groups and sometimes accompanied by lignified
parenchymatous cells with moderately thickened and pitted
walls, sorne of the individual ves seis are very large, with pit
apertures much elongated and the borders difficult to
discern; prism crystals of calcium oxalate up to about 35 ¡Lm
long occur scattered and in parenchymatous tissue; fragments
of cork composed of thin-walled, slightly lignified polygonal
cells. Examine under a microscope using 50% w/w glycerol
in water. The powder shows abundant starch granules,
mostly simple, spherical to ovo id, 3 ¡Lm to 20 ¡Lm in
diameter.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Add 16 mL of water and 4 mL of hydrochloric acid (25 %)
to 0.5 g of the powdered drug, heat on a water-bath under
refiux for 30 minutes, cool and filter. Dry the filter paper and
residue in a fiask at 105° for 60 minutes, add 20 mL of ether,
heat on a water-bath at 40° under refiux for 5 minutes, cool
and filter. Evaporate the filtrate to dryness and dissolve the
residue in 5 mL of ether.
(2) 0.1 % w/v each of glycyrrhetic acid and thymol in ether.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating substance silica gel F254
(Merck 10 x 20 cm plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 10 ¡LL of each solution as a bando
(d) Develop the plate to 15 cm.
(e) Remove the plate and allow it to dry in air for 5 minutes.
Examine under ultraviolet light (254 nm). Spray the plate with
anisaldehyde solution and heat at 100° to 105° for 10 minutes
and examine in daylight.
MOBILE PHASE
1 volume of concentrated ammonia, 9 volumes of water,
25 volumes of ethanol (96%) and 65 volumes of ethyl acetate.
SYSTEM SUITABILITY
Under ultra-violet light, the chromatogram obtained with
solution (2) shows in the lower half a quenching zone due to
glycyrrhetic acid. When sprayed, the chromatogram obtained
with solution (2) shows in the lower half the violet zone of
glycyrrhetic acid and in the upper third the red zone of
thymol.
CONFIRMATION
The chromatogram obtained with solution (1) shows in the
lower half of violet zone corresponding to the zone of
glycyrrhetic acid in the chromatogram obtained with solution
(2) and a yellow zone (isoliquiridigenine) in the upper third
under the zone of thymol in the chromatogram obtained with
solution (2) . Further zones may be presento
TESTS
Total Ash
Not more than 5.0%, Appendix XI J, Method II.
2014
Acid-insoluble ash
Not more than 1.0%, Appendix XI K, Method II.
Loss on drying
0
When dried for 2 hours at 100 to 105°, loses not more than
10.0% of its weight. Use 1 g.
Ochratoxin A
Not more than 20 ppb, Appendix XI S2.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Mix 1 g of the powdered drug with 100 mL of 0.8% w/v
of ammonia, place in an ultrasound bath for 30 minutes,
centrifuge, dilute 1 mL of the supernatant solution to
5 mL with 0.8 % w/v of ammonia and filter through
a O.45-¡Lm filter.
(2) 0.0065% w/v of monoammonium glycyrrhizate EPCRS in
0.8% w/v of ammonia.
(3) 0.013% w/v of monoammonium glyCYI1'hizate EPCRS in
0.8% w/v of ammonia.
(4) 0.0195% w/v of monoammonium glycyrrhizate EPCRS in
0.8% w/v of ammonia.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (12.5 cm x 4 mm) packed
with octadecylsilyl silica gel for chromatography (5 ¡Lm) (Hypersil
ODS SS is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a fiow rate of 1.5 mL per minute.
(d) Use ambient column temperature.
(e) Use a detection wavelength of 254 nm.
(f) Injection 10 ¡LL of each solution.
MOBILE PHASE
6 volumes of glacial acetic acid, 30 volumes of acewnitrile and
64 volumes of water.
SYSTEM SUITABILITY
The assay is not valid unless (a) the column efficiency,
determined on the peak due to glycyrrhizic acid in the
chromatogram obtained with solution (2), is at least 30,000
theoretical plates per metre and (b) the symmetry factor of the
peak is not more than 1.3.
DETERMINATION OF CONTENT
Inject solution (4). Adjust the sensitivity of the system so that
the height of the peaks is at least 50% of the full scale of the
recorder. Inject solutions (2), (3) and (4) and determine the
peak areas. Prepare a calibration curve with the concentration
of the solutions (g per 100 mL) as the abscissa and the
corresponding areas as the ordinate. Inject solution (1).
Using the retention time and the peak area from the
chromatograms obtained with solutions (2), (3) and (4),
locate and integrate the peak due to glycyrrhizic acid in the
chromatogram obtained with solution (1).
Calculate the percentage content of glycyrrhizic acid from the
following expression:
5 822
Ax-xBx-
m 840
A concentration of monoammonium glycyrrhizate in
the solution (1) determined from the calibration
curve, in g per 100 mL,
2014 Long Pepper IV-251

B dec1ared percentage content of monoammonium
glycyrrhizate EPCRS,
m weight of the substance being examined in grams,
822 molecular weight of glycyrrhizic acid,
840 molecular weight of the monoarnmonium
glycyrrhizate (without any water of crystallisation).
STORAGE
Processed Liquorice Root for use in TCM should be
protected from moisture.
vascular tissue with spiral or striated vessels. Examine under
a microscope using a 50 per cent V/V solution of glycerol R.
Rounded, compound starch granules about 20 /lm in
diameter made up of tiny individual granules, ovoid or
polyhedral by compression, free or inc1uded in the
parenchymatous cells of the seed.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R. So ni cate for 10 min,
centrifuge and use the supematant.
Reference solution Dissolve 10 mg of borneol R and 15 mg of
piperine R in 10 mL of methanol R.

*****
Plate TLC silica gel F 254 plate R (5-40 11m) [or TLC silica gel
Long Pepper F 254 plate R (2-10 ¡.¡m)] .
** **
(Ph Eu/' monograph 2453) *** Mobz7e phase ethyl acetate R, cyclohexane R (30:50 V/V) .
____________________________________________
~E~

Application 10 IlL [or 5 IlL] as bands of 10 mm [or 8 mm].

DEFINITION Development Over a path of 15 cm [or 6 cm].
Dried, ripe or nearly ripe fruiting spikes of Piper longum L. Drying In airo
or Piper retrofractum Vahl (syn. P. chaba Hunter and P. Detection A Examine in ultraviolet light at 254 nm.
officinarum (Miq.) C.DC.) or a mixture of both species. Results ASee below the sequence of zones present in the
Content: chromatograms obtained with the reference solution and the
-- essentialoil: minimum 6.0 mUkg (dried drug); test solution. Furthermore, other faint quenching zones may
-- piperine (C17H'9N03; M r 285.3): minimum 3.0 per cent be present in the chromatogram obtained with the test
(dried drug). solution.
IDENTIFICA TION
A. P. longum. The fruiting spikes are cylindrical or irregularly Top oC the plate
cylindrical, 1-2.5 cm long (rarely longer than 2.5 cm), - -- ---
3-5 mm in diameter, blackish-brown or almost black.
The spikes are quite compact, tough, composed of small 2 strong quenching zones
fruits firrnly fixed on the receptacle in regular or oblique A quenching zone
rows. The berries are spherical, about 1 mm in diameter.
The bracts are black, small, punctiforrn, confined 10 --- ---
depressions between adjacent berries . The remains of the A quenching zone
peduncle may be present at the base of the cylinder. Spikes
Piperine: a quenching zone A strong quenching zone (piperine)
can be easily broken; the fracture is irregular and granular.
P. retrofractum. The fruiting spikes are similar to those of
P. longum but c1early more robust, straight and cylindrical,
2.5-4 cm long (rarely smaller than 2.5 cm), 5-8 mm in
ReCerence solution Test solution
diameter, brown or reddish-brown. The berries are also
firrnly fixed on the receptacle but, in contrast 10 those of
p. longum, arranged more obviously in spiral rows. Detection B Treat with anisaldehyde solution R and heat at
The bracts are more prominent than those of P. longum. 100 oC for 5 min; examine in daylight.
B. Microscopic examination (2.8.23). The powder is greyish- Results B See below the sequence of zones present in the
beige . Examine under a microscope using chloral hydrate chroma1Ograms obtained with the reference solution and the
solution R. The powder shows the following diagnostic test solutíon. Furtherrnore, other zones may be present in the
characters: fragments of the endocarp in surface view, chroma1Ogram obtained with the test solution.
consisting of more or less elongated sc1ereids about 75 11m
long, which have irregularly thickened walls and wide
channels and which are sometimes associated with the brown Top oC the plate
pigment layer of the testa; fragments of the endocarp, in A purple-grey zone
transverse section, showing sclereids with thickened inner
---- ---
walls on the 3 lower sides, usually associated with the testa;
fragments of the testa consisting of a layer of reddish-brown A purple zone
pigmented cells and a layer of very thin-walled polygonal Borneol: a yellowish·brown zone A violet zone
cells constituting the 'hyaline layer'; fragments of the
parenchyma of the mesocarp containing more or less A purple-grey zone
polygonal sc1ereids, isolated or in groups, and oil cells about - -- ---
50 11m in diameter; numerous thin-walled, ovoid or polygonal
cells of the parenchyma of the seed; fragments of the epicarp Piperine: a green or brownish zone A green or brownish zone
(piperine)
with extremely thin-walled, reddish-brown pigmented cells
associated with the outer layers of the mesocarp consisting of
groups of sc1ereids with strongly thickened walls; rare,
elongated sclereids about 400 11m long with slightly thickened ReCerence solution Test solution
walls, from the centre of the spike; a few fragments of
 IV-252 Loosestrife 2014

TESTS Al x mz x p
Foreign matter (2.8.2) A z x ml x 2
Maximum 3 per cent.
Loss on drying (2.2.32) area of the peak due to piperine in the
Maximum 11.0 per cent, determined on l.000 g ofthe chromatogram obtained with the test solution;
freshly powdered herbal drug (355) (2.9.12) by drying in an area of the peak due to piperine in the
oven at 105 oC for 2 h. chromatogram obtained with reference solution (a);
mass of the herbal drug to be examined used to
Total ash (2.4.16)
prepare the test solution, in grams;
Maximum 5.0 per cent.
mass of piperine CRS used to prepare reference
ASSAY solution (a), in grams;
Essential oi! (2.8.12) p percentage content of piperine in piperine CRS.
Use 25.0 g of the freshly, coarsely powdered herbal drug _ _ __ _ __ _ _ _ __ _ _ _ __ __ _ _ _ Ph Eur
(1400) (2.9.12), a 1000 mL round-bottomed fiask, 400 mL
of water R as the distillation liquid and 0.5 mL of xylene R in
the graduated tube. Distil at arate of 2-3 mUmin for 3 h.
Piperine
Loosestrife ***
Liquid chromatography (2.2.29) . Carry out the assay protected
from light. *** ***
Test solution Disperse 0.250 g of the powdered herbal drug
(Ph Eur monograph 1537) ***
~E~ _ __ _ __ __ _ _ _ _ _ __ _ __ _ _ _ _
(355) (2.9.12) in 40 mL of ethanol (96 per cent) R. Sonicate
for 20 min and filter. Rinse the fiask and the filter with 5 mL DEFINITION
of ethanol (96 per cent) R, combine the filtrate and washings Dried fiowering tops, whole or cut, of Lythrum salicaria L.
and dilute to 50.0 mL with the same solvent. Filter through
Content
a membrane filter (nominal pore size 0.45 ¡.1m) .
Minimum 5.0 per cent of ta=ins, expressed as pyrogallol
Reference soluLÍon (a) Dissolve 15.0 mg of piperine CRS in (C 6H 6 0 3 ; M r 126.1) (dried drug).
ethanol (96 per cent) R and dilute to 100.0 mL with the same
solvent. IDENTIFICATION
A. The stems are rigid, 4-angled, branching at the top,
Reference solution (b) Disperse 0.250 g of long pepper for
system suitability HRS (355) (2.9.12) in 40 mL of ethanol brownish-green, longitudinally wrink1ed and pubescent.
(96 per cent) R. Sonicate for 20 min and filter. Rinse the fiask The leaves are opposite, decussate, rarely verticillate in threes
and the filter with 5 mL of ethanol (96 per cent) R, combine and sometimes alternate at the infiorescence which forms a
long terminal spike. The leaves are sessile, lanceolate and
the filtrate and washings and dilute to 50.0 mL with the
same solvent. Filter through a membrane filter (nominal pore cordate at the base, 5-15 cm long and 1-2.5 cm wide,
size 0.45 ¡.1m). pubescent on the lower surface; the subsidiary veins form
arcs that anastomose near the leaf margino The fiowers have
Column:
a pubescent, tubular, persistent gamosepalous calyx, 4-8 mm
- size: 1 = 0.15 m, 0 = 4 .6 mm;
long, consisting of 6 sepals bearing 6 small, triangular teeth
- stationary phase: end-capped octadecylsilyl si/ica gel for
alternating with 6 large, acute teeth at least half as long as
chromatography R (5 ¡.1m).
the tube; a polypetalous corolla consisting of 6 violet-pink
Mobile phase: petals, each expanded at the top with a wavy outline and
- mobile phase A: water R; narrowing at the base. The androecium consists of 2 verticils
- mobi/e phase B: acetonitrile R; of 6 stamens (1 verticil with short, barely emerging stamens,
Time Mobile phase A Mobile phase B the other with long stamens extending well out of the
(min) (percent VM (percent VM corolla). The fruit, if formed, is a small capsule included in
0-5 50 50 the persistent calyx.
5·20 50 ~5 50 ~ 95 B. Microscopic examination (2.8.23). The powder is
5~0
greenish-yellow. Examine under a microscope using eh/oral
20·22 95 ~ 100
hydrate solution R . The powder shows the following diagnostic
Flow rate l.0 mUmin. characters (Figure 1537.-1): unicellular [Ea] or bicellular
Detection Spectrophotometer at 343 nm. [Aa], uniseriate, thick-walled, finely pitted covering trichomes
from the epidermis of the leaf [A] and stem [E]; numerous
Injection 1O ~1L.
uniseriate, unicellular [Ga] or bicellular [Gb], thin-walled,
RetenLÍon time Piperine = about 10 min. finely pitted, a=ularly striated covering trichomes from the
Identification of peaks Use the chromatogram supplied with calyx, in side view [G]; transparent violet-pink fragments
long pepper for system suitability HRS and the chromatogram from the petals [F] consisting of epidermal cells with sinuous
obtained with reference solution (b) to identify the peak due walls and a grainy cuticle [Fa], covering fine spiral vessels
to piperine and peak 2. [Fb]; fragments of parenchyma from the leaf [D] with
System suitability Reference solution (b): numerous cells containing cluster crystals of calcium oxalate
- peak-to-valley ratio: minimum 4, where H p = height aboye [Da], associated with spiral vessels [Db]; pollen grains with 3
the baseline of peak 2 and H v = height aboye the baseline pores and a thin and slightly granular exine [C] ; fragments of
of the lowest point of the curve separating the peak due the upper epidermis of the leaf [A] with large polygonal cells
to piperine from peak 2. and sinuous walls, covered by a finely striated cuticle [Ab];
Calculate the percentage content of piperine using the fragments of the lower epidermis of the leaf [B] with smaller
following expression: polygonal cells [Ba] and anomocytic stomata [Bb] ( 2.8.3);
2014
Figure 1537.-1. - Illustration for identification test B of
powdered herbal drug of loosestrife
fragments of the stem [E) consisting of polygonal cells with
straight antidinal walls and a striated cutide [Eb) .
C . Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R and heat in a water-bath
at 65 oC for 5 min with frequent shaking. Cool and filter.
Dilute the filtrate to 10 mL with methanol R.
Referenee solution Dissolve 0.5 mg of ehlorogenie acid R, 1 mg
of hyperoside R, 1 mg of rutin R and 1 mg of vitexin R in
10 mL of methanol R.
Plate TLC siliea gel plate R.
Mobile phase anhydrous aeetie acid R, anhydrous forrnie acid R,
water R , ethyl aeetate R (7.5:7.5:18:67 VIVIV/V).
Applieation 10 ¡.LL as bands.
Development Over a path of 15 cm.
Drying At 100-105 0e.
Detection Treat the still-warm plate with a 10 giL solution
of diphenylborie acid aminoethyl ester R in methanol R.
Subsequently treat with a 50 gIL solution of maerogol 400 R
in rnethanol R. Allow to dry in air for 30 min and examine in
ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower third a yellowish-brown
ftuorescent zone due to rutin and in the middle third a light
blue ftuorescent zone due to chlorogenic acid, aboye it a
yellowish-brown ftuorescent zone due to hyperoside and a
green ftuorescent zone due to vitexin. The chromatogram
obtained with the test solution shows a bright green
ftuorescent zone slightly aboye the zone due to rutin in the
chromatogram obtained with the reference solution, a yellow
ftuorescent zone similar in position to the zone due to
Lovage Root IV-253
chlorogenic acid in the chromatogram obtained with the
reference solution, a yellow ftuorescent zone similar in
position to the zone due to hyperoside in the chromatogram
obtained with the reference solution, and a bright green
ftuorescent zone corresponding to the zone due to vitexin in
the chromatogram obtained with the reference solution.
TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 0e.
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14). Use 0.750 g ofthe powdered herbal drug (180)
(2.9.12) .
____________________________________________ ~E~
Lovage Root ****
** *
(Ph Bur monograph 1233)
**** *
~E~ ____________________________________________
DEFINITION
Whole or cut, dried rhizome and root of L evistieum offieinale
Koch.
Content
Minimum 4.0 mUkg of essential oil for the whole drug and
minimum 3.0 mUkg of essential oil for the cut drug (dried
drug).
IDENTIFICATION
A. The rhizome and the large roots are often split
longitudinally. The rhizome is short, up to 5 cm in diameter,
light greyish-brown or yellowish-brown, simple or with
several protuberances; the roots, showing little ramification,
are the same colour as the rhizome; they are usually up to
1.5 cm thick and up to about 25 cm long; the fracture is
usually smooth and shows a very wide yellowish-white bark
and a narrow brownish-yellow wood.
B. Microscopic examination (2.8.23). The powder is
brownish-yellow. Examine under a microscope using ehloral
hydrate solution R. The powder shows the following diagnostic
characters: cork cells, polygonal or rounded in surface view,
with brown contents; abundant parenchyma, mostly thin-
walled and rounded but sorne with thicker walls; groups of
small, reticulately thickened ves seis embedded in small-celled,
unlignified parenchyma; fragments of larger vessels with
reticulate thickening, up to 125 ¡.Lm in diameter; fragments of
secretory canals up to 180 ¡.tm wide. Examine under a
microscope using a 50 per cent V/V solution of glyeerol R.
The powder shows starch granules, simple, rounded or
ovoid, up to about 12 ¡.tm, and numerous larger, compound
granules, many with several components .
e. Examine the chromatograms obtained in the test for
species of Angelica and Ligusticum described in the European
Pharmacopoeia.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other weak ftuorescent zones may
be present in the chromatogram obtained with the test
solution.
 IV-254 Magnolia Officinalis Bark 2014

Top of the plate Application 4 ~IL, as bands of 8 mm.

Development Over a path of 6 cm.
(Z)-Ligustilide: a bluish-white A bluish-white fluorescent zone
fluorescent zone Drying In air.
--- --- Detection A Examine in ultraviolet light at 365 nm.
Results A The chromatogram obtained with the test solution
Osthole: a blue fluorescent zone
shows no blue ftuorescent zone just below or aboye the zone
Imperatorin: a whitish fluorescent A weak whitish fluorescent zone due to irnperatorin in the chrornatogram obtained with the
zone reference solution.
--- --- Detection B Examine in ultraviolet light at 254 nm.
A weak whitish fluorescent zone R esults B The chromatograrn obtained with the test solution
shows no zone at or just below the zone due to imperatorin
Reference soIution Test solution
in the chromatogram obtained with the reference solution.
Detection C Treat the plate with a solution of 20 mL of
Results B See below the sequence of zones present in the sulfuric acid R in 180 mL of ice-cooled methanol R ; heat at
chromatograms obtained with the reference solution and the 100-105 oC for 5 min and examine in daylight.
test solution. Furthermore, other weak quenching zones may Results C The chromatogram obtained with the test solution
be present in the chrornatogram obtained with the test shows no purple zone between the 2 reddish zones at the top
solution. of the chromatogram and the zone due to (Z)-ligustilide in
the chromatograrn obtained with the reference solution;
Top of the plate the chromatogram obtained with the test solution shows no
purple zone between the zones due to (Z)-ligustilide and
(Z)-Ligustilide: a bluish fluorescent A bluish fluorescent zone
zone osthole in the chromatogram obtained with the reference
--- --- solution.
Osthole: a quenching zone A weak quenching zone
Foreign matter (2.8_2)
Maximum 3 per cent, determined on 50 g.
Imperatorin : a quenching zone
Loss on drying (2.2.32)
--- - - - Maximum 12.0 per cent, determined on 1.000 g of the
Reference solution Test solution
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Results C See below the sequence of zones present in the Maxirnum 8.0 per cent.
chromatograms obtained with the reference solution and the
test solution. Furtherrnore, other faint zones may be present Ash insoluble in hydroch1oric acid (2.8.1)
in the chromatogram obtained with the test solution. Maximum 2.0 per cent.
ASSAY
Top of the plate Carry out the determination of essential oils in herbal drugs
(2.8. 12) _ Use a 2 L ftask, 10 drops of liquid paraffin R,
2 prominent reddish zones
500 mL of water R as the distillation liquid and 0.50 mL of
(Z)-Ligustilide: a grey zone A grey zone xylene R in the graduated tube_ Reduce the herbal drug to a
powder (500) (2.9.12) and irnmediately use 40.0 g for the
--- ---
determination. Distil at arate of 2-3 mUmin for 4 h.
Osthole: a violet zone ____________________________________________ ~E~

Imperatorin: a grey zone

--- - --

2 purple zones
Magnolia Officinalis Bark *****
** *
*** *
A distinct brown zone
(Ph Eur monograph 2567)
Reference solution Test solution ~E~ ____________________________________________

TESTS
Species of Angelica and Ligusticum described in the
European Phannacopoeia
Thin-Iayer chromatography (2_ 2. 27).
Test solutWn To 1 g of the freshly powdered herbal drug
(355) (2.9.12) add 4 rnL of heptane R and sonicate for 5 mino
Centrifuge the mixture and use the supernatant.
R eference solution Dissolve 1 mg of imperatorin R , 1 rng of
(Z) -ligustilide R and 1 mg of osthole R in 10 mL of
methanol R.
Plate TLC silica gel F 254 plate R (2-10 ¡lm) .
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
(1 :10:90 VIVIV).
DEFINITION
Dried stem and branch bark of Magnolia officinalis Rehder et
E.H.Wilson.
Content
Minimurn 2.0 per cent of the sum of magnolol (C lsH1 SOZ;
M r 266.3) and honokiol (ClsH1S02; M r 266.3) (dried drug).
IDENTIFICATION
A. Fragments of stem and branch bark, quilled singly or
double quilled, about 30 cm long and 2-7 mm thick.
The outer surface is brownish-grey, rough, sometimes scaly,
easily exfoliated, with distinct lenticels and longitudinal
striations. The inner surface is reddish-brown or dark brown,
smooth, with numerous fine longitudinal striations.
The texture is hard and difficult to break. The fracture is
 2014 Magnolia Officinalis Bark IV-255

granular, brownish-grey in the outer layers and reddish- Top of the pI ate
brown or dark brown in the inner layers.
A bluish-violet zone
B. Microscopic examination (2.8.23) . The powder is
yellowish-brown. Examine under a microscope using chloral - -- ---
hydrate solution R . The powder shows the following diagnostic Eugenol: a brown zone
characters: numerous sclereids of varying shape and size, up
to 100 ¡lm long, often branched, free or in groups, with
conspicuous pit canals; oval or rounded oil cells, about Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol)
60 ~lm in diameter, with orange-yellow contents; naITOW Honokiol: a dark violet zone A dark violet 7.One (honokiol)
fibres with thick walls and often in bundles; brown cork
fragments. A bluish-violet zone
C . Thin-Iayer chromatography (2.2.27). - -- ---
Test solution Reduce to a powder (355) (2.9. 12), avoiding Reference solution Test solution
heating. To 0.5 g of the powdered herbal drug add 5 mL of
methanol R, sonicate for 5 min, centrifuge, and use the
supernatant. Filter through a membrane filter (nominal pore
size 0.45 ¡lm) if necessary. ASSAY
Reference solution Dissolve 1 mg of honokiol R, 1 mg of Liquid chromatography (2.2.29).
magnolol R and 2 mg of eugenol R in 1 mL of methanol R. Test solution To 0.500 g of the powdered herbal drug (355)
Plate TLC silica gel F254 plate R (5-40 ¡lm) [or TLC silica gel (2.9.12) add 80 mL of methanol R and heat in a water-bath
F 254 plate R (2-10 ¡lm)] . under a refiux condenser for 30 mino Cool, then dilute to
100.0 mL with methanol R. Filter through a membrane filter
Mobile phase methanol R, ethyl acetate R, toluene R
(nominal pore size 0.45 ¡lm) .
(4:8:120 V/V/V) .
Reference solution (a) Dissolve 4.0 mg of honokiol CRS and
Application 5 ¡lL [or 2 ¡lL] as bands of 15 mm [or 8 mm].
4.0 mg of magnolol CRS in methanol R and dilute to 20.0 mL
Development Over a path of 15 cm [or 7 cm]. with the same solvent.
Drying In airo Reference solution (b) Dissolve 2.0 mg of honokiol CRS in
Detection A Examine in ultraviolet Iight at 254 nm. 2.0 mL of acetonitrile R. To 1.0 mL of the solution add
Results ASee below the sequence of zones present in the 15 ¡lL of acetic anhydride R and mix. Heat at 50 oC for
chromatograms obtained with the reference solution and the 60 min. Coo!. Add successively, mixing after each addition,
test solution. Furthermore, other faint zones may be present 16 ¡lL of concentrated ammonia R, 1.0 mL of acetonitrile R and
in the chromatogram obtained with the test solution. 2.0 mL of water R. Filter through a membrane filter
(nominal pore size 0.45 ¡lm).
Column:
Top of the plate
- size: 1 = 0.25 m, 0 = 4.6 mm;
--- --- - stationary phase: end-capped octadecylsilyl silica gel for
Eugenol : a fain! quenching zone
chromatography R1 (5 ~lm);
- temperature: 30 oc.
Mobile phase 0.5 per cent V/V solution of acetic acid R,
Magnolol: a dark blue fluorescent A dark blue fluorescent zone acetonitrile for chromatography R (40:60 V/V).
zone (magnolol) Flow rate 1.0 mUmin.
Honokiol: a quenching zone A quenching zone (honokiol)
Detection Spectrophotometer at 290 nm.
--- --- Injectian 10 ¡lL.
Reference solution Test solution Run time Twice the retention time of honokiol for the test
solution and reference solution (a); 3 times the retention time
of honokiol for reference solution (b).
Detection B Treat with vanillin reagent R, heat at
Relative retention With reference to honokiol (retention
100-105 oC for 5-10 min and examine in daylight.
time = about 8 min): magnolol = about 1.4;
Results B See below the sequence of zones present in the honokiol monoacetate isomer 1 = about 1.5;
chromatograms obtained with the reference solution and the honokiol monoacetate isomer 2 = about 1.6;
test solution. Furthermore, other faint zones of various honokiol diacetate = about 2.6.
colours may be present in the chromatogram obtained with
System suitability Reference solution (b):
the test solution.
- resolutian: minimum 1.8 between the peaks due to
TESTS honokiol monoacetate isomers 1 and 2.
Loss on drying (2.2.32) Calculate the sum of the percentage contents of honokiol and
Maximum 11.0 per cent, determined on 1.000 g of the magnolol using the following expression:
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h. Al x m2 x Pl X 5 A3 x m3 x P2 x 5
---~-~--+ --~-~--
Total ash (2.4.16) 2 A x ml A4 x ml
Maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1) Al area of the peak due to honokiol in the
Maximum 3.0 per cent. chromatogram obtained with the test solution;
A2 area of the peak due to honokiol in the
chromatogram obtained with reference solution (a);
IV-256 Magnolia Officinalis F lower 2014

are a of the peak due to magnolol in the Top of the plate

chromatogram obtained with the test solution; - -- ---
area of the peak due to magnolol in the
chromatogram obtained with reference solution (a); Eugenol: a faint quenching zone
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of honokiol CRS used to prepare reference Magnolol: a dark blue fluorescent A dark blue fluorescent zone
m2
zone (magnolol)
solution (a), in grams;
Honokiol: a quenching zone A quenching zone (honokiol)
mass of magnolol CRS used to prepare reference
solution (a), in grams; --- ---
PI percentage content of honokiol in honokiol CRS; Reference soIution Test soIution
P2 percentage content of magnolol in magnolol CRS.
_ _ _ __ _ _ _ _ _ _ _ _ __ _ _ __ __ _ PhEur
Detection B Treat with vanillin reagent R, heat at
100-105 oC for 5-10 min and examine in daylight.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
Magnolia Officinalis Flower test solution. Furthermore, other faint zones of various
colours may be present in the chromatogram obtained with
(Ph Eur monograph 2568)
the test solution.
~E~ _ __ _ _ __ __ _ _ __ _ __ __ _ __ __

DEFINITION Top of the plate

Steamed and dried, unopened fiower of Magnolia officinalis
Rehder et E.H. Wilson. A bluish-violet zone

Content --- ---

Minimum 0.20 per cent of the sum of magnolol (CIsHIS02;
M r 266 .3) and honokiol (CIsHI S02; M r 266.3) (dried drug) .
IDENTIFICATION
A. The greyish-yellow pedicel is short (0.5-2 cm) and densely
tomentose. The brown or reddish-brown fiower bud is
elongated, conical, 4-7 cm long and 1.5-2.5 cm in diameter
at the base; it usually consists of 12 perianth segments in
several whorls. The stamens are numerous with a fine, short
filament and a linear, yellowish-brown anther. The carpels
are free and numerous, spirally arranged on a conical
receptaele.
B. Microscopic examination (2.8.23). The powder is reddish-
brown. Examine under a microscope using chloral hydrate
solution R . The powder shows the following diagnostic
characters: fragments of the perianth segments with
polyhedral or elliptical epidermal cells, with irregularly
thickened walls and anomocytic stomata (4-6 subsidiary cells)
(2.8.3), accompanied by parenchyma that ineludes oval or
rounded oil cells about 50 ¡1m in diameter with orange-
yellow contents; certain fragments contain epidermal cells
with rounded papillae; numerous, branched selereids, with
cha=elled walls and a large lumen, about 15 ~lm in
diameter; numerous elliptical pollen grains about 50 ¡1m long
and 40 ¡1m wide, with a smooth exine.
C. Examine the chromatograms obtained in the test for other
Magnolia species.
Detection A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Eugenol: a brown zone
Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol)
Honokiol: a dark violet zone A dark violet zone (honokiol)
A bluish-violet zone
--- ---
Reference soIution Test soIution
TESTS
Other Magnolia species
Thin-Iayer chromatography (2.2.27).
Test solution Reduce the herbal drug to a powder (710)
(2.9.12), avoiding heating. To 0.5 g ofthe powdered herbal
drug add 2.5 mL of methanol R. Sonicate for 15 min at a
power of 80 W and a frequency of 37 kHz (sonication time
may be adapted according to the power and frequency used),
then centrifuge at 1500-2000 g for 10 min and transfer the
supematant to a 5 mL fiask. Add 2 mL of methanol R to the
residue, sonicate for 15 min and centrifuge. Transfer the
supematant into the same 5 mL fiask. Dilute to 5 mL with
methanol R. Filter through a membrane filter (nominal pore
size 0.45 ~lm) if necessary.
Reference solution Dissolve 1 mg of honokiol R, 1 mg of
magnolol R and 2 mg of eugenol R in 4 roL of methanol R.
Plate TLC silica gel F 254 plate R (2-10 ~lm).
Mobile phase methanol R, ethyl acetate R, toluene R
(1 :5:30 VIV/V).
Application 8 ¡lL as bands of 8 mm.
Development Over a path of 7 cm.
Drying In air.
Detection Examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the test solution
shows no blue fiuorescent zone in the lower part of the plate
and no green fiuorescent zone in the upper part, nor any
other fiuorescent zone.
2014
Loss on drying (2.2.32)
Maximum 11.0 per cent, determined on 1.000 g of the
powdered herbal drug (7 10) (2.9. 12) by drying in an oven at
105 oc.
Total ash (2.4.16)
Maximum 8.0 per cent.
ASSAY
Liquid chroma1Ography (2.2.29).
Test solution Reduce the herbal drug to a powder (710)
(2.9.12) using a blade grinder equipped with a double-walled
grinding chamber cooled to a temperature of about 10 °C.
To 0.500 g ofthe powdered herbal drug add 10 mL of
methanol R. Sonicate for 1 h at a power of 80 W and a
frequency of 37 kHz (sonication time may be adapted
according to the power and frequency used) . Change the
water of the ultrasonic bath after 30 min of sonication to
prevent heating. Centrifuge at 1500-2000 g for 15 mino
Transfer the supernatant to a 20.0 mL flask. Add 9.5 mL of
methanol R to the residue. Repeat the sonication for 1 h.
Change the water of the ultrasonic bath after 30 min of
sonication 10 prevent heating. Centrifuge. Transfer the
supernatant to the same 20.0 mL flask. Cool, then dilute to
20.0 mL with methanol R. Filter through a membrane filter
(nominal pore size 0.45 ¡1m).
Referenee solution (a) Dissolve 5.0 mg of honokiol CRS in
methanol R and dilute to 5.0 mL with the same solvento
Dilute 1.0 mL of the solution to 25.0 mL with methanol R.
Reference solution (b) Dissolve 6.0 mg of magnolol CRS in
methanol R and dilute to 20.0 mL with the same solvento
Referenee solution (e) Dissolve 2.0 mg of honokiol R in
2.0 mL of aeetonitrile R. Add 30 ~lL of aeetie anhydn"de R and
mix. H eat at 50 oC for 60 mino Coo!. Add successively,
mixing after each addition, 32 ¡lL of eoneentrated ammonia R,
2.0 mL of acetonitrile R and 4.0 mL of water R. Filter
through a membrane filter (nominal pore size 0.45 ¡1m) .
Column:
- size: 1 = 0.15 m, 0 = 4.6 mm;
- stationary phase: end-capped polar-embedded octadecylsil;yl
amorphous organosilica polymer R (3.5 ¡1m);
- temperature: 25 ± 2 oC.
Mobz7e phase:
- mobile phase A : anhydrous fonnic acid R, water R
(0.1:99.9 V/V);
- mobz7e phase B: acetonitrile for chromatography R;
Time Mobile phase A Mobile phase B
(mio)
(per cent VM (per cent VIII)
0·20 47 53
20·22 47 ..... 5 53 ..... 95
22 - 27 5 95
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 292 nm.
lnjection 20 pL.
Relative retention With reference to honokiol (retention
time = about 10 min): magnolol = about 1.3;
honokiol monoacetate isomer 1 = about 1.4;
honokiol monoacetate isomer 2 = about 1.5;
honokiol diacetate = about 1.9.
System suitabz7ity Reference solution (c):
- resolution: minimum 2.0 between the peaks due to
honokiol monoacetate isomers 1 and 2.
Mallow Flower IV-257
If necessary, dilute the test solution 10 obtain peaks of
honokiol and magnolol that are similar in height to the
corresponding peaks in reference solutions (a) and (b).
Calculate the sum of the percentage contents of honokiol and
magnolol using the following expression:
Al x m2 x 0.16 x PI x d + A3 x m 3 x P2 x d
A 2 x mI A4 x mI
area of the peak due to honokiol in the
chromatogram obtained with the test solution;
area of the peak due 10 honokiol in the
chromatogram obtained with reference solution (a);
area of the peak due to magnolol in the
chromatogram obtained with the test solution;
area of the peak due to magnolol in the
chromatogram obtained with reference solution (b);
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of honokiol CRS used to prepare reference
solution (a), in grams;
mass of magnolol CRS used to prepare reference
solution (b), in grams;
PI percentage content of honokiol in honokiol CRS;
P2 percentage content of magnolol in magnolol CRS;
d dilution factor of the test solution.
____________________________________________
~Em
Mallow Flower ***
*** ***
(Ph Eur monograph 1541) ***
~Em __________________________________________
DEFINITION
Whole or fragmented dried flower of Malva sylvestris L. or its
cultivated varieties .
IDENTIFICATION
A. The flower consists of an epicalyx with 3 oblong or
elliptical-lanceolate parts that are shorter than those of the
calyx and situated immediately below it; a calyx with 5
pubescent triangular lobes, gamosepalous at the base;
a corolla 3-4 times longer than the calyx with 5 wedge-
shaped, notched petals fused to the staminal tube at their
base; numerous stamens, the filaments of which fuse into a
staminal tube covered by small star-shaped trichomes and
occasional simple trichomes visible using a lens; numerous
wrinkled carpels, glabrous or sometimes pubescent, enelosed
in the staminal tube and arranged into a cirele around a
central style ending with numerous filiform stigmas .
In cultivated varieties, the epicalyx is 3-7 partite, the calyx
5-8 partite and the corolla 5-10 partite.
B. Reduce 10 a powder (355) (2.9.12). The powder is bluish-
grey. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1541.-1): unicellular, thick-walled,
ftexuous covering trichomes, from the calyx and the epicalyx,
up to 2 mm in length, whole [L) or, most often, fragmented
[Ol; fragments of the epidermis of the sepals in surface view
[D, TI with anomocytic stomata (2.8.3) [Dc]; elub-shaped
glandular trichomes with multicellular heads [Db) and short
unicellular covering trichomes, somewhat curved, either
isolated [TI or in star-shaped groups of 2-6 [Da]; fragments
of covering trichomes [N]; isolated glandular trichomes in
 IV-258 Mallow Leaf
surface view, [F]; or in transverse section [G]; fragments of
the mesophyll of the calyx and the epicalyx whose cells
contain small cluster crystals of calcium oxalate [K]; veins of
the sepals [P] with ves seis [Pa] accompanied by cells with
cluster crystals of ealcium oxalate [Pb]; fragments of petal
epidermis, with elongated eells and sinuous margins, narrow
in the wild plant [A], shorter and broader in the eultivated
varieties [B], bearing sessile glandular trichomes with
multieellular club-shaped heads [Ba, C, E]; fragments of
petal mesophyll [H] consisting of large mueilage eells [He],
sometimes eells with small cluster erystals of ealcium oxalate
[Hb] and spiral vessels [Ha]; spherieal pollen grains, about
150 ~m in diameter, with a roughly spiny exine [M].
Ha
I tJI---+-H- Hb
\ fiattened, longitudinally slightly grooved, green or brownish-
He
Figure 1541.-1.- Illustration for identification test B of
powdered herhal drug of mallow flower
C. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the powdered drug (355) (2.9.12)
add 10 mL of ethanol (60 per eent V/V) R. Stir for 15 min
and filter.
Reference solution 0.5 gIL solution of quinaldine red R in
ethanol (96 per eent) R.
Plate TLC siliea gel plate R .
Mobz7e phase glacial aeetie acid R, water R, butanol R
(15:30:60 VIV/V).
Application 1O ~L of the test solution and 5 ~L of the
reference solution, as bands.
Development Over a path of 10 cm.
Drying In airo
Deteetion Examine in daylight.
Results The chromatogram obtained with the reference
solution shows an orange-red zone in the upper part of the
middle third ; the ehromatogram obtained with the test
solution shows, below the zone in the ehromatogram
obtained with the reference solution, 2 violet zones in the
2014
middle third, with the principal zone (6"-malonyl malvin)
situated just below the other violet zone (malvin).
TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug by drying in an oven at 105 oc.
Total ash (2.4. 16)
Maximum 14.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
Swelling index (2.8.4)
Minimum 15, determined on 0.2 g ofthe powdered drug
(710) (2.9. 12) moistened with 0.5 mL of anhydrous ethanol R
____________________________________________ PhE~
Mallow Leaf
(Ph Bur monograph 2391)
~E~ ____________________________________________
DEFINITlON
Whole or fragmented, dried leaf of Malva sylvestris L., Malva
negleeta Wallr. or a mixture of both speeies .
IDENTIFICATlON
A. The leaves of M. sylvestris are up to 12 cm long and up to
15 cm wide with 3, 5 or 7 lobes and sinuate at the base;
the leaves of M. negleeta are up to 9 cm long and wide,
round or kidney-shaped with 5-7 indistinct lobes. The leaves
of both species have irregular dentate margins and are green
or brownish-green. The abaxial surface of the lamina bears
more hairs and shows a more prominent venation than the
adaxial surface. The major veins on the upper surface of the
leaves and the petioles may be violeto The petioles are as long
as the leaves, up to 2 mm wide, rounded and somewhat
green or vio let. The fragmented drug eonsists of oeeasionally
agglomerated, erumpled pieces of leaves showing prominent
vems.
B. Microscopic examination (2.8.23). The powder is green or
yellowish-green. Examine under a microseope using ehloral
hydrate solution R. The powder shows the following diagnostie
characters (Figure 239l.-1): fragments ofthe lamina, in
transverse section [F], consisting of the lower epidermis, in
surfaee view [C], and the upper epidermis, in surfaee view
[D] or in transverse seetion [Fb], with cells that show
straight, or more or less sinuous anticlinal walls; stomata
mostly anisoeytic (2.8.3) on both surfaces [Ca, Da]; long
covering trichomes with thiekened walls and tapering to a
point at the apex, usually unicellular, whole [A, Fa] or
fragmented [Db], but in M. Sylvestn's they may be stellate
with 2-8 components [H], each strongly pitted at the base;
club-shaped glandular trichomes composed of 2-6 eells [E]
occur in both species; fragments of the mesophyll consisting
of palisade parenchyma, in surfaee view [Dc] or in transverse
section [Fc], and spongy mesophyll eells containing mucilage,
cells containing cluster erystals of ealcium oxalate, often
associated with vessels [B]; occasional spherical pollen grains,
110-170 ~m in diameter, with a spiny exine [G].
 2014 Mandarin Oil IV-259

Top of the plate

--- ---
Hyperoside: a yellow fluorescent
zone
A yellow fluorescent zone
--- ---
Rutin: a yellow fluorescent zone

A yellow fluorescent zone

A light blue fluorescent zone

An orange fluorescent zone

An orange f1uorescent zone

Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of foreign organs, maximum 5 per cent
of leaves with blisters of spores of Puccinia malvacearum and
maximum 2 per cent of foreign elements.
Foreign organs can be flowers, fruits and parts of the stem.
The blisters of spores on the leaves are mostly 1 mm wide,
and red or brown. Examine under a microscope using chloral
hydrate solution R. The spores of Puccinia malvacearum are
oblong or oval with brownish walls and a small appendage.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g of the
powdered drug (710) (2.9.12) by drying in an oven at
105 oC for 2 h.
Figure 2391.-1. - Illustration for identification test B of Total ash (2.4.16)
powdered herbal drug of mallow leaf Maximum 17.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
C. Thin-Iayer chromatography (2.2.27). Maximum 3.0 per cent.
Test solution To 2.0 g of the powdered drug (710) (2.9.12) Swelling index (2.8.4)
add 20 mL of an 80 per cent VIV solution of Minimum 7, determined on l.0 g of the powdered drug
tetrahydr(>furan R; extract for 10 min using sonication and (710) (2.9.12) .
filter. PhEuf
Reference solution Dissolve 3 mg of hyperoside R and 3 mg of
ruán R in 20 mL of methanol R.
Plate TLC silica gel plate R (5-40 flm) [or TLC silica gel
plate R (2-10 flm)].
Mandarin Oil ***
Mobile phase anhydrous formic acid R, anhydrous acetic acid R, *** ***
water R, ethyl formate R, 3-pentanone R (Ph Eur monograph 2355) ***
(4: 11: 14:20:50 VIVIVIVIV) . PhEuf _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ __
Application 10 flL [or 4 flL] as bands of 10 mm [or 8 mm].
DEFINITION
Development Over a path of 10-12 cm [or 6 cm]. Essential oil obtained without heating, by suitable mechanical
Drying In air. treatment, from the peel of the fresh fruit of Citrus reticulata
Detection Reat at 100 oC for 10 mini spray or dip the warm Blanco.
plate in a 10 gIL solution of diphenylboric acid aminoethyl CHARACTERS
ester R in methanol R; remove the solvent with cold air; spray Appearance
or dip the plate in a 50 gIL solution of macrogol 400 R in Greenish, yellow or reddish orange liquid showing blue
methanol R, dry in air and examine afrer 15 min in ultraviolet fluorescence .
light at 365 nm.
Characteristic odour.
Results See below the sequence of fluorescent zones present
in the chromatograms obtained with the reference solution IDENTIFICATION
and the test solution. Furthermore, other faint fluorescent First identijication B.
zones may be present in the chromatogram obtained with the Second identijication A.
test solution. A. Thin-Iayer chromatography (2.2.27).
Test solution Dilute 0.1 mL of the substance to be examined
to 1 mL with toluene R.
 IV-260 Mandarin Oil 2014

Reference solution Dissolve 2 pL of methyl 5 ~IL of methyl N -methylanthramlate R to 5.0 mL with

N-methylanthrani/ate R, 4 mg of guaiaz ulene R and 10 mg of heptane R .
a-terpineol R in 10 mL of toluene R. Reference solution (b) Dissolve 5 ~lL of limonene R in 50 mL
Plate TLC si/ica gel plate R (5-40 pm) [or TLC silica gel of heptane R. Dilute 0.5 mL ofthis solution to 5.0 mL with
plate R (2-10 pm)). heptane R.
Mobile phase ethyl acetate R, toluene R (15:85 V/V). Column:
Application 10 pL [or 2 pL) as bands. - material: fused silica;
- size: l = 60 m, 0 = 0.25 mm;
Development Over a path of 15 cm [or 6 cm).
- stationary phase: poly(dimethyl) (diphenyl)siloxane R (film
Drying In airo thickness 0.25 pm).
Detection A Examine in ultraviolet light at 365 nm. Cam'er gas helium for chromatography R .
Results A The intense blue fiuorescent zone in the Flow rate 1.4 mL/min.
chromatogram obtained with the test solution is similar in
Split ratio 1:70.
position and fiuorescence to the zone due to methyl
N-methylanthranilate in the chromatogram obtained with the Temperature:
reference solution. Furthermore, other fiuorescent zones may Time Temperature
be present in the chromatogram obtained with the test (min) (oC)
solution. Colurnn 0-90 50 -> 230

Detection B Spray with a 200 gIL solution of Injection port 250

phosphomolybdic acid R in ethanol (96 per cent) R and heat at Detector 250
100 oC for 10 min; examine in daylight.
Results B See below the sequence of the zones present in Detection Flame ionisation.
the chromatograms obtained with the reference solution and Injection 1 pL.
the test solution. Furthermore, other zones may be present in Elution order Order indicated in the composition of
the chromatogram obtained with the test solution. reference solution (a); record the retention times of these
substances .
Top of the plate System suitability Reference solution (a):
- resolution: minimum 1.5 between the peaks due to
A blue zone sabinene and ~-pinene and minimum 1.5 between the
Guaiazulene: a blue zone A blue zone peaks due to p-cymene and limonene.
Identification of components Using the retention times
A blue zone
determined from the chromatogram obtained with reference
--- --- solution (a), locate the components of reference solution (a)
A blue zone in the chromatogram obtained with the test solution.
Disregard the peak due to heptane.
- -- ---
Determine the percentage content of each of these
a.-Terpineol: a blue zone A blue zone (ex-terpineol) components. The lirnits are within the following ranges:
Reference solution Test solution - a-pinene: 1.6 per cent to 3.0 per cent;
- sabinene: maximum 0.3 per cent;
- {J-pinene: 1.2 per cent to 2.0 per cent;
B. Examine the chromatograms obtained in the test for - {J-myrcene: 1.5 per cent to 2.0 per cent;
chromatographic profile. - p-cymene: maximum 1.0 per cent;
Results The characteristic peaks in the chromatogram - limonene: 65.0 per cent to 75.0 per cent;
obtained with the test solution are similar in retention time to - y-terpinene: 16.0 per cent to 22.0 per cent;
those in the chromatogram obtained with the reference - methyl N-methylanthranilate: 0.30 per cent to
solution. 0.60 per cent;
- disregard limit: area of the principal peak in the
TESTS chromatogram obtained with reference solution (b).
Relative density (2. 2. S)
Residue on evaporation (2.8.9)
0.848 to 0.855.
1.6 per cent to 4.0 per cent, determined after heating on a
Refractive index (2.2.6) water-bath for 4 h.
1.474 to 1.478.
STORAGE
Optical rotation (2.2. 7)
At a temperature not exceeding 25 oC.
+ 64° to + 75°. _ _ __ _ _ _ __ __ __ __ _ __ __ _ _ ~Ew

Fatty oils and resinified essential oils (2.8.7)

It complies with the test.
Chromatographic pro file
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Dilute 0.20 g of the substance to be examined
to 10.0 mL with heptane R.
Reference solution (a) Dilute 5 pL of a-pinene R, 5 pL of
sabinene R, 5 pL of fi-pinene R, 5 pL of fi-myrcene R, 5 pL of
p-cymene R, 70 pL of limonene R, 20 pL of y-terpinene R and
 2014 Marshmallow Leaf IV-261

Marshmallow Leaf
(Ph Eur monograph 1856)
~E~ ____________________________________________
Ab
I
DEFINITION
Whole or cut, dried leaf of Althaea officinalis L.
IDENTIFICATION e
A. The lea ves have long petioles and are about 7-10 cm long;
the lamina is cordate or ovate with 3-5 shallow lobes and
crenate or dentate margins; the venation is palma te.
The petioles and both surfaces of the lamina are greyish-
green and densely pubescent. Rarely, fragments of the
inflorescence or immature fruits may be presento
B. Microscopic examination (2.8.23) . The powder is greyish-
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1856.-1) : numerous long, rigid, unicellular
covering trichomes with thick walls, pointed at the apex,
often fragmented [C], angular and pitted at the base where
they are sometimes still united to form stellate structures with
up to 8 components, in surface view [B] or in transverse
section [E]; few secretory trichomes, isolated, with unicellular
stalks and globular, multicellular heads [F]; fragments of the
lower [A] and upper [D] leaf epidermises in surface view
with anomocytic [Aa] or paracytic [Da] stomata ( 2.8.3),
glandular trichomes [Ab] and basal cells of covering
trichomes [Ac], often accompanied by palisade parenchyma
[Db]; cluster crystals of calcium oxalate, isolated [H] or
included in the parenchyma of the mesophyll [Gc, Kb]; Figure 1856.-1. - Illustration for identification test B of
fragments ofveins [G] with small, spiral [Gb] or annular powdered herbal drug of marshmallow leaf
[Ga] vessels, often accompanied by sheaths containing cluster
crystals of ca1cium oxalate [Gel; fragments ofthe lamina, in
transverse section [K], showing the epidermis es bearing
broken covering trichomes [Ka], a symmetrical, Top of the plate
heterogeneous mesophyll with sorne cells containing cluster A blue fIuorescent zone
crystals of ca1cium oxalate [Kb]; occasional pollen grains,
spherical, with a roughly spiny exine, about 150 )lm in A yeIlow fIuorescent zone
diameter m. Examine under a microscope using ruthenium Quercitrin: an orange zone
red solution R . The powder shows groups of parenchyma
containing mucilage, which stains orange-red.
----- ---
C. Thin-layer chromatography (2.2.27). An orange fluorescent zone
Test solution To 1 g of the powdered herbal drug (355) An orange fIuorescent zone
(2.9.12) add 10 mL of methanol R. Heat in a water-bath ----- ---
under a reflux condenser for 5 mino Allow to cool and filter.
Distil the filtrate under reduced pressure until the total Chlorogenic acid: a blue
f1uorescent zone
volume is about 2 mL.
A blue f1uorescent zone
Reference solution Dissolve 2.5 mg of chlorogenic acid R and
2.5 mg of quercitrin R in 10 mL of methanol R. An orange fIuorescent zone
Plate TLC silica gel plate R. An intense yeIlow fIuorescent zone
Mobile phase anhydrous formic acid R, glacial acetic acid R,
Reference solution Test solution
water R, ethyl acetate R (11:11:27:100 VIVIV/V).
Application10 [lL as bands.
Development Over a path of 15 cm. TESTS
Drying At 100-105 oC. Foreign matter (2.8.2)
Detection Spray with a 10 giL solution of diphenylboric acid Maximum 4 per cent of leaves infected by Puccinia
aminoethyl ester R in methanol R, then with a 50 gIL solution malvacearum, showing red spots, and maximum 2 per cent of
of macrogol 400 R in methanol R; allow to dry in air for other foreign matter.
30 min and examine in ultraviolet light at 365 nm. Loss on drying (2.2.32)
Results See below the sequence of zones present in the Maximum 10.0 per cent, determined on 1.000 g of the
chromatograms obtained with the reference solution and the powdered herbal drug (355) (2.9.12) by drying in an oven at
test solution. Furthermore, other fluorescent zones may be 105 oC for 2 h .
present in the chromatogram obtained with the test solution. Total ash (2.4.16)
Maximum 18.0 per cent.
 IV-262 Marshmallow Root 2014

Ash insoluble in hydrochloric acid (2.8.1)

Maximurn 2.0 per cent.
Swelling index (2.8.4)
Minimum 12, determined on 0.2 g ofthe powdered herbal
drug (355) (2.9.12).
_ _ __ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ PhEur
~B
***
Marshmallow Root
*** ***
(Ph Eur monograph 112Ó) ***
~Eif_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

DEFINITION
Peeled or unpeeled, whole or cut, dried root of Althaea
offieinalis L.
IDENTIFICATION
A. The unpeeled, non-fragmented drug consists of
cylindrical, slightly twisted roots, up to 2 cm thick, with deep
longitudinal furrows. The outer surface is greyish-brown and
bears numerous rootlet scars. The fracture is fibrous
externally, rugged and granular intemally. The section shows
a more or less thick, whitish bark with brownish periderm,
separated by the well-marked, brownish cambiurn from a
white xylem. The stratified structure of the bark and the
radiate structure of xylem become more distinct when
moistened.
The peeled drug has a greyish-white, finely fibrous outer Figure 1126.-1. - Illustration for identification test B of
surface. eork and external cortical parenchyma are absent. powdered herbal drug of marshmallow root
B. Microscopic examination (2.8.23). The powder is greyish-
brown (unpeeled root) or whitish (peeled root) . Examine
under a microscope using ehloral hydrate solution R . Swelling index (2.8.4)
The powder shows the following diagnostic characters Minimum 10, determined on the powdered herbal drug
(Figure 1126.-1): fragments of colourless, mainly unlignified, (710) (2. 9.12).
thick-walled fibres [e, D, M] with split or pointed ends [D], _ _ _ _ _ _ _ _ _ _ _____ _ _ _ _ _____ ~Eif

sometimes accompanied by parenchymatous cells of the

medullary rays [M], or grouped [C]; fragments of vessels,
bordered-pitted or with reticulate or scalariform thickenings
[G, H]; cluster crystals of calcium oxalate about 20-35 /lm,
mostly 25-30 11m in size, isolated [K] or included in
parenchymatous cells [B]; fragments of parenchyma [E] with
cells containing mucilage [Ea, F]; fragments of cork with
thin-walled, tabular cells in surface view [A] and transverse
section [L] (unpeeled root). Examine under a microscope
using ruthenium red solution R . The powder shows groups of
parenchyma containing mucilage, which stains orange-red.
Examine under a microscope using water R . The powder
shows numerous starch granules m, about 3-25 /lm in size,
occasionally with a longitudinal hilurn. The starch granules
are mostly simple ITa], a few being 2-4 compound Ub].
TESTS
Foreign matter (2.8.2)
Maximurn 2 per cent of brown deteriorated drug.
Loss on drying (2.2.32)
Maximurn 12.0 per cent, determined on l.000 g of the
powdered herbal drug (710) (2.9.12) by drying in an oven at
105 ce for 2 h .
Total ash (2.4.1Ó)
Maximurn 6.0 per cent for the peeled root and maximum
8.0 per cent for the unpeeled root.
Mastic *****
** **
(Ph Eur monograph 187Ó) ***
~Eif____________ _ _ _ _ ________________ _
DEFINITION
Dried resinous exudate obtained from stems and branches of
Pistacia lentiseus L. var. latifolius eoss.
Content
Minimum 10 mlJkg of essential oil (anhydrous drug) .
IDENTIFICATION
A. Smalllight yellow to greenish-yellow, non-uniform,
spherical or pyriform, clear or opaque, hard glassy fragments.
B. Thin-layer chromatography (2.2.27) .
Test solution Dissolve 1 g of the substance to be examined
in 10 mL of methylene ehloride R and fiIter after 1-2 mino
Referenee solution Dissolve 25 mg of eugenol R and 25 mg of
borneol R in 3 mL of methylene ehloride R.
Plate TLC si/iea gel plate R.
Mobile phase light petroleum R, toluene R (5:95 V IV) .
Applieation 1 /lL, as bands.
Development Over a path of 10 cm.
Drying In air.
2014 Matricaria Flowers IV-263

Detection Spray with vanillin reagent R and heat at several dozen yellow central tubular ftorets. The involucre
100-105 oC for 5 min. bracts are ovate or lanceolate, with a brownish-grey scarious
R esults See below the sequence of the zones present in the margino The receptacle is hollow, without paleae. The coralla
chromatograms obtained with the reference solution and the of the ligulate ftorets has a brownish-yellow tube at the base
test solution. Furthermore, other zones of various colours extending to form a white, elongated-ovalligule. The inferior
may be present in the chromatogram obtained with the test ovary is dark brown, ovoid or spherical, and has a long style
solution. and bifid stigma. The tubular ftorets are yellow and have a
five-toothed corolla tube, 5 syngenesious, epipetalous
stamens and a gynoecium similar to that of the ligulate
Top of the plate
ftorets.
A violet zone B. Separate the capitulum into its different parts. Examine
- -- --- under a microscope using chloral hydrate solution R .
The bracts have a margin composed of thin-walled cells and
A pale violet zone
a central region composed of elongated sclereids with
A very pale violet zone occasional stomata (2.8.3). The inner epidermis of the
coralla of the ligulate ftorets, in surface view, consisting of
--- ---
thin-walled, polygonal cells, slightly papillose, those of the
Eugenol: a brown zone A blue zone outer epidermis markedly sinuous and strongly striated;
Borneol : a greenish-blue zone A bluish-violet zone corolla of the tubular ftorets with longitudinally elongated
epidermal cells, and with small groups of papillae near the
A dark violet zone apex of the lobes . Glandular trichomes each consisting of a
Reference solution Test solution short stalk and a head of 2-3 tiers of 2 cells each occur on
the outer surfaces of the bracts and on the corollas of both
types of ftorets. The ovaries have a sclerous ring at the base
TESTS and the wall is composed of vertical bands of thin-walled,
Acid value (2.5.1) longitudinally elongated cells with numerous glandular
50 to 70, determined on 1.0 g. trichomes, alternating with fusiform groups of small, radially
Water (2.2.13) elongated cells containing mucilage. The cells at the apex of
Maximum 10 mUkg, determined on 25 .0 g ofthe drug the stigmas are extended to form rounded papillae.
reduced to a coarse powder (1400) (2.9.12). Numerous small, cluster crystals of calcium oxalate occur in
the inner tissues of the ovaries and the anther lobes . Pollen
Total ash (2.4.16)
grains spherical to triangular, about 30 ¡.tm in diameter with
Maximum 0.5 per cent.
3 pores and a spiny exine.
ASSAY C . Thin-Iayer chromatography (2.2.27).
Essential oil (2.8.12)
Test solutwn Dilute 50 ¡.tL of essential oi! obtained in the
Use a 500 mL round-bottomed ftask and 200 mL of water R
assay of essential oi! in 1 mL of xylene R .
as the distillation liquido Reduce the drug to a coarse powder
(1400) (2. 9.12) and immediately use 20.0 g for the R eference solution Dissolve 2 ¡.tL of chamazulene R, 5 ¡.tL of
determination. Introduce 0.50 mL of xylene R in the (-)-a-bisabolol R and 10 mg of bornyl acetate R in 5 mL of
graduated tube. Distil at arate of 2-3 mUmin for 2 h. toluene R .
Plate TLC silica gel plate R.
STORAGE
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Do not powder.
____________________________________________ ~Em
Application 10 ¡.tL, as bands.
Development Over a path of 10 cm.
Drying In air.
Detection Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 mino Examine immediately in daylight.
Matricaria Flowers
Results See below the sequence of zones present in the
(Matricaria Flower, Ph Bur monograph 0404) chromatograms obtained with the reference solution and the
Preparation test solution. Furthermore, other zones are present in the
Matricaria Liquid Extract chromatogram obtained with the test solution.
~Em ____________________________________________ TESTS
DEFINITION Broken drug
Maximum 25 per cent, determined on 20.0 g, passes through
Dried capitula of Matrican'a recutita L. (Chamomilla recutita
a sieve (710) (2.9.12).
(L.) Rauschert).
Loss on drying (2.2.32)
Content:
Maximum 12.0 per cent, determined on 1.000 g of the
-- blue essential oil: minimum 4 mUkg (dried drug);
powdered drug (355) (2.9.12) by drying in an oven at
-- total apigenin 7-glucoside (C21 H 2001O): minimum
105 oC for 2 h.
0.25 per cent (dried drug).
Total ash (2.4.16)
IDENTIFICATION Maximum 13.0 per cent.
A. Capitula, when spread out, consisting of an involucre
made up of many bracts arranged in 1-3 rows; an elongated-
conical receptacle, occasionally hemispherical (young
capitula); 12-20 marginalligulate ftorets with a white ligule;
 IV-264 Matricaria Oil 2014

Top of the plate Time Mobile phase A Mobile phase B

(min) (per cent V/Vl (per cent V/Vl
1 or 2 blue or bluish·violet zones
0·9 75 25
Chamazulene: a red or A red or reddish·violet zone
9·19 75 .... 25 25 -7 75
reddish·violet zone (chamazulene)
--- --- 19·24 25 75

Bornyl acetate: a yellowish·brown

zone
Flow rate 1 mUmin.
A brown zone (en·yne-dicycloether) Detection Spectrophotometer at 340 nm.
--- ---
Injection 20 flL.
System suitability Reference solution (b):
(-)-a·Bisabolol: a reddish·violet or A reddish·violet or bluish·violet
bluish·violet zone zone «-l-a·bisabolon
resolution: minimum 1.8 between the peaks due to
Reference solution Test solution
apigenin 7-glucoside and 5,7-dihydroxy-
4-methylcoumarin.
Calculate the percentage content of total apigenin 7-glucoside
using the following expression:
ASSAY
Essential oH (2.8.12)
Use 30 g ofwhole drug, a 1000 mL flask, 300 mL of water R
as distillation liquid and 0.50 mL of xylene R in the
graduated tube. Distil at arate of 3-4 mUmin for 4 h .
Al area of the peak due to apigenin 7-glucoside in the
Towards the end of this period, stop the flow of water to the
chromatogram obtained with the test solution;
condenser assembly but continue distilling until the blue,
A2 area of the peak due to apigenin 7-glucoside in the
steam-volatile components have reached the lower end of the
chromatogram obtained with the reference solution;
condenser. Immediately re-start the fiow of water to the
mi mass of the drug in the test solution, in grams;
condenser assembly to avoid warming the separation space.
m2 mas s of apigenin 7-glucoside R in reference solution
Stop the distillation after a further 10 mino
(a), in grams;
Total apigenin 7-glucoside P percentage content of apigenin 7-glucoside in the
Liquid chromatography (2.2.29) . reagent.
Test solution Reduce 40 g of the drug to a powder (500) ____________________________________________ ~Ew

(2. 9.12). Place 2.00 g ofthe powdered drug in a 500 mL

round-bottomed fiask. Add 200 mL of ethanol
(96 per cent) R. Heat the mixture under a refiux condenser
on a water-bath for 15 mino Cool and filter. Rinse the filter
Matricaria OH ****
***
and the residue with a few millilitres of ethanol
*
*** *
(96 per cent) R . To the filtrate add 10 mL offreshly prepared
(Ph Eur monograph 1836)
dilute sodium hydroxide solution R and heat the mixture under
PhEw ____________________________________________
a reflux condenser on a water-bath for about 1 h. Coo!.
Dilute to 250.0 mL with ethanol (96 per cent) R. To 50.0 mL DEFINITION
of the solution add 0.5 g of citric acid R. Shake for 5 min and Blue essential oil obtained by steam distillation from the fresh
filter. Dilute 5.0 mL of this solution to 10.0 mL with the or dried fiower-heads or fiowering tops of Matn·caria recutita
mobile phase (initial mixture). L. (Chamomilla recutita L. Rauschert) . There are 2 types of
Reference solution (a) Dissolve 10.0 mg of apigenin matricaria oil which are characterised as rich in bisabolol
7-glucoside R in 100.0 mL of methanol R. Dilute 25.0 mL of oxides, or rich in (- )-ex-bisabolol.
this solution to 200.0 mL with the mobile phase (initial
CHARACTERS
mixture).
Appearance
Reference solution (b) Dissolve 10.0 mg of 5,7-dihydroxy- Clear, intensely blue, viscous liquido
4-methylcoumarin R in 100.0 mL of methanol R . Dilute
25.0 mL of this solution to 100.0 mL of the mobile phase Intense characteristic odour.
(initial mixture). To 4.0 mL ofthis solution add 4.0 mL of IDENTIFICAnON
reference solution (a) and dilute to 10.0 mL with the mobile First identification B.
phase (initial mixture). Second identification A .
Precolumn: A. Thin-layer chromatography (2.2.27) .
-- size: 1 = 8 mm, 0 = 4.6 mm;
Test solution Dissolve 20 JlL of the substance to be
-- stationary phase: octadecylsi1y1 silica gel for chromatography R
examined in 1.0 mL of toluene R.
(5 flm).
Column: Reference solution Dissolve 2 mg of guaiazulene R, 5 flL of
( - ) -Ci -bisabolol R and 10 mg of bornyl acetate R in 5. O mL of
-- size: 1 = 0.25 m, 0 = 4.6 mm;
-- stationary phase: octadecylsi1y1 silica gel for chromatography R toluene R.
(5 flm). Plate TLC silica gel plate R.
Mobile phase: Mobile phase ethyl acetate R, toluene R (5:95 V/V).
-- mobile phase A: phosphoric acid R, water R (0.5:99 .5 V/V); Application 10 JlL, as bands.
mobile phase B: phosphoric acid R, acetonitrile R Development Over a path of 10 cm.
(0.5:99.5 V/V);
Drying In airo
Detection A Examine in daylight.
2014 Matricaria Oil IV-265

Results ASee below for the sequence of the zones present Top of the plate
in the chromatograms obtained with the reference solution 1 or 2 blue to bluish-violet zones
and the test solution.
Guaiazulene : a red to reddish-violet A red to reddish-violet zone
zone (chamazulene)
Top of the plate --- ---
Guaiazulene : a blue zone A blue zone (chamazulene) Bornyl acetate: a yellowish-brown A brown zone (en-yne-dicycloether)
to greyish-green zone
- -- --- ---
---
--- --- (-}-a-Bisabolol: a reddish-violet to A reddish-violet to bluish-violet
Reference solution Test solution bluish-violet zone zone ((-)-a-bisabolol)
A brownish zone

Reference solution Test solution

Detection B Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 mino Examine immediately in daylight.
R esults B See below for the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, yellowish-brown to greenish-
yellow zones (lower third), violet zones (lower third) and
further weak zones may be present in the chromatogram
obtained with the test solution.
B. Examine the chromatograms obtained in the test for
chromatographic profile.
Results The characteristic peaks due to (- )-ex-bisabolol and
chamazulene in the chromatogram obtained with the test
solution are similar in retention time to those in the
chromatogram obtained with the reference solution.
TESTS
Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Dissolve 20 llL of the oil to be examined in
cyclohexane R and dilute to 5.0 mL with the same solvento
Reference solution Dissolve 20 llL of (- ) -C/. -bisabolol R, 5 mg
of chamazulene R and 6 mg of guaiazulene R in cyclohexane R
and dilute to 5.0 mL with the same solvento
Column:
- material: fused silica,
- size: l = 30 m (a film thickness of 1 11m may be used) to
60 m (a film thickness of 0.2 11m may be used),
o= 0.25-0.53 mm, when using a column longer than
30 m, an adjustment of the temperature programme may
be necessary,
- stationary phase: macrogol 20 000 R .
Carrier gas helium for chromatography R .

3
2

4 5

: ~~.~I
J~~L~ljl.~~~¡.~~I~l
J\

o 5 10 15 20 25 30 35 40 45

1. (}-farnesene 3. bisabolone 5. chamazulene

2. bisabolol oxide B 4. (-)-a.-bisabolol 6. bisabolol oxide A

Figure 1836.-1. - Chromatogram oi matricaria oi! rich in bisabololoxides

 IV-266 Matricaria Oil 2014

5
2

3 I
)-- .1 1

o 5 10 15 20 25 30 35 40 45 mm

1. ¡l-farnesene 3. bisabolone 5. charnazulene

2. bisabolol oxide B 4. (-)-a-bisabolol 6. bisabolol oxide A

Figure 1836.-2. - Chromatogram of matricaria oi! rich in (-)-~bisabolol

Flow rate 1-2 mUmin. solution does not show a peak with the retention time of
Split ratio 1:1OO. guaiazulene.
Temperature: Determine the percentage content of the components.
The limits are within the following ranges.
Time Temperature
(min) (oC)
Colurnn 0-40 70 -7 230 Matricaria 011 rich in Matricaria 011 rich in
hisabolol oxides (-kx-bisabolol
40 - 50 230 (per cent) (per cent)
Injection port 250 Bisabolol oxides 29 - 81
Detector 250 (-)-{l-Bisabolol 10 - 65

Detection Flame ionisation. Chamazulene ;, 1.0 ;, 1.0

Injection 1.0 1lL. Total ofbisabolol oxides ;, 20
and (-WBisabolol
Elution order Order indicated in the composition of the
reference solution. Record the retention times of these
substances. STORAGE
Relative retention With reference to chamazulene At a temperature not exceeding 25 oc.
(retention time = about 34.4 min): ~-farnesene = about 0.5; ____________________________________________ ffiE~

bisabolol oxide B = about 0.8; bisabolone = about 0.87;

(- )-o:-bisabolol = about 0.9; bisabolol oxide A = about 1.02.
System suitability Reference solution:
-- resolution: minimum 1.5 between the peaks due to
chamazulene and guaiazulene.
Using the retention times determined from the
chromatogram obtained with the reference solution, locate
(- )-o:-bisabolol and chamazulene in the chromatogram
obtained with the test solution; locate bisabolol oxides
(bisabolol oxide B, bisabolone and bisabolol oxide A) using
Figures 1836.-1 and 1836.-2 (disregard the peak due to
cyclohexane) . The chromatogram obtained with the test
2014
*****
Matricaria Liquid Extract
** **
(Ph Bur monograph 154'f) ***
~E~ ____________________________________________
DEFINITION
Liquid extract produced from Matricaria fiower (0404).
Content
Minimum 0.30 per cent of blue residual oi!.
PRODUCTION
The extract is produced fram the herbal drug by a suitable
procedure for liquid extracts using a mixture of 2.5 volumes
of a 10 per cent m /m solution of arnmonia (NH 3) ,
47 .5 volumes of water and 50 volumes of ethanol
(96 per cent) .
CHARACTERS
Appearance
Brownish, clear liquido
Intense characteristic odour and characteristic bitter taste.
Solubility
Miscible with water and with ethanol (96 per cent) with
development of turbidity, soluble in ethanol
(50 per cent V/V).
IDENTIFICATION
A. Thin-layer chromalOgraphy (2.2.27) .
Test solution Place 10 mL of the extract to be examined in a
separating funnel and shake with 2 quantities, each of
10 mL, of pentane R. Combine the pentane layers, dry over
2 g of anhydrous sodium sulfate R and filter. Evaporate the
filtra te lO dryness on a water-bath and dissolve the residue in
0.5 mL of toluene R.
Reference solution Dissolve 4 mg of guaiaz ulene R , 20 mg of
(-) -(f.-bisabolol R and 20 mg of bornyl acetate R in 10 rnL of
toluene R.
Plate TLC silica gel F254 plate R .
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 10 ¡.tL as bands.
Development Over a path of 10 cm.
Drying In air.
DeteclÍon A Examine in ultraviolet light at 254 nm.
Results A The chromalOgram obtained with the test solution
shows several quenching zones, of which 2 main zones are in
the middle third (en-yne-dicycloether).
Detection B Examine in ultraviolet light at 365 nm.
Results B The chromatogram obtained with the test solution
shows in the middle part an intense blue fiuorescent zone
(herniarin) .
DeteclÍon C Spray with anisaldehyde solution R and examine
in daylight while heating at 100-105 cC for 5-10 mino
Results C The chromalOgram obtained with the reference
solution shows in the lower third a reddish-violet or bluish-
violet zone ((-)-a-bisabolol), in the middle third a yellowish-
brown or greyish-green zone (bornyl aceta te) and in the
upper third a red or reddish-violet zone (guaiazulene) .
The chromalOgram obtained with the test solution shows in
the lower third yellowish-brown or greenish-yellow and violet
zones and a reddish-violet or bluish-violet zone due to
(-)-a-bisabolol in the chromatogram obtained with the
reference solution; a brownish zone (en-yne-dicycloether)
similar in position lO the zone due lO bornyl acetate in the
chromalOgram obtained with the reference solution; a red or
reddish-violet zone (chamazulene) corresponding to
Matricaria Preparations IV-267
guaiazulene in the chromatogram obtained with the reference
solution and immediately aboye it 1 or 2 blue or bluish-violet
zones; further weak zones may be present in the
chromatogram obtained with the test solution.
B. Thin-layer chramatography (2.2.27).
Test solution The extract lO be examined.
Reference solution Dissolve 1.0 mg of chlorogenic acid R,
2.5 mg of hyperoside R and 2.5 mg of rutin R in 10 mL of
methanol R.
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R, glacial acetic acid R ,
water R, ethyl acetate R (7.5:7.5:18 :67 VIVIV/V).
Application 10 ¡.tL as bands.
D evelopment Over a path of 15 cm.
Drying At 100-105 oC.
Detection Spray the warm plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R;
subsequently spray with a 50 giL solution of macrogol 400 R
in methanol R; allow to dry in air for about 30 min and
examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the midd1e part a light blue fiuorescent
zone (chlorogenic acid), below it a yellowish-brown
fiuorescent zone (rutin) and aboye it a yellowish-brown
fiuorescent zone (hyperoside). The chromatogram obtained
with the test solution shows a yellowish-brown fiuorescent
zone corresponding lO the zone of rutin in the chromatogram
obtained with the reference solution, a light blue fiuorescent
zone corresponding to the zone of chlorogenic acid in the
chromatogram obtained with the reference solution, a
yellowish-brown fiuorescent zone similar in position to the
zone of hyperoside in the chromalOgram obtained with the
reference solution; it also shows aboye the yellowish-brown
fiuorescent zone a green fiuorescent zone, then several bluish
or greenish fiuorescent zones and near the solvent front a
yellowish fiuorescent zone.
TESTS
Ethano1 (2.9.10)
38 per cent V/V lO 53 per cent VIV.
Dry residue (2.8.16)
Minimum 12.0 per cent.
ASSAY
Place 20.0 g in a 1000 mL round-bottomed fiask, add
300 mL of water R and distil until 200 mL has been
collected in a fiask. Transfer the distillate into a separating
funne!. Dissolve 65 g of sodium chloride R in the distillate and
shake with 3 quantities, each of 30 mL, of pentane R
previously used lO rinse the refiux condenser and the fiask.
Combine the pentane layers, dry over 2 g of anhy drous sodium
sulfate R and filter into a tared 100 mL round-bottomed fiask
which has been dried in a desiccator for 3 h. Rinse the
anhydrous sodium sulfate and the filter with 2 quantities,
each of 20 mL, of pentane R. Evaporate the pentane in a
water-bath at 45 oC . The residue of pentane is eliminated in
a current of air for 3 mino Dry the fiask in a desiccalOr for
3 h and weigh. The residual oil is blue (chamazulene).
____________________________________________ ~ E~
 IV-268 Meadowsweet 2014

Meadowsweet
(Ph Eur monograph 1868)
Ph E~ ____________________________________________

DEFINITlON
Whole or cut, dried ftowering tops of Filipendula ulmaria (L.)
Maxim. (syn. Spiraea ulmaria L.).
Content
Minimum 1 mIJkg of essential oi! (dried drug).
CHARACTERS
Aromatic odour of methyl salicylate, after crushing.
IDENTIFICATION
A. The stem, up to 5 mm in diameter, is greenish-brown,
stif!, angular, hollow except at the apex, and has regular,
straight, longitudinal furrows. The petiolate leaf, compound
imparipinnate, has 2 reddish-brown angular stipules.
It consists of 3-9 pairs of leaftets, unevenly dentate, sorne of
which are small and fan-shaped. The leaftets are dark green
and glabrous on the upper surface, tomentose and lighter,
sometimes silvery on the lower surface. The terminal leaftet,
the largest, is divided into 3 segments. The veins are
prominent and brown on the lower surface.
The inflorescence is complex and composed of very
numerous ftowers arranged in irregular cymose panicles.
The ftowers are creamish-white and about 3-6 mm in
diameter; the calyx consists of 5 dark green, reftexed and
hairy sepals fused at the base to a concave receptacle; the 5
free petals, which are readily detached, are pale yellow, Figure 1868.-1. - Illustration for identification test B of
obovate and distinctly narrowed at the base; the stamens are powdered herbal drug of meadowsweet
numerous with rounded anthers and they extend beyond the
petals; the gynoecium consists of about 4-6 carpels, each with
a short style and a globular stigma; the carpels become of vascular tissue [L) with annular, spiral or pitted vessels
twisted together spirally to form yellowish-brown fruits with a from the leaves and stems.
helicoidal twist. Unopened ftower buds are frequently
C. Thin-Iayer chromatography (2.2.27).
presento If the fruit is present, it has a helicoidal twist and
contains brownish seeds. Test solution Xylene solution obtained in the assay.
B. Microscopic examination (2.8.23). The powder is green or Reference solution Dissolve 0.1 mL of methyl salieylate R and
yellowish-green. Examine under a microscope using ehloral 0.1 mL of salicylaldehyde R in xylene R and dilute to 5 mL
hydrate solution R. The powder shows the following diagnostic with the same solvent.
characters (Figure 1868.-1): fragments ofthe epidermis es of Plate TLC siliea gel plate R .
the leaves and sepals [C, E, F] with sinuous or wavy cells Mobile phase hexane R, toluene R (50:50 V/V) .
[Ca, Ea, Fa], short, thick-walled, conical covering trichomes Application 10 JlL as bands.
thickened at the base, in surface view [Eb] and in side view
Development Over a path of 10 cm.
m, unicellular covering trichomes, thin-walled, very long and
ftexuous, with pointed ends in surface view [Fc] and in side Drying In air.
view [A], or their scars (ftexuous trichome [Fd], conical Detection Treat with 3 mL offem'c ehloride solution R3 and
trichome [Fe]) and occasional c1avate glandular trichomes examine in daylight.
with a 1- to 3-celled ([Ed] and [G], respectively), uniseriate Results See below the sequence of zones present in the
stalk, a multicellular head and dense brown contents; chromatograms obtained with the reference solution and the
fragments of the upper epidermis often accompanied by test solution. Furthermore, other zones are present in the
palisade parenchyma [Cb] including sorne hypertrophied cells chromatogram obtained with the test solution.
containing a cluster crystal of calcium oxalate [Cc];
fragments of the lower epidermis with anomocytic stomata
(2.8.3) [Ec, Fb], sometimes accompanied by spongy Top of the plate
parenchyma [Ff] with sorne cells containing cluster crystals of
--- - --
calcium oxalate [Fg]; fragments of the petals [H) with thin-
walled epidermal cells, sorne showing rounded papillae [Ha]; Methyl salicylate : a violet-brown A violet-brown zone (methyl
numerous spherical pollen grains with 3 pores and a faintly zone salicylate)
pitted exine [Bb] ; fragments ofthe anther [B, D) whose
Salicylaldehyde : a violet-brown A violet-brown zon e
fibrous layer shows specific thickenings, in surface view [D) zone (salicylaldehyde)
and in side view [Ba]; fragments ofthe ovary [K) with an
epidermis bearing stomata [Ka) and with parenchyma - -- - --
containing prism crystals of calcium oxalate [Kb); fragments
Reference solution Test solution
2014 Melilot IV-269

TESTS calcium oxalate Ua]; fragments of the stem epidermis with

Foreign matter (2.8.2)
Maximum 5.0 per cent of stems with a diameter greater than
5 mm and maximum 2.0 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4. 16)
elongated, straight-walled cells and anomocytic (2.8.3)
sto mata [L]; fragments of the fibrous layer of the anthers in
surface view [E] and in transverse section [K]; spherical or
ovoid pollen grains about 25 ~m long with 3 germinal pores
and a smooth exine [C].
>
, "• J

Maximum 7.0 per cent.

ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 50.0 g of the cut herbal drug, a 1000 mL flask,
300 mL of dzlute hydrochloric acid R as the distillation liquid,
6€J
\S) O
and 0.5 mL of xylene R in the graduated tube. Distil at arate Oc
of 2-3 mUmin for 2 h.

~
____________________________________________ ~E~

Melilot ¿jI..

(Ph Bur monograph 2120)

~E~ ____________________________________________

DEFINITION
Whole or cut, dried aerial parts of Melilotus officinalis (L.)
Lam.
Content
Minimum 0.3 per cent of coumarin (C 9 H 6 0 2 ; M r 146.1) Ja
(dried drug) .
IDENTIFICATION
A. The stem is green, cylindrical, glabrous and finely ridged.
The leaves are alternate, petiolate and trifoliate with 2 M
lanceolate stipules; the leaflets are up to about 3 cm long and
20 mm wide, elongated or ovate with a finely denta te margin, Figure 2120.-1. - Illustration for identification test B of
acute at the apex and base; the upper surface is dark green powdered herbal drug of melilot
and glabrous, the lower surface paler green with short, fine
hairs, especially at the base. The inflorescence is racemose
C. Thin-Iayer chromatography (2.2.27) .
with numerous pale yellow flowers, about 7 mm long, each
having a hairy calyx with 5 deeply-divided, unequal teeth, Test solution To 0.3 g of the powdered herbal drug (355)
and a papilionate corolla. The fruit is an indehiscent pod, (2.9. 12) add 3 mL of methanol R. Heat on a water-bath at
often persistent within the calyx, yellowish-brown, short and 100 oC for 1 min and filter.
tapering at the apex; the surface is glabrous and transversely Reference solution Dissolve 50 mg of coumarin CRS and
wrinkled. 20 mg of o-coumaric acid R in 50 mL of methanol R.
B. Microscopic examination (2.8.23). The powder is Plate TLC silica gel plate R (5-40 ~m) [or TLC silica gel
yellowish-green. Examine under a microscope using chloral plate R (2-10 ~m)] .
hydrate solution R . The powder shows the following diagnostic Mobile phase dzlute acetic acid R , ether R , toluene R
characters (Figure 2120.-1): fragments of the leaf lamina in (10 :50:50 VIVIV); use the upper layer.
surface view [D] showing unevenly thickened, slightly
Application 25 ~L [or 3 ~L] as bands of 10 mm [or 8 mm] .
sinuous epidermal cells; numerous stomata [Db], mostly
Development Over a path of 12 cm [or 6 cm] .
anomocytic (2.8.3) with 3-6 subsidiary cells [Da] and
frequently, underlying palisade parenchyma [Dc]; uniseriate Drying In airo
covering trichomes with 2 short, smooth-walled basal cells Detection Spray with 2 M alcoholic potassium hydroxide R and
and a long terminal cell, bent at right angles, with a thick examine in ultraviolet light at 365 nm.
wall and a warty cuticle [A, B]; occasional glandular Results See below the sequence of zones present in the
trichomes with a short, 2- or 3- celled stalk and ovo id, chromatograms obtained with the reference solution and the
biseriate head with 4 indistinct cells [H]; fragments ofthe test solution. Furthermore, other faint zones of various
petals composed of cells with wavy walls [M]; fragments of colours may be present in the chromatogram obtained with
vascular tissue from the stem [F, G], including large vessels the test solution.
[G], sometimes associated with unlignified septate fibres [Fa]
and a sheath of parenchymatous cells containing prisms of
calcium oxalate [Fb]; fragments of mesophyll m including
sorne cells which may occasionally contain cluster crystals of
 IV-270 Menthol Preparations 2014

Top of the plate

Menthol and Benzoin Inhalation
Coumarin: a greenish-yellow A greenish-yellow fIuorescent zone Menthol and Benzoin Inhalation Vapour
fluorescent zone (coumarin)
--- ---
DEFINITION
A blue f1uorescent zone Menthol and Benzoin Inhalation is an inhalation vapour,
solution.
o-Coumaric acid: a greenish-yellow A greenish-yellow f1uorescent zone
f1uorescent zone (o-coumaric acid) may be present Raeementhol or 20 g
--- - -- Levomenthol
Benzoin Inhalation Suffieient to produce 1000 mL
Reference soIution Test solution
Extemporaneous preparation
The following direetions apply.
TESTS Dissolve the Raeementhol or the Levomenthol in a portion of
Foreign matter (2.8.2) the Benzoin Inhalation, add suffieient Benzoin Inhalation to
Maximum 2 per cent of stems with a diameter greater than produce 1000 mL and mix.
3 mm and maximum 2 per cent of other foreign matter.
The inhalarion complies with the requirements stated under
Loss on drying (2.2.32)
Preparations for Inhalation and with the following requirements.
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at Content of total balsamic acids
105 oC for 2 h. Not les s than 2.8% w/v, ealculated as einnamie aeid,
Total ash (2.4. 16) C 9H s0 2 .
Maximum 10.0 per cent.
Total solids
ASSAY 9.0 to 12.0% w/v when determined by drying at 105 for 0

Liquid ehromatography (2.2.29). 4 hours, Appendix XI A. Use 2 mL.

Test solution Completely reduce about 50 g of the herbal
drug to a powder (500) (2.9.12). To 5.00 g ofthe powdered
herbal drug add 90 mL of methanol R and boil under a refiux
eondenser for 30 mino Allow to eoo!. Filter under vaeuum
through a fibre-glass filter. Take up the residue and the
fragmented filter with 90 mL of methanol R . Treat in the
same manner as before. Combine the filtrates and dilute to
250 .0 mL with methanol R .
Reference solurion Dissolve 25.0 mg of coumann CRS in
methanol R and dilute to 250.0 mL with the same solvent.
Column:
- size: 1 = 0.25 m, 0 = 4 mm;
- stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (5 ¡.tm).
Mobile phase acetonitnle R, 5 gIL solution of phosph0l1C
acid R (22: 78 V/V).
Flow rate 1.7 mUmin.
Detection Spectrophotometer at 275 nm.
Injecrion 20 ¡.tL.
System suitability:
- retenrion time: eoumarin = about 7.8 mino
Calculate the pereentage eontent of eoumarin using the
following expression:
ASSAY
Boil 10 mL with 25 mL of 0.5M ethanolic potassium hydroxide
under a refiux eondenser for 1 hour. Evaporate the ethanol,
disperse the residue in 50 mL of hot water, eool, add 80 mL
of water and 1.5 g of magnesium sulfate dissolved in 50 mL of
water. Mix thoroughly and allow to stand for 10 minutes.
Filter, wash the residue on the filter with 20 mL of water,
aeidify the eombined filtrate and washings with hydl'ochlonc
acid and extraet with four 40 mL quantities of ether. Diseard
the aqueous solution, combine the ether extraets and extraet
with suceessive quantities of 20, 20, 10, 10 and 10 mL of
sodium hydrogen carbonate solution, washing each aqueous
extraet with the same 20 mL of ether. Discard the ether
layers, earefully aeidify the eombined aqueous extraets with
hydrochlonc acid and extraet with sueeessive quantities of 30,
20, 20 and 10 mL of chloroform, filtering eaeh extraet through
anhydrous sodium sulfate supported on absorbent eotton. Distil
the chloroform from the eombined filtra tes until 10 mL
remains and remove the remainder in a current of airo
Dissolve the residue, with the aid of gentle heat, in 10 mL of
ethanol (96%), previously neutralised to phenol red solution,
eool and titrate with O.lM sodium hydroxide VS using phenol
red solution as indieator. Eaeh mL of 0.1 M sodium hydroxide
VS is equivalent to 14.82 mg of total balsamie aeids,
ealculated as einnamie aeid, C 9H g 0 2 •

AJ area of the peak due to eoumarin in the

ehromatogram obtained with the test solution;
A2 area of the peak due to eoumarin in the
ehromatogram obtained with the referenee solution;
m1 mass of the herbal drug to be examined used to
prepare the test solution, in grams;
1112 mass of coumann CRS used to prepare the referenee
solution, in grams;
p pereentage eontent of eoumarin in coumann CRS.
_ _ __ _ __ _ _ _ _ __ _ _ _ _ __ __ _ ~Ew
 2014 Milk-thistle Fruit IV-271

Milk-thistle Fruit *** fluorescent zones are present between the zones of silibinin
*** *** and taxifolin in the chroma1Ogram obtained with the test
(Ph Eur monograph 1860) *** solution.
Preparation
Refined and Standardised Milk Thistle Dry Extract Top oí the plate
~E~ ____________________________________________
Silibinin: a yellowish·green A yellowish·green fluorescent zone
DEFINITION fluorescent zone (silibinin)

Mature fruit, devoid of the pappus, of Silybum marianum L. --- ---

Gaertner. Taxifolin: an orange f1uorescent An orange fluorescent zone
zone (taxifolin)
Content
Minimum 1.5 per cent of silymarin, expressed as silibinin A yellowish·green fluorescent zone
(silicristin)
(C25H2201O; M r 482 .4) (dried drug).
--- ---
CHARACTERS
A light blue fluorescent zone (line
No rancid odour. of application)
IDENTIFICATION Reíerence solution Test solution
A. The achene is strongly compressed, elongate-obovate,
about 6-8 mm long, 3 mm broad and 1.5 mm thick;
the outer surface is smooth and shiny with a grey or pale TESTS
brown ground colour variably streaked dark brown Loss on drying (2.2.32)
10ngitudinaIly to give an overaIl pale greyish or brown colour; Maximum 8.0 per cent, determined on 1.000 g of the
the fruit is tapering at the base and crowned at the apex with powdered drug (500) (2.9.12) by drying in an oven at
a glistening, pale yeIlow extension forming a collar about 105 oC for 2 h .
1 mm high surrounding the remains of the style. Total asb (2.4.16)
Cut transversely, the fruit shows a narrow, brown outer are a Maximum 8.0 per cent.
and 2 large, dense, white oily cotyledons.
ASSAY
B. Reduce to a powder (355) (2.9.12) . The powder is
Liquid chromatography (2.2.29).
brownish-yeIlow with darker specks. Examine under a
microscope using ehloral hydrate solution R. The powder Test solution Place 5.00 g of the powdered drug (500)
shows the folIowing diagnostic characters: fragments of the (2.9.12) in a continuous-extraction apparatus. Add 100 mL
epicarp composed of colourIess celIs, polygonal in surface of light petroleum R and heat in a water-bath for 8 h. AIIow
view, the lumen appearing fairIy large or as a smaIl slit, the defatted drug to dry at room temperature. In a
depending on the orientation; groups of parenchymatous celIs continuous-extraction apparatus, extract the latter with
from the pigment layer, sorne of them containing colouring 100 mL of methanol R in a water-bath for 5 h. Evaporate the
matter which appears bright red; very abundant groups of methanolic extract in vaeuo to a volume of about 30 mL.
large sclereids from the testa with bright yeIlow pitted walls Filter into a 50 mL volumetric flask, rinsing the extraction
and a narrow lumen; occasionaIly fragments of smaIl-ceIled flask and the filter, and diluting to 50.0 mL with methanol R.
parenchyma with pitted and beaded waIls; abundant thin- Dilute 5.0 mL of this solution 10 50.0 mL with methanol R.
waIled parenchyma10us ceIls from the cotyledons containing Referenee solution Dissolve a quantity of milk thistle
oil globules and scattered cluster crystals of caIcium oxalate; standardised dry extraet CRS equivalent 10 5.0 mg of silibinin
a few larger, prismatic crystals of calcium oxalate. (mI g) in methanol R and dilute 10 50.0 mL with the same
C. Thin-Iayer chroma1Ography (2.2.27). solvent.
Test solution To l.0 g of powdered drug (500) (2.9.12) add Column:
10 mL of methanol R. Heat under reflux in a water-bath at -- size: 1 = 0.125 m, 0 = 4 mm;
70 oC for 5 mino Cool and filter. Evaporate the filtrate 10 -- stationary phase: octadeeylsily! silica gel for ehromatography R
dryness and dissolve the residue in 1.0 mL of methanol R. (5 ¡.¡m) .
Referenee solution Dissolve 2 mg of silibinin R and 5 mg of Mobile phase:
taxifolin R in 10 mL of methanol R. -- mobile phase A : phosphorie acid R; methanol R, water R
(0.5:35:65 V/V/V);
Plate TLC si/iea gel plate R.
-- mobile phase B: phosphoric acid R; methanol R, water R
Mobile phase anhydrous formic aeid R, aeetone R, methylene (0.5:50:50 V/V/V);
ehloride R (8.5:16.5:75 V/V/V).
Time Mobile phase A Mobile phase B
Applieation 30 ¡.¡L of the test solution and 10 ¡.¡L of the (min) (percent VM (pereent VM
reference solution, as bands. 0·28 100 --7 O O --7 100
Development Over a path of 10 cm.
28·35 O 100
Drying At 100-105 oC.
35 - 36 O --7 100 100 --7 O
Deteetion Spray the warm plate with a 10 gIL solution of
diphenylborie aeid aminoethyl ester R in methanol R and 36 - 51 100 O
subsequently spray with a 50 gIL solution of macrogol 400 R
Flow rate 0.8 mUmin.
in methanol R. AIIow 10 dry for 30 min and examine in
ultraviolet light at 365 nm. Detection Spectropho1Ometer at 288 nm.
Results See below the sequence of zones present in the Injeetion 10 ¡.¡L.
chroma1Ograms obtained with the reference solution and the Retention time Silibinin B = about 30 mino If necessary,
test solution. Furthermore, other orange and yeIlowish-green adjust the time periods of the gradient.
 IV-272 Milk Thistle Preparations
System suitability Reference solution:
-- resolution: minimum 1.8 between the peaks due ro
silibinin A and silibinin B;
-- the chromatogram obtained is similar ro the
chromatogram supplied with mük thistle standardised dry
extraet CRS.
Locate silicristin, silidianin, silibinin A, silibinin B, isosilibinin
A and isosilibinin B by comparison with the chromarogram
supplied with milk thistle standardised dry extract CRS. In the
chromatogram obtained with the test solution the peak due
to silidianin may vary in size, be absent or be present as the
principal peak. Determine the area of the peaks due to
silicristin, silidianin, silibinin A, silibinin B, isosilibinin A and
isosilibinin B.
Calculate the percentage content of silymarin, calculated as
silibinin, using the following expression:
(Al + A2 + A3 + A4 + AS + A6) x m 1 X P x 1000
(A7 + A8) x m 2 x (100 - d)
Al area of the peak due ro silicristin in the
chromatogram obtained with the test solution;
A2 area of the peak due ro silidianin in the
chromarogram obtained with the test solution;
A3 area of the peak due ro silibinin A in the
chromarogram obtained with the test solution;
A4 area of the peak due to silibinin B in the
chromarogram obtained with the test solution;
A5 area of the peak due to isosilibinin A in the
chromatogram obtained with the test solution;
A6 =" area of the peak due to isosilibinin B in the
chromarogram obtained with the test solution;
A7 area of the peak due ro silibinin A in the
chromatogram obtained with the reference solution;
A8 area of the peak due to silibinin B in the
chromarogram obtained with the reference solution;
mi mass of milk thistle standardised dry extraet CRS used
to prepare the reference solution, in grams;
m2 mas s of the drug ro be examined, in grams;
p combined percentage content of silibinin A and
silibinin B in milk thistle standardised dry extraet CRSj
d percentage loss on drying of the drug.
_ _ __ _ _ __ _ _ _ __ _ _ __ __ _ ___
Refined and Standardised Milk *****
** **
Thistle Dry Extract ***
(Ph Eur monograph 2071)
~E~ ____ __ _ _ _ _ _ __ _ _ _ _ _ _ __ ___
DEFlNITION
Dry extract, refined and standardised, produced from Milk-
rhistle fruit (1860).
Content
90 per cent to 110 per cent of the nominal content of
silymarin, expressed as silibinin (C2sH220!O; M r 482.4),
stated on the labe!' The nominal content of silymarin is
within the range 30 per cent m/m to 65 per cent m /m (dried
extract).
The content of silymarin corresponds ro:
-- sum of the eontents of silieristin and silidianin (both
C2sH220IO; M r 482.4): 20 per cent to 45 per cent,
calculated with reference to total silymarin;
2014
-- sum of the eontents of silibinin A and silibinin B (both
C2sH2Z0IO; M r 482.4): 40 per cent to 65 per cent,
calculated with reference ro total silymarin;
-- sum of the eontents of isosüibinin A and isosüibinin B (both
C2sH22010; M r 482.4): 10 per cent ro 20 per cent,
calculated with reference to total silymarin.
PRODUCTION
The extract is produced from the herbal drug by an
appropriate procedure, using one or more of the following
solvents:
-- ethyl aceta te;
-- acetone or mixture of acetone and water;
-- ethanol or mixture of ethanol and water;
-- methanol or mixture of methanol and water.
CHARACTERS
Appearance
Yellowish-brown, amorphous powder.
IDENTIFICATION
Thin-Iayer chromarography (2.2.27).
Test solution Dissolve 0.250 g of the extract to be examined
in 5 mL of methanol R.
Referenee solution Dissolve 2 mg of silibinin R and 5 mg of
taxifolin R in 10 rnL of methanol R.
Plate TLC silica gel plate R (5-40 fllTI) [or TLC siliea gel
plate R (2-10 J.1m)].
Mobile phase anhydrous formic acid R, acetone R , methylene
chloride R (8.5:16.5:75 V/V/V).
Application 10 J.1L [or 8 J.1L] of the test solution and 10 J.1L
[or 2 J.1L] of the reference solution, as bands.
Development Over a path of 10 cm [or 6 cm].
Drying At 100-105 oc.
Deteetion Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R and subsequently spray with
a 50 giL solution of maerogol 400 R in methanol R. Allow the
plate to dry in air for about 30 min and examine in
ultraviolet light at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other yellowish-green ftuorescent
zones may be present berween the zones due to silibinin and
~E~ taxifolin in the chromatogram obtained with the test solution.
Top of the plate
Silibinin: a yellowish·green A yellowish-green f1uorescent zone
fluorescent zone (silibinin)
-- -
Taxifolin: an orange fluorescent An orange f1uorescent zone (taxifolin)
zone
A yellowish-green f1uorescent zone
-- -
A fluorescent zone (line of
aoolicationl
Reference solution Test solution
TESTS
Loss on drying (2.8.17)
Maximum 5.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution Dissolve 60.0 mg of the extract to be examined
in methanol R and dilute to 100.0 mL with the same solvent.
2014 Mint Oil IV-273

Referenee solution Dissolve a quantity of milk thistle

standardised dry extraet CRS corresponding to 10.0 mg (mi g)
of silibinin in methanol R and dilute to 100.0 mL with the
same solvento area of the peak due to silieristin in the
Column: chromatogram obtained with the test solution;
- size: l = 0.125 m, 0 = 4 mm; area of the peak due to silidianin in the
- stationary phase: oetadeeylsilyl siliea gel for ehromatography R chromatogram obtained with the test solution;
(5 ¡.¡m). area of the peak due to silibinin A in the
Mobile phase: ehromatogram obtained with the test solution;
- mobile phase A: phosphorie acid R, methanol R, water R area of the peak due to silibinin B in the
(0.5:35:65 VIVIV); chromatogram obtained with the test solution;
- mobile phase B: phosphoric acid R, methanol R, water R area of the peak due to isosilibinin A in the
(0 .5:50:50 VIVIV); chromatogram obtained with the test solution;
Time Mobile phase A Mohile phase B area of the peak due to isosilibinin B in the
(min) (percent VII') (percent VII') chromatogram obtained with the test solution;
0·28 100 ~ O O ~ 100 area of the peak due to silibinin A in the
chromatogram obtained with the reference solution;
28·35 O 100
area of the peak due to silibinin B in the
35·36 O ~ 100 100 ~O chromatogram obtained with the reference solution;
36·51 100 O mas s of silibinin in the reference solution, in grams;
mas s of the extraet to be examined, in grams.
Flow rate 0.8 mLlmin. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ PhEur
Deteetion Spectrophotometer at 288 nm.
Injection 10 ¡.¡L.
Retention time Silibinin B = about 30 min; if necessary,
adjust the time periods of the gradient.
System suitabi/ity Reference solution:
Dementholised Mint Oil
- resolution: minimum 1.8 between the peaks due to (Partly Dementholised Mint Oil)
silibinin A and silibinin B. Ph Eur monograph 1838)
- the chromatogram obtained with the referenee solution is ~Ew _ _ _ _ _ _ _ _ _ _ _ __ _ __ _ _ __ __
similar to the chromatogram supplied with milk thistle
standardised dry extract CRS. DEFINITION
Essential oil obtained by steam distillation from the fresh,
Locate the peaks due to silicristin, silidianin, silibinin A,
flowering aerial parts, recently gathered from Mentha
silibinin B, isosilibinin A and isosilibinin B using the
canadensis L. (syn. M. arvensis L. varo glabrata (Benth) Pern.,
ehromatogram supplied with mi/k thistle standardised dry
M. arvensis varo piperascens Malinv. ex Holmes), followed by
extract CRS. In the chromatogram obtained with the test
partial separation of menthol by crystallisation.
solution the peak due to silidianin may vary in size or be
absent. CHARACTERS
Calculate the percentage content of total silymarin, expressed Appearance
as silibinin, using the following expression: Colourless, pale yellow or greenish-yellow liquido
Characteristic odour.
(H + F2 + F3 + F4 + F5 + F6) x mI IDENTIFICATION
(H +F8) x m 2 First identifieation B.
Second identification A.

Calculate the percentage content of the sum of silicristin and
silidianin, with referenee to total silymarin, using the
following expression:
Calculate the percentage content of the sum of silibinin A
and silibinin B, with reference to total silymarin, using the
following expression:
Calculate the percentage content of the sum of isosilibinin A
and isosilibinin B, with reference to total silymarin, using the
following express ion:
A. Thin-layer chromatography (2.2.27) .
Test solution Dissolve 0.1 mL of the substance to be
examined in 1.0 mL of toluene R.
Reference solution Dissolve 4 ¡.¡L of carvone R, 4 ¡.¡L of
pulegone R, 10 ¡.¡L of menthyl acetate R, 20 ¡.¡L of cineole R and
50 mg of menthol R in 5 mL of toluene R .
Plate TLC silica gel F 254 plate R .
Mobile phase ethyl acetate R, toluene R (5:95 VIV).
Applieation 10 ¡.¡L, as bands.
Development Over a path of 15 cm.
Drying In airo
Detection A Examine in ultraviolet light at 254 nm.
Results ASee below the sequenee of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Purthermore, a quenching zone may be
present in the upper third of the chromatogram obtained
with the test solution.
IV-274 Mint Oil 2014

Top of the plate 40 mg of menthyl acetate R, 20 mg of isopulegol R, 60 mg of

menthol R, 20 mg of pulegone R and 10 mg of carvone R in
Carvone and pulegone: a A quenching zone
quenching zone hexane R and dilute to 10.0 mL with the same solvento
A quenching zone Column:
Reference solution Test solution -- material: fused silica,
-- size: 1 = 30 m (a film thickness of 1 Ilm may be used) to
60 m (a film thickness of 0.2 Ilm may be used),
Detection B Spray with anisaldehyde solution R and heat at o = 0.25-0.53 mm,
100-105 oC for 5-10 mino Examine immediately in daylight.
-- stationary phase: macrogol 20 000 R.
Results B See below the sequence of the zones present in Carrier gas helium for chromatography R.
the chromatograms obtained with the reference solution and
the test solution. Furthermore, the zone due to cineole in the Flow rate 1.5 mUmin.
reference solution is absent in the chromatogram obtained Split ratio 1:100.
with the test solution. No yellowish-brown zone below the
intense reddish-violet zone is present in the chromatogram Temperature:
obtained with the test solution. Time Temperature
(min) (oC)
Column 0·10 60
Top of the plate
10·70 60 -¡ 180
An intense reddish·violet zone
(near the solvent [ront) 70 · 75 180

Menthyl acetate: a bluish·violet A bluish·violet zone (menthyl Injection port 200

zone acetate)
Detector 220
A strongly greenish zone

A greenish zone Detection Flame ionisation.

Carvone and pulegone: a reddish A reddish zone Injection 1.0 J.!L.
zone
Elution order Order indicated in the composition of the
Cineole: a violet zone
reference solution. Record the retention times of these
substances.
A distinctly violet zone System suitability Reference solution:
Menthol: an intense blue zone A very intense blue zone (mentho!)
-- resolution: minimum 1.5 between the peaks due to
limonene and cineole.
Reference solution Test solution
U sing the retention times determined from the
chromatogram obtained with the reference solution, locate
B. Examine the chromatograms obtained in the test for the components of the reference solution in the
chromatographic pro file. chromatogram obtained with the test solution.
Results The characteristic peaks in the chromatogram Determine the percentage content of these components.
obtained with the test solution are approximately similar in The percentages are within the following ranges:
retention time to those in the chromatogram obtained with -- limonene: 1.5 per cent to 7.0 per cent,
the reference solution. Carvone may be absent from the -- cineole: maximum 1.5 per cent,
chromatogram obtained with the test solution. -- menthone: 17.0 per cent to 35.0 per cent,
TESTS -- isomenthone: 5.0 per cent to 13.0 per cent,
Relative density (2.2.S) -- menthyl acetate: 1.5 per cent to 7.0 per cent,
0.888 to 0.910. -- isopulegol: 1.0 per cent to 3.0 per cent,
Refractive index (2.2.6) -- menthol: 30.0 per cent to 50.0 per cent,
1.456 to 1.470. -- pulegone: maximum 2.5 per cent,
-- carvone: maximum 2.0 per cent.
Optical rotation (2.2.7)
- 16.0° to - 34.0°. The ratio of cineole content to limonene content is less than
1.
Acid value (2.5.1)
Maximum 1.0, determined on 5.00 g of the substance to be STORAGE
examined dissolved in 50 mL of the prescribed mixture of At a temperature not exceeding 25 oC.
solvents. ______________________________________________ Ph Ew

Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Dissolve 0.20 g of the substance to be
examined in hexane R and dilute to 10.0 mL with the same
solvento
Reference solution Dissolve 10 mg of limonene R, 20 mg of
cineole R, 40 mg of menthone R, 10 mg of isomenthone R,
2014 Motherwort IV-275

Motherwort ***
*** ***
(Ph Eur monograph 1833) ***
~Ew ____________________________________________

DEFINITION
Whole or cut, dried ftowering aerial parts of Leonurus cardiaca
L.
Cooteot
Minimum 0.2 per cent of ftavonoids, expressed as hyperoside
(C21HZOOI2; M r 464.4) (dried drug) .
IDENfIFICATION
A. The stem pieces are hairy, longitudinally striated,
quadrangular, hollow, and up to about 10 mm wide; they
bear opposite and decussate, petiolate leaves and, in the axils
of the upper leaves, about 6-12 small ftowers, arranged in
sessile whorls forming a long, leafy spike. The lower leaves
are ovate-orbicular, palmately 3- 10 5-lobed, rarely 7-lobed,
the lobes irregularly dentate. The upper leaves are entire or
slightly trifid, lanceolate with a serrate margio and cuneate at
the base. The upper surface of the lea ves is green with
scattered hairs, the lower surface is paler green, densely
pubescent and shows a prominent palmate and reticulate
venation. The ftowers have a funnel-shaped calyx, 3 mm 10
5 mm long with 5 stiff, recurved teeth; the corolla is
2-lipped, the upper lip pink and pubescent 00 the outer
surface, the lower !ip white with purplish spots; stamens 4,
densely pubescent.
B. Microscopic examination (2.8.23). The powder is green.
Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters Figure 1833.-1. - Illustration for identification test B of
(Figure 1833.-1): numerous covering trichomes [A], whole powdered herbal drug of motherwort
[Aa) or fragmented [Ab), uniseriate, with warty walls,
composed of 2-8 cells with slight swellings at the junctions, C. Thin-Iayer chromatography (2.2.27).
up 10 1500 ¡.¡m long; fragments of the upper epidermis, in
Test solution To 0.5 g of the powdered herbal drug (355)
surface view [B), with cells with straight or sinuous anticlinal
(2.9.12) add 5 mL of methanol R. Heat on a water-bath at
walls [Ba), often accompanied by palisade parenchyma [Bb);
65 oC for 5 min with shaking. Cool and filter.
fragments of the lower epidermis, in surface view [C), with
cells with sinuous anticlinal walls [Ca), diacytic stomata Reference solution Dissolve 5 mg of naphthol yellow S R and
(2.8.3) [Cb), bearing glandular trichomes with a short 2.0 mg of catalpol R in 5.0 mL of methanol R.
unicellular stalk and a globular head composed of 8-16 cells Plate TLC silica gel plate R .
[Cc), glandular trichomes with a uni- or bicellular stalk and a Mobile phase glacial acetic acid R, water R, ethyl acetate R
bi- or tetracellular head [Cd) and sometimes covering (20:20:60 VIV/V) .
trichomes; fragments of the lamina, in transverse section [D), Application 20 ¡.¡L as bands.
composed of epidermis es bearing glandular trichomes with a
Development Over a path of 10 cm.
globular head consisting of 8-16 cells [Da) or a bi- or
tetracellular head [Db), a 1-layered palisade mesophyll Drying In airo
extending almost halfway across the section [Dc], and a Detection Treat with dimethylaminobenzaldehyde solution R2,
loosely arranged spongy parenchyma [Dd); fragmeots of the using about 5 mL for a plate 200 mm square; heat at
calyx [G) with an epidermis consisting of polygonal cells 100-105 oC for 10 min until the spots appear; examine in
bearing uni- or bicellular conical covering trichomes, with daylight.
spiny walls [Ga), often associated with fusiform mesophyll Results See below the sequence of zones present in the
cells with thick walls and containing small prism crystals of chromatograms obtained with the reference solurion and the
calcium oxalate [Gb); isolated glandular trichomes [H), test solution. Furthermore, other weak greyish-blue zones
either with a multicellular stalk and a unicellular head from may be present in the chroma1Ogram obtained with the test
the anthers [Ha) or a uni- or multicellular stalk and bi- 10 solution.
tetracellular head [Hb); spherical pollen graios, about
25-30 ¡.¡m in diameter, with 3 pores and 3 furrows and a
smooth exine [E); thick-walled, lignified fibres [F]; fragments
from the stem with spirally and annularly thickened vessels
[K); occasional fragments of pericarp rn consisting of lobed
cells with thick, pitted walls, each containing a single prism
crystal of calcium oxalate ITa).
 IV-276 Mullein Flower
Top of the plate
A wide white zone
A greyish·blue zone (iridoid)
---
Naphthol yellow s: an intense 1 or 2 greyish·blue zones (iridoid)
yellow zone
Catalpol: a greyish·blue zone
---
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximurn 2 per cent of brown or yellow leaves and
maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 12.0 per cent.
ASSAY
Stock solution In a 100 mL round-bottomed flask place
1.00 g ofthe powdered herbal drug (355) (2.9.12), add
1 mL of a 5 gIL solution of hexamethylenetetramine R, 20 mL
of acetone R and 2 mL of hydrochlonc acid R1 . Boil the
mixture under a reflux condenser for 30 min. Filter the
liquid through a plug of absorbent cotton into a flask.
Add the absorbent cotton to the residue in the round-
bottomed flask and extract with 2 quantities, each of 20 mL,
of acetone R, each time boiling under a reflux condenser for
10 min. Allow to cool and filter each extract through the
plug of absorbent cotton into the flask. After cooling, filter
the combined acetone extracts through a paper filter into a
volumetric flask and dilute to 100.0 mL with acetone R by
rinsing the flask and the paper filter. Introduce 20.0 mL of
the solution into a separating funnel, add 20 mL of water R
and shake the mixture with 1 quantity of 15 mL and then
3 quantities, each of 10 mL, of ethyl acetate R . Combine the
ethyl aceta te extracts in a separating funnel, wash with
2 quantities, each of 50 mL, of water R, filter the extracts
over 10 g of anhydrous sodium sulfate R into a volumetric flask
and dilute to 50.0 mL with ethyl acetate R.
Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent VIV solution of glacial acetic acid R in methanol R.
Compensation liquid Dilute 10.0 mL ofthe stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic
acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after
30 min, by comparison with the compensation liquid at
425 nm. Calculate the percentage content of flavonoids,
ca1culated as hyperoside, using the following expression:
A x 1.25
m Drying
Le. taking the specific absorbance of hyperoside to be 500.
A absorbance at 425 nm;
m = mass of the substance to be examined, in grams.
------------------- - - -- -___________________
2014
Mullein Flower ***
** **
(Ph Eur monograph 1853) *****
~Ew ____________________________________________
---
DEFINITION
Dried flower, reduced to the corolla and the androecium, of
Verbascum thapsus L., V. densifiorum BertoL (V. thapslforme
Schrad), and V. phlomoides L.
---
IDENTIFICATlON
A. The corolla of V. thapsus is pale yellow, yellow or brown,
funnel-shaped, about 20 mm in diameter, with 5 slightly
unequal and spreading lobes. The corolla lobes are densely
hairy on the outer surface, glabrous on the inner surface,
with a fine network of light brown veins. There are 5
stamens, altemating with the petallobes; 2 of these are long,
with glabrous filaments, the other 3 shorter, with densely
tomentose filaments. The anthers are attached transversely.
In V. phlomoides the corolla is up to about 30 mm in
diameter, bright yellow or orange, and the anthers are
obliquely attached to the filaments . The corolla of
V. densifiorum, about 30 mm in diameter, is almost flat and
deeply divided into 5 slightly unequallobes, with rounded
apices.
B. Reduce to a powder (355) (2.9.12). The powder is yellow
or yellowish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1853.-1): many covering
trichomes from the corolla, whole and fragrnented,
pluricellular, of the candelabra type, with a central uniseriate
axis from which whorls of branch cells arise at the position of
the cross walls and at the apex, in side view [A, B] or in
surface view [F]; the covering trichomes from the stamen
filaments [G] are unicellular, long, thin-walled and tubular,
have a distinctly granular or striated surface with a sharp tip
[Ga] or sometimes with a c1ub-shaped tip [Gb, Gc];
numerous pollen grains, ovoid with a finely granular exine
with 3 pores [D]; fragrnents of the fibrous layer of the anther
with thickened walls giving a characteristic star-shaped
appearance [C]; yellow fragrnents of the petals, in surface
view [E], the epidermal cells polygonal and isodiametric [Ea];
fragrnents of the underlying mesophyll consisting of irregular
parenchymatous cells [Eb] sometimes accompanied by spiral
vessels [Ec].
e. Thin-Iayer chromatography (2.2.27).
Test solution Heat 1.0 g of the powdered drug (355)
(2.9.12) in 10 mL of methanol R in a water-bath at 60 oC for
5 min, with stirring. Cool and filter.
Reference solution Dissolve 1 mg of caffeic acid R, 2.5 mg of
hyperoside R and 2.5 mg of rutin R in methanol R and dilute
to 10 mL with the same solvento
Place TLC silica gel place R.
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (10:10:30:50 VIVIV/V).
Application 10 ¡.tL of the reference solution and 30 ¡.tL of
the test solution, as bands.
Development Over a path of 15 cm.
At 100-105 0e.
Detection Spray the warm plate with a 10 gIL solution of
diphenylboric acid aminoethyl ester R in methanol R, then with a
50 gIL solution of macrogol 400 R in methanol R; allow to dry
in air for 30 min and examine in ultraviolet light at 365 nm.
~Ew
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
 2014 Myrrh IV-277

Swelling index (2.8.4)

Minimum 9, determined on the powdered drug (710)
(2.9.12), moistened with 2 mL of ethanol (96 per eent) R.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug (710) (2.9.12) by drying in an oven at
105 De for 2 h.
Total ash (2.4.16)
Maximum 6.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
STORAGE
In an airtight container.
____________________________________________
Ea ~Ew

Myrrh ***
Eb -00 *** ***
~'S; (Ph Eur monograph 1349) ***
G Gb Preparation
Myrrh Tincture
~Ew ____________________________________________

Gc
Figure 1853.-1.- Illustration for identification test B of
powdered herbal drug of mullein flower
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
A yellow or yellowish-green
fluorescent zone
Caffeic acid: a greenish-blue
fluorescent zone
A bluish fluorescent zone
A greenish fluorescent zone
A yellowish-green fluorescent zone
A bluish fluorescent zone
Hyperoside: a yellowish-brown
fluorescent zone
A greenish fluorescent zone
DEFINITlON
Gum-resin, hardened in air, obtained by incision or produced
by spontaneous exudation from the stem and branches of
Commiphora molmol Engler and/or other species of
Commiphora.
CHARACTERS
Bitter taste.
IDENTIFICATlON
A. The light or dark orange-brown, irregular or roundish
grains or pieces of different size show components of various
colours. Their surface is mostIy covered with grey or
yeIlowish-brown dust.
B. Reduce to a powder (355) (2.9.12). The powder is
brownish-yeIlow or reddish-brown. Examine under a
microscope, using ehloral hydrate solution R. The powder
shows the foIlowing diagnostic characters: a few tissue
fragments from the original plants including: reddish-brown
cork fragments; single or grouped polyhedral or elongated
stone ceIls with partIy strongly thickened, pitted and lignified
waIls with a brownish content; fragments of thin-waIled
parenchyma and sclerenchymatous fibres; irregular prisma tic
or polyhedral crystals of caIcium oxalate, about 10-25 ¡.1m in
size.

Rutin: a yellowish-brown
e. Examine the chromatograms obtained in the test for
fluorescent zone Commiphora mukul.
Reference solution Test solution Detection Spray with anisaldehyde solution R, and examine in
daylight while heating at 100-105 oC for 10 min.
Results The chromatogram obtained with the reference
D . Boil 1.0 g ofthe powdered drug (355) (2.9.12) with solution shows in the lower third an orange-red zone
15 mL of water R for 1 mino Filter. Add 1 mL of hydroehlor1e (thymol) and in the middle third a violet zone (anethole).
acid R and boil for l min. A greenish-blue colour develops The chromatogram obtained with the test solution shows an
and, after a few minutes, cloudiness appears and then a intense violet zone (furanoeudesma-I,3-diene), exceeding the
blackish precipitate (iridoids). other zones in size and intensity, above the zone of anethole
TESTS in the chromatogram obtained with the reference solution;
Foreign matter (2.8.2) a violet zone similar in position to the zone of anethole in the
Maximum 5 per cent of brown petals and maximum chromatogram obtained with the reference solution; 2 intense
2 per cent of fragments of the calyx and other foreign matter, violet zones similar in position to the zone of thymol in the
determined on 20 g. chromatogram obtained with the reference solution, the
upper one due to curzerenone and the lower one to
 IV-278 Myrrh Preparations 2014

2-methoxyfuranodiene. Further mostly violet zones are Development Over a path of 15 cm.
present in the chromatogram obtained with the test solution. Drying In airo
TESTS Detection Spray with anisaldehyde solution R and examine in
Cornrniphora mukul daylight whilst heating at 100-105 oC for 10 mino
Thin-Iayer chromatography (2.2.27) . Results See below the sequen ce of the zones present in the
Test solutlon To 0.5 g of the powdered drug (355) (2.9.12) chroma1Ograms obtained with the reference solution and the
add 5.0 mL of ethanol (96 per cent) R and warm the mixture test solution. Furthermore, other zones most1y violet, are
on a water-bath for 2-3 mino Cool and filter. present in the chromatogram obtained with the test solution.
Reference solution Dissolve 10 mg of thymol R and 40 ¡.¡L of
anethole R in 10 mL of ethanol (96 per cent) R. Top oí the plate
Plate TLC silica gel plate R .
An intense violet zone exceeding
Mobile phase ethyl acetate R, toluene R (2:98 V/V). the others in size and intensity
(furanoeudesma'),3-diene)
Application 10 ¡.¡L, as bands.
Anethole : a violet zone A violet zone
Development Over a path of 15 cm.
Thymol: an orange·red zane Two intense violet zones
Drying In air. (curzerenone and below
2·methoxyfuranodiene)
Detection Examine in ultraviolet light at 365 nm.
Reíerence solution Test solution
Results The chromatogram obtained with the test solution
shows no blue or violet fluorescent zones in the lower third
of the chromatogram. TESTS
Matter insoluble in ethanol Ethanol content (2.9.10)
Maximum 70 per cent. 82 per cent V/V 10 88 per cent VIV.
Place 1.00 g of the powdered drug (250) (2.9.12) in a flask. Methanol and 2-propanol (2.9.11)
Add 30 mL of ethanol (96 per cent) R and shake vigorously MaxÍmum 0.05 per cent V /V of methanol and maximum
for 10 mino Filter the supernatant through a tared sintered- 0.05 per cent V/V of 2-propanol.
glass filter (16) (2.1.2) avoiding the transfer ofsediment from
the flask. Repeat the extraction with 2 quantities, each of
Dry residue (2.8.16)
Minimum 4.0 per cent mim o
20 mL, of ethanol (96 per cent) R. Quantitatively transfer the
sediment 10 the filter by rinsing the flask with ethanol STORAGÉ
(96 per cent) R. Dry the filter and the residue in an oven at Plastic containers are not recommended.
100-105 oC and weigh. ____________________________________________ PhE~

Loss on drying (2.2.32)

MaxÍmum 15.0 per cent, determined on 1.000 g of
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Cineole Type Niaouli Oil ***
Total ash (2.4.16) * **
*
Maximum 7.0 per cent. (Ph Bur monograph 2468) *****
- - - - - - - - - - - -- - - - - - - - - -- - - -_________________ Ph Eur Ph Eur ____________________________________________

DEFINITION
Essential oil obtained by steam distillation from young leafy
branches of Melaleuca quinquenervia (Cav.) S.T.Blake.
Myrrh Tincture ***
*
* *
* CHARACTERS
(Ph Bur monograph 1877) **** * Appearance
~E~ ___________________________________________ Colourless or pale yellow liquido
Aromatic odour of cineole .
DEFINITION
Tincture produced from Myrrh (1349). IDENTIFICATION
First identification B.
PRODUCTION
The tincture is produced from 1 part of the drug and 5 parts S econd identification A.
of ethanol (90 per cent V/V) by a suitable procedure . A. Thin-layer chromatography (2.2.27).
CHARACTERS Test solution Dissolve 100 ¡.¡L of the oil to be examined in
Clear yellowish-brown or orange-brown liquid. toluene R and dilute to 10.0 mL with the same solvento
Reference solution Dissolve 25 ¡.¡L of trans-nerolidol R and
IDENTIFICATION 50 ¡.¡L of cineole R in toluene R and dilute to 5.0 mL with the
Thin-Iayer chromatography (2.2.27) . same solvent.
Test solution Dilute 5 mL of the tincture to be examined to Plate TLC silica gel plate R (5-40 ¡.¡m) [or TLC silica gel
10 mL with alcohol R. plate R (2-10 ¡.¡m)].
R eference solution Dissolve 10 mg of thymol R and 40 ¡.¡L of Mobile phase ethyl acetate R, toluene R (5:95 V/V) .
anethole R in 10 mL of ether R .
Application 10 ¡.¡L [or 2 ¡.¡L] as bands of 10 mm [or 8 mm] .
Plate TLC silica gel plate R.
Development Over a path of 15 cm [or 6 cm] .
Mobile phase ethyl aceta te R , toIuene R (2:98 V/V) .
Drying In air.
Application 1O ~lL, as bands.
2014 Niaouli Oil, Cineole Type IV-279

Detection Treat with anisaldehyde solution R and heat at
100-105 oC for 3 min; examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
Reference solutwn (b) Dissolve 5 ¡.tL of limonene R in
heptane R and dilute to 50.0 mL with the same solvent.
Dilute 0.5 mL of the solution to 5.0 mL with heptane R.
Column:
-- material: fused silica;
-- size: 1 = 60 m, 0 = 0.25 mm;
-- stationary phase: macrogol 20 000 R (film thickness
0.25 ¡.tm).

- - - --- Carner gas helium for chromatography R.

A faint grey zone

Flow rate 1.3 mUmin.
Split ratio 1:50.
Temperature:
A purple zone
Time Temperature
1,8·Cineole: a violet-brown zone An intense violet-brown zone (min) ( oC)
(l,8-cineole)
Colurnn 0-5 65

frans-Nerolidol : a dark violet zone
- --
An intense violet-brown zone
A violet-brown zone
Reference solution Test solution
TESTS
Relative density (2.2.5)
0.904 to 0.925.
Refractive index (2.2.6)
1.463 to 1.472.
Optical rotation (2.2.7)
_ 4° to + 1°.
Methyleugenol and isomethy1eugenol
Gas chromatography (2.2.28) as described in the test for
chromatographic profile with the following modifications .
Reference solutwn Dissolve 5 ¡.tL of methyleugenol R and 5 ¡.tL
of isomethyleugenol R in heptane R and dilute to 50.0 mL with
the same solvento Dilute 0.5 mL ofthe solution to 5.0 mL
with heptane R.
Elution order Order indicated in the composition of the
reference solution; record the retention times of
methyleugenol and isomethyleugenol.
Identification of peaks Using the retention times determined
from the chromatogram obtained with the reference solution,
locate the components of the reference solution in the
chromatogram obtained with the test solution.
Limits:
-- methyleugenol: maximum 0.05 per cent;
-- isomethyleugenol: maximum 0.05 per cent.
Chromatographic pro file
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solutwn Dilute 0.2 mL of the oil to be examined to
5 - 65 65 -+ 185
65 - 80 185 -+ 230
---
Injection port 230
Detector 250
Detection Flame ionisation.
Injection 1 ¡.tL.
Elution order Order indicated in the composition of
reference solution (a); record the retention times of these
substances.
Identificaríon of peaks Using the retention times determined
from the chromatogram obtained with reference solution (a),
locate the components of reference solution (a) in the
chromatogram obtained with the test solution; the peak due
to viridiflorol elutes with a relative retention of about 1.02
with reference to trans-nerolidol.
System suitabz1ity Reference solution (a):
-- resolution: minimum 1.5 between the peaks due to
limonene and 1,8-cineole.
D etermine the percentage content of each of the following
components. The limits are within the following ranges:
-- rx-pinene: 5.0 per cent to 15.0 per cent;
-- f3-pinene: 1.0 per cent to 4.0 per cent;
-- limonene: 5.0 per cent to 10.0 per cent;
-- 1,8-cineole: 45 .0 per cent to 65 .0 per cent;
-- p-cymene: 0.05 per cent to 4.0 per cent;
-- benzaldehyde: 0.05 per cent to 0.5 per cent;
-- rx-terpineol: 3.0 per cent to 8.0 per cent;
-- trans-nerolidol: 0.05 per cent to 1.5 per cent;
-- viridifiorol: 2.5 per cent to 9.0 per cent;
-- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent) .
STORAGE
At a temperature not exceeding 25 oc.
____________________________________________ ~Ew

10.0 mL with heptane R.

Reference solution (a) Dilute 10 ¡.tL of rx-pinene R, 5 ¡.tL of
f3-pinene R, 10 ¡.tL of limonene R, 50 ¡.tL of cineole R, 5 ¡.tL of
p-cymene R, 5 ¡.tL of benzaldehyde R, 5 mg of rx-terpineol R and
5 ¡.tL of trans-nerolidol R in heptane R and dilute to lO mL
with the same solvent.
 IV-280 Neroli Oil 2014

Neroli Oil *** B. Examine the chromatograms obtained in the test for
*** *** chromatographic profile.
(Ph Eur monograph 1175) *** Results The principal peaks in the chromatogram obtained
PhE~ ____________________________________________ with the test solution are similar in retention time to the
principal peaks in the chromatogram obtained with the
DEFINITION reference solution.
Neroli oil is obtained by steam distillation from the fresh
fiowers of Citrus aurantium L. subsp. aurantium L. TESTS
(e. aurantium L. subsp. amara Engl.). Re1ative density (2.2.5)
0.863 to 0.880.
CHARACTERS
Appearance Refractive index (2.2. 6)
Clear, pale-yellow or dark-yellow Iiquid. 1.464 to 1.474.
Characteristic odour. Optical rotation (2.2. 7)
+ 1S to + US.
IDENTIFICATION
Acid value (2.5. 1)
First identification B.
Maximum 2.0.
Second identification A.
Bergapten
A. Examine the chromatograms obtained in the test for Thin-layer chromatography (2.2.27).
bergapten.
Test solution Dissolve 0.1 g of the substance to be examined
Results ASee below the sequence of zones present in the
in ethanol (96 per cent) R and dilute to 5.0 mL with the same
chromatograms obtained with the reference solution and the solvent.
test solution. Furthermore other zones may be present in the
chromatograms obtained with the test solution. Reference solution Dissolve 2 ~L of methyl anthranilate R,
1O ~L of linalyl acetate R, 20 ~L of linalol R and 5 mg of
bergapten R in ethanol (96 per cent) R and dilute to 10.0 mL
Top of the plate with the same solvento
--- --- Plate TLC silica gel plate R (5-40 ~m) [or TLC silica gel
plate R (2-10 ~m)].
Methyl anthranilate: a blue A faint blue fluorescent zone
fluorescent zone (methy! anthranilate) Mobile phase ethyl acecate R, toluene R (15:85 V/V) .
Application 10 ~L [or 2 ~L] as bands.
Bergapten: a greenish-yeIlow Development Over a path of 15 cm [or 8 cm] .
fluorescent zone Drying In airo
--- ---
Detection Examine in ultraviolet light at 365 nm.
Reference solution Test solution Results The chromatogram obtained with the test solution
do es not show a zone corresponding to the zone due to
bergapten in the chromatogram obtained with the reference
Detection B Spray with anisaldehyde solution R; heat at solution.
100-105 oC for 10 min; examine the chromatograms in
ultraviolet light at 365 nm. Chromatographic pro file
Gas chromatography (2.2. 28): use the normalisation
Results B See below the sequence of zones present in the
procedure.
chromatograms obtained with the reference solution and the
test solution. In the chromatogram obtained with the test Test solution The substance to be examined.
solution the zone due to linalol is more intense than the zone Reference solution (a) Dissolve 20 ~L of f3-pinene R, 5 mg of
due to linalyl acetate. sabinene R, 40 ~L of limonene R, 40 ~L of linalol R, 20 ~L of
linalyl acetare R, 5 mg of rx-terpineol R, 5 ~L of neryl acetate R,
5 ~L of geranyl acetate R, 5 ~L of trans-nerolidol R, 5 ~L of
Top of the plate
methyl anthranilate R and 5 ~L (E,E) -farnesol R in 2 mL of
A brown fluorescent zone heptane R.
Lina!y! aceta te : a brownish-red An intense brownish-red Reference solution (b) Dissolve 5 ~L of methyl anthranilate R
fluorescent zone fluorescent zone (lina!y! acetate) in heptane R and dilute to 10 mL with the same solvent.
- -- --- Column:
Methy! anthranilate : a b!ue A faint b!ue f!uorescent zone
-- material: fused silica,
fluorescent zone (methyl anthranilate) -- size: l = 60 m, 0 = 0.25 mm,
A faint brownish-red fluorescent -- stationary phase: macrogol 20 000 R (film thickness
zone 0.25 ~m).
Lina!ol : a brownish-red fluorescent A brownish-red fluorescent zone Carrier gas helium for chromatography R.
zone (linalol)
Bergapten: a greenish-yeIlow Flow rate 1.5 mUmin.
fluorescent zone Split ratio 1:100 .
--- ---
Severa! blue and brownish-red
fluorescent zones
Reference solution Test solution
 2014 Nettle Leaf IV-281

Temperature: Calculate the percentage content of the specified

(S)-enantiomers from the folIowing expression:
Time Temperature
(min) (OC)
Column 0·4 75 Al
A
4·42.8 75 -4 230 1 + A2 x 100
42.8·63 230
Injection port 270 Al = area of the corresponding (S)-enantiomer,
Detector 270 A 2 = area of the corresponding (R)-enantiomer.
Detection Flame ionisation. Limits:
-- (S) (+)-linalol: maximum 30 per cent,
Injection 0.2)lL
-- (S) (+ )-linalyl acetate: maximum 5 per cent.
Elution order Order indicated in the composition of
reference solution (a). Record the retention times of these STORAGE
substances. At a temperature not exceeding 25 oc.
____________________________________________ ~E~

System suitability Reference solution (a):

-- resolution: minimum 1.5 between the peaks due to
p-pinene and sabinene.
Using the retention times determined from the
chromatogram obtained with reference solution (a), locate
the components of reference solution (a) in the
chromatogram obtained with the test solution.
Limits:
-- p-pinene: 7.0 per cent to 17.0 per cent,
-- limonene: 9.0 per cent to 18.0 per cent,
-- linalol: 28.0 per cent to 44.0 per cent,
-- linalyl acetate: 2.0 per cent to 15.0 per cent,
-- rx-terpineol: 2.0 per cent to 5.5 per cent,
-- neryl acetate: maximum 2.5 per cent,
-- geranyl acetate: 1.0 per cent to 5.0 per cent,
-- trans-nerolidol: 1.0 per cent to 5.0 per cent,
-- methyl anthranilate: 0.1 per cent to 1.0 per cent,
-- (E,E)-farnesol: 0.8 per cent to 4.0 per cent,
-- disregard limit: area of the peak in the chromatogram
obtained with reference solution (b).
Chira! purity
Gas chromatography (2.2.28).
Test solution Dissolve 20 mg of the substance to be
examined in pentane R and dilute to 10.0 mL with the same
solvent.
Reference solution To 10 )lL of linaZol R add 10 )lL of tinalyl
acetate R . Dilute to 10.0 mL with pentane R .
Column:
-- material: fused silica,
-- size: l = 25 m, 0 = 0.25 mm,
-- stationary phase: modified p-cyclodextrin for chiral
chromatography R (film thickness 0.25 )lm).
Carrier gas helium for chromatography R.
Flow rate 1.3 mUmin.
Split ratio 1:30.
Temperature:
Time Temperature
(mln) (oC)
Column 0·65 50 -4 180
Injection port 230
Detector 230
Detection Flame ionisation.
Injection 1)lL
System suitability Reference solution:
-- resolution: minimum 5.5 between the peaks due to
(R)(-)-linalol (1 st peak) and (S) (+ )-linalol (2 nd peak);
minimum 2.7 between the peaks due to (R)(-)-linalyl
acetate (3 rd peak) and (S)(+)-linalyl acetate 4 th peak).
Nettle Leaf *****
** *
*** *
(Ph Eur monograph 1897)
~E~ ____________________________________________
DEFINITION
Whole or cut dried leaves of Urtica dioica L, Urtica urens L,
or a mixture of the 2 species.
Content
Minimum 0.3 per cent for the sum of caffeoylmalic acid and
chlorogenic acid, expressed as chlorogenic acid (CI 6HI S09;
M r 354.3) (dried drug).
IDENTIFICATION
A. The leaves are dark green, dark greyish-green or
brownish-green on the upper surface, paler on the lower
surface; scanered stinging hairs occur on both surfaces, also
smaIl covering trichomes that are more numerous along the
margins and on the veins on the lower surface. The lamina is
strongly shrunken, ovate or oblong, up to 100 mm long and
50 mm wide, with a coarsely serrate margin and a cordate or
rounded base. The venation is reticulate and distinctly
prominent on the lower surface. The petiole is green or
brownish-green, rounded or flattened, about 1 mm wide,
10ngitudinalIy furrowed and twisted; it bears stinging hairs
and covering trichomes.
B. Reduce to a powder (355) (2.9.12). The powder is green
or greyish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the folIowing diagnostic
characters (Figure 1897.-1): fragments ofunicelIular stinging
hairs [A, B, Cj, up to 2 mm long, composed of an elongated
tapering ceIl with a slightly swolIen stinging tip that readily
breaks off, arising from a raised, multicelIular base [Ca] ;
smaII glandular trichomes [F] (35-65 )lm), with a uni- or
biceIlular stalk and a bi- or quadricelIular head, isolated [Fa],
or on fragments of the epidermis [Fb]; fragments of the
upper epidermis of the leaves in surface view [G] or in
transverse section [D] showing slightly sinuous ceIls [Da,
Gc], unicelIular, straight or slightly curved covering
trichomes, enlarged at the base, up to 700 )lm long [De, Ga]
and abundant large cystoliths [Db, Ea, Gb], empty or
containing dense, granular masses of calcium carbonate;
palisade parenchyma in surface view [E], with rounded ceIls
[Eb] surrounding cystoliths (Ea), or in transverse section
[Dd]; fragments of lower epidermis of leaves showing sinuous
or wavy-walIed celIs [H], anomocytic [Ha] or anisocytic
stomata [Hb] (2.8.3) accompanied by spongy mesophylI in
surface view [He] and in transverse section [De] containing
smaII cluster crystals of calcium oxalate in surface view [Hd]
 IV-282 Nettle Leaf 2014

and in transverse section [Df]; occasional small groups of Top of the plate
vessels, accompanied by parenchyma containing cluster
2 red zones
crystals of calcium oxalate UJ.
Seopoletin: an intense blue A blue fluoreseent zone
fluoreseent zone (seopoletin)
A blue fluoreseent zone

--- - --

- -- ---
Chlorogenic acid: a blue A blue fluoreseent zone
fluoreseent zone (ehlorogenie acid)
A brownish-yellow zone

Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems and maximum 5 per cent of
other foreign matter (including infloreseenees).
Loss on drying (2.2.32)
Maximum 12.0 per eent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 20.0 per cent.
Ash insoluble in hydrochIoric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
Liquid ehromatography (2.2.29).
Test solution To 0.200 g of the powdered drug (355)
Figure 1897.-1.- Illustration for identification test B of (2.9.12) add 25.0 mL of a 40 per cent VIV solution of
powdered herbal drug of nettle leaf methanol R. Extraet for 30 min in an ultrasonie bath at 40 oC
and filter.
Reference solutwn Dissolve 10.0 mg of chlorogenic acid CRS
C. Thin-Iayer chromatography (2.2.27).
in 100.0 mL of a 40 per eent VIV solution of methanol R.
Test solution To 1 g of the powdered drug (355) (2.9.12) Dilute 5.0 mL ofthis solution to 25.0 mL with a
add 10 mL of methanol R. Boil under a reflux condenser for 40 per cent V/V solution of methanol R.
15 mino Cool and filter. Evaporate to dryness in vacuo at Precolumn:
40 oc. Dissolve the residue in 2 mL of methanol R.
- size: 1 = 4 mm, 0 = 4 mm;
Reference solution Dissolve 1 mg of scopoletin R and 2 mg of - stationary phase: end-capped octadecylsilyl silica gel for
chlorogenic acid R in 20 mL of methanol R. chromatography R (5 11m).
Plate TLC silica gel plate R (5-40 flIIl) [or TLC silica gel Column:
plate R (2-10 11m)]. - size: 1 = 0.125 m, 0 = 4 mm;
Mobile phase anhydrous formic acid R, methanol R, water R, - stationary phase: end-capped octadecylsilyl silica gel for
ethyl acetate R (2.5:4:4:50 VIVIV/V). chromatography R (5 11m);
Application 10 IlL [or 4 IlL] as bands of 10 mm [or 8 mm]. - temperature: 25 oC .
Development Over a path of 8 cm [or 6 cm] . Mobile phase:
- mobile phase A: mix 15 volumes of methanol R and
Drying In air.
85 volumes of water R and adjust to pH 2.0 with dz1ute
Detection Heat at 100 oC for 5 minó spray the still-warm phosphoric acid R;
plate with a 10 gIL solution of diphenylboric acid aminoethyl - mobile phase B: methanol R;
ester R in methanol R; examine in ultraviolet light at 365 nm.
Time Mobile phase A Mobile phase B
Results See below the sequence of zones present in the (min) (per cen! V/l? (per cen! V/l?
chromatograms obtained with the reference solution and the 100 O
0 ·1
test solution. Furthermore, other faint blue or yellow
fluorescent zones may be present in the lower half of the 1 - 25 100 ~ 85 O ~ 15
chromatogram obtained with the test solution. 25 - 35 85 15
35 - 36 85 ~ O 15 ~ 100

Flow rate 1 mUmin.

Detection Speetrophotometer at 330 nm.
 2014 Notoginseng Root IV-283

lnJeetion 2O )lL. test solution. Furthermore, other faint zones may be present
Relative retention With reference to chlorogenic acid in the chromatogram obtained with the test solution.
(retention time = about 13 min):
caffeoyImalic acid = about 2.2.
Top oC the plate
Calculate the percentage content of caffeoylmalic acid and
chlorogenic acid, expressed as chlorogenic acid, using the A violet zone (at the solvent front)
following expression: A violet zone

Arbutin: a brown zone

----- -----
A violet zone (ginsenosides Rg l
+ Rg2)
sum of the areas of the peaks due to caffeoylmalic
2 violet zones
acid and chlorogenic acid in the chromatogram
obtained with the test solution; 2 faint violet zones
area of the peak due to chlorogenic acid in the ----- -----
chromatogram obtained with the reference solution;
Aescin: a grey zone A violet zone
mas s of the drug to be examined used to prepare the
test solution, in grams; Several violet and greenish zones
mas s of ehlorogenie aeid CRS used to prepare the
ReCerence solution Test solution
reference solution, in grams;
p percentage content of chlorogenic acid in ehlorogenie
acid CRS.
TESTS
____________________________________________ ~E~

Panax ginseng or Panax quinquefolium

Notoginseng Root *****
** **
(Ph Eur monograph 2383) ***
Ph fur ____________________________________________
DEFINITION
Whole or fragmented taproot, without secondary roots, of
Panax pseudoginseng Wall. var. notoginseng (Burk.) Hoo et
Tseng [Panax notoginseng (Burk.) F.H. Chen ex C .Y. Wu et
K.M. Feng] treated with steam and dried.
Content
Minimum 3.8 per cent for the sum of ginsenosides Rg1
(C42H 7201 4,2H 20 ; M r 837) and Rb1 (Cs4H92023,3H 20; M r
1163) (dried drug).
IDENTIFICATION
A. The primary root is conical, subconical or cylindrical, up
to 6 cm long and 4 cm in diameter. The outer surface,
showing shallow transverse striations and secondary root
scars, is brownish-grey or yellowish-grey. The aerial stem scar
is surrounded by warty protuberances at the crown.
The texture of the root is compacto The fracture is smooth,
shiny, brownish-grey and shows a yellowish-grey ring
(cambial zone) and many radial striations.
B. Reduce to a powder (355) (2.9.12) . The powder is light
yellowish-grey. Examine under a microscope using ehloral
hydrate solution R . The powder shows the following diagnostic
characters: abundant fragments of thin-walled
parenchymatous cells; fragments of secretory canals
containing yellowish-brown resin; rare lignified vessels about
30 )lm in diameter, reticulate or pitted; rare cork fragments.
Examine under a microscope using a 50 per cent V/V
solution of glyeerol R. The starch granules, often deformed,
are very abundant, single or in groups of2-3, and 1-10)lm
in diameter.
C. Examine the chromatogram obtained in the test for Panax
ginseng or Panax quinquefolium.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
Thin-layer chromatography (2.2.27).
Test solution To l.0 g of the powdered drug (3 55) (2.9.12)
add 10 mL of a 70 per cent V/V solution of methanol R and
boil under a refiux condenser for 15 mino Filter after cooling
and dilute to 10.0 mL with methanol R.
Referenee solution Dissolve 5.0 mg of aesein R and 5.0 mg of
arbutin R in 1 mL of methanol R.
Plate TLC siliea gel plate R (5-40 ¡.tm) [or TLC siliea gel
plate R (2-10 ¡.tm)].
Mobz7e phase ethyl aeetate R, water R, butanol R
(25:50:100 VIV/V); allow to stand for 10 min and use the
upper layer.
Applieation 20 ¡.tL, as bands of 15 mm [or 4 ¡.tL of the test
solution and 2 ~lL of the reference solution, as bands of
8 mm].
Development In an unsaturated tank, over a path of 10 cm
[or 5 cm].
Drying In air for 30 mino
Detection Spray with anisaldehyde solution R and heat at
105-110 oC for 5-10 min; examine in daylight.
Results In the chromatogram obtained with the test
solution, the absence of a violet zone immediately aboye the
zone due to arbutin in the chromatogram obtained with the
reference solution suggests the presence of Panax ginseng;
in the chromatogram obtained with the test solution, the
presence of a brown zone immediately below the violet zone
due to the ginsenosides Rg1 + Rg2 suggests the presence of
Panax quinquefolium.
Loss on drying (2.2.32)
Maxirnum 12.0 per cent, determined on l.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 6.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
IV-284 Nutmeg Oil 2014

Test solution Reduce about 50 g to a powder (355) (2.9.12). PI percentage content of ginsenoside Rb1 in
Place 0.250 g of the powdered drug and 70 mL of a ginsenoside Rb1 R;
50 per cent V/V solution of methanol R in a 250 mL round- P2 percentage content of ginsenoside Rg1 in
bottomed fiask. After adding a few grains of pumice, boil on ginsenoside Rg1 R
a water-bath under a refiux condenser for 1 h. After cooling, ____________________________________________ ~E~

centrifuge and collect the supernatant liquid. Treat the

residue as described aboye. Mix the collected liquids and
evaporate to dryness under reduced pressure at a temperature
not exceeding 60 oc. Take up the residue with 10.0 mL of a
buffer solution, adjusted to pH 4.5, containing 3.5 g of Nutmeg Oi! *****
sodium dihydrogen phosphate R and 7.2 g of potassium ** **
dihydrogen phosphate R in 1000 mL of water R (solution A). (Ph Eur monograph 1552) ***
Wash a cartridge containing about 0.36 g of oetadecylsilyl ~E~ ____________________________________________

siliea gel for ehromatography R with 5 mL of methanol R

followed by 20 mL of water for ehromatography R. Apply
5.0 mL of solution A to the cartridge. Elute with 20 mL of
water for ehromatography R, followed by 15 mL of a
30 per cent V/V solution of methanol R. Discard the eJuates
after confirming that no ginsenosides are present, otherwise
repeat the assay with another type of cartridge. Elute the
cartridge with 20 mL of methanol R and evaporate the eluate
to dryness. Take up the residue with 5.0 mL of methanol R.
Referenee solution Dissolve 3.0 mg of ginsenoside Rb1 R,
3.0 mg of ginsenoside Rg1 R and 3.0 mg of ginsenoside Rf R in
methanol R and dilute to 5.0 mL with the same solvent.
Column:
-- size: 1 = 0.10 m, 0 = 4.6 mm;
-- stationary phase: aminopropylsilyl silica gel for
ehromatography R (3 ¡.¡m).
Mobile phase:
-- mobile phase A: aeetonitrile R;
-- mobile phase B: water for ehromatography R;
Time Mobile phase A Mobile phase B
(mio) (perceot VM (perceot VM
0·14 90 10
14·18 90 -4 80 10 -4 20
18·55 80 20
Flow rate 2 mUmin.
Deteetion Spectrophotometer at 203 nm.
Injeetion 20 1lL.
System suitability Reference solution:
-- resolutian: minimum 3.0 between the peaks due to
ginsenosides Rf and Rg1 .
Calculate the sum of the percentage contents of ginsenosides
Rb1 and Rg1 using the following expression:
area of the peak due to ginsenoside Rb1 in the
chromatogram obtained with the test solution;
area of the peak due to ginsenoside Rg1 in the
chromatogram obtained with the test solution;
area of the peak due to ginsenoside Rb 1 in the
chromatogram obtained with the reference solution;
area of the peak due to ginsenoside Rg1 in the
chromatogram obtained with the reference solution;
mass of the dried drug to be examined, in grams;
mas s of ginsenoside Rb1 R in the reference solution, in
grams;
mass of ginsenoside Rg1 R in the reference solution, in
grams;
DEFINITION
Essential oil obtained by steam distillation of the dried and
crushed kemels of Myristiea fragran s Houtt.
CHARACTERS
Appearance
Colourless or pale yellow Iiquid.
Spicy odour.
IDENTIFICATION
First identijieation B.
Seeand identijieation A.
A. Thin-Iayer chromatography (2.2.27) .
Test solution Dissolve 1 mL of the substance to be examined
in toluene R and dilute to 10 mL with the same solvento
Referenee solution Dissolve 20 IlL of myristicine R in 10 mL
of toluene R.
Plate TLC si/iea gel plate R.
Mobi/e phase ethyl acetate R, toluene R (5:95 V/V).
Applieation 10 IlL as bands.
Development Over a path of 15 cm.
Drying In airo
Deteetion Spray with vanillin reagent R, heat at 100-105 oC
for 10 min and examine in daylight.
Results The chromatogram obtained with the reference
solution shows in the upper third a pink or reddish-brown
zone (myristicine); the chromatogram obtained with the test
solution shows a series of zones of which 1 is similar in
position and colour to the zone in the chromatogram
obtained with the reference solution; aboye this zone a
brownish zone (s afro le) and a violet zone (hydrocarbons) are
present; below the myristicine zone, 5 blue zones of variable
intensity are presento
B. Examine the chromatograms obtained in the test for
chromatographic profile.
Results The principal peaks in the chromatogram obtained
with the test solution are similar in retention time to those in
the chromatogram obtained with the reference solution.
TESTS
Re1ative density (2.2.5)
0.885 to 0.905.
Refractive index (2.2.6)
l.475 to l.485.
Optical rotation (2.2.7)
+ 8° to + 18°.
Chromatographic profile
Gas chromatography (2.2.28) : use the normalisation
procedure.
Test solution The substance to be examined.
2014 Olive Leaf IV-285

Reference solution Dissolve 15 ¡.¡L of a-pinene R, 15 ~IL of IDENTIFICATION

{J-pinene R, 15 ¡.¡L of sabinene R, 5 ¡.¡L of car-3-ene R, 5 ¡.¡L of A. The bark occurs in cha=elled or quilled pieces, not more
limonene R, 5 ¡.¡L of y-terpinene R, 5 ¡.¡L of terpinen-4-ol R, than 3 mm thick. The outer surface is light grey or greenish-
5 ¡.¡L of safrole R and 10 ¡.¡L of myristicine R in 1 mL of grey, rather smooth, with occasionallenticels. The inner
hexane R. surface is dull brown or reddish-brown and has slightly raised
Column: longitudinal striations about 0.5-1 mm wide. The fracture is
-- material: fused silica; splintery and fibrous.
-- size: l = 25-60 m, 0 = about 0.3 mm; B. Reduce to a powder (355) (2.9. 12). The powder is light
-- stationary phase: bonded macrogol 20 000 R. brown or reddish-brown and fibrous. Examine under a
Carrier gas helium for chromatography R. microscope using chloral hydrate solution R. The powder
Flow rate 1.5 mUmin. shows the following diagnostic characters: groups of thick-
walled fibres surrounded by a moderately thickened
Split ratio 1: 1OO .
parenchymatous sheath containing prism crystals of calcium
Temperature: oxalate; fragments of cork composed of thin-walled tabular
Time Temperature cells filled with brownish or reddish contents; abundant
(min) (OC) sclereids, isolated and in gtoups, sorne large with thick,
Column 0·10 50 stratified walls and branching pits, others smaller and
10 -75 50 -+ 180 thi=er-walled with simple pits, often with dense brown
contents; fragments of parenchyma containing cluster crystals
75 ·130 180
of calcium oxalate; occasional fragments of sieve tissue, thin-
Injection port 200 - 220 walled, sorne showing sieve areas on the oblique end-walls.
Detector 240 - 250 C. To 1 g of the powdered drug (710) (2.9. 12) add 10 mL
of ethanol (30 per cent V/V) R and heat the mixture under a
Detection Flame ionisation. reflux condenser on a water-bath for 30 mino Cool and filter.
Injection 0.2 ¡.¡L. To 1 mL of this solution add 2 mL of a 10 gIL solution of
Elution order Order indicated in the composition of the vanillin R in hydrochloric acid R. A red colour develops.
reference solution; record the retention times of these
TESTS
substances.
Loss on drying (2.2.32)
System suitability Reference solution: Maximum 10.0 per cent, determined on 1.000 g ofthe
-- resolution: minimum 1.5 between the peaks due to powdered drug (710) (2.9.12) by drying in an oven at
~-pinene and sabinene. 105 oC for 2 h .
Identification of components Using the retention times Total ash (2.4.16)
determined from the chromatogram obtained with the Maximum 8.0 per cent.
reference solution, locate the components of the reference
solution in the chromatogtam obtained with the test solution. ASSAY
Determine the percentage content of each of these Carry out the determination of tannins in herbal drugs
components. The percentages are within the following (2.8.14) . Use 0.700 g ofthe powdered drug (710) (2.9.12).
ranges: _ _ __ _ _ _ _ _ _ _ __ _ __ _ __ ____ ~Em

-- a-pinene: 15 per cent to 28 per cent;

-- {J-pinene: 13 per cent to 18 per cent;
-- sabinene: 14 per cent to 29 per cent;
-- car-3-ene: 0.5 per cent to 2.0 per cent; Olive Leaf ***
-- limonene: 2.0 per cent to 7.0 per cent; *** ***
-- y-terpinene: 2.0 per cent to 6.0 per cent; (Ph Eur monograph 1878) ***
-- terpinen-4-ol: 2.0 per cent to 6.0 per cent; Preparation
-- safrole: maximum 2.5 per cent; Olive Leaf Dry Extract
-- myristicine: 5.0 per cent to 12.0 per cent. ~Em _ _ __ _ _ _ _ _ _ _ _ __ _ __ _ __ ___
STORAGE DEFINITION
Protected from heat. Dried leaf of Olea europaea L.
_ _ _ _ _ __ _ _ _ _ __ __ __ __ _ ___ ~Em

Content
Minimum 5.0 per cent of oleuropein (CZSH 3Z0 13; M r 540.5)
(dried drug).
IDENTIFICATION
Oak Bark A. The leaf is simple, thick and coriaceous, lanceo late to
obovate, 30-50 mm long and 10-15 mm wide, with a
(Ph Eur monograph 1887)
mucronate apex and tapering at the base to a short petiole;
~Em _ _ _ _ _ _ _ _ _ __ __ __ _ __ _ _ _ __
the margins are entire and reflexed abaxially. The upper
DEFINITION surface is gteyish-gteen, smooth and shiny, the lower surface
Cut and dried bark from the fresh young branches of Quercus paler and pubescent, particularly along the midrib and main
robur L., Q. petraea (Matt.) Liebl. and Q. pubescens Willd. lateral veins.
Content B. Reduce ro a powder (355) (2.9. 12). The powder is
Minimum 3.0 per cent of ta=ins, expressed as pyrogallol yellowish-gteen. Examine under a microscope using chloral
(C 6 H 6 0 3; M r 126.1) (dried drug). hydrate solution R . The powder shows the following diagnostic
characters: fragments of the epidermis in surface view with
 IV-286 Olive Leaf 2014

smaIl, thick-walled polygonal cells and, in the lower

epidermis only, smaIl anomocytic stomata (2.8.3); fragments
of the lamina in sectional view showing a thick cuticle, a
palisade composed of 3 layers of ceIls and a small-celled
spongy parenchyma; numerous sclereids, very thick-walled

#It..
and mostly fibre-like with blunt or, occasionaIly, forked ends,
isolated or associated with the parenchyma of the mesophyIl; . :;:'4
Fa
abundant, very large peltate trichomes, with a central • n "

uniceIlular stalk from which radiate sorne 10-30 thin-waIled ' C

cells that become free from the adjoining cells at the margin
of the shield, giving an uneven, jagged appearance.
, .~

*
C. Thin-layer chromatography (2.2.27).
.:.. ' , ..'" :.,' ,. ,
Test solution To 1.0 g of the powdered drug (355) (2.9.12) •. .. E
add 10 mL of methanol R. Boil under a reflux condenser for
15 mino Cool and filter.
Reference solution Dissolve 10 mg of oleuropein R and 1 mg
of rutin R in 1 mL of methanol R.
Plate TLC silica gel plate R.
Mobzle phase water R, methanol R, methylene chloride R
( 1.5:15 :85 VIVIV).
Application 10 ¡.tL, as bands.
Development Over a path of 10 cm.
Drying In air.
Detection Spray with vanillin reagent R and heat at
100-105 oC for 5 min; examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present A. Pelta te trichome, seen lrom aboye F. Fragment 01 the lamina, in
in the chromatogram obtained with the test solution. a. Peltate trichome, seen lrom below transverse section, showing a thick
cuticle (Fa), palisade parenchyma
C. Palisade parenchyma
composed 013 layers 01 cells (Fb),
D, G, H and L. Fibre·like and spongy parenchyma (Fe)
Top of the plate sclereids, sorne accompanied by
parenchymatous Iragments 01 the J. Fragment 01 lower epidermis with
A dark violet·blue zone (solvent anomocytic stomata (Ja) and cicatrix
spongy mesophyll
01 peltate trichome (Jb)
front) E. Spongy parenchyma
K. Fragment 01 upper epidermis, in
A dark violet·blue zone surlace view, with underlying
--- --- palisade parenchyma (Ka)
and sclereids 01 the spongy
Oleuropein: a brownish·green A brownish·green zone mesophyll (Kb)
zone (oleuropein)
Figure 1878.·l. - Illustration of powdered herbal drug of olive
--- --- leaf (see Identification B)
Rutin : a brownish·yellow zone

Reference solution Test solution

Column:
- size: 1 = 0.15 m, 0 = 3.9 mm;
TESTS - stationary phase: octadecylsilyl silica gel for chromatography R
Loss on drying (2.2.32) (5 ¡.tm);
Maximum 10.0 per cent, determined on 1.000 g of the - tempera tu re: 25 oc.
powdered drug (355) (2.9.12) by drying in an oven at Mobile phase:
105 oC for 2 h . - mobile phase A: dilute 1.0 mL of glacial acetic acid R to
Total ash (2.4. 16) 100 mL with water R;
Maximum 9.0 per cent.
- mobile phase B : methanol R;

ASSAY Time Mobile phase A Mobile phase B

(min) (pereent VM (pereent VM
Liquid chromatography (2.2.29).
0-5 85 -+ 40 15 -+ 60 .
Test solution In a f1ask, place 1.000 g of the powdered drug
5-12 40 -+ 20 60 -+ 80
(355) (2.9.12) and add 50 mL of methanol R. Heat in a
water-bath at 60 cC for 30 min with shaking. Allow 10 cool 12- 15 20 -+ 85 80 -+ 15
and filter into a 100 mL volumetric f1ask. Rinse the f1ask and
the filter with methanol R and dilute to 100.0 mL with the Flow rate 1 mUmin.
same solvento Dilute 2.5 mL of this solution 10 25.0 mL with Detection Spectropho1Ometer at 254 nm .
water R . Injection 20 ¡.¡L.
Reference solution Dissolve 5.0 mg of oleuropein CRS in Retention time Oleuropein = about 9 mino
5.0 mL of methanol R . Dilute 1.0 mL of this solution 10
Calculate the percentage content of oleuropein using the
25.0 mL with water R.
following expression:
2014 Opium IV-287

area of the peak due to oleuropein in the
ehromatogram obtained with the test solution;
area of the peak due to oleuropein in the
ehromatogram obtained with the referenee solution;
mI mass of the drug to be examined in the test solution,
in grams;
mass of oleuropein CRS in the referenee solution, in
grams;
p pereentage eontent of oleuropein in oleuropein CRS.
____________________________________________
TESTS
Loss on drying (2.8.17)
Maximum 8.0 per cent.
ASSAY
Liquid ehromatography (2.2.29) . Prepare the solutions
immediately before use.
Test solution To 0.250 g of the extraet to be examined add
50 mL of methanol R. Sonieate for 15 min and filter ínto a
100 mL volumetrie flask. Rinse the flask and the filter with
2 mL of methanol R and dilute to 100.0 mL with water R.
Reference solution (a) Dissolve 10.0 mg of oleuropein CRS in
10.0 mL of methanol R and dilute to 25.0 mL with water R.
PhE~

Reference solution (b) Dissolve 4 mg of rutin R in 10 mL of

referenee solution (a).
Column:
-- size: 1 = 0.15 m, 0 = 4.6 mm;
***
Olive Leaf Dry Extraet -- stationary phase: end-capped octadeeylsilyl silica gel for
** ** chromatography R (5 ¡.tm);
(Ph Eur monograph 2313) ***** -- temperature: 25 oC.
~E~ ____________________________________________ Mobile phase trifiuoroacetic aeid R, methanol R, water R
(1:400:600 V!VIV) .
DEFINITlON
Dry extraet produeed from Olive leaf (1878). Flow rate 1 mUmin.
Content Detection Speetrophotometer at 233 nm.
Minimum 16.0 per eent of oleuropein (Cz5H3Z013; M r Injection 20 ¡.tL.
540.5) (dried extraet) . Run time Twice the retention time of oleuropein.
PRODUCTlON Relative retention With reference to oleuropein
The extraet is produeed from the herbal drug by a suitable (retention time = about 11 min): rutin = about 0.7.
proeedure using ethanol (65-96 per eent VIV) . System suitability Referenee solution (b) :
-- resolution: mínimum 3.0 between the peaks due to rutin
CHARACTERS
and oleuropein.
Appearance
Greenish-brown or brown, amorphous powder. Calculate the pereentage content of oleuropein using the
following expression:
IDENTIFICATlON
Thin-Iayer ehromatography (2.2.27).
Al X m2 xp X 4
Test solutwn To 0.25 g ofthe extraet to be examined add A2 X ml
10 mL of methanol R. Sonieate for 15 min and filter.
Referenee solution Dissolve 5 mg of oleuropein R and 1 mg of
area of the peak due to oleuropein in the
rutin R in 1 mL of methanol R . chromatogram obtained with the test solution;
Plate TLC siliea gel plate R (5-40 ¡.tm) [or TLC silica gel area of the peak due to oleuropein in the
plate R (2-10 ¡.tm)]. ehromatogram obtained with referenee solution (a);
Mobile phase water R, anhydrous formic acid R, ethyl aeetate R mI mass of the extract to be examined used to prepare
(7 :13 :80 V!VIV) . the test solution, in grams;
Application 10 ¡.tL [or 2 ¡.tL] as bands of 10 mm [or 8 mm]. m2 mass of oleuropein CRS used to prepare referenee
solution (a), in grams;
Development Over a path of 10 em [or 6 em] .
p percentage content of oleuropeín in oleuropein CRS.
Drying In airo
____________________________________________ ~E~

Detection Spray with anisaldehyde solution R and heat at

100-105 cC for 5 min; examine in daylight.
Results See below the sequenee of zones present in the
ehromatograms obtained with the reference solution and the
***
test solution. Furthermore, other faint zones may be present Opium * *
in the chromatogram obtained with the test solution. * *
(Raw Opium, Ph Eur monograph 0777) *****
Preparations
Top of tbe plate
Opium Tincture
A dark violet-blue zone Prepared Opium
--- ----- Standardised Opium
OIeuropein: a brownish-green zone A brownish-green zone (oIeuropein) Standardised Opium Tincture
____________________________________________
----- --- ~E~

Rutin: a yeIlow zone DEFINITION

Reference solution Test solution Raw opium is intended only as starting material for the
manufacture of galenical preparations. It is not dispensed as such.
 IV-288 Opium
Air-dried latex obtained by incision from the unripe capsules
of Papaver somniferum L.
Content:
- morphine (C17H19N03; M r 285 .3) : minimum
10.0 per cent (dried drug)j
- codeine (C18H21N03; M r 299.4): minimum 2.0 per cent
(dried drug).
CHARACTERS
Characteristic odour.
Appearance
Blackish-brown masses of various sizes, which tend to be soft
and shiny and, after drying, become hard and brittle.
IDENTIFICATION
Strip off any covering, cut the substance to be examined into thin
slices, dry ar about 60 oC jor 48 h, if necessary, and reduce to a
powder (500) (2.9.12).
A. Examined under a microscope, a suspension of raw opium
in a 20 giL solution of potassium hydroxide R shows the
following diagnostic characters: granules of latex
agglomerated in irregular masses, and light-brown elongated
filaments. Sorne fragments of vessels and rather elongated,
refringent crystals are also visible, as well as a smaller
number of round pollen grains and fragments of elongated
fibres. Hairs of various lengths with sharp points and a few
grains of starch introduced during the handling of the latex
may be presento Fragments of epicarp consisting of polygonal
cells with thick walls defining a stellate lumen may also be
presento
B. Thin-Iayer chromatography (2.2.27).
Test solution Triturate 0.10 g of the powdered drug with
5 mL of ethanol (70 per cent V/V) R, add 3 mL of ethanol
(70 per cent V/V) R, transfer to a 25 mL conical fiask and
heat in a water-bath at 50-60 oC with stirring for 30 mino
Cool, filter, wash the filter with ethanol (70 per cent V/V) R
and dilute the filtra te to 10 mL with the same solvent.
Rejerence solurion Dissolve 2.0 mg of papaverine
hydrochloride R, 12.0 mg of codeine phosphate R, 12.0 mg of
noscapine hydrochloride R and 25 .0 mg of morphine
hydrochloride R in ethanol (70 per cent V/V) R and dilute to
25.0 mL with the same solvent.
Piare TLC silica gel G piare R .
Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
acetone R, toluene R (2:6:40:40 V/V/V/V)j use a freshly
prepared mixture.
Application 20 ¡.¡L, as bands of 20 mm by 3 mm.
Development Over a path of 15 cm.
Drying At 100-105 oC for 15 min, then allow to cool.
Detection Spray with potassiwn iodobismurhate solution R2 and
then with a 4 giL solution of su/furic acid R.
Results The chroma1Ogram obtained with the reference
solution shows in the lower part an orange-red or red zone
(morphine) with a similarly coloured zone (codeine) aboye it,
and in the upper part an orange-red or red zone (papaverine)
with a similarly coloured zone (noscapine) aboye thatj
the chromatogram obtained with the test solution shows
orange-red or red zones corresponding to those in the
chromatogram obtained with the reference solution, and may
also show a dark red zone (thebaine) situated between those
due 10 codeine and papaverine.
C. To 1.0 g of the powdered drug add 5 mL of water R,
shake for 5 min and filter. To the filtrate add 0.25 mL of
jerric chloride solurion R2. A red colour develops that does not
2014
disappear upon the addition of 0.5 mL of dilute hydrochloric
acid R.
TESTS
Thebaine
Liquid chromatography (2.2.29).
Test solution Suspend 1.00 g of the substance 10 be
examined, cut into thin slices, in 50 mL of ethanol
(50 per cent V/V) R, mix with the aid of ultrasound for 1 h,
allow to cool and dilute 10 100.0 mL with the same solvent.
Allow 10 stand. To 10.0 mL of the supematant liquid add
5 mL of ammoniwn chloride buffer solurion pH 9.5 R, dilute 10
25.0 mL with water R and mix. Transfer 20.0 mL of this
solution to a chromatography colurnn about 0.15 m long and
about 30 mm in intemal diameter containing 15 g of
kieselguhr jor chromatography R. Allow to stand for 15 mino
Elute with 2 quantities, each of 40 mL, of a mixture of
15 volumes of 2-propanol R and 85 volumes of methylene
chloride R. Evaporate the eluate 10 dryness in vacuo at 40 oC.
Transfer the residue 10 a volumetric fiask with the aid of the
mobile phase and dilute to 25.0 mL with the mobile phase.
Rejerence solution Dissolve 25 .0 mg of thebaine R in the
mobile phase and dilute to 25.0 mL with the mobile phase.
Dilute 10.0 mL of this solution 10 100.0 mL with the mobile
phase.
Precolumn:
- size: 1 = 4 mm, 0 = 4.0 mm;
- stationary phase: octylsilyl silica gel jor chromatography R
(5 ¡.¡m).
Column:
- size: 1 = 0.25 m, 0 = 4.0 mm;
- stationary phase: octylsilyl silica gel jor chromatography R
(5 ¡.¡m).
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust 10 pH 3.2 with
phosphoric acid (4.9 giL H 3P0 4 ) (about 5 mL) and add
180 mL of acetonitrile R.
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 280 nm.
Injection A suitable volume with a loop injector.
System suitability Reference solution:
- number oj theoretical piates: minimum 3000;
- mass distribution ratio: minimum 3.0 for the peak due 10
thebaine.
Calculare the percentage content of the alkaloid using the
following expression:
mI x A 2 X 625 100
x 100 - h
m2 x A 1 x 5
mI mass of the alkaloid used 10 prepare the reference
solution, in grams;
»22 mass of the substance 10 be examined used to
prepare the test solution, in grams;
Al area of the peak due to the alkaloid in the
chromatogram obtained with the reference solutionj
A2 area of the peak due to the alkaloid in the
chroma1Ogram obtained with the test solution;
h percentage loss on drying.
Limit:
- thebaine: maximum 3.0 per cent (dried drug).
 2014 Opium IV-289

Loss on drying (2.2.32)
Maximum 15.0 per cent, determined on l.000 g ofthe
substance to be examined cut into thin slices, by drying in an
oven at 105 oC for 4 h .
Total ash (2.4.16)
Maximum 6.0 per cent.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
thebaine with the following modifications.
Reference solution Dissolve 0.100 g of morphine
hydrochloride R and 25.0 mg of codeine R in the mobile phase
and dilute to 25.0 mL with the mobile phase. Dilute
10.0 mL ofthis solution to 100 .0 mL with the mobile phase.
System suitability Reference solution:
-- resolution: minimum 2.5 between the peaks due to
morphine and codeine; if necessary, adjust the volume of
acetonitrile in the mobile phase;
-- repeatability: maximum relative standard deviation of
1.0 per cent for the area of the peak due to morphine
after 6 injections.
Calculate the percentage content of morphine and codeine
from the expression given in the test for thebaine.
For the calculation, 1 mg of mO/phine hydrochloride R is
equivalent to 0.759 mg ofmorphine, and 1 mg of codeine R
is equivalent to 0.943 mg of codeine.
____________________________________________ ~Em
ethanol (70 per cent V/V) R, transfer to a 25 mL conical ftask.
Heat in a water-bath at 50-60 oC with stirring for 30 mino
Cool, filter, wash the filter with ethanol (70 per cent VIV) R
and dilute the filtrate to 10 mL with the same solvento
Reference solution Dissolve 2.0 mg of papaverine
hydrochloride R, 12.0 mg of codeine phosphate R, 12.0 mg of
noscapine hydrochloride R and 25.0 mg of morphine
hydrochloride R in ethanol (70 per cent VIV) R and dilute to
25.0 mL with the same solvento
Plate TLC silica gel G plate R .
Mobüe phase concentrated ammonia R, ahanol (96 per cene) R,
acewne R , wluene R (2:6:40:40 V/V/V/V). Use a fresh ly
prepared mixture.
Application 20 ~lL, as bands of 20 mm by 3 mm.
Development Over a path of 15 cm.
Drying At 100-105 oC for 15 mino
Detection Allow to cool and spray with potassium
iodobismuthate solution R2 and then with a 4 gIL solution of
sulfuric acid R, examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, a dark red zone (thebaine)
situated between the codeine zone and the papaverine zone
may be present in the chromatogram obtained with the test
solution.

Top oC the plate

Noscapine: an orange·red or red An orange·red or red zone

zone (noscapine)
Prepared Opium ***** ----- -----
** ** Papaverine: an orange·red or red An orange·red or red zone
(Ph Eur monograph 1840) *** zone (papaverine)
~E~ ____________________________________________ ----- - - - --

DEFINITION Codeine: an orange·red or red An orange·red or red zone

Raw opium powdered (180) (2.9.12), and dried at a
temperature not exceeding 70 oC.
Content:
-- morphine (C 17H 19 N0 3; M r 285.3): 9.8 per cent to
10.2 per cent (drug dried at 100-105 oC for 4 h),
-- codeine (ClsH21N03; M r 299.4): minimum l.0 per cent
(drug dried at 100-105 oC for 4 h).
Content adjusted if necessary by adding a suitable excipient
or raw opium powder.
CHARACTERS
Appearance
Yellowish-brown or dark brown powder.
IDENTIFICATION
A. Examine under a microscope using a 20 gIL solution of
potassium hydroxide R. It is seen to consist of granules of latex
agglomerated in irregular masses, and of light brown
elongated filaments. Sorne fragments of vessels and rather
elongated, refringent crystals are also visible, as well as a
smaller number of round pollen grains and fragments of
elongated fibres. Hairs of various lengths with sharp points
and fragments of epicarp consisting of polygonal cells with
thick walls defining a stellate lumen may be presento Examine
under a microscope using glycerol (85 per cene) R. Particles of
excipient and a few grains of starch introduced during the
handling of the latex may be seen.
B. Thin-layer chromatography (2.2.27) .
Test solution Triturate 0.10 g of the drug to be examined
with 5 mL of ethanol (70 per cent VIV) R, rinse with 3 mL of
zone (codeine)
Morphine: an orange·red or red An orange·red or red zone
zone (morphine)
ReCerence solution Test solution
C. To 1.0 g of the drug to be examined add 5 mL of
water R, shake for 5 mm and filter. To the filtrate add
0.25 mL offenic chloride solution R2. A red colour develops
which does not disappear on the addition of 0.5 mL of dilute
hydrochloric acid R.
TESTS
Thebaine
Liquid chromatography (2.2.29).
Test solution Suspend 1.00 g of the drug to be examined in
50 mL of ethanol (50 per cent V/V) R, mix using sonication
for 1 h, allow to cool and dilute to 100.0 mL with the same
solvento Allow to stand. To 10.0 mL of the supernatant
liquid, add 5 mL of ammonium chloride buffer solution
pH 9.5 R , dilute to 25.0 mL with water R and mix. Transfer
20.0 mL of the solution to a chromatography column about
0.15 m long and about 30 mm in internal diameter
containing 15 g of kieselguhr for chromatography R . Allow to
stand for 15 mino Elute with 2 quantities, each of 40 mL, of
a mixture of 15 volumes of 2-propanol R and 85 volumes of
methylene chloride R. Evaporate the eluate to dryness in vacuo
at 40 oC. Transfer the residue to a volumetric ftask with the
aid of the mobile phase and dilute to 25.0 mL with the
mobile phase.
 IV-290 Opium Preparations
Reference solution
Dissolve 25.0 mg of thebaine R in the
mobile phase and dilute to 25.0 mL with the mobile phase.
Dilute 10.0 mL of the solution to 100.0 mL with the mobile
phase.
Precolumn:
- size: 1 = 4 mm, 0 = 4.0 mm,
- stationary phase: octylsilyl silica gel for chromatography R
(5 ¡.¡m).
Column:
- size: 1 = 0.25 m, 0 = 4.0 mm,
- stationary phase: octylsilyl silica gel for chromatography R
(5 ¡.¡m).
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust to pH 3.2 with
phosphoric acid (4.9 giL H 3P0 4 ) (about 5 mL) and add
180 mL of acetonitrile R.
Flow rate 1.5 mlJmin.
Detection Spectrophotometer at 280 nm.
Injection A suitable volume with a loop injector.
System suitability Reference solution:
- mass distribution ratio: minimum 3.0 for the peak due to
thebaine.
Calculate the percentage content of alkaloid from the
expression:
mi x A 2 x 125 100
m2 X Al X 100 - h
mI mass of the alkaloid in the reference solution, in
grams,
m2 mas s of the substance to be examined in the test
solution, in grams,
Al area of the peak due to the alkaloid in the
chromatogram obtained with the reference solution,
A2 area of the peak due to the alkaloid in the
chromatogram obtained with the test solution,
h percentage loss on drying.
Limit:
- thebaine: maximum 3.0 per cent (dried drug).
Loss on drying (2.2.32)
Maximum 8.0 per cent, determined on 1.000 g by drying in
an oven at 105 oC for 4 h.
Total ash (2.4.16)
Maximum 6.0 per cent.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
thebaine with the following modifications.
Reference solution Dissolve 0.100 g of morphine
hydrochloride R and 25.0 mg of codeine R in the mobile phase
and dilute to 25.0 roL with the mobile phase. Dilute
10.0 mL ofthe solution to 100.0 mL with the mobile phase.
System suitability Reference solution:
- resolution: minimum 2.5 between the peaks due to
morphine and codeine; if necessary, adjust the volume of
acetonitrile in the mobile phase,
- repeatability: maximum relative standard deviation of
1.0 per cent for the peak area due to morphine,
determined on 6 replicate injections.
Calculate the percentage content of morphine and codeine
from the expression given in the test for thebaine. For the
calculation, 1 mg of morphine hydrochloride R is taken to be
2014
equivalent to 0.759 mg of morphine and 1 mg of codeine R is
taken to be equivalent to 0.943 mg of codeine.
_________________________________________ ~E~
Standardised Opium Dry Extract ****
**
*
(Ph Eur monograph 1839) *****
_________________________________________
~E~
DEFINITION
Standardised dry extract produced from Raw opium (0777).
Content:
- morphine (C 17H I9N0 3; M r 285 .3): 19.6 per cent to
20.4 per cent (dried extract);
- codeine (ClsH21N03; M r 299.4): minimum 2.0 per cent
(dried extract).
Content adjusted if necessary by adding a suitable excipient
(e.g. lactose, dextrin).
PRODUCTION
It is produced from the drug and water by a suitable
procedure.
CHARACTERS
Appearance: brown, amorphous powder.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution Triturate 0.05 g of the extract to be examined
with 5 mL of ethanol (70 per cent V/V? R. Transfer to a
25 mL conical fiask. Rinse with 3 mL of ethanol (70 per cent
V/V? R and transfer to the same 25 mL conical fiask. Heat in
a water-bath at 50-60 oC, with stirring, for 30 mino Cool,
filter, wash the filter with ethanol (70 per cent V/V? R and
dilute the combined filtra te and washings to 10 mL with the
same solvento
Reference solution Dissolve 5 mg of morphine hydrochloride R
in the solution prepared as follows and dilute to 5 mL with
the same solution: dissolve 2 mg of papaverine
hydrochloride R, 12 mg of codeine phosphate R and 12 mg of
noscapine hydrochloride R in ethanol (70 per cent V/V? R and
dilute to 25 mL with the same solvento
Plate TLC silica gel plate R (5-40 ¡.¡m) [or TLC silica gel
plate R (2-10 ¡.¡m)] .
Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
acetone R, toluene R (2:6:40:40 VIVIV/V) . Use a freshly
prepared mixture.
Application 20 ¡.¡L [or 6 ¡.¡L] as bands.
Development Over a path of 15 cm [or 8 cm].
Drying At 100-105 oC for 15 mino
Detection Allow to cool and spray with potassium
iodobismuthate solution R2 and then with a 4 gIL solution of
sulfun·c acid R; examine in daylight.
Results See below the sequences of zones present in the
chromatograms obtained with the reference solution and the
test solution. A dark red zone (thebaine) situated between
the zone due to codeine and the zone due to papaverine may
be present in the chromatogram obtained with the test
solution. Furthermore, other faint zones may be present in
the chromatogram obtained with the test solution.
2014
Top of the plate
Noscapine : an orange-red or red An orange-red or red zone
zone (noscapine)
-- - -
Papaverine: an orange-red or red An orange-red or red zone
zone (papaverine)
---
Codeine : an orange-red or red An orange-red or red zone
zone (codeine)
Morphine : an orange-red or red An orange·red or red zone
zone (morohine)
Reference solution Test solution
B. To 0.5 g of the extraet to be examined add 5 mL of
water R, shake for 5 min and filter. To the filtrate add
0.25 mL of ferric chloride solution R2. A red eolour develops,
whieh do es not disappear on the addition of 0.5 mL of dilute
hydrochloric acid R.
TESTS
Thebaine
Liquid ehromatography (2.2.29).
Test solution Suspend 0.500 g of the extraet to be examined
in 50 mL of ethanol (50 per cent V/V) R, mix with the aid of
ultrasound for 1 h, allow to eool and dilute to 100.0 mL with
the same solvento Allow to stand. To 10.0 mL of the
supernatant liquid, add 5 mL of ammonium chloride buffer
solution pH 9.5 R, dilute to 25.0 mL with water R and mix.
Transfer 20.0 mL of this solution to a ehromatography
eolumn about 0.15 m long and about 30 mm in internal
diameter eontaining 15 g of kieselguhr for chromatography R .
Allow to stand for 15 min. Elute with 2 quantities, eaeh of
40 mL, of a mixture of 15 volumes of 2-propanol R and
85 volumes of methylene chloride R. Evaporate the eluate to
dryness in vacuo at 40 oc. Transfer the residue to a
volumetrie fiask with the aid of the mobile phase and dilute
to 25 .0 mL with the mobile phase.
Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Reference solution (b) Dissolve 12.0 mg of morphine
hydrochloride CRS in the mobile phase and dilute to 15. O mL
with the mobile phase (solution A). Dissolve 10.0 mg of
codeine CRS in the mobile phase and dilute to 50.0 mL with
the mobile phase. To 10.0 mL ofthis solution add 10.0 mL
of solution A and mix.
Precolumn:
-- size: I = 4 mm, 0 = 4.0 mm;
-- stationary phase: octylsilyl silica gel for chromatography R
(5 [.lm) .
Column:
-- size: 1= 0.25 m, 0 = 4.0 mm;
-- stationary phase: octylsilyl siliea gel for ehromatography R
(5 [.lm).
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
4.9 giL solution of phosphorie aeid R and add 180 mL of
aeetonitnle R.
Flow rate 1.5 mUmin.
Detection Seetrophotometer at 280 nm.
Injection 20 [.lL.
System suitability:
-- resolution: minimum 2.5 between the peaks due to
morphine and eodeine in the ehromatogram obtained
with referenee solution (b);
Opium Preparations IV-291
-- mass distribution ratio: minimum 3.0 for the peak due to
thebaine in the ehromatogram obtained with referenee
solution (a).
--- Calculate the pereentage eontent of thebaine using the
following expression:
---
area of the peak due to the relevant alkaloid in the
ehromatogram obtained with the test solution;
area of the peak due to the relevant alkaloid in the
ehromatogram obtained with the referenee solution;
mass of the extraet to be examined in the test
solution, in grams;
m2 mass of the relevant alkaloid in the referenee solution,
in grams;
p pereentage eontent of the alkaloid in the relevant
alkaloid CRS;
F 6.250 for the determination of thebaine.
Limit:
-- thebaine: maximum 6.0 per eent (dried extraet).
Loss on drying (2.2.32)
Maximum 5.0 per eent, determined on 1.000 g by drying in
an oven at 105 oC for 4 h.
ASSAY
Liquid ehromatography (2.2.29) as deseribed in the test for
thebaine with the following modifieations.
Injection Test solution and referenee solution (b) .
System suitability:
-- repeatability: maximum relative standard deviation of
1.0 per cent for the area of the peak due to morphine
after 6 injeetions of referenee solution (b).
Calculate the pereentage eontent of morphine and eodeine
from the expression given in the test for thebaine, assigning F
as 10.417 for morphine and as 3.125 for eodeine.
_ _ __ _ _ _ _ _ __ _ _ _ _ __ _ _ _ _ _ Ph Eur
Standardised Opium Tincture ***
*** ***
(Ph Bur monograph 1841) ***
~ E~ _ _ _ _ _ __ _ _ _ _ _ _ __ __ _ _ _ ___
DEFINITION
Standardised tineture produeed from Raw opium (0777).
Content:
-- morphine (C 17H 19N0 3; M r 285.3): 0 .95 per eent to
1.05 per eent;
-- eodeine (Cl sH 2IN03; M r 299.4): minimum 0.1 per eent.
PRODUCTION
Ir is produeed from the drug and equal volumes of ethanol
(70 per eent V/V) and water by an appropriate proeedure.
CHARACTERS
Appearance
Reddish-brown liquido
IDENTIFlCATION
Thin-layer ehromatography (2.2.27) .
Test so/ution Dilute 1.0 mL of the tincture to be examined
to 10 mL with ethanol (70 per eent V/V) R.
IV-292 Opium Preparations 2014

Reference solution Dissolve 5 mg of morphine hydrochloride R Precolumn:

in the solution prepared as follows and dilute to 5 mL with - size: l = 4 mm, 0 = 4.0 mm;
the same solution: dissolve 2 mg of papaverine - stationary phase: octylsilyl silica gel for chrommography R
hydrochloride R, 12 mg of codeine phosphate R and 12 mg of (5 J.lm) .
noscapine hydrochloride R in ethanol (70 per cent V/V) R and Colunm:
dilute to 25 mL with the same solvento - size: 1 = 0.25 m, 0 = 4.0 mm;
Plate TLC silica gel plate R (5-40 J.lm) [or TLC silica gel - stationary phase: octylsilyl silica gel for chromatography R
plate R (2-10 J.lm)]. (5 J.lm).
Mobz1e phase concentrated ammonia R, ethanol (96 per cenl) R, Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
acetone R, toluene R (2:6:40:40 VIVIV/V). Use a freshly monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
prepared mixture. 4.9 gIL solution of phosphoric acid R and add 180 mL of
Application 20 J.lL [or 6 ~IL] as bands. acetonitrile R.
Development Over a path of 15 cm [or 8 cm]. Flow rate 1.5 mL/min.
Drying At 100-105 cC for 15 mino Detection Spectrophotometer at 280 nm.
Detection Allow to cool and spray the plate with potassium InJection 20 J1L.
iodobismuthate solution R2 and then with a 4 gIL solution of System suitabilily Reference solution (b):
sulfuric acid R; examine in daylight. - resolution: minimum 2.5 between the peaks due to
Results See below the sequence of zones present in the morphine and codeine.
chromatograms obtained with the reference solution and the Calculate the percentage content of thebaine using the
test solution. A dark red zone (thebaine) situated between following expression:
the zone due to codeine and the zone due to papaverine may
be present in the chromatogram obtained with the test
solution. Furthermore, other faint zones may be present in
the chromatogram obtained with the test solution.

Al area of the peak due to the relevant alkaloid in the

Top of the plate
chromatogram obtained with the test solution;
Noscapine: an orange·red or red An orange·red or red zone A2 area of the peak due to the relevant alkaloid in the
zone (noscapine) chromatogram obtained with the reference solution;
--- ---
mi mass of the tincture to be examined in the test
Papaverine: an orange·red or red An orange·red or red zone solution, in grams;
zone (papaverine)
m2 mass of the relevant alkaloid in the reference solution,
--- --- in grams;
Codeine: an orange·red or red zone An orange·red or red zone (codeine) p percentage content of the alkaloid in the relevant
Morphine: an orange·red or red zone An orange·red or red zone
alkaloid CRS;
(morphine) F 1.563 for the determination of thebaine.
Reference solution Test solution Limit:
- thebaine: maximum 0.3 per cent.
TESTS Dry residue (2.8.16)
Ethano1 (2.9.10) Minimum 4.0 per cent mlm, determined on 3.00 g.
31 per cent VIV to 34 per cent VIV. ASSAY
Thebaine Liquid chromatography (2.2.29) as described in the test for
Liquid chromatography (2.2.29). thebaine with the following modifications.
Test solution Dilute 2.000 g of the tincture to be examined Injection Test solution and reference solution (b).
to 25.0 mL with ethanol (50 per cent V/V) R. To 10.0 mL of System suitability:
the solution add 5 mL of ammonium chloride buffer solution - repeatability: maximum relative standard deviation of
pH 9.5 R, dilute to 25.0 mL with water R and mix. Transfer 1.0 per cent for the area of the peak due to morphine
20.0 mL of this solution to a chromatography column about after 6 injections of reference solution (b) .
0.15 m long and about 30 mm in intemal diameter Calculate the percentage content of morphine and codeine
containing 15 g of kieselguhr for chromatography R. Allow to from the expression given in the test for thebaine, assigning F
stand for 15 minoElute with 2 quantities, each of 40 mL, of as 2.604 for morphine and 0.781 for codeine.
a mixture of 15 volumes of 2-propanol R and 85 volumes of _ _ _ __ __ _ _ __ _ _ _ _ __ _ __ _ _ ~Ew

methylene chloride R. Evaporate the eluate to dryness in vacuo

at 40 oc. Transfer the residue to a volumetric fiask with the
aid of the mobile phase and dilute to 25.0 mL with the
mobile phase .
Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Reference solution (b) Dissolve 12.0 mg of morphine
hydrochloride CRS in the mobile phase and dilute to 15.0 mL
with the mobile phase (solution A). Dissolve 10.0 mg of
codeine CRS in the mobile phase and dilute to 50.0 mL with
the mobile phase. To 10.0 mL ofthis solution add 10.0 mL
of solution A and mix.
2014 Opium Preparations IV-293

Opium Tincture Concentrated Camphorated Opium

DEFINITION Tincture
Opium, slieed 200 g
DEFINITION
Ethanol (90 per eent) A suffieient quantity
Opium Tineture 400 mL
Purified Water A sufficient quantity
Benzoie Acid 40 g
Extemporaneous preparation Raeemic Camphor 24 g
The following direetions apply. Anise Oil or Star Anise Oil 24mL
Pour 500 mL ofboiling Purified Water on to the Opium and Ethanol (96 per eent) 400 mL
allow to stand for 6 hours; add 500 mL of Ethanol Water Suffieient to produce 1000 mL
(90 per eent), mix thoroughly and allow to stand in a Extemporaneous preparation
eovered vessel for 24 hours; strain, press the mare, mix the The following direetions apply.
liquids and allow to stand for not les s than 24 hours; filter.
Dissolve the Benzoic Aeid, the Raeemic Camphor and the
Determine the eoneentration of morphine, ealculated as Anise Oil or Star Anise Oil in the Ethanol (96 per eent), add
anhydrous morphine, in the tineture so prepared by the the Opium Tineture and sufficient Water to produce
Assay. To the remainder of the liquid add suffieient of a 1000 mL, mix and filter if neeessary.
mixture of equal volumes of Ethanol (90 per eent) and
The tincture complies with the requirements for Tinctures stated
Purified Water to produce an Opium Tineture eontaining
under Extracts and with the following requirements.
1% w/v of anhydrous morphine.
The tincture complies with the requirements for Tinctures stated Content of anhydrous morphine, C17H19N03
under Extracts and with the following requirements. 0.36 to 0.44% w/v.

Content of anhydrous morphine, C17Hl~03 TESTS

0.925 to 1.075% w/v. Ethanol content
54 to 59% v/v, Appendix VIII F, Method III.
TESTS
Ethano1 content Relative density
41 to 46% v/v, Appendix VIII F, Method III. 0.912 to 0.930, Appendix V G .

Relative density ASSAY

0.898 to 0.969, Appendix V G. Dilute 10 mL to 100 mL with ethanol (50%) and carry out
the Assay described under Camphorated Opium Tincture
ASSAY using 10 mL of the diluted solution.
Dilute 5 mL to 100 mL with ethanol (45%). To 10 mL of
the resulting solution add 5 mL of water and 1 mL of
5M ammonia and extraet with 30 mL of a mixture of equal
volumes of ethanol (96%) and chloroform and then with two
22.5 mL quantities of a mixture of 2 volumes of chloroform Camphorated Opium Tincture
and 1 volume of ethanol (96%), washing eaeh extraet with the DEFINITION
same 20 mL of a mixture of equal volumes of ethanol (96%) Opium Tineture 50 mL
and water. Evaporate the eombined extraets just to dryness, Benzoic Acid 5g
extraet the residue with two 5 mL quantities of calcium Racemic Camphor 3g
hydroxide solution, filter and wash the filter with 10 mL of Anise Oil or Star Anise Oil 3 mL
calcium hydroxide solution. To the eombined filtrate and Ethanol (60 per eent) Sufficient to produce 1000 mL
washings add 0.1 g of ammonium sulfate, extraet with two
Extemporaneous preparation
10 mL quantities of ethanol-free chloroform, wash the
The following directions apply.
eombined extraets with 10 mL of water and diseard the
ehloroform solution. To the eombined alkaline liquid and Dissolve the Benzoic Acid, the Racemic Camphor and the
aqueous washings add 10 mL of 1M hydrochloric acid, heat on Anise Oil or Star Anise Oil in 900 mL of Ethanol
a water bath to remove any ehloroform, eool and dilute to (60 per cent), add the Opium Tincture and sufficient
100 mL with water. To 10 mL of this solution add 10 mL of Ethanol (60 per cent) to produce 1000 mL and mix. Filter, if
0.1 M hydrochlO1ic acid and 8 mL of a freshly prepared necessary.
1.0% w/v solution of sodium nitrite, allow to stand for The tincture complies with the requirements for Tinctures stated
15 minutes, add 12 mL of 5M ammonia, dilute to 50 mL under Extracts and with the following requirements.
with water and measure the absorbance of a 4-em layer of the Content of anhydrous morphine, C 17H 19N0 3
resulting solution at the maximum at 442 nm, 0.045 to 0.055% w/v.
Appendix II B, using in the referenee eell a solution prepared
at the same time and in the same manner but using 8 mL of
TESTS
water in place of the solution of sodium nitrite. Calculate the Ethanol content
eontent of C17H19N03 taking 124 as the value of 56 to 60% v/v, Appendix VIII F, Method III.
A(l %, 1 cm) at the maximum at 442 nm. Relative density
0.90 to 0.92, Appendix V G.
ASSAY
To 10 mL add 5 mL of water and 1 mL of 5M ammonia and
extraet with 30 mL of a mixture of equal volumes of ethanol
(96%) and chloroform and then with two 22.5-mL quantities
of a mixture of 2 volumes of chloroform and 1 volume of
ethanol (96%), washing eaeh extraet with the same 20 mL of
 IV-294 Bitter-Orange Flower 2014

a mixture of equal volumes of ethanol (96%) and water. The mounting medium becomes yellow beeause of the
Evaporate the eombined extraets almost to dryness, extraet presenee of hesperidin in the drug.
the residue with 10 mL of calcium hydroxide solution, filter
and wash the filter with 10 mL of calcium hydroxide solution.
To the eombined filtrate and washings add 0.1 g of
ammonium sulfate, extraet with two 10 mL quantities of
ethanol-free chloroform, wash the eombined extraets with
10 mL of water and diseard the chloroform solution. To the
combined alkaline liquid and aqueous washings add 10 mL
of 1M hydrochloric acid, heat on a water bath to remove any
ehloroform, cool and dHute to 100 mL with water. To 10 mL
of this solution add 10 mL of O.IM hydrochloric acid and
8 mL of a fresh!y prepared 1.0% w/v solution of sodium
nitrite, allow to stand for 15 minutes, add 12 mL of
5M ammonia, dHute to 50 mL with water and measure the
absorban ce of a 4-cm layer of the resulting solution at the
maximum at 442 nm, Appendix 11 B, using in the reference
cell a solution prepared at the same time and in the same
manner but using 8 mL of water in place of the solution of
sodium nitrite. Calculate the content of C 17H 19N0 3 taking
124 as the value of A(l %, 1 cm) at the maximum at
442 nm.

Bitter-Orange Flower ***

*** ***
(Ph Eur monograph 1810) ***
~ E~ ____________________________________________

DEFINITlON
Whole, dried, unopened fiower of Citrus aurantium L. ssp.
aurantium (e. aurantium L. ssp . amara EngI.) .
Content
Minimum 8.0 per cent of total fiavonoids, expressed as Figure 1810.-1. - Illustration for identification test B of
naringin (C27H32014; M r 580.5) (dried drug). powdered herbal drug of bitter-orange flower
IDENTIFICATlON
A. The fiower buds are white or yellowish-white and may C. Examine the chromatograms obtained in the test for
reaeh up to 25 mm in length. The dialypetalous corolla is sweet-orange flower.
composed of 5 thick, oblong and concave peta!s dotted with Results See below the sequence of zones present in the
oil glands visible under a hand lens; the short, yellowish- chromatograms obtained with the reference so!ution and the
green persistent gamosepalous ealyx has 5 spreading sepals, test solution.
connate at the base and forming a star-shaped structure
attached to the yellowish-green peduncIe, which is about Top of the plate
5-10 mm long. The fiower buds contain at least 20 stamens
with yeIlow anthers and with filaments fused at the base into A weak yellow f1u orescent zone
groups of 4 or 5; the ovary is superior, brownish-black and A weak yellow f1uorescent zone
spherieal, eonsists of 8-10 multi-ovular loculi and is
Hesperidin: a greenish·yellow A greenish-yellow flu orescent zone
surrounded at the base by an annular granular hypogynous fluorescent zone (hesperidin)
disc; the thiek, eylindrical style ends in a eapitate stigma. Naringin : a yellow fluorescent A yellow f1uorescent zone
B. Microseopic examination (2.8.23). The powder is zone (naringin)
brownish-yellow. Examine under a mieroscope using chloral A red f1uorescent zone
hydrate solution R. The powder shows the following diagnostic (neoeriocitrin)
charaeters (Figure 1810.-1): very numerous spherical pollen A yellow f1uorescent zone (diosmin
and neodiosminJ
grains, with a finely pitted exine and 3-5 germinal pores [H,
Reference solution Test solution
K]; fragments of the epidermis of the sepals in surface view
[D] and in transverse section [A, C], aecompanied by
underlying mesophyll [B], sorne cells ofwhieh contain prisms TESTS
of calcium oxalate [Aa, Ba, Db], unicellular covering Sweet-orange flower
triehomes [Ca] and numerous anomoeytic stomata (2.8.3) Thin-Iayer chromatography (2.2.27).
[Da]; fragments of the epidermis of the petals in surface view
Test solution To 0.5 g of the powdered herbal drug (355)
[F, G, TI, with a distinctly striated euticIe; fragments of large
(2.9.12) add 5 mL of methanol R . Heat with stirring at 40 oC
sehizolysigenous oi! glands in transverse section [E], which
. measure up to 100 ¡.1m in diameter. Examine under a for 10 min. Filter.
microscope using a 20 giL solution of potassium hydroxide R. Reference solution Dissolve 3.0 mg of naringin R and 3.0 mg
of hesperidin R in 10 mL of methanol R.
2014 Orange Oil IV-295

Plate TLC silica gel plate R .

Orange Oil
Mobile phase water R, anhydrous 10rmic acid R, ethyl acetate R
(10 :15:75 VIV/V). DEFINITlON
Application 10 flL as bands. Orange Oil is obtained by mechanical means from the fresh
peel of the sweet orange, Citrus sinensis (L.) Osbeck.
Development Over a path of 10 cm.
Drying In air; heat in an oven at 110-120 oC for 5 mino CHARACTERISTlCS
A yellow to yellowish brown liquid, visibly free from water;
Detection Spray the hot plate with a 10 gIL solution of
odour, that of orange.
diphenylbonc acid aminoethyl ester R in methanol R and then
with a 50 gIL solution of macrogol 400 R in methanol R; after TESTS
at least 1 h, examine in ultraviolet light at 365 nm. Optical rotation
Results The chromatogram obtained with the test solution + 94° to + 99°, Appendix V F . On distillation, the first 10%
shows a yellow zone similar in position to the zone of of the distillate has an optical rotation the same as, or only
naringin in the chromatogram obtained with the reference slightly lower than, the original oil.
solution, and immediately below it a red zone (neoeriocitrin) . Refractive index
Loss on drying (2.2.32) 1.472 to 1.476, Appendix V E.
Maxirnum 11 .0 per cent, determined on 1.000 g of the Residue on evaporation
powdered herbal drug (355) (2.9. 12) by drying in an oven at 1.0 to 5.0% when determined by the method for residue on
105 oc. evaporation 01 volatile oils, Appendix X M . Use 2 g and heat
Total ash (2.4.16) for 4 hours.
Maxirnum 10.0 per cent. Solubility in ethanol
ASSAY Soluble at 20°, in 7 parts of ethanol (90%), Appendix X M.
A bright solution is rarely obtained due to the presence of
Stock solution To 0.175 g of the powdered herbal drug
waxy non-volatile substances.
(355) (2.9.12) add 95 mL of ethanol (50 per cent V/V? R.
Heat on a water-bath under a reflux condenser for 30 mino Weight per mL
AlIow to cool and filter through a sintered-glass filter (2.1.2) . 0.842 to 0.848 g, Appendix V G.
Rinse the filter with 5 mL of ethanol (50 per cent V/V? R. Content of aldehydes
Combine the filtrate and the rinsings in a volumetric flask Not les s than 1.0% w/w, calculated as decanal, CJOHzoO.
and dilute to 100.0 mL with ethanol (50 per cent V/V? R. Carry out the method for the determination 01 aldehydes,
Test solution Into a test tube (10 mm x 180 mm) introduce Appendix X K, using 10 g, omitting the toluene and using a
0.150 g ofpowdered magnesium R (250) (2.9.12), a magnetic volume, not less than 7 mL, of alcoholic hydroxylamine solution
stirring bar 25 mm long and 2.00 mL of the stock solution. that exceeds by 1 to 2 mL the volume of O.5M potassium
Maintain the test tube upright, centrifuge at 125 g and hydroxide in ethanol (60%) VS required. Each mL of
carefully add dropwise, especially at the beginning, 2.0 mL of O.5M potassium hydroxide in ethanol (60%) VS is equivalent to
hydrochlonc acid R, and then 6.0 mL of ethanol (50 per cent 78.76 mg of CIOHzoO .
VIV) R . Stopper the tube and mix by inverting. STORAGE
Compensation solution Into a 2nd test tube, introduce Orange Oil should be kept in a well-filled container and
2.00 mL of the stock solution and carefully add dropwise, protected from light.
especially at the beginning, 2.0 mL of hydrochlonc acid R and
then 6.0 mL of ethanol (50 per cent V/V? R.
After 10 min, measure the absorbance (2.2.25) of the test
solution at 530 nm. ***
Sweet Orange Oi!
Calculate the percentage content of total flavonoids, *** ***
expressed as naringin, using the following expression: (Ph Eur monograph 1811) ***
~Ew __________________________________________
A x 9.62
DEFINITlON
m
Essential oil obtained without heating, by suitable mechanical
treatment from the fresh peel of the fruit of Citrus sinensis
i.e. taking the specific absorbance of the reaction product of (L.) Osbeck (Citrus aurantium L. var. dulcis L.) . A suitable
naringin to be 52. antioxidant may be added.
A absorbance at 530 nm;
m mass of the substance to be examined, in grams. CHARACTERS
__________________________________________ PhE~
Appearance
Clear, pale yellow or orange, mobile liquid, which may
become cloudy when chilled.
Characteristic odour of fresh orange peel.
IDENTIFICATlON
First identification B.
Second identification A .
A. Thin-Iayer chromatography (2.2.27).
Examine the chromatograms obtained in the test for
bergapten.
 IV-296 Orange Oil 2014

Results ASee below the sequence of the zones present in Development Over a path of 15 cm.
the chromatograms obtained with the reference solution and Drying In airo
the test solution. Detection A Examine in ultraviolet light at 365 nm.
Results A The chromatogram obtained with the test solution
Top of the plate
shows no greenish-yellow fiuorescent zone present in the
--- --- chromatogram obtained with the reference solution.
Bergaptene: a greenish·yellow Detection B Spray with anisaldehyde solution R and heat at
fluorescent zone 100-105 oC for 10 min; examine the plate in ultraviolet light
--- --- at 365 nm.
Many blue fluorescent zones Chromatographic profile
Gas chromatography (2.2.28) : use the normalisation
Reference solution Test solution
procedure.
Test solutwn Dilute 300 ~L of the substance to be examined
Results B See below the sequence of the zones present in to 1 mL with acetone R.
the chromatograms obtained with the reference solution and Reference solutwn (a) Dilute 10 ~L of a-pinene R, 10 ~L of
the test solution. {J-pinene R, 1O ~L of sabinene R, 20 ~L of {J-myrcene R,
800 ~ of limonene R, 1O ~L of octanal, 1O ~L of decanal R,
Top of the plate 1O ~L of linalol R, 1O ~L of citral R (composed of neral and
geranial) and 1O ~L of valencene R in 1 mL of acetone R.
A brown fluorescent zone
Reference solution (b) Dissolve 5 ~L of {J-pinene R in 10 mL
Linalyl aceta te: a brownish-orange A faint brownish-orange of acetone R . Dilute 0.5 mL to 10 mL with acetone R.
fluorescent zone fluorescent zone (linalyl acetate)
--- Column:
---
- material: fused silica,
Many orange f1uorescent zones - size: l = 30 m, 0 = 0.53 mm,
Linalol: a brownish·orange A brownish-orange fluorescent - stationary phase: macrogol 20 000 R (film thickness 1 ~m).
f1uorescent zone zone (linalol) Carrier gas helium for chromatography R.
Bergaptene : a faint greenish·yellow
f1uorescent zone
Flow rate 1 mUmin.
Many brownish-orange fluorescent Split ratio 1:1 OO.
zones Temperature:
- -- ---
Time Temperature
Many blue fluorescent zones (mio) (OC)
Reference solution Test solution Column 0·6 50
6·31 50 ~ 150
31·41 150 ~ 180
B. Examine the chromatograms obtained in the test for 41·55 180
chromatographic profile. Injection port 250

Results The characteristic peaks in the chromatogram Detector 250

obtained with the test solution are similar in retention time to Detection Flame ionisation.
those in the chromatogram obtained with the reference
solution. 1njection 0.5 ~L.
Elution order Order indicated in the composition of
TESTS
reference solution (a). Record the retention times of these
Relative density (2.2.5) substances.
0.842 to 0.850.
System suitability Reference solution (a)
Refractive index (2.2.6) - resolution: minimum 3.9 between the peaks due to
1.470 to 1.476. ~-pinene and sabinene and minimum 1.5 between the
Optical rotation (2.2.7) peaks due to valencene and geranial.
+ 94° to + 99°. Using the retention times determined from the
Peroxide value (2.5.5, Method B) chromatogram obtained with reference solution (a), locate
Maximum 20. the components of reference solution (a) in the
Fatty oils and resinified essential oils (2.8.7)
chromatogram obtained with the test solution.
It complies with the test for fatty oils and resinified essential Determine the percentage content of these components.
oils. The limits are within the following ranges:
- a-pinene: 0.4 per cent to 0.6 per cent,
Bergaptene
- {J-pinene: 0.02 per cent to 0.3 per cent,
Thin-Iayer chromatography (2.2.27) .
- sabinene: 0.2 per cent to 1.1 per cent,
Test solution Dilute 0.2 mL of the substance to be examined - {J-myrcene: 1.7 per cent to 2.5 per cent,
in 1 mL of alcohol R. limonene: 92.0 per cent to 97.0 per cent,
Reference solution Dissolve 2 mg of bergaptene R, 1O ~L of - octanal: 0.1 per cent to 0.4 per cent,
linalol R and 20 ~L of linalyl acetate R in 10 mL of alcohol R. - decanal: 0.1 per cent to 0.4 per cent,
Plate TLC silica gel plate R. - linalol: 0.2 per cent to 0.7 per cent,
Mobile phase ethyl acetate R, toluene R (15:85 V/V). neral: 0.02 per cent to 0.10 per cent,
- valencene: 0.02 per cent to 0.5 per cent,
Application 1O ~L, as bands.
2014 Bitter-Orange Pe el IV-297

-- geranial: 0.03 per cent to 0.20 per cent.

Dried Bitter-Orange Peel ***
-- disregard limit: area of the peak in the chromatogram *** ***
obtained with reference solution (b) (0.01 per cent). (Bitter-Orange Epicarp and Mesocarp, ***
Residue on evaporation Ph Eur monograph 1603)
1.0 per cent to 5.0 per cent. Preparations
Evaporate 5.0 g to dryness on a water-bath and dry at Orange Peel Infusion
100-105 oC for 4 h. Orange Tincture
STORAGE ~Ew ____________________________________________
At a temperature not exceeding 25 0e.
____________________________________________ ~Ew
DEFINITION
Dried epicarp and mesocarp of the ripe fruit of Citrus
aurantium L. ssp. aurantium (e. aurantium L. ssp. amara
Engl.) partly freed from the white spongy tissue of the
Terpeneless Orange Oil mesocarp and endocarp .
Preparation Content
Compound Orange Spirit Minimum 20 mUkg of essential oil (anhydrous drug).
DEFINITION CHARACTERS
Terpeneless Orange Oil may be prepared by concentrating Aromatic odour and spicy bitter taste.
orange oil under reduced pressure until most of the terpenes
have been removed or by solvent partition. IDENTIFICATION
A. The drug consists of elliptical or irregular pieces 5-8 cm
CHARACTERISTICS
long, 3-5 cm broad and about 3 mm thick. The outer surface
A clear, yellow or orange-yellow liquid, visibly free from
is yellowish or reddish-brown and distinctly punctate, the
water. inner surface is yellowish or brownish-white.
TESTS B. Reduce to a powder (355) (2.9.12). The powder is light
Optical rotation brown. Examine under a microscope using chloral hydrate
Not more than +60°, Appendix V F. solution R. The powder shows the following diagnostic
Refractive index characters: small polygonal cells with slightly thickened
1.461 to 1.473, Appendix V E. anticlinal walls, filled with orange-red chromatophores, and
Solubility in ethanol very occasional anomocytic stomata (2.8.3); fragments of the
Soluble, at 20°, in 1 part of ethanol (90%), Appendix X M. hypodermis showing collenchymatous thickening; groups of
parenchyma with each cell containing a prism crystal of
Weight per roL
ca1cium oxalate; fragments of lysigenous oil glands;
0.855 to 0.880 g, Appendix V G.
parenchyma containing crystals of hesperidin which dissolve
Content of aldehydes in a 20 giL potassium hydroxide R solution giving a yellow
Not less than 18% w/w, ca1culated as de canal, C lOH 20 0. colour.
Carry out the method for the determination of aldehydes,
Appendix X K, using 1.5 g, omitting the toluene and using a
e. Thin-layer chromatography (2.2.27).
volume, not less than 7 mL, of alcoholic hydroxylamine solution Test solution To 1.0 g of the powdered drug (710) (2.9.12)
that exceeds by 1 to 2 mL the volume of O.5M potassium add 10 mL of methanol R and heat in a water-bath at 65 oC
hydroxide in ethanol (60%) VS required. Each mL of for 5 min shaking frequently. Allow to cool and filter.
O.5M potassium hydroxide in ethanol (60%) VS is equivalent to Reference solution Dissolve 1.0 mg of naringin R and 1.0 mg
78.76 mg of C IOH 2 0 0 . of caffeic acid R in 1 mL of methanol R .
STORAGE Plate TLC silica gel plate R.
Terpeneless Orange Oil should be kept in a well-filled
Mobile phase water R, anhydrous formic acid R, ethyl acetate R
container and protected from light.
(10:15:75 VIV/V).
Application 20 ¡.¡L as bands.

Compound Orange Spirit Development Over a path of 10 cm.

DEFINITION Drying In air, and heat in an oven at 110-120 oC for 5 mino

Terpeneless Orange Oil 2.5 mL Detection Spray the warm plate with a 10 gIL solution of
Terpeneless Lemon Oil 1.3 mL diphenylboric acid aminoethyl ester R in methanol R and then
Anise Oil or Star Anise Oil 4.25 mL with a 50 gIL solution of macrogol 400 R in methanol R . After
Coriander Oil 6.25 mL at least 1 h, examine in ultraviolet light at 365 run.
Ethanol (90 per cent) Sufficient to produce 1000 mL
Results See below the sequence of zones present in the
The spirit complies with the requirements stated under Spirits and chromatograms obtained with the reference and test
with the following requirements. solutions. Furthermore, other fluorescent zones are present in
TESTS the chromatogram obtained with the test solution.
Ethanol content
86 to 90% v/v, Appendix VIII F .
Weight per roL
0.828 to 0.841 g, Appendix V G.
IV-298 Orange Peel Preparations
Top of the plate
Caffeic acid: a Iight blue
f1uorescent zone
Naringin: a dark green f1uorescent
zone
Reference solution
Ac
Ad
o~
L DEFINITION
A. Fragment in transverse section
showing epicarp with thick
cuticule (Aa), collenchymatous
hypodermis (Ab) and part of the
mesocarp parenchyma containing
prism crystals (Ac) of calcium
oxalate and fragment of an oil
gland (Ad)
B. Fragment of epicarp with
anomocytic stoma, in surface view
C. Group of cells of the mesocarp,
sorne containing calcium oxalate
crystals
Figure 1603.-1. - Illustration of powdered herbal drug of
bitter-orange epicarp and mesocarp (see Identification B)
TESTS
Water (2.2.13)
Maximum 10.0 per cent, determined by distillation on 20.0 g
ofpowdered drug (355) (2.9.12).
A Iight blue f1uorescent zone
A light blue f1uorescent zone
A Iight blue f1uorescent zone
A light blue fluorescent zone
A dark green fluorescent zone
(naringin)
A red f1uorescent zone
(neoeriocitrin)
An orange f1uorescent zone
Test solution
G Water
~
'~
D, E, F, H, K and M, Fragments of
mesocarp
G, Su~picarpal collenchymatous
cells
J, Prism crystals of calcium oxalate
L. Group of parenchymatous cells
N. Fragment of epicarp with thick
cuticle and hypodermis showing
collenchymatous thickening, in
transverse section
2014
Total ash (2.4.16)
Maxirnum 7.0 per cent.
Extractable matter
Minimum 6.0 per cent.
To 2.000 g of the powdered drug (250) (2.9.12) add a
mixture of 3 mL of water R and 7 mL of ethanol
(96 per cene) R and extract for 2 h, shaking frequently. Filter,
evaporate 2.000 g of the filtrate to dryness on a water-bath
and dry in an oven at 100-105 oC for 3 h . Allow to cool in a
desiccator over diphosphorus pentoxide R and weigh.
The residue weighs a minimum of 120 mg.
ASSAY
Carry out the determination of essential oil in herbal drugs
(2.8.12). Use a 500 mL round-bottomed flask, 200 mL of
water Ras the distillation liquid and 0.5 mL of xylene R in
the graduated tube. Reduce the drug to a powder (710)
(2.9.12) and immediately use 15.0 g for the determination.
Distil at arate of 2-3 mUmin for 90 mino
____________________________________________ ~E~
Orange Peel Infusion
DEFINITION
Concentrated Orange Peel Infusion 100 mL
Sufficient to produce 1000 mL
The infusion complies with the requirements stated under Infusions.
CONCENTRATED ORANGE PEEL
INFUSION
Dried Bitter-Orange Peel, cut small 500 g
Ethanol (25 per cent) 1350 mL
Extemporaneous prepararlon
The following directions apply.
Macerate the Dried Bitter-Orange Peel in a covered vessel for
48 hours with 1000 mL ofthe Ethanol (25 per cent) and
press out the liquid. To the pressed mare add 350 mL of the
Ethanol (25 per cent), macerate for 24 hours, press and add
the liquid to the product of the first pressing. Allow to stand
for not les s than 14 days and filter.
TESTS
Ethanol content
18 to 23% v/v, Appendix VIII F.
Total solids
10 to 15% w/v, Appendix XI A. Use 1 mL.
Weight per mL
l.01 to l.04 g, Appendix V G .
 2014 Oregano IV-299

Orange Tincture ***** Orange Syrup

** **
(Bitter-Orange Epicarp and Mesocarp Tincture, *** DEFINITION
Ph Eur monograph 1604) Orange Tincture 60mL
~E~ ____________________________________________ Syrup Sufficient to produce 1000 mL
The syrup complies with the requirements stated under Oral
DEFINITION
Liquids and with the following requirement.
Tincture produced from Bitter-orange epicarp and
mesocarp (1603). TESTS
Weight per mL
PRODUCTION
l.29 to l.31 g, Appendix V G.
The tincture is produced from 1 part of the freshly powdered
drug (2000) (2.9.12) and 5 parts of alcohol
(70 per cent V/V /V) by an appropriate procedure.
CHARACTERS Oregano ****
Liquid with a bitter taste. *** *
IDENTIFICATION
(Ph Eur monograph 1880) ****
~E~ ____________________________________________
Examine by thin-Iayer chromatography (2.2.27).
Test solution The tincture to be examined. DEFINITION
Reference solution Dissolve l.0 mg of naringin R and l.0 mg Dried lea ves and flowers separated from the stems of
of caffeic acid R in 1 mL of methanol R. Origanum onites L. or Onganum vulgare L. subsp. hirtum
(Link) Ietsw., or a mixture of both species.
Plate TLC silica gel plate R .
Content:
Mobile phase water R, anhydrous formic acid R, ethyl acetate R
-- essentialoil: minimum 25 mUkg (anhydrous drug);
(10:15:75 VIV/V).
-- sum of the contents of carvacrol and thymol (both C IO H 14 0;
Application 20 ¡.tL, as bands . M r 150.2) : minimum 60 per cent in the essential oil.
Development Over a path of 10 cm.
IDENTIFICATION
Drying In air, and heat in an oven at 110-120 oC for 5 mino
A. O. onites. The leaf is yellowish-green, usually 4-22 mm
Detection Spray the warm plate with a 10 gIL solution of long and 3-14 mm wide. It has a long or short petiole or is
diphenylboric acid aminoethyl ester R in methanol R and then sessiIe. The lamina is ovate, elliptic or ovate-lanceolate.
with a 50 gIL solution of macrogol 400 R in methanol R. After Margins are entire or serrate, the apex is acute or obtuse.
1 h, examine in ultraviolet light at 365 nm. The veins are yellowish and conspicuous on the adaxial
Results See below the sequence of the zones present in the surface. Flowers are solitary or seen as broken parts of the
chromatograms obtained with the reference and test corymb. The calyx is bract-like and inconspicuous .
solutions. Furthermore, other zones are present in the The corolla is white, on top of inflorescences or single
chromatogram obtained with the test solution. flowers, or inconspicuous. The bracts are imbricate and
green like the leaves. The drug contains yellowish or
Top oí the plate
yellowish-brown stem parts.
O. v ulgal'e (subsp. hirtum). The leaf is green and usuaIly
A light blue fluoreseent zone
3-28 mm long and 2.5-19 mm wide. It is petiolate or sessile.
A light blue fluoreseent zone The lamina is ovate or ovate-eliptic. The margins are entire
Caffeie aeid: a light blue fluoreseent or serrate, the apex is acute or obtuse. Flowers are rare,
zone found as broken parts of the corymbs. Bracts are greenish-
A light blue fluoreseent zone yeIlow and imbricate. The calyx is coroIla-like and
A light blue fluoreseent zone inconspicuous. The corolla is white, on top of inflorescences,
slightly conspicuous or inconspicuous.
Naringin: a dark green fluoreseent A dark green fluoreseent zone
zone (naringin) B. Reduce to a powder (710) (2.9. 12). The powder is green
A red fluoreseent zone (O. vulgare) or yeIlowish-green (O. onites). Examine under a
(neoeriocitrin)
An orange fluoreseent zone microscope using chloral hydrate solution R (Figure 1880.-1).
O. onites powder shows fragments of leaf epidermis [A, D, G]
Reíerenee solution Test solution
composed of celIs with sinuous walIs, diacytic stomata (2.8.3)
[Ga], covering trichomes and glandular trichomes; there are
2 types of glandular trichomes: sorne of lamiaceous type with
TESTS 8-16 cells, in surface view [Da], and a very common type
Ethanol content (2.9.10) with a unicellular head and uni- [Gc], bi- [H] or tricellular
63 per cent to 67 per cent VIV. stalk; the covering trichomes have smooth, thick walls; sorne
Methanol and 2-propanol (2.9.11) are multicellular [B, Gb], often broken [Aa], and contain
Maximum 0.05 per cent V /V of methanol and maximum prisms of calcium oxalate, while others, which are rare, are
0.05 per cent V /V of 2-propanol. unicellular and conical [C]; scars from covering and
Dry residue glandular trichomes are visible on the epidermis es [Gd, Ge];
Minimum 6.0 per cent m/m, determined on 2.00 g of pollen grains, with smooth exine, are frequent [E, F].
tincture to be examined. O. vulgare subsp. hirtum powder shows fragments of the
____________________________________________ upper epidermis with celIs with sinuous, beaded walls,
m;
~E~

accompanied by palisade parenchyma fragments of the

lower epidermis [N] composed of cells with finely and
 IV-300 Oregano 2014

irregularly thickened walls, diacytic sto mata (2.8.3) [Na], Top of the plate
covering trichomes and glandular trichomes; there are 2 types
A bluish·purple zone
of glandular trichomes: sorne of lamiaceous type with 12
cells, in surface view [Nb] , and arare type with a unicellular --- ---
head [Nc] and bi- or tricellular stalk; the covering trichomes A pale green zone
have thick, warty walls and contain fine needles of calcium
oxalate; sorne are conical, multicellular and serrate [L, M], Thyrnol : a pink zone A pink zone (thyrnol)
while others, which are rare, are unicellular [K]; there are Carvacrol: a pale violet zone A pale violet zone (carvacrol)
occasional pollen grains, with smooth exine [E, F].
- - - ---
A pale purple zone

A grey zone

A pale green zone

A bluish·purple zone

An intense brown zone

Reference solution Test solution

TESTS
Water (2.2.13)
Maximum 120 mUkg, determined on 20.0 g of the
powdered drug (355) (2.9.12).
Total ash (2.4.16)
Maximum 15.0 per cenr.
Ash insoluble in hydrochloric acid (2.8. 1)
Maximum 4.0 per cent.
ASSAY
Essential oil (2.8.12)
Use 30.0 g ofthe drug to be examined, a 1000 mL round-
bottomed fiask and 400 mL of water R as the distillation
liquido Distil at arate of 2-3 mUmin for 2 h without xylene R
in the graduated tube.
Carvacrol and thymol
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Filter the essential oil obtained in the assay of
essential oil over a small amount of anhydrous sodium sulfate R
Figure 1880.-1.- Illustration for identification test B of and dilute to 5.0 mL with heptane R by rinsing the apparatus
powdered herbal drug of oregano and the anhydrous sodium sulfate.
Rejerence solution Dissolve 0.20 g of thymol R and 50 mg of
C. Thin-layer chromatography (2.2.27). carvacrol R in heptane R and dilute to 5.0 mL with the same
Test solution To 1.0 g of the powdered drug (355) (2.9.12) solvent.
add 5 mL of methylene chloride R and shake for 3 min, then Column:
filter through about 2 g of anhydrous sodium sulfate R. - material: fused silica;
Rejerence solution Dissolve 1 mg of thymol R and 10 IlL of - size: 1 = 60 m, 0 = 0.25 mm;
carvacrol R in 10 mL of methylene chloride R. - stationary phase: macrogol 20 000 R (film thickness
0.25 ¡¡m).
Plate TLC silica gel plate R.
Carrier gas nitrogen jor chromatography R or helium jor
Mobile phase methylene chloride R.
chromatography R.
Application 20 IlL as bands.
Flow rate 1.5 mUmin.
Development Over a path of 15 cm.
Split ratio 1: 1OO.
Drying In air.
Temperature:
Detection Spray with anisaldehyde solution R using 10 mL for
Time Temperature
a plate 200 mm square and heat at 100-105 oC for 10 mino
(min) ('C)
Results See below the sequence of zones present in the Column 0-45 40 -7 250
chromatograms obtained with the reference solution and the
Injection port 190
test solution. Furthermore, other zones are present in the
lower third and upper part of the chromatogram obtained Detector 210
with the test solution.
Detection Flame ionisation.
Injection 0.2 1lL.
 2014 Orientvine Stem IV-301

Elution order Order indicated in the composition of the
reference solution; record the retention times of these
substances.
System suitability Reference solution:
-- resolution: minimum 1.5 between the peaks due to thymol
and carvacrol.
Using the retention times determined from the
chromatogram obtained with the reference solution, locate
the components of the reference solution in the
chromatogram obtained with the test solution.
Determine the percentage content of the sum of carvacrol
and thymol.
____________________________________________
Orientvine Stem ***
*
* **
pitted vessels, up 10 200 ¡¡m in diameter, accompanied by
ligneous parenchyma with slightly and regularly thickened
and pitted cells. Examine under a microscope using a
50 per cent V/V solution of glycerol R . The powder shows
simple, spherical starch granules about 10 ¡¡m in diameter,
free or contained in parenchymatous cells .
C . Thin-layer chromatography (2.2.27) .
Test solution To 2 g of the powdered herbal drug (355)
(2.9.12) add 25 mL of ethanol (96 per cent) R and heat under
reflux for 1 h. Filter and evaporate the filtrate to dryness.
Dissolve the residue in 2 mL of ethanol (96 per cene) R .
Reference solution Dissolve 5 mg of sinomenine R and 5 mg of
Ph E~
papaverine hydrochloride R in 5 mL of ethanol (96 per cent) R .
Plate TLC silica gel F 254 plate R (2-10 ~lm).
Mobile phase concentrated ammonia R, water R, toluene R ,
methanol R, ethyl acetate R (2: 10:20:30:40 VIVIVIV/V).
Application 8 ¡¡L as bands of 8 mm.

(Ph Eur monograph 2450) **** * Developmene Over a path of 6 cm.

Drying In airo
Ph EUf ____________________________________________
Detection A Examine in ultraviolet light at 254 nm.
DEFlNITION R esults ASee below the sequence of zones present in the
Dried, whole or fragmented stem of Sinomenium acutum chromatograms obtained with the reference solution and the
(Thunb.) Rehder et E.H.Wilson, collected in late autumn test solution. Furthermore, other faint fluorescent zones may
and early winter. be present in the chroma1Ogram obtained with the test
Content solution.
Minimum 0.5 per cent of sinomenine (CI9H 23N04; M r
329.4) (dried drug).
Top of the plate
IDENTIFICATION
A. W'hole drug. Long cylindrical stem, somewhat curved, Papaverine: a quenching zone
60 cm long or more, 0.5-2 cm in diameter. The outer bark is A quenching zone
greenish-brown or brown, sometimes greyish-brown, with --- - --
relatively wide longitudinal striations and prominent
verrucose lenticels; the nodes are slightly swollen and
branched. The texture is light, hard and difficult 10 break; Sinomenine: a quenching zone A quenching zone (sinomenine)
the fracture is uneven, greyish-yellow or pale greyish-brown;
----- - - - --
the bark is thin (about 1/10 ofthe diameter); the medullary
rays are very conspicuous; the pith is yellowish-white or pale
yellowish-brown.
A dark blue fluorescent zone
Fragmented drug. Fragments of stems, in discs, about 1.5 cm
in diameter and 0.3 cm thick, with greenish-brown, brown or Reference solution Test solution
greyish-brown outer surface; a transverse section shows a
narrow, pale yellow cortical zone, it is mainly occupied by
Detection B Treat with a 10 gIL solution of sodium nim"te R
the vascular system (about 3/4 of the section) consisting of
in potassium iodobismuthate solution R5 and allow 10 dry in air.
very numerous vascular bundles (about 15-20) in a circle
Examine in daylight.
around the yellowish-white or pale yellowish-brown, small,
circular pith; each bundle is delirnited on the outside by a R esults B See below the sequence of zones present in the
narrow and continuous, wavy, light brown zone and is chromatograms obtained with the reference solution and the
separated from the next bundle by a narrow, light brown test solution. Furthermore, other faint zones may be present
medullary ray; the xylem vessels with a relatively wide in the chroma1Ogram obtained with the test solution.
interior lumen are clearly visible.
B. Microscopic examination (2.8.23). The powder is Top of the plate
yellowish-brown or greyish-brown. Examine under a
An orange zone
microscope using chloral hydrate solution R. The powder Papaverine: an orange zone
shows the following diagnostic characters: rare fragments of --- - -- - -

epidermis with polyhedral cells, in surface view, covered with 3 light orange zones
a thick, pale yellow cuticle about 50 ¡¡m in diameter;
sclereids isolated or in groups, of various sizes and shapes Sinomenine: an orange zone An orange zone (sinomenine)
(subsquare, fusiform, elliptical or irregular), with thickened, - -- ---
pitted walls with conspicuous pit canals, free or included in
2 orange zones
fragments of parenchyma; pale yellow or yellow fibres,
30-70 ¡¡m in diameter with thick, distinctly channelled walls A light orange zone
and a very narrow lumen; fragments of parenchyma with
Reference solution Test solution
thin-walled cells containing fine, needle-shaped crystals of
calcium oxalate; fragments of xylem consisting of reticulate or
IV-302 Wild Pansy
TESTS
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 3 h .
Total ash (2.4.16)
Maximum 6.0 per cent.
Aristolochic acids (2.8.21, Method A)
It complies with the test.
ASSAY
Liquid chromatography (2.2.29).
Test solution Disperse 0.500 g of the powdered herbal drug
(355) (2.9.12) in 20.0 mL of ethanol (70 per cent V/V! R in a
conical fiask and weigh. Sonicate for 20 mino Cool and weigh
again. Compensate the loss of solvent with ethanol
(70 per cent VIV) R and stopper the fiask. Shake thoroughly
and filter through a membrane filter (nominal pore size
0.45 11m).
Reference solution (a) Dissolve 3.0 mg of sinomenine CRS in
methanol R and dilute to 10.0 mL with the same solvento
Reference solution (b) Disperse 0.250 g of orientvine stem
HRS in 10.0 mL of ethanol (70 per cent V/V! R in a conical
fiask and weigh. Sonicate for 20 mino Cool and weigh again.
Compensate the loss of solvent with ethanol (70 per cent
V/V! R and stopper the fiask. Shake thoroughly and filter
through a membrane filter (nominal pore size 0.45 11m) .
Column:
- size: l = 0.15 m, 0 = 4.6 mm;
- stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (5 ~lm) .
Moblle phase Adjust a 1.8 gIL solution of disodium hydrogen
phosphate R to pH 8.0 with a 0.8 gIL solution of sodium
dihydrogen phosphate R, then adjust to pH 9.0 with a 10 gIL
solution of triethylamine R. Mix 60 volumes of this solution
with 40 volumes of acetonitrile R .
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 262 nm.
Injection 20 J1L.
Retention time Sinomenine = about 3 mino
System suitability Reference solution (b):
- resolution: minimum 1.5 between peak 1 and the peak due
to sinomenine; identify peak 1 using the chromatogram
supplied with orientvine stem HRS.
Calculate the percentage content of sinomenine using the
following expression:
Al area of the peak due to sinomenine in the
chromatogram obtained with the test solution;
Az area of the peak due to sinomenine in the
chromatogram obtained with reference solution (a);
mI mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mz mass of sinomenine CRS used to prepare reference
solution (a), in grams;
p assigned percentage content of sinomenine in
sinomenine CRS.
_ _ __ __ __ _ _ __ _ __ _ _ _ __ _ _ PhEw
2014
Wild Pansy ***
*** ***
(Wild Pansy (Flowering Aerial Pans), ***
Ph Bur monograph 1855)
PhEw _ _ _ __ _ __ __ __ _ __ __ _ __ ___
DEFINITION
Dried fiowering aerial parts of Viola arvensis Murray and/or
Viola tricolor L.
Content
Minimum 1.5 per cent of fiavonoids, expressed as violanthin
(C27H300I4; M r 578.5) (dried drug).
IDENTIFICATION
A. The stem is angular and hollow. The leaves are oval,
petiolate, with a cordate base or elongated and obtuse, with
lyrate stipules, divided in the middle. The fiowers, with a
long peduncle, are zygomorphic, with 5 oval, lanceo late
sepals, an appendage pointed outwards and 5 petals of which
the lower one bears a spur; in Viola arvensis, the petals are
shorter than the calyx, the lower petal is cream coloured,
with black lines, the 4 upper petals may be cream coloured
or violet blue; in Viola tricolor, the petals are longer than the
calyx and violet coloured, more or les s tinged with yellow.
The androecium consisting of 5 stamens bears at the apex a
membranous connective appendage with 2 spurs.
The trilocular ovary shows a short style and globular
stigmata. The fruit are navicular capsules, three-lobed,
yellowish brown, 5 mm to 10 mm long. The pale yellow,
pyriform seeds are about 1 mm long, bearing a caruncle.
B. Reduce to a powder (355) (2.9.12). The powder is
greenish. Examine under a microscope using chloral hydrate
solution R . The powder shows the following diagnostic
characters: fragments of the epidermis of the leaves in surface
view with wavy-walled cells and anomocytic stomata (2.8.3);
conical unicellular covering trichomes, widened at the base
and sharply pointed at the apex, with a striated cuticle;
glandular trichomes with a multicellular head, and a short,
multicellular stalk in the indentations of the leaf margins;
cluster crystals of calcium oxalate, sometimes included in
parenchyma; fragments of the corolla with wavy-walled
epidermal cells, those from the mid-region papillose and with
sorne extended to form fiask or bottle-shaped projections,
those from the base of the petals with covering trichomes up
to about 300 11m long with characteristic hump-like swellings
along their length; spherical or polyhedral pollen grains,
60 11m to 80 11m in diameter, with finely pitted exines and 5
pores (Viola arvensis) or 4 pores (Viola tricolor) ; occasional
fragments of spiral and reticulate vessels and groups of fibres
from the stem.
C . Thin-layer chromatography (2.2.27).
Test solution Heat in a water-bath at 65 oC for 5 min, with
frequent stirring, 2.0 g ofthe powdered drug (355) (2.9.12)
in 10 mL of alcohol (70 per cent V/V! R . Cool and filter.
Reference solution Dissolve 2.5 mg of rutin R, 2.5 mg of
hyperoside R and 1.0 mg of caffeic acid R in methanol R and
dilute to 10 mL with the same solvento
Plate TLC silica gel plate R.
Moblle phase anhydrous formic acid R , acetic acid R, water R,
ethyl acetate R (11 :11:27:100 VIVIV/V).
Application 10 IlL, as bands.
Development Over a path of 12 cm.
Drying At 100-105 oC .
Detection Spray with a solution containing 10 gIL of
diphenylboric acid aminoethyl ester R and 50 gIL of macrogol
2014
400 R in methanol R. Allow the plate to dry in air for 30 mino
Examine in ultraviolet light at 365 nm.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top ol the plate
Caffeic acid: a greenish·blue to
light blue fluorescent zone
A blue fluorescent zone
---
Hyperoside: a yellowish·brown
fluorescent zone
A yellowish·green fluorescent zone
---
Rutin: a yellowish·brown An in tense yellowish·brown
fluorescent zone fluorescent zone (rutin)
A yellowish·green fluorescent zone
A yellowish-green fluorescent zone
A yellowish·green fluorescent zone
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent.
Swelling index (2.8.4)
Minimum 9, determined on the powdered drug (355)
(2.9.12) .
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9. 12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maxirnum 15.0 per cent.
ASSAY
Stock solution In a 200 mL flask, introduce 0.300 g of the
powdered drug (250) (2.9.12) and 40 mL of alcohol
(60 per cent V/V? R. Heat in a water-bath at 60 oC for
10 min, shaking frequently. AlIow to cool and filter through a
plug of absorbent cotton into a 100 mL volumetric flask.
Transfer the absorbent cotton with the drug residue back
into the 200 mL flask, add 40 mL of alcohol (60 per cent
V/V? R and heat again in a water-bath at 60 oC for 10 min,
shaking frequently. AlIow to cool and filter into the same
100 mL volumetric flask as used previously. Rinse the
200 mL flask with a further quantity of alcohol (60 per cent
V/V? R, filter and transfer to the same 100 mL volumetric
flask. Dilute to volume with alcohol (60 per cent V/V? R and
filter.
Test solution Introduce 5.0 mL of the stock solution into a
round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
Passion Flower IV-303
flask. Rinse the round-bottomed flask with 3 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the same 25 mL
volumetric flask as used previously. Add 10.0 mL of a
solution containing 25 .0 giL of boric acid R and 20.0 gIL of
oxalic acid R in anhydrous formic acid R and dilute to 25.0 mL
with anhydrous acetic acid R .
Compensation liquid Introduce 5.0 mL of the stock solution
into a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
flask. Rinse the round-bottomed flask with 3 mL of a
- -- mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the same 25 mL
volumetric flask as used previously. Add 10.0 mL of
anhydrous formic acid R and dilute to 25.0 mL with anhydrous
acetic acid R .
Measure the absorbance (2.2.25) of the test solution at
405 nm after 30 min.
Calculate the percentage content of total flavonoids,
- --
expressed as violanthin from the expression:
A x 1.25
m
taking the specific absorbance of violanthin to be 400.
A measured absorbance at 405 nm
m = mass of the drug to be examined, in grams
____________________________________________ ~E~
Passion Flower
*
******
Passiflora **** *
(Ph Eur monograph 1459)
Preparation
Passion Flower Dry Extract
~E~ ____________________________________________
DEFINITION
Fragmented or cut, dried aerial parts of Passiflora incarnata L.
It may also contain flowers and/or fruits .
Content
Minimum 1.5 per cent of total flavonoids, expressed as
vitexin (C2 1H2001O; M r 432.4) (dried drug).
IDENTIFICAnON
A. The green or greenish-grey or brownish stem is ligneous,
hollow, longitudinally striated, glabrous or very slightly
pubescent, with a diameter that is generally les s than 8 mm.
The green or greenish-brown lea ves are alterna te, finely
dentate and pubescent, deeply divided into 3 acute lobes of
which the centrallobe is the largest. The midrib is much
more prominent on the lower surface. The petiole is
pubescent and bears 2 dark nectaries near the lamina.
The tendrils are very numerous and grow from the axils of
the leaves; they are fine, smooth, round and terminated in
cylindrical spirals. The radiate flowers, if present, have 3
small bracts and a corolla consisting of 5 white, elongated
petals with several rows of filiform, petaloid appendices.
If present, the greenish or brownish fruit is flattened and
oval; it contains several flattened, brownish-yellow, pitted
seeds .
 IV-304 Passion Flower Preparations
B. Reduce to a powder (355) (2.9.12). The powder is light
green. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
characters: fragments of the leaf epidermis with sinuous walls
and anomocytic stomata (2.8.3); numerous cluster crystals of
calcium oxalate isolated or aligned along the veins; many
isolated or grouped fibres from the stems associated with
pitted ves seIs and tracheids; uniseriate trichomes with
1-3 thin-walled cells, straight or slightly curved, ending in a
point or sometimes a hook. In addition, the powder shows, if
fiowers are present, papillose epidermises of the petals and
appendages and pollen grains with a reticulate exine; and if
mature fruits are present, scattered brown tannin cells and
brownish-yellow, pitted fragments of the testa.
C. Examine the chromatograms obtained in the test for other
species of Passiflora.
Results The chromatogram obtained with the test solution
shows below the zone due to rutin in the chromatogram
obtained with the reference solution a zone of intense yellow
fiuorescence, aboye it a zone of green fiuorescence
(diglycosylfiavone), below the zone due to hyperoside in the
chromatogram obtained with the reference solution a zone of
yellow fiuorescence (iso-orientin) and aboye a zone of green
fiuorescence (isovitexin), aboye the zone due to hyperoside in
the chromatogram obtained with the reference solution a
zone of brownish-yellow fiuorescence (orientin) and aboye it
a zone of green fiuorescence (vitexin). These latter 2 zones
may be absent. Further zones may be presento
TESTS
Other species of Passiflora
Thin-layer chromatography (2.2.27).
Test solution To 1.0 g ofthe powdered drug (355) (2.9.12)
add 5 mL of methanol R. Heat to boiling under a refiux
condenser for 10 min o Cool and filter.
Referenee solution Dissolve with heating 2.0 mg of rutin R
and 2.0 mg of hyperoside R in 10 mL of methanol R.
Plate TLC si/ica gel plate R .
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (10:10:30:50 VIVIV/V).
Applieation 10 ¡.tL, as bands.
Development Over a path of 15 cm.
Drying In air.
Detection Spray with a 10 gIL solution of diphenylboric acid
mninoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R. Allow to dry in air
for 30 mino Examine in ultraviolet light at 365 nm.
Results The chromatogram obtained wirh the reference
solurion shows in the lower third a zone of yellowish-brown
fiuorescence due to rutin and in the middle third a zone of
yellowish-brown fiuorescence due to hyperoside.
The chromatogram obtained wirh rhe test solurion shows no
intense zones of greenish-yellow or orange-yellow
fiuorescence between the zone due ro diglycosylfiavones and
that due to iso-orientin (P. coerulea and P. edulis).
Total ash (2.4.16)
Maximum 13.0 per cent.
Loss on drying (2.2.32)
Maximum 10.0 per cent, derermined on 1.000 g of rhe
powdered drug (355) (2. 9.12) by drying in an oven at
105 oC for 2 h.
ASSAY
Stock solution In a 100 mL round-borromed fiask, introduce
0.200 g ofthe powdered drug (250) (2.9.12) and add 40 mL
2014
of ethanol (60 per cent V/V! R. Heat in a water-bath at 60 oC
under a refiux condenser for 30 min while shaking
frequently. Allow to cool and filter the mixture through a
plug of absorbent cotton in a 100 mL fiask. Transfer the
absorbent corton with the drug residue into the round-
bottomed fiask. Add 40 mL of ethanol (60 per cent V/V! R
and heat again in a water-bath at 60 oC under refiux for
10 mino Allow to cool and filter the mixture and the first
filtrate from the 100 mL fiask through a filter paper into the
100 mL volumetric fiask. Dilute to 100 mL with the same
solvent, while rinsing the fiask, round-bottomed fiask and
filter.
Test solution Introduce 5.0 mL of stock solution into a fiask.
Evaporate to dryness under reduced pressure and take up the
residue with 10 mL of a mixture of 10 volumes of methanol R
and 100 volumes of glacial acetic acid R . Add 10 mL of a
solution consisting of 25 gIL of borie acid R and 20 gIL of
oxalic acid in anhydrous formic acid R and dilute to 25.0 mL
with anhydrous acetic acid R.
Compensation liquid Introduce 5.0 mL of the stock solution
into a second fiask. Evaporate to dryness under reduced
pressure and take up the residue with 10 mL of a mixture of
10 volumes of methanol R and 100 volumes of glacial acetic
acid R. Add 10 mL of anhydrous formic acid R and dilute to
25.0 mL with anhydrous aeetie acid R.
After 30 min, measure the absorbance (2.2.25) of the test
solution at 401 nm, by comparison with the compensation
liquido
Calculate the percentage content of total fiavonoids,
expressed as vitexin, using the following expression:
A x 0.8
m
i.e. taking the specific absorbance of vitexin to be 628.
A absorbance at 401 nm,
m =: mas s of the drug to be examined, in grams.
____________________________________________ ~Ew
Passion Flower Dry Extract *****
** **
(Ph Eur monograph 1882) ***
~Ew ____________________________________________
DEFINITION
Dry extract produced from Passion fiower (1459).
Content
Minimum 2.0 per cent of fiavonoids, expressed as vitexin
(C2¡H200!O; M r 432.4) (dried extract).
PRODUCTION
The extract is produced from the herbal drug and ethanol
(40 per cent V/V to 90 per cent V/V), methanol
(60 per cent V/V) or acetone (40 per cent V/V) by an
appropriate procedure.
CHARACTERS
Appearance
Greenish-brown amorphous powder.
IDENTIFICATION
Thin-layer chromatography (2.2.27) .
Test solution To 0.25 g of the extract to be examined add
methanol R. Shake, filter and dilute to 5 mL with methanol R .
 2014
R eference solulion Dissolve 2.0 mg of hyperoside R and
2.0 mg of rutin R in methanol R and dilute to 10 mL with the
same solvento
Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel
plate R (2-1 0 ¡.tm)).
Mobile phase anhydrous formic acid R, water R, methyl ethyl
ketone R, ethyl acetate R (10 :10:30:50 VIVIV/V).
Application 10 ¡.tL [or 5 ¡.tL) as bands .
D evelopment Over a path of 15 cm [or 5 cm) .
Drying At 100- 105 oc.
Detection Spray with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R . Subsequently spray with a
50 gIL solution of macrogol 400 R in methanol R . Allow the
plate to dry in air for about 30 mino Examine in ultraviolet
light at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Other fiuorescent zones may be present in the
chromatogram obtained with the test solution.
Top oC the plate
---
Hyperoside : a yellowish-orange
fluoreseent zone
A green fluoreseent zone
A yellow fluoreseen! zone
Rutin: a yellowish-orange
fluorescent zone
---
A green fluorescent zone
ReCerenee solution Test solution
TESTS
Loss on drying (2.8.17)
Maximum 5.0 per cent, determined on 0.500 g.
ASSAY
S tock solution To 50 mg of the extract to be examined add
ethanol (60 per cent V/V? R. Shake, filter and dilute to
100.0 mL with ethanol (60 per cent V/V? R .
Test solution Introduce 5.0 mL of the stock solution into a
round-bottomed fiask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
fiask. Rinse the round-bottomed fiask with 3 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the 25 mL volumetric
fiask. Add 10.0 mL of a solution containing 25 .0 gIL of boric
acid R and 20.0 giL of oxalic acid R in anhydrous formic acid R
and dilute to 25.0 mL with anhydrous acetic acid R .
Compensation lU¡uid Introduce 5.0 mL of the stock solution
into a round-b ottomed fiask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into a 25 mL volumetric
fiask. Rinse the round-bottomed fiask with 3 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
Pelargonium Root IV-30S
glacial acetic acid R and transfer into the 25 mL volumetric
fiask. Add 10.0 mL of anhydrousformic acid R and dilute to
25 .0 mL with anhydrous acetic acid R .
After 30 min, measure the absorbance (2.2.25) of the test
solution at 40 1 nm.
Calculate the percentage content of total fiavonoids,
expressed as vitexin, from the following expression:
A x 0.8
m
i.e. taking the specific absorbance of vitexin to be 628.
A absorbance at 401 nm,
m = mass of the extract to be examined, in grams.
_ _ _ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ __ Ph Eur
Pelargonium Root
(Ph Eur monograph 2264)
~E~ _ __ _ _ _ __ _ _ __ _ _ _ _ __ _ _ __
---
DEFINITION
Dried, usually fragm ented, underground organs of
Pelargonium sidoides DC and/or Pelargonium renifonne Curto
Content
Minimum 2.0 per cent of ta=ins, expressed as pyrogallol
(CóHó03; M r 126 .1) (dried drug).
IDENTIFICATION
A. The root is covered with dark, partly reddish-brown,
--- longitudinally fissured bark. The transverse section shows,
underneath the cork layer, yellow or white wood, which
clearly shows partly brownish medullary rays.
B. Reduce to a powder (355 (2.9.12)). The powder is
brownish-red. Examine under a microscope using chloral
hydrate solution R . The powder shows the following diagnostic
characters: multilayer cork cells consisting of almost uniform,
rectangular cells; fragments of parenchyma underneath the
cork containing sclereids with a wide lumen; numerous
calcium oxalate cluster crystals. Examine under a microscope
using a 50 per cent VIV solution of glycerol R . The powder
shows simple starch granules without striations or cracks.
C . Thin-layer chromatography (2.2.27).
Test solution To 0.5 g ofthe powdered drug (355) (2.9.12)
add 10 mL of methanol R , shake for 15 min and filter.
R eference solution Dissolve 1 mg of scopoletin R and 2 mg of
esculin R in 20 mL of methanol R .
Plate TLC silica gel F 254 plate R (5-40 ¡.tm) [or TLC silica gel
F 254 plate R (2-1 0 ¡.tm)).
Mobile phase water R, methanol R, ethyl aceta te R
(10:14:76 VIV/V).
Application 10 ¡.tL [or 5 ¡.tL) as bands.
Development Over a path of 10 cm [or 6 cm).
Drying In air.
D etection Spray with alcoholic potassium hydroxide solution R .
Examine in ultraviolet light at 365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other blue fiuorescent zones may
be present in the chromatogram obtained with the test
solution.
IV-306 White Peony Root
Top of the plate
A blue fluorescent zone
Scopoletin: a very bright blue A weak blue fluorescent zone
fluorescent zone (scopoletin)
---
One or two bright blue fluorescent
zones
Esculin: a very bright blue
fluorescent zone
A blue fluorescent zone
A weak blue fluorescent zone
---
A blue fluorescent zone
Reference solution Test solution
TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on l.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14). Use 0.750 g ofthe powdered drug (180) (2.9.12).
____________________________________________
White Peony Root
DEFINITION
White Peony Root is the dried whole root of Paeonia lactijiora
Pallas (Paeonia albijiora Pallas; Paeonia edulis Salisb.; Paeonia
officinalis Thunb .). It contains not les s than l.6% of
paeonitlorin (C 23 H 2S O¡¡), calculated with reference to the
dried material.
It is collected in summer and autumn, washed c1ean, with
the two ends and roodet removed and dried.
IDENTIFICATION
A. Cylindrical, straight or slightly curved, two ends trunca te,
5 to 20 cm long, 1 to 2.5 cm in diameter. Extemally light
brown to reddish brown, with longitudinal wrinkles, rootlet
scars and with laterally elongated lenticels. Texture compact,
easily broken, fracture relatively even, intemally whitish or
pale brownish-red. Cambium ring distinct and rays radial.
B. Reduce to a powder. The powder is pale yellow. Examine
under a microscope using chloral hydrate solution. The powder
shows abundant rounded, rectangular or elongated
parenchymatous cells which occur singly or in groups, sorne
with pitted or slightly beaded walls; single cluster crystals of
calcium oxalate, 11 to 35 ¡¡m in diameter, isolated or packed
in rows in parenchymatous cells, sometimes with several
crystals in each cell; fragments of lignified vessels, 20 to
65 ¡¡m in diameter, usually reticulately thickened but
occasionally bordered-pitted; fibres occur rarely, usually
accompanying the vessel fragments, they are 15 to 40 ¡¡m in
diameter, with thickened, slightly lignified and frequendy
pitted walls. Examine under a microscope using a 50% v/v
2014
solution of glycerol in water. Many of the parenchymatous
cells contain mas ses of starch grains which may also be found
scattered throughout the powder.
C. In the Assay, the chromatogram obtained with solution
(1) shows a peak with the same retention time as the
---
principal peak in the chromatogram obtained with
solution (2) .
D. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions .
(1) Shake 0.5 g of the powdered root with 10 mL of ethanol
for 5 minutes, filter and evaporate the filtrate to dryness and
dissolve the residue in 1 mL of absolute ethanol.
--- (2) 0.1 % w/v each of paeonijiorin BPCRS and
4'-hydroxyacetophenone BPCRS in ethanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel (Merck silica gel 60 precoated
plates are suitable).
(b) Use the mobile phase described below.
(c) Apply 10 ¡¡L for each solution, as a bando
(d) Develop the plate to 15 cm.
(e) Dry the plate in airo Spray with a 5% w/v solution of
vanillin in sulfuric acid, heat at 105° for 5 minutes and
examine irnmediately.
MOBILE PHASE
0.2 volumes ofjormic acid, 5 volumes of ethyl acetate,
10 volumes of methanol and 40 volumes of dichloromethane.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
PhE~
with solution (2), two clearly separated bands are observed.
CONFIRMA TION
The bluish-purple band with an Rf value of approximately
0.4 in the chromatogram obtained with solution (1)
corresponds in colour and position to that in the
chromatogram obtained with solution (2). Other bands are
present in the chromatogram obtained with solution (1) as
shown below.
T Op of the plate
A dark pink band
Several bluish bands 4' -hydroxyacetophenone:
an orange band
A bluish-purple band Paeoniflorin:
a bluish-purple band
Solution (1) Solution (2)
TESTS
Tree Peony
In the Assay, the chromatogram obtained with solution (1)
shows no peak with a relative retention of approximately 1.2
with reference to paeonifiorin.
2014
Loss 00 drying
When dried for 3 hours at 100° to 105°, loses not more than
12.0% of its weight. Use 1 g.
Acid-insolub1e ash
Not more than 0.5%, Appendix XI K.
Ash
Not more than 6.5%, Appendix XI J.
Cadmiwn
Maximum 3 ppm, Appendix VII.
Lead
Maximum 5 ppm, Appendix VII.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the foIlowing solutions prepared in
0.05M potassium dihydrogen orthophosphate (solution A).
(1) Finely powder not less than 5 g of the herbal drug being
examined. Mix 0.15 g ofthe powdered drug with 3 mL of
methanol and place in an ultrasonic bath for 30 minutes.
Dilute to 10 mL with solution A, mix, filter through a
0.45-f.lm filter and use the filtrate .
(2) Dissolve a quantity of paeonifiorin BPCRS in methanol and
dilute with solution A to produce a solution containing
0.05% w/v in 3 volumes of methanol and 7 volumes of
solution A and mix.
(3) Dissolve a quantity of 4' -hydroxyacetophenone BPCRS in
solution (2) to produce a solution containing 0.05% w/v of
4' -hydroxyacetophenone.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
with octadecylsilyl siliea gel for chromatography (5 f.lm) (Luna
C 18 is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a fIow rate of 1 mL per minute.
(d) Use a column temperature of 30°.
(e) Use a detection wavelength of 230 nm.
(f) Inject 10 f.lL of each solution.
MOBILE PHASE
30 volumes of methanol and 70 volumes of solution A.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (3), the number of theoretieal plates for the peak
due to paeonifIorin is at least 3000, the symmetry factor for
the peak due to paeonifIorin is not more than 1.3 and the
resolution factor between the two peaks is not less than 1.7.
DETERMINATION OF CONTENT
Using the retention time and the peak area from the
chromatograms obtained with solution (2), locate and
integrate the peak due to paeonifIorin in the chromatogram
obtained with solution (1).
Calculate the content of CZ3HzsOll in the sample, using the
declared content of paeonifIorin (C 23 H zs O ll ) in
paeonifiorin BPCRS and the foIlowing expression:
A, rn;, V, 100
- x - x - xpx - -
A 2 V2 m, 100-d
Area of the peak due to paeonifIorin in the
chromatogram obtained with solution (1).
White Peony Root IV-307
Az Area of the peak due to paeonifIorin in the
chromatogram obtained with solution (2).
m¡ Weight of the drug being examined in mg.
mz Weight of paeonifiorin BPCRS in mg.
V¡ Dilution volume of solution (1) in mL.
Vz Dilution volume of solution (2) in mL.
p Percentage content of C 23 H zs O ¡¡ in
paeonifiorin BPCRS.
d Percentage loss on drying of the herbal drug being
examined.
STORAGE
White Peony Root should be protected from moisture.
Processed White Peony Root
DEFINITlON
Processed White Peony Root is White Peony Root that has
been the boiled, peeled and dried. It contains not less than
1.6% ofpaeonifIorin (C 23 H 2S O¡¡), calculated with reference
to the dried material.
PRODUCTlON
It is coIlected in summer and autumn, washed clean, with
the two ends and rootlets removed, either peeled after boiling
in water or boiled in water after peeling and dried in the sun.
The dried root is washed and soaked thoroughly prior to
being sliced transversely or 10ngitudinaIly and dried.
IDENTIFICATlON
A. The root slices are greyish-white to pale brown, darker at
the edges, and up to about 2 mm thick. They are smooth
and compact with a short and even fracture. Those that have
been cut transversely occur in oval pieces up to about 4.5 cm
in diameter and the cut surface shows a distinctly radiate
xylem with narrow lines of vascular tissue separated by wide
meduIlary rays. The 10ngitudinaIly cut slices are up to 10 cm
long and 1.5 cm wide and the cut surface shows raised
strands of vascular tissue running 10ngitudinaIly.
B. Reduce to a powder. The powder is pale yeIlow. Examine
under a microscope using chloral hydrate solurion. The powder
shows abundant rounded, rectangular or elongated
parenchymatous ceIls which occur singly or in groups, sorne
with pitted or slightly beaded waIls; many of the ceIls contain
pale yeIlow or pinkish mas ses of gelatinised starch which are
also found scattered throughout the powder; single cluster
crystals of calcium oxalate, 11 to 35 f.lm in diameter, isolated
or packed in rows in parenchymatous ceIls, sometimes with
several crystals in each ceIl; fragments of lignified ves seis,
20 to 65 f.lm in diameter, usuaIly reticulately thickened but
occasionalIy bordered-pitted; fibres occur rarely, usualIy
accompanying the vessel fragments, they are 15 to 40 f.lm in
diameter, with thickened, slightly lignified and frequently
pitted waIls.
C. In the Assay, the chromatogram obtained with solution
(1) shows a peak with the same retention time as the
principal peak in the chromatogram obtained with
solution (2).
D. Carry out the method for thin-layer chromatography,
Appendix III A, using the folIowing solutions.
(1) Shake 0.5 g of the powdered root with 10 mL of ethanol
for 5 minutes, filter, evaporate the filtrate to dryness and
dissolve the residue in 1 mL of ethano!.
(2) 0.1 % w/v each of paeonifiorin BPCRS and
4' -hydroxyacetophenone BPCRS in ethanol.
 IV-308 White Peony Root 2014

CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel (Merck silica gel 60 precoated
plates are suitable).
(b) Use the mobile phase described below.
(c) Apply 10 ~L of each solution, as a bando
(d) Develop the plate to 15 cm.
(e) Dry the plate in airo Spray with a 5% w/v solution of
vanzllin in sulfuric acid, heat at 105° for 5 minutes and
examine immediately.
MOBILE PHASE
A mixture of 0.2 volumes ofjormic acid, 5 volumes of elhyl
acerale, 10 volumes of melhanol and 40 volumes of
dichloromelhane.
SYSTEM SUlTABILITY
The test is not valid unless, in the chromatogram obtained
with solution (2), two c1early separated spots are observed.
CONFIRMATION
The bluish-purple spot with an Rf value of approximately 0.4
in the chromatogram obtained with solution (1) corresponds
(1) Finely powder not less than 5 g of the herbal drug being
examined. Mix 0.15 g of the powdered drug with 3 mL of
melhanol and place in an ultrasonic bath for 30 minutes.
Dilute to 10 mL with solution A, mix, filter through a
0.45-~ filter and use the filtrate.
(2) Dissolve a quantity of paeonifiorin BPCRS in melhanol and
dilute with solution A to produce a solution containing
0.05% w/v in 3 volumes of methanol and 7 volumes of
solution A and mix.
(3) Dissolve a quantity of 4'-hydroxyacetophenone BPCRS in
solution (2) to produce a solution containing 0.05% w/v of
4'-hydroxyacetophenone.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel colurnn (15 cm x 4.6 mm) packed
with octadecylsilyl silica gel jor chromatography (5 ~m) (Luna
C18 is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a ftow rate of 1 mL per minute.
0
(d) Use a column temperature of 30 •

in colour and position to that in the chromatogram obtained (e) Use a detection wavelength of 230 nm.
with solution (2) . Other spots are present in the (f) Inject 10 flL of each solution.
chromatogram obtained with solution (1) as shown below.
MOBILE PHASE
A mixture of 30 volumes of methanol and 70 volumes of
T op of the plate solution A.
A dark pink band SYSTEM SUlTABILITY
The test is not valid unless, in the chromatogram obtained
with solution (3), the number of lheoretical piares for eaeh
Several bluish bands 4' -hyd roxyacetophenone: peak due to paeoniflorin is at least 3000, the symmelry jactor
an orange band
for the peak due to paeoniftorin is not more than 1.3 and the
resolution jactor between the two peaks is not less than 1.7.
DETERMINATION OF CONTENT
A bluish-purple band Paeoniflorin:
a bluish-purple band Using the retention time and the peak area from the
ehromatograms obtained with solution (2), loe ate and
integrate the peak due to paeoniftorin in the ehromatogram
obtained with solution (1) .
Calculate the content of C 23 H 2SO II in the sample, using the
Solution (1) Solution (2) dec1ared content of paeoniftorin (C23H2S011) in
paeonifiorin BPCRS and the following expression:

TESTS
Tree Peony
In the Assay, the chromatogram obtained with solution (1)
shows no peak with a relative retention of approximately 1.2
Al Area of the peak due to paeoniftorin in the
with reference to paeoniflorin.
chromatogram obtained with solution (1) .
Loss on drying A2 Area of the peak due to paeoniftorin in the
When dried for 3 hours at 100° to 105°, loses not more than chromatogram obtained with solution (2).
12.0% ofits weight. Use 1 g. mI Weight ofthe drug being examined in mg.
Acid-inso1ub1e ash m2 Weight of paeonifiorin BPCRS in mg.
Not more than 0.5%, Appendix XI K. VI Dilution volume of solution (1) in mL.
Ash V2 Dilution volume of solution (2) in mL.
Not more than 6.5%, Appendix XI J. p Pereentage content of C 23 H 2S O II in
paeonifiorin BPCRS.
Cadmium d Pereentage los s on drying of the herbal drug being
Maximum 3 ppm, Appendix VII. examined.
Lead
Maximum 5 ppm, Appendix VII. STORAGE
Proeessed White Peony Root should be proteeted from
ASSAY moisture.
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions prepared in
0.05M porassium dihydrogen orlhophosphate (solution A).
2014 Pepper IV-309

Pepper ****
(Ph Bur monograph 2477)
~Eif ____________________________________________
*** *
*** * ~.
~B
.' . .' e
"' "
.:.

