PHARMEUROPA 18.1 January 2006

PHARMEUROPA 18.
1
CONTENTS
January 2006
New: Pharmeuropa, Pharmeuropa Bio and

Pharmeuropa Scientific Notes Online
25
Publication of Supplements 5.5 and 5.6
General Information
Electronic version of the 5th Edition of the

European Pharmacopoeia
CRS: conditions of sale
List of codes of groups of experts
List of texts published in Supplement 5.5
Comments concerning some revised / corrected texts
published in Supplement 5.5
Elaboration / Revision of a monograph (Procedure 1)
Technical Guide: 4th Edition - 2005 (new)
Pharmeuropa Scientific Notes (new)
Pharmeuropa Bio
Proceedings of conferences of the EDQM
List of Standard Terms: 5th Edition
Proficiency testing studies (PTS): 2006
Knowledge database
Press releases
Certification of suitability of monographs of the European
Pharmacopoeia (Istanbul, Turkey, 27-28 October 2005)
Pharmacopoeial Discussion Group (PDG)
(Chicago, USA, 7-11 November 2005)
International Conferences
Training Sessions on the 5 Edition of the European
Pharmacopoeia: Chemicals
2-3 March 2006, London, UK
27-28 April 2006, Chicago, USA
5
6
11
12
14
18
19
17
21
22
23
24
11
13
25
26
th
Certification of Suitability of the

Monographs of the Ph. Eur.
List of certificates
Scientific Notes
27
32
37
37
41
Effect of Temperature on Plasma Freezing under Industrial

Conditions
41
Draft Monographs and General Texts

for Comment
Allopurinol
Bisoprolol hemifumarate
Cefamandole nafate
Chlortalidone
Cimetidine
Cimetidine hydrochloride
Cisplatin
Clonidine hydrochloride
Clotrimazole
Colony-forming cell assay for human haematopoietic
progenitor cells (2.7.28)
Dacarbazine
Devils claw dry extract
Diethylcarbamazine citrate
PHARMEUROPA Vol. 18, No. 1, January 2006
47
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49
53
55
57
60
63
65
67
69
71
73
75
Dimenhydrinate
Dipyridamole
Dopamine hydrochloride
Dorzolamide hydrochloride
Ethambutol hydrochloride
Fenoterol hydrobromide
Fexofenadine hydrochloride
Fluorescein
Fluorodopa (18F) injection
Fluorouracil
Glucagon, human
Glycerol monocaprylate
Glycerol monocaprylocaprate
Human anti-D immunoglobulin
for intravenous administration
Human plasma for fractionation
Human prothrombin complex
Influenza vaccine (surface antigen,
inactivated, virosome)
Isotretinoin
Liquorice dry extract, quantified
Magnesium citrate, anhydrous
Meclozine hydrochloride
Molsidomine
Moxidectin for veterinary use
Norgestimate
Nucleated cell count and viability (2.7.29)
Paraffin, white soft
Paraffin, yellow soft
Pentaerythrityl tetranitrate, diluted
Perindopril tert-butylamine
Potassium clavulanate
Potassium clavulanate, diluted
Rectal preparations
Ropivacaine hydrochloride monohydrate
Sertraline hydrochloride
Sodium acetate trihydrate
Sodium fluoride
Vaginal preparations
Vinpocetine
Warfarin sodium
Warfarin sodium clathrate
Wettability of porous solids including powders (2.9.45)
Willow bark dry extract
Illustrations of powdered drugs in herbal monographs:
Calendula flower
Ribwort plantain
International Harmonisation
Carmellose
White petrolatum jelly
Yellow petrolatum jelly
PDG state of work (November 2005)
Projected timetable for publication and implementation
of texts signed off by the PDG (November 2005)
Contents of the JP Forum (Vol. 14, No. 3)
Contents of the USP Forum (Vol. 31, No. 6)
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THE EUROPEAN PHARMACOPOEIA

5th Edition: initial volume 5.0 (2 volumes) + 8 Supplements (5.1 - 5.8)
Supplements 5.6, 5.7, 5.8 published in 2006
The 5th Edition 5.0 (2 volumes) has been available since June 2004 (for prices and ordering
information please consult http://book.pheur.org). It is comprised of texts that were implemented
on 1st January 2005 and a cumulative list of reagents.
Publication of Supplements
The supplements are not cumulative and are to be kept for the duration of the 5th Edition.
Modifications (revisions/corrections) to texts are indicated by a line in the margin.
Supplement 5.1 has been available since September 2004; it is comprised of texts that were
implemented on 1st April 2005.
Supplement 5.2 has been available since December 2004; it is comprised of texts that were
implemented on 1st July 2005.
Supplement 5.3 has been available since June 2005; it is comprised of texts implemented on
1st January 2006.
Supplement 5.4 has been available since September 2005; it is comprised of texts that will
be implemented on 1st April 2006 and a cumulative list of reagents.
Supplement 5.5 has been available since December 2005; it is comprised of texts that will be
implemented on 1st July 2006.
Supplement 5.6 will be available in June 2006; it is comprised of texts that will be
implemented on 1st January 2007.
_____________________________________________________________________________
Online access is available to the database giving names of reagents,
especially chromatographic columns. The address is:
http://www.pheur.org/knowledge.htm
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General Information
General Information
5th EDITION OF THE EUROPEAN PHARMACOPOEIA
ELECTRONIC VERSION
1920 New and Revised Monographs and 293 General Texts
With the electronic version of the 5th Edition of the European Pharmacopoeia you can view 1920 monographs,
293 general texts (including general monographs and methods of analysis), 2297 reagents, and also have a direct
internet link to the most recent catalogue of reference substances, which contains 1730 references.
The electronic format has the following convenient features:
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substances used in the monograph;
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________________________________________________________________________________
FREQUENTLY ASKED QUESTIONS:

WHAT IS THE ROLE OF
THE EUROPEAN PHARMACOPOEIA?
The European Pharmacopoeia is the original source of harmonised quality standards for medicines; all of its
published texts have undergone European harmonisation. These texts are mandatory in 34 European countries* and
in the European Union and replace any pre-existing national texts on the same subject.
As laid down in their national legislation, certain member states** may continue to issue a national pharmacopoeia
that republishes all or some of the harmonised European texts (most often General Chapters), translated if necessary
into the national language. In all cases it is the European text that is implemented and made legally binding in all of
the member states.
*Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Portugal, Romania, Serbia and Montenegro, Slovak
Republic, Slovenia, Spain, Sweden, Switzerland, The Former Yugoslav Republic of Macedonia, Turkey, United Kingdom.
**Austria, Bulgaria, Czech Republic, Germany, Greece, Hungary, Portugal, Romania, Spain, Switzerland, United Kingdom.
General Information
REFERENCE SUBSTANCES, PREPARATIONS AND

SPECTRA OF THE EUROPEAN PHARMACOPOEIA
The reference substances and preparations are selected
and verified batches suitable for use as prescribed in the
European Pharmacopoeia. The European Pharmacopoeia
Commission does not guarantee their use for purposes
other than those prescribed. Each vial supplied contains a
quantity sufficient for the prescribed use.
It is recommended that the products be used as soon as
possible.
The stability of the contents of opened vials or ampoules
cannot be guaranteed.
It should be noted that no certificates of analysis,
expiry dates, nor any data not relevant to the use of
the products as defined by the Ph. Eur. monograph
are provided with the reference material. The products
comply with the requirements of the monograph and are
monitored regularly.
For complete information, the catalogue can be
downloaded from our website.
CONDITIONS OF SALE
1730 reference substances, reference preparations
and reference spectra are supplied by the Technical
Secretariat of the European Pharmacopoeia Commission.
Prices
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Prices are identied for each product in the catalogue.

However, please note that prices and package sizes are
subject to change without notice.
The European Directorate for the Quality of Medicines
(EDQM) does not operate a discount policy. The sale
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In the European Union (EU), there is no VAT identication number for organisations with diplomatic status.
The Council of Europe (EDQM) therefore has no VAT
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The goods remain the property of the Council of Europe
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Unless otherwise stated below or specically agreed with

the customer, the goods are shipped to the buyer on
a DDU (Incoterms 2000) basis, namely, delivered duty

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The Council of Europe (EDQM) delivers the goods to
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The Council of Europe (EDQM) bears the cost and
risks of packing, transport to the delivery site and
insurance.
In no event shall the Council of Europe (EDQM) be
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due to their delayed delivery by the carrier.
The buyer is responsible for the cost of import
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if the goods are held up at customs at the time of
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buyer is responsible for any risks associated with use
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Where the shipping costs are paid by the customer,
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In no event shall the Council of Europe (EDQM) be
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The extra charges are applied per shipment. A shipment
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General Information
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Other European countries: 180 EUR per item
France: no extra charge, price is inclusive of

Outside Europe: 250 EUR per item
packaging and postage. At the clients request, Express
f) Dangerous goods in excepted quantities: sent by
Courier delivery is charged at 18 EUR per shipment
carrier chosen by EDQM: Isosorbide dinitrate CRS,
EU: 18 EUR per shipment
Glyceryl trinitrate CRS, Oxaliplatin and its impurities
CRS, Cisplatin CRS, Dichlorodiaminocyclohexane
Other European countries : 80 EUR per shipment
platinum CRS
Outside Europe: 120 EUR per shipment (note: for
(Note: for countries inside the EU (with the exception
India, South America and Africa, our shipment is by
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airport consignment only)
door basis. For all other countries (and the exceptions
Shipping costs paid by the customer: 10 EUR per
above), our shipment is by airport consignment only)
shipment
EU: 100 EUR per item
b) Shipment under ice: sent in cooled freight containers
Other European countries: 125 EUR per item
and by express courier
(Note: for all countries inside the EU (with the
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countries (and the exceptions above), our shipment is
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Outside Europe: 125 EUR per item

g) Dangerous goods sent by road: carrier chosen
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Pentaerythrityl tetranitrate diluted CRS
EU: 150 EUR per item (door to door)
Other European countries: 70 EUR per shipment
Other European countries: 180 EUR per item (door

to door)
Outside Europe: 120 EUR per shipment

shipment
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containers (dry ice) and by express courier
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shipment
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+ dangerous goods from 5 to 100 vials (from 1 to
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BRP, Diphtheria toxin BRP, Bleomycin sulphate CRS,
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j) Narcotics (controlled drugs: sent by carrier chosen by
EDQM)

France: 50 EUR per shipment
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General Information
Outside EU: 160 EUR per shipment

k) Reference spectra
contact name, telephone number, fax number and

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conrmation and shipping notication purposes
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Union)

your order reference/purchase order reference
item order code
shipment.
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The reference substances, reference preparations and
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Please send your order using the CRS order form (see
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General Information
CRS Order Form
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CATALOGUE OF
- CHEMICAL REFERENCE SUBSTANCES - BIOLOGICAL REFERENCE PREPARATIONS - INFRARED REFERENCE SPECTRA - MISCELLANEOUS REAGENTSThe catalogue of reference substances of the European Directorate for the Quality of Medicines is
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adopted by the European Pharmacopoeia Commission.
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and return this form.
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Consult KNOWLEDGE, the new free database at www.pheur.org (under Tools section) which
will tell you if a substance or a method of analysis is part of the work programme of the
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Also available:
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downloadable reference chromatograms
technical information explaining how to carry out the described tests
the list of reference substances used in the monograph
the list of certificates granted.
________________________________________________________________________________
LIST OF CODES OF GROUPS OF EXPERTS

(November 2004)
Groups of experts
1
6
6B
7
10A
10B
10C
10D
Microbiology
Biological substances
Human blood and blood products
Antibiotics
Organic chemistry - synthetic products
Organic chemistry - synthetic products
Organic chemistry - synthetic Products
Organic chemistry - synthetic Products
11
12
13A
13B
13H
14
15
15V
Organic chemistry - natural products

Galenical products
Phytochemistry
Phytochemistry
Fatty oils and derivatives
Radioactive compounds
Sera and vaccines
Veterinary sera and vaccines
Working parties
BOT
BSR
CEL
CRB
CTP
FRC
GEL
GTP
HFA
HOM
ICP
INC
Botulinum toxin
Bovine serum
Cellulose derivatives
Carbohydrates
Cell therapy products
Functionality-related characteristics
Gelatin
Gene therapy products
Propellants
Homeopathy
Inductively coupled plasma spectrometry
Inorganic chemistry
INH
LEC
MAB
MMM
MYC
P4
POW
RGN
ST
STA
VIT
WAT
Inhalations
Lecithins for pharmaceutical purposes
Monoclonal antibodies
Alternative microbiological methods
Mycoplasmas
Procedure 4
Powder characterisation techniques
Reagents
Standard terms
Statistics
Vitamins
Water
11
General Information
LIST OF TEXTS PUBLISHED IN SUPPLEMENT 5.5

A vertical line in the margin indicates where part of a text has been revised or corrected. A horizontal line in the
margin indicates where part of a text has been deleted. It is to be emphasised that these indications, which are not
necessarily exhaustive, are given for information and do not form an official part of the texts. Editorial changes are
not indicated.
Individual copies of texts will not be supplied.
NEW TEXTS
GENERAL CHAPTERS
Monographs
5.1.6. Alternative methods for control of microbiological

quality
E-Acetyldigoxin (2168)
Artichoke leaf (1866)
Chondroitin sulphate sodium (2064)
Clobetasol propionate (2127)
Danaparoid sodium (2090)
Doxazosin mesilate (2125)
Etidronate disodium (1778)
Febantel for veterinary use (2176)
Fumitory (1869)
Iotrolan (1754)
Nandrolone decanoate (1992)
Pine silvestris oil (1842)
Silica, hydrophobic colloidal anhydrous (2208)
Thioctic acid (1648)
Venlafaxine hydrochloride (2119)
MONOGRAPHS
The monographs below appear for the rst time in the
European Pharmacopoeia. They will be implemented on
1 July 2006 at the latest.
Vaccines for human use
Inuenza vaccine (surface antigen, inactivated, prepared
in cell cultures) (2149)
Inuenza vaccine (whole virion, inactivated, prepared in
cell cultures) (2308)
Radiopharmaceutical preparations
Sodium iodide (123I) solution for radiolabelling (2314)
Technetium (99mTc) bicisate injection (2123)
REVISED TEXTS
GENERAL CHAPTERS
Vaccines for human use
2.4.29. Composition of fatty acids in oils rich in omega3-acids

2.6.15. Prekallikrein activator
2.6.21. Nucleic acid amplication techniques
2.6.22. Activated coagulation factors
2.7.4. Assay of human coagulation factor VIII
2.7.11. Assay of human coagulation factor IX
2.7.21. Assay of human von Willebrand factor
2.7.22. Assay of human coagulation factor XI
4.
Reagents (new, revised, corrected)
5.10. Control of impurities in substances for
pharmaceutical use
Pneumococcal polysaccharide conjugate vaccine

(adsorbed) (2150)
MONOGRAPHS
The monographs below have been technically revised
since their last publication. They will be implemented on
1 July 2006.
General monographs
Substances for pharmaceutical use (2034)
Dosage forms
Capsules (0016)
Ear preparations (0652)
Liquid preparations for cutaneous application (0927)
Liquid preparations for oral use (0672)
Rectal preparations (1145)
Semi-solid preparations for cutaneous application (0132)
Tablets (0478)
Vaginal preparations (1164)
12
Monographs
Aluminium hydroxide, hydrated, for adsorption (1664)
Beclometasone dipropionate, anhydrous (0654)
Beclometasone dipropionate monohydrate (1709)
Benzyl alcohol (0256)
Centaury (1301)
Colestyramine (1775)
Digoxin (0079)
Dipivefrine hydrochloride (1719)
Evening primrose oil, rened (2104)
Formoterol fumarate dihydrate (1724)
Gembrozil (1694)
Glutathione (1670)
Human coagulation factor XI (1644)
Ketorolac trometamol (1755)
Lauroyl macrogolglycerides (1231)
Linoleoyl macrogolglycerides (1232)
Liquorice root (0277)
Macrogol 20 glycerol monostearate (2044)
Maltitol, liquid (1236)
Methadone hydrochloride (0408)
Oleoyl macrogolglycerides (1249)
Oxazepam (0778)
Pancuronium bromide (0681)
Star anise (1153)
Sucrose (0204)
Sumatriptan succinate (1573)
General Information
CORRECTED TEXTS
The texts below have been corrected and are republished in their entirety. These corrections are to be taken into
account from the publication date of Supplement 5.5.
GENERAL CHAPTERS
2.3.1. Identication reactions of ions and functional
groups
MONOGRAPHS
Dosage forms
Eye preparations (1163)
Monographs
Arachis oil, rened (0263)
Carboprost trometamol (1712)
Cefradine (0814)
Chloroquine sulphate (0545)
Desogestrel (1717)
Heparin calcium (0332)

Heparin sodium (0333)
Ipratropium bromide (0919)
Levothyroxine sodium (0401)
Norethisterone (0234)
Oxytetracycline dihydrate (0199)
Oxytetracycline hydrochloride (0198)
Propranolol hydrochloride (0568)
Sulbactam sodium (2209)
Sulfaguanidine (1476)
Tetracycline (0211)
Tetracycline hydrochloride (0210)
all-rac-D-Tocopherol (0692)
all-rac-D-Tocopheryl acetate (0439)
Zinc oxide (0252)
SUPPRESSION OF TEXTS
The following text is deleted on 1 April 2006.
MONOGRAPHS
Monographs
Glucagon (0612)
_______________________________________________________________________________________
CERTIFICATION OF SUITABILITY OF MONOGRAPHS OF

THE EUROPEAN PHARMACOPOEIA*
Istanbul, Turkey, 27-28 October 2005
Two days of exchanges and lively discussions conrmed
that the procedure for the certication of monographs of
the European Pharmacopoeia is a major tool of growing
importance for guaranteeing the quality of substances
for pharmaceutical use in the context of constantly
developing world trade. The procedure also plays an
important role in the implementation of the revised
European Directives (2001/83/EEC as amended by
2004/27/EEC and 2001/82/EEC as amended by
2004/28/EEC).
Several ideas for future development have become
apparently necessary in terms of:
communication between the different partners and
the transparency of the procedure;
optimisation of the timeline for the assessment of
dossiers;
reinforcement of the importance of dialogue between
the authorities to guarantee the recognition of the
certicates of suitability and of inspections in the
light of the new legislation on inspections of active
primary ingredients.
The objectives of this conference were particularly
important since they involved reviewing the results

obtained since the last conference (4 years ago), and
sharing points of view and experiences with the principal
users. The proposed programme facilitated dialogue
with users, indeed 12 workshop sessions (covering
the procedure for renewals and revisions, deciencies
in dossiers, sterile products and inspections) and
56 individual consultations (or One-to-One sessions)
were organised, giving each attendee the opportunity
to express and exchange views with European authority
representatives and the assessors who evaluate the
certication dossiers.
Over 180 representatives involved in the quality of
medicines, from 32 countries including Canada, India,
China, South Korea, Israel and the United States,
participated in this international conference organised
by the European Directorate for the Quality of Medicines
(EDQM) of the Council of Europe. The success of the
conference is evidence of the dynamism of international
activities in the domain of the quality of medicines, and
the importance of co-operation between the different
partners implicated (EDQM, European Commission,
EMEA, national licensing authorities, inspection and
industries).
* The procedure for the certication of suitability of monographs of the European Pharmacopoeia
The European directives 2001/83/EEC and 2001/83 EEC amended, on the criteria for the quality, safety and efcacy of medicines on the market,
refer to the specications of the European Pharmacopoeia to dene quality criteria for medicines for human and veterinary use respectively.
Within this legal framework, a supplier of raw materials must provide clients in the pharmaceutical industry with proof that the purity of its
product is suitably controlled by the monographs of the European Pharmacopoeia; this is the role of the certicate of suitability. Since the
beginning of the procedure, more than 1860 certicates, including 502 concerning the evaluation of the reduction of the TSE risk, have been
granted by the EDQM following evaluation of dossiers by assessors designated by the various national licensing authorities.
13
General Information
COMMENTS CONCERNING SOME REVISED/

CORRECTED TEXTS PUBLISHED IN SUPPLEMENT 5.5
Here follows information concerning certain technical modifications to some revised/corrected texts adopted
by the European Pharmacopoeia Commission at the June 2005 session. This information completes the
modifications indicated by lines in the margin in the supplement. Therefore, the information below is not
necessarily exhaustive.
ANALYTICAL METHODS
2.4.29. Composition of fatty acids in oils rich in omega3-acids
In the test for system suitability, as in the case of the
test for oligomers in the omega-3-acid ethyl esters 90
monograph, the rst 3 requirements are considered
sufcient; the 4th requirement is not routinely performed
by the producers who proposed the test, and is deleted.
2.6.15. Prekallikrein activator
This general chapter has been revised based on the
outcome of the international collaborative study BSP049
organised by the EDQM, to mention that a microtitre
plate-based method, which is nowadays the most
frequently used, may also be used instead of methods
using autoanalysers, which were more appropriate when
a large number of samples had to be analysed.
2.6.21. Nucleic acid amplication techniques
This general chapter has been revised to make
it applicable to new applications such as the test
for mycoplasmas (see revised chapter 2.6.7) and
a quantitative test system used to control anti-D
plasma for B19 virus (see monograph Human anti-D
immunoglobulin (0557)).
2.6.22. Activated coagulation factors
This general chapter has been revised together with
general chapters 2.7.11 and 2.7.22 to:
change protamine sulphate R to being an example of
a suitable substance to neutralise the heparin;
replace the reference to cephalin R and platelet
substitute R, which were obsolete, by a phospholipid
preparation to act as a platelet substitute.
2.7.4. Assay of human coagulation factor VIII
The assay of human coagulation factor VIII using a
chromogenic substrate was rst included in the European
Pharmacopoeia in 1993, replacing the two-stage assay,
in line with the recommendation of the Scientic
and Standardisation Committee of the International
Society on Thrombosis and Haemostasis (SSC ISTH).
Commercial kits are used for the assay and the
description of the method is generic to allow the use of
all currently available kits with acceptable performance.
The revision brings no essential major changes to the
method. Work has been carried out recently to dene
critical aspects of the method, particularly with respect
14
to B domain-deleted factor VIII. Problems encountered

with the latter product are best resolved by the use
of a B domain-deleted factor VIII reference standard
for routine assay. The experimental work carried out
recently indicates that it is best for these products to halt
factor Xa generation when the factor Xa concentration
has reached approximately 50 per cent of the maximum
(plateau) level. Furthermore, since it has been shown
that, from a statistical point of view, the potency found
with independent or serial dilutions is not signicantly
different, independent dilutions are no longer required.
2.7.11. Assay of human coagulation factor IX
This general chapter has been revised to:
mention factor IX-decient plasma as a predilution
medium, since this is used routinely;
allow the use of commercial APTT reagents and omit
the reference to cephalin-based reagents, which are now
obsolete.
2.7.21. Assay of human von Willebrand factor
This general chapter has been revised to improve and
supplement the description of ristocetin cofactor activity,
and in particular to introduce general statements on
quantitative assays. Furthermore, since it has been shown
that, from a statistical point of view, the potency found
with independent or serial dilutions is not signicantly
different, independent dilutions are no longer required.
2.7.22. Assay of human coagulation factor XI
The general chapter has been revised to:
mention factor XI-decient plasma as a predilution
medium since this is used routinely;
allow the use of commercial APTT reagents and omit
the reference to cephalin-based reagents, which are now
obsolete.
5.10. Control of impurities in substances for
pharmaceutical use
The section on Interpretation of the test for related
substances in the monographs on active substances has
been modied to replace the examples by a decision
tree, which better illustrates the interpretation of
monographs.
General Information
GENERAL MONOGRAPHS
Substances for pharmaceutical use (2034)
In the section dealing with related substances, the
possibility of exemptions to the general provisions has
been introduced, since it is now seen to be appropriate to
make exceptions in some specic monographs.
The section on residual solvents has been modied to

state explicitly that the content of residual solvents is
taken into account for calculation of specic optical
rotation and specic absorbance.
DOSAGE FORMS
Semi-solid preparations for cutaneous application
(0132)
Capsules (0016)
In order to take account of the new harmonised chapter
on Disintegration, adaptations have been introduced to
the relevant sections.
Ear preparations (0652)
The test for Deliverable mass or volume has been the
cause of some misunderstanding amongst users: it was
not a quality control test, and aimed only to ensure
that the lling was such that the labelled dose could be
withdrawn; furthermore, it has been considered to be
vague and impractical. It has therefore been replaced by
an additional sentence under Production.
Liquid preparations for cutaneous application (0927)
Liquid preparations for oral use (0672)
The test for Deliverable mass or volume has been the
The test for deliverable mass or volume has been the

Tablets (0478)
This monograph was published in Pharmeuropa 15.2
and 16.2 for enquiry related to uniformity of subdivided
tablets and to oral lyophilisates. Due to the comments
received, it was necessary to carry out a new enquiry
as regards oral lyophilisates (Pharmeuropa 17.4). As
regards subdivision of tablets, uniformity of mass is
now tested on 30 parts; no 2nd test is required in case
of failure. This revision is the result of the enquiry
and the study performed by the OMCLs on the basis of
Pharmeuropa 16.2. This text also takes into account
the new harmonised chapters on Disintegration and
Dissolution.
Vaginal preparations (1164)
Rectal preparations (1145)

Also, in order to take account of the new harmonised
chapters on Dissolution and Disintegration, adaptations
have been introduced to the relevant sections.

Also, in order to take account of the new harmonised
chapters on Dissolution and Disintegration, adaptations
have been introduced to the relevant sections.
VACCINES FOR HUMAN USE

Pneumococcal polysaccharide conjugate vaccine
(adsorbed) (2150)
The monograph has been revised to clarify that the test
for sterility that is carried out on intermediates (the
pneumococcal polysaccharides, the carrier protein and
the monovalent bulk conjugate) uses 10 ml for each
medium or the equivalent of 100 doses, whichever is
less. Furthermore, it harmonises this monograph with
the monograph on Meningococcal group C conjugate
vaccine (2112).
15
General Information
MONOGRAPHS
Aluminium hydroxide, hydrated, for adsorption (1664)
Gembrozil (1694)
Based on batch data, the limit for iron has been increased
from 10 ppm to 15 ppm.
Following a study in the EDQM laboratory, method C has

been replaced by method F in the test for heavy metals.
Furthermore, impurity D has been corrected.
Beclometasone dipropionate, anhydrous (0654)

Beclometasone dipropionate monohydrate (1709)
Following the establishment of beclometasone
dipropionate for system suitability CRS and
beclometasone dipropionate for peak identication CRS,
the identication of impurities D and M has been
modied. In addition, relative retentions of the other
detectable impurities have been deleted according to
usual practice.
Benzyl alcohol (0256)
In the test for residue on evaporation, the temperature
of 100 C indicated for evaporation on a water-bath was
too low in view of the boiling point of benzyl alcohol
(205 C), therefore the evaporation method has been
changed.
In the assay, it is now specied that the mixture is heated
on a water-bath.
Digoxin (0079)
This monograph has been revised to replace the TLC
test for related substances by LC and to replace the assay
with an LC test using the same system. For this product
of natural origin having a complex impurity prole, it
has not proved possible to apply the general policy for
impurities shown in the monograph on Substances for
Pharmaceutical Use (2034). It has been necessary to give
an acceptance criterion equivalent to 0.2 per cent for any
other impurities. An acceptance criterion for the sum of
impurities (specied and unspecied) has been dened
and in order to provide a further degree of control, an
acceptance criterion for the subtotal of unspecied
impurities has been added.
Dipivefrine hydrochloride (1719)
The following changes have been made:
an increase in the amount of concentrated ammonia
solution used in the mobile phase to improve the
symmetry of the peaks;
the introduction of a higher upper limit for the
symmetry factor in the assay, i.e. 2.0 for the principal
peak in the chromatogram obtained with reference
solution (c).
Evening primrose oil, rened (2104)
The unsaponiable matter is largely composed of natural
vitamins. It is therefore desirable that the oil is smoothly
rened so that the unsaponiable matter remains rather
high. Based on batch data, the limit for unsaponiable
matter has been increased from 2.0 per cent to 2.5 per
cent.
16
Glutathione (1670)
Following the establishment of the CRS for glutathione,
several batches have been tested and it appeared that the
limits for impurity D and for the total of impurities were
too strict. These 2 limits have therefore been increased.
Human coagulation factor XI (1644)
This monograph has been revised to harmonise the
total protein test with other monographs. Reference to
general chapter 2.5.33 is not appropriate since 7 different
methods are described and it has not been shown that
these 7 methods are equivalent. Since the determination
of nitrogen by sulphuric acid digestion (Kjeldahl method)
is the only method that can be performed without using
a reference preparation, this is the method of choice.
Indeed, this is a big advantage since results obtained
can be compared directly. Other methods can also be
used provided that they have been validated against the
determination of nitrogen by the sulphuric acid digestion
method described in the monograph.
Ketorolac trometamol (1755)
Following a study in the EDQM laboratory, method C has
been replaced by method F in the test for heavy metals.
Lauroyl macrogolglycerides (1231)
Linoleoyl macrogolglycerides (1232)
By analogy with the revision made to the monograph
Stearoyl macrogolglycerides (1268) in 2004, the
conditions used for the test for free glycerol have been
modied.
Macrogol 20 glycerol monostearate (2044)
In the test for hydroxyl value the method has been
changed: method A yields better results, within the limits
of 65 to 85.
Methadone hydrochloride (0408)
The silver nitrate titration in the assay has been replaced
by the acid-base titration, which is more specic as it
determines the active moiety; in addition, the titrant is
low-cost, odourless, and easy to handle. This titration has
been shown to be equivalent to the silver nitrate titration
and to the previous perchloric acid titration.
Oleoyl macrogolglycerides (1249)
By analogy with the revision made to the monograph
stearoyl macrogolglycerides (1268) in 2004, the
conditions used for the test for free glycerol have been
modied.
General Information
Pancuronium bromide (0681)
Sumatriptan succinate (1573)
2 new impurities have been added to the transparency

list. Pancuronium bromide and its impurities cannot
be analysed by LC because of insufcient sensitivity
(little or no chromophores). For this reason, the current
TLC method has been optimised to allow detection of
impurities at 0.1 per cent.
As it is difcult to obtain samples of the individual

impurities to prepare replacement CRS batches, a method
has been developed based on the injection of mixtures of
sumatriptan with impurities obtained by evaporation, to
identify the specied impurities in the test for impurity A
and impurity H and in the test for related substances.
Sucrose (0204)
As the amount of lead in the test solution is in practice
extremely low, the system suitability criterion in the test
for lead has been changed.
________________________________________________________________________________
NEW: PHARMEUROPA SCIENTIFIC NOTES

As from Pharmeuropa 17.4, Scientic Notes are principally published in a new publication called Pharmeuropa
Scientic Notes.
Articles published in Pharmeuropa Scientic Notes are indexed in the PubMed database of the National Library of
Medecine, available on the internet site (www.ncbi.nlm.nih.gov).
The rst edition of Pharmeuropa Scientic Notes (Pharmeuropa SN 2005-1) became available in August 2005.
This issue is included in the subscription to Pharmeuropa, and is not available separately.
SCIENTIFIC NOTES 2005-1

Few Bicyclic Acetals at Reducing End of LowMolecular-Weight Heparins: Might they Restrict
Specication of Pharmacopoeia?
The Control of Impurities in Chlortalidone
Using a Reversed-Phase Stationary Phase
Development of an in vivo Test Procedure for the Ease

of Breaking of Scored Tablets
Chromogenic Assay of Human Coagulation Factor VIII:
Statistical Comparison of 2 Working Dilution
Procedures
Factor VIII Test in Reference Preparations:

Compensation for Different Dilutions
Impurity Profile of Amino Acids?
The Control of Impurities in Amitriptyline

Hydrochloride Using a Reversed-Phase
Hybrid Stationary Phase
Quality Criteria of Homoeopathic Mother Tinctures:

Considerations Regarding Suitable Tests for
Homoeopathic Monographs
A Precise Colour Determination Method for Tablets an Application of Instrumental Colour Measurement
in the Pharmaceutical Development
Instructions for Authors
Batch Variability of Bacitracin: HPLC versus MEKC
Available now (English only)
For prices and ordering information please consult the catalogue on our internet site http://book.pheur.org
17
General Information
ELABORATION / REVISION OF A MONOGRAPH

(Procedure 1)
The European Pharmacopoeia Commission decides to
elaborate/revise a monograph
j
Group of experts:
a rapporteur prepares a draft monograph, which is
evaluated by the experts
j
j
Pharmeuropa (4 issues per year):

the draft monograph is published for public enquiry,
which lasts 3 months
j
The national pharmacopoeia authorities process the
comments received on the draft
The EDQM-Division 1 compiles the comments sent by

the national authorities
The revised draft is

published for further
enquiry, if necessary
j
j
The group of experts examines the comments and

revises the draft monograph accordingly
j
The draft is proposed to the
Commission
j
European Pharmacopoeia Commission
j
- adopts the monograph, if necessary with slight
modications
- adopts the implementation date (about 1 year
after the adoption of the monograph)
j
does not adopt the monograph
j
EUROPEAN PHARMACOPOEIA (3 supplements per year):
the monograph is published about 6 months after adoption
18
General Information
ETHICAL EYE - BIOMEDICAL RESEARCH (2004)

What are the rules and underlying values governing biomedical research in Europe? What form do these values take
and where do they originate? Does biomedical research pose a threat to individuals and their rights? What balance
should be struck between freedom of research and protection of the individual? All these questions are examined in
this book from a pan-European perspective.
The authors look at various international and European standards, including the Helsinki Declaration of the World
Medical Association, EU Directive 2001/20 on pharmaceutical research and the Council of Europes Convention on
Human Rights and Biomedicine. The last named was signed in Oviedo in 1997 and is the rst binding international
treaty on the subject, with a special chapter on scientic research on human beings. The Convention establishes a
common minimum level of protection of fundamental rights throughout Europe. It will soon be supplemented by an
additional protocol specically concerned with biomedical research.
The book contains a glossary and a list of relevant international conventions and treaties, web sites and publications.
It is aimed at both specialists and a wider public interested in this subject.
Contents
BIOMEDICAL RESEARCH IN EUROPE
Preface
Introduction by Claude Huriet (France)
History and denitions by Povl Riis (Denmark)
Germany: current legislation by Jochen Taupitz (Germany)

Central and eastern Europe: research-related problems for
transition countries by Eugenijus Gefenas (Lithuania)
Italy: some shortcomings of biomedical research by Stphane
Bauzon (Italy)
United Kingdom: data protection and condentiality by
Michel Coleman and Vivienne Harpwood (United Kingdom)
ETHICAL DILEMMAS IN RESEARCH

Uses and abuses of biomedical research
by Jan Helge Solbakk (Norway)
Selection and recruitment of participants: European standards
by Herman Nys (Belgium)
Placebo: its action and place in health research
by Andrzej Grski (Poland)
Cancer clinical trials by Maxime Seligmann (France)
Some ethical considerations in industry-sponsored clinical
trials by Tom Gallacher and N. Sreeharan (United Kingdom)
Women in biomedical research by Outi Leena L. Hovatta
(Sweden)
EUROPE AND BIOMEDICAL RESEARCH

European law and biomedical research by Peteris Zilgalvis
(Council of Europe)
APPENDICES
Selected websites
Draft protocol on biomedical research, Council of Europe
The Helsinki Declaration, World Medical Association
ISBN 92-871-5462-7. Price: 15 EUR (Europe) / US$ 23 (outside Europe), + 10 % postage.

This book is available in French and English from:
Council of Europe Publishing - Sales Unit
Ms Sophie Lobey, F- 67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 25 81 - Fax: +33 (0)3 88 41 39 10
Email: publishing@coe.int - Web site http://book.coe.int
________________________________________________________________________________
NEW: TECHNICAL GUIDE FOR THE

ELABORATION OF MONOGRAPHS
4th Edition (2005) available soon on the EDQM internet site
The Technical Guide for the Elaboration of Monographs describes the scientific approach used for the elaboration of
monographs and the establishment of specifications of the European Pharmacopoeia. The guide also describes how
to scientifically elaborate the various sections that must be included in each monograph, for example, definition,
characters, the physical and chemical reactions constituting the identification section, purity tests, assay methods
and storage conditions. It is continually being updated.
Available soon as a free download on the EDQM internet site http://www.pheur.org (bilingual version)
19
General Information
GUIDE TO SAFETY AND QUALITY ASSURANCE

FOR ORGANS, TISSUES AND CELLS
2nd Edition (2004)
The purpose of this guide is to provide guidance for all those involved in transplantation to maximise the quality,
and thereby the success rate, of transplants, and to minimise the risks to all involved in this complex procedure. It
includes safety and quality standards for the procurement, preservation, processing and distribution of organs, tissues
and cells of human origin (allogeneic and autologous) used for transplantation purposes. This guide will be regularly
updated, in line with the latest technical advances.
As the European Union Directive on Tissues and Cells (2004/23/EC) was recently adopted, the European Commission
will build on the Council of Europes guide when establishing technical standards under the directive. This
co-operation will ensure that the same standards are applied throughout Europe.
The 2nd Edition of the Guide can be obtained in English and French from:
Ms Sophie Lobey, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 25 81 - Fax : +33 (0)3 88 41 39 10
E-mail: publishing@coe.int - Web site: http://book.coe.int
Questions and comments on the content should be sent directly to the division in charge:
Council of Europe, Health Division
Mr Karl-Friedrich Bopp, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 22 14 - Fax: +33 (0)3 88 41 27 26
E-mail: karl-friedrich.bopp@coe.int
Council of Europe Portal: www.coe.int
Health and Ethics: www.coe.int/T/E/Social_Cohesion/Health
________________________________________________________________________________
VISIT OUR INTERNET SITE http://www.pheur.org

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access the latest news on official publications

consult the list of adopted monographs and CRS after each session of the Commission
download more than 1500 safety datasheets and about 170 leaflets
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access the work programme database of the European Pharmacopoeia (KNOWLEDGE DATABASE), the
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Please send us your comments and suggestions to help us develop this site!
20
General Information
PHARMEUROPA BIO
These issues are included in the subscription to Pharmeuropa.
BIOLOGICALS 2005-1
Collaborative Study to Establish a New Biological
Reference Preparation for Prekallikrein Activator
Collaborative Study for the Establishment of the Ph.
Eur. BRP Batch 1 for Anti-Vaccinia Immunoglobulin
Feasibility Study to Develop a Common in vitro
D-Antigen Assay for Inactivated Poliomyelitis Vaccines
Allergy Vaccines: a Need for Standardisation in Mass
Units of Major Allergen
Efficacy Demonstration of Tetanus Vaccines by double
antigen ELISA
International Symposium on Alternatives to Whole Cell
Pertussis Vaccine Potency Assay
Collaborative Study for the Validation of Serological

Methods for Potency Testing of Diphtheria Toxoid
Vaccines: Part 1
Collaborative Study for the Validation of Serological
Methods for Potency Testing of Diphtheria Toxoid
Vaccines: Extended study: Correlation of Serology with
In Vivo Toxin Neutralisation
Establishment of European Pharmacopoeia Biological
Reference Preparations Batch 2 for rDNA Hepatitis B
vaccine (Method A and B)
Control of Clostridium Perfringens Vaccines by Means
of an Indirect Competitive ELISA for the Epsilon
Toxin Component Examination of the Assay by a
Collaborative Study
BIOLOGICALS 2004-1
BIOLOGICALS 2003-1
Validation Study to Evaluate the Reproducibility of a

Candidate In Vitro Potency Assay of Newcastle Disease
Vaccines and to Establish the Suitability of a Candidate
Biological Reference Preparation
Establishment of Batch 4 of the Biological Reference
Preparation (BRP) for Rabies Vaccine (Inactivated) for
Veterinary Use
Collaborative Study for the Establishment of
Erythropoietin BRP Batch 2
Somatropin and its Variants: Structural
Characterization and Methods of Analysis
Capillary Electrophoresis for the Control of Impurities
of rDNA Somatropin
Collaborative Study to Establish the Low-MolecularMass Heparin for Assay European Pharmacopoeia
Biological Reference Preparation (BSP060)
Collaborative Study for the Establishment of The

European Pharmacopoeia BRP Batch 1 for
Diphtheria Toxin
Collaborative Study for the Establishment of
European Pharmacopoeia BRP Batch 2 for Inactivated
Poliomyelitis Vaccine for In Vitro D Antigen Assay
Collaborative Study for the Establishment of A Global
(WHO International / US/Ph. Eur.) Standard for the
Potency Assay of Human Anti-D Immunoglobulin
Feasibility Study to Evaluate the Correlation Between
Results of a Candidate In Vitro Assay and Established
In Vivo Assays for Potency Determination of Newcastle
Disease Vaccines
BIOLOGICALS 2003-2
Collaborative Studies for the Establishment of
Reference Substances for the Microbiological Assay of
Antibiotics
Collaborative Study for Establishment of a Global
Standard for the Potency Assay of Human Anti-D
Immunoglobulin
Collaborative Study for Establishment of a European
Pharmacopoeia Biological Reference Preparation (BRP)
For B19 Virus DNA Testing of Plasma Pools by Nucleic
Acid Amplication Technique
BIOLOGICALS 2002-1
Collaborative Study for Establishment of an HPLCMethod for Batch Consistency Control of Recombinant
Interferon-Alfa-2
Calibration of European Pharmacopoeia BRP Batch 3/
Mega 2 (US/FDA) Standard for Human Coagulation
Factor VIII Concentrate for Use in the Potency Assay
Collaborative Study for the Establishment of the
European Pharmacopoeia BRP for Oral Poliomyelitis
Vaccine (OPV) Batch 3 for Use in the Potency Assay
Establishment of the European Pharmacopoeia BRP for
Hepatitis A Vaccine Type B (Aventis Pasteur) Batch 2
For prices and ordering information please consult the catalogue on our internet site http://book.pheur.org
21
General Information
EDQM CONFERENCE PROCEEDINGS

The following free Conference Proceedings are available to download from the EDQM internet site
http://www.pheur.org/site/page_601.php
Certicates of Suitability of Monographs of the
European Pharmacopoeia: Implementation of the 5th
Edition New Procedures for Revision and Renewal of
Certicates
27-28 October 2005, Istanbul, Turkey
Quality on the Move: Dynamics of the European

Pharmacopoeia
4-6 October 2004, Budapest, Hungary
Process Analytical Technologies International Symposium
3-4 May 2004, Cannes, France
OMCL Information Day: Place and Role of the European Microbiological Control Methods in the European
Pharmacopoeia: Present and Future
OMCL Network within the Regulatory Framework in
Europe
5-6 May 2003, Copenhagen, Denmark
27 May 2005, Rome, Italy
Foot and Mouth Disease Vaccines: Current Situation

17-18 March 2003, Strasbourg, France
Alternatives to Whole Cell Pertussis Vaccine Potency

Assay
Standardisation and Quality Control Cell and Gene

Therapy Products
24-25 February 2003, Strasbourg, France
16 March 2005, Geneva, Switzerland
Quality of Homoeopathic Products in the New European

Replacement, Reduction and Renement of the Use of
Legislative Framework
Animals in the Quality Control of Vaccines
15 February 2005, Strasbourg, France
7-8 November 2002, Strasbourg, France
Serological Potency Tests for Diphtheria and Other
Excipients: Classical Requirements and Functionality
Vaccines
Related Testing
4-5 April 2002, Brussels, Belgium
6-7 October 2004, Budapest, Hungary
******
The following Conference Proceedings can be ordered from the EDQM. For prices and ordering please consult the
catalogue on our internet site http://book.pheur.org
Certication of Suitability of Monographs of the
European Pharmacopoeia (CEP) - New Developments
of the Procedure, How to Apply for a CEP
8-9 November 2001, Athens, Greece
The Future Face of the European Pharmacopoeia Current Concerns in Pharmaceutical Analysis
8-9 February 2001, Cannes, France
Herbal Medicinal Products: Quality Evaluation Contribution of the European Pharmacopoeia
16-17 November 2000, Nice, France
Tetanus Vaccine for Human Use
22-23 June 2000, Strasbourg, France
Mycoplasma Testing: The Potentialities and Role
of PCR Tests
13-14 March 2000, Paris, France
Biologicals beyond 2000: Challenge for Quality

Standards in an Evolving Field
27-29 September 1999, Strasbourg, France
General Monographs on Dosage Forms and PharmacoTechnological Test Methods
26-27 October 1998, Seville, Spain
The Vision of the European Pharmacopoeia in the
21st Century - The Dynamics of Quality of Medicines in
Europe
4-7 December 1996, Prague, Czech Republic
Sterility Tests and Efcacy of Antimicrobial
Preservation
5-6 February 1996, Barcelona, Spain
All above proceedings are available in English only and are not included in the subscription to Pharmeuropa.
22
General Information
LIST OF STANDARD TERMS

5th EDITION (printed version available)
(27 European languages)
The present list of Standard Terms is a revised list that was drawn up in response to a request from the European
Commission. It covers medicines for both human and veterinary use. These Standard Terms are to be used in
answering the questions in Module 1 (item 1.2 and 1.3) of the EU application form.
The list of Standard Terms is composed of:
an introduction:
a section of general principles and instructions for the use of Standard Terms,
the summary of the changes (amendments, additions, deletions) performed since the last publication
(December 2002),
the procedure for the addition, deletion or modification of terms in the list of Standard Terms (requests
restricted to licensing authorities);
3 lists of standard terms:
list of pharmaceutical forms,
list of routes and/or methods of administration,
list of containers, closures and administration devices.
The 5th Edition contains translations in 27 European languages: Bulgarian, Croatian, Czech, Danish, Dutch, English,
Finnish, French, German, Greek, Hungarian, Icelandic, Italian, Macedonian, Norwegian, Polish, Portuguese, Slovak,
Slovenian, Spanish, Swedish and Turkish. 5 new languages have been added compared to the printed version
published in December 2002: Lithuanian, Estonian, Latvian, Romanian and Maltese.
The corresponding online version is available only to people who ordered the printed version of the 5th Edition
(December 2004).
Price: see the catalogue on our internet site http://book.pheur.org
________________________________________________________________________________
GUIDE TO THE PREPARATION, USE AND QUALITY

ASSURANCE OF BLOOD COMPONENTS
11th Edition (2005) of the technical appendix to Recommendation No. R (95) 15
This guide contains a compendium of measures designed to ensure the safety, efcacy and quality of blood
components, and is particularly intended for all those working in blood transfusion services. It describes the different
blood components and gives information on their clinical indications and possible side effects.
This guide continues to be the golden standard for blood transfusion services and forms the basis for many national
guidelines in Europe and elsewhere.
During the preparation of this 11th edition, the Council of Europe and the European Commission worked closely
together to ensure that the requirements under Article 29 of the European Union Directive 2002/98/EC and those
of this guide were compatible. In particular, the section in Part A on the quality system for blood transfusion
establishments has been completely overhauled.
Where necessary, chapters have also been revised to take into account what can now be achieved with new advances
in technology.
This reference book will be of interest to blood transfusion centres, legislators, health personnel and all those
working in the eld of blood transfusion.
The European Pharmacopoeia monograph on human plasma for fractionation refers inter alia to the
recommendations made in this Guide.
The 11th Edition of the Guide can be obtained in English and French from:
Ms Sophie Lobey, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 25 81 - Fax: +33 (0)3 88 41 39 10
E-mail: publishing@coe.int - Web site: http://book.coe.int
Questions and comments on the content should be sent directly to the division in charge:
Council of Europe, Health Division
Mr Karl-Friedrich Bopp, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 22 14 - Fax: +33 (0)3 88 41 27 26
E-mail: karl-friedrich.bopp@coe.int
Council of Europe Portal: www.coe.int
Health and Ethics: www.coe.int/T/E/Social_Cohesion/Health
23
General Information
Please complete and return this form to: Josiane Bourrly, Division IV, EDQM before 10 February 2006
by post: EDQM, BP 907, 67029 Strasbourg Cedex 1, France
by fax: +33 (0)3 88 41 27 71;
by E-mail: josiane.bourrely@pheur.org
Year 2006 Physico-Chemical Studies
REGISTRATION FORM: Proficiency Testing Studies (PTS)
REGISTRATION DETAILS
PARTICIPANT DETAILS*(Delivery address)
INVOICING DETAILS (if different from participant/delivery details)
First Name
First Name
Last Name
Last Name
Company/
Institution
Company/
Institution
Name of Unit/Section (to be mentioned in the attestation of the participant)
Address
Address
(No PO
Boxes)
Postcode
Postcode
Town
Town
Country
Country
VAT No
(EU only)
VAT No
(EU only)
Tel
Tel
Fax
Fax
E-mail
E-mail
Purchase Order Reference (to be mentioned on the invoice)
*Please note that all related information, documentation or material (e-mails, protocols, samples, reports, attestations of participation) will be
sent to the above-mentioned registered participant at the above-mentioned address.
PTS ref
Name of Study
Date of
Availability
Participation
Dispatch
Conditions
Delivery
1
Charges
PTS081
Loss on drying (2.2.32)
End Feb 06
YES
NO
Ambient
Temperature
(a)
PTS082
Dissolution test (extended-release tablets, paddle
End April 06
YES
NO
Ambient
Temperature
(a)
apparatus, spectrophometric determination)
PTS083
Potentiometric titration (2.2.20)
End June 06
YES
NO
Ambient
Temperature
(a)
PTS084
Assay and related substances by LC (active
End Sept 06
YES
NO
Ambient
Temperature
(a)
End Nov 06
YES
NO
Ambient
Temperature
(a)
substance for human and veterinary use, isocratic RP-LC,

UV-detection)
PTS085
Microbiological assay of antibiotics (2.7.2)
AREA OF ACTIVITY:
OMCL
Private QC pharmaceutical laboratory
Other (please specify)__________________
FEES
The amount due per study is 230 Euros for laboratories not belonging to the OMCL Network.
1
In addition to these costs, extra charges for delivery will be added for each PTS study dispatched. These charges are set out in our Official
Catalogue of Pharmaceutical Reference Substances and Preparations see Section 2.2: Delivery and Related Costs Prices - Delivery
charges (a).
DELIVERY
Each PTS will be shipped either on a DDU or CIP basis (Incoterms 2000) as set out in our Official Catalogue of Pharmaceutical Reference
Substances and Preparations see Section 2.2: Delivery and Related Costs Prices - Delivery charges (a). A copy of our catalogue is
available on our website www.pheur.org
CANCELLATION AND PAYMENT

About 2 weeks before the PTS study becomes available, we will send you an order confirmation by e-mail and an invoice. The cancellation of
an invoiced PTS is only possible if we have been informed within 3 working days of that order confirmation. In all cases the payment should
be net of charges for the Council of Europe and paid within 30 days from the date of invoice. Details of how to pay are available on our
website and will be outlined on our invoice.
Date
24
Signature
General Information
NEW: PHARMEUROPA ONLINE

Pharmeuropa, Pharmeuropa Bio and Pharmeuropa Scientic Notes Online are now available as a complementary
service for subscribers to the printed edition of Pharmeuropa, and will be offered for a trial period without an
additional fee for those who ordered the printed version of Pharmeuropa Vol. 18 (2006). All issues stretching back to
volume 10 (1998) are stored as Acrobat PDF-les, and can be searched with a search engine identical to the one used
for the online versions of the European Pharmacopoeia and the Standard Terms.
A username and a password are required to access Pharmeuropa Online, and instructions for creating these using the
EDQM Certicate of Authenticity can be found on the inside-front cover of this issue of Pharmeuropa.
________________________________________________________________________________
PHARMACOPOEIAL DISCUSSION GROUP (PDG)

Chicago, USA, 7-10 November 2005
The Pharmacopoeial Discussion Group [European
Pharmacopoeia (EP), Japanese Pharmacopoeia (JP),
United States Pharmacopeia (USP)] met in association
with the Expert Working Groups of the International
Conference on Harmonisation (ICH). The World Health
Organisation attended as an observer.
Calcium disodium edetate, Calcium phosphate dibasic
dihydrate, Calcium phosphate dibasic anhydrous: these
harmonised monographs were signed off.
Benzyl alcohol, Lactose anhydrous, Methylcellulose:
revisions of these harmonised monographs were signed
off.
Microbial contamination: the PDG has worked on
the harmonisation of 3 important general chapters
with relevance for the ICH Q6A guideline: microbial
enumeration methods, tests for specied microorganisms, and acceptance criteria for pharmaceutical
preparations. These harmonised chapters were signed off.
Implementation of harmonised texts: to accelerate
inter-regional implementation of harmonised texts,
the JP has implemented a new procedure for rapid
implementation of harmonised general chapters related
to the Q6A guideline, since full harmonisation is achieved
only when all 3 pharmacopoeias have published and
implemented a monograph/general chapter.
ICH Expert Working Group Q4B (Regulatory Acceptance
of Pharmacopoeial Interchangeability): on November
9, 2005, the PDG held a joint meeting with Q4B to
discuss the regulatory acceptance of harmonised
monographs and general chapters, particularly those
of relevance for the ICH Q6A guideline; the document
detailing the roles and responsibilities of the PDG and
Q4B EWG was discussed and further renement was
necessary; 5 packages for harmonised general chapters
have been submitted by the PDG to the Q4B group,
including dissolution, extractable volume, particulate
matter in parenterals, residue on ignition/sulphated

ash, and sterility test. Examination of the test for
residue on ignition/sulphated ash has been completed
by the Q4B group and tests, analytical procedures,
and acceptance criteria of the 3 pharmacopoeias will
be recognised as interchangeable by the regulatory
authorities in the 3 regions once the harmonised text has
been published and implemented in all 3 regions. The
package for dissolution was submitted to the Q4B group
in August 2005; the PDG is awaiting feedback on this
topic. The USP has revised its text for extractable volume
and the PDG is awaiting feedback from the Q4B group.
Particulate matter was discussed at the November 9
meeting and Q4B provided a preliminary report outlining
a number of issues remaining to be resolved in order
to achieve regulatory interchangeability. A number of
issues remain to be resolved for the sterility test in order
to achieve regulatory interchangeability. Additionally,
packages for the PDG harmonised texts on disintegration
and uniformity of dosage units are in preparation for
submission to Q4B.
Industry Associations: a meeting with industry
associations from the 3 ICH regions was held on
November 8, 2005, to exchange information on progress
with the current work program and future harmonisation
needs. Industry associations were encouraged to play an
active role in the harmonisation process as important
stakeholders.
Excipients Councils: a meeting was held on November 10,
2005, with Tri-PEC (IPEC Americas, IPEC Europe,
Japanese Pharmaceutical Excipients Council) to discuss
the work program on harmonisation of excipient
monographs. Current issues include the policy for
functionality-related characteristics, use of additives and
processing aids in excipients, co-processed excipients,
impurities in excipients, and the future of harmonisation.
The PDG will hold its next meeting on 5-8 June, 2006, in
Yokohama, Japan.
25
AGENDA 2006
INTERNATIONAL CONFERENCES & SYMPOSIUM
***
TRAINING SESSIONS ON THE 5th EDITION
OF THE EUROPEAN PHARMACOPOEIA
CHEMICALS NEW PROGRAMME
London, UK, 2-3 March 2006
Chicago, USA, 27-28 April 2006
***
The EDQM is pleased to announce a new training programme on the European Pharmacopoeia 5th Edition. The
new programme aims to provide professionals with an in-depth and up-to-date knowledge of the most important
and practical aspects of the European Pharmacopoeia. New additions for 2006 include practical examples and case
studies, question and answer sessions giving you the opportunity to clarify any issues that may arise and the chance
to meet one-to-one with the speakers who work in a certain area, thus providing you with more meaningful and
worthwhile interactions.
Do not miss these opportunities to meet the EDQM/European Pharmacopoeia.
More information will be available on the EDQM website www.pheur.org and in the next issues of Pharmeuropa.
GENERAL CONDITIONS FOR REGISTRATION AT THE EDQM CONFERENCES

AND TRAINING SESSIONS
HOW TO REGISTER
Register as soon as possible: places are limited.
We recommend using a separate form for each participant. Please ll in the registration form* and return it to the
EDQM Public Relations Unit, by post, by fax or by e-mail duly completed with the selected method of payment.
NEW Online Registration: Online conference registration is now possible. To use the online registration form, just
click on the icon EDQM Events - Register online and follow the steps described.
REGISTRATION FEE
A special rate is given to permanent staff of national authorities, university, R&D public centres.
The registration fee is not subject to VAT and covers attendance at the lectures, working documents, lunches,
coffee breaks and the ofcial dinner.
The registration fee does not include your hotel accommodation costs (see hotel reservation form) and travel
expenses.
METHOD OF PAYMENT
You can make your payment: by bank transfer, by enclosed cheque or by credit card.
The relevant bank transfer information is given on the invoice.
CANCELLATION POLICY
One month before the event 80% of the registration fee will be refunded; no refunds will be given after this date.
However, registration may be transferred to another person at any time. In case of no show, the registration fee is
due.
* All information requested on the registration form is necessary and used only for the organisation of the seminar.
26
2006 TRAINING SESSION: LONDON

HOW TO USE THE EUROPEAN PHARMACOPOEIA 5TH EDITION
CHEMICAL PRODUCTS
Duration: 1,5 days, Location: Local Government House, Smith Square,
London SW1P 3HZ, UK
2-3 March 2006 Location: Local Government House, London, UK
NEW PROGRAMME: TRAINING SESSION 5TH EDITION
Working language: English
***
NEW PROGRAMME
THURSDAY 2 MARCH 2006
8:45 - 9:15 Registration and Welcome Coffee
Opening remarks and general introduction
9:15 - 9:30 Dr Michael Morris, Chair of the European Pharmacopoeia Commission
SESSION 1: GENERAL INTRODUCTION
The European Regulatory System: Interactive and complementary relationship between EU/European
Commission/European Council/EMEA/EDQM/Council of Europe, place of the EDQM and the European
Pharmacopoeia
9:30 - 9:50 Dr Agns Artiges, Director of the European Directorate for the Quality of Medicines,
EDQM, Council of Europe
9:50 - 10:00 Discussion
SESSION 2: HOW TO FIND YOUR WAY THROUGH THE EUROPEAN PHARMACOPOEIA
- General concepts in the European Pharmacopoeia: theory and rationale
General notice, General monographs and chapters (chromatographic separation techniques, ..)
- Role and status of the European Pharmacopoeia
10:00 - 10:45 Mr Peter Castle, Secretary to the European Pharmacopoeia Commission, EDQM, Council
of Europe
11:00 - 11:30 Coffee Break
- A practical approach: Cases studies of specific monographs (active substances and excipients),
use of reference standards and links with the Certificate of suitability of monographs of the
European Pharmacopoeia (CEP)
11:30 - 12:15 Speaker to be confirmed
SESSION 3: CURRENT PROGRESS IN THE FIELD OF INTERNATIONAL
HARMONISATION
- Regulatory interchangeability between FDA (US) / EU / *MHLW (J)
12:30 - 12:50 Dr Michael Morris
13:00 - 14:00 Lunch break
SESSION 4: HOW TO PARTICIPATE IN THE ELABORATION & REVISION
OF THE EUROPEAN PHARMACOPOEIA
14:00 14:20 Mr Peter Castle
14:20 14:30 Discussion
SESSION 5: THE EUROPEAN PHARMACOPOEIA PUBLICATIONS AND SERVICES
- Publications: The 5th Edition of the European Pharmacopoeia: Implementation and the
publication schedule, Pharmeuropa and list of standard terms
14:30 - 14:50 Dr Hans-Joachim Bigalke, Publications Division, EDQM, Council of Europe
15:00 - 15:30 Coffee break
*Ministry of Health, Labour and Welfare
27
- European Pharmacopoeia Reference Standards

15:30 - 15:50 Speaker to be confirmed
SESSION 6: THE EUROPEAN PHARMACOPOEIA INTERNET SITES, HOW TO ACCESS

THE ONLINE DATABASES AND THE NEW HELPDESK
16:00 - 16:20 Mrs Caroline Larsen Le Tarnec, Public Relations Officer, EDQM, Council of Europe
NEW! SESSION 7: ONE TO ONE CONSULTATIONS
16:30 - 17:30
Topics under which a consultation can be arranged: General questions on the EDQM, the European
regulatory framework and harmonisation; Technical questions on PhEur monographs and texts;
Publications and services; Electronic version of the European Pharmacopoeia
FRIDAY 3 MARCH 2006 (MORNING ONLY)
8:30 - 9:00 Welcome Coffee
During the morning, the electronic/online version of the European Pharmacopoeia will be set up
and demonstrated to participants interested in learning more about this version. The EDQM will
give advice on how to search, retrieve and print documents and illustrate where to find
information such as chromotograms, reference substances and Certificates of suitability.
SESSION 8: GENERAL INTRODUCTION TO THE CERTIFICATION PROCEDURE AND
INSPECTIONS
- General considerations on the Certification procedure and Inspections
Origin, scope, how it works, who is involved
9:00 - 9:15 Dr Agns Artiges
- How to apply for a Certificate
Content of the dossier for chemical substances
Comments on the main deficiencies found in dossiers
9:25 - 11:05 Dr Andrew McMath, Certification Unit, EDQM, Council of Europe
10:10 - 10:40 Coffee break
- Revisions and renewals
11:05 - 11:15 Dr Andrew McMath
11:25 Final addresses and Closure of the meeting
NEW! SESSION 9: ONE TO ONE CONSULTATIONS
11:45 - 12:45
Topics covered: General questions on the EDQM, the European regulatory framework and
harmonisation; Technical questions on PhEur monographs and texts; Certification procedure;
Who should attend?
This training session should be attended by professionals from industry, in particular persons involved in
the manufacture and control of drug substances/products or the preparation of registration dossiers; from
inspectorates, regulatory agencies and academic institutions.
Contact details:
Further information on the conference, accommodation and registration forms will be made available on
the EDQM internet site: http://www.pheur.org. Address: Public Relations Unit, EDQM, 226 avenue de
Colmar BP 907, F-67029, Strasbourg, Cedex 1:
Tel: +33 (0) 3 88 41 30 30 (Dial 4 for Conferences); Fax: + 33 (0) 3 88 41 27 71;
Internet: See under HELPDESK on our website: http://www.pheur.org/site/page_630.php)
FOR MORE INFORMATION PLEASE VISIT THE WEBSITE: http://www.pheur.org
28
Please complete and send this form to: Caroline Larsen Le Tarnec, Public Relations, EDQM Fax: +33 (0)3 88 41
27 71; Internet: Please attach the registration form to our enquiry form (see section Helpdesk on the website:
http://www.pheur.org/site/page_630.php) or register online.
REGISTRATION FORM:
TRAINING SESSION 5th EDITION
DATE OF REGISTRATION (DD/MM/YY) :
_ __ __ _
REGISTRATION FEE (*See general conditions)

600 * or 200 *
Yes, I am interested in reserving a meeting
ONE TO ONE MEETING
Which topic(s)?
EU Regulations Monographs, revisions
No, I am not interested
Publications Certification
Internet
PARTICIPANT DETAILS Please complete one form per participant

Title (Dr., Mr, Mrs, Ms, )
First Name
Family Name
Company/Institution
Address for
Correspondence
Postcode
Town
Country
Telephone
Fax
E-mail
AREA OF ACTIVITY/ Manufacturer of raw material
Manufacturer of medicines
OCCUPATION:
QA
QC
Regulatory authority:
Licensing
Inspection
OMCL
Other (please specify)
for human use for veterinary use

R&D
Regulatory affairs
Pharmacopoeias
University
PAYMENT (NEW)
Following receipt of your registration form, we will send you an invoice. Please note that we must receive payment
before the conference takes place. Details of payment methods will be outlined on the invoice. However, you will
be able to settle your invoice by:
1. PERSONAL OR COMPANY CHEQUE made payable to Council of Europe/EDQM
2. BANK TRANSFER
3. CREDIT CARD
DETAILS FOR INVOICING PURPOSES (if different from participant details)
Company/Institution
Address
Postcode
Town
Country
VAT Number (EU only)
Contact Name
Telephone
Fax
E-mail
PO Number/ Reference
CANCELLATION CHARGES: I have read and accept the cancellation terms as stated on our web-site.
Date
Signature
FOR MORE INFORMATION PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
29
ONE-TO-ONE RESERVATION FORM:
2006 TRAINING SESSION: LONDON

EUROPEAN PHARMACOPOEIA 5TH EDITION CHEMICALS
2-3 MARCH 2006
ONE TO ONE CONSULTATION QUESTION FORM

(Thursday 2 March 2006 from 16:30 17:30
AND Friday 3 March 2006 from 11:30 12:30)
If you would like to register for a one-to-one consultation (15 minutes) with a member of the EDQM
scientific team during the training course, please complete this consultation request form and send it
to the EDQM by fax to +33 3 88 41 27 71 or via the Helpdesk on the website:
http://www.pheur.org/site/page_630.php)
Priority will be given to those who book in advance.
PARTICIPANT DETAILS
Title (Dr., Mr, Mrs, Ms)
First Name
Family Name
Company/Institution
E-mail
The time slots available for one-to-one consultations will be limited. The EDQM shall do all it can to
accommodate all interested participants.
If it is on a specific application, please mention the reference number
Your question:
Date
Signature
FOR MORE INFORMATION ABOUT TRAINING SESSIONS AND CONFERENCES ORGANISED BY THE EDQM
PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
30
HOTEL RESERVATION FORM

2006 TRAINING COURSE LONDON: 2-3 MARCH 2006
Location: Local Government House, London
2-3 MARCH 2006
HOTEL REGISTRATION FORM:
Location: Local Government House, London, UK

NOVOTEL LONDON WATERLOO HOTEL RESERVATION FORM
Fax: +44 (0)207 793 0202
To be filled in and sent by fax before the 15 February 2006
General information:
20 rooms reserved from Wednesday 1st March to Friday 3rd March included.
NOVOTEL LONDON WATERLOO HOTEL: 113 Lambeth Palace Road, London,
SE1 7LS, UK, Contact: Assita Kone Tel +44 (0)207 793 5718
Fax +44 (0)207 793 0202; E-mail : H1785-RM@accor.com
The quotas of rooms reserved will be available until 15 February 2006.
After this date the availability and the negotiated prices will not be guaranteed. The
rooms should be reserved individually by each participant and are available on a
first come, first served basis. Cancellation policy: Any room cancelled 15 days prior
to arrival 50% will be charged and 7 days before arrival 100% will be charged on the
credit card provided.
Participant:
Surname
Forename
Company/ employer
Address
City
Postal Code
Country
E-mail
Tel N
Fax N
Global reservation:
Contact reservations:
Hotel reservation:
NOVOTEL
LONDON
WATERLOO
HOTEL
Arrival day/hour .....

