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EUROPEAN PHARMACOPOEIA - SUPPLEMENT 6.3 TO THE 6th EDITION
published 10 June 2008
The 6th Edition of the European Pharmacopoeia consists of volumes 1 and 2 of the publication 6.0, and
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EUROPEAN PHARMACOPOEIA
SIXTH EDITION
Supplement 6.3
 EUROPEAN
PHARMACOPOEIA
SIXTH EDITION
Supplement 6.3

Published in accordance with the

Convention on the Elaboration of a European Pharmacopoeia
(European Treaty Series No. 50)

Council of Europe
Strasbourg
The European Pharmacopoeia is published by the Directorate for the Quality of Medicines & HealthCare
of the Council of Europe (EDQM).

© Council of Europe, 67075 Strasbourg Cedex, France - 2008

All rights reserved. Apart from any fair dealing for the purposes of research or private study, this
publication may not be reproduced, stored or transmitted in any form or by any means without the
prior permission in writing of the publisher.

ISBN: 978-92-871-6312-7
 CONTENTS
CONTENTS OF SUPPLEMENT 6.3 xxxvii
GENERAL CHAPTERS 3907
2. Methods of Analysis 3907
2.2. Physical and physicochemical methods 3907
2.2.33. Nuclear magnetic resonance spectrometry 3909
2.2.42. Density of solids 3912
2.5. Assays 3913
2.5.24. Carbon dioxide in gases 3915
2.5.25. Carbon monoxide in gases 3915
2.5.27. Oxygen in gases 3916
2.6. Biological tests 3917
2.6.1. Sterility 3919
2.6.12. Microbiological examination of non-sterile products: microbial enumeration tests 3923
2.6.13. Microbiological examination of non-sterile products: test for specified
micro-organisms 3927
2.7. Biological assays 3933
2.7.2. Microbiological assay of antibiotics 3935
2.9. Pharmaceutical technical procedures 3941
2.9.1. Disintegration of tablets and capsules 3943
2.9.33. Characterisation of crystalline and partially crystalline solids by X-ray powder
diffraction (XRPD) 3945
4. Reagents 3951
4.1.1. Reagents 3953
4.1.2. Standard solutions for limit tests 3954
4.1.3. Buffer solutions 3954
4.2.2. Volumetric solutions 3954
5. General Texts 3955
5.1.4. Microbiological quality of non-sterile pharmaceutical preparations and substances
for pharmaceutical use 3957
5.1.5. Application of the F0 concept to steam sterilisation of aqueous preparations 3958
5.1.9. Guidelines for using the test for sterility 3958
5.2.3. Cell substrates for the production of vaccines for human use 3963
GENERAL MONOGRAPHS 3967
MONOGRAPHS ON DOSAGE FORMS 3975
MONOGRAPHS ON VACCINES FOR HUMAN USE 3981
MONOGRAPHS ON VACCINES FOR VETERINARY USE 3995
MONOGRAPHS ON RADIOPHARMACEUTICAL PREPARATIONS 3999
MONOGRAPHS 4011
INDEX 4353

Note : on the first page of each chapter/section there is a list of contents.

EUROPEAN PHARMACOPOEIA 6.3 Contents of Supplement 6.3

CONTENTS OF SUPPLEMENT 6.3

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 Calcium gluconate, anhydrous (2364)

5.1.9. Guidelines for using the test for sterility Citalopram hydrobromide (2288)
Citalopram hydrochloride (2203)
MONOGRAPHS Cod-liver oil, farmed (2398)
The monographs below appear for the first time in the Dydrogesterone (2357)
European Pharmacopoeia. They will be implemented on Esomeprazole magnesium trihydrate (2372)
1 January 2009 at the latest.
Filgrastim concentrated solution (2206)
Vaccines for human use Interferon beta-1a concentrated solution (1639)
Shingles (herpes zoster) vaccine (live) (2418) Lamotrigine (1756)
Radiopharmaceutical preparations Lauromacrogol 400 (2046)
Pentetate sodium calcium for radiopharmaceutical Mallow leaf (2391)
preparations (2353) Meloxicam (2373)
Technetium (99mTc) mebrofenin injection (2393) Methylphenidate hydrochloride (2235)
Tetra-O-acetyl-mannose triflate for radiopharmaceutical Omeprazole magnesium (2374)
preparations (2294) Pea starch (2403)
Monographs Saquinavir mesilate (2267)
Aluminium sodium silicate (1676) Schisandra fruit (2428)
Artichoke leaf dry extract (2389) Sevoflurane (2269)
Benazepril hydrochloride (2388) Teicoplanin (2358)

REVISED TEXTS

GENERAL CHAPTERS MONOGRAPHS

The monographs below have been technically revised
2.2.33. Nuclear magnetic resonance spectrometry since their last publication. They will be implemented on
2.2.42. Density of solids 1 January 2009.
2.5.24. Carbon dioxide in gases General monographs
2.5.25. Carbon monoxide in gases Substances for pharmaceutical use (2034)
Vaccines for human use (0153)
2.5.27. Oxygen in gases
Dosage forms
2.6.1. Sterility Powders for cutaneous application (1166)
2.6.12. Microbiological examination of non-sterile products: Semi-solid preparations for cutaneous application (0132)
microbial enumeration tests
Vaccines for human use
2.6.13. Microbiological examination of non-sterile products: BCG for immunotherapy (1929) (published in error under
test for specified micro-organisms letter B in the monographs section)
2.7.2. Microbiological assay of antibiotics Diphtheria, tetanus, pertussis (acellular, component),
2.9.1. Disintegration of tablets and capsules poliomyelitis (inactivated) and haemophilus type b conjugate
vaccine (adsorbed) (2065)
2.9.33. Characterisation of crystalline and partially Haemophilus type b conjugate vaccine (1219)
crystalline solids by X-ray powder diffraction (XRPD)
Poliomyelitis vaccine (inactivated) (0214)
4. Reagents (new, revised, corrected) Varicella vaccine (live) (0648)
5.1.4. Microbiological quality of non-sterile pharmaceutical Radiopharmaceutical preparations
preparations and substances for pharmaceutical use Technetium (99mTc) colloidal rhenium sulphide
5.1.5. Application of the F0 concept to steam sterilisation injection (0126)
of aqueous preparations Technetium (99mTc) macrosalb injection (0296)
5.2.3. Cell substrates for the production of vaccines for Technetium (99mTc) microspheres injection (0570)
human use Technetium (99mTc) tin pyrophosphate injection (0129)

xxxvii
Contents of Supplement 6.3 EUROPEAN PHARMACOPOEIA 6.3

Monographs Human plasma (pooled and treated for virus inactivation)

Acacia (0307) (1646)
Acacia, spray-dried (0308) Hydroxypropylbetadex (1804)
N-Acetyltryptophan (1383) Kaolin, heavy (0503)
Adenosine (1486) Lactitol monohydrate (1337)
Agar (0310) Lactose, anhydrous (1061)
Air, medicinal (1238) Lactose monohydrate (0187)
Alginic acid (0591) Lactulose (1230)
Almagate (2010) Lactulose, liquid (0924)
Aluminim magnesium silicate (1388) Levodropropizine (1535)
Aluminium oxide, hydrated (0311) Lynestrenol (0558)
Magnesium carbonate, light (0042)
Aluminium phosphate gel (2166)
Magnesium oxide, heavy (0041)
Amphotericin B (1292)
Magnesium oxide, light (0040)
Aprotinin (0580)
Magnesium stearate (0229)
Aprotinin concentrated solution (0579)
Maize starch (0344)
Ascorbic acid (0253)
Maltitol (1235)
BCG for immunotherapy (1929)
Maltodextrin (1542)
Beclometasone dipropionate, anhydrous (0654)
Mannitol (0559)
Beclometasone dipropionate monohydrate (1709)
Mefenamic acid (1240)
Belladonna leaf dry extract, standardised (1294)
Methacrylic acid - ethyl acrylate copolymer (1:1) dispersion
Bentonite (0467) 30 per cent (1129)
Betamethasone valerate (0811) Methotrexate (0560)
Bitter-orange epicarp and mesocarp (1603) Mianserin hydrochloride (0846)
Bitter-orange flower (1810) Naphazoline hydrochloride (0730)
Calcium folinate (0978) Nicotine (1452)
Calcium gluconate (0172) Nicotine resinate (1792)
Calcium gluconate for injection (0979) Olive leaf (1878)
Calcium stearate (0882) Omega-3-acid ethyl esters 60 (2063)
Cellulose acetate (0887) Omega-3-acid ethyl esters 90 (1250)
Cellulose acetate phthalate (0314) Omega-3-acid triglycerides (1352)
Cellulose, microcrystalline (0316) Oxaliplatin (2017)
Cellulose, powdered (0315) Oxymetazoline hydrochloride (0943)
Charcoal, activated (0313) Paclitaxel (1794)
Chondroitin sulphate sodium (2064) Pancreas powder (0350)
Cisplatin (0599) Pepsin powder (0682)
Cod-liver oil (type A) (1192) Perphenazine (0629)
Cod-liver oil (type B) (1193) Polyacrylate dispersion 30 per cent (0733)
Croscarmellose sodium (0985) Poly(vinyl acetate) dispersion 30 per cent (2152)
Crospovidone (0892) Potassium citrate (0400)
Dextran 1 for injection (1506) Potato starch (0355)
Dextran 40 for injection (0999) Pravastatin sodium (2059)
Dextran 60 for injection (1000) Rice starch (0349)
Dextran 70 for injection (1001) Senna leaf dry extract, standardised (1261)
Erythritol (1803) Sodium alginate (0625)
Ferrous gluconate (0493) Sodium ascorbate (1791)
Frangula bark dry extract, standardised (1214) Sodium glycerophosphate, hydrated (1995)
Galactose (1215) Sodium hyaluronate (1472)
Gelatin (0330) Sodium polystyrene sulphonate (1909)
Glucose, liquid, spray-dried (1525) Sodium stearate (2058)
Guar (1218) Sorbitol (0435)
Guar galactomannan (0908) Sorbitol, liquid, partially dehydrated (2048)
Haemodialysis solutions, concentrated, water for diluting Starch, pregelatinised (1267)
(1167) Sucrose (0204)
Hard fat (0462) Sugar spheres (1570)
Human normal immunoglobulin for intravenous Sumatriptan succinate (1573)
administration (0918) Talc (0438)

xxxviii
EUROPEAN PHARMACOPOEIA 6.3 Contents of Supplement 6.3

Tetracosactide (0644) Water, highly purified (1927)

Tragacanth (0532) Water, purified (0008)
Tributyl acetylcitrate (1770)
Wheat starch (0359)
Trypsin (0694)
Tryptophan (1272) Xanthan gum (1277)
Water for injections (0169) Xylitol (1381)

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 6.3 (10 June 2008).

MONOGRAPHS Glycerol mono-oleate (1430)

Granisetron hydrochloride (1695)
Dosage forms
Human haematopoietic stem cells (2323)
Intrauterine preparations for veterinary use (1806)
Hypromellose (0348)
Vaccines for veterinary use Hypromellose phthalate (0347)
Clostridium chauvoei vaccine for veterinary use (0361) Ichthammol (0917)
Monographs Lemon verbena leaf (1834)
Acemetacin (1686) Macrogol 40 sorbitol heptaoleate (2396)
Magaldrate (1539)
Amiodarone hydrochloride (0803)
Methylcellulose (0345)
Amitriptyline hydrochloride (0464)
Methyltestosterone (0410)
Arnica flower (1391)
Moxidectin for veterinary use (1656)
Arnica tincture (1809)
Phenol (0631)
Atropine (2056)
Pholcodine (0522)
Atropine sulphate (0068)
Pilocarpine hydrochloride (0633)
Azithromycin (1649)
Pilocarpine nitrate (0104)
Buserelin (1077)
Polysorbate 20 (0426)
Carprofen for veterinary use (2201)
Polysorbate 40 (1914)
Cholecalciferol concentrate (oily form) (0575) Polysorbate 60 (0427)
Cholecalciferol concentrate (powder form) (0574) Polysorbate 80 (0428)
Cholecalciferol concentrate (water-dispersible form) (0598) Potassium dihydrogen phosphate (0920)
Clonidine hydrochloride (0477) Racecadotril (2171)
Codergocrine mesilate (2060) Sertraline hydrochloride (1705)
Dexamethasone acetate (0548) Sesame oil, refined (0433)
Disodium phosphate, anhydrous (1509) Sodium alendronate (1564)
Ergocalciferol (0082) Sodium molybdate dihydrate (1565)
Ethacridine lactate monohydrate (1591) St. John’s wort dry extract, quantified (1874)
Fluvoxamine maleate (1977) Sultamicillin tosilate dihydrate (2212)
Glucose, anhydrous (0177) Telmisartan (2154)
Glucose monohydrate (0178) Triamterene (0058)

TEXTS WHOSE TITLE HAS CHANGED

The title of the following texts has been changed in Supplement 6.3.

GENERAL CHAPTERS
2.6.12. Microbiological examination of non-sterile products: microbial enumeration tests (previously Microbiological
examination of non-sterile products: total viable aerobic count)
5.1.4. Microbiological quality of non-sterile pharmaceutical preparations and substances for pharmaceutical use
(previously Microbiological quality of pharmaceutical preparations)

xxxix
Contents of Supplement 6.3 EUROPEAN PHARMACOPOEIA 6.3

DELETED TEXTS

The following text was deleted on 1 April 2008.

MONOGRAPHS
Vaccines for human use
Pertussis vaccine (0160)

REINSTATED TEXTS

The following text was deleted on 1 April 2008, but has been reinstated and is reintroduced unchanged in
Supplement 6.3. It is to be taken into account from the publication date of Supplement 6.3 (10 June 2008)
MONOGRAPHS
Monographs
Stanozolol (1568)

xl
EUROPEAN PHARMACOPOEIA 6.3

2.2. PHYSICAL AND

PHYSICOCHEMICAL
METHODS
2.2.33. Nuclear magnetic resonance spectrometry.. ........3909 2.2.42. Density of solids.. ....................................................... 3912

General Notices (1) apply to all monographs and other texts 3907
 EUROPEAN PHARMACOPOEIA 6.3

3908 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.2.33. Nuclear magnetic resonance spectrometry

01/2009:20233 APPARATUS
A high-resolution NMR spectrometer consists of at least the
2.2.33. NUCLEAR MAGNETIC following parts :
— a magnet to deliver the constant magnetic field B0 ;
RESONANCE SPECTROMETRY
— a temperature-controlled probe to contain the sample, to
INTRODUCTION deliver the radiofrequency pulse and to detect radiation
emitted by the sample ;
Nuclear magnetic resonance (NMR) spectrometry is an
— an electronic console to generate high-power
analytical method in particular suitable for the elucidation
radiofrequency pulses and to collect and digitise the
of the chemical structure of organic molecules by means
FID signal ; this unit also maintains the stability of the
of interpretation of their NMR spectra, arising from, for
1 13 19 15 31 instrument electronics ;
example, H or the X-nuclei C, F, N, P. The spectra can
be used for qualitative and quantitative purposes. — a data acquisition and processing unit (computer) ;
Under suitable experimental conditions, the integrated NMR and may also include :
intensities of the signals are directly proportional to the — a continuous flow cell for coupled liquid
number of nuclear spins of the molecular group responsible chromatographic-NMR or flow injection analysis ;
for the signal. These integrals can be used for quantitative — a system for pulsed field gradient NMR.
analysis. The high magnetic field is generated by a superconducting
coil in a Dewar flask filled with liquid helium. The probe
GENERAL PRINCIPLES typically contains the sample in a 5 mm-outer-diameter
Placing an ensemble of nuclei with angular momentum sample tube or in a flow cell, and is connected to the
and a magnetic moment in a static magnetic field (B0) electronics cabinet by RF cables carrying lock, 1H-, and
causes the nuclei to arrange themselves in different, X-nucleus frequencies. Additional devices for tuning and
quantum-mechanically controlled orientations in relation matching the electronic circuits are essential, and sample
to the axis of the magnetic field. These orientations are temperature control is often used.
different in energy. An oscillating high-frequency magnetic The NMR spectrometer should be demonstrated to be
field (B1), perpendicular to B0, will cause transitions between operating correctly. Appropriate tests to demonstrate this
these orientations with net energy absorption. According to are, typically, measurement of linewidths at half height
the resonance condition ω0 = γB0 (γ = gyromagnetic ratio, for defined peaks under defined acquisition conditions,
ω0 = Larmor frequency), either the B0 magnetic field or the signal-to-noise ratios (S/N) for standard mixtures, pulse
frequency (ω1) of the B1 field may be varied to achieve a power (measured as a 90° pulse width), and pulse
spectrum (continuous wave (CW) method). Nowadays the B1 reproducibility. All instrument manufacturers publish
irradiation is achieved by the use of a radiofrequency (RF) specifications and measurement protocols for these
pulse (Fourier transform (FT) method). The coherent parameters for specific instrument/probe combinations,
radiation emitted during the return to the initial state and compliance with these specifications should be
is observed in the form of a decay curve, called the free demonstrated.
induction decay (FID). Subsequent Fourier transformation
gives the spectrum in the frequency domain, providing FOURIER TRANSFORM NMR (FT-NMR)
information about the molecular structure. Additional Contemporary spectrometers generally operate according
radiofrequency fields may be applied during acquisition to the Fourier transform (FT) principle : after exciting the
of the FID signal to suppress scalar (through-bond) sample with a radiofrequency pulse of appropriate frequency
interactions between nuclei (called ‘decoupling’). One- and (ν), amplitude (B1) and duration (τp) and a succeeding
multi-dimensional techniques can be applied for qualitative short dead time (t ) (to enable the electronics to recover),
d
and quantitative purposes, on samples in either the liquid the amplified analogue FID signal is sampled during the
or the solid state. acquisition time (tac) and digitised with an analogue-to-digital
Important structural information is derived from the converter (ADC), and the results are stored in the
following spectroscopic features : spectrometer memory. The receiver output is amplified prior
to digitisation to maximise sensitivity without saturating
resonance frequency kind of nuclei observed the ADC. In case of observation of X-nuclei, the standard
1
number of resonance signals number of chemically distinct groups experiment includes, if necessary, broadband H decoupling,
(singlets, multiplets) of nuclei i.e. irradiation of all the protons during the experiment. To
chemical shift δ (ppm) chemical nature and environment of increase the S/N, multiple FID signals may be accumulated
the structural group observed coherently and summed. Fourier transformation of this
intensity of resonance signals relative number of resonant nuclei time-domain data gives the frequency-domain spectrum.
per chemically distinct group PARAMETERS
multiplicity of coupling pattern number of nuclei that are scalar The following acquisition parameters influence the result of
coupled to the observed nucleus
an FT experiment, and should be adjusted and controlled.
coupling constant nJ (Hz) number of bonds in the coupling
pathway, and its geometry Pulse width (τp). The excitation pulse is directed along the
x-axis of the so-called rotating frame, its duration (or ‘width’,
Correlations of different spectral parameters (e.g. chemical τp) determines the flip angle (θ) and thus the intensity (I) of
shift and coupling constant, or chemical shifts of different the resonance signal :
nuclei within one molecular system) can be performed by
homo- and hetero-nuclear two- and higher-dimensional
methods. Information about the relaxation times T1 and (1)
T2, nuclear Overhauser effects (NOEs) and the kinetics
of time-dependent processes are also accessible from
appropriate experiments. (2)

General Notices (1) apply to all monographs and other texts 3909
2.2.33. Nuclear magnetic resonance spectrometry
The observed magnetisation My is maximum at θ = 90°. The
pulse duration should be short to guarantee that all signals
in the spectral width (SW) are excited to a similar degree.
The magnetisation decays due to relaxation processes.
Dead time (td). The dead time is the time between the end
of the pulse and start of the acquisition, it is necessary
for technical reasons and care should be taken as it may
influence signal intensities and peak phase. Rapidly decaying
signals (giving rise to broad spectral lines) are reduced in
intensity by more than slowly decaying signals (which give
rise to narrow spectral lines).
Acquisition time (tac). The acquisition time (tac) is related to
the spectral width (i.e. the whole observed region) and the
number of digital data points (DP) collected during signal
acquisition.
(3)
Maximal signal intensity and signal-to-noise ratio will be
achieved if tac ≈ 1.2/(πν1/2), where ν1/2 is the full width
at half-height (fwhh), but it should be set to greater than
5/(πν1/2) to minimise signal distortion.
Repetition time (tr). The spin-lattice relaxation (T1) governs
the time required for the spin system to return to equilibrium
after a pulse. Relaxation can be reduced by the use of special
reagents. For quantitative purposes, the repetition time used
should be set relative to T1 and θ to avoid saturation effects.
Receiver gain. The analogue signal detected by the probe is
amplified prior to digitisation and storage. The amplification,
or receiver gain, should be set, either automatically or
manually, so that the signal does not overload the ADC,
which causes signal distortion, but allows random noise
generated in the probe to be digitised (i.e. is non-zero).
OPTIMISATION OF ACQUISITION AND PROCESSING
PARAMETERS FOR QUANTITATIVE PURPOSES
Besides the acquisition parameters, signal intensity is also
influenced by several processing parameters. After collecting
a sufficient number of scans, the resulting FID is Fourier
transformed. For reliable quantitative purposes the following
parameters have to be optimised.
Digital resolution. The digital resolution is the frequency
separation between data points. The processed signal should
have at least 5 digital points above half-height of the signals
to be integrated. To improve the digital resolution additional
points of zero intensity may be added to the end of the
experimental FID before transformation (‘zero filling’).
Signal-to-noise ratio (S/N). This is the ratio between the
intensities (as peak height) of a specified signal in the
NMR spectrum and the random fluctuations in that signal,
which is usually measured in a region of the spectrum that
contains no signals from the analyte. A poor signal-to-noise
ratio (S/N) limits the accuracy of peak integrations and
quantitative analyses. An S/N equal to or greater than
150:1 allows peak integrations with a standard deviation
of less than 1 per cent. Contemporary spectrometers have
software algorithms to report the S/N of appropriate peaks.
A sufficiently high S/N can be difficult to obtain when
analysing dilute solutions, and especially when detecting
nuclei other than 1H. Methods to enhance the S/N include :
— increasing the number of accumulated scans (n), as S/N
increases with ;
— use of exponentional multiplication on the FID signal
before Fourier transformation ; the exponentional
multiplication factor should be in the order of the peak
full width at half-height (fwhh) ;
3910 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
— use of spectrometers with a higher magnetic field B0,
since S/N is proportional to B03/2 ;
— use of digital filtering to reduce noise ;
— use of probes that maximise the filling factor ;
— use of cryoprobes that reduce thermal noise.
Integration region. The intensity of the NMR signals is
obtained by a quasi-analogue signal integration either by
a stepped-line plot or, more accurately, by separate line
integration and digital data presentation. In liquid state,
NMR signals have Lorentzian line shape. Unless otherwise
specified in the monograph or when peak overlap occurs,
the same integration range, expressed as a multiple of the
peak fwhh, should be used for the monitored peak and the
reference peak.
Dynamic range. The dynamic range of the analogue-to-digital
converter (ADC) determines the minimum intensity line that
can be observed or quantified when integrating 2 signals
with the same linewidth in a spectrum. A 16-bit ADC allows
identification of a signal of 0.003 per cent intensity relative
to a strong signal completely filling the dynamic range of
the ADC.
NMR OF SAMPLES IN SOLUTION
Most NMR experiments are performed on dilute solutions
(about 1 per cent) of the analyte in an appropriate solvent,
which can be spiked with a suitable standard for chemical
shift calibration.
Solvents. The solvent should be able to dissolve the analyte
without further interaction if not otherwise intended.
To minimise the intense solvent signals, fully deuterated
solvents (deuterium oxide R, deuterated chloroform R,
deuterated dimethyl sulphoxide R, deuterated acetone R,
deuterated methanol R, etc.) should be used. The solvent
atoms give signals that are easily identified by their chemical
shift and can be used to calibrate the chemical shift axis
(secondary reference).
Referencing. The spectral feature most dependent on the
chemical environment of the atom in the molecule is the
chemical shift, designated as δ and reported in parts per
million. The chemical shift between the resonance for an
NMR active nucleus X (δX,sample) is measured in parts per
million as the difference between the resonance frequency of
that nucleus (νX,sample) and that of an internal shift reference
standard (νX,reference), both in hertz, divided by the basic
spectrometer operating frequency (νX,reference), in megahertz,
at a given B0 :
(4)
By convention, the standard for exact chemical shift
referencing is the 1H resonance of tetramethylsilane R
(TMS), setting δTMS = 0 ppm. Formally, once the 1H shift scale
has been referenced relative to TMS, the exact frequency of
any other X resonance can be calculated and its chemical
shift scale calibrated. The frequency of a (secondary)
reference standard at δX = 0 ppm (νX,reference) is calculated from
the 1H frequency of TMS (νH,TMS) and a tabulated value of the
ratio ( X,reference) of the isotope-specific frequency in relation
to that of 1H in TMS:
(5)
EUROPEAN PHARMACOPOEIA 6.3 2.2.33. Nuclear magnetic resonance spectrometry

Reference standards at δX = 0 ppm and corresponding spectrometer is easily checked by comparing exact intensities
X,reference values are shown below : within a spectrum of any suitable organic compound of
known structure.
Other In addition to the fact that the intensities of signals arising
Nucleus Watera X,reference
solvents X,reference

1
from each component in a mixture are related to each
H DSSb 1.00000000 TMS 1.00000000
other by small integer numbers, the relative molar amounts
13
C DSSb 0.25144953 TMS 0.25145020 of these components can be measured by comparison of
15
N NH3 0.10132912 CH3NO2 0.10136767
the normalised intensities of resonances from different
components. The molar ratio of 2 components of a mixture
19
F CF3COOH not reported CCl3F 0.94094011 is calculated according to the following equation :
31
P H3PO4 0.40480742 (CH3O)3PO 0.40480864
(85 per cent)
a
chemical shift depends on pH
(8)
b
DSS = sodium 2,2-dimethyl-2-silapentane-5-sulfonate The determination is only valid in cases where the structure
of the molecules for which IA and IB are determined are
In practice, X chemical shifts are referenced directly using known (or at least the values of N for the monitored groups).
an appropriate standard. In 1H and 13C NMR, internal Determinations are made using either an internal standard
referencing is mainly used, where the reference is added method or a peak-normalisation procedure.
directly to the system under study. In 15N, 19F and 31P NMR, Internal standard method. The mass (mA) of an analyte (A)
external referencing is often suitable, involving sample and can be determined if a known mass (mB) of a substance (B)
reference contained separately in coaxial cylindrical sample with a known percentage content (PB) is added to the
tubes. solution as an intensity standard. Equation (8) can be
Lock. In order to prevent drifting of the spectrum over converted to equation (9) :
time, a stabilising procedure, called field-frequency locking,
is performed. The 2H (deuterium) signal arising from
deuterated solvents is used to achieve this, unless otherwise
(9)
specified in the monograph.
QUALITATIVE ANALYSIS Here, Mi are the molecular masses.
The principal use for qualitative NMR spectra is as an
The intensity standard has to be carefully chosen ; it should
identity test, in which the 1H or 13C spectrum of a test sample
be completely soluble in the solvent used for the analyte,
is compared to the spectrum of a reference sample or, less
should produce only a small number of signals, and the
commonly, with a published reference spectrum. Spectra
‘monitor group’ should have a signal in an empty spectral
of reference and test samples should be acquired using the
region. A compound of high purity and with a relatively high
same procedure and operational conditions. The peaks
molecular mass is recommended for this purpose.
in the 2 spectra, or characteristic regions of the spectra,
should correspond in position, intensity and multiplicity. Normalisation procedure. The relative proportions of
In appropriate cases, mathematical comparison, such as components in a mixture, the degree of substitution
calculation of a correlation coefficient, may be appropriate. in a structurally modified polymer, or the amount of a
In the absence of a reference standard, an in-house reference contaminant can be determined by comparison of the relative
may be used, whose identity has been demonstrated by intensities of resonances present.
alternative methods, or the demonstration that the NMR The experimental method should be validated to ensure
spectrum is fully consistent with the reported structure for that there is no overlap of the relevant signals. When the
that material. contaminant is of poorly defined structure or molecular
QUANTITATIVE ANALYSIS mass (e.g. an emulsifier), addition of known amounts of that
material to the NMR tube will allow a calibration curve to be
Signal intensity in the basic NMR experiment is the
constructed.
integrated area under the signal curve measured. Only
when 2 signals have equal fwhh and the same multiplicity METHOD
may signal height serve as a measure of intensity. Under
conditions of essentially complete relaxation between scans, Sample handling. Dissolve the sample in the solvent to
the signal intensity (IA) is a true measure of the number (NA) which the appropriate reference material may have been
of nuclei responsible for the respective signal : added to calibrate chemical shift, as prescribed in the
monograph. For quantitative analysis, the solutions must
be free from solid particles. Some quantitative analyses
(6) may require an internal standard to be included, so that
The constant KS includes fundamental constants, properties integrations of resonances from the test sample and the
of the sample and receiver parameters, and can be omitted in reference material can be compared. Appropriate references
cases where signal intensities are compared, giving the direct and concentrations are indicated in the specific monographs.
relation between the numbers of nuclei in the 2 compared In other quantitative analyses, the result is obtained by
structure groups A and B : comparing the relative intensities of 2 or all of the resonances
that arise from the test sample. After loading the sample
into a tube and capping, the sample is introduced into the
(7) NMR magnet, the experimental parameters are loaded and
the experiment is executed. Key experimental parameters
The numbers (Ni) of nuclei belonging to different structure are indicated in the monograph.
groups of 1 molecule are small integers. The values measured The measurement procedure. Equilibrate the sample in
are rounded to the closest integer numbers. However, the probe, and optimise the instrument to achieve best
the proper operation of acquisition and processing of the resonance conditions and to maximise the S/N by tuning

General Notices (1) apply to all monographs and other texts 3911
2.2.42. Density of solids
and matching the probe, and make adjustments to maximise
magnetic field homogeneity over the sample volume (called
‘shimming’). Record, or save to computer, the parameter
settings. An experiment may be composed of multiple
pulse-acquisition-delay sequences, and the individual FIDs
are summed in the computer memory, with random noise
being averaged out. When an appropriate S/N has been
achieved, the FID is stored and the frequency-domain
spectrum is generated by Fourier transformation of the
summed FIDs.
NMR IN THE SOLID STATE
Samples in the solid state can be analysed using NMR
spectrometers specially equipped for that purpose. Certain
technical procedures make observable individual lines for
individual atomic sites with a valuable extension of the
applicability of NMR to inorganic materials as well.
One technique is the rapid rotation (4-30 kHz) of the
powdered sample in a rotor (about 4 mm outer diameter)
inclined at an angle of 54.7° (the ‘magic angle’) to the
direction of the B0 magnetic field axis. This technique
is named magic angle spinning (MAS). Another effective
tool is high-power decoupling and a 3rd method is the
transfer of polarisation from easily excitable nuclei
towards less-polarisable nuclei, i.e. cross polarisation (CP).
The combination of these techniques makes available
high-resolution spectra containing much information
about chemical and structural details in solid glassy,
amorphous, and crystalline materials of ceramic, polymeric
or mineralogical origin.
If NMR is applied to a solid, full details of the procedure are
provided in the monograph.
01/2009:20242
2.2.42. DENSITY OF SOLIDS
The density of solids corresponds to their average mass
per unit volume and typically is expressed in grams per cubic
centimetre (g/cm3) although the International Unit is the
kilogram per cubic meter (1 g/cm3 = 1000 kg/m3).
Unlike gases and liquids whose density depends only on
temperature and pressure, the density of a solid particle also
depends on its molecular assembly and therefore varies with
the crystal structure and degree of crystallinity.
When a solid particle is amorphous or partially amorphous,
its density may further depend upon the history of
preparation and treatment.
Therefore, unlike fluids, the densities of 2 chemically
equivalent solids may be different, and this difference
reflects a difference in solid-state structure. The density of
constituent particles is an important physical characteristic
of pharmaceutical powders.
The density of a solid particle can assume different values
depending on the method used to measure the volume of
the particle. It is useful to distinguish 3 levels of expression
of density :
— the crystal density, which only includes the solid fraction
of the material ; the crystal density is also called true
density ;
— the particle density, which also includes the volume due
to intraparticulate pores ;
— the bulk density, which further includes the
interparticulate void volume formed in the powder bed ;
the bulk density is also called apparent density.
3912 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
CRYSTAL DENSITY
The crystal density of a substance is the average mass
per unit volume, exclusive of all voids that are not a
fundamental part of the molecular packing arrangement. It
is an intrinsic property of the substance, and hence should
be independent of the method of determination. The crystal
density can be determined either by calculation or by simple
measurement.
A. The calculated crystal density is obtained using
crystallographic data (size and composition of the unit
cell) of a perfect crystal, from for example X-ray diffraction
data, and the molecular mass of the substance.
B. The measured crystal density is the mass-to-volume ratio
after measuring the monocrystal mass and volume.
PARTICLE DENSITY
The particle density takes into account both the crystal
density and the intraparticulate porosity (sealed and/or open
pores). Thus, particle density depends on the value of the
volume determined, which in turn depends on the method of
measurement. The particle density can be determined using
one of the 2 following methods.
A. The pycnometric density is determined by measuring
the volume occupied by a known mass of powder, which
is equivalent to the volume of gas displaced by the
powder using a gas displacement pycnometer (2.9.23).
In pycnometric density measurements, the volume
determined includes the volume occupied by open pores ;
however, it excludes the volume occupied by sealed
pores or pores inaccessible to the gas. Due to the high
diffusivity of helium, which is the preferred choice of gas,
most open pores are accessible to the gas. Therefore, the
pycnometric density of a finely milled powder is generally
not very different from the crystal density.
B. The mercury porosimeter density is also called granular
density. With this method the volume determined also
excludes contributions from sealed pores ; however, it
includes the volume only from open pores larger than
some size limit. This pore-size limit or minimal access
diameter depends on the maximal mercury intrusion
pressure applied during the measurement, and under
normal operating pressures the mercury does not
penetrate the finest pores accessible to helium. Various
granular densities can be obtained from one sample since,
for each applied mercury intrusion pressure, a density
can be determined that corresponds to the pore-size limit
at that pressure.
BULK AND TAPPED DENSITY
The bulk density of a powder includes the contribution
of interparticulate void volume. Hence, the bulk density
depends on both the density of powder particles and the
space arrangement of particles in the powder bed.
The bulk density of a powder is often very difficult to
measure since the slightest disturbance of the bed may
result in a new density. Thus, it is essential in reporting bulk
density to specify how the determination was made.
A. The bulk density is determined by measuring the volume
of a known mass of powder that has been passed through
a screen into a graduated cylinder (2.9.34).
B. The tapped density is achieved by mechanically tapping
a measuring cylinder containing a powder sample. After
observing the initial volume, the cylinder is mechanically
tapped, and volume readings are taken until little further
volume change is observed (2.9.34).
EUROPEAN PHARMACOPOEIA 6.3

2.5. ASSAYS
2.5.24. Carbon dioxide in gases............................................ 3915 2.5.27. Oxygen in gases.. ........................................................ 3916
2.5.25. Carbon monoxide in gases....................................... 3915

General Notices (1) apply to all monographs and other texts 3913
 EUROPEAN PHARMACOPOEIA 6.3

3914 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.5.25. Carbon monoxide in gases

01/2009:20524 01/2009:20525

2.5.25. CARBON MONOXIDE IN GASES

2.5.24. CARBON DIOXIDE IN GASES
Gases absorb light at unique wavelengths. This property is
widely used to allow highly selective measurement of their
concentrations.
Description and principle of measurement. The
concentration of carbon dioxide in other gases can be
determined using an infrared analyser.
The infrared analyser generally consists of a light source
emitting broadband infrared radiation, an optical device,
a sample cell and a detector. The optical device may be
positioned either before or after the sample cell and it
consists of one or several optical filters, through which the
broadband radiation is passed. The optical device in this case
is selected for carbon dioxide. The measurement light beam
passes through the sample cell and may also pass through a
reference cell if the analyser integrates such a feature (some
use an electronic system instead of a reference cell).
When carbon dioxide is present in the sample cell, absorption
of energy in the measurement light beam will occur
according to the Beer-Lambert law and this produces a
change in the detector signal. This measurement signal is
compared to a reference signal to generate an output related 0.002 M sodium thiosulphate. Continue flushing until not
to the concentration of carbon dioxide. The generated
signal is linearised in order to obtain the carbon dioxide
concentration. To prevent the entry of particles into the
sensors, which could cause stray-light phenomena, the
apparatus is fitted with a suitable filter.
Required technical specifications. When used for a limit
test, the infrared analyser meets the following technical
specifications :
— limit of detection : (generally defined as a signal-to-noise in the titrations is not greater than the prescribed limit.
ratio of 2) maximum 20 per cent of the maximum
admissible concentration ;
— repeatability : maximum RSD of 10 per cent of the
maximum admissible concentration, determined on 6
measurements ;
— linearity : maximum 10 per cent of the maximum
admissible concentration.
The technical specifications must be met in the presence of The infrared analyser generally consists of a light source
the other gas impurities in the sample.
METHOD I
Apparatus. The apparatus (see Figure 2.5.25.-1) consists of
the following parts connected in series :
— a U-tube (U1) containing anhydrous silica gel R
impregnated with chromium trioxide R ;
— a wash bottle (F1) containing 100 ml of a 400 g/l solution
of potassium hydroxide R ;
— a U-tube (U2) containing pellets of potassium hydroxide R ;
— a U-tube (U3) containing diphosphorus pentoxide R
dispersed on previously granulated, fused pumice ;
— a U-tube (U4) containing 30 g of recrystallised iodine
pentoxide R in granules, previously dried at 200 °C and
kept at a temperature of 120 °C (T) during the test. The
iodine pentoxide is packed in the tube in 1 cm columns
separated by 1 cm columns of glass wool to give an
effective length of 5 cm ;
— a reaction tube (F2) containing 2.0 ml of potassium iodide
solution R and 0.15 ml of starch solution R.
Method. Flush the apparatus with 5.0 litres of argon R and,
if necessary, discharge the blue colour in the iodide solution
by adding the smallest necessary quantity of freshly prepared
more than 0.045 ml of 0.002 M sodium thiosulphate is
required after passage of 5.0 litres of argon R. Pass the gas to
be examined from the cylinder through the apparatus, using
the prescribed volume and the flow rate. Flush the last traces
of liberated iodine into the reaction tube by passing through
the apparatus 1.0 litre of argon R. Titrate the liberated
iodine with 0.002 M sodium thiosulphate. Carry out a blank
test, using the prescribed volume of argon R. The difference
between the volumes of 0.002 M sodium thiosulphate used
METHOD II
Gases absorb light at unique wavelengths. This property is
widely used to allow highly selective measurement of the
concentrations of gases.
Description and principle of measurement. The
concentration of carbon monoxide in other gases can be
determined using an infrared analyser.
emitting broadband infrared radiation, an optical device,

Figure 2.5.25.-1. – Apparatus for the determination of carbon monoxide

Dimensions in millimetres

General Notices (1) apply to all monographs and other texts 3915
2.5.27. Oxygen in gases
a sample cell and a detector. The optical device may be
positioned either before or after the sample cell ; it consists of
one or several optical filters, through which the broadband
radiation is passed. The optical device in this case is selected
for carbon monoxide. The measurement light beam passes
through the sample cell and may also pass through a
reference cell if the analyser integrates such a feature (some
use an electronic system instead of a reference cell).
When carbon monoxide is present in the sample cell,
absorption of energy in the measurement light beam will
occur according to the Beer-Lambert law and this produces
a change in the detector signal. This measurement signal is
compared to a reference signal to generate an output related
to the concentration of carbon monoxide. The generated
signal is linearised in order to obtain the carbon monoxide
concentration. To prevent the entry of particles into the
sensors, which could cause stray-light phenomena, the
apparatus is fitted with a suitable filter.
Required technical specifications. When used for a limit
test, the carbon monoxide infrared analyser meets the
following technical specifications :
— limit of detection : (generally defined as a signal-to-noise
ratio of 2) maximum 20 per cent of the maximum
admissible concentration ;
— repeatability : maximum RSD of 10 per cent of the
maximum admissible concentration, determined on 6
measurements ;
— linearity : maximum 10 per cent of the maximum
admissible concentration.
The technical specifications must be met in the presence of
the other gas impurities in the sample.
3916 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:20527
2.5.27. OXYGEN IN GASES
Oxygen in gases is determined using a paramagnetic
analyser.
The principle of the method is based on the high
paramagnetic sensitivity of the oxygen molecule. Oxygen
exerts a strong interaction on magnetic fields, which is
measured electronically, amplified and converted to a
reading of oxygen concentration. The measurement of
oxygen concentration is dependent upon the pressure
and temperature and, if the analyser is not automatically
compensated for variations in temperature and pressure,
it must be calibrated immediately prior to use. As the
paramagnetic effect of oxygen is linear, the instrument must
have a suitable range with a readability of 0.1 per cent or
better.
Calibration of the instrument. Make the setting in the
following manner :
— set the zero by passing nitrogen R1 through the
instrument until a constant reading is obtained ;
— set the scale to 100 per cent by passing oxygen R through
the instrument at the same flow rate as for nitrogen R1
until a constant reading is obtained.
Assay. Pass the gas to be examined through the instrument
at a constant flow rate until a constant reading is obtained.
Record the concentration of oxygen in the gas to be
examined.
EUROPEAN PHARMACOPOEIA 6.3

2.6. BIOLOGICAL TESTS

2.6.1. Sterility............................................................................ 3919 2.6.13. Microbiological examination of non-sterile products :
2.6.12. Microbiological examination of non-sterile products : test for specified micro-organisms.. ...................................3927
microbial enumeration tests ...............................................3923

General Notices (1) apply to all monographs and other texts 3917
 EUROPEAN PHARMACOPOEIA 6.3

3918 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.1. Sterility

01/2009:20601 taking care to prevent the introduction of non-sterile air into

the container. Do not use the medium for a longer storage
2.6.1. STERILITY period than has been validated.
Fluid thioglycollate medium is to be incubated at 30-35 °C.
The test is applied to substances, preparations or articles For products containing a mercurial preservative that
which, according to the Pharmacopoeia, are required to be cannot be tested by the membrane-filtration method, fluid
sterile. However, a satisfactory result only indicates that thioglycollate medium incubated at 20-25 °C may be used
no contaminating micro-organism has been found in the instead of soya-bean casein digest medium provided that it
sample examined in the conditions of the test. has been validated as described in growth promotion test.
PRECAUTIONS AGAINST MICROBIAL CONTAMINATION Where prescribed or justified and authorised, the following
alternative thioglycollate medium may be used. Prepare
The test for sterility is carried out under aseptic conditions. a mixture having the same composition as that of the
In order to achieve such conditions, the test environment fluid thioglycollate medium, but omitting the agar and the
has to be adapted to the way in which the sterility test is resazurin sodium solution, sterilise as directed above. The
performed. The precautions taken to avoid contamination pH after sterilisation is 7.1 ± 0.2. Heat in a water-bath prior
are such that they do not affect any micro-organisms which to use and incubate at 30-35 °C under anaerobic conditions.
are to be revealed in the test. The working conditions in
which the tests are performed are monitored regularly by Soya-bean casein digest medium
appropriate sampling of the working area and by carrying Pancreatic digest of casein 17.0 g
out appropriate controls. Papaic digest of soya-bean meal 3.0 g
CULTURE MEDIA AND INCUBATION TEMPERATURES Sodium chloride 5.0 g
Media for the test may be prepared as described below, or Dipotassium hydrogen phosphate 2.5 g
equivalent commercial media may be used provided that
Glucose monohydrate/anhydrous 2.5 g/2.3 g
they comply with the growth promotion test.
The following culture media have been found to be suitable Water R 1000 ml
for the test for sterility. Fluid thioglycollate medium is pH after sterilisation 7.3 ± 0.2
primarily intended for the culture of anaerobic bacteria ;
however, it will also detect aerobic bacteria. Soya-bean Dissolve the solids in water R, warming slightly to effect
casein digest medium is suitable for the culture of both fungi solution. Cool the solution to room temperature. Add 1 M
and aerobic bacteria. sodium hydroxide, if necessary, so that after sterilisation
the solution will have a pH of 7.3 ± 0.2. Filter, if necessary,
Fluid thioglycollate medium to clarify, distribute into suitable vessels and sterilise using a
L-Cystine 0.5 g validated process. Store at a temperature between 2 °C and
25 °C in a sterile well-closed container, unless it is intended
Agar 0.75 g for immediate use. Do not use the medium for a longer
Sodium chloride 2.5 g storage period than has been validated.
Glucose monohydrate/anhydrous 5.5 g/5.0 g
Soya-bean casein digest medium is to be incubated at
20-25 °C.
Yeast extract (water-soluble) 5.0 g
The media used comply with the following tests, carried
Pancreatic digest of casein 15.0 g out before or in parallel with the test on the product to be
0.5 g
examined.
Sodium thioglycollate or
Sterility. Incubate portions of the media for 14 days. No
Thioglycollic acid 0.3 ml
growth of micro-organisms occurs.
Resazurin sodium solution (1 g/l of 1.0 ml Growth promotion test of aerobes, anaerobes and fungi.
resazurin sodium), freshly prepared
Water R
Test each batch of ready-prepared medium and each batch
1000 ml
of medium prepared either from dehydrated medium or
pH after sterilisation 7.1 ± 0.2 from ingredients. Suitable strains of micro-organisms are
indicated in Table 2.6.1.-1.
Mix the L-cystine, agar, sodium chloride, glucose,
water-soluble yeast extract and pancreatic digest of casein Inoculate portions of fluid thioglycollate medium with a
with the water R and heat until solution is effected. Dissolve small number (not more than 100 CFU) of the following
the sodium thioglycollate or thioglycollic acid in the solution micro-organisms, using a separate portion of medium for
and, if necessary, add 1 M sodium hydroxide so that, after each of the following species of micro-organism : Clostridium
sterilisation, the solution will have a pH of 7.1 ± 0.2. If sporogenes, Pseudomonas aeruginosa, Staphylococcus
filtration is necessary, heat the solution again without aureus. Inoculate portions of soya-bean casein digest
boiling and filter while hot through moistened filter paper. medium with a small number (not more than 100 CFU) of
Add the resazurin sodium solution, mix and place the the following micro-organisms, using a separate portion of
medium in suitable vessels which provide a ratio of surface medium for each of the following species of micro-organism :
to depth of medium such that not more than the upper half Aspergillus niger, Bacillus subtilis, Candida albicans.
of the medium has undergone a colour change indicative of Incubate for not more than 3 days in the case of bacteria and
oxygen uptake at the end of the incubation period. Sterilise not more than 5 days in the case of fungi.
using a validated process. If the medium is stored, store at Seed lot culture maintenance techniques (seed-lot systems)
a temperature between 2 °C and 25 °C in a sterile, airtight are used so that the viable micro-organisms used for
container. If more than the upper one-third of the medium inoculation are not more than 5 passages removed from the
has acquired a pink colour, the medium may be restored once original master seed-lot.
by heating the containers in a water-bath or in free-flowing The media are suitable if a clearly visible growth of the
steam until the pink colour disappears and cooling quickly, micro-organisms occurs.

General Notices (1) apply to all monographs and other texts 3919
2.6.1. Sterility EUROPEAN PHARMACOPOEIA 6.3

Table 2.6.1.-1 – Strains of the test micro-organisms suitable for use in the growth promotion test and the method
suitability test
Aerobic bacteria

Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134
Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275
Anaerobic bacterium
Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293
Fungi
Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594
Aspergillus niger ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455

METHOD SUITABILITY TEST preparations and for preparations miscible with or soluble in
aqueous or oily solvents provided these solvents do not have
Carry out a test as described below under Test for sterility of
the product to be examined using exactly the same methods an antimicrobial effect in the conditions of the test.
except for the following modifications. Membrane filtration. Use membrane filters having a nominal
Membrane filtration. After transferring the contents of the pore size not greater than 0.45 μm whose effectiveness
container or containers to be tested to the membrane add an to retain micro-organisms has been established. Cellulose
inoculum of a small number of viable micro-organisms (not nitrate filters, for example, are used for aqueous, oily and
more than 100 CFU) to the final portion of sterile diluent weakly alcoholic solutions and cellulose acetate filters, for
used to rinse the filter. example, for strongly alcoholic solutions. Specially adapted
filters may be needed for certain products, e.g. for antibiotics.
Direct inoculation. After transferring the content of the The technique described below assumes that membranes
container or containers to be tested (for catgut and other about 50 mm in diameter will be used. If filters of a different
surgical sutures for veterinary use : strands) to the culture diameter are used the volumes of the dilutions and the
medium add an inoculum of a small number of viable washings should be adjusted accordingly. The filtration
micro-organisms (not more than 100 CFU) to the medium. apparatus and membrane are sterilised by appropriate
In both cases use the same micro-organisms as those means. The apparatus is designed so that the solution to
described above under Growth promotion test of aerobes, be examined can be introduced and filtered under aseptic
anaerobes and fungi. Perform a growth promotion test as conditions ; it permits the aseptic removal of the membrane
a positive control. Incubate all the containers containing for transfer to the medium or it is suitable for carrying out
medium for not more than 5 days. the incubation after adding the medium to the apparatus
If clearly visible growth of micro-organisms is obtained itself.
after the incubation, visually comparable to that in the Aqueous solutions. If appropriate, transfer a small quantity
control vessel without product, either the product possesses of a suitable, sterile diluent such as a 1 g/l neutral solution
no antimicrobial activity under the conditions of the test of meat or casein peptone pH 7.1 ± 0.2 onto the membrane
or such activity has been satisfactorily eliminated. The in the apparatus and filter. The diluent may contain suitable
test for sterility may then be carried out without further neutralising substances and/or appropriate inactivating
modification. substances for example in the case of antibiotics.
If clearly visible growth is not obtained in the presence of Transfer the contents of the container or containers to be
the product to be tested, visually comparable to that in tested to the membrane or membranes, if necessary after
the control vessels without product, the product possesses diluting to the volume used in the method suitability test
antimicrobial activity that has not been satisfactorily with the chosen sterile diluent but in any case using not less
eliminated under the conditions of the test. Modify the than the quantities of the product to be examined prescribed
conditions in order to eliminate the antimicrobial activity in Table 2.6.1.-2. Filter immediately. If the product has
and repeat the method suitability test. antimicrobial properties, wash the membrane not less than
3 times by filtering through it each time the volume of the
This method suitability test is performed : chosen sterile diluent used in the method suitability test. Do
a) when the test for sterility has to be carried out on a new not exceed a washing cycle of 5 times 100 ml per filter, even
product ; if during method suitability test it has been demonstrated
b) whenever there is a change in the experimental conditions that such a cycle does not fully eliminate the antimicrobial
of the test. activity. Transfer the whole membrane to the culture
medium or cut it aseptically into 2 equal parts and transfer
The method suitability test may be performed simultaneously one half to each of 2 suitable media. Use the same volume of
with the test for sterility of the product to be examined. each medium as in the method suitability test. Alternatively,
transfer the medium onto the membrane in the apparatus.
TEST FOR STERILITY OF THE PRODUCT TO BE Incubate the media for not less than 14 days.
EXAMINED Soluble solids. Use for each medium not less than the
The test may be carried out using the technique of membrane quantity prescribed in Table 2.6.1.-2 of the product dissolved
filtration or by direct inoculation of the culture media with in a suitable solvent such as the solvent provided with the
the product to be examined. Appropriate negative controls preparation, water for injections, saline or a 1 g/l neutral
are included. The technique of membrane filtration is solution of meat or casein peptone and proceed with the test
used whenever the nature of the product permits, that is, as described above for aqueous solutions using a membrane
for filterable aqueous preparations, for alcoholic or oily appropriate to the chosen solvent.

3920 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.1. Sterility

Table 2.6.1.-2 — Minimum quantity to be used for each medium

Minimum quantity to be used for each medium unless
Quantity per container
otherwise justified and authorised
Liquids
— less than 1 ml The whole contents of each container
— 1-40 ml Half the contents of each container but not less than 1 ml
— greater than 40 ml and not greater than 100 ml 20 ml
— greater than 100 ml 10 per cent of the contents of the container but not less than 20 ml
Antibiotic liquids 1 ml
Insoluble preparations, creams and ointments to be suspended or
The whole contents of each container to provide not less than 200 mg
emulsified
Solids
— less than 50 mg The whole contents of each container
— 50 mg or more but less than 300 mg Half the contents of each container but not less than 50 mg
— 300 mg to 5 g 150 mg
— greater than 5 g 500 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)

Oils and oily solutions. Use for each medium not less than Ointments and creams. Prepare by diluting to about 1 in
the quantity of the product prescribed in Table 2.6.1.-2. Oils 10 by emulsifying with the chosen emulsifying agent in a
and oily solutions of sufficiently low viscosity may be filtered suitable sterile diluent such as a 1 g/l neutral solution of
without dilution through a dry membrane. Viscous oils may meat or casein peptone. Transfer the diluted product to a
be diluted as necessary with a suitable sterile diluent such as medium not containing an emulsifying agent.
isopropyl myristate shown not to have antimicrobial activity Incubate the inoculated media for not less than 14 days.
in the conditions of the test. Allow the oil to penetrate Observe the cultures several times during the incubation
the membrane by its own weight then filter, applying the period. Shake cultures containing oily products gently each
pressure or suction gradually. Wash the membrane at least day. However when fluid thioglycollate medium is used for
3 times by filtering through it each time about 100 ml of the detection of anaerobic micro-organisms keep shaking
a suitable sterile solution such as 1 g/l neutral meat or or mixing to a minimum in order to maintain anaerobic
casein peptone containing a suitable emulsifying agent at conditions.
a concentration shown to be appropriate in the method Catgut and other surgical sutures for veterinary use. Use
suitability test, for example polysorbate 80 at a concentration for each medium not less than the quantities of the product
of 10 g/l. Transfer the membrane or membranes to the prescribed in Table 2.6.1.-2. Open the sealed package using
culture medium or media or vice versa as described above for aseptic precautions and remove 3 sections of the strand for
aqueous solutions, and incubate at the same temperatures each culture medium. Carry out the test on 3 sections, each
and for the same times. 30 cm long, cut off from the beginning, the centre and the
Ointments and creams. Use for each medium not less than end of the strand. Use whole strands from freshly opened
the quantities of the product prescribed in Table 2.6.1.-2. cassette packs. Transfer each section of the strand to the
Ointments in a fatty base and emulsions of the water-in-oil selected medium. Use sufficient medium to cover adequately
type may be diluted to 1 per cent in isopropyl myristate as the material to be tested (20 ml to 150 ml).
described above, by heating, if necessary, to not more than OBSERVATION AND INTERPRETATION OF RESULTS
40 °C. In exceptional cases it may be necessary to heat to not
more than 44 °C. Filter as rapidly as possible and proceed as At intervals during the incubation period and at its
described above for oils and oily solutions. conclusion, examine the media for macroscopic evidence of
microbial growth. If the material being tested renders the
Direct inoculation of the culture medium. Transfer the medium turbid so that the presence or absence of microbial
quantity of the preparation to be examined prescribed in growth cannot be readily determined by visual examination,
Table 2.6.1.-2 directly into the culture medium so that the 14 days after the beginning of incubation transfer portions
volume of the product is not more than 10 per cent of the (each not less than 1 ml) of the medium to fresh vessels
volume of the medium, unless otherwise prescribed. of the same medium and then incubate the original and
transfer vessels for not less than 4 days.
If the product to be examined has antimicrobial activity, carry If no evidence of microbial growth is found, the product to be
out the test after neutralising this with a suitable neutralising examined complies with the test for sterility. If evidence of
substance or by dilution in a sufficient quantity of culture microbial growth is found the product to be examined does
medium. When it is necessary to use a large volume of the not comply with the test for sterility, unless it can be clearly
product it may be preferable to use a concentrated culture demonstrated that the test was invalid for causes unrelated
medium prepared in such a way that it takes account of the to the product to be examined. The test may be considered
subsequent dilution. Where appropriate, the concentrated invalid only if one or more of the following conditions are
medium may be added directly to the product in its container. fulfilled :
Oily liquids. Use media to which have been added a suitable a) the data of the microbiological monitoring of the sterility
emulsifying agent at a concentration shown to be appropriate testing facility show a fault ;
in the method suitability test, for example polysorbate 80 b) a review of the testing procedure used during the test in
at a concentration of 10 g/l. question reveals a fault ;

General Notices (1) apply to all monographs and other texts 3921
2.6.1. Sterility EUROPEAN PHARMACOPOEIA 6.3

c) microbial growth is found in the negative controls ;
d) after determination of the identity of the micro-organisms
isolated from the test, the growth of this species or these
species may be ascribed unequivocally to faults with respect
to the material and/or the technique used in conducting
the sterility test procedure.
If the test is declared to be invalid it is repeated with the
same number of units as in the original test.
If no evidence of microbial growth is found in the repeat test
the product examined complies with the test for sterility.
If microbial growth is found in the repeat test the product
examined does not comply with the test for sterility.
APPLICATION OF THE TEST TO PARENTERAL
PREPARATIONS, OPHTHALMIC AND OTHER
NON-INJECTABLE PREPARATIONS REQUIRED TO
COMPLY WITH THE TEST FOR STERILITY
When using the technique of membrane filtration, use,
whenever possible, the whole contents of the container,
but not less than the quantities indicated in Table 2.6.1.-2,
diluting where necessary to about 100 ml with a suitable
sterile solution, such as 1 g/l neutral meat or casein peptone.
When using the technique of direct inoculation of media,
use the quantities shown in Table 2.6.1.-2, unless otherwise
justified and authorised. The tests for bacterial and fungal
sterility are carried out on the same sample of the product to
be examined. When the volume or the quantity in a single
container is insufficient to carry out the tests, the contents
of 2 or more containers are used to inoculate the different
media.
MINIMUM NUMBER OF ITEMS TO BE TESTED
The minimum number of items to be tested in relation to the
size of the batch is given in Table 2.6.1.-3.
Guidelines on the test for sterility are given in general
chapter 5.1.9.

Table 2.6.1.-3. — Minimum number of items to be tested

Minimum number of items to be tested for each medium, unless
Number of items in the batch*
otherwise justified and authorised**
Parenteral preparations
— Not more than 100 containers 10 per cent or 4 containers, whichever is the greater
— More than 100 but not more than 500 containers 10 containers
2 per cent or 20 containers (10 containers for large-volume parenterals)
— More than 500 containers
whichever is less
Ophthalmic and other non-injectable preparations
— Not more than 200 containers 5 per cent or 2 containers, whichever is the greater
— More than 200 containers 10 containers
— If the product is presented in the form of single-dose containers, apply
the scheme shown above for preparations for parenteral use
2 per cent or 5 packages whichever is the greater, up to a maximum total
Catgut and other surgical sutures for veterinary use
of 20 packages
Bulk solid products
— Up to 4 containers Each container
— More than 4 containers but not more than 50 containers 20 per cent or 4 containers, whichever is the greater
— More than 50 containers 2 per cent or 10 containers, whichever is the greater

* If the batch size is not known, use the maximum number of items prescribed.
**If the contents of one container are enough to inoculate the 2 media, this column gives the number of containers needed for both the media together.

3922 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.12. Microbial enumeration tests

01/2009:20612 micro-organisms used for inoculation are not more than

5 passages removed from the original master seed-lot. Grow
each of the bacterial and fungal test strains separately as
2.6.12. MICROBIOLOGICAL described in Table 2.6.12.-1.
EXAMINATION OF NON-STERILE
PRODUCTS : MICROBIAL Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions ;
ENUMERATION TESTS to suspend A. niger spores, 0.05 per cent of polysorbate 80
may be added to the buffer. Use the suspensions within 2 h or
within 24 h if stored at 2-8 °C. As an alternative to preparing
1. INTRODUCTION and then diluting a fresh suspension of vegetative cells of
A. niger or B. subtilis, a stable spore suspension is prepared
The tests described hereafter will allow quantitative and then an appropriate volume of the spore suspension is
enumeration of mesophilic bacteria and fungi that may grow used for test inoculation. The stable spore suspension may
under aerobic conditions. be maintained at 2-8 °C for a validated period of time.
The tests are designed primarily to determine whether a
substance or preparation complies with an established 4-3. NEGATIVE CONTROL
specification for microbiological quality. When used for such To verify testing conditions, a negative control is performed
purposes follow the instructions given below, including the using the chosen diluent in place of the test preparation.
number of samples to be taken, and interpret the results as There must be no growth of micro-organisms.
stated below. 4-4. GROWTH PROMOTION OF THE MEDIA
The methods are not applicable to products containing Test each batch of ready-prepared medium and each batch of
viable micro-organisms as active ingredients. medium, prepared either from dehydrated medium or from
Alternative microbiological procedures, including automated the ingredients described.
methods, may be used, provided that their equivalence to the
Pharmacopoeia method has been demonstrated. Inoculate portions/plates of casein soya bean digest broth
and casein soya bean digest agar with a small number (not
2. GENERAL PROCEDURES more than 100 CFU) of the micro-organisms indicated in
Table 2.6.12.-1, using a separate portion/plate of medium
Carry out the determination under conditions designed to
for each. Inoculate plates of Sabouraud-dextrose agar
avoid extrinsic microbial contamination of the product to be
with a small number (not more than 100 CFU) of the
examined. The precautions taken to avoid contamination
micro-organisms indicated in Table 2.6.12.-1, using a separate
must be such that they do not affect any micro-organisms
plate of medium for each. Incubate in the conditions
that are to be revealed in the test.
described in Table 2.6.12.-1.
If the product to be examined has antimicrobial activity, this
is insofar as possible removed or neutralised. If inactivators For solid media, growth obtained must not differ by a factor
are used for this purpose, their efficacy and their absence of greater than 2 from the calculated value for a standardised
toxicity for micro-organisms must be demonstrated. inoculum. For a freshly prepared inoculum, growth of the
If surface-active substances are used for sample preparation, micro-organisms comparable to that previously obtained
their absence of toxicity for micro-organisms and their with a previously tested and approved batch of medium
compatibility with inactivators used must be demonstrated. occurs. Liquid media are suitable if clearly visible growth of
the micro-organisms comparable to that previously obtained
3. ENUMERATION METHODS with a previously tested and approved batch of medium
occurs.
Use the membrane filtration method or the plate-count
methods, as prescribed. The most-probable-number (MPN) 4-5. SUITABILITY OF THE COUNTING METHOD IN THE
method is generally the least accurate method for microbial PRESENCE OF PRODUCT
counts, however, for certain product groups with a very low
4-5-1. Preparation of the sample. The method for sample
bioburden, it may be the most appropriate method.
preparation depends upon the physical characteristics of the
The choice of method is based on factors such as the nature product to be tested. If none of the procedures described
of the product and the required limit of micro-organisms. The below can be demonstrated to be satisfactory, an alternative
chosen method must allow testing of a sufficient sample size procedure must be developed.
to judge compliance with the specification. The suitability of
the method chosen must be established. Water-soluble products. Dissolve or dilute (usually a 1 in
10 dilution is prepared) the product to be examined in
4. GROWTH PROMOTION TEST AND SUITABILITY OF buffered sodium chloride-peptone solution pH 7.0, phosphate
THE COUNTING METHOD buffer solution pH 7.2 or casein soya bean digest broth.
4-1. GENERAL CONSIDERATIONS If necessary, adjust to pH 6-8. Further dilutions, where
The ability of the test to detect micro-organisms in the necessary, are prepared with the same diluent.
presence of product to be tested must be established.
Non-fatty products insoluble in water. Suspend the product
Suitability must be confirmed if a change in testing
to be examined (usually a 1 in 10 dilution is prepared) in
performance, or the product, which may affect the outcome
buffered sodium chloride-peptone solution pH 7.0, phosphate
of the test is introduced.
buffer solution pH 7.2 or casein soya bean digest broth. A
4-2. PREPARATION OF TEST STRAINS surface-active agent such as 1 g/l of polysorbate 80 may be
Use standardised stable suspensions of test strains or added to assist the suspension of poorly wettable substances.
prepare them as stated below. Seed lot culture maintenance If necessary, adjust to pH 6-8. Further dilutions, where
techniques (seed-lot systems) are used so that the viable necessary, are prepared with the same diluent.

General Notices (1) apply to all monographs and other texts 3923
2.6.12. Microbial enumeration tests EUROPEAN PHARMACOPOEIA 6.3

Table 2.6.12.-1. – Preparation and use of test micro-organisms

Micro-organism Preparation of test Growth promotion Suitability of counting method in the
strain presence of the product
Total aerobic Total yeasts and Total aerobic Total yeasts and
microbial count moulds count microbial count moulds count
Staphylococcus Casein soya bean Casein soya bean Casein soya bean
aureus digest agar or casein digest agar and casein digest agar/MPN
such as : soya bean digest broth soya bean digest broth casein soya bean
30-35 °C ≤ 100 CFU digest broth
ATCC 6538 - -
18-24 h 30-35 °C ≤ 100 CFU
NCIMB 9518
≤ 3 days 30-35 °C
CIP 4.83
≤ 3 days
NBRC 13276
Pseudomonas Casein soya bean Casein soya bean Casein soya bean
aeruginosa digest agar or casein digest agar and casein digest agar/MPN
such as : soya bean digest broth soya bean digest broth casein soya bean
30-35 °C ≤ 100 CFU digest broth
ATCC 9027 - -
18-24 h 30-35 °C ≤ 100 CFU
NCIMB 8626
≤ 3 days 30-35 °C
CIP 82.118
≤ 3 days
NBRC 13275
Bacillus subtilis Casein soya bean Casein soya bean Casein soya bean
such as : digest agar or casein digest agar and casein digest agar/MPN
ATCC 6633 soya bean digest broth soya bean digest broth casein soya bean
30-35 °C ≤ 100 CFU - digest broth -
NCIMB 8054
18-24 h 30-35 °C ≤ 100 CFU
CIP 52.62
≤ 3 days 30-35 °C
NBRC 3134
≤ 3 days
Candida albicans Sabouraud-dextrose Casein soya bean Sabouraud-dextrose Casein soya bean Sabouraud-dextrose
such as : agar or Sabouraud- digest agar agar digest agar agar
dextrose broth ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU
ATCC 10231 20-25 °C
NCPF 3179 30-35 °C 20-25 °C 30-35 °C 20-25 °C
2-3 days
IP 48.72 ≤ 5 days ≤ 5 days ≤ 5 days ≤ 5 days
NBRC 1594 MPN : not applicable
Aspergillus niger Sabouraud-dextrose Casein soya bean Sabouraud-dextrose Casein soya bean Sabouraud-dextrose
such as : agar or potato- digest agar agar digest agar agar
dextrose agar ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU
ATCC 16404
20-25 °C 30-35 °C 20-25 °C 30-35 °C 20-25 °C
IMI 149007
5-7 days, or until ≤ 5 days ≤ 5 days ≤ 5 days ≤ 5 days
IP 1431.83 good sporulation is
NBRC 9455 achieved MPN : not applicable

Fatty products. Dissolve in isopropyl myristate, sterilised
by filtration or mix the product to be examined with the
minimum necessary quantity of sterile polysorbate 80 or
another non-inhibitory sterile surface-active agent, heated if
necessary to not more than 40 °C, or in exceptional cases
to not more than 45 °C. Mix carefully and if necessary
maintain the temperature in a water-bath. Add sufficient of
the pre-warmed chosen diluent to make a 1 in 10 dilution
of the original product. Mix carefully whilst maintaining
the temperature for the shortest time necessary for the
formation of an emulsion. Further serial tenfold dilutions
may be prepared using the chosen diluent containing a
suitable concentration of sterile polysorbate 80 or another
non-inhibitory sterile surface-active agent.
Fluids or solids in aerosol form. Aseptically transfer the
product into a membrane filter apparatus or a sterile
container for further sampling. Use either the total contents
or a defined number of metered doses from each of the
containers tested.
Transdermal patches. Remove the protective cover sheets
(‘release liners’) of the transdermal patches and place them,
adhesive side upwards, on sterile glass or plastic trays.
Cover the adhesive surface with a sterile porous material,
for example sterile gauze, to prevent the patches from
sticking together, and transfer the patches to a suitable
volume of the chosen diluent containing inactivators such
as polysorbate 80 and/or lecithin. Shake the preparation
vigorously for at least 30 min.
4-5-2. Inoculation and dilution. Add to the sample prepared
as described above (4-5-1) and to a control (with no test
material included) a sufficient volume of the microbial
suspension to obtain an inoculum of not more than 100 CFU.
The volume of the suspension of the inoculum should not
exceed 1 per cent of the volume of diluted product.
To demonstrate acceptable microbial recovery from the
product, the lowest possible dilution factor of the prepared
sample must be used for the test. Where this is not possible
due to antimicrobial activity or poor solubility, further
appropriate protocols must be developed. If inhibition of
growth by the sample cannot otherwise be avoided, the
aliquot of the microbial suspension may be added after
neutralisation, dilution or filtration.
4-5-3. Neutralisation/removal of antimicrobial activity.
The number of micro-organisms recovered from the prepared
sample diluted as described in 4-5-2 and incubated following
the procedure described in 4-5-4, is compared to the number
of micro-organisms recovered from the control preparation.
If growth is inhibited (reduction by a factor greater than 2),
then modify the procedure for the particular enumeration
test to ensure the validity of the results. Modification of the
procedure may include, for example, (1) an increase in the
volume of the diluent or culture medium, (2) incorporation
of specific or general neutralising agents into the diluent,
(3) membrane filtration, or (4) a combination of the above
measures.

3924 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.12. Microbial enumeration tests

Neutralising agents. Neutralising agents may be 4-5-4-2-1. Pour-plate method

used to neutralise the activity of antimicrobial agents For Petri dishes 9 cm in diameter, add to the dish 1 ml
(Table 2.6.12.-2). They may be added to the chosen diluent of the sample prepared as described under 4-5-1 to
or the medium preferably before sterilisation. If used, their 4-5-3 and 15-20 ml of casein soya bean digest agar or
efficacy and their absence of toxicity for micro-organisms Sabouraud-dextrose agar, both media being at not more
must be demonstrated by carrying out a blank with than 45 °C. If larger Petri dishes are used, the amount
neutraliser and without product. of agar medium is increased accordingly. For each of
the micro-organisms listed in Table 2.6.12.-1, at least 2
Table 2.6.12.-2. – Common neutralising agents for Petri dishes are used. Incubate the plates as indicated in
interfering substances Table 2.6.12.-1. Take the arithmetic mean of the counts per
medium and calculate the number of CFU in the original
Interfering substance Potential neutralising inoculum.
method 4-5-4-2-2. Surface-spread method
Glutaraldehyde, mercurials Sodium hydrogensulphite For Petri dishes 9 cm in diameter, add 15-20 ml of casein
(sodium bisulphite) soya bean digest agar or Sabouraud-dextrose agar at about
Phenolics, alcohol, aldehydes, sorbate Dilution 45 °C to each Petri dish and allow to solidify. If larger
Aldehydes Glycine Petri dishes are used, the volume of the agar is increased
accordingly. Dry the plates, for example in a laminar-air-flow
Quaternary Ammonium Compounds Lecithin cabinet or an incubator. For each of the micro-organisms
(QACs), parahydroxybenzoates (parabens),
bis-biguanides listed in Table 2.6.12.-1, at least 2 Petri dishes are used.
QACs, iodine, parabens Polysorbate
Spread a measured volume of not less than 0.1 ml of the
sample prepared as described under 4-5-1 to 4-5-3 over the
Mercurials Thioglycollate surface of the medium. Incubate and count as prescribed
Mercurials, halogens, aldehydes Thiosulphate under 4-5-4-2-1.
4-5-4-3. Most-probable-number (MPN) method. The
EDTA (edetate) Mg2+ or Ca2+ ions
precision and accuracy of the MPN method is less than
that of the membrane filtration method or the plate-count
If no suitable neutralising method can be found, it can be method. Unreliable results are obtained particularly for the
assumed that the failure to isolate the inoculated organism enumeration of moulds. For these reasons the MPN method
is attributable to the microbicidal activity of the product. is reserved for the enumeration of TAMC in situations where
This information serves to indicate that the product is not no other method is available. If the use of the method is
likely to be contaminated with the given species of the justified, proceed as follows.
micro-organism. However, it is possible that the product Prepare a series of at least 3 serial tenfold dilutions of the
only inhibits some of the micro-organisms specified herein, product as described under 4-5-1 to 4-5-3. From each level
but does not inhibit others not included amongst the test of dilution, 3 aliquots of 1 g or 1 ml are used to inoculate
strains or for which the latter are not representative. Then, 3 tubes with 9-10 ml of casein soya bean digest broth. If
perform the test with the highest dilution factor compatible necessary, a surface-active agent such as polysorbate 80 or
with microbial growth and the specific acceptance criterion. an inactivator of antimicrobial agents may be added to the
4-5-4. Recovery of micro-organism in the presence of medium. Thus, if 3 levels of dilution are prepared, 9 tubes
product. For each of the micro-organisms listed, separate are inoculated.
tests are performed. Only micro-organisms of the added test Incubate all tubes at 30-35 °C for not more than 3 days.
strain are counted. If reading of the results is difficult or uncertain owing to
the nature of the product to be examined, subculture in the
4-5-4-1. Membrane filtration. Use membrane filters having same broth, or in casein soya bean digest agar, for 1-2 days
a nominal pore size not greater than 0.45 μm. The type of at the same temperature and use these results. Determine
filter material is chosen such that the bacteria-retaining the most probable number of micro-organisms per gram or
efficiency is not affected by the components of the sample millilitre of the product to be examined from Table 2.6.12.-3.
to be investigated. For each of the micro-organisms listed, 4-6. RESULTS AND INTERPRETATION
one membrane filter is used. When verifying the suitability of the membrane filtration
method or the plate-count method, a mean count of any
Transfer a suitable amount of the sample prepared as of the test organisms not differing by a factor greater than
described under 4-5-1 to 4-5-3 (preferably representing 1 g of 2 from the value of the control defined in 4-5-2 in the
the product, or less if large numbers of CFU are expected) absence of the product must be obtained. When verifying
to the membrane filter, filter immediately and rinse the the suitability of the MPN method the calculated value from
membrane filter with an appropriate volume of diluent. the inoculum must be within 95 per cent confidence limits
of the results obtained with the control.
For the determination of total aerobic microbial count If the above criteria cannot be met for one or more of the
(TAMC), transfer the membrane filter to the surface of organisms tested with any of the described methods, the
casein soya bean digest agar. For the determination of method and test conditions that come closest to the criteria
total combined yeasts/moulds count (TYMC), transfer are used to test the product.
the membrane to the surface of Sabouraud-dextrose agar.
Incubate the plates as indicated in Table 2.6.12.-1. Perform 5. TESTING OF PRODUCTS
the counting. 5-1. AMOUNT USED FOR THE TEST
Unless otherwise prescribed, use 10 g or 10 ml of the product
4-5-4-2. Plate-count methods. Perform plate-count methods to be examined taken with the precautions referred to above.
at least in duplicate for each medium and use the mean For fluids or solids in aerosol form, sample 10 containers.
count of the result. For transdermal patches, sample 10 patches.

General Notices (1) apply to all monographs and other texts 3925
2.6.12. Microbial enumeration tests EUROPEAN PHARMACOPOEIA 6.3

The amount to be tested may be reduced for active substances Table 2.6.12.-3. – Most-probable-number values of
that will be formulated in the following conditions : the micro-organisms
amount per dosage unit (e.g. tablet, capsule, injection) is less Observed combinations of numbers of
than or equal to 1 mg or the amount per gram or millilitre tubes showing growth in each set MPN per
(for preparations not presented in dose units) is less than 95 per cent
Number of grams or millilitres of gram or per
confidence
1 mg. In these cases, the amount to be tested is not less than product per tube millilitre of
limits
the amount present in 10 dosage units or 10 g or 10 ml of product
0.1 0.01 0.001
the product.
For materials used as active substances where sample 0 0 0 <3 0-9.4
quantity is limited or batch size is extremely small (i.e. less 0 0 1 3 0.1-9.5
than 1000 ml or 1000 g), the amount tested shall be 1 per
0 1 0 3 0.1-10
cent of the batch unless a lesser amount is prescribed or
justified and authorised. 0 1 1 6.1 1.2-17
For products where the total number of entities in a batch 0 2 0 6.2 1.2-17
is less than 200 (e.g. samples used in clinical trials), the
0 3 0 9.4 3.5-35
sample size may be reduced to 2 units, or 1 unit if the size
is less than 100. 1 0 0 3.6 0.2-17
Select the sample(s) at random from the bulk material or 1 0 1 7.2 1.2-17
from the available containers of the preparation. To obtain
1 0 2 11 4-35
the required quantity, mix the contents of a sufficient
number of containers to provide the sample. 1 1 0 7.4 1.3-20
5-2. EXAMINATION OF THE PRODUCT 1 1 1 11 4-35
5-2-1. Membrane filtration 1 2 0 11 4-35
Use a filtration apparatus designed to allow the transfer of 1 2 1 15 5-38
the filter to the medium. Prepare the sample using a method
that has been shown suitable as described in section 4 and 1 3 0 16 5-38
transfer the appropriate amount to each of 2 membrane 2 0 0 9.2 1.5-35
filters and filter immediately. Wash each filter following the
2 0 1 14 4-35
procedure shown to be suitable.
For the determination of TAMC, transfer one of the 2 0 2 20 5-38
membrane filters to the surface of casein soya bean digest 2 1 0 15 4-38
agar. For the determination of TYMC, transfer the other
2 1 1 20 5-38
membrane to the surface of Sabouraud-dextrose agar.
Incubate the plate of casein soya bean digest agar at 2 1 2 27 9-94
30-35 °C for 3-5 days and the plate of Sabouraud-dextrose 2 2 0 21 5-40
agar at 20-25 °C for 5-7 days. Calculate the number of CFU
per gram or per millilitre of product. 2 2 1 28 9-94
When examining transdermal patches, filter 10 per cent 2 2 2 35 9-94
of the volume of the preparation described under 4-5-1 2 3 0 29 9-94
separately through each of 2 sterile filter membranes.
Transfer one membrane to casein soya bean digest agar for 2 3 1 36 9-94
TAMC and the other membrane to Sabouraud-dextrose agar 3 0 0 23 5-94
for TYMC.
3 0 1 38 9-104
5-2-2. Plate-count methods
3 0 2 64 16-181
5-2-2-1. Pour-plate method
3 1 0 43 9-181
Prepare the sample using a method that has been shown to be
suitable as described in section 4. Prepare for each medium 3 1 1 75 17-199
at least 2 Petri dishes for each level of dilution. Incubate the 3 1 2 120 30-360
plates of casein soya bean digest agar at 30-35 °C for 3-5 days
and the plates of Sabouraud-dextrose agar at 20-25 °C for 3 1 3 160 30-380
5-7 days. Select the plates corresponding to a given dilution 3 2 0 93 18-360
and showing the highest number of colonies less than 250
for TAMC and 50 for TYMC. Take the arithmetic mean per 3 2 1 150 30-380
culture medium of the counts and calculate the number of 3 2 2 210 30-400
CFU per gram or per millilitre of product.
3 2 3 290 90-990
5-2-2-2. Surface-spread method
3 3 0 240 40-990
Prepare the sample using a method that has been shown
to be suitable as described in section 4. Prepare at least 2 3 3 1 460 90-1980
Petri dishes for each medium and each level of dilution. For 3 3 2 1100 200-4000
incubation and calculation of the number of CFU proceed as
described for the pour-plate method. 3 3 3 > 1100

3926 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
5-2-3. Most-probable-number method
Prepare and dilute the sample using a method that has been
shown to be suitable as described in section 4. Incubate all
tubes at 30-35 °C for 3-5 days. Subculture if necessary, using
the procedure shown to be suitable. Record for each level
of dilution the number of tubes showing microbial growth.
Determine the most probable number of micro-organisms
per gram or millilitre of the product to be examined from
Table 2.6.12.-3.
5-3. INTERPRETATION OF THE RESULTS
The total aerobic microbial count (TAMC) is considered to be
equal to the number of CFU found using casein soya bean
digest agar ; if colonies of fungi are detected on this medium,
they are counted as part of the TAMC. The total combined
yeasts/mould count (TYMC) is considered to be equal to the
number of CFU found using Sabouraud-dextrose agar ; if
colonies of bacteria are detected on this medium, they are
counted as part of the TYMC. When the TYMC is expected to
exceed the acceptance criterion due to the bacterial growth,
Sabouraud-dextrose agar containing antibiotics may be used.
If the count is carried out by the MPN method the calculated
value is the TAMC.
When an acceptance criterion for microbiological quality is
prescribed it is interpreted as follows:
— 101 CFU : maximum acceptable count = 20 ;
— 102 CFU : maximum acceptable count = 200 ;
— 103 CFU : maximum acceptable count = 2000, and so forth.
The recommended solutions and media are described in
general chapter 2.6.13.
01/2009:20613
2.6.13. MICROBIOLOGICAL
EXAMINATION OF NON-STERILE
PRODUCTS : TEST FOR SPECIFIED
MICRO-ORGANISMS
1. INTRODUCTION
The tests described hereafter will allow determination of the
absence or limited occurrence of specified micro-organisms
that may be detected under the conditions described.
The tests are designed primarily to determine whether a
substance or preparation complies with an established
specification for microbiological quality. When used for such
purposes, follow the instructions given below, including the
number of samples to be taken, and interpret the results as
stated below.
Alternative microbiological procedures, including automated
methods, may be used, provided that their equivalence to the
Pharmacopoeia method has been demonstrated.
2. GENERAL PROCEDURES
The preparation of samples is carried out as described in
general chapter 2.6.12.
If the product to be examined has antimicrobial activity, this
is insofar as possible removed or neutralised as described in
general chapter 2.6.12.
If surface-active substances are used for sample preparation,
their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated
as described in general chapter 2.6.12.
General Notices (1) apply to all monographs and other texts
2.6.13. Test for specified micro-organisms
3. GROWTH-PROMOTING AND INHIBITORY PROPERTIES
OF THE MEDIA AND SUITABILITY OF THE TEST
The ability of the test to detect micro-organisms in the
presence of the product to be tested must be established.
Suitability must be confirmed if a change in testing
performance, or the product, which may affect the outcome
of the test is introduced.
3-1. PREPARATION OF TEST STRAINS
Use standardised stable suspensions of test strains or
prepare them as stated below. Seed lot culture maintenance
techniques (seed-lot systems) are used so that the viable
micro-organisms used for inoculation are not more than
5 passages removed from the original master seed-lot.
3-1-1. Aerobic micro-organisms. Grow each of the bacterial
test strains separately in casein soya bean digest broth or
on casein soya bean digest agar at 30-35 °C for 18-24 h.
Grow the test strain for Candida albicans separately on
Sabouraud-dextrose agar or in Sabouraud-dextrose broth at
20-25 °C for 2-3 days.
— Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83 or NBRC 13276 ;
— Pseudomonas aeruginosa such as ATCC 9027,
NCIMB 8626, CIP 82.118 or NBRC 13275 ;
— Escherichia coli such as ATCC 8739, NCIMB 8545,
CIP 53.126 or NBRC 3972 ;
— Salmonella enterica ssp. enterica serotype typhimurium,
such as ATCC 14028 or, as an alternative, Salmonella
enterica ssp. enterica serotype abony such as
NBRC 100797, NCTC 6017 or CIP 80.39 ;
— Candida albicans such as ATCC 10231, NCPF 3179,
IP 48.72 or NBRC 1594.
Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions.
Use the suspensions within 2 h or within 24 h if stored at
2-8 °C.
3-1-2. Clostridia. Use Clostridium sporogenes such as
ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651)
or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293.
Grow the clostridial test strain under anaerobic conditions
in reinforced medium for clostridia at 30-35 °C for 24-48 h.
As an alternative to preparing and then diluting down a
fresh suspension of vegetative cells of Cl. sporogenes, a
stable spore suspension is used for test inoculation. The
stable spore suspension may be maintained at 2-8 °C for a
validated period.
3-2. NEGATIVE CONTROL
To verify testing conditions, a negative control is performed
using the chosen diluent in place of the test preparation.
There must be no growth of micro-organisms.
3-3. GROWTH PROMOTION AND INHIBITORY
PROPERTIES OF THE MEDIA
Test each batch of ready-prepared medium and each batch of
medium prepared either from dehydrated medium or from
ingredients.
Verify suitable properties of relevant media as described in
Table 2.6.13.-1.
Test for growth promoting properties, liquid media :
inoculate a portion of the appropriate medium with a
small number (not more than 100 CFU) of the appropriate
micro-organism. Incubate at the specified temperature
for not more than the shortest period of time specified
in the test. Clearly visible growth of the micro-organism
comparable to that previously obtained with a previously
tested and approved batch of medium occurs.
3927
2.6.13. Test for specified micro-organisms EUROPEAN PHARMACOPOEIA 6.3

Table 2.6.13.-1 – Growth promoting, inhibitory and indicative properties of media

Medium Property Test strains
Test for bile-tolerant gram-negative Enterobacteria enrichment Growth promoting E. coli
bacteria broth-Mossel P. aeruginosa
Inhibitory S. aureus
Violet red bile glucose agar Growth promoting + indicative E. coli
P. aeruginosa
Test for Escherichia coli MacConkey broth Growth promoting E. coli
Inhibitory S. aureus
MacConkey agar Growth promoting + indicative E. coli
Test for Salmonella Rappaport Vassiliadis Salmonella Growth promoting Salmonella enterica ssp. enterica
enrichment broth serotype typhimurium or Salmonella
enterica ssp. enterica serotype abony
Inhibitory S. aureus
Xylose, lysine, deoxycholate agar Growth promoting + indicative Salmonella enterica ssp. enterica
serotype typhimurium or Salmonella
enterica ssp. enterica serotype abony
Indicative E. coli
Test for Pseudomonas aeruginosa Cetrimide agar Growth promoting P. aeruginosa
Inhibitory E. coli
Test for Staphylococcus aureus Mannitol salt agar Growth promoting + indicative S. aureus
Inhibitory E. coli
Test for clostridia Reinforced medium for clostridia Growth promoting Cl. sporogenes
Columbia agar Growth promoting Cl. sporogenes
Test for Candida albicans Sabouraud dextrose broth Growth promoting C. albicans
Sabouraud dextrose agar Growth promoting + indicative C. albicans

Test for growth promoting properties, solid media : perform
the surface-spread method, inoculating each plate with a
small number (not more than 100 CFU) of the appropriate
micro-organism. Incubate at the specified temperature for
not more than the shortest period of time specified in the
test. Growth of the micro-organism comparable to that
previously obtained with a previously tested and approved
batch of medium occurs.
Test for inhibitory properties, liquid or solid media:
inoculate the appropriate medium with at least 100 CFU of
the appropriate micro-organism. Incubate at the specified
temperature for not less than the longest period of time
specified in the test. No growth of the test micro-organism
occurs.
Test for indicative properties : perform the surface-spread
method, inoculating each plate with a small number (not
more than 100 CFU) of the appropriate micro-organism.
Incubate at the specified temperature for a period of
time within the range specified in the test. Colonies are
comparable in appearance and indication reactions to those
previously obtained with a previously tested and approved
batch of medium.
3-4. SUITABILITY OF THE TEST METHOD
For each product to be tested, perform the sample
preparation as described in the relevant paragraph in
section 4. Add each test strain at the time of mixing, in
the prescribed growth medium. Inoculate the test strains
individually. Use a number of micro-organisms equivalent to
not more than 100 CFU in the inoculated test preparation.
Perform the test as described in the relevant paragraph in
section 4 using the shortest incubation period prescribed.
The specified micro-organisms must be detected with the
indication reactions as described in section 4.
Any antimicrobial activity of the product necessitates a
modification of the test procedure (see 4-5-3 of general
chapter 2.6.12).
If for a given product the antimicrobial activity with respect
to a micro-organism for which testing is prescribed cannot
be neutralised, then it is to be assumed that the inhibited
micro-organism will not be present in the product.
4. TESTING OF PRODUCTS
4-1. BILE-TOLERANT GRAM-NEGATIVE BACTERIA
4-1-1. Sample preparation and pre-incubation. Prepare a
sample using a 1 in 10 dilution of not less than 1 g of the
product to be examined as described in general chapter
2.6.12, but using casein soya bean digest broth as the chosen
diluent, mix and incubate at 20-25 °C for a time sufficient
to resuscitate the bacteria but not sufficient to encourage
multiplication of the organisms (usually 2 h but not more
than 5 h).
4-1-2. Test for absence. Unless otherwise prescribed, use the
volume corresponding to 1 g of the product, as prepared in
4-1-1, to inoculate enterobacteria enrichment broth-Mossel.
Incubate at 30-35 °C for 24-48 h. Subculture on plates of
violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.
The product complies with the test if there is no growth of
colonies.
4-1-3. Quantitative test
4-1-3-1. Selection and subculture. Inoculate suitable
quantities of enterobacteria enrichment broth-Mossel with
the preparation as described under 4-1-1 and/or dilutions
of it containing respectively 0.1 g, 0.01 g and 0.001 g (or
0.1 ml, 0.01 ml and 0.001 ml) of the product to be examined.
Incubate at 30-35 °C for 24-48 h. Subculture each of the
cultures on a plate of violet red bile glucose agar. Incubate
at 30-35 °C for 18-24 h.