DEFINITION
Dried, ripe or nearly ripe fruit of Pipa nigrum L. with an
unbroken pericarp (black pepper) or with the outer layers of
the pericarp removed (white pepper).
Content:
-- essentialoil: minimum 25 mlJkg (anhydrous drug);
-- piperine (C 17 H I9 N0 3 ; M r 285.3): minimum 3.0 per cent
(anhydrous drug) . Fa
IDENTIFICATION
A. White pepper. Spheroid berries, 3-5 mm in diameter,
slightly fiattened at one pole and with a small protuberance
at the other, with smooth, externally matt, brownish-grey,
greyish-white or pale yellowish-white surface, with numerous
pale, linear striations between apex and base.
'.
e-
,A.,'.,
W_.:Qo -"
Hb

Blaek pepper. Spheroid berries, 3-6 mm in diameter,

externally blackish-brown, with raised reticular wrinkles,
bearing fine remains of the style at the apex and a scar of the
peduncle at the base. The texture is hard, the epicarp can be
stripped, the endocarp is greyish-white or pale yellow.
The fracture is greyish-white, starchy, possessing a small
space at the centre. Figure 2477.-1. - Illustration for identification test B of
B. Microscopic examination (2.8.23). powdered herbal drug of pepper
White pepper. The powder is light grey. Examine under a
microscope using ehloral hydrare solution R. The powder
shows the following diagnostic charaeters (Figure 2477 .-1) : C. Thin-layer chromatography (2.2.27).
fragments of the endoearp in surface view, consisting of more Test solution To 0.5 g of the powdered herbal drug (355)
or less polygonal sclereids about 20-30 ¡.¡m in diameter, (2.9.12) add 5 mL of methanol R . Sonieate for 10 min,
whieh have irregularly thickened walls [Ac, C, Fa] and which eentrifuge and use the supernatant.
may or may not be associated with the testa [A, F] , Referenee solution Dissolve 10 mg of borneol R and 15 mg of
consisting of a layer of indistinet, reddish-brown pigmented piperine R in 10 mL of methanol R .
eells constituting the 'pigmented layer' [Ab, Fb] and a layer Plate TLC silica gel F 254 plate R (5-40 ¡.¡m) [or TLC siliea gel
of very thin-walled polygonal eells eonstituting the 'hyaline F 254 plate R (2-10 ¡.¡m)].
layer' [Aa]; fragments of the endocarp, in transverse section
Mobile phase ethyl acetare R, cyclohexane R (30:50 V/V).
[G], showing sclereids with thickened iuner walls on the 3
lower sides [Ga], usually assoeiated with the testa (pigmented Applieation 10 ¡.¡L [or 5 ¡.¡L] as bands of 10 mm [or 8 mm].
layer [Gb] and hyaline layer [Gel); fragments ofthe Development Over a path of 15 cm [or 6 cm].
parenchyma of the mesoearp [D] eontaining large oil cells Drying In airo
50-75 ¡.¡m in diameter [Da]; numerous thin-walled, ovoid or Detection A Examine in ultraviolet light at 254 nm.
polygonal cells of the parenchyma of the seed [E]; rare,
Results ASee below the sequence of zones present in the
elongated sclereids, with thickened walls, from the fruit
ehromatograms obtained with the reference solution and the
peduncle [B]; a few fragments of vascular tissue with narrow
test solution. Furthermore, other faint quenehing zones may
m.
spiral ves seIs Examine under a microseope using a
be present in the chromatogram obtained with the test
50 per cent V/V solution of glyeerol R. Rounded, compound
solution.
stareh granules [H], about 30 ¡.¡m in diameter, made up of
tiny individual granules, ovoid or polyhedral by compression,
Top of the plate
free [Hb] or incJuded in the parenchymatous cells of the seed
[Ha]. - -- - --
Black pepper. The powder is grey. Examine under a 3 quenching zones
mieroseope using ehloral hydrate solution R . In addition to the
diagnostie eharacters deseribed for white pepper, the - -- ---
powdered blaek pepper shows the following diagnostic A quenching zone
eharacters (Figure 2477.-1): fragments ofthe epiearp [K]
Piperine: a quenching zone A strong quenching zone (piperine)
with extremely thin-walled, brownish-red pigmented,
polygonal or ovo id eells, whieh eontain small prisms of
calcium oxalate [Ka], and which are associated with the
outer layers of the mesocarp eonsisting of groups of scJereids
with strongly thiekened walls [Kb]. Reference solution Test solution

Detection B Treat with anisaldehyde solwion R and heat at

100 oC for 5 min; examine in daylight.
 IV-310 Peppermint Leaf 2014

Resulls B See below the sequence of zones present in the Mobile phase:
chromatograms obtained with the reference solution and the - mobile phase A : water R;
test solution. Furthermore, other zones may be present in the - mobile phase B: acetonitrile R;
chromarogram obtained with the test solution. Time Mobile phase A Mobile phase B
(min) (per cen! V!JI) (per cen! V!JI)
O-S 50 50
Top of the plate
5 - 20 50 --7 5 50 --7 95
A strong purple zone
20 - 22 5--70 95 --7 100
A purple zone

- - - --- Flow rate 1.0 mlJmin.

Borneol: a yellowish-brown zone Detection Spectrophotometer at 343 nm.
1njection 1O ¡lL.
A purple-grey zone
Retention time Piperine = about 10 mino
--- ---
ldentijication of peaks Use the chromatogram supplied with
A violet-grey zone long pepper for system suitability HRS and the chromatogram
A grey zone
obtained with reference solution (b) to identify the peak due
ro piperine and peak 2.
Piperine: a green or brownish zone A green or brownish zone
(piperine)
System suitability Reference solution (b):
- peak-to-valley ratio: minimum 4, where Hp = height above
the baseline of peak 2 and H v = height above the baseline
A grey zone of the lowest point of the curve separating the peak due
Reference solution Test solution to piperine from peak 2.
Calculate the percentage content of piperine using the
following expression:
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent.
Water (2.2.13)
area of the peak due ro piperine in the
Maximum 120 mlJkg, determined on 20.0 g of the freshly,
chromatogram obtained with the test solution;
coarsely powdered herbal drug (1400) (2. 9.12) reduced using
a knife milI. area of the peak due ro piperine in the
chromatogram obtained with reference solution (a);
Total ash (2.4.16) mi mass of the herbal drug to be examined used to
Maximum 6.0 per cent. prepare the test solution, in grams;
ASSAY mass of piperine CRS used to prepare reference
Essential oil (2.8.12) solution (a), in grams;
U se 10. O g of the freshly, coarsely powdered herbal drug p percentage content of piperine in piperine CRS.
(1400) (2.9.12), a 1000 mL round-bottomed fiask, 400 mL _ _ _ __ __ _ _ __ _ _ _ __ __ __ __ Ph fur
of water R as the distillation liquid and 0.5 mL of xylene R in
the graduated tube. Distil at arate of 2-3 mlJmin for 3 h.
Piperine
Liquid chromatography (2.2.29). Cany out the assay protected Peppermint Leaf *****
from light. ** **
Test solution Disperse 0.250 g of the powdered herbal drug (Ph Eur monograph 0406) ***
(355) (2.9.12) in 40 mL of ethanol (96 per cent) R. Sonicate Preparation
for 20 min and filter. Rinse the fiask and the filter with 5 mL Peppermint Leaf Dry Extract
of ethanol (96 per cent) R, combine the filtrate and washings ~fw _ _ __ _ _ _ __ __ __ _ _ _ _ __ __ _
and dilute ro 50.0 mL with the same solvento Filter through
a membrane filter (nominal pore size 0.45 ¡lm). DEFINITION
Whole or cut dried leaves of M entha x piperita L.
Reference solution (a) Dissolve 15. O mg of piperine CRS in
ethanol (96 per cene) R and dilute to 100.0 mL with the same Content
solvento Minimum 12 mlJkg of essential oil for the whole drug and
minimum 9 mlJkg of essential oil for the cut drug.
Reference solution (b) Disperse 0.250 g of long pepper for
system suitability HRS (355) (2.9.12) in 40 mL of ethanol CHARACTERS
(96 per cene) R . Sonicate for 20 min and filter. Rinse the fiask Characteristic and penetrating odour.
and the filter with 5 mL of ethanol (96 per cene) R, combine Characteristic aromatic taste.
the filtrate and washings and dilute ro 50.0 mL with the
Peppermint leaf is green or brownish-green, with brownish-
same solvento Filter through a membrane filter (nominal pore
violet veins in sorne varieties. The petioles are green or
size 0.45 ¡lm).
brownish-violet.
Column:
- size: 1 = 0.15 m, 0 = 4.6 mm; IDENTIFICATION
- stationary phase: end-capped octadecylsilyl silica gel for A. The leaf is entire, broken or cut, thin, fragile and ofien
chromatography R (5 ¡lm). crumpled; the entire leaf is 3-9 cm long and 1-3 cm wide.
The lamina is oval or lanceolate, the apex acuminate, the
2014 Peppermint Leaf IV-3II

margin sharply dentate and the base asymmetrical. Venation
is pinnate, prominent on the lower surface, with lateral veins
leaving the midrib at about 45°. The lower surface is slightly
pubescent and secretory trichomes are visible under a lens
(6 x) as bright yelIowish points. The petiole is grooved,
usualIy up to 1 mm in diameter and 0.5-1 cm long.
B. Reduce to a powder (355) (2.9.12). The powder is
brownish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the folIowing diagnostic
characters (Figure 0406.-1): fragments of epidermis es bearing
covering and glandular trichomes; adaxial epidermis, in
surface view [B, H], having celIs with sinuous-wavy walIs
[Ha] and cutide striated over the veins (B) associated with
palisade parenchyma [Hb] ; abaxial epidermis [C] with
diacytic stomata (2.8.3) [Ca]; covering trichomes are usualIy
fragmented, elongated, uniseriate with 3-8 celIs with striated
cutide [A, E]; glandular trichomes of 2 types: a) unicelIular
stalk with smalI, rounded unicelIular head 15-25 !lm in
diameter, in surface view [Ba, Cb] or in transverse section
[D], b) unicelIular stalk with enlarged oval head 55-70 !lm in
diameter composed of 8 radiating celIs, in surface view [Bb]
or in transverse section [Ga]; fragments from near the leaf
margin [F] with isodiametric celIs whose antidinal walIs are
more-or-less straight and beaded [Fa] and short, conical,
unicelIular or bicelIular covering trichomes [Fb]; dorsiventral
mesophyIl fragments, in transverse section [G], with a single
palisade layer [Gc] and 4-6 layers of spongy parenchyma
[Gb] . YelIowish crystals of menthol under the cutide of
secretory ceIls may be presento
Test solution To 0 .2 g of the recently powdered drug add
2 mL of methylene chloride R, shake for a few minutes and
filter. Evaporate the filtrate to dryness at about 40 oC and
dissolve the residue in 0.1 mL of toluene R.
Reference solution Dissolve 50 mg of menthol R, 20 !lL of
cineole R, 10 mg of thymol R and 10 !lL of menthyl acetate R
in toluene R and dilute to 10 mL with the same solvento
Plate TLC silica gel GF254 plate R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application lO!lL of the reference solution and 20 ¡.¡L of
the test solution, as bands.
Development Over a path of 15 cm.
Drying In air until the solvent has evaporated.
Detection A Examine in ultraviolet light at 254 nm.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other weak quenching zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
--- ---
Thyrnol : a quenching zone
Quenching zones rnay be present
(carvone. pulegone)
--- ---

Reference solution Test solution

Detection B Spray with anisaldehyde solution R and examine

in daylight while heating for 5-10 min at 100-105 0e.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.

Top of the plate

An intense violet·red zone (near

---
Menthyl acetate: a violet-blue zone
Thyrnol: a pink zone
the solvent front) (hydrocarbons)
---
A violet-blue zone (rnenthyl
acetate)
A greenish-blue zone (menthone)

Light pink or greyish-blue or

greyish-green zones may be
present (carvone. pulegone,
isornenthone)
Cineole: a violet-blue or brown A faint violet-blue or brown zone
zone (cineole)
--- - --
Menthol: an intense blue or violet An intense blue or violet zone
zone (rnenthol)
Reference solution Test solution

Figure 0406.-1.- Illustration for identification test B of
powdered herbal drug of peppermint leaf
e. Thin-layer chromatography (2.2.27) .
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent stems, whose diameter is not greater
than 1.5 mm; maximum 2 per cent foreign elements;
not more than 8 per cent of the leaves show brown stains
due to Puccinia menthae.
 IV-312 Peppermint Oil 2014

Carry out the determination using 10 g of the drug. Top of the plate
Water (2.2.13) An in tense violet·red zone (near
Maximum 110 mUkg, determined on 20.0 g. the solvent front) (hydrocarbons)
A brownish-yellow zone
Total ash (2.4.16) (menthofuran)
Maximum 15.0 per cent. ----- -----
Ash insoluble in hydrochloric acid (2.8.1)
Menthyl acetate: a violet-blue zone A violet-blue zone (rnenthyl
Maximum 1.5 per cent. acetate)
ASSAY A greenish-blue zone (rnenthone)
Carry out the determination of essential oils in herbal drugs Thyrnol: a pink zone
(2.8.12). Use 20.0 g of crushed drug, a 500 mL fiask, Light pink or greyish-blue or
200 mL of water R as the distillarion liquid and 0.50 mL of greyish-green zones rnay be
xylene R in the graduated tube. Distil at arate of 3-4 mUmin present (carvone, pulegone,
isomenthone)
for 2 h.
1,8-Cineole: a violet-blue or brown A faint violet-blue or brown zone
____________________________________________ ~Ew
zone (l,8-cineole)
----- -----

Menthol: an intense blue or violet An intense blue or violet zone

zone (menthoJ)
Peppermint Oil ***** Reference solution Test solution
** **
(Ph Eur monograph 0405) ***
Preparations B. Examine the chromarograms obtained in the test for
Gastro-resistant Peppermint Oil Capsules chromatographic profile.
Concentrated Peppermint Emulsion Results Tb,e characteristic peaks due ro limonene,
1,8-cineole, menthone, menthofuran, isomenthone, menthyl
Peppermint Spirit
acetate and menthol in the chromatogram obtained with the
~Ew ____________________________________________
test solution are similar in retention time ro those in the
DEFINITION chromarogram obtained with reference solution (a).
Essential oil obtained by steam distillation from the fresh Isopulegol, pulegone and carvone may be present in the
aerial parts of the fiowering plant of Mentha x piperita L. chromatogram obtained with the test solution.
CHARACTERS TESTS
Appearance Relative density (2.2.5)
Colourless, pale yellow or pale greenish-yellow liquido 0.900 to 0.916.
Characteristic odour and taste followed by a sensation of Refractive index (2.2.6)
cold. 1.457 to 1.467.
Solubility Optical rotation (2.2.7)
Miscible with ethanol (96 per cent) and with methylene -30° to -10°.
chloride. Acid value (2.5.1)
IDENTIFICATION Maximum 1.4, determined on 5.0 g diluted in 50 mL of the
First identification B. prescribed mixture of solvents.
Second identification A. Fatty oils and resinified essential oils (2.8.7)
It complies with the test for fatty oils and resinified essential
A. Examine the chromatograms obtained in test A for mint
oils.
oi!.
Results ASee below the sequence of zones present in the
Mintoil
chromatograms obtained with the reference solution and the A. Thin-layer chromatography (2.2.27).
test solution. Test solution Mix 0.1 g of the substance to be exarnined
with toluene R and dilute to 10 mL with the same solvento
Top of the plate Reference solution Dissolve 50 mg of menthol R, 20 f.lL of
cineole R, 10 mg of thymol R and 10 flL of menthyl acetate R
----- ----- in toluene R and dilute ro 10 mL with the same solvento
Thyrnol: a quenching zone Plate TLC silica gel F 254 plate R (5-40 flm) [or TLC silica gel
F 254 plate R (2-10 flm)] .
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Quenching zones rnay be present
(carvone, pulegone) Application 10 flL [or 1 flL] of the reference solution and
----- ----- 20 flL [or 2 flL] of the test solution, as bands of 10 mm [or
8 mm].
Reference solution Test solution
Development Over a path of 15 cm [or 6 cm].
Drying In air.
Results B See below the sequence of zones present in the
Detection A Examine in ultraviolet light at 254 nm.
chromatograms obtained with the reference solution and the
test solution. Furthermore, other less intensely coloured Detection B Treat with anisaldehyde solution R and heat at
zones may be present in the chromatogram obtained with the 100-105 oC for 5-10 min; examine immediately in daylight.
test solution.
 2014 Peppermint Preparations IV- 313

Results B The chromatogram obtained with the test solution - cal'Vone: maximum 1.0 per cent;
shows no blue zone between the zones due to 1,8-cineole - disregard limit: the area of the principal peak in the
and mentho!. chromatogram obtained with reference solution (b)
B. Examine the chromatograms obtained in the test for (0.05 per cent).
chromatographic profile. The ratio of 1,8-cineole content to limonene content is
Results The chromatogram obtained with the test solution minimum 2.
do es not show a peak with the retention time of isopulegol STORAGE
that has an area of more than 0.2 per cent of the total area. At a temperature not exceeding 25 oc.
Chromatographic profile Gas chromatography (2.2.28): __________________________________________ ~E~

use the normalisation procedure.

Test solution Mix 0.20 mL of the substance to be examined
with heptane R and dilute to 10.0 mL with the same solvent.
Reference solution (a) Dissolve 10 ¡.tL of limonene R, 20 ¡.tL
of cineole R, 40 ¡.tL of menthone R, 10 ¡.tL of menthofuran R, Concentrated Peppermint Emulsion
10 ¡.tL of isomenthone R, 40 ¡.tL of menthyl acetate R, 20 ¡.tL of DEFINITION
isopulegol R, 60 mg of menthol R, 20 ¡.tL of pulegone R, 1O ~lL Concentrated Peppermint Emulsion is a 2% v/v dispersion of
of piperitone R and 10 ¡.tL of carvone R in heptane R and dilute Peppermint Oil in a suitable vehicle containing a non-ionic
to 10.0 mL with the same solvent. surface-active agent.
Reference solution (b) Dissolve 5 ¡.tL of isopulegol R in Extemporaneous prepararlon
heptane R and dilute to 10.0 mL with the same solvent. The following formula and directions apply.
Dilute 0.1 mL of the solution to 5.0 mL with heptane R . Peppermint Oil 20mL
Column: Polysorbate 20 1 mL
- material: fused silica; Double-strength 500 mL
- size: l = 60 m, 0 = 0.25 mm; Chloroform Water
- stationary phase: macrogol20 000 R (film thickness Purified Water, freshly Sufficient to produce 1000 mL
0.25 ¡.tm). boiled and cooled
Carrier gas helium for chromatography R. Shake the Peppermint Oil with the Polysorbate 20 and add
Flow rate 1.5 mUmin. gradually, shaking well after each addition, the Double-
Split ratio 1:50. strength Chloroform Water and sufficient Purified Water to
produce 1000 mL.
Temperature:
Time Temperature
(min) (oC)
Column 0·10 60

10·70 60 -+ 180 Peppermint Spirit

Peppermint Essence
70·75 180

lnjection port 200

DEFINITION
Peppermint Oil 100 mL
Detector 220 Ethanol (90 per cent) Sufficient to produce 1000 mL
Detection Flame ionisation. Extemporaneous prepararlon
Injection 1 ¡.tL. The following directions apply.
Elution order Order indicated in the composition of Dissolve the Peppermint Oil in Ethanol (90 per cent) and
reference solution (a); record the retention times of these add sufficient Ethanol (90 per cent) to produce 1000 mL.
substances. If the solution is not c1ear, shake with previously sterilised
Purified Tale and filter.
ldentification of peaks Using the retention times determined
from the chromatogram obtained with reference solution (a), The spirit complies with the requirements stated under Spirits and
locate the components of reference solution (a) in the with the following requirements.
chromatogram obtained with the test solution. TESTS
System suitability Reference solution (a) : Ethanol content
- resolution: minimum 1.5 between the peaks due to 78 to 82% v/v, Appendix VIII F.
limonene and 1,8-cineole; minimum 1.5 between the Weight per mL
peaks due to piperitone and carvone. 0.830 to 0.840 g, Appendix V G.
Determine the percentage content of each of the following Content of oil
components. The limits are within the following ranges: 9.0 to 11.0% v/v when determined by the following method.
- limonene: 1.0 per cent to 3.5 per cent; Add 25 mL of the spirit and 5 mL of xylene to 90 mL of a
- l,8-cineole: 3.5 per cent to 8.0 per cent; 10% w/v solution of sodium chloride containing 1.0% v/v of
- menthone: 14.0 per cent to 32.0 per cent; hydrochloric acid in a flask of about 150 mL capacity with a
- menthofuran: 1.0 per cent to 8.0 per cent; long neck graduated in 0.1 mL increments and of such a
- isomenthone: 1.5 per cent to 10.0 per cent; diameter that a 15-cm length has a capacity of 10 mL. Shake
- menthyl acetate: 2.8 per cent to 10.0 per cent; the mixture for about 30 minutes, allow to separate and raise
- isopulegol: maximum 0.2 per cent; the undissolved oily layer into the graduated part of the neck
- menthol: 30.0 per cent to 55.0 per cent; of the flask by the gradual addition of more of the acidified
- pulegone: maximum 3.0 per cent, sodium chloride solution, allow to stand for 2 hours or until
 IV-314 Peppermint Oil Preparations
there is no further change in volume of the oily layer and
measure the volume of the oily layer. The volume of the oily
layer, after the subtraction of 5 mL, is taken to be the
volume of oi!.
Gastro-resistant Peppermint Oil
Capsules
DEFINITION
Gastro-resistant Peppermint Oil Capsules contain
Peppermint Oi!. They are enteric capsules.
The capsules comply with the requirements stated under Capsules
and with the following requirements.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography,
Appendix III A, using a TLC silica gel GF254 plate and a
mixture of 5 volumes of ethyl acetate and 95 volumes of
toluene as the mobile phase. Apply separately to the plate as
bands 20 /lL of solution (1) and 10 /lL of solution (2).
For solution (1) dissolve a quantity of the contents of the
capsules containing 0.1 g of Peppermint Oil in sufficient
toluene to produce 10 mL. For solution (2) dissolve 10 mg of
thymol, 10 /lL of menthyl aceta te, 20 IJ.L of cineole and 50 mg
of menthol in toluene and dilute to 10 mL with the same
solvent. Allow the plate to dry in air until the solvent has
evaporated and examine under ultraviolet light (254 nm). The
chromatogram obtained with solution (1) may show
quenching zones (carvone, pulegone) situated just below the
level of the zone (thymol) in the chromatogram obtained
with solution (2) . Spray with anisaldehyde solution and
examine in daylight for 5 to 10 minutes while heating at 100°
to 105°. The chromatogram obtained with solution (2)
shows, in order of increasing Rf value, an intense blue to
violet zone (menthol) in the lower third; a violet-blue to
brown zone (cineole); a pink zone (thymol); and a violet-blue
zone (menthyl acetate). In the chromatogram obtained with
solution (1) there is a zone due to menthol (the most
intense) and a faint zone due to cineole; at Rf values between
those of the cineole and thymol zones in the chromatogram
obtained with solution (2), there may be light pink or
greyish-blue or greenish-grey zones (carvone, pulegone,
isomenthone); in the middle of the chromatogram, there is a
violet-blue zone (menthyl acetate) and just below it a
greenish-blue zone (menthone); an intense violet-red zone
(hydrocarbons) appears near the solvent front and below it
there may be a brownish-yellow zone (menthofuran); other
less intensely coloured zones may also appear.
B. Examine the chromatograms obtained in the test for
Chromatographic profile. The retention time of the principal
peaks in the chromatogram obtained with solution (1) is
similar to that of the principal peaks in the chromatogram
obtained with solution (2). Carvone and pulegone may be
absent from the chromatogram obtained with solution (1).
TESTS
Disintegration test
Complies with the requirements stated under Gastro-resistant
Capsules.
Refractive index
1.457 to 1.467, determined on the contents of the capsules,
Appendix V G.
2014
Relative density
0.900 to 0.916, determined on the contents ofthe capsules,
Appendix V E.
Composition of peppermint oil
Carry out the method for gas chromatography,
Appendix 111 B, using the following solutions. For solution
(1) use the contents of the capsules. For solution (2) dissolve
0.1 g of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of
menthofuran, 0.1 g of isomenthone, 0.4 g of menthyl acetate,
0.6 g of menthol, 0.2 g of pulegone and 0.1 g of carvone in
1 mL of hexane. Prepare irnmediately before use.
The chromatographic procedure may be carried out using
(a) a fused-silica capillary column (60 m x 0.25 mm) coated
with macrogol20,OOO as the bonded phase, maintaining the
temperature of the colurnn at 60° for 10 minutes, then
raising the temperature at arate of 2° per minute to 180°
and maintaining at 180° for 5 minutes, a split injection
system with a split ratio of 100 to 1 and maintaining the
temperature of the injection port and of the detector at 220 0
and (b) helium as the carrier gas at a flow rate of 1.5 mL per
minute.
Inject about 0.2 ¡.¡L of solution (2). When the
chromatograms are recorded in the prescribed conditions, the
components elute in the order indicated in the composition
of solution (2); a type chromatogram is reproduced in the
monograph for Peppermint Oi!. Record the retention times
of these substances.
The test is not valid unless the number of theoretical plates
ca1culated from the limonene peak at 110° is at least 30,000
and the resolution factor between the peaks corresponding to
limonene and cineole is at least 1.5.
Inject about 0.2 ¡.tL of solution (1 ). Using the retention times
determined from the chromatogram obtained with solution
(2), locate the components of solution (2) on the
chromatogram obtained with solution (1) (disregard the peak
due to hexane).
Determine the percentage content of the components by the
normalisation procedure.
The percentages are within the following ranges:
Limonene 1.0 to 5.0%,
Cineole 3.5 to 14.0%,
Menthone 14.0 to 32.0%,
Menthofuran 1.0 to 9.0%,
Isomenthone 1.5 to 10.0%,
Menthyl acetate 2.8 to 10.0%,
Menthol 30.0 to 55.0%,
Pulegon Not more than 4.0%,
Carvone Not more than 1.0% .
The ratio of cineole content to limonene content is greater
than 2.
STORAGE
Gastro-resistant Peppermint Oil Capsules should be
protected from light.
 2014 Peru Balsam IV-315

*** Reference solution (a) Dissolve 10.0 mg of rosmarinic

Peppermint Leaf Dry Extraet * *
** ** acid CRS in ethanol (50 per cent V/V) R and dilute to
(Ph Eur monograph 2382) *** 100.0 mL with the same solvento
~E~ ____________________________________________ Reference solution (b) Dissolve 5 mg of ferulic acid R in
referenee solution (a) and dilute to 50 mL with the same
DEFINITION solution.
Dry extraet produeed from Peppermint leaf (0406). Column:
Content -- size: 1 = 0.25 m, 0 = 4.6 mm;
Minimum 0.5 per eent of rosmarinie aeid (ClsHI60S; M r -- stationary phase: octadecylsilyl si/ica gel for chromatography R
360.33) (dried extraet) . (5 flm).
PRODUCTION Mob¡Je phase:
The extraet is produeed from the herbal drug by a suitable -- mobile phase A: phosphoric acid R, acetonitrile R, water R
proeedure using ethanol (30-50 per eent V/V) or water of (1:19:80 V/V/V);
minimum 60 oc. -- mobile phase B: phosphoric acid R, methanol R, acetonitrlle R
(1:40:59 V/V/ V);
CHARACTERS
Appearance Time Mobile phase A Mobile phase B
Brown, amorphous powder. (min) (percent VM (percent VM
O o20 100 -7 55 0-745
IDENTIFICATION
20 o25 55 -7 O 45 -7100
Thin-Iayer ehromatography (2.2.27).
25 o30 0-7100 100 -7 O
Test solution To 0.2 g of the extraet to be examined add
5 mL of methanol R . Sonieate for 5 min and filter.
Flow rate 1.2 mUmin.
Reference solution Dissolve 5 mg of rosmarinic acid R, 1 mg
Detection Speetrophotometer at 330 nm.
of hyperoside R and 1 mg of rutin R in 10 mL of methanol R.
Injection 20 ¡.¡Lo
Piare TLC silica gel piare R (5-40 ¡.un) [or TLC si/ica gel
piare R (2-10 flm)]. Relative retention With referenee to rosmarinie aeid
(retention time = about 11 min): ferulic aeid = about 0.8.
Mob¡Je phase anhydrous formic acid R, water R, ethyl acetare R
(6:6:90 V/V/V). System suitability Referenee solution (b):
-- resolution: minimum 4.0 between the peaks due to ferulie
Application 10 flL [or 4 flL] as bands of 15 mm [or 8 mm].
aeid and rosmarinie aeid.
Development Over a path of 8 em [or 6 em] .
Calculate the pereentage eontent of rosmarinie acid using the
Drying In air. following expression:
Detection Heat at 100 oC for 5 min and spray the hot plate
Al x m2 x p x 0.2
with a 5 gIL solution of diphenylboric acid aminoethyl ester R in
ethyl acetare R; examine in ultraviolet light at 365 nm. A 2 x mI
Results See below the sequenee of zones present in the
Al area of the peak due to rosmarinie aeid in the
ehromatograms obtained with the referenee solution and the
ehromatogram obtained with the test solution;
test solution. Furthermore, other faint zones may be present
A2 area of the peak due to rosmarinie aeid in the
in the ehromatogram obtained with the test solution.
ehromatogram obtained with referenee solution (a);
mI mas s of the extraet to be examined used to prepare
Top of the plate the test solution, in grams;
A light blue fluorescent zone
m2 mass of rosmarinic acid CRS used to prepare referenee
Rosmarinic acid: a light blue
fluorescent zone (rosmarinic acid) solution (a), in grams;
----- ----- p pereentage eontent of rosmarinie aeid in rosmarinic
acid CRS.
----- ---
____________________________________________ ~E~

Hyperoside: an orange fluorescent

zone
A yellow fluorescent zone
***
Peru Balsam
*** ***
A brown fluorescent zone (Ph Eur monograph 0754) ***
Rutin: an orange fluorescent zone A yellow fluorescent zone ~E~ ____________________________________________

Reference solution Test solution DEFINITION

ASSAY
Liquid ehromatography (2.2.29).
Test solution Use brown glass flasks. To 0.400 g of the
extraet to be examined add 15 mL of ethanol (50 per cent
V/V) R, sonieate for 10 min and filter into a 20 mL
volumetrie flask. Rinse the flask and the filter with ethanol
(50 per cent V/V) R and dilute to 20.0 mL with the same
solvento
Balsam obtained from the seorehed and wounded trunk of
Myroxylon balsamum (L.) Harms var. pereirae (Royle) Harms.
Content
45 .0 per eent m/m to 70.0 per eent m/m of esters, mainly
benzyl benzoate and benzyl einnamate .
CHARACTERS
Appearance
Dark brown, viseous liquid whieh is transparent and
yellowish-brown when viewed in a thin layer; it is not stieky,
non-drying and does not form threads.
IV- 316 Phyllanthus Emblica Pericarp
Solubility
Practically insoluble in water, freely soluble in anhydrous
ethanol, not miscible with fatty oils, except for castor oi!.
IDENTIFICATION
A. Dissolve 0.20 g in lO mL of ethanol (96 per eent) R.
Add 0.2 mL offerrie ehlonde solution RI . A green or
yellowish-green colour develops.
B. Thin-Iayer chromatography (2.2.27).
Test solution Dissolve 0.5 g of the substance to be examined
in 10 mL of ethyl aeetate R.
Referenee solution Dissolve 4 mg of thymol R, 30 mg of
benzyl cinnamate R and 80 ¡.tL of benzyl benzoate R in 5 mL
of ethyl acera te R.
Plate TLC siliea gel GF254 plate R.
Mobile phase glacial aeetie acid R, ethyl aeetate R, hexane R
(0 .5:10:90 VIV/V).
Applieation 10 ¡.tL, as bands of 20 mm by 3 mm.
Development Twice over a path of 10 cm.
Drying In airo
Deteetion A Examine in ultraviolet light at 254 nm and
mark the quenching zones.
Results A The chromatogram obtained with the reference
solution shows in the upper third 2 quenching zones, the
higher one due to benzyl benzoate and the lower one due to
benzyl cinnamate. The chromatogram obtained with the test
solution shows 2 quenching zones at the same levels and of
approximately the same size.
Deteetion B Spray with a freshly prepared 200 gIL solution
of phosphomolybdie aeid R in ethanol (96 per eent) R, using
10 mL for a plate 200 mm square and examine in daylight
while heating at 100-105 oC for 5-10 mino
Results B The zones due to benzyl benzoate and benzyl
cinnamate are blue against a yellow background.
The chroma1Ogram obtained with the reference solution
shows at about the middle a violet-grey zone (thymol). In the
chromatogram obtained with the test solution, a blue zone
(nerolidol) is seen just below the leve! of the zone due to
thymol in the chromatogram obtained with the reference
solution. Just below the zone due to nerolidol, no blue zone
is seen corresponding 10 a quenching zone seen when
examined in ultraviolet light at 254 nm (colophony). In the
upper and lower part of the chromatogram obtained with the
test solution, other faint blue zones may be seen.
TESTS
Relative density (2.2.5)
1.14 to l.l7.
Saponification value (2.5.6)
230 to 255, determined on the residue obtained in the assay.
Artificial balsams
Shake 0.20 g with 6 mL of light petroleum RI. The light
petroleum solution is c1ear and colourless and the whole of
the insoluble parts of the balsam stick to the wall of the
test-tube.
Fatty oils
Shake 1 g with 3 mL of a 1000 gIL solution of ehloral
hydrate R . The resulting solution is as c1ear as the 1000 gIL
solution of ehloral hydrate R .
Turpentine
Evaporate 10 dryness 4 mL of the solution obtained in the
test for artificial balsams. The residue has no odour of
turpentine.
2014
ASSAY
To 2.50 g in a separating funnel add 7.5 mL of di/ute sodium
hydroxide solution R and 40 mL of peroxide-free ether R and
shake vigorously for 10 mino Separate the lower layer and
shake it with 3 quantities, each of 15 mL, of peroxide-free
ether R . Combine the ether layers, dry over 10 g of anhydrous
sodium sulfate R and filter. Wash the sodium sulfate with
2 quantities, each of 10 mL, of peroxide-free ether R. Combine
the ether layers and evaporate to dryness. Dry the residue
(esters) at 100-105 oC for 30 min and weigh.
STORAGE
Protected from light.
____________________________________________ ~ Ew
Phyllanthus Emblica Pericarp
DEFINITION
Phyllanthus Emblica Pericarp is the pericarp of dried mature
fruits of Phyllanthus embliea L. (syn. Emblica offieinalis
Gaertn.)
It contains not les s than 6.0 % tannins, expressed as
pyrogallol (C 6H 60 3 . M r 126.1), calculated with reference 10
the dried drug.
IDENTIFICATION
A. Irregular pieces of the pericarp showing a dark brownish
to black outer surface, much wrinkled and grooved with
occasional greyish-white patches; the underlying brown
mesocarp is about 2 to 3 mm wide surrounding a thin, paler
brown endocarp. Infrequently whole seeds, or portions of the
creamish white testa may be present, attached to the
endocarp; whole seeds are subspherical, about 1 cm in
diameter and the testa is marked with 6 equidistant
longitudinal ridges. Occasional whole fruits may also be
present; they are spherical 10 ovoid or irregular, about 2 cm
in diameter and show a depression at one end.
B. The powder is yellow-green or pale brown. It shows small
polygonal cells from the exocarp covered with a thick cutic1e,
numerous mesocarp, sorne filled with amorphous grey
birefractive masses, sorne with mucilage, and sorne with
calcium oxalate crystals in the form of needles, large prism or
microsphenoids; thick-walled, pitted sc1ereids from the
mesocarp found singly and usually surrounded by
parenchyma, and fragments of the endocarp consisting of a
thick layer of pitted fibres and sc1ereids.
C. Carry out the method for thin-layer ehromatography,
Appendix JlI A, using the following solutions.
(1) Add 25 mL of ethanol to l.0 g of the powdered herbal
drug, sonicate for 30 minutes and filter.
(2) 0.1 % w/v of ellagie acid and 0.1 % w/v of gallie acid in
methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating siliea gel 60 F 254 or high-performance
si/iea gel 60 F 254 (Merck silica gel 60 F 254 HPTLC plates are
suitable).
(b) Use the mobile phase as described below.
(c) Apply 10 ¡.tL [or 2 ¡.tL] of each solution, as bands.
(d) Develop the plate 10 15 cm [or 7 cm].
(e) After removal of the plate, dry in air, spray with a 5% w/v
solution of iron(m) ehlonde in ethanol and examine in daylight.
 2014 Dwarf Pine Oil IV-317

MOBILE PHASE IDENTIFICATION

1.5 volumes of toluene, 3 volumes of acetic acid, 4 volumes of
formic acid and 30 volumes of ethyl acetate.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated bands.
CONFIRMA TION
The chromatogram obtained with solution (1) shows a dark
blue band with an Rf value of 0.6 corresponding in colour
and position to the band obtained with ellagic acid in
solution (2) and a dark blue band with an Rf value of
approximately 0.9 corresponding in position to the dark blue
band obtained with gallic acid in solution (2). Two or three
separated blue or faint blue bands with Rf values of between
0.2 and 0.3 are also presento Other bands may be present in
solution (1) .
Top of the plate
Dark blue band Gallic acid: a dark blue band
Dark blue band Ellagic acid: a dark blue band
First identijication B.
Second identijication A.
A. Thin-layer chromatography (2.2.27) .
Test solution Dilute 1 mL of the substance to be examined
to 10 mL with toluene R.
Reference solution Dissolve 10 mg of borneol R and 10 j.1L of
bornyl acetate R in toluene R and dilute to 10 mL with the .
same solvento
Plate TLC silica gel plate R (5-40 j.1m) [or TLC silica gel
plate R (2-10 j.1m)].
Mobüe phase ethyl acetate R, toluene R (5:95 V/V).
Application 10 j.1L [or 2 j.1L], as bands.
Development Over a path of 15 cm [or 6 cm] .
Drying In air.
Detection Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 min; examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate

A pink zone

Blue/faint blue band --- ---

Blue/faint blue band Bornyl acetate: a brown or A brown or greyish-brown zone
greyish-brown zone (bornyl acetatel
A pink zone
Solution (1) Solution (2)
--- ---
Borneol: a brown or greyish-brown A cluster of violet zones
TESTS zone
Ethanol-soluble extractive
Not less than 15.0%, Appendix XI Bl
Reference solution Test solution
Foreign matter
Not more than 5% of foreign matter including seed material,
Appendix XI D . B. Examine the chromatograms obtained in the test for
Water-soluble extractive chromatographic pro file.
Not less than 50%, Appendix XI B2. Results The characteristic peaks in the chromatogram
Ash obtained with the test solution are similar in retention time to
Not more than 7.0%, Appendix XI J, method Il. those in the chromatogram obtained with reference
solution (a) .
Loss on drying
Not more than 10.0%, Appendix IX D. Use 1 g. TESTS
Relative density (2.2.5)
ASSAY
0.857 to 0.868.
Carry out the determination of tannins in herbal drngs,
Appendix XI M. Use 1.0 g of powdered drug. Refractive index (2.2.6)
1.474 to 1.480.
Optical rotation (2.2.7)

*****
- 7° to - 15°.
Dwarf Pine Oil
** ** Acid value (2.5.1)
(Ph Bu/" monograph 2377) *** Maximum 1.0.
____________________________________________
~E~

Peroxide value (2.5.5)

DEFINITION Maximum 20.
Essential oil obtained by steam distillation of the fresh leaves Fatty oils and resinified oils (2.8.7)
and twigs of Pinus mugo Turra. A suitable antioxidant may be It complies with the test.
added. Chromatographic pro file
CHARACTERS Gas chromatography (2.2.28): use the normalisation
Appearance procedure.
Clear, colourless or pale yellow liquido Test solution Dilute 200 j.1L of the substance to be examined
to 10.0 mL with heptane R .
IV- 318 Pine Silvestris Oil 2014

Reference solution (a) Dilute 30 [.lL of rx-pinene R, 5 mg of

camphene R, 10 [.lL of {J-pinene R, 20 [.lL of car-3-ene R, 5 [.lL
Pine Silvestris Oil
of {J-myrcene R, 10 [.lL of limonene R, 5 [.lL of p-cymene R, (Ph Eur monograph 1842)
10 [!L of terpinolene R, 5 [.lL of bornyl acetate R and 5 J.lL of ~E~ _ __ _ __ _ __ _ _ __ _ _ _ __ _ __ _
{J-caryophyllene R in heptane R and dilute to 5 mL with the
same solvento DEFINITION
Reference solurion (b) Dissolve 5 mg of camphene R in Essential oil obtained by steam distillation of the fresh leaves
heptane R and dilute to 50.0 mL with the same solvento and branches of Pinus sylvestris L. A suitable antioxidant may
Dilute 1.0 mL of this solution to 10.0 mL with heptane R. be added.
Column: CHARACTERS
- material: fused silica; Appearance
- size: 1 = 60 m, 0 = 0.25 mm; Clear, colourless or pale yellow liquido
- stationary phase: macrogol 20 000 R (film thickness Characteristic odour.
0.25 [.lm) .
IDENTIFICATION
Carrier gas helium for chromatography R.
First identification B.
Flow rate 1.5 mlJmin.
Second identification A.
Split ratio 1:50.
A. Thin-layer chromatography (2.2.27).
Temperature:
Test solurion Dilute 1 mL of the substance to be examined
Time Temperature to 10 mL with toluene R.
(mio) (oC)
Reference solution Dissolve 10 mg of borneol R and 10 [!L of
Column 0·10 65
bornyl acetate R in toluene R and dilute to 10 mL with the
10·41 65 ~ 220 same solvento
41·50 220 Plate TLC silica gel plate R (5-40 [.lm) [or TLC silica gel
Injedion port 220
plate R (2-10 [.lm)].
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Detector 250
Application 10 [.lL [or 2 J.lL] as bands.
Detection Flame ionisation. Development Over a path of 15 cm [or 6 cm].
1njection 1 [.lL. Drying In air.
Elution order Order indicated in the composition of Detection Treat with anisaldehyde solution R, heat at
reference solution (a); record the retention times of these 100-105 oC for 5-10 min and examine in daylight.
substances. Results See below the sequence of the zones present in the
System suitability Reference solution (a): chromatograms obtained with the reference solution and the
- resolution: minimum 1.5 between the peaks due to test solution. Furthermore other faint zones may be present
car-3-ene and ~-myrcene. in the chromatogram obtained with the test solution.
Identification of components Using the retention times
determined from the chromatogram obtained with reference
Top of the plate
solution (a), locate the components of reference solution (a)
in the chromatogram obtained with the test solution; A pink zone (hydrocarbons)
the peak due to ~-phellandrene is eluted after the peak due --- ---
to limonene with a relative retention of about 1.03 with
reference to limonene. Bornyl aceta te: a brown or A brown or grey-brown zone
grey-brown zone (bornyl acetate)
Determine the percentage content of each of these A pink zone
components. The limits are within the following ranges:
- rx-pinene: 10.0 per cent to 30.0 per cent; --- ---

- camphene: maximurn 2.0 per cent; Borneol: a brown or grey-brown A cluster of violet zanes
- fJ-pinene: 3.0 per cent to 14.0 per cent; zone
- car-3-ene: 10.0 per cent to 20.0 per cent;
- fJ-myrcene: 3.0 per cent to 12.0 per cent; Reference solutioo Test solution
- limonene: 8.0 per cent to 14.0 per cent;
- {J-phellandrene: 10.0 per cent to 19.0 per cent;
- p-cymene: maximum 2.5 per cent; B. Examine the chromatograms obtained in the test for
- terpinolene: maximum 8.0 per cent; chromatographic profile.
- bornyl acetate: 0.5 per cent to 5.0 per cent; Results The characteristic peaks in the chromatogram
- fJ-caryophyllene: 0.5 per cent to 5.0 per cent; obtained with the test solution are similar in retention time to
- disregard limit: the area of the principal peak in the those in the chromatogram obtained with reference
chromatogram obtained with reference solution (b) solution (a).
(0.05 per cent).
TESTS
STORAGE
Relative density (2.2.5)
In an inert container and at a temperature not exceeding
0.855 to 0.875.
25 oc.
_ __ _ __ __ _ _ __ __ _ __ _ _ __ _ PhEur
Refractive index (2.2.6)
1.465 to 1.480.
2014 Plantain IV-319

Optical rotation (2.2.7) -- {J-caryophyllene: 1.0 per cent to 6.0 per cent,
- 9° to - 30°. -- disregard limi!: the area of the principal peak in the
Acid value (2.5.1) chromatogram obtained with reference solution (b).
Maximum 1.0. STORAGE
Peroxide value (2.5.5) At a temperature not exceeding 25 oc.
_______________________
Maximum 20. ~E~

Fatty oils and resinified oils (2.8.7)

It complies with the test.
Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution The substance to be examined.
Reference solution (a) Dissolve 30 ~L of a-pinene R, 10 mg
of camphene R, 20 ~L of {J-pinene R, lO ~L of car-3-ene R,
1O ~L of {J-myrcene R, 20 ~L of limonene R, 1O ~L of
p-cymene R, 1O ~L of terpino lene R, 1O ~L of bornyl aceta te R
and 1O ~L of {J-caryophyllene R in 1 mL of heptane R.
Reference solution (b) Dissolve 10 mg of camphene R in
heptane R and dilute to 2 mL with the same solvent. Dilute
0.1 mL of the solution to 1 mL with heptane R.
Column:
-- material: fused silica,
-- size: 1 = 60 m, 0 = 0.22 mm,
-- stationary phase: macrogol 20 000 R (0.2 ~lm).
Carrier gas helium for chromatography R.
Flow rate 1.5 mUmin.
Split ratio 1: 1OO.
Temperature:
Time Temperature
(min) (OC)
Column 0-10 65
10 - 41 65 ~ 220
41 - 50 220
Injection port 220
Detector 250
Detection Flame ionisation.
Irljection 0.2 ~L.
Elution order Order indicated in the preparation of reference
solution (a). Record the retention times of these substances.
System suitability Reference solution (a) :
-- resolution: minimum 1.5 between the peaks due to car-3-
ene and ~-myrcene.
Identijication of components Using the retention times
determined from the chromatogram obtained with reference
solution (a), locate the components of reference solution (a)
in the chromatogram obtained with the test solution. The
peak due to ~-phellandrene is eluted after the peak due to
limonene with a relative retention of about 1.03 with
reference to limonene.
Determine the percentage content of these components.
The limits are within the following ranges:
-- a-pinene: 32.0 per cent to 60.0 per cent,
-- camphene: 0.5 per cent to 2.0 per cent,
-- {J-pinene: 5.0 per cent to 22.0 per cent,
-- car-3-ene: 6.0 per cent to 18.0 per cent,
-- {J-myrcene: l.5 per cent to 10.0 per cent,
-- limonene: 7. O per cent to 12.O per cent,
-- {J-phellandrene: maximum 2.5 per cent,
-- p-cymene: maximum 2.0 per cent,
-- terpinolene: maximum 4.0 per cent,
-- bornyl acetate: 1.0 per cent to 4.0 per cent,
Plantain ****
***
*
*** *
(Ribwort Plantain, Ph Eur monograph 1884)
~E~ _ _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ ___
DEFINITION
Whole or fragmented, dried leaf and scape of Plamago
lanceolata L. s.l.
Content
Minimum 1.5 per cent of total ortho-dihydroxycinnamic acid
derivatives expressed as acteoside (C29H3601 5; M r 624.6)
(dried drug).
IDENTIFICATION
A. The leaf is up to 30 cm long and 4 cm wide, yellowish-
green to brownish-green, with a prominent, whitish-green,
almost parallel venation on the abaxial surface. It consists of
a lanceolate lamina narrowing at the base into a channelled
petiole. The margin is indistinctly dentate and often
undulate. It has 3, 5 or 7 primary veins, nearly equal in
length and running almost parallel. Hairs may be almost
absent, sparsely scattered or sometimes abundant, especially
on the lower surface and over the veins. The scape is
brownish-green, longer than the leaves, 3-4 mm in diameter
and is deeply grooved longitudinally, with 5-7 conspicuous
ribs. The surface is usually covered with fine hairs.
B. Microscopic examination (2.8.23). The powder is
yellowish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1884.-1): fragments of epidermis,
composed of cells with irregularly sinuous anticlinal walls, the
fragments of the upper epidermis of the lamina in surface
view [H) and in transverse section [D) are accompanied by
palisade parenchyma [Da, Ha], and those of the lower
epidermis in surface view [G) show sto mata (2.8.3) mostly of
the diacytic type [Ga] and sometimes of the anomocytic type
[Gb]; the multicellular, uniseriate, conical covering trichomes
are highly characteristic, whole [C] or mostly fragmented [A],
with a basal cell larger than the other epidermal cells
followed by a short cell supporting 2 or more elongated cells
with the lumen narrow and variable, occluded at intervals
corresponding to slight swellings in the trichome and giving a
jointed appearance, the terminal cell has an acute apex and a
filiform lumen; the glandular trichomes have a unicellular,
cylindrical stalk and a multicellular, elongated, conical head
consisting of several rows of small cells and a single terminal
cell [B, Gc]; dense groups of lignified fibro-vascular tissue
with narrow, spirally and annularly thickened vessels and
slender, moderately thickened fibres [F]; fragments of the
scape [E) with cells with thickened walls and a coarsely
ridged cuticle, sto mata [Ec] , multicellular, uniseriate covering
trichomes [Eb] and glandular trichomes [Ea] of the type
previously described.
C. Examine the chromatograms obtained in the test for
Digitalis lanata leaves.
Results ASee below the sequence of zones present in the
chromatogram obtained with the reference solution and the
 IV-320 Plantain 2014

Development Over a path of 8 cm; heat immediately after

B
development at about 120 oC for 5-10 mino
Detection A Examine in daylight.

A Detection B Examine in ultraviolet light at 365 nm.

Results B The chromatogram obtained with the test solution
Eb shows no bright blue ftuorescent zone just below the reddish-
Da brown ftuorescent zone corresponding lO aucubin in the
chromalOgram obtained with the reference solution.
Foreign matter (2.8.2)
Maximum 5 per cent of leaves of different colour and
maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 14.0 per cent.
ASSAY
Stock solution In a ftask, place 1.000 g of the powdered
herbal drug (355) (2.9.12) and add 90 mL of ethanol
(50 per cent V/Vj R. Boil in a water-bath under a reftux
condenser for 30 mino Allow to cool and filter into a 100 mL
volumetric ftask. Rinse the ftask and the filter with 10 mL of
~

40 ~m ethanol (50 per cent V/Vj R. Combine the filtrate and the
rinsings and dilute to 100.0 mL with ethanol (50 per cent
V/Vj R.
Test solution To a 10 mL volumetric ftask add, mixing after
each addition, 1.0 mL of the stock solution, 2 mL of 0.5 M
Figure 1884.-1. - Illustration for identification test B of
hydrochloric acid, 2 mL of a solution prepared by dissolving
powdered herbal drug of ribwort plantain
10 g of sodium nitrite R and 10 g of sodium molybdate R in
100 mL of water R, and 2 mL of dilute sodium hydroxide
solution R . Dilute lO 10.0 mL with water R.
test solution. Furthermore, other zones may be present in the
Immediately measure the absorbance (2.2.25) of the test
chromatogram obtained with the test solution.
solution at 525 nm using as compensation liquid a solution
prepared as follows: to a 10 mL volumetric ftask add 1.0 mL
Top of the plate of the stock solution, 2 mL of 0.5 M hydrochloric acid and
2 mL of dilute sodium hydroxide solution R, and dilute to
--- ---
10.0 mL with water R.
Acteoside: a yelJow zone A yelJow zone (acteoside) Calculate the percentage content of total ortho-
dihydroxycinnamic acid derivatives, expressed as acteoside,
using the following expression:
--- ---
Aucubin: a blue zone A blue zone (aucubin) A x 1000
Reference solution Test solution 185 x m

i.e. taking the specific absorbance lO be 185 for acteoside at

TESTS 525 nm.
Digitalis lanata leaves A absorbance of the test solution at 525 nm;
Thin-Iayer chromalOgraphy (2.2.27). m = mass of the substance lO be examined, in grams.
Solvent mixture water R, methanol R (30:70 VIV). ____________________________________________ ~E~

Test solution Use a freshly prepared solution. To 1 g of the

powdered herbal drug (355) (2.9.12) in a 25 mL ftask, add
10 mL of the solvent mixture and shake for 30 mino Filter,
rinse the ftask and the filter with 2 quantities, each of 5 mL,
of the solvent mixture. Dilute lO 25 mL with the solvent
mixture.
Reference solution Dissolve 1 mg of acteoside R and 1 mg of
aucubin R in 1 mL of the solvent mixture.
Plate TLC silica gel F 254 plate R.
Mobile phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (11: 11:27: 100 VIVIVIV).
Application 10 J.lL as bands.
2014
Podophyllum Resin
Podophyllin
Action and use
Used in treatment ofwarts.
Preparation
Compound Podophyllin Paint
DEFINITION
Podophyllum Resin is the resin obtained from rhizomes and
roots of Podophy llum hexandrum Royle (P. emodi Wall. ).
It contains not less than 50.0% of total aryltetralin lignans,
calculated as podophyllotoxin.
CHARACTERISTICS
An amorphous powder varying in colour from light brown to
greenish yellow, or brownish grey mas ses; odour,
characteristic; caustic.
Partly soluble in hot water, from which it is precipitated on
cooling, in ehloroform, in ether and in 5M ammonia.
IDENTIFICATION
Carry out the method for thin-layer ehromatography,
Appendix III A, using the following solutions in methanol.
(1) 1% w/v of the substance being examined.
(2) 0.5 % w/v of podophyllotoxin.
(3) 0.1 % w/v of phenazone.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating siliea gel GF254 •
(b) Use the mobile phase as described below.
(c) Apply as bands 10 ~L of each solution.
(d) Develop the plate to 10 cm.
(e) After removal of the plate, allow it to dry in air and
examine under ultraviolet light (254 nm). Spray the plate with
methanolie sulfurie acid (50%) and heat at 130 0 for
10 minutes .
MOBILE PHASE
1 volume of methanol and 25 volumes of ehloroform.
CONFIRMATION
When viewed under ultraviolet light (254 nm), the
chromatogram obtained with solution (1) exhibits quenching
zones corresponding in position to the principal quenching
zones in the chromatograms obtained with solutions (2) and
(3). Other quenching zones may be presento
When viewed after spraying, the chromatogram obtained
with solution (1) exhibits a purplish zone (podophyllotoxin)
corresponding in position and colour to the principal zone in
solution (2) and a purplish zone
(4 '-demethylpodophyl1otoxin) corresponding in position to
the quenching zone found in solution (3). Other coloured
zones may be presento
TESTS
Matter insoluble in ethanol (96%)
Shake 1 g, finely powdered, with 20 mL of ethanol (96%) for
5 minutes, filter through a sintered-glass crucib1e (ISO 4793,
porosity grade 2, is suitable), wash the filter with ethanol
(96%) and dry at 1050. The residue weighs not more than
25 mg.
Matter insoluble in 5m ammonia
Shake 0.5 g, finely powdered, with 30 mL of 5M ammonia for
30 minutes at about 20 0; filter through a sintered-glass
crucible (ISO 4793, porosity grade 2, is suitable) and wash
the fiask and filter with 30 mL of water, the time taken for
Podophyllin Paint IV- 321
filtering and washing being not more than 10 minutes.
Dry the filter and residue to constant weight at 1050.
The residue weighs not les s than 0.18 g and not more than
0.30 g.
Loss on drying
When dried to constant weight at lOS ", loses not more than
5.0% of its weight. Use 1 g.
SuIfated ash
Not more than 1.0%, Appendix IX A.
ASSAY
Dissolve 0.5 g in sufficient ethanol (96%) to produce
100 mL. To 10 mL of this solution in a separating funnel
add 190 mL of water and extract with six 30-mL quantities
of diehloromethane. Combine the dichloromethane layers,
extract with 10 mL of O.2M sodium hydroxide followed by five
10-mL quantities of water and wash each of the six aqueous
layers separately with the same 20-mL quantity of
diehloromethane. Combine the dichloromethane solutions,
filter through absorbent cotton and evaporate the filtrate to
dryness . Dissolve the residue in sufficient ethanol (96%) to
produce 100 mL, dilute 10 mL of this solution to 50 mL
with ethanol (96%) and measure the absorbanee of the
resulting solution at the maximum at 292 nm,
Appendix II B. Calculate the content of total aryltetralin
lignans expressed as podophyllotoxin, taking 105.4 as the
value of A( l %, 1 cm) at the maximum at 292 nm.
STORAGE
Podophyl1um Resin should be protected from light.
On exposure to light, or 10 temperatures aboye 25 0, it
becomes darker in colour.
LABELLING
The label states the botanical source .
Compound Podophyllin Paint
Compound Podophyllin Cutaneous Solution
DEFINITION
Compound Podophyllin Paint is a eutaneous solution.
Podophyl1um Resin 150 g
Compound Benzoin Tincture Sufficient to produce 1000 mL
In making the Compound Benzoin Tincture used 10 prepare
Compound Podophyllin Paint, the Ethanol (90 per cent)
may be replaced by Industrial Methylated Spirit 1 diluted so
as to be of equivalent ethanolic strength.
The paint eomplies with the requirements stated under Liquids for
Cutaneous Applieation and with the following requirements.
IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions in methanol.
(1) 7% v/v of the paint.
(2) 0.5 % w/v of podophyllotoxin.
(3) 0.1 % w/v of phenazone.
CHROMATOGRAPHIC CON DITIONS
(a) Use as the coating siliea gel GF254 •
(b) Use the mobile phase as described below.
(c) Apply 1O ~L of each solution.
(d) Develop the plate to 10 cm.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (254 nm) to locate the quenching zone due to
 IV-322 Red Poppy Petals 2014

phenazone in solution (3). Spray the plate with methanolic

sulfuric acid (50%) and heat at 130° for 10 minutes.
MOBILE PHASE A~
1 volume of methanol and 25 volumes of chlorofonn.
CONFIRMA TION
The chromatogram obtained with solution (1) exhibits a
purplish zone (podophyllotoxin) corresponding in position
and colour to the principal zone in solution (2) and a
purplish zone (4 ' -demethylpodophyllotoxin) corresponding in
position to the quenching zone found in solution (3). Other
coloured zones are present in the chromatogram obtained
with solution (1).

~
TESTS
Weight per mL
H
0.925 to 0.975 g, Appendix V G.
Total solids
27.0 to 33.0% w/v when determined by evaporating 1 mL to Figure 1881.-1.- Illustration for identification test B of
dryness on a water bath and drying the residue at 1050 for powdered herbal drug of red poppy petals
4 hours.

lThe law and the statuwry regulations governing che use oi Industrial Detection Examine in daylight.
Methylated Spiric must be observed.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Red Poppy Petals ***
** ***
*
Top oí the plate
(Ph Eur monograph 1881) ***
Ph E~ ____________________________________________ 2 yellow zones

DEFINITION Quinaldine red: an orange-red

zone
Dried, whole or fragmented petals of Papaver rhoeas L.
--- ---
IDENTIFICATION
A violet principal zone
A. The petal is dark red or dark violet-brown, very thin,
A violet zone
floppy, wrinkled, often crumpled into a ball and velvety to
Ayellow zone
the touch. It is broadly ovate with an entire margin, about
Sulfan blue: a blue zone
6 cm long and 4-6 cm wide, narrowing at the base where
there is a black spot. The vascular bundles radiate from the --- ---
base and they anastomose in a continuous arc, all at the A compact group of violet zones
same short distance from the margino
Reference solution Test solution
E. Reduce to a powder (355) (2.9. 12). Examine under a
microscope using chloral hydrate solution R. The powder has
an intense reddish-pink colour and shows the following
TESTS
diagnostic characters (Figure 1881.-1) : fragments of
Foreign matter (2.8.2)
epidermis composed of elongated, sinuous-walled cells [E, D,
Maximum 2.0 per cent of capsules and maximum
G] with small, rounded, anomocytic stomata (2.8.3) [Ea];
1.0 per cent of other foreign matter.
numerous vascular bundles with spiral vessels [E] embedded
in the parenchyma; occasional fragments of the fibrous layer Loss on drying (2.2.32)
of the anthers [F]; rounded pollen grains, about 30 Jlm in Maximum 12.0 per cent, determined on 1.000 g of the
diameter, with 3 pores and a finely verrucose exine [A, C, powdered drug (355) (2.9. 12) by drying in an oven at
H] . 105 oC for 2 h.
C. Thin-Iayer chromatography (2.2.27). Total ash (2.4.16)
Test solution To 1.0 g of the powdered drug (355) (2. 9.12) Maximum 11.0 per cent.
add 10 mL of ethanol (60 per cent VIV) R. Stir for 15 mino Colouring intensity
Filter through a filter paper. Place 1.0 g of the powdered drug (355) (2. 9. 12) in a 250 mL
Reference solution Dissolve 1 mg of quinaldine red R and flask and add 100 mL of ethanol (30 per cent V /V) R. Allow
1 mg of sulfan blue R in 2 mL of methanol R. to macerate for 4 h with frequent stirring. Filter and discard
the first 10 mL. To 10.0 mL ofthe filtrate add 2 mL of
Plate TLC silica gel plate R.
hydrochloric acid R and dilute to 100.0 mL with ethanol
Mobile phase anhydrous fonnic acid R, water R, butanol R (30 per cent V /V) R. Allow to stand for 10 mino
(10 :12:40 VIV/V).
The absorbance (2.2.25) measured at 523 nm using ethanol
Application 10 JlL as bands. (30 per cent V/V) R as the compensation liquid is not less
Development Over a path of 10 cm. than 0.6.
Drying In airo ____________________________________________ ~E~
2014
Poria
(Ph Eur monograph 2475)
~Ew ____________________________________________
DEFINITION
Dried sderotium without skin of Wolfiporia extensa (Peck)
Ginns (syn. Poria cocos (Schw.) Wolf; Wolfiporia cocos (F.A.
Wolf) Ryvarden & Gilb.).
IDENTIFICATION
A. Square, rectangular or polyhedral pie ces, or slices, varying
in length and thickness; whitish with a pale brown hue, fiat
and smooth, square, rectangular or polyhedral pieces, with
no brown skin, difficult to break; slices easily broken, rough
fracture with granular or farinaceous texture.
B. Microscopic examination (2.8.23) . The powder is whitish
with a pale brown hue. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters: irregularly shaped and occasionally
branched colourless partides, which dissolve gradually in
chloral hydrate solution R. Examine under a microscope using
a 50 gIL solution of potassium hydroxide R . The powder
shows the following diagnostic characters: fragments of
hyphae, colourless, slender, slightly curved, som etimes with
septa, branched, 3-1 6 f.Lm in diameter.
C. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (250)
(2.9. 12) add a mixture of 2 mL of ethyl aceta te R and 3 mL
of methanol R. Sonicate for 10 min, centrifuge and use the
supernatant.
Reference solUlion Dissolve 10 mg of 4-aminobenzoic acid R,
10 mg of coumarin R and 10 mg of thymol R in 10 mL of
methanol R .
Plate TLC silica gel F 254 plate R (2- 10 f.Lm) .
Mobile phase glacial acetic acid R, 2-propanol R, cyclohexane R
(10:10 :80 VIV/V).
Application 5 f.LL as bands of 8 mm.
Developmenl Over a path of 6 cm.
Drying In air.
Detection Examine in ultraviolet light at 254 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint quenching zones may
be present in the chromatogram obtained with the test
solution between the zones due to 4-aminobenzoic acid and
coumarin in the chromatogram obtained with the reference
solution.
Top of the plate
-----
Thyrnol: a quenching zone
2 quench ing zones
-----
Cournarin: a quenching zone
4-Arninobenzoic acid: a quenching
zone
Reference solution Test solution
Primula Root IV-323
D . The herbal drug sticks to the pestle when moistened with
water R and pressed into a mortar.
E. To a small piece of the herbal drug add 1 drop of
iodinated potassium iodide solution R1. A deep red colour is
produced.
TESTS
F oreign matter (2.8.2)
Maximum 0.1 per cent of brown skins and roots of conifer
and maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 13.0 per cent, determined on l.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.1 6)
Maximum 1.0 per cent.
Water-soluble extractive
Minimum 1.5 per cent.
To 5.00 g ofthe powdered herbal drug (355) (2.9. 12) add
100 mL of boiling water R. Allow to stand for 10 min,
shaking occasionally. Allow to cool, dilute to 100.0 mL with
water R and filter. Evaporate 25.0 mL of the filtrate to
dryness on a water-bath. Dry the residue in an oven at
100-105 oc. The residue weighs a minimum of 18.75 mg.
____________________________________________ ~ Ew
Primula Root *****
** **
(Ph Eur monograph 1364) ***
~Ew ____________________________________________
DEFINITION
Whole or cut, dried rhizome and root of Primula veris L.
or Primula elatior HilI.
IDENTIFICATION
A. The coarsely toros e, greyish-brown rhizome is straight or
slightly curved, about 1-5 cm long and about 2-4 mm thick.
The rhizome crown often bears the remains of stems and
leaves. Attached to the rhizome are numerous brittle roots,
about 1 mm thick and usually 6-8 cm long. The root of P.
elatior is light brown or reddish-brown, that of P. veris light
yellow or yellowish-white. The fracture is smooth.
B. Microscopic examination (2.8.23). The powder is greyish-
brown. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1364.-1 ): fragments of parenchyma from
the bark of the root or the rhizome and from the m edulla of
the rhizome [G, H], consisting of rounded or ovoid cells with
irregularly thickened and pitted walls; brownish fragments
from the dermal tissue of the root showing absorbent hairs
--- [e]; yellow or brownish fragments of the epidermis of the
rhizome covered by a striated cutide, in surface view [A] , or
in transverse section [F] accompanied by parenchyma from
the bark [Fa); reticulate vessels [B] sometimes accompanied
---
m;
by spiral ves seis groups of large, strongly pitted,
yellowish-green sdereids from the medullary parenchyma of
the rhizome [E], which are characteristic of P. elatior.
Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows simple or
compound starch granules of various shapes and sizes [D] .
 IV-324 Psyl1ium Seed
Figure 1364.-1. - Illustration for identification test B of
powdered herbal drug of primula root
e. Thin-layer chromatography (2.2.27) as described in the
test for Vincetoxicum hirundinaria Medik. root with the
following modifications.
Detection Treat with anisaldehyde solution R, heat at
100-105 oC for 5-10 min and examine in dayligbt.
Results The main zone (aescin) in the chromatogram
obtained with the reference solution is bluish-violet and is
situated near the boundary between the lower and middle
thirds. The chromatogram obtained with the test solution
shows 1-2 strong dark violet zones a little below the zone due
to aescin in the chromatogram obtained with the reference
solution; further pale violet, yellowish or brownish-green
zones may be visible.
TESTS
Vincetoxicum hirundinaria Medik. root
Trun-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the powdered herbal drug (500)
(2.9.12) add 10 mL of ethanol (70 per cent V/V! R and heat
under a refiux condenser for 15 mino Cool and filter.
Reference solution Dissolve 10 mg of aescin R in 1.0 mL of
ethanol (70 per cent V /V! R .
Plate TLC silica gel F 254 plate R .
Mobzle phase glacial acetic acid R, water R, butanol R
(10:40:50 VIV/V); use the upper layer.
Application 20 ¡.iL as bands.
Development Over a path of 12 cm.
Drying In an oven at 100-105 0e.
Detection A Examine in ultraviolet light at 254 nm.
Results A The chromatograms obtained with the reference
solution and the test solution show a quenching zone (aescin)
2014
near the boundary between the lower and the middle thirds.
Mark this zone.
Detection B Examine in ultraviolet light at 365 nm.
Results B In the chromatogram obtained with the test
solution no zones of ligbt-blue or greenish fiuorescence occur
below the main zone due to aescin in the chromatogram
obtained with the reference solution.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 9.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
____________________________________________ nEw
Psyllium Seed *****
** **
(Ph Eur monograph 0858) ***
Ph Eur ___________________________________________
DEFINITION
Ripe, whole, dry seeds of Plantago afra L. (Plantago psyllium
L.) or Plantago indica L. (Plantago arenaria Waldstein and
Kitaibel).
CHARACTERS
Sweet taste.
IDENTIFICATION
P. afra seeds are light brown to very dark brown but never
black, smooth and sruny having an elliptical oblong shape .
They are 2-3 mm long and 0.8-1.0 mm wide, one end being
wider than the other. Towards the middle of the dorsal
surface there is a fairly marked transverse constriction of light
colour. On the ventral surface, there is a linear lighter-
coloured gro ove in the middle of wruch is a clear spot
corresponding to the hilum and bounded by swollen edges .
P. indica seeds are almost identical to the seeds of P. afra,
but a little less shiny; they are 2-3 mm long and have a
maximum diameter of 1.5 mm.
TESTS
Swelling index (2.8.4)
Minimum 10.
Foreign matter (2.8.2)
Maximum 1.0 per cent, determined on 10.0 g of the drug,
including greenish unripe seeds. Psyllium seed does not
contain seeds having a dark central spot on the groove
(Plantago lanceolata L. and P. major L.) or seeds with
brownish-grey or pinkish outer coats (P. ovata Forssk. and P.
sempervirens Crantz).
Loss on drying (2.2.32)
Maximum 14.0 per cent, determined on 1.000 g of drug by
drying in an oven at 105 oC for 2 h.
Total ash (2.4.16)
Maximum 4.0 per cent.
STORAGE
Store protected from moisture.
___________________________________________ PhEw
2014
Pygeum Bark
(Pygeum Africanum Bark, Ph Bur monograph 1886)
~Ew ____________________________________________
DEFINITION
Whole or cut, dried bark of the stems and branches of Prunus
africana (Rook f.) Kalkm. (syn. Pygeum africanum Rook f.) .
IDENTIFICATION
A. The dark brown to reddish-brown bark occurs in curved,
hard, irregular pieces. The outer surface has a wrinkled dark
reddish-brown cork with areas of adhering lichen.
The reddish-brown to dark brown i=er surface bears
longitudinal striations. It may also occur in rolled fragments
with a fibrous fracture.
B. Reduce to a powder (355) (2.9.12). The powder is
reddish-brown. Examine under a microscope using chloral
hydrate solution R. The powdered drug shows thick-walled
sclereids, solitary or in groups; calcium oxalate cluster
crystals of different size; numerous lignified fibres, thick-
walled and with narrow lumen, sorne of them solitary and
most in groups with forked ends; fragments of pigmented
polygonal cells of reddish-brown colour; fragments of cork.
Examine under a microscope using a 50 per cent V/V
solution of g1ycerol R, the powder shows sorne isolated small
starch grains that stain bluish-black against iodine solution R1.
C. Thin-Iayer chromatography (2.2.27).
Test solution Extract 15.0 g ofpowdered drug (250) (2.9. 12)
with methylene chloride R for 30 min in a continuous
extraction apparatus (Soxhlet type) . Filter. Evaporate the
solvent to dryness under reduced pressure. Dissolve the
residue in 1 mL of methylene chloride R.
Reference solution Dissolve 20 mg of f3-sitosterol R and 20 mg
of ursolic acid R in 10 mL of a mixture of equal volumes of
methanol R and methylene chloride R .
Plate TLC silica gel plate R .
Mobile phase methanol R, methylene chloride R (10:90 V/V).
Applicarion 10 )lL, as 1 cm bands.
Development Over a path of 15 cm.
Drying In airo
Detection Spray with vanillin reagent R. Reat the plate at
100-105 oC for 10 min and allow to cool; examine in
daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
A violet zone
Several weak violet, blue or grey
zones
--- ---
f}-Sitosterol: a violet zone A violet zone (f}-sitosterol)
Ursolic acid: a blue zone A blue zone (ursolic acid)
Several weak violet, blue or grey
zones
--- -----
A violet zone (f}-sitosterol
!!lucosidel
Reference solution Test solutiou
Quillaia IV-325
TESTS
Foreign matter (2.8.2)
Maximum 3.0 per cent.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 10.0 per cent.
Extractab1e matter
Minimum 0.5 per cent.
Extract 20.0 g of the powdered drug (250) (2.9.12) with
methylene chloride R for 4 h in a continuous extraction
apparatus (Soxhlet type) . Evaporate the solution to dryness
on a water-bath in vacuo and then dry the residue at 80 uC
for 2 h . The residue weighs a minimum of 0.10 g.
____________________________________________
~Ew
Quillaia
Quillaia Bark
Preparation
Quillaia Liquid Extract
When Powdered Quillaia is prescribed or demanded, material
complying with the appropriate requirements below shall be
dispensed or supplied.
DEFINITION
Quillaia is the dried inner part of the bark of Quillaja
saponaria Molina and of other species of Quillaja.
IDENTIFICATION
A. Pieces fiat, up to about 1 metre long, lOto 20 cm broad
and 3 to 10 mm, usually 6 mm, thick. Outer surface
brownish white or pale reddish brown, longitudinally striated
or coarsely reticulated, with occasional blackish brown
patches of adherent outer bark; inner surface yellowish white,
smooth and very hard; fracture splintery and laminated, the
broken surface showing numerous large prisms of calcium
oxalate as glistening points. Smoothed transversely cut
surface appearing chequered, with delicate radial lines
representing medullary rays and tangential lines formed by
altemating tangential bands of fibrous and non-fibrous
phloem.
B. Outer bark, when present, consisting of reddish brown
cork cells alternating with bands of brown parenchyma
containing numerous groups of phloem fibres and large
prisms of calcium oxalate. Inner bark consisting of altemating
bands of tortuous fibres, irregularly enlarged at intervals,
about 500 to 1000 )lm long and 20 to 50 )lm wide and of
sieve tissue mixed with parenchyma. Medullary rays mostly
three to four, but sometimes up to six cells wide, with
occasional pitted, sub rectangular sclereids adjacent to the
bundle s of phloem fibres. Starch granules 5 to 20 ¡.tm,
usually about 10 )lm, in diameter and prisms of calcium
oxalate usually 50 to 170 ~lm long and up to 30 )lm wide
present in the parenchymatous cells .
TESTS
Extractive soluble in ethanol (45%)
Not less than 22.0%, Appendix XI Bl.
Acid-insoluble ash
Not more than 1.0%, Appendix XI K.
Foreign matter
Complies with the test for foreign matter, Appendix XI D.
 IV-326 Quillaia Bark 2014

o
Quillaia Bark *****
* *
(Ph Eur monograph 1843) *****
~E~ ____________________________________________

DEFINITION
Whole or fragmented, dried bark, with the cork and
underlying parenchyma removed, of Quillaja saponaria
Molina s.l.
Content
Minimum 6.5 per cent of triterpene gIycosides, expressed as
quillaia saponin III (C104H1680SS; M r 2298) (dried drug).
IDENTIFICATION
A. Large, flat pieces ofvariable length and width, 3-10 mm
thick, or smaller, splintered pieces. The outer surface is
brownish-white or pale reddish-brown, longitudinally striated
or coarsely reticulated, with occasional blackish-brown
patches of incompletely removed outer bark. The iuner
surface is yellowish-white and smooth. The fracture is
splintery and laminated, the surface often glistening due to
the presence of numerous large prisms of calcium oxalate.
B. Microscopic examination (2.8.23). The powder is pale
pinkish-yellow. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 1843.-1): abundant phloem fibres [E, F],
up to 1 mm long, isolated or, more usually, in groups, each 25 iJm
1-----<
fibre irregular in outline with lignified walls of varying
thickness and an uneven lumen; numerous, multiseriate
medullary rays, spindle-shaped in tangential section [Ca, Fb],
accompanied by either phloem fibres [Fa] or phloem Figure 1843.-1. - Illusfration for identification test B of
parenchyma [Cb]; very numerous prisms of calcium oxalate,
powdered herbal drug of quillaia bark
up to 200 ¡.tm long, free, whole or, more usually, fragmented
[A] or included in phloem parenchyma cells [Ce, Cd];
occasional sclereids of 2 types: the 1sr type is sub-rectangular Top of the pIate
with pitted, slightly thickened walls, isolated [G] or included --- - - -
in phloem parenchyma cells [H], while the 2nd type has an
Quillaia saponins: 3 or more green 3 or more green or brown zones
irregularly shaped outline and very thick walls UJ, sometimes or brown zones (quillaia saponins)
adjacent to the bundles of phloem fibres; occasional dark A blue zone
brown or reddish-brown fragments of cork [D] . Examine
under a microscope using a 50 per cent V/V solution of Sucrose : a brown or bIue zone A brown or blue zone (sucrose)
glycerol R. The powder shows numerous, small (5-20 ¡.tm), --- - --
mainly simple, spherical starch granules, either scattered or as
compacted mas ses in parenchyma cells [B].
C. Thin-Iayer chromatography (2.2.27) . Reference soIution Test soIution
Test solution To 1.0 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R and 5 mL of water R.
Sonicate for 10 min and filter. TESTS
Reference solution Dissolve 10 mg of purified quillaia Loss on drying (2.2.32)
saponins R and 2 mg of sucrose R in 1 mL of water R and mix Maximum 10.0 per cent, determined on 1.000 g of the
with 1 mL of methanol R. powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Plate TLC silica gel plate R (2-10 ¡.tm).
Mobile phase anhydrous acetic acid R, ethyl acetate R, water R,
Total ash (2.4.16)
propanol R (1.5:30:30:40 VIVIV/V) . Maximum 10.0 per cent.
Application 5 ¡.tL as bands of 6 mm. Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
Development Over a path of 6 cm.
Drying In hot airo ASSAY
Liquid chromatography (2.2.29).
Detection Treat with a 10 per cent V/V solution of sulfuric
acid R in methanol R; heat at 120 oC for 5 min and examine Test solutwn Introduce 0.500 g of the powdered herbal drug
in daylight. (355) (2.9.12) into a round-bottomed ftask, add 20 mL of a
Results See below the sequence of zones present in the
20 gIL solution of potassium hydroxide R and heat under a
chromatograms obtained with the reference solution and the reftux condenser in a water-bath for 2 h. After cooling, add
test solution. Furthermore, other faint zones may be present 2 mL of phosphoric acid R and filter through a plug of
in the chromatogram obtained with the test solution. absorbent cotton. Add the absorbent cotton to the residue,
add 25 mL of ethanol (96 per cent) R and shake thoroughly.
2014 Restharrow Root IV-327

Filter. Combine the filtrates and dilute to 50.0 mL with

water R . Filter through a membrane filter (nominal pore size
Quillaia Liquid Extract
0.45 ~m). DEFINITION
Reference solution (a) Dissolve 12.0 mg of quillaia saponinfor Quillaia Liquid Extract is prepared by extracting Quillaia
assay CRS (containing monoammonium glycyrrhizate) in a with Ethanol (45 per cent).
mixture of equal volumes of ethanol (96 per cent) R and a Extemporaneous preparation
10 gIL solution of phosphoric acid R, and dilute to 50.0 mL The following formula and directions apply.
with the same mixture of solvents. Quillaia, in moderate/y fine powder 1000 g
Reference solution (b) Introduce 12 mg of purified quillaia Ethanol (45 per cent) A sufficient quantity
saponins HRS into a 50 mL round-bottomed fiask, add Exhaust the Quillaia in moderate/y fine powder with Ethanol
20 mL of a 20 gIL solution of potassium hydroxide R and heat (45 per cent) by percolation, Appendix XI F, and reserve the
under a refiux condenser in a water-bath for 2 h . After first 850 mL of percolate. Evaporate the subsequent
cooling, add 2 mL of phosphon'c acid R. Add 25 mL of percolate to the consistence of a soft extract, dissolve it in the
ethanol (96 per cent) R and shake thoroughly. Dilute to reserved portion and add sufficient Ethanol (45 per cent) to
50.0 mL with water R. Filter through a membrane filter produce 1000 roL. Allow to stand for not less than 24 hours;
(nominal pore size 0.45 ~m). filter.
Column: The extract complies with the requirements stated under Extracts
- size: 1 = 0.25 m, 0 = 4.6 mm; and with the following requirements.
- stationary phase: octadecylsi/yl silica gel for chromatography R
(5 ~m); TESTS
- temperature: 30 ± 2 oc. Ethanol content
28 to 34% v/v, Appendix VIII F, Method III.
Mobile phase acetonitrile R1, 1 giL solution of phosphoric
acid R (35:65 V/V) . Dry residue
Flow rate 1.0 mUmin. 20 to 30% w/v.
Detection Spectrophotometer at 210 nm. Relative density
1.02 to 1.06, Appendix V G .
Injection 50 ~L.
Run lime 1.2 times the retention time of glycyrrhizic acid.
Identification of peaks Use the chromatogram supplied with
purified quillaia saponins HRS and the chromatogram obtained
with reference solution (b) to identify the peaks due to
Quillaia Tincture
monodesm osidic quillaia saponins 1 and 3; a minor peak due DEFINITION
to monodesm osidic quillaia saponin 2 may be present Quillaia Liquid Extract 50mL
between the peaks due to monodesmosidic quillaia saponins Ethanol (45 per cent) Sufficient to produce 1000 mL
1 and 3. Extemporaneous preparation
Retention time Monodesmosidic quillaia saponin 1 = about The following directions apply.
9 min; monodesmosidic quillaia saponin 3 = about 10 min; Mix, allow to stand for not less than 12 hours and filter.
glycyrrhizic acid = about 13 mino
The tincture complies with the requirements for Tinctures stated
Calculate the percentage content of triterpene glycosides, under Extracts and with the following requirements.
expressed as quillaia saponin I1I, using the following
expression: TESTS
Ethanol content
Al x m2 x p x 2298 x 0.6 43 to 45% v/v, Appendix VIII F, Method III.
A 2 x mI x 957 Dry residue
1.0 to 1.5% w/v. Use 10 mL.
sum of the areas of the peaks due to Relative density
monodesmosidic quillaia saponins (1, 2 and 3) in 0 .940 to 0 .955, Appendix V G.
the chromatogram obtained with the test solution;
area of the peak due to glycyrrhizic acid derived
from monoarnmonium glycyrrhizate in the
chromatogram obtained with reference solution Restharrow Root *****
(a); * *
mas s of the herbal drug to be examined used to (Ph Eur monograph 1879) *****
prepare the test solution, in grams; ~E~ __________________________________________
mas s of quillaia saponin for assay CRS used to
prepare reference solution (a), in grams; DEFINITION
p percentage content of monoammonium Whole or cut, dried root of Ononis spinosa L.
glycyrrhizate in quillaia saponin for assay CRS; IDENTIFICATION
0.6 response factor between monoammonium A. The root is more or less fiattened, twisted and branched,
glycyrrhizate and monodesmosidic quillaia deeply wrinkled, brown and grooved longitudinally.
saponin 3; The transversely cut surface shows a thin bark and a xylem
2298 molecular mass of quillaia saponin IJI; cylinder with a conspicuously radiate structure. The fracture
957 molecular mass of monodesmosidic quillaia of the root is short and fibrous.
saponin 3. B. Reduce to a powder (355) (2. 9.12) . The powder is light
__________________________________________ PhE~

brown or brown. Examine under a microscope using chloral

 IV-328 Rhatany Root 2014

hydrate solution R. The powder shows the following diagnostic
characters: brown fragments of cork composed of thin-walled
polygonal cells; groups of thick-walled narrow fibres, often
accompanied by a parenchyma10us crystal sheath containing
prisms of ca1cium oxalate; fragments of ves seIs with
numerous small bordered pits; parenchyma10us cells with
single prisms of calcium oxalate. Examine under a
microscope using a mixture of equal volumes of glyeerol R
and water R. The powder shows numerous simple, round
Extractab1e matter
Minimum 15.0 per cent.
To 2.00 g of the powdered drug (250) (2.9.12) add a
mixture of 8 g of water R and 12 g of ethanol (96 per eene) R
and allow 10 macerate for 2 h, shaking frequently. Filter,
evaporate 5 g of the filtrate 10 dryness on a water-bath and
dry in an oven at 100-105 oC for 2 h. The residue weighs a
minimum of 75 mg.
____________________________________________ ~E~

starch granules, 5-1 O ~m in diameter.

C. Thin-Iayer chroma1Ography (2.2.27).
Test solution To l.0 g of the powdered drug (180) (2.9.12)
add 15 .0 mL ofmethanol R and boil under a reflux
Rhatany Root ***
condenser for 30 mino Cool and filter.
** **
Referenee solution Dissolve 10 mg of resoreinol R and 50 mg Krameria *****
of vanillin R in 10 mL of methanol R.
(Ph Bur monograph 0289)
Plate TLC siliea gel F 254 plate R .
Preparation
Mobile phase ethanol (96 per eent) R, methylene ehloride R, Rhatany Tincture
caluene R (10:45:45 VIV/V).
When Powdered Rhatany Root is prescribed or demanded,
Applieation 20 ~L, as bands. material complying with the requirements below with the
Development Over a path of 15 cm. exception of Identification test A and the test for Foreign
Drying In airo matter shall be dispensed or supplied.
____________________________________________
Deteetion A Examine in ultraviolet light at 254 nm and Ph E~

365 nm.
DEFINITION
Results ASee below the sequence of the zones present in Dried, usually fragmented, underground organs of Kramen·a
the chroma1Ograms obtained with the reference solution and triandra Ruiz and Pavon, known as Peruvian rhatany.
the test solution. Furthermore, other fluorescent zones are
Content
present in the middle third of the chromatogram obtained
Minimum 5.0 per cent of tannins, expressed as pyrogallol
with the test solution.
(C 6H 6 0 3 ; M r 126.1) (dried drug).
IDENTIFICATION
Top of the plate
A. The taproot is dark reddish-brown and has a thick, knotty
Vanillin: a zone visible at 254 nm crown. The secondary roots are the same colour and nearly
--- --- straight or somewhat 1Ortuous. The bark is rugged or scaly in
the older pieces and smooth with sharp, transverse fissures in
Resorcinol: a zone visible at An intense blue fluorescent zone the younger pieces; it separa tes readily from the wood.
254nm visible at 365 nm
The fracture is fibrous in the bark and splintery in the wood.
--- --- The smooth, transversely cut surface shows a dark brownish-
Reference solution Test solution red bark about one third of the radius in thickness; a dense,
pale reddish-brown and finely porous wood is present with
numerous fine medullary rays; the central heartwood is often
Deteetion B Spray with anisaldehyde solution R. Heat at darker.
100-105 oC for 5-10 mino Examine in daylight.
B. Reduce 10 a powder (355) (2.9.12). The powder is
Results B See below the sequence of the zones present in brownish-red. Examine under a microscope using ehloral
the chromatograms obtained with the reference solution and hydrate solution R. The powder shows the following diagnostic
the test solution. characters: cork cells containing dark brown phlobaphenes;
fragments of unlignified phloem fibres, usually 12-30 ~m in
diameter with moderately thick walls; phloem parenchyma
Top of the plate
cells in files containing prisms and microcrystals of ca1cium
Vanillin: a greyish-violet zone oxalate; fragments of vessels usually 20-60 ~m in diameter
A violet zone (onocol) with bordered pits; fragments of tracheids up to 20 ~m wide
with slit-shaped pits. Examine under a microscope using a
Resorcinol: a red zone
50 per cent V/V solution of glyeerol R. The powder shows
Reference solution Test solution rounded starch granules, simple or 2- 10 4-compound, an
individual granule measuring up to 30 ¡.tm in diameter and
some granules being found in the cells of the medullary rays
TESTS and in the parenchyma.
Loss on drying (2.2.32) C. Thin-Iayer chromatography (2.2.27).
Maximum 10.0 per cent, determined on l.000 g ofthe Test solution To 1.0 g of the powdered drug (355) (2.9.12)
powdered drug (355) (2.9.12) by drying in an oven at add 10 mL of a mixture of 3 volumes of water R and
105 oC for 2 h. 7 volumes of ethanol (96 per eene) R, shake for 10 min and
Total ash (2.4.16) filter. To the filtrate add 10 mL of light petroleum R and
Maximum 8.0 per cent. shake. Separate the light petroleum layer, add 2 g of
anhydrous sodium sulfate R, shake and filter. Evaporate the
 2014 Rhubarb IV-329

filtra te to dryness. Dissolve the residue in 0.5 mL of and filter. Evaporare the filtrate to dryness. Dissolve the
methanol R. residue in 0.5 mL of methylene chloride R.
Reference solution Dissolve 5.0 mg of Sudan red G R in Reference solution Dissolve 5 mg of thymol R and 10 mg of
10 mL of methanol R. dichlorophenolindophenol, sodium salt R in 10 mL of alcohol
Plate TLC silica gel plate R. (60 per cent V/ JI) R.
Mobile phase ethyl acetate R, toluene R (2:98 V/V). Plate TLC silica gel plate R.
Application 10 ~L, as bands. Mobile phase methylene chloride R.
Development Over a path of 15 cm. Application 10 ~L, as bands.
Drying In airo Development Over a path of 10 cm.
Detection Spray with a 5 gIL solution of fast blue B sale R . Dlying In airo
Allow to dry in air and spray with 0.1 M ethanolic sodium Detection Spray with a 5 gIL solution of fast blue B sale R;
hydroxide; examine in daylight. allow the plate to dry in air and spray with 0.1 M ethanolic
Results The chromatogram obtained with the reference sodium hydroxide; examine in daylight.
solution shows in the lower third a red zone due to Sudan Results See below the sequence of the zones present in the
red G. The ehromatogram obtained with the test solution chromatograms obtained with the referenee solution and the
shows a violet zone due to rhatany phenol 1 similar in test solution. Furthermore, other zones may be present in the
position to the zone of Sudan red G in the chromatogram chromatogram obtained with the test solution.
obtained with the referenee solution, below it the brownish
zone due to rhatany phenol JI and below this the bluish-grey Top of the plate
zone due to rhatany phenol III. Further zones may be
presento --- ---
TESTS Thyrnol: an orange brownish-yellow A viole! zone
zone
Foreign matter (2.8.2) --- ---
Maximum 2 per cent of foreign matter and maximum
A greenish·grey zone
5 per eent of fragments of erown or root exceeding 25 mm in
diameter. Root without bark may be present in very small A bluish·grey zone
quantities. A yellowish-brown zone
Loss on drying (2.2.32) Dichlorophenolindophenol: a A viole! zone
Maximum 12.0 per cent, determined on 1.000 g of the ~revish-bluezone
powdered drug (355) (2.9.12) by drying in an oven at Reference solution Test solution
105 oc.
Total ash (2.4.16)
TESTS
Maximum 5.5 per cent.
Ethano1 (2.9.10)
ASSAY 63 per cent V/V to 67 per eent V/V.
Carry out the determination of tannins in herbal drugs Methano1 and 2-propanol (2.9.11)
(2.8.14). Use 0.750 g ofpowdered drug (180) (2.9.12). Maximum 0.05 per eent V/V of methanol and maximum
_ _ _ _ _ _ _ _ __ _ __ _ _ _ _ __ _ __ Ph Eur
0.05 per cent V/V of 2-propanol.
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14) using 2.500 g ofthe tineture to be examined.
***
Rhatany Tincture
** **
_ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~E~

(Ph Eur monograph 1888) *****

~E~ _ _ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ __ __

DEFINITION
Tincture produced from Rhatany root (0289).
Content
Minimum 1.0 per cent m/m of tannins, expressed as
pyrogallol (C 6H 6 0 3; M r 126.1) .
PRODUCTION
The tincture is produced from 1 part of the drug and 5 parts
of ethanol (70 per cent V/V) by a suitable procedure.
CHARACTERS
Appearance
Reddish-brown liquido
IDENTIFICATION
Thin-layer chromatography (2.2.27).
Test solution To 5 mL of the tineture to be examined, add
10 mL of light petroleum R and shake. Separate the light
petroleum layer, add 2 g of anhydrous sodium sulfate R, shake
***
Rhubarb
** *
**** **
(Ph Eur monograph 0291)
Preparation
Compound Rhubarb Tincture
When Powdered Rhubarb is prescribed or demanded,
material complying with the requirements below with the
exception of Identifieation test A and the test for Foreign
matter shall be dispensed or supplied.
~E~ _ __ _ _ _ __ __ _ __ _ _ _ _ _ _ __ __
DEFINITION
Rhubarb eonsists of the whole or cut, dried underground
parts of Rheum palmatum L. or of Rheum officinale Baillon or
of hybrids of these two species or of a mixture.
The underground parts are ofren divided; the stem and most
of the bark with the rootlets are removed. It contains not les s
than 2.2 per cent of hydroxyanthraeene derivatives, expressed
IV-330 Rhubarb
as rhein (C 1s H s 0 6, M r 284.2), calculated with reference to
the dried drug.
CHARACTERS
Characteristic, aromatic odour.
IDENTIFICATION
A. The appearance is variable: disc-shaped pieces up to
10 cm in diameter and 1 cm 10 5 cm in thickness; cylindrical
pie ces; oval or planoconvex pieces. The surface has a pinkish
tinge and is usually covered with a layer of brownish-yellow
powder. It shows, especially after moistening, a reticulum of
darker lines. This structure causes the marbled appearance of
the drug. The fracture is granular. The transverse section of
the rhizome shows a narrow outer zone of radiating
brownish-red lines. These medullary rays are crossed
perpendicularly by a dark cambial ringo Inside this zone is a
ring of small star-spot forrnations of anomalous vascular
bundles. The root shows a more radiate structure.
B. Reduce to a powder (355) (2.9.12). The powder is orange
10 brownish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters: large calcium oxalate cluster crystals,
which may measure more than 100 Ilm, and their fragments;
reticulately thickened non-lignified vessels measuring up to
175 Ilm. Numerous groups of rounded or polygonal, thin-
walled parenchyma cells. Sclereids and fibres are absent.
Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows simple, rounded or
compound (2 10 4) starch granules with a star-shaped hilum.
C. Examine by thin-Iayer chromatography (2.2.27), using a
suitable silica gel as the coating substance.
Test solution Heat 50 mg of the powdered drug (180)
(2.9.12) in a water-bath for 15 min with a mixture of 1 rnL
of hydrochloric acid R and 30 mL of water R . Allow to cool
and shake the liquid with 25 mL of ether R. Dry the ether
layer over anhydrous sodium sulfate R and filter. Evaporate the
ether layer to dryness and dissolve the residue in 0.5 mL of
ether R .
Rejerence solution Dissolve 5 mg of emodin R in 5 mL of
ether R.
Apply separately to the plate as bands 20 IlL of each
solution. Develop over a path of 10 cm using a mixture of
1 volume of anhydrous jormic acid R, 25 volumes of ethyl
acetare R and 75 volumes of light petroleum R . Allow the plate
to dry in air and examine in ultraviolet light at 365 nm.
The chromatogram obtained with the reference solution
shows in its central pan a zone of orange fluorescence
(emodin). The chromatogram obtained with the test solution
shows: a zone due to emodin; aboye the emodin zone, two
zones of similar fluorescence (physcione and chrysophanol, in
order of increasing RF value); below the emodin zone, also
two zones of similar fluorescence (rhein and aloe-emodin, in
order of decreasing RF value). Spray with a 100 gIL solution
of potassium hydroxide R in methanol R. All the zones become
red to violet.
D . To about 50 mg ofthe powdered drug (180) (2.9.12) add
25 mL of dilute hydrochloric acid R and heat the mixture on a
water-bath for 15 mino Allow to cool, shake with 20 mL of
erher R and discard the aqueous layer. Shake the ether layer
with 10 mL of dilute ammonia R1 . The aqueous layer
becomes red to violet.
TESTS
Rheum rhaponticum
Examine by thin-Iayer chromatography (2.2.27), using silica
gel G R as the coating substance.
2014
Test solution To 0.2 g of the powdered drug (180) (2.9.12)
add 2 mL of merhanol R and boil for 5 min under a reflux
condenser. Allow to cool and filter. Use the filtrate as the test
solution.
Rejerence solution Dissolve 10 mg of rhaponticin R in 10 mL
of methanol R.
Apply separately to the plate, as bands not more than 20 mm
by 3 mm, 20 IlL of each solution. Develop over a path of
12 cm using a mixture of 20 volumes of methanol R and
80 volumes of methylene chloride R . Allow the plate to dry in
air and spray with phosphomolybdic acid solution R.
The chromatogram obtained with the test solution does not
show a blue zone near the line of application (rhaponticin)
corresponding to the zone in the chromatogram obtained
with the reference solution.
Loss on drying (2.2.32)
Not more than 12.0 per cent, deterrnined on 1.000 g ofthe
powdered drug (180) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.16)
Not more than 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Not more than 2.0 per cent.
ASSAY
Carry out the assay protected from bright light.
Introduce 0.100 g ofthe powdered drug (180) (2. 9.12) into a
100 mL flask. Add 30.0 mL of water R, mix and weigh. Heat
in a water-bath under a reflux condenser for 15 mino Allow
to cool, add 50 mg of sodium hydrogen carbonate R, weigh and
adjust to the original mas s with water R. Centrifuge and
transfer 10.0 mL of the liquid to a 100 mL round-bottomed
flask with a ground-glass neck. Add 20 mL of jerric chloride
solution R1 and mix. Heat under a reflux condenser on a
water-bath for 20 min, add 1 mL of hydrochloric acid R and
heat for a further 20 min, shaking frequently. Cool, transfer
to a separating funnel and shake with three quantities, each
of 25 mL, of ether R previously used to rinse the flask.
Combine the ether extracts and wash with two quantities,
each of 15 mL, of water R . Filter the ether extracts through a
plug of absorbent cotton into a volumetric flask and dilute to
100.0 mL with ether R. Evaporate 10.0 mL carefully to
dryness on a water-bath and dissolve the residue in 10.0 mL
of a 5 gIL solution of magnesium acetate R in methanol R.
Measure the absorbance (2.2.25) at 515 nm, using
methanol R as the compensation liquido
Calculate the percentage content of rhein from the
expression:
A x 0.64
m
i.e. taking the specific absorbance of rhein to be 468,
calculated on the basis of the specific absorbance of
barbaloin.
A absorbance at 515 nm;
m = mass of the drug used, in grams.
____________________________________________ ~E~
 2014
Compound Rhubarb Tincture
DEFINITION
Rhubarb, in moderate1y 100 g
coarse powder
Cardamom Oil 0.40
Coriander Oil 0.03
Glycerol 100
Ethanol (60 per cent) Sufficient to produce 1000
Extemporaneous preparation
The following directions apply.
Moisten the Rhubarb with a sufficient quantity of Ethanol
(60 per cent) and prepare 850 mL of tincture by percolalion,
Appendix XI F. Add the Cardamom Oil, the Coriander Oil
and the Glycerol and sufficient Ethanol (60 per cenr) ro
produce 1000 mL. Mix and filter, if necessary.
The tincture complies with the requirements for Tinctures stated
under Extracts and with the following requirements.
TESTS
Ethanol content
48 to 53% v/v, Appendix VIII F, Method III.
Glycerol
9.0 to 11.0% v/v when determined by the following method.
Dilute 20 mL to 100 mL with water; to 10 mL of this
solution add 100 mL of water and 1 g of aetivated eharcoal
and boil under a reflux condenser for 15 minutes. Filter,
wash the filter and charcoal with sufficient water to produce
150 mL, add 0.25 mL of bromocresol purple solution and
neutralise with O.IM sodium hydroxide or 0.05M sulfurie acid to
the blue colour of the indicator. Add 1.4 g of sodium
periodate, allow to stand for 15 minutes, add 3 mL of
propane-l,2-diol, shake and allow to stand for 5 minutes.
Add 0.25 mL of bromocresol purple solurion and titrate with
O.IM sodium hydroxide VS to rhe same blue colour. Each mL
of O.IM sodium hydroxide VS is equivalent to 9.210 mg of
glycerol. Calculate the percentage v/v of glycerol, taking
1.260 g as its weight per mL.
Relative density
0.958 to 0.977, Appendix V G.
Roselle ***
*** ***
(Ph Eur monograph 1623) ***
PhEur _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut dried calyces and epicalyces of Hibiseus
sabdariffa L. collected during fruiting.
Content
Minimum 13.5 per cent of acids, expressed as citric acid
(C 6H s0 7 ; M r 192.1) (dried drug).
CHARACTERS
Acidic taste.
IDENTIFICATION
A. The calyx is joined in the lower half to form an urceolate
structure, the upper half dividing to form 5 long acuminate
recurved tips. The tips have a prominent, slightly protruding
midrib and a large, thick nectary gland about 1 mm in
diameter. The epicalyx consists of 8-12 small, obovate
leaflets, which are adnate to the base of the calyx. The calyx
and epicalyx are fleshy, dry, easily fragmented and bright red
Roselle IV- 3 31
or deep purple, somewhat lighter at the base of the inner
side.
B. Microscopic examination (2.8.23). The powder is red or
violet-red. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
mL characters (Figure 1623.-1): predominantly red fragments [A,
mL F] consisting of polygonal epidermal cells with very
mL irregularly thickened walls, in surface view [Ac, Fa], sorne
mL containing cluster crystals of calcium oxalate [Fb], with
underlying parenchyma consisting of ovoid cells with slightly
thickened walls [Aa], sorne containing cluster crystals of
calcium oxalate [Ab] whilst others are filled with mucilage,
unicellular, long, flexuous, twisted covering trichomes [Ad],
rigid, straight, unicellular covering trichomes, simple or in
groups of 2-4 [Fd], glandular trichomes with a unicellular
stalk and a globular or oval, multicellular and biseriate head
[Fe] and stomata usually of the anisocytic type (2.8.3) [Fc];
numerous fragments of vascular bundles [D] with spiral or
reticulate vessels [Da], sometimes accompanied by
sclerenchymatous fibres with a wide lumen [Db], and
parenchyma [Dc] , of which sorne cells contain cluster crystals
of calcium oxalate [Dd], whilst others are mucilage-filled
[De]; rare, rectangular, parenchymatous sclereids [H];
numerous fragments of rigid [C, G] or flexuous m
covering
trichomes; free cluster crystals of calcium oxalate [B] and
glandular trichomes [E]; exceptionally, spherical pollen
grains, about 200 ~lm in diameter, with a spiny exine .
C. Thin-layer chromatography (2.2.27).
Test so/ution To 1 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of ethanol (60 per eent V/Ji) R. Shake for
15 min and filter.
Referenee solution Dissolve 2.5 mg of quinaldine red R and
2.5 mg of sulfan blue R in 10 mL of methanol R .
Plate TLC si/iea gel plate R (5-40 ~lm) [or TLC siliea gel
plate R (2-10 )lm)].
Mobile phase anhydrous formie acid R, water R, butanol R
(10:12:40 VIV/V).
Applieation 5)lL [or 2 ~lL] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 6 cm].
Drying In air.
Examine immediately in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
--- ---
Quinaldine red: an orange-red
zone
An intense violet zone
Sulfan blue: a blue zone
An intense violet·blue zone
--- ---
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 2 per cent of fragments of fruits (red funicles and
parts of the 5-caverned capsule with yellowish-grey pericarp,
whose thin walls consist of several layers of differently
 IV-332 Rosemary Leaf
B$De
Figure 1623.-1. - Illustration for identification test B of
powdered herbal drug of roselle
directed fibres; flattened, reniform seeds with a dotted
surface) and maximum 2 per cent of other foreign matrero
Loss on drying (2.2.32)
Maximum 11.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2. 9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4. 16)
Maximum 10.0 per cent.
Colouring intensity
Reduce 100 g to a coarse powder (1400) (2.9. 12) and
homogenise. Reduce about 10 g of this mixture to a very fine
powder (355) (2. 9.12). To 1.0 g ofthis powder in a 100 mL
flask add 25 mL of boiling water R and heat for 15 min on a
water-bath with frequent shaking. Filter the hot mixture into
a 50 mL graduated flask; rinse successively the 100 mL flask
and the filter with 3 quantities, each of 5 mL, of warm
water R. After cooling, dilute to 50 mL with water R. Dilute
5 mL of this solution to 50 mL with water R. Measure the
absorbance (2.2.25) at 520 nm using water Ras the
compensation liquido The absorbance is not less than 0.350
for the whole drug and not less than 0.250 for the cut drug.
ASSAY
Shake 1.00 g of the powdered herbal drug (355) (2.9. 12)
with 100.0 mL of carbon dioxide-free water R for 15 mino
Filter. To 50.0 mL ofthe filtrate add 100 mL of carbon
dioxide-free water R. Titrate with 0.1 M sodium hydroxide to
pH 7.0, determining the end-point potentiometrically
(2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 6.4 mg of
citric acid.
- -__________________________________________ ~E~
2014
Rosemary Leaf *****
** **
(Ph Eur monograph 1560) ***
~E~ ____________________________________________
DEFINITION
Whole, dried leaf of Rosmarinus officinalis L.
Content:
-- minimum 12 mUkg of essential oil (anhydrous drug);
-- minimum 3 per cent of total hydroxycinnamic derivatives,
expressed as rosmarinic acid (ClsH160S; M r 360 .3)
(anhydrous drug) .
CHARACTERS
Strongly aromatic odour.
IDENTIFICATION
A. The leaves are sessile, tough, linear or linear-lanceolate,
1-4 cm long and 2-4 mm wide, with recurved edges.
The upper surface is dark green, glabrous and grainy, the
lower surface is greyish-green and densely tomentose with a
prominent midrib.
B. Microscopic examination (2.8.23) . The powder is greyish-
green or yellowish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1560.-1): fragments ofthe
n
lower epidermis in surface view [B, with straight or
sinuous-walled cells [Ba] and numerous diacytic stomata
(2.8.3) [Bb] and glandular trichomes ITa] or covering
trichomes or their scars [Bc, Bd]; numerous multicellular,
mostly branched, covering trichomes of the lower epidermis,
usually fragmented [A, C, D]; fragments of the upper
epidermis in surface view [F] with cells with straight,
thickened and pitted walls [Fa], and an underlying
hypodermis composed of large, irregular cells with thickened
and beaded anticlinal walls [Fb]; fragments of the lamina in
transverse section [G] , showing the epidermis covered by a
very thick cuticle [Ga], hypodermal cells extending across the
mesophyll [Gb] at intervals, separating 1 or 2 layers of
palisade parenehyma into large, creseent-shaped areas [Ge];
glandular triehomes of 2 types, the majority with a short,
unieellular stalk and a radiate head eomposed of 8 eells, in
surface view [E] and in side view [H], others, less abundant,
with a uni- or bicellular stalk and a spherieal, unieellular
head ITa, K].
C. Thin-layer chromatography (2.2.27).
Test solution Dissolve 20 ¡,¡L of the oil obtained in the assay
in 1 mL of hexane R .
Reference solution Dissolve 5 mg of borneol R, 5 mg of bornyl
acetate R and 10 ¡,¡L of cineole R in 1 mL of hexane R.
Plate TLC silica gel plate R .
Mobile phase ethyl acetate R, toluene R (5:95 V/V) .
Application 1O ~lL as bands.
Development Over a path of 15 cm.
Drying In airo
Detection Treat with anisaldehyde solurion R, heat at
100-105 oC for 10 min and examine in daylight.
Results See below the sequenee of zones present in the
chromatograms obtained with the referenee solution and the
test solution.
 2014 Rosemary Leaf IV-333

Top of the plate

A pink fluorescent zone
Caffeic acid : a Iight blue A blue fluorescent zone of low
fluorescent zone intensity
Rosmarinic acid: a Iight blue An intense light blue f1uorescent
fluorescent zone zone
Reference solution Test solution

TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems and maximum 2 per cent of
other foreign matter.
Water (2.2.13)
Maximum 100 mUkg, determined on 20.0 g ofthe
powdered herbal drug (355) (2.9. 12).
Total ash (2.4.16)
Maximum 9.0 per cent.
ASSAY
Total hydroxycinnamic derivatives
Stock solution To 0.200 g of the powdered herbal drug

oOK (355) (2.9.12) add 80 mL of ethanol (50 per cent V/V> R . Boil
in a water-bath under a reftux condenser for 30 minoAIIow
to cool and filter. Rinse the filter with 10 mL of ethanol
(50 per cent V/V> R. Combine the filtrate and the rinsings in
a volumetric ftask and dilute to 100.0 mL with ethanol
(50 per eent V/V> R .
Test solution To 1.0 mL ofthe stock solution add 2 mL of
Figure 1560.-1. - Illustration for identification test B of 0.5 M hydroehloric acid, 2 mL of a solution prepared by
powdered herbal drug of rosemary leaf dissolving 10 g of sodium nitrite R and 10 g of sodium
molybdate R in 100 mL of water R , and then add 2 mL of
dilute sodium hydroxide solution R and dilute to 10.0 mL with
Top of the plate water R; mix.
A red zone Compensation solution Dilute 1.0 mL of the stock solution to
10.0 mL with water R.
Bornyl aceta te : a yellowish-brown A yellowish-brown zone of low
zone intensity Measure immediately the absorbance (2.2.25) of the test
A coloured zone of low intensity solution at 505 nm.
Cineole: a violet zone A violet zone Calculate the percentage content of total hydroxycinnamic
derivatives, expressed as rosmarinic acid, using the foIlowing
Coloured zones of low intensity expression:
Borneo!: a violet-brown zone A violet·brown zone
A coloured zone of low intensity
A x 2.5
m
Reference solution Test solution
i.e. taking the specific absorbance of rosmarinic acid to be
D. Thin-Iayer chromatography (2.2.27). 400 .
A = absorbance of the test solution at 505 nm;
Test solution Grind 1.0 g of the herbal drug in 10 mL of
m = mass of the substance to be examined, in grams .
methanol R and filter.
Reference solution Dissolve 1.0 mg of caffeic acid R and Essential oH (2.8.12)
5.0 mg of rosmarinie acid R in 10 mL of methanol R. Use 25 .0 g ofthe crushed herbal drug, a 1000 mL ftask and
300 mL of water R as the distilIation liquid. Distil at arate of
Plate TLC silica gel piare R.
2-3 mLlmin for 3 h.
Mobile phase anhydrous formic acid R, acetone R, methylene
Ph Eur
ehloride R (8.5:25 :85 VIV/V).
Application 10 ¡.¡L of the test solution and 20 ¡.¡L of the
reference solution, as bands.
Development Over a path of 8 cm.
Drying In airo
Detection Examine in ultraviolet Iight at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution.
IV-334 Rosemary Oil 2014

Refractive index (2.2.6)

Rosemary Oi! 1.464 to 1.473 .
(Ph Eur monograph 1846) Optical rotation (2.2.7)
~E~ ____________________________________________ - 5° to + 8°.
DEFINITION Acid value (2.5.1)
Essential oil obtained by steam distillation from the fiowering Maximum 1.0.
aerial parts of Rosmarinus officinalis L. Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
CHARACTERS
procedure.
Appearance
Clear, mobile, colourless or pale yellow liquido Test solution Dissolve 0.20 mL of the substance to be
examined in hexane R and dilute to 10.0 mL with the same
Characteristic odour.
solvento
IDENTIFICAnON Reference solution Dissolve 20 ~L of rx-pinene R, 10 mg of
First identification B. camphene R, 20 IlL of fi-pinene R, 1O ~ of fi-myrcene R,
Second identification A. 20 ~L of limonene R, 50 ~L of cineole R, 10 ~L of p-cymene R,
A. Thin-Iayer chromatography (2.2.27). 50 mg of camphor R, 30 mg of bornyl acetate R, 10 mg of
Test solution Dissolve 0.5 mL of the substance to be rx-terpineol R, 10 mg of borneol R and 1O ~L of verbenone R in
examined in toluene R and dilute to 10 mL with the same hexane R and dilute to 10.0 mL with the same solvento
solvento Column:
-- material: fused silica,
Reference solution Dissolve 50 mg of borneol R, 50 mg of
-- size: 1 = 30 m (a film thickness of 1 ~m may be used) to
bornyl acetate R and 100 ~L of cineole R in toluene R and
60 m (a film thickness of 0.2 ~m may be used),
dilute to 10 mL with the same solvento
o = 0.25-0.53 mm,
Plate TLC silica gel plate R. -- stationary phase: macrogol 20 000 R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V). Carrier gas helium for chromatography R.
Application 10~, as bands. Flow rate 1 mUmin.
Development Over a path of 15 cm. Split ratio 1:50.
Drying In air. Temperature:
Detection Spray the plate with vanillin reagent R and heat the
Time Temperature
plate at 100-105 oC for 10 minoExamine irnrnediately in (min) (oC)
daylight. Column 0-10 50
Results See below the sequence of the zones present in the
10 ·85 50 -t 200
chromatograms obtained with the reference solution and the
test solution. Furthermore, several violet-blue to violet-grey 85 - 110 200
zones of medium intensity (terpene aleohols) are present in Injection port 200
the lower third of the chromatogram obtained with the test
Detector 250
solution.
Detection Flame ionisation.
Top of the plate
Injection 1 ~L.
Elution order Order indicated in the composition of the
An intense violet zone
reference solution. Record the retention times of these
A violet-grey zone substances.
--- --- System suitabl1ity Reference solution:
-- resolution: minimum 1.5 between the peaks due to
Bornyl acetate: a bluish-grey zone A bluish-grey zone of low intensity
of low intensity (bornyl acetate)
limonene and cineole and minimum 1.5 between the
A violet-pink zone
peaks due to ex-terpineol and borneo!.
Using the retention times determined trom the
--- --- chromatogram obtained with the reference solution, locate
Cine ole : an intense blue zone An intense blue zone (cineole) the components of the reference solution in the
chromatogram obtained with the test solution.
Borneol: a violet-blue zone of A violet -blue zone of medium
medium intensity intensity (borneo)) Determine the percentage content of these components.
Reference solution Test solution For rosemary oil, Spanish type, the percentages are within
the following ranges:
-- rx-pinene: 18 per cent to 26 per cent,
B. Examine the chromatograms obtained in the test for -- camphene: 8.0 per cent to 12.0 per cent,
chromatographic profile. -- fi-pinene: 2.0 per cent to 6.0 per cent,
Results The characteristic peaks in the chromatogram -- fi-myrcene: 1.5 per cent to 5.0 per cent,
obtained with the test solution are similar in retention time to -- limonene: 2.5 per cent to 5.0 per cent,
those in the chromatogram obtained with the reference -- cineole: 16.0 per cent to 25 .0 per cent,
solution. -- p-cymene: 1.0 per cent to 2.2 per cent,
TESTS -- camphor: 13.0 per cent to 21.0 per cent,
Relative density (2.2.5) -- bornyl acetate: 0.5 per cent to 2.5 per cent,
0.895 to 0.920 . -- rx-terpineol: 1.0 per cent to 3.5 per cent,
2014 Saffiower Flower IV-335

-- borneol: 2.0 per cent to 4.5 per cent, Plate TLC silica gel plate R (5-40 )lm) [or TLC silica gel
-- verbenone: 0.7 per cent to 2.5 per cent. plate R (2-10 )lm)] .
For rosemary oil, Moroccan and Tunisian type, the Mobile phase acetic acid R, anhydrous formic acid R, water R,
percentages are within the following ranges: ethyl acetate R (11 :11 :27:100 V/V/VIV).
-- rx-pinene: 9.0 per cent to 14.0 per cent, Application 25)lL as bands of 15 mm [or 10 )lL as bands of
-- camphene: 2.5 per cent to 6.0 per cent, 8 mm].
-- p-pinene: 4.0 per cent to 9.0 per cent, Development Over a path of 12 cm [or 7 cm] .
-- p-myrcene: 1.0 per cent to 2.0 per cent,
Drying In airo
-- limonene: 1.5 per cent to 4.0 per cent,
-- cineole: 38.0 per cent to 55.0 per cent, Detection A Examine in daylight.
-- p-cymene: 0.8 per cent to 2.5 per cent, Results ASee below the sequence of the zones present in
-- camphor: 5.0 per cent to 15.0 per cent, the chromatograms obtained with the reference solution and
-- bornyl acetate: 0.1 per cent to 1.5 per cent, the test solution. Furtherrnore, other faint zones may be
-- rx-terpineol: 1.0 per cent to 2.6 per cent, present in the chromatogram obtained with the test solution.
-- borneol: 1.5 per cent to 5.0 per cent,
-- verbenone: maximum 0.4 per cent.
Top of the plate
STORAGE
At a temperature not exceeding 25 oC. Quercetin: a light yellow zone
----- -----
LABELLING
The labe! states that the content is Spanish type or ----- -----
Moroccan and Tunisian type. Rutin: a light yellow zone
____________________________________________ PhEw
A red zone

Safflower Flower *** A yellow zone

*** *** A yellow zone
(Ph Eur monograph 2386) ***
~Ew ____________________________________________ Reference solution Test solution

DEFINITION
Dried flower of Carthamus tinctorius L. Detection B Heat at 100 oC for 3 min; spray the plate whilst
Cantent still hot with a 10 gIL solution of diphenylboric acid aminoethyl
Minimum 1.0 per cent of total flavonoids, expressed as ester R in methanol R and then with a 50 gIL solution of
hyperoside (C21H20012; M r 464.4) (dried drug) . macrogol 400 R in methanol R; allow to dry in air for about
30 min; examine in ultraviolet light at 365 nm.
IDENTIFICATION
Results B See below the sequence of zones present in the
A. The orange-yellow or reddish-orange, tubular, gametalous,
chromatograms obtained with the reference solution and the
actinomorphic florets are separate from the capitulum. Each
test solution. Furthermore, other faint zones may be present
consists of a long, filiform tube, about 1 cm long divided into
in the chromatogram obtained with the test solution.
5 equa!, narrow, lanceolate lobes, about 0.5 cm long. From
the opening of the tube emerges the hollow cylinder forrned
by the fused yellow anthers, in which the filiform style Top of the plate
persists, thickened near the apex.
Quercetin: an orange fluorescent
B. Reduce to a powder (355) (2.9.12) . The powder is zone
orange-yellow. Examine under a microscope using chloral A bIue fluorescent zone
hydrate solution R . The powder shows fragments of the corolla --- ---
tube with epidermis consisting of elongated, thin-walled
A green fluorescent zone
polygonal cells; fragments of the lobes of the corolla showing
at their apices a large number of small, rounded, very A brown fluorescent zone
prominent papillae; fragments of parenchyma containing
vascular bundles surrounded by secretory canals with
reddish-brown contents; fragments of anthers consisting of A green fluorescent zone
irregularly shaped cells whose walls show thickenings in --- ---
characteristic bands; fragments of the style, whose lower part
Rutin: a yellow fluorescent zone
consists of elongated cells and which ends in a stigma,
bristling with rather long, conical, confluent papillae; A yellow fluorescent zone
rounded or elliptical triporate pollen grains up to 60 )lm in
diameter with an echinulate exine; calcium oxalate prisms,
either isolated or present in parenchyma cells . A green fluorescent zone

e. Thin-Iayer chromatography (2.2.27) . A brown fluorescent zone

Test solution To 1.0 g ofthe powdered drug (355) (2.9.12) Reference solution Test solution
add 10 mL of methanol R. Sonicate for 10 min and
centrifuge.
Reference solution Dissolve 1 mg of rutin R and 5 mg of
quercetin dihydrate R in 50 mL of methanol R.
 IV-336 Sage Leaf
TESTS
Absorbance (2.2.25)
A. Yellow pigment: macerate 0.1 g of the powdered drug
(355) (2.9.12) in 150 mL of water R, stir for 1 h, filter
through a sintered-glass filter (40) (2.1 .2) and dilute to
500.0 mL, washing the residue, with water R.
The absorbance is not less than 0.40 at 401 nm.
B. Red pigment: to 0.25 g of the powdered drug (355)
(2.9.12) add 50 mL of a mixture of 20 volumes of water R
and 80 volumes of acetone R. Heat on a water-bath at 50 oC
for 90 min. Allow to cool, filter through a sintered-glass filter
(40) (2.1.2) and dilute to 100.0 mL, washing the residue
with a mixture of 20 volumes of water R and 80 volumes of
acetone R. The absorbance is not less than 0.40 at 518 nm.
Loss on drying (2.2.32)
Maximum U .O per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
Solution A Place 0.250 g of the powdered drug (180)
(2.9.12) in a 250 mL fiask and add 95 mL of methanol R.
Heat under a refiux condenser on a water-bath for 30 min.
Allow to cool and filter. Rinse the filter with 5 mL of
methanol R . Combine the filtrate and the rinsing solution in a
volumetric fiask and dilute to 100.0 mL with methanol R.
Test solution Place 5.0 mL of solution A in a volumetric
fiask and dilute to 20.0 mL with a 20 gIL solution of
aluminium chloride R in methanol R.
Compensation solution Place 5.0 mL of solution A in a
volumetric fiask and dilute to 20.0 mL with methanol R .
After exactly 15 min, measure the absorbance (2.2.25) of the
test solution at 420 nm by comparison with the
compensation solution. Calculate the percentage content of
total fiavonoids, expressed as hyperoside, using the following
expression:
A
m
taking the specific absorbance of hyperoside at 420 nm to be
400.
A absorbance of the test solution, at 420 nm;
m = mass of the substance to be examined, in grams.
____________________________________________ PhEw
Sage Leaf
(Sage Leaf (Salvia officinalis),
Ph Eur monograph 1370)
Preparation
Sage Tincture
~Ew ____________________________________________
DEFINITION
Whole or cut dried leaves of Salvia officinalis L.
Content
Minimum 15 mUkg of essential oil for the whole drug and
minimum 10 mUkg of essential oil for the cut drug
(anhydrous drug).
2014
CHARACTERS
Sage leaf (Salvia officinalis) oil is rich in thujone.
IDENTIFICATION
A. The lamina of whole sage leaf (Salvia officinalis) is about
2 cm to 10 cm long and 1 cm to 2 cm wide, oblong-ovate,
elliptical. The margin is finely crenate to smooth. The apex is
rounded or subacute and the base is shrunken at the petiole
and rounded or cordate. The upper surface is greenish-grey
and finely granular; the lower surface is white and pubescent
and shows a dense network of raised veinlets.
B. Reduce to a powder (355) (2.9.12). The powder is light
grey to brownish-green. Examine under a rnicroscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters: very numerous articulated and bent
trichomes with narrow elongated cells and a very thick cell at
the base as well as fragments of these trichomes; fragments of
the upper epidermis with pitted, somewhat polygonal cells;
fragments of the lower epidermis with sinuous cells and
numerous diacytic stomata (2.8.3); rare single glandular
trichomes with a uni- or bicellular head and a stalk consisting
of 1 to 4 cells; abundant glandular trichomes with a
unicellular stalk and a head composed of 8 radiating cells
with a raised common cuticle.
C. Thin-Iayer chromatography (2.2.27).
Test solution Shake 0.5 g of the freshly powdered drug (355)
(2.9.12) with 5 mL of ethanol R for 5 min.
Reference solution Dissolve 20 llL of thujone R and 25 llL of
cineole R in 20 mL of ethanol R.
Plate TLC silica gel plate R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 20 llL, as bands.
Development Over a path of 15 cm.
Drying In airo
Detection Spray the plate with a 200 gIL solution of
phosphomolybdic acid R in ethanol R and heat at 100-105 oC
for 10 min. Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other zones are present in the
chromatogram obtained with the test solution.
Top of the plate
A blue zone (near the solvent
front)
--- -----
u-Thuione and ~-thujone: 2 pinkish-violet zones (u-thujone
2 pinkish-violet zones and ~-thujone)
Cine ole : a blue zone A blue zone (cineole)
- -- -----
Blue zones
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of stems and maximum 2 per cent of
other foreign matter.
Water (2.2.13)
Maximum 100 mUkg, determined on 20.0 g.
Total ash (2.4.16)
Maximum 10.0 per cent.
2014 Sage Leaf IV-337

ASSAY Methanol and 2-propanol (2.9.11)

Carry out the detennination of essential oils in herbal drugs Maximum 0.05 per cent V/V of methanol and maximum
(2.8.12). Use 20.0 g ofthe substance to be examined, cut, if 0.05 per cent of 2-propanoI.
necessary, immediately before the assay, a 500 mL fiask, Dry residue (2.8.16)
250 mL of water Ras the distillation liquid and 0.5 mL of Minimum 2.0 per cent m/m, detennined on 3.00 g.
xylene R in the graduated tube. Distil at arate of 2-3 mUmin
for 2 h. ASSAY
____________________________________________ ~E~
In a 500 mL round-bottomed fiask, place 30.0 g of the
tincture and add 100 mL of water R. Distil, using a
descending condenser, into a separating funnel which has
been marked beforehand at 50 mL. Stop the distillation
process as soon as the distillate reaches the 50 mL mark.
Sage Tincture *** Rinse the condenser with 10 mL of pentane R . Dissolve in
*** *** the distillate sufficient sodium chloride R to produce a
(Ph Eur monograph 1889) *** saturated solution. Shake with 3 quantities, each of 20 mL,
~E~ ____________________________________________ of pentane R. Dry the combined pentane layers, inc1uding the
pentane from rinsing the condenser, over anhydrous sodium
DEFINITION sulfate R and filter through a plug of absorbent cotton into a
Tincture produced from Sage leaf (Salvia officinalis) (1370). weighed 100 mL round-bottomed fiask. Wash the sodium
Content sulfate several times with small quantities of pentane R.
Minimum 0.1 per cent m/m of essential oiI. Remove the pentane carefully at a temperature not exceeding
40 oC. Dry the residue in a desiccator over diphosphorus
PRODUCTION
pentoxide R and hard paraffin at atmospheric pressure and at
The tincture is produced from 1 part of comminuted drug
room temperature for 2 h. Weigh the residue (essential oil) .
and 10 parts of ethanol (70 per cent V/V) by a suitable
____________________________________________ Ph Eur
procedure.
CHARACTERS
Appearance
Brownish liquid with a characteristic odour.
IDENTIFICATION Three-Iobed Sage Leaf *****
* *
Thin-Iayer chromatography (2.2.27). (Ph Eur monograph 1561) **** *
Test solution The tincture to be examined. ~E~ ____________________________________________
Reference solution Dissolve 20 ¡.tL of thujone R and 25 ).lL of
DEFINITION
cineole R in 20 mL of ethanol R.
Whole or cut, dried leaves of Salvia fructicosa MilI. (S. triloba
Plate TLC silica gel plate R. L. fil).
Mobile phase ethyl acetate R, toluene R (5:95 V/V) .
Content
Application 20 ).lL, as bands. Minimum 18 mUkg of essential oil in the whole drug
Development Over a path of 15 cm. (anhydrous drug) and minimum 12 mUkg of essential oil in
Drying In air. the cut drug (anhydrous drug).
Detection Spray with a 200 gIL solution of phosphomolybdic CHARACTERS
acid R in ethanol R and heat at 100-105 oC for 10 mino Spicy odour when ground, similar to eucalyptus oil.
Examine in daylight.
IDENTIFICATION
Results See below the sequence of the zones present in the A. The lamina of the whole three-Iobed sage leaf is about
chromatograms obtained with the reference solution and the 8-50 mm long and about 4-20 mm wide, and oblong-ovate
test solution. Furthermore, other zones are present in the or lanceolate. The margin is finely crenate and undulate but
chromatogram obtained with the test solution. indistinct owing to the dense hairy covering on both surfaces.
The base is obtuse and sometimes bears 1 or 2 more or less
Top oi the plate developed lobes. The upper surface is grey-tomentose
pubescent, the lower surface is densely white-tomentose
A blue zone (near the solvent front)
pubescenti the venation is indistinct. The densely white-
----- ----- tomentose pubescent petiole is about 1 mm in diameter.
a·Thujone and f3-thujone : 2 2 pinkish·violet zones (a·thujone B. Reduce to a powder (355) (2.9.12). The powder is
pinkish·violet zones and f3-thujone) greyish-green and tomentose. Examine under a microscope
Cineole: a blue zone A blue zone (cineole) using chloral hydrate solution R. The powder shows the
----- -----
following diagnostic characters: very numerous, whole or
Blue zones fragmented, covering and glandular trichomes, scattered and
Reierence solution Test solution
attached to fragments of the epidennisesi covering trichomes
articulated, uniseriate, thick-walled and bluntly tapering,
those on the upper epidennis straight, those on the lower
TESTS epidennis longer, tortuous and more densely packedi
Ethanol content (2.9.10) glandular trichomes, sorne with a unicellular or bicellular
64 per cent VIV to 69 per cent V/V. head and a stalk consisting of from 1-4 cells, the majority
having a short, unicellular stalk and a head composed of 8
radiating cells with a raised common cutic1ei the upper
 IV-338 Sage Oil 2014

epidennis with pitted and beaded cells, somewhat polygonal, A. Thin-Iayer chromatography (2.2.27).
with a few diacytic stomata (2.8.3); the lower epidennis with Test solution Dissolve 1 mL of the substance to be examined
sinuous or wavy-walled cells and numerous diacytic stomata. in toluene R and dilute to 10 mL with the same solvento
C. Examine the chromatogram obtained in the test for Reference solution Dissolve 60 J..lL of linalol R, 200 J..lL of
thujone. linalyl aceta te R and 60 J..lL of rt.-terpineol R in toluene R and
Results The chromatogram obtained with the test solution dilute to 10 mL with the same solvento
shows a blue zone due to cineole, equal or greater in size and Plate TLC silica gel plate R .
intensity to the zone in the chromatogram obtained with the Mobile phase ethyl acetate R, toluene R (5:95 V/V).
reference solution. Further zones are presento
Application 5 J..lL of the test solution and 10 J..lL of the
TESTS reference solution, as bands.
Thu;one Development Over a path of 15 cm.
Thin-Iayer chromatography (2.2.27) .
Drying In air.
Test solution Shake 0.3 g of the freshly powdered drug (355)
Detection Spray with vanillin reagent R and heat at
(2.9.12) with 5.0 mL of anhydrous ethanol R for 5 min.
100-105 oC for 5-10 minó examine in daylight within 5 min.
Reference solution Dissolve 20 J..lL of thujone R and 25 J..lL of
Results See below the sequence of the zones present in the
cineole R in 20 mL of anhydrous ethanol R .
chromatograms obtained with the reference solution and the
Plate TLC silica gel plate R. test solution. Furthermore, other faint zones are present in
Mobile phase ethyl acetate R, toluene R (5:95 V/V). the chromatogram obtained with the test solution.
Application 20 J..lL, as bands.
Development Over a path of 15 cm. Top of the plate
Drying In airo
a·Terpineol: a dark violet zone A dark violet zone
D etection Spray with a 200 gIL solution of phosphomolybdic
acid R in anhydrous ethanol R and heat at 100-105 oC for ----- -----
10 min. Examine in daylight. Linalyl aceta te : a dark violet zone A dark violet zone
Results The chromatogram obtained with the reference ----
---
solution shows in the middle part a blue zone (cineole) and
in the upper part a pink-blue zone (thujone). Linalol: a dark violet zone A dark violet zone
The chromatogram obtained with the test solution shows no Reference solution Test solution
zone or a very faint pink-blue zone due to thujone.
Foreign matter (2.8.2)
Maximum 8 per cent of stems and maximum 2 per cent of B. Examine the chromatograms obtained in the test for
other foreign matter. chromatographic profile.
Water (2.2.13) Results The chromatogram obtained with the test solution
Maximum 100 mUkg, detennined on 20.0 g. shows 5 peaks similar in position to the 5 peaks in the
chromatogram obtained with the reference solution. The 2
Total ash (2.4.16) peaks corresponding to (X- and ~-thujone may be absent.
Maximum 10.0 per cent.
TESTS
ASSAY Re1ative density (2.2.5)
Carry out the detennination of essential oils in herbal drugs 0.890 to 0.908.
(2.8.12). Use 20.0 g ofdrug, ifnecessary cut immediately
before the assay, a 500 mL ftask, 250 mL of water R as the Refractive index (2.2.6)
distillation liquido Add 0.50 mL of xylene R in the graduated 1.456 to 1.466.
tube. Distil at arate of 2-3 mUmin for 2 h. Optical rotation (2.2.7)
____________________________________________ PhEm - 26° to - 10°.
Acid value (2.5.1)
Maximum 1.0.
Chromatographic pro file
Gas chromatography (2.2.28): use the nonnalisation
Sage Oil procedure.
(Clary Sage Oil, Ph Eur monograph 1850) Test solution The substance to be examined.
____________________________________________
~Em

Reference solution To 1 g of hexane R, add 5 J..lL of thujone R,

DEFINITION 5 J..lL of linalol R, 100 J..lL of linalyl acetate R, 10 J..lL of
rt.-terpineol R and 25 mg (± 20 per cent) of sclareol R.
Essential oil obtained by steam distillation from the fresh or
Mix thoroughly by stirring.
dried ftowering stems of Salvia sclarea L.
Column:
CHARACTERS -- material: fused silica,
Appearance -- size: l = 30 m (a film thickness of 1 J..lm may be used) to
Colourless or brownish-yellow liquid, usually pale yellow. 60 m (a film thickness of 0.2 J..lm may be used),
Characteristic odour. o = 0.25-0.53 mm,
-- stationary phase: macrogol 20 000 R.
IDENTIFICATION
First identification B. Carrier gas helium for chromatography R.
Second identification A. Split ratio 1:100.
 2014 Sage Oil IV-339

Temperature: Reference solution Dissolve 20 f.l.L of thujone R and 30 f.l.L of

Time Temperature
cineole R in 10 mL of toluene R.
(mio) (oC) Plate TLC silica gel plate R (5-40 f.l.m) [or TLC silica gel
Columo 0·10 60 plate R (2-10 f.l.m)].
10·75 60 -; 190 Mobile phase ethyl acetate R, toluene R (5 :95 V/V).
75·120 190 Application 10 f.l.L [or 3 f.l.L] as bands of 10 mm [or 6 mm] .
Development Over a path of 15 cm [or 6 cm] .
1njection port 220
Drying In airo
Detector 240
Detection Spray with a freshly prepared 200 gIL solution of
Detection Flame ionisation. phosphomolybdic acid R in ethanol (96 per cent) R and heat at
Injection 0.2 f.l.L 105 oC for 10 min; examine in daylight.
Elution order Order indicated in the composition of the Results See below the sequence of zones present in the
reference solution. Record the retention times of these chromatograms obtained with the reference solution and the
substances. test solution. Furthermore, other faint zones may be present
System suitability Reference solution: in the chromatogram obtained with the test solution.
- resolution: minimum 1.5 between the peaks due to linalol
and linalyl aceta te, Top of the plate
U sing the retention times determined from the
A blue zone
chroma1Ogram obtained with the reference solution, locate
the components of the reference solution in the --- ---
chroma1Ogram obtained with the test solution (disregard any Thujone: 2 pinkish·violet zones
peak due to hexane). Thujone R is a mixture of [X- and
~-thujone . [X- Thujone elures before ~-thujone under the CineoIe: a bIue zone A bIue zone (cineoIe)
described conditions . --- ---
Determine the percentage content of each of these
3 bIue zones
components.
Reference soIution Test solution
AIso determine the percentage content of germacrene-D.
The germacrene-D peak can be identified in the
chroma1Ogram obtained with the test solution by its relative
B. Examine the chroma1Ograms obtained in the test for
retention of 1.23 with reference to linalol under the described
chromatographic profile.
operating conditions.
Results The characteristic peaks in the chromatogram
The percentages are within the following ranges:
obtained with the test solution are similar in retention time to
- rx- and P-thujone: maximum 0.2 per cent,
those in the chromatogram obtained with reference
- linalol: 6.5 per cent to 24 per cent,
solution (a).
- linalyl acetate: 56 per cent to 78 per cent,
- rx-terpineol: maximum 5.0 per cent, TESTS
- germacrene-D: 1.0 per cent to 12 per cent, Relative density (2.2.5)
- sclareol: OA per cent to 2.6 per cent. 0.907 to 0.932.
STORAGE Refractive index (2.2.6)
At a temperature not exceeding 25 oC. 1.465 to 1.473.
___________________________________________ ~Ew Optical rotation (2.2.7)
+ 7° to + 17 0
•

Acid value (2.5.1)

Spanish Sage Oi! *** Maximum 2.0, determined on 5.00 g.
*** *** Solubility in alcohol (2.8.10)
(Ph Eur monograph 1849) *** 1 volume is soluble in 2 volumes and more of ethanol
~Ew ___________________________________________ (80 per cent V/V; R.
DEFINITION Chromatographic pro file
Essential oil obtained by steam distillation from the aerial Gas chromatography (2.2.28): use the normalisation
parts of Salvia lavandulijolia Vahl, collected at the fiowering procedure.
stage. Test solution Dissolve 0.200 g of the oil 10 be examined in
heptane R and dilute to 10.0 mL with the same solvent.
CHARACTERS
Appearance Reference solution (a) Dissolve 0.200 g of Spanish sage oil for
Clear, colourless or pale yellow, mobile liquid. peak identification CRS in heptane R and dilure to 10.0 mL
with the same solvent.
Camphor-like odour.
Reference solution (b) Dissolve 5 f.l.L of limonene R in
IDENTIFICATION heptane R and dilute to 50.0 mL with the same solvento
First identification B. Dilute 0.5 mL of this solution to 5.0 mL with heptane R.
Second identification A. Column:
A. Thin-Iayer chromatography (2.2.27) . - material: fused silica;
Test solution Dissolve 0.1 mL of the oil to be examined in - size: 1 = 60 m, 0 = 0.25 mm;
10 mL of toluene R.
 IV-340 Salvia Miltiorrhiza Root and Rhizome
-- stationmy phase: macrogol20 000 R (film thickness
0.25 ).lm) .
Canier gas helium for chromatography R.
Flow rate 1.5 mUmin.
Split ratio 1:50.
Temperature:
Time Temperature
(min) (oC)
Column 0·43 60 --t 232
Injeclion por! 250
Detector 250
Detection Flame ionisation.
1njection 1).lL.
System suitability Reference solution (a):
-- the chromatogram obtained is similar to the
chromatogram supplied with Spanish sage oil for peak
identification CRS;
-- resolution: minimum 1.5 between the peaks due to
limonene and 1,S-cineole and minimum 1.5 between the
peaks due to o:-terpinyl acetate and borneo!.
Use the chromatogram supplied with Spanish sage oil for peak
identification CRS and the chromatogram obtained with
reference solution (a) to locate the peaks due to o:-pinene,
sabinene, limonene, 1,S-cineole, thujone, camphor, linalol,
linalyl acetate, terpinen-4-ol, sabinyl acetate, o:-terpinyl
acetate and borneo!.
Determine the percentage content of each of these
components. The percentages are within the following
ranges:
-- rx-pinene: 4.0 per cent to 11.0 per cent;
-- sabinene: 0.1 per cent to 3.5 per cent;
-- limonene: 2.0 per cent to 6.5 per cent;
-- l,8-cineole: 10.0 per cent to 30.5 per cent;
-- thujone: maximum 0.5 per cent;
-- camphor: 11.0 per cent to 36.0 per cent;
-- linalol: 0.3 per cent to 4.0 per cent;
-- linalyl acetate: maximum 5.0 per cent;
-- terpinen-4-ol: maximum 2.0 per cent;
-- sabinyl acetate: 0.5 per cent to 9.0 per cent;
-- rx-terpinyl acetate: 0.5 per cent to 9.0 per cent;
-- borneol: 1.0 per cent to 7.0 per cent;
-- disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent).
STORAGE
At a temperature not exceeding 25 oC.
____________________________________________
Salvia Miltiorrhiza Root and
*** ***
Rhizome
(Ph Eur monograph 2663)
~E~ ___________________________________________
DEFINITION
Dried, whole or fragmented rhizome and root of Salvia
miltiorrhiza Bunge, collected in spring or autumn.
Content:
-- salvianolic acid B (C36H30016; M r 719): minimum
3.0 per cent (dried drug);
-- tanshinone 1lA (C19H1S03; M r 294.3): minimum
0.12 per cent (dried drug).
2014
IDENTIFICATION
A. The rhizome is short and thick, sometimes with stem
remnants at the apex. The roots are numerous, about
10-20 cm long and 0.3-1 cm in diameter, cylindrical and
slightly curved; sorne are branched, with secondary roots and
rootlets. The outer surface is reddish-brown or dark reddish-
brown, marked with longitudinal striations. The bark of old
roots comes off usually as purplish-brown scales. The texture
is hard and fragile. The fracture is soft, fissured or slightly
even and dense, with a reddish-brown outer part and a
greyish-yellow or purplish-brown wood, showing bundles of
yellowish-white ves seIs, arranged radially.
Cultivars are relatively stout, about 0.5-1.5 cm in diameter.
The outer surface is brownish-red, longitudinally wrink1ed.
The bark adheres closely to the wood and is difficult to
remove. The texture is compact; the fracture is relatively
even.
B. Microscopic examination (2.8.23). The powder is
brownish-red. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: fragments of cork in surface view, consisting of
subrectangular or polygonal cells, up to 150 ).lm in diameter,
containing yellowish-brown pigment; fragments of
parenchyma consisting of polygonal or elongated, thin-walled
cells that may contain yellowish-brown pigment; xylem fibres
usually in bundles, long and fusiform, with pitted walls
showing oblique or criss-cross striations; very numerous
reticulate or pitted vessels, 3-120 ).lm in diameter, free, in
bundles or sometimes accompanying the fibres.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (355)
(2.9.12) add 40 mL of methanol R. Sonicate for 15 mino
Filter. Evaporate the filtrate to 1 mL.
Reference solution Dissolve 2 mg of salvianolic acid BRand
2 mg of tanshinone lIA R in 1 mL of methanol R.
Plate TLC silica gel F 254 plate R (5-40 ).lm) [or TLC silica gel
F254 plate R (2-10 ).lm)] .
Mob¡Je phase methanol R, anhydrous fonnic acid R, toluene R,
methylene chloride R, ethyl aceta te R
(5:20 :20:30:40 VIVIVIV/V).
Application 5).lL [or 5 ¡.tL) as bands of S mm [or S mm].
Development Over a path of S cm [or 6 cm) .
Drying In airo
Detection A Examine in daylight.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
PhE~ in the upper third and middle pan of the chromatogram
obtained with the test solution.
***
Top of the plate
*** Tanshinone JI,,: a prominent red
zone
A prominent red zone
(tanshinone 11)
An orange zone
- -- ---
A faint brownish·green zone
Salvianolic acid B: a fainl grey A fainl grey zone (salvianolic
zone acid B)
- -- ----
Reference solution Test solution
 2014 Salvia Miltiorrhiza rhizome and Root IV-341

Detection B Examine in ultraviolet light at 254 nm. Deteetion Speetrophotometer at 280 nm.
Results See below the sequence of zones present in the lnjeetion 10 flL.
chromatograms obtained with the reference solution and the Relative retention With reference 10 tanshinone IIA
test solution. Furthermore, other faint zones may be present (retention time = about 33 min):
in the upper third and middle part of the chromatogram rosmarinic acid = about 0.3; salvianolic acid B = about 0.4.
obtained with the test solution. System suitability Reference solution (e):
-- resolution : minimum 5.0 between the peaks due to
Top of the plate rosmarinic acid and salvianolic acid B.
Tanshinone JI,, : a prominent A prominent quenching zone Calculate the percentage eontent of tanshinone HA using the
que nching zone (tanshinone JI,,) following expression:
A quenching zone

--~
- --
A quenching zone
Al area ofthe peak due to tanshinone HA in the
Salvianolic acid B: a prominent A prominent quenching zone chromatogram obtained with the test solution;
quenching zone (salvianolic acid B)
- - -
Az area of the peak due to tanshinone HA in the
- --
chromatogram obtained with reference solution (a);
mi mass of the herbal drug to be examined used to
Reference solution Test solution
prepare the test solution, in grams;
mz mass of tanshinone llA CRS used 10 prepare
reference solution (a), in grams;
TESTS PI percentage content of tanshinone HA in tanshinone
Loss on drying (2.2.32) IIA CRS.
Maximum 10.0 per cent, deterrnined on 1.000 g ofthe Calculate the percentage content of salvianolic acid B using
powdered herbal drug (355) (2.9.12) by drying in an oven at the following expression:
105 oC for 2 h.
A3 x m3 x P2 x 2
Total ash (2.4.16)
Maximum 10.0 per cent. A4 x mi
Ash insoluble in hydrochloric acid (2.8.1) A3 area of the peak due to salvianolic acid B in the
Maximum 3.0 per cent. chromatogram obtained with the test solution;
A4 area of the peak due to salvianolic acid B in the
ASSAY
chromatogram obtained with reference solution (b);
Liquid chroma1Ography (2.2.29). Protect the solutions from mi mass of the herbal drug 10 be examined used 10
light. prepare the test solution, in grams;
Test solution Disperse 0.30 g of the powdered herbal drug m3 mass of salvianolie acid B CRS used 10 prepare
(355) (2.9.12) in 50.0 mL of a 70 per cent VIV solution of reference solution (b), in grams;
methanol R . Sonicate for 1 h. Filter through a membrane pz percentage content of salvianolie aeid B in
filter (nominal pore size 0.45 ~lm) . salvianolie acid B CRS.
Referenee solution (a) Dissolve 5.0 mg of tanshinone IIA CRS _ _ _ _ _ _ _ __ _ _ _ __ _ _ _ __ _ __ PhE~

in methanol R and dilute to 50.0 mL with the same solvent.