Departure day/hour .....
Please indicate the room and nights required by a circle
SINGLE
OCCUPANCY
145 GBP*
DOUBLE
OCCUPANCY
165 GBP*
NIGHT
01/03/2006
YES
NIGHT
02/03/2006
YES
* Taxes and breakfast inclusive
Method of payment:
Credit card:
Credit card number:
Visa
EuroCard / MasterCard
Amex
Expiry date:
Cardholder: ______________________________________________________
I authorise the NOVOTEL LONDON WATERLOO HOTEL to charge against my credit card the
amount equivalent to one night in order to block my reservation for the duration of the seminar.
Date: _____________________
Signature: ________________________________________
HOTEL CONFIRMATION RESERVATION N:___________
Signature:____________________
31
32
27-28 April 2006 Location: Radisson Hotel, 160E Huron St., Chicago, USA
2006 TRAINING SESSION: UNITED STATES

THE EUROPEAN PHARMACOPOEIA 5TH EDITION
CHEMICAL DRUG PRODUCTS AND SUBSTANCES
Duration: 2 days, Location: Radisson SAS, 160E Huron Street, Chicago, USA
Working language: English
***
NEW PROGRAMME
THURSDAY 27 APRIL 2006
8:00-8:30 Registration and Welcome Coffee
8:30-8:45 Opening remarks and general introduction
8:45-9:05 European regulations for medicines: How does the system work? Relationship between
EU/EMEA and the EDQM of the Council of Europe. Place and roles of the EDQM and the European
Pharmacopoeia
9:05-9:25 EDQM, general organisation and process of elaboration & revision of the European
Pharmacopoeia. How to submit a new monograph/revision proposal.
9:25-10:10 Pharmacopoeias and international harmonisation process
10:10-10:30 Open discussion
10:30-11:00 Coffee Break
11:00-12:00 How to use the European Pharmacopoeia: General Notices, General chapters, general
monographs and monographs on dosage forms. Frequently asked questions.
12:30-13-20 Lunch Break
13:20-14:20 Cases studies of specific monographs (active substances and excipients), use of reference
standards, links with the Certificate of suitability of monographs of the European Pharmacopoeia (CEP)
14:20-15:30 The European Pharmacopoeia Publications (printed and electronic publications) and
Reference Standards
15:30-16:30 The European Pharmacopoeia Internet sites, How to access to the online databases and to
the new users support: the HELPDESK
17:00-18:00 One to One consultations.
Subject to a prior appointment, questions has to be sent prior the session to guarantee an efficient
answer/advice. Topics under which a consultation can be arranged: General questions on the EDQM, the
European regulatory framework and harmonisation; Technical questions on PhEur monographs and texts;
FRIDAY 28 APRIL 2006

8:00-8:30 Welcome Coffee
27-28 April 2006 Location: Radisson Hotel, 160E Huron St., Chicago, USA
8:30-9:30 Presentation of PHARMEUROPA online and PHARMEUROPA Scientific Notes

9:30-10:30 The certification procedure and inspections: General considerations, How to apply for
a Certificate
10:50-11:10 Coffee break
11:10-12:10 Revisions and renewals in the procedure of certification
12:30-13:30 Lunch break
13:30-14:30 General Chapters in the European Pharmacopoeia: How to understand and to use
them in practice. Decisions tree for impurities.
14:30-15:30 How to use the European Pharmacopoeia in your daily laboratory work
Case of alternative methods, Test procedures in a monograph, How to perform Specific tests,
How to interpret chromatograms and list of impurities
16:00 Final addresses and Closure of the meeting
16:00-17:15 One to One consultations
Topics covered: General questions on the EDQM, the European regulatory framework and
harmonisation; Technical questions on PhEur monographs and texts; Certification procedure;
During coffee breaks, the electronic/online version of the European Pharmacopoeia will be set up
and demonstrated to participants interested in learning more about this version.
Who should attend?
This conference should be attended by professionals from industry, in particular persons involved in
the manufacture and control of drug substances/products or the preparation of registration dossiers;
from inspectorates, regulatory agencies and academic institutions.
Contact details:
Further information on the training course, accommodation and registration forms will be made available
on the EDQM internet site: http://www.pheur.org
Register at: http://www.pheur.org/site/page_597.php or contact the Public Relations Unit, EDQM;
226 avenue de Colmar, BP907, F-67029, Strasbourg, Cedex 1, FRANCE; Tel: 00 33 3 88 41 30 30 (Dial 4);
Fax: 00 33 3 88 41 27 71;
Email: Via the EDQM Helpdesk - go to: http://www.pheur.org/site/page_521.php
FOR MORE INFORMATION PLEASE VISIT THE WEBSITE : http://www.pheur.org
33
Please complete and send this form to: Caroline Larsen Le Tarnec, Public Relations, EDQM Fax: +33 (0)3 88 41
27 71; Internet: Please attach the registration form to our enquiry form (see section Helpdesk on the website:
http://www.pheur.org/site/page_630.php) or REGISTER ON-LINE
27-28 April 2006 Radisson Hotel, 160E Huron St., Chicago, USA
REGISTRATION FORM:
DATE OF REGISTRATION (DD/MM/YY) :
_ __ __ _
REGISTRATION FEE (*See general conditions)

950 * or 400 *
Yes, I am interested in reserving a meeting
ONE TO ONE MEETING
Which topic(s)?
EU Regulations Monographs, revisions
No, I am not interested
Publications Certification
Internet
PARTICIPANT DETAILS Please complete one form per participant

Title (Dr., Mr, Mrs, Ms, )
First Name
Family Name
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Address for
Correspondence
Postcode
Town
Country
Telephone
Fax
E-mail
AREA OF ACTIVITY/ Manufacturer of raw material
Manufacturer of medicines
OCCUPATION:
QA
QC
Regulatory authority:
Licensing
Inspection
OMCL
Other (please specify)
for human use for veterinary use

R&D
Regulatory affairs
Pharmacopoeias
University
PAYMENT (NEW)
Following receipt of your registration form, we will send you an invoice. Please note that we must receive payment
before the conference takes place. Details of payment methods will be outlined on the invoice. However, you will
be able to settle your invoice by:
1. PERSONAL OR COMPANY CHEQUE made payable to Council of Europe/EDQM
2. BANK TRANSFER
3. CREDIT CARD
DETAILS FOR INVOICING PURPOSES (if different from participant details)
Company/Institution
Address
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VAT Number (EU only)
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CANCELLATION CHARGES: I have read and accept the cancellation terms as stated on our web-site.
Date
Signature
FOR MORE INFORMATION PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
34
27-28 April 2006 Location: Radisson Hotel, 160E Huron Street, Chicago, USA
ONE-TO-ONE RESERVATION FORM:
2006 TRAINING SESSION: UNITED STATES

EUROPEAN PHARMACOPOEIA 5TH EDITION CHEMICALS
27-28 APRIL 2006
ONE TO ONE CONSULTATION QUESTION FORM

If you would like to register for a one-to-one consultation (15 minutes) with a member of the EDQM
scientific team during the training course, please complete this consultation request form and send
it to the EDQM by fax to +33 3 88 41 27 71 or via the Helpdesk on the website:
http://www.pheur.org/site/page_630.php)
Priority will be given to those who book in advance.
PARTICIPANT DETAILS
Title (Dr., Mr, Mrs, Ms)
First Name
Family Name
Company/Institution
E-mail
The time slots available for one-to-one consultations will be limited. The EDQM shall do all it can to
accommodate all interested participants.
If it is on a specific application, please mention the reference number
Your question:
Date
Signature
FOR MORE INFORMATION ABOUT TRAINING SESSIONS AND CONFERENCES ORGANISED BY THE EDQM
PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
35
HOTEL RESERVATION FORM

2006 TRAINING COURSE CHICAGO: 27-28 APRIL 2006
27-28 APRIL 2006 Location: Radisson Hotel, 160E Huron St. Chicago, USA
HOTEL REGISTRATION FORM:
Location: Radisson Hotel & Suites Hotel, Chicago, USA

To be filled in and sent by fax before 27 MARCH 2006
Fax: +1-312-757-5158
Global reservation:
40 rooms reserved from Wednesday 26th April to Friday 28th April included.
Contact reservations: RADISSON HOTEL & SUITES: 160E Huron St., Chicago, IL 60611
Contact: Reservations and indicate you are part of the EDQM/European Pharmacopoeia training course;
Tel: +1-312-787-2900; Fax: +1-312-757-5158; E-mail: radchicago@ihrco.com
General information:
The quotas of rooms reserved will be available until 27 March 2006.

After this date the availability and the negotiated prices will not be guaranteed. The rooms should be reserved
individually by each participant and are available on a first come, first served basis. Cancellation policy: Before
26 April 2006 at no further cost, after this date and in case of no show, one night will be charged to your credit
card.
Participant:
Family name:
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City:
Postcode:
State/Country:
Tel:
Fax:
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Hotel reservation:
Arrival day/hour:
Departure day/hour:
Please tick the box indicating the type of room you wish to reserve:
Standard Single Room at 149USD per night*
Standard Double Room at 149USD per night*
Single Corner Suite at 189USD per night*
Double Corner Suite at 189USD per night
*Note: Room rates are subject to state and local taxes and excludes breakfast
Please tick the box(es) indicating number of nights accommodation you require:
Yes Night 26 April
Yes Night 27 April
Yes, Night 28 April
Method of payment:
Credit card: Visa
EuroCard/ MasterCard
Amex
Credit card number:
Diners Card
Expiry date:
Cardholder: ______________________________________________________
I authorise the RADISSON HOTEL & SUITES to charge against my credit card the amount equivalent to one
night in order to block my reservation for the duration of the training session.
HOTEL CONFIRMATION RESERVATION N: ____________ Signature: ______________________
36
Certification of suitability
Certification of Suitability
of Monographs of the Ph. Eur.
LIST OF CERTIFICATES
CERTIFICATION SECTION OF THE EDQMS
INTERNET SITE
Visit the certication section of the EDQMs internet site
(www.pheur.org) and nd:
warnings/news;
answers to frequently asked questions;
general information on the certication procedure;
daily updated list of certicates granted.
LIST OF CERTIFICATES
Granted certicates
Voided or suspended certicates

A list of voided certicates is available online in the
certication section of our internet site (www.pheur.org)
and is also published in Pharmeuropa. This list includes:
the certicates not renewed after the 5-year validity
period (expired certicates);
the certicates withdrawn at the request of the
applicant (production stopped, site closed etc.);
the certicates suspended for a dened period

or withdrawn by EDQM (for GMP deciencies or
A daily updated list (more than 1800 certicates) is
insufcient information no longer in line with
available online in the certication section of our internet
regulatory requirements). They are identied by an
site (www.pheur.org). The database can be searched by
asterisk.
substance name, certicate number or holder name. TSE
certicates can also be searched selectively. To print the
Warning: in the online search of the database, no
full list of certicates: select certicate number in the
difference is made between suspended, expired or
database and search for CEP.
withdrawn certicates.
Chemical purity and microbiological quality

These certicates have not been renewed / have been withdrawn.
Substance
Acetylcysteine
Certificate
R0-CEP 1997-071-Rev 00
Acetylsalicylic acid (CrystalsPowder-Granules 7017)

Allopurinol
Alprazolam
Amiloride Hydrochloride
Amoxicillin Sodium
R0-CEP 2000-013-Rev 00
***
R0-CEP 1997-016-Rev 00
R0-CEP 1998-111-Rev 02
R0-CEP 1995-045-Rev 02
R1-CEP 1996-096-Rev 00
Amoxicillin Sodium
R1-CEP 1998-002-Rev 01
Amoxicillin trihydrate
Amoxicillin trihydrate; Compacted
Amoxicillin trihydrate
Amoxillin Sodium sterile
R0-CEP 1993-001-Rev 00
R0-CEP 1995-032-Rev 02
R0-CEP 1998-127-Rev 00
R1-CEP 1996-096-Rev 00
Ampicillin Sodium
Ampicillin Sodium; Sterile
Ampicillin Trihydrate
Ampicillin Trihydrate
Ampicillin, Anhydrous
Aspartic acid
Ascorbic acid
Ascorbic acid
Atenolol
R0-CEP 1995-004-Rev 01
R0-CEP 1998-132-Rev 00
R0-CEP 1994-010-Rev 00
R0-CEP 1992-001-Rev 02
R0-CEP 1994-009-Rev 00
R0-CEP 2000-295-Rev 02
R0-CEP 1995-019-Rev 01
R0-CEP 1997-035 Rev 02
R0-CEP 1998-033 Rev 02
Beclometasone Dipropionate
Benzylpenicillin potassium
R1-CEP 1992-013-Rev 00
R1-CEP 1992-003-Rev 00
Holder/Detenteur
Sterling SNIFF Italia; I 06073 Solomeo Di
Corciano (Perugia)
Alta Laboratories Ltd ; IND Maharashtra
Siegfried Cms Ag/Ltd; CH 4800 Zofingen
Degussa AG; D 01445 Radebeul
Ribbon SRL Pharmaceutical and Chemical;
I 20145 Milano
Sandoz Industrial Products S.A.; E 08520
Barcelona
Flamma SpA; I 24125 Bergamo
Gist-Brocades BV; NL 2600 MA Delft
Smithkline Beecham; FR 35380 Pllan le Grand
Ribbon Srl Pharmaceutical and Chemical I-20145
Milano
Gist Brocades B.v; NL 2600 AK Delft
Gist-Brocades B V; NL 2600 MA Delft
Biochemie SA; E 08400 Granollers - Barcelona
Gist-Brocades B V; NL 2600 MA Delft
Bim Sifram Group; FR 75010 Paris
Pliva D D Zagreb; CRO 10000 Zagreb
Merck Kgaa D 64271 Darmstadt
Teva Pharmaceutical Industrie Ltd; IL 49131 Petah
Tiqva
Hoechst Marion Roussel; F 92800 Puteaux
* suspended, withdrawn or not renewed by EDQM following an inspection

** withdrawn by CEP Holder after suspension by EDQM as a result of an inspection
*** CEP restored
37
Benzylpenicillin potassium; sterile

Benzylpenicillin sodium; Sterile
Benzylpenicillin, benzathine;
Sterile, ( FA Process, sterile,
lecithin and polysorbate 80 coated;
Material Code Numbers 451028
and 451387)
(Soya lecithin coated 1,2 %)
Sterile, (Soya lecithin coated
1.2%)
Benzylpenicillin, procaine
Benzylpenicillin, procaine
Benzylpenicillin, procaine; (Soya
Lecithin Coated 1.2%), sterile
Benzylpenicillin, procaine; (Soya
lecithin coated)
Caffeine
R0-CEP 1996-086-Rev 01
R0-CEP 1996-085-Rev 01
R1-CEP 1994-018-Rev 04
Gist-Brocades Bv; NL 2600 MA Delft

Sandoz GmbH; A 6250 Kundl, Tyrol
R0-CEP 1995-013-Rev 01
R0-CEP 1996-087-Rev 01
R0-CEP 1995-012-Rev 02
R0-CEP 1996-011-Rev 02
R0-CEP 1996-037-Rev 02

Gist-Brocades Bv; NL 2600 MA Delft
Gist Brocades BV; S 645 41 Strngnas
R0-CEP 1996-010-Rev 01
R1-CEP 1997-047-Rev 00
Caffeine
Captopril
R0-CEP 2000-178-Rev 01
R0-CEP 1997-120-Rev 00
Carbamazepine
Carbamazepine
R0-CEP 1997-117-Rev 00
R0-CEP 1996-089-Rev 00
Carbasalate calcium
Cefadroxil monohydrate
R0-CEP 1997-056Rev 01
R0-CEP 1998-007 Rev 01
Cefadroxil monohydrate
R1-CEP 1998-019-Rev 00
Cefazolin sodium
Cefradine
R0-CEP 1998-054-Rev 03
R1-CEP 1997-106-Rev 00
Ceftriaxone Sodium; sterile
R0-CEP 1997-095-Rev 00
Cholecalciferol
Desmopressin
Diclofenac sodium
Diclofenac sodium
Dicloxacillin Sodium sterile
R0-CEP 1996-046-Rev 00
R0-CEP 1994-015-Rev 02
R0-CEP 1996-034-Rev 02
R0-CEP 1998-072-Rev 01
R0-CEP 1996-092- Rev 00
Digoxin
R0-CEP 1992-011-Rev 02
Hangzhou Minsheng Pharmaceutical Group Co;

RC 310 011 Hangzhou
KW Pfaffenschmidt GmbH; D 22459 Hamburg
Sinova Pharmaceuticals Co (Pte) Ltd; SGP 629534
Singapore
Vis Farmaceutici; I 35129 Padova
Fis-Fabbrica Sintetici SpA I-36041 Alte Di
Montecchio Maggi
DSM Minera BV, NL 3600 AC Maarssen
Dsm Anti-infectives Chemferm SA E 08130 Santa
Perpetua De Mogoda
Ranbaxy Laboratories Ltd; IND 110 019 New
Delhi
Amifarma S L; E 08389 Palafolls, Barcelona
Delhi
Delhi
Roche Vitamins Ltd; CH 4070 Basel
Ferring SA; F 94250 Gentilly
Degussa-Hls AG; D 01445 Radebeul
Klinge Pharma & Co Ltd; IRL County Kerry
Ribbon Srl Pharmaceutical and Chemical I-20145
Milano
Procter & Gamble Pharmaceuticals; F 92201
Neuilly Sur Seine Cdex
Dr Reddys Laboratories Ltd IND 500 016
Hyderabad
Ganes Chemicals Inc; USA NJ 08070 Pennsville
Wacker Chemie GmbH; D 81737 Munchen
Dow Corning France; F 69432 Lyon Cedex 03
Kraemer And Martin Pharma Handels Gmbh; D
47804 Krefeld
Eli Lilly SA; IRL County Cork
Biochemie SA; E 08520 Les Franqueses Del
Valls
Diltiazem Hydrochloride Process I R0-CEP 1997-081 Rev 01

Dimenhydrinate
Dimeticone
Dimeticone; (20, 50)
Doxycycline hyclate
R0-CEP 1998-081-Rev 00
R0-CEP 1998-016-Rev 00
R0-CEP 1995-048-Rev 02
R0-CEP 1996-063-Rev 00
Fluoxetine hydrochloride
Flucloxacillin Sodium; n-butyl
acetate process; powder;
compacted
Folic acid
R0-CEP 1999-046-Rev 02
R0-CEP 1996-029-Rev 04
R0-CEP 1994-002-Rev 00
BASF Aktiengesellschaft; D 67056 Ludwigshafen

*** CEP restored
38
Fructose
Gelatin limed hide gelatin
Guaifenesin
Haloperidol
Hydrocortisone
Hydrocortisone Acetate
Ibuprofen
Iopamidol
Isosorbide Monohydrate, diluted
70% and 80%
Isosorbide Mononitrate, Diluted
Isoleucine
Itraconazole
Ketoconazole
Ketoprofen
Ketoprofen
Levamisole Hydrochloride
Mebendazole; Polymorph C
Metamizole sodium
R0-CEP 1997-013-Rev 00
R0-CEP 2000-386-Rev 00
Danisco Sweeteners Ltd; GB RH1 6YS Redhill

Gelita Group (DGF Stoess, Kind & Knox), D
69412 Eberbach
R0-CEP 2002-194-Rev 00*
Schtz & Co (GmbH & Co); D 20095 Hamburg
R0-CEP 2000-179-Rev 01
Janssen Pharmaceutical Ltd IRL County Cork
R0-CEP 1996-043-Rev 00
Diosynth BV; NL 5340 BH Oss
R0-CEP 1995-042-Rev 00
Diosynth BV; NL 5340 BH Oss
R0-CEP 1997-124- Rev 02
BASF PLC ; GB NE23 9JL Cramlington
R0-CEP-1999-130 Rev 02
Recordati SpA; I 20148 Milano
R0-CEP 2001-329-Rev 00 ** Calao SRL, I 20151 Milano
R0-CEP 1999-107-Rev 00
R0-CEP 1998-041-Rev 03
R0-CEP 2001-150-Rev 01
R0-CEP 1997-105-Rev 02
R0-CEP 1994-019-Rev 01
R0-CEP 1995-046-Rev 02
R1-CEP 1993-006-Rev 00
R0-CEP 1997-036-Rev 00
R0-CEP 2001-220-Rev 00 *
Methotrexate
Metronidazole
Miconazole nitrate
Midazolam
Minocycline Hydrochloride
Nimodipine
Paracetamol
Paracetamol
Paracetamol
R0-CEP 1996-026-Rev 00
R0-CEP 1993-004-Rev 00
R0-CEP 1998-153-Rev 01
R0-CEP 1998-114-Rev 01
R0-CEP-1999-072 Rev 02
R0-CEP-1999-105 Rev 02
R0-CEP 2000-002-Rev 00 *
R0-CEP 1996-057-Rev 01
R0-CEP 2002-020-Rev 01*
Paracetamol
Pentoxifylline
Phenazone
R0-CEP 2001-092-Rev 02
R0-CEP 1996-035-Rev 01
R0-CEP 2001-042-Rev 00 *
Phenoxymethylpenicillin
potassium
Phenylephrine Hydrochloride
R1-CEP-1994-017 Rev 02
R0-CEP 1995-044-Rev 02
R0-CEP 1999-112-Rev 00
Potassium clavulanate; (The

Netherlands)
Propyphenazone
R0-CEP 1996-012-Rev 02
Quinidine sulphate
Quinine hydrochloride
Quinine hydrochloride
Quinine sulphate
Quinine sulphate
Selegiline Hydrochloride
R1-CEP 1992-007-Rev 00
R0-CEP 1997-030-Rev 01
R1-CEP 1992-009 Rev 03
R0-CEP 1998-003-Rev 01
R1-CEP 1992-006- Rev 02
R0-CEP 1997-020-Rev 00
Sodium Valproate
Sulfadiazine
Sulfadiazine
R0-CEP 1996-015-Rev 02
R0-CEP 1992-008-Rev 00
R0-CEP 1995-040-Rev 01
R0-CEP 2001-041-Rev 00 *
Whyte Chemicals; GB N3 2UA Finchley, London

Amino GmbH; D 38373 Frellstedt
Janssen Pharmaceutical Ltd; IRL County Cork
Janssen Pharmaceutical Ltd; IRL County Cork
Aventis Pharma Ltd; GB RM10 7XS Dagenham
Nordic Synthesis Ab; S 69185 Karlskoga
Janssen Pharmaceutica NV; B 2340 Beerse
Janssen Pharmaceutica NV; B 2340 Beerse
Vani Chemicals & Intermediates Ltd ;
IND 500 055 Hyderabad
Pfizer And Pfizer Mack; D 89252 Illertissen
Rhne-Poulenc Rorer SA; F 92165 Antony Cedex
Janssen Pharmaceutical Ltd IRL County Cork
Diosynth BV; NL 5349 AB Oss
Nippon Kayaku Co. Ltd; J 102-8172 Tokyo
Siegfried Ltd; CH 4800 Zofingen
Indukern Chemie AG; CH 8952 Schlieren
BASF Corporation; USA 78343 Bishop, Texas
Farmson Analgesics (Unit of Farmson
Pharmaceutical Gujarat Pvt Ltd); IND 391 340
Nandesari, Dist Vadodara, Gujarat
Bristol Laboratories Ltd; GB HA1 2EN Middlesex
Degussa-Hls AG; D 01445 Radebeul
DSM Anti-Infectives; NL 2613 AX Delft
Smithkline Beecham Pharmaceuticals; GB BN14
8QH Worthing
Roche Diagnostics GmbH; D 68298 Mannheim
Roche Diagnostics GmbH; D 68298 Mannheim
Teva Group Bulk Pharmaceuticals Division; IL
49131 Petah Tiqva
DSM Minera BV; NL 3600 AC Maarssen
Southwest Synthetic Pharmaceutical General
Factory; RC 400 025 Chongqing

*** CEP restored
39
Sulfamethoxazole
Tamoxifen Citrate
Terfenadine
Terfenadine
Theophylline
Trimethoprim
Trimethoprim
Vinblastine Sulphate
R0-CEP 1995-026-Rev 02
R0-CEP 1997-024-Rev 01
R0-CEP 1997-003-Rev 00
R0-CEP 1995-037-Rev 01
R1-CEP 1998-018-Rev 00 *
R0-CEP 1999-132-Rev 02
R1-CEP 1994-017-Rev 02
R1-CEP 1992-005-Rev 00
Shionogi & Co Ltd; J 541-0045 Osaka

Ranbaxy Laboratories Ltd; IND 144 533 Toansa
Avik Pharmaceutical Ltd; IND 396 195 Vapi
KW Pfannenschmidt GmbH; D 22459 Hamburg
Welding GmbH & Co KG; D 20354 Hamburg
Welding GmbH & Co KG; D 20354 Hamburg
Eli Lilly SA - Irish Branch; IRL County Cork

*** CEP restored
Transmissible spongiform encephalopathy risk products

The following certicates have been withdrawn.
Substance
Brain Heart Infusion
Gelatin, Alkaline-Processed
Chromium-Tanned Hide GelatinSPA
Gelatin, Acid bone gelatin
Certificate
R0-CEP 2000-267-Rev 00
R0-CEP 2000-113-Rev 02
Holder/Detenteur
BD Diagnostic Systems ; USA 21152 Sparks
Croda Colloids Ltd; GB WA8 8UB Widnes,
Cheshire
R0-CEP 2000-227-Rev 03
Gelatin, Limed bone gelatin
R0-CEP 2000-228-Rev 03
Gelatin limed hide gelatin
R0-CEP 2000-386-Rev 00
Gelatin US Enzymatic Bone

Gelatin Hydrolysate
Gelatin acid bone gelatin,
American origin
American origin
including NaOH treatment
Gelatin; / Acid bone gelatin;
(French [European] origin)
R0-CEP 2002-153-Rev 01

Cheshire
Cheshire
Gelita Group (DGF Stoess, Kind & Knox), D69412 Eberbach
Gelita Group; D 69412 Eberbach
R0-CEP 2000-139-Rev 05
PB Gelatins; B 1800 Vilvoorde
R0-CEP-2000-026-Rev 03
Rousselot SAS; F 92641 Boulogne Billancourt

Cedex
Rousselot SAS; F 92641 Boulogne Billancourt
Cedex
Skw Biosystems; F 92641 Boulogne Billancourt
Cedex
40
R0-CEP-2001-411-Rev 02
R0-CEP 2000-030-Rev 00
Effect of temperature on plasma freezing
Scientific Notes
The Secretariat of the European Pharmacopoeia recalls that articles reflect the authors opinions. In order that all
opinions are taken into account, concerned users and readers of Pharmeuropa are invited to send their comments
to the Secretariat. Readers are reminded that details regarding contributions made to Pharmeuropa are cited on
the rear cover of this issue.
(IIHFW RI 7HPSHUDWXUH RQ 3ODVPD )UHH]LQJ XQGHU

,QGXVWULDO &RQGLWLRQV
M.I. Bravo, S. Grancha, J.I. Jorquera
6800$5<
The European Pharmacopoeia monograph on Human plasma for fractionation does not define the freezing process
time but does define the freezing temperature ( 30 C or below). Initial freezing conditions are crucial for the quality
of plasma. These conditions were intended to preserve labile proteins such as fVIII, but they can also be considered
favourable for the plasma quality in general.
This study evaluates the way the industrial plasma freezing affects labile coagulation factors. We have studied the
freezing of plasma in industrial-size chambers at temperatures close to 30 C, 25 C and 20 C, and the possible
differences between performing the freezing process in a chamber or in a freezer, in order to elucidate whether or not
these parameters affect the quality of plasma.
For this study, plasma bottles were frozen in industrial chambers set at 30 C, 25 C and 20 C, and in a freezer
set at 20 C. The freezing rates were followed by means of probes in plasma control bottles. From this plasma,
coagulation factors (fVIII, fIX and fibrinogen) were analysed before and after freezing, and cryoprecipitate was obtained
in some cases. Statistically significant differences exist in fVIII:C recovery in thawed plasma between freezing at
30 C and at 20 C (n = 11; 85.4 4.3 % versus 74.6 6.0 % (chamber) or 79.3 6.3 % (freezer)). There is no
difference between 30 C and 25 C, or between freezing at 20 C in a chamber or in a freezer. No significant
loss of activity in thawed plasma is observed for fIX and fibrinogen at 25 C or 20 C versus 30 C. The fVIII
and vWF recovery in cryoprecipitates does not show differences (464.2 IU fVIII/ml at 30 C, 446.7 IU fVIII/ml at
25 C, and 475.8 IU fVIII/ml at 20 C).
The results obtained from this study support that plasma might also be frozen at 25 C or below without any impact on
its quality, and that sporadic and short term deviations, from 30 C or below up to 25 C, in the currently required
freezing temperature, would not have an effect on the labile factors recovery.
.(<:25'6
Plasma, freezing, coagulation factor, cryoprecipitate.
,1752'8&7,21
Currently, the European Pharmacopoeia monograph on
Human plasma for fractionation [1] defines: When obtained
by plasmapheresis, plasma intended for the recovery of
proteins that are labile in plasma is frozen by cooling
rapidly at 30 C or below as soon as possible and at the
latest within 24 h of collection. The monograph on Human
plasma for fractionation does not define the freezing process
time but does define the freezing temperature ( 30 C
or below); in the last revision, the concept in a chamber
was added in order to avoid measuring the temperature in
the plasma. But this last proposal may be open to different
interpretations because it does not explicitly indicate the
core plasma temperature that plasma should reach before it
is stored at 20 C (storage conditions).
Once frozen, the monograph defines the plasma storage
temperature at 20 C or below. The DGTI multicenter
trial initiated by the section Blood Plasma Constituents
determined that the stability of frozen fresh plasma (FFP)
during 2 and 3 years of storage at 20 C, 25 C, 30 C,
and 40 C shows no detectable protein changes in the
FFP [2].
Initial freezing conditions are crucial for the quality of
plasma. Early studies [3], assessing the preservation of labile
proteins such as coagulation factor VIII (fVIII), show that the

freezing conditions should be such that at a low surrounding
temperature (e.g. 40 C in those experiments) freezing to
the core of the plasma container is reached as rapidly as
possible. The study of kerblom et al. [4], found a highly
significant difference of fVIII activity comparing freezing at
20 C with freezing within 40 minutes at 40 C. This
suggests that the freezing should proceed in a short time at
temperatures below the theoretical eutectic point ( 23 C
for plasma) [5, 6]. These freezing conditions were shown to
increase the yield of labile products such as fVIII, but they
can also be considered favourable for the plasma quality in
general.
This study evaluates the way the industrial plasma freezing
affects labile coagulation factors. When a chamber is set at a
temperature of 30 C or below, it might happen that this
temperature temporarily rises slightly over the target during
the day due to opening, loading of the equipment etc. There
is no scientific data available stating that a difference exists
between the plasma freezing performed at temperatures
below 30 C or at temperatures slightly higher than
30 C. For this reason, we have also studied the plasma
freezing in industrial-size chambers at temperatures close to
30 C, 25 C and 20 C. Finally, possible differences
between performing the freezing process in a chamber or
M.I. Bravo. (Corresponding author: e-mail: isabel.bravo@grifols.com), Instituto Grifols, S.A. Pol. Levante. Can Guasch, 2. 08150 Parets del Valls,
Barcelona, Spain
S. Grancha. Instituto Grifols, S.A. Pol. Levante. Can Guasch, 2. 08150 Parets del Valls, Barcelona, Spain
J.I. Jorquera. Instituto Grifols, S.A. Pol. Levante. Can Guasch, 2. 08150 Parets del Valls, Barcelona, Spain
Pharmeuropa Vol 18, No. 1, January 2006
41
in a freezer were studied, to elucidate whether or not the

increase in temperature occurring when loading a small-size
freezer affects the plasma quality.
0$7(5,$/6 $1' 0(7+2'6
3UHSDUDWLRQ RI SODVPD
Plasmapheresis plasma for fractionation was collected in

bottles of approximately 800 ml of plasma volume containing
sodium citrate as anticoagulant. All plasma was introduced
into a chamber at 30 C or below immediately after
collection (within 30 min). The freezing process in the
plasmapheresis centres takes less than 24 h for the plasma
to reach 30 C or below.
The bottles of plasma were thawed in a water bath at
37 C, the content was homogenised by gentle stirring and,
immediately after, plasma was assayed without additional
freezing for fVIII and factor IX (fIX) activity, and fibrinogen
(clottable protein). Aliquots of all samples were separated
and immediately frozen at 70 C or below. Occasionally,
these aliquots were used to confirm results. No apparent
fVIII loss happened because of the additional freezing of
these samples.
Plasma was then frozen under different study conditions
( 30 C as control, 25 C and 20 C). Two independent
runs of freezing at 25 C (n = 11 bottles each) were
performed on different days. Once the core plasma
temperature was obtained (as measured with thermocouple
probes in control plasma units), the plasma bottles were
maintained at this temperature for a period of 6-30 h, and
then changed to 70 C or below until tested (within
5 days).
Some bottles (n = 11) were used to assay the coagulation
factors content and others (n = 6) for cryoprecipitate
preparation.
In order to assay fVIII, fIX and fibrinogen recovery at the
different freezing temperatures, plasma bottles stored at
70 C or below were thawed again as described before

and immediately assayed for the factors content. In this
case, aliquots of all thawed bottles were also separated and
immediately frozen at 70 C or below. Occasionally, these
aliquots were used to confirm the results.
$QDO\WLFDO 0HWKRGV
FVIII activity was determined by a chromogenic

method in an automatic system with S-2765 substrate
(N-a-Z-D-Arg-Gly-Arg-pNA) from Chromogenix; fIX activity
by a coagulation method based in the determination of
activated partial thromboplastin time, and the fibrinogen
(clottable protein) by the von Clauss coagulation method
in an automatic system. Von Willebrand factor ristocetin
cofactor (vWF:RCo) was assayed by monitoring platelet
aggregation using BC von Willebrand Reagent (Behring).
)UHH]LQJ UDWHV
The plasma temperature was monitored by means of probes

placed in the middle of control plasma bottles, which
were connected to a recorder (KM1242 from Comark, or
Testostor 171 from Logger).
)UHH]LQJ FRQGLWLRQV
Bottles (coming from room temperature: 23 3 C, after

being thawed in the water bath at 37 C) were uniformly
distributed into the freezer ( 20 C) or on the chamber
shelves ( 30 C, 25 C and 20 C; Figure 1). The bottles
entered the chambers in 2 groups and the freezer in 4 groups
to minimise the impact on surrounding temperature; the
evolution of plasma freezing was followed up in each group
by means of a probe placed in the center of a bottle.
Freezing at 30 C was performed in a chamber with a
volume of 2388 m3. The study at 25 C and 20 C was
carried out in a chamber with a volume of 87.52 m3. An
additional study at 20 C was performed in a freezer with
a volume of 0.5 m3. At the time of this study, the chambers
Figure 1 Photograph of the general set up of bottles and probes in the chambers
42
were filled up to 90 % of their capacity and the freezer was

empty.
&U\RSUHFLSLWDWH SUHSDUDWLRQV
Cryoprecipitate was obtained by thawing the bottles of

plasma (n = 6) in a water bath at 5 3 C. After thawing,
the content was transferred into 400 ml centrifuge beakers
and centrifuged at 2890 g for 30 min at 2 C. After
centrifugation, the obtained cryoprecipitate was separated
from the supernatant and pooled before processing. All
cryosupernatants were also pooled for fVIII:C assay.
Subsequently, fVIII was extracted from the cryoprecipitate
by weighing the cryoprecipitate and dissolving it in 3 times
its mass of an aqueous extraction solution containing
0.16 g/l NaCl and 80 IU/ml sodium heparin. This process
was carried out in a water bath at 30 C, for 20 min. Then,
the material was adjusted to pH 7.00 0.05 with 0.2 M HCl,
cooled to 25 C and centrifuged at 2890 g for 10 min at
room temperature to clarify the solution from undissolved
material. The clarified solution was then assayed for fVIII:C
and vWF:RCo.
5(68/76
)UHH]LQJ UDWHV
During the freezing study the chamber temperatures were

constant ( 2.2 C from the target temperatures); no effect
was observed by the introduction of plasma bottles. This
is due to the great capacity of these chambers and to the
fact that they were filled up to 90 % of their capacity. In
the second run at 25 C, the temperature increased to
18.4 C due to the malfunction of a compressor, but this
increase occurred when the core plasma had reached the
target temperature and the plasma temperature did not

change.
The 20 C freezer conditions were altered by the
introduction of plasma bottles, increasing the temperature
because of its lower capacity and because it was empty. The
freezer temperature recovered as the plasma froze. Table 1
shows the freezer and chamber temperatures during the
study.
The mean freezing rates of the plasma core obtained under
different conditions are shown in Figure 2. Freezing rates
in the core plasma are defined by the parameters shown in
Table 2.
(IIHFW RI IUHH]LQJ RQ WKH FRDJXODWLRQ IDFWRUV
The effect of different plasma freezing conditions on the

recovery of coagulation factors (fVIII, fIX and fibrinogen)
is summarised in Tables 3-5, including differences versus
control temperature ( 30 C).
Results for fVIII levels are statistically different when plasma
is frozen at 20 C (chamber or freezer) and do not show
differences when plasma is frozen at 25 C. For fIX and
fibrinogen levels, results are similar for all temperatures
assayed; when comparing results in the chamber at 20 C
with results at 30 C, a slight statistically significant
difference is noticed, but it is possibly due to variability of
results, as the apparent recovery is higher at lower freezing
temperatures (increase of 11.8 % in the average for fIX and
3.8 % for fibrinogen).
When comparing results obtained for coagulation factors
(fVIII, fIX and fibrinogen) after freezing at 20 C in the
chamber or in the freezer, there are no statistical differences
detected.
Figure 2 Mean freezing rates of the plasma core obtained under different conditions
43
(two separate runs). FIX and fibrinogen activity recoveries

are not decreased because of freezing at 25 C or 20 C.
The results obtained for fVIII recovery in plasma (85.4 %
at 30 C; 85.5-83.8 % at 25 C and 74.6-79.3 % at 20
C) are similar to those obtained by kerblom et al. [4]
when freezing plasma in bags (200 ml) at temperatures of
',6&866,21
20 C and 40 C, obtaining fVIII recoveries of 80 % and
Freezing of plasma in a chamber at 30 C or below is the 86 %, respectively.
FVIII and vWF recoveries in the cryoprecipitates obtained
usual process currently performed to meet the European
from plasma frozen at 30 C, 25 C or 20 C do
Pharmacopoeias requirements on plasma for fractionation
not show relevant differences. The fVIII yields obtained
intended for the production of labile coagulation factors.
(475.8-446.7 IU fVIII/l plasma) are similar to those obtained
As observed in the study, freezing of plasma in bottles (of
by Farrugia et al. [3] (425 IU fVIII/kg plasma) when freezing
approximately 800 ml) at a temperature of 30 C was
plasma quickly at 70 C.
complete after 9 h. This period was prolonged up to 11-14 h
&21&/86,216
at 25 C, 14 h at 20 C in the chamber and up to 20 h
in the study performed at 20 C in the freezer. Under all When the plasma freezing is performed at 25 C, the
conditions studied, the freezing curves have similar profiles recovery of labile coagulation factors in thawed plasma
but different maximum speeds (see Table 2).
is identical to that observed when the plasma is frozen
at 30 C. The recovery of fVIII and vWF activities in
This study was aimed at analysing whether or not these
cryoprecipitates obtained from plasma frozen at 30 C,
differences in temperature during plasma freezing,
25 C or 20 C is also equivalent.
involving longer freezing times and different final plasma
The results obtained from this study support that plasma
temperatures, affect coagulation factors (fVIII, fIX and
might also be frozen at 25 C or below without any impact
fibrinogen) and the cryoprecipitate production. From the
results of fVIII recovery obtained after plasma freezing under on its quality, and that sporadic and short term deviations,
from 30 C or below, up to 25 C, in the currently
the studied conditions, it is observed that, although the
required freezing temperature, would not have an effect on
difference in fVIII recovery is not highly relevant (between
the labile factors recovery.
85.5 % and 74.6 %), a statistically significant difference
exists between freezing at 30 C and at 20 C. By
contrast, this difference does not exist when comparing the
results obtained at 30 C with those obtained at 25 C
(IIHFW RI IUHH]LQJ RQ FU\RSUHFLSLWDWH
Cryoprecipitate was obtained from plasma frozen at different

temperatures; the results are shown in Table 6. These results
show that fVIII and vWF recoveries in the cryoprecipitate
are comparable for all temperatures assayed.
Table 1 Summary of freezer and chamber temperatures during the study

Chamber at
- 30 C
- 25 C** (1st run)
- 25 C** (2nd run)
- 20 C
- 25.7 C
- 26.5 C
- 21.2 C
Freezer at - 20 C
A*
B*
Mean
- 30.5 C
- 30.3 C
Maximum
- 27.9 C
- 28.7 C
- 24.8 C
- 25.8 C
- 20.3 C
- 11.1 C
Minimum
- 31.5 C
- 31.3 C
- 26.4 C
- 27.2 C
- 21.7 C
- 21.0 C
- 17.9 C
* The A probe is placed at the top of the entrance door, and the B probe is placed at the inner part of the chamber; for this study the plasma was
placed at the inner part of the chamber.
** The temperatures after the malfunction of the compressor are not taken into account.
Table 2 Freezing rate parameters in the core plasma

Chamber at
Freezing rate
parameters
Time to reach 0 C
(minutes)
Time to reach target
temperature (hours)
Maximum freezing
rate* (C/hour)
Freezer at - 20 C
- 30 C
- 25 C (1st run)
- 25 C (2nd run)
- 20 C
60
80
70
75
105
14
11
14
20
- 8.6
- 4.1
- 5.5
- 3.2
- 4.8
*Obtained by calculation from the linear part (gradient) of the freezing curve.
44
Table 3 Summary data on the fVIII activity recovery at different plasma freezing conditions
Chamber at
FVIII:C recovery
Freezer at - 20 C
- 30 C
- 25 C (1st run)
- 25 C (2nd run)
- 20 C
Mean SD (%)
85.4 4.3
85.5 8.7
83.8 5.0
74.6 6.0
79.3 6.3
Range (%)
80.1 - 91.7
74.0 - 92.3
n.s.
p = 0.491
70.9 - 88.8
---
73.7 - 98.7
n.s.
p = 0.974
67.8 - 88.8
Difference versus
control
p < 0.001
p = 0.028
n.s.: not significant.

p (Mann-Whitney Test).
Table 4 Summary data on the fIX activity recovery at different plasma freezing conditions
FIX:C recovery
Chamber at
Freezer at - 20 C
- 30 C
- 25 C
- 20 C
Mean SD (%)
87.9 9.4
90.9 4.1
99.7 5.4
93.9 8.7
Range (%)
71.4 - 100.0
93.3 - 109.5
Difference versus control
---
86.0 - 98.0
n.s.
p = 0.481
81.1 - 103.0
n.s.
p = 0.105
p = 0.009

Table 5 Summary data on the fibrinogen (clottable protein) recovery at different plasma freezing conditions
Fib:C recovery
Chamber at
Freezer at - 20 C
- 30 C
- 25 C
- 20 C
Mean SD (%)
97.2 4.4
99.4 1.6
101.0 2.8
100.1 2.6
Range (%)
87.1 102.5
97.5 105.1
Difference versus control
---
96.9 102.0
n.s.
p = 0.291
97.9 106.1
n.s.
p = 0.057
p = 0.020

Table 6 FVIII and vWF recoveries in the cryoprecipitate at different plasma freezing conditions
Temperatures
- 30 C
- 25 C
- 20 C
Plasma units
Plasma weight (g)
4695
4888
4870
Supernatant weight (g)
4599
4585
4783
Cryoprecipitate weight (g)
96
103
87
FVIII in Cryosupernatant (IU fVIII/ml)
0.10
0.09
0.15
FVIII in cryoprecipitate (IU fVIII/g crio)
22.3
19.6
26.3
FVIII in cryoprecipitate (IU fVIII/l plasma)
464.2
446.7
475.8
VWF:RCo in cryoprecipitate (IU

vWF:RCo/g crio)
38.1
37.1
45.1
VWF:RCo in cryoprecipitate (IU

vWF:RCo/l plasma)
793.1
843.9
816.7
45
rate and storage conditions on cryoprecipitate quality.

5()(5(1&(6
J Clin Pathol 1985; 38:433-7.
[1] Human plasma for fractionation, monograph 0853. Ph.
Eur. Suppl 5.3. Strasbourg, France: Council of Europe [4] kerblom O, Bremme K, Dackland AL et al. Freezing
technique and quality of fresh frozen plasma.
2005.
Infusionstherapie 1992; 19:283-7.
[2] Kotitschke R, Morfeld F, Kirchmaier CM et al. Stability
[5] Carlebjrk G, Blombck M, Philstedt P. Freezing of
of fresh frozen plasma: results of 36-month storage at
plasma and recovery of factor VIII. Transfusion 1986;
20 C, 25 C, 30 C and 40 C. Multicenter
26:159-62.
Study of the Section Blood Plasma Constituents of
the DGTI. Infusion Therapy and Transfusion Medicine [6] Farrugia A, Hill R, Douglas S et al. Factor VIII/von
2000; 27:174-80.
Willebrand factor levels in plasma frozen to 30 degrees
C in air or halogenated hydrocarbons. Thromb Res
[3] Farrugia A, Prowse C. Studies on the procurement of
blood coagulation factor VIII: effects of plasma freezing
1992; 68(1):97-102.
46
Allopurinol
Draft monographs and

general texts for comment
IMPORTANT NOTICE
This section contains proposals for new and revised monographs and general texts, intended for inclusion in the
European Pharmacopoeia and submitted for public comment. You can comment through the appropriate national
authority at the address listed on the back cover page of the present issue. Readers from countries not members of the
European Pharmacopoeia Commission should send their comments directly to the European Directorate for the Quality
of Medicines. In order to facilitate the work of the Secretariats of the National Authorities and the European Directorate
for the Quality of Medicines who collect the comments, please mention in any correspondence the PA/PH reference
number indicated at the beginning of each text. Comments that propose modifications of limits should be supported by
analytical data obtained on a significant number of batches. Proposed changes of methodology should be supported by
experimental results of a comparative trial of the method published in Pharmeuropa for comment and the proposed
alternative. Only comments sent before 31 March 2006 will be considered before the final version is prepared.
It is stressed that these proposals have not been adopted by the European Pharmacopoeia Commission and must not
be regarded as official standards.
In the case of proposals for revision, text to be deleted is crossed out and replacements or additions are underlined.
In certain cases, draft monographs require, for appropriate checking, the use of a reference material that is not yet
commercially available; in exceptional circumstances, we will try to make the necessary substance available; please
submit enquiries to the European Directorate for the Quality of Medicines.
Reference: PA/PH/Exp. 10A/T (05) 85 ANP
A. Dissolve 10 mg in 1 ml of a 4 g/l solution of sodium

hydroxide R and dilute to 100.0 ml with a 10.3 g/l
NOTE ON THE MONOGRAPH
solution of hydrochloric acid R. Dilute 10.0 ml of
this solution to 100.0 ml with a 10.3 g/l solution of
Hydrazine can be used as starting material in the synthesis
hydrochloric acid R. Examined between 220 nm and
of allopurinol and a test has been introduced to limit any
350 nm (2.2.25), the solution shows an absorption
residue which could be present. A proposal was published
maximum at 250 nm and an absorption minimum at
in Pharmeuropa 16.2. This new proposal includes an LC
231 nm. The ratio of the absorbance measured at the
method that can limit hydrazine to a lower content.
absorption minimum at 231 nm to that measured at the
XXXX:0576
absorption maximum at 250 nm is 0.52 to 0.62.
B. Infrared absorption spectrophotometry (2.2.24).
ALLOPURINOL
Preparation : discs.
Comparison : allopurinol CRS.
Allopurinolum
C. Dissolve 0.3 g in 2.5 ml of dilute sodium hydroxide
solution R and add 50 ml of water R. Add slowly and
with shaking 5 ml of silver nitrate solution R1. A white
precipitate is formed which does not dissolve on the
addition of 5 ml of ammonia R.
D. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in concentrated ammonia R and dilute to
C 5H 4N4O
Mr 136.1
10 ml with the same solvent.
Reference solution. Dissolve 20 mg of allopurinol CRS
DEFINITION
in concentrated ammonia R and dilute to 10 ml with the
1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one.
same solvent.
Content : 98.0 97.0 per cent to 102.0 per cent (dried
Plate : TLC silica gel F254 plate R.
substance).
Mobile phase : anhydrous ethanol R, methylene
chloride R (40:60 V/V).
CHARACTERS
Application : 10 l.
Appearance : white or almost white powder.
Development : over 2/3 of the plate.
Solubility : very slightly soluble in water and in ethanol
(96 per cent). It dissolves in dilute solutions of alkali
Drying : in air.
hydroxides.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
IDENTIFICATION
with the test solution is similar in position and size to
First identification : B.
the principal spot in the chromatogram obtained with
the reference solution.
Second identification : A, C, D.
47
Allopurinol
TESTS
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use. Use freshly
prepared solutions, and store and inject them at 8 C,
using a cooled autosampler.
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in 2.5 ml of a 4 g/l solution of sodium hydroxide R
and dilute immediately to 50.0 ml with the mobile phase.
Test solution (b). Dissolve 20.0 mg of the substance to be
and dilute immediately to 250.0 ml with the mobile phase.
Reference solution (a). Dilute 2.0 ml of test solution (a)
to 100.0 ml with the mobile phase. Dilute 5.0 ml of this
solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 5.0 mg of allopurinol
impurity A CRS, 5.0 mg of allopurinol impurity B CRS and
5.0 mg of allopurinol impurity C CRS in 5.0 ml of a 4 g/l
solution of sodium hydroxide R and dilute immediately to
100.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
Reference solution (c). Dissolve 20.0 mg of allopurinol CRS
in 5.0 ml of a 4 g/l solution of sodium hydroxide R and
dilute immediately to 250.0 ml with the mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m)(1).
Mobile phase : 1.25 g/l solution of potassium dihydrogen
phosphate R.
Flow rate : 1.4 ml/min.
Detection : spectrophotometer at 230 nm.
Injection: 20 l of test solution (a) and reference solutions (a)
and (b).
Run time : twice the retention time of allopurinol.
Elution order : impurity A, impurity B, impurity C,
allopurinol.
Retention time : allopurinol = about 10 min.
System suitability : reference solution (b) :
resolution : minimum 1.1 between the peaks due to
impurities B and C.
Limits :
impurity A : not more than twice the area of the principal
solution (a) (0.2 per cent) ;
impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
impurity C : not more than the area of the corresponding
solution (b) (0.1 per cent) ;
unspecified 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 of impurities other than A, B and C : not more than
3 times the area of the principal peak in the chromatogram
disregard limit : 0.5 times the area of the principal peak
(0.05 per cent).
Impurities D and E. Liquid chromatography (2.2.29).

Prepare the solutions immediately before use. Use freshly
prepared solutions, and store and inject them at 8 C,
using a cooled autosampler.
Solution A : 1.25 g/l solution of potassium dihydrogen
phosphate R.
Test solution. Dissolve 50.0 mg of the substance to be
and dilute immediately to 100.0 ml with solution A.
Reference solution. Dissolve 5.0 mg of allopurinol
impurity D CRS and 5.0 mg of allopurinol impurity E CRS
in 5.0 ml of a 4 g/l solution of sodium hydroxide R and
dilute immediately to 100.0 ml with solution A. Dilute 1.0 ml
of this solution to 100.0 ml with solution A.
Column :
size : l = 0.05 m, = 4.6 mm ;
stationary phase : base-deactivated octadecylsilyl silica
gel for chromatography R (3 m)(2).
Mobile phase : methanol R, 1.25 g/l solution of potassium
dihydrogen phosphate R (10:90 V/V).
Flow rate : 2 ml/min.
Injection : 20 l.
Run time : 1.5 times the retention time of impurity E.
Retention times : impurity D = about 3.6 min ;
impurity E = about 4.5 min.
System suitability : reference solution :
impurities D and E.
Limits :
impurity D : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.1 per cent),
impurity E : not more than the area of the corresponding
solution (0.1 per cent).
Impurity F. Liquid chromatography (2.2.29).
Under the following conditions, any hydrazine in the sample
reacts with benzaldehyde to give benzalazine.
Solvent mixture. Mix equal volumes of dilute sodium
hydroxide solution R and methanol R.
Solution A. Dissolve 2.0 g of benzaldehyde R in the solvent
mixture and dilute to 50.0 ml with the solvent mixture.
Test solution. Dissolve 250.0 mg of the substance to
be examined in 5 ml of the solvent mixture. Add 4 ml
of solution A, mix and allow to stand for 2.5 h at room
temperature. Add 5.0 ml of hexane R and shake for 1 min.
Allow the layers to separate and use the upper layer.
Reference solution. Dissolve 10.0 mg of hydrazine
sulphate R in the solvent mixture by sonicating for about
2 min and dilute to 50.0 ml with the solvent mixture. Dilute
1.0 ml to 20.0 ml with the solvent mixture. Dilute 1.0 ml of
this solution to 20.0 ml with the solvent mixture. To 5.0 ml
of the solution obtained, add 4 ml of solution A, mix and
allow to stand for 2.5 h at room temperature. Add 5.0 ml of
hexane R and shake for 1 min. Allow the layers to separate
and use the upper layer.
Blank solution. To 5 ml of the solvent mixture add 4 ml
of solution A, mix and allow to stand for 2.5 h at room
temperature. Add 5.0 ml of hexane R and shake for 1 min.
Allow the layers to separate and use the upper layer.
(1) Hypersil ODS is suitable.

(2) Hypersil BDS C18 is suitable.
48
Column :
size : l = 0.250 m, = 4.0 mm ;
stationary phase : cyanosilyl silica gel for
chromatography R (5 m) with a pore size of 100 nm(3) ;
temperature : 30 C.
Mobile phase : 2-propanol R, hexane R (5:95 V/V).
Injection : 20 l.
Relative retention with reference to benzaldehyde (retention
time = about 2.8 min) : benzalazine = about 0.8.
resolution : minimum 2 between the peaks due to
benzalazine and benzaldehyde ;
signal-to-noise-ratio : minimum 20 for the peak due to
benzalazine.
Limit :
impurity F : the area of the peak due to benzalazine in
the chromatogram obtained with the test solution is
not more than the area of the corresponding peak in
the chromatogram obtained with the reference solution
(2.5 ppm).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 100-105 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection: test solution (b) and reference solution (c).
Calculate the percentage content of C5H4N4O from the
declared content of allopurinol CRS.
IMPURITIES
Specified impurities : A, B, C, D, E, F.
A. R1 = NH2, R2 = H : 5-amino-1H-pyrazole-4-carboxamide,
B. R1 = NH2, R2 = CHO : 5-(formylamino)-1H-pyrazole-4carboxamide,
D. R1 = O-C2H5, R2 = H : ethyl 5-amino-1H-pyrazole-4carboxylate,
E. R1 = O-C2H5, R2 = CHO : ethyl 5-(formylamino)-1Hpyrazole-4-carboxylate,
C. 5-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide,
F. H2N-NH2 : diazane (hydrazine).
Reference: PA/PH/Exp. 10B/T (01) 67 ANP

Related substances tests A and B correspond to different
impurity profiles. As indicated in general chapter 5.10.
Control of impurities in substances for pharmaceutical
use, only the test or tests relevant for the known impurity
profile need to be carried out.
XXXX:1710
BISOPROLOL HEMIFUMARATE
Bisoprololi hemifumaras
C20H33NO6
Mr 383.5
DEFINITION
(RS)-1-[4-[[2-(1-Methylethoxy)ethoxy]methyl]phenoxy]-3-[(1methylethyl)amino]propan-2-ol hemifumarate.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Solubility : very soluble in water, freely soluble in methanol.
It shows polymorphism.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : bisoprolol hemifumarate CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness
and record new spectra using the residues.
TESTS
Related substances.
A. Impurities A, B, C, D, E and F. Liquid chromatography
(2.2.29).
examined in mobile phase A and dilute to 25.0 ml with
mobile phase A.
(3) Lichrospher 100 CN is suitable.
49
The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.
1. fumarate
3. impurity D
5. impurity F
7. impurity B
2. impurity A
4. impurity C
6. bisoprolol
8. impurity E
9. blank
Figure 1710.-1. Chromatogram for test A for related substances of bisoprolol hemifumarate : test solution spiked
with impurities A to F
bisoprolol and impurity B.
Limits :
Reference solution (b). Dissolve 5 mg of bisoprolol
hemifumarate for peak identification CRS (containing
impurity E : not more than twice the area of the
impurities D and E) in 5.0 ml of mobile phase A.
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
Column :
unspecified impurities : for each impurity, not
size : l = 0.25 m, = 4.6 mm ;
more than the area of the principal peak in the
stationary phase: octadecylsilyl silica gel for
chromatogram obtained with reference solution (a)
chromatography R (5 m)(4) ;
(0.10 per cent) ;
total : not more than 3 times the area of the principal
temperature : 30 C.
Mobile phase :
mobile phase A : mix 10 volumes of acetonitrile R1
disregard limit : 0.5 times the area of the principal
and 90 volumes of a solution containing 3.12 g/l of
sodium dihydrogen phosphate R and 0.4 ml/l of
solution (a) (0.05 per cent).
triethylamine R1 ; adjust the apparent pH to 4.2 with B. Impurities A, G, H, I, J, K, L, M, N, O, P and Q. Liquid
phosphoric acid R ;
chromatography (2.2.29).
mobile phase B : mix 25 volumes of a solution
containing 3.12 g/l of sodium dihydrogen
examined in a mixture of 20 volumes of acetonitrile R and
phosphate R and 0.4 ml/l of triethylamine R1 and
80 volumes of water for chromatography R and dilute to
75 volumes of acetonitrile R1 ; adjust the apparent pH
25.0 ml with the same mixture of solvents.
to 4.2 with a 9.8 g/l solution of phosphoric acid R ;
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with a mixture of 20 volumes of acetonitrile R
Time
Mobile phase A
Mobile phase B
and 80 volumes of water for chromatography R. Dilute
(min)
(per cent V/V)
(per cent V/V)
2.0 ml of this solution to 10.0 ml with a mixture of
0 - 40
95 10
5 90
20 volumes of acetonitrile R and 80 volumes of water for
90
40 - 45
10
chromatography R.
Reference solution (b). Dissolve 5 mg of bisoprolol for
45 - 50
10 95
90 5
system suitability CRS (containing impurity G) in a
50 - 60
95
5
mixture of 20 volumes of acetonitrile R and 80 volumes
of water for chromatography R and dilute to 5.0 ml with
a mixture of solvents.
Column :
Injection: 10 l.
size : l = 0.25 m, = 4.6 mm ;
Relative retention with reference to bisoprolol (retention
time = about 14.5 min) : impurity B = about 1.1 ;
temperature : 30 C.
impurity E = about 1.3.
to 100.0 ml with mobile phase A. Dilute 1.0 ml of this
solution to 10.0 ml with mobile phase A.
(4) Spherisorb C18, Nucleosil 100-5C18 HD are suitable.