3928 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.13. Test for specified micro-organisms

4-1-3-2. Interpretation. Growth of colonies constitutes a corresponding to 1 patch of the preparation described
positive result. Note the smallest quantity of the product under 4-5-1 in general chapter 2.6.12 through a sterile filter
that gives a positive result and the largest quantity that membrane and place in 100 ml of casein soya bean digest
gives a negative result. Determine from Table 2.6.13.-2 the broth. Incubate at 30-35 °C for 18-24 h.
probable number of bacteria. 4-4-2. Selection and subculture. Subculture on a plate of
cetrimide agar and incubate at 30-35 °C for 18-72 h.
Table 2.6.13.-2 – Interpretation of results
4-4-3. Interpretation. Growth of colonies indicates the
Results for each quantity of product Probable possible presence of P. aeruginosa. This is confirmed by
number of
0.1 g or 0.01 g or 0.001 g or bacteria per
identification tests.
0.1 ml 0.01 ml 0.001 ml gram or
millilitre of The product complies with the test if colonies are not present
product or if the confirmatory identification tests are negative.
+ + + > 103 4-5. STAPHYLOCOCCUS AUREUS
+ + − < 103 and > 102 4-5-1. Sample preparation and pre-incubation. Prepare a
+ − − < 102 and > 10 sample using a 1 in 10 dilution of not less than 1 g of the
− − − product to be examined as described in general chapter
< 10
2.6.12, and use 10 ml or the quantity corresponding to 1 g or
4-2. ESCHERICHIA COLI 1 ml to inoculate a suitable amount (determined as described
under 3-4) of casein soya bean digest broth and mix. When
4-2-1. Sample preparation and pre-incubation. Prepare a testing transdermal patches, filter the volume of sample
sample using a 1 in 10 dilution of not less than 1 g of the corresponding to 1 patch of the preparation described
product to be examined as described in general chapter under 4-5-1 in general chapter 2.6.12 through a sterile filter
2.6.12, and use 10 ml or the quantity corresponding to membrane and place in 100 ml of casein soya bean digest
1 g or 1 ml to inoculate a suitable amount (determined as broth. Incubate at 30-35 °C for 18-24 h.
described under 3-4) of casein soya bean digest broth, mix
and incubate at 30-35 °C for 18-24 h. 4-5-2. Selection and subculture. Subculture on a plate of
mannitol salt agar and incubate at 30-35 °C for 18-72 h.
4-2-2. Selection and subculture. Shake the container,
transfer 1 ml of casein soya bean digest broth to 100 ml of 4-5-3. Interpretation. The possible presence of S. aureus is
MacConkey broth and incubate at 42-44 °C for 24-48 h. indicated by the growth of yellow/white colonies surrounded
Subculture on a plate of MacConkey agar at 30-35 °C for by a yellow zone. This is confirmed by identification tests.
18-72 h. The product complies with the test if colonies of the types
4-2-3. Interpretation. Growth of colonies indicates described are not present or if the confirmatory identification
the possible presence of E. coli. This is confirmed by tests are negative.
identification tests. 4-6. CLOSTRIDIA
The product complies with the test if no colonies are present 4-6-1. Sample preparation and heat treatment. Prepare
or if the identification tests are negative. the product to be examined as described in general chapter
4-3. SALMONELLA 2.6.12. Take 2 equal portions corresponding to not less than
1 g or 1 ml of the product to be examined. Heat 1 portion
4-3-1. Sample preparation and pre-incubation. Prepare the at 80 °C for 10 min and cool rapidly. Do not heat the other
product to be examined as described in general chapter portion.
2.6.12, and use the quantity corresponding to not less than
10 g or 10 ml to inoculate a suitable amount (determined as 4-6-2. Selection and subculture. Transfer 10 ml of each of
described under 3-4) of casein soya bean digest broth, mix the mixed portions to 2 containers (38 mm × 200 mm, or
and incubate at 30-35 °C for 18-24 h. other suitable containers) containing 100 ml of reinforced
medium for clostridia. Incubate under anaerobic conditions
4-3-2. Selection and subculture. Transfer 0.1 ml of casein at 30-35 °C for 48 h. After incubation, make subcultures
soya bean digest broth to 10 ml of Rappaport Vassiliadis from each tube on Columbia agar and incubate under
Salmonella enrichment broth and incubate at 30-35 °C for anaerobic conditions at 30-35 °C for 48 h.
18-24 h. Subculture on plates of xylose, lysine, deoxycholate
agar. Incubate at 30-35 °C for 18-48 h. 4-6-3. Interpretation. The occurrence of anaerobic growth of
rods (with or without endospores) giving a negative catalase
4-3-3. Interpretation. The possible presence of Salmonella is reaction indicates the presence of clostridia.
indicated by the growth of well-developed, red colonies, with
or without black centres. This is confirmed by identification If no anaerobic growth of micro-organisms is detected on
tests. Columbia agar or the catalase test is positive, the product
complies with the test.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification 4-7. CANDIDA ALBICANS
tests are negative. 4-7-1. Sample preparation and pre-incubation. Prepare the
4-4. PSEUDOMONAS AERUGINOSA product to be examined as described in general chapter
4-4-1. Sample preparation and pre-incubation. Prepare a 2.6.12, and use 10 ml or the quantity corresponding
sample using a 1 in 10 dilution of not less than 1 g of the to not less than 1 g or 1 ml to inoculate 100 ml of
product to be examined as described in general chapter Sabouraud-dextrose broth and mix. Incubate at 30-35 °C
2.6.12, and use 10 ml or the quantity corresponding to 1 g or for 3-5 days.
1 ml to inoculate a suitable amount (determined as described 4-7-2. Selection and subculture. Subculture on a plate
under 3-4) of casein soya bean digest broth and mix. When of Sabouraud-dextrose agar and incubate at 30-35 °C for
testing transdermal patches, filter the volume of sample 24-48 h.

General Notices (1) apply to all monographs and other texts 3929
2.6.13. Test for specified micro-organisms EUROPEAN PHARMACOPOEIA 6.3

4-7-3. Interpretation. Growth of white colonies may Potato dextrose agar

indicate the presence of C. albicans. This is confirmed by Infusion from potatoes 200 g
identification tests.
Dextrose 20.0 g
The product complies with the test if such colonies are
Agar 15.0 g
not present or if the confirmatory identification tests are
negative. Purified water 1000 ml
The following section is given for information. Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at
25 °C. Sterilise in an autoclave using a validated cycle.
5. RECOMMENDED SOLUTIONS AND CULTURE MEDIA Sabouraud-dextrose broth
The following solutions and culture media have been Dextrose 20.0 g
found to be satisfactory for the purposes for which they Mixture of peptic digest of animal tissue and pancreatic 10.0 g
are prescribed in the test for microbial contamination in digest of casein (1:1)
the Pharmacopoeia. Other media may be used if they have Purified water 1000 ml
similar growth promoting and inhibitory properties.
Stock buffer solution. Place 34 g of potassium dihydrogen Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at
phosphate in a 1000 ml volumetric flask, dissolve in 500 ml 25 °C. Sterilise in an autoclave using a validated cycle.
of purified water, adjust to pH 7.2 ± 0.2 with sodium Enterobacteria enrichment broth-Mossel
hydroxide, dilute to 1000.0 ml with purified water and mix. Pancreatic digest of gelatin 10.0 g
Dispense into containers and sterilise. Store at 2-8 °C. Glucose monohydrate 5.0 g
Phosphate buffer solution pH 7.2. Prepare a mixture of stock Dehydrated ox bile 20.0 g
buffer solution and purified water (1:800 V/V) and sterilise.
Potassium dihydrogen phosphate 2.0 g
Buffered sodium chloride-peptone solution pH 7.0
Disodium hydrogen phosphate dihydrate 8.0 g
Potassium dihydrogen phosphate 3.6 g
Brilliant green 15 mg
Disodium hydrogen phosphate 7.2 g, equivalent to 0.067 M phosphate
dihydrate Purified water 1000 ml
Sodium chloride 4.3 g
Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C.
Peptone (meat or casein) 1.0 g
Heat at 100 °C for 30 min and cool immediately.
Purified water 1000 ml Violet red bile glucose agar
Yeast extract 3.0 g
Sterilise in an autoclave using a validated cycle.
Pancreatic digest of gelatin 7.0 g
Casein soya bean digest broth
Bile salts 1.5 g
Pancreatic digest of casein 17.0 g
Sodium chloride 5.0 g
Papaic digest of soya bean 3.0 g
Glucose monohydrate 10.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Dipotassium hydrogen phosphate 2.5 g
Neutral red 30 mg
Glucose monohydrate 2.5 g
Crystal violet 2 mg
Purified water 1000 ml
Purified water 1000 ml
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at
25 °C. Sterilise in an autoclave using a validated cycle. Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C.
Heat to boiling ; do not heat in an autoclave.
Casein soya bean digest agar
MacConkey broth
Pancreatic digest of casein 15.0 g
Pancreatic digest of gelatin 20.0 g
Papaic digest of soya bean 5.0 g
Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Dehydrated ox bile 5.0 g
Agar 15.0 g
Bromocresol purple 10 mg
Purified water 1000 ml
Purified water 1000 ml
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at
25 °C. Sterilise in an autoclave using a validated cycle. 25 °C. Sterilise in an autoclave using a validated cycle.
Sabouraud-dextrose agar MacConkey agar
Dextrose 40.0 g Pancreatic digest of gelatin 17.0 g
Mixture of peptic digest of animal tissue and pancreatic 10.0 g Peptones (meat and casein) 3.0 g
digest of casein (1:1)
Agar 15.0 g Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Purified water 1000 ml
Bile salts 1.5 g
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at Agar 13.5 g
25 °C. Sterilise in an autoclave using a validated cycle.

3930 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.6.13. Test for specified micro-organisms

Neutral red 30.0 mg Heat to boiling for 1 min with shaking. Adjust the pH so
Crystal violet 1 mg that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an
autoclave using a validated cycle.
Purified water 1000 ml Mannitol salt agar
Pancreatic digest of casein 5.0 g
Adjust the pH so that after sterilisation it is 7.1 ± 0.2 at
25 °C. Boil for 1 min with constant shaking then sterilise in Peptic digest of animal tissue 5.0 g
an autoclave using a validated cycle.
Beef extract 1.0 g
Rappaport Vassiliadis Salmonella enrichment broth
D-Mannitol 10.0 g
Soya peptone 4.5 g
Sodium chloride 75.0 g
Magnesium chloride hexahydrate 29.0 g
Agar 15.0 g
Sodium chloride 8.0 g
Phenol red 0.025 g
Dipotassium phosphate 0.4 g
Purified water 1000 ml
Potassium dihydrogen phosphate 0.6 g
Malachite green 0.036 g
Heat to boiling for 1 min with shaking. Adjust the pH so
that after sterilisation it is 7.4 ± 0.2 at 25 °C. Sterilise in an
Purified water 1000 ml autoclave using a validated cycle.
Reinforced medium for clostridia
Dissolve, warming gently. Sterilise in an autoclave using a
Beef extract 10.0 g
validated cycle, at a temperature not exceeding 115 °C. The
pH is to be 5.2 ± 0.2 at 25 °C after heating and autoclaving. Peptone 10.0 g
Xylose, lysine, deoxycholate agar Yeast extract 3.0 g
Xylose 3.5 g Soluble starch 1.0 g
L-Lysine 5.0 g Glucose monohydrate 5.0 g
Lactose monohydrate 7.5 g Cysteine hydrochloride 0.5 g
Sucrose 7.5 g Sodium chloride 5.0 g
Sodium chloride 5.0 g Sodium acetate 3.0 g
Yeast extract 3.0 g Agar 0.5 g
Phenol red 80 mg Purified water 1000 ml
Agar 13.5 g Hydrate the agar, dissolve by heating to boiling with
Sodium deoxycholate 2.5 g continuous stirring. If necessary, adjust the pH so that after
sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave
Sodium thiosulphate 6.8 g
using a validated cycle.
Ferric ammonium citrate 0.8 g Columbia agar
Purified water 1000 ml Pancreatic digest of casein 10.0 g
Meat peptic digest 5.0 g
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C.
Heat to boiling, cool to 50 °C and pour into Petri dishes. Heart pancreatic digest 3.0 g
Do not heat in an autoclave. Yeast extract 5.0 g
Cetrimide agar Maize starch 1.0 g
Pancreatic digest of gelatin 20.0 g
Sodium chloride 5.0 g
Magnesium chloride 1.4 g
Agar, according to gelling power 10.0-15.0 g
Dipotassium sulphate 10.0 g
Purified water 1000 ml
Cetrimide 0.3 g
Hydrate the agar, dissolve by heating to boiling with
Agar 13.6 g continuous stirring. If necessary, adjust the pH so that after
Purified water 1000 ml sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave
using a validated cycle. Allow to cool to 45-50 °C ; add, where
Glycerol 10.0 ml
necessary, gentamicin sulphate corresponding to 20 mg of
gentamicin base and pour into Petri dishes.

General Notices (1) apply to all monographs and other texts 3931
 EUROPEAN PHARMACOPOEIA 6.3

3932 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

2.7. BIOLOGICAL ASSAYS

2.7.2. Microbiological assay of antibiotics..........................3935

General Notices (1) apply to all monographs and other texts 3933
 EUROPEAN PHARMACOPOEIA 6.3

3934 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:20702 In order to assess the validity of the assay, use not fewer
2.7.2. MICROBIOLOGICAL ASSAY
OF ANTIBIOTICS
The potency of an antibiotic is estimated by comparing the
inhibition of growth of sensitive micro-organisms produced in all cases of dispute, a three-point assay as described above
by known concentrations of the antibiotic to be examined
and a reference substance.
The reference substances used in the assays are substances rectangular dish according to a statistically suitable design,
whose activity has been precisely determined with reference except for small Petri dishes that cannot accommodate more
to the corresponding international standard or international than 6 solutions, arrange the solutions of the antibiotic to
reference preparation.
The assay must be designed in a way that will permit
examination of the validity of the mathematical model on
which the potency equation is based. If a parallel-line model Incubate at a suitable temperature for about 18 h. A period
is chosen, the 2 log dose-response (or transformed response) of diffusion prior to incubation, usually 1-4 h, at room
lines of the preparation to be examined and the reference
preparation must be parallel ; they must be linear over the
range of doses used in the calculation. These conditions
must be verified by validity tests for a given probability,
usually P = 0.05. Other mathematical models, such as the
slope ratio model, may be used provided that proof of validity
is demonstrated.
Unless otherwise stated in the monograph, the confidence
limits (P = 0.95) of the assay for potency are not less than
95 per cent and not more than 105 per cent of the estimated
potency.
Carry out the assay by method A or method B.
A. DIFFUSION METHOD
Liquefy a medium suitable for the conditions of the assay
and inoculate it at a suitable temperature, for example 48 °C
to 50 °C for vegetative forms, with a known quantity of a
suspension of micro-organisms sensitive to the antibiotic to
be examined, such that clearly defined zones of inhibition
of suitable diameter are produced with the concentrations
of the antibiotic used for the assay. Immediately pour into
Petri dishes or large rectangular dishes a quantity of the
inoculated medium to form a uniform layer 2-5 mm thick.
Alternatively, the medium may consist of 2 layers, only the
upper layer being inoculated.
Store the dishes so that no appreciable growth or death of
the micro-organisms occurs before the dishes are used and
so that the surface of the medium is dry at the time of use.
Using the solvent and the buffer solution indicated in
Table 2.7.2.-1, prepare solutions of the reference substance
and of the antibiotic to be examined having known
concentrations and presumed to be of equal activity. Apply
the solutions to the surface of the medium, for example,
in sterile cylinders of porcelain, stainless steel or other
suitable material, or in cavities prepared in the agar. The
same volume of solution must be added to each cylinder or
cavity. Alternatively, use sterile absorbent paper discs of
suitable quality ; impregnate the discs with the solutions of
the reference substance or the solutions of the antibiotic to
be examined and place on the surface of the agar.
General Notices (1) apply to all monographs and other texts
2.7.2. Microbiological assay of antibiotics
than 3 doses of the reference substance and 3 doses of
the antibiotic to be examined having the same presumed
activity as the doses of the reference substance. It is
preferable to use a series of doses in geometric progression.
In routine assays when the linearity of the system has been
demonstrated over an adequate number of experiments
using a three-point assay, a two-point assay may be sufficient,
subject to agreement by the competent authority. However,
must be applied.
Arrange the solutions on each Petri dish or on each
be examined and the solutions of the reference substance
in an alternate manner to avoid interaction of the more
concentrated solutions.
temperature or at about 4 °C, as appropriate, may be used
to minimise the effects of the variation in time between the
application of the solutions and to improve the regression
slope.
Measure the diameters with a precision of at least 0.1 mm
or the areas of the circular inhibition zones with a
corresponding precision and calculate the potency using
appropriate statistical methods.
Use in each assay the number of replications per dose
sufficient to ensure the required precision. The assay may be
repeated and the results combined statistically to obtain the
required precision and to ascertain whether the potency of
the antibiotic to be examined is not less than the minimum
required.
B. TURBIDIMETRIC METHOD
Inoculate a suitable medium with a suspension of the chosen
micro-organism having a sensitivity to the antibiotic to be
examined such that a sufficiently large inhibition of microbial
growth occurs in the conditions of the test. Use a known
quantity of the suspension chosen so as to obtain a readily
measurable opacity after an incubation period of about 4 h.
Use the inoculated medium immediately after its preparation.
Using the solvent and the buffer solution indicated in
Table 2.7.2.-2 prepare solutions of the reference substance
and of the antibiotic to be examined having known
concentrations presumed to be of equal activity.
In order that the validity of the assay may be assessed,
use not fewer than 3 doses of the reference substance and
3 doses of the antibiotic to be examined having the same
presumed activity as the doses of the reference substance. It
is preferable to use a series of doses in geometric progression.
In order to obtain the required linearity, it may be necessary
to select from a large number 3 consecutive doses, using
corresponding doses for the reference substance and the
antibiotic to be examined.
3935
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 6.3

Table 2.7.2.-1. — Diffusion assay

Solvent to be used
Buffer solution Medium and final Incubation
Antibiotic Reference substance in preparing the Micro-organism
(pH) pH (± 0.1 pH unit) temperature
stock solution
Saccharomyces
Dimethyl cerevisiae
Amphotericin B Amphotericin B CRS pH 10.5 (0.2 M) F - pH 6.1 35-37 °C
sulphoxide R ATCC 9763
IP 1432-83
Micrococcus luteus
0.01 M hydrochloric NCTC 7743
Bacitracin zinc Bacitracin zinc CRS pH 7.0 (0.05 M) A - pH 7.0 35-39 °C
acid CIP 53.160
ATCC 10240
Mycobacterium
Bleomycin smegmatis
Bleomycin sulphate Water R pH 6.8 (0.1 M) G - pH 7.0 35-37 °C
sulphate CRS
ATCC 607
Bordetella
bronchiseptica
NCTC 8344
CIP 53.157
Colistimethate Colistimethate ATCC 4617
Water R pH 6.0 (0.05 M) B - pH 7.3 35-39 °C
sodium sodium CRS Escherichia coli
NCIB 8879
CIP 54.127
ATCC 10536

Bacillus subtilis E - pH 7.9 30-37 °C

NCTC 10400
CIP 52.62
Framycetin ATCC 6633
Framycetin sulphate Water R pH 8.0 (0.05 M)
sulphate CRS
Bacillus pumilus E - pH 7.9 30-37 °C
NCTC 8241
CIP 76.18
Bacillus pumilus A - pH 7.9 35-39 °C
NCTC 8241
CIP 76.18
Gentamicin Staphylococcus A - pH 7.9 35-39 °C
Gentamicin sulphate Water R pH 8.0 (0.05 M)
sulphate CRS epidermidis
NCIB 8853
CIP 68.21
ATCC 12228
Bacillus subtilis
Methanol R (see the CIP 52.62
Josamycin Josamycin CRS pH 5.6 A - pH 6.6 35-37 °C
monograph) ATCC 6633
NCTC 10400
Bacillus subtilis
Josamycin Josamycin Methanol R (see the CIP 52.62
pH 5.6 A - pH 6.6 35-37 °C
propionate propionate CRS monograph) ATCC 6633
NCTC 10400
Bacillus subtilis A - pH 7.9 30-37 °C
Kanamycin NCTC 10400
monosulphate CIP 52.62
ATCC 6633
Kanamycin
Water R pH 8.0 (0.05 M) Staphylococcus A - pH 7.9 35-39 °C
monosulphate CRS
aureus
Kanamycin acid NCTC 7447
sulphate
CIP 53.156
ATCC 6538 P
Bacillus pumilus E - pH 7.9 30-37 °C
NCTC 8241
CIP 76.18
Neomycin sulphate
Neomycin sulphate for microbiological Water R pH 8.0 (0.05 M) Bacillus subtilis E - pH 7.9 30-37 °C
assay CRS
NCTC 10400
CIP 52.62
ATCC 6633

3936 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.7.2. Microbiological assay of antibiotics

Solvent to be used
Buffer solution Medium and final Incubation
Antibiotic Reference substance in preparing the Micro-organism
(pH) pH (± 0.1 pH unit) temperature
stock solution
Staphylococcus
Netilmicin aureus
Netilmicin sulphate Water R pH 8.0 ± 0.1 A - pH 7.9 32-35 °C
sulphate CRS ATCC 6538P
CIP 53.156
Candida tropicalis F - pH 6.0 30-37 °C
CIP 1433-83
NCYC 1393
pH 6.0 (0.05 M)
Dimethylforma- containing 5 per Saccharomyces F - pH 6.0 30-32 °C
Nystatin Nystatin CRS
mide R cent V/V of dimeth- cerevisiae
ylformamide R
NCYC 87
CIP 1432-83
ATCC 9763
Micrococcus luteus
Rifamycin NCTC 8340
Rifamycin sodium Methanol R pH 7.0 (0.05 M) A - pH 6.6 35-39 °C
sodium CRS CIP 53.45
ATCC 9341
Bacillus subtilis
NCTC 10400
Spiramycin Spiramycin CRS Methanol R pH 8.0 (0.05 M) A - pH 7.9 30-32 °C
CIP 52.62
ATCC 6633
Bacillus subtilis A - pH 7.9 30-37 °C
NCTC 8236
CIP 1.83
Streptomycin Streptomycin
Water R pH 8.0 (0.05 M) Bacillus subtilis A - pH 7.9 30-37 °C
sulphate sulphate CRS
NCTC 10400
CIP 52.62
ATCC 6633
Bacillus subtilis
NCTC 10400
Teicoplanin Teicoplanin CRS pH 6.0 (0.05 M) pH 6.0 (0.05 M) H - pH 7.8-8.0 35-37 °C
CIP 5262
ATCC 6633
Tylosin for A mixture of
2.5 per cent V/V Micrococcus luteus
veterinary use 40 volumes of
solution of NCTC 8340
methanol R and
Tylosin CRS methanol R in 0.1 M A - pH 8.0 32-35 °C
60 volumes of 0.1 M CIP 53.45
Tylosin tartrate for phosphate buffer
phosphate buffer ATCC 9341
veterinary use solution pH 7.0 R
solution pH 8.0 R
Bacillus subtilis
Vancomycin Vancomycin NCTC 8236
Water R pH 8.0 A - pH 8.0 37-39 °C
hydrochloride hydrochloride CRS CIP 52.62
ATCC 6633

Distribute an equal volume of each of the solutions into
identical test-tubes and add to each tube an equal volume of
inoculated medium (for example, 1 ml of the solution and
9 ml of the medium). For the assay of tyrothricin add 0.1 ml
of the solution to 9.9 ml of inoculated medium.
using suitable optical apparatus. Alternatively use a method
which allows the opacity of each tube to be measured after
exactly the same period of incubation.
Calculate the potency using appropriate statistical methods.

Prepare at the same time 2 control tubes without antibiotic, Linearity of the dose-response relationship, transformed or
both containing the inoculated medium and to one of which untransformed, is often obtained only over a very limited
is added immediately 0.5 ml of formaldehyde R. These tubes range. It is this range which must be used in calculating the
are used to set the optical apparatus used to measure the activity and it must include at least 3 consecutive doses in
growth. order to permit linearity to be verified. In routine assays
when the linearity of the system has been demonstrated
Place all the tubes, randomly distributed or in a Latin square over an adequate number of experiments using a three-point
or randomised block arrangement, in a water-bath or other assay, a two-point assay may be sufficient, subject to
suitable apparatus fitted with a means of bringing all the agreement by the competent authority. However, in all cases
tubes rapidly to the appropriate incubation temperature of dispute, a three-point assay must be applied.
and maintain them at that temperature for 3-4 h, taking
precautions to ensure uniformity of temperature and Use in each assay the number of replications per dose
identical incubation time. sufficient to ensure the required precision. The assay may be
repeated and the results combined statistically to obtain the
After incubation, stop the growth of the micro-organisms by required precision and to ascertain whether the potency of
adding 0.5 ml of formaldehyde R to each tube or by heat the antibiotic to be examined is not less than the minimum
treatment and measure the opacity to 3 significant figures required.

General Notices (1) apply to all monographs and other texts 3937
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 6.3

Table 2.7.2.-2. – Turbidimetric assay

Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Escherichia coli
NCIB 8666
Colistimethate Colistimethate
Water R pH 7.0 CIP 2.83 C - pH 7.0 35-37 °C
sodium sodium CRS
ATCC 9637

Staphylococcus
aureus
Framycetin NCTC 7447
Framycetin sulphate Water R pH 8.0 C - pH 7.0 35-37 °C
sulphate CRS
CIP 53.156
ATCC 6538 P
Staphylococcus
aureus
Gentamicin NCTC 7447
Gentamicin sulphate Water R pH 7.0 C - pH 7.0 35-37 °C
sulphate CRS
CIP 53.156
ATCC 6538 P
Enterococcus hirae
CIP 58.55
* ATCC 10541
Gramicidin CRS Methanol R pH 7.0 C - pH 7.0 35-37 °C
Gramicidin Staphylococcus
aureus
ATCC 6538 P
*
Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions, for example 0.1 mg/ml
of polysorbate 80 R
Staphylococcus
aureus
Josamycin CRS Methanol R (see the CIP 53.156
Josamycin pH 5.6 C - pH 8.0 35-37 °C
monograph)
ATCC 6538 P
NCTC 7447
Staphylococcus
aureus
Josamycin Josamycin Methanol R (see the CIP 53.156
pH 5.6 C - pH 8.0 35-37 °C
propionate propionate CRS monograph)
ATCC 6538 P
NCTC 7447
Kanamycin Staphylococcus
monosulphate aureus
Kanamycin NCTC 7447
Water R pH 8.0 C - pH 7.0 35-37 °C
monosulphate CRS
Kanamycin acid CIP 53.156
sulphate ATCC 6538 P
Staphylococcus
Neomycin sulphate aureus
Neomycin sulphate for microbiological Water R pH 8.0 NCTC 7447 C - pH 7.0 35-37 °C
assay CRS CIP 53.156
ATCC 6538 P
Escherichia coli
Rifamycin NCIB 8879
Rifamycin sodium Methanol R pH 7.0 C - pH 7.0 35-37 °C
sodium CRS CIP 54.127
ATCC 10536
Staphylococcus
aureus
Spiramycin Spiramycin CRS Methanol R pH 7.0 NCTC 7447 C - pH 7.0 35-37 °C
CIP 53.156
ATCC 6538 P
Klebsiella
pneumoniae
Streptomycin Streptomycin NCTC 7427
Water R pH 8.0 C - pH 7.0 35-37 °C
sulphate sulphate CRS
CIP 53.153
ATCC 10031

3938 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.7.2. Microbiological assay of antibiotics

Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Tylosin for 2.5 per cent V/V Staphylococcus
veterinary use solution of aureus
Tylosin CRS methanol R in 0.1 M pH 7.0 NCTC 6571 C - pH 7.0 37 °C
Tylosin tartrate for phosphate buffer ATCC 9144
veterinary use solution pH 7.0 R CIP 53.154
Enterococcus hirae
Tyrothricin Gramicidin CRS Alcohol R Alcohol R C - pH 7.0 37 °C
ATCC 10541
Staphylococcus
Vancomycin Vancomycin aureus
Water R pH 8.0 C - pH 7.0 37-39 °C
hydrochloride hydrochloride CRS CIP 53.156
ATCC 6538 P

The following section is published for information. Buffer solutions. Buffer solutions having a pH between 5.8
and 8.0 are prepared by mixing 50.0 ml of 0.2 M potassium
dihydrogen phosphate R with the quantity of 0.2 M sodium
Recommended micro-organisms hydroxide indicated in Table 2.7.2.-3. Dilute with freshly
prepared distilled water R to produce 200.0 ml.
The following text details the recommended micro-organisms Table 2.7.2.-3.
and the conditions of use. Other micro-organisms may be
used provided that they are shown to be sensitive to the pH 0.2 M Sodium hydroxide (ml)
antibiotic to be examined and are used in appropriate media
5.8 3.72
and appropriate conditions of temperature and pH. The
concentrations of the solutions used should be chosen so 6.0 5.70
as to ensure that a linear relationship exists between the 6.2 8.60
logarithm of the dose and the response in the conditions
of the test. 6.4 12.60

Preparation of inocula. Bacillus cereus var. mycoides ; 6.6 17.80

Bacillus subtilis ; Bacillus pumilus. Spore suspensions of 6.8 23.65
the organisms to be used as inocula are prepared as follows.
7.0 29.63
Grow the organism at 35-37 °C for 7 days on the surface 7.2 35.00
of a suitable medium to which has been added 0.001 g/l
of manganese sulphate R. Using sterile water R, wash 7.4 39.50
off the growth, which consists mainly of spores. Heat 7.6 42.80
the suspension at 70 °C for 30 min and dilute to give an
7.8 45.20
appropriate concentration of spores, usually 10 × 106 to
100 × 106 per millilitre. The spore suspensions may be stored 8.0 46.80
for long periods at a temperature not exceeding 4 °C.
Alternatively, spore suspensions may be prepared by These buffer solutions are used for all microbiological assays
cultivating the organisms in medium C at 26 °C for 4-6 days, shown in Table 2.7.2.-1 with the exception of bleomycin
then adding, aseptically, sufficient manganese sulphate R to sulphate and amphotericin B.
give a concentration of 0.001 g/l and incubating for a further For bleomycin sulphate, prepare the buffer solution
48 h. Examine the suspension microscopically to ensure that pH 6.8 as follows : dissolve 6.4 g of potassium dihydrogen
adequate spore formation has taken place (about 80 per cent) phosphate R and 18.9 g of disodium hydrogen phosphate R
and centrifuge. Re-suspend the sediment in sterile water R in water R and dilute to 1000 ml with water R.
to give a concentration of 10 × 106 to 100 × 106 spores per
millilitre, and then heat to 70 °C for 30 min. Store the For amphotericin B, prepare the 0.2 M phosphate buffer
suspension at a temperature not exceeding 4 °C. solution pH 10.5 as follows : dissolve 35 g of dipotassium
hydrogen phosphate R in 900 ml of water R, add 20 ml of
Bordetella bronchiseptica. Grow the test organism on 1 M sodium hydroxide and dilute to 1000.0 ml with water R.
medium B at 35-37 °C for 16-18 h. Wash off the bacterial
growth with sterile water R and dilute to a suitable opacity. Culture media. The following media or equivalent media
may be used.
Staphylococcus aureus ; Klebsiella pneumoniae ;
Escherichia coli ; Micrococcus luteus ; Staphylococcus Medium A
epidermidis. Prepare as described above for 6g
B. bronchiseptica but using medium A and adjusting Peptone
the opacity to one which has been shown to produce a Pancreatic digest of casein 4g
satisfactory dose-response relationship in the turbidimetric 1.5 g
Beef extract
assay, or to produce clearly defined zones of inhibition of
convenient diameter in the diffusion assay, as appropriate. Yeast extract 3g

Glucose monohydrate 1g
Saccharomyces cerevisiae ; Candida tropicalis. Grow the
test organism on medium F at 30-37 °C for 24 h. Wash off Agar 15 g
the growth with a sterile 9 g/l solution of sodium chloride R. 1000 ml
Water to produce
Dilute to a suitable opacity with the same solution.

General Notices (1) apply to all monographs and other texts 3939
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 6.3

Medium B Medium E
Pancreatic digest of casein 17 g Peptone 5g

Papaic digest of soya bean 3g Meat extract 3g

Sodium chloride 5g 26.9 g

Disodium hydrogen phosphate,12H2O
Dipotassium hydrogen phosphate 2.5 g Agar 10 g

Glucose monohydrate 2.5 g 1000 ml

Water to produce
Agar 15 g
The disodium hydrogen phosphate is added as a sterile
Polysorbate 80 10 g solution after sterilisation of the medium.
Water to produce 1000 ml Medium F
Peptone 9.4 g
The polysorbate 80 is added to the hot solution of the other
ingredients after boiling, and immediately before adjusting Yeast extract 4.7 g
to volume. Beef extract 2.4 g
Medium C
Sodium chloride 10.0 g
Peptone 6g
Glucose monohydrate 10.0 g
Beef extract 1.5 g
Agar 23.5 g
Yeast extract 3g
Water to produce 1000 ml
Sodium chloride 3.5 g

Glucose monohydrate 1g Medium G

Dipotassium hydrogen phosphate 3.68 g Glycerol 10 g

Potassium dihydrogen phosphate 1.32 g Peptone 10 g

Water to produce 1000 ml Meat extract 10 g

Sodium chloride 3g
Medium D
1.5 g Agar 15 g
Heart extract
1.5 g Water to produce 1000 ml
Yeast extract
Peptone-casein 5g pH 7.0 ± 0.1 after sterilisation.
Glucose monohydrate 1g
Medium H
Sodium chloride 3.5 g Peptone 5.0 g
Dipotassium hydrogen phosphate 3.68 g Agar 15.0 g
Potassium dihydrogen phosphate 1.32 g 3.0 g
Beef extract powder
Potassium nitrate 2g
Water to produce 1000 ml
Water to produce 1000 ml
pH 7.8 - 8.0 adjusted with 0.1 M sodium hydroxide.

3940 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

2.9. PHARMACEUTICAL
TECHNICAL PROCEDURES
2.9.1. Disintegration of tablets and capsules.....................3943 2.9.33. Characterisation of crystalline and partially
crystalline solids by X-ray powder diffraction (XRPD).. 3945

General Notices (1) apply to all monographs and other texts 3941
 EUROPEAN PHARMACOPOEIA 6.3

3942 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.9.1. Disintegration of tablets and capsules

01/2009:20901 lines perpendicular to the axis and parallel to each other.

4 identical trapezoidal-shaped planes are cut into the wall
2.9.1. DISINTEGRATION OF TABLETS of the cylinder, nearly perpendicular to the ends of the
cylinder. The trapezoidal shape is symmetrical ; its parallel
AND CAPSULES sides coincide with the ends of the cylinder and are parallel
This test is provided to determine whether tablets or capsules to an imaginary line connecting the centres of 2 adjacent
disintegrate within the prescribed time when placed in a holes 6 mm from the cylindrical axis. The parallel side of
liquid medium under the experimental conditions presented the trapezoid on the bottom of the cylinder has a length of
below. 1.6 ± 0.1 mm and its bottom edges lie at a depth of 1.5 mm
to 1.8 mm from the cylinder’s circumference. The parallel
For the purposes of this test, disintegration does not side of the trapezoid on the top of the cylinder has a length
imply complete dissolution of the unit or even of its active of 9.4 ± 0.2 mm and its centre lies at a depth of 2.6 ± 0.1 mm
constituent. Complete disintegration is defined as that from the cylinder’s circumference. All surfaces of the disc
state in which any residue of the unit, except fragments of are smooth.
insoluble coating or capsule shell, remaining on the screen
of the test apparatus or adhering to the lower surface of the If the use of discs is specified, add a disc to each tube and
discs, if used, is a soft mass having no palpably firm core. operate the apparatus as directed under Procedure. The
discs conform to the dimensions shown in Figure 2.9.1.-1.
Use apparatus A for tablets and capsules that are not
greater than 18 mm long. For larger tablets or capsules use The use of automatic detection employing modified discs
apparatus B. is permitted where the use of discs is specified or allowed.
Such discs must comply with the requirements of density
TEST A - TABLETS AND CAPSULES OF NORMAL SIZE and dimension given in this chapter.
Apparatus. The apparatus consists of a basket-rack assembly, Procedure. Place 1 dosage unit in each of the 6 tubes of the
a 1 litre, low-form beaker, 149 ± 11 mm in height and having basket and, if prescribed, add a disc. Operate the apparatus
an inside diameter of 106 ± 9 mm for the immersion fluid, using the specified medium, maintained at 37 ± 2 °C, as
a thermostatic arrangement for heating the fluid between the immersion fluid. At the end of the specified time, lift
35 °C and 39 °C, and a device for raising and lowering the the basket from the fluid and observe the dosage units :
basket in the immersion fluid at a constant frequency rate all of the dosage units have disintegrated completely. If
between 29 and 32 cycles per minute, through a distance 1 or 2 dosage units fail to disintegrate, repeat the test on
of 55 ± 2 mm. The volume of the fluid in the vessel is such 12 additional dosage units. The requirements of the test are
that at the highest point of the upward stroke the wire mesh met if not less than 16 of the 18 dosage units tested have
remains at least 15 mm below the surface of the fluid, and disintegrated.
descends to not less than 25 mm from the bottom of the
vessel on the downward stroke. At no time should the top TEST B – LARGE TABLETS AND LARGE CAPSULES
of the basket-rack assembly become submerged. The time Apparatus. The main part of the apparatus (Figure 2.9.1.-2)
required for the upward stroke is equal to the time required is a rigid basket-rack assembly supporting 3 cylindrical
for the downward stroke, and the change in stroke direction transparent tubes 77.5 ± 2.5 mm long, 33.0 mm ± 0.5 mm in
is a smooth transition, rather than an abrupt reversal of internal diameter, and with a wall thickness of 2.5 ± 0.5 mm.
motion. The basket-rack assembly moves vertically along its Each tube is provided with a cylindrical disc 31.4 ± 0.13 mm
axis. There is no appreciable horizontal motion or movement in diameter and 15.3 ± 0.15 mm thick, made of transparent
of the axis from the vertical. plastic with a relative density of 1.18-1.20. Each disc is
Basket-rack assembly. The basket-rack assembly consists of pierced by 7 holes, each 3.15 ± 0.1 mm in diameter, 1 in
6 open-ended transparent tubes, each 77.5 ± 2.5 mm long the centre and the other 6 spaced equally on a circle of
and having an inside diameter of 21.85 ± 1.15 mm and a wall radius 4.2 mm from the centre of the disc. The tubes are
1.9 ± 0.9 mm thick ; the tubes are held in a vertical position held vertically by 2 separate and superimposed rigid plastic
by 2 plates, each 90 ± 2 mm in diameter and 6.75 ± 1.75 mm plates 97 mm in diameter and 9 mm thick, with 3 holes.
in thickness, with 6 holes, each 24 ± 2 mm in diameter, The holes are equidistant from the centre of the plate and
equidistant from the centre of the plate and equally spaced equally spaced. Attached to the under side of the lower plate
from one another. Attached to the under surface of the is a piece of woven gauze made from stainless steel wire
lower plate is a woven stainless steel wire cloth, which has a 0.63 ± 0.03 mm in diameter and having mesh apertures of
plain square weave with 2.0 ± 0.2 mm mesh apertures and 2.0 ± 0.2 mm. The plates are held rigidly in position and
with a wire diameter of 0.615 ± 0.045 mm. The parts of the 77.5 mm apart by vertical metal rods at the periphery. A
apparatus are assembled and rigidly held by means of 3 bolts metal rod is also fixed to the centre of the upper plate to
passing through the 2 plates. A suitable means is provided enable the assembly to be attached to a mechanical device
to suspend the basket-rack assembly from the raising and capable of raising and lowering it smoothly at a constant
lowering device using a point on its axis. frequency of between 29 and 32 cycles per minute, through
The design of the basket-rack assembly may be varied a distance of 55 ± 2 mm.
somewhat provided the specifications for the glass The assembly is suspended in the specified liquid medium in
tubes and the screen mesh size are maintained. The a suitable vessel, preferably a 1 litre beaker. The volume of
basket-rack assembly conforms to the dimensions shown in the liquid is such that when the assembly is in the highest
Figure 2.9.1.-1. position the wire mesh is at least 15 mm below the surface
Discs. The use of discs is permitted only where specified of the liquid, and when the assembly is in the lowest position
or allowed. Each tube is provided with a cylindrical disc the wire mesh is at least 25 mm above the bottom of the
9.5 ± 0.15 mm thick and 20.7 ± 0.15 mm in diameter. The beaker and the upper open ends of the tubes remain above
disc is made of a suitable, transparent plastic material the surface of the liquid. A suitable device maintains the
having a specific gravity of 1.18-1.20. 5 parallel 2 ± 0.1 mm temperature of the liquid at 35-39 °C.
holes extend between the ends of the cylinder. One of The design of the basket-rack assembly may be varied
the holes is centered on the cylindrical axis. The other provided the specifications for the tubes and wire mesh are
holes are centered 6 ± 0.2 mm from the axis on imaginary maintained.

General Notices (1) apply to all monographs and other texts 3943
2.9.1. Disintegration of tablets and capsules EUROPEAN PHARMACOPOEIA 6.3

Method. Test 6 tablets or capsules either by using the beaker containing the specified liquid. Operate the
2 basket-rack assemblies in parallel or by repeating the apparatus for the prescribed period, withdraw the assembly
procedure. In each of the 3 tubes, place 1 tablet or capsule and examine the state of the tablets or capsules. To pass the
and, if prescribed, add a disc ; suspend the assembly in test, all 6 o f the tablets or capsules must have disintegrated.

Figure 2.9.1.-1. – Disintegration apparatus A

Dimensions in millimetres

3944 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.9.33. Characterisation of crystalline solids by XRPD

Figure 2.9.1.-2. – Disintegration apparatus B

Dimensions in millimetres

01/2009:20933 particle orientation within the sample) ; and diffraction line

profiles (depending on instrumental resolution, crystallite
size, strain and specimen thickness).
2.9.33. CHARACTERISATION OF
Experiments giving angular positions and intensities of
CRYSTALLINE AND PARTIALLY lines can be used for applications such as qualitative phase
CRYSTALLINE SOLIDS BY X-RAY analysis (for example, identification of crystalline phases)
and quantitative phase analysis of crystalline materials. An
POWDER DIFFRACTION (XRPD) estimate of the amorphous and crystalline fractions(1) can
Every crystalline phase of a given substance produces a also be made.
characteristic X-ray diffraction pattern.
The X-ray powder diffraction (XRPD) method provides an
Diffraction patterns can be obtained from a randomly advantage over other means of analysis in that it is usually
oriented crystalline powder composed of crystallites or non-destructive in nature (specimen preparation is usually
crystal fragments of finite size. Essentially 3 types of limited to grinding to ensure a randomly oriented sample).
information can be derived from a powder diffraction XRPD investigations can also be carried out under in situ
pattern : angular position of diffraction lines (depending on conditions on specimens exposed to non-ambient conditions,
geometry and size of the unit cell) ; intensities of diffraction such as low or high temperature and humidity.
lines (depending mainly on atom type and arrangement, and

(1) There are many other applications of the X-ray powder diffraction technique that can be applied to crystalline pharmaceutical substances such as : determination of crystal structures,
refinement of crystal structures, determination of crystallographic purity of crystalline phases, characterisation of crystallographic texture, etc. These applications are not described in this chapter.

General Notices (1) apply to all monographs and other texts 3945
2.9.33. Characterisation of crystalline solids by XRPD
PRINCIPLE
X-ray diffraction results from the interaction between
X-rays and electron clouds of atoms. Depending on the
atomic arrangement, interferences arise from the scattered
X-rays. These interferences are constructive when the path
difference between 2 diffracted X-ray waves differs by an
integral number of wavelengths. This selective condition
is described by the Bragg equation, also called Bragg’s law
(see Figure 2.9.33.-1) :
The wavelength λ of the X-rays is of the same order of
magnitude as the distance between successive crystal lattice
planes, or dhkl (also called ‘d-spacings’). θhkl is the angle
between the incident ray and the family of lattice planes,
and sinθhkl is inversely proportional to the distance between
successive crystal planes or d-spacings.
The direction and spacing of the planes with reference to
the unit cell axes are defined by the Miller indices {hkl}.
These indices are the reciprocals, reduced to the next-lower
integer, of the intercepts that a plane makes with the unit
cell axes. The unit cell dimensions are given by the spacings
a, b and c and the angles between them, α, β, and γ.
The interplanar spacing for a specified set of parallel hkl
planes is denoted by dhkl. Each such family of planes may
show higher orders of diffraction where the d values for the
related families of planes nh, nk, nl are diminished by the
factor 1/n (n being an integer : 2, 3, 4, etc.).
Every set of planes throughout a crystal has a corresponding
Bragg diffraction angle, θhkl, associated with it (for a specific
wavelength λ).
A powder specimen is assumed to be polycrystalline so that
at any angle θhkl there are always crystallites in an orientation
allowing diffraction according to Bragg’s law(2). For a given
X-ray wavelength, the positions of the diffraction peaks (also
referred to as ‘lines’, ‘reflections’ or ‘Bragg reflections’)
are characteristic of the crystal lattice (d-spacings), their
theoretical intensities depend on the crystallographic unit
cell content (nature and positions of atoms), and the line
profiles on the perfection and extent of the crystal lattice.
Figure 2.9.33.-1. – Diffraction of X-rays by a crystal according to Bragg’s law
(2) An ‘ideal’ powder for diffraction experiments consists of a large number of small, randomly oriented spherical crystallites (coherently diffracting crystalline domains). If this number is
sufficiently large, there are always enough crystallites in any diffracting orientation to give reproducible diffraction patterns.
3946
EUROPEAN PHARMACOPOEIA 6.3
Under these conditions the diffraction peak has a finite
intensity arising from atomic arrangement, type of atoms,
thermal motion and structural imperfections, as well as from
instrument characteristics.
The intensity is dependent upon many factors such
as structure factor, temperature factor, crystallinity,
polarisation factor, multiplicity and Lorentz factor.
The main characteristics of diffraction line profiles
are 2θ position, peak height, peak area and shape
(characterised by, for example, peak width or asymmetry,
analytical function, empirical representation). An example
of the type of powder patterns obtained for 5 different solid
phases of a substance are shown in Figure 2.9.33.-2.
In addition to the diffraction peaks, an X-ray diffraction
experiment also generates a more-or-less uniform
background, upon which the peaks are superimposed.
Besides specimen preparation, other factors contribute to
the background, for instance the sample holder, diffuse
scattering from air and equipment, other instrumental
parameters such as detector noise, general radiation from
the X-ray tube, etc. The peak-to-background ratio can be
increased by minimising background and by choosing
prolonged exposure times.
INSTRUMENT
Instrument set-up. X-ray diffraction experiments are usually
performed using powder diffractometers or powder cameras.
A powder diffractometer generally comprises 5 main parts :
an X-ray source ; incident beam optics, which may perform
monochromatisation, filtering, collimation and/or focusing
of the beam ; a goniometer ; diffraction beam optics, which
may perform monochromatisation, filtering, collimation
and focusing or parallelising of the beam ; and a detector.
Data-collection and data-processing systems are also
required and are generally included in current diffraction
measurement equipment.
Depending on the type of analysis to be performed
(phase identification, quantitative analysis, lattice
parameters determination, etc.), different XRPD instrument
configurations and performance levels are required. The
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 2.9.33. Characterisation of crystalline solids by XRPD

Figure 2.9.33.-2. – X-ray powder diffraction patterns collected for 5 different solid phases of a substance
(the intensities are normalised)
simplest instruments used to measure XRPD patterns θ/2θ scans the goniometer rotates the specimen about the
are powder cameras. The replacement of photographic same axis as that of the detector, but at half the rotational
film as the detection method by photon detectors has led speed, in a θ/2θ motion. The surface of the specimen thus
to the design of diffractometers in which the geometric remains tangential to the focusing circle. The parallel plate
arrangement of the optics is not truly focusing but collimator limits the axial divergence of the beam and hence
parafocusing, such as in the Bragg-Brentano geometry. The partially controls the shape of the diffracted line profile.
Bragg-Brentano parafocusing configuration is currently the A diffractometer may also be used in transmission mode.
most widely used and is therefore briefly described here. The advantage with this technology is to lessen the effects
due to preferred orientation. A capillary of about 0.5-2 mm
A given instrument may provide a horizontal or vertical
thickness can also be used for small sample amounts.
θ/2θ geometry or a vertical θ/θ geometry. For both
geometries, the incident X-ray beam forms an angle θ with X-ray radiation. In the laboratory, X-rays are obtained
the specimen surface plane and the diffracted X-ray beam by bombarding a metal anode with electrons emitted by
forms an angle 2θ with the direction of the incident X-ray the thermionic effect and accelerated in a strong electric
beam (an angle θ with the specimen surface plane). The basic field (using a high-voltage generator). Most of the kinetic
geometric arrangement is represented in Figure 2.9.33.-3. energy of the electrons is converted to heat, which limits the
The divergent beam of radiation from the X-ray tube (the power of the tubes and requires efficient anode cooling. A
so-called ‘primary beam’) passes through the parallel plate 20- to 30-fold increase in brilliance can be obtained using
collimators and a divergence slit assembly and illuminates rotating anodes and by using X-ray optics. Alternatively,
the flat surface of the specimen. All the rays diffracted by X-ray photons may be produced in a large-scale facility
suitably oriented crystallites in the specimen at an angle 2θ (synchrotron).
converge to a line at the receiving slit. A second set of The spectrum emitted by an X-ray tube operating at
parallel plate collimators and a scatter slit may be placed sufficient voltage consists of a continuous background
either behind or before the receiving slit. The axes of the line of polychromatic radiation and additional characteristic
focus and of the receiving slit are at equal distances from radiation that depends on the type of anode. Only
the axis of the goniometer. The X-ray quanta are counted this characteristic radiation is used in X-ray diffraction
by a radiation detector, usually a scintillation counter, a experiments. The principal radiation sources utilised
sealed-gas proportional counter, or a position-sensitive for X-ray diffraction are vacuum tubes utilising copper,
solid-state detector such as imaging plate or CCD detector. molybdenum, iron, cobalt or chromium as anodes ; copper,
The receiving slit assembly and the detector are coupled molybdenum or cobalt X-rays are employed most commonly
together and move tangentially to the focusing circle. For for organic substances (the use of cobalt anodes can be

General Notices (1) apply to all monographs and other texts 3947
2.9.33. Characterisation of crystalline solids by XRPD EUROPEAN PHARMACOPOEIA 6.3

A. X-ray tube C. sample E. receiving slit G. detector receiving slit J. diffractometer circle
B. divergence slit D. anti-diffusion slit F. monochromator H. detector K. focusing circle

Figure 2.9.33.-3. – Geometric arrangement of the Bragg-Brentano parafocusing geometry

especially preferred to separate distinct X-ray lines). The regulations or recommendations in a country, the latest
choice of radiation to be used depends on the absorption recommendations of the International Commission on
characteristics of the specimen and possible fluorescence Radiological Protection should be applied.
by atoms present in the specimen. The wavelengths used in
powder diffraction generally correspond to the Kα radiation SPECIMEN PREPARATION AND MOUNTING
from the anode. Consequently, it is advantageous to make The preparation of the powdered material and mounting
the X-ray beam ‘monochromatic’ by eliminating all the other of the specimen in a suitable holder are critical steps in
components of the emission spectrum. This can be partly many analytical methods, and are particularly so for XRPD
obtained using Kβ filters, i.e. metal filters selected as havinganalysis, since they can greatly affect the quality of the data
an absorption edge between the Kα and Kβ wavelengths to be collected(3). The main sources of error due to specimen
emitted by the tube. preparation and mounting are briefly discussed here for
instruments in Bragg-Brentano parafocusing geometry.
Such a filter is usually inserted between the X-ray tube
SPECIMEN PREPARATION
and the specimen. Another, more-and-more-commonly
used way to obtain a monochromatic X-ray beam is via In general, the morphology of many crystalline particles
a large monochromator crystal (usually referred to as a tends to give a specimen that exhibits some degree of
‘monochromator’). This crystal is placed before or behind preferred orientation in the specimen holder. This is
the specimen and diffracts the different characteristic peaks particularly evident for needle-like or plate-like crystals when
of the X-ray beam (i.e. Kα and Kβ) at different angles, so that size reduction yields finer needles or platelets. Preferred
only one of them may be selected to enter into the detector. orientation in the specimen influences the intensities of
It is even possible to separate Kα1 and Kα2 radiations by various reflections, so that some are more intense and others
using a specialised monochromator. Unfortunately, the are less intense, compared to what would be expected from
gain in getting a monochromatic beam by using a filter or a completely random specimen. Several techniques can
a monochromator is counteracted by a loss in intensity. be employed to improve randomness in the orientation of
Another way of separating Kα and Kβ wavelengths is crystallites (and therefore to minimise preferred orientation),
by using curved X-rays mirrors that can simultaneously but further reduction of particle size is often the best and
monochromate and focus or parallelise the X-ray beam. simplest approach. The optimum number of crystallites
depends on the diffractometer geometry, the required
RADIATION PROTECTION. Exposure of any part of the resolution and the specimen attenuation of the X-ray
human body to X-rays can be injurious to health. It is beam. In some cases, particle sizes as large as 50 μm
therefore essential that whenever X-ray equipment is used, will provide satisfactory results in phase identification.
adequate precautions are taken to protect the operator However, excessive milling (crystallite sizes less than
and any other person in the vicinity. Recommended approximately 0.5 μm) may cause line broadening and
practice for radiation protection as well as limits for the significant changes to the sample itself such as :
levels of X-radiation exposure are those established by — specimen contamination by particles abraded from the
national legislation in each country. If there are no official milling instruments (mortar, pestle, balls, etc.) ;

(3) Similarly, changes in the specimen can occur during data collection in the case of a non-equilibrium specimen (temperature, humidity).

3948 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 2.9.33. Characterisation of crystalline solids by XRPD

— reduced degree of crystallinity ; QUALITATIVE PHASE ANALYSIS (IDENTIFICATION OF

PHASES)
— solid-state transition to another polymorph ;
The identification of the phase composition of an unknown
— chemical decomposition ; sample by XRPD is usually based on the visual or
— introduction of internal stress ; computer-assisted comparison of a portion of its XRPD
pattern to the experimental or calculated pattern of a
— solid-state reactions. reference material. Ideally, these reference patterns are
Therefore, it is advisable to compare the diffraction pattern collected on well-characterised single-phase specimens.
of the non-ground specimen with that corresponding to a This approach makes it possible in most cases to identify
specimen of smaller particle size (e.g. a milled specimen). If a crystalline substance by its 2θ diffraction angles or
the XRPD pattern obtained is of adequate quality considering d-spacings and by its relative intensities. The computer-aided
its intended use, then grinding may not be required. comparison of the diffraction pattern of the unknown sample
It should be noted that if a sample contains more than one to the comparison data can be based either on a more-or-less
phase and if sieving is used to isolate particles to a specific extended 2θ-range of the whole diffraction pattern or on a
size, the initial composition may be altered. set of reduced data derived from the pattern. For example,
the list of d-spacings and normalised intensities (Inorm), a
SPECIMEN MOUNTING
so-called (d, Inorm)-list extracted from the pattern, is the
Effect of specimen displacement. A specimen surface that crystallographic fingerprint of the material, and can be
is offset by D with reference to the diffractometer rotation compared to (d, Inorm)-lists of single-phase samples compiled
axis causes systematic errors that are very difficult to avoid in databases.
entirely ; for the reflection mode, this results in absolute For most organic crystals, when using Cu Kα radiation, it is
D·cosθ shifts(4) in 2θ positions (typically of the order of appropriate to record the diffraction pattern in a 2θ-range
0.01° in 2θ at low angles (cosθ 1) for a displacement from as near 0° as possible to at least 40°. The agreement
D = 15 μm) and asymmetric broadening of the profile towards in the 2θ-diffraction angles between specimen and reference
low 2θ values. Use of an appropriate internal standard allows is within 0.2° for the same crystal form, while relative
the detection and correction of this effect simultaneously intensities between specimen and reference may vary
with that arising from specimen transparency. This is by far considerably due to preferred orientation effects. By their
the largest source of errors in data collected on well-aligned very nature, variable hydrates and solvates are recognised
diffractometers. to have varying unit cell dimensions and as such shifting
Effect of specimen thickness and transparency. When occurs in peak positions of the measured XRPD patterns
the XRPD method in reflection mode is applied, it is often for these materials. In these unique materials, variance
preferable to work with specimens of ‘infinite thickness’. in 2θ-positions of greater than 0.2° is not unexpected. As
To minimise the transparency effect, it is advisable to use such, peak position variances such as 0.2° are not applicable
a non-diffracting substrate (zero background holder), for to these materials. For other types of samples (e.g. inorganic
example a plate of single crystalline silicon cut parallel to salts), it may be necessary to extend the 2θ-region scanned
the 510 lattice planes(5). One advantage of the transmission to well beyond 40°. It is generally sufficient to scan past the
mode is that problems with sample height and specimen 10 strongest reflections identified in single phase XRPD
transparency are less important. The use of an appropriate database files.
internal standard allows the detection and correction of It is sometimes difficult or even impossible to identify phases
this effect simultaneously with that arising from specimen in the following cases :
displacement. — non-crystallised or amorphous substances ;
— the components to be identified are present in low mass
CONTROL OF THE INSTRUMENT PERFORMANCE fractions of the analyte amounts (generally less than
Goniometers and the corresponding incident and diffracted 10 per cent m/m) ;
X-ray beam optics have many mechanical parts that need — pronounced preferred orientation effects ;
adjustment. The degree of alignment or misalignment — the phase has not been filed in the database used ;
directly influences the quality of the results of an XRPD
investigation. Therefore, the different components of — formation of solid solutions ;
the diffractometer must be carefully adjusted (optical — presence of disordered structures that alter the unit cell ;
and mechanical systems, etc.) to minimise adequately — the specimen comprises too many phases ;
systematic errors, while optimising the intensities received — presence of lattice deformations ;
by the detector. The search for maximum intensity and — structural similarity of different phases ;
maximum resolution is always antagonistic when aligning a
diffractometer. Hence, the best compromise must be sought QUANTITATIVE PHASE ANALYSIS
whilst performing the alignment procedure. There are many
If the sample under investigation is a mixture of 2 or more
different configurations and each supplier’s equipment
known phases, of which not more than 1 is amorphous,
requires specific alignment procedures.
the percentage (by volume or by mass) of each crystalline
The overall diffractometer performance must be tested and phase and of the amorphous phase can, in many cases, be
monitored periodically using suitable certified reference determined. Quantitative phase analysis can be based on
materials. Depending on the type of analysis, other the integrated intensities, on the peak heights of several
well-defined reference materials may also be employed, individual diffraction lines(6), or on the full pattern. These
although the use of certified reference materials is preferred. integrated intensities, peak heights or full-pattern data
(4) Note that a goniometer zero alignment shift would result in constant shift on all observed 2θ-line positions, in other words, the whole diffraction pattern is in this case translated by an
offset of Z° in 2θ.
(5) In the case of a thin specimen with low attenuation, accurate measurements of line positions can be made with focusing diffractometer configurations in either transmission or reflection
geometry. Accurate measurements of line positions on specimens with low attenuation are preferably made using diffractometers with parallel beam optics. This helps to reduce the
effects of specimen thickness.
(6) If the crystal structures of all components are known, the Rietveld method can be used to quantify them with good accuracy. If the crystal structures of the components are not known,
the Pawley or least squares methods can be used.

General Notices (1) apply to all monographs and other texts 3949
2.9.33. Characterisation of crystalline solids by XRPD
points are compared to the corresponding values of reference
materials. These reference materials shall be single-phase
or a mixture of known phases. The difficulties encountered
during quantitative analysis are due to specimen preparation
(the accuracy and precision of the results require in
particular homogeneity of all phases and a suitable particle
size distribution in each phase) and to matrix effects. In
favourable cases, amounts of crystalline phases as small as — if the sample consists of crystalline fractions and an
10 per cent may be determined in solid matrices.
POLYMORPHIC SAMPLES
For a sample composed of 2 polymorphic phases a and b, the
following expression may be used to quantify the fraction Fa
of phase a :
The fraction is derived by measuring the intensity ratio
between the 2 phases, knowing the value of the constant K.
K is the ratio of the absolute intensities of the 2 pure
polymorphic phases Ioa/Iob. Its value can be determined by
measuring standard samples.
METHODS USING A STANDARD
The most commonly used methods for quantitative analysis
are :
— the ‘external standard method’ ;
— the ‘internal standard method’ ;
— the ‘spiking method’ (often also called the ‘standard
addition method’).
The ‘external standard method’ is the most general method
and consists of comparing the X-ray diffraction pattern of
the mixture, or the respective line intensities, with those
measured in a reference mixture or with the theoretical
intensities of a structural model, if it is fully known.
To limit errors due to matrix effects, an internal reference
material with crystallite size and X-ray absorption coefficient
comparable to those of the components of the sample, and
with a diffraction pattern that does not overlap at all that of
the sample to be analysed, can be used. A known quantity
of this reference material is added to the sample to be
analysed and to each of the reference mixtures. Under these
conditions, a linear relationship between line intensity and
concentration exists. This application, called the ‘internal
standard method’, requires a precise measurement of
diffraction intensities.
In the ‘spiking method’ (or ‘standard addition method’),
some of the pure phase a is added to the mixture containing
the unknown concentration of a. Multiple additions are
made to prepare an intensity-versus-concentration plot in
which the negative x intercept is the concentration of the
phase a in the original sample.
3950 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
ESTIMATE OF THE AMORPHOUS AND CRYSTALLINE
FRACTIONS
In a mixture of crystalline and amorphous phases, the
crystalline and amorphous fractions can be estimated in
several ways. The choice of the method used depends on the
nature of the sample :
amorphous fraction of different chemical compositions,
the amounts of each of the individual crystalline phases
may be estimated using appropriate standard substances
as described above ; the amorphous fraction is then
deduced indirectly by subtraction ;
— if the sample consists of one amorphous and one
crystalline fraction, either as a 1-phase or a 2-phase
mixture, with the same elemental composition, the
amount of the crystalline phase (‘the degree of
crystallinity’) can be estimated by measuring 3 areas of
the diffractogram :
A = total area of the peaks arising from diffraction
from the crystalline fraction of the sample ;
B = total area below area A ;
C = background area (due to air scattering,
fluorescence, equipment, etc).
When these areas have been measured, the degree of
crystallinity can be roughly estimated using the following
formula :
It is noteworthy that this method does not yield absolute
degree-of-crystallinity values and hence is generally used for
comparative purposes only.
More sophisticated methods are also available, such as the
Ruland method.
SINGLE CRYSTAL STRUCTURE
In general, the determination of crystal structures is
performed from X-ray diffraction data obtained using single
crystals. However, crystal structure analysis of organic
crystals is a challenging task, since the lattice parameters are
comparatively large, the symmetry is low and the scattering
properties are normally very low.
For any given crystalline form of a substance, knowledge
of the crystal structure allows the calculation of the
corresponding XRPD pattern, thereby providing a
‘preferred-orientation-free’ reference XRPD pattern, which
may be used for phase identification.
EUROPEAN PHARMACOPOEIA 6.3

4. REAGENTS
4.1.1. Reagents.. .......................................................................3953 4.1.3. Buffer solutions.. ..........................................................3954
4.1.2. Standard solutions for limit tests..............................3954 4.2.2. Volumetric solutions....................................................3954

General Notices (1) apply to all monographs and other texts 3951
 EUROPEAN PHARMACOPOEIA 6.3

3952 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:40101 Lithium trifluoromethanesulphonate. CF3LiO3S. (Mr 156.0).
1173400. [33454-82-9].
4.1.1. REAGENTS
Adenine. 1172800. [73-24-5].
See Adenine (0800).
Aescin. 1001700. [6805-41-0].
A mixture of related saponins obtained from the seeds of
Aesculus hippocastanum L.
A fine, almost white or slightly reddish or yellowish,
amorphous powder.
Chromatography. Examine as prescribed in the monograph
on Senega root (0202) but apply 20 μl of the solution.
After spraying with anisaldehyde solution R and heating,
the chromatogram shows a principal band with an RF of
about 0.4.
Cadmium. Cd. (Ar 112.4). 1014100. [7440-43-9].
A silvery-white, lustrous metal, practically insoluble in water,
freely soluble in nitric acid and in hot hydrochloric acid.
Chromium(III) acetylacetonate. C15H21CrO6. (Mr 349.3).
1172900. [21679-31-2]. (OC-6-11)-Tris(2,4-pentanedionato-
κO,κO′)chromium.
Coomassie staining solution R1. 1173000.
Dissolve 0.275 g of brilliant blue R in 200 ml of methanol R.
Stir until complete dissolution of the crystals (for about 2 h).
Add 750 ml of water R and 50 ml of glacial acetic acid R.
Stir overnight (for at least 16 h) ; filter.
Deuterated acetonitrile. C22H3N. (Mr 44.1). 1173100.
[2206-26-0].
The degree of deuteration is not less than 99.8 per cent.
Clear, colourless liquid, miscible with water, with acetone
and with methanol.
: about 0.78.
: about 1.344.
Endoprotease LysC. 1173200.
Microbial extracellular proteolytic enzyme secreted by
Achromobacter lyticus. A lyophilised powder, free of salts.
Formamide. CH3NO. (Mr 45.0). 1039200. [75-12-7].
A clear, colourless, oily liquid, hygroscopic, miscible with
water and with ethanol (96 per cent). It is hydrolysed by
water.
: about 1.134.
bp : about 210 °C.
Content : minimum 99.5 per cent.
Storage : in an airtight container.
Glutamyl endopeptidase for peptide mapping. 1173300.
[137010-42-5]. Endoproteinase Glu-C of high purity from
Staphylococcus aureus strain V8 (EC 3.4.21.19).
Lauryl alcohol. C12H26O. (Mr 186.3). 1119900. [112-53-8].
Dodecan-1-ol.
: about 0.820.
mp : 24 °C to 27 °C.
Content : minimum 98.0 per cent of C12H26O, determined by
gas chromatography.
General Notices (1) apply to all monographs and other texts
4.1.1. Reagents
Methylal. C3H8O2. (Mr 76.1). 1173500. [109-87-5].
Dimethoxymethane. Dioxapentane. Formaldehyde dimethyl
acetal. Methylene dimethyl ether.
Clear, colourless, volatile, flammable liquid, soluble in water
and miscible with ethanol (96 per cent).
: about 0.860.
: about 1.354.
bp : about 41 °C.
Methylal used in gas chromatography complies with the
following additional test.
Content : minimum 99.5 per cent, determined by gas
chromatography.
3-Pentanone. C5H10O. (Mr 86.13). 1173600. [96-22-0].
Diethyl ketone.
β-Pinene. C10H16. (Mr 136.2). 1109000. [127-91-3].
6,6-Dimethyl-2-methylenebicyclo[3.1.1]heptane.
A colourless, oily liquid, odour reminiscent of turpentine,
practically insoluble in water, miscible with ethanol (96 per
cent).
β-Pinene used in gas chromatography complies with the
following additional test.
Assay. Examine by gas chromatography (2.2.28) as
prescribed in the monograph Bitter-orange-flower oil (1175),
using the substance to be examined as the test solution.
Content : minimum 99.0 per cent.
Poly(cyanopropylphenyl)(14)(methyl)(86)siloxane.
1173700.
Stationary phase for chromatography.
Contains 14 per cent of cyanopropylphenyl groups and
86 per cent of methyl groups.
Schisandrin. C24H32O7. (Mr 432.5). 1173800.
[7432-28-2]. Schisandrol A. Wuweizichun A.
(6S,7S,12aRa)-5,6,7,8-Tetrahydro-1,2,3,10,11,12-
hexamethoxy-6,7-dimethyldibenzo[a,c]cyclooctan-6-ol.
White or almost white, crystalline powder.
Schisandrin used in the assay in the monograph Schisandra
fruit (2428) complies with the following additional
requirements.
Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Schisandra fruit (2428) : use the normalisation
procedure.
Content : minimum 95 per cent.
Storage : in an airtight container, at − 20 °C or below.
γ-Schisandrin. C23H28O6. (Mr 400.5). 1173900.
[61281-37-6]. Schisandrin B. Wuweizisu B.
rac-(6R,7S,13aRa)-1,2,3,13-Tetramethoxy-6,7-dimethyl-5,6,7,
8-tetrahydrobenzo[3,4]cycloocta[1,2-f][1,3]benzodioxole.
White or almost white, crystalline powder.
Storage : in an airtight container, at − 20 °C or below.
Sodium calcium edetate. 1174000. [62-33-9].
See sodium calcium edetate (0231).
3953
4.1.2. Standard solutions for limit tests
1,3,4,6-Tetra-O-acetyl-β-D-mannopyranose. C14H20O10.
(Mr 348.3). 1174100. [18968-05-3].
Colourless or white powder or crystals.
mp : 160 °C to 161 °C.
: − 68, determined on a 7 g/l solution in methylene
chloride R.
Tetrazolium salt. C20H17N5O6S2. (Mr 487.5). 1174200.
[138169-43-4]. 5-(3-Carboxymethoxyphenyl)-3-(4,5-
dimethylthiazol-2-yl)-2-(4-sulphophenyl)-2H-tetrazolium,
inner salt. MTS.
Water. 1095500.
Water, distilled, deionised. 1095508.
Deionised water R prepared by distillation with a
resistivity of not less than 0.18 Mohm·m.
01/2009:40102
4.1.2. STANDARD SOLUTIONS FOR
LIMIT TESTS
Ammonium standard solution (3 ppm NH4). 5006100.
Immediately before use, dilute with water R to 100 times
its volume a solution containing ammonium chloride R
equivalent to 0.889 g of NH4Cl in 1000.0 ml.
Magnesium standard solution (1000 ppm Mg). 5006200.
Dissolve 5.275 g of magnesium nitrate R in 16 ml of dilute
nitric acid R and dilute to 500.0 ml with water R.
3954
EUROPEAN PHARMACOPOEIA 6.3
Standardisation : carry out the determination of magnesium
by complexometry (2.5.11).
01/2009:40103
4.1.3. BUFFER SOLUTIONS
0.05 M Tris-hydrochloride buffer solution pH 9.0.
4013500.
Dissolve 0.605 g of tris(hydroxymethyl)aminomethane R in
water R. Adjust the pH (2.2.3) with 1 M hydrochloric acid
and dilute to 100.0 ml with water R.
01/2009:40202
4.2.2. VOLUMETRIC SOLUTIONS
0.1 M Lanthanum nitrate. 3010100.
Dissolve 43.30 g of lanthanum nitrate R in water R and
dilute to 1000.0 ml with the same solvent.
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.
1 ml of 0.1 M sodium edetate is equivalent to 43.30 mg of
La(NO3)3,6H2O.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3

5.1. GENERAL TEXTS ON

MICROBIOLOGY
5.1.4. Microbiological quality of non-sterile pharmaceutical 5.1.9. Guidelines for using the test for sterility.................3958
preparations and substances for pharmaceutical use.. .3957
5.1.5. Application of the F0 concept to steam sterilisation of
aqueous preparations.. .........................................................3958

General Notices (1) apply to all monographs and other texts 3955
 EUROPEAN PHARMACOPOEIA 6.3

3956 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 5.1.4. Microbiological quality of non-sterile products for pharmaceutical use
01/2009:50104 — 101 CFU : maximum acceptable count = 20 ;
5.1.4. MICROBIOLOGICAL QUALITY
OF NON-STERILE PHARMACEUTICAL
PREPARATIONS AND SUBSTANCES
FOR PHARMACEUTICAL USE
The presence of certain micro-organisms in non-sterile
preparations may have the potential to reduce or even
inactivate the therapeutic activity of the product and has
a potential to adversely affect the health of the patient.
Manufacturers therefore have to ensure a low bioburden of
finished dosage forms by implementing current guidelines
on Good Manufacturing Practice during the manufacture,
storage and distribution of pharmaceutical preparations.
Microbial examination of non-sterile products is performed
according to the methods given in general chapters
2.6.12 and 2.6.13. Acceptance criteria for non-sterile
pharmaceutical products based upon the total aerobic
microbial count (TAMC) and the total combined
yeasts/moulds count (TYMC) are given in Tables 5.1.4.-1 and
5.1.4.-2. Acceptance criteria are based on individual results
or on the average of replicate counts when replicate counts
are performed (e.g. direct plating methods).
When an acceptance criterion for microbiological quality is
prescribed it is interpreted as follows:
Table 5.1.4.-1. – Acceptance criteria for microbiological quality of non-sterile dosage forms
TAMC
Route of administration
(CFU/g or CFU/ml)
Non-aqueous preparations for oral use 103
2 1
Aqueous preparations for oral use 10
Rectal use 103
Oromucosal use
Gingival use
Cutaneous use 102
Nasal use
Auricular use
Vaginal use 102
Transdermal patches (limits for one patch
102
including adhesive layer and backing)
Inhalation use (special requirements apply to
102
liquid preparations for nebulisation)
Special Ph. Eur. provision for oral dosage
forms containing raw materials of natural
(animal, vegetal or mineral) origin for which
antimicrobial pretreatment is not feasible and 104
for which the competent authority accepts
TAMC of the raw material exceeding 103 CFU
per gram or per millilitre
Special Ph. Eur. provision for herbal medicinal
products consisting solely of one or more
herbal drugs (whole, reduced or powdered) :
— herbal medicinal products to which boiling
107
water is added before use
— herbal medicinal products to which boiling
105
water is not added before use
General Notices (1) apply to all monographs and other texts
— 102 CFU : maximum acceptable count = 200 ;
— 103 CFU : maximum acceptable count = 2000, and so forth.
Table 5.1.4.-1 includes a list of specified micro-organisms for
which acceptance criteria are set. The list is not necessarily
exhaustive and for a given preparation it may be necessary
to test for other micro-organisms depending on the nature of
the starting materials and the manufacturing process.
If it has been shown that none of the prescribed tests will
allow valid enumeration of micro-organisms at the level
prescribed, a validated method with a limit of detection as
close as possible to the indicated acceptance criterion is
used.
In addition to the micro-organisms listed in Table 5.1.4.-1,
the significance of other micro-organisms recovered is
evaluated in terms of :
— use of the product : hazard varies according to the route
of administration (eye, nose, respiratory tract) ;
— nature of the product : its ability to support growth, the
presence of adequate antimicrobial preservation ;
— method of application ;
— intended recipient : risk may differ for neonates, infants,
the debilitated ;
— use of immunosuppressive agents, corticosteroids ;
— presence of disease, wounds, organ damage.
TYMC
Specified micro-organisms
(CFU/g or CFU/ml)
102 Absence of Escherichia coli (1 g or 1 ml)
10 Absence of Escherichia coli (1 g or 1 ml)
102 -
Absence of Staphylococcus aureus (1 g or 1 ml)
101
Absence of Pseudomonas aeruginosa (1 g or 1 ml)
Absence of Pseudomonas aeruginosa (1 g or 1 ml)
101 Absence of Staphylococcus aureus (1 g or 1 ml)
Absence of Candida albicans (1 g or 1 ml)
Absence of Staphylococcus aureus (1 patch)
101
Absence of Pseudomonas aeruginosa (1 patch)
Absence of Staphylococcus aureus (1 g or 1 ml)
Absence of Pseudomonas aeruginosa (1 g or 1 ml)
101
Absence of bile-tolerant gram-negative
bacteria (1 g or 1 ml)
Not more than 102 CFU of bile-tolerant
gram-negative bacteria (1 g or 1 ml)
102 Absence of Salmonella (10 g or 10 ml)
Absence of Escherichia coli (1 g or 1 ml)
Absence of Staphylococcus aureus (1 g or 1 ml)
Not more than 102 CFU of Escherichia coli
105
(see Appendix) (1 g or 1 ml)
Not more than 103 CFU of bile-tolerant
104
gram-negative bacteria (1 g or 1 ml)
Absence of Escherichia coli (1 g or 1 ml)
Absence of Salmonella (10 g or 10 ml)
3957
5.1.5. Application of the F0 concept to steam sterilisation EUROPEAN PHARMACOPOEIA 6.3

Where warranted, a risk-based assessment of the relevant The total F0 of a process takes account of the heating up and
factors is conducted by personnel with specialised training cooling down phases of the cycle and can be calculated by
in microbiology and the interpretation of microbiological integration of lethal rates with respect to time at discrete
data. For raw materials, the assessment takes account of temperature intervals.
processing to which the product is subjected, the current When a steam sterilisation cycle is chosen on the basis
technology of testing and the availability of materials of the of the F0 concept, great care must be taken to ensure
desired quality. that an adequate assurance of sterility is consistently
Table 5.1.4.-2. – Acceptance criteria for microbiological achieved. In addition to validating the process, it may also be
quality of non-sterile substances for pharmaceutical use necessary to perform continuous, rigorous microbiological
monitoring during routine production to demonstrate that
TAMC TYMC the microbiological parameters are within the established
(CFU/g or CFU/ml) (CFU/g or CFU/ml) tolerances so as to give an SAL of 10− 6 or better.
Substances for In connection with sterilisation by steam, the Z-value
103 102 relates the heat resistance of a micro-organism to changes
pharmaceutical use
in temperature. The Z-value is the change in temperature
required to alter the D-value by a factor of 10.
Appendix : Special Ph. Eur. provision The D-value (or decimal reduction value) is the value of
for herbal medicinal products consisting a parameter of sterilisation (duration or absorbed dose)
solely of one or more herbal drugs (whole, required to reduce the number of viable organisms to 10 per
cent of the original number. It is only of significance under
reduced or powdered): quantificative test precisely defined experimental conditions.
for E. coli The following mathematical relationships apply :
Use the following protocol.
Sample preparation and pre-incubation. Prepare a sample
using a 10-fold dilution of not less than 1 g of the product D121 = D-value of the reference spores (5.1.2) at 121 °C ;
to be examined as described in general chapter 2.6.12, and NO = initial number of viable micro-organisms ;
use the quantities corresponding respectively to 0.1 g, 0.01 g
and 0.001 g (or 0.1 ml, 0.01 ml and 0.001 ml) to inoculate N = final number of viable micro-organisms ;
a suitable amount (determined as described under 3-4 of IF = inactivation factor.
general chapter 2.6.13) of casein soya bean digest broth, mix
and incubate at 30-35 °C for 18-24 h.
Selection and subculture. Shake the container, transfer 1 ml
of casein soya bean digest broth to 100 ml of MacConkey
broth and incubate at 42-44 °C for 24-48 h. Subculture on a D1 = D-value of the micro-organism at temperature T1 ;
plate of MacConkey agar at 30-35 °C for 18-72 h.
D2 = D-value of the micro-organism at temperature T2.
Interpretation. Growth of colonies indicates the possible
presence of E. coli. This is confirmed by identification tests.
Note the smallest quantity of the product that gives a positive
result and the largest quantity that gives a negative result.
Determine from the following table the probable number t = exposure time ;
of bacteria. D = D-value of micro-organism in the exposure
Results for each quantity of product Probable number of conditions.
bacteria per gram or
0.1 g or 0.01 g or 0.001 g or millilitre of product
0.1 ml 0.01 ml 0.001 ml
+ + + > 103
+ + - 01/2009:50109
< 103 and > 102
+ - - < 102 and > 10
- - - < 10
5.1.9. GUIDELINES FOR USING THE
TEST FOR STERILITY
The purpose of the test for sterility (2.6.1), as that of all
01/2009:50105 pharmacopoeial tests, is to provide an independent control
analyst with the means of verifying that a particular material
meets the requirements of the European Pharmacopoeia. A
5.1.5. APPLICATION OF THE F0 manufacturer is neither obliged to carry out such tests nor
CONCEPT TO STEAM STERILISATION precluded from using modifications of, or alternatives to, the
OF AQUEOUS PREPARATIONS stated method, provided he is satisfied that, if tested by the
official method, the material in question would comply with
The following chapter is published for information. the requirements of the European Pharmacopoeia.
The F0 value of a saturated steam sterilisation process is
the lethality expressed in terms of the equivalent time PRECAUTIONS AGAINST MICROBIAL CONTAMINATION
in minutes at a temperature of 121 °C delivered by the Aseptic conditions for performance of the test can be
process to the product in its final container with reference to achieved using, for example, a class A laminar-air-flow
micro-organisms possessing a theoretical Z-value of 10. cabinet located within a class B clean room, or an isolator

3958 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
GUIDANCE TO MANUFACTURERS
The level of assurance provided by a satisfactory result
of a test for sterility (the absence of contaminated units
in the sample) as applied to the quality of the batch is a
function of the homogeneity of the batch, the conditions
of manufacture and the efficiency of the adopted sampling
plan. Hence for the purpose of this text a batch is defined
as a homogeneous collection of sealed containers prepared
in such a manner that the risk of contamination is the same
for each of the units contained therein.
In the case of terminally sterilised products, physical
proofs, biologically based and automatically documented,
showing correct treatment throughout the batch during
sterilisation are of greater assurance than the sterility test.
The circumstances in which parametric release may be
considered appropriate are described under 5.1.1. Methods
of preparation of sterile products. The method of media-fill
runs may be used to evaluate the process of aseptic
production. Apart from that, the sterility test is the only
analytical method available for products prepared under
aseptic conditions and furthermore it is, in all cases, the only
analytical method available to the authorities who have to
examine a specimen of a product for sterility.
The probability of detecting micro-organisms by the test for
sterility increases with their number present in the sample
tested and varies according to the readiness of growth of
micro-organism present. The probability of detecting very
low levels of contamination even when it is homogenous
General Notices (1) apply to all monographs and other texts
5.1.9. Guidelines for using the test for sterility
throughout the batch is very low. The interpretation of
the results of the test for sterility rests on the assumption
that the contents of every container in the batch, had
they been tested, would have given the same result. Since
it is manifest that every container cannot be tested, an
appropriate sampling plan should be adopted. In the case of
aseptic production, it is recommended to include samples
filled at the beginning and at the end of the batch and after
significant intervention.
OBSERVATION AND INTERPRETATION OF RESULTS
Conventional microbiological/biochemical techniques are
generally satisfactory for identification of micro-organisms
recovered from a sterility test. However, if a manufacturer
wishes to use condition (d) as the sole criterion for
invalidating a sterility test, it may be necessary to
employ sensitive typing techniques to demonstrate
that a micro-organism isolated from the product test
is identical to a micro-organism isolated from the test
materials and/or the testing environment. While routine
microbiological/biochemical identification techniques can
demonstrate that 2 isolates are not identical, these methods
may not be sufficiently sensitive or reliable enough to
provide unequivocal evidence that 2 isolates are from the
same source. More sensitive tests, for example molecular
typing with RNA/DNA homology, may be necessary to
determine that micro-organisms are clonally related and have
a common origin.
3959
 EUROPEAN PHARMACOPOEIA 6.3

3960 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

5.2. GENERAL TEXTS ON

BIOLOGICAL PRODUCTS
5.2.3. Cell substrates for the production of vaccines for
human use...............................................................................3963

General Notices (1) apply to all monographs and other texts 3961
 EUROPEAN PHARMACOPOEIA 6.3

3962 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 5.2.3. Cell substrates for the production of vaccines for human use

01/2009:50203 Cells of neural origin, such as neuroblastoma and P12 cell

lines, may contain substances that concentrate agents of
spongiform encephalopathies and such cells are not used
5.2.3. CELL SUBSTRATES FOR THE for vaccine production.
PRODUCTION OF VACCINES FOR History of the cell seed. The following information is
HUMAN USE recorded : the method used to isolate the cell seed, culture
methods and any other procedures used to establish the
master cell bank, notably any that might expose the cells
This general chapter deals with diploid cell lines and to extraneous agents.
continuous cell lines used for the production of vaccines for
human use ; specific issues relating to vaccines prepared by Full information may not be available on the ingredients of
recombinant DNA technology are covered by the monograph media used in the past for cultivation of cells, for example on
on Products of recombinant DNA technology (0784). the source of substances of animal origin ; where justified
Testing to be carried out at various stages (cell seed, master and authorised, cell banks already established using such
cell bank, working cell bank, cells at or beyond the maximum media may be used for vaccine production.
population doubling level used for production) is indicated in Characterisation of the cell seed. The following properties
Table 5.2.3.-1. General provisions for the use of cell lines and are investigated :
test methods are given below. Where primary cells or cells
that have undergone a few passages without constitution of (1) the identity of the cells (for example, isoenzymes,
a cell bank are used for vaccine production, requirements are serology, nucleic acid fingerprinting) ;
given in the individual monograph for the vaccine concerned. (2) the growth characteristics of the cells and their
Diploid cell lines. A diploid cell line has a high but finite morphological properties (optical and electron microscopes) ;
capacity for multiplication in vitro. (3) for diploid cell lines, karyotype ;
Continuous cell lines. A continuous cell line has the (4) for diploid cell lines, the in vitro life span in terms of
capacity to multiply indefinitely in vitro ; the cells often have population doubling level.
differences in karyotype compared to the original cells ; they Cell substrate stability. Suitable viability of the cell line in
may be obtained from healthy or tumoral tissue. the intended storage conditions must be demonstrated. For
For injectable vaccines produced in continuous cell lines, a given product to be prepared in the cell line, it is necessary
the purification process is validated to demonstrate removal to demonstrate that consistent production can be obtained
of substrate-cell DNA to a level equivalent to not more than with cells at passage levels at the beginning and end of the
10 ng per single human dose, unless otherwise prescribed. intended span of use.
Cell-bank system. Production of vaccines in diploid or Infectious extraneous agents. Cell lines for vaccine
continuous cell lines is based on a cell-bank system. The production shall be free from infectious extraneous agents.
in vitro age of the cells is counted from the master cell Tests for extraneous agents are carried out as shown in
bank. Each working cell bank is prepared from one or more Table 5.2.3.-1.
containers of the master cell bank. The use, identity and Depending on the origin and culture history of the cell line,
inventory control of the containers is carefully documented. it may be necessary to carry out tests for selected, specific
Media and substances of animal and human origin. The potential contaminants, particularly those that are known
composition of media used for isolation and all subsequent to infect latently the species of origin, for example simian
culture is recorded in detail and if substances of human or virus 40 in rhesus monkeys. For cell lines of rodent origin,
animal origin are used they must be free from extraneous antibody-production tests are carried out in mice, rats and
agents. hamsters to detect species-specific viruses.
Cell lines are examined for the presence of retroviruses
If human albumin is used, it complies with the monograph
as described below. Cell lines that show the presence of
on Human albumin solution (0255).
retroviruses capable of replication are not acceptable for
Bovine serum used for the preparation and maintenance production of vaccines.
of cell cultures is tested and shown to be sterile and free Tumorigenicity. For the preparation of live vaccines, the
from bovine viruses, notably bovine diarrhoea virus and cell line must not be tumorigenic at any population doubling
mycoplasmas. level used for vaccine production. Where a tumorigenic cell
Trypsin used for the preparation of cell cultures is examined line is used for the production of other types of vaccine, the
by suitable methods and shown to be sterile and free purification process is validated to demonstrate that residual
from mycoplasmas and viruses, notably pestiviruses and substrate-cell DNA is reduced to a level equivalent to not
parvoviruses. more than 10 ng per single human dose of the vaccine,
unless otherwise prescribed, and that substrate-cell protein
Cell seed. The data used to assess the suitability of the is reduced to an acceptable level.
cell seed comprise information, where available, on source,
history and characterisation. A cell line which is known to have tumorigenic potential
does not have to be tested further. If a cell line is of
Source of the cell seed. For human cell lines, the following unknown tumorigenic potential, it is either regarded as
information concerning the donor is recorded : ethnic and being tumorigenic or it is tested for tumorigenicity using
geographical origin, age, sex, general physiological condition, an in vitro test as described below ; if the result of the in
tissue or organ used, results of any tests for pathogens. vitro test is negative or not clearly positive, an in vivo test
For animal cell lines, the following information is recorded as described below is carried out. The tests are carried out
concerning the source of the cells : species, strain, using cells at or beyond the maximum population doubling
breeding conditions, geographical origin, age, sex, general level that will be used for vaccine production.
physiological condition, tissue or organ used, results of any The MRC-5, WI-38 and FRhL-2 cell lines are recognised as
tests for pathogens. being non-tumorigenic and further testing is not necessary.