Dilute 2.0 mL of the solution to 10.0 mL with methanol R .
Reference solution (b) Dissolve 5.0 mg of salvianolie acid
B CRS in methanol R and dilute 10 25 .0 mL with the same
Processed Salvia Miltiorrhiza Rhizome
solvent. and Root
Referenee solution (e) Dissolve 1 mg of rosmarinie acid R in DEFINITlON
methanol R, add 5 mL of reference solution (b) and dilute 10 Processed Salvia Miltiorrhiza Rhizome and Root is Salvia
10.0 mL with methanol R. Miltiorrhiza Rhizome and Root that has been processed.
Column: It contains not less than 0.04% of tanshinone HA
-- siz e: 1 = 0.25 m, 0 = 4.6 mm; (CI9HIS03), not les s than 0.17% of rosmarinie acid
-- stationary phase: end-capped oetadeeylsilyl siliea gel for (CIsHI60S) and not less than 3.0% of salvianolic acid B
ehromatography R (5 flm). (C36H36016), calculated with referenee to the dried material.
Mobile phase: PRODUCTlON
-- mobile phase A : 0.1 per cent V/V solution of anhydrous It is collected in spring or autumn, separated from soil,
fomúe acid R; washed clean, softened thoroughly, sliced longitudinally or
mobile phase B: aeetonitrile for ehromatography R; transversely and dried. It may be stir baked with wine .
Time Mobile phase A Mobile phase B IDENTIFICATlON
(min) (per cent V!JI) (per cent V!JI) A. The longitudinally-sliced pieces are up to about 5 cm
0-10 79 --+ 71 21 --+ 29 long, 1.5 cm wide and 1 10 2 mm thick; those cut from the
thi=er roots show the dark brown striated cork covering one
10 - 15 71 --+ 65 29 --+ 35
longitudinal surface and the yellowish to cream i=er tissues
15 - 25 65 --+ 28 35 --+ 72 on the other; slices cut from the thieker rhizomes are usually
25 - 37 28 --+ O 72 --+ 100 cut obliquely so that parts of the outer and i=er tissues are
included on both longitudinal surfaces. The transversely-cut
Flow rate 1.0 mUmin. slices are irregularly elliptical to nearly circular, 4 to 12 mm
IV-342 Salvia Miltiorrhiza rhizome and Root
wide and 2 to 3 mm thick; the outer surface is dark brown
and uneven; the smoothed transverse surface shows the outer
layers about 1 to 2 mm wide separated by a darker line from
the yellowish white, radia te vascular tissue; sorne pieces show
a small, light brown central pith.
B. Reduce to a powder (355) . The powder is reddish-brown.
Examine under a microscope using chloral hydrate solution.
The powder shows a surface view of cork cells almost
rectangular or polygonal, containing yellowish-brown
pigment, 12 to 151 pm in diameter. Parenchymatous cells in
cortex squarish or polygonal, containing reddish-brown
pigmental sediments. Xylem fibres usually in bundles, long
fusiform, with oblique or criss-cross striations, 11 to 60 pm
in diameter, vivid yellow when examined under a polarizing
microscope. Numerous mainly bordered or reticulated
ves seis, 3 to 120 pm in diameter.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Place 2 g of the powdered drug (355) in a cellulose
fingerstall in a continuous extraction apparatus (Soxhlet
type). Add 75 mL of methanol and heat for 1 hour.
Evaporate the extract to 20 mL, cool and filter if necessary.
(2) 0.1 % w/v each of tanshinone IIA CRS, rosmarinic acid CRS
and salvianolic acid B CRS in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating si/iea gel F 254 .
(b) Use the mobile phase described below.
(c) Apply as bands 8 pL of solution (1) and 5 ~ of
solution (2).
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (366 nm).
MOBILE PHASE
10 volumes of water, 13.5 volumes of methanol and
100 volumes of ethyl acetate.
SYSTEM SUITABILITY
The chromatogram obtained with solution (2) shows three
clearly separated bands.
CONFIRMATION
The blue fiuorescent bands with Rf values of approximately
0.7 (tanshinone lIA!, 0.2 (rosmarinic acid) and 0.06
(salvianolic acid B) in the chromatogram obtained with
solution (1) correspond in colour and position to those in the
chromatogram obtained with solution (2) . Other bands may
be present in the chromatogram obtained with solution (1) as
shown below.
Top of the plate
A fluorescent band Tanshinone II A : a fl uorescent
band
Several fluorescent bands
A fluorescent band Rosmarinic acid: a
fluorescent band
A fluorescent band Salvianolic acid B: a
fluorescent band
Solution (1) Solution (2)
2014
TESTS
Total ash
Not more than 10%, Appendix XI J.
Acid-inso1uble ash
Not more than 2.0%, Appendix XI K.
Loss on drying
When dried for 2 hours at 105°, loses not more than 12.0%
of its weight. Use 1 g.
ASSAY
For tanshinone IlA
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Finely powder about 5.0 g of the herbal drug being
examined. Transfer 0.5 g of the powder into a 25 mL
volumetric fiask and add 20 mL of the mobile phase given
below. Shake and mix with the aid of ultrasound for
30 minutes, shaking intermittently. Add the mobile phase to
give a total volume of 25 mL. Filter through a 0.45-pm filter.
(2) 0.002% w/v of tanshinone IIA CRS in the mobile phase.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with octadecylst1yl silica gel for chromatography (5 pm)
(Nucleosil ODS is suitable) .
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a fiow rate of 1 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 270 nm.
(!) Inject 20 pL of each solution.
MOBILE PHASE
O.OIM sodium octyl sulfonate in a mixture of 25 volumes of
water and 75 volumes of methanol adjusted to pH 5.0 with
acetic acid.
SYSTEM SUITABILITY
The test is not valid, unless in the chromatogram obtained
with solution (2):
- the symmetry factor of the peak due to tanshinone lIA is
les s than 1.2;
- the number of theoretical plates is not less than 4500.
DETERMINATION OF CONTENT
Using the retention time and peak area from the
chromatograms obtained with solution (2) , locate and
integrate the peak due to tanshinone lIA in the
chromatogram obtained with solution (1) .
Calculate the content of tanshinone HA in the sample using
the declared content of tanshinone HA (CI9HIS03) in
tanshinone IIA CRS and the following expression:
Al lTh VI lOO
-- x -- x -- x p x-----
A, V, ffiI 100 - d
Al Area of the peak due to tanshinone HA in the
chromatogram obtained with solution (1).
A2 Area of the peak due to tanshinone HA in the
chromatogram obtained with solution (2).
mI Weight of the drug in mg.
m2 Weight of tanshinone IIA CRS in mg.
VI Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content of tanshinone HA in tanshinone
IIA CRS.
2014
d = Percentage loss on drying of the herbal drug being
examined.
Por rosrnarinic acid and salvianolic acid B
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions prepared
immediately before use.
(1) Finely powder about 5.0 g of the herbal drug being
examined. Transfer 0.5 g of the powder into a 25-mL
volumetric fiask and add 20 mL of the mobile phase given
below. Shake and mix with ultrasound for 30 minutes,
shaking intermittently. Add the mobile phase to give a total
volume of 25 mL. Filter through a 0.45-¡.tm filter.
(2) 0.003% w/v each of rosmarinic acid CRS andferulic acid in
water.
(3) 0.06% w/v of salvianolic acid B CRS in the mobile phase.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with octadecylszlyl silica gel for chromatography (5 ¡.tm)
(Nucleosil ODS is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a fiow rate of 1 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 330 nm.
(f) Inject 20 ¡.tL of each solution.
MOBILE PHASE
22 volumes of acetonitrile and 78 volumes of 0.4 % v/v of
fonnic acid.
SYSTEM SUITABILITY
The test is not valid unless, in the chroma1Ogram obtained
with solution (2), the resolution factor between the two main
peaks, rosmarinic acid and ferulic acid, is at least 5.0.
DETERMINATION OF CONTENT
Rosmarinic acid U sing the retention time and peak area
from the chromatogram obtained with solution (2) , locate
and integrate the peak due to rosmarinic acid in the
chroma1Ogram obtained with solution (1).
Calculate the content of rosmarinic acid in the sample using
the declared content of rosmarinic acid (CIsHI60S) in
rosmarinic acid CRS and the following expression:
Al m2 VI 100
-x -x - x p x
~ V2 m, 100-d
Al Area of the peak due 10 rosmarinic acid in the
chromatogram obtained with solution (1) .
A2 Area of the peak due 10 rosmarinic acid in the
chromatogram obtained with solution (2).
m] Weight of the drug in mg.
m2 Weight of rosmarinic acid CRS in mg.
VI Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content of rosmarinic acid in rosmarinic
acid CRS.
d Percentage loss on drying of the herbal drug being
examined.
SaIvianolic acid B Using the retention time and peak area
from the chromatogram obtained with solution (3), locate
and integrate the peak due to salvianolic acid B in the
chroma1Ogram obtained with solution (1).
Saw Palmetto Fruit IV-343
Calculate the content of salvianolic acid B in the sample
using the declared content of salvianolic acid B (C36H 3601 6)
in salvianolic acid B CRS and the following expression:
Al m2 V I 100
- x - x -xpx-- -
A2 V2 m, 100-d
Al Area of the peak due to salvianolic acid in the
chromatogram obtained with solution (1) .
A2 Area of the peak due to salvianolic acid in the
chroma1Ogram obtained with solution (3).
mi Weight of the drug in mg.
m2 Weight of salvianolic acid B CRS in mg.
VI Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content of salvianolic acid B in salvianolic
acidB CRS.
d Percentage loss on drying of the herbal drug being
examined.
STORAGE
Processed Salvia Miltiorrhiza Rhizome and Root should be
protected from moisture.
Saw Palmetto Fruit ****
*** *
(Ph Eur monograph 1848) ****
~E~ ____________________________________________
DEFINITION
Dried ripe fruit of Serenoa repens (W. Bartram) SmalI (Syn.
Sabal serrulata (Michaux) T. Nuttal ex Schultes & Schultes).
Content
Minimum 11.0 per cent oftotal fatty acids (dried drug).
CHARACTERS
Odour
Strong but not rancid.
IDENTIFICATION
First identification A, B, D.
Second identificarion A, B, C.
A. The fruit is an ovoid or subspherical drupe, with a dark
brown or blackish, roughly wrinkled surface and more or less
coppery sheen, up to 2.5 cm long and 1.5 cm in diameter.
The apex sometimes bears the remains of the style and
tubular calyx, with 3 teeth, and the base bears a smalI
depression with the scar of the stalk. The epicarp and
underlying mesocarp form a thin fragile layer, which panially
peels off, revealing the thin, hard, pale brown endocarp,
which is fibrous and easi1y separable. The seed is inegularly
spherical or ovoid, up 10 12 mm long and 8 mm in diameter,
with a hard, smooth or finely pitted surface which is reddish-
brown with a paler, raised and membranous area over the
raphe and micropyle; cut transversely, the seed has a thin
testa, narrow perisperm and a large area of dense, horny,
greyish-white endosperm, with the embryo positioned to one
side.
B. Microscopic examination (2.8.23). Reduce 10 a powder
(710) (2.9.12). The powder is reddish or blackish-brown and
oily. Examine under a microscope using chioral hydrate
solution R. The powder shows the foIlowing diagnostic
characters: fragments of epicarp composed of several layers of
thin-walled, reddish-brown, pigmented, polyhedral ceIls
 lV-344 Saw Palmetto Fruit 2014
~~~~~==~~~----------------------------------

00-40 /lm) which are strongly cuticularised; those of the TESTS

Outer Iayers are much smaIler than those of the inner layers. Loss on drying (2.2.32)
Parenehyma eeIls of the mesoearp may be large and filled Maximum 12.0 per eent, determined on l.000 g of the
with oil droplets, or smaller and eontaining nodules of siliea. powdered herbal drug (710) (2.9.12) by drying in an oven at
Groups of xylem tissue of the mesoearp show smalllignified, 105 oC for 2 h.
annular or spirally thickened vessels. Stone eells of the Total ash (2.4.16)
mesoearp (20-200 Jlm) may be found seattered, usually Maximum 5.0 per eent.
singly but sometimes in small groups, the walls are
moderately thiekened, distinctly striated and finely pitted. ASSAY
Fragments of endoearp contain groups of elongated sc1ereids Total fatty acids
about 300 Jlm long, with strongly thiekened walls and Gas ehromatography (2.2.28).
numerous pits. The seed testa eonsists of small, thin-walled Internal standard solutian Dissolve 0.47 g of methyl
eells with brownish eontents and underlying sc1ereids; margarate R in 20.0 mL of dimethylformamide R and dilute to
albumen eells are thick-walled with large eonspieuous pits 100.0 mL with the same solvento
and eontain aleurone grains and fixed oil. Test solution Reduce 50 g of the herbal drug to a powder
C. Thin-layer ehromatography (2.2.27). (200) (2.9.12). Disperse 4.00 g ofthe powdered herbal drug
Test solution To l.5 g of the powdered herbal drug (710) in 60 mL of dimethylformamide R . Sonieate for 15 min and
(2.9.1 2), add 20 mL of ethanol (96 per cent) R and stir for then shake for 30 mino Dilute to 100.0 mL with
15 mino Filter. dimethylformamide R. AlIow to stand for a few minutes and
R eference solution Dissolve 4 mg of p-amyrin R and 10 mg of
filter. To 20.0 mL of this solution add 4.0 mL of the internal
P-sitosterol R in 10 mL of ethanol (96 per cent) R .
standard solution and dilute to 25 .0 mL with
dimethylformamide R. Mix 0.4 mL of this solution and
Plate TLC silica gel plate R (5-40 Jlm) [or TLC silica gel
0.6 mL of an 18.84 gIL solution of trimethylsulfonium
plate R (2-10 Jlm)].
hydroxide R in methanol R .
Mobile phase acetic acid R, ethyl aceta te R, toluene R
Reference solution (a) Dissolve 0.699 g of laurie acid CRS
(1:30:70 VIV/V).
and 0.870 g of oleic acid CRS in dimethylformamide R and
Application 10 JlL [or 2 ~tL] as bands of 10 mm [or 8 mm] . dilute to 10.0 mL with the same solvento To l.0 mL of the
Development Over a path of 10 cm [or 6 cm]. solution add 4.0 mL of the internal standard solution and
Drying In airo dilute to 25.0 mL with dimethylformamide R. Mix 0.4 mL of
Detection Treat with anisaldehyde solution R; heat at this solution and 0.6 mL of an 18.84 gIL solution of
trimethylsulfonium hydroxide R in methanol R.
100-105 oC for 5-10 min; examine in daylight.
Referenee solution (b) Disperse 0.25 g of saw palmetto extraet
Results See below the sequenee of zones present in the
HRS in 10 mL of dimethylformamide R . Add 4.0 mL of the
ehromatograms obtained with the referenee solution and the
test solution. Furthermore, other faint zones may be present, internal standard solution and dilute to 25.0 mL with
dimethylformamide R . Mix 0.4 mL of this solution and
espeeially in the lower third, in the ehromatogram obtained
with the test solution. 0.6 mL of an 18.84 giL solution of trimethylsulfonium
hydroxide R in methanol R .
Column:
Top oC the plate - material: fused siliea;
A strong blue zone - size: 1 = 25 m, 0 = 0.20 mm;
- stationary phase: poly(dimethyl)siloxane R (film thickness
--- --- 0.33 Jlm).
2 faint blue zones Carrier gas helium for ehromatography R .
/3.Amyrin: a blue zone Flow rate 0.5 mUmin.
A strong bluish-violet zone Split ratio 1:40.
Temperature:
/3-Sitosterol: a blue zone A faint blue zone
Time Temperature
(min) (OC)
--- - -- Column 0-2 150

A faint blue zone 2·7 150 ---> 190

ReCerence solution 7 - 12 190

Test soIution
12 - 22 190 ---> 220

22 - 32 220
D . Examine the ehromatograms obtained in the assay of total
fatty aeids. Injection port 300
Results The peaks due to eaproie, eaprylie, eaprie, laurie, Detector 300
myristie, palmitoleie, palmitie, linoleic, linolenie, oleie and
stearie acids in the ehromatogram obtained with the test Deteetion Flame ionisation.
solution are similar in retention time to the eorresponding Injeetion 1 JlL.
peaks in the ehromatogram obtained with referenee solution Identifieation of peaks Use the chromatogram supplied with
(b); the principal peaks are due to laurie acid and oleie aeid. saw palmetto extract HRS and the ehromatogram obtained
with referenee solution (b) to identify the peaks due to
eaproie, eaprylie, eaprie, laurie, myristic, palmitoleie,
2014 Schisandra Fruit IV-345

palmitie, linoleie, linolenie, oleie and stearie aeids and methyl
margarate.
System suitabzlity Referenee solution (b):
- peak-to-valley ratio: minimurn 1.2, where Hp = height
aboye the baseline of the peak due to linolenie aeid and
H v = height aboye the baseline of the lowest point of the
curve separating this peak from the peak due to linoleie
acid.
Calculate the percentage eontent of total fatty aeids, where
caproic, eaprylie, capric, lauric, myristie, palmitoleie, palmitic
and stearie acids are expressed as lauric acid (ClzH 240 2; M r
200.3) and linoleie, linolenie and oleic aeids are expressed as
oleic aeid (C IsH3402; M r 282.5), using the following
expression:
Al X A4 x m2 x PI X 0.5 A5 x A4 xm3 x P2 x 0.5
A 2 x A3 x mi + Ae x A3 x mi
Al surn of the areas of the peaks due to eaproie,
eaprylic, caprie, laurie, myristic, palmitoleic,
palmitie and stearic acids in the chromatogram
obtained with the test solution;
A2 area of the peak due to laurie acid in the
ehromatogram obtained with referenee solution (a);
A3 area of the peak due to methyl margarate in the
chromatogram obtained with the test solution;
A4 area of the peak due 10 methyl margarate in the
ehromatogram obtained with referenee solution (a);
As surn of the areas of the peaks due 10 linoleie,
linolenic and oleie acids in the ehroma1Ogram
obtained with the test solution;
A6 area of the peak due 10 oleie aeid in the
ehromatogram obtained with reference solution (a);
mi mass of the herbal drug 10 be examined used 10
prepare the test solution, in grams;
m2 mas s of ¡aurie aeid CRS used 10 prepare reference
solution (a), in grams;
m3 mass of oleie aeid CRS used to prepare reference
solution (a), in grams;
PI percentage eontent of laurie acid in laurie acid CRS;
P2 pereentage eontent of oleie acid in oleie aezd CRS.
_ __________________________________________ PhE~
eharacters: reddish-brown fragments of periearp, eonsisting of
1 layer of thin-walled epiearp cells, accompanied by sparse oi!
cells and severallayers of ovoid, more-or-less flattened
mesocarp eells; fragments of the outer testa of the seed
eonsisting of thiek-walled, finely ehannelled sclereids,
polygonal in surface view (15-50 Ilm in diameter) and in
palisade arrangement in side view; fragments of the inner
testa with sclereids, isolated or in small groups, about 80 Ilm
in diameter, with slightly thickened and markedly channelled
walls; fragments of endosperm eonsisting of polyhedral cells
eontaining oil droplets and aleurone grains. Examine under a
microscope using a 50 per eent V/V solution of glyeerol R: the
powder shows parenchymatous cells of the mesoearp
eontaining numerous small, round starch granules.
C. Examine the chromatograms obtained in the test for
Sehisandra sphenanthera.
Results ASee below the sequence of quenching zones
present in the ehromatograms obtained with the reference
solution and the test solution. Furthermore, other weak
quenching zones may be present in the chromatogram
obtained with the test solution.
Top of the plate
)'-Schisandrin: a quenching zone A quenching zone ()'-schisandrin)
- -- -----
A weak quenching zone
--- - - --
Schisandrin: a quenching zone A quenching zone (schisandrin)
Reference solution Test solution
Results B See below the sequenee of zones present in the
ehromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the ehromatogram obtained with the test solution.
Top of the plate
)'-Schisandrin : a brown zone A brown zone ()'-schisandrin)

---- - --

----- - --

Schisandra Fruit ***

*** *** Schisandrin : an intense,
brownish-l1reen zone
tn intense, brownish-green zone
schisandrin)
(Ph Eur monograph 2428) *** Reference solution Test solution
~E~ _ _________________________________________

DEFINITION
Whole, dried or steamed and dried, ripe fruit of Sehisandra
ehinensis (Turez.) Baill.
Content
Minimum 0.40 per eent of schisandrin (C24H 320 7; M r
432.5) (dried drug).
IDENTIFICATION
A. The berry is more or les s spherical, up to 8 mm in
diameter; red, reddish-brown or blackish outer surfaee,
sometimes eovered in a whitish frost; strongly shrivelled
pericarp; presence of 1-2 reniform, yellowish-brown, lustrous
seeds, with thin seed-coat.
B. Reduce to a powder (355) (2.9.12). The powder is
reddish-brown. Examine under a mieroseope using ehloral
hydrate solution R. The powder shows the following diagnostic
TESTS
Schisandra sphenanthera
Thin-layer ehromatography (2.2.27).
Test solution To 2.5 g of the powdered drug (355) (2.9.12)
add 10 mL of methanol R. Extraet at 25 oC in an ultrasonic
bath for 5 min and centrifuge.
Referenee solution Dissolve 5 mg of sehisandrin R and 5 mg
of y-sehisandrin R in 5 mL of methanol R .
Plate TLC siliea gel F254 plate R (5-40 Ilm) [or TLC siliea gel
F254 plate R (2-10 Ilm)].
Mobile phase aeetie aeid R, ethyl aeetate R, toluene R
(2:22:46 VIV/V) .
Application 5 IlL [or 2 IlL] as bands of 10 mm [or 6 mm].
Development Over a path of 10 cm [or 7 cm] .
 IV-346 Scutellariae Baicalensis Root 2014

*****
DryingIn air.
Scutellariae Baicalensis Root
Deteetion AExamine in ultraviolet light at 254 nm. ** **
Deteetion B Spray with a 100 gIL solution of sulfurie acid R (Baieal Skulleap Root, Ph Eur monograph 2438) ***
____________________________________________
in methanol R and heat in an oven at 120 oC for 7 min; ~Ew

examine in daylight.
DEFINITION
Results B The chromatogram obtained with the test solution Dried, peeled, usually fragmented root of Seutellaria
shows a zone due to schisandrin and a zone due to baiealensis Georgi without rootlets. It is collected in spring or
y-schisandrin; the chromatogram shows no intense violet- autumn.
pink zone in the middle tlUrd.
Content
Loss on drying (2.2.32) Not less than 9.0 per cent ofbaicalin (C21 R1SOll ; M r 446.4)
Maximum 10.0 per cent, determined on 1.000 g ofthe (dried drug).
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h. IDENTIFICATION
A. The root is conical, twisted and, if not reduced in size,
Total ash (2.4.16)
8-25 cm long and 1-3 cm in diameter. The outer surface is
Maximum 6.0 per cent.
brownish-yellow or dark yellow, bearing sparse, warty traces
ASSAY of rootlets, the upper part rough, with twisted longitudinal
Liquid chromatography (2.2.29) . wrinkles or irregular reticula, the lower part with longitudinal
Test solution Weigh 1.250 g of the powdered drug (355) striations and fine wrink1es. Texture hard and fragile, easily
(2.9.12) into a 250 mL conical ftask, add 90 mL of broken, fracture yellow, reddish-brown in the centre;
methanol R and sonicate for 30 mino Filter the solution into a the central part of an old root dark brown or brownish-black,
volumetric ftask, add 10 mL of methanol R whilst rinsing the withered or hollowed.
filter and dilute to 100.0 mL with the same solvento B. Microscopic examination (2.8.23). The powder is yellow
Referenee solution Dissolve 5.0 mg of sehisandrin R in or light brown. Examine under a microscope using ehloral
methanol R and dilute to 100.0 mL with the same solvento hydrate solution R. The powder shows the following diagnostic
Column: characters: phloem fibres, single or in bundles, fusiform,
-- size: 1 = 0.25 m, 0 = 4.6 mm; 60-250 Jlm long, 9-33 Jlm in diameter, with thick, channelled
-- stationary phase: end-eapped octadecylsilyl siliea gel for walls; stone cells sub-spherical, square or rectangular with
ehromatography R; rounded edges, with thickened walls, sometimes heavily; cork
-- temperature: 25 oC. cells polygonal and brownish-yellow; numerous reticulated
vessels, 24-72 Jlm in diameter; lignified fibres frequently
Mobile phase:
broken, about 12 Jlm in diameter, with sparse, oblique pits.
-- mobile phase A: water R, methanol R (35:65 V/V);
Examine under a microscope using a 50 per cent V/V
-- mobile phase B: methanol R;
solution of glyeerol R. The powder shows abundant starch
Time Mobile phase A Mobile phase B granules, simple, spheroidal, 2-10 Jlm in diameter, with a
(min) (per cent V(!I) (per cent V(!I) distinct hilum, or compound with 2-3 components .
0·10 100 O
C. Thin-layer chromatography (2.2.27).
10·16 100 --7 58 O --7 42 Test solution To 1 g of the powdered herbal drug (3 55)
16·26 58 42 (2.9.12) add 10 mL of methanol R and sonicate for 10 mino
Centrifuge and use the supernatant.
Flow rate 1 mUmin. Referenee solution Dissolve 1 mg of baicalin R and 1 mg of
Deteetion Spectrophotometer at 250 nm. acteoside R in 10 mL of methanol R.
1njeetion 1O JlL. Plate TLC siliea gel F 254 plate R (2-10 Jlm).
Retention time Schisandrin = about 8 mino Mobile phase aeetie acid R, formie acid R, water R, ethyl
System suitability: aeetate R (1:1:2:15 VIVIV/V).
-- number of theoretical plates: minimum 5000, calculated for Applieation 10 JlL as bands.
the peak due to schisandrin in the chromatogram
Development Over a path of 6 cm.
obtained with the reference solution.
Drying In air.
Calculate the percentage content of schisandrin using the
following expression: Deteetion Reat at 100-105 oC for 3 min, treat with a 10 gIL
solution of diphenylborie aeid aminoethyl ester R in methanol R ,
then treat with a 50 gIL solution of maerogol 400 R in
methanol R, allow to dry in air for 30 min and examine in
ultraviolet light at 365 nm.
Results See below the sequence of zones present in the
area of the peak due to schisandrin in the
chromatograms obtained with the reference solution and the
chromatogram obtained with the test solution;
test solution. Furthermore, other faint blue ftuorescent zones
area of the peak due to schisandrin in the
may be present in the chromatogram obtained with the test
chromatogram obtained with the reference solution; solution.
mas s of the drug to be examined used to prepare the
test solution, in grams;
mass of sehisandrin R used to prepare the reference
solution, in grams;
p percentage content of schisandrin in sehisandrin R.
____________________________________________ ~Ew
 2014 Selfheal Fruit-Spike IV-347

Top of the plate Calculate the percentage content of baicalin using the
following expression:
3-4 f1uorescent zones

--- ---
2 f1uorescent zones
- -- --- mI = mass of the herbal drug, in grams;
Verbascoside: a blue f1uorescent A strong blue f1uorescent zone m2 = mass of baicalin used to prepare reference solution (a), in
zone grams;
A blue f1uorescent zone SI = area of the peak due to baicalin in the chromatogram
Baicalin: a black zone A black zone obtained with the test solution;
S2 = area of the peak due to baicalin in the chromatogram
obtained with reference solution (a);
A weak yellow f1uorescen t zone p = percentage content of baicalin in baicalin CRS.

Reference solutioo Test solutioo STORAGE

Protected from moisture.
____________________________________________ ~E~

TESTS
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
Selfheal Fruit-Spike ***
*** ***
105 cC for 2 h.
Total ash (2.4.16) (Common Selfheal Fruit-Spike, ***
Maximum 6.0 per cent. Ph Eur monograph 2439)
Ash insoluble in hydrochloric acid (2.8.1) ~E~ ____________________________________________
Maximum 2.0 per cent.
DEFINITION
ASSAY Dried fruit-spike of Prunella vulgaris L.
Liquid chromatography (2.2.29).
Content
Test solution To 0.3 00 g of the powdered herbal drug (355) Minimum 0.12 per cent ofthe sum of oleanolic acid
(2.9.12) add 40 mL of ethanol (70 per cent V/V] R, heat (C30H4S03; M r 456 .7) and ursolic acid (C30H4S03; M r
under a re flux condenser on a water bath for 3 h, cool and 456.7), expressed as ursolic acid, of which not less than
filter. Transfer the filtrate to a 100 mL volumetric flask. 70.0 per cent consists ofursolic acid (dried drug) .
Wash both the container and the residue several times with a
small volume of ethanol (70 per cene V/V] R and filter the IDENTIFICATION
washings into the same flask. Dilute to 100.0 mL with A. Cylindrical, somewhat flattened, 1.5-8 cm long,
ethanol (70 per cene V/V) R. Mix well. Dilute 1.0 mL of the 0.8-1.5 cm in diameter, accompanied by remains of the stem
solution to 10.0 mL with methanol R. Mix well. up to 15 cm long, pale brown or brownish-red. The whole
spike is composed of up to lOor more whorls of persistent
Reference solurion (a) Dissolve 5.0 mg of baicalin CRS in
calyx and bracts, each whorl with 2 opposite bracts, fan-
methanol R and dilute to 100.0 mL with the same solvent.
shaped, apex acuminate, striations of vein distinct, the outer
Reference solution (b) Dissolve 2 mg of methyl surface with white hairs. Each bract is accompanied by
pamhydroxybenzoate R in methanol R, add 20 mL of reference 3 flowers, with a persistent bilabiate calyx, and whose corolla
solution (a) and dilute to 100 mL with methanol R. is often missing, and by 4 small brown ovoid nutlets, white
Column: and convex at the acute end. Calyx c10sed in the fruit stage.
-- size: 1 = 0.125 m, 0 = 4 mm; B. Microscopic examination (2.8.23). The powder is reddish-
-- stationary phase: octadecylsilyl s¡Jica gel for chromatography R brown or brown. Examine under a microscope using ehloml
(5 ¡.Lm).
hydrate solution R. The powder shows the following diagnostic
Mobile phase: characters: very numerous covering trichomes, multicellular,
-- mobile phase A: 0.1 per cent V/V solution of phosphoric scattered, usually broken, sometimes exceeding 1 mm long
acid R; and 125 ¡.Lm wide at the base, with spiny walls, upper cell
-- mobile phase B: acetonitrzle R; usually shott and acuminate, fine needle-shaped crystals may
Time Mobile phase A Mobile phase B be visible in the cells; fragments of the bracts, in surface
(mio) (per cent V!l'l (per ceot V!l'l view, with lobed epidermal cells, trichomes mostly unicellular
0-30 90 ---? 60 10 ---? 40 and occasionally bi- or tricellular, conical, acute, shott,
serrate; diacytic stomata (2.8.3) usually accompanied by
Flow rate 1.0 mUmin. 2 subsidiary cells very unequal in size and rare glandular
Deteetion Spectrophotometer at 280 nm. trichomes with a unicellular stalk and a bicellular head;
Injection 10 ¡.LL. ftagments of the bracts and/or calyx margins with numerous
Reteneion time Methyl parahydroxybenzoate = about serrate trichomes pointing towards the same direction;
15 min; baicalin = about 16 mino fragments of the calyx, in surface view, composed of lobed
cells strongly thickened and deeply grooved; fragments of
System suitability Reference solution (b):
reticulate or bordered pitted vessels from the stems; rare
-- resolution: minimum 3 between the peaks due to methyl
fragments of the nucules having a pericarp composed of
parahydroxybenzoate and baicalin.
palisade-like mucilaginous cells accompanied by polygonal
cells with thickened walls and granular coloured contents;
IV-348 Selfheal Fruit-Spike 2014

fragments of endosperrn with oily contents; very numerous
oil droplets; glandular trichomes of laminaceous type with
4 secretory cells may be presento
e. Thin-layer chromatography (2.2.27).
Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R, sonicate for 10 min and
centrifuge; use the supernatant.
Reference solution Dissolve 1 mg of f3-sitosterol R and 1 mg of
ursolic acid R in 2 mL of methanol R .
Plate TLC silica gel F 254 plate R (5-40 ¡.tm) [or TLC s¡lica gel
F 254 plate R (2-10 ).1m)].
Mobile phase glacial acetic acid R, ethyl acetate R,
cyclohexane R (0.5:8:20 VIV/V).
Application 10 ¡.tL [or 4 ¡.tL] as bands of 10 mm [or 8 mm].
D evelopment Over a path of 12 cm [or 6 cm].
Drying In air.
D etection Treat with a 10 per cent VIV solution of sulfuric
acid R in anhydrous ethanol R and heat at 100 oC for 3 min;
examine in ultraviolet light at 365 nm.
R esults See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
A pale violel fluorescent zone
---
2 fainl yellow fluorescent zones
¡)-sitoslerol: a violet fluorescent
zone
Ursolic acid: a yellowish-orange A yellowish-orange fluorescent
fluorescent zone zone (ursolic acid)
---
2 faint green fluorescent zones
Reference solution Test solution
if
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems longer than 15 cm and
maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 12.0 per cent, deterrnined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC.
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydroch1oric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Solvent mixture methanol R, 1,1-dimethylethyl methyl ether R
(20:80 V/V).
Test solution Disperse 2.000 g of the powdered herbal drug
(355) (2.9.12) in 20 mL of the solvent mixture, heat under
refiux at 80 oC for 30 min and filter. Repeat the extraction
twice . Combine the filtrates and dilute to 100.0 mL with the
solvent mixture. Evaporate 50.0 mL of this solution to
dryness at 40 oC. Dissolve the residue in 1.0 mL of
1,1-dimethylethyl methyl ether R. Rinse the fiask 4 times with
1.0 mL of 1,1-dimethylethyl methyl ether R . Pre-condition a
3 mL solid phase extraction column, containing 500 mg of
aminopropylsilyl silica gel for chromatography R1, using 2 mL of
methanol R followed by 2 mL of 1,1-dimethylethyl methyl
ether R. Subsequently apply the solution and the washings to
the pre-conditioned column. Wash the column with 1.0 mL
of 1,1-dimethylethyl methyl ether R followed by 5 quantities,
each of 1.0 mL, of methanol R. Apply 1.0 mL of a
2 per cent VIV solution of anhydrous formic acid R in
methanol R and elute after 5 mino Repeat the elution 3 times
and dilute the eluates to 5.0 mL with a 2 per cent V/V
solution of anhydrous formic acid R in methanol R.
Solution A Dissolve 10.0 mg of ursolic acid CRS in
methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a) Dilute 1.0 mL of solution A to
10.0 mL with a 2 per cent VIV solution of anhydrous formic
acid R in methanol R .
Reference solution (b) Dissolve 10.0 mg of oleanolic acid R in
methanol R and dilute to 10.0 mL with the same solvent.
Mix 1.0 mL of the solution and 1.0 mL of solution A and
dilute to 10.0 mL with a 2 per cent VIV solution of
anhydrous formic acid R in methanol R .
Column:
- size: 1 = 0.15 m, 0 = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 ¡.tm).
Mobile phase Mix 25 volumes of a 4.6 gIL solution of
ammonium dihydrogen phosphate R adjusted to pH 6.0 with
---
strong sodium hydroxide solution R, 35 volumes of methanol R1
and 40 volumes of acetonitrile R1.
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 205 nm.
InJection 20 ¡.tL.
Run time 1.1 times the retention time of ursolic acid.
--- Elution order Oleanolic acid, ursolic acid.
Relative retention With reference to ursolic acid (retention
time = about 28 min) : oleanolic acid = about 0.9.
System suitability Reference solution (b) :
- resolution: minimum 1.5 between the peaks due to
oleanolic acid and ursolic acid.
Calculate the percentage contents of ursolic acid and
oleanolic acid, expressed as ursolic acid, using the following
equations:
A¡ x m2 x p x 0.1
n¡=
A 2 x m¡
nI percentage content of ursolic acid;
n2 percentage content of oleanolic acid;
Al area of the peak due to ursolic acid in the
chromatogram obtained with the test solution;
A2 area of the peak due to ursolic acid in the
chromatogram obtained with reference solution (a);
A3 area of the peak due to oleanolic acid in the
chromatogram obtained with the test solution;
mi mass of the herbal drug to be examined used to
prepare the test solution, in grams;
m2 mass of ursolic acid CRS used to prepare solution A,
in grams;
 2014
p assigned percentage content of ursolic acid in ursolic
acid CRS.
Calculate the sum of the percentage contents of ursolic acid
and oleanolic acid (nI + n2) and the relative content of
ursolic acid using the following expression:
____________________________________________ PhEw
Senega Root
Senega
(Ph Eur monograph 0202)
When Powdered Senega Root is prescribed or demanded,
material complying with the requirements below with the
exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
~Ew ____________________________________________
DEFINITION
Dried and usually fragmented root and root crown of
Polygala senega L. or of certain other c10sely related species or
of a mixture of these Polygala species.
CHARACTERS
Faint, sweet odour, slightly rancid or reminiscent of methyl
salicylate.
Reduced to a powder, it is irritant and sternutatory. Shaken
with water, the powder produces a copious froth.
IDENTIFICATION
A. The root crown is greyish-brown and wider than the root;
it forms an irregular head consisting of numerous remains of
stems and tightly packed purplish-brown buds. The taproot
is brown or yellow, occasionally branched, sometimes
fiexuous, usually tortuous and without secondary roots,
except in the Japanese varieties and species, which contain
numerous fibrous rootlets. The diameter is usually 1-8 mm
at the crown, gradually tapering to the tip; the surface is
transversely and longitudinally striated and often shows a
more or less distinct decurrent, elongated spiral kee!.
The fracture is short and shows a yellowish cortex of varying
thickness surrounding a paler central woody area somewhat
circular or irregular in shape depending on the species.
B. Examine under a microscope using chloral hydrate
solution R. The transverse section of the root shows the
following diagnostic characters: cork formed from several
layers of thin-walled cells, phelloderm of slightly
collenchymatous cells containing droplets of oi!; the phloem
and xylem arrangement is usually normal, especially near the
crown but where a keel is present tbis is formed by increased
development of phloem; other anomalous secondary
development sometimes occurs, resulting in the formation of
1 or 2 large wedge-shaped rays in the phloem and xylem, the
parenchymatous cells of which contain droplets of oi!.
The xylem is usually central and consists of vessels up to
60 ~m in diameter associated with numerous thin-walled
tracheids and a few small lignified parenchymatous cells.
C. Reduce to a powder (355) (2.9.12). The powder is light
brown. Examine under a microscope using chlora! hydrate
solution R. The powder shows the following diagnostic
characters: longitudinal fragments of lignified tissue made up
of pitted tracheids and somewhat larger ves seis with
numerous bordered pits or with reticulate thickening;
Senna Fruit IV- 349
yellowish parenchyma and collenchymatous cells containing
droplets of oil; occasional fragments of cork, and of
epiderrnal tissue with stomata and unicellular trichomes from
the bud scales. Crystals and stone cells are absent.
D . Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of the powdered drug (355) (2.9.12)
add 10 mL of ethanol (70 per cent V/V> R, boil under a refiux
condenser for 15 min, filter and allow to coo!.
R eference solution Dissolve 10 mg of aescin R in ethanol
(70 per cent VIV) R and dilute to 10 mL with the same
solvento
Plate TLC silica gel G plate R.
M obile phase The upper layer of a mixture of 10 volumes of
glacial acetic acid R , 40 volumes of water R and 50 volumes of
butanol R .
Application 10 ¡.tL of the test solution and 1O ~L and 40 IlL
of the reference solution, as bands of 20 mm by 3 mm.
Development Over a path of 12 cm.
Dlying At 100-105 oc.
Detection A Spray with about 10 mL of anisaldehyde
solution R for a plate 200 mm square and heat again at
100-105 oC until red zones due to saponosides appear in the
chromatogram obtained with the test solution.
R esults A In the chromatogram obtained with the test
solution, 3-5 red zones appear in the lower and midd1e parts,
similar in po sitio n to the grey-violet zones due to aescin in
the chromatogram obtained with the reference solution.
Detection B Spray with about 10 mL of a 200 gIL solution
of phosphomolybdic acid R in anhydrous ethanol R and heat at
100-105 oC until the zones due to saponosides become blue.
R esults B The intensity and size of the zones in the
chromatogram obtained with the test solution are between
those of the 2 bands due to aescin in the chromatograms
obtained with 1O ~L and 40 IlL of the reference solution.
TESTS
Total ash (2.4.16)
Maximum 6.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
STORAGE
Store protected from humidity.
____________________________________________ ~Ew
Alexandrian Senna Fruit ***
*** ***
(A lexandrian Senna Pods, Ph Eur monograph 0207) ***
Preparations
Senna Liquid Extract
Standardised Senna Granules
Senna Tablets
When Powdered Alexandrian Senna Fruit is prescribed or
demanded, material complying with the requirements below,
with the exception of Identification test A and the test for
Foreign matter, shall be dispensed or supplied.
~Ew ____________________________________________
DEFINITION
Dried fruit of Cassia senna L. (e. acutifolia Delile).
Content
Minimum 3.4 per cent ofhydroxyanthracene glycosides,
expressed as sennoside B (C4zH3S0Z0; M r 863) (dried drug).
IV-350 Senna Fruit
CHARACTERS
Slight odour.
IDENTIFICATION
A. Flattened renifonn pods, green or greenish-brown with
brown patches at the positions corresponding to the seeds,
usuaIly 40-50 mm long and at least 20 mm wide. At one end
is a stylar point and at the other a short stalk. The pods
contain 6-7 ftattened and obovate seeds, green or pale
brown, with a continuous network of prominent ridges on
the testa.
B. Reduce to a powder (355) (2.9.12). The powder is brown.
Examine under a microscope using chloral hydrate solution R.
The powder shows the foIlowing diagnostic characters:
epicarp with polygonal ceIls and a smaIl number of conical
warty trichomes and occasional anomocytic or paracytic
stomata (2.8.3); fibres in 2 crossed layers accompanied by a
crystal sheath of calcium oxalate prisms; characteristic
palisade ceIls in the seed and stratified ceIls in the
endospenn; c1usters and prisms of calcium oxalate.
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g of the powdered drug (180) (2.9.12)
add 5 mL of a mixture of equal volumes of ethanol
(96 per cent) R and water R and heat to boiling. Centrifuge
and use the supernatant liquido
R ejerence solution Dissolve 10 mg of senna extract CRS in
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
and water R (a slight residue remains) .
Plate TLC silica gel G plate R .
Mobile phase glacial acetic acid R, water R, ethyl acetate R,
propanol R (1:30:40:40 VIVIV/V).
Application 10 )lL, as bands of 20 mm by 2 mm.
Development Over a path of 10 cm.
Drying In airo
Detection Spray with a 20 per cent VIV solution of nitric
acid R and heat at 120 oC for 10 minó aIlow to cool and
spray with a 50 gIL solution of potassium hydroxide R in
ethanol (50 per cent V/T-] R until the zones appear.
Results The principal zones in the chromatogram obtained
with the test solution are similar in position (se=osides B, A,
D and C in order of increasing RF value), colour and size to
the principal zones in the chromatogram obtained with the
reference solution; between the zones due to se=osides D
and C, a red zone due to rhein-8-glucoside may be visible;
the zones due to se=osides D and C are faint in the
chromatogram obtained with the test solution.
D . Place about 25 mg of the powdered drug (180) (2.9.12)
in a conical ftask and add 50 mL of water R and 2 mL of
hydrochloric acid R. Heat in a water-bath for 15 min, cool and
shake with 40 mL of ether R. Separate the ether layer, dry
over anhydrous sodium sulfate R, evaporate 5 mL to dryness
and to the cooled residue add 5 mL of dilute ammonia R1.
A yeIlow or orange colour develops. Heat on a water-bath for
2 minoA reddish-violet colour develops.
TESTS
Foreign matter (2.8.2)
Maximum 1 per cent.
Loss on drying (2.2.32)
Maximum 12.0 per cent, detennined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 9.0 per cent.
2014
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
ASSAY
Carry out the assay protecred !rom bnght light.
Place 0.150 g of the powdered drug (180) (2.9.12) in a
100 mL ftask. Add 30.0 mL of water R, mix, weigh and place
in a water-bath. Heat under a reftux condenser for 15 mino
AIIow to cool, weigh and adjust to the original mass with
water R. Centrifuge and transfer 20.0 mL of the supernatant
liquid to a 150 mL separating funnel. Add 0.1 mL of dilute
hydrochloric acid R and shake with 3 quantities, each of
15 mL, of chlorojorm R. AIIow to separate and discard the
chlorofonn layer. Add 0.10 g of sodium hydrogen carbonate R
and shake for 3 mino Centrifuge and transfer 10.0 mL ofthe
supernatant liquid to a 100 mL round-bottomed ftask with a
ground-glass neck. Add 20 mL ofjemc chloride solution R1
and mix. Place the ftask in a water-bath so that the water
level is aboye that of the liquid in the ftask, and heat under a
reftux condenser for 20 mino Add 1 mL of hydrochloric acid R
and heat for a further 20 min, with frequent shaking, to
dissolve the precipitate. Cool, transfer the mixture to a
separating funnel and shake with 3 quantities, each of
25 mL, of ether R previously used to rinse the ftask. Combine
the 3 ether layers and wash with 2 quantities, each of 15 mL,
of water R . Transfer the ether layers to a volumetric ftask and
dilute to 100.0 mL with ether R. Evaporate 10.0 mL carefuIly
to dryness and dissolve the residue in 10.0 mL of a 5 gIL
solution of magnesium acetate R in methanol R. Measure the
absorbance (2.2.25) at 515 nm using methanol Ras the
compensation liquido
Calculate the percentage content of hydroxyanthracene
glycosides, expressed as sennoside B, using the foIlowing
expression:
A x 1.25
m
i.e. taking the specific absorbance of sennoside B to be 240.
A absorbance at 515 nm;
m = mas s of the substance to be examined, in grams.
STORAGE
Protected from moisture.
____________________________________________ ~ E~
Tinnevelly Senna Fruit *****
** **
(Tinnevelly Senna Pods, Ph Bur monograph 0208) ***
Preparations
Se=a Liquid Extract
Se=a Tablets
When Powdered TinneveIly Se=a Fruit is prescribed or
demanded, material complying with the requirements below
with the exception of Identification test A and the test for
Foreign matter shaIl be dispensed or supplied.
~E~ ____________________________________________
DEFINITION
Dried fruit of Cassia angustijolia Vahl.
Content
Minimum 2.2 per cent of hydroxyanthracene glycosides,
expressed as sennoside B (C4zH3S0Z0; M r 863) (dried drug) .
 2014
CHARACTERS
Slight odour.
IDENTIFICATION
A. Flattened, slightly reniform pods, yellowish-brown or
brown with dark brown patches at the positions
corresponding to the seeds, usually 35-60 mm long and
14-18 mm wide. At one end is a stylar point and at the other
a short stalk. The pods contain 5-8 flattened and obovate
seeds, green or pale brown, with incomplete, wavy, transverse
ridges on the testa.
B. Reduce to a powder (355) (2.9.12) . The powder is brown.
Examine under a microscope using chloral hy drate soludon R.
The powder shows the following diagnostic characters:
epicarp with polygonal cells and a small number of conical
warty trichomes and occasional anomocytic or paracytic
stomata (2.8.3); fibres in 2 crossed layers accompanied by a
crystal sheath of calcium oxalate prisms; characteristic
palisade cells in the seed and stratified cells in the
endosperm; clusters and prisms of calcium oxalate.
C. Thin-layer chromatography (2.2.27) .
Test solution To 0.5 g of the powdered drug (180) (2.9.12)
add 5 mL of a mixture of equal volumes of ethanol
(96 per cent) R and water R and heat to boiling. Centrifuge
and use the supematant liquido
Rejerence solution Dissolve 10 mg of senna extraa CRS in
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
and water R (a slight residue remains).
Plate TLC si/ica gel G plate R.
Mobile phase glacial acetic acid R, water R, ethyl acetate R,
propanol R (1:30:40:40 VIVIV/V).
Application 10 ¡lL, as bands of 20 mm by 2 mm.
Development Over a path of 10 cm.
Drying In airo
Detection Spray with a 20 per cent VIV solution of nitric
acid R and heat at 120 oC for 10 min; allow to cool and
spray with a 50 gIL solution of potassium hydroxide R in
ethanol (50 per cent V/V? R until the zones appear.
Results The principal zones in the chromatogram obtained
with the test solution are similar in position (sennosides B, A,
D and C in the order of increasing RF value), colour and size
to the principal zones in the chromatogram obtained with the
reference solution. Between the zones due to sennosides D
and C a red zone due to rhein-8-g1ucoside may be visible.
The zones due to sennosides D and C are faint in the
chromatogram obtained with the test solution.
D. Place about 25 mg of the powdered drug (180) (2.9.12)
in a conical flask and add 50 mL of water R and 2 mL of
hydrochloric acid R. Heat in a water-bath for 15 min, cool and
shake with 40 mL of ether R. Separate the ether layer, dry
over anhydrous sodium sulfate R, evaporate 5 mL to dryness
and to the cooled residue add 5 mL of dilute ammonia R1.
A yellow or orange colour develops. Heat on a water-bath for
2 mino A reddish-violet colour develops.
TESTS
Foreign matter (2.8.2)
Maxirnum 1 per cent.
Loss on drying (2.2.32)
Maxirnum 12.0 per cent, determined on 1.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 9.0 per cent.
Senna Preparations IV-351
Ash insoluble in hydrochloric acid (2.8.1)
Maxirnum 2.0 per cent.
ASSAY
Carry out the assay protected jrom bright light.
Place 0.150 g of the powdered drug (180) (2.9.12) in a
100 mL flask. Add 30.0 mL of water R, mix, weigh and place
in a water-bath. Heat under a reflux condenser for 15 mino
Allow to cool, weigh and adjust to the original mass with
water R . Centrifuge and transfer 20.0 mL of the supematant
liquid to a 150 mL separating funnel. Add 0.1 mL of dilute
hydrochloric acid R and shake with 3 quantities, each of
15 mL, of chlorojorm R. Allow to separate and discard the
chloroform layer. Add 0.10 g of sodium hydrogen carbonate R
and shake for 3 mino Centrifuge and transfer 10.0 mL ofthe
supematant liquid to a 100 mL round-bottomed flask with a
ground-glass neck. Add 20 mL of jerric chloride solUlion R1
and mix. Heat for 20 min in a water-bath under a reflux
condenser, with the water level above that of the liquid in the
flask; add 1 mL of hydrochloric acid R and heat for a further
20 min, with frequent shaking, to dissolve the precipita te.
Cool, transfer the mixture to a separating funnel and shake
with 3 quantities, each of 25 mL, of ether R previously used
to rinse the flask. Combine the 3 ether layers and wash with
2 quantities, each of 15 rnL, of water R. Transfer the ether
layer to a volumetric flask and dilute to 100.0 mL with
ether R. Carefully evaporate 10.0 mL to dryness and dissolve
the residue in 10.0 mL of a 5 giL solution of magnesium
acetate R in methanol R. Measure the absorbance (2.2.25) at
515 nm using methanol R as the compensation liquido
Calculate the percentage content of hydroxyanthracene
glycosides, expressed as sennoside B, using the following
expression:
A x 1.25
m
taking the specific absorbance of sennoside B to be 240 .
A = absorbance at 515 nm;
m = mass of the substance to be examined, in grams.
STORAGE
Protected from moisture.
____________________________________________ ~Em
Senna Liquid Extract
DEFINITION
Senna Fruit, Alexandrian or 1000 g
Tinnevelly, crushed
Coriander Oil 6 mL
Ethanol (90 per cent) 250 mL
Purified Water, freshly boiled and A sufficient quantity
cooled
Extemporaneous preparation
The following directions apply.
Macerate the crushed Senna Fruit in 5 litres of Purified
Water for 8 hours, decant the clear liquid and strain; repeat
the process twice using 2 litres of Purified Water for each
maceration. Lightly press the marc, strain the expressed
liquid, mix the strained liquid with the previously decanted
0
liquid and heat the combined liquids at 80 for 3 minutes in
a covered vessel. Allow to stand for not less than 24 hours;
filter.
IV-352 Senna Preparations
Evaporate the filtrate to 750 mL under reduced pressure at a
temperarure not exceeding 60°. Separately, dissolve the
Coriander Oil in the Ethanol (90 per cent), add the solution
to the evaporated filtra te and add sufficient Purified Water to
produce 1000 mL. Allow to stand for not less than 24 hoursi
filter.
The extract complies with the requirements stated under Extracts
and with the following requiremems.
TESTS
Ethanol content
21 to 24% v/v, Appendix VIII F, Method III.
Dry residue
17 to 25 % w/v.
Relative density
1.02 to 1.09, Appendix V G .
Standardised Senna Granules
DEFINITION
Standardised Senna Granules contain Alexandrian Senna
Fruit in powder form with suitable excipients. The granules
contain 0.55% w/w of sennosides, calculated as sennoside B.
The granules comply with the requirements stated under Granules
and with the following requirements.
Content of sennosides calculated as sennoside B
0.467 to 0.633% w/w.
IDENTIFICATION
A. To 25 mg, in No. 180 powder, add 50 mL of water and
2 mL of hydrochloric acid, heat in a water bath for
15 minutes, allow to cool and shake with 40 mL of ether.
Dry the ether layer over anhydrous sodium sulfate, filter,
evaporate 5 mL of the filtrate to dryness, cool and add 5 mL
of 6M ammonia to the residuei a yellow or orange colour is
produced. Heat on a water bath for 2 minutes; a reddish
violet colour is produced.
B. In the Assay, the chromatogram obtained with solution
(1) exhibits two peaks corresponding to the peaks due to
sennoside A and sennoside B in the chromatogram obtained
with solution (2).
Loss on drying
0 Maximum time, 60 minutes, Appendix XII A.
When dried at 105 for 5 hours, lose not more than 2.0% of
their weight. Use 5 g.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Shake 2 g of the granules with 50 mL of a 0.3% v/v
solution of aeetic acid adjusted to pH 5.9 with 1M sodium
hydroxide for 30 minutes, centrifuge, filter through a glass
fibre paper (Whatman GF/C is suitable) and use the filtrate.
(2) Prepare solution (2) in the same manner as solution (1)
but using a quantity of Alexandrian senna fntit powder BPCRS
1 mg of sennoside B.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
with end-capped octadeeylsilyl silica gel for chromatography
(5 11m) (Spherisorb ODS 2 is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(e) Use a ftow rate of 2 mL per minute.
(d) Use an ambient column temperarure.
2014
(e) Use a detection wavelength of 350 nm.
(!) Inject 20 ~lL of each solution.
MOB ILE PHASE
17 volumes of acetonitn"le and 83 volumes of a 1% v/v
solution of glacial acetic acid.
DETERMINATION OF CONTENT
Calculate the total content of sennosides A and B as
sennoside B in the granules using the declared content of
sennosides in Alexandrian senna fruit powder BPCRS.
STORAGE
Standardised Senna Granules should be kept in an ainight
container.
LABELLING
The quantity of active ingredient is stated in terms of the
equivalent amount of sennoside B.
Senna Tablets
DEFlNITION
Senna Tablets contain the powdered pericarp of Senna Fruit,
Alexandrian or Tinnevelly.
The tablets comply with the requirements stated under Tablets and
with the following requirements.
Content of total sennosides, calculated as sennoside B
85.0 to 115.0% of the stated amount.
IDENTIFICATION
The powdered tablets exhibit diagnostic strucrures of senna
pericarp. External epidermis of isodiametric ceIls with very
thick outer waIls. Occasional stomata. Trichomes few,
unicellular, conical and wany. Parenchymatous cells from
inner part of a two-to five-layered zone subjacent to the
epidermis, each containing a single prism of calcium oxalate.
Thick-walled fibres in two to four layers, the fibres of the
outer and inner zones respectively with their long axes at
right angles to each other. Surural vascular strands sheathed
by cells containing prisms of calcium oxalatei elements of
seed tissue may also be presento
TESTS
Disintegration
ASSAY
Weigh and powder 20 tablets. Carry out the following
procedure protected from light. To a quantity of the powder
containing the equivalent of 7.5 mg of total sennosides add
30 mL of water, weigh, heat under a reftux condenser on a
water bath for 15 minutes, allow to cool, weigh and restore
the original weight with water. Centrifuge, transfer 20 mL of
the supernatant liquid to a separating funnel, add 0.1 mL of
2M hydrochloric acid, shake with two 15 mL quantities of
ehloroform, allow to separate and discard the chloroform
layers. Add 0.10 g of sodium hydrogen carbonate and shake for
3 minutesi centrifuge and transfer 10 mL of the liquid to a
round-bonomed ftask fitted with a ground-glass neck. Add a
mixrure of 8 mL of iron(m) chloride solution and 12 mL of
water and mix. Heat under a reftux condenser on a water
bath for 20 minutes, add 1 mL of hydrochloric acid and
continue heating for a further 20 minutes, shaking frequently,
until the precipitate is dissolved. Allow to cool, transfer the
mixture to a separating funnel and extract with three 25-mL
quantities of ether previously used to rinse the ftask. Wash the
combined ether extracts with two 15-mL quantities of water
2014
and add sufficient ethe/' to produce 100 mL. Evaporate
10 mL just to dryness on a water-bath and dissolve the
residue in 10 mL of 1M potassium hydroxide, filtering if
necessary through a sintered-glass filter (ISO 4793, porosity
grade 3, is suitable). Measure the absorbance of the resulting
solution without delay at the maximum at 500 nm,
Appendix II B. Calculate the content of total sennosides, as
sennoside B, taking 200 as the value of A(l %, 1 cm) at the
maximum at 500 nm.
LABELLING
The quantiry of active ingredient is stated in terms of total
sennosides expressed as the equivalent content of
sennoside B.
Senna Leaf ***
***
(Ph Bur monograph 0206) ****
Preparation
Standardised Senna Leaf Dry Extract
When Powdered Senna Leaf is prescribed or demanded,
material complying with the requirements below with the
exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _
DEFINITION
Dried leafiets of Cassia senna L. (C. acutijolia Delile), known
as Alexandrian or Khartoum senna, or Cassia angustijolia
Vahl, known as Tinnevelly senna, or a mixture ofthe 2
species .
Content
Minimum 2.5 per cent of hydroxyanthracene glycosides,
expressed as sennoside B (C42H3S020; M r 863) (dried drug).
CHARACTERS
Slight characteristic odour.
IDENTIFICATION
A. C. senna occurs as greyish-green or brownish-green, thin,
fragile leafiets, lanceolate, mucronate, asymmetrical at the
base, usually 15-40 mm long and 5-15 mm wide, the
maximum width being at a point slightly below the centre;
the lamina is slightly undulant with both surfaces covered
with fine, short trichomes. Pinnate venation is visible mainly
on the lower surface, with lateral veins leaving the midrib at
an angle of about 60° and anastomosing to form a ridge near
the margino
Stomatal index (2.8.3) 10-12.5-15.
C. angustifolia occurs as yellowish-green or brownish-green
leaflets, elongated and lanceolate, slightly asymmetrical at the
base, usually 20-50 mm long and 7-20 mm wide at the
centre. Both surfaces are smooth with a very small number of
short trichomes and are frequently marked with transverse or
oblique lines.
Stomatal index (2.8.3) 14-17.5-20
B. Reduce to a powder (355) (2.9.12). The powder is light
green or greenish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters: polygonal epidermal cells showing
paracytic stomata (2.8.3); unicellular trichomes, conical in
shape, with warted walls, isolated or attached to fragments of
epidermis; fibres with a crystal sheath of prismatic crystals of
calcium oxalate; cluster crystals isolated or in fragments of
parenchyma.
Senna Leaf IV-353
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.5 g ofthe powdered drug (180) (2.9.12)
add 5 mL of a mixture of equal volumes of ethanol
(96 per cent) R and water R and heat to boiling. Centrifuge
and use the supematant liquido
Reference solution Dissolve 10 mg of senna extract CRS in
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
and water R (a slight residue remains) .
Plate TLC silica gel G plate R .
Mobile phase glacial acetic acid R, water R, ethyl acetate R,
propanol R (1 :30:40:40 VIVIV/V).
Application 10 flL, as bands of 20 mm by 2 mm.
Development Over a path of 10 cm.
Drying In airo
Detection Spray with a 20 per cent V/V solution of nitric
acid R and heat at 120 oC for 10 mino Allow to cool and
*
* spray with a 50 gIL solution of potassium hydroxide R in
alcohol (50 per cent V/V! R until the zones appear.
Results The principal zones in the chromatogram obtained
with the test solution are sitnilar in position (sennosides B, A,
D and C in the arder of increasing Rp value), colour and size
to the principal zones in the chromatogram obtained with the
reference solution. Between the zones due to sennosides D
and C a red zone due to rhein-8-glucoside may be visible.
D. Place about 25 mg of the powdered drug (180) (2.9.12)
in a conical flask and add 50 mL of water R and 2 mL of
hydrochloric acid R . Heat in a water-bath for 15 min, cool and
shake with 40 mL of ether R . Separate the ether layer, dry
over anhydrous sodium sulfate R, evaporate 5 mL to dryness
and to the cooled residue add 5 mL of dilute ammonia R1.
A yellow or orange colour develops. Heat on a water-bath for
2 mino A reddish-violet colour develops.
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of foreign organs and maximum
1 per cent of foreign elements .
Loss on drying (2.2. 32)
Maximum 12.0 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.5 per cent.
ASSAY
Carry out the assay protected fmm bright light.
Place 0.150 g of the powdered drug (180) (2.9.12) in a
100 mL flask. Add 30.0 mL of water R, mix, weigh and place
in a water-bath. Heat under a reflux condenser for 15 mino
Allow to cool, weigh and adjust to the original mas s with
water R. Centrifuge and transfer 20.0 mL of the supematant
liquid to a 150 mL separating funnel. Add 0.1 mL of dilute
hydrochloric acid R and shake with 3 quantities, each of
15 mL, of chloroform R . Allow to separate and discard the
chloroform layer. Add 0.10 g of sodium hydrogen carbonate R
and shake for 3 mino Centrifuge and transfer 10.0 mL of the
supematant liquid to a 100 mL round-bottomed flask with a
ground-glass neck. Add 20 mL of fem'c chloride solution R1
and mix. Heat for 20 min in a water-bath under a reflux
condenser with the water level aboye that of the liquid in the
flask; add 1 mL of hydrochloric acid R and heat for a further
20 min, with frequent shaking, to dissolve the precipitate.
IV-354 Senna Preparations
Cool, transfer the mixture to a separating funnel and shake
with 3 quantities, each of 25 mL, of ether R previously used
to rinse the ftask. Combine the 3 ether layers and wash with
2 quantities, each of 15 mL, of water R. Transfer the ether
layer to a volumetric ftask and dilute to 100.0 mL with
ether R. Evaporate 10.0 mL carefully to dryness and dissolve
the residue in 10.0 mL of a 5 giL solution of magnesium
acetate R in methanol R. Measure the absorbance (2.2.25) at
515 run, using methanol R as the compensation liquido
Calculate the percentage content of hydroxyanthracene
glycosides, expressed as sennoside B, using the following
expression:
A x 1.25
m hydrochloric acid R. Heat in a water-bath for 15 min, cool and
i. e. taking the specific absorbance of sennoside B to be 240.
A absorbance at 515 nm,
m mas s of the substance to be examined, in grams.
STORAGE
Protected from moisture.
____________________________________________
***
Standardised Senna Leaf Dry
** **
Extraet *****
(Ph Eur monograph 1261)
Ph fUf ___________________________________________
DEFINITION
Standardised dry extract produced from Senna leaj (0206).
Content
5.5 per cent to 8.0 per cent of hydroxyanthracene glycosides,
expressed as sennoside B (C4zH 3S0Z0; M r 863) (dried
extract). The measured content does not deviate from the
value stated on the label by more than ± 10 per cent.
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-80 per cent V/V).
CHARACTERS
Appearance
Brownish or brown powder.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Solvent mixture ethanol (96 per cent) R, water R (50:50 V/V).
Test solution To 0.1 g of the extract to be examined add
5 mL of the solvent mixture and heat to boiling. Cool and
centrifuge. Use the supernatant liquido
Rejerence solution Dissolve 10 mg of senna extract CRS in
1 mL of the solvent mixture (a slight residue remains).
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, water R , ethyl acetate R ,
l-propanol R (1:30:40:40 VIVIV/V).
Application 10 ¡.tL as bands.
Development Over a path of 10 cm.
Drying In air.
Detection Spray with a 20 per cent VIV solution of nitric
acid R and heat at 120 oC for 10 min; allow to cool and
spray with a 50 gIL solution of potassium hydroxide R in
ethanol (50 per cent VIV) R until the zones appear.
2014
Results The principal zones in the chromatogram obtained
with the test solution are similar in position, colour and size
to the principal zones in the Chromatogram obtained with the
reference solution. The chromatograms show in the lower
third a prominent brown zone due to sennoside B and above
it a yellow zone followed by another prominent brown zone
due to sennoside A. In the upper half of the chromatograms
are visible, in order of increasing RF value, a prominent
reddish-brown zone and an orange-brown zone followed by a
faint pink zone and 2 yellow zones. Close to the solvent front
a dark pink zone appears, which may be followed by several
faint zones.
B. Place about 25 mg of the extract to be examined in a
conical fiask and add 50 mL of water R and 2 mL of
shake with 40 mL of ether R . Separate the ether layer, dry
over anhydrous sodium sulfate R, evaporate 5 mL to dryness
and to the cooled residue add 5 mL of dilute ammonia Rl.
A yellow or orange colour develops. Heat on a water-bath for
2 mino A reddish-violet colour develops.
TESTS
Phf~
Loss on drying (2.8.17)
Maximum 5.0 per cent.
Microbial contamination
TAMC: acceptance criterion 104 CFU/g (2.6.12).
TYMC: acceptance criterion 10 2 CFU/g (2. 6.12) .
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6. 13).
ASSAY
Carry out the assay protected from bright light.
Place 0.150 g of the extract to be examined in a 100 mL
ftask, dissolve in water R and dilute to 100.0 mL with the
same solvento Filter the solution, discard the first 10 mL of
the filtrate. Transfer 20.0 mL of the filtrate to a 150 mL
separating funnel. Add 0.1 mL of dilute hydrochloric acid R
and shake with 3 quantities, each of 15 mL, of ether R. Allow
the layers to separate and discard the ether layer. Add 0.10 g
of sodium hydrogen carbonate R to the aqueous layer and shake
for 3 mino Centrifuge and transfer 10. O mL of the
supernatant liquid to a 100 mL round-bottomed fiask with a
ground-glass neck. Add 20 mL ofjerrie ehloride solution Rl
and mix. Heat for 20 min under a reftux condenser in a
water-bath with the water level above that of the liquid in the
ftask; add 3 mL of hydroehlorie acid R and heat for a further
30 min with frequent shaking to dissolve the precipitate.
Cool, transfer the mixture to a separating funnel and shake
with 3 quantities, each of 25 mL, of ether R previously used
to rinse the ftask. Combine the ether layers and wash with
2 quantities, each of 15 mL, of water R. Transfer the ether
layers to a volumetric ftask and dilute to 100.0 mL with
ether R . Evaporate 10.0 mL carefully to dryness and dissolve
the residue in 10.0 mL of a 5.0 giL solution of magnesium
aeetate R in methanol R. Measure the absorbance (2.2.25) at
515 nm using methanol R as the compensation liquido
Calculate the percentage content of hydroxyanthracene
glycosides expressed as sennoside B using the following
expression:
A x 4.167
m
i.e. taking the specific absorbance of sennoside B to be 240.
A absorbance at 515 nm;
111 = mass of the herbal drug to be examined, in grams.
2014
LABELLING
The label sta tes the content of hydroxyanthracene glycosides.
_ _ __ _ _ _ _ _ _ _ __ _ __ _ _ _ _ __ PhEur
Sophora Flower ***
*** ***
(Ph Eur monograph 2639) ***
PhEw _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _
DEFINITlON
Dried, opened flower of Styphnolobium japonicum (L.) Schott
(syn. Sophora japonica L.).
Content:
- minimum 8.0 per cent of total flavonoids, expressed as
rutin (C27H30016; M r 611) (dried drug);
- minimum 6.0 per cent ofrutin (C27H30016; M r 611)
(dried drug).
IDENTIFICATlON
A. The opened flower is crumpled, rolled, and has a very
thin and short pedicel. The dark green or brown,
campanulate calyx is about 3-4 mm long and consists of 5
fused sepals with longitudinal striations at the base, divided
at the apex into 5 slightly bilabiate lobes. The pale yellow or
light yellowish-brown, papilionaceous type corolla is often
broken and measures about 10-15 mm; the upper petal is the
largest, subrounded, with a reflexed apex and a bright yellow
unguis at its interna! base. The other 4 petals are oblongo
There are 10 free stamens surrounding a cylindrical and
curved central style.
Figure 2639.-1. - Illustration for identification test B of
powdered herbal drug of sophora flower
Sophora Flower IV-355
B. Microscopic examination (2.8.23) . The powder is
yellowish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters (Figure 2639.-1): roundish [La] or triangular [Lb]
pollen grains [L] with 3 pores and a smooth exine, about
18 ¡.lm in diameter; isolated covering trichomes [A, B, F, O]
of varying lengths (60-660 [lm), slightly flexed, usually
consisting of 1 or 2 basal cells and a long pointed distal cell,
with smooth or slightly warty walls; fragments of sepals [C]
composed of anomocytic stomata ( 2.8.3) with 4-8 subsidiary
cells [Ca], covering trichomes [Cb] or their scars [Cc];
fragments of petals [G, H] with cells covered by a fine1y
striated cmiele [Ga, Ha], sometimes accompanied by fine
annular or spiral vessels [Hb] and parenchyma with sorne
cells containing crystalline masses of rutin [Hc]; fragments of
parenchyma [E] from the sepals containing prisms of calcium
oxalate [Ea] and crystalline mas ses ofrutin [Eb]; fragments
of anthers [N] showing the characteristic fibrous layer, in
transverse section [Na] or in surface view [K], and immature
pollen grains [Nb]; free prisms of calcium oxalate [M].
Examine under a microscope using chloral hydrate solution R,
without heating the preparation: brownish-yellow rutin
crystals are visible, free or ineluded in cells, as crystalline
masses [Eb, Hc, TI or in fan-shaped aggregates of very fine
needles [D, Ec, Gb].
C . Thin-layer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (355)
(2.9. 12) add 5.0 mL of methanol R, sonicate for 10 min and
filter.
Reference solution Dissolve 10 mg of hyperoside R and 10 mg
of rutin R in 10 mL of methanol R.
Plate TLC siliea gel plate R (5-40 [lm) [or TLC silica gel
plate R (2-10 [lm)].
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
(10:10 :80 VIV/V) .
Application 10 [lL [or 5 [lL] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 6 cm].
Drying In air.
Detection Treat with a 10 giL solution of diphenylboric acid
aminoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R, allow to dry in air
for about 30 min, and examine in ultraviolet light at 365 nm.
Results See be10w the sequence of fluorescent zones present
in the chromatograms obtained with the reference solution
and the test solution. Furthermore, other faint fluorescent
zones may be present in the chromatogram obtained with the
test solution.
Top of the plate
An orange-yellow zone
--- - --
A brown zone
Hyperoside: a yellowish·orange
zone
--- ---
2 green zones
Rutin: an orange·yellow zone A very intense orange·yellow zone
JrutiT!l
Reference solution Test solution
 IV-356 Sophora Flower-Bud 2014

TESTS -- stationary phase: octadeeylsilyl siliea gel for ehromatography R

Foreign matter (2.8.2) (5 ¡.lm).
Maximum 5 per cent of fiower buds and maximum Mobile phase:
2 per cent of other foreign matter. -- mobile phase A: 1 per cent V/V solution of glacial aeetic
Loss on drying (2.2.32) acid R;
Maximum 11.0 per cent, determined on 1.000 g of the -- mobile phase B: methanol R;
powdered herbal drug (355) (2.9.12) by drying in an oven at Time Mobile phase A Mobile phase B
105 oC for 2 h. (min) (per een! VM (pereen! VM
0·5 68 32
Total ash (2.4.16)
Maximum 9.0 per cent. 5 - 20 68 ..... 50 32 ..... 50

ASSAY 20·30 50 ..... O 50 ..... 100

Total ftavonoids
Stoek solution Place 2.000 g of the powdered herbal drug
(355) (2.9. 12) in the cartridge of a continuous-extraction
apparatus (Soxhlet type) . Add 100 mL of heptane R and heat
under a refiux condenser until the extraction liquid is
colourless. Allow to cool and discard the heptane.
Add 90 mL of methanol R and continue the extraction with
heating under a refiux condenser until the extraction liquid is
colourless. Allow to coo!. Transfer the methanolic solution to
a 100 mL volumetric fiask. Rinse the extraction fiask with a
few millilitres of methanol R. Combine the methanolic
solutions and dilute to 100.0 mL with methanol R. Dilute
10.0 mL of this solution to 100.0 mL with water R and shake
vigorously.
Test solution Dilute 10.0 mL of the stock solution to
100.0 mL with a 20 gIL solution of aluminium ehloride R in
methanol R .
Compensation solution Dilute 10.0 mL of the stock solution
to 100.0 mL with methanol R .
Measure the absorbance (2.2.25) of the test solution after
15 min by comparison with the compensation solution at
425 nm.
Calculate the percentage content of total fiavonoids,
expressed as rutin, using the following expression:
A x 1000
m x 37
30 - 35 O 100
Flow rate 1.3 mUmin.
Detection Spectrophotometer at 350 nm.
Injeetion 20 ~1L.
R elative retention With reference to rutin (retention
time = about 17 min): apigenin 7-glucoside = about 1.1.
System suitability Reference solution (b):
-- resolution: minimum 1.5 between the peaks due to rutin
and apigenin 7-glucoside.
Calculate the percentage content of rutin using the following
expression:
area of the peak due to rutin in the chromatogram
obtained with the test solution;
area of the peak due to rutin in the chromatogram
obtained with reference solution (a);
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mass of rutoside trihydrate CRS used to prepare
reference solution (a), in grams;
p assigned percentage content of rutin in rutoside
trihydrate CRS.
____________________________________________ nEm

i.e. taking the specific absorbance of rutin to be 370.

A absorbance of the test solution at 425 nm;
m mas s of the herbal drug to be examined, in grams .
Rutin
Liquid chromatography (2.2.29) .
Test solution Place 0.500 g of the powdered herbal drug
(355) (2.9.12) in a conical fiask and add 50.0 mL of
methanol R . Weigh, sonicate for 30 min and allow to coo!.
Weigh and compensate for the loss of solvent with
methanol R. Shake vigorously, filter, and dilute 2.0 mL of the
filtrate to 10.0 mL with methanol R.
Referenee solurion (a) Dissolve 10.0 mg of rutoside
trihydrate CRS in 2 mL of methanol R and dilute to 10.0 mL
with a 50 per cent V/V solution of methanol R. Dilute 2.0 mL
of this solution to 10.0 mL with a 50 per cent VIV solution
of methanol R.
R eferenee solution (b) Dissolve 10.0 mg of apigenin
7-glueoside R and 10.O mg of rutin R in 2 mL of methanol R
and dilute to 10.0 mL with a 50 per cent V/V solution of
methanol R. Dilute 2.0 mL ofthis solution to 10.0 mLwith a
50 per cent VIV solution of methanol R.
Column:
-- size: 1 = 0.25 m, 0 = 4.6 mm;
Sophora Flower-Bud *****
** **
(Ph Bur monograph 2427) ***
n Em ___________________________________________
DEFINITION
Whole, dried fiower bud of Styphnolobium japonicum (L.)
Schott (syn. Sophorajaponica L.).
Content:
-- mínimum 20.0 per cent of total fiavonoids, expressed as
rutin (C27H30016; Mr 611) (dried drug);
-- minimum 15.0 per cent ofrutin (C27H30016; Mr 611)
(dried drug).
IDENTIFICATION
A. The fiat fiower bud, ovoid or ellipsoid, has a very thin and
short pedicel and is about 7-10 mm long and 3-4 mm thick.
The dark green or brown calyx, forming the lower pan of the
bud, is about 3-4 mm long and consists of 5 fused sepals
with longitudinal striations at the base. The pale yellow or
brownish-yellow corolla, unopened, delicate, extends beyond
the calyx and contains 10 free stamens surrounding a central
style.
2014 Sophora Flower-Bud IV-357

E. Microscopic examination (2.8.23). The powder is pale Plate TLC silica gel plate R (5-40 ¡.lm) [or TLC silica gel
yellow. Examine under a microscope using chloral hydrate plate R (2-10 ¡.lm)].
solution R. The powder shows the following diagnostic Mobile phase anhydrous formic acid R, water R, ethyl acetate R
characters (Figure 2427.-1): roundish [La] or triangular [Lb] (10:10:80 VIV/V) .
pollen grains [L] with 3 pores and a smooth exine, about Application 10 ¡.lL [or 5 ¡.lL] as bands of 10 mm [or 8 mm].
18 ¡.lm in diameter; isolated covering trichomes [A, E, F, O]
Development Over a path of 10 cm [or 6 cm].
ofvarying lengths (60-660 ¡.lm), slightly flexed, usually
consisting of 1 or 2 basal cells and a long pointed distal cell, Drying In airo
with smooth or slightly warty walls; fragments of sepals [C] Detection Treat with a 10 giL solution of diphenylboric acid
composed of anomocytic stomata (2.8.3) with 4-8 subsidiary aminoethyl ester R in methanol R and then with a 50 gIL
cells [Ca], covering trichomes [Cb] or their scars [Ce]; solution of macrogol 400 R in methanol R, allow to dry in air
fragments of petals [G, H] with cells covered by a finely for about 30 min, and examine in ultraviolet light at 365 nm.
striated cuticle [Ga, Ha], sometimes accompanied by fine Results See below the sequence of fluorescent zones present
annular or spiral ves seis [Hb] and parenchyma with sorne in the chromatograms obtained with the reference solution
cells containing crystalline mas ses of rutin [He]; fragments of and the test solution. Furthermore, other faint fluorescent
parenchyma [E] from the sepals containing prisms of calcium zones may be present in the chromatogram obtained with the
oxalate [Ea] and crystalline masses of rutin [Eb]; fragments test solution.
of anthers [N] showing the characteristic fibrous layer, in
transverse section [Na] or in surface view [K], and immature
pollen grains [Nb]; free prisms of calcium oxalate [M] . Top of the pIate
Examine under a microscope using chloral hydrate solution R, An orange-yellow zone
without heating the preparation: brownish-yellow rutin
crystals are visible, free or included in cells, as crystalline
--- ---
masses [Eb, He, TI or in fan-shaped aggregates of very fine
A brown zone
needles [D, Ec, Gb] .
Hyperoside: a yellowish·orange
zone
--- ---
2 green zones

Rutin: an orange-yellow zone A very intense orange-yellow zone

(rutin)
Reference soIution Test soIution

Figure 2427.-1. - Illustration for identification test B of
powdered herbal drug of sophora flower-bud
C. Thin-Iayer chromatography (2.2.27).
Test solution To 0.2 g of the powdered herbal drug (355)
(2. 9.12) add 5.0 mL of methanol R, sonicate for 10 min and
filter.
Reference solution Dissolve 10 mg of hyperoside R and 10 mg
of rutin R in 10 mL of methanol R.
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of opened flowers and maximum
2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 11.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maxirnum 9.0 per cent.
ASSAY
Total flavonoids
Stock solution Place 1.00 g of the powdered herbal drug
(355) (2.9.12) in the cartridge of a continuous-extraction
apparatus (Soxhlet type). Add 100 mL of heptane R and heat
under a reflux condenser until the extraction liquid is
colourless. Allow to cool and discard the heptane.
Add 90 mL of methanol R and continue the extraction with
heating under a reflux condenser until the extraction liquid is
colourless. Allow to cool. Transfer the methanolic solution to
a 100 mL volumetric flask. Rinse the extraction flask with a
few millilitres of methanol R. Combine the methanolic
solutions and dilute to 100.0 mL with methanol R. Dilute
10. O mL of this solution to 100. O mL with water R and shake
vigorously.
Test solution Dilute 10.0 mL of the stock solution to
100.0 mL with a 20 gIL solution of aluminium chloride R in
methanol R.
Compensation solution Dilute 10.0 mL of the stock solution
to 100.0 mL with methanol R.
IV-358 Spearmint Oil 2014

Measure the absorbance (2.2.25) of the test solution after
15 min by comparison with the compensation solution at
425 nm.
Calculate the percentage content of total ftavonoids,
expressed as rutin, using the following expression:
A x 1000
mass of the herbal drug to be examined used to
prepare the test solution, in grams;
mas s of rutoszde tn'hydrate CRS used to prepare
reference solution (a), in grams;
p assigned percentage content of rutin in rutoside
trihydrate CRS.
____________________________________________ ~E~

m x 37

i.e. taking the specific absorbance of rutin to be 370.

A = absorbance of the test solution at 425 nm; Spearmint Oil
m = mass of the herbal drug to be examined, in grams. DEFINITION
Rutin Spearrnint Oil is obtained by distillation from fresh ftowering
Liquid chromatography (2.2.29) . plants of Mentha spicata L. and Mentha x cardiaca (Gray)
Bak.
Test solution Place 0.200 g of the powdered herbal drug
(355) (2.9.12) in a conical ftask and add 50.0 mL of CHARACTERISTICS
methanol R . Weigh, sonicate for 30 min and allow to coo1. A c1ear, colourless, pale yellow or greenish yellow liquid
Weigh and compensate for the loss of solvent with when recently distilled, but becoming darker and viscous on
methanol R. Shake vigorously, filter, and dilute 2.0 mL of the keeping; visibly free from water; odour, that of spearmint.
filtrate to 10.0 mL with methanol R. IDENTIFICATION
Reference solurian (a) Dissolve 10.0 mg of rutoside Examine the chromatograms obtained in the test for
trihydrate CRS in 2 mL of methanol R and dilute to 10.0 mL Chromatographic profile. The retention times of the principal
with a 50 per cent V/V solution of methanol R. Dilute 2.0 mL peaks in the chromatogram obtained with solution (1) are
of this solution to 10.0 mL with a 50 per cent VIV solution similar to those of the principal peaks in the chromatogram
of methanol R. obtained with solution (2).
Reference solution (b) Dissolve 10.0 mg of apigenin
TESTS
7-glucoside R and 10. O mg of rutin R in 2 mL of methanal R
Optical rotation
and dilute to 10.0 mL with a 50 per cent V/V solution of
American-type oil, -45° to - 60°; Chinese-type oil, -50 0
methanol R. Dilute 2.0 mL of this solution to 10.0 mL with a
to -62°; Appendix V F.
50 per cent VIV solution of methanol R.
Column: Refractive index
-- size: 1 = 0.25 m, 0 = 4.6 mm; 1.484 to 1.491, Appendix V E.
-- stationary phase: octadecylsilyl silica gel for chromatography R Solubility in ethanol
(5 ~m). Soluble, at 20°, in 1 part of ethanol (80%), Appendix X M .
Mobile phase: The solution may become c10udy when diluted.
-- mobile phase A: 1 per cent VIV solution of glacial acetic Weight per mL
acid R; American-type oil, 0.917 to 0.934 g; Chinese-type oil,
mobile phase B: methanol R; 0.935 to 0.952 g, Appendix V G .
Time Mobile phase A Mobile phase B Chromatographic profile
(min) (per cen! VIJI) (per cen! VIJI) Carry out the method for gas chromatography,
0·5 68 32 Appendix III B, using the following solutions. Solution (1) is
5·20 68 --750 32 --7 50 the substance being examined. For solution (2) mix carefully
0.1 g of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of
20·30 50 --7 O 50 --7 100
(+ )-isomenthane, 0.4 g of menthyl acetate, 0.2 g of pulegone,
30 - 35 O 100 0.6 g of menthol and 0.1 g of carvane with 1 g of hexane.
The chromatographic procedure may be carried out using
Flow rate 1.3 mUmin.
(a) a glass capillary column (25 m to 60 m x about
Detection Spectrophotometer at 350 nm. 0.25 mm) coated with polyethylene glycol 20, 000 as bonded
Injection 20 ~L. phase (Carbowax 20M is suitable) and (b) helium as the
Relative retention With reference to rutin (retention carrier gas at a ftow rate of 1.5 mL per minute. Maintain the
time = about 17 min): apigenin 7-glucoside = about 1.1. temperature of the column at 55° for 6 minutes then increase
System suitability Reference solution (b): it at the rate of 4° per minute to 180°; keep the injection port
-- resolution: minimum 1.5 between the peaks due to rutin temperature at 220° and the detector at 230°.
and apigenin 7-glucoside. Inject 0.1 ~L ofsolution (2). When the chromatograms are
Calculate the percentage content of rutin using the following recorded in the prescribed conditions, the components elute
expression: in the order indicated in the composition of the reference
solution. Record the retention times of these substances.
The test is not valid unless the number of theoretical plates
calculated from the limonene peak is at least 30,000 and the
resolution factor between the peaks corresponding to limonene
and cineole is at least 1.5.
Al area of the peak due to rutin in the chromatogram
obtained with the test solution; Inject 0.1 ~L ofsolution (1). Using the retention times
A2 area of the peak due to rutin in the chromatogram deterrnined from the chromatogram obtained with solution
obtained with reference solution (a); (2) locate the components of the reference solution on the
2014
chromatogram obtained with solution (1) (disregard the peak
due to hexane). Determine the percentage content of the
components by normalisation. The percentages are within the
following ranges:
Limonene 2.0 to 25 .0% .
Cineole less than 2.5% .
Menthone les s than 2.5%.
Isomenthone less than 1.0%.
Menthyl aceta te less than 1.0% .
Pulegone less than 0.5%.
Mentholless than 2.0% .
Carvone Not less than 55.0%.
STORAGE
Spearmint Oil should be kept in a well-filled container and
protected from light.
LABELLING
The label states whether the oil is American-type oil or
Chinese-type oil.
Squill
Preparations
Squill Liquid Extract
Squill Oxymel
When Powdered Squill is prescribed or demanded, material
complying with the appropriate requirements below shall be
dispensed or supplied.
DEFINITION
Squill consists of the bulb of Dnmia mantima (L.) Stearn,
collected soon after the plant has flowered, divested of its
dry, outer, membranous coats, cut into transverse slices and
dried. Ir is known in commerce as white squill.
IDENTIFlCATION
A. Transverse slices, about 5 to 8 mm thick, occurring as
straight or curved triangular pie ces about 5 to 50 mm long
and 3 to 8 mm wide at mid-point, tapering towards each
end, yellowish white, texture horny, somewhat translucent,
breaking with an almost glassy fracture when quite dry, but
readily absorbing moisture when exposed to the air and
becoming tough and flexible; transversely cut surface showing
a single row of prominent, vascular bundles near the concave
edge and numerous smaller bundles scattered throughout the
mesophyll.
B. Epidermis: cells polygonal and axially elongated, 1 to
2 times longer than wide, cuticle thick, stratified; stomata
very rare, anomocytic, Appendix XI R, and nearly circular in
outline, about 50 to 60 ¡.lm in diameter; mesophyll of
colourless, thin-walled parenchyma containing very
occasional starch granules, many cells containing bundles of
acicular crystals of calcium oxalate embedded in mucilage,
crystals up to about 1 mm long and about 1 to 15 ¡.lm wide;
other cells containing sinistrin; vascular bund1es collateral,
scattered throughout the mesophyll; xylem vessels with spiral
and annular wall thickening; trichomes absent.
C. The mucilage contained in the cells of the mesophyll is
stained red with alkaline corallin solution bUl produces no red
colour with ruthenium red solution and no purple colour with
O.OIM iodine.
TESTS
Acid-insoluble ash
Not more than l.5%, Appendix XI K, Method 1.
Squill Preparations IV-359
Extractive soluble in ethanol (60%)
Not less than 68.0%, Appendix XI Bl. Use material that has
been dried for 1 hour at 105° and powdered.
STORAGE
Squill should be stored in a dry place.
Indian Squill
Preparation
Squill Oxymel
When Powdered lndian Squill is prescribed or demanded,
material complying with the appropriate requirements below
shall be dispensed or supplied.
DEFINITION
lndian Squíll consists of the bulb of Drimia indica (Roxb.) J
P Jessop, collected soon after the plant has flowered, divested
of dry, outer membranous coats and usually cut
longitudinally into slices and dried.
CHARACTERISTICS
Odourless or almost odourless.
Macroscopical
Curved or irregularly shaped strips, about lOto 50 mm long,
3 to 10 mm wide and 1 to 3 mm thick, frequently tapering
towards the ends, occasionally grouped three or four together
and attached to a portion of the axis; ridged in the direction
of their length and varying in colour from pale yellowish
brown to buff; brittle when dry, but tough and flexible when
exposed to airo
Microscopical
Epidermis: cells tetrahedral to hexahedral, thin-walled, three
to five times longer than wide, having a thick, striated cuticle;
stomata rare, anomocytic, Appendix XI R, circular in outline,
40 to 42 ¡.tm in diameter; mesophyll of thin-walled polygonal
cells containing mucilage, sorne cells also containing bundles
of acicular crystals of calcium oxalate, 20 to 900 ¡.tm in
length; vascular bundles collateral, scattered throughout the
mesophyll; xylem vessels with spiral and annular wall
thickening; trichomes and starch absent.
IDENTIFICATION
The mucilage contained in the cells of the mesophyll is
stained red with alkaline corallin solution and reddish purple
with O.OIM iodine.
TESTS
Ash
Not more than 6.0%, Appendix XI J.
STORAGE
lndian Squill should be stored in a dry place.
Squill Liquid Extract
DEFINITION
Squill Liquid Extract is prepared by extracting Squill with
Ethanol (70 per cent) .
Extemporaneous preparation
The following formula and directions apply.
Squill, in coarse powder 1000 g
Ethanol (70 per cent) A sufficient quantity
Exhaust the Squill, in coarse powder, with Ethanol
(70 per cent) by percolation, Appendix XI F. Reserve the first
850 mL of the percolate; evaporate the subsequent percolate
to the consistence of a soft extract and dissolve it in the
IV-360 Squill Preparations 2014

reserved portion. Add sufficient Ethanol (70 per cent) to Tolu Syrup 300 mL
produce 1000 mL and filter. The linctus complies with the requiremenlS stated under Oral
The extract complies with the requiremenrs stated under Extracts Liquids and with the following requirements.
and with the following requiremenlS. Content of anhydrous morphine, CI7Hl~03
TESTS 0.013 to 0.020% w/v.
Ethanol content TESTS
34 to 50% v/v, Appendix VIII F, Method III. Ethanol content
Dry residue 18.0 to 22.0% v/v, Appendix VIII F.
40 to 55 % w/v.
ASSAY
Relative density To 12 g add 5 mL of water and 1 mL of 5M ammonia and
1.00 to 1.14, Appendix V G. extract with 30 mL of a mixture of equal volumes of ethanol
(96%) and chloroform and then with two 22.5-mL quantities
of a mixture of 2 volumes of chloroform and 1 volume of
ethanol (96%) , washing each extract with the same 20 mL of
Squill Oxymel a mixture of equal volumes of ethanol (96%) and water.
Evaporate the combined extracts, extract the residue with
DEFINITION 10 mL of calcium hydroxide solucion, filter and wash the filter
Squill, bruised or lndian 50 g with 10 mL of calcium hydroxide soluuon. To the combined
Squill, bruised filtrate and washings add 0.1 g of ammonium sulfate, extract
Acetic Acid (33 per cent) 90 mL or a sufficient quantity with two 10 mL quantities of ethanol-free chloroform, wash the
Purified Water, freshly 250 mL combined extracts with 10 mL of water and discard the
boiled and cooled chloroform solution. To the combined alkaline liquid and
Purified Honey A sufficient quantity aqueous washings add 10 mL of 1M hydrochloric acid, heat on
Extemporaneous preparation a water bath 10 remove any chloroform, cool and dilute to
The following directions apply. 100 mL with water. To 20 mL of this solution add 8 mL of a
Macerate the Squill or the lndian Squill with the Acetic Acid freshly prepared 1.0% w/v solution of sodium nirrite, allow 10
(33 per cent) and the Purified Water for 7 days with stand for 15 minutes, add 12 mL of 5M ammonia, dilute to
occasional agitation, strain, press out the liquid, heat the 50 mL with water and measure the absorban ce of a 4-cm layer
mixed liquids to boiling, filter whilst hot, cool, determine the of the resulting solution at the maximum at 442 nm,
content of acetic acid, add sufficient Acetic Acid Appendix II B, using in the reference cell a solution prepared
(33 per cent) to the remainder of the filtrate to produce a in the same manner and at the same time but using 8 mL of
solution containing about 8.5 % w/v of acetic acid and mix. water in place of the solution of sodium nitrite. Calculate the
To every three volumes of the resulting solution add seven content of C17H19N03 from a calibration curve prepared
volumes of Purified Honey and mix thoroughly. using quantities of 2,4, 6 and 8 mL of a 0.008% w/v
solution of anhydrous morphine in O.IM hydrochloric acid, each
Content of acetic acid, C 2H 40 2 diluted to 20 mL with O.lM hydrochloric acid and using the
2.210 2.7% w/v. method described aboye beginning at the words 'add 8 mL
TESTS .. .'. Determine the weight per mL of the linctus,
Optical rotation Appendix V G, and calculate the content of C 17 H 19 N0 3,
+ 0.6° to _3.0 Appendix V F, when measured in a 25% w/v
0
, weight in volume.
solution in water decolourised, if necessary, with activated
charcoal.
Weight per mL
1.260 to 1.270 g, Appendix V G .
Paediatric Opiate Squill Linctus
ASSAY Opiate Linctus for lnfants; Paediatric Opiate Squill Oral
Dilute 20 mL with 20 mL of carbon dioxide-free water and Solution
titrate with 1M sodium hydroxide VS using phenolphthalein
DEFINITION
solution Rl as indica1Or. Each mL of 1M sodium hydroxide VS
is equivalent 10 60.05 mg of C2 H 4 0 2 • Paediatric Opiate Squill Linctus is an oral soluuon containing
6% v/v each of Squill Oxymel and Camphorated Opium
Tincture in a suitable vehicle with a tolu ftavour.
Extemporaneous preparation
The following formula applies .
Opiate Squill Linctus Squill Oxymel 60 mL
Compound Squill Linctus; Gee's Linctus; Opiate Squill Oral Camphorated Opium Tincture 60mL
Solution Tolu Syrup 60 mL
DEFINITION Glycerol 200 mL
Opiate Squill Linctus is an opalescent oral solution containing Syrup Sufficient to produce
33% v/v each of Squill Oxymel and Camphorated Opium 1000 mL
Tincture in a suitable vehicle with a tolu ftavour. The linclUs complies wirh the requirements s[ated undel' Oral
Extemporaneous preparation Liquids and with the following requirements.
The following formula applies. Content of anhydrous morphine, C17Hl~03
Squill Oxymel 300 mL 0.002410 0.0036% w/v.
Camphorated Opium Tincture 300 mL
 2014
ASSAY
To 32 g add 5 mL of water and 1 mL of 5M ammonia and
extract with 30 mL of a mixture of equal volurnes of ethanol
(96%) and ehloroform and then with two 22.5-rnL quantities
of a mixture of 2 volumes of chloroform and 1 volume of
ethanol (96%), washing each extract with the same 20 mL of
a mixture of equal volumes of ethanol (96%) and water.
Evaporate the combined extracts, extract the residue with
10 mL of calcium hydroxide solution, filter and wash the filter
with 10 mL of ealeium hydroxide solution. To the combined
filtrate and washings add 0.1 g of ammonium sulfate, extract
with two 10 mL quantities of ethanol-free ehloroform, wash the
combined extracts with 10 mL of water and discard the
chloroform solution. To the combined alkaline liquid and
aqueous washings add 5 mL of 1M hydroehlorie aeid, heat on
a water bath to remove any chloroform, cool and dilute to
50 mL with water. To 20 mL of this solution add 8 mL of a
freshly prepared 1.0% w/v solution of sodiwn nitn"te, aIlow to
stand for 15 minutes, add 12 mL of 5M ammonia, dilute to
50 mL with water and measure the absorbanee of a 4-cm layer
of the resulting solution at the maximum at 442 nm,
Appendix II B, using in the reference ceIl a solution prepared
in the same manner and at the same time but using 8 mL of
water in place of the solution of sodium nitrite. Calculate the
content of C 17H 19 N0 3 from a calibration curve prepared
using quantities of 2, 4, 6 and 8 mL of a 0.008% w/v
solution of anhydrous morphine in O.IM hydroehlorie acid, each
diluted to 20 mL with O.IM hydroehlorie acid and using the
method described aboye beginning at the words 'add 8 mL
... '. Determine the weight per mL of the linctus,
Appendix V G, and calculate the content of C17H19N03,
weight in volume.
St. John's Wort
Hypericum
(Ph Bur monograph 1438)
Preparation
Sto John's Wort Dry Extract, Quantified
~E~ ____________________________________________
DEFINITION
Whole or fragrnented, dried fiowering tops of Hyperiewl1
perforatum L., harvested during fiowering time.
Content
Minimum 0.08 per cent of total hypericins, expressed as
hypericin (C30HI60S; M r 504.4) (dried drug).
IDENTIFICATION
A. The branched and bare stem shows 2 more or less
prominent longitudinal ridges. The leaves are opposite,
sessile, exstipulate, oblong-oval and 15-30 mm long; present
on the leaf margins are glands which appear as black dots
and over aIl the surface of the lea ves many smaIl, strongly
translucent excretory glands which are visible in transmitted
light. The fiowers are regular and form corymbose c1usters at
the apex of the stem. They have 5 green, acute sepals, with
black secretory glands on the margins; 5 orange-yeIlow
petals, also with black secretory glands on the margins;
3 sta minal blades, each divided into many orange-yeIlow
stamens and 3 carpels surmounted by red styles.
Sto John's Wort IV-361
The drug may also show the folIowing: irnrnature and ripe
fruits and seeds. Immature fruits are green or yelIowish, seeds
are whitish . Occasional ripe fruits may be present; these are
dry trilocular capsules containing numerous seeds, brown,
broad or smalI-ovate, 5-10 mm long, with broad linear or
punctiform glands, irregularIy striated ducts, conducting
secretions. Ripe seeds are 1-1.3 mm long, cylindrical or
trigonous, shortly pointed at both ends, brown or almost
black, minutely pitted 10ngitudinalIy.
B. Microscopic examination (2.8.23). The powder is
greenish-yelIow. Examine under a microscope using ehloral
hydrate solution R. The powder shows the folIowing diagnostic
characters (Figure 1438.-1): fragrnents of the leaf epidermis
[A, B) or stems [H) with paracytic [Ab, Ha), anisocytic
[Ac, Bb, Hb) or anomocytic [Ae) stomata ( 2.8.3); fragrnents
of the leaf epidermis often accompanied by palisade
parenchyma [Ad, Be); polygonal celIs of the upper epidermis
with thickened and beaded walls [Ba); more or less sinuous,
thin-waIled ceIls of the lower epidermis [Aa); fragrnents of
the leaf and sepal [E) with large, red-pigmented oil glands
[Ea) associated with palisade parenchyrna [Eb) and smalI
ves seIs [Ec); elongated ceIls of fragrnents of the petal
epidermis with straight or wavy anticlinal walIs ill; vessels
[D) with reticulate or pitted walIs [Da) and groups of thick-
waIled fibres [Db); fragrnents of the central parenchyma of
the stems [K) with lignified and pitted rectangular celIs [Ka)
sometimes associated with ves seIs [Kb); fragrnents ofthe
anthers [F) showing the central part consisting of smaIl celIs
containing cluster crystals of calcium oxalate [Fb) and celIs
from the fibrous layer [Fa); fragrnents of the staminal
filament with elongated, thin-walIed celIs with a striated
cuticle [C); numerous polIen grains with 3 germinal pores
and a smooth exine, occurring singly [G) or in dense groups.
Figure 1438.-1. - Illustration for identification test B of
powdered herbal drug of Sto John 's wort
IV-362 Sto John's Wort Preparations
C. Thin-Iayer chromatography (2.2.27).
Test solutwn Stir 0.5 g of the powdered herbal drug (500)
(2.9.12) in 10 mL of methanol R in a water-bath at 60 oC for
10 min and filter.
Reference solution Dissolve 5 mg of hyperoside R and 5 mg of
rutin R in methanol R, then dilute to 5 mL with the same
solvento
Plate TLC silica gel plate R.
Moblle phase anhydrous formic acid R, water R, ethyl acetate R
(6:9:90 VIV/V).
Applicatwn 10 JlL of the test solution and 5 JlL of the
reference solution, as bands of 10 mm.
Development Over a path of 10 cm.
Drying At 100-105 oC for 10 min.
Detection Treat with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R. After abour 30 min,
examine in ultraviolet light at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower third a zone due to rutin and
aboye it a zone due to hyperoside, both with yellow-orange
fiuorescence. The chromatogram obtained with the test
solution shows in the lower third 2 reddish-orange
fiuorescent zones due to rutin and hyperoside, and in the
lower part of the upper third a zone due ro pseudohypericin
and aboye it a zone due to hypericin, both with red
fiuorescence. Other yellow or blue fiuorescent zones are
visible.
TESTS
Foreign matter (2.8.2)
Maxirnum 3 per cent of stems with a diameter greater than
5 mm and maximum 2 per cent of other foreign matter.
Loss 00 dryiog (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (500) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Test solution In a 100 mL round-bottomed fiask, introduce
0.800 g of the powdered herbal drug (500) (2.9.12), 60 mL
of a mixture of 20 volumes of water R and 80 volumes of
tetrahydrofuran R and a magnetic stirrer. Boil the mixture in a
water-bath at 70 oC under a refiux condenser for 30 min.
Centrifuge (2 min at 700 g) and decant the supematant into
a 250 mL fiask. Take up the residue with 60 mL of a
mixture of 20 volumes of water R and 80 volumes of
tetrahydrofuran R. Heat again under a refiux condenser for
30 min. Centrifuge (2 min at 700 g) and decant the
supematant. Combine the extracts and evaporate to dryness.
Take up the residue with 15 mL of methanol R with the help
of ultrasound and transfer to a 25 mL measuring fiask. Rinse
the 250 mL fiask wirh methanol R and dilute to 25.0 mL with
the same solvento Centrifuge again, filter 10 mL through a
syringe filter (0.2 Jlm). Discard the first 2 millilitres of the
filtrate. Introduce 5.0 mL of the filtrate into a measuring
fiask and dilute to 25.0 mL with methanol R.
Compensation IUjuid methanol R.
Measure the absorbance (2.2.25) at 590 nm of the test
solution, by comparison with the compensation liquido
Calculate the percentage content of total hypericins,
expressed as hypericin, using the following expression:
2014
A x 125
m x 870
i.e. taking the specific absorbance of hypericin to be 870.
A absorbance at 590 nm;
m = mas s of the herbal drug to be examined, in grams.
__________________________________________
~E~
Quantified St. John's Wort Dry
Extract
(Ph Bur monograph 1874)
~E~ __________________________________________
DEFINITION
Quantified dry extract obtained from Sto John 's wort (1438).
Conteot:
-- total hypericins, expressed as hypericin (C30H160S; M r
504.5): 0.10 per cent to 0.30 per cent (anhydrous
extract);
-- fiavonoids, expressed as rutin (C27H30016; M r 610.5) :
minimum 6.0 per cent (anhydrous extract);
-- hyperforin (C3sHs204; M r 536.8): maximum 6.0 per cent
(anhydrous extract) and not more than the content stated
on the labe!'
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-80 per cent V/V) or methanol
(50-80per cent V/V) .
CHARACTERS
Appearance: brownish-grey powder.
IDENTIFICATION
Thin-Iayer chromatography (2.2.27).
Test solutwn Disperse 0.25 g of the extract to be examined
in 5 mL of methanol R.
Reference solution Dissolve 5 mg of rutin R and 5 mg of
hyperoside R in methanol R and dilute to 10 mL with the same
solvente
Plate TLC silica gel plate R (5-40 ¡.¡m) [or TLC silica gel
plate R (2-10 Jlm)] .
Mobile phase anhydrous formic acid R, water R, ethyl aceta te R
(6:9:90 VIV/V).
Application 10 ¡.¡L [or 5 JlL] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 7.5 cm] .
Drying At 100-105 oC for 10 min.
Detection Treat with a 10 gIL solution of diphenylboric acid
aminoethyl ester R in methanol R and then with a 50 gIL
solution of macrogol 400 R in methanol R. Examine after
about 30 min in ultraviolet light at 365 nm.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other fiuorescent zones may be
present in the chromatogram obrained with the test solution.
2014 Sto John's Wort Preparations IV-363

Top of the plate area of the peak due 10 hypericin in the

Ayellowish-orange fluorescent zone
ehroma1Ogram obtained with the test solution;
area of the peak due to hyperiein in the
2 red fluorescent zones (hypericin ehromatogram obtained with the referenee solution;
and pseudohypericin)
--- --- mas s of the extraet to be examined used to prepare
the test solution, in grams;
mass of St. John's wort dry extraet HRS used to
3 yellowish-orange fluorescent zones prepare the referenee solution, in grams;
p pereentage eontent of hyperiein in St. John's wort
dry extraet HRS.
--- ---
Hyperforin and flavonoids
Hyperoside: a yellowish-orange A yellowish-orange fluorescent zone Liquid ehroma1Ography (2.2.29). Carry out the assay protected
fluorescent zone (hyperoside)
from light.
Yellow and blue possibly
superimposed fluorescent zones Solvent mixture water R, methanol R (20:80 V/V).
Rutin: a yellowish-orange A yellowish-orange fluorescent zone Test solution Dissolve 75.0 mg ofthe extraet 10 be examined
fluorescent zone (rutin)
in 20.0 mL of the solvent mixture. Sonieate and eentrifuge.
Reference solution Test solution
Referenee solution (a) Dissolve 20.0 mg of rutoside
trihydrate CRS in 200.0 mL of the solvent mixture.
TESTS Referenee solution (b) Dissolve 75.0 mg of St. John's wort dry
Water (2.5.12) extract HRS in 20.0 mL of the solvent mixture. Sonicate and
Maximum 4.0 per eent, determined on 0.5 g. eentrifuge.
ASSAY Column:
T otaI hypericins - size: 1 = 0.15 m, 0 = 4.6 mm;
Liquid ehromatography (2.2.29). - stationary phase: octadecylsilyl siliea gel for ehromatography R
(3 ¡.tm).
Test solution Dissolve 70.0 mg of the extraet to be examined
in 25.0 mL of methanol R. Sonieate and eentrifuge the Mobile phase:
solution. Expose the solution to a xenon lamp at about 765 - mobile phase A: phosphorie acid R, water R (3:1000 V/V);
W/m 2 for 8 min. - mobile phase B: phosphon'e acid R, acetonitrile R
(3:1000 V/V);
Reference solution Dissolve a quantity of St. John's wort dry
extract HRS eorresponding to 0.15 mg of hyperiein in Time Mobile phase A Mobile phase B Flow rate
25.0 mL of methanol R. Sonieate and eentrifuge. Expose the (min) (per cent V!JI) (per cent V!JI) (mL/min)
solution to a xenon lamp at about 765 W/m 2 for 8 min. 0-8 82 18 0.8

Column: 8·18 82 ---> 47 18 ---> 53 0.8

- size: 1 = 0.15 m, 0 = 4.6 mm; 0.8
18 - 18.1 47 ---> 3 53 ---> 97
- stationary phase: octadecylsilyl sz1ica gel for chromatography R
(5 ¡.tm); 18.1 - 19 3 97 0.8 ---> 1.2
- temperature: 40 oc. 19 - 31 3 97 1.2
Mobile phase Mix 39 volumes of ethyl acetate R, 41 volumes
of a 15.6 gIL solution of sodium dihydrogen phosphate R
adjusted to pH 2 with phosphoric acid R and 160 volumes of Deteetion Spectrophotometer at 360 nm, then at 275 nm
methanol R. after the elution of biapigenin (about 22 min).
Flow rate 1.0 mUmin. Injeetion 10 ¡.tL.
Deteetion Speetrophotometer at 590 nm. Identification of peaks Use the ehromatogram supplied with
Injeetion 20 ¡.tL. Sto John's wort dry extraet HRS and the ehromatogram
Run time 15 mino obtained with referenee solution (b) to identify the peaks due
to rutin, hyperoside, isoquereitroside, quereitroside,
Identifieation of peaks Use the ehromatogram supplied with
quereetin, biapigenin, hyperforin and adhyperforin.
St. John's wort dry extract HRS and the ehromatogram
obtained with the referenee solution to identify the peaks due System suitability Referenee solution (b):
to pseudohyperiein and hyperiein. - the ehromatogram obtained is similar 10 the
ehroma1Ogram supplied with Sto John's wort dry extraet
System suitability Referenee solution:
HRS;
- the ehromatogram obtained is similar to the
- resolution: minimum 2.0 between the peaks due 10 rutin
ehroma1Ogram supplied with St. John's wort dry extraet
and hyperoside, and minimum 2.0 between the peaks due
HRS;
to hyperforin and adhyperforin.
- resolution: minimum 2 between the peaks due to
pseudohypericin and hyperiein. Calculate the pereentage eontent of hyperforin using the
following expression:
Calculate the pereentage content of total hyperieins,
expressed as hyperiein, using the following expression:
A4 x m4 x p x 2.3
(Al + A2 ) x ffi2 X P A5 x m3 x 10
A3 x mI
area of the peak due 10 hyperforin in the
area of the peak due 10 pseudohyperiein in the ehroma1Ogram obtained with the test solution;
chromatogram obtained with the test solution;
 IV-364 Stephania Tetrandra Root
As area of the peak due to rutin in the chromatogram
obtained with reference solution (a);
1113 mass of the extract to be examined used to prepare
the test solution, in grams;
1114 mas s of rutoside trihydrate CRS used to prepare
reference solution (a), in grams;
2.3 correction factor for hyperforin with respect to
rutin;
p percentage content of rutin in rutoside
trihydrate CRS.
Calculate the percentage content of fiavonoids, expressed as
rutin, using the following expression:
m4 x p x (A6 + A7 + As + Ag + AlO + All)
m3 x A5 x 10
As area of the peak due to rutin in the chromatogram
obtained with reference solution (a);
A6 area of the peak due to rutin in the chromatogram
obtained with the test solution;
A7 area of the peak due to hyperoside in the
chromatogram obtained with the test solution;
As area of the peak due to isoquercitroside in the
chromatogram obtained with the test solution;
Ag area of the peak due to quercitroside in the
chromatogram obtained with the test solution;
AlO area of the peak due to quercetin in the
chromatogram obtained with the test solution;
A 11 area of the peak due to biapigenin in the
chromatogram obtained with the test solution;
1113 mass of the extract to be examined used to prepare
the test solution, in grams;
1114 mass of rutoside trihydrate CRS used to prepare
reference solution (a), in grams;
p percentage content of rutin in rutoside
tnhydrate CRS.
LABELLING
The label sta tes the content of hyperforin.
____________________________________________ ~Ew
***
Stephania Tetrandra Root
*** ***
(Foursta111en Stephania Root, ***
Ph Eur 111onograph 2478)
PhEw ____________________________________________
DEFINITION
Scraped, cut and dried root of Stephania tetrandra S.Moore.
Content
Minimum 1.6 per cent of the sum of tetrandrine and
fangchinolin~, expressed as tetrandrine (C3sH42N206; M r
623) (dried drug).
IDENTIFICATION
A. The root is found as slices or irregularly cylindrical or
semi-cylindrical pieces, mostly tortuous, about 0.5-1 cm thick
and 1-5 cm in diameter. The greyish-yellow outer surface
usually shows deep and sinuous transversal striations;
the curved parts are knotty and bumpy. The texture is dense
and compactoThe cut surface is greyish-white and shows
radial striations.
B. Reduce to a powder (355) (2.9.12). The powder is
whitish-grey. Examine under a microscope using ehloral
hydrate solution R. The powder shows the following diagnostic
characters: numerous fragments of parenchyma with cells
2014
having slightly thickened and moniliform walls; reticulate or
pitted xylem ves seis accompanied by fibres; fragments of
phelloderm containing sc1ereids; rare cork fragments; rare,
fine, rod-shaped calcium oxalate crystals. Examine under a
microscope using a 50 per cent V/V solution of glyeerol R.
The powder shows very many round or truncated, simple or
2- or 3-compound starch granules, 10-20 ~lm in diameter,
with a punctiform hilum.
C. Thin-layer chromatography (2.2.27).
Test solution To 0.4 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of a mixture of 1 volume of anhydrous
formie acid R, 9 volumes of water R and 40 volumes of
methanol R. Sonicate at 25 oC for 10 min and filter.
Referenee solution Dissolve 10 mg of protopine hydroehloride R
and 10 mg of tetrandrine R in methanol R and dilute to
10 mL with the same solvento
Plate TLC siliea gel plate R (5-40 ¡.¡m) [or TLC siliea gel
plate R (2-10 ¡.¡m)] .
Mobile phase eoneentrated am1110nia R, 111ethanol R, ethyl
aeetate R, toluene R (0.3:5: 10: 1O VIVIV/V).
Applieation 10 ¡.¡L [or 5 ¡.¡L] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 6 cm].
Drying In a current of warm air for 5 mino
Deteetion Treat with a 5 gIL solution of iodine R in ethanol
(96 per cent) R until the background becomes yellow;
examine in daylight after the yellow colour has disappeared.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
Protopine: an orange zone
- -- ---
Tetrandrine: an orange zone An orange zone (tetrandrine)
An orange zone
--- --- -
Reference solution Test solution
TESTS
Aristolochia fangchi
Test for aristolochic acids in herbal drugs (2.8.21). The drug
to be examined complies with method A.
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 4.0 per cent.
Ash insoluble in hydrochloric acid (2.8. 1)
Maximum 1.0 per cent.
ASSAY
Tetrandrine and fangchinoline
Liquid chromatography (2.2.29).
Test solution In a 50 mL round-bonomed ftask, weigh
0.500 g ofthe powdered herbal drug (355) (2.9.12).
Add 25 mL of a 2 per cent VIV solution of hydrochloric
acid R in methanol R. Weigh. Heat under a reftux condenser
on a water-bath at 60 oC for 30 mino Cool and weigh. Adjust
2014
to the inirial weight using a 2 per cent VIV solution of
hydroehlonc acid R in methanol R. Filter. Dilute 5.0 mL of the
filtrate to 10.0 mL with the mobile phase.
Referenee solution Dissolve 10.0 mg of tetrandnne CRS in
5 mL of methanol R and dilute to 10.0 mL with the mobile
phase.
Column:
-- size: 1 = 0.25 m, 0 = 4.6 mm;
-- stationary phase: oetadeeylsilyl silica gel for ehromatography R
(5 ¡lm).
Mobile phase 4.1 gIL solution of sodium laurylsulfonate for
ehromatography R in a mixture of 1 volume of glacial acetie
acid R, 30 volumes of methanol R, 30 volumes of water R and
40 volumes of acetonitnle R.
Flow rate 2.0 mlJmin.
Deteetion Spectrophotometer at 280 nm.
Injection 20 ¡lL.
Run time 30 min.
Relative retention With reference to tetrandrine (retention
time = about 18 min): fangchinoline = about 0.7 .
System suitability Test solution:
-- resolution: minimum 3.0 between the peaks due to
fangchinoline and tetrandrine.
Calculate the percentage content of tetrandrine and
fangchinoline, expressed as tetrandrine, using the following
expression:
(Al + A3) x m2 x p x 5
A 2 x mI
area of the peak due to tetrandrine in the
chromatogram obtained with the test solution;
area of the peak due to tetrandrine in the
chromatogram obtained with the reference solution;
area of the peak due to fangchinoline in the
chromatogram obtained with the test solution;
mas s of the herbal drug to be examined used to
prepare the test solution, in grams;
mas s of tetrandnne CRS used to prepare the
reference solution, in grams;
p assigned percentage content of tetrandrine in
tetrandnne CRS.
____________________________________________ PhE~
Sterculia
Sterculia Gum; Karaya Gum
Preparation
Sterculia Granules
When Powdered Sterculia is prescribed or demanded,
material complying with the appropriate requirements below
and containing not less than 10.0% of volatile acid shall be
dispensed or supplied.
DEFINITION
Sterculia is the gum obtained from Sterculia urens Roxb.
and other species of Stereulia.
CHARACTERlSTICS
It has the macroscopical and microscopical characters
described under Identification tests A and B.
Sterculia Preparations IV-365
Sparingly soluble in water, but swells into a homogeneous,
adhesive, gelatinous mass. Practically insoluble in ethanol
(96%).
IDENTIFICATION
A. Irregular or vermiforrn pieces, about 5 to 20 mm thick;
greyish white with a brown or pink tinge; surface striated.
B. When powdered and mounted in ethanol (96%) it appears
as small, transparent, angular particles of various sizes and
shapes; the particles lose their sharp edges when water is
added and each gradually swells until a large, indefinite,
almost structureless mass results; when mounted in ruthenium
red solution the particles are stained red; no blue coloured
particles (starch) are visible when mounted in iodine
solution Rl.
C. Add 1 g to 80 mL of water and allow to stand for
24 hours, shaking occasionally. A tacky and viscous granular
mucilage is produced. Retain the mucilage for use in test D.
D. Boil4 mL ofthe mucilage obtained in test C with
0.5 mL of hydroehlone acid, add 1 mL of 5M sodium hydroxide,
filter, add 3 mL of cupn-tartan'e solution Rl to the filtrate and
heat. A red precipitate is produced.
E. Warm 0.5 g with 2 mL of 5M sodium hydroxide. A brown
colour is produced.
TESTS
Acid-Ínsoluble ash
Not more than 1.0%, Appendix XI K.
Foreign matter
Complies with the testfor foreign matter, Appendix XI D .
Ash
Not more than 7.0%, Appendix XI J.
Volatile acid
Not less than 14.0%, calculated as acetic acid, C 2H 4 0z,
when deterrnined by the following method. To 1 g contained
in a 700 mL Kjeldahl ftask add 100 mL of water and 5 mL
of orthophosphonc acid, allow to stand for several hours, or
until the gum is completely swollen, and boil gently under a
reftux condenser for 2 hours. Steam distil until 800 mL of
distillate is obtained and the acid residue mea sures about
20 mL and titrate the distillate with O.IM sodium hydroxide
VS using phenolphthalein solution Rl as indicator. Repeat the
operation without the substance being examined.
The difference between the titrations represents the amount
of alkali required to neutralise the volatile acid. Each mL of
O.IM sodium hydroxide VS is equivalent to 6.005 mg of
volatile acid, calculated as C ZH 4 0z.
Microbial contamination
1.0 gis free from Eschenchia coli, Appendix XVI Bl.
STORAGE
Sterculia should be stored in a dry place.
Sterculia Granules
DEFINITION
Sterculia Granules are Sterculia in granule form o
The granules comply with the requirements stated under Granules
and with the following requirements.
CHARACTERlSTICS
White or buff with a distinct odour of acetic acid;
transparent, irregular shaped granules of about 1 to 4 mm
which swell when treated with water.
 IV-366 Stramonium Leaf
IDENTIFICATION
A. Irregular or vennifonn pie ces, about 5 to 20 mm thick;
greyish white with a brown or pink tinge; surface striated.
B. When powdered and mounted in ethanol (96%) it appears
as small, transparent, angular particles of various sizes and
shapes; the particles lose their sharp edges when water is
added and each gradually swells until a large, indefinite,
almost structureless mass results; when mounted in ruthenium
red solution the particles are stained red; no blue coloured
particles (starch) are visible when mounted in iodine
solution Rl.
C. Add 1 g to 80 mL of water and allow to stand for
24 hours, shaking occasionally. A tacky and viscous granular
mucilage is produced. Retain the mucilage for use in test D .
D. Boil 4 mL of the mucilage obtained in test C with
0.5 mL of hydroehlorie acid, add 1 mL of 5M sodium hydroxide,
filter, add 3 mL of eupri-tartarie soluuon Rl to the filtrate and
heat. A red precipitate is produced.
TESTS
Acid-insoluble ash
Not more than 1.0%, Appendix XI K.
Ash
Not more than 7.0%, Appendix XI J.
Volatile acid
Not less than 13.0%, calculated as acetic acid, C 2H 4 02,
when detennined by the following method. To 1 g contained
in a 700 mL Kjeldahl fiask add 100 mL of water and 5 mL
of orthophosphorie acid, allow to stand for several hours, or
until the granules are completely swollen, and boil gently
under a refiux condenser for 2 hours. Steam distil until
800 mL of the distillate is obtained and the acid residue
measures about 20 mL and titrate the distillate with
0.1 M sodium hydroxide VS using phenolphthalein solution R 1 as
indicator. Repeat the operation without the substance being
examined. The difference between the titrations represents
the amount of alkali required to neutralise the volatile acid.
Each mL of O.lM sodium hydroxide VS is equivalent to
6.005 mg of volatile acid, calculated as C 2H 4 0 2.
Loss on drying
The powdered granules, when dried to constant weight at
105°, lose not more than 20.0% of their weight. Use 1 g.
Microbial contamination
1.0 g is free from Eseheriehia eoli, Appendix XVI Bl.
STORAGE
Sterculia Granules should be stored in a dry place.
***
Stramonium Leaf
** **
(Ph Eur monograph 0~46) *****
Preparation
Prepared Stramonium
When Stramonium Leaf or Powdered Stramonium Leaf is
prescribed, Prepared Stramonium shall be dispensed.
PhE~ ____________________________________________
DEFINITION
Dried leaf or dried leaf and fiowering, and occasionally fruit-
bearing, tops of Datura stramonium L. and its varieties.
Content
Minimum 0.25 per cent of total alkaloids, expressed as
hyoscyamine (C 17H 23 N0 3; M r 289.4) (dried drug).
2014
The alkaloids consist mainly of hyoscyamine with varying
proportions of hyoscine (scopolamine).
CHARACTERS
Unpleasant odour.
IDENTIFICATION
A. The leaves are dark brownish-green or dark greyish-green
with a short petiole, often much twisted and shrunken during
drying, thin and brittle, ovate or triangular-ovate, dentately
lobed with an acuminate apex and often unequal at the base.
Young leaves are pubescent on the veins, older leaves are
nearly glabrous. Stems are green or purplish-green, slender,
curved and twisted, wrinkled longitudinally and sometimes
wrinkled transversely, branched dichasially, with a single
fiower or an immature fruit in the fork. Flowers, on short
pedicels, have a gamosepalous calyx with 5 lobes and
trumpet-shaped brownish-white or purplish corolla. The fruit
is a capsule, usually covered with numerous short, stiff
emergen ces; seeds are brown or black with a minutely pitted
testa.
B. Microscopic examination (2.8.23) . The powder is greyish-
green. Examine under a microscope using ehloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 0246.-1): fragments ofupper [A] and
lower [C] epidennises of the lamina, in surface view, showing
cells with slighdy wavy anticlinal walls and a smooth cuticle
accompanied by palisade [Aa] and spongy [Ca] parenchyma;
anisocytic [Ac, Cb] and anomocytic [Ab] stomata (2.8.3),
more frequent on the lower epidennis; fragments of covering
trichomes, conical [E], uniseriate with 3-5 cells with warty
walls, sorne of them collapsed [Ea]; glandular trichomes,
short and clava te, in side view [B] with heads formed by 2-7
cells; dorsiventtal mesophyll in ttansverse section [F], with a
single layer of palisade cells [Fa] and a spongy parenchyma
[Fb] containing cluster crystals of calcium oxalate [Fc];
fragments of spongy parenchyma [D] with sorne cells
containing small cluster crystals of calcium oxalate [Db],
associated with annularly and spirally thickened vessels [Da],
in surface view. The powdered drug may also show: fibres
and reticulately thickened ves seis from the stemsj
subspherical pollen grains about 60-80 ¡¡m in diameter with
3 genninal pores and a nearly smooth exine [G]; fragments
of the corolla [H] with wavy-walled cells [Ha] and underlying
mesophyll [Hb] with sorne cells containing prisms [Hc] or
cluster crystals [Hd] of calcium oxalate; seed fragments
containing yellowish-brown, sinuous, thick-walled sclereids of
the testam, and occasional prisms and microsphenoidal
crystals of calcium oxalate.
C. Examine the chromatograms obtained in the
chromatography test.
Results The principal zones in the chromatograms obtained
with the test solution are similar in position, colour and size
to the principal zones in the chromatogram obtained with the
same volume of the reference solution.
D. Shake 1 g of the powdered herbal drug (180) (2.9.12)
with 10 mL of 0.05 M sulfurie acid for 2 mino Filter and add
to the filtrate 1 mL of concentrated ammonia R and 5 mL of
water R. Shake cautiously with 15 mL of peroxide-free ether R,
avoiding the fonnation of an emulsiono Separate the ether
layer and dry over anhydrous sodium sulfate R. Filter and
evaporate the ether in a porcelain dish. Add 0.5 mL of nitric
acid R and evaporate to dryness on a water-bath. Add 10 mL
of acetone R and, dropwise, a 30 gIL solution of potassium
hydroxide R in ethanol (96 per cent) R. A deep violet colour
develops.
2014
Figure 0246.-1. - Illustration for identification test B of
powdered herbar drug of stramonium (eaf
TESTS
Chromatography
Thin-Iayer chromatography (2.2.27) .
Test solution To 1.0 g of the powdered herbal drug (180)
(2.9.12) add 10 mL of 0.05 M sulfuric acid, shake for 15 min
and filter. Wash the filter with 0.05 M sulfuric acid until
25 mL of filtra te is obtained. To the filtrate add 1 mL of
concentrated ammonia R and shake with 2 quantities, each of
10 mL, of peroxide-free ether R. If necessary, separate by
centrifugation. Dry the combined ether layers over anhydrous
sodium sulfate R, filter and evaporate to dryness on a water-
bath. Dissolve the residue in 0.5 mL of methanol R .
R eference solution Dissolve 50 mg of hyoscyamine sulfate R in
9 mL of methanol R. Dissolve 15 mg of hyoscine
hydrobromide R in 10 mL of methanol R. Mix 3.8 mL of the
hyoscyamine sulfate solution and 4.2 mL of the hyoscine
hydrobromide solution and dilute to 10 mL with methanol R.
Plate TLC silica gel G plate R.
Mobile phase concentrated ammonia R, water R, acetone R
(3:7:90 VIVIV) .
Application 10 ¡.¡L and 20 ¡.¡L, as bands of 20 mm by 3 mm,
leaving 1 cm between the bands .
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 min; allow to cool.
Detection A Spray with potassium iodobismuthate solution R2,
using about 10 mL for a plate 200 mm square, until the
orange or brown zones become visible against a yellow
background.
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the
Stramonium Leaf IV-367
lower third, hyoseine in the upper third of the
chromatograms) and colour to those in the ehromatograms
obtained with the reference solution. The zones in the
ehromatograms obtained with the test solution are at least
equal in size to the corresponding zones in the chromatogram
obtained with the same volume of the reference solution.
Faint secondary zones may appear, particularly in the middle
of the chromatogram obtained with 20 ~lL of the test solution
or near the point of applieation in the chromatogram
obtained with 10 ¡.¡L of the test solution.
Detection B Spray with sodium nitrite solution R until the
eoating is transparent; examine after 15 mino
Results B The zones due to hyoscyamine in the
ehromatograms obtained with the reference solution and the
test solution change from brown to reddish-brown but not to
greyish-blue (atropine) and any secondary zones disappear.
Foreign matter (2.8.2)
Maximum 3 per cent of stems with a diameter greater than
5 mm.
Total ash (2.4.16)
Maximum 20.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
a) Determine the loss on drying (2.2.32) on 2.000 g ofthe
powdered herbal drug (180) (2.9.12) by drying in an oven at
105 oC.
b) Moisten 10.0 g ofthe powdered herbal drug (180)
(2.9.12) with a mixture of 5 mL of ammonia R, 10 mL of
ethanol (96 per cene) R and 30 mL of peroxide-free ether R and
mix thoroughly. Transfer the mixture to a suitable percolator,
if neeessary with the aid of the extraeting mixture. Allow to
macerate for 4 h and pereolate with a mixture of 1 volume of
chloroform R and 3 volumes of peroxide-free ether R until the
alkaloids are completely extracted. Evaporate to dryness a
few millilitres of the liquid flowing from the percolator,
dissolve the residue in 0.25 M sulfuric acid and verify the
absence of alkaloids using potassium tetraiodomercurate
solution R . Concentrate the percolate to about 50 mL by
distilling on a water-bath and transfer it to a separating
funnel, rinsing with peroxide-free ether R . Add a quantity of
peroxide-free ether R equal to at least 2.1 times the volume of
the pereolate to produce a liquid of a density well below that
of water. Shake the solution with no fewer than 3 quantities,
eaeh of 20 mL, of 0.25 M sulfuric acid, separate the 2 layers
by eentrifugation if necessary and transfer the acid layers to a
2nd separating funnel. Make the acid layer alkaline with
ammonia R and shake with 3 quantities, eaeh of 30 mL, of
chloroform R . Combine the ehloroform layers, add 4 g of
anhydrous sodium sulfate R and allow to stand for 30 min with
oeeasional shaking. Decant the chloroform and wash the
anhydrous sodium sulfate with 3 quantities, each of 10 mL,
of chloroform R. Add the washings to the chloroform extraet,
evaporate to dryness on a water-bath and heat in an oven at
100-105 oC for 15 minoDissolve the residue in a few
millilitres of chloroform R, add 20.0 mL of 0.01 M sulfuric acid
and remove the ehloroform by evaporation on a water-bath.
Titrate the excess of acid with 0.02 M sodium hydroxide using
methyl red mixed solution R as indicator.
Calculate the pereentage content of total alkaloids, expressed
as hyoscyamine, using the following expression:
57.88 x (20 - n)
(100 - d) x m
 IV-368 Stramonium
d loss on drying, as a percentage;
n volume of 0.02 M sodium hydroxide, in millilitres;
m mas s of the powdered herbal drug, in grams.
STORAGE
Protected from moisture.
____________________________________________ ~E~
Prepared Stramonium ****
*** * Dissolve the residue in 0.5 mL of methanol R .
(Ph Eur monograph 0247) *** *
~E~ ____________________________________________
DEFINITION
Stramonium leaf powder (180) (2. 9.12) adjusted, if
necessary, by the addition of powdered lactose or
stramonium leaf of lower content of total alkaloids.
Content
0.23 per cent to 0.27 per cent of total alkaloids, expressed as
hyoscyamine (C 17H 23N0 3 ; M r 289.4) (dried drug) .
CHARACTERS
Appearance
Greyish-green powder.
Unpleasant odour.
IDENTIFICATION
A. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters: fragments of leaf lamina showing epidermal cells
with slightIy wavy anticlinal walls and smooth cuticle;
stomata are more frequent on the lower epidermis (anisocytic
and anomocytic) (2.8.3); covering trichomes are conical,
uniseriate with 3-5 cells and warty walls; glandular trichomes
are short and clavate with heads formed by 2-7 cells;
dorsiventral mesophyll, with a single layer of palisade cells
and a spongy parenchyma containing cluster crystals of
calcium oxalate; annularIy and spirally thickened vessels.
The powdered drug may also show the following diagnostic
characters: fibres and reticulately thickened vessels from the
stems; subspherical pollen grains usually about 60-80 11m in
diameter with 3 germinal pores and nearly smooth exine;
fragments of the corolla with papillose epidermis; seed
fragments containing yellowish-brown, sinuous, thick-walled
sclereids of testa; occasional prisms and microsphenoidal
crystals of calcium oxalate. Examined in glycerol
(85 per cent) R, it may be seen to contain lactose crystals.
B. Examine the chromatograms obtained in the
Chromatography test.
Results The principal zones in the chromatogram obtained
with the test solution are similar in position, colour and size
to the principal zones in the chromatogram obtained with the
same volume of the reference solution.
C. Shake 1 g with 10 mL of 0.05 M sulfun·c acid for 2 mino
Filter and add to the filtrate 1 mL of concenlrated ammonia R
and 5 mL of water R. Shake cautiously with 15 mL of
peroxide-free ether R, avoiding the formation of an emulsiono
Separate the ether layer and dry over anhydrous sodium
sulfate R. Filter and evaporate the ether in a porcelain dish.
Add 0.5 mL of nitric acid R and evaporate to dryness on a
water-bath. Add 10 mL of acewne R and, dropwise, a 30 gIL
solution of potassium hydroxide R in ethanol (96 per cent) R.
A deep violet colour develops.
2014
TESTS
Chromatography
Thin-layer chromatography (2.2.27).
Test solutwn To 1.0 g of the drug to be examined add
10 mL of 0.05 M sulfuric acid, shake for 15 min and filter.
Wash the filter with 0.05 M sulfuric acid until 25 mL of
filtrate is obtained. To the filtrate add 1 mL of concentrated
ammonia R and shake with 2 quantities, each of 10 mL, of
peroxide-free ether R . If necessary, separate by centrifugation.
Dry the combined ether layers over anhydrous sodium
sulfate R, filter and evaporate to dryness on a water-bath.
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in
9 mL of methanol R. Dissolve 15 mg of hyoscine hydrobromide
R in 10 mL of methanol R. Mix 3.8 mL of the hyoscyamine
sulfate solution and 4.2 mL of the hyoscine hydrobromide
solution and dilute to 10 mL with methanol R .
Plate TLC silica gel G plate R.
Mobile phase concentrated ammonia R, water R, acewne R
(3:7:90 VIVIV) .
Application 10 llL and 20 llL of each solution as bands of
20 mm by 3 mm, leaving 1 cm between the bands.
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 min and alIow to coo!.
D etection A Spray with potassium iodobismuthate solution R2,
using about 10 mL for a pi ate 200 mm square, until the
orange or brown zones become visible against a yellow
background.
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the
lower third, hyoscine in the upper third of the
chromatogram) and colour to those in the chromatograms
obtained with the reference solution. The zones in the
chromatograms obtained with the test solution are at least
equal in size to the corresponding zones in the chromatogram
obtained with the same volume of the reference solution.
Faint secondary zones may appear, particularly in the middle
of the chromatogram obtained with 20 ¡.¡L of the test solution
or near the point of application in the chromatogram
obtained with 10 ¡.¡L of the test solution.
Detection B Spray with sodium nitrite solution R until the
coating is transparent; examine after 15 mino
Results B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
reference solution change from brown to reddish-brown but
not to greyish-blue (atropine) and any secondary zones
disappear.
Loss on drying (2.2.32)
Maximum 5.0 per cent, determined on 1.000 g by drying in
an oven at 105 oC.
Total ash (2.4.16)
Maximum 20.0 per cent.
Ash insoluble in hydrochIoric acid (2.8.1)
Maximum 4.0 per cent.
ASSAY
a) Determine the loss on drying (2.2.32) on 2.000 g by
drying in an oven at 105 oC.
b) Moisten 10.0 g with a mixture of 5 mL of ammonia R,
10 mL of ethanol (96 per cent) R and 30 mL of peroxide-free
ether R and mix thoroughly. Transfer the mixture to a
suitable percolator, if necessary with the aid of the extracting
mixture. AIlow to macerate for 4 h and percolate with a
mixture of 1 volume of chloroform R and 3 volumes of
 2014 Tea-tree Oíl IV-369

peroxide-free ether R until the alkaloids are completely Referenee solution Dissolve 30 fll- of eineole R, 60 ~lL of
extracted. Evaporate to dryness a few millilitres of the Iiquid terpinen-4-ol R and 10 mg of r:J.-terpineol R in 10 mL of
ftowing from the percolator, dissolve the residue in 0.25 M heptane R .
sulfurie acid and verify the absence of alkaloids using Plate TLC siliea gel plate R.
potassium tetraiodomereurate solution R. Concentrate the Mobile phase ethyl aeetate R, heptane R (20:80 V/V).
percolate to about 50 mL by distilling on a water-bath and
transfer it to a separating funnel, rinsing with peroxzde-free Application 10 ¡.tL, as bands.
ether R. Add a quantity of peroxide-free ether R equal to at Development Over a path of 10 cm.
least 2.1 times the volume of the percolate to produce a Drying In airo
liquid of a density well below that of water. Shake the Detection Spray with anisaldehyde solution R. Heat at
solution with no fewer than 3 quantities, each of 20 mL, of 100-105 oC for 5-10 min while observing. Examine in
0.25 M sulfurie acid, separate the 2 layers by centrifugation if daylight.
necessary and transfer the acid layers to a 2nd separating
Results See below the sequence of the zones present in the
funne!. Make the acid layer alkaline with ammonia R and
chromatograms obtained with the reference solution and the
shake with 3 quantities, each of 30 mL, of ehlorofomz R .
test solution. Furthermore, other zones are present in the
Combine the chloroform layers, add 4 g of anhydrous sodium
chromatogram obtained with the test solution.
sulfate R and allow to stand for 30 min with occasional
shaking. Decant the chloroform and wash the sodium sulfate
with 3 quantities, each of 10 mL, of chloroform R . Add the Top of the plate
washings to the chloroform extract, evaporate to dryness on a
Cineole: a violet·brown zone A violet·brown zone, less intense
water-bath and heat in an oven at 100-105 oC for 15 min. (cineole)
Dissolve the residue in a few millilitres of ehloroform R, add Terpinen4·o1: a brownish·violet A brownish·violet zone
20.0 mL of 0.01 M sulfuric acid and remove the chloroform zone terpinen4·o1)
by evaporation on a water-bath. Titrate the excess of acid (l·terpineol: a violet or A violet or brownish·violet zone
with 0.02 M sodium hydroxide using methyl red mixed brownish-violet zone ((l-terpineol)
solution R as indicator.
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, using the following expression: Reference solution Test solution

57.88 (20 - n)
(100 - d) m B. Examine the chromatograms obtained in the test for
chromatographic pro file.
d loss on drying, as a percentage; Results The characteristic peaks in the chromatogram
n volume of 0.02 M sodium hydroxide, in millilitres; obtained with the test solution are similar in retention time to
m mas s of drug, in grams. those in the chromatogram obtained with the reference
solution.
STORAGE
In an airtight container. TESTS
____________________________________________ ~Ew
Relative density (2.2.5)
0.885 to 0.906.
Refractive index (2.2.6)
1.475 to 1.482.
Optical rotation (2.2.7)
Tea Tree Oil ***
*** *** + 5° to+15°.
Melaleuca Oil *** Chromatographic profile
(Ph Bur monograph 1837) Gas chromatography (2.2.28): use the normalisation
~Ew ____________________________________________ procedure.
Test solution Dissolve 0.15 mL of the substance to be
DEFINITION examined in 10 mL of hexane R.
Essential oil obtained by steam distillation from the foliage Reference solution Dissolve 5 ¡.tL of r:J. -pinene R, 5 ¡.tL of
and terminal branchlets of Melaleuca alternifolia (Maiden and sabinene R, 15 ¡.tL of r:J.-terpinene R, 5 ¡.tL of limonene R, 5 ¡.tL
Betch) Cheel, M. linariifolia Smith, M. dissitifiora F. Mueller of cineole R, 30 ¡.tL of y-terpinene R, 5 ¡.tL of p-cymene R, 5 ¡.tL
and/or other species of Melaleuea. of terpinolene R, 60 ¡.tL of terpinen-4-ol R, 5 ¡.tL of
CHARACTERS aromadendrene R and 5 mg of r:J.-terpineol R in 10 mL of
Appearance hexane R .
Clear, mobile, colourless or pale yellow liquido Column:
Characteristic odour. -- material: fused silica,
-- size: l = 30 m (a film thickness of 1 ¡.tm may be used) to
IDENTIFICATION 60 m (a film thickness of 0.2 ¡.tm may be used),
Fint identifieation B. o= 0.25-0.53 mm,
Second identification A. -- stationary phase: macrogol 20 000 R.
A. Thin-layer chromatography (2.2.27). Carrier gas helium for chromatography R.
Test solution Dissolve 0.1 mL of the substance to be Flow rate 1.3 mUmin.
examined in 5 mL of heptane R. Split ratio 1:50.
IV-370 Terminalia Arjuna Stem Bark
Temperature:
Temperature
Time
(min) (oC)
Column 0·1 50
1·37 50 ---t 230
37·45 230
Injection port 240
Detector 240
Detection Flame ionisation.
Injection 1 ~lL
Elution order Order indicated in the composition of the
reference solution. Record the retention times of these
substances.
System suitability Reference solution:
-- resolution: minimum 2.7 between the peaks due to
terpinen-4-01 and aromadendrene.
U sing the retention times determined from the
chromatogram obtained with the reference solution, locate
the components of the reference solution in the
chromatogram obtained with the test solution. Disregard the
peak due to hexane.
Determine the percentage content of these components.
The percentages are within the following ranges:
-- rx-pinene: 1.0 per cent to 6.0 per cent,
-- sabinene: maximum 3.5 per cent,
-- rx-terpinene: 5.0 per cent to l3.0 per cent,
-- limonene: 0.5 per cent to 4.0 per cent,
-- cineole: maximum 15.0 per cent,
-- y-terpinene: 10.0 per cent to 28 .0 per cent,
-- p-cymene: 0.5 per cent to 12.0 per cent,
-- terpinolene: 1.5 per cent to 5.0 per cent,
-- terpinen-4-ol: minimum 30.0 per cent,
-- aromadendrene: maximum 7.0 per cent,
-- rx-terpineol: 1.5 per cent to 8.0 per cent.
STORAGE
At a temperature not exceeding 25 oc.
____________________________________________ ~E~
Terminalia Arjuna Stem Bark
DEFINITION
Terminalia Arjuna Stem Bark consists of cut dried bark of
the stems of Terminalia arjuna W. and A. It contains not less
than 6% of tannins, expressed as pyrogallol, calculated with
reference to the dried drug.
IDENTIFICATION
A. Irregularly fiattened or slightly curved or recurved pieces,
up to about 8 cm long, 4 cm wide and 1 cm thick; outer
surface uneven, dark brown or sometimes mottled greyish-
brown, smooth or, more frequently, irregularly striated
longitudinally with occasional transverse ridges; inner surface
pink to reddish brown with longitudinal striations and
occasional paler brown patches. Fracture short and starchy in
the inner part, the outer part frequently laminated.
B. Reduce to a powder (355) . The powder is reddish-brown.
Examine under a microscope using chloral hydrate solution.
The powder shows a variety of parenchymatous cells, sorne
thin-walled, square or round and others yellowish, polygonal
and thick-walled. Rectangular or polygonal, pitted, thin-
2014
walled, light brown, lightly lignified cells from the cork layer
or outer areas of the cortex are presento
Fibres occur singly and in small or large groups, individual
cells narrowing to highly pointed ends, and possibly showing
wavy invaginations of the walls where surrounding cells have
become detached; degree of lignification varies; walls are
yellowish brown, sorne pitted, others noto Single fibres may
be complete, but those in groups are usually fragmented,
individual cells being straight or noticeably curved in places.
Rounded cells of the medullary rays, which are one cell wide,
intersperse the fibres.
Small, calcium oxalate cluster crystals occur scattered
throughout as well as being found within parenchymatous
cells, sorne forming a crystal sheath alongside the fibres.
Other crystals are very large and less well defined, and
usually free.
Examine under a microscope using 50% v/v of glycerol.
Starch granules are frequent, but not abundant, mainly free,
but sorne in parenchymatous cells. They are small, simple,
round, oval or irregular in shape, and occasionally in 2 to 3
compound granules, without visible hila. More or less
frequent scattered lumps of brown pigment may be found
sometimes in parenchymatous cells .
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Shake 1.0 g ofthe powdered drug with 10 mL of absolute
ethanol, centrifuge at 3000 rpm for 5 minutes and filter
(Whatman GF/C is suitable).
(2) 0.01 % w/v each of arjunolic acid and gallic acid in absolute
ethanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating high performance silica gel (Merck
silica gel 60 HPTLC plates are suitable).
(b) Use the mobile phase described below.
(c) Apply as bands 8 J.!L of each solution.
(d) Develop the plate to 8 cm.
(e) Remove the plate and allow it to dry in air for 5 minutes.
0
Spray the plate with anisaldehyde solution, heat at 100 to
105° for 5 minutes and examine in daylight.
MOBILE PHASE
15 volumes of ethyl aceta te, 15 volumes of formic acid and
70 volumes of toluene.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated bands.
CONFIRMATION
The chromatogram obtained with solution (1) shows a band
with an Rf value of approximately 0.30 corresponding in
colour and position to the band obtained for arj).lilolic acid in
solution (2); two clearly separated dark bands with
an Rf value of approximately 0.2; a band with an Rf value of
0.63 is presento Other bands may be present.
TESTS
Ash
Not more than 25%, Appendix XI J.
Loss on drying
When dried for 2 hours at 100° to 105 0
, loses not more than
10% of its weight. Use 1 g.
Water soluble extractive
Not less than 20%, Appendix XI B2.
2014 Terminalia Belerica Fruit IV-371

Top of the plate (1) Add 10 mL of absolute ethanol to l.0 g of the powdered
drug, centrifuge at 3000 rpm for 5 minutes and filter
(Whatman GF/C is suitable).
(2) 0.01 % w/v each of mjunolic acid, gallic acid and ellagic acid
in absolute ethanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating high performance silica gel F 254
(Merck silica gel 60 F 254 HPTLC plates are suitable).
Dark band
(b) Use the mobile phase described below.
(c) Apply as bands 8 ¡lL of each solution.
(d) Develop the plate to 8 cm.
(e) Remove the plate and allow it to dry in air for 5 minutes.
Dark band Dark band: arjunolic
Examine under ultraviolet light (254 nm). Spray the plate with
acid
anisaldehyde solution, heat at 100° to 105° for 5 minutes and
Two separated dark Yellow band: gallic
bands examine in daylight.
acid
MOBILE PHASE

Solution (1) Solution (2)

15 volumes of ethyl acetate, 15 volumes of formic acid and
70 volumes of toluene.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
ASSAY solution (2) shows two c1early separated bands under both
Carry out the determination of tannins in herbal drugs, ultraviolet light (254 nm) and daylight.
Appendix XI M. Use l.0 g of powdered drug.
CONFIRMATION
Under ultraviolet light (254 nm) the chromatogram obtained
with solution (1) shows bands with Rf values of
approximately 0.11 and 0.15 corresponding in colour and
Terminalia Belerica Fruit position to the bands obtained with ellagic acid and gallic
DEFINITION acid in solution (2) and light blue bands with Rf values of
Terminalia Belerica Fruit consists of pericarp of dried ripe approximately 0.04 and 0.36. Other bands may be presento
fruits of Terminalia belenea Roxb. It contains not less than
10% of tannins, expressed as pyrogallol, ca1culated with Top of the plate
reference to the dried drug.
IDENTIFICATION
A. The dried fruits are spherical to subspherical, about 3 to
5 cm in diameter, slightly depressed at the upper end and
more or less tapering to the scar of the pedicel at the lower
end. The surface is brown to yellowish brown with a grey
velvety sheen, irregularly wrinkled and sometimes with faint,
incomplete, longitudinal ridges. Cut transversely, the fruit
shows the pericarp about 4 to 5 mm thick enc10sing a very
hard, yellowish-white seed.
B. Reduce to a powder (355). The powder is light-brown.
Examine under a microscope using chloral hydrate solution.
The powder shows many free, unicellular, straight or slightly Light blue band
bent trichomes from the epicarp . A variety of thick-walled,
heavily pitted, lignified fibro-sc1ereids of elongated and
spherical shapes occur in large and small groups; occasional Blue band Slue band : gallic acid
medium sized reticulate ves seIs; heavily pitted, lignified Dark band Dark band: ellagic acid
parenchymatous cells and others with reticulate thickenings; Light blue band
many lignified, pitted, thin walled sc1ereids and groups of Solutíon (1) Solutíon (2)
fragmented, thick-walled, pitted, fibres are presento Rarely oil
globules, starch granules, and ca1cium oxalate crystals may be
found in the parenchymatous cells from the embryo.
Examine under a microscope using 50 % v/v of glycerol.
The powder shows minute, either single or 2 to 4 compound Under daylight afier spraying with anisaldehyde solution the
starch granules, sorne scattered, but mainly filling chromatogram obtained with solution (1) shows a band with
parenchymatous cells . Sorne have slit or stellate hila; simple an Rf value of approximately 0.15 corresponding in colour
granules are ofien not perfectly spherical. Larger ca1cium and position to the band obtained gallic acid in solution (2);
oxalate crystals, with fewer small ones, are scattered or in a dark band with an Rf value of 0.36; several dark bands in
parenchymatous cells. the upper part of the plate.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
 IV-372 Terminalia Chebula Fruit 2014

Top of the plate parenchyma, or thinner-waIled, less lignified and pitted

sclereids, with broad lumens, are also found . There are very
occasional small, spiral, Iignified vessel fragments . SmaIl,
greenish, thick-walIed polygonal epicarp ceIls are seen in
Several dark bands surface view. Parenchymatous ceIls with reticulate thickenings
across the surface are rare, as are others without such
thickenings, but containing oil globules. Examine under a
microscope using 50% v/v of glyeerol. The powder shows
minute, either single or 2 to 4 compound granules, sorne
scattered, but mainly filling parenchymatous ceIls. Sorne have
slit or steIlate hila; simple granules are often not perfectIy
spherical. Larger calcium oxalate crystals, with fewer small
ones, are scattered or in parenchymatous ceIls.
Dark band C. Carry out the method for thin-layer chromatography,
Dark band : arjunolic Appendix III A, using the foIlowing solutions.
acid
(1) To 1. O g of the powdered drug, add 10 mL of absolute
Light brown band Light brown band: ethanol, centrifuge at 3000 rpm for 5 minutes and filter
gallic acid (Whatman GF/C is suitable).
(2) 0.01 % w/v each of arjunolic aeid, gallie acid and ellagie aeid
in absolute ethanol.
Solution (1) Solution (2)
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating high performance silica gel F 254
TESTS (Merck silica gel 60 F 254 HPTLC plates are suitable).
Ash (b) Use the mobile phase described below.
Not more than 7%, Appendix XI J. (c) Apply as bands 8 ¡¡L of each solution.
Loss on drying
(d) Develop the plate to 8 cm.
When dried for 2 hours at 100° to 105°, loses not more than
5.0% of its weight. Use 1 g. (e) Remove the plate and aIlow it to dry in air for 5 minutes.
Water soluble extractive Examine under ultraviolet light (254 nm). Spray the plate with
Not less than 45%, Appendix XI B2. anisaldehyde solution, heat at 100° to 105° for 5 minutes and
examine in daylight.
ASSAY
Carry out the determination of wnnins in herbal drugs, MOBILE PHASE
Appendix XI M . Use 1.0 g of powdered drug. A mixture of 15 volumes of ethyl acetate, 15 volumes of formic
acid and 70 volumes of toluene.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two c1earIy separated bands under both
Terminalia Chebula Fruit ultraviolet light (254 nm) and daylight.

DEFINITION VALIDITY
Terminalia Chebula Fruit consists of pericarp of mature When examined under ultraviolet light (254 nm) the
fruits of Terminalia ehebula Retz. It contains not less than chromatogram obtained with solution (1) shows bands
20% of tannins, expressed as pyrogallol, caIculated with with Rf values of approximately 0.11 and 0.15 corresponding
reference to the dried drug. in colour and position to the bands obtained with gallic acid
and eIlagic acid in solution (2).
IDENTIFICATION
A. The dried fruits are sub-globular to ovo id, 3 to 4 cm long
and 1.5 to 2 cm wide, bluntly pointed at the tip and tapering
towards the base. The surface is yellowish to greenish,
sometimes brown, shiny and more or less wrinkled and has
distinct longitudinal ridges. Cut transversely, the fruit shows
the pericarp about 3 to 4 mm thick, non-adherent to the very
hard, creamy-white seed.
B. Reduce to a powder (355). The powder is yeIlowish
brown. Examine under a microscope using ehloral hydrate
solution. The powder shows round, oval or elongated, thin-
waIled parenchymatous ceIls in groups. Occasional narrow-
walIed, unpitted, lightly lignified fibres occur in small or
larger groups, sorne forming wave-like arrangements. Large
groups of fragmented, heavily lignified and pitted fibres also
occur. A variety of thick- waIled, heavily pitted, lignified
fibro-sclereids of elongated, rectangular, and irregular shapes
occur in large and small groups. Fewer pitted, lignified
 2014 Thyme IV-373

Top of the plate Thyme *****

** **
(Ph Bur monograph 0865) ***
~E~ ____________________________________________

DEFINITION
Whole leaves and ftowers separated from the previously dried
stems of Thymus vulgaris L. or Thymus zygis L. or a mixture
of both species.
Content:
-- essentialoil: minimum 12 mUkg (anhydrous drog);
-- sum oi the eontents oi thymol and earvaerol (both ClQH I4 0;
M r 150.2): minimum 40 per cent in the essential oi!.
CHARACTERS
Blue band Blue band: gallic acid Strong aromatic odour reminiscent of thymol.
Dark band Dark band: ellagic acid
IDENTIFICATION
A. The leaf of Thymus vulgan's is usually 4-12 mm long and
Solution (1) Solution (2)
up to 3 mm wide, sessile or with a very short petiole.
The lamina is tough, entire, lanceolate or ova te, covered on
both surfaces by a grey or greenish-grey indumentum;
When examined under daylight after spraying with the edges are markedly rolled up towards the abaxial surface.
anisaldehyde solurion the chromatogram obtained with solution The midrib is depressed on the adaxial surface and is very
(1) shows a band with an Rfvalue ofapproximately 0.15 prominent on the abaxial surface. The calyx is green, often
corresponding in colour and position to the band obtained with violet spots and is tubular; at the end are 2 Iips of which
for gallic acid in solution (2) and a dark band with the upper one is bent back and at the end has 3 lobes, the
an Rf value of approximately 0.30. There may be sorne faint lower is longer and has 2 hairy teeth. After ftowering, the
brown bands with Rf values of approximately 0.40 and 0.70. calyx tube is cIosed by a crown of long, stiff hairs.
The corolla, about twice as long as the calyx, is usually
brownish in the dry state and is slightly bilabiate.
Top of the plate
The leaf of Thymus zygis is usually 1.7-6.5 mm long and
0.4-1.2 mm wide; it is acicular or linear-Ianceolate and the
edges are markedly rolled towards the abaxial surface. Both
surfaces of the lamina are green or greenish-grey and the
midrib is sometimes violet; the edges, in particular at the
base, have long, white hairs. The dried ftowers are very
similar to those of Thymus vulgaris.
B. Reduce to a powder (355) (2.9.12). The powder of both
species is greyish-green or greenish-brown. Examine under a
microscope using ehloral hydrate solution R. The epidermis es
of the leaves have cells with anticIinal walls which are sinuous
and beaded and the stomata are diacytic (2.8.3); numerous
secretory trichomes made up of 12 secretory cells, the cuticIe
Dark band Dark band: arjunolic of which is generally raised by the secretion to form a
acid globular or ovoid bladder-Iike covering; the glandular
Light brown band Light brown band :
trichomes have a unicellular stalk and a globular or ovoid
gallic acid head; the covering trichomes of the adaxial surface are
common to both species; they have warty walls and are
shaped as pointed teeth; the warty covering trichomes of the
abaxial surface are of many types: unicellular, straight or
Solution (1) Solution (2)
slightly curved, and bicellular or tricellular, articulated and
often elbow-shaped (Thymus vulgans); bicellular or tricelIular,
TESTS more or less straight (Thymus zygis). Fragments of calyx are
Ash covered by numerous, uniseriate, articulated trichomes with
Not more than 5%, Appendix XI J. 5-6 cells and with a weakly striated cuticIe. Fragments of the
corolla have numerous uniseriate covering trichomes, often
Loss on drying collapsed, and secretory trichomes generally with 12 cells.
When dried for 2 hours at 100° to 105°, loses not more than Pollen grains are relatively rare, spherical and smooth with
10% of its weight. Use 1 g. 6 germinal slit-like pores, measuring about 35 ¡lm in
Water soluble extractive diameter. The powder of Thymus zygis also contains
Not less than 50%, Appendix XI B2. numerous thick bundles of tibres from the main veins and
ASSAY from fragments of stems.
Carry out the determination of tannins in herbal drugs,
Appendix XI M. Use 1.0 g of powdered drogo
 IV-374 Thyme 2014

"
,,

A. Epidermis of the outer surface D. Secretory trichome with 12 cells

of the corolla, in surface view, A. Upper epidermis, in surface view, D. Upper epidermis, in surface view,
E. Outer epidermis of the upper with beaded cells (Aa), diacytic with beaded cells (Da), secretory
showing a covering trichome corolla, in surface view, with diacytic
with one cell collapsed (Aa), and stomata (Ab) and covering trichomes trichome made up of 12 secretory
stomata (Ea) and glandular trichome with warty walls (Ac) and underlying cells (Db), and glandular trichome
a unicellular-headed glandular (Eb)
trichomes (Ab) palisade parenchyma (Ad) with a unicellular head (De) and
F. Epidermis of the calyx, in surface B and E. Epidermis, in transverse underlying palisade parenchyma
B. Pollen grain with 6 germinal view, with covering trichomes (Dd)
pores (of which only 3 are visible in section, with unicellular covering
the illustration) trichomes (Ba, Ea) and articulated F. Multicellular covering trichome
bicellular covering trichome (Bb) from the base of the lamina (T. zygis)
C. Epidermis of the lower corolla
with glandular trichome (Ca) C. Articulated tricellular covering G. Epidermis, in transverse section,
trichome with bicellular (Ga) and tricellular
(Gb) covering trichomes (T. zygis)
Figure 0865.-1. - Illustration of powdered herbal drug of
Thymus vulgaris L_ (see Identification B)
Figure 0865.-2. - Illustration of powdered herbal drug of
Thymus zygis L. (see Identification B)
C. Thin-Iayer chromatography (2_2.27).
Test solution To LO g of the powdered drug (355) (2.9_12)
add 5 mL of methylene ehloride R and shake for 3 min, filter
Top of the plate
through about 2 g of anhydrous sodium sulfate R.
Referenee solution Dissolve 5 mg of thymol R and 10 ¡.¡L of --- ---

earvaerol R in 10 mL of methylene ehloride R . A prominent quenching zone

Plate TLC siliea gel F 254 plate R.
Thymol : a quenching zone A quenching zone (thymol)
Mobile phase methylene ehloride R .
--- ---
Applieation 20 ¡.¡L as bands.
Development Over a path of 15 cm.
Drying In air. Quenching zones
Detection A Examine in ultraviolet light at 254 nm. Reference solution Test solution
Results ASee below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution_ Deteetion B Spray with anisaldehyde solution R using 10 mL
for a plate 200 mm square and heat at 100-105 oC for
10 min o
Results B See below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones are present in the
lower third of the chromatogram obtained with the test
solution. The intensity of the zones due to thymol and
carvacrol depends upon the species examined.
2014 Thyrne Oil, Thyrnol Type IV-375

Top oC the plate Temperature:

--- --- Time Temperature

(min) (oC)
Thymol: a brownish-pink zone A brownish-pink zone (thymol) Column 0·45 40 ..... 220
Carvacrol: a pale violet zone A pale violet zone (carvacrol)
Injection port 190
--- ---
Detector 210

A greyish-pink zone
Detection Flame ionisation.
A violet zone (cineole and linalol) Injection 0.2 ¡¡L.
A greyish-brown zone (borneol) Elution order Order indicated in the composition of the
A violet-blue zone reference solution. Record the retention times of these
An in tense violet zone substances.
ReCerence solution Test solution System suitabz7ity Reference solution:
-- resolution : minimum 1.5 between the peaks due ro thymol
and carvacrol.
D. Examine the chromatograms obtained in the assay for
thymol and carvacrol. Using the retention times determined from the
chromatogram obtained with the reference solution, locate
Results The characteristic peaks in the chromatogram
tbe components of the reference solution in the
obtained with the test solution are similar in retention time to
chromatogram obtained with the test solution.
those in the chromatogram obtained with the reference
solution. Determine the percentage content of thymol and carvacrol.
____________________________________________ ~Em

TESTS
Foreign matter (2.8.2)
Maximum 10 per cent of stems and maximum 2 per cent of
other foreign matter. Stems must not be more than 1 mm in
diameter and 15 mm in length. Leaves with long trichomes
Thyme Oil, Thymol Type ***
at their base and with weak1y pubescent other parts (Thymus *** ***
serpyllum L.) are absent. (Ph Eur monograph 1374) ***
Water (2.2.13) ~Em ____________________________________________
Maximum 100 mUkg, determined on 20.0 g ofthe
DEFINITlON
powdered drug (355) (2.9.12).
Essential oil obtained by steam distillation from the fresh
Total ash (2.4.16) fiowering aerial parts of Thymus vulgaris L., T. zygis L. or a
Maximum 15.0 per cent. mixture of both species.
Ash insoluble in hydrochloric acid (2.8.1) CHARACTERS
Maximum 3.0 per cent. Appearance
ASSAY Clear, yellow or very dark reddish-brown, mobile liquido
Essential oi! (2.8.12) Odour reminiscent of thymol.
U se 3 O.O g of the drug, a 1000 mL round-bottomed fiask
Solubility
and 400 mL of water R as the distillation liquido Disti! at a
Miscible with anhydrous ethanol and with light petroleum.
rate of 2-3 mUmin for 2 h without xylene R in the graduated
tube. IDENTIFICATlON
First identification B.
Thymol and carvacrol
Gas chromatography (2.2.28): use the normalisation Second identificatían A.
procedure. A. Thin-Iayer chromatography (2.2.27) .
Test solution Filter the essential oi! obtained in the Test solutían Dissolve 0.2 mL of the substance to be
determination of essential oil over a small amount of examined in methylene chloride R and dilute ro 10 mL with
anhydrous sodium sulfate R and dilute to 5.0 mL with the same solvento
hexane R by rinsing the apparatus and the anhydrous sodium Reference solution Dissolve 5 mg of thymol R and 10 ¡¡L of
sulfate. carvacrol R in methylene chloride R and dilute ro 10 mL with
Reference solution Dissolve 0.20 g of thymol R and 50 mg of the same solvento
carvacrol R in hexane R and dilute to 5.0 mL with the same Plate TLC silica gel plate R (5-40 ¡¡m) [or TLC silica gel
solvento plate R (2-10 ¡¡m)].
Column: Mobile phase methylene chloride R .
-- material: fused silica;
Application 10 ¡¡L [or 4 ¡¡L] as bands of 10 mm [or 8 mm].
-- size: l :=: 30-60 m, 0 :=: 0.25 mm;
-- stationary phase: macrogol 20 000 R (film thickness Development Over a path of 12 cm [or 6 cm] .
0.25 ¡¡m). Drying In airo
Carrier gas nitrogen for chromatography R or helium for Detection Treat with anisaldehyde solution R and heat at
chromatography R. 100-105 oC for 5-10 min; examine in daylight.
Flow rate 1-2 mUmin. Results See below the sequence of zones present in the
Split ratio 1: 1OO . chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromarogram obtained with the test solution.
 IV-376 Thyme 2014

Top of the plate - resolution: minimum 1.5 between the peaks due to thymol
A pink zone
and carvacrol.
Identijication of peaks
- -- - --
Using the retention times determined from the
Thymol: an orange-brown zone An intense orange-brown zone chromarogram obtained with reference solution (a), locate
(thymol) the components of reference solution (a) in the
Carvacrol: an orange-grey zone A faint orange-grey zone chromarogram obtained with the test solution. The peak due
(carvacrol) may be present
ro ex-thujene elutes with a relative retention of about 0.8 with
--- ---
reference to ~-myrcene. The peak due to carvacrol methyl
A pink zone ether elutes with a relative retention of about 0.9 with
A violet zone
reference to thymol.
Determine the percentage content of these components.
A brownish-grey zone
The limits are within the following ranges:
Reference solution Test solution - rx-thufene: 0.2 per cent to 1.5 per cent;
- f3-myreene: 1.0 per cent ro 3.0 per cent;
- rx-terpinene: 0.9 per cent to 2.6 per cent;
B. Examine the chromatograms obtained in the test for - p-cymene: 14.0 per cent ro 28.0 per cent;
chromatographic profile. - y-terpinene: 4.0 per cent to 12.0 per cent;
Results The characteristic peaks in the chromatogram - linalol: 1.5 per cent to 6.5 per cent;
obtained with the test solution are similar in retention time to - terpinen-4-ol: 0.1 per cent to 2.5 per cent;
those in the chromatogram obtained with reference - carvacrol methyl ether: 0.05 per cent to 1.5 per cent;
solution (a) . - thymol: 37.0 per cent to 55.0 per cent;
- earvacrol: 0.5 per cent to 5.5 per cent;
TESTS
- disregard limit: the area of the principal peak in the
Relative density (2.2.5)
chromatogram obtained with reference solution (b)
0.915 to 0.935.
(0.05 per cent) .
Refractive index (2.2. 6)
1.490 to 1.505 . STORAGE
Chromatographic pro file
At a temperature not exceeding 25 oc.
_ _ _ _ _ _ __ __ _ __ _ _ _ _ __ _ _ _ PhEur
Gas chromatography (2.2.28): use the normalisation
procedure.
Test solution Dissolve 200 ~L of the substance to be
examined in heptane R and dilute to 10.0 mL with the same
solvento Wild Thyme
Reference solution (a) Dissolve 5 ~L of f3-myrcene R, 5 ~ of (Ph Eur monograph 1891)
rx-terpinene R, 20 ~L of p-cymene R, 1O ~L of y-terpinene R, PhE~ _ _ _ _ __ __ __ _ _ _ _ _ _ __ _ __ _
5 ~L of linalol R, 5 ~L of terpinen-4-ol R, 40 mg of thymol R
and 5 ~lL of carvacrol R in 5 mL of heptane R. DEFINITION
Reference solution (b) Dissolve 10 ~L of carvacrol R in Whole or cut, dried, ftowering aerial parts of Thymus
heptane R and dilute ro 10.0 mL with the same solvento serpyllum L.s.!.
Dilute 100 ~L of the solution to 10.0 mL with heptane R. Content
Column: Minimum 3.0 mlJkg of essential oil (dried drug).
- material: fused silica; IDENTIFICATION
- size: l = 60 m, 0 = 0.25 mm; A. The stem is much branched, up to about 1.5 mm in
- stationary phase: poly(dimethyl) (diphenyl)siloxane R (film diameter, cy!indrical or indistinctly quadrangular, green,
thickness 0.25 ~m). reddish or purplish, the older stems brown and woody, the
Carrier gas helium for chromatography R. younger stems pubescent. The leaves are opposite, 3 mm to
Flow rate 1.5 mlJmin. 12 mm long and up to 4 mm wide, elliptical to ovate-
Split ratio 1:50. lanceo late with an obtuse apex, cuneate and shortly petiolate
Temperature: at the base; the margin is entire and markedly ciliate,
especially near the base; both surfaces are more or less
Time Temperature glabrous but distinctly punctate. The inftorescence is
(min) (OC)
composed of about 6 ro 12 ftowers in rounded to ovoid,
Column 0 · 75 65 -) 215 terminal heads. The calyx is tubular, two-lipped with the
Injection port 230 upper !ip dividing to form 3 teeth, rhe lower lip with 2 teeth,
Detector edged with long hairs; inner surfaces strongly pubescent, the
250
hairs forming a closed tube after ftowering. The corolla is
Detection Flame ionisation. purp!ish-violet to red, two-lipped, the lower !ip with 3 lobes,
ln:;"eetion 1 ~L. upper lip notched, inner surface strongly pubescent; stamens
4, epipetalous, projecting from the corolla tube.
Elution order Order indicated in the composition of
reference solution (a); record the retention times of these B. Reduce to a powder (355) (2.9.12). The powder is
substances . greyish-green to brownish-green. Examine under a
microscope using ehloral hydrate solution R. The powder
System suitability Reference solution (a):
shows the following diagnostic characters: fragments of rhe
leaf epidermis es with sinuous, slightly thickened anticlinical
2014 Tolu IV-377

waJls and stomata of the diacytic type (2.8.3); numerous TESTS

covering trichomes on both epidermis es and along the leaf
margins, the majority short, conical, uniceJlular, with
thickened and warty waJls, fewer long, uniseriate, composed
of up to 8 ceJls, slightly swoJlen at the joints, with moderately
thickened waJls; abundant glandular trichomes, mostly
multiceJlular with a smaJl, rounded, uniceJlular stalk and a
large globular head composed of a number of indistinct,
radiating ceJls containing brown secretion, others smaJler,
capitate, with unicellular stalk and a unicellular, globoid or
ovoid head; purplish-violet fragments of the corolla, the outer
epidermis with numerous covering and glandular trichomes,
inner epidermis papillose; pollen grains spherical 10 eJliptical,
30 ¡¡m 10 40 ¡¡m in diameter, with a finely grained exine and
6 germinal pores.
C. Thin-layer chromatography (2.2.27).
Test solution T o 1.0 g of the powdered drug (3 55) (2.9.12)
add 5 mL of methylene ehlonde R and shake for 3 mino Filter
through about 2 g of anhydrous sodium sulfate R .
Referenee solution Dissolve 5 mg of thymol R and 10 ¡¡L of
earvaerol R in 10 mL of methylene ehlonde R .
Plate TLC siliea gel F 254 plate R .
Mobile phase methylene ehloride R.
Applieation 20 ¡¡L, as bands.
Development Over a path of 15 cm.
Drying In airo
Foreign matter (2.8.2)
Maximum 3 per cent, determined on 30 g.
Foreign maner may also consist of acicular to linear-
lanceolate leaves with a strongly bent margin, the adaxial
surface showing covering trichomes shaped as pointed teeth
with warty walls, the abaxial surface showing many types of
warty covering trichomes: unicellular, straight or slightly
curved, bicellular or tricellular, ofien elbow-shaped, and
bicellular or triceJlular, more or less straight (Thymus vulgans,
Thymus zygis).
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 1.000 g ofthe
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4. 16)
Maximum 10.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3.0 per cent.
ASSAY
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 50.0 g ofthe cut drug, a 1000 mL round-
bottomed ftask and 500 mL of water R as the distillation
liquidoDistil at arate of 2-3 mUmin for 2 h without xylene R
in the graduated tube.
_ _ _ _ _ _ _ __ __ _ _ __ _ __ _ __ _ Ph Eur

Deteetion A Examine in ultraviolet light at 254 nm.

Results ASee below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test sol ution. Tolu Balsam ***
** **
(Ph Bur monograph 1596) *****
Top of the plate ~E~ ____ __ _ _ __ _ _ _ __ __ _ __ _ __
- -- - --
DEFINITION
A prorninent quenching zone OIeo-resin obtained from the trunk of Myroxylon balsamum
Thyrnol: a quenching zone A quenching zone (thyrnol) (L.) Harms varo balsamum.
--- --- Content
25.0 per cent to 50.0 per cent of free or combined acids,
Quenching zones expressed as cinnamic acid (C 9 H s0 2 ; M r 148.2) (dried
Reference solution Test solution drug).
CHARACTERS
Appearance
Deteetion B Spray with anisaldehyde solution R using 10 mL
Hard, friable, brownish 10 reddish-brown mass; thin
for a plate 200 mm square and heat at 100-105 oC for
fragments are brownish-yellow when examined against the
10 mino
light.
R esults B See below the sequen ce of the zones present in
Reminiscent odour of vaniJlin.
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones are present in the Solubility
lower third of the chromatogram obtained with the test PracticaJly insoluble in water, very soluble to freely soluble in
solution. The intensity of the zones due to thymol and alcohol, practicaJly insoluble in light petroleum.
carvacrol depends upon the sample examined (chemotypes). IDENTIFICATION
Thin-Iayer chromatography (2.2.27) .
Top of the plate Test solurion Stir 0.40 g of the fragmented drug with 10 mL
of methylene eh/onde R for 5 min and filter.
--- ---
Referenee so/ution Dissolve 50 mg of benzyl einnamate R in
Thyrnol: a brownish·pink zone A brownish·pink zone (thyrnol) methylene chlonde R, add 50 ¡¡L of benzyl benzoate R and
Carvacrol: a pale violet zone A pale violet zone (carvacrol) dilute 10 10 mL with methylene ehlonde R.
Plate TLC siliea gel G plate R.
--- ---
Mobile phase light petroleum R, toluene R (5:95 V/V).
Reference solution Test solution
Applieation 20 ¡¡L, as bands.
Development Over a path of 15 cm.
Drying In airo
 IV-378 Tolu Preparations 2014

Detection Spray with vanillin reagent R and heat at
100-105 oC for 5 mino Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the test and reference
solutions. Furthermore, other coloured zones are present in
1 mL of 0.1 M sodium hydroxide is equivalent to 14.82 mg of
total acids, expressed as cinnamic acid.
STORAGE
Do not store in powdered formo
____________________________________________ ~Ew

the chromatogram obtained with the test solution.

Top of the plate

Benzyl benzoate: a greyish-blue a greyish-blue zone
zone Tolu-flavour Solution
Benzyl cinnamate: a greyish-green a greyish-green zone
zone DEFINITION
Reference solution Test solution Cinnamic Acid 5.0 g
Benzoic Acid 2.5 g
Ethyl Cinnamate 0.3 g
TESTS Vanillin 0.1 g
Acid value Cinnamon Oil 0.02 mL
100 to 160. Sucrose 500 g
Dissolve 0.5 g of the fragmented drug in 50 mL of alcohol R. EthanoI (96 per cent) 350 mL
Add 0.5 mL of acid blue 93 solution R and 5.0 mL of 0.5 M Water Sufficient to produce 1000 mL
alcoholic potassium hydroxide. Stir vigorously and titrate with Extemporaneous preparation
0.5 M hydrochloric acid until the colour changes from The following directions apply.
brownish-red to blackish-green (nI mL of 0.5 M hydrochloric
DissoIve the Sucrose in 320 mL ofWater. Add 250 mL of
acid). Carry out a blank test in the same manner (n2 mL of the Ethanol (96 per cent), with mixing. DissoIve the
0.5 M hydrochloric acid). Calculate the acid value in the same Cinnamic Acid, Benzoic Acid, EthyI Cinnamate, Vanillin and
manner as the saponification vaIue (2.5.6).
Cinnamon Oil in the remaining 100 mL of Ethanol
Matter insoluble in alcohol (96 per cent), add this solution to the sucrose solution with
Maximum 5 per cent. mixing, dilute to 1000 mL with Water and mix. Allow to
Boil 2.0 g of the fragmented drug with 25 mL of alcohol stand for a few hours before use.
(90 per cent V/V? R and filter. Wash the residue with alcohol IDENTIFICAnON
(90 per cent VIV) R, boiIing until compIeteIy extracted, then Carry out the method for thin-layer chromatography,
dry the residue at 100-105 oc. Weigh the residue. Appendix III A, using the following solutions.
Loss on drying (2.2.32) (1) The solution being examined.
Maximum 5.0 per cent, determined on 2.000 g of the
(2) 0.5% w/v of cinnamic acid, 0.25% w/v of benzoic acid and
fragmented drug by spreading on a fiat evaporating dish
0.03% v/v of ethyl cinnamate in ethanol (90%).
9 cm in diameter and allowing to dry in vacuo for 4 h.
CHROMATOGRAPHIC CONDITIONS
Total ash (2.4.16)
Maximum 0.3 per cent. (a) Use as the coating silica gel GF254.
(b) Use the mobile phase as described below.
ASSAY
Boil 1.500 g under a refiux condenser with 25 mL of 0.5 M (c) Apply 5 ¡.¡L of each solution.
alcoholic potassium hydroxide for 1 h. Evaporate the ethanoI (d) Develop the plate to 15 cm.
and heat the residue with 50 mL of water R until the (e) After removal ofthe plate, dry in air for 15 minutes and
substance is homogeneously distributed. After cooling, add repeat the development using the same mobile phase.
80 mL of water R and a solution of 1.5 g of magnesium Remove the plate, allow the solvent to evaporate and
sulfate R in 50 mL of water R. Mix, and allow to stand for examine under ultraviolet light (254 nm).
10 mino Filter through a pIeated filter paper and wash the
MOBILE PHASE
residue with 20 mL of water R . Combine the fi1trate and the
washings, acidify with hydrochloric acid R and extract with 15 volumes of glacial acetic acid, 25 volumes of hexane and
4 quantities, each of 40 mL, of ether R. Discard the aqueous 75 volumes of n-pentane.
Iayer. Combine the organic extracts and wash with CONFIRMATION
2 quantities, each of 20 mL, and with 3 quantities, each of The spots in the chromatogram obtained with solution (1)
10 mL, of a 50 gIL soIution of sodium bicarbonate R. Discard are similar in size and correspond in position to those in the
the ether layer. Combine the aqueous extracts, acidify with chromatogram obtained with solution (2).
hydrochloric acid R and stir once with 30 mL, twice with
TESTS
20 mL and once with 10 mL of methylene chloride R. Dry the
combined methylene chIoride extracts over anhydrous sodium Ethanol content
sulfate R. Filter through a pIeated filter and wash the residue 31 to 36% v/v, Appendix VIII F.
with 10 mL of methylene chloride R. Reduce the combined Weight per mL
methyIene chloride extracts to 10 mL by distil1ation and 1.125 to l.155 g, Appendix V G.
eliminate the remaining methylene chloride in a current of
airo DissoIve the residue with heating in 10 mL of alcohol R
previously neutralised to phenol red solution R . After cooling,
titrate with 0.1 M sodium hydroxide, using the same indicator.
2014
Tolu Syrup
DEFINITION
Tolu-flavour Solution 100 mL
Syrup Sufficient to produce 1000 mL
The syrup complies with the requirements stated under Oral
Liquids and with the following requirement.
Weight per mL
1.29 to 1.32 g, Appendix V G.
Paediatric Compound Tolu Linctus
Paediatric Compound Tolu Oral Solution
DEFINITION
Paediatric Compound Tolu Linctus is an oral solution
containing 0.6% w/v of Citric Acid Monohydrate in a
suitable vehicle with a tolu flavour.
The linctus complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of total acid, calculated as citric acid
monohydrate, C 6H 8 0 7 ,H2 0
0.60 to 0.66% w/v.
ASSAY
To 15 g add 100 mL of water and titrate with O.IM sodium
hyd1'Oxide VS using phenolphthalein solution R1 as indicator.
Each mL of 0.1 M sodium hydroxide VS is equivalent to
7.005 mg of C 6H s0 7 ,HzO. Determine the weight per mL of
the linctus, Appendix V G, and calculate the content of
C 6 H s 0 7 ,H 2 0, weight in volume.
Tormentil ***
*** ***
(Ph Eur monograph 1478) ***
Preparation
Tormentil Tincture
~E~ ____________________________________________
DEFINITION
Whole or cut, dried rhizome, freed from the roots, of
Potentilla erecta (L.) Raeusch. (P. tormentilla Stokes).
Content
Minimum 7 per cent of tannins, expressed as pyrogallol
(C 6 H 6 0 3; M r 126.1) (dried drug).
IDENTIFICATION
A. The rhizome is cylindrically spindle-shaped, with a very
irregular appearance, often forming, twisted, knotty tubers,
up to 10 cm long and 1-2 cm thick, very hard and scarcely
branched. The surface is brown to reddish-brown, rugose
and has remains of roots and transversely elongated
depressed whitish scars from the stems. At the top of the
rhizome the remains of numerous aerial stems may be
presento The fracture is short and granular, dark red to
brownish-yellow.
B. Reduce to a powder (355) (2.9.12) . The powder is
reddish-brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic
characters: coarsely serrate cluster crystals of calcium oxalate,
up to 60 ~lm in diameter; fragments of thin-walled
parenchyma containing reddish-brown tannin; groups of
narrow, bordered-pitted ves seis with lateral pores; thick-
Tormentil IV-379
walled and pitted, polygonal parenchyma; groups and
fragments of sclerenchymatous thick-walled fibres; occasional
fragments of cork with thin-walled, brown, tabular cells.
Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows spherical or
elliptical starch granules, up to about 20 ¡.tm in length.
C. Thin-Iayer chromatography (2.2.27) .
Test solution To 0.5 g of the powdered drug (355) (2.9.12)
add 10 mL of water R, shake for 10 min and filter. Shake the
filtrate with 2 quantities, each of 10 mL, of ethyl acetate R
and filter the combined upper phases over 6 g of anhydrous
sodium sulfate R . Evaporate the filtrate to dryness under
reduced pressure and dissolve the residue in 1.0 mL of ethyl
acetate R.
Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of
methanol R.
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, ether R, hexane R, ethyl
acetate R (20:20:20:40 V/V/V/V).
Application 10 ¡.tL as bands.
Development Over a path of 10 cm.
Drying In air for 10-15 min.
Detection Spray with a freshly prepared 5 gIL solution of fast
blue B salt R . Reddish zones appear. Expose the plate to
arnmonia vapour, the zones become more intense turning
reddish-brown. Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other fainter zones are present in
the chromatogram obtained with the test solution.
Top of the plate
Catechin: an intense
reddish-brown zone
A more intense reddish-brown zone
(catechin)
A fainter zone
An intense zone
Fainter zones
Reference solution Test solution
TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of root and stems as well as rhizomes
with black fracture and maximum 2 per cent of other foreign
matter.
Cadmium (2.4.27)
Maximum 2.0 ppm.
Loss on drying (2.2.32)
Maximum 12.0 per cent, deterrnined on l.000 g of the
powdered drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
ASSAY
Carry out the deterrnination of tannins in herbal drugs
(2.8.14). Use 0.500 g ofthe powdered drug (180) (2.9.12).
____________________________________________ Ph Eur
 IV-380 Tormentil Preparations 2014

Tormentil Tincture ***** Trachyspermum Ammi

** **
(Ph Eur monograph 1895) *** DEFINITION
~E~ __________________________________________ Trachyspermum Ammi is the dried ripe fruit of
Traehyspermum ammi (L.) Sprague (syn. C. [Carum] coptieum
DEFINITION (L) Benth. & Hook.f. ex C.B. Clarke) .
Tincture produced from Tormentil (1478). Content
Content It contains not les s than 2.5% v/w of essential oil ca1culated
Minimum 1.5 per cent m /m of tannins, expressed as with reference to the anhydrous drug.
pyrogallol (C 6H 60 3; M r 126.1).
IDENTIFICATION
PRODUCTION A. The dried fruits occur mainly as emire cremocarps with
The tincture is produced from 1 part of comminuted drug carpophore present, yellowish green, ovo id, laterally
and 5 parts of ethanol (70 per cent V/V) by a suitable compressed, 1 to 3 mm in length and 1 to 2.8 mm in
procedure. diameter, usually with pedicel attached; styles remaining as a
CHARACTERS curved, bifid stylopod at the apex. Each fruit composed of
Red or reddish-brown liquido two mericarps, dorsal surface convex with five distinct ridges,
surface warty; commissural surface flat; vittae visible as two
IDENTIFICATION darker longitudinal bands.
Thin-layer chromatography (2.2.27). B. Reduce to a powder (355) . The powder is greenish
Test solution Mix 1.0 mL of the tincture to be examined brown. Examine under a microscope using ehloral hydrate
with 1.0 mL of alcohol (70 per cent V/V? R. solution. The powder contains numerous fragments of the
Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of papillose epicarp, also showing cuticular striations, with
methanol R. attached or detached whole or fragmented unicellular, warty-
Plate TLC silica gel plate R. walled trichomes; parquetry layer of endocarp in surface
view; endosperm of thick-walled cells containing oi! globules
Mobile phase ether R, glacial acetic acid R, hexane R, ethyl
and aleurone grains with embedded microrosette crystals of
acetate R (20:20:20:40 VIVIV/V).
ca1cium oxalate; fragments of yellowish-brown septate vittae;
Applieation 10 I1L as bands. bicollateral vascular bundles with associated lignified,
Development Over a path of 10 cm. reticulate or pitted parenchyma.
Drying In air for 10-15 min. C. Examine the chromatograms obtained in the test for
Detection Spray with a freshly prepared 5 gIL solution of fast Chromatographic profile. The retention times of the principal
blue B salt R. Reddish zones appear. Expose the plate to peaks in the chromatogram obtained with solution (1) are
ammonia vapour, the zones become more intense, tuming similar to those in the chromatogram obtained with
reddish-brown. Examine in daylight. solution (3).
Results See below the sequence of the zones present in the TESTS
chromatograms obtained with the reference solution and the Water
test solution. Not more than 10% w/w, Appendix IX C. Use 10.0 g.
Total Ash
Top of the plate Not more than 10%, Appendix XI J, Method II.
-- ----- Chromatographic profile
Catechin: an intense zane
Carry out the method for gas chromatography,
An in tense zane (catechin)
Appendix III B, using the following solutions.
-- -- (1) Use the essential oil-toluene mixture obtained in the
A fainter zane determination of essential oi!.
An intense zane (2) 0.4% v/v each of y-terpinene and p-cymene in toluene.
Fainter zanes (3) 0.1 % v/v each of p-eymene and y-terpinene and 0.1 % w /v
of thymol in toluene.
Reference solution Test solution
(4) 0.01% v/v of y-terpinene in toluene.
CHROMATOGRAPHIC CONDITIONS
TESTS (a) Use a fused si!ica column (30 m x 0.53 mm) bonded
Ethanol content (2.9.10) with a 1 11m film thickness and coated with polyethylene glycol
64 per cent VIV to 69 per cent VIV. 20,000 as the bonded phase (DB-Wax is suitable).
Methanol and 2-propanol (2.9.11) (b) Use helium as the carrier gas at 1.5 mL per minute.
Maximum 0.05 per cent VIV of methanol and maximum (c) Use the temperature gradient described below.
0.05 per cent V/V of 2-propano!. (d) Inject 1.0 I1L of each solution.
ASSAY (e) Use a split ratio of 1:50.
Carry out the determination of tannins in herbal drugs (t) Record the chromatogram for a sufficient length of time
(2.8.14). Use 2.50 g of the tincture to be examined. to elute all the peaks in the
__________________________________________ ~E~

chromatogram obtained with solution (1).

2014 Tragacanth IV-38 1

Time Temperature (0) longitudinal striae and concentric transverse ridges. It may
also contain pieces similar in shape but somewhat thicker,
(Minutes) more opaque and more difficult to fracture .
column 0---+5 60 B. Reduce to a powder (35 5) (2.9.12). The powder is white
or almost white and forms a mucilaginous gel with about
5---+68 60---+250 10 times its mass of water R. Examine under a microscope
using a 50 per cent V/V solution of glyeerol R. The powder
68---+ 75 250 shows in the gummy mass numerous stratified cellular
Inject part 250 membranes that tum slowly violet when treated with
iodinated zinc ehloride solution R. The gummy mass includes
Detector 260 starch grains, isolated or in small groups, usually rounded in
shape and sometimes deformed, with diameters varying
between 4 ¡lm and 10 ¡lm, occasionally up to 20 ¡lm, and a
central hilum visible between crossed nicol prisms.
SYSTEM SUITABILITY C. Examine the chromatograms obtained in the test for
The test is not valid unless in the chromatogram obtained acacia.
with solution (2), the resolution factor between the peaks due Results The chromatogram obtained with the test solution
to y-terpinene and p-cymene is at least 2.5. shows 3 zones due to galactose, arabinose and xylose. A faint
In the chromatogram obtained with solution (3), the peaks yellowish zone at the solvent front and a greyish-green zone
e1ute in the following order: between the zones due to galactose and arabinose may be
y-terpinene, p-cymene and thymol. presento
DETERMINATION OF CONTENT D. Moisten 0.5 g ofthe powdered drug (355) (2.9. 12) with
Using the retention times determined from the 1 mL of ethanol (96 per eent) R and add gradually, while
chromatogram obtained with solution (3), locate the shaking, 50 mL of water R until a homogeneous mucilage is
components of solution (3) in the chromatogram obtained obtained. To 5 mL of the mucilage add 5 mL of water R and
with solution (1) and calculate the content of p-cymene, y- 2 mL of barium hydroxide solution R. A slight f10cculent
terpinene and thymol by normalisation. precipitate is formed. Heat on a water-bath for 10 mino
Limits: An intense yellow colour develops.
-- p-eymene 10 to 25%, TESTS
-- y-terpinene 10 to 30%, Acacia
-- thymol 45 to 70%. Thin-Iayer chromatography (2.2.27).
Disregard any peak with an area less than the peak in the Test solution To 100 mg of the powdered drug (355)
chromatogram obtained with solution (4). (2.9. 12) in a thick-walled centrifuge test-tube, add 2 mL of a
ASSAY 100 giL solution of trifiuoroaeetie acid R, shake vigorously to
Essential oH dissolve the forming gel, stopper the test-tube and heat the
Carry out the method for Bssential Oils in Herbal Drugs, mixture at 120 oC for 1 h. Centrifuge the resulting
Appendix XI E, using 15 g of the powdered drug with hydrolysate, transfer the clear supematant carefully into a
1000 mL of water as distillation liquido Distil at arate of 2 to 50 mL f1ask, add 10 mL of water R and evaporate the
3 mL per minute for 2 hours using 0.5 mL of toluene in the solution to dryness under reduced pressure. To the resulting
graduated tube. Measure the quantity of essential oil distilled clear film add 0.1 mL of water R and 0.9 mL of methanol R.
and use for the test for Chromatographic profile. Centrifuge to separate the amorphous precipitate, collect the
supematant and, if necessary, dilute 10 1 mL with
methanol R .
Referenee solution Dissolve 10 mg of arabinose R, 10 mg of
galaetose R, 10 mg of rhamnose R and 10 mg of xylose R in
Tragacanth 1 mL of water R and dilute 10 10 mL with methanol R.
(Ph Bur monograph 0532) Piate TLC silica gel plate R .
Mobile phase 16 gIL solution of sodium dihydrogen
9000-65-1
phosphate R, butanol R, acetone R (10:40:50 VIV/V).
When Powdered Tragacanth is prescribed or demanded,
Applieation 10 ¡lL as bands.
material complying with the requirements below with the
exception of Identification test A shall be dispensed or Development A Over a path of 10 cm.
supplied. Drying A In a current of warm air for a few minutes.
PhE~ ____________________________________________ Development B Over a path of 15 cm using the same mobile
phase.
DEFINITION
Air-hardened, gummy exudate, f10wing naturally or obtained Drying B At 110 oC for 10 min.
by incision from the trunk and branches of Astragalus Deteetion Spray with anisaldehyde solution R and dry at
gummifer Labill. and cenain other species of ASlragalus from 110 oC for 10 min.
westem Asia. Results The chromatogram obtained with the reference
IDENTIFICATION solution shows 4 clearly separated coloured zones due to
galactose (greyish-green or green), arabinose (yellowish-
A. Tragacanth occurs in thin, f1attened, ribbon-like, white or
green), xylose (greenish-grey or yellowish-grey) and rhamnose
pale yellow, translucent strips, about 30 mm long and
(yellowish-green), in order of increasing RF value;
10 mm wide and up to 1 mm thick, more or less curved,
the chromatogram obtained with the test solution does not
homy, with a shon fracture; the surface is marked by fine
IV-382 Turmeric
show a yelIowish-green zone corresponding to the zone of
rhamnose in the chromatogram obtained with the reference
solution.
Methylcellulose
Examine the chromatograms obtained in the test for acacia.
Results The chromatogram obtained with the test solution
does not show a red zone near the solvent front.
Sterculia gum
A. Place 0.2 g ofthe powdered drug (355) (2.9.12) in a
10 mL ground-glass-stoppered cylinder graduated in 0.1 mL.
Add 10 mL of ethanol (60 per cent V/V) R and shake. Any gel
formed occupies not more than 1.5 mL.
B. To l.0 g ofthe powdered drug (355) (2.9.12) add
100 mL of water R and shake. Add 0.1 mL of methyl red
solution R. Not more than 5.0 mL of 0.01 M sodiwn hydroxide
is required to change the colour of the indicator.
Foreign matter
Maximum 1.0 per cent.
Place 2.0 g ofthe powdered drug (355) (2.9.12) in a 250 mL
round-bottomed fiask and add 95 mL of methanol R. Swirl to
moisten the powder and add 60 mL of hydrochloric acid R1.
Add a few glass beads about 4 mm in diameter and heat on
a water-bath under a refiux condenser for 3 h, shaking
occasionalIy. Remove the glass beads and filter the hot
suspension in vacuo through a sintered-glass filter (160)
(2.1.2). Rinse the fiask with a smalI quantity of water R and
pass the rinsings through the filter. Wash the residue on the
filter with about 40 mL of methanol R and dry to constant
mass at 110 oC (about 1 h) . AlIow to cool in a desiccator
and weigh. The residue weighs a maximum of 20 mg.
Flow time
Minimum lOs, or minimum 50 s if the substance to be
examined is to be used for the preparation of emulsions.
Place l.0 g of the powdered drug (125-250) (2.9.12) in a
1000 mL round-bottomed fiask with a ground-glass stopper,
add 8.0 mL of ethanol (96 per cene) R and close the fiask.
Disperse the suspension over the inner surface of the fiask by
shaking, taking care not to wet the stopper. Open the fiask
and add as a single portion 72.0 mL of water R. Stopper the
fiask and shake vigorously for 3 mino AlIow to stand for 24 h
and shake vigorously again for 3 mino Eliminate air bubbles
by applying vacuum aboye the mucilage for 5 mino Transfer
the mucilage to a 50 mL cylinder. Dip in the mucilage a
piece of glass tubing 200 mm long and 6.0 mm in internal
diameter and graduated at 20 mm and 120 mm from the
lower end; the tubing must not be rinsed with surface-active
substances. When the mucilage has reached the upper mark,
close the tube with a finger. Withdraw the c10sed tube,
remove the finger and measure with a stop-watch the time
needed for the meniscus to reach the lower graduation. Carry
out this operation 4 times and determine the average value of
the last 3 determinations.
Total ash (2.4.16)
Maximum 4.0 per cent.
Microbial contamination
TAMC: acceptance criterion 104 CFU/g (2.6.12).
TYMC: acceptance criterion 10 2 CFU/g (2.6.12) .
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
LABELLING
The label states whether or not the contents are suitable for
preparing emulsions.
_ _ __ _ _ __ __ _ __ _ __ _ _ _ _ _ _ PhEur
2014
*****
Javanese Turmeric
** **
(Ph Eur monograph 1441) ***
~E~ ___ _ _ __ __ _ _ _ _ _ __ _ _ __ _ _ _
DEFINITION
Dried rhizome, cut in slices, of Curcuma xanthorrhiza Roxb.
(e. x anthorrhiza D. Dietrich).
Content:
-- essentialoil: minimum 50 mUkg (anhydrous drug);
-- dicinnamoyl methane derivatives, expressed as curcumin
(C21H2006; M r 368.4): minimum l.0 per cent
(anhydrous drug).
CHARACTERS
Aromatic odour.
IDENTIFICATION
A. Orange-yelIow or yelIowish-brown or greyish-brown slices,
mostly peeled l.5-6 mm thick and 15-50 mm, more rarely
up to 70 mm, in diameter. Fragments of the brownish-grey
cork are sporadically presento The transverse surface is yelIow
with dark spots in the paler centre. The fracture is short and
finely grained.
B. Reduce 10 a powder (355) (2.9.12). The powder is
reddish-brown. Examine under a microscope, using chloral
hydrate solution R. The powder shows the folIowing diagnostic
characters: fragments of colourless parenchyma with orange-
yelIow or yelIowish-brown secretory celIs; fragments of
reticulate and other vessels; rare fragments of cork and
epidermis and fragments of thick-walIed unicelIular acute
trichomes. Examine under a microscope using a
50 per cent V/V solution of glycerol R . The powder shows
numerous stratified, ovo id or irregular starch granules, about
30-50 ¡.tm long and about 10-30 ¡.tm wide, with an eccentric
hilum and marked, concentric striations.
C. Thin-Iayer chromatography (2.2.27) as described in the
test for Curcuma domestica with the following modifications.
Detection Spray with a freshly prepared 0.4 gIL solution of
dichloroquinonechlorimide R in 2-propanol R. Expose to
arnmonia vapour until the zone due to thymol becomes
bluish-violet.
Results The chromatogram obtained with the reference
solution shows almost in the middle a bluish-violet zone
(thymol) and in the lower part a yelIow zone (fiuorescein) .
The chromatogram obtained with the test solution shows a
blue zone (xanthorrhizol) slightly aboye the zone due to
thymol in the chromatogram obtained with the reference
solution and 2 yelIowish-brown or brown zones (curcumin
and demethoxycurcumin) between the zones due to thymol
and fiuorescein in the chromatogram obtained with the
reference solution.
TESTS
Curcuma domestica
Thin-Iayer chromatography (2.2.27).
Test solution Shake 0.5 g of the freshly powdered drug (500)
(2.9.12) with 5 mL of methanol R for 30 min and filter.
Reference solution Dissolve 5 mg of fiuorescein R and 10 mg
of thymol R in 10 mL of methanol R.
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, toluene R (20:80 V/V).
Application 10 ¡.tL, as bands.
Development Over a path of 10 cm.
Drying In airo
 2014
Deteenon Spray with a mixture of 1 volume of sulfuric acid R
and 9 volumes of aeene anhydride R . Examine in ultraviolet
light at 365 nm.
Results In the chromatogram obtained with the test
solution, no yellowish-red fluorescent zone
(bisdemethoxycurcumin) appears slightly above the greenish-
blue fluorescent zone due to fluorescein in the chromatogram
obtained with the reference solution.
Water (2.2.13)
Maximum 120 mUkg, determined on 20.0 g of the
powdered drug (500) (2.9.12).
Total ash (2.4.16)
Maximum 8.0 per cent.
ASSAY
Essential oH
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use a 500 mL round-bonomed flask, 200 mL of
water Ras the distillation liquid and 0.5 mL of xylene R in
the graduated tube. Reduce the drug to a powder (500)
(2.9. 12) and irnmediately use 5.0 g for the determination.
Distil at arate of 3-4 mUmin for 3 h.
DicinnamoyI methane derivatives
To 0.100 g ofthe powdered drug (180) (2. 9.12) add 60 mL
of glacial aeene acid R and heat in a water-bath at 90 oC for
60 min. Add 2.0 g of boric acid R and 2.0 g of oxalie acid R
and heat in a water-bath at 90 oC for 10 min. Allow to cool,
dilute to 100.0 mL with glacial acetie acid R and shake.
Dilute 5.0 mL of the c1ear supernatant to 50.0 mL with
glacial aeene acid R. Measure the absorbance (2.2.25) at
530 nm, using glacial aeetie acid R as the compensation
liquid.
Calculate the percentage content of dicinnamoyl methane
derivatives, expressed as curcumin, using the following
expression:
A x 0.426
m test solution. Furthermore, other faint zones may be presem
i.e. taking the specific absorbance of curcumin to be 2350.
A absorbance at 530 nm
m = mass of the drug to be examined, in grams
____________________________________________ ~Em
Turmeric Rhizome ***
**** * ** **
(Ph Eur monograph 2543)
~Em ____________________________________________
DEFINITION
Whole, cured (by boiling or steaming), dried rhizome of
Cureuma longa L. (syn. C. domestica Valeton) with roots and
outer surface removed.
Content:
-- essentialoil: minimum 25 mUkg (anhydrous drug);
-- dicinnamoyl methane derivatives, expressed as eureumin
(CzlHzo06; M r 368.4): minimum 2.0 per cent
(anhydrous drug) .
CHARACTERS
Spicy odour.
Turmeric Rhizome IV-383
IDENTIFICATION
A. The rhizome is ovate, oblong-ovoid, pyriform or
cylindrical, often shortly branched, up to 6 cm long and
15 mm thick. The primary rhizome shows scars from the
lateral branches. The surface is slightly dusty, spotted and
brownish-yellow, yellow or brownish-grey, finely striated.
The fracture is granular, smooth, non-fibrous, slightly glossy,
uniformly orange-yellow; it shows a narrow cortex that is
darker on the outside.
B. Microscopic examination (2.8.23). The powder is orange-
yellow. Examine under a microscope using ehloral hydrate
solwion R . The powder shows the following diagnostic
characters: fragments of parenchyma sometimes coloured
yellow by curcumin; reticulate or pined vessels; rare
fragments of brown cork; rare oil droplets. Examine under a
microscope using a 50 per cent V/V solution of glyeerol R .
The powder shows starch granules, free or included in
parenchymatous cells, usually gelatinised and agglomerated;
rare ovoid starch granules, with a punctiform hilum in the
narrow part, are also presento
e. Thin-Iayer chromatography (2.2.27).
Test solution To 1 g of the freshly powdered herbal drug
(355) (2.9.12) add 10 mL of ethanol (96 per eem) R, shake,
allow to stand for 30 min with occasional shaking and filter;
use the filtrate.
Referenee solution Dissolve 20 mg of eureuminoids R and
10 mg of thymol R in 10 mL of ethanol (96 per eem) R .
Place TLC siliea gel place R (5-40 ¡.¡m) [or TLC silica gel
plate R (2- 10 ~lm)] .
Mobile phase glacial aeetic aeid R, toluene R (20:80 V/V).
Applieation 10 ~lL [or 3 ¡.tL] as bands of 10 mm [or 8 mm].
Developmem Over a path of 10 cm [or 6 cm].
Drying In air.
Deceenon A Examine in ultraviolet light at 365 nm.
Results ASee below the sequence of zones present in the
chromatograms obtained with the reference solution and the
in the chromatogram obtained with the test solution.
Top of the plate
- -- ---
Curcuminoids: a greenish A greenish fluorescent zone
fluorescent zone (curcuminoids)
- -- ---
Curcuminoids: 2 greenish 2 greenish fluorescent zones
fluorescent zones (curcuminoids)
Reference solution Test solution
Deteetion B Treat with anisaldehyde solution R and heat at
100-105 oC for 10 min; examine in ultraviolet light at
365 nm.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faim zones may be present
in the chromatogram obtained with the test solution.
 IV-384 Turpentine Oil 2014

Top of the plate

Turpentine Oil
A faint pink zone
Preparation
An in tense reddish zone White Liniment
--- - -- DEFINITION
Thyrnol: a dark zone
Turpentine Oil is obtained by distillation from the oleoresin
obtained from various species of Pinus and rectified.
A pinkish·red zone
CHARACTERISTICS
Curcurninoids : a brown zone A brown zone (curcurninoids) A clear, bright, colourless liquid, visibly free from water;
--- --- odour, characteristic.
Curcuminoids: 2 yellow zones 2 yellow zones (curcurninoids) TESTS
Refractive index
1.467 to 1.477, Appendix V E.
Reference solution Test solution Solubility in ethanol
Soluble, at 20°, in 7 volumes of ethanol (90%) and in
3 volumes of ethanol (96%), Appendix X M.
TESTS
Weight per mL
Curcuma zanthorrhiza Roxb
0.855 to 0.868 g, Appendix V G.
Examine the chromatogram obtained in Identification C,
detection B. Residue on evaporation
R esults B The chromatogram obtained with the test solution
Not more than 1.0% when determined by the method for
residue on evaporation of volatile oils, Appendix X M. U se 2 g
shows no dark zone just above the zone due to thymol in the
chromatogram obtained with the reference solution. and heat for 4 hours.

Water (2.2.13) STORAGE

Maximum 120 mUkg, determined on 15.0 g ofthe Turpentine Oil should be kept in a well-filled container and
powdered herbal drug (500) (2.9.12). protected from light.
Total ash (2.4.16)
Maximum 7.0 per cent.
ASSAY
Pinus Pinaster Type ***
Essential oil
** **
Carry out the determination of essential oils in herbal drugs Turpentine Oil *****
(2.8.12). Use 2.5 g of the freshly powdered herbal drug (500)
(Turpentine Oil, Pinus Pinaster Type,
(2.9.12), a 2 L round-bottomed fiask, 400 mL of water Ras
Ph Eur monograph 1627)
the distillation liquid and 0.5 mL of xylene R in the
~Ew ____________________________________________
graduated tube. Distil at arate of 2 mUmin for 3 h.
Dicinnamoy1 methane derivatives DEFINITION
Disperse 0.500 g ofthe powdered herbal drug (500) (2.9.12) Essential oil obtained by steam distillation, followed by
in 30 mL of ethanol (96 per cent) R in a 100 mL round- rectification at a temperature below 180 oC, from the
bottomed fiask. Heat under a refiux condenser for 2.5 h. oleoresin obtained by tapping Pinus pinaster Aiton. A suitable
Cool and filter into a volumetric fiask, rinse the round- antioxidant may be added.
bottomed fiask and the filter with ethanol (96 per cent) R and CHARACTERS
dilute to 100.0 mL with the same solvento Dilute 1.0 mL of
Appearance
the solution to 50.0 mL with ethanol (96 per cent) R. Measure
Clear, colourless or pale yellow liquido
the absorbance (2.2.25) at 425 nm using ethanol
(96 per cent) R as the compensation liquido Characteristic odour.
Calculate the percentage content of dicinnamoyl methane IDENTIFICATION
derivatives, expressed as curcumin, using the following B.
First identification
expression: Second identification A.
A. Thin-layer chromatography (2.2.27).
A x 5000 Test solution Mix 1 mL of the substance to be examined
1607 x m with toluene R and dilute to 10 mL with the same solvento
Reference solution Mix 10 ¡.¡L of {J-pinene R and 10 ¡.¡L of
Le. taking the specific absorbance of curcumin to be 1607. linalol R with toluene R and dilute to 10 mL with the same
A absorbance at 425 nm; solvento
m = mass of the herbal drug to be examined, in grams.
Plate TLC silica gel plate R .
____________________________________________ ~Ew

Mobile phase ethyl acetate R, toluene R (5:95 V/V).

Application 10 ¡.¡L, as bands .
Development Over a path of 15 cm.
Drying In airo
Detection Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 min. Examine in daylight.
2014 Valerian IV-385

Results See below the sequence of the zones present in the Temperature:
chtomatograms obtained with the reference solurion and the
Time Temperature
test solution. (min) (oC)
Column 0·10 60
10·80 60 -; 200
Top of the plate
80· 120 200
--- --- Injection port 200

/>-Pinene: a pink zone A pink zone (~·pinene) Detector 250

A pink zone Detection Flame ionisation.

--- --- Injection 0.5 ¡.tL.
Linalol: a pinkish·grey zone
Elution order Order indicated in the composition of the
reference solution (a); record the retention times of these
3 faint violet zones substances.
A faint yellow zone System suitability:
-- resolution: minimum 1.5 berween the peaks due to
Reference solution Test solution
car-3-ene and ~-myrcene in the chtomatogram obtained
with reference solurion (a).
B. Examine the chtomatograms obtained in the test for Using the retention times determined from the
chtomatographic pro file. chtomatogram obtained with reference solution (a), locate
Results The peaks in the chtomatogram obtained with the the components of reference solution (a) in the
test solution are similar in retention time to those in the chromatogram obtained with the test solution.
chromatogram obtained with the reference solution. Determine the percentage content of these components.
The limits are within the following ranges:
TESTS -- rx-pinene: 70.0 per cent to 85.0 per cent;
Relative density (2.2.5) -- camphene: 0.5 per cent to 1.5 per cent;
0.856 to 0.872. -- f3-pinene: 11.0 per cent to 20.0 per cent;
Refractive index (2.2.6) -- car-3-ene: maximum 1.0 per cent;
1.465 to 1.475. -- f3-myrcene: 0.4 per cent to 1.5 per cent;
Optical rotation (2.2.7) -- limonene: 1.0 per cent to 7.0 per cent;
- 40° to - 28°. -- longifolene: 0.2 per cent to 2.5 per cent;
-- f3-caryophyllene: 0.1 per cent to 3.0 per cent;
Acid value (2.5.1)
-- caryophyllene oxide: maximum 1.0 per cent;
Maximum 1.0.
-- disregard limit: area of the peak in the chromatogram
Peroxide value (2.5.5, Method B) obtained with reference solurion (b) (0.05 per cent).
Maximum 20.
Residue on evaporation (2.8.9)
Fatty oils and resinified essential oils (2.8.7) Maximum 2.5 per cent, determined after heating on a water-
Ir corrÍ.plies with the test for fatty oils and resinified essential bath for 3 h .
oils.
STORAGE
Chromatographic profile At a temperature not exceeding 25 oc.
Gas chromatography (2.2.28): use the normalisation
____________________________________________ ffiEm
procedure.
Test solutwn The substance to be examined.
Reference solution (a) Dissolve 30 ¡.tL of rx-pinene R, 10 mg
of camphene R, 20 ¡.tL of f3-pinene R, 10 IlL of car-3-ene R,
10 ¡.tL of f3-myrcene R, 20 ~lL of limonene R, 10 IlL of Valerian *****
longifolene R, 10 ¡.tL of {J-caryophyllene R and 10 mg of ** **
caryophyllene oxide R in 1 mL of hexane R. (Valerian Root, Ph Eur monograph 0453) ***
Reference solution (b) Dissolve 5 ¡.tL of f3-caryophyllene R in Preparations
hexane R and dilute to 1 mL with the same solven!. Dilute Valerian Dry Extract
0.1 mL to 1 mL with hexane R. Valerian Dry Hydroalcoholic Extract
Column: Valerian Tincture
-- material: fused silica; When Powdered Valerian is prescribed or demanded,
-- size: l = 60 m, 0 = 0.25 mm; material complying with the appropriate requirements below
-- stationary phase: macrogol 20 000 R (film thickness shall be dispensed or supplied.
0.25 ¡.tm). ffiEm __________________________. __________________
Carrier gas helium for chromatography R .
DEFINITION
Flow rate 1.0 mUmin.
Dried, whole or fragmented underground parts of Valeriana
Split ratio 1:63. officinalis L. s.l., including the rhizome surrounded by the
roots and stolons.
Content:
-- essentialoil: minimum 4 mUkg (dried drug);
IV-386 Valerian 2014

- sesquiterpenie acids: minimum 0.17 per cent m/¡n, Top of the plate
expressed as valerenic acid (C lsH2202; M r 234.3) (dried
--- - --
drug);
Valerenic acid : a violet zone A violet zone (valerenic acid)
IDENTIFICATION
A. The rhizome is yellowish-grey or paJe brownish-grey, Acetoxyvalerenic acid : a violet A violet zone (acetoxyvalerenic
zone acid)
obconical or cylindrical, up to about 50 mm long and 30 mm
in diameter; the base is elongated or compressed, usually --- ---
entirely covered by numerous roots. The apex usually 2 faint or very faint violet zones
exhibits a cup-shaped scar from the aerial parts; stem bases
Referenee solution Test solution
are rarely presento When cut longitudinally, the pith exhibits
a central cavity transversed by septa. The roots are
numerous, almost cylindrical, of the same colour as the
TESTS
rhizome, 1-3 mm in diameter and sometimes more than
Foreign matter (2.8.2)
100 mm long. A few filiform fragile secondary roots are
Maximum 5 per cent of stem bases and maximum 2 per cent
presento The fracture is short. The stolons show prominent
of other foreign matter.
nodes separated by longitudinally striated internodes, each
20-50 mm long, with a fibrous fracture. Loss on drying (2.2.32)
B. Reduce to a powder (355) (2.9.12) . The powder is pale Maximum 12.0 per cent, determined on 1.000 g ofwell
yellowish-grey or pale greyish-brown. Examine under a homogenised powdered drug (3 55) (2.9.12) by drying in an
microscope using ehloral hydrate solution R. The powder oven at 105 oC for 2 h.
shows the following diagnostic characters: cells containing a Total ash (2.4. 16)
pale brown resin or droplets of essential oil; groups of small, Maximum 12.0 per cent.
rectangular sclereids with thick walls and a narrow, Ash insoluble in hydrochloric acid (2.8.1)
channelled branched lumen; occasional groups of larger, Maximum 5.0 per cent.
thinner-walled sclereids from the stem bases; lignified,
reticulately-thickened ves seIs, singly or in small groups; thin- ASSAY
walled, elongated cells of the piliferous layer, sorne with root Essential oil (2.8. 12)
hairs; occasional fragments of cork. Examine under a Use 40.0 g offreshly powdered drug (500) (2.9. 12), a
microscope using a 50 per cent V/V solution of glyeerol R. 2000 mL flask, 500 mL of water R as the distillation liquid
The powder shows abundant starch granules, mainly and 0.50 mL of xylene R in the graduated tube. Distil at a
compound with up to 4-6 components but frequently rate of 3-4 mUmin for 4 h.
separated to form single granules, rounded or irregular and Sesquiterpenic acids
up to about 15 11m in diameter; most of the granules show a Liquid chromatography (2.2.29) .
rather indistinct cleft or radiate hilum. Test solution Place 1.50 g of the powdered drug (7 10)
C. Thin-Iayer chromatography (2.2.27). (2.9.12) in a 100 mL round-bottomed flask with a ground-
Test solution Suspend 1 g of the powdered drug (355) glass neck. Add 20 mL of methanol R1. Mix and heat on a
(2. 9.12) in 10 mL of methanol R and sonicate for 10 mino water-bath under a reflux condenser for 30 minoAlIow to
Filter the supernatant through a membrane filter (nominal cool and filter. Place the filter with the residue in the 100 mL
pore size 0.45 11m). Use the filtrate as the test solution. round-bottomed flask. Add 20 mL of methanol R1 and heat
on a water-bath under the reflux condenser for 15 mino
Referenee solution Dissolve 5 mg of aeetoxyvalerenie acid R
AlIow to cool and filter. Combine the filtrates and dilute to
and 5 mg of valerenie acid R in 20 mL of methanol R.
50.0 mL with methanol R1, rinsing the round-bottomed flask
Plate TLC siliea gel plate R (5-40 ilffi) [or TLC siliea gel and the filter.
plate R (2-10 11m)].
Referenee solution Dissolve an amount of valerian dry extraet
Mobile phase glacial aeetie acid R, ethyl aeetate R, HRS corresponding to 1.0 mg of valerenic acid in
eyclohexane R (2:38:60 VIV/V). methanol RI and dilute to 10.0 mL with the same solvento
Applieation 20 I1L [or 5 ~LL] as bands of 10 mm [or 8 mm]. Sonicate for 10 min and filter through a membrane filter
Development Over a path of 10 cm [or 6 cm] . (nominal pore size 0.45 11m).
Drying In air. Column:
Deteetion Spray with anisaldehyde solution R and heat at - size: 1 = 0.25 m, 0 = 4.6 mm;
100-105 oC for 5-10 min; examine in daylight. - stationary phase: oetadecylsilyl silica gel for ehromatography R
(5 11m).
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the Mobile phase:
test solution. Furthermore, other violet zones may be present - mobile phase A: aeetonitrile RI, 5 giL solution of phosphorie
in the chromatogram obtained with the test solution. acid R (20:80 V/V);
- mobile phase B: 5 gIL solution of phosphorie acid R,
aeetonitrile R1 (20:80 V/V);
Time Mobile phase A Mobile phase B
(min) (pereent VM (pereent VM
0-5 55 45

5 - 18 55 ~ 20 45 ~ 80

18 - 22 20 80

Flow rate 1.5 mUmin.

2014 Valerian IV-387

Detection Spectrophotometer at 220 nm. B. Thin-Iayer chromatography (2.2.27).

Injection 20 ~1L.
Peak identification Use the chromatogram supplied with
valerian dry extract HRS and the chromatogram obtained with
the reference solution to identify the peaks due to
acetoxyvalerenic acid and valerenic acid.
System suitability Reference solution:
-- relative retention with reference to valerenic acid
(retention time = about 19 min):
acetoxyvalerenic acid = about 0.5.
Calculate the percentage content of sesquiterpenic acids,
expressed as valerenic acid, using the following expression:
(Al + A2 ) x m2 X p X 5
A3 X m1
area of the peak due to acetoxyvalerenic acid in the
chromatogram obtained with the test solutioni
area of the peak due to valerenic acid in the
chromatogram obtained with the test solutioni
area of the peak due to valerenic acid in the
chromatogram obtained with the reference solutioni
mass of the drug to be examined used to prepare the
test solution, in gramsi
mas s of valerian dry extract HRS, used to prepare the
reference solution, in gramsi
p percentage content of valerenic acid in valerian dry
extract HRS.
____________________________________________
Test solution Suspend 1 g of the powdered drug (355)
(2.9.12) in 10 mL of methanol R and sonicate for 10 min.
Filter the supernatant through a membrane filter (nominal
pore size 0.45 ¡lm). Use the filtra te as the test solution.
Reference solution Dissolve 5 mg of acewxyvalerenic acid R
and 5 mg of valerenic acid R in 20 mL of methanol R.
Plate TLC silica gel plate R (5-40 ¡lm) [or TLC silica gel
plate R (2-10 ¡lm)] .
Mobile phase glacial acetic acid R, ethyl acetate R,
cyclohexane R (2:38:60 VIV/V).
Application 20 ¡lL [or 5 ¡.tL] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 6 cm].
Drying In airo
Detection Spray with anisaldehyde solution R and heat at
100-105 oC for 5-10 mini examine in daylight.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other violet zones may be present
in the chromatogram obtained with the test solution.
Top oC the plate
--- ---
Valerenic acid: a violet zone A violet zone (valerenic acid)
Acetoxyvalerenic acid: a violet A violet zone (acetoxyvalerenic
zone acid)
~E~
--- ---
2 faint or very faint violet zones
Reference solution Test solution

Cut Valerian ***

*** ***
(Valerian Root, Cut Ph Eur monograph 2526) ***
PhE~ ____________________________________________
DEFINITION
Dried, cut underground parts of Valeriana officinalis L. s.l.,
including the rhizome, roots and stolons.
It is produced from Valerian root (0453) for the purpose of
being used in herbal teas.
Content:
-- essentialoil: minimum 3 mUkg (dried drug)i
-- sesquiterpenic acids: minimum 0.10 per cent m/m expressed
as valerenic acid (ClsHzzOzi M r 234.3) (dried drug).
IDENTIFICATION
A. Reduce to a powder (355) (2.9.12). The powder is pale
yellowish-grey or pale greyish-brown. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters: cells containing a
pale brown res in or droplets of essential Oili groups of small,
rectangular sclereids with thick walls and a narrow,
chaunelled branched lumeni occasional groups of larger,
thin-walled sc1ereids from the stem basesi lignified,
reticulately-thickened vessels, singly or in small groupsi thin-
walled, elongated cells of the piliferous layer, some with root
hairs; occasional fragments of cork. Examine under a
microscope using a 50 per cent V/V solution of glycerol R.
The powder shows abundant starch granules, mainly
compound with up to 4-6 components but frequently
separated to form single granules, rounded or irregular and
up to about 15 ¡lm in diameteri most of the granules show a
rather indistinct cleft or radiate hilum.
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stem bases and maximum 2 per cent
of other foreign matter, deterrnined on the herbal drug prior
to cutting.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g ofwell
homogenised powdered drug (355) (2.9.12) by drying in an
oven at 105 oC for 2 h.
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 5.0 per cent.
ASSAY
Essential oH (2.8.12)
Use 40.0 g offreshly powdered drug (500) (2.9.12), a
2000 mL flask, 500 mL of water R as the distillation liquid
and 0.50 mL of xylene R in the graduated tube. Distil at a
rate of 3-4 mUmin for 4 h.
Sesquiterpenic acids
Liquid chromatography (2.2.29).
Test solution Place 1.50 g of the powdered drug (710)
(2.9.12) in a 100 mL round-bottomed flask with a ground-
glass neck. Add 20 mL of methanol Rl. Mix and heat on a
water-bath under a reflux condenser for 30 min. Allow to
cool and filter. Place the filter with the residue in the 100 mL
round-bottomed flask. Add 20 mL of methanol Rl and heat
on a water-bath under the reflux condenser for 15 min.
Allow to cool and filter. Combine the filtrates and dilute to
 IV-388 Valerian Preparations 2014

SO.O mL with methanol R1, rinsing the round-bottomed flask

and the filter.
Valerian Dry Aqueous Extraet
Reference solution Dissolve an amount of valerian dry extract (Ph Bur monograph 2400)
HRS eorresponding to 1.0 mg of valerenie aeid in ~E~ _ _ _ __ __ _ _ __ _ _ __ _ _ _ __ ___
methanol R1 and dilute to 10.0 mL with the same solvent.
Sonieate for 10 min and filter through a membrane filter DEFINITION
(nominal pore size O.4S ~m) . Extraet produeed from Valerian root (0453).
Column: Content
- size: l = 0.2S m, 0 = 4.6 mm; Minimum 0.02 per eent of sesquiterpenie aeids, expressed as
- stationary phase: octadecylsilyl silica gel for chromatography R valerenic acid (ClsH2202; M r 234.3) (dried extraet).
(S ~m) . PRODUCTION
Mobile phase: The extraet is produeed from the herbal drug by a suitable
- mobile phase A: acetonitrile R1, S giL solution of phosphoric proeedure using water at not les s than 60 ce.
acid R (20:80 V/V);
- mobile phase B: S gIL solution of phosphoric acid R,
CHARACTERS
acetonitrile R1 (20:80 V/V); Appearance
Brown or brownish, hygroseopie powder.
IDENTIFICATION
Time Mobile phase A Mobile phase B
(min) (percent Vil? (percent Vil?
Thin-Iayer ehromatography (2.2.27).
0-5 55 45 Test solution Suspend 1.0 g of the extraet to be examined in
10 mL of methanol R and sonieate for 10 mino Filter the
5 - 18 55 ~ 20 45 ~80
supernatant through a membrane filter (nominal pore size
18 - 22 20 80 O.4S ~m). Use the filtrate as the test solution.
Reference solution Dissolve S mg of acetoxyvalerenic acid R
Flow rate I.S mUmin.
and S mg of valerenic acid R in 20 mL of methanol R.
Detection Speetrophotometer at 220 nm.
Plate TLC silica gel plate R (S-40 ~m) [or TLC silica gel
Injection 20 ~L. plate R (2-10 ~m)].
Peak identification Use the ehromatogram supplied with Mobile phase glacial acetic acid R , ethyl acetate R,
valerian dry extract HRS and the ehromatogram obtained with cyclohexane R (2:38:60 VIV/V) .
the referenee solution to identify the peaks due to Application 20 ~L [or S ~L] as bands of 10 mm [or 8 mm] .
aeetoxyvalerenie acid and valerenic acid.
Development Over a path of 10 cm [or 6 cm].
System suitability Referenee solution:
Drying In airo
- relative retention with referenee to valerenie aeid
(retention time = about 19 min): Detection Spray with anisaldehyde solution R and heat at
aeetoxyvalerenie aeid = about O.S. lOO-lOS oC for S-lO minó examine in daylight.
Calculate the pereentage eontent of sesquiterpenie acids, Results See below the sequenee of zones present in the
expressed as valerenie aeid, using the foIlowing expression: ehromatograms obtained with the referenee solution and the
test solution. A faint violet zone due to valerenie aeid may be
present in the ehromatogram obtained with the test solution.
(Al + Az) x mz x p x 5 Furthermore, other zones may be present in the
A3 x mi
ehromatogram obtained with the test solution.

Al area of the peak due to aeetoxyvalerenie acid in the

Top of the plate
ehromatogram obtained with the test solution;
Az area of the peak due to valerenie aeid in the
--- ---
ehromatogram obtained with the test solution; Valerenic acid: a violet zone
A3 area of the peak due to valerenie aeid in the
Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic acid)
ehromatogram obtained with the referenee solution;
mI mas s of the drug to be examined used to prepare the --- ---
test solution, in grams; A violet zone (hydroxyvalerenic acid)
m2 mass of valerian dry extract HRS, used to prepare the
referenee solution, in grams;
p pereentage eontent of valerenie aeid in valerian dry Reference solution Test solution
extract HRS.
_ _____________________________________ ~E~ TESTS
Loss on drying (2.8.17)
Maximum 6.0 per eent.
ASSAY
Liquid ehromatography (2.2.29).
Solvent mixture methanol R, water R (SO:SO V/V).
Test solution In a 300 mL eonieal flask suspend 1.00 g of
the extraet to be examined in 40 mL of water R whilst
swirling. Add 40 mL of methanol R and swirl for 1 h at 200
r/min. Filter the suspension into a volumetric flask and rinse
2014 Valerian Preparations IV-389

the eonieal fiask with 3 quantities, eaeh of 5 mL, of the

solvent mixture. Dilute to 100.0 mL with the solvent
Valerian Dry Hydroalcoholic ******
*
mixture. Extract *****
Reference solution (a) Dissolve a quantity of valerian dry (Ph Eur monograph 1898)
extract HRS eorresponding to 1.0 mg of valerenie acid in ffiEw ____________________________________________
methanol R and dilute to 10.0 mL with the same solvento
Sonieate for 10 min and filter through a membrane filter DEFINITION
(nominal pore size 0.45 ¡.tm). Extraet produeed from Valerian root (0453).
Reference solution (b) Dilute 1.0 mL of referenee solution (a) Content
to 50.0 mL with methanol R. Minimum 0.25 per eent m/m of sesquiterpenic aeids,
Column: expressed as valerenic acid (C15H2202; M r 234.3) (dried
-- size: 1 = 0.25 m, 0 = 4 mm; extraet).
-- stationary phase: octadecylsi1y1 silica gel for chromatography R PRODUCTION
(5 ¡.tm). The extraet is produeed from the herbal drug by a suitable
Mobile phase: proeedure using ethanol (30-90 per eent V/V) or methanol
-- mobile phase A : acetonitrile Rl, 5 giL solution of phosphoric (40-55 per eent V/V) .
acid R (20:80 V/V);
CHARACTERS
-- mobile phase B: 5 gIL solution of phosphoric acid R,
Appearance
acetonitrile Rl (20 :80 V/V);
Brown, hygroseopic powder.
Time Mobile phase A Mobile phase B
(min) (per cent V111 (per cent V111
IDENTIFICATION
0-5 55 45 Thin-Iayer ehromatography (2.2.27).
5 - 18 55 -720 45 -780
Test solution Suspend 1 g of the extraet to be examined in
10 mL of methanol R and sonicate for 10 mino Filter the
18 - 22 20 80 supematant through a membrane filter (nominal pore size
0.45 ¡.tm). Use the filtrate as the test solution.
Flow rate 1.5 mUmin.
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R
Detection Speetrophotometer at 220 nm. and 5 mg of valerenic acid R in 20 mL of methanol R.
Injection 20 ¡.tL.
Plate TLC silica gel plate R (5-40 ¡.tm) [or TLC silica gel
Identification of peaks Use the ehromatogram supplied with plate R (2-10 ¡.tm)].
valerian dry extract HRS and the ehromatogram obtained with
Mobile phase glacial acetic acid R, ethyl acetate R,
referenee solution (a) to identify the peaks due to
cyclohexane R (2:38:60 VIV/V) .
aeetoxyvalerenie aeid and hydroxyvalerenie aeid.
Application 20 ¡.tL [or 5 ¡.tL] as bands of 10 mm [or 8 mm] .
Relative retention With referenee to valerenie aeid
(retention time = about 19 min): Development Over a path of 10 em [or 6 em] .
hydroxyvalerenie aeid = about 0.2; Drying In airo
aeetoxyvalerenie acid = about 0.5. Detection Spray with anisaldehyde solution R and heat at
Calculate the pereentage eontent of sesquiterpenic acids, 100-105 oC for 5-10 min; examine in daylight.
expressed as valerenic acid, using the following express ion: Results See below the sequenee of zones present in the
ehromatograms obtained with the referenee solution and the
(Al + A2 ) x m2 X p xO.2 test solution. Furthermore, other violet zones may be present
A3 x ml in the ehromatogram obtained with the test solution.

area of the peak due to hydroxyvalerenic aeid in the Top oC the plate

ehromatogram obtained with the test solution; --- ---

area of the peak due to aeetoxyvalerenic acid in the A violet zone (valerenic acid)
Valerenic acid: a violet zone
ehromatogram obtained with the test solution;
area of the peak due to valerenie acid in the Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic acid)
ehromatogram obtained with referenee solution (b); --- ---
mass of the extraet 10 be examined used to prepare
2 faint or very faint violet zones
the test solution, in grams;
mass of valerian dry extract HRS used to prepare ReCerence solution Test solution
referenee solution (a), in grams;
p pereentage eontent of valerenic acid in valerian dry
extract HRS. TESTS
____________________________________________ ffiEw
Loss on d.rying (2.8.17)
Maximum 6.0 per eent.
ASSAY
Liquid ehromatography (2.2.29).
Test solution Suspend 1.00 g of the extraet to be examined
in 50.0 mL of methanol Rl, sonicate for 10 min and filter
through a membrane filter (nominal pore size 0.45 ¡.tm).
Reference solution Dissolve an amount of valerian dry extract
HRS eorresponding to 0.5 mg ofvalerenie aeid in
IV-390 Valerian Preparations 2014

methanol R1 and dilute 10 10.0 mL with the same solvento PRODUCTION

Sonicate for 10 min and filter through a membrane filter The tincture is produced from 1 part of the drug and 5 parts
(nominal pore size 0.45 J.lm). of ethanol (60 10 80 per cent VN) by an appropriate
Column: procedure.
-- size: 1 = 0.25 m, (2) = 4.6 mm; CHARACTERS
-- stationary phase: oetadeeylsilyl silica gel for ehromatography R Appearance
(5 11m). Brown liquido
Mobile phase:
-- mobile phase A: aeetonitrile R1, 5 gIL solution of phosphorie IDENTIFICATION
acid R (20:80 V/V); Thin-layer chromatography (2.2.27).
-- mobile phase B: 5 gIL solution of phosphoric acid R, Test solutwn Dilute 5 mL of the tincture to be examined
aeetonitrile R1 (20:80 V/V); with 5 mL of ethanol (70 per eent V/JI) R.
Time Mobile phase A Mobile phase B Reference solutwn Dissolve 5 mg of aeetoxyvalerenie acid R
(min) (per cent VM (per cent VM and 5 mg of valerenic acid R in 20 mL of methanol R.
0·5 55 45 Plate TLC silica gel plate R (5-40 11m) [or TLC siliea gel
5·18 55 --. 20 45 --.80 plate R (2-10 J.lm)].
Mobile phase glacial acetie acid R, ethyl aeetate R,
18·22 20 80
eyelohexane R (2:38:60 VIV/V).
Flow rate 1.5 mUmin. Applieation 20 J.lL [or 5 ~LL] as bands of 10 mm [or 8 mm].
Detection Spectrophotometer at 220 nm. Development Over a path of 10 cm [or 6 cm].
Injection 20 J.lL. Drying In airo
Identification of peaks Use the chroma1Ogram supplied with Deteetion Spray with anisaldehyde solution R and heat at
valerian dry extract HRS and the chroma1Ogram obtained with 100-105 oC for 5-10 mini examine in daylight.
the reference solution to identify the peaks due to Results See below the sequence of zones present in the
hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic chromatograms obtained with the reference solution and the
acid. test solution. Furthermore, other violet zones may be present
System suitability Reference solution: in the chromatogram obtained with the test solution.
-- relative retention with reference to valerenic acid
(retention time = about 19 min): hydroxyvalerenic
Top of the plate
acid = about 0.2; acetoxyvalerenic acid = about 0.5.
Calculate the percentage content of sesquiterpenic acids, ---- -----
expressed as valerenic acid, using the following expression: Valereoic acid: a viole! zooe A viole! zooe (valereoic acid)

Ace!oxyvalereoic acid: a violet zooe A violet zooe (acetoxyvalereoic acid)

(Al + A 2 + A 3 ) x m2 x P x 5 ----- -----
A4 x mI
2 faio! or very faiot violet zooes

area of the peak due to hydroxyvalerenic acid in the
chromatogram obtained with the test solution;
area of the peak due to acetoxyvalerenic acid in the
chromatogram obtained with the test solution;
area of the peak due to valerenic acid in the
chromatogram obtained with the test solution;
area of the peak due to valerenic acid in the
chroma1Ogram obtained with the reference solution;
mass of the extract to be examined used to prepare
the test solution, in grams;
mass of valerian dry extraet HRS used to prepare the
reference solution, in grams;
p percentage content of valerenic acid in valerian dry
extract HRS.
____________________________________________
Valerian Tincture
(Ph Bur monograph 1899)
~E~ ____________________________________________
DEFINITION
Tincture produced from Valerian root (0453).
Content
Minimum 0.015 per cent m/m of sesquiterpenic acids,
expressed as valerenic acid (ClsH2202; M r 234.3).
Reference solution Test solution
TESTS
Ethanol (2.9.10)
95 per cent to 105 per cent of the quantity stated on the
labe!'
ASSAY
Liquid chromatography (2.2.29).
Test solutwn Dilute 10.0 g of the tincture 10 be examined to
50.0 mL with methanol R1.
Referenee solution Dissolve an amount of valerian dry extract
HRS corresponding 10 1.0 mg of valerenic acid in
~E~
methanol R1 and dilute to 10.0 mL with the same solvento
Sonicate for 10 min and filter through a membrane filter
(nominal pore size 0.45 J.lm).
Column:
-- size: 1 = 0.25 m, (2) = 4.6 mm;
-- stationary phase: octadeeylsilyl siliea gel for ehromatography R
(5 J.lm).
Mobile phase:
-- mobile phase A: aeetonitrile R1, 5 giL solution of phosphorie
acid R (20:80 V/V);
-- mobile phase B: 5 gIL solution of phosphoric acid R,
aeetonitrile RJ (20:80 V/V);
2014 Verbena Herb IV-391

Time Mobile pbase A Mobile pbase B hydrate solution R. The powder shows the following diagnostic
(min) (percent VM (percent VM characters (Figure 1854.-1): fragments of the leaves, which in
0·5 55 45 surface view [C] show sinuous-walled epidermal cells [Ca)
5· 18 55 -7 20 45 -7 80 with anisocyúc [Cb) or anomocyúc [Cc) stomata (2.8.3),
more nurnerous on the lower epidermis; fragments of stem
18·22 20 80
epidermis [A) consisting of long, polygonal or rectangular
epidermal cells [Aa) with thickened walls and sto mata [Ab);
Flow rate 1.5 mUmin.
covering trichomes, unicellular, thick-walled, up to 500 11m
Deteetion Spectrophotometer at 220 nm. long, wide at the base and arising from the centre of a single
Injection 20 ¡¡L. ring of domed, spherical epidermal cells, in surface view [B)
Peak identifieation Use the chromatogram supplied with or in side view [D); occasional glandular trichomes of 2
valerian dry extract HRS and the chromatogram obtained with types: (a) long stalk with a ftattened head about 35 11m in
the reference solution 10 identify the peaks due 10 diameter and consisting of 4-8 radiaúng cells in side view [E)
acetoxyvalerenic acid and valerenic acid. or in surface view of the head [G) , and (b) short unicellular
System suitability Reference solution: stalk and an enlarged ovate head composed of 4 radiaúng
-- relative retention with reference 10 valerenic acid cells in surface view [Cd) or in transverse secúon [K);
(retention time = about 19 min): triangular-ovo id or rounded pollen grains about 30 11m in
acetoxyvalerenic acid = about 0.5 . diameter, with 3 pores and a sm ooth exine m; many
fragments of stems [F] consisting of groups of fibres [Fblo
Calculate the percentage content of sesquiterpenic acids,
vessels [Fa) and fragments of parenchyma [Fc); isolated
expressed as valerenic acid, using the following expression:
fragments of fibres [H).
(Al + A2 )x m2 x p x 5
A3 x mI

Al area of the peak due to acetoxyvalerenic acid in the

chromatogram obtained with the test solution;
A2 area of the peak due 10 valerenic acid in the
chromatogram obtained with the test solution;
A3 area of the peak due to valerenic acid in the
chromatogram obtained with the reference solution;
mi mas s of the tincture to be examined used 10 prepare
the test solution, in grams;
m2 mas s of valerian dry extraet HRS used to prepare the
reference solution, in grams;
p percentage content of valerenic acid in valerian dry
extraet HRS.
____________________________________________ PhEw

Verbena Herb *****

** **
(Ph Eur monograph 1854) ***
nEw ____________________________________________

DEFINITION
Whole or fragmented, dried aerial parts of Verbena officinalis
L. collected during ftowering.
Content
Minimum l.5 per cent ofverbenalin (C17H240!O; M r 388.4)
(dried drug).
IDENTIFICAnON Figure 1854.·1. - Illustration for identification test B of
A. The stem is greenish-brown, quadrangular, longitudinally powdered herbal drug of verbena herb
grooved and roughly hairy, especially on the angles.
The larger leaves are petiolate and deeply pinnately lobed,
C. Examine the chromatograms obtained in test B for Aloysia
with bluntly dentate margins, the smaller leaves are sessile,
citrodora.
not lobed, with crenate or denta te margins; the surfaces are
rough and covered with bristly hairs, parúcularly over the Results See below the sequence of zones present in the
veins, which are prominent on the lower surface . The ftowers chromatograms obtained with the reference solution and the
are numerous, arranged in a slender spike in the axils of leaf- test solution. Furthermore, other zones may be present in the
like bracts; the tubular calyx has 5 acutely pointed lobes with chromatogram obtained with the test solution.
the pale pink or lilac corolla forming a tube about twice as
long as the calyx.
B. Microscopic examination (2.8.23). The powder is
greenish-brown. Examine under a microscope using ehloral
IV-392 Verbena Herb 2014

Top of the plate Loss on drying (2.2.32)

Maximum 10.0 per cent, determined on 1.000 g of the
--- ---
powdered herbal drug (710) (2.9.12) by drying in an oven at
A brown or green zone 105 oC for 2 h.
Arbutin: a blue or brown zone Total ash (2.4.16)
Maxirnum 10.0 per cent.
An intense brownish·grey zone
Ash insoluble in hydrochloric acid (2.8.1)
Rutin: a dark brownish·yellow
zone
Maximum 2.0 per cent.
--- --- ASSAY
Reference solution Test solution Liquid chromatography (2.2.29).
Internal standard solution Dissolve 10.0 mg offerulic acid R
in ethanol (60 per cent V/Ji] R and dilute to 100.0 mL with
TESTS the same solvento
Aloysia citrodora Test solution To 1.00 g of the powdered herbal drug (710)
A. A lemon-like odour indicates the presence of Aloysia (2.9.12) add 50.0 mL of the internal standard solution and
citrodora. stir with a magnetic stirrer for 2 h . Centrifuge for 15 min and
B. Thin-Iayer chromatography (2.2.27). filter the supernatant using a membrane filter (nominal pore
Test solution To 0.5 g of the powdered herbal drug (710) size 0.45 J-lm).
(2.9.12) add 5 mL of methanol R. Heat in a water-bath at Reference solution Dissolve the contents of a vial of
60 oC for 10 mino Cool and filter. verbenalin CRS in the internal standard solution and dilute to
Reference solution Dissolve 10 mg of arbutin R and 10 mg of 5.0 mL with the same solution.
rutin R in methanol R and dilute to 10 mL with the same Precolumn:
solvento - size: 1 = 0.01 m,0 = 4.0 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
Plate TLC si/ica gel plate R (5-40 J-lm) [or TLC si/ica gel
(5 J-lm) .
plate R (2-10 J-lm)].
Column:
Mobile phase anhydrous formic acid R, glacial acetic acid R,
- size: 1 = 0.25 m, 0 = 4.0 mm;
water R, ethyl acetate R (11 :11:27:100 VIVIV/V) .
- stationary phase: octadecylsilyl si/ica gel for chromatography R
Application 20 J-lL [or 5 J-lL] as bands of 10 mm [or 8 mm]. (5 J-lm);
Development Over a path of 12 cm [or 6 cm]. - temperature: 20 oC.
Drying In airo Mobile phase:
Detection Spray with anisaúlehyde solution R and heat at - mobile phase A: 0.3 per cent VIV solution of phosphoric
100-105 oC for about 10 min; examine in daylight. acid R;
Results The chromatogram obtained with the test solution - mobile phase B: acetonitnle R;
shows no intense blue or violet zone approximately at the Time Mobile phase A Mobile phase B
position of rutin in the chromatogram obtained with the (min) (petcenl VM (per ce nI VM
reference solution. 0·20 93 ~ 83 7 ~ 17

20·30 83 17

30·35 83 ~ 75 17 ~ 25

JJI- A
I
o 5 10 15 20 25 30 35 40 45 min

l. verbenalin 2. ferulic acid 3. unknown substance (may be absent) 4. acteoside

Figure 1854.·2. - Chromatogram for the assay of verbena herb; test solution
 2014 Willow Bark IV-393

Flow mte 1.0 mUmin . crystals of calcium oxalate [Ga, Ja]; sorne parenchyma cells
Detection Spectrophotometer at 240 nm. are collenchymatous [G]; uniseriate medullary rays, in
Injection 20 flL. tangential section [Db]; thickened cork cells, in surface view
[F]; numerous scattered prism crystals [E] and cluster
System suitability Test solution:
crystals [A] of calcium oxalate; fragments of brownish
-- the chromatogram obtained is similar to the
collenchyma from the buds may also be presento Twigs show,
chromatogram shown in Figure 1854.-2;
additionally, wood fragments [H] composed of lignified fibres
-- resolution: minimum 3.5 between the peaks due to ferulic
[Ha] and ves seis [Hb], sometimes accompanied by medullary
acid and acteoside. rays [He].
Calculate the percentage content of verbenalin using the
following expression:

Al X A4 x m2 x 1000
A2 X A3 x mI

area of the peak due to verbenalin in the

chromatogram obtained with the test solution;
area of the peak due to verbenalin in the
chromatogram obtained with the reference solution;
area of the peak due to ferulic acid in the
chromatogram obtained with the test solution;
area of the peak due to ferulic acid in the
chromatogram obtained with the reference solution;
mas s of the herbal drug used to prepare the test
solution, in grams;
mass of verbenalin in the reference solution, in
grams.
____________________________________________ ~E~

Willow Bark ***

*** ***
(Ph Eur monograph 1583) ***
Preparation
Willow Bark Dry Extract
~E~ ___________________________________________

DEFINITION
Whole or fragmented dried bark of young branches or whole
dried pieces of current -year twigs of various species of genus
Salix including S. purpurea L., S. daphnoides Vill. and S.
fmgilis L.
Content
Minimum 1.5 per cent of total salicylic derivatives, expressed
as salicin (C1 3Hl S0 7; M r 286.3) (dried drug) .
IDENTIFICATION
A. The bark is 1-2 mm thick and occurs in flexible,
elongated, quilled or curved pieces. The outer surface is
smooth or slightly wrinkled longitudinally and greenish-
yellow or brownish-grey. The i=er surface is smooth or
finely striated longitudinally and white, pale yellow or
reddish-brown, depending on the species. The fracture is
short in the outer part and coarsely fibrous in the inner
region. The diameter of current-year twigs is not greater than
10 mm. The wood is white or pale yellow.
B. Microscopic examination (2.8.23). The powder is pale
yellow, greenish-yellOW or light brown. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters (Figure 1583.-1) :
bundles [B, C] of narrow fibres [Ba, Ca], up to about
600 flm long, with very thick walls and surrounded by a
crystal sheath containing prisms of calcium oxalate [Bb, Cb];
parenchymatous cells of the cortex [D, TI, with thick, pitted
and deeply beaded walls [Da], and containing large cluster
Figure 1583.-1. - Illustration for identification test B of
powdered herbal drug of willow bark
C. Thin-Iayer chromatography (2.2.27).
Test solution (a) To 1.0 g of the powdered herbal drug
(355) (2. 9. 12) add 10 mL of methanol R . Heat on a water-
bath at about 50 oC, with frequent shaking, for 10 min o Cool
and filter.
T est solurion (b) To 5.0 mL of test solution (a) add 1.0 mL
of a 50 gIL solution of anhydrous sodium carbonate R and heat
in a water-bath at about 60 oC for 10 mino Cool and filter if
necessary.
Reference solurion Dissolve 2 mg of salicin R and 2 mg of
chlorogenic acid R in 1.0 mL of methanol R.
Plate TLC silica gel plate R (5-40 flm) [or TLC si/iea gel
plate R (2-10 pm)].
Mobile phase water R, methanol R, ethyl acetate R
(8:15:77 VIVIV).
Application 10 flL [or 2 flL] as bands.
Development Over a path of 15 cm [or 6 cm].
Drying In a current of warm airo
Detection Treat with a mixture of 5 volumes of sulfuric
acid R and 95 volumes of methanol R. Heat at 100-105 oC
for 5 min and examine in daylight.
 IV-394 Willow Preparations 2014

Results See bclow the sequence of zones present in the Time Mobile phase A Mobile phase B
chromatograms obtained with the reference solution and test (min) (per cenl VIV) (per cenl VIV)
solutions (a) and (b). Furthermore, other zones may be
present in the chromatograms obtained with test solutions (a) 0-15 100 O
and (b). 15·17 lOO ~ 90 O ~ 10

17·23 90 lO
Top of the plate

--- ---
Flow rate 1.0 mUmin.
Several reddish·violet Detection Spectrophotometer at 270 nm.
zones may be present
Injection 1O ¡LL.
Salicin: a reddish· A weak reddish·violet A reddish-violet zone Retention time Salicin = about 6.4 min; picein = about
violet zone zone (salicin) (salicin)
7.7 mino
--- --- System suitability Reference solution:
- resolution: minimum 1.5 between the peaks due to salicin
Chlorogenic acid: a
brown zone and picein.
Calculate the percentage content of total salicylic derivatives,
Reference solution Test solution (a) Test solution (b) expressed as salicin, using the following expression:

TESTS
Foreign matter (2.8.2)
Maximum 3 per cent of twigs with a diameter greater than
area of the peak due to salicin in the chromatogram
10 mm and maximum 2 per cent of other foreign matrero
obtained with the test solution;
Cadmium (2.4.27) area of the peak due to salicin in the chromatogram
Maximum 2.0 ppm. obtained with the reference solution;
Loss on drying (2.2.32) m¡ mass of the herbal drug to be examined used to
Maximum 11 per cent, determined on 1.000 g of the prepare the test solution, in grams;
powdered herbal drug (355) (2.9.12) by drying in an oven at mass of salicin CRS used to prepare the reference
105 oC for 2 h . solution, in grams;
Total ash (2.4.16) p percentage content of saliein in salicin CRS.
Maximum 10 per cent. _ _ __ _ __ _ _ __ _ _ _ _ __ _ _ _ _ _ PhEur

ASSAY
Liquid chromatography (2.2.29).
Test solution To 1.000 g of the powdered herbal drug (355)
(2.9.12) add 40 mL of methanol R and 40.0 mL of a 4.2 gIL Willow Bark Dry Extract *****
solution of sodium hydroxide R. Heat in a water-bath at about ** **
60 oC under a refiux condenser, with frequent shaking, for (Ph Eur monograph 2312) ***
about 1 h. After cooling, add 4.0 mL of a 103.0 giL solution PhEur
of hydrochloric acid R. Filter the suspension into a 100 mL
DEFINITION
volumetric fiask, wash and dilute to 100.0 mL with a mixture
Dry extraet produeed from Willow bark (1583).
of equal volumes of methanol R and water R . Filter through a
membrane filter (nominal pore size 0.45 ¡Lm). Content
Reference solution Dissolve 5.0 mg of picein R in 25.0 mL of Minimum 5.0 per eent of total salieylie derivatives, expressed
a mixture of 20 volumes of water R and 80 volumes of as saliein (C 13H¡ S0 7; M r 286.3) (dried extraet).
methanol R (solution A) . Dissolve 15.0 mg of salicin CRS in PRODUCTION
25 mL of a mixture of 20 volumes of water R and The extraet is produeed from the herbal drug by a suitable
80 volumes of methanol R; add 5.0 mL of solution A and proeedure using either water or a hydroalcoholie solvent
dilute to 50.0 mL with water R. equivalent in strength to a maximum of 80 per eent V/V
Column: ethano!.
- size: 1 = 0.10 m, 0 = 4.6 mm; CHARACTERS
- stationary phase: octadecylsilyl silica gel for chromatography R Appearance
(3 ¡LID).
Yellowish-brown amorphous powder.
Mobile phase:
- mobile phase A: tetrahydrofuran R, 0.5 per cent VIV IDENTIFICATION
solution of phosphoric acid R (1.8:98.2 V/V); Thin-layer ehromatography (2.2.27).
- mobile phase B: tetrahydrofuran R; Test solution (a) To 0.200 g of the extraet to be examined
add 5 mL of methanol R. Sonieate for 5 min, filter and dilute
to 10 mL with methanol R .
Test solution (b) To 5.0 mL of test solution (a) add 1.0 mL
of a 50 gIL solution of anhydrous sodium carbonate R and heat
in a water-bath at about 60 oC for 10 mino Cool and filter if
neeessary.
2014 Withania Somnifera Root IV-395

Reference solution Dissolve 2.0 mg of saliein R and 2.0 mg of Flow rate 1.0 mUmin.
ehlorogenie acid R in 1.0 mL of methanol R. Detection Spectrophotometer at 270 nm.
Plate TLC siliea gel plate R (5-40 ¡lm) [or TLC siliea gel Injeetion 10 ¡lL.
plate R (2-10 ¡lm)) . Retention time Salicin = about 6.4 min;
Mobile phase water R, methanol R, ethyl aeetate R picein = about 7.7 mino
(8:15:77 VIV/V).
System suitabtlity Reference solution:
Applieation 10 ¡lL [or 2 ¡lL) as bands.
- resolution: minimum 1.5 between the peaks due to salicin
Development Over a path of 15 cm [or 6 cm). and picein.
Drying In a current of warm air. Calculate the percentage content of total salicylic derivatives,
Deteetion Spray with a mixture of 5 volumes of sulfurie expressed as salicin, from the following expression:
acid R and 95 volumes of methanol R. Heat at 100-105 oC
for 5 min and examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference solution and test
solutions (a) and (b). Furthermore, other zones may be are a of the peak due to salicin in the chromatogram
present in the chromatogram obtained with test solutions (a) obtained with the test solution
and (b). area of the peak due to salicin in the chromatogram
obtained with the reference solution;
mas s of the extract to be examined used to prepare
Top of the plate
the test solution, in grams
--- ---
m2 mass of saliein CRS used to prepare the reference
Severa! reddish-vio!et solution, in grams
zones may be present p percentage content of salicin in saliein CRS.
Salicin: a reddish-violet A weak reddish-vio!et A reddish-violet zone
_ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
zone zone (salicin) (salicin)
- - - ---

Ch!orogenic acid: a
brown zone
Reference solution Test solution (a) Test solution (b) Withania Somnifera Root
DEFINITlON
ASSAY Withania Somnifera Root consists of the dried mature roots
Liquid chromatography (2.2.29) . of Withania somnífera (L.) Duna!.
Test solution To 0.300 g ofthe extract to be examined add It contains not less than 0.01 % withaferin A (C2sH3S06) and
40 mL of methanol R and 40.0 mL of 0.1 M sodium not less than 0.01 % withanolide A (C 29 H 42 0 7 ) .
hydroxide. Heat in a water-bath at about 60 oC under a reflux PRODUCTlON
condenser, with frequent shaking, for about 1 h. After It is collected in winter, washed, dried and cut into short
cooling, add 4.0 mL of 1 M hydroehlorie aeid. Filter the pieces.
suspension into a 100 mL volumetric flask, then wash and
IDENTIFICATlON
dilute to 100.0 mL with a mixture of equal volumes of
A. Pieces of root, cut into lengths of up to 8 cm, varying in
water R and methanol R. Filter through a membrane filter
diameter from 2 mm to 1 cm, with sorne narrower pie ces of
(nominal pore size 0.45 ¡lm).
rhizome, often cut at the transition zone. Outer surface pale
Referenee solution Dissolve 5.0 mg of pieein R in 25.0 mL of greyish-brown, somewhat darker brown in larger specimens.
a mixture of 20 volumes of water R and 80 volumes of Fracture short, showing a whitish interior. The cut surface of
methanol R (solution A). Dissolve 15.0 mg of saliein CRS in the root may show a distinction between the xylem and other
25 mL of a mixture of 20 volumes of water R and tissues marked by a faint yellow-green cambial ringo
80 volumes of methanol R. Add 5.0 mL of solution A and
B. Reduce to a powder (355). Examine under a microscope
dilute to 50.0 mL with water R.
using ehloral hydrate solution. Cork cells in surface view
Column:
polygonal, in sectional view rectangular, thin-walled,
- size: 1 = 0.10 m, 0 = 4.6 mm;
yellowish brown, often broken. Parenchymatous cells in
- stationary phase: octadeeylsilyl stliea gel for ehromatography R
groups, elongated, rectangular, or oval to round, filled with
(3 ¡lm).
starch; sorne pitted, lightly lignified, found alongside vascular
Mobile phase: fragments; parenchyma of the medullary rays, one or two
- mobile phase A: tetrahydrofuran R, 0.5 per cent V/V cells wide shown crossing xylem elements at right angles;
solution of phosphorie aeid R (1.8:98.2 V/V); Occasional fragments of spiral, scalariform or pitted ves seis
- mobile phase B: tetrahydrofuran R; with broad lumen; tracheids and vessels usually heavily
Time Mobile phase A Mobile phase B lignified, reticulate or bordered pitted, single or in small
(min) (per cent V!JI) (per cent V!JI) groups. Fibres often accompanying ves seis, thick walled,
0-15 lOO O heavily pitted, and lignified; others less pitted and lignified,
15 - 17 lOO --> 90 0---710
thin walled, either found singly or in groups of two or three.
Microcrystals of calcium oxalate scattered or occasionally in
17 - 23 90 10 idioblasts. Examine under a microscope using a 50% v/v
23 - 25 90 --> lOO 10 ---7 O solution of glyeerol. Starch granules abundant, simple or 2 to
25 - 40 lOO
4 compound, round to oval, with a point, stellate or cleft
O
hilum.
IV-396 Wíthanía Somnífera Root 2014

C. Carry out the method for thin-layer ehromatography, CHROMATOGRAPHIC CONDITIONS

Appendix III A, using the foIlowing solutions. (a) Use a stainless steel column (15 cm x 4 .6 mm) packed
(1) Shake 0.5 g of freshly powdered (355) drug with 1 mL of with dodeeylsilyl si/iea gel for ehromatography (4 ~m)
dilute ammonia R4, add 10 mL of methanol, sonicate for (Phenomenex Synergi Max-RP 80Á is suitable) .
10 seconds, heat on a water-bath for 3 minutes, cool, filter, (b) Use gradient elution and the mobile phases described
evaporate the filtrate to dryness at 60 0 and dissolve the dried below. Equilibrate the column with a mixture of 65% mobile
residue in 1 mL of methanol. Filter through a 0.45 ~m filter. phase A and 35% mobile phase B for at least 15 minutes.
(2) 0.1 % w/v each of withaferin A CRS, withanolide B CRS (c) Use a fiow rate of 1 mL per minute.
and f3-siwsterol in methanol. (d) Use a column temperature of 50°.
CHROMATOGRAPHlC CONDITIONS (e) Use a detection waveIength of 230 nm.
(a) Use a siliea gel F 254 precoated high performance plate (±) Inject 20 pL of each solution.
(Merck silica gel F 254 HPTLC plates are suitable).
MOBILE PHASE
(b) Use the mobile phase described below.
Mobile phase A water.
(c) Apply 2 pL of each of solution, as 6 mm bands.
Mobile phase B Equal volumes of ethanol (96%) and
(d) Develop the plate to 8 cm. methanol.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (254 nm). Immerse the plate in 5% v/v of
methanolie sulfurie acid for 1 second, aIlow to dry in air, heat
at 110 0 for 2 minutes and examine irnmediately in daylight. Time Mobile phase A Mobile phase B Comments
(Minutes) %v/v %v/v
Examine the derivatised plate under ultraviolet light (366 nm).
0-5 65 35 isocratic
MOBILE PHASE
5-30 65 .... 55 35 ....45 linear gradient
5 volumes of anhydrous formie acid, 15 volumes of ethyl acetate
and 50 volumes of wluene. 30-31 55- ,0 45- ,100 linear gradient

SYSTEM SUITABILITY 31-36 O 100 isocratic

The test is not valid unless the chromatogram obtained with 36-37 0.... 65 100.... 35 linear gradient
solution (2) shows three cJearly separated bands. 37-45 65 35 re-equilibration

CONFIRMATION
The bands with Rf values of approximately 0.1 (withaferin
A), 0.26 (withanolide B) and 0.57 (P-sitosterol) in the
chromatogram obtained with solution (1) correspond in
colour and position to those in the chromatogram obtained
with solution (2).
TESTS
Absence of Withania coagulans
In Identification test C, the derivatised plate under ultraviolet
light (366 nm) shows no orange band with an Rf of
approximately 0.2 in the chromatogram obtained with
solution (1) .
Ash
Not more than 7.0%, Appendix XI J.
Acid-insoluble ash
Not more than 1.0%, Appendix XI K
Loss on drying
When dried for 2 hours at 105 0 , loses not more than 12.0%
of its weight. Use 1 g.
ASSAY
Carry out the method for liquid ehromawgraphy,
Appendix III D, using the foIlowing solutions.
(1) Extract 1 g of the powdered drug with 3.0 mL of
methanol with the aid of ultrasound for 10 minutes, centrifuge
at 3000 rpm for 5 minutes and retain the supernatant
extracto The extraction is repeated twice as described.
Combine the three supematant extracts, adjust the total
volume of the combined extracts to 20.0 mI with methanol
and filter through a 0.45-~m filter.
(2) 0.02% w/v each of withaferin A CRS and withanolick A
CRS and 0.01 % w/v of withanolide B CRS in methanol.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (2), the resolution faewr between the first two
main peaks, of withaferin A and withanolide A is at least 5.0
and the symmetry factor for both peaks is less than 1.3.
DETERMINATION OF CONTENT
Withaferin A Using the retention time and peak area of the
peak due to withaferin A in the chromatogram obtained with
solution (2), locate and integra te the peak due to withaferin
A in the chromatogram obtained with solution (1).
Calculate the content of withaferin A in the sample using the
decJared content withaferin A (C2sH3S06) in withaferin
A CRS and the foIlowing expression:
Al m, V I 100
- x - x - xpx-- -
A, V, m 100-d
Area of the peak due to withaferin A in the
chromatogram obtained with solution (1).
Area of the peak due to withaferin A in the
chromatogram obtained with solution (2).
m¡ Weight of the drug being examined in mg.
m2 Weight of withaferin A CRS in mg.
V¡ Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content of withaferin A in withaferin
A CRS.
d Percentage los s on drying of the herbal drug being
examined.
Withanolide A Using the retention time and peak area of
the peak due to withanolide A in the chromatogram obtained
with solution (2), locate and integrate the peak due to
2014 Wormwood IV-397

withanolide A in the chromatogram obtained with head with 2-4 cells; free glandular trichomes in side view [C] ;
solution (1). fragments of the corollas of the tubular and ray florets, sorne
Calculate the content of withanolide A in the sample using containing small cluster crystals of calcium oxalate [H];
the declared content of withanolide A (Cz9H4207) in numerous paleae each composed of a small cell forming a
withanolide A CRS and the following expression: stalk and a very long, cylindrical and thin-walled terminal cell
about 1-1.5 mm long, either whole [E] or limited to the
distal part [B]; spheroidal pollen grains, about 30 !lm in
Al ill2 VI 100 diameter, with 3 pores and a finely warty exine [G];
-x-x-xpx---
A, V, ffi¡ 100-d fragments of vascular tissue from the leaves [F] or the stems
UJ consisting of ves seIs with spiral or annular thickenings
[Fa], or with bordered pits ITa], fibres [Fb, Jb] and
Al Area of the peak due to withanolide A in the
parenchymatous cells with pitted, moderately thickened walls
chromatogram obtained with solution (1) .
[Fc, Jc].
Az Area of the peak due to withanolide A in the
chromatogram obtained with solution (2).
m¡ Weight of the drug in mg.
m2 Weight of withanolide A CRS in mg.
V¡ Dilution volume of solution (1) in mL.
V2 Dilution volume of solution (2) in mL.
p Percentage content withanolide A in withanolide
A CRS.
d Percentage loss on drying of the herbal drug being
examined.
STORAGE
Withania Somnifera Root should be protected from moisture.

Wormwood *****
** **
(Ph Eur monograph 1380) ***
~Ew ____________________________________________

DEFINITION
Basal leaves or slightly leafY, flowering tops, or mixture of
these dried, whole or cut organs of Artemisia absinthium L.
Content
Minimum 2 mUkg of essential oil (dried drug).
IDENTIFICATION
A. The leaves are greyish or greenish, densely tomentose on
both surfaces. The basalleaves, with long petioles, have
triangular or oval bipinnatisect or tripinnatisect lamina, with
rounded or lanceolate segments. The cauline leaves are less
segmented and the apical leaves are lanceolate. The stem of Figure 1380.·1. - Illustration for identification test B of
the flower-bearing regíon is greenish-grey, tomentose, up to powdered herbal drug of wormwood
2.5 mm in diameter and usually with 5 flattened longitudinal
grooves. The capitula are arranged as loo se, axillary panicles,
C. Thin-layer chromatography (2.2.27) .
inserted at the level of the lanceolate or slightly pinnatisect
leaves; they are spherical or flattened hemispherical, 2-4 mm Test solution Place 2 g ofthe powdered drug (355) (2.9.12)
in diameter and consist of a grey, tomentos e involucre, the in 50 mL of boiling water R and allow to stand for 5 min,
outer bracts linear, inner layer ova te, blunt at the apices with shaking the flask several times. After cooling, add 5 mL of a
scarious margíns, a receptacle with very long paleae up to 100 giL solution of lead acetate R. Mix and filter. Rinse the
1 mm or more long, numerous yellow, tubular, flask and the residue on the filter with 20 mL of water R.
hermaphroditic florets about 2 mm long and few yellow, ray Shake the filter with 50 mL of methylene chloride R. Separate
florets . the organic layer, dry over anhydrous sodium sulfate R, filter
and evaporate the filtrate to dryness on a water-bath.
B. Microscopic examination (2.8.23). The powder is
Dissolve the residue in 0.5 mL of ethanol (96 per cene) R.
greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic Reference solution Dissolve 2 mg of methyl red R and 2 mg of
characters (Figure 1380.-1.): many T-shaped trichomes [A] resorcinol R in 10.0 mL of methanol R.
with a short uniseriate stalk consisting of 1-5 small cells, Plate TLC siliea gel plate R.
perpendicularly capped by a very long, undulating terminal Mobile phase acetone R, glacial acetic acid R, toluene R,
cell tapering at the ends; fragments of epiderrnises in surface methylene chloride R (10:10:30:50 V/V/VIV) .
view [D] with sinuous or wavy walls, anomocytic stomata Application 10 ~lL as bands.
(2.8.3) [Da], covering trichomes [Db] and glandular
Development Over a path of 15 cm.
trichomes containing oil [Dc] or not containing oil [Dd],
each with a short, biseriate, 2-celled stalk and a biseriate Drying In air.
IV-398 Yarrow 2014

D etection A Spray with acetic anhydride - sulfuric acid tubular disk-florets with a radial, five-Iobed, yellowish or light
solution R and examine in daylight. brownish corolla . The pubescent green, partly brown or
Results A The chromatogram obtained with the test solution violet stems are longitudinally furrow ed, up to 3 mm thick
shows a blue zone due to artabsin shortly aboye a red zone with a light-coloured medulla.
due to methyl red in the chromatogram obtained with the B. Microscopic examination (2.8.23) . The powder is green or
reference solution. greyish-green. Examine under a microscope using chloral
Detection B Examine in daylight while heating at hydrate soluLÍon R . The powder shows the following diagnostic
100-105 oC for 5 min. characters (Figure 1382.-1 ): fragments ofthe stem epidermis,
in surface view [K], with cells having a smooth cuticle and
R esults B The chromatogram obtained with the reference
anomocytic stomata (2.8.3); fragments of leaf and bract
solution shows in the middle third a red zone due to methyl
epidermis es, in surface view [B], with cells having wavy and
red and below it a light pink zone due to resorcinol.
irreguIarly thickened walls, a finely striated cuticle and
The chromatogram obtained with the test solution shows an
anomocytic stomata (2.8.3); very rare glandular trichomes
intense red or brownish-red zone due to absinthin with a
with a short stalk and a head formed of 2 rows of 3-5 cells
similar RF value to that of the zone due to resorcinol in the
enclosed in a bladder-like membrane [H]; uniseriate, whole
chromatogram obtained with the reference solution . Other
or fragmented covering trichomes [A] consisting of 4-6 small,
zones are visible, but less intense than that due to absinthin.
more or less isodiametric cells at the base and a thick-walled,
TESTS often somewhat tortuous terminal cell, about 400 [lm to
Foreign matter (2.8.2) greater than 1000 [lm long; fragments of the liguIate corolla
Maximum 5 per cent of stems with a diameter greater than with papillary epidermal cells [D] ; fragments of the corolla
4 mm and maximum 2 per cent of other foreign marter. tubes, in surface view, with sinuous epidermal cells, covered
Bitterness value (2.8.15) by a thin striated cuticle [F]; small-celled parenchyma from
Minimum 10 000. the corolla tubes containing cluster crystals of ca1cium oxalate
[E]; groups oflignified and pitted cells from the bracts [G] ;
Loss on drying (2.2.32)
spherical pollen grains, about 30 [lm in diameter, with
Maximum 10.0 per cent, determined on 1.000 g of the
3 germinal pores and a spiny exine [C]; groups of
powdered herbal drug (355) (2.9.12) by drying in an oven at
sclerenchymatous fibres and small ves seis with spiral or
105 oC for 2 h.
a=ular thickening, from the stem m.
Total ash (2.4.16)
Maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.0 per cent.
ASSAY
C arry out the determination of essential oil in herbal drugs
(2.8.12). Use 50.0 g of the cut drug, a 1000 mL round-
bottomed ftask and 500 mL of water R as the distillation
liquid. Add 0.5 mL of xylene R in the graduated tube. Distil

~ .
at arate of 2-3 mUmin for not les s than 3 h. .
____________________________________________ PhE~

.. ..
•
E

Yarrow *****
* *
(Ph Eur monograph 1382) *****
~E~ ____________________________________________
...;.,:.~
,
, "
DEFINITION
Whole or cut, dried ftowering tops of Achillea millefolium L.
H
Content:
-- essentialoil: minimum 2 mUkg (dried drug);
-- proazulenes, expressed as chamazulene (C 14H 16; Mr 184.3): ... " ~
minimum 0.02 per cent (dried drug).

~
IDENfIFICATION
A. The leaves are green or greyish-green, faintly pubescent
on the upper surface and more pubescent on the lower
surface, 2-3 pinnately divided with linear lobes and a finely
pointed whitish tip oThe capitula are arranged in a corymb at
the end of the stem. Each capitulum, 3-5 mm in diameter, Figure 1382.-1. - Illustration for identification test B of
consists of the receptacle, usually 4-5 ligulate ray-ftorets and powdered herbal drug of yarrow
3-20 tubular disk-ftorets. The involucre consists of 3 rows of
imbricate lanceolate, pubescent green bracts arranged with a C. To 2_0 g of the powdered herbal drug (7 10) (2. 9.12) add
brownish or whitish, membranous margino The receptacle is 25 mL of ethyl acetate R , shake for 5 min and filter_
slightly convex and, in the axillae of paleae, bears ligulate Evaporate to dryness on a water-bath and dissolve the
ray-ftorets with a three-Iobed, whitish or reddish ligule and residue in 0.5 mL of toluene R (solution A). To 0.1 mL of
2014 Yarrow IV-399

this solution add 2.5 mL of dimethylaminobenzaldehyde Proazulenes

solution R8 and heat on a water-bath for 2 mino Allow to
coo1. Add 5 mL of light petroleum R and shake the mixture
vigorously. The aqueous layer shows a blue or greenish-blue
colour.
D . Thin-Iayer chromatography (2.2.27).
Test solution Use solution A prepared in identification test
C.
R eference solution Dissolve 10 mg of cineole R and 10 mg of
guaiazulene R in 20 mL of toluene R.
Plate TLC silica gel plate R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 20 ¡.tL as bands.
Development Over a path of 10 cm.
Drying In airo
Detection Spray with anisaldehyde solution R, heat at
To ensure that as little water as possible is transferred,
transfer the blue essential oil-xylene mixture obtained in the
assay of essential oil into a 50 mL volumetric ftask with the
aid of small portions of xylene R, rinsing the graduated tube
of the apparatus with xylene R and dilute to 50.0 mL with
the same solvento Measure the absorbance (2.2.25) at
608 nm using xylene R as the compensation liquido
Calculate the percentage content of proazulenes, expressed as
chamazulene, using the following expression:
A x 2.1
m
i.e. taking the specific absorbance of chamazulene to be 23.8.
A absorbance at 608 nrn;
m = mass of the herbal drug to be examined, in grams.
____________________________________________ ~E~

100-105 cC for 5-10 min and examine in daylight.

Results The chromatogram obtained with the reference
solution shows in the upper part a red zone (guaiazulene)
and in the middle part a blue or greyish-blue zone (cineole).
The chromatogram obtained with the test solution shows a
violet zone a little aboye the zone due to guaiazulene in the
chromatogram obtained with the reference solution; below
this zone a reddish-violet zone; below which, 1-2 not clearly
separated greyish-violet or greyish zones (which changes to
greenish-grey after a few hours) and a reddish-violet zone a
littIe aboye the zone due to cineole in the chromatogram
obtained with the reference solution. Further faint zones may
be presento
TESTS
Foreign matter (2.8.2)
Maximum 5 per cent of stems with a diameter greater than
3 mm and maximum 2 per cent of other foreign matter.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 0.500 g of
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 oC for 2 h.
Total ash (2.4.16)
Maximum 10.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.5 per cent.
ASSAY
Essential oil
Carry out the determination of essential oils in herbal drugs
(2.8.12). Use 20.0 g of cut drug, a 1000 mL round-bottomed
ftask and 500 mL of a mixture of 1 volume of water R and
9 volumes of ethylene glycol R as the distillation liquido
Add 0.2 mL of xylene R in the graduated tube. Distil at a
rate of 2-3 mUmin for 2 h.
Stop cooling at the end of distillation and continue distilling
until the blue, steam-volatile components have reached the
lower end of the cooler. Immediately start cooling again,
taking care to avoid warming the separation space. Stop the
distillation after 5 mino Replace the 1000 mL round-
bottomed ftask by a 250 mL round-bottomed ftask
containing a mixture of 0.4 mL of xylene R and 50 mL of
water R . Distil for 15 mino After 10 min read the total
volume. To determine the blank value, use 0.2 mL of
xylene R in the graduated tube and distil a mixture of 0.4 mL
of xylene R and 50 mL of water R for 15 mino
Monographs

Materials for use in the

Manufacture of Homoeopathic
Preparations
2014
Homoeopathic Preparations *****
** **
(Ph Eur monograph 1038) ***
____________________________________________
~E~
DEFINITION
Homoeopathic preparations are prepared from substances,
products or preparations called stocks, in accordance with a
homoeopathic manufacturing procedure. A homoeopathic
preparation is usually designated by the Latin name of the
stock, followed by an indication of the degree of dilution.
Raw materials
Raw materials for the production of homoeopathic
preparations may be of natural or synthetic origino
For raw materials of zoological or human origin, adequate
measures are taken to minimise the risk of agents of
infection, inc1uding virus es (5.1.7), in the homoeopathic
preparations. For this purpose, it is demonstrated that:
-- the method of production inc1udes a step or steps that
have been shown to remove or inactivate agents of
infection;
-- where applicable, raw materials of zoological origin
comply with the monograph Products with risk of
transmitting agents of animal spongiform encephalopathies
(1483);
-- where applicable, the animals and the tissues used to
obtain the raw materials comply with the health
requirements of the competent authorities for animals for
human consumption;
-- for materials of human origin, the donor follows the
recommendations applicable to human blood donors and
to donated blood (see Human plasma for fractionation
(0853)), unless otherwise justified and authorised.
A raw material of botanical, zoological or human origin may
be used either in the fresh state or in the dried state. Where
appropriate, fresh material may be kept deep-frozen.
Raw materials of botanical origin comply with the
requirements of the monograph Herbal drugs for homoeopathic
preparations (2045).
Where justified and authorised for transportation or storage
purposes, fresh plant material may be kept in ethanol
(96 per cent) or in alcohol of a suitable concentration,
provided the whole material inc1uding the storage medium is
used for processing.
Raw materials comply with any requirements of the relevant
monographs of the European Phannacopoeia.
Vehicles
Vehic1es are excipients used for the preparation of certain
stocks or for the potentisation process. They may inc1ude, for
example: purified water, alcohol of a suitable concentration,
glycerol and lactose.
Vehic1es comply with any requirements of the relevant
monographs of the European Phannacopoeia.
Stocks
Stocks are substances, products or preparations used as
starting materials for the production of homoeopathic
preparations. A stock is usually one of the following: a
mother tincture or a glycerol macerate, for raw material s of
botanical, zoological or human origin, or the substance itself,
for raw materials of chemical or mineral origino
Mother tinctures comply with the requirements of the
monograph Mother tinctures for homoeopathic preparations
(2029).
Glycerol macerates are Iiquid preparations obtained from raw
materials of botanical, zoological or human origin by using
Homoeopathic Preparations IV-403
glycerol or a mixture of glycerol and either alcohol of a
suitable concentration or a solution of sodium chloride of a
suitable concentration.
Potentisation
Dilutions and triturations are obtained from stocks by a
process of potentisation in accordance with a homoeopathic
manufacturing procedure: this means successive dilutions and
succussions, or successive appropriate triturations, or a
combination of the 2 processes.
The potentisation steps are usually one of the following:
-- 1 part of the stock plus 9 parts of the vehic1e; they may
be designated as 'D', 'DH' or 'X' (decimal);
-- 1 part of the stock plus 99 parts of the vehic1e; they may
be designated as 'e' or 'eH' (centesimal) .
The number of potentisation steps defines the degree of
dilution; for example, 'D3', '3 DH' or '3X' means 3 decimal
potentisation steps, and 'e3', '3 eH' or '3C' means 3
centesimal potentisation steps.
'LM-' (or 'Q-') potencies are manufactured according to a
specific procedure.
Dosage fonns
A dosage fonn of a homoeopathic preparation complies with
any relevant dosage form monograph in the European
Phannacopoeia, and with the following:
-- for the purpose of dosage fonns for homoeopathic use,
'active substances' are considered to be 'dilutions or
triturations of homoeopathic stocks'j
-- these dosage forms are prepared using appropriate
excipientsj
-- the test for uniformity of content (2.9. 6) or uniformity of
dosage units (2.9.40) is nonnally not appropriate;
however, in certain circumstances, it is required by the
competent authority.
Homoeopathic dosage form 'pillule '
PilIules for homoeopathic use are solid preparations obtained
from sucrose, lactose or other suitable excipients. They may
be prepared by impregnation of preformed pillules with a
dilution or dilutions of homoeopathic stocks or by progressive
addition of these excipients and the addition of a dilution or
dilutions of homoeopathic stocks. They are intended for oral
or sublingual use.
Homoeopathic dosage form 'tablet'
Tablets for homoeopathic use are solid preparations obtained
from sucrose, lactose or other suitable excipients according to
the monograph Tablets (0478) . They may either be prepared
by compressing one or more solid active substances with the
excipients or by impregnating preformed tablets with a
dilution or dilutions of homoeopathic stocks. The preformed
tablets for impregnation are obtained from sucrose, lacto se or
other suitable excipients according to the monograph Tablets
(0478). They are intended for oral or sublingual use.
Manufacturing methods
Homoeopathic preparations are manufactured using a range
of methods of preparation and are presented in various
dosage forms (covered by general dosage form monographs).
The methods of preparation are described in the monograph
Methods of preparation of homoeopathic stocks and potentisation
(2371). The use of certain preparations obtained using the
methods listed below is restricted to certain dosage forms as
indicated in Table 1038.-l.
 IV-404 Homoeopathic Preparations 2014

Table 1038.-1. PRODUCTION

Manufacturing methods Dosage forms
2.1.2 Eye drops
Solutions for injection
Nasal preparations
2.2.1, 2.2.2, 2.2.3 Eye drops
Pillules (globuli vela tí)
Solutions for injection
Nasal preparations
Ointments, creams and gels
Oral powders (triturations)
Suppositories
2.2.4 Solutions for injection
3.1.2.3.2.2 Eye drops
Pillules (globuli velatí)
Solutions for injection
Nasal preparations
Ointments, creams and gels
Suppositories
The competent authority has the right to accept or reject
particular combinations of manufacturing method and
substance.
____________________________________________
Herbal drugs for homoeopathic preparations are obtained
from cultivated or wild plants . Suitable collection, cultivation,
harvesting, sorting, drying, fragmentation and storage
conditions are essential to guarantee the quality of herbal
drugs for homoeopathic preparations.
Herbal drugs for homoeopathic preparations are, as far as
possible, free from impurities such as soil, dust, dirt and
other contaminants such as fungal, insect and other animal
contaminants. They do not present signs of decay.
If a decontaminating treatment has been used, it is necessary
to demonstrate that the constituents of the plant are not
affected and that no harrnful residues remain. The use of
ethylene oxide is prohibited for the decontamination of
herbal drugs for homoeopathic preparations.
Fresh herbal drugs are processed as rapidly as possible after
harvesting. Where justified and authorised for transportation
or storage purposes, fresh plant material may be deep-frozen;
it may also be kept in ethanol (96 per cent) or in ethanol of a
suitable concentration, provided the whole material ineluding
the storage medium is used for processing.
Adequate measures have to be taken in order to ensure that
the microbiological quality of homoeopathic preparations
~Ew

containing 1 or more herbal drugs comply with the

recommendations given in general chapter 5.1.4.
Microbiological qualiry of non-sterile pharmaceutical preparations
and substances for pharmaceutical use.

Herbal Drugs for Homoeopathic IDENTIFICATION

Preparations
Herbal Drugs for Homoeopathic Use
(Ph Eur monograph 2045)
PhEw ____________________________________________
DEFINITION
Herbal drugs for homoeopathic preparations are mainly
whole plants or parts of plants, fragmented or broken, and
inelude algae, fungi or lichens, in an unprocessed state,
usually in fresh formo The sta te, fresh or dried, in which the
drug is used, is defined in the individual monograph of the
European Pharmacopoeia or, in its absence, in the individual
monograph of an official national pharmacopoeia of a
member state. In the absence of such a monograph, the state
in which the herbal drug is used has to be defined. Certain
exudates that have not been subjected to a specific treatment
are also considered to be herbal drugs for homoeopathic
preparations. Herbal drugs for homoeopathic preparations
are precisely defined by the botanical scientific name of the
source species according to the binomial system (genus,
species, variety and author).
Whole describes a herbal drug for homoeopathic preparations
that has not been reduced in size and is presented, dried or
undried, as harvested.
Fragmented describes a herbal drug for homoeopathic
preparations that has been reduced in size after harvesting to
permit ease of handling, drying andJor packaging.
Broken describes a herbal drug for homoeopathic
preparations in which the more fragile parts of the plant have
broken during drying, packaging or transportation.
For dried herbal drugs for homoeopathic preparations, cut
describes size reduction, other than powdering, that reduces
the partiele size below that which is described in the
macroscopic identity of the herbal drug for homoeopathic
preparations.
Herbal drugs for homoeopathic preparations are identified
using their macroscopic and, where necessary, microscopic
descriptions and any further tests that may be required (for
example, thin-Iayer chromatography).
TESTS
The tests for foreign matter and loss on drying should be
performed before any further processing of the fresh plant.
Foreign matter (2.8.2)
Where a fresh plant is used as a starting material for the
manufacture of homoeopathic preparations, the content of
foreign matter is as low as possible; if necessary, the
maximum content of foreign matter is indicated in the
individual monograph.
Where a dried plant is used as a starting material for the
manufacture of homoeopathic preparations, carry out a test
for foreign matter, unless otherwise prescribed in the
individual monograph. The content of foreign matter is not
more than 2 per cent mlm, unless otherwise prescribed or
justified and authorised.
Adulteration
A specific appropriate test may apply to herbal drugs for
homoeopathic preparations liable to be falsified.
Loss on drying (2.2.32)
Carry out a test for los s on drying on dried herbal drugs for
homoeopathic preparations.
If a fresh plant is processed more than 24 h after harvesting,
a test for loss on drying should be carried out. The minimum
limit is indicated in the individual monograph.
Water (2.2.13)
A determination of water is carried out on herbal drugs for
homoeopathic preparations with a high essential oil contento
Pesticides (2.8. 13)
Herbal drugs for homoeopathic preparations comply with the
requirements for pesticide residues. The requirements take
into account the origin and the nature of the plant, where
 2014
necessary the preparation in which the plant might be used
and, where available, knowledge of the complete record of
treatment of the batch of the plant. Wnere justified, the test
for pesticides may be performed on the mother tincture
according to the requirements of the general monograph
Mother tinctures for homoeopathic preparations (2029).
1f appropriate, herbal drugs for homoeopathic preparations comply
with other tests, such as the following, for example.
Total ash (2.4.16).
Bitterness value (2.8.15).
Heavy metals (2.4.27)
Unless otherwise stated in an individual monograph or unless
otherwise justified and authorised:
-- cadmium: maximum 1.0 ppm;
-- lead: maximum 5.0 ppm;
-- mercury: maximum 0.1 ppm.
If justified by the nature or origin of the herbal drug or if
required by the competent authority, suitable limits for the
content of other heavy metals such as arsenic or nickel are
defined.
Where justified, the test for heavy metals may be performed
on the mother tincture according ro the requirements of the
general monograph Mother tinctures for homoeopathic
preparations (2029).
Aflatoxin B¡ (2.8.18)
Where appropriate, Jimits for aflatoxins may be required.
Ochratoxin A (2.8.22)
Where appropriate, a limit for ochratoxin A may be required.
Radioactive contarnination
In some specific circumstances, the risk of radioactive
contamination is to be considered.
ASSAY
Where applicable, herbal drugs for homoeopathic
preparations are assayed by an apprapriate method.
STORAGE
Store dried herbal drugs protected from light.
____________________________________________ PhEif
Methods of Preparation of
Homoeopathic Stocks and
Potentisation
(Ph Eur monograph 2371)
~Ew ____________________________________________
Homoeopathic stocks are prepared, using suitable methods,
from raw materials that comply with the requirements of the
monograph Homoeopathic preparations (1038). The methods
described below, combined with established methods for
potentisation, are examples of methods, but other methods
described in an official national pharmacopoeia of a Member
State may equaIly be used.
Where material of animal origin is to be used, particular
reference is made to the requirements conceming the use of
raw material of zoological or human origin in the monograph
Homoeopathic preparations (1038).
In the preparation of liquid dilutions, the ethanol of the
concentration prescribed in the method may, if necessary, be
replaced by ethanol (36 per cent V/V) [ethanol (30 per cent
Homoeopathic Preparations IV-405
m/m)] or ethanol (18 per cent V/V) [ethanol (15 per cent
m/m)].
When the individual monograph aIlows that the mother
tincture be prepared fram more than one plant species, the
mother tincture can be prepared from the specified parts of
an individual plant species or from any mixture thereof.
Unless otherwise stated, mother tinctures are prepared by
maceration. Maceration lasts not less than 10 days and not
more than 30 days.
Maceration may be replaced by long maceration (maximum
60 days) or very long maceration (maximum 180 days),
provided it is demonstrated that the quality of the resulting
mother tincture is the same as that of the mother tincture
prepared by maceration.
Unless otherwise stated in the individual monograph, the
term 'partes), denotes 'mass part(s)'. Unless otherwise stated
in the method, the maximum temperature for the preparation
is 25 oC.
1. MOTHER TINCTURES
METHOD 1.1
Method 1.1.1 (EQUIVALENT TO
HOMÓOPATHlSCHES ARZNEIBUCH (HAB) la:
MOTHER TINCTURES AND LIQUID DILUTIONS)
Method 1.1.1 is used for fresh herbal drugs containing
generaIly more than 70 per cent of expressed juice and no
essential oil or resin or mucilage. Mother tinctures prepared
according to Method 1.1.1 are mixtures of equal parts of
expressed juices and ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)].
Express the comminuted herbal drug. Immediately mix the
expressed juice with an equal mass of ethanol
(90 per cent V/V) [ethanol (86 per cent m/m)]. AIIow ro
stand in a cJosed container at a temperature not exceeding
20 oC for not less than 5 days, then filter.
Adjustment to any value specified in the individual
monograph
Determine the percentage dry residue (2.8.16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al), in kilograms,
of ethanol (50 per cent V/V) [ethanol (43 per cent m/m)]
required, using the foIlowing expression:
m x (Nx - No)
No
m mas s of filtrate, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the filtrate.
Mix the filtrate with the calculated amount of ethanol
(50 per cent V/V) [ethanol (43 per cent m/m)] . AIIow to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1st 'decimal' dilution (DI) is made from:
-- 2 parts of the mother tincture;
-- 8 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
m/m)].
The 2nd decimal dilution (D2) is made from:
-- 1 part of the I sr 'decimal' dilution;
-- 9 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
m/m)].
Subsequent decimal dilutions are produced as stated for D2 .
IV-406 Homoeopathic Preparations
The 1st 'centesimal' dilution (C 1) is made from:
2 parts of the mother tincture;
- 98 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)].
The 2nd centesimal dilution (C2) is made from:
- 1 part of the 1st 'centesimal' dilution;
- 99 parts of ethanol (50 per cent V/V) [ethanol
43 per cent m/m)].
Subsequent centesimal dilutions are produced as stated for
C2.
Method 1.1.2 (EQUIVALENT TO HAB lb: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1.2 is used where the latex of a herbal drug is to
be processed.
Mother tinctures prepared according to Method 1.1.2 are
mixtures of tresh plant latex with ethanol (36 per cent V/V)
[ethanol (30 per cent m/m)] . Mix the fresh latex with 2 parts
by mass of ethanol (36 per cent V/V) [ethanol (30 per cent
m/m)] and filter.
AdjustInent to any value specified in the individual
monograph
Determine the percentage dry residue (2.8. 16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al), in kilograms,
of ethanol (36 per cent V/V) [ethanol (30 per cent m/m)]
required, using the following expression:
m x (N x - No)
No
m mass of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the filtrate.
Mix the filtrate with the calculated amount of ethanol
(36 per cent V/V) [ethanol (30 per cent m/m)]. A1low to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1st 'decimal' dilution (DI) is made from:
- 3 parts of the mother tincture;
- 7 parts of ethanol (36 per cent V/V) [ethanol (30 per cent
m/m) ].
The 2n d decimal dilution (D2) is made from:
- 1 part of the 1st 'decimal' dilution;
- 9 parts of ethanol (18 per cent V/V) [ethanol (15 per cent
m/m) ].
Subsequent decimal dilutions are produced as stated for D2.
Method 1.1.3 (EQUIVALENT TO HAB 2a: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1.3 is used for fresh herbal drugs containing
generally less than 70 per cent of expressed juice and more
than 60 per cent moisture (loss on drying) and no essential
oil or resino
Mother tinctures prepared according to Method 1.1.3
(ethanol content approximately 50 per cent V/V or
43 per cent m/m) are prepared by maceration as described
below.
Comminute the herbal drug. Take a sample and determine
the loss on drying (2.2.32). Unless otherwise prescribed,
determine the loss on drying on 2.00-5.00 g of comminuted
raw material in a fiat-bottomed tared vessel, 45-55 mm in
diameter, that has been previously dried as indicated for the
2014
raw material. Dry the raw material at 105 oC for 2 h then
allow to cool in a desiccator.
To the comminuted herbal drug immediately add not less
than half the mass of ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)] and store in well-c1osed containers at a
temperature not exceeding 20 oC.
Use the following expression to calculate the amount (A 2 ), in
kilograms, of ethanol (90 per cent V/V) [ethanol (86 per cent
m/m)] required for the mas s (m) of raw material, then
subtract the amount of ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)] already added and add the difference to
the mixture.
m xT
100
m = mas s of raw material, in kilograms;
T = percentage loss on drying of the sample.
AlIow to stand at a temperature not exceeding 20 oC for not
less than 10 days, swirling from time to time, then express
the mixture and filter the resulting liquido
AdjustInent to any value specified in the individual
monograph
Determine the percentage dry residue (2.8.16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al), in kilograms,
of ethanol (50 per cent V/V) [ethanol (43 per cent m/m)]
required, using the following expression:
m x (Nx - No)
No
m mass of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the filtra te.
Mix the filtra te with the calculated amount of ethanol
(50 per cent V/V) [ethanol (43 per cent m/m)]. Allow to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1s t 'decimal' dilution (DI) is made from :
- 2 parts of the mother tincture;
- 8 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
m/111)] .
The 2nd decimal dilution (D2) is made trom:
- 1 part of the 1st 'decimal' dilution;
- 9 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
m/m)].
Subsequent decimal dilutions are produced as stated for D2.
The 1st 'centesimal' dilution (C1) is made from:
2 parts of the mother tincture;
- 98 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/111)].
The 2nd centesimal dilution (C2) is made from:
- 1 part of the 1sr 'centesimal' dilution;
- 99 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent 111/m)].
Subsequent centesimal dilutions are produced as stated for
C2.
 2014
Method 1.1.4 (EQUIVALENT TO HAB 2b: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1.4 is used for fresh herbal drugs containing
generally less than 70 per cent of expressed juice and more
than 60 per cent moisture (loss on drying) and no essential
oil or resino
Mother tinctures prepared according to Method 1.1.4
(ethanol content approximately 36 per cent V/V or
30 per cent m/m) are prepared by maceration as described
below.
Comminute the herbal drug. Take a sample and determine
the los s on drying (2.2.32). Unless otherwise prescribed,
determine the loss on drying on 2.00-5.00 g of comminuted
raw material in a fiat-bottomed tared vessel, 45-55 mm in
diameter, that has been previously dried as indicated for the
raw material. Dry the raw material at 105 oC for 2 h then
allow to cool in a desiccator.
To the comminuted herbal drug immediately add not less
than half the mas s of ethanol (70 per cent V/V) [ethanol
(62 per cent m/m)] and store in well-closed containers at a
temperature not exceeding 20 oc.
Use the following expression to calculate the amount (A 2 ), in
kilograms, of ethanol (70 per cent V/V) [ethanol (62 per cent
m/m)] required for the mas s (m) of raw material, then
subtract the amount of ethanol (70 per cent V/V) [ethanol
(62 per cent m/m)] already added and add the difference to
the mixture.
mxT
100
m = mass of raw material, in kilograms;
T = percentage los s on drying of the sample.
AlIow to stand at a temperature not exceeding 20 oC for not
les s than 10 days, swirling from time to time, then express
the mixture and filter the resulting liquido
Adjustment to any value specified in the individual
monograph
Determine the percentage dry residue (2.8.16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate . Calculate the amount (Al), in kilograms,
of ethanol (36 per cent V/V) [ethanol (30 per cent m/m)]
required, using the following expression:
m x (N x - No)
No
m mas s of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
N< percentage dry residue or percentage assay content of
the filtrate.
Mix the filtrate with the calculated amount of ethanol
(36 per cent V/V) [ethanol (30 per cent m/m)]. AlIow to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1sr 'decimal' dilution (DI) is made from:
- 2 parts of the mother tincture;
- 8 parts of ethanol (36 per cent V/V) [ethanol (30 per cent
m/m)].
The 2nd decimal dilution (D2) is made from:
- 1 part of the 1sr 'decimal' dilution;
- 9 parts of ethanol (18 per cent V/V) [ethanol (15 per cent
m/m)] .
Homoeopathic Preparations IV-407
Subsequent decimal dilutions are produced as stated for D2.
Method 1.1.5 (EQUIVALENT TO HAB 3a: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1.5 is used for fresh herbal drugs containing
essential oil or resin, or generally less than 60 per cent
moisture (loss on drying).
Mother tinctures prepared according to Method 1.1. 5
(ethanol content approximately 68 per cent V/V or
60 per cent m/m) are prepared by maceration as described
below.
Comminute the herbal drug. Take a sample and determine
the loss on drying (2.2.32). Unless otherwise prescribed,
determine the loss on drying on 2.00-5.00 g of comminuted
raw material in a fiat-bottomed tared vessel, 45-55 mm in
diameter, that has been previously dried as indicated for the
raw material. Dry the raw material at 105 oC for 2 h then
allow to cool in a desiccator.
To the comminuted herbal drug immediately add not less
than half the mass of ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)] and store in well-closed containers at a
temperature not exceeding 20 oc.
Use the following expression to calcula te the amount (A 3 ), in
kilograms, of ethanol (90 per cent V/V) [ethanol (86 per cent
m/m)] required for the mass (m) of raw material, then
subtract the amount of ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)] already added and add the difference ro
the mixture.
2x m xT
100
m = mass of raw material, in kilograms;
T = percentage loss on drying of the sample.
AlIow to stand at a temperature not exceeding 20 oC for not
less than 10 days, swirling from time to time, then express
the mixture and filter the resulting liquido
Adjustment to any value specified in the individual
monograph
Determine the percentage dry residue (2.8.16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al), in kilograms,
of ethanol (70 per cent V/V) [ethanol (62 per cent m/m)]
required, using the following expression:
m x (Nx - No)
No
m mass of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the filtrate.
Mix the filtra te with the calculated amount of ethanol
(70 per cent V/V) [ethanol (62 per cent m/m)]. AlIow to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1st 'decimal' dilution (DI) is made from:
3 parts of the mother tincture;
- 7 parts of ethanol (70 per cent V/V) [ethanol (62 per cent
m/m)].
The 2 nd decimal dilution (D2) is made from:
- 1 part of the 1sr ' decimal' dilution;
 IV -408 Homoeopathic Preparations
- 9 parts of ethanol (70 per cent V/V) [ethanol (62 per cent
m/m)] .
Subsequent decimal dilutions are produced as stated for D2.
Use ethanol (50 per cent V/V) [ethanol (43 per cent m/m)]
for dilutions from D4 onwards.
The 1st 'centesimal' dilution (C1) is made from:
- 3 parts of the mother tincture;
- 97 parts of ethanol (70 per cent V/V) [ethanol
(62 per cent m/m)].
The 2nd centesimal dilution (C2) is made from :
- 1 part of the 1st 'centesimal' dilution;
- 99 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)].
Subsequent centesimal dilutions are produced as stated for
C2.
Method 1.1.6 (EQUIVALENT TO HAB 3b: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1. 6 is used for fresh herbal drugs containing
essential oils or resins or generally less than 60 per cent
moisture (Ioss on drying).
Mother tinctures prepared according to Method 1.1.6
(ethanol content approximately 50 per cent V/V or
43 per cent m/m) are prepared by maceration as described
below.
Comminute the herbal drug. Take a sample and determine
the loss on drying (2.2.32) . Unless otherwise prescribed,
determine the loss on drying on 2.00-5.00 g of comminuted
raw material in a flat-bottomed tared vessel, 45-55 mm in
diameter, that has been previously dried as indicated for the
raw material. Dry the raw material at 105 oC for 2 h then
allow to cool in a desiccator.
To the comminuted herbal drug immediately add not les s
than half the mas s of ethanol (80 per cent V/V) [ethanol
(73 per cent m/m)] and store in well-closed containers at a
temperature not exceeding 20 oc.
Use the following expression to calculate the amount (A 3 ), in
kilograms, of ethanol (80 per cent V/V) [ethanol (73 per cent
m/m)] required for the mass (m) of raw material, then
subtract the amount of ethanol (80 per cent V/V) [ethanol
(73 per cent m/m)] already added and add the difference to
the mixture.
2x m xT
100
m = mass of raw material, in kilograms;
T = percentage loss on drying of the sample.
Allow to stand at a temperature not exceeding 20 oC for not
less than 10 days, swirling from time ro time, then express
the mixture and filter the resulting liquid.
Adjustment to any value specified in the individual
monograph
Determine the percentage dry residue (2.8.16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al), in kilograms,
of ethanol (50 per cent V/V) [ethanol (43 per cent m/m)]
required, using the following expression:
m x (Nx - No)
No
m mass of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
2014
N x = percentage dry residue or percentage assay content of
the filtrate.
Mix the filtrate with the calculated amount of ethanol
(50 per cent V/V) [ethanol (43 per cent m/m)]. Allow to
stand at a temperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1St 'decimal' dilution (DI) is made from:
- 3 parts of the mother tincture;
- 7 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
m/m)] .
The 2 nd decimal dilution (D2) is made from:
- 1 part of the 1St 'decimal' dilution;
- 9 parts of ethanol (36 per cent V/V) [ethanol (30 per cent
m/m)] .
The 3rd decimal dilution (D3) is made from:
- 1 part of the 2nd decimal dilution;
- 9 parts of ethanol (18 per cent V/V) [ethanol (15 per cent
m/m)] .
Subsequent decimal dilutions are produced as stated for D3.
Method 1.1.7 (EQUIVALENT TO HAB 3c: MOTHER
TINCTURES AND LIQUID DILUTIONS)
Method 1.1. 7 is used for fresh herbal drugs containing
generally less than 60 per cent moisture (Ioss on drying).
Mother tinctures prepared according to Method 1.1. 7
(ethanol content approximately 36 per cent V/V or
30 per cent m/m) are prepared by maceration as described
below.
Comminute the herbal drug. Take a sample and determine
the los s on drying (2.2.32) . Unless otherwise prescribed,
determine the loss on drying on 2.00-5.00 g of comminuted
raw material in a flat-bottomed tared vessel, 45-55 mm in
diameter, that has been previously dried as indicated for the
raw material. Dry the raw material at 105 oC for 2 h then
allow to cool in a desiccator.
To the comminuted herbal drug immediately add not less
than half the mass of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)] and store in well-closed containers at a
temperature not exceeding 20 oC.
Use the following expression to calculate the amount (A 3 ), in
kilograms, of ethanol (50 per cent V/V) [ethanol (43 per cent
m/m)] required for the mass (m) of raw material, then
subtract the amount of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)] already added and add the difference to
the mixture.
2xm xT
100
m = mas s of raw material, in kilograms;
T = percentage loss on drying of the sample.
Allow to stand at a temperature not exceeding 20 oC for not
less than 10 days, swirling from time ro time, then express
the mixture and filter the resulting liquido
Adjustment to any value specified in the individual
monograph
Determine the percentage dry residue (2.8. 16) or, where
prescribed, the percentage assay content of the above-
mentioned filtrate . Calculate the amount (Al), in kilograms,
of ethanol (36 per cent V/V) [ethanol (30 per cent m/m)]
required, using the following expression:
m x (Nx - No)
No
 2014
m mass of filtra te, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the filtrate.
Mix the filtra te with the calculated amount of ethanol
(36 per cent V/V) [ethanol (30 per cent m/m)]. AIIow to
stand at a remperature not exceeding 20 oC for not less than
5 days, then filter if necessary.
Potentisation
The 1st 'decimal' dilution (DI) is made from:
- 3 parts of the mother tincture;
- 7 parrs of ethanol (36 per cent V/V) [ethanol (30 per cent
m/m)].
The 2nd decimal dilution (D2) is made from:
- 1 part of rhe 1st 'decimal' dilution;
- 9 parts of ethanol (18 per cent V/V) [ethanol (15 per cent
m/m)].
Subsequent decimal dilutions are produced as stated for D2.
METHOD 1.1.8 (EQUIVALENT TO HAB 4a:
MOTHER TINCTURES AND LIQUID DILUTIONS)
Method 1.1.8 is generaIly used for dried herbal drugs.
Mother tincrures prepared according to Method 1.1.8 are
prepared by maceration or percolation as described beIow,
using 1 part of dried herbal drug and 10 parts of ethanol of
the appropriate concentration (anhydrous, 96 per cent V/V-
94 per cent m/m, 90 per cent V/V - 86 per cenr m/m,
80 per cent VIV - 73 per cent m/m, 70 per cent V/V-
62 per cent m/m, 50 per cent V/V - 43 per cent m/m,
36 per cent VIV - 30 per cent m/m, 18 per cent V/V-
15 per cent m/m), unless otherwise prescribed in the
individual monograph.
Production by maceration Unless otherwise prescribed,
comminute the herbal drug, mix thoroughly with ethanol of
the appropriate concentration and aIlow to stand in a c10sed
container for an appropriate time. Separate the residue from
the ethanol and, if necessary, press out. In the latter case,
combine the 2 liquids obrained.
Production by percolation If necessary, comminute the herbal
drug. Mix thoroughly with a portion of ethanol of the
appropriate concentration and aIlow ro stand for an
appropriate time. Transfer to a percolator and aIlow the
percolate to fiow slowly, at room remperature, making sure
that the herbal drug ro be extracted is always covered with
the remaining ethano!. The residue may be pressed out and
the expressed liquid combined with the percolare.