(5) Nucleosil 100-5 C18 HD is suitable.
50
1. fumarate
4. impurity I
7. impurity N
10. impurity J
2. impurity A
5. impurities H and L
8. bisoprolol
11. impurity O
3. impurity P
6. impurity Q
9. impurity G
Figure 1710.-2. Chromatogram for test B for related substances of bisoprolol hemifumarate : test solution spiked
with impurities A, G, H, I, J, L, N, O, P, Q
Mobile phase :
mobile phase A : 10 g/l solution of phosphoric acid R ;
mobile phase B : dissolve 10 g of phosphoric acid R in
1000 ml of acetonitrile R ;
Time
(min)
0 - 35
Mobile phase A
(per cent V/V)
90 20
Mobile phase B
(per cent V/V)
10 80
35 - 40
20 90
80 10
40 - 50
90
10

Injection: 10 l.
Relative retention with reference to bisoprolol (retention
time = about 13.4 min) : impurity G = about 1.02.
peak-to-valley ratio : minimum 2.5, where Hp = height
above the baseline of the peak due to impurity G, and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
bisoprolol.
Limits :
impurity G : not more than twice the area of the
than 0.5 times the area of the principal peak in the
(0.10 per cent) ;
total: not more than 2.5 times the area of the principal
Water (2.5.12) : maximum 0.5 per cent, determined on

1.000 g.
on 1.0 g.
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 38.35 mg
of C20H33NO6.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : E, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
use) : A, B, C, D, F, H, I, J, K, L, M, N, O, P, Q.
A. R = H : (RS)-1-(4-hydroxymethyl-phenoxy)-3isopropylaminopropan-2-ol,
B. R = CH2-CH2-O-[CH2]2-CH3 : (RS)-1-isopropylamino-3-[4-(2propoxy-ethoxymethyl)phenoxy]propan-2-ol,
51
C. Ar-CH2-Ar : (RS)-1-[4-[4-(2-hydroxy-3-isopropylaminopropoxy)benzyl]phenoxy]-3-isopropylaminopropan-2-ol,
J. (2RS)-3-[4-((2-isopropoxyethoxy)methyl)phenoxy]-1,2propanediol,
D. Ar-CH2-O-CH2-Ar : (RS)-1-[4-[4-(2-hydroxy-3isopropylaminopropoxy)benzyloxylmethyl]phenoxy]-3isopropylaminopropan-2-ol,
K. 2-isopropoxyethyl 4-[((2RS)-2-hydroxy-3(isopropylamino)propyl)oxy]benzoate,
E. EZ-[3-[4-(2-isopropoxy-ethoxymethyl)phenoxy]allyl]isopropylamine,
L. R = H : 4-[((2RS)-2-hydroxy-3-(isopropylamino)propyl)oxy]benzaldehyde,
F. (RS)-2-[4-(2-isopropoxy-ethoxymethyl)phenoxy]-3isopropylaminopropan-2-ol,
P. R = OH : (2RS)-1-(isopropylamino)-3-[4-((2methoxyethoxy)methyl)phenoxy]-2-propanol,
M. 4-[(2-isopropoxyethoxy)methyl]phenol,
G. (2RS)-1-[4-(((2-isopropoxyethoxy)methoxy)methyl)phenoxy]-3-(isopropylamino)2-propanol,
N. R = C2H5 : (2RS)-1-[4-((2-ethoxyethoxy)methyl)phenoxy]-3(isopropylamino)-2-propanol,
Q. R = CH3 : (2RS)-1-(isopropylamino)-3-[4-((2methoxyethoxy)methyl]phenoxy-2-propanol,
H. 2-hydroxyethyl 4-[((2RS)-2-hydroxy-3-(isopropylamino)propyl)oxy]benzoate,
I. (2RS)-1-[4-((2-hydroxyethoxy)methyl)phenoxy]-3(isopropylamino)-2-propanol,
52
O. (2RS)-1-[4-((4-((2-isopropoxyethoxy)methyl)phenoxy)methyl)phenoxy]-3-(isopropylamino)-2-propanol.
Cefamandole nafate
Reference: PA/PH/Exp. 7/T (05) 61 ANP
Appearance of solution. Solution S is clear (2.2.1) and its

absorbance (2.2.25) at 475 nm is maximum 0.03.
pH : 6.0 to 8.0 for solution S, measured after 30 min.
The expression of content has been revised in order to
Specific optical rotation (2.2.7) : 25.0 to 33.0 35.0 to
reflect the fact that cefamandole is not to be considered
45.0 (anhydrous and sodium carbonate-free substance).

as an impurity but as a participant to the activity of the
Dissolve
1.00 g in acetate buffer solution pH 4.7 R and
substance. A new acceptance range for specific optical
dilute to 10.0 ml with the same solvent.
rotation has been included.
Readers are kindly requested to send information on
Prepare the solutions immediately before use.
the relative retentions of impurities A, C, D and E to the
Secretariat.
Solvent mixture. Mix 18 volumes of acetonitrile R and
XXXX:1402 75 volumes of a 10 per cent V/V solution of triethylamine R
previously adjusted to pH 2.5 with phosphoric acid R.
CEFAMANDOLE NAFATE
Test solution. Dissolve 0.100 g of the substance to be
examined in the solvent mixture and dilute to 10.0 ml with
the solvent mixture.
Cefamandoli nafas
Reference solution (a). Dilute 1 ml of the test solution to
10 ml with the solvent mixture, then heat at 60 C for 30 min.
Reference solution (b). Dilute 1.0 ml of the test solution to
100.0 ml with the solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
chromatography R (5 m).
Mobile phase :
triethylamine phosphate solution : dissolve 2.0 g of
sodium pentanesulphonate R in 350 ml of water R,
add 40 ml of triethylamine R, adjust to pH 2.5 with
phosphoric acid R and dilute to 700 ml with water R ;
mobile phase A : mix 1 volume of triethylamine phosphate
DEFINITION
solution and 2 volumes of water R ;
Sodium Mixture of sodium (6R,7R)-7-[[(2R)-2 mobile phase B : mix equal volumes of triethylamine
(formyloxy)-2-phenylacetyl]amino]-3-[[(1-methylphosphate solution, methanol R and acetonitrile R ;
1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate and
Time
Mobile phase A
Mobile phase B
(6R,7R)-7-[[(2R)-2-hydroxy-2-phenylacetyl]amino]-3(min)
(per cent V/V)
(per cent V/V)
[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia-10
0-1
100
azabicyclo[4.2.0]oct-2-ene-2-carboxylate (cefamandole), with
1 - 35
100 0
0 100
sodium carbonate. Semi-synthetic product derived from a
fermentation product.
0
35 - 45
100
Content :
45 - 50
0 100
100 0
cefamandole (C18H18N6O5S2) : 84.0 per cent to 93.0 per
cent (anhydrous and sodium carbonate-free substance),
cefamandole nafate (C19H17N6NaO6S2) : 93.0 per cent to
102.0 per cent (anhydrous and sodium carbonate-free
Injection : 20 l loop injector.
substance), for the sum of the content of cefamandole
Relative retention with reference to cefamandole nafate
nafate and cefamandole expressed as cefamandole nafate ; (retention time = about 24 min) : cefamandole = about 0.8.
cefamandole (C18H18N6O5S2) : maximum 9.5 per cent
System suitability : reference solution (a) :
(anhydrous and sodium carbonate-free substance) ;
sodium carbonate (Na2CO3) : 4.8 per cent to 6.4 per cent.
cefamandole and cefamandole nafate.
Limits
:
CHARACTERS
any impurity : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
Solubility : freely soluble in water, sparingly soluble in
reference solution (b) (1.0 per cent) ;
methanol.
total : not more than 5 times the area of the principal peak
IDENTIFICATION
in the chromatogram obtained with reference solution (b)
(5.0 per cent) ;
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : cefamandole nafate CRS.
(0.1 per cent).
B. It gives reaction (a) of sodium (2.3.1).
Cefamandole free acid : maximum 9.5 per cent (anhydrous
TESTS
and sodium carbonate-free substance), determined by liquid
chromatography as described under Assay.
Solution S. Dissolve 2.5 g in carbon dioxide-free water R
2-Ethylhexanoic acid (2.4.28) : maximum 0.3 per cent m/m.
and dilute to 25 ml with the same solvent.
53
Cefamandole nafate
LABELLING

The label states :

0.500 g.
Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if
intended for use in the manufacture of parenteral dosage
forms without a further appropriate procedure for the
removal of bacterial endotoxins.
that the substance contains sodium carbonate ;

where applicable, that the substance is free from bacterial
endotoxins.
IMPURITIES
ASSAY
Cefamandole Cefamandole nafate. Liquid chromatography
(2.2.29). Prepare the solutions immediately before use.
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dissolve 50.0 mg of cefamandole
nafate CRS in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (b). Dilute 1 ml of the test solution to
10 ml with the mobile phase, then heat at 60 C for 30 min.
Column :
size : l = 0.25 m, = 4.6 mm ;
A. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid (formylmandeloyl-7-amino-desacetoxycephalosporanic acid),
B. R = H : (6R,7R)-7-[[(2R)-2-hydroxy-2-phenylacetyl]amino]3-[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(cefamandole), deleted,
Mobile phase : mix 25 volumes of acetonitrile R and

75 volumes of a 10 per cent V/V solution of triethylamine R
Injection: 20 l loop injector.
System suitability :
resolution : minimum 7.0 between the 2 principal peaks in
C. (6R,7R)-7-[[(2R)-2-(acetyloxy)-2-phenylacetyl]amino]the chromatogram obtained with reference solution (b) ;
3-[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
repeatability : maximum relative standard deviation
(O-acetylcefamandole),
of 0.8 per cent after a series of single injections of a
minimum 3 freshly prepared reference solutions (a).
Calculate the percentage content of cefamandole
(C18H18N6O5S2) from the sum of the contents of cefamandole
nafate and cefamandole free acid using the declared contents
of cefamandole nafate CRS. 1 mg of cefamandole nafate is
equivalent to 0.9025 mg of cefamandole.
Calculate the percentage content of cefamandole nafate
from the sum of the content of cefamandole nafate and
D. 1-methyl-1H-tetrazole-5-thiol,
cefamandole expressed as cefamandole nafate using the
declared content of cefamandole nafate CRS.
Sodium carbonate. Dissolve 0.500 g in 50 ml of water R.
Titrate with 0.1 M hydrochloric acid, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1 M hydrochloric acid is equivalent to 10.6 mg
of Na2CO3.
STORAGE
In an airtight container, protected from light. If the
substance is sterile, store in a sterile, airtight, tamper-proof
container.
54
E. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3[(acetyloxy)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid (formylmandeloyl-7-ACA).

Chlortalidone
Reference solution (b). Dissolve 10 mg of

chlortalidone CRS and 10 mg of hydrochlorothiazide CRS
in acetone R and dilute to 10 ml with the same solvent.
The TLC test for related substances has been replaced by
Plate : TLC silica gel GF254 plate R.
an LC test which allows improved control of impurities.
Mobile phase : water R, ethyl acetate R (1.5:98.5 V/V).
The same chromatographic method is proposed for the
Application : 5 l.
assay, as practical difficulties in the titration have been
reported by users. More details on the development work is
Development : over a path of 10 cm.
presented in Pharmeuropa Scientific Notes 2005-1. Only
Drying : in air.
the first identification has been kept as the substance is
not used in pharmacies. The appearance of solution test
has been suppressed because no formulations for injection
exist on the market. The test for optical rotation has been
the chromatogram obtained shows 2 clearly separated
deleted as no data on the optical rotation of the single
spots.
enantiomer are available.
Results
: the principal spot in the chromatogram obtained
XXXX:0546
CHLORTALIDONE
reference solution (a).
D. Dissolve about 10 mg in 1 ml of sulphuric acid R. An
Chlortalidonum
intense yellow colour develops.
E. It complies with the test for optical rotation (see Tests).
TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than intensity 6 of the range of
C14H11ClN2O4S
Mr 338.8 reference solutions of the most appropriate colour (2.2.2,
Method II).
DEFINITION
Dissolve 1.0 g in dilute sodium hydroxide solution R and
2-Chloro-5-[(1RS)-1-hydroxy-3-oxo-2,3-dihydro-1H-isoindoldilute to 10 ml with the same solvent.
1-yl]benzenesulphonamide.
Acidity. Dissolve 1.0 g with heating in a mixture of 25 ml
Content : 98.0 per cent to 102.0 per cent (dried substance).
of acetone R and 25 ml of carbon dioxide-free water R.
CHARACTERS
Cool. Titrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2.2.20). Not more than 0.75 ml
Appearance : white or yellowish-white powder.
of
0.1 M sodium hydroxide is required.
Solubility : practically very slightly soluble in water, soluble
Optical rotation (2.2.7) : 0.15 to + 0.15
in acetone and in methanol, slightly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride. It Dissolve 0.20 g in methanol R and dilute to 20 ml with the
dissolves in dilute solutions of alkali hydroxides.
same solvent.
Melting point : about 220 C, with decomposition. It shows
Related substances. Examine by thin-layer chromatography
polymorphism.
(2.2.27), using as the coating substance a suitable silica gel
with a fluorescent indicator having an optimal intensity at
IDENTIFICATION
254 nm.
Test solution. Dissolve 0.2 g of the substance to be examined
Second identification : A, C, D, E.
in a mixture of 1 volume of water R and 4 volumes of
A. Dissolve 50.0 mg in ethanol (96 per cent) R and dilute
acetone R and dilute to 5 ml with the same mixture of
to 50.0 ml with the same solvent. Dilute 10.0 ml of
solvents.
this solution to 100.0 ml with ethanol (96 per cent) R.
Reference solution (a). Dissolve 4 mg of chlortalidone
Examined between 230 nm and 340 nm (2.2.25), the
impurity B CRS and 4 mg of chlortalidone CRS in a mixture
solution shows 2 absorption maxima, at 275 nm and
of 1 volume of water R and 4 volumes of acetone R and
284 nm. The ratio of the absorbance measured at the
absorption maximum at 284 nm to that measured at the dilute to 10 ml with the same mixture of solvents.
absorption maximum at 275 nm is 0.73 to 0.88.
200 ml with a mixture of 1 volume of water R and 4 volumes
of acetone R.
Preparation : discs of potassium bromide R.
Apply separately to the plate 5 l of each solution.
Comparison : chlortalidone CRS.
Develop over a path of 15 cm using a mixture of 5 volumes
If the spectra obtained show differences, dissolve the
of toluene R, 10 volumes of xylene R, 20 volumes of
substance to be examined and the reference substance
concentrated ammonia R, 30 volumes of dioxan R and
separately in methanol R, evaporate to dryness and
30 volumes of 2-propanol R. Allow the plate to dry in a
record new spectra using the residues.
current of warm air and examine in ultraviolet light at
C. Thin-layer chromatography (2.2.27).
254 nm. In the chromatogram obtained with the test
solution : any spot corresponding to impurity B is not more
examined in acetone R and dilute to 10 ml with the same intense than the corresponding spot in the chromatogram
solvent.
obtained with reference solution (a) (1 per cent) ; and
Reference solution (a). Dissolve 10 mg of
any spot, apart from the principal spot and the spot
chlortalidone CRS in acetone R and dilute to 10 ml with corresponding to impurity B, is not more intense than
the same solvent.
the spot in the chromatogram obtained with reference
55
Chlortalidone
solution (b) (0.5 per cent). The test is not valid unless the
chromatogram obtained with reference solution (a) shows
2 clearly separated spots.
Time
(min)
Solvent mixture : a 2 g/l solution of sodium hydroxide R,

mobile phase A, mobile phase B (2:48:50 V/V/V).
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 10.0 ml with the solvent mixture.
Reference solution (b). Dissolve 5 mg of chlortalidone for
peak identification CRS (containing impurities A, B, I, J, K
and L) in the solvent mixture and dilute to 50.0 ml with the
solvent mixture.
Reference solution (c). Dissolve 50.0 mg of
chlortalidone CRS in the solvent mixture and dilute
to 50.0 ml with the solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : octylsilyl silica gel for
Temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 1.32 g of ammonium
phosphate R in about 900 ml of water R and adjust to
pH 5.5 with dilute phosphoric acid R ; dilute to 1000 ml
with water R ;
mobile phase B : methanol R2 ;
0 - 16
Mobile phase A
(per cent V/V)
65
Mobile phase B
(per cent V/V)
35
16 - 21
65 50
35 50
21 - 35
50
50
35 - 40
50 65
50 35

Injection : 20 l of the test solution and reference
solutions (a) and (b).
Relative retention with reference to chlortalidone
(retention time = about 7 min) : impurity A = about 0.4 ;
impurity B = about 0.7 ; impurity J = about 0.9 ;
impurity K = about 2.7 ; impurity L = about 3.0 ;
impurity I = about 4.2.
impurity J and chlortalidone.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 1.4 ;
impurity I = 1.5 ;
impurity B : not more than 6 times the area of the
impurities J, K : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
impurity L : not more than the area of the principal peak
(0.1 per cent) ;
1. impurity A
4. chlortalidone
7. impurity Fb
10. impurity D
13. impurity I
2. impurity B
5. impurity E
8. impurity K
11. impurity C
14. impurity G
3. impurity J
6. impurity Fa
9. impurity L
12. impurity H
Figure 0546.-1 Chromatogram of a sample of chlortalidone spiked with impurities

(6) ZORBAX SB-C8 is suitable.
56
Cimetidine

total: not more than 12 times the area of the principal
(0.05 per cent).
Chlorides (2.4.4) : maximum 350 ppm.
Triturate 0.3 g, add 30 ml of water R, shake for 5 min and
filter. 15 ml of the filtrate complies with the test. Prepare
the standard using 10 ml of chloride standard solution
(5 ppm Cl) R.
on 1.0 g.
ASSAY
Dissolve 0.200 g in 50 ml of acetone R. In an atmosphere of
nitrogen, titrate with 0.1 M tetrabutylammonium hydroxide,
determining the end-point potentiometrically (2.2.20). Carry
out a blank titration.
1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent
to 33.88 mg of C14H11ClN2O4S.
related substances with the following modifications.
Injection : 20 l of the test solution and reference solution (c).
repeatability : maximum relative standard deviation of
2.0 per cent after 6 injections of reference solution (c).
Calculate the percentage content of C14H11ClN2O4S from the
declared content of chlortalidone CRS.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I, J, K, L.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : A, C, D, E, F, G, H, I.
D. R = OC2H5, R = SO2-NH2 : 2-chloro-5-[(1RS)-1-ethoxy-3oxo-2,3-dihydro-1H-isoindol-1-yl]benzenesulphonamide,

E. R = H, R = SO2-NH2 : 2-chloro-5-[(1RS)-3-oxo-2,3-dihydro1H-isoindol-1-yl]benzenesulphonamide,
G. R = OH, R = Cl : (3RS)-3-(3,4-dichlorophenyl)-3-hydroxy-2,
3-dihydro-1H-isoindol-1-one,
H. R = OCH(CH3)2, R = SO2-NH2 : 2-chloro-5-[(1RS)1-(1-methylethoxy)-3-oxo-2,3-dihydro-1H-isoindol-1yl]benzenesulphonamide,
F. bis[2-chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1yl)benzenesulphonyl]amine,
J. impurity of unknown structure with a relative retention
of about 0.9,
K. impurity of unknown structure with a relative retention
of about 2.7,
L. impurity of unknown structure with a relative retention
of about 3.0.
Reference: PA/PH/Exp. 10C/T (05) 26 ANP

The revision includes the replacement of the TLC for
related substances by 2 LC methods which allow the control
of substances synthesized by different routes and also cover
the degradation products. It is also proposed to delete the
second identification series as the substance is probably
not used in pharmacies.
XXXX:0756
CIMETIDINE
Cimetidinum
A. R = H, R = OH : 2-(4-chloro-3-sulphobenzoyl)benzoic acid,
C10H16N6S
Mr 252.3
B. R = H, R = NH2 : 2-(4-chloro-3-sulphamoylbenzoyl)benzoic
DEFINITION
acid,
2-Cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphanyl]ethyl]guanidine.
C. R = C2H5, R = NH2 : ethyl 2-(4-chloro-3sulphamoylbenzoyl)benzoate,
I. R = CH(CH3)2, R = NH2 : 1-methylethyl
2-(4-chloro-3-sulphamoylbenzoyl)benzoate,
CHARACTERS
57
Cimetidine
Solubility : slightly soluble in water, soluble in ethanol

(96 per cent), practically insoluble in methylene chloride. It
dissolves in dilute mineral acids.
Limit :
impurity F : not more than the area of the corresponding
IDENTIFICATION
(2.2.27), using silica gel GF254 R as the coating substance.
Test solution (a). Dissolve 0.50 g of the substance to be
examined in methanol R and dilute to 10 ml with the same
solvent.
A. Melting point (2.2.14) : 139 C to 144 C. If necessary,
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml
dissolve the substance to be examined in 2-propanol R,
with methanol R.
evaporate to dryness and determine the melting point
again.
Reference solution (a). Dilute 1 ml of test solution (a) to
100 ml with methanol R. Dilute 20 ml of this solution to
100 ml with methanol R.
Comparison : cimetidine CRS.
Reference solution (b). Dilute 5 ml of reference solution (a)
If the spectra obtained in the solid state show differences, to 10 ml with methanol R.
Reference solution (c). Dilute 5 ml of reference solution (b)
substance separately in 2-propanol R, evaporate to
to 10 ml with methanol R.
dryness and record new spectra using the residues.
Reference solution (d). Dissolve 10 mg of cimetidine CRS
C. Examine the chromatograms obtained in the test
in 2 ml of methanol R.
for related substances. The principal spot in the
A. Apply separately to the plate 4 l of each solution. Allow
chromatogram obtained with test solution (b) is similar
the plate to stand for 15 min in the chromatographic
in position, colour and size to the principal spot in the
tank saturated with vapour from the mobile phase which
chromatogram obtained with reference solution (d).
consists of a mixture of 15 volumes of concentrated
D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and
ammonia R, 20 volumes of methanol R and 65 volumes
5 ml of a freshly prepared 20 g/l solution of citric acid R
of ethyl acetate R and develop immediately over a path
in acetic anhydride R. Heat in a water-bath for 10 min to
of 15 cm using the same mixture of solvents. Dry the
15 min. A reddish-violet colour develops.
plate in a stream of cold air, expose to iodine vapour
until maximum contrast of the spots has been obtained
TESTS
and examine in ultraviolet light at 254 nm. Any spot
Appearance of solution. The solution is clear (2.2.1) and not
in the chromatogram obtained with test solution (a),
more intensely coloured than reference solution Y5 (2.2.2,
apart from the principal spot, is not more intense than
Method II).
reference solution (a) (0.2 per cent) and not more than
Dissolve 3.0 g in 12 ml of 1 M hydrochloric acid and dilute
two such spots are more intense than the principal
to 20 ml with water R.
spot in the chromatogram obtained with reference
chromatogram obtained with reference solution (c) shows
a clearly visible spot.
examined in the mobile phase and dilute to 50 ml with the
mobile phase.
B. Apply separately to the plate 4 l of each solution.
Reference solution. Dissolve 4.0 mg of cimetidine CRS and
of concentrated ammonia R, 8 volumes of methanol R
4.0 mg of cimetidine impurity F CRS in methanol R and
and 84 volumes of ethyl acetate R. Dry the plate in a
dilute to 50 ml with the same solvent. Dilute 1.0 ml of this
stream of cold air, expose to iodine vapour until maximum
contrast of the spots has been obtained and examine in
Column :
ultraviolet light at 254 nm. Any spot in the chromatogram
size : l = 0.25 m, = 4.6 mm ;
obtained with the test solution (a), apart from the
principal spot, is not more intense than the principal spot
(0.2 per cent) and not more than two such spots are more
Mobile phase : dissolve 661 mg of sodium
intense than the principal spot in the chromatogram
hexanesulphonate R in 760 ml of water R. Add
obtained with reference solution (b) (0.1 per cent). The
240 ml of methanol R and mix. Adjust to pH 2.5 with
test is not valid unless the chromatogram obtained with
phosphoric acid R.
reference solution (c) shows a clearly visible spot.
Liquid chromatography (2.2.29).
Injection : 20 l.
mobile phase.
Run time : 25 min.
cimetidine and impurity F ;
Reference solution (b). Dissolve 5 mg of cimetidine for peak
signal-to-noise ratio : minimum 10 for the peak due to
identification CRS in the mobile phase and dilute to 10 ml
impurity F.
with the mobile phase.
(7) Bondapack C18 is suitable.
58
Cimetidine
1. impurity G
5. cimetidine
9. impurity A
2. impurity I
6. impurity D
10. impurity H
3. impurity E
7. impurity C
4. impurity J
8. impurity B
Figure 0756.-1. Chromatogram for the test for related substances of cimetidine
Column :
size : l = 0.25 m, = 4.6 mm ;
Mobile phase : mix 250 volumes of methanol R with a
mixture of 0.4 volumes of diethylamine R and 780 volumes
of a 1.1 g/l solution of sodium hexanesulphonate R,
Injection : 50 l.
Run time : 3 times the retention time of cimetidine.
Identification of impurities : use the chromatogram
supplied with cimetidine for peak identification CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to the impurities.
Relative retention with reference to cimetidine (retention
time = about 17 min) : impurity G = about 0.26 ;
impurity E = about 0.38 ; impurity D = about 1.27 ;
impurity C = about 1.37 ; impurity B = about 1.86 ;
impurity A = about 2.28 ; impurity H = about 2.56.
impurities D and C.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity G by 0.6 ;
(1.0 per cent) ;
(0.05 per cent).
on 1.0 g.
ASSAY
Dissolve 0.200 g in 60 ml of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid determining the end-point
of C10H16N6S.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.

impurities A, B, C, D, E, G, H : for each impurity, not more or other of the tests in the monograph. They are limited
than the area of the principal peak in the chromatogram by the general acceptance criterion for other/unspecified
(0.10 per cent) ;
pharmaceutical use) : I, J.
(8) Nucleosil 100 C18 is suitable.
59
A. R1 = CN, R2 = SCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isothiourea,

B. R1 = CN, R2 = OCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isourea,
C. R1 = CO-NH2, R2 = NH-CH3 : 1-[(methylamino)[[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethyl]amino]methylene]urea,

The revision includes the replacement of the TLC for
related substances by 2 LC methods which allow the control
of substances synthesized by different routes and also cover
the degradation products. It is also proposed to delete the
second identification series as the substance is probably
not used in pharmacies.
XXXX:1500
CIMETIDINE HYDROCHLORIDE
Cimetidini hydrochloridum
D. R1 = H, R2 = NH-CH3 : 1-methyl-3-[2-[[(5-methyl-1Himidazol-4-yl)methyl]sulphanyl]ethyl]guanidine,
C10H17ClN6S
E. 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphinyl]ethyl]guanidine,
F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphanyl]ethyl]guanidine,
G. 2-cyano-1,3-dimethylguanidine,
H. 2-cyano-3-[2-[[2-[[(1Z)-(cyanoamino)(methylamino)methylene]amino]ethyl]disulphanyl]ethyl]-1-methylguanidine,
I. (4-ethyl-1H-imidazol-5-yl)methanol,
J. 2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethanamine.
60
Mr 288.8
DEFINITION
2-Cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphanyl]ethyl]guanidine hydrochloride.
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, sparingly soluble in
anhydrous ethanol.
IDENTIFICATION
First identification : B, E.
A. Dissolve 70 mg in 0.2 M sulphuric acid and dilute
to 100.0 ml with the same acid. Dilute 2.0 ml of the
solution to 100.0 ml with 0.2 M sulphuric acid. Measure
the absorbance (2.2.25) at the absorption maximum at
218 nm. The specific absorbance at the maximum is 650
to 705.
B. A. Infrared absorption spectrophotometry (2.2.24).
Comparison : cimetidine hydrochloride CRS.
chromatogram obtained with reference solution (d).
D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and
5 ml of a freshly prepared 20 g/l solution of citric acid R
in acetic anhydride R. Heat on a water-bath for 10 min to
15 min. A reddish-violet colour develops.
E. B. It gives reaction (a) of chlorides (2.3.1).
TESTS
Method II).
Dissolve 3.0 g in 12 ml of 1 M hydrochloric acid and dilute
to 20 ml with water R.
pH (2.2.3) : 4.0 to 5.0.
Dissolve 100 mg in carbon dioxide-free water R and dilute
to 10.0 ml with the same solvent.

mobile phase.
Reference solution. Dissolve 4.0 mg of cimetidine CRS and
4.0 mg of cimetidine impurity F CRS in methanol R and
dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of this
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : octadecysilyl silica gel for
Mobile phase : dissolve 661 mg of sodium
hexanesulphonate R in 760 ml of water R. Add
240 ml of methanol R and mix. Adjust to pH 2.5 with
phosphoric acid R.
Injection : 20 l.
Run time : 25 min.
cimetidine and impurity F ;
impurity F.
Limit :
impurity F : not more than the area of the corresponding
(2.2.27), using a TLC silica gel GF254 plate R.
solvent.
with methanol R.
Reference solution (a). Dilute 2 ml of test solution (b) to
100 ml with methanol R.
Reference solution (c). Dilute 5 ml of reference solution (b)

Reference solution (d). Dissolve 10 mg of cimetidine
hydrochloride CRS in 2 ml of methanol R.
A. Apply to the plate 4 l of each solution. Allow the plate
to stand for 15 min in a chromatographic tank saturated
with vapour from the mobile phase, which consists of
a mixture of 15 volumes of concentrated ammonia R,
20 volumes of methanol R and 65 volumes of ethyl
acetate R, and develop immediately over a path of 15 cm
using the same mixture of solvents. Dry the plate in a
stream of cold air, expose to iodine vapour until maximum
contrast of the spots has been obtained and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal
spot, is not more intense than the principal spot in the
(0.2 per cent) and at most two such spots are more intense
than the principal spot in the chromatogram obtained
with reference solution (b) (0.1 per cent). The test is not
valid unless the chromatogram obtained with reference
solution (c) shows a clearly visible spot.
B. Apply to the plate 4 l of each solution. Develop
over a path of 15 cm using a mixture of 8 volumes of
concentrated ammonia R, 8 volumes of methanol R and
84 volumes of ethyl acetate R. Dry the plate in a stream of
cold air, expose to iodine vapour until maximum contrast
of the spots has been obtained and examine in ultraviolet
light at 254 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not
more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) and at
most two such spots are more intense than the principal
chromatogram obtained with reference solution (c) shows
a clearly visible spot.
mobile phase.
1. impurity G
5. cimetidine
9. impurity A
2. impurity I
6. impurity D
10. impurity H
3. impurity E
7. impurity C
4. impurity J
8. impurity B
Figure 1500.-1. Chromatogram for the test for related substances of cimetidine hydrochloride
(9) Bondapack C18 is suitable.
61

Reference solution (b). Dissolve 5 mg of cimetidine for peak
identification CRS in the mobile phase and dilute to 10 ml
Column :
size : l = 0.25 m, = 4.6 mm ;
Mobile phase : mix 250 volumes of methanol R with a
mixture of 0.4 volumes of diethylamine R and 780 volumes
of a 1.1 g/l solution of sodium hexanesulphonate R,
Injection : 50 l.
Run time : 3 times the retention time of cimetidine.
supplied with cimetidine for peak identification CRS and
Relative retention with reference to cimetidine (retention
time = about 17 min) : impurity G = about 0.26 ;
impurity E = about 0.38 ; impurity D = about 1.27 ;
impurity C = about 1.37 ; impurity B = about 1.86 ;
impurity A = about 2.28 ; impurity H = about 2.56.
impurities D and C.
Limits :
the peak area of impurity G by 0.6 ;
impurities A, B, C, D, E, G, H : for each impurity, not more
(0.10 per cent) ;
total: not more than 5 times the area of the principal peak
(1.0 per cent) ;
(0.05 per cent).
on 1.0 g.
1 ml of 0.1 M sodium hydroxide is equivalent to 28.88 mg

of C10H17ClN6S.
STORAGE
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.
pharmaceutical use) : I, J.
A. R1 = CN, R2 = SCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isothiourea,

B. R1 = CN, R2 = OCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isourea,
C. R1 = CO-NH2, R2 = NH-CH3 : 1-[(methylamino)[[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethyl]amino]methylene]urea,
D. R1 = H, R2 = NH-CH3 : 1-methyl-3-[2-[[(5-methyl-1Himidazol-4-yl)methyl]sulphanyl]ethyl]guanidine,
E. 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphinyl]ethyl]guanidine,
F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethyl]guanidine,
ASSAY
Dissolve 0.200 g in a mixture of 5 ml of 0.01 M hydrochloric
acid and 50 ml of ethanol (96 per cent) R. Carry out
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
G. 2-cyano-1,3-dimethylguanidine,
(10) Nucleosil 100 C18 is suitable.
62
Cisplatin
H. 2-cyano-3-[2-[[2-[[(1Z)-(cyanoamino)(methylamino)methylene]amino]ethyl]disulphanyl]ethyl]-1-methylguanidine,
I. (4-ethyl-1H-imidazol-5-yl)methanol,
J. 2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethanamine.
Reference: PA/PH/Exp. 10D/T (04) 36 ANP 1R
B. Thin-layer chromatography (2.2.27).

Test solution. Dilute 1 ml of solution S2 (see Tests) to
10 ml with dimethylformamide R.
Reference solution. Dissolve 10 mg of cisplatin CRS in
5 ml of dimethylformamide R.
Plate : cellulose for chromatography R1 as the coating
substance.
Pretreatment: activate the plate by heating at 150 C for
1 h.
Mobile phase : acetone R, dimethylformamide R
(10:90 V/V).
Application : 2 l.
Drying : in air.
Detection : spray with a 50 g/l solution of stannous
chloride R in a mixture of equal volumes of dilute
hydrochloric acid R and water R. Examine after 1 h.
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
C. Add 50 mg to 2 ml of dilute sodium hydroxide solution R
in a glass dish. Evaporate to dryness. Dissolve the residue
in a mixture of 0.5 ml of nitric acid R and 1.5 ml of
hydrochloric acid R. Evaporate to dryness. The residue
is orange. Dissolve the residue in 0.5 ml of water R and
add 0.5 ml of ammonium chloride solution R. A yellow,
crystalline precipitate is formed.

Following the publication of the previous draft revision
in Pharmeuropa 17.1, a new LC method has been
developed to quantify transplatin (impurity A) and
TESTS
trichloroamineplatinate (impurity B) in the same run.
XXXX:0599 Solution S1. Dissolve 25 mg in a 9 g/l solution of sodium
chloride R prepared with carbon dioxide-free water R and
dilute to 25 ml with the same solvent.
CISPLATIN
Solution S2. Dissolve 0.20 g in dimethylformamide R and
Cisplatinum
Appearance of solution S1. Solution S1 is clear (2.2.1) and
not more intensely coloured than reference solution GY5
(2.2.2, Method II).
Appearance of solution S2. Solution S2 is clear (2.2.1).
pH (2.2.3) : 4.5 to 6.0 for solution S1, measured immediately
[PtCl2(NH3)2]
Mr 300.0 after preparation.
DEFINITION
(2.2.27), using cellulose for chromatography R1 as the
cis-Diaminedichloroplatinum (II).
coating substance. Activate the plate by heating at 150 C
Content : 97.0 per cent to 102.0 per cent.
for 1 h.
CHARACTERS
Test solution (a). Dilute 1 ml of solution S2 to 10 ml with
Appearance : yellow powder or yellow or orange-yellow
dimethylformamide R.
crystals.
Test solution (b). Use solution S2.
Solubility : slightly soluble in water, sparingly soluble in
dimethylformamide, practically insoluble in ethanol (96 per Reference solution (a). Dissolve 10 mg of cisplatin CRS in
5 ml of dimethylformamide R.
cent).
Reference solution (b). Dilute 1 ml of solution S2 to 50 ml
It decomposes with blackening at about 270 C.
with dimethylformamide R.
Carry out identification test B, the tests (except that for
Apply separately to the plate 2.5 l of test solution (a), 2.5 l
silver) and the assay protected from light.
of reference solution (a), 5 l of test solution (b) and 5 l of
IDENTIFICATION
reference solution (b). Develop over a path of 15 cm using
a mixture of 10 volumes of acetone R and 90 volumes of
First identification : A, B.
dimethylformamide R. Allow the plate to dry in air and
Second identification : B, C.
spray with a 50 g/l solution of stannous chloride R in a
mixture of equal volumes of dilute hydrochloric acid R and
water R. After 1 h, the chromatogram obtained with test
Comparison : cisplatin CRS.
solution (b) shows no spot with an Rf value less than that of
63
Cisplatin
the principal spot and any spot with an Rf value greater than
that of the principal spot is not more intense than the spot
in the chromatogram obtained with reference solution (b).
Liquid chromatography (2.2.29). Carry out the test protected
from light. Do not heat or sonicate any platinum-containing
solution. All solutions are to be used within 4 h.
examined in a 9.0 g/l solution of sodium chloride R and
dilute to 25.0 ml with the same solution.
Reference solution (a). Dissolve 25.0 mg of cisplatin CRS in
a 9.0 g/l solution of sodium chloride R and dilute to 25.0 ml
with the same solution.
Reference solution (b). Dissolve 5.0 mg of cisplatin
impurity A CRS in a 9.0 g/l solution of sodium chloride R
and dilute to 50.0 ml with the same solution.
Reference solution (c). Dissolve 5.6 mg of cisplatin
impurity B CRS in a 9.0 g/l solution of sodium chloride R
and dilute to 100.0 ml with the same solution.
Reference solution (d). Mix 0.050 ml of the test solution
with 5.0 ml of reference solution (b) and 5.0 ml of reference
solution (c) and dilute to 25.0 ml with a 9.0 g/l solution of
sodium chloride R.
Reference solution (e). Dilute 5.0 ml of reference solution (d)
to 20.0 ml with a 9.0 g/l solution of sodium chloride R.
Blank solution: 9.0 g/l solution of sodium chloride R.
Column :
size : l = 0.25 m, = 4.0 mm;
chromatography, base-deactivated R (4 m)(11);
temperature : 30 C.
Mobile phase: dissolve 1.08 g of sodium octanesulphonate R,
1.70 g of tetrabutylammonium hydrogen sulphate R and
2.72 g of potassium dihydrogen phosphate R in water for
chromatography R and dilute to 950 ml with the same
solvent. Adjust to pH 5.9 with 1 M sodium hydroxide and
dilute to 1000 ml with water for chromatography R.

Injection : 20 l of the test solution, reference solutions (d)
and (e), and the blank solution.
Run time : 3 times the retention time of cisplatin.
Relative retention with reference to cisplatin (retention
time = about 3.8 min): displacement peak = about 0.5 ;
impurity A = about 0.6 ; impurity B = about 0.7.
System suitability : reference solution (d) :
resolution : minimum 2.5 between the displacement
peak and the peak due to impurity A, and minimum 3.5
between the peaks due to impurities A and B.
Limits :
impurity A : not more than the area of the corresponding
solution (d) (2.0 per cent);
impurity B : not more than the area of the corresponding
solution (d) (1.0 per cent);
than 0.5 times the area of the peak due to cisplatin in
the chromatogram obtained with reference solution (d)
(0.10 per cent);
sum of impurities other than A and B : not more than
2.5 times the area of the peak due to cisplatin in the
chromatogram obtained with reference solution (d)
(0.5 per cent);
disregard limit : the area of the peak due to cisplatin in
the chromatogram obtained with reference solution (e)
(0.05 per cent).
Silver : maximum 250 2.50 102 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
examined in 15 ml of nitric acid R, heating to 80 C. Cool
and dilute to 25.0 ml with water R.
1. displacement peak
2. impurity A
3. impurity B
4. cisplatin
Figure 0599.-1. Chromatogram for the test for related substances of cisplatin: cisplatin spiked with 2 per cent
of impurity A and 1 per cent of impurity B
(11) Superspher 60 RP select B is suitable.
64
Reference solutions. To suitable volumes (10 ml to 30 ml) of

silver standard solution (5 ppm Ag) R add 50 ml of nitric
acid R and dilute to 100.0 ml with water R.
Source : silver hollow-cathode lamp, preferably with a
spectral slit width of 0.5 nm.
Wavelenth : 328 nm.
Atomisation device : fuel-lean air-acetylene flame.
Carry out a blank determination.
ASSAY
Examine by liquid chromatography (2.2.29).
examined in a 9 g/l solution of sodium chloride R and dilute
to 100.0 ml with the same solvent.
Reference solution. Dissolve 50.0 mg of cisplatin CRS in a
9 g/l solution of sodium chloride R and dilute to 100.0 ml
with the same solvent.
The chromatographic procedure may be carried out using :
a column 0.25 m long and 4.6 mm in internal diameter
packed with strong-anion-exchange silica gel for
chromatography R (10 m),
as mobile phase at a flow rate of 1.2 ml/min a mixture of
10 volumes of a 9 g/l solution of sodium chloride R and
90 volumes of methanol R,
as detector a spectrophotometer set at 220 nm.
Use a sample loop. Inject separately 20 l of the test solution
and 20 l of the reference solution.
Injection : 10 l of the test solution and reference solution (a).
Calculate the percentage content of [PtCl2(NH3)2] from the
declared content of cisplatin CRS.

It is proposed to revise the monograph to replace the TLC
by an LC in the test for related substances.
The second identification series is not needed, so it is
deleted.
XXXX:0477
CLONIDINE HYDROCHLORIDE
Clonidini hydrochloridum
C9H10Cl3N3
Mr 266.6
DEFINITION
2,6-Dichloro-N-(imidazolidin-2-ylidene)aniline.
CHARACTERS
Solubility : soluble in water and in anhydrous ethanol.
IDENTIFICATION
First identification : B, D.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 30.0 mg in 0.01 M hydrochloric
STORAGE
acid and dilute to 100.0 ml with the same acid.
Spectral range : 245-350 nm.
Absorption maxima : at 272 nm and 279 nm.
IMPURITIES
Point of inflexion : at 265 nm.
Specified impurities : A, B.
Specific absorbances at the absorption maxima :
at 272 nm : about 18,
at 279 nm : about 16.
Comparison : clonidine hydrochloride CRS.
C. Examine the chromatograms obtained in the test for
related substances.
pharmaceutical use) : C.
with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with
D. B. It gives reaction (a) of chlorides (2.3.1).
A. trans-diaminedichloroplatinum (II) (transplatin),
TESTS
B. aminetrichloroplatinate(),
C. tetrachloroplatinate(2).

Appearance of solution. Solution S is clear (2.2.1) and not
Method II).
pH (2.2.3) : 4.0 to 5.0 for solution S.
(2.2.27), using silica gel G R as the coating substance.
solvent.
65

with methanol R.
Reference solution (a). Dissolve 10 mg of clonidine
hydrochloride CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dilute 1 ml of test solution (a) to
10 ml with methanol R. Dilute 5 ml of this solution to 100 ml
with methanol R.
Apply to the plate 10 l of each solution. Shake a mixture of
10 volumes of glacial acetic acid R, 40 volumes of butanol R
and 50 volumes of water R. Allow to separate. Filter the
upper layer and use the filtrate as the mobile phase. Develop
over a path of 15 cm. Allow the plate to dry in air and
spray with potassium iodobismuthate solution R2. Allow
the plate to dry in air for 1 h, spray again with potassium
iodobismuthate solution R2 and then immediately spray
with a 50 g/l solution of sodium nitrite R. Any spot in the
chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent).
mobile phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A.
Reference solution (b). Dissolve 10 mg of clonidine
hydrochloride impurity B CRS in mobile phase A and dilute
to 10.0 ml with mobile phase A. To 1 ml of the solution, add
1 ml of the test solution and dilute to 10.0 ml with mobile
phase A.
Column :
size : l = 0.15 m, = 3.0 mm ;
stationary phase : propylsilyl silica gel for
temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 4 g of potassium dihydrogen
phosphate R in 1000 ml of water R, and adjust to pH 4.0
with phosphoric acid R ;
mobile phase B : mobile phase A, acetonitrile R
(25:75 V/V) ;
Time
(min)
0
Mobile phase A
(per cent V/V)
90
Mobile phase B
(per cent V/V)
10
0 - 15
90 30
10 70
15 - 15.1
30 90
70 10
15.1 - 20
90
10

Injection : 5 l.
clonidine and impurity B ;
symmetry factor : maximum 2.5 for the peak due to
clonidine.
Limits :
(0.10 per cent) ;
total : not more than 0.2 times the area of the principal
obtained with reference solution (a) (0.05 per cent).
on 1.0 g.
ASSAY
Dissolve 0.200 g in 70 ml of ethanol (96 per cent) R. Titrate
with 0.1 M ethanolic sodium hydroxide determining the
end-point potentiometrically (2.2.20).
of C9H10Cl3N3.
IMPURITIES
pharmaceutical use) : A, B, C.
A. 1-acetylimidazolidin-2-one,
B. 1-acetyl-N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazole2-amine,
C. 2,6-dichloroaniline.
Reagents
Silica gel for chromatography, propylsilyl. XXXXXXX.
A very finely divided silica gel (3-10 m), chemically modified
at the surface by the bonding of propylsilyl groups. The
particle size is indicated after the name of the reagent in the
test where it is used.
(12) Zorbax SB-C3 is suitable.
66
Clotrimazole
Reference: PA/PH/Exp. 10A/T (98) 68 ANP 2R

D. Dissolve about 10 mg in 3 ml of sulphuric acid R. The
solution is pale yellow. Add 10 mg of mercuric oxide R
and 20 mg of sodium nitrite R. Allow to stand with
occasional shaking. An orange colour develops, becoming
orange-brown.

A revision of the monograph is proposed to introduce
an LC for the test for related substances in replacement
of the 2 current TLCs. Another LC method was
proposed previously in Pharmeuropa, the one now
proposed allows the control of one additional impurity
(deschloroclotrimazole). It is also proposed to revise the
section Identification and to delete the test for appearance
of solution because the substance is not used in parenteral TESTS
preparations.
XXXX:0757 not more intensely coloured than reference solution BY6
(2.2.2, Method II).
CLOTRIMAZOLE
Dissolve 1.25 g in alcohol R and dilute to 25 ml with the
same solvent.
(2-Chlorophenyl)diphenylmethanol. Examine by thin-layer
Clotrimazolum
chromatography (2.2.27), using a TLC silica gel GF254 plate R.
examined in alcohol R and dilute to 5 ml with the same
solvent.
with alcohol R.
Reference solution (a). Dissolve 50 mg of clotrimazole CRS
in alcohol R and dilute to 5 ml with the same solvent.
C22H17ClN2
Mr 344.8 (2-chloro-phenyl)diphenylmethanol CRS in alcohol R and
dilute to 5 ml with the same solvent. Dilute 1 ml of the
DEFINITION
solution to 10 ml with alcohol R.
1-[(2-Chlorophenyl)diphenylmethyl]-1H-imidazole.
Apply to the plate 10 l of each solution. Develop over a path
of 15 cm using a mixture of 0.5 volumes of concentrated
ammonia R1, 10 volumes of propanol R and 90 volumes
CHARACTERS
of toluene R. Allow the plate to dry in air. Spray the plates
Appearance : white or pale yellow, crystalline powder.
with a 10 per cent V/V solution of sulphuric acid R in
Solubility : practically insoluble in water, soluble in ethanol alcohol R and heat at 100 C to 105 C for 30 min. Any spot
corresponding to (2-chlorophenyl)diphenylmethanol in the
(96 per cent) and in methylene chloride.
chromatogram obtained with test solution (a) is not more
IDENTIFICATION
intense than the spot in the chromatogram obtained with
reference solution (b) (0.2 per cent).
Imidazole. Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel G plate R.
A. Melting point (2.2.14) : 141 C to 145 C.
examined in alcohol R and dilute to 10 ml with the same
Comparison : clotrimazole CRS.
solvent.
C. Examine before spraying in ultraviolet light at 254 nm,
Reference solution. Dissolve 10 mg of imidazole R in
the chromatograms obtained in the test for related
alcohol R and dilute to 10 ml with the same solvent. Dilute
substances. The principal spot in the chromatogram
1 ml of the solution to 10 ml with alcohol R.
obtained with test solution (b) is similar in position and
of 15 cm using a mixture of 0.5 volumes of concentrated
with reference solution (a).
ammonia R1, 10 volumes of propanol R and 90 volumes of
toluene R. Allow the plate to dry in air. At the bottom of a
chromatography tank, place an evaporating dish containing
a mixture of 1 volume of hydrochloric acid R1, 1 volume of
examined in ethanol (96 per cent) R and dilute to 5 ml
water R and 2 volumes of a 15 g/l solution of potassium
Reference solution. Dissolve 50 mg of clotrimazole CRS permanganate R, close the tank and allow to stand for
15 min. Place the dried plate in the tank and close the
in ethanol (96 per cent) R and dilute to 5 ml with the
tank. Leave the plate in contact with the chlorine vapour
same solvent.
for 5 min. Withdraw the plate and place it in a current
Plate : TLC silica gel GF254 plate R.
of cold air until the excess of chlorine is removed and an
Mobile phase : concentrated ammonia R1, propanol R,
area of coating below the points of application does not
toluene R (0.5:10:90 V/V/V).
give a blue colour with a drop of potassium iodide and
starch solution R. Spray with potassium iodide and starch
Application : 10 l.
solution R. Any spot corresponding to imidazole in the
chromatogram obtained with the test solution is not more
Drying : in air.
the reference solution (0.2 per cent).
67
Clotrimazole

examined in acetonitrile R1 and dilute to 50.0 ml with the
same solvent.
100.0 ml with acetonitrile R1. Dilute 1.0 ml of this solution
to 10.0 ml with acetonitrile R1.
Reference solution (b). Dissolve 5 mg of clotrimazole for
peak identification CRS (containing impurities A, B and F)
in acetonitrile R1 and dilute to 5 ml with the same solvent.
Reference solution (c). Dissolve 5.0 mg of clotrimazole
impurity D CRS and 5.0 mg of clotrimazole impurity E CRS
in acetonitrile R1 and dilute to 100.0 ml with the same
solvent. Dilute 1.0 ml of this solution to 25.0 ml with
acetonitrile R1.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : spherical end-capped octylsilyl silica
gel for chromatography R (5 m)(13) ;
temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R and 0.5 g of tetrabutylammonium hydrogen
sulphate R1 in water R and dilute to 1000 ml with the
same solvent ;
mobile phase B : acetonitrile R1 ;
Time
(min)
0-3
Mobile phase A
(per cent V/V)
75
Mobile phase B
(per cent V/V)
25
3 - 25
75 20
25 80
25 - 30
20
80
30 - 30.1
20 75
80 25
30.1 - 40
75
25

Injection : 10 l.
Relative retention with reference to clotrimazole
(retention time = about 12 min) : impurity D = about 0.1 ;
impurity F = about 0.9 ; impurity B = about 1.1 ;
impurity E = about 1.5 ; impurity A = about 1.8.

impurity F and clotrimazole ;
the chromatogram obtained is similar to the
chromatogram supplied with clotrimazole for peak
identification CRS.
Limits :
impurities A, B : for each impurity, not more than twice
impurities D, E : for each impurity, not more than the
area of the corresponding peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ;
impurity F : not more than the area of the principal peak
(0.1 per cent) ;
(0.5 per cent) ;
(0.05 per cent).
on 1.0 g.
ASSAY
with 0.1 M perchloric acid using 0.3 ml of naphtholbenzein
solution R as indicator until the colour changes from
brownish-yellow to green.
of C22H17ClN2.
STORAGE
IMPURITIES
Specified impurities : A, B, D, E, F.
1. impurity D
3. clotrimazole
5. impurity E
2. impurity F
4. impurity B
6. impurity A
Figure 0757.-1. Chromatogram for the test for related substances of clotrimazole
(13) Zorbax Eclipse XDB- C8 is suitable.
68
2.7.28. CFC assay for human haematopoietic progenitor cells

A. R = OH : (2-chlorophenyl)diphenylmethanol,
C. R = Cl : 1-chloro-2-(chlorodiphenylmethyl)benzene,
B. 1-[(4-chlorophenyl)diphenylmethyl]-1H-imidazole,
D. imidazole,
E. (2-chlorophenyl)phenylmethanone,
F. 1-triphenylmethyl-1H-imidazole (deschloroclotrimazole).
expect that, in a steady-state situation, variations detected

in the more mature compartments will faithfully reflect
those occurring in stem cells. Thus, in a unperturbed
haematopoietic system, the presence of colony-forming cells
is an indirect indicator of a functional haematopoietic stem
cell compartment. However, all of the compartments are
controlled by different factors, some of them genetically
determined, and their ratio may vary independently from
each other, especially in the presence of disease or medical
treatments. This warrants the need for assays that evaluate
each compartment independently. All functional assays
measure 2 principal parameters : cell proliferation (measured
by the number of cells produced) and differentiation
potential (estimated by the number of different lineages
represented in its progeny).
A hierarchy of haematopoietic progenitor cells (HPCs) has
been established, based on the time required in vitro to
obtain a colony of mature cells. HPCs with a progressively
increasing proliferation capacity and a multilineage nature
will give rise to bigger colonies composed of mature cells
later in the assay. Due to physical constraints in matrix-based
assays, the HPCs with the highest proliferation capacity
can only be functionally tested in a limiting dilution of
purified cell populations in liquid cultures. When evaluating
haematopoiesis, 3 parameters have to be taken into account :
the lack of reliable correlation between the phenotype of
a given cell and its function ;
the possibility that a single assay might score together
functionally heterogeneous progenitor cells ;
the ontogeny-related changes in haematopoietic cell
proliferation and self-renewal.
CELL-SURFACE MARKERS
The capacity of colony-forming cells to give rise to
haematopoietic colonies in vitro and/or to reconstitute
the haematopoietic system has been correlated with the
expression of specific cell-surface antigens. The expression
of the membrane antigen CD34 is an accepted marker for
most of the haematopoietic progenitors and stem cells.
The expression of other antigens, such as CD33 or CD38,
can identify specific subpopulations with a restricted
lineage-differentiation capacity. Percentage contents of
colony-forming cells among human CD34+ bone marrow
cells average 15-30 per cent for CD34/CD38+ and 5 per cent
for the more immature CD34+/CD38neg population.
The total number of transplanted CD34+ cells is used as a
predictive factor of engraftment and of the expected length
of post-transplant aplasia. However, large scale sampling on
neonatal blood collections has shown a less-than-perfect
correlation between the percentage of CD34+ cells and the
functional capacity to give rise to colonies in vitro and to
engraftment in vivo. Similar results have been obtained with
the adult bone marrow, especially in individuals treated with
irradiation or chemotherapy.
COLONY ASSAY SPECIFICITY

Colony-forming cells are identified with a nomenclature
XXXX:20728 based on the lineages of mature cells present in the colony
(for example, CFU-Mix, CFU-GEMM, CFU-GM, CFU-G,
CFU-M, BFU-E, CFU-E, CFU-Meg) and are a population of
2.7.28. COLONY-FORMING
progenitors able to give rise to colonies containing one
CELL ASSAY FOR HUMAN
or more lineages of haematopoietic cells. No or very low
HAEMATOPOIETIC PROGENITOR
capacity for self-renewal has been ascribed to this population
of human HPCs compared to less-differentiated cellular
CELLS
elements. However, alternative pathways modulate the
The haematopoietic system represents a continuum of cells extent to which a single colony-forming cell will proliferate
before terminally differentiating, especially in response to
whose phenotype and properties change as they progress
stress stimuli, such as anaemia or infection.
from stem cells to differentiated cells. One could therefore
Reference: PA/PH/Exp. CTP/T (05) 27 ANP
69
2.7.28. CFC assay for human haematopoietic progenitor cells
The amount and type of growth factors supplied during

the culture partially direct the type of colonies that will be
formed. Thus, depending on the amounts and combination
of added or contaminant growth factors, different types of
progenitors may give rise to colonies that will be scored in
the same group on the basis of their morphology and cell
content.
Greater specificity on the general class of HPCs and on
their relative proliferative potential is provided by the time
required to differentiate in vitro into mature cells. The time
required by colony-forming cells to give rise to a colony
formed of mature cells in vitro differs between foetal and
adult progenitors (9-10 days and 12-14 days respectively).
Neonatal cells progenitor mature according to more adultthan foetal-like kinetics. However, neonatal progenitor cells
are characterised by a higher proliferation, and possibly
self-renewal capacity compared to the adult progenitor cells.
Differences in the sensitivity to growth factors have also
been reported between neonatal and adult progenitor cells.
Overall, the in vitro assay to assess the presence of
colony-forming cells, their number and their proliferation
capacity is a useful, although indirect, evaluation of the
overall HPC health status, and hence of their capacity to
engraft and reconstitute within a host.
MATERIALS
Matrix. Using methylcellulose as a semi-solid phase instead
of agar improves the efficiency of growth of the colonies
in this assay. This gives tighter colonies that are easier to
evaluate and count. The possibility of finely adjusting the
viscosity of the medium allows the plated cells to sediment
at the bottom of the dish and the colonies to develop
on a single plane. A methylcellulose with a viscosity of
4000 mPas is suitable. Prepare a 20 g/l solution in the
same culture medium as that used for the assay. The powder
is not easily dissolved in water and care must be taken to
obtain a product of homogeneous viscosity. The final in
vitro concentration ranges around 8-9 g/l.
Culture medium. Various media have been developed for
the clonogenic assay, the latest being the Iscoves Modified
Dulbecco Medium (IMDM), which includes the addition of
sodium selenite and HEPES to improve pH stability during
the 13-14 days required for the assay. Commercially available
media usually give more reproducible results.
Serum. Although serum-deprived culture conditions for the
CFC clonogenic assay have often been described, the absence
of detailed information on commercially available media
supports the continuing use of the serum-supplemented
version. The most common serum used as a source of
unspecified nutrients and growth factors is foetal calf serum
QUALITY ASSURANCE FOR A CFC ASSAY
It is paramount for the overall quality of the colony-forming (FCS). As discussed before it is still advisable to screen this
material to avoid skewing the results towards a specific
cell assay (CFC assay) to apply a strictly standardised
haematopoietic lineage. The use of FCS at concentrations
approach. The source of the materials, including reagents,
ranging from 20 to 40 per cent V/V has been described in the
growth factors and disposables, is codified and reference
literature.
The exact concentration needs to be determined
standards are identified. The apparatus and its condition
based
on
the
characteristics of the particular lot.
of use are clearly stated and validated over time against a
Albumin. Some formulae include the use of albumin
cellular standard. Fresh bone marrow cells from a specific
together with serum in the CFC assay. The albumin molecule
strain of laboratory animals, usually mice, are the most
suitable reference standard, as healthy animals of the same acts as an aspecific carrier for many small molecules with
a cellular-proliferation regulatory activity, such as lipids
strain, sex and age have a consistent colony-forming cell
and vitamins, and as a generic buffer. A deionised solution
content in their bone marrow. Human donors are too
of BSA obtained by the cold precipitation technique
variable to be useful as a standard, especially over a time
(Cohns Fraction V) will usually be added to obtain a final
span of years or across several laboratories.
concentration of 10 g/l.
The sources of the highest variability in the CFC assay are
the number of cells plated and the scoring procedure. Even Growth factors. Due to commercial availability of
trained individuals in the same laboratory may generate
haematopoietic growth factors, it is no longer necessary
a 5 per cent intra-laboratory variability for the same test.
to stimulate progenitor cell proliferation using a
However, if it is necessary to evaluate the colony-forming cell conditioned medium from accessory cells or cell lines. Both
content in a purified cell population, it is possible to use a
multilineage (Kit-ligand or SCF, interleukin-3, GM-CSF) and
limiting dilution approach where the number of wells positive lineage-specific (erythropoietin, G-CSF) growth factors are
for cell proliferation is measured with an automated system. required to obtain the highest number of colonies from a
population containing a mixed population of HPCs. The
The other main source of variability stems from the use of
undefined materials (for example, foetal bovine serum (FBS) amount of each growth factor must be high enough to give
the maximal stimulation of colony formation when added to
or bovine serum albumin (BSA)) in the CFC assay. These
products derive from the processing of large pools of source the assay cells alone. Concentrations of 10-100 ng/ml are
usually sufficient to reach the plateau.
materials and provide an undefined stimulation of cellular
proliferation. However, it is not uncommon to have batches Cells. Single cell suspensions are required for this assay.
with particular characteristics that selectively stimulate the In the case of bone marrow aspirates, such suspensions
proliferation of specific haematopoietic lineages.
can be obtained by forcing the bone marrow through a
sieve or through progressively smaller calibre needles.
Finally, a low level of endotoxins (less than 0.01 IU/ml
Repeated passages through a 21-gauge needle are usually
or less than 0.01 IU/mg) in all the materials used for the
sufficient to separate the bone marrow pieces into a single
clonogenic assay is advisable, as higher levels result first
cell suspension.
in a progressive skewing of the haematopoietic lineages
expression in the cultures and afterwards in a more general Most CFC assays are performed with the mononuclear
inhibition of cell proliferation and clonogenesis.
cell fraction of the sample as the presence of granulocytes
and red cells may interfere in the readout. Sterile media
CFC CLONOGENIC ASSAY
with a relative density higher than 1.077 intended for the
The CFC assay is based on the capacity of progenitor cells
separation of the mononuclear cells from the other mature
to form a colony when plated in a semi-solid medium or in a elements are commercially available.
gel in the presence of specific growth factors. Different types
of matrices may be used (for example, agar, plasma-clot and
methylcellulose) depending on the desired readout.
70
Dacarbazine
PLATING
1 ml of the solution containing the liquid medium,
the methylcellulose and the cells is usually plated in a
bacteriological sterile dish ( 35 mm).
Because of the viscosity of the medium, the solution cannot
be plated with air displacement pipettes and the use of
syringes equipped with large bore ( 18-gauge) blunt-end
needles is required.
The number of cells to be plated depends on the HPC
concentration in the sample to be tested. So that no colony
is derived from 2 different HPCs, the number of cells plated
is kept low enough to avoid an excessive number of colonies
( 100) per plate ( 35 mm). However, too low a number
of colonies ( 10) increases the measurement error. A
number of colonies per plate between 40 and 80 and a test
performed in triplicate usually allow a robust measure of the
colony-forming cell content in the sample.
The plates are incubated in aerobic conditions with a carbone
dioxide concentration of 5 per cent, at 37 C for 13-14 days,
and the number of colonies is then scored under an inverted
microscope. Care must be taken when manipulating the
dishes containing the colonies as the methylcellulose-based
medium is viscous but not jellified. An inclined plate will
result in mixed and comet-shaped colonies making the
scoring likely to be incorrect.
IDENTIFICATION OF THE COLONIES
The size and structure of the colonies depend on the type
of mature cells that are their constituents. 50 cells per
colony is usually considered a minimum. The presence of
haemoglobinised cells identifies progenitors of the erythroid
lineage. As the amount of mature cells for each lineage
largely depends on the growth factors added to the cultures,
performing differentiated counts is not recommended unless
otherwise prescribed.
Second identification : A, C.
(2.2.25).
Test solution. Dissolve 6.0 mg in 100.0 ml of 0.1 M
hydrochloric acid. Dilute 10.0 ml of this solution to
100.0 ml with 0.1 M hydrochloric acid.
Absorption maximum : at 323 nm.
Shoulder : at 275 nm.
Specific absorbance at the absorption maximum : 1067
to 1088.
Comparison : dacarbazine CRS.
examined in methanol R and dilute to 5.0 ml with the
same solvent.
Reference solution. Dissolve 2.0 mg of dacarbazine CRS
in methanol R and dilute to 5.0 ml with the same solvent.
Mobile phase : glacial acetic acid R, water R, butanol R
(1:2:5 V/V/V).
Application : 10 l.
Drying : in air.
TESTS
not more intensely coloured than reference solution BY6
(2.2.2, Method II).
Dissolve 0.5 g in a 210 g/l solution of citric acid R and dilute
XXXX:1691 to 50.0 ml with the same solution.
Impurity A. Liquid chromatography (2.2.29). Use freshly
prepared solutions and protect them from light.
DACARBAZINE
examined and 75.0 mg of citric acid R in distilled water R
Dacarbazinum
and dilute to 5.0 ml with the same solvent.
Reference solution (a). Dissolve 5.0 mg of dacarbazine
impurity A CRS in distilled water R and dilute to 50.0 ml
with the same solvent. Dilute 5.0 ml of this solution to
25.0 ml with distilled water R.
Reference solution (b). Dissolve 5.0 mg of dacarbazine
impurity B CRS in distilled water R and dilute to 10.0 ml
with the same solvent. Dilute 1.0 ml of this solution to
C6H10N6O
Mr 182.2 50.0 ml with distilled water R.
Column :
DEFINITION
size : l = 0.25 m, = 4.5 mm ;
5-(3,3-Dimethyltriazeno)imidazole-4-carboxamide.
substance).
Mobile phase : a 15.63 g/l solution of glacial acetic acid R
CHARACTERS
containing 2.33 g/l of sodium dioctyl sulfosuccinate R. As
Appearance : white or slightly yellowish crystalline powder. the mobile phase contains sodium dioctyl sulfosuccinate, it
must be freshly prepared every day, and the column must be
Solubility : slightly soluble in water and in anhydrous
flushed with a mixture of equal volumes of methanol R and
ethanol.
water R after all tests have been completed or at the end of
IDENTIFICATION
the day, for at least 2 h.
(14) Nucleosil or Symmetry C18 are suitable.
71
Dacarbazine
Temperature :
Injection: 25 l of the test solution and reference solution (a).

Run time : 3 times the retention time of impurity A.
2.0 per cent after 5 injections.
Limit :
impurity A : not more than the area of the principal peak
(0.2 per cent).
Impurity D. Head-space gas chromatography (2.2.28).
Test solution. Introduce 200.0 mg of the substance to be
examined into a 20 ml vial and firmly fix the septum and cap.
Using a 10 l syringe, inject 5 l of water R into the vial.
Reference solution (a). Dilute 556.2 mg of dimethylamine
solution R (impurity D) to 25.0 ml with water R (solution A).
Firmly affix the septum and cap to a 20 ml vial. Using a 10 l
syringe, inject 10 l of solution A into the vial.
Reference solution (b). Firmly affix the septum and cap to a
20 ml vial. Using a 10 l syringe, inject 10 l of solution A
and 10 l of a 10 g/l solution of triethylamine R into the vial.
Column :
material : fused silica ;
size : l = 30.0 m, = 0.53 mm ;
stationary phase : base-deactivated polyethylene glycol R
(film thickness 1.0 m)(15).
Carrier gas : helium for chromatography R.
Split flow : 5.1 ml/min.
Split ratio : 1:1.
Static head-space conditions that may be used:
equilibration temperature : 60 C ;
equilibration time : 10 min ;
transfer-line temperature : 90 C ;
pressurisation time : 30 s.
Column
Time
(min)
0-3
Temperature
(C)
35
3 - 11
35 65
Injection port
180
Detector
220
Detection : flame ionisation.