General Notices (1) apply to all monographs and other texts 3963
5.2.3. Cell substrates for the production of vaccines for human use EUROPEAN PHARMACOPOEIA 6.3

Table 5.2.3.-1 — Testing of cell lines

Test Cell seed Master cell bank Working cell bank Cells at or beyond the
(MCB) (WCB) maximum population
doubling level used for
production
1. IDENTITY AND PURITY
Morphology + + + +

Nucleic acid fingerprinting and a relevant selection + + + +

of the following tests : biochemical (e.g. isoenzymes),
immunological (e.g. histocompatibility), cytogenetic
markers
Karyotype (diploid cell lines) + + +(1) +(1)

Life span (diploid cell lines) − + + −

2. EXTRANEOUS AGENTS
Bacterial and fungal contamination − + + −

Mycoplasmas − + + −

Tests in cell cultures − − + −

Co-cultivation − − + (2)
+(2)

Tests in animals and eggs − − +(2) +(2)

Specific tests for possible contaminants depending − − +(2) +(2)

on the origin of the cells (see above under Infectious
extraneous agents)
Retroviruses − +(3) − +(3)

3. TUMORIGENICITY
Tumorigenicity +(5) − − +(4)

(1) The diploid character is established for each working cell bank but using cells at or beyond the maximum population doubling level used for production.
(2) Testing is carried out for each working cell bank, but using cells at or beyond the maximum population doubling level used for production.
(3) Testing is carried out for the master cell bank, but using cells at or beyond the maximum population doubling level used for production.
(4) The MRC-5, WI-38 and FRhL-2 cell lines are recognised as being non-tumorigenic and they need not be tested. Tests are not carried out on cell lines
that are known or assumed to be tumorigenic.

(5) Testing is carried out on the cell seed, but using cells at or beyond the maximum population doubling level used for production.

Chromosomal characterisation. Diploid cell lines shall be
shown to be diploid. More extensive characterisation of
a diploid cell line by karyotype analysis is required if the
removal of intact cells during processing after harvest has
not been validated. Samples from four passage levels evenly
spaced over the life-span of the cell line are examined. A
minimum of 200 cells in metaphase are examined for exact
count of chromosomes and for frequency of hyperploidy,
hypoploidy, polyploidy, breaks and structural abnormalities.
The MRC-5, the WI-38 and the FRhL-2 cell lines are
recognised as being diploid and well characterised ; where
they are not genetically modified, further characterisation
is not necessary.
TEST METHODS FOR CELL CULTURES
Identification. Nucleic acid fingerprint analysis and a
relevant selection of the following are used to establish the
identity of the cells :
(1) biochemical characteristics (isoenzyme analysis) ;
(2) immunological characteristics (histocompatibility
antigens) ;
(3) cytogenetic markers.
Contaminating cells. The nucleic acid fingerprint analysis
carried out for identification also serves to demonstrate
freedom from contaminating cells.
Bacterial and fungal contamination. The master cell
bank and each working cell bank comply with the test for
sterility (2.6.1), carried out using for each medium 10 ml of
supernatant fluid from cell cultures. Carry out the test on
1 per cent of the containers with a minimum of 2 containers.
Mycoplasmas (2.6.7). The master cell bank and each
working cell bank comply with the test for mycoplasmas by
the culture method and the indicator cell culture method.
Use one or more containers for the test.
Test for extraneous agents in cell cultures. The cells comply
with the test for haemadsorbing viruses and with the tests
in cell cultures for other extraneous agents given in chapter
2.6.16 under Production cell culture : control cells. If the
cells are of simian origin, they are also inoculated into rabbit
kidney cell cultures to test for herpesvirus B (cercopithecid
herpesvirus 1).
Co-cultivation. Co-cultivate intact and disrupted cells
separately with other cell systems including human cells
and simian cells. Carry out examinations to detect possible
morphological changes. Carry out tests on the cell culture
fluids to detect haemagglutinating viruses. The cells comply
with the test if no evidence of any extraneous agent is found.
Retroviruses. Examine for the presence of retroviruses
using :
(1) product-enhanced reverse transcriptase (PERT) assay
(2.6.21) carried out for cell bank supernatants using cells
at or beyond the maximum population doubling level that
will be used for production ;

3964 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 5.2.3. Cell substrates for the production of vaccines for human use
(2) transmission electron microscopy.
If test (1) and/or test (2) gives a positive result, test (3) is
carried out ;
(3) infectivity assays carried out on human cells with an
endpoint PERT assay on the supernatant.
Since the sensitivity of PERT assays is very high,
interpretation of a positive signal may be equivocal and a
decision on the acceptability of a cell substrate is based on
all available data.
Tests in animals. Inject intramuscularly (or, for suckling
mice, subcutaneously) into each of the following groups of
animals 107 viable cells divided equally between the animals
in each group :
(1) 2 litters of suckling mice less than 24 h old, comprising
not fewer than 10 animals ;
(2) 10 adult mice.
Inject intracerebrally into each of 10 adult mice 106
viable cells to detect the possible presence of lymphocytic
choriomeningitis virus.
Observe the animals for at least 4 weeks. Investigate animals
that become sick or show any abnormality to establish the
cause of illness. The cells comply with the test if no evidence
of any extraneous agent is found. The test is invalid if fewer
than 80 per cent of the animals in each group remain healthy
and survive to the end of the observation period.
For cells obtained from a rodent species (for example,
Chinese hamster ovary cells or baby hamster kidney cells),
tests for antibodies against likely viral contaminants of the
species in question are carried out on animals that have
received injections of the cells.
Tests in eggs. Using an inoculum of 106 viable cells per
egg, inoculate the cells into the allantoic cavity of 10 SPF
embryonated hens’ eggs (5.2.2) 9-11 days old and into the
yolk sac of 10 SPF embryonated hens’ eggs 5-6 days old.
Incubate for not less than 5 days. Test the allantoic fluids for
the presence of haemagglutinins using mammalian and avian
red blood cells ; carry out the test at 5 ± 3 °C and 20-25 °C
and read the results after 30-60 min. The cells comply with
the test if no evidence of any extraneous agent is found. The
test is invalid if fewer than 80 per cent of the embryos remain
healthy and survive to the end of the observation period.
Tests for tumorigenicity in vitro. The following test systems
may be used :
(1) colony formation in soft agar gels ;
(2) production of invasive cell growth following inoculation
into organ cultures ;
(3) study of transformation activity using, for example, the
3T3 assay system for active oncogenes.
General Notices (1) apply to all monographs and other texts
Tests for tumorigenicity in vivo. The test consists in
establishing a comparison between the continuous cell line
and a suitable positive control (for example, HeLa or Hep2
cells).
Animal systems that have been shown to be suitable for this
test include :
(1) athymic mice (Nu/Nu genotype) ;
(2) newborn mice, rats or hamsters that have been treated
with antithymocyte serum or globulin ;
(3) thymectomised and irradiated mice that have been
reconstituted (T–, B+) with bone marrow from healthy mice.
Whichever animal system is selected, the cell line and the
reference cells are injected into separate groups of 10 animals
each. In both cases, the inoculum for each animal is 107 cells
suspended in a volume of 0.2 ml, and the injection may be by
either the intramuscular or subcutaneous route. Newborn
animals are treated with 0.1 ml of antithymocyte serum or
globulin on days 0, 2, 7 and 14 after birth. A potent serum or
globulin is one that suppresses the immune mechanisms of
growing animals to the extent that the subsequent inoculum
of 107 positive reference cells regularly produces tumours
and metastases. Severely affected animals showing evident
progressively growing tumours are killed before the end of
the test to avoid unnecessary suffering.
At the end of the observation period all animals, including
the reference group(s), are killed and examined for gross
and microscopic evidence of the proliferation of inoculated
cells at the site of injection and in other organs (for example
lymph nodes, lungs, kidneys and liver).
In all test systems, the animals are observed and palpated
at regular intervals for the formation of nodules at the
sites of injection. Any nodules formed are measured
in 2 perpendicular directions, the measurements being
recorded regularly to determine whether there is progressive
growth of the nodule. Animals showing nodules which
begin to regress during the period of observation are killed
before the nodules are no longer palpable, and processed
for histological examination. Animals with progressively
growing nodules are observed for 1-2 weeks. Among those
without nodule formation, half are observed for 3 weeks
and half for 12 weeks before they are killed and processed
for histological examination. A necropsy is performed on
each animal and includes examination for gross evidence of
tumour formation at the site of injection and in other organs
such as lymph nodes, lungs, brain, spleen, kidneys and liver.
All tumour-like lesions and the site of injection are examined
histologically. In addition, since some cell lines may give rise
to metastases without evidence of local tumour growth, any
detectable regional lymph nodes and the lungs of all animals
are examined histologically.
The test is invalid if fewer than 9 of 10 animals injected
with the positive reference cells show progressively growing
tumours.
3965
 EUROPEAN PHARMACOPOEIA 6.3

3966 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

GENERAL MONOGRAPHS
Substances for pharmaceutical use.. ...................................3969 Vaccines for human use..........................................................3971

General Notices (1) apply to all monographs and other texts 3967
 EUROPEAN PHARMACOPOEIA 6.3

3968 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Substances for pharmaceutical use

01/2009:2034 — is a recombinant protein or another substance obtained

as a direct gene product based on genetic modification,
where applicable, the substance also complies with the
SUBSTANCES requirements of the general monograph Products of
FOR PHARMACEUTICAL USE recombinant DNA technology (0784) ;
— is obtained from animals susceptible to transmissible
Corpora ad usum pharmaceuticum spongiform encephalopathies other than by experimental
challenge, where applicable, the substance also complies
DEFINITION with the requirements of the general monograph Products
Substances for pharmaceutical use are any organic or with risk of transmitting agents of animal spongiform
inorganic substances that are used as active substances encephalopathies (1483) ;
or excipients for the production of medicinal products for — is a substance derived from a fermentation process,
human or veterinary use. They may be obtained from natural whether or not the micro-organisms involved are
sources or produced by extraction from raw materials, modified by traditional procedures or recombinant DNA
fermentation or synthesis. (rDNA) technology, where applicable, the substance also
complies with the requirements of the general monograph
This general monograph does not apply to herbal drugs, Products of fermentation (1468).
herbal drugs for homoeopathic preparations, herbal
drug preparations, extracts, or mother tinctures for If solvents are used during production, they are of suitable
homoeopathic preparations, which are the subject of quality. In addition, their toxicity and their residual level
separate general monographs (Herbal drugs (1433), Herbal are taken into consideration (5.4). If water is used during
drugs for homoeopathic preparations (2045), Herbal drug production, it is of suitable quality.
preparations (1434), Extracts (0765), Mother tinctures for If substances are produced or processed to yield a certain
homoeopathic preparations (2029)). It does not apply to form or grade, that specific form or grade of the substance
raw materials for homoeopathic preparations, except where complies with the requirements of the monograph. Certain
there is an individual monograph for the substance in the functionality-related tests may be described to control
non-homoeopathic part of the Pharmacopoeia. properties that may influence the suitability of the substance
Where a substance for pharmaceutical use not described and subsequently the properties of dosage forms prepared
in an individual monograph of the Pharmacopoeia is used from it.
in a medicinal product prepared for the special needs Powdered substances may be processed to obtain a certain
of individual patients, the need for compliance with the degree of fineness (2.9.35).
present general monograph is decided in the light of a risk Compacted substances are processed to increase the particle
assessment that takes account of the available quality of the size or to obtain particles of a specific form and/or to obtain
substance and its intended use. a substance with a higher bulk density.
Where medicinal products are manufactured using Coated active substances consist of particles of the active
substances for pharmaceutical use of human or animal substance coated with one or more suitable excipients.
origin, the requirements of chapter 5.1.7. Viral safety apply. Granulated active substances are particles of a specified
Substances for pharmaceutical use may be used as such or size and/or form produced from the active substance by
as starting materials for subsequent formulation to prepare granulation directly or with one or more suitable excipients.
medicinal products. Depending on the formulation, certain If substances are processed with excipients, these excipients
substances may be used either as active substances or as comply with the requirements of the relevant monograph or,
excipients. Solid substances may be compacted, coated, where no such monograph exists, the approved specification.
granulated, powdered to a certain fineness, or processed Where active substances have been processed with excipients
in other ways. A monograph is applicable to a substance to produce, for example, coated or granulated substances,
processed with an excipient only where such processing is the processing is carried out under conditions of good
mentioned in the definition section of the monograph. manufacturing practice and the processed substances are
Substance for pharmaceutical use of special grade. regarded as intermediates in the manufacture of a medicinal
Unless otherwise indicated or restricted in the individual product.
monographs, a substance for pharmaceutical use is intended
for human and veterinary use, and is of appropriate quality CHARACTERS
for the manufacture of all dosage forms in which it can be The statements under the heading Characters (e.g.
used. statements about the solubility or a decomposition point)
Polymorphism. Individual monographs do not usually are not to be interpreted in a strict sense and are not
specify crystalline or amorphous forms, unless bioavailability requirements. They are given for information.
is affected. All forms of a substance for pharmaceutical use Where a substance may show polymorphism, this may be
comply with the requirements of the monograph, unless stated under Characters in order to draw this to the attention
otherwise indicated. of the user who may have to take this characteristic into
consideration during formulation of a preparation.
PRODUCTION
IDENTIFICATION
Substances for pharmaceutical use are manufactured
by procedures that are designed to ensure a consistent Where under Identification an individual monograph
quality and comply with the requirements of the individual contains subdivisions entitled First identification and
monograph or approved specification. Second identification, the test or tests that constitute the
First identification may be used in all circumstances. The
The provisions of general chapter 5.10 apply to the control test or tests that constitute the Second identification may
of impurities in substances for pharmaceutical use. be used for identification, provided it can be demonstrated
Whether or not it is specifically stated in the individual that the substance is fully traceable to a batch certified to
monograph that the substance for pharmaceutical use : comply with all the other requirements of the monograph.

General Notices (1) apply to all monographs and other texts 3969
Substances for pharmaceutical use
TESTS
Polymorphism (5.9). If the nature of a crystalline or
amorphous form imposes restrictions on its use in
preparations, the nature of the specific crystalline or
amorphous form is identified, its morphology is adequately
controlled and its identity is stated on the label.
Related substances. Unless otherwise prescribed or justified
and authorised, organic impurities in active substances are
to be reported, identified wherever possible, and qualified
as indicated in Table 2034.-1.
Specific thresholds may be applied for impurities known
to be unusually potent or to produce toxic or unexpected
pharmacological effects.
If the individual monograph does not provide suitable
control for a new impurity, a suitable test for control must
be developed and included in the specification for the
substance.
The requirements above do not apply to biological and
biotechnological products, peptides, oligonucleotides,
radiopharmaceuticals, products of fermentation and
semi-synthetic products derived therefrom, to crude products
of animal or plant origin or herbal products.
Residual solvents are limited according to the principles
defined in chapter 5.4, using general method 2.4.24
or another suitable method. Where a quantitative
determination of a residual solvent is carried out and a test
for loss on drying is not carried out, the content of residual
solvent is taken into account for calculation of the assay
content of the substance, the specific optical rotation and
the specific absorbance.
Microbiological quality. Individual monographs give
acceptance criteria for microbiological quality wherever such
control is necessary. Table 5.1.4.-2. – Acceptance criteria
for microbiological quality of non-sterile substances for
pharmaceutical use in chapter 5.1.4. Microbiological
quality of non-sterile pharmaceutical preparations and
substances for pharmaceutical use gives recommendations
on microbiological quality that are of general relevance for
substances subject to microbial contamination. Depending
on the nature of the substance and its intended use, different
acceptance criteria may be justified.
Sterility (2.6.1). If intended for use in the manufacture
of sterile dosage forms without a further appropriate
sterilisation procedure, or if offered as sterile grade, the
substance for pharmaceutical use complies with the test for
sterility.
Bacterial endotoxins (2.6.14). If offered as bacterial
endotoxin-free grade, the substance for pharmaceutical use
complies with the test for bacterial endotoxins. The limit
and test method (if not gelation method A) are stated in the
individual monograph. The limit is calculated in accordance
with Test for bacterial endotoxins : guidelines in chapter
2.6.14. Bacterial endotoxins, unless a lower limit is justified
from results from production batches or is required by the
competent authority. Where a test for bacterial endotoxins is
prescribed, a test for pyrogens is not required.
Table 2034.-1. – Reporting, identification and qualification of organic impurities in active substances
Use Maximum daily Reporting
dose threshold
Human use or human and ≤ 2 g/day > 0.05 per cent
veterinary use
Human use or human and > 2 g/day > 0.03 per cent
veterinary use
Veterinary use only Not applicable > 0.1 per cent
3970 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Pyrogens (2.6.8). If the test for pyrogens is justified rather
than the test for bacterial endotoxins and if a pyrogen-free
grade is offered, the substance for pharmaceutical use
complies with the test for pyrogens. The limit and test
method are stated in the individual monograph or approved
by the competent authority. Based on appropriate test
validation for bacterial endotoxins and pyrogens, the test for
bacterial endotoxins may replace the test for pyrogens.
Additional properties. Control of additional properties (e.g.
physical characteristics, functionality-related characteristics)
may be necessary for individual manufacturing processes
or formulations. Grades (such as sterile, endotoxin-free,
pyrogen-free) may be produced with a view to manufacture of
preparations for parenteral administration or other dosage
forms and appropriate requirements may be specified in an
individual monograph.
ASSAY
Unless justified and authorised, contents of substances for
pharmaceutical use are determined. Suitable methods are
used.
LABELLING
In general, labelling is subject to supranational and national
regulation and to international agreements. The statements
under the heading Labelling therefore are not comprehensive
and, moreover, for the purposes of the Pharmacopoeia
only those statements that are necessary to demonstrate
compliance or non-compliance with the monograph are
mandatory. Any other labelling statements are included
as recommendations. When the term ‘label’ is used in the
Pharmacopoeia, the labelling statements may appear on the
container, the package, a leaflet accompanying the package
or a certificate of analysis accompanying the article, as
decided by the competent authority.
Where appropriate, the label states that the substance is :
— intended for a specific use ;
— of a distinct crystalline form ;
— of a specific degree of fineness ;
— compacted ;
— coated ;
— granulated ;
— sterile ;
— free from bacterial endotoxins ;
— free from pyrogens ;
— containing gliding agents.
Where applicable, the label states :
— the degree of hydration ;
— the name and concentration of any added substance (for
example, an antimicrobial preservative or an antioxidant).
Where an active substance is processed with addition of an
excipient or excipients, the label states the excipient(s) used
and the content of active substance and excipient(s).
Identification Qualification
threshold threshold
> 0.10 per cent or a daily > 0.15 per cent or a daily
intake of > 1.0 mg (whichever intake of > 1.0 mg (whichever
is the lower) is the lower)
> 0.05 per cent > 0.05 per cent
> 0.2 per cent > 0.5 per cent
EUROPEAN PHARMACOPOEIA 6.3 Vaccines for human use

01/2009:0153 intended to protect against different strains or types of

the same organism and/or against different organisms. A
VACCINES FOR HUMAN USE combined vaccine may be supplied by the manufacturer
either as a single liquid or freeze-dried preparation or as
several constituents with directions for admixture before
Vaccina ad usum humanum use. Where there is no monograph to cover a particular
combination, the vaccine complies with the monograph for
DEFINITION each individual component, with any necessary modifications
Vaccines for human use are preparations containing antigens approved by the competent authority.
capable of inducing a specific and active immunity in man Adsorbed vaccines are suspensions and may form a sediment
against an infecting agent or the toxin or antigen elaborated at the bottom of the container.
by it. Immune responses include the induction of the innate
and the adaptive (cellular, humoral) parts of the immune PRODUCTION
system. Vaccines for human use shall have been shown to General provisions. The production method for a given
have acceptable immunogenic activity and safety in man product must have been shown to yield consistently batches
with the intended vaccination schedule. comparable with the batch of proven clinical efficacy,
Vaccines for human use may contain : whole micro-organisms immunogenicity and safety in man. Product specifications
(bacteria, viruses or parasites), inactivated by chemical including in-process testing should be set. Specific
or physical means that maintain adequate immunogenic requirements for production including in-process testing are
properties ; whole live micro-organisms that are naturally included in individual monographs. Where justified and
avirulent or that have been treated to attenuate their authorised, certain tests may be omitted where it can be
virulence whilst retaining adequate immunogenic properties ; demonstrated, for example by validation studies, that the
antigens extracted from the micro-organisms or secreted by production process consistently ensures compliance with
the micro-organisms or produced by genetic engineering or the test.
chemical synthesis. The antigens may be used in their native
Unless otherwise justified and authorised, vaccines
state or may be detoxified or otherwise modified by chemical
are produced using a seed-lot system. The methods of
or physical means and may be aggregated, polymerised or
preparation are designed to maintain adequate immunogenic
conjugated to a carrier to increase their immunogenicity.
properties, to render the preparation harmless and to
Vaccines may contain an adjuvant. Where the antigen is
prevent contamination with extraneous agents.
adsorbed on a mineral adjuvant, the vaccine is referred to
as ‘adsorbed’. Where vaccines for human use are manufactured
Terminology used in monographs on vaccines for human use using materials of human or animal origin, the general
is defined in chapter 5.2.1. requirements of chapter 5.1.7. Viral safety apply in
conjunction with the more specific requirements relating to
Bacterial vaccines containing whole cells are suspensions viral safety in this monograph, in chapters 5.2.2. Chicken
of various degrees of opacity in colourless or almost flocks free from specified pathogens for the production and
colourless liquids, or may be freeze-dried. They may be quality control of vaccines, 5.2.3. Cell substrates for the
adsorbed. The concentration of living or inactivated bacteria production of vaccines for human use and 2.6.16. Tests for
is expressed in terms of International Units of opacity or, extraneous agents in viral vaccines for human use, and in
where appropriate, is determined by direct cell count or, for individual monographs.
live bacteria, by viable count.
Unless otherwise justified and authorised, in the production
Bacterial vaccines containing bacterial components are of a final lot of vaccine, the number of passages of a virus, or
suspensions or freeze-dried products. They may be adsorbed. the number of subcultures of a bacterium, from the master
The antigen content is determined by a suitable validated seed lot shall not exceed that used for production of the
assay. vaccine shown to be satisfactory in clinical trials with respect
Bacterial toxoids are prepared from toxins by diminishing to safety and efficacy or immunogenicity.
their toxicity to an acceptable level or by completely Vaccines are as far as possible free from ingredients known
eliminating it by physical or chemical procedures whilst to cause toxic, allergic or other undesirable reactions in
retaining adequate immunogenic properties. The toxins man. Suitable additives, including stabilisers and adjuvants
are obtained from selected strains of micro-organisms. The may be incorporated. Penicillin and streptomycin are neither
method of production is such that the toxoid does not revert used at any stage of production nor added to the final
to toxin. The toxoids are purified. Purification is performed product ; however, master seed lots prepared with media
before and/or after detoxification. Toxoid vaccines may be containing penicillin or streptomycin may, where justified
adsorbed. and authorised, be used for production.
Viral vaccines are prepared from viruses grown in animals,
in fertilised eggs, in suitable cell cultures or in suitable Consistency of production is an important feature of vaccine
tissues, or by culture of genetically engineered cells. They production. Monographs on vaccines for human use give
are liquids that vary in opacity according to the type of limits for various tests carried out during production and on
preparation or may be freeze-dried. They may be adsorbed. the final lot. These limits may be in the form of maximum
Liquid preparations and freeze-dried preparations after values, minimum values, or minimum and maximum
reconstitution may be coloured if a pH indicator such as tolerances around a given value. While compliance with
phenol red has been used in the culture medium. these limits is required, it is not necessarily sufficient to
ensure consistency of production for a given vaccine. For
Synthetic antigen vaccines are generally clear or colourless relevant tests, the manufacturer must therefore define for
liquids. The concentration of the components is usually each product a suitable action or release limit or limits to
expressed in terms of specific antigen content. be applied in view of the results found for batches tested
Combined vaccines are multicomponent preparations clinically and those used to demonstrate consistency of
formulated so that different antigens are administered production. These limits may subsequently be refined on a
simultaneously. The different antigenic components are statistical basis in light of production data.

General Notices (1) apply to all monographs and other texts 3971
Vaccines for human use
Substrates for propagation. Substrates for propagation
comply with the relevant requirements of the Pharmacopoeia
(5.2.2, 5.2.3) or in the absence of such requirements with
those of the competent authority. Processing of cell banks
and subsequent cell cultures is done under aseptic conditions
in an area where no other cells are being handled. Serum
and trypsin used in the preparation of cell suspensions shall
be shown to be free from extraneous agents.
Seed lots/cell banks. The master seed lot or cell bank is
identified by historical records that include information on
its origin and subsequent manipulation. Suitable measures
are taken to ensure that no extraneous agent or undesirable
substance is present in a master or working seed lot or a
cell bank.
Culture media. Culture media are as far as possible free
from ingredients known to cause toxic, allergic or other
undesirable reactions in man ; if inclusion of such ingredients
is necessary, it shall be demonstrated that the amount
present in the final lot is reduced to such a level as to render
the product safe. Approved animal (but not human) serum
may be used in the growth medium for cell cultures but
the medium used for maintaining cell growth during virus
multiplication shall not contain serum, unless otherwise
stated. Cell culture media may contain a pH indicator such
as phenol red and approved antibiotics at the lowest effective
concentration, although it is preferable to have a medium
free from antibiotics during production.
Propagation and harvest. The seed cultures are propagated
and harvested under defined conditions. The purity of
the harvest is verified by suitable tests as defined in the
monograph.
Control cells. For vaccines produced in cell cultures, control
cells are maintained and tested as prescribed. In order to
provide a valid control, these cells must be maintained in
conditions that are essentially equivalent to those used
for the production cell cultures, including use of the same
batches of media and media changes.
Control eggs. For live vaccines produced in eggs, control
eggs are incubated and tested as prescribed in the
monograph.
Purification. Where applicable, validated purification
procedures may be applied.
Inactivation. Inactivated vaccines are produced using a
validated inactivation process whose effectiveness and
consistency have been demonstrated. Where it is recognised
that extraneous agents may be present in a harvest, for
example in vaccines produced in eggs from healthy, non-SPF
flocks, the inactivation process is also validated with respect
to a panel of model extraneous agents representative of the
potential extraneous agents. A test for effectiveness of the
inactivation process is carried out as soon as possible after
the inactivation process.
Final bulk. The final bulk is prepared by aseptically blending
the ingredients of the vaccine. For non-liquid vaccines for
administration by a non-parenteral route, the final bulk is
prepared by blending the ingredients of the vaccine under
suitable conditions.
Adjuvants. One or more adjuvants may be included in the
formulation of a vaccine to potentiate and/or modulate
the immune response to the antigen(s). Adjuvants may be
included in the formulation of the final vaccine or presented
separately. Suitable characterisation and quality control
of the adjuvant(s), alone and in combination with the
antigen(s), is essential for consistent production. Quality
specifications are established for each adjuvant, alone and in
combination with the antigen(s).
3972 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Adsorbents as adjuvants. Vaccines may be adsorbed on
aluminium hydroxide, aluminium phosphate, calcium
phosphate or other suitable adsorbents. The adsorbents are
prepared in special conditions that confer the appropriate
physical form and adsorptive properties.
Where an adsorbent is used as an adjuvant and is
generated in situ during production of the vaccine, quality
specifications are established for each of the ingredients
and for the generated adsorbent in the vaccine. Quality
specifications are intended to control, in particular :
— qualitative and quantitative chemical composition ;
— physical form and associated adsorptive properties, where
relevant, and particularly where the adjuvant will be
present as an adsorbent ;
— interaction between adjuvant and antigen ;
— purity, including bacterial endotoxin content and
microbiological quality ;
— any other parameters identified as being critical for
functionality.
The stability of each adjuvant, alone and in combination
with the antigen(s), particularly for critical parameters, is
established during development studies.
Antimicrobial preservatives. Antimicrobial preservatives
are used to prevent spoilage or adverse effects caused by
microbial contamination occurring during the use of a
vaccine. Antimicrobial preservatives are not included in
freeze-dried products. For single-dose liquid preparations,
inclusion of antimicrobial preservatives is not normally
acceptable. For multidose liquid preparations, the need for
effective antimicrobial preservation is evaluated taking into
account likely contamination during use and the maximum
recommended period of use after broaching of the container.
If an antimicrobial preservative is used, it shall be shown
that it does not impair the safety or efficacy of the vaccine.
Addition of antibiotics as antimicrobial preservatives is not
normally acceptable.
During development studies, the effectiveness of the
antimicrobial preservative throughout the period of validity
shall be demonstrated to the satisfaction of the competent
authority.
The efficacy of the antimicrobial preservative is evaluated as
described in chapter 5.1.3. If neither the A criteria nor the
B criteria can be met, then in justified cases the following
criteria are applied to vaccines for human use : bacteria, no
increase at 24 h and 7 days, 3 log reduction at 14 days, no
increase at 28 days ; fungi, no increase at 14 days and 28 days.
Stability of intermediates. During production of vaccines,
intermediates are obtained at various stages and are stored,
sometimes for long periods. Such intermediates include :
— seed lots and cell banks ;
— live or inactivated harvests ;
— purified harvests that may consist of toxins or toxoids,
polysaccharides, bacterial or viral suspensions ;
— purified antigens ;
— adsorbed antigens ;
— conjugated polysaccharides ;
— final bulk vaccine ;
— vaccine in the final closed container stored at a
temperature lower than that used for final-product
stability studies and intended for release without re-assay.
Except where they are used within a short period of time,
stability studies are carried out on the intermediates in the
intended storage conditions to establish the expected extent
of degradation. For final bulk vaccine, stability studies
may be carried out on representative samples in conditions
EUROPEAN PHARMACOPOEIA 6.3
equivalent to those intended to be used for storage. For each
intermediate (except for seed lots and cell banks), a period
of validity applicable for the intended storage conditions is
established, where appropriate in light of stability studies.
Final lot. The final lot is prepared by aseptically distributing
the final bulk into sterile, tamper-proof containers, which,
after freeze-drying where applicable, are closed so as
to exclude contamination. For non-liquid vaccines for
administration by a non-parenteral route, the final lot is
prepared by distributing the final bulk under suitable
conditions into sterile, tamper-proof containers. Where
justified and authorised, certain tests prescribed for the
final lot may be carried out on the final bulk, if it has been
demonstrated that subsequent manufacturing operations do
not affect compliance.
Appearance. Unless otherwise justified and authorised,
each container (vial, syringe or ampoule) in each final lot is
inspected visually or mechanically for acceptable appearance.
Degree of adsorption. For an adsorbed vaccine, unless
otherwise justified and authorised, a release specification
for the degree of adsorption is established in light of results
found for batches used in clinical trials. From the stability
data generated for the vaccine it must be shown that at the
end of the period of validity the degree of adsorption is not
less than for batches used in clinical trials.
Stability. During development studies, maintenance of
potency of the final lot throughout the period of validity shall
be demonstrated ; the loss of potency in the recommended
storage conditions is assessed. Excessive loss even within the
limits of acceptable potency may indicate that the vaccine
is unacceptable.
Expiry date. Unless otherwise stated, the expiry date is
calculated from the beginning of the assay or from the
beginning of the first assay for a combined vaccine. For
vaccines stored at a temperature lower than that used for
stability studies and intended for release without re-assay,
the expiry date is calculated from the date of removal from
cold storage. If, for a given vaccine, an assay is not carried
out, the expiry date for the final lot is calculated from the
date of an approved stability-indicating test or, failing this,
from the date of freeze-drying or the date of filling into the
final containers. For a combined vaccine where components
are presented in separate containers, the expiry date is that
of the component which expires first.
The expiry date applies to vaccines stored in the prescribed
conditions.
Animal tests. In accordance with the provisions of the
European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific
Purposes, tests must be carried out in such a way as to use
the minimum number of animals and to cause the least pain,
suffering, distress or lasting harm. The criteria for judging
tests in monographs must be applied in light of this. For
example, if it is indicated that an animal is considered to be
positive, infected etc. when typical clinical signs or death
occur, then as soon as sufficient indication of a positive result
is obtained the animal in question shall be either euthanised
or given suitable treatment to prevent unnecessary suffering.
In accordance with the General Notices, alternative test
methods may be used to demonstrate compliance with
the monograph and the use of such tests is particularly
encouraged when this leads to replacement or reduction of
animal use or reduction of suffering.
General Notices (1) apply to all monographs and other texts
Vaccines for human use
TESTS
Vaccines comply with the tests prescribed in individual
monographs including, where applicable, the following :
pH (2.2.3). Liquid vaccines, after reconstitution where
applicable, comply with the limits for pH approved for the
particular preparation.
Adjuvant. If the vaccine contains an adjuvant, the amount
is determined and shown to be within acceptable limits
with respect to the expected amount (see also the tests for
aluminium and calcium below).
Aluminium (2.5.13) : maximum 1.25 mg of aluminium (Al)
per single human dose where an aluminium adsorbent has
been used in the vaccine, unless otherwise stated.
Calcium (2.5.14) : maximum 1.3 mg of calcium (Ca) per
single human dose where a calcium adsorbent has been used
in the vaccine, unless otherwise stated.
Free formaldehyde (2.4.18) : maximum 0.2 g/l of free
formaldehyde in the final product where formaldehyde has
been used in the preparation of the vaccine, unless otherwise
stated.
Phenol (2.5.15) : maximum 2.5 g/l in the final product where
phenol has been used in the preparation of the vaccine,
unless otherwise stated.
Water (2.5.12) : maximum 3.0 per cent m/m for freeze-dried
vaccines, unless otherwise stated.
Extractable volume (2.9.17). Unless otherwise justified and
authorised, it complies with the requirement for extractable
volume.
Bacterial endotoxins. Unless otherwise justified and
authorised, a test for bacterial endotoxins is carried out
on the final product. Where no limit is specified in the
individual monograph, the content of bacterial endotoxins
determined by a suitable method (2.6.14) is less than the
limit approved for the particular product.
STORAGE
Store protected from light. Unless otherwise stated, the
storage temperature is 5 ± 3 °C ; liquid adsorbed vaccines
must not be allowed to freeze.
LABELLING
The label states :
— the name of the preparation ;
— a reference identifying the final lot ;
— the recommended human dose and route of
administration ;
— the storage conditions ;
— the expiry date ;
— the name and amount of any antimicrobial preservative ;
— the name of any antibiotic, adjuvant, flavour or stabiliser
present in the vaccine ;
— where applicable, that the vaccine is adsorbed ;
— the name of any constituent that may cause adverse
reactions and any contra-indications to the use of the
vaccine ;
— for freeze-dried vaccines :
— the name or composition and the volume of the
reconstituting liquid to be added ;
— the time within which the vaccine is to be used after
reconstitution.
3973
 EUROPEAN PHARMACOPOEIA 6.3

3974 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

DOSAGE FORMS
Intrauterine preparations for veterinary use.. ...................3977 Semi-solid preparations for cutaneous application.. ........3979
Powders for cutaneous application......................................3978

General Notices (1) apply to all monographs and other texts 3975
 EUROPEAN PHARMACOPOEIA 6.3

3976 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2008:1806 Uniformity of content (2.9.6). Unless otherwise prescribed
corrected 6.3 or justified and authorised, solid single-dose preparations
INTRAUTERINE PREPARATIONS FOR 2tablets)
VETERINARY USE
Praeparationes intra-uterinae
ad usum veterinarium
DEFINITION
Intrauterine preparations for veterinary use are liquid,
semi-solid or solid preparations intended for the direct
administration to the uterus (cervix, cavity or fundus),
usually in order to obtain a local effect. They contain 1 or
more active substances in a suitable basis.
Where appropriate, containers for intrauterine preparations for veterinary use, for example one of the tests described in
for veterinary use 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 intrauterine preparations for veterinary
use may be distinguished :
— intrauterine tablets,
— intrauterine capsules,
— intrauterine solutions, emulsions and suspensions,
concentrates for intrauterine solutions,
— tablets for intrauterine solutions and suspensions,
— semi-solid intrauterine preparations,
— intrauterine foams,
— intrauterine sticks.
PRODUCTION
During the development of an intrauterine preparation for
veterinary use, the effectiveness of any added antimicrobial
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 under Efficacy of antimicrobial
preservation (5.1.3).
In the manufacture, packaging, storage and distribution of
intrauterine preparations for veterinary use, suitable means uterus.
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), see
Table 5.1.4.-1. – Cutaneous use.
Sterile intrauterine preparations for veterinary use are
prepared using materials and methods designed to ensure
sterility and to avoid the introduction of contaminants and
the growth of microorganisms ; recommendations on this
aspect are provided in the text on Methods of preparation of
sterile products (5.1.1).
During development, it must be demonstrated that the
nominal content can be withdrawn from the container of
liquid and semi-solid intrauterine preparations for veterinary their shape and size. Intrauterine capsules have various
use presented in single-dose containers.
TESTS
Uniformity of dosage units. Single-dose intrauterine
preparations for veterinary use 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 and pessaries (2.9.2). Examine the state of the capsules after
to the provisions of this paragraph.
General Notices (1) apply to all monographs and other texts
Intrauterine preparations for veterinary use
with a content of active substance less than 2 mg or less than
per cent of the total mass comply with test A (intrauterine
or test B (intrauterine capsules) for uniformity of
content of single-dose preparations. If the preparation has
more than 1 active substance, the requirement applies only to
those substances which correspond to the above conditions.
Uniformity of mass (2.9.5). Solid single-dose intrauterine
preparations for veterinary use comply with the test for
uniformity of mass of single-dose preparations. If the
test for uniformity of content is prescribed or justified
and authorised 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 intrauterine preparations
Dissolution test for solid dosage forms (2.9.3).
When a dissolution test is prescribed, a disintegration test
may not be required.
Sterility (2.6.1). Sterile intrauterine preparations for
veterinary use comply with the test for sterility. Applicators
supplied with the preparation also comply with the test for
sterility. Remove the applicator with aseptic precautions
from its package and transfer it to a tube of culture medium
so that it is completely immersed. Incubate and interpret the
results as described in the test for sterility.
LABELLING
The label states :
— the name of any added antimicrobial preservative,
— where applicable, that the preparation is sterile.
Intrauterine tablets
DEFINITION
Intrauterine tablets are solid preparations each containing a
single dose of 1 or more active substances. They generally
conform to the definition given in the monograph on
Tablets (0478).
A suitable applicator may be used for application into the
TESTS
Disintegration. Unless intended for prolonged local action,
they comply with the test for disintegration of suppositories
and pessaries (2.9.2). Examine the state of the tablets after
30 min, unless otherwise justified and authorised.
Intrauterine capsules
DEFINITION
Intrauterine capsules are solid, single-dose preparations.
They are generally similar to soft capsules, differing only in
shapes, usually ovoid. They are smooth and have a uniform
external appearance.
A suitable applicator may be used for application into the
uterus.
TESTS
Disintegration. Unless intended for prolonged local action,
they comply with the test for disintegration of suppositories
30 min, unless otherwise justified and authorised.
3977
Powders for cutaneous application EUROPEAN PHARMACOPOEIA 6.3

Intrauterine solutions, They are often supplied in single-dose containers. The

suspensions and emulsions container is adapted to deliver the preparation to the uterus
or it may be accompanied by a suitable applicator.
Concentrates for intrauterine solutions
DEFINITION Intrauterine foams
Intrauterine solutions, suspensions and emulsions are liquid DEFINITION
preparations. Concentrates for intrauterine solutions are
intended for administration after dilution. Intrauterine foams comply with the requirements of the
monograph on Medicated foams (1105).
They may contain excipients, for example to adjust the
viscosity of the preparation, to adjust or stabilise the pH, They are supplied in multi-dose containers. The container is
to increase the solubility of the active substance(s) or to adapted to deliver the preparation to the uterus or it may be
stabilise the preparation. The excipients do not adversely accompanied by a suitable applicator.
affect the intended medical action, or, at the concentrations
used, cause undue local irritation. Intrauterine sticks
Intrauterine emulsions may show evidence of phase
separation, but are readily redispersed on shaking. DEFINITION
Intrauterine suspensions may show a sediment that is readily Intrauterine sticks comply with the requirements of the
dispersed on shaking to give a suspension which remains monograph on Sticks (1154). They often produce a foam
sufficiently stable to enable a homogeneous preparation to when coming into contact with physiological fluids.
be delivered.
They may be supplied in single-dose containers. The
container is adapted to deliver the preparation to the uterus 01/2009:1166
or it may be accompanied by a suitable applicator.
PRODUCTION POWDERS FOR CUTANEOUS
In the manufacture of intrauterine suspensions, measures APPLICATION
are taken to ensure a suitable and controlled particle size
with regard to the intended use. Pulveres ad usum dermicum
Tablets for intrauterine solutions and Where justified and authorised, the requirements of
this monograph do not apply to powders for cutaneous
suspensions application intended for veterinary use.
DEFINITION DEFINITION
Tablets intended for the preparation of intrauterine solutions Powders for cutaneous application are preparations
and suspensions are single-dose preparations which are consisting of solid, loose, dry particles of varying degrees of
dissolved or dispersed in water at the time of administration. fineness. They contain one or more active substances, with
They may contain excipients to facilitate dissolution or or without excipients and, if necessary, colouring matter
dispersion or to prevent caking. authorised by the competent authority.
Tablets for intrauterine solutions or suspensions conform Powders for cutaneous application are presented as
with the definition given in the monograph on Tablets (0478). single-dose powders or multidose powders. They are free
After dissolution or dispersion, they comply with the from grittiness. Powders specifically intended for use on
requirements for intrauterine solutions or intrauterine large open wounds or on severely injured skin are sterile.
suspensions, as appropriate. Multidose powders for cutaneous application may be
TESTS dispensed in sifter-top containers, containers equipped with
a mechanical spraying device or in pressurised containers.
Disintegration. Tablets for intrauterine solutions or
suspensions disintegrate within 3 min when tested according Powders dispensed in pressurised containers comply
to the test for disintegration of tablets and capsules (2.9.1), with the requirements of Pressurised pharmaceutical
but using water R at 15-25 °C. preparations (0523).
Where applicable, containers for powders comply with the
LABELLING requirements of Materials used for the manufacture of
The label states : containers (3.1 and subsections) and Containers (3.2 and
subsections).
— the method of preparation of the intrauterine solution or
suspension, PRODUCTION
— the conditions and duration of storage of the solution or In the manufacture of powders for cutaneous application,
suspension after reconstitution. measures are taken to ensure a suitable particle size with
regard to the intended use.
Semi-solid intrauterine preparations In the manufacture, packaging, storage and distribution of
powders for cutaneous application, suitable means are taken
DEFINITION to ensure their microbial quality ; recommendations on this
Semi-solid preparations for intrauterine use are ointments, aspect are provided in the text Microbiological quality of
creams or gels. pharmaceutical preparations (5.1.4).
Semi-solid preparations for intrauterine use comply with the Sterile powders for cutaneous application are prepared
requirements of the monograph on Semi-solid preparations using materials and methods designed to ensure sterility
for cutaneous application (0132). and to avoid the introduction of contaminants and the

3978 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Semi-solid preparations for cutaneous application

growth of micro-organisms ; recommendations on this aspect The basis may consist of natural or synthetic substances
are provided in the text Methods of preparation of sterile and may be single phase or multiphase. According to the
products (5.1.1). nature of the basis, the preparation may have hydrophilic or
hydrophobic properties ; it may contain suitable excipients
TESTS such as antimicrobial preservatives, antioxidants, stabilisers,
Fineness. If prescribed, the fineness of a powder is emulsifiers, thickeners and penetration enhancers.
determined by the sieve test (2.9.35) or another appropriate Semi-solid preparations for cutaneous application intended
method. for use on severely injured skin are sterile.
Uniformity of dosage units. Single-dose powders for Where applicable, containers for semi-solid preparations
cutaneous application comply with the test for uniformity of for cutaneous application comply with the requirements of
dosage units (2.9.40) or, where justified and authorised, with Materials used for the manufacture of containers (3.1 and
the tests for uniformity of content and/or uniformity of mass subsections) and Containers (3.2 and subsections).
shown below. Herbal drugs and herbal drug preparations Several categories of semi-solid preparations for cutaneous
present in the dosage form are not subject to the provisions application may be distinguished :
of this paragraph.
— ointments ;
Uniformity of content (2.9.6). Unless otherwise prescribed — creams ;
or justified and authorised, single-dose powders for
cutaneous application with a content of active substance — gels ;
less than 2 mg or less than 2 per cent of the total mass — pastes ;
comply with test B for uniformity of content of single-dose — poultices ;
preparations. If the preparation has more than one active
substance, the requirement applies only to those substances — medicated plasters.
that correspond to the above conditions. According to their structure, ointments, creams and gels
Uniformity of mass (2.9.5). Single-dose powders for generally show viscoelastic behaviour and are non-Newtonian
cutaneous application comply with the test for uniformity of in character e.g. plastic, pseudoplastic or thixotropic type
mass of single-dose preparations. If the test for uniformity of flow at high shear rates. Pastes frequently exhibit dilatancy.
content is prescribed for all the active substances, the test PRODUCTION
for uniformity of mass is not required.
During development of semi-solid preparations for cutaneous
Sterility (2.6.1). Where the label indicates that the application whose formulation contains an antimicrobial
preparation is sterile, it complies with the test for sterility. preservative, the need for and the efficacy of the chosen
preservative shall be demonstrated to the satisfaction of
LABELLING the competent authority. A suitable test method together
The label states : with criteria for judging the preservative properties of
— that the preparation is for external use ; the formulation are provided in Efficacy of antimicrobial
preservation (5.1.3). In the manufacture, packaging,
— where applicable, that the preparation is sterile. storage and distribution of semi-solid preparations for
cutaneous application, suitable steps are taken to ensure
their microbiological quality ; recommendations on this are
provided in Microbiological Quality of Pharmaceutical
01/2009:0132 Preparations (5.1.4). Sterile semi-solid preparations
for cutaneous application are prepared using materials
and methods designed to ensure sterility and to avoid
SEMI-SOLID PREPARATIONS FOR the introduction of contaminants and the growth of
CUTANEOUS APPLICATION micro-organisms ; recommendations on this are provided in
Methods of Preparation of Sterile Products (5.1.1).
Praeparationes molles ad usum dermicum During development, it must be demonstrated that the
nominal content can be withdrawn from the container of
The requirements of this monograph apply to all semi-solid preparations for cutaneous application presented
semi-solid preparations for cutaneous application. Where in single-dose containers.
appropriate, additional requirements specific to semi-solid In the manufacture of semi-solid preparations for cutaneous
preparations intended to be applied to particular surfaces application, suitable measures are taken to ensure that
or mucous membranes may be found in other general the defined rheological properties are fulfilled. Where
monographs, for example Ear preparations (0652), Nasal appropriate, the following non-mandatory tests may be
preparations (0676), Rectal preparations (1145), Eye carried out : measurement of consistency by penetrometry
preparations (1163) and Vaginal preparations (1164). (2.9.9), viscosity (apparent viscosity) (2.2.10) and a suitable
test to demonstrate the appropriate release of the active
DEFINITION substance(s).
Semi-solid preparations for cutaneous application are In the manufacture of semi-solid preparations for cutaneous
intended for local or transdermal delivery of active application containing 1 or more active substances that are
substances, or for their emollient or protective action. They not dissolved in the basis (e.g. emulsions or suspensions),
are of homogeneous appearance. measures are taken to ensure appropriate homogeneity of
Semi-solid preparations for cutaneous application consist the preparation to be delivered.
of a simple or compound basis in which, usually, 1 or more In the manufacture of semi-solid preparations for cutaneous
active substances are dissolved or dispersed. According to application containing dispersed particles, measures are
its composition, the basis may influence the activity of the taken to ensure a suitable and controlled particle size with
preparation. regard to the intended use.

General Notices (1) apply to all monographs and other texts 3979
Semi-solid preparations for cutaneous application EUROPEAN PHARMACOPOEIA 6.3

TESTS Hydrophilic Creams

Uniformity of dosage units. Semi-solid preparations
supplied in single-dose containers that represent one dose
of medicinal product and are intended for transdermal
delivery of the active substance(s) in view of a systemic effect
comply with the test for uniformity of dosage units (2.9.40).
Semi-solid preparations in which the active substance(s) are
dissolved comply with the test for mass variation ; semi-solid
preparations in which the active substance(s) are suspended
comply with the test for content uniformity. Follow the
procedure described for liquid dosage forms. Herbal drugs
and herbal drug preparations present in the dosage form are
not subject to the provisions of this paragraph.
Sterility (2.6.1). Where the label indicates that the
preparation is sterile, it complies with the test for sterility.
STORAGE
If the preparation contains water or other volatile
ingredients, store in an airtight container. If the preparation
is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states :
— the name of any added antimicrobial preservative ;
— where applicable, that the preparation is sterile.
Ointments
Hydrophilic creams have as the continuous phase the
aqueous phase. They contain oil-in-water emulsifying agents
such as sodium or trolamine soaps, sulphated fatty alcohols,
polysorbates and polyoxyl fatty acid and fatty alcohol esters
combined, if necessary, with water-in-oil emulsifying agents.
Gels
DEFINITION
Gels consist of liquids gelled by means of suitable gelling
agents.
Lipophilic Gels
Lipophilic gels (oleogels) are preparations whose bases
usually consist of liquid paraffin with polyethylene or fatty
oils gelled with colloidal silica or aluminium or zinc soaps.
Hydrophilic Gels
Hydrophilic gels (hydrogels) are preparations whose bases
usually consist of water, glycerol or propylene glycol gelled
with suitable gelling agents such as poloxamers, starch,
cellulose derivatives, carbomers and magnesium-aluminium
silicates.
Pastes
DEFINITION
Pastes are semi-solid preparations for cutaneous application
containing large proportions of solids finely dispersed in
the basis.

DEFINITION Poultices
An ointment consists of a single-phase basis in which solids
DEFINITION
or liquids may be dispersed.
Poultices consist of a hydrophilic heat-retentive basis in
Hydrophobic Ointments which solid or liquid active substances are dispersed. They
Hydrophobic ointments can absorb only small amounts of are usually spread thickly on a suitable dressing and heated
water. Typical bases used for their formulation are hard, before application to the skin.
liquid and light liquid paraffins, vegetable oils, animal fats,
synthetic glycerides, waxes and liquid polyalkylsiloxanes. Medicated plasters
Water-emulsifying Ointments DEFINITION
Water-emulsifying ointments can absorb larger amounts Medicated plasters are flexible preparations containing 1 or
of water and thereby produce water-in-oil or oil-in-water more active substances. They are intended to be applied
emulsions after homogenisation, depending on the nature of to the skin. They are designed to maintain the active
the emulsifiers : water-in-oil emulsifying agents such as woolsubstance(s) in close contact with the skin such that these
alcohols, sorbitan esters, monoglycerides and fatty alcohols, may be absorbed slowly, or act as protective or keratolytic
or oil-in-water emulsifying agents such as sulphated fatty agents.
alcohols, polysorbates, macrogol cetostearyl ether or esters Medicated plasters consist of an adhesive basis, which may
of fatty acids with macrogols may be used for this purpose. be coloured, containing 1 or more active substances, spread
Their bases are those of the hydrophobic ointments. as a uniform layer on an appropriate support made of natural
Hydrophilic Ointments or synthetic material. It is not irritant or sensitising to the
Hydrophilic ointments are preparations having bases that are skin. The adhesive layer is covered by a suitable protective
miscible with water. The bases usually consist of mixtures of liner, which is removed before applying the plaster to the
liquid and solid macrogols (polyethylene glycols). They may skin. When removed, the protective liner does not detach
contain appropriate amounts of water. the preparation from the outer, supporting layer.
Medicated plasters are presented in a range of sizes directly
adapted to their intended use or as larger sheets to be cut
Creams before use. Medicated plasters adhere firmly to the skin
when gentle pressure is applied and can be peeled off without
DEFINITION causing appreciable injury to the skin or detachment of the
Creams are multiphase preparations consisting of a lipophilic preparation from the outer, supporting layer.
phase and an aqueous phase. TESTS
Lipophilic Creams Dissolution. A suitable test may be required to demonstrate
Lipophilic creams have as the continuous phase the lipophilic the appropriate release of the active substance(s), for
phase. They usually contain water-in-oil emulsifying agents example one of the tests described in Dissolution test for
such as wool alcohols, sorbitan esters and monoglycerides. transdermal patches (2.9.4).

3980 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

VACCINES FOR HUMAN USE

Diphtheria, tetanus, pertussis (acellular, component), Poliomyelitis vaccine (inactivated).. .....................................3988
poliomyelitis (inactivated) and haemophilus type b Shingles (herpes zoster) vaccine (live)................................ 3991
conjugate vaccine (adsorbed)..............................................3983 Varicella vaccine (live).............................................................3992
Haemophilus type b conjugate vaccine...............................3985

General Notices (1) apply to all monographs and other texts 3981
 EUROPEAN PHARMACOPOEIA 6.3

3982 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 DIP-TET-PERa-IPV-HIB

01/2009:2065 on the label into each of 5 healthy guinea-pigs, each weighing

250-350 g, that have not previously been treated with any
DIPHTHERIA, TETANUS, PERTUSSIS material that will interfere with the test. If within 42 days of
the injection any of the animals shows signs of or dies from
(ACELLULAR, COMPONENT), diphtheria toxaemia or tetanus, the vaccine does not comply
POLIOMYELITIS (INACTIVATED) AND with the test. If more than 1 animal dies from non-specific
causes, repeat the test once ; if more than 1 animal dies in
HAEMOPHILUS TYPE b CONJUGATE the second test, the vaccine does not comply with the test.
VACCINE (ADSORBED) Bacterial endotoxins (2.6.14). The content of bacterial
endotoxins in bulk purified diphtheria toxoid, tetanus toxoid,
Vaccinum diphtheriae, tetani, pertussis pertussis components, purified, inactivated monovalent
sine cellulis ex elementis praeparatum, poliovirus harvests and bulk PRP conjugate is determined to
monitor the purification procedure and to limit the amount
poliomyelitidis inactivatum et haemophili in the final vaccine. For each component, the content of
stirpi b coniugatum adsorbatum bacterial endotoxins is less than the limit approved by the
competent authority for the particular vaccine.
DEFINITION Development and consistency studies. During development
Diphtheria, tetanus, pertussis (acellular, component), studies and wherever revalidation is necessary, it shall be
poliomyelitis (inactivated) and haemophilus type b conjugate demonstrated by tests in animals that the vaccine induces a
vaccine (adsorbed) is a combined vaccine composed of : T-cell-dependent B-cell immune response to PRP.
diphtheria formol toxoid ; tetanus formol toxoid ; individually Where the haemophilus component is presented in a
purified antigenic components of Bordetella pertussis ; separate container, and as part of consistency studies, the
suitable strains of human poliovirus types 1, 2 and 3 grown assays of the diphtheria, tetanus, pertussis and poliomyelitis
in suitable cell cultures and inactivated by a suitable method ; components are carried out on a suitable number of batches
polyribosylribitol phosphate (PRP) covalently bound to a of vaccine reconstituted as for use. For subsequent routine
carrier protein ; a mineral adsorbent such as aluminium control, the assays of these components may be carried out
hydroxide or hydrated aluminium phosphate. The product without mixing with the haemophilus component.
is presented either as a pentavalent liquid formulation in Reference vaccine(s). Provided valid assays can be
the same container, or as a tetravalent liquid formulation performed, monocomponent reference vaccines may be used
with the freeze dried haemophilus component in a separate for the assays on the combined vaccine. If this is not possible
container, the contents of which are mixed with the other because of interaction between the components of the
components immediately before use. combined vaccine or because of the difference in composition
The formol toxoids are prepared from the toxins produced between monocomponent reference vaccine and the test
by the growth of Corynebacterium diphtheriae and vaccine, a batch of combined vaccine shown to be effective
Clostridium tetani respectively. in clinical trials or a batch representative thereof is used as
The vaccine contains either pertussis toxoid or a a reference vaccine. For the preparation of a representative
pertussis-toxin-like protein free from toxic properties batch, strict adherence to the production process used for
produced by expression of a genetically modified form of the batch tested in clinical trials is necessary. The reference
the corresponding gene. Pertussis toxoid is prepared from vaccine may be stabilised by a method that has been shown
pertussis toxin by a method that renders the toxin harmless to have no effect on the assay procedure.
while maintaining adequate immunogenic properties PRODUCTION OF THE COMPONENTS
and avoiding reversion to toxin. The acellular pertussis The production of the components complies with the
component may also contain filamentous haemagglutinin, requirements of the monographs on Diphtheria vaccine
pertactin (a 69 kDa outer-membrane protein) and other (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452),
defined components of B. pertussis such as fimbrial-2 and Pertussis vaccine (acellular, component, adsorbed) (1356),
fimbrial-3 antigens. The latter 2 antigens may be co-purified. Poliomyelitis vaccine (inactivated) (0214) and Haemophilus
The antigenic composition and characteristics are based type b conjugate vaccine (1219).
on evidence of protection and freedom from unexpected FINAL BULKS
reactions in the target group for which the vaccine is
intended. The final tetravalent bulk of the diphtheria, tetanus, pertussis
and poliomyelitis components is prepared by adsorption,
PRP is a linear copolymer composed of repeated units of separately or together, of suitable quantities of bulk purified
3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], diphtheria toxoid, bulk purified tetanus toxoid and bulk
with a defined molecular size and derived from a suitable purified acellular pertussis components onto a mineral
strain of Haemophilus influenzae type b. The carrier carrier such as aluminium hydroxide or hydrated aluminium
protein, when conjugated to PRP, is capable of inducing phosphate, and admixture of suitable quantities of purified,
a T-cell-dependent B-cell immune response to the monovalent harvests of human poliovirus types 1, 2 and 3
polysaccharide. or a suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
PRODUCTION
Where the vaccine is presented with all 5 components in the
GENERAL PROVISIONS same container, the final bulk is prepared by addition of
The production method shall have been shown to yield a suitable quantity of the haemophilus bulk conjugate to
consistently vaccines comparable with the vaccine of proven the tetravalent bulk. Where the haemophilus component is
clinical efficacy and safety in man. presented in a separate container, the final bulk is prepared
Specific toxicity of the diphtheria and tetanus components. by dilution of the bulk conjugate with suitable diluents for
The production method is validated to demonstrate that freeze drying. A stabiliser may be added.
the product, if tested, would comply with the following test : Only final bulks that comply with the following requirements
inject subcutaneously 5 times the single human dose stated may be used in the preparation of the final lot.

General Notices (1) apply to all monographs and other texts 3983
DIP-TET-PERa-IPV-HIB
Bovine serum albumin. Determined on the poliomyelitis
components by a suitable immunochemical method (2.7.1)
during preparation of the final bulk vaccine, before addition
of the adsorbent, the amount of bovine serum albumin is
such that the content in the final vaccine will be not more
than 50 ng per single human dose.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical method. The amount is not less than 85 per cent
and not greater than 115 per cent of the intended content.
Sterility (2.6.1). Carry out the test for sterility using 10 ml
for each medium.
FINAL LOT
Where the haemophilus component is presented in a
separate container, the final bulk of the haemophilus
component is freeze-dried.
Only a final lot that is satisfactory with respect to the test
for osmolality shown below and with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use.
Provided that the test for absence of residual pertussis
toxin and irreversibility of pertussis toxoid, the test for
antimicrobial preservative and the assay have been carried
out with satisfactory results on the final bulk vaccine, they
may be omitted on the final lot.
Provided that the free formaldehyde content has been
determined on the bulk purified antigens and the purified
monovalent harvests or the trivalent pool of polioviruses or
the final bulk and it has been shown that the content in the
final lot will not exceed 0.2 g/l, the test for free formaldehyde
may be omitted on the final lot.
If the in vivo assay for the poliomyelitis component is used,
provided it has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given product
and for each poliovirus type that the acceptance criteria
for the D-antigen determination are such that it yields the
same result as the in vivo assay in terms of acceptance or
rejection of a batch. This demonstration must include testing
of subpotent batches, produced experimentally if necessary,
for example by heat treatment or other means of diminishing
the immunogenic activity. Where there is a significant
change in the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35). The osmolality of the vaccine,
reconstituted where applicable, is within the limits approved
for the particular preparation.
Free PRP. Where the haemophilus component is presented
in liquid formulation, the presence of other components may
interfere in the assay and it may not be possible to separate
the PRP from the adjuvant. The presence of free PRP may
be determined on the bulk conjugate prior to the addition
of other components or on the non-adsorbed fraction in the
final combination.
Where the haemophilus component is presented in a separate
container, a number of methods have been used to separate
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation. The
free PRP can then be quantified by a range of techniques,
including high-performance anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD) and
immunoassays with anti-PRP antibodies. The amount of free
3984 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
PRP is not greater than that approved for the particular
product.
IDENTIFICATION
Identification tests A, B, C and D are carried out using
the vial containing the diphtheria, tetanus, pertussis and
poliomyelitis components ; identification test E is carried
out either on the vial containing all 5 components, or on
the vial containing the haemophilus component alone.
A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 g/l solution. Maintain at 37 °C for
about 16 h and centrifuge until a clear supernatant liquid
is obtained. The clear supernatant liquid reacts with a
suitable diphtheria antitoxin, giving a precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to
certain vaccines, is given as an example. The clear
supernatant liquid obtained during identification
test A reacts with a suitable tetanus antitoxin, giving a
precipitate.
C. The pertussis components are identified by suitable
immunochemical methods (2.7.1). The following method,
applicable to certain vaccines, is given as an example. The
clear supernatant liquid obtained during identification
test A reacts with specific antisera to the pertussis
components of the vaccine.
D. The vaccine is shown to contain human poliovirus types 1,
2 and 3 by a suitable immunochemical method (2.7.1),
such as determination of D-antigen by enzyme-linked
immunosorbent assay (ELISA).
E. The haemophilus component is identified by a suitable
immunochemical method (2.7.1) for PRP.
TESTS
Where the haemophilus component is presented in a
separate container, the tests for absence of residual
pertussis toxin and irreversibility of pertussis toxoid,
aluminium, free formaldehyde, antimicrobial preservative
and sterility are carried out on the container with
the diphtheria, tetanus, pertussis and poliomyelitis
components ; the tests for PRP, water, sterility and
pyrogens are carried out on the container with the
haemophilus component alone.
Where the haemophilus component is presented in a
separate container, some tests may be carried out on the
freeze-dried product rather than on the bulk conjugate
where the freeze-drying process may affect the component
to be tested.
Absence of residual pertussis toxin and irreversibility of
pertussis toxoid. This test is not necessary for the product
obtained by genetic modification. Use 3 groups each of not
fewer than 5 histamine-sensitive mice. Inject intraperitoneally
into the 1st group twice the single human dose of the vaccine
stored at 2-8 °C. Inject intraperitoneally into the 2nd group
twice the single human dose of the vaccine incubated at
37 °C for 4 weeks. Inject diluent intraperitoneally into
the 3rd group of mice. After 5 days, inject into each mouse
2 mg of histamine base intraperitoneally in a volume not
exceeding 0.5 ml and observe for 24 h. The test is invalid
if 1 or more control mice die following histamine challenge.
The vaccine complies with the test if no animal in the 1st or
2nd group dies following histamine challenge. If 1 mouse dies
in either or both of the 1st and 2nd groups, the test may be
repeated with the same number of mice or with a greater
number and the results of valid tests combined ; the vaccine
EUROPEAN PHARMACOPOEIA 6.3
complies with the test if, in both of the groups given the
vaccine, not more than 5 per cent of the total number of
mice die following histamine challenge.
The histamine sensitivity of the strain of mice used is
verified at suitable intervals as follows : inject intravenously
threefold dilutions of a reference pertussis toxin preparation
in phosphate-buffered saline solution containing 2 g/l of
gelatin and challenge with histamine as above ; the strain is
suitable if more than 50 per cent of the animals are sensitised
by 50 ng of pertussis toxin and none of the control animals
injected with only diluent and challenged similarly with
histamine show symptoms of sensitisation.
Pertussis toxin BRP is suitable for use as a reference
pertussis toxin.
PRP : not less than 80 per cent of the amount of PRP stated
on the label. PRP is determined either by assay of ribose
(2.5.31) or phosphorus (2.5.18), by an immunochemical
method (2.7.1) or by anion-exchange liquid chromatography
(2.2.29) with pulsed-amperometric detection.
Aluminium (2.5.13) : maximum 1.25 mg per single human
dose, if aluminium hydroxide or hydrated aluminium
phosphate is used as the adsorbent.
Free formaldehyde (2.4.18) : maximum 0.2 g/l.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical method. The content is not less than the minimum
amount shown to be effective and is not greater than 115 per
cent of the quantity stated on the label.
Water (2.5.12) : maximum 3.0 per cent for the freeze-dried
haemophilus component.
Sterility (2.6.1). It complies with the test for sterility.
Pyrogens (2.6.8). It complies with the test for pyrogens.
Inject per kilogram of the rabbit’s mass a quantity of the
vaccine equivalent to : 1 μg of PRP for a vaccine with
diphtheria toxoid or CRM 197 diphtheria protein as carrier ;
0.1 μg of PRP for a vaccine with tetanus toxoid as carrier ;
0.025 μg of PRP for a vaccine with OMP as a carrier.
ASSAY
Diphtheria component. Carry out one of the prescribed
methods for the assay of diphtheria vaccine (adsorbed)
(2.7.6).
Unless otherwise justified and authorised, the lower
confidence limit (P = 0.95) of the estimated potency is not
less than 30 IU per single human dose.
Tetanus component. Carry out one of the prescribed
methods for the assay of tetanus vaccine (adsorbed) (2.7.8).
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 40 IU per single human dose.
Pertussis component. It complies with the assay of pertussis
vaccine (acellular) (2.7.16).
Poliomyelitis component
D-antigen content. As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7.1) following desorption, using a reference
preparation calibrated in European Pharmacopoeia Units
of D-antigen. For each type, the content, expressed with
reference to the amount of D-antigen stated on the label,
is within the limits approved for the particular product.
Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
the International Unit are equivalent.
General Notices (1) apply to all monographs and other texts
Haemophilus type b conjugate vaccine
In vivo test. The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label states :
— the minimum number of International Units of diphtheria
and tetanus toxoid per single human dose ;
— the names and amounts of the pertussis components per
single human dose ;
— the nominal amount of poliovirus of each type (1, 2
and 3), expressed in European Pharmacopoeia Units of
D-antigen, per single human dose ;
— the type of cells used for production of the poliomyelitis
component ;
— the number of micrograms of PRP per single human dose ;
— the type and nominal amount of carrier protein per single
human dose ;
— where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults ;
— the name and the amount of the adsorbent ;
— that the vaccine must be shaken before use ;
— that the vaccine is not to be frozen ;
— where applicable, that the vaccine contains a
pertussis-toxin-like protein produced by genetic
modification.
01/2009:1219
HAEMOPHILUS TYPE b CONJUGATE
VACCINE
Vaccinum haemophili stirpi b coniugatum
DEFINITION
Haemophilus type b conjugate vaccine is a liquid or
freeze-dried preparation of a polysaccharide, derived
from a suitable strain of Haemophilus influenzae type b,
covalently bound to a carrier protein. The polysaccharide,
polyribosylribitol phosphate, referred to as PRP, is
a linear copolymer composed of repeated units of
3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n],
with a defined molecular size. The carrier protein, when
conjugated to PRP, is capable of inducing a T-cell-dependent
B-cell immune response to the polysaccharide.
PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
consistently haemophilus type b conjugate vaccines of
adequate safety and immunogenicity in man. The production
of PRP and of the carrier protein are based on seed-lot
systems.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
During development studies and wherever revalidation of the
manufacturing process is necessary, it shall be demonstrated
by tests in animals that the vaccine consistently induces a
T-cell-dependent B-cell immune response.
The stability of the final lot and relevant intermediates is
evaluated using one or more indicator tests. Such tests may
include determination of molecular size, determination of
free PRP in the conjugate and the immunogenicity test in
mice. Taking account of the results of the stability testing,
3985
Haemophilus type b conjugate vaccine EUROPEAN PHARMACOPOEIA 6.3

release requirements are set for these indicator tests to
ensure that the vaccine will be satisfactory at the end of the
period of validity.
BACTERIAL SEED LOTS
The seed lots of H. influenzae type b are shown to be free
from contamination by methods of suitable sensitivity. These
may include inoculation into suitable media, examination
of colony morphology, microscopic examination of
Gram-stained smears and culture agglutination with suitable
specific antisera.
No complex products of animal origin are included in the
medium used for preservation of strain viability, either for
freeze-drying or for frozen storage.
It is recommended that PRP produced by the seed lot
be characterised using nuclear magnetic resonance
spectrometry (2.2.33).
H. INFLUENZAE TYPE b POLYSACCHARIDE (PRP)
H. influenzae type b is grown in a liquid medium that
does not contain high-molecular-mass polysaccharides ;
if any ingredient of the medium contains blood-group
substances, the process shall be validated to demonstrate
that after the purification step they are no longer detectable.
The bacterial purity of the culture is verified by methods
of suitable sensitivity. These may include inoculation
into suitable media, examination of colony morphology,
microscopic examination of Gram-stained smears and culture
agglutination with suitable specific antisera. The culture
may be inactivated. PRP is separated from the culture
medium and purified by a suitable method. Volatile matter,
including water, in the purified polysaccharide is determined
by a suitable method ; the result is used to calculate the
results of certain tests with reference to the dried substance,
as prescribed below.
Only PRP that complies with the following requirements
may be used in the preparation of the conjugate.
Identification. PRP is identified by an immunochemical
method (2.7.1) or other suitable method, for example 1H
nuclear magnetic resonance spectrometry (2.2.33).
Molecular-size distribution. The percentage of PRP eluted
before a given K0 value or within a range of K0 values is
determined by size-exclusion chromatography (2.2.30) ; an
acceptable value is established for the particular product
and each batch of PRP must be shown to comply with this
limit. Limits for currently approved products, using the
indicated stationary phases, are shown for information
in Table 1219.-1. Where applicable, the molecular-size
distribution is also determined after chemical modification
of the polysaccharide.
Liquid chromatography (2.2.29) with multiple-angle laser
light-scattering detection may also be used for determination
of molecular-size distribution.
A validated determination of the degree of polymerisation or
of the weight-average molecular weight and the dispersion of
molecular masses may be used instead of the determination
of molecular size distribution.
Ribose (2.5.31) : within the limits approved by the competent
authority for the particular product, calculated with
reference to the dried substance.
Phosphorus (2.5.18) : within the limits approved by the
competent authority for the particular product, calculated
with reference to the dried substance.
Protein (2.5.16) : maximum 1.0 per cent, calculated with
reference to the dried substance. Use sufficient PRP to
allow detection of proteins at concentrations of 1 per cent
or greater.
Nucleic acid (2.5.17) : maximum 1.0 per cent, calculated with
reference to the dried substance.
Bacterial endotoxins (2.6.14) : less than 25 IU per microgram
of PRP.
Residual reagents. Where applicable, tests are carried out
to determine residues of reagents used during inactivation
and purification. An acceptable value for each reagent is
established for the particular product and each batch of PRP
must be shown to comply with this limit. Where validation
studies have demonstrated removal of a residual reagent, the
test on PRP may be omitted.

Table 1219.-1. – Product characteristics and specifications for PRP and carrier protein in currently approved products
Carrier Haemophilus Conjugation
polysaccharide
Type Purity Nominal Type of PRP Nominal Coupling method Procedure
amount per amount per
dose dose
Diphtheria toxoid > 1500 Lf per 18 μg Size-reduced PRP 25 μg cyanogen bromide activated diphtheria
milligram of K0 : 0.6-0.7, using activation of PRP toxoid (D-AH+),
nitrogen cross-linked cyanogen bromide-
agarose for activated PRP
chromatography R
Tetanus toxoid > 1500 Lf per 20 μg PRP ≥ 50 % 10 μg carbodi-imide ADH-activated
milligram of ≤ K0 : 0.30, using mediated PRP (PRP-cov.-AH)
nitrogen cross-linked + tetanus toxoid
agarose for + EDAC
chromatography R
CRM 197 > 90 % of 25 μg Size-reduced PRP 10 μg reductive amination direct coupling of
diphtheria protein diphtheria protein Dp = 15-35 or 10-35 (1-step method) or PRP to CRM 197
N-hydroxysuccinim- (cyanoborohydride
ide activation activated)
Meningococcal outer membrane 125 μg or Size-reduced PRP 7.5 μg or 15 μg thioether bond PRP activation by
group B outer protein 250 μg K0 < 0.6, using CDI PRP-IM + BuA2
membrane protein vesicles : ≤ 8 % of cross-linked + BrAc = PRP-BuA2-
(OMP) lipopolysaccharide agarose for BrAc + thioactivated
chromatography R OMP
or Mw > 50 × 103
ADH = adipic acid dihydrazide Dp = degree of polymerisation
BrAc = bromoacetyl chloride EDAC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
BuA2 = butane-1,4-diamide IM = imidazolium
CDI = carbonyldiimidazole Mw = weight-average molecular weight

3986 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
CARRIER PROTEIN
The carrier protein is chosen so that when the PRP is
conjugated it is able to induce a T-cell-dependent B-cell
immune response. Currently approved carrier proteins and
coupling methods are listed for information in Table 1219.-1.
The carrier proteins are produced by culture of suitable
micro-organisms ; the bacterial purity of the culture is
verified ; the culture may be inactivated ; the carrier protein
is purified by a suitable method.
Only a carrier protein that complies with the following
requirements may be used in the preparation of the
conjugate.
Identification. The carrier protein is identified by a suitable
immunochemical method (2.7.1).
Sterility (2.6.1). Carry out the test using for each medium
10 ml or the equivalent of 100 doses, whichever is less.
Diphtheria toxoid. Diphtheria toxoid is produced as
described in Diphtheria vaccine (adsorbed) (0443) and
complies with the requirements prescribed therein for bulk
purified toxoid.
Tetanus toxoid. Tetanus toxoid is produced as described
in Tetanus vaccine (adsorbed) (0452) and complies with
the requirements prescribed therein for bulk purified toxoid,
except that the antigenic purity is not less than 1500 Lf per
milligram of protein nitrogen.
Diphtheria protein CRM 197 : minimum 90 per cent,
determined by a suitable method. Suitable tests are carried
out, for validation or routinely, to demonstrate that the
product is non-toxic.
OMP (meningococcal group B outer membrane protein
complex). OMP complies with the following requirements
for lipopolysaccharide and pyrogens.
Lipopolysaccharide : maximum 8 per cent of
lipopolysaccharide, determined by a suitable method.
Pyrogens (2.6.8). Inject into each rabbit 0.25 μg of OMP
per kilogram of body mass.
BULK CONJUGATE
PRP is chemically modified to enable conjugation ; it is
usually partly depolymerised either before or during this
procedure. Reactive functional groups or spacers may
be introduced into the carrier protein or PRP prior to
conjugation. As a measure of consistency, the extent of
derivatisation is monitored. The conjugate is obtained by
the covalent binding of PRP and carrier protein. Where
applicable, unreacted but potentially reactogenic functional
groups are made unreactive by means of capping agents ; the
conjugate is purified to remove reagents.
Only a bulk conjugate that complies with the following
requirements may be used in the preparation of the final bulk
vaccine. For each test and for each particular product, limits
of acceptance are established and each batch of conjugate
Table 1219.-2. – Bulk conjugate requirements for currently approved products
Test
Diphtheria toxoid Tetanus toxoid
Free PRP < 37 %
Free protein <4% < 1 %, where applicable
PRP to protein ratio 1.25 - 1.8
Molecular size (Ko) :
cross-linked agarose for 95 % < 0.75
chromatography R
cross-linked agarose for 0.6 - 0.7
chromatography R1
General Notices (1) apply to all monographs and other texts
Haemophilus type b conjugate vaccine
must be shown to comply with these limits. Limits applied
to currently approved products for some of these tests are
listed for information in Table 1219.-2. For a freeze-dried
vaccine, some of the tests may be carried out on the final lot
rather than on the bulk conjugate where the freeze-drying
process may affect the component being tested.
PRP. The PRP content is determined by assay of
phosphorus (2.5.18) or by assay of ribose (2.5.31) or by an
immunochemical method (2.7.1).
Protein. The protein content is determined by a suitable
chemical method (for example, 2.5.16).
PRP to protein ratio. Determine the ratio by calculation.
Molecular-size distribution. Molecular-size distribution is
determined by size-exclusion chromatography (2.2.30).
Free PRP. A number of methods have been used to separate
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation. The
free PRP can then be quantified by a range of techniques,
including high-performance anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD) and
immunoassays with anti-PRP antibodies.
Free carrier protein. Determine the content by a suitable
method, either directly or by deriving the content by
calculation from the results of other tests. The amount is
within the limits approved for the particular product.
Unreacted functional groups. No unreacted functional
groups are detectable in the bulk conjugate unless process
validation has shown that unreacted functional groups
detectable at this stage are removed during the subsequent
manufacturing process (for example, owing to short half-life).
Residual reagents. Removal of residual reagents such as
cyanide, EDAC (ethyldimethylaminopropylcarbodi-imide)
and phenol is confirmed by suitable tests or by validation of
the process.
Sterility (2.6.1). Carry out the test using for each medium
10 ml or the equivalent of 100 doses, whichever is less.
FINAL BULK VACCINE
An adjuvant, an antimicrobial preservative and a stabiliser
may be added to the bulk conjugate before dilution to the
final concentration with a suitable diluent.
Only a final bulk vaccine that complies with the following
requirements may be used in preparation of the final lot.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical or physico-chemical method. The content is not
less than 85 per cent and not greater than 115 per cent of
the intended amount.
Sterility (2.6.1). It complies with the test for sterility, carried
out using 10 ml for each medium.
Protein carrier
CRM 197 OMP
< 20 % < 25 % < 15 %
< 1 % or < 2 %, depending not applicable
on the coupling method
0.30 - 0.55 0.3 - 0.7 0.05 - 0.1
60 % < 0.2 50 % 0.3 - 0.6 85 % < 0.3
85 % < 0.5
3987
Poliomyelitis vaccine (inactivated)
FINAL LOT
Only a final lot that is satisfactory with respect to each of
the following requirements and the requirements given
below under Identification and Tests may be released for use.
Provided the test for antimicrobial preservative has been
carried out on the final bulk vaccine, it may be omitted on
the final lot.
pH (2.2.3). The pH of the vaccine, reconstituted if necessary,
is within the range approved for the particular product.
Free PRP. A number of methods have been used to separate
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation. The
free PRP can then be quantified by a range of techniques,
including HPAEC-PAD and immunoassays with anti-PRP
antibodies. The amount of free PRP is not greater than that
approved for the particular product.
IDENTIFICATION
The vaccine is identified by a suitable immunochemical
method (2.7.1) for PRP.
TESTS
PRP : minimum 80 per cent of the amount of PRP stated
on the label. PRP is determined either by assay of ribose
(2.5.31) or phosphorus (2.5.18), by an immunochemical
method (2.7.1) or by anion-exchange liquid chromatography
with pulsed amperometric detection (2.2.29).
Aluminium (2.5.13) : maximum 1.25 mg per single human
dose, if aluminium hydroxide or hydrated aluminium
phosphate is used as the adsorbent.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical or physico-chemical method. The content is not
less than the minimum amount shown to be effective and not
greater than 115 per cent of the quantity stated on the label.
Water (2.5.12) : maximum 3.0 per cent for freeze-dried
vaccines.
Sterility (2.6.1). It complies with the test for sterility.
Pyrogens (2.6.8). It complies with the test for pyrogens.
Inject per kilogram of the rabbit’s mass a quantity of the
vaccine equivalent to : 1 μg of PRP for a vaccine with
diphtheria toxoid or CRM 197 diphtheria protein as carrier ;
0.1 μg of PRP for a vaccine with tetanus toxoid as carrier ;
0.025 μg of PRP for a vaccine with OMP as carrier.
LABELLING
The label states :
— the number of micrograms of PRP per human dose ;
— the type and nominal amount of carrier protein per single
human dose.
01/2009:0214
POLIOMYELITIS VACCINE
(INACTIVATED)
Vaccinum poliomyelitidis inactivatum
DEFINITION
Poliomyelitis vaccine (inactivated) is a liquid preparation
of suitable strains of human poliovirus types 1, 2 and 3
grown in suitable cell cultures and inactivated by a validated
method. It is a clear liquid that may be coloured owing to
the presence of a pH indicator.
3988 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
PRODUCTION
The production method shall have been shown to
yield consistently vaccines of acceptable safety and
immunogenicity in man.
Production of the vaccine is based on a virus seed-lot system.
Cell lines are used according to a cell-bank system. If
primary, secondary or tertiary monkey kidney cells are used,
production complies with the requirements indicated below.
Unless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
the master seed lot than was used to prepare the vaccine
shown in clinical studies to be satisfactory with respect to
safety and efficacy.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a human diploid cell line (5.2.3),
in a continuous cell line (5.2.3) or in primary, secondary or
tertiary monkey kidney cells.
Primary, secondary or tertiary monkey kidney cells. The
following special requirements for the substrate for virus
propagation apply to primary, secondary or tertiary monkey
kidney cells.
Monkeys used in the preparation of kidney cell cultures for
production and control of the vaccine. The animals used are
of a species approved by the competent authority, in good
health and, unless otherwise justified and authorised, have
not been previously employed for experimental purposes.
Kidney cells used for vaccine production and control are
derived from monitored, closed colonies of monkeys bred in
captivity, not from animals caught in the wild ; a previously
approved seed lot prepared using virus passaged in cells from
wild monkeys may, subject to approval by the competent
authority, be used for vaccine production if historical data
on safety justify this.
Monitored, closed colonies of monkeys. The monkeys are
kept in groups in cages. Freedom from extraneous agents is
achieved by the use of animals maintained in closed colonies
that are subject to continuous and systematic veterinary
and laboratory monitoring for the presence of infectious
agents. The supplier of animals is certified by the competent
authority. Each monkey is tested serologically at regular
intervals during a quarantine period of not less than 6 weeks
imposed before entering the colony, and then during its stay
in the colony.
The monkeys used are shown to be tuberculin-negative and
free from antibodies to simian virus 40 (SV40) and simian
immunodeficiency virus. The blood sample used in testing
for SV40 antibodies must be taken as close as possible to
the time of removal of the kidneys. If Macaca sp. monkeys
are used for production, the monkeys are also shown to
be free from antibodies to herpesvirus B (cercopithecine
herpesvirus 1) infection. Human herpesvirus 1 has been used
as an indicator for freedom from herpesvirus B antibodies
on account of the danger of handling herpesvirus B
(cercopithecine herpesvirus 1).
Monkeys from which kidneys are to be removed
are thoroughly examined, particularly for evidence
of tuberculosis and herpesvirus B (cercopithecine
herpesvirus 1) infection. If a monkey shows any pathological
lesion relevant to the use of its kidneys in the preparation
of a seed lot or vaccine, it is not to be used nor are any of
the remaining monkeys of the group concerned unless it
is evident that their use will not impair the safety of the
product.
EUROPEAN PHARMACOPOEIA 6.3
All the operations described in this section are conducted
outside the area where the vaccine is produced.
Monkey cell cultures for vaccine production. Kidneys
that show no pathological signs are used for preparing cell
cultures. Each group of cell cultures derived from a single
monkey forms a separate production cell culture giving rise
to a separate single harvest.
The primary monkey kidney cell suspension complies with
the test for mycobacteria (2.6.2) ; disrupt the cells before
carrying out the test.
If secondary or tertiary cells are used, it shall be
demonstrated by suitable validation tests that cell cultures
beyond the passage level used for production are free from
tumorigenicity.
SEED LOTS
Each of the 3 strains of poliovirus used shall be identified by
historical records that include information on the origin of
the strain and its subsequent manipulation.
Only a working seed lot that complies with the following
requirements may be used for virus propagation.
Identification. Each working seed lot is identified as human
poliovirus types 1, 2 or 3 by virus neutralisation in cell
cultures using specific antibodies.
Virus concentration. The virus concentration of each
working seed lot is determined to define the quantity of virus
to be used for inoculation of production cell cultures.
Extraneous agents. The working seed lot complies with the
requirements for seed lots for virus vaccines (2.6.16). In
addition, if primary, secondary or tertiary monkey kidney
cells have been used for isolation of the strain, measures
are taken to ensure that the strain is not contaminated
with simian viruses such as simian immunodeficiency virus,
simian virus 40, filoviruses and herpesvirus B (cercopithecine
herpesvirus 1). A working seed lot produced in primary,
secondary or tertiary monkey kidney cells complies with
the requirements given below under Virus propagation and
harvest for single harvests produced in such cells.
PROPAGATION AND HARVEST
All processing of the cell bank and cell cultures is done
under aseptic conditions in an area where no other cells or
viruses are being handled. Approved animal serum (but not
human serum) may be used in the cell culture media. Serum
and trypsin used in the preparation of cell suspensions
and media are shown to be free from extraneous agents.
The cell culture media may contain a pH indicator such as
phenol red and approved antibiotics at the lowest effective
concentration. Not less than 500 ml of the cell cultures
employed for vaccine production is set aside as uninfected
cell cultures (control cells) ; where continuous cell lines in a
fermenter are used for production, 200 × 106 cells are set
aside to prepare control cells ; where primary, secondary or
tertiary monkey kidney cells are used for production, a cell
sample equivalent to at least 500 ml of the cell suspension,
at the concentration employed for vaccine production, is
taken to prepare control cells.
Only a single harvest that complies with the following
requirements may be used in the preparation of the
vaccine. The tests for identification and bacterial and
fungal contamination may be carried out instead on the
purified, pooled monovalent harvest. After demonstration of
consistency of production at the stage of the single harvest,
the test for virus concentration may be carried out instead
on the purified, pooled monovalent harvest.
Control cells. The control cells of the production cell
culture comply with a test for identification (if a cell-bank
system is used for production) and with the requirements
General Notices (1) apply to all monographs and other texts
Poliomyelitis vaccine (inactivated)
for extraneous agents (2.6.16 ; where primary, secondary
or tertiary monkey kidney cells are used, the tests in cell
cultures are carried out as shown below under Test in rabbit
kidney cell cultures and Test in cercopithecus kidney cell
cultures).
Test in rabbit kidney cell cultures. Test a sample of at least
10 ml of the pooled supernatant fluid from the control
cultures for the absence of herpesvirus B (cercopithecine
herpesvirus 1) and other viruses by inoculation onto rabbit
kidney cell cultures. The dilution of supernatant in the
nutrient medium is not greater than 1/4 and the area of
the cell layer is at least 3 cm2 per millilitre of inoculum. Set
aside one or more containers of each batch of cells with the
same medium as non-inoculated control cells. Incubate the
cultures at 37 °C and observe for at least 2 weeks. The test is
not valid if more than 20 per cent of the control cell cultures
are discarded for non-specific, accidental reasons.
Test in cercopithecus kidney cell cultures. Test a sample
of at least 10 ml of the pooled supernatant fluid from the
control cultures for the absence of SV40 virus and other
extraneous agents by inoculation onto cell cultures prepared
from the kidneys of cercopithecus monkeys, or other cells
shown to be at least as sensitive for SV40, by the method
described under Test in rabbit kidney cell cultures. The
test is not valid if more than 20 per cent of the control cell
cultures are discarded for non-specific, accidental reasons.
Identification. The single harvest is identified as containing
human poliovirus types 1, 2 or 3 by virus neutralisation in
cell cultures using specific antibodies.
Virus concentration. The virus concentration of each single
harvest is determined by titration of infectious virus in cell
cultures.
Bacterial and fungal contamination. The single harvest
complies with the test for sterility (2.6.1), carried out using
10 ml for each medium.
Mycoplasmas (2.6.7). The single harvest complies with the
test for mycoplasmas, carried out using 10 ml.
Test in rabbit kidney cell cultures. Where primary,
secondary or tertiary monkey kidney cells are used for
production, test a sample of at least 10 ml of the single
harvest for the absence of herpesvirus B (cercopithecine
herpesvirus 1) and other viruses by inoculation onto rabbit
kidney cell cultures as described above for the control cells.
Test in cercopithecus kidney cell cultures. Where primary,
secondary or tertiary monkey kidney cells are used for
production, test a sample of at least 10 ml of the single
harvest for the absence of SV40 virus and other extraneous
agents. Neutralise the sample by a high-titre antiserum
against the specific type of poliovirus. Test the sample in
primary cercopithecus kidney cell cultures or cells that have
been demonstrated to be at least as susceptible for SV40.
Incubate the cultures at 37 °C and observe for 14 days. At
the end of this period, make at least one subculture of fluid
in the same cell culture system and observe both primary
cultures and subcultures for an additional 14 days.
PURIFICATION AND PURIFIED MONOVALENT HARVEST
Several single harvests of the same type may be pooled and
may be concentrated. The monovalent harvest or pooled
monovalent harvest is purified by validated methods. If
continuous cell lines are used for production, the purification
process shall have been shown to reduce consistently the
content of substrate-cell DNA to not more than 100 pg per
single human dose.
Only a purified monovalent harvest that complies with the
following requirements may be used for the preparation of
the inactivated monovalent harvest.
3989
Poliomyelitis vaccine (inactivated)
Identification. The virus is identified by virus neutralisation
in cell cultures using specific antibodies or by determination
of D-antigen.
Virus concentration. The virus concentration is determined
by titration of infectious virus.
Specific activity. The ratio of the virus concentration or the
D-antigen content, determined by a suitable immunochemical
method (2.7.1), to the total protein content (specific activity)
of the purified monovalent harvest is within the limits
approved for the particular product.
INACTIVATION AND INACTIVATED MONOVALENT
HARVEST
Several purified monovalent harvests of the same type may
be mixed before inactivation. To avoid failures in inactivation
caused by the presence of virus aggregates, filtration is
carried out before and during inactivation ; inactivation
is started within a suitable period, preferably not more
than 24 h and in any case not more than 72 h, of the prior
filtration. The virus suspension is inactivated by a validated
method that has been shown to inactivate poliovirus without
destruction of immunogenicity ; during validation studies,
an inactivation curve with at least 4 points (for example,
time 0 h, 24 h, 48 h and 96 h) is established showing
the decrease in concentration of live virus with time. If
formaldehyde is used for inactivation, the presence of an
excess of formaldehyde at the end of the inactivation period
is verified. The inactivation kinetics tests mentioned below
are carried out on each batch to ensure consistency of the
inactivation process.
Only an inactivated monovalent harvest that complies with
the following requirements may be used in the preparation
of a trivalent pool of inactivated monovalent harvests or a
final bulk vaccine.
Test for effective inactivation. After neutralisation of the
formaldehyde with sodium bisulphite (where applicable),
verify the absence of residual live poliovirus by inoculation
on suitable cell cultures of 2 samples of each inactivated
monovalent harvest, corresponding to at least 1500 human
doses. Cells used for the test must be of optimal sensitivity
regarding residual infectious poliovirus, for example kidney
cells from certain monkey species (Macaca, Cercopithecus
or Papio), or Hep-2 cells. If other cells are used, they must
have been shown to possess at least the same sensitivity as
those specified above. Take one sample not later than 3/4
of the way through the inactivation period and the other
at the end. Inoculate the samples in cell cultures such that
the dilution of vaccine in the nutrient medium is not greater
than 1/4 and the area of the cell layer is at least 3 cm2 per
millilitre of inoculum. Set aside one or more containers
with the same medium as non-inoculated control cells.
Observe the cell cultures for at least 3 weeks. Make not
fewer than 2 passages from each container, one at the end
of the observation period and the other 1 week before ; for
the passages, use cell culture supernatant and inoculate as
for the initial sample. Observe the subcultures for at least
2 weeks. No sign of poliovirus multiplication is present in
the cell cultures. At the end of the observation period, test
the susceptibility of the cell culture used by inoculation
of live poliovirus of the same type as that present in the
inactivated monovalent harvest.
Inactivation kinetics. Kinetics of inactivation are established
and approved by the competent authority. Adequate data
on inactivation kinetics are obtained and consistency of the
inactivation process is monitored.
Sterility (2.6.1). The inactivated monovalent harvest
complies with the test for sterility, carried out using 10 ml
for each medium.
3990 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
D-antigen content. The content of D-antigen determined
by a suitable immunochemical method (2.7.1) is within the
limits approved for the particular preparation.
FINAL BULK VACCINE
The final bulk vaccine is prepared directly from the
inactivated monovalent harvests of human poliovirus types 1,
2 and 3 or from a trivalent pool of inactivated monovalent
harvests. A suitable stabiliser and a suitable antimicrobial
preservative may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Sterility (2.6.1). The final bulk vaccine complies with the
test for sterility, carried out using 10 ml for each medium.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical or physicochemical method. The amount is not
less than 85 per cent and not greater than 115 per cent of
the intended amount.
FINAL LOT
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may
be released for use. Provided that the tests for free
formaldehyde and antimicrobial preservative and the in vivo
assay have been performed with satisfactory results on the
final bulk vaccine, they may be omitted on the final lot.
The in vivo assay may be omitted once it has been
demonstrated for a given product and for each poliovirus
type that the acceptance criteria for the D-antigen
determination are such that it yields the same result as
the in vivo assay in terms of acceptance or rejection of a
batch. This demonstration must include testing of subpotent
batches, produced experimentally if necessary, for example
by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change
in the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered.
Provided that the test for bovine serum albumin has been
performed with satisfactory results on the trivalent pool of
inactivated monovalent harvests or on the final bulk vaccine,
it may be omitted on the final lot.
IDENTIFICATION
The vaccine is shown to contain human poliovirus types 1,
2 and 3 by a suitable immunochemical method (2.7.1)
such as the determination of D-antigen by enzyme-linked
immunosorbent assay (ELISA).
TESTS
Free formaldehyde (2.4.18) : maximum 0.2 g/l.
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable
chemical or physicochemical method. The amount is not less
than the minimum amount shown to be effective and is not
greater than 115 per cent of that stated on the label.
Protein nitrogen content (2.5.33, Method 2) : maximum
10 μg per single human dose.
Bovine serum albumin : maximum 50 ng per single human
dose, determined by a suitable immunochemical method
(2.7.1).
Sterility (2.6.1). It complies with the test.
Bacterial endotoxins (2.6.14) : less than 5 IU per single
human dose.
EUROPEAN PHARMACOPOEIA 6.3
ASSAY
D-antigen content. As a measure of consistency of
production, determine the D-antigen content for human
poliovirus types 1, 2 and 3 by a suitable immunochemical
method (2.7.1) using a reference preparation calibrated
in European Pharmacopoeia Units of D-antigen. For
each type, the content, expressed with reference to the
amount of D-antigen stated on the label, is within the limits
approved for the particular product. Poliomyelitis vaccine
(inactivated) BRP is calibrated in European Pharmacopoeia
Units and intended for use in the assay of D-antigen. The
European Pharmacopoeia Unit and the International Unit
are equivalent.
In vivo test. The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2.7.20).
LABELLING
The label states :
— the types of poliovirus contained in the vaccine ;
— the nominal amount of virus of each type (1, 2 and 3),
expressed in European Pharmacopoeia Units of D-antigen,
per single human dose ;
— the cell substrate used to prepare the vaccine.
01/2009:2418 human) serum may be used in the culture media. Serum
SHINGLES (HERPES ZOSTER)
VACCINE (LIVE)
Vaccinum zonae vivum
DEFINITION
Shingles (herpes zoster) vaccine (live) is a freeze-dried
preparation of a suitable attenuated strain of human
herpesvirus 3. The vaccine is reconstituted immediately
before use, as stated on the label, to give a clear or slightly Only a virus harvest that complies with the following
opalescent liquid, almost white suspension or pale yellow
liquid that may be coloured owing to the presence of a pH
indicator. It is intended for administration to adults.
PRODUCTION
The production of vaccine is based on a virus seed-lot system in cell culture, using specific antibodies.
and a cell-bank system. The production method shall have
been shown to yield consistently live shingles vaccines of
adequate immunogenicity and safety in man. The virus
in the final vaccine shall not have been passaged in cell
cultures beyond a defined number of passages approved by
the competent authority from the original isolated virus.
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on
available epidemiological data on neurovirulence and
neurotropism, primarily for the wild-type virus. In light of
this, a risk analysis is carried out. Where necessary and if
available, a test is carried out on the vaccine strain using an
animal model that differentiates wild-type and attenuated
virus ; tests on strains of intermediate attenuation may also
be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3).
General Notices (1) apply to all monographs and other texts
Shingles (herpes zoster) vaccine (live)
VIRUS SEED LOT
The strain of human herpesvirus 3 shall be identified as
being suitable by historical records that include information
on the origin of the strain and its subsequent manipulation.
The virus shall at no time have been passaged in continuous
cell lines. Seed lots are prepared in the same kind of cells
as those used for the production of the final vaccine. Virus
seed lots are prepared in large quantities and stored at
temperatures below − 20 °C if freeze-dried, or below − 60 °C
if not freeze-dried.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification. The master and working seed lots are
identified as human herpesvirus 3 by serum neutralisation
in cell culture, using specific antibodies.
Virus concentration. The virus concentration of the master
and working seed lots is determined as prescribed under
Assay to monitor consistency of production.
Extraneous agents (2.6.16). The working seed lot complies
with the requirements for seed lots for live virus vaccines ; a
sample of 50 ml is taken for the test in cell cultures.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures
is done under aseptic conditions in an area where no other
cells or virus are being handled. Approved animal (but not
and trypsin used in the preparation of cell suspensions and
media are shown to be free from extraneous agents. The
cell culture medium may contain a pH indicator such as
phenol red and approved antibiotics at the lowest effective
concentration. It is preferable to have a substrate free from
antibiotics during production. 5 per cent, but not less than
50 ml, of the cell cultures employed for vaccine production
is set aside as uninfected cell cultures (control cells). The
infected cells constituting a single harvest are washed,
released from the support surface and pooled. The cell
suspension is disrupted by sonication.
requirements may be used in the preparation of the final
bulk vaccine.
Identification. The virus harvest contains virus that is
identified as human herpesvirus 3 by serum neutralisation
Virus concentration. The concentration of infective virus
in virus harvests is determined as prescribed under Assay
to monitor consistency of production and to determine the
dilution to be used for the final bulk vaccine.
Extraneous agents (2.6.16). Use 50 ml for the test in cell
cultures.
Control cells. The control cells of the production cell culture
from which the single harvest is derived comply with a
test for identity and with the requirements for extraneous
agents (2.6.16).
FINAL BULK VACCINE
Virus harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Bacterial and fungal contamination. Carry out the test for
sterility (2.6.1) using 10 ml for each medium.
3991
Varicella vaccine (live) EUROPEAN PHARMACOPOEIA 6.3