If adjustment to a given concentration is necessary, calculate
the amount (Al), in kilograms, of ethanol of the appropriate
concentration required ro obtain the concentration specified
or used for production, using the foIlowing expression:
m x (Nx - No)
No
m mass of percolare or macerare, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
Nx percentage dry residue or percentage assay content of
the percolate or macerate.
Mix the macerate or percolate with the calculated amount of
ethanol of the appropriate concentration. AIIow ro stand at a
temperature nor exceeding 20 oC for not less than 5 days,
then filter if necessary.
Homoeopathic Preparations IV-409
Potentisation
The mother tincture corresponds to the 1st decimal dilution
(0 = DI).
The 2nd decimal dilution (D2) is made from:
- 1 part ofthe mother tincture (DI);
- 9 parts of ethanol of the same concentration.
The 3rd decimal dilution (D3) is made from:
- 1 part of the 2nd decimal dilution;
- 9 parts of ethanol of the same concentration.
Unless a different ethanol concentration is specified, use
ethanol (50 per cent V/V) [ethanol (43 per cent m/m)] for
subsequent decimal dilutions from D4 onwards and proceed
as stated for D3.
The 1st 'centesimal' dilution (Cl) is made from:
- 10 parts ofthe mother tincture (DI);
- 90 parts of ethanol of the same concentration.
The 2nd centesimal dilution (C2) is made from:
- 1 part of the 1st 'centesimal' dilution;
- 99 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)], unless a different ethanol
concentration is specified.
Subsequent centesimal dilutions are produced as stated for
C2.
METHOD 1.1.9 (EQUIVALENT TO HAB 4b:
MOTHER TINCTURES AND LIQUID DILUTIONS)
Method 1.l.9 is generaIly used for animal matter.
Mother tinctures prepared according to Method 1.1.9 are
prepared by maceration or percolation as described below,
using 1 part of animal matter and 10 parts of ethanol of the
appropriate concentration (anhydrous, 96 per cent V/V -
94 per cent m/m, 90 per cent V/V - 86 per cent m/m,
80 per cent V/V - 73 per cent m/m, 70 per cent V/V-
62 per cent m/m, 50 per cent VN - 43 per cent m/m,
36 per cent VN - 30 per cent m/m, 18 per cent VN-
15 per cent mlm), unless otherwise prescribed in the
individual monograph.
Production by maceration. Unless otherwise prescribed,
comminute the animal matter, mix thoroughly with ethanol
of the appropriate concentration and aIlow to stand in a
c10sed container for an appropriate time. Separate the residue
from the ethanol and, if necessary, press out. In the latter
case, combine the 2 liquids obtained .
Production by percolation. If necessary, comminute the animal
matter. Mix thoroughly with a portion of ethanol of the
appropriate concentration and aIlow ro stand for an
appropriate rime. Transfer ro a percolator and aIlow the
percolate ro fiow slowly at room temperature, making sure
that the animal matter ro be extracted is always covered with
the remaining ethano!. The residue may be pressed out and
the expressed liquid combined with the percolate.
If adjustment to a given concentration is necessary, calculate
rhe amount (A 1), in kilograms, of ethanol of the appropriate
concentration required to obtain the concentration specified
or used for production, using the foIlowing expression:
m x (N x - No)
No
m mass of percolate or macerate, in kilograms;
No percentage dry residue or percentage assay content as
required in the individual monograph;
percentage dry residue or percentage assay content of
the percolate or macerate.
 IV-410 Homoeopathic Preparations
Mix the macerate or percolate with the calculated amount of
ethanol of the appropriate concentration. AlIow to stand at a
temperature not exceeding 20 °e for not les s than 5 days,
then filter if necessary.
Potentisation
The mother tincture corresponds to the 1sr decimal dilution
(0 = DI).
The 2nd decimal dilution (D2) is made from:
- 1 part of the mother tincture (Dl);
- 9 parts of ethanol of the same concentration.
The 3rd decimal dilution (D3) is made from:
- 1 part of the 2nd decimal dilution;
- 9 parts of ethanol of the same concentration.
Unless a different ethanol concentration is specified, use
ethanol (50 per cent V/V) [ethanol (43 per cent m/m)] for
subsequent decimal dilutions from D4 onwards and proceed
as stated for D3 .
The 1sr 'centesimal' dilution (el) is made from:
- 10 parts ofthe mother tincture (DI);
- 90 parts of ethanol of the same concentration.
The 2nd centesimal dilution (e2) is made from:
1 part of the 1st 'centesimal' dilution;
- 99 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)], unless a different ethanol
concentration is specified.
Subsequent centesimal dilutions are produced as stated for
e2.
METHOD 1.1.10 (FRENCH PHARMAeOPOEIA)
Method 1.1.10 is generally used for herbal drugs. The state
of the herbal drug, fresh or dried, is specified in the
individual monograph.
Mother tinctures prepared according to Method 1.1.10 are
prepared by maceration.
eomminute appropriately the herbal drug. Take a sample
and determine the loss on drying at 105 °e for 2 h (2.2.32)
or the water content (2.2.13) . Taking this value into account,
calculate and add to the herbal drug the quantities of ethanol
of the appropriate concentration required to produce, unless
otherwise prescribed, a 1 in 10 mother tincture (1: 10 mother
tincture) with a suitable ethanol contento AlIow to macerate
for at least 10 days, with sufficient shaking.
Separate the residue from the ethanol and strain under
pressure if necessary. AlIow the combined liquids to stand for
48 h and filter. For mother tinctures with a required assay
content, adjustment may be carried out, if necessary, by
adding ethanol of the same concentration as used for the
preparation of the tincture.
Potentisation
The 1sr decimal dilution (D 1) is made from:
- 1 part of the mother tincture;
- 9 parts of ethanol of the appropriate concentration.
The 2nd decimal dilution (D2) is made from:
- 1 part of the 1sr decimal dilution;
- 9 parts of ethanol of the appropriate concentration.
Subsequent decimal dilutions are produced as stated for D2,
using ethanol of the appropriate concentration.
The 1sr centesimal dilution (el) is made from:
- 1 part of the mother tincture;
- 99 parts of ethanol of the appropriate concentration.
The 2nd centesimal dilution (e2) is made from:
- 1 part of the 1sr centesimal dilution;
- 99 parts of ethanol of the appropriate concentration.
2014
Subsequent centesimal dilutions are produced as stated for
e2, using ethanol of the appropriate concentration.
METHOD 1.1.11 (FRENCH PHARMACOPOEIA)
Method 1.1.11 is generally used for animal matter.
Mother tinctures prepared according to Method 1.1.11 are
prepared by maceration.
The mass ratio of raw material to mother tincture is usually
1 to 20. To the raw material, appropriately comminuted, add
the quantity of ethanol of the appropriate concentration
required to produce a 1 in 20 mother tincture. AlIow to
macerate for at least 10 days, with sufficient shaking. Decant
and filter. Allow to stand for 48 h and filter again.
For mother tinctures with a required assay content,
adjustment may be carried out, if necessary, by adding
ethanol of the same concentration as used for the preparation
of the tincture.
Potentisation
The 1sr decimal dilution (D 1) is made from:
- 1 part of the mother tincture;
- 9 parts of ethanol of the appropriate concentration.
The 2nd decimal dilution (D2) is made from:
- 1 part of the 1st decimal dilution;
- 9 parts of ethanol of the appropriate concentration.
Subsequent decimal dilutions are produced as stated for D2,
using ethanol of the appropriate concentration.
The 1st centesimal dilution (el) is made from:
- 1 part of the mother tincture;
- 99 parts of ethanol of the appropriate concentration.
The 2nd centesimal dilution (e2) is made from:
- 1 part of the 1sr centesimal dilution;
- 99 parts of ethanol of the appropriate concentration.
Subsequent centesimal dilutions are produced as stated for
e2, using ethanol of the appropriate concentration.
2. GLYCEROL MACERATES
METHOD 2.1
Method 2.1 is used for maceration of raw materials of animal
or herbal origin in glycerol (85 per cent) or glycerol/ethanol
mixtures of appropriate concentration. Pathological material
is excluded.
The raw material s are finely minced before use, where
appropriate.
METHODS 2.1.1,2.1.2 (EQUIVALENT TO HAB 42a
AND 42b: MOTHER TINCTURES AND LIQUID
DILUTIONS THEREOF)
Raw materials of animal origin - freshly killed animals or
parts thereof - are used. Animals are processed immediately
after being killed.
Maceration
Disperse 1 part of finely minced animal material in:
- 9 parts (decimal dilutions) or 99 parts (centesimal
dilutions) of glycerol (85 per cent) for Method 2.1.1,
- or 2.1 parts of glycerol (85 per cent) for Method 2. 1.2.
AlIow to macerate for at least 2 h, then succusS. Filter when
necessary.
Where justified, 1 part of glycerol (85 per cent) may be
added to 1 part of animal material before mincing. Where
very small amounts of animal material are used, the dilution
may be prepared by dispersing 1 part of finely minced animal
material in 99 parts of glycerol (85 per cent) (el or 'D2' if
to be used for further decimal dilutions).
Potentisation
M ethod 2.1.1
2014
The 2nd decimal dilution (D2) is made from:
1 part of the glycerol macerate D 1;
- 9 parts of glycerol (85 per cent) or ethanol
(18 per cent V/V) [ethanol (15 per cent m/m)].
Subsequent decimal dilutions are produced as stated for D2
but with ethanol (18 per cent V/V) [ethanol (15 per cent
m/m)] as the vehicle .
The 2nd centesimal dilution (e2) is made from:
- 1 part of the glycerol macerate el;
- 99 parts of ethanol (18 per cent V/V) [ethanol
15 per cent m/m) ].
Subsequent centesimal dilutions are produced as stated for
e2.
Mechad 2.1.2
The l sr 'decimal' dilution (DI) is made from:
- 3 parts of the glycerol macerate;
- 7 parts of water for injections.
The 2nd decimal dilution (D2) is made from:
- 1 part of DI;
- 9 parts of water for injections.
Subsequent decimal dilutions are produced as stated for D2.
METHOD 2.1.3 (FRENCH PHARMACOPOEIA)
Raw materials of herbal or animal origin are used.
Maceration
eomminute the raw material appropriately. Take a sample
and determine the loss on drying at 105 °e for 2 h (2.2.32)
or the water content (2.2.13). Taking this value into account,
calculate and add lO the raw material the quantity of the
ethanol/glycerol mixture of the appropriate concentration to
produce, unless otherwise prescribed, a 1 in 20 glycerol
macerate. Allow to macerate for at least 3 weeks, with
sufficient shaking. Decant and strain under pressure if
necessary. Allow the combined liquids to stand for 48 h and
filter.
Potentisation
The l sr decimal dilution (DI) is made from:
- 1 part of the glycerol macera te;
- 9 parts of a water/ethanol/glycerol mixture of appropriate
concentration.
The 2nd decimal dilution (D2) is made from:
- 1 part of the 1st decimal dilution;
- 9 parts of a water/ethanol/glycerol mixture of appropriate
concentration.
Subsequent decimal dilutions are produced as stated for D2
or using another appropriate vehicle .
The 1sr centesimal dilution (el) is made from:
- 1 part of the glycerol macerate;
- 99 parts of a water/ethanol/glycerol mixture of appropriate
concentration.
The 2nd centesimal dilution (e2) is made from:
- 1 part of the 1sr centesimal dilution;
- 99 parts of a water/ethanol/glycerol mixture of appropriate
concentration.
Subsequent centesimal dilutions are produced as stated for
e2 or using another appropriate vehicle.
METHOD 2.2
METHODS 2.2.1, 2.2.2, 2.2.3, 2.2.4 (EQUIVALENT TO
HAB 41a, 41h, 41c AND 41d: GL MOTHER
TINCTURES AND LIQUID DILUTIONS THEREOF)
Method 2.2 is used for maceration of raw materials of animal
origin in a glycerol solution containing sodium chloride.
Pathological material is excluded.
Homoeopathic Preparations IV-411
Raw materials from freshly killed animals, parts or secretions
thereof are used in Methods 2.2 .1, 2.2.2 and 2.2.3. Lower
animals are killed with carbon dioxide in a covered vessel.
All animal s are processed irnmediately after being killed.
Blood components from live horses are used in method
2.2.4.
Sample collection andlor pre-treatment
The raw materials used in Methods 2.2.1, 2.2.2 and 2.2.3
are finely minced before use, where appropriate.
The blood used in Method 2.2.4 is collected by a
veterinarian. Blood obtained from animals killed by bleeding
must not be used. Take 200 mL of this blood and add 15 IV
of heparin sodium and 0.625 mL of a 9 g/kg solution of
sodium chloride per millilitre. Separate the blood
components by fractional centrifugation and resuspend each
individual cell sediment in 1.1 mL of a 9 g/kg solution of
sodium chloride. These cell suspensions are processed into
the glycerol macerate.
Maceration
Mix 1 pan of finely minced animal material, secretions or
blood cell suspensions, according to the method used, with 5
parts of a sodium chloride solution of the appropriate
concentration (see table 2371.-1) and 95 parts of glycerol.
Allow to stand protected from light for at least 7 days, then
decanto Ifnecessary for Methods 2.2 .1, 2.2.2 and 2.2.3,
centrifuge before decanting, then filter the supematant if
necessary. The decanted liquid or the filtrate respectively is
the glycerol macerate.
Any sediment present must be resuspended before processing
the glycerol macerate.
TabIe 2371.·1
Methods 2.2.1 and
Method 2.2.2 Method 2.2.3
2.2.4
15 g/kg soIution of 40 g/kg soIution of 80 g/kg sol ution of
sodium chloride in sodium chloride in sodium chloride in
purified water purified water purified water
Vehic1e
0.2 parts of sodium hydrogen carbonate and 8.8 parts of
sodium chloride in 991 parts of water for injections or
purified water as appropriate.
Potentisation
The glycerol macera te corresponds lO the 2nd decimal
dilution ('D2') or the 1sr centesimal dilution (el).
The 3rd decimal dilution (D3) is made from:
- 1 part of the 2nd decimal dilution;
- 9 parts of the appropriate vehicle.
Subsequent decimal dilutions are produced as stated for D3.
Where appropriate, the 4th decimal dilution (D4) is made
from 1 part of the 3rd decimal dilution, 5.6 parts of the
vehicle and 3.4 parts of water for injections.
The 2nd centesimal dilution (e2) is made from:
- 1 part of the 1st centesimal dilution;
- 99 parts of the appropriate vehicle.
Subsequent centesimal dilutions are produced as stated for
e2.
3. LIQUID DILUTIONS
METHOD 3.1
Methods 3.1.1, 3.1.2 and 3.1.3 are used for dissolution of
any suitable inorganic or organic starting material, for
example minerals or venoms.
IV-412 Homoeopathic Preparations 2014

Unless otherwise specified, dissolve 1 part of the starting Additives

material in 9 parts (DI) or 99 parts (el) ofthe liquid vehicle For Method 3.1.1, if a reaction such as precipitation is
and succuss. observed in the final dilution, the following additives may be
Where justified and authorised, in case of insufficient used to enhance stability and/or solubility, unless otherwise
solubility of the starting material in the specified vehicle, specified:
direct1y produce the first possible dilution. For example, if - glacial acetic acid;
the starting material is slight1y soluble, dissolve 1 part of the - concentrated hydrochloric acid;
starting material in 99 parts of the vehicle (el or 'D2' if to - lactic acid;
be used for further decimal dilutions). - sodium hydroxide.
METHODS 3.1.1, 3.1.2 (EQUIVALENT TO HAB Sa, Where solutions or dilutions have been pH-adjusted, they
Sb: SOLUTIONS, AQUEOUS SOLUTIONS) must not be potentised further.
Vehic1es METHOD 3.1.3
The vehicles in Table 2371.-2 may be used. Vehic1es
Suitable vehicles, for example, ethanol of an appropriate
Table 2371-2 concentration, glycerol or purified water may be used alone
or combined.
Method 3.1.1 Method 3.1.2
Potentisation
Anhydrous ethanol Water for injections
Unless otherwise specified, the 2nd decimal dilution (D2) is
Ethanol (96 per cent V/Vl [ethanol (94 per Purified water made from:
cent m/ m))
- 1 part of the 1st decimal dilution (D 1);
Ethanol (90 per cent V/Vl [ethanol (86 per
centm/ m))
- 9 parts of the appropriate vehicle .
Ethanol (80 per cent V/Vl [ethanol (73 per Subsequent decimal dilutions are produced as stated for D2.
cent m/ m)) Unless otherwise specified, the 2nd centesimal dilution (e2)
Ethanol (70 per cent V/Vl [ethanol (62 per is made from :
cent m/ m))
- 1 part ofthe 1st centesimal dilution (el);
Ethanol (50 per cent V/Vl [ethanol (43 per
cent m/ m))
- 99 parts of the appropriate vehicle.
Ethanol (36 per cent V/Vl [ethanol (30 per Subsequent centesimal dilutions are produced as stated for
cent m/ m)) e2.
Ethanol (18 per cent V/Vl [ethanol (15 per METHOD3.2
cent m/ m)]
Method 3.2 is generally used to produce liquid dilutions of
Purifi ed water
triturations of substances that for the most part are sparingly
Glycerol (85 per cen t) soluble to practically insoluble.
METHODS 3.2.1, 3.2.2 (EQUIVALENT TO HAB Sa,
For Method 3.1.1, if ethanol (18 per cent V/V) [ethanol Sb: LIQUID PREPARATIONS MADE FROM
(15 per cent m/m)) is used, the starting material may be TRITURATIONS, AQUEOUS PREPARATIONS
dissolved in 7.58 parts of purified water and the ethanol MADE FROM TRITURATIONS)
concentration adjusted by adding 1.42 parts of ethanol Preparations made according to Method 3.2.1 and Method
(96 per cent V/V) [ethanol (94 per cent m/m)) to the 3.2.2 are produced from triturations D4, D5 and D6 or from
solution, for decimal dilutions . For centesimal dilutions, use triturations e4, es and e6, prepared according to method
83.4 parts ofpurified water for 15.6 parts ofethanol 4.1.1 by at least 2 potentisation steps.
(96 per cent V/V) [ethanol (94 per cent m/m)). Vehic1es
For Method 3.1.2, if the starting material is not stable andlor The vehicles in Table 2371.-3 may be used.
soluble in water, glycerol (85 per cent) may be added at a
concentration of not more than 35 per cent of the vehicle, for Table 2371.-3
potentisation up to D4.
Method 3.2.1 Method 3.2.2
Potentisation
1" potentisation: All potentisations:
Unless otherwise specified, the 2nd decimal dilution (D2) is Purified water Water for injections
made from: Purified water
- 1 part ofthe 1st decimal dilution (DI);
- 9 parts of ethanol (50 per cent V/V) [ethanol (43 per cent
2" potentisation:
m/m)) for Method 3.1.1 or 9 parts ofwater for injections Ethanol (36 per cent V/Vl [ethanol
(or purified water, as appropriate) for Method 3.1.2. (30 per cent m/m)]
Subsequent decimal dilutions are produced as stated for D2.
Unless otherwise specified, the 2nd centesimal dilution (e2) Further potentisations:
Ethanol (50 per cent V/Vl [ethanol
is made from: (43 per cent m/ m)]
- 1 part of the 1st centesimal dilution (el);
- 99 parts of ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)) for Method 3.1.1 or 99 parts ofwater
for injections (or purified water, as appropriate) for Potentisation
Method 3.1.2. For the first liquid potentisation, dissolve 1 part of the
Subsequent centesimal dilutions are produced as stated for trituration in 9 parts (decimal dilutions) or 99 parts
e2. (centesimal dilutions) of the specified vehicle (se e
Table 2371.-3) and succuss. For further potentisations,
2014 Homoeopathic Preparations IV-413

proceed in the same manner with 1 part of the previous (centesimal trituration) from 1 part of the raw material
dilution. (replace the mass of water lost from the fresh plant by an
The D6, D7, C6 and C7 dilutions produced by the aboye equivalent amount of the vehic1e). A suitable gentle drying
method are not to be used for the preparation of further process may need 10 be applied 10 the solid dilution.
dilutions. Where justified and authorised, it may be necessary to
METHOD 3.2.3 directly produce a C1 or 'D2' if to be used for further
Preparations made according to Method 3.2.3 are produced decimal triturations as the first solid trituration, made from 1
from triturations D2 onwards and from triturations C1, C2, part of raw material and 99 parts of vehic1e.
C3 and C4, prepared according to method 4.1.2. Trituration
Vehic1es Unless otherwise justified and authorised, the method
Suitable vehic1es such as ethanol of an appropriate consists of dividing the vehic1e into 3 equal parts and adding
concentration or purified water may be used. the raw material to the first part, then adding the second and
third part of the vehic1e, thoroughly triturating after each
Potentisation addition.
Unless otherwise specified, the first liquid decimal dilution
(Dn-1 ) is made from: For mechanical trituration, use a machine allowing the
- 1 part of the decimal trituration Dn-2; requirements for partic1e size of the first decimal or
- 9 parts of purified water or of another suitable vehic1e in centesimal solid trituration to be meto A machine fitted with
appropriate proportions. a scraping device may be used to ensure even trituration.
The time required to prepare one trituration is at least 1 h,
The following decimal dilution (Dn) is made from: unless otherwise justified and authorised.
- 1 part of the first Iiquid decimal dilution Dn-1 ;
- 9 parts of a suitable vehic1e. For manual trituration, divide the vehic1e into 3 equal parts
and briefiy triturate the first part in a porcelain mortar.
Subsequent decimal dilutions are produced as stated for Dn. Add the raw material, triturate the mixture for 6 min, scrape
Unless otherwise specified, the first Iiquid centesimal dilution down for 4 min with an appropriate non-metallic device (for
(Cn-1) is made from: example, a porcelain spatula). Triturate for a further 6 min,
- 1 part of the centesimal trituration Cn-2; scrape down again for a further 4 min, then add the second
- 99 parts of purified water or of another suitable vehic1e in part of the vehic1e and continue as aboye. Proceed in the
appropriate proportions. same manner with the rest of the vehic1e. The minimum time
The following centesimal dilution (Cn) is made from: required for the whole process is thus 1 h. Carry out the
- 1 part of the first Iiquid centesimal dilution Cn-1; whole process again for each subsequent solid dilution.
- 99 parts of a suitable vehic1e. Triturations from DS or CS onwards may also be prepared
Subsequent centesimal dilutions are produced as stated for by intense mechanical treatrnent by a suitable mixing
Cn. machine as follows: add the solid trituration to one third of
the vehic1e and mix. Add the second third of the vehic1e, mix
4. TRITURATIONS
and proceed in the same manner with the last third of the
METHOD 4.1
vehic1e. The whole process lasts minimum 1 hour, unless
Method 4.1 is used for triturations, that is solid dilutions, of
otherwise justified and authorised.
raw material s or of triturations prepared according to
Methods 4.2.1 or 4.2.2. The duration and intensity of the In all cases, it is possible to change to a liquid medium from
trituration are such that homogeneity and potentisation are the 4th, 5th and 6th decimal or centesimal triturations, as
achieved. described in Methods 3.2.1 and 3.2.2.
Vehic1e METHOD 4.1.2 (FRENCH PHARMACOPOEIA)
Unless otherwise specified, lacto se monohydrate is used. Trituration
METHOD 4.1.1 (EQUIVALENT TO HAB 6: Triturations are prepared as follows:
TRITURATIONS) Decimal triturations
Triturations are prepared manually or mechanically. Reduce 1 part of the homoeopathic stock to a powder.
Mechanical trituration must be used for quantities exceeding Triturate carefully with a small quantity of the vehic1e.
1 kg. The resulting partic1e size of the raw material in the Add the vehic1e in small quantities until 9 parts of this
first decimal or centesimal dilution does not exceed 100 ¡lm, vehic1e have been used. The resulting trituration is the 1st
unless otherwise prescribed in the individual monograph. decimal trituration (DI ).
Ratios of raw material to vehic1e Triturate as described aboye 1 part of this trituration with 9
parts of the vehic1e. The resulting trituration is the 2nd
Decimal triturations Centesimal triturations decimal trituration (D2).
The 1" decimal trituration (DI) is The 1" centesimal trituration (Cl) is In all cases, it is possible to change to a Iiquid medium after
made from: made from: the 7th decimal trituration (D7) as described in Method
l part of the raw material 1 part oE the raw material 3.2.3 .
9 parts oE Ihe vehiele 99 parts oE the vehiele Centesimal triturations
Subsequent decimal triturations (Dn)
Proceed in the same manner but following a centesimal
Subsequent centesimal
are produced as stated Eor DI, using triturations (Cn) are produced senes.
1 part oE the previous trituration as stated for CI , using 1 part of the
(Dn·!). previo us trituration (Cn·I).
In all cases, it is possible to change to a liquid medium after
the 3rd centesimal trituration (C3) as described in Method
3.2.3.
Where fresh plant material is used, the quantity of vehic1e
added is such so as to obtain 10 parts of the trituration
(decimal trituration) or 100 parts of the trituration
IV-414 Homoeopathic Preparations 2014

METHOD4.2 METHOD 4.2.2

Method 4.2 is used for triturations, that is solid dilutions, of Ratios of starting material to vehicle
liquid preparations such as mother tinctures and solutions,
their dilutions, mixtures and co-potentised mixtures. Mother tinclures prepared according to Methods 1.1.10 and 1.1.11
Gradually impregnate the total amount of vehicle, gently dry
The 1" decimal trituration (DI) is The 1" centesimal trituration (C1) is
the moist mixture, mil! and sieve if necessary, then mix and made from: made from :
triturate until homogeneity and potentisation are achieved. 1 part of the mother tincture 1 part of the moth er tincture
Trituration is further carried out as described for Method
10 parts of the vehicle 100 parts of the vehicle
4.1.1 or Method 4.1.2.
Vehicle
Unless otherwise specified, lactose monohydrate is used. 5. OTHER PREPARATIONS
METHOD 4.2.1 (EQUIVALENT TO HAB 7: METHOD 5.1
TRITURATIONS) Method 5.1 is used for preparing homoeopathic preparations
by co-potentising 2 or more stocks and/or dilutions thereof,
Ratios of starting material to vehicle
where co-potentisation consists of mixing several stocks or
The quantity of vehicle added must always be such so as to
dilutions of stocks then potentising them together in one or
obtain 10 parts of the trituration (decimal trituration) or 100
more potentisation steps.
parts of the trituration (centesimal trituration) from the
required number of parts of the liquid preparation (see METHODS 5.1.1, 5.1.2, 5.1.3 (EQUIVALENT TO HAB
Table 2371.-4), taking the mas s of the dry residue into 40a, 40b, 40c: CO-POTENTISED MIXTURES)
consideration. Where the dry residue is considered negligible, The stocks and/or dilutions in Table 2371.-5 may be used.
the quantity of vehicle added is 10 parts (decimal trituration)
or 100 parts (centesimal trituration), for 1 part of the liquid Table 2371.·5
preparation. Method 5.1.1 Method 5.1.2 Method 5.1.3

Stocks Aqueous preparations Triturations

Table 2371.·4
Solutions Glycerol macera tes
Decimal triturations Centesimal triturations and aqueous dilutions
thereof
Mother !inclures prepared according to Methods 1.1.1,1.1.3 and 1.1.4 Triturations Triturations
The 1" 'decimal' trituration (DI) is The 1" 'centesimal' trituration (C1) Liquid dilulions
made from: is made from:
2 parts of the mother tincture 2 parts of the mother tincture Mother tinctures
whose method of
maximum 10 parts of the vehicle, maximum 100 parts of the vehic1e, production specifies
taking the mass of the dry residue taking the mass of !he dry residue a l/ lO (or l/lOO)
into consideration into consideration dilution
Mother tinctures prepared according to Methods 1.1.2, 1.1.5, 1.1.6 and
1.1.7 Vehicles
The 1" 'decimal' trituration (DI) is The 1" 'centesimal' trituration (C1) The choice of the vehicle is determined by and must comply
made from: is made from:
with any special requirement for the particular stock as well
3 parts of the mother tincture 3 parts of the mother tincture
as the dosage form (see table Table 2371.-6).
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle,
taking the mass of the dry residue taking the mass of the dry residue
into consideration into consideration Table 2371·6
Mother !inctures prepared according to Methods 1.1.8 and 1.1.9 Method 5.1.1 Method 5.1.2 Method 5.1.3

The molher linelure eorresponds to Ihe 1" decimal dilulion (DI) Ethanol (96 per cent V/I'1lethanol Water for Ladose
(94 per cent m/ m)] injections monohydrate
The 2" decimal trituration (D2) is The 1" 'centesimal' trituration (C1) Ethanol (90 per cent V/I'1lethanol Purified water
made from: is made from: (86 per cent m/ m)]
1 part of the mother tincture 10 parts of the mother tindure Ethanol (80 per cent V/I'1lethanol Sugar syrup
(73 per cent m/ m) ] (sucrose,
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle, purified water
taking the mass of the dry residue taking the mass of the dry residue (64:36))
into consideration into consideration
Ethanol (70 per cent V/1'1 [ethanol
Solutions prepared according to Method 3.1.1 or liquid dilutions, mixtures (62 per cent m/ m)]
and co-potentised mixtures
Ethanol (50 per cent V/I'1lethanol
Decimal trituration n+ 1 (Dn+ 1) is Centesimal tri turation n+ 1 (Cn+ 1) is (43 per cent m/ m)]
made from: made from:
Ethanol (36 per cent V/1'1 [ethanol
1 part of !he dilution (Dn) 1 part of the dilution (Cn) (30 per cent m/ m)]
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle, Ethanol (18 per c,~ft V/1'1 [ethanol
taking the mass of the dry residue taking the mass of the dry residue (15 per cent m/ m)
into consideration into consideration

For Method 5.1.1, when starting from a trituration and

where justified, purified water is used for the 1st
potentisation sÚ:p.
For Method 5.1.2, when starting from a glycerol macerate
containing sodium chloride, unless otherwise justified and
authorised, the following vehicle is used: 0.2 parts of sodium
hydrogen carbonate and 8.8 parts of sodium chloride in 991
parts of water for injections.
 2014
Potentisation
For each potentisation step, combine and succuss or triturate
1 part of the given mixture with 9 parts (decimal dilutions)
or 99 parts (centesimal dilutions) of the appropriate vehicle.
METHOD 5.1.4
Vehicles
Ethanol of an appropriate concentration, purified water or
lacto se monohydrate may, for example, be used.
Potentisation
Potentisation may be performed as prescribed for Methods
5.1.1,5.1.2 and 5.1.3, either on the last step or on several
successive steps.
METHOD 5.1.5
Vehicle
Ethanol of an appropriate concentration, purified water or
lactose monohydrate may, for example, be used.
Potentisation
For a co-potentisation of centesimal dilutions, each dilution
(Cn-l) represents 1 per cent of the final product and the
proportion of vehicle to be added is reduced by the
proportion of the active substances [i.e. 100 per cent -
(1 per cent x the number of active substances)] . The same
procedure applies, in the appropriate proportions, when
co-potentising decimal dilutions.
____________________________________________ ~Ew
***
Mother Tinctures for
*** ***
Homoeopathic Preparations ***
(Ph Bur monograph 2029)
~Ew ____________________________________________
DEFINITION
Mother tinctures for homoeopathic preparations are liquid
preparations obtained by the solvent action of a suitable
vehicle upon raw materials. The raw materials are usually in
the fresh form but may be dried. Mother tinctures for
homoeopathic preparations may also be obtained from plant
juices, with or without the addition of a vehiele. For sorne
preparations, the matter to be extracted may undergo a
preliminary treatment.
PRODUCTION
Mother tinctures for homoeopathic preparations are prepared
by maceration, digestion, infusion, decoction, fermentation or
as described in the individual monographs, usually using
alcohol of suitable concentration.
Mother tinctures for homoeopathic preparations are obtained
using a fixed proportion of raw material to solvent, taking the
moisture content of the raw material into account, unless
otherwise justified and authorised.
If fresh plants are used, suitable procedures are used to
ensure freshness. The competent authorities may require that
the freshness is demonstrated by means of a suitable test.
Mother tinctures for homoeopathic preparations are usually
elear. A slight sediment may form on standing and that is
acceptable as long as the composition of the tincture is not
changed significantly.
The manufacturing process is defined so that it is
reproducible.
Homoeopathic Preparations IV-415
Production by maceration
Unless otherwise preseribed, reduce the matter to be
extracted to pieces of suitable size, mix thoroughly and
extract according to the prescribed extraction method with
the prescribed extraetion solvento AlIow to stand in a elosed
vessel for the prescribed time. The residue is separated from
the extraction solvent and, if necessary, pressed out. In the
latter case, the 2 liquids obtained are combined.
Adjustment of the contents
Adjustment of the eontent of constituents may be carried out
if necessary, either by adding the extraetion solvent of
suitable concentration, or by adding another mother tincture
for homoeopathic preparations of the vegetable or animal
matter used for the preparation.
IDENTIFICATION
Where applicable, at least 1 chromatographic identification
test is carried out.
TESTS
The limits in an individual monograph are set to inelude
official methods of production. Specific limits will apply to
each defined method of production.
Jf the test for relative density is carried out, the test for ethanol
need not be carried out, and vice versa.
Relative density (2.2.5)
The mother tincture for homoeopathic preparations complies
with the limits prescribed in the monograph.
Ethanol (2.9.10)
The ethanol content eomplies with that prescribed in the
monograph.
Methanol and 2-propanol (2.9.11)
Maximum 0.05 per cent V/V of methanol and maximum
0.05 per cent V/V of 2-propanol, unless otherwise preseribed.
Dry residue (2.8. 16)
Where applicable, the mother tincture for homoeopathic
preparations complies with the limits prescribed in the
monograph.
Pesticides (2.8. 13)
Where applicable, the mother tincture for homoeopathic
preparations complies with the test. This requirement is met
if the herbal drug has been shown to comply with the test.
Justification is provided in cases where the test for pesticides
is performed on the mother tincture, instead of on the herbal
drug according to the requirements of the general monograph
Herbal drugs for homoeopathic preparations (2045). Limits will
be set, taking into consideration the nature and the origin of
the herbal drug. The dilution factor of the mother tincture
and the limit of detection of the method are also taken into
aecount when fixing these limits.
Heavy metals (2.4.27)
Justifieation is provided in cases where the test for heavy
metals is performed on the mother tincture, instead of on the
herbal drug according to the requirements of the general
monograph Herbal drugs for homoeopathic preparations (2045).
Limits will be set, taking into consideration the nature and
the origin of the herbal drug. The dilution factor of the
mother tincture and the limit of detection of the method are
also taken into account when fixing these limits.
If required by the eompetent authority, suitable limits for the
content of other heavy metals such as arsenic or nickel may
be defined .
 IV-416 Homoeopathic Preparations 2014

ASSAY ***
Pillules for Homoeopathic * *
Where applicable, an assay with quantitative limits is ** **
performed. Preparations ***
STORAGE (Ph Eur monograph 2153)
Protected from light. A maximum storage temperature may ~E~ _ _ _ _ _ __ _ _ _ _ __ _ _ __ _ _ __ __

be specified. DEFINITION
LABELLING Preparations of solid consistency obtained from sucrose,
The labe! sta tes: lactose or other suitable excipients. They possess a suitable
-- that the product is a mother tincture for homoeopathic mechanical strength to resist handling without crumbling or
preparations (designated as 'TM' or '0'); breaking. They are intended for impregnation or coating with
-- the name of the raw material using the Latin title of the one or more homoeopathic preparations. The impregnated
European Pharmacopoeia monograph where one exists; pillules comply with the requirements of the monograph
-- the method of preparation; Homoeopathic pillules) impregnated (2079).
-- the ethanol content or other solvent content, in PRODUCTION
per cent V/V, in the mother tincture;
In the manufacture, packaging, storage and distribution of
-- the ratio of raw material to mother tincture;
pillules for homoeopathic preparations, suitable measures are
-- where applicable, the storage conditions.
taken to ensure their microbiological quality;
___ __ __ _ _ _ _ __ __ _ _ _ _ __ _ __ PhEur
recommendations on this aspect are provided in chapter
5.1 .4. Microbiological quality of non-sterile pharmaceutical
preparations and substances for phannaceutical use.
If a system of sizing is used, the indications in Table 2153.-1
*** are used.
Impregnated Homoeopathic
*** ***
Pillules *** Table 2153.-1. - Classification of pillules according lo lheir
(Ph Eur monograph 2079) mass and size
~E~ ____ _ __ __ _ _ _ _ _ _ _ __ _ _ _ ___ Category Number of pillules Mass Fineness
for homoeopathic (g) (11m)
DEFINITION preparations
Preparations of solid consistency obtained from sucrose, 470 - 530 1.0 1000 - 1600
lactose or other suitable excipients. They possess a suitable 2 160 - 333 1.0 1400 · 2000
mechanical strength to resist handling without crumbling or
breaking. Impregnated homoeopathic pillules are prepared by 3 no - 130 1.0 1800 - 2500

impregnation of Pillules for homoeopathic preparations (2153) 4 70 - 90 1.0 2000 - 2800

with one or more liquid homoeopathic preparations. They 5 40·50 1.0 2500·3350
are intended for sublingual or oral use.
6 16·30 1.0 3150 - 4500
PRODUCTION
7 10 0.9· 1.1 4000·5600
Impregnation takes place using liquid preparations containing
ethanol usually at a concentration of at least 68 per cent V/V 8 5 0.9· 1.1 5600·6700
(60 per cent m/m) in proportions of 1 mass part of liquid to 9 3 0.9 - 1.1 7100 - 8000
100 mass parts of pillules.
10 0.9· 1.1 8000·9500
In the manufacture, packaging, storage and distribution of
homoeopathic pillules, suitable measures are taken to ensure NOTE: for categories 7-10. the mass is obtained by weighing the
specified number of pillules.
their microbiological quality; recommendations on this aspect
are provided in chapter 5.1.4. Microbiological quality of non-
sterile pharmaceutical preparations and substances for CHARACTERS
pharmaceutical use. Appearance
CHARACTERS White or almost white spheroids.
Appearance Solubility
White, almost white or slightly coloured spheroids . Usually freely soluble in water.
Solubility IDENTIFICATION
Usually freely soluble in water. The excipients used for the manufacture of pillules for
TESTS homoeopathic preparations are identified by one or more
Microbial contamination suitable testes).
Unless otherwise justified, authorised and labelled, the TESTS
pillules are intended for sublingual administration and the 1f the test for fineness is carried out) the test for umfonnity of mass
following acceptance criteria apply. need not be cam'ed out) and vice versa.
TAMC: acceptance criterion 102 CFU/g (2.6.12). Uniformity of mass
TYMC: acceptance criterion lO! CFU/g (2.6.12) . Carry out the test using 20 pillules to constitute 1 unit.
Absence of Staphylococcus aureus (2.6.13). Weigh individually 20 units taken at random and determine
Absence of Pseudomonas aeruginosa (2.6. 13) . the individual and average masses. Not more rhan 2 of the
_ _ _ _ _ _ __ _ _ _ _ _ __ _ _ _ __ _ __ ~E~
individual masses deviate from the average mass by more
than 10 per cent and none deviate by more than 20 per cent.
2014
Fineness (2.9.35)
Not less than 90 per cent m/m of the pillules are between the
lower and upper limits of the corresponding category as
indicated in Table 2153.-1.
Impregnation
Use an approved method. The average for the results is
within a validated range.
Microbial contamination
T AMC: acceptance criterion 10 2 CFU/g (2.6.12).
TYMC : acceptance criterion 10 1 CFU/g (2.6.12) .
Absence of Staphylococcus aureus (2.6.13).
Absence of Pseudo111onas aeruginosa (2.6.13).
LABELLING
The label states:
- the composition of the pillules;
- where applicable, the size of the pillules.
____________________________________________
Apomorphine Hydrochloride for
Homoeopathic Preparations
Apo111orphinU1n Muriaticu111 for H0111oeopathic Preparations
DEFINITION
Apomorphine Hydrochloride for Homoeopathic Preparations
contains Apomorphine Hydrochloride Hemihydrate.
PRODUCTION OF STOCK
The first trituration of Apomorphine Hydrochloride for
Homoeopathic Preparations is prepared using a suitable
quantity of Lacrose or Anhydrous Lacrose as the vehicle and
a validated trituration method that ensures homogeneity is
achieved. The vehicle complies with the statement under
Vehicles in the monograph for Homoeopathic Preparations.
Content of apomorphine hydrochIoride,
C 17 H 17 NO z,HCI
The first decimal trituration contains 9.5 % to 10.5% of
C 17H I7 N0 2,HCl (dried substance).
CHARACTERISTICS
The first decimal trituration is a white powder.
IDENTIFICATION
Dissolve 2.5 g of the substance being examined without
heating in water and dilute ro 25 mL with the same solvent
(solution S).
A. To 5 mL of solution S add a few millitres of sodiu111
hydrogen carbonate solution until a permanent, white
precipitate is formed. The precipitate slowly becomes a
greenish colour. Add 0.25 mL of 0.05M iodine and shake.
The precipitate becomes a greyish-green colour. Collect the
precipita te. The precipitate dissolves in ether giving a purple
solution, dissolves in dichloromethane chloride giving a violet-
blue solution and dissolves in alcohol giving a blue solution.
B. To 2 mL of solution S add 0.1 mL of nitric acid, mix and
filter. The filtrate yields reaction A characteristic of chlondes,
Appendix VI.
C. Dissolve 0.25 g of the substance being examined in 5 mL
of water. Add 5 mL of ammonia and heat in a water-bath at
80° for 10 minutes. A red colour develops.
ASSAY
Disperse 2.5 g of the substance being examined in a mixture
of 5.0 mL of O.OIM hydrochloric acid and 50 mL of ethanol
Homoeopathic Preparations IV-417
(96%) . Carry out the method for potentiometric titration,
Appendix VIII B, using O.IM sodiu111 hydroxide. Measure the
titrant between the first 2 points of infiexion. Each rnL of
O.IM sodiu111 hydroxide is equivalent ro 30.38 mg of
C 17H 17N02,HCI.
Arsenious Trioxide for *****
** **
Homoeopathic Preparations ***
(Ph Eur 111onograph 1599)
197.8 1327-53-3
~E~ ___________________________________________
DEFINITION
Content
PhE~ 99.5 per cent to 100.5 per cent of AS 2 0 3 .
CHARACTERS
Appearance
White or almost white powder.
Solubility
Practically insoluble ro sparingly soluble in water. It dissolves
in solutions of alkali hydroxides and carbonates.
IDENTIFICATION
A. Dissolve 20 mg in 1 mL of dilute hydrochlon'c acid R, add
4 mL of water R and 0.1 mL of sodiu111 sulfide solution R .
The resulting yellow precipitate is soluble in dilute
a111monia Rl.
B. Dissolve 20 mg in 1 mL of hydrochloric acid Rl, add 5 mL
of hypophosphorous reagent R and heat for 15 min on a water-
bath. A black precipitate develops.
TESTS
Appearance of solution
A 100 gIL solution in dilute a1111110nia R1 is clear (2.2.1) and
colourless (2.2.2, Method 11).
Sulfides
Dissolve 1.0 g in 10.0 mL of dilute sodiu111 hydroxide
solution R. Add 0.05 mL of lead acetate solution R. Any colour
in the test solution is not more intense than that in a
standard prepared at the same time and in the same manner
using a mixture of 10.0 mL of a 0.015 giL solution of sodiu111
sulfide R in dilute sodiu111 hydroxide solutwn R and 0.05 mL of
lead acetate solutwn R (20 ppm).
ASSAY
Dissolve 40.0 mg in a mixture of 10 mL of water R and
10 mL of dilute sodiu111 hydroxide solution R. Add 10 mL of
dilute hydrochloric acid R and 3 g of sodiU1n hydrogen
carbonate R and mix. Add 1 mL of starch solution R and
titrate with 0.05 M iodine.
1 mL of 0.05 M iodine is equivalent ro 4.946 mg of AS2 0 3 .
____________________________________________ PhE~
IV-418 Homoeopathic Preparations
Artemisia Cina for Homoeopathic
Preparations
DEFINITION
Artemisia Cina for Homoeopathic Preparations is the dried,
unexpanded flower heads of Seriphidium cinum (Berg ex
Poljakov) Poljakov (Syn Artemisia cina Berg ex Poljakov)
Artemisia cina O.C.Berg et C.F. Schmidt.
It contains not les s than 1.0% ofsantonin (ClsH1 S0 3)
caJculated with reference to the dried material.
IDENTIFICATlON
A. The dried, slightly shiny capitula are conical to elongated-
ovoid, 2 to 4 mm long and 1 to 2 mm wide, yellow-green to
brownish and composed of 3 to 6 hermaphrodite florets
enclosed in an involucre of 14 to 20 imbricated ovate to
lanceolate bracts. Each bract has a distinct keel which is most
pronounced in the ovate outer bracts near the base; the keel
forms the midrib and it branches freely, the veinlets
becoming contorted and frequently anastomising. The outer
surface of the bracts is covered with glistening glandular
hairs. The fiorets are about 1 mm long and 0.5 mm wide;
the corolla is contracted at the base and divides at the apex
into 5 short, triangular teeth.
B. Reduce to a powder and examine under a microscope
using chloral hydrace solution. Abundant fragments of the
involucral bracts in surface view are seen. Fragments from
the margins are usually only one or two cells thick and are
composed of very thin-walled, elongated cells; fragments
from the central region of the bracts show polygonal,
isodiametric epidermal cells with thickened and beaded walls;
fairly numerous anomocytic stomata are present on the outer
surface only. Very small cluster crystals of caJcium oxalate
may be present in the parenchymatous cells underlying the
epidermis.
Groups of sclereids from the central region of the bracts
show: individual cells varying in shape but usually
considerably elongated; the ends are square or bluntly
tapering or, occasionally, somewhat enlarged; the walls are
strongly thickened and have scanered pits. Small groups of
these sclereids are occasionally found anached to fragments
of the epidermis of the bracts.
The unicellular covering trichomes are nearly always found
detached; they are usually very thin-walled although slight
thickening may occur in the basal region; sorne of these
trichomes are very long and can be found in groups forming
loosely felted, cottony masses. The typically labiate glandular
trichomes are abundant on the bracts and are also found
detached; each has a short, biseriate stalk, and a biseriate
head of two or four cells around which the cuticle is raised to
form a bladder-like covering.
The abundant pollen grains are fairly small, spherical, with
three pores and three furrows; the exine is finely warted.
A large number of immature pollen grains are present,
forming elongated, c10sely packed masses.
C. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Add 10 mL of ethanol (90%) to 1 g of the coarsely
powdered drug, stir for one hour and filter.
(2) 0.1% w/v each of santonin and cineole in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel H.
(b) Use the mobile phase as described below.
(c) Apply 10 ~L of each solution.
2014
(d) Develop the plate to 10 cm.
(e) After removal of the plate, allow to dry in air, spray with
ethanolic phosphomolybdic acid solulion, heat at 100 0 to 1050 for
about 5 minutes and examine in daylight.
mobile phase
5 volumes of glacial acetic acid, 45 volumes of hexane and
50 volumes ethyl acetate.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows the grey-blue santonin band just between
the lower third and middle third and the grey-blue cineole
band in the upper third.
CONFIRMATION
The chromatogram obtained with solution (1) shows a grey-
blue band below the band obtained for santonin in solution
(2), a strong grey-blue band level with that of santonin in
solution (2) and one or two grey-blue bands just aboye
santonin; one or two grey-blue bands between the bands
obtained for santonin and cine ole in solution (2) and a
strong grey-blue band level with the band obtained with
cineole.
Top of the plate
A grey-blue band Cineole: a grey-blue band
1 or 2 a grey-blue bands
1 or 2 grey-blue bands
a grey-blue band Santonin: a grey-blue band
a grey-blue band
Solution (1) Solution (2)
TESTS
Foreign matter
Not more than 5% of sections of stem and pieces of narrow-
linear hairy leaves; not more than 2% of other foreign matter,
Appendix XI D.
Ash
Not more than 11.0%, Appendix XI J, Method n.
Loss on drying
Not more than 10.0%, Appendix IX D.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) To 1.0 g of the powdered herbal drug add 50 mL of
methanol and stir for 2hours. Filter the solution using a dry
filter paper into a 100 mL volumetric flask, wash the filtrate
with methanol, add the washings to the filtrate and dilute to
100 mL with methanol and mix. Weigh approximately 5 g
(6.5 mL) of the solution and add 20 mL of methanol in a
50 mL volumetric fiask and dilute to volume with water.
(2) 0.005% w/v of santonin BPCRS prepared by dissolving
100 mg santonin BPCRS in 100 mi methanol and diluting
5 mL of the resulting solution to 100 mL with the mobile
phase.
(3) 0.005 % w/v each of santonin BPCRS and methyl4-
hydroxybenzoate in the mobile phase.
2014 Homoeopathic Preparations IV-419

CHROMATOGRAPHIC CONDITIONS Dry residue

(a) Use a stainless steel column (15 cm x 4.6 mm) packed Not less than l.8% w/w, Appendix XI P .
with octadecylsilyl siliea gel for chromatography (5 ¡.1m) Relative density
(Kromasil C18 is suitable) fined with a stainless steel 0.835 to 0.855, Appendix V G.
guard column packed with the same material.
ASSAY
(b)Use isocratic elution and the mobile phase described Carry out the method for liquid chromatography,
below.
Appendix III D, as described for the herbal drug using as
(c) Use a fiow rate of l.0 mL per minute. solution (1) the mother tincture.
(d)Use a column temperature of 25°. DETERMINATION OF CONTENT
(e) Use a detection wavelength of 236 nm. Calculate the content of Cl5HlS03 in the mother tincture
(f) Inject 10 ¡.IL of each solution. using the declared content of ClsHlS03 in santonin BPCRS
MOBILE PHASE using the following expression:
Equal volumes of methanol and water.
SYSTEM SUITABILITY A m
2 X_
_1 X _ 1 v: xp
The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peaks due ~ Vz ~
to methyl4-hydroxybenzoate and santonin is not less than 2.0.
DETERMINATION OF CONTENT area of the peak due 10 santonin in the
Calculate the content of Cl5HlS03 in the herbal drug using chromatogram obtained with solution (1);
the declared content of Cl5HlS03 in santonin BPCRS using area of the peak due 10 santonin in the
the following expression: chromatogram obtained with solution (2);
weight of the herbal drug being examined in mg;
weight of santonin BPCRS in mg;
dilution volume of solution (1) in mL;
dilution volume of solution (2) in mL;
percentage content of santonin (CI5HlS03) in
santonin BPCRS.

area of the peak due 10 santonin in the

chromatogram obtained with solution (1);
Barium Chloride Dihydrate tor ***
*** ***
area of the peak due to santonin in the
chromatogram obtained with solution (2);
weight of the herbal drug being examined in mg; Homoeopathic Preparations ***
weight of santonin BPCRS in mg; (Ph Eur monograph 2142)
dilution volume of solution (1) in mL; 244.3 10326-27-9
dilution volume of solution (2) in mL; PhEur _ _ _ _ _ _ _ __ __ _ __ __ _ _ _ __
percentage content of santonin (CI5HlS03) in
santonin BPCRS; DEFINITION
d percentage loss on drying of the herbal drug being Content
examined. 99.0 per cent 10 10l.0 per cent ofBaCI2,2H 20.
CHARACTERS
MOTHER TINCTURE Appearance
White or almost white, crystalline powder or colourless
The mother tincture complies with the requirements stated under crystals.
Mother Tinctures for Homoeopathic Preparations and with the
following requirements. Solubility
Freely soluble in water, very slightly soluble or practically
DEFINITION insoluble in ethanol (96 per cent).
It contains not les s than 0.1 % of santonin (CI5HlS03)'
IDENTIFICATION
PRODUCTION A. Dissolve 0.1 g in 1 mL of water R. Add 0.3 mL of dilute
The mother tincture of Artemisia cina is prepared from the sulfuric acid R. A white precipitate is formed; it is insoluble in
powdered drug using Method 1.1.8 described in the dilute hydrochloric acid R and in dilute nitric acid R.
monograph for Methods of Preparation of Homoeopathic B. It gives reaction (a) of chlorides (2.3.1).
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol.
TESTS
CHARACTERISTICS
Solution S
The mother tincture is a golden yellow to greenish liquido Dissolve 10.0 g in water R and dilute to 100 mL with the
IDENTIFICATION same solvento
The mother tincture complies with Identification test C Appearance of solution
aboye using the mother tincture as solution (1). Solution S is clear (2.2.1) and colourless (2.2.2, Method lI).
TESTS Acidity or alkalinity
Ethano1 To 10 mL ofsolution S add 0.1 mL ofphenolphthalein
40% to 46% w/w (47% 10 54% v/v), Appendix VIII F. solution R. Not more than 0.2 mL of 0.01 M hydrochloric acid
 IV-420 Homoeopathic Preparations 2014

or O. 01 M sodium hydroxide is required to change the colour Zinc sulfate, aIkaline-earth sulfates, rare-earth sulfates
of the indicator. Dissolve 1.0 g in 17 mL of water R. Add 0.5 mL of
Heavy metals (2.4.8) hydroehlorie acid R and 1 g of thioaeetamide R. Heat in a
Maximum 10 ppm. water-bath for 10 min. Dilute to 20.0 mL with water R and
filter. Evaporate 10.0 mL of this solution to dryness in an
12 mL of solution S complies with limit test A. Prepare the
oven. Ignite the residue at about 800 ± 50 oC to constant
reference solution using lead standard solution (J ppm Pb) R.
mass. The residue weighs a maximum of 2 mg.
ASSAY Arsenic (2.4.2, Method A)
Dissolve 0.200 g in 100 mL of water R. Add 100 mL of Maximum 2 ppm, determined on 5 mL of solution S.
methanol R, 10 mL of eoneentrated ammonia R and 2 mg of
phthalein purple R. Titrate with 0.1 M sodium edetate until the Water (2.5.12)
colour changes from violet to colourless. 16.0 per cent to 20.0 per cent, determined on 80 mg. Shake
for 10 min before carrying out the determination.
1 mL of 0.1 M sodium edetate is equivalent to 24.43 mg of
BaCI2,2H 2 0 . ASSAY
__________________________________________ ~Ew
Dissolve 0.200 g in 50 mL of water R. Add 10 mL of
ammonium ehloride buffer solution pH 10. O R and 50 mg of
mordant blaek 11 triturate R1 . Titrate with 0.1 M sodium
edetate until the colour changes from red to green.
*** 1 mL of 0.1 M sodium edetate is equivalent to 20.85 mg of
Cadmium Sulfate Hydrate for
*** ***
CdS0 4 ·
__________________________________________
Homoeopathic Preparations ***
~ Ew

Cadmium Sulphate Hydrate for Homoeopathic

Preparations
(Ph Eur monograph 2143) ***
Calcium lodide Tetrahydrate for * **
256.5 10124-36-4 **
~Ew _________________________________________ Homoeopathic Preparations ****
(Ph Eur monograph 2144)
DEFINITION
Content 366.0 13640-62-5
98.0 per cent to 102.0 per cent (anhydrous substance). ~Ew __________________________________________

CHARACTERS DEFINITION
Appearance Content
White or almost white, crystalline powder. 97.0 per cent to 102.0 per cent ofCaI2 (anhydrous
Solubility substance).
Freely soluble in water, practically insoluble in ethanol CHARACTERS
(96 per cent). Appearance
IDENTIFICATION White or almost white powder, very hygroscopic.
A. It gives reaction (a) of sulfates (2.3.1) . Solubility
B. To 2 mL of solution S (see Tests) add 2 mL of sodium Very soluble to freely soluble in water and in ethanol
sulfide solution R . A yellow precipitate is formed. (96 per cent).

TESTS
Solution S
Dissolve 5.0 g in earbon dioxide-free water R and dilute to
50 mL with the same solvent.
Appearance of solution
Solution S is c1ear (2.2.1) and colourless (2.2.2, Method JJ).
Acidity or aIkalinity
To 10 mL of solution S add 0.3 mL of methyl orange
solution R. Not more than 0.5 mL of 0.01 M hydrochlorie acid
or O. 01 M sodium hydroxide is required to change the colour
of the indicator.
Nitrates
Maximum 100 ppm.
Dissolve 1.0 g in water R and dilute to 20 .0 mL with the
same solvento To 1.0 mL of this solution add 0.2 mL of a
10 giL solution of sulfanilie acid R in acetic aeid R and 0.2 mL
of a recently prepared 3 gIL solution of naphthylamine R in
acetic acid R. Add a tuming of zine R. A pink colour is
produced within 5 mino It is not more intense than that of a
mixture of 0.5 mL of nitrate standard solution (JO ppm
NO.,) R and 0.5 mL of water R, prepared at the same time.
IDENTIFICATION
A. Solution S (se e Tests) gives reaction (a) of calcium
(2.3.1).
B. Solution S (se e Tests) gives reaction (b) of iodides (2.3.1).
TESTS
Solution S
Dissolve 10.0 g in distilled water R and dilute to 100.0 mL
with the same solvent.
Appearance of solution
Solution S is c1ear (2.2.1) and not more intensely coloured
than reference solution GY s (2.2.2, M ethod IJ).
Free iodine, iodates
To 5 mL of solution S add 2 mL of methylene chl0/1'de R.
Shake and allow to stand. The organic layer is colourless
(2.2.2, Method J) (free iodine) . Add 0.2 mL of dilute sulfuric
acid R. Shake and allow to stand. The organic layer remains
colourless (2.2.2, Methad J) (iodates).
Sulfates (2.4. 13)
Maximum 150 ppm.
Dilute 10 mL of solution S to 15 mL with distilled water R.
Iron (2.4.9)
Maximum 10 ppm, determined on 10 mL of solution S.
 2014
Heavy metals (2.4.8)
Maximum 10 ppm.
12 mL of solution S complies with limit test A. Prepare the
reference solution using lead standard solunon (1 ppm Pb) R.
Water (2.5.12)
18.0 per cent to 22.0 per cent, determined on 0.100 g.
ASSAY
Dissolve 0.300 g in 50 mL of water R. Add 5 mL of dilute
nitn'c acid R and 25. O mL of 0.1 M silver nitrate. Shake.
Add 2 mL of ferric ammoníum sulfate solutíon R2 and titrate
with 0.1 M ammoníum thíocyanate until the colour changes to
reddish-yellow.
1 mL of 0.1 M silver nitrate is equivalent to 14.70 mg of
CaI 2 ·
STORAGE
In an airtight container.
__________________________________________
Calcium Phosphate for Homoeopathic
Preparations
Calcium Phosphoricum for Homoeopathic Preparanons
DEFINITION
Calcium Phosphate for Homoeopathic Preparations contains
Calcium Phosphate.
PRODUCTION OF STOCK
The first trituration of Calcium Phosphate for Homoeopathic
Preparations is prepared using a suitable quantity of a
vehicle, such as Lactose, Anhydrous Lactose or Sucrose, and
a validated method for trituration that ensures homogeneity
is achieved. The vehicle complies with the statement under
Vehicles in the monograph for Homoeopathic Preparations.
Content of calcium Ca
The first decimal trituration contains 3.5% to 4.0% of Ca.
CHARACTERISTICS
The first decimal trituration is a white powder.
IDENTIFICATION
Wash 5 g of the first decimal trituration of the substance
being examined with three 10-mL quantities of water. The
dried residue complies witb the following tests.
A. Dissolve 0.1 g of the dried residue in 5 mL of a 25% v/v
solution of nitric acid. The resulting solution yields reaction B
of phosphates, Appendix VI.
B. The dried residue yields reaction B characteristic of
calcium salts, Appendix VI. Filter before adding potassium
ferrocyanide solution.
C. The dried residue complies with the limits of the Assay.
D. If the preparation includes Lactose as the vehicle, it
complies with the following test. Dissolve 0.25 g in 5 mL of
water. Add 5 mL of ammonia and heat in a water-bath at 80
for 10 minutes. A red colour develops.
E. If the preparation includes Sucrose as the vehicle, it
complies with the following test. Dissolve 5.0 g in carbon
dioxide-free water and dilute to 10 mL with the same solvent.
Dilute 1 mL of the solution to 100 mL with water. To 5 mL
of the solution add 0.15 mL of freshly prepared copper sulfate
solurion and 2 mL of freshly prepared dilute sodium hydroxide
solution. The solution is blue and clear and remains so after
boiling. To the hot solution add 4 mL of dilute hydrochloric
Homoeopathic Preparations IV-421
acid and boil for 1 minute. Add 4 mL of dilute sodium
hydroxide solunon. An orange precipitate is formed
immediately.
ASSAY
Dissolve 0.2 g of the residue in a mixture of 1.0 mL of
hydrochloric acid R1 and 5 mL ofwater. Add 25.0 mL of
O.lM disodium edetate and dilute to 200 mL with water.
Adjust to about pH 10 with concentrated ammonia.
Add 10 mL of ammonia buffer pH 10.0 and a few
milligrams of mordant black 11 tritura te. Titrate the excess
disodiurn edetate with O.IM zinc sulfate until the colour
changes from blue to vio let. Each mL ofO.lM sodium
hydroxide is equivalent to 4.008 mg of Ca.
Oriental Cashew for *****
PhE~
** **
Homoeopathic Preparations ***
(Ph Bur monograph 2094)
ffiE~ __________________________________________
DEFINITION
Dried fruit of Semecarpus anacardium L. (Anacardium orientale
L).
Content
Minimum 6.0 per cent m/m of total phenol derivatives
expressed as eugenol (C IO H 12 0 2; M r 164.2) (dried drug).
IDENTIFICATION
A. The dried fruit is oval and more or less heart-shaped;
about 2 cm long, nearly 2 cm wide and 0.5 cm thick.
Its surface is smooth, shiny and blackish. A transverse section
shows a rather well developed, tough pericarp riddled with
rather wide lacunae containing an abundant thick reddish-
brown juice. The pericarp covers a white kernel under a
reddish skin. The fruit may include the blackish, fleshy,
wrinkled, cupuliferous receptacle.
B. Thin-Iayer chromatography (2.2.27).
Test solution To 1.0 g of suitably cut drug, add 10 mL of
ethanol (90 per cent V/V? R. Heat under reflux on a water-
bath at 60 oC for 15 mino Allow to cool and filter.
Reference solution Dissolve 5 mg of gallic aeid R and 5 mg of
eaffeic acid R in methanol R and dilute to 10 mL with the
same solvent.
Plate TLC siliea gel plate R.
Mobile phase methanol R, toluene R (15:85 V/V) .
Applieation 20 IlL of the test solution and 10 IlL of the
reference solution, as bands.
Development Over a path of 15 cm.
Drying In airo
Detection Spray with a solution containing 10 gIL of
diphenylboric aeid aminoethyl ester R and 50 gIL of maerogol
400 R in methanol R. Examine in ultraviolet light at 365 nm.
0
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other fainter zones may be
present in the chromatogram obtained with the test solution.
 IV-422 Homoeopathic Preparations
Top of the plate
A ¡¡reenish·blue fluoreseent zone
---
Several violet·blue fluoreseent
zones
A yellow fluoreseent zone
Caffeie acid: a violet·blue
fluoreseent zone
---
Gallie acid: a violet·blue A violet·blue fluoreseent zone
fluoreseent zone (¡¡allie acid)
Reference solution Test solution
TESTS
Anacardium occidentale L.
Fruits of Anacardium occidentale L are not presento These are
up to 35 mm long, 30 mm large, 20 mm thick, light brown
and distinctly kidney-shaped. The pericarp is smooth or
slightly crinkled with dark marbling in places.
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on LOOO g ofthe finely
divided drug by drying in an oven at 105 oC for 2 h.
Total ash (2.4.16)
Maximum 5.0 per cent.
ASSAY
Total phenol derivatives
Absorption spectrophotometry (2.2.25).
Stock solution Place 4.500 g of the crushed drug in a fiask.
Add 200 mL of ethanol (90 per cent VIl--? R. Boil in a water-
bath under refiux for 4 h. Cool the fiask. Quantitatively
transfer into a volumetric fiask. Dilute to 250.0 mL with
ethanol (90 per cent VIl--? R. Filter the liquid through a paper
filter 125 mm in diameter. Discard the first 50 mL of the
filtrate. Dilute 5.0 mL of filtrate to 50.0 mL with ethanol
(90 per cent VIl--? R and shake. Dilute 5.0 mL of this solution
to 10.0 mL with ethanol (90 per cent VIl--? R and shake.
Test solution To 2.0 mL of stock solution add 1.0 mL of
phosphomolybdotungstic reagent R and 10 mL of water R, mix
and dilute to 25.0 mL with a 290 gIL solution of sodium
carbonate R. Wait exactly 3 min then filter the solution
through a fibre-glass filter with a 1 ¡.lm mesh aperture,
discarding the first 5 mL
Reference solution Dissolve 80.0 mg of eugenol R in ethanol
(90 per cent VIl--? R and dilute to 250.0 mL with the same
solvent. Dilute 5.0 mL of the solution to 25.0 mL with
ethanol (90 per cent VIl--? R. To 2.0 mL of this solution add
1 .O mL of phosphomolybdotungstic reagent R and 10 mL of
water R, mix and dilute to 25.0 mL with a 290 gIL solution
of sodium carbonate R. Wait exactly 3 min then filter the
solution through a fibre-glass filter with a 1 ¡.lm mesh
aperture, discarding the first 5 mL
Measure the absorbance (2.2.25) of the test solution and the
reference solution at 755 nm after 30 min using water Ras
compensation liquido
Calculate the percentage content mlm of total phenol
derivatives, expressed as eugenol, from the following
expression:
2014
absorbance of the test solution;
absorbance of the reference solution;
mas s of the drug to be examined, in milligrams;
--- mass of eugenol in the reference solution, in
milligrams.
MOTHER TINCTURE
The mother tincture complies with the requirements of the
--- general monograph on Mother tinctures for homoeopathic
preparations (2029).
DEFINITION
The mother tincture of oriental cashew is prepared by
maceration using ethanol of a suitable concentration from the
dried fruit of Semecarpus anacardium L (Anacardium on'entale
L).
Content
0.5 per cent mlm to LO per cent mlm of total phenol
derivatives expressed as eugenol.
CHARACTERS
Appearance
Yellowish-brown or reddish-brown liquido
IDENTIFICATION
Thin-layer chromatography (2.2.27) as described under
Identification B of the drug with the following modification.
Test solution The tincture to be examined.
Results See identification B for the drug.
TESTS
Relative density (2.2.5)
0.815 to 0.845 .
Ethanol (2.9.10)
85 per cent VIV to 95 per cent VIV.
Dry residue (2.8.16)
Minimum 1.50 per cent mlm.
ASSAY
Total phenol derivatives
Absorption spectrophotometry (2.2.25) as described in the
assay of the drug to be examined with the following
modifications.
Stock solution Place 8.000 g of the mother tincture to be
examined in a volumetric fiask and dilute to 250.0 mL with
ethanol (90 per cent VIl--? R. Dilute 5.0 mL of this solution to
20.0 mL with ethanol (90 per cent VIV) R.
Test solution To 2.0 mL of stock solution add 1.0 mL of
phosphomolybdotungstic reagent R and 10 mL of water R, mix
and dilute to 25.0 mL with a 290 gIL solution of sodium
carbonate R. Wait exactly 3 min then filter the solution
through a fibre-glass filter with a 1 ¡.lm mesh aperture,
discarding the first 5 mL
Reference solution Dissolve 80.0 mg of eugenol R in ethanol
(90 per cent VIl--? R and dilute to 250.0 mL with the same
solvento Dilute 5.0 mL of the solution to 25 .0 mL with
ethanol (90 per cent VIl--? R . To 2.0 mL of this solution add
1.0 mL of phosphomolybdotungstic reagent R and 10 mL of
water R, mix and dilute to 25.0 mL with a 290 gIL solution
of sodium carbonate R. Wait exactly 3 min then filter the
solution through a fibre-glass filter with a 1 ~lm mesh
aperture, discarding the first 5 mL
Measure the absorbance (2.2.25) of the test solution and the
reference solution at 755 nm after 30 min, using water R as
compensation liquido
2014
Calculate the percentage content m/m of total phenol
derivatives expressed as eugenol, using the following
expression:
absorbance of the test solution;
absorbanee of the reference solution;
mass of the mother tincture to be examined, in
milligrams;
mass of eugenol in the reference solution, in
milligrams.
_ _ __ _ _ _ _ _ _ _ __ _ _ _ _ __ __ _ PhEur
Cineraria Maritima for Homoeopathic
Preparations
DEFINITION
Cineraria Maritima for Homoeopathic Preparations is the
fresh aerial parts of Cineraria maritima L. harvested before
ftowering.
IDENTIFICATION
Plant Low growing, woody-based perennial 25 to 30 cm
occasionally up to 100 cm high, with strong, white tomentose
shoots up to 20 mm in diameter. The shoots are mueh
branched and those bearing the ftowers are elongated with
sorne smaller leaves in the upper part; the shorter, non-
ftowering shoots remain compressed with the leaves forming
a rosette at the topo
Leaves The leaves are alternate, up to 25 cm long and
12 cm wide, ovate or oblong-ovate, the lowest eoarsely
toothed, the upper ones deeply pinnatified or pinnate with
4 to 6 oblong to blunt, often 3 to 5 lobed, unequal segments.
The under surfaee is eovered with a dense white felt, the
upper surface is green with scattered cottony hairs.
MOTHER TINCTURE
The mother tincture complies with the requirements stated under
Mother Tinctures for Homoeopathic Preparations and with the
following requirements.
PRODUCTION
The mother tincture of Cineraria maritima L. is prepared
from the cut drug using Method 1.1.7 described in the
monograph for Methods of Preparation of Homoeopathic
Stoeks and Potentisation. Use 43% w/w (50% v/v) of ethanol.
CHARACTERISTICS
The mother tincture is a dark yellow, clear to slightly turbid
liquido
IDENTIFICATION
A. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Dilute 5 mL of the mother tincture with 15 mL of water
and transfer to a cartridge containing octadecyl-bonded silica
sorbent (a Sep-pak C18 cartridge is suitable) previously
washed with 10 mL of methanol followed by 10 mL of water.
Elute with 10 mL of methanol, evaporate the eluant and
dissolve the residue in 0.5 mL of methanol.
(2) 0.05% w/v each of hyperoside and rutin and 0.01 % w/v of
scopoletin in methanol.
Homoeopathic Preparations IV-423
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F254 .
(b) Use the mobile phase described below.
(e) Apply 30 J.IL of solution (1) and 10 J.IL of solution (2) as
10 mm bands.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and spray the plate
with a 1% w/v solution of diphenylboric acid aminoethyl ester in
methanol and then spray with a 5% w/v solution of
polyethylene glycol 400 in methanol. Heat at 100° to 105° for
5 minutes, allow to dry in air and examine immediately in
ultraviolet light (365 nm).
MOBILE PHASE
10 volumes of water, 10 volumes of formic acid and
80 volumes of ethyl acetate.
SYSTEM SUIT ABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows three ftuoreseent bands: an orange
ftuorescent band with a low Rf value (rutin), an orange
ftuoreseent band with an Rf value in the middle region
(hyperoside) and a blue ftuoreseent band with an Rfvalue in
the upper regíon (scopoletin).
CONFIRMATION
The chromatogram obtained with solution (1) shows an
orange ftuorescent band in a similar position to rutin, another
ftuorescent band aboye this orange band, another orange
ftuoreseent band in a similar position to hyperoside with a
green ftuorescent band just below, one or two green
ftuorescent bands between the bands in similar positions to
hyperoside and seopoletin, one blue-green ftuoreseent band
in a similar position to seopoletin and one yellow-green to
orange ftuorescent band aboye the blue-green ftuoreseent
bando
Top of the plate
A yellow-green to
orange band
A blue-green band Scopoletin: a blue band
An orange band Hyperoside: an orange
band
A green band
A band
An orange band Rutin: an orange band
Solution (1) Solution (2)
B. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Evaporate offthe ethanol from 50 mL ofthe mother
tincture. Make the residue alkaline with dilute ammonia Rl
and extract with three 20-mL quantities of chloroform.
Evaporate the eombined ehloroform extracts to dryness and
dissolve the residue in 1 mL of ethanol (60%).
(2) 0.1 % w/v of reserpine in acetone.
IV-424 Homoeopathic Preparations 2014

CHROMATOGRAPHIC CONDITIONS and the placentae. The external surface is marked by spiral,
(a) Use as the coaúng silica gel F254 • ftatúsh, knife marks where the peel has been removed.
(b) Use the mobile phase described below. In cross secúon, three conspicuous fissures can be seen
radiaúng from the centre and dividing the fruit into three
(c) Apply 30 pL of solution (1) and 20 pL of solution (2) as
pans. Each part contains two groups of seeds near the
10 mm bands. periphery, the remaining space being filled with pithy
(d) Develop the plate to 10 cm. parenchyma. Each fruit contains 200 to 300 seeds .
(e) After removal of the plate, dry in air and spray the plate The inferior ovary is initially tripartite but as the placentae
with a 2% w/v solution of dimethylaminobenzaldehyde in grow out from the centre towards the circurnference, each
ethanol and then spray with a solution of sulfuric acid. Heat at divides into two, half curving backwards, and giving the
100° to 105° for 5 minutes and examine in daylight. appearance of a hexapartite ovary.
MOBILE PHASE B. Reduce to a powder. The powder is pale yellowish-buff.
10 volumes of methanol and 90 volumes of chlorofonn. Examine under a microscope using chloral hydrate solution.
The powder shows abundant, large, partly lignified, thin-
SYSTEM SUITABILITY walled, finely pitted, usually fragmented parenchyma; smaller
The test is not valid unless the chromatogram obtained with cells with slightly collenchymatous thickening and more
solution (2) shows one blue band with an Rf value of 0.80 . disúnct pitted circular to oval areas, lignified, spirally or
CONFIRMATION annularly thickened vessels.
The chromatogram obtained with solution (1) shows a series C. Carry out the method for thin-layer chromatography,
of violet bands between the line of applicaúon and Rf value Appendix In A, using the following solutions.
0.65, one pink band at Rfvalue 0.75 and one red band (1) Add 30 mL of 86% v/v ethanol to 3 g of the eoarsely
at Rfvalue 0.90. powdered drug and heat under reftux for 2 hours. Allow to
cool and filter. Evaporate 20 mL of the filtrate to about
Top of the plate 5 mL.
(2) 0.1 % w/v eaeh of caffeine, coumarin and resorcinol in
A red band methanol.
Reserpine: a blue CHROMATOGRAPHIC CONDITIONS
A pink band band (a) Use as the eoating silica gel F254 .
(b) Use the mobile phase described below.
(e) Apply 20 ~lL of each solution.
A series of violet (d) Develop the plate to 10 cm.
bands between the
(e) Remove the plate, dry it in air and examine under
line of application ultraviolet light (254 nm).
and Rf 0.65
MOBILE PHA SE
1 volume of 13.5M ammonia, 9 volumes of methanol and
90 volumes of dichloromethane.
Solution (1) Solution (2)
SYSTEM SUITABILITY

TESTS The test is not valid unless the ehromatogram obtained with
Ethanol solution (2) shows three c1early separated bands
25 to 35% w/w (31 to 42% v/v), Appendix VIII F. (approximate Rfvalues: resoreinol 0.31, eaffeine 0.67 and
coumarin 0.87) .
Dry residue
Not less than 1.0%, determined on 2 mL, Appendix XI P. CONFIRMATION

Relative density The chromatogram obtained with solution (1) shows two
0.957 to 0.977, Appendix V G. dark bands at Rfvalues ofO .08 and 0.1 respectively between
the line of applicaúon and the band due to resorcinol, one
STORAGE dark band at an Rf value of 0.56 positioned between the
Cineraria Maritima for Homoeopathic Preparations should band due to resorcinol and that due to eaffeine, and one dark
be protected from light. band at approximately Rf value of 0.78 positioned between
the band due to eaffeine and that due to eoumarin. Other
bands may be presento
TESTS
Citrullus Colocynthis Fruit for Foreign matter
Not more than 2.0% of the outer part of the periearp;
Homoeopathic Preparations not more than 5.0% of seeds; not more than 2.0% of other
DEFINITION foreign matter, Appendix XI D.
Citrullus Colocynthis Fruit for Homoeopathic Preparaúons is Loss on drying
the dried, peeled fruits of Citrullus colocynthis (L.) Schrad. When dried at 100° to 105° for 2 hours, loses not more than
with the seeds removed. 22.0% of its weight. Use 1 g.
IDENTIFICATION Total ash
A. The peeled fruits are spherical with a diameter of 5 to Not more than 13.0%, Appendix XI J, Method n.
10 cm, white to pale yellow and very light in texture,
consisting mainly of soft, spongy tissue from the inner cupule
 2014 Homoeopathic Preparations IV-425

IDENTIFICATION
Top of the plate
First identification A, B, D.
Second identification A, B, C.
A. The fruits are dark greyish-brown or black, reniform sub-
Coumarin : a dark band
spherical, about 6-1 0 mm in diameter and 9-12 mm long;
the outer surface is irregularly wrink1ed with a ridge about
4-6 mm long running between the pale, circular scar left by
A dark band
the stalk and a small beak of the remains of the stigma.
The pericarp is hard, about 1 mm thick and the inner surface
is brownish-grey, hard and woody. Cut transversely, the fruit
Caffeine: a dark band
shows a single, cup-shaped seed inro the hollow of which an
A dark band ingrowth of the mesocarp and endocarp projects.
Cut longitudinally, the endosperm shows the presence of 2
narrow cavities in each of which is enclosed 1 of the
Resorcinol: a dark foliaceous cotyledons.
band B. Microscopic exarnination (2.8.23). The powder (7 10) is
brown. Examine under a microscope using chloral hydrate
A dark band solution R . The powder shows brownish, thin-walled,
A dark band elongated cells with brown granular contents; rather large
vascular bundles; very thick lignified fibres; sclereids; large,
thin-walled, cubic or polygonal cells containing fatty oil and
large protein granules; cells containing small needle-shaped
crystals and, in the largest cavities, prism crystals.
Sol ution (1) Solution (2)
C. Thin-Iayer chromatography (2.2.27).
Test solution To 2.00 g of the powdered herbal drug (7 10)
MOTHER TINCTURE (2.9.12) add 20 mL of ethanol (90 per cent VIV) R, shake for
The mother tincture complies with the requirements stated under 2 h and then centrifuge (1000 g). Use the supematant.
Mother Tinctures for Homoeopathic Preparations and with the Reference solution Dissolve 10 mg of picrotin CRS and 10 mg
following requirements. of picrotoxinin CRS in ethanol (96 per cent) R and dilute to
10 mL with the same solvent.
PRODUCTION
Plate TLC silica gel plate R (5-40 )lm) [or TLC silica gel
The mother tincture of Citrullus colocynthis (L.) Schrad.
plate R (2-10 )lm)] .
is prepared from the powdered drug using Method 4a
described in the monograph for Methods of Preparation of Mobile phase methanol R, ethyl acerate R , heptane R
Homoeopathic Stocks and Potentisation. Use 86% w/w ( 10:40:50 VIV/V).
(90% v/v) ethanol. Application 40)lL [or 10 )lL], as bands of 20 mm [or
10 mm] .
CHARACTERISTICS
The mother tincture is a light yellow ro yellow liquid. Development Over a path of 10 cm [or 6 cm].
Drying In air.
IDENTIFICATION
The mother tincture complies with Identification test C Detection Spray with anisaldehyde solution R, hea t at
above using the mother tincture as solution (1). 100-105 oC for 5-10 min and examine in daylight within
5-10 mino
TESTS
Results See below the sequence of zones present in the
Ethanol chromatograms obtained with the reference solution and the
81 % to 91 % w/w (86% ro 94% v/v), Appendix VIII F. test solution. Above the zone due to picrotoxinin, several
Dry residue pink or violet zones may also be visible in the chromatogram
1.0% ro 2.5% w/w, Appendix XI P. obtained with the test solution.
Relative density
0.830 to 0.850, Appendix V G.
Top of the plate

----- -----
Cocculus Indicus for Picrotoxinin: a blue zone A blue zone (picrotoxinin)

Homoeopathic Preparations ----- -----

(Anamina Cocculus for Homoeopathic Preparations,
Picrotin: a blue zone A blue zone (picrotin)
Ph Eur monograph 2486)
~E~ __________________________________________ Reference solution Test solution

DEFINITION
Dried, ripe fruit of Anamina paniculata Colebr. (A. cocculus D. Examine the chromarograms obtained in the assay.
Wight et Am.). Results The peaks due to picrotoxinin and picrotin in the
Content chromatogram obtained with the test solution are similar in
Minimum 0.80 per cent ofpicrotoxinin (ClsHI606; M r retention time to the corresponding peaks in the
292.3) (dried drug). chromatogram obtained with the reference solution.
IV-426 Homoeopathic Preparations 2014

TESTS - method l.l.8 using the powdered herbal drug (710)

Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on l.000 g ofthe
powdered drug (710) (2.9.12) by drying in an oven at
105 oC for 2 h .
Total ash (2.4.16)
Maximum 6.0 per cent.
ASSAY
Liquid chromatography (2.2.29) .
Test solution To 2.00 g of the powdered drug (710) (2.9.12)
add 20.0 mL of ethanol (90 per cent V/V? R, shake for 2 h
and then centrifuge at 1000 g for 5 mino Dilute 2.0 mL of
the supernatant to 20.0 mL with the mobile phase and filter
through a membrane filter (nominal pore size 0.45 ¡.lm).
Reference solution Dissolve 5.0 mg of picrotin CRS and
5.0 mg of picrotoxinin CRS in 10.0 mL of acetonitrile R.
Dilute 2.0 mL of the solution to 20.0 mL with the mobile
phase.
Column:
- size: 1 = 0.125 m, 0 = 4.0 mm;
- stationary phase: octadecylsilyl s¡Jica gel for chromatography R
(5 ¡.lm).
Mobüe phase acetonitrile for chromatography R, water R
(30:70 V/V).
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 200 nm.
Injection 10 ¡.lL.
Run time Twice the retention time of picrotoxinin CRS.
Retention time Picrotin = about 6 min;
picrotoxinin = about 10 mino
System suitab¡Jity Reference solution:
- resolution: minimum 2.0 between the peaks due to picrotin
and picrotoxinin.
Calculate the percentage content of picrotoxinin using the
following expression:
(2.9.12) and ethanol (90 per cent V/V? R; use ethanol
(70 per cent V/V? R to prepare the 4 th decimal dilution
and ethanol (50 per cent VIV) R for subsequent dilutions;
- method l.l.10 using the crushed drug in fragments of
about 2-3 mm, ethanol (90 per cent V/V? R and a
maceration time of about 3 weeks.
CHARACTERS
Appearance
Yellow or dark yellow liquido
IDENTIFICATION
A. Thin-layer chromatography (2.2.27) as described in
identification test C of the herbal drug with the following
modification.
Test solurion The mother tincture to be examined.
B. Examine the chromatograms obtained in the assay.
Results The peaks due to picrotoxinin and picrotin in the
chromatogram obtained with the test solution are similar in
retention time to the corresponding peaks in the
chromatogram obtained with the reference solution.
TESTS
Relative density (2.2.5)
0.830 to 0.845 (method 1.1.8).
Ethanol (2.9.10)
85 per cent VlVto 95 per cent VIV(method l. 1.1 O).
Dry residue (2.8.16)
Minimum 0.7 per cent.
ASSAY
Liquid chromatography (2.2.29) as described in the assay for
the herbal drug with the following modification.
Test solution Dilute 0.500 g of the mother tincture to be
examined to 10.0 mL with the mobile phase and filter using
a membrane filtre (nominal pore size 0.45 ¡.lm).
Calculate the percentage content of picrotoxinin using the
following expression:

Al x m2 x p
A 2 x ml x 10
A¡ area of the peak due to picrotoxinin in the
A¡ area of the peak due to picrotoxinin in the
chromatogram obtained with the test solution;
chromatogram obtained with the test solution;
A2 area of the peak due to picrotoxinin in the
A2 area of the peak due to picrotoxinin in the
chromatogram obtained with the reference solution;
chromatogram obtained with the reference solution;
m¡ mass of the herbal drug to be examined to prepare
m¡ mass of the mother tincture to be examined used to
the test solution, in grams;
prepare the test solution, in grams;
m2 mass of picrotoxinin CRS used to prepare the
m2 mass of picrotoxinin CRS used to prepare the
reference solution, in grams;
reference solution, in grams;
p assigned percentage content of picrotoxinin in
p assigned percentage content of picrotoxinin in
picrotoxinin CRS.
picrotoxinin CRS.
MOTHER TINCTURE _ _ _ __ _ _ _ __ __ _ __ _ _ __ _ _ _ PhEur
The mother tincture complies with the requirements of the
general monograph on M other tinctures for homeopathic
preparations (2029).
DEFINITION
Content
0.07 per cent m/m to 0.15 per cent mlm ofpicrotoxinin
(C¡sH¡óOó)·
PRODUCTION
The mother tincture is prepared from the dried, ripe fruit of
A. paniculata Colebr. according to the following methods
prescribed in the monograph Methods of preparation of
homoeopathic stocks and potentisation (2371):
2014 Homoeopathic Preparations IV-427

Copper for Homoeopathic *** Reference solutions Prepare the reference solutions using lead
*** *** standard solution (0.1 per cent Pb) R, diluted as necessary with
Preparations *** a 1 per cent V/V solution of nitric acid R.
Copper for Homoeopathic Use Source Lead holIow-cathode lamp o
(Ph Eur monograph 1610) Wavelength 283 .3 nm.
Cu 63.5 7440-50-8 Flame Air-acetylene.
~E~ __________________________________________ Zinc
Maximum 50 ppm.
DEFINITION
Aromic absorption spectrometry (2.2.23, Method 1).
Content
99.0 per cent to 101.0 per cent of Cu. Test solution Use the test solution prepared for the test for
¡ron.
CHARACTERS
Reference solutions Prepare the reference solutions using zinc
Appearance
standard solution (100 ppm Zn) R, diluted as necessary with a
Reddish-brown powder.
I per cent V/V solution of nitric acid R.
Solubility Source Zinc hollow-cathode lamp o
PracticalIy insoluble in water, soluble in hydrochloric acid
and in nitric acid, practicalIy insoluble in alcohol.
Wavelength 213.9 nm.
Flame Air-acetylene.
IDENTIFICATION
A. To 2 mL of solution S (see Tests) add 0.5 mL of ASSAY
potassium ferrocyanide solution R . A reddish-brown precipitate Dissolve 0.100 g in 5 mL of nitnc acid R . Heat to expel the
is formed. nitrous fumes. Add 200 mL of water R and neutralise (2.2.3)
with dilute ammonia R1. Add 1 g of ammonium chloride R and
B. To 5 mL of solution S add 0.6 mL of ammonia R. A blue
precipitate is formed. Add 2 mL of ammonia R.
3 mg of murexide R. Titrate with 0.1 M sodium edetate until
the colour changes from green ro vio let.
The precipitate disappears; the solution has an intense blue
colour. 1 mL of 0.1 M sodium edetate is equivalent ro 6.354 mg of
Cu.
TESTS __________________________________________ ~E~

Solution S
Dissolve 2.0 g in 10 mL of nitric acid R. After nitrous fumes
are no longer evolved, dilute to 60 mL with distilled water R.
Acidity or aIkalinity
Copper Acetate Monohydrate for ****
To 5.0 g add 20 mL of carbon dioxide-free water R. Boil for
** *
1 mino Cool. Filter and dilute to 25.0 mL with carbon dioxide Homoeopathic Preparations *****
free water R. To 10 mL of the solution add 0.1 mL of (Ph Bur monograph 2146)
bromothymol blue solution R1. Not more than 0.5 mL of
O. 01 M hydrochloric acid or O. 01 M sodium hydroxide is 199.7 6046-93-1
required to change the colour of the indicator. ~E~ __________________________________________

Chlorides (2.4.4) DEFINITION

Maximum 100 ppm. Content
15 mL of solution S complies with the limit test for 99 .0 per cent to 101.0 per cent of Cu(C 2H 3 0 2 )2,H 20.
chlorides. CHARACTERS
Sulfates (2.4.13) Appearance
Maximum 300 ppm. Greenish-blue crystals or green powder.
15 mL of solution S complies with the limit test for sulfates. Solubility
Iron Soluble in water, slightly soluble or very slightly soluble in
Maximum 50 ppm. ethanol (96 per cent).
Atomic absorption spectrometry (2.2.23, Method 1). IDENTIFlCATION
Test so/ution Dissolve 1.00 g in 5 mL of nitnc acid R and A. It gives reaction (a) of acetates (2.3.1).
dilute to 50.0 mL with water R. B. Dissolve 0.1 g in 10 mL of water R and add dilute
Reference solutions Prepare the reference solutions using iron ammonia R1 dropwise. A dark blue colour is produced.
standard solurion (20 ppm Fe) R, diluted as necessary with a TESTS
1 per cent VIV solution of nitric acid R.
Solution S
Source Iron holIow-cathode lampo Dissolve 3.0 g in a mixture of 40 mL of distilled water R and
Wavelength 248 .3 nm. 0.6 mL of glacial acetic acid R, with heating at 70 oC. Cool
Flame Air-acetylene. and dilute to 45 mL with distilled water R.
Lead Appearance of solution
Maximum 100 ppm . Solution S is clear (2.2.1) .
Atomic absorption spectrometry (2.2.23, Method 1) . Impurities not precipitating with hydrogen sulfide
Test solution Use the test solution prepared for the test for Maximum 0.1 per cent, calculated as sulfates.
iron. To 2.000 g add 92 mL of water R and 8.0 mL of dilute
sulfuric acid R. Heat to 70 oC. Pass a current of hydrogen
sulfide R until there is no longer precipitation of copper
IV-428 Homoeopathic Preparations
sulfide. Allow to cool and stand, then filter. Evaporate to
dryness 50.0 mL of the filtrate in a crucible. Ignite the
residue at about 600 ± 50 oC to constant mass.
Chlorides (2.4.4)
Maximum 50 ppm, determined on 15 mL of solution S.
Sulfates (2.4.13)
Maximum 150 ppm, determined on 15 mL of solution S.
Iron (2.4.9)
Maximum 20 ppm.
Dissolve 0.500 g in 10 mL of water R. Transfer to a
separating funnel. Add 20 mL of hydrochloric acid R1 and
10 mL of methyl isobutyl ketone R. Shake vigorously for
3 mino Allow to stand. Transfer the organic layer to a second
separating funnel and add 10 mL of water R. Shake
vigorously for 3 mino Allow to stand. The aqueous layer
complies with the limit test for iron.
Nickel
Maximum 10 ppm.
To the residue obtained in the test for impurities not
precipitating with hydrogen sulfide, add 2.0 mL of
hydrochloric acid R and 1.0 mL of sulfuric acid R. Evaporate to
dryness. Dissolve the residue in a mixture of 3.0 mL of dilute
sulfuric acid R and 17. O mL of water R. To 4. O mL of this
solution add 4.0 mL of water R, 5.0 mL of bromine water R,
7.0 mL of dilute ammonia R1 and 3.0 mL of a 10 gIL
solution of dimethylglyoxime R in ethanol (90 per cent VIV) R.
This solution is not more intensely coloured within 1 min
than a solution prepared as follows: mix 4.0 mL of a 1 ppm
solution of nickel (Ni) prepared from nickel standard solution
(la ppm Ni) R, 4.0 mL of water R and 5.0 mL of bromine
water R; carefully add 7.0 mL of dilute ammonia RJ and
3.0 mL of a 10 giL solution of dimethylglyoxime R in ethanol
(90 per cent VIV) R.
ASSAY
Dissolve 0.400 g in water R and dilute to 50 mL with the
same solvent. Add 6.0 mL of glacial acetic acid R, 10.0 g of
potassium iodide R and 1 mL of starch solurion R. Titrate with
O. J M sodium thiosulfate.
1 mL of 0.1 M sodium thiosulfate is equivalent to 19.97 mg of
CU(C ZH 3 0 Z)z,H zO.
____________________________________________ PhE~
Cydonia Oblonga for Homoeopathic
Preparations
DEFINITION
Cydonia Oblonga for Homoeopathic Preparations is the
seeds of Cydonia oblonga MilI.
IDENTIFICATION
The seeds are 6 to 7 mm long, reddish-brown to dark-
brown, frequently cohering by a white mucilage appearing in
fiakes on the surface and in the spaces between the seeds;
four-sided, one arched, one often distinctly ridged and two
larger and fiattened; pointed at one end, where the hilum
oeeurs as a paler spot, obtuse at the other extremity, where
the chalaza is situated. Cut transversely, the seed shows a
very narrow endosperm surrounding two yellowish-white
eotyledons.
TESTS
Total ash
Not more than 5%, Appendix XI J, Method n.
2014
MOTHER TINCTURE
The mother tincture complies with the requirements stated under
Mother Tinctures for Homoeopathic Preparations and with the
following requirements.
PRODUCTION
The mother tineture of Cydonia oblonga Mili. is prepared
from the powdered drug using Method 1.1.8 deseribed in the
monograph for Methods of Preparation of Homoeopathie
Stoeks and Potentisation. Use glycerol.
CHARACTERISTICS
The mother tineture is a pale yellow, clear or slightly turbid
viseous liquido
IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Dilute 5 mL of the mother tineture with 5 mL of water,
mix thoroughly and transfer the diluted tineture to a
cartridge eontaining octadecyl-bonded silica sorbent (a Sep-pak
C18 eartridge is suitable) previously washed with 10 mL of
methanol followed by 10 mL of water. Wash the eartridge
with 15 mL of water and elute with 10 mL of methanol.
Evaporate the eluant to dryness using a rotary evaporator.
Dissolve the residue in 0.5 mL of methanol.
(2) 0.1 % w/v of hyperoside, 0.1 % w/v of rutin and 0.01% w/v
of scopoletin in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the eoating silica gel 60 F254 .
(b) Use the mobile phase as deseribed below.
(e) Apply 40 ¡.tL of solution (1) and 10 ¡.tL of solution (2), as
12 mm bands.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and spray the plate
with a 1% w/v solution of diphenylboric acid aminoethyl ester in
methanol, and then with a 5% w/v solution of polyethylene
glycol 400 in methanol and examine under ultraviolet light
(365 nm).
MOBILE PHASE
15 volumes of anhydrous formic acid, 15 volumes of water and
70 volumes of ethyl acetate.
SYSTEM SUIT ABILITY
The test is not valid unless the ehromatogram obtained with
solution (2) shows two clearly separated orange fiuoreseent
bands and one blue fiuoreseent band at a higher Rf value.
In order of inereasing Rf value the bands are: rutin,
hyperoside and seopoletin.
CONFIRMATION
The ehromatogram obtained with solution (1) shows three
yellow fiuorescent bands in the lower third, a blue fiuoreseent
band just below the rutin standard, a blue fiuoreseent band
just below the hyperoside standard and a blue fiuoreseent
band with the same Rf value of the seopoletin standard.
Other bands may be presento
TEST
Refractive index
1.468 to 1.475, Appendix V E.
 2014 Homoeopathic Preparations IV-429

It has a peculiar and unpleasant aroma tic odour.

Top of the plate
IDENTIFICATION
A. To 2 mL of the mother tincture to be examined, add
0.2 mL of dilute sodium hydroxide solution R. A yellowish-
A blue band Scopoletin: a blue
white precipitate develops.
band
B. Thin-layer chromatography (2.2.27).
Test solution Extract 5 mL of the mother tincture to be
Hyperoside: an orange examined with 2 quantities, each of 10 mL, of ether R.
band Combine the ether layers and dry over anhydrous sodiwn
A blue band sulfate R . Filter and evaporate the filtrate in a water-bath at
low temperature. Dissolve the residue in 0.4 mL of
methanol R.
Referenee solution Dissolve 10 mg of resorcinol R, 10 mg of
Rutin: an orange band thymol R and 30 mg of gallie acid R in 10 mL of methanol R .
A blue band
Plate TLC siliea gel F 254 place R.
A yellow band
Mobile phase anhydrous formic acid R, toluene R, di-isopropyl
A yellow band ether R (10:40:50 VIV/V).
A yellow band Applieation 40!lL of the test solution and 10 !lL of the
reference solution.
Development Over a path of 10 cm.
Drying In air.
Sol ution (1) Solution (2) Detection Examine in ultraviolet light at 254 nm and
identify gallic acidi spray with anisaldehyde solurion R, heat to
105-110 oC for 5-10 mino Examine in daylight within
10 min.
Garlie for Homoeopathie ***** R esults See below the sequence of the zones present in the
* *
Preparations **** * chromatograms obtained with the reference solution and the
test solution. Other zones may also be visible in the
(Ph Eur monograph 2023)
chromatogram obtained with the test solution.
~E~ ____________________________________________

DEFINITION Top of the plate

Fresh bulb of Allium sativum L.
An intense reddish-violet zone
CHARACTERS
It has a characteristic odour after cutting. Thymol: an orange-red zone

IDENTIFICATION An intense reddish-violet zone

The bulb is generally 3 cm to 5 cm broad and almost A violet zone
sphericali the fiat base bears the remnants of numerous short A yellowish or greenish zone
greyish-brown adventitious roots. The bulb consists of about
10 daughter bulbs (cloves) arranged roughly in a circle --- - --
around a central axis. Individual daughter bulbs are 1 cm to Resorcinol: an intense orange-red
3 cm long, laterally compressed and convex on the dorsal zone
side. Each daughter bulb has a tough, white or reddish skin --- - --
around a fieshy tubular leaf, investing a more or les s rounded A violet zone
Gallic acid: a yellow zone
elongated cone of leaf primordia and vegetative apex.
(UV at 254 nm: a f1uorescent A greenish-yellow zone
TESTS quenching zone)
Water (2.2.13) A violet zone may be present
Minimum 55.0 per cent, determined on 10.0 g ofthe finely
Reference solution Test solution
cut drug, if performed to demonstrate the freshness of the
drug.
TESTS
MOTHER TINCTURE Relative density (2.2.5)
The mother tincture complies with the requirements of the 0.885 to 0.960.
general monograph on Mother tinctures for homoeopathie Ethanol (2.9. JO)
preparations (2029). 50 per cent VIV to 70 per cent V/V.
PRODUCTION Dry residue (2.8.16)
The mother tincture of Allium sativum L. is prepared by Minimum 4.0 per cent.
maceration of the cut drug using alcohol of a suitable
STORAGE
concentration.
In an airtight container.
CHARACTERS ____________________________________________ ~E~

Appearance
Brownish-yellow liquido
IV-430 Homoeopathic Preparations 2014

Results See below the sequence of the zones present in the

Hedera Helix for Homoeopathic chromatograms obtained with the reference solution and the
Preparations test solution. Other faint zones may also be present in the
(Ph Eur monograph 2092) chromatogram obtained with the test solution.
PhE~ ____________________________________________

DEFINITION Top of the plate

Fresh, young, fully developed but not yet lignified branch of ----- -----
Hedera he/ix L., harvested immediately before or at the a·Hederin: a violet zone A violet zone
beginning of flowering. (a·hederin)

IDENTIFICATION Hederacoside e: a brown zone A brown zone

(hederacoside C)
The fresh, young branches of Hedera helix L. are thin and ----- A greyish·brown zone -----
flexible, climbing; they cling to their support by stem-roots.
Ayellow zone
The leaves are alternate, simple and petiolate. The petiole
shows a cylindrical section. The upper surface of the leaves is
glabrous and shiny, darker than the lower surface. Reference solution Test solution
The lamina is usually divided into 3-5 more or les s deeply
cut lobes on sterile branches; it is oval, with a pointed apex
on fertile branches. The inflorescences are arranged in a TESTS
simple semi-globular corymb and grouped in terminal Relative density (2.2.5)
clusters. The pedicels of the umbel are covered in whitish 0.890 10 0.925.
hairs. Each flower shows 5 small teeth formed by the upper
part of the sepals and 5 petals covered in very small inverted Ethanol (2.9.10)
hairs. 60 per cent V/V to 70 per cent V/V.
Dry residue (2.8.16)
TESTS
Minimum 2.0 per cent.
Foreign matter (2.8.2)
If required by the competent authority, maximum 5 per cent. ASSAY
Loss on drying (2.2.32) Liquid chromatography (2.2.29).
If required by the competent authority, minimum Test solution In a 20.0 mL volumetric flask, dilute 3.000 g
50 per cent, determined on 5.0 g of the finely cut drug by ofthe mother tincture to be examined to 20.0 mL with the
drying in an oven at 105 oC for 2 h. mobile phase.
Reference solution In a 50.0 mL volumetric flask, dissolve
20.0 mg of hederacoside CRin the mobile phase and dilute 10
MOTHER TINCTURE
50.0 mL with the mobile phase.
The mother tincture complies with the requirements of the Column:
general monograph on Mother tinctures for homoeopathic -- size: 1 = 0.25 m, 0 = 4 mm;
preparations (2029). -- stationary phase: octadecylsilyl silica gel for chromatography R
PRODUCTION (5 ¡.¡m) .
The mother tincture of Hedera he/ix L. is prepared by Mobile phase Mix 35 volumes of water R, adjusted 10 pH 3
maceration using ethanol of a suitable concentration. with phosphoric acid R, and 65 volumes of methanol R.
Content Flow rate 1 mUmin.
Minimum 0.15 per cent m/m of hederacoside C (Cs9H96026; Detection Spectrophotometer at 205 nm.
M r 1221). Injection 20 ¡.¡L.
CHARACTERS Retention time Hederacoside C = about 8 mino
Appearance Calculate the percentage content m/m of hederacoside C
Dark greenish-brown liquido using the following expression:
IDENTIFICATION
Thin-layer chromatography (2.2.27). Al x m2 x e x 0.4
Test solution The mother tincture to be examined. A 2 x mI
Reference solution Dissolve 1 mg of ex-hederin R and 1 mg of
hederacoside CRin methanol R and dilute to 2 mL with the area of the peak due 10 hederacoside C in the
same solvento chromatogram obtained with the test solution;
Plate TLC silica gel plate R. area of the peak due to hederacoside C in the
Mobi1e phase glacial acetic acid R, water R, butanol R chromatogram obtained with the reference solution;
(1:1:4 V/V/V). mass of the mother tincture in the test solution, in
Application 20 ¡.¡L as bands. grams;
mass of hederacoside CRin the reference solution, in
Development Over half of the plateo grams;
Drying In air. C percentage content of hederacoside C R.
Detection Spray with a 10 per cent V/V solution of sulfuric ____________________________________________ ~E~

acid R in methanol R and heat at 100-105 oC for 10 mino

Examine in daylight.
2014 Homoeopathic Preparations IV-431

***
Honey Bee for Homoeopathic * * Top of Ihe plate
** ** -- - -
Preparations ***
A pink zone
(Ph Bur monograph 2024)
~E~ __________________________________________ Leucine: a pink zone A pink zone
A pink zone
DEFINITION
Live worker honey bee (Apis mellifera L.). A pink zone
-- --
CHARACTERS
Characters described under Identification. Proline: an orange-yellow zone An orange-yellow zone

PRODUCTION 4·Aminobutanoic acid: a pink zone A pink zone

If the bee has been exposed to treatment to prevent or cure
diseases, appropriate steps are taken to ensure that the levels
of residues are as low as possible.
Reference solution Test solution
IDENTIFICATION
The body of a honey bee is about 15 mm long, black, with a
silky sheen, and covered with red hairs with a rouch of grey.
***
The broad tibiae are without spines. The posterior margins Hydrastis Canadensis for
of the segments and legs are brown, with gradual transition *** ***
ro orange-red. The claws are two-membered, the maxillary Homoeopathic Preparations ***
palps single-membered. On the hind legs are baskets or (Ph Bur monograph 2500)
scoops invested with bristles. The wings have 3 complete ~E~ __________________________________________
cubital cells, with the radial cell twice as long as it is wide;
The herbal drug complies with the requirements of the
the 3 cells on the lower margin and the 3 middle cells are
monograph Goldenseal rhizome (1831).
c1osed. A duct connects the barbed sting with the poison saco
MOTHER TINCTURE
The mother tincture complies with the requirements of the
MOTHER TINCTURE general monograph Mother tinccures for homoeopathic
The mother tincture complies with the requirements of the preparations (2029).
general monograph on Mother tinctures for homoeopathic
DEFINITION
preparations (2029).
The mother tincture is prepared from the whole or cut, dried
PRODUCTION rhizome and roots of Hydrastis canadensis L.
The mother tincture of Apis mellifera L. is prepared by Content:
maceration using alcohol of a suitable concentration. -- hydrastine (C 21 H 2I N0 6 ; M r 383.4): 0.10 per cent to
CHARACTERS 0.40 per cent;
Pale yellow liquid that may darken on storage. -- berberine (C2oHl SN04; M r 336.4): 0.20 per cent to
0.50 per cent.
IDENTIFICATION
Thin-layer chromatography (2.2.27). PRODUCTION
Test solution The mother tincture ro be examined. The mother tincture is prepared by the following methods
prescribed in the monograph Methods of preparation of
Reference solution Dissolve 12 mg of 4-aminobutanoic acid R,
homoeopathic stocks and potentisation (2371):
12 mg of leucine R and 12 mg of proline R in 5 mL of water R
-- Method 1.1.8, using the powdered herbal drug (710)
and dilute ro 50 mL with alcohol R.
(2.9.12) and ethanol (70 per cent V/V) [or ethanol
Plate TLC silica gel plate R. (62 per cent m/m)l ;
Mobile phase water R, ethanol R (17:63 V/V). -- Method 1.1.10, using the fragmented herbal drug (pieces
Application 20 ¡.¡L, as bands. about 1 cm in diameter), ethanol (65 per cent V/V) and
Development Over a path of 10 cm. maceration for 3-5 weeks.
Drying In airo CHARACTERS
Detection Spray with ninhydrin solution R and heat at Appearance
100-105 oC for 10 mini examine in daylight. Yellowish-brown liquido
Results See below the sequence of the zones present in the IDENTIFICATION
chromatograms obtained with the reference and test Thin-layer chromarography (2.2.27).
solutions. Other zones may also be visible. Test solution The mother tincture to be examined.
TESTS Reference solution Immediately before use, dissolve 5 mg of
Relative density (2.2.5) hydrastine hydrochloride R and 5 mg of berberine chloride R in
0.890 ro 0.910. 10 mL of methanol R.
E thanol (2.9. JO) Plate TLC silica gel plate R (5-40 ¡.¡m) [or TLC silica gel
60 per cent V/V to 70 per cent VIV. plate R (2-10 ¡.¡m)] .
Dry residue (2.8.16) Mobile phase anhydrous formic acid R, water R, ethyl acetate R
Minimum 0.30 per cent. (10:10:80 VIV/V).
__________________________________________ PhE~
Application 20 ~lL [or 5 ¡.¡Ll as bands.
Development Over a path of 15 cm [or 6 cm] .
 IV-432 Homoeopathic Preparations 2014

Drying In air. Al x m2 x p
- : - - ---=-- X 0.905
Detection Examine in ultraviolet light at 365 nm.
A 2 x mI x 5
Results See below the sequence of fiuorescent zones present
in the chromatograms obtained with the reference solution area of the peak due ro hydrastine or ro berberine in
and the test solution. Furthermore, other faint fiuorescent the chromatogram obtained with the test solution;
zones may be present in the chromatogram obtained with the area of the peak due to hydrastine or ro berberine in
test solution. the chromatogram obtained with the reference
solution;
mass of the mother tincture to be examined used to
Top of the plate prepare the test solution, in grams;
--- --- m2 mas s of hydrastine hydrochloride CRS or mass of
berberine chloride CRS used to prepare the reference
Berberine : a bright yellow A bright yellow fluorescent zone
fluorescent zone (berberine) solution, in grams;
Hydrastine: a deep blue fluorescent A deep blue fluorescent zone p percentage content of hydrastine hydrochloride CRS
zone (hydrastine) or percentage content of berberine chloride CRS.
- -- - -- ___ _ _ _ __ __ __ __ _ __ _ __ _ ___ ~Ew

Referenee solution Test solution

TESTS
Hyoscyamus for Homoeopathic ***
Relative density (2.2.5)
*** ***
0.890 ro 0.905, where Method 1.1.8 is used. Preparations ***
Ethanol (2.9.10) (Ph Eur monograph 2091)
60 per cent VIV ro 70 per cent VIV, where Method 1.1.10 is ~Ew ____ _ _ __ _ _ _ __ _ __ __ _ __ _ __
used.
Dry residue (2.8.16) DEFINITION
Minimum 1.2 per cent mlm. Whole, fresh fiowering plant of Hyoscyamus niger L.

ASSAY IDENTIFICATION
Liquid chromarography (2.2.29). Hyoscyamus is an annual or biennial plant, with a well
developed taproot. The robust, erect stem is hollow and
Test solution Dilute about 1.000 g, accurately weighed, of
subcylindrical and up ro 80 cm long. The soft, viscid, dull
the mother tincture to be examined to 20.0 mL with the
dark-green lea ves are densely pubescent on both surfaces,
mobile phase.
especially on the veins. The lower leaves are petiolate and are
Reference solution Immediately before use, dissolve 10.0 mg arranged in a rosette; the lower cauline leaves are semi-
of hydrastine hydrochloride CRS and 10.0 mg of berberine amplexicaul and the upper ones are completely amplexicaul.
chloride CRS in methanol R and dilute to 100.0 mL with the The lamina, up ro 25 cm long, is oblong to ovate with 2 ro 5
same solvent. broadly dentate lobes on each side. The midrib is well
Column: developed. The secondary veins arise at a wide angle from
-- size: 1 = 0.125 m, (2) = 4 mm; the midrib and terminate in the apices of the lobes.
-- stationary phase: end-capped octadecylsilyl silica gel for The fiowering tops are densely pubescent and form a short
chromatography R (5 ¡un). drooping cluster. Each fiower arises in the axils of a large
Mobile phase Dissolve 9.93 g of potassium dihydrogen bract. The gamosepalous calyx is covered with dense cotton-
phosphate R in 730 mL of water R, add 270 mL of like hairs and has 5 triangular-ovate lobes, each ending in a
acetonitrile R and mix. short point that becomes spiny. The gamopetalous corolla,
Flow rate 1.2 mUmin. with 5 nearly equal lobes, is yellowish and with a delicate,
Detection Spectrophorometer at 235 nm. brown to blackish-violet venation. The fruit, sometimes
present at the base of the infiorescences, is a pyxis distinctly
lny'ection 10 ¡.tL.
swollen at the base.
Elution order Hydrastine, berberine.
TESTS
ldentification of peaks Use the chromatogram obtained with
Foreign matter (2.8.2)
the reference solution to identify the peaks due to hydrastine
If required by the competent authority, maximum 5 per cent.
and berberine.
System suitability Reference solution: Loss on drying (2.2.32)
If required by the competent authority, minimum
-- resolution: minimum 1.5 between the peaks due ro
hydrastine and berberine. 50 per cent, determined on 5.0 g of the finely cut drug by
drying in an oven at 105 oC for 2 h .
Calculate the percentage contents mlm of hydrastine using
the following expression: Hyoscyamus albus L.
The presence of middle and upper leaves with a petiole and
of fruits barely swollen at the base indicates adulteration by
Al x m2 x p
- : - - -- -=-- X 0.913 Hyoscyamus albus L.
A 2 x mI x 5

Calculate the percentage contents mlm of berberine using the

following expression:
 2014 Homoeopathic Preparations IV-433

MOTHER TINCTURE TESTS

The mother tincture complies with the requirements of the
general monograph on Mother tinctures for homoeopathic
preparations (2029).
PRODUCTION
The mother tincture of Hyoscyamus niger L. is prepared by
maceration of the drug, using ethanol of a suitable
concentration.
Content
0.002 per cent m/m to 0.01 per cent m/m of total alkaloids,
expressed as hyoscyamine (C 17H Z3 N0 3; M r 289.4) .
CHARACTERS
Appearance
Dark greenish-brown liquid.
IDENTIFICATION
Thin-Iayer chromatography (2.2.27).
Test solution Evaporate 10 mL of the mother tincture to be
examined in a water-bath at 40 oC, under reduced pressure.
Take up the residue with 1 mL of ammonia R, and shake
with 2 quantities, each of 10 mL, of ether R . Combine the
ether layers, dry over anhydrous sodium sulfate R and filter.
Evaporate on a water-bath and dissolve the residue in
0.50 mL of methanol R .
Reference solution (a) Dissolve 50 mg of hyoscyamine
sulfate R in 10 mL of methanol R (solution A). Dissolve
15 mg of hyoscine hydrobromide R in 10 mL of methanol R
(solution B). Mix 4 mL of solution A and 2 mL of
solution B and dilute to 10 mL with methanol R .
Reference solution (b) Dissolve 20 mg of atropine sulfate R in
methanol R and dilute to 10 mL with the same solvent.
Plate TLC si/iea gel plate R.
Mobile phase coneentrated ammonia R, water R, aeetone R
(3:7:90 V/V/V).
Applieation 20 ~lL, as bands.
Development Over a path of 10 cm.
Drying At 100-105 oC for 15 mino
Detection A Spray with dilute potassium iodobismuthate
solution R until orange zones become visible. Examine in
daylight.
Results ASee below the sequence of the zones present in
the chromatograms obtained with the reference solutions and
the test solution. Other faint zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
Hyascine: an arange An arange zane
zane (hyascine)
--- - --
Relative density (2.2.5)
0.930 to 0.9 60.
Atropine
Examine the chromatograms obtained in the test for
identification.
Results The zone due to hyoscyamine in the chromatogram
obtained with the test solution changes from orange to
reddish-brown but not to greyish-blue (atropine).
Ethanol (2.9.10)
40 per cent V/V to 50 per cent V/V.
Dry residue (2.8.16)
Minimum 1.2 per cent.
ASSAY
Evaporate 100.0 g of the mother tincture to be examined, at
a low temperature under reduced pressure, until a residue of
about 10 gis obtained. Quantitatively transfer the residue to
a separating funnel using a few millilitres of ethanol
(70 per cent V/V? R. Add 5 mL of coneentrated ammonia R
and 25 mL of water R . Extract wirh successive fractions of a
mixture of 1 volume of ehloroform R and 3 volumes of
peroxide-free ether R until the alkaloids are completely
extracted. Evaporate to dryness a few millilitres of the last
organic fraction. Take up the residue in 0.25 M sulfurie acid
and verifY rhe absence of alkaloids using potassium
tetraiodomereurate solution R. Combine the organic layers and
extract several times with 0.25 M sulfmie acid. Separate the
layers by centrifugation if necessary and transfer the acid
layers to a second separating funnel. Make the acid layer
alkaline with ammonia R and shake with at least 3 quantities,
each of 30 mL, of ehloroform R . Combine the chloroform
layers, add 4 g of anhydrous sodium sulfate R and allow to
stand for 30 min with occasional shaking. Decant the
chloroform and wash the anhydrous sodium sulfate with
3 quantities, each of 10 mL, of chloroform R . Combine the
chloroform fractions, evaporate to dryness on a water-bath
and dry in an oven at 100-105 oC for 15 mino Dissolve the
residue in a few millilitres of chloroform R, add 10.0 mL of
0.005 M sulfurie acid and remove the chloroform by
evaporation on a water-bath. Titrate the excess of acid with
O. 01 M sodium hydroxide using methyl red mixed solution R as
indicator.
Calculate the percentage content m/m of total alkaloids,
expressed as hyoscyamine, from the expression:
0.2894 (10 - n)
m
n volume of 0.01 M sodium hydroxide used, in millilitres,
m mas s of the mother tincture used, in grams.
--- _ _ _ __ _ _ __ _ __ _ __ _ _ _ _ _ _ _ _ ~Ew

Hyascyamine: an Atropine: an arange A arange zane (hyascy·

orange zane zone amine/atropine)

--- --- - --
Faint arange zanes
(!ine ef aDDlicatian)
Reference solution (a) Reference solution (b) Test solution

Detection B Subsequently spray with sodium nitrite solution R

until the yellow background disappears. Examine in daylight
after 15 mino
Results B See test for atropine.
IV-434 Homoeopathic Preparations 2014

*** Detection Spray with a 10 gIL solution of diphenylboric acid

Hypericum tor Homoeopathic * *
** ** aminoethyl ester R in methanol R and then a 50 gIL solution of
Preparations *** macrogol 400 R in methanol R. Examine the plates after
(Ph Eur monograph 2028) 30 min in ultraviolet light at 365 nm.
PhE~ ____________________________________________ Results See below the sequence of the zones present in the
chroma1Ograms obtained with the reference solution and the
DEFINITION test solution. In the chromatogram obtained with the test
Whole, fresh plant of Hypericum perforatum L., at the solution, the zone due to rutin may be weak or even absent.
beginning of the flowering periodo The chromatogram obtained with the test solution shows a
IDENTIFICATION group of zones that may be blue or yellow, with a RF similar
The perennial plant consists of a spindle-shaped root and a to that of the zone due to hyperoside in the chroma1Ogram
branched rhizome, giving rise 10 long, decumbent runners. obtained with the reference solution. Other weak zones may
The cylindrical, erect stem is woody at the base, 0.2 m to also be visible.
1 m long, branched in the upper part, with 2 raised
longitudinallines. Top of the plate
The leaves are opposite, sessile, exstipulate, oblong-oval and A yellow to blue zone
15 mm to 30 mm long. The leaf margins show black
Hypericin: a red zone 2 red zones
glandular dots, and many small translucent oil glands are
present on the entire surface and are visible by transmitted ----- -----
light. Several zones
The flowers are regular and form corymbose clusters at the
----- -----
apex of the stem. They have 5 green, lanceolate sepals with
acuminate apices, and black oil glands near the entire Hyperoside: a yellow to orange zone Blue or yellow zones
margins; 5 orange-yellow petals, much longer than the sepals,
with black oil glands near the terminal margins only;
Rutin: a yellow to orange zone A yellow to orange zone
3 staminal blades, each divided into many orange-yellow
stamens and 3 carpels surmounted by red styles. Each petal Reference solution Test solution
is asymmetrically linear-ovate in shape, with one of the
margin entire and the other denta te.
TESTS
TESTS
Re1ative density (2.2. 5)
Foreign matter (2.8.2)
0.900 to 0.920 .
Maximum 4 per cent of fruits and maximum 1 per cent of
other foreign matter. Ethanol (2.9.10)
60 per cent V/V to 75 per cent V/V.
Loss on drying (2.2.32)
If performed to demonstrate the freshness of the drug, Dry residue (2.8.16)
mínimum 55 per cent, determined on 5.0 g offinely cut drug Minimum 1.3 per cent.
by drying in an oven at 105 oc. ____________________________________________ ~E~

MOTHER TINCTURE
The mother tincture complies with the requirements of the ***
Iron tor Homoeopathic
general monograph on Mother tinctures for homoeopathic
*** ***
preparations (2029) . Preparations ***
PRODUCTION Iron for Homoeopathic Use
The mother tincture of Hypericum perforatum L. is prepared (Ph Eur monograph 2026)
by maceration using alcohol of a suitable concentration.
Fe 55.85 7439-89-6
CHARACTERS PhE~ ____________________________________________
Dark red 10 brownish red liquid.
DEFINITION
IDENTIFICATION
Obtained by reduction or sublimation as a fine blackish-grey
Thin-Iayer chromatography (2.2.27).
powder.
Test solution The mother tincture to be examined.
Content
Reference solution Dissolve 5 mg of rutin R, 1 mg of 97.5 per cent to 101.0 per cent.
hypericin R and 5 mg of hyperoside R in methanol R and dilute
10 5 mL with the same solvent. CHARACTERS
Plate TLC silica gel plate R. Appearance
Fine, blackish-grey powder, without metallic lustre.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
(6:9:90 VIV/V). Solubility
Practically insoluble in water and in alcohol. It dissolves with
Application 1O ~L of the test solution and 5 ~L of the
heating in dilute mineral acids.
reference solution, as 10 mm bands.
Development Over a path of 10 cm. IDENTIFICATION
Dissolve 50 mg in 2 mL of dilute sulfuric acid R and dilute 10
Drying At 100-105 oC for 10 mino
10 mL with water R . The solution gives reaction (a) of iron
(2.3.1).
2014
TESTS
Solution S
To 10.0 g add 40 mL of water R. Boil for 1 mino Cool, filter
and dilute to 50.0 mL with water R.
A1kalinity
To 10 mL of solution S add 0.1 mL of bromothymol blue
solution Rl. Not more than 0.1 mL of 0.01 M hydrochloric
acid is required to change the colour of the indicator to
yellow.
Substances insoluble in hydrochloric acid
Dissolve 2.00 g in 40 mL of hydrochloric acid R. Heat on a
water-bath. As soon as fumes are no longer evolved, filter
through a sintered-glass filter (16) (2.1.2). Rinse with
water R. Dry the residue in an oven at 100-105 oC for 1 h.
The residue weighs a maximum of 20 mg (1.0 per cent).
Substances soluble in water
Evaporate 10.0 mL of solution S on a water-bath and dry at
100-105 oC for 1 h. The residue weighs a maximum of 2 mg
(0.1 per cent).
Chlorides (2.4.4)
Maximum 50 ppm.
Dilute 5 mL of solution S 10 15 mL with water R.
The solution complies with the limit test for chlorides.
Sulfides and phosphides
In a 100 mL conical fiask carefully mix 1.0 g with 10 mL of
dilute hydrochloric acid R. Within 30 s lead acetate paper R
moistened with water R and placed over the mouth of the
fiask is not coloured more intensely than light brown by the
resulting fumes.
Arsenic (2.4.2)
Maximum 5 ppm.
Boil 0.2 g in 25 mL of dilute hydrochloric acid R until
completely dissolved. The solution complies with limit test A.
Copper
Maximum 50 ppm.
Atomic absorption spectrometry (2.2.23, Method J).
Test solurion Dissolve 1.00 g in a mixture of 60 mL of dllute
hydrochloric acid R and 10 mL of dilute hydrogen peroxide
solution R. Reduce to a volume of 5 mL and dilute to
50.0 mL with water R.
Reference solutions Prepare the reference solutions using
copper standard solution (0.1 per cent Cu) R, diluted as
necessary with a 1 per cent VIV solution of hydrochloric
acid R.
Source Copper hollow-cathode lampo
Wavelength 324.8 nm.
Flame Air-acetylene .
Lead
Maximum 50 ppm.
Atomic absorption spectrometry (2.2.23, Method J).
Test solurion In a separating funnel, place 20 mL of the test
solution prepared for the test for copper. Add 25 mL of lead-
free hydrochloric acid R. Stir with 3 quantities, each of 25 mL,
of di-isopropyl ether R. Collect the aqueous layer. Add 0.10 g
of sodium sulfate decahydrate R. Evaporate to dryness. Take
up the residue with 1 mL of lead-free nitric acid R and dilute
10 20 mL with water R.
Reference solutions Prepare the reference solutions using lead
standard solurion (0.1 per cent Pb) R, diluted as necessary with
a 10 per cent VIV solution of nitric acid R containing 5 giL of
sodium sulfate decahydrate R.
Source Lead hollow-cathode lampo
Homoeopathic Preparations IV-435
Wavelength 217 nm.
Flame Air-acetylene.
ASSAY
Stir for 10 min 0.100 g in a hot solution of 1.25 g of copper
sulfate R in 20 mL of water R in a 100 mL conical fiask with
a ground-glass s1Opper. Filter rapidly and wash the filter.
Combine the filtrate and the washings, acidifY with dilure
sulfuric acid R and titrate with 0.02 M potassium permanganate
until a pink colour is obtained.
1 mL of 0.02 M potassium permanganate is equivalent 10
5.585 mg of Fe.
LABELLING
The label indica tes whether the iron for homoeopathic
preparations is obtained by reduction or sublimation.
__________________________________________ ~Ew
Hydrated Iron(lII) Phosphate for
Homoeopathic Preparations
FeP0 4 ,4H2 0 222.8 10045-86-0
(anhydrous)
DEFINITlON
Hydrated Iron(m) Phosphate for Homoeopathic Preparations
contains hydrated iron(m) phosphate. It contains not less
than 96.0% and not more than 106.5% ofFeP0 4 ,4H 2 0.
CHARACTERISTlCS
A yellow to pale ochre powder.
Insoluble in water; soluble in dilute mineral acids.
IDENTIFICATlON
Dissolve 0.5 g of the substance being examined in 5 mL of
dilute hydrochloric acid, with warming. Dilute the resulting
solution to 35 mL with water and filter if necessary (solution
S).
A. Solution S yields reactions B and C characteristic of iron
and iron salts, Appendix VI.
B. Solution S yields reaction B characteristic of phosphates,
Appendix VI.
TESTS
Clarity of solution
Solution S is clear, Appendix IV A, Method n.
Chloride
To 0.05 g ofthe substance being examined add 1 mL of
dilure nitric acid. Heat, dilute with 14 mL of water and filter.
The filtrate complies with the limit test for chlorides,
Appendix VII (0.1 %).
Heavy metals
Dissolve 1.0 g of the substance being examined in 20 mL of
hydrochloric acid if necessary with heating. Extract the solution
using five 20-rnL quantities of a mixture of 100 mL of
freshly distilled methyl isobutyl ketone and 1 mL of hydrochloric
acid R1. A1low to stand, separate the aqueous layer and
evaporate to half its volume, allow to cool and dilute to
35 mL with water. Neutralise 7.5 mL of this solution to
litmus paper using dilure ammonia Rl and dilute 10 15 mL
with water. 12 mL of the resulting solution complies with
limit test heavy metals, Appendix VII, Method A (70 ppm).
Use lead standard solution (1 ppm Pb) 10 prepare the standard.
 IV-436 Homoeopathic Preparations
Loss on drying
When dried to constant weight at 200°, loses not less than
28% and not more than 33% of its weight, Appendix IX D .
Use 1 g.
ASSAY
Dissolve 0.45 g in 3 mL of hydrochloric acid RI in an iodine
fiask, add 10 mL of water and 6.0 g of potassium iodide, close
the fiask and allow to stand protected from light for
30 minutes. Add 100 mL of water and 1 mL of starch solution
and titrate with O.IMsodium thiosulfate VS. Each mL of O.I M
sodium thiosulfate VS is equivalent to 22.29 mg of
FeP0 4,4H2 0 .
PRODUCTION OF STOCK
The first decimal trituration of Hydrated Iron(m) Phosphate
for Homoeopathic Preparations is prepared using a suitable
quantity of Lactose or Anhydrous Lactose as the vehicle and
a validated trituration method that ensures homogeneity is
achieved. The vehicle complies with the statement under
Vehicles in the monograph for Homoeopathic Preparations.
Content ofhydrated iron(ili) phosphate FeP0 4 , 4H2 0
The first decimal trituration contains 9.0% to 11.0% of
FeP0 4 , 4H zO.
CHARACTERlSTICS
The first decimal trituration is a yellowish powder.
IDENTIFICATION
Dissolve, with warming, 1.5 g of the first decimal trituration
in a mixture of 1.5 mL of dilute hydrochloric acid and 9 mL of
water (solution SI).
A. Solution SI yields reactions B and C characteristic of iron
and iron salts, Appendix VI.
B. Solution SI yields reaction B characteristic of phosphates,
Appendix VI.
C. Dissolve 0.25 g of the substance being examined in 5 mL
of water. Add 5 mL of ammonia and heat in a water-bath at
80° for 10 minutes. A red colour develops.
ASSAY
Dissolve 4.0 g of the first decimal trituration in 3 mL of
hydrochloric acid RI in an iodine fiask, add 10 mL of water
and 8.0 g of potassium iodide, close the fiask and allow to
stand protected from light for 30 minutes. Add 100 mL of
water and 1 mL of starch solution and titrate with O.IM sodium
thiosulfate VS. Each mL of 0.1 Msodium thiosulfate VS is
equivalent to 22.29 mg of FeP0 4,4H zO.
STORAGE
Hydrated Iron(Ill) Phosphate for Homoeopathic Preparations
should be protected from light.
Hydrated Iron(lI) and Iron(lII) Phosphate
for Homoeopathic Preparations
DEFINITION
Hydrated Iron(n) and lron(m) Phosphate for Homoeopathic
Preparations contains a mixture of hydrated iron(n)
phosphate and iron(m) phosphate and sorne hydrated oxides
of iron. It contains not less than 16.0% of Fe 2 +, equivalent to
not less than 47.9% ofFe3(P04h,8HzO.
CHARACTERlSTICS
A slate blue amorphous powder.
Insoluble in water; soluble in hydrochloric acid.
2014
IDENTIFICATION
Dissolve 0.5 g in 5 mL of dilute hydrochloric acid with
warming. Dilute the resulting solution to 35 mL with water
and filter if necessary (solution S) .
A. Solution S yields the reactions characteristic of iron and
iron salts, Appendix VI.
B. Solution S yields the reactions characteristic of phosphates,
Appendix VI.
TESTS
Heavy metals
Dissolve 1.0 g of the substance being examined in 20 mL of
hydrochloric acid. Extract the solution using five 20-mL
quantities of a mixture of 100 mL of freshly distilled methyl
isobutyl ketone with 1 mL of hydrochloric acid RI. Allow to
stand, separate the aqueous layer and evaporate to half its
volume, allow to cool and dilute to 35 mL with water.
N eutralise 7.5 mL of this solution to litmus paper using dilute
ammonia RI and dilute to 15 mL with water. 12 mL of the
resulting solution complies with limit test A jor heavy metals,
Appendix VII (70 ppm). Use lead standard solution
(1 ppm Pb) to prepare the standard.
Sulfates
Dissolve 0.25 g of the substance being examined in water.
Add 3 mL of dilute hydrochloric acid and dilute to 15 mL with
water. The resulting solution complies with the limit test jor
sulfates, Appendix VII.
ASSAY
Dissolve 0.3 g ofthe substance being examined in 3 mL of
orthophosphoric acid and 10 mL of a 14% v/v solution of
sulfuric acid in water. Add 100 mL of water and titrate with
0.1 M potassium permanganate. Each mL of 0.1 M potassium
permanganate VS is equivalent to 27.925 mg of Fe 2 +.
PRODUCTION OF STOCK
The first decimal trituration of Hydrated Iron(n) and Iron(m)
Phosphate for Homoeopathic Preparations is prepared using
a suitable quantity of Lactose or Anhydrous Lactose as the
vehicle and a validated trituration method that ensures
homogeneity is achieved. The vehicle complies with the
statement under Vehicles in the monograph for
Homoeopathic Preparations.
Content ofhydrated iron(ii) and iron(iii) phosphate
The first decimal trituration contains 4.5 % to 5.0% of
Fe3(P04)z, 8H 2 0.
CHARACTERlSTICS
The first decimal trituration is a light grey powder.
IDENTIFICATION
A. Yields the reactions characteristic of iron and iron salts,
Appendix VI.
B. Yields the reactions characteristic of phosphates,
Appendix VI.
C. Dissolve 0.25 g in 5 mL of water. Add 5 mL of ammonia
and heat in a water-bath at 80° for 10 minutes. A red colour
develops.
ASSAY
Dissolve 3.0 g of the first decimal trituration in 3 mL of
orthophosphoric acid and 10 mL of a 14% v/v solution of
sulfuric acid in water. Add 100 mL of water and titrate with
O.IM potassium permanganate. Each mL of O.IM potassium
permanganate VS is equivalent to 27.925 mg of Fe 2 +.
STORAGE
Hydrated Iron(n) and Iron(m) Phosphate for Homoeopathic
Preparations should be protected from light.
 2014 Homoeopathic Preparations IV-437

and a red band between the bands obtained for formononetin

Medicago Sativa for Homoeopathic and coumarin in solution (2). Other bands may be present in
Preparations the chromatogram obtained with solution (1).
DEFINITION Ultraviolef lighf (365 nm)
Medicago Sativa for Homoeopathic Preparations is the fresh Top of the plate
whole fiowering plant of Medicago sativa L.
IDENTIFICATION
Plant A herbaceous perennial, reaching up to 100 cm.
Leaves Alternate, petiolate, trifoliolate, leafiets mucronate,
approximately 2 cm long, 1 cm broad, typically oblanceolate
or oblong, with a toothed or entire margin, glabrous on the
upper surface and sparsely pubescent on the lower surface;
stipules lanceolate, up to 1 cm long, toothed to entire.
Coumarin : a turquoise blue
Stems 4-angled, branching, glabrous to pubescent. fluorescent band
Flowers Compact, axillary, racemes of up to 40 fiowers,
Red fluorescent band
peduncle up to 3 cm long, typically pubescent; corolla
papilionaceous, up to 1 cm long, 5 mm broad, purple to
whitish; calyx tube approximately 5 mm in length, 2 mm in One or two blue
fluorescent bands Formononetin : a turquoise
diameter, 5-lobed, typically glabrous, lobes equal or
blue fluorescent band with
subequal, up to 4 mm long. slight tailing
MOTHER TINCTURE Blue fluorescent band

The nlOther tincture complies with the requirements stated under Blue or pink fluorescent
band
Mother TinClures for Homoeopathic Preparations and with the
folÚJwing requirements. Blue or pink fluorescent
band
PRODUCTION
Solution (1) Solution (2)
The mother tincture of Medicago sativa L. is prepared from
the herbal drug using method 1.1.5 described in the The chromatogram obtained with solution (1) sprayed with
monograph for Methods of Preparation of Homoeopathic the anisaldehyde solution shows the following fiuorescent
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol. bands: a faint blue band close to the origin, followed by a
CHARACTERISTICS faint purple or blue f1uorescent band and a blue band below
The mother tincture is a greenish-brown liquid. the band obtained for formononetin in solution (2), followed
by three yellow to orange bands between the bands obtained
IDENTIFICATION for formononetin and coumarin in solution (2) and an orange
Carry out the method for thin-layer chl"Omatography, band between the band obtained for coumarin and the
Appendix III A, using the following solutions. solvent front. Other bands may be present in the
(1) The mother tincture. chromatogram obtained with solution (1) .
(2) 0.1 % w/v coumarin BPCRS and 0.05 % w/v of
Anisaldehyde solution spray and ultraviolef lighf (365 nm)
formononetin BPCRS in methanol.
Top of the plate
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel 60 F 254 (Merck silica gel 60
precoated plates are suitable).
Orange fluorescent
(b) Use the mobile phase described below. band
(c) Apply 10 ¡lL of each solution as 3 mm bands.
(d) Deve10p the plate to 15 cm.
(e) After removal of the plate dry in air and examine under
ultra-violet light (365 nm).
(f) Spray the plate with anisaldehyde solution and heat at 105° Coumarin : a faint indigo
for 5 minutes and examine under ultra-violet light (365 nm) . blue fluorescent band
MOBILE PHASE Yellow orange
fluorescent band
10 volumes of methanol and 90 volumes of toluene.
Faint orange
SYSTEM SUITABILITY fluorescent band
The test is not valid unless, in the chromatogram obtained
Formononetin : a yellow
with solution (2), two clearly separated bands are observed Orange fluorescent fluorescent band
under ultra-violet light (3 65 nm) before and after spraying band
with anisaldehyde solution.
CONFIRMATION Blue fluorescent band
Under ultra-violet light, the chromatogram obtained with Faint purple or blue
fluorescent band
solution (1) shows the following fiuorescent bands: one blue
or pink fiuorescent band close to the origin, followed by a Faint blue band
blue or pink fiuorescent band and then a blue band below the Solution (1) Solution (2)
band obtained for formononetin in solution (2), followed by
one or two blue bands approximately level with formononetin
IV-438 Homoeopathic Preparations 2014

CHARACTERISTICS the dark for 10 mino Add 150 mL of water R. Titrate with
The mother tineture is a greenish-brown liquid. 0.1 M sodium chiosulfate until the eolour ehanges from blue to
green, adding 1 mL of starch solution R near the end of the
TESTS
titration.
Ethanol
55% to 65% w/w (63% to 72% v/v), Appendix VIII F. 1 mL of 0.1 M sodium thiosulfate is equivalent to 4.903 mg of
K 2 Cr2 0 7·
Dry residue __________________________________________ ~E~

Not less than 1.0% w/w, Appendix XI P.

Reladve density
0.880 to 0.950, Appendix V G.

Prunus Spinosa Fruit for Homoeopathic

Preparations
Potassium Dichromate for *****
** ** DEFINITION
Homoeopathic Preparations *** Prunus Spinosa Fruit for Homoeopathie Preparations is the
(Ph Eur monograph 2501) fresh ripe fruit of Prunus spinosa L.
294.2 7778-50-9 IDENTIFICATION
~E~ __________________________________________ The ripe fruit is nearly a globose drupe, 8 to 15 mm in
diameter, bluish-blaek with a blue-grey bloom. The dense
DEFINITION green pulp surrounds a hard, spherical to ovoid and fiattened
Content stone, 6 to 9 mm long, 6 to 8 mm wide and 4 to 6 mm
99.0 per eent to 101.0 per eent ofKzCrZ07. thick. The stone bulges slight1y and has a sharp edge.
CHARACTERS
Appearance MOTHER TINCTURE
Orange erystals.
The mother tincture complies with che requirements stated under
Solubility Mother Tinctures for Homoeopathic Preparations and with che
Freely soluble in water, praetieally insoluble in ethanol following requirements.
(96 per eent) .
PRODUCTION
IDENTIFICATION The mother tineture of Prunus spinosa L. is prepared from
A. It gives reaetion (b) of potassium (2.3.1). the herbal drug using Method 1.1.7 deseribed in the
B. Dissolve 10 mg in 5 mL of water R . Add 0.25 mL of monograph for Methods of Preparation of Homoeopathie
dilute sulfuric acid R, 0.5 mL of strong hydrogen peroxide Stoeks and Potentisation. Use 43% w/w (50% v/v) of ethanol.
solution R and 1 mL of ether R. Shake. The upper layer is
CHARACTERISTICS
blue.
The mother tineture is a purple-red to red, clear to slight1y
TESTS turbid liquid.
Soludon S1
IDENTIFICATION
Dissolve 5.0 g in distilled water R and dilute to 50.0 mL with
the same solvent. Carry out the method for thin-layer chromatography,
Appendix !II A, using the following solutions.
Soludon S2
(1) Centrifuge 10 mL of the mother tineture at about 3000
To 20.0 mL of solution SI add 20 mL of hydrochloric acid R
rpm for 5 minutes. Preeondition a eartridge eontaining
and 50 mL of tributyl phosphate R . Stir for 2 minoRemove
oetadeeyl-bonded siliea sorbent (a Sep-pak C18 eartridge is
the lower layer and shake it with 10 mL of ether R . Evaporate
suitable) with 10 mL of methanol followed by 10 mL of
the lower layer to dryness under redueed pressure. Dissolve
water. Apply 4.5 mL of the clear supernatant to the eolumn,
the residue in 10 mL of discilled water R. Add dilute
wash with 5 mL water and elute with 5 mL of methanol.
ammonia R1 until the solution is neutral to blue litmus paper R
Evaporate me eluant on a rotary evaporator at 50°. Dissolve
and dilute to 20.0 mL with distilled water R .
the residue in 1 mL of methanol.
Appearance of solution
(2) 0.025 % w/v of chlorogenic acid and 0.005% w/v of
Solution SI is clear (2.2.1).
scopoletin in methanol.
Calcium (2.4.3)
CHROMATOGRAPHIC CONDITIONS
Maximum 500 ppm.
(a) Use as the eoating silica gel F254 .
Dilute 2.0 mL of solution S2 to 15 mL with distilled water R.
(b) Use the mobile phase deseribed below.
Chlorides (2.4.4)
Maximum 50 ppm. (e) Apply 40 ¡.tL of eaeh solution as 12 mm bands.
Dissolve 1.0 g in 15 mL of dilute nitric acid R. Use 1 mL of (d) Develop the plate to 15 cm.
nitric acid R instead of the preseribed dilute nitric acid R. (e) After removal of the plate, dry it in air and spray with a
1% w/v solution of diphenylboric acid aminoethyl ester in
Sulfates (2.4.13)
methanol, followed by a 5% w/v solution of polyethylene glycol
Maximum 150 ppm.
400 in methanol and examine under ultraviolet light (365 nm) .
Dilute 10 mL of solution S2 to 15 mL with distilled water R.
MOBILE PHASE
ASSAY
15 volumes of anhydrous formic acid, 15 volumes of water and
Dissolve 0.100 g in 25 mL of water R. Add 2 g of potassium
70 volumes of ethyl acetare.
iodide R and 25 mL of dilute sulfuric acid R . Allow to stand in
2014 Homoeopathic Preparations IV-439

SYSTEM SUIT ABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows a yellow-green fiuorescent band
(chlorogenic acid) in the middle third of the plate and a blue
fiuorescent band (scopoletin) in the upper third of the plateo
CONFIRMATION
The chromatogram obtained with solution (1) shows at least
one orange fiuorescent band below chlorogenic acid, one
yellow-green fiuorescent band with the Rf of chlorogenic acid
and one orange fiuorescent band below scopoletin.
In addition the following bands may be present: a blue
fiuorescent band with the same Rf as scopoletin and an
orange fiuorescent band with an Rf slightly higher than that
of scopoletin. Two or more orange fiuorescent bands may be
present between the two standards.
Top of the plate
Scopoletin: a blue band
Orange band
A yellow-green band Chlorogenic acid: a
yellow-green band
Orange band
Solution (1) Solution (2)
TESTS
Ethanol
24 to 34% w/w (29.3 to 40.8% v/v), Appendix VIII F.
Dry residue
Not les s than 3.5% w/w, Appendix XI P.
Relative density
0.955 to 0.995, Appendix V G.
Common Stinging Nettle for
Homoeopathic Preparations
(Ph Eur monograph 2030)
ffiEw ____________________________________________
DEFINITION
Whole, fresh, fiowering plant of Urtica dioica L.
CHARACTERS
The plant causes an itching, burning sensation on the skin.
IDENTIFICAnON
A. Common stinging nettle is perennial. The taproot sends
out creeping subterranean rhizomes, more or les s 4-angled in
transverse section, from which extend adventious secondary
roots and very numerous brownish hairy rootlets. The stipes
are erect, generally unbranched, 3-5 mm in diameter and
0.3-1.5 m high, rarely up to 2.5 m high, 4-angled, greyish-
green and covered in short hairs and stinging hairs.
The decussate leaves are 30-150 mm long and 20-80 mm
wide. The petiole is hispid and usually slightly les s than one-
third the length of the lamina. The leaf blade is ova te,
acuminate, corda te or rounded at the base, and coarsely
dentate; the apical tooth is distinctly larger than the lateral
teeth. The upper side of the leaves is dark green and usually
matt, both sides bear short serried hairs intermingled with
long stinging hairs. The 2 stipules are linear-subulate and
free. The infiorescences growing from the leafaxils are
complex, the fiowers unisexual, and, particularIy in male
plants, generally distinctly longer than the petiole. After
shedding their pollen, male infiorescences are erect at an
oblique angle or horizontal; female infiorescences are pendent
when the fruit is ripe. All fiowers have long stalks.
The perianth of the male fiowers is divided half-way down
into equal green lobes, widest at their base, with short bristIes
and stinging hairs at the margins. The stamens are equal and
opposite to the perianth segments, each with a long, whitish
filament that curves inwards before pollen is shed and
spreads out afterwards. The ovary is rudimentary, button or
cup-shaped. The perianth of the female fiowers is downy or
bristIy on the outside and consists of outer, and 2 inner
segments; the inner segments are about twice the length of
the outer ones. The hypogynous, ova te, unilocular ovary
bears a large capitate stigma with a brush-like shock of hair.
As the one-seeded fruit grows ripe, the 2 inner segments of
the perianth fold around it like wings.
B. It complies with the test for Urtica urens (se e Tests).
TESTS
Urtica urens
The margin of the lamina is not serrate with teeth twice as
long as wide. The clusters of fiowers in the axils are longer
than the petiole of the leaf. Unisexual, apetalous fiowers are
not together on the same plant and in the same cluster.
Foreign marter (2.8.2)
Maxirnum 5 per cent.
Loss on drying (2.2.32)
Minimum 65.0 per cent, deterrnined on 5.0 g of finely cut
drug by drying in an oven at 105 oC for 2 h, if performed to
demonstrate the freshness of the drug.
MOTHER TINCTURE
The mother rincture complies with the requirements of the
general monograph on Mother tinctures for homoeopathic
preparations (2029).
PRODUCTION
The mother rincture of Urtica dioica L. is prepared by
maceration using alcohol of a suitable concentration.
CHARACTERS
Appearance
Greenish-brown or orange-brown liquido
IDENTIFICATION
Thin-Iayer chromatography (2.2.27) .
Test solution The mother tincture to be examined.
Reference solution Dissolve 10 mg of phenylalanine R and
10 mg of serine R in a mixture of equal volumes of
methanol R and water R and dilure ro 10 mL with the same
mixture of solvents.
Plate TLC silica gel plate R.
 IV-440 Homoeopathic Preparations 2014

Mobile phase glacial acetic acid R, water R, acetone R, vascular bundles with small spirally thickened ves seis and no
butanol R (10:20:35:35 VIVIVIV) . fibres.
Application 20 ¡.tL, as bands. C. Carefully crush pieces of the drug to coarse particles and
Development Over a path of 10 cm. moisten with 0.2 mL of phosphomolybdic acid solution R .
Drying In air. The particles mm blue within 1-2 min or they have a blue
areole around them.
Detection Spray with a 1 gIL solution of ninhydrin R in
alcohol R. Heat the plate at 105-110 DC for 5-10 min then D . Examine by thin-layer chromatography (2.2.21).
examine in daylight within 10 mino Test solution Carefully crush 0.1 g of the drug with a glass
Results See below the sequence of the zones present in the rod and moisten with 0.2 mL of water R . After 3 min add
chromatograms obtained with the reference solution and the 5 mL of methanol R, allow to stand for 20 min, protected
test solution. from light, and filter through a plug of glass wool.
Reference solution Dissolve 5 mg of naphthol yellow R in
5 mL of methanol R and add a solution of 5 mg of Sudan red
Top of the plate G R in 5 roL of methylene chlonde R.
--- --- Plate TLC si/ica gel F254 plate R.
Phenylalanine: a violet to Mobtle phase water R, 2-propanol R, ethyl acetate R
reddish·brown zone (10:25 :65 VIVIV).
4 red to viole! zones
Application 10 ¡.tL of the test solution and 5 ¡.tL of the
--- --- reference solution as bands .
Serine: a reddish·violet zone A viole! zone Development Over a path of 10 cm.
A viole! zone Drying In airo
Reference solution Test solution Detection Examine in daylight.
Results See below the sequence of the zones present in the
chromatograms obtained with the reference and test
TESTS solutions.
Relative density (2.2.5)
0.930 to 0.950 .
Top of the plate
Ethanol (2.9. 10)
A red zone
40 per cent VIV to 56 per cent VIV.
Methanol (2.9.11) Ayellow zone
Maximum 0.10 per cent VIV. 2 yellow zones
Dry residue (2.8. 16) An intense yellow zone (crocine)
Minimum 1.1 per cent.
Reference solution Test solution
_ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _________ PhEm

Detection In ultraviolet light at 254 nm.

Saffron for Homoeopathic *** Results See below the sequence of the zones present in the
*** *** chromatograms obtained with the reference and test
Preparations *** solutions.
Saffron for Homoeopathic Use
(Ph Bur monograph 1624) Top of the plate
PhEm ___________ _ __ ___ ___________ _ ___
A red zone 1 or 2 quenching zones
DEFINITION Ayellow zone A quenching zone
Dried stigmas of Crocus sativus L. usually joined by the base
to a short style. Reference solution Test solu!ion

CHARACTERS
Characteristic, aromatic odour. Detection Spray with anisaldehyde solution R and examine in
daylight while heating at 100-105 oC for 5-10 mino
IDENTIFICATION
A. The dark brick-red stigmas, when dry, are 20 mm to Results See below the sequence of the zones present in the
40 mm long and after soaking with water, about 35 mm to chromatograms obtained with the reference and test
50 mm long. The tubes, gradually widening at the top, are solutions.
incised on one side, the upper margin is open and finely
crenated. The style connecting the 3 stigmas is pale yellow Top of the plate
and not more than 5 mm long.
A red zone 1 or 2 red to reddish·violet zones
B. Examine under a microscope using chloral hydrate
solution R. Ir shows the following diagnostic characters: A blue to bluish·green zone A red to reddish·vioJet zone
elongated epidermal cells, frequently with a short, central 2 blue to bluish·green zones
papilla; in water they release a yellow colouring matter;
An intense bJue to bJuish-green
the upper border of the stigma has finger-shaped papillae, up zone (crocine)
to 150 !lm long; berween them are single, globular pollen Reference solution Test solution
grains, about 100 !lm wide, with a finely pitted exine,
2014 Homoeopathic Preparations IV-441

E. Dilute 0.1 mL of the test solution (see Identification test Nitrates

D) with 1 mL of methanol R. Deposit 0.1 mL of this solution
on a filter paper, allow to dry and spray with a 10 giL
solution of diphenylborie acid aminoethyl ester R in methanol R .
Examine in ultraviolet light at 365 nm. The spot shows an
intense orange-yellow fluorescence.
TESTS
Colouring intensity
Introduce 0.10 g into a 5 mL volumetric flask and add to
5.0 mL with distilled water R. Close the flask and shake every
30 min for 8 h . Then allow to stand for 16 h. Dilute 1.0 mL
ro 500.0 mL with distilled water R. The absorbance (2.2.25)
measured at 440 nm using distilled water R as the
compensation liquid, is not less than 0.44.
Foreign matter
Examine the drug microscopically. No parts with rough
walls, no crystals and no pollen grains containing 3 germinal
pores are presento
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 0.200 g by drying in
an oven at 105 oc.
Total ash (2.4.16)
Maximum 7.0 per cent, determined on the residue obtained
in the test for los s on drying.
__________________________________________ ~E~
***
Sodium Tetrachloroaurate * *
**
Dihydrate for Homoeopathic ****
Preparations
(Ph Eur monograph 2141)
Maximum 200 ppm.
Dissolve 0.20 g in 10 mL of nitrate-free water R. Add 0.2 g of
oxalie acid R . Heat the solution on a water-bath for 30 min,
allow to cool and filter. Rinse the filter with nitrate-free
water R and dilute the filtra te ro 20 mL with the same
solvent. To 1.0 mL of the solution obtained add 4.0 mL of
nitrate-free water R, 0.4 mL of a 100 gIL solution of potassium
ehloride R, 0.1 mL of diphenyfamine sofution R and, dropwise
with shaking, 5 mL of nitrogen-free sulfurie acid R. Transfer
the tube ro a water-bath at 50 oc. After 15 min, any blue
colour in the solution is not more intense than that in a
reference solution prepared at the same time in the same
manner using a mixture of 0.2 mL of nitra te standard sofution
(10 ppm NOy R and 4.8 mL of nitrate-free water R .
Heavy metals (2.4.8)
Maximum 100 ppm.
Dissolve 0.20 g in 15 mL of water R. Add 0.25 g of hydrazine
sulfate R. Heat the solution on a water-bath for 30 min, allow
to cool and filter. Rinse the filter with water R and dilute the
filtra te to 20 mL with the same solvent. 12 mL of the
solution complies with test A. Prepare the reference solution
using fead standard sofution (1 ppm Pb) R.
ASSAY
Dissolve 40.0 mg in 10 mL of potassium iodide sofution R .
Allow to stand for 5 minoTitrate with 0.01 M sodium
thiosulfate until decolourised. Shortly before reaching the
endpoint, add 0.5 mL of stareh sofution R .
1 mL of 0.01 M sodium thiosulfate is equivalent to 1.989 mg
* of Na [AuCl 4 J,2H2 0.
STORAGE
In an airtight container, protected from light.
Ph Eur

Na[AuCI4l,2H 20 397.8
~E~ __________________________________________

DEFINITlON ***
Sulfur for Homoeopathic
Sodium tetrachloroaurate( 1-) dihydrate. **
**** **
*
Content Preparations
97 .0 per cent to 101.0 per cent ofNa[AuCI 4l,2H2 0 . (Ph Eur monograph 2515)
CHARACTERS S 32.07 7704-34-9
Appearance ~E~ __________________________________________
Orange-yellow, hygroscopic powder or crystals.
DEFINITION
Solubility Obtained by sublimation.
Very soluble or freely soluble in water and in ethanol
(96 per cent). Content
99.0 per cent to 101.0 per cent.
IDENTIFICATlON
A. Dissolve 20 mg in 2.0 mL of 0. 1 M nitrie aeid. Add 0.1 g CHARACTERS
of oxalie aeid R and boil in a water-bath for 1 h. A deposit of Appearance
metallic gold is formed. Yellow powder.
B. Solution S (se e Tests) gives reaction (a) of chlorides Solubility
(2.3.1). Practically insoluble in water, soluble in carbon disulfide,
C. Solution S gives reaction (b) of sodium (2.3.1). slightly soluble in vegetable oils.
mp
TESTS
About 120 oc.
Solution S
Ignite 0.20 g in a porcelain crucible at 600 oC ± 50 cC for IDENTIFICATlON
30 minoAllow to cool and extract with 3 mL of water R, A. Heated in the presence of air, it burns with a blue flame,
heating if necessary. Use the supernatant. emining sulfur dioxide, which changes the colour of
Free hydrochIoric acid moistened bfue litmus paper R to red.
When a glass rod impregnated with eoneentrated ammonia R is E. Heat 0.1 g with 0.5 mL of bromine water R until
held close to the substance to be examined, no white fumes decolourised. Add 5 mL of water R and filter. The solution
are produced. gives reaction (a) of sulfates (2.3.1).
IV-442 Homoeopathic Preparations
TESTS
Solution S
To 5.0 g add 50 mL of carbon dioxide-free water R prepared
from disti/led water R. Allow to stand for 30 min with
frequent shaking and filter.
Appearance of solution
Solution S is colourless (2.2.2, Method ll).
Odour (2.3.4)
It has no perceptible odour of hydrogen sulfide.
Acidity or aIkalinity
To 5 mL of solution S add 0.1 mL of phenolphthalein
soluNon R1 . The solution is colourless. Add 0.2 mL of
0.01 M sodium hydroxide. The solution is red. Add 0.3 mL of
o. 01 M hydrochloric acid. The solution is colourless.
Add 0.15 mL of methyl red solution R. The solution is orange-
red.
Chlorides (2.4.4)
Maximum 100 ppm.
Dilute 5 mL of solution S to 15 mL with water R.
Sulfates (2.4.13)
Maximum 100 ppm, determined on solution S.
Sulfides
To 10 mL of solution S add 2 mL of buffer solution pH 3.5 R
and 1 mL of a freshly prepared 1.6 gIL solution of lead
nitra te R in carbon dioxide-free water R. Shake. After 1 min
any colour in the solution is not more intense than that in a
reference solution prepared at the same time using 1 mL of
lead standard solution (IO ppm Pb) R, 9 mL of carbon dioxide-
free water R, 2 mL of buffer solution pH 3.5 R and 1.2 mL of
thioacetamide reagent R.
Arsenic (2.4.2, Method B)
Maximum 8 ppm.
Shake 2.5 g with 50 mL of dilute ammonia Rl for 1 h and
filter. Evaporate 25 mL of the filtrate to dryness . Add 2 mL
of water R and 3 mL of nitric acid R to the residue and
evaporate to dryness. The residue complies with the test.
Prepare the standard using 1 mL of arsenic standard solution
(IO ppm As) R.
Sulfated ash (2.4.14)
Maximum 0.2 per cent, determined on 1.0 g.
ASSAY
Carry out the oxygen-fiask method (2.5.10), using 60.0 mg
in a 1000 mL combustion fiask with a tefion joint. Absorb
the combustion products in a mixture of 5 mL of dz1ute
hydrogen peroxide solution R and 10 mL of water R . Heat to
boiling, boil gently for 2 min and coo!. Using 0.2 mL of
phenolphthalein solution R as indicator, titrate with 0.1 M
sodium hydroxide until the colour changes from colourless to
red. Carry out a blank titration under the same conditions.
1 mL of 0.1 M sodium hydroxide is equivalent to 1.603 mg of
S.
STORAGE
Protected from light.
__________________________________________ PhE~
2014
Symphytum Officinale Root for
Homoeopathic Preparations
DEFINITION
Symphytum Officinale Root for Homoeopathic Preparations
is the fresh root of Symphytum officinale L.
IDENTIFICATION
The strong, spirally-formed rootstock with a smooth, black-
brown cortex, subdivides at the base. It is about 5 to 8 cm in
diameter and 17 to 30 cm long, surmounted by several tops,
close together, consisting of the black residues of the previous
year's rosettes of leaves between the current year's new
growths. A ring of lOto 15 horizontally-running, secondary,
glabrous smooth roots grows from the base of the rosettes,
the roots often reaching a length of more than 50 cm and a
diameter of approximately 1.5 cm. The middle portion of the
rootstock, which is more or less glabrous, branches at the
lower end into several clearly separated, straight downwards
pointing, smooth roots, each about 1.5 cm thick and bearing
a few secondary rootlets 1 to 2 cm long.
The rootstock and the thick secondary roots are non-
lignified, succulent and break off easily. In the yellowish-
white, glassy, slightly differentiated cross section, there is a
very thin, black rhizodermis, and an easily detachable layer of
cortex, 5-8 mm thick. Within this is a single dark pigmented
vascular ringo The cut surface and particularly the richly
exuding mucilagenous substance tum yellow to red-brown on
exposure.
TESTS
Foreign matter
Not more than 2%, Appendix XI D.
MOTHER TINCTURE
The mother tincture complies with the requirements stated under
Mother Tinctures for Homoeopathic PreparaNons and with the
following requirements.
PRODUCTION
The mother tincture of Symphytum officinale L., is prepared
from the herbal drug using Method 1.1.3 described in the
monograph for Methods of Preparation of Homoeopathic
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol.
CHARACTERISTICS
The mother tincture is a brown liquido
IDENTIFICATION
Carry out the method for thin-layer ehromatography,
Appendix III A, using the following solutions.
(1) The mother tincture.
(2) 0.1% w/v of allantoin in ethanol (45 %).
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating si/iea gel.
(b) Use the mobile phase as described below.
(c) Apply 20 ¡.tL of solution (1) and 10 ¡.tL of solution (2) as
10 mm bands.
(d) Develop the plate to 10 cm.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (365 nm).
(f) Spray the plate with a 5% w/v solution of
dimethylaminobenzaldehyde in hydroehlorie acid and examine in
daylight.
 2014
MOBILE PHASE
10 volumes of anhydrous formic acid, 10 volumes of water and
80 volumes of ethyl aceta te.
SYSTEM SUITABILITY
The test is not valid unless under ultraviolet light (365 nm)
the chromatogram of solution (1) shows two bluish
fiuorescent bands at Rf 0.25 and Rf 0.55 and one light-green
fiuorescent band at the solvent front.
CONFIRMATION
After spraying, the chromatogram obtained with solution (2)
shows the yellow allantoin band at Rf 0.35.
The chromatogram obtained with solution (1) shows one
yellow band with the same Rf of 0.35 as that for allantoin.
TESTS
Ethanol
35 to 45% w/w (42 to 53% v/v), Appendix VIII F .
Dry residue
Not less than 1.5% w/w, Appendix XI P.
Relative density
0.920 to 0.970, Appendix V G.
Symphytum Officinale Root for Ethanol
Decoction for Homoeopathic
Preparations
DEFINITION
Symphytum Officinale Root for Ethanol Decoction for
Homoeopathic Preparations is the fresh root of Symphytum
officinale L.
IDENTIFICATION
The strong, spirally-formed rootstock with a smooth, black-
brown cortex, subdivides at the base. It is about 5 to 8 cm in
diameter and 17 to 30 cm long, surmounted by several tops,
close together, consisting of the black residues of the previous
year's rosettes of leaves between the current year's new
growths. A ring of lOto 15 horizontally-running, secondary,
glabrous smooth roots, grows from the base of the rosettes,
the roots often reaching a length of more than 50 cm and a
diameter of approximately 1.5 cm. The middle portion of the
rootstock, which is more or less glabrous, branches at the
lower end into several clearly separated, straight downwards
pointing, smooth roots, each about 1.5 cm thick and bearing
a few secondary rootlets 1 to 2 cm long.
The rootstock and the thick secondary roots are non-
lignified, succulent and break off easily. In the yellowish-
white, glassy, slightly differentiated cross section, there is a
very thin, black rhizodermis, and an easily detachable layer of
cortex, 5-8 mm thick. Within this is a single dark pigmented
vascular ringo The cut surface and particularly the richly
exuding mucilagenous substance turn yellow to red-brown on
exposure.
MOTHER TINCTURE
The mother tincture c01nplies with the requirements stated under
Mother Tinctures for H omoeopathic Preparations and with the
following requirements.
PRODUCTION
The mother tincture of Symphytum officinale L. is prepared
by decoction of the herbal drug using 43% w/w (50% v/v) of
ethanol.
Homoeopathic Preparations IV-443
CHARACTERISTICS
The mother tincture is a brown liquido
IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) The mother tincture.
(2) 0.1 % w/v of allantoin in ethanol (45%).
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating si/iea gel.
(b) Use the mobile phase as described below.
(e) Apply 20 ¡.¡L ofsolution (1) and 10 ¡.¡L ofsolution (2) as
10 mm bands.
(d) Develop the plate 10 10 cm.
(e) After removal of the plate, dry it in air and examine
under ultraviolet light (365 nm).
(f) Spray the plate with a 5% w/v solution of
dimethylaminobenzaldehyde in hydrochloric acid and examine in
daylight.
MOBILE PHASE
10 volumes of anhydrous formic acid, 10 volumes of water and
80 volumes of ethyl acetate.
SYSTEM SUITABILITY
The test is not valid unless under ultraviolet light (365 nm),
the chromatogram obtained with solution (1) shows two
bluish fiuorescent bands at Rf value of 0.25 and Rf value of
0.55 and one light-green fiuorescent band at the solvent
front.
CONFIRMATION
After spraying, the chromatogram obtained with solution (1)
shows a yellow band at Rf value of 0.35 corresponding in
position and colour to that obtained with solution (2) .
TESTS
Ethanol
25 to 35% w/w (3 1 to 42% v/v), Appendix VIII F.
Dry residue
Not les s than 3.0% w/w, Appendix XI P.
Relative density
0.973 to 0.982, Appendix V G.
Toxicodendron Quercifolium for
Homoeopathic Preparations
Rhus Toxicodendron for Homoeopathic Preparations
DEFINITION
Toxicodendron Quercifolium for Homoeopathic Preparations
is the fresh, young, not yet lignified shoots, with leaves, of
Toxicodendron quercifolium (Michx.) Greene.
CAUTION The shoots contain a yellowish-white mi/ky sap that
is a strong cutaneous irritant and darkens the skin. Contact with
the skin and mucous membranes is to be avoided.
IDENTIFICATION
Shoots The thin shoots have a downy to cottony
indumentum and may bear at their ends, and in the axils of
the alternate leaves, pointed buds covered in brown, woolly
hairs.
Leaves Dark green on the upper surface, lighter green on the
lower surface and have scattered hairs, more numerous on
the veins; they are tripi=ate with petioles 15 to 20 cm long.
 IV-444 Homoeopathic Preparations 2014

The leaflets are ovate to slightly rhombic and of varying size, Top of the plate
the middle leaflet being the largest, up to 20 cm long and
11 cm wide with long petiolule; the two lateralleaflets are A reddish-brown band Gallic acid: a reddish-brown
smaller, up 10 16 cm long and 9 cm wide with short A reddish-brown band band
petiolules. The margins of the laminae may be entire or
broadly dentate with up to 3 or more short triangular lobes
on each side, particularly in the apical region of the middle 2 or 3 closely positioned
leaflets; on the lateralleaflets the margin is frequently reddish-brown bands
asymmetrical with the lobes on one side only; all the leaflets
are cuneate at the base and acute at the apex.
Arbutin: a reddish-brown
MOTHER TINCTURE band

The mother tincture complies with the requirements stated under

Mother Tinctures for Homoeopathic Preparations and with the
following requirements.
DEFINITION
It contains not les s than 0.1 % w/w of total flavonoids
expressed as quercitrin (C21H20011)'
PRODUCTION
The mother tincture of Toxicodendron quercijolium (Michx.)
Greene is prepared from the herbal drug using Method 1.1.3
described in the monograph for Methods of Preparation of
Homoeopathic S10cks and Potentisation. Use 86% w/w
(90% v/v) of ethanol.
CHARACTERISTICS
The mother tincture is a yellowish-brown to reddish-brown
liquid.
IDENTIFICATION
A. To 1 mL of the mother tincture add 1 granule of zinc, a
few tumings of magnesium and 1 mL of hydrochloric acid.
A dark red colour is produced which can be extracted with
tert -pentyl alcohol.
B. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Use the mother tincture.
(2) Dissolve 20 mg of arbutin and 10 mg of gallic acid in
10 mL of methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel.
(b) Use the mobile phase as described below.
(e) Apply 10 ~LL of each solution as bands.
(d) Develop the plate 10 10 cm.
(e) Remove the plate, dry in air, spray with a 0.5% w/v
solution of fast blue B salt in water, dry briefty and spray with
0.1 M alcoholic sodium hydroxide. Examine in daylight.
MOBILE PHASE
10 volumes of anhydrous formic acid, 10 volumes of water and
80 volumes of ethyl aceta te.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two well separated bands.
CONFIRMATION
The chroma1Ogram obtained with solution (1) shows the
following reddish-brown bands: two or three bands, which lie
close 10gether at a position between those obtained for
arbutin and gallic acid in solution (2) and two further
pronounced bands at about the same leve! as that obtained
for gallic acid in solution (2). Other bands may be present in
the chromatogram obtained with solution (1).
Solution (1) Solutionj2)
TESTS
Ethanol
36% 10 46% w/w (43% to 54% v/v), Appendix VIII F.
Dry residue
Not less than 3.5% w/w, Appendix Xl P.
Relative density
0.945 10 0.965, Appendix V G.
Urushiols
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
(1) Evaporate 10.00 g of the mother tincture to dryness using
a rotary evapora1Or at a temperature not exceeding 40°.
Add 10 mL water 10 the residue and mix with the aid of
ultrasound for 20 minutes. Add 10 mL of heptane and shake
vigorously for 15 minutes. Allow to separate, remove and
retain the heptane layer avoiding any suspended particles.
Perform the extraction twice more on the aqueous phase,
then discard the remaining aqueous layer and rinse the flask
with 10 mL of heptane. Combine the extracts and washings
and filter through anhydrous sodium sulfate and evaporate the
filtrate to dryness using a rotary evapora1Or at a temperature
not exceeding 40°. Dissolve the residue in 2.0 mL of
methanol.
(2) To 0.5 mL of a 0.175% w/v solution of 4-dodecylresorcinol
in methanol add 0.5 mL of a solution containing 0.1 % w/v
each of urushiol 1 and urushiol II in methanol and dilute to
5 mL with methanol.
(3) Dilute 1 volume of solution (2) to 20 volumes with
methanol.
(4) 0.00025% w/v of 4-dodecylresorcinol in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with octadecylsz1yl silica gel for chromatography (5 ¡.un) (Waters
Symmetry C18 is suitable).
(b) Use gradient elution and the mobile phase described
below.
(e) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 30°.
(e) Use a detection wave!ength of 276 nm.
(f) Inject 20 ¡.¡L of each solution.
MOBILE PHASE
Mobile phase A 0.2% v/v orthophosphoric acid.
Mobz1e phase B methanol.
2014 Homoeopathic Preparations IV-445

Time Mobile phase Mobile phase Comment CHROMATOGRAPHIC CONDITIONS

(Minutes) A B (a) Use a stainless steel column (25 cm x 4.6 mm) packed
(%v/v) (%v/v) with octadecylsilyl siliea gel for ehromatography (5 ¡.tm) (Waters
Symmetry C18 is suitable) .
0-2 20 80 isocratic
(b) Use gradient elution and the mobile phase described
2-82 20-->0 80-->100 linear gradient
below.
82-83 0-->20 100-->80 linear gradient (c) Use a flow rate of 1 mL per minute.
83-100 20 80 re-equilibration (d) Use an ambient column temperature.
(e) Use a detection wavelength of 340 nm.
(f) Inject 20 ¡.tL of each solution.
When the chromatograms are recorded under the prescribed
conditions the relative retentions with reference to 4- MOBILE PHASE
dodecylresorcinol (retention time about 27 minutes) are Mobile phase A water adj usted to pH to 2.3 with
urushiolI about 2.0 and urushiol II about 2.4. The relative orthophosphorie acid.
retention of urushiol 1 to urushiol II is about 0.8 . Mobile phase B aeetonimle.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained Time Mobile phase A Mobile phase B Comment
with solution (2), the eolumn efficieney, determined using the (Minutes) (%v/v) (%v/v)
peak due to 4-dodecyl resorcinol, is not less than 20,000
0-2 95 5 isocratic
theoretical plates, and
2-18 95--t87 5--t13 linear gradient
in the chromatogram obtained with solution (3) the signal-to-
noise ratio of the peaks due to urushiol 1 and urushiol II is at 18-32 87--t74 13--t26 linear gradient
least 10. 32-42 74 26 isocratic

LIMITS 42-43 74--t95 26--t5 linear gradient

Calculate the percentage w/w content of urushiols in solution
(1), expressed as 4-dodecylresorcinol, using the following
expression:
A 1 X C2 X P X 1.20
A2 X Cl X 100
Al sum of the peak areas due to urushiols in the
chromatogram obtained with solution (1);
A2 area of the peak due to 4-dodecylresorcinol in the
chromatogram obtained with solution (2);
el concentration of the mother tincture sample in
solution (1) in % w/v;
e2 concentration of 4-dodecylresorcinol in solution (2)
in % w/v;
p percentage content of 4-dodecylresorcinol in 4-
dodeeylresoreinol;
1.20 average molar mass ratio of urushiol 1 and II to 4-
dodeeylresorcinol.
In the chromatogram obtained with solution (1) :
The total content of urushiols, expressed as
4-dodecylresorcinol, is not more than 0.05 %.
Disregard any peak:
with an area less than the area of the peak due to 4-dodecyl
resorcinol in solution (4) (0.00005%);
with a retention time less than 0.25 times that of the peak
due to 4-dodecylresorcinol, or with a retention time greater
than the peak due to urushiol II in the chromatogram
obtained with solution (2).
ASSAY
Carry out the method for liquid ehromatography,
Appendix III D, using the following solutions in 50 volumes
of methanol and 50 volumes of water.
(1) 10% w/v of the mother tincture.
(2) 0.018 % w/v each of isoquercitrin and quercitrin BPCRS.
(3) 0.0000018 % w/v of quercitrin BPCRS.
43-60 95 5 re-equilibration
When the chromatograms are recorded using the prescribed
conditions, the retention time of isoquercitrin is about
31 minutes and that of quercitrin is about 34 minutes, the
relative retention is about 0.9.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (2):
the symmetry factor for both isoquercitrin and quercitrin is not
less than 0.8 and not more than 1.2;
the number of theoretical plates with respect to isoquercitrin
is at least 200,000.
DETERMINATION OF CONTENT
Calculate the content of total flavonoids, expressed as
quercitrin from the chromatograms obtained and using the
declared content of C21H2001 1 in quercitrin BPCRS and the
following expression. Disregard any peak with an area less
than that in solution (3).
Sum of the peak areas due to quercitrin and
flavonoids in the chromatogram obtained with
solution (1);
area of the peak due to quercitrin in the
chromatogram obtained with solution (2);
el concentration of the mother tincture sample in
solution (1) in % w/v;
concentration of quereitrin BPCRS in solution (2) in
% w/v;
p percentage content of quercitrin in quercitrin BPCRS.
 IV-446 Homoeopathic Preparations 2014

C. Carry out the method for thin-layer chromatography,

Urtica Urens Herb for Homoeopathic
Preparations
DEFINITION
Urtica Urens Herb for Homoeopathic Preparations is the
fresh leaves and flowers of Urtica urens L.
CHARACTERISTICS
The plant produces an itchy, burning sensation.
IDENTIFICATION
Plant Annual.
Leaves Decussate with diffusely haired petiole, which in the
lower lea ves is mostly as long as the lamina; ovate to elliptic,
I to 5 cm long and 1 to 4 cm wide lamina with incised
serrated leaf margin, blunt to cuneate at the base, acuminate
towards the apex. The leaves are dark-green on the upper
surface, slightly shiny and paler green on the lower surface;
prominent stinging hairs occur scattered all over the upper
surface, on the lower surface they occur mostly over the
veins . The two stipules on each side are lanceolate and the
margins are entire.
Flowers The complicated inflorescences consist mainly of
female flowers and only a few male flowers; they arise from
the leafaxils and are about 1.5 to 2 cm long and usually
shorter than the leaf petioles. The perigonium of the male
flowers is split into four pale green lobes of equal size; each
one of the four stamens situated in front of one of the
perigonium lobes and has a long filament which at first is
incurved and then widens out before the anther releases the
pollen. The perigonium of the female flowers consists of two
outer, short, bract-like segments and two longer, inner ones,
all with ciliated margins and scattered hairs over the surfaces;
the superior ovary is ovoid with a short style and a
conspicuous, brushlike stigma. The ripe fruits are
monospermic and enclosed by the two inner segments of the
perigonium.
TESTS
Urtica dioica
The plant is dioecious as follows :
the male and female flowers occur on separate plants;
the inflorescences are longer than the leaf petioles;
the leaves, especially those on the lower part of the stem, are
longer than their petioles.
MOTHER TINCTURE
Appendix III A, using the following solutions.
(1) The mother tincture.
(2) Dissolve 10 mg of leucine and 10 mg of threonine in
10 mL of 50% v/v ethanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel G.
(b) Use the mobile phase described below.
(e) Apply 20 J.lL of each solution as bands.
(d) Develop the plate to 15 cm.
(e) After removal ofthe plate, dry in a current ofwarm air or
in an oven at 100° to 105°. Spray the chromatogram with a
0.5% w/v solution of ninhydrin in butan-I-ol. Heat for
10 minutes at 100° to 105° and examine in daylight.
MOBILE PHASE
10 volumes of glacial acetic acid, 20 volumes of water,
35 volumes of acetone and 35 volumes of butan-I-ol.
SYSTEM SUITABILITY
The test is not valid unless the chroma1Ogram obtained with
the solution (2) shows two clearly separated coloured zones
due to threonine (reddish pink) in the lower region and
leucine (pink) in the middle region.
CONFIRMATION
The chroma1Ogramobtained with solution (1 ) shows a pink
spot (which may be separated into two spots) corresponding
10 leucine. A reddish pink spot occurs in the lower region
corresponding 10 threonine. Between the spots corresponding
to threonine and leucine there is an orange pink spot and a
pink spot present, in order of increasing Rf value. Several
spots are present below the spot corresponding to threonine.
Top of the plate
A pink band (may be two Leucine: a pink band
bands)
A pink band
An orange-pink band

The mother tincture complies with the requirements stated under

Mother Tinctures for Homoeopathic Preparations and with the A reddish-pin k band Threonine: a reddish-pink
band
following requirements.
Several bands may be
PRODUCTION observed
The mother tincture of Urtica urens L., is prepared from the
herbal drug using Method 2b described in the monograph for Solution (1) Solution (2)

Methods of Preparation of Homoeopathic Stocks and

Potentisation. Use 62% w/w (70% v/v) of ethanol.
CHARACTERISTICS
TESTS
The mother tincture is a green 10 brownish-green liquid.
Ethanol
IDENTIFICATION 25% 10 35% w/w (30% to 42% v/v), Appendix VIII F.
A. To 1 mL of potassium hydroxide solution add 1 mL of the Dry residue
mother tincture and heat to boiling. Red litmus paper held Not less than 1.0% w/w, Appendix XI P.
over the mouth of the test tube turns blue.
Relative density
B. Add 1 mL of hydrochloric acid and a few crystals of 0.956 to 0.968, Appendix V G.
resorcinol 10 1 mL of the mother tincture. Heat to boiling.
A red colour is produced.
Monographs

Blood-related Products
 2014
BLOOD-RELATED PRODUCTS
Anticoagulant and Preservative
*** ***
Solutions for Blood
(Anticoagulant and Preservative Solutions for Human
Elood, Ph Eur monograph 0209)
PhE~ ____________________________________________
DEFINITlON
Anticoagulant and preservative solutions for human blood are
sterile and pyrogen-free solutions prepared with water for
injections, filtered, distributed in the final containers and
sterilised. The content of sodium citrate
(C6HsNa307,2HzO), glucose monohydrate (C6H1Z06,HzO)
or anhydrous glucose (C 6H 12 0 6) and sodium dihydrogen
phosphate dihydrate (NaH zP0 4 ,2H zO) is not less than
95 .0 per cent and not more than 105.0 per cent ofthat
stated in the formulae below. The content of citric acid
monohydrate (C 6H s0 7,HzO) or anhydrous citric acid
(C 6H s07) is not less than 90.0 per cent and not more than
110.0 per cent of that stated in the formulae below. Subject
to agreement by the competent authority, other substances,
such as red-cell preservatives, may be included in the formula
provided that their name and concentration are stated on the
labe!.
Anticoagulant and preservative solutions for human blood are
presented in airtight, tamper-proof containers of glass (3.2.1)
or plastic (3.2.3).
ANTICOAGULANT ACID-CITRATE-GLUCOSE
SOLUTlONS (ACD)
A B
Sodium ci/ra/e (0412) 22.0 g 13.2 g
Citrie aeid monohydra/e (0456) 8.0 g 4.8 g
or Citrie aeid, anhydrous (0455) 7.3 g 4.4 g
G/ueose monohydra/e (0178)' 24.5 g 14.7 g
or G/ucose, anhydrous (0177)' 22.3 g 13.4 g
Water for injeetions (0169) to 1000.0 roL 1000.0 roL
Voluroe to be used per 100 roL of blood 15.0 roL 25.0 roL
'The competent authority may require that the substances coroply with the
test for pyrogens given in the roonographs on Glucose monohydrate (0178)
and Glucose, anhydrous (0177), respectively.
CHARACTERS
A colourless or faintly yellow, clear liquid, practically free
from particles.
IDENTIFICATlON
A. Examine by thin-Iayer chromatography (2.2.27), using
silica gel G R as the coating substance.
Test solution Dilute 2 mL of the solution to be examined
(for formula A) or 3 mL (for formula B) 10 100 mL with a
mixture of 2 volumes of water R and 3 volumes of
methanol R.
Reference solution (a) Dissolve 10 mg of glucose CRS in a
mixture of 2 volumes of water R and 3 volumes of methanol R
and dilute 10 20 mL with the same mixture of solvents.
Reference solution (b) Dissolve 10 mg each of glucose CRS,
lactase CRS, fructose CRS and sucrose CRS in a mixture of
Blood-related Products IV-449
2 volumes of water R and 3 volumes of methanol R and dilute
10 20 mL with the same mixture of solvents.
Apply separately to the plate 2 ¡.tL of each solution and
thoroughly dry the points of application. Develop over a path
of 15 cm using a mixture of 10 volumes of water R ,
***
15 volumes of methanol R, 25 volumes of anhydrous acetic
acid R and 50 volumes of ethylene chloride R. The volumes of
*** solvents have to be measured accurately since a slight excess
of water produces cloudiness . Dry the plate in a current of
warm airo Repeat the development immediately, after
renewing the mobile phase. Dry the plate in a current of
warm air and spray evenly with a solution of 0.5 g of
thymol R in a mixture of 5 mL of sulfuric acid R and 95 mL
of alcohol R. Heat at 130 oC for 10 mino The principal spot
in the chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the
ehromatogram obtained with referenee solution (a) . The test
is not valid unless the ehromatogram obtained with referenee
solution (b) shows 4 clearly separated spots.
B. To 2 mL add 5 mL of cupri-citric solution R . Heat to
boiling. An orange precipitate is formed and the solution
beeomes yellow.
C. To 2 mL (for formula A) add 3 mL of water R or 10
4 mL (for formula B) add 1 mL of water R. The solution
gives the reaction of citrates (2.3. 1).
D . 0.5 mL gives reaetion (b) ofsodium (2.3.1).
TESTS
pH (2.2.3)
The pH of the solution to be examined is 4.7 to 5.3.
Hydroxymethylfurfural
To 2.0 mL add 5.0 mL of a 100 gIL solution of p-toluidine R
in 2-propanol R eontaining 10 per eent V/V of glacial acetic
acid R and 1.0 mL of a 5 giL solution of barbituric acid R.
The absorbanee (2.2.25), determined at 550 nm after
allowing the mixture to stand for 2 min to 3 min, is not
greater than that of a standard prepared at the same time in
the same manner using 2.0 mL of a solution containing
5 ppm of hydroxymethylfurfural R for formula A or 3 ppm of
hydroxymethylfurfural R for formula B.
Sterility (2. 6.1)
They comply with the test for sterility.
Pyrogens (2.6.8)
They eomply with the test for pyrogens. Dilute with a
pyrogen-free, 9 gIL solution of sodium chloride R 10 obtain a
solution eontaining approximately 5 gIL of sodium citrate.
Injeet 10 mL of the diluted solution per kilogram of the
rabbit's mass.
ASSAY
Citricacid
To 10.0 mL (for formula A) or to 20.0 mL (for formula B)
add 0.1 mL of phenolphthalein solution R1. Titrate with 0.2 M
sodium hydroxide until a pink eolour is obtained.
1 mL of 0.2 M sodium hydroxide is equivalent to 14.01 mg of
C6H807,HzO or 10 12.81 mg of C 6H s07'
Sodium citrate
Prepare a ehromatography eolumn 0.10 m long and 10 mm
in intemal diameter and filled with strongly acidic ion-exchange
resin R (300 ¡.tm 10 840 ~lm). Maintain a 1 em layer of liquid
aboye the resin at all times. Wash the column with 50 mL of
de-ionised water R at a f10w rate of 12-14 mUmin.
Dilute 10.0 mL ofthe solution 10 be examined (for formula
A) or 15.0 mL (for formula B) to about 40 mL with
de-ionised water R in a beaker and transfer 10 the eolumn
IV-450 Blood-related Products 2014

reservo ir, washing the beaker 3 times with a few millilitres of LABELLING
de-ionised water R . Allow the solution to run through the The label states:
column at a flow rate of 12-14 mUmin and callect the - the composition and volume of the solution,
eluate. Wash the column with 2 quantities, each of 30 mL, - the maximum amount of blood to be collected in the
and with one quantity of 50 mL, of de-ionised water R. container.
The column can be used for 3 successive determinations ANTICOAGULANT CITRATE-PHOSPHATE-
before regeneration with 3 times its volume of dilute
GLUCOSE SOLUTION (CPD)
hydrochloric acid R. Titrate the combined eluate and washings
(about 150 mL) with 0.2 M sodium hydroxide, using 0.1 mL
Sadium citrate (0412) 26.3 g
of phenolphthalein solution RI as indicator.
Citric acid monohydrate (0456) 3.27 g
Calculate the content of sodium citrate in grams per litre
from the following expressions: or Cilric acid, anhydrous (0455) 2.99 g

G/ucase manohydrate (0178)" 25.5 g

For formula A: 1.961n - 1.40C
or G/ucase, anhydrous (0177)' 23.2 g
or 1.961n - 1.53C' Sodium dihydrogen phosphate dihydrate (0194) 2.51 g

Water for injeclions (0169) to 1000.0 rnL

For formula B : 1.307n - 1.40C
VoIume to be used per 100 mL o( bIood 14.0mL
or 1.307n - 1.53C' "The competent au thority may require that the substances comply with the
test for pyrogens given in the monographs on G/ucose monohydrate (0178)
n number of millilitres of 0.2 M sodium hydroxide used and G/ucose, anhydrous (0177), respectively.
in the titration,
C content of citric acid monohydrate in grams per litre CHARACTERS
determined as prescribed aboye,
A colourless or faintly yellow, clear liquid, practically free
C' content of anhydrous citric acid in grams per litre
from particles.
determined as prescribed aboye.
IDENTIFICATION
Reducing sugars
A. Examine by thin-Iayer chromatography (2.2.27) , using
Dilute 5.0 mL (for formula A) or 10.0 mL (for formula B) to
silica gel G R as the coating substance.
100.0 mL with water R. Introduce 25.0 mL of the solution
into a 250 mL conical flask with ground-glass neck and add Test solution Dilute 2 mL of the solution to be examined to
25 .0 mL of cupri-citric solution R1. Add a few pieces of 100 mL with a mixture of 2 volumes of water R and
porous material, attach a reflux condenser, heat so that 3 volumes of metha/wl R.
boiling begins within 2 min and boil for exactly 10 mino Cool R eference solution (a) Dissolve 10 mg of glucose CRS in a
and add 3 g of potassium iodide R dissolved in 3 mL of mixture of 2 volumes of water R and 3 volumes of methanol R
water R. Add 25 mL of a 25 per cent m /m solution of sulfuric and dilute to 20 mL with the same mixture of solvents.
acid R with caution and in small quantities. Titrate with R eference solution (b) Dissolve 10 mg each of glucose CRS,
O. 1 M sodium thiosulfate using 0.5 mL of starch solution R, lactase CRS, fructase CRS and sucrose CRS in a mixture of
added towards the end of the titration, as indicator (ni mL). 2 volumes of water R and 3 volumes of methanol R and dilute
Carry out a blank titration using 25.0 mL of water R (n2 to 20 mL with the same mixture of solvents.
mL). Apply separately to the plate 2 J-lL of each solution and
Calculate the content of reducing sugars as anhydrous thoroughly dry the starting points. Develop over a path of
glucose or as glucose monohydrate, as appropriate, from 15 cm using a mixture of 10 volumes of water R, 15 volumes
Table 0209.-l. of methanol R , 25 volumes of anhydrous acetic acid R and
50 volumes of ethylene chloride R. The volumes of solvents
Table 0209.-1 have to be measured accurately since a slight excess of water
Volume of 0.1 M Anhydrous glucose Glucose mono-
produces cloudiness. Dry the plate in a current of warm air.
sodium thiosulfate in milligrams bydrate in Repeat the development immediately, after renewing the
(n 2 -n¡ rnL) milligrams mobile phase. Dry the plate in a current of warm air and
8 19.8 21.6 spray evenly with a solution of 0.5 g of thymol R in a mixture
of 5 mL of sulfuric acid R and 95 mL of alcohol R . Reat at
9 22.4 24.5
130 oC for 10 mino The principal spot in the chromatogram
10 25.0 27.2 obtained with the test solution is similar in position, colour
11 27.6 30.2
and size to the principal spot in the chromatogram obtained
with reference solution (a). The test is not valid unless the
12 30.3 33.1 chromatogram obtained with reference solution (b) shows 4
13 33.0 36.1 clearly separated spots.
14 35.7 39.0 B. To 2 mL add 5 mL of cupri-citric solution R. Reat to
boiling. An orange precipitate is formed and the solution
15 38.3 42.1
becomes yellow.
16 41.3 45.2 C. To 2 mL add 3 mL of water R . The solution gives the
reaction of citrates (2.3.1) .
STORAGE D. 1 mL gives reaction (b) of phosphates (2.3.1).
Store in an airtight, tamper-proof container, protected from E. 0.5 mL gives reaction (b) of sodium (2.3.1) .
light.
 2014
TESTS
pH (2.2. 3)
The pH of the solution is 5.3 to 5.9.
Hydroxyrnethylfurfural
To 2.0 mL add 5.0 mL of a 100 gIL solution of p-toluidine R
in 2-propanol R containing 10 per cent VIV of glacial acetic
acid R and 1.0 mL of a 5 gIL solution of barbituric acid R.
The absorbance (2.2.25), determined at 550 nm after
allowing the mixture to stand for 2 min to 3 min, is not
greater than that of a standard prepared at the same time in
the same manner using 2.0 mL of a solution containing
5 ppm of hydroxymethylfurfural R .
Sterility (2.6. 1)
They comply with the test for sterility.
Pyrogens (2.6.8)
They comply with the test for pyrogens. Dilute with a
pyrogen-free, 9 giL solution of sodium chloride R to obtain a
solution containing approximately 5 gIL of sodium citrate.
Inject 10 mL of the diluted solution per kilogram of the
rabbit's mass.
ASSAY
Sodium dihydrogen phosphate
Dilute 10.0 mL to 100.0 mL with water R. To 10.0 mL of
this solution add 10.0 mL of nitro-vanado-molybdic reagent R .
Mix and allow to stand at 20 oC to 25 oC for 30 mino At the
same time and in the same manner, prepare a reference
solution using 10.0 mL of a standard solution containing
0.219 g of potassium dihydrogen phosphate R per litre. Measure
the absorbance (2.2.25) of the 2 solutions at 450 nm using as
the compensation liquid a solution prepared in the same
manner using 10 mL of water R. Calculate the content of
sodium dihydrogen phosphate dihydrate (P) in grams per
litre from the expression:
e = concentration of potassium dihydrogen phosphate R in
the standard solution in grams per litre,
Al = absorbance of the test solution,
A 2 = absorbance of the reference solution.
Citric acid
To 20.0 mL add 0.1 mL of phenolphthalein solution R1 and
titrate with 0.2 M sodium hydroxide.
Calculate the content of citric acid monohydrate (C), or
anhydrous citric acid (e '), in grams per litre from the
equations:
e = 0.7005n - 0.4490P
e' = 0.6404n - 0.4105P
n number of millilitres of 0.2 M sodium hydroxide used in
the titration,
P content of sodium dihydrogen phosphate dihydrate in
grams per litre determined as prescribed aboye.
Sodium citrate
Prepare a chromatography column 0.10 m long and 10 mm
in intemal diameter and filled with strongly acidic ion-exchange
resin R (300 ¡.¡m to 840 ¡.¡m). Maintain a 1 cm layer of liquid
aboye the res in at all times. Wash the column with 50 mL of
de-ionised water R at a flow rate of 12-14 mUmin.
Blood-related Products IV-451
Dilute 10.0 mL of the solution to be examined to about
40 mL with de-ionised water R in a beaker and transfer to
the column reservoir, washing the beaker 3 times with a few
millilitres of de-ionised water R. Allow the solution to run
through the column at a flow rate of 12-14 mUmin and
collect the eluate. Wash the column with 2 quantities, each
of 30 mL, and with one quantity of 50 mL, of de-ionised
water R . The column can be used for 3 successive
determinations before regeneration with 3 times its volume of
dilute hydrochloric acid R. Titrate the combined eluate and
washings (about 150 mL) with 0.2 M sodium hydroxide, using
0.1 mL of phenolphthalein solution R1 as indicator.
Calculate the content of sodium citrate in grams per litre
from the following expressions:
1.961n - 1.257P - 1.40e
1.961n - 1.257P - 1.53e'
n number of millilitres oi 0.2 M sodium hydroxide used
in the titration,
P content of sodium dihydrogen phosphate dihydrate in
grams per litre determined as prescribed aboye,
e content of citric acid monohydrate in grams per litre
determined as prescribed aboye
e' content of anhydrous citric acid in grams per litre
determined as prescribed aboye.
Reducing sugars
Dilute 5.0 mL to 100.0 mL with water R. Introduce 25.0 mL
of the solution into a 250 mL conical flask with ground-glass
neck and add 25.0 mL of cupri-citric solution Rl . Add a few
pieces of porous material, attach a reflux condenser, heat so
that boiling begins within 2 min and boil for exactly 10 mino
Cool and add 3 g of potassium iodide R dissolved in 3 mL of
water R. Add 25 mL of a 25 per cent m/m solution of sulfuric
acid R with caution and in small quantities . Titrate with
0.1 M sodium thiosulfate using 0.5 mL of starch solution R,
added towards the end of the titration, as indicator (nI mL).
Carry out a blank titration using 25.0 mL of water R (n2
mL).
Calculate the content of reducing sugars as anhydrous
glucose or as glucose monohydrate, as appropriate, from
Table 0209 .- 1.
STORAGE
Store in an airtight, tamper-proof container, protected from
light.
LABELLING
The label states:
- the composition and volume of the solution,
- the maximum amount of blood to be collected in the
container.
__________________________________________ PhE~
 IV-452 Blood-related Products
Plasma for Fractionation
(Human Plasma for Fractionation,
Ph Eur monograph 0853)
~E~ ____________________________________________
DEFINITION
Liquid part of human blood remaining after separation of the
cellular elements from blood collected in a receptacle
containing an anticoagulant, or separated by continuous
filtration or centrifugation of anticoagulated blood in an
apheresis procedure; it is intended for the manufacture of
plasma-derived products.
PRODUCTlON
DONORS
Only a carefully selected, healthy donor who, as far as can be
ascertained after medical examination, laboratory blood tests
and a study of the donor's medical history, is free from
detectable agents of infection transmissible by plasma-derived
products may be used. Recommendations in this field are
made by the Council of Europe [Recommendation No. R (95)
15 on the preparation, use and qualily assurance of blood
components, or subsequent revision]; a directive of the
European Union also deals with the matter: Commission
Directive 20041331EC of 22 March 2004 implementing Directive
20021981EC of the European Parliament and of the Councü as
regards certain technical requirements for blood and blood
components.
Irnmunisation of donors
Irnmunisation of donors to obtain immunoglobulins with
specific activities may be carried out when sufficient supplies
of material of suitable quality caunot be obtained from
naturally immunised donors. Recommendations for such
immunisations are formulated by the World Health
Organization (Requirements for the collection, processing and
qualily control oi blood, blood components and plasma derivatives,
WHO Technical Report Series, No. 840, 1994 or subsequent
revision).
Records
Records of donors and donations made are kept in such a
way that, while maintaining the required degree of
confidentiality concerning the donor's identity, the origin of
each donation in a plasma pool and the results of the
corresponding acceptance procedures and laboratory tests
can be traced.
Laboratory tests
Laboratory tests are carried out for each donation to detect
the following viral markers :
1. antibodies against human irnmunodeficiency virus 1 (anti-
HN-l);
2. antibodies against human immunodeficiency virus 2 (anti-
HN-2);
3. hepatitis B surface antigen (HBsAg);
4. antibodies against hepatitis C virus (anti-HCV).
The test methods used are of suitable sensitivity and
specificity and comply with the regulations in force. If a
repeat-reactive result is found in any of these tests, the
donation is not accepted.
INDIVIDUAL PLASMA UNITS
The plasma is prepared by a method that removes cells and
cell debris as completely as possible. Whether prepared from
whole blood or by plasmapheresis, the plasma is separated
from the cells by a method designed to prevent the
introduction of micro-organisms. No antibacterial or
2014
antifungal agent is added to the plasma. The containers
comply with the requirements for glass containers (3.2.1) or
for plastic containers for blood and blood components
(3.2.3) . The containers are closed so as to prevent any
possibility of contamination.
If 2 or more units are pooled prior to freezing, the operations
are carried out using sterile connecring devices or under
aseptic conditions and using containers that have nor
previously been used.
When obtained by plasmapheresis or from whole blood (after
separation from cellular elements), plasma intended for the
recovery of proteins that are labile in plasma is frozen within
24 h of collection by cooling rapidly in conditions validated
to ensure that a temperature of - 25 oC or below is attained
at the core of each plasma unit within 12 h of placing in the
freezing apparatus.
When obtained by plasmapheresis, plasma intended solely for
the recovery of proteins that are nor labile in plasma is frozen
by cooling rapidly in a chamber at - 20 oC or below as soon
as possible and at the latest within 24 h of collection.
When obtained from whole blood, plasma intended solely for
the recovery of proteins that are not labile in plasma is
separated from cellular elements and frozen in a chamber at
- 20 oC or below as soon as possible and at the latest within
72 h of collection.
It is not intended that the determination of total protein and
human coagulation factor VIII shown below be cam'ed out on
each unit of plasma. They are rather given as guidelines for good
manufacturing practice, the test for human coagulation factor VIII
being relevant for plasma intended for use in the preparation of
concentrates oi labile proteins.
The total protein content of a unit of plasma depends on the serum
protein content of the donor and the degree of dilution inherent in
the donation procedure. When plasma is obtained from a suitable
donor and using the in tended proportion of anticoagulant solution,
a total protein content complying with the limit of 5OgiL is
obtained. If a volume of blood or plasma smaller than intended is
collected into the anticoagulant solution, the resulting plasma is not
necessarily unsuitable for pooling for fractionation. The aim of
good manufacturing practice must be to achieve the prescribed limÍt
for all normal donations.
Preservation of human coagulation factor VIII in the donation
depends on the collection procedure and the subsequent handling of
the blood and plasma. With good practice, O. 7 IU/mL can usually
be achieved, but units of plasma with a lower activity may still be
suitable for use in the production of coagulation factor
concentrates. The aim of good manufacturing practice is to
conserve labüe proteins as much as possible.
Total protein
Carry out the test using a pool of not fewer than 10 units.
Dilute an appropriate volume of the preparation with a 9 gIL
solution of sodium chloride R to obtain a solution containing
about 15 mg of protein in 2 mL. To 2.0 mL of this solution
in a round-bottomed centrifuge tube, add 2 mL of a 75 gIL
solution of sodium molybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
and allow the inverted tube to drain on filter paper.
Determine the nitro gen in the residue by the method of
sulfuric acid digestion (2.5.9) and calculate the protein
content by multiplying the quantity of nitrogen by 6.25 .
The total protein content is not less than 50 gIL.
 2014
Human coagulation factor VIII (2.7.4)
Carry out the test using a pool of not fewer than 10 units.
Thaw the samples to be examined, if necessary, at 37 oC.
Carry out the assay using a reference plasma calibrated
against the International Standard for human coagulation
factor VIII in plasma. The activity is not less than
0.7 IU/rnL.
STORAGE AND TRANSPORT
Frozen plasma is stored and transported in conditions
designed to rnaintain the temperarure at or below -20 oC;
for accidental reasons, the storage temperarure may rise
aboye -20 oC on one or more occasions during storage and
transport but the plasma is nevertheless considered suitable
for fractionation if aH the foHowing conditions are fulfilled:
-- the total period of time during which the temperature
exceeds - 20 oC do es not exceed 72 h;
-- the temperature does not exceed - 15 oC on more than 1
occasion;
-- the temperature at no time exceeds -5 oC.
POOLED PLASMA
During the manufacrure of plasma products, the first
homogeneous pool of plasma (for example, after removal of
cryoprecipitate) is tested for HBsAg and for HIV antibodies
using test methods of suitabJe sensitivity and specificity;
the pool must give negative results in these tests.
The plasma pool is also tested for hepatitis C virus RNA
using a validated nucleic acid amplification technique
(2.6.21). A positive control with 100 IV/mL ofhepatitis
C virus RNA and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the
positive control is non-reactive or if the result obtained with
the internal control indicates the presence of inhibitors.
The plasma pool complies with the test if it is found non-
reactive for hepatitis C virus RNA.
Hepatitis e virus RNA for NAT testing BRP is suitable for use
as a positive control.
CHARACTERS
Before freezing: clear or slightly turbid liquid without visible
signs of haemolysis; it may vary in colour from light yellow to
green.
LABELLING
The label enables each individual unit to be traced to a
specific donor.
____________________________________________ PhEw
Plasma (Pooled and Treated for ***
*** ***
Virus Inactivation) ***
(Human Plasma (Pooled and Treated for Virus
Inactivation), Ph Eur monograph 1646)
PhEw ____________________________________________
DEFINITION
Human plasma (po oled and treated for virus inactivation) is
a frozen or freeze-dried, sterile, non-pyrogenic preparation
obtained from human plasma derived from donors belonging
to the same ABO blood group. The preparation is thawed or
reconstiruted before use to give a solution for infusion.
The human plasma used complies with the monograph
Human plasma for fractionation (0853).
Blood-related Products IV-453
PRODVCTION
The units of plasma to be used are cooled to - 30 oC or
lower within 6 h of separation of cells and always within 24 h
of collection.
The pool is prepared by mixing units of plasma belonging to
the same ABO blood group.
The pool of plasma is tested for hepatitis B surface antigen
(HBsAg) and for HIV antibodies using test methods of
suitable sensitivity and specificity; the pool must give negative
results in these tests.
Hepatitis A virus RNA
The plasma pool is tested using a validated nucleic acid
amplification technique (2.6.21). A positive control with 1.0
x 102 IV of hepatitis A virus RNA per millilitre and, to test
for inhibitors, an internal control prepared by addition of a
suitable marker to a sample of the plasma pool are included
in the test. The test is invalid if the positive control is non-
reactive or if the result obtained with the internal control
indicates the presence of inhibitors. The pool complies with
the test if it is found non-reactive for hepatitis A virus RNA.
Hepatitis C virus RNA
The plasma pool is tested using a validated nucJeic acid
amplification technique (2.6.21). A positive control with 1.0
x 10 2 IV of hepatitis C virus RNA per millilitre and, to test
for inhibitors, an internal control prepared by addition of a
suitable marker to a sample of the plasma pool are incJuded
in the test. The test is invalid if the positive control is non-
reactive or if the result obtained with the internal control
indica tes the presence of inhibitors. The pool complies with
the test if it is found non-reactive for hepatitis C virus RNA.
Hepatitis e virus RNA for NAT testing BRP is suitable for use
as a positive control.
To limit the potential burden of B19 virus in plasma pools,
the plasma pool is also tested for B19 virus using a validated
nucleic acid amplification technique (2.6.21).
B19 virus DNA
The plasma pool contains not more than 10.0 IV/¡.tL.
A positive control with 10.0 IV of B19 virus DNA per
micro litre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the
positive control is non-reactive or if the result obtained with
the internal control indicates the presence of inhibitors.
B19 virus DNA for NAT testing BRP is suitable for use as a
positive control.
The method of preparation is designed to minimise activation
of any coagulation factor (to minimise potential
thrombogenicity) and incJudes a step or steps that have been
shown to inactivate known agents of infection; if substances
are used for the inactivation of viruses during production, the
subsequent purification procedure must be validated to
demonstrate that the concentration of these substances is
reduced to a suitable leve! and that any residues are such as
not to compromise the safety of the preparation for patients.
Inactivation process
The solvent-detergent process, which is one of the methods
used to inactivate enveloped viruses, uses treatrnent with a
combination of tributyl phosphate and octoxinol 10; these
reagents are subsequently removed by oi! extraction or by
solid phase extraction so that the amount in the final product
is les s than 2 ¡.tg/mL for tributyl phosphate and less than
5 ¡.tg/mL for octoxinol 10.
No antimicrobial preservative is added.
IV-454 Blood-related Products
The solution is passed through a bacteria-retentive filter,
distributed aseptically into the final containers and
immediately frozen; it may subsequently be freeze-dried.
Plastic containers comply with the requirements for sterile
plastic containers for human blood and blood components
(3.2.3).
Glass containers comply with the requirements for glass
containers for pharmaceutical use (3.2.1).
CHARACTERS
Frozen preparation: clear or slightly opalescent liquid, free
from solid and gelatinous particles after thawing.
Freeze-dried preparation: almost white or slightly yellow
powder or friable solido
Thaw or reconstitute the preparatian to be examined as stated on
the label immediately before canying out the identification, tests
and assay.
IDENfIFICATION
A. Examine by electrophoresis (2.2.31) comparing with
normal human plasma. The electropherograms show the
same bands.
B. It complies with the test for anti-A and anti-B
haemagglutinins (see Tests).
TESTS
pH (2.2.3)
6.5 to 7.6.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
Minimum 45 gIL.
Dilute if necessary with a 9 gIL solution of sodium chloride R
to obtain a protein concentration of about 7.5 mglmL. Place
2.0 mL of this solution in a round-bottomed centrifuge tube
and add 2 mL of a 75 giL solution of sodium molybdate R and
2 mL of a mixture of 1 volume of nürogen-free sulfuric acid R
and 30 volumes of water R. Shake, centrifuge for 5 min,
decant the supematant and allow the inverted tube to drain
on filter papero Determine the nitrogen in the residue by the
method of sulfuric acid digestion (2.5.9) and calculate the
quantity of protein by multiplying the result by 6.25.
Activated coagulation factors (2.6.22)
It complies with the test for activated coagulation factors.
Carry out the test with 0.1 mL of the preparation to be
examined instead of 10-fold and 100-fold dilutions .
The coagulation time for the preparation to be examined is
not les s than 150 S.
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
The presence of haemagglutinins (anti-A or anti-B)
corresponds to the blood group stated on the labe!'
Hepatitis A virus antibodies
Minimum 1.0 IU/mL, determined by a suitable
immunochemical method (2.7.1).
Human hepatitis A immunoglobulin BRP is suitable for use as a
reference preparation.
Irregular erythrocyte antibodies
The preparation to be examined do es not show the presence
of irregular erythrocyte antibodies when examined without
dilution by an indirect antiglobulin test.
Citrate
Liquid chromatography (2.2.29).
2014
Test solution Dilute the preparation to be examined with an
equal volume of a 9 gIL solution of sodium chloride R . Filter
the solution using a filter with 0.45 ~m pores.
Reference solution Dissolve 0.300 g of sodium citrate R in
water R and dilute to 100.0 mL with the same solvento
Column:
- size: l = 0.3 m, 0 = 7.8 mm;
- stationary phase: cation exchange resin R (9 ~lm).
Mobile phase 0.51 gIL solution of sulfuric acid R.
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 215 nm.
Equilibration 15 mino
Injection 1O ~L.
Retention time Citrate = about 10 mino
Limit:
- cÚrate: maximum 25 mmol/L.
Calcium
Maximum 5.0 mmol/L.
Atomic absorption spectrometry (2.2.23, Method 1).
Source Calcium hollow-cathode lamp using a transmission
band preferably of 0.5 nm.
Wavelength 622 nm.
Atomisation device Air-acetylene or acetylene-propane flame o
Potassium
Maximum 5.0 mmol/L.
Atomic emission spectrometry (2.2.22, Method 1).
Wavelength 766.5 nm.
Sodium
Maximum 200 mmollL.
Atomic emission spectrometry (2.2.22, Method 1) .
Wavelength 589 nm.
Water
Determined by a suitable method, such as the semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrometry (2.2.40), the water content is
within the limits approved by the competent authority
(freeze-dried product).
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject 3 mL per kilogram of the rabbit's
mass.
Where the bacterial endotoxin test is used, the preparation to
be examined contains less than 0.1 IU of endotoxin per
millilitre.
ASSAY
Assay ofhuman coagulation factor VIII (2.7.4)
Use a reference plasma calibrated against the Intemational
Standard for blood coagulation factor VIII in plasma.
The estimated potency is not les s than 0.5 IU/mL.
The confidence limits (P = 0.95) are not les s than
80 per cent and not more than 120 per cent of the estimated
potency.
Assay of human coagulation factor V
Carry out the assay of human coagulation factor V described
below using a reference plasma calibrated against the
2014
Intemational Standard for blood coagulation factor V in
plasma.
Using imidazole buffer solunon pH 7.3 R, prepare at least 3
twofold dilutions of the preparation ro be examined,
preferably in duplicate, from 1 in lOto 1 in 40. Test each
dilution as folIows: mix 1 volume of plasma substrate defieient
in factor V R, 1 volume of the dilution to be examined,
1 volume of thromboplastin R and 1 volume of a 3.5 gIL
solution of calcium chlonde R; measure the coagulation times,
i.e. the interval between the moment at which the calcium
chloride solution is added and the 1st indication of the
formation of fibrin, which may be observed visualIy or by
means of a suitable apparatus.
In the same manner, determine the coagulation time of 4
twofold dilutions (1 in lOro 1 in 80) of human normal
plasma in imidazole buffer solution pH 7.3 R.
Check the validity of the assay and calculate the potency of
the test preparation by the usual statistical methods (for
example, 5.3).
The estimated potency is not less than 0.5 IU/mL.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
Assay ofhuman coagu1ation factor XI (2. 7.22)
Use a reference plasma calibrated against the Intemational
Standard for blood coagulation factor XI in plasma.
The estimated potency is not less than 0.5 IU/mL.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
Assay ofhuman protein C (2.7.30)
Use a reference plasma calibrated against the Intemational
Standard for human protein C in plasma.
The estimated potency is not less than 0.7 IU/mL.
The confidence limits (P == 0.95) are not les s than
80 per cent and not more than 120 per cent of the estimated
potency.
Assay ofhuman protein S (2. 7.31)
Use a reference plasma calibrated against the Intemational
Standard for human protein S in plasma.
The estimated potency is within the limits approved for the
particular product. The confidence limits (P = 0.95) are not
less than 80 per cent and not more than 120 per cent of the
estimated potency.
Assay ofhuman plasmin inhibitor (2. 7.25) (az-
antiplasmin)
Use a reference plasma calibrated against human normal
plasma.
1 unit of human plasmin inhibitor is equal to the activity of
1 mL of human normal plasma. Human normal plasma is
prepared by pooling plasma units from not fewer than 30
donors and sroring at - 30 oC or lower.
The estimated potency is not less than 0.2 units/mL.
The confidence limits (P == 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
Activated partia! thromboplastin time (APTT)
Use an apparatus suitable for measurement of coagulation
times or perform the assay with incubation tubes maintained
in a water-bath at 37 cc. Place in each tube 0.1 mL ofthe
preparation to be examined and 0.1 mL of a suitable APTT
reagent (containing phospholipid and contact activator), both
previously heated ro 37 oC, and incubate the mixture for a
Blood-related Products IV-455
recommended time at 37 oc. To each tube add 0.1 mL of a
3.7 gIL solution of ealcium ehlonde R previously heated to
37 oc. Using a timer, measure the coagulation time,
i.e. the interval between the moment of the addition of the
calcium chloride and the 1st indication of the formation of
fibrin, which may be observed visualIy or by means of a
suitable apparatus. The volumes given aboye may be adapted
to the APTT reagent and apparatus used. The coagulation
time complies with the agreed specification for the producto
LABELLING
The label states:
-- the ABO blood group;
-- the method used for virus inactivation.
___________________________________________ ~E~
Albumin Solution ***
*** ***
Albumin; Human Albumin ***
(Human Albumin SolutionJ Ph Eur monograph 0255)
~E~ ________________________________________
DEFINITION
Sterile liquid preparation of a plasma protein fraction
containing human albumin. It is obtained from plasma that
complies with the monograph Human plasma for
fractionation (0853). The preparation may contain excipients
such as sodium caprylate (sodium octano ate) or
N-acetyltryptophan or a combination of the two.
PRODUCTION
Separation of the albumin is carried out under controlled
conditions, particularly of pH, ionic strength and temperature
so that in the final product not less than 95 per cent of the
total protein is albumin. Human albumin solution is prepared
as a concentrated solution containing 150-250 gIL of total
protein or as an isoronic solution containing 35-50 gIL of
total protein. No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
filter and distributed aseptically into sterile containers which
are then closed so as to prevent contamination. The solution
in its final container is heated to 60 ± 1.0 oC and
maintained at this temperature for not les s than 10 h.
The containers are then incubated at 30-32 oC for not less
than 14 days or at 20-25 oC for not les s than 4 weeks and
examined visually for evidence of microbial contamination.
CHARACTERS
Appearance
Clear, slightly viscous liquid, almost colourless, yelIow, amber
or green.
IDENTIFICATION
Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal human serum, compare normal
human serum and the preparation to be examined, both
diluted to contain 10 giL of protein. The main component of
the preparation to be examined corresponds to the main
component of normal human serum. The preparation may
show the presence of smalI quantities of other plasma
proteins.
TESTS
pH (2.2.3)
6.7 ro 7.3.
 IV-456 Blood-related Products
Dilute the preparation to be examined with a 9 gIL solution
of sodium ehloride R to obtain a solution containing 10 gIL of
protein.
Total protein
If necessary, dilute an accurately measured volume of the
preparation to be examined with a 9 gIL solution of sodium
ehloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of this solution in a round-
bottomed centrifuge rube add 2 mL of a 75 gIL solution of
sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfurie acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
inverted rube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the quantity of protein by multiplying by 6.25.
The protein content is not les s than 95 per cent and not
more than 105 per cent of the stated content.
Protein composition
Zone electrophoresis (2.2.31).
Use strips of suitable cellulose acetate gel or agarose gel as
the supporting medium and barbital buffer solution pH 8.6 Rl
as the electrolyte solution.
If cellulose acetate is the supporting material, the method
described below can be used. If agarose gels are used, and
because they are normally part of an automated system, the
manufacrurer's instructions are followed instead.
Test solution Dilute the preparation to be examined with a
9 giL solution of sodium ehloride R to a protein concentration
of 20 gIL.
Referenee solution Dilute human albumin for
electrophoresis BRP with a 9 gIL solution of sodium chloride R
to a protein concentration of 20 gIL.
To a strip apply 2.5 ¡.¡L of the test solution as a 10 mm band
or apply 0.25 ¡.¡L per millimetre if a narrower strip is used.
To another strip, apply in the same manner the same volume
of the reference solution. Apply a suitable electric field such
that the most rapid band migrates at least 30 mm. Treat the
strips with amido black 10B solution R for 5 mino DecoJorise
with a mixture of 10 volumes of glacial acetie ac¡d R and
90 volumes of methanol R until the background is just free of
colour. Develop the transparency of the strips with a mixrure
of 19 volumes of glacial acetic acid R and 81 volumes of
melhanol R . Measure the absorbance of the bands at 600 nm
in an instrument having a linear response over the range of
measurement. Calculate the result as the mean of
3 measurements of each strip.
System suitability In the electropherogram obtained with the
reference solution on cellulose acetate or on agarose gels, the
proportion of protein in the principal band is within the
limits stated in the leaftet accompanying the reference
preparation.
Results In the electropherogram obtained with the test
solution on cellulose acetate or on agarose gels, not more
than 5 per cent of the protein has a mobility different from
that of the principal bando
Molecular-size distribution
Size exclusion chromatography (2.2.30).
Test solution Dilute the preparation to be examined with a
9 gIL solution of sodium ehloride R to a concentration suitable
for the chromatographic system used. A concentration in the
range of 4-12 gIL and injection of 50-600 ¡.¡g of protein are
usually suitable.
Column:
- size: 1 = 0.6 m, 0 = 7.5 mm, or l = 0.3 m, 0 = 7.8 mm;
2014
- stationary phase: hydrophilie silica gel for chromatography R,
of a grade suitable for fractionation of globular proteins
with relative molecular masses in the range 10 000 to
500000.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
dzhydrate R, 1.741 g of sodium dihydrogen phosphate
monohydrale R, 11.688 g of sodium ehloride R and 50 mg of
sodium azide R in 1 L of water R .
Flow rate 0.5 mUmin.
Deleaion Spectrophotometer at 280 nm.
The peak due to polymers and aggregates is located in the
part of the chromatogram representing the void volume.
Disregard the peak due to the stabiliser. The area of the peak
due to polymers and aggregates is not greater than
10 per cent of the total area of the chromatogram. This
represents not more than 5 per cent when expressed in
percentage of protein considering the difference in response
factor between the albumin monomer and the polymers and
aggregates.
Haem
Dilute the preparation to be examined using a 9 gIL solution
of sodium ehloride R to obtain a solution containing 10 giL of
protein. The absorbance (2.2.25) of the solution measured at
403 nm using water R as the compensation liquid is not
greater than 0.15.
Prekallikrein activator (2.6.15)
Maximum 35 IU/mL.
Aluminium
Maximum 200 ¡.¡gIL.
Atomic absorption spectrometry (2.2.23, Method 1 or 1l).
Use a furnace as atomic generator.
Use plastic containers for preparation of the solutions and use
plastie equipment where possible. Wash glassware (or equipment)
in nitrie acid (200 giL HNO:;) before use.
Test solution Use the preparation to be examined, diluted if
necessary.
Referenee solutions Prepare at least 3 reference solutions in a
range spanning the expected aluminium concentration of the
preparation to be examined, for example by diJuting
aluminium standard solution (10 ppm Al) R with a 1 gIL
solution of oetoxinol 10 R .
Monitor solution Add aluminium standard solution (10 ppm
Al) R or a suitable certified reference material to the test
solution in a sufficient amount to in crease the aluminium
concentration by 20 ¡.¡gIL.
Blank solution 1 gIL solution of oetoxinol 10 R.
Wavelenglh 309.3 nm or other suitable wavelength.
Slil widlh 0.5 nm.
Tube Pyrolytically coated, with integrated platform.
Background eorreaor Off.
Atomisation deviee Furnace; fue between readings.
The operating conditions in Table 0255.-1 are cited as an
example of conditions found suitable for a given apparatus;
they may be modified to obtain optimum conditions .
1njeetion Each of the following solutions 3 times: blank
solution, reference solutions, test solution and monitor
solution.
Syslem suitability:
- the recovery of aluminium added in preparation of the
monitor solution is within the range 80-120 per cent.
Prepare a calibration curve from the mean of the readings
obtained with the reference solutions and determine the
2014 Blood-related Products IV-457

Table 0255.-1. - Operating conditions found suitable, cited

Antithrombin 111 Concentrate ***
** **
as an example
Step Final Ramp time Hold time Gas (Human Amith¡-ombin III Concentrate, **** *
temperature (s) (s)
(OC) Ph Eur monograph 0878)
120 10 80 argon
Action and use
2 200 5 20 argon
Anticoagulant factor.
3 650 5 10 argon
~E~ ____________________________________________
4 1300 5 10 argo n
DEFINITION
5 1300 10 no gas
Sterile, freeze-dried preparation of a plasma glycoprotein
6 2500 0.7 4 no gas fraction that inactivates thrombin in the presence of an excess
7 2600 0.5 3 argo n of heparin. Ir is obtained from human plasma that complies
with the monograph on Human plasma for
8 20 12.9 3 no gas
fractionation (0853). The preparation may contain excipients
such as stabilisers.
When reconstituted in the volume of solvent stated on the
aluminium content of the preparation to be examined using label, the potency is not less than 25 IU of antithrombin III
the calibration curve. per millilitre.
Potassium PRODUCTION
Maximum 0.05 mmol of K per gram of protein. The method of preparation is designed to maintain
Atomic emission spectrometry (2.2.22, Method 1). functional integrity of antithrombin III. It incIudes a step or
Wavelength 766.5 nm. steps that have been shown to remove or to inactivate known
agents of infection; if substances are used for inactivation of
Sodium
viruses during production, the subsequent purification
Maximum 160 mmoIIL and 95 per cent to 105 per cent of
the content of N a stated on the labe!' procedure must be validated to demonstrate that the
concentration of these substances is reduced to a suitable
Atomic emission spectrometry (2.2.22, Method 1). level and any residues are such as not to compromise the
Wavelength 589 nm. safety of the preparation for patients .
Sterility (2.6.1) The specific activity is not less than 3 IU of antithrombin III
It complies with the test. per milligram of total protein, excIuding albumin.
Pyrogens (2.6.8) or Bacteria! endotoxins (2.6. 14) The antithrombin III is purified and concentrated.
It complies with the test for pyrogens or, preferably and No antimicrobial preservative or antibiotic is added.
where justified and authorised, with a validated in vitro test The antithrombin III concentrate is passed through a
such as the bacterial endotoxin test. bacteria-retentive filter, distributed aseptically into its final,
For the pyrogen test, for a solution with a protein content of sterile containers and immediately frozen. Ir is then freeze-
35-50 gIL, inject 10 mL per kilogram of the rabbit's mass; dried and the containers are cIosed under vacuum or in an
for a solution with a protein content of 150-250 gIL, inject atmosphere of inen gas.
5 mL per kilogram of the rabbit's mass. It shalI be demonstrated that the manufacturing process
Where the bacterial endotoxin test is used, the preparation to yields a product with a consistent fraction of antithrombin III
be examined contains less than 0.5 IU of endotoxin per able to bind to heparin. It is evaluated by a suitable analytical
millilitre for solutions with a protein content not greater than procedure which is detennined during process development,
50 gIL, less than 1.3 IU of endotoxin per millilitre for such as:
solutions with a protein content greater than 50 gIL but not Heparin-binding fraction Examine by agarose gel
greater than 200 gIL, and less than 1.7 IU of endotoxin per electrophoresis (2.2.31) . Prepare a 10 giL solution of agarose
millilitre for solutions with a protein content greater than for electrophoresis R containing 15 IU of heparin R per millilitre
200 giL but not greater than 250 giL. in barbital buffer solucion Ph 8.4 R. Pour 5 mL of this solution
onto a glass plate 5 cm square. Cool at 4 oC for 30 mino
STORAGE
Cut 2 wells 2 mm in diameter 1 cm and 4 cm from the side
Protected from Iight.
of the plate and 1 cm from the cathode. Introduce into
LABELLING one well 5 ~lL of the preparation to be examined, diluted to
The label states: an activity of about 1 IU of antithrombin III per millilitre.
-- the name of the preparation; Introduce into the other well 5 flL of a solution of a marker
-- the volume of the preparation; dye such as bromophenol blue R . Allow the electrophoresis to
-- the content of protein expressed in grams per litre; proceed at 4 oC, using a constant electric field of 7 V/cm,
-- the content of sodium expressed in millimoles per litre; until the dye reaches the anode.
-- that the product is not to be used if it is cIoudy or if a Cut across the agarose gel 1.5 cm from that side of the plate
deposit has formed; on which the preparation to be examined was applied and
-- the name and quantity of any added substance remove the larger portion of the gel leaving a band 1.5 cm
____________________________________________ ~E~
wide containing the material to be examined . Replace the
removed portion with an even layer consisting of 3.5 mL of a
10 gIL solution of agarose for electrophoresis R in barbital
buffer solution pH 8.4 R, containing a rabbit anti-human
antithrombin III antiserum at a suitable concentration,
previously detennined, to give adequate peak heights of at
IV-458 Blood-related Products
least 1.5 cm. Place the plate with the original gel at the
cathode so that a 2 nd electrophoretic migration can occur at
right angles to the 1sto Allow this 2 nd electrophoresis to
proceed using a constant electric field of 2 V/cm for 16 h.
Cover the plates with filter paper and severallayers of thick
lint soaked in a 9 gIL solution of sodium chloride R and
compress for 2 h, renewing the saline several times. Rinse
with water R, dry the plates and stain with acid
blue 92 solution R.
Calculate the fraction of antithrombin III bound to heparin,
which is the peak closest to the ano de, with respect to the
total amount of antithrombin III, by measuring the area
defined by the 2 precipitation peaks.
The fraction of antithrombin III able to bind to heparin is
not less than 60 per cent.
CHARACTERS
Appearance
White or almost white, hygroscopic, friable solid or powder.
Reconstitute the preparation to be examined as stated on the label
immediately before carrying out the identijication, tests (except
those for solubility, total protein and water) and assay.
IDENTIFICATION
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined add the
volume of liquid stated on the label at the recommended
temperature. The preparation dissolves completely under
gentle swirling within 10 min in the volume of the solvent
stated on the label, forming a clear or slightly turbid,
colourless or almost colourless solution.
pH (2.2.3)
6.0 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmollkg.
Total protein
If necessary, dilute an accurately measured volume of the
reconstituted preparation to obtain a solution containing
about 15 mg of protein in 2 mL. To 2.0 mL of the solution
in a round-bottomed centrifuge tube add 2 mL of a 75 gIL
solution of sodium molybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
and allow the inverted tube to drain on filter papero
Determine the nitrogen in the residue by the method of
sulfuric acid digestion (2.5.9) and calculate the amount of
protein by multiplying the result by 6.25.
Heparin (2.7.5)
Maximum 0.1 IU ofheparin per Intemational Unit of
antithrombin III.
Ir is necessary to validate the method for assay of heparin for
each preparation to be examined to allow for interference by
antithrombin III.
Water
Determined by a suitable method, such as semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
2014
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to 50 IU of antithrombin III.
Where the bacterial endotoxin test is used, the preparation to
be examined contains less than 0.1 IU of endotoxin per
Intemational Unit of antithrombin III.
ASSAY
Human antithrombin III (2.7.17)
The estimated potency is not less than 80 per cent and not
more than 120 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
90 per cent and not more than 11 O per cent of the estimated
potency.
STORAGE
Protected from light, in an airtight container.
LABELLING
The label sta tes:
-- the number of International Units of antithrombin III in
the container;
-- the name and volume of the liquid to be used for
reconstitution;
-- where applicable, the amount of albumin added as a
stabiliser.
____________________________________________ ~E~
*****
Dried Factor VII Fraction
** **
(Human Coagulation Factor VII, ***
Ph Bur monograph 1224)
Action and use
Coagulation factor VII substitute.
~E~ ____________________________________________
DEFINITION
Sterile, liquid or freeze-dried preparation of a plasma protein
fraction containing the single-chain glycoprotein human
coagulation factor VII and may also contain small amounts
of the activated form, the 2-chain derivative human
coagulation factor Vlla. It may also contain human
coagulation factors II, IX and X, protein C and protein S.
Ir is obtained from human plasma that complies with the
monograph on Human plasma for fractionation (0853).
The preparation may contain excipients such as stabilisers,
heparin and antithrombin.
The potency of the preparation, reconstituted as stated on
the label, is not less than 15 IU of human coagulation
factor VII per millilitre.
PRODUCTION
GENERAL PROVISIONS
The method of preparation is designed to maintain
functional integrity of human coagulation factor VII and to
minimise activation of any coagulation factor (to minimise
potential thrombogenicity). It includes a step or steps that
have been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of virus es
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of
these substances is reduced to a suitable leve! and that any
2014
residues are such as not to compromise the safety of the
preparation for patients.
The specific activity is not les s than 2 IU of human
coagulation factor VII per milligram of total protein, before
the addition of any protein stabiliser.
The human coagulation factor VII fraction is dissolved in a
suitable liquido No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
filter, distributed aseptically into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas.
CONSISTENCY OF THE METHOD OF PRODUCTION
It shall be demonstrated that the manufacturing process
yields a product with consistent activities of human
coagulation factors II, IX and X, expressed in International
Units relative to the activity of human coagulation factor VII.
This is evaluated by suitable analytical procedure(s) that is
(are) determined during process development.
It shall be demonstrated that the manufacturing process
yields a product with a consistent activity of human
coagulation factor VIla. This is evaluated by suitable
analytical procedure(s) that is (are) determined during
process development.
Activity of human coaguIation factor VIIa
It may be determined, for example, using a recombinant
soluble tissue factor that does not activate human coagulation
factor VII but possesses a cofactor function specific for
human coagulation factor VIIa; after incubation of a mixture
of the recombinant soluble tissue factor with phospholipids
reagent and the dilution of the test sample in human
coagulation factor VII-deficient plasma, calcium chloride is
added and the clotting time determined; the clotting time is
inversely related to the human coagulation factor VIIa activity
of the test sample.
CHARACTERS
Appearance
White or almost white, pale yellow, green or blue,
hygroscopic powder or friable solid.
Reconstitute the preparation to be examined as stated on the label
immediately before canying out the identijication, tests (except
those for solubility and water) and assay.
IDENTIFICAnON
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined add the
volume of liquid stated on the label at the recommended
temperature. The preparation dissolves completely with
gentle swirling within lO min, giving a clear or slightly
opalescent solution that may be coloured.
pH (2.2.3)
6.5 to 7.5 .
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
If necessary, dilute an accurately measured volume of the
reconstituted preparation with a 9 gIL solution of sodium
chloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of the solution in a round-
bottomed centrifuge tube, add 2 mL of a 75 gIL solution of
sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
Blood-related Products IV-459
inverted tube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of protein by multiplying the result
by 6.25.
Activated coagulation factors (2.6.22)
For each of the dilutions, the coagulation time is not less
than 150 s.
Heparin (2.7.12)
If heparin has been added, the preparation to be examined
contains not more than the amount of heparin stated on the
label and in any case not more than 0.5 IU of heparin
per International Unit of human coagulation factor VII.
Thrombin
If the preparation to be examined contains heparin,
determine the amount present as described in the test for
heparin and neutralise the heparin by addition of protamine
sulfate R (lO Jlg of protamine sulfate neutralises 1 IU of
heparin). In each of 2 test-tubes, mix equal volumes of the
reconstituted preparation and of a 3 gIL solution of
fibrinogen R. Keep one of the tubes at 37 oC for 6 h and the
other at room temperature for 24 h . In a 3rd tube, mix equal
volumes of the fibrinogen solution and of a solution of
human thrombin R (1 IU/mL) and place the tube in a water-
bath at 37 oc. No coagulation occurs in the tubes containing
the preparation to be examined. Coagulation occurs within
30 s in the tube containing thrombin.
Human coagulation factor 11 (2.7.18)
The estimated content is not more than 125 per cent of the
stated content. The confidence limits (P = 0.95) are not less
than 90 per cent and not more than 111 per cent of the
estimated potency.
Human coagulation factor IX (2.7.11)
The estimated content is not more than 125 per cent of the
stated contento The confidence limits (P = 0.95) are not less
than 80 per cent and not more than 125 per cent of the
estimated potency.
Human coagulation factor X (2.7.19)
The estimated content is not more than 125 per cent of the
stated contento The confidence limits (P = 0.95) are not less
than 90 per cent and not more than 111 per cent of the
estimated potency.
Water
Determined by a suitable method, such as the semi-micro
determinatíon ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrometry (2.2.40), the water content is
within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the test for bacterial endotoxins.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to not les s than 30 IU of human
coagulation factor VII.
Where the test for bacterial endotoxins is used, the
preparation to be examined contains less than 0.1 IU of
endotoxin per International Unit of human coagulation
factor VII.
ASSAY
Human coagulation factor VII (2.7.10) . The estimated
potency is not les s than 80 per cent and not more than
125 per cent of the stated potency. The confidence limits
 IV-460 Blood-related Products
(P = 0.95) are not les s than 80 per cent and not more than
125 per cent of the estimated potency.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
-- the number of International Units of human coagulation
factor VII per container;
-- the maximum content of human coagulation factor II,
human coagulation factor IX and human coagulation
factor X per container, in International Units;
-- the amount of protein per container;
-- the name and quantity of any added substances,
including, where applicable, heparin;
-- the name and volume of the liquid to be used for
reconstitution;
-- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
____________________________________________ Ph
Dried Factor VIII (rDNA) ****
*** * tests may altematively be carned out on the finished producto
(Human Coagulation Factor VIII (rDNA), *** *
Ph Eur monograph 1643)
Action and use
Coagulation factor VIII substitute.
~E~ ____________________________________________
DEFINITION
Human coagulation factor VIII (rDNA) is a freeze-dried
preparation of glycoproteins having the same activity as
coagulation factor VIII in human plasma. It acts as a cofactor
of the activation of factor X in the presence of factor IXa,
phospholipids and calcium ions.
Human coagulation factor VIII circulates in plasma mainly as
a two-chain glycosylated protein with 1 heavy (relative
molecular mass of about 200 000) and 1 light (relative
molecular mass 80 000) chain held together by divalent
metal ions. Human coagulation factor VIII (rDNA) is
prepared as full-Iength factor VIII (octocog alfa), or as a
shortened two-chain structure (relative molecular mas s
90 000 and 80 000), in which the B-domain has been
deleted from the heavy chain (moroctocog alfa).
Full-Iength human rDNA coagulation factor VIII contains 25
potential N-glycosylation sites, 19 in the B domain of the
heavy chain, 3 in the remaining part of the heavy chain
(relative molecular mass 90 000) and 3 in the light chain
(relative molecular mass 80 000). The different products are
characterised by their molecular size and post-translational
modification ami/or other modifications.
PRODUCTION
Human coagulation factor VIII (rDNA) is produced by
recombinant DNA technology in mammalian cell culture.
It is produced under conditions designed to minimise
microbial contamination.
Purified bulk factor VIII (rDNA) may contain added human
albumin ami/or other stabilising agents, as well as other
auxiliary substances to provide, for example, correct pH and
osmolality.
2014
The specific activity is not less than 2000 IU of factor VIII:C
per milligram of total protein before the addition of any
protein stabiliser, and varies depending on purity and the
type of modification of molecular structure of factor VIII.
The quality of the bulk preparation is controlled using one or
more manufacturer's reference preparations as reference.
MANUFACTURER'S REFERENCE PREPARATIONS
During development, reference preparations are established
for subsequent verification of batch consistency during
production, and for control of bulk and final preparation.
They are derived from representative batches of purified bulk
factor VIII (rDNA) that are extensively characterised by tests
including those described below and whose procoagulant and
other relevant functional properties have been ascertained
and compared, wherever possible, with the International
Standard for factor VIII concentra te. The reference
preparations are suitably characterised for their intended
purpose and are stored in suitably sized aliquots under
conditions ensuring their stability.
E~
PURIFIED BULK FACTOR VIII (rDNA)
The purified bulk complies with a suitable combination of the
following tests for characterisation of integrity of the factor VIII
(rDNA). Where any substance added during preparation of the
purified bulk interferes with a test, the test is carried out before
addition of that substance. Where applicable, the characterisation
Specific biological activity or ratio of factor VIII
activity to factor VIII antigen
Carry out the assay of human coagulation factor VIII (2. 7.4).
The protein content, or where a protein stabiliser is present,
the factor VIII antigen content, is determined by a suitable
method and the specific biological activity or the ratio of
factor VIII activity to factor VIII antigen is calculated.
Protein composition
The protein composition is determined by a selection of
appropriate characterisation techniques which may include
peptide mapping, Western blots, HPLC, gel electrophoresis,
capillary electrophoresis, mass spectrometry or other
techniques to monitor integrity and purity. The protein
composition is comparable to that of the manufacturer's
reference preparation.
Molecular size distribution
Using size-excIusion chromatography (2.2.30) , the molecular
size distribution is comparable to that of the manufacturer's
reference preparation.
Peptide mapping (2.2.55)
There is no significant difference between the test protein
and the manufacturer's reference preparation.
Carbohydrates/sialic acid
To monitor batch-to-batch consistency, the monosaccharide
content and the degree of sialylation or the oligosaccharide
profile are monitored and correspond to those of the
manufacturer's reference preparation.
FINALLOT
Ir complies with the requirements under Identification, Tests
and Assay.
Excipients
80 per cent to 120 per cent of the stated content, determined
by a suitable method, where applicable.
CHARACTERS
Appearance
White or slightly yellow powder or friable mass.
 2014
IDENTIFICATION
A. It complies with the limits of the assay.
B. The distribution of characteristic peptide bands
corresponds with that of the manufacturer's reference
preparation (SDS-PAGE or Western blot).
TESTS
Reconstitute the preparation as stated on the label immediately
before canying out the tests (except those for solubility and water)
and assay.
Solubility
It dissolves within 5 min at 20-25 oC, giving a clear or
slightly opalescent solution.
pH (2.2.3)
6.5 to 7.5 .
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Water
Determined by a suitable method, such as the semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test for sterility.
Bacteria! endotoxins (2.6.14)
Less than 3 IU in the volume that contains 100 IU of factor
VIII activity.
ASSAY
Carry out the assay of human coagulation factor VIII (2.7.4).
The estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
STORAGE
Protected from light.
LABELLING
The label states:
-- the factor VIII content in International Units,
-- the name and amount of any excipient,
-- the composition and volume of the liquid to be used for
reconstitution.
____________________________________________
Dried Factor VIII Fraction *****
** **
(Human Coagulation Factor VIII, ***
Ph Bur monograph 0275)
Action and use
Coagulation factor VIII substitute.
~E~ ____________________________________________
DEFINITION
Sterile, freeze-dried preparation of a plasma protein ftaction
containing the glycoprotein human coagulation factor VIII
together with varying amounts of human von Willebrand
factor, depending on the method of preparation. Ir is
prepared from human plasma that complies with the
monograph on Human plasma for fractionation (0853) .
The preparation may contain excipients such as stabilisers.
Blood-related Products IV-461
The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU offactor VIII:C per millilitre.
PRODUCTION
GENERAL PROVISIONS
The method of preparation is designed to maintain
functional integrity of human coagulation factor VIII and to
minimise potential neoantigenicity. Ir inc1udes a step or steps
that have been shown to remove or to inactivate known
agents of infection; if substances are used for the inactivation
of viruses, the subsequent purification procedure must be
validated to demonstrate that the concentration of these
substances is reduced to a suitable leve! and that any residues
are such as not to compromise the safety of the preparation
for patients.
The specific activity is not less than 1 IU of factor VIII:C per
milligram of total protein before the addition of any protein
stabiliser.
The human coagulation factor VIII fraction is dissolved in a
suitable liquido No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
filter, distributed aseptically into the final containers and
immediately frozen. Ir is subsequently freeze-dried and the
containers are c10sed under vacuum or under an inert gas.
CONSISTENCY OF THE METHOD OF PRODUCTION
Products stated to have human von Willebrand factor activity
(products intended for treatment of von Willebrand's disease).
Ir shall be demonstrated by suitable analytical procedures
determined during process development that the
manufacturing process yields a product with a consistent
composition with respect to human von Willebrand factor.
This composition may be characterised in a number of ways.
For example, the distribution of the different human von
Willebrand factor multimers may be determined by sodium
dodecyl sulfate (SDS) agarose gel electrophoresis (about
1 per cent agarose) with or without Western blot analysis,
using a normal human plasma pool as reference.
Visualisation of the multimeric pattern may be performed
using, for example, an immunoenzymatic technique and
quantitative evaluation may be carried out by densitometric
analysis.
Products that show fiakes or particles after reconstitution for
use. If a few small flakes or partic1es remain when the
preparation is reconstituted, it shall be demonstrated during
validation studies that the potency is not significantly affected
PhE~
after passage of the preparation through the filter provided.
CHARACTERS
Appearance
White or pale yellow, hygroscopic powder or friable solido
Reconstitute the preparation to be examined as stated on the label
immediately before canying out the identification, tests (except
those for solubility and water) and assay.
IDENTIFICATION
Ir complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined, add the
volume of the liquid stated on the label at the recommended
temperature. The preparation dissolves completely with
gentle swirling within 10 min, giving a c1ear or slightly
opalescent, colourless or slightly yellow solution.
Where the label states that the product may show a few small
flakes or partic1es after reconstitution, reconstitute the
preparation as described on the label and pass it through the
IV-462 Blood-related Products
filter provided: the filtered solution is clear or slightly
opalescent.
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
If necessary, dilute an accurately measured volume of the
reconstituted preparation with a 9 giL solution of sodium
chloMe R to obtain a protein concentration of about
7.5 mglmL. Place 2.0 mL of this solution in a round-
bottomed centrifuge tube and add 2 mL of a 75 giL solution
of sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
inverted tube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of protein by multiplying the result
by 6.25. For some products, especially those without a protein
stabiliser such as albumin, this method may not be applicable and
another validated method jor protein determination must therejore
be performed.
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
The 1 to 64 dilution does not show agglutination. Dilute the
reconstituted preparation with a 9 giL solution of sodium
chloride R to contain 3 IU of factor VIII:C per millilitre.
Water
Determined by a suitable method, such as semi-micro
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2. 6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitra test
such as the test for bacterial endotoxins.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to not less than 50 IU of factor VIII:C.
Where the test for bacterial endotoxins is used, the
preparation to be examined contains less than 0.03 IU of
endotoxin per International Unit of factor VIII:C.
ASSAY
Human coagulation factor VIII (2.7.4)
The estimated potency is not les s than 80 per cent and not
more than 120 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
Human von Willebrand factor (2.7.21)
If preparations are intended for the treatment of von
Willebrand's di seas e, the estimated potency is not less than
60 per cent and not more than 140 per cent of the stated
potency.
Pending the availability oj an International Standard jor human
von Willebrand jactor concentra te calibrated jor use in the
collagen-binding assay, only the ristocetin cojactor assay may be
used.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
2014
-- the number of International Units of factor VIII:C and,
where applicable, of human von Willebrand factor in the
container;
-- the amount of protein in the container;
-- the name and quantity of any added substance;
-- the name and volume of the liquid to be used for
reconstitution;
-- where applicable, that the preparation may show the
presence of a few small fiakes or particles after
reconstitution;
-- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
____________________________________________ ~Ew
Dried Factor IX Fraction ***
** **
(Human Coagulation Factor IX, **** *
Ph Eur monograph 1223)
Action and use
Coagulation factor IX substitute.
~Ew ___________________________________________
DEFINITION
Sterile freeze-dried preparation of a plasma protein fraction
containing coagulation factor IX. It is obtained from human
plasma that complies with the monograph on Human plasma
jor jractionation (0853), by a method that effectively separates
human coagulation factor IX from other prothrombin
complex factors (human coagulation factors Il, VII and X).
The preparation may contain excipients such stabilisers,
heparin and antithrombin.
The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU of human coagulation
factor IX per rnillilitre.
PRODUCTION
GENERAL PROVISIONS
The method of preparation is designed to maintain
functional integrity of human coagulation factor IX and to
rninimise activation of any coagulation factor (to minimise
potential thrombogenicity). It includes a step or steps that
have been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of
these substances is reduced to a suitable level and that any
residues are such as not to compro mise the safety of the
preparation for patients.
The specific activity is not less than 50 IU of human
coagulation factor IX per milligram of total protein, before
the addition of any protein stabiliser.
The human coagulation factor IX fraction is dissolved in a
suitable liquido No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
filter, distributed asepticaIly into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas.
CONSISTENCY OF THE METHOD OF PRODUCTION
It shall be demonstrated that the manufacturing process
yields a product having a consistent composition. This is
evaluated by suitable analyticaI procedures that are
2014
determined during process development and that normally
inc1ude:
-- assay of human coagulation factor IX;
-- determination of activated coagulation factors;
-- determination of activities of human coagulation factors
II, VII and X, which shall be shown to be not more than
5 per cent of the activity of human coagulation factor IX.
CHARACTERS
Appearance
White or pale yellow, hygroscopic powder or friable solid.
Reconstitute the preparation to be examined as stated on the labe!
immediately before canying out the identification, tests (except
those for solubility and water) and assay.
IDENTIFlCATION
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined add the
volume of the liquid stated on the label at the recommended
temperature. The preparation dissolves complete!y with
gentle swirling within 10 min, giving a c1ear or slightly
opalescent, colourless solution.
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
If necessary, dilute an accurately measured volume of the
reconstituted preparation with a 9 gIL solution of sodium
chloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of the solution in a round-
bottomed centrifuge tube, add 2 mL of a 75 gIL solution of
sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
inverted tube to drain on filter papero Determine the nitro gen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of protein by multiplying the result
by 6.25 . For some products, especially those without a protein
stabiliser such as albumin, this method may not be applicable.
Another validated method for protein determination must therefore
be performed.
Activated coagulation factors (2.6.22)
If necessary, dilute the reconstituted preparation to contain
20 IU of human coagulation factor IX per millilitre. For each
of the dilutions, the coagulation time is not less than 150 S.
Heparin (2. 7.12)
If heparin has been added, the preparation to be examined
contains not more than the amount of heparin stated on the
labe! and in all cases not more than 0.5 IU of heparin per
International Unit of human coagulation factor IX.
Water
Determined by a suitable method, such as semi-micro
determination of water (2.5.12), los s on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2. 6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the test for bacterial endotoxins.
Blood-related Products IV-463
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to not less than 50 IU of human
coagulation factor IX.
Where the test for bacterial endotoxins is used, the
preparation to be examined contains les s than 0.03 IU of
endotoxin per International Unit of human coagulation
factor IX.
ASSAY
Human coagulation factor IX (2.7.11). The estimated
potency is not less than 80 per cent and not more than
125 per cent of the stated potency. The confidence limits
(P = 0.95) are not less than 80 per cent and not more than
125 per cent of the estimated potency.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
-- the number of International Units of human coagulation
factor IX per container;
-- the amount of protein per container;
-- the name and quantity of any added substances inc1uding,
where applicable, heparin;
-- the name and volume of the liquid to be used for
reconstitution;
-- that the transmission of infectious agents cannot be totally
exc1uded when medicinal products prepared from human
blood or plasma are administered.
_ _ _ __ _ _ _ __ __ _ _ _ _ _ _ __ _ __ Ph Eur
Dried Factor XI Fraction ***
*** ***
(Human Coagulation Factor XI, ***
Ph Eur monograph 1644)
Action and use
Coagulation factor XI substitute.
PhE~ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ __ _ __
DEFINITION
Sterile plasma protein fraction containing coagulation factor
XI. It is prepared from Human plasma for fractionation
(0853) . The preparation may contain excipients such as
heparin, Cl-esterase inhibitor and antithrombin III.
The potency of the preparation, reconstituted as stated on
the label, is not less than 50 units per millilitre.
PRODUCTION
The method of preparation is designed to maintain
functional integrity of human coagulation factor XI and to
minimise activation of any coagulation factor (to minimise
potential thrombogenicity). It inc1udes a step or steps that
have been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of
these substances is reduced to a suitable leve! and any
residues are such as not to compromise the safety of the
preparation for patients.
After preparation, the factor XI fraction is dissolved in a
suitable liquid. No antimicrobial preservative or antibiotic is
added. The solution is distributed into the final containers
and immediately frozen. It is subsequently freeze-dried and
the containers are c10sed under vacuum or under inert gas.
 IV-464 Blood-related Products
CHARACTERS
Appearance
White or almost white powder or friable solido
Reconstilute Ihe preparation 10 be examined as stated on the label
immediately befare earrying out the identifieation, tests (exeept
those for solubilily and water) and assay.
IDENTIFICATlON
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined, add the
volume of liquid stated on the label at room temperature.
The preparation dissolves completely with gentle swirling
within lO mino
pH (2.2.3)
6.8 to 7.4 .
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
If necessary, dilute an accurately measured volume of the
preparation to be examined with a 9 gIL solution of sodium
ehlonde R to obtain a protein concentration of about
7.5 mglmL. Place 2.0 mL of this solution in a round-
bottomed centrifuge tube and add 2 mL of a 75 giL solution
of sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free suifurie aeid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
inverted tube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of protein by multiplying the result
by 6.25.
Activated coagulation factors (2.6.22)
For each of the dilutions, the coagulation time is not less
than 150 S.
Heparin (2.7.12)
If heparin has been added, the preparation to be examined
contains not more than the amount of heparin stated on the
label and in all cases not more than 0.5 IU of heparin per
unit of factor XI.
Antithrombin III (2.7.17)
If antithrombin nI has been added, the preparation to be
examined contains not more than the amount of
antithrombin III stated on the labe!.
Cl-esterase inhibitor
If C¡-esterase inhibitor has been added, the preparation to be
examined contains not more than the amount of C¡-esterase
inhibitor stated on the labe!.
The C ¡-esterase inhibitor content of the preparation to be
examined is determined by comparing its ability to inhibit
C¡-esterase with the same ability of a reference preparation
consisting of human normal plasma. 1 unir of C ¡-esterase is
equal to the activity of 1 mL of human normal plasma.
Varying quantities of the preparation to be examined are
mixed with an excess of C¡-esterase and the remaining
C ¡-esterase activity is determined using a suitable
chromogenic substrate.
Method Reconstitute the preparation as stated on the labe!.
Prepare an appropriate series of 3 or 4 independent dilutions
from 1 unit/mL of factor XI, for both the preparation to be
examined and the reference preparation, using a solution
containing 9 gIL of sodium ehloride R and either 10 gIL of
human albumin R or 10 gIL of bovine albumin R. Warm all
solutions to 37 oC in a water-bath for 1-2 min before use.
2014
Place a suitable amount of C ¡-esterase solution in tubes or in
microtitre plate wells and incubate at 37 oC. Add a suitable
amount of one of the dilutions of the reference preparation
or of the preparation to be examined and incubate at 37 oC
for 5 mino Add a suitable amount of a suitable chromogenic
substrate such as methoxycarbonyl-L-Iysyl(e-
benzyloxycarbonyl)-glycyl-L-arginine 4-nitroanilide. Read the
rate of increase of absorbance (LlA/min) at 405 nm. Carry
out a blank test using tris(hydroxymethy1)aminomethane sodium
ehlonde buffer solution pH 7.4 R instead of the C ¡-esterase and
the substrate.
Calculate the C¡-esterase inhibitor content using the usual
statistical methods (for example, 5.3) .
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
The 1 to 64 dilution does not show agglutination.
Water
Determined by a suitable method, such as the semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40) , the water content
is within the limits approved by the competent authority.
Sterility (2.6. 1)
It complies wirh the test.
Pyrogens (2.6.8) or Bacteria! endotoxins (2.6. 14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to 100 IU of factor XI.
Where the bacterial endotoxin test is used, the preparation to
be examined contains less than 0.1 IU of endotoxin per
International Unit of factor XI.
ASSAY
Carry out the assay of human coagulation factor XI (2.7.22).
The estimated potency is not less than 80 per cent and not
more than 120 per cent of the stated potency.
The confidence limits (P = 0.95) are not les s than
80 per cent and not more than 125 per cent of the estimated
potency.
STORAGE
Protected from light, at a temperature of 2 oC to 8 oc.
LABELLING
The labe! states:
-- the number of units per container;
-- the maximum amount of protein per container;
-- where applicable, the amount of heparin per container;
-- where applicable, the amount of antithrombin In per
container;
-- where applicable, the amount of C¡-esterase inhibitor per
container;
-- the name and volume of the liquid to be used for
reconstitution.
____________________________________________ ~Em
 2014
*****
Dried Prothrombin Complex
** **
(Human Prothrombin Complex, ***
Ph Eur monograph 0554)
Action and use
Coagulation factor IX substitute. Preparations with
appropriate activity may be used to correct deficiencies of
coagulation factors II or X.
~E~ ____________________________________________
DEFINITION
Sterile plasma protein fraction containing human coagulation
factor IX together with variable amounts of human
coagularion factors II, VII and X; the presence and
proportion of these additional factors depends on the method
of fractionarion. It is obtained from human plasma that
complies with the monograph on Human plasma for
fraetionation (0853). The preparation may contain excipients
such as stabilisers, heparin and antithrombin.
The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU of human coagulation factor
IX per millilitre.
If the content of any of the factors is stated as a single value,
the estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency; if the content
of any of the factors is stated as a range, the estimated
potency is not less than the lower Iimit and not greater than
the upper limit of the stated range.
PRODUCTION
The method of preparation is designed to maintain
functional integrity of the relevant coagulation factors it
contains and to minimise activation of any coagularion factor
(to minimise potential thrombogenicity) . It incIudes a step or
steps that have been shown to remove or to inactivate known
agents of infection; if substances are used for inactivation of
viruses during production, the subsequent purification
procedure must be validated to demonstrate that the
concentration of these substances is reduced to a suitable
level and that any residues are such as not to compromise the
safety of the preparation for patients.
The specific activity is not less than 0.6 IV of human
coagulation factor IX per milligram of total protein, before
the addition of any protein stabiliser.
The prothrombin complex fraction is dissolved in a suitable
liquid. No antimicrobial preservative or antibiotic is added.
The solution is passed through a bacteria-retentive filter,
distributed aseptically into the final containers and
immediately frozen. It is subsequentIy freeze-dried and the
containers are cIosed under vacuum or under an inert gas .
CHARACTERS
Appearance
White or slightly coloured, very hygroscopic powder or friable
solid.
Reeonstitute the preparation to be examined as stated on the label
immediately before carrying out the identijieation, tests (exeept
those for solubility and water) and assay.
IDENTIFICATION
It complies with the limits of the assays for human
coagulation factors IX and II and, where applicable, those for
human coagulation factors VII and X.
Blood-related Products IV-465
TESTS
Solubility
To a container of the preparation to be examined add the
volume of the liquid stated on the label at the recommended
temperature. The preparation dissolves completely with
gentle swirling within 10 min, giving a cIear solution that
may be coloured.
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
If necessary, dilute an accurately measured volume of the
reconstituted preparation with a 9 gIL solution of sodium
ehloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of the solution in a round-
bottomed centrifuge tube add 2 mL of a 75 gIL solution of
sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfurie acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and aIlow the
inverted tube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of protein by mulriplying the result
by 6.25.
Activated coagulation factors (2.6.22)
If necessary, dilute the reconstituted preparation to contain
20 IU of human coagulation factor IX per millilitre. For each
of the dilutions, the coagulation time is not less than 150 S.
Heparin (2.7.12)
If heparin has been added during preparation, the
preparation to be examined contains not more than the
amount of heparin stated on the labe! and in all cases not
more than 0.5 IU of heparin per International Unit of human
coagulation factor IX.
Thrombin
If the preparation to be examined contains heparin,
detennine the amount present as described in the test for
heparin and neutralise it by addition of protamine sulfate R
(10 Jlg of protamine sulfate neutralises 1 IU of heparin) .
In each of 2 test-tubes, mix equal volumes of the
reconstituted preparation and of a 3 gIL solution of
fibrinogen R. Keep one of the tubes at 37 oC for 6 h and the
other at room temperature for 24 h. In a 3 rd tube, mix equal
volumes of the fibrinogen solution and of a solution of
human thrombin R (1 IU/mL) and place the tube in a water-
bath at 37 oC. No coagulation occurs in the tubes containing
the preparation to be examined. Coagularion occurs within
30 s in the tube containing thrombin.
Water
Detennined by a suitable method, such as semi-micro
detennination ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrometry (2.2.40), the water content is
within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6. 14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to not less than 30 IU of human
coagulation factor IX.
IV-466 Blood-related Products
Where the bacterial endotoxin test is used, the preparation to
be examined contains less than 0.05 IU of endotoxin per
International Unit of human coagulation factor IX.
ASSAY
Human coagulation factor IX (2.7.11)
The estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency.
The confidence interval (P = 0.95) is not greater than
80 per cent to 125 per cent of the estimated potency.
Human coagulation factor 11 (2.7.18)
The estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency.
The confidence interval (P = 0.95) is not greater than
90 per cent to 111 per cent of the estimated potency.
The estimated human coagulation factor II potency is not
les s than 70 per cent and not more than 165 per cent of the
estimated human coagulation factor IX potency.
Human coagulation factor VII (2.7.10)
If the label states that the prepanition contains human
coagulation factor VII, the estimated potency is not less than
80 per cent and not more than 125 per cent of the stated
potency. The confidence interval (P = 0.95) is not greater
than 80 per cent to 125 per cent of the estimated potency.
Human coagulation factor X (2.7.19)
If the label states that the preparation contains human
coagulation factor X, the estimated potency is not less than
80 per cent and not more than 125 per cent of the stated
potency. The confidence interval (P = 0.95) is not greater
than 90 per cent to 111 per cent of the estimated potency.
STORAGE
In an airtight container, protected from light.
LABELLING
The label sta tes:
-- the number of International Units of human coagulation
factor IX, and the number or range of International Units
of human coagulation factor II per container;
-- where applicable, the number or range of Intemational
Units of human coagulation factor VII and human
coagulation factor X per container;
-- the amount of protein per container;
-- the name and quantity of any added substances,
including, where applicable, heparin and antithrombin;
-- the name and quantity of the liquid to be used for
reconstitution;
-- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
___________________________________________ PhEw
Dried Fibrinogen ***
*** ***
(Human Fibrinogen, Ph Eur monograph 0024) ***
~Ew ___________________________________________
DEFINITION
Sterile, freeze-dried preparation of a plasma protein fraction
containing the soluble constituent of human plasma that is
transformed to fibrin on the addition of thrombin. It is
obtained from human plasma that complies with the
monograph on Human plasma for fractionation (0853).
The preparation may contain excipients such as salts, buffers
and stabilisers.
2014
When reconstituted as stated on the label, the solution
contains not less than 10 gIL of fibrinogen.
PRODUCTION
The method of preparation is designed to maintain
functional integrity of human fibrinogen. It includes a step or
steps that have been shown to remove or to inactivate known
agents of infection; if substances are used for inactivation of
virus es during production, the subsequent purification
procedure must be validated to demonstrate that the
concentration of these substances is reduced to a suitable
level and any residues are such as not to compromise the
safety of the preparation for patients.
The specific activity (fibrinogen content with respect to total
protein content) is not les s than 80 per cent before addition
of any protein stabiliser. The fibrinogen content is
determined by a suitable method such as that described
under Assay, and the total protein content is determined by a
suitable method such as that described under Total protein
in Human albumin solution (0255). Albumin may also be
obtained with fibrinogen during fractionation, in which case a
specific determination of albumin is carried out by a suitable
immunochemical method (2. 7.1) and the quantity of albumin
determined is subtracted from the total protein content for
the calculation of the specific activity.
The protein fraction is dissolved in a suitable liquido
No antimicrobial preservative or antibiotic is added.
The solution is passed through a bacteria-retentive filter,
distributed asepticalIy into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas.
CHARACTERS
Appearance
White or pale yellow, hygroscopic powder or friable solido
Reconstitute the preparation to be examined as stated on the label
immediately before carrying out the identification, tests (except
those for solubility and water) and assay.
IDENTIFICATION
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined add the
volume of liquid stated on the label at the recommended
temperature. The preparation dissolves within 30 min at
20-25 oC, forming an almost colourless, slightly opalescent
solution.
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Stability of solution
No gel formation appears at 20-25 oC within 60 min
following reconstitution.
Water
Determined by a suitable method, such as semi-micro
determination ofwater (2.5.12), loss on dtying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
2014
Pyrogens (2.6.8) or Bacteria! endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the test for bacterial endotoxins.
For the pyrogen test, inject per kilogram of the rabbit's mas s
a volume equivalent to not les s than 30 mg of fibrinogen.
Where the test for bacterial endotoxins is used, the
preparation to be examined contains less than 0.03 IV of
endotoxin per milligram of fibrinogen .
ASSAY
Mix 0.2 mL of the reconstituted preparation with 2 mL of a
suitable buffer solution (pH 6.6-6.8) containing sufficient
thrombin (approximately 3 IU/mL) and calcium
(0.05 mollL). Maintain at 37 oC for 20 min, separate the
precipitate by centrifugation (5000 g, 20 min) and wash
thoroughly with a 9 gIL solution of sodium chloride R.
Determine the nitro gen content by sulfuric acid digestion
(2.5. 9) and calculate the fibrinogen (clottable protein)
content by multiplying the result by 6.0. The content is not
less than 70 per cent and not more than 130 per cent of the
stated content of fibrinogen.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
- the content of fibrinogen in the container;
- the name and volume of the liquid to be used for
reconstitution;
- where applicable, the name and amount of protein
stabiliser added in the preparation.
_ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Fibrin Sealant Kit ***
*** ***
(Ph Eur monograph 0903) ***
~E~ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ __ _ ___
DEFINITION
Sterile, freeze-dried, frozen or liquid preparation of plasma
protein fractions containing essentially 2 components, namely
fibrinogen concentrate (component 1), a protein fraction
containing human fibrinogen, and a preparation containing
human thrombin (component 2). A fibrin clot is rapidly
formed when the 2 thawed or reconstituted components are
mixed. Other ingredients (for example, human coagulation
factor XIII, a fibrinolysis inhibitor or calcium ions) and
stabilisers (for example, Human albumin solution (0255) may
be added.
Human constituents are obtained from plasma that complies
with the monograph on Human plasma for fractionation
(0853).
When thawed or reconstituted as stated on the label,
component 1 contains not les s than 40 gIL of c10ttable
protein; the thrombin activity of component 2 varies over a
wide range (approximately 4-1000 IV/rnL).
PRODVCTION
The method of preparation is designed to maintain
functional integrity of the components. Ir includes a step or
steps that have been shown to remove or to inactivate known
agents of infection; if substances are used for inactivation of
virus es during production, the subsequent purification
procedure must be validated to demonstrate that the
Blood-related Products IV-467
concentration of these substances is reduced to a suitable
level and any residues are such as not to compromise the
safety of the preparation for patients.
The constituents or mixtures of constituents are dissolved in
a suitable liquido No antimicrobial preservative or antibiotic is
added. Constituents or mixtures of constituents are passed
through a bacteria-retentive filter and distributed aseptically
into sterile containers. Containers of freeze-dried constituents
are c10sed under vacuum or filled with a suitable inert gas,
such as oxygen-free nitrogen, before being closed.
If the human coagulation factor XIII content in component 1
is greater than 10 units/mL, the assay of human coagulation
factor XIII is carried out.
CHARACTERS
Appearance:
- freeze-dried constituents: white or pale yellow, hygroscopic
powder or friable solid,
- frozen constituents: colourless or pale yellow, opaque solid,
- liquid constituents: colourless or pale yellow liquido
Por the freeze-dried or frozen constituents, reconstitute or thaw as
stated on the label immediately before carrying out the
identification and the tests, except those for solubility and water.
COMPONENT 1 (FIBRINOGEN CONCENTRATE)
IDENTIFICATION
A. It complies with the limits of the assay of fibrinogen.
B. It complies with the limits of the assay of human
coagulation factor XIII (where applicable) .
TESTS
Solubility
Freeze-dried concentrates dissolve within 20 min in the
volume of liquid and at the temperature stated on the label,
forming an almost colourless, clear or slightly turbid solution.
pH (2.2.3)
6.5 to 8.0.
Stability of solution
No gel formation appears at room temperature during
120 min following thawing or reconstitution.
Water
Determined by a suitable method, such as semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
ASSAY
Fibrinogen (clottable protein)
Mix 0.2 mL of the reconstituted concentrate with 2 mL of a
suitable buffer solution (pH 6.6-7.4) containing sufficient
human thrombin R (approximately 3 IV/mL) and calcium
(0.05 mollL). Maintain at 37 oC for 20 min, separate the
precipitate by centrifugation at 5000 g for 20 min, wash
thoroughly with a 9 gIL solution of sodium chloride R and
determine the protein as nitrogen by sulfuric acid digestion
(2.5.9). Calculate the clottable protein content by multiplying
the result by 6.0. The estimated content in milligrams of
clottable protein is not les s than 70 per cent and not more
than 130 per cent of the stated contento If for a particular
preparation this method cannot be applied, use another
validated method for determination of fibrinogen.
Human coagulation factor XIII
Where the label indicates that the human coagulation factor
XIII potency is greater than 10 units/mL, the estimated
 IV-468 Blood-related Products
potency is not less than 80 per cent and not more than
120 per cent of the stated potency.
Make at least 3 suitable dilutions of thawed or reconstituted
concentrate and of human normal plasma (reference
preparation) using human coagulation factor XIII-deficient
plasma or another suitable diluent. Add to each dilution
suitable amounts of the following reagents:
-- activator reagent, containing bovine or human thrombin,
a suitable buffer, calcium chloride and a suitable inhibitor
such as Gly-Pro-Arg-Pro-Ala-NH2 which inhibits clotting
of the sample but does not prevent human coagulation
factor XIII activation by thrombin;
-- detection reagent, containing a suitable factor XIIIa-
specific peptide substrate, such as Leu-Gly-Pro-Gly-Glu-
Ser-Lys-Val-Ile-Gly-NH2 and glycine ethyl ester as 2nd
substrate in a suitable buffer solution;
-- NADH reagent, containing glutamate dehydrogenase,
ex-ketoglutarate and NADH in a suitable buffer solution.
After mixing, the absorbance changes (M/min) are measured
at a wavelength of 340 nm, after the linear phase of the
reaction is reached.
1 unit of human coagulation factor XIII is equal to the
potency of 1 mL of human normal plasma.
Calculate the potency of the test preparation by the usual
statistical methods (5.3, for example). The confidence limits
(P = 0.95) are not less than 80 per cent and not more than
125 per cent of the estimated potency.
COMPONENT 2 (THROMBIN PREPARATION)
IDENTIFICATION
It complies with the limits of the assay of thrombin.
TESTS
Solubility
Freeze-dried preparations dissolve within 5 min in the
volume of liquid stated on the label, forming a colourless,
clear or slightly turbid solution.
pH (2.2.3)
5.0 to 8.0.
Water
Determined by a suitable method, such as semi-micro
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2. 6. 1)
It complies with the test.
ASSAY
Thrombin
If necessary, dilute the reconstituted preparation to be
examined to approximately 2-20 IV of thrombin per millilitre
using as diluent a suitable buffer solution (pH 7.3-7.5), such
as imidazole buffer solution pH 7.3 R containing 10 gIL of
human albumin R or bovine albumin R. To a suitable volume
of the dilution, add a suitable volume of fibrinogen solution
(1 gIL of clottable protein) warmed to 37 oC and stan
measurement of the clotting time immediately. Repeat the
procedure with each of at least 3 dilutions, in the range
stated aboye, of a reference preparation of thrombin,
calibrated in Intemational Units.
Calculate the activity of the test preparation by the usual
statistical methods (5.3, for example). The estimated activity is
not less than 80 per cent and not more than 125 per cent of
the stated activity. The confidence limits (P = 0.95) are not
less than 80 per cent and not more than 125 per cent of the
estimated activity.
2014
STORAGE
Protected from light and, for freeze-dried components, in an
airtight container.
LABELLING
The label states:
-- the amount of fibrinogen (milligrams of clottable protein),
thrombin (Intemational Units) per container, and of
human coagulation factor XIII, if the latter is greater than
10 units/mL,
-- where applicable, the name and volume of liquid to be
used to reconstitute the components .
____________________________________________ ~E~
Human Haematopoietic Stem Cells *****
** **
(Ph Eur monograph 2323) ***
~E~ ____________________________________________
This monograph provides a standard for the preparation and
control of human haematopoietic stem cells for use in therapy.
It does not exclude the use of altemative preparatíon and control
methods that are acceptable to the competent authority.
DEFINITION
Human haematopoietic stem cells are primitive multipotent
cells capable of self-renewal as well as differentiation and
maturation into all haematopoietic lineages. They are found
in small numbers in bone marrow, in the mononuclear cell
fraction of circulating blood and in umbilical cord blood.
The preparation also contains haematopoietic progenitor
cells, which are capable of differentiation but not self-
renewal. The numbers of haematopoietic stem cells and
haematopoietic progenitor cells are correlated.
This monograph applies to haematopoietic stem cells that
have not undergone expansion or genetic modification, and
that are intended to provide a successful engraftment leading
to a permanent restoration of alllineages of blood cell
production to a sufficient level and function in a recipient
whose haematopoiesis has been compromised by, for
example, disease or high doses of chemotherapy and/or
radiation therapy, or has to be replaced in certain congenital
diseases. The infused haematopoietic stem cells can originate
from the recipient (autologous) or from another individual
(allogeneic).
Haematopoietic stem cells are recognised by their ability to
reconstitute human haematopoiesis in vivo. They also have
the capacity to differentiate into colony-forming cells, which
are able to give rise to colonies in the presence of various
growth factors . The membrane marker CD34 is commonly
used for the successful isolationlpurification of
haematopoietic stem cells from crude preparations and as an
indicator of haematopoietic stem cell content in routine
quality control.
PRODUCTION
DONORS
Where allogeneic cells are used, they are derived from
carefully selected donors in accordance with donor selection
criteria. Directive 2004/23/EC of the European Union deals
with the criteria for donor selection.
COLLECTION
Peripheral blood stem cells These are collected by cytapheresis
after mobilisation from the bone marrow by administration of
growth factors and/or treatment of autologous donors with
 2014
cytotoxic substances. The cells may be processed to select a
population of interest and may be cryopreserved.
Bone marrow Bone marrow is harvested by aspirating the
cells from the cavities of hoIlow bones, then removing bone
fragments by filtration and, if necessary, separating the buftY
coat ceIls after centrifugation or with commercial kits based
on the cytapheresis principie. The cells may be processed to
select a population of interest and may be cryopreserved.
Umbzlieal eord blood Placental blood haematopoietic ceIls are
coIlected from placentae via the vein of the umbilical cord.
The ceIls are then cryopreserved.
CRYOPRESERVAnON
Cryopreservation allows storage for long periods. The ceIls
are suspended in a validated medium containing a suitable
cryoprotectant (for example, dimethyl sulfoxide) and
macromolecules (for example, autologous plasmaJalbumin)
and are frozen in cryobags in a manner designed to maintain
viability of the ceIls by controIled cooling according to a
validated method. They are stored at a temperature of
- 140 cC or lower. Where cryobags are stored under other
conditions of temperature and duration, the functionality of
the preparation must be validated. Cryobags from donors
that test positive for any infectious disease marker must be
stored in such a way as to avoid cross-contamination.
SUBSTANCES USED IN PRODUCTION
The quality of substances used in production may be critical
with respect to the quality, safety and efficacy of the final
product, particularIy for substances of biological origino This
is of particular importance for:
- proteins, incIuding enzymes and antibodies;
- cryopreservation reagents;
- purification reagents.
Quality assurance
AIl substances must be produced within a recognised quality
management system using suitable production facilities.
Quality specifications
A suitable quality specification must be presented for each
substance, incIuding notably:
- identity;
- potency (where applicable);
- purity;
- determination of bacterial endotoxins (2.6.14) (where
applicable);
- microbiological quality (total viable count, tests for
specified micro-organisms);
- sterility (2.6.1) (where applicable).
Viral safety
The requirements of chapter 5.1.7 apply.
Transmissible spongiforrn encephalopathies
A risk assessment of the product with respect to transmissible
spongiform encephalopathies is carried out, and suitable
measures are taken to minimise any such risk (5.2.8).
Water
Water used in the preparation of ceIlular products complies
with the relevant monograph (Water fOl· injections (0169),
Water, highly purified (1927), Purified water (0008). Water
incorporated into the final product complies with the section
on Water for injections in bulk in the monograph Water for
injections (0169), and in addition is sterile.
TESTS
Target speeifications are established for the different tests, but these
are not used as rigid acceptance eriteria.
Blood-re1ated Products IV-469
Tests carried out incIude the foIlowing (further tests, such as
purging, ceIl depletion, aIlogeneic application, may be
necessary depending on any treatment applied to the ceIls
and on the intended recipient) :
Nuc1eated cell count (2.7.29).
Viability (2.7.29)
Viability is assessed for products that are not infused within
24 h of coIlection.
CD34+ cell count
For peripheral blood stem ceIls, CD34+ ceIl count is
determined using a validated automated apparatus to analyse
ceIls labeIled with anti-CD34 antibodies. The apparatus and
method employed must be able to determine the number of
CD34+ ceIls with a sensitivity, accuracy and reproducibility
comparable with those of irnmunophenotyping (2.7.23),
where ceIls are labelled using anti-CD34 and anti-CD45
antibodies conjugated to a fiuorochrome and analysed by
fiow cytometry (2.7.24).
Colony-forming cell (CFC) assay (2.7.28)
Proliferative capacity is established by a suitable assay.
The test is not necessarily carried out on each unit.
The correlation between the dose of CD34 and the number
of CFCs in a given situation (pathology, packaging,
mobilisation) is determined. The CFC assay is carried out
periodicaIly; whenever a change that could affect the quality
of CD34+ cells is made to the protocol for packaging or
mobilisation, it is carried out on a suitable number of units.
Microbiological control
Examine as prescribed in general method 2.6.27.
Mierobiologieal control of cellular products. Where justified, the
product may be released before completion of the test.
__________________________________________ ffiE~
Normal Immunoglobulin
*
******
Normal Immunoglobulin Injection *****
(Human Normal Immunoglobulin, Ph Eur monograph 0338)
Ph Eur ___________________________________________
DEFINITION
Human normal immunoglobulin is a sterile liquid or freeze-
dried preparation containing immunoglobulins, mainly
immunoglobulin G (IgG). Other proteins may be presento
Human normal irnmunoglobulin contains the IgG antibodies
of normal subjects. It is intended for intramuscular or
subcutaneous administration. The preparation may contain
excipients such as stabilisers. Multidose preparations contain
an antimicrobial preservative.
Human normal irnmunoglobulin is obtained from plasma
that complies with the requirements of the monograph
Human plasma for fractionation (0853).
PRODUCTION
The method of preparation incIudes a step or steps that have
been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses, it
shaIl have been shown that any residues present in the final
product have no adverse effects on the patients treated with
the immunoglobulin.
For preparations intended for subcutaneous administration,
the method of preparation also incIudes a step or steps that
have been shown to remove thrombosis-generating agents.
Emphasis is given to the identification of activated
IV-470 Blood-related Products
coagulation factors and their zymogens and process steps that
may cause their activation. Consideration is also to be given
to other procoagulant agents that could be introduced by the
manufacturing process.
The product shall have been shown, by suitable tests in
animals and evaluation during clinical trials, to be well
tolerated when administered intramuscularly or
subcutaneously. Any antimicrobial preservative or stabilising
agent used shall have been shown to have no deleterious
effect on the final product in the amount presento
Human normal immunoglobulin is prepared from pooled
material from at least 1000 donors by a method that has
been ShOWll to yield a product that:
- does not transmit infection;
- at a protein concentration of 160 gIL, contains antibodies
for at least 2 of which (1 viral and 1 bacterial) an
International Standard or Reference Preparation is
available, the concentration of such antibodies being at
least 10 times that in the initial pooled material;
- has a defined distribution of IgG subclasses;
- complies with the test for Fc function of irnmunoglobulin
(2. 7.9), if the preparation is intended for subcutaneous
administration.
Human normal irnmunoglobulin is prepared as a stabilised
solution, for example in a 9 gIL solution of sodium chloride,
a 22.5 giL solution of glycine or, if the preparation is to be
freeze-dried, a 60 giL solution of glycine . No antibiotic is
added to the plasma used. Single-dose preparations do not
contain an antirnicrobial preservative. The solution is passed
through a bacteria-retentive filter. The preparation may
subsequently be freeze-dried and the containers closed under
vacuum or under an inert gas.The stability of the preparation
is demonstrated by suitable tests carried out during
development studies.
CHARACTERS
Appearance:
- liquid preparation: clear and colourless or pale-yellow or
light-broWll; during storage it may show formation of
slight turbidity or a small amount of particulate matter.
- freeze-dried preparation: powder or solid, friable mass,
hygroscopic, white or slightly yellow.
For the freeze-dried preparation, reconstitute as stated on the label
immediate1:y before carrying out the identijication and the tests,
except those for solubility and water.
IDENTIFICATION
Examine by a suitable irnmunoelectrophoresis technique.
Using antiserum to normal human serum, compare normal
human serum and the preparation to be examined, both
diluted to a protein concentration of 10 gIL. The main
component of the preparation to be examined corresponds to
the IgG component of normal human serum. The solution
may show the presence of small quantities of other plasma
proteins.
TESTS
Solubility
For the freeze-dried preparation, to a container of the
preparation to be examined add the volume of the liquid
stated on the label at the recommended temperature.
The preparation dissolves completely within 20 min at
20-25 oc.
pH (2.2. 3)
5.0 to 7.2.
Dilute the preparation to be examined with a 9 gIL solution
of sodium chloride R to a protein concentration of 10 giL.
2014
Total protein
The preparation has a protein concentration of not less than
100 giL and not more than 180 gIL and contains not less
than 90 per cent and not more than 110 per cent of the
quantity of protein stated on the labe!'
Dilute the preparation to be examined with a 9 gIL solution
of sodium chloride R to obtain a solution containing about
15 mg of protein in 2 mL. To 2.0 mL of this solution in a
round-bottomed centrifuge tube add 2 mL of a 75 giL
solution of sodium mo1:ybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
liquid and allow the inverted tube to drain on filter papero
Determine the nitro gen in the residue by the method of
sulfuric acid digestion (2.5.9) and calculate the content of
protein by multiplying the result by 6.25.
Protein composition
Examine by zone electrophoresis (2.2.31) .
Use strips of suitable cellulose acetate gel or suitable agarose
gel as the supporting medium and barbital buffer
solution pH 8.6 R1 as the electrolyte solution.
If cellulose acetate is the supporting material, the method
described below can be used. If agarose gels are used, and
because they are normally part of an automated system, the
manufacturer's instructions are followed instead.
Test solution Dilute the preparation to be examined with a
9 giL solution of sodium chloride R to a protein concentration
of 50 giL.
Reference solution Reconstitute human immunoglobulin for
electrophoresis BRP and dilute with a 9 giL solution of sodium
chlonde R to a protein concentration of 50 gIL.
To a strip apply 2.5 ¡.tL of the test solution as a 10 mm band
or apply 0.25 ¡.tL per millimetre if a narrower strip is used.
To another strip apply in the same manner the same volume
of the reference solution. Apply a suitable electric field such
that the albumin band of normal human serum applied on a
control strip migra tes at least 30 mm. Stain the strip with
amido black 10B solution R for 5 min. Decolourise with a
mixture of 10 volumes of glacial acetic acid R and 90 volumes
of methanol R so that the b ackground is just free of colour.
Develop the transparency of the strips with a mixture of
19 volumes of glacial acetic acid R and 81 volumes of
methanol R. Measure the absorbance of the bands at 600 nm
in an instrument having a linear response over the range of
measurement. Calcula te the result as the mean of
3 measurements of each strip .
System suitability In the elecrropherogram obtained with the
reference solution, the proportion of protein in the principal
band is within the limits stated in the leaflet accompanying
the reference preparation.
Results In rhe electropherogram obtained with the test
solution, not more than 10 per cent of protein has a mobility
different from that of the principal bando
Distribution of molecular size
Size exclusion chromatography (2.2.30) .
Test solution Dilute the preparation to be examined with a
9 giL solution of sodium chloride R to a concentration suitable
for the chromatographic system used. A concentration in the
range of 4-12 gIL and injection of 50-600 ¡.tg of protein are
usually suitable.
Reference solution Dilute human immunoglobulin (molecular
size) BRP with a 9 gIL solution of sodium ehloride R to the
same protein concentration as the test solution.
 2014
Column:
- size: 1 = 0.6 m, 0 = 7.5 mm, or 1 = 0.3 m, 0 = 7.8 mm;
- stationary phase: hydrophilic silica gel for chromatography R,
of a grade suitable for fractionation of globular proteins
with relative molecular masses in the range 10 000 to
500000.
Mobtle phase Dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1.741 g of sodium dihydrogen phosphate
monohydrate R, 11.688 g of sodium chloride R and 50 mg of
sodium azide R in 1 L of water R.
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 280 nm.
In the chromatogram obtained with the reference solution,
the principal peak corresponds to the IgG monomer and
there is a peak corresponding to the dimer with a relative
retention to the principal peak of about 0.85. Identify the
peaks in the chromatogram obtained with the test solution by
comparison with the chromatogram obtained with the
reference solution; any peak with a retention time less than
that of the dimer corresponds to polymers and aggregates.
Results In the chromatogram obtained with the test
solution:
- retention time: for the monomer and for the dimer, the
retention time relative to the corresponding peak in the
chromatogram obtained with the reference solution is 1
± 0.02;
- peak area: the sum of the peak areas of the monomer and
the dimer represent not less than 85 per cent of the total
area of the chromatogram and the sum of the peak areas
of polymers and aggregates represents not more than
10 per cent of the total area of the chromatogram.
Anti-A and anti-B haemagglutinins (2.6.20, method B)
If human normal immunoglobulin is intended for
subcutaneous administration, it complies with the test.
Anti-D antibodies (2.6.26)
If human normal immunoglobulin is intended for
subcutaneous administration, it complies with the test.
Antibody to hepatitis B surface antigen
Minimum 0.5 IU per gram of immunoglobulin, determined
by a suitable irnmunochemical method (2.7.1).
Antibody to hepatitis A virus
If intended for use in the prophylaxis of hepatitis A, it
complies with the following additional requirement.
Determine the antibody content by comparison with a
reference preparation calibrated in Intemational Units, using
an immunoassay of suitable sensitivity and specificity (2.7.1).
The Intemational Unit is the activity contained in a stated
amount of the Intemational Standard for anti-hepatitis A
immunoglobulin. The equivalence in Intemational Units of
the Intemational Standard is stated by the World Health
Organization.
Human hepatitis A immunoglobulin BRP is calibrated in
Intemational Units by comparison with the Intemational
Standard.
The stated potency is not les s than 100 IU/mL.
The estimated potency is not les s than the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
Immunoglobulin A
As determined by a suitable irnmunochemical method
(2.7.1), the content of irnmunoglobulin A is not greater than
the maximum content stated on the labe!'
Blood-related Products IV-471
Water
Determined by a suitable method, such as the semi-micro
determination ofwater (2.5.12), loss on drying (2.2.32) or
near infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test for sterility.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject 1 mL per kilogram of the rabbit's
mass.
Where the bacterial endotoxin test is used, the product
contains less than 5 IU of endotoxin per millilitre.
STORAGE
Liquid preparation In a colourless glass container, protected
from light.
Freeze-dried preparatíon In an airtight, colourless glass
container, protected from light.
LABELLING
The labe! sta tes:
- for liquid preparations, the volume of the preparation in
the container and the protein content expressed in grams
per litre;
- for freeze-dried preparations, the quantity of protein in
the container;
- the route of administration;
- for freeze-dried preparations, the name or composition
and the volume of the reconstituting liquid to be added;
- the distribution of subclasses of IgG present in the
preparation;
- where applicable, that the preparation is suitable for use
in the prophylaxis of hepatitis A infection;
- where applicable, the anti-hepatitis A virus activity in
Intemational Units per millilitre;
- where applicable, the name and amount of antimicrobial
preservative in the preparation;
- the maximum content of irnmunoglobulin A.
__________________________________________ ~E~
Normal Immunoglobulin for ***
*** ***
Intravenous Use ***
(Human Normal Immunoglobulin for Intravenous
Administration, Ph Eur monograph 0918)
~E~ ____________________________________________
DEFINITION
Human normal irnmunoglobulin for intravenous
administration is a sterile liquid or freeze-dried preparation
containing irnmunoglobulins, mainly immunoglobulin G
(IgG). Other proteins may be presento Human normal
immunoglobulin for intravenous administration contains the
IgG antibodies of normal subjects. This monograph does not
apply to products intentionally prepared to contain fragments
or chemically modified IgG.
Human normal immunoglobulin for intravenous
administration is obtained from plasma that complies with
the requirements of the monograph Human plasma for
fractionatíon (0853) . The preparation may contain excipients
such as stabilisers .
 IV-472 Blood-related Products
PRODUCTION
The method of preparation includes a step or steps that have
been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses, it
shall have been shown that any residues present in the final
product have no adverse effects on the patients treated with
the immunoglobulin. The method of preparation also
includes a step or steps that have been shown to remove
thrombosis-generating agents. Emphasis is given to the
identification of activated coagulation factors and their
zymogens and process steps that may cause their activation.
Consideration is also to be given to other procoagulant
agents that could be introduced by the manufacruring
process .
The product shall have been shown, by suitable tests in
animals and evaluation during clinical trials, to be well
tolerated when administered intravenously.
Human normal immunoglobulin for intravenous
administration is prepared from pooled material from not
fewer than 1000 donors by a method that has been shown to
yield a product that:
- does not transmit infection;
- at an immunoglobulin concentration of 50 gIL, contains
antibodies for at least 2 of which (1 viral and 1 bacterial)
an International Standard or Reference Preparation is
available, the concentration of such antibodies being at
least 3 times that in the initial pooled material;
- has a defined distribution of immunoglobulin G
subclasses;
- complies with the test for Fc function of immunoglobulin
(2.7.9);
- do es not exhibit thrombogenic (procoagulant) activity.
Human normal immunoglobulin for intravenous
administration is prepared as a stabilised solution or as a
freeze-dried preparation. In both cases the preparation is
passed through a bacteria-retentive filter. The preparation
may subsequently be freeze-dried and the containers closed
under vacuum or under an inert gas. No antibiotic is added
to the plasma used. No antimicrobial preservative is added
either during fractionation or at the stage of the final bulk
solution.
The stability of the preparation is demonstrated by suitable
tests carried out during development studies.
CHARACTERS
Appearance:
- liquid preparation: clear or slightly opalescent and
colourless or pale yellow liquidó
- freeze-dried preparation: hygroscopic, white or slightly
yellow powder or solid friable mass.
For the freeze-dried preparation, reeonstitute as stated on the label
immediately before earrying out the identifieation and the tests,
exeept those for solubility and water.
IDENTIFICATION
Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal human serum, compare normal
human serum and the preparation to be examined, both
diluted to contain 10 giL of protein. The main component of
the preparation to be examined corresponds to the IgG
component of normal human serum. The preparation to be
examined may show the presence of small quantities of other
plasma proteins; if human albumin has been added as a
stabiliser, it may be seen as a major component.
2014
TESTS
SoIubility
For the freeze-dried preparation, add to the container the
volume of the liquid stated on the label at the recommended
temperarure. The preparation dissolves completely within
30 min at 20-25 oc.
pH (2.2.3)
4.0 to 7.4.
Dilute the preparation to be examined with a 9 gIL solution
of sodium ehloride R to obtain a solution containing 10 giL of
protein.
Osmolality (2.2.35)
Minimum 240 mosmol/kg.
Total protein
The preparation contains not less than 30 gIL and between
90 per cent and 1 10 per cent of the quantity of protein
stated on the labe!'
Dilute the preparation to be examined with a 9 gIL solution
of sodium ehloride R to obtain a solution containing about
15 mg of protein in 2 mL. To 2.0 mL of this solution in a
round-bottomed centrifuge tube add 2 mL of a 75 giL
solution of sodium molybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfurie aeid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
liquid and allow the inverted rube to drain on filter paper.
Determine the nitro gen in the centrifugation residue by the
method of sulfuric acid digestion (2.5.9) and calculate the
content of protein by multiplying the result by 6.25 .
Protein composition
Zone electrophoresis (2.2.31) .
U se strips of suitable cellulose acetate gel or suitable agarose
gel as the supporting medium and barbital buffer solution
pH 8.6 Rl as the electrolyte solution.
If cellulose acetate is the supporting material, the method
described below can be used. If agarose gels are used, and
because they are normally part of an automated system, the
manufacrurer's instructions are followed instead.
Test solution Dilute the preparation to be examined with a
9 giL solution of sodium ehloride R to an immunoglobulin
concentration of 30 gIL.
Referenee solution Reconstitute human immunoglobulin for
electrophoresis BRP and dilute with a 9 giL solution of sodium
ehloride R to a protein concentration of 30 gIL.
To a strip apply 4.0 ¡.¡L of the test solution as a 10 mm band
or apply 0.4 ¡.¡L per millimetre if a narrower strip is used.
To another strip apply in the same manner the same volume
of the reference solution. Apply a suitable electric field such
that the albumin band of normal human serum applied on a
control strip migra tes at least 30 mm. Stain the strips with
amido blaek lOB solution R for 5 mino Decolourise with a
mixture of 10 volumes of glacial aeetie acid R and 90 volumes
of methanol R so that the background is just free of colour.
Develop the transparency of the strips with a mixture of
19 volumes of glacial aeetie acid R and 81 volumes of
methanol R. Measure the absorbance of the bands at 600 nm
in an instrument having a linear response over the range of