Injection : 1 l.
impurity D and triethylamine.
Limit :
Related substances. Liquid chromatography (2.2.29) as
described in the test for impurity A with the following
modifications.
Mobile phase : mix 45 volumes of a 15.63 g/l solution of
glacial acetic acid R containing 2.33 g/l of sodium dioctyl
sulfosuccinate R with 55 volumes of methanol R.
Injection : 10 l of the test solution and reference solution (b).
2.5 per cent after 6 injections.
Limits :
(0.1 per cent) ;
obtained with reference solution (b) (0.10 per cent) ;
(0.5 per cent) ;
(0.05 per cent).
1. impurity A
2. impurity C
Figure 1691.-1. Chromatogram for the test for impurity A of dacarbazine

(15) Stabilwax-DB (Restek, N10855) is suitable.
72
1. impurity B
2. dacarbazine
Figure 1691.-2. Chromatogram for the test for related substances of dacarbazine
2.0 g complies with test A. Prepare the reference solution
0.500 g.
on 1.0 g.
ASSAY
of C6H10N6O.
IMPURITIES
Specified impurities : A, B, D.
D. dimethylamine.
Reagents
Sodium dioctyl sulfosuccinate. C20H37NaO7S. (Mr 444.6).
XXXXXXX. [577-11-7].
White or almost white, waxy solid.
Dimethylamine solution. XXXXXXX.
A 400 g/l solution.
Clear colourless solution.
Density : about 0.89.
bp : about 54 C.
mp : about 37 C.
Base-deactivated polyethylene glycol. XXXXXXX.
Stationary phase for gas chromatography.
Cross-linked base-deactivated polyethylene glycol specially
designed for amine analysis.
Reference: PA/PH/Exp. 13B/T (02) 65 ANP 1R

The monograph has already been published in
Pharmeuropa 16.1 ; it is republished to introduce a new
method for the assay. Only comments on this updated
assay are requested.
XXXX:1871
A. 2-azahypoxanthine,
DEVILS CLAW DRY EXTRACT

Harpagophyti extractum siccum
B. 5-amino-1H-imidazole-carboxamide,
C. 5-diazoimidazol-4-carboxamide,
DEFINITION
Dry extract obtained from Devils claw root (1095).
Content : minimum 1.5 per cent of harpagoside (C24H30O11 ;
Mr 494.5) (dried extract).
PRODUCTION
The extract is produced from the herbal drug by an
appropriate procedure using either water or a hydroalcoholic
solvent equivalent in strength to a maximum of 95 per
cent V/V ethanol.
73
CHARACTERS
The following chromatogram is shown for information but

will not be published in the European Pharmacopoeia.
Appearance : light brown powder.
IDENTIFICATION
Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the extract to be examined add
10 ml of methanol R and heat at 60 C in a water-bath for
10 min. Cool and filter.
Reference solution. Dissolve 1.0 mg of harpagoside R and
2.5 mg of fructose R in 1.0 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, methanol R, ethyl acetate R
(8:15:77 V/V/V).
Application : 20 l, as bands.
Drying : in a current of warm air.
Detection : spray with a 10 g/l solution of phloroglucinol R
in ethanol (96 per cent) R and then with hydrochloric
acid R ; heat at 80 C for 5-10 min and examine in daylight.
1. harpagoside
Figure 1871.-1. Chromatogram for the harpagoside assay

of Devils claw dry extract
Results : see below the sequence of the zones present in the

chromatograms obtained with the reference solution and the Column :
test solution. Furthermore, other zones may be present in
size : l = 0.10 m, = 4.0 mm ;
the chromatogram obtained with the test solution.
Top of the plate
_______
Harpagoside : a green zone
_______
A green zone (harpagoside)
_______
_______
A yellow zone
A light green zone
Fructose : a yellowish-grey zone
A yellowish-grey zone may be

present (fructose)
A brown zone
Reference solution
Test solution
TESTS

Mobile phase : methanol R, water R (50:50 V/V).
Injection : 10 l.
Run time : 3 times the retention time of harpagoside.
Retention time : harpagoside = about 7 min.
Calculate the percentage content of harpagoside using the
following expression :
Loss on drying (2.8.17) : maximum 5.0 per cent.
ASSAY
Test solution. Transfer 0.350 g of the extract to be examined F1
to a 100 ml volumetric flask, add 90 ml of methanol R and
sonicate for 20 min. After cooling down to room temperature F2
dilute to 100.0 ml with methanol R and filter through a
membrane filter (0.2 m).
m1
Reference solution. Dissolve 0.015 g of harpagoside R in
m2
100.0 ml of methanol R. Dilute 5.0 ml of this solution to
10.0 ml with methanol R.
74
=
=
=
=
area of the peak due to harpagoside in the

chromatogram obtained with the test solution ;
area of the peak due to harpagoside in the
chromatogram obtained with the reference
solution ;
mass of the extract to be examined, in grams ;
mass of harpagoside R in the reference solution,
in grams.
Diethylcarbamazine citrate

A revision proposal was published in Pharmeuropa 16.2.
It is now proposed to use the LC method for the related
substances test and the assay. The TLC for impurities A and
B is maintained as these impurities are not UV absorbent.
XXXX:0271
DIETHYLCARBAMAZINE CITRATE
Diethylcarbamazini citras
C16H29N3O8
Mr 391.4
DEFINITION
N,N-Diethyl-4-methylpiperazine-1-carboxamide dihydrogen
2-hydroxypropane-1,2,3-tricarboxylate.
Content : 98.0 per cent to 101.0 per cent 102.0 per cent
(dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder,
slightly hygroscopic.
Solubility : very soluble in water, soluble in ethanol (96 per
cent), practically insoluble in acetone.
mp : about 138 C, with decomposition.
IDENTIFICATION
First identification : A, C.
Comparison : diethylcarbamazine citrate CRS.
B. Examine the chromatograms obtained in the test for
impurities A and B.
with reference solution (a).
C. Dissolve 0.1 g in 5 ml of water R. The solution gives the
reaction of citrates (2.3.1).
TESTS
Solution S. Shake 2.5 g with water R until dissolved and
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY6 (2.2.2, Method II).
Dimethylpiperazine and methylpiperazine Impurities A
and B. Thin-layer chromatography (2.2.27).
in methanol R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 0.1 g of diethylcarbamazine
citrate CRS in methanol R and dilute to 2.0 ml with the
same solvent.

methylpiperazine R in methanol R and dilute to
Reference solution (c). Dissolve 10 mg of
dimethylpiperazine R in methanol R and dilute to
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, methyl ethyl
ketone R, methanol R (5:30:65 V/V/V).
Application : 10 l.
Drying : at 100-105 C.
Detection : expose to iodine vapour for 30 min.
Limits :
impurity A : any spot due to impurity A is not more
impurity B : any spot due to impurity B is not more
reference solution (c) (0.2 per cent).
Solution A. Dissolve 31.24 g of potassium dihydrogen
phosphate R in water R and dilute to 1000 ml with the same
solvent.
Test solution (a). Suspend 0.300 g of the substance to
be examined in solution A and dilute to 100.0 ml with
solution A. Filter or centrifuge and use the clear filtrate or
supernatant.
Test solution (b). Dissolve 5.0 mg of the substance to be
examined in solution A and dilute to 50.0 ml with solution A.
Reference solution (a). Dilute 1.0 ml of test solution (a) to
100.0 ml with solution A. Dilute 1.0 ml of this solution to
10.0 ml with solution A.
Reference solution (b). Dissolve 10 mg of citric acid R in
solution A and dilute to 10 ml with solution A.
Reference solution (c). To 3 ml of test solution (a) add 0.5 ml
of strong hydrogen peroxide solution R and heat at 80 C
for 3 h. Dilute to 100.0 ml with solution A.
Reference solution (d). Dissolve 5.0 mg of
diethylcarbamazine citrate CRS in solution A and
dilute to 50.0 ml with solution A.
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 m)(16).
Mobile phase : mix 100 volumes of methanol R and
900 volumes of a 10 g/l solution of potassium dihydrogen
phosphate R.
Injection : 20 l of test solution (a) and reference
solutions (a), (b) and (c).
Run time : twice the retention time of diethylcarbamazine.
obtained with reference solution (b) to identify the peak due
to the citrate.
Relative retention with reference to diethylcarbamazine
(retention time = about 7 min) : citrate = about 0.2.
System suitability : reference solution (c) :
diethylcarbamazine and the degradation product.
(16) Kromasil C18 is suitable.
75
Dimenhydrinate
Limits :
(0.5 per cent) ;
(0.05 per cent) ; disregard the peak due to the citrate.
12 ml of solution S complies with test A. Prepare the
reference solution using 10 ml of lead standard solution
(2 ppm Pb) R.
on 1.000 g by drying in vacuo at 60 C for 4 h.
on 1.0 g.
ASSAY
Dissolve 0.350 g in 25 ml of anhydrous acetic acid R and
add 25 ml of acetic anhydride R. Using 0.2 ml of crystal
violet solution R as indicator, titrate with 0.1 M perchloric
acid until a greenish-blue colour is obtained.
of C16H29N3O8.
Injection : test solution (b) and reference solution (d).
Calculate the percentage content of C16H29N3O8 from the
declared content of diethylcarbamazine citrate CRS.
STORAGE
In an airtight container.
IMPURITIES
DEFINITION
Content :
diphenhydramine [2-(diphenylmethoxy)-N,Ndimethylethylamine] (C17H21NO, Mr 255.4) : 53.0 per cent
to 55.5 per cent (dried substance) ;
8-chlorotheophylline [8-chloro-3,7-dihydro-1,3-dimethyl1H-purine-2,6-dione] (C7H7ClN4O2, Mr 214.6) : 44.0 per
cent to 46.5 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : slightly soluble in water, freely soluble in ethanol
(96 per cent).
IDENTIFICATION
First identification : C.
Second identification : A, B, D.
B. Dissolve 0.1 g in a mixture of 3 ml of water R and 3 ml of
ethanol (96 per cent) R, add 6 ml of water R and 1 ml
of dilute hydrochloric acid R and cool in iced water for
30 min, scratching the wall of the tube with a glass rod if
necessary to initiate crystallisation. Dissolve about 10 mg
of the precipitate obtained in 1 ml of hydrochloric acid R,
add 0.1 g of potassium chlorate R and evaporate to
dryness in a porcelain dish. A reddish residue is obtained
which becomes violet-red when exposed to ammonia
vapour.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : dimenhydrinate CRS.
D. Dissolve 0.2 g in 10 ml of ethanol (96 per cent) R. Add
10 ml of picric acid solution R and initiate crystallisation
by scratching the wall of the tube with a glass rod. The
precipitate, washed with water R and dried at 100-105 C,
melts (2.2.14) at 130 C to 134 C.
TESTS
colourless (2.2.2, Method II).
Dissolve 1.0 g in ethanol (96 per cent) R and dilute to 20 ml
A. R = H : 1-methylpiperazine,
pH (2.2.3) : 7.1 to 7.6 for the filtrate.
B. R = CH3 : 1,4-dimethylpiperazine.
To 0.4 g add 20 ml of carbon dioxide-free water R, shake for
2 min and filter.
Theophylline and substances related to diphenhydramine.
Examine by thin-layer chromatography (2.2.27), using silica
gel
GF254 R as the coating substance.
It is proposed to revise the monograph to replace the TLC Test solution. Dissolve 0.40 g of the substance to be
examined in methylene chloride R and dilute to 10 ml with
by an LC in the test for related substances.
XXXX:0601 the same solvent.
Reference solution (a). Dissolve 20 mg of theophylline R in
methylene chloride R and dilute to 100 ml with the same
DIMENHYDRINATE
solvent.
Dimenhydrinatum
100 ml with methylene chloride R. Dilute 10 ml of this
solution to 100 ml with methylene chloride R.
Apply separately to the plate 5 l of each solution. Develop
over a path of 15 cm using a mixture of 1 volume of
concentrated ammonia R, 9 volumes of methanol R and
90 volumes of methylene chloride R. Dry the plate in
a current of cold air and examine in ultraviolet light at
254 nm. Any spot corresponding to theophylline in the
chromatogram obtained with the test solution is not more
C24H28ClN5O3
Mr 470.0 intense than the spot in the chromatogram obtained with
76
Dimenhydrinate
reference solution (a) (0.5 per cent). Spray with potassium

iodobismuthate solution R. Allow the plate to dry in air and
spray with dilute hydrogen peroxide solution R. Any spot
in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot
(0.5 per cent). Disregard any spot extending from the
starting point to an Rf of about 0.1.
the mobile phase.

on 1.000 g by drying in vacuo.
on 1.0 g.
ASSAY
Diphenhydramine. Dissolve 0.200 g in 60 ml of anhydrous
acetic acid R. Titrate with 0.1 M perchloric acid, determining
the end-point potentiometrically (2.2.20).
of C17H21NO.
8-Chlorotheophylline. To 0.800 g add 50 ml of water R, 3 ml

Reference solution. Dilute 1.0 ml of the test solution to
100.0 ml with the mobile phase. Dilute 1.0 ml of this solution of dilute ammonia R1 and 0.6 g of ammonium nitrate R
and heat on a water-bath for 5 min. Add 25.0 ml of 0.1 M
silver nitrate and continue heating on a water-bath for
15 min with frequent swirling. Cool, add 25 ml of dilute
Column :
nitric acid R and dilute to 250.0 ml with water R. Filter
and discard the first 25 ml of the filtrate. Using 5 ml of
size : l = 0.25 m, = 4.6 mm ;
ferric ammonium sulphate solution R2 as indicator, titrate
100.0 ml of the filtrate with 0.1 M ammonium thiocyanate
stationary phase : diisopropyl cyanopropylsilyl silica
until a yellowish-brown colour is obtained.
gel for chromatography R (5 m)(17).
Mobile phase. Mix 37 volumes of acetonitrile R and
63 volumes of a solution prepared as follows : mix
0.5 volumes of triethylamine R and 99.5 volumes of a
freshly prepared 1.4 g/l solution of potassium dihydrogen
phosphate R and adjust to pH 3.0 with phosphoric acid R.
Injection : 10 l.
Run time : 4 times the retention time of diphenhydramine.
Retention time : diphenhydramine = about 6 min ;
8-chlorotheophylline = about 5 min.
1 ml of 0.1 M silver nitrate is equivalent to 21.46 mg

of C7H7ClN4O2.
IMPURITIES
pharmaceutical use) : A, B.
System suitability : test solution :

diphenhydramine and 8-chlorotheophylline.
A. theophylline,
Limits :
unspecified impurities : not more than the sum of the
areas of the 2 principal peaks in the chromatogram
obtained with the reference solution (0.10 per cent) ;
total: not more than 5 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with the
reference solution (0.5 per cent) ;
disregard limit : 0.5 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with the
reference solution (0.05 per cent).
Dissolve 2.5 g in a mixture of 15 volumes of water R and
85 volumes of acetone R and dilute to 25 ml with the same
mixture of solvents. The solution complies with test B.
Prepare the reference solution using lead standard solution
(2 ppm Pb) prepared by diluting lead standard solution
(100 ppm Pb) R with a mixture of 15 volumes of water R
and 85 volumes of acetone R.
B. 8-[(2-(diphenylmethoxy)ethyl)(methyl)amino]-1,3dimethyl-3,7-dihydro-1H-purine-2,6-dione.
Reagents
Silica gel for chromatography, diisopropyl cyanopropylsilyl.
XXXXXXX.
A very finely divided silica gel chemically modified at the
surface by the bonding of diisopropyl cyanopropylsilyl
groups. The particle size is indicated after the name of the
reagent in the tests where it is used.
(17) Nucleosil-CN or Zorbax SB-CN is suitable.
77
Dipyridamole

A revision of the test for related substances is proposed to
replace the isocratic LC by a gradient LC that allows the
control of additional impurities.
XXXX:1199
DIPYRIDAMOLE
Dipyridamolum
C24H40N8O4
Mr 504.6
DEFINITION
2,2,2,2-[[4,8-Di(piperidin-1-yl)pyrimido[5,4-d]pyrimidine2,6-diyl]dinitrilo]tetraethanol.
CHARACTERS
Appearance : bright yellow, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone, soluble in anhydrous ethanol. It dissolves in dilute
mineral acids.
IDENTIFICATION
First identification : C.
Second identification : A, B, D.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 10 mg in a mixture of 1 volume of
0.1 M hydrochloric acid and 9 volumes of methanol R
and dilute to 50.0 ml with the same mixture of solvents.
Dilute 5.0 ml of this solution to 100.0 ml with a mixture
of 1 volume of 0.1 M hydrochloric acid and 9 volumes
of methanol R.
Absorption maxima: at 232 nm and 284 nm.
Absorbance ratio : A284/A232 = 1.25 to 1.45.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : dipyridamole CRS.
D. Dissolve about 5 mg in a mixture of 0.1 ml of nitric acid R
and 2 ml of sulphuric acid R. An intense violet colour
is produced.
TESTS
Related substances. Examine by liquid chromatography
(2.2.29).
Test solution. Dissolve 10.0 mg in the mobile phase and
dilute to 20.0 ml with the mobile phase.

Reference solution (b). Dissolve 10.0 mg of diltiazem
hydrochloride CRS in the mobile phase and dilute to 10.0 ml
with the mobile phase. Dilute 1.0 ml of this solution to
20.0 ml with reference solution (a).
a stainless steel column 0.25 m long and 4.6 mm in
internal diameter packed with octylsilyl silica gel for
as mobile phase at a flow rate of 1.3 ml/min a mixture
prepared as follows : dissolve 0.504 g of potassium
dihydrogen phosphate R in 370 ml of water R and
adjust to pH 3.0 with phosphoric acid R ; add 80 ml of
acetonitrile R and 550 ml of methanol R,
as detector a spectrophotometer set at 290 nm,
maintaining the temperature of the column at 30 C.
Inject 20 l of each solution and continue the
chromatography of the test solution for nine times the
retention time of dipyridamole. The test is not valid unless,
in the chromatogram obtained with reference solution (b),
the resolution between the peaks corresponding respectively
to diltiazem and dipyridamole is at least 2.0. In the
chromatogram obtained with the test solution : the area of
any peak, apart from the principal peak is not greater than
the area of the peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ; and the sum of the
areas of all the peaks, apart from the principal peak, is not
greater than twice the area of the peak in the chromatogram
obtained with reference solution (a) (1 per cent). Disregard
any peak with an area less than 0.1 times that of the peak in
the chromatogram obtained with reference solution (a).
solvent.
to 100.0 ml with methanol R. Dilute 1.0 ml of this solution
to 10.0 ml with methanol R.
Reference solution (b). Dissolve 4 mg of dipyridamole
impurity D CRS in methanol R and dilute to 10 ml with
the same solvent. To 1 ml of this solution, add 200 mg of
the substance to be examined and dilute to 100 ml with
methanol R.
Reference solution (c). Dissolve 5 mg of dipyridamole for
peak identification CRS (containing impurities A, B, C, D
and E) in methanol R and dilute to 10 ml with the same
solvent.
Column :
size : l = 0.10 m, = 4.0 mm ;
stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 m)(18) ;
temperature : 45 C.
Mobile phase :
phosphate R in 900 ml of water R, adjust to pH 7.0 with
0.5 M sodium hydroxide and dilute to 1000 ml with
water R ;
mobile phase B : methanol R ;
(18) Nucleosil C18 is suitable.
78
Dipyridamole
1. impurity B
3. impurity D
5. impurity E
2. impurity F
4. dipyridamole
6. impurity C
7. impurity A
Figure 1199.-1. Chromatogram for the test for related substances of dipyridamole
Time
(min)
0-1
Mobile phase A
(per cent V/V)
40
Mobile phase B
(per cent V/V)
60
1 - 15
40 5
60 95
15 - 20
5 40
95 60
20 - 25
40
60

Injection : 5 l.
supplied with dipyridamole for peak identification CRS
and the chromatogram obtained with reference solution (c)
to identify the peaks due to impurities A, B, C, D and E.
Relative retention with reference to dipyridamole
(retention time = about 6 min) : impurity B = about 0.3 ;
impurity D = about 0.9 ; impurity E = about 1.3 ;
impurity C = about 1.5 ; impurity A = about 2.2.
impurity D and dipyridamole ;
symmetry factor : maximum 2.5 for the peak due to
dipyridamole.
Limits :
the peak area of impurity B by 1.7 ;
total : not more than 10 times the area of the principal

(0.05 per cent).
To 0.250 g add 10 ml of water R and shake vigorously. Filter,
rinse the filter with 5 ml of water R and dilute to 15 ml with
water R.
on 1.0 g.
ASSAY
Dissolve 0.400 g in 70 ml of methanol R. Titrate with
0.1 M perchloric acid, determining the end-point
of C24H40N8O4.
STORAGE
IMPURITIES
Specified impurities : A, B, C, D, E.
impurities A, B, C : for each impurity, not more than
5 times the area of the principal peak in the chromatogram Other detectable impurities (the following substances
impurities D, E : for each impurity, not more than twice
than the area of the principal peak in the chromatogram See also 5.10. Control of impurities in substances for
pharmaceutical use) : F, G.
79
A.
B.
C.
D.
E.
F.
(2.2.25).
Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M
hydrochloric acid.
R1 = R2 = R3 = NC5H10, R4 = N(CH2-CH2-OH)2 :
2,2-[[4,6,8-tri(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-2Spectral range : 230-350 nm.
yl]nitrilo]diethanol,
R1 = R2 = R4 = N(CH2-CH2-OH)2, R3 = NC5H10 :
2,2,2,2,2,2-[[8-(piperidin-1-yl)pyrimido[5,4to 150.
d]pyrimidine-2,4,6-triyl]trinitrilo]hexaethanol,
R1 = R3 = NC5H10, R2 = Cl, R4 = N(CH2-CH2-OH)2 :
Preparation : discs of potassium chloride R.
2,2-[[2-chloro-4,8-di(piperidin-1-yl)pyrimido[5,4Comparison : dopamine hydrochloride CRS.
d]pyrimidin-6-yl]nitrilo]diethanol,
C. Dissolve about 5 mg in a mixture of 5 ml of 1 M
R1 = R3 = NC5H10, R2 = N(CH2-CH2-OH)2, R4 = NH-CH2hydrochloric acid and 5 ml of water R. Add 0.1 ml
CH2-OH : 2,2-[[6-[(2-hydroxyethyl)amino]-4,8-dipiperidin-1of sodium nitrite solution R containing 100 g/l of
ylpyrimido[5,4-d]pyrimidin-2-yl]imino]diethanol,
ammonium molybdate R. A yellow colour develops which
becomes red on the addition of strong sodium hydroxide
R1 = R4 = N(CH2-CH2-OH)2, R2 = R3 = NC5H10 :
solution R.
2,2,2,2-[(6,8-dipiperidin-1-ylpyrimido[5,4-d]pyrimidineD. Dissolve about 2 mg in 2 ml of water R and add 0.2 ml
2,4-diyl)dinitrilo]tetraethanol,
of ferric chloride solution R2. A green colour develops
R1 = NC5H10, R2 = R4 = N(CH2-CH2-OH)2, R3 = NH-CH2which changes to bluish-violet on the addition of 0.1 g of
CH2-OH : 2,2,2,2-[[4-[(2-hydroxyethyl)amino]hexamethylenetetramine R.
8-piperidin-1-ylpyrimido[5,4-d]pyrimidine-2,6E. It gives reaction (a) of chlorides (2.3.1).
diyl]dinitrilo]tetraethanol,
G. R1 = R3 = NC5H10, R2 = R4 = Cl : 2,6-dichloro-4,8dipiperidin-1-ylpyrimido[5,4-d]pyrimidine.

It is proposed to replace the current TLC test for related
substances by an LC test. In addition, the limits of
content have been tightened based on batch data, and the
recommendations for storage have been supplemented.
XXXX:0664
DOPAMINE HYDROCHLORIDE
Dopamini hydrochloridum
C8H12ClNO2
Mr 189.6
DEFINITION
4-(2-Aminoethyl)benzene-1,2-diol hydrochloride.
Content : 98.0 99.0 per cent to 102.0 101.0 per cent (dried
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder
Solubility : freely soluble in water, soluble in ethanol (96 per
cent), sparingly soluble in acetone and in methylene chloride.
IDENTIFICATION
80
TESTS
not more intensely coloured than reference solution B6 or
Y6 (2.2.2, Method II).
Dissolve 0.4 g in water R and dilute to 10 ml with the same
solvent.
Acidity or alkalinity. Dissolve 0.5 g in carbon dioxide-free
water R and dilute to 10 ml with the same solvent. Add
0.1 ml of methyl red solution R and 0.75 ml of 0.01 M
sodium hydroxide. The solution is yellow. Add 1.5 ml of
0.01 M hydrochloric acid. The solution is red.
solvent.
Reference solution (a). Dissolve 7.5 mg of
4-O-methyldopamine hydrochloride R in methanol R and
Reference solution (b). Dissolve 7.5 mg each
of 3-O-methyldopamine hydrochloride R and
4-O-methyldopamine hydrochloride R in methanol R and
of 15 cm using a mixture of 2 volumes of anhydrous formic
acid R, 7 volumes of water R, 36 volumes of methanol R
and 52 volumes of chloroform R. Allow the plate to dry in
air for 15 min. Spray evenly and abundantly with a mixture
of equal volumes of potassium ferricyanide solution R and
ferric chloride solution R1, prepared immediately before
use. Any spot in the chromatogram obtained with the test
solution with an Rf value higher than that of the principal
spot is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.25 per cent). The
test is not valid unless the chromatogram obtained with
reference solution (b) shows two clearly separated spots.

Protect the solutions from light.
Buffer solution. Dissolve 21 g of citric acid R in 200 ml of
1 M sodium hydroxide and dilute to 1000 ml with water R.
To 600 ml of this solution add 400 ml of 0.1 M hydrochloric
acid.
mobile phase A.
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
3-O-methyldopamine hydrochloride R (impurity B)
and 10 mg of 4-O-methyldopamine hydrochloride R
(impurity A) in mobile phase A and dilute to 100.0 ml with
mobile phase A. Dilute 6.0 ml of this solution to 25.0 ml with
mobile phase A.
Column :
size : l = 0.15 m, = 3.9 mm ;
silica gel for chromatography R (4 m)(19).
Mobile phase :
mobile phase A : dissolve 1.08 g of sodium
octanesulphonate R in 880 ml of the buffer solution and
add 50 ml of methanol R and 70 ml of acetonitrile R ;
mobile phase B : dissolve 1.08 g of sodium
octanesulphonate R in 700 ml of the buffer solution and
add 100 ml of methanol R and 200 ml of acetonitrile R ;
Time
(min)
0 - 5.0
Mobile phase A
(per cent V/V)
90
Mobile phase B
(per cent V/V)
10
5.0 - 20.0
90 40
10 60
20.0 - 25.0
40
60

Injection : 10 l.
Retention time : dopamine = about 5 min.

impurities B and A.
Limits :
unspecificied impurities : for each impurity, not more
total : not more than twice the area of the principal peak
(0.2 per cent) ;
(0.05 per cent).
on 1.000 g by drying in an oven at 100-105 C for 2 h.
on 1.0 g.
ASSAY
In order to avoid overheating in the reaction medium, mix
thoroughly throughout the titration and stop the titration
immediately after the end-point has been reached.
Dissolve 0.1500 g in 10 ml of anhydrous formic acid R. Add
50 ml of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
of C8H12ClNO2.
STORAGE
In an airtight container, under nitrogen, protected from light.
IMPURITIES
1. dopamine
2. impurity B
3. impurity A
4. impurity C
Figure 0664.-1. Chromatogram for the test for related substances of dopamine hydrochloride
(19) Novapak C18 is suitable.
81
TESTS
Impurity A. Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile R, glacial acetic acid R,
1,1-dimethylethyl methyl ether R (3:10:87 V/V/V).
Test solution. In a centrifuge tube, dissolve 20.0 mg of the
substance to be examined in 4 ml of dilute ammonia R4,
add 4 ml of ethyl acetate R, and mix. Separate the organic
layer and transfer it to a separate centrifuge tube. Add
4 ml of ethyl acetate R to the aqueous layer, mix, separate
the organic layer, and combine it with the 1st extract.
A. 5-(2-aminoethyl)-2-methoxyphenol (4-O-methyldopamine), Evaporate the combined organic layers to dryness in a water
bath at 50 C under a stream of nitrogen R. Dissolve the
residue in 3 ml of acetonitrile R, add 0.06 ml (3 drops) of
(S)-(-)--methylbenzyl isocyanate R, and heat in a water
bath at 50 C for 5 min. Evaporate to dryness in a water
bath at 50 C under a stream of nitrogen R. Dissolve the
B. 4-(2-aminoethyl)-2-methoxyphenol (3-O-methyldopamine), residue in 10 ml of the solvent mixture.
Reference solution. In a centrifuge tube, dissolve 18.0 mg of
dorzolamide hydrochloride CRS and 2.0 mg of dorzolamide
impurity A CRS in 4 ml of dilute ammonia R4, and proceed
as indicated for the test solution beginning with add 4 ml
of ethyl acetate R, and mix.
C. 2-(3,4-dimethoxyphenyl)ethanamine.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : silica gel for chromatography R
(5 m), with a pore size of 8 nm, a specific area of
180 m2/g and a porosity of 60 per cent(20).
XXXX:2359 Mobile phase : water R, acetonitrile R, heptane R,
1,1-dimethylethyl methyl ether R (0.2:2:35:63 V/V/V/V).
DORZOLAMIDE HYDROCHLORIDE Flow rate : 2 ml/min.
Injection : 10 l.
Dorzolamidi hydrochloridum
Run time : twice the retention time of dorzolamide.
Relative retention with reference to dorzolamide (retention
time = about 10 min) : impurity A = about 1.4.
dorzolamide and impurity A.
Calculate the percentage content of impurity A using the
C10H17N2O4S3Cl
Mr 360.9
DEFINITION
Hydrochloride of (4S,6S)-4-(ethylamino)-5,6-dihydro6-methyl-4H-thieno[2,3-]thiopyrane-2-sulphonamide
7,7-dioxide.
CHARACTERS
Solubility : soluble in water, slightly soluble in methanol,
very slightly soluble in anhydrous ethanol.
area of the peak due to impurity A in the

chromatogram obtained with the test solution,
area of the peak due to dorzolamide in the
chromatogram obtained with the test solution.
Limit :
impurity A : maximum 0.5 per cent.
IDENTIFICATION
mobile phase A.
Reference solution (a). Dissolve 1.0 ml of the test solution to
Comparison : dorzolamide hydrochloride CRS.
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
If the spectra obtained in the solid state show differences, to 10.0 ml with mobile phase A.
dissolve the substance to be examined and the reference Reference solution (b). Dissolve 2 mg of dorzolamide
substance separately in methanol R, evaporate to dryness hydrochloride for system suitability CRS (containing
impurities B, C and D) in mobile phase A and dilute to 2 ml
B. It gives reaction (a) of chlorides (2.3.1).
with mobile phase A.
(20) Zorbax Rx-SIL is suitable.
82
1. impurity D
2. impurity C
3. dorzolamide
4. impurity B
Figure 2359.-1. Chromatogram for the test for related substances of dorzolamide hydrochloride : test solution spiked
with impurities B, C and D
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: end-capped octadecylsilyl silica gel
for chromatography R (5 m)(21) ;
temperature : 35 C.
Mobile phase :
mobile phase A : mix 65 ml of acetonitrile R and 935 ml of
a 3.7 g/l solution of potassium dihydrogen phosphate R ;
mobile phase B : acetonitrile R ;
Time
(min)
0 - 15
Mobile phase A
(per cent V/V)
100
Mobile phase B
(per cent V/V)
0
15 - 30
100 50
0 50
30 - 37
50 100
50 0
37 - 44
100

on 1.000 g by drying in an oven at 105 C.
on 1.0 g.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric
acid and 50 ml of ethanol (96 per cent) R, using sonication
if necessary. Carry out a potentiometric titration (2.2.20),
using 0.1 M sodium hydroxide. Read the volume added
between the 1st and the 3rd point of inflexion.
of C10H17N2O4S3Cl.
IMPURITIES
Specified impurities : A, C.
pharmaceutical use) : B, D.

Injection: 10 l.
Relative retention with reference to dorzolamide
(retention time = about 11 min) : impurity C = about 0.9 ;
impurity B = about 1.1.
impurity C and dorzolamide ;
peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity B and
dorzolamide.
Limits :
impurity C : not more than the area of the principal peak A. (4R,6R)-4-(ethylamino)-5,6-dihydro-6-methyl-4H-thieno
[2,3-b]thiopyran-2-sulphonamide 7,7-dioxide,
(0.1 per cent) ;
total: not more than twice the area of the principal peak
(0.2 per cent) ;
in the chromatogram obtained with reference solution (a) B. (4RS,6SR)-4-(ethylamino)-5,6-dihydro-6-methyl-4H(0.05 per cent).
thieno[2,3-b]thiopyran-2-sulphonamide 7,7-dioxide,
(21) Inertsil ODS-2 and Kromasil C18 are suitable.
83
CHARACTERS
hygroscopic.
Solubility : freely soluble in water, soluble in ethanol (96 per
cent).
It melts at about 202 C.
C. [2-[[(4S,6S)-2-(aminosulphonyl)-6-methyl-7,7-dihydro-4Hthieno[2,3-b]thiopyran-4-yl]amino]ethyl]boronic acid,
D. (4S,6S)-4-(amino)-5,6-dihydro-6-methyl-4H-thieno
[2,3-b]thiopyran-2-sulphonamide 7,7-dioxide.
Reagents
Ammonia, dilute R4. XXXXXXX.
Content : 8.4 g/l to 8.6 g/l of NH3 (Mr 17.03).
Dilute 3.5 g of concentrated ammonia R to 100 ml with
water R.
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
Comparison : ethambutol hydrochloride CRS.
Examine the substances prepared as discs.
2-aminobutanol impurity A.
with test solution (b) is similar in position, colour and size
reference solution (b).
C. Dissolve 0.1 g in 10 ml of water R and add 0.2 ml of
copper sulphate solution R. Add 0.5 ml of dilute sodium
hydroxide solution R. A blue colour is produced.
D. B. It gives reaction (a) of chlorides (2.3.1).
TESTS
pH (2.2.3) : 3.7 to 4.0.
Dissolve 0.2 g in 10 ml of carbon dioxide-free water R.
2-Aminobutanol Impurity A. Thin-layer chromatography
(2.2.27).
solvent.
with methanol R.
2-aminobutanol R (impurity A) in methanol R and
dilute to 10 ml with the same solvent. Dilute 1 ml of this
The control of impurities has been improved by the
introduction of an LC method which uses a derivatisation solution to 10 ml with methanol R.
with a chiral reagent. Only the first identification series
Reference solution (b). Dissolve 50 mg of ethambutol
has been kept as the substance is not used in pharmacies. hydrochloride CRS and 5 mg of 2-aminobutanol R in
The assay by optical rotation has been replaced by a
methanol R and dilute to 10 ml with the same solvent.
titration method.
XXXX:0553 Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, water R,
methanol R (10:15:75 V/V/V).
ETHAMBUTOL HYDROCHLORIDE
Application : 2 l.
Ethambutoli hydrochloridum
Drying : in air ; heat at 110 C for 10 min.
Detection : cool and spray with ninhydrin solution R1 ; heat
the plate at 110 C for 5 min.
the chromatogram shows 2 clearly separated spots.
Limit :
C10H26Cl2N2O2
Mr 277.2 impurity A : any spot corresponding to 2-aminobutanol
impurity A in the chromatogram obtained with test
DEFINITION
solution (a) is not more intense than the spot in the
2,2-(Ethylenediimino)bis[(2S)-butan-1-ol] dihydrochloride
(1.0 per cent).
(2S,2S)-2,2-(Ethane-1,2-diyldiimino)dibutan-1-ol
dihydrochloride.
Related susbstances. Liquid chromatography (2.2.29).
(S)-(-)--Methylbenzyl isocyanate. C9H9NO. (Mr 147.2).

XXXXXXX. [14649-03-7]. ((1S)-1-Nitrosoethyl)benzene.
A colourless liquid.
: about 1.044.
: about 1.514.
bp : 55 C to 56 C.
NOTE : do not use the reagent if it is coloured.
84
Test solution. Suspend 4.0 mg of the substance to be

examined in 4.0 ml of acetonitrile R1 and add 100 l of
triethylamine R. Sonicate the mixture for 5 min. Add 15 l
of R-(+)-phenylethylisocyanate R and heat the mixture for
20 min at 70 C.
100.0 ml with acetonitrile R1.
Reference solution (b). Treat 4.0 mg of ethambutol for
system suitability CRS (containing impurity B) as described
under test solution.
Column :
size : l = 0.10 m, = 4.6 mm ;
temperature : 40 C.
Mobile phase :
mobile phase A : methanol R, water R (50:50 V/V) ;
mobile phase B : methanol R ;
Time
(min)
0 - 30
Mobile phase A
(per cent V/V)
71
Mobile phase B
(per cent V/V)
29
30 - 35
71 0
29 100
35 - 37
100
37 - 38
0 71
100 29

Injection : 10 l.
Relative retention with reference to ethambutol (retention

time = about 14 min) : impurity B = 1.3.
ethambutol and impurity B.
Limits :
impurity B : not more than twice the area of the principal
unspecified impurities with a relative retention with
reference to ethambutol of 0.75 to 1.5 : for each impurity,
not more than 0.20 times the area of the peak due to
ethambutol in the chromatogram obtained with reference
(1.0 per cent) ;
disregard limit : 0.1 times the area of the peak due to
ethambutol in the chromatogram obtained with reference
solvent. 12 ml of the solution complies with test A. Prepare
the reference solution using 2 ml of lead standard solution
(1 ppm Pb) R.
on 1.0 g.
1. impurity C
2. ethambutol
3. impurity B
Figure 0553.-1. Chromatogram for the test for related substances of ethambutol hydrochloride : test solution spiked
with impurities B and C
(22) LUNA C18(2) is suitable
85
ASSAY
To 70 ml of dilute ammonia R2 add 4.0 ml of copper
sulphate solution R, mix, add 5.0 ml of dilute sodium
hydroxide solution R and dilute to 100 ml with water R.
Dissolve 0.100 g of the substance to be examined in 20 ml of
this solution and dilute to 25.0 ml with the same solution.
Prepare a reference solution in the same manner using
0.100 g of ethambutol hydrochloride CRS. Measure the
angle of optical rotation (2.2.7) of the solutions at 436 nm.
Calculate the content of C10H26Cl2N2O2 from the angles of
optical rotation measured and the concentrations of the
solutions.
Dissolve 0.150 g in 50 ml of water R and add 1.0 ml of 0.1 M
hydrochloric acid. Carry out a potentiometric titration
(2.2.20), using 0.1 M sodium hydroxide. Read the volume
added between the 2 points of inflection.
of C10H26Cl2N2O2.

Currently, the control of impurities of fenoterol
hydrobromide is performed using a UV-photometric test for
impurity B and an LC test for impurity A (diastereoisomers).
In the framework of the special revision programme it is
proposed to modify slightly the existing LC test conditions
to cover impurity A and other related substances. A new
detection wavelength has been chosen. Only comments
on the tests for impurity A and related substances are
requested.
XXXX:0901
FENOTEROL HYDROBROMIDE
Fenoteroli hydrobromidum
STORAGE
IMPURITIES
A. 2-aminobutan-1-ol,
B. (2R,2S)-2,2-(ethane-1,2-diyldiimino)dibutan-1-ol
(meso-ethambutol),
C. (2R,2R)-2,2-(ethane-1,2-diyldiimino)dibutan-1-ol
((R,R)-ethambutol).
Reagents
R-(+)-1-Phenylethyl isocyanate. C9H9NO. (Mr 147.18).
XXXXXXX. [33375-06-3]. R-(+)--Methylbenzyl isocyanate.
Content : minimum 98.0 per cent.
A colourless liquid.
bp : about 74 C to 76 C.
86
C17H22BrNO4
Mr 384.3
DEFINITION
(1RS)-1-(3,5-Dihydoxyphenyl)-2-[[(1RS)-2-(4-hydroxyphenyl)1-methylethyl]amino]ethanol hydrobromide.
CHARACTERS
Solubility : soluble in water and in ethanol (96 per cent).
IDENTIFICATION
(2.2.25).
Test solution. Dissolve 50.0 mg in dilute hydrochloric
acid R1 and dilute to 50.0 ml with the same acid. Dilute
5.0 ml to 50.0 ml with dilute hydrochloric acid R1.
Shoulder : at about 280 nm.
to 86.
Comparison : Ph. Eur. reference spectrum of fenoterol
hydrobromide.
examined in ethanol (96 per cent) R and dilute to 10 ml
Reference solution. Dissolve 10 mg of fenoterol
hydrobromide CRS in ethanol (96 per cent) R and dilute
to 10 ml with the same solvent.
Mobile phase : concentrated ammonia R, water R,
aldehyde-free methanol R (1.5:10:90 V/V/V).
Application : 2 l.
Drying : in air.
Detection : spray with a 10 g/l solution of potassium
permanganate R.
D. Dissolve about 10 mg in a 20 g/l solution of disodium
tetraborate R and dilute to 50 ml with the same solution.
Add 1 ml of a 10 g/l solution of aminopyrazolone R,
10 ml of a 2 g/l solution of potassium ferricyanide R
and 10 ml of methylene chloride R. Shake and allow to
separate. A reddish-brown colour develops in the lower
layer.
E. It gives reaction (a) of bromides (2.3.1).
Column :
size : l = 0.25 m 0.15 m, = 4.6 mm;
chromatography R (5 m to 10 m)(23).
Mobile phase. Dissolve 24 g of disodium hydrogen
phosphate R in 1000 ml of water R and adjust to pH 8.5
with phosphoric acid R. Mix 69 volumes of this solution
with 1 volume of a 9 g/l solution of potassium of hydrogen
phosphate R and 35 volumes of methanol R2. to a mixture
of 1 volume of a 9 g/l solution of potassium dihydrogen
phosphate R and 69 volumes of a 24 g/l solution of
disodium hydrogen phosphate R, adjusted to pH 8.5 using
phosphoric acid R, add 30 volumes of methanol R.
Detection : spectrophotometer at 280 nm 215 nm.
Injection : 20 l loop injector of the test solution and
TESTS
Run time : 3 times the retention time of fenoterol.
Relative retention with reference to fenoterol (retention
Appearance of solution. Solution S is clear (2.2.1) and not time = about 7 min) : impurity A = about 1.3.
Sensitivity : the height of the peak due to the
diastereoisomers eluting immediately after the principal peak
Method II).
is not less than 10 per cent of the full scale of the recorder.
Phenone : maximum 0.2 per cent. The absorbance (2.2.25)
the height of the trough separating the peak due to the
of solution S at 330 nm has a maximum of 0.42.
diastereoisomers from the principal peak is less than
Impurity A Diastereoisomers. Liquid chromatography
4 per cent of the full scale of the recorder,
(2.2.29). Prepare the solutions immediately before use.
the retention time of the principal peak is less than
20 min.
Test solution. Dissolve 25.0 mg 24.0 mg of the substance to
be examined in water R and dilute to 10.0 ml 20.0 ml with
the same solvent.
fenoterol and impurity A.
Reference solution (a). Dissolve 25.0 mg 24.0 mg of
Calculate the content of diastereoisomers by determining
fenoterol hydrobromide CRS (containing impurity A) in
the height of a perpendicular dropped from the apex of the
water R and dilute to 10.0 ml 20.0 ml with the same solvent. peak to a line drawn from the trough between the 2 peaks to
the baseline, and taking into account the declared content of
Reference solution (b). Dissolve 6 mg of fenoterol
diastereoisomers in fenoterol hydrobromide CRS.
hydrobromide for peak identification CRS (containing
Calculate the content of impurity A from the areas of the
impurities B and C) in water R and dilute to 5 ml with the
peaks and the declared content of impurity A in fenoterol
same solvent.
hydrobromide CRS.
Reference solution (c). Dilute 10.0 ml of the test solution
Limit :
to 50.0 ml with water R. Dilute 1.0 ml of this solution to
100.0 ml with water R.
impurity A : maximum 4.0 per cent.
1. fenoterol
3. impurity B
2. impurity A
4. impurity C
Figure 0901.-1. Chromatogram for the test for impurity A and for the test for related substances of fenoterol
hydrobromide
(23) Kromasil C18 is suitable.
87
Related substances. Liquid chromatography (2.2.29) as

described in the test for impurity A with the following
modifications.
Injection : 20 l of the test solution and reference
solutions (b) and (c).
Relative retention with reference to fenoterol (retention
time = about 7 min) : impurity B = about 2.0 ;
B. 1-(3,5-dihydoxyphenyl)-2-[[(1RS)-2-(4-hydroxyphenyl)-1impurity C = about 2.2.
methylethyl]amino]ethanone (phenone),
impurities B and C.
Limits :
in the chromatogram obtained with reference solution (c) C. (1RS)-1-(3,5-dihydoxyphenyl)-2-[[(1RS)-2-(4-hydroxy-3(0.2 per cent) ;
methylphenyl)-1-methylethyl]amino]ethanol.
impurity C : not more than 1.5 times the area of the
reference solution (c) (0.3 per cent) ;
Reference: PA/PH/Exp. P4/T (04) 21 ANP
chromatogram obtained with reference solution (c)
XXXX:2280
(0.10 per cent) ;
FEXOFENADINE HYDROCHLORIDE
total: maximum 0.3 per cent ;
Fexofenadini hydrochloridum
in the chromatogram obtained with reference solution (c)
(0.05 per cent) ; disregard the peak due to impurity A.
Iron (2.4.9) : maximum 5 ppm.
Dissolve the residue obtained in the test for sulphated ash
in 2.5 ml of dilute hydrochloric acid R and dilute to 10 ml
with water R.
on 1.0 g.
C32H40ClNO4
Mr 538.1
ASSAY
DEFINITION
Dissolve 0.600 g in 50 ml of water R and add 5 ml of dilute
2-[4-[(1RS)-1-Hydroxy-4-(4-hydroxydiphenylmethyl-1nitric acid R, 25.0 ml of 0.1 M silver nitrate and 2 ml of
piperidinyl)butyl]phenyl]-2-methylpropanoic
acid.
ferric ammonium sulphate solution R2. Shake and titrate
with 0.1 M ammonium thiocyanate until an orange colour Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
is obtained. Carry out a blank titration.
1 ml of 0.1 M silver nitrate is equivalent to 38.43 mg
of C17H22BrNO4.
STORAGE
IMPURITIES
Specified impurities: A, B, C.
A. (1RS)-1-(3,5-dihydoxyphenyl)-2-[[(1SR)-2-(4hydroxyphenyl)-1-methylethyl]amino]ethanol,
88
CHARACTERS
Solubility : slightly soluble in water, freely soluble in
methanol, very slightly soluble in acetone.
IDENTIFICATION
Comparison : fexofenadine hydrochloride CRS.
B. Dissolve 30 mg in a mixture of equal volumes of
methanol R and water R. Sonicate if necessary and dilute
to 2 ml with the same mixture of solvents. The solution
gives reaction (a) of chlorides (2.3.1).
TESTS
Impurity B. Liquid chromatography (2.2.29).
the mobile phase.
Reference solution (a). Dissolve the contents of a vial of

fexofenadine hydrochloride impurity B CRS in the test
solution and dilute to 2.0 ml with the test solution.
Reference solution (b). Dilute 1.0 ml of the test solution
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: silica gel BC for chiral
chromatography R1 (5 m)(24).
Mobile phase : mix 20 volumes of acetonitrile for
chromatography R and 80 volumes of a buffer solution
prepared as follows : dilute 1.15 ml of glacial acetic acid R
to 1000 ml with water for chromatography R and adjust to
pH 4.0 0.1 with dilute ammonia R1.
Injection: 20 l.
Run time : 1.2 times the retention time of fexofenadine.
Relative retention with reference to fexofenadine (retention
time = 15-23 min) : impurity B = about 0.7.
fexofenadine and impurity B.
Limits :
(0.1 per cent).
Buffer solution. Dissolve 6.64 g of sodium dihydrogen
phosphate monohydrate R and 0.84 g of sodium
perchlorate R in water for chromatography R and dilute
to 1000.0 ml with the same solvent ; adjust to pH 2.0 0.1
with phosphoric acid R.
Solvent mixture. Mix equal volumes of acetonitrile for
chromatography R and the buffer solution.
examined in 25.0 ml of the solvent mixture.
Test solution (b). Dilute 3.0 ml of test solution (a) to 50.0 ml
Reference solution (a). Dissolve 25.0 mg of fexofenadine
hydrochloride CRS in the solvent mixture and dilute to
25.0 ml with the solvent mixture. Dilute 3.0 ml of this
Reference solution (b). Dilute 1.0 ml of test solution (a) to
Reference solution (c). Dissolve 1 mg each of fexofenadine
hydrochloride impurity A CRS and fexofenadine
hydrochloride impurity C CRS in 20 ml of reference
solution (a) and dilute to 200.0 ml with the mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : phenylsilyl silica gel for
Mobile phase : mix 350 volumes of acetonitrile for
chromatography R and 650 volumes of the buffer solution ;
add 3 volumes of triethylamine R and mix.

Injection : 20 l of test solution (a) and reference solutions (b)
and (c).
Relative retention with reference to fexofenadine
impurity D = about 2.3 ; impurity C = about 3.2.
Run time : 6 times the retention time of fexofenadine for test
solution (a) and reference solution (c), twice the retention
time of fexofenadine for reference solution (b).
fexofenadine and impurity A.
Limits :
correction factor: for the calculation of content, multiply
the peak area of impurity A by 1.4 ;
impurity A : not more than 1.8 times the area of the
impurity D : not more than the area of the principal peak
(0.1 per cent) ;
(0.3 per cent) ;
(0.05 per cent).
85 volumes of methanol R, and dilute to 20 ml with the
same mixture of solvents. 12 ml of this solution complies
with test B. Prepare the reference solution using 1 ml of lead
standard solution (10 ppm Pb) R.
Water (2.5.32) : maximum 0.5 per cent.
Dissolve 1.000 g in 5.0 ml of anhydrous methanol R. Use
1.0 ml of this solution.
on 1.0 g.
ASSAY
related substances with the following modifications.
Injection : test solution (b) and reference solution (a).
Run time : twice the retention time of fexofenadine.
Calculate the percentage content of fexofenadine
hydrochloride from the declared content of fexofenadine
hydrochloride CRS.
STORAGE
At a temperature not exceeding 30 C.
IMPURITIES
Specified impurities : A, B, C, D.
(24) Astec Cyclobond I 2000 is suitable.

(25) Agilent Technologies Zorbax SB phenyl is suitable.
89
Fluorescein

pharmaceutical use) : E, F, G.
E. 4-hydroxydiphenylmethylpiperidine,
A. 2-[4-[4-(4-hydroxydiphenylmethyl-1-piperidinyl)butanoyl]phenyl]-2-methylpropanoic acid,
G. 2-[4-[1-hydroxy-4-(4-diphenylmethylidene-1piperidinyl)butyl]phenyl]-2-methylpropanoic acid.
Reagents
Silica gel BC for chiral chromatography R1. XXXXXXX.
A very finely divided silica gel for chromatography (5 m)
coated with -cyclodextrin.
B. 2-[3-[(1RS)-1-hydroxy-4-(4-hydroxydiphenylmethyl-1piperidinyl)butyl]phenyl]-2-methylpropanoic acid,
Reference: PA/PH/Exp. 10D/T (05) 19 ANP

XXXX:2348
FLUORESCEIN
Fluoresceinum
C. (1RS)-4-[4-(hydroxydiphenylmethyl)piperidin-1-yl]-1-[4-(1methylethyl)phenylbutan-1-ol,
C20H12O5
Mr 332.3
DEFINITION
3,6-Dihydroxy-3H-spiro[2-benzofuran-1,9-xanthen]-3-one.
D. R = CH3, R = CO-OCH3 : methyl 2-[4-[(1RS)-1-hydroxy4-(4-hydroxydiphenylmethyl-1-piperidinyl)butyl]phenyl]2-methylpropanoate,

F. R = H, R = CO2H : 2-[4-[(1RS)-1-hydroxy-4-(4-hydroxydiphenylmethyl-1-piperidinyl)butyl]phenyl]propanoic acid,
90
CHARACTERS
Appearance : orange-red, fine powder.
Solubility : practically insoluble in water, soluble in hot
ethanol (96 per cent). It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
Comparison : fluorescein CRS.
Fluorescein
Reference solution (b). Dissolve 10.0 mg phthalic acid CRS

(impurity B) and 10.0 mg resorcinol CRS (impurity A) in
the solvent mixture and dilute to 100.0 ml with the solvent
mixture. Dilute 1.0 ml of this solution to 100.0 ml with the
solvent mixture.
Reference solution (c). Dilute 5.0 ml of test solution (b) to
B. Dilute 0.1 ml of solution S (see Tests) to 10 ml
with water R. The solution shows a yellowish-green
fluorescence. The fluorescence disappears on addition of Reference solution (d). Dilute 10.0 ml of reference
solution (c) to 100.0 ml with the solvent mixture.
0.1 ml of dilute hydrochloric acid R and reappears on
addition of 0.2 ml of dilute sodium hydroxide solution R. Column :
size : l = 0.25 m, = 4.6 mm ;
C. The absorption by a piece of filter paper of 0.05 ml of
solution prepared for identification B (before the addition stationary phase : octylsilyl silica gel for
chromatography R3 (5 m)(26) ;
of dilute hydrochloric acid R) colours the paper yellow.
On exposing the moist paper to bromine vapour for 1 min temperature : 35 C.
and then to ammonia vapour, the colour becomes deep
Mobile phase :
pink.
phosphate R in water for chromatography R, adjust to
pH 2.0 with phosphoric acid R and dilute to 1000.0 ml
TESTS
with water for chromatography R ;
Solution S. Suspend 1.0 g in 35.0 ml of water R and add
mobile
phase B : acetonitrile for chromatography R ;
dropwise with shaking 4.5 ml of 1 M sodium hydroxide.
Adjust to pH 8.5-9.0 with 1 M sodium hydroxide and dilute
Time
Mobile phase A
Mobile phase B
to 50.0 ml with water R to obtain a clear solution.
(min)
(per cent V/V)
(per cent V/V)
If the spectra obtained in the solid state show differences,
substance separately in the minimum volume of ethanol
(96 per cent) R, evaporate to dryness and record new
spectra using the residues.
Appearance of solution. Solution S is clear (2.2.1) and

orange-yellow with yellowish-green fluorescence.
0 - 20
85 20
15 80
20 - 29
20
80
29 - 30
20 85
80 15
Solvent mixture : acetonitrile for chromatography R, mobile

phase A (30:70 V/V).
30 - 40
85
15

solutions (b), (c) and (d).
Test solution (b). Dilute 5.0 ml of test solution (a) to 250.0 ml Relative retention with reference to fluorescein (retention
with the solvent mixture.
time = about 15 min) : impurity A = about 0.42 ;
Reference solution (a). Dissolve 50.0 mg of fluorescein CRS impurity B = about 0.48 ; impurity C = about 0.86.
in 15.0 ml of ethanol (96 per cent) R. Sonicate and dilute
to 50.0 ml with water R. Dilute 5.0 ml of this solution to
impurities A and B.

examined in 15.0 ml of ethanol (96 per cent) R. Sonicate
and dilute to 50.0 ml with the solvent mixture.
1. impurity A
2. impurity B
3. impurity C
4. fluorescein
Figure 2348.-1. Chromatogram for the test for related substances of fluorescein spiked with impurities A, B and C
(26) Zorbax SBC8 is suitable.
91
Limits :
the peak area of impurity C by 1.9 ;
impurities A, B : for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with reference solution (b) (0.1 per cent) ;
reference solution (c) (0.6 per cent) ;

XXXX:1918
FLUORODOPA (18F) INJECTION

Fluorodopae (18F) solutio iniectabilis

(0.10 per cent) ;
sum of impurities other than A, B and C : not more
(0.2 per cent) ;
DEFINITION
Sterile solution of (2S)-2-amino-3-[2-([18F]fluoro)-4,5dihydroxyphenyl]propanoic acid (6-[18F]fluorolevodopa). It
may
contain stabilisers such as ascorbic acid and edetic acid.
disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (d)
This monograph applies to an injection containing
(0.05 per cent).
6-[18F]fluorolevodopa produced by electrophilic substitution.
Chlorides (2.4.4) : maximum 0.25 per cent.
Content : 90 per cent to 110 per cent of the declared
To 10.0 ml of solution S add 90.0 ml of water R and 1.0 ml of fluorine-18 radioactivity at the date and time stated on the
dilute nitric acid R, wait for at least 10 min and filter. Dilute label.
Content of levodopa : maximum 1 mg per maximum
10.0 ml of the filtrate to 15.0 ml with water R.
recommended dose in milliliters.
Content of 6-fluorolevodopa: maximum 15 mg per maximum
recommended dose in milliliters.
ASSAY
Injection: test solution (b) and reference solution (a).
Calculate the percentage content of C20H12O5 from the

declared content of fluorescein CRS.
STORAGE
IMPURITIES
Specified impurities : A, B, C.
A. resorcinol,
B. benzene-1,2-dicarboxylic acid (phthalic acid),
C. 2-(2,4-dihydroxybenzoyl)benzoic acid.
92
PRODUCTION
RADIONUCLIDE PRODUCTION
Fluorine-18 is a radioactive isotope of fluorine 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 fluorine-18 in a chemical form suitable for
electrophilic substitution reactions as fluorine gas or gaseous
acetylhypofluorite, a small amount of non-radioactive
fluorine gas (0.3-0.8 per cent of the target gas volume) must
be added as a carrier at some step in the production process.
RADIOCHEMICAL SYNTHESIS
6-[18F]Fluorolevodopa may be prepared by various
radiochemical synthetic pathways, which lead to different
products in terms of yields, specific radioactivity, by-products
and possible impurities. Electrophilic pathways for
production of 6-[18F]fluorolevodopa may proceed by
fluorodemetalation of a stannylated derivative of levodopa,
with molecular [18F]fluorine or [18F]acetylhypofluorite,
followed by hydrolysis of protecting groups and final
purification by semipreparative liquid chromatography.
Pathways using demercuration and dethallation must not
be used.
CHARACTERS
Appearance : clear, colourless solution.
Half-life and nature of radiation of fluorine-18 : see Table of
physical characteristics of radionuclides (5.7).
IDENTIFICATION
A. Test A for radionuclidic purity (see Tests).
B. Half-life : 105 min to 115 min, determined by 3
measurements of the activity of a sample in the same
geometrical conditions at intervals of about 15 min.
radiochemical purity (see Tests).
impurity B : not more than the area of the peak due to

Results : the principal peak in the radiochromatogram
levodopa in the chromatogram obtained with reference
obtained with the test solution is similar in retention time
solution (b) (1.0 mg/V) ;
to the peak due to 6-fluorolevodopa in the chromatogram
obtained with reference solution (a).
impurity C : not more than the area of the corresponding
TESTS
solution (c) (0.05 mg/V) ;
pH (2.2.3) : 4.0 to 5.5.
Sterility. It complies with the test for sterility prescribed
in the monograph on Radiopharmaceutical preparations
solution (e) (0.5 mg/V).
(0125). The injection may be released for use before
Residual solvents are limited according to the principles
completion of the test.
defined in the general chapter (5.4), using the general
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml,
method (2.4.24). The preparation may be released for use
V being the maximum recommended dose in millilitres. The before completion of the test.
injection may be released for use before completion of the
RADIONUCLIDIC PURITY
test.
Fluorine-18 : minimum 99.9 per cent of the total
Impurities A, B, C and D. Liquid chromatography (2.2.29).
radioactivity.
Prepare the reference solutions immediately before use.
The preparation may be released for use before completion
Test solution. The preparation to be examined.
of test B.
Reference solution (a). Dissolve 15 mg of fluorodopa
A. Gamma-ray spectrometry.
impurity A CRS in 5 ml of the mobile phase and dilute
Results : the only gamma photons have an energy of
to V ml with the mobile phase, V being the maximum
0.511 MeV and, depending on the measurement geometry,
recommended dose in millilitres.
a sum peak of 1.022 MeV may be observed.
Reference solution (b). Dissolve 1.0 mg of levodopa R in 5 ml
B.
Gamma-ray spectrometry.
of the mobile phase and dilute to V ml with the mobile phase,
V being the maximum recommended dose in millilitres.
Determine the amount of fluorine-18 and radionuclidic
impurities with a half-life longer than 2 h. For the
Reference solution (c). Dissolve 1 mg of 6-hydroxydopa
detection and quantification of impurities, retain the
hydrochloride R(27) in 5 ml of the mobile phase and dilute
preparation to be examined for a sufficient time to
0.25 ml of this solution to V ml with the mobile phase,
allow the fluorine-18 to decay to a level that permits the
V being the maximum recommended dose in millilitres.
detection of impurities.
Reference solution (d). Mix equal volumes of reference
Results : the spectrum obtained with the preparation to
be examined does not differ significantly from that of a
Reference solution (e). Dissolve 1 mg of trimethyltin
background spectrum.
chloride R in 2 ml of the mobile phase and dilute 1.0 ml of
RADIOCHEMICAL PURITY
this solution to V ml with the mobile phase, V being the
maximum recommended dose in millilitres.
impurities A, B, C and D.
Column :
Examine the chromatogram recorded using the radioactivity
size : l = 0.25 m, = 4.0 mm ;
detector and locate the peak due to 6-[18F]fluorolevodopa by
stationary phase: spherical end-capped octadecylsilyl
comparison
with the chromatogram obtained with reference
silica gel for chromatography R(28) ;
solution (a).
temperature : maintain at a constant temperature between Limit :
20 C and 30 C.
6-[18F]fluorolevodopa : minimum 95 per cent of the total
Mobile phase : a 6.9 g/l solution of sodium dihydrogen
radioactivity due to fluorine-18.
phosphate R adjusted to pH 2.4 with a 4.8 g/l solution of
Enantiomeric purity, impurities E and F. Thin-layer
phosphoric acid R.
chromatography (2.2.27).
Test solution. The preparation to be examined.
Detection : spectrophotometer at 200 nm and radioactivity
Reference solution (a). Dissolve 2 mg of DL-fluorodopa R(29)
detector connected in series.
in water R and dilute to 10 ml with the same solvent.
Injection: 20 l.
Reference
solution (b). Dissolve 2 mg of fluorolevodopa R
Run time : 15 min.
in water R and dilute to 10 ml with the same solvent.
Relative retention with reference to impurity A
Plate : TLC octadecylsilyl silica gel plate for chiral
(retention time = about 7 min) : impurity C = about 0.7 ;
separations R(30).
impurity B = about 0.8.
Mobile phase : methanol R, water R (50:50 V/V).
Application : 2 l.
impurities B and C.
Drying : in air for 5 min.
Limits : examine the chromatograms obtained with the
spectrophotometer :
Detection : spray with a 2 g/l solution of ninhydrin R
impurity A : not more than the area of the corresponding in anhydrous ethanol R and heat at 60 C for 10 min.
Determine the distribution of radioactivity using a suitable
solution (a) (15 mg/V) ;
detector.
(27)
(28)
(29)
(30)
Available from RBI and Sigma-Aldrich.

Merck Rp-18e is suitable.
The substance is available from ABX Germany.
Chiralplate from Macherey-Nagel is suitable.
93
Fluorouracil
Retention factors : impurity E = about 0 ;

[18F]fluorodopa = about 0.34 ; impurity F = about 0.56.
the chromatogram shows two clearly separated spots.
Limits :
[18F]fluorolevodopa: minimum 92 per cent of the total
radioactivity due to fluorine-18 ;
impurity E : maximum 4 per cent of the total radioactivity
due to fluorine-18 ;
impurity F : maximum 4 per cent of the total radioactivity
due to fluorine-18.