FINAL LOT — that contact between the vaccine and disinfectants is to

The final bulk vaccine is distributed aseptically into sterile, be avoided ;
tamper-proof containers and freeze-dried to a moisture — that the vaccine is not to be administered to pregnant
content shown to be favourable to the stability of the women.
vaccine. The containers are then closed so as to prevent
contamination and the introduction of moisture. 01/2009:0648
Only a final lot that is satisfactory with respect to the test
for water and each of the requirements given below under VARICELLA VACCINE (LIVE)
Identification, Tests and Assay may be released for use.
Provided that the test for bovine serum albumin has been Vaccinum varicellae vivum
carried out with satisfactory results on the final bulk vaccine,
it may be omitted on the final lot. DEFINITION
Water (2.5.12). Not more than the amount shown to ensure Varicella vaccine (live) is a freeze-dried preparation of a
stability of the vaccine as approved by the competent suitable attenuated strain of human herpesvirus 3. The
authority, determined by the semi-micro determination of vaccine is reconstituted immediately before use, as stated on
water. the label, to give a clear liquid that may be coloured owing
to the presence of a pH indicator.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is PRODUCTION
mixed with specific human herpesvirus 3 antibodies, it is no The production of vaccine is based on a virus seed-lot system
longer able to infect susceptible cell cultures. and a cell-bank system. The production method shall have
been shown to yield consistently live varicella vaccines of
TESTS adequate immunogenicity and safety in man. The virus
Bacterial and fungal contamination. The reconstituted in the final vaccine shall not have been passaged in cell
vaccine complies with the test for sterility (2.6.1). cultures beyond a defined number of passages approved by
the competent authority from the original isolated virus.
Bovine serum albumin : maximum 0.65 μg per human dose,
The potential neurovirulence of the vaccine strain is
determined by a suitable immunochemical method (2.7.1).
considered during preclinical development, based on
ASSAY available epidemiological data on neurovirulence and
neurotropism, primarily for the wild-type virus. In light of
Titrate the vaccine for infective virus, using at least this, a risk analysis is carried out. Where necessary and if
3 separate vials of vaccine. Titrate 1 vial of an appropriate available, a test is carried out on the vaccine strain using an
virus reference preparation in triplicate to validate each animal model that differentiates wild-type and attenuated
assay. The virus concentration of the reference preparation virus ; tests on strains of intermediate attenuation may also
is monitored using a control chart and a titre is established be needed.
on a historical basis by each laboratory. Calculate the
individual virus concentration for each vial of vaccine and The production method is validated to demonstrate that the
for each replicate of the reference preparation as well as the product, if tested, would comply with the test for abnormal
corresponding combined virus concentrations, using the toxicity for immunosera and vaccines for human use (2.6.9).
usual statistical methods (for example, 5.3). The combined SUBSTRATE FOR VIRUS PROPAGATION
estimate of the virus concentration for the 3 vials of vaccine The virus is propagated in human diploid cells (5.2.3).
is not less than that stated on the label. VIRUS SEED LOT
The assay is not valid if : The strain of human herpesvirus 3 used shall be identified as
— the confidence interval (P = 0.95) of the estimated being suitable by historical records that include information
virus concentration of the reference preparation for the on the origin of the strain and its subsequent manipulation.
3 replicates combined is greater than ± 0.3 log PFU ; The virus shall at no time have been passaged in continuous
— the virus concentration of the reference preparation cell lines. Seed lots are prepared in the same kind of cells
differs by more than 0.5 log PFU from the established as those used for the production of the final vaccine. Virus
value. seed lots are prepared in large quantities and stored at
temperatures below − 20 °C if freeze-dried, or below − 60 °C
The assay is repeated if the confidence interval (P = 0.95) if not freeze-dried.
of the combined virus concentration of the vaccine is
greater than ± 0.3 log PFU ; data obtained from valid assays Only a virus seed lot that complies with the following
only are combined by the usual statistical methods (for requirements may be used for virus propagation.
example, 5.3) to calculate the virus concentration of the Identification. The master and working seed lots are
sample. The confidence interval (P = 0.95) of the combined identified as human herpesvirus 3 by serum neutralisation
virus concentration is not greater than ± 0.3 log PFU. in cell culture, using specific antibodies.
Where justified and authorised, different assay designs may Virus concentration. The virus concentration of the master
be used ; this may imply the application of different validity and working seed lots is determined as prescribed under
and acceptance criteria. However, the vaccine must comply Assay to monitor consistency of production.
if tested as described above. Extraneous agents (2.6.16). The working seed lot complies
LABELLING with the requirements for seed lots for live virus vaccines ; a
sample of 50 ml is taken for the test in cell cultures.
The label states :
VIRUS PROPAGATION AND HARVEST
— the strain of virus used for the preparation of the vaccine ;
All processing of the cell bank and subsequent cell cultures
— the type and origin of the cells used for the preparation is done under aseptic conditions in an area where no other
of the vaccine ; cells or viruses are being handled. Approved animal (but not
— the minimum virus concentration ; human) serum may be used in the culture media. Serum

3992 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
and trypsin used in the preparation of cell suspensions and
media are shown to be free from extraneous agents. The
cell culture medium may contain a pH indicator such as
phenol red and approved antibiotics at the lowest effective
concentration. It is preferable to have a substrate free from
antibiotics during production. 5 per cent, but not less than
50 ml, of the cell cultures employed for vaccine production
is set aside as uninfected cell cultures (control cells). The
infected cells constituting a single harvest are washed,
released from the support surface and pooled. The cell
suspension is disrupted by sonication.
Only a virus harvest that complies with the following
requirements may be used in the preparation of the final
bulk vaccine.
Identification. The virus harvest contains virus that is
identified as human herpesvirus 3 by serum neutralisation
in cell culture, using specific antibodies.
Virus concentration. The concentration of infective virus
in virus harvests is determined as prescribed under Assay
to monitor consistency of production and to determine the
dilution to be used for the final bulk vaccine.
Extraneous agents (2.6.16). Use 50 ml for the test in cell
cultures.
Control cells. The control cells of the production cell culture The assay is not valid if :
from which the single harvest is derived comply with a
test for identity and with the requirements for extraneous
agents (2.6.16).
FINAL BULK VACCINE
Virus harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot. greater than ± 0.3 log PFU ; data obtained from valid assays
Bacterial and fungal contamination. Carry out the test for example, 5.3) to calculate the virus concentration of the
sterility (2.6.1) using 10 ml for each medium.
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile, Where justified and authorised, different assay designs may
tamper-proof containers and freeze-dried to a moisture
content shown to be favourable to the stability of the
vaccine. The containers are then closed so as to prevent
contamination and the introduction of moisture.
Only a final lot that is satisfactory with respect to the test
for water and each of the requirements given below under
Identification, Tests and Assay may be released for use.
Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the final bulk vaccine,
it may be omitted on the final lot.
Water (2.5.12). Not more than the amount shown to ensure — that contact between the vaccine and disinfectants is to
stability of the vaccines as approved by the competent
authority, determined by the semi-micro determination of
water.
General Notices (1) apply to all monographs and other texts
Varicella vaccine (live)
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific human herpesvirus 3 antibodies, it is no
longer able to infect susceptible cell cultures.
TESTS
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).
Bovine serum albumin : maximum 0.5 μg per human dose,
determined by a suitable immunochemical method (2.7.1).
ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine. Titrate 1 vial of an appropriate
virus reference preparation in triplicate to validate each
assay. The virus concentration of the reference preparation
is monitored using a control chart and a titre is established
on a historical basis by each laboratory. Calculate the
individual virus concentration for each vial of vaccine and
for each replicate of the reference preparation as well as the
corresponding combined virus concentrations, using the
usual statistical methods (for example, 5.3). The combined
estimate of the virus concentration for the 3 vials of vaccine
is not less than that stated on the label.
— the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than ± 0.3 log PFU ;
— the virus concentration of the reference preparation
differs by more than 0.5 log PFU from the established
value.
The assay is repeated if the confidence interval (P = 0.95)
of the combined virus concentration of the vaccine is
only are combined by the usual statistical methods (for
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than ± 0.3 log PFU.
be used ; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply
if tested as described above.
LABELLING
The label states :
— the strain of virus used for the preparation of the vaccine ;
— the type and origin of the cells used for the preparation
of the vaccine ;
— the minimum virus concentration ;
be avoided ;
— that the vaccine is not to be administered to pregnant
women.
3993
 EUROPEAN PHARMACOPOEIA 6.3

3994 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

VACCINES FOR
VETERINARY USE
Clostridium chauvoei vaccine for veterinary use..............3997

General Notices (1) apply to all monographs and other texts 3995
 EUROPEAN PHARMACOPOEIA 6.3

3996 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Clostridium chauvoei vaccine for veterinary use

01/2008:0361 3-2. Bacteria and fungi. The vaccine and, where applicable,
corrected 6.3 the liquid supplied with it comply with the test for sterility
prescribed in the monograph on Vaccines for veterinary
use (0062).
CLOSTRIDIUM CHAUVOEI VACCINE 3-3. Safety. Use 2 animals of one of the species for which the
FOR VETERINARY USE vaccine is intended and that have not been vaccinated against
C. chauvoei. Administer to each animal at a single site, by
a recommended route, twice the maximum recommended
dose. Observe the animals at least daily for 7 days.
Vaccinum Clostridii chauvoei The vaccine complies with the test if no animal shows
ad usum veterinarium notable signs of disease or dies from causes attributable to
the vaccine.
1. DEFINITION 3-4. Potency
Use for the test not fewer than 10 healthy guinea-pigs, each
Clostridium chauvoei vaccine for veterinary use is prepared weighing 350-450 g. Administer to each animal by the
from liquid cultures of one or more suitable strains of subcutaneous route a quantity of the vaccine not greater
Clostridium chauvoei. The whole culture is inactivated than the minimum dose stated on the label as the first dose.
to eliminate its toxicity while maintaining adequate After 28 days, administer into the same animals a quantity
immunogenic properties. This monograph applies to of the vaccine not greater than the minimum dose stated
vaccines intended for active immunisation of animals against on the label as the second dose. 14 days after the second
disease caused by C. chauvoei. vaccination, inoculate by the intramuscular route into each
of the vaccinated guinea-pigs and into each of 5 control
animals a suitable quantity of a virulent culture, or of a spore
2. PRODUCTION suspension, of C. chauvoei, activated if necessary with an
2-1. PREPARATION OF THE VACCINE activating agent such as calcium chloride.
C. chauvoei used for production is grown in an appropriate The vaccine complies with the test if not more than 10 per
liquid medium. Inactivated cultures may be treated with a cent of the vaccinated guinea-pigs die from C. chauvoei
suitable adjuvant. infection within 5 days and all the control animals die from
C. chauvoei infection within 48 h of challenge or within
2-2. CHOICE OF VACCINE COMPOSITION 72 h if a spore suspension was used for the challenge. If
The vaccine is shown to be satisfactory with respect to safety more than 10 per cent but not more than 20 per cent of
(5.2.6) and efficacy (5.2.7) for the animals for which it is the vaccinated animals die, repeat the test. The vaccine
intended. complies with the test if not more than 10 per cent of the
second group of vaccinated animals die within 5 days and
all of the second group of control animals die within 48 h
3. BATCH TESTS of challenge or within 72 h if a spore suspension was used
3-1. Identification. The vaccine protects susceptible animals for the challenge. To avoid unnecessary suffering following
against infection with C. chauvoei. The potency test may virulent challenge, moribund animals are euthanised and are
also serve for identification. then considered to have died from C. chauvoei infection.

General Notices (1) apply to all monographs and other texts 3997
 EUROPEAN PHARMACOPOEIA 6.3

3998 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
RADIOPHARMACEUTICAL
PREPARATIONS
Pentetate sodium calcium for radiopharmaceutical
preparations............................................................................ 4001
Technetium (99mTc) colloidal rhenium sulphide injection
.. .................................................................................................4002
Technetium (99mTc) macrosalb injection..............................4003
General Notices (1) apply to all monographs and other texts
Technetium (99mTc) mebrofenin injection.. .........................4004
Technetium (99mTc) microspheres injection........................4005
Technetium (99mTc) tin pyrophosphate injection...............4006
Tetra-O-acetyl-mannose triflate for radiopharmaceutical
preparations............................................................................4008
3999
 EUROPEAN PHARMACOPOEIA 6.3

4000 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Pentetate sodium calcium for radiopharmaceutical preparations

01/2009:2353 add 1 ml of reference solution (a) and dilute to 100.0 ml with

the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml
PENTETATE SODIUM CALCIUM with the solvent mixture.
Column :
FOR RADIOPHARMACEUTICAL
— size: l = 0.10 m, Ø = 4.6 mm ;
PREPARATIONS — stationary phase : spherical graphitised carbon for
chromatography R1 (5 μm) with a specific surface area of
Natrii calcii pentetas ad radiopharmaceutica 120 m2/g and a pore size of 25 nm.
Mobile phase : dissolve 50 mg of ferric sulphate
pentahydrate R in 50 ml of 0.5 M sulphuric acid and add
750 ml of water R ; adjust to pH 1.5 with 0.5 M sulphuric
acid or 1 M sodium hydroxide, add 20 ml of ethylene
glycol R and dilute to 1000 ml with water R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 273 nm.
Injection : 20 μl of the test solution and reference
solution (b) ; filter the solutions and inject immediately.
Run time : 4 times the retention time of the iron complex
of impurity A.
Retention time : iron complex of impurity A = about 5 min ;
C14H18CaN3Na3O10,xH2O Mr 497.4 (anhydrous substance) iron complex of edetic acid = about 10 min ; the iron complex
of pentetic acid elutes with the void volume.
DEFINITION
System suitability : reference solution (b) :
Trisodium [1,1′,1″,1′′′-[[(carboxylatomethyl)imino]bis(ethyle-
nenitrilo)]tetraacetato]calciate(3-). — resolution : minimum 7 between the peaks due to the iron
complex of impurity A and the iron complex of edetic acid ;
It is a starting material for the preparation of technetium
99m
( Tc) pentetate injection. — signal-to-noise ratio : minimum 50 for the peak due to
the iron complex of impurity A.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance). Limit :
— impurity A : not more than the area of the corresponding
CHARACTERS peak in the chromatogram obtained with reference
Appearance : white or almost white, hygroscopic powder solution (b) (0.1 per cent).
or crystals. Impurity B : maximum 1.0 per cent.
Solubility : freely soluble in water, practically insoluble in Dissolve 5.0 g of the substance to be examined in 250 ml of
ethanol (96 per cent). water R. Add 10 ml of ammonium chloride buffer solution
IDENTIFICATION pH 10.0 R and 50 mg of mordant black 11 triturate R. Not
more than 1.3 ml of 0.1 M magnesium chloride is required
A. Infrared absorption spectrophotometry (2.2.24). to change the colour of the indicator to violet.
Comparison : pentetate sodium calcium CRS. Chlorides : maximum 0.1 per cent.
B. Ignite. The residue gives reaction (b) of calcium (2.3.1). Dissolve 0.7 g in water R and dilute to 20 ml with the same
C. The substance to be examined gives reaction (a) of sodium solvent. Add 30 ml of dilute nitric acid R, allow to stand
(2.3.1). for 30 min and filter. Dilute 10 ml of the filtrate to 50 ml
with water R. Use this solution as the test solution. Prepare
TESTS the reference solution using 0.40 ml of 0.01 M hydrochloric
Solution S. Dissolve 5.0 g in carbon dioxide-free water R acid, add 6 ml of dilute nitric acid R and dilute to 50 ml with
and dilute to 25.0 ml with the same solvent. water R. Filter both solutions if necessary. Add 1 ml of silver
Appearance of solution. Solution S is clear (2.2.1) and nitrate solution R2 to the test solution and the reference
colourless (2.2.2, Method II). solution. Mix and allow to stand for 5 min protected from
light. Any opalescence in the test solution is not more
pH (2.2.3) : 8.0 to 9.5 for solution S. intense than that in the reference solution.
Impurity A. Liquid chromatography (2.2.29). Carry out the Iron (2.4.9) : maximum 20 ppm.
test protected from light.
Dilute 2.5 ml of solution S to 10 ml with water R. Add 0.25 g
Solvent mixture. Dissolve 10 g of ferric sulphate of calcium chloride R to the test solution and the standard
pentahydrate R in 20 ml of 0.5 M sulphuric acid and add before the addition of the thioglycollic acid R.
780 ml of water R. Adjust to pH 2.0 with 1 M sodium
hydroxide and dilute to 1000 ml with water R. Heavy metals (2.4.8) : maximum 20 ppm.
Test solution. Dissolve 0.100 g of the substance to be 1.0 g complies with test F. For the digestion replace sulphuric
examined in the solvent mixture and dilute to 25.0 ml with acid R by nitric acid R. Prepare the reference solution using
the solvent mixture. 2 ml of lead standard solution (10 ppm Pb) R.
Reference solution (a). Dissolve 0.100 g of sodium calcium Water (2.5.12) : maximum 15.0 per cent, determined on
edetate R in the solvent mixture and dilute to 25.0 ml with 0.100 g.
the solvent mixture. Bacterial endotoxins (2.6.14) : less than 0.1 IU/mg,
Reference solution (b). Dissolve 40.0 mg of nitrilotriacetic if intended for use in the manufacture of parenteral
acid R (impurity A) in the solvent mixture and dilute to preparations without a further appropriate procedure for the
100.0 ml with the solvent mixture. To 10.0 ml of the solution removal of bacterial endotoxins.

General Notices (1) apply to all monographs and other texts 4001
Technetium (99mTc) colloidal rhenium sulphide injection
ASSAY
Dissolve 0.100 g in water R and dilute to 50.0 ml with
the same solvent. To 25.0 ml of this solution add 80 ml
of water R and adjust to pH 2.3 with dilute nitric acid R.
Titrate with 0.01 M bismuth nitrate using 0.1 ml of a 1 g/l
solution of xylenol orange R as indicator. The colour of the
solution changes from yellow to red.
1 ml of 0.01 M bismuth nitrate is equivalent to 4.974 mg
of C14H18CaN3Na3O10.
STORAGE
In an airtight container, protected from light.
LABELLING
The label recommends testing the substance in a production
test before its use for the manufacture of radiopharmaceutical B. Examine the chromatogram obtained in the test for
preparations. This ensures that, under specified production
conditions, the substance yields the radiopharmaceutical
preparation in the desired quantity and of the quality
specified.
IMPURITIES
Specified impurities : A, B.
A. nitrilotriacetic acid,
B. [[(carboxymethyl)imino]bis(ethylenenitrilo)]tetraacetic
acid (pentetic acid).
01/2009:0126 absorbances obtained with the reference solutions, draw a
TECHNETIUM ( Tc) COLLOIDAL99m
RHENIUM SULPHIDE INJECTION
Rhenii sulfidi colloidalis et technetii (99mTc) injection, remove the liver, spleen and lungs and measure
solutio iniectabilis
DEFINITION
99m
Technetium ( Tc) colloidal rhenium sulphide injection
is a sterile colloidal dispersion of rhenium sulphide the
micelles of which are labelled with technetium-99m. It
is stabilised with gelatin. The injection contains not less
than 90.0 per cent and not more than 110.0 per cent of
the declared technetium-99m radioactivity at the date and
hour stated on the label. Not less than 92 per cent of the
radioactivity corresponds to technetium-99m in colloidal
form. The pH of the injection may be adjusted by the
addition of a suitable buffer such as a citrate buffer solution. In each of the 3 mice at least 80 per cent of the radioactivity
The injection contains a variable concentration of colloidal
rhenium sulphide, not exceeding 0.22 mg of rhenium (Re)
per millilitre, according to the method of preparation.
It is prepared from sodium pertechnetate (99mTc) injection
(fission or non-fission) using suitable sterile ingredients
and calculating the ratio of radionuclidic impurities with
reference to the date and hour of administration.
4002 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
CHARACTERS
A light-brown liquid.
Technetium-99m has a half-life of 6.02 h and emits gamma
radiation.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrument. The spectrum does not differ significantly
from that of a standardised technetium-99m solution
either by direct comparison or by using an instrument
calibrated with the aid of such a solution. Standardised
technetium-99m and molybdenum-99 solutions are
available from laboratories recognised by the competent
authority. The most prominent gamma photon of
technetium-99m has an energy of 0.140 MeV.
radiochemical purity. The distribution of radioactivity
contributes to the identification of the injection.
C. To 1 ml add 5 ml of hydrochloric acid R, 5 ml of a 50 g/l
solution of thiourea R and 1 ml of a 200 g/l solution of
stannous chloride R in hydrochloric acid R. A yellow
colour is produced.
TESTS
pH (2.2.3). The pH of the injection is 4.0 to 7.0.
Rhenium
Test solution. Use 1 ml of the injection to be examined.
Reference solutions. Using a solution containing 100 μg of
potassium perrhenate R (equivalent to 60 ppm of Re) and
240 μg of sodium thiosulphate R per millilitre, prepare a
range of solutions and dilute to the same final volume with
water R.
To the test solution and to 1 ml of each of the reference
solutions add 5 ml of hydrochloric acid R, 5 ml of a 50 g/l
solution of thiourea R and 1 ml of a 200 g/l solution of
stannous chloride R in hydrochloric acid R and dilute
to 25.0 ml with water R. Allow to stand for 40 min and
measure the absorbance (2.2.25) of each solution at 400 nm,
using a reagent blank as the compensation liquid. Using the
calibration curve and calculate the concentration of rhenium
in the injection to be examined.
Physiological distribution. Inject a volume not greater
than 0.2 ml into a caudal vein of each of three mice each
weighing 20 g to 25 g. Euthanise the mice 20 min after the
the radioactivity in the organs using a suitable instrument.
Measure the radioactivity in the rest of the body after having
removed the tail. Determine the percentage of radioactivity
in the liver, the spleen and the lungs from the following
expression :
A = radioactivity of the organ concerned,
B = total radioactivity in the liver, the spleen, the
lungs and the rest of the body.
is found in the liver and spleen and not more than 5 per cent
in the lungs. If the distribution of radioactivity in 1 of the
3 mice does not correspond to the prescribed proportions,
repeat the test on a further three mice. The preparation
complies with the test if the prescribed distribution of
radioactivity is found in 5 of the 6 mice used. The injection
may be released for use before completion of the test.
EUROPEAN PHARMACOPOEIA 6.3
Sterility. It complies with the test for sterility
prescribed in the monograph on Radiopharmaceutical
preparations (0125). The injection may be released for use
before completion of the test.
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml,
V being the maximum recommended dose in millilitres.
RADIOCHEMICAL PURITY
Examine by ascending paper chromatography (2.2.26).
Apply to the paper 10 μl of the injection. Develop
immediately over a path of 10 cm to 15 cm using a 9 g/l
solution of sodium chloride R. Allow the paper to dry.
Determine the distribution of radioactivity using a suitable
detector. Technetium-99m in colloidal form remains at the
starting-point and pertechnetate ion migrates with an RF of
about 0.6. There may be other impurities with an RF of 0.8
to 0.9. The radioactivity corresponding to technetium-99m
in colloidal form represents not less than 92 per cent of the
total radioactivity of the chromatogram.
RADIOACTIVITY
Measure the radioactivity using suitable counting equipment
by comparison with a standardised technetium-99m solution
or by measurement in an instrument calibrated with the aid
of such a solution.
LABELLING
The label states, in particular, the concentration of rhenium Non-filterable radioactivity. Use a polycarbonate membrane
expressed in milligrams per millilitre.
01/2009:0296 a suitable holder. Place 0.2 ml of the injection on the
TECHNETIUM ( Tc) MACROSALB99m
INJECTION
Technetii (99mTc) macrosalbi
suspensio iniectabilis
DEFINITION
Technetium (99mTc) macrosalb injection is a sterile
suspension of human albumin in the form of irregular
insoluble aggregates obtained by denaturing human
albumin in aqueous solution ; the particles are labelled
with technetium-99m. The injection contains reducing
substances, such as tin salts in a concentration not
exceeding 3 mg of Sn per millilitre ; it may contain a suitable maximum dimension greater than 150 μm is present.
buffer such as acetate, citrate or phosphate buffer and
also non-denatured human albumin and an antimicrobial
preservative such as benzyl alcohol. The human albumin
employed complies with the requirements prescribed in
the monograph on Human albumin solution (0255). The
injection contains not less than 90.0 per cent and not
more than 110.0 per cent of the declared technetium-99m
radioactivity at the date and hour stated on the label. Not
less than 90 per cent of the technetium-99m radioactivity is liquid. Resuspend the residue in 5.0 ml of sodium carbonate
bound to the particles of the suspension as determined by
the test for non-filterable radioactivity. The particles have a dissolve the aggregated albumin. Allow to cool, transfer
typical diameter between 10 μm and 100 μm. The specific
radioactivity is not less than 37 MBq of technetium-99m
per milligram of aggregated albumin at the date and hour
of administration.
99m
It is prepared from sodium pertechnetate ( Tc) injection
(fission or non-fission) using suitable sterile ingredients
and calculating the ratio of radionuclidic impurities with
reference to the date and hour of administration.
CHARACTERS
A white suspension which may separate on standing.
General Notices (1) apply to all monographs and other texts
Technetium (99mTc) macrosalb injection
Technetium-99m has a half-life of 6.02 h and emits gamma
radiation.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrument. The spectrum does not differ significantly
from that of a standardised technetium-99m solution
either by direct comparison or by using an instrument
calibrated with the aid of such a solution. Standardised
technetium-99m and molybdenum-99 solutions are
available from laboratories recognised by the competent
authority. The most prominent gamma photon of
technetium-99m has an energy of 0.140 MeV.
B. The tests for non-filterable radioactivity and particle size
contribute to the identification of the preparation.
C. Transfer 1 ml of the injection to a centrifuge tube
and centrifuge at 2500 g for 5 min to 10 min. Decant
the supernatant liquid. To the residue add 5 ml of
cupri-tartaric solution R2, mix and allow to stand for
10 min. If necessary, heat to dissolve the particles
and allow to cool. Add rapidly 0.5 ml of dilute
phosphomolybdotungstic reagent R, mixing immediately.
A blue colour develops.
TESTS
pH (2.2.3). The pH of the injection is 3.8 to 7.5.
filter 13 mm to 25 mm in diameter, 10 μm thick and with
circular pores 3 μm in diameter. Fit the membrane into
membrane and filter, adding 20 ml of a 9 g/l solution of
sodium chloride R during the filtration. The radioactivity
remaining on the membrane represents not less than 90 per
cent of the total radioactivity of the injection.
Particle size. Examine using a microscope. Dilute the
injection if necessary so that the number of particles is just
low enough for individual particles to be distinguished.
Using a syringe fitted with a needle having a calibre not less
than 0.35 mm, place a suitable volume in a suitable counting
chamber such as a haemocytometer cell, taking care not
to overfill the chamber. Allow the suspension to settle for
1 min and, carefully add a cover slide without squeezing
the sample. Scan an area corresponding to at least 5000
particles. Not more than 10 particles have a maximum
dimension greater than 100 μm. No particle having a
Aggregated albumin
Test solution. Transfer a volume of the injection expected to
contain about 1 mg of aggregated albumin to a centrifuge
tube and centrifuge at about 2500 g for 5 min to 10 min.
Decant the supernatant liquid. Resuspend the residue in
2.0 ml of a 9 g/l solution of sodium chloride R. Centrifuge
at 2500 g for 5 min to 10 min. Decant the supernatant
solution R1. Heat in a water-bath at 80 °C to 90 °C to
to a volumetric flask and dilute to 10.0 ml with sodium
carbonate solution R1.
Reference solutions. Prepare a range of solutions containing
0.05 mg to 0.2 mg of human albumin per millilitre in sodium
carbonate solution R1.
Introduce 3.0 ml of each solution separately into 25 ml flasks.
To each flask add 15.0 ml of cupri-tartaric solution R2,
mix and allow to stand for 10 min. Add rapidly 1.5 ml
of dilute phosphomolybdotungstic reagent R and mix
immediately. Allow to stand for 30 min and measure the
absorbance (2.2.25) of each solution at 750 nm using sodium
4003
Technetium (99mTc) mebrofenin injection EUROPEAN PHARMACOPOEIA 6.3

carbonate solution R1 as the compensation liquid. Using 01/2009:2393

the absorbances obtained with the reference solutions, draw
a calibration curve and calculate the content of aggregated
albumin in the injection.
TECHNETIUM (99mTc) MEBROFENIN
Tin
INJECTION
Test solution. To 1.0 ml of the injection, add 1.0 ml of 2 M Technetii (99mTc) mebrofenini solutio
hydrochloric acid. Heat in a water-bath for 30 min. Cool
and centrifuge for 10 min at 300 g. Dilute 1.0 ml of the iniectabilis
supernatant liquid to 25.0 ml with 1 M hydrochloric acid.
Reference solution. Dissolve 0.115 g of stannous chloride R
in 1 M hydrochloric acid and dilute to 1000.0 ml with the
same acid.
To 1.0 ml of each solution, add 0.4 ml of a 20 g/l solution of
sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml
of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid.
Mix. Measure the absorbance (2.2.25) of each solution at
540 nm, using 0.2 M hydrochloric acid as the compensation DEFINITION
liquid. The absorbance of the test solution is not greater Sterile solution of a complex of technetium-99m with
than that of the reference solution (3 mg of Sn per millilitre). mebrofenin. It may contain stabilisers and inert additives.
Physiological distribution. Inject a volume not greater than Content : 90 per cent to 110 per cent of the declared
0.2 ml into a caudal vein of each of three rats weighing 150 g technetium-99m radioactivity at the date and time stated on
to 250 g. Euthanise the rats 15 min after the injection, the label.
remove the liver, the spleen and the lungs and measure
the radioactivity in the organs using a suitable instrument. PRODUCTION
Measure the radioactivity in the rest of the body, including It is prepared by dissolving [[[(3-bromo-2,4,6-
the blood, after having removed the tail. Determine the trimethylphenyl)carbamoyl]methyl]imino]diacetic
percentage of radioactivity in the lungs, the liver and the acid (mebrofenin) in the presence of a reducing agent such
spleen from the following expression : as a stannous salt in Sodium pertechnetate (99mTc) injection
(fission) (0124) or Sodium pertechnetate (99mTc) injection
(non-fission) (0283).
CHARACTERS
A = radioactivity of the organ concerned, Appearance : clear, colourless solution.
B = total radioactivity in the liver, the spleen, the Half-life and nature of radiation of technetium-99m : see
lungs and the rest of the body. general chapter 5.7. Table of physical characteristics of
radionuclides.
In not fewer than 2 of the 3 rats used, at least 80 per cent of
the radioactivity is found in the lungs and not more than a IDENTIFICATION
total of 5 per cent in the liver and spleen. The injection may A. Gamma-ray spectrometry.
be released for use before completion of the test. Results : the most prominent gamma photon of
Sterility. It complies with the test for sterility technetium-99m has an energy of 0.141 MeV.
prescribed in the monograph on Radiopharmaceutical B. Examine the chromatogram obtained in the test for other
preparations (0125). The injection may be released for use radiochemical impurities (see Tests).
before completion of the test.
Results : the principal peak in the chromatogram obtained
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, with the test solution is similar in retention time to
V being the maximum recommended dose in millilitres. the peak due to technetium-99m mebrofenin in the
chromatogram obtained with the reference solution.
TESTS
RADIOACTIVITY
pH (2.2.3) : 4.0 to 7.5.
Measure the radioactivity using suitable counting equipment
by comparison with a standardised technetium-99m solution Sterility. It complies with the test for sterility prescribed
or by measurement in an instrument calibrated with the aid in the monograph on Radiopharmaceutical preparations
of such a solution. (0125). The injection may be released for use before
completion of the test.
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml,
LABELLING V being the maximum recommended dose in millilitres.
The label states : RADIOCHEMICAL PURITY
— the concentration of tin expressed in milligrams per Impurity A. Thin-layer chromatography (2.2.27).
millilitre, if any, Test solution. The preparation to be examined.
— that the preparation is to be shaken before use, Reference solution (a). To 1 ml of a 1 g/l solution of
stannous chloride R in 0.05 M hydrochloric acid in a closed
— that the preparation is not to be used if after shaking, the vial, add 2 ml of sodium pertechnetate (99mTc) injection
suspension does not appear homogeneous. (fission or non-fission). Use within 30 min after preparation.

4004 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Reference solution (b). Dissolve 40 mg of mebrofenin CRS
in 2 ml of water R and adjust to pH 6.5 with a 40 g/l
solution of sodium hydroxide R. To this solution add
25 μl of a 20 mg/ml solution of stannous chloride R
in 0.05 M hydrochloric acid and 400 MBq of sodium
pertechnetate (99Tc) injection (fission or non-fission) in a
volume of 2 ml. Allow to stand for 15 min.
Plate : TLC silica gel plate R; use a glass-fibre plate.
Mobile phase : water R, acetonitrile R (40:60 V/V).
Application : about 5 μl.
Development : immediately, over 4/5 of the plate.
Drying : in air.
Detection : determine the distribution of radioactivity using
a suitable detector.
Retardation factor : impurity A = 0-0.1.
System suitability : the retardation factor of the principal
peak in the chromatogram obtained with reference
solution (a) is not more than 0.1. The retardation factor
of the principal peak in the chromatogram obtained with
reference solution (b) is more than 0.7.
Other radiochemical impurities. Liquid chromatography
(2.2.29).
Test solution. The preparation to be examined.
Reference solution. Use reference solution (b) of the test
for impurity A.
Column:
— size : l = 0.25 m, Ø = 4.0 mm;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography with polar incorporated groups R
(5 μm).
Mobile phase A : 3.85 g/l solution of ammonium acetate R.
Mobile phase B : acetonitrile R.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 20 70 30
20 - 25 70 → 0 30 → 100
25 - 30 0 100
Flow rate : 1.0 ml/min.
Detection : radioactivity detector.
Injection : 20 μl.
Relative retention with reference to technetium-99m
mebrofenin (retention time = about 20 min) :
impurity B = about 0.17.
Limits :
— technetium-99m mebrofenin : minimum 94 per cent of
the total radioactivity.
Calculate the percentage of radioactivity due to
technetium-99m mebrofenin using the following
expression :
A = percentage of radioactivity due to impurity A
determined in the test for impurity A under
radiochemical purity ;
T = proportion of the radioactivity in the peak due
to technetium-99m mebrofenin relative to the
total eluted radioactivity in the chromatogram
obtained with the test solution.
General Notices (1) apply to all monographs and other texts
Technetium (99mTc) microspheres injection
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument.
IMPURITIES
A. technetium-99m in colloidal form,
B. [99mTc]pertechnetate ion.
01/2009:0570
TECHNETIUM (99mTc) MICROSPHERES
INJECTION
Technetii (99mTc) microsphaerarum
suspensio iniectabilis
DEFINITION
Technetium (99mTc) microspheres injection is a sterile
suspension of human albumin which has been denatured
to form spherical insoluble particles ; the particles are
labelled with technetium-99m. The injection contains
reducing substances, such as tin salts in a concentration
not exceeding 3 mg of Sn per millilitre ; it may contain a
suitable buffer such as acetate, citrate or phosphate and
additives such as wetting agents. The human albumin
used complies with the requirements of the monograph on
Human albumin solution (0255). The injection contains not
less than 90.0 per cent and not more than 110.0 per cent of
the declared technetium-99m radioactivity at the date and
hour stated on the label. Not less than 95 per cent of the
technetium-99m radioactivity is bound to the particles of
the suspension as determined by the test for non-filterable
radioactivity. The particles have a typical diameter between
10 μm and 50 μm. The radioactivity is not less than 185 MBq
of technetium-99m per million particles at the date and hour
of administration.
Technetium (99mTc) microspheres injection is prepared from
sodium pertechnetate (99mTc) injection (fission or non-fission)
using suitable sterile ingredients and calculating the ratio of
radionuclidic impurities with reference to the date and hour
of administration.
CHARACTERS
A suspension of white, yellow or artificially coloured particles
which may separate on standing.
Technetium-99m has a half-life of 6.02 h and emits gamma
radiation.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrument. The spectrum does not differ significantly
from that of a standardised technetium-99m solution
either by direct comparison or by using an instrument
calibrated with the aid of such a solution. Standardised
technetium-99m and molybdenum-99 solutions are
available from laboratories recognised by the competent
authority. The most prominent gamma photon of
technetium-99m has an energy of 0.140 MeV.
B. The tests for non-filterable radioactivity and particle size
contribute to the identification of the preparation.
C. Transfer 1 ml of the injection to a centrifuge tube
and centrifuge at 2500 g for 5 min to 10 min. Decant
the supernatant liquid. To the residue add 5 ml of
cupri-tartaric solution R2, mix and allow to stand for
10 min. If necessary, heat to dissolve the particles
4005
Technetium (99mTc) tin pyrophosphate injection EUROPEAN PHARMACOPOEIA 6.3

and allow to cool. Add rapidly 0.5 ml of dilute A = radioactivity of the organ concerned,
phosphomolybdotungstic reagent R, mix immediately. A
blue colour develops. B = total radioactivity in the liver, the spleen, the
lungs and the rest of the body, including voided
urine.
In not fewer than 2 of the 3 rats used, not less than 80 per
TESTS cent of the radioactivity is found in the lungs and not more
pH (2.2.3). The pH of the injection is 4.0 to 9.0. than a total of 5 per cent in the liver and spleen. The injection
may be released for use before completion of the test.
Non-filterable radioactivity. Use a polycarbonate membrane
filter 13 mm to 25 mm in diameter, 10 μm thick and with Sterility. It complies with the test for sterility
circular pores 3 μm in diameter. Fit the membrane into prescribed in the monograph on Radiopharmaceutical
a suitable holder. Place 0.2 ml of the injection on the preparations (0125). The injection may be released for use
membrane and filter, adding 20 ml of a 9 g/l solution of before completion of the test.
sodium chloride R during the filtration. The radioactivity Bacterial endotoxins (2.6.14) : less than 175/V IU/ml,
remaining on the membrane represents not less than 95 per V being the maximum recommended dose in millilitres.
cent of the total radioactivity of the injection.
Particle size. Examine using a microscope. Dilute the RADIOACTIVITY
injection if necessary so that the number of particles is just Measure the radioactivity using suitable counting equipment
low enough for individual particles to be distinguished. by comparison with a standardised technetium-99m solution
Using a syringe fitted with a needle having a calibre not less or by measurement in an instrument calibrated with the aid
than 0.35 mm, place a suitable volume in a suitable counting of such a solution.
chamber such as a haemocytometer cell, taking care not to
overfill the chamber. Allow the suspension to settle for 1 min LABELLING
and carefully add a cover slide without squeezing the sample. The label states :
Scan an area corresponding to at least 5000 particles. The — the concentration of tin expressed in milligrams per
particles have a uniform spherical appearance. Not more millilitre, if any,
than 10 particles have a maximum dimension greater than — that the preparation is to be shaken before use.
75 μm. No particle having a maximum dimension greater
than 100 μm is present.
01/2009:0129
Number of particles. Examine using a microscope. Fill a
suitable counting chamber such as a haemocytometer cell
with a suitable dilution of the injection taking care that
TECHNETIUM (99mTc) TIN
particles do not separate during the transfer. Count the PYROPHOSPHATE INJECTION
number of particles in the chamber. Repeat this procedure
twice and calculate the number of particles per millilitre of Stanni pyrophosphatis et technetii (99mTc)
the injection.
solutio iniectabilis
Tin
DEFINITION
Test solution. To 1.0 ml of the injection add 0.5 ml of Technetium (99mTc) tin pyrophosphate injection is a sterile
sulphuric acid R and 1.5 ml of nitric acid R. Heat and solution which may be prepared by mixing solutions of
evaporate to approximately 1 ml. Add 2 ml of water R and sodium pyrophosphate and stannous chloride with sodium
evaporate again to approximately 1 ml. Repeat this procedure pertechnetate (99mTc) injection (fission or non-fission). The
twice, cool and dilute to 25.0 ml with 1 M hydrochloric acid. injection contains not less than 90.0 per cent and not
more than 110.0 per cent of the declared technetium-99m
Reference solution. Dissolve 0.115 g of stannous chloride R radioactivity at the date and hour stated on the label. Not
in 1 M hydrochloric acid and dilute to 1000.0 ml with the less than 90 per cent of the radioactivity corresponds to
same acid. technetium-99m complexed with tin pyrophosphate. The
injection contains a concentration of sodium pyrophosphate
To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of (Na4P2O7,10H2O) that may vary from 1 mg to 50 mg per
sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml millilitre and a variable concentration of tin (Sn) not
of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. exceeding 3.0 mg per millilitre.
Mix. Measure the absorbance (2.2.25) of each solution at It is prepared from sodium pertechnetate (99mTc) injection
540 nm, using 0.2 M hydrochloric acid as the compensation (fission or non-fission) using suitable sterile ingredients
liquid. The absorbance of the test solution is not greater and calculating the ratio of radionuclidic impurities with
than that of the reference solution (3 mg of Sn per millilitre). reference to the date and hour of administration.
Physiological distribution. Inject a volume not greater than CHARACTERS
0.2 ml into a caudal vein of each of three rats weighing 150 g
A clear, colourless solution.
to 250 g. Euthanise the rats 15 min after the injection,
remove the liver, the spleen and the lungs and measure Technetium-99m has a half-life of 6.02 h and emits gamma
the radioactivity in the organs using a suitable instrument. radiation.
Measure the radioactivity in the rest of the body, including IDENTIFICATION
the blood and voided urine, after having removed the tail.
Determine the percentage of radioactivity in the liver, the A. Record the gamma-ray spectrum using a suitable
spleen and the lungs from the following expression : instrument. The spectrum does not differ significantly
from that of a standardised technetium-99m solution
either by direct comparison or by measurement in an
instrument calibrated with the aid of such a solution.

4006 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Technetium (99mTc) tin pyrophosphate injection

Standardised technetium-99m and molybdenum-99 stand for 30 min and measure the absorbance (2.2.25) of
solutions are available from laboratories recognised by each solution at 530 nm, using as the compensation liquid
the competent authority. The most prominent gamma a reagent blank containing the same quantity of sodium
photon of technetium-99m has an energy of 0.140 MeV. pyrophosphate R as the injection to be examined. Using the
absorbances obtained with the reference solutions, draw a
B. Examine the chromatograms obtained in the test for calibration curve and calculate the concentration of tin in
radiochemical purity. The distribution of radioactivity the injection to be examined.
contributes to the identification of the injection. Sterility. lt complies with the test for sterility
prescribed in the monograph on Radiopharmaceutical
C. To 1 ml add 1 ml of acetic acid R. Heat on a water-bath preparations (0125). The injection may be released for use
for 1 h. After cooling, add 10 ml of nitro-vanadomolybdic before completion of the test.
reagent R and allow to stand for 30 min. A yellow colour
develops. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml,
V being the maximum recommended dose in millilitres.
D. To 1 ml add 2 ml of a 30 per cent V/V solution of
sulphuric acid R, 1 ml of hydrochloric acid R, 0.05 ml of RADIOCHEMICAL PURITY
thioglycollic acid R, 0.4 ml of a 20 g/l solution of sodium (a) Examine by thin-layer chromatography (2.2.27) using
laurilsulfate R and 0.1 ml of dithiol reagent R and allow silica gel as the coating substance on a glass-fibre sheet.
to stand for 30 min. A pink colour develops. Heat the plate at 110 °C for 10 min. The plate used should
be such that during development the mobile phase migrates
over a distance of 10 cm to 15 cm in about 10 min.
TESTS
Apply to the plate 5 μl to 10 μl of the injection and dry
pH (2.2.3). The pH of the injection is 6.0 to 7.0. in a stream of nitrogen. Develop over a path of 10 cm to
Sodium pyrophosphate 15 cm using methyl ethyl ketone R through which nitrogen
has been bubbled in the chromatography tank for 10 min
Test solution. Use 1 ml of the injection to be examined or a immediately before the chromatography. Allow the plate
suitable dilution of it. to dry. Determine the distribution of radioactivity using a
suitable detector. The technetium-99m tin pyrophosphate
complex remains at the starting-point and pertechnetate ion
Reference solutions. Using a solution containing sodium
migrates with an RF of 0.95 to 1.0.
pyrophosphate R and stannous chloride R in the same
proportions as in the injection to be examined, prepare a (b) Examine by thin-layer chromatography (2.2.27) using
range of solutions and dilute to the same final volume with silica gel as the coating substance on a glass-fibre sheet.
water R. Heat the plate at 110 °C for 10 min. The plate used should
be such that during development the mobile phase migrates
To the test solution and to 1 ml of each of the reference over a distance of 10 cm to 15 cm in about 10 min.
solutions add successively 10 ml of a 1 g/l solution of
disodium hydrogen phosphate R, 10 ml of iron standard Apply to the plate 5 μl to 10 μl of the injection. Develop
solution (8 ppm Fe) R, 5 ml of glacial acetic acid R and immediately over a path of 10 cm to 15 cm using a
5 ml of a 1 g/l solution of hydroxylamine hydrochloride R. 136 g/l solution of sodium acetate R. Allow the plate to
Dilute each solution to 40 ml with water R and heat in a dry. Determine the distribution of radioactivity using a
water-bath at 40 °C for 1 h. To each solution, add 4 ml of a suitable detector. Impurities in colloidal form remain at
1 g/l solution of phenanthroline hydrochloride R and dilute the starting-point and technetium-99m tin pyrophosphate
to 50.0 ml with water R. Measure the absorbance (2.2.25) of complex and pertechnetate ion migrate with an R of 0.9
F
each solution at 515 nm using as the compensation liquid to 1.0.
a reagent blank containing hydrochloric acid (1.1 g/l HCl)
instead of the iron standard solution (8 ppm Fe) R. Using Add together the percentages of radioactivity corresponding
the absorbances obtained with the reference solutions, to impurities in the chromatograms obtained in test (a) and
draw a calibration curve and calculate the concentration of test (b). The sum does not exceed 10 per cent.
sodium pyrophosphate in the injection to be examined.
Tin
RADIOACTIVITY
Test solution. Use 1 ml of the injection to be examined or a
suitable dilution of it. Measure the radioactivity using suitable counting equipment
by comparison with a standardised technetium-99m solution
Reference solutions. Using a solution in hydrochloric or by measurement in an instrument calibrated with the aid
acid (6.2 g/l HCl) containing sodium pyrophosphate R of such a solution.
and stannous chloride R in the same proportions as in
the injection to be examined, prepare a range of solutions
and dilute to the same final volume with hydrochloric acid LABELLING
(6.2 g/l HCl).
The label states :
To the test solution and to 1 ml of each of the reference
solutions add 2 ml of a 300 g/l solution of sulphuric — the concentration of sodium pyrophosphate expressed
acid R, 1 ml of hydrochloric acid R, 0.05 ml of thioglycollic in milligrams per millilitre ;
acid R, 0.4 ml of a 20 g/l solution of sodium laurilsulfate R
and 0.1 ml of dithiol reagent R and dilute to 15 ml with — the concentration of tin expressed in milligrams per
hydrochloric acid (6.2 g/l HCl). Allow the solutions to millilitre.

General Notices (1) apply to all monographs and other texts 4007
Tetra-O-acetyl-mannose triflate for radiopharmaceutical preparations EUROPEAN PHARMACOPOEIA 6.3

01/2009:2294 Mobile phase :

— mobile phase A : water R ;
TETRA-O-ACETYL-MANNOSE — mobile phase B : acetonitrile R1 ;
TRIFLATE FOR Time Mobile phase A Mobile phase B
RADIOPHARMACEUTICAL (min) (per cent V/V) (per cent V/V)
0-1 80 20
PREPARATIONS
1 - 20 80 → 55 20 → 45
Tetra-O-acetylmannosi triflas 20 - 35 55 45
ad radiopharmaceutica 35 - 45 55 → 0 45 → 100
45 - 50 0 100

Flow rate : 1 ml/min.

C15H19F3O12S Mr 480.4 — resolution : minimum 5.0 between the peaks due to
DEFINITION
1,3,4,6-Tetra-O-acetyl-2-O-trifluoromethanesulphonyl-β-D-
mannopyranose.
Content : 97.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white crystalline hygroscopic
powder.
Solubility : practically insoluble in water, very soluble in
acetonitrile, freely soluble in methylene chloride, slightly
soluble in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : tetra-O-acetyl-mannose triflate CRS.
TESTS
Specific optical rotation (2.2.7) : − 18.0 to − 22.0 (dried
substance), measured at 25 °C.
Dissolve 40.0 mg in methylene chloride R and dilute to
10.0 ml with the same solvent.
Melting point (2.2.14) : 117 °C to 122 °C.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Dissolve 10.0 mg of the substance to be
examined in acetonitrile R and dilute to 5.0 ml with the
same solvent.
Reference solution (a). Dissolve 10.0 mg of
tetra-O-acetyl-mannose triflate CRS in acetonitrile R and
dilute to 5.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to area identified in the spectrum obtained with reference
10.0 ml with acetonitrile R. Dilute 1.0 ml of this solution to solution (c) at the same chemical shift (0.2 per cent).
100.0 ml with acetonitrile R.
Reference solution (c). Dissolve 10 mg of
1,3,4,6-tetra-O-acetyl-β-D-mannopyranose R (impurity A) in 25 mg by thermogravimetry (2.2.34). Heat to 80 °C at a rate
5 ml of acetonitrile R. Mix 1 ml of this solution and 1 ml of of 2.5 °C/min.
reference solution (a).
Column:
— size : l = 0.25 m, Ø = 4.0 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 25 °C.
Detection : spectrophotometer at 220 nm.
Injection : 20 μl of the test solution and reference
solutions (b) and (c).
Relative retention with reference to tetra-O-acetyl-
mannose triflate (retention time = about 29 min) :
impurity A = about 0.2.
System suitability : reference solution (c) :
tetra-O-acetyl-mannose triflate and impurity A.
Limits :
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
— any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.1 per cent) ;
— total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Impurity B. Nuclear magnetic resonance (2.2.33), using
the 19F-NMR technique. Prepare the solutions immediately
before use.
Test solution. Dissolve 10.0 mg of the substance to be
examined in deuterated acetonitrile R and dilute to 5.0 ml
with the same solvent.
Reference solution (a). Dissolve 10.0 mg of
tetra-O-acetyl-mannose triflate CRS in deuterated
acetonitrile R and dilute to 5.0 ml with the same solvent.
Reference solution (b). Dissolve 2.0 mg of lithium
trifluoromethanesulphonate R (lithium salt of impurity B)
in deuterated acetonitrile R and dilute to 5.0 ml with the
same solvent.
Reference solution (c). Mix 1.0 ml of reference solution (a)
and 10 μl of reference solution (b).
Limit : the peak area identified in the spectrum obtained
with the test solution at − 78 ppm is smaller than the peak
Loss on drying : maximum 0.6 per cent, determined on
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Calculate the percentage content of C15H19F3O12S using the
declared content of tetra-O-acetyl-mannose triflate CRS.

4008 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Tetra-O-acetyl-mannose triflate for radiopharmaceutical preparations

STORAGE
At a temperature of 2 °C to 8 °C, in an airtight container,
protected from light.

LABELLING
The label recommends testing the substance in a production
run before its use for the manufacture of radiopharmaceutical
preparations. This ensures that under specified production
conditions, the substance yields the radiopharmaceutical
preparation in the desired quantity and of the quality A. 1,3,4,6-tetra-O-acetyl-β-D-mannopyranose,
specified.

IMPURITIES
Specified impurities : A, B. B. trifluoromethanesulphonic acid.

General Notices (1) apply to all monographs and other texts 4009
 EUROPEAN PHARMACOPOEIA 6.3

4010 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
A
Acacia.......................................................................................... 4013
Acacia, spray-dried.. ................................................................. 4014
Acemetacin.. .............................................................................. 4015
N-Acetyltryptophan.................................................................. 4016
Adenosine.. ................................................................................ 4018
Agar............................................................................................. 4019
Air, medicinal.. ..........................................................................4020
Alginic acid................................................................................4022
Almagate.. ..................................................................................4023
Aluminium magnesium silicate.............................................4024
Aluminium oxide, hydrated....................................................4025
Aluminium phosphate gel.. ....................................................4026
Aluminium sodium silicate.. ..................................................4026
General Notices (1) apply to all monographs and other texts
Amiodarone hydrochloride.. ..................................................4028
Amitriptyline hydrochloride.. ................................................4029
Amphotericin B.. ...................................................................... 4031
Aprotinin.. ..................................................................................4033
Aprotinin concentrated solution...........................................4035
Arnica flower.............................................................................4038
Arnica tincture..........................................................................4040
Artichoke leaf dry extract.. .................................................... 4041
Ascorbic acid.............................................................................4042
Atropine.. ...................................................................................4044
Atropine sulphate.....................................................................4045
Azithromycin.............................................................................4047
4011
 EUROPEAN PHARMACOPOEIA 6.3

4012 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Acacia

01/2009:0307 0.1 ml of water R and 0.9 ml of methanol R. Centrifuge to

separate the amorphous precipitate. Dilute the supernatant,
ACACIA if necessary, to 1 ml with methanol R.
Reference solution. Dissolve 10 mg of arabinose R, 10 mg
Acaciae gummi of galactose R, 10 mg of glucose R, 10 mg of rhamnose R
and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml
DEFINITION with methanol R.
Air-hardened, gummy exudate flowing naturally from or Plate : TLC silica gel plate R.
obtained by incision of the trunk and branches of Acacia Mobile phase : 16 g/l solution of sodium dihydrogen
senegal L. Willdenow, other species of Acacia of African phosphate R, butanol R, acetone R (10:40:50 V/V/V).
origin and Acacia seyal Del. Application : 10 μl as bands.
CHARACTERS Developement A : over a path of 10 cm.
Acacia is almost completely but very slowly soluble, after Drying A : in a current of warm air for a few minutes.
about 2 h, in twice its mass of water leaving only a very Development B : over a path of 15 cm using the same mobile
small residue of vegetable particles ; the liquid obtained is phase.
colourless or yellowish, dense, viscous, adhesive, translucent Drying B : at 110 °C for 10 min.
and weakly acid to blue litmus paper. Acacia is practically
insoluble in ethanol (96 per cent). Detection : spray with anisaldehyde solution R and heat
at 110 °C for 10 min.
IDENTIFICATION Results : the chromatogram obtained with the reference
A. Acacia occurs as yellowish-white, yellow or pale amber, solution shows 5 clearly separated coloured zones due to
sometimes with a pinkish tint, friable, opaque, spheroidal, galactose (greyish-green or green), glucose (grey), arabinose
oval or reniform pieces (tears) of a diameter from about (yellowish-green), xylose (greenish-grey or yellowish-grey)
1-3 cm, frequently with a cracked surface, easily broken and rhamnose (yellowish-green), in order of increasing RF
into irregular, whitish or slightly yellowish angular value. The chromatogram obtained with the test solution
fragments with conchoidal fracture and a glassy and shows no grey zone and no greyish-green zone between
transparent appearance. In the centre of an unbroken the zones corresponding to galactose and arabinose in the
tear there is sometimes a small cavity. chromatogram obtained with the reference solution.
B. Reduce to a powder (355) (2.9.12). The powder is white Starch, dextrin and agar. To 10 ml of solution S previously
or yellowish-white. Examine under a microscope using a boiled and cooled add 0.1 ml of 0.05 M iodine. No blue or
50 per cent V/V solution of glycerol R. The powder shows reddish-brown colour develops.
the following diagnostic characters : angular, irregular, Sterculia gum
colourless, transparent fragments. Only traces of starch
or vegetable tissues are visible. No stratified membrane A. Place 0.2 g of the powdered drug (355) (2.9.12) in a 10 ml
is apparent. ground-glass-stoppered cylinder graduated in 0.1 ml. Add
10 ml of ethanol (60 per cent V/V) R and shake. Any gel
C. Examine the chromatograms obtained in the test for formed occupies a maximum of 1.5 ml.
glucose and fructose.
B. To 1.0 g of the powdered drug (355) (2.9.12) add 100 ml of
Results : the chromatogram obtained with the test water R and shake. Add 0.1 ml of methyl red solution R.
solution shows 3 zones due to galactose, arabinose Not more than 5.0 ml of 0.01 M sodium hydroxide is
and rhamnose. No other important zones are visible, required to change the colour of the indicator.
particularly in the upper part of the chromatogram.
D. Dissolve 1 g of the powdered drug (355) (2.9.12) in 2 ml Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride
of water R by stirring frequently for 2 h. Add 2 ml of solution R1. A gelatinous precipitate is formed, but neither
ethanol (96 per cent) R. After shaking, a white, gelatinous the precipitate nor the liquid are dark blue.
mucilage is formed which becomes fluid on adding 10 ml Tragacanth. Examine the chromatograms obtained in the
of water R. test for glucose and fructose.
Results : the chromatogram obtained with the test solution
TESTS shows no greenish-grey or yellowish-grey zone corresponding
Solution S. Dissolve 3.0 g of the powdered drug (355) to the zone of xylose in the chromatogram obtained with
(2.9.12) in 25 ml of water R by stirring for 30 min. Allow to the reference solution.
stand for 30 min and dilute to 30 ml with water R. Loss on drying (2.2.32) : maximum 15.0 per cent, determined
Insoluble matter : maximum 0.5 per cent. on 1.000 g of the powdered drug (355) (2.9.12) by drying
To 5.0 g of the powdered drug (355) (2.9.12) add 100 ml of in an oven at 105 °C.
water R and 14 ml of dilute hydrochloric acid R, boil gently Total ash (2.4.16) : maximum 4.0 per cent.
for 15 min, shaking frequently and filter while hot through a
tared sintered-glass filter (2.1.2). Wash with hot water R and Microbial contamination
4
dry at 100-105 °C. The residue weighs a maximum of 25 mg. TAMC : acceptance criterion 10 CFU/g (2.6.12).
2
Glucose and fructose. Thin-layer chromatography (2.2.27). TYMC : acceptance criterion 10 CFU/g (2.6.12).
Test solution. To 0.100 g of the powdered drug (355) Absence of Escherichia coli (2.6.13).
(2.9.12) in a thick-walled centrifuge tube add 2 ml of a Absence of Salmonella (2.6.13).
100 g/l solution of trifluoroacetic acid R, shake vigorously
to dissolve the forming gel, stopper the tube and heat the FUNCTIONALITY-RELATED CHARACTERISTICS
mixture at 120 °C for 1 h. Centrifuge the hydrolysate, This section provides information on characteristics
transfer the clear supernatant carefully into a 50 ml flask, that are recognised as being relevant control parameters
add 10 ml of water R and evaporate the solution to dryness for one or more functions of the substance when used
under reduced pressure. To the resulting clear film add as an excipient (see chapter 5.15). This section is a

General Notices (1) apply to all monographs and other texts 4013
Acacia, spray-dried
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristic may be relevant for acacia
used as a viscosity-increasing agent and/or suspending
agent in aqueous preparations.
Apparent viscosity. Determine the dynamic viscosity using a
capillary viscometer (2.2.9) or a rotating viscometer (2.2.10)
on a 100 g/l solution of acacia (dried substance).
01/2009:0308
ACACIA, SPRAY-DRIED
Acaciae gummi dispersione desiccatum
DEFINITION
Spray-dried acacia is obtained from a solution of acacia.
CHARACTERS
It dissolves completely and rapidly, after about 20 min, in
twice its mass of water. The liquid obtained is colourless or
yellowish, dense, viscous, adhesive, translucent and weakly
acid to blue litmus paper. Spray-dried acacia is practically
insoluble in ethanol (96 per cent).
IDENTIFICATION
A. Examined under a microscope, in ethanol (96 per cent) R,
the powder is seen to consist predominantly of spheroidal
particles about 4-40 μm in diameter, with a central cavity
containing 1 or several air-bubbles ; a few minute flat
fragments are present. Only traces of starch granules are
visible. No vegetable tissue is seen.
B. Examine the chromatograms obtained in the test for
glucose and fructose.
Results : the chromatogram obtained with the test
solution shows 3 zones due to galactose, arabinose
and rhamnose. No other important zones are visible,
particularly in the upper part of the chromatogram.
C. Dissolve 1 g of the drug to be examined in 2 ml of
water R by stirring frequently for 20 min. Add 2 ml of
ethanol (96 per cent) R. After shaking a white gelatinous
mucilage is formed which becomes fluid on adding 10 ml
of water R.
TESTS
Solution S. Dissolve 3.0 g of the drug to be examined in
25 ml of water R by stirring for 10 min. Allow to stand for
20 min and dilute to 30 ml with water R.
Glucose and fructose. Thin-layer chromatography (2.2.27).
Test solution. To 0.100 g in a thick-walled centrifuge tube
add 2 ml of a 100 g/l solution of trifluoroacetic acid R,
shake vigorously to dissolve the forming gel, stopper the
tube and heat the mixture at 120 °C for 1 h. Centrifuge the
hydrolysate, transfer the clear supernatant carefully into a
50 ml flask, add 10 ml of water R and evaporate to dryness
under reduced pressure. To the resulting clear film add
0.1 ml of water R and 0.9 ml of methanol R. Centrifuge to
separate the amorphous precipitate. Dilute the supernatant,
if necessary, to 1 ml with methanol R.
4014 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Reference solution. Dissolve 10 mg of arabinose R, 10 mg
of galactose R, 10 mg of glucose R, 10 mg of rhamnose R
and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml
with methanol R.
Plate : TLC silica gel plate R.
Mobile phase : 16 g/l solution of sodium dihydrogen
phosphate R, butanol R, acetone R (10:40:50 V/V/V).
Application : 10 μl as bands.
Development A : over a path of 10 cm.
Drying A : in a current of warm air for a few minutes.
Development B : over a path of 15 cm using the same mobile
phase.
Detection : spray with anisaldehyde solution R and heat
at 110 °C for 10 min.
Results : the chromatogram obtained with the reference
solution shows 5 clearly separated coloured zones due to
galactose (greyish-green or green), glucose (grey), arabinose
(yellowish-green), xylose (greenish-grey or yellowish-grey)
and rhamnose (yellowish-green), in order of increasing RF
value. The chromatogram obtained with the test solution
shows no grey zone and no greyish-green zone between
the zones corresponding to galactose and arabinose in the
chromatogram obtained with the reference solution.
Starch, dextrin and agar. To 10 ml of solution S previously
boiled and cooled add 0.1 ml of 0.05 M iodine. No blue or
reddish-brown colour develops.
Sterculia gum
A. Place 0.2 g in a 10 ml ground-glass-stoppered cylinder
graduated in 0.1 ml. Add 10 ml of ethanol (60 per
cent V/V) R and shake. Any gel formed occupies not
more than 1.5 ml.
B. To 1.0 g add 100 ml of water R and shake. Add 0.1 ml
of methyl red solution R. Not more than 5.0 ml of
0.01 M sodium hydroxide is required to change the
colour of the indicator.
Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride
solution R1. A gelatinous precipitate is formed, but neither
the precipitate nor the liquid shows a dark blue colour.
Tragacanth. Examine the chromatograms obtained in the
test for Glucose and fructose.
Results : the chromatogram obtained with the test solution
shows no greenish-grey or yellowish-grey zone corresponding
to the zone of xylose in the chromatogram obtained with
the reference solution.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Total ash (2.4.16). : maximum 4.0 per cent.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
EUROPEAN PHARMACOPOEIA 6.3 Acemetacin

Where control methods are cited, they are recognised as Reference solution (e). Dissolve the contents of a vial of
being suitable for the purpose, but other methods can also acemetacin impurity mixture CRS (containing impurities C,
be used. Wherever results for a particular characteristic are D, E and F) in 1.0 ml of the test solution.
reported, the control method must be indicated. Column :
The following characteristic may be relevant for spray-dried — size: l = 0.25 m, Ø = 4 mm ;
acacia used as a viscosity-increasing agent and/or — stationary phase : spherical end-capped octadecylsilyl
suspending agent in aqueous preparations. silica gel for chromatography R (5 μm) ;
Apparent viscosity. Determine the dynamic viscosity using a — temperature : 40 °C.
capillary viscometer (2.2.9) or a rotating viscometer (2.2.10)Mobile phase :
on a 100 g/l solution of spray-dried acacia (dried substance).
— mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R in 900 ml of water R, adjust to pH 6.5
04/2008:1686 with 1 M sodium hydroxide and dilute to 1000 ml with
corrected 6.3 water R ;
— mobile phase B : acetonitrile for chromatography R ;
ACEMETACIN Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Acemetacinum 0-5 95 5
5-9 95 → 65 5 → 35
9 - 16 65 35
16 - 28 65 → 20 35 → 80
28 - 34 20 80

Flow rate : 1.0 ml/min.

C21H18ClNO6 Mr 415.8
[53164-05-9]
DEFINITION
[[[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetyl]oxy]acetic acid.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : yellow or greenish-yellow, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone,
slightly soluble in anhydrous ethanol.
It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : acemetacin CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be
examined in acetonitrile for chromatography R and dilute
to 20.0 ml with the same solvent.
Reference solution (a). Dilute 5.0 ml of the test solution
to 50.0 ml with acetonitrile for chromatography R. Dilute
1.0 ml of this solution to 100.0 ml with acetonitrile for
chromatography R.
Reference solution (b). Dissolve 5.0 mg of acemetacin
impurity A CRS and 10.0 mg of indometacin CRS
(impurity B) in acetonitrile for chromatography R, and
dilute to 50.0 ml with the same solvent.
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 20.0 ml with acetonitrile for chromatography R.
Reference solution (d). To 1 ml of reference solution (b),
add 10 ml of the test solution and dilute to 20 ml with
acetonitrile for chromatography R.
Detection : spectrophotometer at 235 nm.
Injection : 20 μl.
Identification of impurities :
— use the chromatogram supplied with acemetacin
impurity mixture CRS and the chromatogram obtained
with reference solution (e) to identify the peaks due to
impurities C, D, E and F ;
— use the chromatogram obtained with reference
solution (b) to identify the peak due to impurity B.
Relative retention with reference to acemetacin
(retention time = about 15 min) : impurity A = about 0.7 ;
impurity B = about 0.9 ; impurity F = about 1.2 ;
impurity C = about 1.3 ; impurity D = about 1.5 ;
impurity E = about 2.2.
System suitability : reference solution (d) :
— peak-to-valley ratio : minimum 15, 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
acemetacin.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity C = 1.3 ;
impurity D = 1.4 ; impurity F = 1.3 ;
— impurity E : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
— impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.2 per cent) ;
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.1 per cent) ;
— impurities C, D, F : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (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) ;

General Notices (1) apply to all monographs and other texts 4015
N-Acetyltryptophan
— total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.4 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals : maximum 20 ppm.
Solvent mixture : methanol R, acetone R (10:90 V/V).
Test solution. Dissolve 0.250 g of the substance to be
examined in 20 ml of the solvent mixture.
Reference solution. Dilute 0.5 ml of lead standard solution
(10 ppm Pb) R to 20 ml with the solvent mixture.
Blank solution : 20 ml of the solvent mixture.
Monitor solution. Dissolve 0.250 g of the substance to be
examined in 0.5 ml of lead standard solution (10 ppm Pb) R
and dilute to 20 ml with the solvent mixture.
To each solution, add 2 ml of buffer solution pH 3.5 R.
Mix and add to 1.2 ml of thioacetamide reagent R. Mix
immediately. Filter the solutions through a membrane filter
(nominal pore size 0.45 μm) (2.4.8). Compare the spots on
the filters obtained with the different solutions. The test is
invalid if the reference solution does not show a slight brown
colour compared to the blank solution. The substance to be
examined complies with the test if the brown colour of the
spot resulting from the test solution is not more intense than
that of the spot resulting from the reference solution.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.350 g in 20 ml of acetone R and add 10 ml of
water R. Titrate with 0.1 M sodium hydroxide, determining
the end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 41.58 mg
of C21H18ClNO6.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F.
A. 4-chlorobenzoic acid,
B. R1 = R2 = R3 = H : indometacin,
C. R1 = Cl, R2 = H, R3 = CH2-CO2H : [[[1-(3,4-dichlorobenzoyl)-
5-methoxy-2-methyl-1H-indol-3-yl]acetyl]oxy]acetic acid,
4016 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
D. R1 = H, R2 = C(CH3)3, R3 = CH2-CO2H :
[[[1-(4-chlorobenzoyl)-6-(1,1-dimethylethyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetyl]oxy]acetic acid,
E. R1 = R2 = H, R3 = CH2-CO-O-C(CH3)3 : 1,1-dimethylethyl
[[[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetyl]oxy]acetate,
F. R1 = R2 = H, R3 = CH2-CO-O-CH2-CO2H :
[[[[[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetyl]oxy]acetyl]oxy]acetic acid.
01/2009:1383
N-ACETYLTRYPTOPHAN
N-Acetyltryptophanum
C13H14N2O3 Mr 246.3
[87-32-1]
DEFINITION
(RS)-2-Acetylamino-3-(1H-indol-3-yl)propanoic acid.
Content : 99.0 per cent to 101.0 per cent (dried substance).
PRODUCTION
Tryptophan used for the production of N-acetyltryptophan
complies with the test for impurity A and other related
substances in the monograph on Tryptophan (1272).
CHARACTERS
Appearance : white or almost white, crystalline powder, or
colourless crystals.
Solubility : slightly soluble in water, very soluble in ethanol
(96 per cent). It dissolves in dilute solutions of alkali
hydroxides.
mp : about 205 °C.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D, E.
A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : N-acetyltryptophan CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 50 mg of the substance to be
examined in 0.2 ml of concentrated ammonia R and
dilute to 10 ml with water R.
Reference solution (a). Dissolve 50 mg of
N-acetyltryptophan CRS in 0.2 ml of concentrated
ammonia R and dilute to 10 ml with water R.
Reference solution (b). Dissolve 10 mg of tryptophan R in
the test solution and dilute to 2 ml with the test solution.
Plate : TLC silica gel F254 plate R.
Mobile phase : glacial acetic acid R, water R, butanol R
(25:25:40 V/V/V).
Application : 2 μl.
Development : over a path of 10 cm.
Drying : in an oven at 100-105 °C for 15 min.
EUROPEAN PHARMACOPOEIA 6.3
Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) :
— the chromatogram shows 2 clearly separated spots.
Results : 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).
D. Dissolve about 2 mg in 2 ml of water R. Add 2 ml of
dimethylaminobenzaldehyde solution R6. Heat on a
water-bath. A blue or greenish-blue colour develops.
E. It gives the reaction of acetyl (2.3.1). Proceed as described
for substances hydrolysable only with difficulty.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution Y7 or
GY7 (2.2.2, Method II).
Dissolve 1.0 g in a 40 g/l solution of sodium hydroxide R
and dilute to 100 ml with the same alkaline solution.
Optical rotation (2.2.7) : − 0.1° to + 0.1°.
Dissolve 2.50 g in a 40 g/l solution of sodium hydroxide R
and dilute to 25.0 ml with the same alkaline solution.
Related substances. Liquid chromatography (2.2.29).
Prepare the test and reference solutions immediately before
use.
Buffer solution pH 2.3. Dissolve 3.90 g of sodium
dihydrogen phosphate R in 1000 ml of water R. Add about
700 ml of a 2.9 g/l solution of phosphoric acid R and adjust Ammonium (2.4.1, Method B) : maximum 200 ppm,
to pH 2.3 with the same acidic solution.
Solvent mixture : acetonitrile R, water R (10:90 V/V).
Test solution. Dissolve 0.10 g of the substance to be
examined in a mixture of 50 volumes of acetonitrile R and
50 volumes of water R and dilute to 20.0 ml with the same
mixture of solvents.
Reference solution (a). Dilute 1.0 ml of the test solution to 3 quantities, each of 10 ml, of methyl isobutyl ketone R1,
100.0 ml with the solvent mixture.
Reference solution (b). Dilute 4.0 ml of reference solution (a) add 10 ml of water R and shake for 3 min. Examine the
to 100.0 ml with the solvent mixture.
Reference solution (c). Dissolve the contents of a vial of
1,1′-ethylidenebistryptophan CRS in 1 ml of reference
solution (b).
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase :
— mobile phase A : acetonitrile R, buffer solution pH 2.3
(115:885 V/V) ;
— mobile phase B : acetonitrile R, buffer solution pH 2.3
(350:650 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 10 100 0 Protected from light.
10 - 45 100 → 0 0 → 100
45 - 65 0 100
65 - 66 0 → 100 100 → 0
66 - 80 100 0
General Notices (1) apply to all monographs and other texts
N-Acetyltryptophan
Flow rate : 0.7 ml/min.
Detection : spectrophotometer at 220 nm.
Injection : 20 μl of the test solution and reference
solutions (a) and (c).
Retention time : N-acetyltryptophan = about 29 min ;
1,1′-ethylidenebis(tryptophan) = about 34 min.
System suitability : reference solution (c) :
— resolution : minimum 8.0 between the peaks due to
N-acetyltryptophan and 1,1′-ethylidenebis(tryptophan) ;
if necessary, adjust the time programme for the elution
gradient (an increase in the duration of elution with
mobile phase A produces longer retention times and a
better resolution) ;
— symmetry factor : maximum 3.5 for the peak due to
1,1′-ethylidenebistryptophan in the chromatogram
obtained with reference solution (c).
Limits :
— impurities A, B, C, D, E, F, G, H, I, J, K, L: for each
impurity, not more than 0.25 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.25 per cent) ;
— total : not more than 0.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) ;
— disregard limit : 0.01 times the area of the principal peak
the chromatogram obtained with reference solution (a)
(0.01 per cent).
determined on 0.10 g.
Prepare the standard using 0.2 ml of ammonium standard
solution (100 ppm NH4) R.
Iron (2.4.9) : maximum 10 ppm.
Dissolve 1.0 g in 50 ml of hydrochloric acid R1, with heating
at 50 °C. Allow to cool. In a separating funnel, shake with
shaking for 3 min each time. To the combined organic layers
aqueous layer.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 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 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.200 g in 5 ml of methanol R. Add 50 ml of
anhydrous ethanol R. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 24.63 mg
of C H N O .
13 14 2 3
STORAGE
IMPURITIES
Specified impurities: A, B, C, D, E, F, G, H, I, J, K, L.
A. tryptophan,
4017
Adenosine
B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-
3-yl]propanoic acid (dioxyindolylalanine),
C. R = H : (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid
(kynurenine),
E. R = CHO : (S)-2-amino-4-[2-(formylamino)phenyl]-4-
oxobutanoic acid (N-formylkynurenine),
D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
(5-hydroxytryptophan),
F. (S)-2-amino-3-(phenylamino)propanoic acid
(3-phenylaminoalanine),
G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid
(2-hydroxytryptophan),
H. R = H : (3RS)-1,2,3,4-tetrahydro-9H-β-carboline-3-
carboxylic acid,
I. R = CH3 : 1-methyl-1,2,3,4-tetrahydro-9H-β-carboline-3-
carboxylic acid,
J. R = CHOH-CH2-OH : (S)-2-amino-3-[2-[2,3-dihydroxy-1-(1H-
indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid,
K. R = H : (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-3-
yl]propanoic acid,
4018
EUROPEAN PHARMACOPOEIA 6.3
L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H-β-carboline-
3-carboxylic acid.
01/2009:1486
ADENOSINE
Adenosinum
C10H13N5O4 Mr 267.2
[58-61-7]
DEFINITION
9-β-D-Ribofuranosyl-9H-purin-6-amine.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water, soluble in hot water,
practically insoluble in ethanol (96 per cent) and in
methylene chloride. It dissolves in dilute mineral acids.
mp : about 234 °C.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : adenosine CRS.
TESTS
Solution S. Suspend 5.0 g in 100 ml of distilled water R and
heat to boiling. Allow to cool, filter with the aid of vacuum
and dilute to 100 ml with distilled water R.
Appearance of solution. Solution S is colourless (2.2.2,
Method II).
Acidity or alkalinity. To 10 ml of solution S, add 0.1 ml
of bromocresol purple solution R and 0.1 ml of 0.01 M
hydrochloric acid. The solution is yellow. Add 0.4 ml of
0.01 M sodium hydroxide. The solution is violet-blue.
Specific optical rotation (2.2.7) : − 45 to − 49 (dried
substance).
Dissolve 1.25 g in 1 M hydrochloric acid and dilute to
50.0 ml with the same acid. Examine within 10 min of
preparing the solution.
Related substances
Liquid chromatography (2.2.29).
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 Agar

Solvent mixture. Dissolve 6.8 g of potassium hydrogen Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
sulphate R and 3.4 g of tetrabutylammonium hydrogen on 1.0 g.
sulphate R in water R, adjust to pH 6.5 with a 60 g/l solution
of potassium hydroxide R and dilute to 1000 ml with the ASSAY
same solvent. Use a freshly prepared solvent mixture. Dissolve 0.200 g, warming slightly if necessary, in a mixture
Test solution. Dissolve 20 mg of the substance to be of 20 ml of acetic anhydride R and 30 ml of anhydrous acetic
examined in the mobile phase and dilute to 20 ml with the acid R. Titrate with 0.1 M perchloric acid, determining the
mobile phase. end-point potentiometrically (2.2.20).
Reference solution (a). Dilute 1.0 ml of the test solution 1 ml of 0.1 M perchloric acid is equivalent to 26.72 mg
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this of C10H13N5O4.
solution to 10.0 ml with the mobile phase. IMPURITIES
Reference solution (b). Dissolve 5 mg of adenine R Specified impurities: A, G.
(impurity A) and 5 mg of inosine R (impurity G) in the mobile
phase and dilute to 50 ml with the mobile phase. Dilute 4 ml Other detectable impurities (the following substances
of this solution to 100 ml with the mobile phase. would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
Column: by the general acceptance criterion for other/unspecified
— size : l = 0.25 m, Ø = 4.6 mm ; impurities and/or by the general monograph Substances for
— stationary phase : end-capped octadecylsilyl silica gel pharmaceutical use (2034). It is therefore not necessary to
for chromatography R (5 μm). identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
Mobile phase : water R, solvent mixture (40:60 V/V). pharmaceutical use) : F, H.
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 254 nm. A. adenine,
Injection : 20 μl.
Run time : 1.5 times the retention time of adenosine.
Relative retention with reference to adenosine
(retention time = about 13 min) : impurity A = about 0.3 ;
impurity G = about 0.4.
System suitability : reference solution (b) :
— resolution : minimum 1.5 between the peaks due to
impurities A and G.
Limits :
— correction factors : for the calculation of content, F. 1-β-D-ribofuranosylpyrimidine-2,4(1H,3H)-dione (uridine),
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.6 ;
impurity G = 1.4 ;
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
— impurity G : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(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) ; G. R = H : 9-β-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one
— total : not more than 5 times the area of the principal peak (inosine),
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ; H. R = NH2 : 2-amino-9-β-D-ribofuranosyl-1,9-dihydro-6H-
purin-6-one (guanosine).
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Chlorides (2.4.4) : maximum 100 ppm. 01/2009:0310
Dilute 10 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 200 ppm, determined on AGAR
solution S.
Ammonium (2.4.1, Method B) : maximum 10 ppm, Agar
determined on 0.5 g. DEFINITION
Prepare the standard using 5 ml of ammonium standard Polysaccharides from various species of Rhodophyceae
solution (1 ppm NH4) R. mainly belonging to the genus Gelidium. It is prepared by
Loss on drying (2.2.32) : maximum 0.5 per cent, determined treating the algae with boiling water ; the extract is filtered
on 1.000 g by drying in an oven at 105 °C. whilst hot, concentrated and dried.