measurement. Calculate the result as the mean of 3
measurements of each strip.
System suitability In the electropherogram obtained with the
reference solution, the proportion of protein in the principal
band is within the limits stated in the leaflet accompanying
the reference preparation.
 2014
Results In the electropherogram obtained with the test
solution, not more than 5 per cent of protein has a mobility
different from that of the principal bando This limit is not
applicable if albumin has been added to the preparation as a
stabiliser; for such preparations, a test for protein
composition is carried out during manufacture before
addition of the srabiliser.
Molecular size distribution
Size exclusion chromatography (2.2.30).
Test solution Dilute the preparation to be examined with a
9 gIL solution of sodium chloride R to a concentration suitable
for the chromatographic system used. A concentrarion in the
range of 4-12 gIL and injection of 50-600 ~lg of protein are
usually suitable.
Reference solution Dilute human immunoglobulin (molecular
size) BRP with a 9 gIL solution of sodium chloride R to the
same prorein concentration as the test solution.
Column:
- size: 1 = 0.6 m, 0 = 7.5 mm, or 1= 0.3 m, 0 = 7.8 mm;
- stationary phase: hydrophilic siliea gel for chromatography R
of a grade suitable for fractionation of globular proteins
with relative molecular mas ses in the range 10 000 to
500000.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1.741 g of sodium dihydrogen phosphate
monohydrate R, 11.688 g of sodium chloride R and 50 mg of
sodium azide R in 1 L of water R.
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 280 nm.
Identification of peaks In the chromatogram obtained with
the reference solution, the principal peak corresponds to the
IgG monomer and there is a peak corresponding to the
dimer with a relative retention to the principal peak of about
0.85; identify the peaks in the chromatogram obtained with
the test solution by comparison with the chromatogram
obtained with the reference solution; any peak with a
retention time shorter than that of the dimer corresponds to
polymers and aggregates.
Results In the chromatogram obtained with the test
solution:
- retention time: for the monomer and for the dimer, the
retention time relative to the corresponding peak in the
chromatogram obtained with the reference solution is 1
± 0.02;
- peak area: the sum of the peak areas of the monomer and
the dimer represent not less than 90 per cent of the total
area of the chromatogram and the sum of the peak areas
of polymers and aggregates represents not more than
3 per cent of the total are a of the chromatogram. This
requirement does not apply to products where albumin
has been added as a stabiliser; for products stabilised with
albumin, a test for distribution of molecular size is carried
out during manufacture before addition of the stabiliser.
Anticomplementary activity (2.6. 17)
The consumption of complement is not greater than
50 per cent (1 CH 50 per milligram of immunoglobulin).
Prekallikrein activator (2.6.15)
Maximum 35 IU!mL, calculated with reference to a dilution
of the preparation to be examined containing 30 gIL of
irnmunoglobulin.
Anti-A and anti-B haemagglutinins (2.6.20, method B)
It complies with the test for anti-A and anti-B
haemagglutinins (direct method).
Blood-related Products IV-473
Anti-D antibodies (2.6.26)
It complies with the test for anti-D antibodies in human
immunoglobulin.
Antibody to hepatitis B surface antigen
Mínimum 0.5 IU per gram of irnmunoglobulin, determined
by a suitable irnmunochemical method (2. 7. 1) .
Immunoglobulin A
As determined by a suitable irnmunochemical method
(2.7.1), the content of irnmunoglobulin A is not greater than
the maximum content stated on the labe!'
Water
Determined by a suitable method, such as the semi-micro
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacteria! endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
For the pyrogen test, inject per kilogram of the rabbit's mas s
a volume equivalent to 0.5 g of immunoglobulin, but not
more than 10 mL per kilogram of the rabbit's mass.
Where the bacterial endotoxin test is used, the preparation to
be examined contains less than 0.5 IU of endotoxin per
millilitre for solutions with a protein content not greater than
50 gIL, and less than 1.0 IU of endotoxin per millilitre for
solutions with a protein content greater than 50 gIL but not
greater than 100 gIL.
STORAGE
Liquid preparation In a colourless glass container, protected
from light, at the temperature stated on the labe!'
Freeze-dried prepararion In an airtight colourless glass
container, protected from light, at a temperature not
exceeding 25 oC.
LABELLING
The label states:
- for liquid preparations, the volume of the preparation in
the container and the protein content expressed in grams
per litre;
- for freeze-dried preparations, the quantity of prorein in
the container;
- the amount of immunoglobulin in the container;
- the route of administration;
- for freeze-dried preparations, the name or composition
and the volume of the reconstituting liquid to be added;
- the distribution of subclasses of immunoglobulin G
present in the preparation;
- where applicable, the amount of albumin added as a
stabiliser;
- the maximum content of immunoglobulin A.
__________________________________________ ~E~
 IV-474 Blood-re1ated Products
*****
Anti-D (Rh o) Immunoglobulin
** **
(Human Anti-D Immunoglobulin, ***
Ph Bur monograph 0557)
~E~ ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
irnmunoglobulins, mainly irnmunoglobulin G .
The preparation is intended for intramuscular administration.
It contains specific antibodies against erythrocyte D-antigen
and may also contain small quantities of other blood-group
antibodies. Human normal immunoglobulin (0338) andlor
Human albumin solution (0255) may be added.
It complies with the monograph Human normal
immunoglobulin (0338), except for the minimum number of
donors and the minimum total protein contento
The test for anti-D antibodies (2.6.26) prescribed in the
monograph Human normal immunoglobulin (0338) is not
carried out, since it is replaced by the assay of human anti-D
irnmunoglobulin (2.7.13) as prescribed below under Potency.
For products prepared by a method that eliminates
irnmunoglobulins with specificities other than anti-D, where
authorised, the test for antibodies to hepatitis B surface
antigen is not required.
PRODUCTION
Human anti-D irnmunoglobulin is preferably obtained from
the plasma of donors with a sufficient titre of previously
acquired anti-D antibodies. Where necessary, in order to
ensure an adequate supply of human anti-D
irnmunoglobulin, it is obtained from plasma derived from
donors immunised with D-positive erythrocytes that are
compatible in relevant blood group systems in order to avoid
formation of undesirable antibodies.
ERYTHROCYTE DONORS
Erythrocyte donors comply with the requirements for donors
prescribed in the monograph Human plasma for fractionation
(0853).
IMMUNISATION
Irnmunisation of the plasma donor is carried out under
proper medical supervision. Recommendations concerning
donor immunisation, including testing of erythrocyte donors,
have been formulated by the World Health Organization
(Requirements for the collection, processing and quality control of
blood, blood components and plasma derivatives, WHO
Technical Report Series, No. 840, 1994 or subsequent
revision).
POOLED PLASMA
To limit the potential B19 virus burden in plasma pools used
for the manufacture of anti-D immunoglobulin, the plasma
pool is tested for B 19 virus using validated nucleic acid
amplification techniques (2.6.21).
B19 virus DNA
Maximum 10.0 IU/IlL.
A positive control with 10.0 IU ofB19 virus DNA per
microlitre and, to test for inhibitors, an intemal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the
positive control is non-reactive or if the result obtained with
the intemal control indica tes the presence of inhibitors.
B19 virus DNA for NAT testing BRP is suitable for use as a
positive control.
If Human normal immunoglobulin (0338) andlor Human
albumin solution (0255) are added to the preparation, the
2014
plasma pool or pools from which they are derived comply
with the aboye requirement for B19 virus DNA.
POTENCY
Human anti-D immunoglobulin (2.7.13, Method A)
The estimated potency is not less than 90 per cent of the
stated potency. The confidence limits (P = 0.95) are not les s
than 80 per cent and not more than 120 per cent of the
estimated potency.
Method B or e (2.7.13) may be used for potency
determination if a satisfactory correlation with the results
obtained by Method A has been established for the particular
producto
STORAGE
See Human normal immunoglobulin (0338).
LABELLING
See Human normal immunoglobulin (0338).
The label states the number of Intemational Units per
container.
____________________________________________ ~E~
Anti-D Immunoglobulin for
Intravenous Use
(Human Anti-D Immunoglobulin for Intravenous
Administration, Ph Bur monograph 1527)
Ph Eur ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G. It contains
specific antibodies against erythrocyte D-antigen and may
also contain small quantities of other blood-group antibodies.
Human normal immunoglobulin for intravenous
administration (0918) and/or Human albumin solution (0255)
may be added.
It complies with the monograph Human normal
immunoglobulin for intravenous administration (0918), except
for the minimum number of donors, the minimum total
protein content, the limit for osmolality and the limit for
prekallikrein activator.
The test for anti-D antibodies (2.6.26) prescribed in the
monograph Human normal immunoglobulin for intravenous
administration (0918) is not carried out, since it is replaced by
the assay of human anti-D immunoglobulin (2.7.13) as
prescribed below under Potency.
For products prepared by a method that eliminates
immunoglobulins with specificities other than anti-D, where
authorised, the test for antibodies to hepatitis B surface
antigen is not required; a suitable test for Fc function is
carried out instead of that described in general chapter 2.7.9,
which is not applicable to such a product.
PRODUCTION
Human anti-D immunoglobulin is preferably obtained from
the plasma of donors with a sufficient titre of previously
acquired anti-D antibodies. Where necessary, in order to
ensure an adequate supply of human anti-D
immunoglobulin, it is obtained from plasma derived from
donors immunised with D-positive erythrocytes that are
compatible in relevant blood group systems in order to avoid
formation of undesirable antibodies .
2014
ERYTHROCYTE DONORS
Erythrocyte donors comply with the requirements for donors
prescribed in the monograph Human plasma for fractionation
(0853).
IMMUNISA TION
Immunisation of the plasma donor is carried out under
proper medical supervision. Recommendations concerning
donor immunisation, including testing of erythrocyte donors,
have been formulated by the World Health Organization
(R equirements for the collection, processing and quality control of
blood, blood componems and plasma derivatives, WHO
T echnical Report Series, No. 840, 1994 or subsequent
revision) .
POOLED PLASMA
To limit the potential B19 virus burden in plasma pools used
for the manufacture of anti-D immunoglobulin, the plasma
pool is tested for B 19 virus using validated nucleic acid
amplification techniques (2. 6.21).
B19 virus DNA
Maximum 10.0 IU/¡.tL.
A positive control with 10.0 IU ofB19 virus DNA per
micro litre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the
positive control is non-reactive or if the resuIt obtained with
the internal control indicates the presence of inhibitors.
B 19 virus DNA for NA T testing BRP is suitable for use as a
positive control.
If Human normal immunoglobulin for imravenous
administration (0918) and/or Human albumin solution (0255)
are added to the preparation, the plasma pool or pools from
which they are derived comply with the aboye requirement
for B 19 virus DNA.
ASSAY
Human anti-D immunoglobulin (2.7.13, Method A)
The estimated potency is not less than 90 per cent of the
stated potency. The confidence limits (P = 0.95) are not less
than 80 per cent and not more than 120 per cent of the
estirnated potency.
Method B or e (2.7.13) may be used for potency
determination if a satisfactory correlation with the resuIts
obtained by Method A has been established for the particular
product.
STORAGE
See Human normal immunoglobulin for intravenous
administration (0918).
LABELLING
See Human normal immunoglobulin for imravenous
administration (0918).
The label states the number of International Units per
container.
____________________________________________ PhE~
Blood-related Products IV-475
***
Hepatitis A Immunoglobulin
*** ***
(Human Hepatitis A Immunoglobulin, ***
Ph Eur monograph 0769)
~E~ ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma from selected donors having
antibodies against hepatitis A virus. Human normal
immunoglobulin (0338) may be added.
Ir complies with the monograph on Human normal
immunoglobulin (0338), except for the minimum number of
donors and the minimum total protein contento
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
reference preparation calibrated in International Units, using
an immunoassay of suitable sensitivity and specificity (2.7.1).
The International Unit is the activity contained in a stated
amount of the International Standard for anti-hepatitis A
immunoglobulin. The equivalence in International Units of
the International Standard is stated by the World Health
Organization.
Human hepatitis A immunoglobulin BRP is calibrated in
International Units by comparison with the International
Standard.
The stated potency is not less than 600 IU/mL.
The estirnated potency is not less than the stated potency.
The confidence lirnits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
STORAGE
See Human normal immunoglobulin (0338).
LABELLING
See Human normal immunoglobulin (0338).
The label states the number of International Units per
container.
____________________________________________ ~E~
***
Hepatitis B Immunoglobulin * *
(Human Hepatitis B Immunoglobulin,
* ******
Ph Eur monograph 0722)
PhE~ ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma from selected and/or immunised
donors having antibodies against hepatitis B surface antigen.
Human normal immunoglobulin (0338) may be added.
It complies with the monograph on Human normal
immunoglobulin (0338), except for the minimum number of
donors and the minimum total protein contento
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin ro be examined with that of a
IV-476 Blood-related Products
reference preparation calibrated in Intemational Units, using
an immunoassay of suitable sensitivity and specificity (2.7.1).
The Intemational Unit is the activity contained in a stated
amount of the Intemational Reference Preparation of
hepatitis B immunoglobulin. The equivalence in Intemational
Units of the Intemational Reference Preparation is stated by
the World Health Organization.
The stated potency is not less than 100 IU/mL.
The estimated potency is not les s than the stated potency.
The confidence limits (P = 0.95) are not les s than
80 per cent and not more than 125 per cent of the estimated
potency.
STORAGE
See Human normal immunoglobulin (0338).
LABELLING
See Human nonnal immunoglobulin (0338).
The label states the number of Intemational Units per
container.
____________________________________________ PhEw
Hepatitis B Immunoglobulin for
Intravenous Use
(Human Hepatitis B Immunoglobulin for Intravenous
Administration, Ph Bur monograph 1016)
~ Ew ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G. It is obtained
from plasma from selected andlor immunised donors having
antibodies against hepatitis B surface antigen. Human nonnal
immunoglobulin for intravenous administration (0918) may be
added.
It complies with the monograph Human nonnal
immunoglobulin for intravenous administration (0918), except
for the minimum number of donors, the minimum total
protein content and the limit for osmolality.
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
reference preparation calibrated in Intemational Units, using
an immunoassay (2.7.1) of suitable sensitivity and specificity.
The Intemational Unit is the activity contained in a stated
amount of the Intemational Reference Preparation of
hepatitis B immunoglobulin. The equivalence in Intemational
Units of the Intemational Reference Preparation is stated by
the World Health Organization.
The stated potency is not less than 50 IU/rnL. The estimated
potency is not less than the stated potency. The confidence
limits (P = 0.95) are not less than 80 per cent and not more
than 125 per cent of the estimated potency.
STORAGE
See Human normal immunoglobulin for intravenous
administration (0918).
LABELLING
See Human nonnal immunoglobulin for intravenous
administration (0918).
The label states the minimum number of Intemational Units
of hepatitis B immunoglobulin per container.
____________________________________________ ~Ew
2014
Measles Immunoglobulin
(Human Measles Immunoglobulin,
Ph Bur monograph 0397)
~Ew ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma containing specific antibodies
against measles virus. Human nonnal immunoglobulin (0338)
may be added.
It complies with the monograph on Human nonnal
immunoglobulin (0338), except for the minimum number of
donors and the minimum total protein contento
POTENCY
The potency of the liquid preparation and of the freeze-dried
preparation after reconstitution as stated on the label is not
less than 50 IU per millilitre of neutralising antibody against
measles virus.
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
reference preparation calibrated in Intemational Units, using
a challenge dos e of measles virus in a suitable cell culture
system. A method of equal sensitivity and precision may be
used providing that the competent authority is satisfied that it
correlates with neutralising activity for the measles virus by
comparison with the reference preparation.
The Intemational Unit is the specific neutralising activity for
measles virus contained in a stated amount of the
Intemational Standard for human anti-measles serum.
The equivalence in Intemational Units of the Intemational
Reference Preparation is stated by the World Health
Organization.
Method
Prepare serial 2-fold dilutions of the immunoglobulin to be
examined and of the reference preparation. Mix each dilution
with an equal volume of a suspension of meas les virus
containing about 100 CCID so in 0.1 mL and incubate
protected from light at 37 oC for 2 h. Using not fewer than
6 cell cultures per mixture, inoculate 0.2 mL of each mixture
into each of the cell cultures allocated to that mixture and
incubate for not les s than 10 days. Examine the cultures for
viral activity and compare the dilution containing the smallest
quantity of the immunoglobulin which neutralises the virus
with that of the corresponding dilution of the reference
preparation.
Calculate the potency of the immunoglobulin to be examined
in Intemational Units per millilitre of neutralising antibody
against measles virus.
STORAGE
See Human nonnal immunoglobulin (0338).
LABELLING
See Human nonnal immunoglobulin (0338) .
The labe! states the number of Intemational Units per
container.
____________________________________________ ~Em
 2014
******
Human cx-1-proteinase Inhibitor
**
(Ph Eur monograph 2387) *** *
~E~ ____________________________________________
DEFINITION
Human cx-l-proteinase inhibitor is a plasma protein fraction
containing mainly human cx-l-proteinase inhibitor (also
known as human a-l-antitrypsin or a-l-antiproteinase) .
Human a-l-proteinase inhibitor is a glycoprotein existing in
isoforms with different isoelectric points and is the most
abundant multifunctional serine proteinase inhibitor in
human plasma. It is obtained from human plasma that
complies with the monograph Human plasma for fractionation
(0853), using a suitable fractionation process and further
purification steps. Other plasma proteins may be presento
PRODUCTION
GENERAL PROVISIONS
The method of preparation ineludes steps that have been
shown to remove or to inactivate known agents of infection.
The subsequent purification procedure must be validated to
demonstrate that the concentration of any substances used
for inactivation of virus es during production is reduced to a
suitable level and that any residues are such as not to
compromise the safety of the preparation for patients.
The specific activity is not less than 0.35 mg of active human
a-l-proteinase inhibitor per milligram of total protein. Ratio
of human a-l proteinase inhibitor activity to human a-l-
proteinase inhibitor antigen is not less than 0.7.
Buffering and other auxiliary substances such as a stabiliser
may be ineluded. No antimicrobial preservative is added.
The solution is passed through a bacteria-retentive filter and
distributed aseptically into the final containers. The product
may be freeze-dried.
CONSISTENCY OF THE METHOD OF PRODUCTION
The consistency of the method of production, ineluding
demonstration that the manufacturing process yields a
product with a consistent composition and maintains the
functional integrity of human a-l-proteinase inhibitor, is
evaluated by suitable analytical procedures that are
determined during process development, and which inelude:
-- assay of human a-l-proteinase inhibitor activity;
-- determination ofspecific human a-l-proteinase inhibitor
activity, expressed as the ratio of active human li-l-
proteinase inhibitor to total protein;
-- characterisation of isoform composition and protein
structure by suitable methods such as isoelectric focusing
(2.2.54), spectrometric methods (for example, mass
spectrometry) or capillary electrophoresis (2.2.47);
determination of the ratio of human cx-l-proteinase
inhibitor activity to human a-l-proteinase inhibitor
antigen;
-- characterisation of accompanying plasma proteins that
might be present, by a set of suitable methods su eh as
SDS-PAGE, cellulose acetate electrophoresis or capillary
zone electrophoresis (2.2.31) and quantitative
determination of relevant accompanying plasma proteins;
-- determination of molecular-size distribution, used to
quantify the polymeric forms of human a-l-proteinase
inhibitor; consideration is given to the potential presence
of accompanying proteins that might affect the results.
CHARACTERS
Appearance
Freeze-dried products are hygroscopic, white or pale yellow
or pale brown powders or friable solids; liquid products are
Blood-related Products IV-477
elear or slightly opalescent, colourless or pale yellow or pale
green or pale brown.
1f the preparation ca be examined is freez e-dried, reconstitute it as
staced on the label immediately befare carrying out the
identification, tests (except those for solubility and water) and
assay.
IDENTIFICATION
The assay of human a-l-proteinase inhibitor activity serves to
identify the preparation.
TESTS
pH (2.2.3)
6.5 to 7.8.
Solubility
To a container of the preparation to be examined add the
volume of the liquid stated on the label at room temperature.
The preparation dissolves completely when reconstituted
according to the instructions for use, giving a elear, colourless
or pale green or pale yellow or pale brown solution.
Osmolality (2.2.35)
Minimum 210 mosmol/kg.
Total protein
Dilute the preparation to be examined with a 9 gIL solution
of sodium chloride R to obtain a solution containing about
15 mg of protein in 2 mL. To 2.0 mL of this solution in a
round-bottomed centrifuge tube add 2 mL of a 75 gIL
solution of sodium molybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
and allow the inverted tube to drain on filter paper.
Determine the nitrogen in the residue by the method of
sulfuric acid digestion (2.5.9) and calculate the protein
content by multiplying by 6.25.
Water
Determined by a suitable method, such as the semi-micro
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8)
It complies with the test. Inject per kilogram of the rabbit's
mass a volume equivalent to not les s than 60 mg of human
a-l-proteinase inhibitor.
ASSAY
Carry out the assay of human a-l-proteinase inhibitor
(2.7.32). The estimated potency is not less than 80 per cent
and not more than 120 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
STORAGE
Unless otherwise justified and authorised, in an airtight and
sterile container, at a temperature not exceeding 25 oC.
LABELLING
The label states:
-- the potency of active (functional) human cx-l-proteinase
inhibitor per container;
-- the name and quantity of any added substances;
-- the quantity of protein per container;
-- where applicable, the name and volume of the liquid to
be used for reconstitution;
 IV-478 Blood-related Products
-- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
____________________________________________ ~Ew
Rabies Irnrnunoglobulin *****
** **
(Human Rabies Immunoglobulin> ***
Ph Eur monograph 0723)
____________________________________________
~Ew
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma from donors irnmunised against
rabies. It contains specific antibodies neutralising the rabies
virus. Human normal immunoglobulin (0338) may be added.
It complies with the monograph on Human normal
immunoglobulin (0338), except for the minimum number of
donors and the minimum total protein content.
POTENCY
The potency is determined by comparing the dose of
irnmunoglobulin required to neutralise the infectivity of a
rabies virus suspension with the dose of a reference
preparation, calibrated in Intemational Units, required to
produce the same degree of neutralisation (2. 7.1). The test is
performed in sensitive cell cultures and the presence of
unneutralised virus is revealed by immunofluorescence.
The Intemational Unit is the specific neutralising activity for
rabies virus in a stated amount of the Intemational Standard
for anti-rabies irnmunoglobulin. The equivalence in
Intemational Units of the Intemational Standard is stated by
the World Health Organization.
Human rabies immunoglobulin BRP is calibrated in
Intemational Units by comparison with the Intemational
Standard.
Method
Carry out the test in suitable sensitive cells. It is usual to use
the BHK-21 cellline, grown in the medium described below,
between the 18th and 30th passage levels counted from the
ATCC seed lot. Harvest the cells after 2 to 4 days of growth,
treat with trypsin and prepare a suspension containing
500 000 cells per millilitre (cell suspension). 10 min before
using this suspension add 10 flg of diethylamino-ethyldextran R
per millilitre, if necessary, to increase the sensitivity of the
cells.
Use a fixed virus strain grown in sensitive cells, such as the
CVS strain of rabies virus adapted to growth in the BHK-
21 cellline (seed virus suspension). Estimate the titre of the
seed virus suspension as follows.
Prepare a series of dilutions of the viral suspension. In the
chambers of cell-culture slides (8 chambers per slide), place
0.1 mL of each dilution and 0.1 mL of medium and add
0.2 mL of the cell suspension. Incubate in an atrnosphere of
carbon dioxide at 37 oC for 24 h. Carry out fixation,
irnmunofluorescence staining and evaluation as described
below. Determine the end-point titre of the seed virus
suspension and prepare the working virus dilution
corresponding to 100 CCIDso per 0.1 mL.
For each assay, check the amount of virus used by
performing a control titration: from the dilution
2014
corresponding to 100 CCID so per 0.1 mL, make 3 tenfold
dilutions. Add 0.1 mL of each dilution to 4 chambers
containing 0.1 mL of medium and add 0.2 mL of the cell
suspension. The test is not valid unless the titre lies between
30 CCID so and 300 CCID so .
Dilute the reference preparation to a concentration of
2 IU/mL using non-supplemented culture medium (stock
reference dilution, stored below - 80 OC). Prepare 2 suitable
predilutions (1:8 and 1: 1O) of the stock reference dilution so
that the dilution of the reference preparation that reduces the
number of fluorescent fields by 50 per cent lies within the
4 dilutions of the cell-culture slide. Add 0.1 mL of the
medium to each chamber, except the first in each of 2 rows,
to which add respectively 0.2 mL of the 2 predilutions of the
stock reference dilution transferring successively 0.1 mL to
the other chambers.
Dilute the preparation to be examined 1 in 100 using non-
supplemented medium (stock immunoglobulin dilution) - to
reduce to a minimum errors due to viscosity of the undiluted
preparation - and make 3 suitable predilutions so that the
dilution of the preparation to be examined that reduces the
number of fluorescent fields by 50 per cent lies within the
4 dilutions of the cell-culture slide. Add 0.1 mL of the
medium to all the chambers except the first in each of 3
rows, to which add respectively 0.2 mL of the 3 predilutions
of the stock irnmunoglobulin dilution. Prepare a series of
2-fold dilutions transferring successively 0.1 mL to the other
chambers.
To all the chambers containing the dilutions of the reference
preparation and the dilutions of the preparation to be
examined, add 0.1 mL of the virus suspension corresponding
to 100 CCID so per 0.1 mL (working virus dilution), shake
manually, allow to stand in an atrnosphere of carbon dioxide
at 37 oC for 90 min, add 0.2 mL of the cell suspension,
shake manually and allow to stand in an atrnosphere of
carbon dioxide at 37 oC for 24 h.
After 24 h, discard the medium and remove the plastic walls .
Wash the cell monolayer with phosphate buffered sahne
pH 7.4 R and then with a mixture of 20 volumes of water R
and 80 volumes of acetone R and fix in a mixture of
20 volumes of water R and 80 volumes of acetone R at
-20 oC for 3 mino Spread on the slides fluorescein-conJugated
rabies antiserum R ready for use. Allow to stand in an
atrnosphere with a high level of moisture at 37 oC for
30 mino Wash with phosphate buffered saline pH 7.4 R and
dry. Examine 20 fields in each chamber at a magnification of
250 x, using a microscope equipped for fluorescence
readings. Note the number of fields with at least 1
fluorescent cell. Check the test dose used in the virus
titration slide and determine the dilution of the reference
preparation and the dilution of the preparation to be
examined that reduce the number of fluorescent fields by
50 per cent, calculating the 2 or 3 dilutions together using
probit analysis. The test is not valid unless the statistical
analysis shows a significant slope of the dose-response curve
and no evidence of deviation from linearity or parallelism.
The stated potency is not less than 150 IU/mL.
The estirnated potency is not les s than the stated potency
and is not greater than twice the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
2014 Blood-related Products IV -479

CULTURE MEDIUM FOR GROWTH OF BHK-21 It complies with the monograph on H uman normal
CELLS immunoglobulin (0338), except for the minimum number of
Commercially available media that have a slightly difierent donors and the minimum total protein contento
composition ¡rom that shown below may also be used. POTENCY
Sodium ehloride 6.4 g The potency is determined by comparing the activity of the
Potassium ehloride 0.40 g preparation 10 be examined in a suitable haemagglutination-
Calcium ehloride, anhydrous 0.20 g inhibition test with that of a reference preparation calibrated
Magnesium sulfate, heptahydrate 0.20 g in Intemational Units .
Sodium dihydrogen phosphate, monohydrate 0.124 g
The Intemational Unit is the activity contained in a stated
Glueose monohydrate 4.5 g
amount of the Intemational Standard for anti-rubella
Ferrie nitra te, nonahydrate 0.10 mg
immunoglobulin. The equivalence in Intemational Units of
L-Arginine hydroehloride 42.0 mg
the Intemational Reference Preparation is stated by the
L-Cystine 24.0 mg
World Health Organization.
L-Histidine 16.0 mg
L-Isoleueine 52.0 mg The estimated potency is not les s than 4500 IU/mL.
L-Leueine 52.0 mg The confidence limits (P = 0.95) of the estimated potency
L-Lysine hydroehloride 74.0 mg are not less than 50 per cent and not more than 200 per cent
L-Phenylalanine 33.0 mg of the stated potency.
L-Threonine 48.0 mg STORAGE
L-Tryptophan 8.0 mg See Human normal immunoglobulin (0338).
L-Tyrosine 36.0 mg
L-Valine 47.0 mg LABELLING
L-Methionine 15.0 mg See Human nonnal immunoglobulin (0338) .
L-Glutamine 0.292 g The label states the number of Intemational Units per
i-Inositol 3.60 mg millilitre.
Choline ehloride 2.0 mg ____________________________________________ ~E~

Folie aeid 2.0 mg

Nicotinamide 2.0 mg
Calcium pantothenate 2.0 mg
Pyridoxal hydrochloride 2.0 mg ***
Thiamine hydrochloride 2.0 mg Tetanus Immunoglobulin * *
** **
Riboftavine 0.2 mg ***
(Human Tetanus Immunoglobulin,
Phenol red 15. O mg
Ph Eur monograph 0398)
Sodium hydrogen carbonate 2.75 g
~E~ ____________________________________________
Water to
1000 mL DEFINITION
The medium is supplemented with: Sterile liquid or freeze-dried preparation containing
Foetal calf serum (heated at 56 oC for 30 min) 10 per cent immunoglobulins, mainly immunoglobulin G.
Tryptose phosphate broth 10 per cent The preparation is intended for intramuscular administration.
Benzylpenicillin sodium 60 mg/L It is obtained from plasma containing specific antibodies
Streptomycin 0.1 gIL against the toxin of Clostridium tetani. Human normal
immunoglobulin (0338) may be added.
STORAGE
It complies with the monograph Human normal
See Human normal immunoglobulin (0338).
immunoglobulin (0338), except for the minimum number of
LABELLING donors and the minimum total protein contento
See Human normal immunoglobulin (0338).
PRODUCTION
The label states the number of Intemational Units per During development, a satisfactory relationship shall be
container. established between the potency determined by immunoassay
____________________________________________ ~E~

as described under Potency and that determined by means of

the following test for toxin-neutralising capacity in mice.
Toxin-neutralising capacity in mice The potency is
determined by comparing the quantity necessary to protect
*** mice against the paralytic effects of a fixed quantity of
Rubella Immunoglobulin * *
******* tetanus toxin with the quantity of a reference preparation of
(Human Rubella Immunoglobulin, human tetanus immunoglobulin, calibrated in Intemational
Ph Eur monograph 0617) Units, necessary to give the same protection.
~E~ __~ ________________________________________ The Intemational Unit of antitoxin is the specific neutralising
activity for tetanus toxin contained in a stated amount of the
DEFINITION Intemational Standard, which consists of freeze-dried human
Sterile liquid or freeze-dried preparation containing immunoglobulin. The equivalence in Intemational Units of
immunoglobulins, mainly immunoglobulin G. the International Standard is stated by the World Health
The preparation is intended for intramuscular administration. Organization.
It is obtained from plasma containing specific antibodies
Human tetanus immunoglobulin BRP is calibrated in
against rubella virus. Human normal immunoglobulin (0338)
Intemational Units by comparison with the Intemational
may be added.
Standard.
 IV-480 Blood-related Products
Method
Selection of animals Use mice weighing 16-20 g.
Prepararíon of the test toxin Prepare the test toxin by a
suitable method fram the sterile filtrate of a culture in liquid
medium of C. tetani. The 2 methods shown below are given
as examples and any other suitable method may be used .
(1) To the filtrate of an approximately 9-day culture, add
1-2 volumes of glycerol R and store the mixture in the liquid
state at a temperature slightly below O oC.
(2) Precipitate the toxin by addition to the filtra te of
ammonium sulfate R, dry the precipitate in vacuo over
diphosphorus pentoxide R, reduce to a powder and store dry,
either in sealed ampoules or in vacuo over diphosphorus
pentoxide R.
Determination of test dose of toxin (Lp/l Odose) Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 0.5 IU of antitoxin per millilitre. If the test
toxin is stored dry, reconstitute it using a suitable liquido
Prepare mixtures of the solution of the reference preparation
and the test toxin such that each contains 2.0 mL of the
solution of the reference preparation, one of a graded series
of volumes of the test toxin and sufficient of a suitable liquid
to bring the volume to 5.0 mL. AIlow the mixtures to stand,
protected from light, for 60 min. Using 6 mice for each
mixture, inject adose of 0.5 mL subcutaneously into each
mouse. Observe the mice for 96 h. Mice that become
paralysed may be euthanised. The test dose of toxin is the
quantity in 0.5 mL of the mixture made with the smaIlest
amount of toxin capable of causing, despite partial
neutralisation by the reference preparation, paralysis in aIl 6
mice injected with the mixture, within the observation
periodo
Determination of potency of the immunoglobulin Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 0.5 IU of antitoxin per miIIilitre. Prepare a
solution of the test toxin in a suitable liquid such that it
contains 5 test doses per millilitre. Prepare mixtures of the
solution of the test toxin and the immunoglobulin to be
examined such that each contains 2.0 mL ofthe solution of
the test toxin, one of a graded series of volumes of the
irnmunoglobulin to be examined and sufficient of a suitable
liquid to bring the total volume to 5.0 mL. AIso prepare
mixtures of the solution of the test toxin and the solution of
the reference preparation such that each contains 2.0 mL of
the solution of the test toxin, one of a graded series of
volumes of the solution of the reference preparation centred
on that volume (2.0 mL) that contains 1 IU and sufficient of
a suitable liquid to bring the total volume to 5.0 mL. AIlow
the mixtures to stand, protected from light, for 60 mino
Using 6 mice for each mixture, inject subcutaneously adose
of 0.5 mL into each mouse. Observe the mice for 96 h. Mice
that become paralysed may be euthanised. The mixture that
contains the largest volume of irnmunoglobulin that fails to
protect the mice from paralysis contains 1 IU. This quantity
is used to calculate the potency of the immunoglobulin in
Intemational Units per miIIilitre.
The test is not valid unless aIl the mice injected with
mixtures containing 2.0 mL or less of the solution of the
reference preparation show paralysis and aIl those injected
with mixtures containing more do noto
POTENCY
The potency is determined by comparing the antibody titre
of the preparation to be examined with that of a reference
preparation calibrated in Intemational Units, using suitable
immunochemical methods (2.7.1) such as enzyme-linked
2014
immunosorbent assay (ELISA) or toxoid inhibition assay
(TIA).
The Intemational Unit is the activity contained in a stated
amount of the Intemational Standard for anti-tetanus
irnmunoglobulin. The equivalence in Intemational Units of
the Intemational Standard is stated by the WorId Health
Organization.
Human tetanus únmunoglobulin BRP is calibrated in
Intemational Units and is suitable for use as a reference
preparation.
The stated potency is not less than 100 IU/mL of tetanus
antitoxin. The estimated potency is not less than the stated
potency. The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
The description of methods A and B below are provided as
examples.
Method A: direct enzyme immunoassay
The amount of tetanus irnmunoglobulin bound to tetanus
toxoid, which is coated to a microtitre plate, is determined by
means of a peroxidase-conjugated polyclonal anti-human IgG
antibody.
Materials
- Phosphate-buffered saline pH 7.1 (PBS). Dissolve 0.2 g of
potassium chloride R, 0.2 g of pOlassium dihydrogen
phosphate R, 1.15 g of anhydrous disodium hydrogen
phosphate R and 8.0 g of sodium chloride R in water R and
adjust the pH (2.2.3) if necessary. Dilute to 1000 mL
with water R.
- PBS-T PBS containing 0.05 per cent VIV of polysorbate
20R.
- Carbonate buffer pH 9.6. Dissolve 1.4 g of anhydrous
sodium carbonate R and 3.0 g of sodium hydrogen
carbonate R in water R and adjust the pH (2.2.3) if
necessary. Dilute to 1000 mL with water R.
- Tetanus toxoid Purified and chemicaIly inactivated tetanus
toxin.
- Microtitre plate Use a flat-bottomed microtitre plate with
high protein-binding capacity.
Method
Distribute 100 IlL of a 0.2 Lfi'mL solution of tetanus toxoid
in carbonate buffer pH 9.6 into each of the welIs of the
microtitre plate. Incubate at 4 oC for approximately 18 h.
Wash the plate 5 times with PBS-T. To block unbound
binding sites add 200 IlL of PBS containing 5 gIL of bovine
albumin R to each of the weIls and incubate for 1 h at 37 oC
on a plate shaker set at 120 r/min. Wash 5 times with PBS-
T.
Reconstitute the reference preparation and the preparation to
be examined according to the instructions. For each
preparation, prepare 2 independent predilutions of
0.004 IU/rnL in PBS by applying severa! dilution steps.
Using PBS, prepare from each predilution 5 serial dilutions
with a dilution factor of 1.5 resulting in a dilution series of 6
dilutions in the range of 0.0005-0.004 IU/mL. Depending on
the reagents used, a smaIl modification of the dilution series
might be necessary to meet the conditions of the statistical
model used.
Apply 100 pL of each of the samples of the dilution series to
the plate. Incubate for 2 h at 37 oC on a plate shaker set at
120 r/min and wash the plate 5 times with PBS-T. Apply
100 IlL of a peroxidase-conjugated anti-human IgG antibody
diluted to a suitable concentration with PBS-T containing
5 giL of bovine albumin R to each of the welIs and incubate
2014
for 1 h at 37 oC on a plate shaker set at 120 r/min. Wash the
plate 5 times with PBS-T and apply 100 JlL of a suitable
3,3/,5,5/-tetramethylbenzidine (TMB) substrate to each of
the wells and incubate at room temperature for 10 min in the
dark. To stop the reaction, add 100 JlL of a 196.2 giL
solution of sulfuric acid R to each of the wells. Measure the
absorbances at 450 nm and at the reference wavelength of
630 nm. Calculate the potencies of the preparations by the
usual statistical methods (5.3).
Method B: indirect determination by toxoid-binding
inhibition assay
The amount of unbound toxoid in a mixture of toxoid and
tetanus immunoglobulin is determined by an enzyme
immunoassay and is inversely proportional to the amount of
tetanus immunoglobulin presento The method is performed
over 2 consecutive days.
Materials
- Phosphate-buffered saline pH 7.1 (PBS) See under Method
A.
-- PBS-T See under Method A.
- Carbonate buffer pH 9.6 See under Method A.
- Tetanus toxoid See under Method A.
-- Mab Mouse monoclonal tetanus toxoid antibody.
Use according to the instructions. Prepare a suitable
dilution of Mab, e.g. 1/5000, in PBS.
- Peroxidase-conjugated antibody Peroxidase-conjugated anti-
mouse IgG (H + L) antibody, affinity-purified F(ab)2
fragment without cross-reactivity to human serum
proteins. Use according to the instructions. Prepare a
suitable dilution of the peroxidase-conjugated antibody in
PBS-T containing 5 gIL of bovine albumin R.
- Microtitre plate Use a round-bottomed microtitre plate
with medium protein-binding capacity.
-- ELISA plate Use a ftat-bottomed microtitre plate with
high protein-binding capacity.
Method
Day 1
To block the protein-binding sites of the microtitre plate, add
200 ~lL of PBS containing 5 giL of bovine albumin R to each
of the wells of the microtitre plate a~d incubate for 1 h at
37 oC on a plate shaker set at 120 r/min. Wash the plate
5 times with PBS-T.
Reconstitute the reference preparation and the preparation to
be examined according to the instructions. For each
preparation, prepare 2 independent predilutions of
0.4 IU/mL in PBS by applying several dilution steps. Prepare
from each predilution a dilution series of dilutions containing
0.04 IU/rnL, 0.10 IU/mL, 0.12 IU/mL, 0.14 IU/mL,
0.16 IU/mL, 0.18 IU/mL and 0.20 IU/mL. Prepare each
dilution directly from the 0.4 IU/mL predilution.
Transfer 100 JlL of each dilution of the dilution series to a
well of the blocked plate and add 50 JlL of a 0.2 Lfi'mL
solution of tetanus toxoid in carbonate buffer pH 9.6 into
each ofthe wells. Incubate for approximately 18 h at 37 oC
on a plate shaker set at 120 r/min.
To coat the ELISA plate, distribute 100 !lL of a solution of a
human tetanus irnmunoglobulin diluted to 1 IU/rnL in
carbonate buffer pH 9.6 into each of the wells of the ELISA
plate. Incubate for approximately 18 h at 37 oC on a plate
shaker set at 120 r/min.
Day 2
Wash the coated ELISA plate 5 times with PBS-T. To block
unbound binding sites add 200 !lL of PBS containing 5 gIL
of bovine albumin R to each of the wells and incubate for 1 h
Blood-related Products IV-481
at 37 oC on a plate shaker set at 120 r/min. Wash the plate
5 times with PBS-T. Transfer 100 JlL of each mixture of
toxoid and tetanus immunoglobulin from the microtitre plate
to the coated ELISA plate and incubate for 2 hours at 37 oC
on a plate shaker set at 120 r/min. Wash the plate 5 times
with PBS-T. Add 100 JlL of diluted Mab to each of the
wells, incubate the plate for 1 h at 37 oC on a plate shaker
set at 120 r/min and wash the plate 5 times with PBS-T.
Add 1 00 ~lL of the diluted peroxidase-conjugated antibody to
each of the wells, incubate the plate for 1 h at 37 oC on a
plate shaker set at 120 r/min and wash the plate 5 times with
PBS-T. Apply 100 JlL of a suitable 3,3/,5,5/-
tetramethylbenzidine (TMB) substrate to each of the wells
and incubate at room temperature for 10 min in the dark.
To stop the reaction, add 100 ~lL of a 196.2 gIL solution of
sulfun'c acid R to each of the wells. Measure the absorbances
at 450 nm and at the reference wavelength of 630 nm.
Calculate the potencies of the preparations by the usual
statistical methods (5.3).
STORAGE
See Human nonnal immunoglobulin (0338).
LABELLING
See Human normal immunoglobulin (0338).
The label sta tes the number of International Units per
container.
___________________________________________
~Em
***
Varicella Immunoglobulin
*** ***
(Human Varicella Immunoglobulin, ***
Ph Eur monograph 0724)
~Em ____________________________________________
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma from selected donors having
antibodies against Herpesvirus varicellae. Human normal
immunoglobulin (0338) may be added.
It complies with the monograph on Human normal
immunoglobulin (0338) except for the minimum number of
donors, the minimum total protein content and, where
authorised, the test for antibody to hepatitis B surface
antigen.
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
reference preparation calibrated in International Units, using
an immunoassay of suitable sensitivity and specificity (2.7.1).
The International Unit is the activity contained in a stated
amount of the International Standard for anti varicella-zoster.
The equivalence in International Units of the International
Standard is stated by the World Health Organization.
The stated potency is not less than 100 IU/mL.
The estimated potency is not less than the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
STORAGE
See Human normal immunoglobulin (0338).
 IV-482 Blood-related Products
LABELLING
See Human nomal immunoglobulin (0338).
The labe! sta tes the number of Intemational Units per
container.
_ _ __ _ _ _ __ __ _ __ _ _ __ __ _ _ Ph Eur
***
Varicella Irnrnunoglobulin for
***
Intravenous Use *** *
(Human Varicella Immunoglobulin for Intravenous
Administration, Ph Eur monograph 1528)
Ph E~ _ _ __ _ __ _ __ _ _ _ _ _ __ __ _ _ _
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G. It is obtained
from plasma from selected donors having antibodies against
human herpesvirus 3 (varicella-zoster virus 1). Human nomal
immunoglobulin for intravenous administration (0918) may be
added.
It complies with the monograph on Human nomal
immunoglobulin for intravenous administration (0918), except
for the minimum number of donors, the minimum total
protein content and the limit for osmolality.
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
reference preparation calibrated in Intemational Units, using
an immunoassay of suitable sensitivity and specificity (2.7.1) .
The Intemational Unit is the activity contained in a stated
amount of the Intemational Standard for anti varicella-zoster
immunoglobulin. The equivalence in Intemational Units of
the Intemational Standard is stated by the World Health
Organization.
The stated potency is not less than 25 IU/mL. The estimated
potency is not less than the stated potency. The confidence
limits (P = 0.95) are not less than 80 per cent and not more
than 125 per cent of the estimated potency.
STORAGE
See Human nomal immunoglobulin for intravenous
administration (0918).
LABELLING
See Human normal immunoglobulin for intravenous
administration (0918).
The labe! sta tes the number of Intemational Units per
container.
_ __ _ __ __ _ _ _ _ _ __ __ _ __ _ _
***
von Willebrand Factor * *
** **
(Human von Willebrand factor, ***
Ph Eur m onograph 2298)
~E~ _ _ __ _ _ _ _ __ __ _ _ __ __ _ _ _ _
DEFINITION
Sterile, freeze-dried preparation of a plasma protein fraction
containing the glycoprotein human von Willebrand factor
with varying amounts of human coagulation factor VIII,
depending on the method of preparation. It is prepared from
human plasma that complies with the monograph on Human
2014
plasma for fractionation (0853). The preparation may contain
excipients such as stabilisers.
This monograph applies to preparations formulated
according to the human von Willebrand factor activity.
The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU of human von Willebrand
factor per millilitre.
PRODUCTION
* GENERAL PROVISIONS
* The method of preparation is designed to maintain
functional integrity of human von Willebrand factor.
It ineludes steps that have been shown to remove or to
inactivate known agents of infection; if substances are used
for the inactivation of viruses, the subsequent purification
procedure must be validated to demonstrate that the
concentration of these substances is reduced to a suitable
leve! and that any residues are such as not to compromise the
safety of the preparation for patients.
The specific activity is not less than 1 IU of human von
Willebrand factor per milligram of total protein, before the
addition of any protein stabiliser.
The human von Willebrand factor fraction is dissolved in a
suitable liquido No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
filter, distributed aseptically into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are elosed under vacuum or under an inert gas.
CONSISTENCY OF THE METHOD OF PRODUCTION
It shall be demonstrated that the manufacturing process
yields a product having a consistent composition with respect
to human von Willebrand factor, human coagulation factor
VIII and the proportions of human von Willebrand factor
and human coagulation factor VIII. This is evaluated by
suitable analytical procedures that are determined during
process development, and that inelude the following checks:
Human von Willebrand factor multimers
The distribution of the different human von Willebrand
factor multimers is determined by a suitable method such as
sodium dodecyl sulfate (SDS) agarose gel electrophoresis
with or without Westem blot analysis, using a suitable
normal human plasma as standard. Visualisation of the
multimeric pattem may be performed using, for example, an
immunoenzymatic technique and quantitative evaluation may
be carried out by densitometric analysis.
Human von Willebrand factor activity (2.7.21)
The human von Willebrand factor activity is estimated by
determining the ristocetin cofactor activity and by one or
more other suitable assays such as determination of collagen-
binding activity using a suitable reference preparation.
~ E~
Human von Willebrand factor activity/antigen ratio
Consistency of the manufacturing process with respect to the
ratio of human von Willebrand factor activity to human von
Willebrand factor antigen content is demonstrated.
Products that show particles after reconstitution If a few
partieles remain when the preparation is reconstituted, it shall
be demonstrated during validation studies that the potency is
not significantly affected after passage of the preparation
through the filter to be provided with the preparation.
CHARACTERS
Appearance
Hygroscopic, white or pale yellow, powder or friable solido
2014 Blood-related Products IV-483

Reconstitute the preparation to be examined as stated on the label ASSAY

immediately before canying out the identification, tests (except
those for solubility and water) and assay.
IDENTIFICAnON
It complies with the limits of the assay.
TESTS
Solubility
To a container of the preparation to be examined, add the
volume of the liquid stated on the label at the recommended
temperature. The prepararion dissolves completely with
gentle swirling within 10 min, forming a clear or slightly
opalescent, colourless or slightly yellow solution.
In addition, where the label sta tes that the product may show
a few particles after reconstitution, reconstitute the
prepararion as described on the labe! and pass it through the
filter provided: the filtered solution is clear or slightly
opalescent.
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 240 mosmoVkg.
T ota! protein
If necessary, dilute an accurately measured volume of the
reconsrituted preparation with a 9 gIL solurion of sodium
chloride R to obtain a protein concentration of about
7.5 mglmL. Place 2.0 mL ofthis solution in a round-
bottomed centrifuge tube and add 2 mL of a 75 gIL solution
of sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant and allow the
inverted tube to drain on filter paper. Determine the nitrogen
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the amount of pro te in by multiplying the result
by 6.25 . For some products, especially those without a protein
stabiliser, this method may not be applicable. Another validated
method for protein determination must therefore be peiformed.
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
The 1 to 64 dilution do es not show agglutination. Dilute the
reconstituted preparation with a 9 gIL solution of sodium
chloride R to contain 6 IU of human von Willebrand factor
Human von Willebrand factor (2. 7.21)
The esrimated potency is not less than 80 per cent and not
more than 120 per cent of the stated potency.
The confidence limits (P = 0.95) are not les s than
80 per cent and not more than 120 per cent of the estimated
potency.
Pending the availability of an International Standard for human
von Willebrand factor concentrate calibrated for use in the
collagen-binding assay, only the ristocetin cofactor assay may be
used.
Human coagulation factor VIII (2.7.4)
The assay is carried out where the human coagulation factor
VIII content is greater than 10 IU of human coagulation
factor VIII per 100 IU of human von Willebrand factor
activity. The estimated potency is not less than 60 per cent
and not more than 140 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
- the number of Intemational Units of human von
Willebrand factor in the container;
- the number of International Units of human coagularion
factor VIII in the container, or that the content of human
coagulation factor VIII is less than or equal to 10 IU of
human coagulation factor VIII per 100 IU of human von
Willebrand factor activity;
- the amount of protein in the container;
- the name and quantity of any added substance;
- the name and volume of the liquid to be used for
reconstiturion;
- where applicable, that the prepararion may show the
presence of a few particles after reconstitution;
- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
_ _________________________________________ ~E~

activity per millilitre.

Water
Determined by a suitable method, such as semi-micro
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectrophotometry (2.2.40), the water content
is within the limits approved by the competent authority.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacteria! endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and
where jusrified and authorised, with a validated in vitro test
such as the test for bacterial endotoxins.
For the pyrogen test, inject per kilogram of the rabbit's mass
a volume equivalent to not less than 100 IU ofhuman von
Willebrand factor.
Where the test for bacterial endotoxins is used, the
preparation to be examined contains less than 0.05 IU of
endotoxin per International Unit of human von Willebrand
factor.
Monographs

Immunological Products
2014
Monoclonal Antibodies for Human *****
** **
Use ***
(Ph Eur monograph 2031)
Monoclonal Antibodies for Human Use comply wÍlh zhe
requirements of zhe European Pharmacopoeia. These requirements
are reproduced below.
The requirements of zhe monograph for Immunosera do not
necessarily apply LO zhe monograph for Monoclonal Anzibodies for
Human Use.
~E~ ____________________________________________
DEFINITION
Monoclonal antibodies for human use are preparations of an
immunoglobulin or a fragment of an immunoglobulin, for
example, F (ab')2, with defined specificity, produced by a
single clone of cells . They may be conjugated to other
substances, including for radiolabelling.
They can be obtained from immortalised B lymphocytes that
are cloned and expanded as continuous cell lines or from
rDNA-engineered celllines.
Examined under suitable conditions of visibility, they are
practically free from partieles.
Currently available rDNA-engineered antibodies inelude the
following antibodies.
Chimeric monoclonal antibodies The variable heavy- and
light-chain domains of a human antibody are replaced by
those of a non-human species that possess the desired
antigen specificity.
Humanised monoclonal antibodies The 3 short hypervaciable
sequences (the complementarity-determining regions)
of non-human variable domains for each chain are
engineered into the variable domain framework of a human
antibody; other sequence changes may be made to improve
antigen binding.
Recombinant human monoclonal antibodies The variable
heavy- and light-chain domains of a human antibody are
combined with the constant region of a human antibody.
Monoelonal antibodies obtained from celllines modified by
recombinant DNA technology also comply with the
requirements of the monograph Products of recombinant DNA
technology (0784).
This monograph applies to monoelonal antibodies, including
conjuga tes, for therapeutic and prophylactic use and for use
as in vivo diagnostics. It does not apply to monoclonal
antibodies used as reagents in the manufacture of medicinal
products. Nor do es it apply to monoclonal antibodies
produced in ascites, for which requirements are decided by
the competent authority.
PRODUCTION
GENERAL PROVlSIONS
Production is based on a seed-lot system using a master cell
bank and, if applicable, a working cell bank derived from the
cloned cells. The production method is validated during
development studies in order to prevent transmission of
infectious agents by the final product. All biological materials
and cells used in the production are characterised and are in
compliance with chapter 5.2.8. Minimising the risk of
transmitting animal spongiform encephalopathy agents via human
and veterinary medicinal products. Where monoclonal
antibodies for human use are manufactured using materials
of human or animal origin, the requirements of chapter
5.1.7. Viral safety also apply. Where an immunogen is used,
it is characterised and the method of immunisation is
documented .
Irnrnunosera IV-487
Process validation
During development studies, the production method is
validated for the following aspects:
consistency of the production process including
cell-culture/fermentation, purification and, where
applicable, fragmentation method;
-- removal or inactivation of infectious agents;
-- adequate removal of product- and process-related
impurities (for example, host-cell protein and DNA,
protein A, antibiotics, cell-culture components);
-- specificity and biological activity of the monoelonal
antibody;
-- absence of non-endotoxin pyrogens, where applicable;
-- reusability of purification components (for example,
column material), limits or acceptance critecia being set as
a function of the validation;
-- methods used for conjugation, where applicable.
Product characterisation
The product is characterised to obtain adequate information
including: structural integrity, isotype, amino-acid sequence,
secondary structure, carbohydrate moiety, disulfide bridges,
conformation, specificity, affinity, biological activity and
heterogeneity (characterisation of isoforms) .
A battery of suitable analytical techniques is used including
chemical, physical, irnmunochemical and biological tests (for
example, peptide mapping, N- and C-terminal amino-acid
sequencing, mass spectrometry, chromatographic,
electrophoretic and spectroscopic techniques) . Additional
tests are performed to obtain information on cross-reactivity
with human tissues.
For those products that are modified by fragmentation or
conjugation, the infiuence of the methods used on the
antibody is characterised.
Process intermediates
Where process intermediates are stored, an expiry date or a
storage period justified by stability data is established for
each.
Biological assay
The biological assay is chosen in terms of its correlation with
the intended mode of action of the monoclonal antibody.
Reference preparation
A batch shown to be stable and shown to be suitable in
clinical trials, or a batch representative thereof, is used as a
reference preparation for the identification, tests and assay.
The reference preparation is appropriately characterised as
defined under Product characterisation, except that it is not
necessary to examine cross-reactivity for each batch of
reference preparation.
Definition of a batch
Definition of a batch is required throughout the process.
SOURCE CELLS
Source cells include fusion partners, lymphocytes, myeloma
cells, feeder cells and host cells for the expression of the
recombinant monoelonal antibody.
The origin and characteristics of the parental cell are
documented, including information on the health of the
donors, and on the fusion partner used (for example,
myeloma cellline, human lymphoblastoid B-cellline).
Wherever possible, source cells undergo suitable screening
for extraneous agents and endogenous agents. The choice
of viruses for the tests is dependent on the species and tissue
of origino
 IV-488 Immunosera
CELL UNE PRODUCING THE MONOCLONAL ANTIBODY
The suitability of the cell line producing the monoelonal
antibody is demonstrated by:
- documentation on the hisrory of the cellline ineluding
description of the cell fusion, irnmortalisation or
transfection and eloning procedure;
characterisation of the cell line (for example, phenotype,
isoenzyme analysis, immunochemical markers and
cyrogenetic markers);
characterisation of relevant features of the antibody;
consistency of critical quality attributes for the antibody
up to or beyond the population doubling leve! or
generation number used for routine production;
for recombinant DNA products, consistency of the coding
sequence of the expression construct in cells cultivated ro
the limit of in vitro cell age for production use or beyond,
by either nueleic acid testing or product analysis.
CELLBANKS
The master cell bank is a homogeneous suspension of the
cell line producing the monoelonal antibody, distributed in
equal volumes in a single operation into individual containers
for storage.
A working cell bank is a homogeneous suspension of the cell
material derived from the master cell bank at a finite passage
level, distributed in equal volumes in a single operation into
individual containers for srorage.
Post-production cells are cells cultured up to or beyond the
population doubling level or generation number used for
routine production.
The following tests are performed on the master cell bank:
viability, identity, absence of bacterial, fungal and
mycoplasmal contamination, characterisation of the
monoelonal antibody produced. Adventitious viral
contamination is tested with a suitable range of in vivo and in
vitro tests. Retrovirus and other endogenous viral
contamination is tested using a suitable range of in vitro tests.
The following tests are performed on the working cell bank:
viability, identity, absence of bacterial, fungal and
mycoplasmal contamination. Adventitious viral
contamination is tested with a suitable range of in vivo and in
vitro tests. For the first working cell bank, these tests are
performed on post-production cells, generated from that
working cell bank; for working cell banks subsequent to the
first working cell bank, a single in vitro and in vivo test can
be done either directly on the working cell bank or on
post-production cells.
For the master cell bank and working cell bank, tests for
specific virus es are carried out when potentially contarninated
biological material has been used during preparation of the
cell banks, taking into account the species of origin of this
material. This may not be necessary when this material is
inactivated using validated procedures.
The following tests are performed on the post-production
cells: absence of bacterial, fungal and mycoplasmal
contamination. Adventitious viral contamination is tested
with a suitable range of in vivo and in vitro tests. Retrovirus
and other endogenous viral contamination is tested using a
suitable range of in vitro tests.
CULTURE AND HARVEST
Production at finite passage level (single harvest)
Cells are cultivated up ro a defined maximum number of
passages or population doublings, or up to a fixed harvest
time (in accordance with the stability of the cellline).
Product is harvested in a single operation.
2014
Continuous-culture production (multiple harvest)
Cells are continuously cultivated for a defined period (in
accordance with the stability of the system and production
consistency) . Moniroring is necessary throughout the life of
the culture; the required frequency and type of monitoring
will depend on the nature of the production system.
Each harvest is tested for antibody content, bioburden,
endoroxin and mycoplasmas. General or specific tests for
adventitious viruses are carried out at a suitable stage
depending on the nature of the manufacturing process and
the materials used. For processes using production at finite
passage leve! (single harvest), at least 3 harvests are tested for
adventitious viruses using a suitable range of in vitro
methods.
The acceptance criteria for harvests for further processing are
elearly defined and linked ro the schedule of monitoring
applied. If any adventitious virus es are detected, the process
is carefully investigated to determine the cause of the
contamination and the harvest is not further processed.
Harvests in which an endogenous virus has been detected are
not used for purification unless an appropriate action plan
has been defined to prevent transmission of infectious agents.
PURIFlCATION
Harvests or intermediate pools may be pooled before further
processing. The purification process ineludes steps that
remove and/or inactivate non-enveloped and enveloped
viruses. A validated purification process, for which removal
and/or inactivation of infectious agents and removal of
product- and process-related impurities has been
demonstrated, is used. Defined steps of the process lead ro a
purified monoelonal antibody (active substance) of consistent
quality and biological activity.
ACTIVE SUBSTANCE
The test programme for the active substance depends on the
validation of the process, on demonstration of consistency
and on the expected leve! of product- and process-related
impurities. The active substance is tested for appearance,
identity, bioburden and bacterial endoroxins, product-related
substances, product- and process-related impurities ineluding
tests for host-cell-derived proteins and host-cell- and vector-
derived DNA, as well as structural integrity, protein content
and biological activity by suitable analytical methods,
comparing with the reference preparation where necessary.
When the active substance is a conjugated or transformed
antibody, appropriate tests must be performed before and
after the antibody conjugation/modification.
If storage of intermediates is intended, adequate stability of
these preparations and its impact on quality or shelf-life of
the finished product are evaluated.
FINAL BULK
One or more batches of active substance may be combined
ro produce the final bulk. Suitable stabilisers and other
excipients may be added during preparation of the final bulk.
The final bulk must be stored under validated conditions
with respect to bioburden and stability.
FINALLOT
The final bulk is sterile-filtered and distributed under aseptic
conditions into sterile containers, which may subsequently be
freeze-dried.
As part of the in-pro ces s control each container (vial, syringe
or ampoule) is inspected after filling to eliminate containers
that contain visible partieles. During development of the
product it must be demonstrated that either the process will
not generate visible proteinaceous partieles in the finallot or
 2014
such partieles are reduced to a low level as justified and
authorised.
CHARACTERS
Liquid preparations are elear or slightly opalescent, colourless
or slightly coloured liquids. Freeze-dried products are white
or slightly coloured powders or solid friable masses. After
reconstitution they show the same characteristics as liquid
preparations.
IDENTIFICATION
The identity is established by suitable validated methods
comparing the product with the reference preparation, where
appropriate. The assay also contributes te identification.
TESTS
Appearance
Liquid or reconstituted freeze-dried preparations comply with
the limits approved for the particular product with regard te
degree of opalescence (2.2.1) and degree of coloration
(2.2.2). They are without visible partieles, unless otherwise
justified and authorised.
Solubility
Freeze-dried preparations dissolve completely in the
prescribed volume of reconstituting liquid, within a defined
time, as approved for the particular product.
pH (2.2.3)
It complies with the limits approved for the particular
product.
Osmolality (2.2.35)
Minimum 240 mosmoVkg, unless otherwise justified and
authorised.
Extractable volurne (2.9.17)
It complies with the test for extractable volume.
Total protein (2.5.33)
It complies with the limits approved for the particular
producto
Molecular-size distribution
Molecular-size distribution is determined by a suitable
method, for example size-exelusion chromategraphy (2.2.30) .
It complies with the limits approved for the particular
product.
Molecular identity and structural integrity
Depending on the nature of the monoelonal antibody, its
microheterogeneity and isoforms, a number of different tests
can be used to demonstrate molecular identity and structural
integrity. These tests may inelude peptide mapping,
isoelectric focusing, ion-exchange chromatography,
hydrophobic interaction chromatography, oligosaccharide
mapping, monosaccharide content and mass spectrometry.
Purity
Tests for process- and product-related impurities are carried
out by suitable validated methods. Provided that tests for
process-related impurities have been carried out on the active
substance or on the final bulk with satisfactory results, they
may be omitted on the finallot.
Stabiliser
Where applicable, it complies with the limits approved for
the particular producto
Water (2.5.12)
Freeze-dried products comply with the limits approved for
the particular product.
Sterility (2.6.1)
It complies with the test for sterility.
Irnrnunosera IV-489
Bacterial endotoxins (2.6.14)
It complies with the limits approved for the particular
producto
Tests applied to modified antibodies
Suitable tests are carried out depending on the type of
modification.
ASSAY
Carry out a suitable biological assay compared te the
reference preparation. Design of the assay and ca1culation of
the results are made according te the usual principies (for
example, 5.3).
STORAGE
As stated on the labe!'
Expiry date The expiry date is ca1culated from the date of
sterile filtration, the date of filling (for liquid preparations) or
the date offreeze-drying (where applicable).
LABELLING
The label states:
- the number of units per millilitre, where applicable;
- the quantity of protein per container;
-- the quantity of monoelonal antibody in the container;
-- for liquid preparations, the volume of the preparation in
the container;
-- for freeze-dried preparations:
- the name and the volume of the reconstitution liquid
to be added;
-- the period of time within which the monoclonal
antibody is te be used after reconstitution;
-- the dilution to be made before use of the product, where
applicable.
___________________________________________ ~Em
Immunosera ***
*** ***
Antisera ***
(lmmunosera for Human Use, Animal, Ph Eur monograph 0084)
Immunosera comply wúh the requirements of the European
Pharmacopoeia monograph for Immunosera for Human Use,
Animal. These requirements are reproduced below.
~Ew ____________________________________________
DEFINITION
Animal immunosera for human use are liquid or freeze-dried
preparations containing purified immunoglobulins or
immunoglobulin fragments obtained from serum or plasma
of immunised animals of different species.
The immunoglobulins or immunoglobulin fragments have
the power of specifically neutralising or binding to the
antigen used for immunisation . The antigens include
microbial or other toxins, human antigens, suspensions of
bacterial and viral antigens and venoms of snakes, scorpions
and spiders. The preparation is intended for intravenous or
intramuscular administration, after dilution where applicable.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown te yield
consistently irnmunosera of acceptable safety, potency in man
and stability.
Any reagent of biological origin used in the production of
immunosera shall be free of contamination with bacteria,
fungi and viruses. The general requirements of chapter 5.1.7.
IV-490 Immunosera
Viral safety apply to the manufacture of animal immunosera
for human use, in conjunction with the more specific
requirements relating to viral safety in this monograph.
The method of preparation includes a step or steps that have
been shown to remove or inactivate known agents of
infection.
Methods used for production are validated, effective,
reproducible and do not impair the biological activity of the
product.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
Referenee preparatial1 A batch shown to be suitable in clinical
trials, or a batch representative thereof, is used as the
reference preparation for the tests for high molecular mass
proteins and purity.
ANIMALS
The animals used are of a species approved by the competent
authority, are healthy and are exclusively reserved for
production of immunoserum. They are tested and shown to
be free from a defined Iist of infectious agents.
The introduction of animals into a closed herd follows
specified procedures, including definition of quarantine
measures. Where appropriate, additional specific agents are
considered depending on the geographicallocalisation of the
establishment used for the breeding and production of the
animals. The feed originates from a controlled source and no
animal proteins are added. The suppliers of animals are
certified by the competent authority.
If the animals are treated with antibiotics, a suitable
withdrawal period is allowed before collection of blood or
plasma. The animals are not treated with penicillin
antibiotics. If a Iive vaccine is administered, a suitable waiting
period is imposed between vaccination and collection of
serum or plasma for immunoserum production.
IMMUNISATION
The antigens used are identified and characterised, where
appropriate; where relevant, they are shown to be free from
extraneous infectious agents. They are identified by their
names and a batch number; information on the source and
preparation are recorded.
The selected animals are isolated for at least 1 week before
being irnmunised according to a defined schedule, with
booster injections at suitable intervals. Adjuvants may be
used.
Animals are kept under general health surveillance and
specific antibody production is controlled at each cycle of
immunisation.
Animals are thoroughly examined before collection of blood
or plasma. If an animal shows any pathologicallesion not
related to the immunisation process, it is not used, nor are
any other of the animals in the group concerned, unless it is
evident that their use will not impair the safety of the
producto
COLLECTION OF BLOOD OR PLASMA
Collection of blood is made by venepuncture or
plasmapheresis. The puncture area is shaved, cleaned and
disinfected. The animals may be anaesthetised under
conditions that do not infiuence the quality of the product.
Unless otherwise prescribed, an antimicrobial preservative
may be added. The blood or plasma is collected in such a
manner as to maintain sterility of the product. The blood or
plasma collection is conducted at a site separate from the
area where the animals are kept or bred and the area where
2014
the immunoserum is purified. If the serum or plasma is
stored before further processing, precautions are taken to
avoid microbial contamination.
Several single plasma or serum samples may be pooled before
purification. The single or pooled samples are tested before
purification for the following tests.
Tests for contaminating virus es
If an antimicrobial preservative is added, it must be
neutralised before carrying out the tests, or the tests are
carried out on a sample taken before addition of the
antimicrobial preservative. Each pool is tested for
contaminating viruses by suitable in vitra tests.
Each pool is tested for virus es by inoculation to cell cultures
capable of detecting a wide range of viruses relevant for the
particular product.
Potency
Carry out a biological assay as indicated in the monograph
and express the result in International Units per millilitre,
where applicable. A validated in vitro method may also be
used.
Protein content
Dilute the product to be examined with a 9 gIL solution of
sadium ehlaride R to obtain a solution containing about 15 mg
of protein in 2 mL. To 2 mL of this solution in a round-
bottomed centrifuge tube add 2 mL of a 75 gIL solution of
sadium malybdate R and 2 mL of a mixture of 1 volume of
nitragen-free sulfurie acid R and 30 volumes of water R. Shake,
centrifuge for 5 min, decant the supernatant liquid and allow
the inverted tube to drain on filter paperoDetermine the
nitrogen in the residue by the method of sulfuric acid
digestion (2.5.9) and calculate the content of protein by
multiplying by 6.25 . The protein content is within approved
Iimits.
PURIFICATION AND VIRAL INACTIVATION
The immunoglobulins are concentrated and purified by
fractional precipitation, chromatography, immunoadsorption
or by other chemical or physical methods. They may be
processed further by enzyrne treatment. The methods are
selected and validated to avoid contamination at all steps of
processing and to avoid formation of protein aggregates that
affect the immunobiological characteristics of the product.
For products intended to consist of irnmunoglobulin
fragments, the methods are validated to guarantee total
fragmentation. The methods of purification used are such
that they do not generate additional components that
compro mise the quality and the safety of the product.
Unless otherwise justified and authorised, validated
procedures are applied for removal andJor inactivation of
viruses. The procedures are selected to avoid the formation
of polyrners or aggregates and, unless the product is intended
to consist of Fab' fragments, to minimise the splitting of
F(ab ')2 into Fab ' fragments.
After purification and treatment for removal andJor
inactivation of virus es, a stabiliser may be added to the
intermediate product, which may be stored for a period
defined in light of stability data.
Only an intermediate product that complies with the
following requirements may be used in the preparation of the
final bulk.
Purity
Examine by non-reducing polyacrylamide gel electrophoresis
(2.2.31), by comparison with the reference preparation.
The bands are compared in intensity and no additional
bands are found.
2014
FINALBULK
The final bulk is prepared from a single intermediate product
or from a pool of intermediate products obtained from
animals of the same species. Intermediate products with
different specificities may be pooled.
An antimicrobial preservative and a stabiliser may be added.
If an antimicrobial preservative has been added to the blood
or plasma, the same substance is used as the antimicrobial
preservative in the final bulk.
Only a final bulk that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable physico-chemical method.
Ir contains not les s than 85 per cent and not more than
115 per cent of the amount stated on the labe!'
Sterility (2.6.1)
It complies with the test for sterility.
FINALLOT
The final bulk of immunoserum is distributed aseptically into
sterile, tamper-proof containers . The containers are closed so
as to prevent contamination.
Only a final lot that complies with the requirements
prescribed below under Identification, Tests and Assay may
be relea sed for use. Provided that the tests for osmolality,
protein content, molecular-size distribution, antimicrobial
preservative, stabiliser, purity, foreign proteins and albumin
and the assay have been carried out with satisfacrory results
on the final bulk, they may be omitted on the finallot.
Reconstitute the preparation to be examined as stated on the label
immediately before canying out the identification, tests (except
those for solubiliry and water) and assay.
IDENTIFICATION
The identity is established by immunological tests and, where
necessary, by determination of biological activity. The assay
may also serve for identification.
CHARACTERS
Irnmunosera are clear to opalescent and colourless to very
faintly yellow liquids . They are free from turbidity.
Freeze-dried products are white or slightly yellow powders or
solid friable masses. After reconstitution they show the same
characteristics as liquid preparations.
TESTS
Solubility
To a container of the preparation to be examined, add the
volume of the liquid for reconstitution stated on the labe!'
The preparation dissolves completely within the time stated
on the labe!'
Extractable volume (2.9. 17)
It complies with the requirement for extractable volume.
pH (2.2.3)
The pH is within the limits approved for the particular
producto
Osmolality (2.2.35)
Minimum 240 mosmol/kg after dilution, where applicable.
Protein content
90 per cent to 110 per cent of the amount stated on the
labe!, and, unless otherwise justified and authorised, not
more than 100 gIL.
Dilute the preparation to be examined with a 9 gIL solution
of sodium chloride R to obtain a solution containing about
15 mg of protein in 2 mL. To 2 mL of this solution in a
Immunosera IV-491
round-bottomed centrifuge tube add 2 mL of a 75 gIL
solution of sodium molybdate R and 2 mL of a mixture of
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
water R . Shake, centrifuge for 5 min, decant the supematant
liquid and allow the inverted tube to drain on filter papero
Determine the nitro gen in the residue by the method of
sulfuric acid digestion (2.5.9) and calculate the content of
protein by multiplying by 6.25.
Molecular-size distribution
Examine by liquid chromatography (2.2.29 or 2.2.30) .
It complies with the specification approved for the particular
producto
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable physicochemical method.
The amount is not less than the minimum amount shown to
be effective and is not greater than 115 per cent of that
stated on the labe!'
Phenol (2.5.15)
Maximum 2.5 gIL for preparations containing pheno!.
Stabiliser
Determine the amount of stabiliser by a suitable physico-
chemical method. The preparation contains not less than
80 per cent and not more than 120 per cent of the quantity
stated on the labe!'
Purity
Examine by non-reducing polyacrylamide gel electrophoresis
(2.2.31), by comparison with the reference preparation.
No additional bands are found for the preparation to be
examined.
Foreign proteins
When examined by precipitation tests with specific antisera,
only protein from the declared animal species is shown to be
present, unless otherwise prescribed, for example where
material of human origin is used during production.
Albumin
Unless otherwise prescribed in the monograph, when
examined electrophoretically, the content of albumin is not
greater than the limit approved for the particular product
and, in any case, is not greater than 3 per cent.
Water (2.5.12)
Maximum 3 per cent.
Sterility (2. 6.1)
It complies with the test for sterility.
Pyrogens (2.6.8)
Unless otherwise justified and authorised, it complies with
the test for pyrogens. Unless otherwise prescribed, inject
1 mL per kilogram of the rabbit's body mass.
ASSAY
Carry out a biological assay as indicated in the monograph
and express the result in Intemational Units per millilitre,
where appropriate. A validated in vitro method may also be
used.
STORAGE
Protected from light, at the temperature stated on the labe!'
Do not allow liquid preparations ro freeze.
Expiry date The expiry date is calculated from the beginning
of the assay.
LABELLING
The label states:
- the number of Intemational Units per millilitre, where
applicable;
IV-492 Immunosera
- the amount of protein per container;
- for freeze-dried preparations:
- the na me and volume of the reconstituting liquid to
be added;
- that the immunoserum is to be used immediately after
reconstitution;
- the time required for complete dissolution;
- the route of administration;
- the storage conditions;
- the expiry date, except for containers of less than 1 mL
which are individually packed; the expiry date may be
omitted from the label on the container, provided it is
shown on the package and the labe! on the package states
that the container must be kept in the package until
required for use;
- the animal species of origin;
- the name and amount of any antimicrobial preservative,
any stabiliser and any other excipient.
_ __________________________________________ ~Ew
*****
Anti-T Lymphocyte
** **
Immunoglobulin for Human Use, ***
Animal
(Ph Eur monograph 1928)
____________________________________________
~Ew
DEFINITION
Sterile liquid or freeze-dried preparation containing
immunoglobulins, obtained from serum or plasma of
animals, mainly rabbits or horses, immunised with human
lymphocytic antigens.
The immunoglobulin has the property of diminishing the
number and function of immunocompetent cells, in
particular T -lymphocytes. The preparation contains
principally immunoglobulin G. It may contain antibodies
against other lymphocyte subpopulations and against other
cells. The preparation is intended for intravenous
administration, after dilution with a suitable diluent where
applicable. The preparation may contain excipients such as
stabilisers.
Applicable provisions of the monograph on Immunosera for
human use, animal (0084) are stated be!ow.
PRODUCTION
GENERAL PROVISIONS
The production method has been shown to yield consistently
immunoglobulins of acceptable safety, potency in man and
stability.
Any reagent of biological origin used in production shall be
free of contamination with bacteria, fungi and viruses.
The method of preparation includes a step or steps that have
been shown to remove or inactivate known agents of
infection.
During development studies, it shall be demonstrated that
the production method yields a product that:
- does not transmit infectious agents,
- is characterised by a defined pattern of immunological
activity, notably: antigen binding, complement-dependent
and independent cytotoxicity, cytokine re!ease, induction
of T -cell activation, cell death,
does not contain antibodies that cross-react with human
tissues to a degree that would impair clinical safety,
2014
- has a defined maximum content of anti-thrombocyte
antibody activity,
- has a defined maximum content of haemoglobin.
The product has been shown, by suitable tests in animals
and evaluation during clinical trials, to be well tolerated.
Reference preparation A batch shown to be suitable for
checking the validity of the assay and whose efficacy has been
demonstrated in clinical trials, or a batch representative
thereof.
ANIMALS
The animals used are of a species approved by the competent
authority, are healthy and exclusively reserved for production
of anti-T lymphocyte immunoglobulin. They are tested and
shown to be free from a defined list of infectious agents.
The introduction of animals into a closed herd follows
specified procedures, including definition of quarantine
measures. Where appropriate, tests for additional specific
agents are considered depending on the geographical
localisation of the establishment used for the breeding and
production of the animals. The feed origina tes from a
controlled source and no animal proteins are added .
The suppliers of animals are certified by the
competent authority.
If the animal s are treated with antibiotics, a suitable
withdrawal period is allowed before collection of blood or
plasma. The animals are not treated with penicillin
antibiotics. If a live vaccine is administered, a suitable waiting
period is imposed between vaccination and collection of
serum or plasma for immunoglobulin production.
The species, origin and identification number of the animals
are specified.
IMMUNISATION
The antigens used are identified and characterised, where
appropriate. They are identified by their names and a batch
number; information on the source and preparation are
recorded.
The selected animals are isolated for at least 1 week before
being irnmunised according to a defined schedule with
booster injections at suitable intervals. Adjuvants may be
used.
Animals are kept under general health surveillance and
specific antibody production is controlled at each cyc!e of
immunisation.
Animals are thoroughly examined before collection of blood
or plasma. If an animal shows any pathological lesion not
related to the immunisation process, it is not used, nor are
any other of the animal s in the group con cerned, unless it is
evident that their use will not impair the safety of the
producto
Human antigens such as continuously growing T -lymphocyte
celllines or thymocytes are used to immunise the animals.
Cells may be subjected to a sorting procedure.
The immunising antigens are shown to be free from
infectious agents by validated methods for relevant blood-
borne pathogens, notably hepatitis B virus (HBV), hepatitis
C virus (HCV) and human immunodeficiency virus (HIV)
and other relevant adventitious agents originating from the
preparation of the antigen. The cells used comply with
defined requirements for purity of the cell population and
freedom from adventitious agents.
COLLECTION OF BLOOD OR PLASMA
Collection of blood is made by venepuncture or
plasmapheresis. The puncture area is shaved, c!eaned and
 2014
disinfected. The animals may be anaesthetised under
conditions that do not inftuence the quality of the producto
No antimicrobial preservative is added to the plasma and
serum samples. The blood or plasma is collected in such a
manner as to maintain sterility oi the productoThe blood or
plasma collection is conducted at a site separate from the
area where the animals are kept or bred and the area where
the immunoglobulin is purified. If the serum or plasma is
stored before further processing, precautions are taken to
avoid microbial contamination.
Several single plasma or serum samples may be pooled before
purification. The single or pooled samples are tested before
purification for the following tests.
Tests for contanúnating virus es
Each pool is tested for contaminating virus es by suitable in
vitro tests ineluding inoculation to cell cultures capable of
detecting a wide range of viruses relevant for the particular
producto Where applicable, in vitro tests for contaminating
virus es are carried out on the adsorbed pool, after the last
production stage that may introduce viral contaminants.
PURIFICATlON AND VIRAL INACTlVATlON
The immunoglobulins are concentrated and purified by
fractional precipitation, chromatography, irnmuno-adsorption
or by other suitable chernical or physical methods.
The methods are selected and validated to avoid
contamination at all steps of processing and to avoid
formation of protein aggregates that effect immunobiological
characteristics of the producto
Unless otherwise justified and authorised, validated
procedures are applied for removal and/or inactivation of
viruses.
After purification and treatment for removal and/or
inactivation of virus es, a stabiliser may be added to the
intermediate product, which may be stored for a period
defined in the light of stability data.
Only an intermediate product that complies with the
following requirements may be used in the preparation of the
final bulk.
If the method of preparation ineludes a step for adsorption of
cross-reacting anti-human antibodies using material from
human tissues and/or red blood cells, the human materials
are submitted to a validated procedure for inactivation of
infectious agents, unless otherwise justified and authorised.
If erythrocytes are used for adsorption, the donors for such
materials comply with the requirements for donors of blood
and plasma of the monograph on Human plasma for
fractionation (0853) . If other human material is used, it is
shown by validated methods to be free from relevant blood-
borne pathogens, notably HBV, HCV and HIV. If substances
are used for inactivation or removal of virus es, it shall have
been shown that any residues present in the final product
have no adverse effects on the patients treated with the anti-
T lymphocyte immunoglobulin.
FINAL BULK
The final bulk is prepared from a single intermediate product
or from a pool of intermediate products obtained from
animals of the same species. No antimicrobial preservative is
added either during the manufacturing procedure or for
preparation of the final bulk solution. During manufacturing,
the solution is passed through a bacteria-retentive filter.
FINALLOT
The final bulk of anti-T-Iymphocyte immunoglobulin is
distributed aseptically into sterile, tamper-proof containers.
The containers are closed as to prevent contamination.
Immunosera IV-493
Only a final lot that complies with the requirements
prescribed below under Identification, Tests and Assay may
be released for use.
CHARACTERS
Appearance:
- liquid preparation: clear or slightly opalescent, colourless or
pale yellow liquid;
- freeze-dried preparation: white or slightly yellow powder or
solid friable mass, which after reconstitution gives a liquid
preparation corresponding to the description aboye.
IDENTIFICAnON
A. Using a suitable range of species-specific antisera, carry
out precipitation tests on the preparation to be examined.
It is recommended that the test be carried out using antisera
specific to the plasma proteins of each species of domes tic
animal commonly used in the preparation of materials of
biological origin in the country concerned and antisera
specific to human plasma proteins. The preparation is shown
to contain proteins originating from the animal used for the
anti-T Iymphocyte irnmunoglobulin production.
B. Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal serum ofthe animal used for
production, compare this serum and the preparation to be
examined, both diluted to a concentration that will allow a
clear gammaglobulin precipitation are to be obtained on the
ge!. The main component of the preparation to be examined
corresponds to the IgG component of normal serum of the
animal used for production.
C. The preparation complies with the assay.
TESTS
Solubility
For the freeze-dried preparation, to a container add the
volume of the liquid stated on the labe!' The preparation
dissolves completely within the time stated on the labe!'
Extractable volume (2.9.17)
It complies with the requirement for extractable volume.
pH (2.2.3)
The pH is within the limits approved for the particular
product.
Osmolality (2.2.35)
Minimum 240 mosmol/kg after dilution, where applicable.
Total protein (2.5.33)
90 per cent to 110 per cent of the amount stated on the
labe!'
Stabiliser
Determine the amount of stabiliser by a suitable physico-
chemical method. The preparation contains not less than
80 per cent and not more than 120 per cent of the quantity
stated on the labe!'
Distribution of molecular size
Size-exclusion chromatography (2.2.30).
Test solution Dilute the preparation to be examined with a
9 giL solution of sodium chloride R to a concentration suitable
for the chromatographic system used. A concentration in the
range 2-20 giL is usually suitable.
Reference solution Dilute human immunoglobulin (molecular
size) BRP with a 9 gIL solution of sodium chloride R to the
same protein concentration as the test solution.
Column:
- size: l = 0.6 m, 0 = 7.5 mm,
- stationary phase: silica gel for size-exclusion
chromatography R, a grade suitable for fractionation of
IV-494 Irnrnunosera
globular proteins in the molecular mass range of
20 000 to 200 000.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1.741 g of sodium dihydrogen phosphate
monohydrate R and 11.688 g of sodium chloride R in 1 litre of
water R.
Flow rate 0.5 mUmin.
Detection Spectrophotometer at 280 nm.
Injection 50-600 J.lg of protein.
Retention time Identify the peaks in the chromatogram
obtained with the test solution by comparison with the
chromatogram obtained with the reference solution; any peak
with a retention time shorter than that of dimer corresponds
to polymers and aggregates.
System suitability:
- reference solution: the principal peak corresponds to IgG
monomer and there is a peak corresponding to dimer
with a retention time relative to monomer of 0.85 ±
0.05,
- test solution: the relative retentions of monomer and dimer
are 1 ± 0.05 with reference to the corresponding peaks
in the chromatogram obtained with the reference solution.
Limits:
- total monomer and dimer. at least 95 per cent of the total
area of the peaks;
- total polymers and aggregates: maximum 5 per cent of the
total area of the peaks.
Purity
Polyacrylamide gel electrophoresis (2.2.31), under
non-reducing and reducing conditions.
Resolving gel Non-reducing conditions: 8 per cent
acrylamide; reducing conditions: 12 per cent acrylamide.
Test solution Dilute the preparation to be examined to a
protein concentration of 0.5-2 mglmL.
Reference solution Dilute the reference preparation to the
same protein concentration as the test solution.
Application 10 J.lL.
Detection Coomassie staining.
Results Compared with the electropherogram of the
reference solution, no additional bands are found in the
electropherogram of the test solution.
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
The 1 to 64 dilution do es not show agglutination.
Where applicable, dilute the preparation to be examined as
prescribed for use before preparing the dilutions for the test.
Haemolysins
Prepare alto 64 dilution of the preparation to be examined,
diluted if necessary as stated on the labe!. Take 6 aliquots of
the 1 to 64 dilution. To 1 volume of 3 of the aliquots, add
1 volume of a 10 per cent V/V suspension of group Al,
group B and group O erythrocytes in a 9 giL solution of
sodium chloride R, respectively. To 1 volume of the remaining
3 aliquots, add 1 volume of a 10 per cent V/V suspension of
group Al, group B and group O erythrocytes in a 9 gIL
solution of sodium chloride R, respectively, and to each aliquot
1 volume of fresh group AB serum (as a source of
complement). Mix and incubate at 37 oC for 1 h. Examine
the supernatant liquids for haemolysis. No signs of
haemolysis are presento
Thrombocyte antibodies
Examined by a suitable method, the level of thrombocyte
antibodies is shown to be below that approved for the
specific producto
2014
Water (2.5.12)
Maximum 3 per cent.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8)
Unless otherwise justified and authorised, it complies with
the test for pyrogens. Unless otherwise prescribed, inject
1 mL per kilogram of the rabbit's body mass.
ASSAY
The biological activity is determined by measuring the
complement-dependent cytotoxicity on target cells. Flow
cytometry is performed with read-out of dead cells stained
using propidium iodide. The activity is expressed as the
concentration of anti-T lymphocyte irnmunoglobulin in
milligrams per millilitre which mediates 50 per cent
cytotoxicity .
Lymphocyte separation medium Cornmercial separation media
with low viscosity and a density of 1.077 g/mL.
Complement Commercial complement is suitable.
Buffered salt solution pH 7.2 Dissolve 8.0 g of sodium
chloride R, 0.2 g of potassium chloride R, 3.18 g of disodium
hydrogen phosphate R and 0.2 g of potassium dihydrogen
phosphate R in water R and dilute to 1000.0 mL with the
same solvent.
Buffer solution for fiow cytometry Add 40 mL of
0.1 per cent VIV sodium azide R and 10 mL of foetal calf
serum to 440 mL ofbuffered salt solution pH 7.2. The foetal
calf serum is inactivated at 56 oC for 30 min prior to use.
Store at 4 oC.
Propidium iodide solution Dissolve propidium iodide R in
buffered salt solution pH 7.2, to a concentration of
1 mglroL. Store this stock solution at 2-8 oC and use within
1 month. For the assay, dilute this solution with buffer
solution for fiow cytometry, to obtain a concentration of
5 J.lglmL. Store at 2-8 oC and use within 3 h .
Microtitre plates Plates used to prepare irnmunoglobulin
dilutions are U- or V-bottomed polystyrene or poly(vinyl
chloride) plates without surface treatment.
Micronic tubes Suitable for fiow cytometry measurement.
Cell suspension Collect blood in anticoagulant from at least
one healthy donor. Immediately iso late the peripheral blood
mononuclear cells (PBMC) by gradient centrifugation in
Iymphocyte separation medium so that the PBMC form a
visible c1ean interface between the plasma and the separation
medium. Collect the layer containing the cells and dispense
into centrifuge tubes containing buffered salt solution
pH 7.2. Centrifuge at 400 g at 2-8 oC for 10 min. Discard
the supernatant. Suspend the cell pellet in buffer solution for
fiow cytometry. Repeat the centrifugation and resuspension
procedure of the cells twice. After the third centrifugation,
resuspend the cell pellet in 1 mL of buffer solution for fiow
cytometry. Determine the number and vitality of the cells
using a haemocytometer. Cell viability of at least 90 per cent
is required. Adjust the cell number to 7 x 106/roL by adding
buffer solution for fiow cytometry. Store the cell suspension
at 4 oC and use within 12 h .
If necessary, the first PBMC pellet may be resuspended in
buffered salt solution pH 7.2 containing 20 per cent foetal
calf serum and stored overnight at 2 oC. Centrifuge at 400 g
at 2-8 oC for 10 min. Discard the supernatant. Suspend the
cell pellet in buffer solution for fiow cytometry. Determine
the number and vitality of the cells using a haemocytometer.
Cell viability of at least 90 per cent is required. Adjust the
2014
cell number to 7 x 1Q6/mL by adding buffer solution for
fiow cytometry.
It is also possible for cells to be immediately frozen and
stored in nitro gen using the following method.
Buffer solution for freezing To 20 mL of cell culture medium,
add 25 mL of foetal calf serum and 5 mL of dimethyl
sulfoxide (DMSO). Store this solution at 2-8 oC and use
within 3 h.
20 x 10 6 cells per ampoule are frozen. These ampoules are
stored in liquid nitrogen.
Buffer solution for thawing To 450 mL of cell culture
medium, add 50 mL of foetal calf serum. Store this solution
at 2-8 oC and use within 3 h.
Each ampoule is thawed in a water-bath at 37 oC with
shaking. Cell suspension is repeated in a buffer solution for
thawing. Centrifuge at 200 g at 2-8 oC for 10 mino Discard
the supematant. Suspend the cell pellet in buffer solution for
fiow cytometry. Repeat the procedure for centrifugation and
resuspension of cells once. After the second centrifugation,
resuspend the cells pellet in 1 mL of buffer solution for fiow
cytometry. Determine the number and vitality of the cells
using a haemocytometer. Cell viability of at least 90 per cent
is required. Adjust the cell number to 7 x 106/mL by adding
buffer solution for flow cytometry. Store the cell suspension
at 4 oC and use within 3 h.
Test solutions For freeze-dried preparations, reconstitute as
stated on the labe!' Prepare 3 independent series of not fewer
than 7 dilutions using buffer solution for flow cytometry as
diluent.
Reference solutions For freeze-dried preparations, reconstitute
according to the instructions for use. Prepare 3 independent
dilution series of not fewer than 7 dilutions using buffer
solution for flow cytometry as diluent.
Distribute 75 J..lL of each of the dilutions of the test solution
or reference solution to each of a series of wells of a
microtitre plate. Add 25 J..lL of the cell suspension of PBMC
into each wel!. Add 25 J..lL of rabbit complement to each of
the wells. Incubate at 37 cC for 30 mino
Centrifuge the plates at 200 g at 4 oC for 8 min, discard the
supernatant and keep the plate on ice. Preparation for flow
cytometry measurement is done step-wise by using a certain
number of wells in order to allow labelling with propidium
iodide R solution and measurement within a defined time
periodo Resuspend carefully the cell pellet of a certain
number of wells with 200 J..lL of propidium iodide solution.
Transfer the suspension into tubes. Incubate at 25 oC for
10 min then place immediately on ice.
Proceed with fluorescence measurement in a flow cytometer.
Define a region including all propidium iodide-positive cells
on the basis of Forward-Scattered, light (FSC) and
fiourescence (FL2 or FL3 for propidium iodide). Measure
the percentage of propidium iodide-positive cells, without
gating but excluding debris. Analyse at least 3000 cells for
each of the test and reference solutions.
Use the percentages of dead cells to estimate the potency as
the concentration in milligrams per millilitre of the
preparation to be examined necessary to induce 50 per cent
of cytotoxicity by fitting a sigmoidal dose response curve to
the data obtained with the test and the reference preparations
and by using a 4-parameter logistic model (see, for example,
chapter 5.3) and suitable sofrware. The test is not valid
unless the percentage of propidium iodide-positive cells at the
lower asymptote of the curve is less then 15 per cent and the
Immunosera IV-495
percentage of propidium iodide-positive cells at the upper
asymptote of the curve is at least 80 per cent.
The estimated activity is 70 per cent to 130 per cent of the
activity approved for the particular producto
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
potency.
STORAGE
Protected from light at the temperature stated on the labe!'
Expiry date The expiry date is calculated from the beginning
of the assay.
LABELLING
The label states:
-- for liquid preparations, the volume of the preparation in
the container and the protein content,
-- for freeze-dried preparations:
-- the name and the volume of the reconstitution liquid
10 be added,
-- the quantity of protein in the container,
-- that the immunoserum is to be used immediately after
reconstitution,
-- the time required for complete dissolution,
-- the animal species of origin,
-- the name and amount of stabiliser, where applicable,
-- the dilution to be made before use of the product.
____________________________________________ ~Ew
Botulinum Antitoxin ***
*** ***
(Ph Eur monograph 0085) ***
The label may state 'BotlSer' followed by a letter or letters
indicating the type or types presento
When Mixed Botulinum Antitoxin or Botulinum Antitoxin is
prescribed or demanded and the types to be present are not
stated, Botulinum Antitoxin prepared from types A, B and E
shall be dispensed or supplied.
____________________________________________
~Ew
DEFINITION
Botulinum antitoxin is a preparation containing antitoxic
globulins that have the power of specifically neutralising the
toxins formed by Glostridium botulinum type A, type B or type
E, or any mixture of these types .
PRODUCTION
It is obtained by fractionation from the serum of horses, or
other mammals, that have been immunised against
Gl. botulinum type A, type B and type E toxins.
IDENTIFICATION
It specificaIly neutralises the types of Gl. botulinum toxins
stated on the label, rendering them harmless to susceptible
animals.
POTENCY
Not less than 500 IU of antitoxin per millilitre for each of
types A and B and not less than 50 IU of antitoxin per
millilitre for type E.
The potency of botulinum antitoxin is determined by
comparing the dos e necessary to protect mice against the
lethal effects of a fixed dose of botulinum toxin with the
quantity of the standard preparation of botulinum antitoxin
necessary to give the same protection. For this comparison a
reference preparation of each type of botulinum antitoxin,
 IV-496 Irnrnunosera
calibrated in International Units, and suitable preparations of
botulinum toxins, for use as test toxins, are required.
The potency of each test toxin is determined in relation to
the specific reference preparation; the potency of the
botulinum antitoxin to be examined is determined in relation
to the potency of the test toxins by the same method.
International Units of the antitoxin are the specific
neutralising activity for botulinum toxin type A, type B and
type E contained in stated amounts of the International
Standards which consist of dried irnmune horse sera of types
A, B and E. The equivalence in Intemational Units of the
Intemational Standard is stated from time to time by the
World Health Organisation.
S election of animals Use mice having body masses such that
the difference between the lightest and the heaviest does not
exceed 5 g.
Preparation of test toxins eAUTION: B otulinum toxin is
extremely toxic: exceptional care must be taken in any procedure
in which it is employed. Prepare type A, B and E toxins from
sterile filtrates of approximately 7-day cultures in liquid
medium of el. botulinum types A, B and E. T o the filtrates,
add 2 volumes of glycerol, concentrate, if necessaty, by
dialysis against glycerol and store at or slightly below o oc.
Selection of test toxins Select toxins of each type for use as
test toxins by determining for mice the L+11 O dose and the
LD so, the observation period being 96 h. The test toxins
contain at least 1000 LD so in an L+/10 dose.
D eterminatian af test dases af the toxins (L +/l O dase) Prepare
solutions of the reference preparations in a suitable liquid
such that each contains 0.25 IU of antitoxin per millilitre.
Using each solution in turn, determine the test dose of the
corresponding test toxin.
Prepare mixtures of the solution of the reference preparation
and the test toxin such that each contains 2.0 mL of the
solution of the reference preparation, one of a graded series
of volumes of the test toxin and sufficient of a suitable liquid
to bring the total volume to 5.0 mL. AlIow the mixtures to
stand at room temperature, protected from light, for 60 mino
Using four mice for each mixture, inject adose of 1.0 mL
intraperitoneally into each mouse. Observe the mice for 96 h.
The test dose of toxin is the quantity in 1.0 mL of the
mixture made with the smallest amount of toxin capable of
causing, despite partial neutralisation by the reference
preparation, the death of all four mice injected with the
mixture within the observation periodo
D eterminatian af potency af the antitoxin Prepare solutions of
each reference preparation in a suitable liquid such that each
contains 0.25 IU of antitoxin per millilitre.
Prepare solutions of each test toxin in a suitable liquid such
that each contains 2.5 test doses per millilitre.
Using each toxin solution and the corresponding reference
preparation in turn, determine the potency of the antitoxin.
Prepare mixtures of the solution of the test toxin and the
antitoxin to be examined such that each contains 2.0 mL of
the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined, and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. AIso
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
2.0 mL of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
centred on that volume (2.0 mL) that contains 0.5 IU, and
sufficient of a suitable liquid to bring the total volume to
5.0 mL. AlIow the mixtures to stand at room temperature,
2014
protected from light, for 60 min o Using four mice for each
mixture, inject adose of 1.0 mL intraperitoneally into each
mouse. Observe the mice for 96 h .
The mixture that contains the largest volume of antitoxin
that fails to protect the mice from death contains 0.5 IU.
This quantity is used to calculate the potency of the antitoxin
in International Units per millilitre.
The test is not valid unless all the mice injected with
mixtures containing 2.0 mL or less of the solution of the
reference preparation die and all those injected with mixtures
containing more survive.
LABELLING
The label sta tes the types of el. botulinum toxin neutralised
by the preparation.
____________________________________________ ~Ew
Diphtheria Antitoxin ***
*** ***
(Ph E ur manograph 0086) ***
The label may state 'Dip/Ser'.
~Ew ____________________________________________
DEFINITlON
Diphtheria antitoxin is a preparation containing antitoxic
globulins that have the power of specifically neutralising the
toxin formed by eorynebacterium diphtheriae.
PRODUCTlON
It is obtained by fractionation from the serum of horses, or
other mammals, that have been immunised against diphtheria
toxin.
IDENTIFICATlON
It specifically neutralises the toxin formed by C. diphtheriae,
rendering it harmless to susceptible animals.
ASSAY
Not less than 1000 IU of antitoxin per millilitre for antitoxin
obtained from horse serum. Not less than 500 IU of
antitoxin per millilitre for antitoxin obtained from the serum
of other mammals.
The potency of diphtheria antitoxin is determined by
comparing the dose necessary to protect guinea-pigs or
rabbits against the erythrogenic effects of a fixed dose of
diphtheria toxin with the quantity of the standard preparation
of diphtheria antitoxin necessary to give the same protection.
For this comparison a reference preparation of diphtheria
antitoxin, calibrated in International Units, and a suitable
preparation of diphtheria toxin, for use as a test toxin, are
required. The potency of the test toxin is determined in
relation to the reference preparation; the potency of the
diphtheria antitoxin to be examined is determined in relation
to the potency of the test toxin by the same method.
The International Unit of antitoxin is the specific neutralising
activity for diphtheria toxin contained in a stated amount of
the International Standard, which consists of a quantity of
dried immune horse serum. The equivalence in International
Units of the Intemational Standard is stated by the World
Health Organisation.
Preparation af test toxin Prepare diphtheria toxin from
cultures of C. diphtheriae in a liquid medium . Filter the
culture to obtain a sterile toxic filtra te and store at 4 oC.
S election of test toxin Select a toxin for use as a test toxin by
determining for guinea-pigs or rabbits the Ir/ lOO dos e and the
minimal reacting dose, the observation period being 48 h.
2014
The test toxin has at least 200 minimal reacting doses in the
Ir/ lOO dose .
Minimal reacting dase This is the smallest quantity of toxin
which, when injected intracutaneously into guinea-pigs or
rabbits, causes a small, characteristic reaction at the site of
injection within 48 h.
The test toxin is allowed to stand for sorne months before
being used for the assay of antitoxin. During this time its
toxicity declines and the Ir/1 00 dose may be increased.
Determine the minimal reacting dose and the Ir/ lOO dose at
frequent intervals. When experiment shows that the Ir/lOO
dose is constant, the test toxin is ready for use and may be
used for a long periodo Store the test toxin in the dark at
O oC to 5 ce. Maintain its sterility by the addition of toluene
or other antimicrobial preservative that do es not cause a
rapid decline in specific toxicity.
Determinatian 01 test dase 01 toxin (lr/lOO dase) Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 0.1 IU of antitoxin per millilitre.
Prepare mixtures of the solution of the reference preparation
and of the test toxin such that each contains 1.0 mL of the
solution of the reference preparation, one of a graded series
of volumes of the test toxin and sufficient of a suitable liquid
to bring the total volume to 2.0 mL. Allow the mixtures to
stand at room temperature, protected from light, for 15 min
to 60 mino Using two animals for each mixture, inject adose
of 0.2 mL intracutaneously into the shaven or depilated
fianks of each animal. Observe the animals for 48 h.
The test dose of toxin is the quantity in 0.2 mL of the
mixture made with the smallest amount of toxin capable of
causing, despite partial neutralisation by the reference
preparation, a small but characteristic erythematous lesion at
the site of injection.
Determinarian al patency al the antitoxin Prepare a solution of
the reference preparation in a suitable liquid such that it
contains 0.125 IU of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that it contains 12.5 test doses per millilitre.
Prepare mixtures of the solution of the test toxin and of the
antitoxin to be examined such that each contains 0.8 mL of
the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 2.0 mL. AIso
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
0.8 mL of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
centred on that volume (0.8 mL) that contains 0.1 IU and
sufficient of a suitable liquid to bring the total volume to
2.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 15 min to 60 mino U sing two
animals for each mixture, inject adose of 0.2 mL
intracutaneously into the shaven or depilated fianks of each
animal. Observe the animals for 48 h.
The mixture that contains the largest volume of antitoxin
that fails to protect the guinea-pigs from the erythematous
effects of the toxin contains 0.1 ID. This quantity is used to
calculate the potency of the antitoxin in Intemational Units
per millilitre.
The test is not valid unless all the sites injected with mixtures
containing 0.8 mL or less of the solution of the reference
preparation show erythematous lesions and at all those
injected with mixtures containing more there are no lesions.
______________________________________________ PhE~
Irnrnunosera IV-497
European Viper Venom Antiserum
(Ph Eur managraph 0145)
The only poisonous snake native to the British Isles is the
adder or common viper, Vipera berus. In a geographical
region where other species of snake (including elapids) are
found, antisera able to neutralise the venoms of the species of
snake indigenous to the regíon should be used. When the
preparation is intended to neutralise the venom or venoms of
one or more snakes other than vipers, the title Snake Venom
Antiserum is used.
PhE~ ______________________________________________
DEFINITION
European viper venom antiserum is a preparation containing
antitoxic globulins that have the power of neutralising the
venom of one or more species of viper. The globulins are
obtained by fractionation of the serum of animals that have
been immunised against the venom or venoms.
IDENTIFICATION
Ir neutralises the venom of Vipera ammadytes, or Vipera aspis,
or Vipera berus, or Vipera ursinii or the mixture of these
venoms stated on the label, rendering them harmless to
susceptible animals.
ASSAY
Each millilitre of the preparation to be examined contains
sufficient antitoxic globulins to neutralise not less than 100
mouse LDso of Vipera ammodytes venom or Vipera aspis
venom and not less than 50 mouse LDso of the venoms of
other species of viper.
The potency of European viper venom antiserum is
determined by estimating the dos e necessary to protect mice
against the lethal effects of a fixed dose of venom of the
relevant species of viper.
Selectian al test venams Use venoms which have the normal
physicochemical, toxicological and immunological
characteristics of venoms from the particular species of
vipers. They are preferably freeze-dried and stored in the
dark at 5 ± 3 oC.
Select a venom for use as a test venom by determining the
LDso for mice, the observation period being 48 h.
Determinatian al the test dase 01 venam Prepare graded
dilutions of the reconstituted venom in a 9 gIL solution of
sadium chlaride R or other isotonic diluent in such a manner
that the middle dilution contains in 0.25 mL the dose
expected to be the LDso. Dilute with an equal volume of the
same diluent. Using at least four mice, each weighing 18 g to
20 g, for each dilution, inject 0.5 mL intravenously into each
mouse. Observe the mice for 48 h and record the number of
deaths. Calculate the LDso using the usual statistical
methods.
Determination 01 the patency al the antiserum to be
examined Dilute the reconstituted test venom so that
0.25 mL contains the test dose of 5 LDso (test venom
solution) .
Prepare serial dilutions of the antiserum to be examined in a
9 gIL solution of sadium chlaride R or other isotonic diluent,
the dilution factor being l.5 to 2.5 . Use a sufficient number
and range of dilutions to enable a mortality curve between
20 per cent and 80 per cent mortality to be established and
to permit an estimation of the statistical variation.
Prepare mixtures such that 5 mL of each mixture contains
2.5 mL of one of the dilutions of the antiserum to be
examined and 2.5 mL of the test venom solution. Allow the
IV-498 Irnrnunosera
mixtures to stand in a water-bath at 37 oC for 30 mino Using
not fewer than six mice, each weighing 18 g to 20 g, for each
mixture, inject 0.5 mL intravenously into each mouse.
Observe the mice for 48 h and record the number of deaths.
Calculate the PD so, using the usual statistical methods.
At the same time verify the number of LDso in the test dose
of venom, using the method described aboye. Calculate the
potency of the antiserum using the following expression:
(Tv - 1)
PDso
Tv = number of LDso in the test dose of venom.
In each mouse dose of the venom-antiserum mixture at the
end point there is one LDso of venom remaining
unneutralised by the antiserum and it is this unneutralised
venom that is responsible for the deaths of 50 per cent of the
mice inoculated with the mixture. The amount of venom
neutralised by the antiserum is thus one LDso less than the
total amount contained in each mouse dose. Therefore, as
the potency of the antiserum is defined in terms of the
number of LDso of venom that are neutralised. rather than
the number of LDso in each mouse dose, the expression
required in the calculation of potency is Tv - 1 rather than
Tv ·
Altemative!y, the quantity of test venom in milligrams that is
neutralised by 1 mL or sorne other defined volume of the
antiserum to be examined may be calculated.
LABELLING
The labe! sta tes the venom or venoms against which the
antiserum is effective.
eA UTION Because 01 the allergenic properties 01 viper venoms,
inhalation 01 venom dust should be avoided by suitable
precautions.
____________________________________________ PhEw
Gas-gangrene Antitoxin (Novyi) ***
*** ***
Gas-gangrene Antitoxin (Oedematiens) ***
(Ph Eur monograph 0087)
The labe! may state 'Nov/Ser'.
Preparation
Mixed Gas-gangrene Antitoxin
~Ew ____________________________________________
DEFINITION
Gas-gangrene antitoxin (novyi) is a preparation containing
antitoxic globulins that have the power of neutralising the
alpha toxin formed by elostridium novyi (Former
nomenclature: elostridium oedematiens). Ir is obtained by
fractionation from the serum of horses, or other mammals,
that have been immunised against el. novyi alpha toxin.
IDENfIFICATION
Ir specifically neutralises the alpha toxin formed by el. novyi,
rendering it harrnless to susceptible animals .
ASSAY
Not less than 3750 IU of antitoxin per millilitre.
The potency of gas-gangrene antitoxin (novyi) is determined
by comparing the dos e necessary to protect mi ce or other
suitable animals against the lethal effects of a fixed dose of
el. novyi toxin with the quantity of the standard preparation
2014
of gas-gangrene antitoxin (novyi) necessary to give the same
protection. For this comparison a reference preparation of
gas-gangrene antitoxin (novyi), calibrated in Intemational
Units, and a suitable preparation of el. novyi toxin for use as
a test toxin are required. The potency of the test toxin is
determined in relation to the reference preparation;
the potency of the gas-gangrene antitoxin (novyi) to be
examined is determined in relation to the potency of the test
toxin by the same method.
The Interrtational Unit of antitoxin is the specific neutralising
activity for el. novyi toxin contained in a stated amount of
the Intemational Standard, which consists of a quantity of
dried irnmune horse serum. The equivalence in International
Units of the International Standard is stated by the World
Health Organisation.
Selection 01 animals Use mice having body masses such that
the difference between the lightest and the heaviest do es not
exceed 5 g.
Preparation 01 test toxin Prepare the test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of
el. novyi. Treat the filtrate with ammonium sulfate R, collect
the precipitate, which contains the toxin, dry in vacuo over
diphosphorus pentoxide R, powder and store dry.
Selection 01 test toxin Select a toxin for use as a test toxin by
determining for mice the L+ dose and the LD so, the
observation period being 72 h. The test toxin has an L+ dose
of 0.5 mg or les s and contains not less than 25 LDso in each
L+ dose.
Determination 01 test dose 01 toxin (L+ dose) Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 12.5 ID of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that 1 mL contains a precisely known amount such as
10 mg.
Prepare mixtures of the solution of the reference preparation
and the solution of the test toxin such that each contains
0.8 mL of the solution of the reference preparation, one of a
graded series of volumes of the solution of the test toxin and
sufficient of a suitable liquid to bring the total volume to
2.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 mino Using six mice for each
mixture, inject adose of 0.2 mL intramuscularly into each
mouse. Observe the mice for 72 h.
The test dose of toxin is the quantity in 0.2 mL of the
mixture made with the smallest amount of toxin capable of
causing, despite partial neutralisation by the reference
preparation, the death of all six mice injected with the
mixture within the observation periodo
Determination 01 potency 01 the antitoxin Prepare a solution of
the reference preparation in a suitable liquid such that it
contains 12.5 IU of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that it contains 12.5 test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the
antitoxin to be examined such that each contains 0.8 mL of
the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 2.0 mL. Also
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
0.8 mL of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
centred on that volume (0.8 mL) that contains 10 IU and
sufficient of a suitable liquid to bring the total volume to
2014
2.0 mL. Al!ow the mixtures to stand at room temperature,
protected from light, for 60 mino Using six mice for each
mixture, inject adose of 0.2 mL intramuscularly into each
mouse. Observe the mice for 72 h.
The mixture that contains the largest volume of antitoxin
that fails to protect the mice from death contains 10 IU. This
quantity is used to calculate the potency of rhe antitoxin in
Intemational Units per millilitre.
The test is not valid unless al! the mice injected wirh
mixtures containing 0.8 mL or less of the solution of the
reference preparation die and al! rhose injected wirh mixtures
containing a larger volume survive.
____________________________________________ ~E~
Gas-gangrene Antitoxin
(Perfringens)
(Ph Eur managraph 0088)
The label may state 'PertlSer'.
Preparation
Mixed Gas-gangrene Antitoxin
~E~ ____________________________________________
DEFINITION
Gas-gangrene antitoxin (perfringens) is a preparation
containing antitoxic globulins that have the power of
specifically neutralising the alpha toxin formed by elastridium
perfringens. It is obtained by fractionation from rhe serum of
horses, or other mammals, that have been immunised against
el. perfringens alpha toxin.
IDENTIFICATION
It specifically neutralises the alpha toxin formed by
el. perfringens, rendering it harmless to susceptible animals.
ASSAY
Not less rhan 1500 IU of antitoxin per millilitre.
The potency of gas-gangrene antitoxin (perfringens) is
determined by comparing rhe dos e necessary to protect mice
or other suitable animals against the lethal effects of a fixed
dose of el. perfringens toxin with the quantity of rhe standard
preparation of gas-gangrene antitoxin (perfringens) necessary
to give rhe same protection. For this comparison a reference
preparation of gas-gangrene antitoxin (perfringens), calibrated
in Intemational Units, and a suitable preparation of
el. perfringens toxin for use as a test toxin are required.
The potency of the test toxin is determined in relation to rhe
reference preparation; the potency of rhe gas-gangrene
antitoxin (perfringens) to be examined is determined in
relation to the potency of rhe test toxin by rhe same merhod.
The Intemational Unit of antitoxin is rhe specific neutralising
activity for el. perfringens toxin contained in a stated amount
of me Intemational Standard, which consists of a quantity of
dried irnmune horse serum. The equivalence in Intemational
Units of the Intemational Standard is stated by me World
Healm Organisation.
Selectian ai animals Use mice havmg body masses such mat
the difference between the lightest and the heaviest do es not
exceed 5 g.
Preparatian ai test taxin Prepare me test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of
el. perfringens. Treat me filtrate with ammanium sulfate R,
collect the precipita te, which contains me toxin, dry in vacuo
over diphaspharus pentoxide R, powder and store dry.
Irnrnunosera IV-499
Selectian ai test toxin Select a toxin for use as a test toxin by
determining for mice the L+ dose and me LD so, me
observation period being 48 h. The test toxin has an L+ dose
of 4 mg or les s and contains not less man 20 LDso in each
L+ dose.
Determinatian ai test dase ai toxin (L+ dase) Prepare a
solution of the reference preparation in a suitable liquid such
mat it contains 5 IU of antitoxin per millilitre.
Prepare a solution of me test toxin in a suitable liquid such
mat 1 mL contains a precisely known amount such as 10 mg.
Prepare mixtures of me solution of the reference preparation
and me solution of me test toxin such mat each contains
2.0 mL of me solution of the reference preparation, one of a
graded series of volumes of me solution of the test toxin and
sufficient of a suitable liquid to bring me total volume to
5.0 mL. Allow me mixtures to stand at room temperature,
protected from light, for 60 mino Using six mice for each
mixture, inject adose of 0.5 mL intravenously into each
mouse. Observe me mice for 48 h.
The test dos e of toxin is the quantity in 0.5 mL of me
mixture made wim me smallest amount of toxin capable of
causing, despite partial neutralisation by me reference
preparation, the deam of all six mice injected with the
mixture within me observation periodo
Determinatian ai patency ai the antitoxin Prepare a solution of
me reference preparation in a suitable liquid such that it
contains 5 IU of antitoxin per millilitre.
Prepare a solution of me test toxin in a suitable liquid such
mat it contains five test doses per millilitre.
Prepare mixtures of me solution of the test toxin and me
antitoxin to be examined such mat each contains 2.0 mL of
me solution of me test toxin, one of a graded series of
volumes of me antitoxin to be examined and sufficient of a
suitable liquid to bring me total volume to 5.0 mL. Also
prepare mixtures of the solution of me test toxin and me
solution of the reference preparation such that each contains
2.0 mL of me solution of the test toxin, one of a graded
series of volumes of me solution of me reference preparation
centred on mat volume (2.0 mL) mat contains 10 IU and
sufficient of a suitable liquid to bring rhe total volume to
5.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 mino Using six mice for each
mixture, inject adose of 0.5 mL intravenously into each
mouse. Observe me mice for 48 h.
The mixture mat contains me largest volume of antitoxin
mat fails to protect the mice from deam contains 10 IU. This
quantity is used to calculate the potency of the antitoxin in
Intemational Units per millilitre.
The test is not valid unless all me mice injected wim
mixtures containing 2.0 mL or less of the solution of me
reference preparation die and all those injected with mixtures
containing a larger volume survive.
____________________________________________ PhE~
 IV-SOO Irnrnunosera
***
Gas-gangrene Antitoxin
*** ***
(Septicum) ***
(Ph Eur monograph 0089)
The labe! may state 'Sep/Ser'.
Preparation
Mixed Gas-gangrene Antitoxin
____________________________________________
PhE~
DEFINITION
Gas-gangrene antitoxin (septicum) is a preparation
containing antitoxic globulins that have the power of
specifically neutralising the alpha toxin formed by Glostridium
septicum. It is obtained by fractionation from the serum of
horses, or other mammals, that have been immunised against
Gl. septicum alpha toxin.
IDENTIFICATION
It specifically neutralises the alpha toxin formed by
Gl. septicum, rendering it harmless to susceptible animals.
ASSAY
N ot les s than 1500 IU of antitoxin per millilitre.
The potency of gas-gangrene antitoxin (septicum) is
determined by comparing the dose neeessary to protect mice
or other suitable animals against the lethal effects of a fixed
dose of Gl. septicum toxin with the quantity of the standard
preparation of gas-gangrene antitoxin (septicum) necessary to
give the same protection. For this comparison a reference
preparation of gas-gangrene antitoxin (septicum), calibrated
in International Units, and a suitable preparation of
Gl. septicum toxin for use as a test toxin are required.
The potency of the test toxin is determined in relation to the
reference preparation; the potency of the gas-gangrene
antitoxin (septicum) to be examined is determined in relation
to the potency of the test toxin by the same method.
The Intemational Unit of antitoxin is the specific neutralising
activity for Gl. septicum toxin contained in a stated amount of
the International Standard, which consists of a quantity of
dried immune horse serum. The equivalence in International
Units of the International Standard is stated by the World
Health Organisation.
Selection oi animals Use mice having body mas ses such that
the difference between the lightest and the heaviest do es not
exceed 5 g.
Preparation oi test toxin Prepare the test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of
Gl. septicum. Treat the filtrate with ammonium sulfate R,
colleet the precipita te, which contains the toxin, dry in vacuo
over diphosphorus pentoxide R, powder and store dry.
Selection oi test toxin Select a toxin for use as a test toxin by
determining for mice the L+ dose and the LD so, the
observation period being 72 h. The test toxin has an L+ dose
of 0.5 mg or less and contains not less than 25 LDso in each
L+ dose.
Determination oi test dose oi toxin (L+ dose) Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 5 IU of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that 1 mL contains a precisely known amount such as
20 mg.
Prepare mixtures of the solution of the reference preparation
and the solution of the test toxin such that each contains
2.0 mL of the solution of the reference preparation, one of a
graded series of volumes of the solution of the test toxin and
2014
sufficient of a suitable liquid to bring the total volume to
5.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 min. Using six mice for each
mixture, inject adose of 0.5 mL intravenously into each
mouse. Observe the mice for 72 h .
The test dose of toxin is the quantity in 0.5 mL of the
mixture made with the smallest amount of toxin capable of
causing, des pite partial neutralisation by the reference
preparation, the death of all six mice injected with the
mixture within the observation periodo
Determination oi potency oi the antitoxin Prepare a solution of
the reference preparation in a suitable liquid such that it
contains 5 IU of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that it contains five test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the
antitoxin to be examined such that each contains 2.0 mL of
the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. AIso
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
2.0 mL of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
eentred on that volume (2 .0 mL) that contains 10 IU and
sufficient of a suitable liquid to bring the total volume to
5.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 min. Using six mice for each
mixture, inject adose of 0.5 mL intravenously into each
mouse. Observe the mice for 72 h.
The mixture that contains the largest volume of antitoxin
that fails to protect the mice from death contains 10 IU. This
quantity is used to calculate the potency of the antitoxin in
International Units per millilitre.
The test is not valid unless all the mice injected with
mixtures containing 2.0 mL or les s of the solution of the
reference preparation die and all those injected with mixtures
containing more survive.
____________________________________________ ~E~
***
Mixed Gas-gangrene Antitoxin * *
** **
(Ph Eur monograph 0090) ***
The label may state 'Gas/Ser'.
~E~ ____________________________________________
DEFINITION
Mixed gas-gangrene antitoxin is prepared by mixing gas-
gangrene antitoxin (novyi), gas-gangrene antitoxin
(perfringens) and gas-gangrene antitoxin (septicum) in
appropriate quantities.
IDENTIFICATION
It specifically neutralises the alpha toxins formed by
Glostridium novyi (former nomenclature: Glostridium
oedematiens), Glostridium perfringens and Glostridium septicum,
rendering them harmless to susceptible animals.
ASSAY
Gas-gangrene antitoxin (novyi), not less than 1000 IU of
antitoxin per millilitre; gas-gangrene antitoxin (perfringens),
not less than 1000 IU of antitoxin per millilitre; gas-gangrene
antitoxin (septicum) not less than 500 IU of antitoxin per
millili tre.
2014
Carry out the assay for each component, as prescribed in the
monographs on Gas-gangrene antitoxin (novyi) (0087),
Gas-gangrene antitoxin (perfringens) (0088) and Gas-gangrene
antitoxin (septicum) (0089) .
_ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ PhEur
Tetanus Antitoxin *****
** **
(Tetanus Antitoxin ior Human Use, ***
Ph Eur monograph 0091)
The label may state 'TetlSer'.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ __
DEFINITION
Tetanus antitoxin for human use is a preparation containing
antitoxic globulins that have the power of specificalIy
neutralising the toxin formed by elostridium tetani.
PRODUCTION
It is obtained by fractionation from the serum of horses, or
other mammals, that have been immunised against tetanus
toxin.
IDENTIFICATION
It specificalIy neutralises the toxin formed by el. tetani,
rendering it harmless to susceptible animals.
POTENCY
Not les s than 1000 IU of antitoxin per milIilitre when
intended for prophylactic use. Not les s than 3000 IU of
antitoxin per millilitre when intended for therapeutic use.
The potency of tetanus antitoxin is determined by comparing
the dose necessary to protect guinea-pigs or mice against the
paralytic effects of a fixed dose of tetanus toxin with the
quantity of the standard preparation of tetanus antitoxin
necessary to give the same protection. In countries where the
paralysis method is not obligatory the lethal method may be
used. For this method the number of animals and the
procedure are identical with those described for the paralysis
method but the end-point is the death of the animal rather
than the onset of paralysis and the L+1l O dos e is used
instead of the Lpll O dose. For this comparison a reference
preparation of tetanus antitoxin, calibrated in International
Units, and a suitable preparation of tetanus toxin, for use as
a test toxin, are required. The potency of the test toxin is
determined in relation to the reference preparation;
the potency of the tetanus antitoxin to be examined is
determined in relation to the potency of the test toxin by the
same method.
The Intemational Unit of antitoxin is the specific neutralising
activity for tetanus toxin contained in a stated amount of the
International Standard which consists of a quantity of dried
immune horse serum. The equivalence in Intemational Units
of the Intemational Standard is stated by the World Health
Organisation.
Seleetion oi animals If mice are used, the body masses
should be such that the difference between the lightest and
the heaviest does not exceed 5 g.
Preparation oi test toxin Prepare the test toxin from a sterile
filtra te of an approximately 9-day culture in liquid medium of
el. terani. To the filtrate add 1 to 2 volumes of glycerol and
store slight1y below O oc. Altematively, treat the filtrate with
ammonium sulfate R, colIect the precipita te, which contains
the toxin, dry in vaeuo over diphosphorus pentoxt'de R, powder
Immunosera IV-50l
and store dry, either in sealed ampoules or in vacuo over
diphosphorus pentoxide R.
Determination oi test dose oi toxin (Lp/lO dose) Prepare a
solution of the reference preparation in a suitable liquid such
that it contains 0.5 IU of antitoxin per milIilitre.
If the test toxin is stored dry, reconstitute it using a suitable
liquido
Prepare mixtures of the solution of the reference preparation
and the test toxin such that each contains 2.0 mL of the
solution of the reference preparation, one of a graded series
of volumes of the test toxin and sufficient of a suitable liquid
to bring the volume to 5.0 mL. AlIow the mixtures 10 stand
at room temperature, protected from light, for 60 minoUsing
six mice for each mixture, inject adose of 0.5 mL
subcutaneously into each mouse. Observe the mice for 96 h.
Mice that become paralysed may be euthanised.
The test dose of toxin is the quantity in 0.5 mL of the
mixture made with the smalIest amount of toxin capable of
causing, despite partial neutralisation by the reference
preparation, paralysis in alI six mice injected with the mixture
within the observation periodo
Determination oi potency oi the antitoxin Prepare a solution of
the reference preparation in a suitable liquid such that ir
contains 0.5 IU of antitoxin per millilitre.
Prepare a solution of the test toxin in a suitable liquid such
that it contains five test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the
antitoxin to be examined such that each contains 2.0 mL of
the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. Also
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
2.0 mL of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
centred on that volume (2.0 mL) that contains 1 IU and
sufficient of a suitable liquid to bring the total volume to
5.0 mL. AIlow the mixtures to stand at room temperature,
protected from light, for 60 mino Using six mice for each
mixture, inject into each mouse subcutaneously adose of
0.5 mL. Observe the mice for 96 h. Mice that become
paralysed may be euthanised.
The mixture that contains the largest volume of antitoxin
that fails to protect the mice from paralysis contains 1 IU.
This quantity is used to calculate the potency of the antitoxin
in International Units per millilitre.
The test is not valid unless aIl the mice injected with
mixtures containing 2.0 mL or les s of the solution of the
reference preparation show paralysis and alI those injected
with mixtures containing more do noto
_ _ _ _ _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ ~E~
IV-502 Vaccines
*****
VACCINES
** **
(Vaccines for Human Use, Ph Bur monograph 0153) ***
Vaccines comply with the requirements of the European
Pharmacopoeia monograph for Vaccines for Human Use. These
requirements are reproduced below.
~Ew ____________________________________________
DEFINITION
Vaccines for human use are preparations containíng antigens
capable of inducing a specific and active irnmunity in man
against an infecting agent or the toxin or antigen elaborated
by it. Irnmune responses include the induction of the innate
and the adaptive (cellular, humoral) parts of the irnmune
system. Vaccines for human use shall have been shown to
have acceptable immunogenic activity and safety in man with
the intended vaccination schedule.
Vaccines for human use may contain: whole micro-organisms
(bacteria, viruses or parasites), inactivated by chemical or
physical means that maintain adequate irnmunogenic
properties; whole live micro-organisms thar are naturally
avirulent or that have been treated to attenuate their
virulence whilst retaining adequate irnmunogenic properties;
antigens extracted from the micro-organisms or secreted by
the micro-organisms or produced by genetic engineering or
chemical synthesis. The antigens may be used in their native
state or may be detoxified or otherwise modified by chemical
or physical means and may be aggregated, polymerised or
conjugated to a carrier to increase their irnmunogenicity.
Vaccines may contain an adjuvant. Where the antigen is
adsorbed on a mineral adjuvant, the vaccine is referred to as
'adsorbed' .
Terminology used in monographs on vaccines for human use
is defined in chapter 5.2.1.
Bacterial vaccines containing whole cells are suspensions of
various degrees of opacity in colourless or almost colourless
liquids, or may be freeze-dried. They may be adsorbed.
The concentration of living or inactivated bacteria is
expressed in terms of Intemational Units of opacity or, where
appropriate, is determined by direct cell count or, for live
bacteria, by viable count.
Bacterial vaccines containing bacterial components are
suspensions or freeze-dried products. They may be adsorbed.
The antigen content is determined by a suitable validated
assay.
Bacterial toxoids are prepared from toxins by diminishing their
toxicity to an acceptable level or by completely eliminating it
by physical or chemical procedures whilst retaining adequate
immunogenic properties. The toxins are obtained from
selected strains of micro-organisms. The method of
production is such that the toxoid do es not revert to toxin.
The toxoids are purified. Purification is performed before
and/or after detoxification. Toxoid vaccines may be adsorbed.
Viral vaccines are prepared from virus es grown in animals, in
fertilised eggs, in suitable cell cultures or in suitable tissues,
or by culture of genetically engineered cells. They are liquids
that vary in opacity according to the type of preparation or
may be freeze-dried. They may be adsorbed. Liquid
preparations and freeze-dried preparations after reconstitution
may be coloured if a pH indicator such as phenol red has
been used in the culture medium.
Synthetic antigen vaccines are generally clear or colourless
liquids. The concentration of the components is usually
expressed in terms of specific antigen contento
2014
Combined vaccines are multicomponent preparations
formulated so that different antigens are administered
simultaneously. The different antigenic components are
intended to protect against different strains or types of the
same organism and/or against different organisms.
A combined vaccine may be supplied by the manufacturer
either as a single liquid or freeze-dried preparation or as
several constituents with directions for admixture before use.
Where there is no monograph to cover a particular
combination, the vaccine complies with the monograph for
each individual component, with any necessary modifications
approved by the competent authority.
Adsorbed vaccines are suspensions and may form a sediment
at the bottom of the container.
PRODUCTION
General provisions
The production method for a given product must have been
shown to yield consistently batches comparable with the
batch of proven clinical efficacy, immunogenicity and safety
in mano Product specifications including in-pro ces s testing
should be set. Specific requirements for production including
in-pro ces s testing are included in individual monographs.
Where justified and authorised, certain tests may be omitted
where it can be demonstrated, for example by validation
studies, that the production process consistently ensures
compliance with the test.
Unless otherwise justified and authorised, vaccines are
produced using a seed-Iot system. The methods of
preparation are designed to maintain adequate immunogenic
properties, to render the preparation harmless and to prevent
contamination with extraneous agents.
Where vaccines for human use are manufactured using
materials of human or animal origin, the general
requirements of chapter 5.1.7. Viral safel'y apply in
conjunction with the more specific requirements relating to
viral safety in this monograph, in chapters 5.2.2. Chicken
flocks free from specified pathogens for the production and qualil'y
control of vaccines, 5.2.3. Cell substrates for the production of
vaccines for human use and 2.6.16. Tests for extraneous agents in
viral vaccines for human use, and in individual monographs.
Unless otherwise justified and authorised, in the production
of a final lot of vaccine, the number of passages of a virus, or
the number of sub cultures of a bacterium, from the master
seed lot shall not exceed that used for production of the
vaccine shown to be satisfactory in clinical trials with respect
to safety and efficacy or immunogenicity.
Vaccines are as far as possible free from ingredients known to
cause toxic, allergic or other undesirable reactions in mano
Suitable additives, including stabilisers and adjuvants may be
incorporated. Penicillin and streptomycin are neither used at
any stage of production nor added to the final product;
however, master seed lots prepared with media containing
penicillin or streptomycin may, where justified and
authorised, be used for production.
Consistency of production is an important feature of vaccine
production. Monographs on vaccines for human use give
limits for various tests carried out duríng production and on
the final lot. These limits may be in the form of maximum
values, mínimum values, or minimum and maximum
tolerances around a given value. While compliance with these
limits is required, it is not necessarily sufficient to ensure
consistency of production for a given vaccine. For relevant
tests, the manufacturer must therefore define for each
product a suitable action or release limit or limits to be
applied in view of the results found for batches tested
2014
clinically and those used to demonstrate consistency of
production. These limits may subsequently be refined on a
statistical basis in light of production data.
Substrates for propagation
Substrates for propagation comply with the relevant
requirements of the Pharmacopoeia (5.2.2, 5.2.3) or in the
absence of such requirements with those of the competent
authority. Processing of cell banks and subsequent cell
cultures is done under aseptic conditions in an area where no
other cells are being handled. Serum and trypsin used in the
preparation of cell suspensions shall be shown to be free froro
extraneous agents.
Seed lots/cell banks
The master seed lot or cell bank is identified by historical
records that include information on its origin and subsequent
manipulation. Suitable mea sures are taken to ensure that no
extraneous agent or undesirable substance is present in a
master or working seed lot or a cell bank.
Culture media
Culture media are as far as possible free from ingredients
known to cause toxic, allergic or other undesirable reactions
in man; if inclusion of such ingredients is necessary, it shall
be demonstrated that the amount present in the finallot is
reduced to such a level as to render the product safe.
Approved animal (but not human) serum may be used in the
growth medium for cell cultures but the medium used for
maintaining cell growth during virus multiplication shall not
contain serum, unless otherwise stated. Cell culture media
may contain a pH indicator such as phenol red and approved
antibiotics at the lowest effective concentration, although it is
preferable to have a medium free from antibiotics during
production.
Propagation and harvest
The seed cultures are propagated and harvested under
defined conditions. The purity of the harvest is verified by
suitable tests as defined in the monograph.
Control cells
For vaccines produced in cell cultures, control cells are
maintained and tested as prescribed. In order to provide a
valid control, these cells must be maintained in conditions
that are essentially equivalent to those used for the
production cell cultures, including use of the same batches of
media and media changes.
Control eggs
For live vaccines produced in eggs, control eggs are
incubated and tested as prescribed in the monograph.
Purification
Where applicable, validated purification procedures may be
applied.
Inactivation
Inactivated vaccines are produced using a validated
inactivation process whose effectiveness and consistency have
been demonstrated. Where it is recognised that extraneous
agents may be present in a harvest, for example in vaccines
produced in eggs from healthy, non-SPF flocks, the
inactivation process is also validated with respect to a panel
of model extraneous agents representative of the potential
extraneous agents. A test for effectiveness of the inactivation
process is carried out as soon as possible after the
inactivation process.
Test for sterility of intermediates prior to final buIk
Individual monographs on vaccines for human use may
prescribe a test for sterility for intermediates.
Vaccines IV-503
In agreement with the competent authority, replacement of
the sterility test by a bioburden test with a low bioburden
limit based on batch data and process validation may be
acceptable for intermedia tes preceding the final bulk,
provided that a sterilising filtration is performed later in the
production process .
It is a prerequisite that the intermediate is filtered through a
bacteria-retentive filter prior to storage, that authorised pre-
filtration bioburden limits have been established for this
filtration, and that adequate mea sures are in place to avoid
contamination and growth of micro-organisms during storage
of the intermediate.
Final buIk
The final bulk is prepared by aseptically blending the
ingredients of the vaccine. For non-liquid vaccines for
administration by a non-parenteral route, the final bulk is
prepared by blending the ingredients of the vaccine under
suitable conditions .
Acijuvants One or more adjuvants may be included in the
formulation of a vaccine to potentiate and/or modulate the
immune response to the antigen(s). Adjuvants may be
included in the formulation of the final vaccine or presented
separately. Suitable characterisation and quality control of the
adjuvant(s), alone and in combination with the antigen(s), is
essential for consistent production. Quality specifications are
established for each adjuvant, alone and in combination with
the antigen(s).
Adsorbents as ad;juvants Vaccines may be adsorbed on
aluminium hydroxide, aluminium phosphate, calcium
phosphate or other suitable adsorbents. The adsorbents are
prepared in special conditions that confer the appropriate
physical form and adsorptive properties.
Where an adsorbent is used as an adjuvant and is generated
in situ during production of the vaccine, quality specifications
are established for each of the ingredients and for the
generated adsorbent in the vaccine. Quality specifications are
intended to control, in particular:
- qualitative and quantitative chemical composition;
- physical form and associated adsorptive properties, where
relevant, and particularly where the adjuvant will be
present as an adsorbent;
interaction between adjuvant and antigen;
- purity, including bacterial endotoxin content and
microbiological quality;
- any other parameters identified as being critical for
functionality.
The stability of each adjuvant, alone and in combination with
the antigen(s), particularly for critical parameters, is
established during development studies.
Antimicrobial preservatives Antimicrobial preservatives are
used to prevent spoilage or adverse effects caused by
microbial contamination occurring during the use of a
vaccine. Antimicrobial preservatives are not included in
freeze-dried products . For single-dos e liquid preparations,
inclusion of antimicrobial preservatives is not normally
acceptable. For multidose liquid preparations, the need for
effective antimicrobial preservation is evaluated taking into
account likely contamination during use and the maximum
recommended period of use after broaching of the container.
If an antimicrobial preservative is used, it shall be shown that
it does not impair the safety or efficacy of the vaccine .
Addition of antibiotics as antimicrobial preservatives is not
normally acceptable.
During development studies, the effectiveness of the
antimicrobial preservative throughout the period of validity
 IV-504 Vaccines
shalJ be demonstrated to the satisfaction of the competent
authority.
The efficacy of the antimicrobial preservative is evaluated as
described in chapter 5.1.3. If neither the A criteria nor the B
criteria can be met, then in justified cases the following
criteria are applied to vaccines for human use: bacteria, no
increase at 24 h and 7 days, 3 log reduction at 14 days, no
increase at 28 daysj fungi, no increase at 14 days and
28 days.
Stability of intermediates
During production ofvaccines, intermediates are obtained at
various stages and are stored, sometimes for long periods.
Such intermediates include:
- seed lots and cell banksj
- live or inactivated harvestsj
- purified harvests that may consist of toxins or toxoids,
polysaccharides, bacterial or viral suspensionsj
- purified antigensj
- adsorbed antigensj
- conjugated polysaccharidesj
- final bulk vaccinej
- vaccine in the final closed container stored at a
temperature lower than that used for final-product
stability studies and intended for release without re-assay.
Except where they are used within a short period of time,
stability studies are carried out on the intermedia tes in the
intended storage conditions to establish the expected extent
of degradation. For final bulk vaccine, stability studies may
be carried out on representative samples in conditions
equivalent to those intended to be used for storage. For each
intermediate (except for seed lots and celJ banks), a period of
validity applicable for the intended storage conditions is
established, where appropriate in light of stability studies.
Finallot
The final lot is prepared by aseptically distributing the final
bulk into sterile, tamper-proof containers, which, after freeze-
drying where applicable, are closed so as to exclude
contamination. For non-liquid vaccines for administration by
a non-parenteral route, the final lot is prepared by
distributing the final bulk under suitable conditions into
sterile, tamper-proof containers. Where justified and
authorised, certain tests prescribed for the final lot may be
carried out on the final bulk, if it has been demonstrated that
subsequent manufacturing operations do not affect
compliance.
Appearance
UnIess otherwise justified and authorised, each container
(vial, syringe or ampoule) in each finallot is inspected
visualJy or mechanically for acceptable appearance.
Degree 01 adsorption For an adsorbed vaccine, unIess
otherwise justified and authorised, a release specification for
the degree of adsorption is established in light of results
found for batches used in clinical trials. From the stability
data generated for the vaccine it must be shown that at the
end of the period of validity the degree of adsorption is not
less than for batches used in clinical trials.
Thermal stabiliry When the thermal stability test is
prescribed in a monograph for a live attenuated vaccine, the
test is carried out on the finallot to monitor the lot-to-Iot
consistency in heat-sensitivity of viral/bacterial particles in the
product. Suitable conditions are indicated in the individual
monograph. The test may be omitted as a routine test for a
given product once the consistency of the production process
has been demonstrated, in agreement with the competent
authority, using relevant parameters, such as consistency in
2014
yield, ratio of infectious viruses (viable bacteria) before and
after freeze-drying, potency at release and real-time stability
under the prescribed conditions as welJ as thermal stability.
Where there is a significant change in the manufacturing
procedure of the antigen(s) or formulation, the need for
re-introduction of the test is considered.
Stabiliry During development studies, maintenance of
potency of the final lot throughout the period of validity shalJ
be demonstratedj the loss of potency in the recommended
storage conditions is assessed. Excessive loss even within the
limits of acceptable potency may indicate that the vaccine is
unacceptable.
Expiry date UnIess otherwise stated, the expiry date is
calculated from the beginning of the assay or from the
beginning of the first assay for a combined vaccine.
For vaccines stored at a temperature lower than that used for
stability studies and intended for release without re-assay, the
expiry date is calculated from the date of removal from cold
storage. If, for a given vaccine, an assay is not carried out,
the expiry date for the final lot is calculated from the date of
an approved stability-indicating test or, failing this, from the
date of freeze-drying or the date of filling into the final
containers. For a combined vaccine where components are
presented in separate containers, the expiry date is that of the
component which expires first.
The expiry date applies to vaccines stored in the prescribed
conditions.
Animal tests
In accordance with the provisions of the European
Convention for the Protection of Vertebra te Animals U sed
for Experimental and Other Scientific Purposes, tests must
be carried out in such a way as to use the minimum number
of animals and to cause the least pain, suffering, distress or
lasting harm. The criteria for judging tests in monographs
must be applied in light of this. For example, if it is indicated
that an animal is considered to be positive, infected, etc.
when typical clinical signs or death occur, then as soon as
sufficient indication of a positive result is obtained the animal
in question shall be either euthanised or given suitable
treatment to prevent unnecessary suffering. In accordance
with the General Notices, alternative test methods may be
used to demonstrate compliance with the monograph and the
use of such tests is particularly encouraged when this leads to
replacement or reduction of animal use or reduction of
suffering.
TESTS
Vaccines comply with the tests prescribed in individual
monographs including, where applicable, the following:
pH (2.2.3)
Liquid vaccines, after reconstitution where applicable,
comply with the limits for pH approved for the particular
preparation.
Adjuvant
If the vaccine contains an adjuvant, the amount is
determined and shown to be within acceptable limits with
respect to the expected amount (see also the tests for
aluminium and calcium below).
Aluminium (2.5.13)
Maximum 1.25 mg of aluminium (Al) per single human dose
where an aluminium adsorbent has been used in the vaccine,
unless otherwise stated.
 2014
Calcium (2.5.14)
Maximum 1.3 mg of calcium (Ca) per single human dose
where a calcium adsorbent has been used in the vaccine,
unless otherwise stated.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL of free formaldehyde in the final product
where formaldehyde has been used in the preparation of the
vaccine, unless otherwise stated.
Phenol (2.5.15)
Maximum 2.5 gIL in the final product where phenol has
been used in the preparation of the vaccine, unless otherwise
stated.
Water (2.5.12)
Maximum 3.0 per cent m/m for freeze-dried vaccines, unless
otherwise stated.
Extractable volume (2.9.17)
Unless otherwise justified and authorised, it complies with
the requirement for extractable volume.
Bacterial endotoxins
Unless otherwise justified and authorised, a test for bacterial
endotoxins is carried out on the final product. Where no
limit is specified in the individual monograph, the content of
bacterial endotoxins determined by a suitable method
(2. 6.14) is les s than the limit approved for the particular
product.
STORAGE
Store protected from light. Unless otherwise stated, the
storage temperature is 5 ± 3 oC; liquid adsorbed vaccines
must not be allowed to freeze.
LABELLING
The label states:
-- the name of the preparation;
-- a reference identifying the finallot;
-- the recommended human dose and route of
administration;
-- the storage conditions;
-- the expiry date;
-- the name and amount of any antimicrobial preservative;
-- the name of any antibiotic, adjuvant, fiavour or stabiliser
present in the vaccine;
-- where applicable, that the vaccine is adsorbed;
-- the name of any constituent that may cause adverse
reactions and any contra-indications to the use of the
vaccine;
-- for freeze-dried vaccines:
-- the na me or composition and the volume of the
reconstituting liquid to be added;
-- the time within which the vaccine is to be used after
reconstitution.
____________________________________________ Ph
Vaccines IV-50S
*****
Anthrax Vaccine for Human Use
* *
(Adsorbed, Prepared from Culture **** *
Filtrates)
(Ph Eur monograph 2188)
The label may state 'Anthrax'.
~E~ ____________________________________________
DEFINITION
Anthrax vaccine for human use (adsorbed, prepared from
culture filtra tes) is a preparation of Bacillus anthracis antigens
precipitated by aluminium potassium sulfate. The antigens
are prepared from a sterile culture filtrate produced by a
non-encapsulated strain, either avirulent or anenuated,
of B. anthracis.
The main virulence components of B . anthracis are the
polyglutamic aicd capsule and 2 binary anthrax toxins,
namely lethal toxin and redema toxin, formed from the
respective combination of protective antigen (PA) with either
lethal factor (LF) or redema factor (EF).
LF is a zinc-dependent endopeptidase and EF is a potent
calmodulin and calcium-dependent adenylate cyclase.
Cell-free cultures of B. anthracis contain PA and because
expression of the 3 toxin-component genes is co-ordinately
regulated, LF and EF are also presento In addition, the
vaccine is likely to contain many other B. anthracis antigens,
including membrane proteins, secreted proteins, cytoplasmic
proteins, peptidoglycans, nucleic acids and carbohydrates.
PRODUCTION
GENERAL PROVISIONS
Cultures are managed in a seed-Iot system. The vaccine
strain is toxigenic but lacks the plasmid with the necessary
genes for synthesis of the capsule, an important virulence
factor.
The production method must be shown to yield a consistent
and active product with a safety and efficacy profile that is
adequate or equivalent to previous lots. The vaccine must
show a level of protection against a virulent strain of B.
anthracis, in a suitable animal infection model, that is equal
to or greater than that of a reference vaccine. The vaccine
must not show a level of toxicity that exceeds that of a
reference vaccine.
The production method and stability of the finallot and
relevant intermediates are evaluated using one or more
indicator tests. Such tests include potency and specific
toxicity, and may be supported by tests confirming the
presence of relevant antigens and associated proteins. Release
and shelf-life specifications are established based upon the
results of stability testing so as 10 ensure satisfactory product
E~
performance during the approved period of validity.
SEED LOTS
The attenuated non-encapsulated strain of B . anthracis used
is identified by historical records that include information on
its origin and subsequent manipulation and the tests used to
characterise the strain. These include morphological, cultural,
biochemical and genetic properties of the strain. Only a
master seed lot or, where applicable, working seed lots, that
comply with the following requirements may be used.
Identification
Each seed lot is identified as containing B. anthracis.
Phenotypic parameters
Each seed lot must have a known biochemical and enzymatic
profile and have a known history of absence of antibiotic
resistance.
 IV-506 Vaccines
Microbial purity
Each seed lot complies with the requirements for absence of
contaminating organisms. Purity of bacterial cultures is
verified by methods of suitable sensitivity.
Virulence test
The absence of bacterial capsule is demonstrated for each
seed lot by McFadyean stain and the specific toxicity
(oedema) test.
REFERENCE PREPARATION
The potency and toxicity of the vaccine bulk are verified
using reference standards derived from representative vaccine
batches. These batches are extensively characterised for their
intended purpose and are stored in suitably sized aliquots
under conditions ensuring their stability.
PROPAGATION ANO HARVEST
The atrenuated strain is grown using suitable liquid media.
At the end of cultivation, the purity of the culture is tested.
The culture medium is separated from the bacterial mass by
filtration. The pH of the filtra te is determined after dilution
with a 0.9 gIL solution of sodium chloride R and is shown to
be within limits suitable for stability. A suitable test for
absence of live B. anthracis, including spores, is carried out.
Aluminium potassium sulfate or an alternative adjuvant may
be added at this stage. An antimicrobial preservative may be
added to the suspension to form the purified harvest.
Only a purified harvest that complies with the following
requirements may be used in the preparation of the finallot.
Immunological identity
Confirm the presence of B. anthracis protective antigen by a
suitable irnmunochemical method (2.7.1).
Antimicrobial preservative
Determine the amount of antimicrobial preservative by a
suitable chemical method . The amount is not less than
85 per cent and not greater than 115 per cent of the
intended contento
FINAL BULK VACCINE
The purified harvest is diluted aseptically with sterile saline
solution to make the final bulk vaccine.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof glass ampoules and heat-sealed to prevent
contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided the potency assay,
the specific toxicity (o edema) test and the test for
antimicrobial preservative have been carried out with
satisfactory results on the purified harvest, they may be
omitted on the finallot.
IDENTIFICATION
The presence of B. anthracis protective antigen is confirmed
by a suitable irnmunochemical method (2.7. 1) .
TESTS
Abnorrnal toxicity
Inject intraperitoneally up to 4 human doses of vaccine into
each of at least 10 healthy mice, each weighing 17-22 g.
Observe the mice daily for 7 days. The vaccine complies with
the test if none of the animals shows signs of ill health.
2014
Specific toxicity (oedema) test
Use not fewer than 2 rabbits per test. Prepare serial two-fold
dilutions of vaccine with normal saline, corresponding to 4,
2, 1, 0.5 and 0.25 human doses. Inject intradermally 0.1 mL
of each dilution of the test and of the reference vaccine into
the shaved flanks of 2 rabbits. Each rabbit receives the 10
previously prepared injections (5 dilutions of the test vaccine
and 5 dilutions of the reference vaccine). In one of the
rabbits, the lower concentrations are injected at the anterior
end and the higher concentrations at the posterior end.
The reverse is used for the 2 nd rabbit. The rabbits are
monitored for 24 h for signs of oedema at the injection site.
The vaccine complies with the test if the oedematous
reaction is not greater than that observed with the reference
vaccine.
Alternatively, specific in virra assays for lethal factor and
adenylate cyclase activity may be used, subject to validation.
Antimicrobial preservative
Determine the amount of antimicrobial preservative by a
suitable chemical method. The content is not less than the
minimum amount shown to be effective and is not greater
than 115 per cent of the intended contento
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
The potency of the anthrax vaccine is determined by
comparing the dose required to protect guinea-pigs against
intradermal challenge by a virulent strain of B. anthracis with
the dose of a suitable reference preparation that gives the
same protection. Use 9 groups of not fewer than 16 female
guinea-pigs, each weighing 250-350 g. Prepare 4 dilutions of
the vaccine and of the reference preparation containing 1.5,
0.5, 0.17 and 0.05 human doses in 0.5 mL. Allocate each
dilution to a separate group. The remaining group receives
0.5 mL of saline and is used to verify the challenge dose.
Inject subcutaneously into each guinea-pig 0.5 mL of the
dilution allocated to its group on each of 2 occasions, 1 week
apart. 7 days after the 2 nd injection, inject intradermally into
each guinea-pig 2000 spores of a virulent strain of B.
anthracis (Vollum) in 0.1 mL. Observe the animals for
10 days and record the number of deaths per group. The test
is not valid unless all the control animals die within 5 days of
challenge. Using the proportions of animals that survive in
each of the vaccinated groups, calculate the potency of the
vaccine relative to the reference preparation using the usual
statistical methods (5.3). The vaccine complies with the test
if:
- the relative potency estimate exceeds 1.0, or;
- the 95 per cent confidence interval for the relative
potency includes 1.0, and the lower 95 per cent
confidence limit is not less than 50 per cent of the relative
potency estimate.
LABELLlNG
The label states that the vaccine is not to be frozen.
_ _ _ __ _ __ _ _ __ _ _ _ __ _ _ _ __ Ph Eur
2014
Bacillus Calmette-Guérin Vaccine ***
*** ***
BCG Vaccine ***
(BCG Vaccine, Freeze-dried, Ph Eur monograph 0163)
The label may state 'BCG'.
~Ew ____________________________________________
DEFINITION
Freeze-dried BCG vaccine is a preparation of live bacteria
derived from a culture of the bacillus of Calmene and Guérin
(Mycobacterium bovis BCG) whose capacity to protect against
tuberculosis has been established.
PRODUCTION
GENERAL PROVISIONS
BCG vaccine shall be produced by a staff consisting of
healthy persons who do not work with other infectious
agents; in panicul~r they shall not work with virulent strains
of Mycobacterium tuberculosis, nor shall they be exposed to a
known risk of tuberculosis infection. Staff are examined
periodically for tuberculosis. BCG vaccine is susceptible to
sunlight: the procedures for the preparation of the vaccine
shall be designed so that all cultures and vaccines are
protected from direct sunlight and from ultraviolet light at all
stages of manufacture, testing and storage.
Production of the vaccine is based on a seed-Iot system.
The production method shall have been shown to yield
consistently BCG vaccines that induce adequate sensitivity to
tuberculin in man, that have acceptable protective potency in
animal s and are safe. The vaccine is prepared from cultures
which are derived from the master seed lot by as few
sub cultures as possible and in any case not more than 8
sub cultures. During the course of these sub cultures the
preparation is not freeze-dried more than once.
If a bioluminescence test or other biochemical method is
used instead of viable count, the method is validated against
the viable count for each stage of the process at which it is
used.
BACTERIAL SEED LOTS
The strain used to establish the master seed lot is chosen for
and maintained to preserve its characteristics, its capacity to
sensitise man to tuberculin and to protect animals against
tuberculosis, and its relative absence of pathogenicity for man
and laboratory animals. The strain used shall be identified by
historical records that inelude information on its origin and
subsequent manipulation.
A suitable batch of vaccine is prepared from the first working
seed lot and is reserved for use as the comparison vaccine.
When a new working seed lot is established, a suitable test
for delayed hypersensitivity in guinea-pigs is carried out on a
batch of vaccine prepared from the new working seed lot;
the vaccine is shown to be not significantly different in
activity from the comparison vaccine. Antimicrobial agent
sensitivity testing is also carried out.
Only a working seed lot that complies with the following
requirements may be used for propagation.
IDENTIFICATION
The bacteria in the working seed lot are identified as
Mycobacterium bovis BCG using microbiological techniques,
which may be supplemented by molecular biology techniques
(for example, nueleic acid amplification and restriction-
fragment-Iength polymorphism).
Vaccines IV-507
Bacterial and funga1 contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium. The working seed lot complies with the test for
sterility except for the presence of mycobacteria.
Virulent mycobacteria
Examine the working seed lot as prescribed under Tests,
using 10 guinea-pigs.
PROPAGATlON AND HARVEST
The bacteria are grown in a suitable medium for not more
than 21 days by surface or submerged culture. The culture
medium do es not contain substances known to cause toxic or
allergic reactions in humans or to cause the bacteria to
become virulent for guinea-pigs. The culture is harvested and
suspended in a sterile liquid medium that protects the
viability of the vaccine as determined by a suitable method of
viable count.
FINAL BULK VACCINE
The final bulk vaccine is prepared from a single harvest or by
pooling a number of single harvests. A stabiliser may be
added; if the stabiliser interferes with the determination of
bacterial concentration in the final bulk vaccine, the
determination is carried out before addition of the stabiliser.
Only final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bacterial and funga1 contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium. The final bulk vaccine complies with the test for
sterility except for the presence of mycobacteria.
Count of viable units
Determine the number of viable units per millilitre by viable
count on solid medium using a method suitable for the
vaccine to be examined or by a suitable biochemical method.
Carry out the test in parallel on a reference preparation of
the same strain.
Bacterial concentration
Determine the total bacterial concentration by a suitable
method, either directly by determining the mas s of the micro-
organisms, or indirectly by an opacity method that has been
calibrated in relation to the mas s of the organisms; if the
bacterial concentration is determined before addition of a
stabiliser, the concentration in the final bulk vaccine is
established by calculation. The total bacterial concentration is
within the limits approved for the particular product.
The ratio of the count of viable units to the total bacterial
concentration is not les s than that approved for the panicular
product.
FINALLOT
The final bulk vaccine is distributed into sterile containers
and freeze-dried to a moisture content favourable to the
stability of the vaccine; the containers are e10sed either under
vacuum or under an inen gas.
Except where the filled and e10sed containers are stored at a
temperature of -20 oC or lower, the expiry date is not later
than 4 years from the date of harvest.
Only a final lot that complies with the following requirement
for count of viable units and with each of the requirements
given below under Identification, Tests and Assay may be
relea sed for use. Provided the test for virulent mycobacteria
has been carried out with satisfactory results on the final bulk
vaccine, it may be omined on the finallot. Provided the test
for excessive dermal reactivity has been carried out with
satisfactory results on the working seed lot and on
5 consecutive finallots produced from ir, rhe test may be
omitted on the final lot.
 IV-50S Vaccines
Count of viable units
Determine the number of viable units per millilitre of the
reconstituted vaccine by viable count on solid medium using
a method suitable for the vaccine to be examined or by a
suitable biochemical method. The ratio of the count of viable
units after freeze-drying to that before is not less than that
approved for the particular producto
Thennal stability
Maintain containers of the final lot of freeze-dried vaccine in
the dry state at 37 ± 1 oC for 4 weeks. Determine the
number of viable units as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. The number of viable
units in the heated vaccine is not less than 20 per cent of
that in the unheated vaccine.
IDENTIFICATION
BCG vaccine is identified by microscopic examination of the
bacilli in stained smears demonstrating their acid-fast
property and by the characteristic appearance of colonies
grown on solid medium. Altematively, molecular biology
techniques (for example nueleic acid amplification) may be
used.
TESTS
Virulent rnycobacteria
Inject subcutaneously or intramuscularly into each of
6 guinea-pigs, each weighing 250-400 g and having received
no treatrnent likely to interfere with the test, a quantity of
vaccine equivalent to at least 50 human doses. Observe the
animals for at least 42 days. At the end of this period,
euthanise the guinea-pigs and examine by autopsy for signs
of infection with tuberculosis, ignoring any minor reactions at
the site of injection. Animals that die during the observation
period are also examined for signs of tuberculosis.
The vaccine complies with the test if none of the guinea-pigs
shows signs of tuberculosis and if not more than 1 animal
dies during the observation periodo If 2 animals die during
this period and autopsy does not reveal signs of tuberculosis
repeat the test on 6 other guinea-pigs. The vaccine complies
with the test if not more than 1 animal dies during the
42 days following the injection and autopsy do es not reveal
any sign of tuberculosis.
Bacterial and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1) except for the presence of mycobacteria.
Excessive dennal reactivity
Use 6 healthy, white or pale-coloured guinea-pigs, each
weighing not les s than 250 g and having received no
treatment likely to interfere with the test. Inject intradermally
into each guinea-pig, according to a randomised plan,
0.1 mL of the reconstituted vaccine and of 2 tenfold serial
dilutions of the vaccine and identical doses of the comparison
vaccine. Observe the lesions formed at the site of the
injection for 4 weeks. The vaccine complies with the test if
the reaction it produces is not markedly different from that
produced by the comparison vaccine.
Water
Not more than the limit approved for the particular product,
determined by a suitable method.
ASSAY
Determine the number of viable units in the reconstituted
vaccine by viable count on solid medium using a method
suitable for the vaccine to be examined or by a suitable
validated biochemical method. The number is within the
2014
range stated on the labe!' Determine the number of viable
units in the comparison vaccine in parallel.
LABELLING
The label states:
-- the minimum and maximum number of viable units per
millilitre in the reconstituted vaccine,
-- that the vaccine must be protected from direct sunlight.
___________________________________________ PhE~
BCG for Immunotherapy ***
** **
(Ph Eur monograph 1929) *****
___________________________________________
~E~
DEFINITION
BCG for immunotherapy is a freeze-dried preparation of live
bacteria derived from a culture of the bacillus of Calmette
and Guérin (Mycobacterium bovis BCG) whose capacity for
treatment has been established.
It complies with the monograph Vaccines for human use
(0153).
PRODUCTION
GENERAL PROVISIONS
BCG for immunotherapy shall be produced by a staff
consisting of healthy persons who do not work with other
infectious agents; in particular they shall not work with
virulent strains of Mycobacterium tuberculosis, nor shall they be
exposed to a known risk of tuberculosis infection. Staff are
examined periodically for tuberculosis. BCG for
immunotherapy is susceptible to sunlight: the procedures for
production shall be so designed that all products are
protected from direct sunlight and from ultraviolet light at all
stages of manufacture, testing and storage.
Production is based on a seed-Iot system. The production
method shall have been shown to yield consistently BCG
products that can be used for treatrnent of superficial bladder
cancer and are safe. The product is prepared from cultures
which are separated from the master seed lot by as few
subcultures as possible and in any case not more than 8
subcultures. During the course of these sub cultures the
preparation is not freeze-dried more than once.
If a bioluminescence test or other biochemical method is
used instead of viable count, the method is validated against
the viable count for each stage of the process at which it is
used.
SEED LOTS
The strain used to establish the master seed lot is chosen for
and maintained to preserve its characteristics, its capacity to
treat and prevent superficial bladder cancer, and its relative
absence of pathogenicity for man and laboratory animals.
The strain used shall be identified by historical records that
inelude information on its origin and subsequent
manipulation.
Before establishment of a working seed lot a batch is
prepared and reserved for use as the comparison product.
When a new working seed lot is established, a suitable test
for delayed hypersensitivity in guinea-pigs is carried out on a
batch of product prepared from the new working seed lot;
the product is shown to be not significantly different in
activity from the comparison producto Antimicrobial agent
sensitivity testing is also carried out.
 2014
Only a working seed lot that complies with the following
requirements may be used for propagation.
Identification
The bacteria in the working seed lot are identified as
Mycobacterium bovis BCG using microbiological techniques,
which may be supplemented by molecular biology techniques
(for example, nucleic acid amplification and restriction-
fragment-Iength polyrnorphism).
Bacterial and fungal contamination
Carry out the test for steriliry (2.6.1), using 10 mL for each
medium. The working seed lot complies with the test for
steriliry, except for the presence of mycobacteria.
Virulent rnycobacteria
Examine the working seed lot as prescribed under Tests,
using 10 guinea-pigs.
PROPAGATION AND HARVEST
The bacteria are grown in a suitable medium for not more
than 21 days by surface or submerged culture. The culture
medium does not contain substances known to cause toxic or
allergic reactions in human beings or to cause the bacteria to
become virulent for guinea-pigs. The culture is harvested and
suspended in a sterile liquid medium that protects the
viabiliry of the culture as determined by a suitable method of
viable count.
FINAL BULK
The final bulk is prepared from a single harvest or by pooling
a number of single harvests. A stabiliser may be added; if the
stabiliser interferes with the determination of bacterial
concentration on the final bulk, the determination is carried
out before addition of the stabiliser.
Only final bulk that complies with the following requirements
may be used in the preparation of the final lot.
Bacterial and fungal contamination
Carry out the test for steriliry (2.6. 1), using 10 mL offinal
bulk for each medium. The final bulk complies with the test
for steriliry, except for the presence of mycobacteria.
Count of viable units
Determine the number of viable units per millilitre by viable
count on solid medium using a method suitable for the
product to be examined or by a suitable biochemical method.
Carry out the test in parallel on a reference preparation of
the same strain.
Bacterial concentration
Determine the total bacterial concentration by a suitable
method, either directly by determining the mass of the micro-
organisms, or indirectly by an opacity method that has been
calibrated in relation to the mas s of the micro-organisms;
if the bacterial concentration is determined before addition of
a stabiliser, the concentration in the final bulk is established
by calculation. The total bacterial concentration is within the
limits approved for the particular product.
The ratio of the count of viable units to the total bacterial
concentration is not less than that approved for the particular
product.
FINALLOT
The final bulk is distributed into sterile containers and freeze-
dried to a moisture content favourable to the stability of the
product; the containers are c10sed either under vacuum or
under an inert gas.
Except where the filled and c10sed containers are stored at a
temperature of - 20 oC or lower, the expiry date is not later
than 4 years from the date of harvest.
Vaccines IV-509
Only a finallot that complies with the following requirement
for count of viable units and with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided the test for virulent mycobacteria
has been carried out with satisfactory results on the final
bulk, it may be omitted on the final lot.
Count of viable units
Determine the number of viable units per millilitre of the
reconstituted product by viable count on solid medium using
a method suitable for the product 10 be examined, or by a
suitable biochemical method. The ratio of the count of viable
units after freeze-drying to that before is not less than that
approved for the particular producto
IDENTIFICATION
BCG for immunotherapy is identified by microscopic
examination of the bacilli in stained smears demonstrating
their acid-fast property and by the characteristic appearance
of colonies grown on solid medium. Altematively, molecular
biology techniques (for example, nucleic acid amplificarion)
may be used.
TESTS
Virulent rnycobacteria
Inject subcutaneously or intramuscularly into each of
6 guinea-pigs, each weighing 250-400 g and having received
no treatment Iikely to interfere with the test, a quantity of the
product to be examined equivalent to at least 1/25 of 1
human dose. Observe the animals for at least 42 days. At the
end of this period, euthanise the guinea-pigs and examine by
autopsy for signs of infection with tuberculosis, ignoring any
minor reactions at the site of injection. Animals that die
during the observation period are also examined for signs of
tuberculosis. The product complies with the test if none of
the guinea-pigs shows signs of tuberculosis and if not more
than 1 animal die s during the observation periodo
If 2 animals die during this period and autopsy does not
reveal signs of tuberculosis, repeat the test on 6 other guinea-
pigs. The product complies with the test if not more than
1 animal dies during the 42 days following the injection and
autopsy do es not reveal any sign of tuberculosis.
Bacterial and fungal contamination
The reconstituted product complies with the test for sterility
(2.6.1) except for the presence of mycobacteria.
Water
Not more than the limit approved for the particular product,
determined by a suitable method.
ASSAY
Determine the number of viable units in the reconstituted
product by viable count on solid medium using a method
suitable for the product to be examined or by a suitable
validated biochemical method. The number is within the
range stated on the labe!' Determine the number of viable
units in the comparison control in paralle!.
LABELLING
The label sta tes:
- the minimum and the maxirnum number of viable units
per dose in the reconstituted product;
- that the product must be protected from direct sunlight.
___________________________________________ nE~
IV-510 Vaccines
Cholera Vaccine *****
** **
(Ph Eur monograph 0154) ***
The label may state 'Cholera'.
~E~ ____________________________________________
DEFINITION
Cholera vaccine is a homogeneous suspension of a suitable
strain or strains of Vibrio cholerae containing not les s than 8
x 10 9 bacteria in each human dose. The human dose does
not exceed 1.0 mL.
PRODUCTION
The vaccine is prepared using a seed-lot system. The vaccine
consists of a mixture of equal parts of vaccines prepared from
smooth strains of the 2 main serological types, Inaba and
Ogawa. These may be of the classical biotype with or without
the El-Tor biotype. A single strain or several strains of each
type may be included. AH strains must contain, in addition ro
their type O antigens, the heat-stable O antigen common to
Inaba and Ogawa. If more than one strain each of Inaba and
Ogawa are used, these may be selected so as to contain other
O antigens in addition. The World Health Organisation
recommends new strains which may be used if necessary, in
accordance with the regulations in force in the signatory
States of the Convention on the Elaboration of a European
Pharmacopoeia. In order to comply with the requirements for
vaccination certifica tes required for intemational travel, the
vaccine must contain not les s than 8 x 10 9 organisms of the
classical biotype. Each strain is grown separately.
The bacteria are inactivated either by heating the suspensions
(for example, at 56 oC for 1 h) or by treatment with
formaldehyde or phenol or by a combination of the physical
and chemical methods.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9)
modified as follows: inject 0.5 mL of the vaccine into each
mouse and 1.0 mL into each guinea pig.
IDENTIFICATION
It is identified by specific agglutination tests.
TESTS
Phenol (2.5.15)
If phenol has been used in the preparation, the concentration
is not more than 5 giL.
Antibody production
Test the ability of the vaccine ro induce antibodies (such as
agglutinating, vibriocidal or haemagglutinating antibodies) in
the guinea-pig, the rabbit or the mouse. Administer the
vaccine to a group of at least 6 animals. At the end of the
interval of time necessary for maximum antibody formation,
determined in preliminary tests, collect sera from the animals
and titrate them individually for the appropriate antibody
using a suitable method. The vaccine ro be examined passes
the test if each serotype has elicited a significant antibody
response.
Sterility (2.6.1)
It complies with the test for sterility.
LABELLING
The label states:
- the method used to inactivate the bacteria,
- the number of bacteria in each human dose.
__________________________________________ ~E~
2014
Cholera Vaccine, Freeze-dried *****
** *
(Ph Eur monograph 0155) *** *
The label may state 'DriedlCholera'.
~E~ ____________________________________________
DEFINITION
Freeze-dried cholera vaccine is a preparation of a suitable
strain or strains of Vibrio cholerae. The vaccine is
reconstituted as stated on the labe! ro give a uniform
suspension containing not less than 8 x 109 bacteria in each
human dose. The human dose do es not exceed 1.0 mL of
the reconstituted vaccine.
PRODUCTION
The vaccine is prepared using a seed-Iot system. The vaccine
consists of a mixture of equal parts of vaccines prepared from
smooth strains of the 2 main serological types, Inaba and
Ogawa. These may be of the classical biotype with or without
the El-Tor biotype. A single strain or several strains of each
type may be included. All strains must contain, in addition to
their type O antigens, the heat-stable O antigen common to
Inaba and Ogawa. If more than one strain each of Inaba and
Ogawa are used, these may be selected so as to contain other
O antigens in addition. The World Health Organisation
recommends new strains which may be used if necessary in
accordance with the regulations in force in the signarory
Sta tes of the Convention on the Elaboration of a European
Pharmacopoeia. In order to comply with the requirements for
vaccination certificates required for intemational travel, the
vaccine must contain not less than 8 x 10 9 organisms of the
classical biotype. Each strain is grown separately.
The bacteria are inactivated either by heating the suspensions
(for example, at 56 oC for 1 h) or by treatment with
formaldehyde or by a combination of the physical and
chemical methods . Phenol is not used in the preparation.
The vaccine is distributed into sterile containers and freeze-
dried ro a moisture content favourable to the stability of the
vaccine. The containers are then closed so as to exclude
contamination.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9)
modified as follows: inject 0.5 mL of the vaccine into each
mouse and 1.0 mL inro each guinea pig.
IDENTIFICATION
The vaccine reconstituted as stated on the label is identified
by specific agglutination tests.
TESTS
Phenol (2.5.15)
If phenol has been used in the preparation, the concentration
is not more than 5 gIL.
Antibody production
Test the abiliry of the vaccine to induce antibodies (such as
agglutinating, vibriocidal or haemagglutinating antibodies) in
the guinea-pig, the rabbit or the mouse. Administer the
reconstituted vaccine ro a group of at least 6 animals. At the
end of the interval of time necessary for maximum antibody
formation, determined in preliminary tests, collect sera from
the animals and titrate them individually for the appropriate
antibody using a suitable method. The vaccine to be
examined passes the test if each serorype has elicited a
significant antibody response.
Sterility (2.6.1)
The reconstituted vaccine complies with the test for sterility.
2014
LABELLING
The labe! states:
-- the method used to inactivate the bacteria,
-- the number of bacteria in each human dose.
____________________________________________ ~Em
Cholera Vaccine (Inactivated, Oral) *****
* * **** *
(Ph Eur 111onograph 2327)
The label may state Cholera(oral)
Ph Eur ____________________________________________
DEFINITION
Cholera vaccine (inactivated, oral) is a homogeneous
suspension of inactivated suitable strains of Vibrio cholerae
serogroup 01, representing serotypes and biotypes of
epidemic strains. The vaccine may contain the B subunit of
cholera toxin (CTB). Just prior to ingestion, one dose of
vaccine suspension is mixed with a suitable buffer as stated
on the labe!'
PRODUCTION
GENERAL PROVISIONS
The production method must be validated to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
The production process must be validated to show that no
clinically significant quantities of active toxin are present in
the product.
CHOICE OF VACCINE STRAIN
The vaccine consists of a mixture of epidemic V. cholerae
strains inactivated by a suitable method such as heat or
fonnalin inactivation. AII strains express smooth
lipopolysaccharide (LPS). The CTB is produced by
recombinant DNA technology in a strain that lacks the gene
for cholera toxin subunit A (ctxA-). Se!ected V. cholerae
strains are low cholera-toxin producers.
The World Health Organisation (WHO) can recommend
new vaccine strains or antigens that may be used if necessary,
in accordance with the regulations in force in the signatory
states of the Convention on the Elaboration of a European
Phannacopoeia.
SEEDLOTS
The strains of V. cholerae used shall be identified by historical
records that inc1ude infonnation on the origin of the strains
and their subsequent manipulation. Characterisation and
maintenance of the recombinant strains and plasmids used
for production of the recombinant B subunit of cholera toxin
(rCTB) and the origin of the gene for cholera toxin
subunit B (ctxB) are documented. The stability of the rCTB
plasmid in the recombinant strain during storage and beyond
the passage level used in production is confirmed.
Characterisation of the rCTB is undertaken using a variety of
analytical techniques including detennination of molecular
size, charge and amino acid composition. Techniques
suitable for such purposes include sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and
different liquid chromatographies. The identity of the
product is confirmed by at least partial N-tenninal and
C-tenninal amino acid sequencing.
Master seed lots are grown on agar plates, which may
contain appropriate antibiotics. Colonies are used to produce
working seed lots in liquid media that are free from
Vaccines IV -511
antibiotics. Cultures derived from the working seed lot must
have the same characteristics as the cultures of the strain
from which the master seed lot was derived.
Only a seed lot that complies with the following requirements
may be used in the preparation of the monovalent cell
harvest.
Identification
Master seed lots are identified by colony morphology, and by
biochemical characterisation, using suitable molecular assays
or immunoassays. Working seed lots are identified by colony
morphology and by molecular assays or irnmunoassays.
Purity
Purity of master seed lots and working seed lots is verified by
methods of suitable sensitivity.
PROPAGATION AND HARVEST
Each strain is grown separately from the working seed loto
Cultures are checked at different stages of fermentation
(subcultures and main culture) for purity, identity, cell
opacity, pH and biochemical characteristics. Unsatisfactory
cultures must be discarded.
Production cultures are shown to be consistent in respect of
growth rate, pH and yield of cells or cell products .
MONOVALENT CELL HARVEST
Only a monovalent harvest that complies with established
specifications for the following tests may be used.
pH (2.2.3)
Within the range approved for the particular producto
Identification
Relevant antigenic characteristics are verified by suitable
immunological or biochemical assays.
Purity
Samples of culture are examined by microscopy of Gram-
stained smears, by inoculation of appropriate culture media
or by another suitable procedure.
Opacity
The absorbance at 600 nm (2.2.25) is within the range
approved for the particular producto
INACTIVATED MONOVALENT CELL BULK
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. Bacteria are
inactivated after washing, either by treatrnent with
formaldehyde or by heating under conditions that ensure
inactivation.
Only an inactivated monovalent cell bulk that complies with
established specifications for the following tests may be used
in the preparation of the final bulk.
pH (2.2.3)
Within the range approved for the particular producto
Identification
Verified by slide agglutination.
Inactivation
Complete inactivation is verified by a suitable culture
method.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
Opacity
The inactivation process may affect the accuracy of opacity
measurements .
IV -512 Vaccines
Purity
Samples of culture are examined by microscopy of Gram-
stained smears, by inoculation of appropriate culture media
or by another suitable procedure.
Smooth LPS content
Verified by a suitable immunoassay (2.7.1).
Residual cholera toxin
The absence of residual cholera toxin is verified by a suitable
irnmunoassay (2.7. 1) or biochemical assay.
Free formaldehyde (2.4.18)
Content to be determined where formaldehyde is used for
inactivation.
PURIFIED RCTB
Production of the reTB follows the guidelines for assuring
the quality of pharmaceutical and biological products
prepared by recombinant technology and is covered by the
monograph Products 01 recombinant DNA technology (0784).
Prior to harvest, the cell culture is checked for purity and
opacity. rCTB is harvested by suitable filtration, concentrated
by diafiltration, purified by chromatography, filter-sterilised
and stored under suitable conditions. The pH of the pooled
eluate is adjusted prior to buffer exchange.
On1y purified reTB that complies with established
specifications for the following tests may be used in the
preparation of the final bulk.
pH (2.2.3)
Within the range approved for the particular product.
Purity
Verified by SDS-PAGE (2.2.31) and an appropriate liquid
chromatography method (2.2.29).
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
rCTB
The amount of reTB is determined by a suitable
irnmunoassay (2.7.1) .
FINAL BULK
The final bulk vaccine is prepared by aseptically mixing a
suitable buffer with monovalent cell bulks. Where used, the
rCTB bulk is added in appropriate amounts. Preservatives, if
used, may be added at this stage.
Only a final bulk that complies with the following
requirements may be used in the preparation of the finallot.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
FINALLOT
The final bulk is mixed to homogeneity and filled aseptically
into suitable containers .
On1y a final lot that is within the Iimits approved for the
particular product and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
Assay may be released for use.
IDENTIFICATION
Serotypes are detected by a suitable irnmunoassay (2.7.1) or
molecular assay. reTB is detected by a suitable irnmunoassay
2014
(2.7.1). The antigen-content assays may also serve as an
identity test.
TESTS
pH (2.2.3)
Within the range approved for the particular product.
Sterility (2.6.1)
It complies with the test for sterility.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
ASSAY
Antigen content
The amount of smooth LPS, and where applicable, the
amount of reTB, are within the limits approved for the
particular product, determined by a suitable immunoassay
(2.7.1).
LABELLING
The label states:
- the method of inactivation;
- the serogroup, serotypes and biotypes of vaccine strains;
- the number of bacteria per human dose;
- the amount of rCTB.
___________________________________________ ~Elf
Adsorbed Diphtheria Vaccine *****
** **
(Diphtheria Vaccine (Adsorbed), ***
Ph Eur monograph 0443)
Ph Eur __________________________________________
DEFINITION
Diphtheria vaccine (adsorbed) is a preparation of diphtheria
formol toxoid with a mineral adsorbent. The formol toxoid is
prepared from the toxin produced by the growth of
Corynebacterium diphtheriae.
PRODUCTION
GENERAL PROVISIONS
Specific toxicity
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia, the vaccine do es not comply with the
test. If more than 1 animal dies from non-specific causes,
repeat the test once; if more than 1 animal dies in the second
test, the vaccine does not comply with the test.
BULK PURIFlED TOXOID
For the production of diphtheria toxin, from which toxoid is
prepared, seed cultures are managed in a defined seed-Iot
system in which toxinogenicity is conserved and, where
necessary, restored by deliberate reselection. A highly
toxinogenic strain of Corynebacterium diphtheriae with known
origin and history is grown in a suitable liquid medium.
At the end of cultivation, the purity of each culture is tested
 2014
and contaminated cultures are discarded. Toxin-containing
culture medium is separated aseptically from the bacterial
mass as soon as possible. The toxin content (ti per miJlilitre)
is checked (2.7.27) to monitor consistency of production.
Single harvests may be pooled to prepare the bulk purified
toxoid. The toxin is purified to remove components likely to
cause adverse reactions in humans. The purified toxin is
detoxified with formaldehyde by a method that avoids
destruction of the irnmunogenic potency of the toxoid and
reversion of the toxoid to toxin, particularly on exposure to
heat. Altematively, purification may be carried out after
detoxification.
Only bulk purified toxoid that complies with the following
requirements may be used in the preparation of the final bulk
vaccme.
Sterility (2.6.1)
Carry out the test for steriJity using 10 mL for each medium.
Absence of toxin and irreversibility of toxoid
Using the same buffer solution as for the final vaccine,
without adsorbent, prepare a solution of bulk purified toxoid
at 100 Lfi'mL. Divide the solution into 2 equal parts.
Maintain 1 part at 5 ± 3 oC and the other at 37 °C for
6 weeks . Carry out a test in Vero cells for active diphtheria
toxin using 50 J..lUwell of both samples. The sample should
not contain antimicrobial preservatives and detoxifying agents
should be determined to be below the concentration toxic to
Vero cells. Non-specific toxicity may be eJiminated by
dialysis.
Use freshly trypsinised Vero cells at a suitable concentration,
for example 2.5 x 105 mL- 1 and a reference diphtheria
toxin diluted in 100 Lti'mL diphtheria toxoid. A suitable
reference diphtheria toxin will contain either not less than
100 LDso/mL or 67 to 133 Ir/lOO in 1 ti and 25 000 to
50 000 minimal reacting doses for guinea-pig skin in 1 ti
(diphtheria toxin BRP is suitable for use as the reference
toxin) . Dilute the toxin in 100 Lfi'mL diphtheria toxoid to a
suitable concentration, for example 2 x 10- 4 Lti'mL.
Prepare serial twofold dilutions of the diluted diphtheria
toxin and use undiluted test samples (50 ~lUwell). Distribute
them in the wells of a sterile tissue culture plate containing a
medium suitable for Vero cells. To ascertain that any
cytotoxic effect noted is specific to diphtheria toxin, prepare
in parallel dilutions where the toxin is neutralised by a
suitable concentration of diphtheria antitoxin, for example
100 IU/mL. Inelude control wells without toxoid or toxin
and with non-toxic toxoid at 100 Lti'rnL on each plate to
verify normal cell growth. Add cell suspension to each well,
seal the plates and incubate at 37 oC for 5-6 days. Cytotoxic
effect is judged to be present where there is complete
metaboJic inhibition of the Vero cells, indicated by the pH
indicator of the medium. Confirm cytopathic effect by
microscopic examination or suitable staining such as MTT
dye. The test is invaJid if 5 x 10- 5 Lti'mL of reference
diphtheria toxin in 100 Lti'mL toxoid has no cytotoxic effect
on Vero cells or if the cytotoxic effect of this amount of toxin
is not neutralised in the wells containing diphtheria antitoxin.
The bulk purified toxoid complies with the test if no toxicity
neutraJisable by antitoxin is found in either sample.
Antigenic purity (2. 7.27)
Not les s than 1500 ti per milligram of protein nitrogen.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of a suitable
quantity of bulk purified toxoid onto a mineral carrier such
as hydrated aluminium phosphate or aluminium hydroxide;
the resulting mixture is approximately isotonic with blood.
Vaccines IV-513
Suitable antimicrobial preservatives may be added . Certain
antimicrobial preservatives, particularly those of the phenoJic
type, adversely affect the antigenic activity and must not be
used.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where appJicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
Carry out the test for steriJity using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into steriJe,
tamper-proof containers. The containers are cJosed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be relea sed for use. Provided the test for
antimicrobial preservative and the assay have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the final lot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot .
IDENTIFICATION
Diphtheria toxoid is identified by a suitable immunochemical
method (2. 7.1). The following method, appJicable to certain
vaccines, is given as an example. Dissolve in the vaccine to
be examined sufficient sodium citrate R to give a 100 gIL
solution. Maintain at 37 oC for about 16 h and centrifuge
until a cJear supematant liquid is obtained. The elear
supematant liquid reacts with a suitable diphtheria antitoxin,
giving a precipita te.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
absorbent.
Free forrnaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antirnicrobial
preservative by a suitable chemical method. The content is
not les s than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence lirnit (P = 0.95) of the estimated
potency is not les s than 30 IU per single human dose.
LABELLING
The label sta tes:
- the minimum number of Intemational Units per single
human dose,
IV-514 Vaccines
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suirable for
reinforcing doses or for administration 10 adults,
-- rhe name and the amount of the adsorbent,
-- thar the vaccine must be shaken before use,
-- rhat the vaccine is not to be frozen.
____________________________________________ ~Ew
Diphtheria Vaccine (Adsorbed, *****
** **
Reduced Antigen Content) ***
Adsorbed Diphtheria Vaccine for Adults and
Adolescents
(Ph Eur monograph 0646)
For a vaccine for use in the United Kingdom, the amount of
toxoid used is adjusred so thar the final vaccine contains nor
more than 2.0 fiocculation equivalents (2.0 Lf) per dose.
~ Ew ____________________________________________
DEFINITION
Diphtheria vaccine (adsorbed, reduced antigen contenr) is a
preparation of diphtheria formol toxoid with a mineral
adsorbent. The formol toxoid is prepared from the toxin
produced by the growth of C01ynebactenum diphthenae.
It shall have been demonstrated to the competent authority
that the quantity of diphtheria toxoid used does not produce
adverse reactions in subjects from the age groups for which
the vaccine is intended.
PRODUCTION
GENERAL PROVISIONS
Specific toxicity
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia, the vaccine does not comply with the
test. If more than one animal dies from non-specific causes,
repeat the test once; if more than one animal dies in the
second test, the vaccine do es not comply with the test.
BULK PURIFIED TOXOID
The bulk purified toxoid is prepared as described in the
monograph on Diphthena v accine (adsorbed) (0443) and
complies with the requirements prescribed therein.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of a suitable
quantity of bulk purified toxoid onto a mineral carrier such
as hydrated aluminium phosphate or aluminium hydroxide;
the resulting mixture is approximately isotonic with blood.
Suitable antimicrobial preservatives may be added. Certain
antimicrobial preservatives, particularly those of the phenolic
type, adversely affect the antigenic activity and must not be
used.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable che mi cal method. The amount is
nor les s than 85 per cent and not greater than 115 per cent
of the intended amount.
2014
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a finallot thar is satisfactory wirh respecr 10 each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided the test for
antimicrobial preservative and the assay have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the final lot.
Provided the free formaldehyde content has been determined
on the bulk purified toxoid or on the final bulk and it has
been shown that the content in the final lot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
IDENTIFICATION
Diphtheria toxoid is identified by a suitable immunochemical
method (2.7.1) . The following method, applicable to certain
vaccines, is given as an example. Dissolve in the vaccine to
be examined sufficient sodium citrate R to give a 100 gIL
solution. Maintain at 37 oC for abour 16 h and centrifuge
until a clear supematant liquid is obtained. The clear
supematant liquid reacts with a suitable diphtheria antitoxin,
giving a precipitate. If a satisfactory result is not obtained
with a vaccine adsorbed on aluminium hydroxide, carry out
the test as follows. Centrifuge 15 mL of the vaccine to be
examined and suspend the residue in 5 mL of a freshly
prepared mixture of 1 volume of a 56 gIL solution of sodium
edetate R and 49 volumes of a 90 gIL solution of disodium
hydrogen phosphate R. Maintain at 37 oC for not less than 6 h
and centrifuge. The clear supematant liquid reacts with a
suitable diphtheria antitoxin, giving a precipitate.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable che mi cal method. The content is
not les s than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limÍ! (P = 0.95) of the estimated
potency is not less than 2 IU per single human dose.
LABELLING
The label sta tes:
-- the minimum number of Intemational Units per single
human dose;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________
~Ew
2014
Adsorbed Diphtheria and Tetanus *****
** **
Vaccine ***
(Diphtheria and Tetanus Vaccine (Adsorbed),
Ph Eur monograph 0444)
The label may state 'DT'.
~E~ ____________________________________________
DEFINITlON
Diphtheria and tetanus vaccine (adsorbed) is a preparation of
diphtheria formol toxoid and tetanus formol toxoid with a
mineral adsorbent. The formol toxoids are prepared from the
toxins produced by the growth of Corynebacterium diphtheriae
and Clostridium tetani, respectively.
PRODUCTlON
GENERAL PROVISIONS
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dos e stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal die s in the
second test, the vaccine do es not comply with the test.
BULK PURIFIED DlPHTIfERIA AND TETANUS TOXOIDS
The bulk purified diphtheria and tetanus toxoids are
prepared as described in the monographs on Diphtheria
vaccine (adsorbed) (0443) and Tetanus vaccine (adsorbed)
(0452) and comply with the requirements prescribed therein.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of suitable
quantities of bulk purified diphtheria toxoid and tetanus
toxoid onto a mineral carrier such as hydrated aluminium
phosphate or aluminium hydroxide; the resulting mixture is
approximately isotonic with blood. Suitable antimicrobial
preservatives may be added. Certain antimicrobial
preservatives, particularly those of the phenolic type,
adversely affect the antigenic activity and must not be used.
OnIy a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antirnicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not les s than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed asepticaIly into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
OnIy a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided the test for
antimicrobial preservative and the assay have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the final loto
Vaccines IV-515
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the finallot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
IDENTIFlCATlON
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The foIlowing method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a clear supernatant liquid is
obtained. The clear supernatant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable irnmunochemical
method (2.7.1) . The foIlowing method, applicable to certain
vaccines, is given as an example. The clear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
TESTS
Alurninium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantiry stated on the
labe!'
Sterility (2.6. 1)
The vaccine complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7. 6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 30 IV per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IV per single human dose.
LABELLING
The label states:
- the minimum number of Intemational Vnits of each
component per single human dose,
- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults,
- the name and the amount of the adsorbent,
- that the vaccine must be shaken before use,
- that the vaccine is not to be frozen.
__________________________________________ Ph Eur
IV-516 Vaccines
Diphtheria and Tetanus Vaccine ***
** **
(Adsorbed, Reduced Antigen(s) **** *
Content)
Adsorbed Diphtheria and Tetanus Vaccine for
Adults and Adolescents
(Ph Eur monograph 0647)
The label may state 'dT' .
For a vaccine for use in the Vnited Kingdom, the amount of
diphtheria toxoid used is adjusted so that the final vaccine
contains not more than 2.0 f1occulation equivalents (2.0 Lf)
of diphtheria 1Oxoid per dose.
~ Ew ____________________________________________
DEFINITION
Diphtheria and tetanus vaccine (adsorbed, reduced antigen(s)
content) is a preparation of diphtheria formol toxoid and
tetanus formol toxoid with a mineral adsorbent. The formol
toxoids are prepared from the toxins produced by the growth
of Corynebacterium diphtheriae and Clostridium tetani,
respectively. It shall have been demonstrated to the
competent authority that the quantity of diphtheria toxoid
used does not produce adverse reactions in subjects from the
age groups for which the vaccine is intended.
PRODUCTION
GENERAL PROVISIONS
Specific toxicity of the diphtheria and tetanus
components
The production method is validated 10 demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animal s shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine does not comply
with the test. If more than one animal dies from non-specific
causes, repeat the test once; if more than one animal dies in
the second test, the vaccine does not comply with the test.
BULK PURIFIED DIPHTHERIA TOXOID ANO TETANUS
TOXOIDS
The bulk purified diphtheria and tetanus toxoids are
prepared as described in the monographs on Diphtheria
vaccine (adsorbed) (0443) and Tetanus vaccine (adsorbed)
(0452) and comply with the requirements prescribed therein.
FINAL BULK VACCINE
The vaccine is prepared by adsorption of suitable quantities
of bulk purified diphtheria toxoid and tetanus toxoid onto a
mineral carrier such as hydrated aluminium phosphate or
aluminiurn hydroxide; the resulting mixture is approximately
isotonic with blood. Suitable antimicrobial preservatives may
be added. Certain antimicrobial preservatives, particularly
those of the phenolic type, adversely affect the antigenic
activity and must not be used.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable che mi cal method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2. 6.1)
Carry out the test for sterility using 10 mL for each medium.
2014
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as 10
prevent contamination.
Only a finallot that is satisfactory with respect 10 each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided the test for
antimicrobial preservative and the assay have been carried
out with satisfac10ry results on the final bulk vaccine, they
may be omined on the finallot.
Provided the free formaldehyde content has been determined
on the buIk purified toxoids or on the final bulk and it has
been shown that the content in the final lot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
IDENTIFICATION
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2. 7.1). The following method,
applicable 10 certain vaccines, is given as an example.
Dissolve in the vaccine 10 be examined sufficient sodium
citrate R 10 give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a clear supematant liquid is
obtained. The clear supematant liquid reacts with a suitable
diphtheria anti1Oxin, giving a precipitate. If a satisfac10ry
resuIt is not obtained with a vaccine adsorbed on alurniniurn
hydroxide, carry out the test as follows . Centrifuge 15 mL of
the vaccine 10 be examined and suspend the residue in 5 mL
of a freshly prepared mixture of 1 volume of a 56 giL
solution of sodium edetate R and 49 volurnes of a 90 gIL
solution of disodium hydrogen phosphate R. Maintain at 37 oC
for not less than 6 h and centrifuge. The clear supematant
liquid reacts with a suitable diphtheria antitoxin, giving a
precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2. 7.1). The following method, applicable to certain
vaccines, is given as an example. The clear supematant liquid
obtained during identification test A reacts with a suitable
tetanus anti1Oxin, giving a precipitate.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximurn 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown 10 be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not les s than 2 IV per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
2014
The lower confidence limit (P = 0.95) ofthe estimated
potency is not less than 20 IV per single human dose.
LABELLING
The label states:
-- the minimum number of International Vnits of each
component per single human dose;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not 10 be frozen.
___________________________________________ PhE~
Diphtheria, Tetanus and
Hepatitis 8 (rDNA) Vaccine
(Adsorbed)
(Ph Eur monograph 2062)
The label may state 'DTlHepB'.
~E~ ___________________________________________
DEFINITION
Diphtheria, tetanus and hepatitis B (rDNA) vaccine
(adsorbed) is a combined vaccine composed of: diphtheria
formol toxoid; tetanus formol toxoid; hepatitis B surface
antigen (HBsAg); a mineral adsorbent such as aluminium
hydroxide or hydrated aluminium phosphate.
The formol 1Oxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetaní, respectively.
HBsAg is a component protein of hepatitis B virus;
the antigen is obtained by recombinant DNA technology.
PRODVCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
The content of bacterial endotoxins (2.6.14) in the bulk
purified diphtheria toxoid and tetanus toxoid is determined
to monitor the purification procedure and to limit the
amount in the final vaccine. For each component, the
content of bacterial endotoxins is less than the limit approved
for the particular vaccine and in any case the contents are
such that the final vaccine contains less than 100 IV per
single human dose.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of the difference in composition
between monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
c1inical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence 10 the production process used for the
batch tested in c1inical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dos e stated
Vaccines IV-51 7
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine does not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal die s in the
second test, the vaccine do es not comply with the test.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs on Diphtheria vaccine
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and
Hepatitis B vaccine (rDNA) (1056) .
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
or together, of suitable quantities of bulk purified diphtheria
toxoid, tetanus toxoid and HBsAg onto a mineral carrier
such as aluminium hydroxide or hydrated aluminium
phosphate. Suitable antimicrobial preserva ti ves may be
added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Only a finallot that is satisfactory with respect to the test for
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the test for antimicrobial preservative and the assays
for the diphtheria and tetanus components have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the finallot.
Provided the content of free formaldehyde has been
determined on the bulk purified antigens or on the final bulk
and it has been shown that the content in the final lot will
not exceed 0.2 gIL, the test for free formaldehyde may be
omitted on the finallot.
If an in vivo assay is used for the hepatitis B component,
provided it has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the particular preparation.
IDENTIFICATION
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1) . The following method,
applicable 10 certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supernatant liquid is
obtained. The c1ear supernatant liquid reacts with a suitable
diphtheria anti1Oxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to certain
vaccines, is given as an example. The c1ear supernatant liquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipitate.
IV-518 Vaccines
C. The assay or, where applicable, the electrophoretic pro file,
serves also to identifY the hepatitis B component of the
vaccine.
TESTS
Alunúnium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
It complies with the test for sterility.
Pyrogens (2.6.8)
It complies with the test for pyrogens. In;ect the equivalent of
1 human dos e into each rabbit.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2. 7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 30 IV per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IV per single human dose.
Hepatitis B component
It complies with the assay ofhepatitis B vaccine (2.7.15).
LABELLING
The label sta tes:
- the minimum number of Intemational Vnits of diphtheria
and tetanus toxoid per single human dose,
- the amount of HBsAg per single human dose,
- the type of cells used for production of the HBsAg
component,
- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults,
- the name and the amount of the adsorbent,
- that the vaccine must be shaken before use,
- that the vaccine is not to be frozen.
_ _ __ __ _ __ _ _ _ _ _ __ _ _ __ _ _ PhEur
2014
Diphtheria, Tetanus and Pertussis *****
** **
(Whole Cell) Vaccine (Adsorbed) ***
Diphtheria, Tetanus and Perrussis Vaccine
(Adsorbed)
(Ph Eur monograph 0445)
The label may state 'DTwP'.
~E~ _ __ __ _ __ _ _ __ _ __ _ _ _ _ __ _
DEFINITION
Diphtheria, tetanus and perrussis (whole cell) vaccine
(adsorbed) is a preparation of diphtheria formol toxoid and
tetanus formol toxoid with a mineral adsorbent to which a
suspension of inactivated Bordetella pertussis has been added.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani, respectively.
PRODUCTION
GENERAL PROVlSIONS
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonsrrate that the
product, if tested, would comply with the following test:
in;ect subcutaneously 5 times the single human dos e stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the in;ection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine does not comply with the test.
BULK PURIFIED DIPHTHERIA AND TETANUS TOXOIDS,
BULK INACTIVATED B. PERTUSSIS SUSPENSION
The bulk purified diphtheria and tetanus toxoids and the
inactivated B. pertussis suspension are prepared as described
in the monographs Diphtheria vaccine (adsorbed) (0443),
Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine (whole
cell, adsorbed) (0161), respectively, and comply with the
requirements prescribed therein.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of suitable
quantities of bulk purified diphtheria toxoid and tetanus
toxoid onto a mineral carrier such as hydrated aluminium
phosphate or aluminium hydroxide and admixture of an
appropriate quantity of a suspension of inactivated B.
pertussis; the resulting mixture is approximately isotonic with
blood. The B. pertussis concenrration of the final bulk vaccine
does not exceed that corresponding to an opacity of 20 IV
per single human dose. If 2 or more srrains of B. pertussis are
used, the composition of consecutive lots of the final bulk
vaccine shall be consistent with respect to the proportion of
each srrain as measured in opacity units . Suitable
antimicrobial preservatives may be added to the bulk vaccine.
Certain antimicrobial preservatives, particularly those of the
phenolic type, adversely affect the antigenic activity and must
not be used.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
 2014
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are c!osed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use . Provided the tests for specific
toxicity of the pertussis component, antimicrobial preservative
and the assay have been carried out with satisfactory results
on the final bulk vaccine, they may be omitted on the final
lot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the finallot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
IDENTIFICATION
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c!ear supernatant liquid is
obtained; reserve the precipitate for identification test C.
The c!ear supernatant liquid reacts with a suitable diphtheria
antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to certain
vaccines, is given as an example. The c!ear supernatant liquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipitate.
C. Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 °C for
about 16 h and centrifuge to obtain a bacterial precipitate.
Other suitable methods for separating the bacteria from the
adsorbent may also be used. Identify pertussis vaccine by
agglutination of the bacteria from the resuspended precipitate
by antisera specific to B. pertussis or by the assay.
TESTS
Specific toxicity of the pertussis component
Use not fewer than 5 mice each weighing 14 - 16 g for the
vaccine group and for the saline contro!. Use mice of the
same sex or distribute males and females equally between the
groups. AlIow the animals access to food and water for at
least 2 h before injection and during the test. Inject each
mouse of the vaccine group intraperitoneally with 0.5 mL,
containing a quantity of the vaccine equivalent to not less
than half the single human dose . Inject each mouse of the
control group with 0.5 mL of a 9 gIL sterile solution of
sodium chloride R, preferably containing the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 h and 7 days after the injection. The vaccine
complies with the test if: (a) at the end of 72 h the total mass
of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mass per vaccinated mouse is not less than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test.
The test may be repeated and the results of the tests
combined.
Vaccines IV-519
Aluminium (2.5. 13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
If the test is carried out in guinea-pigs, the lower confidence
limit (P = 0.95) of the estimated potency is not les s than
40 IU per single human dose; if the test is carried out in
mice, the lower confidence limit (P = 0.95) of the estimated
potency is not les s than 60 IU per single human dose.
Pertussis component
Carry out the assay of pertussis vaccine (whole cell) (2.7.7).
The estimated potency is not less than 4.0 IU per single
human dose and the lower confidence limit (P = 0.95) of the
estimated potency is not less than 2.0 IU per single human
dose.
LABELLING
The label states:
-- the minimum number of International Units of each
component per single human dose;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ ~E~
Adsorbed Diphtheria, Tetanus and *****
** **
Pertussis (Acellular Component) ***
Vaccine
(Diphtheria, Tetanus and Pertussis (Acellular,
Component) Vaccine (Adsorbed),
Ph Eur monograph 1931)
The label may state 'DTaP'.
~E~ ____________________________________________
DEFINITION
Diphtheria, tetanus and pertussis (acellular, component)
vaccine (adsorbed) is a combined vaccine composed of:
diphtheria formol toxoid; tetanus formol toxoid; individually
purified antigenic components of Bordetella pertussis; a mineral
IV-520 Vaccines
adsorbent such as aluminium hydroxide or hydrated
aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacrm'wn diphtheriae and Clostn'dium
tetani, respectively.
The vaccine contains either penussis toxoid or a pertussis-
toxin-like protein free from toxic propenies, produced by
expression of a geneticalIy modified form of the
corresponding gene. Perrussis toxoid is prepared from
perrussis toxin by a method that renders the latter harmless
while maintaining adequate immunogenic propenies and
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa outer-
membrane protein) and other defined components of B.
pertussis such as fimbóal-2 and fimbóal-3 antigens. The latter
2 antigens may be co-purified. The antigenic composition
and characteóstics are based on evidence of protection and
freedom from unexpected reactions in the target group for
which the vaccine is intended.
PRODVCTION
GENERAL PROVISIONS
The production method shaIl have been shown ro yield
consistentIy vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the folIowing test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that wiII interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine does not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine does not comply with the test.
The content of bacteóal endotoxins (2.6.14) in the bulk
purified diphtheóa toxoid, tetanus toxoid and pertussis
components is determined to monitor the purification
procedure and ro limit the amount in the final vaccine.
For each component, the content ofbacteóal endotoxins is
less than the limit approved for the panicular vaccine and, in
any case, the contents are such that the final vaccine contains
less than 100 IV per single human dose.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
c1inical tóals or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence ro the production process used for the
batch tested in c1inical tóals is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria v accine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine
(acellular, component, adsorbed) (1356).
2014
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of suitable
quantities of bulk purified diphtheria toxoid, tetanus toxoid
and perrussis components separately or rogether onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate. Suitable antimicrobial preservatives
may be added.
Only a final bulk vaccine that complies with the folIowing
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for steólity using 10 mL for each medium.
FINALLOT
Only a final lot that is satisfactory with respect to the test for
osmolality and with respect ro each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the tests for residual pertussis toxin and
irreversibility of pertussis toxoid, free formaldehyde and
antimicrobial preservative and the assay have been carried
out with satisfactory resuIts on the final bulk vaccine, they
may be omitted on the final lot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the final lot wiII not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot .
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the panicular preparation.
IDENTIFICATION
A. Diphtheria roxoid is identified by a suitable
immunochemical method (2.7.1) . The folIowing method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supernatant liquid is
obtained. The c1ear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1) . The folIowing method, applicable to certain
vaccines, is given as an example. The c1ear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
C. The perrussis components are identified by a suitable
immunochemical method (2. 7.1). The folIowing method,
applicable to certain vaccines, is given as an example.
The c1ear supernatant liquid obtained as described in
identification test A reacts with specific antisera to the
pertussis components of the vaccine.
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The final lot complies with the test.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
2014
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6. 1)
The vaccine complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than the minimum potency stated on the
labe!'
Unless otherwise justified and authorised, the minimum
potency stated on the label is 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16).
The capacity of the vaccine to induce antibodies for each
inc1uded acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine .
LABELLING
The label states:
- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
- the names and amounts of the pertussis components per
single human dose;
- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
- the name and the amount of the adsorbent;
- that the vaccine must be shaken before use;
- that the vaccine is not to be frozen;
- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.
____________________________________________
~E~
*****
Adsorbed Diphtheria, Tetanus,
** **
Pertussis (Acellular Component) ***
and Haemophilus Type b
Conjugate Vaccine
(Diphtheria, Tetanus, Pertussis (Acellular, Component)
and Haemophilus Type b Conjugate Vaccine
(Adsorbed), Ph Eur monograph 1932)
The label may state 'DTaPlHib'.
Ph Eur _____________ _ _ _ ________________
DEFINITION
Diphtheria, tetanus, pertussis (acellular, component) and
haemophilus type b conjuga te vaccine (adsorbed) is a
Vaccines IV-52 1
combined vaccine composed of: diphtheria formol toxoid;
tetanus formo l toxoid; individually purified antigenic
components of Bordetella pertussis; polyribosylribitol phosphate
(PRP) covalently bound to a carrier protein; a mineral
absorbent such as aluminium hydroxide or hydrated
aluminium phosphate. The product is presented either as a
tetravalent liquid formulation in the same container, or as a
trivalent liquid formulation with the haemophilus component
in a separate container, the contents of which are mixed with
the other components immediately before use.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphthen'ae and Clostridium
tetani respectively.
The vaccine contains either pertussis toxoid or a pertussis-
toxin-like protein free from toxic properties produced by
expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the toxin harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The acellular pertussis
component may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other
defined components of B . pertussis such as fimbrial-2 and
fimbrial-3 antigens. The latter 2 antigens may be co-purified.
The antigenic composition and characteristics are based on
evidence of protection and freedom from unexpected
reactions in the target group for which the vaccine is
intended.
PRP is a linear copolymer composed of repeated units of
3-~-D-ribofuranosyl-(l ..... 1)-ribitol-5-phosphate
[(CIOH1 9012P)n], with a defined molecular size and derived
from a suitable strain of Haemophilus infiuenzae type b.
The carrier protein, when conjugated to PRP, is capable of
inducing a T-cell-dependent B-cell immune response to the
polysaccharide.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
Where the haemophilus component is presented in a separate
container, as part of consistency studies the assays of the
diphtheria, tetanus and pertussis components are carried out
on a suitable number of batches of vaccine reconstituted as
for use. For subsequent routine control, the assays of these
components may be carried out without mixing with the
haemophilus component.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal die s in the
second test, the vaccine does not comply with the test.
The content of bacterial endotoxins (2.6.14) in bulk purified
diphtheria toxoid, tetanus toxoid, pertussis components and
bulk PRP conjugate is determined to monitor the purification
procedure and to limit the amount in the final vaccine.
For each component, the content of bacterial endotoxins is
 IV-522 Vaccines
less than the limit approved for the particular vaccine; where
the haemophilus component is presented in a separate
container, the contents of the diphtheria, tetanus and
perrussis antigens are in any case such that the final vial for
these components contains less than 100 IU per single
human dose.
The production method is validated ro demonstrate that the
product, if tested, would comply with the test for abnormal
roxicity for immunosera and vaccines for human use (2.6.9).
During deveIopment srudies and wherever revalidation is
necessary, it shall be demonstrated by tests in animaIs that
the vaccine induces a T-cell dependent B-ceIl immune
response ro PRP.
Where the haemophilus component is presented in a separate
container, the production method is vaIidated to demonstrate
that the haemophilus component, if tested, wouId comply
with the test for pyrogens (2.6.8), carried out as follows:
inject per kilogram of the rabbit's mass a quantity of the
vaccine equivalent to: 1 Ilg of PRP for a vaccine with
diphtheria toxoid or CRM 197 diphtheria protein as carrier;
0.1 Ilg of PRP for a vaccine with tetanus toxoid as carrier;
0.025 Ilg of PRP for a vaccine with OMP (meningococcal
group B outer membrane protein complex) as carrier.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possibIe
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
c1inicaI triaIs or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in c1inicaI trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Haemophilus type b
conjugate vaccine (1219).
FINAL BULK VACCINE
Different methods of preparation may be used : a final bulk
vaccine may be prepared by adsorption, separately or
together, of suitable quantities of bulk purified diphtheria
toxoid, tetanus toxoid, acellular pertussis components and
PRP conjugate onto a mineral carrier such as aluminium
hydroxide or hydrated aluminium phosphate; or 2 final bulks
may be prepared and filled separately, one containing the
diphtheria, tetanus and perrussis components, the other the
haemophilus component, which may be freeze-dried. Suitable
antimicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
2014
FINALLOT
Only a final lot that is satisfactory with respect to the test for
osmolality shown below and with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use.
Provided the test for residual perrussis toxin and
irreversibility of perrussis toxoid, the test for antimicrobial
preservative and the assay have been carried out with
satisfacrory results on the final bulk vaccine, they may be
omitted on the finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or the final bulk and it has been
shown that the content in the final lot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
Osmolality (2.2.35)
The osmolality of the vaccine, reconstituted where applicable,
is within the limits approved for the particular preparation.
pH (2.2.3)
The pH of the vaccine, reconstituted if necessary, is within
the range approved for the particular producto
Free PRP
Unbound PRP is determined after removal of the conjugate,
for example by anion-exchange, size-exclusion or
hydrophobic chromarography, ultrafiltration or other
validated methods. The amount of free PRP is not greater
than that approved for the particular product.
IDENTIFICATION
Where the haemophilus component is presented in a separate
e
container: identification tests A, B and are carried out using the
container containing the diphthelia, tetanus and pertussis
componentsj identification test D is carried out on the container
containing the haemophilus component.
A. Diphtheria roxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supernatant liquid is
obtained. The c1ear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1) . The following method, applicable ro certain
vaccines, is given as an example. The c1ear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antiroxin, giving a precipitate.
C. The perrussis components are identified by a suitable
immunochemical method (2.7.1). The following method,
applicable ro certain vaccines, is given as an example.
The c1ear supematant liquid obtained as described in
identification test A reacts with specific antisera to the
perrussis components of the vaccine.
D. The haemophilus component is identified by a suitable
immunochemical method (2.7.1) for PRP.
TESTS
Where the product is presented with the haemophilus component in
a separate container: the tests for residual pertussis roxin and
irreversibiliry of pertussis toxoid, aluminiwn, free formaldehyde,
antimicrobial preservative and steriliry are cam'ed out on the
container with the diphtheria, tetanus and pertussis componentsj
the tests for PRP content, water (where applicable), sterilityand
bacterial endoroxins are cam'ed out on the container with the
haemophilus component.
 2014
Jf the haemophilus component is freeze-dried, some tests may be
carried out on the freeze-dried product rather than on the bulk
conjugate where the freeze-drying process may affect the component
ca be tested.
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
PRP
Minimum 80 per cent of the amount of PRP stated on the
labe!' PRP is determined either by assay of ribose (2.5.31) or
phosphorus (2.5.18), by an irnmunochemical method (2.7.1)
or by anion-exchange liquid chromatography (2.2.29) with
pulsed-amperometric detection.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Water (2.5.12)
Maximum 3.0 per cent for the freeze-dried haemophilus
component.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
The content is within the limits approved by the competent
authority for the haemophilus component of the particular
producto If any components of the vaccine prevent the
determination of endotoxin, a test for pyrogens is carried out
as described under General provisions.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than the minimum potency stated on the
labe!'
Unless otherwise justified and authorised, the minimum
potency stated on the label is 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16) .
The capacity of the vaccine to induce antibodies for each
inc1uded acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
LABELLING
The labe! states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the names and amounts of the pertussis components per
single human dose;
Vaccines IV-523
-- the number of micrograms of PRP per single human
dose;
-- the type and nominal amount of carrier protein per single
human dose;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen;
-- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.
____________________________________________ Ph E~
Adsorbed Diphtheria, Tetanus, ***
*** ***
Pertussis (Acellular Component) ***
and Hepatitis B (rDNA) Vaccine
(Diphtheria, Tetanus, Pertussis (Aeellular, Compone/u)
and H epatitis B (rDNA) Vaccine (Adsorbed),
Ph Bur monograph 1933)
The label may state 'DTaP/HepB'.
~E~ ____________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (acellular, component) and
hepatitis B (rDNA) vaccine (adsorbed) is a combined vaccine
composed of: diphtheria formol toxoid; tetanus formol
toxoid; individually purified antigenic components of
Bordetella p ertussis; hepatitis B surface antigen; a mineral
adsorbent such as aluminium hydroxide or hydrated
aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani, respectively.
The vaccine contains either pertussis toxoid or a pertussis-
toxin-like protein free from toxic properties, produced by
expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the latter harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa
outer-membrane protein) and other defined components of
B . pertussis such as fimbrial-2 and fimbrial-3 antigens.
The latter 2 antigens may be co-purified. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the
target group for which the vaccine is intended.
Hepatitis B surface antigen is a component protein of
hepatitis B virus; the antigen is obtained by recombinant
DNA technology.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
IV-524 Vaccines
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or die s from
diphtheria toxaemia or tetanus, the vaccÍne does not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine do es not comply with the test.
The content of bacterial endotoxins (2.6.14) in the bulk
purified diphtheria toxoid, tetanus toxoid and pertussis
components is determined to monitor the purification
procedure and to limit the amount in the final vaccine.
For each component, the content of bacterial endotoxins is
less than the limit approved for the particular vaccine.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccÍne. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccÍne and the test
vaccine, a batch of combined vaccine shown to be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in clinical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
PRODUCTlON OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Hepatitis B vaccine
(rDNA) (1056).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
or together, of suitable quantities of bulk purified diphtheria
toxoid, tetanus toxoid, acellular pertussis components and
hepatitis B surface antigen onto a mineral carrier such as
aluminium hydroxide or hydrated aluminium phosphate.
Suitable antimicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not les s than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Only a final lot that is satisfactory with respect to the test for
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the test for residual pertussis toxin and
irreversibility of pertussis toxoid, the test for antimicrobial
preservative and the assays for the diphtheria, tetanus and
pertussis components have been carried out with satisfactory
results on the final bulk vaccine, they may be omitted on the
finallot.
Provided the content of free formaldehyde has been
determined on the bulk purified antigens or on the final bulk
and it has been shown that the content in the finallot will
2014
not exceed 0.2 gIL, the test for free formaldehyde may be
omitted on the final lot.
If an in vivo assay is used for the hepatitis B component,
provided it has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limirs approved
for the particular preparation.
IDENTIFICAnON
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a clear supernatant liquid is
obtained. The clear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to certain
vaccines, is given as an example. The clear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
C. The pertussis components are identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
The clear supernatant liquid obtained as described in
identification test A reacts with specific antisera to the
pertussis components of the vaccine.
D. The assay or, where applicable, the electrophoretic profile,
serves also to identifY the hepatitis B component of the
vaccme.
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not les s than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
label.
Sterility (2.6.1)
The vaccine complies with the test for sterility.
Pyrogens (2.6.8)
The vaccine complies with the test for pyrogens. Inject the
equivalent of 1 human dose into each rabbit.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P == 0.95) of the estimated
potency is not less than the minimum potency stated on the
label.
Unless otherwise justified and authorised, the minimum
potency stated on the label is 30 IU per single human dose.
 2014
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acelIular) (2.7.16).
The capacity of the vaccine to induce antibodies for each
included acelIular pertussis antigen is not significantIy
(P = 0.95) less than that ofthe reference vaccine.
Hepatitis B component
The vaccine complies with the assay of hepatitis B vaccine
(2.7.15).
LABELLING
The label states:
- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
- the names and amounts of the pertussis components per
single human dose;
- the amount of HBsAg per single human dose;
- the type of celIs used for production of the hepatitis B
component;
- where applicable, that the vaccine is intended for primaty
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
- the name and the amount of the adsorbent;
- that the vaccine must be shaken before use;
- that the vaccine is not to be frozen;
- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.
_ _________________________________________ ~Ew
Adsorbed Diphtheria, Tetanus, ***
*** ***
Pertussis (Acellular Component) ***
and Inactivated Poliomyelitis
Vaccine
(Diphtheria, Tetanus, Pertussis (Acellular, Component)
and Poliomyelitis (Inactivated) Vaccine (Adsorbed),
Ph Eur monograph 1934)
The label may state 'DTaPIIPV'.
PhEw __________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (acelIular, component) and
poliomyelitis (inactivated) vaccine (adsorbed) is a combined
vaccine containing: diphtheria formol toxoid; tetanus formol
toxoid; individualIy purified antigenic components of
Bordetella pertussis; suitable strains of human poliovirus types
1, 2 and 3 grown in suitable celI cultures and inactivated by
a validated method; a mineral adsorbent such as aluminium
hydroxide or hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
The vaccine contains either pertussis toxoid or a pertussis-
toxin-like protein free from toxic properties produced by
expression of a geneticalIy modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the toxin harmless
Vaccines IV-525
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa
outer-membrane protein) and other defined components of
B. pertussis such as fimbrial-2 and fimbrial-3 antigens .
The latter 2 antigens may be co-purified. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the
target group for which the vaccine is intended.
PRODUCTION
GENERAL PROVISIONS
The production method shaII have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the folIowing test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that wiII interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine does not comply with the test.
The content of bacterial endotoxins (2.6.14) in bulk purified
diphtheria toxoid, tetanus toxoid, pertussis components and
purified, inactivated monovalent poliovirus harvests is
determined to monitor the purification procedure and to
limit the amount in the final vaccine. For each component,
the content of bacterial endotoxins is less than the limit
approved for the particular vaccine and, in any case, the
contents are such that the final vaccine contains less than
100 IU per single human dose .
R eference vaccine(s) rovided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
c1inical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in c1inical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure .
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Poliomyelitis
vaccine (inactivated) (0214).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid, tetanus toxoid,
acelIular pertussis components and admixture of suitable
quantities of purified monovalent harvests of human
poliovirus types 1, 2 and 3 or a suÍtable quantity of a
trivalent pool of such purified monovalent harvests. SuÍtable
antimicrobial preservatives may be added.
IV-526 Vaccines
Onlya final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
immunochemical method (2.7.1) after virus harvest and
before addition of the adsorbent in the preparation of the
final bulk vaccine, the amount of bovine serum albumin is
such that the content in the final vaccine will be not more
than 50 ng per single human dose.
Antimicrobial preservative
Where applicable, determine the amount of antirnicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contenr.
Sterility (2.6. 1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Only a finallot that is satisfactory with respect to the test for
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the test for residual pertussis toxin and
irreversibility of pertussis roxoid, the test for antirnicrobial
preservative and the assays for the diphtheria, tetanus and
pertussis components have been carried out with satisfactory
results on the final bulk vaccine, they may be omitted on the
finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the finallot will not exceed
0.2 gIL, the test for free formaldehyde may be oITÚtted on the
finallot.
Provided that the determination of D-antigen content has
been carried out with satisfactory results during preparation
of the final bulk before addition of the adsorbent, it may be
omitted on the finallot.
Provided that the in vivo assay for the poliomyelitis
component has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the finallot.
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must include testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in viera assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the particular preparation.
IDENfIFICATION
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
abour 16 h and centrifuge until a clear supematant liquid is
obtained. The clear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
2014
B. Tetanus roxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable ro certain
vaccines, is given as an example. The clear supematant liquid
obtained as described in identification test A reacts with a
suitable tetanus antiroxin, giving a precipitate.
C. The pertussis components are identified by a suitable
irnmunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
The clear supematant liquid obtained as described in
identification test A reacts with specific antisera to the
pertussis components of the vaccine.
D. The vaccine is shown to contain human poliovirus types
1,2 and 3 by a suitable immunochemical method (2.7.1)
such as the determination of D-antigen by enzyme-linked
irnmunosorbent assay (ELISA).
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies~ith the test.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dos e if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antirnicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown ro be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence lirnit (P = 0.95) of the estimated
potency is not less than the minirnum potency stated on the
labe!'
Unless otherwise justified and authorised, the minimum
potency stated on the label is 30 IU per single human dose .
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence lirnit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16).
The capacity of the vaccine to induce antibodies for each
included acellular pertussis antigen is not significantly
(P = 0.95) less than thar ofthe reference vaccine.
Poliomyelitis component
D-antigen content As a measure of consisrency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7.1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the lirnits approved for the particular producto
2014
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Phannacopoeia Unit and
the International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the names and amounts of the pertussis components per
single human dose;
-- the types of poliovirus contained in the vaccine;
-- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
-- the type of cells used for production of the poliomyelitis
component;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen;
-- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.
____________________________________________ ~Ew
Diphtheria, Tetanus and ***
*** ***
Poliornyelitis (Inactivated) Vaccine ***
(Adsorbed, Reduced Antigen(s)
Content)
(Ph Eur monograph 2328)
The label may state 'TdlIPV'.
~Ew ____________________________________________
DEFINITION
Diphtheria, tetanus and poliomyelitis (inactivated) vaccine
(adsorbed, reduced antigen(s) content) is a combined vaccine
containing: diphtheria formol toxoid; tetanus formol toxoid;
suitable strains of human poliovirus types 1, 2 and 3 grown
in suitable cell cultures and inactivated by a validated
method; a mineral adsorbent such as aluminium hydroxide
or hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
The amount of diphtheria toxoid per single human dose is
reduced compared to vaccines generally used for primary
vaccination; the amount of tetanus toxoid may also be
reduced.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
Vaccines IV-527
combined vaccine or because of the difference in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in clinical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than one animal dies from non-specific
causes, repeat the test once; if more than one animal dies in
the second test, the vaccine does not comply with the test.
The content of bacterial endotoxins (2.6.14) in bulk purified
diphtheria toxoid, tetanus toxoid and inactivated monovalent
poliovirus harvests is determined to monitor the purification
procedure and to limit the amount in the final vaccine.
For each component, the content of bacterial endotoxins is
less than the limit approved for the particular vaccine and, in
any case, the contents are such that the final vaccine contains
less than 100 IU per single human dose.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs on Diphtheria vaccine
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and
Poliomyelitis vaccine (inactivated) (0214) .
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid and tetanus
toxoid, and an admixture of suitable quantities of purified
monovalent harvests of human poliovirus types 1, 2 and 3 or
a suitable quantity of a trivalent pool of such purified
monovalent harvests. Suitable antimicrobial preservatives may
be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
immunochemical method (2.7.1) after virus harvest and
before addition of the adsorbent in the preparation of the
final bulk vaccine, the amount of bovine serum albumin is
such that the content in the final vaccine will be not more
than 50 ng per single human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
IV-528 Vaccines
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are c10sed so as to
prevent contamination.
Only a finallot that is satisfactory with respect to the test for
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be relea sed
for use.
Provided the test for antimicrobial preservative and the assays
for the diphtheria and tetanus components have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
been shown that the content in the finallot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
Provided the determination of D-antigen content cannot be
carried out on the finallot, it is carried out during
preparation of the final bulk before addition of the adsorbent.
Provided the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulk
vaccine, it may be omitted on the final lot.
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given vaccine
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must inc1ude testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the particular preparation.
IDENTIFICAnON
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supernatant liquid is
obtained. The c1ear supernatant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate. If a satisfactory
result is not obtained with a vaccine adsorbed on aluminium
hydroxide, carry out the test as follows. Centrifuge 15 mL of
the vaccine to be examined and suspend the residue in 5 mL
of a freshly prepared mixture of 1 volume of a 56 giL
solution of sodium edetate R and 49 volumes of a 90 gIL
solution of disodiwl1 hydrogen phosphate R . Maintain at 37 cc
for not less than 6 h and centrifuge. The c1ear supernatant
liquid reacts with a suitable diphtheria antitoxin,
giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7. 1). The following method, applicable to certain
vaccines, is given as an example. The c1ear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
C. The vaccine is shown to contain human poliovirus types
1, 2 and 3 by a suitable immunochemical method (2.7.1)
2014
such as the determination of D-antigen by enzyme-linked
immunosorbent assay (ELISA).
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
1abe!'
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 2 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.9 5) of the estimated
potency is not less than 20 IU per single human dose.
Poliomyelitis component
D-antigen contem As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7. 1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the limits approved for the particular producto
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
the International Unit are equivalent.
In vivo test. The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the types of poliovirus contained in the vaccine;
-- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
-- the type of cells used for production of the poliomyelitis
component;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ ~Ew
 2014
*****
Diphtheria, Tetanus, Pertussis
** **
(Acellular, Component) and ***
Poliomyelitis (lnactivated) Vaccine
(Adsorbed, Reduced Antigen(s)
Content)
(Ph Eur monograph 2329)
The label may state ' dTaPIIPV'.
~E~ ____________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (acellular, component) and
poliomyelitis (inactivated) vaccine (adsorbed, reduced
antigen(s) content) is a combined vaccine containing:
diphtheria formol toxoid; tetanus formol toxoid; individually
purified antigenic components of Bordetella pertussis; suitable
strains of human poliovirus types 1, 2 and 3 grown in
suitable cell cultures and inactivated by a validated method;
a mineral adsorbent such as aluminium hydroxide or
hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of COlynebacterium diphtheriae and Clostridium
tetani respectively.
The amount of diphtheria toxoid per single human dose is
reduced compared to vaccines generally used for primary
vaccination; the amounts of tetanus toxoid and pertussis
components may also be reduced.
The vaccine contains either pertussis toxoid or a pertussis-
1Oxin-like protein free from 10xic properties produced by
expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the 10xin harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa
outer-membrane protein) and other defined components of
B. pertussis such as fimbrial-2 and fimbrial-3 antigens.
The latter 2 antigens may be co-purified. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the
target group for which the vaccine is intended.
PRODVCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
R e/erence vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown 10 be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in clinical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
10 have no effect on the assay procedure.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated 10 demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
Vaccines IV-529
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or die s from
diphtheria 10xaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal die s in the
second test, the vaccine do es not comply with the test.
The content of bacterial endo1Oxins (2.6.14) in bulk purified
diphtheria toxoid, tetanus toxoid, pertussis components and
inactivated monovalent poliovirus harvests is determined to
monitor the purification procedure and 10 limit the amount
in the final vaccine. For each component, the content of
bacterial endotoxins is less than the limit approved for the
particular vaccine and, in any case, the contents are such that
the final vaccine contains less than 100 IV per single human
dose.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Poliomyelitis
vaccine (inactivated) (0214) .
FINAL BULK VACCINE
The fin al bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid, tetanus 1Oxoid
and acellular pertussis components, and an admixture of
suitable quantities of purified monovalent harvests of human
poliovirus types 1, 2 and 3 or a suitable quantity of a
trivalent pool of such purified monovalent harvests . Suitable
antirnicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
immunochemical method (2.7. 1) after virus harvest and
before addition of the adsorbent in the preparation of the
final bulk vaccine, the amount of bovine serum albumin is
such that the content in the final vaccine will be not more
than 50 ng per single human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to the test for
osmolality and with respect 10 each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the test for residual pertussis 10xin and
irreversibility of pertussis toxoid, the test for antimicrobial
preservative and the assays for the diphtheria, tetanus and
pertussis components have been carried out with satisfactory
results on the final bulk vaccine, they may be omitted on the
finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulk and it has
IV-530 Vaccines
been shown that the content in the final lot will not exceed
0.2 gIL, the test for free formaldehyde may be omined on the
finallot.
Provided the determination of D-antigen content cannot be
carried out on the finallot, it is carried out during
preparation of the final bulk before addition of the adsorbent.
Provided the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulk
vaccine, it may be omined on the final loto
The in vivo assay for the poliomyelitis component may be
omined once it has been demonstrated for a given vaccine
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must inc1ude testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatrnent or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vilro assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the particular preparation.
IDENTIFICATION
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example. Dissolve
in the vaccine to be examined sufficient sodium cilrate R to
give a 100 giL solution. Maintain at 37 °C for about 16 h and
centrifuge until a c1ear supernatant liquid is obtained.
The c1ear supernatant liquid reacts with a suitable diphtheria
antitoxin, giving a precipita te. If a satisfactory result is not
obtained with a vaccine adsorbed on aluminium hydroxide,
carry out the test as follows . Centrifuge 15 mL of the vaccine
to be examined and suspend the residue in 5 mL of a freshly
prepared mixture of 1 volume of a 56 gIL solution of sodium
edetate R and 49 volumes of a 90 gIL solution of disodium
hydrogen phosphate R. Maintain at 37 oC for not less than 6 h
and centrifuge. The c1ear supernatant liquid reacts with a
suitable diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable irnmunochemical
method (2.7.1) . The following method, applicable to certain
vaccines, is given as an example. The c1ear supernatant liquid
obtained as described in identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
C. The pertussis components are identified by a suitable
irnmunochemical method (2. 7.1). The following method,
applicable to certain vaccines, is given as an example.
The c1ear supernatant liquid obtained as described in
identification test A reacts with a specific antisera to the
pertussis components of the vaccine.
D. The vaccine is shown to contain human poliovirus types
1, 2 and 3 by a suitable immunochemical method (2.7.1)
such as the determination of D-antigen by enzyme-linked
irnmunosorbent assay (ELISA).
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
2014
Free formaldehyde (2.4.18)
Maximum 0.2 giL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7. 6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 2 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2. 7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 20 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16) .
The capacity of the vaccine to induce antibodies for each
inc1uded acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
Poliomyelitis component
D-antigen cantent As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1,2 and 3 by a suitable immunochemical
method (2. 7.1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the limits approved for the particular product.
Poliomyelitis vaccine (inactivated) BRPis calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
the International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20) .
LABELLING
The label states:
- the minimum number of Intemational Units of diphtheria
and tetanus toxoid per single human dose;
- the names and amounts of the pertussis components per
single human dose;
- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification;
- the types of poliovirus contained in the vaccine;
- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
- the type of cells used for production of the poliomyelitis
component;
- the name and the amount of the adsorbent;
- that the vaccine must be shaken before use;
- that the vaccine is not to be frozen.
_ _ __ __ _ _ _ _ _ _ _ __ _ __ _ _ _ _ Ph Eur
2014
Diphtheria, Tetanus, Pertussis ***
*** ***
(Acellular, Component), ***
Poliomyelitis (Inactivated) and
Haemophilus Type b Conjugate
Vaccine (Adsorbed)
(Ph Eur monograph 2065)
The label may state 'DTaPIIPVlHib'.
Ph EUf _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ __ _ __
DEFINITION
Diphtheria, tetanus, pertussis (acellular, component),
poliomyelitis (inactivated) and haemophilus type b conjugate
vaccine (adsorbed) is a combined vaccine composed of:
diphtheria formol toxoid; tetanus formol toxoid; individually
purified antigenic components of Bordetella pertussis; suitable
strains of human poliovirus types 1, 2 and 3 grown in
suitable cell cultures and inactivated by a suitable method;
polyribosylribitol phosphate (PRP) covalently bound to a
carrier protein; a mineral adsorbent such as aluminium
hydroxide or hydrated aluminium phosphate. The product is
presented either as a pentavalent liquid formulation in the
same container, or as a tetravalent liquid formulation with
the freeze-dried haemophilus component in a separate
container, the contents of which are mixed with the other
components immediately before use.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
The vaccine contains either pertussis toxoid or a
pertussis-toxin-like protein free from toxic properties
produced by expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the toxin harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The acellular pertussis
component may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other
defined components of B. pertussis such as fimbrial-2 and
fimbrial-3 antigens. The latter 2 antigens may be co-purified.
The antigenic composition and characteristics are based on
evidence of protection and freedom from unexpected
reactions in the target group for which the vaccine is
intended.
PRP is a linear copolymer composed of repeated units of
3-~-D-ribofuranosyl-( 1-> 1)-ribitol-5-phosphate
[(CIOHI 9012P) n], with a defined molecular size and derived
from a suitable strain of Haemophilus infiuenzae type b.
The carrier protein, when conjugated to PRP, is capable of
inducing a T -cell-dependent B-cell immune response to the
polysaccharide.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dos e stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
Vaccines IV-531
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or die s from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal die s in the
second test, the vaccine do es not comply with the test.
Bacteria! endotoxins (2.6.14)
The content of bacterial endotoxins in bulk purified
diphtheria toxoid, tetanus toxoid, pertussis components,
purified, inactivated monovalent poliovirus harvests and bulk
PRP conjugate is determined to monitor the purification
procedure and to limit the amount in the final vaccine.
For each component, the content of bacterial endotoxins is
less than the limit approved by the competent authority for
the particular vaccine.
Development and consistency studies
During development studies and wherever revalidation is
necessary, it shall be demonstrated by tests in animals that
the vaccine induces a T-cell-dependent B-cell immune
response to PRP .
Where the haemophilus component is presented in a separate
container, and as part of consistency studies, the assays of the
diphtheria, tetanus, pertussis and poliomyelitis components
are carried out on a suitable number of batches of vaccine
reconstituted as for use. For subsequent routine control, the
assays of these components may be carried out without
mixing with the haemophilus component.
Where the haemophilus component is presented in a separate
container, the production method is validated to demonstrate
that the haemophilus component, if tested, would comply
with the test for pyrogens (2.6.8), carried out as follows:
inject per kilogram of the rabbit's mass a quantity of the
vaccine equivalent to: 1 ~g of PRP for a vaccine with
diphtheria toxoid or CRM 197 diphtheria protein as carrier;
0.1 ~g of PRP for a vaccine with tetanus toxoid as carrier;
0.025 ~g of PRP for a vaccine with OMP (meningococcal
group B outer membrane protein complex) as carrier.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in clinical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356), Poliomyelitis vaccine
(inactivated) (0214) and Haemoph¡lus type b conjugate vaccine
(1219).
FINAL BULKS
The final tetravalent bulk of the diphtheria, tetanus, pertussis
and poliomyelitis components is prepared by adsorption,
separately or together, of suitable quantities of bulk purified
diphtheria toxoid, bulk purified tetanus toxoid and bulk
purified acellu1ar pertussis components onto a mineral carrier
such as aluminium hydroxide or hydrated aluminium
phosphate, and admixture of suitable quantities of purified,
 IV-532 Vaccines
monovalent harvests of human poliovirus types 1, 2 and 3 or
a suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
Where the vaccine is presented with all 5 components in the
same container, the final bulk is prepared by addition of a
suitable quantity of the haemophilus bulk conjugate to the
tetravalent bulk. Where the haemophilus component is
presented in a separate container, the final bulk is prepared
by dilution of the bulk conjugate with suitable diluents for
freeze-drying. A stabiliser may be added.
OnIy final bulks that comply with the following requirements
may be used in the preparation of the final loto
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
immunochemical method (2.7.1) during preparation of the
final bulk vaccine, before addition of the adsorbent, the
amount of bovine serum albumin is such that the content in
the final vaccine will be not more than 50 ng per single
human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Where the haemophilus component is presented in a separate
container, the final bulk of the haemophilus component is
freeze-dried.
Only a final lot that is satisfactory with respect to the test for
osmolality shown below and with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use.
Provided that the test for residual pertussis toxin and
irreversibility of pertussis toxoid, the test for antimicrobial
preservative and the assay have been carried out with
satisfactory resuIts on the final bulk vaccine, they may be
omitted on the finallot.
Provided that the free formaldehyde content has been
determined on the bulk purified antigens and the purified
monovalent harvests or the trivalent pool of polioviruses or
the final bulk and it has been shown that the content in the
finallot will not exceed 0.2 gIL, the test for free
formaldehyde may be omitted on the finallot.
If the in vivo assay for the poliomyelitis component is used,
provided it has been carried out with satisfactory resuIts on
the final bulk vaccine, it may be omitted on the finallot.
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
resuIt as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must include testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine, reconstituted where applicable,
is within the limits approved for the particular preparation.
2014
Free PRP
Where the haemophilus component is presented in liquid
formulation, the presence of other components may interfere
in the assay and it may not be possible to separate the PRP
from the adjuvant. The presence of free PRP may be
determined on the bulk conjugate prior to the addition of
other components or on the non-adsorbed fraction in the
final combination.
Where the haemophilus component is presented in a separate
container, a number of methods have been used to separate
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion exchange and hydrophobic
chromatography, ultrafiltration and uItracentrifugation.
The free PRP can then be quantified by a range of
techniques, including high-performance anion-exchange
chromatography with pulsed amperometric detection
(HPAEC-PAD) and immunoassays with anti-PRP
antibodies. The amount of free PRP is not greater than that
approved for the particular producto
IDENTIFICAnON
Identification tests A, B, e and D are carried out using the vial
containing the diphtheria, tetanus, pertussis and poliomyelitis
components; identification test E is carried out either on the vial
containing all 5 components, or on the vial containing the
haemophilus component alone.
A. Diphtheria toxoid is identified by a suitable
irnmunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 DC for
about 16 h and centrifuge until a clear supernatant liquid is
obtained. The clear supernatant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to certain
vaccines, is given as an example. The clear supernatant liquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipita te.
C. The pertussis components are identified by a suitable
irnmunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
The clear supernatant liquid obtained during identification
test A reacts with specific antisera to the pertussis
components of the vaccine.
D. The vaccine is shown to contain human poliovirus types
1, 2 and 3 by a suitable immunochemical method (2.7.1),
such as determination of D-antigen by enzyme-linked
irnmunosorbent assay (ELISA).
E. The haemophilus component is identified by a suitable
irnmunochemical method (2.7.1) for PRP.
TESTS
Where the haemophilus component is presented in a separate
container, the tests for residual pertussis toxin and irreversibiliry of
pertussis toxoid, aluminium, free formaldehyde, antimicrobial
preservative and sterility are carried out on the container with the
diphtheria, tetanus, pertussis and poliomyelitis components;
the tests for PRP, water, steriliry and bacterial entodoxins are
carried out on the container with the haemophilus component
alone.
Where the haemophilus component is presented in a separate
container, some tests may be carried out on the freeze-dried
product rather than on the bulk conjugate where the freeze-dlying
process may affecl the component to be tested.
2014
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The final lot complies with the test.
PRP
Not less than 80 per cent of the amount of PRP stated on
the labe!' PRP is determined either by assay of ribose
(2.5.31) or phosphorus (2.5.18), by an immunochemical
method (2.7.1) or by anion-exchange liquid chromatography
(2.2.29) with pulsed-amperometric detection.
Alurninium (2.5.13)
Maximum 1.25 mg per single human dos e, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antirnicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Water (2.5.12)
Maximum 3.0 per cent for the freeze-dried haemophilus
component.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
The content is within the limits approved by the competent
authority for the haemophilus component of the particular
product. If any components of the vaccine prevent the
determination of endotoxin, a test for pyrogens is carried out
as described under General provisions.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
Unless otherwise justified and authorised, the lower
confidence limit (P = 0.95) of the estimated potency is not
less than 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16).
The capacity of the vaccine to induce antibodies for each
inc1uded acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
Poliomyelitis component
D-amigen contem As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2. 7. 1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the limits approved for the particular product.
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
Vaccines IV-533
assay of D-antigen. The European Pharmacopoeia Unit and
the International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The labe! states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the names and amounts of the pertussis components per
single human dos e;
-- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
-- the type of cells used for production of the poliomyelitis
component;
-- the number of micrograms of PRP per single human
dose;
-- the type and nominal amount of carrier protein per single
human dose;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administratian to adults;
-- the na me and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen;
-- where applicable, that the vaccine contains a pertussis-
toxin-like protein produced by genetic modification.
____________________________________________
~Eif
Diphtheria, Tetanus, Pertussis
(Acellular, Component),
Hepatitis B (rDNA), Poliomyelitis
(Inactivated) and Haemophilus
Type b Conjugate Vaccine
(Adsorbed)
(Ph Eur monograph 2067)
The label may state 'DTaPlHepBLIPVlHib'.
~Eif ___________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (acellular, component),
hepatitis B (rDNA), poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) is a
combined vaccine composed of: diphtheria formol toxoid;
tetanus formol toxoidj individually purified antigenic
components of Bordetella pertussisj hepatitis B surface antigen
(HBsAg); human poliovirus types 1,2 and 3 grown in
suitable cell cultures and inactivated by a suitable method;
polyribosylribitol phosphate (PRP) covalently bound to a
carrier protein. The antigens in the vaccine may be adsorbed
on a mineral carrier such as aluminium hydroxide or
hydrated aluminium phosphate. The product is presented
either as a hexavalent liquid formulation in the same
container, or as a pentavalent liquid formulation with the
haemophilus component in a separate container, the contents
of which are mixed with the other components immediately
before or during use.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacten'um diphthen"ae and Clostn"dium
tetani respectively.
 IV-534 Vaccines
The vaccine contains either pertussis toxoid or a
pertussis-toxin-like protein free from toxic properties
produced by expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the toxin harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The acellular pertussis
component may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other
defined components of B. pertussis such as fimbrial-2 and
fimbrial-3 antigens. The latter 2 antigens may be co-purified.
The antigenic composition and characteristics are based on
evidence of protection and freedom from unexpected
reactions in the target group for which the vaccine is
intended.
Hepatitis B surface antigen is a component protein of
hepatitis B virus; the antigen is obtained by recombinant .
DNA technology.
PRP is a linear copolymer composed of repeated units of
3-~-D-ribofuranosyl-(1-> 1)-ribitol-5-phosphate
[(CloH1 901 ZP)n), with a defined molecular size and derived
from a suitable strain of Haemophilus infiuenzae type b.
The carrier protein, when conjugated to PRP, is capable of
inducing a T-cell-dependent B-cell immune response to the
polysaccharide.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
If the vaccine is presented with the haemophilus component
in a separate container, as part of consistency studies the
assays of the diphtheria, tetanus, pertussis, hepatitis B and
poliomyelitis components are carried out on a suitable
number of batches of vaccine reconstituted as for use.
For subsequent routine control, the assays of these
components may be carried out without mixing with the
haemophilus component.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dos e stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or die s from
diphtheria toxaemia or tetanus, the vaccine does not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine does not comply with the test.
The content of bacterial endotoxins (2.6.14) in bulk purified
diphtheria toxoid, tetanus toxoid and pertussis components,
hepatitis B surface antigen, purified, inactivated monovalent
poliovirus harvests and bulk PRP conjugate is determined to
monitor the purification procedure and to limit the amount
in the final vaccine. For each component, the content of
bacterial endotoxins is not greater than the limit approved.
During development studies and wherever revalidation is
necessary, a test for pyrogens in rabbits (2.6.8) is carried out
by injection of a suitable dose of the final loto The vaccine is
shown to be acceptable with respect to absence of pyrogenic
activity.
2014
During development studies and wherever revalidation is
necessary, it shall be demonstrated by tests in animals that
the vaccine induces a T-cell-dependent B-cell immune
response to PRP.
The stability of the final lot and relevant intermediates is
evaluated using one or more indicator tests. For the
haemophilus component, such tests may inc1ude
determination of molecular size, determination of free PRP in
the conjugate and kinetics of depolymerisation. Taking
account of the results of the stability testing, release
requirements are set for these indicator tests to ensure that
the vaccine will be satisfactory at the end of the period of
validity.
R eference vaccine(s) Provided val id assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
c1inical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in c1inical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356), Hepatitis B vaccine
(rDNA) (1056), Poliomyelitis vaccine (inactivated) (0214) and
Haemophilus type b conjugate vaccine (1219).
FINAL BULKS
Vaccine with all components in the same container The final
bulk is prepared by adsorption, separately or together, of
suitable quantities of bulk purified diphtheria toxoid, tetanus
toxoid, acellular pertussis components and hepatitis B surface
antigen onto a mineral carrier such as aluminium hydroxide
or hydrated aluminium phosphate and admixture of a
suitable quantity of PRP conjugate and suitable quantities of
purified and inactivated, monovalent harvests of human
poliovirus types 1, 2 and 3 or a suitable quantity of a
trivalent pool of such monovalent harvests. Suitable
antimicrobial preservatives may be added.
Vaccine with the haemophilus component in a separate
container The final bulk of diphtheria, tetanus, pertussis,
hepatitis B and poliovirus component is prepared by
adsorption, separately or together, of suitable quantities of
bulk purified diphtheria toxoid, tetanus toxoid, acellular
pertussis components and hepatitis B surface antigen onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate and admixture of suitable quantities of
purified and inactivated, monovalent harvests of human
poliovirus types 1, 2 and 3 or a suitable pool of such
monovalent harvests. This final bulk is filled separately.
Suitable antimicrobial preservatives may be added. The final
bulk of the haemophilus component is prepared by dilution
of the bulk conjugate to the final concentration with a
suitable diluent. A stabiliser may be added.
Only final bulks that comply with the following requirements
may be used in the preparation of the final lot.
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
immunochemical method (2.7.1) after purification of the
2014
harvests and before preparation of the final bulk vaccine,
before addition of the adsorbent, the amount of bovine
serum albumin is such that the content in the final vaccine
will be not more than 50 ng per single human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not les s than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Where the haemophilus component is in a separate
container, the final bulk of the haemophilus component is
freeze-dried. Only a final lot that is satisfactory with respect
to the test for osmolality shown below and with respect to
each of the requirements given below under Identification,
Tests and Assay may be released for use.
Provided that the test for osmolality, the test for residual
pertussis toxin and irreversibility of pertussis toxoid, the test
for antimicrobial preservative and the assays for the
diphtheria, tetanus and pertussis components have been
carried out with satisfactory results on the final bulk vaccine,
they may be omitted on the finallot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens and the purified monovalent
harvests or the trivalent pool of polioviruses or the final bulk
and it has been shown that the content in the finallot will
not exceed 0.2 gIL, the test for free formaldehyde may be
omitted on the final lot.
Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the trivalent pool of
inactivated monovalent harvests of polioviruses or on the
final bulk vaccine, it may be omitted on the final loto
If an in vivo assay is used for the hepatitis B component,
provided it has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the finallot.
Provided the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulk
vaccine, it may be omitted on the finallot.
The in vivo assay for the poliomyelitis component may be
ornitted once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must include testing of
subpotent batch es, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Free PRP
For vaccines with all components in the same container, the
free PRP content is determined on the non-absorbed
fraction. Unbound PRP is determined on the haemophilus
component after removal of the conjugate, for examp1e by
anion-exchange, size-exclusion or hydrophobic
chromatography, ultrafiltration or other validated methods.
The amount of free PRP is not greater than that approved
for the particular producto
Bacterial endotoxins (2.6.14)
Less than the limit approved for the product concemed.
Vaccines IV-535
Osmolality (2.2.35)
The osmolality of the vaccine, reconstituted where applicable,
is within the limits approved for the particular preparation.
IDENTIFICATION
1f the vaccine is presented with the haemophilus component in a
separate container: identification tests A, B, C, D and E are
carried out using the container with the diphtheria, tetanus,
pertussis, hepatitis B and poliomyelitis componentsj identification
test F is carried out on the container with the haemophilus
components.
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method is
given as an example. Dissolve in the vaccine to be examined
sufficient sodium citrate R to give a 100 gIL solution.
Maintain at 37 oC for about 16 h and centrifuge until a clear
supematant liquid is obtained. The clear supematant liquid
reacts with a suitable diphtheria antitoxin, giving a
precipita te.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method is given as an
example. The clear supematant liquid obtained during
identification test A reacts with a suitable tetanus antitoxin,