This monograph has already been published in
Pharmeuropa 16.1 to revise the test for related substances
(replacement of TLC by LC). It has been revised a second
time to reintroduce the TLC to cover 2 additional impurities
F and G, not detected by LC. Only comments on this test
are requested.
XXXX:0611
FLUOROURACIL
Fluorouracilum
RADIOACTIVITY
Measure the radioactivity using a calibrated instrument.
LABELLING
The label states the maximum recommended dose in
millilitres.
IMPURITIES
C4H3FN2O2
Mr 130.1
DEFINITION
5-Fluoropyrimidine-2,4(1H,3H)-dione.
A. (2S)-2-amino-3-[2-fluoro)-4,5-dihydroxyphenyl]propanoic
acid (6-fluorolevodopa),
B. (R,S)-2-amino-3-[2-fluoro-4,5-dihydroxyphenyl]propanoic
acid (DL-dopa),
C. (2S)-2-amino-3-(2,4,5-trihydroxyphenyl)propanoic acid
(6-hydroxydopa),
D. trimethyltin,
E. (6-[18F]fluorodextrodopa),
F. [18F]fluoride.
Reagents
6-Hydroxydopa hydrochloride. C8H11NO3.
(Mr 205.6). XXXXXXX. [28094-15-7]. 2-(2,4,5trihydroxyphenyl)ethylamine hydrochloride.
mp : about 233 C.
Trimethyltin chloride. C3H9ClSn. (Mr 199.3). XXXXXXX.
[1066-45-1].
DL-Fluorodopa.
XXXXXXX.
Fluorolevodopa. XXXXXXX.
94
CHARACTERS
Solubility : sparingly soluble in water, slightly soluble in
ethanol (96 per cent).
IDENTIFICATION
First identification : A.
Comparison : fluorouracil CRS.
related substances in ultraviolet light at 254 nm. The
principal spot in the chromatogram obtained with test
solution (b) is similar in position and size to the principal
solution (a).
C. In a test-tube, heat 0.5 ml of chromic acid cleansing
mixture R in a naked flame until white fumes appear in
the upper part of the tube. The solution wets the side of
the tube and there is no appearance of greasiness. Add
about 2 mg of the substance to be examined and heat
again in a naked flame until white fumes appear. The
solution does not wet the sides of the tube.
TESTS
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY7 or Y7
(2.2.2, Method II).
Impurities F and G. Thin-layer chromatography (2.2.27).
examined in a mixture of equal volumes of methanol R and
water R and dilute to 10.0 ml with the same mixture of
solvents.
Fluorouracil

2-ethoxy-5-fluorouracil CRS (impurity F) in a mixture of
equal volumes of methanol R and water R and dilute to
200.0 ml with the same mixture of solvents.
Reference solution (b). Dissolve 200.0 mg of urea CRS
(impurity G) in methanol R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml of the solution to 100.0 ml with
methanol R.
Mobile phase : methanol R, water R, ethyl acetate R
(15:15:70 V/V/V).
Application : 10 l.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Limit A :
impurity F : any spot due to impurity F is not more
reference solution (a) (0.25 per cent).
Detection B : spray with a mixture of 200 ml of a 10 g/l
solution of dimethylaminobenzaldehyde R in anhydrous
ethanol R and 20 ml of hydrochloric acid R ; dry in an oven
at 80 C for 3-4 min, then examine in daylight (impurity G
produces a yellow spot and fluorouracil is not detected by
the spray).
Limit B :
impurity G : any spot due to impurity G is not more
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
examined in the mobile phase and dilute to 50.0 ml with the
mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with
the mobile phase.
Reference solution (a). Dissolve 5.0 mg of fluorouracil
impurity C CRS in the mobile phase and dilute to 50.0 ml
Reference solution (b). Dilute 1.0 ml of reference solution (a)

Reference solution (c). Dissolve 5.0 mg of fluorouracil
impurity A CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 1.0 ml of the solution to
Reference solution (d). Dissolve 5.0 mg of fluorouracil
impurity B CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 1.0 ml of the solution to
Reference solution (e). Dilute 1.0 ml of the test solution
Reference solution (f). To 1 ml of reference solution (a) add
1 ml of the test solution and dilute to 10 ml with the mobile
phase.
Column :
size : l = 0.25 m, = 4.6 mm ;
Mobile phase : a 6.805 g/l solution of potassium dihydrogen
phosphate R adjusted to pH 5.7 0.1 with 5 M potassium
hydroxide.
Injection : 20 l.
Run time : 3 times the retention time of fluorouracil.
Relative retention with reference to fluorouracil
impurity B = about 0.7 ; impurity C = about 0.9 ;
impurity D = about 1.6 ; impurity E = about 1.9.
System suitability : reference solution (f) :
fluorouracil and impurity C.
1. impurity A
3. impurity C
5. impurity D
2. impurity B
4. fluorouracil
6. impurity E
Figure 0611.-1. Chromatogram for the test for related substances of fluorouracil spiked with impurities A, B, C, D, E
(31) Spherisorb ODS 2 is suitable.
95
Glucagon, human
Limits :
the corresponding correction factor : impurity D = 1.5 ;
impurity E = 1.3 ;
impurity A : not more than the area of the principal peak
(0.1 per cent) ;
in the chromatogram obtained with reference solution (d)
(0.1 per cent) ;
impurity C : not more than the area of the principal peak
(0.1 per cent) ;
impurities D, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (e) (0.1 per cent) ;
obtained with reference solution (e) (0.10 per cent) ;
in the chromatogram obtained with reference solution (e)
(0.5 per cent) ;
in the chromatogram obtained with reference solution (e)
(0.05 per cent).
1.0 g complies with test C. Use a platinum crucible. Prepare
the reference solution using 2 ml of lead standard solution
(10 ppm Pb) R.
on 1.000 g by drying in vacuo at 80 C for 4 h.
on 1.0 g in a platinum crucible.
C. R = H : pyrimidine-2,4(1H,3H)-dione (uracil),
D. R = OCH3 : 5-methoxypyrimidine-2,4(1H,3H)-dione
(5-methoxyuracil),
E. R = Cl : 5-chloropyrimidine-2,4(1H,3H)-dione
(5-chlorouracil),
F. 2-ethoxy-5-fluoropyrimidin-4(1H)-one,
G. urea.

A general revision of the monograph is proposed to
simplify the test procedures. A change in composition
of mobile phase A in the test for related proteins is also
proposed to improve the resolution between glucagon and
carbamoylglucagon.
XXXX:1635
GLUCAGON, HUMAN
Glucagonum humanum
ASSAY
Dissolve 0.100 g in 80 ml of dimethylformamide R, warming
gently. Cool and titrate with 0.1 M tetrabutylammonium
hydroxide, using 0.25 ml of a 10 g/l solution of thymol
blue R in dimethylformamide R as indicator. Carry out a
blank titration.
1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent C H N O S
Mr 3483
153 225 43 49
to 13.01 mg of C4H3FN2O2.
DEFINITION
STORAGE
Polypeptide having the same structure (29 amino acids)
as the hormone produced by the -cells of the human
pancreas, which increases the blood-glucose concentration
by promoting rapid breakdown of liver glycogen.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G.
substance).
A. X1 = H2, X2 = O : pyrimidine-2,4,6(1H,3H,5H)-trione
(barbituric acid),
B. X1 = O, X2 = H2 : dihydropyrimidine-2,4,5(3H)-trione
(isobarbituric acid or 5-hydroxyuracil),
96
PRODUCTION
Human glucagon is produced by a method based on
recombinant DNA (rDNA) technology. During the course
of product development it must be demonstrated that
the manufacturing process produces a product having a
biological activity of not less than 1 IU/mg using a suitable
validated bioassay, based on hyperglycaemia measurement.
Host-cell-derived proteins : the limit is approved by the
competent authority.
Host-cell- and vector-derived DNA : the limit is approved
by the competent authority.
Glucagon, human
CHARACTERS
Solubility : practically insoluble in water and in most
organic solvents, soluble in dilute mineral acids and in dilute
solutions of alkali hydroxides.
IDENTIFICATION
A. Peptide mapping. Liquid chromatography (2.2.29).
Test solution. Prepare a 10 5 mg/ml solution of the
substance to be examined in 0.01 M hydrochloric
acid. Mix 200 l of the solution with 800 l of 0.1 M
ammonium carbonate buffer solution pH 10.3 R (diluted
stock solution). Freshly pPrepare a 2.0 mg/ml solution
of -chymotrypsin for peptide mapping R in 0.1 M
ammonium carbonate buffer solution pH 10.3 R and add
25 l of this solution to the diluted stock solution of the
substance to be examined. Place the test solution in a
closed vial at 37 C for 2 h. Remove the vial and stop the
reaction immediately by the addition of 120 l of glacial
acetic acid R. The -chymotrypsin solution is stable for
12 months at 70 C or below.
Reference solution. Prepare at the same time and in
the same manner as for the test solution but using
human glucagon CRS instead of the substance to be
examined. a 1 mg/ml solution of human glucagon CRS
in 0.1 M ammonium carbonate buffer solution pH 10.3 R
(diluted stock solution) and continue as described for the
test solution.
Column :
size : l = 0.05 m, = 4 mm ;
TESTS
Specific absorbance (2.2.25) : 21 to 25, determined at the
maximum at 276 nm (anhydrous substance).
Dissolve 2.5 mg in 0.01 M hydrochloric acid and dilute to
10.0 ml with the same solvent.
Deamidated glucagon. Liquid chromatography (2.2.29) : use
the normalisation procedure.
Test solution. Dissolve the substance to be examined in
0.01 M hydrochloric acid to obtain a concentration of
1.0 mg/ml.
Resolution solution. Dissolve the substance to be examined
in 0.1 M hydrochloric acid to obtain a concentration
of 1.0 mg/ml. Incubate in an oven at 60 C for 2 h.
Immediately after degradation, adjust to pH 2.5 with 1 M
sodium hydroxide.
Column :
material : glass ;
size : l = 0.05 m, = 5 mm ;
stationary phase : anion exchange resin R2.
Mobile phase :
mobile phase A : mix 1000 ml of tris-hydrochloride buffer
solution pH 8.3 R and 1000 ml of anhydrous ethanol R ;
mobile phase B : dissolve 29.2 g of sodium chloride R in
1000 ml of tris-hydrochloride buffer solution pH 8.3 R ;
add 1000 ml of anhydrous ethanol R ;
Time
(min)
0-4
Mobile phase A
(per cent V/V)
100
Mobile phase B
(per cent V/V)
0
stationary phase: octadecylsilyl silica gel for

chromatography R (5 m )(32).
4 - 30
100 78
0 22
30 - 34
78 45
22 55
Mobile phase :
34 - 38
45 20
55 80
mobile phase A : mix 500 l of trifluoroacetic acid R

and 1000 ml of water R ;
38 - 40
20 100
80 0
40 - 60
100
mobile phase B : mix 500 l of trifluoroacetic acid R

with 600 ml of anhydrous ethanol R and add 400 ml
of water R ;

Equilibration : mobile phase A for at least 15 min.
Time
Mobile phase A
Mobile phase B
(min)
(per cent V/V)
(per cent V/V)
Injection : 60 l.
0 47
0 - 35
100 53
System suitability : resolution solution :
35 - 45
53 0
47 100
retention time : glucagon = about 10 min ; 4 deamidated
45 - 46
0 100
100 0
forms : between 15 min and 40 min ;
0
46 - 75
100
resolution : baseline separation of the 4 deamidated
forms and glucagon.
Limit :
total of the 4 deamidated forms : maximum 0.5 per cent,
calculated from the peaks eluting between 10 15 min and
Equilibration: mobile phase A for at least 15 min.
40 min.
Injection: 10 20 l.
Related proteins. Liquid chromatography (2.2.29) : use the
Results : the profile of the chromatogram obtained with
normalisation procedure.
the test solution corresponds to that of the chromatogram
2.8 M urea solution. Dissolve 16.8 g of urea R in 0.01 M
obtained with the reference solution.
hydrochloric acid and dilute to 100 ml with the same solvent
B. Examine the chromatograms obtained in the assay.
100 ml of water R.
Results : the principal peak in the chromatogram obtained Test solution. Dissolve the substance to be examined in
0.01 M hydrochloric acid to obtain a concentration of 1.0
with the test solution is similar in retention time to the
principal peak in the chromatogram obtained with the
0.5 mg/ml. Maintain the solution at 2-8 C and use within
reference solution.
24 h.
(32) Lichrosphere C-18 is suitable.
97
Reference solution. Dissolve the contents of a vial of human ASSAY

glucagon CRS in 0.01 M hydrochloric acid to obtain a
concentration of 1.0 0.5 mg/ml. Maintain the solution at
related proteins with the following modifications.
2-8 C and use within 24 h.
Injection : test solution and reference solution.
Resolution solution. Dissolve 10 mg of the substance to
Calculate the content of human glucagon (C153H225N43O49S)
be examined in 10 20 ml of 2.8 M urea solution. Adjust to
from the declared content of C153H225N43O49S in human
pH 7 with 1 M sodium hydroxide and place the sealed vial
glucagon CRS.
at about 50 C for about 2 h. Cool and adjust to pH 2.5
with 1 M hydrochloric acid. Heat at 50 C for 2 h. Cool
STORAGE
and adjust to pH 2.2 with 1 M hydrochloric acid. Maintain
In an airtight container, protected from light, at a
the solution at 2-8 C and use within 2 h or maintain the
temperature lower than 15 C.
solution below 15 C, then thaw and filter through a
0.22 m filter before use.
Column :
size : l = 0.25 m, = 4.6 mm ;
Reference: PA/PH/Exp. 13H/T (03) 60 ANP

chromatography R (5 m) with a pore size of 30 nm(33) ;
XXXX:2213
GLYCEROL MONOCAPRYLATE
temperature : 45 C.
Mobile phase :
mobile phase A : dissolve 14.2 13.6 g of anhydrous
sodium sulphate R potassium dihydrogen phosphate R
in 400 ml of water R, add 1.35 ml of phosphoric acid R
and adjust to pH 2.5 (2.2.3) with ethanolamine R
phosphoric acid R, and add 100 ml of acetonitrile for
chromatography R ;
mobile phase B : acetonitrile for chromatography R,
water R (40:60 V/V) ;
Time
(min)
0 - 23
Mobile phase A
(per cent V/V)
57
Mobile phase B
(per cent V/V)
43
23 - 29
57 10
43 90
29 - 30
10
90
30 - 31
10 57
90 43
31 - 75
57
43

Injection: 25 50 l of the test solution and the resolution
solution.
Relative retention with reference to glucagon (retention
time = about 20 min) : carbamoylglucagon = about 1.1.
System suitability : resolution solution :
retention time : glucagon = about 20 min ;
carbamoylglucagon = about 22 min,
resolution : minimum 1.3 1.5 between the peaks due to
glucagon and carbamoylglucagon.
symmetry factor : 0.6 to 1 for the peak due to glucagon.
Limit :
total of all impurities : maximum 2.5 per cent.
Water (2.5.12) : maximum 10 per cent, determined on 20.0
50 mg.
Bacterial endotoxins (2.6.14) : less than 10 IU/mg.
Glyceroli monocaprylas
DEFINITION
Mixture of monoacylglycerols, mainly monocaproylglycerol,
containing variable quantities of di- and triacylglycerols,
obtained by direct esterification of glycerol with caprylic acid.
Content :
glycerol monocaprylate (type I) :
monoacylglycerols : 45.0 per cent to 75.0 per cent ;
diacylglycerols : 20.0 per cent to 50.0 per cent ;
triacylglycerols : maximum 10.0 per cent ;
glycerol monocaprylate (type II) :
monoacylglycerols : minimum 80.0 per cent ;
diacylglycerols : maximum 20.0 per cent ;
triacylglycerols : maximum 5.0 per cent.
CHARACTERS
Appearance : colourless or slightly yellow, oily liquid or soft
mass.
Solubility : practically insoluble in water, very soluble
in ethanol (96 per cent) and freely soluble in methylene
chloride.
IDENTIFICATION
A. Composition of fatty acids (see Tests).
B. It complies with the limits of the assay (monoacylglycerols).
TESTS
Acid value (2.5.1) : maximum 4.0.
Iodine value (2.5.4, Method A) : maximum 1.0.
Composition of fatty acids (2.4.22, Method C). Use the
mixture of calibrating substances in Table 2.4.22.-2.
Composition of the fatty acid fraction of the substance :
caproic acid : maximum 1.0 per cent ;
caprylic acid : minimum 80.0 per cent ;
capric acid : maximum 20.0 per cent ;
lauric acid : maximum 1.0 per cent ;
myristic acid : maximum 0.5 per cent.
(33) Waters Symmetry C18 and Phenomenex Jupiter C18 are suitable.
98
Free glycerol : maximum 3.0 per cent.
Temperature :
Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat

to about 50 C then allow to cool. Add 100 ml of water R.
Shake and add 25.0 ml of periodic acetic acid solution R.
Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l
solution of potassium iodide R. Allow to stand for 1 min.
Add 1 ml of starch solution R. Titrate with 0.1 M sodium
thiosulfate. Carry out a blank titration.
1 ml of 0.1 M sodium thiosulfate is equivalent to 2.3 mg
of glycerol.
Column
Time
(min)
0-3
Temperature
(C)
60
3 - 38
60 340
38 - 50
340
Injection port
350
Detector
370
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g. Detection : flame ionisation.
Total ash (2.4.16) : maximum 0.5 per cent.
Injection : 1 l.
ASSAY
Gas chromatography (2.2.28) : use the normalisation
procedure.
Identification of peaks : use the chromatogram supplied

with glycerol monocaprylate CRS and the chromatogram
obtained with reference solution (a) to identify the peaks
due to mono-, di- and triacylglycerols.
Test solution. To 0.25 g of the substance to be examined,

add 5.0 ml of tetrahydrofurane R and shake to dissolve.
Reference solution (a). To 0.25 g of glycerol

monocaprylate CRS, add 5.0 ml of tetrahydrofurane R and
shake to dissolve.
resolution : minimum 5 between the peaks due to glycerol

mono-octanoate and glycerol monodecanoate.
For the calculation of content of mono-, di- and

triacylglycerols, disregard the peaks with a retention time
Reference solution (b). To 50 mg of glycerol
mono-octanoate R and 50 mg of glycerol monodecanoate R, less than that of the monoacylglycerols, which are due to
impurities of the solvent and to the free fatty acids.
add 2.5 ml of tetrahydrofurane R and shake to dissolve.
Calculate the content of free fatty acids (B) using the
Column :
size : l = 10 m, = 0.32 mm ;
stationary phase : poly(dimethyl)(diphenyl)siloxane R(34)
(film thickness 0.1 m).

IA
Split ratio : 1:50.
acid value of the substance to be examined.
1. monoacylglycerols C8
2. diacylglycerols C8
3. triacylglycerols C8
Figure 2213.-1. Chromatogram for the assay of glycerol monocaprylate
(34) OPTIMA 5 or equivalent : HP5 or CP Sil 8.
99
Calculate the content of mono-, di- and triacylglycerols using

the following expressions :
diacylglycerols : maximum 20.0 per cent ;

triacylglycerols : maximum 5.0 per cent.
CHARACTERS
Appearance : colourless or slightly yellow, oily liquid or soft
mass.
Solubility : practically insoluble in water, very soluble
in ethanol (96 per cent) and freely soluble in methylene
chloride.
IDENTIFICATION
A. Composition of fatty acids (see Tests).
B. It complies with the limits of the assay (monoacylglycerols).
percentage content of free glycerol ;
monoacylglycerols content obtained by

normalisation ;
diacylglycerols content obtained by normalisation ;
triacylglycerols content obtained by

normalisation.
LABELLING
The label states the type of glycerol monocaprylate (type I
or II).
Reagents
Glycerol mono-octanoate. C11H22O4. (Mr 218.3). XXXXXXX.
[19670-49-6]. 1-Octanoyl-rac-glycerol(35).
Content : about 99 per cent.
Glycerol monodecanoate. C13H26O4. (Mr 246.3). XXXXXXX.
[26402-22-2]. 1-Decanoyl-rac-glycerol(36).
Reference: PA/PH/Exp. 13H/T (05) 67 ANP
TESTS
Acid value (2.5.1) : maximum 4.0.
Iodine value (2.5.4, Method A) : maximum 1.0.
Composition of fatty acids (2.4.22, Method C). Use the
mixture of calibrating substances in Table 2.4.22.-2.
Composition of the fatty acid fraction of the substance :
caproic acid : maximum 3.0 per cent ;
caprylic acid : 50.0 per cent to 90.0 per cent ;
capric acid : 10.0 per cent to 50.0 per cent ;
lauric acid : maximum 3.0 per cent ;
myristic acid : maximum 1.0 per cent.
Free glycerol : maximum 3.0 per cent.
Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat
to about 50 C then allow to cool. Add 100 ml of water R.
Shake and add 25.0 ml of periodic acetic acid solution R.
Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l
solution of potassium iodide R. Allow to stand for 1 min.
Add 1 ml of starch solution R. Titrate with 0.1 M sodium
thiosulphate. Carry out a blank titration.
1 ml of 0.1 M sodium thiosulphate is equivalent to 2.3 mg
of glycerol.
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
Total ash (2.4.16) : maximum 0.5 per cent.
XXXX:2392 ASSAY
Gas chromatography (2.2.28) : use the normalisation
GLYCEROL MONOCAPRYLOCAPRATE procedure.
Test solution. To 0.25 g of the substance to be examined,
add 5.0 ml of tetrahydrofuran R and shake to dissolve.
Glyceroli monocaprylocapras
Reference solution (a). To 0.25 g of glycerol
monocaprylocaprate CRS, add 5.0 ml of tetrahydrofuran R
DEFINITION
and shake to dissolve.
Mixture of monoacylglycerols, mainly monocaproylglycerol
and monocaprilylglycerol, containing variable quantities of
Reference solution (b). To 50 mg of glycerol
di- and triacylglycerols, obtained by direct esterification of
mono-octanoate R and 50 mg of glycerol monodecanoate R,
glycerol with caprylic and capric acids.
add 2.5 ml of tetrahydrofuran R and shake to dissolve.
Content :
Column :
glycerol monocaprylocaprate (type I) :
size : l = 10 m, = 0.32 mm ;
monoacylglycerols : 45.0 per cent to 75.0 per cent ;
stationary phase : poly(dimethyl)(diphenyl)siloxane R(37)
(film thickness 0.1 m).
diacylglycerols : 20.0 per cent to 50.0 per cent ;
triacylglycerols : maximum 10.0 per cent ;
glycerol monocaprylocaprate (type II):
Split ratio : 1:50.
monoacylglycerols : minimum 80.0 per cent ;
(35) Sigma Ref. M-2265.
(36) Sigma Ref. M-2140.
(37) OPTIMA 5 or equivalent : HP 5 or CP Sil 8.
100
5. triacylglycerols
Figure 2392.-1. Chromatogram for the assay of glycerol monocaprylocaprate

Temperature :
Column
Time
(min)
0-3
Temperature
(C)
60
3 - 38
60 340
38 - 50
340
Injection port
350
Detector
370

Injection: 1 l.
Identification of peaks: use the chromatogram supplied with
glycerol monocaprylocaprate CRS and the chromatogram
obtained with reference solution (a) to identify the peaks
due to mono-, di- and triacylglycerols.
resolution : minimum 5 between the peaks due to glycerol
mono-octanoate and glycerol monodecanoate.
For the calculation of content of mono-, di- and
triacylglycerols, disregard the peaks with a retention time
less than that of the monoacylglycerols, which are due to
impurities of the solvent and to the free fatty acids.
Calculate the content of free fatty acids (B) using the
IA
acid value of the substance to be examined.
Calculate the content of mono-, di- and triacylglycerols using

the following expressions :
percentage content of free glycerol ;
monoacylglycerols content obtained by

normalisation ;
diacylglycerols content obtained by normalisation ;
triacylglycerols content obtained by

normalisation.
LABELLING
The labelling states the type of glycerol monocaprylocaprate
(type I or II).
Reagents
Glycerol mono-octanoate. C11H22O4. (Mr 218.3). XXXXXXX.
[19670-49-6]. 1-Octanoyl-rac-glycerol(38).
Glycerol monodecanoate. C13H26O4. (Mr 246.3). XXXXXXX.
[26402-22-2]. 1-Decanoyl-rac-glycerol(39).
(38) Sigma Ref. M-2265.

(39) Sigma Ref. M-2140.
101
If Human normal immunoglobulin (0338) is added to

the preparation, the plasma pool from which it is derived
complies with the above requirement for B19 virus DNA.
It is proposed in the revised draft below, in order to increase If Human albumin solution (0255) is added to the
the level of safety with regard to B19 virus contamination, preparation, it complies with the above requirement for
B19 virus DNA.
to require to carry out the test for B19 virus DNA on
albumin when it is used as stabiliser.
POTENCY
XXXX:0557
Carry out the assay of human anti-D immunoglobulin
(2.7.13, Method A). The estimated potency is not less than
HUMAN ANTI-D IMMUNOGLOBULIN 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 estimated potency.
Immunoglobulinum humanum anti-D
Method B or C (2.7.13) may be used for potency
determination if a satisfactory correlation with the results
DEFINITION
obtained by Method A has been established for the particular
Human anti-D immunoglobulin is a liquid or freeze-dried
product.
preparation containing immunoglobulins, mainly
immunoglobulin G. The preparation is intended for
STORAGE
intramuscular administration. It contains specific antibodies See Human normal immunoglobulin (0338).
against erythrocyte D-antigen and may also contain small
quantities of other blood-group antibodies. Human normal LABELLING
immunoglobulin (0338) may be added.
See Human normal immunoglobulin (0338).
It complies with the monograph on Human normal
The label states the number of International Units per
immunoglobulin (0338), except for the minimum number of container.
donors and the minimum total protein content. 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.
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.
ERYTHROCYTE DONORS
Erythrocyte donors comply with the requirements for
donors prescribed in the monograph Human plasma for
fractionation (0853).
IMMUNISATION
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 Organisation
(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 B19 virus using validated nucleic acid
amplification techniques (2.6.21).
B19 virus DNA : maximum 10.0 IU/l.
A positive control with 10.0 IU of B19 virus DNA per
microlitre 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.
102

It is proposed in the revised draft below, in order to increase
the level of safety with regard to B19 virus contamination,
to require to carry out the test for B19 virus DNA on
albumin when it is used as stabiliser.
XXXX:1527
HUMAN ANTI-D IMMUNOGLOBULIN

FOR INTRAVENOUS
ADMINISTRATION
Immunoglobulinum humanum anti-D
ad usum intravenosum
DEFINITION
Human anti-D immunoglobulin for intravenous
administration is a 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) may be added.
It complies with the monograph on 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. 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
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 NOTE ON THE MONOGRAPH
blood group systems in order to avoid formation of
In order to clarify the requirements for freezing plasma
undesirable antibodies.
intended for the recovery of labile proteins, whether it is
obtained by plasmapheresis or from whole blood, and in
ERYTHROCYTE DONORS
order to ensure homogeneous quality, it is proposed to
Erythrocyte donors comply with the requirements for
replace the chamber temperature indication ( 30 C or
donors prescribed in the monograph Human plasma for
below) by the following conditions : freezing conditions
have to be validated to ensure that a temperature of 25 C
or lower is attained at the core of each plasma unit within
IMMUNISATION
12 h of placing in the freezing chamber. These data are
Immunisation of the plasma donor is carried out under
based on scientific studies on the freezing conditions for
proper medical supervision. Recommendations concerning
donor immunisation, including testing of erythrocyte donors, plasma. One of these studies is published in the Scientific
Notes section of this edition of Pharmeuropa, and will also
have been formulated by the World Health Organisation
be published in the next Pharmeuropa Scientific Notes.
(Requirements for the collection, processing and quality
XXXX:0853
control of blood, blood components and plasma derivatives,
WHO Technical Report Series, No. 840, 1994 or subsequent
revision).
HUMAN PLASMA FOR
FRACTIONATION
POOLED PLASMA
To limit the potential B19 virus burden in plasma pools used
for the manufacture of anti-D immunoglobulin, the plasma
Plasma humanum ad separationem
pool is tested for B19 virus using validated nucleic acid
DEFINITION
amplification techniques (2.6.21).
Human plasma for fractionation is the liquid part of human
B19 virus DNA : maximum 10.0 IU/l.
blood remaining after separation of the cellular elements from
blood collected in a receptacle containing an anticoagulant,
A positive control with 10.0 IU of B19 virus DNA per
or separated by continuous filtration or centrifugation
microlitre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the of anticoagulated blood in an apheresis procedure ; it is
plasma pool are included in the test. The test is invalid if the intended for the manufacture of plasma-derived products.
PRODUCTION
the internal control indicates the presence of inhibitors.
DONORS
B19 virus DNA for NAT testing BRP is suitable for use as a
Only a carefully selected, healthy donor who, as far as can
positive control.
be ascertained after medical examination, laboratory blood
tests and a study of the donors medical history, is free from
If Human normal immunoglobulin for intravenous
detectable agents of infection transmissible by plasma-derived
administration (0918) is added to the preparation, the
plasma pool from which it is derived complies with the above products may be used. Recommendations in this field
are made by the Council of Europe [Recommendation
requirement for B19 virus DNA.
No. R (95) 15 on the preparation, use and quality assurance
If Human albumin solution (0255) is added to the
of blood components, or subsequent revision] ; a directive of
preparation, it complies with the above requirement for
the European Union also deals with the matter : Commission
B19 virus DNA.
Directive 2004/33/EC of 22 March 2004 implementing
Directive 2002/98/EC of the European Parliament and of
the Council as regards certain technical requirements for
POTENCY
blood and blood components.
Carry out the assay of human anti-D immunoglobulin
Immunisation of donors. Immunisation of donors to obtain
(2.7.13, Method A). The estimated potency is not less than
immunoglobulins with specific activities may be carried
90 per cent of the stated potency. The confidence limits
out when sufficient supplies of material of suitable quality
(P = 0.95) are not less than 80 per cent and not more than
cannot be obtained from naturally immunised donors.
120 per cent of the estimated potency.
Recommendations for such immunisations are formulated
by the World Health Organisation (Requirements for the
Method B or C (2.7.13) may be used for potency
collection, processing and quality control of blood, blood
determination if a satisfactory correlation with the results
obtained by Method A has been established for the particular components and plasma derivatives, WHO Technical Report
Series, No. 840, 1994 or subsequent revision).
product.
Records. Records of donors and donations made are kept in
such a way that, while maintaining the required degree of
STORAGE
confidentiality concerning the donors identity, the origin
See Human normal immunoglobulin for intravenous
of each donation in a plasma pool and the results of the
administration (0918).
corresponding acceptance procedures and laboratory tests
can be traced.
Laboratory tests. Laboratory tests are carried out for each
LABELLING
donation to detect the following viral markers :
See Human normal immunoglobulin for intravenous
1. antibodies against human immunodeficiency virus 1
administration (0918).
(anti-HIV-1) ;
2. antibodies against human immunodeficiency virus 2
The label states the number of International Units per
(anti-HIV-2) ;
container.
103
3. hepatitis B surface antigen (HBsAg) ;

4. antibodies against hepatitis C virus (anti-HCV).
Pending complete harmonisation of the laboratory tests to
be carried out, the competent authority may require that a
test for alanine aminotransferase (ALT) also be carried out.
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
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 connecting devices or under
aseptic conditions and using containers that have not
previously been used.
When obtained by plasmapheresis, plasma intended for the
recovery of proteins that are labile in plasma is frozen by
cooling rapidly in a chamber at 30 C or below as soon as
possible in conditions validated to ensure that a temperature
of 25 C or below is attained at the core of each plasma
unit within 12 h of placing in the freezing apparatus and
at the latest within 24 h of collection.
When obtained by plasmapheresis, plasma intended solely
for the recovery of proteins that are not labile in plasma is
frozen by cooling rapidly in a chamber at 20 C or below
as soon as possible and at the latest within 24 h of collection.
When obtained from whole blood, plasma intended for the
recovery of proteins that are labile in plasma is separated
from cellular elements and is frozen by cooling rapidly
in a chamber at 30 C or below as soon as possible in
conditions validated to ensure that a temperature of 25 C
or below is attained at the core of each plasma unit within
12 h of placing in the freezing apparatus 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 C 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 factor VIII shown below be carried out on each unit
of plasma. They are rather given as guidelines for good
manufacturing practice, the test for factor VIII being
relevant for plasma intended for use in the preparation of
concentrates of 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 intended
proportion of anticoagulant solution, a total protein content
complying with the limit of 50 g/l 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 limit for all normal donations.
104
Preservation of factor VIII in the donation depends on

the collection procedure and the subsequent handling
of the blood and plasma. With good practice, 0.7 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 labile proteins as
much as possible.
Total protein. Carry out the test using a pool of not fewer
than 10 units. Dilute the pool with a 9 g/l 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 g/l solution
of sodium molybdate R and 2 ml of a mixture of 1 volume
of nitrogen-free sulphuric 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 paper.
Determine the nitrogen in the residue by the method of
sulphuric 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 g/l.
Factor VIII. Carry out the test using a pool of not fewer than
10 units. Thaw the samples to be examined, if necessary,
at 37 C. Carry out the assay of factor VIII (2.7.4), 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/ml.
STORAGE AND TRANSPORT
Frozen plasma is stored and transported in conditions
designed to maintain the temperature at or below 20 C ;
for accidental reasons, the storage temperature may rise
above 20 C on one or more occasions during storage and
transport but the plasma is nevertheless considered suitable
for fractionation if all the following conditions are fulfilled :
the total period of time during which the temperature
exceeds 20 C does not exceed 72 h ;
the temperature does not exceed 15 C on more than
one occasion ;
the temperature at no time exceeds 5 C.
POOLED PLASMA
During the manufacture 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 suitable 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 IU/ml of hepatitis 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
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.
CHARACTERS
Before freezing, a clear to 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.
Human prothrombin complex

The main indication of the human prothrombin complex
has changed : it is now more linked to the factor II content
than to the factor IX content. Furthermore, a factor II
(prothrombin) overload could lead to complications like
thrombosis. For both reasons, this revised draft includes a
ratio limit between factor II and factor IX, with factor II
content between 0.7 and 1.5 times factor IX content. This
makes it possible to guarantee a better level of safety for
the product.
CHARACTERS
A white or slightly coloured powder or friable solid, very
hygroscopic.
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 for coagulation
factor IX activity and, where applicable, those for factors II,
VII and X.
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 clear
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
HUMAN PROTHROMBIN COMPLEX volume of the reconstituted preparation with a 9 g/1
solution of sodium chloride R to obtain a solution expected
to contain about 15 mg of protein in 2 ml. To 2.0 ml of the
Prothrombinum multiplex humanum
solution in a round-bottomed centrifuge tube add 2 ml of
a 75 g/l solution of sodium molybdate R and 2 ml of a
mixture of 1 volume of nitrogen-free sulphuric acid R and
DEFINITION
30 volumes of water R. Shake, centrifuge for 5 min, decant
Human prothrombin complex is a plasma protein fraction
the supernatant liquid and allow the inverted tube to drain
containing blood coagulation factor IX together with variable on filter paper. Determine the nitrogen in the residue by the
amounts of coagulation factors II, VII and X ; the presence
method of sulphuric acid digestion (2.5.9) and calculate the
and proportion of these additional factors depends on the
amount of protein by multiplying the result by 6.25.
method of fractionation. It is obtained from human plasma
Activated coagulation factors (2.6.22). If necessary, dilute
that complies with the monograph on Human plasma for
the preparation to be examined to contain 20 IU of factor IX
per millilitre. For each of the dilutions, the coagulation time
The potency of the preparation, reconstituted as stated on
is not less than 150 s.
the label, is not less than 20 IU of factor IX per millilitre.
Heparin. If heparin has been added during preparation,
If a factor content is stated as a single value, the estimated
determine the amount present by the assay of heparin in
potency is not less than 80 per cent and not more than
coagulation factor concentrates (2.7.12). The preparation to
125 per cent of the stated potency ; if a factor content is
be examined contains not more than the amount of heparin
stated as a range, the estimated potency is not less than the stated on the label and in any case not more than 0.5 IU of
lower limit and not greater than the upper limit of the range. heparin per International Unit of factor IX.
Thrombin. If the preparation to be examined contains
PRODUCTION
heparin, determine the amount present as described in the
test for heparin and neutralise it by addition of protamine
The method of preparation is designed to minimise
sulphate R (10 g of protamine sulphate neutralises 1 IU
activation of any coagulation factor (to minimise potential
of heparin). In each of 2 test tubes, mix equal volumes
thrombogenicity) and includes a step or steps that have
of the reconstituted preparation and a 3 g/l solution of
been shown to remove or to inactivate known agents of
fibrinogen R. Keep one of the tubes at 37 C for 6 h and the
infection ; if substances are used for inactivation of viruses
other at room temperature for 24 h. In a third tube, mix a
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of volume of the fibrinogen solution with an equal volume of a
these substances is reduced to a suitable level and that any solution of human thrombin R (1 IU/ml) and place the tube
in a water-bath at 37 C. No coagulation occurs in the tubes
residues are such as not to compromise the safety of the
containing the preparation to be examined. Coagulation
preparation for patients.
occurs within 30 s in the tube containing thrombin.
The specific activity is not less than 0.6 IU of factor IX per
milligram of total protein, before the addition of any protein Water. Determined by a suitable method, such as the
semi-micro determination of water (2.5.12), loss on drying
stabiliser.
(2.2.32) or near-infrared spectrometry (2.2.40), the water
The prothrombin complex fraction is dissolved in a suitable content is within the limits approved by the competent
liquid. Heparin, antithrombin and other auxiliary substances authority.
such as a stabiliser may be added. No antimicrobial
Sterility (2.6.1). It complies with the test for sterility.
preservative is added. The solution is passed through a
bacteria-retentive filter, distributed aseptically into the
Pyrogens (2.6.8). It complies with the test for pyrogens.
final containers and immediately frozen. It is subsequently
Inject per kilogram of the rabbits mass a volume of the
freeze-dried and the containers are closed under vacuum
reconstituted preparation equivalent to not less than 30 IU
or under an inert gas.
of factor IX.
Moreover, in order to clarify the assays requirements, it
is specifically stated under Definition that limits of the
confidence interval apply only when the coagulation factor
content is stated by a single value. When the content is
stated as a range, the estimated potency is not less than
the lower limit and not greater than the upper limit of the
range stated.
XXXX:0554
105
Influenza vaccine (surface antigen, inactivated, virosome)
ASSAY
Factor IX. Carry out the assay of human coagulation

factor IX (2.7.11).

It is proposed to revise this monograph so that it covers
2 slightly different manufacturing processes. Furthermore,
The estimated potency is not less than 80 per cent and
the virosome size test has been revised in order to comply
not more than 125 per cent of the stated potency. The
with the ISO guide 13321, and an upper limit on the
confidence interval (P = 0.95) of the estimated potency is not polydispersity index has been included.
greater than 80 per cent to 125 per cent.
XXXX:2053
Factor II. Carry out the assay of human coagulation factor II
(2.7.18).
INFLUENZA VACCINE (SURFACE
confidence interval (P = 0.95) of the estimated potency is not
ANTIGEN, INACTIVATED, VIROSOME)
Vaccinum influenzae inactivatum ex corticis

antigeniis praeparatum virosomale
DEFINITION
Influenza vaccine (surface antigen, inactivated, virosome) is a
sterile, aqueous suspension of a strain or strains of influenza
Factor VII. If the label states that the preparation contains virus, type A or B, or a mixture of strains of the 2 types
grown individually in fertilised hens eggs, inactivated and
factor VII, carry out the assay of human coagulation
treated so that the preparation consists predominantly of
factor VII (2.7.10).
haemagglutinin and neuraminidase antigens reconstituted
to virosomes with phospholipids and without diminishing
the
antigenic properties of the antigens. The stated amount
of
haemagglutinin
antigen for each strain present in the
confidence interval (P = 0.95) of the estimated potency is not
vaccine
is
15
g
per
dose, unless clinical evidence supports
the use of a different amount.
Factor X. Carry out the assay of human coagulation factor X The vaccine is a slightly opalescent liquid.
(2.7.19).
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
confidence interval (P = 0.95) of the estimated potency is not consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in man.
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).
STORAGE
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.
LABELLING
Such strains are used in accordance with the regulations
in force in the signatory states of the Convention on the
The label states :
Elaboration of a European Pharmacopoeia. It is now
common practice to use reassorted strains giving high yields
the number of International Units of factor IX, factor II
of the appropriate surface antigens. The origin and passage
and factor X per container ;
history of virus strains shall be approved by the competent
authority.
where applicable, the number of International Units of
SUBSTRATE FOR VIRUS PROPAGATION
factor VII per container ;
Influenza virus seed to be used in the production of vaccine
where applicable, that the preparation contains protein C is propagated in fertilised eggs from chicken flocks free
from specified pathogens (SPF) (5.2.2) or in suitable cell
and/or protein S ;
cultures (5.2.4), such as chick-embryo fibroblasts or chick
kidney cells obtained from SPF chicken flocks (5.2.2). For
the amount of protein per container ;
production, the virus of each strain is grown in the allantoic
the name and quantity of any added substances, including cavity of fertilised hens eggs from healthy flocks.
where applicable, heparin ;
VIRUS SEED LOT
The production of vaccine is based on a seed lot system.
the name and quantity of the liquid to be used for
Working seed lots represent not more than 15 passages from
reconstitution ;
the approved reassorted virus or the approved virus isolate.
The final vaccine represents 1 passage from the working
that the transmission of infectious agents cannot be
seed lot. The haemagglutinin and neuraminidase antigens of
totally excluded when medicinal products prepared from each seed lot are identified as originating from the correct
human blood or plasma are administered.
strain of influenza virus by suitable methods.
The measured factor II potency is not less than 70 per cent
and not more than 150 per cent of the measured factor IX
potency.
106
Influenza vaccine (surface antigen, inactivated, virosome)
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 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 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
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. 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 a
temperature of 5 3 C. If formaldehyde solution is used,
the concentration does not exceed 0.2 g/l of CH2O at any
time during inactivation ; if betapropiolactone is used, the
concentration does not exceed 0.1 per cent V/V at 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 for the preparation of
virosomes.
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 immunodiffusion test (2.7.1),
by comparison with a haemagglutinin antigen reference
preparation or with an antigen preparation calibrated against
it(40). Carry out the test at 20-25 C.
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.
PREPARATION OF MONOVALENT VIROSOMES
Virus particles are disrupted into component subunits
by a suitable detergent approved procedures and further
purified so that the monovalent bulk consists mainly of
haemagglutinin and neuraminidase antigens. After addition
of suitable phospholipids, solubilisation by ultrasonication
and sterile filtration, virosomal preparations 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.
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(40). Carry out the test at 20-25 C.
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 present.
Residual chemicals Chemicals 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.
Phospholipid. The content and identity of the
phospholipids is determined by suitable immunochemical or
physico-chemical methods.
Ratio of haemagglutinin to phospholipid. The ratio of
haemagglutinin content to phospholipid content is within
the limits approved for the particular product.
Virosome size distribution. The size of the virosomes
average virosome diameter, determined by a suitable
method such as laser light scattering photon correlation
spectroscopy, is not less than 100 nm and not greater than
500 300 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 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.
FINAL LOT
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 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
(40) Reference haemagglutinin antigens are available from the National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire
EN6 3QC, United Kingdom.
107
Isotretinoin
LABELLING
phospholipid, ratio of haemagglutinin to phospholipid,
free formaldehyde, ovalbumin and total protein have been
The label states :
performed with satisfactory results on the final bulk vaccine,
that the vaccine has been prepared on eggs ;
they may be omitted on the final lot.
the strain or strains of influenza virus used to prepare
the vaccine ;
IDENTIFICATION
the
method of inactivation ;
The assay serves to confirm the antigenic specificity of the
vaccine.
the haemagglutinin content, in micrograms per virus
strain per dose ;
TESTS
the maximum amount of ovalbumin ;
Residual infectious virus. Inoculate 0.2 ml of the vaccine
the season during which the vaccine is intended to
into the allantoic cavity of each of 10 fertilised eggs and
protect.
incubate at 33-37 C 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
Reference: PA/PH/Exp. VIT/T (05) 3 ANP 1R
10 fertilised eggs and incubate at 33-37 C for 3 days.
The test is not valid unless at least 8 of the 10 embryos
survive. Harvest about 0.1 ml of the allantoic fluid from each NOTE ON THE MONOGRAPH
It is proposed to revise the test for related substances in
surviving embryo and examine each individual harvest for
live virus by a haemagglutination test. If haemagglutination order to obtain a better separation between impurities
B and C and the principal peak. In order to reduce the
is found for any of the fluids, carry out for that fluid a
amount of toxic solvents/reagents used, it is proposed to
further passage in eggs and test for haemagglutination ; no
delete the 2nd identification series, and to modify the assay
haemagglutination occurs.
slightly. As IR alone is sufficient for identification, test A
pH (2.2.3) : 6.5 to 7.8
is also deleted. It is also proposed that the determination
Phospholipid. The content and identity of the phospholipids of water replaces the test for loss on drying, which is
is determined by a suitable immunochemical or
unreliable due to the potential of the oxidation process to
physico-chemical method.
cause a weight increase ; in addition, the drying time is
very long (16 h).
Ratio of haemagglutinin to phospholipid. The ratio of
XXXX:1019
haemagglutinin content to phospholipid content is within
the limits approved for the particular product.
ISOTRETINOIN
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical method. The content is not less than the minimum
Isotretinoinum
amount shown to be effective and is not greater than 115 per
cent of the quantity stated on the label.
Free formaldehyde (2.4.18) : maximum 0.2 g/l, 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)
C20H28O2
Mr 300.4
using a suitable reference preparation of ovalbumin.
DEFINITION
Total protein. Not more than 40 g of protein other than
(2Z,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1haemagglutinin per virus strain per human dose and
enyl)nona-2,4,6,8-tetraenoic acid.
not more than a total of 120 g of protein other than
Content : 98.0 per cent to 102.0 per cent (dried anhydrous
hemagglutinin per human dose.
substance).
Sterility (2.6.1). It complies with the test for sterility.
Virosome size distribution. The size of the virosomes
average virosome diameter, determined by a suitable
method such as laser light scattering photon correlation
spectroscopy, is not less than 100 nm and not greater than
500 300 nm. The polydispersity index is not greater than 0.4.
Bacterial endotoxins (2.6.14) : maximum less than 100 IU
per human dose.
CHARACTERS
Appearance : yellow or light orange, crystalline powder.
Solubility : practically insoluble in water, soluble in
methylene chloride, slightly soluble in ethanol (96 per cent).
It is sensitive to air, heat and light, especially in solution.
Carry out all operations as rapidly as possible and avoid
exposure to actinic light ; use freshly prepared solutions.
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(40). Carry out the
test at 20-25 C. 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 content. The lower
confidence limit (P = 0.95) is not less than 80 per cent of the
amount stated on the label for each strain.
IDENTIFICATION
First identification : A, B.
(2.2.25).
Test solution. Dissolve 75.0 mg in 5 ml of methylene
chloride R and dilute immediately to 100.0 ml with
acidified 2-propanol (prepared by diluting 1 ml of 0.01 M
hydrochloric acid to 1000 ml with 2-propanol R). Dilute
108
Isotretinoin
5.0 ml of this solution to 100.0 ml with the acidified

2-propanol (solution A). Dilute 5.0 ml of solution A to
50.0 ml with the acidified 2-propanol.
to 1420.
Comparison : isotretinoin CRS.
Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel GF254 plate R.
examined in methylene chloride R and dilute to 10 ml
Reference solution (a). Dissolve 10 mg of
isotretinoin CRS in methylene chloride R and dilute to
Reference solution (b). Dissolve 10 mg of isotretinoin CRS
and 10 mg of tretinoin CRS in methylene chloride R and
Apply separately to the plate 5 l of each solution.
of glacial acetic acid R, 4 volumes of acetone R,
40 volumes of peroxide-free ether R and 54 volumes of
cyclohexane R. Allow the plate to dry in air and examine
in ultraviolet light at 254 nm. The principal spot in
the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the
chromatogram obtained with reference solution (a).
The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated
principal spots.
D. Dissolve about 5 mg in 2 ml of antimony trichloride
solution R. An intense red colour develops and later
becomes violet.
TESTS
(2.2.29).
examined in methanol R and dilute to 50.0 ml with the same
solvent.
Reference solution (a). Dissolve 10.0 mg of tretinoin CRS in
methanol R and dilute to 10.0 ml with the same solvent.
to 25.0 ml with methanol R.
Reference solution (c). Mix 1.0 ml of reference solution (a)
with 0.5 ml of the test solution and dilute to 25.0 ml with
methanol R.
Reference solution (d). Dilute 0.5 ml of the test solution to
100.0 ml with methanol R.
internal diameter packed with octadecylsilyl silica gel for
as mobile phase at a flow rate of 1.0 ml/min a mixture
of 5 volumes of glacial acetic acid R, 225 volumes of
water R and 770 volumes of methanol R.
as detector a spectrophotometer set at 355 nm.
Inject separately 10 l of each of reference solutions (b),
(c) and (d) and of the test solution. Adjust the sensitivity
of the detector so that the height of the principal peak in
the chromatogram obtained with reference solution (b) is

not less than 70 per cent of the full scale of the recorder.
The test is not valid unless the resolution between the
peaks due to isotretinoin and tretinoin in the chromatogram
obtained with reference solution (c) is at least 2.0. In the
chromatogram obtained with the test solution : the area of
any peak due to tretinoin is not greater than the area of the
principal peak in the chromatogram obtained with reference
solution (b) (2.0 per cent) ; the sum of the areas of any peaks,
apart from the principal peak and any peak due to tretinoin,
is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (d) (0.5 per
cent).
in 10 ml of a 1.0 g/l solution of butylhydroxytoluene R in
tetrahydrofuran R and dilute to 100.0 ml with acetonitrile R.
100.0 ml with acetonitrile R. Dilute 1.0 ml of this solution to
100.0 ml with acetonitrile R.
to 50 ml with methanol R. Heat to 50 C in daylight for
2 h. Allow to cool, then dilute to 100.0 ml with methanol R
(in situ degradation to obtain impurities A, B and C).
Column :
size : l = 0.25 m, = 3.0 mm ;
temperature : 35 C.
Mobile phase : glacial acetic acid R, water R, methanol R
(2.5:200:1800 V/V/V).
Injection : 10 l.
Run time : twice the retention time of isotretinoin.
obtained with reference solution (b) to identify the peaks
due to impurities A and B + C.
Relative retention with reference to isotretinoin (retention
time = about 16 min) : impurity B + C = about 1.2 ;
impurity A = about 1.6.
isotretinoin and impurities B + C.
Limits :
impurity A : not more than 5 times the area of the
sum of impurities B and C : not more than twice the area
(0.7 per cent) ;
(0.05 per cent).
(41) MERCK Purospher RP18 non-endcapped is suitable.
109
1. isotretinoin
2. impurities B + C
3. impurity A
Figure 1019.-1. Chromatogram for the test for related substances of isotretinoin : test solution
0.5 g complies with test D. Prepare the reference solution
on 1.000 g by drying in vacuo for 16 h.
C. (2Z,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexWater (2.5.12) : maximum 0.5 per cent, determined on
1-enyl)nona-2,4,6,8-tetraenoic acid (11,13-di-cis-retinoic
1.000 g.
acid).
E. oxidation products of isotretinoin. deleted.
on 1.0 g.
ASSAY
Dissolve 0.200 g in 70 ml of acetone R. Titrate with 0.1 M
tetrabutylammonium hydroxide in 2-propanol determining
the end-point potentiometrically (2.2.20).
1 ml of 0.1 M tetrabutylammonium hydroxide in 2-propanol The monograph presented below is a quality high in
is equivalent to 30.04 mg of C20H28O2.
glycyrrhizic acids suitable for flavouring purposes. The
readers are requested to inform us, if there is a necessity
STORAGE
for a quality low in glycyrrhizic acids suitable for other
purposes.
In an airtight container, protected from light, at a
XXXX:2378
temperature not exceeding 25 C.
It is recommended that the contents of an opened container
be used as soon as possible and any unused part be protected
by an atmosphere of an inert gas.
IMPURITIES
Specified impurities : A, B, C.
A. tretinoin,
LIQUORICE DRY EXTRACT,

QUANTIFIED
Liquiritiae extractum siccum quantificatum
DEFINITION
Dried extract produced from Liquorice root (0277).
Content : 5.0 per cent to 7.0 per cent of glycyrrhizic acid
(C42H62O16 ; Mr 823) (dried extract).
PRODUCTION
The extract is prepared from the cut herbal drug by water
extraction according to the monograph Extracts (0765).
CHARACTERS
Appearance : yellowish-brown or brown powder.
Very sweet taste.
B. R = CO2H, R = H : (2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,
6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid
(9,13-di-cis-retinoic acid),
D. R = H, R = CO2H : (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,
6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid
(9-cis-retinoic acid), deleted,
110
IDENTIFICATION
Thin layer chromatography (2.2.27).
Test solution. Add 30 ml of hydrochloric acid R1 to 0.30 g
of the extract to be examined and heat on a water-bath under
a reflux condenser for 60 min. After cooling, extract the
mixture with 2 quantities, each of 20 ml, of ethyl acetate R.
Combine the organic layers and filter through a filter covered
ASSAY
with anhydrous sodium sulphate R. Evaporate the filtrate
to dryness in vacuo and dissolve the residue in 2.0 ml of a
mixture of equal volumes of ethyl acetate R and methanol R.
Reference solution. Dissolve 5.0 mg of glycyrrhetic acid R
and 5.0 mg of thymol R in 5.0 ml of ether R.
Plate : TLC silica gel F254 plate R (5-40 m).
Mobile phase : concentrated ammonia R, water R, ethanol
(96 per cent) R, ethyl acetate R (1:9:25:65 V/V/V/V).
Application : 20 l, as bands.
Test solution. Place 0.250 g of the extract to be examined in

a 150 ml ground glass conical flask. Add 100.0 ml of an 8 g/l
solution of ammonia R and treat in an ultrasonic bath for
30 min. Centrifuge a part of the supernatant layer and dilute
1.0 ml of the supernatant to 5.0 ml with an 8 g/l solution of
ammonia R. Filter through a 0.45 m filter.
Reference solution (a). Dissolve 0.130 g of monoammonium
glycyrrhizate CRS in an 8 g/l solution of ammonia R and
dilute to 100.0 ml with the same solvent (solution A). Dilute
5.0 ml of solution A to 100.0 ml with an 8 g/l solution of
ammonia R (solution A).

Drying : in air for 5 min.
Detection A : examine in ultraviolet light at 254 nm.
Results A : see below the sequence of the zones present in
the chromatograms obtained with the reference solution
and the test solution. The zone due to glycyrrhetic acid in
the chromatogram obtained with the test solution is similar
in intensity to the zone due to glycyrrhetic acid in the
chromatogram obtained with the reference solution.
Reference solution (b). Dilute 10.0 ml of solution A to

100.0 ml with an 8 g/l solution of ammonia R.
Reference solution (c). Dilute 15.0 ml of solution A to
100.0 ml with an 8 g/l solution of ammonia R.
Column :
Top of the plate
size : l = 0.10 m, = 4.0 mm ;

Thymol: a quenching zone
_______
_______
_______
_______
Glycyrrhetic acid : a quenching

zone
Reference solution
A quenching zone (glycyrrhetic

acid)

Mobile phase : glacial acetic acid R, acetonitrile R, water R
(6:30:64 V/V/V).
Test solution
Detection B : spray with anisaldehyde solution R, and heat

at 100-105 C for 5-10 min ; examine in daylight.

Injection : 10 l.
Results B : see below the sequence of the zones present in

the chromatograms obtained with the reference solution and Run time : 3 times the retention time of monoammonium
the test solution. Furthermore, other zones may be present glycyrrhizate.
in the chromatogram obtained with the test solution.
Retention time : monoammonium glycyrrhizate = about
9 min.
Top of the plate
Thymol: a red zone
A yellow zone
_______
_______
_______
_______
Glycyrrhetic acid : a violet zone
Reference solution
Establish a calibration curve with the concentration of the

reference solutions as the abscissa and the corresponding
areas as the ordinate. Calculate the percentage content of
glycyrrhizic acid using the following expression :
A violet zone (glycyrrhetic acid)
Test solution
TESTS
concentration of monoammonium
glycyrrhizate in the test solution determined
from the calibration curve, in g/100 ml ;
declared percentage content of
monoammonium glycyrrhizate CRS ;
mass of the extract to be examined, in grams ;
822
molecular weight of glycyrrhizic acid ;
840
molecular weight of the monoammonium

glycyrrhizate (anhydrous substance).
Water-insoluble substances : maximum 1.0 per cent.