General Notices (1) apply to all monographs and other texts 4019
Air, medicinal
CHARACTERS
Appearance : powder or crumpled strips 2-5 mm wide or
sometimes flakes, colourless or pale yellow, translucent,
somewhat tough and difficult to break, becoming more
brittle on drying.
Mucilaginous taste.
IDENTIFICATION
A. Examine under a microscope. When mounted in
0.005 M iodine, the strips or flakes are partly stained
brownish-violet. Magnified 100 times, they show the
following diagnostic characters : numerous minute,
colourless, ovoid or rounded grains on an amorphous
background ; occasional brown, round or ovoid spores
with a reticulated surface, measuring up to 60 μm,
may be present. Reduce to a powder, if necessary. The
powder is yellowish-white. Examine under a microscope
using 0.005 M iodine. The powder presents angular
fragments with numerous grains similar to those seen in
the strips and flakes ; some of the fragments are stained
brownish-violet.
B. Dissolve 0.1 g with heating in 50 ml of water R. Cool. To
1 ml of the mucilage carefully add 3 ml of water R so as
to form 2 separate layers. Add 0.1 ml of 0.05 M iodine. A
dark brownish-violet colour appears at the interface. Mix.
The liquid becomes pale yellow.
C. Heat 5 ml of the mucilage prepared for identification
test B on a water-bath with 0.5 ml of hydrochloric acid R
for 30 min. Add 1 ml of barium chloride solution R1. A
white turbidity develops within 30 min.
D. Heat 0.5 g with 50 ml of water R on a water-bath until
dissolved. Only a few fragments remain insoluble. During
cooling, the solution gels between 35 °C and 30 °C. Heat
the gel thus obtained on a water-bath ; it does not liquefy
below 80 °C.
TESTS
Swelling index (2.8.4) : minimum 10 and within 10 per cent
of the value stated on the label, determined on the powdered
drug (355) (2.9.12).
Insoluble matter : maximum 1.0 per cent.
To 5.00 g of the powdered drug (355) (2.9.12) add 100 ml
of water R and 14 ml of dilute hydrochloric acid R. Boil
gently for 15 min with frequent stirring. Filter the hot liquid
through a tared, sintered-glass filter (160) (2.1.2), rinse the
filter with hot water R and dry at 100-105 °C. The residue
weighs a maximum of 50 mg.
Gelatin. To 1.00 g add 100 ml of water R and heat on a
water-bath until dissolved. Allow to cool to 50 °C. To 5 ml of
this solution add 5 ml of picric acid solution R. No turbidity
appears within 10 min.
Loss on drying (2.2.32) : maximum 20.0 per cent, determined
on 1.000 g of the powdered drug (355) (2.9.12) by drying
in an oven at 105 °C.
Total ash (2.4.16) : maximum 5.0 per cent.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
LABELLING
The label states the swelling index.
4020 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:1238
AIR, MEDICINAL
Aer medicinalis
DEFINITION
Compressed ambient air.
Content : 20.4 per cent V/V to 21.4 per cent V/V of
oxygen (O2).
CHARACTERS
Appearance : colourless gas.
Solubility : at 20 °C at a pressure of 101 kPa, 1 volume
dissolves in about 50 volumes of water.
PRODUCTION
Carbon dioxide : maximum 500 ppm V/V, determined using
an infrared analyser (2.5.24).
Gas to be examined. Filter the substance to be examined to
avoid stray light phenomena.
Reference gas (a). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
less than 1 ppm V/V of carbon dioxide R1.
Reference gas (b). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
500 ppm V/V of carbon dioxide R1.
Calibrate the apparatus and set the sensitivity using
reference gases (a) and (b). Measure the content of carbon
dioxide in the gas to be examined.
Carbon monoxide : maximum 5 ppm V/V, determined using
an infrared analyser (2.5.25).
Gas to be examined. Filter the substance to be examined to
avoid stray light phenomena.
Reference gas (a). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
less than 1 ppm V/V of carbon monoxide R.
Reference gas (b). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
5 ppm V/V of carbon monoxide R.
Calibrate the apparatus and set the sensitivity using
reference gases (a) and (b). Measure the content of carbon
monoxide in the gas to be examined.
Sulphur dioxide : maximum 1 ppm V/V, determined using
an ultraviolet fluorescence analyser (Figure 1238.-1).
The apparatus consists of the following :
— a system generating ultraviolet radiation with a
wavelength of 210 nm, made up of an ultraviolet lamp,
a collimator, and a selective filter ; the beam is blocked
periodically by a chopper rotating at high speeds ;
— a reaction chamber, through which flows the gas to be
examined ;
— a system that detects radiation emitted at a wavelength of
350 nm, made up of a selective filter, a photomultiplier
tube and an amplifier.
Gas to be examined. Filter the substance to be examined.
Reference gas (a). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1.
Reference gas (b). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
0.5 ppm V/V to 2 ppm V/V of sulphur dioxide R1.
Calibrate the apparatus and set the sensitivity using
reference gases (a) and (b). Measure the content of sulphur
dioxide in the gas to be examined.
EUROPEAN PHARMACOPOEIA 6.3
Figure 1238.-1. – UV fluorescence analyser
Oil : maximum 0.1 mg/m3, determined using an oil detector
tube (2.1.6), when an oil-lubricated compressor is used for
the production.
Nitrogen monoxide and nitrogen dioxide : maximum
2 ppm V/V in total, determined using a chemiluminescence
analyser (2.5.26).
Gas to be examined. The substance to be examined.
Reference gas (a). Use a mixture of 21 per cent V/V of
oxygen R and 79 per cent V/V of nitrogen R1, containing
less than 0.05 ppm V/V of nitrogen monoxide and nitrogen
dioxide.
Reference gas (b). Use a mixture of 2 ppm V/V of nitrogen
monoxide R in nitrogen R1.
Calibrate the apparatus and set the sensitivity using
reference gases (a) and (b). Measure the content of nitrogen
monoxide and nitrogen dioxide in the gas to be examined.
Water : maximum 67 ppm V/V, determined using an
electrolytic hygrometer (2.5.28), except where the competent
authority decides that the following limit applies to medicinal
air generated on-site and distributed in pipe-line systems
operating at a pressure not greater than 10 bars and a
temperature not less than 5 °C : maximum 870 ppm V/V,
determined using an electrolytic hygrometer (2.5.28).
Assay. Determine the concentration of oxygen in air using a
paramagnetic analyser (2.5.27).
IDENTIFICATION
First identification : C.
Second identification : A, B.
A. In a conical flask containing the substance to be
examined, place a glowing wood splinter. The splinter
remains glowing.
B. Use a gas burette (Figure 1238.-2) of 25 ml capacity in
the form of a chamber in the middle of which is a tube
graduated in 0.2 per cent between 19.0 per cent and
23.0 per cent, and isolated at each end by a tap with a
conical barrel. The lower tap is joined to a tube with an
olive-shaped nozzle and is used to introduce the gas into
the apparatus. A cylindrical funnel above the upper tap
General Notices (1) apply to all monographs and other texts
Air, medicinal
is used to introduce the absorbent solution. Wash the
burette with water R and dry. Open the 2 taps. Connect
the nozzle to the source of the gas to be examined and set
the flow rate to 1 litre/min. Flush the burette by passing
the gas to be examined through it for 1 min. Close the
lower tap of the burette and immediately afterwards
the upper tap. Rapidly disconnect the burette from the
source of the gas to be examined. Rapidly give a half turn
to the upper tap to eliminate any excess pressure in the
burette. Keeping the burette vertical, fill the funnel with a
freshly prepared mixture of 21 ml of a 560 g/l solution of
potassium hydroxide R and 130 ml of a 200 g/l solution
of sodium dithionite R. Open the upper tap slowly. The
solution absorbs the oxygen and enters the burette. Allow
to stand for 10 min without shaking. Read the level of
the liquid meniscus on the graduated part of the burette.
This figure represents the percentage V/V of oxygen. The
value read is 20.4 to 21.4.
C. It complies with the limits of the assay.
TESTS
Carbon dioxide : maximum 500 ppm V/V, determined using
a carbon dioxide detector tube (2.1.6).
Sulphur dioxide : maximum 1 ppm V/V, determined using a
sulphur dioxide detector tube (2.1.6).
Oil : maximum 0.1 mg/m3, determined using an oil detector
tube (2.1.6), when an oil-lubricated compressor is used for
the production.
Nitrogen monoxide and nitrogen dioxide : maximum
2 ppm V/V, determined using a nitrogen monoxide and
nitrogen dioxide detector tube (2.1.6).
Carbon monoxide : maximum 5 ppm V/V, determined using
a carbon monoxide detector tube (2.1.6).
Water vapour : maximum 67 ppm V/V, determined using
a water vapour detector tube (2.1.6), except where the
competent authority decides that the following limit applies
to medicinal air generated on-site and distributed in pipe-line
systems operating at a pressure not greater than 10 bars and
a temperature not less than 5 °C : maximum 870 ppm V/V,
determined using a water vapour detector tube (2.1.6).
4021
Alginic acid EUROPEAN PHARMACOPOEIA 6.3

01/2009:0591

ALGINIC ACID
Acidum alginicum
DEFINITION
Mixture of polyuronic acids [(C6H8O6)n] composed of residues
of D-mannuronic and L-guluronic acids, obtained mainly from
algae belonging to the Phaeophyceae. A small proportion of
the carboxyl groups may be neutralised.
Content : 19.0 per cent to 25.0 per cent of carboxyl groups
(-CO2H) (dried substance).
CHARACTERS
Appearance : white or pale yellowish-brown, crystalline or
amorphous powder.
Solubility : very slightly soluble or practically insoluble
in ethanol (96 per cent), practically insoluble in organic
solvents. It swells in water but does not dissolve ; it dissolves
in solutions of alkali hydroxides.
IDENTIFICATION
A. To 0.2 g add 20 ml of water R and 0.5 ml of sodium
carbonate solution R. Shake and filter. To 5 ml of the
filtrate add 1 ml of calcium chloride solution R. A
voluminous gelatinous mass is formed.
B. To 5 ml of the filtrate obtained in identification test A add
0.5 ml of a 123 g/l solution of magnesium sulphate R.
No voluminous gelatinous mass is formed.
C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared
10 g/l solution of 1,3-dihydroxynaphthalene R in
ethanol (96 per cent) R and 5 ml of hydrochloric acid R.
Boil gently for 3 min, cool, add 5 ml of water R, and shake
with 15 ml of di-isopropyl ether R. Carry out a blank
test. The upper layer obtained with the substance to be
examined exhibits a deeper bluish-red colour than that
obtained with the blank.
TESTS
Chlorides : maximum 1,0 per cent.
To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and
Figure 1238.-2. – Gas burette dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml
of the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of
STORAGE toluene R. Titrate with 0.1 M ammonium thiocyanate, using
As a gas, in suitable containers complying with the legal 2 ml of ferric ammonium sulphate solution R2 as indicator
regulations or as a gas supplied by a pipe network. and shaking vigorously towards the end-point.
1 ml of 0.1 M silver nitrate is equivalent to 3.545 mg of Cl.
LABELLING Heavy metals (2.4.8) : maximum 20 ppm.
Where applicable, the label states the production method, as 1.0 g complies with test F. Prepare the reference solution
regards to the use of an oil - lubricated compression. using 2 ml of lead standard solution (10 ppm Pb) R.
IMPURITIES Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 0.1000 g by drying in an oven at 105 °C for 4 h.
A. carbon dioxide, Sulphated ash (2.4.14) : maximum 8.0 per cent (dried
substance), determined on 0.100 g.
B. sulphur dioxide, Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
C. nitrogen monoxide, Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
D. nitrogen dioxide,
ASSAY
E. oil, To 0.2500 g add 25 ml of water R, 25.0 ml of 0.1 M sodium
hydroxide and 0.2 ml of phenolphthalein solution R. Titrate
F. carbon monoxide, with 0.1 M hydrochloric acid.
1 ml of 0.1 M sodium hydroxide is equivalent to 4.502 mg
G. water. of carboxyl groups (-CO2H).

4022 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Almagate

FUNCTIONALITY-RELATED CHARACTERISTICS TESTS

This section provides information on characteristics pH (2.2.3) : 9.1 to 9.7.
that are recognised as being relevant control parameters
for one or more functions of the substance when used Disperse 4.0 g in 100 ml of carbon dioxide-free water R, stir
as an excipient (see chapter 5.15). This section is a for 2 min and filter.
non-mandatory part of the monograph and it is not Neutralising capacity. Carry out the test at 37 °C. Disperse
necessary to verify the characteristics to demonstrate 0.5 g in 100 ml of water R, heat, add 100.0 ml of 0.1 M
compliance. Control of these characteristics can however hydrochloric acid, previously heated and stir continuously ;
contribute to the quality of a medicinal product by the pH (2.2.3) of the solution between 5 min and 20 min is
improving the consistency of the manufacturing process not less than 3.0 and not greater than 4.5. Add 10.0 ml of
and the performance of the medicinal product during use. 0.5 M hydrochloric acid, previously heated, stir continuously
Where control methods are cited, they are recognised as for 1 h and titrate with 0.1 M sodium hydroxide to pH 3.5 ;
being suitable for the purpose, but other methods can also not more than 20.0 ml of 0.1 M sodium hydroxide is
be used. Wherever results for a particular characteristic are required.
reported, the control method must be indicated. Chlorides (2.4.4) : maximum 0.1 per cent.
The following characteristics may be relevant for alginic Dissolve 0.33 g in 5 ml of dilute nitric acid R and dilute to
acid used as disintegrant and/or binder. 100 ml with water R. 15 ml of the solution complies with the
Particle-size distribution (2.9.31 or 2.9.38). limit test for chlorides. Prepare simultaneously the standard
Settling volume. Place 75 ml of water R in a 100 ml by diluting 0.7 ml of dilute nitric acid R to 5 ml with water R
graduated cylinder and add 1.5 g of the substance to be and adding 10 ml of chloride standard solution (5 ppm Cl) R.
examined in 0.5 g portions, shaking vigorously after each Sulphates (2.4.13) : maximum 0.4 per cent.
addition. Dilute to 100.0 ml with water R and shake again
until the substance is homogeneously distributed. Allow to Dissolve 0.25 g in 5 ml of dilute hydrochloric acid R
stand for 4 h and determine the volume of the settled mass. and dilute to 100 ml with distilled water R. 15 ml of the
solution complies with the limit test for sulphates. Prepare
The following characteristic may be relevant for alginic simultaneously the standard by adding 0.8 ml of dilute
acid used as gelling agent or viscosity-increasing agent. hydrochloric acid R to 15 ml of sulphate standard solution
Apparent viscosity. Determine the dynamic viscosity using (10 ppm SO4) R.
a rotating viscometer (2.2.10). Sodium : maximum 1.50 × 102 ppm.
Prepare a 20 g/l suspension of alginic acid (dried substance)
Atomic absorption spectrometry (2.2.23, Method I).
and add 0.1 M sodium hydroxide until a solution is obtained.
Test solution. Dissolve 0.25 g in 50 ml of a 103 g/l solution
of hydrochloric acid R.
01/2009:2010 Reference solutions. Prepare the reference solutions using
sodium standard solution (200 ppm Na) R, diluted as
ALMAGATE necessary with a 103 g/l solution of hydrochloric acid R.
Heavy metals (2.4.8) : maximum 20 ppm.
Almagatum Dissolve 1.0 g in dilute hydrochloric acid R and dilute to
20.0 ml with the same acid. 12 ml of the solution complies
Al2Mg6C2O20H14,4H2O Mr 630 with limit test A. Prepare the reference solution using lead
[66827-12-1] standard solution (1 ppm Pb) R.
DEFINITION Loss on ignition : 43.0 per cent to 49.0 per cent, determined
Hydrated aluminium magnesium hydroxycarbonate. on 1.000 g by ignition at 900 ± 50 °C.
Content : Microbial contamination
— aluminium : 15.0 per cent to 17.0 per cent (calculated TAMC : acceptance criterion 103 CFU/g (2.6.12).
as Al2O3), TYMC : acceptance criterion 102 CFU/g (2.6.12).
— magnesium : 36.0 per cent to 40.0 per cent (calculated
Absence of Escherichia coli (2.6.13).
as MgO),
— carbonic acid : 12.5 per cent to 14.5 per cent (calculated Absence of Pseudomonas aeruginosa (2.6.13).
as CO2).
ASSAY
CHARACTERS Aluminium. Dissolve 1.000 g in 5 ml of hydrochloric acid R,
Appearance : white or almost white, fine, crystalline powder. heating if necessary. Allow to cool to room temperature
Solubility : practically insoluble in water, in ethanol and dilute to 100.0 ml with water R (solution A). Introduce
(96 per cent) and in methylene chloride. It dissolves with 10.0 ml of solution A into a 250 ml conical flask, add 25.0 ml
effervescence and heating in dilute mineral acids. of 0.05 M sodium edetate, 20 ml of buffer solution pH 3.5 R,
40 ml of ethanol R and 2 ml of a freshly prepared 0.25 g/l
IDENTIFICATION solution of dithizone R in ethanol R. Titrate the excess of
A. Infrared absorption spectrophotometry (2.2.24). sodium edetate with 0.05 M zinc sulphate until the colour
Comparison : Ph. Eur. reference spectrum of almagate. changes from greenish-violet to pink.
B. Dissolve 0.15 g in dilute hydrochloric acid R and dilute 1 ml of 0.05 M sodium edetate is equivalent to 2.549 mg
to 20 ml with the same acid. 2 ml of the solution gives the of Al2O3.
reaction of aluminium (2.3.1). Magnesium. Introduce 10.0 ml of solution A prepared in the
C. 2 ml of the solution prepared under identification test B assay of aluminium into a 500 ml conical flask, add 200 ml of
gives the reaction of magnesium (2.3.1). water R, 20 ml of triethanolamine R with shaking, 10 ml of

General Notices (1) apply to all monographs and other texts 4023
Aluminium magnesium silicate EUROPEAN PHARMACOPOEIA 6.3

ammonium chloride buffer solution pH 10.0 R and 50 mg Content :

of mordant black 11 triturate R. Titrate with 0.05 M sodium — aluminium (Al ; Ar 26.98) : 95.0 per cent to 105.0 per cent
edetate until the colour changes from violet to pure blue. of the value stated on the label ;
1 ml of 0.05 M sodium edetate is equivalent to 2.015 mg — magnesium (Mg ; Ar 24.30) : 95.0 per cent to 105.0 per
of MgO. cent of the value stated on the label.
Carbonic acid : 12.5 per cent to 14.5 per cent.
CHARACTERS
Test sample. Place 7.00 mg of the substance to be examined Appearance : almost white powder, granules or plates.
in a tin capsule. Seal the capsule.
Solubility : practically insoluble in water and in organic
Reference sample. Place 7.00 mg of almagate CRS in a tin solvents.
capsule. Seal the capsule.
It swells in water to produce a colloidal dispersion.
Introduce separately the test sample and the reference
sample into a combustion chamber of a CHN analyser purged IDENTIFICATION
with helium for chromatography R and maintained at a A. Fuse 1 g with 2 g of anhydrous sodium carbonate R.
temperature of 1020 °C. Simultaneously, introduce oxygen R Warm the residue with water R and filter. Acidify the
at a pressure of 40 kPa and a flow rate of 20 ml/min and filtrate with hydrochloric acid R and evaporate to dryness
allow complete combustion of the sample. Sweep the on a water-bath. 0.25 g of the residue gives the reaction
combustion gases through a reduction reactor and separate of silicates (2.3.1).
the gases formed by gas chromatography (2.2.28).
B. Dissolve the remainder of the residue obtained in
Column: identification test A in a mixture of 5 ml of dilute
— size : l = 2 m, Ø = 4 mm ; hydrochloric acid R and 10 ml of water R. Filter and add
— stationary phase : ethylvinylbenzene-divinylbenzene ammonium chloride buffer solution pH 10.0 R. A white,
copolymer R1. gelatinous precipitate is formed. Centrifuge and keep the
Carrier gas: helium for chromatography R. supernatant for identification C. Dissolve the remaining
precipitate in dilute hydrochloric acid R. The solution
Flow rate : 100 ml/min. gives the reaction of aluminium (2.3.1).
Temperature : C. The supernatant liquid obtained after centrifugation in
— column : 65 °C ; identification test B gives the reaction of magnesium
— detector : 190 °C. (2.3.1).
Detection : thermal conductivity. TESTS
Run time : 16 min. pH (2.2.3) : 9.0 to 10.0.
System suitability : Disperse 5.0 g in 100 ml of carbon dioxide-free water R.
— average percentage of carbon in 5 reference samples must Arsenic (2.4.2, Method A) : maximum 3 ppm.
be within ± 0.2 per cent of the value assigned to the CRS ;
the difference between the upper and the lower values of Transfer 16.6 g to a 250 ml beaker containing 100 ml of
the percentage of carbon in these samples must be below dilute hydrochloric acid R. Mix, cover with a watch glass
0.2 per cent. and boil gently, with occasional stirring, for 15 min. Allow
the insoluble matter to settle and decant the supernatant
Calculate the percentage content of carbonic acid in the testliquid through a rapid-flow filter paper into a 250 ml
sample according to the following formula : volumetric flask, retaining as much sediment as possible
in the beaker. To the residue in the beaker add 25 ml of
hot dilute hydrochloric acid R, stir, heat to boiling, allow
the insoluble matter to settle and decant the supernatant
C = percentage content of carbonic acid in the liquid through the filter into the volumetric flask. Repeat
reference sample ; the extraction with 4 additional quantities, each of 25 ml, of
hot dilute hydrochloric acid R, decanting each supernatant
K = mean value for the 5 reference samples of the liquid through the filter into the volumetric flask. At the
ratio of the mass in milligrams to the area of the last extraction, transfer as much of the insoluble matter
peak due to carbonic acid ; as possible onto the filter. Allow the combined filtrates to
A = area of the peak due to carbonic acid in the cool to room temperature and dilute to 250.0 ml with dilute
chromatogram obtained with the test sample ; hydrochloric acid R. Dilute 5.0 ml of this solution to 25.0 ml
m = sample mass, in milligrams. with dilute hydrochloric acid R.
Lead : maximum 15.0 ppm.
STORAGE Atomic absorption spectrometry (2.2.23, Method I).
In an airtight container. Test solution. Transfer 10.0 g to a 250 ml beaker containing
100 ml of dilute hydrochloric acid R. Mix, cover with a
01/2009:1388 watch glass and boil for 15 min. Allow to cool to room
temperature and allow the insoluble matter to settle. Decant
the supernatant liquid through a rapid-flow filter paper into
ALUMINIUM MAGNESIUM SILICATE a 400 ml beaker. To the insoluble matter in the 250 ml
beaker add 25 ml of hot water R. Stir, allow the insoluble
Aluminii magnesii silicas matter to settle and decant the supernatant liquid through
the filter into the 400 ml beaker. Repeat the extraction with
DEFINITION 2 additional quantities, each of 25 ml, of water R, decanting
Mixture of particles with colloidal particle size of each time the supernatant liquid through the filter into the
montmorillonite and saponite, free from grit and 400 ml beaker. Wash the filter with 25 ml of hot water R,
non-swellable ore. collecting this filtrate in the 400 ml beaker. Concentrate

4024 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
the combined filtrates to about 20 ml by gently boiling. If a
precipitate appears, add about 0.1 ml of nitric acid R, heat
to boiling and allow to cool to room temperature. Filter the
concentrated extracts through a rapid-flow filter paper into a
50 ml volumetric flask. Transfer the remaining contents of
the 400 ml beaker through the filter paper and into the flask
with water R. Dilute this solution to 50.0 ml with water R.
Reference solutions. Prepare the reference solutions using
lead standard solution (10 ppm Pb) R, diluted as necessary
with water R.
Source : lead hollow-cathode lamp.
Wavelength : 217 nm.
Atomisation device : oxidising air-acetylene flame.
Loss on drying (2.2.32) : maximum 8.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
ASSAY
Aluminium. Atomic absorption spectrometry (2.2.23,
Method I).
Test solution. In a platinum crucible mix 0.200 g with
1.0 g of lithium metaborate R. Heat slowly at first and
ignite at 1000-1200 °C for 15 min. Allow to cool, then
place the crucible in a 100 ml beaker containing 25 ml of
dilute nitric acid R and add an additional 50 ml of dilute
nitric acid R, filling and submerging the crucible. Place
a polytetrafluoroethylene-coated magnetic stirring bar in
the crucible and stir gently with a magnetic stirrer until
dissolution is complete. Pour the contents into a 250 ml
beaker and remove the crucible. Warm the solution and
transfer through a rapid-flow filter paper into a 250 ml
volumetric flask, wash the filter and beaker with water R
and dilute to 250.0 ml with water R (solution A). To 20.0 ml
of solution A add 20 ml of a 10 g/l solution of sodium
chloride R and dilute to 100.0 ml with water R.
Reference solutions. Dissolve, with gentle heating, 1.000 g
of aluminium R in a mixture of 10 ml of hydrochloric acid R
and 10 ml of water R. Allow to cool, then dilute to 1000.0 ml
with water R (1 mg of aluminium per millilitre). Into 3
identical volumetric flasks, each containing 0.20 g of sodium
chloride R, introduce 2.0 ml, 5.0 ml and 10.0 ml of this
solution respectively, and dilute to 100.0 ml with water R.
Source : aluminium hollow-cathode lamp.
Wavelength : 309 nm.
Atomisation device : oxidising acetylene-nitrous oxide flame.
Magnesium. Atomic absorption spectrometry (2.2.23,
Method I).
Test solution. Dilute 25.0 ml of solution A, prepared in the
assay for aluminium, to 50.0 ml with water R. To 5.0 ml of
this solution add 20.0 ml of lanthanum nitrate solution R
and dilute to 100.0 ml with water R.
Reference solutions. Place 1.000 g of magnesium R in a
250 ml beaker containing 20 ml of water R and carefully
add 20 ml of hydrochloric acid R, warming if necessary to
dissolve. Transfer the solution to a volumetric flask and
dilute to 1000.0 ml with water R (1 mg of magnesium per
millilitre). Dilute 5.0 ml of this solution to 250.0 ml with
water R. Into 4 identical volumetric flasks, introduce 5.0 ml,
10.0 ml, 15.0 ml and 20.0 ml of the solution respectively. To
each flask add 20.0 ml of lanthanum nitrate solution R and
dilute to 100.0 ml with water R.
Source : magnesium hollow-cathode lamp.
General Notices (1) apply to all monographs and other texts
Aluminium oxide, hydrated
Wavelength : 285 nm.
Atomisation device : reducing air-acetylene flame.
LABELLING
The label states the content of aluminium and magnesium.
01/2009:0311
ALUMINIUM OXIDE, HYDRATED
Aluminii oxidum hydricum
DEFINITION
Content : 47.0 per cent to 60.0 per cent of Al2O3 (Mr 102.0).
CHARACTERS
Appearance : white or almost white, amorphous powder.
Solubility : practically insoluble in water. It dissolves in
dilute mineral acids and in solutions of alkali hydroxides.
IDENTIFICATION
Solution S (see Tests) gives the reaction of aluminium (2.3.1).
TESTS
Solution S. Dissolve 2.5 g in 15 ml of hydrochloric acid R,
heating on a water-bath. Dilute to 100 ml with distilled
water R.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution GY6 (2.2.2, Method II).
Alkaline impurities. Shake 1.0 g with 20 ml of carbon
dioxide-free water R for 1 min and filter. To 10 ml of the
filtrate add 0.1 ml of phenolphthalein solution R. Any
pink colour disappears on the addition of 0.3 ml of 0.1 M
hydrochloric acid.
Neutralising capacity. Carry out the test at 37 °C. Disperse
0.5 g in 100 ml of water R, heat, add 100.0 ml of 0.1 M
hydrochloric acid, previously heated, and stir continuously ;
the pH (2.2.3) of the solution after 10 min, 15 min and
20 min is not less than 1.8, 2.3 and 3.0 respectively and is at
no time greater than 4.5. Add 10.0 ml of 0.5 M hydrochloric
acid, previously heated, stir continuously for 1 h and titrate
with 0.1 M sodium hydroxide to pH 3.5 ; not more than
35.0 ml of 0.1 M sodium hydroxide is required.
Chlorides (2.4.4) : maximum 1 per cent.
Dissolve 0.1 g with heating in 10 ml of dilute nitric acid R
and dilute to 100 ml with water R. Dilute 5 ml of the solution
to 15 ml with water R.
Sulphates (2.4.13) : maximum 1 per cent.
Dilute 4 ml of solution S to 100 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined
on 10 ml of solution S.
Heavy metals (2.4.8) : maximum 60 ppm.
Neutralise 20 ml of solution S with concentrated ammonia R,
using metanil yellow solution R as an external indicator.
Filter, if necessary, and dilute to 30 ml with water R. 12 ml
of the solution complies with test A. Prepare the reference
solution using 10 ml of lead standard solution (1 ppm Pb) R.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of bile-tolerant gram-negative bacteria (2.6.13).
Absence of Escherichia coli (2.6.13).
4025
Aluminium phosphate gel
ASSAY
Dissolve 0.800 g in 10 ml of hydrochloric acid R1, heating
on a water-bath. Cool and dilute to 50.0 ml with water R.
To 10.0 ml of the solution add dilute ammonia R1 until
a precipitate begins to appear. Add the smallest quantity
of dilute hydrochloric acid R needed to dissolve the
precipitate and dilute to 20 ml with water R. Carry out the
complexometric titration of aluminium (2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 5.098 mg
of Al2O3.
STORAGE
In an airtight container, at a temperature not exceeding 30 °C. Cool and centrifuge. Separate the supernatant. Wash the
01/2009:2166
ALUMINIUM PHOSPHATE GEL
Aluminii phosphatis liquamen
DEFINITION
Hydrated AlPO4 in gel form.
Content : 19.0 per cent to 21.0 per cent of AlPO4.
CHARACTERS
Appearance : gel.
Solubility : practically insoluble in water, in ethanol (96 per while shaking. Determine the pH after 15 min. The pH
cent) and in methylene chloride. It dissolves in dilute
solutions of mineral acids.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (b) of phosphates
(2.3.1).
B. Solution S gives the reaction of aluminium (2.3.1).
C. It complies with the assay.
TESTS
Solution S. Dissolve 2.00 g in dilute hydrochloric acid R
and dilute to 100 ml with the same acid.
pH (2.2.3) : 6.0 to 8.0.
Peroxides: maximum 150 ppm, expressed as hydrogen
peroxide.
Test solution. Dissolve with heating 1.0 g of the substance
to be examined in 5 ml of dilute hydrochloric acid R, then
add 5 ml of water R and 2 ml of divanadium pentoxide
solution in sulphuric acid R.
Reference solution. Dilute 1.0 ml of dilute hydrogen
peroxide solution R to 200.0 ml with water R. To 1 ml of
this solution add 9 ml of water R and 2 ml of divanadium
pentoxide solution in sulphuric acid R.
The test solution is not more intensely coloured than the
reference solution.
Chlorides (2.4.4) : maximum 500 ppm.
Dissolve 1.3 g in 5 ml of dilute nitric acid R and dilute to
200 ml with water R.
Soluble phosphates : maximum 0.5 per cent, expressed
as PO4.
Test solution. Centrifuge 10.0 g until a clear supernatant
is obtained. To 2.00 ml of the supernatant add 20.0 ml of
a 10.3 g/l solution of hydrochloric acid R and dilute to
100.0 ml with water R. To 10.0 ml of this solution add 10.0 ml
of nitro-molybdovanadic reagent R and dilute to 50.0 ml
with water R. Allow to stand protected from light for 15 min.
4026 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Reference solution. Add 10.0 ml of nitro-molybdovanadic
reagent R to 10.0 ml of a 143 mg/l solution of potassium
dihydrogen phosphate R and dilute to 50.0 ml with water R.
Allow to stand protected from light for 15 min.
Measure the absorbances (2.2.25) of the 2 solutions at
400 nm. The absorbance of the test solution is not greater
than that of the reference solution.
Sulphates (2.4.13) : maximum 0.2 per cent.
Dilute 25 ml of solution S to 100 ml with distilled water R.
Soluble aluminium : maximum 50 ppm.
To 16.0 g add 50 ml of water R. Heat to boiling for 5 min.
residue with 20 ml of water R and centrifuge. Separate
the supernatant and add to the first supernatant. To the
combined supernatants add 5 ml of hydrochloric acid R and
20 ml of water R. Introduce all of this solution into a 500 ml
conical flask and carry out the complexometric titration of
aluminium (2.5.11) using 0.01 M sodium edetate.
Arsenic (2.4.2, Method A) : maximum 1 ppm, determined
on 1.0 g.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 4.0 g in dilute hydrochloric acid R and dilute to
20 ml with the same acid. 12 ml of the solution complies with
test A. Prepare the reference solution using lead standard
solution (2 ppm Pb) R.
Acid neutralising capacity. Add 2.0 g to 30 ml of 0.1 M
hydrochloric acid R heated to 37 °C and maintain at 37 °C
(2.2.3) of the mixture is 2.0 to 2.5.
Residue on ignition : 19.0 per cent to 23.0 per cent.
Heat 0.500 g at 50 °C for 5 hours, then ignite at 500 ± 50 °C
until constant mass.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of bile-tolerant gram-negative bacteria (2.6.13).
Absence of Escherichia coli (2.6.13).
ASSAY
Dissolve with heating 0.300 g in 5 ml of dilute hydrochloric
acid R. Add 45 ml of water R, 10.0 ml of 0.1 M sodium edetate
and 30 ml of a mixture of equal volumes of ammonium
acetate solution R and dilute acetic acid R. Heat to boiling
and maintain boiling for 3 min. Cool, then add 25 ml of
ethanol (96 per cent) R. Titrate with 0.1 M zinc sulphate,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M zinc sulphate is equivalent to 12.2 mg of AlPO4.
STORAGE
In an airtight container.
01/2009:1676
ALUMINIUM SODIUM SILICATE
Aluminii natrii silicas
DEFINITION
Silicic acid aluminium sodium salt of synthetic origin.
Content :
— aluminium (Al ; Mr 26.98) : 2.7 per cent to 7.9 per cent
(dried substance) ;
— sodium (Na ; Mr 22.99) : 3.7 per cent to 6.3 per cent (dried
substance).
EUROPEAN PHARMACOPOEIA 6.3
CHARACTERS
Appearance : white or almost white, fine, light, amorphous
powder.
Solubility : practically insoluble in water and in organic
solvents.
IDENTIFICATION
A. Transfer 1.0 g to a 100 ml beaker and add 10 ml of dilute
hydrochloric acid R. Mix, cover with a watch glass and
boil for 15 min. Allow to cool to room temperature, mix
and centrifuge the solution. 2 ml of the supernatant gives TAMC : acceptance criterion 103 CFU/g (2.6.12).
the reaction of aluminium (2.3.1).
B. 2 ml of the supernatant obtained in identification test A TYMC : acceptance criterion 10 CFU/g (2.6.12).
gives reaction (a) of sodium (2.3.1).
C. 0.2 g gives the reaction of silicates (2.3.1).
TESTS
pH (2.2.3) : 9.5 to 11.5.
Disperse 5.0 g in 100 ml of carbon dioxide-free water R.
Arsenic (2.4.2, Method A) : maximum 3 ppm.
Transfer 8.3 g to a 250 ml beaker containing 50 ml of dilute
hydrochloric acid R. Mix, cover with a watch glass and boil
gently, with occasional stirring, for 15 min. Centrifuge,
and decant the supernatant through a rapid-flow filter
paper into a 250 ml volumetric flask. To the residue in
the beaker, add 25 ml of hot dilute hydrochloric acid R,
stir, centrifuge, and decant the supernatant through the
same filter into the volumetric flask. Repeat the extraction
with 3 additional quantities, each of 25 ml, of hot dilute
hydrochloric acid R, filtering each supernatant through this
filter into the volumetric flask. Allow the combined filtrates
to cool to room temperature and dilute to 250.0 ml with
dilute hydrochloric acid R. Dilute 10.0 ml of the solution to
25.0 ml with water R.
Lead : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Transfer 5.0 g to a 250 ml beaker containing
50 ml of dilute hydrochloric acid R. Mix, cover with a watch
glass and boil for 15 min. Allow to cool to room temperature.
Centrifuge, and decant the supernatant through a rapid-flow
filter paper into a 250 ml beaker. To the insoluble matter add
25 ml of hot water R. Stir vigorously, centrifuge, and decant
the supernatant through the same filter into the beaker.
Repeat the extraction with 2 additional quantities, each of
25 ml, of hot water R, decanting each supernatant through
the filter into the beaker. Wash the filter with 25 ml of hot
water R, collecting the filtrate in the beaker. Concentrate
the combined filtrates by gently boiling to about 15 ml. Add
about 0.05 ml of heavy metal-free nitric acid R, heat to
boiling and allow to cool to room temperature. Filter the
concentrated extracts through a rapid-flow filter paper into a
25 ml volumetric flask. Transfer the remaining contents of
the beaker through the filter paper and into the volumetric
flask with water R and dilute to 25.0 ml with the same
solvent.
Reference solutions. Into 4 separate 100 ml volumetric
flasks, introduce respectively 3.0 ml, 5.0 ml, 10.0 ml and
15.0 ml of lead standard solution (10 ppm Pb) R, add 0.20 ml (200 ppm Na) R and dilute to 200.0 ml with water R.
of heavy metal-free nitric acid R and dilute to 100.0 ml with
water R.
General Notices (1) apply to all monographs and other texts
Aluminium sodium silicate
Source : lead hollow-cathode lamp.
Wavelength : 217.0 nm.
Atomisation device : air-acetylene flame.
Loss on drying (2.2.32) : maximum 8.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Loss on ignition : 5.0 per cent to 11.0 per cent (dried
substance), determined on 1.000 g by ignition in a platinum
crucible to constant mass at 1000 ± 25 °C.
Microbial contamination
2
Absence of Escherichia coli (2.6.13).
ASSAY
Aluminium. Atomic absorption spectrometry (2.2.23,
Method I).
Acid mixture. Add 50 ml of nitric acid R to 500 ml of
water R. Dissolve in this solution 17 g of tartaric acid R and
dilute to 1000 ml with water R.
Blank solution. Dissolve 1.4 g of anhydrous lithium
metaborate R in 60 ml of the acid mixture and dilute to
200 ml with water R.
Test solution. In a platinum crucible mix 0.200 g with 1.4 g
of anhydrous lithium metaborate R. Heat slowly at first
and ignite at 1100 ± 25 °C for 15 min. Cool, then place the
crucible in a 100 ml beaker containing 60 ml of the acid
mixture. Place a polytetrafluoroethylene-coated magnetic
stirring bar in the crucible and stir gently with a magnetic
stirrer for 16 h. Transfer the contents of the crucible into a
200 ml volumetric flask. Wash the crucible, the magnetic
stirring bar and the beaker with water R and dilute to
200.0 ml with the same solvent (solution A). To 10.0 ml of
this solution, add 1.0 ml of lanthanum chloride solution R
and dilute to 50.0 ml with water R.
Reference solutions. Into 5 separate 50 ml volumetric flasks,
introduce respectively 1.0 ml, 2.5 ml, 5.0 ml, 7.5 ml and
10.0 ml of aluminium standard solution (100 ppm Al) R,
add 1 ml of lanthanum chloride solution R and 10 ml of the
blank solution, and dilute to 50.0 ml with water R.
Source : aluminium hollow-cathode lamp.
Wavelength : 309.3 nm.
Atomisation device : acetylene-nitrous oxide flame.
Sodium. Atomic emission spectrometry (2.2.22, Method I).
Test solution. To 2.0 ml of solution A, prepared in the assay
of aluminium, add 1 ml of a 12.5 g/l solution of caesium
chloride R and dilute to 20.0 ml with water R.
Reference solutions. Into 5 separate 200 ml volumetric
flasks, each containing 10 ml of a 12.5 g/l solution of
caesium chloride R, introduce respectively 1.0 ml, 2.0 ml,
4.0 ml, 6.0 ml and 10.0 ml of sodium standard solution
Wavelength : 589.0 nm.
4027
Amiodarone hydrochloride EUROPEAN PHARMACOPOEIA 6.3

01/2008:0803 System suitability : reference solution (b):

corrected 6.3 — the spot due to impurity H is clearly visible.
Limit :
AMIODARONE HYDROCHLORIDE — impurity H : any spot with the same RF as the spot
due to impurity H in the chromatogram obtained with
Amiodaroni hydrochloridum reference solution (b), is not more intense than the spot
in the chromatogram obtained with reference solution (a)
(0.02 per cent).
Related substances. Liquid chromatography (2.2.29).
Buffer solution pH 4.9. To 800 ml of water R add 3.0 ml
of glacial acetic acid R, adjust to pH 4.9 with dilute
ammonia R1 and dilute to 1000 ml with water R.
Test solution. Dissolve 0.125 g of the substance to be
C25H30ClI2NO3 Mr 682 examined in a mixture of equal volumes of acetonitrile R
[19774-82-4] and water R and dilute to 25.0 ml with the same mixture
DEFINITION of solvents.
(2-Butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3,5- Reference solution. Dissolve 5 mg of amiodarone
diiodophenyl]methanone hydrochloride. impurity D CRS, 5 mg of amiodarone impurity E CRS and
5.0 mg of amiodarone hydrochloride CRS in methanol R
Content : 98.5 per cent to 101.0 per cent (dried substance). and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of
CHARACTERS this solution to 20.0 ml with a mixture of equal volumes of
Appearance : white or almost white, fine, crystalline powder. acetonitrile R and water R.
Solubility : very slightly soluble in water, freely soluble inColumn :
methylene chloride, soluble in methanol, sparingly soluble — size: l = 0.15 m, Ø = 4.6 mm ;
in ethanol (96 per cent). — stationary phase : octadecylsilyl silica gel for
IDENTIFICATION chromatography R (5 μm) ;
A. Infrared absorption spectrophotometry (2.2.24). — temperature : 30 °C.
Comparison : amiodarone hydrochloride CRS. Mobile phase : buffer solution pH 4.9, methanol R,
acetonitrile R (30:30:40 V/V/V).
B. It gives reaction (b) of chlorides (2.3.1).
Flow rate : 1 ml/min.
TESTS Detection : spectrophotometer at 240 nm.
Appearance of solution. The solution is clear (2.2.1) and not Injection : 10 μl.
more intensely coloured than reference solution GY5 or BY5
(2.2.2, Method II). Run time : twice the retention time of amiodarone.
Dissolve 1.0 g in methanol R and dilute to 20 ml with the Relative retention with reference to amiodarone
same solvent. (retention time = about 24 min) : impurity A = about 0.26 ;
impurity D = about 0.29 ; impurity E = about 0.37 ;
pH (2.2.3) : 3.2 to 3.8. impurity B = about 0.49 ; impurity C = about 0.55 ;
Dissolve 1.0 g in carbon dioxide-free water R, heating at impurity G = about 0.62 ; impurity F = about 0.69.
80 °C, cool and dilute to 20 ml with the same solvent.
System suitability: reference solution :
Impurity H. Thin-layer chromatography (2.2.27). Prepare — resolution : minimum 3.5 between the peaks due to
the solutions immediately before use and keep protected impurities D and E.
from bright light.
Test solution. Dissolve 0.500 g of the substance to be Limits :
examined in methylene chloride R and dilute to 5.0 ml with — impurities A, B, C, D, E, F, G : for each impurity, not
the same solvent. more than the area of the peak due to amiodarone in
Reference solution (a). Dissolve 10.0 mg of the chromatogram obtained with the reference solution
(2-chloroethyl)diethylamine hydrochloride R (impurity H) (0.2 per cent) ;
in methylene chloride R and dilute to 50.0 ml with the — unspecified impurities : for each impurity, not more
same solvent. Dilute 2.0 ml of this solution to 20.0 ml with than 0.5 times the area of the peak due to amiodarone in
methylene chloride R. the chromatogram obtained with the reference solution
Reference solution (b). Mix 2.0 ml of the test solution and (0.10 per cent) ;
2.0 ml of reference solution (a). — total : not more than 2.5 times the area of the peak due
Plate : TLC silica gel F254 plate R. to amiodarone in the chromatogram obtained with the
Mobile phase : anhydrous formic acid R, methanol R, reference solution (0.5 per cent) ;
methylene chloride R (5:10:85 V/V/V). — disregard limit : 0.25 times the area of the peak due
Application : 50 μl of the test solution and reference to amiodarone in the chromatogram obtained with the
solution (a) ; 100 μl of reference solution (b). reference solution (0.05 per cent).
Development : over 2/3 of the plate. Iodides : maximum 150 ppm.
Drying : in a current of cold air. Prepare the test and reference solutions simultaneously.
Detection : spray with potassium iodobismuthate Solution A. Add 1.50 g of the substance to be examined
solution R1 and then with dilute hydrogen peroxide to 40 ml of water R at 80 °C and shake until completely
solution R ; examine immediately in daylight. dissolved. Cool and dilute to 50.0 ml with water R.

4028 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Amitriptyline hydrochloride

Test solution. To 15.0 ml of solution A add 1.0 ml of

0.1 M hydrochloric acid and 1.0 ml of 0.05 M potassium
iodate. Dilute to 20.0 ml with water R. Allow to stand
protected from light for 4 h.
Reference solution. To 15.0 ml of solution A add 1.0 ml of
0.1 M hydrochloric acid, 1.0 ml of an 88.2 mg/l solution of
potassium iodide R and 1.0 ml of 0.05 M potassium iodate.
Dilute to 20.0 ml with water R. Allow to stand protected D. R1 = R2 = I : (2-butylbenzofuran-3-yl)(4-hydroxy-3,5-
from light for 4 h. diiodophenyl)methanone,
Measure the absorbances (2.2.25) of the solutions at 420 nm,
using a mixture of 15.0 ml of solution A and 1.0 ml of E. R1 = R2 = H : (2-butylbenzofuran-3-yl)(4-
0.1 M hydrochloric acid diluted to 20.0 ml with water R as hydroxyphenyl)methanone,
the compensation liquid. The absorbance of the test solution
F. R1 = I, R2 = H : (2-butylbenzofuran-3-yl)(4-hydroxy-3-
is not greater than half the absorbance of the reference
iodophenyl)methanone,
solution.
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 at 50 °C at a pressure not exceeding H. 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine,
0.3 kPa for 4 h. (2-chloroethyl)diethylamine).
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
01/2008:0464
ASSAY corrected 6.3
Dissolve 0.600 g in a mixture of 5.0 ml of 0.01 M hydrochloric
acid and 75 ml of ethanol (96 per cent) R. Carry out AMITRIPTYLINE HYDROCHLORIDE
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion. Amitriptylini hydrochloridum
1 ml of 0.1 M sodium hydroxide is equivalent to 68.18 mg
of C25H30ClI2NO3.

STORAGE
Protected from light, at a temperature not exceeding 30 °C.

IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.
A. R1 = R2 = R4 = H, R3 = C2H5 : (2-butylbenzofuran-3-yl)[4-
[2-(diethylamino)ethoxy]phenyl]methanone,
B. R1 = R2 = I, R3 = R4 = H : (2-butylbenzofuran-3-yl)[4-[2-
(ethylamino)ethoxy]-3,5-diiodophenyl]methanone,
C. R1 = I, R2 = R4 = H, R3 = C2H5 : (2-butylbenzofuran-3-
yl)[4-[2-(diethylamino)ethoxy]-3-iodophenyl]methanone,
G. R1 = R2 = I, R3 = C2H5, R4 = OCH3 :
[4-[2-(diethylamino)ethoxy]-3,5-
diiodophenyl][2-[(1RS)-1-methoxybutyl]benzo-
furan-3-yl]methanone,
C20H24ClN Mr 313.9
[549-18-8]
DEFINITION
3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-N,N-
dimethylpropan-1-amine hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder or colourless
crystals.
Solubility : freely soluble in water, in ethanol (96 per cent)
and in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : amitriptyline hydrochloride CRS.
B. 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution B7 (2.2.2,
Method II).
Dissolve 1.25 g in water R and dilute to 25 ml with the same
solvent.

General Notices (1) apply to all monographs and other texts 4029
Amitriptyline hydrochloride EUROPEAN PHARMACOPOEIA 6.3

Acidity or alkalinity. Dissolve 0.20 g in carbon dioxide-free ASSAY

water R and dilute to 10 ml with the same solvent. Add Dissolve 0.250 g in 30 ml of ethanol (96 per cent) R. Titrate
0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium with 0.1 M sodium hydroxide, determining the end-point
hydroxide. The solution is yellow. Add 0.4 ml of 0.01 M potentiometrically (2.2.20).
hydrochloric acid. The solution is red.
1 ml of 0.1 M sodium hydroxide is equivalent to 31.39 mg
Related substances. Liquid chromatography (2.2.29). of C20H24ClN.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the STORAGE
mobile phase. Protected from light.
Reference solution (a). Dissolve 5.0 mg of
dibenzosuberone CRS (impurity A) and 5.0 mg of IMPURITIES
cyclobenzaprine hydrochloride CRS (impurity B) in 5.0 ml Specified impurities: A, B.
of the test solution and dilute to 100.0 ml with the mobile Other detectable impurities (the following substances
phase. would, if present at a sufficient level, be detected by one
Reference solution (b). Dilute 1.0 ml of reference solution (a) or other of the tests in the monograph. They are limited
to 50.0 ml with the mobile phase. by the general acceptance criterion for other/unspecified
Column: impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
— size : l = 0.15 m, Ø = 4.6 mm ; identify these impurities for demonstration of compliance.
— stationary phase : end-capped polar-embedded See also 5.10. Control of impurities in substances for
octadecylsilyl amorphous organosilica polymer R pharmaceutical use) : C, D, E, F, G.
(5 μm) ;
— temperature : 40 °C.
Mobile phase : mix 35 volumes of acetonitrile R and
65 volumes of a 5.23 g/l solution of dipotassium hydrogen
phosphate R previously adjusted to pH 7.0 with phosphoric
acid R.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 220 nm. A. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one
Injection : 10 μl. (dibenzosuberone),
Run time : 3 times the retention time of amitriptyline.
Relative retention with reference to amitriptyline
(retention time = about 14 min) : impurity B = about 0.9 ;
impurity A = about 2.2.
System suitability : reference solution (a) :
— resolution : minimum 2.0 between the peaks due to
impurity B and amitriptyline.
Limits : B. 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,N-
— impurity B : not more than the area of the corresponding dimethylpropan-1-amine (cyclobenzaprine),
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent) ;
— impurity A : not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.05 per cent) ;
— unspecified impurities: for each impurity, not more
than the area of the peak due to amitriptyline in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ; C. 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-N-
— total : not more than 3 times the area of the peak due methylpropan-1-amine (nortriptyline),
to amitriptyline in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
— disregard limit: 0.5 times the area of the peak due
to amitriptyline in the chromatogram obtained with
reference solution (b) (0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
D. R = CH2-CH2-CH2-N(CH3)2 : 5-[3-(dimethylamino)propyl]-
Loss on drying (2.2.32) : maximum 0.5 per cent, determined 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol,
on 1.000 g by drying in an oven at 105 °C for 2 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined G. R = H : 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol
on 1.0 g. (dibenzosuberol),

4030 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Amphotericin B

IDENTIFICATION
First identification : B, D.
Second identification : A, C.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 25 mg in 5 ml of dimethyl
sulphoxide R and dilute to 50 ml with methanol R. Dilute
E. N,N-dimethyl-3-(1,2,3,4,4a,10,11,11a-octahydro-5H- 2 ml of the solution to 200 ml with methanol R.
dibenzo[a,d][7]annulen-5-ylidene)propan-1-amine,
Spectral range : 300-450 nm.
Absorption maxima: at 362 nm, 381 nm and 405 nm.
Absorbance ratios :
— A362/A381 = 0.57 to 0.61 ;
— A381/A405 = 0.87 to 0.93.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : amphotericin B CRS.
F. (5EZ,10RS)-5-[3-(dimethylamino)propylidene]-10,11- If the spectra obtained show differences, dry the
dihydro-5H-dibenzo[a,d][7]annulen-10-ol. substance to be examined and reference substance at
60 °C at a pressure not exceeding 0.7 kPa for 1 h and
record new spectra.
01/2009:1292 C. To 1 ml of a 0.5 g/l solution in dimethyl sulphoxide R,
add 5 ml of phosphoric acid R to form a lower layer,
avoiding mixing the 2 liquids. A blue ring is immediately
AMPHOTERICIN B produced at the junction of the liquids. Mix, an intense
blue colour is produced. Add 15 ml of water R and mix ;
Amphotericinum B the solution becomes pale yellow.
D. Examine the chromatograms obtained in the test for
related substances.
Results : the principal peak in the chromatogram obtained
with the test solution at 383 nm is similar in retention
time to the principal peak in the chromatogram obtained
with reference solution (a).

TESTS

Related substances. Liquid chromatography (2.2.29).

Protect the solutions from light and use within 24 h of
preparation, except for reference solution (c) which should
be injected immediately after its preparation.
Solvent mixture : 10 g/l solution of ammonium acetate R,
N-methylpyrrolidone R, methanol R (1:1:2 V/V/V).
Test solution. Dissolve 20.0 mg of the substance to be
C47H73NO17 Mr 924 examined in 15 ml of N-methylpyrrolidone R and within 2 h
[1397-89-3] dilute to 50.0 ml with the solvent mixture. Dilute 5.0 ml of
this solution to 25.0 ml with the solvent mixture.
DEFINITION Reference solution (a). Dissolve 20.0 mg of
Mixture of antifungal polyenes produced by the growth of amphotericin B CRS in 15 ml of N-methylpyrrolidone R and
certain strains of Streptomyces nodosus or obtained by any within 2 h dilute to 50.0 ml with the solvent mixture. Dilute
other means. It consists mainly of amphotericin B which is 5.0 ml of this solution to 25.0 ml with the solvent mixture.
(1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E,25E, Reference solution (b). Dilute 1.0 ml of reference solution (a)
27E,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6-dideoxy-β- to 100.0 ml with the solvent mixture.
D-mannopyranosyl)oxy]-1,3,5,6,9,11,17,37-octahydroxy-15,16,
18-trimethyl-13-oxo-14,39-dioxabicyclo[33.3.1]nonatriaconta- Reference solution (c). Dissolve 20.0 mg of nystatin CRS
19,21,23,25,27,29,31-heptaene-36-carboxylic acid. in 15 ml of N-methylpyrrolidone R and within 2 h dilute
to 50.0 ml with the solvent mixture. Dilute 5.0 ml of the
Content : minimum 750 IU/mg (dried substance). solution to 25.0 ml with solution A. Dilute 2.0 ml of this
CHARACTERS solution to 100.0 ml with the solvent mixture.
Appearance : yellow or orange, hygroscopic powder. Reference solution (d). In order to prepare impurities B
and C, dissolve 10 mg of the substance to be examined in
Solubility : practically insoluble in water, soluble in dimethyl 5 ml of N-methylpyrrolidone R and within 2 h add 35 ml
sulphoxide and in propylene glycol, slightly soluble in of a mixture of 1 volume of methanol R and 4 volumes of
dimethylformamide, very slightly soluble in methanol, anhydrous ethanol R. Add 0.10 ml of dilute hydrochloric
practically insoluble in ethanol (96 per cent). acid R, mix and incubate at 25 °C for 2.5 h. Add 10 ml of
It is sensitive to light in dilute solutions. 10 g/l solution of ammonium acetate R and mix.

General Notices (1) apply to all monographs and other texts 4031
Amphotericin B EUROPEAN PHARMACOPOEIA 6.3

Reference solution (e). Dissolve 4 mg of amphotericin B for — impurity B at 383 nm : not more than 4 times the area
peak identification CRS (containing impurities A and B) of the principal peak in the chromatogram obtained with
in 5 ml of N-methylpyrrolidone R and within 2 h dilute to reference solution (b) (4.0 per cent) ;
50 ml with the solvent mixture.
— any other impurity at 383 nm : for each impurity, not
Blank solution. The solvent mixture. more than 2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
Column: (2.0 per cent) ;
— size : l = 0.15 m, Ø = 4.6 mm ; — total at 303 and 383 nm : maximum 15.0 per cent ;
— stationary phase : base-deactivated end-capped — disregard limit at 303 nm : 0.05 times the area of the
octadecylsilyl silica gel for chromatography R (3 μm) ; principal peak in the chromatogram obtained with
reference solution (c) (0.1 per cent) ;
— temperature : 20 °C.
— disregard limit at 383 nm : 0.1 times the area of the
Mobile phase : principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent).
— mobile phase A : mix 1 volume of methanol R, 3 volumes
of acetonitrile R and 6 volumes of a 4.2 g/l solution Loss on drying (2.2.32) : maximum 5.0 per cent, determined
of citric acid R previously adjusted to pH 4.7 using on 1.000 g by drying in an oven at 60 °C at a pressure not
concentrated ammonia R ; exceeding 0.7 kPa.
— mobile phase B : mix 12 volumes of methanol R, Sulphated ash (2.4.14) : maximum 3.0 per cent, determined
20 volumes of a 4.2 g/l solution of citric acid R previously on 1.0 g ; if intended for use in the manufacture of parenteral
adjusted to pH 3.9 using concentrated ammonia R and preparations : maximum 0.5 per cent.
68 volumes of acetonitrile R ; Bacterial endotoxins (2.6.14) : less than 1.0 IU/mg,
if intended for use in the manufacture of parenteral
Time Mobile phase A Mobile phase B preparations without a further appropriate procedure for the
(min) (per cent V/V) (per cent V/V) removal of bacterial endotoxins.
0-3 100 0
3 - 23 100 → 70 0 → 30 ASSAY
23 - 33 70 → 0 30 → 100 Protect all solutions from light throughout the assay.
33 - 40 0 100 Dissolve 25.0 mg in dimethyl sulphoxide R and dilute,
with shaking, to 25.0 ml with the same solvent. Under
Flow rate : 0.8 ml/min. constant stirring of this stock solution, dilute with
dimethyl sulphoxide R to obtain solutions of appropriate
Detection : spectrophotometer : concentrations (the following concentrations have been
found suitable : 44.4, 66.7 and 100 IU/ml). Prepare final
— at 303 nm : detection of tetraenes ; solutions by diluting 1:20 with 0.2 M phosphate buffer
solution pH 10.5 so that they all contain 5 per cent V/V of
— at 383 nm : detection of heptaenes.
dimethyl sulphoxide. Prepare the reference and the test
Injection : 20 μl of the test solution and reference solutions simultaneously. Carry out the microbiological
solutions (b), (c), (d) and (e). assay of antibiotics (2.7.2).

Identification of impurities: use the chromatograms

supplied with amphotericin B for peak identification CRS STORAGE
and the chromatograms obtained with reference solution (e) Protected from light, at a temperature of 2 °C to 8 °C in
to identify the peaks due to impurities A and B. an airtight container. If the substance is sterile, store in a
Relative retention with reference to amphotericin B sterile, tamper-proof container.
(retention time = about 16 min) : impurity B = about 0.75 ;
impurity A = about 0.8 ; nystatin = about 0.85. LABELLING
System suitability at 383 nm : reference solution (d) : The label states, where applicable, that the substance
is suitable for use in the manufacture of parenteral
— resolution : minimum 1.5 between the 2 peaks presenting preparations.
a relative retention of about 0.7.
Limits : IMPURITIES
— impurity A at 303 nm : not more than 2.5 times the area Specified impurities : A, B.
of the principal peak in the chromatogram obtained with
reference solution (c) (5.0 per cent) ; if intended for use in Other detectable impurities (the following substances
the manufacture of parenteral preparations : not more would, if present at a sufficient level, be detected by one
than the area of the principal peak in the chromatogram or other of the tests in the monograph. They are limited
obtained with reference solution (c) (2.0 per cent) ; by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
— any other impurity at 303 nm : for each impurity, not pharmaceutical use (2034). It is therefore not necessary to
more than 0.5 times the area of the principal peak in identify these impurities for demonstration of compliance.
the chromatogram obtained with reference solution (c) See also 5.10. Control of impurities in substances for
(1.0 per cent) ; pharmaceutical use) : C.

4032 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Aprotinin

DEFINITION
Aprotinin is a polypeptide consisting of a chain of 58 amino
acids. It inhibits stoichiometrically the activity of several
proteolytic enzymes such as chymotrypsin, kallikrein,
plasmin and trypsin. It contains not less than 3.0 Ph. Eur. U.
of aprotinin activity per milligram, calculated with reference
to the dried substance.
PRODUCTION
The animals from which aprotinin is derived must fulfil the
requirements for the health of animals suitable for human
consumption to the satisfaction of the competent authority.
The method of manufacture is validated to demonstrate that
the product, if tested, would comply with the following tests.
A. amphotericin A (28,29-dihydro-amphotericin B), Abnormal toxicity (2.6.9). Inject into each mouse a quantity
of the substance to be examined containing 2 Ph. Eur. U.
dissolved in a sufficient quantity of water for injections R to
give a volume of 0.5 ml.
Histamine (2.6.10) : maximum 0.2 μg of histamine base per
3 Ph. Eur. U.
CHARACTERS
Appearance : almost white hygroscopic powder.
Solubility : soluble in water and in isotonic solutions,
practically insoluble in organic solvents.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Solution S (see Tests).
Reference solution. Dilute aprotinin solution BRP in
water R to obtain a concentration of 15 Ph. Eur. U./ ml.
B. amphotericin X1 (13-O-methyl-amphotericin B), Plate : TLC silica gel G plate R.
Mobile phase : water R, glacial acetic acid R (80:100 V/V)
containing 100 g/l of sodium acetate R.
Application : 10 μl.
Development : over a path of 12 cm.
Drying : in air.
Detection : spray with a solution of 0.1 g of ninhydrin R
in a mixture of 6 ml of a 10 g/l solution of cupric
chloride R, 21 ml of glacial acetic acid R and 70 ml of
anhydrous ethanol R. Dry the plate at 60 °C.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Determine the ability of the substance to be examined to
C. amphotericin X2 (13-O-ethyl-amphotericin B). inhibit trypsin activity using the method described below.
Test solution. Dilute 1 ml of solution S to 50 ml with
01/2009:0580 buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin BRP in
APROTININ 0.002 M hydrochloric acid and dilute to 100 ml with the
same acid.
Aprotininum Casein solution. Dissolve 0.2 g of casein R in buffer
solution pH 7.2 R and dilute to 100 ml with the same
buffer solution.
Precipitating solution : glacial acetic acid R, water R,
anhydrous ethanol R (1:49:50 V/V/V).
Mix 1 ml of the test solution with 1 ml of the trypsin
solution. Allow to stand for 10 min and add 1 ml of the
casein solution. Incubate at 35 °C for 30 min. Cool in
iced water and add 0.5 ml of the precipitating solution.
Shake and allow to stand at room temperature for 15 min.
The solution is cloudy. Carry out a blank test under the
same conditions using buffer solution pH 7.2 R instead of
C284H432N84O79S7 Mr 6511 the test solution. The solution is not cloudy.

General Notices (1) apply to all monographs and other texts 4033
Aprotinin
TESTS
Solution S. Prepare a solution of the substance to be
examined containing 15 Ph. Eur. U./ml, calculated from the
activity stated on the label.
Appearance of solution. Solution S is clear (2.2.1).
Absorbance (2.2.25) : maximum 0.80 by measuring at the
absorption maximum at 277 nm.
Prepare a solution of the substance to be examined
containing 3.0 Ph. Eur. U./ml.
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin. Capillary
zone electrophoresis (2.2.47) : use the normalisation
procedure.
Test solution. Prepare a solution of the substance
to be examined in water R containing not less than
1 Ph. Eur. U./ml.
Reference solution. Dilute aprotinin solution BRP in
water R to obtain the same concentration as the test solution.
Capillary :
— material: uncoated fused silica ;
— size : effective length = 45-60 cm, Ø = 75 μm.
Temperature : 25 °C.
CZE buffer. Dissolve 8.21 g of potassium dihydrogen
phosphate R in 400 ml of water R, adjust to pH 3.0 with
phosphoric acid R, dilute to 500.0 ml with water R and filter
through a membrane filter (nominal pore size 0.45 μm).
Detection : spectrophotometer at 214 nm.
Between-run rinsing : rinse the capillary for at least 1 min
with 0.1 M sodium hydroxide filtered through a membrane
filter (nominal pore size 0.45 μm) and for 2 min with the
CZE buffer.
Injection : under pressure or vacuum (for example, 3 s at a
differential pressure of 3.5 kPa).
Migration : apply a field strength of 0.2 kV/cm, using the
CZE buffer as the electrolyte in both buffer reservoirs.
Run time : 30 min.
Identification of impurities: use the electropherogram
supplied with aprotinin solution BRP and the
electropherogram obtained with the reference solution to
identify the peaks due to impurities A and B.
Relative migration with reference to aprotinin (migration
time = about 22 min) : impurity A = about 0.98 ;
impurity B = about 0.99.
System suitability : reference solution after at least
6 injections :
— migration time : aprotinin = 19.0 min to 25.0 min ;
— resolution : minimum 0.8 between the peaks due to
impurities A and B ; minimum 0.5 between the peaks due
to impurity B and aprotinin ;
— peak distribution : the electrophoregram obtained
is qualitatively and quantitatively similar to the
electropherogram supplied with aprotinin solution BRP ;
— height of the principal peak : at least 1000 times the
height of the baseline noise. If necessary, adjust the
sample load to give peaks of sufficient height.
Limits :
— impurity A : maximum 8.0 per cent ;
— impurity B : maximum 7.5 per cent.
Pyroglutamyl-aprotinin and related compounds. Liquid
chromatography (2.2.29) : use the normalisation procedure.
Test solution. Prepare a solution of the substance
to be examined in mobile phase A, containing about
5 Ph. Eur. U./ml.
4034 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Reference solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS in mobile phase A to
obtain the same concentration as the test solution.
Column :
— size: l = 0.075 m, Ø = 7.5 mm ;
— stationary phase : strong cation-exchange silica gel for
chromatography R (10 μm) ;
— temperature : 40 °C.
Mobile phase :
— mobile phase A : dissolve 3.52 g of potassium dihydrogen
phosphate R and 7.26 g of disodium hydrogen phosphate
dihydrate R in 1000 ml of water ; filter and degas ;
— mobile phase B : dissolve 3.52 g of potassium dihydrogen
phosphate R, 7.26 g of disodium hydrogen phosphate
dihydrate R and 66.07 g of ammonium sulphate R in
1000 ml of water ; filter and degas ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 21 92 → 64 8 → 36
21 - 30 64 → 0 36 → 100
30 - 31 0 → 92 100 → 8
31 - 40 92 8
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 40 μl.
Relative retention with reference to aprotinin (retention
time = 17.0 min to 20.0 min) : impurity C = about 0.9.
System suitability : reference solution :
— resolution : minimum 1.5 between the peaks due to
impurity C and aprotinin ;
— symmetry factor : maximum 1.3 for the peak due to
aprotinin.
Limits :
— impurity C : maximum 1.0 per cent ;
— any other impurity: maximum 0.5 per cent ;
— sum of impurities other than C : maximum 1.0 per cent.
Aprotinin oligomers. Size-exclusion chromatography
(2.2.30) : use the normalisation procedure.
Test solution. Prepare a solution of the substance to be
examined in water R containing about 5 Ph. Eur. U./ml.
Reference solution. Treat the substance to be examined to
obtain about 2 per cent aprotinin oligomers. For example,
heat freeze-dried aprotinin at about 110 °C for about 4 h.
Then dissolve in water R to obtain a concentration of about
5 Ph. Eur. U./ml.
Column : 3 columns coupled in series :
— size: l = 0.30 m, Ø = 7.8 mm ;
— stationary phase : hydrophilic silica gel for
chromatography R of a grade suitable for fractionation of
globular proteins in the relative molecular mass range of
20 000 to 10 000 000 (8 μm).
Mobile phase : acetonitrile R, glacial acetic acid R, water R
(2:2:6 V/V/V) ; filter and degas.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 277 nm.
Injection : 100 μl.
Run time : 40 min.
Relative retention with reference to aprotinin monomer
(retention time = 24.5 min to 25.5 min) : aprotinin
dimer = about 0.9.
EUROPEAN PHARMACOPOEIA 6.3 Aprotinin concentrated solution

System suitability : reference solution : under the same conditions, a titration using 1.0 ml of the
— resolution : minimum 1.3 between the peaks due to dilute trypsin solution. Determine the number of millilitres
aprotinin dimer and monomer ; of 0.1 M sodium hydroxide used per second (n2 ml).
— symmetry factor : maximum 2.5 for the peak due to Calculate the aprotinin activity in European Pharmacopoeia
aprotinin monomer. Units per milligram using the following expression :
Limit :
— total : maximum 1.0 per cent.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined The estimated activity is not less than 90 per cent and not
on 0.100 g by drying in vacuo. more than 110 per cent of the activity stated on the label.
Bacterial endotoxins (2.6.14) : less than 0.14 IU per STORAGE
European Pharmacopoeia Unit of aprotinin, if intended for In an airtight, tamper-proof container, protected from light.
use in the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial LABELLING
endotoxins. The label states :
ASSAY — the number of European Pharmacopoeia Units of
aprotinin activity per milligram ;
The activity of aprotinin is determined by measuring its
inhibitory action on a solution of trypsin of known activity. — where applicable, that the substance is suitable for use in
The inhibiting activity of the aprotinin is calculated from the manufacture of parenteral preparations.
the difference between the initial activity and the residual IMPURITIES
activity of the trypsin.
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml, provided
with :
— a device that will maintain a temperature of 25 ± 0.1 °C ;
— a stirring device, such as a magnetic stirrer ;
— a lid with 5 holes for accommodating the electrodes, the
tip of a burette, a tube for the admission of nitrogen and
the introduction of the reagents. A. Ra = H, Rb = OH : aprotinin-(1-56)-peptide,
An automatic or manual titration apparatus may be used. In B. Ra = H, Rb = Gly-OH : aprotinin-(1-57)-peptide,
the latter case the burette is graduated in 0.05 ml and the
pH-meter is provided with a wide reading scale and glass and C. Ra = Glp, Rb = Gly-Ala-OH : (5-oxoprolyl)aprotinin
calomel or glass-silver-silver chloride electrodes. (pyroglutamylaprotinin).
Test solution. Prepare a solution of the substance to be
examined in 0.0015 M borate buffer solution pH 8.0 R
expected to contain 1.67 Ph. Eur. U./ml (about 0.6 mg 01/2009:0579
(m mg) per millilitre).
Trypsin solution. Prepare a solution of trypsin BRP APROTININ CONCENTRATED
containing about 0.8 microkatals per millilitre (about SOLUTION
1 mg/ml), using 0.001 M hydrochloric acid as the solvent.
Use a freshly prepared solution and keep in iced water.
Aprotinini solutio concentrata
Trypsin and aprotinin solution. To 4.0 ml of the trypsin
solution add 1.0 ml of the test solution. Dilute immediately
to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water. Use within 6 h of preparation.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution
to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and
stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer
solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l C284H432N84O79S7 Mr 6511
solution of benzoylarginine ethyl ester hydrochloride R.
Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the DEFINITION
temperature has reached equilibrium at 25 ± 0.1 °C, add Aprotinin concentrated solution is a solution of aprotinin, a
1.0 ml of the trypsin and aprotinin solution and start a polypeptide consisting of a chain of 58 amino acids, which
timer. Maintain at pH 8.0 by the addition of 0.1 M sodium inhibits stoichiometrically the activity of several proteolytic
hydroxide and note the volume added every 30 s. Continue enzymes such as chymotrypsin, kallikrein, plasmin and
the reaction for 6 min. Determine the number of millilitres of trypsin. It contains not less than 15.0 Ph. Eur. U. of aprotinin
0.1 M sodium hydroxide used per second (n1 ml). Carry out, activity per millilitre.

General Notices (1) apply to all monographs and other texts 4035
Aprotinin concentrated solution
PRODUCTION
The animals from which aprotinin is derived must fulfil the
requirements for the health of animals suitable for human
consumption to the satisfaction of the competent authority.
The method of manufacture is validated to demonstrate that
the product, if tested, would comply with the following tests.
Abnormal toxicity (2.6.9). Inject into each mouse a quantity
of the preparation to be examined containing 2 Ph. Eur. U.
diluted with a sufficient quantity of water for injections R to
give a volume of 0.5 ml.
Histamine (2.6.10) : maximum 0.2 μg of histamine base per
3 Ph. Eur. U.
CHARACTERS
Appearance : clear, colourless liquid.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Solution S (see Tests).
Reference solution. Dilute aprotinin solution BRP in
water R to obtain a concentration of 15 Ph. Eur. U./ ml.
Plate : TLC silica gel G plate R.
Mobile phase : water R, glacial acetic acid R (80:100 V/V)
containing 100 g/l of sodium acetate R.
Application : 10 μl.
Development : over a path of 12 cm.
Drying : in air.
Detection : spray with a solution of 0.1 g of ninhydrin R
in a mixture of 6 ml of a 10 g/l solution of cupric
chloride R, 21 ml of glacial acetic acid R and 70 ml of
anhydrous ethanol R. Dry the plate at 60 °C.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Determine the ability of the preparation to be examined to
inhibit trypsin activity using the method described below.
Test solution. Dilute 1 ml of solution S to 50 ml with
buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin BRP in
0.002 M hydrochloric acid and dilute to 100 ml with the
same acid.
Casein solution. Dissolve 0.2 g of casein R in buffer
solution pH 7.2 R and dilute to 100 ml with the same
buffer solution.
Precipitating solution : glacial acetic acid R, water R,
anhydrous ethanol R (1:49:50 V/V/V).
Mix 1 ml of the test solution with 1 ml of the trypsin
solution. Allow to stand for 10 min and add 1 ml of the
casein solution. Incubate at 35 °C for 30 min. Cool in
iced water and add 0.5 ml of the precipitating solution.
Shake and allow to stand at room temperature for 15 min.
The solution is cloudy. Carry out a blank test under the
same conditions using buffer solution pH 7.2 R instead of
the test solution. The solution is not cloudy.
TESTS
Solution S. Prepare a solution containing 15 Ph. Eur. U./ml,
if necessary by dilution, on the basis of the activity stated
on the label.
Appearance of solution. Solution S is clear (2.2.1).
Absorbance (2.2.25) : maximum 0.80 by measuring at the
absorption maximum at 277 nm.
Prepare a solution containing 3.0 Ph. Eur. U./ml.
4036 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin. Capillary
zone electrophoresis (2.2.47) : use the normalisation
procedure.
Test solution. Dilute the preparation to be examined in
water R to obtain a concentration of not less than 1 Ph Eur.
U./ml.
Reference solution. Dilute aprotinin solution BRP in
water R to obtain the same concentration as the test solution.
Capillary :
— material: uncoated fused silica ;
— size: effective length = 45-60 cm, Ø = 75 μm.
Temperature : 25 °C.
CZE buffer. Dissolve 8.21 g of potassium dihydrogen
phosphate R in 400 ml of water R, adjust to pH 3.0 with
phosphoric acid R, dilute to 500.0 ml with water R and filter
through a membrane filter (nominal pore size 0.45 μm).
Detection : spectrophotometer at 214 nm.
Between-run rinsing : rinse the capillary for at least 1 min
with 0.1 M sodium hydroxide filtered through a membrane
filter (nominal pore size 0.45 μm) and for 2 min with the
CZE buffer.
Injection : under pressure or vacuum (for example, 3 s at a
differential pressure of 3.5 kPa).
Migration : apply a field strength of 0.2 kV/cm, using the
CZE buffer as the electrolyte in both buffer reservoirs.
Run time : 30 min.
Identification of impurities : use the electropherogram
supplied with aprotinin solution BRP and the
electropherogram obtained with the reference solution to
identify the peaks due to impurities A and B.
Relative migration with reference to aprotinin (migration
time = about 22 min) : impurity A = about 0.98 ;
impurity B = about 0.99.
System suitability : reference solution after at least 6
injections :
— migration time : aprotinin = 19.0 min to 25.0 min ;
— resolution : minimum 0.8 between the peaks due to
impurities A and B ; minimum 0.5 between the peaks due
to impurity B and aprotinin ;
— peak distribution: the electrophoregram obtained
is qualitatively and quantitatively similar to the
electropherogram supplied with aprotinin solution BRP ;
— height of the principal peak : at least 1000 times the
height of the baseline noise. If necessary, adjust the
sample load to give peaks of a sufficient height.
Limits :
— impurity A : maximum 8.0 per cent ;
— impurity B : maximum 7.5 per cent.
Pyroglutamyl-aprotinin and related compounds. Liquid
chromatography (2.2.29) : use the normalisation procedure.
Test solution. Dilute the preparation to be examined in
mobile phase A to a concentration of about 5 Ph. Eur. U./ml.
Reference solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS in mobile phase A to
obtain the same concentration as the test solution.
Column :
— size: l = 0.075 m, Ø = 7.5 mm ;
— stationary phase : strong cation-exchange silica gel for
chromatography R (10 μm) ;
— temperature : 40 °C.
EUROPEAN PHARMACOPOEIA 6.3
Mobile phase :
— mobile phase A : dissolve 3.52 g of potassium dihydrogen
phosphate R and 7.26 g of disodium hydrogen phosphate
dihydrate R in 1000 ml of water ; filter and degas ;
— mobile phase B : dissolve 3.52 g of potassium dihydrogen
phosphate R, 7.26 g of disodium hydrogen phosphate
dihydrate R and 66.07 g of ammonium sulphate R in
1000 ml of water ; filter and degas ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 21 92 → 64 8 → 36
21 - 30 64 → 0 36 → 100
30 - 31 0 → 92 100 → 8
31 - 40 92 8 ASSAY
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 40 μl.
Relative retention with reference to aprotinin (retention
time = 17.0 min to 20.0 min) : impurity C = about 0.9.
System suitability : reference solution :
— resolution : minimum 1.5 between the peaks due to
impurity C and aprotinin ;
— symmetry factor : maximum 1.3 for the peak due to
aprotinin.
Limits :
— impurity C : maximum 1.0 per cent ;
— any other impurity : maximum 0.5 per cent;
— sum of impurities other than C : maximum 1.0 per cent.
Aprotinin oligomers. Size-exclusion chromatography
(2.2.30) : use the normalisation procedure.
Test solution. Dilute the preparation to be examined in
water R to obtain a concentration of about 5 Ph. Eur. U./ml. pH 8.0 R prepare an appropriate dilution (D) of the aprotinin
Reference solution. Treat the substance to be examined to
obtain about 2 per cent aprotinin oligomers. For example,
heat freeze-dried aprotinin at about 110 °C for about 4 h.
Then dissolve in water R to obtain a concentration of about containing about 0.8 microkatals per millilitre (about
5 Ph. Eur. U./ml.
Column: 3 columns coupled in series :
— size : l = 0.30 m, Ø = 7.8 mm ;
— stationary phase : hydrophilic silica gel for
chromatography R of a grade suitable for fractionation of Allow to stand at room temperature for 10 min and then
globular proteins in the relative molecular mass range of keep in iced water. Use within 6 h of preparation.
20 000 to 10 000 000 (8 μm).
Mobile phase : acetonitrile R, glacial acetic acid R, water R to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
(2:2:6 V/V/V) ; filter and degas.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 277 nm.
Injection : 100 μl.
Run time : 40 min.
Relative retention with reference to aprotinin monomer
(retention time = 24.5 min to 25.5 min) : aprotinin
dimer = about 0.9.
System suitability : reference solution :
— resolution : minimum 1.3 between the peaks due to
aprotinin dimer and monomer ;
— symmetry factor : maximum 2.5 for the peak due to
aprotinin monomer.
General Notices (1) apply to all monographs and other texts
Aprotinin concentrated solution
Limit :
— total : maximum 1.0 per cent.
Specific activity of the dry residue : minimum 3.0 Ph. Eur. U.
of aprotinin activity per milligram of dry residue.
Evaporate 25.0 ml to dryness in a water-bath, dry the residue
at 110 °C for 15 h and weigh. From the mass of the residue
and the activity determined as described below, calculate the
number of European Pharmacopoeia Units per milligram
of dry residue.
Bacterial endotoxins (2.6.14) : less than 0.14 IU per
European Pharmacopoeia Unit of aprotinin, if intended for
use in the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial
endotoxins.
The activity of aprotinin is determined by measuring its
inhibitory action on a solution of trypsin of known activity.
The inhibiting activity of the aprotinin is calculated from
the difference between the initial activity and the residual
activity of the trypsin.
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml, provided
with :
— a device that will maintain a temperature of 25 ± 0.1 °C ;
— a stirring device, such as a magnetic stirrer ;
— a lid with 5 holes for accommodating the electrodes, the
tip of a burette, a tube for the admission of nitrogen and
the introduction of the reagents.
An automatic or manual titration apparatus may be used. In
the latter case the burette is graduated in 0.05 ml and the
pH-meter is provided with a wide reading scale and glass and
calomel or glass-silver-silver chloride electrodes.
Test solution. With 0.0015 M borate buffer solution
concentrated solution expected, on the basis of the stated
potency, to contain 1.67 Ph. Eur. U./ml.
Trypsin solution. Prepare a solution of trypsin BRP
1 mg/ml), using 0.001 M hydrochloric acid as the solvent.
Use a freshly prepared solution and keep in iced water.
Trypsin and aprotinin solution. To 4.0 ml of the trypsin
solution add 1.0 ml of the test solution. Dilute immediately
to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution
Allow to stand at room temperature for 10 min and then
keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and
stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer
solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l
solution of benzoylarginine ethyl ester hydrochloride R.
Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the
temperature has reached equilibrium at 25 ± 0.1 °C, add
1.0 ml of the trypsin and aprotinin solution and start a
timer. Maintain at pH 8.0 by the addition of 0.1 M sodium
hydroxide and note the volume added every 30 s. Continue
the reaction for 6 min. Determine the number of millilitres of
0.1 M sodium hydroxide used per second (n1 ml). Carry out,
under the same conditions, a titration using 1.0 ml of the
dilute trypsin solution. Determine the number of millilitres
of 0.1 M sodium hydroxide used per second (n2 ml).
4037
Arnica flower
Calculate the aprotinin activity in European Pharmacopoeia
Units per millilitre using the following expression :
D = dilution factor of the aprotinin concentrated
solution to be examined in order to obtain a
solution containing 1.67 Ph. Eur. U./ml.
The estimated activity is not less than 90 per cent and not
more than 110 per cent of the activity stated on the label.
STORAGE
In an airtight, tamper-proof container, protected from light.
LABELLING
The label states :
— the number of European Pharmacopoeia Units of
aprotinin activity per millilitre ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
A. Ra = H, Rb = OH : aprotinin-(1-56)-peptide,
B. Ra = H, Rb = Gly-OH : aprotinin-(1-57)-peptide,
C. Ra = Glp, Rb = Gly-Ala-OH : (5-oxoprolyl)aprotinin
(pyroglutamylaprotinin).
04/2008:1391
corrected 6.3
ARNICA FLOWER
Arnicae flos
DEFINITION
Whole or partially broken, dried flower-heads of Arnica
montana L.
Content : minimum 0.40 per cent m/m of total sesquiterpene
lactones, expressed as dihydrohelenalin tiglate (dried drug). B. Separate the capitulum into its different parts. Examine
CHARACTERS
Aromatic odour.
The capitulum, when spread out, is about 20 mm in diameter
and about 15 mm deep, and has a peduncle 2-3 cm long.
The involucre consists of 18-24 elongated lanceolate bracts,
with acute apices, arranged in 1-2 rows : the bracts, about
8-10 mm long, are green with yellowish-green external hairs
visible under a lens. The receptacle, about 6 mm in diameter,
is convex, alveolate and covered with hairs. Its periphery
bears about 20 ligulate florets 20-30 mm long ; the disc
bears a greater number of tubular florets about 15 mm long.
The ovary, 4-8 mm long, is crowned by a pappus of whitish
bristles 4-8 mm long. Some brown achenes, crowned or not
by a pappus, may be present.
4038 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
IDENTIFICATION
A. The involucre consists of elongated oval bracts with acute
apices ; the margin is ciliated. The ligulate floret has a
reduced calyx crowned by fine, shiny, whitish bristles,
bearing small coarse trichomes. The orange-yellow
corolla bears 7-10 parallel veins and ends in 3 small
lobes. The stamens, with free anthers, are incompletely
developed. The narrow, brown ovary bears a stigma
divided into 2 branches curving outwards. The tubular
floret is actinomorphic. The ovary and the calyx are
similar to those of the ligulate floret. The short corolla
has 5 reflexed triangular lobes ; the 5 fertile stamens are
fused at the anthers.
A. Epidermis of the ligulate corolla
with covering trichome in surface
E. Epidermis of the corolla with
striated cuticle and biseriate secretory
view (Aa) and in side view (Ab) trichome in surface view (Ea) and in
B. Secretory trichome with side view (Eb)
multicellular head F. Covering trichome of the ovary in
C. Bristles of the calyx surface view (Fa) and in side view (Fb)
D. Pollen grain G. Secretory trichome of the ovary
Figure 1391.-1. – Illustration of powdered herbal drug of
arnica flower (see Identification B)
under a microscope using chloral hydrate solution R. The
powder shows the following diagnostic characters : the
epidermises of the bracts of the involucre have stomata
and trichomes, more abundant on the outer (abaxial)
surface. There are several different types of trichomes :
uniseriate multicellular covering trichomes, varying in
length from 50-500 μm, particularly abundant on the
margins of the bract ; secretory trichomes with uni- or
biseriate multicellular stalks and with multicellular,
globular heads, about 300 μm long, abundant on the
outer surface of the bract ; secretory trichomes with
uniseriate multicellular stalks and with multicellular,
globular heads, about 80 μm long, abundant on the
inner surface of the bract. The epidermis of the ligulate
corolla consists of lobed or elongated cells, a few stomata
EUROPEAN PHARMACOPOEIA 6.3
and trichomes of different types : covering trichomes,
with very sharp ends, whose length may exceed 500 μm,
consisting of 1-3 proximal cells with thickened walls and
2-4 distal cells with thin walls ; secretory trichomes with
biseriate multicellular heads ; secretory trichomes with
multicellular stalks and multicellular globular heads. The
ligule ends in rounded papillose cells. The epidermis
of the ovary is covered with trichomes : secretory
trichomes with short stalks and multicellular globular
heads ; twinned covering trichomes usually consisting of
2 longitudinally united cells, with common punctuated
walls ; their ends are sharp and sometimes bifid. The
epidermises of the calyx consist of elongated cells bearing
short, unicellular, covering trichomes pointing towards
the upper end of the bristle. The pollen grains have a
diameter of about 30 μm, are rounded, with a spiny exine,
and have 3 germinal pores.
A. Epidermis of the bracts of D. Epidermis of bracts of the
the involucre with covering involucre with stomata and
trichomes and stomata biseriate secretory trichome
B. Multicellular stalk of covering E. Secretory trichome
trichome
C. Pollen grain
Figure 1391.-2. – Illustration of powdered herbal drug of
arnica flower (see Identification B)
C. Examine the chromatograms obtained in the test for
Calendula officinalis L. - Heterotheca inuloides Cass.
Results : the chromatogram obtained with the test
solution shows, in the middle, a fluorescent blue zone
corresponding to the zone due to chlorogenic acid in the
chromatogram obtained with the reference solution ; it
shows, above this zone, 3 fluorescent yellowish-brown
or orange-yellow zones, and above these 3 zones a
fluorescent greenish-yellow zone due to astragalin.
The zone located below the astragalin zone is due to
isoquercitroside ; the zone located just below this zone is
General Notices (1) apply to all monographs and other texts
Arnica flower
due to luteolin-7-glucoside. It also shows a fluorescent
greenish-blue zone below the zone due to caffeic acid in
the chromatogram obtained with the reference solution.
TESTS
Foreign matter (2.8.2) : maximum 5.0 per cent.
Calendula officinalis L. - Heterotheca inuloides Cass .
Thin-layer chromatography (2.2.27).
Test solution. To 2.00 g of the powdered drug (710) (2.9.12)
add 10 ml of methanol R. Heat in a water-bath at 60 °C for
5 min with shaking. Cool and filter.
Reference solution. Dissolve 2.0 mg of caffeic acid R, 2.0 mg
of chlorogenic acid R and 5.0 mg of rutin R in methanol R
and dilute to 30 ml with the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : anhydrous formic acid R, water R, methyl
ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
Application : 15 μl, as bands.
Development : over a path of 15 cm.
Drying : in air for a few minutes.
Detection : spray with a 10 g/l solution of diphenylboric
acid aminoethyl ester R in methanol R, and then with a
50 g/l solution of macrogol 400 R in methanol R. Heat at
100-105 °C for 5 min. Allow to dry in air and examine in
ultraviolet light at 365 nm.
Results : the chromatogram obtained with the reference
solution shows in the lower part an orange-yellow fluorescent
zone due to rutin, in the middle part a fluorescent zone
due to chlorogenic acid and in the upper part a light bluish
fluorescent zone due to caffeic acid. The chromatogram
obtained with the test solution does not show a fluorescent
orange-yellow zone corresponding to the zone due to rutin
in the chromatogram obtained with the reference solution,
nor does it show a zone below this.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered drug (355) (2.9.12) by drying
in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 10.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Internal standard solution. Dissolve immediately before use
0.010 g of santonin CRS, accurately weighed in 10.0 ml of
methanol R.
Test solution. Introduce 1.00 g of the powdered drug (355)
(2.9.12) into a 250 ml round-bottomed flask, add 50 ml of a
mixture of equal volumes of methanol R and water R and
heat under a reflux condenser in a water-bath at 50-60 °C
for 30 min, shaking frequently. Allow to cool and filter
through a paper filter. Add the paper filter, cut into pieces,
to the residue in the round-bottomed flask, add 50 ml of a
mixture of equal volumes of methanol R and water R and
heat under a reflux condenser in a water-bath at 50-60 °C
for 30 min, shaking frequently. Repeat this procedure twice.
To the combined filtrate add 3.00 ml of the internal standard
solution and evaporate to 18 ml under reduced pressure.
Rinse the round-bottomed flask with water R and dilute,
with the washings, to 20.0 ml. Transfer the solution to
a chromatography column about 0.15 m long and about
30 mm in internal diameter containing 15 g of kieselguhr
for chromatography R. Allow to stand for 20 min. Elute
with 200 ml of a mixture of equal volumes of ethyl acetate R
and methylene chloride R. Evaporate the eluate to dryness
in a 250 ml round-bottomed flask. Dissolve the residue in
10.0 ml of methanol R and add 10.0 ml of water R. Add 7.0 g
of neutral aluminium oxide R, shake for 120 s, centrifuge at
4039
Arnica tincture EUROPEAN PHARMACOPOEIA 6.3

5000 g for 10 min and filter through a paper filter. Evaporate CHARACTERS
10.0 ml of the filtrate to dryness. Dissolve the residue in Appearance : yellowish-brown liquid.
3.0 ml of a mixture of equal volumes of methanol R and
water R and filter. IDENTIFICATION
Column: Examine the chromatograms obtained in the test for
— size : l = 0.12 m, Ø = 4 mm ; Calendula officinalis - Heterotheca inuloides.
— stationary phase : octadecylsilyl silica gel for Chromatogram obtained with the test solution :
chromatography R (4 μm). — in the middle, a fluorescent blue zone corresponding to
Mobile phase : the zone due to chlorogenic acid in the chromatogram
— mobile phase A : water R ; obtained with the reference solution ;
— mobile phase B : methanol R ; — above this zone, 3 fluorescent yellowish-brown to
orange-yellow zones, and above these 3 zones a
Time Mobile phase A Mobile phase B fluorescent greenish-yellow zone corresponding to
(min) (per cent V/V) (per cent V/V) astragalin ; the zone located below the astragalin zone
0-3 62 38 corresponds to isoquercitrin ; the zone located just below
3 - 20 62 → 55 38 → 45 this zone corresponds to luteolin-7-glucoside ;
20 - 30 55 45 — a fluorescent greenish-blue zone below the zone due
to caffeic acid in the chromatogram obtained with the
30 - 55 55 → 45 45 → 55 reference solution.
55 - 57 45 → 0 55 → 100
TESTS
57 - 70 0 100
Calendula officinalis - Heterotheca inuloides. Thin-layer
70 - 90 62 38 chromatography (2.2.27).
Test solution. The tincture to be examined.
Flow rate : 1.2 ml/min.
Reference solution. Dissolve 2.0 mg of caffeic acid R, 2.0 mg
Detection : spectrophotometer at 225 nm.
of chlorogenic acid R and 5.0 mg of rutin R in methanol R
Injection : a 20 μl loop injector. and dilute to 30.0 ml with the same solvent.
Calculate the percentage content of total sesquiterpene Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
lactones, expressed as dihydrohelenalin tiglate, using the plate R (2-10 μm)].
following expression :
Mobile phase : anhydrous formic acid R, water R, methyl
ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
Application : 30 μl [or 8 μl] as bands.
Development : over a path of 15 cm [or 8 cm].
SLS = area of all peaks due to sesquiterpene lactones Drying : at 80-105 °C.
appearing after the santonin peak in the
chromatogram obtained with the test solution ; Detection : spray the plate whilst still hot with a 10 g/l
solution of diphenylboric acid aminoethyl ester R in
SS = area of the peak due to santonin in the methanol R and then with a 50 g/l solution of macrogol
chromatogram obtained with the test solution ; 400 R in methanol R ; heat 5 min at 100-105 °C, allow the
m = mass of the drug to be examined, in grams ; plate to dry in air and examine in ultraviolet light at 365 nm.
C = concentration of santonin in the internal Results : the chromatogram obtained with the reference
standard solution used for the test solution, in solution shows in the lower part an orange-yellow
milligrams per millilitre ; fluorescent zone (rutin), in the middle part a fluorescent
V = volume of the internal standard solution used zone due to chlorogenic acid and in the upper part a light
for the test solution, in millilitres ; bluish fluorescent zone (caffeic acid). The chromatogram
obtained with the test solution does not show any
1.187 = peak correlation factor between dihydrohelenalin fluorescent orange-yellow zone corresponding to rutin in the
tiglate and santonin. chromatogram obtained with the reference solution and no
zone below the zone corresponding to rutin.
01/2008:1809 Ethanol (2.9.10) : the final ethanol concentration is not less
corrected 6.3 than 90 per cent of that of the initial extraction solvent.
Methanol and 2-propanol (2.9.11) : maximum 0.05 per
ARNICA TINCTURE cent V/V of methanol and maximum 0.05 per cent V/V of
2-propanol.
Arnicae tinctura Dry residue (2.8.16) : minimum 1.7 per cent.