giving a precipitate.
C. The clear supematant liquid obtained during
identification test A reacts with specific antisera to the
pertussis components of the vaccine when examined by
suitable immunochemical methods (2.7.1).
D. The hepatitis B component is identified by a suitable
immunochemical method (2. 7.1), for example the in vitro
assay, or by a suitable electrophoretic method (2.2.31) .
E. The vaccine is shown to contain human poliovirus types
1, 2 and 3 by a suitable immunochemical method (2. 7. 1),
such as determination of D-antigen by enzyme-linked
immunosorbent assay (ELISA).
F. The PRP and its carrier protein are identified by a suitable
immunochemical method (2.7.1) .
TESTS
1f the product is presented with the haemophilus component in a
separate container, the tests for residual pertussis toxin and
irreversibility of pertussis toxoid, free formaldehyde, aluminium,
antimicrobial preservative and sterilir:y are carried out on the
container with the diphtheria, tetanus, pertussis, poliomyelitis and
hepatitis B componentSj the tests for PRP, water, antimicrobial
preservative (where applicable) , aluminium (where applicable)
and sterilir:y are carried out on the container with the haemophilus
component.
Some tests for the haemophilus component are carried out on the
freeze-dried product rather than on che bulk conjugate where the
freeze-drying process may affect the component to be tested.
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
PRP
Minimum 80 per cent of the amount of PRP stated on the
label, for a vaccine with the haemophilus component in a
separate container.
For a vaccine with all components in the same container: the
PRP content determined on the non-absorbed fraction is not
less than that approved for the producto
PRP is determined either by assay of ribose (2.5.31) or
phosphorus (2.5.18), by an immunochemical method (2.7.1)
or by anion-exchange liquid chromatography (2.2.29) with
pulsed-amperometric detection.
IV-536 Vaccines
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL of free formaldehyde per single human
dose.
AntimicrobiaI preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Water (2.5.12)
Maximum 3.0 per cent for the freeze-dried haemophilus
component.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than the minimum potency stated on the
labe!'
Unless otherwise justified and authorised, the minimum
potency stated on the labe! is 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2.7.16) .
The capacity of the vaccine to induce antibodies for each
included acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
Hepatitis B component
The vaccine complies with the assay of hepatitis B vaccine
(2. 7.15).
Poliomyelitis component
D-antigen content As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7.1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the labe!, is
within the limits approved for the particular producto
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
the International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the names and amounts of the pertussis components per
single human dose;
2014
-- the amount of HBsAg per single human dose;
-- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
-- the types of cells used for production of the poliomyelitis
and the hepatitis B components;
-- the number of micrograms of PRP per single human
dose;
-- the type and nominal amount of carrier protein per single
human dose;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the na me and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen;
-- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.
____________________________________________ nE~
Diphtheria, Tetanus, Pertussis *****
* *
(Whole Cel!), Poliomyelitis *****
(Inactivated) and Haemophilus
Type b Conjugate Vaccine
(Adsorbed)
Diphtheria, Tetanus, Pertussis, Poliomyelitis
(Inactivated) and Haemophilus Type b Conjugate
Vaccine (Adsorbed)
(Ph Eur monograph 2066)
The label may state 'DTwPIIPV/Hib' .
nE~ ___________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (whole cell), poliomye!itis
(inactivated) and haemophilus type b conjugate vaccine
(adsorbed) is a combined vaccine composed of: diphtheria
formol toxoid; tetanus formol toxoid; an inactivated
suspension of Bordetella pertussis; suitable strains of human
poliovirus types 1, 2 and 3 grown in suitable cell cultures and
inactivated by a suitable method; polyribosylribitol phosphate
(PRP) covalentIy bound to a carrier protein; a mineral
adsorbent such as aluminium hydroxide or hydrated
aluminium phosphate. The product is presented with the
haemophilus component in a separate container, the contents
of which are mixed with the other components immediately
before use.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphthenae and Clostridium
tetani respectively.
PRP is a linear copolymer composed of repeated units of
3-~-D-ribofuranosyl-(l---> 1)-ribitol-5-phosphate
[(CIOH1901ZP)n], with a defined molecular size and derived
from a suitable strain of Haemophilus infiuenzae type b.
The carrier protein, when conjugated to PRP, is capable of
inducing a T -ceIl-dependent B-ce!1 immune response to the
polysaccharide.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
 2014
During development studies and wherever revalidation is
necessary, it shall be demonstrated by tests in animals that
the vaccine induces a T -celI-dependent B-ceJI immune
response to PRP.
As pan of consistency studies the assays of the diphtheria,
tetanus, pertussis and poliomyelitis components are carried
out on a suitable number of batches of vaccine reconstituted
as for use . For subsequent routine control, the assays of these
components may be carried out without mixing with the
haemophilus component.
For the haemophilus component, the production method is
validated to demonstrate that the haemophilus component, if
tested, would comply with the test for pyrogens (2.6.8),
carried out as folIows : inject per kilogram of the rabbit's mass
a quantity of the vaccine equivalent 10: 1 ~lg of PRP for a
vaccine with diphtheria toxoid or CRM 197 diphtheria
protein as carrier; 0.1 Ilg of PRP for a vaccine with tetanus
toxoid as carrier; 0.025 Ilg of PRP for a vaccine with OMP
(meningococcal group B outer membrane protein complex)
as carrier.
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of the difference in composition
between monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown 10 be effective in
c1inical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in c1inical trials is necessary. The reference
vaccine may be stabilised by a method that has been shown
10 have no effect on the assay procedure.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated 10 demonstrate that the
product, if tested, would comply with the folIowing test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that wiII interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
diphtheria toxaemia or tetanus, the vaccine does not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine do es not comply with the test.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphthena vaccine (adsorbed)
(0443) , Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(whole cell, adsorbed) (0161), Poliomyelitis vaccine (inactivated)
(02 14) and Haemophilus type b conjugate vaccine (1219) .
FINAL BULKS
The final bulk of the diphtheria, tetanus, pertussis and
poliomyelitis components is prepared by adsorption,
separately or together, of suitable quantities of bulk purified
diphtheria toxoid, and bulk purified tetanus toxoid onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate and admixture of suitable quantities of
an inactivated suspension of B . pertussis and of purified,
monovalent harvests of human poliovirus types 1, 2 and 3 or
a suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
Vaccines IV-537
The final bulk of the haemophilus component is prepared by
dilution of the bulk conjugate 10 the final concentration with
a suitable diluent. A stabiJiser may be added.
Only final bulks that comply with the folIowing requirements
may be used in the preparation of the final lot.
Bovine serum a1bumin
Deterrnined on the poliomyelitis components by a suitable
immunochemical method (2.7.1) during preparation of the
final bulk vaccine, before addition of the adsorbent, the
amount of bovine serum albumin is such that the content in
the final vaccine wiII be not more than 50 ng per single
human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk of the haemophilus component is freeze-dried.
Only a finallot that is satisfactory with respect 10 the test for
osmolality shown below and with respect to each of the
requirements given below under Identification, Tests and
Assay may be relea sed for use.
Provided the tests for specific toxicity of the pertussis
component and antirnicrobial preservative, and the assays for
the diphtheria, tetanus and pertussis components have been
carried out with satisfactory results on the final bulk vaccine,
they may be omined on the finallot.
Provided the free forrnaldehyde content has been determined
on the bulk purified antigens, the inactivated B . pertussis
suspension and the purified monovalent harvests or the
trivalent pool of polioviruses or on the final bulk and it has
been shown that the content in the final lot wiII not exceed
0.2 gIL, the test for free formaldehyde may be omined on the
finallot.
Provided the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulk
vaccine, it may be omined on the finallot.
The in vivo assay for the poliomyelitis component may be
omined once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must include testing of
subpotent batches, produced experimentalIy if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine, reconstituted where applicable,
is within the limits approved for the particular preparation.
Free PRP
Unbound PRP is determined on the haemophilus component
after removal of the conjugate, for example by anion-
exchange, size-exclusion or hydrophobic chromatography,
ultrafiltration or other validated methods. The amount of free
PRP is not greater than that approved for the particular
product.
IV-538 Vaccines
IDENTIFICATION
Identijication tests A, B, e and D are camed out using the vial
containing the diphtheria, tetanus, pertussis and poliomyelitis
componentsj identijication test E is camed out on the vial
containing the haemophilus component.
A. Diphtheria lOxoid is identified by a suitable
irnmunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supematant liquid is
obtained. The c1ear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable irnmunochemical
method (2.7.1). The following method, applicable to certain
vaccines, is given as an example. The c1ear supematant liquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipita te.
C. The centrifugation residue obtained in identification A
may be used. Other suitable methods for separating the
bacteria from the adsorbent may also be used. Identify
pertussis vaccine by agglutination of the bacteria from the
resuspended precipitate by antisera specific 10 B. pertussis or
by the assay of the pertussis component prescribed under
Assay.
D . The vaccine is shown 10 contain human poliovirus types
1,2 and 3 by a suitable immunochemical method (2.7.1),
such as determination of D-antigen by enzyme-linked
irnmunosorbent assay (ELISA) .
E. The haemophilus component is identified by a suitable
immunochemical method (2.7.1) for PRP.
TESTS
The tests for specijic toxicity of the pertussis component,
aluminium, free formaldehyde, antimicrobial preservative and
sterility are camed out on the container with diphthen·a, tetanus,
pertussis and poliomyelitis componentsj the tests for PRP, water,
sterility and bacterial endotoxins are camed out on the container
with the haemophilus component.
Some tests for the haemophilus component may be cam·ed out on
the freeze-dried product rather than on the bulk conjugate where
the freeze-drying process may affect the component to be tested.
Specific toxicity of the pertussis component
Use not fewer than 5 healthy mice each weighing 14-16 g,
for the vaccine group and for the saline control. Use mice of
the same sex or distribute males and females equally between
the groups. AlIow the animals access to food and water for at
least 2 h before injection and during the test. Inject each
mouse of the vaccine group intraperitoneally with 0.5 mL,
containing a quantity of the vaccine equivalent to not less
than half the single human dose. Inject each mouse of the
control group with 0.5 mL of a 9 gIL sterile solution of
sodium chloride R, preferably containing the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 h and 7 days after the injection. The vaccine
complies with the test if: (a) at the end of 72 h the total mas s
of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mas s per vaccinated mouse is not less than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test.
The test may be repeated and the results of the tests
combined.
2014
PRP
Minimum 80 per cent of the amount of PRP stated on the
label. PRP is determined either by assay of ribo se (2.5.31) or
phosphorus (2.5.18), by an irnmunochemical method (2.7.1)
or by anion-exchange liquid chromatography (2.2.29) with
pulsed-amperometric detection.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is nor greater than 115 per cent of the quantity stated on the
label.
Water (2.5.12)
Maximum 3.0 per cent for the haemophilus component.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2. 6. 14)
The content is within the limits approved by the competent
authority for the haemophilus component of the particular
product. If any components of the vaccine prevent the
determination of endotoxin, a test for pyrogens is carried out
as described under General provisions.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
If the test is carried out in guinea-pigs, the lower confidence
limit (P = 0.95) of the estimated potency is not less than
40 IU per single human dose; if the test is carried out in
mice, the lower confidence limit (P = 0.95) of the estimated
potency is not less than 60 IU per single human dose.
Pertussis component
Carry out the assay of pertussis vaccine (whole cel!) (2.7.7) .
The estimated potency is not less than 4.0 IU per single
human dose and the lower confidence limit (P = 0.95) of the
estimated potency is not less than 2.0 IU per single human
dose.
Poliomyelitis component
D-antigen content As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1,2 and 3 by a suitable immunochemical
method (2.7.1) following desorption using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the limits approved for the particular product.
Poliomyelitis vaccine (inacrivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
the Intemational Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20) .
 2014
LABELLING
The label states:
-- the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose;
-- the minimum number of International Units of pertussis
vaccine per single human dose;
-- the nominal amount ofpoliovirus ofeach type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
the type of cells used for production of the poliomyelitis
component;
-- the number of micrograms of PRP per single human
dose;
-- the type and nominal amount of carrier protein per single
human dose;
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ ffiEw
Diphtheria, Tetanus, Pertussis ***
*** ***
(Whole Cell) and Poliomyelitis ***
(lnactivated) Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis and Poliomyelitis
(Inactivated) Vaccine (Adsorbed)
(Ph Eur monograph 2061)
The label may state 'DTwPIIPV'.
ffiEw ____________________________________________
DEFINITION
Diphtheria, tetanus, pertussis (whole cell) and poliomyelitis
(inactivated) vaccine (adsorbed) is a combined vaccine
containing: diphtheria formol toxoid; tetanus formol toxoid;
an inactivated suspension of Bordetella pertussis; suitable
strains of human poliovirus types 1, 2 and 3 grown in
suitable cell cultures and inactivated by a validated method;
a mineral adsorbent such as aluminium hydroxide or
hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
Reference vaccine(s) Provided valid assays can be performed,
monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
because of interaction between the components of the
combined vaccine or because of the difference in composition
between monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in clinical trials is necessary. The reference
Vaccines IV-539
vaccine may be stabilised by a method that has been shown
to have no effect on the assay procedure.
Specific toxicity of the diphtheria and tetanus
components
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or die s from
diphtheria toxaemia or tetanus, the vaccine do es not comply
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the
second test, the vaccine does not comply with the test.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the
requirements of the monographs Diphtheria vaccine (adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(whole cell, adsorbed) (0161) and Poliomyelitis vaccine
(inactivated) (0214).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid and bulk purified
tetanus toxoid and admixture of suitable quantities of an
inactivated suspension of B. pertussis and purified monovalent
harvests of human poliovirus types 1, 2 and 3 or a suitable
quantity of a trivalent pool of such purified monovalent
harvests. Suitable antimicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bovine serum albumin
Determined on the poliomyelitis components by a suitable
irnrnunochemical method (2.7.1) during preparation of the
final bulk vaccine, before addition of the adsorbent, the
amount of bovine serum albumin is such that the content in
the final vaccine will be not more than 50 ng per single
human dose.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not les s than 85 per cent and not greater than 115 per cent
of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
Only a finallot that is satisfactory with respect to the test for
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released
for use.
Provided that the tests for specific toxicity of the pertussis
component and antimicrobial preservative, and the assays for
the diphtheria, tetanus and pertussis components have been
carried out with satisfactory results on the final bulk vaccine,
they may be omitted on the finallot.
Provided that the free formaldehyde content has been
determined on the bulk purified antigens, the inactivated B.
pertussis suspension and the purified monovalent harvests or
the trivalent pool of polioviruses or on the final bulk and it
has been shown that the content in the finallot will not
exceed 0.2 gIL, the test for free formaldehyde may be
omitted on the finallot.
 IV-540 Vaccines
Provided that the in vivo assay for the poliomyelitis
component has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the finallot.
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must inc1ude testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
the manufacturing process of the antigens or their
formulation, any impact on the in v ivo and in viero assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35)
The osmolality of the vaccine is within the limits approved
for the particular preparation.
IDENfIFICAnON
A. Diphtheria toxoid is identified by a suitable
irnmunochemical method (2.7.1). The following method,
applicable ro certain vaccines, is given as an example.
Dissolve in the vaccine ro be examined sufficient sodium
cúrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge until a c1ear supernatant liquid is
obtained. The c1ear supematant liquid reacts with a suitable
diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable irnmunochemical
method (2.7.1). The following method, applicable ro certain
vaccines, is given as an example. The c1ear supernatant Iiquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipitate.
C. The centrifugation residue obtained in identification A
may be used. Other suitable methods for separating the
bacteria from the adsorbent may also be used.
Identify pertussis vaccine by agglutination of the bacteria
from the resuspended precipitate by antisera specific to
B. pertussis or by the assay of the pertussis component
prescribed under Assay.
D. The vaccine is shown to contain human poliovirus types
1,2 and 3 by a suitable immunochemical method (2.7.1)
such as the determination of D-antigen by enzyme-Iinked
irnmunosorbent assay (ELISA).
TESTS
Specific toxicity of the pertussis component
Use not fewer than 5 healthy mice each weighing 14-16 g for
the vaccine group and for the saline contro!. Use mice of the
same sex or distribute males and females equally between the
groups. Allow the animals access to food and water for at
least 2 h before injection and during the test. Inject each
mouse of the vaccine group intraperitoneally with 0.5 mL,
containing a quantity of the vaccine equivalent to not less
than half the single human dose. Inject each mouse of the
control group with 0.5 mL of a 9 gIL sterile solution of
sodium chloride R , preferably containing the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 h and 7 days after the injection. The vaccine
complies with the test if: (a) at the end of 72 h the total mass
of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mass per vaccinated mouse is not les s than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test.
2014
The test may be repeated and the results of the tests
combined.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Diphtheria component
Carry out one of the prescribed methods for the assay of
diphtheria vaccine (adsorbed) (2. 7. 6).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 30 IU per single human dose.
Tetanus component
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2. 7.8).
If the test is carried out in guinea pigs, the lower confidence
limit (P = 0.95) of the estimated potency is not less than
40 IU per single human dose; if the test is carried out in
mice, the lower confidence limit (P = 0.95) of the estimated
potency is not less than 60 IU per single human dose.
Pertussis component
Carry out the assay of pertussis vaccine (whole cel!) (2.7. 7).
The estimated potency is not les s than 4.0 IU per single
human dose and the lower confidence limit (P = 0.95) of the
estimated potency is not less than 2.0 IU per single human
dose.
Poliomyelitis component
D-antigen content As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7.1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the ¡abel, is
wirhin rhe limits approved for rhe particular product.
Poliomyelitis vaccine (inactiv ated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
rhe International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label sta tes:
- rhe minimum number of International Units of diphtheria
and tetanus toxoid per single human dos e;
- the minimum number of International Units of pertussis
vaccine per single human dose;
- the nominal amount of poliovirus of each type (1, 2 and
3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
- the type of cells used for production of rhe poliomyelitis
component;
2014
-- where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
-- the na me and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ ~Em
****
Haemophilus Type b Conjugate
*** *
Vaccine ****
(Ph Eur monograph 1219)
The labe! may state ' Hib' .
Ph Eur ____________________________________________
DEFINITlON
Haemophilus type b conjugate vaccine is a liquid or freeze-
dried preparation of a polysaccharide, derived from a suitable
strain of Haemophilus il1fiuenzae type b, covalently bound to a
carrier protein. The polysaccharide, polyribosylribitol
phosphate, referred to as PRP, is a linear copolymer
composed of repeated units of 3-~-D-ribofuranosyl-(I --> 1)-
ribitol-5-phosphate [(ClOH1901ZP)n], with a defined
molecular size. The carrier protein, when conjugated to PRP,
is capable of inducing a T-cell-dependent B-ce!l irnmune
response to the polysaccharide.
PRODUCTlON
GENERAL PROVlSIONS
The production method shall have been shown to yield
consistently haemophilus type b conjugate vaccines of
adequate safety and immunogenicity in mano The production
of PRP and of the carrier protein are based on seed-lot
systems.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9)
and also with the test for pyrogens (2.6.8), carried out as
follows: inject per kilogram of the rabbit's mass a quantity of
the vaccine equivalent to: 1 ¡.¡g of PRP for a vaccine with
diphtheria toxoid or CRM 197 diphtheria protein as carrier;
0.1 ¡.¡g of PRP for a vaccine with tetanus toxoid as carrier;
0.025 ¡.¡g ofPRP for a vaccine with OMP (meningococcal
group B outer membrane protein complex) as carrier.
During development studies and wherever revalidation of the
manufacturing process is necessary, it shall be demonstrated
by tests in animals that the vaccine consistently induces a
T-cell-dependent B-ce!l irnmune response.
The stability of the final lot and relevant intermediates is
evaluated using one or more indicator tests. Such tests may
include determination of molecular size, determination of free
PRP in the conjugate and the immunogenicity test in mice.
Taking account of the results of the stability testing, re!ease
requirements are set for these indicator tests to ensure that
the vaccine will be satisfactory at the end of the period of
validity.
BACTERIAL SEED LOTS
The seed lots of H. influenzae type b are shown to be free
from contamination by methods of suitable sensitivity. These
may include inoculation into suitable media, examination of
colony morphology, microscopic examination of
Gram-stained smears and culture agglutination with suitable
specific amisera.
Vaccines IV-541
No complex products of animal origin are included in the
medium used for preservation of strain viability, either for
freeze-drying or for frozen storage.
Ir is recommended that PRP produced by the seed lot be
characterised using nuclear magnetic resonance spectrometry
(2.2.33).
H. INFLUENZAE TYPE B POLYSACCHARlDE (PRP)
H. il1fluel1zae type b is grown in a liquid medium that does
not contain high-molecular-mass polysaccharides; if any
ingredient of the medium contains blood-group substances,
the process shall be validated to demonstrate that after the
purification step they are no longer detectable. The bacterial
purity of the culture is verified by methods of suitable
sensitivity. These may include inoculation into suitable
media, examination of colony morphology, microscopic
examination of Gram-stained smears and culture
agglutination with suitable specific antisera. The culture may
be inactivated. PRP is separated from the culture medium
and purified by a suitable method. Volatile matrer, including
water, in the purified polysaccharide is determined by a
suitable method; the result is used to calcula te the results of
certain tests with reference to the dried substance, as
prescribed below.
Only PRP that complies with the following requirements may
be used in the preparation of the conjuga te.
Identification
PRP is idemified by an irnmunochemical method (2. 7.1) or
other suitable method, for example lH nuclear magnetic
resonance spectrometry (2.2.33).
Molecular-size distribution
The percentage of PRP eluted before a given Ka value or
within a range of Ka values is determined by size-exclusion
chromatography (2.2.30); an acceptable value is established
for the particular product and each batch of PRP must be
shown to comply with this limitoLimits for currently
approved products, using the indicated stationary phases, are
shown for information in Table 1219.-1. Where applicable,
the molecular-size distribution is also determined after
chemical modification of the polysaccharide.
Liquid chromatography (2.2.29) with multiple-angle laser
light-scattering detection may also be used for determination
of molecular-size distribution.
A validated determination of the degree of polymerisation or
of the weight-average molecular weight and the dispersion of
molecular masses may be used instead of the determination
of molecular size distribution.
Ribose (2.5.31)
Within the limits approved by the competent authority for
the particular product, calculated with reference to the dried
substance.
Phosphorus (2.5.18)
Within the limits approved by the competent authority for
the particular product, calculated with reference to the dried
substance.
Protein (2.5.16)
Maximum 1.0 per cent, calculated with reference to the dried
substance. Use sufficient PRP to allow detection of proteins
at concentrations of 1 per cent or greater.
Nuc1eic acid (2.5.17)
Maximum 1.0 per cent, calculated with reference to the dried
substance.
Bacterial endotoxins (2.6.14)
Less than 10 IU per microgram ofPRP.
 IV-542 Vaccines 2014

Table 1219.-1. - Product characteristics and specifications for PRP and carrier protein in currently approved products
Carrier Haemophilus Conjugation
polysaccharide

Type Purity Nominal Type of PRP Nominal amount Coupling method Procedure
amount per per dose
dose

Diphtheria toxoid > 1500 Lf per 181.lg Size·reduced PRP 25 I.lg cyanogen bromide activated diphtheria
milligram of Ko: 0.6·0.7, using activatian oi PRP loxoid (D·AW),
nitrogen cross-linked cyanogen bromide·
agarose for activated PRP
chromatography R

Tetanus toxoid > 1500 Lf per 20 I.lg PRP" 50 % 10 iJg carbodiimide ADH·activated
milligram of ,; Ko: 0.30, using mediated PRP (PRP-cov.·AH)
nitrogen cross·linked + tetanus toxoid
agarose for + EDAC
chromatography R

CRM 197 diphtheria > 90 % oi diphtheria 25 iJg Size·reduced PRP 10 iJg reductive amination direct coupling of
protein protein Dp = 15·35 or 10-35 (l·step method) or PRP to CRM 197
Nhydroxysuccinimide (cyanoborohydride
activation activated)

Meningococcal outer membrane 125 iJg or 250 I.lg Size·reduced PRP 7.5 I.lg or 15 I.lg thioether bond PRP activation by
group B outer protein Ko < 0.6, using CDI PRp·IM + BuA2
membrane protein vesicles: ~ 8 % of cross-linked + BrAc = PRp·BuA2·
(OMP) lipopolysaccharide agarose for BrAc + thioactivated
chromatography R OMP
or Mw> 50 X lO'

ADH = adipic acid dihydrazide Dp = degree of polymerisation

BrAc = bromoacetyl chloride EDAC = l·ethyl·3·(3·dimethylaminopropyl)carbodiimide
BuA2 = butane·l,4·diamide 1M = imidazolium
CDI = carbonyldiimidazole Mw= weight·average molecular weight

Residual reagents
Where applicable, tests are carried out to determine residues
of reagents used during inactivation and purification.
An acceptable value for each reagent is established for the
particular product and each batch of PRP must be shown to
comply with this limit. Where validation studies have
demonstrated removal of a residual reagent, the test on PRP
may be omitted.
CARRIER PROTEIN
The carrier protein is chosen so that when the PRP is
conjugated it is able to induce a T-cell-dependent B-cell
immune response. Currently approved carrier proteins and
coupling methods are listed for information in Table 1219.-1.
The carrier proteins are produced by culture of suitable
micro-organisms; the bacterial purity of the culture is
verified; the culture may be inactivated; the carrier protein is
purified by a suitable method.
Only a carrier protein that complies with the following
requirements may be used in the preparation of the
conjugate.
Identification
The carrier protein is identified by a suitable
immunochemical method (2. 7.1).
Diphtheria toxoid
Diphtheria toxoid is produced as described in the monograph
Diphtheria vaccine (adsorbed) (0443) and complies with the
requirements prescribed therein for bulk purified toxoid
except that the test for sterility (2.6.1) is not required.
Tetanus toxoid
Tetanus toxoid is produced as described in the monograph
Tetanus vaccine (adsorbed) (0452) and complies with the
requirements prescribed therein for bulk purified toxoid,
except that the antigenic purity is not less than
1500 Lf per milligram of protein nitrogen and that the test
for sterility (2.6.1) is not required.
Diphtheria protein CRM 197
Minimum 90 per cent, determined by a suitable method.
Suitable tests are carried out, for validation or routinely, to
demonstrate that the product is non-toxico
OMP (meningococcal group B outer membrane
protein complex)
OMP complies with the following requirements for
lipopolysaccharide and pyrogens.
Lipopolysaccharide Maximum 8 per cent of
lipopolysaccharide, determined by a suitable method.
Pyrogens (2.6.8) Inject into each rabbit 0.25 f.lg of OMP per
kilogram of body mass.
BULK CONJUGATE
PRP is chemicaIly modified to enable conjugation; it is
usuaIly partly depolymerised either before or during this
procedure. Reactive functional groups or spacers may be
introduced into the carrier protein or PRP prior to
conjugation. As a measure of consistency, the extent of
derivatisation is monitored. The conjugate is obtained by the
covalent binding of PRP and carrier protein. Where
applicable, unreacted but potentially reactogenic functional
groups are made unreactive by means of capping agents;
the conjugate is purified to remove reagents.
Only a bulk conjugate that complies with the following
requirements may be used in the preparation of the final bulk
vaccine. For each test and for each particular product, limits
of acceptance are established and each batch of conjugate
must be shown to comply with these limits. Limits applied to
currently approved products for sorne of these tests are listed
for information in Table 1219.-2. For a freeze-dried vaccine,
sorne of the tests may be carried out on the finallot rather
 2014
Table 1219.-2. - Bulk conjugate requirements for currently approved products
Test
Diphtheria toxoid
Free PRP < 37 %
Free protein <4% < 1 %, where applicable
PRP to protein ratio 1.25 - 1.8
Molecular size (Kol:
cross-linked agarose for 95 % < 0.75
chromatography R
cross-linked agarose for 0.6 - 0.7
chromatoQraphlJ Rl
than on the bulk conjugate where the freeze-drying process
may affect the component being tested .
PRP
The PRP content is determined by assay of phosphorus
(2.5.18) or by assay of ribose (2.5.31) or by an
irnrnunochemical method (2.7.1).
Protein
The protein content is determined by a suitable chemical
method (for example, 2.5.16).
PRP to protein ratio
Determine the ratio by calculation.
Molecular-size distribution
Molecular-size distribution is determined by size-exc1usion
chromatography (2.2.30).
Free PRP
A number of methods have been used to separate free PRP
from the conjugate, inc1uding precipitation, gel filtration,
size-exc1usion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation.
The free PRP can then be quantified by a range of
techniques, inc1uding high-performance anion-exchange
chromatography with pul sed amperometric detection
(HPAEC-PAD) and irnrnunoassays with anti-PRP
antibodies.
Free carrier protein
Determine the content by a suitable method, either directly
or by deriving the content by calculation from the results of
other tests. The amount is within the limits approved for the
particular product.
Unreacted functional groups
No unreacted functional groups are detectable in the bulk
conjugate unless process validation has shown that unreacted
functional groups detectable at this stage are removed during
the subsequent manufacturing process (for example, owing to
short half-life) .
Residual reagents
Removal of residual reagents such as cyanide,
EDAC (ethyldimethylaminopropylcarbodiimide) and phenol
is confirmed by suitable tests or by validation of the process.
Sterility (2.6.1)
Carry out the test using for each medium 10 mL or the
equivalent of 100 doses, whichever is less.
FINAL BVLK VACCINE
An adjuvant, an antimicrobial preservative and a stabiliser
may be added to the bulk conjugate before dilution to the
final concentration with a suitable diluent.
Only a final bulk vaccine that complies with the following
requirements may be used in preparation of the finallot.
Vaccines IV-543
Protein carrier
Tetanus toxoid CRM 197 OMP
< 20 % < 25 % < 15 %
< 1 % or < 2 %, depending
not applicable
on the couolin!1 method
0.30·0.55 0.3·0.7 0.05·0.1
60 % < 0.2 50 % 0.3·0.6 85 % < 0.3
85 % < 0.5
Antirnicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
FINALLOT
Only a finallot that is satisfactory with respect to each of the
following requirements and the requirements given below
under Identification and Tests may be relea sed for use.
Provided the test for antimicrobial preservative has been
carried out on the final bulk vaccine, it may be omitted on
the finallot.
pH (2.2.3)
The pH of the vaccine, reconstituted if necessary, is within
the range approved for the particular product.
Free PRP
A number of methods have been used to separate free PRP
from the conjuga te, inc1uding precipitation, gel filtration,
size-exc1usion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation.
The free PRP can then be quantified by a range of
techniques, inc1uding HPAEC-PAD and irnrnunoassays with
anti-PRP antibodies. The amount of free PRP is not greater
than that approved for the particular product.
IDENTIFICATION
The vaccine is identified by a suitable immunochemical
method (2.7.1) for PRP.
TESTS
PRP
Minimum 80 per cent of the amount of PRP stated on the
labe!' PRP is determined either by assay of ribose (2.5.31) or
phosphorus (2.5.18), by an immunochemical method (2.7.1)
or by anion-exchange liquid chromatography with pulsed
amperometric detection (2.2.29).
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not less than the minimum amount
shown to be effective and not greater than 115 per cent of
the quantity stated on the ¡abe!'
Water (2.5.12)
Maximum 3.0 per cent for freeze-dried vaccines.
IV-544 Vaccines
Sterility (2.6.1
It complies with the test for sterility.
Bacteria! endotoxins (2.6.14)
The content is within the limits approved by the competent
authority for the particular product. If any components of the
vaccine prevent the determination of endotoxin, a test for
pyrogens is carried out as described under General
provisions.
LABELLING
The label states:
-- the number of micrograms of PRP per human dose;
-- the type and nominal amount of carrier protein per single
human dose.
____________________________________________ ~E~
Haemophilus Type b and Meningococcal
Group C Conjugate Vaccine
The label may state 'Hib/MenC'.
DEFINITlON
Haemophilus Type b and Meningococcal Group C
Conjugate Vaccine is a combined vaccine. It is prepared from
purified polysaccharides derived from a suitable strain of
Neissena meningitidis group C and from a suitable strain of
Haemophilus influenzae, type b covalently bound to a carrier
protein.
The product is presented as a combined lyophilisate of the
haemophilus type b and meningococcal group C components
together with the solvent in a separate container. The final
product is prepared by mixing the contents of the two
containers irnmediately before use.
PRODUCTlON
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines, Appendix XIV E.
The stability of the finallot and relevant intermediates is
evaluated using one or more indicator tests. Such tests may
include determination of molecular size, determination of free
saccharides in the conjugate or an immunogenicity test in
animals. Taking account of the results of the stability testing,
release requirements are set for these indicator tests to ensure
that the vaccine will be satisfactory at the end of the period
of validity.
During development studies and wherever revalidation of the
manufacturing process is necessary, it shall be demonstrated
by tests in animals that the vaccine consistently induces a
T -cell-dependent B-cell irnrnune responses to PRP and to
meningococcal group C polysaccharides.
PRODUCTION OF THE COMPONENTS
The production of the vaccine components complies with the
requirements of the monographs for Haemophilus Type b
Conjugate Vaccine and Meningococcal Group C Conjugate
Vaccine.
FINAL BULK VACCINE
A final bulk may be prepared by mixing suitable quantities of
the haemophilus and meningococcal C bulk conjuga tes and
dilution to the final concentration with a suitable diluent.
2014
Different methods of preparation may be used. A suitable
adjuvant, a suitable antimicrobial preservative and a stabiliser
may be added to the components bulk conjugates.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chernical
method. The content is not less than the minimum amount
shown to be effective and not greater than 115 % of the
intended amount.
Sterility
Carry out the test for sterility, Appendix XVI A, using 10 mL
for each medium.
FINALLOT
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification and Tests may
be released for use. Provided the test for antimicrobial
preservative has been carried out on the final bulk vaccine, it
may be omitted on the final lot.
Acidity or aIkalinity
pH of the reconstituted vaccine is within the range approved
by the competent authority for the particular product,
Appendix V L.
Free PRP
Unbound PRP is determined after removal of the conjugate,
for example by anion-exchange, size-exclusion or
hydrophobic chromatography, ultrafiltration or other
validated methods. The amount of free PRP is not greater
than that approved by the competent authority for the
particular product.
Free meningococcal C saccharide
Unbound saccharide is determined after removal of the
conjuga te, for example by anion-exchange liquid
chromatography, size-exclusion or hydrophobic
chromatography, ultrafiltration or other validated methods.
The amount of free meningococcal C saccharide is not
greater than that approved for the particular product.
The vaccine complies with the requirements stated under Vaccines
and with the following requirements.
IDENTIFICATlON
The haemophilus and menigococcal components are
identified by a suitable immunochemical method,
Appendix XIV B.
TESTS
PRP
Not less than 80% of the amount of PRP stated on the labe!'
PRP is determined either by assay of ribose or phosphorus,
Appendix XV G, by an irnrnunochemical method,
Appendix XIV B, or by anion-exchange liquid
chromatography, Appendix III D, with pulsed-amperometric
detection.
~eningococcalsaccharide
Not les s than 80% of the amount of meningococcal group C
polysaccharide stated on the labe!' The saccharide content is
determined by a suitable validated assay, for example sialic
acid assay, Appendix XV G or anion-exchange liquid
chromatography, Appendix III D, with pulsed-amperometric
detection.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not less than the minimum amount
2014
shown to be effective and not greater than 115% of the
quantity stated on the labe!'
Bacterial endotoxins
Carry out the test for bacten"al endotoxins, Appendix XVI C.
Less than 25 ID per single human dose.
Sterility
Complies with the test for sterility, Appendix XVI A.
LABELLING
The labe! states per human dose: (1) the number of
micrograms of PRP; (2) the number of micrograms of
meningococcal group C polysaccharide; (3) the type and
nominal amount of carrier protein.
Inactivated Hepatitis A Vaccine
(Hepatitis A Vaccine (Inactivated, Adsorbed),
Ph Eur monograph 1107)
The label may state 'HepA'.
~Ew ____________________________________________
DEFINITION
Hepatitis A vaccine (inactivated, adsorbed) is a suspension
consisting of a suitable strain of hepatitis A virus grown in
cell cultures, inactivated by a validated method and adsorbed
on a mineral carrier.
PRODUCTION
GENERAL PROVISIONS
Production of the vaccine is based on a virus seed-lot system
and a cell-bank system. The production method shall have
been shown to consistently yield vaccines that comply with
the requirements for immunogenicity, safety and stability.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
Dnless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
the master seed lot than were used to prepare the vaccine
shown in clinical studies to be satisfactoty with respect to
safety and efficacy.
Reference prepararíon A part of a batch shown to be at least
as immunogenic in animals as a batch that, in clinical studies
in young healthy adults, produced not less than 95 per cent
seroconversion, corresponding to a level of neutralising
antibody accepted to be protective, afier a full-course primary
immunisation is used as a reference preparation. An antibody
level of 20 mIU/mL determined by enzyme-linked
immunosorbent assay is recognised as being protective.
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a human diploid cellline (5.2.3)
or in a continuous cell line approved by the competent
authority.
SEED LOTS
The strain of hepatitis A virus used to prepare the master
seed lot shall be identified by historical records that include
information on the origin of the strain and its subsequent
manipulation.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
Each master and working seed lot is identified as hepatitis A
virus using specific antibodies.
Vaccines IV-545
Virus concentration
The virus concentration of each master and working seed lot
is determined to monitor consistency of production.
Extraneous agents
The working seed lot complies with the requirements for
seed lots for virus vaccines (2.6.16). In addition, if primaty
monkey cells have been used for isolation of the strain,
measures are taken to ensure that the strain is not
contaminated with simian virus es such as simian
immunodeficiency virus and filoviruses.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are being handled. Animal serum (but not human serum)
may be used in the cell culture media. Serum and trypsin
used in the preparation of cell suspensions and media are
shown to be free from extraneous agents. The cell culture
media may contain a pH indicator, such as phenol red, and
antibiotics at the lowest effective concentration. Not less than
500 mL of the cell cultures employed for vaccine production
is set aside as uninfected cell cultures (control cells). Multiple
harvests from the same production cell culture may be
pooled and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the vaccine.
When the determination of the ratio of virus concentration to
antigen content has been carried out on a suitable number of
single harvests to demonstrate production consistency, it may
subsequently be omitted as a routine test.
Identification
The test for antigen content also serves to identify the single
harvest.
Bacterial and fungal contamination
The single harvest complies with the test for sterility (2.6.1),
carried out using 10 mL for each medium.
Mycoplasmas (2.6. 7)
The single harvest complies with the test for mycoplasmas,
carried out using 1 mL for each medium.
Control cells
The control cells of the production cell culture comply with a
test for identification and the requirements for extraneous
agents (2.6.16) .
Antigen content
Determine the hepatitis A antigen content by a suitable
immunochemical method (2.7.1) to monitor production
consistency; the content is within the limits approved for the
particular producto
Ratio of virus concentration to antigen content
The consistency of the ratio of the concentration of infectious
virus, determined by a suitable cell culture method, to
antigen content is established by validation on a suitable
number of single harvests.
PURIFICATION AND PURlFIED HARVEST
The harvest, which may be a pool of several single harvests,
is purified by validated methods. If continuous celllines are
used for production, the purification process shall have been
shown to reduce consistently the level of host-cell DNA.
Only a purified harvest that complies with the following
requirements may be used in the preparation of the
inactivated harvest.
Virus concentration
The concentration of infectious virus in the purified harvest
is determined by a suitable cell culture method to monitor
 IV-546 Vaccines
production consistency and as a starting point for monitoring
the inactivation curve.
Antigen:total protein ratio
Determine the hepatitis A virus antigen content by a suitable
immunochemical method (2.7.1). Determine the total protein
by a validated method. The ratio of hepatitis A virus antigen
content to total protein content is within the limits approved
for the particular producto
Bovine serum albumin
Not more than 50 ng in the equivalent of a single human
dose, determined by a suitable immunochemical method
(2.7.1). Where appropriate in view of the manufacturing
process, other suitable protein markers may be used to
demonstrate effective purification.
Residual host-cell DNA
If a continuous cellline is used for virus propagation, the
content of residual host-cell DNA, determined using a
suitable method, is not greater than 100 pg in the equivalent
of a single human dose.
Residual chemicals
If chemical substances are used during the purification
process, tests for these substances are carried out on the
purified harvest (or on the inactivated harvest), unless
validation of the process has demonstrated total clearance.
The concentration must not exceed the limits approved for
the particular producto
INACTIVATION AND INACTIVATED HARVEST
Several purified harvests may be pooled before inactivation.
In order to avoid interference with the inactivation process,
virus aggregation must be prevented or aggregates must be
removed irnmediately before and/or during the inactivation
process. The virus suspension is inactivated by a validated
method; the method shall have been shown to be consistently
capable of inactivating hepatitis A virus without destroying
the antigenic and immunogenic activity; for each inactivation
procedure, an inactivation curve is plotted representing
residual live virus concentration measured at not fewer than
3 points in time (for example, on days O, 1 and 2 of the
inactivation process). If formaldehyde is used for inactivation,
the presence of excess free formaldehyde is verified at the
end of the inactivation process.
Only an inactivated harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Inactivation
Carry out an amplification test for residual infectious
hepatitis A virus by inoculating a quantity of the inactivated
harvest equivalent to 5 per cent of the batch or, if the harvest
contains the equivalent of 30 000 doses or more, not les s
than 1500 doses of vaccine into cell cultures of the same type
as those used for production of the vaccine; incubate for a
total of not les s than 70 days making not fewer than one
passage of cells within that periodo At the end of the
incubation period, carry out a test of suitable sensitivity for
residual infectious virus. No evidence of hepatitis A virus
multiplication is found in the samples taken at the end of the
inactivation process. Use infectious virus inocula concurrently
as positive controls to demonstrate cellular susceptibility and
absence of interference.
Sterility (2.6.1)
The inactivated viral harvest complies with the test for
sterility, carried out using 10 mL for each medium.
Bacterial endotoxins (2.6.14)
Less than 2 IU in the equivalent of a single human dose.
2014
Antigen content
Determine the hepatitis A virus antigen content by a suitable
irnmunochemical method (2.7.1).
Residual chemicals
See under Purification and purified harvest.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
inactivated harvests. Approved adjuvants, stabilisers and
antimicrobial preservatives may be added.
OnIy a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile
containers. The containers are then closed so as to avoid
contamination.
OnIy a finallot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the tests for free formaldehyde
(where applicable) and antimicrobial preservative content
(where applicable) have been carried out on the final bulk
vaccine with satisfactory results, these tests may be omitted
on the finallot. If the assay is carried out using mice or other
animals, then provided it has been carried out with
satisfactory results on the final bulk vaccine, it may be
omitted on the finallot.
IDENTIFICATION
The vaccine is shown to contain hepatitis A virus antigen by
a suitable immunochemical method (2.7.1) using specific
antibodies or by the in vivo assay (2.7.14).
TESTS
Aluminium (2.5.13)
Maximum 1.2 5 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
The vaccine complies with the assay of hepatitis A vaccine
(2.7.14).
LABELLING
The label sta tes the biological origin of the cells used for the
preparation of the vaccine.
____________________________________________ ~E~
2014
*****
Hepatitis A Vaccine (Inactivated,
**
Virosome) ***
(Ph Eur monograph 1935)
The label may state 'HepA'.
ffiE~ ____________________________________________
DEFINITION
Hepatitis A vaccine (inactivated, virosome) is a suspension of
a suitable strain of hepatitis A virus grown in cell cultures
and inactivated by a validated method. Viro so mes composed
of influenza proteins of a strain approved for the particular
product and phospholipids are used as adjuvants.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
Reference preparation A reference preparation of inactivated
hepatitis A antigen is calibrated against a batch of hepatitis A
vaccine (inactivated, virosome) that, in clinical studies in
young healthy adults, produced not less than 95 per cent
seroconversion, corresponding to a leve! of neutralising
antibody accepted to be protective, after a full-course primary
immunisation. An antibody level not less than 20 mIU/mL
determined by enzyme-linked immunosorbent assay is
recognised as being protective.
PREPARATION OF HEPATITIS A ANTIGEN
Production of the hepatitis A antigen is based on a virus
seed-Iot system and a cell-bank system. The production
method shall have been shown to consistently yield vaccines
that comply with the requirements for immunogenicity, safety
and stability.
Unless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
the master seed lot than were used to prepare the vaccine
shown in clinical studies to be satisfactory with respect to
safety and efficacy.
SUBSTRATE FOR PROPAGATION OF HEPATITIS A VIRUS
The virus is propagated in a human diploid cellline (5.2.3) .
SEED LOTS OF HEPATITIS A VIRUS
The strain of hepatitis A virus used to prepare the master
seed lot shall be identified by historical records that include
information on the origin of the strain and its subsequent
manipulation.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
Each master and working seed lot is identified as hepatitis A
virus using specific antibodies.
Virus concentration
The virus concentration of each master and working seed lot
is determined to monitor consistency of production.
Extraneous agents
The working seed lot complies with the requirements for
seed lots for virus vaccines (2.6.16).
PROPAGATION AND HARVEST OF HEPATITIS A VIRUS
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cel!s
are handled. Animal serum (but not human serum) may be
Vaccines IV-547
used in the cell culture media. Serum and trypsin used in the
**
preparation of cel! suspensions and media are shown to be
free from extraneous agents. The cel! culture media may
contain a pH indicator such as phenol red and antibiotics at
the lowest effective concentration. Not les s than 500 mL of
the cell cultures employed for vaccine production is set aside
as uninfected cell cultures (control cells) . Multiple harvests
from the same production cell culture may be pooled and
considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the vaccine.
When the determination of the ratio of virus concentration to
antigen content has been carried out on a suitable number of
single harvests to demonstrate consistency, it may
subsequently be omitted as a routine test.
Identification
The test for antigen content also serves to identify the single
harvest.
Bacterial and fungal contamination
The single harvest complies with the test for sterility (2.6.1),
carried out using 10 rnL for each medium.
Mycoplasmas (2.6.7)
The single harvest complies with the test for mycoplasmas.
Control cells
The control cells of the production cel! culture comply with a
test for identity and the requirements for extraneous agents
(2.6.16).
Antigen content
Determine the hepatitis A antigen content by a suitable
immunochemical method (2.7.1) to monitor production
consistency; the content is within the limits approved for the
particular producto
Ratio of virus concentration to antigen content
The consistency of the ratio of the concentration of infectious
virus, as determined by a suitable cell culture method, to
antigen content is established by validation on a suitable
number of single harvests.
PURIFICATION AND PURIFIED HARVEST OF HEPATITIS A
VIRUS
The harvest, which may be a pool of several single harvests,
is purified by validated methods. If continuous cell lines are
used for production, the purification process shall have been
shown to reduce consistently the level of host-cel! DNA.
Only a purified harvest that complies with the following
requirements may be used in the preparation of the
inactivated harvest.
Virus concentration
The concentration of infective virus in the purified harvest is
determined by a suitable cell culture method to monitor
production consistency and as a starting point for monitoring
the inactivation curve.
Ratio of antigen to total protein
Determine the hepatitis A virus antigen content by a suitable
immunochemical method (2.7.1). Determine the total protein
by a validated method. The ratio of hepatitis A virus antigen
content to total protein content is within the limits approved
for the particular product.
Bovine serum albumin
Maximum 50 ng per single human dose if foetal bovine
serum is used, determined by a suitable immunochemical
method (2.7.1). Where appropriate in view of the
manufacturing process, other suitable protein markers may
be used to demonstrate effective purification.
IV-548 Vaccines
Residual chemicals
If chemical substances are used during the purification
process, tests for these substances are carried out on the
purified harvest (or on the inactivated harvest), unless
validation of the process has demonstrated total clearance.
The concentration must not exceed the limits approved for
the particular product.
INACTIVATION AND INACTIVATED HARVEST OF
HEPATITIS A VIRUS
Several purified harvests may be pooled before inactivation.
In order to avoid interference with the inactivation process,
virus aggregation must be prevented or aggregates must be
removed immediately before and/or during the inactivation
process. The virus suspension is inactivated by a validated
method; the method shall have been shown to be consistently
capable of inactivating hepatitis A virus without destroying
the antigenic and immunogenic activity; for each inactivation
procedure, an inactivation curve is plotted representing
residuallive virus concentration measured on at least 3
occasions (for example, on days O, 1 and 2 of the inactivation
process). If formaldehyde is used for inactivation, the
presence of excess free formaldehyde is verified at the end of
the inactivation process.
Only an inactivated harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Inactivation
Carry out an amplification test for residual infectious
hepatitis A virus by inoculating a quantity of the inactivated
harvest equivalent to 5 per cent of the batch or, if the harvest
contains the equivalent of 30 000 doses or more, not less
than 1500 doses of vaccine into cell cultures of the same type
as those used for production of the vaccine; incubate for a
total of not less than 70 days making not fewer than 1
passage of cells within that periodo At the end of the
incubation period, carry out a test of suitable sensitivity for
residual infectious virus. No evidence of hepatitis A virus
multiplication is found in the samples taken at the end of the
inactivation process. Use infective virus inocula concurrently
as positive controls to demonstrate cellular susceptibility and
absence of interference.
Sterility (2.6.1)
The inactivated viral harvest complies with the test for
sterility, carried out using 10 mL for each medium.
Bacterial endotoxins (2.6.14)
Less than 2 IU of endotoxin in the equivalent of a single
human dose.
Antigen content
Determine the hepatitis A virus antigen content by a suitable
immunochemical method (2.7.1).
Residual chemica!s
See under Purification and purified harvest.
PREPARATION OF INACTIVATED INFLUENZA VIRUS
The production of influenza virus es is based on a seed-Iot
system. Working seed lots represent not more than 15
passages from the approved reassorted virus or the approved
virus isolate. The final production represents 1 passage from
the working seed lot. The strain of influenza virus to be used
is approved by the competent authority.
SUBSTRATE FOR PROPAGATION OF INFLUENZA VIRUS
Influenza virus seed to be used in the production of vaccine
is propagated in fertilised eggs from chicken flocks free from
specified pathogens (5.2.2) or in suitable cell cultures (5.2.4),
such as chick-embryo fibroblasts or chick kidney cells
2014
obtained from chicken flocks free from specified pathogens
(5.2.2) . For production, the virus is grown in the allantoic
cavity of fertilised hens' eggs from healthy flocks.
SEED LOTS OF INFLUENZA VIRUS
The haemagglutinin and neuraminidase antigens of each seed
lot are identified as originating from the correct strain of
influenza virus by suitable methods.
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the
monovalent pooled harvest.
Bacteria! and fungal contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
PROPAGATION AND HARVEST OF INFLUENZA VIRUS
An antimicrobial agent may be added ro the inoculum. After
incubation at a controlled temperature, the allantoic fluids
are harvested and combined to form the monovalent pooled
harvest. An antimicrobial agent may be added at the time of
harvest. At no stage in the production is penicillin or
streptomycin used.
POOLED HARVEST OF INFLUENZA VIRUS
To lirnit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
3 consecutive batches ro be consistently effective for the
manufacturero The inactivation process shall have been
shown ro be capable of inactivating the influenza virus
without destroying antigenicity of haemagglutinin.
The inactivation process shall also have been shown ro be
capable of inactivating avian leucosis viruses and
mycoplasmas. If the monovalent po oled harvest is stored
after inactivation, it is held at a temperature of 5 ± 3 oc.
If formaldehyde solution is used, the concentration does not
exceed 0.2 giL of CH2 0 at any time during inactivation;
if betapropiolactone is used, rhe concentration does not
exceed 0.1 per cent V/Vat any time during inactivation.
Only a pooled harvest that complies with the following
requirements may be used in the preparation of the
virosomes.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it. Carry out the test at
20-25 oc.
Sterility (2.6. 1)
Carry out the test for sterility, using 10 mL for each
medium.
Vira! inactivation
Inoculate 0.2 mL of the harvest into the allantoic cavity of
each of 10 fertilised eggs and incubate at 33-37 oC for
3 days. The test is not valid unless at least 8 of the 10
embryos survive. Harvest 0.5 mL of the allantoic fluid from
each surviving embryo and pool the fluids. Inoculate 0.2 mL
of the pooled fluid into a further 10 fertilised eggs and
incubate at 33-37 oC for 3 days. The test is nor valid unless
at least 8 ofthe 10 embryos survive. Harvest about 0.1 mL
of the allantoic fluid from each surviving embryo and
examine each individual harvest by a haemagglutination test.
If haemagglutination is found for any of the fluids, carry out
for that fluid a further passage in eggs and test for
haemagglutination; no haemagglutination occurs .
2014
Ovalbumin
Maximum 1 ¡.¡g of ovalbumin in the equivalent of 1 human
dos e, determined by a suitable technique using a suitable
reference preparation of ovalbumin.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Residual chemicals
Tests are carried out on the monovalent pooled harvest for
the chemicals used for inactivation, the limits being approved
by the competent authority.
PREPARATION OF VIROSOMES
Inactivated influenza virions are solubilised using a suitable
detergent and are purified by high-speed centrifugation in
order to obtain supernatants containing mainly influenza
antigens. After the addition of suitable phospholipids,
virosomes are formed by removal of the detergent either by
adsorption chromatography or another suitable technique.
Only virosomes that comply with the following requirements
may be used in the preparation of the final bulk vaccine.
Haemagglutinin content
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7. 1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it.
Phospholipids
The content and identity of the phospholipids are determined
by suitable irnmunochemical or physico-chemical methods.
Ratio of phospholipid to haemagglutinin
The ratio of phospholipid content to haemagglutinin content
is within the limits approved for the particular producto
Residual chemicals
Tests are carried out for the chemicals used during the
process. The concentration of each residual chemical is
within the limits approved for the particular producto
FINAL BULK VACCINE
The bulk vaccine is prepared by adding virosomes to
inactivated hepatitis A viruses to yield an approved hepatitis
A antigen:haemagglutinin ratio. Several bulks may be pooled,
and approved stabilisers and antimicrobial preservatives may
be added.
OnIy a final bulk vaccine that complies with the folIowing
requirements may be used in the preparation of the finallot.
Protein content
The amount of protein is determined using a suitable
technique, the limits being approved by the competent
authorithy.
Phospholipids
The content and identity of the phospholipids are determined
by suitable immunochemical or physico-chemical methods.
The amount of phospholipids complies with the limits
approved for the particular producto
Haemagglutinin content
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1). The amount of haemagglutinin
must not exceed the limits approved for the particular
product.
Hepatitis A antigen content
Determine the hepatitis A antigen content by a suitable
immunochemical method. The amount of antigen must not
exceed the limits approved for the particular product.
Vaccines IV-549
Ratio of hepatitis A antigen to haemagglutinin
The ratio of hepatitis A antigen content to haemagglutinin
content is within the limits approved for the particular
product.
Ovalbumin
Maximum 1 ¡.¡g of ovalbumin per human dose, determined
by a suitable technique using a suitable reference preparation
of ovalbumin.
Virosome size
The size distribution of the virosome-hepatitis A virus
mixture is within the limits approved for the particular
product.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Residual chemicals
If chemical substances are used during the formulation
process, tests for these substances are carried out, the limits
being approved by the competent authorithy.
FINALLOT
The final bulk vaccine is distributed asepticalIy into sterile
containers. The containers are then closed so as to avoid
contamination.
OnIy a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the tests for free formaldehyde
(where applicable) and antimicrobial preservative content
(where applicable) have been carried out on the final bulk
vaccine with satisfactory results, these tests may be omitted
on the final loto If the assay is carried out in vivo, provided it
has been carried out with satisfactory results on the final bulk
vaccine, it may be omitted on the finallot.
IDENfIFICATION
The vaccine is shown to contain hepatitis A virus antigen by
a suitable immunochemical method (2.7.1) using specific
antibodies.
TESTS
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!.
Sterility (2.6. 1)
The vaccine complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than 2 IU of endotoxin per human dose.
ASSAY
Determine the antigen content of the vaccine using a suitable
irnmunochemical method (2.7. 1) by comparison with the
reference preparation. The acceptance criteria are approved
for a given reference preparation by the competent authority.
LABELLING
The label states:
IV-550 Vaccines
-- the biological origin of the cells used for the preparation
of the vaccine,
-- that the carrier contains influenza proteins prepared in
eggs,
-- that the vaccine is not to be frozen,
-- that the vaccine is to be shaken before use.
_ _ _ _ _ __ _ __ _ _ __ _ __ ______
~Ew
Hepatitis A (Inactivated, Adsorbed)
and Typhoid Polysaccharide
Vaccine
(Ph Eur monograph 2597)
The label may state 'HepA!fyphoid'
~Ew _ __ __ __ _ _ __ _ __ _ _ _ __ _ ___
DEFINITION
Hepatitis A (inactivated, adsorbed) and typhoid
polysaccharide vaccine is a suspension consisting of a suitable
strain of hepatitis A virus, grown in cell cultures and
inactivated by a validated method, and of purified Vi capsular
polysaccharide obtained from Salmonella typhi Ty 2 strain or
sorne other suitable strain that has the capacity to produce Vi
polysaccharide.
The hepatitis A antigen is adsorbed on a mineral carrier,
such as aluminium hydroxide, and the Vi capsular
polysaccharide consists of partiy 3-0-acetylated repeated
units of 2-acetylamino-2-deoxY-D-galactopyranuronic acid
with ex-(l -.4) linkages.
The product is presented either as a liquid mixture
containing the hepatitis A component and the typhoid Vi
polysaccharide component or as 2 separate liquids, one
containing the hepatitis A component and the other the
typhoid Vi polysaccharide component, which are mixed
together immediately before use.
PRODUCnON
GENERAL PROVISIONS
The 2 components are prepared as described in the
monographs Hepatitis A vaccine (inactivated, adsorbed) (1107)
and Typhoid polysaccharide vaccine (1160) and comply with
the requirements prescribed therein.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
Reference preparation The hepatitis A reference preparation
is part of a representative batch shown to be at least as
irnmunogenic in animals as a batch that, in clinical studies in
young healthy adults, produced not less than 95 per cent
seroconversion, corresponding to a level of neutralising
antibody accepted to be protective, after a full-course primary
irnmunisation. An antibody level not les s than 20 mIU/mL
determined by enzyme-linked immunosorbent assay is
recognised as being protective.
FINALBULKS
The hepatitis A final bulk is prepared from 1 or more
inactivated harvests of hepatitis A virus. Approved adjuvants,
stabilisers and antimicrobial preservatives may be added.
The Vi polysaccharide final bulk is prepared from 1 or more
batches of purified Vi polysaccharide which are dissolved in a
suitable solvent, which may contain an antimicrobial
preservative, so that the volume corresponding to 1 dose
2014
contains 25 J..lg of polysaccharide and the solution is isotonic
with blood (250-350 mosmollkg) .
Where the vaccine is presented as a liquid mixture of both
components, the final bulk is prepared by addition of a
suitable quantity of the Vi capsular polysaccharide bulk to
the hepatitis A bulk.
0nly final bulks that comply with the following requirements
may be used in the preparation of the finallot.
Antitnicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Sterility (2.6. 1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulks are distributed aseptically into sterile
containers. The containers are then closed so as to avoid
contamination.
0nly a finallot that complies with each of the requirements
given below under Identification, Tests and Assay may be
relea sed for use. Provided that the tests for free formaldehyde
(where applicable), antimicrobial preservative (where
applicable) and bacterial endotoxins have been carried out on
the final bulks with satisfactory results, they may be omitted
on the final lot. If the assay of the hepatitis A component is
carried out in vivo, then provided it has been carried out with
satisfactory results on the final bulk containing the hepatitis
A component, it may be omitted on the final loto
CHARACTERS
lf the vaccine is presented as 2 separate liquids test A is cam·ed out
using the hepatitis A component and test B is carried out using the
typhoid Vi polysaccharide component. Test e is carried out if the
vaccine is presented as a liquid mixture of both components or
immediately after mixing both components if the vaccine is
presented as 2 separate liquids.
A. Whitish, cloudy suspension.
B. Clear, colourless liquid, free from visible particles.
C. Turbid liquid with a slow settling white deposito
IDENTIFICAnON
lf the vaccine is presented as 2 separate liquids, identification test
A is carried out using the hepatitis A component and identification
test B is cam·ed out using the typhoid Vi polysaccharide
component. lf the vaccine is presented as a liquid mixture, tests A
and B are carried out.
A. Hepatitis A virus antigen is identified by a suitable
immunochemical method (2.7.1) using specific antibodies or
by the in vivo assay (2.7.14).
B. Typhoid Vi polysaccharide is identified by a suitable
immunochemical method (2.7.1) using specific antibodies.
TESTS
lf the vaccine is presented as 2 separate liquids, the tests for pH,
antimicrobial preservative and bacteria! endotoxins are carried out
on both componentsj the test for aluminium is cam·ed out using the
hepatitis A component and the test for O-acetyl groups is carried
out using the typhoid Vi polysaccharide componentj the tests for
pH, free formaldehyde, osmolality and sterility are cam·ed out
immediately after mixing both components. lf the vaccine is
presented as a liquid mixture, the test for O-acetyl groups is carried
out before the 2 components are mixed.
2014
pH (2.2.3)
6.8 to 7.8 for the hepatitis A component and 6.5 to 7.5 for
the typhoid Vi polysaccharide component; 6.6 to 7.6 for the
vaccine presented as a liquid mixture or immediately after
mixing both components if the vaccine is presented as
2 separate liquids.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide is used as the adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
the amount stated on the labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
Osmolality (2.2.35)
Where applicable, the osmolality of the vaccine is within the
limits approved for the particular producto
Bacterial endotoxins (2.6.14)
The bacterial endotoxins content is less than 2 IU per
human dose for the hepatitis A component and within the
limit approved for the typhoid Vi polysaccharide component.
If the vaccine is presented as a liquid mixture of hepatitis A
component and typhoid Vi polysaccharide component the
bacterial endotoxins content is within the limit approved for
the specific producto
O-Acetyl groups (2.5.19)
0.085 flmol (± 25 per cent) per dose (25 flg of
polysaccharide) .
ASSAY
Hepatitis A component
The vaccine complies with the assay of hepatitis A vaccine
(2.7.14).
Typhoid Vi polysaccharide component
Determine Vi polysaccharide by a suitable immunochemical
method (2.7.1), using a reference purified polysaccharide.
The estimated amount of polysaccharide per dose is
80 per cent to 120 per cent of the content stated on the
labe!' The confidence limits (P = 0.95) of the estimated
amount of polysaccharide are not less than 80 per cent and
not more than 120 per cent.
LABELLING
The label states:
-- the amount of hepatitis A virus antigen per human dose;
-- the number of micrograms of polysaccharide per human
dose (25 flg);
-- the total quantity of polysaccharide in the container;
-- the type of cells used for production of the vaccine;
-- the name and amount of the adsorbent used;
-- that the vaccine must be shaken before use;
-- that the vaccine must not be frozen.
____________________________________________ ~Ew
Vaccines IV-551
*****
Hepatitis A (Inactivated) and
** **
Hepatitis 8 (rDNA) Vaccine ***
(Hepatitis A (Inactivated) and Hepatitis B (rDNA)
Vaccine (Adsorbed), Ph Eur monograph 1526)
The label may state 'HepAIHepB'.
PhEw ____________________________________________
DEFINITION
Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine
(adsorbed) is a suspension consisting of a suitable strain of
hepatitis A virus, grown in cell cultures and inactivated by a
validated method, and of hepatitis B surface antigen
(HBsAg), a component protein of hepatitis B virus obtained
by recombinant DNA technology; the antigens are adsorbed
on a mineral carrier, such as aluminium hydroxide or
hydrated aluminium phosphate.
PRODUCTION
GENERAL PROVISIONS
The two components are prepared as described in the
monographs on Hepatitis A vaccine (inactivated, adsorbed)
(1107) and Hepatitis B vaccine (rDNA) (1056) and comply
with the requirements prescribed therein.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2. 6.9) .
Reference preparation The reference preparation is part of a
representative batch shown to be at least as immunogenic in
animals as a batch that, in clinical studies in young healthy
adults, produced not les s than 95 per cent seroconversion,
corresponding to a level of neutralising antibody recognised
to be protective, after a full-course primary immunisation.
For hepatitis A, an antibody level not less than 20 mIU/mL
determined by enzyme-linked immunosorbent assay is
recognised as being protective. For hepatitis B, an antibody
leve! not less than 10 mIU/mL against HBsAg is recognised
as being protective.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
inactivated harvests of hepatitis A virus and one or more
batches of purified antigen.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot .
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
FINALLOT
Only a finallot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the tests for free formaldehyde
(where applicable) and antimicrobial preservative content
(where applicable) have been carried out on the final bulk
vaccine with satisfactory results, they may be omitted on the
finallot. If the assay of the hepatitis A and/or the hepatitis B
component is carried out in vivo, then provided it has been
carried out with satisfactory results on the final bulk vaccine,
it may be omitted on the final loto
IV-552 Vaccines
IDENTIFICATION
The vaccine is shown to contain hepatitis A virus antigen and
hepatitis B surface antigen by suitable irnmunochemical
methods (2. 7.1), using specific antibodies or by the mouse
irnmunogenicity tests described under Assay.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, dete=ine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!'
Sterility (2. 6.1)
The vaccine complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than 2 IV per human dose.
ASSAY
Hepatitis A component
The vaccine complies with the assay of hepatitis A vaccine
(2.7.14) .
Hepatitis B component
The vaccine complies with the assay of hepatitis B vaccine
(rDNA) (2.7.15) .
LABELLING
The label sta tes:
- the amount of hepatitis A virus antigen and hepatitis B
surface antigen per container,
- the type of cells used for production of the vaccine,
- the name and amount of the adsorbent used,
- that the vaccine must be shaken before use,
- that the vaccine must not be frozen .
_ _ _ __ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ Ph Eur
Hepatitis B Vaccine (rDNA)
(Ph Eur monograph 1056)
The labe! may state 'HepB' .
~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ ___
DEFINITION
Hepatitis B vaccine (rDNA) is a preparation of hepatitis B
surface antigen (HBsAg), a component protein of hepatitis B
virus; the antigen may be adsorbed on a mineral carrier such
as aluminium hydroxide or hydrated aluminium phosphate.
The vaccine may also contain the adjuvant 3-0-desacyl-4 '-
monophosphoryllipid A. The antigen is obtained by
recombinant DNA technology.
PRODVCTION
GENERAL PROVISIONS
The vaccine shall have been shown to induce specific,
protective antibodies in mano The production method shall
have been shown to yield consistently vaccines that comply
with the requirements for immunogenicity and safety.
2014
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abno=al
toxicity for immunosera and vaccines for human use (2.6.9).
Hepatitis B vaccine (rDNA) is produced by the expression of
the viral gene coding for HBsAg in yeast (Saccharomyces
cerevisiae) or mammalian cells (Chinese hamster ovary
(CHO) cells or other suitable celllines), purification of the
resulting HBsAg and the rendering of this antigen into an
immunogenic preparation. The suitability and safety of the
cells are approved by the competent authority.
The vaccine may contain the product of the S gene (major
protein), a combination of the S gene and pre-S2 gene
products (middle protein) or a combination of the S gene,
the pre-S2 gene and pre-S1 gene products (large protein) .
Reference preparation Part of a representative batch shown to
be at least as immunogenic in animal s as a batch that, in
dinical studies in young, healthy adults, produced not less
than 95 per cent seroconversion, corresponding to a level of
HBsAg neutralising antibody recognised to be protective,
after a full-course primary immunisation. An antibody leve!
not less than 10 mIU/mL is recognised as being protective.
CHARACTERISATION OF THE SUBSTANCE
Development studies are carried out to characterise the
antigen. The complete protein, lipid and carbohydrate
structure of the antigen is established. The morphological
characteristics of the antigen partides are established by
electron microscopy. The mean buoyant density of the
antigen partides is determined by a physico-chemical
method, such as gradient centrifugation. The antigenic
epitopes are characterised. The protein fraction of the antigen
is characterised in terms of the primary srructure (for
example, by dete=ination of the amino-acid composition, by
partial amino-acid sequence analysis and by peptide
mapping).
CULTURE AND HARVEST
Identity, microbial purity, plasmid retention and consistency
of yield are dete=ined at suitable production stages.
If mammalian cells are used, tests for extraneous agents and
mycoplasmas are performed in accordance with general
chapter 2.6.16. Tests for extraneous agents in viral vaccines for
human use, but using 200 mL of harvest in the test in cell
culture for other extraneous agents.
PURlFIED ANTIGEN
Only a purified antigen that complies with the following
requirements may be used in the preparation of the final bulk
vaccme.
Total protein
The total protein is determined by a validated method.
The content is within the limits approved for the specific
producto
Antigen content and identification
The quantity and specificity of HBsAg is determined in
comparison with the International Standard for HBsAg
subtype ad or an in-house reference, by a suitable
immunochemical method (2. 7.1) such as radio-irnmunoassay
(RIA), enzyme-linked immunosorbent assay (ELISA),
immunoblot (preferably using a monodonal antibody
directed against a protective epitope) or single radial
diffusion. The antigen/protein ratio is within the limits
approved for the specific product.
The molecular weight of the major band revealed following
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) performed under reducing conditions
 2014
corresponds to the value expected from the known nuc1eic
acid and polypeptide sequences and possible glycosylation.
Antigenic purity
The purity of the antigen is determined by comparison with
a reference preparation using liquid chromatography or other
suitable methods such as SDS-PAGE with staining by acid
blue 92 and silver. A suitable method is sensitive enough to
detect a potential contaminant at a concentration of
1 per cent of total protein. Not less than 95 per cent of the
total protein consists of hepatitis B surface antigen.
Composition
The content of proteins, lipids, nuc1eic acids and
carbohydrates is determined.
Host-celI- and vector-derived DNA
If mammalian cells are used for production, not more than
10 pg of DNA in the quantity of purified antigen equivalent
to a single human dose of vaccine.
Caesium
If a caesium salt is used during production, a test for residual
caesium is carried out on the purified antigen. The content is
within the limits approved for the specific product.
Sterility (2.6.1)
The purified antigen complies with the test, carried out using
10 mL for each medium.
Additional tests on the purified antigen may be required
depending on the production method used: for example, a
test for residual animal serum where mammalian cells are
used for production or tests for residual chemicals used
during extraction and purification.
ADSORBED 3-0-DESACYL-4'-MONOPHOSPHORYL LIPID A
BULK
If 3-0-desacyl-4'-monophosphoryl lipid A is inc1uded in the
vaccine it complies with the monograph 3-0-desacyl-4'-
monophosphoryllipid A (2537). Where 3-0-desacyl-4 '-
monophosphoryl lipid A liquid bulk is adsorbed prior to
inc1usion in the vaccine, the adsorbed 3-0-desacyl-4'-
monophosphoryl lipid A bulk complies with the following
requirements.
Degree ofadsorption of3-0-desacyI-4 '-
monophosphoryllipid A
The content of non-adsorbed 3-0-desacyl-4'-
monophosphoryl lipid A in the adsorbed 3-0-desacyl-4'-
monophosphoryllipid A bulk is determined by a suitable
method, for example gas chromatographic quantification of
the 3-0-desacyl-4'-monophosphoryllipid A (2537) fatty acids in
the supernatant, evaporated to dryness, after centrifugation.
pH (2.2.3)
The pH is within the limits approved for the particular
preparation.
Sterility (2.6.1)
It complies with the test, carried out using 10 mL for each
medium.
FINAL BULK VACCINE
An antimicrobial preservative, a mineral carrier, such as
aluminium hydroxide or hydrated aluminium phosphate, and
the adjuvant 3-0-desacyl-4'-monophosphoryllipid A may be
inc1uded in the formulation of the final bulk.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
AntimicrobiaI preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
Vaccines IV-553
method . The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Sterility (2.6.1)
The final bulk vaccine complies with the test, carried out
using 10 mL for each mediurn.
FINALLOT
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the tests for free formaldehyde
(where applicable) and antimicrobial preservative content
(where applicable) have been carried out on the final bulk
vaccine with satisfactory results, they may be omitted on the
final lot. If the assay is carried out in vivo, then provided it
has been carried out with satisfactory results on the final bulk
vaccine, it may be omitted on the final lot.
Degree of adsorption
The degree of adsorption of the antigen and, where
applicable, 3-0-desacyl-4'-monophosphoryllipid A is
assessed.
IDENTIFICAnON
The assay or, where applicable, the electrophoretic profile,
serves also to identify the vaccine. In addition, where
applicable, the test for 3-0-desacyl-4'-monophosphoryl
lipid A content also serves to identify the
3-0-desacyl-4'-monophosphoryl lipid A-containing vaccine.
TESTS
ALUMINIUM (2.5. 13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
3-0-DesacyI-4'-monophosphoryllipid A
Minimum 80 per cent and maximum 120 per cent of the
intended amount.
Where applicable, determine the content of
3-0-desacyl-4 '-monophosphoryllipid A by a suitable
method, for example gas chromatography (2.2.28).
FREE FORMALDEHYDE (2.4.18)
Maximum 0.2 gIL.
ANTIMICROBIAL PRESERVATIVE
Where applicable, determine the content of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not les s than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!'
Sterility (2.6.1)
The vaccine complies with the test.
PYROGENS (2.6.8)
The vaccine complies with the test for pyrogens . Inject the
equivalent of one human dose into each rabbit or, if the
vaccine contains 3-0-desacyl-4' -monophosphoryl lipid A,
inject per kilogram of the rabbit's mass an amount of the
vaccine containing 2.5 ¡.¡g of 3-0-desacyl-4' -monophosphoryl
lipid A.
ASSAY
The vaccine complies with the assay of hepatitis B vaccine
(rDNA) (2.7.15).
LABELLING
The label states:
- the amount of HBsAg per container;
- the type of cells used for production of the vaccine;
- the na me and amount of the adjuvant and/or adsorbent
used;
IV-554 Vaccines
- that the vaccine must be shaken before use;
- that the vaccine must not be frozen.
_ _________________________________________ ~E~
Inactivated Influenza Vaccine ***
***
(Whole Virion) ***
(Influenza Vaccine (W'hole Virion, Inactivated),
Ph Eur monograph 0159)
The label may state 'Flu'.
When Inactivated Influenza Vaccine or Influenza Vaccine is
prescribed or demanded and the form is not stated,
Inactivated Influenza Vaccine (Whole Virion), Inactivated
Influenza Vaccine (Split Virion) or Inactivated Influenza
Vaccine (Surface Antigen) may be dispensed or supplied.
Ph Eur ___________________________________________
DEFINITION
Influenza vaccine (whole virion, inactivated) is a sterile,
aqueous suspension of a strain or strains of influenza virus,
type A or B, or a mixture of strains of the 2 types grown
individually in fertilised hens' eggs and inactivated in such a
manner that their antigenic properties are retained.
The stated amount of haemagglutinin antigen for each strain
present in the vaccine is 15 Ilg per dose, unless clinical
evidence supports the use of a different amount.
The vaccine is a slightly opalescent liquid.
PRODUCTION
The production method is validated 10 demonstrate that the
product, if tested, would comply with the test for abnormal
10xicity for irnmunosera and vaccines for human use (2.6.9).
CHOICE OF VACCINE STRAIN
The World Health Organisation reviews the world
epidemiological situation a=ually and if necessary
recommends the strains that correspond to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signatory States of the Convention on the
Elaboration of a European Pharmacopoeia. It is now
common practice 10 use reassorted strains giving high yields
of the appropriate surface antigens. The origin and passage
history of virus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus seed to be used in the production of vaccine
is propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
(5.2.4), such as chick-embryo fibroblasts or chick kidney cells
obtained from SPF chicken flocks (5.2.2). For production,
the virus of each strain is grown in the allantoic cavity of
fertilised hens' eggs from healthy flocks.
VIRUS SEED LOT
The production of vaccine is based on a seed-Iot system.
Working seed lots represent not more than 15 passages from
the approved reassorted virus or the approved virus iso late.
The final vaccine represents 1 passage from the working seed
lot. The haemagglutinin and neuraminidase antigens of each
seed lot are identified as originating from the correct strain of
influenza virus by suitable methods.
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the
monovalent pooled harvest.
2014
Bacteria! and fungal contaminarlon
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
VIRUS PROPAGATION AND HARVEST
An antimicrobial agent may be added to the inoculum. After
**
*
incubation at a controlled temperature, the allantoic fluids
are harvested and combined to form a monovalent pooled
harvest. An antimicrobial agent may be added at the time of
harvest. At no stage in the production is penicillin or
strep10mycin used.
MONOVALENT POOLED HARVEST
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
3 consecutive batches to be consistently effective for the
manufacturero The inactivation process shall have been
shown to be capable of inactivating the influenza virus
without destroying its antigenicity; the process should cause
minimum alteration of the haemagglutinin and
neuraminidase antigens. The inactivation process shall also
have been shown to be capable of inactivating avian leucosis
viruses and mycoplasmas. If the monovalent pooled harvest is
stored after inactivation, it is held at 5 ± 3 oC.
If formaldehyde solution is used, the concentration do es not
exceed 0.2 giL of CHzO at any time during inactivation;
if betapropiolactone is used, the concentration do es not
exceed 0.1 per cent V/Vat any time during inactivation.
Before or after the inactivation process, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or other suitable method.
Only a monovalent pooled harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it 1. Carry out the test
at 20-25 oC.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent pooled harvests from each working
seed lot.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
Residual infecrlous virus
Carry out the test described below under Tests.
FINAL BULK VACCINE
Appropriate quantities of the monovalent pooled harvests are
blended to make the final bulk vaccine.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lo!.
Antimicrobial preservative
Where applicable, determine the amount of antirnicrobial
preservative by a suitable chemical method. The content is
1 Reference haemagglutinin antigens are available from the National Insritute
for Bi%gieal Standards and Control, Blanche Lane, South Mimms, Potrers
Bar, Herifordshire, EN6 3QG, Great Britain
 2014
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a finallot that is satisfactory with respect to each of the
requirements given below under Tests and Assay may be
relea sed for use. Provided that the test for residual infectious
virus has been performed with satisfactory results on each
monovalent pooled harvest and that the tests for free
formaldehyde, ovalbumin and total protein have been
performed with satisfactory results on the final bulk vaccine,
they may be omitted on the final loto
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccine.
TESTS
Residual infectious virus. Inoculate 0.2 mL of the vaccine
into the allantoic cavity of each of 10 fertilised eggs and
incubate at 33-37 oC for 3 days. The test is not valid unless
at least 8 of the 10 embryos survive. Harvest 0.5 mL of the
alIantoic fluid from each surviving embryo and pool the
fluids . Inoculate 0.2 mL of the pooled fluid into a further 10
fertilised eggs and incubate at 33-37 oC for 3 days. The test
is not valid unless at least 8 of the 10 embryos survive.
Harvest about 0.1 mL of the al!antoic fluid from each
surviving embryo and examine each individual harvest for live
virus by a haemagglutination test. If haemagglutination is
found for any of the fluids, carry out for that fluid a further
passage in eggs and test for haemagglutination;
no haemagglutination occurs.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Ovalbumin
Not more than the quantity stated on the label and in any
case not more than 1 ¡¡g per human dose, determined by a
suitable immunochemical method (2.7.1) using a suitable
reference preparation of ovalbumin.
T ota! protein
Not more than 6 times the total haemagglutinin content of
the vaccine as determined in the assay, but in any case, not
more than 100 ~tg of protein per virus' strain per human dos e
and not more than a total of 300 ~tg of protein per human
dose.
Sterility (2. 6. 1)
It complies with the test for sterility.
Bacteria! endotoxins (2.6.14)
Less than 100 IU per human dose.
ASSAY
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
Vaccines IV-555
antigen preparation calibrated against it 1. Carry out the test
at 20-25 oC. The confidence limits (P = 0.95) are not less
than 80 per cent and not more than 125 per cent of the
estimated haemagglutinin antigen contentoThe lower
confidence limit (P = 0.95) is not less than 80 per cent of
the amount stated on the label for each strain.
LABELLING
The label states:
- that the vaccine has been prepared on eggs,
- the strain or strains of influenza virus used to prepare the
vaccine,
- the method of inactivation,
- the haemagglutinin content in micrograms per virus strain
per dose,
- the maximum amount of ovalbumin,
- the season during which the vaccine is intended to
protect.
__________________________________________ ~Ew
Influenza Vaccine (Whole Virion, ***
*** ***
Inactivated, Prepared in Ce" ***
Cultures)
(Ph Eur monograph 2308)
The label may state 'Flu' or 'Flu(adj)' as appropriate.
__________________________________________ ~Ew
DEFINITION
Influenza vaccine (whole virion, inactivated, prepared in cel!
cultures) is a sterile, aqueous suspension of a strain or strains
of influenza virus, type A or B, or a mixture of strains of the
2 types grown individually in cell cultures and inactivated in
such a manner that their antigenic properties are retained.
The stated amount of haemagglutinin antigen for each strain
present in the vaccine is 15 ¡¡g per dose, unless clinical
evidence supports the use of a different amount. The vaccine
is a slightly opalescent or opalescent liquido The vaccine may
contain an adjuvant. This monograph applies to vaccines
produced in diploid or continuous cell lines of mammalian
origino
PRODUCTION
GENERAL PROVISIONS
Production of the vaccine is based on a virus seed-lot system
and a cell-bank system. The production method shall have
been shown to yield consistently vaccines that comply with
the requirements for irnmunogenicity, safety and stability.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
The production method is validated to demonstrate suitable
reduction of residual host-cell protein. With the agreement of
the competent authority and for each specific product,
routine testing for residual host-cel! proteins may be omitted
based on the results of validation studies for the product.
Guidance on the principies of such validation studies is
given, for example, in the monograph Products of recombinant
DNA technology (0784), in particular in the sections
'Validation of the production process - Extraction and
1 Reference haemagglwinin antigens are available jrom ¡he Nacional Institute
for Biological Standards and Control, Blanche Lane, South Mimms, Porters
Bar, Hertfordshire, EN6 3QG, Great Britain
 IV-556 Vaccines
purification' and 'Production consistency - Host-cell-derived
proteins '.
CHOICE OF VACCINE STRAIN
The World Health Organisation reviews the world
epidemiological situation annually and if necessary
recommends new strains corresponding to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signa10ry sta tes of the Convention on the
Elaboration of a European Pharmacopoeia. It is now
common practice to use reassorted strains giving high yields
of the appropriate surface antigens . The origin and passage
his10ry of virus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus used in the preparation of seed lots is
propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cen cultures
(5.2.3), such as chick-embryo fibroblasts, chick kidney cens
obtained from SPF chicken flocks (5.2.2), or a diploid or
continuous cen lineo The final passage for establishment of
the working seed lot is prepared in the cen line used for
routine production. For this production, the virus of each
strain is propagated in a diploid or continuous cen line
(5.2.3) .
VIRUS SEED LOT
The production of vaccine is based on a seed-Iot system.
Each of the strains of influenza virus used shan be identified
by historical records that inelude information on the origin of
the strain and its subsequent manipulation. Working seed
lots represent not more than 15 passages from the approved
reassorted virus or the approved virus isolate. The final
vaccine represents 1 passage from the working seed lot.
Only a seed lot that complies with the fonowing requirements
may be used for virus propagation.
Identification
The haemagglutinin and neuraminidase antigens of each
master and working seed lot are identified as originating from
the correct strain of influenza virus by suitable methods.
Virus concentration
The virus concentration of each working seed lot is
determined. Where applicable, the virus concentration of
each master seed lot is determined.
Extraneous agents (2.6.16)
The working seed lots comply with the requirements for seed
lots. It is recognised that due to a seasonal change in one or
more of the influenza vaccine strains, timely testing of a virus
seed for extraneous agents according to general chapter
2.6.16 may be problematic (e.g. duration of in m·vD tests,
tirnely availability of specific neutralising antisera).
In agreement with the competent authority, and in light of a
risk assessment, rapid assays (e.g. multiplex PCR) may be
applied as alternatives 10 general chapter 2.6.16 fonowing
validation.
Such risk assessment and validation ineludes more general
considerations on potential contaminants of the virus isolates,
the susceptibility of the cen substrate to such virus es and the
capacity of the production process for viral removal or
inactivation; validation ineludes also comparative data on
testing of seeds according to general chapter 2.6.16 and the
proposed rapid assays . Each applied PCRlNAT test (2.6.21)
must be shown to be suitable for its intended use by
appropriate analytical validation. The risk assessment is
reviewed when new information becomes available on
2014
potential viral contaminants, and the justification of the
chosen PCR panel of extraneous agents tested for is provided
to the competent authority within the annual update. This
update also ineludes vaccine strain-specific aspects such as
specific PCR inhibitory effects.
If an agent is detected in a virus seed and the mammalian
cens used for production are shown to be susceptible to this
agent, the virus seed is not used for vaccine production.
If an agent is detected in a virus seed and the marnmalian
cens are not susceptible to the agent, validation of the
production process 10 demonstrate removal or inactivation of
the agent is carried out. If removal or inactivation cannot be
demonstrated, the inactivated monovalent harvest is tested to
demonstrate absence of any contaminant identified in the
virus seed.
PROPAGATION AND SINGLE HARVEST
AH processing of the cen bank and subsequent cen cultures is
done under aseptic conditions in an area where no other cens
are being handled at the same time. Approved animal serum
(but not human serum) may be used in the cen culture
media. Serum and trypsin used in the preparation of cell
suspensions or media are shown to be free from extraneous
agents. The cen culture media may contain a pH indicator,
such as phenol red, and antibiotics at the lowest effective
concentration. A sufficient quantity of the cen cultures
employed for vaccine production are set aside as uninfected
cen cultures (control cens).
Only a single harvest that complies with the fonowing
requirements may be used in the preparation of the vaccine.
Identification
The test for antigen content also serves to identify the single
harvest.
Bacteria! and fungal contarnination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL for each
medium.
Control ceIls
The control cens of the production cen culture comply with a
test for identification and the requirements for extraneous
agents (2.6.16) .
Haemagglutinin antigen
Determine the haemagglutinin antigen content by a suitable
immunochemical method (2. 7.1) .
INACTIVATED AND PURIFIED MONOVALENT HARVEST
The harvest, which may be a pool of several single harvests
of the same strain, is inactivated and purified by validated
methods. Before or after the inactivation process, the
monovalent harvest is concentrated and purified by
high-speed centrifugation or another suitable method.
The influenza virus is inactivated by a method that has been
demonstrated on 3 consecutive batches to be consistently
effective for the manufacturer. The inactivation process shan
have been shown to be capable of inactivating the influenza
virus without destroying its antigenicity; the process is
designed so as 10 cause minimum alteration of the
haemagglutinin and neuraminidase antigens .
If continuous cen lines are used for production, the
purification process shan have been validated to reduce
consistently host-cen DNA to a suitable leve!.
 2014 Vaccines IV-557

Only an inactivated, purified monovalent harvest that TESTS

complies with the following requirements may be used in the Residual infectious virus. Carry out an amplification test for
preparation of the final bulk vaccine. residual infectious influenza virus by inoculating not less than
Haemagglutinin antigen 4 mL of the vaccine into cell cultures of the same type as
Determine the haemagglutinin antigen content by a suitable used for production of the vaccine; incubate for not less than
immunochemical method (2.7.1). 7 days at 32 ± 2 oc. Inoculate not less than 10 mL of the
cell culture harvested medium into a new semi-confluent cell
Antigenltotal protein ratio
culture and incubate as before. At the end of the incubation
Determine the haemagglutinin antigen content by a suitable
period, examine for live virus by a haemagglutination test.
immunodiffusion test. Determine the total protein by a
If haemagglutination is found for any of the fluids, carry out
validated method. The ratio of haemagglutinin antigen
for that fluid a further passage on cell cultures and test for
content to total protein content is within the limits approved
haemagglutination; no haemagglutination occurs.
for the particular product.
Antimicrobial preservative
Neuraminidase antigen
Where applicable, determine the amount of antimicrobial
The presence and type of neuraminidase antigen are
preservative by a suitable chemical method. The content is
confirmed by suitable enzymatic or immunological methods
not les s than the minimum amount shown to be effective and
on the first 3 monovalent harvests from each working seed
is not greater than 115 per cent of the quantity stated on the
lot.
labe!'
Sterility (2.6.1)
Free formaldehyde (2.4.18)
Carry out the test for sterility, using 10 mL for each
Maximum 0.2 gIL, where applicable.
medium.
Bovine serum albumin
Residual infectious virus
Maximum 50 ng per human dose, determined by a suitable
Carry out the test described below under Tests.
immunochemical method (2.7.1).
FINAL BULK VACCINE
Total protein
Appropriate quantities of the inactivated, purified monovalent
Not more than 6 times the total haemagglutinin content of
pooled harvests are blended to make the final bulk vaccine.
the vaccine as determined in the assay, but in any case, not
An adjuvant may be added.
more than 100 ¡.¡g of protein per virus strain per human dose.
Only a final bulk vaccine that complies with the following
Sterility (2.6.1)
requirements may be used in the preparation of the finallot.
It complies with the test for sterility.
Antimicrobial preservative
Bacterial endotoxins (2.6. 14)
Where applicable, determine the amount of antimicrobial
Less than 25 IU per human dose.
preservative by a suitable chemical method. The content is
not less than 85 per cent and not greater than 115 per cent ASSAY
of the intended amount. Determine the content of haemagglutinin antigen by an
Sterility (2.6.1) immunodiffusion test (2.7.1), by comparison with a
Carry out the test for sterility, using 10 mL for each haemagglutinin antigen reference preparation l or with an
medium. antigen preparation calibrated against it. Carry out the test at
20-25 oc. The confidence limits (P = 0.95) are not less than
Residual host-cell DNA
80 per cent and not more than 125 per cent of the estimated
If a continuous cell line is used for virus propagation, the
contento The lower confidence limit (P = 0.95) is not les s
content of residual host-cell DNA, determined using a
than 80 per cent of the amount stated on the label for each
suitable method, is not greater than 10 ng in the equivalent
strain.
of a single human dose.
FINALLOT
LABELLING
The final bulk vaccine is distributed aseptically into sterile, The label states:
tamper-proof containers. The containers are closed so as to - the biological origin of the cells used for the preparation
prevent contamination. of the vaccine;
- the strain or strains of influenza virus used to prepare the
Only a final lot that is satisfactory with respect to each of the
vaccine;
requirements given below under Tests and Assay may be
- the method of inactivation;
released for use. Provided that the test for residual infectious
- the haemagglutinin antigen content in micrograms per
virus has been performed with satisfactory results on each
virus strain per dose;
inactivated and purified monovalent harvest and that the tests
- the season during which the vaccine is intended to
for free formaldehyde, bovine serum albumin and total
protect;
protein have been performed with satisfactory results on the
- where applicable, the name and the quantity of adjuvant
final bulk vaccine, they may be omitted on the finallot.
used.
If the vaccine contains an adjuvant, suitable tests for identity __________________________________________ ~E~

and other relevant quality criteria are carried out on the final
lot. These tests may include chemical and physical analysis,
determination of particle size and determination of the
number of particles per unit volume.
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccine. 1 Reference haemagglutimi¡ antigens are avat1able from the Nacional Inscitute
for Biological Standards and Control, Blanche Lane, South Mimms, Potters
Bar, Hertfordshire, EN6 3QG, Great Britain
 IV-558 Vaccines
*****
Inactivated Influenza Vaccine
* *
(Split Virion) *****
(Influenz a Vaccine (Split Virion, Inactivated),
Ph Eur monograph 0158)
The label may state 'Flu'.
When Inactivated Influenza Vaccine or Influenza Vaccine is
prescribed or demanded and the form is not stated,
Inactivated Influenza Vaccine (Whole Virion), Inactivated
Influenza Vaccine (Split Virion) or Inactivated Influenza
Vaccine (Surface Antigen) may be dispensed or supplied.
~E~ ____________________________________________
DEFINITION
Influenza vaccine (split virion, inactivated) is a sterile,
aqueous suspension of a strain or strains of influenza virus,
type A or B, or a mixture of strains of the 2 types grown
individually in fertilised hens' eggs, inactivated and treated so
that the integrity of the virus particles has been disrupted
without diminishing the antigenic properties of the
haemagglutinin and neuraminidase antigens. The stated
amount of haemagglutinin antigen for each strain present in
the vaccine is 15 Jlg per dose, unless clinical evidence
supports the use of a different amount.
The vaccine is a slightly opalescent liquido
PRODUCTION
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
CHOICE OF VACCINE STRAIN
The World Health Organisation reviews the world
epidemiological situation annually and if necessary
recommends the strains that correspond to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signatory States of the Convention on the
Elaboration of a European Pharmacopoeia. It is now
common practice to use reassorted strains giving high yields
of the appropriate surface antigens. The origin and passage
history of virus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus seed to be used in the production of vaccine
is propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
(5.2.4), such as chick-embryo fibroblasts or chick kidney cells
obtained from SPF chicken flocks (5.2.2). For production,
the virus of each strain is grown in the allantoic cavity of
fertilised hens' eggs from healthy flocks.
VIRUS SEED LOT
The production of vaccine is based on a seed-Iot system.
Working seed lots represent not more than 15 passages from
the approved reassorted virus or the approved virus isolate.
The final vaccine represents 1 passage from the working seed
lot. The haemagglutinin and neurarninidase antigens of each
seed lot are identified as originating from the correct strain of
influenza virus by suitable methods.
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the
monovalent pooled harvest.
Bacterial and funga1 contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
2014
Mycop1asmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
VIRUS PROPAGATlON AND HARVEST
An antimicrobial agent may be added to the inoculum. After
incubation at a controlled temperature, the allantoic fluid s
are harvested and combined to form a monovalent pooled
harvest. An antimicrobial agent may be added at the time of
harvest. At no stage in the production is penicillin or
streptomycin used.
MONOVALENT POOLED HARVEST
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
3 consecutive batches to be consistently effective for the
manufacturero The inactivation process shall have been
shown to be capable of inactivating the influenza virus
without destroying its antigenicity; the process should cause
minimum alteration of the haemagglutinin and
neuraminidase antigens. The inactivation process shall also
have been shown to be capable of inactivating avian leucosis
viruses and mycoplasmas. If the monovalent pooled harvest is
stored after inactivation, it is held at 5 ± 3 oc.
If formaldehyde solution is used, the concentration does not
exceed 0.2 giL of CH2 0 at any time during inactivation;
if betapropiolactone is used, the concentration does not
exceed 0.1 per cent V/Vat any time during inactivation.
Before or after the inactivation procedure, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or other suitable method and the virus
particles are disrupted into component subunits by the use of
approved procedures. For each new strain, a validation test is
carried out to show that the monovalent bulk consists
predominantly of disrupted virus particles.
Only a monovalent pooled harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2. 7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it l . Carry out the test
at 20-25 oc.
For sorne vaccines, the physical form of the haemagglutinin
particles prevents quantitative determination by
immunodiffusion after inactivation of the virus. For these
vaccines, a determination of haemagglutinin antigen is made
on the monovalent pooled harvest before inactivation.
The production process is validated to demonstrate suitable
conservation of haemagglutinin antigen and a suitable tracer
is used for formulation, for example, protein contento
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent pooled harvests from each working
seed lot.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
Residual infectious virus
Carry out the test described below under Tests.
1 Reference haemaggluunin amigens are available from che Nacional Institute
for Biological Standards and Control, Blanche Lane, SOUlh M imms, Pocters
Bar, Hertfordshire, EN6 3QG, Great Britain
2014
Chemicals used for disruption
Tests are carried out on the monovalent pooled harvest for
the chemicals used for disruption, the limits being approved
by the competent authority.
FINAL BULK VACCINE
Appropriate quantities of the monovalent pooled harvests are
blended to make the final bulk vaccine.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2. 6.1)
Carry out the test for sterility, using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are c10sed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Tests and Assay may be
released for use. Provided that the test for residual infectious
virus has been performed with satisfactory results on each
monovalent pooled harvest and that the tests for free
formaldehyde, ovalbumin and total protein have been
performed with satisfactory results on the final bulk vaccine,
they may be omined on the final lot.
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccme.
TESTS
Residual infectious virus
Inoculate 0.2 mL of the vaccine into the allantoic cavity of
each of 10 fertilised eggs and incubate at 33-37 oC for
3 days. The test is not valid unless at least 8 of the 10
embryos survive. Harvest 0.5 mL of the allantoic fluid from
each surviving embryo and pool the fluids. Inoculate 0.2 mL
of the pooled fluid into a further 10 fertilised eggs and
incubate at 33-37 oC for 3 days. The test is not valid unless
at least 8 ofthe 10 embryos survive. Harvest about 0.1 mL
of the allantoic fluid from each surviving embryo and
examine each individual harvest for live virus by a
haemagglutination test. If haemagglutination is found for any
of the fluids, carry out for that fluid a further passage in eggs
and test for haemagglutination; no haemagglutination occurs.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Ovalbumin
Not more than the quantity stated on the label and in any
case not more than 1 ¡.tg per human dos e, determined by a
suitable immunochemical method (2.7.1) using a suitable
reference preparation of ovalbumin.
Total protein
Not more than 6 times the total haemagglutinin content of
the vaccine as determined in the assay, but in any case, not
Vaccines IV-559
more than 100 Ilg of protein per virus strain per human dose
and not more than a total of 300 ~lg of protein per human
dose.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6. 14)
Less than 100 ID per human dose.
ASSAY
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it l . Carry out the test
at 20-25 oC . The confidence limits (P = 0.95) are not less
than 80 per cent and not more than 125 per cent of the
estimated haemagglutinin antigen contento The lower
confidence limit (P = 0.95) is not less than 80 per cent of
the amount stated on the label for each strain.
For sorne vaccines, quantitative determination of
haemagglutinin antigen with respect to available reference
preparations is not possible. An immunological identification
of the haemagglutinin antigen and a semi-quantitative
determination are carried out instead by suitable methods.
LABELLING
The label sta tes:
-- that the vaccine has been prepared on eggs,
-- the strain or strains of influenza virus used to prepare the
vaccine,
-- the method of inactivation,
-- the haemagglutinin content in micrograms per virus strain
per dose,
-- the maximum amount of ovalbumin,
-- the season during which the vaccine is intended to
protect.
____________________________________________ PhEw
Inactivated Influenza Vaccine
(Surface Antigen)
(Influenza Vaccine (Suiface Antigen, Inactivated),
Ph Eur monograph 0869)
The label may state 'Flu' or 'Flu(adj)' as appropriate.
When Inactivated Influenza Vaccine or Influenza Vaccine is
prescribed or demanded and the form is not stated,
Inactivated Influenza Vaccine (Whole Virion), Inactivated
Influenza Vaccine (Split Virion) or Inactivated Influenza
Vaccine (Surface Antigen) may be dispensed or supplied.
~Ew ____________________________________________
DEFINITION
Influenza vaccine (surface antigen, inactivated) is a sterile
suspension of a strain or strains of influenza virus, type A
or B, or a mixture of strains of the 2 types grown individually
in fertilised hens' eggs, inactivated and treated so that the
preparation consists predorninantly of haemagglutinin and
neuraminidase antigens, without diminishing the antigenic
properties of these antigens. The stated amount of
haemagglutinin antigen for each strain present in the vaccine
1 Reference haemagglutinin antigens are available from the National Institute
for Biological Standards and Control, Blanche Lane, South Mimms, Potters
Bar, Hertfordshire, EN6 3QG, Great Britain
 IV-560 Vaccines
is 15 flg per dose, unless clinical evidence supports the use of
a different amount. The vaccine may contain an adjuvant.
PRODUCTION
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
CHOICE OF VACCINE STRAIN
The World Health Organisarion reviews the world
epidemiological situation annually and if necessary
recommends the strains that correspond to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signatory states of the Convention on the
Elaboration of a European Pharmacopoeia. Ir is now
common practice to use reassorted strains giving high yields
of the appropriate surface antigens. The origin and passage
history ofvirus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus seed to be used in the production of vaccine
is propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
(5.2.4), such as chick-embryo fibroblasts or chick kidney cells
obtained from SPF chicken flocks (5.2.2). For production,
the virus of each strain is grown in the allantoic cavity of
fertilised hens' eggs from healthy flocks.
VIRUS SEED LOT
The production of vaccine is based on a seed-Iot system.
Working seed lots represent not more than 15 passages from
the approved reassorted virus or the approved virus isolate.
The final vaccine represents one passage from the working
seed lot. The haemagglutinin and neuraminidase antigens of
each seed lor are identified as originating from the correcr
strain of influenza virus by suitable methods.
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the
monovalent pooled harvest.
Bacterial and fungal contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
VIRUS PROPAGATION ANO HARVEST
An antimicrobial agent may be added to the inoculum. After
incubation at a controlled temperature, the allantoic fluids
are harvested and combined to form a monovalent pooled
harvest. An antimicrobial agent may be added at the time of
harvest. At no stage in the production is penicillin or
streptomycin used.
MONOVALENT POOLED HARVEST
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
3 consecutive batches to be consistently effective for the
manufacturero The inactivation process shall have been
shown to be capable of inactivating the influenza virus
without destroying its antigenicity; the process should cause
minimum alteration of the haemagglutinin and
neuraminidase antigens . The inactivation process shall also
have been shown ro be capable of inactivating avian leucosis
viruses and mycoplasmas. If the monovalent pooled harvest is
stored after inactivation, it is held at 5 ± 3 oc.
If formaldehyde solution is used, the concentration does not
exceed 0.2 giL of CH 2 0 at any time during inactivation;
2014
if betapropiolacrone is used, the concentration does not
exceed 0.1 per cent V/Vat any time during inactivation.
Before or after the inactivation process, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or other suitable method. Virus particles are
disrupted into component subunits by approved procedures
and further purified so that the monovalent bulk consists
mainly of haemagglutinin and neuraminidase antigens.
Only a monovalent pooled harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it 1. Carry out the test
at 20-25 oC.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent pooled harvests from each working
seed lot.
Sterility (2.6. 1)
Carry out the test for sterility, using 10 mL for each
medium.
Residual infectious virus
Carry out the test described below under Tests .
Purity
The purity of the monovalent pooled harvest is examined by
polyacrylamide gel electrophoresis or by other approved
techniques. Mainly haemagglutinin and neuraminidase
antigens shall be presento
Chemicals used for disruption and purification
Tests are carried out on the monovalent pooled harvest for
the chemicals used for disruption and purification, the limits
being approved by the competent authority.
FINAL BULK VACCINE
Appropriate quantities of the monovalent pooled harvests are
blended ro make the final bulk vaccine. An adjuvant may be
added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not les s than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6. 1)
Carry out the test for sterility, using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a final lot that is satisfactory with respecr to each of the
requirements given below under Tests and Assay may be
released for use. Provided that the test for residual infectious
virus has been performed with satisfactory results on each
I Referenee haemagglurinin antigens are available f1"Om lhe Nazional InsLÍtule
for Biological Szandards and ConLrol, Blanehe Lane, Sourh Mi11l111S, Pouers
Bar, Herifordshire, EN6 3QG, Greal BriLain
 2014
monovalent pooled harvest and that the tests for free
formaldehyde, ovalbumin and total protein have been
performed with satisfactory results on the final bulk vaccine,
they may be omitted on the final lot.
If the ovalbumin and formaldehyde content cannot be
determined on the final lot, owing to interference from the
adjuvant, they are determined on the monovalent pooled
harvest, the acceptance limits being set to ensure that the
limits for the final product wiII not be exceeded.
If the vaccine contains an adjuvant, suitable tests for identity
and other relevant quality criteria are carried out on the final
lot. These tests may incIude chemical and physical analysis,
determination of particIe size and determination of the
number of particIes per unit volume.
IDENTIFICATION
The assay serves to confitm the antigenic specificity of the
vaccine.
TESTS
Residual infectious virus
Inoculate 0.2 mL of the vaccine into the aIlantoic cavity of
each of 10 fertilised eggs and incubate at 33-37 oC for
3 days. The test is not valid unless at least 8 of the 10
embryos survive. Harvest 0.5 mL of the aIlantoic fluid from
each surviving embryo and pool the fluids. Inoculate 0.2 mL
of the po oled fluid into a further 10 fertilised eggs and
incubate at 33-37 cC for 3 days. The test is not valid unless
at least 8 ofthe 10 embryos survive. Harvest about 0.1 mL
of the aIlantoic fluid from each surviving embryo and
examine each individual harvest for Iive virus by a
haemagglutination test. If haemagglutination is found for any
of the fluids, carry out for that fluid a further passage in eggs
and test for haemagglutination; no haemagglutination occurs.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservaríve by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Free fonnaldehyde (2.4.18)
M aximum 0.2 gIL, where applicable.
Ovalbumin
Not more than the quantity stated on the label and in any
case not more than 1 ¡lg per human dose, determined by a
suitable immunochemical method (2.7.1) using a suitable
reference preparation of ovalbumin.
Total protein
Not more than 40 ¡lg of protein other than haemagglutinin
per virus strain per human dose and not more than a total of
120 ¡lg of protein other than haemagglutinin per human
dose.
Sterility
It complies with the test for sterility (2.6. 1).
Bacterial endotoxins (2.6.14)
Less than 100 IU per human dose.
ASSAY
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it 1. Carry out the test
¡ Referenee haemagglutinin antigens are available frol1l rhe Nalional Insútute
for Biologieal Standards and Control, Blanehe Lane, South Mimms, Poners
Bar, Herifordshire, EN6 3QG, Grear Blitain
Vaccines IV-561
at 20-25 oc. The confidence Iimits (P = 0.95) are not less
than 80 per cent and not more than 125 per cent of the
estimated contentoThe lower confidence limit (P = 0.95)
haemagglutinin antigen is not less than 80 per cent of the
amount stated on the labe! for each strain.
LABELLING
The label states:
- that the vaccine has been prepared on eggs,
- the strain or strains of influenza virus used to prepare the
vaccine,
- the method of inactivation,
- the haemagglutinin content in micrograms per virus strain
per dose,
- the sea son during which the vaccine is intended to
protect,
- the maximum amount of ovalbumin,
- where applicable, the name and the quantity of adjuvant
used.
_ _________________________________________ ~E~
*****
Influenza Vaccine (Surface
* *
Antigen, Inactivated, Prepared in *****
Cell Cultures)
(Ph Bur monograph 2149)
The labe! may state 'Flu' or 'Flu(adj)' as appropriate.
~E~ ____________________________________________
DEFINITION
Influenza vaccine (surface anrígen, inactivated, prepared in
ceIl cultures) is a sterile, aqueous suspension of a strain or
strains of influenza virus, type A or B, or a mixture of strains
of the 2 types grown individuaIly in ceIl cultures, inactivated
and treated so that the prepararíon consists predominantly of
haemagglutinin and neuraminidase antigens, preserving
adequate antigenic properties of these antigens. The stated
amount of haemagglutinin antigen for each strain present in
the vaccine is 15 ¡lg per dose, unless clinical evidence
supports the use of a different amount. The vaccine is a clear
or slightIy opalescent liquid. The vaccine may contain an
adjuvant. This monograph applies to vaccines produced in
diploid or continuous ceIl lines of mammalian origino
PRODUCTION
GENERAL PROVISIONS
Production of the vaccine is based on a virus seed-Iot system
and a ceIl-bank system. The production method shaIl have
been shown to yield consistentIy vaccines that comply with
the requirements for immunogenicity, safety and stability.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
The production method is validated to demonstrate suitable
reduction of residual host-ceIl protein. With the agreement of
the competent authority and for each specific product,
routine testing for residual host-ceIl proteins may be omitted
based on the resuIts of validaríon studies for the producr.
Guidanee on the principIes of such validation studies is
given, for example, in the monograph Produces oi recombinant
DNA technology (0784), in particular in the seetions
'Validation of the production proeess - Extraetion and
purification' and 'Produetion eonsisteney - Host-eeIl-derived
proteins '.
IV-562 Vaccines
CHOICE OF VACCINE STRAIN
The World Health Organisation reviews the world
epidemiological situation annually and if necessary
recommends new strains corresponding to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signatory sta tes of the Convention on the
Elaboration of a European Pharmacopoeia. It is now
common practice to use reassorted strains giving high yields
of the appropriate surface antigens. The origin and passage
history of virus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus used in the preparation of seed lots is
propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
(5.2.3), such as chick-embryo fibroblasts, chick kidney cells
obtained from SPF chicken flocks (5.2.2), or a diploid or
continuous cellline. The final passage for establishment of
the working seed lot is prepared in the ceHline used for
routine production. For this production, the virus of each
strain is propagated in a diploid or continuous cellline
(5.2.3).
VIRUS SEED LOT
The production of vaccine is based on a seed-Iot system.
Each of the strains of influenza virus used shall be identified
by historical records that include information on the origin of
the strain and its subsequent manipulation. Working seed
lots represent not more than 15 passages from the approved
reassorted virus or the approved virus isolate. The final
vaccine represents 1 passage from the working seed lot.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
The haemagglutinin and neuraminidase antigens of each
master and working seed lot are identified as originating from
the correct strain of influenza virus by suitable methods.
Virus concentration
The virus concentration of each working seed lot is
determined. Where applicable, the virus concentration of
each master seed lot is determined.
Extraneous agents (2.6.16)
The working seed lots comply with the requirements for seed
lots. It is recognised that due to a seasonal change in one or
more of the influenza vaccine strains, timely testing of a virus
seed for extraneous agents according to general chapter
2.6.16 may be problematic (e.g. duration of in vivo tests,
timely availability of specific neutralising antisera).
In agreement with the competent authority, and in light of a
risk assessment, rapid assays (e.g. multiplex PCR) may be
applied as altematives to general chapter 2.6.16 following
validation.
Such risk assessment and validation incJudes more general
considerations on potential contaminants of the virus isolates,
the susceptibility of the cell substrate to such virus es and the
capacity of the production process for viral removal or
inactivation; validation incJudes also comparative data on
testing of seeds according to general chapter 2.6.16 and the
proposed rapid assays. Each applied PCRlNAT test (2.6.21)
must be shown to be suitable for its intended use by
appropriate analytical validation. The risk assessment is
reviewed when new information becomes available on
potential viral contaminants, and the justification of the
chosen PCR panel of extraneous agents tested for is provided
2014
to the competent authority within the annual update. This
update also incJudes vaccine strain-specific aspects such as
specific PCR inhibitory effects.
If an agent is detected in a virus seed and the marnmalian
cells used for production are shown to be susceptible to this
agent, the virus seed is not used for vaccine production.
If an agent is detected in a virus seed and the marnmalian
cells are not susceptible to the agent, validation of the
production process to demonstrate removal or inactivation of
the agent is carried out. If removal or inactivation cannot be
demonstrated, the inactivated monovalent harvest is tested to
demonstrate absence of any contaminant identified in the
virus seed.
PROPAGATION AND SINGLE HARVEST
AH processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are being handled at the same time. Approved animal serum
(but not human serum) may be used in the cell culture
media. Serum and trypsin used in the preparation of cell
suspensions or media are shown to be free from extraneous
agents. The cell culture media may contain a pH indicator,
such as phenol red, and antibiotics at the lowest effective
concentration. Not less than 500 mL of the cell cultures
employed for vaccine production are set aside as uninfected
ceH cultures (control cells).
Only a single harvest that complies with the following
requirements may be used in the preparation of the vaccine .
Identification
The test for antigen content also serves to identify the single
harvest.
Bacteria! and fungal contamination
Carry out the test for sterility (2.6. 1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL for each
medium.
Control cells
The control cells of the production ceH culture comply with a
test for identification and the requirements for extraneous
agents (2.6.16).
Haemagglutinin antigen
Determine the haemagglutinin antigen content by a suitable
irnmunochemical method (2.7.1).
INACTIVATED AND PURIFIED MONOVALENT HARVEST
The harvest, which may be a pool of several single harvests
of the same strain, is inactivated and purified by vaJidated
methods. Before or after the inactivation process, the
monovalent harvest is concentrated and purified by high-
speed centrifugation or another suitable method.
The influenza virus is inactivated by a method that has been
demonstrated on 3 consecutive batches to be consistently
effective for the manufacturer. The inactivation process shall
have been shown to be capable of inactivating the influenza
virus without destroying its antigenicity; the process is
designed so as to cause minimum alteration of the
haemagglutinin and neuraminidase antigens.
Virus particJes are disrupted into component subunits by
approved pro ce dures and further purified so that the
monovalent bulk consists mainly of haemagglutinin and
neuraminidase antigens.
If continuous celllines are used for production, the
purification process shall have been validated to reduce
consistently host-cell DNA to a suitable leve!.
 2014
Only an inactivated, purified monovalent harvest that
complies with the following requirements may be used in the
preparation of the final bulk vaccine .
Haemagglutinin antigen
Determine the haemagglutinin antigen content by a suitable
immunochemical method (2.7.1).
Antigenltotal protein ratio
Determine the haemagglutinin antigen content by a suitable
immunodiffusion test. Determine the total protein by a
validated method. The ratio of haemagglutinin antigen
content to total protein content is within the limits approved
for the particular product.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent harvests from each working seed
lot.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
Residual infectious virus
Carry out the test described below under Tests.
Purity
The purity of the monovalent harvest is examined by
polyacrylamide gel electrophoresis or by other approved
techniques. Mainly haemagglutinin and neuraminidase
antigens are presento
Chemicals used for disruption and purification
Tests are carried out on the monovalent harvest for the
chemicals used for disruption and purification, unless
validation of the process has demonstrated total clearance.
The concentration must not exceed the limits approved by
the competent authority for the particular producto
FINAL BULK VACCINE
Appropriate quantities of the inactivated, purified monovalent
pooled harvests are blended to make the final bulk vaccine.
An adjuvant may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
Residual host-cell DNA
If a continuous cell line is used for virus propagation, the
content of residual host-cell DNA, determined using a
suitable method, is not greater than lOng in the equivalent
of a single human dose.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Tests and Assay may be
released for use. Provided that the test for residual infectious
virus has been performed with satisfactory results on each
inactivated and purified monovalent harvest and that the tests
for free formaldehyde, bovine serum albumin and total
Vaccines IV-563
protein have been performed with satisfactory results on the
final bulk vaccine, they may be omitted on the finallot.
If the vaccine contains an adjuvant, suitable tests for identity
and other relevant quality criteria are carried out on the final
lot. These tests may include chemical and physical analysis,
determination of particle size and determination of the
number of particles per unit volume.
IDENTIFICAnON
The assay serves to confirm the antigenic specificity of the
vaccine.
TESTS
Residual infectious virus
Carry out an amplification test for residual infectious
influenza virus by inoculating not less than 0.2 mL of the
vaccine into cell cultures of the same type as used for
. production of the vaccine; incubate for not less than 4 days
at 37 oC. Inoculate not les s than 0.2 mL of the cell culture
harvested medium into a new semiconfluent cel! culture and
incubate as before. At the end of the incubation period,
examine for live virus by a haemagglutination test.
If haemagglutination is found for any of the fluids, carty out
for that fluid a further passage on cel! cultures and test for
haemagglutination; no haemagglutination occurs .
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Bovine serum albumin
Maximum 50 ng per human dose, determined by a suitable
immunochemical method (2. 7.1).
Total protein
Maximum 40 Jlg of protein other than haemagglutinin per
virus strain per human dose.
Sterility (2. 6. 1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than 25 IV per human dose.
ASSAY
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation 1 or with an
antigen preparation calibrated against it. Carry out the test at
20-25 oc. The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
contento The lower confidence limit (P = 0.95) is not less
than 80 per cent of the amount stated on the label for each
strain.
LABELLING
The label states:
- the biological origin of the cells used for the preparation
of the vaccine;
- the strain or strains of influenza virus used to prepare the
vaccine;
- the method of inactivation;
1 Reference hae11lagglutinin antigens are avaüable fro112 ¡he N ational InsiÍtute
for Biological Srandards and Control, Blanche Lane, Sou¡h Mi11211lS, Pouers
Bar, Hertfordshire, EN6 3QG, Great Brirain
 IV-564 Vaccines
-- the haemagglutinin antigen content in micrograms per
virus strain per dose;
-- the sea son during which the vaccine is intended to
protect;
-- where applicable, the name and the quantity of adjuvant
used.
___________________________________________ ~Ew
Influenza Vaccine (Surface
Antigen, Inactivated, Virosome)
(Ph Eur monograph 2053)
The label may state 'Flu'.
~Ew ___________________________________________
DEFINITION
Influenza vaccine (surface antigen, inactivated, virosome) is a
sterile, aqueous suspension of a strain or strains of influenza
virus, type A or B, or a mixture of strains of the 2 types
grown individually in fertilised hens' eggs, inactivated and
treated so that the preparation consists predominantly of
haemagglutinin and neuraminidase antigens reconstituted to
virosomes and without diminishing the antigenic properties of
the antigens. The stated amount of haemagglutinin antigen
for each strain present in the vaccine is 15 ~g per dose,
unless clinical evidence supports the use of a different
amount.
The vaccine is a slightly opalescent liquido
PRODUCTION
GENERAL PROVISIONS
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
CHOICE OF VACCINE STRAIN
The World Health Organisation reviews the world
epidemiological situation annually and if necessary
recommends the strains that correspond to this
epidemiological evidence.
Such strains are used in accordance with the regulations in
force in the signatory states of the Convention on the
Elaboration of a European Pharmacopoeia. It is now
common practice to use reassorted strains giving high yields
of the appropriate surface antigens. The origin and passage
history of virus strains shall be approved by the competent
authority.
SUBSTRATE FOR VIRUS PROPAGATION
Influenza virus seed to be used in the production of vaccine
is propagated in fertilised eggs from chicken flocks free from
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
(5.2.4), such as chick-embryo fibroblasts or chick kidney cells
obtained from SPF chicken flocks (5.2.2). For production,
the virus of each strain is grown in the allantoic cavity of
fertilised hens' eggs from healthy flocks.
VIRUS SEED LOT
The production of vaccine is based on a seed lot system.
Working seed lots represent not more than 15 passages from
the approved reassorted virus or the approved virus isolate.
The final vaccine represents 1 passage from the working seed
lot. The haemagglutinin and neuraminidase antigens of each
seed lot are identified as originating from the correct strain of
influenza virus by suitable methods.
2014
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the
monovalent pooled harvest.
Bacteria! and fungal contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
VIRUS PROPAGATION AND HARVEST
An antimicrobial agent may be added to the inoculum. After
incubation at a controlled temperature, the allantoic fluids
are harvested and combined to form a monovalent pooled
harvest. An antimicrobial agent may be added at the time of
harvest.
MONOVALENT PO OLED HARVEST
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
3 consecutive batches to be consistently effective for the
manufactureroThe inactivation process shall have been
shown to be capable of inactivating the influenza virus
without destroying its antigenicity; the process is designed so
as to cause minimum alteration of the haemagglutinin and
neuraminidase antigens. The inactivation process shall also
have been shown to be capable of inactivating avian leucosis
virus es and mycoplasmas. If the monovalent pooled harvest is
stored after inactivation, it is held at a temperature of
5 ± 3 oc. If formaldehyde solution is used, the
concentration does not exceed 0.2 giL of CH2 0 at any time
during inactivation; if betapropiolactone is used, the
concentration do es not exceed 0.1 per cent V/Vat any time
during inactivation.
Before or after the inactivation process, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or another suitable method.
Only a monovalent pooled harvest that complies with the
following requirements may be used for the preparation of
vrrosomes.
Provided the tests for haemagglutinin antigen, neuraminidase
antigen and residual infectious virus have been carried out
with satisfactory results on the monovalent virosomal
preparation, they may be omitted on the monovalent pooled
harvest when the manufacturing process is continuous
between the monovalent pooled harvest and the monovalent
virosomal preparation.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
irnmunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation 1 or with an
antigen preparation calibrated against it. Carry out the test at
20-25 oc.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent pooled harvests from each working
seed lot.
Residual infectious virus
Carry out the test described under Tests.
1 Reference haemagglucinin antigens are available from che Nacional Inscitute
for Biological Scandards and Control, Blanche Lane, Souch Mimms, Potters
Bar, Herrjordshire, EN6 3QG, Great Britain
2014
PREPARATION OF MONOVALENT VIROSOMES
Virus partic1es are disrupted into component subunits by
approved procedures and further purified so that the
monovalent bulk consists mainly of haemagglutinin and
neuraminidase antigens. Additional phospholipids may be
added and virosomes may be formed by removal of the
detergent either by adsorption chromatography or another
suitable technique . Several monovalent virosomal
preparations may be pooled.
Only a monovalent virosomal preparation that complies with
the following requirements may be used in the preparation of
the final bulk vaccine.
Haemagg1utinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation 1 or with an
antigen preparation calibrated against it. Carry out the test at
20-25 oC.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
confirmed by suitable enzymatic or immunological methods
on the first 3 virosomal preparations from each working seed
lot.
Residual infectious virus
Carry out the test described under Tests. Provided this test
has been carried out with satisfactory results on the
monovalent pooled harvest, it may be omitted on the
preparation of monovalent virosomes.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
Purity
The purity of the monovalent virosomal preparation is
examined by polyacrylamide gel electrophoresis (2.2.31) or
by other approved techniques. Mainly haemagglutinin and
neuraminidase antigens are presento
Chemica1s used for disruption and purification
Tests for the chemicals used for disruption and purification
are carried out on the monovalent virosomal preparation, the
limits being approved by the competent authority.
Phospholipids
The content and identity of the phospholipids are determined
by suitable irnmunochemical or physico-chemical methods.
Ratio of haemagg1utinin to phospholipid
The ratio of haemagglutinin content to phospholipid content
is within the limits approved for the particular product.
Virosome size
The average virosome diameter, determined by a suitable
method such as photon-correlation spectroscopy, is not les s
than 100 nm and not greater than 300 nm.
The polydispersity index is not greater than 0.4.
FINAL BULK VACCINE
Appropriate quantities of the monovalent virosomal
preparations are blended to make the final bulk vaccine.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
1 Reference haemagglutinin antigem are available fr0111 the N ational lmtitute
for Bi% gical Standards and Control, Blanche Lane, South Mirn111s, Potters
Bar, Herr;Jordsh,:re, EN6 3QG, Great Britain
Vaccines IV-565
preservative by a suitable chemical or physico-chemical
method. The content is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility, using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are c10sed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given under Tests and Assay may be released
for use. Provided that the test for residual infectious virus has
been performed with satisfactory results on each monovalent
pooled harvest or, where appropriate, on the monovalent
virosomal preparations, and that the tests for phospholipids,
ratio of haemagglutinin to phospholipid, free formaldehyde,
ovalbumin and total protein have been performed with
satisfactory results on the final bulk vaccine, they may be
ornitted on the finallot.
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccine.
TESTS
Residual infectious virus
Inoculate 0.2 mL of the vaccine into the allantoic cavity of
each of 10 fertilised eggs and incubate at 33-37 oC for
3 days. The test is not valid unless at least 8 of the 10
embryos survive. Harvest 0.5 mL of the allantoic fluid from
each surviving embryo and pool the fluids. Inoculate 0.2 mL
of the pooled fluid into a further 10 fertilised eggs and
incubate at 33-37 oC for 3 days. The test is not val id unless
at least 8 ofthe 10 embryos survive. Harvest about 0.1 mL
of the allantoic fluid from each surviving embryo and
examine each individual harvest for live virus by a
haemagglutination test. If haemagglutination is found for any
of the fluid s, carry out for that fluid a further passage in eggs
and test for haemagglutination; no haemagglutination occurs.
pH (2.2.3)
6.5 to 7.8
Phospholipids
The content and identity of the phospholipids is determined
by a suitable immunochemical or physico-chemical method.
Ratio of haemagg1utinin to phospholipid
The ratio of haemagglutinin content to phospholipid content
is within the limits approved for the particular product.
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
the quantity stated on the labe!'
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Ova1bumin
Not more than the quantity stated on the label and in any
case not more than 1 Ilg per human dose, determined by a
suitable immunochemical method (2.7.1) using a suitable
reference preparation of ovalbumin.
Total protein
Not more than 40 Ilg of protein other than haemagglutinin
per virus strain per human dose, and not more than a total of
120 Ilg of protein other than hemagglutinin per human dose.
 IV-566 Vaccines
Sterility (2.6.1)
It complies with the test for sterility.
Virosome size
The average virosome diameter, determined by a suitable
method such as photon-correlation spectroscopy, is not less
than 100 nm and not greater than 300 nm.
The polydispersity index is not greater than 0.4.
Bacteria! endotoxins (2.6.14)
Less than 100 IV per human dose.
ASSAY
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.7.1), by comparison with a
haemagglutinin antigen reference preparation 1 or with an
antigen preparation calibrated against it. Carry out the test at
20-25 oc. The confidence limits (P = 0.95) are not less than
80 per cent and not more than 125 per cent of the estimated
haemagglutinin antigen contento The lower confidence limit
(P = 0.95) is not less than 80 per cent of the amount stated
on the label for each strain.
LABELLING
The label sta tes:
- that the vaccine has been prepared on eggs;
- the strain or strains of influenza virus used to prepare the
vaccine;
- the method of inactivation;
- the haemagglutinin content, in micrograms per virus
strain per dose;
- the maximum amount of ovalbumin;
- the season during which the vaccine is intended to
protect.
_ _ _ _ _ _ _ __ __ _ _ __ _ __ _ _ __
Measles Vaccine, Live
*** ***
(Measles Vaccine (Live), Ph Eur monograph 0213)
The label may state 'Measles'.
PhEif _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ __
DEFINITION
Measles vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of measles virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.
PRODUCTION
The production ofvaccine is based on a virus seed-Iot system
and, if the virus is propagated in human diploid cells, a
cell-bank system. The production method shall have been
shown to yield consistently live measles vaccines of adequate
immunogenicity and safety in mano Vnless otherwise justified
and authorised, the virus in the final vaccine shall have
undergone no more passages from the master seed lot than
were used to prepare the vaccine shown in clinical studies to
be satisfactory with respect to safety and efficacy; even with
authorised exceptions, the number of passages beyond the
level used for clinical studies shall not exceed 5.
The potential neurovirulence of the vaccine strain is
1 Reference haemagglutinin antigens are available from the Nationallnstitute
for Biological Standards and Control, Blanche Lane, South Mimms, Potters
Bar, Herifordshire, EN6 3QG, Great Britain
2014
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk
analysis is carried out. Where necessary and if available, a
test is carried out on the vaccine strain using an animal
model that differentiates wild-type and attenuated virus; tests
on strains of intermediate attenuation may also be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3) or in
cultures of chick-embryo cells derived from a chicken flock
free from specified pathogens (5.2.2).
SEED LOT
The strain of measles virus used shall be identified by
historical records that include information on the origin of
the strain and its subsequent manipulation. Virus seed lots
are prepared in large quantities and stored at temperatures
below - 20 oC if freeze-dried, or below - 60 oC if not
freeze-dried.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
The master and working seed lots are identified as measles
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentration
The virus concentration of the master and working seed lots
is determined to monitor consistency of production.
PhEur Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots.
PROPAGATION AND HARVEST
*** All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
*** are handled during production. Suitable animal (but not
human) serum may be used in the growth medium, but the
_ _
final medium for maintaining cells during virus multiplication
does not contain animal serum. Serum and trypsin used in
the preparation of cell suspensions and culture media are
shown to be free from extraneous agents. The cell culture
medium may contain a pH indicator such as phenol red and
suitable antibiotics at the lowest effective concentration. It is
preferable to have a substrate free from antibiotics during
production. Not les s than 500 mL of the production cell
cultures is set aside as uninfected cell cultures (control cells).
The viral suspensions are harvested at a time appropriate to
the strain of virus being used.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccme.
Identification
The single harvest contains virus that is identified as measles
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentration
The virus concentration in the single harvest is determined as
prescribed under Assay to monitor consistency of production
and to determine the dilution to be used for the final bulk
vaccine.
2014
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous
agents.
Control cells
If human diploid cells are used for production, the control
cells comply with a test for identification. They comply with
the tests for extraneous agents (2.6.16).
FINAL BULK VACCINE
Virus harvests that comply with the aboye tests are po oled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Bacteria! and funga! contamination
The final bulk vaccine complies with the test for sterility
(2. 6.1), carried out using 10 mL for each medium.
FINALLOT
A minimum virus concentration for release of the product is
established such as to ensure, in light of stability data, that
the minimum concentration stated on the label will be
present at the end of the period of validity.
Only a finallot that complies with the requirements for
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
requirements given below under Identification and Tests may
be released for use. Provided that the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, it may be omitted on the final loto
Thennal stability
Maintain at least 3 vials of the finallot of freeze-dried
vaccine in the dry state at 37 ± 1 oC for 7 days. Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. The virus
concentration of the heated vaccine is not more than 1.0 log
lower than that of the unheated vaccine.
IDENTIFICAnON
When the vaccine reconstituted as stated on the label is
mixed with specific measles antibodies, it is no longer able to
infect susceptible cell cultures .
TESTS
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1) .
Bovine serum albumin
Not more than 50 ng per single human dose, determined by
a suitable irnmunochemical method (2.7.1).
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine and inoculating a suitable number
of wells for each dilution step. Titrate 1 vial of an
appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory.
The relation with the appropriate European Pharmacopoeia
Biological Reference Preparation is established and
monitored at regular intervals if a manufacturer's reference
preparation is used. Calculate the individual virus
concentration for each vial of vaccine and for each replicate
Vaccines IV-567
of the reference preparation as well as the corresponding
combined virus concentrations, using the usual statistical
methods (for example, 5.3). The combined estimate of the
virus concentration for the 3 vials of vaccine is not less than
that stated on the label; the minimum virus concentration
stated on the label is not less than 3.0 log CCID so per single
human dose.
The assay is not val id if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so;
-- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so; data obtained from valid assays only
are combined by the usual statistÍcal methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log CCIDso .
Measles vaccine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance critería. However, the vaccine must comply if
tested as descríbed aboye.
LABELLING
The label states:
-- the strain of virus used for the preparation of the vaccine;
-- the type and origin of the cells used for the preparation of
the vaccine;
-- the minimum virus concentration;
-- that contact between the vaccine and disinfectants is to be
avoided.
___________________________________________ ~Em
Measles, Mumps and Rubella *****
** **
Vaccine, Uve ***
(Measles, Mumps and Rubella Vaccine (Live),
Ph Eur monograph 1057)
The label may state 'MMR' .
~Em ___________________________________________
DEFINITION
Measles, mumps and rubella vaccine (Iive) is a freeze-dríed
preparation of suitable attenuated strains of measles virus,
mumps virus and rubella virus.
The vaccine is reconstituted immediately before use, as
stated on the label, to give a clear liquid that may be
coloured owing to the presence of a pH indicator.
PRODucnON
The 3 components are prepared as described in the
monographs Measles vaccine (live) (0213), Mumps vaccine
(live) (0538) and Rubella vaccine (live) (0162) and comply
with the requirements prescríbed therein.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for irnmunosera and vaccines for human use (2.6.9) .
FINAL BULK VACCINE
Virus harvests for each component are pooled and clarífied to
remove cells. A suitable stabiliser may be added and the
 IV-568 Vaccines
pooled harvests diluted as appropriate. Suitable quantities of
the pooled harvest for each component are mixed.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Bacteria! and fungal contaminatíon
Carry out the test for sterility (2.6.1), using 10 mL for each
medium .
FINAL LOT
For each component, a minimum virus concentration for
release of the product is established such as ro ensure, in
light of stability data, that the minimum concentration stated
on the labe! will be present at the end of the period of
validity.
Only a final lot that complies with the requirements for
minimum virus concentration of each component for re!ease,
with the following requirement for thermal stability and with
each of the requirements given below under Identification
and Tests may be re!eased for use. Provided that the tests for
bovine serum albumin and, where applicable, for ovalbumin
have been carried out with satisfactory results on the final
bulk vaccine, they may be omitted on the final loto
Thennal stability
Maintain at least 3 vials of the finallot of freeze-dried
vaccine in the dry state at 37 ± 1 oC for 7 days. Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. For each component,
the virus' concentration of the heated vaccine is not more
than 1.0 log lower than that of the unheated vaccine.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with antibodies specific for measles virus, mumps virus
and rubella virus, it is no longer able ro infect cell cultures
susceptible to these viruses. When the vaccine reconstituted
as stated on the labe! is mixed with quantities of specific
antibodies sufficient to neutralise any 2 viral components, the
3rd viral component infects susceptible cell cultures.
TESTS
Bacteria! and fungal contaminatíon
The reconstituted vaccine complies with the test for sterility
(2.6.1).
Bovine serum albumin
Not more than 50 ng per single human dose, determined by
a suitable immunochemical method (2.7.1).
Ovalbumin
If the mumps component is produced in chick embryos, the
vaccine contains not more than 1 ~g of ovalbumin per single
human dose, determined by a suitable immunochemical
method (2.7.1).
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
ASSAY
The celllines ancl/or neutralising antisera are chosen to
ensure that each component is assayed without interference
from the other 2 components.
Titrate the vaccine for infective measles, mumps and rubella
virus, using at least 3 separate vials ofvaccine and
inoculating a suitable number of wells for each dilution step .
Titrate 1 vial of the appropriate virus reference preparation in
triplicate ro valida te each assay. The virus concentration of
the reference preparation is monirored using a control chart
and a titre is established on a historical basis by each
2014
laborarory. The relation with the appropriate European
Pharmacopoeia Biological Reference Preparation is
established and monirored at regular intervals if a
manufacturer's reference preparation is used. Calculate the
individual virus concentration for each vial of vaccine and for
each replicate of the reference preparation as well as the
corresponding combined virus concentrations, using the usual
statistical methods (for example, 5.3).
The combined estima tes of the meas les, mumps and rubella
virus concentrations for the 3 vials of vaccine are not less
than that stated on the label; the minimum measles virus
concentration stated on the label is not less than 3.0 log
CCID so per single human dose; the minimum mumps virus
concentration stated on the label is not less than 3.7 log
CCID so per single human dose; the minimum rubella virus
concentration stated on the label is not less than 3.0 log
CCID so per single human dose.
The assay is not valid if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so;
-- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) ro calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log CCID so .
Measles vaccine (live) BRP is suitable for use as a reference
preparation.
Mumps vaccine (live) BRP is suitable for use as a reference
preparation.
Rubella vaccine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
-- the strains of virus used in the preparation of the vaccine;
-- where applicable, that chick embryos have been used for
the preparation of the vaccine;
-- the type and origin of the cells used for the preparation of
the vaccine;
-- the minimum virus concentration for each component of
the vaccine;
-- that contact between the vaccine and disinfectants is ro be
avoided.
____________________________________________ ~E~
 2014
*****
Measles, Mumps, Rubella and
** **
Varicella Vaccine (Live) ***
(Ph Eur monograph 2442)
The label may state 'MMRVar'.
~Ew ____________________________________________
DEFINITION
Measles, mumps, rubella and varicella vaccine (live) is a
freeze-dried preparation of suitable attenuated strains of
measles virus, mumps virus, rubella virus and human
herpesvirus 3. The vaccine is reconstituted immediately
before use, as stated on the label, to give a clear liquid that
may be coloured owing to the presence of a pH indicator.
PRODUCTION
The 4 components are prepared as described in the
monographs Measles vaccine (live) (0213), Mumps vaccine
(live) (0538), Rubella vaccine (live) (0162) and Varicella
vaccine (live) (0648) and comply with the requirements
prescribed therein.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
FINAL BULK VACCINE
Virus harvests for each component are pooled and c1arified to
remove cells. A suitable stabiliser may be added and for each
component the pooled harvests diluted as appropriate.
Suitable quantities of the pooled harvest for each component
are mixed.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacteria! and fungal contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium.
FINALLOT
For each component, a minimum virus concentration for
release of the product is established such as to ensure, in
light of stability data, that the minimum concentration stated
on the label will be present at the end of the period of
validity. The final bulk vaccine is distributed aseptically into
sterile, tamper-proof containers and freeze-dried to a
moisture content shown to be favourable to the stability of
the vaccine. The containers are then closed so as to prevent
contamination and the introduction of moisture .
Only a final lot that complies with the requirements for
minimum virus concentration of each component for releas e,
with the following requirements for thermal stability, bovine
serum albumin and water, and with each of the requirements
given under Identification and Tests may be released for use.
Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the final bulk vaccine,
it may be omitted on the final loto
Thermal stability
For the measles, mumps and rubella components maintain at
least 3 containers of the finallot of freeze-dried vaccine in
the dry state at 37 ± 1 oC for 7 days. Determine the virus
concentration as described under Assay in parallel for the
heated vaccine and for vaccine stored at the temperature
recommended for storage. For each component, the virus
concentration of the heated vaccine is not more than 1.0 log
lower than that of the unheated vaccine.
Vaccines IV-569
Bovine serum albumin
Not more than the amount approved by the competent
authority, determined by a suitable irnmunochemical method
(2.7.1).
Water (2.5.12)
Not more than the amount shown to ensure stability of the
vaccines as approved by the competent authority, determined
by the semi-micro determination of water.
IDENTIFlCATION
When the vaccine reconstituted as stated on the labe! is
mixed with antibodies specific for measles virus, mumps
virus, rubella virus and human herpesvirus 3, it is no longer
able to infect cell cultures susceptible to these viruses. When
the vaccine reconstituted as stated on the label is mixed with
quantities of specific antibodies sufficient to neutralise any 3
viral components, the 4 th viral component infects susceptible
cell cultures.
TESTS
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6. 1).
ASSAY
The cell lines and/or neutralising antisera are chosen to
ensure that each component is assayed without interference
from the other 3 components.
Titrate the vaccine for infective measles virus, mumps virus,
rubella virus and human herpesvirus 3 using at least
3 separate containers of vaccine and inoculating a suitable
number of wells for each dilution step. Titrate 1 container of
the appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory. Unless
otherwise justified and authorised, for the measles, mumps,
rubella and human herpesvirus 3 virus es the relation with the
appropriate European Pharmacopoeia Biological Reference
Preparation is established and monitored at regular intervals
if a manufacturer's reference preparation is used. Calculate
the individual virus concentration for each container of
vaccine and for each replicate of the reference preparation as
well as the corresponding combined virus concentrations,
using the usual statistical methods (for example, 5.3).
The combined estima tes of the measles virus, mumps virus,
rubella virus and human herpesvirus 3 concentrations for the
3 containers of vaccine are not les s than that stated on the
label; the minimum measles virus concentration stated on the
label is not less than 3.0 log CCID so per single human dos e;
the minimum mumps virus concentration stated on the label
is not less than 3.7 log CCID so per single human dose;
the minimum rubella virus concentration stated on the label
is not less than 3.0 log CCID so per single human dose.
The assay is not valid if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so
(measles virus, mumps virus and rubella virus) or ± 0.3
log PFU (human herpesvirus 3);
-- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so (measles virus, mumps
virus and rubella virus) or 0.5 log PFU (human
herpesvirus 3) from the established value.
The assay is repeated ifthe confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so (measles virus, mumps virus and
IV-570 Vaccines
rubella virus) or ± 0.3 log PFU (human herpesvirus 3); data
obtained from valid assays only are combined by using the
usual statistical methods (for example, 5.3) 10 calculate the
virus concentration of the sample. The confidence interval
(P == 0.95) ofthe combined virus concentration is not greater
than ± 0.3 log CCIDso (measles virus, mumps virus and
rubella virus) or ± 0.3 log PFU (human herpesvirus 3).
Measles vaccine (live) BRP is suitable for use as a reference
preparation.
Mumps vaccine (live) BRP is suitable for use as a reference
preparation.
Rubella vaccine (live) BRP is suitable for use as a reference
preparation.
Varicella vaccine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
-- the strains of virus used in the preparation of the vaccine;
-- the type and origin of the cells used for the preparation of
the vaccine;
-- the minimum virus concentration for each component of
the vaccine;
-- that contact between the vaccine and disinfectants is 10 be
avoided.
____________________________________________ ~E~
*****
Meningococcal Group C Conjugate
** **
Vaccine ***
(Ph Eur monograph 2112)
The label may state 'MenC(conJ)'.
~E~ ____________________________________________
DEFINITION
Meningococcal group C conjugate vaccine is a liquid or
freeze-dried preparation of purified capsular polysaccharide
derived from a suitable strain of Neisseria meningitidis group C
covalently linked to a carrier protein. Meningococcal group C
polysaccharide consists of partly O-acetylated or
O-deacetylated repeating units of sialic acids, linked with
20:->9 glycosidic bonds. The carrier protein, when
conjugated 10 group C polysaccharide, is capable of inducing
a T-cell-dependent B-cell irnmune response to the
polysaccharide. The vaccine may contain an adjuvant.
PRODUCTION
GENERAL PROVISIONS
The production method shall consistently have been shown
10 yield meningococcal group C conjugate vaccines of
satisfactory irnmunogenicity and safety in mano
The production of meningococcal group C polysaccharide
and of the carrier protein are based on seed-Iot systems.
During development studies and wherever revalidation is
necessary, a test for pyrogens in rabbits (2.6.8) is carried out
by injection of a suitable dose of the finallot. The vaccine is
shown 10 be acceptable with respect 10 absence of pyrogenic
activity. The production method is validated to demonstrate
that the vaccine, if tested, would comply with the test for
2014
abnormal toxicity for immunosera and vaccines for human
use (2.6.9) .
During development studies and wherever revalidation of the
manufacturing process is necessary, it shall be demonstrated
by tests in animals that the vaccine consistently induces a
T-cell-dependent B-ce11 immune response.
The stability of the finallot and relevant intermedia tes is
evaluated using 1 or more indica10r tests. Such tests may
inc1ude determination of molecular size, determination of free
saccharide in the conjugate or an immunogenicity test in
animals.
BACTERIAL SEED LOTS
The bacterial strains used for master seed lots shall be
identified by his10rical records that inc1ude information on
their origin and the tests used 10 characterise the strain.
Cultures from the working seed lot shall have the same
characteristics as the strain that was used to prepare the
master seed lot.
Purity of bacterial cultures is verified by methods of suitable
sensitivity. These may inc1ude inoculation into suitable
media, examinaríon of colony morphology, microscopic
examination of Gram-stained smears and culture
agglutination with suitable specific antisera.
MENINGOCOCCAL GROUP C POLYSACCHARIDE
N. meningitidis is grown in a liquid medium that does not
contain high-molecular-mass polysaccharides and is free from
ingredients that will form a precipitate upon addition of
cetyltrimethylammonium bromide (CTAB). The culture may
be inactivated by heat and filtered before the polysaccharide
is precipitated by addition of CTAB. The precipitate is
further purified using suitable methods to remove nuc1eic
acids, proteins and lipopolysaccharides and the final
purification step consists of ethanol precipitation.
An O-deacetylation step may also be inc1uded. Volatile
matter, inc1uding water, in the purified polysaccharide is
determined by a suitable method such as thermogravimetry
(2.2.34). The value is used 10 calcula te the results of other
tests with reference 10 the dried substance, as prescribed
below.
Only meningococcal group C polysaccharide that complies
with the following requirements may be used in the
preparation of the conjugate .
Protein (2.5. 16)
Maximum 1.0 per cent, calculated with reference 10 the dried
substance.
Nucleic acid (2.5.17)
Maximum 1.0 per cent, calculated with reference to the dried
substance.
O-acetyl groups
Examine by a suitable method (for example 2.5.19) .
An acceptable value is established for the particular product
and each batch of meningococcal group C polysaccharide
must be shown to comply with this limit.
Sialic acid (2.5.23)
Minimum 0.800 g of sialic acid per gram of meningococcal
group C polysaccharide using N-acer:ylneuraminic acid R 10
prepare the reference solution.
Residual reagents
Where applicable, tests are carried out to determine residues
of reagents used during inactivation and purification.
An acceptable value for each reagent is established for the
particular product and each batch of meningococcal group C
polysaccharide must be shown 10 comply with this limit.
Where validation studies have demonstrated removal of a
 2014
residual reagent, the test on purified meningococcal group C
polysaccharide may be omitted.
Molecular-size distribution
Examine by size-exclusion chromatography (2.2.30).
An acceptable value is established for the particular product
and each batch of meningococcal group C polysaccharide
must be shown to comply with this limit. Where applicable,
the molecular-size distribution is also determined after
chemical modification of the meningococcal group C
polysaccharide.
Identification and serological specificity
The identity and serological specificity are determined by a
suitable irnmunochemical method (2.7.1) or other suitable
method, for example lH nuclear magnetic resonance
spectrometry (2.2.33).
Bacterial endotoxins (2.6.14)
Less than 100 IU per microgram of meningococcal group C
polysaccharide.
CARRIER PROTEIN
The carrier protein is chosen so that when meningococcal
group C polysaccharide is conjugated it is able 10 induce a
T-cell-dependent immune response. Tetanus toxoid and the
non-toxic mutant of diphtheria toxin-like protein, CRM 197,
are suitable. The carrier protein is produced by culture of a
suitable microorganism, the bacterial purity of which is
verified.
Only a carrier protein that complies with the following
requirements may be used in preparation of the conjugate.
Identification
The carrier protein is identified by a suitable
immunochemical method (2.7.1).
CRM 197
Where CRM 197 is used as the carrier protein, it is not less
than 90 per cent pure, determined by a suitable method.
Suitable tests are carried out, for validation or routinely, to
demonstrate that the product is non-toxico
T etanus toxoid
Where tetanus toxoid is used as the carrier, it is produced as
described in the monograph Tetanus vaccine (adsorbed)
(0452) and complies with the requirements prescribed
therein for bulk purified toxoid, except that the antigenic
purity (2.7.27) is not less than 1500 Lf per milligram of
protein nitrogen and that the test for sterility (2.6.1) is not
required.
BULK CONJUGATE
Meningococcal group C polysaccharide is chemically
modified to enable conjugation; it is usually partly
depolymerised either before or during this procedure.
The conjugate is obtained by the covalent bonding of
activated meningococcal group C oligosaccharide and carrier
protein. The conjugate purification procedures are designed
to remove residual reagents used for conjugation.
The removal of residual reagents and reaction by-products is
confirmed by suitable tests or by validation of the purification
process.
Only a bulk conjugate that complies with the following
requirements may be used in the preparation of the final bulk
vaccin<l. For each test and for each particular product, limits
of acceptance are established and each batch of conjugate
must be shown to comply with these limits.
Molecular-size distribution
Examine by size-exclusion chromatography (2.2.30).
An acceptable value is established for the particular product
Vaccines IV-571
and each batch of bulk conjugate must be shown to comply
with this limit.
Saccharide
The saccharide content is determined by a suitable validated
assay (for example 2.5.23). Anion-exchange liquid
chromatography with pulsed amperometric detection (2.2.29)
may also be used for determination of saccharide contento
An acceptable value is established for the particular product
and each batch of bulk conjugate must be shown 10 comply
with this limito
Protein
The protein content is determined by a suitable chemical
method (for example 2.5.16). An acceptable value is
established for the particular product and each batch of bulk
conjugate must be shown to comply with this limit.
Saccharide-to-protein ratio
Determine the ratio by calculation.
Free saccharide
Unbound saccharide is determined after removal of the
conjugate, for example by anion-exchange liquid
chromatography, size-exclusion or hydrophobic
chromatography, ultrafiltration or other validated methods.
An acceptable value is established for the particular product
and each batch of bulk conjugate must be shown to comply
with this limit.
Free carrier protein
Determine the content, either directly by a suitable method
or by deriving the content by calculation from the results of
other tests. An acceptable value is established for the
particular product and each batch of bulk conjugate must be
shown 10 comply with this limito
Residual reagents
Removal of residual reagents such as cyanide is confirmed by
suitable tests or by validation of the process.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium or the equivalent of 100 dos es, whichever is
less.
FINAL BULK VACCINE
An adjuvant and a stabiliser may be added to the bulk
conjugate before dilution 10 the final concentration with a
suitable diluent.
Only a final bulk vaccine that complies with the following
requirement and is within the limits approved for the
particular product may be used in preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
FINALLOT
Only a final lot that is within the limits approved for the
particular product and is satisfactory with respect to each of
the requirement given below under Identification, Tests, and
Assay may be released for use.
IDENTIFICATION
The vaccine is identified by a suitable immunochemical
method (2.7.1).
TESTS
pH (2.2.3)
The pH of the vaccine, reconstituted if necessaty, is within
± 0.5 pH units of the limit approved for the particular
product.
 IV-572 VaccÍnes
Aluminium (2.5.13)
Maximum 1.25 mg per single human dos e, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Water (2.5.12)
Maximum 3.0 per cent for freeze-dried vaccines.
Free saccharide
Vnbound saccharide is determined after removal of the
conjugate, for example by anion-exchange liquid
chromatography, size-exelusion or hydrophobic
chromatography, ultrafiltration or other validated methods.
An acceptable value consistent with adequate
immunogenicity, as shown in elinical trials, is established for
the particular product and each final lot must be shown to
comply with this limit.
Sterility (2.6.1)
It complies with the test for sterility.
Bacteria! endotoxins (2.6.14)
Less than 25 IV per single human dose .
ASSAY
Saccharide
Minimum 80 per cent of the amount of meningococcal
group C polysaccharide stated on the labe!' The saccharide
content is determined by a suitable validated assay, for
example sialic acid assay (2.5.23) or anion-exchange liquid
chromatography with pulsed amperometric detection
(2.2.29).
LABELLING
The label sta tes:
-- the number of micrograms of meningococcal group C
polysaccharide per human dos e;
-- the type and number of micrograms of carrier protein per
human dose.
____________________________________________ PhEw
Meningococcal Polysaccharide
Vaccine
(Ph Eur monograph 0250)
The label may state 'Men' plus relevant antigen.
For example, 'MenAC'.
____________________________________________
~Ew
DEFINITION
Meningococcal polysaccharide vaccine is a freeze-dried
preparation of one or more purified capsular polysaccharides
obtained from one or more suitable strains of Neisseria
meningitidis group A, group C, group Y and group W135 that
are capable of consistently producing polysaccharides.
N. meningitidis group A polysaccharide consists of partly
O-acetylated repeating units of N-acetylmannosarnine, linked
with 10:--->6 phosphodiester bonds.
N. meningitidis group C polysaccharide consists of partly
O-acetylated repeating units of sialic acid, linked with
2:x--->9 glycosidic bonds.
N. meningitidis group Y polysaccharide consists of partly
O-acetylated alternating units of sialic acid and D-glucose,
linked with 20:--->6 and 10:---> 4 glycosidic bonds.
N. meningitidis group W135 polysaccharide consists of partly
O-acetylated alternating units of sialic acid and D-galactose,
linked with 211 --->6 and 111--->4 glycosidic bonds.
2014
The polysaccharide component or components stated on the
label together with calcium ions and residual moisture
account for over 90 per cent of the mass of the preparation.
PRODVCTION
Production of the meningococcal polysaccharides is based on
a seed-Iot system. The production method shall have been
shown to yield consistently meningococcal polysaccharide
vaccines of satisfactory immunogenicity and safety in mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SEED LOTS
The strains of N. meningitidis used for the master seed lots
shall be identified by historical records that inelude
information on their origin and by their biochemical and
serological characteristics.
Cultures ftom each working seed lot shall have the same
characteristics as the strain that was used 10 prepare the
master seed lot. The strains have the following
characteristics:
-- colonies obtained from a culture are rounded, uniform in
shape and smooth with a mucous, opalescent, greyish
appearance,
-- Gram staining reveals characteristic Gram-negative
diplococci in "coffee-bean" arrangement,
-- the oxidase test is positive,
-- the culture utilises glucose and maltose,
-- suspensions of the culture agglutinate with suitable
specific antisera.
Purity of bacterial strains used for the seed lots is verified by
methods of suitable sensitivity. These may inelude
inoculation into suitable media, examination of colony
morphology, microscopic examination of Gram-stained
smears and culture agglutination with suitable specific
antisera.
PROPAGATION ANO HARVEST
The working seed lots are cultured on solid media that do
not contain blood-group substances or ingredients of
mammalian originoThe inoculum may undergo 1 or more
sub cultures in liquid medium before being used for
inoculating the final medium. The liquid media used and the
final medium are semisynthetic and free from substances
precipitated by cetrimonium bromide
(hexadecyltrimethylarnmonium bromide) and do not contain
blood-group substances or high-molecular-mass
polysaccharides.
The bacterial purity of the culture is verified by methods of
suitable sensitivity. These may inelude inoculation into
suitable media, examination of colony morphology,
microscopic examination of Gram-stained smears and culture
agglutination with suitable specific antisera.
The cultures are centrifuged and the polysaccharides
precipitated from the supernatant by addition of cetrimonium
brornide. The precipitate obtained is harvested and may be
stored at - 20 oC awaiting further purification.
PURIFIED POLYSACCHARIDES
The polysaccharides are purified, after dissociation of the
complex of polysaccharide and cetrimonium bromide, using
suitable procedures 10 remove successively nueleic acids,
proteins and lipopolysaccharides.
The final purification step consists of ethanol precipitation of
the polysaccharides which are then dried and stored
at - 20 oc. The los s on drying is determined by
thermogravimetry (2.2.34) and the value is used to calculate
2014
the results of the other chemical tests with reference to the
dried substance.
Only purified polysaccharides that comply with the following
requirements may be used in the preparation of the final bulk
vaccine.
Protein (2.5.16)
Not more than 10 mg of protein per gram of purified
polysaccharide, calculated with reference to the dried
substance.
Nucleic acids (2.5.17)
Not more than 10 mg of nucleic acids per gram of purified
polysaccharide, calculated with reference to the dried
substance.
O-Acetyl groups (2.5.19)
Not less than 2 mmol of O-acetyl groups per gram of
purified polysaccharide for group A, not less than 1.5 mmol
per gram of polysaccharide for group C, not less than
0.3 mmol per gram of polysaccharide for groups Y and
W135, all calculated with reference to the dried substance.
Phosphorus (2.5.18)
Not less than 80 mg of phosphorus per gram of group A
purified polysaccharide, calculated with reference to the dried
substance.
Sialic acid (2.5.23)
Not less than 800 mg of sialic acid per gram of group C
polysaccharide and not les s than 560 mg of sialic acid per
gram of purified polysaccharide for groups Y and W135, all
calculated with reference to the dried substance. Use the
following reference solutions.
Group C polysaccharide: a 150 mg/L solution of
N-acet:ylneuraminic acid R.
Group Y polysaccharide: a solution containing 95 mg/L of
N-acet:ylneuraminic acid R and 55 mg/L of glucose R.
Group W135 polysaccharide: a solution containing 95 mg/L
of N-acetylneuraminic acid R and 55 mg/L of galactose R.
Calcium
If a calcium salt is used during purification, a determination
of calcium is carried out on the purified polysaccharide;
the content is within the Iimits approved for the particular
producto
Distribution of molecular size
Examine by size-exclusion chromatography (2.2.30) using
agarose for chromatography R or cross-linked agarose for
chromatography R. Use a column about 0.9 m long and
16 mm in internal diameter equilibrated with a solvent
having an ionic strength ofO.2 mol/kg and a pH of7.0-7.5.
Apply to the column about 2.5 mg of polysaccharide in a
volume of about 1.5 mL and elute at about 20 mUh. Collect
fractions of about 2.5 mL and determine the content of
polysaccharide by a suitable method. At least 65 per cent of
group A polysaccharide, 75 per cent of group C
polysaccharide, 80 per cent of group Y polysaccharide and
80 per cent of group W135 polysaccharide is eluted before a
distribution coefficient (Ka) of 0.50 is reached. In addition,
the percentages eluted before this distribution coefficient are
within the limits approved for the particular producto
Identification and serological specificity
The identity and serological specificity are determined by a
suitable immunochemical method (2.7.1). Identity and purity
of each polysaccharide shall be confirmed; it shall be shown
that there is not more than 1 per cent m/m of
group-heterologous N. meningitidis polysaccharide.
Vaccines IV-573
Pyrogens (2.6.8)
The polysaccharide complies with the test for pyrogens.
Inject into each rabbit per kilogram of body mass 1 mL of a
solution containing 0.025 flg of purified polysaccharide per
millilitre.
FINAL BULK VACCINE
One or more purified polysaccharides of 1 or more N.
meningitidis groups are dissolved in a suitable solvent that
may contain a stabiliser. When dissolution is complete, the
solution is filtered through a bacteria-retentive filter.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile
containers. The containers are then c10sed so as to avoid
contamination.
Only a final lot that is satisfactory with respect to each of the
requirements prescribed below under Identification, Tests
and Assay may be released for use.
CHARACTERS
A white or cream-coloured powder or pellet, freely soluble in
water.
IDENTIFlCATION
Carry out an identification test for each polysaccharide
present in the vaccine by a suitable immunochemical method
(2.7.1).
TESTS
Distribution of molecular size
Examine by size-exclusion chromatography (2.2.30) . Use a
column about 0.9 m long and 16 mm in internal diameter
equilibrated with a solvent having an ionic strength of 0.2
mol/kg and a pH of 7.0-7.5. Apply to the column about
2.5 mg of each polysaccharide in a volume of about 1.5 mL
and elute at about 20 mlJh. Collect fractions of about
2.5 mL and determine the content of polysaccharide by a
suitable method.
For a divalent vaccine (group A + group C), use cross-linked
agarose for chromatography R. The vaccine complies with the
test if:
- 65 per cent of group A polysaccharide is eluted before
Ko = 0.50,
- 75 per cent of group C polysaccharide is eluted before
Ka = 0.50.
For a tetravalent vaccine (group A + group C + group Y +
group WI35), use cross-linked agarosefor chromatography R1
and apply a suitable immunochemical method (2.7.1) to
establish the elution pattern of the different polysaccharides.
The vaccine complies with the test if Ka for the principal
peak is:
- not greater than 0.70 for group A and group C
polysaccharide,
- not greater than 0.57 for group Y polysaccharide,
- not greater than 0.68 for group W135 polysaccharide.
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
Sterility (2.6.1)
It complies with the test for sterility.
IV-574 Vaccines
Pyrogens (2.6.8)
Ir complies with the test for pyrogens. Inject per kilogram of
the rabbit's mas s 1 mL of a solution containing:
- 0.025 Ilg of polysaccharide for a monovalent vaccine,
- 0.050 Ilg of polysaccharide for a divalent vaccine,
- 0.10 Ilg ofpolysaccharide for a tetravalent vaccine.
ASSAY
Carry out an assay of each polysaccharide present in the
vaccine.
For a divalent vaccine (group A + group e), use
measurement of phosphorus (2.5.18) to determine the
content of polysaccharide A and measurement of sialic acid
(2.5.23) to determine the content of polysaccharide C.
To determine sialic acid, use as reference solution a
150 mg!L solution of N-acezylneuraminic acid R.
For a tetravalent vaccine (group A + group e + group Y +
group W135) a suitable immunochemical method (2. 7.1) is
used with a reference preparation of purified polysaccharide
for each group.
The vaccine contains not les s than 70 per cent and not more
than 130 per cent of the quantity of each polysaccharide
stated on the labe!'
LABELLING
The label sta tes:
- the group or groups of polysaccharides (A, e, y or
W135) present in the vaccine,
- the number of micrograms of polysaccharide per human
dose.
_ _________________________________________
~E~
Mumps Vaccine, Uve ****
*** *
****
(Mumps Vaccine (Live), Ph Eur monograph 0538)
The label may state 'Mumps'.
Ph Eur _ _________________________________________
DEFINITION
Mumps vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of mumps virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a elear liquid that may be coloured owing to the
presence of a pH indicator.
PRODUCTION
The production of vaccine is based on a virus seed-Iot system
and, if the virus is propagated in human diploid cells, a
cell-bank system. The production method shall have been
shown to yield consistently live mumps vaccines of adequate
immunogenicity and safety in mano Unless otherwise justified
and authorised, the virus in the final vaccine shall have
undergone no more passages from the master seed lot than
were used to prepare the vaccine shown in elinical studies to
be satisfactory with respect to safety and efficacy.
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk
analysis is carried out. Where necessary and if available, a
test is carried out on the vaccine strain using an animal
model that differentiates wild-type and attenuated virus; tests
on strains of intermediate attenuation may also be needed.
2014
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3) or in
chick-embryo cells or in the amniotic cavity of chick embryos
derived from a chicken ftock free from specified pathogens
(5.2.2).
SEED LOT
The strain of mumps virus used shall be identified by
historical records that ¡nelude information on the origin of
the strain and its subsequent manipulation. Virus seed lots
are prepared in large quantities and stored at temperatures
below - 20 ce if freeze-dried, or below - 60 oc if not
freeze-dried.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identificarlon
The master and working seed lots are identified as mumps
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentrarlon
The virus concentration of the master and working seed lots
is determined to ensure consistency of production.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots.
PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are handled during the production. Suitable animal (but not
human) serum may be used in the culture media. Serum and
trypsin used in the preparation of cell suspensions and
culture media are shown to be free from extraneous agents.
The cell culture medium may contain a pH indicator such as
phenol red and suitable antibiotics at the lowest effective
concentration. Ir is preferable to have a substrate free from
antibiotics during production. Not less than 500 mL of the
production cell cultures is set aside as uninfected cell cultures
(control cells). If the virus is propagated in chick embryos,
2 per cent but not les s than 20 eggs are set aside as
uninfected control eggs. The viral suspensions are harvested
at a time appropriate to the strain ofvirus being used.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Identificarlon
The single harvest contains virus that is identified as mumps
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentrarlon
The virus concentration in the single harvest is determined as
prescribed under Assay to monitor consistency of production
and to determine the dilution to be used for the final bulk
vaccine.
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous
agents.
Control cells or eggs
If human diploid cells are used for production, the control
cells comply with a test for identification; the control cells
and the control eggs comply with the tests for extraneous
agents (2.6.16).
2014
FINAL BULK VACCINE
Single harvests that comply with the aboye tests are pooled
and c1arified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacteria! and fungal contamination
The final bulk vaccine complies with the test for sterility
(2.6.1), carried out using 10 mL for each medium.
FINALLOT
A minimum virus concentration for releas e of the product is
established such as to ensure, in light of stability data, that
the minimum concentration stated on the label will be
present at the end of the period of validity.
Only a finallot that complies with the requirements for
minimum virus concentration for releas e, with the following
requirement for thermal stability and with each of the
requirements given below under Identification and Tests may
be relea sed for use. Provided that the tests for bovine serum
albumin and, where applicable, for ovalbumin have been
carried out with satisfactory results on the final bulk vaccine,
they may be omitted on the finallot.
Thermal stability
Maintain at least 3 vials of the finallot of freeze-dried
vaccine in the dry state at 37 ± 1 oC for 7 days. Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. The virus
concentration of the heated vaccine is not more than 1.0 log
lower than that of the unheated vaccine.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific mumps antibodies, it is no longer able to
infect susceptible cell cultures.
TESTS
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1).
Bovine serum albumin
Not more than 50 ng per single human dose, determined by
a suitable immunochemical method (2.7.1).
Ovalbumin
If the vaccine is produced in chick embryos, it contains not
more than 1 Jlg of ovalbumin per single human dose,
determined by a suitable irnmunochemical method (2.7.1).
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine and inoculating a suitable number
of wells for each dilution step. Titrate 1 vial of an
appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory.
The relation with the appropriate European Pharmacopoeia
Biological Reference Preparation is established and
monitored at regular intervals if a manufacturer's reference
preparation is used. Calculate the individual virus
concentration for each vial of vaccine and for each replicate
of the reference preparation as well as the corresponding
combined virus concentrations, using the usual statistical
Vaccines IV-575
methods (for example, 5.3). The combined estimate of the
virus concentration for the 3 vials of vaccine is not less than
that stated on the label; the minimum virus concentration
stated on the label is not les s than 3.7 log CCID so per single
human dose.
The assay is not valid if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so;
-- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log CCID so .
Mumps vaccine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label sta tes:
-- the strain of virus used for the preparation of the vaccine;
-- that the vaccine has been prepared in chick embryos or
the type and origin of cells used for the preparation of the
vaccine;
-- the minimum virus concentration;
-- that contact between the vaccine and disinfectants is to be
avoided.
___________________________________________ ffiE~
Human Papillomavirus Vaccine *****
** **
(rDNA) ***
(Ph Eur monograph 2441)
The label may state 'HPV'.
___________________________________________
ffiE~
DEFINITION
Human papillomavirus vaccine (rDNA) is a preparation of
purified virus-like partic1es (VLPs) composed of the major
capsid protein (Ll) of one or more human papillomavirus
(HPV) genotypes; the antigens may be adsorbed on a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate. The vaccine may also contain the
adjuvant 3-0-desacyl-4'-monophosphoryl lipid A.
The antigens are obtained by recombinant DNA technology.
PRODUCTION
GENERAL PROVISIONS
The vaccine shall have been shown to induce specific
neutralising antibodies in manoThe production method shall
have been shown to yield consistently vaccines comparable in
quality with the vaccine of proven clinical efficacy and safety
mman.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
 IV-576 Vaccines
The vaccine is produced by the expression of the viral genes
coding for the capsid proteins in yeast or in an insect
celllbaculovirus expression vector system, purificarion of the
resulring VLPs and the rendering of these partic1es into an
irnmunogenic preparation. The suitability and safety of the
expression systems are approved by the competent authority.
Producrion of the vaccine is based on a seed lotlcell bank
system. Unless otherwise justified and authorised, the virus
and cells used for vaccine producrion shall not have
undergone more passages from the master seed lotlcell bank
than was used to prepare the vaccine shown in c1inical
studies to be satisfactory with respect to safety and efficacy.
Reference preparation A batch of vaccine shown to be
effective in clinical trials or a batch representative thereof is
used as a reference vaccine. The reference vaccine is
preferably stabilised and the stabilisarion method shall have
been shown to have no significant effect on the assay validity.
CHARACTERISAnON
Characterisation of the VLPs is performed on lots produced
during vaccine development, including the process validation
batches. Characterisarion includes protein composirion, for
example using techniques such as sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and Westem
blotting or mass spectrometry, pepride mapping and/or
terminal amino acid sequence analysis. Morphological
characterisrics of the VLPs and degree of aggregation are
determined to confirm the presence of the conformational
epitopes that are essenrial for efficacy. VLP characterisation
may be done by atomic force microscopy and transmission
electron microscopy, dynamic light scattering, epitope
mapping and reactivity with neutralising monoclonal
antibodies. In addition, the protein, lipid, nucleic acid and
carbohydrate content are measured where applicable.
The leve! of residual host-cell protein derived from insect
cells meets acceptable safety criteria as set by the competent
authority.
CELL BANKS AND SEED LOTS
Production in recombinant yeast cells Only cell banks that have
been satisfactorily characterised for identity, microbial purity,
growth characteristics and stability shall be used for
production. Gene homogeneity is studied for the master and
working cell banks. A full description of the biological
characteristics of the host cell and expression vectors is given.
The physiological measures used to promote and control the
expression of the cloned gene in the host cell are described in
detai!. This includes generic markers of the host cell, the
construcrion, genetics and structure of the expression vector,
and the origin and identification of the gene that is being
cloned. The nuc1eotide sequence of the gene insert and of
adjacent segments of the vector and restriction-enzyme
mapping of the vector containing the gene insert are
provided. Data that demonstrates the stability of the
expression system during storage of the recombinant working
cell bank up to or beyond the passage level used for
producrion is provided.
Production in an insect celllbaculovirus expression vector system
- ¡nsect cell substrate. Only cell banks that have been
satisfactorily characterised for identity, purity, growth
characteristics, stability, extraneous agents and
tumorigenicity shall be used for production. Such
characterisation is performed at suitable stages of
production in accordance with general chapters 5.2.3. Gell
substrates for the production of vaccines for human use and
2.6.16. Tests for extraneous agents in viral vaccines for
human use. Special attention is given to insect-bome
2014
viruses, in particular insect-bome potential human
pathogens (e.g. arboviruses). Adventitious infectious
agents of insect cells may be without cytopathic effect.
Tests therefore include nucleic acid amplification
techniques, and other tests such as electron microscopy
and co-cultivation.
- Recombinant baculovirus. The use of the recombinant
baculovirus vector is based on a seed-Iot system with a
defined number of passages between the original virus
and the master and the working seed-Iots, as approved by
the competent authorities. The recombinant baculovirus
expression vector contains the coding sequence of the
HPV Ll antigen. Segments of the expression construct
are analysed using nucleic acid amplification techniques
in conjunction with other tests performed on the purified
recombinant protein for assuring the quality and
consistency of the expressed HPV Ll antigens.
The recombinant baculovirus used in the production of
HPV vaccines is identified by historical records, which
include informarion on the origin and identity of the gene
being cloned as well as the construction, genetics and
structure of the baculovirus expression vectores).
The genetic stability of the expression construct is
demonstrated from the baculovirus master seed up to at
least the highest level used in production and preferably
beyond this leve!.
Recombinant baculovirus seed lots are prepared in large
quantities and stored at temperatures favourable for stability.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
The master and working seed lots are identified by the HPV
type of the inserted gene of origin, by an appropriate method
such as nucleic acid amplification techniques (NA T)
(2.6.21).
Virus concentration
The virus concentrarion of the master and working seed lots
is determined to monitor consistency of producrion.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots and control cells. Special attention is given to
Spiroplasma spp. and insect-bome viruses, in particular
insect-bome potenrial human pathogens (e.g. arboviruses).
PROPAGATION AND HARVEST
AlI processing of the cell banks and baculovirus seed lots and
subsequent cell cultures is done under aseptic condirions in
an area where no other cells are being handled.
Where justified and authorised for production in an insect
celllbaculovirus expression vector system, a stored virus
intermediate culture that complies with the 5 following tests
may be used for virus propagation.
Identification
Each stored virus intermediate culture is identified by HPV
type, by an irnmunological assay using specific antibodies or
by a molecular identity test such as NAT (2.6.21).
Bacteria! and fungal contamination
Each stored virus intermediate culture complies with the test
for sterility (2.6.1), carried out using 10 mL for each
medium.
Virus concentration
The virus concentrarion of each stored baculovirus
intermediate culture is determined by a suitable method such
as plaque assay or NAT (2.6.21) in order to monitor
consistency of production.
2014
Extraneous agents (2.6.16)
Each stored virus intermediate culture complies with the tests
for extraneous agents.
Control cells
The control ceIls of the production ceIl culture from which
each stored virus intermediate is derived comply with a test
for identity and with the requirements for extraneous agents
(2.6.16).
Production in recombinant yeast cells Identity, microbial
purity, plasmid retemion and consistency of yield are
determined at suitable production stages.
Production in an insect celllbaculovirus expression vector
system Insect ceIl cultures are inoculated with recombinant
baculovirus at a defined muItiplicity of infection as approved
by the competent authority. Several single harvests may be
pooled before testing. No antibiotics are added at the time of
harvesting or at any later stage of manufacturing.
SINGLE HARVESTS
Only a single harvest or a pool of single harvests that
complies with the foIlowing requirements may be used in the
preparation of the purified monovalent antigen.
Identification
Each single harvest is identified as the appropriate HPV type
by immunological assay or by a molecular biology-based
assay, for example hybridisation or polymerase chain reaction
(PCR).
Bacterial and fungal contanñnation
In case of production in an insect celllbaculovirus express ion
vector system the single harvest complies with the test for
sterility (2.6.1). In case of production in yeast ceIls the single
harvest is tested for culture purity by inoculation of suitable
medium to ensure no growth other than the host organismo
Extraneous agents (2.6.16)
In case of production in an insect celllbaculovirus expression
vector system the single harvest complies with the tests for
extraneous agents. Special attention is given to insect-borne
viruses as mentioned under CeIl banks and seed lots.
Control cells
In case of production in an insect celllbaculovirus expression
vector system the control ceIls comply with a test for
identification and with the requirements for extraneous
agents (2.6.16). Special attention is given to insect-borne
virus es as memioned under CeIl banks and seed lots.
PURIFIED MONOVALENT ANTIGEN
Harvests are purified using validated methods. When an
insect celllbaculovirus expression vector system substrate is
used, the production process is validated for its capacity to
elimina te (by removal and/or inactivation) adventitious
virus es and recombinant baculoviruses.
Only a purified monovalent antigen that complies with the
foIlowing requiremems may be used in the preparation of the
final bulk. In agreement with the competent authority one or
more of the tests mentioned below may be omitted if
performed on the adsorbed monovalent antigen.
Total protein
The total protein is determined by a validated method.
The content is within the limits approved for the particular
producto
Antigen content and identification
The quantity and specificity of each antigen type is
determined by a suitable immunochemical method (2.7.1)
such as radio-immunoassay (RlA), enzyme-linked
immunosorbent assay (ELISA), immunoblot (preferably
Vaccines IV-577
using a monocIonal antibody directed against a protective
epitope) or single radial diffusion. The antigen/protein ratio
may be determined and is within the Iimits approved for the
particular producto
Antigenic purity
The purity of each purified monovalent antigen is determined
by a suitable method, such as SDS-PAGE with quantification
by densitometric analysis, the limit of detection being
1 per cent of impurities or better with respect to total
protein. A reference preparation is used to validate each test.
The protein purity is calculated as the ratio of the Ll
protein-related bands relative to the total protein bands,
expressed as a percentage. For the genotypes incIuded in the
vaccine, the value calculated for purity is within the limits
approved for the particular producto
Percent intact Ll monomer
The antigenic purity assay serves also to assess the integrity
of the Ll monomer. The percent intact Ll monomer is the
ratio of the intact Ll monomer to the total protein,
expressed as a percentage.
VLP size and structure
The size and structure of the VLPs is established and
monitored by a suitable method such as dynamic Iight
scattering. The size is within the limits approved for the
particular producto
Composition
The protein, lipid, nucleic acid and carbohydrate contents
are determined, where applicable.
Host-cell and vector-derived DNA
Maximum lOng of DNA in a quantity of purified antigen
equivalent to a single human dose of vaccine, determined in
each monovalent purified antigen by sensitive methods.
Residual host-cell proteins
Tests for residual host-ceIl proteins are carried out.
The content is within the limits approved for the particular
producto
Chemicals used for disruption and purification
Tests for the chemicals used for purification or other stages
of production are carried out. The content is within the
limits approved for the particular products.
Sterility (2.6.1)
Each purified monovalent antigen complies with the test,
carried out using 10 mL for each medium.
ADSORBED MONOV ALENT ANTIGEN
The purified monovalent antigens may be adsorbed onto a
mineral vehicIe such as an aluminium salt.
Only an adsorbed monovalent antigen that complies with the
foIlowing requirements may be used in the preparation of the
final bulk.
Sterility (2.6.1)
Each adsorbed monovalent antigen complies with the test,
carried out using 10 mL for each medium.
Bacterial endotoxins (2.6.14)
Each adsorbed monovalent antigen is tested for bacterial
endotoxins. The content is within the limits approved for the
particular producto
Antigen content and identification
Each antigen type is identified by a suitable immunochemical
method (2.7. 1) such as radio-immunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), immunoblot
(preferably using a monoclonal antibody directed against a
protective epitope) or single radial diffusion.
The antigen/protein ratio is determined.
IV-578 Vaccines
Minera! vehic1e concentration
Where applicable, each adsorbed monovalent antigen is
tested for the content of mineral vehicle. The content is
within the limits approved for the particular product.
ADSORBED 3-0-DESACYL-4 ' -MONOPHOSPHORYL LIPID A
BULK
If 3-0-desacyl-4'-monophosphoryllipid A is included in the
vaccine it complies with the monograph 3-0-desacyl-4'-
monophosphoryllipid A (2537). Where 3-0-desacyl-4'-
monophosphoryllipid A is adsorbed prior to inclusion in the
vaccine, the adsorbed 3-0-desacyl-4'-monophosphoryllipid
A bulk complies with the following requirements.
Degree of adsorption of 3-0-desacyI-4'-
monophosphoryllipid A
The content of non-adsorbed 3-0-desacyl-4'-
monophosphoryllipid A in the adsorbed 3-0-desacyl-4 '-
monophosphoryl lipid A bulk is detertnined by a suitable
method, for example gas chromatographic quantification of
the 3-0-desacyl-4'-monophosphoryllipid A (2537) fatty acids in
the supernatant, evaporated to dryness, after centrifugation.
pH (2.2.3)
The pH is within the limits approved for the particular
preparation.
Sterility (2.6.1)
It complies with the test, carried out using 10 mL for each
medium.
FINAL BULK VACCINE
The final bulk vaccine is prepared directly from each purified
monovalent antigen HPV type or adsorbed purified
monovalent antigen HPV type. An antimicrobial preservative,
a mineral vehicle such as an aluminium salt and the adjuvant
3-0-desacyl-4'-monophosphoryllipid A may be included in
the formulation of the final bulk.
Only a final bulk that complies with the following
requirements may be used in the preparation of the finallot.
AntimicrobiaI preservative
Where applicable, detertnine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended content.
Sterility (2.6.1)
The final bulk vaccine complies with the test, carried out
using 10 mL for each medium.
FINALLOT
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
relea sed for use. Provided that the test for antimicrobial
preservative content (where applicable) has been carried out
with satisfactory results on the final bulk vaccine, it may be
omitted on the finallot. If an in vivo assay is carried out,
then provided it has been carried out with satisfactory results
on the final bulk vaccine, it may be omitted on the final lot.
Adjuvants
If the vaccine contains an adjuvant, the amount is
detertnined and shown to be within acceptable limits with
respect to the expected amount. A suitable method for
3-0-desacyl-4'-monophosphoryllipid A is, for example, gas
chromatography.
Degree of adsorption
The degree of adsorption of each antigen and, where
applicable, 3-0-desacyl-4'-monophosphoryllipid A is
assessed.
2014
IDENTIFICATION
The vaccine is shown to contain the different types of HPV
antigen by a suitable irnmunochemÍcal method (2.7.1).
The in vitro assay may serve to identify the vaccine.
In addition, where applicable, the test for 3-0-desacyl-
4 '-monophosphoryl lipid A content also serves to identify the
3-0-desacyl-4'-monophosphoryllipid A-containing vaccine.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
3-0-DesacyI-4'-monophosphoryllipid A
Minimum 80 per cent and maximum 120 per cent of the
intended amount.
Where applicable, detertnine the content of 3-0-desacyl-
4 '-monophosphoryllipid A by a suitable method, for
example gas chromatography (2.2.28).
AntimicrobiaI preservative
Where applicable, detertnine the content of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!'
Sterility (2.6.1)
The vaccine complies with the test.
Bacteria! endotoxins (2.6. 14)
Maximum 5 IU per single human dose. If the adjuvant
prevents the detertnination of endotoxin, a suitable in-process
test is carried out.
ASSAY
The assay is perfortned by an in vivo test or an in vitro test
having acceptance criteria established by correlation studies
against an in vivo test.
In vivo test
A suitable in vivo assay method consists of the injection of
not fewer than 3 dilutions of the vaccine to be examined and
of a reference vaccine preparation, using for each dilution a
group of a suitable number of female mice of a suitable
strain. The vaccine is diluted in a solution of sodium
chloride R containing the aluminium adjuvant used for the
vaccine production. The mice are 6-8 weeks old at the time
of injection, and each mouse is given a 0.5 mL injection.
A preimmunisation serum sample is taken prior to
inoculation, and a final serum sample is taken at a defined
time between days 21 and 28. Assay the individual sera for
specific neutralising antibodies against each HPV type by a
suitable immunochemical method (2.7.1) .
The test is not valid unless:
- for both the vaccine to be examined and the reference
vaccine, the ED 50 lies between the smallest and the
largest doses given to the animals;
- the statistical analysis shows no significant deviation from
linearity or parallelism;
- the confidence limits (P = 0.95) are within the limits
approved for the particular producto
In vitro test
Carry out an immunochemical detertnination (2. 7. 1) of each
antigen genotype contento Enzyrne-linked irnmunosorbent
assay (ELISA) and radio-immunoassay (RIA) using
monoclonal antibodies specific for protection-inducing
epitopes of the Ll protein have been shown to be suitable.
Suitable numbers of dilutions of the vaccine to be examined
2014
and a manufacturer's reference preparation are used and a
suitable model is used to analyse the data. For each type, the
antigen content is within the limits approved for the
particular product.
LABELLING
The label states:
-- the amount of Ll proteins and the genotype of HPV
contained in the vaccine;
-- the cell substrate used for production of the vaccine;
-- the name and amount of the adjuvant and/or adsorbent
used;
-- that the vaccine must not be frozen .
_ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ __ _ _ _ Ph Eur
Pertussis Vaccine (Whole Cell, *** .
** ***
* contain B. pertussis cells with the same characteristics with
Adsorbed) ***
(Ph Eur monograph 0161)
The labe! may state 'wP'.
~Ew _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _
DEFINITION
Pertussis vaccine (whole cell, adsorbed) is a sterile suspension
of inactivated whole cells of one or more strains of Bordetella
pertussis, treated to minimise toxicity and retain potency.
The vaccine contains a mineral adsorbent such as hydrated
aluminium phosphate or aluminium hydroxide.
PRODUCTION
GENERAL PROVISIONS
The production process shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
Levels of pertussis toxin, active heat-Iabile toxin
(dermonecrotic toxin) or tracheal cytotoxin must be
comparable to the levels present in the vaccine of proven
clinical efficacy and safety in man and be approved by the
compete:!t authority.
CHOICE OF VACCINE STRAIN
The vaccine consists of a mixture of one or more strains
of B. pertussis. Strains of B. pertussis used in preparing
vaccines are well characterised and chosen in such a way that
the final vaccine contains predominantly phase 1 cells that
display fimbria e 2 and 3, as determined by an agglutination
test or other suitable immunochemical method (2.7.1) .
SEED LOTS
The production of pertussis vaccine is based on a seed-Iot
system.
The strains of B. pertussis used are identified by a full
historical record, including information on the origin of the
strain and its subsequent manipulation, characteristics on
isolation, and particularly on al! tests carried out periodical!y
to verifY the strain's characters.
The media chosen for growing B. pertussis are carefully
selected and enable the micro-organism to retain phase 1
characteristics.
When animal blood or animal blood products are used, they
are removed by washing the harvested bacteria.
Human blood or human blood products are not used in any
culture media for propagating bacteria, either for seed or for
vaccine .
Vaccines IV-579
PROPAGATION AND HARVEST
Each strain is grown separately from the working seed lot.
Cultures are checked at different stages of fermentation
(subcultures and main culture) for purity, identity, cel!
opacity and pH. Unsatisfactory cultures must be discarded.
Production cultures are shown to be consistent in respect of
growth rate, pH and yield of cells or cell products.
The bacteria are harvested and may be washed to remove
substances derived from the medium and suspended in a
9 giL solution of sodium chloride or other suitable isotonic
solution.
MONOVALENT CELL HARVEST
Consistency of production is monitored in respect of growth
rate, pH, yie!d and demonstration of characteristics of phase
1 organisms in the culture, such as presence of fimbriae 2
and 3 and haemolytic activity. Single harvests are not used
for the final bulk vaccine unless they have been shown to
regard to growth and agglutinogens as the parent strain, and
to be free from contaminating bacteria and fungi .
Only a monovalent harvest that complies with the following
requirements may be used in further production.
Purity
Samples of single harvests taken before inactivation are
examined by microscopy of Gram-stained smears or by
inoculation into appropriate culture media or by another
suitable procedure.
Opacity
The opacity of each single harvest is measured not later than
2 weeks afrer harvest and before the bacterial suspension has
been subjected to any process capable of altering its opacity,
by comparison with the Intemational Reference Preparation
of Opacity, and used as the basis of calculation for
subsequent stages in vaccine preparation. The equivalence in
Intemational Units of the Intemational Reference
Preparation is stated by the World Health Organization.
A spectrophotometric method validated against the opacity
reference preparation may be used and absorbance may, for
example, be measured at 600 nm (2.2.25).
INACTIVATION AND DETOXIFICATION OF B. PERTUSSIS
SUSPENSION
Inactivation is initiated as soon as possible afrer taking
samples of single harvests for purity control and opacity
measurement. The bacteria are killed and detoxified in
controlled conditions by means of a suitable chemical agent
or by heating or by a combination of these methods .
The suspension is maintained at 5 ± 3 oC for a suitable
period to diminish its toxicity.
Only an inactivated monovalent cell bulk that complies with
established specifications for the following tests may be used
in the preparation of the final bulk.
Residual live B. pertussis
Inactivation of the whole cells of B. pertussis is verified by a
suitable culture medium.
Pertussis toxin
Presence of pertussis toxin is measured by a CHO cell
culture assay using a semi-quantitative technique and range
determined for the particular product.
pH (2.2.3)
Within the range approved for the particular product.
Identification
Verified by agglutination assay or suitable immunodiffusion
assay.
IV-580 Vaccines
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
Opacity
The opacity of each single harvest is measured in the final
phase, at the end of the main fermentation process, by
comparison with the International Reference Preparation of
Opacity. The equivalence in International Vnits of the
International Reference Preparation is stated by the World
Health Organization. The absorbance, for example measured
at 600 nm (2.2.25), is within the range approved for the
particular product.
FINAL BULK
The final bulk vaccine is prepared by aseptically mixing
suitable quantities of the inactivated single harvests.
If 2 or more strains of B. pertussis are used, the composition
of consecutive lots of the final bulk vaccine shall be
consistent with respect to the proportion of each strain as
measured in opacity units. The bacterial concentration of the
final bulk vaccine do es not exceed that corresponding to an
opacity of 20 IV per single human dose. The opacity
measured on the single harvests is used to calculate the
bacterial concentration in the final bulk. A mineral adsorbent
such as hydrated aluminium phosphate or aluminium
hydroxide is added to the cell suspension. Suitable
antimicrobial preservatives may be added. Phenol is not used
as a preservative.
Only a final bulk that complies with the following
requirements may be used in the preparation of the finallot.
Fimbriae
Each bulk is examined, before adsorbent is added, for the
presence of fimbriae 2 and 3 to ensure that appropriate
expression has occurred during bacterial growth.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended amount.
FINALLOT
The final bulk is mixed to homogeneity and filled aseptically
into suitable containers.
Only a final lot that is within the limits approved for the
particular product and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
Assay may be released for use. Provided the tests for specific
toxicity, free formaldehyde and antimicrobial preservative and
the determination of potency have been carried out with
satisfactory results on the final bulk vaccine, they may be
omitted on the finallot.
IDENTIFICATION
Dissolve in the vaccine to be examined sufficient sadium
eitrate R to give a 100 gIL solution. Maintain at 37 oC for
about 16 h and centrifuge to obtain a bacterial precipitate.
Identity of pertussis vaccine is based on an irnmunological
reaction, for example agglutination of the resuspended
bacteria with a specific anti-pertussis serum or another
suitable immunochemical method (2.7.1).
2014
TESTS
Specific toxicity
Vse not fewer than 5 healthy mice each weighing 14-16 g for
the vaccine group and for the saline control. Use mi ce of the
same sex or distribute males and females equally between the
groups. Inject each mouse of the vaccine group
intraperitoneally with 0.5 mL, containing a quantity of the
vaccine equivalent to not less than half the single human
dose. Inject each mouse of the control group with 0.5 mL of
a 9 gIL sterile solution of sadium eh/aride R, preferably
containing, where applicable, the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 h and 7 days after the injection. The vaccine
complies with the test if: (a) at the end of 72 h the average
weight of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mass per vaccinated mouse is not less than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test. If the
test is carried out using 5 mice and 1 vaccinated mouse dies,
the test may be repeated using 15 mice and the results of
both tests combined.
AIuminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL, where applicable.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
label.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Carry out the assay of pertussis vaccine (whole cel!) (2.7.7) .
The estimated potency is not less than 4 .0 IV per single
human dos e and the lower confidence limit (P = 0.95) of the
estimated potency is not less than 2.0 IV per single human
dose.
LABELLING
The label states:
-- the minimum number of International Vnits per single
human dos e;
-- the method used for inactivation;
-- the name and the amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ ~Ew
 2014
Adsorbed Pertussis Vaccine ***
*** ***
(Acellular Component) ***
(Pertussis Vaccine (Acellular, Component, Adsorbed),
Ph Eur monograph 1356)
The label may state 'aP' .
~E~ ____________________________________________
DEFINITION
Pertussis vaccine (acellular, component, adsorbed) is a
preparation of individually prepared and purified antigenic
components of Bordetella pertussis adsorbed on a mineral
carrier such as aluminium hydroxide or hydrated aluminium
phosphate.
The vaccine contains either pertussis toxoid or a
pertussis-toxin-like protein free from toxic properties,
produced by expression of a genetically modified form of the
corresponding gene. Pertussis toxoid is prepared from
pertussis toxin by a method that renders the latter harmless
while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa
outer-membrane protein) and other defined components of
B . pertussis such as fimbrial-2 and fimbrial-3 antigens .
The latter 2 antigens may be co-purified. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the
target group for which the vaccine is intended.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
c1inical efficacy and safety in mano
Where a genetically modified form of B. pertussis is used,
production consistency and genetic stability shall be
established in confonnity with the requirements of the
monograph Products of recombinant DNA technology (0784).
R eference vaccine A batch of vaccine shown to be effective in
c1inical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the
batch tested in c1inical trials is necessary. The reference
vaccine is preferably stabilised by a method that has been
shown to have no significant effect on the assay procedure
when the stabilised and non-stabilised batches are compared.
CHARACTERISATION OF COMPONENTS
During development of the vaccine, the production process
shall be validated to demonstrate that it yields consistently
individual components that comply with the following
requirements; after demonstration of consistency, the tests
need not be applied routinely to each batch.
Adenylate cyclise
Not more than 500 ng in the equivalent of 1 dose of the final
vaccine, determined by immunoblot analysis or another
suitable method.
Tracheal cytotoxin
Not more than 2 pmol in the equivalent of 1 dos e of the final
vaccine, determined by a suitable method such as a biological
assay or liquid chromatography (2.2.29) .
Absence of residual dermonecrotic toxin
Inject intradennally into each of 3 unweaned mice, in a
volume of 0.1 mL, the amount of component or antigenic
fraction equivalent to 1 dose of the final vaccine. Observe for
48 h. No dermonecrotic reaction is demonstrable.
Vaccines IV-581
Specific properties
The components of the vaccine are analysed by one or more
of the methods shown below in order to detennine their
identity and specific properties (activity per unit amount of
protein) in comparison with reference preparations.
Pertussis toxin Chinese hamster ovary (CHO) cell-c1ustering
effect and haemagglutination as in vitro methods;
Iymphocytosis-promoting activity, histamine-sensitising
activity and insulin secretory activity as in vivo methods .
The toxin shows ADP-ribosyl transferase activity using
transducin as the acceptor.
Filamentous haemagglurinin Haemagglutination and
inhibition by a specific antibody.
Pertactin, fimbrial-2 and fimbrial-3 antigens Reactivity with
specific antibodies.
Pertussis toxoid The toxoid induces in animals production of
antibodies capable of inhibiting all the properties of pertussis
toxin.
PURIFIED COMPONENTS
Production of each component is based on a seed-Iot system.
The seed cultures from which toxin is prepared are managed
to conserve or, where necessary, restore toxinogenicity by
deliberate selection.
None of the media used at any stage contains blood or blood
products of human origino Media used for the preparation of
seed lots and inocula may contain blood or blood products of
animal origino
Pertussis toxin and, where applicable, filamentous
haemagglutinin and pertactin are purified and, after
appropriate characterisation, detoxified using suitable
chemical reagents, by a method that avoids reversion of the
toxoid to toxin, particularly on storage or exposure to heat.
Other components such as fimbrial-2 and fimbrial-3 antigens
are purified either separately or together, characterised and
shown to be free from toxic substances. The purification
pro ce dure is validated to demonstrate appropriate c1earance
of substances used during culture or purification.
The content of bacterial endotoxins (2.6.14) is detennined to
monitor the purification procedure and to limit the amount
in the final vaccine. The limits applied for the individual
components are such that the final vaccine contains less than
100 IV per single human dose.
Before detoxification, the purity of the components is
detennined by a suitable method such as polyacrylamide gel
electrophoresis (PAGE) or liquid chromatography.
SDS-PAGE or immunoblot analysis with specific monoc1onal
or polyc1onal antibodies may be used to characterise
subunits. Requirements are established for each individual
product.
Only purified components that comply with the fo llowing
requirements may be used in the preparation of the final bulk
vaccme.
Sterility (2.6.1)
Carry out the test for sterility using for each medium a
quantity of purified component equivalent to not less than
100 doses.
Residual pertussis toxin (2. 6.33)
It complies with the test.
A validated test based on the c1ustering effect of the toxin for
Chinese hamster ovary (CHO) cells may be used instead of
the test in mice.
 IV-582 Vaccines
Residual detoxifying agents and other reagents
The content of residual detoxifying agents and other reagents
is determined and shown to be below approved limits unless
validation of the process has demonstrated acceptable
clearance.
Antigen content
Determine the antigen content by a suitable
immunochemical method (2.7.1) and protein nitrogen by
sulfuric acid digesiion (2.5.9) or another suitable method.
The ratio of antigen content to protein nitrogen is within the
limits established for the product.
FINAL BULK VACCINE
The vaccine is prepared by adsorption of suitable quantities
of purified components, separately or together, onto
aluminium hydroxide or hydrated aluminium phosphate.
A suitable antimicrobial preservative may be added.
OnJy a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
Antirnicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended contento
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
OnJy a final lot that is satisfactory with respect to each of the
requirements given be!ow under Identification, Tests and
Assay may be released for use. Provided that the tests for
residual pertussis toxin and irreversibility of pertussis toxoid,
antimicrobial preservative, free formaldehyde and the assay
have been carried out with satisfactory results on the final
bulk vaccine, these tests may be omined on the finallot.
IDENTIFICATION
Subject the vaccine to a suitable desorption procedure such
as the following: dissolve in the vaccine to be examined
sufficient sbdium citrate R ro give a 10 gIL solution; maintain
at 37 oC for about 16 h and centrifuge until a clear
supematant liquid is obtained. Examined by a suitable
immunochemical method (2.7.1), the clear supematant liquid
reacts with specific antisera to the components stated on the
labe!'
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dos e, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not les s than the minimum amount
shown to be effective and is not greater than 115 per cent of
the quantity stated on the labe!'
Sterility (2.6.1)
It complies with the test for sterility.
2014
ASSAY
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2. 7.16).
The capacity of the vaccine to induce antibodies for each
included acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
LABELLING
The label states:
-- the names and amounts of the components present in the
vaccine;
-- where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification;
-- the name and amount of the adsorbent;
-- that the vaccine must be shaken before use;
-- that the vaccine is not to be frozen.
____________________________________________ Ph E~
Adsorbed Pertussis Vaccine ***
* **
*
(Acellular, Co-purified) **** *
(Pertussis Vaccine (Acellular, Co-purified, Adsorbed),
Ph Eur monograph 1595)
The label may state 'aP'.
~E~ ____________________________________________
DEFINITION
Pertussis vaccine (acellular, co-purified, adsorbed) is a
preparation of antigenic components of Bordetella pertussis
adsorbed on a mineral carrier such as aluminium hydroxide
or hydrated aluminium phosphate .
The vaccine contains an antigenic fraction purified without
separation of the individual components. The antigenic
fraction is treated by a method that transforms pertussis toxin
to toxoid, rendering it harmless while maintaining adequate
immunogenic properties of all the components and avoiding
reversion to toxin. The antigenic fraction is composed of
pertussis toxoid, filamentous haemagglutinin, pertactin (a 69
kDa outer-membrane protein) and other defined components
of B. pertussis such as fimbrial-2 and fimbrial-3 antigens.
It may contain residual pertussis toxin up to a maximum
leve! approved by the competent authority. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the
target group for which the vaccine is intended.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano
Reference vaccine A batch of vaccine shown to be effective in
clinical trials or a batch representative thereof is used as a
reference vaccine. For the preparation of a representative
batch, strict adherence to the production pro ces s used for the
batch tested in clinical trials is necessary. The reference
vaccine is preferably stabilised, by a method that has been
shown to have no significant effect on the assay procedure
when the stabilised and non-stabilised batches are compared.
CHARACTERISATION OF COMPONENTS
During development of the vaccine, the production process
shall be validated to demonstrate that it yields consistently an
antigenic fraction that complies with the following
2014
requirements; after demonstration of consistency, the tests
need not be applied routinely to each batch.
Adenylate cyclise
Not more than 500 ng in the equivalent of 1 dose of the final
vaccine, determined by immunoblot analysis or another
suitable method.
Tracheal cytotoxin
Not more than 2 pmol in the equivalent of 1 dose of the final
vaccine, determined by a suitable method such as a biological
assay or liquid chromatography (2.2.29).
Absence of residual dennonecrotic toxin
Inject intradermally into each of 3 unweaned mice, in a
volume of 0.1 mL, the amount of antigenic fraction
equivalent to 1 dose of the final vaccine. Observe for 48 h.
No dermonecrotic reaction is demonstrable.
Specific properties
The antigenic fraction is analysed by one or more of the
methods shown below in order to determine the identity and
specific properties (activity per unit amount of protein) of its
components in comparison with reference preparations.
Pertussis toxin Chinese hamster ovary (CHO) cell-clustering
effect and haemagglutination as in vitro methods;
Iymphocytosis-promoting activity, histamine-sensitising
activity and insulin secretory activity as in vivo methods.
The toxin shows ADP-ribosyl transferase activity using
transducin as the acceptor.
Fzlamenwus haemagglutinin Haemagglutination and
inhibition by a specific antibody.
Pertactin, fimbrial-2 and fimbrial-3 antigens Reactivity with
specific antibodies.
Pertussis wxoid The toxoid induces in animals the
production of antibodies capable of inhibiting all the
properties of pertussis toxin.
PURIFIED ANTIGENIC FRACTION
Production of the antigenic fraction is based on a seed-Iot
system. The seed cultures are managed to conserve or, where
necessary, restore toxinogenicity by deliberate selection.
None of the media used at any stage contains blood or blood
products of human origino Media used for the preparation of
seed batches and inocula may contain blood or blood
products of animal origino
The antigenic fraction is purified and, after appropriate
characterisation, detoxified using suitable reagents by a
method that ensures minimal reversion of toxoid to toxin,
particularly on storage or exposure to heat. The purification
procedure is validated to demonstrate appropriate clearance
of substances used during culture or purification.
The content of bacterial endotoxins (2.6.14) is determined to
monitor the purification procedure and to limit the amount
in the final vaccine. The limits applied are such that the final
vaccine contains not more than 100 IU per single human
dose.
Before detoxification, the purity of the antigenic fraction is
determined by a suitable method such as polyacrylamide gel
electrophoresis (PAGE) or liquid chromatography.
SDS-PAGE or immunoblot analysis with specific monoclonal
or polyclonal antibodies may be used to characterise
subunits. Requirements are established for each individual
product.
Only a purified antigenic fraction that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Vaccines IV-583
Sterility
Carry out the test for sterility (2.6.1) using for each medium
a quantity of purified antigenic fraction equivalent to not less
than 100 doses of the final vaccine.
Residual pertussis toxin (2.6.33)
The purified antigenic fraction complies with the test.
A validated test based on the clustering effect of the toxin for
Chinese hamster ovary (CHO) cells may be used instead of
the test in mice.
Residual detoxifying agents and other reagents
The content of residual detoxifYing agents and other reagents
is determined and shown to be below approved limits unless
validation of the process has demonstrated acceptable
clearance.
Antigen content
Determine the complete quantitative antigen composition of
the antigenic fraction by suitable immunochemical methods
(2.7.1) and protein nitrogen by sulfuric acid digestion (2.5.9)
or another suitable method. The ratio of total antigen
content to protein nitrogen is within the limits established for
the producto
FINAL BULK VACCINE
The vaccine is prepared by adsorption of a suitable quantity
of the antigenic fraction onto aluminium hydroxide or
hydrated aluminium phosphate. A suitable antimicrobial
preservative may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not
greater than 115 per cent of the intended contento
Sterility
The final bulk vaccine complies with the test for sterility
(2.6.1), carried out using 10 mL for each medium.
FINALLOT
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be relea sed for use.
Provided that the tests for residual pertussis toxin and
irreversibility of pertussis toxoid, antimicrobial preservative,
free formaldehyde and the assay have been carried out with
satisfactory results on the final bulk vaccine, these tests may
be omitted on the finallot.
IDENTIFlCATION
Subject the vaccine to a suitable desorption procedure such
as the following: dissolve in the vaccine to be examined
sufficient sodium citrate R to give a 10 gIL solution; maintain
at 37 oC for about 16 h and centrifuge until a clear
supernatant liquid is obtained. Examined by a suitable
immunochemical method (2.7.1), the clear supernatant liquid
reacts with specific antisera to the components in the vaccine.
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The finallot complies with the test.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
the quantity stated on the labe!'
 IV-584 Vaccines
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Sterility
It complies with the test for sterility (2.6.1).
ASSAY
Carry out one of the prescribed methods for the assay of
pertussis vaccine (acellular) (2. 7.16).
The capacity of the vaccine to induce antibodies for each
included acellular pertussis antigen is not significantly
(P = 0.95) les s than that of the reference vaccine.
LABELLING
The label states:
- the names and amounts of the antigenic components
present in the vaccine,
- the maximum amount of residual pertussis toxin present
in the vaccine,
- the maximum degree of reversion of toxoid to toxin
during the period of validity,
- the name and amount of the adsorbent,
- that the vaccine must be shaken before use,
- that the vaccine is not to be frozen.
_ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ __ _ _ PhEur
Pneumococcal Polysaccharide ***
***
Vaccine ***
(Ph Eur monograph 0966)
The label may state 'Pneumo'.
PhE~ _ _ _ _ _ __ __ _ _ _ __ _ _ _ _ _ _ __
DEFINITION
Pneumococcal polysaccharide vaccine consists of a mixture of
equal parts of purified capsular polysaccharide antigens
prepared from suitable pathogenic strains of Streptococcus
pneumoniae whose capsules have been shown to be made up
of polysaccharides that are capable of inducing satisfactory
levels of specific antibodies in mano It contains the 23
immunochemically different capsular polysaccharides listed in
Table 0966-1 .
The vaccine is a c1ear, colourless liquid.
PRODUCTION
Production of the vaccine is based on a seed-Iot system for
each type. The production method shall have been shown to
yield consistently pneumococcal polysaccharide vaccines of
adequate safety and immunogenicity in mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for irnmunosera and vaccines for human use (2.6.9)
modified as follows for the test in guinea-pigs: inject 10
human doses into each guinea-pig and observe for 12 days.
MONOVALENT BULK POLYSACCHARIDES
The bacteria are grown in a suitable liquid medium that does
not contain blood-group substances or high-molecular-mass
polysaccharides. The bacterial purity of the culture is verified
and the culture is inactivated with phenol. Impurities are
removed by such techniques as fractional precipitation,
enzymatic digestion and ultrafiltration. The polysaccharide is
2014
obtained by fractional precipitation, washed, and dried in a
vacuum to a residual moisture content shown to be
favourable to the stability of the polysaccharide. The residual
moisture content is determined by drying under reduced
pressure over diphosphorus pentoxide or by
thermogravimetric analysis and the value obtained is used to
calculate the results of the tests shown below with reference
to the dried substance. The monovalent bulk polysaccharide
is stored at a suitable temperature in conditions that avoid
the uptake of moisture.
Only a monovalent bulk polysaccharide that complies with
the following requirements may be used in the preparation of
the final bulk vaccine. Percentage contents of components,
determined by the methods prescribed below, are shown in
Table 0966-l.
Protein (2.5. 16).
Nuc1eic acids (2.5.17).
Total nitro gen (2.5.9).
Phosphorus (2.5.18).
Molecular size
Determine by size-exclusion chromatography (2.2.30) using
cross-linked agarose for chromatography R or cross-linked agarose
for chromatography R1.
Uronic acids (2.5.22).
Hexosarnines (2.5.20).
Methylpentoses (2.5.21).
O-AcetyI groups (2.5.19).
Identification (2. 7.1)
Confirm the identity of the monovalent bulk polysaccharide
*
*
*
by double immunodiffusion or electroimmunodiffusion
(except for polysaccharides 7F, 14 and 33F), using specific
antisera.
Specificity
No reaction occurs when the antigens are tested against all
the antisera specific for the other polysaccharides of the
vaccine, including factor sera for distinguishing types within
groups. The polysaccharides are tested at a concentration of
50 ¡.tglmL using a method capable of detecting 0.5 ¡.tg/mL.
FINAL BULK VACCINE
The final bulk vaccine is obtained by aseptically mixing the
different polysaccharide powders . The uniform mixture is
aseptically dissolved in a suitable isotonic solution so that one
human dose of 0.50 mL contains 25 ¡.tg of each
polysaccharide. An antimicrobial preservative may be added.
The solution is sterilised by filtration through a
bacteria-retentive filter.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
AntimicrobiaI preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers.
Only a final lot that is satisfactory with respect to each of the
requirements given below under identification, tests and assay
 2014 Vaccines IV-585

Table 0966.-1 - Percentage contents of components of monovalent bulk polysaccharides

Molecular Protein Nuc\eic Total Phosphorus Molecular size Uronic Hexos- Methyl- O-acetyl
type' acids nitrogen (K) acids amines pentoses Groups
-- ---
1 52 52 3.5·6 0·1.5 50. 15 " 45 " 1.8
2 52 $2 0-1 0-1.0 50.15 " 15 " 38
3 55 $2 0-1 0·1.0 50.15 " 40
4 53 52 4-6 0·1.5 50.15 " 40
5 5 7.5 52 2.5·6.0 52 50.60 " 12 ;, 20

68 52 ,;2 0-2 2.5·5.0 ,; 0.50 " 15

7F 55 52 1.5-4.0 0-1.0 50.20 "13

8 52 $2 0·1 0-1.0 $ 0.15 " 25

9N $2 $1 2.2-4 0-1.0 50.20 " 20 ;, 28

9V $2 ,;2 0.5-3 0·1.0 $ 0.45 " 15 ,,13

lOA $ 7 52 0.5-3.5 1.5-3.5 $ 0.65 " 12

llA $3 $2 0-2.5 2.0-5.0 $ 0.40 " 9
12F $3 ,;2 3-5 0·1.0 ,; 0.25 " 25
14 ~5 $2 1.5-4 0-1.0 $ 0.30 " 20
158 53 52 1-3 2.0-4.5 50.55 " 15
17A or17F ,;2 52 0·1.5 0-3.5 $ 0.45 " 20
18e ,;3 ,;2 0·1 2.4-4.9 ,; 0.15 " 14
19A 52 52 0.6-3.5 3.0·7.0 50.45 ;, 12 " 20
19F $3 $2 1.4-3.5 3.0·5.5 $ 0.20 " 12.5 " 20
20 $2 $2 0.5·2.5 1.5-4.0 $ 0.60 " 12
22F ,;2 ,;2 0-2 0·1.0 ,; 0.55 " 15 " 25
23F 52 52 0-1 3.0-4.5 50.15 " 37
33F ,;2.5 52 0·2 0-1.0 50.50

- Th e different types are indicated using the Danish nomenclature.

_. Cross-linked agarose for chromatography R.

_•• Cross-linked agarose for chromatography Rl.

may be relea sed for use. Provided that the tests for phenol
and for antimicrobial preservative have been carried out with
satisfactory results on the final bulk vaccine, they may be
omitted on the final lot. When consistency of production has
been established on a suitable number of consecutive
batches, the assay may be replaced by a qualitative test that
identifies each polysaccharide, provided that an assay has
been performed on each monovalent bulk polysaccharide
used in the preparation of the final lot.
IDENTIFICAnON
The assay serves also to identify the vaccine.
TESTS
pH (2.2.3)
The pH of the vaccine is 4.5 to 7.4.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantiry stated on the
labe!'
Phenol (2.5.1 S)
Not more than 2.5 gIL.
Sterility (2. 6.1)
It complies with the test for sterility.
Pyrogens (2.6.8)
It complies with the test for pyrogens. Inject per kilogram of
the rabbit's mass 1 mL of a dilution of the vaccine
containing 2.5 flg/mL of each polysaccharide.
ASSAY
Determine the content of each polysaccharide by a suitable
immunochemical method (2.7.1), using antisera specific for
each polysaccharide contained in the vaccine, inc1uding factor
sera for types within groups, and purified polysaccharides of
each type as standards.
The vaccine contains not less than 70 per cent and not more
than 130 per cent of the quantity stated on the label for each
polysaccharide. The confidence limits (P = 0.95) are not les s
than 80 per cent and not more than 120 per cent of the
estimated contento
LABELLING
The label states:
- the number of micrograms of each polysaccharide per
human dose,
IV-586 Vaccines
- the total amount of polysaccharide in the container.
_ _ _ _ _ __ _ __ _ __ _ _ __ _ __ _ _ PhEur
Pneumococcal Polysaccharide ***
*** ***
Conjugate Vaccine (Adsorbed) ***
(Ph Eur monograph 2150)
The label may state 'Pneumo(conj)'.
~E~ _ __ _ __ _ __ _ _ __ __ _ _ _ _ __ __
DEFINITION
Pneumococcal polysaccharide conjugate vaccine (adsorbed) is
a sterile suspension of purified capsular polysaccharides
obtained from Streptococcus pneumoniae serotypes individually
conjugated to a carrier protein. The carrier protein used may
vary for the various polysaccharide conjuga tes contained in a
multivalent vaccine. The vaccine may be adsorbed on a
suitable adjuvant or adsorbant.
Each serotype, produced from suitab1e pathogenic strains of
S. pneumoniae, is grown in an appropriate medium.
The individual polysaccharides are purified through suitable
purification methods (for example centrifugation,
precipitation, ultrafiltration and column chromatography).
Each polysaccharide has a defined composition and a defined
molecular size range.
The choice of polysaccharide depends on the frequency of
the serotypes responsib1e for acute pathologies and their
geographical distribution. The vaccine contains
imrnunochemically different capsular polysaccharides.
PRODVCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently S. pneumoniae conjugate vaccines of adequate
safety and immunogenicity in mano The production of
polysaccharides and of the carrier(s) is based on a seed-Iot
system.
During development studies and wherever revalidation is
necessary, a test for pyrogens in rabbits (2.6.8) is carried out.
The vaccine is shown to be acceptable with respect to
absence of pyrogenic activity.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
During development studies and whenever revalidation of the
manufacturing process is necessary, it shall be demonstrated
by tests in animals that the vaccine consistently induces a
T -cell-dependent B-cell immune response.
The stability of the conjugated bulk and/or finallot and
pneumococcal saccharide is evaluated using suitable indicator
tests. Such tests may include determination of molecular size,
quantification of saccharide content and free polysaccharide
content in the conjuga te.
BACTERIAL SEED LOTS
The bacterial strains used for master seed lots shall be
identified by historical records that include information on
their origin and the tests used to characterise the strain.
Cultures obtained from the working seed lot shall have the
same characteristics as the strain that was used to prepare the
master seed lot.
Purity of bacterial cultures is verified by methods of suitable
sensitivity. These may include inoculation into suitable
2014
media, examination of colony morphology, microscopic
examination of Gram-stained smears and culture
agglutination with suitable specific antisera.
PNEUMOCOCCAL POLYSACCHARIDES
Each strain of S. pneumoniae serotypes is individually grown
in a liquid medium that does not contain high-molecular-
mass polysaccharides; if any ingredient of the medium
contains blood-group substances, the process is validated to
demonstrate that after the purification step they are no longer
detectable. The bacterial purity of the culture is verified by
suitable methods. The culture is then inactivated. Each
polysaccharide is separated from the liquid culture and
purified by suitable methods. Volatile matter, including
water, in the purified polysaccharide is determined by a
srutable method such as thermogravimetry (2.2.34),
semi-micro determination of water (2.5.12) or, where
applicable, determination of solvent and/or alcohol content
by spectrometry. The values are used to calcula te the results
of other chemical tests with reference to the dried substance,
as prescribed below.
0nly polysaccharides that comply with the following
requirements may be used in the preparation of the
conjugate.
Identification
Each polysaccharide is identified by an immunochemical
method (2.7.1) or other suitable methods, for example 1H
nuclear magnetic resonance spectrometry (2.2.33).
Protein (2.5.16)
Depending on the serotype used, not more than the limit
approved for the product, calculated with reference to the
dried substance.
Nucleic acid (2.5.17)
Depending on the serotype used, not more than the limit
approved for the product, calculated with reference to the
dried substance.
Molecular size
The molecular size is evaluated by liquid chromatography
(2.2.29) with multiple-angle laser light scattering detection
(MAllS) or other suitable methods, such as size-exclusion
chromatography (2.2.30) using cross-linked agarose lor
chromatography R or cross-linked agarose lor chromatography R1 .
The values are within the limits approved for each serotype.
A validated determination of the degree of polymerisation or
of the weight-average molecular weight and the dispersion of
molecular masses may be used instead of the determination
of molecular-size distribution.
Bacterial endotoxins (2.6.14)
Less than 0.5 IV of endotoxin per microgram of
polysaccharide.
Residual reagents
Where applicable, suitable tests are carried out to determine
residues of reagents used during inactivation and purification.
An acceptable value for each reagent is established for the
particular product and each batch of polysaccharide must be
shown to comply with this lirnit. Where validation studies
have demonstrated removal of residual reagents, the test on
polysaccharides may be omitted.
Water
Where applicable, the values are within the limits approved
for each serotype, determined by a suitable method.
Depending on the chemical composition of a pneumococcal
polysaccharide serotype, not all of the following tests may be
applicable. The values are within the limits approved.
Suitable limits for sorne pneumococcal polysaccharide
 2014
serotypes are given in the monograph Pneumococcal
polysacchande vaccine (0966).
Total nitro gen (2.5.9).
Phosphorus (2.5.18).
Vronic acids (2.5.22).
Hexosamines (2.5.20).
Methylpentoses (2.5.21).
O-Acetyl groups (2.5.19).
MODIFIED PNEUMOCOCCAL POLYSACCHARIDES
Before conjugation, the polysaccharide can be depolymerised
by chemical or mechanical means foIlowed by a
concentration step to obtain polysaccharides of a desired
molecular size range . Polysaccharides or depolymerised
polysaccharides are modified by an activation process.
Only modified polysaccharides that comply with the
foIlowing requirements may be used in the preparation of the
conjugate.
Molecular size
In the case of a size-reduced modified pneumococcal
polysaccharide, the molecular size is evaluated by liquid
chromatography (2.2.29) with MALLS detection or other
suitable methods, such as size-exclusion chromatography
(2.2.30) using cross-linked agarose for chromatography R or
cross-linked agarose for chromatography R1 . The values are
within the lirnits approved for each serotype.
Degree of oxidation
Where applicable, the degree of oxidation is represented by
the ratio of moles of saccharide repeat unit per mole of
aldehyde and determined by a suitable method. The values
are within the limits approved for each serotype.
CARRIER PROTEIN
The carrier protein is produced by culture of suitable
(including inducible recombinant) micro-organisms;
the bacterial purity of the culture is verified. The culture is
inactivated. The carrier protein is purified by a suitable
method. Suitable tests are carried out, for validation or
routinely, to demonstrate that, where applicable, the product
is free from specific toxins.
Where diphtheria toxoid is used, it is produced as described
in the monograph Diphtheria vaccine (adsorbed) (0443) and
complies with the requirements prescribed therein for bulk
purified toxoid, except that the test for sterility (2.6.1) is not
required.
Where CRM 197 is used as the carrier protein, it is not less
than 90 per cent pure, determined by a suitable method.
Where tetanus toxoid is used as the carrier protein, it is
produced as described in the monograph Tetanus vaccine
(adsorbed) (0452) and complies with the requirements
prescribed therein for purified bulk toxoid, except that the
antigenic purity (2.7.27) is not less than 1500 Lfper
milligram of protein nitrogen and that the test for sterility
(2.6.1) is not required.
Where protein D is used, a specific strain of Escherichia coli is
modified by a plasmid carrying the protein D coding
sequence in order to express this outer-surface protein of
Haemophilus infiuenzae. The modified strain is grown in a
suitable liquid medium to express protein D. At the end of
cultivation, protein D is purified and sterilised by suitable
methods. The product contains not less than 95 per cent of
protein D.
Only a carrier protein that complies with the foIlowing
requirements may be used in the preparation of the
conjugate.
VacCÍnes IV-587
Identification
The carrier protein is identified by a suitable
irnmunochemical method (2. 7.1).
Bacterial endotoxins (2.6.14)
Less than 1 IV per microgram of protein.
Carrier protein
Not less than 90 per cent of the total protein content,
determined by a suitable method.
MONOVALENT BULK CONJUGATE
The conjugate is obtained by the covalent binding of
activated polysaccharides to the carrier protein.
The conjugate purification procedures are designed to
remove residual reagents used for conjugation. The removal
of residual reagents is confirmed by suitable tests or by
validation of the purification process.
Only a bulk conjugate that complies with the foIlowing
requirements may be used in the preparation of the final bulk
vaccine. For each test, limits of acceptance are established
and each batch of conjugate must be shown to comply with
these limits .
Saccharide
The polysaccharide content is determined by a suitable
physical or chemical method or by an irnmunochemical
method (2. 7. 1) . The value complies with the requirement
approved for each serotype.
Protein
The protein content is determined by a suitable physical or
chemical method (for exampJe, 2.5.16). The value complies
with the requirement approved for each serotype.
Saccharide-to-protein ratio
Determine the saccharide-to-protein ratio by calculation.
The value complies with the requirement approved for each
serotype.
Free saccharide
Unbound polysaccharide is determined after removal of the
conjuga te, for example by anion-exchange, size-exclusion or
hydrophobic chromatography, ultrafiltration, or other
validated methods. A value consistent with adequate
immunogenicity as shown in clinical trials is established for
each serotype and each lot must be shown to comply with
this limito
Free carrier protein
Determine the content by a suitable method, either directIy
or by deriving the content by calculation from the resuIts of
other tests. The value complies with the requirement
approved for each serotype.
Molecular size
The molecular size is evaluated by liquid chromatography
(2.2.29) with MALLS detection or other suitable methods.
The values are within the limits approved for each serotype.
Residual reagents
Where applicable, suitable tests are carried out to determine
residues of reagents used during inactivation and purification.
An acceptable value for each reagent is established for the
particular product and each batch of conjugate must be
shown to comply with this limil. Where validation studies
have demonstrated removal of residual reagents, the test on
conjugate polysaccharides may be omitted.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium or the equivalent of 100 doses for each
medium, whichever is less.
 IV-588 Vaccines
Bacteria! endotoxins (2.6.14)
Less than 0.75 IU of endotoxin per microgram of
polysaccharide.
ADSORBED MONOVALENT BULK CON}UGATE
An aluminium-containing adjuvant may be added to each of
the monovalent bulk conjuga tes prior to formulation of the
final bulk. Once the conjugates are adsorbed on a sterile
adjuvant, sterility is assured by aseptic processing.
Only an adsorbed monovalent bulk conjugate that complies
with the following requirements may be used in the
preparation of the final bulk vaccine.
Identification
Each adsorbed polysaccharide conjugate is identified by an
immunochernical method (2.7.1) or other suitable methods.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
or the equivalent of 100 doses for each medium, whichever is
less.
Saccharide
The polysaccharide content is determined by a suitable
physical or chemical method or by an immunochernical
method (2.7. 1). The value complies with the requirement
approved for each serotype.
Free saccharide
Centrifuge the adsorbed monovalent bulk conjugate. In the
supernatant the unbound polysaccharide is determined after
removal of the conjugate, for example by anion-exchange,
size-exclusion or hydrophobic liquid chromatography,
ultrafiltration, or other validated methods. An acceptable
value consistent with adequate immunogenicity as shown in
clinical trials is established for each serotype and each lot
must be shown to comply with this limito
Degree of adsorption
The degree of adsorption of each polysaccharide conjugate is
assessed.
FINAL BULK VACCINE
A final bulk vaccine may be formulated from the individually
adsorbed monovalent bulk conjugates, or from the mixture of
the monovalent bulk conjuga tes that is adsorbed on an
aluminium-containing adjuvant.
Where a final bulk vaccine is formulated as a releas e
intermedia te, it complies with the following requirements and
is within the limits approved for the particular producto
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
or the equivalent of 100 doses for each medium, whichever is
less.
FINALLOT
Only a final lot that is within the limits approved for the
particular product and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
Assay may be released for use.
IDENTIFICATION
Each polysaccharide present in the vaccine is identified by a
suitable immunochemical method (2.7.1).
TESTS
Alurninium (2.5.13)
Maximum 1.25 mg per single human dose.
Sterility (2.6.1)
It complies with the test for sterility.
2014
Bacteria! endotoxins (2.6.14)
Less than 12.5 IU per single human dose, unless otherwise
justified and authorised.
ASSAY
Saccharide content
The polysaccharide content for each serotype is determined
by a suitable immunochemical method (for example,
nephelometry assay or enzyme-linked immunosorbent assay
(EUSA)). The vaccine contains not less than 70 per cent
and not more than 130 per cent of the quantity stated on the
label for each polysaccharide. The confidence limits
(P = 0.95) are not less than 80 per cent and not more than
120 per cent of the estimated contento
LABELLING
The label states:
-- the pneumococcal serotype and carrier protein present in
each single human dose;
-- the number of micrograms of each polysaccharide per
single human dose;
-- the number of micrograms of carrier protein per single
human dose;
-- if applicable, the name and amount of adsorbent;
-- if applicable, that the vaccine must be shaken before use;
-- if applicable, that the vaccine must not be frozen.
____________________________________________ ~E~
*****
Inactivated Poliomyelitis Vaccine
** **
(Poliomyelitis Vaccine (Inactivated) , ***
Ph Eur monograph 0214)
The labe! may state 'IPV' .
~E~ ____________________________________________
DEFINITION
Poliomyelitis vaccine (inactivated) is a liquid preparation of
suitable strains of human poliovirus types 1, 2 and 3 grown
in suitable cell cultures and inactivated by a validated
method. It is a clear liquid that may be coloured owing to
the presence of a pH indicator.
PRODUCTION
The production method shall have been shown to yield
consistently vaccines of acceptable safety and immunogenicity
inman.
Production of the vaccine is based on a virus seed-Iot system.
Celllines are used according to a cell-bank system.
If primary, secondary or tertiary monkey kidney cells are
used, production complies with the requirements indicated
below.
Unless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
the master seed lot than was used to prepare the vaccine
shown in clinical studies to be satisfactory with respect to
safety and efficacy.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a human diploid cellline (5.2.3),
in a continuous cellline (5.2.3) or in primary, secondary or
tertiary monkey kidney cells.
 2014
Primary, secondary or tertiary monkey kidney cells
The following special requirements for the substrate for virus
propagation apply to primary, secondary or tertiary monkey
kidney cells.
Monkeys used in the preparation of kidney cel! cultures for
production and control of the vaccine The animals used are of
a species approved by the competent authority, in good
health and, unless otherwise justified and authorised, have
not been previously employed [or experimental purposes.
Kidney cells used for vaccine production and control are
derived from monitored, elosed colonies of monkeys bred in
captivity, not from animals caught in the wild; a previously
approved seed lot prepared using virus passaged in cells from
wild monkeys may, subject to approval by the competent
authority, be used for vaccine production if historical data on
safety justify this.
Monitored, closed colonies of monkeys The monkeys are kept
in groups in cages. Freedom from extraneous agents is
achieved by the use of animals maintained in elosed colonies
that are subject to continuous and systematic veterinary and
laboratory monitoring [or the presence of infectious agents.
The supplier of animals is certified by the competent
authority. Each monkey is tested serologically at regular
intervals during a quarantine period of not less rhan 6 weeks
imposed before entering the colony, and rhen during its stay
in the colony.
The monkeys used are shown to be tuberculin-negative and
free from antibodies to simian virus 40 (SV40) and simian
immunodeficiency virus. The blood sample used in testing
for SV40 antibodies must be taken as elose as possible ro the
time of removal of the kidneys. If Macaca sp. monkeys are
used for production, the monkeys are also shown to be free
from antibodies to herpesvirus B (cercopithecine herpesvirus
1) infection. Human herpesvirus 1 has been used as an
indicator for freedom from herpesvirus B antibodies on
account of the danger of handling herpesvirus B
(cercopithecine herpesvirus 1).
Monkeys from which kidneys are to be removed are
thoroughly examined, particularly for evidence of tuberculosis
and herpesvirus B (cercopithecine herpesvirus 1) infection.
If a monkey shows any pathological lesion relevant to the use
of its kidneys in the preparation of a seed lot or vaccine, it is
not to be used nor are any of the remaining monkeys of the
group concemed unless it is evident that their use will not
impair the safety of the product.
All the operations described in rhis secrion are conducted
outside the area where the vaccine is produced.
Monkey cel! cultures for vaccine production Kidneys that show
no pathological signs are used for preparing cell cultures.
Each group of cell cultures derived from a single monkey
forms a separate production cell culture giving rise to a
separate single harvest.
The primary monkey kidney cell suspension complies with
the test for mycobacteria (2.6.2); disrupt the cells before
carrying out the test.
If secondary or tertiary cells are used, it shall be
demonstrated by suirable validation tests that cell cultures
beyond the passage level used for production are free from
tumorigenicity.
SEED LOTS
Each of the 3 strains of poliovirus used shall be identified by
historical records that inelude information on the origin of
the strain and its subsequent manipulation.
Vaccines IV-589
Only a working seed lot that complies with rhe following
requirements may be used for virus propagation.
Identification
Each working seed lot is identified as human poliovirus types
1, 2 or 3 by virus neutralisation in cell cultures using specific
antibodies.
Virus concentration
The virus concentration of each working seed lot is
determined to define the quantity of virus to be used for
inoculation of production cell cultures.
Extraneous agents
The working seed lot complies with the requirements for
seed lots for virus vaccines (2.6.16). In addition, if primary,
secondary or tertiary monkey kidney cells have been used for
isolation of the strain, mea sures are taken to ensure that the
strain is not contaminated with sÍrnian virus es such as simian
immunodeficiency virus, sÍrnian virus 40, filoviruses and
herpesvirus B (cercopithecine herpesvirus 1). A working seed
lot produced in primary, secondary or tertiary monkey kidney
cells complies with the requirements given below under Virus
propagation and harvest for single harvests produced in such
cells.
PROPAGATlON AND HARVEST
All processing of the cell bank and cell cultures is done under
aseptic conditions in an area where no other cells or virus es
are being handled. Approved animal serum (but not human
serum) may be used in the cell culture media. Serum and
trypsin used in the preparation of cell suspensions and media
are shown to be free from extraneous agents. The cell culture
media may contain a pH indicator such as phenol red and
approved antibiotics at the lowest effective concentration.
Not less than 500 mL of the cell cultures employed for
vaccine production is set aside as uninfected cell cultures
(control cells); where continuous celllines in a fermenter are
used for production, 200 x 10 6 cells are set aside to prepare
control cells; where primary, secondary or tertiary monkey
kidney cells are used for production, a cell sample equivalent
to at least 500 mL of the cell suspension, at the
concentration employed for vaccine production, is taken to
prepare control cells.
Only a single harvest that complies with the following
requirements may be used in the preparation of the vaccine.
The tests for identification and bacterial and fungal
contamination may be carried out instead on the purified,
pooled monovalent harvest. After demonstration of
consistency of production at the stage of the single harvest,
the test for virus concentration may be carried out instead on
the purified, pooled monovalent harvest.
Control cells
The control cells of the production cell culture comply with a
test for identification (if a cell-bank system is used for
production) and with the requirements for extraneous agents
(2.6.16; where primary, secondary or tertiary 111011key kidney ceUs
are used, the tests in cel! cultures are carried out as ShOWI1 below
under Test in rabbit kidney cel! cultures and Test in cercopithecus
kidney cell cultures).
Test in rabbit kidney cell cultures Test a sample of at least
10 mL of the pooled supematant fluid from the control
cultures for the absence of herpesvirus B (cercopithecine
herpesvirus 1) and other virus es by inoculation onto rabbit
kidney cell cultures. The dilution of supematant in the
nutrient medium is not greater than 1/4 and the area of the
celllayer is at least 3 cm 2 per millilitre of inoculum. Ser aside
one or more containers of each batch of cells with the same
medium as non-inoculated control cells. Incubate the
IV-590 Vaccines
cultures at 37 oC and observe for at least 2 weeks. The test is
not valid if more than 20 per cent of the control cell cultures
are discarded for non-specific, accidental reasons.
Test in cercopithecus kidney cel! cultures Test a sample of at
least 10 mL of the po oled supematant fluid from the control
cultures for the absence of SV40 virus and other extraneous
agents by inoculation onto cell cultures prepared from the
kidneys of cercopithecus monkeys, or other cells shown to be
at least as sensitive for SV40, by the method described under
Test in rabbit kidney cell cultures. The test is not valid if
more than 20 per cent of the control cen cultures are
discarded for non-specific, accidental reasons.
Identification
The single harvest is identified as containing human
poliovirus types 1, 2 or 3 by virus neutralisation in cell
cultures using specific antibodies.
Virus concentration
The virus concentration of each single harvest is determined
by titration of infectious virus in cell cultures .
Bacteria! and fungal contamination
The single harvest complies with the test for sterility (2.6.1),
carried out using 10 mL for each medium.
Mycoplasmas (2.6.7)
The single harvest complies with the test for mycoplasmas,
carried out using 10 mL.
Test in rabbit kidney cell cultures
Where primary, secondary or tertiary monkey kidney cells are
used for production, test a sample of at least 10 mL of the
single harvest for the absence of herpesvirus B
(cercopithecine herpesvirus 1) and other viruses by
inoculation onto rabbit kidney cell cultures as described
aboye for the control censo
Test in cercopithecus kidney cell cultures
Where primary, secondary or tertiary monkey kidney cells are
used for production, test a sample of at least 10 mL of the
single harvest for the absence of SV40 virus and other
extraneous agents. Neutralise the sample by a high-titre
antiserum against the specific type of poliovirus. Test the
sample in primary cercopithecus kidney cen cultures or cens
that have been demonstrated to be at least as susceptible for
SV40. Incubate the cultures at 37 oC and observe for
14 days. At the end of this period, make at least one
subculture of fluid in the same cen culture system and
observe both primary cultures and subcultures for an
additional 14 days.
PURIFICATION AND PURIFIED MONOVALENT HARVEST
Several single harvests of the same type may be pooled and
may be concentrated. The monovalent harvest or pooled
monovalent harvest is purified by validated methods.
If continuous cell lines are used for production, the
purification process shall have been shown to reduce
consistently the content of substrate-cell DNA to not more
than 100 pg per single human dose.
Only a purified monovalent harvest that complies with the
following requirements may be used for the preparation of
the inactivated monovalent harvest.
Identification
The virus is identified by virus neutralisation in cell cultures
using specific antibodies or by determination of D-antigen.
Virus concentration
The virus concentration is determined by titration of
infectious virus.
2014
Specific activity
The ratio of the virus concentration or the D-antigen
content, determined by a suitable immunochemical method
(2.7.1), to the total protein content (specific activity) of the
purified monovalent harvest is within the limits approved for
the particular product.
INACTIVATION AND INACTIVATED MONOVALENT
HARVEST
Several purified monovalent harvests of the same type may
be mixed before inactivation. To avoid failures in inactivation
caused by the presence of virus aggregates, filtration is
carried out before and during inactivation; inactivation is
started within a suitable period, preferably not more than
24 h and in any case not more than 72 h, of the prior
filtration. The virus suspension is inactivated by a validated
method that has been shown to inactivate poliovirus without
destruction of immunogenicity; during validation studies, an
inactivation curve with at least 4 points (for example, time
O h, 24 h, 48 h and 96 h) is established showing the decrease
in concentration of live virus with time. If formaldehyde is
used for inactivation, the presence of an excess of
formaldehyde at the end of the inactivation period is verified.
The inactivation kinetics tests mentioned below are carried
out on each batch to ensure consistency of the inactivation
process.
OnIy an inactivated monovalent harvest that complies with
the following requirements may be used in the preparation of
a trivalent pool of inactivated monovalent harvests or a final
bulk vaccine.
Test for effective inactivation
After neutralisation of the formaldehyde with sodium bisulfite
(where applicable), verify the absence of residuallive
poliovirus by inoculation on suitable cell cultures of
2 samples of each inactivated monovalent harvest,
corresponding to at least 1500 human doses. cens used for
the test must be of optimal sensitivity regarding residual
infectious poliovirus, for example kidney cells from certain
monkey species (Macaca, Cercopithecus or Papio), or Hep-2
cells. If other cells are used, they must have been shown to
possess at least the same sensitivity as those specified aboye.
Take one sample not later than 3/4 of the way through the
inactivation period and the other at the end. Inoculate the
samples in cell cultures such that the dilution of vaccine in
the nutrient medium is not greater than 1/4 and the area of
the celllayer is at least 3 cm2 per millilitre of inoculum.
Set aside one or more containers with the same medium as
non-inoculated control cells. Observe the cell cultures for at
least 3 weeks. Make not fewer than 2 passages from each
container, one at the end of the observation period and the
other 1 week before; for the passages, use cell culture
supematant and inoculate as for the initial sample. Observe
the sub cultures for at least 2 weeks. No sign of poliovirus
multiplication is present in the cell cultures. At the end of the
observation period, test the susceptibility of the cell culture
used by inoculation of live poliovirus of the same type as that
present in the inactivated monovalent harvest.
Inactivation kinetics
Kinetics of inactivation are established and approved by the
competent authority. Adequate data on inactivation kinetics
are obtained and consistency of the inactivation process is
monitored.
Sterility (2.6.1)
The inactivated monovalent harvest complies with the test for
sterility, carried out using 10 mL for each medium.
2014
D-antigen content
The content of D-antigen detennined by a suitable
immunochemical method (2.7.1) is within the limits
approved for the particular preparation.
FINAL BULK VACCINE
The final bulk vaccine is prepared directly from the
inactivated monovalent harvests of human poliovirus types 1,
2 and 3 or from a trivalent pool of inactivated monovalent
harvests. A suitable stabiliser and a suitable antimicrobial
preservative may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
Antimicrobial preservative
Where applicable, detennine the amount of antimicrobial
preservative by a suitable chemical or physicochemical
method. The amount is not les s than 85 per cent and not
greater than 115 per cent of the intended amount.
FINALLOT
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
relea sed for use. Provided that the tests for free formaldehyde
and antimicrobial preservative and the in vivo assay have
been perfonned with satisfactory resuIts on the final bulk
vaccine, they may be omitted on the final loto
The in vivo assay may be omitted once it has been
demonstrated for a given product and for ea eh poliovirus
type that the acceptance criteria for the D-antigen
detennination are such that it yields the same resuIt as the in
vivo assay in tenns of acceptance or rejection of a batch. This
demonstration must inelude testing of subpotent batches,
produced experimentally if necessary, for example by heat
treatment or other means of diminishing the immunogenic
activity. Where there is a significant change in the
manufacturing process of the antigens or their fonnulation,
any impact on the in vivo and in vitro assays must be
evaluated, and the need for revalidation considered.
Provided that the protein content has been determined on
the purified monovalent harvests or on the inactivated
monovalent harvests and that it has been shown that the
content in the finallot will not exceed 10 llg per single
human dose, the test for protein content may be omitted on
the final loto
Provided that the test for bovine serum albumin has been
performed with satisfactory resuIts on the trivalent pool of
inactivated monovalent harvests or on the final bulk vaccine,
it may be omitted on the finallot.
IDENfIFICAnON
The vaccine is shown to contain human poliovirus types 1, 2
and 3 by a suitable immunochemical method (2. 7.1) such as
the detennination of D-antigen by enzyme-linked
immunosorbent assay (ELISA).
TESTS
Free fonnaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, detennine the amount of antimicrobial
preservative by a suitable chemical or physicochemical
method . The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of
that stated on the labe!.
Vaccines IV-591
Protein content (2.5.33, Method 2)
Maximum 10 ¡.tg per single human dose.
Bovine serum albumin
Maximum 50 ng per single human dose, detennined by a
suitable immunochemical method (2.7.1).
Sterility (2.6.1)
It complies with the test.
Bacterial endotoxins (2. 6. 14)
Less than 5 IU per single human dose.
ASSAY
D-antigen content
As a measure of consistency of production, detennine the
D-antigen content for human poliovirus types 1, 2 and 3 by
a suitable immunochemical method (2.7.1) using a reference
preparation calibrated in European Phannacopoeia Units of
D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label, is
within the limits approved for the particular producto
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Phannacopoeia Units and intended for use in the
assay of D-antigen. The European Phannacopoeia Unit and
the International Unit are equivalent.
In vivo test
The vaccine complies with the in vivo assay of poliomyelitis
vaccine (inactivated) (2.7.20).
LABELLING
The label states:
-- the types of poliovirus contained in the vaccine;
-- the nominal amount of virus of each type (1, 2 and 3),
expressed in European Pharmacopoeia Units of
D-antigen, per single human dose;
-- the cell substrate used to prepare the vaccine.
____________________________________________ PhE~
Poliomyelitis Vaccine, Uve (Oral) ***
*** ***
(Poliomyelitis Vaccine (Ora!), ***
Ph Eur monograph 0215)
The label may state ' OPV' .
~E~ ____________________________________________
DEFINITION
Oral poliomyelitis vaccine is a preparation of approved strains
of live attenuated poliovirus type 1, 2 or 3 grown in in vitro
cultures of approved cells, containing any one type or any
combination of the 3 types of Sabin strains, presented in a
fonn suitable for oral administration.
The vaccine is a elear liquid that may be coloured owing to
the presence of a pH indicator.
PRODUCnON
The vaccine strains and the production method shall have
been shown to yield consistently vaccines that are both
immunogenic and safe in mano
The production of vaccine is based on a virus seed-Iot
system. Celllines are used according to a cell-bank system.
If primary monkey kidney cell cultures are used, production
complies with the requirements indicated below. Unless
otherwise justified and authorised, the virus in the final
vaccine shall not have undergone more than 2 passages from
the master seed loto
 IV-592 Vaccines
REFERENCESTANDARDS
Poliomyelitis vaccine (oral) BRP is suitable for use as a virus
reference preparation for the assay.
The International Standards for poliovirus type 2 (Sabin) for
MAPREC (Mutant Analysis by PCR and Restriction Enzyrne
Cleavage) assays and poliovirus, type 3 (Sabin) synthetic
DNA for MAPREC assays are suitable for use in the tests for
genetic markers and the molecular tests for consistency of
production.
Reference preparations of each poliovirus type at the Sabin
Original + 2 passage level, namely WHO (SO + 2)/I for type
1 virus, WHO (SO + 2)/II for type 2 virus and WHO
(SO + 2)/I1I for type 3 virus are available for comparison of
the in vivo neurovirulence with that of homotypic vaccines.
Requests for the WHO reference preparations for in vivo
neurovirulence tests are to be directed to WHO, Biologicals,
Geneva, Switzerland.
A suitable reference preparation is to be ineluded in each
test.
SUBSTRATE FOR VIRUS PROPAGATlON
The virus is propagated in human diploid cells (5.2.3), in
continuous celllines (5.2.3) or in primary monkey kidney cell
cultures (ineluding serially passaged celJs from primary
monkey kidney cells).
Primary monkey kidney cel1 cultures
The following special requirements for the substrate for virus
propagation apply to primary monkey kidney cel! cultures.
Monkeys used for preparation of primary monkey kidney cel!
cultures and for testing of virus If the vaccine is prepared in
primary monkey kidney celJ cultures, animals of a species
approved by the competent authority, in good health, kept in
elosed or intensively monitored colonies and not previously
employed for experimental purposes shall be used.
The monkeys shall be kept in welJ-constructed and
adequately ventilated animal rooms in cages spaced as far
apart as possible. Adequate precautions shall be taken to
prevent cross-infection between cages. Not more than
2 monkeys shall be housed per cage and cage-mates shalJ not
be interchanged. The monkeys shalJ be kept in the country of
manufacture of the vaccine in quarantine groups for a period
of not les s than 6 weeks before use. A quarantine group is a
colony of selected, healthy monkeys kept in one room, with
separate feeding and eleaning facilities, and having no contact
with other monkeys during the quarantine periodo If at any
time during the quarantine period the overall death rate of a
shipment consisting of one or more groups reaches
5 per cent (exeluding deaths from accidents or where the
cause was specificalJy determined not to be an infectious
disease), monkeys from that entire shipment shall continue in
quarantine from that time for a minimum of 6 weeks.
The groups shall be kept continuously in isolation, as in
quarantine, even after completion of the quarantine period,
until the monkeys are used. After the last monkey of a group
has been taken, the room that housed the group shall be
thoroughly eleaned and decontaminated before being used
for a fresh group. If kidneys from near-term monkeys are
used, the mother is quarantined for the term of pregnancy.
Monkeys from which kidneys are to be removed shall be
anaesthetised and thoroughly examined, particularly for
evidence of tuberculosis and cercopithecid herpesvirus 1 (B
virus) infection.
If a monkey shows any pathologicallesion relevant to the use
of its kidneys in the preparation of a seed lot or vaccine, it
shall not be used, nor shall any of the remaining monkeys of
2014
the quarantine group con cerned be used unless it is evident
that their use will not irnpair the safety of the product.
AII the operations described in this section shall be
conducted outside the areas where the vaccine is produced.
The monkeys used shall be shown to be free from antibodies
to simia n virus 40 (SV40), simian immunodeficiency virus
and spumaviruses. The blood sample used in testing for
SV40 antibodies must be taken as elose as possible to the
time of removal ofthe kidneys. If Macaca spp . are used for
production, the monkeys shall also be shown to be free from
antibodies to cercopithecid herpesvirus 1 (B virus). Human
herpesvirus has been used as an indicator for freedom from B
virus antibodies on account of the danger of handling
cercopithecid herpesvirus 1 (B virus). Monkeys used for the
production of new seed lots are shown to be free from
antibodies to simia n cytomegalovirus (sCMV) .
Primary monkey kidney cel! cultures for vaccine
production Kidneys that show no pathological signs are used
for preparing celJ cultures. If the monkeys are from a colony
maintained for vaccine production, serially passaged monkey
kidney cell cultures from primary monkey kidney cells may
be used for virus propagation, otherwise the monkey kidney
cells are not propagated in series. Virus for the preparation of
vaccine is grown by aseptic methods in such cultures.
If animal serum is used in the propagation of the cells, the
maintenance medium after virus inoculation shall contain no
added serum.
Each group of cell cultures derived from a single monkey or
from foetuses from no more than 10 near-term monkeys is
prepared and tested as an individual group.
VIRUS SEED LOTS
The strains of poliovirus used shall be identified by historical
records that inelude information on the origin and
subsequent manipulation of the strains.
Working seed lots are prepared by a single passage from a
master seed lot and at an approved passage level from the
original Sabin virus. Virus seed lots are prepared in large
quantities and stored at a temperature below - 60 oc.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification
Each working seed lot is identified as polio virus of the given
type, using specific antibodies.
Virus concentration
Determined by the method described below, the virus
concentration is the basis for the quantity of virus used in the
neurovirulence test.
Extraneous agents (2.6.16)
If the working seed lot is produced in human diploid cells or
in a continuous celJ line, it complies with the requirements
for seed lots for virus vaccines. If the working seed lot is
produced in primary monkey kidney celJ cultures, it complies
with the requirements given below under Virus Propagation
and Harvest and Monovalent Pooled Harvest and with the
tests in adult mice, suckling mice and guinea-pigs given in
chapter 2.6. 16.
In addition to the requirements in chapter 2.6.16, for
vaccines produced in celJ lines and when the seed lot was
produced in primary monkey kidney celJ cultures, a validated
test for sCMV is performed.
Working seed lots shall be free from detectable DNA
sequences from simian virus 40 (SV40).
 2014
N eurovirulence
Each master and working seed lot complies with the test for
neuroviru1ence of poliomye1itis vaccine (oral) in monkeys
(2.6.19). In addition, at 1east the first 4 consecutive batches
of monova1ent poo1ed harvest prepared from these seed lots
shall be shown to comp1y with the test for neuroviru1ence of
poliomye1itis vaccine (oral) in monkeys (2.6.19) before the
seed 10t is deemed suitab1e for use. Furthermore, the seed lot
shall cease to be used in vaccine production if the frequency
of fai1ure of the monova1ent poo1ed harvests produced from it
is greater than predicted statistically. This statistica1
prediction is calcu1ated after each test on the basis of all the
monova1ent poo1ed harvests tested; it is equal to the
probability of fa1se rejection on the occasion of a first test
(i.e.l per cent), the probability of false rejection on retest
being neg1igible. If the test is carried out on1y by the
manufacturer, the test slides are provided to the control
authority for assessment.
Genetic markers
Each working seed 10t is tested for its replicating properties at
temperatures ranging from 36 oC to 40 oC as described
under Monovalent pooled harvest. A profi1e (i.e. percentage
of mutant) of the seed virus using the MAPREC assay is
prepared. Type 3 virus seed lots comply with the MAPREC
assay as described under Monovalent poo1ed harvest.
VIRUS PROPAGATION AND HARVEST
All processing of the cell banks and subsequent cell cultures
is done under aseptic conditions in an area where no other
cells are handled during the production. Suitable animal
(but not human) serum may be used in the culture media,
but the final medium for maintaining cell growth during virus
multiplication does not contain animal serum. Serum and
trypsin used in the preparation of cell suspensions and media
are shown to be free from live extraneous agents. The
cell-cu1ture medium may contain a pH indicator such as
phenol red and suitable antibiotics at the lowest effective
concentration. It is preferab1e to have a substrate free from
antibiotics during production. On the day of inocu1atian with
the virus working seed lot, not less than 5 per cent or
1000 mL, whichever is the less, of the cell cultures employed
for vaccine production are set aside as uninfected cell
cultures (control cells) . Special requirements, given be10w,
app1y to control cells when the vaccine is produced in
primary monkey kidney cell cultures. The virus suspension is
harvested not later than 4 days after virus inoculation. After
inocu1ation of the production cell culture with the virus
working seed 10t, inocu1ated cells are maintained at a fixed
temperature, shown to be suitable, within the range
33-35 DC; the temperature is maintained constant to
± 0.5 cC; control cell cultures are maintained at 33-35 oC
for the relevant incubation periods.
On1y a single virus harvest that complies with the following
requirements may be used in the preparation of the
monovalent pooled harvest.
Virus concentration
The virus concentration of virus harvests is determined as
prescribed under Assay to monitor consistency of production
and to determine the dilution to be used for the final bulk
vaccine.
Molecular tests for consistency of production
The MAPREC assay is performed on each virus harvest.
The acceptancelrejection criteria for consistency of
production are determined for each manufacturer and for
each working seed by agreement with the competent
authority. These criteria are periodically reviewed and
Vaccines IV-593
updated to the satisfaction of the competent authority.
An investigation of consistency occurs if a virus harvest gives
results that are inconsistent with previous production history.
Control cells
The control cells of the production cell culture from which
the virus harvest is derived comp1y with a test for identity
and with the requirements for extraneous agents (2.6.16) or,
where primary monkey kidney cell cultures are used, as
shown be10w.
Primary monkey kidney cell cultures
The following special requirements apply 10 virus propagation and
harvest in primmy monkey kidney cell cultures.
Gel! cultures On the day of inoculation with the virus
working seed lot, each cell culture is examined for
degeneration caused by an infective agent. If, in this
examination, evidence is found of the presence in a cell
culture of any extraneous agent, the entire group of cultures
concemed shall be rejected.
On the day of inoculation with the virus working seed 10t, a
samp1e of at least 30 mL of the pooled fluid removed from
the cell cultures of the kidneys of each single monkey or from
foetuses from not more than 10 near-term monkeys is
divided into 2 equal portions. 1 portion of the pooled fluid is
tested in monkey kidney cell cultures prepared from the same
species, but not the same animal, as that used for vaccine
production. The other portion of the pooled fluid is, where
necessary, tested in monkey kidney cell cultures from another
species so that tests on the pooled fluids are done in cell
cultures from at least 1 species known to be sensitive to
SV40. The pooled fluid is inoculated into bottles of these cell
cultures in such a way that the dilution of the pooled fluid in
the nutrient medium do es not exceed 1 in 4. The area of the
cell sheet is at least 3 cm 2/rnl of pooled fluid. At least 1
bottle of each type of cell culture remains uninoculated to
serve as a control. If the monkey species used for vaccine
production is known to be sensitive to SV40, a test in a 2nd
species is not required. Animal serum may be used in the
propagation of the cells, provided thar it do es not contain
SV40 antibody, but the maintenance medium after
inoculation of test material contains no added serum except
as described below.
The cultures are incubated at a temperature of 35-37 oC and
are observed for a total period of at least 4 weeks. During
this observation period and after not less than 2 weeks'
incubarion, at least 1 sub culture of fluid is made from each
of these cultures in rhe same cell culture sysrem.
The sub cultures are also observed for at least 2 weeks.
Serum may be added to the original culture at the time of
subculturing, provided that the serum does not contain SV40
antibody.
Fluorescent-antibody techniques may be useful for detecting
SV40 virus and other virus es in the cells.
A further sample of at least 10 mL of the pooled fluid is
tesred for cercopithecid herpesvirus 1 (B virus) and other
viruses in rabbit kidney cell cultures. Serum used in the
nutrient medium of these cultures shall have been shown to
be free from inhibitors of B virus. Human herpesvirus has
been used as an indicator for freedom from B virus inhibitors
on account of the danger of handling cercopithecid
herpesvirus 1 (B virus). The sample is inoculared into bottles
of these cell cultures in such a way that the dilution of the
pooled fluid in the nutrient medium does not exceed 1 in 4.
The area of the cell sheet is at least 3 cm 2/ml of pooled fluid.
At least 1 bottle of the cell cultures remains uninoculated to
serve as a control.
 IV-594 Vaccines
The cultures are incubated at a temperature of 35-37 oC and
observed for at least 2 weeks .
A further sample of 10 mL of the pooled fluid removed from
the cell cultures on the day of inoculation with the seed lot
virus is tested for the presence of extraneous agents by
inoculation into human cell cultures sensitive to measles
VIruS.
The tests are not valid if more than 20 per cent of the