Shake 1.00 g with 50 ml of water R for 15 min. Filter the
mixture through a tared sintered glass filter crucible (100).
Wash the residue with 2 quantities, each of 20 ml, of water R
and with 1 quantity of 10 ml of water R. Dry the residue
in an oven at 100-105 C and determine the weight after
cooling. The residue weighs a maximum of 10 mg.
111
Magnesium citrate, anhydrous

Dissolve 2.0 5.0 g in 7 15 ml of dilute hydrochloric acid R
and heat with heating. Adjust to pH 3.5 with ammonia R.
In order to allow a better identification of the substance,
Dilute to 20 50 ml with distilled water R. 12 ml of this
it is proposed to add a cross reference to the tests for pH
solution complies with test A. Prepare the reference solution
and for loss on drying. The testing conditions for the loss
using lead standard solution (1 ppm Pb) R.
on drying have been revised in order to dry the substance
Loss on drying (2.2.32) : maximum 2.5 3.5 per cent,
completely. Limits of content have consequently been
determined on 1.000 g by drying in an oven at 130 180 C
modified.
XXXX:2339 for 16 5 h.
ASSAY
MAGNESIUM CITRATE, ANHYDROUS Dissolve 0.150 g in 50 ml of water R. Carry out the
complexometric titration of magnesium (2.5.11).
Magnesii citras anhydricus
1 ml of 0.1 M sodium edetate is equivalent to 2.431 mg of Mg.
Reference: PA/PH/Exp. INC/T (04) 26 ANP 1R

Mr 451.1 The TLC for related substances has been replaced by an
LC. The corresponding transparency statement has been
DEFINITION
introduced. Based on the low contents found, the impurities
Trimagnesium bis(2-hydroxypropane-1,2,3-tricarboxylate).
have been classified under other detectable impurities.
Content : 15.0 per cent to 17.0 16.5 per cent of Mg (dried
However, the TLC is maintained for identification
substance).
purposes. The 2nd identification series is maintained as the
substance may be used in pharmacies. It is also proposed
CHARACTERS
to tighten the limits of content based on batch results.
Appearance : white or almost white, fine powder.
XXXX:0622
Solubility : soluble in water, practically insoluble in ethanol
(96 per cent). It dissolves in dilute hydrochloric acid.
MECLOZINE HYDROCHLORIDE
Mg3(C6H5O7)2
IDENTIFICATION
A. It gives the reaction of citrates (2.3.1).
B. It gives the reaction of magnesium (2.3.1).
C. pH (see Tests).
D. Loss on drying (see Tests).
TESTS
prepared from distilled water R by heating at 60 C, cool
Appearance of solution. Solution S is not more opalescent
than reference suspension III clear (2.2.1) and not more
intensely coloured than reference solutions Y7 or BY6
(2.2.2, Method II).
Oxalates : maximum 280 ppm.
Dissolve 0.50 g in 4 ml of water R. Add 3 ml of hydrochloric
acid R and 1 g of activated zinc R. Allow to stand for 5 min.
Transfer the liquid to a tube containing 0.25 ml of a 10 g/l
solution of phenylhydrazine hydrochloride R. Heat to
boiling. Cool rapidly, transfer to a graduated cylinder and
add an equal volume of hydrochloric acid R and 0.25 ml
of potassium ferricyanide solution R. Shake and allow to
stand for 30 min. Any pink colour in the solution is not more
intense than that in a standard prepared at the same time
and in the same manner using 4 ml of a 50 mg/l solution
of oxalic acid R.
Sulphates (2.4.13) : maximum 0.2 per cent.
Dilute 1.5 ml of solution S to 15 ml with distilled water R.
Calcium (2.4.3) : maximum 0.2 per cent.
Iron (2.4.9) : maximum 100 ppm.
112
Meclozini hydrochloridum
C25H29Cl3N2
Mr 463.9
DEFINITION
1-[(RS-(4-Chlorophenyl)phenylmethyl]-4-(3methylbenzyl)piperazine dihydrochloride.
Content : 98.0 99.0 per cent to 102.0 101.0 per cent
(anhydrous substance).
CHARACTERS
Appearance : yellow white or yellowish-white, crystalline
powder.
Solubility : slightly soluble in water, soluble in ethanol
(96 per cent) and in methylene chloride.
IDENTIFICATION
First identification : B, D.
(2.2.25).
Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M
hydrochloric acid.
Specific absorbance at the absorption maximum : 345 to

380 (anhydrous substance).
The test solution shows a weak absorbance without a
defined maximum between 260 nm and 300 nm.
Preparation : discs of potassium chloride R.
Comparison : meclozine hydrochloride CRS.
Examine the chromatograms obtained in the test
chromatogram obtained with reference solution (a).
Solvent mixture : methanol R, methylene chloride R
(50:50 V/V).
examined in the solvent mixture and dilute to 10 ml with
Reference solution. Dissolve 50 mg of meclozine
10 ml with the solvent mixture.
Mobile phase : concentrated ammonia R, methanol R,
toluene R, methylene chloride R (0.5:5:30:60 V/V/V/V).
Application : 10 l.
Drying : in air.
Detection : spray with dilute potassium iodobismuthate
solution R.
Dissolve about 15 mg in 2 ml of ethanol (96 per cent) R.
The solution gives reaction (a) of chlorides (2.3.1).
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with

a mixture of equal volumes of methanol R and methylene
chloride R.
Reference solution (a). Dissolve 50 mg of meclozine
hydrochloride CRS in a mixture of equal volumes of
B.
methanol R and methylene chloride R and dilute to 10 ml
with the same mixture of solvents.
Reference solution (b). Dilute 0.5 ml of test solution (b) to
C.
10 ml with a mixture of equal volumes of methanol R and
methylene chloride R.
over a path of 15 cm using a mixture of 0.5 volumes of
concentrated ammonia R, 5 volumes of methanol R,
C.
30 volumes of toluene R and 60 volumes of methylene
chloride R. Allow the plate to dry in air and spray with
dilute potassium iodobismuthate solution R. Any spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent). Disregard any yellowish-white spot at the starting
point.
Solvent mixture : water R, acetonitrile R (30:70 V/V).
examined in the solvent mixture and dilute to 100 ml with
Reference solution (b). Dissolve 10 mg of the substance
to be examined and 10 mg of 4-chlorobenzophenone R
(impurity C) in the solvent mixture and dilute to 100.0 ml
with the solvent mixture. Dilute 5.0 ml of this solution to
Column :
D.
size : l = 0.30 m, = 3.9 mm ;
TESTS
Appearance of solution. The solution is clear (2.2.1) and not Mobile phase : dissolve 1.5 g of sodium heptanesulphonate R
in 300 ml of water R and adjust to pH 4 with dilute sulphuric
acid R ; mix 30 volumes of this solution with 70 volumes of
Method II).
acetonitrile R.
Dissolve 0.50 g in ethanol (96 per cent) R and dilute to
Acidity or alkalinity. Calculate the acidity or alkalinity
Injection : 20 l.
from the titration volumes obtained in the assay using the
Run time : 3 times the retention time of meclozine.
Retention time : meclozine = about 4 min.
V1 = volume of 0.1 M sodium hydroxide added at the
meclozine and impurity C.
1st point of inflexion ;
Limits :
V2 = volume of 0.1 M sodium hydroxide added at the
2nd point of inflexion.
A is not less than 0.3 ml and not more than 0.3 ml for
0.3500 g of the substance to be examined.
(0.10 per cent) ;
Related substances. Examine by thin-layer chromatography total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
examined in a mixture of equal volumes of methanol R and disregard limit : 0.25 times the area of the principal peak
methylene chloride R and dilute to 10 ml with the same
mixture of solvents.
(0.05 per cent).
(42) -Bondapack C18 is suitable.
113
1. impurity A
2. impurity B
3. meclozine
4. impurity C
Figure 0622.-1. Chromatogram for the test for related substances of meclozine hydrochloride

0.200 g.
on 1.0 g.
A. 3-methylbenzaldehyde,
ASSAY
Dissolve 0.3500 g in 50 ml of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
of C25H29Cl3N2.
B. (RS)-(4-chlorophenyl)(phenyl)methanol,
STORAGE
IMPURITIES
C. (4-chlorophenyl)(phenyl)methanone.
Reagents
4-Chlorobenzophenone. C13H9ClO. (Mr 216.7). XXXXXXX.
[134-85-0]. (4-Chlorophenyl)(phenyl)methanone.
114
Molsidomine

A proposal was published in Pharmeuropa 14.1. Following
the comments received, additional experimental work was
performed and the draft monograph has been completely
revised. The lower content limit was tightened based
on batch data, the pH range was enlarged based on the
registered limits, and the limit for sulphated ash was
reduced based on batch data. The gradient LC method
proposed allows the control of impurity B at 3 ppm. A
specific test for the control of impurity E, which is a
potential degradation product, has also been introduced.
XXXX:1701
MOLSIDOMINE
Molsidominum
C9H14N4O4
Mr 242.2
DEFINITION
N-(Ethoxycarbonyl)-3-(morpholin-4-yl)sydnonimine.
CHARACTERS
Solubility : sparingly soluble in water, soluble in anhydrous
ethanol, methylene chloride and ethyl acetate.
mp : about 142 C.
IDENTIFICATION
Comparison : molsidomine CRS.
TESTS
more intensely coloured than reference solution B7 (2.2.2,
Method II).
Dissolve 1.0 g in anhydrous ethanol R and dilute to 20.0 ml
pH (2.2.3) : 5.5 to 7.5.
Dissolve 0.50 g in carbon dioxide-free water R and dilute to
Impurity B. Liquid chromatography (2.2.29) as described
in the test for related substances with the following
modifications.
Injection: 20 l of test solution (a) and reference solution (c).
Relative retention with reference to molsidomine (retention
time = about 9 min) : impurity B = about 0.43.
signal-to-noise ratio : minimum 20 for the principal peak.
Limit :
solution (c) (3 ppm).
Impurity E. Liquid chromatography (2.2.29).

examined in 100.0 ml of the mobile phase.
Reference solution (a). Dissolve 1.000 g of molsidomine
impurity E CRS in 1000 ml of water for chromatography R.
Dilute 25.0 ml of this solution to 500.0 ml with water
for chromatography R. Dilute 20.0 ml of this solution to
500.0 ml with water for chromatography R. Dilute 10.0 ml
of this solution to 100.0 ml with the mobile phase.
Reference solution (b). Mix 10.0 ml of the test solution with
10.0 ml of reference solution (a).
Column :
size : l = 0.25 m, = 4.0 mm ;
stationary phase : resin for reversed-phase ion
chromatography R(43) ;
temperature : 25 C.
Mobile phase : dilute 3.0 ml of methanesulphonic acid R
and 75 ml of acetonitrile R in water for chromatography R
Suppressor regenerant : water for chromatography R.
Expected background conductivity : less than 0.5 S.
Detection : conductivity detector with a sensitivity of 10 S.
Injection : 50 l.
Run time : 20 min.
Retention time : impurity E = about 7 min.
impurity E in the chromatogram obtained with reference
solution (b) ;
recovery : the area of the peak due to impurity E in the
chromatogram obtained with reference solution (b) is not
less than 80 per cent of the peak area of the principal peak
in the chromatogram obtained with reference solution (a).
Limit :
impurity E : not more than the area of the principal peak
(0.01 per cent).
Protect the solutions from light.
Solvent mixture : methanol R, mobile phase B (1:9 V/V).
examined in 2.5 ml of methanol R using an ultrasonic bath
and keeping at 20 C. Dilute to 5.0 ml with mobile phase B.
Reference solution (a). Dilute 1.0 ml of test solution (b)
Reference solution (b). Dissolve 10 mg of the substance to
be examined in 2.0 ml of 2 M hydrochloric acid and boil
for 30 min to produce impurity C. Allow to cool. Neutralise
with 2.0 ml of 2 M sodium hydroxide and dilute to 10.0 ml
with methanol R. Dilute 1.0 ml of this solution to 10.0 ml
Reference solution (c). Dissolve 2.4 mg of molsidomine
impurity B CRS in 80 ml of methanol R using an ultrasonic
bath and keeping at 20 C. Dilute to 100.0 ml with
methanol R. Dilute 2.0 ml of the solution to 100.0 ml with
the solvent mixture. Dilute 5.0 ml of this solution to 20.0 ml
(43) Dionex IonPac CS14 is suitable.
115
Molsidomine
2. impurity D
1. impurity A
3. impurity C
4. molsidomine
Figure 1701.-1. Chromatogram for the test for related substances of molsidomine : test solution spiked with 0.1 per cent
of impurities A, C and D
Reference solution (d). Dissolve 10 mg of linsidomine
hydrochloride R (impurity A) and 5 mg of molsidomine
impurity D CRS in 10 ml of methanol R and dilute to 50.0 ml
with the solvent mixture. Dilute 5.0 ml of this solution to
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase: end-capped octadecylsilyl silica gel
for chromatography R (5 m) with a specific surface area
of 430 m2/g and a pore size of 9.5 nm(44) ;
temperature : 30 C.
Mobile phase :
mobile phase A : methanol R ;
mobile phase B : dissolve 4.0 g of potassium dihydrogen
phosphate R in water for chromatography R and dilute
to 1000 ml with the same solvent ;
Time
(min)
0-3
Mobile phase A
(per cent V/V)
10
Mobile phase B
(per cent V/V)
90
3 - 10
10 80
90 20
10 - 13
80
20
13 - 13.1
80 10
20 90
13.1 - 15
10
90

Injection: 20 l of test solution (b) and reference
solutions (a), (b) and (d).
Relative retention with reference to molsidomine

impurity D = about 0.32 ; impurity C = about 0.86.
impurities A and D.
Limits :
correction factor: for the calculation of content, multiply
the peak area of impurity C by 1.5 ;
impurity C : not more than the area of the peak due
to molsidomine in the chromatogram obtained with
than the area of the peak due to molsidomine in the
(0.10 per cent) ;
total : not more than 3 times the area of the peak due
disregard limit : 0.5 times the area of the peak due
reference solution (a) (0.05 per cent).
on 1.000 g by drying in an oven at 105 C.
on 1.0 g.
(44) Phenomenex Luna 5 C18 100 is suitable.
116
ASSAY
Dissolve 0.200 g in a mixture of 5 ml of acetic anhydride R
and 50 ml of acetic acid R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
of C9H14N4O4.
XXXX:1656
MOXIDECTIN FOR VETERINARY USE

Moxidectinum ad usum veterinarium
STORAGE
IMPURITIES
Specified impurities : B, C, E.
pharmaceutical use) : A, D.
A. 3-(morpholin-4-yl)sydnonimine (linsidomine),
C37H53NO8
Mr 640
DEFINITION
(2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,17aR,20R,20aR,
20bS)-6-[(1E)-1,3-Dimethyl-1-buten-1-yl]-20,20b-dihydroxy5,6,8,19-tetramethyl-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)dione 4-(O-methyloxime). Semi-synthetic product derived
from a fermentation product. It may contain suitable
stabilisers such as antioxidants.
substance).
B. R = NO : 4-nitrosomorpholine,
CHARACTERS
Appearance : white or almost white, amorphous powder.
Solubility : practically insoluble in water, very soluble in
ethanol (96 per cent), slightly soluble in hexane.
D. R = CHO : morpholine-4-carbaldehyde,
IDENTIFICATION
Comparison : moxidectin CRS.
E. R = H : morpholine,
C. (2E)-(morpholin-4-ylimino)acetonitrile.
Reagents
Linsidomine hydrochloride. C6H11ClN4O2. (Mr 206.6).
XXXXXXX. [33876-97-0]. 3-(Morpholin-4-yl)sydnonimine
hydrochloride.
White or almost white powder.
TESTS
not more intensely coloured than reference solution GY6
(2.2.2, Method II).
Dissolve 0.40 g in benzyl alcohol R and dilute to 20 ml with
the same solvent.
A. Test solution. Dissolve 25.0 mg of the substance to be
examined in acetonitrile R and dilute to 25.0 ml with
to 100.0 ml acetonitrile R.
Reference solution (b). Dissolve 5 mg of moxidectin for
system suitability CRS in 5 ml of acetonitrile R.
Reference solution (c). Dissolve 25.0 mg of
moxidectin CRS in acetonitrile R and dilute to 25.0 ml
with acetonitrile R.
117
Column :
size : l = 0.15 m, = 3.9 mm ;
temperature : 50 C.
Mobile phase : dissolve 7.7 g of ammonium acetate R in
400 ml of water R, adjust to pH 4.8 with glacial acetic
acid R, and add 600 ml of acetonitrile R.
Injection: 10 l of the test solution and reference
Run time : 3 times the retention time of moxidectin.
supplied with moxidectin for system suitability CRS and
the chromatogram obtained with reference solution (b)
to identify the peaks due to impurities A, B, C, D, E + F
and G.
Relative retention with reference to moxidectin
impurity B = about 0.7 ; impurity C = about 0.75 ;
impurity D = about 0.94 ; impurities E + F = about 1.3-1.5 ;
impurity G = about 1.6.
peak-to-valley ratio : minimum 3, where Hp = height
above the baseline of the peak due to impurity D and
moxidectin.
Limits :
impurity D : not more than 2.5 times the area of the
sum of impurities E and F : not more than 1.5 times
impurities A, C, G : for each impurity, not more
(1.5 per cent) ;
impurity B : not more than 0.5 times the area of the
any other impurity : for each impurity, not more
(0.5 per cent) ;
solution (a) (0.1 per cent) ; disregard the peak due to
the stabiliser.
B. Test solution. Dissolve 75.0 mg of the substance to be
examined in acetonitrile R and dilute to 25.0 ml with the
same solvent.
to 100.0 ml with acetonitrile R.
Reference solution (b). Dissolve 5 mg of moxidectin for
system suitability CRS in 5 ml of acetonitrile R.
Column :
size : l = 0.15 m, = 3.9 mm ;

temperature : 35 C.
Mobile phase : dissolve 3.8 g of ammonium acetate R in
250 ml of water R, adjust to pH 4.2 with acetic acid R,
and add 750 ml of acetonitrile R.
Injection : 10 l.
Run time : 10 times the retention time of moxidectin.
Identification of impurities: use the chromatogram
supplied with moxidectin for system suitability CRS and
identify the peaks due to impurities H + I, J and K.
Relative retention with reference to moxidectin (retention
time = about 4 min) : impurities H + I = about 2 ;
impurity J = about 2.2 ; impurity K = about 3.4.
impurities H + I and J.
Limits :
sum of impurities H and I : not more than the area of
the principal peak in the chromatogram obtained with
impurities J, K : for each impurity, not more than
0.5 times the area of the principal peak in the
(0.5 per cent) ;
(0.5 per cent) ;
solution (a) (0.1 per cent) ; disregard the peak due to
the stabiliser.
Total of all impurities. Calculate the total of the impurities
eluting from the start of the run to impurity G in test A, and
from impurities H + I to the end of the run in test B. The sum
of all impurities is not more than 7.0 per cent.
1 g complies with test C. Prepare the reference solution
on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in test A for
Injection : test solution and reference solution (c).
Calculate the percentage content of C37H53NO3 using the
declared content of moxidectin CRS.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I, J, K.
(45) Waters Nova-Pak C18 and Waters Pico-Tag C18 are suitable.
(46) Waters Nova-Pak C18 and Waters Pico-Tag C18 are suitable.
118

pharmaceutical use) : L.
D. (2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,17aS,20R,20aR,
20bS)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20b-dihydroxy5,6,8,19-tetramethyl-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
A. (2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,17aR,20R,20aR,
6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)20bS)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20b-dihydroxydione 4-(O-methyloxime),
5,6,8,19-tetramethyl-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)dione 4-(O-methyloxime),
B. (2aE,4E,4Z,6R,6S,8E,11R,13R,15S,17aR,20R,20aR,
20bS)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20b-dihydroxy- E. (2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,19S,20R,20aR,
20bR)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20b-dihydroxy6,8,19-trimethyl-5,6,10,11,14,15,17a,20,20a,20b5,6,8,19-tetramethyl-5,6,10,11,14,15,19,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
decahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)dione 4-(O-methyloxime),
dione 4-(O-methyloxime),
C. (2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,17aR,20R,20aR,
20bS)-20,20b-dihydroxy-5,6,8,19-tetramethyl-6-[(1E)1-methyl-1-buten-1-yl]-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
F. one of R1 to R5 is ethyl, the others are methyl,

119
G. (3S,4E,5R,5S,6S,7R,9E,12R,13E,15E,16aS,18S,20aR)6-[(1E)-1,3-dimethyl-1-buten-1-yl]-16a,18-dihydroxy5,10,12,16,19-pentamethyl-3,4,5,6,7,8,11,12,16a,
17,18,20a-dodecahydro-1H-spiro[3,7-methano[2,
6]benzodioxacyclooctadecine-5,2-pyran]-1,4(3H)-dione
4-(O-methyloxime),
H. (2aE,4E,4E,5S,6R,6S,8E,11R,13R,15S,20aR,
20bR)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20b-hydroxy5,6,8,19-tetramethyl-5,6,10,11,14,15,20a,20boctahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
I. (2aE,4E,4S,5S,6R,6S,8E,11R,13S,15S,17aR,20R,
20aR,20bS)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20bdihydroxy-5,6,8,19-tetramethyl-4-[(methylthio)methoxy]3,4,5,6,6,7,10,11,14,15,17a,20,20a,20b-tetradecahydro2H,17H-spiro[11,15-methanofuro[4,3,2-pq][2,
6]benzodioxacyclooctadecine-13,2-pyran]-17-one,
120
J. R = CH2-S-CH3, R = H : (2aE,4E,4E,5S,6R,6S,8E,11R,
13R,15S,17aR,20R,20aR,20bS)-6-[(1E)-1,3-dimethyl1-buten-1-yl]-20-hydroxy-5,6,8,19-tetramethyl-20b[(methylthio)methoxy]-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
K. R = H, R = CO-C6H4-pNO2 : (2aE,4E,4E,5S,6R,6S,8E,
11R,13R,15S,17aR,20R,20aR,20bS)-6-[(1E)-1,3-dimethyl1-buten-1-yl]-20b-hydroxy-4-(methoxyimino)-5,6,8,19tetramethyl-17-oxo-3,4,5,6,6,10,11,14,15,17,17a,20,20a,
20b-tetradecahydro-2H,7H-spiro[11,15-methanofuro[4,3,
2-pq][2,6]benzodioxacyclooctadecine-13,2-pyran]-20-yl
4-nitrobenzoate,
L. (2aE,4E,4Z,5S,6R,6S,8E,11R,13R,15S,17aR,20R,20aR,
20bS)-6-[(1E)-1,3-dimethyl-1-buten-1-yl]-20,20b-dihydroxy5,6,8,19-tetramethyl-5,6,10,11,14,15,17a,20,20a,20bdecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,
6]benzodioxacyclooctadecine-13,2-pyran]-4,17(3H,6H)dione 4-(O-methyloxime).
Norgestimate

Norgestimate is a steroid with progestative action. The
substance has been known for more than 25 years and is
used in combination with estrogenic compounds as an oral
contraceptive. A typical daily dose is 250 g. Norgestimate
is already described in the USP. This Ph. Eur. proposal is
based on the USP monograph, however, now only 1 related
substance test is proposed. It has been shown that the
impurities detected with the 2nd USP related substance test
are far below the current identification threshold.
XXXX:1732
NORGESTIMATE
Norgestimatum
C23H31NO3
Mr 369.5
DEFINITION
(EZ)-13-Ethyl-3-hydroxyimino-18,19-dinor-17-pregn-4-en20-yn-17-yl acetate.
CHARACTERS
Solubility : practically insoluble in water, freely soluble in
methylene chloride, soluble in acetone.
IDENTIFICATION
Comparison : norgestimate CRS.
TESTS
Specific optical rotation (2.2.7) : + 42.0 to + 50.0 (dried
substance).
Dissolve 0.200 g in methylene chloride R and dilute to
Solvent mixture : water R, methanol R (1:4 V/V).
Reference solution (b). Dissolve 2 mg of norgestimate for
system suitability CRS (containing impurity A) in 4 ml of the
solvent mixture.
Column :
size : l = 0.10 m, = 4.6 mm ;
silica gel for chromatography R (5 m)(47) ;
temperature : 40 C.
Mobile phase : acetonitrile R, tetrahydrofuran for
chromatography R, water R (18:22:60 V/V/V).
Injection : 25 l.
Run time : 3 times the retention time of the (E)-isomer of
norgestimate.
supplied with norgestimate for system suitability CRS and
identify the peak due to impurity A.
Relative retention with reference to the (E)-isomer
of norgestimate (retention time = about 14 min) :
impurity A = about 0.7 ; (Z)-isomer of norgestimate = about 0.9.
resolution : minimum 1.5 between the peaks due to the
(E)- and (Z)-isomers of norgestimate.
1. impurity B
3. impurity D
5. (Z)-isomer of norgestimate
2. impurity C
4. impurity A
6. (E)-isomer of norgestimate
Figure 1732.-1. Chromatogram for the test for related substances of norgestimate : test solution spiked with impurities
A, B, C and D
(47) Luna C18 100A is suitable.
121
2.7.29. Nucleated cell count and viability
Limits :
B. levonorgestrel,
the peak area of the (Z)-isomer of norgestimate by 1.33 ;
impurity A : not more than twice the sum of the areas of
the peaks due to the (Z)- and (E)-isomers of norgestimate
(0.2 per cent) ;
than the sum of the areas of the peaks due to the (Z)and (E)-isomers of norgestimate in the chromatogram
C. (E)-13-ethyl-3-hydroxyimino-18,19-dinor-17-pregn-4-enobtained with reference solution (a) (0.10 per cent) ;
20-yn-17-ol ((E)-norelgestromin),
total: not more than 3 times the sum of the areas of the
peaks due to the (Z)- and (E)-isomers of norgestimate in
(0.3 per cent) ;
disregard limit : 0.5 times the sum of the areas of the
peaks due to the (Z)- and (E)-isomers of norgestimate in
(0.05 per cent).
Ratio of (E)- to (Z)-isomers. Liquid chromatography (2.2.29)
as described in the test for related substances with the
following modification.
D. (Z)-13-ethyl-3-hydroxyimino-18,19-dinor-17-pregn-4-enInjection: test solution (a).
20-yn-17-ol ((Z)-norelgestromin).
Calculate the (E)- to (Z)-isomer ratio by dividing the area of
the peak due to the (E)-isomer by 1.33 times the area of the
peak due to the (Z)-isomer. The ratio is 1.27 to 1.78.
ASSAY
Dissolve 0.300 g in 40 ml of tetrahydrofuran R. Add
10 ml of a 100 g/l solution of silver nitrate R and titrate
with 0.1 M sodium hydroxide, determine the end-point
potentiometrically (2.2.20). Rinse the electrode with
acetone R after each titration.
If necessary, after multiple titrations re-equilibrate the
electrode in water R for 15 min to obtain sharper titration
curves.
of C23H31NO3.
Reference: PA/PH/Exp. CTP/T (05) 28 ANP

XXXX:20729
2.7.29. NUCLEATED CELL COUNT AND

VIABILITY
The determination of the overall health of cell cultures

requires accurate measurements of both cell concentration
and percentage of viable cells. These data are essential to the
decision-making process for basic cell-culture passage and for
IMPURITIES
maintaining optimum culture conditions. The cell count may
Specified impurities : A.
be expressed as the number of cells per volume of medium
or per area of attached surface (for anchorage-dependent
cells). The cell-count procedure may be performed manually
(haemocytometer) or with an apparatus based on light
measurement (for example, particle counter, flow cytometer,
impurities and/or by the general monograph Substances for fluorimeter).
CELL NUMBER
HAEMOCYTOMETER
pharmaceutical use) : B, C, D.
Test principle and description of the apparatus. A
haemocytometer is a specialised microscope slide that has
been etched with a grid over a defined area and is available in
different designs. The most commonly used haemocytometer
model (see example shown in Figure 2.7.29.-1) is a modified
thick slide with 2 counting chambers separated by deep
grooves to avoid cross-filling. The counting chamber
is etched in the glass and contains 9 large squares of
1 mm 1 mm. A large square is bounded by a triple line
and almost fills the field of view when the microscope lens
and the oculars both have a 10 magnification. Different
large-square grid patterns are used for different purposes
A. 13-ethyl-3-oxo-18,19-dinor-17-pregn-4-en-20-yn-17-yl
in haematology laboratories.
acetate (levonorgestrel acetate),
122
A. covership
B. ruled grid area
.
Figure 2.7.29.-1. Counting chamber of an improved
Neubauer haemocytometer
Dimensions in millimetres
The other special feature of a haemocytometer is the
thickened coverslip. When placed on the haemocytometer,
the typical rainbow sheen of Newtons rings may be seen
on the side of the chamber. The haemocytometer is sealed
so that each chamber is maintained at a defined distance
from the grid (0.1 mm). The volume of the chamber is
therefore 0.1 mm3. The haemocytometer can therefore be
used to quantify the number of cells in a given solution by
calculation of the cell concentration per millilitre (C) using
the following expression :
number of cells in the large square ;
dilution factor (where applicable).
Some cells are found on the triple lines delimiting the

large squares : cells crossing the middle line on the top
and left sides are usually counted while those on the right
and bottom sides are ignored. It is possible to distinguish

between mixed cell populations provided they differ in size
or pigmentation (for example, leukocytes and erythrocytes).
However, haemocytometers have a significant intrinsic error.
Different operators analysing the same cell population
obtain a spread of results due to the subjective nature of
the determination.
Materials. The following materials are required :
a haemocytometer with coverslip ;
a hand-held counter ;
a light microscope - low power 40 to 100 magnification ;
hand-held pipettes of a 10-100 l volume range and
corresponding tips.
Test preparation and analysis. Mount the haemocytometer
and the coverslip, cleaned beforehand with deionised water
and ethanol (70 per cent V/V) or a 70 per cent V/V solution
of isopropyl alcohol. Move the coverslip back and forth over
the chamber, pressing slightly until the rainbow sheen is
seen on the sides of the etched counting chambers.
Add 5-8 l of the cell suspension to the counting chamber.
The liquid is added to the border of the coverslip and
is drained inside the chamber by capillarity. For most
haemocytometers, this is sufficient to fill the chamber. The
use of a variable-volume micropipette is suggested to avoid
spilling. Carefully place the haemocytometer under the
microscope and focus on a large-square chamber. Count the
cells for each large square. Calculate the cell concentration
per millilitre in the diluted and original samples.
To increase the accuracy of the measurement, it is important
to respect the following basic precautions :
use only suitably thickened coverslips ;
ensure that the rainbow sheen is present on the sides of
the counting chambers ;
wherever possible, count more than 200 cells (i.e. count
more large squares) ;
where cell clustering is detected (i.e. the cell suspension
is not monocellular), resuspend the cells before sampling
and repeat the procedure ;
avoid underfilling or overflowing the chamber, otherwise
the volume will no longer be accurate ;
count the cells twice.
FLOW CYTOMETER
A flow cytometer (2.7.24)(48) is an instrument in which cells
are made to flow in 1 single file through an orifice (usually
70-100 m in diameter) where they intersect a laser beam.
The cells cause the light to be scattered and may fluoresce
if stained with a fluorescent dye. A set of photomultipliers
positioned around the point of intersection collect the
scattered light and measure the number and intensity of
events. A computer analyses the events and expresses the
results in terms of a ratio between any 2 sets of parameters
measured.
Measurements taken include light scatter, fluorescent
emission (wavelength and intensity) and optical density. This
system has the following advantages :
it allows the counting of different groups of cells in the
same sample at the same time ;
it may count up to 10 000 events per second, allowing the
enumeration of rare populations ;
it may give structural information such as the complexity
of the intracellular organelles or the cells volume.
(48) See Pharmeuropa 16.4 (October 2004).
123
It needs to be calibrated with reference beads of known

concentration to give an absolute cell number per volume.
However, a calibrating solution is no longer necessary in
some instruments using 2 electrodes inserted in the sampling
chamber. The fixed size of the sampling chamber and
distance between the 2 electrodes allow the measurement of
the content of a fixed volume. This type of instrument rarely
needs to be calibrated after the initial setting.
PARTICLE COUNTERS
Particle counters based on conductivity variation.
Electronic particle counting devices measure the size and
number of particles in a solution. The oldest approach is
based on the change in conductivity with respect to that
of the medium alone when a particle crosses an orifice of
a roughly similar size. The cell suspension is drawn by
vacuum through a fine orifice 70-100 m in diameter and the
impulses are recorded by 2 electrodes as an analogue signal,
which is measured and counted. The intensity of the pulse
is used to measure the relative size of the cell, as a fraction
of the orifice is occupied by a non-conductive material (i.e.
the cell). The volume of liquid analysed is measured using
an internal calibrated tube. As cell debris may also generate
pulses that may cause errors, counters are also fitted with
a threshold control allowing only larger particles to be
counted.
Particle counters are calibrated before use with a solution
of synthetic latex beads of known concentration. To allow
the counting of larger particles, tubes fitted with differently
calibrated orifices are available. These apparatuses do not
allow the discrimination between dead and live cells.
Because old staining solutions are prone to aggregation

and polymerisation causes bad staining, freshly prepared
dye solutions are used.
Storage conditions of dye solution : Generally a 0.4 or 0.5 per
cent trypan blue solution in sterile phosphate-buffered
saline is used. Ready-made solutions are also commercially
available. Store protected from light and air, at room
temperature.
Test preparation and analysis. Stain the cell suspension at
the required dilution (usually in phosphate-buffered saline)
with a solution of, for example, 0.1 to 0.2 per cent trypan
blue. Mix gently. Incubate for not more than 2-4 min at
room temperature. Mix gently and place a suitable volume in
a counting chamber. Count without delay. The number of
squares or lines that have to be read for a correct estimation
is validated previously.
Determine the percentage of viable cells from the ratio of the
number of unstained cells to the total number of cells under
a direct light microscope, considering all blue cells as dead
cells. Viability (V) is calculated as a percentage using the
number of unstained (viable) cells ;
total number of cells (blue and unstained).
It is essential that the incubation time be less than 5 min

as the number of stained cells may increase significantly
afterwards. For a new determination, it may therefore be
necessary to prepare a new test.
VIABILITY
AUTOMATED METHODS
This section applies to cell staining by viability dyes and
analysis, under direct microscope light or by flow cytometry, Flow cytometry
of a cell suspension in order to determine the percentage
Test principle. The test is based on the capacity of certain
of viable cells.
dyes to cross damaged membranes and bind DNA by
Viability may be determined by staining with dyes such
intercalating between bases so that dead cells may fluoresce
as trypan blue (manual or automated method) or by flow
and be detected by flow cytometry (2.7.24)(48). Non-viable
cytometry using nucleic acid intercalating agents such as
cells are evaluated and discriminated by focusing on positive
propidium iodide (PI), 7-aminoactinomycin D (7-AAD) or
staining whereas viable cells remain unstained. This analysis
other suitable dyes.
is generally performed with 7-AAD or PI but other suitable
dyes may also be used.
Depending on the type of cells, results obtained with the
Dye. 7-AAD and PI are given as examples of
2 methods may differ.
membrane-impermeants that may be used as viability dyes.
MANUAL DYE-EXCLUSION METHOD
7-AAD is an analogue of actinomycin D that contains a
Test principle. This test is based on the exclusion of the
dye from viable cells whereas dead or damaged cells absorb substituted amino group at position 7 of the chromophore.
It intercalates between cytosine and guanine DNA bases.
the dye and are coloured. It provides information on the
The spectral properties of 7-AAD make this molecule
cytoplasmic membrane integrity but its results do not
particularly suitable for flow-cytometry analysis. The
necessarily reflect cell functionality. Recently trypsinised
maximum absorption of the 7-AAD/DNA complex is situated
living cells or cells thawed after cryopreservation in a
in the green spectral region and is thus suitable for an
medium containing dimethylsulfoxide may have leaky
argon laser-equipped cytometer (excitation wavelength of
membranes, causing them to absorb the dye.
488 nm). The deep red fluorescence emission of the 7-AAD
Dye. Trypan blue is the stain most commonly used to
viability dye (635 nm to 675 nm) eases the use of the probe
distinguish between viable and non-viable cells, but other
in combination with fluorescein isothiocyanate (FITC)
suitable dyes such as erythrosin B or nigrosin may also be
and phycoerythrin (PE)-conjugated antibodies, because in
used. It is an acid dye (Mr 961), an anion with 4 sulphonate contrast to PI, the 7-AAD/DNA complex shows minimal
groups that can easily bind to proteins. Binding to cellulose overlap with FITC and PE.
and proteins may also result from van der Waals forces or
PI binds to double-stranded DNA by intercalating between
from the formation of hydrogen bonds.
bases with little or no sequence preference and with a
stoichiometry of 1 dye molecule per 4-5 DNA base pairs.
Test conditions. Dye fixation is strongly influenced by pH,
Once the dye is bound to nucleic acids, its fluorescence
within a range of 6.6 to 7.6. Fixation is optimal at pH 7.5.
is enhanced 20- to 30-fold, the fluorescence excitation
The other conditions, such as the dye concentration, the
temperature and the staining time are standardised. Because maximum is shifted around 30-40 nm towards the red and
trypan blue is easily fixed on soluble proteins, such as serum the fluorescence emission maximum (615 nm) is shifted
proteins, the protein concentration of the preparation to be around 15 nm towards the blue. Although its absorptivity
is quite low, PI exhibits a sufficiently large Stokes shift
tested must be as low as possible.
124
Paraffin, white soft
to allow simultaneous detection of nucleic acids and

fluorescein-labelled antibodies, provided that the proper
optical filters are used.
Storage conditions of nucleic acid dye solution : Store at
5 3 C.
Test preparation and analysis. In the case of haematopoietic
cells, the dye may be used after CD45 labelling to obtain
a better separation of cells from debris and platelets with
a side scatter (SS)/CD45 gating region. The incubation
conditions (time and concentration) of the cell suspension
with the dye are validated previously.
Incubation is performed at room temperature in the dark.
Where necessary, lysis of red blood cells is performed using,
for example, ammonium chloride. If not, add buffer alone.
Acquisition of sample tubes is performed according to the
standardised operating procedure.
Percentages of viable cells are directly given by the flow
cytometer and deduced from the analysis of positive cells
(dead cells) in the SS/7-AAD or SS/PI cytogram (dot plots).
Positive controls may consist of stabilised cells (dead cells)
mixed with fresh viable cells at a target value.
Other automated dye-exclusion methods. The development
of digital imaging has allowed the automation of
dye-exclusion methods. The cell suspension and viability-dye
solution are directly mixed by a machine. The system, which
allows sample aspiration, reagent handling, and subsequent
instrument cleaning is fully automated. Once the cellular
suspension has been aspirated and mixed with the dye
solution, it is pumped to the flow cell for imaging. The
stained cell suspension is aspirated through a chamber
where stroboscopic light allows a camera to photograph the
flowing cells. The images are digitalised and the number of
dark (dead) or bright (live) cells counted by the software.
The first step is to digitise the collected video image and
transform this from a continuous smooth image into an
array of distinct elements or pixels. Each element is assigned
a grey level or brightness value from 0 (black) to 255 (white).
Thresholds within the software then determine which cells
have absorbed the dye (dead cells) and which have not
(viable cells).
correspond to a range of products, rather than a

tolerance for a given product. They are to be deleted
in accordance with the general policy on FRCs, the
acceptance criterion being a matter for agreement
between supplier and user.
Polycyclic aromatic hydrocarbons. A tighter limit is
applied for oral use. This test is to be maintained as
a local European requirement, since it is required by
regulatory authorities.
XXXX:1799
PARAFFIN, WHITE SOFT

Vaselinum album
DEFINITION
Purified and wholly or nearly decolorised mixture of
semi-solid hydrocarbons, obtained from petroleum. It may
contain a suitable antioxidant stabiliser. White soft paraffin
described in this monograph is not suitable for oral use.
CHARACTERS
Appearance : white or almost white, translucent, soft
unctuous mass, slightly fluorescent in daylight when melted.
methylene chloride, practically insoluble in ethanol (96 per
cent) and in glycerol.
IDENTIFICATION
First identification : A, B, D.
A. The drop point is between 35 C and 70 C and does not
differ by more than 5 C from the value stated on the
label, according to method (2.2.17) with the following
modification to fill the cup : heat the substance to be
examined at a temperature not exceeding 80 C, with
stirring to ensure uniformity. Warm the metal cup at a
temperature not exceeding 80 C in an oven, remove it
from the oven, place on a clean plate or ceramic tile and
pour a sufficient quantity of the melted sample into the
cup to fill it completely. Allow the filled cup to cool for
30 min on the plate or the ceramic tile and place it in a
water bath at 24-26 C for 30-40 min. Level the surface of
the sample with a single stroke of a knife or razor blade,
avoiding compression of the sample.
A.
Melting range : 38 C to 60 C.
The revision proposal for this monograph is published
Melt a quantity of the substance to be examined slowly,
below as part of the framework of harmonisation with the
while stirring, until it reaches a temperature of 90-92 C.
JP and the USP. The stage 4 draft provided by the USP is
Remove the source of the heat and allow the molten
published in the International harmonisation section. This
substance to cool to a temperature of 8-10 C above
draft will not lead to complete harmonisation for reasons
the expected melting point. Chill the bulb of a suitable
described in the following briefing note :
thermometer to 5 C, wipe it dry, and while it is still cold
Definition. The term antioxidants is replaced by
dip it into the molten substance so that approximately
stabilisers. The statement concerning unsuitability for
the lower half of the bulb is submerged. Withdraw it
oral use will be maintained since this reflects regulatory
immediately, and hold it vertically away from the heat
requirements in Europe.
until the wax surface dulls, then dip it for 5 min into a
Identification. The second identification will be
water-bath at a temperature not higher than 16 C.
maintained as a local provision.
Fix the thermometer securely in a test tube so that the
The determination of drop point is replaced by a
lower point is 15 mm above the bottom of the test tube.
determination of melting point by a specific method.
Suspend the test tube in a water-bath adjusted to about
For the identification by IR, the reference spectrum is
16 C, and raise the temperature of the bath at the rate
to be replaced. The test cites a number of bands typical
of 2 C/min to 30 C, then change to a rate of 1 C/min,
of paraffin.
and note the temperature at which the first drop of
Appearance. The reference solution is slightly modified.
melted substance leaves the thermometer. Repeat the
Consistency. This functionality-related characteristic
determination twice on a freshly melted portion of the
(FRC) is placed in the special section according to
test substance. If the variation of 3 determinations is less
current policy. The limits in the existing monograph
than 1 C, take the average of the 3 determinations as the
125
melting point. If the variation of 3 determinations is 1 C

or greater than 1 C, rate 2 additional determinations
and take the average of the 5 determinations.
Preparation : spread a thin film of the melted substance
to be examined between sodium chloride plates.
Comparison : Ph. Eur. reference spectrum of white soft
paraffin white soft paraffin CRS.
High intensity bands are obtained between 3000 cm-1 and
2800 cm-1, medium intensity bands between 1500 cm-1
and 1300 cm-1, and low intensity bands between 750 cm-1
and 700 cm-1.
C. Melt 2 g and when a homogeneous phase is obtained,
add 2 ml of water R and 0.2 ml of 0.05 M iodine. Shake.
Allow to cool. The solid upper layer is violet-pink.
D. Appearance Colour (see Tests).
STORAGE
LABELLING
The label states :
the nominal drop point,
where applicable, the name and concentration of any
added antioxidant stabiliser.
FUNCTIONALITY-RELATED CHARACTERISTICS
The following test is not a mandatory requirement but in
view of its known importance for achieving consistency
in manufacture, quality and performance of medicinal
products, it is recommended that suppliers should verify
this characteristic and provide information on the result
and the analytical method applied to users. The method
indicated below has been found suitable but other methods
may be used.
TESTS
The following characteristic is relevant for white soft paraffin
Appearance. The substance is white. Melt 12 g on a
water-bath. The melted mass is not more intensely coloured used as an emollient in semi-solid dosage forms.
than a mixture of 1 volume of yellow primary solution and
Consistency (2.9.9). Carry out a minimum of 3 tests, each
9 volumes of a 1 per cent m/V solution of hydrochloric
spaced such that there is no overlapping of the areas of
acid R (2.2.2, Method II).
penetration. Where the penetration exceeds 20 mm, use a
separate container of the test substance for each test. Read
Colour. Melt about 10 g on a steam bath, and pour 5 ml of
the resulting liquid into a clear-glass, 16 mm 150 mm test the penetration to the nearest 0.1 mm. Calculate the average
tube : the warm, melted liquid is not more intensely coloured of the 3 or more readings, and conduct further tests to a
than a solution prepared by mixing 1.6 ml of yellow primary total of 10 if the individual results differ from the average
by more than 3 per cent : each millimetre of penetration
solution (2.2.2) and 3.4 ml of water R. The comparison of
corresponds to a consistency value of 10.
the 2 preparations being made in reflected light against a
white background, the tubes being held directly against the
background at such an angle that there is no fluorescence.
Acidity or alkalinity. To 10 g add 20 ml of boiling water R
and shake vigorously for 1 min. Allow to cool and decant. To
10 ml of the aqueous layer add 0.1 ml of phenolphthalein
solution R. The solution is colourless. Not more than 0.5 ml
of 0.01 M sodium hydroxide is required to change the colour
of the indicator to red.
The revision proposal for this monograph is published
Consistency (2.9.9) : 60 to 300.
below as part of the framework of harmonisation with the
Polycyclic aromatic hydrocarbons : maximum 300 ppm.
JP and the USP. The stage 4 draft provided by the USP is
Use reagents for ultraviolet absorption spectrophotometry. published in the International harmonisation section. This
draft will not lead to complete harmonisation for reasons
Dissolve 1.0 g in 50 ml of hexane R which has been
described in the following briefing note :
previously shaken with 2 quantities, each of 10 ml, of
dimethyl sulphoxide R. Transfer the solution to a 125 ml
Definition. The term antioxidants is replaced by
separating funnel with unlubricated ground-glass parts
stabilisers.
(stopper, stopcock). Add 20 ml of dimethyl sulphoxide R.
Identification. The second identification will be

Shake vigorously for 1 min and allow to stand until 2 clear
maintained
as a local provision.
layers are formed. Transfer the lower layer to a second
The determination of drop point is replaced by a
separating funnel. Repeat the extraction with a further 20 ml
determination of melting point by a specific method.
of dimethyl sulphoxide R. Shake vigorously the combined
lower layers with 20 ml of hexane R for 1 min. Allow to stand
For the identification by IR, the reference spectrum is
until 2 clear layers are formed. Separate the lower layer and
to be replaced. The test cites a number of bands typical
dilute to 50.0 ml with dimethyl sulphoxide R. Measure the
of paraffin.
absorbance (2.2.25) between 260 nm and 420 nm using a
Appearance. The reference solution is slightly modified.
path length of 4 cm and using as the compensation liquid
the clear lower layer obtained by vigorously shaking 10 ml
Consistency. This functionality-related
of dimethyl sulphoxide R with 25 ml of hexane R for 1 min.
characteristic (FRC) is placed in the special
Prepare a 6.0 mg/l reference solution of naphthalene R
section according to current policy. The limits in the
in dimethyl sulphoxide R and measure the absorbance of
existing monograph correspond to a range of products,
this solution at the absorption maximum at 278 nm using a
rather than a tolerance for a given product. They are
path length of 4 cm and using dimethyl sulphoxide R as
to be deleted in accordance with the general policy
the compensation liquid. At no wavelength in the range
on FRCs, the acceptance criterion being a matter for
260-420 nm does the absorbance of the test solution exceed
agreement between supplier and user.
that of the reference solution at 278 nm.
Polycyclic aromatic hydrocarbons. This test is to be
maintained as a local European requirement, since it is
required by regulatory authorities.
on 2.0 10.0 g.
126
XXXX:1554
PARAFFIN, YELLOW SOFT

Vaselinum flavum
DEFINITION
Purified mixture of semi-solid hydrocarbons, obtained from
petroleum. It may contain a suitable antioxidant stabiliser.
High intensity bands are obtained between 3000 cm 1 and

2800 cm, 1 medium intensity bands between 1500 cm 1
and 1300 cm 1, and low intensity bands between 750 cm 1
and 700 cm 1.
C. Melt 2 g and when a homogenous phase is obtained, add
2 ml of water R and 0.2 ml of 0.05 M iodine. Shake.
Allow to cool. The solid upper layer is violet-pink.
D. Appearance Colour (see Tests).
TESTS
Appearance. The substance is yellow. Melt 12 g on a
CHARACTERS
water-bath. The melted mass is not more intensely coloured
than a mixture of 7.6 volumes of yellow primary solution and
Appearance : yellow, translucent, unctuous mass, slightly
2.4 volumes of red primary solution (2.2.2, Method II).
fluorescent in daylight when melted.
Colour. Melt about 10 g on a steam bath, and pour 5 ml
methylene chloride, practically insoluble in ethanol (96 per of the resulting liquid into a clear-glass, 16 mm 150 mm
test tube : the warm, melted liquid is not more intensely
cent) and in glycerol.
coloured than a solution made by mixing 3.8 ml of yellow
primary solution and 1.2 ml of red primary solution (2.2.2)
IDENTIFICATION
in a similar tube, the comparison of the 2 being made in
reflected light against a white background, the tubes being
held directly against the background at such an angle that
there is no fluorescence.
A. The drop point (2.2.17) is 40 C to 60 C and does not
differ by more than 5 C from the value stated on the
Acidity or alkalinity. To 10 g add 20 ml of boiling water R
label, with the following modification to fill the cup : heat and shake vigorously for 1 min. Allow to cool and decant. To
the substance to be examined at 118 C to 122 C, with 10 ml of the aqueous layer add 0.1 ml of phenolphthalein
stirring to ensure uniformity, then cool to 100 C to
solution R. The solution is colourless. Not more than 0.5 ml
107 C. Warm the metal cup at 103 C to 107 C in an
of 0.01 M sodium hydroxide is required to change the colour
oven, remove it from the oven, place on a clean plate or
of the indicator to red.
ceramic tile and pour a sufficient quantity of the melted
Consistency (2.9.9). The consistency is 100 to 300.
sample into the cup to fill it completely. Allow the filled
cup to cool for 30 min on the ceramic tile and place it
Polycyclic aromatic hydrocarbons. Use reagents for
in a water-bath at 24 C to 26 C for a further 30 min
ultraviolet absorption spectrophotometry. Dissolve 1.0 g
to 40 min. Level the surface of the sample with a single
in 50 ml of hexane R which has been previously shaken
stroke of a knife or razor blade, avoiding compression of with 2 quantities, each of 10 ml, of dimethyl sulphoxide R.
the sample.
Transfer the solution to a 125 ml separating funnel with
unlubricated ground-glass parts (stopper, stopcock). Add
A. Melting range : 38 C to 60 C.
20 ml of dimethyl sulphoxide R. Shake vigorously for 1 min
Melt a quantity of the substance to be examined slowly,
and allow to stand until 2 clear layers are formed. Transfer
while stirring, until it reaches a temperature of 90-92 C. the lower layer to a second separating funnel. Repeat the
Remove the source of the heat and allow the molten
extraction with a further 20 ml of dimethyl sulphoxide R.
substance to cool to a temperature of 8-10 C above
Shake vigorously the combined lower layers with 20 ml
the expected melting point. Chill the bulb of a suitable
of hexane R for 1 min. Allow to stand until 2 clear layers
thermometer to 5 C, wipe it dry, and while it is still cold are formed. Separate the lower layer and dilute to 50.0 ml
dip it into the molten substance so that approximately
with dimethyl sulphoxide R. Measure the absorbance
the lower half of the bulb is submerged. Withdraw it
(2.2.25) between 260 nm and 420 nm using a path length of
immediately, and hold it vertically away from the heat
4 cm and using as the compensation liquid the clear lower
until the wax surface dulls, then dip it for 5 min into a
layer obtained by vigorously shaking 10 ml of dimethyl
water-bath at a temperature not higher than 16 C.
sulphoxide R with 25 ml of hexane R for 1 min. Prepare a
9.0 mg/l reference solution of naphthalene R in dimethyl
Fix the thermometer securely in a test tube so that the
sulphoxide R and measure the absorbance of this solution at
lower point is 15 mm above the bottom of the test tube.
the absorption maximum at 278 nm using a path length of
Suspend the test tube in a water-bath adjusted to about
4 cm and using dimethyl sulphoxide R as the compensation
16 C, and raise the temperature of the bath at the rate
of 2 C/min to 30 C, then change to a rate of 1 C/min, liquid. At no wavelength in the range 260-420 nm does the
absorbance of the test solution exceed that of the reference
and note the temperature at which the first drop of
solution at 278 nm.
melted substance leaves the thermometer. Repeat the
determination twice on a freshly melted portion of the
test substance. If the variation of 3 determinations is less on 2.0 10.0 g.
than 1 C, take the average of the 3 determinations as the
melting point. If the variation of 3 determinations is 1 C STORAGE
or greater than 1 C, rate 2 additional determinations
and take the average of the 5 determinations.
LABELLING
Preparation : spread a thin film of the related substance The label states :
to be examined between sodium chloride plates.
the nominal drop point,
Comparison : Ph. Eur. reference spectrum of yellow soft where applicable, the name and concentration of any
added antioxidant stabiliser.
paraffin CRS yellow soft paraffin CRS.
127
FUNCTIONALITY-RELATED CHARACTERISTICS
Preparation : separately shake a quantity of the substance
to be examined and a quantity of the reference substance,
The following test is not a mandatory requirement but in
each corresponding to 25 mg of pentaerythrityl
view of its known importance for achieving consistency
tetranitrate, with 10 ml of acetone R for 5 min. Filter,
in manufacture, quality and performance of medicinal
evaporate to dryness at a temperature below 40 C
products, it is recommended that suppliers should verify
and dry the residue, over diphosphorus pentoxide R at
this characteristic and provide information on the result
a pressure of 0.7 kPa for 16 h. Examine the residues
and the analytical method applied to users. The method
prepared as discs.
indicated below has been found suitable but other methods
Comparison : diluted pentaerythrityl tetranitrate CRS.
may be used.
The following characteristic is relevant for yellow soft
paraffin used as an emollient in semi-solid dosage forms.
Test solution. Shake a quantity of the substance to be
examined corresponding to 10 mg of pentaerythrityl
Consistency (2.9.9). Carry out a minimum of 3 tests, each
tetranitrate with 10 ml of ethanol (96 per cent) R for
spaced such that there is no overlapping of the areas of
5 min and filter.
penetration. Where the penetration exceeds 20 mm, use a
Reference solution. Shake a quantity of diluted
separate container of the test substance for each test. Read
pentaerythrityl tetranitrate CRS corresponding to 10 mg
the penetration to the nearest 0.1 mm. Calculate the average
of pentaerythrityl tetranitrate with 10 ml of ethanol
of the 3 or more readings, and conduct further tests to a
(96 per cent) R for 5 min and filter.
total of 10 if the individual results differ from the average
by more than 3 per cent : each millimetre of penetration
corresponds to a consistency value of 10.
Mobile phase : ethyl acetate R, toluene R (20:80 V/V).
Application : 10 l.
Drying : in air.
Reference: PA/PH/Exp. 10D/T (05) 34 ANP
Detection : spray with freshly prepared potassium iodide
and starch solution R. Expose to ultraviolet light at
254 nm for 15 min. Examine in daylight.
The current method in the test for related substances
does not adequately control impurity C. A new method is
proposed in replacement.
XXXX:1355
PENTAERYTHRITYL TETRANITRATE, D. Thin-layer chromatography (2.2.27).
DILUTED
examined corresponding to 0.10 g of lactose or mannitol
with 10 ml of water R. Filter if necessary.
Pentaerythrityli tetranitras dilutus
Reference solution (a) Dissolve 0.10 g of lactose R in
water R and dilute to 10 ml with the same solvent.
Reference solution (b). Dissolve 0.10 g of mannitol R in
water R and dilute to 10 ml with the same solvent.
Reference solution (c). Mix equal volumes of reference
C5H8N4O12
Mr 316.1
Mobile phase : water R, methanol R, anhydrous acetic
DEFINITION
acid R, ethylene chloride R (10:15:25:50 V/V/V/V).
Dry mixture of 2,2-bis(hydroxymethyl)propane-1,3-diol
Measure the volumes accurately since a slight excess of
tetranitrate (pentaerythrityl tetranitrate) and Lactose
water produces cloudiness.
monohydrate (0187) or Mannitol (0559).
Application : 1 l ; thoroughly dry the starting points.
Content : 95.0 per cent m/m to 105.0 per cent m/m of the
Development A : over a path of 15 cm.
declared content of pentaerythrityl tetranitrate.
Drying A : in a current of warm air.
CHARACTERS
Development B : immediately, over a path of 15 cm, after
Appearance of pentaerythrityl tetranitrate : white or slightly
renewing the mobile phase.
yellowish powder.
Drying B : in a current of warm air.
Solubility of pentaerythrityl tetranitrate : practically
Detection : spray with 4-aminobenzoic acid solution R.
insoluble in water, soluble in acetone, slightly soluble in
Dry in a current of cold air until the acetone is removed.
ethanol (96 per cent V/V).
Heat at 100 C for 15 min. Allow to cool and spray with a
The solubility of diluted pentaerythrityl tetranitrate depends
2 g/l solution of sodium periodate R. Dry in a current of
on the diluent and its concentration.
cold air. Heat at 100 C for 15 min.
IDENTIFICATION
with the test solution is similar in position, colour and size
A. Melting point (2.2.14) : 138 C to 142 C, for the
residue obtained with the substance to be examined in
reference solution (a) for lactose or to the principal spot
identification test B.
for mannitol.
128
TESTS
Inorganic nitrates. Thin-layer chromatography (2.2.27).
examined corresponding to 0.10 g of pentaerythrityl
tetranitrate with 5 ml of ethanol (96 per cent) R and filter.
Reference solution. Dissolve 10 mg of potassium nitrate R
in 1 ml of water R and dilute to 100 ml with ethanol (96 per
cent) R.
Mobile phase : glacial acetic acid R, acetone R, toluene R
(15:30:60 V/V/V).
Application : 10 l.
Drying : in a current of air until the acetic acid is completely
removed.
Detection : spray copiously with freshly prepared potassium
iodide and starch solution R. Expose the plate to ultraviolet
light at 254 nm for 15 min. Examine in daylight.
Limit :
nitrate : any spot due to nitrate is not more intense than
the spot in the chromatogram obtained with the reference
solution (0.5 per cent, calculated as potassium nitrate).
(2.2.29) as described under Assay.
Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained with reference
solution (c) is at least 20 per cent of the full scale of the
recorder.
The test is not valid unless in the chromatogram obtained
with reference solution (e), the resolution between the peaks
corresponding to glyceryl trinitrate and to pentaerythrityl
tetranitrate is at least 2.0.
Inject 20 l of test solution (a) and 20 l of reference
solution (c) and record the chromatogram of test solution (a)
for at least five times the retention time of pentaerythrityl
tetranitrate. In the chromatogram obtained with test
solution (a) ; the area of any peak, apart from the principal
peak is not greater than the area of the principal peak in
the chromatogram obtained with reference solution (c)
(0.3 per cent) ; the sum of the areas of all the peaks, apart
from the principal peak, is not greater than twice the area
reference solution (c) (0.6 per cent). Disregard any peak with
an area less than 0.2 times that of the principal peak in the
chromatogram obtained with reference solution (c).
Test solution (a). Sonicate for 15 min a quantity of the
substance to be examined corresponding to 25.0 mg of
pentaerythrityl tetranitrate in 20 ml of the mobile phase and
dilute to 25.0 ml with the mobile phase. Filter through a
suitable membrane filter.
Reference solution (a). Sonicate for 15 min a quantity of
diluted pentaerythrityl tetranitrate CRS corresponding to
25.0 mg of pentaerythrityl tetranitrate in 20 ml of the mobile
phase and dilute to 25.0 ml with the mobile phase. Filter
through a suitable membrane filter.
Reference solution (c). Dilute 0.3 ml of reference solution (b)
Reference solution (d). Dilute 200 l of glyceryl trinitrate
solution CRS to 25.0 ml with the mobile phase.
Reference solution (e). To 1 ml of reference solution (b)
add 1 ml of reference solution (d) and dilute to 10 ml with
the mobile phase.
Reference solution (f). Dilute 1.0 ml of reference solution (a)
Column :
size : l = 0.15 m, = 3.9 mm ;
Mobile phase : water R, acetonitrile R (35:65 V/V).
solutions (c), (e) and (f).
Run time : 5 times the retention time of pentaerythrityl
tetranitrate.
Retention time : pentaerythrityl tetranitrate = about 2.4 min ;
impurity C = about 7.1 min.
System suitability : reference solution (e) :
glyceryl trinitrate and pantaerythrityl tetranitrate.
1. impurity B
3. impurity D
2. pentaerythrityl tetranitrate
4. impurity C
Figure 1355.-1. Chromatogram for the test for related substances of diluted pentaerythrityl tetranitrate : substance
spiked with impurities B, C and D
(49) Symmetry C8 is suitable.
129
Limits :
Reference solution (e). To 1 ml of reference solution (b),

add 1 ml of reference solution (d) and dilute to 10 ml with
the mobile phase.
impurity C : not more than the area of the principal peak

in the chromatogram obtained with reference solution (c) The chromatographic procedure may be carried out using :
(0.3 per cent) ;
internal diameter packed with octadecylsilyl silica gel for
unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (f) (0.10 per cent) ;
as mobile phase at a flow rate of 2 ml/min a mixture of
40 volumes of water R and 60 volumes of methanol R,
in the chromatogram obtained with reference solution (c) as detector a spectrophotometer set at 230 nm.
(0.6 per cent) ;
STORAGE
disregard limit : the area of the principal peak in the
Store protected from light and heat.
chromatogram obtained with reference solution (f)
(0.05 per cent).
LABELLING
The label states :
ASSAY
the content of pentaerythrityl tetranitrate as a percentage ;

the diluent used.
Injection : test solution (b) and reference solution (b).

Calculate the percentage content of C5H8N4O12 from the
declared content of pentaerythrityl tetranitrate CRS.
When the chromatograms are recorded in the prescribed
conditions the retention time of pentaerythrityl tetranitrate
is about 8 min. Inject 20 l of reference solution (b).
Adjust the sensitivity of the system so that the height of
the principal peak in the chromatogram obtained is at least
50 per cent of the full scale of the recorder. Inject reference
solution (b) six times. The assay is not valid unless the
relative standard deviation for the area of the principal peak
is at most 2.0 per cent. Inject test solution (b) and reference
solution (b) alternately.
IMPURITIES
Specified impurities : A, C.
pharmaceutical use) : B, D.
A. inorganic nitrates,
Test solution (a). Sonicate for 15 min a quantity of the

substance to be examined corresponding to 25.0 mg of
pentaerythrityl tetranitrate in 20 ml of methanol R and
dilute to 25.0 ml with the mobile phase. Filter through a
suitable membrane filter.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml B. pentaerythrityl trinitrate,
Reference solution (a). Sonicate for 15 min a quantity of
diluted pentaerythrityl tetranitrate CRS corresponding
to 25.0 mg of pentaerythrityl tetranitrate in 20 ml of
methanol R and dilute to 25.0 ml with the mobile phase.
Filter through a suitable membrane filter.
C. tripentaerythrityl octanitrate,
Reference solution (d). Sonicate for 15 min a quantity of
glyceryl trinitrate solution CRS corresponding to 20.0 mg
of glyceryl trinitrate in 20 ml of methanol R and dilute to
25.0 ml with the mobile phase. Filter through a suitable
membrane filter. Dilute 1.0 ml of the filtrate to 10.0 ml with
the mobile phase.
130
D. dipentaerythrityl hexanitrate.

The current LC method for related substances has been
criticised for lack of ease of laboratory transfer, the
separation of impurity D and perindopril seeming to
be especially critical. Also, new degradation products
have been detected under severe conditions of storage
(temperature and humidity). These degradation products
may be co-eluted with impurity B in the present method. A
more robust and easy-to-use chromatographic system that
also allows separation and detection of these new impurities
was developed and is now proposed. A peak-to-valley ratio
criterion for the separation between impurities B and K
is proposed. The limits for impurities are the same as in
the present monograph. The new degradation products
are listed as impurities J and K under other detectable
impurities, limited by the standard acceptance criterion
of not more than 0.10 per cent. Impurities L, M, N and O,
which are potential synthesis impurities not normally
found in the batches, have also been included in the list as
other detectable impurities for information. In routine
quality control it was also observed that the solubility
in methylene chloride is slightly better that at present
described, therefore an adjustment is proposed.
XXXX:2019

impurity A.
Results : in the chromatogram obtained with the test
solution a spot is observed with the same Rf as the spot
with the higher Rf in the chromatogram obtained with
reference solution (c) (tert-butylamine).
TESTS
Impurity A. Thin-layer chromatography (2.2.27).
solvent.
Reference solution (a). Dissolve 5 mg of perindopril
impurity A CRS in methanol R and dilute to 25.0 ml with
the same solvent.
Reference solution (c). To 5 ml of reference solution (a) add
5 ml of a 20 g/l solution of 1,1-dimethylethylamine R in
methanol R.
Mobile phase : glacial acetic acid R, toluene R, methanol R
(1:40:60 V/V/V).
Application : 10 l of the test solution and reference
Development : in a saturated tank, over 2/3 of the plate.
PERINDOPRIL tert-BUTYLAMINE
tert-Butylamini perindoprilum
Detection : expose to iodine vapour for at least 20 h.
Limit :
impurity A : any spot due to impurity A is not more
Stereochemical purity. Liquid chromatography (2.2.29).
C23H43N3O5
Mr 441.6 Test solution. Dissolve 20 mg of the substance to be
examined in ethanol (96 per cent) R and dilute to 10.0 ml
DEFINITION
2-Methylpropan-2-amine (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1Reference solution (a). Dilute 1.0 ml of the test solution to
(ethoxycarbonyl)butyl]amino]propanoyl]octahydro-1H200.0 ml with ethanol (96 per cent) R.
indole-2-carboxylate.
Reference
solution (b). Dissolve 10 mg of perindopril for
stereochemical purity CRS in ethanol (96 per cent) R and
substance).
CHARACTERS
Reference solution (c). Dilute 10.0 ml of reference
Appearance : white or almost white, slightly hygroscopic,
solution (a) to 50.0 ml with ethanol (96 per cent) R.
crystalline powder.
Column :
Solubility : freely soluble in water and in ethanol (96 per
size : l = 0.25 m, = 4.6 mm ;
cent), soluble or sparingly soluble in methylene chloride.
stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 m)(50) with a specific surface area
of 450 m2/g and a pore size of 10 nm ;
IDENTIFICATION
A. Specific optical rotation (2.2.7) : 66 to 69 (anhydrous temperature : 50 C for the column and at least 30 cm of
the tubing preceding the column.
substance).
Mobile
phase : mix, in the following order, 21.7 volumes of
Dissolve 0.250 g in ethanol (96 per cent) R and dilute to
acetonitrile R, 0.3 volumes of pentanol R and 78 volumes
of a 1.50 g/l solution of sodium heptanesulphonate R,
previously adjusted to pH 2.0 with a mixture of equal
volumes of perchloric acid R and water R.
Comparison : perindopril tert-butylamine CRS.
If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance
separately in methylene chloride R, evaporate to dryness Equilibration : minimum 4 h.
Injection : 10 l.
(50) Inertsil ODS3 is suitable.
131
Run time : 1.5 times the retention time of perindopril.

Retention time : perindopril = about 100 min.
signal-to-noise ratio : minimum 3 for the principal peak in
the chromatogram obtained with reference solution (c) ;
peak-to-valley ratio : minimum 3, where Hp = height above
the baseline of the peak due to impurity I and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to perindopril in
the chromatogram obtained with reference solution (b) ;
the chromatogram obtained with reference solution (b) is
similar to the chromatogram provided with perindopril
for stereochemical purity CRS.
Limits :
any impurity : not more than 0.2 times the area of
reference solution (a) (0.1 per cent) ; disregard any peak
with a retention time less than 0.6 times the retention
time of perindopril and any peak with a retention time
greater than 1.4 times the retention time of perindopril.
Prepare the solutions immediately before use or maintain
them at a temperature lower than 10 C.
mobile phase A.
Reference solution (a). Dissolve 15 mg of perindopril for
system suitability peak identification CRS (containing
impurities B, E, F, H and K) in mobile phase A and dilute to
Reference solution (b). Dilute 1.0 ml of the test solution to
to 10.0 ml with mobile phase A.
Column :
size : l = 0.25 0.15 m, = 4.6 4 mm ;
stationary phase : spherical octylsilyl silica gel for
chromatography R (5 m)(51) with a pore size of 15 nm ;
temperature : 70 60 C.
Mobile phase :
mobile phase A : dissolve 0.92 g of sodium
heptanesulphonate R in 1000 ml of water R, add 1 ml of
triethylamine R and adjust to pH 2.0 with a mixture of
equal volumes of perchloric acid R and water R,
mobile phase B : acetonitrile R1,
Time
(min)
0-1
Mobile phase A
(per cent V/V)
70
Mobile phase B
(per cent V/V)
30
1 - 20
70 40
30 60
20 - 25
40
60
25 - 35
40 20
60 80
35 - 40
20 0
80 100
40 - 45
0 70
100 30
Mobile phase :
mobile phase A : water R adjusted to pH 2.5 with a
mixture of equal volumes of perchloric acid R and
water R ;
mobile phase B : a 0.03 per cent (V/V) solution of
perchloric acid R in acetonitrile R1 ;
Time
(min)
0 - (5 t)
Mobile phase A
(per cent V/V)
95
Mobile phase B
(per cent V/V)
5
(5 t) - (60 t)
95 40
5 60
(60 t) - (65 t)
40 95
60 5
1. impurity J
4. impurity L
7. impurity E
10. impurity N
13. impurity H
2. impurity B
5. impurity M
8. impurity C
11. impurity O
14 - 15. impurity G
3. impurity K
6. perindopril
9. impurity D
12. impurity F
Figure 2019.-1. Chromatogram for the test for related substances of perindopril tert-butylamine : substance with
about 0.5 per cent of the impurities
(51) Lichrospher RP8 ec Inertsil C8 is suitable.
132
The isocratic step is described for a dwell volume of 2 ml

of the chromatographic system. If the dwell volume (D) is
different from 2 ml, correct each time of the gradient with
the value (t), calculated using the following expression :
Flow rate : 1.5 1.0 ml/min.

Injection: 20 l.
Relative retention with reference to perindopril (retention
time = about 8 25 min) : impurity B = about 0.4 ;
impurity C = about 0.8 ; impurity D = about 0.9 ;
impurity E = about 1.4 ; impurity F = about 1.7 ;
impurity G = about 2.2 and 2.3 ; impurity H = about 3.6
and 3.7 impurity B = about 0.68 ; impurity K = about 0.72 ;
impurity E = about 1.16 ; impurity F = about 1.57 ;
impurity H = about 1.80 (impurity H may be eluted as 1 or
2 peaks).
STORAGE
IMPURITIES
Specified impurities : A, B, E, F, H, I.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
use) : C, D, G, J, K, L, M, N, O.

peak-to-valley ratio : minimum 10, where Hp = height
above the baseline of the peak due to impurity D and
perindopril. If necessary, adjust the concentration of
triethylamine R in mobile phase A minimum 3, where
Hp = height above the baseline of the peak due to
impurity B and Hv = height above the baseline of the
lowest point of the curve separating this peak from the
peak due to impurity K.
Limits :
impurity B : not more than 0.6 times the area of the
impurity E : not more than 0.8 times the area of the
A. (2S,3aS,7aS)-octahydro-1H-indole-2-carboxylic acid,
B. R = H : (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-carboxybutyl]amino]propanoyl]octahydro-1H-indole-2-carboxylic acid,
E. R = CH(CH3)2 : (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-[(1-methylethoxy)carbonyl]butyl]amino]propanoyl]octahydro-1H-indole-2-carboxylic acid,
impurities F, H : for each impurity, not more than 0.4 times

chromatogram obtained with reference solution (b)
(0.10 per cent) ;
total: not more than twice the area of the principal peak C. R = H : (2S)-2-[(3S,5aS,9aS,10aS)-3-methyl-1,4in the chromatogram obtained with reference solution (b)
dioxodecahydropyrazino[1,2-a]indol-2(1H)-yl]pentanoic
(1 per cent) ;
acid,
in the chromatogram obtained with reference solution (b) F. R = C2H5 : ethyl (2S)-2-[(3S,5aS,9aS,10aS)-3-methyl-1,4dioxodecahydropyrazino[1,2-a]indol-2(1H)-yl]pentanoate,
(c) (0.05 per cent).
on 1.0 g.
ASSAY
of C23H43N3O5.
D. (2S)-2-[(3S,5aS,9aS,10aR)-3-methyl-1,4-dioxodecahydropyrazino[1,2-a]indol-2(1H)-yl]pentanoic acid,
133
M. (2S,3aS,7aS)-1-((2S)-2-(((1S)-1-((methyloxy)carbonyl)butyl)amino)propanoyl)octahydro-1H-indole-2-carboxylic
acid,
G. (2S,3aS,7aS)-1-[(2S)-2-[(5RS)-3-cyclohexyl-2,4-dioxo-5propylimidazolidin-1-yl]propanoyl]octahydro-1H-indole-2carboxylic acid,
N. (2S)-3-cyclohexyl-2-(((2S)-2-(((1S)-1-((ethyloxy)carbonyl)butyl)amino)propanoyl)amino)propanoic acid,
H. (2S,3aS,7aS)-1-[(2S)-2-[(5RS)-3-cyclohexyl-2(cyclohexylimino)-4-oxo-5-propylimidazolidin-1yl]propanoyl]octahydro-1H-indole-2-carboxylic acid,
O. (2S,3aS,7aS)-1-(((2S,3aS,7aS)-1-((2S)-2-(((1S)-1((ethyloxy)carbonyl)butyl)amino)propanoyl)octahydro-1Hindol-2-yl)carbonyl)octahydro-1H-indole-2-carboxylic acid.