DEFINITION
Tincture produced from Arnica flower (1391).
Content : minimum 0.04 per cent of sesquiterpene lactones
expressed as dihydrohelenalin tiglate (C20H26O5 ; Mr 346.42).
PRODUCTION
The tincture is produced from the herbal drug by a suitable
procedure using 10 parts of ethanol (60-70 per cent V/V)
for 1 part of drug.
ASSAY
Liquid chromatography (2.2.29).
Internal standard solution. Dissolve immediately before use
0.010 g accurately weighed of santonin CRS and 0.02 g of
butyl 4-hydroxybenzoate R in 10.0 ml of methanol R.
Test solution. In a round-bottomed flask introduce 5.00 g
of the tincture to be examined, add 2.00 ml of the internal
standard solution and 3 g of anhydrous aluminium oxide R,
shake for 120 s and filter through a paper filter. Rinse the

4040 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Artichoke leaf dry extract

round-bottomed flask and filter with 5 ml of a mixture Content : minimum 0.6 per cent of chlorogenic acid
of equal volumes of methanol R and water R and filter. (C16H18O9 ; Mr 354.3) (dried extract).
Evaporate the filtrate to dryness. Dissolve the residue in
2.0 ml of a mixture of 20 volumes of water R and 80 volumes PRODUCTION
of methanol R and filter through a membrane filter (porosity : The extract is produced from the herbal drug by an
0.45 μm). appropriate procedure using water of minimum 80 °C.
Reference solution. Dissolve 0.02 g of methyl
CHARACTERS
4-hydroxybenzoate R and 0.02 g of ethyl
4-hydroxybenzoate R in methanol R and dilute to Appearance : light brown or brown amorphous powder.
10.0 ml with the same solvent.
IDENTIFICATION
Column:
Thin-layer chromatography (2.2.27).
— size : l = 0.12 m, Ø = 4 mm ;
Test solution. Dissolve 1.0 g of the extract to be examined
— stationary phase : end-capped octadecylsilyl silica gel in 10 ml of ethanol (60 per cent V/V) R. Sonicate for 5 min
for chromatography R (5 μm) ; and filter.
— temperature : 20 °C. Reference solution. Dissolve 5 mg of luteolin-7-glucoside R
Mobile phase : and 5 mg of chlorogenic acid R in 10 ml of methanol R.
— mobile phase A : water R ; Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
— mobile phase B : methanol R ; plate R (2-10 μm)].
Time Mobile phase A Mobile phase B Mobile phase : acetic acid R, anhydrous formic acid R,
(min) (per cent V/V) (per cent V/V) water R, ethyl acetate R (11:11:27:100 V/V/V/V).
0-3 62 38 Application : 10 μl [or 2 μl] as bands of 10 mm [or 8 mm].
3 - 20 62 → 55 38 → 45 Development : over a path of 13 cm [or 6 cm].
20 - 30 55 45 Drying : in air.
Detection : heat at 100 °C for 5 min ; spray the warm plate
30 - 55 55 → 45 45 → 55
with a 10 g/l solution of diphenylboric acid aminoethyl
Flow rate : 1.2 ml/min. ester R in methanol R followed by a 50 g/l solution of
macrogol 400 R in methanol R ; examine in ultraviolet light
Detector: spectrophotometer at 225 nm. at 365 nm.
Injection : 20 μl.
Results : see below the sequence of fluorescent zones present
Relative retention with reference to santonin (retention in the chromatograms obtained with the reference solution
time = about 9.5 min) : butyl 4-hydroxybenzoate = about 4.6. and the test solution. Furthermore, other fluorescent zones
System suitability: reference solution : may be present in the chromatogram obtained with the test
— resolution : minimum 5 between the peaks due to methyl solution.
4-hydroxybenzoate and ethyl 4-hydroxybenzoate. Top of the plate
Calculate the percentage of lactone sesquiterpenes,
A light blue fluorescent zone
expressed as dihydrohelenalin tiglate, using the following
expression : _______ _______

Luteolin-7-glucoside : a yellow or A yellow or orange fluorescent

orange fluorescent zone zone (luteolin-7-glucoside)

F1 = area of all peaks appearing between the peaks due Chlorogenic acid : a light blue A light blue fluorescent zone
fluorescent zone (chlorogenic acid)
to santonin and butyl 4-hydroxybenzoate in the _______ _______
chromatogram obtained with the test solution ;
F2 = area of the peak due to santonin in the Reference solution Test solution
chromatogram obtained with the test solution ;
m = mass of the tincture to be examined, in grams ; TESTS
C = concentration of santonin in the internal standard Loss on drying (2.8.17) : maximum 6.0 per cent.
solution used to prepare the test solution, in Total ash (2.4.16) : maximum 30.0 per cent.
milligrams per millilitre ;
V = volume of the internal standard solution used to ASSAY
prepare the test solution, in millilitres ; Liquid chromatography (2.2.29).
1.187 = peak correlation factor between dihydrohelenalin Test solution. Dissolve 30.0 mg of the extract to be examined
tiglate and santonin. in a mixture of 30 volumes of methanol R and 70 volumes
of water R and dilute to 25.0 ml with the same mixture of
solvents.
01/2009:2389 Reference solution (a). Dissolve 5.0 mg of chlorogenic
acid CRS in 50.0 ml of methanol R. Transfer 5.0 ml of this
ARTICHOKE LEAF DRY EXTRACT solution to a volumetric flask, add 5 ml of methanol R and
dilute to 20.0 ml with water R.
Cynarae folii extractum siccum Reference solution (b). Dissolve 30 mg of the standardised
artichoke leaf dry extract CRS in a mixture of 30 volumes of
DEFINITION methanol R and 70 volumes of water R and dilute to 25.0 ml
Dry extract produced from Artichoke leaf (1866). with the same mixture of solvents.

General Notices (1) apply to all monographs and other texts 4041
Ascorbic acid EUROPEAN PHARMACOPOEIA 6.3

Column: DEFINITION
— size : l = 0.250 m, Ø = 4.6 mm ; (5R)-5-[(1S)-1,2-Dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-
— stationary phase : octadecylsilyl silica gel for one.
chromatography R (5 μm) ; Content : 99.0 per cent to 100.5 per cent.
— temperature : 40 °C. CHARACTERS
Mobile phase : Appearance : white or almost white, crystalline powder or
— mobile phase A : phosphoric acid R, water R colourless crystals, becoming discoloured on exposure to
(0.5:99.5 V/V) ; air and moisture.
— mobile phase B : phosphoric acid R, acetonitrile R Solubility : freely soluble in water, soluble in ethanol (96 per
(0.5:99.5 V/V) ; cent).
Time Mobile phase A Mobile phase B mp : about 190 °C, with decomposition.
(min) (per cent V/V) (per cent V/V)
IDENTIFICATION
0-1 92 8
First identification : B, C.
1 - 20 92 → 75 8 → 25
Second identification : A, C, D.
20 - 33 75 25
A. Ultraviolet and visible absorption spectrophotometry
33 - 35 75 → 0 25 → 100 (2.2.25).
Test solution. Dissolve 0.10 g in water R and dilute
Flow rate : 1.2 ml/min. immediately to 100.0 ml with the same solvent. Add
Detection : spectrophotometer at 330 nm. 1.0 ml of this solution to 10 ml of 0.1 M hydrochloric
Injection : 25 μl. acid and dilute to 100.0 ml with water R.
System suitability : reference solution (b) : Absorption maximum : at 243 nm, determined
— peak-to-valley ratio : minimum 2.5, where Hp = height immediately after dissolution.
above the baseline of the peak immediately after the Specific absorbance at the absorption maximum : 545
peak due to chlorogenic acid and Hv = height above the to 585.
baseline of the lowest point of the curve separating this B. Infrared absorption spectrophotometry (2.2.24).
peak from the peak due to chlorogenic acid ;
— the chromatogram obtained is similar to the Comparison : ascorbic acid CRS.
chromatogram supplied with the standardised artichoke C. pH (2.2.3) : 2.1 to 2.6 for solution S (see Tests).
leaf dry extract CRS D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R
Calculate the percentage content of chlorogenic acid using and 0.2 ml of silver nitrate solution R2. A grey precipitate
the following expression : is formed.
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R
and dilute to 20 ml with the same solvent.
A1 = area of the peak due to chlorogenic acid in the Appearance of solution. Solution S is clear (2.2.1) and not
chromatogram obtained with the test solution ; more intensely coloured than reference solution BY7 (2.2.2,
A2 = area of the peak due to chlorogenic acid in Method II).
the chromatogram obtained with reference Specific optical rotation (2.2.7) : + 20.5 to + 21.5.
solution (a) ;
m1 = Dissolve 2.50 g in water R and dilute to 25.0 ml with the
mass of the extract to be examined used to same solvent.
prepare the test solution, in milligrams ;
m2 = Impurity E : maximum 0.2 per cent.
mass of chlorogenic acid CRS used to prepare
reference solution (a), in milligrams ; Test solution. Dissolve 0.25 g in 5 ml of water R. Neutralise
p = to red litmus paper R using dilute sodium hydroxide
percentage content of chlorogenic acid in solution R and add 1 ml of dilute acetic acid R and 0.5 ml of
chlorogenic acid CRS. calcium chloride solution R.
Reference solution. Dissolve 70 mg of oxalic acid R in
water R and dilute to 500 ml with the same solvent ; to 5 ml
01/2009:0253 of this solution add 1 ml of dilute acetic acid R and 0.5 ml of
calcium chloride solution R.
ASCORBIC ACID Allow the solutions to stand for 1 h. Any opalescence in the
test solution is not more intense than that in the reference
Acidum ascorbicum solution.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Phosphate buffer solution. Dissolve 6.8 g of potassium
dihydrogen phosphate R in water R and dilute to about
175 ml with the same solvent. Filter (porosity 0.45 μm) and
dilute to 1000 ml with water R.
Test solution. Dissolve 0.500 g of the substance to be
C 6 H8 O 6 Mr 176.1 examined in the mobile phase and dilute to 10.0 ml with the
[50-81-7] mobile phase.

4042 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Ascorbic acid

Reference solution (a). Dissolve 10.0 mg of ascorbic acid Reference solutions. Prepare the reference solutions
impurity C CRS in the mobile phase and dilute to 5.0 ml (0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting iron standard
with the mobile phase. solution (20 ppm Fe) R with 0.1 M nitric acid.
Reference solution (b). Dilute 2.5 ml of reference solution (a) Source : iron hollow-cathode lamp.
to 100.0 ml with the mobile phase. Wavelength : 248.3 nm.
Reference solution (c). Dilute 1.0 ml of the test solution to Atomisation device : air-acetylene flame.
200.0 ml with the mobile phase. Mix 1.0 ml of this solution
with 1.0 ml of reference solution (a). Adjust the zero of the apparatus using 0.1 M nitric acid.
Column: Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in water R and dilute to 20 ml with the same
— size : l = 0.25 m, Ø = 4.6 mm ; solvent. 12 ml of the solution complies with test A. Prepare
— stationary phase : aminopropylsilyl silica gel for the reference solution using lead standard solution (1 ppm
chromatography R (5 μm) ; Pb) R.
— temperature : 45 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Mobile phase : phosphate buffer solution, acetonitrile R1
(30:70 V/V). ASSAY
Flow rate : 1.0 ml/min. Dissolve 0.150 g in a mixture of 10 ml of dilute sulphuric
Detection : spectrophotometer at 210 nm. acid R and 80 ml of carbon dioxide-free water R. Add 1 ml
of starch solution R. Titrate with 0.05 M iodine until a
Injection : 20 μl of the test solution and reference persistent violet-blue colour is obtained.
solutions (b) and (c).
1 ml of 0.05 M iodine is equivalent to 8.81 mg of C6H8O6.
Run time : twice the retention time of ascorbic acid.
Relative retention with reference to ascorbic acid (retention STORAGE
time = about 8 min) : impurity C = about 1.4. In a non-metallic container, protected from light.
System suitability : reference solution (c) :
IMPURITIES
— resolution : minimum 3.0 between the peaks due to
ascorbic acid and impurity C. Specified impurities: C, E.
Limits : Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
— impurity C : not more than the area of the corresponding or other of the tests in the monograph. They are limited
peak in the chromatogram obtained with reference by the general acceptance criterion for other/unspecified
solution (b) (0.1 per cent) ; impurities and/or by the general monograph Substances for
— unspecified impurities: for each impurity, not more pharmaceutical use (2034). It is therefore not necessary to
than the area of the peak due to impurity C in the identify these impurities for demonstration of compliance.
chromatogram obtained with reference solution (b) See also 5.10. Control of impurities in substances for
(0.10 per cent) ; pharmaceutical use) : A, B, D.
— total : not more than twice the area of the peak due to
impurity C in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
— disregard limit: 0.5 times the area of the peak due to
impurity C in the chromatogram obtained with reference A. 2-furaldehyde,
solution (b) (0.05 per cent).
Copper : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 2.0 g in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutions. Prepare the reference solutions B. R = [CH2]3-CH3 : butyl D-sorbosonate,
(0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting copper standard
solution (10 ppm Cu) R with 0.1 M nitric acid.
C. R = H : D-sorbosonic acid,
Source : copper hollow-cathode lamp.
Wavelength : 324.8 nm.
D. R = CH3 : methyl D-sorbosonate,
Atomisation device : air-acetylene flame.
Adjust the zero of the apparatus using 0.1 M nitric acid.
Iron : maximum 2.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 5.0 g in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid. E. oxalic acid.

General Notices (1) apply to all monographs and other texts 4043
Atropine
04/2008:2056 Test solution. Dissolve 24 mg of the substance to be
corrected 6.3 examined in mobile phase A and dilute to 100.0 ml with
ATROPINE
Atropinum
C17H23NO3 Mr 289.4 (impurity C) in mobile phase A and dilute to 10 ml with
[51-55-8]
DEFINITION
(1R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-3-hydroxy-2-phenylpropanoate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very slightly soluble in water, freely soluble in
ethanol (96 per cent) and in methylene chloride.
IDENTIFICATION
First identification : A, B, E.
Second identification : A, C, D, E.
A. Melting point (2.2.14) : 115 °C to 119 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : atropine CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 10 mg of atropine CRS in
methanol R and dilute to 10 ml with the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, water R,
acetone R (3:7:90 V/V/V).
Application : 10 μl.
Development : over half of the plate.
Drying : at 100-105 °C for 15 min.
Detection : after cooling, spray with dilute potassium
iodobismuthate solution R.
Results : the principal spot in the chromatogram obtained impurity H = about 0.93 ; impurity G = about 1.1 ;
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained System suitability : reference solution (b) :
with the reference solution.
D. Place about 3 mg in a porcelain crucible and add 0.2 ml
of fuming nitric acid R. Evaporate to dryness on a
water-bath. Dissolve the residue in 0.5 ml of a 30 g/l
solution of potassium hydroxide R in methanol R ; a
violet colour develops.
E. Optical rotation (see Tests).
TESTS
Optical rotation (2.2.7) : − 0.70° to + 0.05° (measured in
a 2 dm tube).
Dissolve 1.25 g in ethanol (96 per cent) R and dilute to
25.0 ml with the same solvent.
Related substances. Liquid chromatography (2.2.29).
4044 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
mobile phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
Reference solution (b). Dissolve 5 mg of atropine
impurity B CRS in the test solution and dilute to 20 ml with
the test solution. Dilute 5 ml of this solution to 25 ml with
mobile phase A.
Reference solution (c). Dissolve the contents of a vial
of atropine for peak identification CRS (containing
impurities A, B, D, E, F, G and H) in 1 ml of mobile phase A.
Reference solution (d). Dissolve 5 mg of tropic acid R
mobile phase A. Dilute 1 ml of the solution to 100 ml with
mobile phase A. Dilute 1 ml of this solution to 10 ml with
mobile phase A.
Column :
— size: l = 0.10 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
— mobile phase A : dissolve 3.5 g of sodium dodecyl
sulphate R in 606 ml of a 7.0 g/l solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.3
with 0.05 M phosphoric acid, and mix with 320 ml of
acetonitrile R1 ;
— mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 95 5
2 - 20 95 → 70 5 → 30
Flow rate : 1 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μl.
Identification of impurities : use the chromatogram
supplied with atropine for peak identification CRS and
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B, D, E, F, G and H.
Use the chromatogram obtained with reference solution (d)
to identify the peak due to impurity C.
Relative retention with reference to atropine (retention
time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
impurity F = about 0.8 ; impurity B = about 0.89 ;
impurity A = about 1.7.
— resolution : minimum 2.5 between the peaks due to
impurity B and atropine.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.6 ;
impurity C = 0.6 ;
— impurities E, H : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) ;
— impurities A, B, C, D, F, G : for each impurity, not
more than twice the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.2 per cent) ;
EUROPEAN PHARMACOPOEIA 6.3 Atropine sulphate

— 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 : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.2 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
ASSAY
Dissolve 0.250 g in 40 ml of anhydrous acetic acid R,
warming if necessary. Allow the solution to cool. 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 28.94 mg
of C17H23NO3.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.
A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
2-phenylpropenoate (apoatropine),
B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-
phenylpropanoate (noratropine),
C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),
D. R1 = OH, R2 = H : (1R,3S,5R,6RS)-6-hydroxy-8-
methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-
phenylpropanoate (6-hydroxyhyoscyamine),
E. R1 = H, R2 = OH : (1S,3R,5S,6RS)-6-hydroxy-8-
methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-
phenylpropanoate (7-hydroxyhyoscyamine),
F. hyoscine,
(2RS)-2-hydroxy-3-phenylpropanoate (littorine),
H. unknown structure.
04/2008:0068
corrected 6.3
ATROPINE SULPHATE
Atropini sulfas
C34H48N2O10S,H2O Mr 695
[5908-99-6]
DEFINITION
Bis[(1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-3-hydroxy-2-phenylpropanoate] sulphate monohydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent).
IDENTIFICATION
First identification : A, B, E.
Second identification : C, D, E, F.
A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : atropine sulphate CRS.
C. Dissolve about 50 mg in 5 ml of water R and add 5 ml
of picric acid solution R. The precipitate, washed with
water R and dried at 100-105 °C for 2 h, melts (2.2.14)
at 174 °C to 179 °C.
D. To about 1 mg add 0.2 ml of fuming nitric acid R and
evaporate to dryness in a water-bath. Dissolve the residue
in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution
of potassium hydroxide R in methanol R. A violet colour
develops.
E. It gives the reactions of sulphates (2.3.1).
F. It gives the reaction of alkaloids (2.3.1).
TESTS
pH (2.2.3) : 4.5 to 6.2.
Dissolve 0.6 g in carbon dioxide-free water R and dilute to
30 ml with the same solvent.
Optical rotation (2.2.7) : − 0.50° to + 0.05° (measured in
a 2 dm tube).
Dissolve 2.50 g in water R and dilute to 25.0 ml with the
same solvent.

General Notices (1) apply to all monographs and other texts 4045
Atropine sulphate EUROPEAN PHARMACOPOEIA 6.3

Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 24 mg of the substance to be
examined in mobile phase A and dilute to 100.0 ml with
mobile phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
Reference solution (b). Dissolve 5 mg of atropine
impurity B CRS in the test solution and dilute to 20 ml with
the test solution. Dilute 5 ml of this solution to 25 ml with
mobile phase A.
Reference solution (c). Dissolve the contents of a vial
of atropine for peak identification CRS (containing
impurities A, B, D, E, F, G and H) in 1 ml of mobile phase A.
Reference solution (d). Dissolve 5 mg of tropic acid R
(impurity C) in mobile phase A and dilute to 10 ml with
mobile phase A. Dilute 1 ml of the solution to 100 ml with
mobile phase A. Dilute 1 ml of this solution to 10 ml with
mobile phase A.
Column:
— size : l = 0.10 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
— mobile phase A : dissolve 3.5 g of sodium dodecyl
sulphate R in 606 ml of a 7.0 g/l solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.3
with 0.05 M phosphoric acid, and mix with 320 ml of
acetonitrile R1 ;
— mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min)
(per cent V/V) (per cent V/V)
0-2 95 5
2 - 20 95 → 70 5 → 30
— impurities E, H : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) ;
— impurities A, B, C, D, F, G : for each impurity, not
more than twice the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.2 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 : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.12) : 2.0 per cent to 4.0 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.500 g in 30 ml of anhydrous acetic acid R,
warming if necessary. Cool the solution. 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 67.68 mg
of C34H48N2O10S.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.

Flow rate : 1 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 10 μl.
Identification of impurities: use the chromatogram A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
supplied with atropine for peak identification CRS and 2-phenylpropenoate (apoatropine),
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B, D, E, F, G and H.
Use the chromatogram obtained with reference solution (d)
to identify the peak due to impurity C.
Relative retention with reference to atropine (retention
time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
impurity F = about 0.8 ; impurity B = about 0.89 ;
impurity H = about 0.93 ; impurity G = about 1.1 ;
impurity A = about 1.7. B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-
phenylpropanoate (noratropine),
System suitability : reference solution (b) :
— resolution : minimum 2.5 between the peaks due to
impurity B and atropine.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.6 ;
impurity C = 0.6 ; C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),

4046 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Azithromycin

D. R1 = OH, R2 = H : (1R,3S,5R,6RS)-6-hydroxy-8-
methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-
phenylpropanoate (6-hydroxyhyoscyamine),
E. R1 = H, R2 = OH : (1S,3R,5S,6RS)-6-hydroxy-8-
methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-
phenylpropanoate (7-hydroxyhyoscyamine),
F. hyoscine,
G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-2-hydroxy-3-phenylpropanoate (littorine).
H. unknown structure.
01/2008:1649 Test solution. Dissolve 0.200 g of the substance to be
corrected 6.3 examined in the solvent mixture and dilute to 25.0 ml with
AZITHROMYCIN
Azithromycinum
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, freely soluble in
anhydrous ethanol and in methylene chloride.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : azithromycin CRS.
If the spectra obtained in the solid state show differences,
prepare further spectra using 90 g/l solutions in methylene
chloride R.
TESTS
Solution S. Dissolve 0.500 g in anhydrous ethanol R and
dilute to 50.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3) : 9.0 to 11.0.
Dissolve 0.100 g in 25.0 ml of methanol R and dilute to
50.0 ml with carbon dioxide-free water R.
Specific optical rotation (2.2.7) : − 45 to − 49 (anhydrous
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture. Prepare a 1.73 g/l solution of ammonium
dihydrogen phosphate R adjusted to pH 10.0 with
ammonia R. Transfer 350 ml of this solution to a suitable
container. Add 300 ml of acetonitrile R1 and 350 ml of
methanol R1. Mix well.
the solvent mixture.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with the solvent mixture.
Reference solution (b). Dissolve the contents of a vial
of azithromycin for system suitability CRS (containing
impurities F, H and J) in 1.0 ml of the solvent mixture and
sonicate for 5 min.
Reference solution (c). Dissolve 8.0 mg of azithromycin for
peak identification CRS (containing impurities A, B, C, E, F,
G, I, J, L, M, N, O, P) in 1.0 ml of the solvent mixture.

Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl amorphous
organosilica polymer for mass spectrometry R (5 μm) ;
— temperature : 60 °C.
Mobile phase :
— mobile phase A : 1.80 g/l solution of anhydrous disodium
C38H72N2O12,xH2O Mr 749 (anhydrous substance) hydrogen phosphate R adjusted to pH 8.9 with dilute
with x = 1 or 2 phosphoric acid R or with dilute sodium hydroxide
[83905-01-5] solution R ;
— mobile phase B : methanol R1, acetonitrile R1
DEFINITION (250:750 V/V) ;
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy- Time Mobile phase A Mobile phase B
3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl- (min) (per cent V/V) (per cent V/V)
3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6- 0 - 25 50 → 45 50 → 55
trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1-
oxa-6-azacyclopentadecan-15-one. The degree of hydration 25 - 30 45 → 40 55 → 60
is 1 or 2. 30 - 80 40 → 25 60 → 75
Semi-synthetic product derived from a fermentation product. 80 - 81 25 → 50 75 → 50
Content : 96.0 per cent to 102.0 per cent (anhydrous 81 - 93 50 50
substance).

General Notices (1) apply to all monographs and other texts 4047
Azithromycin
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 50 μl.
Relative retention with reference to azithromycin
(retention time = 45-50 min) : impurity L = about 0.29 ;
impurity M = about 0.37 ; impurity E = about 0.43 ;
impurity F = about 0.51 ; impurity D = about 0.54 ;
impurity J = about 0.54 ; impurity I = about 0.61 ;
impurity C = about 0.73 ; impurity N = about 0.76 ;
impurity H = about 0.79 ; impurity A = about 0.83 ;
impurity P = about 0.92 ; impurity O = about 1.23 ;
impurity G = about 1.26 ; impurity B = about 1.31.
Identification of impurities: use the chromatogram
supplied with azithromycin for peak identification CRS and
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B, C, E, F, G, I, J, L, M,
N, O and P and the chromatogram obtained with reference
solution (b) to identify the peak due to impurity H.
System suitability : reference solution (b) :
— peak-to-valley ratio : minimum 1.4, where Hp = height
above the baseline of the peak due to impurity J and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
impurity F.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity F = 0.3 ;
impurity H = 0.1 ; impurity L = 2.3 ; impurity M = 0.6 ;
impurity N = 0.7 ;
— impurity B : not more than twice the area of the principal — temperature : 40 °C.
peak in the chromatogram obtained with reference
solution (a) (2.0 per cent) ;
— impurities A, C, E, F, G, H, I, L, M, N, O, P : for each
impurity, not more than 0.5 times the area of the principal of potassium hydroxide R.
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) ;
— sum of impurities D and J : not more than 0.5 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
— any other impurity : for each impurity, not more
than 0.2 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.2 per cent) ;
— total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a) Calculate the percentage content of C H N O from the
(3.0 per cent) ;
— disregard limit : 0.1 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.1 per cent) ; disregard the peaks eluting
before impurity L and after impurity B.
4048
EUROPEAN PHARMACOPOEIA 6.3
Heavy metals (2.4.8) : maximum 25 ppm.
Dissolve 2.0 g in a mixture of 15 volumes of water R and
85 volumes of anhydrous ethanol 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 (2.5 ppm Pb) obtained by diluting
lead standard solution (100 ppm Pb) R with a mixture
of 15 volumes of water R and 85 volumes of anhydrous
ethanol R.
Water (2.5.12) : 1.8 per cent to 6.5 per cent, determined on
0.200 g.
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
on 1.0 g.
ASSAY
Liquid chromatography (2.2.29).
Solution A. Mix 60 volumes of acetonitrile R1 and
40 volumes of a 6.7 g/l solution of dipotassium hydrogen
phosphate R adjusted to pH 8.0 with phosphoric acid R.
Test solution. Dissolve 53.0 mg of the substance to be
examined in 2 ml of acetonitrile R1 and dilute to 100.0 ml
with solution A.
Reference solution (a). Dissolve 53.0 mg of
azithromycin CRS in 2 ml of acetonitrile R1 and
dilute to 100.0 ml with solution A.
Reference solution (b). Dissolve 5 mg of the substance to
be examined and 5 mg of azithromycin impurity A CRS in
0.5 ml of acetonitrile R1 and dilute to 10 ml with solution A.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl vinyl polymer for
chromatography R (5 μm) ;
Mobile phase : mix 60 volumes of acetonitrile R1 and
40 volumes of a 6.7 g/l solution of dipotassium hydrogen
phosphate R adjusted to pH 11.0 with a 560 g/l solution
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μl.
Run time : 1.5 times the retention time of azithromycin.
Retention time : azithromycin = about 10 min.
System suitability : reference solution (b) :
— resolution : minimum 3.0 between the peaks due to
impurity A and azithromycin.
38 72 2 12
declared content of azithromycin CRS.
STORAGE
In an airtight container.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 Azithromycin

IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I, J, L, M, N,
O, P.
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 G. 3′-N-demethyl-3′-N-[(4-methylphenyl)sulphonyl]azithro-
impurities and/or by the general monograph Substances for mycin,
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) : K.

H. 3′-N-[[4-(acetylamino)phenyl]sulphonyl]-3′-N-
demethylazithromycin,

J. 13-O-decladinosylazithromycin,

A. R1 = OH, R2 = R6 = H, R3 = R4 = R5 = CH3 :
6-demethylazithromycin,

B. R1 = R6 = H, R2 = R3 = R4 = R5 = CH3 :
3-deoxyazithromycin (azithromycin B),

C. R1 = OH, R2 = R3 = R5 = CH3, R4 = R6 = H : L. azithromycin 3′-N-oxide,

3″-O-demethylazithromycin (azithromycin C),

D. R1 = OH, R2 = R3 = R4 = CH3, R5 = CH2OH, R6 = H :

14-demethyl-14-(hydroxymethyl)azithromycin
(azithromycin F),

F. R1 = OH, R2 = R4 = R5 = CH3, R3 = CHO, R6 = H :

3′-N-demethyl-3′-N-formylazithromycin,
M. 3′-(N,N-didemethyl)-3′-N-formylazithromycin,
I. R1 = OH, R2 = R4 = R5 = CH3, R3 = R6 = H :
3′-N-demethylazithromycin,

O. R1 = OH, R2 = R3 = R4 = R5 = R6 = CH3 :
2-desethyl-2-propylazithromycin,

N. 3′-de(dimethylamino)-3′-oxoazithromycin,

K. C14,1″-epoxyazithromycin (azithromycin E),

E. 3′-(N,N-didemethyl)azithromycin (aminoazithromycin), P. unknown structure.

General Notices (1) apply to all monographs and other texts 4049
 EUROPEAN PHARMACOPOEIA 6.3

4050 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
B
BCG for immunotherapy.. ......................................................4053
Beclometasone dipropionate, anhydrous.. .........................4054
Beclometasone dipropionate monohydrate.. .....................4056
Belladonna leaf dry extract, standardised..........................4059
Benazepril hydrochloride.......................................................4060
General Notices (1) apply to all monographs and other texts
Bentonite.. .................................................................................4062
Betamethasone valerate.. .......................................................4062
Bitter-orange epicarp and mesocarp....................................4064
Bitter-orange flower.................................................................4065
Buserelin....................................................................................4067
4051
 EUROPEAN PHARMACOPOEIA 6.3

4052 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 BCG for immunotherapy

01/2009:1929 Virulent mycobacteria. Examine the working seed lot as

prescribed under Tests, using 10 guinea-pigs.
BCG FOR IMMUNOTHERAPY PROPAGATION AND HARVEST
The bacteria are grown in a suitable medium for not more
than 21 days by surface or submerged culture. The culture
BCG ad immunocurationem medium does not contain substances known to cause toxic
or allergic reactions in human beings or to cause the bacteria
DEFINITION
to become virulent for guinea-pigs. The culture is harvested
BCG for immunotherapy is a freeze-dried preparation of live and suspended in a sterile liquid medium that protects the
bacteria derived from a culture of the bacillus of Calmette viability of the culture as determined by a suitable method of
and Guérin (Mycobacterium bovis BCG) whose capacity for viable count.
treatment has been established.
FINAL BULK
It complies with the monograph Vaccines for human The final bulk is prepared from a single harvest or by pooling
use (0153). a number of single harvests. A stabiliser may be added ; if
the stabiliser interferes with the determination of bacterial
PRODUCTION concentration on the final bulk, the determination is carried
GENERAL PROVISIONS out before addition of the stabiliser.
BCG for immunotherapy shall be produced by a staff Only final bulk that complies with the following requirements
consisting of healthy persons who do not work with other may be used in the preparation of the final lot.
infectious agents ; in particular they shall not work with Bacterial and fungal contamination. Carry out the test for
virulent strains of Mycobacterium tuberculosis, nor shall sterility (2.6.1), using 10 ml of final bulk for each medium.
they be exposed to a known risk of tuberculosis infection. The final bulk complies with the test for sterility, except for
Staff are examined periodically for tuberculosis. BCG for the presence of mycobacteria.
immunotherapy is susceptible to sunlight : the procedures
for production shall be so designed that all products are Count of viable units. Determine the number of viable units
protected from direct sunlight and from ultraviolet light at per millilitre by viable count on solid medium using a
all stages of manufacture, testing and storage. method suitable for the product to be examined or by a
suitable biochemical method. Carry out the test in parallel
Production is based on a seed-lot system. The production on a reference preparation of the same strain.
method shall have been shown to yield consistently BCG
products that can be used for treatment of superficial Bacterial concentration. Determine the total bacterial
bladder cancer and are safe. The product is prepared from concentration by a suitable method, either directly by
cultures which are separated from the master seed lot by as determining the mass of the micro-organisms, or indirectly by
few subcultures as possible and in any case not more than an opacity method that has been calibrated in relation to the
8 subcultures. During the course of these subcultures the mass of the micro-organisms ; if the bacterial concentration is
preparation is not freeze-dried more than once. determined before addition of a stabiliser, the concentration
in the final bulk is established by calculation. The total
If a bioluminescence test or other biochemical method is
bacterial concentration is within the limits approved for the
used instead of viable count, the method is validated against
particular product.
the viable count for each stage of the process at which it
is used. The ratio of the count of viable units to the total bacterial
concentration is not less than that approved for the
SEED LOTS
particular product.
The strain used to establish the master seed lot is chosen for
and maintained to preserve its characteristics, its capacity to FINAL LOT
treat and prevent superficial bladder cancer, and its relative The final bulk is distributed into sterile containers and
absence of pathogenicity for man and laboratory animals. freeze-dried to a moisture content favourable to the stability
The strain used shall be identified by historical records of the product ; the containers are closed either under
that include information on its origin and subsequent vacuum or under an inert gas.
manipulation. Except where the filled and closed containers are stored at a
Before establishment of a working seed lot a batch is temperature of − 20 °C or lower, the expiry date is not later
prepared and reserved for use as the comparison product. than 4 years from the date of harvest.
When a new working seed lot is established, a suitable test Only a final lot that complies with the following requirement
for delayed hypersensitivity in guinea-pigs is carried out on for count of viable units and with each of the requirements
a batch of product prepared from the new working seed given below under Identification, Tests and Assay may be
lot ; the product is shown to be not significantly different in released for use. Provided the test for virulent mycobacteria
activity from the comparison product. Antimicrobial agent has been carried out with satisfactory results on the final
sensitivity testing is also carried out. bulk, it may be omitted on the final lot.
Only a working seed lot that complies with the following Count of viable units. Determine the number of viable units
requirements may be used for propagation. per millilitre of the reconstituted product by viable count on
Identification. The bacteria in the working seed lot solid medium using a method suitable for the product to be
are identified as Mycobacterium bovis BCG using examined, or by a suitable biochemical method. The ratio of
microbiological techniques, which may be supplemented the count of viable units after freeze-drying to that before is
by molecular biology techniques (for example, nucleic acid not less than that approved for the particular product.
amplification and restriction-fragment-length polymorphism).
Bacterial and fungal contamination. Carry out the test for IDENTIFICATION
sterility (2.6.1), using 10 ml for each medium. The working BCG for immunotherapy is identified by microscopic
seed lot complies with the test for sterility, except for the examination of the bacilli in stained smears demonstrating
presence of mycobacteria. their acid-fast property and by the characteristic appearance

General Notices (1) apply to all monographs and other texts 4053
Beclometasone dipropionate, anhydrous EUROPEAN PHARMACOPOEIA 6.3

of colonies grown on solid medium. Alternatively, molecular DEFINITION

biology techniques (for example, nucleic acid amplification) 9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-
may be used.
TESTS
Virulent mycobacteria. Inject subcutaneously or
intramuscularly into each of 6 guinea-pigs, each weighing
250-400 g and having received no treatment likely to
interfere with the test, a quantity of the product to be
examined equivalent to at least 1/25 of 1 human dose.
Observe the animals for at least 42 days. At the end of this
period, euthanise the guinea-pigs and examine by autopsy
for signs of infection with tuberculosis, ignoring any minor
reactions at the site of injection. Animals that die during
the observation period are also examined for signs of
tuberculosis. The product complies with the test if none
of the guinea-pigs shows signs of tuberculosis and if not
more than 1 animal dies during the observation period. If
2 animals die during this period and autopsy does not reveal
signs of tuberculosis, repeat the test on 6 other guinea-pigs.
The product complies with the test if not more than 1 animal
dies during the 42 days following the injection and autopsy
does not reveal any sign of tuberculosis.
Bacterial and fungal contamination. The reconstituted
product complies with the test for sterility (2.6.1) except for
the presence of mycobacteria.
Water. Not more than the limit approved for the particular
product, determined by a suitable method.
ASSAY
Determine the number of viable units in the reconstituted
product by viable count on solid medium using a method
suitable for the product to be examined or by a suitable
validated biochemical method. The number is within
the range stated on the label. Determine the number of
viable units in the comparison control in parallel.
LABELLING
The label states :
— the minimum and the maximum number of viable units
per dose in the reconstituted product ;
— that the product must be protected from direct sunlight.
01/2009:0654 solution to dissolve the contents of a vial of beclometasone
BECLOMETASONE DIPROPIONATE, Reference solution (d). Dissolve 50.0 mg of anhydrous
ANHYDROUS
Beclometasoni dipropionas anhydricus Column :
C28H37ClO7 Mr 521.1 — mobile phase B : tetrahydrofuran R, acetonitrile R,
[5534-09-8]
17,21-diyl dipropanoate.
Content : 96.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone, sparingly soluble in ethanol (96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : anhydrous beclometasone
dipropionate CRS.
B. Treat 25 mg by the oxygen-flask method (2.5.10). Use a
mixture of 1 ml of 1 M sodium hydroxide and 20 ml of
water R to absorb the combustion products. The solution
gives reaction (a) of chlorides (2.3.1).
C. Loss on drying (see Tests).
TESTS
Specific optical rotation (2.2.7) : + 108 to + 115 (dried
substance).
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
10.0 ml with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : mobile phase A, mobile phase B
(45:55 V/V).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in 28 ml of mobile phase B and dilute to 50.0 ml
with mobile phase A.
Test solution (b). Dilute 1.0 ml of test solution (a) to 50.0 ml
with the solvent mixture.
Reference solution (a). Dilute 5.0 ml of test solution (b) to
100.0 ml with the solvent mixture.
Reference solution (b). Dissolve 5 mg of beclometasone
dipropionate for system suitability CRS (containing
impurity D) in 3 ml of mobile phase B and dilute to 5 ml with
mobile phase A.
Reference solution (c). Dissolve 5 mg of beclometasone
dipropionate for peak identification CRS (containing
impurities A, B, C, L and M) in 3 ml of mobile phase B
and dilute to 5 ml with mobile phase A. Use 1 ml of this
dipropionate impurities F and N CRS.
beclometasone dipropionate CRS in 28 ml of mobile
phase B and dilute to 50.0 ml with mobile phase A. Dilute
1.0 ml of this solution to 50.0 ml with the solvent mixture.
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : spherical difunctional
bonded end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 50 °C.
Mobile phase :
— mobile phase A : 2.72 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 2.35 with
phosphoric acid R ;
methanol R (5:23:25 V/V/V) ;

4054 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Beclometasone dipropionate, anhydrous

Time Mobile phase A Mobile phase B Injection : test solution (b) and reference solution (d).
(min) (per cent V/V) (per cent V/V) Calculate the percentage content of C28H37ClO7 from
0-4 40 60 the declared content of beclometasone dipropionate
4 - 12 40 → 45 60 → 55 anhydrous CRS.
12 - 59 45 55

Flow rate : 1.4 ml/min. IMPURITIES

Specified impurities : A, B, C, D, F, L, M, N.
Detection : spectrophotometer at 254 nm.
Other detectable impurities (the following substances would,
Injection : 20 μl of test solution (a) and reference if present at a sufficient level, be detected by one or other of
solutions (a), (b) and (c). the tests in the monograph. They are limited by the general
Identification of impurities: use the chromatogram acceptance criterion for other/unspecified impurities and/or
supplied with beclometasone dipropionate for peak by the general monograph Substances for pharmaceutical
identification CRS and the chromatogram obtained use (2034). It is therefore not necessary to identify these
with reference solution (c) to identify the peaks due to impurities for demonstration of compliance. See also 5.10.
impurities A, B, C, F, L, M and N ; use the chromatogram Control of impurities in substances for pharmaceutical
supplied with beclometasone dipropionate for system use) : E, H, I, J, O, Q, R, S, U, V.
suitability CRS and the chromatogram obtained with
reference solution (b) to identify the peak due to impurity D.
Relative retention with reference to beclometasone
dipropionate (retention time = about 25 min) :
impurity A = about 0.3 ; impurity B = about 0.6 ;
impurity D = about 1.1 ; impurity M = about 1.2 ;
impurity L = about 1.3 ; impurity C = about 1.8 ;
impurity N = about 2.0 ; impurity F = about 2.2.
System suitability : reference solution (b) :
— peak-to-valley ratio : minimum 1.5, where Hp = height
above the baseline of the peak due to impurity D and A. R1 = R3 = H, R2 = Cl, R4 = CO-C2H5 : 9-chloro-11β,17-
Hv = height above the baseline of the lowest point of dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-21-yl
the curve separating this peak from the peak due to propanoate (beclometasone 21-propionate),
beclometasone dipropionate.
Limits : B. R1 = H, R2 = Cl, R3 = CO-C2H5, R4 = CO-CH3 :
— correction factors : for the calculation of content, 21-(acetyloxy)-9-chloro-11β-hydroxy-16β-methyl-3,20-
multiply the peak areas of the following impurities by dioxopregna-1,4-dien-17-yl propanoate (beclometasone
the corresponding correction factor : impurity F = 1.3 ; 21-acetate 17-propionate),
impurity M = 2.0 ;
C. R1 = H, R2 = Cl, R3 = CO-C2H5, R4 = CO-CH2-CH2-CH3 :
— impurity L : not more than 6 times the area of the 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxo-17-
principal peak in the chromatogram obtained with (propanoyloxy)-pregna-1,4-dien-21-yl butanoate
reference solution (a) (0.6 per cent) ; (beclometasone 21-butyrate 17-propionate),
— impurities B, F, M : for each impurity, not more than
5 times the area of the principal peak in the chromatogram D. R1 = H, R2 = Br, R3 = R4 = CO-C2H5 : 9-bromo-11β-
obtained with reference solution (a) (0.5 per cent) ; hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl
dipropanoate,
— impurities A, D, N : for each impurity, not more than
twice the area of the principal peak in the chromatogram F. R1 = Br, R2 = Cl, R3 = R4 = CO-C2H5 : 6α-bromo-9-chloro-
obtained with reference solution (a) (0.2 per cent) ; 11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,
— impurity C : not more than 1.5 times the area of the 21-diyl dipropanoate,
principal peak in the chromatogram obtained with
reference solution (a) (0.15 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 : not more than 15 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined E. R1 = Cl, R2 = CO-C2H5 : 6α,9-dichloro-11β-hydroxy-
on 1.000 g by drying in an oven at 105 °C for 3 h. 16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl
dipropanoate,
ASSAY
Liquid chromatography (2.2.29) as described in the test for H. R1 = R2 = H : 9-chloro-11β,21-dihydroxy-16β-methyl-3,20-
related substances with the following modification. dioxopregna-1,4-dien-17-yl propanoate (beclometasone
17-propionate),

General Notices (1) apply to all monographs and other texts 4055
Beclometasone dipropionate monohydrate EUROPEAN PHARMACOPOEIA 6.3

I. 16β-methyl-3,20-dioxopregna-1,4,9(11)-triene-17,21-diyl N. 2-bromo-9-chloro-11β-hydroxy-16β-methyl-3,20-
dipropanoate, dioxopregna-1,4-diene-17,21-diyl dipropanoate,

O. R1 = R2 = Cl : 9,11β-dichloro-16β-methyl-3,20-
J. R1 = R2 = CO-C2H5 : 9,11β-epoxy-16β-methyl-3,20-dioxo- dioxopregna-1,4-diene-17,21-diyl dipropanoate,
9β-pregna-1,4-diene-17,21-diyl dipropanoate,
Q. R1 = R2 = H : 16β-methyl-3,20-dioxopregna-1,4-diene-17,
21-diyl dipropanoate,
R. R1 = R2 = H : 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β- S. R1 = O-CO-C H , R2 = Cl : 9-chloro-16β-methyl-3,20-
pregna-1,4-diene-3,20-dione, 2 5
dioxopregna-1,4-diene-11β,17,21-triyl tripropanoate
(beclometasone tripropionate).
U. R1 = H, R2 = CO-C2H5 : 9,11β-epoxy-21-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-17-yl propanoate,
01/2009:1709
V. R1 = CO-C2H5, R2 = H : 9,11β-epoxy-17-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-21-yl propanoate, BECLOMETASONE DIPROPIONATE
MONOHYDRATE
Beclometasoni dipropionas monohydricus

L. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregn-4-ene- C28H37ClO7,H2O Mr 539.1

17,21-diyl dipropanoate,
DEFINITION
9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-
17,21-diyl dipropanoate monohydrate.
Content : 97.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, freely soluble in
acetone, sparingly soluble in ethanol (96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
M. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-4,6- Comparison : beclometasone dipropionate
diene-17,21-diyl dipropanoate, monohydrate CRS.

4056 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Beclometasone dipropionate monohydrate

B. Treat 25 mg by the oxygen-flask method (2.5.10). Use a Identification of impurities : use the chromatogram
mixture of 1 ml of 1 M sodium hydroxide and 20 ml of supplied with beclometasone dipropionate for peak
water R to absorb the combustion products. The solution identification CRS and the chromatogram obtained with
gives reaction (a) of chlorides (2.3.1). reference solution (c) to identify the peaks due to impurities
B, C, F and L ; use the chromatogram supplied with
C. Loss on drying (see Tests). beclometasone dipropionate for system suitability CRS and
the chromatogram obtained with reference solution (b) to
TESTS identify the peak due to impurity D.
Specific optical rotation (2.2.7) : + 108 to + 115 (dried Relative retention with reference to beclometasone
substance). dipropionate (retention time = about 25 min) :
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to impurity B = about 0.6 ; impurity D = about 1.1 ;
10.0 ml with the same solvent. impurity L = about 1.3 ; impurity C = about 1.8 ;
impurity F = about 2.2.
Related substances. Liquid chromatography (2.2.29).
System suitability: reference solution (b) :
Solvent mixture : mobile phase A, mobile phase B — peak-to-valley ratio : minimum 1.5, where Hp = height
(45:55 V/V).
above the baseline of the peak due to impurity D and
Test solution (a). Dissolve 50.0 mg of the substance to be Hv = height above the baseline of the lowest point of
examined in 28 ml of mobile phase B and dilute to 50.0 ml the curve separating this peak from the peak due to
with mobile phase A. beclometasone dipropionate.
Test solution (b). Dilute 1.0 ml of test solution (a) to 50.0 ml Limits :
with the solvent mixture. — correction factor : for the calculation of content, multiply
Reference solution (a). Dilute 5.0 ml of test solution (b) to the peak area of impurity F by 1.3 ;
100.0 ml with the solvent mixture. — impurity B : not more than 5 times the area of the
Reference solution (b). Dissolve 5 mg of beclometasone principal peak in the chromatogram obtained with
dipropionate for system suitability CRS (containing reference solution (a) (0.5 per cent) ;
impurity D) in 3 ml of mobile phase B and dilute to 5 ml with — impurities C, F, L : for each impurity, not more
mobile phase A. than 1.5 times the area of the principal peak in the
Reference solution (c). Dissolve 5 mg of beclometasone chromatogram obtained with reference solution (a)
dipropionate for peak identification CRS (containing (0.15 per cent) ;
impurities B, C and L) in 3 ml of mobile phase B and dilute — unspecified impurities : for each impurity, not more
to 5 ml with mobile phase A. Use 1 ml of this solution to than the area of the principal peak in the chromatogram
dissolve the contents of a vial of beclometasone dipropionate obtained with reference solution (a) (0.10 per cent) ;
impurities F and N CRS. — total : not more than 10 times the area of the principal
Reference solution (d). Dissolve 50.0 mg of beclometasone peak in the chromatogram obtained with reference
dipropionate anhydrous CRS in 28 ml of mobile phase B solution (a) (1.0 per cent) ;
and dilute to 50.0 ml with mobile phase A. Dilute 1.0 ml of — disregard limit : 0.5 times the area of the principal peak
this solution to 50.0 ml with the solvent mixture. in the chromatogram obtained with reference solution (a)
Column: (0.05 per cent).
— size : l = 0.25 m, Ø = 4.6 mm ; Loss on drying (2.2.32) : 2.8 per cent to 3.8 per cent,
determined on 1.000 g by drying in an oven at 105 °C for 3 h.
— stationary phase : spherical difunctional
bonded end-capped octadecylsilyl silica gel for ASSAY
chromatography R (5 μm) ;
Liquid chromatography (2.2.29) as described in the test for
— temperature : 50 °C. related substances with the following modification.
Mobile phase :
Injection : test solution (b) and reference solution (d).
— mobile phase A : 2.72 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 2.35 with Calculate the percentage content of C28H37ClO7 from
phosphoric acid R ; the declared content of anhydrous beclometasone
dipropionate CRS.
— mobile phase B : tetrahydrofuran R, acetonitrile R,
methanol R (5:23:25 V/V/V) ;
Time Mobile phase A Mobile phase B IMPURITIES
(min) (per cent V/V) (per cent V/V)
0-4 40 60
Specified impurities : B, C, F, L.
4 - 12 40 → 45 60 → 55
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
12 - 59 45 55 the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Flow rate : 1.4 ml/min. by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
Detection : spectrophotometer at 254 nm.
impurities for demonstration of compliance. See also 5.10.
Injection : 20 μl of test solution (a) and reference Control of impurities in substances for pharmaceutical
solutions (a), (b) and (c). use) : A, D, E, H, I, J, M, N, O, Q, R, S, U, V.

General Notices (1) apply to all monographs and other texts 4057
Beclometasone dipropionate monohydrate EUROPEAN PHARMACOPOEIA 6.3

A. R1 = R3 = H, R2 = Cl, R4 = CO-C2H5 : 9-chloro-11β,17- I. 16β-methyl-3,20-dioxopregna-1,4,9(11)-triene-17,21-diyl

dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-21-yl dipropanoate,
propanoate (beclometasone 21-propionate),

D. R1 = H, R2 = Br, R3 = R4 = CO-C2H5 : 9-bromo-11β-

hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl
dipropanoate,

E. R1 = R2 = Cl, R3 = R4 = CO-C2H5 : 6α,9-dichloro-11β- J. R1 = R2 = CO-C2H5 : 9,11β-epoxy-16β-methyl-3,20-dioxo-

hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl 9β-pregna-1,4-diene-17,21-diyl dipropanoate,
dipropanoate,
R. R1 = R2 = H : 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β-
pregna-1,4-diene-3,20-dione,

U. R1 = H, R2 = CO-C2H5 : 9,11β-epoxy-21-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-17-yl propanoate,

H. R1 = R4 = H, R2 = Cl, R3 = CO-C2H5 : 9-chloro-11β,21-

dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl V. R1 = CO-C2H5, R2 = H : 9,11β-epoxy-17-hydroxy-16β-
propanoate (beclometasone 17-propionate), methyl-3,20-dioxo-9β-pregna-1,4-dien-21-yl propanoate,

B. R1 = H, R2 = CO-CH3 : 21-(acetyloxy)-9-chloro-11β- L. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregn-4-ene-

hydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl 17,21-diyl dipropanoate,
propanoate (beclometasone 21-acetate 17-propionate),

C. R1 = H, R2 = CO-CH2-CH2-CH3 : 9-chloro-11β-hydroxy-16β-
methyl-3,20-dioxo-17-(propanoyloxy)-pregna-1,4-dien-21-yl
butanoate (beclometasone 21-butyrate 17-propionate),

F. R1 = Br, R2 = CO-C2H5 : 6α-bromo-9-chloro-11β-

hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl M. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-4,6-
dipropanoate, diene-17,21-diyl dipropanoate,

4058 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Belladonna leaf dry extract, standardised

furthermore, the chromatogram obtained with the test

solution shows a little above the start a yellowish-brown
fluorescent zone and directly above that a yellow
fluorescent zone, and a yellow or yellowish-brown
fluorescent zone between the zone due to rutin and the
zone due to chlorogenic acid. Further zones may be
present.
B. Examine the chromatograms obtained in the test for
atropine.
Results : the principal zones in the chromatogram
N. R1 = Br, R2 = OH, R3 = Cl : 2-bromo-9-chloro-11β- obtained with the test solution are similar in position
hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl and colour to the principal zones in the chromatogram
dipropanoate, obtained with the reference solution.
O. R1 = H, R2 = R3 = Cl : 9,11β-dichloro-16β-methyl-3,20-
dioxopregna-1,4-diene-17,21-diyl dipropanoate, TESTS
Atropine. Thin-layer chromatography (2.2.27).
Q. R1 = R2 = R3 = H : 16β-methyl-3,20-dioxopregna-1,4-diene-
17,21-diyl dipropanoate, Test solution. To 0.20 g of the extract to be examined add
10.0 ml of 0.05 M sulphuric acid, shake for 2 min and
S. R1 = H, R2 = O-CO-C2H5, R3 = Cl : 9-chloro-16β-methyl- filter. Add 1.0 ml of concentrated ammonia R and shake
3,20-dioxopregna-1,4-diene-11β,17,21-triyl tripropanoate with 2 quantities, each of 10 ml, of peroxide-free ether R.
(beclometasone tripropionate). If necessary, separate by centrifugation. Dry the combined
ether layers over about 2 g of anhydrous sodium sulphate R,
filter and evaporate to dryness on a water-bath. Dissolve the
01/2009:1294 residue in 0.5 ml of methanol R.
Reference solution. Dissolve 50 mg of hyoscyamine
BELLADONNA LEAF DRY EXTRACT, sulphate R in 9 ml of methanol R. Dissolve 15 mg of
STANDARDISED hyoscine hydrobromide R in 10 ml of methanol R. Mix
1.8 ml of the hyoscine hydrobromide solution and 8 ml of
the hyoscyamine sulphate solution.
Belladonnae folii extractum siccum
Plate : TLC silica gel plate R.
normatum Mobile phase : concentrated ammonia R, water R, acetone R
DEFINITION (3:7:90 V/V/V).
Standardised dry extract obtained from Belladonna Application : 20 μl as bands.
leaf (0221). Development : over a path of 10 cm.
Content : 0.95 per cent to 1.05 per cent of total alkaloids, Drying : at 100-105 °C for 15 min ; allow to cool.
expressed as hyoscyamine (C17H23NO3 ; Mr 289.4) (dried Detection A : spray with potassium iodobismuthate
extract). solution R2, until orange or brown zones become visible
PRODUCTION against a yellow background.
The extract is produced from the herbal drug by a suitable Results A : the zones in the chromatogram obtained with
procedure using ethanol (70 per cent V/V). the test solution are similar in position (hyoscyamine in the
lower third, hyoscine in the upper third) and colour to those
CHARACTERS in the chromatogram obtained with the reference solution.
Appearance : brown or greenish, hygroscopic powder. Other faint zones may be present in the chromatogram
obtained with the test solution.
IDENTIFICATION Detection B : spray with sodium nitrite solution R until the
A. Thin-layer chromatography (2.2.27). coating is transparent and examine after 15 min.
Test solution. To 1 g of the extract to be examined add Results B : the zones due to hyoscyamine in the
5.0 ml of methanol R. Shake for 2 min and filter. chromatograms obtained with the test solution and
Reference solution. Dissolve 1.0 mg of chlorogenic the reference solution change from orange or brown to
acid R and 2.5 mg of rutin R in 10 ml of methanol R. reddish-brown but not to greyish-blue (atropine).
Plate : TLC silica gel plate R. Loss on drying (2.8.17) : maximum 5.0 per cent.
Mobile phase : anhydrous formic acid R, water R, methyl Microbial contamination
ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V). TAMC : acceptance criterion 104 CFU/g (2.6.12).
Application : 20 μl as bands. TYMC : acceptance criterion 102 CFU/g (2.6.12).
Development : over a path of 15 cm. Absence of Escherichia coli (2.6.13).
Drying : at 100-105 °C.
Absence of Salmonella (2.6.13).
Detection : spray the warm plate with a 10 g/l solution of
diphenylboric acid aminoethyl ester R in methanol R, ASSAY
then spray with a 50 g/l solution of macrogol 400 R in At each extraction stage it is necessary to check that the
methanol R ; allow to dry in air for 30 min and examine in alkaloids have been completely extracted. If the extraction is
ultraviolet light at 365 nm. into the organic phase this is done by evaporating to dryness
Results : the chromatograms obtained with the reference a few millilitres of the last organic layer, dissolving the
solution and the test solution show in the central part a residue in 0.25 M sulphuric acid and verifying the absence
light blue fluorescent zone (chlorogenic acid) and in the of alkaloids using potassium tetraiodomercurate solution R.
lower part a yellowish-brown fluorescent zone (rutin) ; If the extraction is into the acid aqueous phase, this is done

General Notices (1) apply to all monographs and other texts 4059
Benazepril hydrochloride
by taking a few millilitres of the last acid aqueous phase
and verifying the absence of alkaloids using potassium
tetraiodomercurate solution R.
Disperse 3.00 g in a mixture of 5 ml of ammonia R and
15 ml of water R. Shake with no fewer than 3 quantities,
each of 40 ml, of a mixture of 1 volume of methylene
chloride R and 3 volumes of peroxide-free ether R until
the alkaloids are completely extracted. Concentrate the
combined organic layers to about 50 ml by distilling on a
water-bath and transfer the resulting liquid to a separating
funnel, rinsing with peroxide-free ether R. Add a quantity of
peroxide-free ether R equal to at least 2.1 times the volume
of the liquid to produce a layer having a density well below
that of water. Shake the resulting solution with no fewer
than 3 quantities, each of 20 ml, of 0.25 M sulphuric acid
until the alkaloids are completely extracted. Separate the
layers by centrifugation, if necessary, and transfer the acid
layers to a 2nd separating funnel. Make the combined acid
layers alkaline with ammonia R and shake with no fewer
than 3 quantities, each of 30 ml, of methylene chloride R
until the alkaloids are completely extracted. Combine the
organic layers, add 4 g of anhydrous sodium sulphate R and
allow to stand for 30 min with occasional shaking. Decant
the methylene chloride and wash the sodium sulphate
with 3 quantities, each of 10 ml, of methylene chloride R.
Combine the organic extracts and evaporate to dryness on
a water-bath. Heat the residue in an oven at 100-105 °C
for 15 min. Dissolve the residue in a few millilitres of
methylene chloride R, evaporate to dryness on a water-bath
and again heat the residue in an oven at 100-105 °C for
15 min. Dissolve the residue in a few millilitres of methyleneexamined in the mobile phase and dilute to 50.0 ml with the
chloride R, add 20.0 ml of 0.01 M sulphuric acid and remove
the methylene chloride by evaporation on a water-bath.
Titrate the excess of acid with 0.02 M sodium hydroxide
using methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressedhydrochloride CRS in the mobile phase and dilute to 50.0 ml
as hyoscyamine, using the following expression :
n = volume of 0.02 M sodium hydroxide used, in
millilitres ;
m = mass of drug used, in grams.
01/2009:2388 — stationary phase : end-capped octadecylsilyl silica gel
BENAZEPRIL HYDROCHLORIDE
Benazeprili hydrochloridum
C24H29ClN2O5 Mr 461.0 impurity F = about 0.4 ; impurity C = about 0.5 ;
[86541-74-4]
DEFINITION
[(3S)-3-[[(1S)-1-(Ethoxycarbonyl)-3-phenylpropyl]amino]-
2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid
hydrochloride.
4060 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Content : 97.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water, freely soluble in
anhydrous ethanol, very slightly soluble in ethyl acetate,
practically insoluble in cyclohexane.
It shows polymorphism (5.9).
IDENTIFICATION
Carry out either tests A, B, D or tests B, C, D.
A. Specific optical rotation (2.2.7) : − 136 to − 141 (dried
substance).
Dissolve 1.000 g in anhydrous ethanol R and dilute to
50.0 ml with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : benazepril hydrochloride 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.
C. Enantiomeric purity (see Tests).
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
mobile phase.
Test solution (b). Dilute 10.0 ml of test solution (a) to
100.0 ml with the mobile phase.
Reference solution (a). Dissolve 50.0 mg of benazepril
with the mobile phase. Dilute 10.0 ml of this solution to
100.0 ml with the mobile phase.
Reference solution (b). Dissolve the contents of a vial
of benazepril for system suitability CRS (containing
impurities B, C, D, E, F and G) in 1.0 ml of test solution (a).
Reference solution (c). Dilute 1.0 ml of reference solution (a)
to 50.0 ml with the mobile phase.
Column :
— size: l = 0.30 m, Ø = 3.9 mm ;
for chromatography R (10 μm).
Mobile phase : add 0.2 ml of glacial acetic acid R to 1000 ml
of a mixture of 360 volumes of water R and 640 volumes of
methanol R2 ; add 0.81 g of tetrabutylammonium bromide R
and stir to dissolve.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 240 nm.
Injection : 25 μl of test solution (a) and reference solutions (b)
and (c).
Run time : 3 times the retention time of benazepril.
Relative retention with reference to benazepril
(retention time = about 6 min) : impurity E = about 0.3 ;
impurity B = about 1.8 ; impurity D = about 2.0 ;
impurity G = about 2.5.
Identification of impurities : use the chromatogram
supplied with benazepril for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities B, C, D, E, F and G.
EUROPEAN PHARMACOPOEIA 6.3 Benazepril hydrochloride

System suitability : reference solution (b) : System suitability : reference solution (c) :
— resolution : minimum 2.5 between the peaks due to — peak-to-valley ratio : minimum 2.5, where Hp = height
benazepril and impurity B and minimum 1.5 between the above the baseline of the peak due to impurity A and
peaks due to impurities E and F. Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
Limits : benazepril.
— correction factors : for the calculation of content, Limit :
multiply the peak areas of the following impurities by — impurity A : not more than the area of the corresponding
the corresponding correction factor : impurity E = 0.5 ; peak in the chromatogram obtained with reference
impurity F = 0.7 ; solution (b) (0.1 per cent).
— impurity B : not more than 2.5 times the area of the Heavy metals (2.4.8) : maximum 20 ppm.
principal peak in the chromatogram obtained with
reference solution (c) (0.5 per cent) ; 1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
— impurity C : not more than 1.5 times the area of the
Loss on drying (2.2.32) : maximum 0.50 per cent, determined
principal peak in the chromatogram obtained with
on 1.000 g by drying in vacuo at 105 °C for 3 h.
reference solution (c) (0.3 per cent) ;
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
— impurities D, E, F, G : for each impurity, not more than on 1.0 g.
the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ; ASSAY
— unspecified impurities: for each impurity, not more Liquid chromatography (2.2.29) as described in the test for
than 0.5 times the area of the principal peak in the related substances with the following modification.
chromatogram obtained with reference solution (c) Injection : test solution (b) and reference solution (a).
(0.10 per cent) ;
Calculate the percentage content of C24H29ClN2O5 from the
— total : not more than 10 times the area of the principal declared content of benazepril hydrochloride CRS.
peak in the chromatogram obtained with reference
solution (c) (2.0 per cent) ; STORAGE
— disregard limit: 0.25 times the area of the principal peak Protected from light.
in the chromatogram obtained with reference solution (c) IMPURITIES
(0.05 per cent).
Specified impurities : A, B, C, D, E, F, G.
Enantiomeric purity. Liquid chromatography (2.2.29).
Buffer solution pH 6.0. Dissolve 3.58 g of disodium
hydrogen phosphate R and 9.66 g of potassium dihydrogen
phosphate R in water R and dilute to 1000.0 ml with the
same solvent.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the
mobile phase.
A. [(3R)-3-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-2-
Reference solution (a). Dissolve 5.0 mg of benazepril oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid,
impurity A CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase.
Reference solution (b). Dilute 1.0 ml of reference solution (a)
to 100.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of reference solution (a)
to 10.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the test solution.
Column: B. [(3RS)-3-[[(1SR)-1-(ethoxycarbonyl)-3-phenylpropyl]-
— size : l = 0.10 m, Ø = 4.0 mm ; amino]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-
yl]acetic acid,
— stationary phase : spherical silica gel AGP for chiral
chromatography R (5 μm) ;
— temperature : 30 °C.
Mobile phase : methanol R2, buffer solution pH 6.0
(20:80 V/V).
Flow rate : 0.9 ml/min.
Detection : spectrophotometer at 240 nm.
C. R = H : (2S)-2-[[(3S)-1-(carboxymethyl)-2-oxo-2,3,4,5-
Injection : 50 μl of the test solution and reference tetrahydro-1H-1-benzazepin-3-yl]amino]-4-phenylbutanoic
solutions (b) and (c). acid,
Run time : 3.5 times the retention time of benazepril. G. R = C2H5 : ethyl (2S)-2-[[(3S)-1-(2-ethoxy-2-oxoethyl)-2-
Relative retention with reference to benazepril (retention oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl]amino]-4-
time = about 6 min) : impurity A = about 1.9. phenylbutanoate,

General Notices (1) apply to all monographs and other texts 4061
Bentonite
D. [(3S)-3-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl)propyl]-
amino]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-
yl]acetic acid,
E. R = H : [(3S)-3-amino-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-1-yl]acetic acid,
F. R = C(CH3)3 : 1,1-dimethylethyl [(3S)-3-amino-2-oxo-2,3,4, 50.0 ml with water R. To 5 ml of this solution add 5 ml of
5-tetrahydro-1H-1-benzazepin-1-yl]acetate.
01/2009:0467 Dissolve the residue in 1 ml of acetic acid R, dilute to 25 ml
BENTONITE
Bentonitum
DEFINITION
Natural clay containing a high proportion of montmorillonite, TAMC : acceptance criterion 103 CFU/g (2.6.12).
a native hydrated aluminium silicate in which some
aluminium and silicon atoms may be replaced by other atoms
such as magnesium and iron.
CHARACTERS
Appearance : very fine, homogeneous, greyish-white powder
with a more or less yellowish or pinkish tint.
Solubility : practically insoluble in water and in aqueous
solutions.
It swells with a little water forming a malleable mass.
IDENTIFICATION
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R
and 3 g of sodium carbonate R and heat until the mixture
melts. Allow to cool. To this residue add 20 ml of boiling
water R, mix and filter. Wash the insoluble residue with
50 ml of water R. To this residue add 1 ml of hydrochloric
acid R and 5 ml of water R. Filter. To the filtrate add 1 ml
of strong sodium hydroxide solution R and filter. To this
filtrate add 3 ml of ammonium chloride solution R. A
gelatinous white precipitate is formed.
B. Swelling power with water (see Tests).
C. 0.25 g gives the reaction of silicates (2.3.1).
TESTS
Alkalinity. To 2 g add 100 ml of carbon dioxide-free water R
and shake for 5 min. To 5 ml of this suspension add 0.1 ml of
thymolphthalein solution R. The liquid becomes bluish. Add
0.1 ml of 0.1 M hydrochloric acid. The liquid is decolourised
within 5 min.
Coarse particles : maximum 0.5 per cent.
To 20 g add 1000 ml of water R and mix for 15 min using
a high-speed mixer capable of operating at not less than
5000 r/min. Transfer the suspension to a wet sieve (75),
tared after drying at 100-105 °C. Wash with 3 quantities,
4062 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
each of 500 ml, of water R, ensuring that any agglomerates
have been dispersed. Dry the sieve at 100-105 °C and weigh.
The particles on the sieve weigh a maximum of 0.1 g.
Sedimentation volume. To 6.0 g add 200 ml of water R and
mix for 20 min using a high-speed mixer capable of operating
at 10 000 r/min. Transfer 100 ml of this suspension to a
graduated cylinder. Allow to stand for 24 h. The volume of
the clear supernatant liquid is not greater than 2 ml.
Swelling power with water. Add 2.0 g in 20 portions to
100 ml of a 10 g/l solution of sodium laurilsulfate R in a
100 ml graduated cylinder about 30 mm in diameter. Allow
2 min between additions for each portion to settle. Allow to
stand for 2 h. The apparent volume of the sediment is not
less than 22 ml.
Heavy metals (2.4.8) : maximum 50 ppm.
To 5.0 g add 7.5 ml of dilute hydrochloric acid R and
27.5 ml of water R. Boil for 5 min. Centrifuge and filter
the supernatant liquid. Wash the centrifugation residue
with water R and filter. Dilute the combined filtrates to
water R, 10 ml of hydrochloric acid R and 25 ml of methyl
isobutyl ketone R and shake for 2 min. Separate the layers.
Evaporate the aqueous layer to dryness on a water-bath.
with water R and filter. 12 ml of the filtrate complies with
test A. Prepare the reference solution using lead standard
solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 15 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Microbial contamination
01/2009:0811
BETAMETHASONE VALERATE
Betamethasoni valeras
C27H37FO6 Mr 476.6
[2152-44-5]
DEFINITION
9-Fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-1,4-
dien-17-yl pentanoate.
Content : 97.0 per cent to 103.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble
in acetone and in methylene chloride, soluble in ethanol
(96 per cent).
mp : about 192 °C, with decomposition.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : betamethasone 17-valerate CRS.
EUROPEAN PHARMACOPOEIA 6.3 Betamethasone valerate

If the spectra obtained in the solid state show differences, identify the peaks due to impurities C, D, G, H and I; use
dissolve the substance to be examined and the reference the chromatogram obtained with reference solution (c) to
substance separately in the minimum volume of identify the peaks due to impurities A and E.
methylene chloride R, evaporate to dryness on a
water-bath and record new spectra using the residues. Relative retention with reference to betamethasone valerate
(retention time = about 20 min) : impurity A = about 0.3 ;
B. Liquid chromatography (2.2.29). impurity I = about 0.6 ; impurity C = about 0.8 ;
impurity H = about 1.3 ; impurity D = about 1.4 ;
Examine the chromatograms obtained in the test for impurity E = about 1.6 ; impurity G = about 2.0.
related substances.
System suitability : reference solution (b) :
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size — resolution : minimum 1.7 between the peaks due to
to the principal peak in the chromatogram obtained with impurities H and D.
reference solution (b).
Limits :
— impurity A : not more than 7 times the area of the
principal peak in the chromatogram obtained with
TESTS reference solution (a) (0.7 per cent) ;
Specific optical rotation (2.2.7) : + 77 to + 83 (dried
substance). — impurities E, G : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
Dissolve 0.250 g in anhydrous ethanol R and dilute to obtained with reference solution (a) (0.3 per cent) ;
25.0 ml with the same solvent.
— impurities C, H, I : for each impurity, not more
Related substances. Liquid chromatography (2.2.29). Carry than 1.5 times the area of the principal peak in the
out the test protected from light. Prepare the solutions chromatogram obtained with reference solution (a)
immediately before use. (0.15 per cent) ;
Solvent mixture : glacial acetic acid R, mobile phase — unspecified impurities : for each impurity, not more
(1:1000 V/V). than the area of the principal peak in the chromatogram
Test solution. Dissolve 50 mg of the substance to be obtained with reference solution (a) (0.10 per cent) ;
examined in the solvent mixture and dilute to 20.0 ml with — total : not more than 15 times the area of the principal
the solvent mixture. peak in the chromatogram obtained with reference
Reference solution (a). Dilute 1.0 ml of the test solution solution (a) (1.5 per cent) ;
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this — disregard limit : 0.5 times the area of the principal peak
solution to 10.0 ml with the solvent mixture. in the chromatogram obtained with reference solution (a)
Reference solution (b). Dissolve 12.5 mg of betamethasone (0.05 per cent).
valerate for system suitability CRS (containing impurities D Loss on drying (2.2.32) : maximum 0.5 per cent, determined
and G) in 5.0 ml of the solvent mixture. Use 1.0 ml of this on 1.000 g by drying in an oven at 105 °C.
solution to dissolve the contents of a vial of betamethasone
valerate impurity mixture CRS (containing impurities C,
H and I). ASSAY
Reference solution (c). Dissolve 6 mg of betamethasone CRS Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
(impurity A) and 3 mg of betamethasone 21-valerate CRS 100.0 ml with the same solvent. Dilute 2.0 ml of this solution
(impurity E) in 30.0 ml of the solvent mixture. Dilute 1.0 ml to 50.0 ml with ethanol (96 per cent) R. Measure the
of this solution to 10.0 ml with the solvent mixture. absorbance (2.2.25) at the absorption maximum at 240 nm.

Column: Calculate the content of C27H37FO6 taking the specific

absorbance to be 325.
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel STORAGE
for chromatography R (5 μm) ;
Protected from light.
— temperature : 20 °C.
Mobile phase : acetonitrile R, water R (50:50 V/V). IMPURITIES
Flow rate : 1 ml/min. Specified impurities: A, C, E, G, H, I.
Detection : spectrophotometer at 239 nm. Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
Injection : 20 μl. or other of the tests in the monograph. They are limited
Run time : 2.5 times the retention time of betamethasone by the general acceptance criterion for other/unspecified
valerate. impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
Identification of impurities: use the chromatogram supplied identify these impurities for demonstration of compliance.
with betamethasone valerate for system suitability CRS and See also 5.10. Control of impurities in substances for
the chromatogram obtained with reference solution (b) to pharmaceutical use) : B, D, F.

General Notices (1) apply to all monographs and other texts 4063
Bitter-orange epicarp and mesocarp
A. R1 = R3 = R5 = R6 = H, R2 = F, R4 = CH3 : betamethasone,
C. R1 = R4 = R6 = H, R2 = F, R3 = CH3, R5 = CO-[CH2]3-CH3 :
9-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxopregna-
1,4-dien-17-yl pentanoate (dexamethasone 17-valerate),
E. R1 = R3 = R5 = H, R2 = F, R4 = CH3, R6 = CO-[CH2]3-CH3 :
9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
1,4-dien-21-yl pentanoate (betamethasone 21-valerate),
G. R1 = Br, R2 = F, R3 = R6 = H, R4 = CH3, R5 =
CO-[CH2]3-CH3 : 6α-bromo-9-fluoro-11β,21-dihydroxy-16β-
methyl-3,20-dioxopregna-1,4-dien-17-yl pentanoate
(6α-bromo-betamethasone valerate),
H. R1 = R3 = R6 = H, R2 = Cl, R4 = CH3, R5 = CO-[CH2]3-CH3 : mesocarp and endocarp.
9-chloro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-
1,4-dien-17-yl pentanoate (beclomethasone 17-valerate),
I. R1 = R3 = R4 = R6 = H, R2 = F, R5 = CO-[CH2]3-CH3 :
9-fluoro-11β,21-dihydroxy-3,20-dioxopregna-1,4-dien-17-yl
pentanoate (9-fluoro-prednisolone 17-valerate),
B. R1 = F, R2 = R3 = H : 9-fluoro-11β,17-dihydroxy-
16β-methylpregna-1,4-diene-3,20-dione (21-deoxy-
betamethasone),
D. R1 = Br, R2 = CO-[CH2]3-CH3, R3 = OH : 9-bromo-11β,21-
dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl
pentanoate (9-bromo-betamethasone valerate),
4064 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
F. 21-hydroxy-16β-methyl-3,20-dioxopregna-1,4,9(11)-trien-
17-yl pentanoate (betamethasone valerate δ-9(11)).
01/2009:1603
BITTER-ORANGE EPICARP AND
MESOCARP
Aurantii amari epicarpium et mesocarpium
DEFINITION
Dried epicarp and mesocarp of the ripe fruit of Citrus
aurantium L. ssp. aurantium (C. aurantium L. ssp. amara
Engl.) partly freed from the white spongy tissue of the
Content : minimum 20 ml/kg of essential oil (anhydrous
drug).
CHARACTERS
Aromatic odour and spicy bitter taste.
IDENTIFICATION
A. The drug consists of elliptical or irregular pieces 5-8 cm
long, 3-5 cm broad and about 3 mm thick. The outer
surface is yellowish or reddish-brown and distinctly
punctate, the inner surface is yellowish or brownish-white.
B. Reduce to a powder (355) (2.9.12). The powder is light
brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following
diagnostic characters : small polygonal cells with
slightly thickened anticlinal walls, filled with orange-red
chromatophores, and very occasional anomocytic
stomata (2.8.3) ; fragments of the hypodermis showing
collenchymatous thickening ; groups of parenchyma
with each cell containing a prism crystal of calcium
oxalate ; fragments of lysigenous oil glands ; parenchyma
containing crystals of hesperidin which dissolve in a
20 g/l potassium hydroxide R solution giving a yellow
colour.
EUROPEAN PHARMACOPOEIA 6.3 Bitter-orange flower

Results : see below the sequence of zones present in

the chromatograms obtained with the reference and
test solutions. Furthermore, other fluorescent zones
are present in the chromatogram obtained with the test
solution.
Top of the plate
A light blue fluorescent zone
A light blue fluorescent zone
Caffeic acid : a light blue
fluorescent zone
A light blue fluorescent zone
A light blue fluorescent zone
Naringin : a dark green A dark green fluorescent zone
fluorescent zone (naringin)
A red fluorescent zone
(neoeriocitrin)
An orange fluorescent zone
Reference solution Test solution

A. Fragment in transverse section
showing epicarp with thick
cuticule (Aa), collenchymatous
hypodermis (Ab) and part of
the mesocarp parenchyma
containing prism crystals (Ac) of
calcium oxalate and fragment of
an oil gland (Ad)
B. Fragment of epicarp with
anomocytic stoma, in surface
view
C. Group of cells of the mesocarp,
some containing calcium oxalate
crystals
D, E, F, H, K and M. Fragments
of mesocarp
G. Sub-epicarpal
collenchymatous cells
J. Prism crystals of calcium
oxalate
L. Group of parenchymatous
cells
N. Fragment of epicarp with thick
cuticle and hypodermis showing
collenchymatous thickening, in
transverse section
TESTS
Water (2.2.13) : maximum 10.0 per cent, determined by
distillation on 20.0 g of powdered drug (355) (2.9.12).
Total ash (2.4.16) : maximum 7.0 per cent
Extractable matter : minimum 6.0 per cent.
To 2.000 g of the powdered drug (250) (2.9.12) add a mixture
of 3 ml of water R and 7 ml of ethanol (96 per cent) R and
extract for 2 h, shaking frequently. Filter, evaporate 2.000 g
of the filtrate to dryness on a water-bath and dry in an oven
at 100-105 °C for 3 h. Allow to cool in a dessicator over
diphosphorus pentoxide R and weigh. The residue weighs a
minimum of 120 mg.
ASSAY
Carry out the determination of essential oil in herbal drugs
(2.8.12). Use a 500 ml round-bottomed flask, 200 ml of
water R as the distillation liquid and 0.5 ml of xylene R in
the graduated tube. Reduce the drug to a powder (710)
(2.9.12) and immediately use 15.0 g for the determination.
Distil at a rate of 2-3 ml/min for 90 min.