culture vessels have been discarded for non-specific
accidental reasons by the end of the respective test periods.
If, in these tests, evidence is found of the presence of an
extraneous agent, the single harvest from the whole group of
cell cultures concerned is rejected.
If the presence of cercopithecid herpesvirus 1 (B virus) is
demonstrated, the manufacture of oral poliomyelitis vaccine
shall be discontinued and the competent authority shall be
informed. Manufacturing shall not be resumed until a
thorough investigation has been completed and precautions
have been taken against any reappearance of the infection,
and then only with the approval of the competent authority.
If these tests are not done irnmediately, the samples of
pooled cell-culture fluid shall be kept at a temperature of
- 60 oC or below, with the exception of the sample for the
test for B virus, which may be held at 4 oC, provided that the
test is done not more than 7 days after ir has been taken.
Control cel! cultures On the day of inoculation with the virus
working seed lot, 25 per cent (but not more than 2.5 litres)
of the cell suspension obtained from the kidneys of each
single monkey or from not more than 10 near-term monkeys
is taken to prepare uninoculated control cell cultures. These
control cell cultures are incubated in the same conditions as
the inoculated cultures for at least 2 weeks and are examined
during this period for evidence of cytopathic changes.
The tests are not valid if more than 20 per cent of the
control cell cultures have been discarded for non-specific,
accidental reasons. At the end of the observation period, the
control cell cultures are examined for degeneration caused by
an infectious agent. If this examination or any of the tests
required in this section shows evidence of the presence in a
control culture of any extraneous agent, the poliovirus grown
in the corresponding inoculated cultures from the same
group shall be rejected.
Tests for haemadsorbing viruses At the time of harvest or
within 4 days of inoculation of the production cultures with
the virus working seed lot, a sample of 4 per cent of the
control cell cultures is taken and tested for haemadsorbing
viruses. At the end of the observation period, the remaining
control cell cultures are similarly tested. The tests are carried
out as described in chapter 2.6. 16.
Tests for other extraneous agents At the time of harvest, or
within 7 days of the day of inoculation of the production
cultures with the working seed lot, a sample of at least
20 mL of the po oled fluid from each group of control
cultures is taken and tested in 2 kinds of monkey kidney cell
culture, as described aboye.
At the end of the observation period for the original control
cell cultures, similar samples of the pooled fluid are taken
and the tests referred to in this section in the 2 kinds of
monkey kidney cell culture and in the rabbit cell cultures are
repeated, as described aboye under Cell cultures.
If the presence of cercopithecid herpesvirus 1 (B virus) is
demonstrated, the production cell cultures shall not be used
and the mea sures concerning vaccine production described
aboye must be undertaken.
The fluids collected from the control cell cultures at the time
2014
of virus harvest and at the end of the observation period may
be pooled before testing for extraneous agents. A sample of
2 per cent of the pooled fluid is tested in each of the cell
culture systems specified.
Single harvests
Tests for neutralised single harvests in primary monkey kidney cel!
cultures A sample of at least 10 mL of each single harvest is
neutralised by a type-specific poliomyelitis antiserum
prepared in animals other than monkeys . In preparing
antisera for this purpose, the irnmunising antigens used shall
be prepared in non-simian cells.
Half of the neutralised suspension (corresponding to at least
5 mL of single harvest) is tested in monkey kidney cell
cultures prepared from the same species, but not the same
animal, as that used for vaccine production. The other half of
the neutralised suspension is tested, if necessary, in monkey
kidney cell cultures from another species so that the tests on
the neutralised suspension are done in cell cultures from at
least 1 species known to be sensitive to SV40.
The neutralised suspensions are inoculated into bottles of
these cell cultures in such a way that the dilution of the
suspension in the nutrient medium does not exceed 1 in 4.
The area of the cell sheet is at least 3 cmz/ml of neutralised
suspension. At least 1 bottle of each type of cell culture
remains uninoculated to serve as a control and is maintained
by nutrient medium containing the same concentration of the
specific antiserum used for neutralisation.
Animal serum may be used in the propagation of the cells,
provided that it does not contain SV40 antibody, but the
maintenance medium, after the inoculation of the test
material, contains no added serum other than the poliovirus
neutralising antiserum, except as described below.
The cultures are incubated at a temperature of 35-37 oC and
observed for a total period of at least 4 weeks. During this
observation period and after not less than 2 weeks'
incubation, at least 1 subculture of fluid is made from each
of these cultures in the same cell-culture system.
The sub cultures are also observed for at least 2 weeks.
Serum may be added to the original cultures at the time of
subculturing, provided that the serum does not contain SV40
antibody.
Additional tests are made for extraneous agents on a further
sample of the neutralised single harvests by inoculation of
10 mL into human cell cultures sensitive to measles virus.
This test is also validated for the detection of sCMV.
Fluorescent-antibody techniques may be useful for detecting
SV40 virus and other viruses in the cells.
The tests are not valid if more than 20 per cent of the
culture ves seis have been discarded for non-specific
accidental reasons by the end of the respective test periods .
If any cytopathic changes occur in any of the cultures, the
causes of these changes are investigated. If the cytopathic
changes are shown to be due to unneutralised poliovirus, the
test is repeated. If there is evidence of the presence of SV40
or other extraneous agents attributable to the single harvest,
that single harvest is rejected.
MONOVALENf PO OLED HARVEST
Monovalent pooled harvests are prepared by pooling a
number of satisfactory single harvests of the same virus type.
Monovalent pooled harvests from continuous cell lines may
be purified. Each monovalent pooled harvest is filtered
through a bacteria-retentive filter.
Only a monovalent po oled harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
2014
Identification
Each monovalent pooled harvest is identified as poliovirus of
the given type, using specific antibodies.
Virus concentration
The virus concentration is determined by the method
described below and serves as the basis for calculating the
dilutions for preparation of the final bulk, for the quantity of
virus used in the neurovirulence test and to establish and
monitor production consistency.
Genetic markers
For Sabin poliovirus type 3, a validated MAPREC assay is
performed. In this analysis the amount of the mutation at
position 4 n of the genome (4 n-c) is estimated and
expressed as a ratio relative to the Intemational Standard for
MAPREC analysis of poliovirus type 3 (Sabin). A poliovirus
type 3 monovalent pooled harvest found to have significantly
more 4 n-c than the Intemational Standard for MAPREC
analysis of poliovirus type 3 (Sabin) fails in the MAPREC
assay.
The MAPREC analysis of poliovirus type 3 (Sabin) is carried
out using a standard operating procedure approved by the
competent authority. A suitable procedure (Mucant analysis
by PCR and resmaion enzyme cleavage (MAPREC) for oral
poliovirus (Sabin) vaccine) is available from WHO, Quality
and Safety of Biologicals (QSB), Geneva. A laboratory must
demonstrate to the competent authority that it is competent
to perform the assay. The manufacturer and the competent
authority shall agree on the procedure and the criteria for
deciding whether a monovalent pooled harvest contains
significantly more 4 n-c than the Intemational Standard.
Acceptance/rejection criteria for assessment of consistency of
production are determined for each manufaeturer and for
each working seed lot by agreement with the competent
authority. These eriteria are updated as each new bulk is
prepared and analysed. An investigation of consistency occurs
if a monovalent pooled harvest gives results that are
inconsistent with previous production hisrory.
As the MAPREC assay for type 3 poliovirus (Sabin) is highly
predictive of in vivo neurovirulenee, if a filtered monovalent
pooled harvest of type 3 poliovirus (Sabin) fails the
MAPREC assay then this triggers an investigation of the
consisteney of the manufacturing process. This investigation
also ineludes a consideration of the suitability of the working
seed lot.
Monovalent pooled harvests passing the MAPREC assay are
subsequently tested for in vivo neurovirulence.
For poliovirus type 3, results from the MAPREC assay and
the monkey neurovirulence test (2.6.19) are used
concomitantly ro assess the impact of changes in the
production process or when a new manufacturer starts
production.
Pending validation of MAPREC assays for poliovirus types 1
and 2, for these viruses filtered bulk suspension is tested for
the property of reproducing at temperatures of 36 oC and
40 oc. A ratio of the replication capacities of the virus in the
monovalent pooled harvest is obtained over a temperature
range between 36 oC and 40 oC in comparison with the seed
lot or a reference preparation for the marker tests and with
appropriate rct/40 - and rct/40+ strains of poliovirus of the
same type. The incubation temperatures used in this test are
controlled ro within ± 0.1 oC. The monovalent pooled
harvest passes the test if, for both the virus in the harvest and
the appropriate reference material, the titre determined at
36 oC is at least 5.0 log greater than that determined at
40 oc. If growth at 40 oC is so low that a valid comparison
Vaccines IV-595
cannot be established, a temperature in the region of
39.0-39.5 oC is used, at which temperature the reduction in
titre of the reference material must be in the range 3.0-5.0
log of its value at 36 oC; the aeceptable minimum reduction
is determined for each virus strain at a given temperature.
If the titres obtained for 1 or more of the reference viruses
are not concordant with the expected values, the test must be
repeated.
Neuroviru1ence (2.6.19)
Each monovalent pooled harvest complies with the test for
neurovirulence of poliomyelitis vaccine (oral). If the monkey
neurovirulence test is carried out only by the manufacturer,
the test slides are provided to the competent authority for
assessment. The TgPVR21 transgenic mouse model provides
a suitable altemative ro the monkey neurovirulence test for
neurovirulence testing of types 1, 2 or 3 vaccines once a
laboratory qualifies as being competent ro perform the test
and the experience gained is to the satisfaction of the
competent authority. The test is carried out using a standard
operating procedure approved by the eompetent authority.
A suitable procedure (Neurovirulence test of type 1J 2 or 3 live
poliomyelitis vaccines (oral) in transgenic mice susceptible LO
poliovirus) is available from WHO, Quality and Safety of
Biologicals, Geneva.
Primary monkey kidney cell cultures
The following special requirements apply LO monovalent pooled
harvests derived from primary monkey kidney cell cultures.
Retroviruses The monovalent pooled harvest is examined
using a reverse transcriptase assay. No indication of the
presence of retroviruses is found.
Test in rabbits A sample of the monovalent pooled harvest is
tested for cercopithecid herpesvirus 1 (B virus) and other
virus es by injection of not less than 100 mL into not fewer
than 10 healthy rabbits each weighing 1.5-2.5 kg. Each rabbit
receives not less than 10 mL and not more than 20 mL, of
which 1 mL is given intradermally at multiple sites sinee the
maximum volume ro be given intradermally at each site is
0.1 mL, and the remainder subeutaneously. The rabbits are
observed for at least 3 weeks for death or signs of illness.
AII rabbits that die after the first 24 h of the test and those
showing signs of illness are examined by autopsy, and the
brain and organs removed for detailed examination to
establish the cause of death.
The test is not valid if more than 20 per cent of the
inoculated rabbits show signs of intercurrent infection during
the observation periodo The monovalent pooled harvest
passes the test if none of the rabbits shows evidence of
infection with B virus or with other extraneous agents or
lesions of any kind attributable ro the bulk suspension.
If the presence of B virus is demonstrated, the measures
conceming vaccine production described aboye under Cell
cultures are taken.
Test in guinea-pigs 1f the primary monkey kidney cell cultures
are not derived from monkeys kept in a closed colony, the
monovalent pooled harvest shall be shown LO comply with the
following test. Administer to each of not fewer than
5 guinea-pigs, each weighing 350-450 g, 0.1 mL ofthe
monovalent pooled harvest by intracerebral injection
(0.05 mL in each cerebral hemisphere) and 0.5 mL by
intraperitoneal injection. Measure the rectal temperature of
each animal on eaeh working day for 6 weeks. At the end of
the observation period carry out autopsy on each animal.
In addition, administer to not fewer than 5 guinea-pigs
0.5 mL by intraperitoneal injection and observe as described
 IV-596 Vaccines
aboye for 2-3 weeks. At the end of the observation period,
carry out a passage from these animals to not fewer than
5 guinea-pigs using blood and a suspension of liver or spleen
tissue. Measure the rectal temperature of the lalter
guinea-pigs for 2-3 weeks. Examine by autopsy all animals
that, after the first day of the test, die or are euthanised
because they show disease, or show on 3 consecutive days a
body temperature higher than 40.1 oC; carry out histological
examination to detect infection with filoviruses; in addition,
inject a suspension of liver or spleen tissue or of blood
intraperitoneally into not fewer than 3 guinea-pigs. If any
signs of infection with filoviruses are noted, confirmatory
serological tests are carried out on the blood of the affected
animals. The monovalent pooled harvest complies with the
test if not fewer than 80 per cent of the guinea-pigs survive
to the end of the observation period and remain in good
health, and no animal shows signs of infection with
filoviruses .
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
satisfactory monovalent pooled harvests and may contain
more than one virus type. Suitable flavouring substances and
stabilisers may be added.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Bacterial and fungal contamination
Carry out the test for sterility (2. 6.1), using 10 mL for each
medium.
FINALLOT
Only a final lot that complies with the following requirement
for thermal stability and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
Assay may be released for use.
Thennal stability
Maintain not fewer than 3 containers of the finallot at 37 ±
1 oC for 48 h. Determine the total virus concentration as
described under Assay in parallel for the heated vaccine and
for vaccine maintained at the temperature recommended for
storage.The total virus concentration of the heated vaccine is
not more than 0.5 log lower than that of the unheated
vaccine.
IDENTIFICATION
The vaccine is shown 10 contain poliovirus of each type
stated on the label, using specific antibodies .
TESTS
Bacterial and fungal contamination
The vaccine complies with the test for sterility (2.6.1).
ASSAY
Titrate the vaccine for infectious virus, using not fewer than
3 separate containers of vaccine, following the method
described below. Titrate 1 container of an appropriate virus
reference preparation in triplicate to validate each assay.
The virus concentration of the reference preparation is
monitored using a control chart and a titre is established on a
historical basis by each laboratory. If the vaccine contains
more than one poliovirus type, titrate each type separately,
using an appropriate type-specific antiserum (or preferably a
monoclonal antibody) to neutralise each of the other types
presento
Calculate the individual virus concentration for each
container of vaccine and for each replicate of the reference
preparation as well as the corresponding combined virus
concentrations, using the usual statistical methods (for
example, 5.3).
2014
For a trivalent vaccine, the combined estimated virus titres
per single human dose must be:
-- not less than 6.0 log infectious virus units (CCID so) for
type 1;
-- not less than 5.0 log infectious virus units (CCID so) for
type 2; and
-- not less than 5.5 log infectious virus units (CCID so ) for
type 3.
For a monovalent or divalent vaccine, the minimum virus
titres are decided by the competent authority.
Method Inoculate a suitable number of wells in a microtitre
plate with a suitable volume of each of the selected dilutions
of virus followed by a suitable volume of a cell suspension of
the Hep-2 (Cincinnati) lineo Examine the cultures between
days 7 and 9.
The assay is not valid if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so;
-- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so from the established value.
The relation with the appropriate European
Pharmacopoeia Biological Reference Preparation is
established and monitored at regular intervals when a
manufacturer's reference preparation is used.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) ofthe combined virus
concentration is not greater than ± 0.3 log CCID so.
Poliomyelitis vaccine (oral) BRP is suitable for use as a
reference preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
-- the types of poliovirus contained in the vaccine;
-- the minirnum amount of virus of each type contained in a
single human dose;
-- the cell substrate used for the preparation of the vaccine.
____________________________________________ Ph E~
Rabies Vaccine ***
*** ***
(Rabies Vaccine for Human Use Prepared in Cel! ***
Cultures, Ph Eur monograph 0216)
The label may state 'Rab'.
~E~ ____________________________________________
DEFINITION
Rabies vaccine for human use prepared in cell cultures is a
freeze-dried preparation of a suitable strain of fixed rabies
virus grown in cell cultures and inactivated by a validated
method.
The vaccine is reconstituted immediately before use as stated
on the label to give a clear liquid that may be coloured owing
10 the presence of a pH indicator.
2014
PRODUCTION
GENERAL PROVISIONS
The production of the vaccine is based on a virus seed-Iot
system and, if a cell line is used for virus propagation, a
cell-bank system. The production method shall have been
shown to yield consistently vaccines that comply with the
requirements for immunogenicity, safety and stabilíty. Unless
otherwise justified and authorised, the virus in the final
vaccine must not have undergone more passages from the
master seed lot than were used to prepare the vaccine shown
in clinical studies to be satisfactory with respect to safety and
efficacy; even with authorised exceptions, the number of
passages beyond the level used for clinical studies must not
exceed 5.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a human diploid cellline (5.2.3),
in a continuous cell line approved by the competent
authority, or in cultures of chick-embryo cells derived from a
flock free from specified pathogens (5.2.2).
SEED LOTS
The strain of rabies virus used shall be identified by historical
records that include information on the origin of the strain
and its subsequent manipulation.
Working seed lots are prepared by not more than 5 passages
from the master seed lot.
Only a working seed lot that complíes with the following tests
may be used for virus propagation.
Identification
Each working seed lot is identified as rabies virus using
specific antibodies.
Virus concentration
The virus concentration of each working seed lot is
determined by a cell culture method using
immunofluorescence, to ensure consistency of production.
Extraneous agents (2.6.16)
The working seed lot complíes with the requirements for
virus seed lots . If the virus has been passaged in mouse brain,
specific tests for murine viruses are carried out.
VIRUS PROPAGATION ANO HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are handled. Approved animal (but not human) serum may
be used in the media, but the final medium for maintaining
cell growth during virus multiplication does not contain
animal serum; the media may contain human albumin.
Serum and trypsin used in the preparation of cell suspensions
and media are shown to be free from extraneous agents.
The cell culture media may contain a pH indicator such as
phenol red and approved antibiotics at the lowest effective
concentration. Not less than 500 mL of the cell cultures
employed for vaccine production are set aside as uninfected
cell cultures (control cells). The virus suspension is harvested
on one or more occasions during incubation. Multiple
harvests from the same production cell culture may be
pooled and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the
inactivated viral harvest.
Identification
The single harvest contains virus that is identified as rabies
virus using specific antibodies.
Vaccines IV-597
Virus concentration
Titrate for infective virus in cell cultures; the titre is used to
monitor consistency of production.
Control cells
The control cells of the production cell culture from which
the single harvest is derived comply with a test for
identification and with the requirements for extraneous
agents (2.6.16).
PURIFICATION ANO INACTIVATION
The virus harvest may be concentrated ami/or purified by
suitable methods; the virus harvest is inactivated by a
valídated method at a fixed, well-defined stage of the process,
which may be before, during or after any concentration or
purification. The method shall have been shown to be
capable of inactivating rabies virus without destruction of the
immunogenic activity. If betapropiolactone is used, the
concentration shall at no time exceed 1:3500.
OnIy an inactivated viral suspension that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Residual infectious virus
Carry out an amplification test for residual infectious rabies
virus immediately after inactivation or using a sample frozen
irnmediately after inactivation and stored at - 70 cC .
Inoculate a quantity of inactivated viral suspension equivalent
to not les s than 25 human doses of vaccine into cell cultures
of the same type as those used for production of the vaccine.
A passage may be made after 7 days. Maintain the cultures
for a total of 21 days and then examine the cell cultures for
rabies virus using an immunofluorescence test.
The inactivated virus harvest complies with the test if no
rabies virus is detected.
Residual host-cell DNA
If a continuous cellline is used for virus propagation, the
content of residual host-cell DNA, determined using a
suitable method as described in Products 01 recombinant DNA
technology (0784), is not greater than 10 ng per single human
dose.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
inactivated viral suspensions. An approved stabiliser may be
added to maintain the activity of the product during and
after freeze-drying.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Glycoprotein content
Determine the glycoprotein content by a suitable
immunochemical method (2.7.1), for example, single-radial
immunodiffusion, enzyme-linked immunosorbent assay or an
antibody-binding test. The content is within the limits
approved for the particular product.
Sterility (2.6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile
containers and freeze-dried to a moisture content shown to
be favourable to the stability of the vaccine . The containers
are then closed so as to avoid contamination and the
introduction of moisture.
OnIy a finallot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the test for residual infectious
IV-598 Vaccines
virus has been carried out with satisfactory results on the
inactivated viral suspension and the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, these tests may be omitted on the final lot.
IDENTIFICATION
The vaccine is shown to contain rabies virus antigen by a
suitable immunochemical method (2.7.1) using specific
antibodies, preferably monoclonal; alternatively, the assay
serves also to identify the vaccine.
TESTS
Residual infectious virus
Inoculate a quantity equivalent to not les s than 25 human
doses of vaccine into cell cultures of the same type as those
used for production of the vaccine. A passage may be made
after 7 days. Maintain the cultures for a total of 21 days and
then examine the cell cultures for rabies virus using an
immunoftuorescence test. The vaccine complies with the test
if no rabies virus is detected.
Bovine serum albumin
Maximum 50 ng per single human dose, determined by a
suitable immunochemical method (2.7.1).
Sterility (2.6.1)
It complies with the test.
Bacteria! endotoxins (2.6.14)
Less than 25 IU per single human dose.
Pyrogens (2.6.8)
It complies with the test. Unless otherwise justified and
authorised, inject into each rabbit a single human dose of the
vaccine diluted to 10 times its volume.
Water (2.5.12)
Maximum 3.0 per cent.
ASSAY
The potency of rabies vaccine is determined by comparing
the dose necessary to protect mice against the effects of a
lethal dose of rabies virus, administered intracerebrally, with
the quantity of a reference preparation of rabies vaccine
necessary to provide the same protection. For this
comparison a reference preparation of rabies vaccine,
calibrated in International Units, and a suitable preparation
of rabies virus for use as the challenge preparation are
necessary.
The International Unit is the activity contained in a stated
quantity of the International Standard. The equivalence in
International Units of the International Standard is stated by
the World Health Organisation.
The test described below uses a parallel-line model with at
least 3 points for the vaccine to be examined and the
reference preparation. Once the analyst has experience with
the method for a given vaccine, it is possible to carry out a
simplified test using a single dilution of the vaccine to be
examined. Such a test enables the analyst to determine that
the vaccine has a potency significantly higher than the
required minimum, but do es not give full information on the
validity of each individual potency determination. The use of
a single dilution allows a considerable reduction in the
number of animals required for the test and must be
considered by each laboratory in accordance with the
provisions of the European Convention for the Protection of
Vertebrate Animals used for Experimental and other
Scientific Purposes.
Selection and distribution oi the test animals U se healthy
female mice, about 4 weeks old, each weighing 11-15 g, and
from the same stock. Distribute the mice into 6 groups of a
2014
size suitable to meet the requirements for validity of the test
and, for titration of the challenge suspension, 4 groups of 5.
Preparation oi the challenge suspension Inoculate mice
intracerebrally with the Challenge Virus Standard (CVS)
strain of rabies virus and when the mice show signs of rabies,
but before they die, euthanise them, then remove the brains
and prepare a homogenate of the brain tissue in a suitable
diluent. Separate gross particulate matter by centrifugation
and use the supematant liquid as the challenge suspension.
Distribute the suspension in small volumes in ampoules, seal
and store at a temperature below - 60 oC. Thaw one
ampoule of the suspension and make serial dilutions in a
suitable diluent. Allocate each dilution to a group of 5 mice
and inject intracerebrally into each mouse 0.03 mL of the
dilution allocated to its group. Observe the mice for 14 days.
Calculate the LDso of the undiluted suspension using the
number in each group that, between the 5th and 14th days,
die or develop signs of rabies .
Determination oi potency oi the vaccine Prepare 3 fivefold
serial dilutions of the vaccine to be examined and 3 fivefold
serial dilutions of the reference preparation. Prepare the
dilutions such that the most concentrated suspensions may
be expected to protect more than 50 per cent of the animals
to which they are administered and the least concentrated
suspensions may be expected to protect les s than 50 per cent
of the animals to which they are administered. Allocate the 6
dilutions, 1 to each of the 6 groups of mice, and inject by the
intraperitoneal route into each mouse 0.5 mL of the dilution
allocated to its group. After 7 days, prepare 3 identical
dilutions of the vaccine to be examined and of the reference
preparation and repeat the injections. 7 days after the second
injection, prepare a suspension of the challenge virus such
that, on the basis of the preliminary titration, 0.03 mL
contains about 50 LDso. Inject intracerebrally into each
vaccinated mouse 0.03 mL of this suspension. Prepare 3
suitable serial dilutions of the challenge suspension. Allocate
the challenge suspension and the 3 dilutions, 1 to each of the
4 groups of 5 control mice, and inject intracerebrally into
each mouse 0.03 mL of the suspension or dilution allocated
to its group. Observe the animals in each group for 14 days
and record the number in each group that die or show signs
of rabies in the period 5-14 days after challenge.
The test is not valid unless:
- for both the vaccine to be examined and the reference
preparation the 50 per cent protective dose lies berween
the largest and smallest doses given to the mice;
- the titration of the challenge suspension shows that
0.03 mL of the suspension contained not les s than 10
LDso;
- the statistical analysis shows a significant slope and no
significant deviations from linearity or parallelism of the
dose-response curves;
- the confidence limits (P = 0.95) are not less than
25 per cent and not more than 400 per cent of the
estimated potency.
The vaccine complies with the test if the estimated potency is
not less than 2.5 IU per human dose.
Application oi altemative end-points Once a laboratory has
established the aboye assay for routine use, the lethal
end-point is replaced by an observation of clinical signs and
application of an end-point earlier than death to reduce
animal suffering. The following is given as an example.
The progress of rabies infection in mice following
intracerebral injection can be represented by 5 stages defined
by typical clinical signs:
2014
Stage 1: ruffled fur, hunched back;
Stage 2: slow movements, loss of alertness (circular
movements may also occur);
Stage 3: shaky movements, trembling, convulsions;
Stage 4: signs of paresis or paralysis;
Stage 5: moribund state.
Mice are observed at least twice daily from day 4 after
challenge. Clinical signs are recorded using a chart such as
that shown in Table 0216.-l. Experience has shown that
using stage 3 as an end-point yields assay results equivalent
to those found when a lethal end-point is used. This must be
verified by each laboratory by scoring a suitable number of
assays using both the c\inical signs and the lethal end-point.
Table 0216.·1. - Example of a chart used to record clinical
signs in the rabies vaccine potency test
Days after challenge
Clínical signs 4 5 6 7 8 9 10
Ruffled fur
Hunched back
Slow movements
Loss of alertness
Circular movements
Shaky movements
Trembling
Convulsions
Paresis
Paralysis
Moribund state
LABELLING
The label sta tes the biological origin of the cells used for the
preparation of the vaccine.
____________________________________________
Rotavirus Vaccine (Uve, Oral) ***
*** ***
(Ph Eu,. mOl1ograph 2417) ***
The label may state ' Rotavirus (Iive, oral)'.
~E~ ____________________________________________
DEFINITION
Rotavirus vaccine (live, oral) is a preparation of one or more
suitable virus serotypes, grown in an approved cell substrate
and presented in a form suitable for oral administration.
The vaccine is a c\ear liquid or it may be a freeze-dried
preparation to be reconstituted irnmediately before use, as
stated on the label, to give a slightly turbid liquid.
The vaccine ready for administration may be coloured owing
to the presence of a pH indicator.
PRODUCTION
GENERAL PROVISIONS
The vaccine strains and the production method shall have
been shown to yield consistently vaccines comparable with
the vaccine of proven c1inical efficacy and safety in mano
The vaccine is formulated so as to avoid inactivation by
Rotavirus Vaccine IV-599
gastric fluids. Where the vaccine is freeze-dried, the anta cid
capacity of the solvent and its stability are established.
The production of vaccine is based on a virus seed-Iot system
and a cell-bank system. Unless otherwise justified and
authorised, the virus in the final vaccine shall have undergone
no more passages from the master seed lot than were used to
prepare the vaccine shown in c\inical studies to be
satisfactory with respect to safety and efficacy.
If purification steps are present, the reduction of selected
process-related impurities and residuals such as residual
host-cell proteins, residual cellular DNA, endotoxins, bovine
serum, trypsin, and antibiotics is monitored to establish
consistency of the purification process.
REFERENCE PREPARATION
A suitable reference preparation that is representative of
batches of vaccine shown to be effective in c\inical trials is
established for use in tests to determine virus concentration.
The differences in the composition and characteristics of
11 rota virus vaccines mean that there will be a specific reference
preparation for each one.
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a suitable cellline (5. 2.3).
VIRUS SEED LOTS
The strain(s) of rotavirus used shall be identified by historical
records that include information on the origin of each strain
and its subsequent manipulation inc\uding the method of
attenuation, whether the strains have been biologically c\oned
prior to generation of the master seed lot, genetic sequence
information, the phenotypic and genotypic stability of the
master and working seed lots when passaged up to the single
harvest level, and the passage level at which attenuation for
humans was demonstrated by c\inical trials. Virus seed lots
are stored at temperatures below - 20 oC if freeze-dried, or
below - 60 cC if not freeze-dried.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
The master and working seed lots are shown to be of the
required rotavirus type by an immunological assay using
~E~ specific antibodies or by a molecular identity test such as
polyacrylamide gel electrophoresis of RNA, RNNRNA
hybridisation, or restriction-enzyme mapping of genetic
sequences of polymerase chain reaction (PCR)-amplified
VP7 gene segments.
Virus concentration
The virus concentration of the master and working seed lots
is determined to monitor consistency of production. Direct
cell-culture based methods and nuc\eic acid amplification
techniques (NAT) (2.6.21) such as PCR quantification of
virus replication in cell culture may be used.
Extraneous agents (2.6. 16)
Each working seed lot complies with the requirements for
virus seed lots.
VIRUS PROPAGATION, SINGLE HARVEST, MONOVALENT
POOLED HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are being handled . Suitable animal (but not human) serum
may be used in the culture media, but the final medium for
maintaining cell growth during virus multiplication does not
contain animal serum. Serum and trypsin used in the
preparation of cell suspensions and culture media are shown
to be free from extraneous agents. The cell culture medium
may contain a pH indicator such as phenol red and suitable
 IV-600 Rotavirus Vaccine
antibiotics at the lowest effective concentration. Ir is
preferable to have a substrate free from antibiotics during
production.
STO RED VIRUS INTERMEDIATE CULTURE
Where a stored virus intermedia te culture, prepared from the
working seed lot, is used for inoculation, on the day of
inoculation not les s than 5 per cent or 500 mL of the cell
cultures employed, whichever is greater, are set aside as
uninfected cell cultures (control cells). Stored virus
intermediate cultures are harvested at a time appropriate to
the strain of virus and stored at temperatures below - 60°C.
Only a stored virus intermediate culture that complies with
the following requirements may be used for virus
propagation.
Identification
Each stored virus intermediate culture is identified by
rota virus type by an irnmunological assay using specific
antibodies or by a molecular identity test such as NAT
(2.6.21).
Bacterial and fungal contarnination
Each stored virus intermediate culture complies with the test
for sterility (2.6.1), carried out using 10 mL for each
medium.
Virus concentration
The virus concentration of each stored virus intermediate
culture is determined as prescribed under Assay to monitor
consistency of production. Both direct cell-culture based
methods and NAT (2.6.21) such as PCR quantification of
virus replication in cell culture may be used.
Extraneous agents (2.6.16)
Each stored virus intermediate culture complies with the tests
for extraneous agents.
Control cells
The control cells of the production cell culture from which
each sto red virus intermediate culture is derived comply with
a test for identity and with the requirements for extraneous
agents (2.6.16).
VIRUS PROPAGATION AND SINGLE HARVEST
On the day of inoculation with the virus working seed lot or
stored virus intermediate culture, cell cultures employed for
vaccine production are set aside as uninfected cell cultures
(control cells) . If bioreactor technology is used, the size and
handling of the cell sample to be examined is approved by
the competent authority. The virus suspensions are harvested
at a time appropriate to the strain of virus being used.
Only a single virus harvest that complies with the following
requirements may be used for further processing.
Bacterial and fungal contarnination
Each single virus harvest complies with the test for sterility
(2.6.1), carried out using 10 mL for each medium.
Control cells
The control cells of the production cell culture from which
each single harvest is derived comply with a test for identity
and with the requirements for extraneous agents (2.6.16) .
MONOVALENT PO OLED HARVEST
Monovalent pooled harvests are prepared by pooling a
number of single harvests of the same virus type. If no
monovalent po oled harvest is prepared, the tests below are
carried out on each single harvest.
Only a single harvest or a monovalent pooled harvest that
complies with the following requirements may be used in the
preparation of the purified monovalent harvest.
2014
Identification
Each single harvest or monovalent pooled harvest is
identified by rotavirus type by an irnmunological assay using
specific antibodies or by a molecular identity test such as
NAT (2.6.21).
Bacterial and fungal contarnination
Each single harvest or monovalent pooled harvest complies
with the test for sterility (2.6.1), carried out using 10 mL for
each medium.
Virus concentration
The virus concentration of each single harvest or monovalent
pooled harvest is determined as prescribed under Assay to
monitor consistency of production. Both direct cell-culture
based methods and NAT (2.6.21) such as PCR
quantification of virus replication in cell culture may be used.
Extraneous agents (2.6.16)
Each single harvest or monovalent pooled harvest complies
with the tests for extraneous agents.
PURIFIED MONOVALENT HARVEST
The purified monovalent harvest is prepared from a single
harvest or a pooled monovalent harvest. The single harvest or
pooled monovalent harvest is clarified to remove cell debris
and may be further purified.
Only a purified monovalent harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Bacterial and fungal contarnination
The purified monovalent harvest complies with the test for
sterility (2.6.1), carried out using 10 mL for each medium.
Virus concentration
The virus concentration of the purified monovalent harvest is
determined as prescribed under Assay to monitor consistency
of production. Both direct cell-culture based methods and
NAT (2.6.21) such as PCR quantification of virus replication
in cell culture may be used.
Residual cellular DNA
Maxirnum 100 Jlg of cellular DNA per human dose for
viruses grown in continuous cells lines.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
satisfactory purified monovalent harvests and may contain
more than one virus type. Suitable stabilisers may be added.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacterial and fungal contarnination
The final bulk vaccine complies with the test for sterility
(2. 6.1), carried out using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile
containers and may be freeze-dried to a moisture content
shown to be favourable to the stability of the vaccine.
The containers are then closed so as to avoid contamination
and the introduction of moisture.
An approved minimum virus concentration for release of the
product is established for each virus type to ensure, in light
of stability data, that the minimum concentration stated on
the labe! will be present at the end of the period of validity.
For freeze-dried vaccines, tests for identity, pH, volume,
sterility and content of key components are carried out on
the solvent.
Only a final lot that complies with the following requirement
for thermal stability and is satisfac10ry with respect 10 each of
 2014
the requirements given below under Identification, Tests and
Assay may be released for use.
Therrnal stability
Maintain not fewer than 3 containers of the finallot at an
elevated temperature for a defined time period, using
conditions found suitable for the particular product as
approved by the competent authority. Determine the virus
concentration as described under Assay in parallel for the
heated vaccine and for vaccine maintained at the temperature
recornmended for storage. The virus concentration of the
containers that have been heated do es not decrease by more
than an approved amount during the period of exposure.
For a multivalent vaccine, if there is no significant difference
in the virus loss between serotypes, the loss may be
determined from total virus concentration.
IDENTIFICATION
The vaccine is shown to contain rota virus of each type stated
on the label by an immunological assay using specific
antibodies or by a molecular identity test. If PCR is used for
the assay, this may serve as the identity test.
TESTS
Bacterial and fungal contamination
The vaccine complies with the test for sterility (2.6.1) .
Water (2.5.12)
Maximum 3.0 per cent for each finallot of freeze-dried
vaccine.
ASSAY
The assay of rotavirus vaccine is carried out by inoculation of
suitable cell cultures with dilutions of the vaccine and
evaluation of the rotavirus concentration, either by
visualisation of infected areas of a cell monolayer or by
comparison of the capacity of the vaccine to produce viral
RNA following infection of cells with the corresponding
capacity of an approved reference preparation.
Por the assay based on visualisation oi iniected areas oi a cel!
monolayer, titrate the vaccine for infective virus using at least
3 separate containers. Titrate the contents of 1 container of
an appropriate virus reference preparation in triplicate to
valida te each assay. If the vaccine contains more than 1
rotavirus type, titrate each type separately using a method of
suitable specificity. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory.
Calculate the individual virus concentration for each
container of vaccine and for each replicate of the reference
preparation as well as the corresponding combined virus
concentrations, using the usual statistical methods (for
example, 5.3).
The assay is not valid if:
the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log CCID so (or
an equivalent value expressed with a unit suitable for the
method used for the assay);
the virus concentration of the reference preparation differs
by more than 0.5 log CCID so (or an equivalent value
expressed with a unit suitable for the method used for the
assay) from the established value.
The assay is repeated ifthe confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so (or an equivalent value expressed
with a unit suitable for the method used for the assay); data
generated from valid assays only are combined by the usual
statistical methods (for example, 5.3) to calculate the virus
Rotavirus Vaccine IV-601
concentration of the sample. The confidence interval
(P = 0.95) of the combined virus concentration is not greater
than ± 0.3 log CCID so (or an equivalent value expressed
with a unit suitable for the method used for the assay) .
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
Por the assay based on comparison oi the capacity oi rhe vaccine
to produce viral RNA following infection of cells with the
corresponding capacity of an approved reference preparation,
a suitable number of cell cultures in a microtitre plate are
infected in parallel with serial dilutions of the vaccine and the
reference preparation. After incubation to allow virus
replication, viral RNA in the individual wells is relea sed from
the cells and quantified by NAT (2.6.21), such as real-time
quantitative reverse-transcriptase polymerase chain reaction
(RT-PCR) technology.
Not fewer than 3 separate containers of the vaccine are
assayed against a container of the reference preparation
titrated in triplica te.
Calculate the individual virus concentration for each
container of vaccine against the reference preparation as well
as the corresponding combined virus concentrations, using
the usual statistical methods (for example, 5.3).
The combined estimate of the virus concentration for the 3
containers of vaccine is not les s than that stated on the labe!'
The assay is not val id unless:
the negative external NAT control is unambiguously
negative;
- the positive external NAT control is unambiguously
positive;
the negative matrix control (uninfected cells) is
unambiguously negative;
the positive matrix control (ce lis spiked with viral RNA) is
unambiguously positive;
the statistical analysis shows a significant slope and no
significant deviations from linearity or parallelism of the
dose-response curves.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log infectious units; data generated from valid
assays only are combined by the usual statistical methods (for
example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than ± 0.3 log infectious
units.
LABELLING
The label sta tes:
the type or types of rotavirus contained in the vaccine;
- the minimum amount of each type of virus contained in 1
single human dose;
the cell substrate used for the preparation of the vaccine.
_ _ __ _ _ _ _ _ __ _ __ __ __ _ _ _ _ _ PII Eur
IV-602 Vaccines
*****
Rubella Vaccine, Uve
** **
(Ph Eur monograph 0162) ***
The label may state 'Rubella'.
~E~ ____________________________________________
DEFINITION
Rubella vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of rubella virus. The vaccine is
reconstituted irnmediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.
PRODUCTION
The production ofvaccine is based on a virus seed-Iot system
and a cell-bank system. The production method shall have
been shown to yield consistently live rubella vaccines of
adequate immunogenicity and safety in mano Unless
otherwise justified and authorised, the virus in the final
vaccine shall have undergone no more passages from the
master seed lot than were used to prepare the vaccine shown
in clinical studies to be satisfactoty with respect to safety and
efficacy.
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk
analysis is carried out. Where necessary and if available, a
test is carried out on the vaccine strain using an animal
model that differentiates wild-type and attenuated virus; tests
on strains of intermediate attenuation may also be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3).
SEED LOT
The strain of rubella virus used shall be identified by
historical records that include information on the origin of
the strain and its subsequent manipulation. Virus seed lots
are prepared in large quantities and stored at temperatures
below - 20 oC if freeze-dried, or below - 60 oC if not
freeze-dried.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
The master and working seed lots are identified as rubella
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentration
The virus concentration of the master and working seed lots
is determined to ensure consistency of production.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots.
PROPAGATION ANO HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are handled during the production. Suitable animal (but not
human) serum may be used in the growth medium, but the
final medium for maintaining cell growth during virus
multiplication do es not contain animal serum. Serum and
trypsin used in the preparation of cell suspensions and
culture media are shown to be free from extraneous agents.
The cell culture medium may contain a pH indicator such as
2014
phenol red and suitable antibiotics at the lowest effective
concentration. It is preferable to have a substrate free from
antibiotics during production. Not les s than 500 mL of the
production cell cultures is set aside as uninfected cell cultures
(control cells). The temperature of incubation is controlled
during the growth of the virus. The virus suspension is
harvested, on one or more occasions, within 28 days of
inoculation. Multiple harvests from the same production cell
culture may be pooled and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Identification
The single harvest contains virus that is identified as rubella
virus by serum neutralisation in cell culture, using specific
antibodies.
Virus concentration
The virus concentration in the single harvest is determined as
prescribed under Assay to monitor consistency of production
and to determine the dilution to be used for the final bulk
vaccine.
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous
agents.
Control cells
The control cells comply with a test for identification and
with the tests for extraneous agents (2.6.16) .
FINAL BULK VACCINE
Single harvests that comply with the aboye tests are pooled
and clarified to remove cells . A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
Bacterial and fungal contamination
The final bulk vaccine complies with the test for sterility
(2.6.1), carried out using 10 mL for each medium.
FINALLOT
A minimum virus concentration for release of the product is
established such as to ensure, in light of stability data, that
the minimum concentration stated on the label will be
present at the end of the period of validity.
Only a finallot that complies with the requirements for
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
requirements given below under Identification and Tests may
be released for use. Provided that the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, it may be omitted on the finallot.
Therma1 stability
Maintain at least 3 vials of the finallot of freeze-dried
vaccine in the dry state at 37 ± 1 oC for 7 days . Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage . The virus
concentration of the heated vaccine is not more than 1.0 log
lower than that of the unheated vaccine.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific rubella antibodies, it is no longer able to
infect susceptible cell cultures.
 2014
TESTS
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1).
Bovine serum albumin
Not more than 50 ng per single human dose, determined by
a suitable immunochemical method (2.7.1) .
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine and inoculating a suitable number
of wells for each dilution step. Titrate 1 vial of an
appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory.
The re!ation with the appropriate European Pharmacopoeia
Biological Reference Preparation is established and
monitored at regular intervals if a manufacturer's reference
preparation is used. Calculate the individual virus
concentration for each vial of vaccine and for each replicate
of the reference preparation as well as the corresponding
combined virus concentrations, using the usual statistical
methods (for example, 5.3). The combined estimate of the
virus concentration for the 3 vials of vaccine is not less than
that stated on the label; the minimum virus concentration
stated on the label is not less than 3.0 log CCID so per single
human dose.
The assay is not valid if:
- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log eCID so;
- the virus concentration of the reference preparation differs
by more than 0.5 log CCID so from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log CCID so; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log CCID so .
Rubella v accine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The labe! states:
- the strain of virus used for the preparation of the vaccine;
- the type and origin of the cells used for the preparation of
the vaccine;
- the minimum virus concentration;
- that contact between the vaccine and disinfectants is to be
avoided.
_ _ _ __ _ _ _ __ _ _ __ __ __ _ __ _ Ph Eur
Vaccines IV-603
*****
Shingles (Herpes Zoster) Vaccine
** **
(Live) ***
(Ph Eur monograph 2418)
The label may state 'Shingles (live) '.
~E~ _ _ __ __ _ _ _ _ _ _ __ _ _ _ _ _ _ ___
DEFINITION
Shingles (herpes zoster) vaccine (live) is a freeze-dried
preparation of a suitable attenuated strain of human
herpesvirus 3. The vaccine is reconstituted immediately
before use, as stated on the label, to give a clear or slightly
opalescent liquid, almost white suspension or pale yellow
liquid that may be coloured owing ro the presence of a pH
indicator. It is intended for administration to adults.
PRODUCTION
The production of vaccine is based on a virus seed-lot systern
and a cell-bank system. The production method shall have
been shown to yield consistently live shingles vaccines of
adequate immunogenicity and safety in mano The virus in the
final vaccine shall not have been passaged in cell cultures
beyond a defined number of passages approved by the
competent authority from the original isolated virus .
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk
analysis is carried out. Where necessary and if available, a
test is carried out on the vaccine strain using an animal
model that differentiates wild-type and attenuated virus; tests
on strains of intermediate attenuation may also be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9) .
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3) .
VIRUS SEED LOT
The strain of human herpesvirus 3 shall be identified as
being suitable by historical records that include information
on the origin of the strain and its subsequent manipulation.
The virus shall at no time have been passaged in continuous
celllines. Seed lots are prepared in the same kind of cells as
those used for the production of the final vaccine. Virus seed
lots are prepared in large quantities and stored at
temperatures below - 20 ce if freeze-dried, or below
- 60 oc if not freeze-dried.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification
The master and working seed lots are identified as human
herpesvirus 3 by serum neutralisation in cell culture, using
specific antibodies.
Virus concentration
The virus concentration of the master and working seed lots
is determined as prescribed under Assay to monitor
consistency of production.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots for live virus vaccines; a sample of 50 mL is taken
for the test in cell cultures.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
 IV-604 Vaccines
or virus are being handled. Approved animal (but not
human) serum may be used in the culture media. Serum and
trypsin used in the preparation of cell suspensions and media
are shown to be free from extraneous agents. The cell culture
medium may contain a pH indicator such as phenol red and
approved antibiotics at the lowest effective concentration.
It is preferable to have a substrate free from antibiotics
during production. 5 per cent, but not less than 50 mL, of
the cell cultures employed for vaccine production is set aside
as uninfected cell cultures (control cells). The infected cells
constituting a single harvest are washed, released from the
support surface and pooled. The cell suspension is disrupted
by sonication.
Only a virus harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine .
Identificarlon
The virus harvest contains virus that is identified as human
herpesvirus 3 by serum neutralisation in cell culture, using
specific antibodies.
Virus concentrarlon
The concentration of infective virus in virus harvests is
determined as prescribed under Assay to monitor consistency
of production and to determine the dilution to be used for
the final bulk vaccine.
Extraneous agents (2.6.16)
Use 50 mL for the test in cell cultures.
Control cells
The control cells of the production cell culture from which
the single harvest is derived comply with a test for identity
and with the requirements for extraneous agents (2.6.16) .
FINAL BULK VACCINE
Virus harvests that comply with the aboye tests are pooled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bacteria! and fungal contamination
Carry out the test for sterility (2.6. 1) using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers and freeze-dried to a moisture
content shown to be favourable to the stability of the vaccine.
The containers are then closed so as to prevent
contamination and the introduction of moisture.
Only a final lot that is satisfactory with respect to the test for
water and each of the requirements given below under
Identification, Tests and Assay may be relea sed for use.
Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the final bulk vaccine,
it may be omitted on the final lot.
Water (2.5.12)
Not more than the amount shown to ensure stability of the
vaccine as approved by the competent authority, determined
by the semi-micro determination of water.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific human herpesvirus 3 antibodies, it is no
longer able to infect susceptible cell cultures.
TESTS
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1).
2014
Bovine serum albumin
Maximum 0.65 Ilg per human dose, determined by a suitable
immunochemical method (2.7. 1).
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine. Titrate 1 vial of an appropriate
virus reference preparation in triplicate to validate each assay.
The virus concentration of the reference preparation is
monitored using a control chart and a titre is established on a
historical basis by each laboratory. Calculate the individual
virus concentration for each vial of vaccine and for each
replicate of the reference preparation as well as the
corresponding combined virus concentrations, using the usual
statistical methods (for example, 5.3). The combined
estimate of the virus concentration for the 3 vials of vaccine
is not less than that stated on the labe!'
The assay is not valid if:
- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log PFU;
- the virus concentration of the reference preparation differs
by more than 0.5 log PFU from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 log PFU; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log PFU.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
- the strain of virus used for the preparation of the vaccine;
- the type and origin of the cells used for the preparation of
the vaccine;
- the minimum virus concentration;
- that contact between the vaccine and disinfectants is to be
avoided;
- that the vaccine is not to be administered to pregnant
women.
___________________________________________ PhE~
Smallpox Vaccine (Uve) *****
** **
(Ph Eur monograph 0164) ***
The label may state 'SMV(live)' .
~E~ _ _________________________________________
DEFINITION
Smallpox vaccine (live) is a liquid or freeze-dried preparation
of live vaccinia virus grown in ovo in the membranes of the
chick embtyo, in cell cultures or in the skin of living animals.
This monograph applies to vaccines produced using strains of
confirmed efficacy in man, in particular those used during
eradication of smallpox, for example the Lister strain
(sometimes referred to as the ListerlElstree strain) and the
New York City Board of Health (NYCBOH) strain. It does
not apply to non-replicative strains such as Modified Virus
Ankara (MVA).
2014
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently smallpox vaccines of adequate safety and
immunogenicity in man. The strain used shall have been
shown ro produce typical vaccinia skin lesions in man.
Production is based on a seed-Iot system.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity of immunosera and vaccines for human use (2.6.9).
The International Reference Preparation for smallpox vaccine
is suitable for use as a reference preparation in virus titration.
SUBSTRATE FOR VIRUS PROPAGATION
Anirnals used for production of skin-derived vaccines
If the vaccine is prepared in animals skins, the animals used
are of a species approved by the competent authority, are in
good health, are kept in e10sed or intensively monitored
colonies, and have not previously been employed for
experimental purposes. Only animals susceptible to infection
by dermal inoculations with vaccinia virus may be used for
vaccine production.
The animals are kept in well-constructed and adequately
ventilated animals rooms with cages spaced as far apart as
possible. Adequate precautions are taken ro prevent
cross-infection between cages. Not more than 1 large animal
is housed per stall . Not more than 2 small animals are
housed per cage and cage-mates must not be interchanged.
The animals must be kept in the country of production of
the vaccine in quarantine groups for a period of not les s than
6 weeks before use.
If at any time during the quarantine period the overall death
rate of the group reaches 5 per cent, no animals from that
entire group may be used for vaccine production.
The groups are kept continuously in isolation, as in
quarantine, even after completion of the quarantine period,
until the animals are used. After the last animal of a group
has been taken, the room that housed the group is
thoroughly c1eaned and decontaminated before receiving a
new group.
Animals that are to be inoculated are anaesthetised and
thoroughly examined. If an animal shows any pathological
lesion, it is not used in the preparation of a seed lot or a
vaccine, nor are any of the remaining animals of the
quarantine group con cerned unless it is evident that their use
will not impair the safety of the product.
The prophylactic and diagnostic measures adopted to
exelude the presence of infectious disease are approved by
the competent authority. According to the species of animals
used and the diseases ro which that animal is Iiable in the
country where the vaccine is being produced, these measures
may vary. Consideration must also be given ro the danger of
spreading diseases ro other countries ro which the vaccine
may be shipped. Special attention must always be given to
foot-and-mouth disease, brucellosis, Q fever, tuberculosis and
dermatomycosis, and it may also be necessary to consider
diseases such as contagious pustular dermatitis (orf), anthrax,
rinderpest, haemorrhagic septicaemia, Rift valley fever and
others.
Embryonated eggs
Embryonated eggs used for production are obtained from a
ftock free from specified pathogens (SPF) (5.2.2).
Human diploid cells, continuous celllines
Human diploid cells and continuous celllines comply with
the requirements for cell substrates (5.2.3) .
Vaccines IV-605
Primary chick embryo cells
Primary chick embryo cells are derived from an SPF ftock
(5.2.2) .
Primary rabbit kidney cells
Only healthy rabbits derived from a e10sed colony approved
by the competent authority are used as a source.
The animals, preferably 2-4 weeks old, are tested to ensure
freedom from specified pathogens or their antibodies.
Where new animal s are introduced into the colony, they are
maintained in quarantine for a minimum of 2 months and
shown to be free from specified pathogens. Animals to be
used ro provide kidneys shall not have been previously
employed for experimental purposes, especially those
involving infectious agents. The colony is monirored for
zoonotic viruses and markers of contamination at regular
intervals.
At the time the colony is established, all animals are tested ro
determine freedom from antibodies to possible viral
contarninants for which there is evidence of capacity for
infecting humans or evidence of capacity ro replicate in vitra
in cells of human origin. A test for retroviruses using a
sensitive polymerase chain reaction (PCR)-based reverse
transcriptase assay is also ineluded. Nucleic acid
amplification tests (2.6.21) for retroviruses may also be used.
After the colony is established, it is monitored by testing a
representative group of at least 5 per cent of the animals,
which are then b1ed at suitable (for example monthly)
intervals. In addition, the colony is screened for pathogenic
micro-organisms, ineluding mycobacteria, fungi and
mycoplasmas. The screening programme is designed to
ensure that all animal s are tested within a given period of
time.
Any animal that dies is examined to determine the cause of
death. If the presence of a causative infectious agent is
demonstrated in the colony, the production of smallpox
vaccine is discontinued.
At the time of kidney harvest, the animals are examined for
the presence of abnormalities and, if any are noted, the
animals are not used for vaccine production.
Each set of control cultures derived from a single group of
animals used to produce a single virus harvest must remain
identifiable as such until all testing, especially for extraneous
agents, is completed.
VIRUS SEED LOT
The vaccinia virus iso late used for the master seed lot is
identified by historical records that inelude information on its
origin and the tests used in its characterisation.
Virus from the working seed lot must have the same
characteristics as the strain that was used to prepare the
master seed lot. The number of passages required to produce
single harvests from the original iso late is Iimited and
approved by the competent authority. Vaccine is produced
from the working seed with a minimum number of
intervening passages.
Since cell culture production and e10nal selection (for
example, plaque purification) may lead to altered
characteristics of the virus, the master seed virus must be
characterised as fully as possible, for example by comparing
the safety profile and biological characteristics of the strain
with that of the parental isolate. The characterisation shall
inelude the following:
- antigenic analyses using specific antisera and/or
monoelonal antibodies;
 IV-606 Vaccines
biological studies such as infectivity titre, chorioallantoic
membrane (CAM) assay, in vitra yield and in vivo growth
characteristics in a suitable animal model;
genetic analyses such as restriction mapping/southem
blotting, PCR analyses and limited sequencing studies;
- phenotypic and genetic stability upon passage in the
substrate;
- neurovirulence testing and immunogenicity studies.
The characterisation tests are also carried out on each
working seed lot and on 3 batches of vaccine from the first
working seed lot ro verify genetic stability of the vaccine
strain.
Only a virus seed that complies with the foJlowing
requirements may be used for virus propagation.
Identification
Each working seed lot is identified as vaccinia virus using
specific antibodies and molecular tests. Suitable tests are
conducted to exclude the presence of variola virus and other
orthopoxviruses.
Virus concentration
Determine by the CAM assay or by a suitable validated
in vitra assay (plaque assay or CCID so assay) . The virus
concentration is the basis for the quantity of virus used in the
neurovirulence test.
Extraneous agents (2.6.16)
If the working seed lot is produced in embryonated eggs,
human diploid cells, or in a continuous ceJl line, it complies
with the requirements for seed lots for virus vaccines. Seed
lots produced in embryonated eggs and seed lots produced in
primary cell cultures comply with the additional requirements
described below.
Where the tests prescribed cannot be carried out because
complete neutralisation of the seed virus is not possible, the
seed lot may be diluted ro a concentration equivalent to that
of the dilution used as inoculum for production of vaccine
prior ro testing for extraneous viruses. Supplementary specific
testing for extraneous virus es using validated nucleic acid
amplification techniques (2.6.21) or imrnunochemical
methods (2. 7.1) may be envisaged. Where the indicator cell
culture method for mycoplasma detection (2. 6.7) cannot be
carried out, nucleic acid amplification testing is performed
instead.
Seed lots ro be used for embryonated egg or cell culture
production are in addition to be tested for carry-over of
potential extraneous agents from the original seed. Given that
the complete passage history of the original seed is unlikely to
be known and that more than one species may have been
used, this additional testing must at least cover important
extraneous agents of concem.
The bioburden of master and working seed lots prepared in
animal skins is limited by meticulous controls of facilities,
personnel, and animals used for production, and by specific
tests on the seeds. However, it may be difficult ro ensure that
seed lots produced in animal skins are totally free from
extraneous agents, and consideration must be given to
production procedures which remove or reduce them. Such
lots must comply with the requirements indicated below.
The absence of specific human pathogens is confirmed by
additional testing procedures, for example, bacterial and
fungal cultures, virus culture, nucleic acid amplification
testings (2.6.21) for viral agents.
N eurovirulence
The neurovirulence of master and working seed lots is
assessed using a suitable animal model, for example in
2014
monkeys or mice. The parental isolate is used as compararor.
Where the original isolate is not available for this purpose,
equivalent materials may be used.
VIRUS PROPAGATION ANO HARVEST
Vaccine produced in living anirnals
Before inoculation the animals are cleaned and thereafter
kept in scrupulously clean stalls until the vaccinia material is
harvested. For 5 days before inoculation and during
incubation the animals remain under veterinary supervision
and must remain free from any sign of disease; daily rectal
temperatures are recorded. If any abnormal rise in
temperature occurs or any clinical sign of disease is observed,
the production of vaccine from the group of animals
con cerned must be suspended until the cause has been
resolved.
The inoculation of seed virus is carried out on such parts of
the animal that are not liable to be soiled by urine and
faeces. The surface used for inoculation is shaved and
cleaned so as to achieve conditions that are as close as
possible ro surgical asepsis. If any antiseptic substance
deleterious to the virus is used in the cleaning process it is
removed by thorough rinsing with sterile water prior to
inoculation. During inoculation the exposed surface of the
animal not used for inoculation is covered with a sterile
covering. By hisrorical experience the ventral surface of
female animals is appropriate for inoculation and inoculation
of mal e animals is more appropriate on the fiank.
Before the collection of the vaccinia material, any antibiotic is
removed and the inoculated area is cleaned.
The uninoculated surfaces are covered with a sterile covering.
Before harvesting the animals are euthanised and
exsanguinated ro avoid heavy mixtures of the vaccinia
material with blood. The vaccinia material from each animal
is collected separately with aseptic precautions. AII animals
used in the production of vaccine are examined by auropsy.
If evidence of any generalised or systemic disease other than
vaccinia is found, the vaccinia material from that animal is
discarded. If the disease is considered to be a communicable
one, the harvest fram the entire group of animals exposed
must be discarded unless otherwise justified and authorised.
Vaccine produced in eggs
All processing of embryonated eggs is done under aseptic
conditions in an area where no other infectious agents or
cells are handled at the same time. After inoculation and
incubation at a controlled temperature only living and
suitable chick embryos are harvested. The age of the embryos
at the time of virus harvest is reckoned from the initial
introduction of the egg into the incubator and shall be not
more than 12 days. After homogenisation and clarification by
centrifugation, the extract of embryonic pulp is tested as
described below and kept at -70 oC or below until funher
processing. Virus harvests that comply with the prescribed
tests may be pooled. No human protein is added to the virus
suspension at any stage during production. If stabilisers are
added, they shall have been shown to have no antigenic or
sensitising properties for mano
Only a single harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Control eggs
Control eggs comply with the tests for extraneous agents
(2.6.16) . A sample of2 per cent ofuninoculated
embryonated eggs (not less than 20 and not more than 50)
from the batch used for vaccine production shall be
incubated under the same conditions as the inoculated eggs.
 2014
At the time of virus harvest the uninoculated eggs are
processed in the same manner as the inoculated eggs.
Sterility (2.6.1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
Vaccine produced in cell cultUres (prirnary chick
embryo cells, primary rabbit kidney cells, human
diploid ceUs or continuous ceU lines)
AH processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are handled at the same time during production. Suitable
animal (but not human) serum may be used in the culture
media, but the final medium for maintaining cell growth
during virus multiplication does not contain animal serum.
Serum and trypsin used in the preparation of cell suspensions
and media are shown to be free from extraneous agents.
The cell culture medium may contain a pH indicator such as
phenol red and suitable antibiotics at the lowest effective
concentration. It is preferable to have a substrate free from
antibiotics during production. On the day of inoculation with
the virus working seed lot, not less than 5 per cent or
1000 mL, whichever is the least, of the cell cultures
employed for vaccine production are set aside as uninfected
cell cultures (control cells); special requirements, given
below, apply to control cells when the vaccine is produced in
primary rabbit kidney cell cultures.
After inoculation of the production cell culture with the
working seed lot, inoculated cells are maintained at a suitable
fixed temperature, and the virus suspension is harvested after
a suitable incubation periodo
Only a single harvest that complies with the following
requirements may be used in the preparation of the
monovalent pooled harvest.
Control cells
The control cells of the production cell culture from which
the virus harvest is derived comply with a test for identity
and with the requirements for extraneous agents (2.6.16) or,
where primary rabbit kidney cells cultures are used, with
specific tests as mentioned hereafter. The test is invalid if
more than 20 per cent of the control cell cultures have been
discarded at the end of the observation periodo
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous
agents. Complete neutralisation of vaccinia virus may be
difficult to achieve at high virus concentration. In this case
specific tests such as nucleic acid amplification (2. 6.21) and
immunochernical tests (2.7.1) can replace non-specific testing
in cell culture or eggs. To save biological reagents such as
vaccinia neutralising antisera, testing for extraneous agents
may be perforrned on the final bulk instead of on the single
harvests.
Vaccine prepared in primary chick embryo cells A sample of
fluids pooled from the control cultures is tested for
adenoviruses and for avian retroviruses such as avian leukosis
virus. In addition, a volume of each neutralised virus pool
equivalent to 100 human doses of vaccine or 10 mL,
whichever is the greater, is tested in a group of fertilised eggs
by the allantoic route of inoculation, and a similar sample is
tested in a separate group of eggs by the yolk-sac route of
inoculation . In both cases 0.5 mL of inoculum is used per
egg. The virus pool passes the test if, after 3-7 days, there is
no evidence of the presence of any extraneous agent.
Vaccine prepared in primary rabbit kidney cell cultures The
following special requirements apply to virus propagation,
Vaccines IV-607
harvest and testing. On the day of inoculation with virus
working seed, a sample of at least 30 mL of the pooled fluid
is removed from the cell cultures of the kidneys of each
group of animals used to prepare the primary cell suspension.
The pooled fluid is inoculated in primary kidney cell cultures
in such a way that the dilution of the pooled fluid does not
exceed 1 in 4. The cultures are incubated at a temperature of
34-36 oC and observed for a period of at least 4 weeks.
During this observation period and after not less than
2 weeks of incubation, at least 1 sub culture of fluid is made
from each of these cultures and observed also for a period of
2 weeks. The test is invalid if more than 20 per cent of the
cultures are discarded. If evidence is found of the presence of
an extraneous agent, no cell cultures from the entire group
may be used for vaccine production.
Control cell cultures. Cultures prepared on the day of
inoculation with the working virus seed lot from
25 per cent of the cell suspensions obtained from the
kidneys of each group of animals are maintained as
controls. These control cell cultures are incubated under
the same conditions as the inoculated cultures for at least
2 weeks. The test is invalid if more than 20 per cent of
the control cell cultures are discarded for non-specific
reasons.
Test for haemadsorbing viruses. At the time of harvest or
not more than 4 days after the day of inoculation of the
production cultures with the virus working seed, a sample
of 4 per cent of the control cell cultures is tested for
haemadsorbing viruses by addition of guinea-pig red
blood cells.
- Test for other extraneous agents. At the time of harvest or
not more than 7 days after the day of inoculation of the
production cultures with the virus working seed, a sample
of at least 20 mL of the pooled fluid from each group of
control cultures is tested for other extraneous agents.
Tests of neutralised single harvest in primary rabbit kidney cell
cultures. Each neutralised single harvest is additionally
tested in primary kidney cell cultures prepared from a
different group of animals to that used for production.
POOLED HARVEST
Only a pooled harvest that complies with the following
requirements and is within the limits approved for the
product may be used in the preparation of the final loto
Identity
The vaccinia virus in the pooled harvest is identified by
serological methods, which may be supplemented by
molecular methods . Molecular tests such as restriction
fragment length polymorphism or partial sequencing,
especiaHy of terminal DNA sequences which show the
greatest variation between vaccinia strains, may be useful.
Virus concentration
The vaccinia virus concentration of the pooled harvest is
deterrnined by chick egg CAM assay or in cell cultures.
A reference preparation is assayed in the same system in
parallel for validation of the pooled harvest titration.
The virus concentration serves as the basis for the quantity of
virus used in the neurovirulence test in mice.
Consistency of virus characteristics
Vaccinia virus in the pooled harvest or the final bulk is
examined by tests that are able to determine that the
phenotypic and genetic characteristics of the vaccinia virus
have not undergone changes during the multiplication in the
production system. The master seed or an equivalent
preparation is used as a comparator in these tests and the
IV-60S Vaccines
comparator and the tests to be used are approved by the
competent authority.
N eurovirulence
The neurovirulence of the po oled harvest is assessed versus a
comparator original seed (or equivalent) by intracerebral
inoculation into suckling mice. Other tests may be useful ro
discriminate between acceptable and unacceptable batches.
Residual DNA
For viruses grown in continuous cells the po oled harvest is
tested for residual DNA. The production process
demonstrates a leve! of cellular DNA of les s than lOng per
human dose.
Bacteria! and fungal contamination
For vaccines other than those prepared on animal skins, the
final bulk complies with the test for sterility (2.6.1) using
10 mL for each medium.
Mycoplasma (2.6.7)
For vaccines other than those prepared on animal skins, the
final bulk complies with the test for mycoplasma, carried out
using 10 mL.
FINAL BULK VACCINE
A minimum virus concentration for release of the product is
esrablished such as to ensure, in the light of stability data,
that the minimum concentration stated on the label will be
present at the end of the period of validity.
Vaccine produced in living animals
The pooled harvest is centrifuged. If the vaccine is intended
for issue in the liquid form, treatment ro reduce the presence
of extraneous agents may consist of the addition of glycerol
or another suitable diluent, with or without an antimicrobial
substance, and temporary storage at a suitable temperature.
If the vaccine is intended for issue in the dried form, the
treatrnent may consist of the addition of a suitable
antimicrobial substance. The following special requirements
apply to the bulk vaccine for vaccines produced in living
animals.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final loto
T ota! bacteria! count
For vaccines produced on animal skins only, maximum
50 per millilitre, determined by plate count using a suitable
volume of the final bulk vaccine.
Escherichia coli
At least 1 mL samples of al: 100 dilution of the final bulk
vaccine is cultured on plates of a medium suitable for
differentiating E. coli from other bacteria. The plates are
incubated at 35-37 oC for 48 h. If E. coli is detected the final
bulk is discarded or, subject ro approval by the competent
authority, processed further.
Haemolytic streptococci, coagu1ase-positive
staphylococci or any other pathogenic micro-organisms
which are known to be harrnful to man by vaccination
At least 1 mL samples of al: 100 dilution of the final bulk
vaccine are cultured on blood agar. The plates are incubated
at 35-37 oC for 48 h. If micro-organisms are detected, the
final bulk vaccine is discarded.
Bacillus anthracis
Any colony seen on any of the plates that morphologically
resembles B. anthracis is examined. If the organisms
contained in the colony are non-motile, further tests for the
cultural character of B . anthracis are carried out, including
pathogenicity tests in suitable animals. If B. anthracis is found
to be present, the final bulk vaccine and any other associated
2014
bulks are discarded. Additional validated molecular testing
may be performed.
Clostridium tetani and other pathogenic spore-forming
anaerobes
A total volume of not less than 10 mL of the final bulk
vaccine is distributed in equal amounts into 10 tubes, each
containing not les s than 10 mL of suitable medium for the
growth of anaerobic micro-organisms. The tubes are kept at
65 oC for 1 h in order ro reduce the content of
non-spore-forming organisms, after which they are
anaerobically incubated at 35-37 cC for at least 1 week.
From every tube or plate showing growth, sub cultures are
made on plates of a suitable medium. Tubes and plates are
incubated anaerobically at the same temperature.
A1l anaerobic colonies are examined and identified and if C.
tetani or other pathogenic spore-forming anaerobes are
present, the final bulk is discarded.
Vaccine produced in eggs
The pooled harvest is clarified and may be further purified.
Vaccine produced in cell cultures (primary chick
embryos fibroblasts, human diploid cells or continuous
celllines)
The pooled harvest is clarified to remove cells and may be
further purified.
FINALLOT
Only a finallot that complies with the requirements for
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
requirements given be!ow under Identification, Tests and
Assay may be released for use. Provided that the tests for
antimicrobial preservative, protein content, bovine serum
albumin and ovalbumin have been carried out with
satisfactory results on the final bulk vaccine, they may be
omitted on the finallot.
Thermal stability
For liquid products, maintain not fewer than 3 containers of
the final lot at an elevated temperature for a defined time
period, using conditions found suitable for the particular
product as approved by the competent authority. Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. The virus
concentration of the containers that have been heated do es
not decrease by more than an approved amount during the
period of exposure. The conditions of the test and the
requirements are approved by the competent authority.
For freeze-dried products, maintain at least 3 containers of
the finallot in the dry state at 37 ± 1 oC for 28 days.
Determine the virus concentration as described under Assay
in parallel for the heated vaccine and for vaccine stored at
the temperature recornmended for storage. The virus
concentration of the heated vaccine is not more than 1.0 log
lower than that of the unheated vaccine.
IDENTIFICAnON
The vaccinia virus is identified by an appropriate method.
TESTS
Antimicrobia! preservative
Where applicable determine the amount of antimicrobial
preservative by a suirable che mi cal method. The content is
not les s than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!.
 2014
Phenol (2.5. 15)
Maximum 0.5 per cent, ifphenol is used.
Protein content
The protein content of each filling lot, if not done on the
final bulk, is determined and is within the limits approved by
the competent authority.
Bovine serurn albumin
Maximum 50 ng per single human dose, determined by a
suitable immunochemical method (2. 7.1), where bovine
serum albumin is used during cell culture.
Ovalburnin
For vaccines produced in embryonated eggs, the ovalbumin
content is within the limits approved by the competent
aurhority.
Residual moisture
The residual moisture content of each finallot of freeze-dried
vaccines is within the limits approved by the competent
authority.
Bacterial count
For skin-derived vaccines, examine the vaccine by suitable
microscopic and culture methods for micro-organisms
pathogenic for man and, in particular, haemolytic
streptococci, staphylococci, pathogenic spore-bearing
organisms, especially B. anthracis, and E. eolio The vaccine is
free from such contaminants. The total number of
non-pathogenic bacteria does not exceed 50 per millilitre.
Sterility (2.6.1)
Except for skin-derived vaccines, the vaccine complies with
the test for sterility.
Bacterial endotoxins (2.6.14)
The vaccine complies with the specification approved by the
competent authority.
ASSAY
Reconstitue the vaccine if necessary and titrate for infectious
virus using at least 3 separate containers of vaccine. Titrate 1
container of an appropriate virus reference preparation in
triplicate to validate each assay. The virus concentration of
the reference preparation is monitored using a control chart
and a titre is established on a historical basis by each
laboratory. Calculate the individual virus concentration for
each container of vaccine and for each replicate of the
reference preparation as well as the corresponding combined
virus concentrations, using the usual statistical methods (for
example, 5.3) . The combined virus concentration for the 3
containers of vaccine is not les s than 8.0 log pock-forming
units per millilitre or the validated equivalent in
plaque-forming units or 50 per cent cell culture infective
doses, unless a lower titre is justified by clinical studies.
The assay is not valid if:
- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.5 log infectious
unitsj
- the virus concentration of the reference preparation differs
by more than 0.5 log infectious units from the established
value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.5 log infectious unitsj data obtained from valid
assays only are combined by the usual statistical methods (for
example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than ± 0.5 log infectious
units .
Vaccines IV-609
Where justified and authorised, different assay designs may
be usedj this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
- the designation of the vaccinia virus strainj
- the minimum amount of virus per millilitrej
- the substrate used for the preparation of the vaccine;
- the nature and amount of stabiliser, preservative or
additive present in the vaccine andlor in the diluent.
_ _ __ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ PhEuf
Adsorbed Tetanus Vaccine
(Tetanus Vaecine (Adsorbed),
Ph Eur monograph 0452)
The label may state 'Tet'.
When Tetanus Vaccine is prescribed or demanded and the
form is not stated, Adsorbed Tetanus Vaccine may be
dispensed or supplied.
Ph EUf _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ __ __ _
DEFINITION
Tetanus vaccine (adsorbed) is a preparation of tetanus formol
toxoid with a mineral adsorbent. The formol toxoid is
prepared from the toxin produced by the growth of
Clostridium tetani.
PRODUCTION
GENERAL PROVISIONS
Specific toxicity
The production method is validated to demonstrate that the
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 21 days of
the injection any of the animals shows signs of or dies from
tetanus, the vaccine does not comply with the test. If more
than 1 animal dies from non-specific causes, repeat the test
once; if more than 1 animal dies in the second test, the
vaccine does not comply with the test.
BULK PURIFIED TOXOID
For the production of tetanus toxin, from which toxoid is
prepared, seed cultures are managed in a defined seed-Iot
system in which toxinogenicity is conserved and, where
necessary, resto red by delibera te reselection. A highly
toxinogenic strain of Clostridium tetani with known origin and
history is grown in a suitable liquid medium. At the end of
cultivation, the purity of each culture is tested and
contaminated cultures are discarded. Toxin-containing
culture medium is collected aseptically. The toxin content
(Lf per millilitre) is checked (2.7.27) to monitor consistency
of production. Single harvests may be pooled to prepare the
bulk purified toxoid. The toxin is purified to remove
components likely to cause adverse reactions in humans.
The purified toxin is detoxified with formaldehyde by a
method that avoids destruction of the immunogenic potency
of the toxoid and reversion of toxoid to toxin, particularly on
exposure to heat. Altematively, purification may be carried
out after detoxification.
 IV-610 Vaccines
Only bulk purified toxoid that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
Absence of toxin and irreversibility oftoxoid
Using the same buffer solution as for the final vaccine,
without adsorbent, prepare a solution of bulk purified toxoid
at the same concentration as in the final vaccine. Divide the
dilution into 2 equal parts. Keep one of them at 5 ± 3 oC
and the other at 37 oC for 6 weeks. Test both dilutions as
described below. Use 15 guinea-pigs, each weighing
250-350 g and that have not previously been treated with any
material that will interfere with the test. Inject
subcutaneously into each of 5 guinea-pigs 5 mL of the
dilution incubated at 5 ± 3 oc. Inject subcutaneously into
each of 5 other guinea-pigs 5 mL of the dilution incubated at
37 oc. Inject subcutaneously into each of 5 guinea-pigs at
least 500 Lf of the non-incubated bulk purified toxoid in a
volume of 1 mL. The bulk purified toxoid complies with the
test if during the 21 days following the injection no animal
shows signs of or die s from tetanus. If more than 1 animal
dies from non-specific causes, repeat the test; if more than
1 animal dies in the second test, the toxoid does not comply
with the test.
Antigenic purity (2.7.27)
Not less than 1000 Lf per milligram of protein nitrogen.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption of a suitable
quantity of bulk purified toxoid onto a mineral carrier such
as hydrated aluminium phosphate or aluminium hydroxide;
the resulting mixture is approximately isotonic with blood.
Suitable antimicrobial preservatives may be added. Certain
antimicrobial preservatives, particularly those of the phenolic
type, adversely affect the antigenic activity and must not be
used.
Only final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Antimicrobial preservative
Where applicable, detennine the amount of antimicrobial
preservative by a suitable chemical method. The amount is
not less than 85 per cent and not greater than 115 per cent
of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided the test for
antimicrobial preservative and the assay have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the final lot.
Provided the free formaldehyde content has been detennined
on the bulk purified toxoid or on the final bulk and it has
been shown that the content in the finallot will not exceed
0.2 gIL, the test for free formaldehyde may be omitted on the
finallot.
IDENTIFICATION
Tetanus toxoid is identified by a suitable immunochemical
method (2. 7. 1). The following method, applicable to certain
2014
vaccines, is given as an example . Dissolve in the vaccine to
be examined sufficient sodium citrate R to give a 100 gIL
solution. Maintain at 37 oC for about 16 h and centrifuge
until a clear supernatant liquid is obtained. The clear
supernatant liquid reacts with a suitable tetanus antitoxin,
giving a precipitate.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dos e, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobial preservative
Where applicable, detennine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
labe!'
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2. 7.8) .
The lower confidence limit (P = 0.95) ofthe estimated
potency is not less than 40 IU per single human dose.
LABELLING
The label sta tes:
-- the minimum number of Intemational Units per single
human dose,
-- the name and the amount of the adsorbent,
-- that the vaccine must be shaken before use,
-- that the vaccine is not to be frozen.
------______________________________________ ~Ew
Tick-borne Encephalitis Vaccine,
Inactivated
(Tick-borne Encephalitis Vaccine (Inactivated),
Ph Eur monograph 1375)
The label may state 'Tic/enceph'.
~Ew ____________________________________________
DEFINITION
Tick-borne encephalitis vaccine (inactivated) is a liquid
preparation of a suitable strain of tick-borne encephalitis
virus grown in cultures of chick-embryo celIs or other
suitable cell cultures and inactivated by a suitable, validated
method.
PRODUCTION
GENERAL PROVISIONS
Production of the vaccine is based on a virus seed-Iot system.
The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in mano Unless otherwise justified
and authorised, the virus in the final vaccine shall not have
undergone more passages from the master seed lot than the
virus in the vaccine used in clinical trials.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnonnal
toxicity for immunosera and vaccines for human use (2.6.9) .
 2014
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in chick embryo cells prepared from
eggs derived from a chicken flock free from specified
pathogens (5.2.2) or in other suitable cell cultures (5.2.3).
SEED LOTS
The strain of virus used is identified by historical records that
include information on the origin of the strain and its
subsequent manipulation. Virus seed lots are stored at or
below - 60 oc.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification
Each seed lot is identified as containing the vaccine strain of
tick-borne encephalitis virus by a suitable immunochemical
method (2.7.1), preferably using monoclonal antibodies.
Virus concentration
The virus concentration of each seed lot is determined by
titration in suitable cell cultures to monitor consistency of
production.
Extraneous agents (2.6.16)
Each seed lot complies with the requirements for extraneous
agents in viral vaccines for human use. For neutralisation of
the vaccine virus, the use of monoclonal antibodies is
preferable.
VIRUS PROPAGATION AND HARVEST
If the virus has been passaged in mouse brain during
preparation of the master seed lot, not fewer than 2 passages
of the master seed virus in cell culture are made before
inoculation of the production cell culture.
All processing of the cell cultures is performed under aseptic
conditions in an area where no other cells are being handled.
Serum and trypsin used in the preparation of cell suspensions
and media used must be shown to be free from extraneous
agents. The cell culture media may contain a pH indicator
such as phenol red and approved antibiotics at the lowest
effective concentration. At least 500 mL of the cell cultures
employed for vaccine production is set aside as uninfected
cell cultures (control cells) .
Only a single harvest that complies with the following
requirements may be used in the preparation of the
inactivated harvest.
Identification
The single harvest is shown to contain tick-borne encephalitis
virus by a suitable immunochemical method (2. 7.1),
preferably using monoclonal antibodies, or by virus
neutralisation in cell cultures .
Bacteria! and fungal contamination
The single harvest complies with the test for sterility (2.6.1),
carried out using 10 mL for each medium.
Mycoplasmas (2.6. 7)
The single harvest complies with the test for mycoplasmas
carried out using 1 mL for each medium.
Control cells
The control cells comply with the tests for extraneous agents
(2.6.16). If the vaccine is produced using a cell-bank system,
the control cells comply with a test for identification.
Virus concentration
Determine the virus concentration by titration in suitable cell
cultures to monitor consistency of production.
INACTIVATION
To avoid interference, viral aggregates are removed, where
necessary, by tiltration immediately before the inactivation
process . The virus suspension is inactivated by a validated
Vaccines IV -611
method; the method shall have been shown to be consistently
capable of inactivating tick-borne encephalitis virus without
destroying the antigenic and immunogenic activity; as part of
the validation studies, an inactivation curve is plotted
representing residual live virus concentration measured on
not fewer than 3 occasions. If formaldehyde is used for
inactivation, the presence of an excess of free forrnaldehyde is
verified at the end of the inactivation process.
Only an inactivated harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccme.
Residual infective virus
Inoculate a quantity of the inactivated harvest equivalent to
not less than 10 human doses of vaccine in the final lot into
primary chicken fibroblast cell cultures, or other cells shown
to be at least as sensitive to tick-borne encephalitis virus, with
not less than 3 cm2 of cell sheet per millilitre of inoculum.
Incubate at 37 ± 1 oC for 14 days. No cytopathic effect is
detected at the end of the incubation periodo Collect the
culture fluid and examine for the presence of infective
tick-borne encephalitis virus by the following test in mice or
by a validated in vitro method: inoculate 0.03 mL
intracerebrally into each of not fewer than 10 mice about
4 weeks old. Observe the mice for 14 days . They show no
evidence of tick-borne encephalitis virus infection.
PURIFICATION
Several inactivated single harvests may be pooled before
concentration and purification by suitable methods,
preferably by continuous-fiow, sucrose density-gradient
centrifugation.
Several purified inactivated harvests may be pooled.
Only a purified, inactivated harvest that complies with the
following requirements may be used in the preparation of the
final bulk vaccine.
Sterility (2. 6. 1)
The purified, inactivated harvest complies with the test for
sterility carried out using 10 mL for each medium.
Specific activity
Determine the antigen content of the purified, inactivated
harvest by a suitable immunochemical method (2.7.1).
Determine the total protein content by a suitable method.
The specific activity, calculated as the antigen content per
unit mass of protein, is within the lirnits approved for the
specific product.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more purified,
inactivated harvests.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the finallot.
S terility (2. 6. 1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
FINALLOT
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be relea sed for use. Provided that the tests for free
forrnaldehyde, bovine serum albumin (where applicable) and
pyrogens and the assay have been carried out with
satisfactory results on the final bulk vaccine, they may be
omitted on the finallot.
IDENTIFICAnON
The vaccine is shown to contain tick-borne encephalitis virus
antigen by a suitable immunochemical method (2. 7.1) using
 IV-612 Vaccines
specific antibodies. The assay also serves to identify the
vaccme.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium
hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
Free formaldehyde (2.4.18)
Maximum 0.1 gIL.
Bovine serum albumin
Maximum 50 ng per single human dose, determined by a
suitable immunochemical method (2.7.1), ifbovine serum
albumin has been used during production.
Sterility (2.6.1)
The vaccine complies with the test for sterility.
Pyrogens (2.6.8)
The vaccine complies with the test for pyrogens. Inject into
each rabbit, per kilogram of body mass, 1 dose of vaccine.
ASSAY
The potency is determined by comparing the dose necessary
to protect a given proportion of mice against the effects of a
lethal dose of tick-borne encephalitis virus, administered
intraperitoneally, with the quantity of a reference preparation
of tick-borne encephalitis vaccine necessary 10 provide the
same protection. For this comparison an approved reference
preparation and a suitable preparation of tick-borne
encephalitis virus from an approved strain for use as the
challenge preparation are necessary.
The following is cited as an example of a method that has been
found suitable for a given vaeeine.
Selection and distribution of test animals Use healthy mice
weighing 11-17 g and derived from the same stock.
Distribute the mice into not fewer than 6 groups of a suitable
size to meet the requirements for validity of the test;
for titration of the challenge suspension, use not fewer than
4 gtoups of 10 mice. Use mice of the same sex or distribute
males and females equally between groups.
Determination of potency of the vaeeine Prepare not fewer
than 3 suitable dilutions of the vaccine 10 be examined and
of the reference preparation; in order 10 comply with validity
criteria 4 or 5 dilutions will usually be necessary. Prepare
dilutions such that the most concentrated suspension is
expected to protect more than 50 per cent of the animals and
the least concentrated suspension les s than 50 per cent.
Allocate each dilution to a different group of mice and inject
subcutaneously into each mouse 0.2 mL of the dilution
allocated 10 its group. 7 days later make a second injection
using the same dilution scale. 14 days after the second
injection prepare a suspension of the challenge virus
containing not les s than 100 LDso in 0.2 mL. Inject 0.2 mL
of this virus suspension intraperitoneally into each vaccinated
mouse. To verify the challenge dose, prepare a series of not
fewer than 3 dilutions of the challenge virus suspension at
not gteater than one-hundredfold intervals. Allocate the
challenge suspension and all of the dilutions, one to each of
the gtoups of 10 mice, and inject intraperitoneally into each
mouse 0.2 mL of the challenge suspension or the dilution
allocated to its group . Observe the animals for 21 days after
the challenge and record the number of mice that die in the
period between 7 and 21 days after the challenge. Humane
endpoints may be used to avoid unnecessary suffering of
animals after the virulent challenge.
Caleulations Calculate the results for an assay with quantal
responses by the usual statistical methods (for example, 5.3).
2014
Validity eriteria The test is not valid unless:
-- the concentration of the challenge virus is not less than
100 LD so ,
-- for both the vaccine to be examined and the reference
preparation the 50 per cent protective dose (PD so) lies
between the largest and smallest doses given 10 the mice,
-- the statistical analysis shows a significant slope and no
significant deviation from linearity and parallelism of the
dose-response lines,
-- the confidence limits (P = 0.95) are not less than
33 per cent and not more than 300 per cent of the
estimated potency.
Poteney requirement Include all valid tests 10 estimate the
mean potency and the confidence limits (P = 0.95) for the
mean potency; compute weighted means with the inverse of
the squared standard error as weights. The vaccine complies
with the test if the estimated potency is not less than that
approved by the competent authority, based on data from
clinical efficacy trials.
LABELLING
The label states:
-- the strain of virus used in preparation,
-- the type of cells used for production of the vaccine.
_____________________________________________ ~E~
Typhoid Polysaccharide Vaccine ***
*** ***
(Ph Eur monograph 1160) ***
The label may state 'Typhoid'.
~E~ ____________________________________________
DEFINITlON
Typhoid polysaccharide vaccine is a preparation of purified
Vi capsular polysaccharide obtained from Salmonella typhi Ty
2 strain or some other suitable strain that has the capacity to
produce Vi polysaccharide.
Capsular Vi polysaccharide consists of partly 3-0-acetylated
repeated units of 2-acetylamino-2-deoXY-D-
galactopyranuronic acid with ex-(1-->4) linkages.
PRODUCTlON
The production of Vi polysaccharide is based on a seed-Iot
system. The method of production shall have been shown 10
yield consistently typhoid polysaccharide vaccines of adequate
immunogenicity and safety in mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6. 9).
BACTERIAL SEED LOTS
The strain of S. typhi used for the master seed lot shall be
identified by historical records that include information on its
origin and by its biochemical and serological characteristics.
Cultures from the working seed lot shall have the same
characteristics as the strain that was used to prepare the
master seed lot.
Only a strain that has the following characteristics may be
used in the preparation of the vaccine: (a) stained smears
from a culture are typical of enterobacteria; (b) the culture
utilises glucose without production of gas; (c) colonies on
agar are oxidase-negative; (d) a suspension of the culture
agglutinates specifically with a suitable Vi antiserum or
colonies form halo es on an agar plate containing a suitable Vi
antiserum.
2014
Purity of bacterial strain used for the seed lot is verified by
methods of suitable sensitivity. These may include
inoculation into suitable media, examination of colony
morphology, microscopic examination of Gram-stained
smears and culture agglutination with suitable specific
antisera.
CULTURE AND HARVEST
The working seed lot is cultured on a solid medium, which
may contain blood-group substances, or a Iiquid medium;
the inoculum obtained is transferred to a liquid medium
which is used to inoculate the final medium. The liquid
medium used and the final medium are semi-synthetic, free
from substances that are precipitated by cetrimonium
bromide and do not contain blood-group substances or
high-molecular-mass polysaccharides, unIess it has been
demonstrated that they are removed by the purification
process.
The bacterial purity of the culture is verified by methods of
suitable sensitivity. These may include inoculation into
suitable media, examination of colony morphology,
microscopic examination of Gram-stained smears and culture
agglutination with suitable specific antisera.
The culture is then inactivated at the beginning of the
stationary phase by the addition of formaldehyde. Bacterial
cells are eliminated by centrifugation; the polysaccharide is
precipitated from the culture medium by addition of
hexadecyltrimethylammonium bromide (cetrimonium
bromide) . The precipitate is harvested and may be stored at
- 20 oC before purification.
PURIFIED VI POLYSACCHARIDE
The polysaccharide is purified, after dissociation of the
polysaccharide/cetrimonium bromide complex, using suitable
procedures to eliminate successively nucleic acids, proteins
and Iipopolysaccharides. The polysaccharide is precipitated as
the calcium salt in the presence of ethanol and dried at
2-8 oC; the powder obtained constitutes the purified Vi
polysaccharide. The loss on drying is determined by
thermogravimetry (2.2.34) and is used to calculate the results
of the chemical tests shown below with reference to the dried
substance.
OnIy a purified Vi polysaccharide that complies with the
following requirements may be used in the preparation of the
final bulk.
Protein (2.5.16)
Maximum 10 mg per gram of polysaccharide, calculated with
reference to the dried substance.
Nucleic acids (2.5.17)
Maximum 20 mg per gram of polysaccharide, calculated with
reference to the dried substance.
O-Acetyl groups (2.5.19)
Minimum 2 mmol per gram of polysaccharide, calculated
with reference to the dried substance.
Molecular size
Examine by size-exclusion chromatography (2.2.30) using
cross-linked agarosefor chromatography R. Use a column 0.9 m
long and 16 mm in internal diameter equilibrated with a
solvent having an ionic strength of 0.2 mollkg and a pH of
7.0-7 .5. Apply about 5 mg ofpolysaccharide in a volume of
1 mL to the column and elute at about 20 mlJh. Collect
fractions of about 2.5 mL. Determine the point
corresponding to Ko = 0.25 and make 2 pools consisting of
fractions eluted before and after this point. Determine
O-acetyl groups on the 2 pools (2.5.19). Not les s than
Vaccines IV-613
50 per cent of the polysaccharide is found in the pool
containing fractions eluted before Ko = 0.25.
Identification
Carry out an identification test using a suitable
immunochemical method (2.7.1).
Bacteria! endotoxins
The content of bacterial endotoxins determined by a suitable
method (2.6.14) is within the limits approved for the specific
producto
FINAL BULK VACCINE
One or more batches of purified Vi polysaccharide are
dissolved in a suitable solvent, which may contain an
antimicrobial preservative, so that the volume corresponding
to 1 dose contains 25 ¡.tg of polysaccharide and the solution
is isotonic with blood (250 mosmollkg to 350 mosmollkg) .
Only a final bulk vaccine that complies with the following
tests may be used in the preparation of the final lot.
Sterility (2. 6.1)
The final bulk vaccine complies with the test for sterility,
carried out using 10 mL for each medium.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable physicochemical method.
The amount is not less than 85 per cent and not greater than
115 per cent of the intended amount.
FINALLOT
The final bulk vaccine is distributed asepticaIly into sterile
tamper-proof containers that are then c10sed so as to prevent
contamination.
Only a final lot that is satisfactory with respect to each of the
requirements prescribed below under Identification, Tests
and Assay and with the requirement for bacterial endotoxins
may be released for use. Provided the tests for free
formaldehyde and antimicrobial preservative have been
carried out on the final bulk vaccine, they may be omitted on
the final lot.
Bacteria! endotoxins
The content of bacterial endotoxins determined by a suitable
method (2.6.14) is within the limit approved for the specific
product.
CHARACTERS
Clear colourIess Iiquid, free from visible particles.
IDENTIFICATION
Carry out an identification test using a suitable
immunochemical method (2.7.1).
TESTS
pH (2.2.3)
6.5 to 7.5.
O-Acetyl groups
0.085 ( ± 25 per cent) ¡.tmol per dose (25 ¡.tg of
polysaccharide) .
Test solution Place 3 mL of the vaccine in each of 3 tubes
(2 reaction solutions and 1 correction solution) .
Reference solutions Dissolve 0.150 g of acetylcholine chloride R
in 10 mL of water R (stock solution containing 15 gIL of
acetylcholine chloride). Immediately before use, dilute
0.5 mL of the stock solution to 50 mL with water R (working
dilution containing 150 ¡.tg/mL of acetyIcholine chloride) .
In 10 tubes, place in duplicate (reaction and correction
solutions) 0.1 mL, 0.2 mL, 0.5 mL, l.0 mL and l.5 mL of
the working dilution.
Prepare a blank using 3 mL of water R.
IV-614 Vaccines
Make up the volume in each tube to 3 mL with water R.
Add 0.5 mL of a mixture of 1 volume of water R and
2 volumes of di/ute hydroehlarie acid R to each of the
correction tubes and to the blank. Add 1.0 mL of alkaline
hydroxylamine salutian R to each tube. Allow the reaction to
proceed for exactly 2 min and add 0.5 mL of a mixture of
1 volume of water R and 2 volumes of dilute hydroehlarie
aeid R to each of the reaction tubes. Add 0.5 mL of a
200 giL solution of Jerrie ehlaride R in 0.2 M hydraehlarie acid
to each tube, stopper the tubes and shake vigorously to
remove bubbles.
Measure the absorbance (2.2.25) of each solution at 540 nm
using the blank as the compensation liquidoFor each reaction
solution, subtract the absorbance of the corresponding
correction solution. Draw a calibration curve from the
corrected absorbances for the 5 reference solutions and the
corresponding content of acetylcholine chloride and read
from the curve the content of acetylcholine chloride in the
test solution for each volume tested. Calculate the mean of
the 2 values.
1 mole of acetylcholine ch10ride (181.7 g) is equivalent to
1 mole of O-acetyl (43 .05 g).
Free formaldehyde (2.4.18)
Maximum 0.2 gIL.
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable physicochemical method.
The content is not less than the minimum amount shown to
be effective and not more than 115 per cent of the content
stated on the labe!' If phenol has been used in the
preparation, the content is not more than 2.5 giL (2.5.15).
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Determine Vi polysaccharide by a suitable irnmunochemical
method (2. 7.1), using a reference purified polysaccharide.
The estimated amount of polysaccharide per dose is
80 per cent to 120 per cent of the content stated on the
labe!' The confidence limits (P = 0.95) of the estimated
amount of polysaccharide are not less than 80 per cent and
not more than 120 per cent.
LABELLING
The label states:
-- the number of micrograms of polysaccharide per human
dose (25 ~lg),
-- the total quantity of polysaccharide in the container.
_ _ _ _ _ _ __ _ _ __ _ __ __ _ _ __ _ Ph Eur
Typhoid Vaccine
(Ph Eur managraph 0156)
The label may state 'Typhoid'.
~E~ ____________ _ __ _ __ _ ______ _ __
DEFINITION
Typhoid vaccine is a sterile suspension of inactivated
Salmanella typhi containing not less than 5 x 10 8 and not
more than 1 x 109 bacteria (S. typh¡) per human dose.
The human dose do es not exceed 1.0 mL.
PRODUCTION
The vaccine is prepared using a seed-lot system from a
2014
suitable strain, such as Ty 2 1, of S. typhi. The final vaccine
represents not more than 3 sub cultures from the strain on
which were made the laboratory and clinical tests that
showed it to be suitable. The bacteria are inactivated by
acetone, by formaldehyde, by phenol or by heating or by a
combination of the last 2 methods .
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for irnmunosera and vaccines for human use (2.6.9)
modified as follows: inject 0.5 mL of the vaccine into each
mouse and 1.0 mL into each guinea pig.
IDENTIFICATION
It is identified by specific agglutination.
TESTS
Phenol (2.5.15)
If phenol has been used in the preparation, the concentration
is not more than 5 giL.
Antigenic power
When injected into susceptible laboratory animals, it elicits
anti-O, anti-H and, to a les ser extent, anti-Vi agglutinins.
Sterility (2.6.1)
It complies with the test for sterility.
LABELLING
The labe! sta tes:
-- the method used to inactivate the bacteria,
-- the number of bacteria per human dose.
_ _ _ _ _ _ _ _ _ __________ _ _ _ _____ ~E~
Typhoid Vaccine, Freeze-dried ***
*** ***
(Ph Eur monagraph 0157) ***
The label may state 'Typhoid'.
~E~ ____________ __ _ ________ __ __
DEFINITION
Freeze-dried typhoid vaccine is a freeze-dried preparation of
inactivated Salmonella typhi. The vaccine is reconstituted as
stated on the label to give a uniform suspension containing
not less than 5 x 108 and not more than 1 x 109 bacteria
(S. typh¡) per human dose. The human dos e does not exceed
1.0 mL of the reconstituted vaccine.
PRODUCTION
The vaccine is prepared using a seed-lot system from a
suitable strain, such as Ty 2 1, of S. typhi. The final vaccine
represents not more than 3 subcultures from the strain on
which were made the laboratory and clinical tests that
showed it to be suitable. The bacteria are inactivated either
by acetone or by formaldehyde or by heat. Phenol is not used
in the preparation. The vaccine is distributed into sterile
containers and freeze-dried to a moisture content favourable
to the stability of the vaccine. The containers are then closed
so as to exclude contamination.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9)
modified as follows: inject 0.5 mL of the vaccine into each
1 This s¡rain is issued by the World H ealth Organisation Collaborating
Centre for Reference and Research on Bacterial Vaccines, Human Serum
and Vaccine Institute, Szallas Utea 5, H-II07, Budapest, Hungary.
2014
mouse and 1.0 mL into each guinea pig.
IDENTIFICATION
The vaccine reconstituted as stated on the label is identified
by specific agglutination.
TESTS
Phenol (2.5.15)
If phenol has been used in the preparation, the concentration
is not more than 5 giL.
Antigenic power
When injected into susceptible laboratory animal s, the
reconstituted vaccine elicits anti-O, anti-H and, ro a lesser
extent, anti-Vi agglutinins.
Sterility (2. 6.1)
The reconstituted vaccine complies with the test for sterility.
LABELLING
The label sta tes:
-- the method used to inactivate the bacteria,
-- the number of bacteria per human dose,
-- that the vaccine should be used within 8 h of
reconstitution.
____________________________________________ PhEw
***
Typhoid (Strain Ty 21a) Vaccine,
*** ***
Live (Oral) ***
(Typhoid Vaccine (Live, Oral, Strain Ty 21a),
Ph Eur monograph 1055)
The label may state 'Typhoid (live,oral)'.
~Ew ____________________________________________
DEFINITION
Typhoid vaccine (live, oral, strain Ty 21a) is a freeze-dried
preparation of live Sabnonella typhi strain Ty 21a grown in a
suitable medium. When presented in capsules, the vaccine
complies with the monograph on Capsules (0016).
PRODUCTION
CHOICE OF VACCINE STRAIN
The main characteristic of the strain is the defect of the
enzyme uridine diphosphate-galactose-4-epimerase.
The activities of galacropermease, galacrokinase and
galactose-1-phosphate uridyl-transferase are reduced by
50 per cent ro 90 per cent. Whatever the growrh conditions,
the strain do es not contain Vi antigen. The strain
agglutinates ro anti-O:9 antiserum only if grown in medium
containing galactose. It contains the ftagellar H:d antigen and
does not produce hydrogen sulfide on Kligler iron agar.
The strain is nonvirulent for mice. Cells of strain Ty 21a Iyse
if grown in the presence of 1 per cent of galactose.
BACTERIAL SEED LOTS
The vaccine is prepared using a seed-Iot system. The working
seed lots represent not more than one subculture from the
master seed lot. The final vaccine represents not more than
four subcultures from the original vaccine on which were
made the laborarory and clinical tests showing the strain ro
be suitable.
Only a master seed lot that complies with the following
requirements may be used in the preparation of working seed
lots.
Galactose metabolism
In a spectrophotometric assay, no activiry of the enzyme
uridine diphosphate-galactose-4-epimerase is found in the
cytoplasm of strain Ty 21a compared ro strain Ty 2.
Vaccines IV-615
Biosynthesis of lipopolysaccharide
Lipopolysaccharides are extracted by the hot-phenol method
and examined by size-exclusion chromarography. Strain Ty
21a grown in medium free of galactose shows only the rough
(R) type of lipopolysaccharide.
Sero1ogical characteristics
Strain Ty 21a grown in a synthetic medium without
galacrose do es not agglutinate ro specific anti-O:9 antiserum.
Whatever the growth conditions, strain Ty 21a does not
agglutinate to Vi antiserum. Strain Ty 21a agglutinates ro
H:d ftagellar antiserum.
Biochemical markers
Strain Ty 21 a does not produce hydrogen sulfide on Kligler
iron agar. This properry serves ro distinguish Ty 21a from
other galactose-epimerase-negative S. typhi strains.
Cell growth
Strain Ty 21a cells Iyse when grown in the presence of
1 per cent of galactose.
BACTERIAL PROPAGATION AND HARVEST
The bacteria from the working seed lot are multiplied in a
preculture, subcultured once and are then grown in a suitable
medium containing 0.001 per cent of galacrose at 30 oC for
13 h ro 15 h. The bacteria are harvested. The harvest must
be free from contaminating micro-organisms.
Only a single harvest that complies with the following
requirements may be used for the preparation of the
freeze-dried harvest.
pH
The pH of the culture is 6.8 ro 7.5.
Optical density
The optical density of the culture, measured at 546 nm, is
6.5 to 11.0. Before carrying out the measurement, dilute the
culture so that a reading in the range 0.1 to 0.5 is obtained
and correct the reading to take account of the dilution.
Identification
Culture bacteria on an agar medium containing 1 per cent of
galacrose and bromothymol blue. Light blue, concave
colonies, transparent due to Iysis of cells, are formed.
No yellow colonies (galactose-fermenting) are found.
FREEZE-DRIED HARVEST
The harvest is mixed with a suitable stabiliser and
freeze-dried by a process that ensures the survival of at least
10 per cent of the bacteria and to a water content shown ro
be favourable to the stabiliry of the vaccine. No antimicrobial
preservative is added ro the vaccine.
Only a freeze-dried harvest that complies with the following
tests may be used for the preparation of the final bulk.
Identification
Culture bacteria are examined on an agar medium containing
1 per cent of galactose and bromothymol blue. Light blue,
concave colonies, transparent due to Iysis of cells, are
formed. No yellow colonies (galactose-fermenting) are found.
Nwnber of live bacteria
Not fewer than 1 x 10 11 live S. typhi strain Ty 21a per
gramo
Water (2.5.12)
1.5 per cent to 4.0 per cent, determined by the semi-micro
determination of water.
FINAL BULK VACCINE
The final bulk vaccine is prepared by mixing under suitable
conditions one or more freeze-dried harvests with suitable
excipients.
 IV -616 Vaccines
Only a final bulk that complies with the following
requirement may be used in the preparation of the final lot.
Number of live bacteria
Not fewer than 40 x 10 9 live S. typhi strain Ty 21a per
gramo
FINALLOT
The final bulk vaccine is distributed under suitable
conditions iuto capsules with a gastro-resistant shell or into
suitable containers.
Only a finallot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Number of live bacteria may be released for use, except that
in the determination of the number of live bacteria each
dosage unit must contain not fewer than 4 x 10 9 live
bacteria.
IDENTIFICATION
Culture bacteria from the vaccine to be examined on an agar
medium containing 1 per cent of galactose and bromothymol
blue. Light blue, concave colonies, transparent due to lysis of
cells, are formed. No yellow colonies (galactose-fermenting)
are found.
TESTS
Contaminating micro-organisms (2.6.12, 2.6.13)
Carry out the test using suitable selective media. Determine
the total viable count using the plate-count method.
The number of contaminating micro-organisms per dosage
unit is not greater than 102 bacteria and 20 fungi.
No pathogenic bacterium, patticulady Esehenehia eoli,
Staphyloeoeeus aureus, Pseudomonas aeruginosa, and no
salmonella other than strain Ty 21a are found.
Water (2.5.12)
1.5 per cent to 4.0 per cent, determined on the contents of
the capsule or of the container by the semi-micro
determination of water.
NUMBER OF UVE BACTERIA
Carry out the test using not fewer than five dosage units.
Homogenise the contents of the dosage units in a 9 gIL
solution of sodium ehlonde R at 4 oC using a mixer in a cold
room with sufficient glass beads to emerge from the liquido
Irnmediately after homogenisation prepare a suitable dilution
of the suspension using cooled diluent and inoculate brain
heart infusion agari incubate at 36 ± 1 oC for 20 h to 36 h.
The vaccine contains not fewer than 2 x 10 9 live S. typhi Ty
21a bacteria per dosage unit.
LABELLING
The labe! states:
-- the minimum number of live bacteria per dosage unit,
-- that the vaccine is for oral use only.
____________________________________________
Varicella Vaccine (Live)
(Ph Eur monograph 0648)
The !abe! may state 'Var(live)'.
~E~ _____________________________________________
DEFINITION
Varicella vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of human herpesvirus 3.
The vaccine is reconstituted immediately before use, as
stated on the label, to give a elear liquid that may be
coloured owing to the presence of a pH indicator.
2014
PRODUCTION
The production of vaccine is based on a virus seed-Iot system
and a cell-bank system. The production method shall have
been shown to yield consistently live varicella vaccines of
adequate immunogenicity and safety in mano The virus in the
final vaccine shall not have been passaged in cell cultures
beyond a defined number of passages approved by the
competent authority from the original isolated virus.
The potential neurovirulence of the vaccine strain is
considered during prec1inical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk
analysis is carried out. Where necessary and if available, a
test is carried out on the vaccine strain using an animal
model that differentiates wild-type and attenuated virus; tests
on strains of intermediate attenuation may also be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3).
VIRUS SEED LOT
The strain of human herpesvirus 3 used shall be identified as
being suitable by historical records that inelude information
on the origin of the strain and its subsequent manipulation.
The virus shall at no time have been passaged in continuous
celllines. Seed lots are prepared in the same kind of cells as
those used for the production of the final vaccine. Virus seed
lots are prepared in large quantities and stored at
temperatures below - 20 oC if freeze-dried, or below
- 60 oC if not freeze-dried.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification
The master and working seed lots are identified as human
herpesvirus 3 by serum neutralisation in cell culture, using
specific antibodies.
Virus concentration
The virus concentration of the master and working seed lots
is determined as prescribed under Assay to monitor
consistency of production.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
seed lots for live virus vaccines; a sample of 50 mL is taken
for the test in cell cultures.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
or viruses are being handled. Approved animal (but not
~E~
human) serum may be used in the culture media. Serum and
trypsin used in the preparation of cell suspensions and media
are shown to be free from extraneous agents. The cell culrure
medium may contain a pH indicator such as phenol red and
approved antibiotics at the lowest effective concentration.
It is preferable to have a substrate free from antibiotics
during production. 5 per cent, but not less than 50 mL, of
the cell cultures employed for vaccine production is set aside
as uninfected cel! cultures (control cel!s). The infected cells
constituting a single harvest are washed, released from the
support surface and pooled. The cel! suspension is disrupted
by sonication.
Only a virus harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
 2014
Identification
The virus harvest contains virus that is identified as human
herpesvirus 3 by serum neutralisation in cell culture, using
specific antibodies.
Virus concentration
The concentration of infective virus in virus harvests is
determined as prescribed under Assay ro monitor consistency
of production and to determine the dilution ro be used for
the final bulk vaccine.
Extraneous agents (2.6. 16)
Use 50 mL for the test in cell cultures.
Control cells
The control cells of the production cell culture from which
the single harvest is derived comply with a test for identity
and with the requirements for extraneous agents (2.6. 16).
FINAL BULK VACCINE
Virus harvests that comply with the aboye tests are pooled
and c1arified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bacterial and fungal contamination
Carry out the test for sterility (2.6. 1) using 10 mL for each
medium.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers and freeze-dried ro a moisture
content shown to be favourable to the stability of the vaccine.
The containers are then c10sed so as ro prevent
contamination and the introduction of moisture.
Only a final lot that is satisfacrory with respect to the test for
water and each of the requirements given below under
Identification, T ests and Assay may be released for use.
Provided that the test for bovine serum albumin has been
carried out with satisfacrory results on the final bulk vaccine,
it may be omitted on the final lor.
Water (2.5.12)
Not more than the amount shown to ensure stability of the
vaccines as approved by the competent authority, determined
by the semi-micro determinarion of water.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific human herpesvirus 3 antibodies, it is no
longer able ro infect susceptible cell cultures.
TESTS
Bacterial and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1).
Bovine serum albumin
Maximum 0.5 ~lg per human dose, determined by a suitable
immunochemical method (2. 7.1).
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine. Titrate 1 vial of an appropriate
virus reference preparation in triplica te to validate each assay.
The virus concentration of the reference preparation is
monitored using a control chart and a titre is established on a
historical basis by each laborarory. The relation with the
appropriate European Pharmacopoeia Biological Reference
Preparation is established and monitored at regular intervals
if a manufacturer's reference preparation is used. Calculate
the individual virus concentration for each vial of vaccine and
Vaccines IV-617
for each replicate of the reference preparation as well as the
corresponding combined virus con centra tion s, using the usual
statisrícal methods (for example, 5.3). The combined
estimate of the virus concentration for the 3 vials of vaccine
is not less than that stated on the labe!'
The assay is not valid if:
- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replicates combined is greater than ± 0.3 log PFU;
- the virus concentration of the reference preparation differs
by more than 0.5 log PFU from the established value .
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentraríon of the vaccine is greater
than ± 0.3 log PFU; data obtained from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 log PFU.
Vancella vaccine (live) BRP is suitable for use as a reference
preparation.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label states:
- the strain of virus used for the prepararíon of the vaccine;
- the type and origin of the cells used for the preparation of
the vaccine;
- the minimum virus concentration;
- that contact between the vaccine and disinfectants is to be
avoided .
_ _________________________________________ ~E~
Yellow Fever Vaccine, Live ***
** **
(Yellow Fever Vaccine (Live), **** *
Ph Eur 111onograph 0537)
The label may sta te 'Yel(live).'
~E~ ___________________________________________
DEFINITION
Yellow fever vaccine (live) is a freeze-dried preparation of
yellow fever virus derived from the 17D strain and grown in
fertilised hen eggs. The vaccine is reconstituted irnmediately
before use, as stated on the label, to give a c1ear liquido
PRODUCTION
The production of vaccine is based on a virus seed-Iot
system. The production method shall have been shown ro
yield consistently yellow fever vaccine (live) of acceptable
immunogenicity and safety for mano
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
roxicity for immunosera and vaccines for human use (2.6.9)
modified as follows for the test in guinea-pigs: inject 10
human doses into each guinea-pig at 2 different injection
sites and observe for 21 days.
Reference preparation In the test for neurotropism, a suitable
batch of vaccine known ro have sarísfactory properties in man
is used as the reference preparation.
A reference prepararíon calibrated in International Units per
ampoule is used to verify the titre of the virus inoculum in
IV-618 Vaccines
the tests for viraemia (viscerotropism) and immunogenicity,
and to titrate the vaccine batch in the potency assay.
The International Unit is the activity contained in a stated
quantity of the International Standard. The equivalence in
International Units of the International Standard is stated by
the World Health Organization.
SUBSTRATE FOR VIRUS PROPAGATION
Virus for the preparation of master and working seed lots and
of all vaccine batches is grown in the tissues of chick embryos
from a ftock free from specified pathogens (SPF) (5.2.2).
SEED LOTS
The 17D strain shall be identified by historical records that
include inforrnation on the origin of the strain and its
subsequent manipulation. Virus seed lots are prepared in
large quantities and stored at a temperature below -60 oc.
Master and working seed lots shall not contain any human
protein, added serum or antibiotics.
Vnless otherwise justified and authorised, the virus in the
final vaccine shall be between passage levels 204 and 239
from the original isolate of strain 17D. A working seed lot
shall be only 1 passage from a master seed lot. A working
seed lot shall be used without intervening passage as the
inoculum for infecting the tissues used in the production of a
vaccine lot, so that no vaccine virus is more than 1 passage
from a seed lot that has passed all the safety tests.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification
The master and working seed lots are identified as containing
yellow fever virus by serum neutralisation in cell culture
using specific antibodies, or by molecular methods
(e.g. nucleic acid amplification techniques (NAT),
sequencing) .
Extraneous agents (2.6.16)
Each master seed lot complies with the following tests:
- bacterial and fungal sterility (as described in chapter
2.6.16 under Virus seed lot and virus harvests);
- mycoplasmas (as described in chapter 2.6.16 under Virus
seed lot and virus harvests);
- mycobacteria (as described in chapter 2.6.16 under Virus
seed lot and virus harvests).
Avian leucosis viruses (2.6.24)
Each master seed lot complies with the test for avian leucosis
viruses.
Extraneous agents (2.6.16)
Each working seed lot complies with the following tests:
- test in adult mice (intraperitoneal inoculation only) (as
described in chapter 2.6.16 under Virus seed lot);
- test in guinea-pigs (as described in chapter 2.6.16 under
Virus seed lot);
- bacterial and fungal sterility (as described in chapter
2.6.16 under Virus seed lot and virus harvests);
- mycoplasmas (as described in chapter 2.6.16 under Virus
seed lot and virus harvests);
- mycobacteria (as described in chapter 2.6.16 under Virus
seed lor and virus harvests);
- test in cell culture for other extraneous agents: a
neutralised sample of 5 mL of working seed lot,
representing at least 500 000 (5.7 loglo) IU, is tested for
the presence of extraneous agents by inoculation into
continuous simian kidney and human cell cultures as well
as into primary chick-embryo-fibroblast cells; the cells are
incubated at 36 ± 1 oC and observed for a period of
14 days; the working seed lot passes the test if there is no
2014
evidence of the presence of any extraneous agents;
the test is not valid unless at least 80 per cent of the cell
cultures remain viable;
- avian viruses: a neutralised sample of 1 mL of working
seed lot, representing at least 100 000 (5.0 loglo) IU, is
tested for the presence of avian viruses by inoculation by
the allantoic route into a group of at least 20 fertilised, 9-
to ll-day-old, SPF eggs (5.2.2), and by inoculation into
the yolk sac of a group of at least 20 fertilised, 5- to
7-day-old, SPF eggs (5.2.2); incubate for 7 days;
the working seed lot complies if the allantoic and yolk sac
ftuids show no signs of haemagglutinating agents and if
the embryos and chorio-allantoic membranes examined to
detect any macroscopic pathology are typical; the test is
not valid unless at least 80 per cent of the inoculated eggs
survive during the 7-day observation periodo
Avian leucosis viruses (2.6.24)
Each working seed lot complies with the test for avian
leucosis viruses.
Tests in monkeys
Each master and working seed lot complies with the
following tests in monkeys for viraemia (viscerotropism),
immunogenicity and neurotropism.
The monkeys shall be Macaca sp. susceptible to yellow fever
virus and shall have been shown to be non-immune to yellow
fever at the time of injecting the seed virus. They shall be
healthy and shall not have received previously intracerebral or
intraspinal inoculation. Furthermore, they shall not have
been inoculated by other roures with neurotropic viruses or
with antigens related to yellow fever virus. Not fewer than
10 monkeys are used for each test.
Use a test dose of 0.25 mL containing the equivalent of not
less than 5000 (3.7 loglo) IV and not more than 50 000 (4.7
10glO) IU, detennined by an in vitro titration for infectious
virus in cell culture. Inject the test dose into 1 frontallobe of
each monkey under anaesthesia and observe the monkeys for
not less than 30 days.
Viraemia (Viscerotropism) Viscerotropism is indicated by the
amount ofvirus present in serum. Take blood from each of
the test monkeys on the 2nd , 4th and 6th days after
inoculation and prepare serum from each sample. Prepare
1: 1O, 1: 100 and 1: 1000 dilutions from each serum and
inoculate each dilution into a group of at least 4 cell culture
vessels used for the determination of the virus concentration.
The seed lot complies with the test if none of the sera
contains more than the equivalent of 500 (2 .7 loglo) IU in
0.03 mL and at most 1 serum contains more than the
equivalent of 100 (2.0 loglo) IU in 0.03 mL.
lmmunogenicity Take blood from each monkey 30 days after
the injection of the test dos e and prepare serum from each
sample. The seed lot complies with the test if at least
90 per cent of the test monkeys are shown to be irnrnune, as
detennined by examining their sera in the test for
neutralisation of yellow fever virus described below.
It has been shown that a low dilution of serum (for example,
1:10) may contain non-specific inhibitors that inftuence this
test; such serum shall be treated to remove inhibitors.
Mix dilutions of at least 1: 1O, 1:40 and 1: 160 of serum from
each monkey with an equal volume of 17D vaccine virus at a
dilution that will yield an optimum number of plaques with
the titration method used. Incubate the serum-virus mixtures
in a water-bath at 37 oC for 1 h and then cool in iced water;
add 0.2 mL of each serum-virus mixture to each of 4
cell-culture plates and proceed as for the detennination of
virus concentration. Inoculate similarly 10 plates with the
2014
same amount of virus, plus an equal volume of a 1: 1O
dilution of monkey serum known to contain no neutralising
antibodies to yellow fever virus. At the end of the observation
period, compare the mean number of plaques in the pla tes
receiving virus plus non-immune serum with the mean
number of plaques in the plates receiving virus plus dilutions
of each monkey serum. Not more than 10 per cent of the
test monkeys have serum that fails to reduce the number of
plaques by 50 per cent at the 1: 1O dilution.
Neurotropism Neurotropism is assessed from clinical
evidence of encephalitis, from incidence of clinical
manifestations and by evaluation of histological lesions, in
comparison with 10 monkeys injected with the reference
preparation. The seed lot is not acceptable if either the onset
and duration of the febrile reaction or the clinical signs of
encephalitis and pathological findings are such as to indicate
a change in the properties of the virus.
Clinical evaluation
The monkeys are examined daily for 30 days by personnel
familiar with clinical signs of encephalitis in primates (if
necessary, the monkeys are removed from their cage and
examined for signs of motor weakness or spasticity).
The seed lot is not acceptable if in the monkeys injected with
ir the incidence of severe signs of encephalitis, such as
paralysis or inability to stand when stimulated, or mortality is
greater than for the reference vaccine. These and other signs
of encephalitis, such as paresis, incoordination, lethargy,
tremors or spasticity are assigned numerical values for the
severity of symptoms by a grading method. Each day each
monkey in the test is given a score based on the following
scale:
- grade 1: rough coat, not eating;
- grade 2: high-pitched voice, inactive, slow moving;
- grade 3: shaky movements, tremors, incoordination, limb
weakness;
- grade 4: inability to stand, limb paralysis or death (a dead
monkey receives a daily score of 4 from the day of death
until day 30).
A clinical score for a particular monkey is the average of its
daily scores; the clinical score for the group is the arithmetic
mean of the individual monkey scores. The seed lot is not
acceptable if the mean of the clinical severity scores for the
group of monkeys inoculated with it is significantly greater
(P = 0.95) than the mean for the group of monkeys injected
with the reference preparation. In addition, special
consideration is given to any animal showing unusually severe
signs when deciding on the acceptability of the seed loto
Histological evaluation
5 levels of the brain are examined including:
- block 1: the corpus striatum at the level of the optic
chiasma;
- block 11: the thalamus at the leve! of the mamillary
bodies;
- block III: the mesencephalon at the level of the superior
colliculi;
- block IV: the pons and cerebellum at the level of the
superior olives;
- block V: the medulla oblongata and cerebellum at the
level of the mid-inferior olivary nuclei.
Cervical and lumbar enlargements of the spinal cord are each
divided equally into 6 blocks; 15 [.1m sections are cut from
the tissue blocks embedded in paraffin wax and stained with
gallocyanin. Numerical scores are given to each hemisection
of the cord and to structures in each hemisection of the brain
as listed below. Lesions are scored as follows:
Vaccines IV -619
- grade 1 - minimal: 1 to 3 small focal inflammatory
infiltrates; degeneration or loss of a few neurons;
- grade 2 - moderate: 4 or more focal inflammatory
infiltrates; degeneration or loss of neurons affecting not
more than one third of cells;
- grade 3 - severe: moderate focal or diffuse inflarnmatory
infiltration; degeneration or loss of 33-90 per cent of the
neurons;
- grade 4 - overwhelming: variable but often severe
inflammatory reaction; degeneration or loss of more than
90 per cent of neurons.
It has been found that inoculation of yellow fever vaccine
into the monkey brain causes histological lesions in different
anatomical formations of the central nervous system with
varying frequency and severity (1. S. Levenbook et al., Joumal
oi Biological Standardization, 1987, 15, 305-313). Based on
these 2 indicators, the anatomical structures can be divided
into target, spared and discriminator areas. Target areas are
those that show more severe specific lesions in a majority of
monkeys irrespective of the degree of neurovirulence of the
seed loto Spared areas are those that show only minimal
specific lesions and in a minority of monkeys. Discriminator
areas are those where there is a significant increase in the
frequency of more severe specific lesions with seed lots
having a higher degree of neurovirulence. Discriminator and
target areas for Macaca cynomolgus and Macaca rhesus
monkeys are shown in the table below.
Type oí monkey Discriminator areas Target areas
Macaca cynomolgus Globus pallidus Substantia nigra
Putamen
Anterior/ median
thalamic nucleus
Lateral thalamic nucleus
Macaca rhesus Caudate nucleus Substantia nigra
Globus pallidus Cervical enlargement
Putamen Lumbar enlargement
Anterior/ median
thalamic nucleus
Lateral thalamic nucleus
Cervical enlargement
Lumbar enlargement
Scores for discriminator and target areas are used for the
final evaluation of the seed loto The individual monkey score
is caIculated from the sum of individual target area scores in
each hemisection divided by the number of areas examined.
A separate score is caIculated similarly for the discriminator
areas.
Mean scores for the test group are caIculated in 2 ways: (1)
by dividing the sum of the individual monkey discriminator
scores by the number of monkeys; and (2) by dividing the
sum of the individual monkey target and discriminator seo res
by the number of monkeys. These 2 mean scores are taken
into account when deciding on the acceptability of the seed
loto The seed lot is not acceptable if either of the mean lesion
scores is significantly greater (P = 0.95) than for the
reference preparation.
PROPAGATION AND HARVEST
All processing of the fertilised eggs is done under aseptic
conditions in an area where no other infectious agents or
cells are handled at the same time. At least 2 per cent but
IV-620 Vaccines
not fewer than 20 and not more than 80 eggs are maintained
as uninfected control eggs. After inoculation and incubation
at a controlled temperature, only living and typical chick
embryos are harvested. At the time of harvest, the control
eggs are treated in the same way as the inoculated eggs to
obtain a control embryonic pulpo The age of the embryo at
the time of virus harvest is reckoned from the initial
introduction of the egg into the incubator and shall be not
more than 12 days. After homogenisation and clarification by
centrifugation, the extract of embryonic pulp is tested as
described below and kept at -70 oC or colder until further
processing. Virus harvests may be pooled. No human protein
is added to the virus suspension at any stage during
production. If stabilisers are added, they shall have been
shown to have no antigenic or sensitising properties for mano
Only a single harvest or, where applicable, a pool of single
harvests that complies with the following requirements may
be used in the preparation of the final bulk vaccine.
Identification
The single harvest or pool of single harvests contains virus
that is identified as yellow fever virus by serum neutralisation
in cell culture using specific antibodies, or by molecular
methods (e.g. NAT, sequencing).
Bacteria! and fungal contamination
The single harvest complíes with the test for sterility (2.6.1),
carried out using 10 mL for each medium.
Mycoplasmas (2.6.7)
The single harvest or pool of single harvests complies with
the test for mycoplasmas, carried out using 10 mL.
Mycobacteria (2.6.2)
A 5 mL sample of the single harvest or pool of single
harvests is tested for the presence of Mycobactenum spp .
by culture methods known to be sensitive for the detection of
these organisms.
Embryonic pulp of control eggs
The extract of the control eggs shows no evidence of the
presence of any extraneous agents in the tests described
below.
Test in cell culture for other extraneous agents Inoculate a
5 mL sample of embryonic pulp of the control eggs into
continuous simian kidney and human cell cultures as well as
into primary chick-embryo-fibroblast cells. The cells are
incubated at 36 ± 1 oC and observed for a period of
14 days. The embryonic pulp of the control eggs passes the
test if there is no evidence of the presence of any extraneous
agents. The test is not valid unless at least 80 per cent of the
cell cultures remain viable.
Avian viruses Vsing 0.1 mL per egg, inoculate the
embryonic pulp of control eggs: by the allantoic route into a
group of 10 fertilised, 9- to ll-day-old, SPF eggs (5.2.2);
and into the yolk sac of a group of 10 fertilised, 5- to 7-day-
old, SPF eggs (5.2.2). Incubate for 7 days. The embryonic
pulp lot of the control eggs complies if the allantoic and yolk
sac fluids show no signs of haemagglutinating agents and if
the embryos and chorio-allantoic membranes examined to
detect any macroscopic pathology are typical. The test is not
valid unless at least 80 per cent of the inoculated eggs survive
during the 7 day observation periodo
Virus concentration
In order to calcula te the dilution for formulation of the final
bulk, each single harvest is titrated as described under Assay.
FINAL BULK VACCINE
Single harvests or pools of single harvests that comply with
the tests prescribed aboye are pooled and clarified again.
2014
A test for protein nitrogen content is carried out. A suitable
stabiliser may be added and the pooled harvests diluted as
appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the finallot.
Bacteria! and fungal contamination
The final bulk vaccine complies with the test for sterility
(2.6.1), carried out using 10 mL for each medium.
Protein nitrogen content
Maximum 0.25 mg per human dose before the addition of
any stabiliser.
FINALLOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers and freeze-dried to a moisture
content shown to be favourable to the stability of the vaccine.
The containers are then closed so as to prevent
contamination and the introduction of moisture.
Only a final lot that is satisfactory with respect to thermal
stability and each of the requirements given below under
Identification, Tests and Assay may be released for use.
Provided that the test for ovalbumin has been performed
with satisfactory results on the final bulk vaccine, it may be
omitted on the final lot.
Thermal stability
Maintain at least 3 containers of the final lot of freeze-dried
vaccine in the dry state at 37 ± 1 oC for 14 days. Determine
the virus concentration as described under Assay in parallel
for the heated vaccine and for vaccine stored at the
temperature recommended for storage. The virus
concentration of the heated vaccine is not more than 1.0
loglo lower than that of the unheated vaccine.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific yellow fever virus antibodies, there is a
significant reduction in its ability to infect susceptible cell
cultures. Alternatively, the vaccine reconstituted as stated on
the labe! contains virus that is identified as yellow fever virus
by molecular methods (e.g. NAT, sequencing).
TESTS
Ovalbumin
Maximum 5 ¡.tg of ovalbumin per human dose, determined
by a suitable immunochemical method (2.7.1).
Water (2.5.12)
Maximum 3.0 per cent.
Bacteria! and fungal contamination
The reconstituted vaccine complies with the test for sterility
(2.6.1).
Bacteria! endotoxins (2.6.14)
Less than 5 IV per single human dose.
ASSAY
Titrate for infective virus in cell cultures using at least
3 separate containers of vaccine. Titrate 1 container of an
appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory. Calculate
the individual virus concentration for each container of
vaccine and for each replicate of the reference preparation as
well as the corresponding combined virus concentrations
using the usual statistical methods (for example, 5.3).
The combined virus concentration for the 3 containers of
vaccine is compared to the results of the reference
preparation titrated in parallel, to obtain results in
2014 Vaccines IV-621

Intemational Units. The combined virus concentration of the

vaccine is not les s than 3.0 loglo IU per human dose and not
more than the upper limit approved for the particular
product by the competent authority.
The assay is not valid if:
-- the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the 3
replica tes combined is greater than ± 0.3 logl o IU;
-- the virus concentration of the reference preparation differs
by more than 0.5 loglo IU from the established value.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than ± 0.3 logl o IU; data obtaÍned from valid assays only
are combined by the usual statistical methods (for example,
5.3) 10 calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than ± 0.3 logl o ID.
Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if
tested as described aboye.
LABELLING
The label sta tes:
-- the strain of virus used in preparation of the vaccine;
-- that the vaccine has been prepared in chick embryos;
-- the mínimum virus concentration;
-- that contact between the vaccÍne and disinfectants is to be
avoided.
____________________________________________ PhE~
IV-622 Diagnostic Preparations
DIAGNOSTIC PREPARATIONS
Old Tuberculin
(Tuberculin lor Human Use, Old,
Ph Eur monograph 0152)
~ E~ ____________________________________________
DEFINITION
Old tuberculin for human use consists of a filtrate,
concentrated by heating, containing the soluble products of
the culture and lysis of one or more strains of Mycobacterium
bovis ami/or Mycobacterium tuberculosis that is capable of
demonstrating a delayed hypersensitivity in an animal
sensitised to micro-organisms of the same species .
Old tuberculin for human use in concentrated form is a
transparent, viscous, yellow or brown liquido
PRODUCTION
GENERAL PROVISIONS
The production of old tuberculin is based on a seed-Iot
system. The production method shall have been shown to
yield consistently old tuberculin of adequate potency and
safety in mano A batch of old tuberculin, calibrated in
International Units by the method described under Assay
and for which adequate clinical information is available as to
its activity in man, is set aside to serve as a reference
preparation.
The International Unit is the activity of a stated quantity of
the International Standard. The equivalence in International
Units of the International Standard is stated by the World
Health Organisation.
SEED LOTS
The strains of mycobacteria used shall be identified by
historical records that include information on their origin and
subsequent manipulation.
The working seed lots used to inoculate the media for the
production of a concentrated harvest shall not have
undergone more than 4 subcultures from the master seed loto
Only seed lots that comply with the following requirements
may be used for propagation.
Identification
The species of mycobacterium of the master and working
seed lots is identified.
Bacterial and fungal contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium. The working seed lot complies with the test for
sterility except for the presence of mycobacteria.
PROPAGATION AND HARVEST
The bacteria are grown in a liquid medium which may be a
glycerolated broth or a synthetic medium. Growth must be
typical for the strain. The culture is inactivated by a suitable
method, such as treatrnent in an autoclave (121 °C for not
less than 30 min) or in fiowing steam at a temperature not
less than 100 oC for at least 1 h. The culture liquid, from
which the micro-organisms may or may not have been
separated by filtration, is concentrated by evaporation,
usually to one-tenth of its initial volume. The preparation is
free from live mycobacteria. The concentrated harvest is
shown to comply with the test for mycobacteria (2.6.2)
before addition of any antimicrobial preservative or other
substance that might interfere with the test. Phenol (5 gIL)
2014
or another suitable antimicrobial preservative that does not
give rise to false positive reactions may be added.
Only a concentrated harvest that complies with the following
requirements may be used in the preparation of the final bulk
tuberculin.
pH (2.2.3)
The pH of the concentrated harvest is 6.5 to 8.
Glycerol
Where applicable, determine the glycerol content of the
concentrated harvest. The amount is within the limits
approved for the particular product.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not less than 85 per cent and not
more than 115 per cent of the intended amount. If phenol
has been used in the preparation, the concentration is not
more than 5 gIL (2.5.15).
Sensitisation
Carry out the test described under Tests .
Sterility (2.6.1)
The concentrated harvest complies with the test for sterility,
carried out using 10 mL for each medium.
Potency
Determine the potency as described under Assay.
FINAL BULK TUBERCULIN
The concentrated harvest is diluted aseptically.
Only a final bulk tuberculin that complies with the following
requirement may be used in the preparation of the final loto
Sterility (2.6.1)
The final bulk tuberculin complies with the test for sterility,
carried out using 10 mL for each medium.
FINALLOT
The final bulk tuberculin is distributed aseptically into sterile
containers which are then closed so as to prevent
contamination.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use.
The following tests may be omitted on the finallot if they
have been carried out at the stages indicated:
-- live mycobacteria: concentrated harvest,
-- sensitisation: concentrated harvest,
-- toxicity: concentrated harvest or final bulk tuberculin,
-- antimicrobial preservative: final bulk tuberculin.
IDENfIFICATION
Inject increasing doses of the preparation to be examined
intradermally into healthy, white or pale-coloured guinea-
pigs, specifically sensitised (for example, as described under
Assay). A reaction varying from erythema to necrosis is
produced at the site of the injection. Similar injections
administered to non-sensitised guinea-pigs do not stimulate a
reaction. The assay may also serve as identification.
TESTS
Old tuberculin lor human use in concentrated lorm
(::: 100 000 IUlmL) complies with each 01 the tests prescribed
below; the diluted product complies with the tests lor amimicrobial
preservative and sterility.
Toxicity
Inject a quantity equivalent to 50 000 IU subcutaneously
into each of two healthy guinea-pigs weighing 250 g to 350 g
and which have not been subjected to any treatment likely to
2014
interfere with the test. Observe the animals for 7 days.
No adverse effect is produced.
Sensitisation
Use 3 guinea-pigs that have not been subjected to any
treatment likely to interfere with the test. On 3 occasions at
intervals of 5 days, inject intradermally into each guinea-pig
about 500 IU of the preparation to be examined in a volume
of 0.1 mL. 2 to 3 weeks after the third injection, administer
the same dose intradermally to the same animals and to a
control group of 3 guinea-pigs of the same mass that have
not previously received injections of tuberculin. After 24 h to
72 h, the reactions in the 2 groups of animals are not
substantially different.
Antinñcrobial preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not les s than the minimum amount
shown to be effective and not more than 115 per cent of the
amount stated on the labe!' If phenol has been used in the
preparation, the concentration is not more than 5 gIL
(2.5.15) .
Live mycobacteria (2.6.2)
It complies with the test for mycobacteria.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
The potency of old tuberculin is determined by comparing
the reactions produced by the intradermal injection of
increasing doses of the preparation to be examined into
sensitised guinea-pigs with the reactions produced by known
concentrations of the reference preparation.
Prepare a suspension containing a suitable amount (0.1 mg
to 0.4 mglmL) of heat-inactivated, dried mycobacteria in
mineral oil with or without emulsifier; use mycobacteria of a
strain of the same species as that used in the preparation to
be examined. Sensitise not fewer than 6 pale-coloured
guinea-pigs weighing not less than 300 g by injecting
intramuscularly or intradermally a total of about 0.5 mL of
the suspension, divided between several sites if necessary.
Carry out the test after the period of time required for
optimal sensitisation which is usually 4 to 8 weeks after
sensitisation. Depilate the fianks of the animals so that it is
possible to make at least three injections on each side and
not more than a total of 12 injection points per anima!'
Use at least three different dos es of the reference preparation
and at least 3 different doses of the preparation to be
examined. For both preparations, use dos es such that the
highest dose is about 10 times the lowest dose. Choose the
dos es such that when they are injected the lesions produced
have a diameter of not less than 8 mm and not more than
25 mm. In any given test, the order of the dilutions injected
at each point is chosen at random in a Latin square designo
Inject each dose intradermally in a constant volume of
0.1 mL or 0.2 mL. Measure the diameters ofthe lesions
24 h to 48 h later and calculate the results of the test by the
usual statistical methods, assuming that the diameters of the
lesions are directly proportional to the logarithm of the
concentration of the preparation.
The estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
64 per cent and not more than 156 per cent of the stated
potency.
Diagnostic Preparations IV-623
STORAGE
Store protected from light.
LABELLING
The label states:
-- the number of Intemational Units per millilitre,
-- the species of mycobacterium used to prepare the
product,
-- the name and quantity of any antimicrobial preservativé
or any other excipient,
-- the expiry date,
-- where applicable, that old tuberculin is not to be injected
in its concentrated form but diluted so as to administer
not more than 100 IU per dose.
____________________________________________ Ph Ew
Tuberculin Purified Protein ******
*
Derivative *****
Tuberculin P.P.D .
(Tuberculine Purified Protein Derivative for Human Use,
Ph Eur monograph 0151)
Ph Ew ____________________________________________
DEFINITION
Tuberculin purified protein derivative (tuberculin PPD) for
human use is a preparation obtained by precipitation from
the heated products of the culture and Iysis of Mycobacterium
bovis andlor Mycobacterium tuberculosis and capable of
demonstrating a delayed hypersensitivity in an animal
sensitised to micro-organisms of the same species.
Tuberculin PPD is a colourless or pale-yellow liquid;
the diluted preparation may be a freeze-dried powder which
upon dissolution gives a colourless or pale-yellow liquid.
PRODUCTION
GENERAL PROVISIONS
The production of tuberculin PPD is based on a seed-Iot
system. The production method shall have been shown to
yield consistently tuberculin PPD of adequate potency and
safety in mano A batch of tuberculin PPD, calibrated in
Intemational Units by method A described under Assay and
for which adequate clinical information is available as to its
activity in man, is set aside to serve as a reference
preparation.
The Intemational Unit is the activity of a stated quantity of
the Intemational Standard. The equivalence in Intemational
Units of the Intemational Standard is stated by the World
Health Organisation.
SEED LOTS
The strains of mycobacteria used shall be identified by
historical records that include information on their origin and
subsequent manipulation.
The working seed lots used to inoculate the media for
production of a concentrated harvest shall not have
undergone more than 4 sub cultures from the master seed lot.
Only seed lots that comply with the following requirements
may be used for propagation.
Identification
The species of mycobacterium of the master and working
seed lots is identified.
 IV-624 Diagnostic Preparations
Bacteria! and funga1 contamination
Carry out the test for sterility (2.6.1), using 10 mL for each
medium . The working seed lot complies with the test for
sterility except for the presence of mycobacteria.
PROPAGATION AND HARVEST
The bacteria are grown in a liquid synthetic medium.
Growth must be typical for the strain. The culture is
inactivated by a suitable method such as treatment in an
autoclave (121 °C for not less than 30 min) or in fiowing
steam at a temperature not less than 100 oC for at least 1 h
and filtered. The active fraction of the filtrate, consisting
mainly of protein, is isolated by precipitation, washed and
re-dissolved. The preparation is free from mycobacteria.
The concentrated harvest is shown to comply with the test
for mycobacteria (2.6.2) before addition of any antimicrobial
preservative or other substance that might interfere with the
test. Phenol (5 gIL) or another suitable antimicrobial
preservative that do es not give rise to false positive reactions
may be added; a suitable stabiliser intended to prevent
adsorption on glass or plastic surfaces may be added.
The concentrated harvest may be freeze-dried. Phenol is not
added to preparations that are to be freeze-dried.
OnIy a concentrated harvest that complies with the following
requirements may be used in the preparation of the final bulk
tuberculin PPD.
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical or physico-chemical
method. The content is not les s than 85 per cent and not
more than 115 per cent of the intended amount. If phenol
has been used in the preparation, the concentration is not
more than 5 gIL (2.5.15).
Sensitisation
Carry out the test described under Tests.
Sterility (2.6.1)
The concentrated harvest, reconstituted if necessaty,
complies with the test for sterility, carried out using 10 mL
for each medium.
Potency
Determine the potency as described under Assay.
FINAL BULK TUBERCULIN PPD
The concentrated harvest is diluted aseptically, after
reconstitution if necessary.
Only a final bulk tuberculin PPD that complies with the
following requirement may be used in the preparation of the
finallot.
Sterility (2.6.1)
The final bulk tuberculin PPD complies with the test for
sterility, carried out using 10 mL for each medium.
FINALLOT
The final bulk tuberculin PPD is distributed aseptically into
sterile containers which are then closed so as to prevent
contamination. It may be freeze-dried.
Only a final lot that is satisfactory with respect to ea eh of the
requirements given below under Identification, Tests and
Assay may be released for use.
The following tests may be omitted on the final lot if they
have been carried out at the stages indicated:
- live mycobacteria: concentrated harvest
- sensitisation: concentrated harvest
- toxicity: concentrated harvest or final bulk tuberculin
PPD
- antimicrobial preservative: final bulk tuberculin PPD.
2014
IDENfIFICATION
Inject increasing doses of the preparation to be examined
intradermally into healthy, white or pale-coloured guinea-
pigs, specifically sensitised (for example as described under
Assay). A reaction varying from erythema to necrosis is
produced at the site of the injection. Similar injections
administered to non-sensitised guinea-pigs do not stimulate a
reaction. The assay may also serve as identification.
TESTS
Tuberculin purified protein derivative for human use in
concentrated fonn (2: 100 000 IU/mL) complies with each of the
tests prescribed belowj the diluted product complies with the tests for
pH, antimicrobial preservative and sterility.
pH (2.2. 3)
The pH of the preparation, reconstituted if necessary as
stated on the label, is 6.5 to 7.5.
Toxicity
Inject subcutaneously 50 000 IU of the preparation to be
examined into each of two healthy guinea-pigs weighing
250 g to 350 g and which have not been subjected to any
treatment likely to interfere with the test. Observe the
animals for 7 days. No adverse effect is produced.
Sensitisation
Use 3 guinea-pigs that have not been subjected to any
treatment likely to interfere with the test. On 3 occasions at
intervals of 5 days, inject intradermally into each guinea-pig
about 500 IU of the preparation to be examined in a volume
of 0.1 mL. 2 to 3 weeks after the third injection, administer
the same dose intradermally to the same animals and to a
control group of three guinea-pigs of the same mas s that
have not previously received injections of tuberculin. After
24 h to 72 h, the reactions in the 2 groups of animals are not
substantially different.
Antimicrobia1 preservative
Where applicable, determine the amount of antimicrobial
preservative by a suitable che mi cal or physico-chemical
method. The content is not less than the minimum amount
shown to be effective and not more than 115 per cent of the
amount stated on the labe!' If phenol has been used in the
preparation, the concentration is not more than 5 gIL
(2.5.15).
Live mycobacteria (2.6.2)
It complies with the test for mycobacteria.
Sterility (2.6.1)
It complies with the test for sterility.
ASSAY
Use method A or, where the preparation contains 1 IU to
2 IU, use method B.
METHODA
The potency of tuberculin PPD is determined by comparing
the reactions produced by the intradermal injection of
increasing doses of the preparation to be examined into
sensitised guinea-pigs with the reactions produced by known
concentrations of the reference preparation.
Prepare a suspension containing a suitable amount
(0.1 mg/rnL to 0.4 mg/mL) of heat-inactivated, dried
mycobacteria in mineral oil with or without emulsifier;
use mycobacteria of a strain of the same species as that used
in the preparation to be examined. Sensitise not fewer than
six pale-coloured guinea-pigs weighing not less than 300 g by
injecting intramuscularly or intradermally a total of about
0.5 mL of the suspension, divided between several sites if
necessary. Carry out the test after the period of time required
 2014 Diagnostic Preparations IV-625

for optimal sensitisation which is usually 4 to 8 weeks after
sensitisation. Depilate the ftanks of the animals so that it is
possible to make at least 3 injections on each side but not
more than a total of 12 injection points per animal. Prepare
dilutions of the preparation to be examined and of the
reference preparation using isotonic phosphate-buffered saline
(pH 6.5 to 7.5) containing 50 mg/L of polysorbate 80 R. If the
preparation to be examined is freeze-dried and does not
contain a stabiliser, reconstitute it using the liquid described
aboye. Use at least 3 different doses of the reference
preparation and at least 3 different doses of the preparation
to be examined. For both preparations, use doses such that
the highest dose is about 10 times the lowest dose. Choose
the doses such that when they are injected the lesions
produced have a di ame ter of not less than 8 mm and not
more than 25 mm. In any given test, the order of the
di!utions injected at each point is chosen at random in a
Latin square designo Inject each dos e intradermally in a
constant volume of 0.1 mL or 0.2 mL. Measure the
diameters of the lesions 24 h to 48 h later and calculate the
results of the test by the usual statistical methods, assuming
that the diameters of the lesions are directly proportional to
the logarithm of the concentration of the preparation.
The estimated potency is not les s than 80 per cent and not
64 per cent and not more than 156 per cent of the stated
potency.
STORAGE
Store protected from light.
LABELLING
The ¡abe! states:
-- the number of International Units per container,
-- the species of mycobacteria used to prepare the product,
-- the na me and quantity of any antimicrobial preservative
or any other excipient,
-- the expiry date,
-- for freeze-dried products, a statement that the product is
to be reconstituted using the liquid provided by the
manufacturer,
-- where applicable, that tuberculin PPD is not to be
injected in its concentrated form but diluted so as to
adrninister not more than 100 IU per dose.
If the package does not contain a leaftet warning that the
inhalation of concentrated tuberculin PPD may produce
toxic effects, this warning must be shown on the label on the
container together with a statement that the powder must be
handled with careo
____________________________________________ ~E~

more than 125 per cent of the stated potency.

The confidence limits (P = 0.95) are not less than
64 per cent and not more than 156 per cent of the stated
potency.
METHODB
The potency of tuberculin PPD is determined by comparing
the reactions produced by the intradermal injection of the
preparation to be examined into sensitised guinea-pigs with
the reactions produced by known concentrations of the
reference preparation.
Prepare a suspension in mineral oi! with or without emulsifier
and containing a suitable amount (0.1 mg/mL to 0.4 mg/mL)
of heat-inactivated, dried rnycobacteria; use mycobacteria of
a strain of the same species as that used in the preparation to
be examined. Sensitise not fewer than 6 pale-coloured
guinea-pigs weighing not less than 300 g by injecting
intramuscularly or intradermally a total of about 0.5 mL of
the suspension, divided between several sites if necessary.
Carry out the test after the period of time required for
optimal sensitisation which is usually 4 to 8 weeks after
sensitisation. Depilate the ftanks of the animals so that it is
possible to make at least 3 injections on each side but not
more than a total of 12 injection points per animal. Prepare
dilutions of the reference preparation using isotonic
phosphate-buffered saline (pH 6.5 to 7.5) containing
50 mg/L of polysorbate 80 R. Use at least 3 different doses of
the reference preparation such that the highest dose is about
10 times the lowest dose and the median dose is the same as
that of the preparation to be examined. In any given test, the
order of the dilutions injected at each point is chosen at
random in a Latin square designo Inject the preparation to be
examined and each dilution of the reference preparation
intradermally in a constant volume of 0.1 mL or 0.2 mL.
Measure the diameters of the lesions 24 h to 48 h later and
calcula te the results of the test by the usual statistical
methods, assuming that the areas of the lesions are directly
proportional to the logarithm of the concentration of the
preparation to be examined. (This dose relationship applies
to this assay and not necessarily to other test systems.)
The estimated potency is not les s than 80 per cent and not
more than 125 per cent of the stated potency.
The confidence limits (P = 0.95) are not less than
Monographs

Radiopharmaceutical
Preparations
2014
Rad iopharmaceutical Preparations *****
* * **** *
(Ph Bur monograph 0125)
Radiopharmaceutical Preparations comply with the requirements 01
the Buropean Phannacopoeia. These requirements are reproduced
below.
~E~~~~~~~~~ ____________~~~__________
DEFINITIONS
Radiopharmaceutical preparations or radiopharmaceuticals
are medicinal products which, when ready for use, contain 1
or more radionuclides (radioactive iso topes) included for a
medicinal purpose.
For the purpose of this general monograph
radiopharmaceutical preparations also cover:
-- radionuclide generators: any system incorporating a fixed
parent radionuclide from that is produced a daughter
radionuclide that is to be obtained by elution or by any
other method and used in a radiopharmaceutical
preparation;
-- kits for radiopharmaceutical preparation: any preparation
to be reconstituted or combined with radionuclides in the
final radiopharmaceutical preparation, usually prior to its
administration;
-- radionuclide precursors: any radionuclide produced for
radiolabelling of another substance prior to
administration;
-- chemical precursors: non-radio active substances for
combination with a radionuclide.
Radionuclide precursors may be supplied as solutions for
radiolabelling.
A nuclide is a species of atom characterised by the number of
protons and neutrons in its nucleus (and hence by its atomic
number 2, and mas s number A) and also by its nuclear
energy state. Isotopes of an element are nuclides with the
same atomic number but different mass numbers. Nuclides
containing an unstable arrangement of protons and neutrons
will transform spontaneously to either a stable or another
unstable combination of protons and neutrons with a
constant statistical probability. Such nuclides are said to be
radio active and are called radionuclides. The initial unstable
nuclide is referred to as the parent radionuclide and the
resulting nuclide as the daughter nuclide.
Decay or transformation of radionuclides may involve the
emission of charged particles, electron capture (Ee) or
isomeric transition (IT). The charged particles emitted from
nuclei may be alpha particles (nuclei of 4He) or beta
particles (negatively charged, gene rally called electrons, or
positively charged, generally called positrons). Alpha decay
usually concems heavy nuclei (2 > 82). Radionuclides with
a deficit of protons usually decay by emitting electrons.
Radionuclides with a deficit of neutrons usually decay by
electron capture or by emitting positrons. In the latter case,
radionuclides are called positron emitters. Positrons are
annihilated after interaction with electrons in the surrounding
matter. The annihilation results in the emission of 2 gamma
photons, each with energy of 0.511 MeV, generally emitted
at 180 0 to each other (annihilation radiation). All decay
modes may be accompanied by an emission of gamma rays.
The emission of gamma rays may be partly or completely
replaced by the ejection of electrons, known as internal
conversion electrons. This phenomenon, like the process of
electron capture, causes a secondary emission of X-rays (due
to a reorganisation of the electrons in the atom). This
secondary emission may itself be partly replaced by the
ejection of electrons, known as Auger electrons.
Radiopharmaceutical Preparations IV-629
Radioactivity
Generally the term 'radioactivity' is used both to describe the
phenomenon of radioactive decay and to express the physical
quantity of this phenomenon.
The radioactivity of a preparation is the number of nuclear
disintegrations or transformations per unit time .
In the International System (SI), radioactivity is expressed in
becquerel (Bq), which is 1 nuclear transformation per
second. Absolute radioactivity measurements require a
specialised laboratory but identification of radioactivity and
quantitative measurement of radioactivity can be carried out
relatively by comparing the measured samples with
standardised preparations provided by laboratories recognised
by the competent authority or by using a calibrated
instrumento
Radioactive decay
Any radionuclide decays at an exponential rate with its
characteristic decay constant.
The curve of exponential decay (decay curve) is described by
the following expression:
At = Aoe - Át
the radioactivity at time t;
the radioactivity at time t = O;
the decay constant, characteristic of each
radionuclide;
e the base of Napierian logarithms.
The half-life (TII2 ) is the time in which a given radioactivity
(amount) of a radionuclide decays to half its initial value .
It is related to the decay constant (A.) by the following
equation:
in 2
T 1/ 2 = T
The equation of exponential decay can thus be expressed
also in the following way, useful for the fast estimation of the
radioactivity left after elapsing time t:
The penetrating power of each radiation varies considerably
according to its nature and its energy. Alpha particles are
completely absorbed in a thickness of a few micrometres to
sorne tens of micrometres of matter. Beta particles are
completely absorbed in a thickness of several millimetres to
several centimetres of matter. Gamma rays are not
completely absorbed but only attenuated and a tenfold
reduction may require, for example, several centimetres of
lead. The denser the absorbent, the shorter the range of
alpha and beta particles and the greater the attenuation of
gamma rays.
Each radionuclide is characterised by an invariable half-life,
expressed in units of time and by the nature and energy of its
radiation or radiations. The energy is expressed in
electronvolts (eV), kilo-electronvolts (keV) or
mega-electronvolts (Me V).
Radionuclidic purity
The ratio, expressed as a percentage, of the radioactivity of
the radionuclide concerned to the total radioactivity of the
radiopharmaceutical preparation. The relevant potential
IV-630 Radiopharmaceutical Preparations
radionuclidic impurities are listed with their limits in the
individual monographs.
Radiochemical purity
The ratio, expressed as a percentage, of the radioactivity of
the radionuclide con cerned which is present in the
radiopharmaceutical preparation in the stated che mi cal form,
to the total radioactivity of that radionuclide present in the
radiopharmaceutical preparation. The relevant potential
radiochemical impurities are listed with their limits in the
individual monographs.
Chemical purity
In monographs on radiopharmaceutical preparations,
chemical purity is controlled by specifying limits for chemical
impurities.
Isotopic carrier
A stable iso tope of the element concemed either present in or
added to the radio active preparation in the same chemical
form as that in which the radionuclide is presento
Carrier-free preparation
A preparation free from stable isotopes of the same element
as the radionuclide con cerned present in the preparation in
the stated chemical form or at the position of the
radionuclide in the molecule concerned.
No-carrier-added preparation
A preparation to which no stable iso topes of the same
element as the radionuclide concerned are intentionally
added in the stated chemical form or at the position of the
radionuclide in the molecule concerned.
Specific radioactivity
The radioactivity of a radionuclide per unit mas s of the
element or of the chemical form concerned, e.g. becquerel
per gram or becquerel per mole.
Radioactivity concentration
The radioactivity of a radionuclide per unit volume or unit
mass of the preparation. For radiopharmaceutical solutions, it
is expressed as radioactivity per unit volume of the
preparation.
Total radioactivity
The radioactivity of the radionuclide, expressed per unit (vial,
capsule, ampoule, generator, etc).
Chemical precursors for syntbesis of radioactive
substances
If the active substance of a radiopharmaceutical preparation
is not isolated, the chemical precursor for its synthesis is
considered as a substance for pharmaceutical use .
It is recommended to test each batch of chemical precursor
material in production runs before its use for the
manufacture of radiopharmaceutical preparations to ensure
that, under specified production conditions, the substance
yields the radiopharmaceutical preparation in the desired
quantity and of the quality specified.
Period of validity
The time during which specifications described in the
monograph must be fulfilled .
PRODUCTION
A radiopharmaceutical preparation contains its radionuclide:
as an element in atomic or molecular form, e.g. 133Xe,
¡JSO]02;
as an ion, e.g. [131 I]iodide, [99mTc]pertechnetate;
- included in, adsorbed on or attached to molecules by
chelation, e.g. ¡J llIn]indium oxine, or by covalent
bonding, e.g. 2-¡J8F]fluoro-2-deoXY-D-glucose.
Radionuclides can be produced in the following ways:
2014
- in reactions of neutrons (target irradiation in nuclear
reactors);
- in reactions of charged particles (target irradiation using
accelerators, in particular cyclotrons);
- by its separation from radionuclide generators.
The probability of nuclear reaction occurrence depends on
the nature and energy of the incident particles (protons,
neutrons, deuterons etc.) and on the nature of the nucleus
that is irradiated by them. The rate of production (yield) of a
given radionuclide resulting from the irradiation depends in
addition on the isotopic composition of the target material
and its chemical purity, and in the case of neutrons on their
flux, and in the case of charged particles on beam current.
In addition to the desired nuclear reaction, simultaneous
transforrnations usually occur. Probability of their occurrence
is given by the same factors as mentioned in the previous
paragraph. Such simultaneous transforrnations may give rise
to radionuclidic impurities.
The nuclear reaction (transformation) can be written in the
form: target nucleus (incident particle, emitted particle)
produced nucleus.
Examples:
NEUTRON IRRADIATION
Irradiation of stable radionuclides in nuclear reactors usually
results in proton-deficient nuclei, i.e. electron emitters that
are formed in (n,y) reactions (so-called radiative capture) .
The product is isotopic with the target nucleus and it may
thus contain a considerable amount of camer.
A number of nuclides with high atomic number are
fissionable by neutrons. Nuclear fission, denoted as (n, t)
reaction, results in a large number of radionuclides of various
mas ses and half-lives. The most frequently used fission is that
of 23SU. Iodine-131, molybdenum-99 and xenon-133 can be
produced by irradiation of 23SU in nuclear reactors and by
their separation from more than 200 radionuclides forrned in
that process.
CHARGED PARTICLE IRRADIATION
Irradiation of stable radionuclides with charged particles
usually results in neutron-deficient nuclei that decay either by
electron capture or by positron emission. They are formed in
particular in (p, xn) reactions (where x is the number of
emitted neutrons). The product is not isotopic with the
target nucleus and its specific radioactivity might be close to
that of a carrier-free preparation.
RADIONUCLIDE GENERATORS
Radionuclide generator systems use a parent radionuclide
which decays to a daughter radionuclide with a shorter
half-life.
By separating the daughter radionuclide from the parent
radionuclide by a chemical or physical process, it is possible
to use the daughter radionuclide at a considerable distance
from the production site of the generator despite its short
half-life.
TARGET MATERIALS
The isotopic composition and purity of the target material
together with other factors such as the nature and energy of
incident particles will determine the relative percentages of
the principal radionuclide and radionuclidic impurities
produced by irradiation. The use of isotopically enriched
target material in which the abundance of the required target
2014
nuclide has been artificially increased, can improve the
production yield and the purity of the desired radionuclide.
The che mi cal form, the purity and the physical state of the
target material and the chemical additives, as well as the
irradiation conditions and the direct physical and chemical
environment, determine the chemical state and chemical
purity of the radionuclides that are produced. In the
production of radionuclides, and particularly of radionuclides
with a short half-life, it may not be possible ro determine any
of these quality criteria before further processing and
manufacture of radiopharmaceutical preparations. Therefore
the quality of each batch of target material is assessed before
its use in routine radionuclide production and manufacture
of radiopharmaceutical preparations.
The target material is contained in a holder in gaseous, liquid
or solid sta te, in order to be irradiated by a beam of particles.
For neutron irradiation, the target material is commonly
contained in quartz ampoules or high-purity aluminium or
titanium containers. Ir is necessary ro ascertain that no
interaction can occur between the container and its contents
under the irradiation conditions.
For charged particle irradiation, the holder for target material
is constructed of an appropriate metal, possibly with inlet
and outlet ports, a surrounding cooling system and usually a
thin metal foil target window.
To evaluate all effects on the efficiency of the production of
the radionuclide in terms of quality and quantity, the
production procedure must clearly describe and take into
consideration: the target material, the construction of the
holder for target material, method of irradiation and
separation of the desired radionuclide.
CHARACTERS
The Table of physical characterisrics of radionuclides (5.7)
summarises the most commonly accepted physical
characteristics of radionuclides used in preparations that are
the subject of monographs in the European Pharmacopoeia.
In addition, the Table states the physical characteristics of
the main potential radionuclidic impurities of the
radionuclides mentioned in the monographs .
The term 'transition probability' means the probability of the
transformation of a nucleus in a given energy state, via the
transition concemed. Instead of 'probability' the term
'abundance' is also used.
The term 'emission probability' means the probability that an
arom of a radionuclide gives rise to the ernission of the
particles or radiation concemed.
Irrespective of which meaning is intended, probability is
usually stated as a percentage.
IDENTIFICATION
A radionuclide is generally identified by its half-life or by the
nature and energy of its radiation or radiations or by both, as
prescribed in the monograph.
Approximate half-Jife
The half-life as determined over a relatively short time period
to allow release for use of radiopharmaceutical preparations.
The calculated approximate half-life is within the range of
the values stated in the individual monograph.
Deternúnation of the nature and energy of the
radiation
The nature and energy of the radiation emitted are
determined using spectrometry. The nature and energy of the
radiation of positron emitters is usually not determined; their
Radiopharmaceutical Preparations IV-631
identification is performed by determination of their half-life
and gamma-ray spectrum.
TESTS
It is somerimes difficult to carry out some of the following tests
before releasing the batch for use when the half-life of the
radionuclide in the prepararion is short. The individual monograph
indica tes the tests that med not be completed before release for use.
These tests then constitute a control of the qualiry of production.
Non-radioactive substances and related substances
This section prescribes the determination of non-radioactive
substances and related substances that can be presento
Residual solvents
Residual solvents are limited according to general chapter
5.4. Residual solvents, using the methods given in general
chapter 2.4.24. Identificatíon and control of residual solvents or
another suitable method.
RADIONUCUmc PURITY
Radionuclidic impurities may arise during the production and
decay of a radionuclide. Potential radionuclidic impurities
may be mentioned in the monographs and their
characteristics are described in general chapter 5.7. Table of
physical characteristics of radionuclides mentioned in the European
Pharmacopoeia.
In most cases, ro establish the radionuclidic purity of a
radiopharmaceutical preparation, the identity of every
radionuclide present and its radioactivity must be known.
Generally, the most useful method for examination of the
radionuclidic purity of garnma- and X-ray emitting
radionuclides is gamma-ray spectrometry. The use of sodium
iodide detectors may cause a problem: the peaks due to
gamma-ray emitting impurities may be concealed in the
spectrum of the principal radionuclide or left unresolved
from peaks of other radionuclidic impurities in the
preparation. Alpha- and beta-particle emitting impurities that
do not emit gamma- or X-rays cannot be detected in this
way. For alpha- and beta-emitters other methods must be
employed.
The individual monographs prescribe the radionuclidic purity
required and may set limits for specific radionuclidic
impurities (for example, molybdenum-99 in technetium-
99 m ) . While these requirements are necessary, they are not in
themselves sufficient to ensure that the radionuclidic purity of
a preparation is sufficient for its clinical use .
The manufacturer must examine the product in detail and
especially must examine preparations of radionuclides with a
short half-life for impurities with a long half-life after a
suitable period of decay. In this way, information on the
suitability of the manufacturing processes and the adequacy
of the testing procedures is obtained. In cases where 2 or
more positron-emitting radionuclides need to be identified
and/or differentiated, for example the presence of 18F_
impurities in 13N-preparations, half-life determinations need
to be carried out in addition to gamma-ray spectrometry.
Due to differences in the half-lives of the different
radionuclides present in a radiopharmaceutical preparation,
the radionuclidic purity changes with time.
RADIOCHEMICAL PURITY
Radiochemical impurities may originate from:
- radionuclide production;
- subsequent che mi cal procedures;
- incomplete preparative separation;
- chemical changes during storage.
The determination of radiochemical purity requires
separation of the different chemical substances containing the
 IV-632 Radiopharmaceutical Preparations
radionuelide and detennination of the percentage of
radioactivity of the radionuelide concerned associated with
the stated chemical formo The radiochemical purity section of
an individual monograph may inelude limits for specified
radiochemical impurities, ineluding isomers.
In principIe, any method of analytical separation may be used
in the detennination of radiochemical purity. For example,
the monographs for radiophannaceutical preparations may
inelude paper chromatography (2.2.26), thin-layer
chromatography (2.2.27), electrophoresis (2.2.31), size-
exelusion chromatography (2.2.30), gas chromatography
(2.2.28) and liquid chromatography (2.2.29). The technical
description of these analytical methods is set out in the
monographs. Moreover, certain precautions special to
radiopharmaceuticals must also be considered, su eh as
radiation protection, measurement geometry, detector
linearity, use of carriers, dilution of the preparation.
Specific radioactivity
Specific radioactivity is usually calculated taking into account
the radioactivity concentration and the concentration of the
chemical substance being studied, after verification that the
radioactivity is anributable only to the radionuelide
(radionuelidic purity) and the chemical species
(radiochemical purity) con cerned.
Specific radioactivity changes with time. The statement of the
specific radioactivity therefore ineludes reference to a date
and, if necessary, time.
Physiological distribution
Tests involving animals should be avoided wherever possible.
Where the tests for identity and for radiochemical purity are
not adequate to completely define and control the
radiochemical species in a radiopharmaceutical preparation, a
physiological distribution test may be required.
The distribution pattern of radioactivity observed in specified
organs, tissues or other body compartments of an appropriate
animal species can be a reliable indication of the suitability
for the intended purpose.
Altematively, a physiological distribution test can serve to
establish the biological equivalence of the preparation under
test with similar preparations known to be elinically effective.
The individual monograph prescribes the details conceming
the conduct of the test and the physiological distribution
requirements that must be meL
In general, the test is perfonned as follows.
Each of 3 animals is injected intravenously with the
preparation. In sorne cases, dilution immediately before
injection may be necessary.
Immediately after injection each animal is placed in a
separate cage for collection of excreta and prevention of
contamination of the body surface of the animal. At the
specified time after injection, the animals are euthanised by
an appropriate method and dissected. Selected organs and
tissues are assayed for their radioactivity. The physiological
distribution is then calculated and expressed in terms of the
percentage of the administered radioactivity that is found in
each of the selected organs or tissues, taking into account
corrections for radioactive decay. For sorne
radiopharrnaceutical preparations ir is necessary to detennine
the ratio of the radioactivity in weighed samples of selected
tissues (radioactivity/mass).
A preparation meets the requirements of the test if the
distribution of radioactivity in at least 2 of the 3 animals
complies with all the specified criteria.
2014
Disregard the results from any animal showing evidence of
extravasation of the injection (observed at the time of
injection or revealed by subsequent assay of tissue
radioactivity) . In that case the test may be repeated.
Sterility
Radiopharmaceutical preparations for parenteral
administration comply with the test for sterility. They must
be prepared using precautions designed to exelude microbial
contamination and to ensure sterility. The test for sterility is
carried out as described in the general method (2.6.1) .
Special difficulties arise with radiophannaceutical
preparations because of the short half-life of sorne
radionuelides, the small size of batches and the radiation
hazards. In the case that the monograph sta tes that the
preparation can be released for use before completion of the
test for sterility, the sterility test must be started as soon as
practically possible in relation to the radiation. If not started
irnmediately, samples are stored under conditions that are
shown to be appropriate in order to prevent false negarive
results. Parametric release (5.1.1) of the product
manufactured by a fully validared process is the method of
choice in such cases. When aseptic manufacturing is used,
the test for sterility has to be perfonned as a control of the
quality of production.
When the size of a batch of the radiopharmaceutical
preparation is limited to 1 or a few samples, sampling the
batch for sterility testing according to the recommendations
of the general method (2.6.1) may not be applicable.
When the half-life of the radionuelide is less than 5 min, the
administration of the radiophannaceutical preparation to the
patient is generally on-line with a validated production
system.
For safety reasons (high level of radioactivity) ir is not
possible to use the quantity of radiophannaceutical
preparations as required in the test for sterility (2.6.1).
The method of membrane filtration is preferred to limit
irradiation of personnel.
Notwithstanding the requiremenrs conceming the use of
antimicrobial preservatives in the monograph Parenteral
preparations (0520), their addition to radiophannaceutical
preparations in multidose containers is not obligatory, unless
prescribed in the monograph.
Bacteria! endotoxins - pyrogens
Radiopharmaceuticals for parenteral adminisrration comply
with the test for bacterial endotoxins (2.6.14) or with the test
for pyrogens (2.6.8).
Eluates of radionuelide generators, solutions for radiolabelling
and kits for radiopharmaceutical preparations also comply
with the test for bacterial endotoxins if they are intended for
the preparation of radiopharmaceuticals for parenteral
administration without further purification.
Radionuelide precursors and chemical precursors comply
with the test for bacterial endotoxins if intended for use in
the manufacture of parenreral preparations without a further
appropriate procedure for the removal of bacterial
endotoxins.
The test for bacterial endotoxins is carried out as described
in rhe general method (2.6.14), taking the necessary
precautions to limit irradiation of the personnel carrying out
the test. The limit for bacterial endotoxins is indicated in the
individual monograph or calculated according to general
chapter 5.1.10. Guidelinesfor using the testfor bacterial
endotoxins.
When the nature of the radiophannaceutical preparation or
the precursor results in an interference in the test for
 2014
bacterial endotoxins by inhibition or activation and it is not
possible to elimina te the interfering factor(s), the test for
pyrogens (2.6.8) may be specifically prescribed.
STORAGE
Store preparations containing radio active substances in an
airtight container that is sufficiently shielded to protect
personnel from irradiation by primary or secondary emissions
and that complies with national and international regulations
concerning the storage of radioactive substances. During
storage, containers may darken due to irradiation. Such
darkening does not necessarily involve deterioration of the
preparations.
LABELLING
The labelling of radiopharmaceutical preparations complies
with the relevant national and European legislation.
For preparations prepared at the site of use, the labelling can
be modified.
The radioactivity of a preparation is stated at a given date.
If the half-life is less than 70 days the time is also indicated,
with reference to a time zone. The radioactivity at other
times may be calculated from the decay equation or from
tables.
In addition to the aboye, the label on the container, the
package, a leaflet accompanying the package or a certificate
of analysis accompanying the radiopharmaceutical
preparation states:
-- the route of administration;
-- if applicable, the maximum recommended dose in
millilitres;
-- the name and concentration of any added antimicrobial
preservative;
-- where applicable, any special storage conditions.
For chemical precursors, the accompanying information
recommends testing the substance in 1 or more production
runs before its use for the manufacture of
radiopharmaceutical preparations to ensure that, under
specified production conditions, the substance yields the
radiopharmaceutical preparation in the desired quantity and
of the quality specified.
MEASUREMENT OF RADIOACTIVlTY
The radioactivity of a preparation is stated at a given date
and, if necessary, time.
The absolute measurement of the radioactivity of a given
sample may be carried out if the decay scheme of the
radionuclide is known, but in practice many corrections are
required to obtain accurate results. For this reason it is
common to carry out the measurement with the aid of a
primary standard source. Primary standards may not be
available for short-lived radionuclides e.g. positron emitters.
Measuring instruments are calibrated using suitable standards
for the particular radionuclides. Standards are available from
the laboratories recognised by the competent authority.
Ionisation chambers and Geiger-Müller counters may be
used to measure beta and beta/gamma emitters; scintillation
or semiconductor counters or ionisation chambers may be
used for measuring gamma emitters; low-energy beta emitters
require a liquid-scintillation counter. For the detection and
measurement of alpha ernitters, specialised equipment and
techniques are required. For an accurate comparison of
radio active sources, it is essential for samples and standards
to be measured under similar conditions.
Low-energy beta emitters may be measured by liquid-
scintillation counting. The sample is dissolved in a solution
containing one or more often two organic fluorescent
Radiopharmaceutical Preparations IV-633
substances (primary and secondary scintillators), which
convert part of the energy of disintegration into photons of
light, which are detected by a photomultiplier and converted
into electrical impulses. When using a liquid-scintillation
counter, comparative measurements are corrected for light-
quenching effects. Direct measurements are made, wherever
possible, under similar conditions, (e.g. volumes and type of
solutions) for the source to be examined and the standard
source.
AlI measurements of radioactivity must be corrected by
subtracting the background due to radioactivity in the
environment and to spurious signals generated in the
equipment itself.
With sorne equipment, when measurements are made at high
levels of radioactivity, it may be necessary to correct for loss
by coincidence due to the finite resolving time of the detector
and its associated e1ectronic equipment. For a counting
system with a fixed dead time T following each count, the
correction is:
N = N obs
1- N obs T
the true count rate per second;
the observed count rate per second;
the dead time, in seconds.
With sorne equipment this correction is made automatically.
Corrections for loss by coincidence must be made before the
correction for background radiation.
If the time of an individual measurement, tm is not negligible
short compared with the half-life, T IIZ, the decay during this
measurement time must be taken into account. After having
corrected the instrurnent reading (count rate, ionisation
current, etc.) for background and, if necessary, for losses due
to electronic effects, the decay correction during
measurement time is:
Rtmln2
T1/ 2
R corr = ----;(~tm-l:-n-=-2"')
l-exp - - -
T1/ 2
R eorr instrument reading corrected to the beginning of the
individual measurement;
R instrument reading before decay correction, but
already corrected for background, etc.
The results of determinations of radioactivity show variations
which derive mainly from the random nature of nuclear
transformation. A sufficient number of counts must be
registered in order to compensate for variations in the
number of transformations per unit of time. The standard
deviation is the square root of the counts, so at least 10 000
counts are necessary to obtain a relative standard deviation of
not more than 1 per cent (confidence interval: 1 sigma) .
AH statements of radioactive content are accompanied by a
statement of the date and, if necessary, the time at which the
measurement was made. This statement of the radio active
content must be made with reference to a time zone (GMT,
CET). The radioactivity at other times may be calculated
from the exponential decay equation or from tables.
The radioactivity of a solution is expressed per unit volurne
to give the radio active concentration.
_____________________________________________ PhEw
IV-634 Radiopharmaceutical Preparations 2014

Table of Physical Characteristics of

Radionuclides Mentioned in the
European Pharmacopoeia
The following table is given to complete the general
monograph Radiopharmaceutical preparations (0125).
The values are obtained from the databas e of the National
Nuclear Data Center (NNDC) at Brookhaven National
Laboratory, Upton. N .Y., USA, directIy accessible via
Internet at the address:
.http://www.nndc.bnl.gov/nndc/nudat/radform.html •.
In case another source of information is preferred (more
recent values), this source is explicitly mentioned.
Other data sources:
* DAMRI (Département des Applications et de la
Métrologie des Rayonnements Ionisants, CEA Gif-sur-
Yvette, France),
** PTB (Physikalisch-Technische Bundesanstalt,
Braunschweig, Germany),
*** NPL (National Physical Laboratory, Teddington,
Middlesex, UK).
The uncertainty of the half-lives are given in parentheses.
In principIe the digits in parentheses are the standard
uncertainty of the corresponding last digits of the indicated
numerical value ('Guide to the Expression of Uncertainty in
Measurement', International Organisation for Standardisation
(ISO), 1993, ISBN 92-67-10188-9) .
The following abbreviations are used:
eA Auger electrons,
ce conversion electrons,
~- electrons,
~+ positrons,
y gamma rays,
X X-rays.

Eledronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide HaIf-life Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

Tritium (3H) -12.33 (6) years 'f3" '0.006 (1) (max: 0.019) 'lOO

Carbon-ll elC) 20.385 (20) min W 0.386 (1) (max: 0.960) 99.8 Y 0.511 199.5 (11)

Nitrogen-13 (13N) 9.965 (4) min W 0.492 (1) (max: 1.198) 99.8 Y 0.511 199.6 (1)

Oxygen-15 (15 0) 122.24 (16) s W 0.735 (1) (max: 1.732) 99.9 Y 0.511 199.8 (11)

Fluorine-18 (18 F) 109.77 (5) min W 0.250 (1) (max: 0.633) 96.7 Y 0.511 193.5 (11 )

Phosphorus-32 (32 P) 14.26 (4) days p- 0.695 (1) (max: 1.71) 100

Phosphorus-33 (33P) 25.34 (12) days p- 0.076 (1) (max: 0.249) 100

Sulphur-35 (35S) 87.51 (12) days f3" 0.049 (1) (max: 0.167) 100

27.7025 (24) days eA 0.004 67 X 0.005 22.3

Chromium-51 (51Cr)
y 0.320 9.9

(1) Mean energy of the p spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
2014 Radiopharmaceutical Preparations IV-635

Electronic emission Photon emission

Emission Emission
probabill- probabUi-
Radionuclide Half-life Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
dlsintegra- disintegra-
tions) tions)

77.27 (3) days eA 0.006 47 X 0.006-0.007 25

W 0.179(1) 0.9 Y 0.511 38.0 (JI)

0.631 m 18.1 0.847 100.0

1.038 14.1

1.175 2.2

1.238 66.1
Cobalt-56 (56CO)
1.360 4.3

1.771 15.5

2.015 3.0

2.035 7.8

2.598 17.0

3.202 3.1

3.253 7.6

271.79 (9) days eA+ce 0.006-0.007 177.4 X 0.006-0.007 57

ce 0.014 7.4 Y 0.014 9.2

Cobalt-57 (57CO)
0.115 1.8 0.122 85.6

0.129 1.3 0.136 10.7

0.692 0.15

70.86 (7) days eA 0.006 49.4 X 0.006-0.007 26.3

W 0.201 (1) 14.9 Y 0.511 29.9 (11)

Cobalt-58 (saCo)
0.811 99.4

0.864 0.7

1.675 0.5

5.2714 (5) years p- 0.096 (1) (max: 0.318) 99.9 Y 1.173 100.0
Cobalt-60 (6OCo)
1.333 100.0

(1) Mean energy of the p spectrum.

(n) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
IV-636 Radiopharmaceutical Preparations 2014

Electronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-life Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

9.49 (7) hours eA 0.008 21 X 0.009.().010 19.1

W 0.157 (1) 1 Y 0.511 112 (11)

0.331 (1) 0.7 0.834 5.9

0.397 (1) 3.8 1.039 37

0.782 (1) 0.3 1.333 1.2

1.90 (1) 50 1.919 2.1

2.190 5.6
GaJlium-66 (66Ga)
2.423 1.9

2.752 23.4

3.229 1.5

3.381 1.5

3.792 1.1

4.086 1.3

4.295 4.1

4.807 1.8

3.2612 (6) days eA 0.008 62 X 0.00s.o.010 57

ce 0.082.().084 30.4 Y 0.091·0.093 42.4

0.090.0.092 3.6 0.185 21.2

GaJlium-67 (6'Ga)
0.175 0.3 0.209 2.4

0.300 16.8

0.394 4.7

0.888 0.15

270.82 (27) days eA 0.008 42.4 X 0.009'().01O 44.1

Germanium-68 (68Ge)
in equilibrium with
GaJlium-68 (68Ga) W 0.353 (J) 1.2 y 0.511 178.3
(68Ga: 67.629
(24) min) 0.836 m 88.0 1.077 3.0

67.629 (24) min eA 0.008 5.1 X 0.009.().01O 4.7

GaJlium-68 (68Ga)
W 0.353 (1) 1.2 Y 0.511 178.3

0.836 (1) 88.0 1.077 3.0

13.10 (3) s ce 0.176 26.4 X 0.012.().014 17.0

Krypton-81m (81 mKr) 0.189 4.6

Y 0.190 67.6

(1) Mean energy of the ~ spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the so urce per 100 disintegrations.
2014 Radiopharmaceutical Preparations IV-637

Electronlc emlsslon Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-Jife Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

4.576 (5) hours eA 0.011 31.3 X 0.013-0.014 57.2

ee 0.176 25.0 Y 0.190 64

Rubidium-81 (8I Rb) 0.188 4.3 0.446 23.2

in equilibrium with
Krypton-81m (8Im Kr) 0.457 3.0

W 0.253 11
) 1.8 0.510 5.3

0.447 (1) 25.0 0.511 54.2

(8ImKr: 13.10 (3) s) 0.538 2.2

50.53 (7) days p- 0.583 (1) (max: 1.492) 99.99 Y 0.909 0.01
Strontium-89 ("Sr)
in equilibrium with
Yttrium-89m ('9my)
(89my: 16.06 (4) s)

28.74 (4) years p- 0.196 (1 ) (max: 0.546) 100

Strontium-90 (90Sr)
in equilibrium with
Yttrium-90 (90y)
(9'Y: 64.10 (8) hours)

64.10 (8) hours p- 0.934 (1) (max: 2.280) 100

Yttrium-90 (9Oy)

65.94 (1) hours p- 0.133 (1) 16.4 X 0.018-0.021 3.6

0.290 {O 1.1

0.443 (1) 82.4 Y 0.041 1.1

Molybdene-99 (99Mo)
0.141 4.5
in equilibrium with
Teehnetium-99m 0.181 6
(99mTc)
0.366 1.2

(99mTe: 6.01 (1) hours) 0.740 12.1

0.778 4.3

6.01 (1) hours ce 0.002 74 X 0.018-0.021 7.3

eA 0.015 2.1 Y 0.141 89.1

Technetium-99m
(9!"''Te)

ce 0.120 9.4

0.137-0.140 1.3

2.11 x 105 years 13- 0.085 (1) (max: 0.294) 100

Technetium-99 ("Te)

(1) Mean energy of the 13 spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
IV-638 Radiopharmaceutical Preparations 2014

Electronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-Iife Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

39.26 (2) days eA+ce 0.017 12 X 0.020-0.023 9.0

Ruthenium-103 (' 03 Ru) ce 0.030-0.039 88.3 Y 0.497 91

in equilibrium with
Rhodium-103m 0.610 5.8
('o3m Rh)
/3- 0.031 (1) 6.6
('03mRh:
56.114 (20) min) 0.064 (1) 92.2

4.9 (1) hours eA 0.019 13.4 X 0.023-0.026 70.5

Y 0.642 25.9

Indium-110 (UOIn) 0.658 98.3

0.885 92.9

0.938 68.4

0.997 10.5

69.1 (5) min eA 0.019 5.3 X 0.023-0.026 27.8

Indium-llOm (IlOmIn) W 1.015 (1) 61 Y 0.511 123.4 (n)

0.658 97.8

2. 129 2.1

2.8047 (5) days eA 0.019 15.6 X 0.003 6.9

0.023-0.026 82.3

ce 0.145 7.8
Indium-ll1 (lII In)
0.167-0.171 1.3 Y 0.171 90.2

0.219 4.9 0.245 94.0

0.241-0.245 1.0

49.51 (1) days ce 0.162 40 X 0.023-0.027 36.3

0.186-0.190 40
Indium-1l4m (ll4m In)
in equilibrium with Y 0.190 15.6
Indium-1l4 (!"In)
"/3- 0.777 (11 (max: 1.985) 95 0.558 3.2

(ll4In: 71.9 (1) s) 0.725 3.2

154.0 (7) days eA 0.003 88.0 X 0.026-0.031 50.5

0.022-0.023 7.4
Tellurium-121m
y 0.212 81.4
(I21mTe)
in equilibrium with ce 0.050 33.2 1.102 2.5
Tellure-121 (! 2I Te)
(I2!Te: 19.16 (5) days) 0.077 40.0

0.180 6.1

(1) Mean energy of the /3 speetrum.

(JI) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
2014 Radiopharmaceutical Preparations IV-6 39

Electronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-life Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

""19.16 (5) days eA 0.022 11.6 X 0.026-0.030 75.6

y 0.470 1.4
Tellurium·121 (l2lTe)

0.508 17.7

0.573 80.3

13.27 (8) hours eA 0.023 12.3 X 0.004 9.3

0.027.Q.031 86.6

ce 0.127 13.6

0.154 1.8 Y 0.159 83.3

Iodine·123 (1231) 0.158 0.4 0.346 0.1

0.440 0.4

0.505 0.3

0.529 1.4

0.538 0.4

59.402 (14) days eA+ce 0.004 80 X 0.004 15.5

0.023-0.035 33 0.027 114

Iodine-125 (1251) 0.031 26

Y 0.035 6.7

13.11 (5) days eA 0.023 6 X 0.027·0.031 42.2

ce 0.354 0.5 Y 0.388 34

0.634 0.1 0.491 2.9

0.511 2.3 (11)

lodine-126 (1261)
13- 0.109 (1) 3.6 0.666 33

0.290 (1) 32.1 0.754 4.2

0.459 (1) 8.0 0.880 0.8

1.420 0.3

W 0.530 (1) 1

(1) Mean energy of the 13 spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
IV-640 Radiopharmaceutical Preparations 2014

Electronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-Iife Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

8.02070 (U) days ce 0.46 3.5 X 0.029-0.030 3.9

0.330 1.6

Y 0.080 2.6

Iodine-131 (131I) p- 0.069 (I ) 2.1 0.284 6.1

0.097 en 7.3 0.365 81.7

0.192 en 89.9 0.637 7.2

0.723 1.8

11.84 (7) days eA 0.025 6.8 X 0.004 8.3

0.030 44.0

Xenon-131m (I3lmXe) ce 0.129 61 0.034 10.2

0.159 28.5

0.163 8.3 Y 0.164 2.0

20.8 (1) hours p- 0.140 ()) 3.8 Y 0.530 87

Iodine-133 (J331) 0.162 (1) 3.2 0.875 4.5
(decays lo radioactive
Xenon-133) 0.299 (1) 4.2 1.298 2.4

0.441 en 83

5.243 (1) days eA 0.026 5.8 X 0.004 6.3

0.031 40.3
ce 0.045 55.1 0.035 9.4
Xenon-133 (l33Xe)
0.075-0.080 9.9

Y 0.080 38.3

p- 0.101 ()) 99.0

2.19 (1) days eA 0.025 7 X 0.004 7.8

0.030 45.9
Xenon-133m (133mXe)
(decays lo radioactive ce 0.199 64.0 0.034 10.6
Xenon-133)
0.228 20.7

0.232 4.6 Y 0.233 10.0

(1) Mean energy of the p spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
2014 Radiopharmaceutical Preparations IV -641

Electronic emission Photon emission

Emission Emission
probabili- probabili-
Radionuclide Half-life Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

6.57 (2) hours p- 0.140 (1) 7.4 Y '0.527 13.8

0.237 (11 8 0.547 7.2

0.307 (1) 8.8 0.837 6.7

0.352 (1) 21.9 1.039 8.0

lodine-135 (1351)
(decays to radio active 0.399 (1) 8 1.132 22.7
Xenon-135)
0.444(1) 7.5 1.260 28.9
(1
0.529 ) 23.8 1.458 8.7

1.678 9.6

1.791 7.8

9.14 (2) hours ce 0.214 5.5 X 0.031-0.035 5.0

Xenon-135 (' 35Xe)

p- 0.171 3.1 Y 0.250 90.2

0.308 96.0 0.608 2.9

30.04 (3) years eA 0.026 0.8 X 0.005 1

0.032-0.036 7
ce 0.624 8.0
Caesium-137 (137Cs)
in equilibrium with 0.656 1.4 Y 0.662 85.1
Barium-137m ('37rnBa)

p- 0.174 (1) 94.4

(137mBa: 2.552 (1) min) 0.416 O) 5.6

26.1 (1) hours ce 0.285 3.4 X 0.010 32.0

0.353 1.4 0.069-0.071 63.3

0.08 17.5

W 0.495 (1) 0.3

Y 0.368 87.2

0.579 13.8
Thallium-200 (200r1)
0.828 10.8

1.206 29.9

1.226 3.4

1.274 3.3

1.363 3.4

1.515 4.0

(1) Mean energy of the 13 spectrum.

(11) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
IV-642 Radiopharmaceutical Preparations 2014

Electronic emission Pboton emission

Emission Emission
probabili- probabili-
Radionuclide Half-Iife Type Energy (MeV) ty (per 100 Type Energy (MeV) ty (per 100
disintegra- disintegra-
tions) tions)

9.33 (3) hours eA 0.055 3 X 0.07()'0.073 69

0.083 19

ce 0.246 8.5

0.276 2 Y 0.331 79

0.316 2.3 0.361 9.9

0.406 2.0

Lead.201 ('OlPb) 0.585 3.6

(decays to radioactive
Thallium-201) 0.692 4.3

0.767 3.2

0.826 2.4

0.908 5.7

0.946 7.9

1.099 1.8

1.277 1.6

72.912 (17) hours ce 0.016-0.017 17.7 X 0.010 46.0

0.027-0.029 4.1 0.069-0.071 73.7

0.052 7.2 0.080 20.4

Thallium-201 (2OlTI)
0.084 15.4

0.153 2.6 Y 0.135 2.6

0,167 10.0

12.23 (2) days eA 0.054 2.8 X 0.010 31.0

0.069-0.071 61.6

Thallium-202 ('·'TI) ce 0.357 2.4 0.080 17.1

Y 0.440 91.4

51.873 (9) hours eA 0.055 3.0 X 0.010 37.0

0.071-0.073 69.6

ce 0.194 13.3 0.083 19.4

Lead-203 (' 03Pb)

y 0.279 80.8

0.401 3.4

(I) Mean energy of the ~ spectrum.

(II) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.
2014 Radiopharmaceutical Preparations IV-643

lobenguane Sulfate for ***** - resolution: minimum 4.0 between the peaks due to
** ** impurity A and iobenguane.
Radiopharmaceutical Preparations *** Limit:
Iobenguane Sulphate for Radiopharmaceutical - impurity A : not more than the area of the corresponding
Preparations peak in the chromatogram obtained with reference
(Ph Eur monograph 2351) solution (d) (l.O per cent).
Loss on drying (2.2.32)
Maximum 3.0 per cent, determined on 0.100 g by drying in
an oven at 105 oc.
Bacteria! endotoxins (2.6.14)
Less than 10 IU/mg, if intended for use without a further
appropriate procedure for the removal of bacterial
endotoxins .
648 87862-25-7
~Ew __________________________________________
ASSAY
Liquid chromatography (2.2.29) as described in the test for
DEFINITION related substances with the following modification.
Bis [(3-iodobenzyl)guanidine1 sulfate. Injection
Content Test solution and reference solution (a).
98.0 per cent to 102.0 per cent (dried substance). Calculate the percentage content of C16HzzlzN604S from
CHARACTERS the dec1ared content of iobenguane sulfate CRS.
Appearance STORAGE
White or almost white crystals. Protected from light, at a temperature below 25 oC.
IDENTIFICATION LABELLING
A. Infrared absorption spectrophotometry (2.2.24) The label recommends testing the substance in a production
Comparison Ph. Eur. referenee spectrum of iobenguane sulfate. test before its use for the manufacture of radiopharmaceutical
B. Dissolve about 10 mg in 1 mL of water R with gentle preparations. This ensures that, under specified production
heating. The solution gives reaction (a) of sulfates (2.3.1) . conditions, the substance yields the radiopharmaceutical
preparation in the desired quantity and quality specified.
TESTS
Impurity A IMPURITIES
Liquid chromatography (2.2.29). Prepare the solutions and the Specified impurities A.
mobile phase immediately before use.
Test solution Dissolve 10.0 mg of the substance to be
examined in 1 mL of the mobile phase and dilute to 5.0 mL
with the mobile phase.
Referenee solution (a) Dissolve 10.0 mg of iobenguane
sulfate CRS in 1 mL of the mobile phase and dilute to A. 1-(3-iodophenyl)methanamine.
5.0 mL with the mobile phase. _________________________________________ ~Ew

Referenee solution (b) Dissolve 23.1 mg of

3-iodobenzylammonium ehloride R (salt of impurity A) in 1 mL
of the mobile phase and dilute to 10.0 mL with the mobile
phase.
Medronic Acid for ***
Referenee solution (e) Mix 1 mL of reference solution (a) ** **
and l mL of reference solution (b). Radiopharmaceutical Preparations *****
Referenee solution (d) Dilute 0.1 mL ofreference solution (Ph Eur monograph 2350)
(b) to 10.0 mL with the mobile phase.
Column: o O
-- size: 1= 0.25 m, 0 = 4.0 mm; 11 11
-- stationary phase: silica gel for ehromatography R (5 ¡.¡m); HO-t~P\-OH
-- temperature: maintain at a constant temperature between HO OH
20 oC and 30 oC.
Mobile phase Mix 40 mL of an 80 gIL solution of
176 1984-15-2
ammonium nitrate R, 80 mL of dilute ammonia R2 and
1080 mL of methanol R. Action and use
Flow rate 1 mUmin. Bisphosphonate; used for bone scanning.
Detection Spectrophotometer at 254 nm. ~Ew __________________________________________
Injeetion 20 ¡.¡L of the test solution and reference solutions
(c) and (d). DEFINITlON
Run time 15 mino Methylenediphosphonic acid.
Relative retention With reference to iobenguane Content
(retention time = about 7 min): impurity A = about 0.2. 99.0 per cent to 10l.0 per cent (dried substance) .
System suitability Reference solution (c):
IV-644 Radiopharmaceutical Preparations 2014

CHARACTERS Loss on drying (2.2.32)

Appearance Maximum 0.5 per cent, determined on 0.500 g by drying in
White or almost white, amorphous or crystaIline, hygroscopic an oven at 105 oC.
powder. Bacterial endotoxins (2.6.14)
Solubility Less than 2.0 IU/mg.
Very soluble in water, very slightly soluble in anhydrous ASSAY
ethanol, practicaIly insoluble in methylene chloride. Dissolve 75 mg in water R and dilute to 50 mL with the
IDENTIFICATION same solvent. Titrate with 0.1 M sodium hydroxide, using
First identification A. 0.1 mL of bromocresol green solution R as indicator.
Second identification B. 1 mL of 0.1 M sodium hydroxide is equivalent to 8.80 mg of
A. lH Nuclear magnetic resonance spectrometry (2.2.33). CH 6 0 6 P 2 ·
Preparation 100 gIL solution in deuterium oxide R. STORAGE
Companson 100 gIL solution of medronic acid CRS in In an airtight container, protected from light.
deutenum oxide R. LABELLING
B. Infrared absorption spectrophotometry (2.2.24). The label recommends testing the substance in a production
Companson medronic acid CRS. test before its use for the manufacture of radiopharmaceutical
preparations. This ensures that, under specified production
TESTS conditions, the substance yields the radiopharmaceutical
Impurities A and B preparation in the desired quantity and quality specified.
lH Nuclear magnetic resonance spectrometry (2.2.33).
Test solution To 1.0 g of the substance to be examined add
IMPURITIES
10 mL of deuterated chloroform R. Stir for 1 hour. Pass the Specified impurities A, B.
resulting solution through a sintered-glass filter to remove the
precipitate containing medronic acid. Evaporate the filtrate to
about 0.5 mL.
Reference solution (a) Mix 10 IlL of medronic acid
impunty A CRS with 1.0 mL of deuterated chloroform R.
Reference solution (b) Mix 10 IlL of medronic acid
impurity B CRS with 1.0 mL of deuterated chloroform R.
Reference solution (c) After recording the NMR spectrum of
the test solution, add 10 IlL of medronic acid impurity A CRS A. tris ( l-methylethoxy)phosphane,
and 10 IlL of medronic acid impun'ty B CRS to the test
solution.
Apparatus NMR spectrometer operating at minimum
250 MHz.
Record the 1H NMR spectra of the test solution and the
reference solutions, if necessary using tetramethylsilane R as a
che mi cal shift intemal reference compound.
Position of the signals Deuterated chloroform
= about 7.3 ppm; B. tetrakis(l-methylethyl) methylenediphosphonate.
impurity A = about 4.4 ppm and 1.3 ppm; ____________________________________________ ~E~

impurity B = about 4.7 ppm, 2.4 ppm and 1.3 ppm.

System suitability:
-- the positions of the signals due to impurities A and B in
the spectrum obtained with reference solution (c) do not
differ significantIy from those in the spectra obtained with
reference solutions (a) and (b).
Limits:
-- integration: integrate the multiplet at 4.4 ppm due to
impurity A and the multiplet at 2.4 ppm due to
impurity B in the spectra obtained with the test solution
and reference solution (c) to obtain the areas of the peaks
used in the comparison of impurity contents;
-- impurities A, B: for each impurity, not more than
0.5 times the area of the corresponding peak in the
spectrum obtained with reference solution (c)
(1 per cent).
Phosphates (2.4.11)
Maximum 1.0 per cent.
Dissolve 0.100 g in 10 mL of water R and dilute to
100.0 mL with the same solvent. Dilute 1.0 mL of this
solution to 100.0 mL with water R.
2014 Radiopharmaceutical Preparations IV-645

Rejerence solution (b) Dissolve 40.0 mg of nitrilotriaeetie

Pentetate Sodium Calcium for acid R (impurity A) in the solvent mixture and dilute to
Rad iopharmaceutical Preparations 100.O mL with the solvent mixture. To 10. O mL of the
(Ph Bur monograph 2353) solution add 1 mL of reference solution (a) and dilute to
100.0 mL with the solvent mixture. Dilute 1.0 mL ofthis
solution to 10.0 mL with the solvent mixture.
3-
Column:
-- size: l = 0.10 m, 0 = 4.6 mmi
-- stationary phase: spherical graphitised carbon jor
ehromatography R1 (5 /lm) with a specific surface area of
3 Na+ 2
xH 2 0 120 m fg and a pore size of 25 nm.
Mobile phase Dissolve 50 mg ofjerrie sulfate pentahydrate R
in 50 mL of 0.5 M sulfuric acid and add 750 mL of water R;
adjust to pH 1.5 with 0.5 M sulfuric acid or 1 M sodium
hydroxide, add 20 mL of ethylene glyeol R and dilute to
1000 mL with water R.
CI4H1SCaN3Na}OlO,xH20 497.4 Flow rate 1 mUmin.
(anhydrous substance) Deteetion Spectrophotometer at 273 nm.
~Elf __________________________________________ Injection 20 /lL of the test solution and reference solution
(b); filter the solutions and inject immediately.
DEFINITION Run time 4 times the retention time of the iron complex of
Trisodium [1,1',1",1"'- impurity A.
[[(carboxylatomethyl)imino]bis(ethylenenitrilo)]tetraacetato]
Retention time iron complex of impurity A = about 5 mini
ca1ciate(3-) .
iron complex of edetic acid = about 10 mini the iron
It is a starting material for the preparation of technetium complex of pentetic acid elutes with the void volume.
C9mTc) pentetate injection. System suitability Reference solution (b):
Content -- resolution: minimum 7 between the peaks due to the iron
98.0 per cent to 102.0 per cent (anhydrous substance). complex of impurity A and the iron complex of edetic
CHARACTERS acidi
Appearance -- signal-to-noise ratio: minimum 50 for the peak due to the
White or almost white, hygroscopic powder or crystals. iron complex of impurity A.
Solubility Limit:
Freely soluble in water, practically insoluble in ethanol -- impurity A: not more than the area of the corresponding
(96 per cent). peak in the chromatogram obtained with reference
solution (b) (0.1 per cent).
IDENTIFICATlON
Impurity B
A. Infrared absorption spectrophotometry (2.2.24).
Maximum 1.0 per cent.
Comparison pentetate sodium ealeium CRS.
Dissolve 5.0 g of the substance to be examined in 250 mL of
B. Ignite. The residue gives reaction (b) of ca1cium (2.3.1). water R. Add 10 mL of ammonium ehloride buffer solution
C. The substance to be examined gives reaction (a) of pH 10. O R and 50 mg of mordant black 11 m'turate R.
sodium (2.3.1). Not more than 1.3 mL of 0.1 M magnesium chloride is
TESTS required to change the colour of the indicator to violeto
Solution S Chlorides
Dissolve 5.0 g in earbon dioxide-jree water R and dilute to Maximum 0.1 per cent.
25 .0 mL with the same solvento Dissolve 0.7 g in water R and dilute to 20 mL with the same
Appearance of solution solvento Add 30 mL of dilute nitric acid R, allow to stand for
Solution S is clear (2.2.1) and colourless (2.2.2, Method JI). 30 min and filter. Dilute 10 mL of the filtrate to 50 mL with
water R. Use this solution as the test solution. Prepare the
pH (2.2.3)
reference solution using 0.40 mL of O. 01 M hydroehlorie acid,
8.0 to 9.5 for solution S.
add 6 mL of dilute nitric acid R and dilute to 50 mL with
Impurity A water R. Filter both solutions if necessary. Add 1 mL of silver
Liquid chromatography (2.2.29). Cany out the test proteeted nitrate solution R2 to the test solution and the reference
jrom light. solution. Mix and allow to stand for 5 min protected from
Solvent mixture Dissolve 10 g of jeme sulfate pentahydrate R light. Any opalescence in the test solution is not more intense
in 20 mL of 0.5 M sulfuric acid and add 780 mL of water R. than that in the reference solution.
Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to Iron (2.4.9)
1000 mL with water R. Maximum 20 ppm.
Test solution Dissolve 0.100 g of the substance to be Dilute 2.5 mL of solution S to 10 mL with water R.
examined in the solvent mixture and dilute to 25.0 mL with Add 0.25 g of calcium ehloride R to the test solution and the
the solvent mixture. standard before the addition of the thioglycollic acid R.
Rejerence solution (a) Dissolve 0.100 g of sodium calcium Heavy metals (2.4.8)
edetate R in the solvent mixture and dilute to 25.0 mL with Maximum 20 ppm.
the solvent mixture.
IV-646 Radiopharmaceutical Preparations 2014

1.0 g complies with test F. For the digestion replace sulfuric

Sodium lodohippurate Dihydrate ***
acid R by nitric acid R. Prepare the reference solution using ** **
2 mL of lead standard solution (lO ppm Pb) R. for Radiopharmaceutical *****
Water (2.5.12) Preparations
Maximum 15.0 per cent, determined on 0.100 g.
(Ph Bur monograph 2352)
Bacteria! endotoxins (2.6.14)
Less than 0.1 IU/mg, if intended for use in the manufacture
of parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins.
ASSAY
Dissolve 0.100 g in water R and dilute to 50.0 mL with the
same solvent. To 25.0 mL of this solution add 80 mL of 363.1 5990-94-3
C9H7INNa03,2H20
water R and adjust to pH 2.3 with dilute nitric acid R. Titrate
PhE~ _ _ _ __ _ _ __ __ __ _ _ _.___ _ __
with O. 01 M bismuth nitrate using 0.1 mL of a 1 gIL solution
of xylenol orange R as indicator. The colour of the solution DEFINITION
changes from yellow to red. Sodium (2-iodobenzamido)acetate dihydrate.
1 mL of 0.01 M bismuth nitrate is equivalent to 4.974 mg of Content
C¡4H¡sCaN3Na30¡O. 99.0 per cent to 101.0 per cent (anhydrous substance).
STORAGE CHARACTERS
In an airtight container, protected from light. Appearance
LABELLING White or almost white, crystalline powder.
The label recommends testing the substance in a production Solubility
test before its use for the manufacture of radiopharmaceutical Soluble in water and in ethanol (96 per cent), practically
preparations. This ensures that, under specified production insoluble in methylene chloride.
conditions, the substance yields the radiopharmaceutical
IDENTIFICAnON
preparation in the desired quantity and of the quality
A. Infrared absorption spectrophotometry (2.2.24).
specified.
Comparison sodium iodohippurate CRS.
IMPURITIES
B. It gives reaction (b) of sodium (2.3.1).
Specijied impurities A, B.
TESTS
Re1ated substances
Liquid chromatography (2.2.29).
Test solution Dissolve 0.100 g of the substance to be
examined in the mobile phase and dilute to 10.0 mL with
the mobile phase.
A. nitrilotriacetic acid,
Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL ofthis
H02 C (C02H
solution to 10.0 mL with the mobile phase.
i
H0 C'-../ N --..../'- N ~ N '-../ C0 H
2 Reference solution (b) Dissolve 10 mg of 2-iodobenzoic acid R
2
(impurity A) in methanol R and dilute to 100.0 mL with the
l.C0 2H same solvent.
Reference solution (e) Dissolve 10 mg of benzoie acid R in the
mobile phase, add 1 mL of the test solution and dilute to
B. [[(carboxyrnethyl)imino]bis(ethylenenitrilo)]tetraacetic acid
(pentetic acid) . 100 mL with the mobile phase.
_ _ _ __ _ _ __ _ __ _ __ _ _ __ __ PhEur Column:
-- size: l = 0.25 m, (2) = 4.6 mm;
-- stationary phase: end-capped polar-embedded octadeeylsilyl
amorphous organosüica polymer R (5 Jlm).
Mobile phase aeetic aeid R, methanol R, water R
(1 :50:50 VIVIV).
Flow rate 1 mUmin.
Detection Spectrophotometer at 230 nm.
Injection 20 JlL.
Run time 7 times the retention time of 2-iodohippuric acid.
Identijication of impurities Use the chromatogram obtained
with reference solution (b) to identify the peak due to
impurity A.
R elative retention With reference to 2-iodohippuric acid
(retention time = about 4.5 min): benzoic acid = about 1.6;
impurity A = about 2.1.
System suitability Reference solution (e):
2014
- resolution: minimum 5.0 between the peaks due to
2-iodohippuric acid and benzoic acid.
Limits:
- impurity A: not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent);
- unspeeified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent);
- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent);
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.12)
8.0 per cent to 12.0 per cent, determined on 0.100 g.
Bacteria! endotoxins (2.6.14)
Less than 2 IU/mg, if intended for use in the manufacture of
parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins.
ASSAY
Dissolve 0.250 g in 20 mL of glacial aeetie acid R. Titrate
with 0.1 M perchlorie acid, determining the end-point
potentiometrically (2.2.20) .
1 mL of 0.1 M perehlorie aeid is equivalent to 32.71 mg of
C9H7INNa03·
STORAGE
Protected from light.
LABELLING
The label recommends testing the substance in a production
test before its use for the manufacture of radiopharmaceutical
preparations. This ensures that, under specified production
conditions, the substance yields the radiopharmaceutical
preparation in the desired quantity and of the quality
specified.
IMPURITIES
Speeified impurities A.
A. 2-iodobenzoic acid.
_ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ Ph EUf
Radiopharmaceutical Preparations IV -647
Tetra-O-Acetyl-Mannose Triflate ***
*** **
for Radiopharmaceutical ****
Preparations
(Ph Eur monograph 2294)
Ph EUf _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ __
DEFINITION
1,3,4,6-Tetra-O-acetyl-2-0-trifluoromethanesulfonyl-~-D.
mannopyranose.
Content
97.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, crystalline, hygroscopic powder.
Solubility
Practically insoluble in water, very soluble in acetonitrile,
freely soluble in methylene chloride, slightly soluble in
ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison tetra-O-aeetyl-mannose trifiate CRS.
TESTS
Specific optica! rotation (2.2.7)
-12.0 to - 16.0 (dried substance), measured at 20 oc.
Dissolve 0.250 g in aeetonitrile R and dilute to 10.0 mL with
the same solvento
Impurity B
19p Nuclear magnetic resonance spectrometry (2.2.33).
Prepare the solutions immediately before use.
Test solution Dissolve 20.0 mg of the substance to be
examined in deuterated aeetonitrile R and dilute to 1.0 mL
with the same solvento
Referenee solutwn (a). Dissolve 20.0 mg of tetra-O-acetyl-
mannose trifiate CRS in deuterated acetonitrile R and dilute to
1.0 mL with the same solvento
Referenee solution (b) Dissolve 4.0 mg of lithium
trifiuoromethanesulfonate R (lithium salt of impurity B) in
deuterated aeetonitrüe R and dilute to 1.0 mL with the same
solvento
Referenee solution (e) Mix 1.0 mL of reference solution (a)
and 10 ¡.¡L ofreference solution (b).
Limit The peak area identified in the spectrum obtained
with the test solution at about - 78 ppm is smaller than the
peak area identified in the spectrum obtained with reference
solution (c) at the same chemical shift (0.2 per cent).
Related substances
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
 IV-648 Radiopharmaceutical Preparations 2014

Test solution (a) Dissolve 0.200 g of the substance to be Jnjeetion Test solution (b) and reference solution (a) .
examined in acetonitrile R and dilute to 2.0 mL with the same Calculate the percentage content of C1 5H19F3012S from the
solvento declared content of tetra-O-acetyl-mannose trifiate CRS.
Test solution (b) Dissolve 10.0 mg of the substance to be STORAGE
examined in acetonitrile R and dilute to 5.0 mL with the same
In an airtight container, protected from light, at a
solvento
temperature of 2 oC to 8 oc.
Reference solution (a) Dissolve 10.0 mg of tetra-O-acetyl-
mannose trifiate CRS in acetonitrile R and dilute to 5.0 mL LABELLING
with the same solvento The label recommends testing the substance in a production
run before its use for the manufacture of radiopharrnaceutical
Reference solution (b) Dilute 1.0 mL of test solution (a) to
preparations. This ensures that, under specified production
10.0 mL with acetonitrile R. Dilute 1.0 mL of this solution to
conditions, the substance yields the radiopharrnaceutical
100.0 mL with acetonitrile R.
preparation in the desired quantity and of the quality
Referenee solution (e) Dissolve 10 mg of l,3,4,6-tetra-O- specified.
acetyl-f3-D-mannopyranose R (impurity A) in 5 mL of
acetonitrile R. Mix 1 mL of this solution and 1 mL of IMPURITIES
reference solution (a) . Speeified impurities A, B.
Column:
-- size: 1 =: 0.25 m, 0 = 4.0 mm;
-- stationary phase: oetadeeylsilyl sz7iea gel for ehromatography R
(5 ~m);
-- temperature: 25 oc.
Mobile phase:
-- mobile phase A: water R;
-- mobile phase B: aeetonitrile R1;
Time Mobile phase A Mobile phase B
(min) (per cen! V!JI) (per cen! V!JI)
A. 1,3,4,6-tetra-O-acetyl-~-D-mannopyranose,
0· 1 80 20

¡·20 80 --> 55 20 --> 45

20·35 55 45

35 - 45 55 --> O 45 --> 100

B. trifluoromethanesulfonic acid.
45 - 50 O 100
____________________________________________ PhEm

Flow rate 1 mUmin.

Deteetion Spectrophotometer at 220 nm.
Injection 20 llL of test solution (a) and reference solutions
(b) and (c) .
Relative retention With reference to tetra-O-acetyl-mannose
lodinated C251) Albumin Injection
triflate (retention time = about 29 min): impurity A = about (Human Albumin Jnjeetion Jodinated (125J),
0.2 . Ph Bur monograph 1922)
____________________________________________
System suitability Reference solution (c): ~Em

-- resolution: minimum 5.0 between the peaks due to DEFINITION

impurity A and tetra-O-acetyl-mannose triflate.
Limits:
-- impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent);
-- any other impurity: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent);
-- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent);
-- disregard limit: 0.5 times the area ofthe principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent) .
Loss on drying
Maximum 0.6 per cent, deterrnined on 25 mg by
therrnogravimetry (2.2.34). Reat to 80 oC at arate of
2.5 °C/min.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Sterile, endotoxin-free solution of human albumin labelled
with iodine-125. It may contain a suitable buffer and an
antimicrobial preservative. The human albumin used
complies with the requirements of the monograph on Human
albumin solution (0255).
Content
90 per cent to 11 O per cent of the declared iodine-125
radioactivity at the date stated on the labe!'
Purity:
-- minimum of 99 .0 per cent of the total radioactivity
corresponds to iodine-125,
-- minimum of 80 per cent of the total radioactivity is
associated with the albumin fractions II to V,
-- maximum of 5 per cent of the total radioactivity
corresponds to unbound iodide.
Content of albumin
95 per cent to 105 per cent of the declared albumin content
stated on the labe!'
CHARACTERS
Appearance
Clear, colourless to yellowish solution.
2014 Radiopharmaceutical Preparations IV-649

Half-life and nature of radiation of iodine-125 Referenee solution Human albumin solution R or another
See general chapter 5.7. Table of physical eharactensties of appropriate human albumin standard diluted with the mobile
radionuclides. phase to a suitable albumin concentration.
IDENTIFICATION Column:
A. Gamma-ray and X-ray spectrometry. -- size: 1 = 0.6 m, 0 = 7.5 mm,
-- stationary phase: siliea gel for size-exclusion
Companson Standardised iodine-125 solution, or by using a
chromatography R,
calibrated instrumento Standardised iodine-125 solutions -- temperature: 25 oc.
and/or standardisation services are available from the
competent authority. Mobile phase Dissolve 11.24 g of potassium dihydrogen
phosphate R, 42.0 g of disodium hydrogen phosphate R,
Results The spectrum obtained with the preparation to be
11.70 g of sodium ehloride R in 2000 mL of water R .
examined does not differ significant1y from that obtained
with a standardised iodine-125 solution, apart from any Flow rate 0.6 mUmin.
differences attributable to the presence of iodine-126 . Deteetion Spectrophotometer at 280 nm and radioactivity
The most prominent photon has an energy of 0.027 MeV, detector set for iodine-125 connected in series.
corresponding to the characteristic X-ray of tellurium, Injeetion Loop injector.
gamma photons of an energy of 0.035 MeV are also presento Run time 85 mino
Iodine-126 has a half-life of 13.11 days and its most
Retention times:
prominent gamma photons have energies of 0.388 MeV and
0.666 MeV.
Peak Fraclion Description oí the compound Relenlion
B. Examine by a suitable immunoelectrophoresis technique No. time
(2.7.1). Using antiserum to normal human serum, compare (min)
normal human serum and the preparation to be examined,
both diluted if necessary. The main component of the High molecular mass compound 18 - 20
preparation to be examined corresponds to the main 2 1I Poly III albumin 23 - 24
component of the normal human serum. The diluted
solution may show the presence of small quantities of other 3 III Poly 1I albumin 25 · 26
plasma proteins.
4 IV Poly I albumin 28
TESTS
5 V Human serum albumin 29 - 31
pH (2.2.3)
5.0 to 9.0. 6 VI lodide 43 - 45
Albumin
Referenee solution Dilute human albumin solution R with a
9 giL solution of sodium ehloride R to a concentration of 5 mg The main peak in the chromatogram obtained with the
of albumin per millilitre. reference solution corresponds to fraction V.
To 1.0 mL of the preparation to be examined and to 1.0 mL Limits:
of the reference solution add 4.0 mL of biuret reagent R and -- radioaetivity in fraetions 11 to V: minimum 80 per cent of
mix. After exactly 30 min, measure the absorbance (2.2.25) the total radioactivity applied to the column,
of each solution at 540 nm, using as the compensation liquid -- iodine-125 in fraetion VI: maximum 5 per cent of the total
a 9 gIL solution of sodium ehlonde R treated in the same radioactivity .
manner. From the absorbances measured, calculate the RADIOACTIVITY
content of albumin in the injection to be examined in Measure the radioactivity using suitable equipment by
milligrams per millilitre. comparison with a standardised iodine-125 solution or by
Sterility measurement with a calibrated instrumento
It complies with the test for sterility prescribed in the LABELLING
monograph on Radiopharmaeeutieal preparations (0125). The label states:
Bacteria! endotoxins (2.6.14) -- the amount of albumin,
Less than 175/ V IU/mL, Vbeing the maximum -- the maximum volume to be injected.
recommended dos e in millilitres. ____________________________________________ ~E~

RADIONUCLIDlC PURITY
Iodine-125
Minimum 99 .0 per cent ofthe total radioactivity.
Gamma-ray and X-ray spectroscopy.
Companson Standardised solution of iodine-125 .
Ammonia C3N) Injection ***
*** ***
Determine the relative amounts of iodine-125 and iodine-126 (Ph Eur monograph 1492) ***
presento PhE~ ____________________________________________

RADIOCHEMICAL PURITY DEFINITION

Iodine-125 in albumin fractions 11 to V, iodine-125
corresponding to unbound iodide
Size-exclusion chromatography (2.2 .30) .
Test solution Mix 0.25 mL of the preparation to be
examined with 0.25 mL of the mobile phase.
Use immediately after mixing.
Sterile solution of [13 N]ammonia for diagnostic use .
Nitrogen-13
90 per cent to 11 O per cent of the declared nitrogen-13
radioactivity at the date and time stated on the labe!'
 IV-650 Radiopharmaceutical Preparations 2014

CHARACTERS Reference soluLÍon Dilute 1.0 mL of dilute ammonia R2 to

Appearance 10.0 mL with water R.
Clear, colourIess solution. Column:
Half-tife and nature of radiation of nitrogen-13 - size: 1 = 0.04 m, 0 = 4.0 mm;
See general chapter 5.7. Table of physical characteristics of - stationary phase: cation exchange resin R (lO ¡.¡m);
radionuclides. - temperature: constant at 20-30 ce.
M obile phase 0.002 M nitric acid.
IDENTIFICATION
A. Gamma-ray spectrometry. Flow rate 2 mUmin.
Resulls The only gamma photons have an energy of DetecLÍon Suirable radioactivity detector and conductivity
0.511 MeV and, depending on the measurement geometry, a detector.
sum peak of 1.022 MeV may be observed. System suitability The chromatogram obtained with the test
B Test A for radionuclidic purity (se e Tests). solution and the radioactivity detector shows a principal peak
with approximately the same retention time as the peak in
e. Examine the chromatograms obtained in the test for the chromatogram obtained with the reference solution and
radiochemical purity (see Tests).
the conductivity detector.
Result The principal peak in the radiochromatogram
Limit:
obtained with the test solution has approximately the same
- ( 3Njammonia: minimum 99 per cent of the total
retention time as the principal peak in the
radioactivity due to nitrogen-13.
radiochromatogram obtained with the reference solution.
RADIOACTIVITY
TESTS
Determine the radioactivity using a calibrated instrument.
pH (2.2.3)
5.5 to 8.5. IMPURITIES
Sterility A. e 3N] 0 2- '
It complies with the test for sterility prescribed in the B. [13Nj03- '
monograph Radiopharmaceutical preparations (0125). e. [1 8F - j,
The preparation may be released for use before completion D . H 2 [l SOj .
of the test. ____________________________________________ ~E~

Bacterial endotoxins (2.6.14)

Less than 175/V IU/mL, V being the maximum
recommended dose in millilitres. The preparation may be
released for use before completion of the test.
Aluminium
Maximum 2 ppm. The preparation may be released for use
before completion of the test.
Test solution In a test-tube about 12 mm in intemal
diameter, mix 1 mL of acetate buffer solution pH 4.6 R and
2 mL of a 1 in 20 dilution of the preparation to be examined
in water R. Add 0.05 mL of a 10 giL solution of chromazurol
SR .
Reference solution Prepare at the same time and in the same
manner as the test solution using 2 mL of a 1 in 20 dilution
of aluminium standard solution (2 ppm Al) R.
After 3 min, the colour of the test solution is not more
intense than that of the reference solution.
RADIONUCLIDIC PURITY
The preparation may be relea sed for use before completion
of tests A and B.
A. Half-lije. The half-life is between 9 min and 11 min.
B. Gamma emitting impurities: maximum 1.0 per cent of the
total radioactivity.
Gamma-ray spectrometry Retain a sample of the preparation
to be examined for 2 h. Examine the gamma-ray spectrum of
the decayed material for the presence of radionuclidic
impurities, which should, where possible, be identified and
quantified .
RADIOCHEMICAL PURITY
13
[ N]Ammonia
Liquid chromatography (2.2.29).
The preparation may be released for use before completion
of the test.
Test solution The preparation to be examined.
Carbon Monoxide eSO) *****
** **
(Ph Bur monograph 1607) ***
~ E~ ____________________________________________
DEFINITION
Mixture of carbon [lSOjmonoxide in the gaseous phase and a
suitable vehicle such as Medicinal air (1238), for diagnostic
use.
Purity:
- minimum 99 per cent of the total radioactivity
corresponds to oxygen-15,
- minimum 97 per cent of the total radioactivity
corresponds to oxygen-15 in the form of carbon
monoxide (CO) .
PRODUCTION
RADIONUCLIDE PRODUCTION
Oxygen-15 is a radioactive iso tope of oxygen which may be
produced by various nuclear reactions such as proton
irradiation of nitrogen-15 or deuteron irradiation of nitro gen-
14.
RADIOCHEMICAL SYNTHESIS
In order to recover oxygen-15 as molecul ar oxygen from the
nitrogen target gas, carrier oxygen is added at concentrations
generaIly ranging from 0.2 per cent V/V to 1.0 per cent VIV.
After irradiation, the target gas is usuaIly reacted with
activated charcoal at a temperature of about 950 D e.
The activated charco al is preconditioned before use by
ftushing an inert gas at the production ftow rate at a
temperature of about 950 oC for not less than 1 h .
The carbon e SOjmonoxide obtained is purified by passage
through a carbon dioxide scavenger, such as soda lime,
before mixing with the vehicle.
 2014
CHARACTERS
Appearance
Colourless gas.
Half-life and nature of radiation of oxygen-15
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICAnON
A. Gamma spectrometry.
Results The only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. Radionuclidic purity (see Tests).
C. Examine the chromatograms obtained in rhe test for
radiochemical purity.
Results The principal peaks in the chromatogram obtained
with the test gas using the radioactivity detector are similar in
retention times to the principal peaks corresponding to
carbon monoxide in the chromatogram obtained with
reference gas (a) using the thermal conductivity detector.
TESTS
The following tests are peifonned on carbon ¡I 5 0 }monoxide as
described under radiochemical synthesis before mixing with the
vehicle.
Carbon monoxide
Gas chromatography (2.2.28) as described in the test for
radiochemical purity.
The concentrarion of carbon monoxide in the test sample is
determined before administration and is used to calculate the
amount of carbon monoxide to be adminisrered to the
patient.
InJection Test sample, reference gas (b).
Examine the chromatogram obtained with the thermal
conductivity detector and calculare rhe content of carbon
monoxide.
RADIONUCLIDIC PURITY
Oxygen-15
Minimum 99 per cent of the total radioactivity.
A. Gamma spectrometry.
Comparison Standardised fiuorine-18 solution, or by using
an instrument calibrated with the aid of such a solurion.
Standardised fiuorine-18 solutions and/or standardisation
services are available from the competent authority.
Results The spectrum obtained with the solution to be
examined does not differ significantly from that obtained
with a standardised fiuorine-18 solution.
B. Half-life: l.9 min to 2.2 mino
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
Carbon [150]monoxide
Gas chromatography (2.2.28): use the normalisation
procedure.
Test sample Carbon ¡i 5 0]monoxide as described under
radiochemical synthesis.
Reference gas (a) Nitrogen gas mixlure R.
Reference gas (b) Nitrogen R, containing 2.0 per cent VIV of
carbon monoxide Rl.
Colwnn:
- size: 1 = 1.8 m, 01 = 6.3 mm and 02 = 3.2 mm,
-- slationary phase: GC concentrical column R,
Radiopharmaceutical Preparations IV-651
Carrier gas helium for chromalOgraphy R.
Flow rale 65 mLJmin.
Temperalure:
-- column: 40 oC,
-- inJection port: 40 cC,
-- lhennal conductivity delector: 70 oc.
Detection Thermal conductivity detector and radioactivity
detector connected in series.
InJection Loop injector.
Run lime 10 mino
Relention limes Oxygen, nitro gen and carbon monoxide
eluting from the inner column = abour 0.4 min; carbon
dioxide eluting from the inner column = about 0.8 min;
oxygen eluting from the outer column = about 2.1 min;
nirrogen eluting from the outer column = about 3.1 min;
carbon monoxide eluting from the outer column = about
6.2 mino
System suilability Reference gas (a):
-- 5 clearly separated principal peaks are observed in the
chromatogram obtained using the thermal conductivity
detector,
-- resolution: minimum of 1.5 between rhe peaks due to
carbon dioxide eluting from the inner column and oxygen
eluting from the outer column, in the chromatogram
obtained using the thermal conductivity detector.
Limits Examine the chromatogram obrained with the
radioactivity detector and calcula te the percentage content of
oxygen-15 substances from the peak areas.
-- carbon ¡I 5 0}monoxide: minimum 97 per cent of the total
radioactivity.
-- disregard the first peak corresponding to components
co-eluting from the inner column.
RADIOACTIVITY
The radio active concentration is determined before
administration.
Measure the radioactivity using suitable equipment by
comparison with a standardised fiuorine-18 solution or by
measurement in an instrument calibrated with the aid of such
a solution.
___________________________________________ ~E~
Chromium (51 Cr) Edetate Injection *****
** **
(Ph Eur monograph 0266) ***
____________________________________________
~E~
DEFINITION
Sterile solution containing chromium-51 in the form of a
complex of chromium(III) with (ethylenedinitrilo)tetraacetic
acid, the latter being present in excess. Ir may be made
isotonic by the addition of sodium chloride and may contain
a suitable antimicrobial preservative such as benzyl alcohol.
 IV-652 Radiopharmaceutical Preparations
Chromium-51
90 per cent to 110 per cent of the dec1ared chromium-51
radioactivity at the date and time stated on the labe!'
Chromium
Maximum 1 mglmL.
CHARACTERS
Appearance
Clear, violet solution.
Half-life and nature of radiation of chromium-51
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENfIFICATION
A. Radionuc1idic purity (se e Tests).
B. Examine the chromatograms obtained in the test for
radiochemical purity (se e Tests).
Result The principal peak in the radiochromatogram
obtained with the test solution is similar in retardation factor
to the principal peak in the chromatogram obtained with the
reference solution.
TESTS
pH (2.2.3)
3.5 to 6.5.
Chromium
Maximum 1 mglmL.
Ultraviolet and visible absorption spectrophotometry (2.2.25) .
Test solution The preparation to be examined.
Reference solution Dissolve 0.96 g of chromic potassium
sulfale R and 2.87 g of sodium edetate R in 50 mL of water R,
boil for 10 min, cool, adjust to pH 3.5-6.5 with dilule sodium
hydroxide solution R and dilute to 100.0 mL with water R.
Measure the absorbance of the test solution and the reference
solution at the absorption maximum at 560 nm.
Result The absorbance of the test solution is not greater
than that of the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIONUCLIDIC PURITY
Chromium-51
Minimum 99.9 per cent of the total radioactivity.
A. Gamma-ray spectrometry.
Results The only gamma photons have an energy of
0.320 MeV.
B. Gamma-ray spectrometry.
Determine the relative amount of radionuc1idic impurities.
Results The total radioactivity due to radionuc1idic
impurities is not more than 0.1 per cent.
RADIOCHEMICAL PURITY
51
[ Cr]Chromium edetate
Descending paper chromatography (2.2.26).
Test solution The preparation to be examined.
Reference solution Use the reference solution from the test
for chromium.
Chromate carrier solution Dissolve 0.1 g of potassium
chromate R in 1 mL of concentrated ammonia R1 and dilute to
100 mL with water R.
Paper paper for chromatography R.
2014
Mobile phase concentrated ammonia R1, ethanol
(96 per cent) R, water R (1:2:5 VIV/V).
Application Apply a band of a 50 giL solution of lead
acetate R to the paper at about 4 cm from the origin and dry
in hot air. Apply 10 ¡.¡L of the chromate carrier solution at
the origin, followed by 10 IlL of the test solution on the same
spot. On a separate sheet, repeat the aboye procedure,
applying 10 ¡.¡L of the reference solution instead of the test
solution.
Development Irnmediately, over a path of 14 cm.
Drying In air.
Detection Suitable detector to determine the distribution of
radioactivity .
Retardation factors Impurity A = O; impurity B = 0.2 to 0.4;
[slCr]chromium edetate = 0.8 to 0.9.
System suitabz7ity The band of lead acetate tums yellow due
to reaction with the chromate carrier solution.
The retardation factor of the radioactive spot due to
[slCr]chromium edetate in the radiochromatogram obtained
with the test solution is similar to that of the violet spot in
the chromatogram obtained with the reference solution.
Limit:
- f 1CrJchromium edetate: minimum 97.0 per cent of the
total radioactivi ty due to chromium-51.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. [slCr]chromium(llI) ion,
B. [slCr]chromate ion .
_ _ _ _ _ _ _ __ _ _ __ _____ _ __ _ _ Ph Eur
Cyanocobalamin (57 CO) Capsules *****
* *
(Ph Eur monograph 0710) **** *
PhE~ _ _ _ __ _ _ _ __ _ __ __ _ __ _ _ __
DEFINITION
e
Capsules containing 7 Co]-cx-(5,6-dimethylbenzimidazol-l-
yl)cobamide cyanide; they may contain suitable excipients.
The capsules comply with the requirements for hard capsules
prescribed in the monograph Capsules (0016), unless
otherwise justified and authorised.
Cobalt-57
90 per cent to 110 per cent of the dec1ared cobalt-57
radioactivity at the date stated on the labe!'
CHARACTERS
Appearance
Hard, gelatin capsules .
Half-life and nature of radiation of cobalt-57
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENfIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of cobalt-57 has
an energy of 0.122 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity (see Tests).
Result The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
2014 Radiopharmaceutical Preparations IV-653

the principal peak in the chromatogram obtained with the

Cyanocobalamin (57 CO) Solution ***
reference solution. *** ***
TESTS (Ph Eur monograph 0269) ***
__________________________________________
Disintegration
~Em

The capsules comply with the test for disintegration of tablets DEFINITION
and capsules (2.9.1), except that 1 capsule is used in the test Solution of [57 Co] -a-(5,6-dimethylbenzimidazol-1-
instead of 6. yl)cobamide cyanide and may contain a stabiliser and an
Uniformity of content antimicrobial preservative.
Determine, by measurement in a suitable counting assembly Cobalt-57
and under identical geometrical conditions, the radioactivity 90 per cent to 110 per cent of the decIared cobalt-57
of each of not fewer than 10 capsules. Calculate the average radioactivity at the date stated on the labe!'
radioactivity per capsule. The radioactivity of no capsule
differs by more than 10 per cent from the average. CHARACTERS
The relative standard deviation is les s than 3.5 per cent. Appearance
Clear, colourless or slightly pink solution.
RADIONUCLIDIC PURITY
Cobalt-57 Half-life and nature of radiation of cobalt-57
See general chapter 5.7. Table of physical characteristics of
Minimum 99.9 per cent of the total radioactivity.
radwnuclides.
Gamma-ray spectrometry.
Determine the relative amounts of cobalt-57, cobalt-56 and IDENfIFICATION
cobalt-58 presento A. Gamma-ray spectrometry.
Result The most prominent gamma photon of cobalt-57 has
RADIOCHEMICAL PURITY
an energy ofO.122 MeV.
[57 Co ]Cyanocobalamin
B. Examine the chromatograms obtained in the test for
Liquid chromatography (2.2.29) .
radiochemical purity (see Tests) .
Test solutwn Dissolve the contents of a capsule in 1.0 mL of
Result The principal peak in the radiochromatogram
water R and allow to stand for 10 mino Centrifuge at 2000
obtained with the test solution is similar in retention time to
r/min for 10 min. Use the supematant.
the principal peak in the chromatogram obtained with the
Reference solutwn Dissolve 10 mg of cyanocobalamin CRS in reference solution.
the mobile phase and dilute to 100 mL with the mobile
phase. Dilute 2 mL of this solution to 100 mL with the TESTS
mobile phase. Use within 1 h of preparation. pH (2.2.3)
Column: 4.0 to 6.0.
- size: 1 = 0.25 m, 0 = 4.0 mm; RADIONUCLIDIC PURITY
- stationary phase: oCl)llsilyl silica gel for chromatography R Cobalt-57
(5 ¡.tm). Minimum 99.9 per cent of the total radioactivity.
Mobile phase 26.5 volumes of methanol R and 73.5 volumes Gamma-ray spectrometry.
of a 10 gIL solution of disodium hydrogen phosphate R adjusted Determine the relative amounts of cobalt-57, cobalt-56 and
to pH 3.5 using phosphoric acid R. Use within 2 days of cobalt-58 presento
preparation.
RADIOCHEMICAL PURITY
Flow rate l.0 mUmin.
[57Co ]Cyanocobalamin
Detection Radioactivity detector adjusted for cobalt-57 and
Liquid chromatography (2.2.29).
spectrophotometer at 361 nm.
Test solutwn The preparation to be examined.
Injection 100 ¡.tL.
Reference solutwn Dissolve 10 mg of cyanocobalamin CRS in
Run time 3 times the retention time of cyanocobalamin for
the mobile phase and dilute to 100 mL with the mobile
the test solution; 30 min for the reference solution.
phase. Dilute 2 mL of this solution to 100 mL with the
Limit:
mobile phase. Use within 1 h after preparation.
- f 7CoJcyanocobalamin: minimum 90 per cent of the total
radioactivity due to cobalt-57 . Column:
- size: 1 = 0.25 m, 0 = 4.0 mm;
RADIOACTIVITY - stationary phase: oCl)llsilyl silica gel for chromatography R
Determine the radioactivity using a calibrated instrumento (5 ¡.tm) .
STORAGE Mobile phase 26.5 volumes of methanol R and 73 .5 volumes
In an airtight container, protected from light, at a of a 10 gIL solution of disodium hydrogen phosphate R adjusted
temperature of 2 oC to 8 oC. to pH 3.5 using phosphoric acid R (use within 2 days after
preparation) .
IMPURITIES
Flow rate l.0 mUmin.
A. cobalt-56,
Detection Radioactivity detector adjusted for cobalt-57 and
B. cobalt-58.
spectrophotometer at 361 nm.
__________________________________________ PhEm
Injection 100 ¡.tL.
Run time 3 times the retention time of cyanocobalamin for
the test solution; 30 min for the reference solution.
IV-654 Radiopharmaceutical Preparations 2014

Limit: Gamma-ray spectrometry.

-- l7Co}cyanocobalamin: minimum 90 per cent of the Determine the relative amounts of cobalt-58, cobalt-57 and
radioactivity due to cobalt-57 . cobalt-60 presento
RADIOACTIVITY Result:
Determine the radioactivity using a calibrated instrumento -- cobalt-60: maximum 1 per cent of the total radioactivity.
STORAGE RADIOCHEMICAL PURITY
S8
Protected from light, at a temperature of 2 oC to 8 oC. [ CoJ Cyanocobalamin
Liquid chromatography (2.2.29).
IMPURITIES
A. cobalt-56, Test solution Dissolve the contents of a cap su le in 1.0 mL of
water R and allow to stand for 10 min. Centrifuge at 2000
B. cobalt-58 .
r/min for 10 min. Use the supernatant.
__________________________________________ ~E~

Reference solution Dissolve 10 mg of cyanocobalamin CRS in

the mobile phase and dilute to 100 mL with the mobile
phase. Dilute 2 mL of this solution to 100 mL with the
mobile phase. Use within 1 h after preparation.
Cyanocobalamin (S8CO) Capsules Column:
-- size: l = 0.25 m, 0 = 4.0 mm;
(Ph Eur monograph 1505) -- stationary phase: octylsilyl silica gel for chromatography R
~E~ __________________________________________ (5 ¡.¡m).
DEFINITION Mobile phase 26.5 volumes of methanol R and 73.5 volumes
Capsules containing [58 CO ]-a -(5 ,6-dimethylbenzimidazol-1- of a 10 gIL solution of disodium hydrogen phosphate R,
yl)cobamide cyanide; they may contain suitable excipients. adjusted to pH 3.5 with phosphoric acid R (use within 2 days).
The capsules comply with the requirements for hard capsules Flow rate 1.0 mUmin.
in the monograph Capsules (0016), unless otherwise justified Detection Radioactivity detector adjusted for cobalt-58 and
and authorised. spectrophotometer at 361 nm.
Cobalt-58 Injection 100 ¡.¡L.
Average between 90 per cent and 110 per cent of the Run time 3 times the retention time of cyanocobalamin for
declared cobalt-58 radioactivity at the date stated on the the test solution; 30 min for the reference solution.
labe!' Limit:
CHARACTERS -- t 8
Co}cyanocobalamin: minimum 84 per cent of the total
Appearance radioactivity due to cobalt-58 .
Hard gelatin capsules. RADIOACTIVITY
Half-life and nature of radiation of cobalt-58 Determine the radioactivity using a calibrated instrumento
See general chapter 5.7. Table of physical characten'stics of STORAGE
radionuclides. In an airtight container, protected from light, at a
IDENTIFICATION temperature of 2 oC to 8 oc.
A. Gamma-ray spectrometry. IMPURITIES
Results The most prominent gamma photons of cobalt-58 A. cobalt-57,
have energies of 0.511 MeV (annihilation radiation) and B. cobalt-60.
0.811 MeV. __________________________________________ ~E~

B. Examine the chromatograms obtained in the test for

radiochemical purity (se e Tests).
Result The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
Cyanocobalamin (S8CO) Solution ***
*** ***
the principal peak in the chromatogram obtained with the
reference solution.
(Ph Eur monograph 0270) ***
TESTS ~E~ __________________________________________
Disintegration
The capsules comply with the test for disintegration of tablets DEFINITION
and capsules (2.9.1) except that 1 capsule is used in the test Solution of [58 Co]-a-(5,6-dimethylbenzimidazol-l-
instead of 6. yl)cobamide cyanide and may contain a stabiliser and an
antimicrobial preservative.
Uniforrnity of content
Determine by measurement in a suitable counting assembly Cobalt-58
and under identical geometrical conditions the radioactivity 90 per cent to 110 per cent of the declared cobalt-58
of each of not less than 10 capsules. Calculate the average radioactivity at the date stated on the labe!'
radioactivity per capsule. The radioactivity of no capsule CHARACTERS
differs by more than 10 per cent from the average. Appearance
The relative standard deviation is 1ess than 3.5 per cent. Clear, colourless or slightly pink solution.
RADIONUCLIDIC PURITY Half-life and nature of radiation of cobalt-58
Cobalt-58 See general chapter 5.7. Table of physical characten'stics of
Minimum 98 per cent of the total radioactivity. radionuclides.
2014 Radiopharmaceutical Preparations IV-655

IDENTIFICATION
A. Gamma-ray spectrometry.
Fludeoxyglucose C8F) Injection *****
* *
(Ph Bur monograph 1325) *****
Results The most prominent gamma photons of cobalt-58
have energies of 0.511 MeV (annihilation radiation) and
0.811 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity (se e Tests) . H°1;O, OH
Result The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to H~ 18F
the principal peak in the chromatogram obtained with the
reference solution.
PhE~ ___________________________________________
TESTS
pH (2.2. 3) DEPINITION
4.0 to 6.0. Sterile solution containing 2-¡I SPlfluoro-2-deoxY-D-
RADIONUCLIDIC PURITY glucopyranose (2-[l SPlfluoro-2-deoXY-D-glucose) prepared by
nucleophilic substitution. It may also contain 2-¡I sF]fluoro-2-
Cobalt-58
deoXY-D-mannose.
Minimum 98 per cent of the total radioactivity.
Pluorine-18
Gamma-ray spectrometry.
90 per cent to 110 per cent of the declared fluorine-18
Determine the relative amounts of cobalt-58, cobalt-57 and radioactivity at the date and time stated on the labe!.
cobalt-60 presento
2-Pluoro-2-deoxy-d-glucose
Result:
Maximum 0.5 mg per maximum recommended dose, in
-- cobalt-60: maximum 1 per cent of the total radioactivity.
millilitres.
RADIOCHEMICAL PURITY
58
CHARACTERS
[ CO ]Cyanocobalamin Appearance
Liquid chromatography (2.2.29). Clear, colourless or slightly yellow solution.
Test solurion The preparation to be examined.
Half-tife and nature ofradiation offtuorine-18
Reference solution Dissolve 10 mg of cyanocobalamin CRS in See general chapter 5.7. Table of physical characteristics of
the mobile phase and dilute to 100 mL with the mobile radionuclides.
phase. Dilute 2 mL of this solution to 100 mL with the
mobile phase. Use within 1 h after preparation. IDENTIFICATION
A. Test A for radionuclidic purity (see Tests).
Column:
-- size: l = 0.25 m, 0 = 4.0 mm; B. Determine the approximate half-life by no fewer than 3
-- stationary phase: octylsi1yl silica gel for chromatography R measurements of the activity of a sample in the same
(5 ¡.1m). geometrical conditions within a suitable period of time (for
Mobile phase 26.5 volumes of methanol R and 73 .5 volumes example, 30 min).
of a 10 gIL solution of disodium hydrogen phosphate R adjusted Result 105 to 115 mino
to pH 3.5 using phosphoric acid R (use within 2 days). C. Examine the chromatograms obtained in test A for
Flow rate 1.0 mUmin. radiochemical purity (see Tests).
Detection Radioactivity detector adjusted for cobalt-58 and Result The principal peak in the radiochromatogram
spectrophotometer at 361 nm. obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
InJection 100 ¡.IL.
reference solution (a).
Run time 3 times the retention time of cyanocobalamin for
the test solution; 30 min for the reference solution. TESTS
Limit: Particular tests for chemical impurities may be omitted if the
-- l8Cojcyanocobalamin: minimum 90 per cent of the substances mentioned are not used or cannot be form ed in the
radioactivity due to cobalt-58. production process.
RADIOACTIVITY pH (2.2.3)
Determine the radioactivity using a calibrated instrumento 4.5 to 8.5.
2-Pluoro-2-deoxy-d-glucose and impurity A
STORAGE Liquid chromatography (2.2.29).
Protected from light, at a temperature of 2 oC to 8 oc.
Test solurion The preparation to be examined.
IMPURITIES Reference solution (a) Dissolve 1.0 mg of 2-fiuoro-2 -deoxY-D-
A. cobalt-57, glucose R in water R and dilute to 2.0 mL with the same
B. cobalt-60. solvento Dilute 1.0 mL of the solution to V with water R, V
____________________________________________ ~E~ being the maximum recommended dose in millilitres .
Reference solution (b) Dissolve 1.0 mg of 2-chloro-2-deoxY-D-
glucose R (impurity A) in water R and dilute to 2.0 mL with
the same solvento Dilute 1.0 mL of the solution to V with
water R, V being the maximum recommended dose in
millilitres.
 IV-656 Radiopharmaceutical Preparations
Referenee solution (e) Dissolve 1.0 mg of 2-fiuoro-2-deoXY-D-
mannose R in water R and dilute to 2.0 mL with the same
solvent. Mix 0.5 mL of this solution with 0.5 mL of
reference solution (a).
Column:
- size: 1 = 0.25 m, 0 = 4.0 mm;
- stationary phase: strongly basie anion exehange resin for
ehromatography R (10 ¡.tm);
- temperature: constant, between 20-25 oc.
Mobile phase 4 gIL solution of sodium hydroxide R in earbon
dioxide-free water R.
Flow rate 1 mUmin.
DeteetionDetector suitable for carbohydrates in the required
concentration range, such as pulse amperometric detector
and radioactivity detector connected in series.
Injection 20 ¡.tL.
Run time Twice the retention time of 2-fluoro-2-deoxY-D-
glucose.
Relative retention With reference to 2-fluoro-2-deoXY-D-
glucose (retention time = about 12 min): 2-fluoro-2-deoXY-D-
mannose = about 0.9; impurity A = about 1.1.
System suitability Reference solution (c), using the
carbohydrate detector:
- resolution: minimum 1.5 between the peaks due to
2-fluoro-2-deoXY-D-mannose and 2-fluoro-2-deoXY-D-
glucose;
- signal-to-noise ratio: minimum 10 for the peak due to
2-fluoro-2-deoXY-D-glucose.
Limits In the chromatogram obtained with the carbohydrate
detector:
- 2-fiuoro-2-deoxy-n-glucose: not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (a) (0.5 mglV);
- impuriry A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.5 mglV) .
Impurity B
Spot test.
Test solution The preparation to be examined.
Reference solution (a) water R.
Referenee solution (b) Dissolve 11.0 mg of aminopolyether R
(impurity B) in water R and dilute to 5.0 mL with the same
solvent. Dilute 1 mL of the solution to V with water R, V
being the maximum recommended dos e in millilitres.
Plate TLC siliea gel plate for aminopolyether test R.
Application 2.5 ¡.tL; in addition, apply 2.5 ¡.tL of the test
solution and then 2.5 ¡.tL of reference solution (b) at the
same place.
Detection Visually compare the spots 1 min after
application.
System suitability:
- the spot due to the successive application of the test
solution and reference solution (b) is similar in
appearance to the spot due to reference solution (b),
which is characterised by a number of concentric cirdes;
the darker innermost cirde (of intensity proportional to
the concentration of impurity B) may be surrounded by a
bluish-black ring, outside of which is a Iighter cirde
surrounded by a peripheral dark edge;
- the spot due to reference solution (a) has a more diffuse
inner cirde, which is brownish-pink and without a distinct
margin between it and the surrounding lighter zone;
2014
- the spot due to reference solution (b) is c1earIy different
from the spot due to reference solution (a).
Limit:
- the central portion of the spot due to the test solution is
less intense than that of the spot due to reference solution
(b) (2.2 mglV).
ImpurityC
Liquid chromatography (2.2.29) .
Test solution The preparation to be examined .
Reference solution (a) Dilute 2.1 mL of a 25.95 giL solution
of tetraburylammonium hydroxide R (impurity C) to 20.0 mL
with water R. Dilute 1.0 mL of this solution to V with
water R, V being the maximum recommended dose in
millilitres.
Reference solution (b) Dilute 1 mL of a 25.95 gIL solution of
tetraburylammonium hydroxide R to 10 mL with water R .
Dilute 1 mL of this solution to 25 mL with water R.
Column:
- size: 1 = 0.10 m, 0 = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(3 ¡.tm);
- temperature: constant, between 20-25 oc.
Mobz7e phase 25 volumes of a 0.95 gIL solution of
toluenesulfonic acid R and 75 volumes of acetonitrile R.
Flow rate 0.6 mUmin.
Detection Spectrophotometer at 254 nm.
Injection 20 ¡.tL.
Run time Twice the retention time of tetrabutylammonium
ions.
R etention time tetrabutylammonium hydroxide = about
3.3 mino
System suitability Reference solution (b):
- signal-to-noise ratio: minimum 10 for the principal peak;
- symmetry factor: maximum 1.8 for the principal peak.
Limit:
- impurity C: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (2.75 mglV).
Impurity D
Maximum 0.02 mglV.
Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solution The preparation to be examined.
Reference solution Dissolve 20.0 mg of 4- (4-methylpiperidin- l-
yl)pyridine R (impurity D) in water R and dilute to 100.0 mL
with the same solvent. Dilute 0.1 mL of the solution to V
with water R, V being the maximum recommended dose in
milliIitres.
Measure the absorbance of the test solution and the reference
solution at the absorption maximum of 263 nm.
Result The absorbance of the test solution is not greater
than that of the reference solution.
Residual solvents
Limited according to the principIes defined in general
chapter 5.4. The preparation may be released for use before
completion of the test.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaeeutical preparations (0 125).
The preparation may be released for use before completion
of the test.
 2014 Radiopharmaceutical Preparations IV-657

Bacterial endotoxins (2.6.14) derivatives of 2-C sFlfiuoro-2-deoxy-o-glucose and

Less than 175/ V IU/mL, V being the maximum
recommended dose in millilitres. The preparation may be
released for use before completion of the test.
RADIONUCLIDIC PURITY
The preparation may be released for use before completion
of test B.
A. Gamma-ray spectrometry.
R esults The only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. Gamma-ray spectrometry.
Determine the amount of fiuorine-l8 and radionuclidic
impurities with a half-life longer than 2 h . For the detection
and quantification of impurities, reta in the preparation to be
examined for at least 24 h to allow the fiuorine-18 to decay
to a level that permits the detection of impurities.
Results The total radioactivity due to radionuclidic
impurities is not more than 0.1 per cent.
RADIOCHEMICAL PURITY
A. Liquid chromatography (2.2.29) as described in the test
2-CsFlfiuoro-2-deoXY-D-ma=ose = about 0.8 to 0.95.
System suitability Reference solution:
-- the ehromatogram shows 2 c1early separated spots.
Limits:
-- ( 8Plfiuon"ne in the jorm oj 2-(8Plfiuoro-2-deoxY-D-glucose
and 2-(8Plfiuoro-2-deoxY-D-mannose: minimum
95 per cent of the total radioactivity due to fiuorine-18;
-- ( 8Plfiuorine in the jonn oj fiuonde and partially or jully
acetylated derivatives oj 2-(8Plfiuoro-2-deoxY-D-glucose and
2-(8Plfiuoro-2-deoxY-D-mannose: maximum 5 per eent of
the total radioactivity due to fiuorine-18.
RADIOACTIVITY
Determine the radioaetivity using a ealibrated instrumento
LABELLING
The label states the maximum recommended dose, in
millilitres.
IMPURITIES
Specified impurities A, B, C, D, E.

HO~O,
for 2-fiuoro-2-deoxY-D-glucose and impurity A. If necessary,
dilute the test solution with water R to obtain a radioactivity
OH
concentration suitable for the radioactivity detector.
Injection Test solution and reference solutions (a) and (c). HfL{
R elative retention With reference to 2-C sFlfiuoro-2-deoxY-D- el
glucose (retention time = about 12 min): 2-C sFlfiuoro-2-
deoxy-o-ma=ose = about 0.9. Partially or fully acetylated
A. 2-chloro-2-deoXY-D-glucopyranose (2-chloro-2-deoXY-D-
derivatives of both compounds hydrolyse under the
glucose),
chromatographic conditions and therefore elute as
2-CsFlfiuoro-2-deoXY-D-glucose and 2-[ lSFlfiuoro-2-deoXY-D-
mannose.
Locate the peaks due to 2-CsFlfiuoro-2-deoxy-o-glueose and
2-[ lSFlfiuoro-2-deoxy-o-ma=ose using the chromatograms
obtained with the carbohydrate detector and referenee
solutions (a) and (e).
L imits:
-- ( 8Plfiuorine in the jorm oj 2-(8Plfiuoro-2-deoxY-D-glucose
and 2-(8Plfiuoro-2-deoxY-D-mannose: minimum
95 per cent of the total radioaetivity due to fiuorine-18; B.4,7,l3,l6,2l,24-hexaoxa-l,10-
2-(8Plfiuoro-2-deoxY-D-mannose: maximum 10 per cent of
diazabicyclo[8 .8.8lhexaeosane (aminopolyether),
the total radioactivity due to 2-C sFlfiuoro-2-deoxY-D-
glueose and 2-[ l SFlfiuoro-2-deoxY-D-mannose.
B. Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
R ejerence solution Dissolve, with gentle heating, 30 mg of
1,2,3,4-tetra-O-acetyl-fi-D-glucopyranose R and 20 mg of C. N,N,N-tributylbutan-l-aminium (tetrabutylammonium),
glucose R in 1 mL of water R.
Plate TLC siliea gel plate R.
Mobile phase water R, acetonitrile R (5:95 V/V).
Application About 2 ¡.¡L.
Development Over a path of 8 cm.
Drying In air for 15 mino
Detection Suitable detector to determine the distribution of D . 4-( 4-methylpiperidin-l-yl)pyridine,
radioaetivity; immerse the plate in a 75 gIL solution of
E. CSFlfiuoride.
sulfuric acid R in methanol R and dry with a heat gun or at
____________________________________________ PhE~

150 oC until the appearance of dark spots in the

ehromatogram obtained with the reference solution.
ls
Retardation jactors [ Flfiuoride = about O; 2-C sFlfiuoro-2-
deoXY-D-glueose and 2-[ lS Flfiuoro-2-deoXY-D-
mannose = about 0.45; partially or fully aeetylated
IV-658 Radiopharmaceutical Preparations
Flumazenil (J\LC 1C]methyl) *****
** **
Injection ***
(Ph Eur monograph 1917)
~Eif ____________________________________________
DEFINITION
Sterile solution of ethyl 8-fluoro-5-e lC]methyl-6-oxo-5,6-
dihydro-4H-imidazo[I,5-a] [1,4]benzodiazepine-3-carboxylate
which may contain a stabiliser such as ascorbic acid.
Content
90 per cent to 110 per cent of the declared carbon-ll
radioactivity at the date and time stated on the labe!'
Content of flumazenil
Maximum 50 ¡.¡g in the maximum recommended dose in
millili tres.
PRODUCTION
RADIONUCLIDE PRODUCTION
Carbon-ll is a radio active iso tope of carbon which is most
commonly produced by proton irradiation of nitrogen.
Depending on the addition of either trace amounts of oxygen
or small amounts of hydrogen, the radioactivity is obtained as
e1C]carbon di oxide or [llC]methane, respectively.
RADIOCHEMICAL SYNTHESIS
[5-Methyl-llC]flumazenil may be prepared by N-alkylation of
ethyl 8-fluoro-6-oxo-5 ,6-dihydro-4H-imidazo [1 ,5-
a] [1,4] benzodiazepine-3-carboxylate (demethylflumazenil)
with iodo[llC]methane or [llC]methyl
trifluoromethanesulfonate.
Synthesis of iodoe lC]methane
Iodoe1C]methane may be produced from [llC]carbon
dioxide or from [llC]methane. The most frequently used
method is reduction of e lC]carbon dioxide with lithium
aluminium hydride. The [ll C]methanolate formed is reacted
with hydriodic acid. Alternatively [llC]methane, either
obtained directly in the target or by on-line processes from
[1lC]carbon dioxide, is reacted with iodine.
Synthesis of [IlC]methyl trifluoromethanesulfonate
[1lC]methyl trifluoromethanesulfonate may be prepared from
iodo[llC]methane using a solid support such as graphitised
carbon, impregnated with silver trifluoromethanesulfonate.
Synthesis of [5-methyl-llC]flumazenil
The most widely used method to obtain
[5-methyl-llC]flumazenil is the N-alkylation of
demethylflumazenil with iodo[llC]methane in alkaline
conditions in a solvent such as dimethylforrnamide or
acetone. The resulting [5-methyl-llC]flumazenil can be
purified by semi-preparative liquid chromatography.
For example, a column packed with octadecylsilyl silica gel
for chromatography eluted with a mixture of ethanol and
water is suitable.
PRECURSOR FOR SYNTHESIS
Demethylflumazenil
Melting point (2.2.14): 286 oC to 289 oc.
2014
Infrared absorption spectrophotometry (2.2.24).
Comparison Ph. Eur. referenee spectrum of demethylfiumazenil.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of carbon-11
See general chapter 5.7. Table of physieal eharacteristies of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The only gamma photons have an energy of
O.511 MeV and, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. It complies with test B for radionuclidic purity (see Tests).
C. Examine the chromatograms obtained in the test for
radiochemical purity.
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
pH (2.2.3)
6.0 to 8.0.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaeeutieal preparations (0125) .
The injection may be released for use before completion of
the test.
Bacterial endotoxins (2.6.14)
Less than 175/V IU/rnL, V being the maximum
recommended dose in millilitres. The injection may be
released for use before completion of the test.
Flumazenil and impurity A
Liquid chromatography (2.2.29).
Test solution The preparation to be examined.
R eferenee solution (a) Dissolve 2.5 mg ofjlumazenil R in
5 mL of methanol R .
Referenee solution (b) Dissolve 2.5 mg of
demethylfiumazenil R in 50 mL of methanol R.
R eferenee solution (e) To 0.1 mL ofreference solution (a)
add 0.1 mL of reference solution (b) and dilute to V with a
0.9 gIL solution of sodium ehloride R, Vbeing the maximum
recommended dose in millilitres.
Referenee solution (d) Dilute 0.1 mL of reference solution (a)
to 50 mL with methanol R. Dilute 1.0 mL of this solution to
V with a 0.9 gIL solution of sodium ehloride R , V being the
maximum recommended dose in millilitres.
Column:
- size: 1 = 0.15 m, 0 = 3.9 mm,
- stationary phase: spherical octadeeylsilyl siliea gel for
ehromatography R (5 ~lm) with a specific surface area of
440 m 2/g, a pore size of 100 nm and a carbon loading of
19 per cent,
- temperature: maintain at a constant temperature between
20-30 oc.
Mobile phase methanol R, water R (45:55 V/V).
Flow rate 1 mUmin.
Deteetion Spectrophotometer at 260 nm and radioactivity
detector connected in series.
Injeetion 100 ¡.¡L.
Run time 10 mino
2014 Radiopharmaceutical Preparations IV-659

Relative retentíon With reference to flumazenil:

Fluoride C8F) Solution for *****
impurity A = about 0.74. ** **
System suitability Reference solution (c) : Radiolabelling ***
-- resolution: minimum 2.5 between the peaks due to (Ph Bur monograph 2390)
flumazenil and impurity A. ~E~ ____________________________________________
Limits Examine the chromatogram obtained with the
DEFINITION
spectrophotometer:
Alkaline solution containing fiuorine-18 in the form of
-- flumazenil: not more than the area of the corresponding
eSF]fluoride.
peak in the chromatogram obtained with reference
solution (c) (50 flg/V), Content
-- impurity A: not more than the area of the corresponding 90 per cent to 110 per cent of the declared fluorine-18
peak in the chromatogram obtained with reference radioactivity at the date and time stated on the labe!'
solution (c) (5 flg/V), CHARACTERS
-- any other impurity: not more than the area of the principal Appearance
peak in the chromatogram obtained with reference Clear, colourless solution.
solution (d) (1 flg/V).
Half-life and nature ofradiation offluorine-18
Residual solvents See general chapter 5.7. Table oi physical characteristics oi
Are lirnited according to the principies defined in the general radionuclides.
chapter (5.4), using the general method (2.4.24).
The preparation may be released for use before completion IDENTIFICATION
of the test. A. Gamma-ray spectrometry.
RADIONUCLIDIC PURITY Result The principal photons have an energy of 0.511 MeV
and, depending on the measurement geometry, a sum peak
Carbon-ll
of 1.022 MeV may be observed.
Minimum 99 per cent of the total radioactivity.
B. Determine the approximate half-life by no fewer than 3
The preparation may be released for use before completion
measurements of the activity of a sample in the same
of the test. geometrical conditions within a suitable period of time (for
A. Gamma-ray spectrometry. example, 30 min).
Results The spectrum obtained with the solution to be Result 105 min to 115 mino
examined does not differ significantly from that obtained e. Examine the chromatograms obtained in the test for
with a standardised fluorine-18 solution. radiochemical purity (se e Tests) .
B. Half-life: 19.9 min to 20.9 mino
Results The principal peak in the radiochromatogram
RADIOCHEMICAL PURlTY obtained with the test solution is similar in retention time to
Liquid chromatography (2.2.29) as described in the test for the principal peak in the chromatogram obtained with the
flumazenil and impurity A, with the following modifications. reference solution; in the chromatogram obtained with the
Injection Test solution and reference solution (a); reference solution, the signal due to fiuoride is negative.
if necessary, dilute the test solution to a radioactivity TESTS
concentration suitable for the detector. pH
Limit Examine the chromatogram obtained with the 8.0 to 14.0, using a pH indicator strip R.
radioactivity detector:
Bacterial endotoxins (2.6.14)
-- [5-methyl- l1 Clflumazenil: minimum 95 per cent ofthe
Less than 20 IV/mL, if intended for use in the manufacture
total radioactivity.
of parenteral preparations without a further appropriate
RADIOACTIVITY procedure for the removal of bacterial endotoxins.
Determine the radioactivity using a calibrated instrumento The preparation may be released for use before completion
LABELLING of the test.
The labe! states the maxirnum recommended dose in RADIONUCLIDIC PURITY
millilitres. The preparation may be released for use before completion
of test B.
IMPURlTIES
Fluorine-18
(~N o
Minimum 99.9 per cent ofthe total radioactivity.

RX:X;N
I o~ j
CH 3
A. Gamma-ray spectrometry. Preliminary test.
Limit Peaks in the gamma spectrum corresponding to
F "'" NH photons with an energy different from 0.511 MeV or
o l.022 MeV represent not more than 0.1 per cent ofthe total
radioactivity.
B. Gamma-ray spectrometry.
A. R = H : ethyl 8-fiuoro-6-oxo-5,6-dihydro-4H-imidazo[1,5-
Determine the amount of fiuorine-18 and radionuclidic
a] [1 A]benzodiazepine-3-carboxylate (demethylfiumazenil),
impurities with a half-life longer than 2 h. For the detection
B. R = CHz-CO-CH3: ethyl 8-fiuoro-6-oxo-9-(2-oxopropyl)- and quantification of impurities, retain the preparation to be
5,6-dihydro-4H-irnidazo[1,5-a] [1,4]benzodiazepine-3- examined for at least 24 h to allow the fiuorine-18 to decay
carboxylate (acetone addition compound of to a level that permits the detection of impurities.
demethylflumazenil) .
Result The total radioactivity due to radionuclidic impurities
____________________________________________ ~E~
is not more than 0.1 per cent.
 IV-660 Radiopharmaceutical Preparations
RADIOCHEMICAL PURITY
[ 18F]fluoride
Liquid chromatography (2.2.29).
Test solution Dilute the preparation to be examined with
water R to obtain a radioactivity concentration suitable for
the radioactivity detector.
Reference solution Dissolve 10 mg of potassium fiuoride R in
water R and dilute to 10 mL with the same solvent.
Column:
-- size: 1 = 0.25 m, 0 = 4.0 mm;
-- stationary phase: strongly basic anion-exchange resin for
chromatography R (lO ¡tm) .
Mobile phase 4 gIL solution of sodium hydroxide R in carbon
dioxide-free water R, protected from atrnospheric carbon
dioxide.
Plow rate 1 mlJmin.
Detection Spectrophotometer at 220 nm and a radioactivity
detector connected in series.
Injection 20 ¡tL.
Run time 12 mino
System suitability Reference solution:
-- signal-to-noise ratio: minimum 10 for the principal peak;
-- retention time of fiuon'de: minimum 3 times the hold-up
time.
Examine the chromatogram obtained with the test solution
using the radioactivity detector and locate the peak due to
fiuoride by comparison with the chromatogram obtained with
the reference solution using the spectrophotometer.
Limit:
-- ¡JBpJfiuoride: minimum 98.5 per cent of the total
radioactivity due to fiuorine-18.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label states:
-- that the solution is not for direct administration to
humans;
-- where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
____________________________________________ PhE~
Fluorodopa C8F) Injection ***
*** ***
(PluorodiJpa ( Bp) (Prepared By Electrophilic ***
Substitution) Injection, Ph Eur monograph 1918)
____________________________________________
~E~
DEFINITION
Sterile solution of (2S)-2-amino-3-(2-([ lSF)fiuoro)-4,5-
dihydroxyphenyl)propanoic acid (6- [lsF) fiuorolevodopa).
It may contain stabilisers such as ascorbic acid and edetic
acid.
2014
This monograph applies to an injection containing
6-[ ls F)fiuorolevodopa produced by electrophilic substitution.
Content:
-- fiuorine-18: 90 per cent to 110 per cent of the declared
fiuorine-18 radioactivity at the date and time stated on
the rabel;
-- dopa: maximum 1 mg per maximum recommended dos e
in millilitres;
-- 6-fiuorolevodiJpa: maximum 15 mg per maximum
recommended dose in millilitres.
PRODUCTION
RADIONUCLIDE PRODUCTION
Fluorine-18 is a radioactive iso tope of fiuorine that may be
produced by various nuclear reactions induced by proton
irradiation of oxygen-18, deuteron irradiation of neon-20, or
helium-3 or helium-4 irradiation of oxygen-16.
In order to obtain fiuorine-18 in a chemical form suitable for
electrophilic substitution reactions, such as fiuorine gas or
gaseous acetylhypofiuorite, a small amount of non-radioactive
fiuorine gas (0.3-0.8 per cent of the target gas volume) must
be added as a carrier at sorne step in the production process.
RADIOCHEMICAL SYNTHESIS
6-¡IsF]Fluorolevodopa may be prepared by various
radiochemical synthetic pathways, which lead to different
products in terms of yield, specific radioactivity, by-products
and possible impurities. Electrophilic pathways for
production of 6-¡I sF)fiuorolevodopa may proceed by
fiuorodemetallation of a stannylated derivative of levodopa,
with molecular ¡IsF]fiuorine or ¡IsF)acetylhypofiuorite,
followed by hydrolysis of protecting groups and final
purification by semipreparative liquid chromatography.
Pathways using demercuration or dethallation must not be
used.
CHARACTERS
Appearance
Clear, colourless solution.
Half-Jife and nature of radiation of fluorine-18
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Test A for radionuclidic purity (se e Tests).
B. Determine the approximate half-life by at least 3
measurements of the activity of a sample in the same
geometrical conditions over a suitable period of time, for
example 30 mino
Results 105 min to 115 mino
C . Examine the chromatograms obtained in the test for
radiochemical purity (se e Tests).
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the peak due to 6-fiuorolevodopa in the chromatogram
obtained with reference solution (a).
D. Examine the chromatograms obtained in the test for
impurities C and D (see Tests).
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retardation factor
to the peak due to 6-fiuorolevodopa in the chromatogram
obtained with reference solution (b) .
TESTS
pH (2.2.3)
4.0 to 5.5.
 2014
Sterility
It complies with !he test for sterility prescribed in !he
monograph Radiophannaceutical preparations (0125).
The injection may be relea sed for use before completion of
the test.
Bacteria! endotoxins (2.6.14)
Less than 175/V IV/mL, V being the maximum
recommended dose in millilitres. The injection may be
released for use before completion of!he test.
6-Fluorolevodopa, dopa, impurity A and impurity B
Liquid chromatography (2.2.29). Prepare the reference solutions
immediately before use.
Test solution The preparation to be examined.
Reference solurion (a) Dissolve 18.0 mg of 6-fiuorolevodopa
hydrochloride R in 5.0 mL of!he mobile phase and dilute to V
with the m obile phase, V being the maximum recommended
dose in milJilitres.
Reference solution (b) Dissolve 1.0 mg of levodopa R in 5 mL
of!he mobile phase and dilute to V with the mobile phase, V
being !he maximum recommended dose in millilitres.
Referenee solution (e) Dissolve 1.0 mg of trimethyltin
ehloride R (impurity A) in 2.0 mL of!he mobile phase. Dilute
1.0 mL of this solution to V with !he mobile phase, V being
the maximum recommended dose in milJilitres.
Reference solution (d) Mix equal volumes of reference
solutions (b) and (c).
Reference solution (e) Dissolve 2.0 mg of 6-hydroxydopa R
(impurity B) in 20.0 mL of the mobile phase . Dilute
0.25 mL of this solution to V with the mobile phase, V being
the maximum recommended dose in milJilitres.
Column:
- size: 1 = 0.25 m, 0 = 4.0 mm;
- stationQ1Y phase: spherical end-capped oetadecylsilyl siliea gel
for ehromatography R;
- temperacure: maintain at a constant temperature between
20 °e and 30 ce.
Mobile phase 6.9 gIL solution of sodium dihydrogen
phosphate R adjusted to pH 2.4 with a 4.8 gIL solution of
phosphoric acid R.
Plow rate 1 mUmin.
Deteaion Spectrophotometer at 200 nm and radioactivity
detector connected in series.
Injeetion 20 ilL.
Run time 15 mino
Relative retention With reference to 6-fiuorolevodopa
(retention time = about 6 min):
impurity A and impurity B = about 0.7; dopa = about 0.8.
System suitability Reference solution (d):
- resolution: minimum 1.5 between !he peaks due to dopa
and impurity A.
Limits Examine !he chromatograms obtained wi!h !he
spectrophotometer:
- 6-:fluorolevodopa: not more than the area of!he
corresponding peak in !he chromatogram obtained wi!h
reference solution (a) (15 mg/V);
- dopa: not more !han !he area of the peak due to levodopa
in the chromatogram obtained with reference solution (b)
( 1.0 mg/V);
- sum of impurities A and B: not more than the area of!he
principal peak in the chromatogram obtained with
reference solution (e) (corresponding to a limit of
0.5 mg/V of impurity A or a limit of 0.025 mg/Vof
Radiopharmaceutical Preparations IV-661
impurity B, or to lower limits of each if bo!h impurities
are present) .
Residual solvents
Limited according to !he principies defined in general
chapter 5.4. The preparation may be released for use before
completion of!he test.
RADIONUCLIDIC PURITY
Fluorine-18
Minimum 99.9 per cent ofthe total radioactivity.
The preparation may be released for use before completion
of test B.
A. Gamma-ray spectrometry
Results The only gamma photons have an energy of
0.511 MeV and, depending on !he measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. Gamma-ray spectrometry
Determine the amount of fiuorine-18 and radionuc1idic
impurities with a half-life longer !han 2 h. For !he detection
and quantification of impurities, retain !he preparation to be
examined for a sufficient time to alJow the fiuorine-18 to
decay to a level that permits the detection of impurities.
Results The spectrum obtained wi!h !he preparation to be
examined does not differ significantly from a background
spectrum.
RADIOCHEMICAL PURITY
Liquid chromatography (2.2.29) as described in !he test for
6-fiuorolevodopa, dopa, impurity A and impurity B.
Examine the chromatogram recorded using the radioactivity
detector and locate !he peak due to 6-C 8 F]fiuorolevodopa by
comparison wi!h !he chromatogram obtained with reference
solution (a) and !he spectrophotometer.
Limit:
- 6-¡J8Plfiuorolevodopa: minimum 95 per cent of the total
radioactivity due to fiuorine-18 .
Impurities e and D
Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Referenee solution (a) Dissolve 2 mg of DL-6-fiuorodopa
hydroehloride R in water R and dilute to 10 mL with the same
solvent.
Referenee solurion (b) Dissolve 2 mg of 6-fiuorolevodopa
hydroehloride R in water R and dilute to 10 mL with the same
solvent.
Plate oetadecylsilyl TLC siliea gel plate for ehiral separations R.
Mobile phase methanol R, water R (50:50 V/V).
Application 2 ilL.
Development Over a path of 10 cm.
Drying In air for 5 min.
Deteetion Spray wi!h a 2 gIL solution of ninhydrin R in
anhydrous ethanol R and heat at 60 oC for 10 min; determine
the distribution of radioactivity using a suitable detector.
Retardation factors Impurity D = about O;
6-[ 18 F]fiuorolevodopa = about 0.3; impurity e = about 0.5 .
System suitability Reference solution (a):
- !he chromatogram shows 2 c1early separated spots.
Limits:
- impurity C: maximurn 2 per cent of the total radioactivity
due to fiuorine-18;
- impurity D: maximum 4 per cent of!he total radioactivity
due to fiuorine-18 .
IV-662 Radiopharmaceutical Preparations 2014

RADIOACTIVITY TESTS
Measure the radioactivity using a calibrated instrumento pH (2,2,3)
5,0 to 8,0,
LABELLING
The label states the maximum recommended dose in Zinc
millilitres. Maximum 5 ppm.
Test solution To 0,1 mL of the preparation to be examined
IMPURITIES
add 0,9 mL of water R, 1 mL of a 250 giL solution of sodium
A. CI-Sn(CH 3h chlorotrimethylstannane (trimethyltin
thiosulfate R, 5 mL of acetate buffer solution pH 4,7 R and
chloride),
5,0 mL of a dithizone solution prepared as follows: dissolve

HO
0 ,
OH

1.-9'
C0 H
H 'NH
2
2
and enantiomer
°
1 mg of dithizone R in 100 mL of methyl ethyl ketone R allow
to stand for 5 min, filter and irnmediarely before use dilute
the solution to 10 times its volume with methyl ethyl ketone R .
Shake vigorously for 2 min and allow the organic layer to
separare,
OH Rejerence solution 0.1 mL of zinc standard solution (5 ppm
Zn) R treated in the same manner as the test solution,
B, (2RS)-2-amino-3-(2,4,5-trihydroxyphenyl)propanoic acid Measure the absorbance (2.2.25) of the organic layers at
(6-hydroxydopa), 530 nm, using the organic layer of a blank solution as the
compensation liquid,
Results The absorbance of the organic layer obtained with
the test solution is not greater than that of the organic layer
obtained with the reference solution,
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125) ,
The preparation may be relea sed for use before completion
C. (2R)-2-amino-3-(2-¡I SF]fiuoro-4,5- of the test,
dihydroxyphenyl)propanoic acid (6-¡I 8F]fiuorodextrodopa),