I. (2S,3aS,7aS)-1-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)butyl]amino]propanoyl]octahydro-1H-indole-2-carboxylic acid,
J. (2S,3aS,7aS)-1-((2S)-2-aminopropanoyl)octahydro-1Hindole-2-carboxylic acid,

The present transparency statement and impurity limits do
not accurately reflect the quality of potassium clavulanate
in approved products. The list of specified impurities
detected by LC has therefore been reduced to C, E and
G ; the limit for any other impurity has been tightened
to 0.2 per cent ; and a system suitability CRS has been
introduced.
XXXX:1140
POTASSIUM CLAVULANATE
Kalii clavulanas
C8H8KNO5
K. (3S,5aS,9aS,10aS)-3-methyldecahydropyrazino
[1,2-a]indole-1,4-dione,
L. (2S,3aS,7aS)-1-acetyloctahydro-1H-indole-2-carboxylic
acid,
134
Mr 237.3
DEFINITION
Potassium (2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1azabicyclo[3.2.0]heptane-2-carboxylate, the potassium salt of
a substance produced by the growth of certain strains of
Streptomyces clavuligerus or obtained by any other means.
substance).
CHARACTERS
hygroscopic.
Solubility : freely soluble in water, slightly soluble in ethanol Detection : spectrophotometer at 230 nm.
(96 per cent), very slightly soluble in acetone.
Injection : 20 l.
Identification of impurities : use the chromatogram supplied
PRODUCTION
with potassium clavulanate for system suitability CRS and
The methods of production, extraction and purification are
the chromatogram obtained with reference solution (c) to
such that clavam-2-carboxylate is eliminated or present at a identify the peaks due to impurities C, E and G.
level not exceeding 0.01 per cent.
Relative retention with reference to clavulanate
(retention time = about 3 min) : impurity E = about 2.3 ;
IDENTIFICATION
impurity G = about 3.6 ; impurity C = about 7.4.
Comparison : Ph. Eur. reference spectrum of potassium System suitability : reference solution (b) :
clavulanate.
clavulanate (1st peak) and amoxicillin (2nd peak).
B. It gives reaction (b) of potassium (2.3.1).
Limits :
TESTS
any impurity impurities C, E, G : for each impurity,
not more than the area of the principal peak in the
(1.0 per cent) ;
pH (2.2.3) : 5.5 to 8.0.
Dilute 5 ml of solution S to 10 ml with carbon dioxide-free any other impurity : for each impurity, not more
water R.
Specific optical rotation (2.2.7) : + 53 to + 63 (anhydrous
(0.2 per cent) ;
substance), determined on solution S.
Absorbance (2.2.25) : maximum 0.40 at 278 nm.
(2.0 per cent) ;
Dissolve 50.0 mg in 0.1 M phosphate buffer solution
pH 7.0 R and dilute to 50.0 ml with the same buffer solution. disregard limit : 0.05 times the area of the principal peak
Measure the absorbance immediately.
(0.05 per cent).
Aliphatic amines. Gas chromatography (2.2.28).
The method shown below can be used to determine
the following aliphatic amines : 1,1-dimethylethylamine ;
mobile phase A.
diethylamine ; N,N,N,N-tetramethylethylenediamine ;
Reference solution (a). Dilute 1.0 ml of the test solution to 1,1,3,3-tetramethylbutylamine ; N,N-diisopropylethylenediamine ; 2,2-oxydi(N,N)dimethylethylamine.
Internal standard solution : dissolve 50 l of
Reference solution (b). Dissolve 10 mg of lithium
clavulanate CRS and 10 mg of amoxicillin trihydrate CRS 3-methylpentan-2-one R in water R and dilute to 100.0 ml
in mobile phase A and dilute to 100 ml with mobile phase A. with the same solvent.
Test solution. Weigh 1.00 g of the substance to be examined
Reference solution (c). Dissolve 5 mg of potassium
into a centrifuge tube. Add 5.0 ml of the internal standard
clavulanate for system suitability CRS in 1 ml of mobile
solution, 5.0 ml of dilute sodium hydroxide solution R,
phase A.
10.0 ml of water R, 5.0 ml of 2-methylpropanol R and 5 g of
Column :
sodium chloride R. Shake vigorously for 1 min. Centrifuge
size : l = 0.10 m, = 4.6 mm ;
to separate the layers.
Reference solution. Dissolve 80.0 mg of each of the following
chromatography R (5 m) ;
amines : 1,1-dimethylethylamine R ; diethylamine R ;
tetramethylethylenediamine R ; 1,1,3,3-tetramethyl temperature : 40 C.
butylamine R ; N,N-diisopropylethylenediamine R
Mobile phase :
and 2,2-oxybis(N,N-dimethylethylamine) R in dilute
mobile phase A : a 7.8 g/l solution of sodium dihydrogen hydrochloric acid R and dilute to 200.0 ml with the same
phosphate R adjusted to pH 4.0 with phosphoric acid R acid. Introduce 5.0 ml of this solution into a centrifuge tube.
and filtered through a 0.5 m filter ;
Add 5.0 ml of the internal standard solution, 10.0 ml of dilute
mobile phase B : a mixture of equal volumes of mobile
sodium hydroxide solution R, 5.0 ml of 2-methylpropanol R
phase A and methanol R ;
and 5 g of sodium chloride R. Shake vigorously for 1 min.
Centrifuge to separate the layers.
Time
Mobile phase A
Mobile phase B
Column :
(min)
(per cent V/V)
(per cent V/V)
0-4
0
100
size : l = 50 m, = 0.53 mm ;
4 - 15
100 50
0 50
stationary phase : poly(dimethyl)(diphenyl)siloxane R
15 - 18
50
50
(film thickness 5 m)(52).
18 - 24
50 100
50 0
24 - 39
0
100
Split ratio : 1:10.
(52) Chrompack CPSil 8CB is suitable.
135
Temperature :
Time
(min)
0-7
Temperature
(C)
35
7 - 10.8
35 150
10.8 - 25.8
150
Column
Injection port
200
Detector
250
1 mg of clavulanate (C8H9NO5) is equivalent to 1.191 mg

of C8H8KNO5.
STORAGE
In an airtight container, at a temperature of 2 C to
8 C. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
LABELLING
The label states, where applicable, that the substance is free
from bacterial endotoxins.
IMPURITIES
Specified impurities : A, B, C, D, E, G, H, I, J, K, L, M.
Relative retention with reference to 3-methylpentan-2-one
(retention time = about 11.4 min) : impurity H = about 0.55 ; or other of the tests in the monograph. They are limited
impurity I = about 0.76 ; impurity J = about 1.07 ;
impurity K = about 1.13 ; impurity L = about 1.33 ;
impurity M = about 1.57.
Limit :
aliphatic amines : maximum 0.2 per cent.
pharmaceutical use) : A, B, D, E, F.
2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent.
By liquid chromatography : A, B, C, D, E, F, G.
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g. By gas chromatography : H, I, J, K, L, M.
Bacterial endotoxins (2.6.14) : less than 0.03 IU/mg if
intended for use in the manufacture of parenteral dosage
forms without a further appropriate procedure for the
removal of bacterial endotoxins.
Injection: 1 l of the upper layers obtained from the test
solution and the reference solution.
ASSAY
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
A. R = H : 2,2-(pyrazine-2,5-diyl)diethanol,
B. R = CH2-CH2-CO2H : 3-[3,6-bis(2-hydroxyethyl)pyrazin-2yl]propanoic acid,
Test solution. Dissolve 50.0 mg of the substance to

be examined in a 4.1 g/l solution of sodium acetate R
C. R = CH2-CH3 : 2,2-(3-ethylpyrazine-2,5-diyl)diethanol,
previously adjusted to pH 6.0 with glacial acetic acid R, and
Reference solution (a). Dissolve 50.0 mg of lithium
clavulanate CRS in a 4.1 g/l solution of sodium acetate R
previously adjusted to pH 6.0 with glacial acetic acid R and
D. 4-(2-hydroxyethyl)pyrrole-3-carboxylic acid,
Reference solution (b). Dissolve 50.0 mg of lithium
clavulanate CRS and 50.0 mg of amoxicillin trihydrate CRS
in a 4.1 g/l solution of sodium acetate R previously adjusted
to pH 6.0 with glacial acetic acid R and dilute to 50.0 ml
with the same solution.
Column :
size : l = 0.3 m, = 4.6 mm ;
Mobile phase : mix 5 volumes of methanol R1 and
95 volumes of a 15 g/l solution of sodium dihydrogen
phosphate R previously adjusted to pH 4.0 with dilute
phosphoric acid R.
E. (2R,4R,5Z)-2-(carboxymethyl)-5-(2-hydroxyethylidene)3-[[(2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa1-azabibyclo[3.2.0]hept-2-yl]carbonyl]oxazolidine-4carboxylic acid,

Injection: 10 l.
136
F. 4-[[[[4-(2-hydroxyethyl)-1H-pyrrol-3-yl]carbonyl]oxy]methyl]-1H-pyrrole-3-carboxylic acid,
G. 4-[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-4oxobutanoic acid (N-succinyltyrosine),

The present transparency statement and impurity limits do
not accurately reflect the quality of potassium clavulanate
in approved products. The list of specified impurities
detected by LC has therefore been reduced to C, E and
G ; the limit for any other impurity has been tightened
to 0.2 per cent ; and a system suitability CRS has been
introduced.
XXXX:1653
POTASSIUM CLAVULANATE, DILUTED

Kalii clavulanas dilutus
H. 2-amino-2-methylpropane (1,1-dimethylethylamine),
C8H8KNO5
I. diethylamine,
J. 1,2-bis(dimethylamino)ethane (N,N,N,Ntetramethylethylenediamine),
K. 2-amino-2,4,4-trimethylpentane (1,1,3,3tetramethylbutylamine),
L. N,N-bis(1-methylethyl)-1,2-ethanediamine
(N,N-diisopropylethylenediamine),
M. bis(2-dimethylamino)ethyl ether [2,2-oxybis(N,Ndimethylethylamine)].

Mr 237.3
DEFINITION
Dry mixture of Potassium clavulanate (1140) and
Cellulose, microcrystalline (0316) or Silica, colloidal
anhydrous (0434) or Silica, colloidal hydrated (0738).
Content : 91.2 per cent to 107.1 per cent of the content of
potassium clavulanate stated on the label.
CHARACTERS
Appearance of diluted potassium clavulanate : white or
almost white powder, hygroscopic.
Solubility of potassium clavulanate : freely soluble in water,
slightly soluble in ethanol (96 per cent), very slightly soluble
in acetone.
The solubility of the diluted product depends on the diluent
and its concentration.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to
B. It gives reaction (b) of potassium (2.3.1).
C. Depending on the diluent used, carry out the
corresponding identification test (a) or (b).
(a) A quantity of the substance to be examined,
corresponding to 20 mg of cellulose, when placed on
a watch-glass and dispersed in 4 ml of iodinated zinc
chloride solution R, becomes violet-blue.
(b) It gives the reaction of silicates (2.3.1).
TESTS
pH (2.2.3) : 4.8 to 8.0.
Suspend a quantity of the substance to be examined
corresponding to 0.200 g of potassium clavulanate in 20 ml
of carbon dioxide-free water R.
Absorbance (2.2.25) : maximum 0.40 at 278 nm.
Disperse a quantity of the substance to be examined
corresponding to 50.0 mg of potassium clavulanate in 10 ml
of 0.1 M phosphate buffer solution pH 7.0 R, dilute to
50.0 ml with the same buffer solution and filter. Measure
the absorbance immediately.
137

Test solution. Disperse a quantity of the substance to be
examined corresponding to 0.250 g of potassium clavulanate
in 5 ml of mobile phase A, dilute to 25.0 ml with mobile
phase A and filter.
Reference solution (b). Dissolve 10 mg of amoxicillin
trihydrate CRS in 1 ml of the test solution and dilute to
100 ml with mobile phase A.
Reference solution (c). Dissolve 5 mg of potassium
clavulanate for system suitability CRS in 1 ml of mobile
phase A.
Column :
size : l = 0.10 m, = 4.6 mm ;
chromatography R (5 m) ;
temperature : 40 C.
Mobile phase :
mobile phase A : a 7.8 g/l solution of sodium dihydrogen
phosphate R adjusted to pH 4.0 with dilute phosphoric
acid R ;
mobile phase B : a mixture of equal volumes of mobile
phase A and methanol R ;
Time
(min)
0-4
Mobile phase A
(per cent V/V)
100
Mobile phase B
(per cent V/V)
0
4 - 15
100 50
0 50
15 - 18
50
50
18 - 24
50 100
50 0
24 - 39
100

Injection: 20 l.
Identification of impurities : use the chromatogram supplied
with potassium clavulanate for system suitability CRS and
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities C, E and G.
Relative retention with reference to clavulanate
(retention time = about 3 min) : impurity E = about 2.3 ;
impurity G = about 3.6 ; impurity C = about 7.4.
Limits :
any impurity impurities C, E, G : for each impurity,
not more than the area of the principal peak in the
(1.0 per cent) ;
(0.2 per cent) ;
(2.0 per cent) ;
(0.05 per cent).
1.000 g.
138
ASSAY
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
Test solution. Disperse a quantity of the substance to be
examined corresponding to 50.0 mg of potassium clavulanate
in a 4.1 g/l solution of sodium acetate R previously adjusted
to pH 6.0 with glacial acetic acid R, dilute to 50.0 ml with
the same solution and filter.
Reference solution (a). Dissolve 50.0 mg of lithium
clavulanate CRS in a 4.1 g/l solution of sodium acetate R
previously adjusted to pH 6.0 with glacial acetic acid R and
Reference solution (b). Dissolve 10 mg of amoxicillin
trihydrate CRS in 10 ml of reference solution (a).
Column :
size : l = 0.3 m, = 4.6 mm ;
Mobile phase : mix 5 volumes of methanol R1 and
95 volumes of a 15 g/l solution of sodium dihydrogen
phosphate R previously adjusted to pH 4.0 with dilute
phosphoric acid R.
Injection : 10 l.
1 mg of C8H9NO5 is equivalent to 1.191 mg of C8H8KNO5.
STORAGE
LABELLING
The label states the m/m percentage content of potassium
clavulanate and the diluent used to prepare the mixture.
IMPURITIES
Specified impurities : A, B, C, D, E, G.
pharmaceutical use) : A, B, D, E, F.
A. R = H : 2,2-(pyrazine-2,5-diyl)diethanol,
B. R = CH2-CH2-CO2H : 3-[3,6-bis(2-hydroxyethyl)pyrazin-2yl]propanoic acid,
C. R = CH2-CH3 : 2,2-(3-ethylpyrazine-2,5-diyl)diethanol,
D. 4-(2-hydroxyethyl)-1H-pyrrole-3-carboxylic acid,
Rectal preparations
rectal capsules ;
rectal solutions, emulsions and suspensions ;
powders and tablets for rectal solutions and suspensions ;
semi-solid rectal preparations ;
rectal foams ;
rectal tampons.
E. (2R,4R,5Z)-2-(carboxymethyl)-5-(2-hydroxyethylidene)3-[[(2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa1-azabicyclo[3.2.0]hept-2-yl]carbonyl]oxazolidine-4carboxylic acid,
PRODUCTION
During the development of a rectal preparation whose
formulation contains an antimicrobial preservative, the
need for and the efficacy of the chosen preservative shall be
demonstrated to the satisfaction of the competent authority.
A suitable test method together with criteria for judging the
preservative properties of the formulation are provided in
the text on Efficacy of antimicrobial preservation (5.1.3).
During development, it must be demonstrated that the
nominal content can be withdrawn from the container
of liquid and semi-solid rectal preparations presented in
single-dose containers.
F. 4-[[[[4-(2-hydroxyethyl)-1H-pyrrol-3-yl]carbonyl]oxy]methyl]-1H-pyrrole-3-carboxylic acid,
In the manufacture, packaging, storage and distribution of

rectal preparations, suitable measures are taken to ensure
their microbial quality ; recommendations on this aspect
are provided in the text on Microbiological quality of
pharmaceutical preparations (5.1.4).
In the manufacture of semi-solid and liquid rectal
preparations containing dispersed particles, measures are
taken to ensure a suitable and controlled particle size with
regard to the intended use.
G. 4-[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-4oxobutanoic acid (N-succinyltyrosine).

In the current monograph, the test for uniformity of
dosage units applies only to solid single-dose preparations.
However, certain preparations are presented as liquid
or semi-solid preparations in single-dose containers, for
which the total content corresponds to a precise dose of the
medicine (expressed as unit amount of active substance). It
is proposed to modify the corresponding paragraph to take
these preparations into account.
XXXX:1145
RECTAL PREPARATIONS
Rectalia
DEFINITION
Rectal preparations are intended for rectal use in order to
obtain a systemic or local effect, or they may be intended
for diagnostic purposes.
Where applicable, containers for rectal preparations
comply with the requirements for Materials used for the
manufacture of containers (3.1 and subsections) and
Containers (3.2 and subsections).
Several categories of rectal preparations may be
distinguished :
suppositories ;
TESTS
Uniformity of dosage units. Liquid and semi-solid
single-dose rectal preparations comply with the test for
uniformity of dosage units (2.9.40). Solid single-dose
rectal preparations comply with the test for uniformity of
dosage units (2.9.40) or, where justified and authorised, with
the tests for uniformity of content and/or uniformity of mass
shown below. Herbal drugs and herbal drug preparations
present in the dosage form are not subject to the provisions
of this paragraph.
Uniformity of content (2.9.6). Unless otherwise prescribed
or justified and authorised, solid single-dose rectal
preparations with a content of active substance less than
2 mg or less than 2 per cent of the total mass comply with
test A (tablets) or test B (suppositories, rectal capsules)
for uniformity of content of single-dose preparations. If
the preparation contains more than one active substance,
this requirement applies only to those substances that
correspond to the above conditions.
Uniformity of mass (2.9.5). Solid single-dose rectal
preparations comply with the test for uniformity of mass. If
the test for uniformity of content is prescribed for all active
substances, the test for uniformity of mass is not required.
Dissolution. A suitable test may be required to demonstrate
the appropriate release of the active substance(s) from solid
single-dose rectal preparations, for example the Dissolution
test for lipophilic solid dosage forms (2.9.42).
Where a dissolution test is prescribed, a disintegration test
may not be required.
LABELLING
The label states the name of any added antimicrobial
preservative.
139
Rectal preparations
Suppositories
DEFINITION
Rectal solutions, emulsions

and suspensions
Suppositories are prepared by compression or moulding. If

necessary, the active substance(s) are previously ground and
sieved through a suitable sieve. When prepared by moulding,
the medicated mass, sufficiently liquefied by heating, is
poured into suitable moulds. The suppository solidifies on
cooling. Various excipients are available for this process,
such as hard fat, macrogols, cocoa butter, and various
gelatinous mixtures consisting of, for example, gelatin, water
and glycerol. Where applicable, the determination of the
softening time of lipophilic suppositories (2.9.22) and/or the
determination of the resistance to rupture of suppositories
(2.9.24) are carried out.
DEFINITION
Rectal solutions, emulsions and suspensions are liquid
preparations intended for rectal use in order to obtain
a systemic or local effect, or they may be intended for
diagnostic purposes.
Rectal solutions, emulsions and suspensions are supplied
in single-dose containers and contain 1 or more active
substances dissolved or dispersed in water, glycerol or
macrogols or other suitable solvents. Emulsions may show
evidence of phase separation but are readily redispersed on
shaking. Suspensions may show a sediment that is readily
dispersible on shaking to give a suspension that remains
sufficiently stable to enable the correct dose to be delivered.
Rectal solutions, emulsions and suspensions may contain
excipients, for example to adjust the viscosity of the
preparation, to adjust or stabilise the pH, to increase the
solubility of the active substance(s) or to stabilise the
preparation. These substances do not adversely affect the
intended medical action or, at the concentrations used,
cause undue local irritation.
Rectal solutions, emulsions and suspensions are supplied
in containers containing a volume in the range of 2.5 ml to
2000 ml. The container is adapted to deliver the preparation
to the rectum or is accompanied by a suitable applicator.
A suitable test is carried out to demonstrate the appropriate

release of the active substance(s) from suppositories
intended for modified release or for prolonged local action.
Powders and tablets for rectal solutions

and suspensions
Suppositories are solid, single-dose preparations. The

shape, volume and consistency of suppositories are suitable
for rectal administration.
They contain 1 or more active substances dispersed or
dissolved in a suitable basis that may be soluble or dispersible
in water or may melt at body temperature. Excipients such
as diluents, adsorbents, surface-active agents, lubricants,
antimicrobial preservatives and colouring matter, authorised
by the competent authority, may be added if necessary.
PRODUCTION
In the manufacture of suppositories containing dispersed

active substances, measures are taken to ensure a suitable
and controlled particle size.
TESTS
Disintegration. Unless intended for modified release or
for prolonged local action, they comply with the test for
disintegration of suppositories and pessaries (2.9.2). For
suppositories with a fatty base, examine after 30 min, and
for suppositories with a water-soluble base, examine after
60 min, unless otherwise justified and authorised.
Rectal capsules
DEFINITION
Rectal capsules (shell suppositories) are solid, single-dose
preparations generally similar to soft capsules as defined in
the monograph on Capsules (0016) except that they may
have lubricating coatings. They are of elongated shape, are
smooth and have a uniform external appearance.
PRODUCTION
release of the active substance(s) from rectal capsules
intended for modified release or for prolonged local action.
TESTS
Disintegration. Unless intended for modified release
or for prolonged local action, they comply with the test
for disintegration of suppositories and pessaries (2.9.2).
Examine the state of the capsules after 30 min, unless
otherwise justified and authorised.
140
DEFINITION
Powders and tablets intended for the preparation of rectal
solutions or suspensions are single-dose preparations
that are dissolved or dispersed in water at the time of
administration. They may contain excipients to facilitate
dissolution or dispersion or to prevent aggregation of the
particles.
After dissolution or suspension, they comply with the
requirements for rectal solutions or rectal suspensions, as
appropriate.
TESTS
Disintegration. Tablets for rectal solutions or suspensions
comply with the test for disintegration of tablets and capsules
(2.9.1) but using water R at 15-25 C. Carry out the test on
6 tablets. Examine the state of the tablets after 3 min. The
tablets comply with the test if all 6 have disintegrated.
LABELLING
The label states :
the method of preparation of the rectal solution or
suspension ;
the conditions and duration of storage of the solution or
suspension after constitution.
Semi-solid rectal preparations

DEFINITION
Semi-solid rectal preparations are ointments, creams or gels.
They are often supplied as single-dose preparations in
containers provided with a suitable applicator.
Semi-solid rectal preparations comply with the requirements
of the monograph on Semi-solid preparations for cutaneous
application (0132).
Rectal foams
DEFINITION
Rectal foams comply with the requirements of the
monograph on Medicated foams (1105).
Rectal tampons
DEFINITION
Rectal tampons are solid, single-dose preparations intended
to be inserted into the lower part of the rectum for a limited
time.
They comply with the requirements of the monograph on
Medicated tampons (1155).
Comparison : ropivacaine hydrochloride

monohydrate CRS.
B. Specific optical rotation (2.2.7) : 64.0 to 74.0
(anhydrous substance).
Mix 2 ml of a 200 g/l solution of sodium hydroxide R
and 30 ml of water R and dilute to 100.0 ml with ethanol
(96 per cent) R (solution A). Dissolve 0.500 g of the
substance to be examined in solution A and dilute to
50.0 ml with solution A.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 0.50 g of the substance to be examined
in water R and dilute to 25.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1).
pH (2.2.3) : 4.5 to 6.0.
Reference: PA/PH/Exp. P4/T (04) 29 ANP
Dissolve 0.250 g in carbon dioxide-free water R and dilute
XXXX:2335 to 25 ml with the same solvent.
Absorbance (2.2.25) : maximum 0.030 at 405 nm and
maximum 0.025 at 436 nm, determined on solution S,
ROPIVACAINE HYDROCHLORIDE
prepared immediately before use, with a path length of 5 cm
MONOHYDRATE
and using water R as compensation liquid.
Ropivacaini hydrochloridum
monohydricum
mobile phase.
Reference solution (b). Dissolve 5 mg of the substance to be
examined and 5 mg of bupivacaine hydrochloride CRS in
C17H27ClN2O,H2O
Mr 328.9 the mobile phase and dilute to 5 ml with the mobile phase.
Dilute 1.0 ml of this solution to 100.0 ml with the mobile
DEFINITION
phase.
S()-1-Propyl-N-(2,6-dimethylphenyl)piperidine-2Column :
carboxamide hydrochloride monohydrate.
size : l = 0.15 m, = 3.9 mm ;
substance).
for chromatography R (4 m)(53).
Mobile phase : mix 1.3 ml of a 156 g/l solution of sodium
CHARACTERS
dihydrogen phosphate R and 32.5 ml of an 89 g/l solution
of disodium hydrogen phosphate R and dilute to 1000 ml
Solubility : soluble in water and in ethanol (96 per cent),
with water R ; mix equal volumes of this solution and
slightly soluble in methylene chloride.
acetonitrile R.
IDENTIFICATION
Injection : 20 l.
1. impurity B
3. impurity D
5. ropivacaine
2. impurity C
4. impurity E
6. impurity A
Figure 2335.-1. Chromatogram for the test for related substances of ropivacaine hydrochloride monohydrate : solution
of ropivacaine spiked with impurities A, B, C, D and E
(53) Nova-Pak C18 is suitable.
141

Run time : 2.5 times the retention time of ropivacaine.
Relative retention with reference to ropivacaine (retention
time = about 6 min) : impurity A = about 1.6.
ropivacaine and impurity A.
Limits :
impurity A : not more than twice the area of the principal
(0.5 per cent) ;
(0.05 per cent).
Enantiomeric purity. Capillary electrophoresis (2.2.47) : use
the normalisation procedure.
examined in water R and dilute to 25.0 ml with the same
solvent.
Reference solution (b). Dissolve 1.5 mg of the substance to be
examined and 1.5 mg of (R)-ropivacaine hydrochloride CRS
in water R and dilute to 100.0 ml with the same solvent.
Capillary :
size : effective length = about 72 cm, = 50 m.
Temperature : 30 C.
CZE buffer : prepare a 133 g/l solution of
dimethyl--cyclodextrin R in a 9.8 g/l solution of
phosphoric acid R previously adjusted to pH 3.0 with
triethanolamine R. The CZE buffer is prepared and filtered
through a 0.45 m membrane filter immediately before use.

Preconditioning of the capillary : rinse the capillary with
water R for 1 min, with 0.1 M sodium hydroxide for 10 min
and with water R for 3 min. If the capillary is new or dry
increase the sodium hydroxide rinse to 30 min.
Between-run-rinsing : rinse the capillary, at 100 kPa with
water R for 1 min, with 0.1 M sodium hydroxide for 4 min,
with water R for 1 min and with CZE buffer for 4 min.
Injection : under pressure (5 kPa) for 5 s.
Migration : apply a field strength of 375 V/cm, initial
ramping 500 V/s, positive polarity and resulting current
40-45 A.
Run time : 30 min.
(R)-ropivacaine (impurity G) (1st peak) and (S)-ropivacaine.
If necessary increase the dimethyl--cyclodextrin
concentration in the CZE buffer or lower the temperature ;
signal-to-noise ratio : minimum 10 for reference
solution (a).
Limit :
impurity G : maximum 0.5 per cent.
85 volumes of methanol R and dilute to 20 ml with the same
mixture of solvents. 12 ml of the solution complies with
test B. Prepare the reference solution using lead standard
solution (1 ppm Pb) obtained by diluting lead standard
solution R (100 ppm Pb) with a mixture of 15 volumes of
water R and 85 volumes of methanol R.
2,6-Dimethylaniline. Liquid chromatography (2.2.29)
as described in the test for related substances with the
following modifications.
mobile phase.
The following electropherogram is shown for information but will not be published in the European Pharmacopoeia.
1. impurity G
2. ropivacaine
Figure 2335.-2. Electropherogram for the test for enantiomeric purity of ropivacaine hydrochloride monohydrate :
reference solution (b)
142
Reference solution. Dissolve 13.0 mg of 2,6-dimethylaniline

hydrochloride R(54) in the mobile phase and dilute to
100.0 ml with the mobile phase. Dilute 1.0 ml of the solution
Retention time : 2,6-dimethylaniline = about 2-3 min.
Limit :
2,6-dimethylaniline : not more than the area of the
principal peak in the chromatogram obtained with the
reference solution (10 ppm).
Water (2.5.12) : 5.0 per cent to 6.0 per cent, determined on
0.100 g.
on 1.0 g.
ASSAY
Dissolve 0.250 g in a mixture of 10 ml of water R and 40 ml of
ethanol (96 per cent) R. Add 5.0 ml of 0.01 M hydrochloric
acid. Carry out a potentiometric titration (2.2.20) using
0.1 M sodium hydroxide. Read the volume added between
the 2 points of inflexion.
of C17H27ClN2O.
G. (R)-ropivacaine.
Reagents
Dimethyl--cyclodextrin. C56H98O35. (Mr 1331). XXXXXXX.
[51166-71-3]. 2,6-Di-O-methyl--cyclodextrin.
White or almost white powder.
2,6-Dimethylaniline hydrochloride. C8H12ClN. (Mr 157.6).
XXXXXXX. [21436-98-6]. 2,6-Xylidine hydrochloride.

XXXX:1705
SERTRALINE HYDROCHLORIDE
Sertralini hydrochloridum
IMPURITIES
Specified impurities : A.
pharmaceutical use) : B, C, D, E, F.
C17H18Cl3N
A. R = [CH2]3-CH3 : bupivacaine,
Mr 342.7
DEFINITION
(1S,4S)-4-(3,4-Dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1naphthalenamine hydrochloride.
substance).
CHARACTERS
Solubility : slightly soluble in water, freely soluble in
R = CH3 : ()-1-methyl-N-(2,6-dimethylphenyl)piperidine-2- anhydrous ethanol, slightly soluble in acetone and in
carboxamide (mepivacaine),
isopropanol.
R = CH2-CH3 : ()-1-ethyl-N-(2,6-dimethylphenyl)piperidine- It shows polymorphism.
2-carboxamide
IDENTIFICATION
A. Specific optical rotation (2.2.7) : + 39.0 to + 43.0.
R = CH(CH3)-CH3 : ()-1-isopropyl-N-(2,6dimethylphenyl)piperidine-2-carboxamide,
Dissolve 0.250 g in a 103 g/l solution of hydrochloric
acid R diluted to 20 volumes with methanol R and dilute
to 25 ml with the same solution. Measure the specific
optical rotation at 25 C.
Preparation : 10 g/l solutions in methylene chloride R.
Comparison : sertraline hydrochloride CRS.
C. Dissolve 10 mg in 5 ml of anhydrous ethanol R and
add 5 ml of water R. The solution gives reaction (a) of
acetone adduct,
chlorides (2.3.1).
B. R = H : ()-1-H-N-(2,6-dimethylphenyl)piperidine-2carboxamide,
C.
D.
E.
F.
(54) Available from TCI Europe product NX0029, purity > 98 per cent.
143
TESTS
Enantiomeric purity. Liquid chromatography (2.2.29).
Solvent mixture : diethylamine R, hexane R, 2-propanol R
(1:40:60 V/V/V).
Reference solution (a). Dissolve 30.0 mg of sertraline
Reference solution (b). Dissolve 3 mg of sertraline
impurity G CRS in the solvent mixture, add 1 ml of reference
solution (a) and dilute to 2 ml with the solvent mixture.
Reference solution (c). Dilute 1.0 ml of reference solution (a)
to 100.0 ml with the solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: silica gel for chiral separation R
(5 m)(55).
Mobile phase : mix 30 volumes of hexane R and 70 volumes
of a mixture of 1 volume of diethylamine R, 25 volumes of
2-propanol R and 975 volumes of hexane R.
Injection: 20 l.
Run time : 30 min.
Elution order : sertraline, impurity G.
sertraline and impurity G in the chromatogram obtained
with reference solution (b) ;
sertraline in the chromatogram obtained with reference
solution (c).
Limit :
impurity G : not more than the area of the principal peak
(0.5 per cent).
Impurity E. Thin-layer chromatography (2.2.27).
examined in a mixture of equal volumes of methanol R and
methylene chloride R and dilute to 10.0 ml with the same
mixture of solvents.
Reference solution. Dissolve 5.0 mg of sertraline
impurity E CRS in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 50.0 ml with the
same mixture of solvents.
Mobile phase : ammonium hydroxide R, methanol R,
methylene chloride R (15:50:120 V/V/V).
Application : 100 l as bands of about 8 cm. Allow to dry.
Drying : in air.
Limit :
impurity E : any zone due to impurity E is not more
intense than the zone in the chromatogram obtained with
the reference solution (0.2 per cent).
Related substances. Gas chromatography (2.2.28) : use the

normalisation procedure.
Test solution. Introduce 0.250 g of the substance to be
examined into a 15 ml stoppered centrifuge tube, add 2.0 ml
of methanol R and 0.20 ml of a 25 per cent solution of
potassium carbonate R and mix in a vortex mixer for 30 s.
Add 8 ml of methylene chloride R, stopper the tube and mix
in a vortex mixer for 60 s. Add 1 g of anhydrous sodium
sulphate R, mix well and then centrifuge for about 5 min.
Use the methylene chloride layer.
Reference solution. Introduce 50 mg of sertraline for peak
identification CRS into a 5 ml stoppered centrifuge tube,
add 0.5 ml of methanol R and 0.05 ml of a 25 per cent
solution of potassium carbonate R and mix in a vortex mixer
for 30 s. Add 2 ml of methylene chloride R, stopper the tube
and mix in a vortex mixer for 60 s. Add 0.25 g of anhydrous
sodium sulphate R, mix well and then centrifuge for about
5 min. Use the methylene chloride layer.
Column :
size : l = 30 m, = 0.53 mm ;
stationary phase : poly[phenyl(50)methyl(50)]siloxane R(56) (film thickness 1.0 m).
Split ratio : 1:10.
Temperature :
Column
Time
(min)
0-1
Temperature
(C)
200
1 - 31
200 260
31 - 39
260
Injection port
250
Detector
280

Injection : 1 l.
supplied with sertraline for peak identification CRS and
the chromatogram obtained with the reference solution to
Relative retention with reference to sertraline (retention
impurities C and D = about 0.7 ; impurity A = about 1.05 ;
impurity F = about 1.1.
the chromatogram obtained is similar to the
chromatogram supplied with sertraline for peak
identification CRS.
Limits :
impurities A, B, F : for each impurity, maximum 0.2 per
cent ;
sum of impurities C and D : maximum 0.8 per cent ;
unspecified impurities : for each impurity, maximum
0.10 per cent ;
total : maximum 1.5 per cent ;
disregard limit : 0.05 per cent.
(55) Chiralpak Daicel AD is suitable.

(56) OV 17 is suitable.
144

on 1.0 g.
ASSAY
Buffer solution. To 28.6 ml of glacial acetic acid R slowly
add, while stirring and cooling, 34.8 ml of triethylamine R,
and dilute to 100 ml with water R. Dilute 10 ml of this
solution to 1000 ml with water R.
C. ()-cis-4-(4-chlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1Test solution. Dissolve 55.0 mg of the substance to be
naphthalenamine,
mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with
the mobile phase.
Reference solution. Dissolve 55.0 mg of sertraline
hydrochloride CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 5.0 ml of this solution to
100.0 ml with the mobile phase.
Column :
size : l = 0.15 m, = 3.9 mm ;
D. ()-cis-4-(3-chlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1chromatography R (4 m)(57) ;
naphthalenamine,
temperature : 30 C.
Mobile phase : methanol R, buffer solution, acetonitrile R
(15:40:45 V/V/V).
Injection: 20 l.
E. mandelic acid,
Run time : twice the retention time of sertraline.
Calculate the percentage content of C17H18Cl3N from the
declared content of sertraline hydrochloride CRS.
STORAGE
IMPURITIES
Specified impurities : A, B, C, D, E, F, G.
F. 4-(3,4-dichlorophenyl)-3,4-dihydro-1(2H)-naphthalenone
(tetralone),
A. ()-trans-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-Nmethyl-1-naphthalenamine,
G. 4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1naphthalenamine ((1R,4R) enantiomer).
Reagents
B. ()-cis-4-phenyl-1,2,3,4-tetrahydro-N-methyl-1naphthalenamine,
Poly[phenyl(50)methyl(50)]siloxane. XXXXXXX.
Stationary phase for gas chromatography.
Contains 50 per cent of methyl groups and 50 per cent of
phenyl groups.
(57) Nova-Pak Waters Cat. No. 086355 is suitable.
145
Sodium acetate trihydrate
Reference: PA/PH/Exp. INC/T (05) 35 ANP
Calcium and magnesium : maximum 50 ppm, calculated

as Ca.
To 200 ml of water R add 10 ml of ammonium chloride
As the test for reducing substances does not give satisfactory buffer solution pH 10.0 R, 0.1 g of mordant black 11
results, it is proposed to revise it.
triturate R, 2.0 ml of 0.05 M zinc chloride and, dropwise,
XXXX:0411 0.02 M sodium edetate until the colour changes from violet
to blue. Add to the solution 10.0 g of the substance to be
examined and shake to dissolve. Titrate with 0.02 M sodium
SODIUM ACETATE TRIHYDRATE
edetate until the blue colour is restored. Not more than
0.65 ml of 0.02 M sodium edetate is required.
Natrii acetas trihydricus
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Iron (2.4.9) : maximum 10 ppm, determined on 10 ml of
C2H3NaO2,3H2O
Mr 136.1 solution S.
Loss on drying (2.2.32) : 39.0 per cent to 40.5 per cent,
DEFINITION
determined on 1.000 g by drying in an oven at 130 C.
Sodium ethanoate trihydrate.
Introduce the substance to be examined into the oven while
the latter is cold.
CHARACTERS
Appearance : colourless crystals.
Solubility : very soluble in water, soluble in ethanol (96 per
cent).
ASSAY
Dissolve 0.250 g in 50 ml of anhydrous acetic acid R, add
5 ml of acetic anhydride R, mix and allow to stand for
30 min. Using 0.3 ml of naphtholbenzein solution R as
indicator, titrate with 0.1 M perchloric acid until a green
IDENTIFICATION
colour is obtained.
A. 1 ml of solution S (see Tests) gives reaction (b) of acetates 1 ml of 0.1 M perchloric acid is equivalent to 8.20 mg
(2.3.1).
of C2H3NaO2.
B. 1 ml of solution S gives reaction (a) of sodium (2.3.1).
STORAGE
C. Loss on drying (see Tests).
TESTS
LABELLING
prepared from distilled water R and dilute to 100 ml with
The label states, where applicable, that the substance is
the same solvent.
suitable for use in the manufacture of dialysis solutions.
pH (2.2.3) : 7.5 to 9.0.
Dilute 5 ml of solution S to 10 ml with carbon dioxide-free
Reference: PA/PH/Exp. INC/T (05) 36 ANP
water R.
Reducing substances. Dissolve 1.0 5.0 g in 100 50 ml of
boiling water R, then add 5 ml of dilute sulphuric acid R
The current assay method often causes problems due to the
and 0.5 ml of 0.002 M potassium permanganate, mix and
unsatisfactory solubility of the substance in the titration
boil gently for 5 min. The pink colour is not completely
medium. Another method is therefore proposed as a
discharged persists for at least 1 h.
replacement.
XXXX:0514
Dilute 2.5 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 200 ppm.
SODIUM FLUORIDE
Natrii fluoridum
Aluminium (2.4.17) : maximum 0.2 ppm, if intended for use
in the manufacture of dialysis solutions.
Prescribed solution. Dissolve 20 g in 100 ml of water R and NaF
Mr 41.99
adjust to pH 6.0 by the addition of 1 M hydrochloric acid
(about 10 ml).
DEFINITION
Reference solution. Mix 2 ml of aluminium standard
solution (2 ppm Al) R, 10 ml of acetate buffer solution
CHARACTERS
pH 6.0 R and 98 ml of water R.
Blank solution. Mix 10 ml of acetate buffer solution
Appearance : white or almost white powder or colourless
pH 6.0 R and 100 ml of water R.
crystals.
Solubility : soluble in water, practically insoluble in ethanol
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined
(96 per cent).
on 0.5 g.
146
IDENTIFICATION
A. To 2 ml of solution S (see Tests) add 0.5 ml of calcium
chloride solution R. A gelatinous white precipitate is
formed that dissolves on adding 5 ml of ferric chloride
solution R1.
Standardisation. To 20 ml of the lanthanum nitrate solution,

add 15 ml of water R and 25 ml of 0.1 M sodium edetate.
Add about 50 mg of xylenol orange triturate R and about
2 g of hexamethylenetetramine R. Titrate with 0.1 M zinc
sulphate until the colour changes from yellow to violet-pink.
B. To about 4 mg add a mixture of 0.1 ml of alizarin S

solution R and 0.1 ml of zirconyl nitrate solution R and
mix. The colour changes from red to yellow.
C. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
without heating and dilute to 100 ml with the same solvent.
Acidity or alkalinity. Dissolve 2.5 g of potassium nitrate R
in 40 ml of solution S and dilute to 50 ml with carbon
dioxide-free water R. Cool to 0 C and add 0.2 ml of
phenolphthalein solution R. If the solution is colourless,
not more than 1.0 ml of 0.1 M sodium hydroxide is required
to produce a red colour that persists for at least 15 s. If the
solution is red, not more than 0.25 ml of 0.1 M hydrochloric
acid is required to change the colour of the indicator.
Dilute 10 ml of solution S to 15 ml with water R.
Fluorosilicates. Heat to boiling the neutralised solution
obtained in the test for acidity or alkalinity and titrate whilst
hot. Not more than 0.75 ml of 0.1 M sodium hydroxide is
required to change the colour of the indicator to red.
Sulphates (2.4.13) : maximum 200 ppm.
Dissolve 0.25 g in 10 ml of a saturated solution of boric
acid R in distilled water R. Add 5 ml of distilled water R
and 0.6 ml of hydrochloric acid R1. Prepare the standard
by mixing 0.6 ml of hydrochloric acid R1, 5 ml of sulphate
standard solution (10 ppm SO4) R and 10 ml of a saturated
solution of boric acid R in distilled water R.
on 1.000 g by drying in an oven at 130 C for 3 h.
ASSAY
To 80.0 mg add a mixture of 5 ml of acetic anhydride R
and 20 ml of anhydrous acetic acid R and heat to dissolve.
Allow to cool and add 20 ml of dioxan R. Using 0.1 ml of
crystal violet solution R as indicator, titrate with 0.1 M
perchloric acid until a green colour is obtained. Carry out
a blank titration.
Reference: PA/PH/Exp.12/T (05) 48 ANP

In the current monograph, the test for uniformity of
dosage units applies only to solid single-dose preparations.
However, certain preparations are presented as liquid
or semi-solid preparations in single-dose containers, for
which the total content corresponds to a precise dose of the
medicine (expressed as unit amount of active substance). It
is proposed to modify the corresponding paragraph to take
these preparations into account.
XXXX:1164
VAGINAL PREPARATIONS
Vaginalia
DEFINITION
Vaginal preparations are liquid, semi-solid or solid
preparations intended for administration to the vagina
usually in order to obtain a local effect. They contain 1 or
more active substances in a suitable basis.
Where appropriate, containers for vaginal preparations
comply with the requirements for Materials used for the
manufacture of containers (3.1 and subsections) and
Containers (3.2 and subsections).
Several categories of vaginal preparations may be
distinguished :
pessaries ;
vaginal tablets ;
vaginal capsules ;
vaginal solutions, emulsions and suspensions ;
tablets for vaginal solutions and suspensions ;
semi-solid vaginal preparations ;
vaginal foams ;
medicated vaginal tampons.
PRODUCTION
During development, it must be demonstrated that the
nominal content can be withdrawn from the container of
of NaF.
liquid and semi-solid vaginal preparations presented in
Dissolve 0.100 g in water R and dilute to 60 ml with the same single-dose containers.
solvent. Titrate with 0.1 M lanthanum nitrate determining
In the manufacturing, packaging, storage and distribution
the end-point potentiometrically using a fluoride-selective
of vaginal preparations, suitable measures are taken to
indicator electrode and a silver-silver chloride reference
ensure their microbial quality ; recommendations on this
electrode (2.2.20).
aspect are provided in the text on Microbiological quality of
pharmaceutical preparations (5.1.4).
1 ml of 0.1 M lanthanum nitrate is equivalent to 12.60 mg
g of NaF.
TESTS
Uniformity of dosage units. Liquid and semi-solid
Reagents
single-dose vaginal preparations comply with the test for
uniformity of dosage units (2.9.40). Solid single-dose
0.1 M Lanthanum nitrate. XXXXXXX.
vaginal preparations comply with the test for uniformity of
Dissolve 43.30 g of lanthanum nitrate R in water R and
dosage units (2.9.40) or, where justified and authorised, with
the tests for uniformity of content and/or uniformity of mass
147
shown below. Herbal drugs and herbal drug preparations

present in the dosage form are not subject to the provisions
of this paragraph.
Uniformity of content (2.9.6). Unless otherwise prescribed
or justified and authorised, solid single-dose vaginal
preparations with a content of active substance less than
2 mg or less than 2 per cent of the total mass comply with
test A (vaginal tablets) or test B (pessaries, vaginal capsules)
for uniformity of content of single-dose preparations.
If the preparation has more than one active substance,
the requirement applies only to those substances which
correspond to the above conditions.
Uniformity of mass (2.9.5). Solid single-dose vaginal
preparations comply with the test for uniformity of mass
of single-dose preparations. If the test for uniformity of
content is prescribed for all the active substances, the test
for uniformity of mass is not required.
Dissolution. A suitable test may be carried out to
demonstrate the appropriate release of the active
substance(s) from solid single-dose vaginal preparations,
for example one of the tests described in Dissolution test
for solid dosage forms (2.9.3) or in Dissolution test for
lipophilic solid dosage forms (2.9.42).
When a dissolution test is prescribed, a disintegration test
may not be required.
Pessaries
Vaginal tablets
DEFINITION
Vaginal tablets are solid, single-dose preparations.
They generally conform to the definitions of uncoated or
film-coated tablets given in the monograph on Tablets (0478).
PRODUCTION
release of the active substance(s) from vaginal tablets
intended for prolonged local action.
TESTS
Disintegration. Unless intended for prolonged local action,
they comply with the test for disintegration of suppositories
and pessaries (special method for vaginal tablets, 2.9.2).
Examine the state of the tablets after 30 min, unless
otherwise justified and authorised.
Vaginal capsules
DEFINITION
Vaginal capsules (shell pessaries) are solid, single-dose
preparations. They are generally similar to soft capsules
as defined in the monograph on Capsules (0016), differing
only in their shape and size. Vaginal capsules have various
shapes, usually ovoid. They are smooth and have a uniform
external appearance.
PRODUCTION
Pessaries are solid, single-dose preparations. They have
release of the active substance(s) from vaginal capsules
various shapes, usually ovoid, with a volume and consistency intended for prolonged local action.
suitable for insertion into the vagina. They contain 1 or more
active substances dispersed or dissolved in a suitable basis
TESTS
that may be soluble or dispersible in water or may melt at
Disintegration. Unless intended for prolonged local action,
body temperature. Excipients such as diluents, adsorbents,
they comply with the test for disintegration of suppositories
surface-active agents, lubricants, antimicrobial preservatives and pessaries (2.9.2). Examine the state of the capsules after
and colouring matter authorised by the competent authority 30 min, unless otherwise justified and authorised.
may be added, if necessary.
DEFINITION
PRODUCTION
Pessaries are usually prepared by moulding. Where
appropriate in the manufacture of pessaries, measures are
taken to ensure a suitable and controlled particle size of the
active substance(s). If necessary, the active substance(s) are
previously ground and sieved through a suitable sieve.
Vaginal solutions, emulsions

and suspensions
DEFINITION
Vaginal solutions, emulsions and suspensions are liquid
preparations intended for a local effect, for irrigation or
for diagnostic purposes. They may contain excipients, for
When prepared by moulding, the medicated mass, sufficiently example to adjust the viscosity of the preparation, to adjust
liquefied by heating, is poured into suitable moulds. The
or stabilise the pH, to increase the solubility of the active
pessary solidifies on cooling. Various excipients are available substance(s) or to stabilise the preparation. The excipients
for this process, such as hard fat, macrogols, cocoa butter,
do not adversely affect the intended medical action or, at the
and various gelatinous mixtures consisting, for example, of
concentrations used, cause undue local irritation.
gelatin, water and glycerol.
Vaginal emulsions may show evidence of phase separation
A suitable test is carried out to demonstrate the appropriate but are readily redispersed on shaking. Vaginal suspensions
release of the active substance(s) from pessaries intended for may show a sediment that is readily dispersed on shaking to
give a suspension that remains sufficiently stable to enable a
prolonged local action.
homogeneous preparation to be delivered.
Where appropriate, the determination of the resistance to
They are supplied in single-dose containers. The container
rupture of pessaries (2.9.24) is carried out.
is adapted to deliver the preparation to the vagina or it is
accompanied by a suitable applicator.
TESTS
Disintegration. Unless intended for prolonged local action, PRODUCTION
they comply with the test for disintegration of suppositories In the manufacture of vaginal suspensions measures are
and pessaries (2.9.2). Examine the state of the pessaries
taken to ensure a suitable and controlled particle size with
after 60 min, unless otherwise justified and authorised.
regard to the intended use.
148
Vinpocetine
Tablets for vaginal solutions

and suspensions

The test for related substances has been modified to cover
DEFINITION
all the specified impurities.
XXXX:2139
Tablets intended for the preparation of vaginal solutions and
suspensions are single-dose preparations which are dissolved
or dispersed in water at the time of administration. They
VINPOCETINE
may contain excipients to facilitate dissolution or dispersion
or to prevent caking.
Vinpocetinum
Apart from the test for disintegration, tablets for vaginal

solutions or suspensions conform with the definition for
Tablets (0478).
After dissolution or dispersion, they comply with the
requirements for vaginal solutions or vaginal suspensions, as
appropriate.
TESTS
Disintegration. Tablets for vaginal solutions or suspensions
comply with the test for disintegration of tablets and capsules
(2.9.1), but using water R at 15-25 C. Carry out the test on
6 tablets. Examine the state of the tablets after 3 min. The
tablets comply with the test if all 6 have disintegrated.
LABELLING
The label states :
the method of preparation of the vaginal solution or
suspension ;
the conditions and duration of storage of the solution or
suspension after constitution.
Semi-solid vaginal preparations

DEFINITION
C22H26N2O2
Mr 350.5
DEFINITION
Ethyl (13aS,13bS)-13a-ethyl-2,3,5,6,13a,13b-hexahydro1H-indolo[3,2,1-de]pyrido[3,2,1-ij][1,5]naphthyridine-12carboxylate.
CHARACTERS
Appearance : white or slightly yellow, crystalline powder.
methylene chloride, slightly soluble in anhydrous ethanol.
IDENTIFICATION
A. It complies with the test for specific optical rotation (see
Tests).
Comparison : vinpocetine CRS.
TESTS
Specific optical rotation (2.2.7) : + 127 to + 134 (dried
substance).
They are often supplied in single-dose containers. The
Dissolve 0.25 g in dimethylformamide R and dilute to
container is provided with a suitable applicator.
Semi-solid vaginal preparations comply with the requirements Related substances. Liquid chromatography (2.2.29).
of the monograph on Semi-solid preparations for cutaneous
application (0132).
examined in acetonitrile R the mobile phase and dilute to
50.0 ml with the mobile phase.
Vaginal foams
20.0 ml 50.0 ml with acetonitrile R the mobile phase. Dilute
1.0 ml of this solution to 50.0 ml with acetonitrile R.
DEFINITION
Reference solution (b). Dissolve 5.0 mg of vinpocetine
impurity B CRS, 6.0 mg of vinpocetine impurity A CRS,
Vaginal foams comply with the requirements of the
5.0 mg of vinpocetine impurity C CRS and 5.0 mg of
monograph on Medicated foams (1105).
vinpocetine impurity C D CRS in acetonitrile R the mobile
phase and dilute to 50.0 ml with the mobile phase. Dilute
1.0 ml of the solution to 20.0 ml with the test solution.
Medicated vaginal tampons
Reference solution (c). Dissolve 5.0 mg of vinpocetine
impurity A CRS in acetonitrile R and dilute to 50.0 ml with
DEFINITION
the same solvent. Dilute 1.0 ml of the solution to 20.0 ml
Medicated vaginal tampons are solid, single-dose
with acetonitrile R. Dilute 1.0 ml of reference solution (a)
preparations intended to be inserted in the vagina for a
and 1.0 ml of reference solution (b) to 20.0 ml with the
limited time.
mobile phase.
Column :
They comply with the requirements of the monograph on
Medicated tampons (1155).
size : l = 0.25 m, = 4.6 mm ;
Semi-solid vaginal preparations are ointments, creams or
gels.
149
Vinpocetine
1. impurity A
3. impurity B
2. impurity D
4. impurity C
5. vinpocetine
Figure 2139.-1. Chromatogram for the test for related substances of vinpocetine
temperature : 40 C.
Mobile phase : 0.1 M ammonium carbonate a 15.4 g/l
solution of ammonium acetate R, acetonitrile R (32:68 V/V
45:55 V/V).
Injection: 15 l.
Run time : twice 3 times the retention time of vinpocetine.
Relative retention with reference to vinpocetine (retention
time = about 17 min 16 min) : impurity A = about 0.4 ;
impurity D = about 0.68 ; impurity B = about 0.8 0.75 ;
impurity C = about 0.85 0.83.
System suitability : reference solution (b) (c) :
resolution : minimum 1.5 2.0 between the peaks due to
impurities B and C D.
Limits :
impurity A : not more than the area of the principal
corresponding peak in the chromatogram obtained with
reference solution (c) (0.5 per cent 0.6 per cent) ;
solution (b) (c) (0.5 per cent) ;
impurity C : not more than 0.4 times 0.6 times the area
of the corresponding peak in the chromatogram obtained
with reference solution (b) (c) (0.2 per cent 0.3 per
cent per cent) ;

solution (c) (0.5 per cent) ;
any other impurity unspecified impurities : for each
impurity, not more than the area of the principal peak
due to vinpocetine in the chromatogram obtained with
reference solution (a) (c) (0.10 per cent) ;
total : maximum 1.0 per cent not more than 10 times the
area of the peak due to vinpocetine in the chromatogram
obtained with reference solution (c) (1.0 per cent) ;
due to vinpocetine in the chromatogram obtained with
reference solution (a) (c) (0.05 per cent).
on 1.000 g by drying in vacuo in an oven at 100 C for 3 h.
on 1.0 g.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of equal volumes of
acetic anhydride R and anhydrous acetic acid R. Titrate
of C22H26N2O2.
IMPURITIES
Specified impurities : A, B, C, D.
(58) Waters spherisorb S5 ODS2 is suitable. Supelco discovery C18 is suitable.
150
Warfarin sodium
Solubility : very soluble in water and in ethanol (96 per

cent), soluble in acetone, very slightly soluble in methylene
chloride.
A. ethyl (12RS,13aSR,13bSR)-13a-ethyl-12-hydroxy-2,3,5,6,
12,13,13a,13b-octahydro-1H-indolo[3,2,1-de]pyrido[3,2,1ij][1,5]naphthyridine-12-carboxylate (ethyl vincaminate),
B. R1 = CH3, R2 = H : methyl (13aS,13bS)-13a-ethyl-2,3,5,6,

13a,13b-hexahydro-1H-indolo[3,2,1-de]pyrido[3,2,1-ij][1,
5]naphthyridine-12-carboxylate (apovincamine),
C. R1 = C2H5, R2 = OCH3 : ethyl (13aS,13bS)-13a-ethyl9-methoxy-2,3,5,6,13a,13b-hexahydro-1H-indolo[3,2,
1-de]pyrido[3,2,1-ij][1,5]naphthyridine-12-carboxylate
(methoxyvinpocetine),
IDENTIFICATION
First identification : B, D, E.
A. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute
hydrochloric acid R and filter. Reserve the filtrate for
identification test E. The precipitate, washed with water R
and dried at 100 C to 105 C, melts (2.2.14) at 159 C
to 163 C.
Dissolve 1 g in 25 ml of water R, add 2 ml of dilute
identification test E. Examine the precipitate.
Comparison : warfarin sodium CRS.
chromatogram obtained with test solution (b) is
chromatogram obtained with reference solution (b).
D. B. Dissolve 1 g in 10 ml of water R, add 5 ml of nitric
acid R and filter. To the filtrate add 2 ml of potassium
dichromate solution R1 and shake for 5 min. Allow to
stand for 20 min. The solution is not greenish-blue when
compared with a blank.
E. C. The filtrate obtained in identification test A It gives
reaction (b) of sodium (2.3.1).
TESTS
D. dihydrovinpocetine.
solvent.
pH (2.2.3) : 7.6 to 8.6.
It is proposed to replace the TLC test for related substances
by a LC test and to delete the 2nd identification series.
XXXX:0698 examined in acetone R and dilute to 10 ml with the same
solvent.
WARFARIN SODIUM
with acetone R.
Warfarinum natricum
200 ml with acetone R.
Reference solution (b). Dissolve 40 mg of warfarin
sodium CRS in acetone R and dilute to 10 ml with the same
solvent.
acenocoumarol CRS in acetone R, add 1 ml of test
solution (a) and dilute to 10 ml with acetone R.
over a path of 15 cm using a mixture of 20 volumes of glacial
Cl9H15NaO4
Mr 330.3 acetic acid R, 50 volumes of methylene chloride R and
50 volumes of cyclohexane R. Allow the plate to dry in air
DEFINITION
and examine in ultraviolet light at 254 nm. Any spot in the
Sodium 2-oxo-3-[(1RS)-3-oxo-1-phenylbutyl]-2H-1chromatogram obtained with test solution (a), apart from
benzopyran-4-olate.
chromatogram obtained with reference solution (a) (0.1 per
cent). The test is not valid unless the chromatogram obtained
substance).
with reference solution (c) shows two clearly separated spots
CHARACTERS
and the chromatogram obtained with reference solution (a)
shows a clearly visible spot.
Appearance : white or almost white, hygroscopic powder.
151
Warfarin sodium
2. impurity C
1. impurity B
3. warfarin
4. impurity A
Figure 0698.-1. Chromatogram for the test for related substances of warfarin sodium
Solvent mixture : methanol R, water R (25:75 V/V).
Reference solution (a). Dissolve 2.0 mg of warfarin
impurity B CRS and 2.0 mg of warfarin impurity C CRS in
25 ml of methanol R and dilute to 100.0 ml with water R.
Column :
size : l = 0.25 m, = 4.0 mm ;
stationary phase : spherical nitrile silica gel for
temperature : 30 C.
(1:25:75 V/V/V).
Injection : 20 l.
Run time : twice the retention time of warfarin.
Relative retention with reference to warfarin (retention
impurity C = about 0.6.
impurities B and C.
Limits :
the corresponding correction factor : impurity B = 0.5 ;
impurity C = 0.4 ;
impurities B, C : for each impurity, not more than the

(0.3 per cent) ;
(0.05 per cent).
Phenolic ketones : the absorbance (2.2.25) is maximum 0.20
measured at 385 nm within 15 min of preparing the solution.
Dissolve 1.25 g in a 20 g/l solution of sodium hydroxide R
0.750 g.
ASSAY
Dissolve 0.1000 g in 0.01 M sodium hydroxide and dilute to
100.0 ml with the same solvent. Dilute 10.0 ml of the solution
to 100.0 ml with 0.01 M sodium hydroxide. Dilute 10.0 ml
of this solution to 100.0 ml with 0.01 M sodium hydroxide.
Measure the absorbance (2.2.25) at the absorption maximum
at 308 nm.
Calculate the content of C19H15NaO4 taking the specific
absorbance to be 431.
STORAGE
IMPURITIES
Specified impurities : B, C.
pharmaceutical use) : A.
(59) LiChrospher 100 CN is suitable.
152
A. 3-(2-hydroxyphenyl)-5-phenylcyclohex-2-en-1-one,
B. 4-hydroxycoumarin,
C. benzalacetone.
IDENTIFICATION
First identification : B, D, E.
A. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute
identification test E. The precipitate, washed with water R
and dried at 100 C to 105 C, melts (2.2.14) at 159 C
to 163 C.
Dissolve 1 g in 25 ml of water R, add 2 ml of dilute
identification test E. Examine the precipitate.
Comparison : the precipitate prepared in the same
manner from warfarin sodium clathrate CRS.
chromatogram obtained with test solution (b) is
chromatogram obtained with reference solution (b).
D. B. Dissolve 1 g in 10 ml of water R, add 5 ml of nitric
acid R and filter. To the filtrate add 2 ml of potassium
dichromate solution R1 and shake for 5 min. Allow to
stand for 20 min. The solution is greenish-blue when
compared with a blank.
E. C. The filtrate obtained in identification test A It gives
reaction (b) of sodium (2.3.1).
TESTS
It is proposed to replace the TLC test for related substances Dissolve 1.0 g in water R and dilute to 20 ml with the same
by a LC test and to delete the 2nd identification series.
solvent.
Based on batch results, the limit in the test for water has
pH (2.2.3) : 7.6 to 8.6.
been increased to 0.3 per cent, which is also in line with
the USP monograph.
XXXX:0699 100 ml with the same solvent.
WARFARIN SODIUM CLATHRATE
examined in acetone R and dilute to 10 ml with the same
Warfarinum natricum clathratum
solvent.
with acetone R.
200 ml with acetone R.
Reference solution (b). Dissolve 40 mg of warfarin
sodium CRS in acetone R and dilute to 10 ml with the same
solvent.
acenocoumarol CRS in acetone R, add 1 ml of test
DEFINITION
solution (a) and dilute to 10 ml with acetone R.
Mixture, in the form of a clathrate, of warfarin sodium (sodium
2-oxo-3-[(1RS)-3-oxo-1-phenylbutyl]-2H-1-benzopyran-4-olate)
over a path of 15 cm using a mixture of 20 volumes of glacial
and propan-2-ol in molecular proportions 2:1 (equivalent to
acetic acid R, 50 volumes of methylene chloride R and
about 92 per cent of warfarin sodium).
50 volumes of cyclohexane R. Allow the plate to dry in air
Content :
and examine in ultraviolet light at 254 nm. Any spot in the
warfarin sodium : 98.0 per cent to 102.0 per cent
(anhydrous and propan-2-ol-free substance) ;
chromatogram obtained with reference solution (a) (0.1 per
propan-2-ol : 8.0 per cent to 8.5 per cent.
cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated spots
CHARACTERS
and the chromatogram obtained with reference solution (a)
shows a clearly visible spot.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent), soluble in acetone, very slightly soluble in
Solvent mixture : methanol R, water R (25:75 V/V).
methylene chloride.
153
1. impurity B
2. impurity C
3. warfarin
4. impurity A
Figure 0699.-1. Chromatogram for the test for related substances of warfarin sodium clathrate
Reference solution (a). Dissolve 2.0 mg of warfarin
impurity B CRS and 2.0 mg of warfarin impurity C CRS in
25 ml of methanol R and dilute to 100.0 ml with water R.
Column :
size : l = 0.25 m, = 4.0 mm ;
stationary phase : spherical nitrile silica gel for
temperature : 30 C.
(1:25:75 V/V/V).
Injection : 20 l.
Run time : twice the retention time of warfarin.
Relative retention with reference to warfarin (retention
impurity C = about 0.6.
impurities B and C.
Limits :
the corresponding correction factor : impurity B = 0.5 ;
impurity C = 0.4 ;
impurities B, C : for each impurity, not more than the
(0.3 per cent) ;