Figure 1603.-1. — Illustration of powdered herbal drug of

bitter-orange epicarp and mesocarp (see Identification B) 01/2009:1810

C. Thin-layer chromatography (2.2.27). BITTER-ORANGE FLOWER

Test solution. To 1.0 g of the powdered drug (710)
(2.9.12) add 10 ml of methanol R and heat in a water-bath
at 65 °C for 5 min shaking frequently. Allow to cool and
filter.
Reference solution. Dissolve 1.0 mg of naringin R and
1.0 mg of caffeic acid R in 1 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, anhydrous formic acid R, ethyl
acetate R (10:15:75 V/V/V).
Application : 20 μl as bands.
Development : over a path of 10 cm.
Drying : in air, and heat in an oven at 110-120 °C for
5 min.
Detection : spray the warm plate with a 10 g/l solution
of diphenylboric acid aminoethyl ester R in methanol R
and then with a 50 g/l solution of macrogol 400 R in
methanol R. After at least 1 h, examine in ultraviolet light
at 365 nm.
Aurantii amari flos
DEFINITION
Whole, dried, unopened flower of Citrus aurantium L.
ssp. aurantium (C. aurantium L. ssp. amara Engl.).
Content : minimum 8.0 per cent of total flavonoids, expressed
as naringin (C27H32O14 ; Mr 580.5) (dried drug).
IDENTIFICATION
A. The flower buds are white or yellowish-white and may
reach up to 25 mm in length. The dialypetalous corolla
is composed of 5 thick, oblong and concave petals
dotted with oil glands visible under a hand lens ; the
short, yellowish-green persistent gamosepalous calyx has
5 spreading sepals, connate at the base and forming a
star-shaped structure attached to the yellowish-green
peduncle which is about 5-10 mm long. The flower buds
contain at least 20 stamens with yellow anthers and with
filaments fused at the base into groups of 4 or 5 ; the
ovary is superior, brownish-black and spherical, consists

General Notices (1) apply to all monographs and other texts 4065
Bitter-orange flower
of 8-10 multi-ovular loculi and is surrounded at the base
by an annular granular hypogynous disc ; the thick,
cylindrical style ends in a capitate stigma.
B. Reduce to a powder (355) (2.9.12). The powder is
brownish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the
following diagnostic characters : numerous spherical
pollen grains, with a finely pitted exine and 3-5 germinal
pores ; fragments of the epidermis of the sepals with
unicellular trichomes and with large prism crystals of
calcium oxalate in the underlying mesophyll ; fragments
of the epidermis of the petals with a distinctly striated
cuticle ; fragments of large schizolysigenous oil glands
which measure up to 100 μm in diameter, numerous
anomocytic stomata (2.8.3). Examine under a microscope
using a 20 g/l solution of potassium hydroxide R.
The mounting medium becomes yellow because of the
presence of hesperidin in the drug.
A. Epidermis of the sepals and E. Fragment of large
cells of the underlying mesophyll schizolysigenous oil gland
containing prism crystals of
calcium oxalate, in transverse
section
B. Cells of the mesophyll, some F, G and J. Epidermis of the
containing prism crystals of petals in surface view showing the
calcium oxalate striated cuticle
C. Epidermis of the sepals with H and K. Spherical pollen grains
unicellular covering trichome with a finely pitted exine
D. Epidermis of the sepals in
surface view with anomocytic
stomata and part of the underlying
mesophyll containing prism
crystals of calcium oxalate
Figure 1810.-1. — Illustration of powdered herbal drug of
bitter-orange flower (see Identification B)
C. Examine the chromatograms obtained in the test for
sweet-orange flower.
4066
EUROPEAN PHARMACOPOEIA 6.3
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and
the test solution.
Top of the plate
A weak yellow fluorescent zone
A weak yellow fluorescent zone
Hesperidin : a greenish-yellow A greenish-yellow fluorescent
fluorescent zone zone (hesperidin)
Naringin : a yellow fluorescent A yellow fluorescent zone
zone (naringin)
A red fluorescent zone
(neoeriocitrin)
A yellow fluorescent zone
(diosmin and neodiosmin)
Reference solution Test solution
TESTS
Sweet-orange flower. Thin-layer chromatography (2.2.27).
Test solution. To 0.5 g of the powdered drug (355) (2.9.12),
add 5 ml of methanol R. Heat with stirring at 40 °C for
10 min. Filter.
Reference solution. Dissolve 3.0 mg of naringin R and
3.0 mg of hesperidin R in 10 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, anhydrous formic acid R, ethyl
acetate R (10:15:75 V/V/V).
Application : 10 μl as bands.
Development : over a path of 10 cm.
Drying : in air ; heat in an oven at 110-120 °C for 5 min.
Detection : spray the hot plate with a 10 g/l solution of
diphenylboric acid aminoethyl ester R in methanol R
and then with a 50 g/l solution of macrogol 400 R in
methanol R. After at least 1 h, examine in ultraviolet light
at 365 nm.
Results : the chromatogram obtained with the test solution
shows a yellow zone similar in position to the zone of
naringin in the chromatogram obtained with the reference
solution and immediately below it a red zone (neoeriocitrin).
Loss on drying (2.2.32) : maximum 11.0 per cent, determined
on 1.000 g of the powdered drug (355) (2.9.12) by drying
in an oven at 105 °C.
Total ash (2.4.16) : maximum 10.0 per cent.
ASSAY
Stock solution. To 0.175 g of the powdered drug (355)
(2.9.12) add 95 ml of ethanol (50 per cent V/V) R. Heat on
a water-bath under a reflux condenser for 30 min. Allow to
cool and filter through a sintered-glass filter (2.1.2). Rinse
the filter with 5 ml of ethanol (50 per cent V/V) R. Combine
the filtrate and the rinsings in a volumetric flask and dilute
to 100.0 ml with ethanol (50 per cent V/V) R.
Test solution. Into a test tube (10 mm × 180 mm) introduce
0.150 g of powdered (250) (2.9.12) magnesium R, a magnetic
stirring bar 25 mm long and 2.00 ml of the stock solution.
Maintain the test tube upright, centrifuge at 125 g and
carefully add dropwise, especially at the beginning, 2.0 ml
of hydrochloric acid R, and then 6.0 ml of ethanol (50 per
cent V/V) R. Stopper the tube and mix by inverting.
Compensation solution. Into a 2nd tube, introduce 2.00 ml
of the stock solution and carefully add dropwise, especially
at the beginning, 2.0 ml of hydrochloric acid R and then
6.0 ml of ethanol (50 per cent V/V) R.
After 10 min, measure the absorbance (2.2.25) of the test
solution at 530 nm.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Calculate the percentage content of total flavonoids,
expressed as naringin, using the following expression :
i.e. taking the value of the specific absorbance of the
reaction product of naringin to be 52.
A = absorbance at 530 nm ;
m = mass of the substance to be examined, in grams.
01/2008:1077
corrected 6.3
BUSERELIN
Buserelinum
C60H86N16O13 Mr 1239
[57982-77-1]
DEFINITION
5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-O-(1,1-
dimethylethyl)-D-seryl-L-leucyl-L-arginyl-N-ethyl-L-prolinamide.
Synthetic nonapeptide analogue of human
gonadotrophin-releasing hormone GnRH with agonistic
activity to gonadorelin. It is obtained by chemical synthesis
and is available as an acetate.
Content : 95.0 per cent to 102.0 per cent (anhydrous, acetic
acid-free substance).
CHARACTERS
Appearance : white or slightly yellowish powder,
hygroscopic.
Solubility : sparingly soluble in water and in dilute acids.
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 and size
to the principal peak in the chromatogram obtained with
reference solution (b).
B. Nuclear magnetic resonance spectrometry (2.2.33).
Preparation : 4 mg/ml solution in a mixture of 20 volumes
of deuterated acetic acid R and 80 volumes of deuterium
oxide R.
Comparison : 4 mg/ml solution of buserelin CRS in a
mixture of 20 volumes of deuterated acetic acid R and
80 volumes of deuterium oxide R (dissolve the contents
of a vial of buserelin CRS in this solvent mixture to obtain
the desired concentration).
Operating conditions : field strength : minimum 300 MHz.
Results : the 1H NMR spectrum obtained is qualitatively
similar to the 1H NMR spectrum obtained with
buserelin CRS.
C. Amino acid analysis (2.2.56). For protein hydrolysis use
Method 1 and for analysis use Method 1.
Express the content of each amino acid in moles.
Calculate the relative proportions of the amino acids,
taking 1/6 of the sum of the number of moles of glutamic
General Notices (1) apply to all monographs and other texts
Buserelin
acid, histidine, tyrosine, leucine, arginine and proline as
equal to 1. The values fall within the following limits :
serine 1.4 to 2.0 ; proline 0.8 to 1.2 ; glutamic acid
0.9 to 1.1 ; leucine 0.9 to 1.1 ; tyrosine 0.9 to 1.1 ; histidine
0.9 to 1.1 ; arginine 0.9 to 1.1. Not more than traces of
other amino acids are present, with the exception of
tryptophan.
TESTS
Appearance of solution. A 10 g/l solution is clear (2.2.1)
and not more intensely coloured than reference solution Y7
(2.2.2, Method II).
Specific optical rotation (2.2.7) : − 49 to − 58 (anhydrous,
acetic acid-free substance), determined on a 10 g/l solution.
Specific absorbance (2.2.25) : 49 to 56, measured at the
absorption maximum at 278 nm (anhydrous, acetic acid-free
substance).
Dissolve 10.0 mg in 100.0 ml of 0.01 M hydrochloric acid.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 5.0 mg of the substance to be
examined in 5.0 ml of the mobile phase.
Reference solution (a). Dissolve the contents of a vial
of D-His-buserelin CRS in the mobile phase. Dilute an
appropriate volume of this solution in the mobile phase to
obtain a final concentration of 1 mg/ml. Add 1.0 ml of the
test solution to 1.0 ml of this solution.
Reference solution (b). Dissolve the contents of a
vial of buserelin CRS in the mobile phase. Dilute an
appropriate volume of this solution in the mobile phase to
obtain a final concentration of 1.0 mg/ml.
Reference solution (c). Dilute 1.0 ml of the test solution to
100.0 ml with the mobile phase.
Column :
— size: l = 0.25 m, Ø = 4 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 200 ml of acetonitrile R and 700 ml of an
11.2 g/l solution of phosphoric acid R and adjust to pH 2.5
with triethylamine R.
Flow rate : 0.8 ml/min.
Detection : spectrophotometer at 220 nm.
Injection : 10 μl of the test solution, reference solution (a)
and reference solution (c).
Relative retention with reference to buserelin (retention
time = about 36 min) : impurity B = about 0.76 ;
impurity C = about 0.83 ; impurity A = about 0.90 ;
impurity D = about 0.94 ; impurity E = about 0.94.
System suitability : reference solution (a) :
— resolution : minimum 1.5 between the peaks due to
impurity A and buserelin.
Limits :
— sum of impurities D and E : not more than 3 times the
area of the principal peak in the chromatogram obtained
with reference solution (c) (3 per cent) ;
— any other impurity : for each impurity, not more than
3 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (3 per cent) ;
— total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(5 per cent) ;
— disregard limit : 0.1 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.1 per cent).
4067
Buserelin EUROPEAN PHARMACOPOEIA 6.3

Acetic acid (2.5.34) : 3.0 per cent to 7.0 per cent. IMPURITIES
Test solution. Dissolve 20.0 mg of the substance to be Specified impurities : A, B, C, D, E.
examined in a mixture of 5 volumes of mobile phase B and
95 volumes of mobile phase A and dilute to 10.0 ml with the
same mixture of solvents.
Water (2.5.12) : maximum 4.0 per cent, determined on
80.0 mg.
Bacterial endotoxins (2.6.14) : less than 55.5 IU/mg, if
intended for use in the manufacture of parenteral dosage
forms without a further appropriate procedure for the A. X2 = D-His, X4 = L-Ser, X5 = L-Tyr : [2-D-histidine]buserelin,
removal of bacterial endotoxins. B. X2 = L-His, X4 = D-Ser, X5 = L-Tyr : [4-D-serine]buserelin,
ASSAY D. X2 = L-His, X4 = L-Ser, X5 = D-Tyr : [5-D-tyrosine]buserelin,
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : 10 μl of the test solution and reference solution (b).
Calculate the content of buserelin (C60H86N16O13) using the
areas of the peaks in the chromatograms obtained and the
declared content of C60H86N16O13 in buserelin CRS. C. buserelin-(3-9)-peptide,

STORAGE
In an airtight container, protected from light, at a
temperature of 2 °C to 8 °C. If the substance is sterile, store
in an airtight, sterile, tamper-proof container.
LABELLING
The label states the mass of peptide in the container. E. [1-(5-oxo-D-proline)]buserelin.

4068 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
C
Calcium folinate.. .....................................................................4071
Calcium gluconate.. .................................................................4073
Calcium gluconate, anhydrous.. ........................................... 4074
Calcium gluconate for injection............................................ 4074
Calcium stearate....................................................................... 4076
Carprofen for veterinary use.. ...............................................4077
Cellulose acetate.. ....................................................................4078
Cellulose acetate phthalate....................................................4079
Cellulose, microcrystalline.....................................................4080
Cellulose, powdered.. ..............................................................4084
Charcoal, activated.. ................................................................4088
Cholecalciferol concentrate (oily form)...............................4089
Cholecalciferol concentrate (powder form)........................ 4091
General Notices (1) apply to all monographs and other texts
Cholecalciferol concentrate (water-dispersible form).......4093
Chondroitin sulphate sodium................................................4095
Cisplatin.. ...................................................................................4097
Citalopram hydrobromide.. ....................................................4099
Citalopram hydrochloride.. .....................................................4101
Clonidine hydrochloride......................................................... 4102
Codergocrine mesilate.. .......................................................... 4103
Cod-liver oil, farmed.. .............................................................. 4105
Cod-liver oil (type A)................................................................ 4109
Cod-liver oil (type B)................................................................ 4113
Croscarmellose sodium............................................................4117
Crospovidone.. .......................................................................... 4119
4069
 EUROPEAN PHARMACOPOEIA 6.3

4070 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Calcium folinate

01/2009:0978 Detection : examine in ultraviolet light at 254 nm.

Results : the principal spot in the chromatogram obtained
CALCIUM FOLINATE with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
Calcii folinas the reference solution.
D. It gives reaction (b) of calcium (2.3.1).
Carry out the tests and the assay as rapidly as possible,
protected from actinic light.
TESTS
Solution S. Dissolve 1.25 g in carbon dioxide-free water R,
heating at 40 °C if necessary, and dilute to 50.0 ml with the
same solvent.
Appearance of solution. Solution S is clear (2.2.1) and its
C20H21CaN7O7,xH2O Mr 511.5 (anhydrous substance) absorbance (2.2.25) at 420 nm is not greater than 0.60. Use
DEFINITION water R as the compensation liquid.
Calcium (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo- pH (2.2.3) : 6.8 to 8.0 for solution S.
1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]amino]- Specific optical rotation (2.2.7) : + 14.4 to + 18.0 (anhydrous
benzoyl]amino]pentanedioate. substance), determined on solution S.
Content : Acetone, ethanol and methanol. Head-space gas
— calcium folinate (C20H21CaN7O7) : 97.0 per cent to chromatography (2.2.28) : use the standard additions
102.0 per cent (anhydrous substance) ; method.
— calcium (Ca ; Ar 40.08) : 7.54 per cent to 8.14 per cent Test solution. Dissolve 0.25 g of the substance to be
(anhydrous substance). examined in water R and dilute to 10.0 ml with the same
It contains a variable amount of water. solvent.
Reference solution. Dilute 0.125 g of acetone R, 0.750 g of
CHARACTERS anhydrous ethanol R and 0.125 g of methanol R in water R
Appearance : white or light yellow, amorphous or crystalline and dilute to 1000.0 ml with water R.
powder. Column :
Solubility : sparingly soluble in water, practically insoluble — material: fused silica ;
in acetone and in ethanol (96 per cent).
— size: l = 10 m, Ø = 0.32 mm ;
The amorphous form may produce supersaturated solutions
in water. — stationary phase : styrene-divinylbenzene copolymer R.
Carrier gas: nitrogen for chromatography R.
IDENTIFICATION Flow rate : 4 ml/min.
First identification : A, B, D. Static head-space conditions that may be used :
Second identification : A, C, D. — equilibration temperature: 80 °C ;
A. Specific optical rotation (see Tests). — equilibration time : 20 min ;
B. Infrared absorption spectrophotometry (2.2.24).
— pressurisation time : 30 s.
Preparation : discs.
Temperature :
Comparison : calcium folinate CRS.
Time Temperature
If the spectra obtained show differences, dissolve the
(min) (°C)
substance to be examined and the reference substance
Column 0-6 125 → 185
separately in the minimum volume of water R and add
dropwise sufficient acetone R to produce a precipitate. 6 - 15 185
Allow to stand for 15 min, collect the precipitate by Injection port 250
centrifugation, wash the precipitate with 2 small
quantities of acetone R and dry. Record new spectra Detector 250
using the residues.
Detection : flame ionisation.
C. Thin-layer chromatography (2.2.27).
Injection : at least 3 times.
Test solution. Dissolve 15 mg of the substance to be
examined in a 3 per cent V/V solution of ammonia R and Limits :
dilute to 5 ml with the same solvent. — acetone : maximum 0.5 per cent ;
Reference solution. Dissolve 15 mg of calcium — ethanol : maximum 3.0 per cent ;
folinate CRS in a 3 per cent V/V solution of ammonia R — methanol : maximum 0.5 per cent.
and dilute to 5 ml with the same solvent. Related substances. Liquid chromatography (2.2.29).
Plate : cellulose for chromatography F254 R as the coating Test solution. Dissolve 10.0 mg of the substance to be
substance. examined in water R and dilute to 10.0 ml with the same
Mobile phase : the lower layer of a mixture of 1 volume of solvent.
isoamyl alcohol R and 10 volumes of a 50 g/l solution of Reference solution (a). Dissolve 10.0 mg of calcium
citric acid R previously adjusted to pH 8 with ammonia R. folinate CRS in water R and dilute to 10.0 ml with the same
Application : 5 μl. solvent.
Development : over a path of 15 cm. Reference solution (b). Dilute 1.0 ml of reference solution (a)
Drying : in air. to 100.0 ml with water R.

General Notices (1) apply to all monographs and other texts 4071
Calcium folinate EUROPEAN PHARMACOPOEIA 6.3

Reference solution (c). Dissolve 10.0 mg of formylfolic Water (2.5.12) : maximum 17.0 per cent, determined on
acid CRS (impurity D) in the mobile phase and dilute to 0.200 g (ground to a very fine powder). Stir the substance to
100.0 ml with the mobile phase. Dilute 1.0 ml of this solution be examined in the titration solvent for about 6 min before
to 10.0 ml with water R. titrating and use a suitable titrant that does not contain
Reference solution (d). Dilute 1.0 ml of reference solution (b) pyridine.
to 10.0 ml with water R. Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if
Reference solution (e). Dilute 5.0 ml of reference solution (c) intended for use in the manufacture of parenteral dosage
to 10.0 ml with reference solution (b). forms without a further appropriate procedure for the
removal of bacterial endotoxins.
Column:
— size : l = 0.25 m, Ø = 4 mm ; ASSAY
Calcium. Dissolve 0.400 g in 150 ml of water R and dilute to
— stationary phase : octadecylsilyl silica gel for
300 ml with the same solvent. Carry out the complexometric
chromatography R (5 μm) ;
titration of calcium (2.5.11).
— temperature : 40 °C. 1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
Mobile phase : mix 220 ml of methanol R and 780 ml Calcium folinate. Liquid chromatography (2.2.29) as
of a solution containing 2.0 ml of tetrabutylammonium described in the test for related substances with the following
hydroxide solution (400 g/l) R and 2.2 g of disodium modifications.
hydrogen phosphate R, previously adjusted to pH 7.8 with
phosphoric acid R. Injection : test solution and reference solution (a).
Flow rate : 1 ml/min. System suitability :
— repeatability : maximum relative standard deviation of
Detection : spectrophotometer at 280 nm. 2.0 per cent after 6 injections of reference solution (a).
Injection : 10 μl of the test solution and reference Calculate the percentage content of C20H21CaN7O7 from the
solutions (b), (c), (d) and (e). declared content of calcium folinate CRS.
Run time : 2.5 times the retention time of folinate.
STORAGE
System suitability : reference solution (e) :
In an airtight container, protected from light. If the
— resolution : minimum 2.2 between the peaks due to substance is sterile, store in a sterile, airtight, tamper-proof
folinate and impurity D. container.
Limits :
IMPURITIES
— impurity D : not more than the area of the principal peak Specified impurities : A, B, C, D, E, F, G.
in the chromatogram obtained with reference solution (c)
(1 per cent) ;
— impurities A, B, C, E, F, G : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (1 per cent) ;
— sum of impurities other than D : not more than 2.5 times
the area of the principal peak in the chromatogram A. (2S)-2[(4-aminobenzoyl)amino]pentanedioic acid,
obtained with reference solution (b) (2.5 per cent) ;
— disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (d)
(0.1 per cent).
Chlorides : maximum 0.5 per cent.
Dissolve 0.300 g in 50 ml of water R heating at 40 °C
if necessary. Add 10 ml of 2M nitric acid and titrate
with 0.005 M silver nitrate determining the end-point
potentiometrically (2.2.20).
B. (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-
1 ml of 0.005 M silver nitrate is equivalent to 0.177 mg of Cl. hexahydropteridin-6-yl]methyl]formylamino]benzoyl]-
Heavy metals (2.4.8) : maximum 50 ppm. amino]pentanedioic acid (5,10-diformyltetrahydrofolic
acid),
1.0 g complies with test F. Prepare the reference solution
using 5 ml of lead standard solution (10 ppm Pb) R. C. folic acid,
Platinum : maximum 20.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 1.00 g in water R and dilute to
100.0 ml with the same solvent.
Reference solutions. Prepare the reference solutions using
platinum standard solution (30 ppm Pt) R, diluted as
necessary with a mixture of 1 volume of nitric acid R and
99 volumes of water R.
D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
Source : platinum hollow-cathode lamp. yl)methyl]formylamino]benzoyl]amino]pentanedioic acid
Wavelength : 265.9 nm. (10-formylfolic acid),

4072 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
E. 4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-
hexahydropteridin-6-yl]methyl]amino]benzoic acid
(5-formyltetrahydropteroic acid),
F. R = CHO : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydro-
pteridin-6-yl)methyl]formylamino]benzoyl]amino]pentane-
dioic acid (10-formyldihydrofolic acid),
G. R = H : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydro-
pteridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic
acid (dihydrofolic acid).
01/2009:0172
CALCIUM GLUCONATE
Calcii gluconas
C12H22CaO14,H2O Mr 448.4
DEFINITION
Calcium D-gluconate monohydrate.
Content : 98.5 per cent to 102.0 per cent of C12H22CaO14,H2O.
CHARACTERS
Appearance : white or almost white, crystalline or granular
powder.
Solubility : sparingly soluble in water, freely soluble in
boiling water.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in 1 ml of water R, heating if necessary in a
water-bath at 60 °C.
Reference solution. Dissolve 20 mg of calcium
gluconate CRS in 1 ml of water R, heating if necessary in
a water-bath at 60 °C.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application : 5 μl.
General Notices (1) apply to all monographs and other texts
Calcium gluconate
Development : over a path of 10 cm.
Drying : at 100 °C for 20 min. Allow to cool.
Detection : spray with a 50 g/l solution of potassium
dichromate R in a 40 per cent m/m solution of sulphuric
acid R.
Results : after 5 min, the principal spot in the
chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the
chromatogram obtained with the reference solution.
B. Solution S (see Tests) gives the reactions of calcium
(2.3.1).
TESTS
Solution S. Dissolve 1.0 g in water R heated to 60 °C and
dilute to 50 ml with the same solvent.
Appearance of solution. At 60 °C, solution S is not
more intensely coloured than reference solution Y6 (2.2.2,
Method II). After cooling, it is not more opalescent than
reference suspension II (2.2.1).
Organic impurities and boric acid. Introduce 0.5 g into a
porcelain dish previously rinsed with sulphuric acid R and
placed in a bath of iced water. Add 2 ml of cooled sulphuric
acid R and mix. No yellow or brown colour develops.
Add 1 ml of chromotrope II B solution R. A violet colour
develops and does not become dark blue. The solution is not
more intensely coloured than that of a mixture of 1 ml of
chromotrope II B solution R and 2 ml of cooled sulphuric
acid R.
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand. Dilute to 25 ml with water R
and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric
solution R and boil for 1 min. Allow to stand for 2 min. No
red precipitate is formed.
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 12.5 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 100 ppm.
Dissolve 10.0 g with heating in a mixture of 10 ml of acetic
acid R and 90 ml of distilled water R.
Magnesium and alkali metals : maximum 0.4 per cent.
Dissolve 1.00 g in 100 ml of boiling water R, add 10 ml of
ammonium chloride solution R, 1 ml of ammonia R and,
dropwise, 50 ml of hot ammonium oxalate solution R. Allow
to stand for 4 h, dilute to 200 ml with water R and filter.
Evaporate 100 ml of the filtrate to dryness and ignite. The
residue weighs a maximum of 2 mg.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Heat the substance to be
examined gradually and with care until it is almost
completely transformed into a white mass and then ignite.
Prepare the reference solution using 2 ml of lead standard
solution (10 ppm Pb) R.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
ASSAY
Dissolve 0.8000 g in 20 ml of hot water R, allow to cool and
dilute to 300 ml with water R. Carry out the complexometric
titration of calcium (2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 44.84 mg
of C12H22CaO14,H2O.
4073
Calcium gluconate, anhydrous EUROPEAN PHARMACOPOEIA 6.3

01/2009:2364 does not become dark blue. Compare the colour obtained
with that of a mixture of 1 ml of chromotrope II B solution R
and 2 ml of cooled sulphuric acid R.
CALCIUM GLUCONATE, ANHYDROUS
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
Calcii gluconas anhydricus for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand for 10 min. Dilute to 25 ml
with water R and filter. To 5 ml of the filtrate add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 2 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 12.5 ml of solution S to 15 ml with water R.
C12H22CaO14 Mr 430.4 Sulphates (2.4.13) : maximum 100 ppm.
Dissolve 10.0 g with heating in a mixture of 10 ml of acetic
DEFINITION acid R and 90 ml of distilled water R.
Anhydrous calcium D-gluconate. Magnesium and alkali metals : maximum 0.4 per cent
Content : 98.0 per cent to 102.0 per cent (dried substance). (expressed as MgO).
Dissolve 1.00 g in 100 ml of boiling water R, add 10 ml of
CHARACTERS ammonium chloride solution R, 1 ml of ammonia R and,
Appearance : white or almost white, crystalline or granular dropwise, 50 ml of hot ammonium oxalate solution R. Allow
powder. to stand for 4 h, dilute to 200 ml with water R and filter.
Solubility : sparingly soluble in water, freely soluble in Evaporate 100 ml of the filtrate to dryness and ignite. The
boiling water. residue weighs a maximum of 2 mg.
Heavy metals (2.4.8) : maximum 10 ppm.
IDENTIFICATION
2.0 g complies with test D. Heat the substance to be
A. Thin-layer chromatography (2.2.27). examined gradually and with care until it is almost
Test solution. Dissolve 20 mg of the substance to be completely transformed into a white mass, and then ignite.
examined in 1 ml of water R, heating if necessary in a Prepare the reference solution using 2 ml of lead standard
water-bath at 60 °C. solution (10 ppm Pb) R.
Reference solution. Dissolve 20 mg of calcium Loss on drying (2.2.32) : maximum 2.0 per cent, determined
gluconate CRS in 1 ml of water R, heating if necessary in on 1.000 g by drying in an oven at 105 °C for 16 h.
a water-bath at 60 °C. Microbial contamination
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel TAMC : acceptance criterion 103 CFU/g (2.6.12).
plate R (2-10 μm)].
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V). ASSAY
Application : 1 μl. Dissolve 0.350 g in 20 ml of hot water R, allow to cool and
dilute to 300 ml with water R. Carry out the complexometric
Development : over 2/3 of the plate.
titration of calcium (2.5.11).
Drying : at 100 °C for 20 min, then allow to cool. 1 ml of 0.1 M sodium edetate is equivalent to 43.04 mg
Detection : spray with a solution containing 25 g/l of of C12H22CaO14.
ammonium molybdate R and 10 g/l of cerium sulphate R
in dilute sulphuric acid R, and heat at 100-105 °C for
about 10 min. 01/2009:0979
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and CALCIUM GLUCONATE FOR
size to the principal spot in the chromatogram obtained INJECTION
with the reference solution.
B. Solution S (see Tests) gives the reactions of calcium Calcii gluconas ad iniectabile
(2.3.1).
C. Loss on drying (see Tests).
TESTS
Solution S. Dissolve 1.0 g in water R heated to 60 °C and
dilute to 50 ml with the same solvent.
Appearance of solution. At 60 °C, solution S is not C12H22CaO14,H2O Mr 448.4
more intensely coloured than reference solution Y6 (2.2.2,
Method II). After cooling, it is not more opalescent than DEFINITION
reference suspension II (2.2.1). Calcium D-gluconate monohydrate.
Organic impurities and boric acid. Place 0.5 g in a porcelain Content : 99.0 per cent to 101.0 per cent of C12H22CaO14,H2O.
dish previously rinsed with sulphuric acid R and placed in
a bath of iced water. Add 2 ml of cooled sulphuric acid R CHARACTERS
and mix. No yellow or brown colour develops. Add 1 ml of Appearance : white or almost white, crystalline or granular
chromotrope II B solution R. A violet colour develops and powder.

4074 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Solubility : sparingly soluble in water, freely soluble in
boiling water.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in 1 ml of water R, heating if necessary in a
water-bath at 60 °C.
Reference solution. Dissolve 20 mg of calcium
gluconate CRS in 1 ml of water R, heating if necessary in Flow rate of the mobile phase : 2 ml/min.
a water-bath at 60 °C.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, ethyl acetate R, Flow rate of the suppressor regeneration solution :
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V). 4 ml/min.
Application : 5 μl.
Development : over a path of 10 cm.
Drying : at 100 °C for 20 min and allow to cool.
Detection : spray with a 50 g/l solution of potassium
dichromate R in a 40 per cent m/m solution of sulphuric
acid R.
Results : after 5 min, the principal spot in the
chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the
chromatogram obtained with the reference solution.
B. About 20 mg gives reaction (b) of calcium (2.3.1).
TESTS
Solution S. To 10.0 g add 90 ml of boiling distilled water R S
and boil with stirring, for not more than 10 s, until completely
dissolved, then dilute to 100.0 ml with the same solvent.
Appearance of solution. At 60 °C, solution S is not more
intensely coloured than reference solution B7 (2.2.2,
Method II). After cooling to 20 °C, it is not more opalescent
than reference suspension II (2.2.1).
pH (2.2.3) : 6.4 to 8.3.
Dissolve 1.0 g in 20 ml of carbon dioxide-free water R,
heating on a water-bath.
Organic impurities and boric acid. Introduce 0.5 g into a
porcelain dish previously rinsed with sulphuric acid R and
placed in a bath of iced water. Add 2 ml of cooled sulphuric Chlorides (2.4.4) : maximum 50 ppm.
acid R and mix. No yellow or brown colour develops.
Add 1 ml of chromotrope II B solution R. A violet colour
develops and does not become dark blue. The solution is not Phosphates (2.4.11) : maximum 100 ppm.
more intensely coloured than that of a mixture of 1 ml of
chromotrope II B solution R and 2 ml of cooled sulphuric
acid R.
Oxalates. Liquid chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be
examined in water for chromatography R and dilute to
100.0 ml with the same solvent.
Reference solution. Dissolve 1.00 g of the substance to
be examined in water for chromatography R, add 0.5 ml
of a 0.152 g/l solution of sodium oxalate R in water for
chromatography R and dilute to 100.0 ml with the same
solvent.
Guard column :
— size : l = 30 mm, Ø = 4 mm ;
— stationary phase : suitable strong anion exchange resin
(30-50 μm).
Columns 1 and 2 :
— size : l = 0.25 m, Ø = 4 mm ;
— stationary phase : suitable strong anion exchange resin
(30-50 μm).
General Notices (1) apply to all monographs and other texts
Calcium gluconate for injection
Anion-suppresser column : connected in series with
the guard and analytical columns and equipped with a
micromembrane that separates the mobile phase from the
suppressor regeneration solution, flowing countercurrent to
the mobile phase.
Mobile phase : dissolve 0.212 g of anhydrous sodium
carbonate R and 63 mg of sodium hydrogen carbonate R in
water for chromatography R and dilute to 1000.0 ml with
the same solvent.
Suppressor regeneration solution : 1.23 g/l solution of
sulphuric acid R in water for chromatography R.
Detection : conductance.
Injection : 50 μl.
System suitability : reference solution :
— repeatability : maximum relative standard deviation of
the area of the peak due to oxalate of 2.0 per cent after
5 injections.
Inject 50 μl of each solution 3 times. Calculate the content of
oxalates in parts per million using the following expression :
ST = area of the peak due to oxalate in the
chromatogram obtained with the test solution ;
= area of the peak due to oxalate in the
R
chromatogram obtained with the reference
solution.
Limit :
— oxalates : maximum 1.00 × 102 ppm.
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand for 10 min. Dilute to 25 ml
with water R and filter. To 5 ml of the filtrate add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 2 min. No red precipitate is formed.
To 10 ml of previously filtered solution S add 5 ml of water R.
Dilute 1 ml of solution S to 100 ml with water R.
Sulphates (2.4.13) : maximum 50 ppm, determined on
previously filtered solution S.
Prepare the standard using a mixture of 7.5 ml of sulphate
standard solution (10 ppm SO4) R and 7.5 ml of distilled
water R.
Iron : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Introduce 2.0 g into a 100 ml
polytetrafluoroethylene beaker and add 5 ml of
nitric acid R. Boil, evaporating almost to dryness. Add 1 ml
of strong hydrogen peroxide solution R and evaporate again
almost to dryness. Repeat the hydrogen peroxide treatment
until a clear solution is obtained. Using 2 ml of nitric acid R,
transfer the solution into a 25 ml volumetric flask. Dilute to
25.0 ml with dilute hydrochloric acid R. In the same manner,
prepare a compensation solution using 0.65 g of calcium
chloride R1 instead of the substance to be examined.
Reference solutions. Prepare the reference solutions from
iron solution (20 ppm Fe) R diluted with dilute hydrochloric
acid R.
4075
Calcium stearate
Source : iron hollow-cathode lamp.
Wavelength : 248.3 nm.
Atomisation device : air-acetylene flame.
Carry out a basic correction using a deuterium lamp.
Magnesium and alkali metals : maximum 0.4 per cent.
To 0.50 g add a mixture of 1.0 ml of dilute acetic acid R and
10.0 ml of water R and rapidly boil, whilst shaking, until
completely dissolved. To the boiling solution add 5.0 ml of
ammonium oxalate solution R and allow to stand for at
least 6 h. Filter through a sintered-glass filter (1.6) (2.1.2)
into a porcelain crucible. Carefully evaporate the filtrate to
dryness and ignite. The residue weighs not more than 2 mg.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Bacterial endotoxins (2.6.14) : less than 167 IU/g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
ASSAY
Dissolve 0.350 g in 20 ml of hot water R, allow to cool and
dilute to 300 ml with water R. Carry out the complexometric
titration of calcium (2.5.11). Use 50 mg of calconecarboxylic
acid triturate R.
1 ml of 0.1 M sodium edetate is equivalent to 44.84 mg
of C12H22CaO14,H2O.
01/2009:0882
CALCIUM STEARATE
Calcii stearas
DEFINITION
Mixture of calcium salts of different fatty acids consisting
mainly of stearic (octadecanoic) acid [(C17H35COO)2Ca ;
Mr 607] and palmitic (hexadecanoic) acid [(C15H31COO)2Ca ;
Mr 550.9] with minor proportions of other fatty acids.
Content :
— calcium : 6.4 per cent to 7.4 per cent (Ar 40.08) (dried
substance) ;
— stearic acid in the fatty acid fraction : minimum 40.0 per
cent ;
— sum of stearic acid and palmitic acid in the fatty acid
fraction : minimum 90.0 per cent.
CHARACTERS
Appearance : fine, white or almost white, crystalline powder.
Solubility : practically insoluble in water and in ethanol
(96 per cent).
IDENTIFICATION
First identification : C, D.
Second identification : A, B, D.
A. Freezing point (2.2.18) : minimum 53 °C, for the residue
obtained in the preparation of solution S (see Tests).
B. Acid value (2.5.1) : 195 to 210.
Dissolve 0.200 g of the residue obtained in the preparation
of solution S in 25 ml of the prescribed mixture of
solvents.
C. Examine the chromatograms obtained in the test for fatty
acid composition.
4076 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Results : the retention times of the principal peaks in
the chromatogram obtained with the test solution are
approximately the same as those of the principal peaks in
the chromatogram obtained with the reference solution.
D. Neutralise 5 ml of solution S to red litmus paper R using
strong sodium hydroxide solution R. The solution gives
reaction (b) of calcium (2.3.1).
TESTS
Solution S. To 5.0 g add 50 ml of peroxide-free ether R,
20 ml of dilute nitric acid R and 20 ml of distilled water R.
Boil under a reflux condenser until dissolution is complete.
Allow to cool. In a separating funnel, separate the aqueous
layer and shake the ether layer with 2 quantities, each of
5 ml, of distilled water R. Combine the aqueous layers, wash
with 15 ml of peroxide-free ether R and dilute the aqueous
layer to 50 ml with distilled water R (solution S). Evaporate
the ether layer to dryness and dry the residue at 100-105 °C.
Keep the residue for identification tests A and B.
Acidity or alkalinity. To 1.0 g add 20 ml of carbon
dioxide-free water R and boil for 1 min with continuous
shaking. Cool and filter. To 10 ml of the filtrate add 0.05 ml
of bromothymol blue solution R1. Not more than 0.5 ml of
0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
Chlorides (2.4.4) : maximum 0.1 per cent.
Dilute 0.5 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 0.3 per cent.
Dilute 0.5 ml of solution S to 15 ml with distilled water R.
Cadmium : maximum 3.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Place 50.0 mg in a polytetrafluoroethylene
digestion bomb and add 0.5 ml of a mixture of 1 volume
of hydrochloric acid R and 5 volumes of cadmium- and
lead-free nitric acid R. Allow to digest at 170 °C for 5 h.
Allow to cool. Dissolve the residue in water R and dilute to
5.0 ml with the same solvent.
Reference solutions. Prepare the reference solutions using
cadmium standard solution (10 ppm Cd) R, diluted if
necessary with a 1 per cent V/V solution of hydrochloric
acid R.
Source : cadmium hollow-cathode lamp.
Wavelength : 228.8 nm.
Atomisation device : graphite furnace.
Lead : maximum 10.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Use the solution described in the test for
cadmium.
Reference solutions. Prepare the reference solutions using
lead standard solution (10 ppm Pb) R, diluted if necessary
with water R.
Source : lead hollow-cathode lamp.
Wavelength : 283.3 nm ; 217.0 nm may be used depending
on the apparatus.
Atomisation device : graphite furnace.
Nickel : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Use the solution described in the test for
cadmium.
Reference solutions. Prepare the reference solutions using
nickel standard solution (10 ppm Ni) R, diluted if necessary
with water R.
EUROPEAN PHARMACOPOEIA 6.3
Source : nickel hollow-cathode lamp.
Wavelength : 232.0 nm.
Atomisation device : graphite furnace.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Calcium. To 0.500 g in a 250 ml conical flask add 50 ml
of a mixture of equal volumes of anhydrous ethanol R
and butanol R, 5 ml of concentrated ammonia R, 3 ml of
ammonium chloride buffer solution pH 10.0 R, 30.0 ml
of 0.1 M sodium edetate and 15 mg of mordant black 11
triturate R. Heat to 45-50 °C until the solution is clear. Cool
and titrate with 0.1 M zinc sulphate until the colour changes
from blue to violet. Carry out a blank titration.
1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
Composition of fatty acids. Gas chromatography (2.2.28) :
use the normalisation procedure.
Test solution. In a conical flask fitted with a reflux
condenser, dissolve 0.10 g of the substance to be examined
in 5 ml of boron trifluoride-methanol solution R. Boil
under a reflux condenser for 10 min. Add 4 ml of heptane R
through the condenser. Boil under a reflux condenser for
10 min. Allow to cool. Add 20 ml of a saturated sodium
chloride solution R. Shake and allow the layers to separate.
Remove about 2 ml of the organic layer and dry over 0.2 g of
anhydrous sodium sulphate R. Dilute 1.0 ml of the solution
to 10.0 ml with heptane R.
Reference solution. Prepare the reference solution in the
same manner as the test solution using 50.0 mg of palmitic
acid CRS and 50.0 mg of stearic acid CRS instead of calcium
stearate.
Column:
— material: fused silica ;
— size : l = 30 m, Ø = 0.32 mm ;
— stationary phase : macrogol 20 000 R (film thickness
0.5 μm).
Carrier gas: helium for chromatography R.
Flow rate : 2.4 ml/min.
Temperature :
Time Temperature
(min) (°C)
Column 0-2 70
2 - 36 70 → 240
36 - 41 240
Injection port 220
Detector 260
Detection : flame ionisation.
Injection : 1 μl.
Relative retention with reference to methyl stearate : methyl potassium
palmitate = about 0.88.
System suitability : reference solution :
— resolution : minimum 5.0 between the peaks due to
methyl palmitate and methyl stearate.
Calculate the content of palmitic acid and stearic acid.
Disregard the peak due to the solvent.
General Notices (1) apply to all monographs and other texts
Carprofen for veterinary use
07/2008:2201
corrected 6.3
CARPROFEN FOR VETERINARY USE
Carprofenum ad usum veterinarium
C15H12ClNO2 Mr 273.7
[53716-49-7]
DEFINITION
(2RS)-2-(6-Chloro-9H-carbazol-2-yl)propanoic acid.
Content : 98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone, soluble in methanol, slightly soluble in 2-propanol.
It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : carprofen CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.
TESTS
Appearance of solution. The solution is clear and not
more intensely coloured than reference solution BY3 (2.2.2,
Method II).
Dissolve 1.0 g in methanol R and dilute to 25 ml with the
same solvent.
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
Test solution. Dissolve 50 mg of the substance to be
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dissolve 2.5 mg of carprofen for
system suitability CRS (containing impurity C) in the mobile
phase and dilute to 10.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R
(5 μm).
Mobile phase : mix 30 volumes of a 1.36 g/l solution of
dihydrogen phosphate R adjusted to pH 3.0 with
phosphoric acid R and 70 volumes of methanol R2.
Flow rate : 1.3 ml/min.
Detection : spectrophotometer at 235 nm.
Injection : 20 μl.
Run time : 4 times the retention time of carprofen.
Retention time : carprofen = about 10 min.
4077
Cellulose acetate EUROPEAN PHARMACOPOEIA 6.3

System suitability : reference solution (a) :

— resolution : minimum 1.5 between the peaks due to
impurity C and carprofen.
Limits :
— unspecified impurities: for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ; B. R = H, R′ = CO2H : (2RS)-2-(9H-carbazol-2-yl)propanoic
— total : not more than 5 times the area of the principal peak acid,
in the chromatogram obtained with reference solution (b) C. R = Cl, R′ = OH : (1RS)-1-(6-chloro-9H-carbazol-2-
(0.5 per cent) ; yl)ethanol,
— disregard limit: the area of the principal peak in the G. R = Cl, R′ = CO-O-C2H5 : ethyl (2RS)-2-(6-chloro-9H-
chromatogram obtained with reference solution (b) carbazol-2-yl)propanoate,
(0.1 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 1.0 g in ethanol (96 per cent) R and dilute to 20 ml
with the same solvent. 12 ml of the solution complies with
test B. Prepare the reference solution using lead standard
solution (2 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 105 °C for 2 h. D. R = CO-CH3 : 1-(6-chloro-9H-carbazol-2-yl)ethanone,
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined E. R = H : 3-chloro-9H-carbazole,
on 1.0 g. H. R = C2H5 : 6-chloro-2-ethyl-9H-carbazole.

ASSAY
Dissolve 0.200 g in 50 ml of ethanol (96 per cent) R. Add 01/2009:0887
1.0 ml of 0.1 M hydrochloric acid. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically CELLULOSE ACETATE
(2.2.20). Read the volume added between the 2 points of
inflexion. Cellulosi acetas
1 ml of 0.1 M sodium hydroxide is equivalent to 27.37 mg of
C15H12ClNO2. DEFINITION
Partly or completely O-acetylated cellulose.
STORAGE CHARACTERS
Protected from light. Appearance : white, yellowish-white or greyish-white,
hygroscopic powder or granules.
IMPURITIES Solubility : practically insoluble in water, soluble in acetone,
Other detectable impurities (the following substances would, in formic acid and in a mixture of equal volumes of methanol
if present at a sufficient level, be detected by one or other of and methylene chloride, practically insoluble in ethanol
the tests in the monograph. They are limited by the general (96 per cent).
acceptance criterion for other/unspecified impurities and/or IDENTIFICATION
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these Infrared absorption spectrophotometry (2.2.24).
impurities for demonstration of compliance. See also 5.10. Comparison : cellulose acetate CRS.
Control of impurities in substances for pharmaceutical Preparation : prepare a 100 g/l solution of cellulose acetate,
use) : A, B, C, D, E, F, G, H. previously dried, in dioxane R, and spread 1 drop of the
solution between 2 sodium chloride plates ; separate the
plates, heat them both at 105 °C for 1 h, and reassemble
the dried plates.
TESTS
Free acid : maximum 0.1 per cent, calculated as acetic acid
(dried substance).
To 5.00 g in a 250 ml conical flask, add 150 ml of carbon
dioxide-free water R, insert the stopper, swirl the suspension
gently and allow to stand for 3 h. Filter, then wash the flask
and the filter with carbon dioxide-free water R, adding these
A. R = H : 2-(6-chloro-9H-carbazol-2-yl)-2-methylpropanedioic washings to the filtrate. Add 0.1 ml of phenolphthalein
acid, solution R1 and titrate the combined filtrate and washings
with 0.01 M sodium hydroxide until a pale pink colour is
obtained.
F. R = C2H5 : diethyl 2-(6-chloro-9H-carbazol-2-yl)-2- 1 ml of 0.01 M sodium hydroxide is equivalent to 0.6005 mg
methylpropanedioate, of free acid, calculated as acetic acid.

4078 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
STORAGE
In an airtight container.
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for cellulose
acetate used as film former.
Apparent viscosity. Dissolve 10 g in a mixture of 50 ml of
methanol R and 50 ml of methylene chloride R by shaking. Apparent viscosity : see test above.
Determine the viscosity of this solution at 20 ± 0.1 °C using Acetyl groups : see test above.
a rotating viscometer (2.2.10).
Acetyl groups (C2H3O) : typically 29.0 per cent to 44.8 per
cent of acetyl groups (dried substance) and typically 90.0 per Particle-size distribution (2.9.31).
cent to 110.0 per cent of the nominal acetyl content (dried
substance).
A. Cellulose acetate containing not more than 42.0 per cent
of acetyl groups
To 2.000 g in a 500 ml conical flask, add 100 ml of
acetone R and 10 ml of water R. Close the flask and stir
with a magnetic stirrer until dissolution is complete.
Add 30.0 ml of 1 M sodium hydroxide with constant
stirring. A finely divided precipitate of regenerated
cellulose, free from lumps, is obtained. Close the flask
and stir with a magnetic stirrer for 30 min. Add 100 ml
of water R at 80 °C, washing down the sides of the flask,
stir for 2 min and cool to room temperature. Titrate with
0.5 M sulphuric acid, using 0.1 ml of phenolphthalein
solution R as indicator. Carry out a blank titration.
Calculate the percentage content of acetyl groups using
the following expression :
General Notices (1) apply to all monographs and other texts
Cellulose acetate phthalate
d = loss on drying as a percentage ;
m = mass of the substance to be examined, in
grams ;
n1 = number of millilitres of 0.5 M sulphuric acid
used in the test ;
n2 = number of millilitres of 0.5 M sulphuric acid
used in the blank titration.
B. Cellulose acetate containing more than 42.0 per cent of
acetyl groups
To 2.000 g in a 500 ml conical flask, add 30 ml of dimethyl
sulphoxide R and 100 ml of acetone R. Close the flask
and stir with a magnetic stirrer for 16 h. Add 30.0 ml of
1 M sodium hydroxide with constant stirring. Close the
flask and stir with a magnetic stirrer for 6 min. Allow to
stand without stirring for 60 min. Resume stirring and
add 100 ml of water R at 80 °C, washing down the sides
of the flask, stir for 2 min and cool to room temperature.
Titrate with 0.5 M hydrochloric acid, using 0.1 ml of
phenolphthalein solution R as indicator. Add 0.5 ml of
0.5 M hydrochloric acid in excess, stir for 5 min and allow
to stand for 30 min. Titrate with 0.5 M sodium hydroxide,
until a persistent pink colour is obtained, stirring with a
magnetic stirrer. Calculate the net number of millimoles
of 0.5 M sodium hydroxide consumed, taking the mean
of 2 blank titrations into consideration.
Calculate the percentage content of acetyl groups using
the following expression :
d = loss on drying as a percentage ;
m = mass of the substance to be examined, in
grams ;
n = net number of millimoles of 0.5 M sodium
hydroxide consumed.
The following characteristics may be relevant for cellulose
acetate used as matrix former in prolonged-release tablets.
Molecular mass distribution (2.2.30).
Powder flow (2.9.36).
01/2009:0314
CELLULOSE ACETATE PHTHALATE
Cellulosi acetas phthalas
[9004-38-0]
DEFINITION
Partly O-acetylated and O-phthalylated cellulose.
CHARACTERS
Appearance : white or almost white, free-flowing powder or
colourless flakes, hygroscopic.
4079
Cellulose, microcrystalline EUROPEAN PHARMACOPOEIA 6.3

Solubility : practically insoluble in water, freely soluble in
acetone, soluble in diethylene glycol, practically insoluble in
ethanol (96 per cent) and in methylene chloride. It dissolves
in dilute solutions of alkali hydroxides.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : cellulose acetate phthalate CRS.
TESTS
Phthaloyl groups (C8H5O3 ; Mr 149.1) : typically 30.0 per
cent to 36.0 per cent (anhydrous and acid-free substance).
Dissolve 1.000 g in 50 ml of a mixture of 2 volumes of
acetone R and 3 volumes of ethanol (96 per cent) R. Add
0.1 ml of phenolphthalein solution R and titrate with 0.1 M
sodium hydroxide. Carry out a blank titration.
Calculate the percentage content of phthaloyl groups (P)
using the following expression :

Free acid : maximum 3.0 per cent, calculated as phthalic

acid (anhydrous substance). a = percentage content of water ;
Shake 3.0 g for 2 h with 100 ml of a 50 per cent V/V solution m = mass of the substance to be examined, in grams ;
of methanol R and filter. Wash the flask and the filter with
n = volume of 0.1 M sodium hydroxide used, in
2 quantities, each of 10 ml, of a 50 per cent V/V solution
of methanol R. Combine the filtrate and washings, add millilitres ;
phenolphthalein solution R and titrate with 0.1 M sodium S = percentage content of free acid (see Tests).
hydroxide until a faint pink colour is obtained. Carry out
a blank titration on 120 ml of a 50 per cent V/V solution Acetyl groups (C2H3O ; Mr 43.05) : typically 21.5 per cent
of methanol R. to 26.0 per cent (anhydrous and acid free substance). To
0.100 g add 25.0 ml of 0.1 M sodium hydroxide and heat on
1 ml of 0.1 M sodium hydroxide is equivalent to 8.3 mg of a water-bath under a reflux condenser for 30 min. Cool, add
free acid, calculated as phthalic acid. 0.1 ml of phenolphthalein solution R and titrate with 0.1 M
Heavy metals (2.4.8) : maximum 10 ppm. hydrochloric acid. Carry out a blank titration.
2.0 g complies with test C. Prepare the reference solution Calculate the percentage content of acetyl groups using the
using 2 ml of lead standard solution (10 ppm Pb) R. following expression :
Water (2.5.12) : maximum 5.0 per cent, determined on
0.500 g.
Carry out the test using a mixture of 2 volumes of methylene
chloride R and 3 volumes of anhydrous ethanol R.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined a = percentage content of water ;
on 1.0 g. m = mass of the substance to be examined, in grams ;
n1 = volume of 0.1 M hydrochloric acid used in the
STORAGE test, in millilitres ;
In an airtight container. n2 = volume of 0.1 M hydrochloric acid used in the
blank titration, in millilitres ;
FUNCTIONALITY-RELATED CHARACTERISTICS
P = percentage content of phthaloyl groups ;
This section provides information on characteristics
that are recognised as being relevant control parameters S = percentage content of free acid (see Tests).
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a 01/2009:0316
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however CELLULOSE, MICROCRYSTALLINE
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process Cellulosum microcristallinum
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for cellulose
acetate phthalate used as film former in gastro-resistant
tablets and capsules.
Apparent viscosity (2.2.9) : typically 45 mPa·s to 90 mPa·s,
determined at 25 °C.
Dissolve 15 g, calculated with reference to the anhydrous
substance, in 85 g of a mixture of 1 part of water R and C6nH10n+2O5n+1
249 parts of acetone R. DEFINITION
Solubility of a film. Dissolve about 0.15 g in 1 ml of Purified, partly depolymerised cellulose prepared by treating
acetone R and pour onto a clear glass plate. A film is formed. alpha-cellulose, obtained as a pulp from fibrous plant
Take a piece of the film and place it in a flask containing material, with mineral acids.
0.1 M hydrochloric acid. It does not dissolve. Then place
the piece of film in a flask containing phosphate buffer CHARACTERS
solution pH 6.8 R. It dissolves. Appearance : white or almost white, fine or granular powder.

4080 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cellulose, microcrystalline

Solubility : practically insoluble in water, in acetone, in liquid after a stable reading has been obtained and measure
anhydrous ethanol, in toluene, in dilute acids and in a 50 g/l the conductivity of the water used to prepare the test
solution of sodium hydroxide. solution.
IDENTIFICATION Ether-soluble substances: maximum 0.05 per cent (5 mg)
for the difference between the weight of the residue and the
A. Place about 10 mg on a watch-glass and disperse in 2 ml
weight obtained from a blank determination.
of iodinated zinc chloride solution R. The substance
becomes violet-blue. Place 10.0 g in a chromatography column about 20 mm in
B. The degree of polymerisation is not more than 350. internal diameter and pass 50 ml of peroxide-free ether R
through the column. Evaporate the eluate to dryness. Dry
Transfer 1.300 g to a 125 ml conical flask. Add 25.0 ml the residue at 105 °C for 30 min, allow to cool in a dessicator
of water R and 25.0 ml of cupriethylenediamine and weigh. Carry out a blank determination.
hydroxide solution R. Immediately purge the solution
with nitrogen R, insert the stopper and shake until Water-soluble substances: maximum 0.25 per cent (12.5 mg)
completely dissolved. Transfer an appropriate volume of for the difference between the mass of the residue and the
the solution to a suitable capillary viscometer (2.2.9). mass obtained from a blank determination.
Equilibrate the solution at 25 ± 0.1 °C for at least 5 min. Shake 5.0 g with 80 ml of water R for 10 min. Filter through
Record the flow time (t1) in seconds between the 2 marks a filter paper with the aid of vacuum into a tared flask.
on the viscometer. Calculate the kinematic viscosity (ν1) Evaporate to dryness on a water-bath avoiding charring. Dry
of the solution using the following expression : at 105 °C for 1 h and weigh. Carry out a blank determination.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
where k1 is the viscometer constant. using 2 ml of lead standard solution (10 ppm Pb) R.
Dilute a suitable volume of cupriethylenediamine
hydroxide solution R with an equal volume of water R Loss on drying (2.2.32) : maximum 7.0 per cent, determined
and measure the flow time (t2) using a suitable capillary on 1.000 g by drying in an oven at 105 °C for 3 h.
viscometer. Calculate the kinematic viscosity (ν2) of the Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
solvent using the following expression : on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
where k2 is the viscometer constant.
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Determine the relative viscosity (ηrel) of the substance to
be examined using the following expression : Absence of Escherichia coli (2.6.13).
Absence of Pseudomonas aeruginosa (2.6.13).
Absence of Staphylococcus aureus (2.6.13).
Determine the intrinsic viscosity ([η]c) by interpolation, Absence of Salmonella (2.6.13).
using the intrinsic viscosity table (Table 0316.-1).
Calculate the degree of polymerisation (P) using the FUNCTIONALITY-RELATED CHARACTERISTICS
following expression :
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
where m is the mass in grams of the substance to be non-mandatory part of the monograph and it is not
examined and b is the loss on drying as a percentage. necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
TESTS contribute to the quality of a medicinal product by
Solubility. Dissolve 50 mg in 10 ml of ammoniacal solution improving the consistency of the manufacturing process
of copper tetrammine R. It dissolves completely, leaving no and the performance of the medicinal product during use.
residue. Where control methods are cited, they are recognised as
pH (2.2.3) : 5.0 to 7.5 for the supernatant liquid. being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
Shake 5 g with 40 ml of carbon dioxide-free water R for reported, the control method must be indicated.
20 min and centrifuge.
The following characteristics may be relevant for
Conductivity (2.2.38). The conductivity of the test solution microcrystalline cellulose used as binder, diluent or
does not exceed the conductivity of the water by more than disintegrant.
75 μS·cm− 1.
Use as test solution the supernatant liquid obtained in the Particle-size distribution (2.9.31 or 2.9.38).
test for pH. Measure the conductivity of the supernatant Powder flow (2.9.36).
Table 0316.-1. — Intrinsic viscosity table
Intrinsic viscosity [η]c at different values of relative viscosity ηrel
[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180

General Notices (1) apply to all monographs and other texts 4081
Cellulose, microcrystalline EUROPEAN PHARMACOPOEIA 6.3

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 0.268
1.3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 0.342 0.350
1.4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 0.573 0.580
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782

2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285
2.9 1.290 1.295 1.300 1.305 1.310 1.314 1.319 1.324 1.329 1.333

3.0 1.338 1.343 1.348 1.352 1.357 1.362 1.367 1.371 1.376 1.381
3.1 1.386 1.390 1.395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3.3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762

4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116

5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209

4082 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cellulose, microcrystalline

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
5.3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6.2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680

7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
7.2 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915

8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126

9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206

General Notices (1) apply to all monographs and other texts 4083
Cellulose, powdered EUROPEAN PHARMACOPOEIA 6.3

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9.7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9.8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3.300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3.320 3.321

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4.10 4.11 4.13 4.14 4.15 4.17 4.18 4.19 4.20 4.22
16 4.23 4.24 4.25 4.27 4.28 4.29 4.30 4.31 4.33 4.34
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.54 4.55 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4.65 4.66

01/2009:0315 Transfer 0.250 g to a 125 ml conical flask. Add 25.0 ml

of water R and 25.0 ml of cupriethylenediamine
CELLULOSE, POWDERED hydroxide solution R. Immediately purge the solution
with nitrogen R, insert the stopper and shake until
completely dissolved. Transfer an appropriate volume of
Cellulosi pulvis the solution to a suitable capillary viscometer (2.2.9).
Equilibrate the solution at 25 ± 0.1 °C for at least 5 min.
Record the flow time (t1) in seconds between the 2 marks
on the viscometer. Calculate the kinematic viscosity (ν1)
of the solution using the following expression :

where k1 is the viscometer constant.

Dilute a suitable volume of cupriethylenediamine
hydroxide solution R with an equal volume of water R
and measure the flow time (t2) using a suitable capillary
viscometer. Calculate the kinematic viscosity (ν2) of the
C6nH10n+2O5n+1 solvent using the following expression :
DEFINITION
Purified, mechanically disintegrated cellulose prepared by
processing alpha-cellulose obtained as a pulp from fibrous where k2 is the viscometer constant.
plant material. Determine the relative viscosity (ηrel) of the substance to
be examined using the following expression :
CHARACTERS
Appearance : white or almost white, fine or granular powder.
Solubility : practically insoluble in water, slightly soluble in
Determine the intrinsic viscosity ([η]c) by interpolation,
a 50 g/l solution of sodium hydroxide, practically insoluble
using the intrinsic viscosity table (Table 0315.-1).
in acetone, in anhydrous ethanol, in toluene, in dilute acids
and in most organic solvents. Calculate the degree of polymerisation (P) using the
following expression :
IDENTIFICATION
A. Place about 10 mg on a watch-glass and disperse in 2 ml
of iodinated zinc chloride solution R. The substance
becomes violet-blue. where m is the mass in grams of the substance to be
B. The degree of polymerisation is greater than 440. examined and b is the loss on drying as a percentage.

4084 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cellulose, powdered

TESTS Loss on drying (2.2.32) : maximum 6.5 per cent, determined

Solubility. Dissolve 50 mg in 10 ml of ammoniacal solution
of copper tetrammine R. It dissolves completely, leaving no
residue.
pH (2.2.3) : 5.0 to 7.5 for the supernatant liquid.
Mix 10 g with 90 ml of carbon dioxide-free water R and
allow to stand with occasional stirring for 1 h.
Ether-soluble substances : maximum 0.15 per cent (15 mg)
for the difference between the mass of the residue and the
mass obtained from a blank determination.
Place 10.0 g in a chromatography column about 20 mm in
internal diameter and pass 50 ml of peroxide-free ether R
through the column. Evaporate the eluate to dryness in a
previously dried and tared evaporating dish, with the aid
of a current of air in a fume hood. After all the ether has
evaporated, dry the residue at 105 °C for 30 min, allow
to cool in a dessiccator and weigh. Carry out a blank
determination.
Water-soluble substances: maximum 1.5 per cent (15.0 mg)
for the difference between the mass of the residue and the
mass obtained from a blank determination.
Shake 6.0 g with 90 ml of carbon dioxide-free water R for
10 min. Filter with the aid of vacuum into a tared flask.
Discard the first 10 ml of the filtrate and pass the filtrate
through the same filter a second time, if necessary, to obtain
a clear filtrate. Evaporate a 15.0 ml portion of the filtrate to
dryness in a tared evaporating dish without charring. Dry
at 105 °C for 1 h, allow to cool in a desiccator and weigh.
Carry out a blank determination.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Table 0315.-1. – Intrinsic viscosity table
Intrinsic viscosity [η]c at different values of relative viscosity ηrel
ηrel 0.00 0.01 0.02 0.03
on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulphated ash (2.4.14) : maximum 0.3 per cent, determined
on 1.0 g (dried substance).
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Pseudomonas aeruginosa (2.6.13).
Absence of Staphylococcus aureus (2.6.13).
Absence of Salmonella (2.6.13).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited they are recognised as
being suitable for the purpose but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for powdered
cellulose used as diluent or disintegrant.
Particle-size distribution (2.9.31 or 2.9.38).
Powder flow (2.9.36).
[η ]c
0.04 0.05 0.06 0.07 0.08 0.09

1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 0.268
1.3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 0.342 0.350
1.4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 0.573 0.580
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782

2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285

General Notices (1) apply to all monographs and other texts 4085
Cellulose, powdered EUROPEAN PHARMACOPOEIA 6.3

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
2.9 1.290 1.295 1.300 1.305 1.310 1.314 1.319 1.324 1.329 1.333

3.0 1.338 1.343 1.348 1.352 1.357 1.362 1.367 1.371 1.376 1.381
3.1 1.386 1.390 1.395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3.3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762

4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116

5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209
5.3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6.2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680

4086 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cellulose, powdered

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
7.2 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915

8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126

9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9.7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9.8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3.300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3.320 3.321

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4.10 4.11 4.13 4.14 4.15 4.17 4.18 4.19 4.20 4.22
16 4.23 4.24 4.25 4.27 4.28 4.29 4.30 4.31 4.33 4.34

General Notices (1) apply to all monographs and other texts 4087
Charcoal, activated EUROPEAN PHARMACOPOEIA 6.3

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η ]c
ηrel 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.54 4.55 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4.65 4.66

01/2009:0313 Fluorescent substances. In an intermittent-extraction

apparatus, treat 10.0 g with 100 ml of cyclohexane R1 for 2 h.
CHARCOAL, ACTIVATED Collect the liquid and dilute to 100 ml with cyclohexane R1.
Examine in ultraviolet light at 365 nm. The fluorescence of
Carbo activatus the solution is not more intense than that of a solution of
83 μg of quinine R in 1000 ml of 0.005 M sulphuric acid
DEFINITION examined under the same conditions.
Obtained from vegetable matter by suitable carbonisation Sulphides. To 1.0 g in a conical flask add 5 ml of
processes intended to confer a high adsorption power. hydrochloric acid R1 and 20 ml of water R. Heat to boiling.
The fumes released do not turn lead acetate paper R brown.
CHARACTERS
Appearance : black, light powder free from grittiness. Copper : maximum 25.0 ppm.
Solubility : practically insoluble in all usual solvents. Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Use solution S.
IDENTIFICATION
Reference solutions. Prepare the reference solutions using
A. When heated to redness it burns slowly without a flame.
copper standard solution (0.1 per cent Cu) R and diluting
B. Adsorption power (see Tests). with 0.1 M hydrochloric acid.
TESTS Source : copper hollow-cathode lamp.
Solution S. To 2.0 g in a conical flask with a ground-glass Wavelength : 325.0 nm.
neck add 50 ml of dilute hydrochloric acid R. Boil gently Atomisation device : air-acetylene flame.
under a reflux condenser for 1 h, filter and wash the filter
with dilute hydrochloric acid R. Evaporate the combined Lead : maximum 10.0 ppm.
filtrate and washings to dryness on a water-bath, dissolve Atomic absorption spectrometry (2.2.23, Method I).
the residue in 0.1 M hydrochloric acid and dilute to 50.0 ml Test solution. Use solution S.
with the same acid.
Reference solutions. Prepare the reference solutions using
Acidity or alkalinity. To 2.0 g add 40 ml of water R and boil lead standard solution (100 ppm Pb) R and diluting with
for 5 min. Cool, restore to the original mass with carbon 0.1 M hydrochloric acid.
dioxide-free water R and filter. Reject the first 20 ml of the Source : lead hollow-cathode lamp.
filtrate. To 10 ml of the filtrate add 0.25 ml of bromothymol
blue solution R1 and 0.25 ml of 0.02 M sodium hydroxide. Wavelength : 283.3 nm ; 217.0 nm may be used depending
The solution is blue. Not more than 0.75 ml of 0.02 M on the apparatus.
hydrochloric acid is required to change the colour of the Atomisation device : air-acetylene flame.
indicator to yellow. Zinc : maximum 25.0 ppm.
Acid-soluble substances : maximum 3 per cent. Atomic absorption spectrometry (2.2.23, Method I).
To 1.0 g add 25 ml of dilute nitric acid R and boil for 5 min.
Test solution. Use solution S.
Filter whilst hot through a sintered-glass filter (10) (2.1.2)
and wash with 10 ml of hot water R. Evaporate the combined Reference solutions. Prepare the reference solutions using
filtrate and washings to dryness on a water-bath, add to the zinc standard solution (100 ppm Zn) R and diluting with
residue 1 ml of hydrochloric acid R, evaporate to dryness 0.1 M hydrochloric acid.
again and dry the residue to constant mass at 100-105 °C. Source : zinc hollow-cathode lamp.
The residue weighs a maximum of 30 mg. Wavelength : 214.0 nm.
Alkali-soluble coloured substances. To 0.25 g add 10 ml Atomisation device : air-acetylene flame.
of dilute sodium hydroxide solution R and boil for 1 min.
Cool, filter and dilute the filtrate to 10 ml with water R. Loss on drying (2.2.32) : maximum 15 per cent, determined
The solution is not more intensely coloured than reference on 1.00 g by drying in an oven at 120 °C for 4 h.
solution GY4 (2.2.2, Method II). Sulphated ash (2.4.14) : maximum 5.0 per cent, determined
Ethanol (96 per cent) soluble substances : maximum on 1.0 g.
0.5 per cent. Adsorption power. To 0.300 g in a 100 ml
To 2.0 g add 50 ml of ethanol (96 per cent) R and boil under ground-glass-stoppered conical flask add 25.0 ml of
a reflux condenser for 10 min. Filter immediately, cool, and a freshly prepared solution of 0.5 g of phenazone R in 50 ml
dilute to 50 ml with ethanol (96 per cent) R. The filtrate is of water R. Shake thoroughly for 15 min. Filter and reject
not more intensely coloured than reference solution Y6 or the first 5 ml of filtrate. To 10.0 ml of the filtrate add 1.0 g
BY6 (2.2.2, Method II). Evaporate 40 ml of the filtrate to of potassium bromide R and 20 ml of dilute hydrochloric
dryness and dry to constant mass at 100-105 °C. The residue acid R. Using 0.1 ml of methyl red solution R as indicator,
weighs a maximum of 8 mg. titrate with 0.0167 M potassium bromate until the red colour

4088 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
is discharged. Titrate slowly (1 drop every 15 s) towards the
end of the titration. Carry out a blank titration using 10.0 ml
of the phenazone solution.
Calculate the quantity of phenazone adsorbed per 100 g of
activated charcoal from the following expression :
a = number of millilitres of 0.0167 M potassium
bromate used for the blank ;
b = number of millilitres of 0.0167 M potassium
bromate used for the test ;
m = mass in grams of the substance to be examined.
Minimum 40 g of phenazone is adsorbed per 100 g of
activated charcoal, calculated with reference to the dried
substance.
Microbial contamination
3
TAMC : acceptance criterion 10 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
STORAGE
In an airtight container.
01/2008:0575
corrected 6.3
CHOLECALCIFEROL CONCENTRATE
(OILY FORM)
Cholecalciferolum densatum oleosum
DEFINITION
Solution of Cholecalciferol (0072) in a suitable vegetable
fatty oil, authorised by the competent authority.
Content : 90.0 per cent to 110.0 per cent of the
cholecalciferol content stated on the label, which is not less Dissolve 5.0 g in 25 ml of the prescribed mixture of solvents.
than 500 000 IU/g.
It may contain suitable stabilisers such as antioxidants.
CHARACTERS
Appearance : clear, yellow liquid.
Solubility : practically insoluble in water, slightly soluble in
anhydrous ethanol, miscible with solvents of fats.
Partial solidification may occur, depending on the
temperature.
IDENTIFICATION
First identification : A, C.
Second identification : A, B.
A. Thin-layer chromatography (2.2.27). Prepare the
solutions immediately before use.
Test solution. Dissolve an amount of the preparation to
be examined corresponding to 400 000 IU in ethylene
chloride R containing 10 g/l of squalane R and 0.1 g/l
of butylhydroxytoluene R and dilute to 4 ml with the
same solution.
Reference solution (a). Dissolve 10 mg of
cholecalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R to 50.0 ml with the mobile phase. Keep the solution in iced
and dilute to 4 ml with the same solution.
General Notices (1) apply to all monographs and other texts
Cholecalciferol concentrate (oily form)
Reference solution (b). Dissolve 10 mg of
ergocalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R
and dilute to 4 ml with the same solution.
Plate : TLC silica gel G plate R.
Mobile phase : a 0.1 g/l solution of butylhydroxytoluene R
in a mixture of equal volumes of cyclohexane R and
peroxide-free ether R.
Application : 20 μl.
Development : immediately, protected from light, over
a path of 15 cm.
Drying : in air.
Detection : spray with sulphuric acid R.
Results : the chromatogram obtained with the test
solution shows immediately a bright yellow principal spot
which rapidly becomes orange-brown, then gradually
greenish-grey, remaining so for 10 min. This spot is
similar in position, colour and size to the spot in the
chromatogram obtained with reference solution (a). The
chromatogram obtained with reference solution (b) shows
immediately at the same level an orange principal spot
which gradually becomes reddish-brown and remains so
for 10 min.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Prepare a solution in cyclohexane R
containing the equivalent of about 400 IU/ml.
Spectral range : 250-300 nm.
Absorption maximum : at 267 nm.
C. 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
the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
Acid value (2.5.1) : maximum 2.0.
Peroxide value (2.5.5, Method A) : maximum 20.
ASSAY
Carry out the assay as rapidly as possible, avoiding
exposure to actinic light and air.
Liquid chromatography (2.2.29).
Test solution. Dissolve a quantity of the preparation to
be examined, weighed with an accuracy of 0.1 per cent,
equivalent to about 400 000 IU, in 10.0 ml of toluene R and
dilute to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of
cholecalciferol CRS without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of cholecalciferol for
system suitability CRS to 5.0 ml with the mobile phase.
Heat in a water-bath at 90 °C under a reflux condenser for
45 min and cool.
Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS
without heating in toluene R and dilute to 100.0 ml with
the same solvent.
Reference solution (d). Dilute 5.0 ml of reference solution (c)
water.
4089
Cholecalciferol concentrate (oily form) EUROPEAN PHARMACOPOEIA 6.3

Reference solution (e). Place 5.0 ml of reference S′D = area (or height) of the peak due to cholecalciferol
solution (c) in a volumetric flask, add about 10 mg of in the chromatogram obtained with reference
butylhydroxytoluene R and displace air from the flask with solution (a) ;
nitrogen R. Heat in a water-bath at 90 °C under a reflux Sp = area (or height) of the peak due to
condenser protected from light and under nitrogen R for pre-cholecalciferol in the chromatogram
45 min. Cool and dilute to 50.0 ml with the mobile phase. obtained with the test solution ;
Column: f = conversion factor.
— size : l = 0.25 m, Ø = 4.6 mm ;
STORAGE
— stationary phase : silica gel for chromatography R
(5 μm). In an airtight, well-filled container, protected from light.
The contents of an opened container are to be used as
Mobile phase : pentanol R, hexane R (3:997 V/V). soon as possible ; any unused part is to be protected by an
Flow rate : 2 ml/min. atmosphere of nitrogen.

Detection : spectrophotometer at 254 nm. LABELLING

Injection : the chosen volume of each solution (the same The label states :
volume for reference solution (a) and for the test solution) ;
automatic injection device or sample loop recommended. — the number of International Units per gram ;
— the method of restoring the solution if partial solidification
Relative retention with reference to cholecalciferol : pre-
occurs.
cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5.
System suitability : reference solution (b) : IMPURITIES
— resolution : minimum 1.0 between the peaks due to
pre-cholecalciferol and trans-cholecalciferol ; if necessary
adjust the proportions of the constituents and the flow
rate of the mobile phase to obtain this resolution ;
— repeatability : maximum relative standard deviation of
1.0 per cent for the peak due to cholecalciferol after
6 injections.
Calculate the conversion factor (f) using the following
expression :

K = area (or height) of the peak due to cholecalciferol

in the chromatogram obtained with reference A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol
solution (d) ; (trans-cholecalciferol, trans-vitamin D3),
L = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (e) ;
M = area (or height) of the peak due to
pre-cholecalciferol in the chromatogram
obtained with reference solution (e).
The value of f determined in duplicate on different days may
be used during the entire procedure.
Calculate the content of cholecalciferol in International
Units per gram using the following expression :
B. cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol,
provitamin D3),

m = mass of the preparation to be examined in the test

solution, in milligrams ;
m′ = mass of cholecalciferol CRS in reference
solution (a), in milligrams ;
V = volume of the test solution (100 ml) ;
V′ = volume of reference solution (a) (100 ml) ;
SD = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with the test
solution ; C. (9β,10α)-cholesta-5,7-dien-3β-ol (lumisterol3),

4090 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol
(iso-tachysterol3),
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3). B. Ultraviolet and visible absorption spectrophotometry
01/2008:0574
corrected 6.3
CHOLECALCIFEROL CONCENTRATE
(POWDER FORM)
Cholecalciferoli pulvis
DEFINITION
Powder concentrate obtained by dispersing an oily solution
of Cholecalciferol (0072) in an appropriate matrix, which is
usually based on a combination of gelatin and carbohydrates
of suitable quality, authorised by the competent authority.
Content : 90.0 per cent to 110.0 per cent of the
cholecalciferol content stated on the label, which is not less Carry out the assay as rapidly as possible, avoiding
than 100 000 IU/g.
It may contain suitable stabilisers such as antioxidants.
CHARACTERS
Appearance : white or yellowish-white, small particles.
Solubility : practically insoluble, swells, or forms a dispersion accuracy of 0.1 per cent, equivalent to about 100 000 IU.
in water, depending on the formulation.
IDENTIFICATION
First identification : A, C.
Second identification : A, B.
A. Thin-layer chromatography (2.2.27). Prepare the
solutions immediately before use.
Test solution. Place 10.0 ml of the test solution
prepared for the assay in a suitable flask and evaporate
to dryness under reduced pressure by swirling in
a water-bath at 40 °C. Cool under running water
and restore atmospheric pressure with nitrogen R.
Dissolve the residue immediately in 0.4 ml of ethylene
chloride R containing 10 g/l of squalane R and 0.1 g/l
of butylhydroxytoluene R.
General Notices (1) apply to all monographs and other texts
Cholecalciferol concentrate (powder form)
Reference solution (a). Dissolve 10 mg of
cholecalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R
and dilute to 4 ml with the same solution.
Reference solution (b). Dissolve 10 mg of
ergocalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R
and dilute to 4 ml with the same solution.
Plate : TLC silica gel G plate R.
Mobile phase : a 0.1 g/l solution of butylhydroxytoluene R
in a mixture of equal volumes of cyclohexane R and
peroxide-free ether R.
Application : 20 μl.
Development : immediately, protected from light, over
a path of 15 cm.
Drying : in air.
Detection : spray with sulphuric acid R.
Results : the chromatogram obtained with the test
solution shows immediately a bright yellow principal spot,
which rapidly becomes orange-brown, then gradually
greenish-grey, remaining so for 10 min. This spot is
similar in position, colour and size to the spot in the
chromatogram obtained with reference solution (a). The
chromatogram obtained with reference solution (b) shows
immediately at the same level an orange principal spot,
which gradually becomes reddish-brown and remains so
for 10 min.
(2.2.25).
Test solution. Place 5.0 ml of the test solution prepared
for the assay in a suitable flask and evaporate to dryness
under reduced pressure by swirling in a water-bath
at 40 °C. Cool under running water and restore
atmospheric pressure with nitrogen R. Dissolve the
residue immediately in 50.0 ml of cyclohexane R.
Spectral range : 250-300 nm.
Absorption maximum : at 265 nm.
C. 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
the principal peak in the chromatogram obtained with
reference solution (a).
ASSAY
exposure to actinic light and air.
Liquid chromatography (2.2.29).
Test solution. Introduce into a saponification flask a
quantity of the preparation to be examined, weighed with an
Add 5 ml of water R, 20 ml of anhydrous ethanol R, 1 ml of
sodium ascorbate solution R and 3 ml of a freshly prepared
50 per cent m/m solution of potassium hydroxide R.
Heat in a water-bath under a reflux condenser for 30 min.
Cool rapidly under running water. Transfer the liquid to
a separating funnel with the aid of 2 quantities, each of
15 ml, of water R, 1 quantity of 10 ml of ethanol (96 per
cent) R and 2 quantities, each of 50 ml, of pentane R. Shake
vigorously for 30 s. Allow to stand until the 2 layers are clear.
Transfer the lower aqueous-alcoholic layer to a 2nd separating
funnel and shake with a mixture of 10 ml of ethanol (96 per
cent) R and 50 ml of pentane R. After separation, transfer
the aqueous-alcoholic layer to a 3rd separating funnel and
the pentane layer to the 1st separating funnel, washing the
2nd separating funnel with 2 quantities, each of 10 ml, of
pentane R and adding the washings to the 1st separating
4091
Cholecalciferol concentrate (powder form) EUROPEAN PHARMACOPOEIA 6.3

funnel. Shake the aqueous-alcoholic layer with 50 ml of
pentane R and add the pentane layer to the 1st funnel. Wash
the pentane layer with 2 quantities, each of 50 ml, of a
freshly prepared 30 g/l solution of potassium hydroxide R
in ethanol (10 per cent V/V) R, shaking vigorously, then
wash with successive quantities, each of 50 ml, of water R
until the washings are neutral to phenolphthalein. Transfer
the washed pentane extract to a ground-glass-stoppered
flask. Evaporate the contents of the flask to dryness under
reduced pressure by swirling in a water-bath at 40 °C. Cool
under running water and restore atmospheric pressure with
nitrogen R. Dissolve the residue immediately in 5.0 ml of
toluene R and add 20.0 ml of the mobile phase to obtain a
solution containing about 4000 IU/ml.
Reference solution (a). Dissolve 10.0 mg of
cholecalciferol CRS, without heating, in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of cholecalciferol for
system suitability CRS to 5.0 ml with the mobile phase.
Heat in a water-bath at 90 °C under a reflux condenser for
45 min and cool.
Reference solution (c). Dissolve 0.10 g of
cholecalciferol CRS, without heating, in toluene R
and dilute to 100.0 ml with the same solvent.
Reference solution (d). Dilute 5.0 ml of reference solution (c) SD
to 50.0 ml with the mobile phase. Keep the solution in iced
water.
S′D
Reference solution (e). Place 5.0 ml of reference
solution (c) in a volumetric flask, add about 10 mg of
butylhydroxytoluene R and displace the air from the flask
with nitrogen R. Heat in a water-bath at 90 °C under a reflux Sp
condenser, protected from light and under nitrogen R, for
45 min. Cool and dilute to 50.0 ml with the mobile phase.
f = conversion factor.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : silica gel for chromatography R
(5 μm).
Mobile phase : pentanol R, hexane R (3:997 V/V).
Flow rate : 2 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : the chosen volume of each solution (the same
volume for reference solution (a) and for the test solution) ;
automatic injection device or sample loop recommended.
K = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (d) ;
L = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (e) ;
M = area (or height) of the peak due to
pre-cholecalciferol in the chromatogram
obtained with reference solution (e).
The value of f determined in duplicate on different days may
be used during the entire procedure.
Calculate the content of cholecalciferol in International
Units per gram using the following expression :
m = mass of the preparation to be examined in the
test solution, in milligrams ;
m′ = mass of cholecalciferol CRS in reference
solution (a), in milligrams ;
V = volume of the test solution (25 ml) ;
V′ = volume of reference solution (a) (100 ml) ;
= area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with the test
solution ;
= area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (a) ;
= area (or height) of the peak due to
pre-cholecalciferol in the chromatogram
obtained with the test solution ;
STORAGE
In an airtight, well-filled container, protected from light.
The contents of an opened container are to be used as
soon as possible ; any unused part is to be protected by an
atmosphere of nitrogen.
LABELLING
The label states the number of International Units per gram.
IMPURITIES

Relative retention with reference to cholecalciferol : pre-

cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5.
System suitability : reference solution (b) :
— resolution : minimum 1.0 between the peaks due to
pre-cholecalciferol and trans-cholecalciferol ; if necessary,
adjust the proportions of the constituents and the flow
rate of the mobile phase to obtain this resolution ;
— repeatability : maximum relative standard deviation of
1.0 per cent for the peak due to cholecalciferol after
6 injections.
Calculate the conversion factor (f) using the following
expression :
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol
(trans-cholecalciferol, trans-vitamin D3),