RADIONUCLIDIC PURITY
D, ¡ISF]fiuoride,
__________________________________________ ~E~
Gallium-67
Minimum 99,8 per cent of the total radioactivity,
Gamma-ray spectrometry.
Determine the relative amounts of gallium-66 and other
*** radionuclidic impurities present,
Gallium (67 Ga) Citrate Injection * * RADIOACTIVITY
** **
(Ph Bur monograph 0555) *** Determine the radioactivity using a calibrated instrument,
__________________________________________
~E~

IMPURITIES
DEFINITION A. gallium-66.
__________________________________________ Ph Eur
Sterile solution of gallium-67 in the form of gallium citrate ,
It may be made isotonic by the addition of sodium chloride
and sodium citrate and may contain a suitable antimicrobial
preservative such as benzyl alcohol.
Gallium-67
***
90 per cent to 110 per cent ofthe declared gallium-67
Gallium (68 Ga) Chloride Solution * **
radioactivity at the date and time stated on the label. *
CHARACTERS for Radiolabelling **** *
Appearance (Ph Bur monograph 2464)
Clear, colourless solution, 174,3
Half-life and nature of radiadon of gallium-67 ~E~ __________________________________________
See general chapter 5,7, Table oj physical characteristics oj
DEFINITION
radionuchdes.
Solution containing gallium-68 in the form of gallium
IDENTIFICATION chloride in dilute hydrochloric acid, The preparation may
A. Gamma-ray spectrometry, contain acetone,
R esults The most prominent gamma photons have energies Content:
ofO.093 MeV, 0,185 MeV and 0,300 MeV,
B, To 0.2 mL of the preparation to be examined add 0,2 mL
°
-- gallium-68: 9 per cent to 110 per cent of the declared
gallium-68 radioactivity at the date and time stated on the
of a solution containing 1 gIL of jerric chloride R and label.
0,1 per cent VIV of hydrochloric acid R and mix, CHARACTERS
Compare the colour with that of a solution containing 7 gIL Appearance
of sodium chloride R and 9 gIL of benzyl alcohol R treated in Clear, colourless solution,
the same manner. A yellow colour develops in the test
solution only.
2014 Radiopharmaceutical Preparations IV-663

Half-Jife and nature of radiation of gallium-68 Furnace programme

See general chapter 5. 7. Table of physical charactenstics of
radionuclides. Step Final Ramp Hold time Internal
temperature time (s) (s) protective
IDENTIFICAnON (OC) gas flow rate
A. Gamma-ray spectrometry. (mL/ min)
Drying 110 30 250
Result The principal gamma photons have energies of
0.511 MeVand 1.077 MeV and, depending on the Drying 130 15 30 250
measurement geometry, a sum peak of 1.022 MeV may be 1400
Pyrolysis 10 20 250
observed.
Atomisation 2100 O 5 O
B. Determine the approximate half-life by no fewer than 3
measurements of the activity of a sample in the same Cleaning 2450 3 250
geometrical conditions within a suitable period of time (for
example, 15 min).
The solution may be released for use before completion of
Result 62 min to 74 mino the test.
e. pH (see Tests). Zinc
D. To a volume of20-100 ¡.tL ofthe solution to be examined Maximum 10 ¡.tg/GBq.
add 1 mL of a 1.03 gIL solution of hydrochloric acid R. Apply
Atomic absorption spectrometry (2.2.23, Method 1).
this solution to the top of a column containing strong cation-
exchange resin R, push 5 mL of air through the column and Test solution Dilute the solution to be examined with a
coJlect the eluate. Determine the radioactivity of the eluate 1 per cent V/V solution of nitnc acid R to obtain a
(A l ). Elute the column with 1 mL of a 1.03 gIL solution of radioactivity concentration of 50 MBq/mL.
hydrochlonc acid R . Determine the radioactivity of the eluate Reference solutions Prepare the reference solutions using zinc
(A2). Elute the column with 1 mL of a mixture of 2 volumes standard solution (J Oppm Zn) R, diluting with a
of hydrochloric acid R and 98 volumes of acetone R and push 1 per cent VIV solution of nitnc acid R .
5 mL of air through the column. Determine the radioactivity Source zinc hollow-cathode lampo
of the eluate (A3) and the residual activity on the column Wavelength 213.9 nm.
(A4).
Atomisation device Air-acetylene flameo
Calculate the percentage of radioactivity in the A3 eluate
The solution may be released for use before completion of
using the following expression:
the test.
Bacterial endotoxins (2.6.14)
A3 x 100/ (Al + A2 + A3 + A4)
Less than 175 IU/ V, V being the maximum volume to be
used for the preparation of a single patient dose, if intended
Result The percentage of radioactivity in the A3 eluate is for use in the manufacture of parenteral preparations without
not les s than 90 per cent. a further appropriate procedure for the removal of bacterial
E. To 100 ¡.tL of silver nitrate solution R2 add 50 ¡.tL of the endotoxins. The solution may be relea sed for use before
solution to be examined. A white precipitate is formed. completion of the test.
TESTS RADIONUCLIDIC PURITY
pH The solution may be released for use before completion of
Maximum 2, using a pH indicator strip R. test B.
Iron Gallium-68
Maximum 10 ¡.tg/GBq. Minimum 99.9 per cent of the total radioactivity.
Atomic absorption spectrometry (2.2.23, Method 1). A. Gamma-ray spectrometry.
Modijier solution 14 gIL solution of magnesium nitra te R. Limit Peaks in the gamma-ray spectrum corresponding to
Test solution Dilute the solution to be examined with a photons with an energy different from 0.511 MeV,
1 per cent VIV solution of nitnc acid R to obtain a 1.077 MeV, 1.022 MeV and 1.883 MeV represent not more
radioactivity concentration of 2.5 MBq/rnL. than 0.1 per cent of the total radioactivity.
Reference solutions Prepare the reference solutions using iron B. Germanium-68 and garnma-ray-emining impurities.
standard solution (20 ppm Fe) R, diluting with a Gamma-ray spectrometry.
1 per cent VIV solution of nitric acid R. Determine the amount of gallium-68, germanium-68 and
Source lron hollow-cathode lampo radionuclidic impurities with a half-life longer than 5 h .
For the detection and quantification of germanium-68 and
Wavelength 248 .3 nm.
gamma-ray-emitting impurities, retain the solution to be
Atomisation device Graphite furnace. examined for at least 48 h to allow the galJium-68 to decay
An example of the injection and instrument parameters for to a level that permits the detection of impurities.
the graphic fumace atomic absorption analysis is shown Result The total radioactivity due to germanium-68 and
below. gamma-ray-emitting impurities is not more than
Internal and external protective gas argon R. 0.001 per cent.
Injection 20 ¡.tL of the test solution and the reference RADIOCHEMICAL PURITY
solutions, and 1 ¡.tL of the modifier solution. 68
[ Ga]Gallium(I1I) ion
Injection temperature 20°C Thin-layer chromatography (2.2.27).
Test solution Adjust the solution to be examined to obtain a
concentration of hydrochloric acid R of 10.3 gIL.
 IV-664 R adioph arm aceutical preparations 2014

Reference solution (a) To 0.2 mL of the test solution add
0.3 mL of a 4 gIL solution of sodiwn hydroxide R. Use within
30 min of preparation.
Reference solurion (b) To 1 mL of the test solution add
1 mL of a 10 giL solution of pentetic acid R in a 4 gIL
solution of sodiwn hydroxide R. Use within 30 min of
preparation.
Plate TLC silica gel plate R; use a glass-fibre plateo
Mobile phase 77 gIL solution of ammonium acetate R,
methanol R (50:50 V/V).
Application About 5 ~1L.
Development Immediately, over a path of at least 10 cm.
Drying In airo
Detection Suitable detector to determine the distribution of
radioactivity.
Retardation factor [68Ga]Gallium(III) ion = 0-0.2.
System suitability The retardation factor of the principal
peak in the chromatogram obtained with reference solution
(a) is not more than 0.1; the retardation factor of the
principal peak in the chromatogram obtained with reference
solution (b) is not 1ess than 0.7.
LimÍl:
-- [68GaJgallium(IlI) ion: minimum 95 per cent of the total
radioactivity due to gallium-68.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label states:
-- that the solution is not intended for direct administration
to humans;
-- the maximum volume that can be used for the
preparation of a single patient dose;
-- the concentration of hydrochloric acid;
-- the concentration of acetone, if present;
-- that the solutíon is intended for use in the preparation of
gallium-68-labelled radiopharmaceuticals;
-- a procedure to reduce the level of germanium-68 below
0.001 per cent of the total radioactivity.
IMPURITIES
A. germanium-68.
____________________________________________ PhEw
Gallium (GaGa) Edotreotide ***
** * aceta te R in a 10.3 gIL solution of hydroehlorie acid R, V being
Injection **** **
(Ph Eur 1110nograph 2482)
~Ew ____________________________________________
tetraazacycJododecan-1-yl] acetyl) -D-phenylalanyl-L-cysteinyl-
L-tyrosyl-D-tryptophyl-L-lysyl-L-threonyl-N-[(lR,2R)-2-
hydroxy-1-(hydroxyrnethyl)propyl) -L-cysteinamide cycJic
(2--7)-disulfide) (gallium-68 DOTATOC).
Content:
-- galliwn-68: 90 per cent to 110 per cent of the decJared
gallium-68 radioactivity at the date and time stated on the
label;
-- edotreotide: maximum 50 flg per maximum recommended
dos e in millilitres.
CHARACTERS
Appearance
Clear, colourless solution.
Half-Jife and nature of radiation of gallium-68
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The principal gamma photons have energies of
0.511 MeVand 1.077 MeV and, depending on the
measurement geometry, a sum peak of 1.022 MeV may be
observed.
B. Determine the approximate half-life by no fewer than 3
measurements of the activity of a sample in the same
geometrical conditions within a suitable period of time (for
example, 15 min).
Resu!t 62 min to 74 mino
C. Examine the chromatograms obtained in the test for other
radiochemical impurities (see Tests).
Result The principal peak in the radiochromatogram
obtained with the test solution has a relative retention of 1.3
with reference to the principal peak in the chromatogram
obtained with reference solution (a) using the
spectrophotometer.
TESTS
pH
4.0 to 8.0, using a pH indicator strip R.
Edotreotide, gallium edotreotide and other re1ated
substances
Liquid chromatography (2.2.29).
Test solution The preparation to be examined.
Reference solution (a) Prepare a 50 flg/V solution of
edotreotide R in a 10.3 giL solution of hydrochlorie aeid R, V
being the maximum recommended dose in millilitres.
Referenee solution (b) Prepare a 50 flg/V solutíon of oetreotide
the maximum recommended dose in millilitres.
Referenee solution (e) Mix 0.1 mL ofreference solution (a)
and 0.1 mL ofreference solution (b).
Colwnn:
-- size: 1 = 0.15 m, 0 = 3.0 mm;
-- stationary phase: base-deactivated octadeeylsilyl siliea gel for
ehromatography R (3 flm).
Mobüe phase:
-- mobile phase A: trifiuoroaeetie acid R, water R
(0.1:99.9 V/V);
-- mobile phase B: trifiuoroaeetie acid R, aeetonimle R
(0.1:99 .9 V/V)j
Time Mobile phase A Mobile phase B
(min) (per cent V/ V) (per cent V!JI)
0·8 76 24

DEFINITION 76 __ 40 24 __ 60
8·9
Sterile solution of a complex of gallium-68 with edotreotide 9·14 40 60
(N-[[4, 7,10-tris(carboxyrnethyl)-1,4, 7,10-
 2014
Flow rate 0.6 mUmin.
Deteetion Spectrophotometer at 220 nm and radioactivity
detector connected in series.
Injeetion 20 ¡lL.
Relative retentíon With reference to edotreotide (retention
time = about 3.3 min): gallium edotreotide = about l.3;
octreotide = about 2.6 .
System suirabiliry Reference solution (c) using the
spectrophotometer:
- resolution: minimum 5.0 between the peaks due to
edotreotide and octreotide.
Limits In the chromatogram obtained with the
spectrophotometer:
- edotreotide and metal eomplexes of edotreotide (sum of the
areas of the peaks with a relative retention with reference
to edotreotide between 0.8 and l.4): not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (50 ¡lgN);
unspeeijied impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (50 ¡lglV); disregard any peak
with a relative retention with reference to edotreotide of
0.5 or less.
Impurity D
Thin-layer chromatography (2.2.27).
Test solution The preparation to be examined.
Referenee solution Dissolve 10 mg of HEPES R (impurity D)
in water R and dilute to V with the same solvent, V being the
maximum recommended dose in millilitres . Dilute 1.0 mL of
the solution to 50.0 mL with water R.
Plate TLC siliea gel F254 plate R; use an aluminium plateo
Mobz1e phase water R, aeetonitrile R (25:75 V/V).
Applieation (V/2000) mL, Vbeing the maximum
recommended dose in millilitres; apply portions of 1 ¡lL and
dry with a current of warm air after each application.
Development Over 2/3 of the plateo
Deteetion Expose to iodine vapour for 4 min.
Retardation factor Impurity D = about 0.3.
System suitabz1iry Reference solution:
- the chromatogram shows a c1early visible spot.
Limit:
- impurity D: any spot due to impurity D is not more
intense than the corresponding spot in the chromatogram
obtained with the reference solution (200 ¡lglV).
Ethan01
Gas chromatography (2.2.28).
Internal standard solution Dilute 1 mL of propanol R to
1000 mL with water R.
Test solution Dilute 0.10 mL of the preparation to be
examined to 10.0 mL with the intemal standard solution.
Referenee solution Dilute l.0 mL of anhydrous ethanol R to
100.0 mL with the intemal standard solution. Dilute l.0 mL
of this solution to 10.0 mL with the intemal standard
solution.
Column:
- material: fused silica;
- size: 1 = 30 m, 0 = 0.53 mm;
- stationary phase: maerogol 20 000 R (film thickness
l.0 ¡lm) .
Cam'er gas helium for ehromatography R.
Flow rate 10 mUmin.
Split ratio 1: 1O.
Radiopharmaceutical preparations IV-665
Temperature:
- eolumn: 35 oC;
- injeetion port: 140 oC;
- detector: 220 oc.
Deteetion Flame ionisation.
Injeetion l.0 ¡lL.
System suitability Reference solution:
- retentíon time: ethanol = 2 min to 4 min;
- resolution: minimum 5.0 between the peaks due to ethanol
and propano!.
Limit:
- ethanol: maximum 10 per cent V/Vand maximum 2.5 g
per administration, taking the density (2.2.5) to be
0.790 glmL.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophmmaeeutieal preparations (0125).
The preparation may be released for use before completion
of the test.
Bacteria! endotoxins (2.6.14)
Less than 175N IU/mL, V being the maximum
recommended dose in millilitres. The preparation may be
released for use before completion of the test.
RADIONUCLIDIC PURITY
The preparation may be released for use before completion
of test B.
Gallium-68
Minimum 99.9 per cent of the total radioactivity.
A. Gamma-ray spectrometry.
Limit Peaks in the gamma-ray spectrum corresponding to
photons with an energy different from 0.511 MeV,
l.077 MeV, l.022 MeV and l.883 MeV represent not more
than 0.1 per cent of the total radioactivity.
B. Germanium-68 and gamma-ray-emitting impurities.
Gamma-ray spectrometry.
Determine the amount of gallium-68, germanium-68 and
radionuc1idic impurities with a half-life longer than 5 h.
For the detection and quantification of gerrnanium-68 and
gamma-ray-emitting impurities, retain the preparation to be
examined for at least 48 h to allow the gallium-68 to decay
to a level that permits the detection of impurities.
Result The total radioactivity due to gerrnanium-68 and
gamma-ray-emitting impurities is not more than
0.001 per cent.
RADIOCHEMICAL PURITY
- f 8Ga]gallium edotreotide: minimum 91 per cent of the
total radioactivity due to gallium-68.
Impurity A
Thin-layer chromatography (2.2.27).
Test solution The preparation to be examined.
Referenee solution (a) Dilute gallium (8Ga) ehloride solution R
with water R to obtain a final concentration of 10 gIL of
hydroehloric aeid R. To 1 mL of this solution add l.5 mL of a
4 giL solution of sodium hydroxide R . Use within 30 min of
preparation.
Referenee solution (b) Dilute gallium (8Ga) ehloride solution R
with water R to obtain a final concentration of 10 gIL of
hydroehlorie acid R. To 1 mL of this solution add 1 mL of a
solution containing 10 giL of pentetie acid R and 4 gIL of
sodium hydroxide R . Use within 30 min after preparation.
Plate TLC silica gel plate R; use a glass-fibre plateo
Mobl7e phase 77 gIL solution of ammonium acetate R in
water R, methanol R (50:50 V/V).
 IV-666 Radiopharmaceutical Preparations 2014

Application 5 J.lL.
Development Irnrnediately, over 2/3 of the plateo
Indium C11 1n) Chloride Solution
Drying In air. (Ph Bur monograph 1227)
~E~ ____________________________________________
Detection Suitable detector to determine the distribution of
radioactivity. DEFINITION
Retardationfactors Impurity A = 0.0-0.1; [68 Ga]gallium Sterile solution of indium-111 as the chloride in aqueous
edotreotide = 0.8-1.0. hydrochloric acid containing no additives.
System suitability The retardation factor of the principal Indium-ll1
signal in the chromatogram obtained with reference solution 90 per cent to 110 per cent of the declared indium-111
(a) is not more than 0.1; the retardation factor of the radioactivity at the date and time stated on the label.
principal signal in the chromatogram obtained with reference
SPECIFlC RADIOACTIVITY
solution (b) is more than 0.7.
Minimum 1.85 GBq of indium-ll1 per microgram of
Limit: indium.
- impurity A: not more than 3 per cent of the total
radioactivity due to gallium-68. PRODUCTION
No carrier indium is added.
Other radiochemical impurities
Liquid chromatography (2.2.29) as described in the test for CHARACTERS
edotreotide, gallium edotreotide and other related substances. Appearance
If necessary, dilute the test solution with water R to a Clear, colourless solution.
radioactivity concentration suitable for the radioactivity Half-life and nature of radiation of indium-ll1
detector. See general chapter 5.7. Table of physical characteristics of
Examine the chromatogram recorded using the radioactivity radionuclides.
detector and locate the peak due to [68 Ga]gallium
IDENTIFICATION
edotreotide by comparison with the chromatogram obtained
A. Gamma-ray and X-ray spectrometry. Carry out the test
with reference solution (a) and the spectrophotometer.
afier allowing sufficient time for short-lived impurities such as
Relative retention With reference to [68 Ga]gallium indium-ll Om to decay.
edotreotide (retention time = about 4.2 min):
Results The most prominent gamma photons of indium-111
impurity B = about 0.3.
have energies of 0.171 MeV and 0.245 MeV.
Limit:
- impurity B: not more than 2 per cent of the total B. To 100 J.lL of silver nitrate solution R2 add 50 ~lL of the
radioactivity due to gallium-68. preparation to be examined. A white precipitate is formed .
Calculate the percentage of radioactivity due to C. pH (se e Tests) .
68 D. Examine the chromatogram obtained in the test for
[ Ga]gallium edotreotide using the following expression:
radiochemical purity (se e Tests).
(100 - A) x T Result The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is 0.5 to
0.8.
A percentage of radioactivity due to impurity A
determined in the test for impurity A under TESTS
radiochemical purity; pH (2.2.3)
T proportion of the area of the peak due to 1.0 to 2.0.
68
[ Ga]gallium edotreotide relative to the total areas Cadmium
of the peaks in the chromatogram obtained with the Maximum 0.40 J.lg/mL.
test solution. Atomic absorption spectrometry (2.2.23, Method I) .
RADIOACTIVITY Test solution Dilute 0.05 mL of the preparation to be
Determine the radioactivity using a calibrated instrumento examined to a suitable volume with a suitable concentration
LABELLING of hydrochloric acid R.
The label states the percentage content of ethanol in the Reference solutions Prepare the reference solutions using
preparation. cadmium standard solution (0. 1 per cent Cd) R, diluting with
the same concentration of hydrochloric acid R as in the test
IMPURlTIES
solution.
Specified impurities A, B, C, D.
Source Cadmium hollow-cathode lampo
A. [68 Ga]gallium in colloidal form,
Wavelength 228 .8 nm.
B. [68 Ga]gallium(lIl) ion,
Atomisation device Electrothermal.
C. germanium-68,
Copper
Maximum 0.15 J.lg/mL.
HO~Nl
Atomic absorption spectrometry (2.2.23, Method I) .
~ N~S03H Test solution Dilute 0.1 mL ofthe preparation to be
examined to a suitable volume with a suitable concentration
D. 2-[ 4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid of hydrochloric acid R.
(HEPES).
____________________________________________ ~E~
 2014 Radiopharmaceutical Preparations IV-667

Reference solutions Prepare the reference solutions using Limit:

copper standard solution (0.1 per cent) R diluting with the same -- [Il I lnJindium(IlI) ion: minimum 95 per cent of the
concentration of hydrochlon·c acid R as in the test solution. radioactivity due to indium-lll.
Source Copper hollow-cathode lampo RADIOACTIVITY
Wavelength 324.8 nm. Determine the radioactivity using a calibrated instrumento
Atomisation device Electrotherma!. IMPURITIES
Iron A. indium-114m.
Maximum 0.60 J.lg/mL. ____________________________________________ PhE~

Atomic absorption spectrometry (2.2.23, M ethod 1).

Test solution Dilute 0.1 mL of the preparation to be
examined to a suitable volume with a suitable concentration
of hydrochloric acid R.
Reference solutions Prepare the reference solutions using iron
Indium C11 1n) Oxine Solution *****
** **
standard solution (0.1 per cent Fe) R diluting with the same (Ph Eur monograph 1109) ***
concentration of hydrochloric acid R as in the test solution.
Source Iron hollow-cathode lamp
Wavelength 248.3 nm.
Atomisation device Electrotherma!.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
RADIONUCLIDIC PURITY
Indium-ll1
Gamma-ray and X-ray spectrometry. 547.2
____________________________________________
Comparison Standardised indium-lll solution.
~E~

Result The spectrum obtained with the preparation to be DEFINITION

examined do es not differ significantIy from that obtained
with a standardised indium-lll solution apart from any
differences due to the presence of indium-114m.
Impurity A
Maximum 0.25 per cent of the total radioactivity.
Gamma-ray spectrometry. Cany out the test after allowing
sufficient time for short-lived impurities such as indium-ll Om to
decay.
Take a volume equivalent to 30 MBq and record the
gamma-ray spectrum using a suitable detector with a shield
of lead, 6 mm thick, placed between the sample and the
detector.
Results The response in the region corresponding to the
0.558 MeV photon and the 0.725 MeV photon of indium-
114m do es not exceed that obtained using 75 kBq of a
standardised indium-114m solution measured under the
same conditions, when all measurements are calculated with
reference to the date and time of administration.
RADIOCHEMICAL PURITY
[l1l1n]Indium(I1I) ion
Thin-layer chromatography (2.2.27)
Test solution The preparation to be examined.
Plate TLC silica gel plate R. Use silica gel as the coating
substance on a glass-fibre sheet.
Mobile phase 9.0 gIL solution of sodium chlon·de R adjusted
to pH 2.3 ± 0.05 with dilute hydrochloric acid R .
Application 5 J.lL.
Development Immediately over a path of 15 cm.
Drying In a current of cold airo
Detection Suitab1e detector to determine the distribution of
radioactivity.
e
Retardation factor: JJIn]indium(III) ion = 0.5 to 0.8.
Sterile solution of indium-lll in the form of a complex with
8-hydroxyquinoline. It may contain suitable surface active
agents and may be made isotonic by the addition of sodium
chloride and a suitable buffer.
Indium-l11
90 per cent to 110 per cent of the declared indium-lll
radioactivity at the date and time stated on the labe!'
Specific radioactivity
Minimum 1.85 GBq of indium-l11 per microgram of
indium.
PRODUCTION
No carrier indium is added.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation ofindium-ll1
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray and X-ray spectrometry. Cany out the test
after allowing sufficient time for short-lived impurities such as
indium-llOm to decay.
Results The most prominent gamma photons of indium-ll1
have energies of 0.171 MeV and 0.245 MeV.
B. Place 5-10 mg of magnesium oxide R in a glass container
about 20 mm in internal diameter. Add 20 J.lL of the
prepararíon to be examined. Examine in ultraviolet light at
365 nrn. Bright yellow ftuorescence is produced.
C. The distribution of radioactivity between the organic and
aqueous phases in the test for radiochemical purity
(see Tests) contributes to the identification of the
preparation.
 IV-668 Radiopharmaceutical Preparations
TESTS
pH (2.2.3)
6.0 to 7.5.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIONUCLIDIC PURITY
Indium-lll
Gamma-ray and X-ray spectrometry.
Compmison Standardised indium-lll solution.
Result The spectrum obtained with the preparation to be
examined does not differ significantly from that obtained
with a standardised indium-111 solution, apart from any
differences due to the presence of indium-1l4m.
Impurity A
Maximum 0.25 per cent of the total radioactivity.
Gamma-ray specrrometry. Cany out the test afier allowing
sufficient time for short-lived impurities sueh as indium-110m to
deeay.
Take a volume equivalent to 30 MBq and record the
gamma-ray spectrum using a suitable detector with a shield
of lead, 6 mm thick, placed between the sample and the
detector.
Results The response in the region corresponding to the
0.558 MeV photon and the 0.725 MeV photon of indium-
114m does nor exceed thar obtained using 75 kBq of a
standardised indium-114m solution measured under the
same conditions, when all measurements are calculated with
reference to the date and rime of administration.
RADIOCHEMICAL PURITY
[111In]Indium oxine
To a silanised separating funnel containing 3 mL of a 9 gIL
solution of sodium ehloride R add 100 ¡.tL of the preparation
to be examined and mix. Add 6 mL of octanol R and shake
vigorously. Allow the phases to separate and then run the
lower layer into a suitable vial for counting. Allow the upper
layer to drain completely into a similar vial. Add 1 mL of
oetanol R to the separating funnel, shake vigorously and drain
into the vial containing the organic fraction. Add 5 mL of
dilute hydroehlorie acid R to the separating funnel, shake
vigorously and drain these rinsings into a 3rd vial. Seal each
vial and, using a suitable insrrument, measure the
radioactivity in each. Calculate the radiochemical purity by
expressing the radioactivity of the [1l1 In]indium oxine, found
in the organic phase, as a percentage of the total radioactivity
due to indium-111 measured in the 3 solutions.
Result Minimum 90 per cent of the radioactivity due to
indium-111 .
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The labe! states that the solution is not for direct
administration to humans.
IMPURITIES
A. indium-1l4m.
_ _ __ __ __ _ _ _ __ _ __ _ _ __ _ _ PhEur
2014
Indium C11 1n) Pentetate Injection *****
** **
(Ph Eur monograph 0670) ***
~Ew _ _ __ _ _ _ _ _ _ _ _ __ _ __ __ __ _
DEFINITION
Sterile solution containing indium-111 in the form of indium
diethylenetriaminepenta-acetate. It may contain calcium and
may be made isotonic by the addition of sodium chloride and
a suitable buffer.
Indium-ll1
90 per cent to 110 per cent of the declared indium-ll1
radioactivity at the date and time stated on the label.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation ofIndium-l11
See general chapter 5.7. Table of physieal eharaeteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray and X-ray spectrometry.
Results The mosr prominent gamma photons of indium-111
have energies of 0.171 MeV and 0.245 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity (see Tests) . The distribution of
radioactivity contributes to rhe identification of the
preparation.
TESTS
pH (2.2.3)
7.0 to 8.0.
Cadmium
Maximum 5 ¡.tglmL.
Atomic absorption spectrometry (2.2.23, Method 1I).
Test solution Mix 0.1 mL of the preparation to be examined
with 0.9 mL of a mixture of 1 volume of hydroehlon'e acid R
and 99 volumes of water R .
Referenee solutions Prepare the reference solutions using
eadmium standard solution (0.1 per eent Cd) R and diluting
with a mixture of 1 volume of hydroehlorie acid R and
99 volumes of water R.
SouTee Cadmium hollow-cathode lampo
Wavelength 228 .8 nm.
Atomisation deviee Air-acetylene f1ame.
Uncomplexed diethylenetriarninepenta-acetic acid
Maximum 0.4 mglmL.
In a micro test-tube, mix 100 ¡.tL of the preparation to be
examined with 100 ¡.tL of a freshly prepared 1 gIL solution of
hydroxynaphthol blue, sodium salt R in a 42 gIL solution of
sodium hydroxide R. Add 50 ¡.tL of a 0.15 gIL solution of
ealcium ehloride R. The solution remains pinkish-violet or
changes from blue to pinkish-violet.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaeeutieal preparations (0125). The
preparation may be released for use before completion of the
test.
Bacterial endotoxins (2.6.14)
Less than 14N IU/roL, V being the maximum recommended
dose in millilitres.
2014 Radiopharmaceutical Preparations IV-669

RADIONUCLIDIC PURITY suitable buffer, a suitable labelling catalyst such as ionic

Indium-l11 copper, a suitable labelling stabiliser such as ascorbic acid
Gamma-ray and X-ray spectrometry. and antimicrobial preservatives.
Comparison Standardised indium-111 solution. Iodine-123
90 per cent to 110 per cent of the declared iodine-123
Result The spectrum obtained with the preparation to be
radioactivity at the date and time stated on the labe!'
examined does not differ significantly from that obtained
with a standardised indium-111 solution apart from any Specific radioactivity
differences due to the presence of indium-114m. Minimum 10 GBq of iodine-123 per gram of iobenguane
base.
Impurity A
Maximum 0.2 per cent of the total radioactivity at the date CHARACTERS
and time of administration. Appearance
Gamma-ray spectrometry. Clear, colourless or slightly yellow solution.
Retain a sample of the preparation to be examined for a Half-life and nature ofradiation ofiodine-123
sufficient time to allow the indium-111 radioactivity to decay See general chapter 5.7. Table of physical characteristics of
to a sufficiently low level to permit the detection of radionuclides.
radionuclidic impurities. Record the gamma-ray spectrum of IDENTIFICATION
the decayed material in a suitable instrument calibrated with
A. Gamma-ray and X-ray spectrometry.
the aid of a standardised indium-114m solution.
Result The energy of the most prominent gamma photon of
Result Indium-114m has a half-life of 49.5 days and its iodine-123 is 0.159 MeV.
most prominent gamma photon has an energy ofO.190 MeV.
B. Examine the chromatogram obtained in the test for
RADIOCHEMICAL PURITY radiochemical purity (see Tests). The distribution of the
[111 In]Indium pentetate radioactivity contributes to the identification of the
Thin-layer chromatography (2.2.27). preparation.
Test solution The preparation to be examined. TESTS
Plate TLC silica gel plate R; use silica gel as the coating pH (2.2.3)
substance on a glass-fibre sheet heated at 110 oC for 10 mino 3.5 to 8.0.
Mobile phase 9 gIL solution of sodium chloride R. Specific radioactivity
Application 5-10 JlL. The specific radioactivity is calculated from the results
Development Over a path of 10-15 cm in about 10 mino obtained in the test for radiochemical purity. Determine the
content of iobenguane sulfate from the areas of the peaks
Drying In air.
corresponding to iobenguane in the chromatograms obtained
Detection Suitable detector to determine the distribution of with the test solution and reference solution (b). Calculate
radioactivity . the concentration as iobenguane base by multiplying the
ldentification of spotse II In] indium pentetate migrates near result obtained in the test by 0.85.
to the solvent front.
Sterility
Limit: It complies with the test for sterility prescribed in the
- ¡r 1I InJindium pentetate : minimum 95 per cent of the
monograph Radiopharmaceutical preparations (0125).
radioactivity due to indium-111 . The preparation may be released for use before completion
RADIOACTIVITY of the test.
Determine the radioactivity using a calibrated instrumento Bacterial endotoxins (2.6.14)
IMPURITIES Less than 175/ V IU/mL, V being the maximum
A. indium-1l4m. recommended dose in millilitres.
____________________________________________ PhE~
RADIONUCLIDIC PURITY
The preparation may be released for use before completion
of the test.
Radionuclides other than iodine-123
lobenguane C231) Injection Maximum 0.35 per cent of the total radioactivity.
Gamma-ray and X-ray spectrometry.
(Ph Bur monograph 1113) Record the gamma-ray spectrum and the X-ray spectrum
using a suitable instrumento
NH Determine the relative amounts ofiodine-125, tellurium-121
)l
y~
and other radionuc1idic impurities presento For their
NH,
determination, retain the preparation to be examined for a
sufficient time to allow iodine-123 to decay to a level which
1231 permits the detection of radionuc1idic impurities.
No radionuc1ides with a half-life longer than that of iodine-
125 are detected.
Ph Eur _ __________________________________________ RADIOCHEMICAL PURITY

DEFINITION
C23I]Iobenguane
Liquid chromatography (2.2.29).
Sterile, bacterial endotoxin-free solution of
1-(3-[ 123I]iodobenzyl)guanidine or its salts. It may contain a
Test solution The preparation to be examined.
IV-670 Radiopharmaceutical Preparations 2014

Reference solution (a) Dissolve 0.100 g of sodium iodide R in Specific radioactivity

the mobile phase and dilute to 100 mL with the mobile Minimum 20 GBq of iodine-131 per gram of iobenguane
phase. base.
Reference solution (b) Dissolve 10.0 mg of iobenguane CHARACTERS
sulfate CRS in 25 mL of the mobile phase and dilute to Appearance
50.0 mL with the mobile phase. Clear, colourless or slightly yellow solution.
Column:
Half-life and nature of radiation of iodine-131
- size: 1 = 0.25 m, 0 = 4.0 mm;
See general chapter 5.7. Table of physical characteristics of
- stationary phase: silica gel for chromatography R (5 ~lm) .
radionuclides.
Mobile phase 80 gIL solution of ammonium nitra te R, dilute
ammonia R2, methanol R (1:2:27 V/V/V).
IDENTIFICATION
A. Gamma-ray spectrometry.
Flow rate l.0 mUmin.
Result The most prominent gamma photon ofiodine-131
Detection Suitable detector to determine the distribution of
has an energy of 0.365 MeV.
radioactivity and spectrophotometer at 254 nm, provided
with a ftow-cell. B. Examine the chromatogram obtained in the test for
radiochemical purity (se e Tests). The distribution of the
lnjection 10 ¡.¡L.
radioactivity comributes to the identification of the
Limits: preparation.
- [123IJiobenguane: minimum 95 per cent of the radioactivity
due to iodine-123; TESTS
- impurity A: maxirnum 4 per cent of the radioactivity due pH (2.2.3)
to iodine-123; 3.5 to 8.0.
- other impurities: maximum 1 per cent of the radioactivity Specific radioactivity
due to iodine-123 . The specific radioactivity is calculated from the results
RADIOACTIVITY obtained in the test for radiochemical purity. Determine the
Determine the radioactivity using a calibrated instrumento content of iobenguane sulfate from the areas of the peaks
corresponding to iobenguane in the chromatograms obtained
STORAGE with the test solution and reference solution (b). Calculate
Protected from light. the concentration as iobenguane base by multiplying the
LABELLING result obtained in the test by 0.85.
The label states the specific radioactivity expressed in GBq of Sterility
iodine-123 per gram of iobenguane base. It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
IMPURITIES
The preparation may be released for use before completion
A. [123 I]iodide,
of the test.
B. iodine-125,
Bacterial endotoxins (2.6.14)
C. tellurium-12l.
Less than 175/V ID/mL, V being the maxirnum
_ _ _ __ _ __ _ _ _ _ _ _ _ _ __ _ _ __ Ph EUf
recommended dose in millilitres .
RADIONUCLIDIC PURITY
Iodine-131
Minimum 99.9 per cent ofthe total radioactivity.
lobenguane C31 1) Injection for ****
***
Gamma-ray spectrometry.
*
Diagnostic Use *** * Determine the relative amounts of iodine-131, iodine-133,
iodine-135 and other radionuclidic impurities presento
(Ph Bur monograph 1111)
RADIOCHEMICAL PURlTY
[131 I]Iobenguane
Liquid chromatography (2.2.29).
Test solution The preparation to be examined.
R eference solution (a) Dissolve 0.100 g of sodium iodide R in
the mobile phase and dilute to 100 mL with the mobile
phase.
Reference solution (b) Dissolve 10.0 mg of iobenguane
sulfate CRS in 25 mL of the mobile phase and dilute to
~E~ _ __ _ _ __ __ _ _ _ _ _ _ _ _ _ __ _ _
50.0 mL with the mobile phase.
DEFINITION Column:
Sterile, bacterial endotoxin-free solution of - size: 1 = 0.25 m, 0 = 4.0 mm;
1-(3-¡I 31 I]iodobenzyl)guanidine or its salts. It may contain a - stationary phase: silica gel for chromatography R (5 ~lm).
suitable buffer, a suitable labelling catalyst such as ionic Mobile phase 80 gIL solution of ammonium nitrate R, dilute
copper, a suitable labelling stabiliser such as ascorbic acid, ammonia R2, methanol R (1:2:27 VIV/V).
and amirnicrobial preservatives. Flow rate l.0 mUmin.
Iodine-131 Detection Suitable detector to determine the distribution of
90 per cent to 11 O per cent of the declared iodine-131 radioactivity and spectrophotometer at 254 nm, provided
radioactivity at the date and time stated on the labe!. with a ftow-cell.
2014 Radiopharmaceutical Preparations IV-671

Injection 1O ~1L. radioactivity contributes to the identification of the

Limirs: preparation.
-- [131 Ijiobenguane: minimum 94 per cent of the radioactivity TESTS
due to iodine-131; pH (2.2.3)
-- impurity A: maximum 5 per cent of the radioactivity due 3.5 to 8.0.
to iodine-1 31;
-- other impul1úes: maximum 1 per cent of the radioactivity Specific radioactivity
due to iodine-131. The specific radioactivity is calculated from the results
obtained in the test for radiochemical purity. Determine the
RADIOACTIVITY content of iobenguane sulfate fram the areas of the peaks
Determine the radioactivity using a calibrated instrumento corresponding to iobenguane in the chromatograms obtained
STORAGE with the test solution and reference solution (b). Calculate
Protected from light. the concentration as iobenguane base by multiplying the
result obtained in the test by 0.85.
LABELLING
Sterility
The label states the specific radioactivity expressed in GBq of
It complies with the test for sterility prescribed in the
iodine-131 per gram of iobenguane base.
monograph Radiophannaceutical preparations (0125).
IMPURITIES The preparation may be relea sed for use before completion
A. ¡t 3II)iodide, of the test.
B. iodine-133, Bacterial endotoxins (2. 6.14)
C. iodine-1 35. Less than 175 /V IU/mL, V being the maxÍmum
____________________________________________ Ph E~ recommended dose in millilitres.
RADIONUCLImC PURITY
Iodine-131
Minimum 99.9 per cent of the total radioactivity.
lobenguane C31 1) Injection for *** Gamma-ray spectrometry.
** *
Therapeutic Use **** ** Determine the relative amounts of iodine-131, iodine-133,
iodine-135 and other radionucIidic impurities presento
(Ph Bur monograph 1112) RADIOCHEMICAL PURITY
13I
[ I]Iobenguane
Liquid chromatography (2.2.29) .
Test solution The preparation to be examined.
Reference solution (a) Dissolve 0.100 g of sodium iodide R in
the mobile phase and dilute to 100 mL with the mobile
phase.
Reference solution (b) Dissolve 10.0 mg of iobenguane
sulfate CRS in 25 mL of the mobile phase and dilute to
~ E~ ___________________________________________
50.0 mL with the mobile phase.
DEFINITION Column:
Sterile, bacterial endotoxin-free solution of -- size: 1 = 0.25 m, 0 = 4.0 mm;
1-(3-[131I)iodobenzyl)guanidine or its salts. It may contain a -- stationary phase: silica gelfor chromatography R (5 )lm).
suitable buffer, a suitable labeIling catalyst such as ionic Mobile phase 80 gIL solution of ammonium nitrate R, dilute
copper, a suitable labeIling stabiliser such as ascorbic acid, ammonia R2, methanol R (1:2:27 V/V/V) .
and antimicrobial preservatives. Plozo rate 1.0 mL/min.
Iodine-131 Detection Suitable detector to determine the distribution of
90 per cent to 110 per cent ofthe decIared iodine-131 radioactivity and spectraphotometer at 254 nm, provided
radioactivity at the date and time stated on the labe!' with a f1ow-cell.
Specific radioactivity Injection 1O ¡.¡L.
Minimum 400 GBq of iodine-131 per gram of iobenguane Limits:
base. -- ( 3/Ijiobenguane: minimum 92 per cent ofthe radioactivity
CHARACTERS due to iodine-131;
Appearance -- impurity A: maximum 7 per cent of the radioactivity due
Clear, colourless or slightly yeIlow solution. to iodine-131;
-- other i111puritieS: maximum 1 per cent of the radioactivity
Half-life and nature ofradiation ofiodine-131
due to iodine-131.
See general chapter 5.7. Table of physical characteristics of
radionuclides. RADIOACTIVITY
IDENTIFICATION Determine the radioactivity using a calibrated instrumento
A. Gamma-ray spectrometry. STORAGE
Result The most prominent gamma photon of iodine-131 Pratected fram light.
has an energy of 0.365 MeV.
B. Examine the chromatogram obtained in the test for radio-
chemical purity (see Tests). The distribution of the
 IV-672 Radiopharmaceutical Preparations 2014

LABELLING The preparation may be released for use before completion

The label states the specific radioactivity expressed in GBq of of the test.
iodine-131 per gram of iobenguane base. Bacterial endotoxins (2.6.14)
IMPURITIES Less than 175/V IU/mL, V being the maximum
A. [ l3l I]iodide, recommended dose in millilitres.
B. iodine-133, RADIONUCLIDIC PURITY
C. iodine-135. Iodine-131
__________________________________________ PhEw Minimum 99.9 per cent ofthe total radioactivity.
Gamma-ray spectrometry.
Determine the relative amounts ofiodine-131, iodine-133,
iodine-135 and other radionuclidic impurities presento
Radiochemical purity
6~-[131I]Iodomethyl-19-norcholest-5(10)-en-3~-ol
lodomethylnorcholesterol C31 1) *****
** **
Thin-layer chromatography (2.2.27).
Injection *** Test solution The preparation to be examined.
Iodinated (mI) Norcholesterol Injection Carrier solution Dissolve 10 mg of potassium iodide R, 20 mg
(Ph Eur monograph 0939) of potassium iodate R and 0.1 g of sodium hydrogen carbonate R
in distilled water R and dilute to 10 mL with the same solvent.
Plate TLC silica gel GF254 plate R.
Mobile phase chloroform R.
CH 3 Application Up to 5 ¡.tL of the test solution and 10 ¡.tL of the
carrier solution on the same spot.
Development Over a path of 15 cm in about 60 mino
Drying In airo
Detection Ultraviolet light at 254 nm and suitable detector
to determine the distribution of radioactivity.
__________________________________________
Retardation factor 6~-e 3I I]iodomethyl-19-norcholest-5(1 0)-
~Ew

DEFINITION en-3~-01 = about 0.5.

Sterile solution of 6~- e 31 1] iodomethyl-19-norcholest-5(l 0)-
en-3~-0!. Ir may contain a suitable emulsifier such as
polysorbate 80 and a suitable antimicrobial preservative such
as benzyl alcoho!'
Iodine-131
90 per cent to 110 per cent of the declared iodine-131
radioactivity at the date and time stated on the labe!'
Specific radioactivity
3.7 GBq to 37 GBq per gram of
6 ~-iodomethylnorcholestero1.
CHARACTERS
Appearance
Clear or slight1y turbid, colourless or pale yellow solution.
H alf-lzfe and nature of radiation of iodine-131: see general
chapter 5.7. Table of physical characten'stics of radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent photon of iodine-131 has an
energy of 0.365 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity 6~-e 3 II]iodomethyl-19-norcholest-
5(10)-en-3~-01 (se e Tests).
Result The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is about
0.5 .
Identification of spots Impurity C remains near the point of
application.
Limit:
-- 6fJ-[/3/ Ijiodomethyl-19-norcholest-5 (10) -en-3 fJ-ol: minimum
85 per cent of the total radioactivity due to iodine-131.
Impurity C
Thin-layer chromatography (2.2.27).
Test solution The preparation to be examined.
Carrier solution Dissolve 10 mg of potassium iodide R, 20 mg
of potassium iodate R and 0.1 g of sodium hydrogen carbonate R
in distilled water R and dilute to 10 mL with the same solvento
Plate TLC silica gel GF254 plate R.
Mobile phase chloroform R, anhydrous ethanol R (50:50 V/V).
Application 10 ¡.tL of the carrier solution and then up to
5 ¡.tL of the test solution on the same spot.
Development Over a path of 15 cm in about 90 mino
Drying In air.
Detection Ultraviolet light at 254 nm for 5 min and suitable
detector to determine the distribution of radioactivity.
Retardation factor Impurity C (yellow spot) = about 0.5 .
Identifications of spots The principal peak of radioactivity is
near to the solvent front; other iodocholesterols migrate near
the solvent front.
Limit:
-- impurity C: maximum 5 per cent of the total radioactivity.

TESTS RADIOACTIVITY
pH (2.2.3) Determine the radioactivity using a calibrated instrumento
3.5 to 8.5. STORAGE
Sterility Protected from light, at - 18 oC or below.
Ir complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
 2014
IMPURlTlES
A. iodine-133,
B. iodine-135,
C. [ l3l I]iodide.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ PhEur
Krypton (81m Kr) Inhalation Gas
(Ph Bur monograph 1533)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _
~Ew
DEFINITlON
Gaseous mixture of krypton-81m and a suitable vehic1e such
as alT.
PRODUCTlON
Krypton-81m is formed by decay of its parent radionuc1ide
rubidium-81. Rubidium-81 has a half-life of 4.58 h.
The krypton-81m formed is separated from the rubidium-81
with a flow of a suitable gas in a rubidium/krypton generator.
Rubidium-81 is produced by proton irradiation of krypton
iso topes or by helium-3 or helium-4 irradiation of bromine.
After separation of rubidium-81 from the target, it is retained
by a suitable support.
Krypton-81m is eluted at a suitable flow rate with a vehic1e
such as air. The level of moisture required in the eluent
depends on the rype of generator used. The transport tube
for administration has a defined length and inner diameter.
The radioactivity concentration is determined before
administration.
CHARACTERS
Appearance
Clear, colourless gas.
Half-life and nature ofradiation ofkrypton-81m
See general chapter 5. 7. Table oi physical characteristics oi
radionuclides.
IDENTIFICATlON
A. Gamma-ray and X-ray spectrometry.
Result The gamma photon of krypton-81 m has an energy of
0.190 MeV.
B. The half-life of krypton-81m is 11.8 s to 14.4 s.
TESTS
RADIONUCLIDIC PURITY
Radionuclides other than krypton-81m
Maximum 0.1 per cent of the radioactivity passed through
the absorber, calculated with reference to the date and time
of administration.
Gamma-ray and X-ray spectrometry Elute the generator as
prescribed. Pass a sufficient amount (2 L to 10 L) of eluate
at a suitable flow rate through a suitable absorber such as
water. Determine the amount of radioactivity eluted. Allow
the krypton-81m to decay for 5 min and record the gamma
and X-ray spectrum of the residual radioactivity on the
absorber using a suitable instrumentoExamine the gamma-
ray and X -ray spectrum of the absorber for the presence of
radio active impurities, which must be identified and
quantified.
RADIOACTlVITY
Determine the radio active concentration of the preparation
using suitable equipment such as an ionisation chamber or a
gamma ray spectrometer. The radioactivity is measured
Radiopharmaceutical Preparations IV-673
under defined operating conditions, such as gas flow rate and
measurement geometry, that are identical to those used for
the calibration of the instrumento
STORAGE
The storage conditions apply to the generator.
LABELLING
The labelling conditions apply to the generator.
_ _ _ _ _ _ _ _ __ _ __ __ _ _ _ _ _ _ _ ~Ew
L-Methionine ([11C]Methyl) Injection *****
** **
(Ph Bur monograph 1617) ***
~Ew _________ _ _ _ _ __ __ _ _ _ _ ___
DEFINITlON
Sterile solution of (2S)-2-amino-4-
([ lI C]methylsulfanyl)butanoic acid for diagnostic use.
Content
90 per cent to 110 per cent of the dec1ared carbon-ll
radioactivity at the date and time stated on the labe!'
Purity:
-- minimum of 99 per cent of the total radioactivity
corresponds to carbon-11,
-- minimum of 95 per cent of the total radioactivity
corresponds to carbon-ll in the form of
L- [methyl-lI q methionine and D- [methyl-lI q methionine,
-- maximum of 10 per cent of the total radioactivity
corresponds to carbon-ll in the form of
D-[methyl-llqmethionine.
Content of methionine
Maximum of 2 mg per maximum recommended dose in
millili tres.
PRODUCTlON
RADIONUCLIDE PRODUCTION
Carbon-l1 is a radioactive isotope of carbon which is most
commonly produced by proton irradiation of nitrogen.
Depending on the addition of either trace amounts of oxygen
or small amounts of hydrogen, the radioactivity is obtained as
¡I lqcarbon dioxide or [lIqmethane.
RADIOCHEMICAL SYNTHESIS
L-[Methyl-l1qmethionine can be prepared by various
chemical synthetic pathways. All methods rely on the
alkylation of the sulfide anion of L-homocysteine with
[lIqmethyl iodide or [l1qmethyl triflate. Variations in the
procedures used to generate the sulfide anion of
L-homocysteine and methods to obtain [l1qmethyl iodide
lead to negligible differences with respect to quality in terms
of specific radioactivity, enantiomeric purity and possible
chemical and radiochemical impurities.
Synthesis of [l1C]methyl iodide
[lIqMethyl iodide can be obtained either starting from
[llqcarbon dioxide or from [llqmethane. The most
frequently used method is the reduction of [l1qcarbon
dioxide with lithium aluminium hydride. The formed
[l1qmethanol is reacted with hydroiodic acid. Altematively
[llqmethane, either obtained directly in the target or by
IV-674 Radiopharmaceutical Preparations
on-line processes from [llC]carbon dioxide, is reacted with
iodine.
Synthesis of [llC]methyl triflate
e
[llC]methyl triftate can be prepared from lC]methyl iodide
using a silver triftate-impregnated solid support such as
graphitised carbono
Synthesis ofl- [methyl-ll C ]methionine
The most widely used method to obtain L-
[methyl-llC]methionine is the alkylation of the sulfide anion,
e
generated from L-homocysteine thiolactone, with lC]methyl
iodide or [1lC]methyl triftate in alkaline conditions in a
solvent such as acetone. The L-[methyl-llC]methionine
obtained can be purified by semi-preparative liquid
chromatography. For example, a column packed with
octadecylsilyl silica gel for chromatography eluted with a
9 gIL solution of sodium chloride is suitable.
1-Homocysteine thiolactone hydrochloride
Specific optical rotation (2.2.7): + 20.5 to + 21.5,
determined on a 10 giL solution at 25 0e.
Infrared absorption spectrophotometry (2.2.24).
Companson Ph. Eur. reference spectrum of L-homocysteine
thiolactone hydrochlonde.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation of carbon-ll
See general chapter 5.7. Table of physical charactenstics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
R esults The only gamma photons have an energy of
0.511 MeVand, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. Radionuc1idic purity (see Tests) .
e. Examine the chromatograms obtained in the test for
radiochemical purity.
R esults The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (b).
TESTS
pH (2.2.3)
4.5 to 8.5.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaceutical preparations (0 125).
The injection may be released for use before completion of
the test.
Bacterial endotoxins (2.6.14)
Less than 175/V IU/mL, V being the maximum
recommended dose in millilitres. The injection may be
released for use before completion of the test.
CHEMICAL PURITY
Impurity A, impurity B and methionine
Liquid chromatography (2.2.29) .
Test solution The preparation to be examined.
Reference solution (a) Dissolve 0.6 mg of L-homocysteine
thiolactone hydrochlonde R, 2 mg of DL-homocysteine R and
2 mg of DL-methionine R in water R and dilute to V, V being
the maximum recommended dose in millilitres.
2014
R eference solution (b) Dissolve 2 mg of L-111ethionine R in the
same solvent as used in the test solution and dilute to 10 mL
with the same solvento
Colwnn:
- size: l = 0.25 m, 0 = 4.6 mm,
- stationa/Y phase: spherical octadecylsi!yl silica gel for
chromatography R (5 flm) with a specific surfa ce of 220
m 2/g, a pore size of 8 nm and a carbon loading of
6.2 per cent,
temperature: 25 ce.
Mobile phase 1.4 gIL solution of potassiwn dihydrogen
phosphate R .
Flow rate 1 mUmin.
Detection Spectrophotometer at 225 nm and radioactivity
detector connected in series.
Injection Loop inj ector.
Run time 10 mino
Relative retention With reference to methionine
(retention time = about 2.6 min): impurity B = about 0.8,
impurity A = about 2.7.
System suitability Reference solution (a):
- resolution: minimum of 2.5 between the peaks due to
methionine and impurity B.
Limits Examine the chromatogram obtained with the
spectrophotometer:
- impunty A: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (0.6 mglV),
- impunty B: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (2 mglV),
- methionine: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (2 mglV) .
Residual solvents (2.4.24)
Maximum 50 mglV for the concentration of acetone, V being
the maximum recommended dose in millilitres.
The preparation may be released for use before completion
of the test.
RADIONUCLlDIC PURITY
Carbon-ll
Minimum 99 per cent of the total radioactivity.
A. Gamma-ray spectroscopy.
Companson Standardised ftuorine-18 solution, or by using
an instrument calibrated with the aid of such a solution.
Standardised ftuorine-18 solutions and/or standardisation
services are available from the competent authority.
R esults The spectrum obtained with the solution to be
examined does not differ significantly from that obtained
with a standardised ftuorine-1 8 solution.
B. Half-life: 19.9 min to 20.9 mino
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
1-[Methyl-llC]methionine and impurity E
Liquid chromatography (2.2.29) as described in the test for
impurity A, impurity B and methionine.
Injection Test solution and reference solution (b).
Limits Examine the chromatogram obtained with the
radioactivity detector:
total of L-[methyl-llCj methionine and impunty E : minimum
of 95 per cent of the total radioactivity,
2014 Radiopharmaceutical Preparations IV-675

-- other peaks in the chromatogram may be due to H NH 2

H[11 C] V
impurity C, impurity D and impurity F. 3 -"S~C02H andenanliomer
11
ENANTIOMERIC PURITY o
Impurity E
Thin-Iayer chromatography (2.2.27) . D . (2RS)-2-amino-4-(e lC]methylsulfinyl)butanoic acid
Test solution The preparation to be examined. (DL-[methyl-llC]methionine S-oxide),
Reference solution (a) Dissolve 2 mg of L-methionine R in
water R and dilute to 10 mL with the same solvento
Reference solution (b) Dissolve 4 mg of DL-methionine R in
water R and dilute to 10 mL with the same solvento
Plate TLC octadecysilyl silica gel plate for chiral separations R. E. (2R)-2-amino-4-([1lC)methylsulfanyl)butanoic acid
Mobile phase methanol R, water R (50:50 V/V). (D- [methyl_ll C]methionine),
Application 2-10 ¡tL.
Development Over a path of 8 cm.
Drying In air for 5 mino F. [llC]methanol
Detection Spray with a 2 gIL solution of ninhydrin R in ____ _ __ _ _ __ _ _ _ _ _ _ __ _ _ _ _ _ Ph Eur
ethanol R and heat at 60 oC for 10 mino Determine the
distribution of radioactivity using a suitable detector.
Retardationfactors L-[methyl-llC]methionine = about 0.58;
impurity E = about 0.51.
System suitability The chromatogram obtained with
Oxygen eSO) *****
reference solution (b) shows 2 clearly separated spots.
** **
(Ph Eur monograph 1620) ***
Limits: ~Ew _ __ _ _ _ _ __ __ _ _ _ ____ _ _ __ __
-- total of L-[methyl-llC}methionine and impurity E: minimum
95 per cent of the total radioactivity, DEFINITION
-- impurity E: maximum 10 per cent of the total e
Mixture of 5 0Joxygen in the gaseous phase and a suitable
radioactivity. vehicle such as Medicinal air (1238), for diagnostic use.
The preparation may be released for use before completion Purity:
of the test. -- minimum 99 per cent of the total radioactivity
corresponds to oxygen-1S,
RADIOACTIVITY
-- minimum 97 per cent of the total radioactivity
Measure the radioactivity using suitable equipment by
corresponds to oxygen-1S in the form of oxygen (Oz).
comparison with a standardised f1uorine-18 solution or by
measurement in an instrument calibrated with the aid of such PRODUCTION
a solution. RADIONUCLIDIC PRODUCTION
Oxygen-1S is a radio active isotope of oxygen which may be
LABELLING
produced by various nuclear reactions such as pro ton
The accompanying information specifies the maximum
irradiation of nitrogen-1S or deuteron irradiation of nitro gen-
recommended dose in millilitres.
14.
IMPURITIES RADIOCHEMICAL SYNTHESIS
In order to recover oxygen-1S as molecular oxygen from the
nitrogen target gas, carrier oxygen is added at concentrations
generally ranging from 0.2 per cent V/V to 1.0 per cent V/V.
After irradiation, the target gas is usually passed through
activated charco al and a carbon dioxide scavenger, such as
soda lime, before mixing with the vehicle .
A. (3S)-3-aminodihydrothiophen-2(3H)-one (L-homocysteine
thiolactone), CHARACTERS
Appearance
Colourless gas.
Half-life and nature of radiation of oxygen-15
See general chapter 5.7. Table of physical characteristics oi
radionuclides.
B. (2S)-2-amino-4-sulfanylbutanoic acid (L-homocysteine), IDENTIFICATION
A. Gamma spectrometry.
H NH 2 Results The only gamma photons have an energy of
H[11C] V 0.511 MeV and, depending on the measurement geometry, a
3 -.. S ~ CO H and enanliomer
ti \\ 2
o o sum peak of 1.022 MeV may be observed.
B. Radionuclidic purity (se e Tests) .
C . (2RS)-2-amino-4-(e lC]methylsulfonyl)butanoic acid C. Examine the chromatograms obtained in the test for
(DL- [methyl_ll C]methionine S,S-dioxide), radiochemical purity.
Results The retention times of the principal peaks in the
chromatogram obtained with the test gas using the
 IV-676 Radiopharmaceutical Preparations
radioactivity detector are similar to those of the principal
peaks corresponding to oxygen in the chromatogram
obtained with the reference gas using the thermal
conductivity detector.
TESTS
The following tests are performed on / 50joxygen as described
under radiochemical synthesis before mixing with the vehicle.
RADIONUCLIDIC PURITY
Oxygen-15
Minimum 99 per cent of the total radioactivity.
A. Gamma spectrometry.
Comparison Standardised fluorine-18 solution, or by using
an instrument calibrated with the aid of such a solution.
Standardised fluorine-18 solutions and/or standardisation
services are available from the competent authority.
Results The spectrum obtained with the solution to be
examined does not differ significantly from that obtained
with a standardised ftuorine-18 solution.
B. Half-life: 1.9 min to 2.2 mino
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
Oxygen-15 in the forrn of O 2
Gas chromatography (2.2.28): use the normalisation
procedure.
Test sample ¡JsO]oxygen as described under radiochemical
synthesis.
Reference gas Nitrogen gas mixture R.
Column:
-- size: l = 1.8 m, 01 = 6.3 mm and 02 = 3.2 mm,
-- stationary phase: GC concentrical column R.
Carrier gas Helium for chromatography R.
Flow rate 65 mUmin.
Temperature:
-- column: 40 oC,
-- injection port: 40 oC,
-- thermal conductivity detector: 70 oc.
Detection Thermal conductivity detector and radioactivity
detector connected in series.
Injection Loop injector.
Run time 10 min.
Retention times Oxygen, nitrogen and carbon monoxide
eluting from the inner column = about 0.4 min; carbon
dioxide eluting from the inner column = about 0.8 min;
oxygen eluting from the outer column = about 2.1 min;
nitrogen eluting from the outer column = about 3.1 min;
carbon monoxide eluting from the outer column = about
6.2 mino
System suitability Reference gas:
-- 5 c1early separated principal peaks are observed in the
chromatogram obtained using the thermal conductivity
detector,
resolution: minimum of 1.5 between the peaks due to
carbon dioxide eluting from the inner column and oxygen
eluting from the outer column, in the chromatogram
obtained using the thermal conductivity detector.
Limits Examine the chromatogram obtained with the
radioactivity detector and calculate the percentage content of
oxygen-15 substances from the peak areas.
-- oxygen-15 gas in the form of Oi minimum 97 per cent of
the total radioactivity,
2014
-- disregard the first peak corresponding to components
co-eluting from the inner column.
RADIOACTIVITY
The radioactive concentration is determined before
administration.
Measure the radioactivity using suitable equipment by
comparison with a standardised fluorine-18 solution or by
measurement in an instrument calibrated with the aid of such
a solution.
____________________________________________
~Ew
Raclopride (C 1C]Methoxy)
Injection
(Ph Eur monograph 1924)
Ph Ew ____________________________________________
DEFINITION
Sterile solution of 3,5-dichloro-N- [[ (2S)-I-ethylpyrrolidin-2-
yl] methyl]-2-hydroxy-6-( [11 C] methoxy) benzamide.
Content
90 per cent to 11 O per cent of the declared carbon-11
radioactivity at the date and time stated on the labe!'
Purity:
-- minimum of 99 per cent of the total radioactivity
corresponds to carbon-ll,
-- minimum of 95 per cent of the total radioactivity
corresponds to carbon-ll in the form of
[methoxy-II C] raclopride.
Content of raclopride
Maximum of 10 ¡.tg per maximum recommended dose in
millilitres.
PRODUCTION
RADIONUCLIDE PRODUCTION
Carbon-ll is a radio active isotope of carbon most commonly
produced by proton irradiation of nitrogen. Depending on
the addition of either trace amounts of oxygen or small
amounts of hydrogen, the radioactivity is obtained as
[llC]carbon dioxide or ¡JIC]methane, respectively.
RADIOCHEMICAL SYNTHESIS
[Methoxy_llC]raclopride may be prepared by O-alkylation of
the corresponding phenolate anion (S)-3,5-dichloro-2,6-
dihydroxy-N- [( l-ethylpyrrolidin-2-yl)methyl] benzamide with
iodo[llC]methane or [llC]methyl trifluoromethanesulfonate.
Synthesis of iodo[llC]methane
Iodo[llC]methane may be produced from [llC]carbon
dioxide or from ¡I1C]methane. The most frequently used
method is reduction of [11C]carbon dioxide with lithium
aluminium hydride. The lithium aluminium
¡I1C]methanolate formed is reacted with hydroiodic acid to
iodo[llC]methane via ¡JIC]methanol. Alternatively
[llC]methane, either obtained directly in the target or by
on-line processes from [llC]carbon dioxide, is reacted with
iodine.
 2014
Synthesis of [llC]methyl triftuoromethanesulfonate
[llC]Methyl trifluoromethanesulfonate may be prepared from
iodo[llC]methane using a solid support such as graphitised
carbon impregnated with silver trifiuoromethanesulfonate.
Synthesis of [methoxy_llC]raclopride
Methylation with iodo[llC]methane is performed under
alkaline conditions in a solvent such as dimethyl sulfoxide.
The methylation with [llC]methyl trifiuoromethanesulfonate
is performed in a solvent such as dimethylformamide or
acetone. The resulting [metho~_IIC]rac1opride may be
purified by semi-preparative liquid chromatography using, for
example, a column packed with octadecylsilyl silica gel for
chromatography eluted with a mixture of 25 volumes of
acetonitrile and 75 volumes of 0.0 1 M phosphoric acid.
PRECURSOR FOR SYNTHESIS
(S)-3,5-Dichloro-2,6-dihydroxy-N-[(1-ethylpyrrolidin-
2-yl)methyl]benzamide hydrobromide
Melting point (2.2.14): 211 oC to 213 oC.
Specific optical rotation (2.2.7): + 11.3 to + 11.5,
determined on a 15.0 gIL solution in ethanol R at 22 oc.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiadon of carbon-ll
See general chapter 5.7. Table of physieal eharaeteristics of
radionuclides.
IDENTIFICAnON
A. Gamma-ray spectrometry.
Results The only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. It complies with test B for radionuc1idic purity (se e Tests).
C. Examine the chromatograms obtained in the test for
radiochemical purity.
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (d).
TESTS
pH (2.2.3)
4.5 to 8.5.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaeeutieal preparations (0125).
The injection may be released for use before completion of
the test.
Bacterial endotoxins (2.6.14)
Less than 175/VIU/rnL, Vbeing the maximum
recommended dose in millilitres. The injection may be
released for use before completion of the test.
CHEMICAL PURITY
Raclopride and impurity A
Liquid chromatography (2.2.29).
Test solution The preparation to be examined.
Referenee solution (a) Dissolve 7.2 mg of raelopride tartrate R
in water R and dilute to 50 mL with the same solvent.
Referenee solution (b) Dissolve 1.2 mg of (S)-3,5-diehloro-
2,6-dihydroxy-N-[(l-ethylpyrrolidin-2-yl)methyij benzamide
hydrobromide R in methanol R and dilute to 100 mL with the
same solvent.
Radiopharmaceutical Preparations IV-677
Referenee solution (e) To 0.1 mL of reference solution (a)
add 0.1 mL ofreference solution (b) and dilute to Vwith
water R, V being the maximum recommended dose in
miJlilitres.
Referenee solution (d) Dilute 1.0 mL of reference solution (a)
to 10.0 mL with water R.
Column:
- size: 1 = 0.05 m, 0 = 4.6 mm,
- stationary phase: spherical end-eapped octadecylsilyl silica gel
for chromatography R (3 .5 [.1m) with a specific surface are a
of 175 m 2/g, a pore size of 12.5 nm, a pore volume of
0.7 cm 3/g and a carbon loading of 15 per cent,
- temperature: 30 cC.
Mobile phase Dissolve 2 g of sodiUln heptanesulfonate R in
700 mL of water R, adjust to pH 3.9 with phosphoric acid R
and dilute to 1000 mL with aeetonitrile R .
Flow rate 1 mUmin.
Detection Spectrophotometer at 220 nm and radioactivity
detector connected in series.
Injection Loop injector; inject the test solution and reference
solutions (b) and (e) .
Run time 10 mino
Relative retention With reference to rac1opride:
impurity A = about 0.46.
System suitability Reference solution (e):
- resolution: minimum of 5 between the peaks due to
rac10pride and to impurity A.
Limits Examine the ehromatogram obtained with the
spectrophotometer:
- raclopride: not more than the area of the corresponding
peak in the ehromatogram obtained with reference
solution (c) (lO [.Ig/V),
- impurity A: not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (l [.IglV).
Residual solvents
Are limited according to the principies defined in [he general
ehapter (5.4), using the general method (2.4.24).
The preparation may be released for use before completion
of the test.
RADIONUCLIDIC PURITY
Carbon-ll
Minimum 99 per cent of the total radioactivity.
The preparation may be relea sed for use before eompletion
of the test.
A. Garnma-ray spectrometry.
Comparison Standardised fiuorine-18 solution, or by using a
calibrated instrumento Standardised fiuorine-1 8 solutions
and/or standardisation services are available from the
competent authority.
Results The speetrum obtained with the solution to be
examined does not differ signifieantly from that obtained
with a standardised fiuorine-18 solution.
B. Half-life. 19.9 min to 20.9 mino
RADIOCHEMICAL PURITY
Liquid chromatography (2.2.29) as described in the test for
rac10pride and impurity A with the foJlowing modifieations.
Injection Test solution and reference solution (d).
Limits Examine the chromatogram obtained with the
radioactivity detector:
[Methoxy_ll C] raclopride: minimum of 95 per cent of the
total radioactivity.
 IV-678 Radiopharmaceutical Preparations 2014

RADIOACTIVITY Half-life and nature of radiation of carbon-ll

Mesure the radioactivity using suitable equipment by See general chapter 5.7. Table of physical characteristics of
comparison with a standardised ftuorine-18 solution or by radionuclides.
using a calibrated instrumento IDENTIFICATION
LABELLING A. Gamma-ray spectrometry.
The accompanying information specifies the maximum Results The only gamma photons have an energy of
recommended dose in millilitres. 0.511 MeV and, depending on the measurement geometry, a
IMPURlTIES sum peak of 1.022 MeV may be observed.
B. It complies with test B for radionuclidic purity (see Tests).

e'lXN/'.J:)
I
OH

#'
O

OH
H H N
~
C. Examine the chromatograms obtained in the test for
radiochemical purity.
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
el eH 3 the principal peak in the chromatogram obtained with the
reference solution.
A. 3,5-dichloro-N- [[ (2S)-I-ethylpyrrolidin-2-yl]methyl]-2,6- TESTS
dihydroxybenzamide. pH (2.2.3)
____________________________________________ PhEw 4.5 to 8.5.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaceutical preparations (0125).
The injection may be relea sed for use before completion of
Sodium Acetate ([1_ 11 C]) Injection the test.
(Ph Bur monograph 1920) Bacterial endotoxins (2.6.14)
Less than 175/V IU/mL, V being the maximum
CH/ICOONa
PhEw ____________________________________________
recommended dose in millilitres . The injection may be
released for use before completion of the test.
DEFINITION CHEMICAL PURITY
Sterile solution ofsodium [1-11C]acetate, in equilibrium with Acetate
[1_ 11 C] acetic acid. Liquid chromatography (2.2.29).
Content Test solution The preparation to be examined.
90 per cent to 11 O per cent of the declared carbon-ll
Reference solution Dissolve 28 mg of sodium acetate R in
radioactivity at the date and time stated on the labeL
water R and dilute to V, V being the maximum
PRODUCTION recommended dose in millilitres.
RADIONUCUDE PRODUCTION Column:
Carbon-ll is a radioactive isotope of carbon which is most -- size: 1 = 0.25 m, 0 = 4.0 mm;
commonly produced by pro ton irradiation of nitrogen. By the -- stationary phase: strongly basic anion exchange resin for
addition of trace amounts of oxygen, the radioactivity is chromatography R (lO J.lm);
obtained as [llC]carbon dioxide. -- temperature: 25 oc.
RADIOCHEMICAL SYNfHESIS Mobile phase 0.1 M sodium hydroxide protected from
[llC]Carbon dioxide may be separated from the target gas atrnospheric carbon dioxide .
mixture by cryogenic trapping or by trapping on a molecular Flow rate 1 mUmin.
el
sieve at room temperature. C] Carbon dioxide is then
Detection Spectrophotometer at 220 nm and radioactivity
released from the trap using an inert gas such as nitrogen at
detector connected in series.
a temperature higher than the trapping temperature.
Injection Loop injector.
[1- 11 C]Acetate is usuaIly prepared by reaction of [llC]carbon
dioxide with methylmagnesium bromide in organic solvents Run time 10 mino
such as ether or tetrahydrofuran. System suitabz1ity Reference solution:
Hydrolysis of the product yields [1_ 11 C] acetic acid. It is -- resolution: minimum 4.0 between the peaks due to hold-
purified by chromatographic procedures. The eluate is up volume and acetate.
diluted with sodium chloride solution. Limi¡ Examine the chromatograms obtained with the
spectrophotometer:
PRECURSOR FOR SYNfHESIS
-- acetate: not more than the area of the corresponding peak
MethyImagnesium bromide in the chromatogram obtained with the reference solution
The reactivity of methyImagnesium bromide is tested by (20 mg per V).
decomposition of a defined amount with water. The amount
Residual solvents
of methane released during this reaction is not less than
90 per cent of the theoretical value. Are limited according to the principIes defined in the general
chapter (5.4), using the general method (2.4.24) .
CHARACTERS The preparation may be released for use before completion
Appearance of the test.
Clear, colourIess solution.
2014
RADIONUCLIDIC PURITY
Carbon-ll
Minimum 99 per cent of the total radioactivity.
The preparation may be released for use before completion
of the tests .
A. Gamma-ray spectrometry.
Comparison Standardised fiuorine-18 solution, or by using a
calibrated instrumento Standardised fiuorine-18 solutions
and/or standardisation services are available from laboratories
recognised by the competent authority.
Results The spectrum obtained with the solution to be
examined does not differ significantIy from that obtained
with a standardised fiuorine-18 solution.
B. Half-Jife : 19.9 min to 20.9 mino
RADIOCHEMICAL PURITY
[l- Il C]Acetate
Liquid chromatography (2.2.29) as described in the test for
acetate.
Limit Examine the chromatograms obtained with the
spectrophotometer and the radioactivity detector:
-- total of [l- JICjacetate: minimum 95 per cent ofthe total
radioactivity.
RADIOACTIVITY
Measure the radioactivity using suitable equipment by
comparison with a standardised fiuorine-18 solution or by
measurement with a caJibrated instrumento
LABELLING
The accompanying information specifies the maximum
recommended dose in millilitres.
____________________________________________
Sodium Chromate (51 Cr) Sterile
*** ***
Solution
(Ph Eur monograph 0279)
Ph Eur ____________________________________________
DEFINITION
Sterile solution of sodium e1Cr]chromate made isotonic by
the addition of sodium chloride.
Chromium-51
90 per cent to 110 per cent of the declared chromium-51
radioactivity at the date and time stated on the label.
Specific radioactivity
Minimum 370 MBq of chromium-51 per milligram of
chromate ion.
CHARACTERS
Appearance
Clear, colourless or sJightly yellow solution.
Half-life and nature of radiation of chromium-51
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The only gamma photon of chromium-51 has an
energy of 0.320 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity (se e Tests).
Radiopharmaceutical Preparations IV-679
Result The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is about
0.9.
TESTS
pH (2.2.3)
6.0 to 8.5 .
Total chromate
Maximum 2.7 Ilg of chromate ion (CrO/ -) per MBq.
Test solution The preparation to be examined.
Reference solution 1.7 mg/L solution of potassium chromate R.
Measure the absorbance ofthe solutions (2.2.25) at the
absorption maximurn at 370 nm. If necessary, adjust the test
solution and the reference solution to pH 8.0 by adding
sodium hydrogen carbonate solution R. Calculate the content of
chromate in the preparation to be examined using the
measured absorbances.
Sterility
It complíes with the test for steriJity prescribed in the
monograph Radiopharmaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
RADIONUCLIDIC PURITY
Chromium-51
Gamma-ray spectrometry.
Result The spectrum obtained with the preparation to be
examined does not differ significantIy from that obtained
with a standardised chromium-51 solution.
RADIOCHEMICAL PURITY
51
[ Cr]Chromate ion
PhE~ Ascending paper chromatography (2.2.26).
Test solution The preparation to be examined.
Paper paper for chromatography R.
Mobile phase ammonia R, ethanol (96 per cent) R, water R
*** (25 :50:125 V/V/V).
Application A volurne of the solution sufficient for the
*** detection method.
Development Irnmediately, for 2.5 h.
Detection Suitable detector to determine the distribution of
the radioactivity.
Retardationfactor Impurity A = 0.0 to 0.1;
chromate ion = about 0.9.
Limit:
51
- - [ Crjchromate ion: minimum 90 per cent of the total
radioactivity due to chromium-51 .
RADIOACTIVITY
Determine the radioactivity using a calíbrated instrumento
IMPURITIES
A. [5I Cr]chromium(I1I) ion.
____________________________________________ ~E~
 IV-680 Radiopharmaceutical Preparations
Sodium Fluoride C8F) Injection
(Ph Eur monograph 2100)
~E~ ____________________________________________
DEFINITION
Sterile solution containing fiuorine-l S in the form of sodium
fiuoride. It may contain carrier fiuoride and a suitable buffer.
Content:
-- fiuorine-18: 90 per cent to 110 per cent of the declared
fiuorine-18 radioactivity at the date and hour stated on
the label,
-- fiuoride: maximum 4.52 mg per maximum recommended
dos e in millilitres.
PRODUCTION
The radionuclide fiuorine-18 is most commonly produced by
proton irradiation ofwater enriched in oxygen-18 . Fluorine-
18 in the form of fiuoride is recovered from the target water,
generally by adsorption and desorption from anion-exchange
resins or electrochemical deposition and redissolution.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of fluorine-18
See general chapter 5.7. Table 01 physical characteristics
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry, a
sum peak of 1.022 MeV may be observed.
B. It complies with test B for radionuclidic purity (see Tests).
C. Examine the chromatograms obtained in the test for
radiochemical purity (see Tests) .
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with the
reference solution. In the chromatogram obtained with the
reference solution, the peak due to fiuoride is negative.
TESTS
pH (2.2.3)
5.0 to 8.5.
Fluoride
Liquid chromarography (2.2.29) .
Test solution The preparation ro be examined.
Relerence solution Dissolve 10 mg of sodium fiuoride R in
water R and dilute to V with the same solvent, V being the
maximum recommended dos e in millilitres.
Column:
-- size: l = 0.25 m, 0 = 4 mm,
-- stationary phase: strongly basic anion-exchange resin lor
chromatography R (10 )lm),
-- temperature: constant, between 20 oC and 30 oc.
Mobile phase 4 gIL solution of sodium hydroxide R, protected
from atmospheric carbon dioxide.
Plow rate 1 mUmin.
Detection Spectrophotometer at 220 nm and a radioactivity
detector connected in series.
1njection 2 O ¡.¡.L.
Run time 15 mino
2014
*****
** **
System suitability Examine the chromatogram obtained with
the reference solution using the spectrophotometer:
*** -- signal-to-noise ratio: minimum 10 for the principal peak,
-- retention time 01 fiuoride: minimum 3 times the hold-up
time.
Limit Examine the chromatogram obtained with the
spectrophorometer:
-- fiuoride: not more than the area of the corresponding peak
in the chromatogram obtained with the reference solution
(4 .52 mg/V) .
Sterility
Ir complies with the test for sterility prescribed in the
monograph on Radiopharmaceutical preparations (0125) .
The injection may be released for use before completion of
the test.
Bacterial endotoxins (2.6.14)
Less than 175/V IU/mL, V being the maximum
recommended dose in millilitres. The injection may be
released for use before completion of the test.
RADIONUCLIDIC PURITY
Fluorine-18
Minimum 99.9 per cent of the total radioactivity.
The preparation may be released for use before completion
of the tests.
01 A. Gamma-ray spectrometry.
Determine the amount of fiuorine-18 and radionuclidic
impurities with a half-life longer than 2 h. For the detection
and quantification of impurities, retain the preparation ro be
examined for a sufficient time to allow the fiuorine-18 to
decay to a level which permits the detection of impurities.
Results The spectrum obtained with the preparation to be
examined does not differ significantly from that of a
background spectrum.
B. Half-life: 105 min ro 115 mino
RADIOCHEMICAL PURlTY
C8F]fluoride
Liquid chromarography (2.2.29) as described in the test for
fiuoride . Jf necessary, dilute the test solution with water R to
obtain a radioactivity concentration suitable for the
radioactivity detector.
Limit Examine the chromatogram obtained with the
radioactivity detector:
-- ¡T8Plfiuoride: minimum 98.5 per cent of the total
radioactivity .
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label states the maximum recommended dose in
millilitres.
____________________________________________ Ph Eur
 2014
Sodium lodide C23 1) Injection ***
** **
(Ph Bur monograph 0563) **** *
~E~ ____________________________________________
DEFINITION
Sterile solution containing iodine-123 in the form of sodium
iodide; it may contain sodium thiosulfate or sorne other
suitable reducing agent and a suitable buffer.
Content
90 per cent to 110 per cent ofthe declared iodine-123
radioactiviry at the date and hour stated on the labe!'
PRODUCTION
Iodine-123 is obtained by proton irradiation of xenon
enriched in xenon-124 (minimum 98 per cent) followed by
the decay of xenon-123 which is formed directly and by the
decay of caesium-123. No carrier iodide is added.
CHARACTERS
Appearance
Clear, colourless solution.
Half-Jife and nature of radiation of iodine-123
See general chapter 5.7. Table of physieal eharacteristies of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The spectrum obtained with the preparation to be
examined does not differ significantIy from that of a
standardised iodine-123 solution. The most prominent
gamma photon has an energy of O.159 MeV and is
accompanied by the principal X-ray of 0.027 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical puriry.
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
pH (2.2.3)
7.0 to 10.0.
Sterility
It complies with the test for steriliry prescribed in the
monograph on Radiopharmaeeutical preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIONUCLIDIC PURlTY
Iodine-123
Minimum 99.65 per cent of the total radioactiviry.
Gamma-ray spectrometry.
Determine the relative amounts ofiodine-123, iodine-125,
tellurium-121 and other radionuclidic impurities presento
For the detection of tellurium-121 and iodine-125, retain the
preparation to be examined for a sufficient time to allow
iodine-123 to decay to a leve! which permits the detection of
radionuclidic impurities. No radionuclides with a half-life
longer than that ofiodine-125 are detected .
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURlTY
[ 123I]iodide
Liquid chromatography (2.2.29).
Radiopharmaceutical Preparations IV-681
Test solution Dilute the preparation to be examined with a
2 giL solution of sodium hydroxide R to a radioactive
concentration suitable for the detector. Add an equal volume
of a solution containing 1 gIL of potassium iodide R, 2 gIL of
potassium iodate R and 10 gIL of sodium hydrogen carbonate R
and mix.
Reference solution (a) Dilute 1 mL of a 26.2 mglL solution
of potassium iodide R to 10 mL with water R .
Referenee solution (b) Dilute 1 mL of a 24.5 mgIL solution
of potassium iodate R to 10 mL with water R . Mix equal
volumes of this solution and reference solution (a).
Column:
-- size: 1 = 0.25 m, 0 = 4.0 mm,
-- stationary phase: oetadeeylsilyl si/ica gel for chromatography R
(5 ¡.tm),
-- temperature: constant between 20 oC and 30 oC.
Mobile phase Dissolve 5.85 g of sodium ehloride R in
1000 mL of water R, add 0.65 mL of octylamine R and adjust
to pH 7.0 with dilute phosphorie acid R; add 50 mL of
aeetonitrile R and mix.
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 220 nm and a radioactiviry
detector connected in series.
Injeetion 20 ¡.tL.
Run time 12 mino
Relative retention With reference to iodide
(retention time = about 5 min): iodate = 0.2 to 0.3.
System suitability Reference solution (b):
-- resolution: minimum 2 between the peaks due to iodide
and iodate in the chromatogram recorded with the
spectrophotometer.
Limit Examine the chromatogram obtained with the test
solution using the radioactiviry detector and locate the peak
due to iodide by comparison with the chromatogram
obtained with reference solution (a) using the
spectrophotometer:
123
-- [ Ijiodide: minimum 95 per cent of the total
radioactiviry .
RADIOACTIVITY
Determine the radioactiviry using a calibrated instrumento
LABELLlNG
The label states the name of any excipient.
IMPURITIES
A. [ 123 I]iodate ion.
____________________________________________ PhE~
Sodium lodide C23 1) Solution for ***
** **
Radiolabelling **** *
(Ph Bur monograph 2314)
~E~ ____________________________________________
DEFINITION
Strongly alkaline solution containing iodine-123 in the form
of sodium iodide.
Content
90 per cent to 11 O per cent of the declared iodine-123
radioactiviry at the date and hour stated on the labe!'
PRODUCTION
Iodine-1 23 is obtained by proton irradiation ofxenon highly
enriched in xenon-124 followed by the decay of directly
 IV-682 Radiopharmaceutical Preparations 2014

formed xenon-1 23 and by the decay of caesium-123. to pH 7.0 with di/ute phosphoric acid R; add 50 mL of
No carrier iodide or reducing agents are added. acetonitrile R and mix.
CHARACTERS Flow rate 1.5 mUmin.
Appearance Detection Spectrophotometer at 220 nrn and a radioactivity
C lear, colourless solution. detector connected in series.
Half-life and nature ofradiation ofiodine-123 Injection 20 ¡.¡L.
See general chapter 5.7. Table of physieal eharacteristies oi Run time 12 min.
radionuclides. Relative retention With reference to iodide
IDENTIFICATION (retention time = about 5 min): iodate = 0.2 to 0.3.
A. Gamma-ray spectrometry. System suitability Reference solution (b):
Results The most prominent gamma photon of iodine-123 -- resolution: minimum 2 between the peaks due to iodide
has an energy of 0.159 MeV and is accompanied by the and iodate in the chromatogram recorded with the
principal X-ray of 0.027 MeV. spectrophotometer.
B. Examine the chromatograms obtained in the test for Examine the chromatogram obtained with the test solution
radiochemical purity (se e Tests) . using the radioactivity detector and locate the peak due to
Results The principal peak in the radiochromatogram iodide by comparison with the chromatogram obtained with
reference solution (a) using the spectrophotometer.
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with Limit:
reference solution (a). -- ¡I 23I]iodide: minimum 95 per cent of the total
radioactivity.
TESTS
Alkalinity (2.2.4) RADIOACTIVITY
The solution is strongly alkaline. Determine the radioactivity using a calibrated instrumento
RADIONUCLIDIC PURITY LABELLING
Iodine-123 The label states:
Minimum 99.7 per cent of the total radioactivity. -- the name of any excipient;
-- that the solution is not for direct administration to
Gamma-ray spectrometry.
humans.
Determine the relative amounts of iodine-123, iodine-125,
tellurium-121 and other radionuc1idic impurities presento IMPURITIES
For the detection oftellurium-121 and iodine-125, retain the A. iodine-125,
solution to be examined for a sufficient time to allow iodine- B. tellurium-121,
123 to decay to a level which permits the detection of C. [123I]iodate ion.
radionuc1idic impurities. No radionuc1ides with a half-life ____________________________________________ PhE~

longer than that ofiodine-1 25 are detected.

The solution may be released for use before completion of
the test.
RADIOCHEMICAL PURITY
Sodium lodide C31 1) Capsules for ***
[ 123I]iodide *** ***
Liquid chromatography (2.2.29). Diagnostic Use ***
Test solution Dilute the solution to be examined with an (Ph Eur monograph 0938)
equal volume of a solution containing 1 gIL of potassium ~E~ _____________________________________________

iodide R, 2 gIL of potassium iodate R and 10 gIL of sodium DEFINITION

hydrogen carbonate R and mix. If necessary, first dilute the
solution to be examined with a 2 gIL solution of sodium
hydroxide R to ensure that the final mixture has a
radioactivity concentration suitable for the radioactivity
detector.
Referenee solution (a) Dissolve 10 mg of potassium iodide R in
water R and dilute to 10 mL with the same solvento
Referenee solution (b) Dissolve 20 mg of potassium iodate R in
water R and dilute to 10 mL with the same solvent.
Mix equal volumes of this solution and reference
solution (a).
Column:
-- size: l = 0.25 m, 0 = 4.0 mm;
-- stationary phase: octadecylsilyl si/ica gel for chromatography R
(5 ¡.¡m);
-- temperature: constant, between 20 oC and 30 oc.
Use stainless steel tubing.
Mobi/e phase Dissolve 5.85 g of sodium ehloride R in
1000 mL of water R, add 0.65 mL of octylamine R and adjust
Capsules for diagnostic use containing iodine-131 in the form
of sodium iodide on a solid support; they may contain
sodium thiosulfate or sorne other suitable reducing agents
and a suitable buffering substance. A package contains 1 or
more capsules.
Content:
-- iodine-131: maximum 37 MBq per cap su le; the average
radioactivity determined in the test for uniformity of
content is 90 per cent to 110 per cent of the dec1ared
iodine-131 radioactivity at the date and hour stated on
the label;
-- iodide: maximum 20 ¡.¡g per capsule.
PRODUCTION
Iodine-131 is obtained by neutron irradiation oftellurium or
by extraction from uranium fission products. No carrier
iodide is added.
CHARACTERS
Half-life and nature ofradiation ofiodine-131
See general chapter 5.7. Table of physical eharacteristics oi
radionuclides.
2014
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The spectrum obtained with the preparation to be
examined does not differ significantly from that of a
standardised iodine-131 solution. The most prominent
gamma photon has an energy of 0.365 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity.
Results The principal peak in the radiochromatogram
obtained with test solution (b) is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a) .
TESTS
Disintegration
The contents of the capsule dissolve completely within
15 mino
In a water-bath at 37 oC, warm in a small beaker about
20 mL of a 2.0 giL solution of potassium iodide R . Add a
capsule to be examined. Stir magnetically at 20 r/min.
Uniformity of content
Determine the radioactivity of each of not fewer than
10 capsules . Calculate the average radioactivity per capsule.
The radioactivity of no capsule differs by more than
10 per cent from the average, the relative standard deviation
is not greater than 3.5 per cent.
lodide
Liquid chromatography (2.2.29) .
Test solution (a) Dissolve a capsule to be examined in
10 mL of water R . Filter through a 0.2 flm filter.
Test solutwn (b) Dissolve a capsule to be examined in
water R. Filter through a 0.2 flm filter and dilute the filtrate
with a 2 giL solution of sodium hydroxide R to a radio active
concentration suitable for the detector. Add an equal volume
of a solution containing 1 gIL of potassium iodide R, 2 gIL of
potassium iodate R and 10 giL of sodium hydrogen carbonate R
and mix.
Reference solution (a) Dilute 1 mL of a 26.2 mg/L solution
of potassium iodide R to 10 mL with water R .
Reference solution (b) Dilute 1 mL of a 24.5 mg/L solution
of potassium iodate R to 10 mL with water R. Mix equal
volumes of this solution and reference solution (a).
Blank solution Prepare a solution containing 2 mglmL of
each constituent stated on the label, apart from iodide.
Column:
-- size: 1 == 0.25 m, 0 == 4.0 mm,
-- stationary phase: octadecylsilyl si/ica gel for chromatography R
(5 flm),
-- tempera tu re: constant between 20 oC and 30 oc.
Mobile phase Dissolve 5.85 g of sodium ehloride R in
1000 mL of water R, add 0.65 mL of oetylamine R and adjust
to pH 7.0 with dilute phosphoric acid R; add 50 mL of
acetonitrile R and mix.
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 220 nm and radioactivity
detector connected in series.
Injection 20 ~lL of test solution (a) , reference solutions (a)
and (b) and the blank solution.
Run time 12 mino
Relative retention With reference to iodide
(retention time = about 5 min): iodate == 0.2 to 0.3.
Radiopharmaceutical Preparations IV-683
System suitability:
-- in the chromatogram obtained with the blank solution,
none of the peaks has a retention time similar to that of
the peak due to iodide,
-- resolution: minimum 2 between the peaks due to iodide
and iodate in the chromatogram obtained with reference
solution (b) recorded with the spectrophotometer.
Limit Examine the chromatograms obtained with the
spectrophotometer:
-- iodide: not more than the area of the corresponding peak
in the chromatogram obtained with reference solution (a)
(20 ~lg/capsule) .
RADIONUCLIDlC PURITY
l odine-131
Minimum 99.9 per cent ofthe total radioactivity.
Gamma-ray spectrometry.
Determine the relative amounts of iodine-131, iodine-133,
iodine-135 and other radionuclidic impurities present.
RADIOCHEMICAL PURITY
[131 IJiodide
Liquid chromatography (2.2.29) as described in the test for
iodide with the following modifications.
Injection 20 flL of test solution (b) and reference
solution (a) .
Limit Examine the chromatogram obtained with the test
solution using the radioactivity detector and locate the peak
due to iodide by comparison with the chromatogram
obtained with reference solution (a) using the
spectrophotometer:
-- [131 IJiodide: minimum 95 per cent of the total
radioactivity .
RADIOACTIVITY
Determine the radioactivity of the package using a calibrated
instrumento
LABELLING
The label sta tes the name of any excipient and the number of
capsules in the package.
IMPURITIES
A. [I3l I]iodate ion.
____________________________________________ PhE~
Sodium lodide C31 1) Capsules tor ***
*** ***
Therapeutic Use ***
(Ph Eur monograph 2116)
~E~ ____________________________________________
DEFINITION
Capsules for therapeutic use containing iodine-131 in the
form of sodium iodide on a solid support; they contain
sodium thiosulfate or sorne other suitable reducing agents
and a suitable buffering substance .
Content:
-- iodine-131 : 90 per cent to 110 per cent of the declared
radioactivity at the date and hour stated on the label,
-- iodide: maximum 20 flg per capsule.
PRODUCTION
Iodine-131 is obtained by neutron irradiation of tellurium or
by extraction from uranium fission products. No carrier
iodide is added.
IV-684 Radiopharmaceutical Preparations 2014

CHARACTERS System suitability:

Half-life and nature of radiation ofiodine-131
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The spectrum obtained with the preparation to be
examined does not differ significantly from that of a
standardised iodine-131 solution. The most prominent
gamma photon of iodine-131 has an energy of 0.365 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity.
Results The principal peak in the radiochromatogram
obtained with test solution (b) is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
Disintegration
The contents of the capsule dissolve completely within
15 mino
In a water-bath at 37 oC, warm in a small beaker about
20 mL of a 2.0 gIL solution of potassium iodide R. Add a
capsule to be examined. Stir magnetically at a rotation rate of
20 r/min.
Iodide
Liquid chromatography (2.2.29) .
Test solution (a) Dissolve a capsule to be examined in
10 mL of water R. Filter through a 0.2 }lm filter.
Test solution (b) Dissolve a capsule to be examined in
water R. Filter through a 0.2 }lm filter and dilute the filtrate
with an equal volume of a solution containing 1 gIL of
potassium iodide R, 2 gIL of potassium iodate R and 10 gIL of
sodium hydrogen carbonate R. If necessary, first dilute the
filtrate with a 2 gIL solution of sodium hydroxide R to ensure
that the final mixture has a radioactivity concentration
suitable for the radioactivity detector.
Reference solution (a) Dilute 1.0 mL of a 26.2 mg/L solution
of potassium iodide R to 10.0 mL with water R .
Reference solution (b) Dilute 1 mL of a 24.5 mgIL solution
of potassium iodate R to 10 mL with water R. Mix equal
volumes of this solution with reference solution (a).
Blank solution Prepare a solution containing 2 mg/rnL of
each excipient stated on the label, apart from iodide.
Column:
-- size: 1 = 0.25 m, 0 = 4.0 mm,
-- stationary phase: octadecylsilyl silica gel for chromatography R
-- in the chromatogram obtained with the blank solution,
none of the peaks has a retention time similar to that of
the peak due to iodide;
-- resolution: minimum 2 between the peaks due to iodide
and iodate in the chromatogram obtained with reference
solution (b) using the spectrophotometer.
Limits Examine the chromatograms obtained with the
spectrophotometer; locate the peak due to iodide by
comparison with the chromatogram obtained with reference
solution (a):
-- iodide: not more than the area of the corresponding peak
in the chromatogram obtained with reference solution (a)
(20 }lg/capsule).
RADIONUCLIDIC PURITY
Iodine-131
Minimum 99.9 per cent ofthe total radioactivity.
Gamma-ray spectrometry.
Determine the relative amounts ofiodine-130, iodine-131,
iodine-133, iodine-135 and other radionuc1idic impurities
presento
RADIOCHEMICAL PURITY
[ 13l1]iodide
Liquid chromatography (2.2.29) as described in the test for
iodide with the following modifications.
Injection 20}lL of test solution (b) and reference
solution (a).
Limits Examine the chromatogram obtained with test
solution (b) using the radioactivity detector and locate the
peak due to iodide by comparison with the chromatogram
obtained with reference solution (a) using the
spectrophotometer:
131
-- [ Ijiodide: minimum 95 per cent of the total
radioactivity.
RADIOACTIVITY
Determine the radioactivity of each capsule using a calibrated
instrumento
LABELLING
The label states the name of any excipient.
IMPURITIES
A. [l3l I]iodate ion,
B. iodine-130,
C. iodine-133,
D. iodine-135.
____________________________________________ ~E~

(5 }lm),
-- temperature: constant, between 20 oC and 30 oc.
Use stainless steel tubing.
Mobile phase Dissolve 5.85 g of sodium chloride R in
1000 mL of water R, add 0.65 mL of octylamine R and adjust Sodium lodide C31 1) Solution ***
*** ***
to pH 7.0 with dilute phosphoric acid R; add 50 mL of (Ph Bur monograph 0281) ***
acetonitrile R and mix. ~E~ ____________________________________________
Flow rate 1.5 mUmin.
DEFINITION
Detection Spectrophotometer at 220 nm and radioactivity
Solution containing iodine-131 in the form of sodium iodide
detector connected in series.
and also sodium thiosulfate or sorne other suitable reducing
Injection 20}lL of test solution (a), reference solutions (a) agent. It may contain a suitable buffer.
and (b) and the blank solution.
Content:
Run time 12 mino
-- iodine-131: 90 per cent to 110 per cent of the dec1ared
Relative retention With reference to iodide radioactivity at the date and hour stated on the label,
(retention time = about 5 min): iodate = 0.2 to 0.3. -- iodide: maximum 20 }lg in the maximum recommended
dose in millilitres.
2014 Radiopharmaceutical Preparations IV-685

PRODUCTION Detection Spectrophotometer at 220 nm and radioactivity

Iodine-131 is a radioactive isotope of iodine and may be detector connected in series.
obtained by neutro n irradiation of tellurium or by extraction Injection 25 llL; inject test solution (a), the blank solution
from uranium fission products . No carrier iodide is added. and reference solutions (a) and (b).
CHARACTERS Run time 12 mino
Appearance Relative retention With reference to iodide
Clear, colourless solution. (retention time = about 5 min): iodate = 0.2 to 0.3.
HaIf-life and nature ofradiation ofiodine-131 System suitability:
See general chapter 5.7. Table of physical charactenstics of - in the chromatogram obtained with the blank solution,
radionuclides. none of the peaks shows a retention time similar to that
of the peak due to iodide,
IDENTIFICATION
- resolution: minimum 2 between the peaks due to iodide
A. Gamma-ray spectrometry.
and iodate in the chromatogram obtained with reference
Results The spectrum obtained with the preparation to be solution (b) recorded with the spectrophotometer.
examined does not differ significantly from that of a
Limit Examine the chromatogram obtained with the
standardised iodine-131 solution. The most prominent
spectrophotometer; locate the peak due to iodide by
gamma photon has an energy of 0.365 MeV.
comparison with the chromatogram obtained with reference
B. Examine the chromatograms obtained in the test for solution (a):
iodide. - iodide: not more than the area of the corresponding peak
Results The principal peak in the radiochromatogram in the chromatogram obtained with reference solution (a).
obtained with test solution (a) is similar in retention time to RADIONUCLIDIC PURITY
the principal peak in the chromatogram obtained with
reference solution (a).
Iodine-131
Minimum 99 .9 per cent of the total radioactivity.
TESTS Gamma-ray spectrometry.
pH (2.2.3)
Determine the relative amounts ofiodine-131 , iodine-133,
7.0 to 10.0.
iodine-135 and other radionuc1idic impurities presento
Sterility
RADIOCHEMICAL PURITY
If intended for parenteral administration, it complies with the
l3l
test for sterility prescribed in the monograph on [ I]Iodide
Radiopharmaceurical preparations (0125) . The solution may be Liquid chromatography (2.2.29) as described in the test for
released for use before completion of the test. iodide with the following modification.
Iodide Injection Test solution (b).
Liquid chromatography (2.2.29). Limit Examine the chromatogram obtained with the
Test solution (a) The preparation to be examined. radioactivity detector:
- [J3J Ijiodide: minimum 95 per cent of the total
Test solution (b) Dilute the preparation to be examined with
radioactivity.
0.05 M sodium hydroxide until the radioactivity is equivalent
to about 74 MBq/mL. Add an equal volume of a solution RADIOACTIVITY
containing 1 gIL of potassium iodide R, 2 gIL of potassium Measure the radioactivity using suitable equipment by
iodate R and 10 gIL of sodium hydrogen carbonate R and mix. comparison with a standardised iodine-131 solution or by
Reference solution (a) Dilute 1 mL of a 26.2 mg!L solution using a calibrated instrumento
of potassium iodide R to V with water R, V being the LABELLING
maximum recommended dose in millilitres . The label states:
Reference solurion (b) Dilute 1 mL of a 24.5 mg!L solution - the name of any excipient,
of potassium iodate R to V with water R, V being the - the maximum recommended dose, in millilitres,
maximum recommended dose in millilitres. Mix equal - where applicable, that the preparation is suitable for use
volumes of this solution and of reference solution (a). in the manufacture of parenteral preparations.
Blank solution Prepare a solution containing 2 mg/rnL of IMPURITIES
each of the components stated on the label, apart from A. [ J31 I]iodate ion.
iodide.
_ _ _ __ __ __ _ __ _ __ _ _ _ _ _ _ _ Ph fur
Column:
- size: l = 0.25 m, 0 = 4.0 mm,
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 ~lm),
- temperature: maintain at a constant temperature between
Sodium lodide C31 1) Solution For *****
** **
20 oC and 30 oC . Radiolabelling ***
(Ph Bur monograph 2121)
U se stainless steel tubing.
~f~ _ _ _ __ _ _ _ __ _ _ __ __ _ __ __ _
Mobile phase Dissolve 5.844 g of sodium chloride R in
1000 mL of water R, add 650 llL of octylamine R and adjust DEFINITION
to pH 7.0 with phosphonc acid R; add 50 mL of acetonitrzle R Strongly alkaline solution containing iodine-131 in the form
and mix. of sodium iodide. It does not contain a reducing agent.
Flow rate 1.5 mlJmin. Content
90 per cent to 11 O per cent of the dec1ared iodine-131
radioactivity at the date and hour stated on the labe!'
 IV-686 Radiopharmaceutical Preparations 2014

PRODUCTION Detection Spectrophotometer at 220 nm and a radioactivity

Iodine-131 may be obtained by neutron irradiation of detector connected in series.
tellurium or by extraction from uranium fission products. lnjeaion 20 J.lL.
No carrier iodide is added. Run time 12 min.
CHARACTERS Relative retention With reference to iodide
Appearance (retention time = about 5 min): iodate = 0.2 to 0.3.
Clear, colourless solution. System suitability Reference solution (b):
Half-life and nature of radiation of iodine-131 -- resolution: minimum 2 between the peaks due to iodide
See general chapter 5.7. Table of physical characteristics of and iodate in the chromatogram recorded with the
radionuclides. spectrophotometer.
IDENTIFICATION Limit Examine the chromatogram obtained with the
A. Gamma-ray spectrometry. radioactivity detector:
-- [131IJiodide: minimum 95 per cent of the total
Results The spectrum obtained with the preparation to be
radioactivity.
examined does not differ significantly from that of a
standardised iodine-131 solution. The most prominent RADIOACTIVITY
gamma photon of iodine-131 has an energy of 0.365 MeV. Determine the radioactivity using a calibrated instrument.
B. Examine the chromatograms obtained in the test for LABELLING
radiochemical purity (se e Tests). The label sta tes:
Results The principal peak in the radiochromatogram -- the method of production of iodine-131,
obtained with the test solution is similar in retention time to -- the name of any excipient,
the principal peak in the chromatogram obtained with -- that the preparation is not for direct human use .
reference solution (a) .
IMPURITIES
TESTS A. [l3l I]iodate ion.
AIkalinity (2.2.4) _____ _ _ _ __ __ __ _ _ _ _ _ _ __ ___ ~E~

The preparation is strongly alkaline.

RADIONUCLlDlC PURITY
Iodine-131
Minimum 99.9 per cent ofthe total radioactivity.
Gamma-ray spectrometry.
Sodium lodohippurate C23 1) **
****
*
Determine the relative amounts ofiodine-130, iodine-131, Injection *****
iodine-133, iodine-135 and other radionuclidic impurities (Ph Eur monograph 0564)
present. PhE~ ___ __ _ _ _ __ _ __ _ _ _ __ _ __ ___

RADIOCHEMICAL PURITY DEFINITION

[
131
I]iodide
Liquid chromatography (2.2.29).
Test solution Dilute the preparation to be examined with an
equal volume of a solution containing 1 gIL of potassium
iodide R, 2 giL of potassium iodate R and 10 gIL of sodium
hydrogen carbonate R and mix. If necessary, first dilute the
preparation to be examined with a 2 gIL solution of sodium
hy droxide R to ensure that the final mixture has a
radioactivity concentration suitable for the radioactivity
detector.
Reference solution (a) Dissolve 10 mg of potassium iodide R in
water R and dilute to 10 mL with the same solvento
Reference solution (b) Dissolve 20 mg of potassium iodate R in
water R and dilute to 10 mL with the same solvento
Mix equal volumes of this solution and reference
solution (a) .
Colwnn:
-- size: 1 = 0.25 m, 0 = 4.0 mm,
-- stationary phase: octadecylsilyl silica gel for chromatography R
(5 ¡lm),
-- temperature: constant, between 20 oC and 30 oC.
Use stainless steel tubing.
Mobzle phase Dissolve 5.85 g of sodium chloride R in
1000 mL of water R, add 0.65 mL of octylamine R and adjust
to pH 7.0 with dz1ute phosphoric acid R; add 50 mL of
acetonitrzie R and mix.
Flow rate 1.5 mUmin.
Sterile solution of sodium (2-¡I 23 I]iodobenzamido)acetate.
It may contain a suitable buffer and a suitable antimicrobial
preservative such as benzyl alcohol. .
Iodine-123
90 per cent to 110 per cent of the declared iodine-123
radioactivity at the date and time stated on the label.
Specific radioactivity
0.74 GBq to 10.0 GBq ofiodine-123 per gram ofsodium
2-iodohippurate.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of iodine-123
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray and X-ray spectrometry.
Results The most prominent gamma photon has an energy
of 0.159 MeV and is accompanied by an X-ray of
0.027 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity (see Tests) .
Result The principal spot in the radiochromatogram
obtained with the test solution is similar in retardation factor
to the spot corresponding to 2-iodohippuric acid in the
chromatogram obtained with the reference solution.
2014 Radiopharmaceutical Preparations IV-687

TESTS LABELLING
pH (2.2.3) The label states whether or not the preparation is suitable for
3.5 to 8.5. renal plasma-ftow studies.
Sterility IMPURITIES
It complies with the test for sterility prescribed in the A. iodine-125,
monograph Radiophannaceutical preparations (0125). B. tellurium-121,
The preparation may be released for use before completion
of the test. C. [123I]iodide,
D. 2-e z3 I]iodobenzoic acid.
RADIONUCLIDIC PURITY
____________________________________________ Ph E~

The preparation may be released for use before completion

of the test.
Radionuclides other than iodine-123
Maximum 0.35 per cent of the total radioactivity.
Gamma-ray and X-ray spectrometry.
Sodium lodohippurate C 1)
31
*****
** **
Determine the relative amounts of iodine-125, tellurium-121 Injection ***
and other radionuc1idic impurities presento For their (Ph Eur monograph 0282)
detection, retain the preparation to be examined for a ~E~ ____________________________________________
sufficient time to allow iodine-123 to decay to a level which
permits the detection of radionuc1idic impurities. Record the DEFINITION
gamma-ray spectrum and X-ray spectrum of the decayed Sterile solution of sodium 2-(2-[ J31 I]iodobenzamido)acetate.
material. No radionuc1ides with a half-life longer than that of It may contain a suitable buffer and a suitable antimicrobial
iodine-125 are detected. preservative such as benzyl alcohol.
RADIOCHEMICAL PURITY Iodine-131
90 per cent to 110 per cent ofthe dec1ared iodine-131
2-[ 123 I]Iodohippuric acid
radioactivity at the date and time stated on the ¡abel.
Thin-layer chromatography (2.2.27) .
Test solurion Dissolve 1 g of potassium iodide R in 10 mL of Specific radioactivity
water R, add 1 volume of this solution to 10 volumes of the 0.74 GBq to 7.4 GBq ofiodine-131 per gram ofsodium
preparation to be examined and use within 10 min of mixing. 2-iodohippurate.
If necessary, dilute with the reference solution (carrier) to CHARACTERS
give a radioactive concentration sufficient for the detection Appearance
method, for example 3.7 MBq per millilitre. Clear, colourless solution.
Reference solurion (carrier) Dissolve 40 mg of 2-iodobenzoic Half-life and nature ofradiation ofiodine-131
acid R and 40 mg of 2-iodohippuric acid R in 4 mL of a 4 gIL See general chapter 5.7. Table of physical characteristics of
solution of sodium hydroxide R, add 10 mg of potassium radionuclides.
iodide R and dilute to 10 mL with water R.
IDENTIFICATION
Plate TLC siliea gel GF254 plate R.
A. Gamma-ray spectrometry.
Mobile phase water R, glacial acetic acid R, butanol R,
Result The most prominent gamma photon of iodine-131
toluene R (1:4:20:80 VIVIV/V).
has an energy of 0.365 MeV.
Application 1O ~1L.
B. Examine the chromatograms obtained in the test for
Development Over a path of 12 cm in about 75 min o radiochemical purity (see Tests).
Drying In air. Result The principal peak in the radiochromatogram
Detection Examine in ultraviolet light at 254 nm and obtained with the test solution has a similar retardation factor
determine the distribution of radioactivity using a suitable as the spot corresponding to 2-iodohippuric acid in the
detector. chromatogram obtained with the reference solution.
Identification of spots The chromatogram obtained with the TESTS
reference solution shows a spot corresponding to pH (2.2.3)
2-iodohippuric acid and nearer to the solvent front a spot 6.0 to 8.5 .
corresponding to impurity D ; impurity C remains near the
point of application. Sterility
It complies with the test for sterility prescribed in the
Limits:
monograph Radiophannaeeutical preparations (0125).
-- 2-[123Ijiodohippuric acid: minimum 96 per cent of the total
The preparation may be released for use before completion
radioactivity due to iodine-123;
of the test.
-- impurity C: maximum 2 per cent of the total radioactivity
due to iodine-123; RADIONUCLIDIC PURlTY
-- impurity D: maximum 2 per cent of the total radioactivity Iodine-131
due to iodine-123. Minimum 99.9 per cent ofthe total radioactivity.
RADIOACTIVITY Gamma-ray spectrometry.
Determine the radioactivity using a calibrated instrumento Determine the relative amounts of iodine-131, iodine-133,
STORAGE iodine-135 and other radionuc1idic impurities presento
Protected fram light. RADIOCHEMICAL PURITY
2-[ l31 I]Iodohippuric acid
Thin-layer chromatography (2.2.27).
 IV-688 Radiopharmaceutical Preparations
Test solution Dissolve 1 g of potassium iodide R in 10 mL of
water R, add 1 volume of this solution to 10 volumes of the
preparation to be examined and use within 10 min of mixing.
If necessary dilute with the reference solution (carrier) to give
a radioactive concentration sufficient for the detection
method, for example 3.7 MBq/mL.
Referenee solution (cam'er) Dissolve 40 mg of 2-iodobenzoic
acid R and 40 mg of 2-iodohippurie acid R in 4 mL of a 4 gIL
solution of sodium hydroxide R, add 10 mg of potassium
iodide R and dilute to 10 mL with water R.
Plate TLC siliea gel GF254 pIare R.
Mobile phase water R, glacial acetie acid R, butanol R,
toluene R (1 :4:20:80 VIVIV/V) .
Application 10 ¡.tL.
Development Over a path of 12 cm in about 75 min o
Drying In air.
Detection In ultraviolet light at 254 nm and with a suitable
detector to determine the distribution of radioactivity.
ldentifieation of spots The chromatogram obtained with the
reference solution shows a spot corresponding to
2-iodohippuric acid and nearer to the solvent front a spot
corresponding to impurity C; impurity D remains near the
point of application.
Limits:
-- 2_[I3J IJiodohippuric acid: minimum 96 per cent of the total
radioactivity due to iodine-131;
-- impurity C: maximum 2 per cent of the total radioactivity
due to iodine-131;
- impurity D: maximum 2 per cent of the total radioactivity
due to iodine-13l.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument.
STORAGE
Protected from light.
LABELLING
The labe! sta tes that the preparation is not necessarily
suitable for renal plasma-flow studies.
IMPURITIES
A. iodine-133,
B. iodine-135,
C. 2-¡J 3 1I]iodobenzoic acid,
D. [I3l I]iodide.
____________________________________________ PhE~
Sodium Molybdate (99Mo) Solution *****
** **
(Fission) ***
(Ph Eur monograph 1923)
~E~ _ _ _ ___________________________________
DEFINITION
Alkaline solution of sodium [99Mo]molybdate obtained by
extraction of fission products of uranium-235. It may contain
stabilisers.
Content
90 per cent to 110 per cent of the dec1ared molybdenum-99
radioactivity at the date and time stated on the labe!'
PRODUCTION
Molybdenum-99 is usually produced by fission of uranium
enriched in uranium-235, which is caused by the absorption
of a thermal neutron, resulting in high-specific-activity
2014
molybdenum-99 . By the fission of uranium after neutro n
capture, more than 200 different radionuc1ides are produced.
In approximately 6 per cent of the fissions, molybdenum-99
is formed after decay of a number of short-lived parent
radionuc1ides. After dissolution of the target, the
molybdenum-99 is separated from the mixture of nuc1ides
and purified by using chromatographic processes in order to
obtain molybdenum-99 with a high level of radionuc1idic
purity.
CHARACTERS
Appearance
Clear, colourless or almost colourless solution.
Half-life and nature of radiation of molybdenum-99
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICA TION
A. Gamma-ray spectrometry.
Results The most prominent gamma photon of
molybdenum-99 has an energy of 0.740 MeV; a peak with an
energy of 0.141 MeV, due to technetium-99-m is also visible.
B. Examine the chromatograms obtained in the test for
radiochemical purity (see Tests).
Results The principal peak in the radiochromatogram
obtained with the test solution has a similar retardation factor
to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Solution S
Dilute the preparation to be examined to a radioactivity
concentration of approximately 370 MBq/mL with a 2.42 gIL
solution of sodium molybdate R.
AIkalinity
The preparation is alkaline (2.2.4).
RADIONUCLIDlC PURITY
Iodine-131, ruthenium-103 and tellurium-132:
- iodine-131 : maximum 5 x 10- 3 per cent ofthe total
radioactivity;
- ruthenium-103: maximum 5 x 10- 3 per cent ofthe total
radioactivity;
- tellurium-132: maximum 5 x 10- 3 per cent ofthe total
radioactivity.
The following method has been found to be suitable; other
validated methods, approved by the competent authority,
may be used.
Gamma-ray spectrometry.
Condition a column with an internal volume of
approximately 1.5 mL of strongly basic anion exchange resin R
with a mixture of equal volumes of glacial acetic acid R and
water R. All elutions of the column are made at a flow rate
not exceeding 1 mL/min.
Test solution In a test -tube, successive!y add, with mixing,
1 mL of a 24.2 gIL solution of sodium molybdate R, 0.5 mL
of strong hydrogen peroxide solution R, 2.5 mL of glacial acetic
acid R, 1.0 mL of iodine-123 and ruthenium-106 spiking
solution R and 1.0 mL of solution S. Allow to stand for
30 min at room temperature.
Referenee solution Mix 1.0 mL of iodine-123 and ruthenium-
106 spiking solution R and 4.0 mL of water R .
Apply the test solution to the column and elute. Just before
the disappearance of the liquid from the top of the column,
add 6 mL of a mixture of equal volumes of glacial aeetic
aeid R and water R and elute . Transfer 5.0 mL of the
 2014
combined eluates to a counting tube. Determine the
radioactivity of iodine-123, iodine-131 , ruthenium-I03,
ruthenium-I06 and iodine-132 at the gamma-ray energies of
0.159 MeV for iodine-123, 0.365 MeV for iodine-131,
0.497 MeV for ruthenium-I03, 0.512 MeV for ruthenium-
106 and 0.668 MeV for iodine-132 . Determine in the same
way the radioactivity of iodine-123 and ruthenium-I06 in the
reference solution and calculate the recovery of iodine-123
and ruthenium-I06 in the combined eluates.
Calculate the radioactivity ofiodine-131, iodine-132 and
ruthenium-I03 in the combined eluates, taking into account
the recovery, the fraction of eluate used, the counting
efficiency and the radio active decay. From the radioactivity of
iodine-132 (daughter radionuclide oftellurium-132),
calculate the radioactivity of tellurium-132, taking into
account the time of the test and the time of separation of
molybdenum-99.
Total radioactivity due to strontium-89 and strontium-
90
Maxirnum 6 x 10- 5 per cent of the total radioactivity.
The following method has been found to be suitable; other
validated methods, approved by the competent authority,
may be used.
Liquid scintillation spectrometry.
Connect 2 columns, each with an imernal volume of
approximately 1.5 mL of strongly basic anion exchange resin R,
in series and condition the columns with 10 mL of a 4 giL
solution of sodium hydroxide R. A11 elutions of the columns
are made at a fiow rate not exceeding l mUmin.
Test solution In a test-tube, successively add, with mixing, .
1.0 mL of solution S, 50 ¡tL of strontium-85 spiking solution R
and 0.05 mL of strong sodiu111 hypochlorite sollltion R. Allow to
stand for 10 min at room temperature .
Reference solurion Mix 50 ¡tL of stromium-85 spiking
solution R with 5.0 mL of a 9.5 gIL solution of nitric acid R in
a vial for liquid scimillation couming and add 10 mL of
liquid scimillation cocktail R.
Apply the test solution to the upper of the 2 columns and
elute. Just before the disappearance of the liquid from the top
of the upper column, add 3 mL of a 4 giL solution of sodiwn
hydroxide R and elute until the columns are dry. Combine the
eluates and add 4 mL of a 947 gIL solution of nitric acid R
(molybdenum-poor eluate). Determine the radioactivity due
to molybdenum-99 using gamma-ray spectrometry. If the
radioactivity due to molybdenum-99 is higher than 6 x 10-7
per cent of the radioactivity due to molybdenum-99 in 1 mL
of solution S, repeat the aboye procedure using 2 new
columns .
Condition a column with an intemal volume of
approximately 2 mL of strontium selective extraction resin R
with 5 mL of a 473 gIL solution of nitric acid R and dry the
column. AII elutions of the column are made at a fiow rate
not exceeding 1 mUmin . Apply to the column the
molybdenum-poor eluate and elute. Just before the
disappearance of the liquid from the top of the column, add
20 mL of a 473 gIL solution of nicric acid R and elute umil
the column is dry. Rinse the column with 2 mL of a 9.5 gIL
solution of nitric acid R, dry the column and discard the
eluate. Elute the column with 8.0 mL of a 9.5 gIL solution of
nitric acid R until the column is dry. Transfer 5.0 mL of the
eluate imo a vial for liquid scimillation counting and add
10 mL of liquid scintillation cocktail R.
Determine the total radioactivity due to stromium-89 and
strontium-90 in this solution by liquid scintillation
Radiopharmaceutical Preparations IV-689
spectrometry, and the radioactivity due to strontium-85 by
gamma-ray spectrometry. Determine the radioactivity due to
strontium-85 in the reference solution by gamma-ray
spectrometry. Calculate the recovery of strontium-85 in the
eluate. Calculate the measured total radioactivity of
strontium-89 and strontium-90 in the eluate, taking into
account the recovery of strontium and the fraction of eluate
used.
Total radioactivity due to alpha-particle-emitting
impurities
Maximum 1 x 10- 7 per cent of the total radioactivity.
The following method has been found to be suitable; other
validated methods, approved by the competent authority,
may be used.
Alpha-ray spectrometry.
Test solution To 0.2 mL of the preparation to be examined
add 1.0 mL of plutonium-242 spiking solut¡on R, 1.0 mL of
americium-243 spiking solution R and 9.0 mL of a 927 giL
solution of hydrochloric acid R. Evaporate the sample to
dryness. Dissolve the residue in 2 mL of a 927 gIL solution
of hydrochloric acid R . Evaporate again to dryness. Dissolve
the residue in 2 mL of a 10.3 giL solution of hydrochloric
acid R.
Apply the test solution ro a column containing 0.7 g of anion
exchange resin Rl. Collect the eluate and wash the column
with 1 mL of a 10.3 giL solution of hydrochloric acid R.
Evaporate the combined eluates ro dryness and dissolve the
residue in 2 mL of a 10.3 gIL solution of hydrochloric acid R.
Apply this solution to a 2nd column comaining 0.7 g of anion
exchange resin Rl. Collect the eluate and wash the column
with 1 mL of a 10.3 giL solution of hydrochloric acid R.
Evaporate the combined eluates ro dryness and dissolve the
residue in 1 mL of nitric acid R. Evaporate to dryness.
Dissolve the residue again in 1 mL of nicric acid R.
Add 1 mL of a 42.6 giL solution of anhydrous sodiu111
sulfate R and evaporate ro dryness. Add 0.3 mL of sulfuric
acid R. Warm until the residue is dissolved. Add 4 mL of
distilled water R and 0.01 mL of thymol blue solurion R.
Add concentrated a1111110nia R dropwise until the colour
changes from red ro yellow.
Prepare an electrodeposition cell as follows.
An electropolished stainless steel planchet is fitted in the cap
of a 20 mL polyethylene scintillation vial. The bottom of the
vial has been cut off and a hole has been drilled through the
centre of the cap for electrical connection to the planchet
cathode. The planchet, 20 mm in diameter and 0.5 mm
thick, is rinsed with acetone R and water R prior to use.
The ano de, a platinum spiral, is introduced through the
bottom of the vial and fitted 5 mm from the cathode.
Pour the solution prepared as described aboye imo the
electrodeposition cell and rinse the container with a total of
5 mL of a 10 giL solution of sulfuric acid R (the solution
becomes slightly pink). Adjust ro pH 2.1-2.4. with
concemrated ammonia R or with a 200 giL solution of sulfuric
acid R. Electrolyse at 1.2 A for 75 min without stirring.
Add 1 mL of concentrated ammonia R about 1 min prior to
switching off the current. Rinse the planchet with a 57 gIL
solution of ammonia R . Rinse the planchet with acetone R and
remove any residual solvent by patting the planchet with
absorbent paper. Heat the planchet on a hot plate at 180 oC
for 10 mino
Determine the radioactivity of alpha emitters by alpha-ray
spectrometry, taking imo account the recovery of the alpha-
IV-690 Radiopharmaceutical Preparations
particle-emining radionuclides (measured using the
plutonium-242 and americium-243 spiking solutions).
Total of gamma-ray-emitting radionuclides other than
rnolybdenum-99, technetiurn-99rn, iodine-131,
ruthenium-103 and tellurium-132
Maximum 1 x 10- 2 per cent of the total radioactivity.
The following method has been found to be suitablei other
validated methods, approved by the competent authority,
may be used.
Gamma-ray spectrometry.
Allow the preparation to decay for 4-6 weeks. Examine the
gamma-ray spectrum for the presence of other gamma-ray-
emitting impurities. Identify and quantify other gamma-ray-
emining impurities. The preparation may be relea sed for use
before completion of the test.
RADIOCHEMICAL PURITY
[99Mo ]Molybdate
The following method has been found to be suitablei other
validated methods, approved by the competent authority,
may be used.
Thin-layer chromatography (2.2.27).
Test solution Dilute the preparation to be examined with a
4.0 gIL solution of sodium hydroxide R to a radioactivity
concentration suitable for the detector.
R eference solution 50 giL solution of sodium molybdate R in a
4.0 gIL solution of sodium hydroxide R .
Plate TLC silica gel plate R .
Mobile phase 10.6 giL solution of anhydrous sodium
carbonate R.
Application 5 ~lL of the test solution and 2 ~ of the
reference solution.
Development Over 2/3 of the plateo
Drying In a current of warm air.
D etection Determine the distribution of radioactivity using a
suitable detector and spray with a 2 gIL solution of
phenylhydrazine R in glacial acetic acid Ri heat at 100-105 oC
for 5 min.
R etardation factor Molybdate and pertechnetate = about
0.9.
L imit:
- sum of t 9MoJmolybdate and [99mTcJpertechnetate:
minimum 95 per cent of the total radioactivity.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument.
LABELLING
The label states that the preparation is only suitable for the
preparation of technetium-99m generators.
IMPURITIES
A. iodine-131 ,
B. ruthenium-l03,
C. tellurium-132,
D . strontium-89,
E. strontium-90 .
- -_ _ __ _ _ _ __ __ _ __ _ __ __ _ PhEur
2014
Sodium Pertechnetate (99m Tc) *****
* *
Injection (Fission) *****
(Ph Eur monograph 0124)
~E~ _ __ _ __ _ __ _ _ _ __ _ _ _ _ _ _ __
This monograph applies to sodium pertechnetate ~9I11 Tc) injection
obtained from molybdenum-99 extracted from fission products of
uranium. Sodium pertechnetate ~9mTc) injection obtainedfrom
molybdenum-99 produced by mueron irradiation of molybdenum is
described in the monograph Sodium pertechnetate (99mTc)
injection (non-fission) (0283).
DEFINITION
Sterile solution containing technetium-99m in the form of
pertechnetate ion and made isotonic by the addition of
sodium chloride. The injection may be prepared from a
sterile preparation of molybdenum-99 under aseptic
conditions.
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation oftechnetium-99m
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
Gamma-ray spectrometry.
Result: the most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
TESTS
pH (2.2.3)
4.0 to 8.0.
Alurninium
Maximum 5 ppm.
Test solution In a test tube about 12 mm in internal
diameter, mix 1 mL of acetate buffer solution pH 4. 6 R and
2 mL of a 1 in 2.5 dilution of the preparation to be
examined in water R. Add 0.05 mL of a 10 gIL solution of
chromazurol S R.
R eference solution Prepare at the same time and in the same
manner as the test solution and using 2 mL of aluminium
standard solution (2 ppm Al) R.
After 3 min, the colour of the test solution is not more
intense than that of the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
RADIONUCLIDIC PURITY
Preliminary test To obtain an approximate estimate before
use of the preparation, take a volume equivalent to 37 MBq
and determine the gamma-ray spectrum using a sodium
iodide detector with a shield of lead, of thickness 6 mm,
interposed between the sample and the detector.
The response in the region corresponding to the 0.740 MeV
photon of molybdenum-99 does not exceed that obtained
using 37 kBq of a standardised molybdenum-99 solution
measured under the same conditions, when all measurements
are expressed with reference to the date and time of
administration.
 2014 Radiopharmaceutical Preparations IV-691

Definirive test Retain a sample of the preparation to be -- Alpha-emitung impurities: maximum 1 x 10- 7 per cent of
examined for a sufficient time to allow the technetium-99m the total radioactivity.
radioactivity to decay to a sufficiently low level to permit the Measure the alpha radioactivity of the decayed material to
detection of radionuclidic impurities. AII measurements of detect any alpha-emitting radionuclidic impurities, which
radioactivity are expressed with reference to the date and should, where possible, be identified and quantified.
time of administration.
RADIOCHEMICAL PURITY
-- lmpurity A: Maximum 5 x 10- 3 per cent of the total
radioactivi ty. [99m Tc]Pertechnetate ion
Descending paper chromatography (2.2.26).
Gamma-ray spectrometry. Record the spectrum of the decayed
material. Test solution Dilute the preparation to be examined with
water R to a suitable radio active concentration.
Comparison Suitable instrument calibrated with the aid of a
standardised iodine-131 solution. Paper paper for ehromatography R .
R esults The most prominent photon has an energy of Mobile phase water R , methanol R (20:80 V/V).
0.365 MeV; iodine-131 has a half-life of 8.04 days. Applieation 5 1lL.
-- l mpurity B: maximum 0.1 per cent ofthe total Development For 2 h.
radioactivity.
Drying In air.
Gamma-ray spectrometry. Record the spectrum of the decayed
Detection Suitable detector to determine the distribution of
material.
radioactivity.
Comparison Suitable instrument calibrated with the aid of a 99m
Retardau011 factor [ Tc]pertechnetate ion = about 0.6.
standardised molybdenum-99 solution.
Limit:
Results The most prominent photons have energies of
0.181 MeV, 0.740 MeV and 0.778 MeV; molybdenum-99
-- t 9
/1lTejpertechnetate ion: minimum 95 per cent of the total
radioactivity due to technetium-99m.
has a half-life of 66.0 h.
-- lmpurity C: maximum 5 x 10~3 per cent of the total RADIOACTIVITY
radioactivity. Determine the radioactivity using a calibrated instrumento
Gamma-ray spectrometry. R ecord the spectrum of the decayed IMPURITIES
material. A. iodine-131 ,
Comparison Suitable instrument calibrated using a B. molybdenum-99,
standardised ruthenium-l 03 solution. C. ruthenium-103,
Results The most prominent photon has an energy of D . strontium-89,
0.497 MeV; ruthenium-103 has a half-life of 39.3 days.
E . strontium-90.
-- lmpurity D : maximum 6 x 1O ~ 5 per cent of the total
____________________________________________ PhE~

radioactivity.
Determine the presence of strontium-89 in the decayed
material with an instrument suitable for the detection of beta
rays. Ir is usually necessary first to carry out chemical
separation of the strontium so that the standard and the Sodium Pertechnetate (99m Tc) *****
sample may be compared in the same physical and chemical ** **
formo Injection (Non-fission) ***
Comparison Standardised strontium-89 solution. (Ph Eur monograph 0283)
Results Strontium-89 decays with a beta emission of Ph Eur ____________________________________________

1.492 MeV maximum energy and has a half-life of 50.5 days.
-- lmpurity E: maximum 6 x 1O ~6 per cent of the total
radioactivity.
Determine the presence of strontium-90 in the decayed
material with an instrument suitable for the detection of beta
rays. To distinguish strontium-90 from strontium-89,
compare the radioactivity of yttrium-90, the daughter nuclide
of strontium-90, with an yttrium-90 standard after the
chemical separation of the yttrium. If prior chemical
separation of the strontium is necessary, the conditions of
radio active equilibrium must be ensured. The yttrium-90
standard and the sample must be compared in the same
physical and chemical form o
Results Strontium-90 and yttrium-90 decay with respective
beta emissions of 0.546 MeV and 2.284 MeV maximum
energy and half-lives of 29.1 years and 64.0 h .
-- Other gamma-emiuing impurities: maximum 0.01 per cent
of the total radioactivity.
Gamma-ray spectrometry.
Examine the spectrum of the decayed material for the
presence of other radionuclidic impurities, which should,
where possible, be identified and quantified.
This monograph applies to sodium perteehnetate ('9I1/ Te) injeeuon
obtained from molybdenum-99 produeed by neutron úmdiauon of
molybdenum. Sodium perteehnetate (' 9m Te) injeetion obtained
from molybdenum-99 extracted from fission produets of uranium is
deseribed in the monograph Sodium pertechnetate 9mTc) C
injection (fission) (0124).
DEFINITION
Sterile solution containing technetium-99m in the form of
pertechnetate ion and made isotonic by the addition of
sodium ch1oride.
Technetiwn-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physieal eharactenstics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
IV-692 Radiopharmaceutical Preparations 2014

Result The most prominent gamma photon of technetium- Test solution Dilute the preparation to be examined with
99m has an energy of 0.141 MeV. water R to a suitable radio active concentration.
B. Examine the chromatogram obtained in the test for Paper paper for chromatography R.
radiochemical purity (se e Tests) . Mobile phase water R, methanol R (20:80 V/V) .
Result The retardation factor of the principal peak in the Application 5 ~lL.
radiochromatogram obtained with the test solution is about Development For 2 h.
0.6.
Drying In airo
TESTS Detection Suitable detector to determine the distribution of
pH (2.2.3) radioactivity .
4.0 to 8.0.
Aluminium
Retardation fa ctor e 9m
Tc]pertechnetate ion = about 0.6.
Limit:
Maximum 5 ppm. -- f 9m TcJpertechnetate ion: minimum 95 per cent of the total
Test solution In a test tube about 12 mm in internal radioactivity due to technetium-99m.
diameter, mix 1 mL of acetate buffer solution pH 4.6 R and
RADIOACTIVITY
2 mL of a 1 in 2.5 dilution of the preparation to be
examined in water R. Add 0.05 mL of a 10 gIL solution of Determine the radioactivity using a calibrated instrumento
chromazurol S R. IMPURITIES
Reference solution Prepare at the same time and in the same A. molybdenum-99.
manner as the test solution and using 2 mL of aluminium ____________________________________________ ~E~

standard solution (2 ppm Al) R.

After 3 min, the colour of the test solution is not more
intense than that of the reference solution.
Sterility
Sodium Phosphate ep) Injection
2

It complies with the test for sterility prescribed in the (Ph Eur monograph 0284)
____________________________________________
monograph Radiopharmaceutical preparations (0125) . ~E~

The preparation may be relea sed for use before completion DEFINITION
of the test.
RADIONUCLIDIC PURlTY
Sterile solution of disodium and monosodium 2 p) e
orthophosphates made isotonic by the addition of sodium
Preliminary test To obtain an approximate estimate before chloride.
use of the preparation, take a volume equivalent to 37 MBq
Phosphorus-32
and record the gamma-ray spectrum using a sodium iodide
90 per cent to 110 per cent of the declared phosphorus-32
detector with a shield of lead, 6 mm thick, interposed
radioactivity at the date and time stated on the labe!'
between the sample and the detector. The response in the
region corresponding to the 0.740 MeV photon of Specific radioactivity
molybdenum-99 does not exceed that obtained using 37 kBq Minimum 11.1 MBq of phosphorus-32 per milligram of
of a standardised molybdenum-99 solution measured under orthophosphate ion.
the same conditions, when al! measurements are expressed CHARACTERS
with reference to the date and time of administration. Appearance
Definitive test Retain a sample of the preparation to be Clear, colourless solution.
examined for a sufficient time to al!ow the technetium-99m Half-life and nature of radiation of phosphorus-32
radioactivity to decay to a sufficiently low level to permit the See general chapter 5.7. Table of physical characteristics of
detection of radionuclidic impurities. Al! measurements of radionuclides.
radioactivity are expressed with reference to the date and
time of administration. IDENTIFICATION
-- lmpurity A: maximum 0.1 per cent of the total A. Beta-ray spectrometry.
radioactivity. Result The maximum energy of the beta radiation is
Gamma-ray spectrometry. Record the gamma-ray spectrum 1.71 MeV.
of the decayed materia!. B. Examine the chromatogram obtained in the test for
Comparison Standardised molybdenum-99 solution. radiochemical purity (se e Tests).
Results The most prominent gamma photons have energies Result The principal peak in the radiochromatogram
of 0.181 MeV, 0.740 MeV and 0.778 MeV; molybdenum-99 obtained with the test solution is similar in retardation factor
has a half-life of 66.0 h. to the principal peak in the chromatogram obtained with the
-- Other gamma-emitting impurities: maximum 0.01 per cent reference solution.
of the total radioactivity. TESTS
Gamma-ray spectrometry. Examine the gamma-ray spectrum pH (2.2.3)
of the decayed material for the presence of other 6.0 to 8.0.
radionuclidic impurities, which should, where possible, be Phosphates
identified and quantified. Maximum 89 ~lglMBq .
RADIOCHEMICAL PURITY Test solution Dilute the preparation to be examined with
[99m Tc]Pertechnetate ion water R to give a radioactive concentration of 370 kBq of
Descending paper chromatography (2.2.26) . phosphorus-32 per millilitre. Mix in a volumetric flask, with
shaking, 1.0 mL of this solution with a mixture of 0.5 mL of
ammonium molybdate solution R, 0.5 mL of a 2.5 giL solution
 2014
of ammonium vanadate R and 1 mL of perchlOlic acid R, and
dilute to 5.0 mL with water R .
R eference solution Prepare at the same time and in the same
manner as the test solution, using 1.0 mL of a solution
containing 33 mg of orthophosphate ion per litre.
After 30 min, the test solution is not more intensely coloured
than the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
RADIONUCLIDIC PURITY
Beta-ray spectrometry.
R esult The spectrum obtained with the preparation to be
examined does not differ significantly from that obtained
under the same conditions with a standardised phosphorus-
32 solution.
RADIOCHEMICAL PURITY
e p]Phosphate
2
Ascending paper chromatography (2.2.26).
Test solution Dilute the preparation to be examined with
water R until the radioactivity is equivalent to 10000-20 OO@
counts per minute per 1O ~L.
R eference solution A solution of phosphon·c acid R containing
2 mg of phosphorus per millilitre.
Paper Paper for chromatography R; use a strip of paper
25 mm wide and about 300 mm long.
Mobile phase Mixture of 0.3 mL of ammonia R, 5 g of
nichloroacetic acid R, 25 mL of water R and 75 mL of
2-propanol R.
Application 1O ~IL of the reference solution, then apply to
the same point of application 10 ~L of the test solution.
D evelopment For 16 h.
D rying In air.
D etection Determine the position of the non-radioactive
phosphoric acid by spraying with a 50 gIL solution of
perchloric acid R and then with a 10 gIL solution of
ammonium molybdate R . Expose the paper to hydrogen
sulfide R. A blue colour develops . Determine the distribution
of radioactivity using a suitable detector.
Limit:
- f 2p]phosphate: minimum 95 per cent of the total
radioactivity due to phosphorus-32.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
_ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ __ __ PhEur
Strontium (89 Sr) Chloride Injection
(Ph Eur monograph 1475)
PhEur _ _ _ _ _ _ _ __ _ _ _ __ _ _ _ _ __ _ _
DEFINITION
Sterile solution of [89 Sr]strontium chloride.
Strontium-89
90 p er cent to 110 per cent of the dec1ared strontium-89
radioactivity at the date stated on the labe!'
Specific radioactivity
Minimum 1.8 MBq of strontium-89 per milligram of
strontium.
Radiopharmaceutical Preparations IV-693
Strontium
6.0 mglmL to 12.5 mglmL.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of strontium-89
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray and X-ray spectrometry.
R esult The gamma photon detected has an energy of
0.909 MeV and is due to the short-lived daughter product,
yttrium-89m (formed in 0.01 per cent of the disintegrations),
in equilibrium with the strontium-89.
B. To 0.1 mL of the preparation to be examined, add 1 mL
of a freshly prepared 1 gIL solution of sodium rhodizonate R .
Mix and allow to stand for 1 mino A reddish-brown
precipitate is formed.
C. To 0.1 mL of silver nitrate solution R2 add 50 ~L of the
preparation to be examined. A white precipitate is formed.
TESTS
pH (2.2.3)
4.0 to 7.5 .
Note: the following tests for aluminium, iron and lead may be
camed out simultaneously with the test for strontium. 1f this is not
the case, the reference solutions are prepared such that they contain
strontium at approximately the same concentration as in the test
solution.
Aluminium
Maximum 2 ¡.tglmL.
Atomic emission spectrometry (plasma or arc method)
(2.2.22, Method J) .
Test solution Dilute 0.2 mL of the preparation to be
examined to a suitable volume with dilute nitric acid R.
Reference solutions Prepare the reference solutions using
aluminium standard solution (JO ppm Al) R diluted as
necessary with dilute nitric acid R.
Iron
Maximum 5 ~glmL.
Atomic emission spectrometry (plasma or arc method)
(2.2.22, Method J).
Test solution Dilute 0.2 mL of the preparation to be
examined to a suitable volume with dilute nitn·c acid R.
Reference solutions Prepare the reference solutions using iron
standard solution (20 ppm Fe) R diluted as necessary with
dilute nitric acid R.
Lead
Maximum 5 ¡.tglmL.
Atomic emission spectrometry (plasma or arc method)
(2.2.22, Method J).
Test solution Dilute 0.2 mL of the preparation to be
examined to a suitable volume with dilute nitric acid R .
Reference solutions Prepare the reference solutions using lead
standard solution (JO ppm Pb) R diluted as necessary with
d¡Jute nitn·c acid R .
Strontium
6.0 mglmL to 12.5 mglmL.
Atomic emission spectrometry (2.2.22, Method J).
Test solution Dilute 0.2 mL of the preparation to be
examined to a suitable volume with dilute nitric acid R.
IV-694 Radiopharmaceutical Preparations
R eference solutions Prepare the reference solutions using
strontium standard solution (1. Oper cent Sr) R diluted as
necessary with dilute nitric acid R.
Sterility
It complies with the test for sterility prescribed in the
monograph R adiophannaceutical preparations (0 125).
RADIONUCLIDIC PURITY
The total radioactivity due to radionuclides other than
strontium-89 is not more than 0.6 per cent.
Gamma emitters other than yttrium-89m
Maximum 0.4 per cent of the total radioactivity.
Gamma-ray and X-ray spectrometry.
Beta emitters
Evaporate to dryness 100 IlL of the preparation to be
examined under a radiant heat source. Dissolve the residue
in 2 mL of 47 per cent hydrobromic acid R, evaporate to
dryness under the radiant heat source and dissolve the
residue in 2 mL of dilute hydrobromic acid R1 . Transfer the
solution to the top of a column, 5-6 mm in diameter, packed
with approximately 2 mL of cation exchange resin R1
(100-250 ¡.¡m), previously conditioned with dilute hydrobromic
acid R1 and eiute the column with the same solvent until
10 mL of eluate has been collected into a container
containing 50 IlL of a 15 gIL solution of anhydrous sodium
sulfate R in 1 M hydrochloric acid.
To a liquid scintillation cocktail vial add an appropriate
volume of liquid scintillation cocktail R followed by 1 mL of
water R, 0.1 mL of a 15 gIL solution of anhydrous sodium
sulfate R in 1 M hydrochloric acid and 100 IlL of eluate. Shake
to obtain a clear solution. Using suitable counting equipment
determine the radioactivity due to impurities A and B in the
sample.
Taking into account the recovery efficiency of the separation,
counting efficiency and radioactive decay, determine the
radioactive concentration of impurities A and B in the sample
and hence the percentage of total beta emitting impurities in
the injection to be examined.
R esult:
- impurities A and B : maximum 0.2 per cent of the total
radioactivity.
RADIOACnVITY
Determine the radioactivity using a calibrated instrumento
IMPURlTIES
A. sulfur-35,
B. phosphorus-32.
_ __ _ _ _ __ _ __ _ _ __ _ _ _ _ _ _ _ PhEur
Technetium (99m Tc) Albumin ***
** **
Injection *****
( Technetium f9"'Tc) Human Albul1'lin Injection,
Ph Eur monograph 0640)
~E~ _ __ _ _ _ __ _ _ _ _ __ _ _ _ _ _ _ __
DEFINITION
Sterile, apyrogenic solution of human albumin labelled with
technetium-99m. Ir is prepared using Sodium pertechnetate
(99I11 Tc) injection (jission) (0124) or Sodiu111 pertechnetate
9m
f Tc) injection (non jission) (0283). Ir contains a reducing
substance, such as a tin salt in an amount not exceeding
1 mg of Sn per millilitre. Although, at present, no definite
value for a maximum limit of tin can be fixed, available
evidence tends to suggest the importance of keeping the ratio
2014
of tin to albumin as low as possible. Ir may contain a suitable
buffer and an antimicrobial preservative. The human albumin
used complies with the requirements of the monograph
H uman albumin solution (0255).
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
Albumin
90.0 per cent to 110.0 per cent of the quantity of albumin
stated on the labe!'
CHARACTERS
Appearance
Clear, colourless or pale yellow solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physical characteristus of
radionuclides.
IDENTIFICAnON
A. Gamma-ray spectrometry.
R esult The most prominent gamma photon of technetium-
99m has an energy of 0.141 MeV.
B. Using a suitable range of species-specific antisera, carry
out precipitation tests on the preparation to be examined .
The test is to be carried out using antisera specific to the
plasma proteins of each species of domestic animal currently
used in the preparation of materials of biological origin in the
country concemed. The preparation is shown to contain
proteins of human origin and gives negative results with
antisera specific to plasma proteins of other species.
C. Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal human serum, compare normal
human serum and the preparation to be examined, both
diluted if necessary. The main component of the preparation
to be examined corresponds to the main component of the
normal human serum. The diluted preparation may show the
presence of small quantities of other plasma proteins.
TESTS
pH (2.2.3)
2.0 to 6.5 .
Albumin
Test solution The preparation to be examined.
R eference solution Dilute human albumin solution R with a
9 gIL solution of sodium chloride R to a concentration of 5 mg
of albumin per millilitre.
To 1.0 mL of the test solution and to 1.0 mL of the
reference solution add 4.0 mL of biuret reagent R and mix.
After exactly 30 min, measure the absorbance (2.2.25) of
each solution at 540 nm, using as the compensation liquid a
9 giL solution of sodium chloride R treated in the same
manner. From the absorbances measured, calcula te the
content of albumin in the preparation to be examined in
milligrams per millilitre.
Tin
Maximum 1 mglmL.
Test solution To 1.0 mL of the preparation to be examined
add 1.0 mL of a 206 gIL solution of hydrochloric acid R . Heat
in a water-bath at 100 oC for 30 mino Cool and centrifuge at
300 g for 10 mino Dilute 1.0 mL of the supematant liquid to
10 mL with a 103 gIL solution of hydrochl0/1C acid R .
R eferenee solut¡on Dissolve 95 mg of stannous ehloride R in a
103 giL solution of hydrochlorie acid R and dilute to
1000.0 mL with the same acid.
 2014 Radiopharmaceutical Preparations IV-695

To 1.0 mL of each solution add 0.05 mL of thioglycollic Column:

acid R, 0.1 mL of dithiol reagent R, 0.4 mL of a 20 gIL - size: 1 = 0.6 m, 0 = 7.5 mm;
solution of sodium laurilsulfate R and 3.0 mL of a 21 gIL - stationary phase: silica gel for size-exclusion
solution of hydrochloric acid R. Mix. Measure the absorbance chromalOgraphy R .
(2.2.25) of each solution at 540 nm, using a 21 gIL solution Mobile phase mobile phase (concentrated), water R
of hydrochloric acid R as the compensation liquid. (50:50 VIV).
The absorbance of the test solution is not greater than that of Flow rate 0.6 mUmin.
the reference solution.
Detection Radioactivity detector set for technetium-99m.
Physiological distribution
Injection 200 f.lL.
Inject a volume not greater than 0.5 mL and containing not
more than 1.0 mg of alburnin into a suitable vein such as a Run time At least 10 min after background level is reached.
caudal vein or a saphenous vein of each of 3 male rats, each Retention times of eluted peaks:
weighing 150-250 g. Measure the radioactivity in the syringe 1 High molecular mas s compound 19-20 min
before and after the injection. Euthanise the rats 30 min after II Poly II1-albumin 23-24 min
the injection. Take 1 mi of blood by a suitable method and III Poly lI-albumin 25-27 min
remove the liver and, if a caudal vein has been used for the IV Poly I-albumin 28-29 min
injection, the tai!. Using a suitable instrument determine the V Human serum albumin 32-33 min
radioactivity in these organs and blood. Determine the VI Tin colloid 40-47 min
percentage of radioactivity in the liver and in 1 mL of blood VII Pertechnetate 48 min
with respect to the total radioactivity ca1culated as the
Limit:
difference between both measurements made on the syringe - f 9m Tc]technetium albumin fractions II lO V: minimum
minus the activity in the tail (if a caudal vein has been used 80 per cent of the radioactivity due to technetium-99m
for the injection) . Correct the blood radioactivity by
applied to the colurnn.
multiplying by a factor of ml200 where m is the body mass of
the rat in grams. In not fewer than 2 of the 3 rats used, the RADIOACTIVITY
radioactivity in the liver is not more than 15 per cent and Determine the radioactivity using a calibrated instrumento
that in blood, after correction, is not les s than 3.5 per cent. LABELLING
Sterility The label states:
It complies with the test for sterility prescribed in the - the amount of albumin;
monograph Radiopharmaceutical preparations (0125). - the amount of tin, if any.
The preparation may be released for use before completion
IMPURITIES
of the test.
A. C9mTc)pertechnetate ion.
Bacterial endotoxins (2.6.14) __________________________________________ PhEm
Less than 175/VIU/mL, Vbeing the maximum
recommended dose in millilitres.
RADIOCHEMICAL PURITY
Impurity A ***
Technetium (99m Tc) Bicisate
Thin-Iayer chromatography (2.2.27).
*** ***
Test solution The preparation to be examined. Injection ***
Plate TLC silica gel plate R; use silica gel as the coating (Ph Bur monograph 2123)
substance on a glass-fibre sheet, heated at 11 O oC for
10 min.
Moblle phase methyl ethyl kelOne R.
Application 5-10 f.lL and allow to dry.
Development Over a path of 10-15 cm in about 10 min.
Drying In airo
Detection Suitable detector to determine the distribution of ~Em __________________________________________
radioactivity.
DEFINITION
Retardation faclOrs C9mTc)technetium human Sterile solution of a complex of technetium-99m with diethyl
albumin = 0.0 to 0.1; impurity A = 0.9 to l.0 . N,N' -ethylenedi-L-cysteinate. It may contain stabilisers and
Limit: inert additives such as Mannitol (0559) and Disodium edetate
- impurity A: maximum 5.0 per cent of the total (0232).
radioactivity due to technetiurn-99m.
99m
Content
[ Tc]Technetium albumin fractions 11 to V 90 per cent to 11 O per cent of the declared technetium-99m
Size-exclusion chromatography (2.2.30). radioactivity at the date and hour stated on the labe!'
Mobile phase (concentrated) Dissolve 1.124 g of potassium PRODUCTION
dihydrogen phosphate R, 4.210 g of disodium hydrogen
It is prepared from N,N'-(l ,2-ethylenediyl)bis [(2R)-2-amino-
phosphate R, l.17 g of sodium chloride R and 0.10 g of sodium
3-sulfanylpropanoic acid) diethyl ester and Sodium
azide R in water R and dilute to 100 mL with the same
pertechnetate ~9mTc) injection (fission) (0124) or Sodium
solvent.
pertechnetate ~9nlTc) injection (non-fission) (0283) in the
Test solution Mix 0.25 mL of the preparation to be presence of reducing agents such as a stannous salto
examined with 0.25 mL of the mobile phase (concentrated).
Use immediately after dilution.
 IV-696 Radiopharmaceutical Preparations 2014

CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation oftechnetium-99m
See general chapter 5.7. Table of physieal eharactensties of
radionuclides. C. complex of technetium-99m with ethyl hydrogen
N,N' -ethylenedi-L-cysteinate,
IDENTIFICATION
A. Gamma-ray spectrometry.
Results The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatograms obtained in the test for
radiochemical purity (see Tests).
R esults The principal peak in the chromatogram obtained
with the test solution is similar in retardation factor to the D . complex of technetium-99m with N,N'-ethylenedi
principal peak in the chromatogram obtained with reference -L-cysteine,
solution (a). E. complex of technetium-99m with mannitol,
TESTS F. complex of technetium-99m with disodium edetate.
pH (2.2.3) _ _ _ _ _ __ _ _ _ __ _ _ _ __ _ _ _ _ _ Ph Eur
6.5 to 7.5.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaeeutieal preparations (0125). ***
Technetium (99m Tc) Colloidal
The injection may be relea sed for use before completion of *** ***
the test. Rhenium Sulfide Injection ***
RADIOCHEMICAL PURlTY Technetium (99m Tc) Colloidal Rhenium Sulphide
Impurities A, B, C, D, E, F Injection
Thin-layer chromatography (2.2.27). (Ph Bu/" monograph 0126)
Test solution The preparation to be examined. ~E~ _ _ _ _ __ _ _ __ _ _ _ __ _ __ _ _ __

Referenee solution (a) To vial B of bieisate labelling kit CRS in DEFINITION

lead shielding add 2 mL of sodium pertechnetate (99mTc)
injection (fission or non-fission) containing 400-800 MBq.
Dissolve the contents of vial A of bieisate labelling kit CRS in
3 mL of a 9 gIL solution of sodium ehlonde R. Immediately
transfer 1.0 mL of the solution contained in vial A to vial B.
Mix and alJow to stand for 30 min at room temperature.
Referenee solution (b) Sodium pertechnetate (99mTc)
injection (fission or non fission) .
Plate TLC siliea gel plate R.
Mobile phase ethyl aeetate R.
Applieation 5 J.lL, alJow the spots to dry for 5-10 mino
Development Over 4/5 of the plate o
Drying In airo
Detection Determine the distribution of radioactivity using a
suitable detector.
Retardation faetors technetium-99m bicis ate = more than
0.4; impurities A, B, C, D, E and F = less than 0.2.
System suitability The retardation factor of the principal
peak in the chromatogram obtained with reference solution
(a) is cJearly different from the retardation factor of the peak
in the chromatogram obtained with reference solution (b).
Limit:
- sum of impurities A, B, C, D, E and F: not more than
6 per cent of the total radioactivity.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. technetium-99m in colloidal form,
B. [99m Tc]pertechnetate ion,
Sterile colloidal dispersion of rhenium sulfide, the micelles of
which are labelled with technetium-99m. It is prepared using
Sodium perteehnetate (99m Te) injeetion (fission) (0124) or
Sodium perteehnetate (l 9m Tc) injeetion (non fission) (0283). It is
stabilised with gelatin. The pH of the injection may be
adjusted by the addition of a suitable buffer such as citrate
buffer.
Technetium-99m
90 per cent to 110 per cent of the decJared technetium-99m
radioactivity at the date and time stated on the labe!'
Rhenium
Maximum 0.22 mglmL.
CHARACTERS
Appearance
Light brown liquido
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physical eharactensties of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity (se e Tests) .
Result The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is 0.0 to
0.1.
C. To 1 mL add 1 mL of a 200 gIL solution of stannous
ehlonde R in hydroehloric aeid R, 5 mL of hydroehlone aeid R
and 5 mL of a 50 giL solution of thiourea R . A yellow colour
develops.
 2014
TESTS
pH (2.2.3)
4.0 to 7.0.
Rhenium
Maximum 0.22 mglrnL.
Test solution The preparation to be examined.
Reference solutions Using a solution containing 100 ¡.¡g of
potassium perrhenate R (equivalent to 60 ppm of Re) and
240 ¡.¡g of sodium thiosulfate R per millilitre, prepare a range of
solutions and dilute to the same final volume with water R .
To 1 mL of the test solution and to 1 mL of each of the
reference solutions add 1 mL of a 200 gIL solution of
stannous chloride R in hydrochloric acid R, 5 mL of hydrochloric
acid R and 5 mL of a 50 gIL solution of thiourea R and dilute
to 25.0 mL with water R. Allow to stand for 40 min and
measure the absorbance (2.2.25) of each solution at 400 nm,
using a reagent blank as the compensation liquido Using the
absorbances obtained with the reference solutions, draw a
calibration curve and calculate the concentration of rhenium
in the preparation to be examined.
Physiological distribution
Inject a volume not greater than 0.2 mL into a caudal vein of
each of 3 mice each weighing 20-25 g. Euthanise the mice
20 min after the injection, remove the liver, spleen and lungs
and measure the radioactivity in the organs using a suitable
instrumento Measure the radioactivity in the rest of the body
after having removed the tai!. Determine the percentage of
radioactivity in the liver, the spleen and the lungs using the
following expression:
A x 100
B
A radioactivity of the organ concemed,
B total radioactivity in the liver, the spleen, the lungs
and the rest of the body.
In each of the 3 mice at least 80 per cent of the radioactivity
is found in the liver and spleen and not more than 5 per cent
in the lungs. If the distribution of radioactivity in 1 of the 3
mice do es not correspond to the prescribed proportions,
repeat the test on a further 3 mice. The preparation complies
with the test if the prescribed distribution of radioactivity is
found in 5 of the 6 mice used. The preparation may be
released for use before completion of the test.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
Bacterial endotoxins (2.6.14)
Less than 175/V IU/mL, V being the maximum
recommended dose in millilitres.
RADIOCHEMICAL PURITY
99m
[ Tc]Technetium in colloidal form
Ascending paper chromatography (2.2.26).
Test solution The preparation to be examined.
Paper paper for chromatography R.
Mobile phase 9 gIL solution of sodium chloride R .
Application 10 ¡.¡L.
Development Irnrnediately over a path of 10-15 cm.
Drying In air.
Radiopharmaceutical Preparations IV-697
Detection Suitable detector to determine the distribution of
radioactivity.
R etardation factors [99mTc] technetium in colloidal
form = 0.0 to 0.1; impurity A = about 0.6; other
impurities = 0.8 to 0.9 .
Limit:
-- f 9/1lTc]technetium in colloidal form: minimum 92 per cent
of the total radioactivity due to technetium-99m.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label sta tes the concentration of rhenium expressed in
milligrams per millilitre.
IMPURITIES
A. [99mTc]pertechnetate ion.
____________________________________________ ~E~
Technetium (99mTc) Colloidal ***
*** ***
Sulfur Injection ***
Technetium (99mTc) Colloidal Sulphur Injection
(Ph Bur monograph 0131)
~E~ ____________________________________________
DEFINITION
Sterile, apyrogenic colloidal dispersion of sulfur, the micelles
of which are labelled with technetium-99m. It is prepared
Sodium pertechnetate r r
using Sodium pertechnetate 9rn Tc) injection (fission) (0124) or
9rn
Tc) injection (non fissian) (0283).
It may be stabilised with a colloid-protecting substance based
on gelatin. The pH of the injection may be adjusted by the
addition of a suitable buffer, such as an aceta te, citrate or
phosphate buffer solution. The injection contains a variable
amount of colloidal sulfur, according to the method of
preparation.
Technetium-99m
90 per cent to 110 per cent of the dec1ared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear or opalescent, colourless or yellowish liquido
Half-life and nature ofradiation oftechnetium-99 m
See general chapter 5.7. Table af physical characteristics af
radianuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity (see Tests).
R esult The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is 0.0 to
0.1.
e. In a test-tube 100 mm long and 16 mm in intemal
diameter, evaporate 0.2 mL of the preparation to be
examined to dryness. Dissolve the sulfur by shaking the
residue with 0.2 mL of pyridine R and add about 20 mg of
benzain R. Cover the open end of the tube with a filter paper
moistened with lead acetate solutian R . Heat the test-tube in a
bath containing glycerol at 150 0e. The paper slowly
becomes brown.
 IV-698 Radiopharmaceutical Preparations
TESTS
pH (2.2.3)
4.0 to 7.0.
Physiological distribution
Inject a volume not greater than 0.2 mL into the caudal vein
of each of 3 mice, each weighing 20-25 g. Euthanise the
mice 20 min after the injection, remove the liver, spleen and
lungs and measure the radioactivity in these organs using a
suitable instrumento Measure the radioactivity in the rest of
the body of each animal after having removed the tail.
Determine the percentage of radioactivity in the liver, the
spleen and the lungs using the following expression:
A x 100
B Technetium-99m
A radioactivity of the organ concerned;
B total radioactivity in the liver, the spleen, the lungs
and the rest of the body.
In each of the 3 mice at least 80 per cent of the radioactivity
is found in the liver and spleen and not more than 5 per cent
in the lungs. If the distribution of radioactivity in 1 of the 3
mice do es not correspond to the prescribed proportions,
repeat the test on a further 3 mice. The preparation to be
examined complies with the test if the prescribed distribution
of radioactivity is found in 5 of the 6 mice used.
The preparation may be relea sed for use before completion
of the test.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaeeutieal preparations (0125).
The preparation may be released for use before completion
of the test.
Pyrogens
It complies with the test for pyrogens prescribed in the
monograph Radiophannaeeutieal preparations (0125) . Inject,
per kilogram of the rabbit's mass, not les s than 0.1 mL.
The preparation may be relea sed for use before completion
of the test.
RADIOCHEMICAL PURITY
99nt
[ Tc]Technetium in coUoidal form
Ascending paper chromatography (2.2.26).
Test solution The preparation to be examined.
Paper paper for chromatography R.
Mobile phase 9 gIL solution of sodium ehloride R .
Applieation 10 ¡¡L.
Development Irnmediately, over a path of 10-15 cm.
Drying In air.
Deteetion Suitable detector to determine the distribution of
radioactivity .
Retardation faetors [99mTc]technetium in colloidal
form = 0.0 to 0.1; impurity A = about 0.6; other
impurities = 0.8 to 0.9.
Limit:
-- f 9"'Te]technetium in eoUoidal form: minimum 92 per cent
of the total radioactivity due to technetium-99m.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. [99m Tc]perrechnetate ion.
____________________________________________
2014
Technetium (99m Tc) Colloidal Tin *****
** **
Injection ***
(Ph Eur monograph 0689)
Ph E~ ____________________________________________
DEFINITION
Sterile, colloidal dispersion of tin labelled with technetium-
99m. It is prepared using Sodium pertechnetate ~9m Te)
injeetion (fission) (0124) or Sodium perteehnetate ~9''' Te)
injeetion (non fission) (0283). The injection contains a variable
quantity of tin not exceeding 1 mg of Sn per millilitre;
it contains fluoride ions, it may be stabilised with a suitable,
apyrogenic colloid-protecting substance and it may contain a
suitable buffer.
90 per cent to 110 per cent of the dec1ared technetium-99m
radioactivity at the date and time stated on the labe!'
Tin
Maxirnum 1 mglrnL.
CHARACTERS
Appearance
Clear or opalescent, colourless solution.
Half-Jife and nature of radiation oftechnetium-99m
See general chapter 5.7. Table of physieal characteristics of
radionuclides.
IDENTIFICAnON
A. Garnma-ray spectrornetry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Mix 0.05 mL of ú reonyl nitrate solution R with 0.05 mL of
alizarin S solution R . Add 0.05 mL of the preparation to be
examined. A yellow colour is produced.
TESTS
pH (2.2.3)
4.0 to 7.0.
Tin
Maximum 1 mglmL.
Test solution Dilute 3.0 rnL of the preparation to be
examined to 50.0 rnL with a 103 gIL solution of hydroehlorie
aeid R.
Referenee solution Dissolve 0.115 g of stannous ehloride R in a
103 giL solution of hydrochloric acid R and dilute to
1000.0 mL with the same acid.
To 1.0 mL of each solution add 0.05 mL of thioglycollic
acid R, 0.1 rnL of dithiol reagent R, 0.4 mL of a 20 giL
solution of sodium laurilsulfate R and 3.0 rnL of a 21 gIL
solution of hydrochloric acid R. Mix. Measure the absorbance
(2.2.25) of each solution at 540 nm, using a 21 gIL solution
of hydrochloric acid R as the compensarion liquido
The absorbance of the test solution is not greater than that of
the reference solution.
Physiological distribution
Inject not more than 0.2 mL into a caudal vein of each of 3
rnice, each weighing 20-25 g. Euthanise the mice 20 min
after the injection and remove the liver, spleen and lungs .
Measure the radioactivity in the organs using a suitable
instrumento Measure the radioactivity in the rest of the body
of each animal, after having removed the tail. Determine the
percentage of radioactivity in the liver, the spleen and the
lungs with respect to the total radioactivity of all organs and
the rest of the body excluding the tail.
PhE~
 2014
In each of the 3 mice at least 80 per cent of the radioactivity
is found in the liver and spleen and n ot more than 5 per cent
in the lungs . If the distribution of radioactivity in 1 of the 3
mice do es not correspond to the prescribed proportions,
repeat the test on a further 3 mice. The preparation to be
examined complies with the test if the prescribed distribution
of radioactivity is found in 5 of the 6 mice used.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaeeutieal preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIOCHEMICAL PURITY
99rn
[ Tc]Technetium in colloidal forro
Thin-layer chromatography (2.2.21).
Test solution The preparation to be examined.
Plate TLC siliea gel plate R; use silica gel as the coating
substance on a glass-fibre sheet heated at 110 oC for 10 mino
Mobile phase 9 gIL sol~tion of sodium ehloride R purged with
nitrogen R.
A pplieation 5-1 O ~lL.
Development Over a path of 10-15 cm in about 10 mino
Drying In air.
Detection Suitable detector to determine the distribution of
radioactivity.
Retardation faetors [9 9mTc]technetium in colloidal
form = 0.0 to 0.1; impurity A = 0.9 to 1.0.
Limit:
-- f9/11Te]teehnetium in eolloidal for1'l1 : minimum 95 per cent
of the radioactivity due to technetium-99m.
RADIOACTIVITY
D etermine the radioactivity using a calibrated instrumento
IMPURITIES
A. [99m Tc]pertechnetate ion .
____________________________________________ ~Em
Technetium (99mTc) Etifenin ***
** **
Injection *****
(Ph Eur monograph 0585)
~Em ____________________________________________
DEFINITION
Technetium (99mTc) etifenin injection is a sterile solution
which may be prepared by mixing sodium pertechnetate
(
99m
Tc) injection (fission or non-fission) with solutions of
etifenin [[ (2, 6-diethylphenyl)carbamoylmethylimino] di-acetic
acid; C16H22N20S] and stannous chloride. The injection
contains a variable quantity of tin (Sn) not exceeding
0.2 mg!mL. The injection contains not less than
90.0 per cent and not more than 110.0 per cent of the
declared technetium-99m radioactivity at the date and hour
stated on the label. Not les s than 95.0 per cent of the
radioactivity corresponds to technetium-99m complexed with
etifenin.
lt is prepared from sodium pertechnetate (99mTc) injection
(fission or non-fission) using suitable, sterile ingredients and
calculating the ratio of radionuclidic impurities with reference
to the date and hour of administration.
Radiopharmaceutical Preparations IV-699
CHARACTERS
A clear, colourless solution.
Technetium-99m has a half-life of 6.02 h and emits gamma
radiation.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrumento The spectrum does not differ significantly from
that of a standardised technetiurn-99m solution either by
direct comparison or by using an instrument calibrated with
the aid of such a solution. Standardised technetium-99m and
molybdenurn-99 solutions are available from laboratories
recognised by the competent authority. The most prominent
gamma photon of technetium-99m has an energy of
0.140 MeV.
B. Examine by liquid chromatography (2.2.29).
Test solution Dilute the injection to be examined with
methanol R to obtain a solution containing about I mg of
etifenin per millilitre.
Referenee solution Dissolve 5.0 mg of etifenin CRS in
methanol R and dilute to 5.0 mL with the same solvent.
The chromatographic procedure may be carried out using:
-- a colurnn 0.25 m long and 4.6 mm in internal diameter
packed with oetadeeylsilyl slliea gel for ehromatography R
(5 ¡.¡m to 10 ¡.¡m),
-- as mobile phase at a fiow rate of 1 mUmin a mixture of
20 volumes of methanol R and 80 volurnes of a 14 gIL
solution of potassium dihydrogen phosphate R adjusted to
pH 2.5 by the addition of phosphoric aeid R,
-- a spectrophotometer set at 230 nm.
Inject 20 ¡.¡L of each solution. The principal peak in the
chromatogram obtained with the test solution has a similar
retention time to the principal peak in the chromatogram
obtained with the reference solution.
TESTS
pH (2.2.3)
The pH of the injection is 4.0 to 6.0.
Physiological distribution
Inject 0.1 mL (equivalent to about 3.7 MBq) into a caudal
vein of each of three mice, each weighing 20 g to 25 g.
Euthanise the mice I h after the injection. Remove the liver,
gall-bladder, small intestine, large intestine and kidneys,
collecting excreted urine. Measure the radioactivity in the
organs using a suitable instrumento Measure the radioactivity
of the rest of the body, after having removed the tail.
Determine the percentage of radioactivity in each organ from
the expression:
A
13 x 100
A radioactivity of the organ concemed,
B radioactivity of all organs and the rest of the body,
excluding the tail.
In not fewer than two mice the sum of the percentages of
radioactivity in the gall-bladder and small and large intestine
is not less than 80 per cent. Not more than 3 per cent of the
radioactivity is present in the liver, and not more than
2 per cent in the kidneys.
Tin
Test solution Dilute 1.0 mL of the injection to be examined
to 5.0 mL with 1 M hydroehlorie acid.
 IV-700 Radiopharmaceutical Preparations
Relerenee so/ution Prepare a reference solution containing
0.075 mg of stannous ehloride R per millilitre in 1 M
hydroehlorie aeid.
To 1.0 mL of each solution add 0.4 mL of a 20 giL solution
of sodium laurilsulfate R, 0.05 mL of thioglyeollic acid R,
0.1 mL of dithiol reagent R and 3.0 mL of 0.2 M hydroehlorie
acid. Mix. Measure the absorbance (2.2.25) of each solution
at 540 nm, using 0.2 M hydrochlorie aeid as the compensation
liquido The absorbance of the test solution is not greater than
that of the reference solution (0.2 mg of Sn per millilitre).
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaeeutieal preparations (0125).
The injection may be relea sed for use before completion of
the test.
RADIOCHEMICAL PURITY
Examine by thin-Iayer chromatography (2.2.27) using silicic
acid as the coating substance on a glass-fibre sheet. Heat the
plate at 110 oC for 10 mino The plate used should be such
that during development the mobile phase moves over a
distance of 10 cm to 15 cm in about 15 mino
Apply to the pi ate 5 ¡.tL to 10 ¡.tL of the injection to be
examined. Develop immediately over a path of 10 cm to
15 cm using a 9 giL solution of sodium ehloride R . Allow the
plate to dry. Determine the distribution of radioactivity using
a suitable detector. Technetium-99m complexed with etifenin
migrates almost to the middle of the chromatogram and
pertechnetate ion migrates with the solvent front. Impurities
in colloidal form remain at the point of application.
The radioactivity corresponding to technetium-99m
complexed with etifenin represents not les s than
95 .0 per cent of the total radioactivity of the chromatogram.
RADIOACnVITY
Measure the radioactivity using suitable counting equipment
by comparison with a standardised technetium-99m solution
or by measurement in an instrument calibrated with the aid
of such a solution.
____________________________________________ ~Ew
Technetium (99mTc) Exametazime
Injection
(Ph Bur monograph 1925)
~Ew ____________________________________________
DEFINITION
Sterile solution of lipophilic technetium-99m exametazime
which may be prepared by dissolving a racemic mixture of
(3RS,9RS)-4,8-diaza-3,6,6,9-tetramethylundecane-2,10-dione
bisoxime in the presence of a stannous salt in Sodium
t
perteehnetate 9m Te) injection (fission) (0124) or Sodium
t
perteehnetate 9/11Te) injection (non-fission) (0283). Ir may
contain stabilisers and inert additives .
2014
Content
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
Purity
Minimum of 80 per cent of the total radioactivity
corresponds to lipophilic technetium-99m exametazime and
its meso isomer.
CHARACTERS
Appearance
Clear solution.
Half-life and nature of radiation oftechnetium-99m
see general chapter 5.7. Table 01 physieal eharacteristies 01
radionuclides.
IDENTIFICAnON
A. Gamma-ray spectrometry.
Comparison Standardised technetium-99m solution, or by
using a calibrated instrumento Standardised technetium-99m
solutions ami/or standardisation services are available from
the competent authority.
Results The spectrum obtained with the solution to be
examined do es not differ significantly from that obtained
with a standardised technetium-99m solution. The most
prominent gamma photon has an energy ofO.141 MeV.
B. Examine the chromatograms obtained in the test
Impurity A under Radiochemical purity.
Results The principal peak in the chromatogram obtained
with the test solution is similar in retention time to the peak
due to lipophilic technetium-99m exametazime in the
chromatogram obtained with the reference solution.
TESTS
pH (2.2.3)
5.0 to 10.0.
Sterility
It complies with the test for sterility prescribed in the
monograph on Radiopharmaeeutical preparations (0125).
The injection may be released for use before completion of
the test.
RADIOCHEMICAL PURITY
Impurity C
Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC siliea gel piare R; use a glass-fibre plateo
Mobile phase 9 giL solution of sodium ehloride R.
Applieation About 5 ¡.tL.
Development Immediate, over 2/3 of the plateo
Drying In airo
Deteetion Determine the distribution of radioactivity using a
suitable detector.
Retardation lactors impurity C =: 0.8 to 1.0; lipophilic
technetium-99m exametazime and impurities A, B, D and E
do not migrate.
Limits:
-- impurity C; maximum 10 per cent of the total
radioactivity .
Total oflipophilic technetium-99m exametazime and
impurity A
Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plare TLC siliea gel pIare R; use a glass-fibre plateo
Mobile phase methyl ethyl ketone R.
 2014 Radiopharmaceutical Preparations IV- 7 01

Applieation About 5 flL. IMPURITIES

Development Immediate, over 2/3 of the plateo
Drying In airo
Deteetion Determine the distribution of radioactivity using a
suitable detector.
Retardation faetors lipophilic technetium-99m
exametazime = 0.8 to 1.0, impurity A = 0.8 to 1.0,
impurity C = 0.8 to 1.0; impurities B, D and E do not
migrate.
Limits Calculate the percentage of radioactivity due to
A. meso isomer of lipophilic technetium-99m exametazime,
impurities B, D and E from test B (E) and the percentage of
the radioactivity due to impurity C from test A (A) . B. technetium-99m in colloidal form,
Calculate the total percentage of lipophilic technetium-99m C. [99m Tc]pertechnetate ion,
exametazime and impurity A from the expression: D. non lipophilic technetium-99m exametazime complex,
E. meso isomer of non lipophilic technetium-99m
100 - A - E exametazime complexo
____________________________________________ ~Ew

-- total of lipophilie teehnetium-99m exametazime and

impurity A: minimum 80 per cent of the total
radioactivity .

Technetium (99m Tc) Gluconate *****

Impurity A
Liquid chromatography (2.2.29). ** **
Test solution The preparation to be examined. Injection ***
Referenee solution Dissolve the contents of a vial of meso-rieh (Ph Eur monograph 1047)
exametazime CRS in 0.5 mL of a 9 giL solution of sodium ~Ew ___________________________________________
ehloride R and transfer to a lead-shielded, nitrogen-filled vial.
DEFINITION
Add 6 flL of a freshly prepared 1 gIL solution of stannous
ehloride R in 0.05 M hydroehlorie acid and 2.5 mL of sodium Sterile solution of a complex of technetium-99m with
pertechnetate (99mTc) injection (fission or non-fission) calcium gluconate. It is prepared using Sodium perteehnetate
9m
(' Te) injeetion fission (0124) or Sodium perteehnetate ('9/1l Te)
containing 370-740 MBq. Mix carefully and use within
30 min of preparation. injeetion non fission (0283).
Column: Technetium-99m
-- size: 1 = 0.25 m, 0 = 4.6 mm, 90 per cent to 110 per cent of the dec1ared technetium-99m
-- stationary phase: spherieal base-deaetivated end-eapped radioactivity at the date and time stated on the labe!'
oetadeeylsilyl siliea gel for ehromatography R (5 flm) with a CHARACTERS
pore size of 13 nm and a carbon loading of 11 per cent. Appearance
Mobile phase Mix 33 volumes of aeetonitrzle R and Slightly opalescent solution.
67 volumes of 0.1 M phosphate buffer solution pH 3. O R . HaIf-life and nature ofradiation oftechnetium-99m
Flow rate 1.5 mUmin. See general chapter 5. 7. Table of eharacteristies of radionuclides.
Deteetion Radioactivity detector. IDENTIFICATION
Injeetion Loop injector. A. Gamma-ray spectrometry.
Run time 20 mino Result The most prominent gamma photon of technetium-
Relative retention With reference to lipophilic technetium- 99m has an energy of 0.141 MeV.
99m exametazime: impurity A = about 1.2. B. 5 flL of the preparation to be examined complies with
System suitability Reference solution: identification A prescribed in the monograph Calcium
-- chromatogram similar to the chromatogram provided with glueonate (0172).
meso-rieh exametazime CRS, C. Examine the chromatograms obtained in tests A and B for
-- resolution: minimum of 2 between the peaks due to radiochemical purity (se e Tests).
lipophilic technetium-99m exametazime and to Results:
impurity A. -- the retardation factor of the principal peak in the
Limits: radiochromatogram obtained with the test solution in test
-- impurity A: maximum 5 per cent of the radioactivity due A is 0.9 to 1.0;
to lipophilic technetium-99m exametazime and -- the retardation factor of the principal peak in the
impurity A. radiochromatogram obtained with the test solution in
RADIOACTIVITY test B is 0.0 to 0.1.
Measure the radioactivity using suitable equipment by TESTS
comparison with a standardised technetium-99m solution or pH (2.2.3)
by using a calibrated instrumento 6.0 to 8.5.
Physiological distribution
Inject a volume not greater than 0.2 mL into the caudal vein
of each of 3 rats weighing 150-250 g. Measure the
radioactivity of the syringe before and after injection.
IV-702 Radiopharmaceutical Preparations
Euthanise the rats 30 min after the injection. Remove at least
1 g of blood by a suitable method and remove the kidneys,
the Iiver, the bladder plus voided urine and the tail. Weigh
the sample of blood.
D etermine the radioactivity in the organs, the blood sample
and the tail using a suitable instrumento Calculate the
percentage of radioactivity in each organ and in 1 g of blood
with respect to the total radioactivity calculated as the
difference between the 2 measurements made on the syringe
minus the activity in the tail. Correct the blood concentration
by multiplying by a factor of m/200 where m is the body
mass of the rat in grams.
In not fewer than 2 of the 3 rats used, the radioactivity is:
in the kidneys: minimum 15 per cent,
in che bladder plus voided urine: minimum 20 per cent,
in the liver: maximum 5 per cent.
in the blood, after correction: maximum 0.50 per cent.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125).
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
A. Impurity A. Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC siliea gel plate R; use silica gel as the coating
substance on a glass-fibre sheet heated at 110 oC for 10 mino
Mobile phase 9 gIL solution of sodiwn ehloride R .
Applieation 5-10 ¡.IL.
Development Immediately over a path of 10-15 cm in about
10 mino
D rying In air.
Detection Suitable detector to determine the distribution of
radioactivity.
R etardation faclOrs impurity A = o. O to 0.1;
[99mTc]technetium gluconate and impurity B = 0.9 to l.0.
B. Impurity B. Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC silica gel plate R ; use silica gel as the coating
substance on a glass-fibre sheet heated at 110 °C for 10 mino
Mobile phase methyl ethyl kelOne R .
Application 5-10 ¡.IL and allow to dry.
Development Over a path of 10-15 cm in about 10 mino
D rying In a current of warm air.
Deteetion Suitable detector to determine the distribution of
radioactivity.
99m
R etardation factors [ T c]technetium gluconate and
impurity A = 0.0 to 0.1; impurity B = 0.9 to l.0 .
L imit:
- sum of impurities A and B: maximum 10 per cent of the
radioactivity due to technetium-99m in the
chromatograms obtained in tests A and B.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. [99mTc]technetium in colloidal form,
B. [991l1Tc]pertechnetate ion.
____________________________________________
2014
Technetium (99m Tc) Macrosalb *****
** **
Injection ***
(Ph Eur monograph 0296)
~E~ ___________________________________________
DEFINITION
Sterile suspension of human albumin in the form of irregular
insoluble aggregates obtained by denaturing human albumin
in aqueous solution. Ir is prepared using Sodium pertechnetate
~91I1 Tc) injection (fission) (0124) or S odium pertechnetate
~911ITc) injection (non fiss ion) (0283). The particles are
labelled with technetium-99m and have a typical di ame ter
between 10 ¡.1m and 100 pm. The injection contains reducing
substances, such as tin salts; it may contain a suitable buffer
such as acetate, citrate or phosphate buffer and also non-
denature d human albumin and an antimicrobial preservative
such as benzyl alcohol.
The human albumin employed complies with the
requirements prescribed in the monograph H uman albumin
solution (0255).
Technetium-99m 90 per cent 10 110 per cent of the declared
technetium-99m radioactivity at the date and time stated on
the label.
Specific radioactiviry Minimum 37 MBq of technetium-99m
per milligram of aggregated albumin at the date and time of
administration.
CHARACTERS
Appearance
White suspension which m ay separate on standing.
Halj-life and nature of radiation of technetium-99m See
general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
R esult The most prominent gamma photon of technetium-
99m has an energy of 0.141 MeV.
B. The tests for non-filterable radioactivity and particle size
contribute to the identification of the preparation (se e Tests).
C. Transfer 1 mL of the preparation 10 be examined to a
centrifuge tube and centrifuge at 2500 g for 5-10 mino
Decant the supernatant Iiquid. To the residue add 5 mL of
cupri-tartan·c solution R2, mix and allow 10 stand for 10 mino
If necessary, heat 10 dissolve the particles and allow 10 cool.
Add rapidly 0.5 mL of dilute phosphomolybdotungstic reagent R ,
mixing immediately. A blue colour develops.
TESTS
p H (2.2.3)
3.8 10 7.5.
Non-filterable radioactivity
Minimum 90 per cent of the total radioactivity.
Use a polycarbonate membrane filter 13-25 mm in diameter,
10 ¡.1m thick and with circular pores 3 ~lm in diameter.
Fit the membrane into a suitable holder. Place 0.2 mL of the
preparation 10 be examined on the membrane and filter,
adding 20 mL of a 9 gIL solution of sodium chloride R during
the filtration. Determine the radioactivity remaining on the
membrane.
Particle size
~E~ not more than 10 particles have a maximum dimension
greater than 100 pm and no particle having a maximum
dimension greater than 150 ~lm is presento
2014
Examine using a microscope. Dilute the preparation to be
examined if necessary so that the number of partic1es is just
low enough for individual partic1es to be distinguished. Vsing
a syringe fitted with a needle having a calibre not les s than
0.35 mm, place a suitable volume in a suitable counting
chamber such as a haemocytometer cell, taking care not to
overfill the chamber. Allow the preparation to be examined
to settle for 1 min and, carefully add a cover slide without
squeezing the sample. Scan an area corresponding to at least
5000 partic1es .
Aggregated albumin
Test solution Transfer a volume of the preparation to be
examined containing about 1 mg of aggregated albumin to a
centrifuge tube and centrifuge at about 2500 g for 5-10 mino
Decant the supematant liquido Resuspend the residue in
2.0 mL of a 9 gIL solution of sodium chlmide R. Centrifuge at
2500 g for 5-10 mino Decant the supematant liquid.
Resuspend the residue in 5.0 mL of sodium carbonate
solurion R1. Heat in a water-bath at 80-90 oC to dissolve the
aggregated albumin. Allow to cool, transfer to a volumetric
ftask and dilute to 10.0 mL with sodium carbonate solution R1.
Reference solutions Prepare a range of solutions containing
0.05-0.2 mg of human albumin per millilitre by diluting
human albumin solution R with sodium carbonate solution R1 .
Introduce 3.0 mL of each solution separately into 25 mL
ftasks . To each ftask add 15.0 mL of cupn·-tartan·c solution R2,
mix and allow to stand for 10 mino Add rapidly 1.5 mL of
dilute phosphomolybdotungstic reagent R and mix immediately.
Allow to stand for 30 min and measure the absorbance
(2.2.25) of each solution at 750 nm using sodium carbonate
solution R1 as the compensation liquid. Vsing the
absorbances obtained with the reference solutions, draw a
calibration curve and calculate the content of aggregated
albumin in the preparation to be examined.
Tin
Maximum 3 mglmL.
Test solution To 1.0 mL of the preparation to be examined,
add 1.0 mL of a 206 gIL solution of hydrochloric acid R. Heat
in a water-bath for 30 mino Cool and centrifuge for 10 min
at 300 g. Dilute 1.0 mL of the supematant liquid to 25.0 mL
with a 103 gIL solution of hydrochloric acid R .
Reference solution Dissolve 0.115 g of stannous chlon·de R in a
103 gIL solution of hydrochloric acid R and dilute to
1000.0 mL with the same acid.
To 1.0 mL of each solution, add 0.05 mL of thioglycollic
acid R , 0.1 mL of dilhiol reagent R, 0.4 mL of a 20 gIL
solution of sodium laurilsulfate R and 3.0 mL of a 21 gIL
solution of hydrochloric acid R. Mix. Measure the absorbance
(2.2.25) of each solution at 540 nm, using a 21 gIL solution
of hydrochloric acid R as the compensation liquid.
The absorbance of the test solution is not greater than that of
the reference solution.
Physiological distribution
Inject a volume not greater than 0.2 mL into a caudal vein of
each of 3 rats weighing 150-250 g. Euthanise the rats 15 min
after the injection, remove the liver, the spleen and the lungs
and measure the radioactivity in the organs using a suitable
instrumento Measure the radioactivity in the rest of the body,
inc1uding the blood, after having removed the tai!. Determine
the percentage of radioactivity in the lungs, the liver and the
spleen from the following expression:
A See general chapter 5.7. Table of physical characten"stics of
B x 100
Radiopharmaceutical Preparations IV-703
A radioactivity of the organ con cerned;
B total radioactivity in the liver, the spleen, the lungs
and the rest of the body.
In not fewer than 2 of the 3 rats used, at least 80 per cent of
the radioactivity is found in the lungs and not more than a
total of 5 per cent in the liver and spleen. The preparation
may be released for use before cornpletion of the test.
SteriJity
It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125).
The preparation may be released for use before completion
of the test.
Bacterial endotoxins (2.6.14)
Less than 175/ V IV/mL, V being the maximum
recommended dose in millilitres.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label states:
-- the concentration of tin expressed in milligrams per
millilitre, if any;
-- that the preparation is to be shaken before use;
-- that the preparation is not to be used if after shaking, the
suspension does not appear homogeneous.
____________________________________________ ~E~
Technetium (99m Tc) Mebrofenin ***
*** ***
Injection ***
(Ph Eur monograph 2393)
~Ew ____________________________________________
DEFINITION
Sterile solution of a complex of technetium-99m with
mebrofenin. It may contain stabilisers and inert additives.
Content
90 per cent to 110 per cent of the dec1ared technetium-99m
radioactivity at the date and time stated on the labe!.
PRODUCTION
It is prepared by dissolving [[[(3-bromo-2,4,6-
trimethylphenyl)carbamoyl] methyl] imino] diacetic acid
(mebrofenin) in the presence of a reducing agent such as a
stannous salt in Sodium pertechnetate (l 9m Tc) injection (fission)
(0124) or Sodium pertechnetate (l 9nz Tc) injection (non-fission)
(0283).
CHARACTERS
Appearance
Clear, colourless solution.
Half-Jife and nature of radiation of technetium-99m
radionuclides.
 IV-704 Radiopharmaceutical Preparations 2014

IDENTIFICATION Time Mobile phase A Mobile phase B

A. Gamma-ray spectrometry. (min) (pereen! VII') (pereen! VII')
0·20 70 30
Results The most prominent gamma photon of technetium-
99m has an energy ofO .141 MeV. 20·25 70 -7 O 30 -7 100
B. Examine the chromatogram obtained in the test for other 25·30 O 100
radiochemical impurities (se e Tests).
Restdts The principal peak in the chromatogram obtained
with the test solution is similar in retention time to the peak Flow rate 1.0 mUmin.
due to technetium-99m mebrofenin in the chromatogram Detection Radioactivity detector.
obtained with the reference solution. Injection 20 /lL.
TESTS Relative retention With reference to technetium-99m
pH (2.2.3) mebrofenin (retention time = about 20 min):
4.0 to 7.5. impurity B = abour 0.17.
Sterility Limits:
It complies with the test for sterility prescribed in the -- technetium-99m mebrofenin: minimum 94 per cent of the
monograph on Radiopharmaceutical preparations (0125). total radioactivity.
The injection may be released for use before completion of Calculate the percentage of radioactivity due to technetium-
the test. 99m mebrofenin using the following expression:
Bacteria! endotoxins (2.6.14)
Less than 175/V IU/mL, V being the maximum (100 - A) x T
recommended dose in millilitres .
RADIOCHEMICAL PURITY A percentage of radioactivity due to impurity A
Impurity A determined in the test for impurity A under
Thin-Iayer chromatography (2.2.27). radiochemical purity;
Test solurion The preparation to be examined. T proportion of the radioactivity in the peak due to
Reference solution (a) To 1 mL of a 1 giL solution of
technetium-99m mebrofenin relative to the total eluted
radioactivity in the chromatogram obtained with the
stannous chloride R in 0.05 M hydrochloric acid in a closed vial,
add 2 mL of sodium pertechnetate (99mTc) injection (fission test solurion.
or non-fission). Use within 30 min after preparation. RADIOACTIVITY
Reference solution (b) Dissolve 40 mg of mebrofenin CRS in Determine the radioactivity using a calibrated instrumento
2 mL of water R and adjust to pH 6.5 with a 40 gIL solution IMPURITIES
of sodium hydroxide R. To this solution add 25 /lL of a
A. technetium-99m in colloidal form,
20 mglmL solution of stannous chlonde R in 0.05 M
hydrochlon'c acid and 400 MBq of sodium pertechnetate B. [99mTc]pertechnetate ion .
____________________________________________ PhEw
(99Tc) injection (fission or non-fission) in a volume of 2 mL.
Allow to stand for 15 mino
Plate TLC silica gel plate R; use a glass-fibre plateo
Mobile phase water R, acetonirrile R (40:60 V/V).
Technetium (99mTc) Medronate ***
*** ***
Application About 5 /lL.
Development Immediately, over 4/5 of the plateo
Injection ***
Drying In airo
(Ph Eur monograph 0641)
Detection Determine the distribution of radioactivity using a ~Ew ____________________________________________
suitable detector.
Retardation factor impurity A = 0-0 .1. DEFINITION
Sterile solution of a complex of technetium-99m with sodium
System suirabiliry The retardation factor of the principal
peak in the chromatogram obtained with reference solution medronate. Ir is prepared using Sodium pertechnetate (i 9m Tc)
injection (fission) (0124) or Sodium pertechnetate (i 9m Tc)
(a) is not more than 0.1. The retardation factor of the
injection (non fission) (0283). The injection may contain
principal peak in the chromatogram obtained with reference
solution (b) is more than 0.7. antimicrobial preservatives, antioxidants, stabilisers and
buffers.
Other radiochemica! impurities
Liquid chromatography (2.2.29). Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
Test solution The preparation to be examined.
radioactivity at the date and time stated on the labe!'
Reference solution Use reference solurion (b) of the test for
impurity A. CHARACTERS
Appearance
Column:
Clear, colourless solution.
-- size: 1 = 0.25 m, 0 = 4.0 mm;
-- stationary phase: end-capped octadecylsilyl silica gel for Half-life and nature ofradiation oftechnetium-99m
chromatography with polar incorporated groups R (5 /lm). See general chapter 5.7. Table of physical characteristics of
radionuclides.
Mobile phase A 3.85 gIL solution of ammonium acetate R.
Mobile phase B acetonitrile R. IDENTIFICATION
A. Gamma-ray spectrometry.
 2014
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatograms obtained in tests A and B for
radiochemical purity (see Tests) .
Results:
- the retardation factor of the principal peak in the
radiochromatogram obtained with the test solution in test
A is 0.9 to l.0;
- the retardation factor of the principal peak in the
radiochromatogram obtained with the test solution in
test B is 0.0 to O.l.
C. Thin-layer chromatography (2.2.27).
Test solution Dilute the preparation to be examined with
water R to obtain a solution containing about 0.1-0.5 mg/mL
of sodium medronate.
Reference solution Dissolve a suitable quantity (1-5 mg) of
medronic acid CRS in a suitable mixture of a 9.0 gIL solution
of sodium chloride R and water R and dilute to 10 mL with
the same solvent mixture so as to obtain a solution similar to
the test solution with regard to sodium medronate and
sodium chloride concentrations.
Plate Cellulose for chromatography R as the coating substance.
Mobile phase 2-propanol R, 1 M hydroehloric acid, methyl ethyl
ketone R (20 :30:60 VIVIV).
Application 10 llL.
Development Over a path of 12-14 cm in about 4 h.
Drying In airo
Detection Spray with ammonium molybdate solution R4, then
expose to ultraviolet light at 254 nm for about 10 min o
Results The principal spot in the chromatogram obtained
with the test solution is similar in position and colour to the
spot in the chromatogram obtained with the reference
solution.
TESTS
pH (2.2.3)
3.5 to 7.5 .
Tio
Maximum 3 mg/mL.
Test solution Dilute l.0 mL of the preparation to be
examined to 50.0 mL with a 103 giL solution of hydroehloric
acid R.
Reference solution Dissolve 0.115 g of stannous ehloride R in a
103 gIL solution of hydrochlorie acid R and dilute to
1000.0 mL with the same acid.
To 1.0 mL of each solution add 0.05 mL of thioglycollic
acid R, 0.1 mL of dithiol reagent R, 0.4 mL of a 20 gIL
solution of sodium laurilsulfate R and 3.0 mL of a 21 gIL
solution of hydrochloric acid R. Mix. Measure the absorbance
(2.2.25) of each solution at 540 nm, using a 21 gIL solution
of hydroehlorie acid R as compensation liquidoThe absorbance
of the test solution is not greater than that of the reference
solution.
Physiological distributioo
Inject a volume not greater than 0.2 mL, equivalent to not
more than 0.05 mg of sodium medronate, into a suitable vein
such as a caudal vein or the saphenous vein of each of 3 rats,
each weighing 150-250 g. Measure the radioactivity in the
syringe before and after injection. Euthanise the rats 2 h after
the injection. Remove 1 femur, the liver, and sorne blood.
Weigh the blood. Remove the tai! if a caudal vein has been
used for the injection. Using a suitable instrument measure
the radioactivity in the femur, liver and blood, and in the tail
Radiopharmaceutical Preparations IV-70S
if a caudal vein has been used for the injection. Determine
the percentage of radioactivity in each sample using the
foIlowing expression:
A
13 x 100
A radioactivity of the sample concemedí
B total radioactivity, which is equal to the difference
between the two measurements made on the syringe
minus the radioactivity in the tai! if a caudal vein has
been used for the injection.
Calculate the radioactivity per unit mas s in the blood.
Correct the blood concentration by multiplying by a factor
ml200 where m is the body mass of the rat in grams.
In not fewer than 2 of the 3 rats used, the radioactivity is:
- in the liver: maximum 1.0 per ceutí
- in the femur: mínimum 1.5 per centí
- in the blood afier correction: maximum 0.05 per cent per
gramo
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125).
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
A. Impurity A. Thin-layer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC siliea gel plate Rí use silica gel as the coating
substance on a glass-fibre sheet.
Mobile phase 136 gIL solution of sodium acetate R.
Applieation 5-10 llL.
Development Irnmediately, over a path of 10-15 cm in about
10 mino
Drying In airo
Detection Suitable detector to determine the distribution of
radioactivity.
Retardationfaetors impurity A = 0.0 to O.lí
99m
[ Tc]technetium medronate and impurity B = 0.9 to l.0.
B. Impurity B. Thin-layer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC silica gel plate Rí use silica gel as the coating
substance on a glass-fibre sheet.
Mobile phase methyl ethyl ketone R.
Applieation 5-10 llLí dry quickly.
Development Over a path of 10-15 cm in about 10 mino
Drying In airo
Deteetion Suitable detector to determine the distribution of
radioactivity.
Retardation faetors [99m Tc]technetium medronate and
impurity A = 0.0 to O.lí impurity B = 0.9 to l.0 .
Limits:
- impurity B: maximum 2.0 per cent of the radioactivity due
to technetium-99m in the chromatogram obtained in
test Bí
- sum of impurities A and B: maximum 5.0 per cent of the
radioactivity due to technetium-99m in the
chromatograms obtained in tests A and B.
RADIOACTlVITY
Determine the radioactivity using a calibrated instrumento
IV-706 Radiopharmaceutical Preparations 2014

IMPURITIES The preparation may be relea sed for use before completion
A. [99m Tc]technetium in colloidal form; of the test.
B. [99mTc]pertechnetate ion. RADIOCHEMICAL PURITY
____________________________________________ PhE~
Impurity A
Ascending paper chromatography (2.2.26).
Test solution The preparation to be examined.
Paper paper for chromatography R.
Technetium (99m Tc) Mertiatide ***
*** ***
Mobile phase water R, acetonitrile R (40:60 V/V).
Application 2 /lL.
Injection *** Development Over a path of 15 cm.
(Ph Eur monograph 1372)
Drying In air.
2- Detection Suitable detector to determine the distribution of
o radioactivity.
rl Retardation factor impurity A = 0.0-0.1.

Na+ °lN~T!Nl Limit:

2
s /11"-
o NO
yO
O Liquid chromatography (2.2.29).
~E~ ____________________________________________
DEFINITION
Sterile solution of disodium oxo[N-[N-[N-
(sulfanylacetyl)glycyl] glycyl] glycynato (5-)-
4 99m
K N,N',N",S] [ Tc]technetate(V). It may be prepared by
either heating a mixture containing
S-benzoylmercaptoacetyltriglycine (betiatide), a weak
chelating agent such as tartrate, a stannous salt and Sodium
pertechnetate (' 9m Tc) injection fission (0124) or Sodium
pertechnetate (' 9m Tc) injection non-fission (0283), or by mixing
solutions of mercaptoacetyltriglycine (mertiatide), a stannous
salt and Sodium pertechnetate (' 9m Tc) injection fission (0124) or
Sodium pertechnetate (' 9m Tc) injection non-fission (0283) at
alkaline pH. It may contain stabilísers and a buffer.
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5. 7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy of 0.141 MeV.
B. Examine the chromatograms obtained in the test of other
radiochemical impurities in the section Radiochemical purity
(see Tests) .
Result The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with the
reference solution.
TESTS
pH (2.2.3)
5.0 to 7.5.
Sterility
It complíes with the test for sterilíty prescribed in the
monograph Radiopharmaceutical preparations (0125).
-- impurity A: maximum 2.0 per cent of the total
radioactivity.
Other radiochemical impurities
Test solution The preparation to be examined.
Reference solution Dissolve with heating on a water-bath
5 mg of S-benzylmercaptoacetyltriglycine CRS in 5 mL of
water R. To 1 mL of this solution in a closed vial filled with
nitrogen R, add 0.5 mL of a 40 gIL solution of sodium
potassium tartrate R, 25 /lL of a 4 gIL solution of stannous
chloride R in a 5 gIL solution of hydrochloric acid R and
370-740 MBq of sodium pertechnetate (99mTc) injection
(fission or non-fission) in a volume not exceeding 3 mL.
Heat the mixture on a water-bath for 10 min and allow to
cool to room temperature.
Column:
-- size: l = 0.25 m, 0 = 4.0 mm;
-- stationary phase: octadecylsilyl silica gel for chromatography R
(5/lm).
Mobile phase A Mix 7 volumes of anhydrous ethanol R with
93 volumes of a 1.36 gIL solution of potassium dihydrogen
phosphate R, adjusted to pH 6.0 with a 4 gIL solution of
sodium hydroxide R.
Mobile phase B water R, methanol R (10:90 V/V).
Time Mobile phase A Mobile phase B
(min) (per cen! V/12 (per cen! V/12
0-10 100 O
10 - 25 O 100
Flow rate 1.0 mUmin.
Detection Suitable detector to determine the distribution of
radioactivity.
Equilibration With mobile phase A for 20 mino
lnjection 20 /lL.
Limits:
-- sum of the areas preceding the principal peak (corresponding to
hydrophilic impurities) including impurity B): maximum
3.0 per cent of the sum of the areas of all peaks in the
chromatogram obtained with the test solution;
-- sum of the peaks following the principal peak (corresponding to
lipophilic impurities): maximum 4.0 per cent of the sum of
the area of all peaks in the chromatogram obtained with
the test solution;
-- f9111Tc]technetium mertiatide: minimum 94 per cent of the
radioactivity due to technetium-99m.
2014
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. [9 9m Tc]technetium in colloidal form,
B. [99 m Tc]pertechnetate ion.
______________________ ~E~
Technetium (99m Tc) Microspheres ***
*** ***
Injection ***
(Ph Eur monograph 0570)
~E~ ____________________________________________
DEFINITION
Sterile suspension of human albumin which has been
denatured to form spherical insoluble partic1es. The partic1es
are labelled with technetium-99m and have a typical
diameter of 10-50 ~lm. It is prepared using S odium
pertechnetate ~9I1/Tc) injection (fission) (0124) or Sodium
pertechnetate ~9"'Tc) injection (non fission) (0283).
The injection contains reducing substances, such as tin salts.
It may contain a suitable buffer such as aceta te, citrate or
phosphate and additives such as werting agents.
The human albumin used complies with the requirements of
the monograph Human albumin solution (0255).
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
Radioactivity
Minimum 185 MBq of technetium-99m per million particles
at the date and time of administrarion.
CHARACTERS
Appearance
Suspension of white, yellow or artificially coloured particles
which may separate on standing.
Half-life and nature ofradiation oftechnetium-99m
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO .141 MeV.
B. The tests for non-filterable radioactivity and partic1e size
(see Tests) contribute to the identification of the preparation.
C. Transfer 1 mL of the preparation to be examined to a
centrifuge tube and centrifuge at 2500 g for 5-10 mino
Decant the supernatant liquido To the residue add 5 mL of
cupri-tartaric solution R2, mix and allow to stand for 10 mino
If necessary, heat to dissolve the partic1es and allow to coo!.
Add rapidly 0.5 mL of di/ute phosphomolybdolUl'lgstie reagent R,
mix irnmediarely. A blue colour develops.
TESTS
pH (2.2.3)
4.0 to 9.0 .
Non-filterable radioactivity
Minimum 95 per cent of the total radioactivity.
Use a polycarbonate membrane filter 13-25 mm in diameter,
10 ¡.tm thick and with circular pores 3 ¡.tm in diameter.
Fit the membrane into a suitable holder. Place 0.2 mL of the
preparation to be examined on the membrane and filter,
Radiopharmaceutical Preparations IV-707
adding 20 mL of a 9 gIL solution of sodium ehloride R during
the filtration. Determine the radioactivity remaining on the
membrane.
Particle size
Maximum 10 partic1es have a maximum dimension greater
than 75 ~lm but no partic1e have a maximum dimension
greater than 100 ~lm.
Examine using a microscope. Dilute the preparation if
necessary so that the number of particles is just low enough
for individual partic1es to be distinguished. Using a syringe
fitted with a needle having a calibre not less than 0.35 mm,
place a suitable volume in a suitable counting chamber such
as a haemocytometer cell, taking care not to overfill the
chamber. Allow the suspension to settle for 1 min and
carefully add a cover slide without squeezing the sample.
Scan an area corresponding to at least 5000 particles.
The partic1es have a uniform spherical appearance.
Number of particles
Examine using a microscope. Fill a suitable counting
chamber such as a haemocytometer cell with a suitable
dilution of the preparation taking care that particles do not
separate during the transfer. Count the number of partic1es
in the chamber. Repeat this procedure twice and calculate
the number of partic1es per millilitre of the preparation to be
examined.
Tin
Maximum 3 mglmL.
Test solution To 1.0 mL of the preparation to be examined
add 0.5 mL of sulfurie acid R and 1.5 mL of nim·e aeid R.
Heat and evaporate to approximately 1 mL. Add 2 mL of
water R and evaporate again to approximately 1 mL. Repeat
this procedure twice, cool and dilute to 25.0 mL with a
103 gIL solution of hydroehlorie aeid R.
Referenee soluLÍon Dissolve 0.115 g of stannous ehloride R in a
103 giL solution of hydrochlorie acid R and dilute to
1000.0 mL with the same acid.
To 1.0 mL of each solution add 0.4 mL of a 20 gIL solution
of sodium laurilsulfate R, 0.05 mL of thioglyeollie acid R,
0.1 mL of dithiol reagenr R and 3.0 mL of a 21 gIL solution
of hydroehlorie acid R. Mix. Measure the absorbance (2.2.25)
of each solution at 540 nm, using a 21 gIL solution of
hydrochloric acid R as the compensation liquido
The absorbance of the test solution is nor greater than that of
the reference solution.
Physiological distribution
Inject a volume not greater than 0.2 mL into a caudal vein of
each of 3 rats weighing 150-250 g. Euthanise the rats 15 min
after the injection, remove the liver, the spleen and the lungs
and measure the radioactivity in these organs using a suitable
instrumento Measure the radioactivity in the rest of the body,
including the blood and voided urine, after having removed
the tai!. Determine the percentage of radioactivity in the liver,
the spleen and the lungs, using the following expression:
A
13 x 100
A radioactivity of the organ con cerned,
B total radioactivity in the liver, rhe spleen, the lungs
and the rest of the body, including voided urine.
In not fewer than 2 of the 3 rats used, not less than
80 per cent of the radioactivity is found in the lungs and not
more than a total of 5 per cent in rhe liver and spleen.
IV-708 Radiopharmaceutical Preparations
The preparation may be released for use before completion
of the test.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125).
The preparation may be relea sed for use before completion
of the test.
Bacterial endotoxins (2.6.14)
Less than 175/V IU/rnL, V being the maximum
recommended dose in millilitres .
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
LABELLING
The label states:
-- the concentration of tin expressed in milligrams per
millilitre, if any,
-- that the preparation is to be shaken before use.
__________________________________________
Technetium (99mTc) Pentetate
Injection
(Ph Eur monograph 0642)
~E~ __________________________________________
DEFINITION
Sterile solution of a complex of technetium-99m with sodium
pentetate or caIcium trisodium pentetate. It is prepared using
Sodium perteehnetate ~9>IiTe) injeetion (fission) (0124) or
Sodium perteehnetate ~9mTe) injeetion (non fission) (0283).
It may contain suitable antimicrobial preservatives,
antioxidants, stabilisers and buffers.
Technetium-99m
90 per cent to 110 per cent of the decIared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear, colourIess or slightIy yellow solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physieal eharacteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatograms obtained in tests A and B for
radiochemical purity (se e Tests).
Results:
-- the retardation factor of the principal peak in the
radiochromatogram obtained with the test solution in test
A is 0.9 to 1.0;
-- the retardation factor of the principal peak in the
radiochromatogram obtained with the test solution in
test Bis 0.0 to 0.1.
e. Test solution. In a cIean, dry, 10 mL glass tube, place a
volume of the preparation to be examined containing 2 mg of
pentetate. Dilute, if necessary, to 1 mL with water R.
Referenee solution In a cIean, dry, 10 mL glass tube, place
1 rnL of water R.
2014
To each tube add 0.1 mL of a 1 gIL solution of niekel
sulfate R, 0.5 mL of a 50 per cent V/V solution of glacial
aeetie aeid R and 0.75 mL of a 50 giL solution of sodium
hydroxide R. Mix and check that the pH is not aboye 5.
To each tube add 0.1 mL of a 10 gIL solution of
dimethylglyoxime R in ethanol (96 per eent) R. Mix and aIlow
to stand for 2 mino Adjust the pH in each tube to not less
than 12 by adding a 100 gIL solution of sodium hydroxide R.
Mix and check that the pH is not below 12. Allow to stand
for 2 mino Heat the tubes gently on a water-bath for 2 mino
Results:
-- the test solution remains cIear and colourIess throughout;
-- the reference solution becomes red on addition of
dimethylglyoxime solution and a red precipita te is formed
when the tube is heated on the water-bath.
TESTS
pH (2.2.3)
4.0 to 7.5.
PhE~
Tin
Maximum 1 mglmL.
Test solution Dilute 1.5 mL of the preparation to be
examined to 25 .0 mL with a 103 gIL solution of hydroehlorie
acid R.
Referenee solution Dissolve 0.115 g of stannous ehloride R in a
103 gIL solution of hydroehlorie aeid R and dilute to
1000.0 mL with the same acid.
To 1.0 mL of each solution add 0.05 mL of thioglyeollic
acid R, 0.1 mL of dithiol reagent R, 0.4 mL of a 20 giL
solution of sodium laurilsulfate R and 3.0 mL of a 21 giL
solution of hydroehlorie acid R. Mix. Measure the absorbance
(2.2.25) of each solution at 540 nm, using a 21 gIL solution
of hydroehloric acid R as the compensation Iiquid.
The absorbance of the test solution is not greater than that of
the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125) .
The preparation may be relea sed for use before completion
of the test.
RADIOCHEMICAL PURITY
A.
Impurity A
Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC siliea gel plate R; use silica gel as the coating
substance on a glass-fibre sheet, previously heated at 110 oC
for 10 mino
Mobile phase 9 gIL solution of sodium ehloride R .
Applieation 5-10 JlL.
Development Immediately, over a path of 10-15 cm in about
10 mino
Drying In airo
Deteetion Suitable detector to determine the distribution of
radioactivity.
Retardationfaetors impurity A := 0.0 to 0.1;
99m
[ Tc]technetium pentetate and impurity B := 0.9 to 1.0.
B.
Impurity B
Thin-Iayer chromatography (2.2.27) .
Test solution The preparation to be examined.
2014 Radiopharmaceutical Preparations IV-709

Plate TLC siliea gel plate R; use silica gel as the coating Results The spectrum obtained with the solution 10 be
substance on a glass-fibre sheet, previously heated at 110 oC examined does not differ significantly from that of a
for 10 mino standardised technetium-99m solution. The most prominent
Mobile phase methyl ethyl ketone R. gamma photon has an energy of 0.141 MeV.
Applieation 5-10 IlL; allow to dry. B. Examine the chroma1Ograms obtained in the test for
impurity C under Radiochemical purity.
Development Over a path of 10-15 cm in about 10 mino
Results The principal peak in the radiochromatogram
Drying In airo
obtained with the test solution is similar in retention time to
Detection Suitable detector to determine the distribution of the principal peak in the radiochroma1Ogram obtained with
radioactivity. the reference solution.
Retardation factors [99mTc]technetium pentetate and
TESTS
impurity A = 0.0 10 0.1; impurity B = 0.9 to l.0.
pH (2.2.3)
Limit:
5.0 to 6.0.
-- sum of impurities A and B: maximum 5.0 per cent of the
radioactivity due to technetium-99m in the Sterility
chromatograms obtained in tests A and B. It complies with the test for sterility prescribed in the
monograph on Radiophannaeeutieal preparations (0125).
RADIOACTIVITY The injection may be relea sed for use before completion of
Determine the radioactivity using a calibrated insrrument. the test.
IMPURITIES RADIOCHEMICAL PURITY
A. [99mTc]technetium in colloidal form,
Impurity A and other polar impurities
B. [99mTc]pertechnetate ion. Thin-layer chroma1Ography (2.2.27) .
____________________________________________ ~Ew
Test solution The preparation to be examined.
Plate TLC oetadeeylsilyl silica gel plate R.
Mobile phase Mix 10 volumes of tetrahydrofuran R ,
20 volumes of a 38.5 gIL solution of ammonium aeetate R ,
Technetium (99mTc) Sestamibi *** 30 volumes of methanol R and 40 volumes of aeetonitrile R .
*** *** Applieation About 5 1lL.
Injection *** Development Immediately over a path of 6 cm.
(Ph Eur monograph 1926)
Drying In airo
+ Deteetion Determine the distribution of radioactivity using a
radioactivity detector.
Retardation faetors impurity B and apolar impurities = O to
0.1; impurity C and technetium-99m sestamibi = 0.3 10 0.6;
impurity A and other polar impurities = 0.9 to 1.0.
Limit See test for impurity B.
Impurity B
Paper chroma1Ography (2.2.26). 1f no aelÍvity is found at
retardalÍon factor O to 0.1 in the test for impurity A and other
polar impurities, impurity B is absent and the test for impurity B
may be omitted.
Test solutum The preparation to be examined.
Ph Eur ____________________________________________
Paper paper for ehromatography R .
DEFINITION Mobile phase Mix equal volumes of aeetonitrile R, 0.5 M
Sterile solution of (OC-6-11 )-hexakis [1-(isocyano-KC)-2- aeetie aeid and a 20 giL solution of sodium ehloride R.
r
methoxy-2-methylpropane] 9mTc]technetium(I) chloride, Applieation About 5 1lL.
which may be prepared by heating a mixture containing
Development Over a path of 10 cm.
[tetrakis (2-methoxy-2-methylpropyl-1-isocyanide) copper
(1 +)] tetrafiuoroborate, a weak chelating agent, a sta=ous Drying In airo
salt and Sodium perteehnetate (J 9m Te) injeetion (fission) (0124) DeteelÍon Determine the distribution of radioactivity using a
or Sodium perteehnetate (J 9m Te) injeetion (non-fission) (0283) . radioactivity detector.
Content RetardalÍon faetors impurity B = O to 0.1; impurity A,
90 per cent 10 110 per cent of the declared technetium-99m impurity C and technetium-99m sestamibi = 0.8 10 l.0.
radioactivity at the date and hour stated on the labe!' Limit:
-- sum of impurity A and other polar impurities, and impurity B:
CHARACTERS
maximum 5 per cent of the total radioactivity.
Appearance
Clear, colourless solution. Impurity C
Liquid chromatography (2.2.29).
Half-life and nature ofradiation oftechnetium-99m
See general chapter 5.7. Table of physieal eharaeteristics of Test solution The preparation to be examined.
radionuclides. Referenee solution To a vial of sestamibi labelling kit CRS add
3 mL of a 9 gIL solution of sodium ehloride R containing
IDENTIFICATION
700 MBq 10 900 MBq of sodium pertechnetate (99mTc)
A. Gamma-ray spectrometry.
IV- 710 Radiopharmaceutical Preparations
injection (fission or non-fission). Heat the mixture in a water-
bath for 10 min and allow to cool to room temperature.
Column:
- size: 1 = 0.25 m, 0 = 4.6 mm,
- stanonary phase: spherical base-deaenvated end-eapped
octadeeylsilyl siliea gel for ehromatography R (5 ¡.tm).
Mob17e phase Mix 20 volumes of aeetonitrile R, 35 volumes
of a 6.6 giL solution of ammonium sulfate R and 45 volumes
of methanol R.
Flow rate 1.5 mUmin.
Deteenon Radioactivity detector.
lnjeenon 25 ¡.tL.
Run time 25 mino
Relative retention With reference to technetium-99m
sestamibi: impurity C = about 1.3.
System suitability Reference solution:
- the chromatogram is similar to the chromatogram
provided with sestamibi labelling kit CRS,
- relative retention with reference to technetium-99m
sestamibi: impurity C = minimum 1.2.
Limies:
- impurity C: not more than 3 per cent of the total
radioactivity,
- teehnetium-99m sestamibi: minimum 94 per cent of the
total radioactivity.
Calculate the percentage of radioactivity due to technetium-
99m sestamibi from the expression:
(100 - B) x T
100
B percentage of radioactivity due to impurity B
determined in the test for impurity B under
Radiochemical purity,
T area of the peak due to technetium-99m sestamibi in
the chromatogram obtained with the test solution.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A. [99mTcJ04-: C9mTc)pertechnetate ion,
B. technetium-99m in colloidal form,
c. (OC-6-22)-pentakis [1-(isocyano-KC)-2-methoxy-2-
methylpropaneJ [1-(isocyano-KC)-2-methylprop-l-
eneJ [99mTcJtechnetium (1 + ).
_ _ _ _ _ __ _ _ __ _ _ _ _ _ _ _ __ _ _ Ph fur
2014
Technetium (99m Tc) Succimer *****
*
**** **
Injection
(Ph Bur monograph 0643)
~f~ _ _ _ _ __ __ _ _ _ __ _ _ _ __ __ _ _
DEFINITION
Sterile solution of a complex of technetium-99m with meso-
2,3-dimercaptosuccinic acid. It is prepared using Sodium
perteehnerate (19/11 Te) injection (fission) (0124) or Sodium
perteehnetate (191llTe) il1jeetion (non fission) (0283) . It contains
a reducing substance, such as tin salt and may contain
stabilisers, antioxidants such as ascorbic acid, and inert
additives.
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physieal eharaeteristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma photon of technetium-
99m has an energy ofO.141 MeV.
B. Examine the chromatogram obtained in the test for
radiochemical purity (see Tests).
Result The retardation factor of the principal peak in the
radiochromatogram obtained with the test solution is 0.0 to
0.1.
C. Place 1 mL of the preparation to be examined in a test-
tube and add 0.1 mL of glacial aeenc acid R and 1 mL of a
20 gIL solution of sodium nitroprusside R. Mix. Carefully place
a layer of concentrated ammonia R at the top of the solution.
A violet ring develops between the layers.
TESTS
pH (2.2.3)
2.3 to 3.5.
Tin
Maximum 1 mglmL.
Test solution Dilute 1.5 mL of the preparation to be
examined to 25.0 mL with a 103 gIL solution of hydrochloric
acid R.
Referenee solution Dissolve 0.115 g of stannous ehloride R in a
103 gIL solution of hydrochlorie acid R and dilute to
1000.0 mL with the same solution.
To 1.0 mL of each soIution add 0.05 mL of thioglyeollie
acid R, 0.1 mL of dithiol reagent R, 0.4 mL of a 20 gIL
solution of sodium laurilsulfate R and 3.0 mL of a 21 giL
soIution of hydroehloric acid R. Mix. AlIow to stand for
60 mino Measure the absorbance (2.2.25) of each soIution at
540 nm, using a 21 gIL solution of hydroehlorie acid R as the
compensation liquido The absorbance of the test solution is
not greater than that of the reference solution.
Physiological distribution
Inject a volume not greater than 0.2 mL and containing not
more than 0.1 mg of dimercaptosuccinic acid into a suitable
vein, such as a caudal vein or a saphenous vein, of each of 3
rats each weighing 150-250 g. Measure the radioactivity in
the syringe before and after the injection. Euthanise the rats
 2014
1 h after the injection. Remove the kidneys, the liver, the
sromach, the lungs and, if a caudal vein has been used for
the injection, the tai!. Using a suitable instrument determine
the radioactivity in these organs. Determine the percentage of
radioactivity in each organ with respect to the total
radioactivity calculated as the difference between the 2
measurements made on the syringe minus the activity in the
tail (if a caudal vein has been used for the injection).
In not fewer than 2 of the 3 rats used, the radioactivity is:
-- in the kidneys: minimum 40 per cent;
-- in the liver: maximum 10.0 per cent;
-- in the lungs: maximum 5.0 per cent;
-- in the sromach: maximum 2.0 per cent.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiopharmaeeutieal preparations (0125).
The preparation may be released for use before completion
of the test.
RADIOCHEMICAL PURITY
Impurity A
Thin-Iayer chromatography (2. 2.27).
Test solution The preparation to be examined.
Piare TLC siliea gel plate R; use silica gel plate as the coating
substance on a glass-fibre sheet, heated at 110 °C for
10 min o
Mobile phase methyl ethyl ketone R.
Applieation 5-10 ¡.tL.
Development Immediately, over a path of 10-15 cm in about
10 mino
Drying In air.
Deteetion Suitable detector to determine the distribution of
radioactivity .
Retardation faetors [99m Tc)technetium succimer = 0.0 ro
0.1; impurity A = 0.9 to 1.0.
Limits:
-- f9111Te]teehnetium suecimer: minimum 95.0 per cent of the
total radioactivity due to technetium-99m;
-- impurity A: maximum 2.0 per cent of the total
radioactivity due ro technetium-99m.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
STORAGE
Protected from light.
IMPURITIES
A. [99m Tc)pertechnetate ion.
____________________________________________ PhEm
Technetium (99m Tc) Tin ***
*** ***
Pyrophosphate Injection ***
(Ph Bur monograph 0129)
~Em ____________________________________________
DEFINITION
Sterile solution which may be prepared by mixing solutions
of sodium pyrophosphate and stannous chloride with Sodium
perteehnetate ~9mTe) injeetion (fission) (0124) or Sodium
perteehnetate ~9mTe) injeetion (non fission) (0283).
Technetium-99m
90 per cent to 110 per cent of the declared technetium-99m
radioactivity at the date and time stated on the labe!'
Radiopharmaceutical Preparations IV- 711
Sodium pyrophosphate (Na4Pz07,lOHzO)
1 mg/rnL ro 50 mg/mL.
Tin
Maximum 3.0 mg/rnL.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of technetium-99m
See general chapter 5.7. Table of physieal eharaeteristies of
radionuclides.
IDENTIFICATION
A. Gamma-ray spectrometry.
Result The most prominent gamma phoron of technetium-
99m has an energy of 0.141 MeV.
B. Examine the chromarograms obtained in the tests A
and B for radiochemical purity (see Tests) .
Results:
-- the retardation factor of the principal peak in the
radiochromarogram obtained with the test solution in the
test A is 0.9 ro 1.0,
-- the retardation factor of the principal peak in the
radiochromatogram obtained with the test solution in the
test B is 0.0 to 0.1.
C. To 1 mL add 1 mL of aeetie aeid R . Heat on a water-bath
for 1 h. After cooling, add 10 mL of nitro-molybdovanadie
reagent R and allow ro stand for 30 mino A yellow colour
develops.
D. To 1 mL add 0.05 mL of rhioglyeollie acid R, 0.1 mL of
dithiol reagent R, 0.4 mL of a 20 gIL solution of sodium
laurilsulfate R, 1 mL of hydroehlorie acid R, 2 mL of a
30 per cent VIV solution of sulfurie acid R and allow ro stand
for 30 mino A pink colour develops.
TESTS
pH (2.2.3)
6.0 to 7.0.
Sodium pyrophosphate
1 mg/rnL ro 50 mg/mL.
Test solurion Use 1 mL of the preparation ro be examined or
a suitable dilution of it.
Reference solutions Using a solution containing sodium
pyrophosphate R and stannous ehloride R in the same
proportions as in the test solution, prepare a range of
solutions and dilute ro the same final volume with water R.
To the test solution and to 1 mL of each of the reference
solutions add successively 10 mL of a 1 gIL solution of
disodium hydrogen phosphate R, 10 mL of iron standard solution
(8 ppm Fe) R, 5 mL of glacial aeetie acid R and 5 mL of a
1 giL solution of hydroxylamine hydroehloride R . Dilute each
solution to 40 mL with water R and heat in a water-bath at
40 oC for 1 h . To each solution, add 4 mL of a 1 giL
solution of phenanthroline hydroehloride R and dilute to
50.0 mL with water R. Measure the absorbance (2.2.25) of
each solution at 515 nm using as the compensation liquid a
reagent blank containing hydrochloric acid (1.1 gIL HCI)
instead of the iron standard solution (8 ppm Fe) R. U sing the
absorbances obtained with each of the reference solutions,
draw a calibration curve and calculate the concentration of
sodium pyrophosphate in the preparation ro be examined.
Tin
Maximum 3.0 mglmL.
Test solution Use 1 mL of the preparation ro be examined or
a suitable dilution of it.
 IV- 712 Radiopharmaceutical Preparations 2014

Reference solutions Using a solution in hydrochloric acid Limit:

(6.2 gIL He!) containing sodium pyrophosphate R and stannous -- sum of impunúes A and B: maximum 10 per cent of the
chloride R in the same proportions as in the test solution, total radioactivity due to technetium-99m in the
prepare a range of solutions and dilute to the same final chromatograms obtained in tests A and B.
volume with hydrochloric acid (6.2 gIL Hel). RADIOACTIVITY
To the test solution and to 1 mL of each of the reference Determine the radioactivity using a calibrated instrumento
solutions add 0.05 mL of thioglycollic acid R, 0.1 mL of dithiol
reagent R, 0.4 mL of a 20 gIL solution of sodium LABELLING
laun"lsulfate R, 1 mL of hydrochloric acid R and 2 mL of a The label states:
300 gIL solution of sulfuric acid R, and dilute to 15 mL with -- the concentration of sodium pyrophosphate expressed in
hydrochloric acid (6.2 gIL Hel). Allow the solutions to stand milligrams per millilitre;
for 30 min and measure the absorbance (2.2.25) of each -- the concentration of tin expressed in milligrams per
solution at 530 nm, using as the compensation liquid a millili tre.
reagent blank containing the same quantity of sodium IMPURITIES
pyrophosphate R as the test solution. Using the absorbances A. [99mTc]technetium in colloidal form,
obtained with each of the reference solutions, draw a B. [99mTc]pertechnetate ion.
calibration curve and calculate the concentration of tin in the
____________________________________________ ~E~

preparation to be examined.
Sterility
lt complies with the test for sterility prescribed in the
monograph Radiophalmaceutical preparations (O 125) .
The preparation may be released for use before completion
of the test.
Bacterial endotoxins (2.6.14)
Less than 175/VIU/mL, Vbeing the maximum
recommended dose in millilitres.
RADIOCHEMICAL PURITY
A.
Impurity A.
Thin-Iayer chromatography (2.2.27) .
Test solution The preparation to be examined.
Plate TLC silica gel plate R; use silica gel plate as the coating
substance on a glass-fibre sheet heated at 110 °e for 10 mino
Mobile phase 136 gIL solution of sodium acetate R.
Application 5-10 ¡.tL.
Development Immediately, over a path of 10-15 cm in about
10 mino
Drying In airo
Detection Suitable detector to determine the distribution of
radioactivity .
Retardationfactors Impurity A = 0.0 to 0.1;
C9mTc]technetium tin pyrophosphate and
impurity B = 0.9 to 1.0.
B.
Impurity B
Thin-Iayer chromatography (2.2.27).
Test solution The preparation to be examined.
Plate TLC silica gel plate R; use silica gel plate as the coating
substance on a glass-fibre sheet heated at 110 oC for 10 mino
Mobile phase methyl ethyl ketone R through which nitro gen
has been bubbled in the chromatographic tank for 10 min
immediately before the chromatography.
Application 5-10 ¡.tL and dry in a steam of nitrogen.
Development Over a path of 10-15 cm in about 10 mino
Drying In air.
Detection Suitable detector to determine the distribution of
radioactivity.
Retardation factors [99mTc]technetium tin
pyrophosphate = 0.0 to 0.1; impurity B = 0.95 to 1.0.
Thallous e TI) Chloride Injection
01
*****
** **
(Ph Eur monograph 0571) ***
~E~ ____________________________________________
DEFINITION
Sterile solution of thallium-20 1 in the form of thallous
chloride. It may be made isotonic by the addition of Sodium
chloride (0193) and may contain a suitable antimicrobial
preservative such as Benzyl alcohol (0256).
Thallium-201
90 per cent to 110 per cent of the declared thallium-201
radioactivity, at the date and time stated on the labe!'
Specific radioactivity
Minimum 3.7 GBq per milligram ofthallium.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation ofthallium-201
See general chapter 5.7. Table of physical characreristics of
radionuclides.
IDENTIFICATION
A. Gamma-ray and X-ray spectrometry.
Results The most prominent gamma photons of
thal1ium-201 have energies ofO .135 MeV, 0.166 MeV and
0.167 MeV; the X-rays have energies of 0.069 MeV to
0.083 MeV.
B. Examine the electropherogram obtained in the test for
radiochemical purity (see Tests) . The distribution of
radioactivity contributes to the identification of the
preparation.
TESTS
pH (2.2.3)
4.0 to 7.0.
Thallium
Test solution To 0.5 mL of the preparation to be examined
add 0.5 mL ofhydrochloric acid (220 gIL HCI) and
0.05 mL of bromine water R, and mix. Add 0.1 mL of a
30 gIL solution of sulfosalicylic acid R. After decolorisation
add 1.0 mL of a 1 gIL solution of rhodamine B R. Add 4 mL
of toluene R and shake for 60 s. Separate the toluene layer.
Reference solution Prepare at the same time and in the same
manner as the test solution, using 0.5 mL of thallium standard
solution (J Oppm Tl) R.
 2014 Radiopharmaceutical Preparations IV- 713

The toluene layer of the test solution is not more intensely
coloured than the toluene layer of the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIONUCLIDIC PURITY
Thallium-201
Minimum 97 .0 per cent ofthe total radioactivity.
Gamma-ray and X-ray spectrometry.
Determine the relative amounts of thallium-200,
thallium-20 1, thallium-202, lead-201, lead-203 and other
radionuclidic impurities presento
Result The total radioactivity due to thallium-202 is not
more than 2.0 per cent.
RADIOCHEMICAL PURITY
eOITl]Thallous ions
Zone electrophoresis (2.2.31).
Use a suitable cellulose acetate strip as the supporting
medium and a 18.6 gIL solution of sodium edecate R as the
electrolyte solution. Soak the strip in the electrolyte solution
for 45-60 min. Remove the strip with forceps taking care to
handle the outer edges only. Place the strip between 2
absorbent pads and blot to remove excess solution.
Test solution Mix equal volumes of the preparation to be
examined and the electrolyte solution.
Apply not less than 5 ¡.tL of the test solution to the centre of
the strip and mark the point of application. Apply an electric
field of 17 V/cm for at least 10 mino AlIow the strip to dry in
air. D etermine the distribution of radioactivity using suitable
detector.
Result Minimum 95.0 per cent ofthe radioactivity migrates
towards the cathode.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
IMPURITIES
A.lead-201,
B. lead-203,
C. thallium-200,
D. thallium-202,
E. [20 ¡TI] thallic(lII) ion.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Half-life and nature of radiation of tritium
See general chapter 5.7. Table of physical characteristics of
radiol1uclides.
IDENTIFICATION
Beta-ray spectrometry as described in test A for radionuclidic
purity (see Tests).
Result The maximum energy of the beta radiation is
0.019 MeV.
TESTS
pH (2.2.3)
4.5 to 7.0.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125).
RADIONUCLIDIC PURITY
A. Test soluciono Mix 100 ¡.tL of a suitable dilution of the
preparation to be examined with 10 mL of liquid scintillation
cocktail R1.
Reference solution A standardised tritiated e H) water having
approximately the same radioactivity as the test solution.
Measure the radioactivity of the test solution in a liquid
scintillation counter fitted with a discriminator. The count
should be about 5000 impulses per second at the lowest
setting of the discriminator. Record the count at different
discriminator settings . For each measurement, count at least
10000 impulses over a period of at least 1 mino Immediately
determine in me same conditions the count for the reference
solution.
Plot the counts at each discriminator setting, correcting for
background activity, on semi-Iogarithmic paper, the
discriminator settings being in arbitrary units as the abscissae.
The vertical distance between the 2 curves obtained is
constant. They obey me following mathematical relationship :
BI B z x 100 < 20
Al
BI
radioactivity recorded for the reference solution at the
lowest discriminator setting,
radioactivity recorded for the test solution to be
examined at the lowest discriminator setting,
radioactivity recorded for me reference solution at the
discriminator setting such that,

Tritiated (3H) Water Injection *** E 2 = radioactivity recorded for the test solution to be
*** *** examined at me latter discriminator setting.
(Ph Eur mOl1ograph 0112) *** B. Gamma-ray spectrometry. The instrument registers only
_ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ _ __ __
~E~
background activity.
DEFINITION RADIOCHEMICAL PURITY
Water for injections in which sorne of the water molecules Place a quantity of the preparation to be examined equivalent
contain tritium atoms in place of protium atoms. It may be to about 74 kBq, diluted to 50 mL with water R, in an all-
made isotonic by the addition of sodium chloride. glass distillation apparatus of the type used for the
determination of distillation range (2. 2. 11) . Determine the
Tritium
radio active concentration. Distil until about 25 mL of
90 per cent to 110 per cent of the declared tritium
distillate has been collected. Precautions must be taken to
radioactivity at the date stated on the labe!'
avoid contamination of me air. If me test is carried out in a
CHARACTERS fume cupboard, the equipment must be protected from
Appearance draughts. Determine me radio active concentration of the
Clear, colourless solution. distillate and of the liquid remaining in the distillation fiask.
IV- 714 Radiopharmaceutical Preparations
Neither of the radioactive concentrations determined after
distillation differs by more than 5 per cent from the value
determined before distillation.
RADIOACTIVITY
Determine the radioactivity using a Iiquid scintillation
counter.
_ _ _ _ _ __ _ __ __ _ _ _ _ __ _ __ _ Ph Eur
***
Water CSO) Injection
*** ***
(Ph Eur monograph 1582) ***
Ph Eur _ _ _ __ __ _ _ _ _ _ __ _ _ __ __ __
DEFINITION
Sterile solution of ¡I 5 0]water for diagnostic use.
Oxygen-15
90 per cent to 110 per cent ofthe dec1ared oxygen-15
radioactivity at the date and time stated on the label.
CHARACTERS
Appearance
Clear, colourless Iiquid.
Half-life and nature of radiation of oxygen -15
See general chapter 5.7. Table of physical characteristics of
radionuclides.
IDENTIFICATION
A. Gamroa-ray spectrometry.
Results The only gamma photons of ¡I 5 0]water have an
energy of 0.511 MeV and, depending on the measurement
geometry, a sum peak of 1.022 MeV may be observed.
B. Radionuc1idic purity (see Tests) .
C. Examine the chromatogram obtained in the test for
radiochemical purity (see Tests) . The retention time of the
2nd peak is due to the radioactivity eluting in the void
volume.
TESTS
pH (2.2.3)
5.5 to 8.5.
Ammonium (2.4.1)
Maximum 10 ppm, determined on 1 mL. The preparation
may be released for use before completion of the test.
Nitrates
Maximum 10 ppm. The preparation may be released for use
before completion of the test.
Test solution To 1 mL add 49 mL of nitrate-free water R.
Place 5 mL of this solution in a test-tube immersed in iced
water, add 0.4 mL of a 100 giL solution of potassium
chloride R, 0.1 mL of diphenylamine solution R and, dropwise
with shaking, 5 roL of sulfuric acid R. Transfer the tube to a
water-bath at 50 oC.
Reference solution Prepare at the same time in the same
manner as the test solution, using a mixture of 4.5 mL of
nitrate-free water R and 0.5 mL of nitrate standard solution
(2 ppm NO:J R.
After 15 min, any blue colour in the test solution is not more
intense than that in the reference solution.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophamwceutical preparations (0125).
The preparation may be released for use before completion
of the test.
2014
Bacteria! endotoxins (2.6.14)
Less than 175/ V IU/mL, V being the maximum
recommended dose in millilitres. The preparation may be
released for use before completion of the test.
RADIONUCLIDIC PURITY
The preparation may be released for use before completion
of the test.
Oxygen-15
Minimum 99 per cent oftotal radioactivity.
Gamma-ray spectrometry.
Results:
- the spectrum obtained with the preparation to be
examined does not differ significantly from that obtained
with a standardised fluorine-18 solution;
- the half-Iife is between 1.9 min and 2.2 mino
RADIOCHEMICAL PURITY
The preparation may be released for use before completion
of the test.
[150]Water
Liquid chromatography (2.2.29) .
Test solution The preparation to be examined.
Column:
- siz e: 1 = 0.25 m, 0 = 4.0 mm;
- stationary phase: aminopropylsilyl sllica gel ¡or
chromatography R (10 11m);
- temperature: constant, at 20-30 oC .
Mobile phase 10 gIL solution of potassium dihydrogen
phosphate R adjusted to pH 3 with phosphoric acid R.
Flow rate 1 mUmin.
Detection Suitable detector to determine the distribution of
radioactivity and internal recovery detection system,
consisting of a loop of the chromatographic tubing between
the injector and the column through the radioactivity
detector, which has been calibrated for count recovery.
Run time 10 mino
Identification of peaks In the chromatogram obtained with
the test solution, the 1SI peak corresponds to the injected
radioactivity of the test solution, the 2 nd peak corresponds to
the amount ofradioactivity as ¡I 5 0]water.
Limit:
-
J5
[ O]water: minimum 99 per cent of the total radioactivity
due to oxygen-15 .
RADIOACTIVITY
Determine the radioactivity using a calibrated instrumento
_ __ __ _ _ __ __ _ _ _ _ _ _ _ _ _ __ Ph E~
***
Xenon C33Xe) Injection *** ***
***
(Ph Eur monograph 0133)
~E~ _ __ _ __ _ _ _ _ _ _ _ _ __ __ _ _ __
DEFINITION
Sterile solution of xenon-133 that may be made isotonic by
the addition of sodium chloride.
Xenon-133
80 per cent to 130 per cent ofthe dec1ared xenon-133
radioactivity at the date and time stated on the label.
The injection is presented in a container that allows the
contents to be removed without introducing air bubbles.
The container is filled as completely as possible and any gas
bubble present does not occupy more than 1 per cent of the
2014 Radiopharmaceutical Preparations IV-715

volume of the injection as judged by visual comparison with

a suitable standard.
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature ofradiation ofxenon-133
See general chapter 5.7. Table oi physical characteristics oi
radionuclides.
IDENTIFICATION
Gamma-ray and X-ray spectrometry.
Comparison Standardised xenon-133 solution in a 9 gIL
solution of sodium chlol1'de R.
Results The most prominent gamma photon of xenon-133
has an energy of 0.081 MeV and there is an X-ray (resulting
from intemal conversion) of 0.030 MeV to 0.035 MeV.
TESTS
pH (2.2.3)
5.0 to 8.0.
Sterility
It complies with the test for sterility prescribed in the
monograph Radiophannaceutical preparations (0125).
The preparation may be relea sed for use before completion
of the test.
RADIONUCLIDIC PURITY
A. Gamma-ray and X-ray spectrometry.
Comparison Standardised xenon-133 solution in a 9 gIL
solution of sodium chloride R.
Result The spectrum obtained with the preparation to be
examined does not differ significantly from that obtained
with a standardised xenon- 133 solution in a 9 giL solution of
sodium chloride R, apart from any differences attributable to
the presence of xenon-131m and xenon-133m.
B. Transfer 2 mL of the preparation to be examined to an
open fiask and pass a current of air through the solution for
30 min, taking suitable precautions conceming the dispersion
of radioactivity. Measure the residual beta and gamma
activity of the solution. The activity do es not differ
significantly from the background activity detected by the
instrumento
RADIOACTIVITY
Weigh the container with its contents. Determine its total
radioactivity using suitable counting equipment by
comparison with a standardised xenon-133 solution or by
measurement in an instrument calibrated with the aid of such
a solution, operating in strictly identical conditions. If an
ionisation chamber is used its i=er wall should be such that
the radiation is not seriously attenuated. Remove at least half
the contents and re-weigh the container. Measure the total
residual radioactivity of the container and the remaining
contents as described aboye. From the measurements,
calculate the radioactive concentration ofxenon-1 33 in the
preparation to be examined.
CAUTION
Significant amounts oi xenon-133 may be present in the closures
and on the walls oi the container. This must be taken inlO account
in applying the rules concerning the transport and slOrage oi
radioactive substances and in disposing oi used comainers
IMPURITIES
A. xenon-131m.
- -- - - -______________________________________ PhEif
Monographs

Surgical Materials
2014
Absorbent Cotton *****
* *
(Ph Eur monograph 0036) **** *
_____________________________________________
~Ew
DEFlNITION
Absorbent cotton consists of new fibres or good quality
combers obtained from the seed-coat of various species of the
genus Gossypium L., cleaned, purified, bleached and carefully
carded. It may not contain any compensatory colouring
matter.
CHARACTERS
It is white or almost white and is composed of fibres of
average length not less than 10 mm, determined by a suitable
method, and contains not more than traces of leaf residue,
pericarp, seed-coat or other impurities. It offers appreciable
resistance when pulled. It do es not shed any appreciable
quantity of dust when gently shaken.
IDENTIFICATION
A. Examined under a microscope, each fibre is seen to
consist of a single cell, up to about 4 cm long and up to
40 f.lm wide, in the form of a ftattened tube with thick and
rounded walls and often twisted.
B. When treated with iodinated zinc chloride solution R, the
fibres become violeto
C. To 0.1 g add 10 mL of zinc chloride-formic acid solurion R.
Heat to 40 oC and allow to stand for 2 h 30 min, shaking
occasionally. It do es not dissolve.
TESTS
Solution S
Place 15.0 g in a suitable vessel, add 150 mL of water R,
close the vessel and allow to macerate for 2 h. Decant the
solution, squeeze the residualliquid carefully from the
sample with a glass rod and mix. Reserve 10 mL of the
solution for the test for surface-active substances and filter
the remainder.
Acidity or aIkalinity
To 25 mL of solution S add 0.1 mL of phenolphthalein
solution R and to another 25 mL add 0.05 mL of methyl
orange solution R. Neither solution is pink.
Foreign fibres
Examined under a microscope, it is seen to consist
exclusively of typical cotton fibres, except that occasionally a
few isolated foreign fibres may be presento
Fluorescence
Examine a layer about 5 mm in thickness under ultraviolet
light at 365 nm. It displays only a slight brownish-violet
ftuorescence and a few yellow partic1es. It shows no intense
blue ftuorescence, apart from that which may be shown by a
few isolated fibres.
Neps
Spread about 1 g evenly between 2 colourless transparent
plates each 10 cm square. Examine for neps by transmitted
light and compare with Couon wool standard for neps CRS.
The product to be examined is not more neppy than the
standard.
Absorbency
Apparatus A dry cylindrical copper wire basket 8.0 cm high
and 5.0 cm in diameter. The wire of which the basket is
constructed is about 0.4 mm in diameter, the mesh is 1.5 cm
to 2.0 cm wide and the mass of the basket is 2.7 ± 0.3 g.
Sinking time Not more than 10 s. Weigh the basket to the
nearest centigram (mI). Take a total of 5.00 g in
approximately equal quantities from 5 different place s in the
Surgical Ma terials IV-719
product to be examined, place loosely in the basket and
weigh the filled basket to the nearest centigram (m2). Fill a
beaker 11 cm to 12 cm in diameter to a depth of 10 cm with
water at about 20 oc. Hold the basket horizontally and drop
it from a height of about 10 mm into the water. Measure
with a stopwatch the time taken for the basket to sink below
the surface of the water. Calculate the result as the average of
3 tests.
Water-holding capacity Not less than 23.0 g of water per
gramo After the sinking time has been measured, remove the
basket from the water, allow it to drain for exactly 30 s
suspended in a horizontal position over the beaker, transfer it
to a tared beaker (m3) and weigh to the nearest centigram
(m4) . Calculate the water-holding capacity per gram of
absorbent cotton using the following expression:
Calculate the result as the average of 3 tests.
Ether-soluble substances
Not more than 0.50 per cent. In an extraction apparatus,
extract 5.00 g with ether R for 4 h at arate of at least 4
extractions per hour. Evaporate the ether extract and dry the
residue to constant mass at 100 oC to 105 oc.
Extractable colouring matter
In a narrow percolator, slowly extract 10.0 g with alcohol R
until 50 mL of extract is obtained. The liquid obtained is not
more intensely coloured (2.2.2, Method JI) than reference
solution Y5, GY 6 or a reference solution prepared as follows:
to 3.0 mL of blue primary solution add 7.0 mL of
hydrochloric acid (10 gIL HC1). Dilute 0.5 mL of this
solution to 10.0 mL with hydrochloric acid (10 gIL HC1).
Surface-active substances
Introduce the 10 mL portion of solution S reserved before
filtration into a 25 mL graduated ground-glass-stoppered
cylinder with an external diameter of 20 mm and a wall
thickness of not greater than 1.5 mm, previously rinsed
3 times with su/fun·c acid R and then with water R. Shake
vigorously 30 times in 10 s, allow to stand for 1 min and
repeat the shaking. After 5 min, any foam present must not
cover the entire surface of the liquido
Water-soluble substances
Not more than 0.50 per cent. Boil 5.000 g in 500 mL of
water R for 30 min, stirring frequentJy. Replace the water lost
by evaporation. Decant the liquid, squeeze the residual liquid
carefully from the sample with a glass rod and mix. Filter the
liquid whilst hoto Evaporate 400 mL of the filtrate
(corresponding to 4/5 of the mass of the sample taken) and
dry the residue to constant mas s at 100 oC to 105 cc.
Loss on drying (2.2.32)
Not more than 8.0 per cent, determined on 5.000 g by
drying in an oven at 105 oC.
Sulfated ash (2.4.14)
Not more than 0.40 per cent. Introduce 5.00 g into a
previously heated and cooled, tared crucible. Heat cautiously
over a naked ftame and then carefully to dull redness at
600 oc. Allow to cool, add a few drops of di/u te sulfuric
acid R, then heat and incinerate until all the black particles
have disappeared. Allow to cool. Add a few drops of
ammonium carbonate solution R. Evaporate and incinerate
carefully, allow to cool and weigh again. Repeat the
incineration for periods of 5 min to constant mass.
STORAGE
Store in a dust-proof package in a dry place.
_____________________________________________ ~Ew
IV-720 Surgical Materials
Absorbent Viscose Wadding ***
** **
(Ph Bur monograph 0034) **** *
~E~ ____________________________________________
DEFINITION
Absorbent viscose wadding consists of bleached, carefully
carded, new fibres of regenerated cellulose obtained by the
viscose process, with or without the addition of titanium
dioxide, oflinear density 1.0 dtex to 8.9 dtex (dtex = mass
of 10 000 m of fibre, expressed in grams) and cut to a
suitable staple length. It does not contain any compensatory
colouring matter.
CHARACTERS
It is white or very slightly yellow, has a lustrous or matt
appearance, and is soft to the touch.
IDENTIFICATION
A. Viscose rayon fibres may be solid or hollow; hollow fibres
may have a continuous lumen or be compartmented.
The fibres have an average length of 25 mm to 80 mm and
when examined under a microscope in the dry sta te, or when
mounted in alcohol R and water R, the following characters
are observed. They are usually of a more or less uniform
width, with many longitudinal parallel lines distributed
unequally over the width. The ends are cut more or less
straight. Matt fibres contain numerous granular particles of
approximately 1 Jlm average diameter.
Solid fibres In longitudinal view, the surface of the fibres
may be uneven or crenate. Fibres having an approximately
circular or elliptical cross section have a diameter of about
10 Jlm to 20 ~lm and those that are ftattened and twisted
ribbons vary in width from 15 Jlm to 20 Jlm as the twisting
of the filament reveals first the major axis and then the minor
axis. They are about 4 pm in thickness. Other solid cross
sections are Y-shaped and have protruding limbs with the
major axis 5 Jlm to 25 Jlm in length and the minor axis 2 ~lm
to 8 pm wide.
Hollow fibres Fibres with a continuous, hollow lumen have a
diameter of up to about 30 Jlm; they are thin-walled, with a
wall thickness of about 5 flm. When mounted in alcohol R
and water R, the lumen is clearly indicated in many fibres by
the presence of many entrapped air bubbles.
Compartmented fibres These fibres may have a diameter of
up to 80 pm; they are hollow, having a central lumen which
is divided up into several compartments. Individual
compartments vary in size but typically may be up to about
60 Jlm in length and there may be more than one
compartment across the width of each fibre. Sorne
compartments show entrapped air bubbles when the fibres
are mounted in alcohol R and water R.
B. When treated with iodinated zinc chlonde solution R, the
fibres become violet.
e. To 0.1 g add 1O mL of zinc chloride-formic acid solution R.
Heat to 40 oC and allow to stand for 2 h 30 min, shaking
occasionally. Ir dissolves completely except for the matt
variety where titanium dioxide particles remain.
D. Dissolve the residue obtained in the test for sulfated ash
by warrning gently with 5 mL of sulfunc acid R. Allow to cool
and add 0.2 mL of dilute hydrogen peroxide solution R.
The solution obtained from the lustrous variety undergoes no
change in colour; that from the matt variety shows an
orange-yellow colour, the intensity of which depends on the
quantity of titanium dioxide presento
2014
TESTS
Solution S
Place 15.0 g in a suitable vessel, add 150 mL of water R,
close the vessel and allow to macerate for 2 h. Decant the
solution, squeeze the residual liquid carefully from the
sample with a glass rod and mix. Reserve 10 mL of the
solution for othe test for surface-active substances and filter
the remainder.
Acidity or aIkalinity
To 25 mL of solution S add 0.1 mL of phenolphthalein
solution R and to another 25 mL add 0.05 mL of methyl
orange solution R. Neither solution is pink.
Foreign fibres
Examined under a microscope, it is seen to consist
exclusively of viscose fibres, except that occasionally a few
isolated foreign fibres may be present.
Fluorescence
Examine a layer about 5 mm in thickness under ultraviolet
light at 365 nm. Ir displays only a slight brownish-violet
ftuorescence . Ir shows no intense blue ftuorescence, apan
from that which may be shown by a few isolated fibres.
Absorbency
Apparatus A dry cylindrical copper-wire basket 8.0 cm high
and 5.0 cm in diameter. The wire of which the basket is
constructed is about 0.4 mm in diameter, the mesh is 1.5 cm
ro 2.0 cm wide and the mas s ofthe basket is 2.7 ± 0.3 g.
Sinking time Not more than 10 s. Weigh the basket to the
nearest centigram (mI)' Take a total of 5.00 g in
approximately equal quantities from 5 different places in the
product to be examined, place loosely in the basket and
weigh the filled basket to the nearest centigram (m2)' Fill a
beaker 11 cm to 12 cm in diameter to a depth of 10 cm with
water at about 20 oc. Hold the basket horizontally and drop
it from a height of about 10 mm into the water. Measure
with a stopwatch the time taken for the basket to sink below
the surface of the water. Calculate the result as the average of
3 tests.
Water-holding capacity Not less than 18.0 g ofwater per
gramo After the sinking time has been measured, remove the
basket from the water, allow it to drain for exactly 30 s
suspended in a horizontal position over the beaker, transfer it
ro a tared beaker (m3) and weigh to the nearest centigram
(m4)' Calculate the water-holding capacity per gram of
absorbent viscose wadding using the following expression:
Calculate the result as the average of 3 tests.
Ether-soluble substances
Not more than 0.30 per cent. In an extraction apparatus,
extract 5.00 g with ether R for 4 h at arate of at least 4
extractions per hour. Evaporate the ether extract and dry the
residue to constant mass at 100 oC to 105 D e.
Extractable colouring matter
In a narrow percolator, slowly extract 10.0 g with alcohol R
until 50 mL of extract is obtained. The liquid obtained is not
more intensely coloured (2.2.2, M ethod JI) than reference
solution Ys, GY 6 or a reference solution prepared as follows:
to 3.0 mL of blue primary solution add 7.0 mL of
hydrochloric acid (10 giL HCl) and dilute 0.5 mL of this
solution to 10.0 mL with hydrochloric acid (10 gIL HCI).
 2014
Surface-active substances
Introduce the 10 mL portion of solution S reserved before
filtration into a 25 mL graduated ground-glass-stoppered
cylinder with an external di ame ter of 20 mm and a wall
thickness of not greater than 1.5 mm, previously rinsed
3 times with sulfun'c acid R and then with water R. Shake
vigorously 30 times in lOs, allow to stand for 1 min and
repeat the shaking. After 5 min, any foam present does not
cover the en tire surface of the liquid.
Water-soluble substances
Not more than 0.70 per cent. Boil 5.00 g in 500 mL of
water R for 30 min, stirring frequently. Replace the water lost
by evaporation. D ecant the liquid, squeeze the residualliquid
carefully from the sample with a glass rod and mix. Filter the
liquid whilst hoto Evaporate 400 mL of the filtrate
(corresponding to 4/5 of the mass of the sample taken) and
dry the residue to constant mass at 100 oC to 105 oc.
Hydrogen sulfide
To 10 mL of solution S add 1.9 mL of water R, 0.15 mL of
dilute acetic acid R and 1 mL of lead acetate solution R . After
2 min, the solution is not more intensely coloured than a
reference solution prepared at the same time using 0.15 mL
of dilute acetic acid R, 1.2 mL of thioacetamide reagent R ,
1.7 mL of lead standard solution (lO ppm Pb) R ilnd 10 mL of
solution S.
Loss on drying (2.2.32)
Not more than 13.0 per cent, determined on 5.000 g by
drying in an oven at 105 uc.
Sulfated ash (2. 4.14)
Not more than 0.45 per cent for the lustrous variety and not
more than l.7 per cent for the matt variety. Introduce 5.00 g
into a previously heated and cooled, tared crucible. H eat
cautiously over a naked flame and then carefully to dull
redness at 600 oc. AlIow to cool, add a few drops of dilute
sulfuric acid R , then heat and incinerate until all the black
particles have disappeared. Allow to cool. Add a few drops of
ammonium carbonate solution R . Evaporate and incinerate
carefully, allow to cool and weigh again. Repeat the
incineration for periods of 5 min to constant mass.
STORAGE
Store in a dust-proof package in a dry place .
~~~~~~~~~~~~~~~~~~~~~_ Ph Eur
***
SUTURES
*** ***
(Ph E ur monograph 90004) ***
PhEur ~~~~~~~~~~~~~~~~~~~~~_
INTRODUCTION
The following monographs apply to sutures for human use:
Catgut, slerile (03 17), Sutures, sterile non-absorbable ( 0324) ,
Sutures, sterile synchelic absorbable braided (0667) and Sutures,
slerile synthetic absorbable monofilament (0666). They cover
pe¡formance characteristics of sutures and may inelude methods of
identification. Sutures are medical devices as defined in Directive
93/42/EEG.
These monographs can be applied to show compliance with
essential requirements as defined in Aniele 3 of Directive
93/42/EEC coven'ng the following:
Physical peiformance characteristics: diameter, breaking load,
needle auachment, packaging, sterility, information supplied by the
manufacturer (see Section 13 of Annex 1 of Direaive
93/42/EEC), labelling.
Surgical Materials IV-721
To show compliance with other essential requiremencs, the
application of appropriate harmonised standards as defined in
Aniele 5 of D irective 93/42/EEC may be considered.
~~~~~~~~~~~~~~~~~~~~~_~ E~
***
Sterile Catgut * *
** **
(Ph Eur monograph 0317) ***
~E~ ~~~~~~~~~~~~~~~~~~~~~ __
DEFINITION
Sterile catgut consists of sutures prepared from collagen
taken from the intestinal membranes of mammals. After
cleaning, the membranes are split longitudinally into strips of
varying width, which, when assembled in small numbers,
according to the diameter required, are twisted under
tension, dried, polished, selected and sterilised. The sutures
may be treated with chemical substances such as chromium
salts to prolong absorption and glycerol to make them
supple, provided such substances do not reduce tissue
acceptability .
Appropriate harmonised standards may be considered when
assessing compliance with respect to origin and processing of
raw material s and with respect to biocompatibility.
Sterile catgut is a surgical wound-closure device. Being an
absorbable suture it serves to approximate tissue during the
healing period and is subsequently metabolised by proteolytic
activity.
PRODUCTION
Production complies with relevant regulations on the use of
animal tissues in medical devices notably concerning the risk
of transmission of animal spongiform encephalopathy agents.
Appropriate harmonised standards may apply with respect to
appropriate validated methods of sterilisation, environmental
control during manufacturing, labelling and packaging.
It is essential for the effectiveness and the performance
characteristics during use and during the function al lifetime
of catgut that the following physical properties are specified:
consistent diameter, sufficient initial strength and firm needle
attachment.
The requirements outlined below have been established,
taking into account stresses which occur during normal
conditions of use. These requirements can be used to
demonstrate that individual production batches of sterile
catgut are suitable for wound closure according to usual
surgical techniques.
TESTS
Jf stored in a preserving liquid, remove the sutures from the sachet
and measure promptly and in succession the lengeh, diameter and
breaking load. Jf sto red in the dry state, immerse the sutures in
alcohol R or a 90 per cent V/V solution of 2-propanol R for 24 h
and proceed with the measurements as indicated below.
Length
Measure the length without applying to the suture more
tension than is necessary to keep it straight. The length of
each suture is n ot less than 90 per cent of the length stated
on the label and does not exceed 350 cm.
Diameter
Carry out the test on 5 sutures. U se a suitable instrument
capable of measuring with an accuracy of at least 0.002 mm
and having a circular pressor foot 10 mm to 15 mm in
diameter. The pressor foot and the moving parts attached to
IV- 722 Surgical Materials 2014

it are weighted so as to apply a total load of 100 ± 10 g to and no individual result is les s than that given in column D
the suture being tested. When making the measurement, for the gauge number concerned.
lower the pressor foot slowly to avoid crushing the suture.
Measure the diameter at intervals of 30 cm over the whole
length of the suture. For a suture less than 90 cm in length,
measure at 3 points approximately evenly spaced along the
suture. The suture is not subjected to more tension than is
necessary to keep it straight during measurement.
The average of the measurements carried out on the sutures
being tested and not less than two-thirds of the
measurements taken on each suture are within the limits
given in the columns under A in Table 0317.-1 for the gauge
number concerned. None of the measurements is outside the
limits given in the columns under B in Table 0317.-1 for the
gauge number concerned.

Table 0317.-1.- Diamelers and Breaking Loads

Diame!er Breaking load Figure 0317.-1. - Simple knot
(millimet res) (newtons)
Gauge
number A B e D
max.
Soluble chromium compounds
max.
mino mino
I Place 0.25 g in a conical fiask containing 1 mL of water R
0.1 0.010 0.019 0.005 0.025 per 10 mg of catgut. Stopper the fiask, allow to stand at 37
0.2 0.020 0.029 0.015 0.035 ± 0.5 oC for 24 h, cool and decant the liquid. Transfer
5 mL to a small test tube and add 2 mL of a 10 giL solution
0.3 0.030 0.039 0.025 0.045 0.20 0.05
of diphenylcarbazide R in alcohol R and 2 mL of dilute sulfuric
0.4 0.040 0.049 0.035 0.060 0.30 0.10 acid R. The solution is not more intensely coloured than a
0.5 0.050 0.069 0.045 0.085 0.40 0.20 standard prepared at the same time using 5 mL of a solution
containing 2.83 Jlg of potassium dichromate R per millilitre,
0.7 0.070 0.099 0.060 0.125 0.70 0.30
2 mL of dilute sulfuric acid R and 2 mL of a 10 giL solution
l 0.100 0.149 0.085 0.175 1.8 0.40 of diphenylcarbazide R in alcohol R (1 ppm of Cr).
1.5 0.150 0.199 0.125 0.225 3.8 0.70 Needle attachment
2 0.200 0.249 0.175 0.275 7.5 1.8 If the catgut is supplied with an eyeless needle attached that is
not stated to be detachable, it complies with the test for needle
2.5 0.250 0.299 0.225 0.325 10 3.8
attachment. Carry out the test on 5 sutures. Use a suitable
3 0.300 0.349 0.275 0.375 12.5 7.5 tensilometer, such as that described for the determination of
3.5 0.350 0.399 0.325 0.450 20 10 the minimum breaking load. Fix the needle and suture
(without knot) in the clamps of the apparatus in such a way
4 0.400 0.499 0.375 0.550 27.5 12.5
that the swaged part of the needle is completely free of the
5 0.500 0.599 0.450 0.650 38.0 20.0 clamp and in line with the direction of pull on the suture.
6 0.600 0.699 0.550 0.750 45.0 27.5 Set the mobile clamp in motion and note the force required to
break the suture or to detach it from the needle. The average
7 0.700 0.799 0.650 0.850 60.0 38.0
ofthe 5 determinations and all individual values are not les s
8 0.800 0.899 0.750 0.950 70.0 45.0 than the respective values given in Table 0317.-2 for the gauge
number concerned. If not more than one individual value fails
to meet the individual requirement, repeat the test on an
Minimum breaking load additional 10 sutures. The catgut complies with the test if
The minimum breaking load is determined over a simple none of these 10 values is less than the individual value in
knot formed by placing one end of a suture held in the right Table 0317 .-2 for the gauge number concerned.
hand over the other end held in the left hand, passing one
end over the suture and through the loop so formed (see
Table 0317.-2. - Minimum Strengths of Needle Attachmenl
Figure 0317.-1) and pulling the knot tight. Carry out the test
Gauge number Mean value Individual values
on 5 sutures. Submit sutures of length greater than 75 cm to (new!ons) (new!ons)
2 measurements and shorter sutures to one measurement. 0.5 0.50 0.25
Determine the breaking load using a suitable tensilometer.
0.7 0.80 0.40
The apparatus has 2 clamp s for holding the suture, one of
which is mobile and is driven at a constant rate of 1.7 0.80
30 cm/mino The clamp s are designed so that the suture being 1.5 2.3 1.1
tested can be attached without any possibility of slipping.
2 4.5 2.3
At the beginning of the test the length of suture between the
clamps is 12.5 cm to 20 cm and the knot is midway between 2.5 5.6 2.8
the clamps. Set the mobile clamp in motion and note the 3 6.8 3.4
force required to break the suture. If the suture breaks in a
3.5 1l.0 4.5
clamp or within 1 cm of it, the result is discarded and the
test repeated on another suture. The average of all the 4 15.0 4.5
results, excluding those legitimately discarded, is equal to or 5 18.0 6.0
greater than the value given in column C in Table 0317. -1
2014
STORAGE (PACKAGING)
Sterile catgut sutures are presented in individual sachets that
maintain sterility and allow the withdrawal and use of the
sutures in aseptic conditions. Sterile catgut may be stored dry
or in a preserving liquid to which an antimicrobial agent but
not an antibiotic may be added.
Sutures in their individual sachets (primary packaging) are
kept in a protective cover (box) which maintains the physical
and mechanical properties until the time of use.
The application of appropriate harmonised standards for
packaging of medical devices shall be considered.
LABELLING
Reference may be made to the appropriate harmonised
standards for labelling of medical devices.
The details strictly necessary for the user to identify the
product properly are indicated on or in each sachet (primary
packaging) and on the protective cover (box) and include at
least:
-- gauge number,
-- length in centimetres or metres,
-- if appropriate, that the needle is detachable,
-- name of the product,
-- intended use (surgical suture, absorbable).
____________________________________________ PhE~
***
Sterile Synthetic Absorbable
*** ***
Braided Sutures ***
(Ph Eur monograph 0667)
Ph Eur ___________________________________________
DEFINITION
Sterile synthetic absorbable braided sutures consist of sutures
prepared from a synthetic polymer, polymers or copolymers
which, when introduced into a living organism, are absorbed
by that organism and cause no undue tissue irritation. They
consist of completely polymerised material. They occur as
multifilament sutures consisting of elementary fibres which
are assembled by braiding. The sutures may be treated to
facilitate handling and they may be coloured.
Appropriate harmonised standards may be considered when
assessing compliance with respect to origin and processing of
raw materials and with respect to biocompatibility.
Sterile synthetic absorbable braided sutures are wound-
closure devices. Being absorbable they serve to approximate
tissue during the healing period and subsequently lose tensile
strength by hydrolysis.
PRODUCTION
Appropriate harmonised standards may apply with respect to
appropriate validated methods of sterilisation, environmental
control during manufacturing, labelling and packaging.
It is essential for the effectiveness and the performance
characteristics during use and during the functionallifetime
of these sutures that the following physical properties are
specified: consistent diameter, sufficient initial strength and
firm needle attachment.
The requirements below have been established, taking into
account stresses which occur during normal conditions of
use. These requirements can be used to demonstrate that
individual production batches of these sutures are suitable for
wound closure according to usual surgical techniques .
Surgical Materials IV- 723
TESTS
Cany out the following tests on the sutures in the state in zvhich
they are removed from the sachet.
Length
Measure the length of the suture without applying more
tension than is necessary to keep it straight. The length of
eacn suture is not less than 95 per cent of the length stated
on the label and does not exceed 400 cm.
Diameter
Unless otherwise prescribed, measure the diameter by the
following method, using five sutures in the condition in
which they are presented. Use a suitable instrument capable
of measuring with an accuracy of at least 0.002 mm and
having a circular pressor foot 10 mm to 15 mm in diameter.
The pressor foot and the moving parts attached to it are
weighted so as to apply a total load of 100 ± 10 g to the
suture being tested. When making the measurements, lower
the pressor foot slowly to avoid crushing the suture. Measure
the diameter at intervals of 30 cm over the whole length of
the suture. For a suture less than 90 cm in length, measure
at three points approximately evenly spaced along the suture.
During the measurement, submit the sutures to a tension not
greater than one-fifth of the minimum breaking load shown
in column C of Table 0667.-1 appropriate to the gauge
number and type of material or ION whichever is less.
For sutures of gauge number aboye 1.5 make two
measurements at each point, the second measurement being
made after rotating the suture through 90°. The diameter of
that point is the average of the two measurements.
The average of the measurements carried out on the sutures
being tested and not less than two-thirds of the
measurements taken on each suture are within the limits
given in the columns under A in Table 0667.-1 for the gauge
number concerned. None of the measurements is outside the
limits given in the columns under B in Table 0667.-1 for the
gauge number con cerned.
Minimum breaking load
The minimum breaking load is determined over a simple
kilot formed by placing one end of a suture held in the right
hand over the other end held in the left hand, passing one
end over the suture and through the loop so formed (se e
Figure 0667.-1) and pulling the kilot tight.
Carry out the test on five sutures. Submit sutures of length
greater than 75 cm to two measurements and shorter sutures
to one measurement. Determine the breaking load using a
suitable tensilometer. The apparatus has two clamps for
holding the suture, one of which is mobile and is driven at a
constant rate of 25 cm to 30 cm per minute. The clamps are
designed so that the suture being tested can be attached
without any possibility of slipping. At the beginning of the
test the length of suture between the clamp s is 12.5 cm to
20 cm and the kilot is midway between the clamps. Set the
mobile clamp in motion and note the force required to break
the suture. If the suture breaks in a clamp or within 1 cm of
it, the result is discarded and the test repeated on another
suture. The average of all the results excluding those
legitimately discarded is equal to or greater than the value
given in column C in Table 0667.-1 and no individual result
is less than that given in column D for the gauge number
concerned.
 IV-724 Surgical Materials 2014

Table 0667.-1. - Diameters and breaking loads values is less than the individual value in Table 0667.-2 for
Diameter Breaking load the gauge number concerned.
(millimetres) (newtons)
Gauge
number A B e D
Table 0667.-2. - Minimum strengths of needle aftachment
mino max. mino max.
Gauge number Mean value Individual value
0.01 0.001 0.004 0.0008 0.005 (newtons) (newtons)
0.4 0.50 0.25
0.05 0.005 0.009 0.003 0.012
0.5 0.80 0.40
0.1 0.010 0.019 0.005 0.025
0.7 1.7 0.80
0.2 0.020 0.029 0.015 0.035
2.3 1.1
0.3 0.030 0.039 0.025 0.045 0.45 0.23
1.5 4.5 2.3
0.4 0.040 0.049 0.035 0.060 0.70 0.35
2 6.8 3.4
0.5 0.050 0.069 0.045 0.085 1.4 0.7
2.5 9.0 4.5
0.7 0.070 0.099 0.060 0.125 2.5 1.3
3 U.O 4.5
1 0.100 0.149 0.085 0.175 6.8 3.4
3.5 15.0 4.5
1.5 0.150 0.199 0.125 0.225 9.5 4.8
4 18.0 6.0
2 0.200 0.249 0.175 0.275 17.7 8.9
5 18.0 7.0
2.5 0.250 0.299 0.225 0.325 21.0 10.5
3 0.300 0.349 0.275 0.375 26.8 13.4
3.5 0.350 0.399 0.325 0.450 39.0 18.5 STORAGE (PACKAGING)
Sterile synthetic absorbable braided sutures are presented in
4 0.400 0.499 0.375 0.550 50.8 25.4
a suitable sachet that maintains sterility and allows the
5 0.500 0.599 0.450 0.650 63.5 31.8 withdrawal and use of the sutures in aseptic conditions.
6 0.600 0.699 0.550 0.750 The sutures must be stored dry.
7 0.700 0.799 0.650 0.850 They are intended to be used only on the occasion when the
sachet is first opened.
Sutures in their individual sachets (primary packaging) are
kept in a protective cover (box) which maintains the physical
and mechanical properties until the time of use.
The application of appropriate harmonised standards for
packaging of medie al devices may be considered in addition.
LABELLING
Reference may be made to the appropriate harmonised
standards for the labelling of medie al devices.
The details strictly necessary for the user to identify the
product properly are indicated on or in each sachet (primary
packaging) and on the protective cover (box) and include at
least:
- gauge number,
- length in centimetres or metres,
- if appropriate, that the needle is detachable,
Figure 0667.-1. - Simple knot - name of the product,
- intended use (surgical absorbable suture),
- if appropriate, that the suture is coloured,
Needle attachment - the structure (braided).
If the suture is supplied with an eyeless needle anached that _ _ _ _ _ __ __ _ __ _ _ __ _ _ __ _ _ Ph Eur
is not stated to be detachable the attachment, it complies
with the test for needle attachment. Carry out the test on five
sutures. Use a suitable tensilometer, such as that described
for the determination of the minimum breaking load. Fix the
needle and suture (without mot) in the clamps of the Sterile Synthetic Absorbable ***
apparatus in such a way that the swaged part of the needle is *** ***
completely free of the clamp and in line with the direction of Monofilament Sutures ***
pul! on the suture. Set the mobile clamp in motion and note (Ph Eur monograph 0666)
the force required ro break the suture or ro detach it from ~E~ _ __ _ __ __ __ _ _ __ _ _ __ _ __ _
the needle. The average of the five deter-minations and al!
individual values are not less than the respective values given DEFINITION
in Table 0667 .-2 for the gauge number concerned. Ifnot Sterile synthetic absorbable monofilament sutures consist of
more than one individual value fails to meet the individual sutures prepared from a synthetic polymer, polymers or
requirement, repeat the test on an additional ten sutures. copolymers which, when introduced into a living organism,
The attachment complies with the test if none of the ten are absorbed by that organism and cause no undue tissue
irritation. They consist of completely polymerised material.
2014 Surgical Materials IV-725

They occur as monofilament sutures. The sutures may be Table 0666.-1. - Diamelers and breaking loads
treated to facilitate handling and they may be coloured. Diameter Breaking load
(milIlmetres) (newlons)
Appropriate harmonised standards may be considered when Gauge
assessing compliance with respect to origin and processing of number A B e D
raw material s and with respect to biocompatibility. mino max. mino max.

Sterile synthetic absorbable monofilament sutures are wound- 0.5 0.050 0.094 0.045 0.125 1.4 0.7
closure devices. Being absorbable they serve to approximate
0.7 0.095 0.149 0.075 0.175 2.5 1.3
tissue during the healing period and subsequently lose tensile
strength by hydrolysis . 1 0.150 0.199 0.125 0.225 6.8 3.4

PRODUCTION 1.5 0.200 0.249 0.175 0.275 9.5 4.7

The appropriate harmonised standards may apply with 2 0.250 0.339 0.225 0.375 17.5 8.9
respect to appropriate validated methods of sterilisation, 3 0.340 0.399 0.325 0.450 26.8 13.4
environmental control during manufacturing, labelling and
packaging. 3.5 0.400 0.499 0.375 0.550 39.0 18.5

It is essential for the effectiveness and the performance
characteristics during use and during the functionallifetime
of these sutures that the following physical properties are
specified: consistent diameter, sufficient initial strength and
firm needle attachment.
The requirements below have been established, taking into
account stresses which occur during normal conditions of
use . These requirements can be used to demonstrate that
individual production batches of these sutures are suitable for
wound closure according to usual surgical techniques.
TESTS
Carry out che following tests on the sutures in the state in which
they are removed from the sachet.
Length
Measure the length of the suture without applying more
tension than is necessary to keep it straight. The length of
each suture is not less than 95 per cent of the length stated
on the label and does not exceed 400 cm.
Díameter
Unless otherwise prescribed, measure the diameter by the
following method, using five sutures in the condition in
which they are presented. Use a suitable instrument cap-able
of measuring with an accuracy of at least 0.002 mm and
having a circular pressor foot 10 mm to 15 mm in diameter.
The pressor foot and the moving parts attached to it are
weighted so as to apply a total load of 100 ± 10 g to the
suture being tested. When making the measurements, lower
the pressor foot slowly to avoid crushing the suture. Measure
the diameter at intervals of 30 cm over the whole length of
the suture. For a suture less than 90 cm in length, measure
at three points approximately evenly spaced along the suture.
During the measurement, submit the sutures to a tension not
greater than that required to keep them straight. The average
of the measurements carried out on the sutures being tested
and not less than two-thirds of the measurements taken on
each suture are within the limits given in the columns under
A in Table 0666 .-1 for the gauge number concemed. None
of the measurements is outside the limits given in the
columns under B in Table 0666.-1 for the gauge number
concemed.
Mínimum breaking load
The minimum breaking load is determined over a simple
knot formed by placing one end of a suture held in the right
hand over the other end held in the left hand, passing one
end over the suture and through the loop so formed (see
Figure 0666.-1 ) and pulling the knot tight.
4 0.500 0.570 0.450 0.600 50.8 25.4
5 0.571 0.610 0.500 0.700 63.5 31.8
Figure 0666.-1. - Simple knot
Carry out the test on five sutures. Submit sutures of length
greater than 75 cm to two measurements and shorter sutures
to one measurement. Determine the breaking load using a
suitable tensilometer. The apparatus has two c1amps for
holding the suture, one of which is mobile and is driven at a
constant rate of 25 cm to 30 cm per minute. The c1amps are
designed so that the suture being tested can be attached
without any possibility of slipping. At the beginning of the
test the length of suture between the clamps is 12.5 cm to
20 cm and the knot is midway between the clamps . Set the
mobile clamp in motion and note the force required to break
the suture. If the suture breaks in a clamp or within 1 cm of
it, the result is discarded and the test repeated on another
suture. The average of all the results excluding those
legitimately discarded is equal to or greater than the value
given in column C in Table 0666.-1 and no individual result
is less than that given in column D for the gauge number
concemed.
Needle attachment
If the suture is supplied with an eyeless needle attached that
is not stated to be detachab1e, the attachment complies with
the test for needle attachment. Carry out the test on five
sutures. Use a suitable tensilometer, such as that described
for the determination of the minimum breaking load. Fix the
needle and suture (without knot) in the clamps of the
apparatus in such a way that the swaged part of the needle is
completely free of the clamp and in line with the direction of
pull on the suture. Set the mobile clamp in motion and note
the force required to break the suture or to detach it from
IV-726 Surgical Materials 2014

the needle. The average of the five determinations and all

Sterile Non-absorbable Sutures *****
individual values are not less than the respective values given ** **
in Table 0666.-2 for the gauge number concerned. If not Sterile Non-absorbable Ligatures ***
more than one individual value fails to meet the individual (Ph Eur monograph 0324)
requirement, repeat the test on an additional ten sutures.
NOTE : The name Nylon 6 as a synonym for Polyamide 6 and
The attachment complies with the test if non e of the ten
Nylon 6/6 as. a synonym for Polyamide 6/6 may be used freely in
values is less than the individual value in Table 0666.-2 for
many countries, including che United Kingdom, buc exclusive
the gauge number concemed.
proprietary righcs in chis name are claimed in certain other
countries.
Table 0666.-2. - Minimum strengths of needle attachment ~Ew _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ ___
Gauge number Mean value Individual value
(newtons) (newtons) DEFINITION
0.5 0.80 0.40 Sterile non-absorbable sutures are sutures which, when
introduced into a living organism, are not metabolised by
0.7 1.7 0.80
that organismo Sterile non-absorbable sutures vary in origin,
2.3 1.1 which may be animal, vegetable, metallic or synthetic. They
1.5 4.5 2.3 occur as cylindrical monofilaments or as multifilament
sutures consisting of elementary fibres which are assembled
2 6.8 3.4
by twisting, cabling or braiding; they may be sheathed; they
2.5 9.0 4.5 may be treated to render them non-capillary, and they may
3 B.O 4.5 be coloured.
3.5 15.0 4.5 Appropriate hannonised standards may be considered when
assessing compliance with respect to origin and processing of
4 18.0 6.0
raw material s and with respect to biocompatibility.
5 18.0 7.0 Sterile non-absorbable surgical sutures serve to approximate
tissue during the healing period and provide continuing
wound support.
STORAGE (PACKAGING) Commonly used materials include the following:
Sterile synthetic absorbable monofilament sutures are
presented in a suitable sachet that maintains sterility and
Silk
Sterile braided silk suture is obtained by braiding a number
allows the withdrawal and use of the sutures in aseptic
of threads, according to the diameter required, of degummed
conditions. The sutures must be stored dry.
sil k obtained from the cocoons of the silkworm Bombyx mori
They are intended to be used only on the occasion when the L.
sachet is first opened.
Linen
Sutures in their individual sachets (primary packaging) are
Sterile linen thread consists of the pericyclic fibres of the
kept in a protective cover (box) which maintains the physical
stem of Linum usitatissimum L. The elementary fibres, 2.5 cm
and mechanical properties until the time of use.
to 5 cm long, are assembled in bundles 30 cm to 80 cm long
The application of appropriate hartnonised standards for and spun into continuous lengths of suitable diameter.
packaging of medical devices may be considered in addition.
Poly(ethylene terephthalate)
LABELLING Sterile poly(ethylene terephthalate) suture is obtained by
Reference may be made to appropriate harmonised standards drawing poly(ethylene terephthalate) through a suitable die.
for the labelling of medical devices. The suture is prepared by braiding very fine filaments in
The details strictly necessary for the user to identifY the suitable numbers, depending on the gauge required.
product properly are indicated on or in each sachet (primary Polyamide-6
packaging) and on the protective cover (box) and include at Sterile polyamide-6 suture is obtained by drawing through a
least: suitable die a synthetic plastic material fortned by the
-- gauge number, polymerisation of B-caprolactam. It consists of smooth,
-- length in centimetres or metres, cylindrical monofilaments or braided filamems, or lightly
-- if appropriate, that the needle is detachable, twisted sutures sheathed with the same material.
-- name of the product,
Polyamide-6/6
-- intended use (surgical absorbable suture),
Sterile polyamide-6/6 suture is obtained by drawing through
-- if appropriate, that the suture is coloured,
a suitable die a synthetic plastic material formed by the
-- the structure (monofilament).
polycondensation of hexamethylenediamine and adipic acid.
___ _ _ __ __ __ _ __ __ __ _ _ ____ ~Ew

It consists of smooth, cylindrical monofilaments or braided

filaments, or lightly twisted sutures sheathed with the same
material.
Polypropylene
Polypropylene suture is obtained by drawing polypropylene
through a suitable die. It consists of smooth cylindrical
mono-filaments.
Monofilament and multifilament stainless steel
Sterile stainless steel sutures have a chemical composition as
specified in ISO 5832-1 - Metallic Materials for surgical
2014
implants - Part 1: Specification for wrought stainless steel
and comply with ISO 10334 - Implants for surgery -
Malleable wires for use as sutures and other surgical
applications.
Stainless steel sutures consist of smooth, cylindrical
monofilaments or twisted filaments or braided filaments.
Poly(vinylidene difluoride) (PVDF)
Sterile PVDF suture is obtained by drawing through a
suitable die a synthetic plastic material which is formed by
polymerisation of 1, 1-difluorethylene. It consists of smooth
cylindrical monofilaments.
IDENTIFICATION
Non-absorbable sutures may be identified by chemical tests .
Materials from natural origin may also be identified by
microscopic examination of the morphology of these fibres.
For synthetic materials, identification by infrared
spectrophotometry (2.2.24) or by differential scanning
calorimetry may be applied.
Identification of silk
A. Dissect the end of a suture, using a needle or fine
tweezers, to iso late a few individual fibres. The fibres are
sometimes marked with very fine longitudinal striations
parallel to the axis of the suture. Examined under a
microscope, a cross-section is more or les s triangular to semi-
circular, with rounded edges and without a lumen.
B . Impregnate isolated fibres with iodinated potassium iodide
solution R. The fibres are coloured pale yellow.
Identification of linen
A. Dissect the end of a suture, using a needle or fine
tweezers, to iso late a few individual fibres. Examined under a
microscope, the fibres are seen to be 12 ¡lm to 31 ¡lm wide
and, along the greater part of their length, have thick walls,
sometimes marked with fine longitudinal striations, and a
narrow lumen. The fibres gradually narrow to a long, fine
point. Sometimes there are unilateral swellings with
transverse lines.
B. Impregnate isolated fibres with iodinated zinc chloride
solution R. The fibres are coloured violet-blue.
Identification of poly( ethyleneterephthalate)
It is practically insoluble in most of the usual organic
solvents, but is attacked by strong alkaline solutions. It is
incompatible with phenols.
A. 50 mg dissolves with difficulty when heated in 50 mL of
dimethylfonnamide R.
B. To about 50 mg add 10 mL of hydrochlonc acid Rl.
The material remains intact even after immersion for 6 h.
Identification of polyamide-6
It is practically insoluble in the usual organic solvents; it is
not attacked by dilute alkaline solutions (for example a
100 giL solution of sodium hydroxide R) but is attacked by
dilute mineral acids (for example a 20 giL solution of sulfunc
acid R), by hot glacial acetic acid R and by a 70 per cent m/m
solution of anhydrous fonnic acid R.
A. Heat about 50 mg with 0.5 mL of hydrochlonc acid RJ in a
sealed glass tube at 110 DC for 18 h and allow to stand for
6 h. No crystals appear.
B. 50 mg dissolves in 20 mL of a 70 per cent m/m solution
of anhydrous fonnic acid R.
Identification of polyamide-6/6
It is practically insoluble in the usual organic solvents; it is
not attacked by dilute alkaline solutions (for example a
100 gIL solution of sodium hydroxide R) but is attacked by
dilute mineral acids (for example a 20 gIL solution of sulfunc
Surgical Materials IV-727
acid R), by hot glacial acetic acid R and by an 80 per cent m/m
solution of anhydrous fonnic acid R.
A. In contact with a flame it melts and bums, forming a hard
globule of residue and gives off a characteristic odour
resembling that of celery.
B. Place about 50 mg in an ignition tube held vertically and
heat gently until thick fumes are evolved. When the fumes fill
the tube, withdraw it from the flame and insert a strip of
nitrobenzaldehyde paper R. A violet-brown colour slowly
appears on the paper and fades slowly in airó it disappears
almost immediately on washing with dilute sulfuric acid R.
e. To about 50 mg add 10 mL of hydrochlon'de acid RJ.
The material disintegrates in the cold and dissolves within a
few minutes.
D . 50 mg does not dissolve in 20 mL of a 70 per cent m/m
solution of anhydrous fonnic acid R but dissolves in 20 mL of
an 80 per cent m/m solution of anhydrous formic acid R.
Identification of polypropylene
Polypropylene is soluble in decahydronaphthalene,
l-chloronaphthalene and trichloroethylene . It is not soluble
in alcohol, in ether and in cyclohexanone.
A. It softens at temperatures between 160 oC and 170 D e.
It burns with a blue flame giving off an odour of burning
paraffin wax and of octyl alcohol.
B. To 0.25 g add 10 mL of toluene R and boil under a reflux
condenser for about 15 minoPlace a few drops of the
solution on a disc of sodium chlonde R slide and evaporate the
solvent in an oven at 80 0e. Examine by infrared absorption
spectrophotometry (2.2.24), comparing with the spectrum
obtained with polypropylene CRS.
e. To 2 g add 100 mL of water R and boil under a reflux
condenser for 2 h. Allow to cool. The relative density (2.2.5)
of the material is 0.89 g/mL to 0.91 g/mL, determined using
a hydrostatic balance.
Identification of stainless steeI
Stainless steel sutures are identified by confirming that the
composition is in accordance with ISO 5832 Part 1.
Identification of poly(vinylidene difluoride)
It is soluble in warm dimethylformamide. It is insoluble in
ethanol, hot and cold isopropyl alcohol, ethyl aceta te,
tetrachlorethylene.
A. The strand melts between 170 oC and 180 oC. It melts in
a flame and does not bum after removal of the flameo Place a
small piece of suture on an annealed copper wire or sheet.
Heat in an oxidising flameo No green colour is produced.
B. Dissolve 0.25 g of the suture in 10 mL of
dimethylfonnamide R and boil under a reflux condenser for
about 15 mino Place a few drops of the solution on a sodium
ehlonde R slide and evaporate the solvent in an oven at 80 oC
(1 h). Examine by infrared absorption spectrophotometry
(2.2.24) . The spectrum shows absorption maxima at the
following wave-numbers: 838.3 ± 0.5 cm- l , 873.3 ±
1 cm- l , 1070.0 ± 2 cm- l, 1165.0 ± 10 cm- l, 1275 ±
0.5 cm-l, 1399 ± 5 cm- l.
C. To 2 g of suture add 100 mL of water R and boil under a
reflux condenser for 2 h . Allow to cool. The relative density
(2.2.5) ofthe material is 1.71 to 1.78.
PRODUCTION
The appropriate harmonised standards may apply with
respect to appropriate validated methods of sterilisation,
environmental control during manufacturing, labelling and
packaging.
IV-728 Surgical Materials 2014

It is essential for the effectiveness and the perfonnance Diameter

characteristics during use and during the functional Iifetime
of these sutures that the foIlowing physical properties are
specified: consistent diameter, sufficient initial strength and
finn needJe attachment.
The requirements below have been established, taking into
account stresses which occur during nonnal conditions of
use. These requirements can be used to demonstrate that
individual production batches of these sutures are suitable for
wound cJosure in accordance with usual surgical techniques.
TESTS
Remove the sutures ¡rom the sachet and measure prompt/y and in
succession the length, diameter and minimum load.
If linen is tested the sutures are conditioned as foIlows: if
stored in the dry sta te, expose to an atrnosphere with a
relative humidity of 65 ± 5 per cent at 20 ± 2 oC for 4 h
immediately before measuring the diameter and for the
detennination of minimum breaking load immerse in water R
at room temperature for 30 min immediately before carrying
out the test.
Length
Measure the length without applying more tension than is
necessary to keep them straight. The length of the suture is
not less than 95 per cent of the length stated on the label
and do es not exceed 400 cm.
Unless otherwise prescribed, measure the diameter by the
foIlowing method using 5 sutures. Use a suitable mechanical
instrument capable of measuring with an accuracy of at least
0.002 mm and having a circular pressor foot 10-15 mm in
diameter. The pressor foot and the moving parts attached to
it are weighted so as to apply a total load of 100 ± 10 g to
the suture being tested. When making the measurements,
lower the pressor foot slowly to avoid crushing the suture.
Measure the diameter at intervals of 30 cm over the whole
length of the suture. For a suture less than 90 cm in length,
measure at 3 points approximately evenly spaced along the
suture. During the measurement submit monofilament
sutures to a tension not greater than that required to keep
them straight. Submit multifilament sutures to a tension not
greater than one-fifth of the minimum breaking load shown
in column e of Table 0324.-1 appropriate to the gauge
number and type of material concemed or ION whichever is
less. Stainless steel sutures do not require tension to be
applied during the measurement of diameter.
For multifilament sutures of gauge number aboye 1.5 make 2
measurements at each point, the second measurement being
made after rotating the suture through 90°. The diameter of
that point is the average of the 2 measurements. The average
of the measurements carried out on the sutures being tested
and not less than two-thirds of the measurements taken on
each suture are within the limits given in the column under
A in Table 0324.-1 for the gauge number concemed. None

Table 0324.-1. - Diamelers and minimum breaking loads

Diameter Minimum breaking load
(millimetres) (newtons)
AlI other non-absorbable
A B Linen thread Stainless steel
strands
Gauge number
min. max. mino max. e D e D e D
0.05 0.005 0.009 0.003 0.012 0.01
0.1 0.010 0.019 0.005 0.025 0.03
0.15 0.015 0.019 0.012 0.025 0.06 0.01
0.2 0.020 0.029 0.015 0.035 0.1
0.3 0.030 0.039 0.025 0.045 0.35 0.06
0.4 0.040 0.049 0.035 0.060 0.60 0.15 1.1
0.5 0.050 0.069 0.045 0.085 1.0 0.35 1.6
0.7 0.070 0.099 0.060 0.125 1.0 0.3 1.5 0.60 2.7
0.100 0.149 0.085 0.175 2.5 0.6 3.0 1.0 5.3 4.0
1.5 0.150 0.199 0.125 0.225 5.0 1.0 5.0 1.5 8.0 6.0
2 0.200 0.249 0.175 0.275 8.0 2.5 9.0 3.0 13.3 10.0
2.5 0.250 0.299 0.225 0.325 9.0 5.0 13.0 5.0 15.5 11.6
3 0.300 0.349 0.275 0.375 11.0 8.0 15.0 9.0 17.7 13.3
3.5 0.350 0.399 0.325 0.450 15.0 9.0 22.0 13.0 33.4 25.0
4 0.400 0.499 0.375 0.550 18,0 11.0 27.0 15.0 46.7 35.0
5 0.500 0.599 0.450 0.650 26.0 15.0 35.0 22.0 57.9 43.4
6 0.600 0.699 0,.550 0.750 37.0 18.0 50.0 27.0 89.4 67.0
7 0.700 0.799 0.650 0.850 50.0 26.0 62.0 35.0 m.8 83.9
8 0.800 0.899 0.750 0.950 65.0 37.0 73.0 50.0 133.4 100.1
9 0.900 0.999 0.850 1.050 156.0 117.0
10 1.000 1.099 0.950 1.150 178.5 133.9
 2014 Surgical Materials IV-729

of the measurements are outside the limits given in the the individual value in Table 0324.-2 for the gauge number
columns under B in Table 0324.-1 for the gauge number concemed.
concemed.
Minimum breaking load Table 0324.-2. - Minimum strengths of needle atlachment
Unless otherwise prescribed, detelmine the minimum Gauge number Mean value Individual value
breaking load by the fol!owing method using sutures in the (newtons) (newtons)
condition in which they are presented. The minimum 0.4 0.50 0.25
breaking load is detelmined over a simple knot formed by 0.5 0.80 0.40
placing one end of a suture held in the right hand over the
0.7 1.7 0.80
other end held in the left hand, passing one end over the
suture and through the loop so formed (see Figure 0324.-1) 2.3 1.1
and pu11ing the knot tight. For stainless steel sutures gauges 1.5 4.5 2.3
3.5 and aboye, the mínimum breaking load is determined on
2 6.8 3.4
a straight pul!. Carry out the test on 5 sutures. Submit
sutures of length greater than 75 cm to 2 measurements and 2.5 9.0 4.5
shorter sutures ro 1 measurement. D etermine the breaking 3 11.0 4.5
load using a suitable tensilometer. The apparatus has 2
3.5 15.0 4.5
clamps for holding the suture, 1 of which is mobile and is
driven at a constant rate of 30 cm/min. The clamps are 4 18.0 6.0
designed so that the suture being tested can be artached 5 18.0 7.0
without any possibility of slippíng. At the beginning of the
6 25.0 12.5
test the length of suture between the clamps is 12.5 cm ro
20 cm and the knot is midway between the clamps. Set the 7 25.0 12.5
mobile clamp in motion and note the force required ro break 8 50.0 25
the suture. If the suture breaks in a clamp or within 1 cm of
9 50.0 25
it, the result is discarded and the test repeated on another
suture. The average of al! the results, excluding those 10 75.0 37.5
legitimately discarded, is equal to or greater than the value
given in column C in Table 0324.-1 and no value is less than
that given in column D for the gauge number and type of Extractable colour
material concerned. Sutures that are dyed and intended to remain so during use
comply with the test for extractable colour. Place 0.25 g of
the suture ro be examined in a conical flask, add 25.0 mL of
water R and cover the mouth of the flask with a short-
stemmed funnel. Boil for 15 min, cool and adjust ro the
original volume with water R. Depending on the colour of the
suture, prepare the appropriate reference solution as
described in Table 0324. -3 using the primary colour
solutions (2.2.2).
The test solution is not more intensely coloured than the
appropriate reference solution.

Table 0324.-3 . - Colour reference solutions

Colour oí Composition of reference solution
strand (parts by volume)
Red Yellow B1ue WalerR
primary primary primary
Figure 0324.-1. - Simple knot solution solution solution
Yellow-brown 0.2 1.2 8.6

Pink-red 1.0 9.0

Needle attachment
If the sutures are supplied with an eyeless needle attached
that is not stated ro be detachable, they comply with the test
for needle attachment. Carry out the test on 5 sutures. Use a
suitable tensilometer, such as that described for the
determination of the minimum breaking load. Fix the needle
and suture (without knot) in the clamps of the apparatus in
su eh a way that the swaged part of the needle is completely
free of the clamp and in líne with the direction of pu11 on the
suture. Set the mobile clamp in motion and note the force
required to break the suture or to detach it from the needle.
The average of the 5 determinations and a11 individual values
are not less than the respective values given in Table 0324.-2
for the gauge number concemed. If not more than 1
individual value fails ro meet the individual requirement,
repeat the test on an additional 10 sutures. The attachment
complies with the test if non e of these 10 values is less than
Creen-blue 2.0 8.0
Violet 1.6 8.4
Monomer and oligomers
Polyamide-6 suture additiona11y complies with the fo11owing
test for monomer and oligomers. In a continuous-extraction
apparatus, treat 1.00 g with 30 mL of methanol R at arate of
at least 3 extractions per hour for 7 h. Evaporate the extract
to dryness, dry the residue at 110 cC for 10 min, a110w ro
cool in a desiccaror and weigh. The residue weighs not more
than 20 mg (2 per cent).
STORAGE (PACKAGING)
Sterile non-absorbable sutures are presented in a suitable
sachet that maintains sterility and a110ws the withdrawal and
use of a suture in aseptic conditions. They may be srored dry
IV-730 Surgical Materials 2014

or in a preserving liquid ro which an antimicrobial agent but

no antibiotic may be added.
Sterile non-absorbable sutures are imended ro be used only
on the occasion when the sachet is first opened.
Sutures in their individual sachets (primary packaging) are
kept in a protective cover (box) which maintains the physical
and mechanical properties until the time of use.
The application of appropriate harmonised standards for
packaging of medical devices shall be considered in addition.
LABELLING
Reference may be made to the appropriate harmonised
standards for the labelling of medical devices.
The details strictly necessary for the user ro identify the
product properly are indicated on or in each sachet (primary
packaging) and on the protective cover (box) and include at
least:
-- gauge number,
-- length, in centimetres or metres,
-- if appropriate, that the needle is detachable,
-- name of the product,
-- imended use (surgical suture, non-absorbable),
-- if appropriate, that the suture is coloured,
-- if appropriate, the structure (braided, monofilamem,
sheathed).
_ _ _ _ _ _ __ _ _ __ _ _ _ __ __ _ ___ ~E~