(0.05 per cent).
Phenolic ketones : the absorbance (2.2.25) is maximum 0.20
measured at 385 nm within 15 min of preparing the solution.
Dissolve 1.25 g in a 20 g/l solution of sodium hydroxide R
Propan-2-ol. Gas chromatography (2.2.28).
Internal standard solution. Dilute 1.0 ml of propanol R to
examined in water R and dilute to 5.0 ml with the same
solvent.
Test solution (b). Dissolve 0.50 g of the substance to be
examined in the internal standard solution and dilute to
10.0 ml with the internal standard solution.
Reference solution. Dilute 0.50 ml of 2-propanol R to
100.0 ml with the internal standard solution.
Column :
size : l = 1.5 m, = 4 mm ;
stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (125-150 m).
Carrier gas : nitrogen for chromatography R.
Temperature :
column : 150 C ;
injection port : 180 C ;
detector : 200 C.
Injection : the chosen volume of the test solutions and the
reference solution.
Calculate the content of propan-2-ol taking its density at
20 C to be 0.785 g/ml.
Limit :
propan-2-ol : 8.0 per cent to 8.5 per cent.
Water (2.5.12) : maximum 0.1 0.3 per cent, determined on
2.500 g.
(60) LiChrospher 100 CN is suitable.
154
2.9.45. Wettability of porous solids including powders
With the Washburn method and the Wilhelmy plate method

the contact angles are indirectly measured. The methods
are based on the capillary effect of the powder pores. The
effect (mass gain) is recorded instantly by special electronic
balances starting the moment that the powder sample
touches the surface of the liquid. The measurement has
very little or no effect on the state of the powder. However,
the indirect measurements do not yield the true values of
the contact angle. Only apparent values that significantly
depend on the true contact angle are obtained. The
Wilhelmy plate method needs the lowest sample mass and
STORAGE
is therefore important in pharmaceutical development. The
Washburn method and the Wilhelmy plate method can be
carried out on the same type of electronic balance ; only the
IMPURITIES
sample holders differ.
Specified impurities : B, C.
The sessile drop method is a simple method, which makes
it reasonably easy to measure directly the contact angle of
a sessile drop on a compacted powder disc. However, the
drop technique and the analysis of the drop shape are being
further developed.
pharmaceutical use (2034). It is therefore not necessary to Any pre-treatment of the sample to be examined is
disadvantageous, since the properties may be significantly
altered. For example, the compression of a powder as a disc
may decrease the surface free energy when the crystallisation
pharmaceutical use) : A.
behaviour of the powder is changed (metastable forms), or
may increase surface free energy by creating crystal defects.
The wettability of solid surfaces is commonly characterised
by direct or indirect contact angle measurements. The
contact angle () between a liquid and a solid is the angle
naturally formed when a drop of a liquid is placed on a solid
surface. This is depicted in Figure 2.9.45.-1. Wettable solids
show a contact angle of less than 90, non-wettable solids
show a contact angle of 90 or more.
ASSAY
Dissolve 0.1000 g in 0.01 M sodium hydroxide and dilute to
100.0 ml with the same solvent. Dilute 10.0 ml of the solution
to 100.0 ml with 0.01 M sodium hydroxide. Dilute 10.0 ml
of this solution to 100.0 ml with 0.01 M sodium hydroxide.
Measure the absorbance (2.2.25) at the absorption maximum
at 308 nm.
Calculate the content of warfarin sodium (C19H15NaO4)
taking the specific absorbance to be 431.
A. 3-(2-hydroxyphenyl)-5-phenylcyclohex-2-en-1-one,
B. 4-hydroxycoumarin,
Figure 2.9.45.-1. Contact angle () of a sessile drop

observed on a non-porous surface
WASHBURN METHOD
INTRODUCTION
The Washburn method is able to measure the contact
angle of porous solids with a contact angle in the range
C. benzalacetone.
of 0 to 90. Usually the method is applied to examine the
following parameters :
batch-to-batch consistency of substances or formulations
referring to wettability ;
Reference: PA/PH/Exp. POW/T (03) 12 ANP
effect of liquid viscosity on wettability (e.g. reconstitution
of a powder) ;
XXXX:20945 effect of liquid surface tension on wettability ;
alteration of surface properties of formulations (e.g. to
2.9.45. WETTABILITY OF POROUS
provide more complete and/or rapid wetting ; testing of
different formulations of a non-wettable active substance
SOLIDS INCLUDING POWDERS
in a blend with wettable excipients).
INTRODUCTION
The tested material is the combination of the sample, the
holder and the filter system. Therefore, an estimation or
Three methods for the determination of wettability are
determination of the true value is not possible and apparent
described below. The methods are capable of measuring
values of the contact angle are determined instead. However,
the wettability of porous solids like powders or granules.
the contact angle of the sample is the functional property on
All 3 methods express the wettability by a contact angle
which the result is significantly dependent. The outcome of
measurement between the porous solid and a given liquid.
155
the test is a ranking order listing the wettability of different

substances or formulations characterised by an apparent
contact angle.
PRINCIPLE
If a porous solid is brought into contact with a liquid, such
that the solid is not submerged in the liquid, but rather is
just touching the liquid surface, then the rise of liquid into
the pores of the solid due to capillary action will be governed
by the following equations :
(1)
m
mass of liquid sucked into the solid ;
time elapsed since the solid and the liquid were

brought into contact ;
constant, dependent on the properties of the
liquid and the solid to be examined, calculated
using the following equation :
average capillary radius within the porous solid ;
number of capillaries per volumetric unit.
The Washburn equation contains 2 unknowns, the material

constant (c) and the contact angle ().
However, if a Washburn determination is performed
with a liquid that is known to have a contact angle of 0
(cos 0 = 1) on the solid, then the solid material constant (c)
is the only remaining unknown in equation (3) and can
thus be determined. n-Heptane is the liquid of choice for
determining material constants because of its low surface
tension (20.3 mNm 1 at room temperature). n-Hexane may
also be used (18.4 mNm 1 at room temperature) but is more
volatile. If the powder dissolves too quickly in these liquids,
hexamethyldisiloxane may be used instead (15.9 mNm 1 at
25 C).
If a series of liquids (at least 2 liquids in addition to the liquid
used to determine the material constant) is tested against a
given solid then the resultant contact-angle data can be used
to calculate the surface energy of the porous solid.
APPARATUS
(2)
viscosity of the liquid ;
density of the liquid ;
surface tension of the liquid ;
contact angle between the solid and the liquid ;
material constant, dependent on the porous

architecture of the solid.
Combining equations (1) and (2), followed by rearrangement,

leads to equation (3), which is the useful form of Washburns
equation.
(3)
In setting up a Washburn determination, a liquid with known
density (), viscosity (), and surface tension () is used. An
inspection of equation (3) leads to the conclusion that, if this
is the case, and the mass of liquid that rises into the porous
solid can be monitored as a function of time (such that
capillary velocity ( ) is the raw data), then 2 unknowns
remain : the contact angle () of the liquid on the solid, and
the solid material constant (c).
Once the material constant (c) has been determined for the
solid to be examined (see below), a sample of the solid can
be tested for wettability by another liquid. The material
constant determined by the n-heptane test is used in the
Washburn equation, in combination with the capillary
velocity ( ) data obtained while testing the substance
to be examined in the prescribed liquid. This allows the
calculation of the contact angle.
Determination of the material constant (c). The material
constant for a porous solid is theoretically given by the
following formula :
A. electronic balance
D. filter
B. computer
E. immersion liquid
C. sample holder
F. lift
Figure 2.9.45.-2. Apparatus for the contact angle

measurement by the Washburn method
Figure 2.9.45.-2 shows the principal components of the
apparatus. The main device is an electronic balance with a
suitable processor ensuring a suitable resolution in force
measurement and a suitable resolution in lifting up the
immersion liquid towards the sample.
Table 2.9.45.-1 indicates parameters of the electronic balance
that are generally considered suitable.
Table 2.9.45.-1. Technical parameters of the electronic
balance
Range
Resolution
(4)
156
Speed
Lift
Mass measurement
> 110 mm
0 - 210 g
0.1 m
10 g
0.099-500 mm/min
Sample holders. The sample holder may be a small glass

cylinder with a sintered-glass filter at one end.
Other powder material holders (see Figure 2.9.45-3) are
made of aluminium, are less fragile than glass, with small
holes in the bottom that render them easier to clean than a
sintered-glass filter. The cover for the cell is equipped with
2 screw threads. One connects it with the sample chamber
while the other allows the user to guide a piston down onto
the sample itself and compress it. The apparatus is similar to
an automatic tensiometer, except for the sample holder.
The filter does not have to be paper, but it must be a material

that is easily wetted by the liquid to be tested. A black-band
filter (used for reverse osmosis) is recommended because of
its high porosity and minimum flow resistance.
Place a known amount of powder into the cell. This amount
is big enough so that the compressed powder obtained
during the last step of this procedure is sufficient. If this
is the case, then reproducibility of material constants and
contact angles will depend almost solely on the ability to
weigh out the same amount of powder for each test.
For most powders, a correct amount is in the range of 1-2 g,
which normally equals 2/3 of the capacity of the holder.
Place a second piece of filter paper on top of the powder in
the cell. This will prevent powder from rising through the
holes in the piston during the compression process and/or
during the determination.
Tapping/compaction of the powder. A bulk powder bed is
very porous and thus very sensitive to small influences that
can easily alter the porosity. Therefore a tapped powder may
be advantageous and will show more reproducible results.
The appropriate number of taps must first be evaluated :
50 to 100 taps are usually appropriate.
If the aluminium sample holder is used then it may be
mounted in the cylinder of a stamp volumeter, which can
run the evaluated number of taps.
If tapping is not appropriate, the powder bed is compacted
by screwing the piston of the aluminium sample holder or by
a piston weighted down by a specific mass such as 1 kg.
Where applicable, a compacted disc of the powder sample
is mounted on the electronic balance. A sample holder is
omitted in this case.
CRITICAL PARAMETERS
The following points must be considered :
the compaction state of the different powder samples
must be uniform ;
it may be advisable to sieve the sample (e.g. using a
250 m sieve) before testing ;
the optimal compaction parameters (amount of sample,
number of taps or piston mass) must be determined ;
A. fixing
C. thread
E. capillary holes
B. cover
D. plunger
F. capillary holes
uniformity of the results is improved by using a sample

holder made of aluminium ;
Figure 2.9.45.-3. Example of sample holder with plunger

for compaction of a powder
the sample holder or, if used, the glass frit must be

carefully cleaned ;
PROCEDURE
Place the porous solid in an appropriate sample holder
and suspend it just above the surface of a test liquid (see
Figure 2.9.45.-2), using the lift.
specifications of the immersion liquid must be indicated.
The liquid is further raised until it just touches the bottom of

the porous sample. Mass versus time data is then collected
as liquid penetrates into the solid. At the end of the
experiment data can be output in either graphical or tabular
format. The apparatus may perform the whole determination
automatically.
Filling of the sample holder. Place a disc of filter paper in
the bottom of the aluminium or glass sample holder. This
prevents powder from leaking out of the bottom of the cell.
SESSILE DROP METHOD

This method may be used to directly characterise the
wettability of coatings and compacted formulations such
as tablets. Moreover, it is sometimes possible to use the
sessile drop instrument in a dynamic measurement (dynamic
contact angle measurement, Figure 2.9.45.-4) of porous
solid/liquid systems where the contact angle becomes less
than 90. By taking several contact angle measurements as
a function of time, the rate of penetration of a liquid droplet
into a slightly porous solid may be studied.
157
This coating is used afterwards to measure the contact angle

on a smooth plate :
perimeter of the smooth plate, equal to the

geometric perimeter.
With this information, the effective wet perimeter and the

resistance factor can be calculated. Testing can start.
APPARATUS
The apparatus used for the Wilhelmy plate method
is similar to that used for the Washburn method (see
Figure 2.9.45.-2) except that the sample holder is replaced
by a powder-covered glass plate or powder-covered adhesive
tape (see Figure 2.9.45.-5).
Figure 2.9.45.-4. Sessile drop determination with visual

inspection of the droplet : evolution of the contact angle
over time
PROCEDURE
Since powders are unable to form a completely flat surface,
the powder is usually compressed as a disc in an attempt
to make the surface smoother. A drop with a given size is
placed on the disc (see Figure 2.9.45.-4.) allowing a direct
measurement of the contact angle using a goniometer fitted
with an eyepiece protractor, or by geometric construction
on a photomicrograph. Other physical and mathematical
procedures of data analysis may also be appropriate. The
drop size may influence the result.
Figure 2.9.45.-5. Wilhelmy plate just touching the liquid

CRITICAL PARAMETERS
The Wilhelmy plate method is advantageous since less
powder is needed and there is no risk of altering the surface
by compression. Considering the rapid measurement, the
solubility will not disturb the measurement as much as in
other techniques. The measurements are dependent on the
atmospheric humidity at the time of measuring.
Therefore, the reproducibility can be improved by
WILHELMY PLATE METHOD
pre-equilibration of the environment with vapour. By this
PRINCIPLE
method, it is possible to attain a correct ordering of the
This method is a dynamic contact angle (DCA) measurement, samples at all angles irrespective of whether the contact
angle is greater than or less than 90.
where an apparent contact angle between a given liquid
and a system consisting of plate, adhesive and sample is
determined according to the following details.
By immersing the plate in a liquid, it is possible to measure
the immersion load as the force (F0), using the following
equation :
XXXX:2312
WILLOW BARK DRY EXTRACT
surface tension of the immersion liquid ;
effective wet perimeter of the powder-covered

plate ;
contact angle ;
resistance factor for the roughness of the plate,

representing the elastic energy released during
wetting as a result of surface defects.
Both the perimeter and the resistance factor are regarded

to be independent of the liquid and have to be determined
initially. A preliminary determination is carried out with a
liquid of low surface tension, resulting in perfect wetting,
and the contact angle can be assumed to be 0.
Other measurements in liquids exhibiting finite contact
angles (determined using another method such as sessile
drop) on a particular coating material with which the rough
surface is covered must also be carried out.
158
Salicis corticis extractum siccum

DEFINITION
Dry extract produced from Willow bark (1583).
Content : minimum 5.0 per cent of total salicylic derivatives,
expressed as salicin (C13H18O7 ; Mr 286.3) (dried extract).
PRODUCTION
The extract is produced from the herbal drug using ethanol
(60-80 per cent V/V) by an appropriate procedure.
CHARACTERS
Appearance : yellowish-brown amorphous powder.
IDENTIFICATION
Test solution (a). To 0.200 g of the extract to be examined

add 5 ml of methanol R. Sonicate for 5 min, filter and dilute
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of
a 50 g/l solution of anhydrous sodium carbonate R and
heat in a water-bath at about 60 C for 10 min. Cool and
filter if necessary.
Reference solution. Dissolve 2.0 mg of salicin R and 2.0 mg
of chlorogenic acid R in 1.0 ml of methanol R.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : water R, methanol R, ethyl acetate R
(8:15:77 V/V/V).
Application : 10 l [or 2 l] as bands.
Development : over a path of 15 cm [or 6 cm].
Detection : spray with a mixture of 5 volumes of sulphuric
acid R and 95 volumes of methanol R. Heat at 100-105 C
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 the test solutions. Furthermore, several reddish-violet
zones may be present in the chromatogram obtained with
test solution (a).
Test solution. To 0.300 g of the extract to be examined

add 40 ml of methanol R and 40.0 ml of 0.1 M sodium
hydroxide. Heat in a water-bath at about 60 C under a
reflux condenser, with frequent shaking, for about 1 h. After
cooling, add 4.0 ml of 1 M hydrochloric acid. Filter the
suspension into a 100 ml volumetric flask, then wash and
dilute to 100.0 ml with a mixture of equal volumes of water R
and methanol R. Filter through a 0.45 m membrane filter.
Reference solution. Dissolve 5.0 mg of picein R in 25.0 ml
of a mixture of 20 volumes of water R and 80 volumes of
methanol R (solution A). Dissolve 15.0 mg of salicin R in
25 ml of a mixture of 20 volumes of water R and 80 volumes
of methanol R. Add 5.0 ml of solution A and dilute to 50.0 ml
with a mixture of 20 volumes of water R and 80 volumes of
methanol R.
Column:
size : l = 0.10 m, = 4.6 mm ;
Mobile phase:
mobile phase A : tetrahydrofuran R, a 0.5 per cent V/V
solution of phosphoric acid R (1.8:98.2 V/V) ;
mobile phase B : tetrahydrofuran R ;
Top of the plate

_______
_______
Salicin : a reddish-violet A weak reddish-violet

zone
zone (salicin)
_______
Chlorogenic acid : a
brown zone
Reference solution
Test solution (a)
A reddish-violet zone
(salicin)
_______
Test solution (b)
Time
(min)
0 - 15
Mobile phase A
(per cent V/V)
100
Mobile phase B
(per cent V/V)
0
15 - 17
100 90
0 10
17 - 23
90
10
23 - 25
90 100
10 0
25 - 40
100
TESTS
ASSAY
Injection : 10 l.
The following chromatogram is shown for information but

will not be published in the European Pharmacopoeia.
retention time : salicin = about 6.4 min ;

picein = about 7.7 min ;


salicin and picein.
Calculate the percentage content of total salicylic derivatives,
expressed as salicin, from the following expression :
1. salicin
2. picein
Figure 2312.-1. Chromatogram for the assay of willow

bark dry extract : test solution
A1
A2
m1
m2
area of the peak due to salicin in the

chromatogram obtained with the test solution ;
area of the peak due to salicin in the chromatogram
obtained with the reference solution ;
mass of the extract to be examined used in the
preparation of the test solution, in milligrams ;
mass of salicin R in the reference solution, in
milligrams ;
percentage content of salicin in salicin R.
(61) Waters Spherisorb 3 m ODS2 is suitable.
159
160
Ribwort plantain
Illustrations of Powdered Drugs

in Herbal Monographs
It has been decided to progressively introduce illustrations of powdered drugs into herbal monographs in order to
complement the corresponding identification section (usually Identification B). These drawings will be published
in Pharmeuropa as they become available.
Reference: PA/PH/Exp. 3/T (97) 79 DEF
Reference: PA/PH/Exp. 13B/T (03) 19 DEF

This monograph is currently published in the 5th Edition.
This monograph is currently published in the 5th Edition.
01/2005:1297
01/2005:1884
CALENDULA FLOWER
RIBWORT PLANTAIN
Calendulae flos
Plantaginis lanceolatae folium
A. Epidermis of the corolla
D. Stigma fragment
A. Covering trichome of the leaf
D. Vein fragment with vessels
B. Epidermis of the corolla at the

apex, with anomocytic stomata
C. Pollen grains
E. Covering trichomes
B. Lower epidermis of lamina, with

glandular trichomes
C. Upper epidermis of lamina
E. Epidermis of scape
F. Glandular trichomes
Figure 1297.-1. Illustration of powdered herbal drug of

calendula flower (see Identification B)
F. Palisade parenchyma
Figure 1884.-1. Illustration of powdered herbal drug of

ribwort plantain (see Identification B)
161
162
Carmellose
This section contains proposals for monographs and general texts, new or revised, elaborated under the international
harmonisation procedure (see chapter 5.8 of the European Pharmacopoeia). After these texts have undergone the
harmonisation procedure and have been adopted, they will be included in the European Pharmacopoeia and the
pharmacopoeias of the United States and Japan.
The draft harmonised texts are published for comment (stage 4: official public enquiry in the forum of each of the three
pharmacopoeias). You may send your comments through the appropriate national pharmacopoeia authority at the
address listed on the back cover page of this issue. Readers whose country is not a signatory state of the European
Pharmacopoeia Convention can send their comments directly to the European Directorate for the Quality of Medicines
(see address of the EDQM on the cover of this issue). To facilitate the processing of comments received by the Secretariats
of the national authorities and the EDQM please mention in any correspondence the PA/PH reference number at
the beginning of each text. If you are requesting a change in the limits or are proposing other methods of analysis,
please support your proposal by providing appropriate analytical data obtained on a significant number of samples
and the results of a comparative study between the official method and the proposed method. Comments sent before
31 March 2006 will be considered for the preparation of the final version of the harmonised texts.
We wish to emphasise that these draft texts have not yet been adopted by the European Pharmacopoeia Commission
and therefore cannot be considered to be official texts.
It should be noted that for monographs, a version drafted in the European Pharmacopoeia style is usually published at
the same time in the section on Draft monographs and general texts for comments.
Reference: PA/PH/Exp. CEL/T (05) 9 ANP

Stage 4
The JP is the coordinating Pharmacopoeia.
Although there is no production of carmellose actually in
Europe, it seems that the substance could be of interest for
the Pharmaceutical industry in the near future.
XXXX:2360
CARMELLOSE
Carmellosum
DEFINITION
Polycarboxymethylether of cellulose.
CHARACTERS
Appearance : white or almost white powder, hygroscopic.
Solubility : practically insoluble in anhydrous ethanol. It
swells with water to form suspension and becomes viscid in
1 M sodium hydroxide.
IDENTIFICATION
A. pH (2.2.3) : 3.5 to 5.0.
Suspend 1.0 g in 100 ml of water R.
Comparison : carmellose CRS.
C. Shake 0.1 g with 10 ml of water R, add 2 ml of 1 M sodium
hydroxide and allow to stand for 10 min (solution A).
Dilute 1 ml of solution A to 5 ml with water R. To 0.05 ml
of this solution add 0.5 ml of chromotropic acid-sulphuric
acid concentrated solution - acid solution R and heat on
a water bath for 10 min. A red-purple colour develops.
D. Shake 5 ml of solution A (obtained in identification
test C) with 1 ml of a 90 g/l solution of ferric chloride R.
A brown flocculent precipitate is produced.
TESTS
Chlorides: maximum 0.36 per cent.
Shake 0.8 g with 50 ml of water R, dissolve in 10 ml of 1 M
sodium hydroxide and dilute to 100 ml with water R. Heat
on a water-bath a mixture of 10 ml of dilute nitric acid R
and 20 ml of this solution until a flocculent precipitate is
produced. Cool, centrifuge and take out the supernatant
liquid. Wash the precipitate with 3 quantities, each of
10 ml, of water R, by centrifuging each time. Combine the
supernatant liquid and the washings and dilute to 100 ml
with water R. To 25 ml of this solution add 6 ml of dilute
nitric acid R and dilute to 50 ml with water R (test solution).
Prepare the reference solution with 0.40 ml of 0.01 M
hydrochloric acid. Add 1 ml of silver nitrate solution R2 to
the test solution and the reference solution. After standing
protected from light for 5 min, any opalescence in the test
solution is not more intense than that in the reference
solution.
Sulphates : maximum 0.75 per cent.
Shake 0.40 g with 25 ml of water R, dissolve in 5 ml of
1 M sodium hydroxide and add 20 ml of water R. Heat
this solution with 2.5 ml of hydrochloric acid R in a
water-bath until a flocculent precipitate is produced. Cool,
centrifuge, and take out the supernatant liquid. Wash the
precipitate with 3 quantities, each of 10 ml, of water R, by
centrifuging each time. Combine the supernatant liquid and
the washings, and dilute to 100 ml with water R. Filter, and
discard the first 5 ml of the filtrate. To 25 ml of the filtrate
add 1 ml of dilute hydrochloric acid R and dilute to 50 ml
with water R (test solution). Prepare the reference solution
with 1.5 ml of 0.005 M sulphuric acid. Add 2 ml of a 120 g/l
solution of barium chloride R to the test solution and the
reference solutions. Mix and allow to stand for 10 min. The
white turbidy produced in the test solution is not thicker
than that in the reference solution.
Heavy metals : maximum 20 ppm.
Place 1.0 g in a quartz or porcelain crucible. Cover loosely
with a lid and carbonise by gentle ignition. Cool and add
2 ml of nitric acid R and 5 drops of sulphuric acid R. Heat
cautiously until white fumes are no longer evolved and
incinerate by ignition at 500-600 C. Cool and add 2 ml of
hydrochloric acid R. Evaporate to dryness on a water-bath.
163
White Petrolatum Jelly
Moisten the residue with 3 drops of hydrochloric acid R,

add 10 ml of hot water R and heat for 2 min. Add 1 drop
of phenolphthalein solution R1, add dilute ammonia R1
dropwise until the solution develops a pale red color. Add
2 ml of dilute acetic acid R, filter if necessary, and wash
with 10 ml of water R. Transfer the filtrate and washings to
a test tube, and dilute to 50 ml with water R (test solution).
Prepare the reference solution as follows : evaporate a
mixture of 2 ml of nitric acid R, 5 drops of sulphuric acid R
and 2 ml of hydrochloric acid R on a water bath, then
evaporate to dryness on a sand bath. Moisten the residue
with 3 drops of hydrochloric acid R. Proceed as described
for the test solution, then add 2.0 ml of lead standard
solution (10 ppm Pb) R and dilute to 50 ml with water R.
on 1.000 g by drying in an oven at 105 C for 4 h.
Residue on ignition : maximum 1.5 per cent, determined
on 1.000 g.
STORAGE
Reagents
Chromotropic acid-sulphuric acid concentrated solution.
XXXXXXX.
Suspend 0.5 g of chromotropic acid, sodium salt R in 50 ml
of sulphuric acid R, centrifuge and use the supernatant
liquid. Prepare immediately before use.

Stage 4
The USP is the coordinating pharmacopoeia for this
monograph.
The draft for harmonisation presented below is essentially
for information. In the section on monographs for
comment of this issue of Pharmeuropa, a revision proposal
for the European Pharmacopoeia monograph on white soft
paraffin is published. This shows how the stage 4 draft
would affect the European monograph. Comments are
invited on the revision proposal (rather than on the USP
draft).
XXXX:1799
WHITE PETROLATUM JELLY

White Petrolatum is a purified and wholly or nearly
decolorized mixture of semisolid hydrocarbons obtained
from petroleum. It may contain a suitable stabilizer.
Packaging and storage- Preserve in well-closed containers,
protected from light.
Labeling- The labeling indicates the name and concentration
of any added stabilizer. Where the labeling indicates the
consistency, determine compliance using Consistency.
Color- Melt about 10 g on a steam bath, and pour 5 mL of
the liquid into a clear-glass, 16- 150-mm bacteriological test
tube : the warm, melted liquid is not darker than a solution
164
made by mixing 1.6 mL of ferric chloride CS and 3.4 mL of

water in a similar tube, the comparison of the two being
made in reflected light against a white background, the
tubes being held directly against the background at such an
angle that there is no fluorescence.
Identification- Its infrared absorption spectrum, obtained
by spreading a thin film of melted test specimen between
sodium chloride plates, exhibits high-intensity bands between
3000 cm 1 and 2800 cm 1, medium intensity bands between
1500 cm 1 and 1300 cm 1, and low intensity bands between
750 cm 1 and 700 cm 1.
Melting range, Class III <741> : between 38 and 60.
ConsistencyApparatus- Determine the consistency of White Petrolatum
Jelly by means of a penetrometer fitted with a polished
cone-shaped metal plunger weighing 150 g, having a
detachable steel tip of the following dimensions : the tip of
the cone has an angle of 30, the point being truncated
to a diameter of 0.381 0.025 mm, the base of the tip is
8.38 0.05 mm in diameter, and the length of the tip is
14.94 0.05 mm. The remaining portion of the cone has an
angle of 90, is about 28 mm in height, and has a maximum
diameter at the base of about 65 mm. The containers for
the test are flat-bottom metal cylinders that are 100 6 mm
in diameter and not less than 65 mm in height. They are
constructed of at least 1.6-mm (16-gauge) metal, and are
provided with well-fitting, water-tight covers.
Procedure- Place the required number of containers in an
oven, and bring them and a quantity of White Petrolatum
Jelly to a temperature of 82 2.5. Pour the White
Petrolatum Jelly into one or more of the containers, filling
to within 6 mm of the rim. Cool to 25 2.5 over a period
of not less than 16 hours, protected from drafts. Two
hours before the test, place the containers in a water bath
at 25 0.5. If the room temperature is below 23.5 or
above 26.5, adjust the temperature of the cone to 25 0.5
by placing it in the water bath.
Without disturbing the surface of the substance under test,
place the container on the penetrometer table, and lower the
cone until the tip just touches the top surface of the test
substance at a spot 25 mm to 38 mm from the edge of the
container. Adjust the zero setting and quickly release the
plunger, then hold it free for 5 seconds. Secure the plunger,
and read the total penetration from the scale. Make three
or more trials, each so spaced that there is no overlapping
of the areas of penetration. Where the penetration exceeds
20 mm, use a separate container of the test substance for
each trial. Read the penetration to the nearest 0.1 mm.
Calculate the average of the three or more readings, and
conduct further trials to a total of 10 if the individual results
differ from the average by more than 3% : each mm of
penetration corresponds to a consistency value of 10.
Acidity or alkalinity- To 10 g add 20 mL of boiling water,
and shake vigorously for 1 minute. Allow to cool, and decant.
To 10 mL of the aqueous layer add 0.1 mL of a 1 in 10
solution in alcohol of phenolphthalein TS. The solution is
colorless. Not more than 0.5 mL of 0.01 N sodium hydroxide
is required to change the color of the indicator to red.
Residue on ignition <281>- Heat 10 g in an open porcelain
or platinum dish over a Bunsen flame : on ignition it yields
not more than 0.05% of residue.
Yellow Petrolatum Jelly

Stage 4
The USP is the coordinating pharmacopoeia for this
monograph.
The draft for harmonisation presented below is essentially
for information. In the section on monographs for
comment of this issue of Pharmeuropa, a revision proposal
for the European Pharmacopoeia monograph on yellow
soft paraffin is published. This shows how the stage 4 draft
would affect the European monograph. Comments are
invited on the revision proposal (rather than on the USP
draft).
XXXX:1554
YELLOW PETROLATUM JELLY

Petrolatum is a purified mixture of saturated semisolid and
bleached hydrocarbons obtained from petroleum. It may
contain a suitable stabilizer.
Packaging and storage- Preserve in well-closed containers,
protected from light.
Labeling- The labeling indicates the name and concentration
of any added stabilizer. Where the labeling indicates the
consistency, determine compliance using Consistency.
Color- Melt about 10 g on a steam bath, and pour 5 mL of
the liquid into a clear-glass, 16- 150-mm bacteriological
test tube : the warm, melted liquid is not darker than a
solution made by mixing 3.8 mL of ferric chloride CS and
1.2 mL of cobaltous chloride CS in a similar tube, the
comparison of the two being made in reflected light against a
white background, the tubes being held directly against the
background at such an angle that there is no fluorescence.
Identification- Its infrared absorption spectrum, obtained
by spreading a thin film of melted test specimen between
sodium chloride plates, exhibits high-intensity bands between
3000 cm 1 and 2800 cm 1, medium intensity bands between
1500 cm 1 and 1300 cm 1, and low intensity bands between
750 cm 1 and 700 cm 1.
Melting range, Class III <741> : between 38 and 60.
ConsistencyApparatus- Determine the consistency of Yellow Petrolatum
Jelly by means of a penetrometer fitted with a polished
cone-shaped metal plunger weighing 150 g, having a

detachable steel tip of the following dimensions : the tip of
the cone has an angle of 30, the point being truncated
to a diameter of 0.381 0.025 mm, the base of the tip is
8.38 0.05 mm in diameter, and the length of the tip is
14.94 0.05 mm. The remaining portion of the cone has an
angle of 90, is about 28 mm in height, and has a maximum
diameter at the base of about 65 mm. The containers for
the test are flat-bottom metal cylinders that are 100 6 mm
in diameter and not less than 65 mm in height. They are
constructed of at least 1.6-mm (16-gauge) metal, and are
provided with well-fitting, water-tight covers.
Procedure- Place the required number of containers in an
oven, and bring them and a quantity of Yellow Petrolatum
Jelly to a temperature of 82 2.5. Pour the Yellow
Petrolatum Jelly into one or more of the containers, filling
to within 6 mm of the rim. Cool to 25 2.5 over a period
of not less than 16 hours, protected from drafts. Two hours
before the test, place the containers in a water bath at
25 0.5. If the room temperature is below 23.5 or above
26.5, adjust the temperature of the cone to 25 0.5 by
placing it in the water bath.
Without disturbing the surface of the substance under test,
place the container on the penetrometer table, and lower the
cone until the tip just touches the top surface of the test
substance at a spot 25 mm to 38 mm from the edge of the
container. Adjust the zero setting and quickly release the
plunger, then hold it free for 5 seconds. Secure the plunger,
and read the total penetration from the scale. Make three
or more trials, each so spaced that there is no overlapping
of the areas of penetration. Where the penetration exceeds
20 mm, use a separate container of the test substance for
each trial. Read the penetration to the nearest 0.1 mm.
Calculate the average of the three or more readings, and
conduct further trials to a total of 10 if the individual results
differ from the average by more than 3% : each mm of
penetration corresponds to a consistency value of 10.
Acidity or alkalinity- To 10 g add 20 mL of boiling water,
and shake vigorously for 1 minute. Allow to cool, and decant.
To 10 mL of the aqueous layer add 0.1 mL of a 1 in 10
solution in alcohol of phenolphthalein TS. The solution is
colorless. Not more than 0.5 mL of 0.01 N sodium hydroxide
is required to change the color of the indicator to red.
Residue on ignition <281>- Heat 10 g in an open porcelain
or platinum dish over a Bunsen flame : on ignition it yields
not more than 0.05% of residue.
165
166
STATE OF WORK
OF INTERNATIONAL HARMONISATION
(Updated November 2005)
Item
General Methods Relevant to Q6A
Dissolution
Disintegration
Uniformity of Content/Mass
Microbial Contamination
Tests for specied microorganism
Microbial enumeration
Microbial contamination limits for non-sterile products
Bacterial Endotoxins (rev. 1)
Color (instrumental method)
Extractable Volume of Parenterals (rev. 1)
Test for particulate contamination: subvisible particles (rev. 1)
Residue on Ignition (rev. 2)
Sterility test
General Chapters
Analytical sieving
Bulk Density and Tapped Density
Conductivity
Density of Solids
Flowability (Powder Flow)
Tablet Friability
Heavy Metals
Inhalation
Optical Microscopy
Powder Fineness
Specic Surface Area
Porosimetry by Mercury Intrusion
Laser diffraction measurement of particle size
X-Ray powder diffraction
Water-solid interactions
Thermal behaviour of powders
Methods for Biotechnology Products
Amino acid determination
Capillary electrophoresis
Isoelectric focusing
Protein determination
Peptide mapping
Polyacrylamide Gel Electrophoresis
Excipients
Alcohol (rev. 2)
Dehydrated Alcohol (rev. 2)
Benzyl Alcohol (rev. 1)
Calcium Disodium Edetate
Calcium Phosphate Dibasic (and anhydrous)
Carmellose Calcium (rev. 1)
Carmellose Sodium
Croscarmellose Sodium
Microcrystalline Cellulose (rev. 1)
Cellulose, Powdered (rev. 1)
Cellulose Acetate (rev. 1)
CP
Stage
USP
USP
USP
EP
EP
EP
EP
JP
EP
EP
EP
JP
EP
6
6
6
6
6
6
2
2
6
6
6
6
USP
EP
EP
EP
USP
USP
USP
EP
USP
USP
EP
EP
EP
EP
EP
EP
6
4
2
4
6
6
3
4
6
4 rev.
6
4
4
4
3
2
USP
EP
EP
USP
USP
EP
6
6
6
6
6
6
EP
EP
EP
JP
JP
USP
USP
USP
USP
USP
USP
5B
5B
6
6
6
6
4
6
6
6
6
167
Cellulose Acetate Phthalate

Citric Acid, Anhydrous (rev. 1)
Citric Acid, monohydrate (rev. 1)
Crospovidone
Ethylcellulose
Hydroxyethylcellulose
Hydroxypropylcellulose
Hydroxypropylcellulose, Low Substituted
Hydroxypropylmethylcellulose
Hydroxypropylmethylcellulose Phthalate
Lactose, Anhydrous (rev. 2)
Lactose, Monohydrate
Magnesium Stearate
Methylcellulose
Methyl paraben
Petrolatum
Petrolatum, White
Polyethylene Glycol
Polysorbate 80
Povidone
Saccharin
Saccharin, Sodium (rev. 1)
Saccharin, Calcium
Silicon Dioxide
Silicon Dioxide, Colloidal
Sodium Chloride (rev. 2)
Sodium Starch Glycolate (rev. 1)
Starch, Corn (rev. 1)
Starch, Potato
Starch, Rice
Starch, Wheat
Stearic Acid
Sucrose
Talc
Titanium Dioxide
Ethyl Paraben
Propyl Paraben
Butyl Paraben
Glycerin
Carmellose
Calcium carbonate
Copovidone
Gelatin
Glucose monohydrate / anhydrous
Glyceryl monostearate
Mannitol
Propylene glycol
Sodium laurylsulfate
Starch, pregelatinised
Water for Injection in Containers
Stage 1:
Stage 2:
Stage 3:
Stage 4:
Stage 5A:
Stage 5B:
Stage 6:
Stage 7:
168
USP
EP
EP
EP
EP
EP
USP
USP
JP
USP
USP
USP
USP
JP
EP
USP
USP
USP
EP
JP
USP
USP
USP
JP
JP
EP
USP
USP
EP
EP
EP
EP
EP
EP
JP
EP
EP
EP
USP
JP
USP
JP
EP
EP
USP
EP
EP
USP
JP
USP
6
6
6
4
6
4-2
4
4
6
5B
5A
6
4 rev.
6
6
4
4
4
4
5A
6
6
6
4 rev.
4 rev.
6
6
6
6
5A
6
5A
4
6
5A2
6
6
6
3
4
3
4
2
3
2
3
4
3
2
2
Identication
Investigation
Proposal for Expert Committee Review
Ofcial Inquiry
Provisional Consensus
Draft sign-off Consensus
Regional adoption and implementation
Inter-regional implementation
PROJECTED TIMETABLE FOR PUBLICATION AND

IMPLEMENTATION OF TEXTS SIGNED OFF BY
THE PHARMACOPOEIAL DISCUSSION GROUP (PDG)
Effective harmonisation of monographs and general chapters requires that texts signed off by the PDG be published
and implemented by all three pharmacopoeias. This process of adoption and implementation takes place according
to the particular procedure of each pharmacopoeia. The table below shows the projected timetable for publication
and implementation of all signed-off texts, based on information provided by each pharmacopoeia.
Q6A GENERAL CHAPTERS (November 2005)
Item
Sign-off
Dissolution
Disintegration
Content
uniformity
Extractable
volume of
parenterals
Rev. 1
Particulate
matter in
injectables
Rev. 1
Sterility
Microbiological
quality
Bacterial
endotoxins
2004/6
2004/6
2004/2
EP
2005/6
2005/6
2004/12
JP
2006/3
2006/3
2006/3
USP
2005/3
2005/3
2005/1
Stage 6B
Regional Implementation
EP
JP
USP
2006/1
2006/4
2006/4
2006/1
2006/4
2006/4
2005/6
2006/4
2007/1
2000/7
2001/6
2000/7
2002/1
2002/1
2004/6
2001/5
2005/6
2002/6
2005/7`
-
2005/11
2001/3
2006/1
2003/1
2005/7
-
2006/8
2002/1
2007/1
2005/7
2006/8
2004/6
2002/10
2005/6
2003/6
2006/3
2004/12
2005/2
2003/5
2006/1
2004/1
2006/4
2005/1
2005/4
2004/1
2006/7
2006/1
2006/4
2006/4
2006/4
2004/1
2000/1
2000/6
2001/3
2000/7
2001/1
2001/4
2001/1
2000/2
2001/6
2002/12
2002/11
2002/1
2003/1
2002/9
2004/10
2005/6
(2004/12)
2006/3
(2002/11)
2005/5
2006/1
(2005/1)
2006/4
(2003/8)
2006/4
Sulphated ash/
Residue on
ignition
Rev. 1
Rev. 2
Colour and
clarity of
solution
Publication
Stage 7
Inter-regional Implementation
EP
JP
USP
2007/1
2006/4
2006/4
2007/1
2006/4
2006/4
2007/1
2006/4
2007/1
2003/8
2005/4
2006/4
2006/4
OTHER GENERAL CHAPTERS (November 2005)

Item
Publication
Sign-off
EP
JP
USP
Stage 6B
EP
JP
USP
Stage 7
EP
JP
USP
Amino acid
determination
2002/9
2003/6
2004/12
2001/12
2005/1
2005/1
2002/1
TBD
2006/4
TBD
Capillary
electrophoresis
2002/9
2003/6
2004/12
2003/7
2005/1
2005/1
2003/8
TBD
2006/4
TBD
Isoelectric focusing
2002/9
2003/6
2004/12
2003/7
2004/1
2005/1
2003/8
TBD
2006/4
TBD
Protein determination
2002/9
2003/6
2004/12
2002/12
2004/1
2005/1
2003/1
TBD
2006/4
TBD
Peptide mapping
2002/9
2003/6
2004/12
2001/12
2004/1
2005/1
2002/1
TBD
2006/4
TBD
Polyacrylamide gel
electrophoresis
1999/10
2001/9
2002/12
2001/1
2002/1
2003/1
2001/3
TBD
2006/4
TBD
Tablet Friability
2004/2
2004/12
2006/3
2006/8
2005/7
2006/4
2006/8
2007/1
2006/4
2006/8
Specic Surface Area
2003/11
2004/9
2006/3
2004/7
2005/4
2006/4
2005/4
2007/1
2006/4
2006/5
Analytical Sieving
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
TBD
2006/4
TBD
Powder Flow
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
TBD
2006/4
TBD
Optical Microscopy
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
TBD
2006/4
TBD
169
MONOGRAPHS (November 2005)
Item
Sign-off
Alcohol (Rev. 1)
Alcohol,
dehydrated
Benzyl alcohol
Carmellose
calcium (Rev. 1)
Calcium
disodium edetate
Calcium
phosphate,
dibasic,
anhydrous
Calcium
phosphate,
dibasic, dihydrate
Croscarmellose
sodium
Cellulose acetate
(Rev. 1)
Cellulose acetate
phthalate
Cellulose,
microcrystalline
Rev. 1
Cellulose powder
Rev. 1
Citric acid
anhydrous
Rev. 1
Citric acid
monohydrate
Rev .1
Ethylcellulose
Hypromellose
Lactose
anhydrous
Rev. 1
Rev. 2
Lactose
monohydrate
Methyl cellulose
Methyl Paraben
Saccharin
Saccharin
calcium (Rev. 1)
Saccharin
sodium
Rev. 1
Sodium chloride
Rev. 1
Rev. 2
Sodium starch
glycolate
Rev. 1
Starch, corn
Rev. 1
Starch, potato
Starch, wheat
Talc
Ethyl Paraben
Propyl Paraben
Butyl Paraben
2002/9
EP
2003/6
JP
2006/3
USP
2004/9
Stage 6B
EP
JP
USP
2004/1
2006/4
2005/8
2002/9
2003/6
2006/3
2004/9
2004/1
2006/4
2005/8
2006/8
2000/7
2001/10
2004/12
2004/3
2002/4
2005/1
2005/1
2006/8
2003/7
2003/9
2004/12
2004/3
2004/4
2005/1
2005/1
2006/8
2005/11
2006/10
2005/11
2006/10
2005/11
2006/10
2001/10
2001/9
2006/3
2004/3
2002/4
2006/4
2005/1
2006/8
2003/2
2003/9
2004/3
2004/4
2005/1
2006/8
2001/10
2002/6
2004/3
2003/1
2005/1
2006/8
170
Publication
2004/12
2004/2
2005/1
2004/7
2005/5
2004/2
2005/5
2006/6
2006/3
2006/6
2001/5
2003/6
2003/11
Stage 7
EP
JP
USP
2006/8
2005/4
2007/1
2006/4
2006/3
2006/3
2004/7
2006/3
2007/1
2006/4
2007/1
2005/4
2007/1
2002/12
2004/3
2004/1
2003/1
2005/1
2006/3
2004/9
2006/4
2005/8
2006/8
2006/8
2006/8
2001/5
2003/6
2002/12
2004/3
2004/1
2003/1
2005/1
2003/11
2002/2
2003/11
2002/11
2006/6
2006/3
2007/9
2006/3
2004/9
2004/3
2006/4
2003/6
2007/1
2006/4
2007/10
2006/4
2005/8
2005/1
2007/1
2006/8
2006/8
2006/8
2003/2
2003/6
2006/3
2006/4
2004/1
2006/4
2007/1
2006/8
2002/9
2003/6
2006/3
2006/4
2004/1
2006/4
2007/1
2006/8
2003/11
2004/2
2003/2
2006/6
2004/2
2005/6
2006/3
2006/3
2006/3
2006/4
2004/7
2004/7
2007/1
2004/2
2006/1
2006/4
2006/4
2006/4
2007/4
2005/4
2006/4
2006/8
2006/8
2006/4
2006/8
2003/2
2003/2
2004/2
2001/5
2001/10
2003/11
2004/7
2005/6
2003/6
2006/3
2002/12
2004/7
2006/1
2004/3
2004/1
2006/3
2003/11
2004/12
2005/5
2001/10
2004/2
2001/10
2001/10
2003/11
2004/2
2004/2
2004/2
2006/6
2002/6
2002/6
2002/6
2004/9
2004/2
2004/2
2004/2
2007/9
2004/12
2004/12
2004/12
2007/9
2006/3
2006/3
2006/3
2006/4
2006/4
2003/1
2006/8
2005/1
2006/4
2006/8
2004/7
2005/7
2006/4
2005/4
2005/9
2004/3
2007/1
2007/10
2006/4
2005/1
2005/4
2004/2
2004/2
2004/2
2005/1
2005/1
2005/1
2007/10
2006/4
2006/4
2006/4
2004/3
2004/3
2004/9
2004/7
2004/7
2004/7
2005/1
2005/1
2005/8
2005/4
2005/4
2006/1
2006/8
2006/8
2006/8
2006/8
2006/8
2006/8
2006/8
2006/8
Contents of the JP Forum
PUBLICATION OF THE LISTS OF CONTENTS

APPEARING IN THE JP FORUM AND USP FORUM
Partners in International Harmonisation
JAPANESE PHARMACOPOEIAL FORUM (JP)
9.41. Reagents,Teat Solutions

3. Ofcial Monographs
(1) Addition
Vol. 14, No. 3
September 2005
CONTENTS
Revision Drafts for the JP 15

1. General Rules for Preparations
1. General Notices for Preparations
2. Aerosols
3. Aromatic Waters
4. Capsules
5. Cataplasms /Gel Patches
6. Elixirs
7. Extracts
8. Fluidextracts
9. Granules
10. Infusions and Decoctions
11. Injections
12. Lemonades
13. Liniments
14. Liquids and Solutions
15. Lotions
16. Ointments
17. Ophthalmic Ointments
18. Ophthalmic Solutions
19. Pills
20. Plasters and Pressure Sensitive Adhesives Tapes
21. Powders
22. Spirits
23. Suppositories
24. Suspensions and Emulsions
25. Syrups
26. Tablets
27. Tinctures
28. Transdermal Systems
29. Troches
2. General Tests, Processes and Apparatus
(1) Revision
1.11. Arsenic Limit Test
2.57. Boiling Point and Distilling Range Test
2.60. Melting Point Determination
3.02. Specic Surface Area by Gas Adsorption
3.04. Particle Size Determination
4.02. Microbial Assay for Antibiotics
5.01. Crude Drugs Test
5.02. Microbial Limit Test for Crude Drugs
9. Reference Standards; Reagents, Test
Solutions; Standard Solutions for
Volumetric Analysis; Standard Solutions;
Matching Fluids for Color; Optical Filters
for Wavelength and Transmission Rate
Calibration; and Measuring Instruments,
Appliances
L-Aspartic Acid
Benincasa Seed
Celmoleukin (Genetical Recombination)
Croscarmellose Sodium
Dolichos Seed
Eleutherococcus Senticosus Rhizome
Faropenem Sodium for Syrup
Human Menopausal Gonadotrophin
Nelumbo Seed
Polygonatum Rhizome
Pullulan
Rifampicin Capsules
Teceleukin (Genetical Recombination)
Teceleukin for Injection (Genetical
Recombination)
Verapamil Hydrochloride Tablets
(2) Revision
Achyranthes Root
Alisma Rhizome
Powdered Alisma Rhizome
L-Arginine Hydrochloride
Astragalus Root
Atractylodes Lancea Rhizome
Powdered Atractylodes Lancea Rhizome
Benidipine Hydrochloride Tablets
Bupleurum Root
Calcium Gluconate
Calcium Gluconate Hydrate
L-Carbocisteine
Cefalotin Sodium
Cefditoren Pivoxil
Microcrystalline Cellulose
Powdered Cellulose
Clindamycin Hydrochloride
Dexamethasone
Dicloxacillin Sodium
Enviomycin Sulfate
10% Ephedrine Hydrochloride Powder
Erythromycin
Ethanol for Disinfection
Famotidine Powder
Famotidine Tablets
Powdered Fennel
Flopropione Capsules
Ginger
Powdered Ginger
Glycyrrhiza
Powdered Glycyrrhiza
Idarubicin Hydrochloride
Imipenem
Insulin
Insulin Injection
Japanese Angelica Root
Powdered Japanese Angelica Root
171
Contents of the USP Forum
Kitasamycin Tartrate
Lincomycin Hydrochloride
Moutan Bark
Powdered Moutan Bark
Ophiopogon Tuber
Oxytocin
Oxytocin Injection
Powdered Peach Kernel
Peony Root
Powdered Peony Root
Phytonadione
Pinellia Tuber
Pirarubicin
Platycodon Root
Powdered Platycodon Root
Polymixin B Sulfate
Poria Sclerotium
Powdered Poria Sclerotium
Povidone
Powdered Rhubarb
Pueraria Root
Rehmannia Root
Rhubarb
Roxithromycin
Scutellaria Root
Powdered Scutellaria Root
Spectinomycin Hydrochloride
Talampicillin Hydrochloride
Teicoplanin
Tranexamic Acid Capsules
Tranexamic Acid Tablets
Trichosanthes Root
Vasopressin Injection
Wine
Zinostatin Stimalamer
Additional Revision Drafts for JP 15

1. General Tests, Processes and Apparatus
(1) Revision
14. Disintegration Test
2. Ofcial Monographs
(1) Addition
Amphotericin B Tablets
L-Arginine
Betamethasone Tablets
Cefditoren Pivoxil Tablets
Clindamycin Hydrochloride Capsules
Faropenem Sodium Tablets
Metronidazole Tablets
Trimetazidine Hydrochloride Tablets
Voglibose Tablets
(2) Revision
Calcium Para-aminosalicylate Granules
Chlorpheniramine Maleate Tablets
Norepinephrine
Noradrenaline
Norepinephrine Injection
International Harmonization
2. Stage 4 (Ofcial Inquiry Stage Draft)
(1) Characterisation of Crystalline Solids by X-ray
Powder Diffraction Polyethylene Glycol (XRPD)
(by EP)
Contents Pages of Pharmacopeial Forum and Pharmeuropa
Japanese Pharmacopoeia Reference Standards and Ofcial
Non-pharmacopoeial Reference Standards available from
the Society of Japanese Pharmacopoeia
- Ordering Information
4. General Information
(1) Revision
14. Tablet Friability Test
_______________________________________________________________________________
PHARMACOPOEIAL FORUM (USP)

Vol. 31, No. 6
NovDec 2005
CONTENTS*
Call for High Priority Monographs for Drug Substances

and Products, and Excipients
Pharmacopeial Education Courses
Visit the USP Web Site at http://www.usp.org
International Correspondence
How to Submit Comments
Publication Schedules
SIXTH INTERIM REVISION ANNOUNCEMENT
STANDARDS DEVELOPMENT
HOW TO USE PF
Section Descriptions
Committee Designations
Staff Directory
POLICIES AND ANNOUNCEMENTS
USP Seeks Submission of Proposals for Stability
Indicating Assay Procedures for Steroids
NOTICE OF OFFICIAL STATUS

Vinorelbine Injection
NOTICE OF POSTPONEMENT
<905> Uniformity of Dosage Units
GENERAL CHAPTERS
<1> Injections
ERRATA LIST FOR USP 28NF 23
* The USPNF (USP 29NF 24), the Supplement (Supp), or the Interim Revision Announcement (IRA) for which the revision proposal is targeted
is shown in parentheses next to each proposed item.
172
Contents of the USP Forum
IN-PROCESS REVISION
Polyoxyl 35 Castor Oil (NF 25)

Anhydrized Liquid Sorbitol (NF 25)
Tetrauoroethane [new] (NF 25)
USP MONOGRAPHS
Amitriptyline Hydrochloride (USP 30)
Calcium Lactate (USP 30)
Calcium Lactate Tablets (USP 30)
Cladribine [new] (USP 30)
Diphenoxylate Hydrochloride and Atropine Sulfate Oral
Solution (USP 30)
Diphenoxylate Hydrochloride and Atropine Sulfate
Tablets (USP 30)
Diphtheria Toxin for Schick Test (USP 30)
Ensulizole (USP 30)
Estradiol Vaginal Tablets [new] (USP 30)
Synthetic Conjugated Estrogens [new] (USP 30)
Etidronate Disodium (USP 30)
Fentanyl [new] (USP 30)
Flumazenil (USP 30)
Gemcitabine for Injection (USP 30)
Glipizide and Metformin Hydrochloride Tablets [new]
(USP 30)
Glucagon (Proposal for 2nd IRA)
Goserelin Acetate [new] (USP 30)
Hepatitis B Virus Vaccine Inactivated (USP 30)
Sodium Iodide I 123 Capsules (USP 30)
Sodium Iodide I 123 Solution (USP 30)
Sodium Iodide I 131 Solution (USP 30)
Diluted Isosorbide Mononitrate (Proposal for 2nd IRA)
Ivermectin (USP 30)
Levocabastine Hydrochloride [new] (USP 30)
Lindane (USP 30)
Mangafodipir Trisodium(USP 30)
Mirtazapine (USP 30)
Ondansetron Injection (USP 30)
Orphenadrine Citrate Injection (USP 30)
Oxybutynin Chloride Extended-Release Tablets [new]
(USP30)
Prednicarbate Cream [new] (USP30)
Prednicarbate Ointment [new] (USP 30)
Risperidone [new] (USP 30)
Rubella and Mumps Virus Vaccine Live (USP 30)
Schick Test Control (USP 30)
Talc (USP 30)
EXCIPIENTS
NF MONOGRAPHS
Canola Oil [new] (NF 25)
Ethylcellulose Aqueous Dispersion (NF 25)
Glyceryl Monostearate (NF 25)
Oleyl Oleate [new] (NF 25)
Polacrilin Potassium (NF 25)
GENERAL CHAPTERS
<11> USP Reference Standards (USP 30)
<621> Chromatography (USP 30)
<711> Dissolution (Proposal for 2nd IRA) (2nd Supp to
USP 29)
GENERAL INFORMATION CHAPTERS
<1217> Tablet Breaking Force [new] (USP 30)
REAGENTS, INDICATORS AND SOLUTIONS
Reagent Specifications
Geneticin [new] (USP 30)
Hydroxypropyl-beta-cyclodextrin [new] (USP 30)
IsopropylIodide (USP 30)
Sodium Carbonate, Monohydrate [new] (USP 30)
1-Vinyl-2-pyrrolidinone (USP 30)
REFERENCE TABLES
Container Specications for Capsules and Tablets
(USP 30)
Description and Solubility (USP 30)
PENDING PROPOSALS
CANCELED PROPOSALS
HARMONIZATION
GENERAL INFORMATION CHAPTERS
<1216> Tablet Friability (Proposal for 2nd IRA)
PHARMACOPEIAL PREVIEWS
STIMULI TO THE REVISION PROCESS
Instructions to Authors
USP Advisory Panels on the USP Performance Test,
L. Shargel, and T. Foster
Critical Quality and Performance Parameters for
Modied-Release Parenteral Dosage Forms, Diane J.
Burgess, Brian C. Clark, Mary Joan Hampson-Carlin,
and Pankaj Shah
Compendial Calculations: Improving Calculations in
USPNF, Philip Travis, Kerrie Heck, Deborah Teitz,
Luciano Virgili, and Mark Wiggins
Comments on Compendial Calculations: Improving the
Calculations in USPNF, USP Staff
NOMENCLATURE
INDEX
_______________________________________________________________________________
CORRESPONDENCE
Individuals who wish to correspond with the JP or the USP concerning any of the monographs/articles mentioned
can do so at the following addresses:
Pharmacopeial Forum
Japanese Pharmacopoeial Forum
Secretariat of the Japanese Pharmacopoeia
Evaluation and Licensing Division
Pharmaceutical and Medical Safety Bureau
Ministry of Health and Welfare
1-2-2, Kasumigaseki, Chiyoda-ku
Tokyo, 100-8045, JAPAN
The United States Pharmacopeia

Division of Standards Development, USP-NF
12601 Twinbrook Parkway
Rockville, Maryland 20852
USA
173
174
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175
ADDRESSES OF THE NATIONAL PHARMACOPOEIA

AUTHORITIES
Correspondence relating to the draft monographs submitted for comment in
the present issue are to be sent to the relevant Authority.
AUSTRIA
Bundesministerium fr
Gesundheit und Frauen,
Pharmazeutische
Angelegenheiten
Radetzkystrasse 2
A 1030 WIEN
robert.schloegel@bmsg.gv.at
BELGIUM
Service public fdral

Sant publique
Direction gnrale Mdicaments
Place Victor Horta 40
BP 10
B 1060 BRUXELLES
paule.jacqmain@afigp.fgov.be
BOSNIA AND
HERZEGOVINA
Institute for the Medicines

Quality Control
M. Titova, 9
P.O. Box 0413
BA 71000 SARAJEVO
medqcins@bih.net.ba
FINLAND
LITHUANIA
FRANCE
LUXEMBOURG
Lkelaitos Nat. Agency for

Medicines
P.O. Box 55
FIN 00301 HELSINKI
kaarina.sinivuo@nam.fi
Agence franaise de Scurit
Sanitaire des Produits de Sant
Direction des Laboratoires et
des Contrles
Unit Pharmacope
143-147, bld. Anatole France
F 93285 SAINT-DENIS CEDEX
pharmacopeefrancaise@
afssaps.sante.fr
GERMANY
Bundesinstitut fr Arzneimittel
und Medizinprodukte
Geschftsstelle der Deutschen
Arzneibuch-Kommissionen
Kurt-Georg-Kiesinger Allee 3
D 53175 BONN
d.schnaedelbach@bfarm.de
GREECE
BULGARIA
Bulgarian Pharmacopoeia
Commission
Bulgarian Drug Agency
26, Janko Sakazol blvd
BG SOFIA 1504
lkostova@bda.bg
CROATIA
Povjerenstvo za farmakopeju
Hrvatski zavod za koutrolu
lijekova
Ksaverska cesta 4
HR 10000 ZAGREB
blazenka.jurisic@hzkl.hr
CYPRUS
Dr Louis Panayi
Department of Pharmaceutical
Services
Ministry of Health
CY 1475 LEFKOSIA
pkokkinou@phs.moh.gov.cy
CZECH REPUBLIC
Czech Pharmacopoeia
Commission
State Institute for Drug Control
Srobrova 48
CZ 100 41 PRAGUE 10
lekopis@sukl.cz
DENMARK
Danish Medicines Agency

Axel Heides Gade 1
DK 2300 KBENHAVN S
kemkon@dkma.dk
ESTONIA
State Agency of Medicines

19 Ravila Street
EE 50411 TARTU
signe.leito@sam.ee
Pharmacopoeia Section
National Organisation for
Medicines
Mesogeion Avenue, 284
GR 15562 HOLARGOS ATTIKIS
ellinph@eof.gr
HUNGARY
National Institute of Pharmacy

Pharmacopoeia Section
Zrinyi u.3.
P.O. Box 450
H 1372 BUDAPEST V
hszalai@ogyi.hu
ICELAND
Head of the Pharmaceutical

Division, Ministry of Health
and Social Security
Laugavegur 116
IS 150 REYKJAVIK
ingolf.j.petersen@htr.stjr.is
IRELAND
State Medicines Control Agency

Ministry of Health
Traku str. 14
LT 01132 VILNIUS
vvkt@vvkt.lt
Ministre de la Sant
Division de la Pharmacie et
des Mdicaments
Villa Louvigny, Alle Marconi
L 2120 LUXEMBOURG
jacqueline.genoux-hames@
ms.etat.lu
MALTA
Malta Medicines Authority

198, Rue DArgens,
Gzira GZR 03
MT Malta
eloise.xerri@gov.mt
NETHERLANDS
Secretariat of the Dutch

Pharmacopoeia Authority
Lab. for Quality Control of
Medicines
P.O.Box 1
NL 3720 BA BILTHOVEN
farmacopee@rivm.nl
NORWAY
Norwegian Pharmacopoeia
Authority
Statens legemiddelverk
Sven Oftedals vei, 6
N 0950 OSLO 9
farmakope@noma.no
PORTUGAL
Secrt. Pharmacop Portugaise

Faculdade de Farmacia
Rua Anibal Cunha
P 4050 PORTO
slobo@ff.up.pt
ROMANIA
National Medicines Agency

48, av. Sanatescu Str.
RO 71324 BUCHAREST
magdalena.badulescu@anm.ro
Medicines Division
Department of Health and
Children
Hawkins House, Poolbeg Street
IRL DUBLIN 2
sandra_barnes@health.irlgov.ie
SERBIA AND MONTENEGRO
ITALY
SLOVAK REPUBLIC
Segreteria Tecnica della

Farmacopea Ufficiale Italiana
Istituto Superiore di Sanit
Viale Regina Elena, 299
I 00161 ROMA
farmacp@iss.it
LATVIA
Latvia State Agency of

Medicines
Jersikas iela, 15
LV 1003 RIGA
vza@vza.gov.lv
Institute of Pharmacy
of Serbia
Vojvode Stepe 458
11152 BELGRADE
hygia@zzfs.org.yu
Statny ustav pre kontrolu lieciv

Slovenska liekopisna komisia
Kvetn 11
SK 825 08 BRATISLAVA
martincova@sukl.sk
SLOVENIA
Ministrstvo za zdravje
Urad RS za zdravila
Komisija za farmakopejo
Kersnikova 2
SI 1000 LJUBLJANA
urad.zdravila@gov.si
SPAIN
Real Farmacopea Espanola

Agencia Espanola de
Medicamentos y Productos
Sanitarios
C/ Alcala 56
Office n 554
E 28014 MADRID
avardulaki@agemed.es
SWEDEN
Swedish Pharmacopoeia
Commission
Medical Products Agency
P.O. Box 26
S 751 03 UPPSALA
karl-gustav.svensson@mpa.se
SWITZERLAND
Swissmedic, Swiss Agency

for Therapeutic Products,
Pharmacopoeia Unit
Hallerstrasse 7
P. O. Box
CH 3000 BERN 9
pharmacopoeia@swissmedic.ch
THE FORMER YUGOSLAV
REPUBLIC OF MACEDONIA
Ministry of Health
Pharmaceutical Department
Divizija b.b., 50
MK 91000 SKOPJE
farmac@mol.com.mk
TURKEY
Ministry of Health
General Directorate of
Pharmaceuticals
Ilkiz Sokak No. 4
TR 06430 SIHHIYE ANKARA
hmihcak@saglik.gov.tr
UNITED KINGDOM
British Pharmacopoeia
Commission
Market Towers
1, Nine Elms Lane
GB LONDON SW8 5NQ
bpsect@mhra.gsi.gov.uk

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