4092 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
B. cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol,
provitamin D3),
C. (9β,10α)-cholesta-5,7-dien-3β-ol (lumisterol3),
D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol
(iso-tachysterol3),
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3).
01/2008:0598
corrected 6.3 B. Ultraviolet and visible absorption spectrophotometry
CHOLECALCIFEROL CONCENTRATE
(WATER-DISPERSIBLE FORM)
Cholecalciferolum in aqua dispergibile
DEFINITION
Solution of Cholecalciferol (0072) in a suitable vegetable
fatty oil, authorised by the competent authority, to which
suitable solubilisers have been added.
General Notices (1) apply to all monographs and other texts
Cholecalciferol concentrate (water-dispersible form)
Content : 90.0 per cent to 115.0 per cent of the
cholecalciferol content stated on the label, which is not less
than 100 000 IU/g.
It may contain suitable stabilisers such as antioxidants.
CHARACTERS
Appearance : slightly yellowish liquid of variable opalescence
and viscosity.
Highly concentrated solutions may become cloudy at low
temperatures or form a gel at room temperature.
IDENTIFICATION
First identification : A, C, D.
Second identification : A, B, D.
A. Thin-layer chromatography (2.2.27). Prepare the
solutions immediately before use.
Test solution. Place 10.0 ml of the test solution
prepared for the assay in a suitable flask and evaporate
to dryness under reduced pressure by swirling in
a water-bath at 40 °C. Cool under running water
and restore atmospheric pressure with nitrogen R.
Dissolve the residue immediately in 0.4 ml of ethylene
chloride R containing 10 g/l of squalane R and 0.1 g/l
of butylhydroxytoluene R.
Reference solution (a). Dissolve 10 mg of
cholecalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R
and dilute to 4 ml with the same solution.
Reference solution (b). Dissolve 10 mg of
ergocalciferol CRS in ethylene chloride R containing
10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R
and dilute to 4 ml with the same solution.
Plate : TLC silica gel G plate R.
Mobile phase : a 0.1 g/l solution of butylhydroxytoluene R
in a mixture of equal volumes of cyclohexane R and
peroxide-free ether R.
Application : 20 μl.
Development : immediately, protected from light, over
a path of 15 cm.
Drying : in air.
Detection : spray with sulphuric acid R.
Results : the chromatogram obtained with the test
solution shows immediately a bright yellow principal spot,
which rapidly becomes orange-brown, then gradually
greenish-grey, remaining so for 10 min. This spot is
similar in position, colour and size to the principal spot in
the chromatogram obtained with reference solution (a).
The chromatogram obtained with reference solution (b)
shows immediately at the same level an orange principal
spot, which gradually becomes reddish-brown and
remains so for 10 min.
(2.2.25).
Test solution. Place 5.0 ml of the test solution prepared
for the assay in a suitable flask and evaporate to dryness
under reduced pressure by swirling in a water-bath
at 40 °C. Cool under running water and restore
atmospheric pressure with nitrogen R. Dissolve the
residue immediately in 50.0 ml of cyclohexane R.
Spectral range : 250-300 nm.
Absorption maximum : at 265 nm.
C. Examine the chromatograms obtained in the assay.
4093
Cholecalciferol concentrate (water-dispersible form)
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
D. Mix about 1 g with 10 ml of water R previously warmed
to 50 °C, and cool to 20 °C. Immediately after cooling, a
uniform, slightly opalescent and slightly yellow dispersion Injection : the chosen volume of each solution (the same
is obtained.
ASSAY
Carry out the assay as rapidly as possible, avoiding
exposure to actinic light and air.
Liquid chromatography (2.2.29).
Test solution. Introduce into a saponification flask a
quantity of the preparation to be examined, weighed with an
accuracy of 0.1 per cent, equivalent to about 100 000 IU.
Add 5 ml of water R, 20 ml of anhydrous ethanol R, 1 ml of — repeatability : maximum relative standard deviation of
sodium ascorbate solution R and 3 ml of a freshly prepared
50 per cent m/m solution of potassium hydroxide R.
Heat in a water-bath under a reflux condenser for 30 min.
Cool rapidly under running water. Transfer the liquid to
a separating funnel with the aid of 2 quantities, each of
15 ml, of water R, 1 quantity of 10 ml of ethanol (96 per
cent) R and 2 quantities, each of 50 ml, of pentane R. Shake
vigorously for 30 s. Allow to stand until the 2 layers are
clear. Transfer the aqueous-alcoholic layer to a 2nd separating K
funnel and shake with a mixture of 10 ml of ethanol (96 per
cent) R and 50 ml of pentane R. After separation, transfer
the aqueous-alcoholic layer to a 3rd separating funnel and
the pentane layer to the 1st separating funnel, washing the
nd
2 separating funnel with 2 quantities, each of 10 ml, of
pentane R and adding the washings to the 1st separating
funnel. Shake the aqueous-alcoholic layer with 50 ml of
pentane R and add the pentane layer to the 1st funnel. Wash
the pentane layer with 2 quantities, each of 50 ml, of a
freshly prepared 30 g/l solution of potassium hydroxide R The value of f determined in duplicate on different days may
in ethanol (10 per cent V/V) R, shaking vigorously, and then be used during the entire procedure.
wash with successive quantities, each of 50 ml, of water R
until the washings are neutral to phenolphthalein. Transfer Units per gram using the following expression :
the washed pentane extract to a ground-glass-stoppered
flask. Evaporate the contents of the flask to dryness under
reduced pressure by swirling in a water-bath at 40 °C. Cool
under running water and restore atmospheric pressure with
nitrogen R. Dissolve the residue immediately in 5.0 ml of
toluene R and add 20.0 ml of the mobile phase to obtain a
solution containing about 4000 IU/ml.
Reference solution (a). Dissolve 10.0 mg of
cholecalciferol CRS, without heating, in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of cholecalciferol for
system suitability CRS to 5.0 ml with the mobile phase.
Heat in a water-bath at 90 °C under a reflux condenser for
45 min and cool.
Reference solution (c). Dissolve 0.10 g of
cholecalciferol CRS, without heating, in toluene R
and dilute to 100.0 ml with the same solvent.
Reference solution (d). Dilute 5.0 ml of reference solution (c)
to 50.0 ml with the mobile phase. Keep the solution in iced
water.
Reference solution (e). Place 5.0 ml of reference
solution (c) in a volumetric flask, add about 10 mg of
butylhydroxytoluene R and displace the air from the flask
with nitrogen R. Heat in a water-bath at 90 °C under a reflux
condenser, protected from light and under nitrogen R, for
45 min. Cool and dilute to 50.0 ml with the mobile phase.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
4094 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
— stationary phase : silica gel for chromatography R
(5 μm).
Mobile phase : pentanol R, hexane R (3:997 V/V).
Flow rate : 2 ml/min.
Detection : spectrophotometer at 254 nm.
volume for reference solution (a) and for the test solution) ;
automatic injection device or sample loop recommended.
Relative retention with reference to cholecalciferol : pre-
cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5.
System suitability : reference solution (b) :
— resolution : minimum 1.0 between the peaks due to
pre-cholecalciferol and trans-cholecalciferol ; if necessary,
adjust the proportions of the constituents and the flow
rate of the mobile phase to obtain this resolution ;
1.0 per cent for the peak due to cholecalciferol after
6 injections.
Calculate the conversion factor (f) using the following
expression :
= area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (d) ;
L = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (e) ;
M = area (or height) of the peak due to
pre-cholecalciferol in the chromatogram
obtained with reference solution (e).
Calculate the content of cholecalciferol in International
m = mass of the preparation to be examined in the test
solution, in milligrams ;
m′ = mass of cholecalciferol CRS in reference
solution (a), in milligrams ;
V = volume of the test solution (25 ml) ;
V′ = volume of reference solution (a) (100 ml) ;
SD = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with the test
solution ;
S′D = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (a) ;
Sp = area (or height) of the peak due to
pre-cholecalciferol in the chromatogram
obtained with the test solution ;
f = conversion factor.
STORAGE
In an airtight, well-filled container, protected from light, at
the temperature stated on the label.
The contents of an opened container are to be used as
soon as possible ; any unused part is to be protected by an
atmosphere of inert gas.
EUROPEAN PHARMACOPOEIA 6.3
LABELLING
The label states :
— the number of International Units per gram ;
— the storage temperature.
IMPURITIES
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol
(trans-cholecalciferol, trans-vitamin D3),
B. cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol,
provitamin D3),
C. (9β,10α)-cholesta-5,7-dien-3β-ol (lumisterol3),
D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol
(iso-tachysterol3),
General Notices (1) apply to all monographs and other texts
Chondroitin sulphate sodium
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3).
01/2009:2064
CHONDROITIN SULPHATE SODIUM
Chondroitini natrii sulfas
H2O(C14H19NNa2O14S)x
DEFINITION
Natural copolymer based mainly on the 2 disaccharides :
[4)-(β-D-glucopyranosyluronic acid)-(1→3)-[2-
(acetylamino)-2-deoxy-β-D-galactopyranosyl
4-sulphate]-(1→] and [4)-(β-D-glucopyranosyluronic
acid)-(1→3)-[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl
6-sulphate]-(1→], sodium salt. On complete hydrolysis it
liberates D-galactosamine, D-glucuronic acid, acetic acid
and sulphuric acid. It is obtained from cartilage of both
terrestrial and marine origins. Depending on the animal
species of origin, it shows different proportions of 4-sulphate
and 6-sulphate groups.
Content : 95 per cent to 105 per cent (dried substance).
PRODUCTION
The animals from which chondroitin sulphate sodium is
derived must fulfil the requirements for the health of animals
suitable for human consumption.
CHARACTERS
Appearance : white or almost white, hygroscopic powder.
Solubility : freely soluble in water, practically insoluble in
acetone and in ethanol (96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of potassium bromide R.
Comparison : for chondroitin sulphate sodium of
terrestrial origin use chondroitin sulphate sodium CRS
and for chondroitin sulphate sodium of marine origine
use chondroitin sulphate sodium (marine) CRS.
B. Solution S1 (see Tests) gives reaction (b) of sodium
(2.3.1).
4095
Chondroitin sulphate sodium
C. Examine the electropherograms obtained in the test for
related substances.
Results : the principal band in the electropherogram
obtained with the test solution is similar in position to
the principal band in the electropherogram obtained with
reference solution (a).
TESTS
Solution S1. Dissolve 2.500 g in 50.0 ml of carbon
dioxide-free water R.
Solution S2. Dilute 1.0 ml of solution S1 to 10.0 ml with
water R.
pH (2.2.3) : 5.5 to 7.5 for solution S1.
Specific optical rotation (2.2.7) : − 20 to − 30 (terrestrial
origin) or − 12 to − 19 (marine origin) (dried substance),
determined on solution S1.
Intrinsic viscosity : 0.01 m3/kg to 0.15 m3/kg.
Test solution (a). Weigh 5.000 g (m0p) of the substance to be
examined and add about 80 ml of an 11.7 g/l solution of
sodium chloride R at room temperature. Dissolve by shaking
at room temperature for 30 min. Dilute to 100.0 ml with an
11.7 g/l solution of sodium chloride R. Filter through a
0.45 μm filter membrane and discard the first 10 ml. The
concentration of test solution (a) is only indicative and must
be adjusted after an initial measurement of the viscosity of
test solution (a).
Test solution (b). To 15.0 ml of test solution (a) add 5.0 ml of
an 11.7 g/l solution of sodium chloride R.
Test solution (c). To 10.0 ml of test solution (a) add 10.0 ml
of an 11.7 g/l solution of sodium chloride R.
Test solution (d). To 5.0 ml of test solution (a) add 15.0 ml of
an 11.7 g/l solution of sodium chloride R.
Determine the flow-time (2.2.9) for an 11.7 g/l solution
of sodium chloride R (t0) and the flow times for the 4 test
solutions (t1, t2, t3 and t4), at 25.00 ± 0.03 °C. Use an
appropriate suspended level viscometer (specifications :
viscometer constant = about 0.005 mm2/s2, kinematic
viscosity range = 1-5 mm2/s, internal diameter of
tube R = 0.53 mm, volume of bulb C = 5.6 ml, internal
diameter of tube N = 2.8-3.2 mm) with a funnel-shaped lower
capillary end. Use the same viscometer for all measurements ;
measure all outflow times in triplicate. The test is not valid
unless the results do not differ by more than 0.35 per cent
from the mean and if the flow time t1 is not less than 1.6 × t0
and not more than 1.8 × t0. If this is not the case, adjust the
concentration of test solution (a) and repeat the procedure.
Calculation of the relative viscosities
Since the densities of the chondroitin sulphate solutions and
of the solvent are almost equal, the relative viscosities ηri
(being ηr1, ηr2, ηr3 and ηr4) can be calculated from the ratio
of the flow times for the respective solutions ti (being t1, t2,
t3 and t4) to the flow time of the solvent t0, but taking into
account the kinetic energy correction factor for the capillary
(B = 30 800 s3), as shown below :
Calculation of the concentrations
Calculate the concentration c1 (expressed in kg/m3) of
chondroitin sulphate sodium in test solution (a) using the
following expression :
4096 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
x = percentage content of chondroitin sulphate
sodium as determined in the assay ;
h = loss on drying as a percentage.
Calculate the concentration c2 (expressed in kg/m3) of
chondroitin sulphate sodium in test solution (b) using the
following expression :
Calculate the concentration c3 (expressed in kg/m3) of
chondroitin sulphate sodium in test solution (c) using the
following expression :
Calculate the concentration c4 (expressed in kg/m3) of
chondroitin sulphate sodium in test solution (d) using the
following expression :
Calculation of the intrinsic viscosity
The specific viscosity ηsi of the test solution (being ηs1,
ηs2, ηs3 and ηs4) is calculated from the relative viscosities
ηri (being ηr1, ηr2, ηr3 and ηr4) according to the following
expression :
The intrinsic viscosity [η], defined as
is calculated by linear least-squares regression analysis using
the following equation :
ci = concentration of the substance to be examined
expressed in kg/m3 ;
kH = Huggins’ constant.
Related substances. Electrophoresis (2.2.31).
Buffer solution A (0.1 M barium acetate pH 5.0). Dissolve
25.54 g of barium acetate R in 900 ml of water R. Adjust to
pH 5.0 with glacial acetic acid R and dilute to 1000.0 ml
with water R.
Buffer solution B (1 M barium acetate pH 5.0). Dissolve
255.43 g of barium acetate R in 900 ml of water R. Adjust
to pH 5.0 with glacial acetic acid R and dilute to 1000.0 ml
with water R.
Staining solution. Dissolve 1.0 g of toluidine blue R and
2.0 g of sodium chloride R in 1000 ml of 0.01 M hydrochloric
acid. Filter.
Test solution. Prepare a 30 mg/ml solution of the substance
to be examined in water R.
Reference solution (a). Prepare a 30 mg/ml solution of
chondroitin sulphate sodium CRS in water R.
Reference solution (b). Dilute 2.0 ml of reference solution (a)
to 100.0 ml with water R.
Reference solution (c). Mix equal volumes of reference
solution (b) and water R.
Procedure. Allow the electrophoresis support to cool the
plate to 10 °C. Pre-equilibrate the agarose gel for 1 min in
buffer solution A. Remove excess liquid by careful decanting.
Dry the gel for approximately 5 min. Place 400 ml of buffer
solution B into each of the containers of the electrophoresis
equipment. Transfer 1 μl of each solution to the slots
EUROPEAN PHARMACOPOEIA 6.3
of the agarose gel. Pipette a few millilitres of a 50 per
cent V/V solution of glycerol R onto the cooled plate of
the electrophoresis equipment and place the gel in the
middle of the ceramic plate. Place a wick, saturated with
buffer solution B, at the positive and negative sides of the
agarose gel. Ensure that there is good contact between the
electrophoresis buffer and the agarose gel. Perform the
electrophoresis under the following conditions : 75 mA/gel,
resulting in a voltage of 100-150 V (maximum 300-400 V) for
a gel of about 12 cm × 10 cm. Carry out the electrophoresis
for 12 min. Place the gel in a mixture consisting of
10 volumes of anhydrous ethanol R and 90 volumes of
buffer solution A for 2 min. Carry out the electrophoresis for
20 min. Place the gel in a mixture consisting of 30 volumes
of anhydrous ethanol R and 70 volumes of buffer solution A
for 2 min. Carry out the electrophoresis for 20 min. Stain the
gel in the staining solution for 10 min. Destain the gel for
15 min under running tap water followed by 10-15 min with
water R until the band in the electropherogram obtained
with reference solution (c) is visible. Allow the gel to dry.
System suitability :
— the electropherogram obtained with reference solution (c)
shows a visible band ;
— the band in the electropherogram obtained with reference
solution (b) is clearly visible and similar in position to the
band in the electropherogram obtained with reference
solution (a).
Results : any secondary band in the electropherogram
obtained with the test solution is not more intense than
the band in the electropherogram obtained with reference
solution (b) (2 per cent).
Protein (2.5.33, Method 2) : maximum 3.0 per cent (dried
substance).
Test solution. Dilute 1.0 ml of solution S1 to 50.0 ml with
0.1 M sodium hydroxide.
Reference solutions. Dissolve about 0.100 g of bovine
albumin R, accurately weighed, in 0.1 M sodium hydroxide
and dilute to 50.0 ml with the same solvent. Carry out all
additional dilutions using 0.1 M sodium hydroxide.
Chlorides (2.4.4) : maximum 0.5 per cent.
Dilute 1 ml of solution S2 to 15 ml with water R. Do not
add diluted nitric acid. Prepare the standard using 5 ml of
chloride standard solution (5 ppm Cl) and 10 ml of water R.
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 12.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Staphylococcus aureus (2.6.13).
Absence of Pseudomonas aeruginosa (2.6.13).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
Absence of bile-tolerant gram-negative bacteria (2.6.13).
ASSAY
Test solution (a). Weigh 0.100 g (m1) of the substance to be
examined, dissolve in water R and dilute to 100.0 ml with
the same solvent.
Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml
with water R.
General Notices (1) apply to all monographs and other texts
Cisplatin
Reference solution (a). Weigh 0.100 g (m0) of chondroitin
sulphate sodium CRS, previously dried as described in the
test for loss on drying, dissolve in water R and dilute to
100.0 ml with the same solvent.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 50.0 ml with water R.
Titrant solution (a). Weigh 4.000 g of cetylpyridinium
chloride monohydrate R and dilute to 1000 ml with water R.
Titrant solution (b). Weigh 1.000 g of cetylpyridinium
chloride monohydrate R and dilute to 1000 ml with water R.
Perform either visual or photometric titration as follows :
Visual titration. Titrate 40.0 ml of reference solution (a)
and 40.0 ml of test solution (a) with titrant solution (a). The
solution becomes turbid. At the end point, the liquid appears
clear, with an almost-white precipitate in suspension. The
precipitate is more apparent if 0.1 ml of a 1 per cent solution
of methylene blue R is added before starting the titration.
The precipitated particles are more apparent against the
blue background.
Photometric titration. Titrate 50.0 ml of reference
solution (b) and 50.0 ml of test solution (b) with titrant
solution (b). To determine the end point, use a suitable
autotitrator equipped with a phototrode at a suitable
wavelength (none is critical) in the visible range.
Calculate the percentage content of chondroitin sulphate
sodium using the following expression :
v0 = volume of appropriate titrant solution when
titrating the appropriate reference solution, in
millilitres ;
v1 = volume of appropriate titrant solution when
titrating the appropriate test solution, in
millilitres ;
h = loss on drying of the substance to be examined,
as a percentage ;
Z = percentage content of H2O(C14H19NNa2O14S)x in
chondroitin sulphate sodium CRS.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states the origin of the substance (marine or
terrestrial).
01/2009:0599
CISPLATIN
Cisplatinum
PtCl2(NH3)2 Mr 300.0
[15663-27-1]
DEFINITION
cis-Diamminedichloroplatinum(II).
Content : 97.0 per cent to 102.0 per cent.
4097
Cisplatin
CHARACTERS
Appearance : yellow powder, or yellow or orange-yellow
crystals.
Solubility : slightly soluble in water, sparingly soluble in
dimethylformamide, practically insoluble in ethanol (96 per
cent).
Carry out identification test B, the tests (except that for
silver) and the assay protected from light.
IDENTIFICATION
First identification : A, B.
Second identification : B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : cisplatin CRS.
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 μl.
Development : over 2/3 of the plate.
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.
Results : the principal spot in the chromatogram obtained
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.
TESTS
Solution S1. Dissolve 25 mg in a 9 g/l solution of sodium
chloride R in carbon dioxide-free water R and dilute to
25 ml with the same solvent.
Solution S2. Dissolve 0.20 g in dimethylformamide R and
dilute to 10 ml with the same solvent.
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
after preparation.
Related substances. 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.
Test solution. Dissolve 25.0 mg of the substance to be
examined in a 9.0 g/l solution of sodium chloride R and
dilute to 25.0 ml with the same solution.
4098 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
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 0.05 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, Ø = 4.0 mm ;
— stationary phase : base-deactivated octylsilyl silica gel
for chromatography R (4 μm) ;
— 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.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 20 μl of the test solution, reference solutions (d)
and (e), and the blank solution.
Run time : 3 times the retention time of cisplatin.
The displacement peak is the latest eluting peak of the group
of injection peaks in the chromatogram obtained with the
blank solution.
Identification of cisplatin aquo complex : use the
chromatogram supplied with cisplatin CRS and the
chromatogram obtained with reference solution (a) to
identify the peak due to cisplatin aquo complex.
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 ; cisplatin
aquo complex = about 1.2.
System suitability : reference solution (d) :
— resolution : minimum 2.5 between the peaks due to
impurities A and B, the displacement peak and the peak
due to impurity A are well separated.
Limits :
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (d) (2.0 per cent) ;
— impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (d) (1.0 per cent) ;
— unspecified impurities : for each impurity, not more
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) ;
EUROPEAN PHARMACOPOEIA 6.3
— disregard limit: the area of the peak due to cisplatin in
the chromatogram obtained with reference solution (e)
(0.05 per cent). Disregard any peak due to the cisplatin
aquo complex.
Silver : maximum 2.50 × 102 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 0.100 g in 15 ml of nitric acid R,
heating to 80 °C. Cool and dilute to 25.0 ml with water R.
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 using a
transmission band of 0.5 nm.
Wavelenth : 328 nm.
Atomisation device : fuel-lean air-acetylene flame.
Carry out a blank determination.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : 10 μl of the test solution and reference solution (a). CHARACTERS
Calculate the percentage content of PtCl2(NH3)2 from the sum Appearance : white or almost white, crystalline powder.
of the areas of the peaks due to cisplatin and cisplatin aquo
complex and from the declared content of cisplatin CRS.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B.
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) : C.
A. trans-diamminedichloroplatinum(II) (transplatin),
B. amminetrichloroplatinate(–),
C. tetrachloroplatinate(2–).
General Notices (1) apply to all monographs and other texts
Citalopram hydrobromide
01/2009:2288
CITALOPRAM HYDROBROMIDE
Citaloprami hydrobromidum
C20H22BrFN2O Mr 405.3
[59729-32-7]
DEFINITION
(1RS)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbonitrile hydrobromide.
Content : 99.0 per cent to 101.5 per cent (dried substance).
Solubility : sparingly soluble in water and in anhydrous
ethanol.
IDENTIFICATION
A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : citalopram hydrobromide CRS.
C. It gives reaction (a) of bromides (2.3.1).
TESTS
Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 1.0 g in methanol R and dilute to 20 ml with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be
examined in mobile phase A and dilute to 100.0 ml with
mobile phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
Reference solution (b). Dissolve the contents of a vial of
citalopram for system suitability CRS (impurities B, D, F
and G) in 1.0 ml of reference solution (a).
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (4 μm) ;
— temperature : 40 °C.
Mobile phase :
— mobile phase A : dissolve 1.58 g of ammonium formate R
in 500 ml of a mixture of 4 volumes of acetonitrile R,
32 volumes of methanol R and 64 volumes of water R ;
— mobile phase B : dissolve 1.58 g of ammonium formate R
in 500 ml of a mixture of 32 volumes of water R and
68 volumes of acetonitrile R ;
4099
Citalopram hydrobromide EUROPEAN PHARMACOPOEIA 6.3

Time Mobile phase A Mobile phase B 1 ml of 0.1 M sodium hydroxide is equivalent to 40.53 mg
(min) (per cent V/V) (per cent V/V) of C20H22BrFN2O.
0-2 100 0
IMPURITIES
2 - 25 100 → 40 0 → 60
Specified impurities : B, D, F, G.
25 - 30 40 60
Other detectable impurities (the following substances
Flow rate : 1.0 ml/min. would, if present at a sufficient level, be detected by one
Detection : spectrophotometer at 230 nm and at 254 nm. or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Injection : 20 μl. impurities and/or by the general monograph Substances for
Identification of impurities: use the chromatogram pharmaceutical use (2034). It is therefore not necessary to
supplied with citalopram for system suitability CRS and identify these impurities for demonstration of compliance.
the chromatogram obtained with reference solution (b) to See also 5.10. Control of impurities in substances for
identify the peaks due to impurities B, D, F and G. pharmaceutical use) : A, C, E.
Relative retention with reference to citalopram
(retention time = about 19 min) : impurity G = about
0.5 ; impurity B = about 0.7 ; impurity D = about 0.9 ;
impurity F = about 1.6.
System suitability : reference solution (b) :
— resolution : minimum 1.5 between the peaks due to
impurity D and citalopram at 230 nm.
Limits :
— correction factors : for the calculation of content,
multiply the peak area of the following impurities by
the corresponding correction factor : impurity F = 1.4 ; A. R = CO-NH2, X = H2 : (1RS)-1-[3-(dimethylamino)propyl]-1-
impurity G = 0.6 ; (4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide,
— impurity D at 230 nm : not more than twice the area of
the principal peak in the chromatogram obtained with C. R = CN, X = O : (3RS)-6-cyano-3-[3-(dimethylamino)propyl]-
reference solution (a) (0.2 per cent) ; 3-(4-fluorophenyl)isobenzofuran-1(3H)-one,
— impurities B, F at 230 nm: for each impurity, not more
than 1.5 times the area of the principal peak in the E. R = Cl, X = H2 : 3-[(1RS)-5-chloro-1-(4-fluorophenyl)-1,3-
chromatogram obtained with reference solution (a) dihydroisobenzofuran-1-yl]-N,N-dimethylpropan-1-amine,
(0.15 per cent) ;
— impurity G at 254 nm : not more than 1.5 times the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
— unspecified impurities at 230 nm and 254 nm: for each
impurity, not more than the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.10 per cent) ;
— sum of impurities at 230 nm : not more than 5 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ; B. 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3-hydroxy-
— disregard limit at 230 nm : 0.5 times the area of the 1,3-dihydroisobenzofuran-5-carbonitrile,
principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 0.5 g in ethanol (96 per cent) R and dilute to 20 ml
with the same solvent. 12 ml of the solution complies with
test B. Prepare the reference solution using lead standard
solution (0.5 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R with ethanol (96 per cent) R. Filter
the solutions through a membrane filter (nominal pore size
0.45 μm). D. R1 = CN, R2 = H : (1RS)-1-(4-fluorophenyl)-1-[3-
Loss on drying (2.2.32) : maximum 0.5 per cent, determined (methylamino)propyl]-1,3-dihydroisobenzofuran-5-
on 1.000 g by drying in an oven at 105 °C for 4 h. carbonitrile,
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g in a platinum crucible. F. R1 = Br, R2 = CH3 : 3-[(1RS)-5-bromo-1-(4-fluorophenyl)-
1,3-dihydroisobenzofuran-1-yl]-N,N-dimethylpropan-1-
ASSAY amine,
Dissolve 0.300 g in 50 ml of ethanol (96 per cent) R and add
0.5 ml of 0.1 M hydrochloric acid. Carry out a potentiometric G. R1 = CO-[CH2]3-N(CH3)2, R2 = CH3 : 4-(dimethylamino)-1-
titration (2.2.20), using 0.1 M sodium hydroxide. Read the [(1RS)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
volume added between the 2 points of inflexion. dihydroisobenzofuran-5-yl]butan-1-one.

4100 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Citalopram hydrochloride

01/2009:2203 — mobile phase B : dissolve 1.58 g of ammonium formate R

in 500 ml of a mixture of 32 volumes of water R and
68 volumes of acetonitrile R ;
CITALOPRAM HYDROCHLORIDE
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Citaloprami hydrochloridum 0-2 100 0
2 - 25 100 → 40 0 → 60
25 - 30 40 60

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 230 nm.
Injection : 20 μl.
Identification of impurities : use the chromatogram
supplied with citalopram for system suitability CRS and
C20H22ClFN2O Mr 360.9 the chromatogram obtained with reference solution (b) to
[85118-27-0] identify the peaks due to impurities B and D.
Relative retention with reference to citalopram
DEFINITION (retention time = about 19 min) : impurity B = about 0.7 ;
(1RS)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3- impurity D = about 0.9.
dihydroisobenzofuran-5-carbonitrile hydrochloride. System suitability : reference solution (b) :
Content : 99.0 per cent to 101.5 per cent (dried substance). — resolution : minimum 1.5 between the peaks due to
impurity D and citalopram.
CHARACTERS
Limits :
Appearance : white or almost white, crystalline powder.
— impurity B : not more than 1.5 times the area of the
Solubility : very soluble in water, freely soluble in anhydrous principal peak in the chromatogram obtained with
ethanol. reference solution (a) (0.15 per cent) ;
— unspecified impurities : for each impurity, not more
IDENTIFICATION
than the area of the principal peak in the chromatogram
A. Optical rotation (see Tests). obtained with reference solution (a) (0.10 per cent) ;
B. Infrared absorption spectrophotometry (2.2.24). — total : not more than twice the area of the principal peak
Comparison : citalopram hydrochloride CRS. in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
C. It gives reaction (a) of chlorides (2.3.1).
— disregard limit : 0.5 times the area of the principal peak
TESTS in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Solution S. Dissolve 1.0 g in methanol R and dilute to 20 ml
with the same solvent. Heavy metals (2.4.8) : maximum 20 ppm.
Appearance of solution. Solution S, examined immediately Dissolve 1.0 g in 20 ml of water R. 12 ml of the solution
after preparation, is clear (2.2.1) and not more intensely complies with test A. Prepare the reference solution using
coloured than reference solution Y6 (2.2.2, Method II). lead standard solution (1 ppm Pb) R.
Optical rotation (2.2.7) : − 0.10° to + 0.10°, determined on Loss on drying (2.2.32) : maximum 0.5 per cent, determined
solution S. on 1.000 g by drying in an oven at 105 °C for 4 h.
Related substances. Liquid chromatography (2.2.29). Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g in a platinum crucible.
Test solution. Dissolve 50 mg of the substance to be
examined in mobile phase A and dilute to 100.0 ml with ASSAY
mobile phase A. Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R and add
Reference solution (a). Dilute 1.0 ml of the test solution to 0.5 ml of 0.1 M hydrochloric acid. Carry out a potentiometric
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution titration (2.2.20), using 0.1 M sodium hydroxide. Read the
to 10.0 ml with mobile phase A. volume added between the 2 points of inflexion.
Reference solution (b). Dissolve the contents of a vial of 1 ml of 0.1 M sodium hydroxide is equivalent to 36.09 mg
citalopram for system suitability CRS (impurities B and D) of C20H22ClFN2O.
in 1.0 ml of reference solution (a).
IMPURITIES
Column:
Specified impurities : B.
— size : l = 0.25 m, Ø = 4.6 mm ; Other detectable impurities (the following substances
— stationary phase : end-capped octadecylsilyl silica gel would, if present at a sufficient level, be detected by one
for chromatography R (4 μm) ; or other of the tests in the monograph. They are limited
— temperature : 40 °C. by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
Mobile phase : pharmaceutical use (2034). It is therefore not necessary to
— mobile phase A : dissolve 1.58 g of ammonium formate R identify these impurities for demonstration of compliance.
in 500 ml of a mixture of 4 volumes of acetonitrile R, See also 5.10. Control of impurities in substances for
32 volumes of methanol R and 64 volumes of water R ; pharmaceutical use) : A, C, D, E, F.

General Notices (1) apply to all monographs and other texts 4101
Clonidine hydrochloride
A. R1 = CO-NH2, R2 = CH3, X = H2 : (1RS)-1-[3-
(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carboxamide,
C. R1 = CN, R2 = CH3, X = O : (3RS)-6-cyano-3-[3-
(dimethylamino)propyl]-3-(4-fluorophenyl)isobenzofuran-
1(3H)-one,
D. R1 = CN, R2 = H, X = H2 : (1RS)-1-(4-fluorophenyl)-1-
[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5-
carbonitrile,
E. R1 = Cl, R2 = CH3, X = H2 : 3-[(1RS)-5-chloro-1-(4-
fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-N,N-
dimethylpropan-1-amine,
F. R1 = Br, R2 = CH3, X = H2 : 3-[(1RS)-5-bromo-1-(4-
fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-N,N-
dimethylpropan-1-amine,
B. 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3-hydroxy-
1,3-dihydroisobenzofuran-5-carbonitrile.
01/2008:0477
corrected 6.3 TESTS
CLONIDINE HYDROCHLORIDE
Clonidini hydrochloridum
C9H10Cl3N3 Mr 266.6 phase A.
[4205-91-8]
DEFINITION
2,6-Dichloro-N-(imidazolidin-2-ylidene)aniline hydrochloride.
Content : 98.5 per cent to 101.0 per cent (dried substance). impurity B CRS in 2 ml of acetonitrile R and dilute to 5 ml
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : soluble in water and in anhydrous ethanol.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
4102 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 30.0 mg in 0.01 M hydrochloric
acid and dilute to 100.0 ml with the same acid.
Spectral range : 245-350 nm.
Absorption maxima : at 272 nm and 279 nm.
Point of inflexion : at 265 nm.
Specific absorbance at the absorption maxima :
— at 272 nm : about 18 ;
— at 279 nm : about 16.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : clonidine hydrochloride CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 5 mg of the substance to be
examined in methanol R and dilute to 5 ml with the same
solvent.
Reference solution. Dissolve 5 mg of clonidine
hydrochloride CRS in methanol R and dilute to 5 ml
with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : glacial acetic acid R, butanol R, water R
(10:40:50 V/V/V) ; allow to separate, filter the upper layer
and use the filtrate.
Application : 10 μl.
Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with potassium iodobismuthate
solution R2. Allow 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.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
D. It gives reaction (a) of chlorides (2.3.1).
Solution S. Dissolve 1.25 g in carbon dioxide-free water R
and dilute to 25 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2,
Method II).
pH (2.2.3) : 4.0 to 5.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be
examined in mobile phase A and dilute to 50 ml with mobile
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
Reference solution (b). Dissolve 5 mg of clonidine
with mobile phase A. To 1 ml of this solution, add 1 ml of the
test solution and dilute to 10 ml with mobile phase A.
Column :
— size: l = 0.15 m, Ø = 3.0 mm ;
— stationary phase : propylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 40 °C.
EUROPEAN PHARMACOPOEIA 6.3 Codergocrine mesilate

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 R1
(25:75 V/V) ; B. 1-acetyl-2-[(2,6-dichlorophenyl)amino]-4,5-dihydro-1H-
Time Mobile phase A Mobile phase B
imidazole,
(min) (per cent V/V) (per cent V/V)
0 90 10
0 - 15 90 → 30 10 → 70
15 - 15.1 30 → 90 70 → 10
15.1 - 20 90 10
C. 2,6-dichloroaniline.
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 210 nm.
01/2008:2060
Injection : 5 μl.
corrected 6.3
System suitability : reference solution (b) :
— resolution : minimum 5 between the peaks due to CODERGOCRINE MESILATE
clonidine and impurity B.
Limits : Codergocrini mesilas
— 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 : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
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).
1 ml of 0.1 M sodium hydroxide is equivalent to 26.66 mg
of C9H10Cl3N3.

IMPURITIES
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 DEFINITION
pharmaceutical use (2034). It is therefore not necessary to A mixture of :
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for — (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2,5-
pharmaceutical use) : A, B, C. bis(1-methylethyl)-3,6-dioxooctahydro-8H-oxazolo[3,2-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,
10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide
methanesulphonate (dihydroergocornine mesilate) ;
— (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8H-
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,
6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide methanesulphonate (dihydroergocristine
A. 1-acetylimidazolidin-2-one, mesilate) ;

General Notices (1) apply to all monographs and other texts 4103
Codergocrine mesilate
— (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-
(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide methanesulphonate (α-dihydroergocryptine
mesilate) ;
— (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-
methylethyl)-5-[(1RS)-1-methylpropyl]-3,6-dioxooctahydro-
8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide methanesulphonate (β-dihydroergocryptine
mesilate or epicriptine mesilate).
Content : 98.0 per cent to 102.0 per cent (dried substance).
PRODUCTION
The production method must be evaluated to determine
the potential for formation of alkyl mesilates, which is
particularly likely to occur if the reaction medium contains
lower alcohols. Where necessary, the production method
is validated to demonstrate that alkyl mesilates are not
detectable in the final product.
CHARACTERS
Appearance : white or yellowish powder.
Solubility : sparingly soluble in water, sparingly soluble to
soluble in ethanol (96 per cent), slightly soluble in methylene
chloride.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.20 g of the substance to be
examined in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5 ml
with the same mixture of solvents.
Reference solution. Dissolve 0.20 g of methanesulphonic Perform the test as rapidly as possible and protected from
acid R in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5 ml
with the same mixture of solvents.
Plate : TLC silica gel plate R.
Mobile phase : water R, concentrated ammonia R,
butanol R, acetone R (5:10:20:65 V/V/V/V).
Application : 10 μl.
Development : over 2/3 of the plate.
Drying : in a current of cold air for not more than 1 min. chloride R and dilute to 10.0 ml with the same mixture
Detection : spray with a 1 g/l solution of bromocresol
purple R in methanol R, adjusted to a violet-red colour
with 0.05 ml of dilute ammonia R1.
Drying : in a current of hot air at 100 °C.
Results : the principal spot in the chromatogram obtained 9 volumes of methylene chloride R.
with the test solution is similar in position and colour to Reference solution (c). To 1.0 ml of reference solution (a),
the principal spot in the chromatogram obtained with
the reference solution.
B. Examine the chromatograms obtained in the test for
composition.
Results : the 4 principal peaks in the chromatogram
obtained with the test solution are similar in retention
time to the 4 principal peaks in the chromatogram
obtained with the reference solution.
TESTS
pH (2.2.3) : 4.2 to 5.2.
Dissolve 0.10 g in carbon dioxide-free water R and dilute to First development: in an unsaturated tank, over 2/3 of the
20 ml with the same solvent.
4104 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Composition. Liquid chromatography (2.2.29) : use the
normalisation procedure.
Test solution. Dissolve 20 mg of the substance to be
examined in a mixture of 1 volume of anhydrous ethanol R
and 2 volumes of a 10 g/l solution of tartaric acid R and
dilute to 10 ml with the same mixture of solvents.
Reference solution. Dissolve 20 mg of codergocrine
mesilate CRS in a mixture of 1 volume of anhydrous
ethanol R and 2 volumes of a 10 g/l solution of tartaric
acid R and dilute to 10 ml with the same mixture of solvents.
Column :
— size: l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : triethylamine R, acetonitrile R, water R
(2.5:25:75 V/V/V).
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 280 nm.
Injection : 20 μl.
Run time : 20 min.
Elution order : dihydroergocornine, α-dihydroergocryptine,
dihydroergocristine, β-dihydroergocryptine.
System suitability : test solution :
— resolution : minimum 3 between any 2 consecutive
principal peaks.
Composition :
— dihydroergocornine: 30.0 per cent to 35.0 per cent ;
— α-dihydroergocryptine : 20.0 per cent to 25.0 per cent ;
— dihydroergocristine : 30.0 per cent to 35.0 per cent ;
— β-dihydroergocryptine : 10.0 per cent to 13.0 per cent ;
— disregard limit : 1.0 per cent.
Related substances. Thin-layer chromatography (2.2.27).
direct light. Prepare the test solution last and immediately
before application on the plate.
Test solution. Dissolve 0.40 g of the substance to be
examined in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5.0 ml with
the same mixture of solvents.
Reference solution (a). Dissolve 40 mg of
dihydroergocristine mesilate CRS in a mixture of
1 volume of methanol R and 9 volumes of methylene
of solvents. Dilute 3.0 ml of the solution to 50.0 ml with
a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R.
Reference solution (b). To 2.0 ml of reference solution (a),
add 1.0 ml of a mixture of 1 volume of methanol R and
add 2.0 ml of a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R.
Reference solution (d). To 1.0 ml of reference solution (a),
add 5.0 ml of a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, methanol R, ethyl
acetate R, methylene chloride R (1:3:50:50 V/V/V/V).
Application : 10 μl.
Drying : in the dark for 2 min after the application of the
last solution.
plate.
EUROPEAN PHARMACOPOEIA 6.3
Drying : in a current of cold air for not more than 1 min.
Second development : in an unsaturated tank, over 2/3 of
the plate ; use freshly prepared mobile phase.
Drying : in a current of cold air for not more than 1 min.
Detection : spray thoroughly with dimethylaminobenzal-
dehyde solution R7 and dry in a current of hot air until
the spot in the chromatogram obtained with reference
solution (d) is clearly visible.
System suitability : test solution :
— the chromatogram shows at least 3 separated secondary
spots.
Limits :
— any impurity : any spots, apart from the principal spot,
are not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.3 per cent) ; not
more than 4 such spots are more intense than the spot in Peroxide value (2.5.5, Method B) : maximum 5.0.
the chromatogram obtained with reference solution (c)
(0.1 per cent) and 2 of these may be more intense than
the spot in the chromatogram obtained with reference
solution (b) (0.2 per cent).
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 0.500 g by drying at 120 °C under high vacuum.
ASSAY
Dissolve 0.500 g in 60 ml of pyridine R. Pass a stream of
nitrogen R over the surface of the solution and titrate with Test solution. Dissolve 190-210 mg of the substance to be
0.1 M tetrabutylammonium hydroxide, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent Apparatus : high resolution FT-NMR spectrometer operating
to 68.04 mg of codergocrine mesilate (average Mr = 680).
STORAGE
Protected from light.
01/2009:2398
COD-LIVER OIL, FARMED
Iecoris aselli oleum domestici
DEFINITION
Purified fatty oil obtained from the fresh livers of farmed used :
cod, Gadus morhua L., solid substances being removed by
cooling and filtering.
Content :
— sum of the contents of EPA and DHA (expressed as
triglycerides) : 10.0 per cent to 28.0 per cent ;
— vitamin A : 50 IU (15 μg) to 500 IU (150 μg) per gram ;
— vitamin D3 : maximum 50 IU (1.3 μg) per gram.
Authorised antioxidants in concentrations not exceeding the
levels specified by the competent authority may be added.
PRODUCTION
The fish shall only be given feed with a composition that
is in accordance with the relevant EU or other applicable
regulations.
CHARACTERS
Appearance : clear, pale yellowish liquid.
Solubility : practically insoluble in water, slightly soluble in
alcohol (96 per cent), miscible with light petroleum.
General Notices (1) apply to all monographs and other texts
Cod-liver oil, farmed
IDENTIFICATION
A. Examine the 13C NMR spectra obtained in the test for
positional distribution (β(2)-acyl) of fatty acids (see Tests).
The spectra contain peaks between 172 ppm and 173 ppm
with shifts similar to those in the spectrum shown in
Figure 2398-1.
The positional distribution (β(2)-acyl) for cervonic
(docosahexaenoic) acid (C22:6 n-3 ; DHA), timnodonic
(eicosapentaenoic) acid (C20:5 n-3 ; EPA) and moroctic
acid (C18:4 n-3) complies with the limits.
B. Linoleic acid (see Tests).
TESTS
Acid value (2.5.1) : maximum 2.0.
Anisidine value (2.5.36) : maximum 10.0.
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
determined on 2.0 g, and extracting with 3 quantities, each
of 50 ml, of peroxide-free ether R.
Stearin. Heat at least 10 ml to 60-90 °C then allow to cool
for 3 h in a bath of iced water or a thermostatically controlled
bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
filter the sample after heating. The sample remains clear.
Positional distribution (β(2)-acyl) of fatty acids. Nuclear
magnetic resonance spectrometry (2.2.33).
examined in 500 μl of deuterated chloroform R. Prepare at
least 3 samples and examine within 3 days.
at minimum 300 MHz.
Acquisition of 13C NMR spectra. The following parameters
may be used :
— sweep width : 200 ppm (− 5 ppm to 195 ppm) ;
— irradiation frequency offset : 95 ppm ;
— time domain : 64 K ;
— pulse delay : 2 s ;
— pulse program : zgig 30 (inverse gated, 30° excitation
pulse) ;
— dummy scans : 4 ;
— number of scans : 4096.
Processing and plotting. The following parameters may be
— size: 64 K (zero-filling) ;
— window multiplication : exponential ;
— Lorentzian broadening factor: 0.2 Hz.
Use the CDCl3 signal for shift referencing. The shift of the
central peak of the 1:1:1 triplet is set to 77.16 ppm.
Plot the spectral region δ 171.5-173.5 ppm. Compare the
spectrum with the spectrum shown in Figure 2398.-1. The
shift values lie within the ranges given in Table 2398.-1.
Table 2398.-1. – Shift values
Signal Shift range (ppm)
β(2) DHA 172.05 - 172.09
α(2) DHA 172.43 - 172.47
β(2) EPA 172.52 - 172.56
α(2) EPA 172.90 - 172.94
β(2) C18:4 172.56 - 172.60
α(2) C18:4 172.95 - 172.99
4105
Cod-liver oil, farmed EUROPEAN PHARMACOPOEIA 6.3

1. α(2) C18:4 2. α(2) EPA 3. β(2) C18:4 4. β(2) EPA 5. α(2) DHA 6. β(2) DHA

Figure 2398.-1. – 13C NMR spectrum carbonyl region of farmed cod-liver oil

System suitability : Linoleic acid (2.4.29) : 3.0 per cent to 11.0 per cent.
— signal-to-noise ratio : minimum 5 for the smallest relevant ASSAY
peak corresponding to α(2) C18:4 signal (in the range δ
172.95-172.99 ppm) ; EPA and DHA (2.4.29). See the chromatogram shown in
Figure 2398.-2.
— peak width at half-height: maximum 0.02 ppm for the
central CDCl3 signal (at δ 77.16 ppm). Vitamin A. Carry out the test as rapidly as possible,
avoiding exposure to actinic light and air, oxidising agents,
Calculation of positional distribution (β(2)-acyl) : use the oxidation catalysts (for example, copper and iron) and
following expression : acids.
Use method A. If method A is found not to be valid, use
method B.
METHOD A
α(2) = peak area of the corresponding α(2)-carbonyl Ultraviolet absorption spectrophotometry (2.2.25).
peak ; Test solution. To 1.00 g in a round-bottomed flask, add
β(2) = peak area of β(2)-carbonyl peak from C22:6 n-3, 3 ml of a freshly prepared 50 per cent m/m solution of
C20:5 n-3 or C18:4 n-3, respectively. potassium hydroxide R and 30 ml of anhydrous ethanol R.
Boil under reflux in a current of nitrogen R for 30 min.
Limits : Cool rapidly and add 30 ml of water R. Extract with 50 ml
The positional distribution (β(2)-acyl) is 71 per cent to 81 per of ether R. Repeat the extraction 3 times and discard the
cent for cervonic (docosahexaenoic) acid (C22:6 n-3 ; DHA), lower layer after complete separation. Wash the combined
32 per cent to 40 per cent for timnodonic (eicosapentaenoic) upper layers with 4 quantities, each of 50 ml, of water R, and
acid (C20:5 n-3 ; EPA) and 28 per cent to 38 per cent for evaporate to dryness under a gentle current of nitrogen R
moroctic acid (C18:4 n-3). at a temperature not exceeding 30 °C or in a rotary
evaporator at a temperature not exceeding 30 °C under
Composition of fatty acids (2.4.29). For identification of the reduced pressure (water ejector). Dissolve the residue in
peaks, see the chromatogram shown in Figure 2398.-2. sufficient 2-propanol R1 to give an expected concentration
The 24 largest peaks of the methyl esters account for more of vitamin A equivalent to 10-15 IU/ml.
than 90 per cent of the total area (these correspond to, in Measure the absorbances of the solution at 300 nm,
common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1, 310 nm, 325 nm and 334 nm and at the wavelength of
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, maximum absorption with a suitable spectrophotometer in
20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, 20:4 n-3, 1 cm specially matched cells, using 2-propanol R1 as the
20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3). compensation liquid.

4106 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cod-liver oil, farmed

1. C14:0 5. C16:4 n-1 9. C18:2 n-6 13. C20:1 n-9 17. C20:3 n-3 21. C22:1 n-9
2. C15:0 6. C18:0 10 C18:3 n-3 14. C20:1 n-7 18. C20:4 n-3 22. C21:5 n-3
3. C16:0 7. C18:1 n-9 11. C18:4 n-3 15. C20:2 n-6 19. C20:5 n-3 23. C22:5 n-3
4. C16:1 n-7 8. C18:1 n-7 12. C20:1 n-11 16. C20:4 n-6 20. C22:1 n-11 24. C22:6 n-3

Figure 2398.-2. – Chromatogram for the test for composition of fatty acids of farmed cod-liver oil
Calculate the content of vitamin A, as all- trans -retinol, in — the absorbance at 300 nm relative to that at 325 nm is
International Units per gram, using the following expression : at most 0.73.
METHOD B
Liquid chromatography (2.2.29).
Test solution. Prepare duplicates. To 2.00 g in a
A325 = absorbance at 325 nm ; round-bottomed flask, add 5 ml of a freshly prepared 100 g/l
m = mass of the substance to be examined, in grams ; solution of ascorbic acid R, 10 ml of a freshly prepared
= total volume of solution containing 10-15 IU of 800 g/l solution of potassium hydroxide R and 100 ml of
V anhydrous ethanol R. Boil under a reflux condenser on
vitamin A per millilitre ; a water-bath for 15 min. Add 100 ml of a 10 g/l solution
1821 = conversion factor for the specific absorbance of of sodium chloride R and cool. Transfer the solution to a
all- trans -retinol, in International Units. 500 ml separating funnel, rinsing the round-bottomed flask
The above expression can be used only if A325 has a value with about 75 ml of a 10 g/l solution of sodium chloride R
of not greater than A325,corr /0.970, where A325,corr is the and then with 150 ml of a mixture of equal volumes of light
corrected absorbance at 325 nm and is given by the following petroleum R1 and ether R. Shake for 1 min. When the
equation : layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution of
potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R and then with 3 quantities, each of
50 ml, of a 10 g/l solution of sodium chloride R. Filter the
A designates the absorbance at the wavelength indicated
upper layer through 5 g of anhydrous sodium sulphate R
by the subscript.
on a fast filter paper into a 250 ml flask suitable for a rotary
If A325 has a value greater than A325,corr /0.970, calculate the evaporator. Wash the funnel with 10 ml of fresh extraction
content of vitamin A using the following expression : mixture, filter and combine the upper layers. Distil them at
a temperature not exceeding 30 °C under reduced pressure
(water ejector) and fill with nitrogen R when evaporation
is completed. Alternatively, evaporate the solvent under a
The assay is not valid unless : gentle current of nitrogen R at a temperature not exceeding
— the wavelength of maximum absorption lies between 30 °C. Dissolve the residue in 2-propanol R, transfer to a
323 nm and 327 nm ; 25 ml volumetric flask and dilute to 25 ml with 2-propanol R.

General Notices (1) apply to all monographs and other texts 4107
Cod-liver oil, farmed
Gentle heating in an ultrasonic bath may be required. A large
fraction of the white residue is cholesterol, constituting
approximately 50 per cent m/m of the unsaponifiable
matter of cod-liver oil.
Reference solution (a). Prepare a solution of retinol
acetate CRS in 2-propanol R1 so that 1 ml contains about
1000 IU of all-trans-retinol.
The exact concentration of reference solution (a) is assessed
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R1 to a presumed
concentration of 10-15 IU/ml and measure the absorbance
at 326 nm in matched 1 cm cells using 2-propanol R1 as the
compensation liquid.
Calculate the content of vitamin A in International Units
per millilitre of reference solution (a) using the following
expression, taking into account the assigned content of
retinol acetate CRS :
A326 = absorbance at 326 nm ;
V1 = volume of reference solution (a) used ;
V2 = volume of the diluted solution ;
1900 = conversion factor for the specific absorbance of
retinol acetate CRS, in International Units.
Reference solution (b). Proceed as described for the test
solution but using 2.00 ml of reference solution (a) in place
of the substance to be examined.
The exact concentration of reference solution (b) is assessed 10 ml of a freshly prepared 800 g/l solution of potassium
by ultraviolet absorption spectrophotometry (2.2.25). Dilute hydroxide R and 100 ml of anhydrous ethanol R. Boil
reference solution (b) with 2-propanol R1 to a presumed
all-trans-retinol concentration of 10-15 IU/ml and measure
the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International
Units per millilitre of reference solution (b), using the
following expression :
A325 = absorbance at 325 nm ;
V3 = volume of the diluted solution ;
V4 = volume of reference solution (b) used ;
1821 = conversion factor for the specific absorbance of
all-trans-retinol, in International Units.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm).
Mobile phase : water R, methanol R (3:97 V/V).
Flow rate : 1 ml/min.
Detection : spectrophotometer at 325 nm.
Injection : 10 μl ; inject in triplicate the test solution and
reference solution (b).
Retention time : all-trans-retinol = 5 ± 1 min.
System suitability :
— the chromatogram obtained with the test solution shows a Reference solution (b). In a round-bottomed flask, add 2.0 ml
peak that corresponds to the peak due to all-trans-retinol of reference solution (a) and 2.0 ml of the internal standard
in the chromatogram obtained with reference solution (b) ; solution and proceed as described for test solution (a).
4108 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
— the results obtained with the duplicate test solutions do
not differ by more than 5 per cent ;
— the recovery of all-trans-retinol in reference solution (b)
as assessed by direct absorption spectrophotometry is
greater than 95 per cent.
Calculate the content of vitamin A using the following
expression :
A1 = area of the peak due to all-trans-retinol in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to all-trans-retinol in
the chromatogram obtained with reference
solution (b) ;
C = concentration of retinol acetate CRS in
reference solution (a) as assessed prior to the
saponification, in International Units per millilitre
(= 1000 IU/ml) ;
V = volume of reference solution (a) treated (2.00 ml) ;
m = mass of the substance to be examined in the test
solution (2.00 g).
Vitamin D3. Liquid chromatography (2.2.29). Carry out the
assay as rapidly as possible, avoiding exposure to actinic
light and air.
Internal standard solution. Dissolve 0.50 mg of
ergocalciferol CRS in 100 ml of anhydrous ethanol R.
Test solution (a). To 4.00 g in a round-bottomed flask, add
5 ml of a freshly prepared 100 g/l solution of ascorbic acid R,
under a reflux condenser on a water-bath for 30 min. Add
100 ml of a 10 g/l solution of sodium chloride R and cool
the solution to room temperature. Transfer the solution to a
500 ml separating funnel, rinsing the round-bottomed flask
with about 75 ml of a 10 g/l solution of sodium chloride R
and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution of
potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R , and then with 3 quantities, each of
50 ml, of a 10 g/l solution of sodium chloride R. Filter the
upper layer through 5 g of anhydrous sodium sulphate R
on a fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction
mixture, filter and combine the upper layers. Distil them at
a temperature not exceeding 30 °C under reduced pressure
(water ejector) and fill with nitrogen R when evaporation
is completed. Alternatively, evaporate the solvent under a
gentle current of nitrogen R at a temperature not exceeding
30 °C. Dissolve the residue in 1.5 ml of the mobile phase
described under Purification. Gentle heating in an ultrasonic
bath may be required. A large fraction of the white residue
is cholesterol, constituting approximately 50 per cent m/m
of the unsaponifiable matter of cod-liver oil.
Test solution (b). Prepare duplicates. To 4.00 g add 2.0 ml of
the internal standard solution and proceed as described for
test solution (a).
Reference solution (a). Dissolve 0.50 mg of
cholecalciferol CRS in 100.0 ml of anhydrous
ethanol R.
EUROPEAN PHARMACOPOEIA 6.3
PURIFICATION
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : nitrile silica gel for chromatography R
(10 μm).
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
Flow rate : 1.1 ml/min.
Detection : spectrophotometer at 265 nm.
Inject 350 μl of reference solution (b). Collect the eluate
from 2 min before until 2 min after the retention time of
cholecalciferol, in a ground-glass-stoppered tube containing
1 ml of a 1 g/l solution of butylhydroxytoluene R in
hexane R. Repeat the procedure with test solutions (a)
and (b). Evaporate the eluates obtained from reference
solution (b) and from test solutions (a) and (b), separately,
to dryness at a temperature not exceeding 30 °C under a
gentle current of nitrogen R. Dissolve each residue in 1.5 ml
of acetonitrile R.
DETERMINATION
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : phosphoric acid R, 96 per cent V/V solution
of acetonitrile R (0.2:99.8 V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 265 nm.
Injection : 2 quantities not exceeding 200 μl of each of the
3 solutions obtained under Purification.
System suitability :
— resolution : minimum 1.4 between the peaks due to
ergocalciferol and cholecalciferol in the chromatogram
obtained with reference solution (b) ;
— the results obtained with the test solution (b) duplicates
do not differ by more than 5 per cent.
Calculate the content of vitamin D3 in International Units
per gram using the following expression, taking into account
the assigned content of cholecalciferol CRS :
m1
= mass of the sample in test solution (b), in grams ;
m2 = total mass of cholecalciferol CRS used for
the preparation of reference solution (a), in
micrograms (500 μg) ;
A1 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (a) ;
A2
= area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (b) ;
A3 = area (or height) of the peak due to ergocalciferol
in the chromatogram obtained with reference
solution (b) ;
A4
= area (or height) of the peak due to ergocalciferol in
the chromatogram obtained with test solution (b) ;
A5 = area (or height) of a possible peak in the
chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting
with ergocalciferol in test solution (b) ;
General Notices (1) apply to all monographs and other texts
Cod-liver oil (type A)
A6 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (b) ;
V1 total volume of reference solution (a) (100 ml) ;
=
V2 = volume of reference solution (a) used for preparing
reference solution (b) (2.0 ml).
STORAGE
In an airtight and well-filled container, protected from light.
If no antioxidant is added, store under an inert gas.
Once the container has been opened, its contents are used
as soon as possible and any part of the contents not used at
once is protected by an atmosphere of inert gas.
LABELLING
The label states :
— the concentration of EPA and DHA as a sum ;
— the number of International Units of vitamin A per gram ;
— the number of International Units of vitamin D3 per gram.
01/2009:1192
COD-LIVER OIL (TYPE A)
Iecoris aselli oleum A
DEFINITION
Purified fatty oil obtained from the fresh livers of wild
cod, Gadus morhua L. and other species of Gadidae, solid
substances being removed by cooling and filtering. A
suitable antioxidant may be added.
Content : 600 IU (180 μg) to 2500 IU (750 μg) of vitamin A
per gram and 60 IU (1.5 μg) to 250 IU (6.25 μg) of vitamin D3
per gram.
CHARACTERS
Appearance : clear, yellowish, liquid.
Solubility : practically insoluble in water, slightly soluble in
ethanol (96 per cent), miscible with light petroleum.
IDENTIFICATION
First identification : A, B, C.
Second identification : C, D.
A. In the assay for vitamin A using method A, the test
solution shows an absorption maximum (2.2.25) at
325 ± 2 nm. In the assay for vitamin A using method B,
the chromatogram obtained with the test solution shows
a peak corresponding to the peak of all-trans-retinol in
the chromatogram obtained with the reference solution.
B. In the assay for vitamin D3, the chromatogram obtained
with test solution (a) shows a peak corresponding to the
peak of cholecalciferol in the chromatogram obtained
with reference solution (b).
C. Composition of fatty acids (see Tests).
D. To 0.1 g add 0.5 ml of methylene chloride R and 1 ml of
antimony trichloride solution R. Mix. A deep blue colour
develops in about 10 s.
TESTS
Colour : not more intensely coloured than a reference
solution prepared as follows : to 3.0 ml of red primary
solution add 25.0 ml of yellow primary solution and dilute
to 50.0 ml with a 10 g/l solution of hydrochloric acid R
(2.2.2, Method II).
Relative density (2.2.5) : 0.917 to 0.930.
Refractive index (2.2.6) : 1.477 to 1.484.
4109
Cod-liver oil (type A) EUROPEAN PHARMACOPOEIA 6.3

Acid value (2.5.1) : maximum 2.0. — stationary phase : macrogol 20 000 R (film thickness
Anisidine value (2.5.36) : maximum 30.0. 0.25 μm).
Carrier gas : hydrogen for chromatography R or helium for
Iodine value (2.5.4, Method B) : 150 to 180. chromatography R, where oxygen scrubber is applied.
Use starch solution R2. Split ratio : 1:200.
Peroxide value (2.5.5, Method B) : maximum 10.0. Temperature :
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent, Time Temperature
determined on 2.0 g, and extracting with 3 quantities, each (min) (°C)
of 50 ml, of peroxide-free ether R. Column 0 - 55 170 → 225
Stearin. Heat at least 10 ml to 60-90 °C then allow to cool 55 - 75 225
for 3 h in a bath of iced water or a thermostatically-controlled
Injection port 250
bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
filter the sample after heating. The sample remains clear. Detector 280
Composition of fatty acids. Gas chromatography (2.2.28). Detection : flame ionisation.
Trivial name of Nomencla- Lower limit Upper limit Injection : 1 μl, twice.
fatty acid ture area area System suitability :
(per cent) (per cent)
Saturated fatty acids :
— the 15 fatty acids to be tested are satisfactorily identified
from the chromatogram shown in Figure 1192.-1 ;
Myristic acid 14:0 2.0 6.0 — injection of a mixture of equal amounts of methyl
Palmitic acid 16:0 7.0 14.0 palmitate R, methyl stearate R, methyl arachidate R and
Stearic acid 18:0 1.0 4.0 methyl behenate R gives area percentages of 24.4, 24.8,
Mono-unsaturated fatty acids : 25.2 and 25.6 (± 0.5 per cent), respectively ;
Palmitoleic acid 16:1 n-7 4.5 11.5 — resolution : minimum 1.3 between the peaks due to
cis-Vaccenic acid 18:1 n-7 2.0 7.0 methyl oleate and methyl cis-vaccenate ; the resolution
Oleic acid 18:1 n-9 12.0 21.0 between the pair due to methyl gadoleate and methyl
Gadoleic acid 20:1 n-11 1.0 5.5 gondoate is sufficient for purposes of identification and
Gondoic acid 20:1 n-9 5.0 17.0 area measurement.
Erucic acid 22:1 n-9 0 1.5 Calculate the area per cent for each fatty acid methyl ester
Cetoleic acid 22:1 n-11+13 5.0 12.0 using the following expression :
(22:1 n-11)
Poly-unsaturated fatty acids :
Linoleic acid 18:2 n-6 0.5 3.0
α-Linolenic acid 18:3 n-3 0 2.0
Ax = peak area of fatty acid x ;
Moroctic acid 18:4 n-3 0.5 4.5
Timnodonic 20:5 n-3 7.0 16.0 At = sum of the peak areas (up to C22:6 n-3).
(eicosapentaenoic)
acid (EPA) The calculation is not valid unless :
Cervonic 22:6 n-3 6.0 18.0 — the total area is based only on peaks due solely to fatty
(docosahexaenoic) acid methyl esters ;
acid (DHA)
— the number of fatty acid methyl ester peaks exceeding
Test solution. Introduce about 0.45 g of the substance to be 0.05 per cent of the total area is at least 24 ;
examined into a 10 ml volumetric flask, dissolve in hexane R — the 24 largest peaks of the methyl esters account for more
containing 50 mg of butylhydroxytoluene R per litre and than 90 per cent of the total area. (These correspond
dilute to 10.0 ml with the same solvent. Transfer 2.0 ml of to, in common elution order : 14:0, 15:0, 16:0, 16:1 n-7,
this solution into a quartz tube and evaporate the solvent 16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3,
with a gentle current of nitrogen R. Add 1.5 ml of a 20 g/l 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6,
solution of sodium hydroxide R in methanol R, cover with 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3,
nitrogen R, cap tightly with a polytetrafluoroethylene-lined 22:5 n-3, 22:6 n-3).
cap, mix and heat in a water-bath for 7 min. Cool, add
2 ml of boron trichloride-methanol solution R, cover with ASSAY
nitrogen R, cap tightly, mix and heat in a water-bath for Vitamin A. Carry out the test as rapidly as possible,
30 min. Cool to 40-50 °C, add 1 ml of trimethylpentane R, avoiding exposure to actinic light and air, oxidising agents,
cap and vortex or shake vigorously for at least 30 s. oxidation catalysts (for example, copper and iron) and
Immediately add 5 ml of saturated sodium chloride acids.
solution R, cover with nitrogen R, cap and vortex or shake Use method A. If method A is found not to be valid, use
vigorously for at least 15 s. Allow the upper layer to become method B.
clear and transfer it to a separate tube. Shake the methanol
layer once more with 1 ml of trimethylpentane R and METHOD A
combine the trimethylpentane extracts. Wash the combined Ultraviolet absorption spectrophotometry (2.2.25).
extracts with 2 quantities, each of 1 ml, of water R and dry Test solution. To 1.00 g in a round-bottomed flask, add
over anhydrous sodium sulphate R. Prepare 2 solutions for 3 ml of a freshly prepared 50 per cent m/m solution of
each sample. potassium hydroxide R and 30 ml of anhydrous ethanol R.
Column: Boil under reflux in a current of nitrogen R for 30 min.
Cool rapidly and add 30 ml of water R. Extract with 50 ml
— material: fused silica ; of ether R. Repeat the extraction 3 times and discard the
— size : l = 30 m, Ø = 0.25 mm ; lower layer after complete separation. Wash the combined

4110 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Cod-liver oil (type A)

Figure 1192.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type A)

upper layers with 4 quantities, each of 50 ml, of water R, and — the absorbance at 300 nm relative to that at 325 nm is
evaporate to dryness under a gentle current of nitrogen R at most 0.73.
at a temperature not exceeding 30 °C or in a rotary METHOD B
evaporator at a temperature not exceeding 30 °C under
Liquid chromatography (2.2.29).
reduced pressure (water ejector). Dissolve the residue in
sufficient 2-propanol R1 to give an expected concentration Test solution. Prepare duplicates. To 2.00 g in a
of vitamin A equivalent to 10-15 IU/ml. round-bottomed flask, add 5 ml of a freshly prepared 100 g/l
solution of ascorbic acid R, 10 ml of a freshly prepared
Measure the absorbances of the solution at 300 nm, 800 g/l solution of potassium hydroxide R and 100 ml of
310 nm, 325 nm and 334 nm and at the wavelength of anhydrous ethanol R. Boil under a reflux condenser on
maximum absorption with a suitable spectrophotometer in a water-bath for 15 min. Add 100 ml of a 10 g/l solution
1 cm specially matched cells, using 2-propanol R1 as the of sodium chloride R and cool. Transfer the solution to a
compensation liquid. 500 ml separating funnel, rinsing the round-bottomed flask
Calculate the content of vitamin A, as all-trans-retinol, in with about 75 ml of a 10 g/l solution of sodium chloride R
International Units per gram, using the following expression : and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution
of potassium hydroxide R in a 10 per cent V/V solution of
A325 = absorbance at 325 nm ; anhydrous ethanol R and then with 3 quantities, each of
m = mass of the substance to be examined, in grams ; 50 ml, of a 10 g/l solution of sodium chloride R. Filter the
upper layer through 5 g of anhydrous sodium sulphate R
V = total volume of solution containing 10-15 IU of on a fast filter paper into a 250 ml flask suitable for a rotary
vitamin A per millilitre ; evaporator. Wash the funnel with 10 ml of fresh extraction
1821 = conversion factor for the specific absorbance of mixture, filter and combine the upper layers. Distil them at
all-trans-retinol, in International Units. a temperature not exceeding 30 °C under reduced pressure
The above expression can be used only if A325 has a value of (water ejector) and fill with nitrogen R when evaporation
not greater than A325,corr/0.970, where A325,corr is the corrected is completed. Alternatively, evaporate the solvent under a
absorbance at 325 nm and is given by the following equation : gentle current of nitrogen R at a temperature not exceeding
30 °C. Dissolve the residue in 2-propanol R, transfer to a
25 ml volumetric flask and dilute to 25 ml with 2-propanol R.
Gentle heating in an ultrasonic bath may be required. A large
fraction of the white residue is cholesterol, constituting
A designates the absorbance at the wavelength indicated approximately 50 per cent m/m of the unsaponifiable
by the subscript. matter of cod-liver oil.
If A325 has a value greater than A325,corr/0.970, calculate the Reference solution (a). Prepare a solution of retinol
content of vitamin A using the following expression : acetate CRS in 2-propanol R1 so that 1 ml contains about
1000 IU of all-trans-retinol.
The exact concentration of reference solution (a) is assessed
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R1 to a presumed
The assay is not valid unless : concentration of 10-15 IU/ml and measure the absorbance
— the wavelength of the maximum absorption lies between at 326 nm in matched 1 cm cells using 2-propanol R1 as the
323 nm and 327 nm ; compensation liquid.

General Notices (1) apply to all monographs and other texts 4111
Cod-liver oil (type A)
Calculate the content of vitamin A in International Units
per millilitre of reference solution (a) using the following
expression, taking into account the assigned content of
retinol acetate CRS :
A326 = absorbance at 326 nm ;
V1 = volume of reference solution (a) used ;
V2 = volume of the diluted solution ;
1900 = conversion factor for the specific absorbance of
retinol acetate CRS, in International Units.
Reference solution (b). Proceed as described for the test
solution but using 2.00 ml of reference solution (a) in place light and air.
of the substance to be examined.
The exact concentration of reference solution (b) is assessed
by ultraviolet absorption spectrophotometry (2.2.25). Dilute Test solution (a). To 4.00 g in a round-bottomed flask, add
reference solution (b) with 2-propanol R1 to a presumed
all-trans-retinol concentration of 10-15 IU/ml and measure
the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International
Units per millilitre of reference solution (b), using the
following expression :
A325 = absorbance at 325 nm ;
V3 = volume of the diluted solution ;
V4 = volume of reference solution (b) used ;
1821 = conversion factor for the specific absorbance of
all-trans-retinol, in International Units.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm).
Mobile phase : water R, methanol R (3:97 V/V).
Flow rate : 1 ml/min.
Detection : spectrophotometer at 325 nm.
Injection : 10 μl ; inject in triplicate the test solution and the internal standard solution and proceed as described for
reference solution (b).
Retention time : all-trans-retinol = 5 ± 1 min.
System suitability :
— the chromatogram obtained with the test solution shows a Reference solution (b). Into a round-bottomed flask,
peak that corresponds to the peak due to all-trans-retinol add 2.0 ml of reference solution (a) and 2.0 ml of the
in the chromatogram obtained with reference solution (b) ; internal standard solution and proceed as described for test
— the results obtained with the duplicate test solutions do
not differ by more than 5 per cent ;
— the recovery of all-trans-retinol in reference solution (b)
as assessed by direct absorption spectrophotometry is
greater than 95 per cent.
Calculate the content of vitamin A using the following
expression :
4112 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
A1 = area of the peak due to all-trans-retinol in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to all-trans-retinol in
the chromatogram obtained with reference
solution (b) ;
C = concentration of retinol acetate CRS in
reference solution (a) as assessed prior to the
saponification, in International Units per millilitre
(= 1000 IU/ml) ;
V = volume of reference solution (a) treated (2.00 ml) ;
m = mass of the substance to be examined in the test
solution (2.00 g).
Vitamin D3. Liquid chromatography (2.2.29). Carry out the
assay as rapidly as possible, avoiding exposure to actinic
Internal standard solution. Dissolve 0.50 mg of
ergocalciferol CRS in 100 ml of anhydrous ethanol R.
5 ml of a freshly prepared 100 g/l solution of ascorbic acid R,
10 ml of a freshly prepared 800 g/l solution of potassium
hydroxide R and 100 ml of anhydrous ethanol R. Boil
under a reflux condenser on a water-bath for 30 min. Add
100 ml of a 10 g/l solution of sodium chloride R and cool
the solution to room temperature. Transfer the solution to a
500 ml separating funnel, rinsing the round-bottomed flask
with about 75 ml of a 10 g/l solution of sodium chloride R
and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution
of potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R, and then with 3 quantities, each of
50 ml, of a 10 g/l solution of sodium chloride R. Filter the
upper layer through 5 g of anhydrous sodium sulphate R
on a fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction
mixture, filter and combine the upper layers. Distil them at
a temperature not exceeding 30 °C under reduced pressure
(water ejector) and fill with nitrogen R when evaporation
is completed. Alternatively, evaporate the solvent under a
gentle current of nitrogen R at a temperature not exceeding
30 °C. Dissolve the residue in 1.5 ml of the mobile phase
described under Purification. Gentle heating in an ultrasonic
bath may be required. A large fraction of the white residue
is cholesterol, constituting approximately 50 per cent m/m
of the unsaponifiable matter of cod-liver oil.
Test solution (b). Prepare duplicates. To 4.00 g add 2.0 ml of
test solution (a).
Reference solution (a). Dissolve 0.50 mg of
cholecalciferol CRS in 100.0 ml of anhydrous
ethanol R.
solution (a).
PURIFICATION
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : nitrile silica gel for chromatography R
(10 μm).
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
Flow rate : 1.1 ml/min.
Detection : spectrophotometer at 265 nm.
EUROPEAN PHARMACOPOEIA 6.3
Inject 350 μl of reference solution (b). Collect the eluate
from 2 min before until 2 min after the retention time of
cholecalciferol, in a ground-glass-stoppered tube containing
1 ml of a 1 g/l solution of butylhydroxytoluene R in
hexane R. Repeat the procedure with test solutions (a)
and (b). Evaporate the eluates obtained from reference
solution (b) and from test solutions (a) and (b), separately,
to dryness at a temperature not exceeding 30 °C under a
gentle current of nitrogen R. Dissolve each residue in 1.5 ml
of acetonitrile R.
DETERMINATION
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : phosphoric acid R, 96 per cent V/V solution
of acetonitrile R (0.2:99.8 V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 265 nm.
Injection : 2 quantities not exceeding 200 μl of each of the
3 solutions obtained under Purification.
System suitability :
— resolution : minimum 1.4 between the peaks due to
ergocalciferol and cholecalciferol in the chromatogram
obtained with reference solution (b) ;
— the results obtained with test solution (b) duplicates do
not differ by more than 5 per cent.
Calculate the content of vitamin D3 in International Units
per gram using the following expression, taking into account
the assigned content of cholecalciferol CRS :
m1 = mass of the sample in test solution (b), in grams ;
m2 = total mass of cholecalciferol CRS used for
the preparation of reference solution (a), in
micrograms (500 μg) ;
A1 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (a) ;
A2 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (b) ;
A3 = area (or height) of the peak due to ergocalciferol
in the chromatogram obtained with reference
solution (b) ;
A4 = area (or height) of the peak due to ergocalciferol in
the chromatogram obtained with test solution (b) ;
A5 = area (or height) of a possible peak in the
chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting
with ergocalciferol in test solution (b) ;
A6 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (b) ;
V1
= total volume of reference solution (a) (100 ml) ;
V2 = volume of reference solution (a) used for preparing
reference solution (b) (2.0 ml).
STORAGE
In an airtight and well-filled container, protected from light.
If no antioxidant is added, store under an inert gas.
General Notices (1) apply to all monographs and other texts
Cod-liver oil (type B)
Once the container has been opened, its contents are used
as soon as possible and any part of the contents not used at
once is protected by an atmosphere of inert gas.
LABELLING
The label states :
— the number of International Units of vitamin A per gram ;
— the number of International Units of vitamin D3 per gram.
01/2009:1193
COD-LIVER OIL (TYPE B)
Iecoris aselli oleum B
DEFINITION
Purified fatty oil obtained from the fresh livers of wild
cod, Gadus morhua L. and other species of Gadidae, solid
substances being removed by cooling and filtering. A
suitable antioxidant may be added.
Content : 600 IU (180 μg) to 2500 IU (750 μg) of vitamin A
per gram and 60 IU (1.5 μg) to 250 IU (6.25 μg) of vitamin D3
per gram.
CHARACTERS
Appearance : clear, yellowish liquid.
Solubility : practically insoluble in water, slightly soluble in
alcohol, miscible with light petroleum.
IDENTIFICATION
First identification : A, B, C.
Second identification : C, D.
A. In the assay for vitamin A using method A, the test
solution shows an absorption maximum (2.2.25) at
325 ± 2 nm. In the assay for vitamin A using method B,
the chromatogram obtained with the test solution shows
a peak corresponding to the peak of all-trans-retinol in
the chromatogram obtained with the reference solution.
B. In the assay for vitamin D3, the chromatogram obtained
with test solution (a) shows a peak corresponding to the
peak of cholecalciferol in the chromatogram obtained
with reference solution (b).
C. It complies with the test for composition of fatty acids
(see Tests).
D. To 0.1 g add 0.5 ml of methylene chloride R and 1 ml of
antimony trichloride solution R. Mix. A deep blue colour
develops in about 10 s.
TESTS
Colour : not more intensely coloured than a reference
solution prepared as follows : to 3.0 ml of red primary
solution add 25.0 ml of yellow primary solution and dilute
to 50.0 ml with a 10 g/l solution of hydrochloric acid R
(2.2.2, Method II).
Relative density (2.2.5) : 0.917 to 0.930.
Refractive index (2.2.6) : 1.477 to 1.484.
Acid value (2.5.1) : maximum 2.0.
Iodine value (2.5.4, Method B) : 150 to 180.
Use starch solution R2.
Peroxide value (2.5.5, Method B) : maximum 10.0.
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
determined on 2.0 g and extracting with 3 quantities, each
of 50 ml, of peroxide-free ether R.
4113
Cod-liver oil (type B) EUROPEAN PHARMACOPOEIA 6.3

Stearin. Heat at least 10 ml to 60-90 °C then allow to cool Temperature :

for 3 h in a bath of iced water or a thermostatically-controlled
Time Temperature
bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
(min) (°C)
filter the sample after heating. The sample remains clear.
Column 0 - 55 170 → 225
Composition of fatty acids. Gas chromatography (2.2.28).
55 - 75 225

Trivial name Nomencla- Lower limit Upper limit Injection port 250
of fatty acid ture area area
Detector 280
(per cent) (per cent)
Saturated fatty acids : Detection : flame ionisation.
Myristic acid 14:0 2.0 6.0 Injection : 1 μl, twice.
Palmitic acid 16:0 7.0 14.0
1.0 4.0
System suitability :
Stearic acid 18:0
Mono-unsaturated fatty acids : — the 15 fatty acids to be tested are satisfactorily identified
from the chromatogram shown in Figure 1193.-1 ;
Palmitoleic acid 16:1 n-7 4.5 11.5
cis-Vaccenic acid 18:1 n-7 2.0 7.0
— injection of a mixture of equal amounts of methyl
Oleic acid 18:1 n-9 12.0 21.0
palmitate R, methyl stearate R, methyl arachidate R, and
Gadoleic acid 20:1 n-11 1.0 5.5
methyl behenate R give area percentages of 24.4, 24.8,
20:1 n-9 5.0 17.0
25.2 and 25.6 (± 0.5 per cent), respectively ;
Gondoic acid
Erucic acid 22:1 n-9 0 1.5 — resolution : minimum of 1.3 between the peaks due to
Cetoleic acid 22:1 n-11+13 5.0 12.0 methyl oleate and methyl cis-vaccenate ; the resolution
(22:1 n-11) between the pair due to methyl gadoleate and methyl
Poly-unsaturated fatty acids : gondoate is sufficient for purposes of identification and
area measurement.
Linoleic acid 18:2 n-6 0.5 3.0
α-Linolenic acid 18:3 n-3 0 2.0 Calculate the area per cent for each fatty acid methyl ester
Moroctic acid 18:4 n-3 0.5 4.5 from the expression :
Timnodonic 20:5 n-3 7.0 16.0
(eicosapentaenoic)
acid (EPA)
Cervonic 22:6 n-3 6.0 18.0
(docosahexaenoic) Ax = peak area of fatty acid x ;
acid (DHA)
At = sum of the peak areas (up to C22:6 n-3).
Test solution. Introduce about 0.45 g of the substance The calculation is not valid unless :
to be examined into a 10 ml volumetric flask, dissolve in
hexane R containing 50 mg of butylhydroxytoluene R per — the total area is based only on peaks due to solely fatty
litre and dilute to 10.0 ml with the same solvent. Transfer acids methyl esters ;
2.0 ml of the solution into a quartz tube and evaporate the — the number of fatty acid methyl ester peaks exceeding
solvent with a gentle current of nitrogen R. Add 1.5 ml of a 0.05 per cent of the total area is at least 24 ;
20 g/l solution of sodium hydroxide R in methanol R, cover — the 24 largest peaks of the methyl esters account for more
with nitrogen R, cap tightly with a polytetrafluoroethylene than 90 per cent of the total area. (These correspond
lined cap, mix and heat in a water-bath for 7 min. Cool, add to, in common elution order : 14:0, 15:0, 16:0, 16:1 n-7,
2 ml of boron trichloride-methanol solution R, cover with 16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3,
nitrogen R, cap tightly, mix and heat in a water-bath for 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6,
30 min. Cool to 40-50 °C, add 1 ml of trimethylpentane R, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3,
cap and vortex or shake vigorously for at least 30 s. 22:5 n-3, 22:6 n-3).
Immediately add 5 ml of saturated sodium chloride
solution R, cover with nitrogen R, cap and vortex or shake
ASSAY
thoroughly for at least 15 s. Allow the upper layer to become
Vitamin A. Carry out the test as rapidly as possible,
clear and transfer to a separate tube. Shake the methanolavoiding exposure to actinic light and air, oxidising agents,
layer once more with 1 ml of trimethylpentane R and oxidation catalysts (for example, copper and iron) and
combine the trimethylpentane extracts. Wash the combined acids.
extracts with 2 quantities, each of 1 ml, of water R and dry
over anhydrous sodium sulphate R. Prepare 2 solutions forUse method A. If method A is found not to be valid, use
each sample. method B.
METHOD A
Column: Ultraviolet absorption spectrophotometry (2.2.25).
— material: fused silica ; Test solution. To 1.00 g in a round-bottomed flask, add
3 ml of a freshly prepared 50 per cent m/m solution of
— size : l = 30 m, Ø = 0.25 mm ; potassium hydroxide R and 30 ml of ethanol R. Boil under
reflux in a current of nitrogen R for 30 min. Cool rapidly
— stationary phase : macrogol 20 000 R (film thickness and add 30 ml of water R. Extract with 50 ml of ether R.
0.25 μm). Repeat the extraction 3 times and discard the lower layer
after complete separation. Wash the combined upper
Carrier gas: hydrogen for chromatography R or helium for layers with 4 quantities, each of 50 ml, of water R and
chromatography R, where oxygen scrubber is applied. evaporate to dryness under a gentle current of nitrogen R
at a temperature not exceeding 30 °C or in a rotary
Split ratio : 1:200. evaporator at a temperature not exceeding 30 °C under

4114 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Figure 1193.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type B)
reduced pressure (water ejector). Dissolve the residue in
sufficient 2-propanol R1 to give an expected concentration
of vitamin A equivalent to 10-15 IU/ml.
Measure the absorbances of the solution at 300 nm,
310 nm, 325 nm and 334 nm and at the wavelength of
maximum absorption with a suitable spectrophotometer in
1 cm specially matched cells, using 2-propanol R1 as the
compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in
International Units per gram from the expression :
A325 = absorbance at 325 nm ;
m = mass of the substance to be examined, in grams ;
V = total volume of solution containing 10-15 IU of
vitamin A per millilitre ;
1821 = conversion factor for the specific absorbance of
all-trans-retinol, in International Units.
The above expression can be used only if A325 has a value of and combine the upper layers. Distil them at a temperature
not greater than A325,corr/0.970 where A325,corr is the corrected not exceeding 30 °C under reduced pressure (water ejector)
absorbance at 325 nm and is given by the equation :
A designates the absorbance at the wavelength indicated
by the subscript.
If A325 has a value greater than A325,corr/0.970, calculate the
content of vitamin A from the expression :
The assay is not valid unless :
— the wavelength of maximum absorption lies between
323 nm and 327 nm ;
— the absorbance at 300 nm relative to that at 325 nm is
at most 0.73.
General Notices (1) apply to all monographs and other texts
Cod-liver oil (type B)
METHOD B
Liquid chromatography (2.2.29).
Test solution. Prepare duplicates. To 2.00 g in a
round-bottomed flask, add 5 ml of a freshly prepared 100 g/l
solution of ascorbic acid R and 10 ml of a freshly prepared
800 g/l solution of potassium hydroxide R and 100 ml of
ethanol R. Boil under a reflux condenser on a water-bath
for 15 min. Add 100 ml of a 10 g/l solution of sodium
chloride R and cool. Transfer the solution to a 500 ml
separating funnel rinsing the round-bottomed flask with
about 75 ml of a 10 g/l solution of sodium chloride R and
then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution
of potassium hydroxide R in a 10 per cent V/V solution of
ethanol R and then with 3 quantities, each of 50 ml, of a
10 g/l solution of sodium chloride R. Filter the upper layer
through 5 g of anhydrous sodium sulphate R on a fast filter
paper into a 250 ml flask suitable for a rotary evaporator.
Wash the funnel with 10 ml of fresh extraction mixture, filter
and fill with nitrogen R when evaporation is completed.
Alternatively evaporate the solvent under a gentle current of
nitrogen R at a temperature not exceeding 30 °C. Dissolve
the residue in 2-propanol R, transfer to a 25 ml volumetric
flask and dilute to 25 ml with 2-propanol R. Gentle heating
in an ultrasonic bath may be required. (A large fraction of
the white residue is cholesterol, constituting approximately
50 per cent of the unsaponifiable matter of cod-liver oil).
Reference solution (a). Prepare a solution of retinol
acetate CRS in 2-propanol R1 so that 1 ml contains about
1000 IU of all-trans-retinol.
The exact concentration of reference solution (a) is assessed
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R1 to a presumed
concentration of 10-15 IU/ml and measure the absorbance
at 326 nm in matched 1 cm cells using 2-propanol R1 as the
compensation liquid.
4115
Cod-liver oil (type B)
Calculate the content of vitamin A in International Units
per millilitre of reference solution (a) using the following
expression, taking into account the assigned content of
retinol acetate CRS :
A326 = absorbance at 326 nm ;
V1 = volume of reference solution (a) used ;
V2 = volume of the diluted solution ;
1900 = conversion factor for the specific absorbance of
retinol acetate CRS, in International Units.
Reference solution (b). Proceed as described for the test
solution but using 2.00 ml of reference solution (a) in place
of the substance to be examined.
The exact concentration of reference solution (b) is assessed Test solution (a). To 4.00 g in a round-bottomed flask, add
by ultraviolet absorption spectrophotometry (2.2.25). Dilute 5 ml of a freshly prepared 100 g/l solution of ascorbic
reference solution (b) with 2-propanol R1 to a presumed
concentration of 10-15 IU/ml of all-trans-retinol and measure potassium hydroxide R and 100 ml of ethanol R. Boil under
the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International
Units per millilitre of reference solution (b) from the
expression :
A325 = absorbance at 325 nm ;
V3 = volume of the diluted solution ;
V4 = volume of reference solution (b) used ;
1821 = conversion factor for the specific absorbance of
all-trans-retinol, in International Units.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm).
Mobile phase : water R, methanol R (3:97 V/V).
Flow rate : 1 ml/min.
Detection : spectrophotometer at 325 nm.
Injection : 10 μl ; inject in triplicate the test solution and the internal standard solution and proceed as described for
reference solution (b).
Retention time : all-trans-retinol = 5 ± 1 min.
System suitability :
— the chromatogram obtained with the test solution shows solution and proceed as described for test solution (a).
a peak due to that of all-trans-retinol in the chromatogram
obtained with reference solution (b) ;
— the results obtained with the duplicate test solutions do
not differ by more than 5 per cent ;
— the recovery of all-trans-retinol in reference solution (b)
as assessed by direct absorption spectrophotometry is
greater than 95 per cent.
Calculate the content of vitamin A using the following
expression :
4116 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
A1 = area of the peak due to all-trans-retinol in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to all-trans-retinol in
the chromatogram obtained with reference
solution (b) ;
C = concentration of retinol acetate CRS in
reference solution (a) as assessed prior to the
saponification, in International Units per millilitre
(= 1000 IU/ml) ;
V = volume of reference solution (a) treated (2.00 ml) ;
m = mass of the substance to be examined in the test
solution (2.00 g).
Vitamin D3. Liquid chromatography (2.2.29). Carry out the
assay as rapidly as possible, avoiding exposure to actinic
light and air.
Internal standard solution. Dissolve 0.50 mg of
ergocalciferol CRS in 100 ml of ethanol R.
acid R, 10 ml of a freshly prepared 800 g/l solution of
a reflux condenser on a water-bath for 30 min. Add 100 ml
of a 10 g/l solution of sodium chloride R and cool the
solution to room temperature. Transfer the solution to a
500 ml separating funnel rinsing the round-bottomed flask
with about 75 ml of a 10 g/l solution of sodium chloride R
and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution
of potassium hydroxide R in a 10 per cent V/V solution
of ethanol R, and then with 3 quantities, each of 50 ml, of
a 10 g/l solution of sodium chloride R. Filter the upper
layer through 5 g of anhydrous sodium sulphate R on a
fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction
mixture, filter and combine the upper layers. Distil them at
a temperature not exceeding 30 °C under reduced pressure
(water ejector) and fill with nitrogen R when evaporation
is completed. Alternatively evaporate the solvent under a
gentle current of nitrogen R at a temperature not exceeding
30 °C. Dissolve the residue in 1.5 ml of the mobile phase
described under Purification. Gentle heating in an ultrasonic
bath may be required. (A large fraction of the white residue
is cholesterol, constituting approximately 50 per cent m/m
of the unsaponifiable matter of cod-liver oil).
Test solution (b). Prepare duplicates. To 4.00 g add 2.0 ml of
test solution (a).
Reference solution (a). Dissolve 0.50 mg of
cholecalciferol CRS in 100.0 ml of ethanol R.
Reference solution (b). In a round-bottomed flask, add 2.0 ml
of reference solution (a) and 2.0 ml of the internal standard
PURIFICATION
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : nitrile silica gel for chromatography R
(10 μm).
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
Flow rate : 1.1 ml/min.
Detection : spectrophotometer at 265 nm.
Inject 350 μl of reference solution (b). Collect the eluate
from 2 min before until 2 min after the retention time of
cholecalciferol, in a ground-glass-stoppered tube containing
EUROPEAN PHARMACOPOEIA 6.3
1 ml of a 1 g/l solution of butylhydroxytoluene R in
hexane R. Repeat the procedure with test solutions (a)
and (b). Evaporate the eluates obtained from reference
solution (b) and from test solutions (a) and (b), separately,
to dryness at a temperature not exceeding 30 °C under a
gentle current of nitrogen R. Dissolve each residue in 1.5 ml
of acetonitrile R.
DETERMINATION
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : phosphoric acid R, a 96 per cent V/V solution
of acetonitrile R (0.2:99.8 V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 265 nm.
Injection : 2 quantities not exceeding 200 μl of each of the
3 solutions obtained under Purification.
System suitability :
— resolution : minimum 1.4 between the peaks due to
ergocalciferol and cholecalciferol in the chromatogram
obtained with reference solution (b) ;
— the results obtained with the test solution (b) duplicates
do not differ by more than 5 per cent.
Calculate the content of vitamin D3 in International Units
per gram using the following expression, taking into account
the assigned content of cholecalciferol CRS :
m1 = mass of the sample in test solution (b) in grams ;
m2 = total mass of cholecalciferol CRS used for
the preparation of reference solution (a) in
micrograms (500 μg) ;
A1 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (a) ;
A2 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with test
solution (b) ;
A3 = area (or height) of the peak due to ergocalciferol
in the chromatogram obtained with reference
solution (b) ;
A4 = area (or height) of the peak due to ergocalciferol in
the chromatogram obtained with test solution (b) ;
A5 = area (or height) of a possible peak in the
chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting
with ergocalciferol in test solution (b) ;
A6 = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference
solution (b) ;
V1 = total volume of reference solution (a) (100 ml) ;
V2 = volume of reference solution (a) used for preparing
reference solution (b) (2.0 ml).
STORAGE
In an airtight and well-filled container, protected from light.
If no antioxidant is added, store under an inert gas.
Once the container has been opened, its contents are used
as soon as possible and any part of the contents not used at
once is protected by an atmosphere of inert gas.
General Notices (1) apply to all monographs and other texts
Croscarmellose sodium
LABELLING
The label states :
— the number of International Units of vitamin A, per gram ;
— the number of International Units of vitamin D3, per gram.
01/2009:0985
CROSCARMELLOSE SODIUM
Carmellosum natricum conexum
DEFINITION
Cross-linked sodium carboxymethylcellulose.
Sodium salt of a cross-linked, partly O-carboxymethylated
cellulose.
CHARACTERS
Appeareance : white or greyish-white powder.
Solubility : practically insoluble in acetone, in anhydrous
ethanol and in toluene.
IDENTIFICATION
A. Mix 1 g with 100 ml of a solution containing 4 ppm of
methylene blue R, stir the mixture and allow it to settle.
The substance to be examined absorbs the methylene
blue and settles as a blue, fibrous mass.
B. Mix 1 g with 50 ml of water R. Transfer 1 ml of the mixture
to a small test-tube and add 1 ml of water R and 0.05 ml
of a freshly prepared 40 g/l solution of α-naphthol R in
methanol R. Incline the test-tube and carefully add 2 ml
of sulphuric acid R down the side so that it forms a lower
layer. A reddish-violet colour develops at the interface.
C. The solution prepared from the sulphated ash in the test
for heavy metals (see Tests) gives reaction (a) of sodium
(2.3.1).
TESTS
pH (2.2.3) : 5.0 to 7.0 for the suspension.
Shake 1 g with 100 ml of carbon dioxide-free water R for
5 min.
Sodium chloride and sodium glycollate : maximum 0.5 per
cent (dried substance) for the sum of the percentage contents
of sodium chloride and sodium glycollate.
Sodium chloride. Place 5.00 g in a 250 ml conical flask, add
50 ml of water R and 5 ml of strong hydrogen peroxide
solution R and heat on a water-bath for 20 min, stirring
occasionally to ensure total hydration. Cool, add 100 ml of
water R and 10 ml of nitric acid R. Titrate with 0.05 M silver
nitrate, determining the end-point potentiometrically (2.2.20)
using a silver indicator electrode and a double-junction
reference electrode containing a 100 g/l solution of
potassium nitrate R in the outer jacket and a standard filling
solution in the inner jacket, and stirring constantly.
1 ml of 0.05 M silver nitrate is equivalent to 2.922 mg of
NaCl.
Sodium glycollate. Place a quantity of the substance to be
examined equivalent to 0.500 g of the dried substance in a
100 ml beaker. Add 5 ml of glacial acetic acid R and 5 ml
of water R and stir to ensure total hydration (about 15 min).
Add 50 ml of acetone R and 1 g of sodium chloride R. Stir
for several minutes to ensure complete precipitation of the
carboxymethylcellulose. Filter through a fast filter paper
impregnated with acetone R into a volumetric flask, rinse
the beaker and the filter with 30 ml of acetone R and dilute
4117
Croscarmellose sodium
the filtrate to 100.0 ml with the same solvent. Allow to stand
for 24 h without shaking. Use the clear supernatant to
prepare the test solution.
Prepare the reference solutions as follows : in a 100 ml
volumetric flask, dissolve 0.100 g of glycollic acid R,
previously dried in vacuo over diphosphorus pentoxide R,
in water R and dilute to 100.0 ml with the same solvent ; use
the solution within 30 days ; transfer 1.0 ml, 2.0 ml, 3.0 ml
and 4.0 ml of the solution to separate volumetric flasks,
dilute the contents of each flask to 5.0 ml with water R,
add 5 ml of glacial acetic acid R, dilute to 100.0 ml with
acetone R and mix.
Transfer 2.0 ml of the test solution and 2.0 ml of each of
the reference solutions to separate 25 ml volumetric flasks.
Heat the uncovered flasks for 20 min on a water-bath
to eliminate acetone. Allow to cool and add 5.0 ml of
2,7-dihydroxynaphthalene solution R to each flask.
Mix, add a further 15.0 ml of 2,7-dihydroxynaphthalene
solution R and mix again. Close the flasks with aluminium
foil and heat on a water-bath for 20 min. Cool and dilute to
25.0 ml with sulphuric acid R.
Measure the absorbance (2.2.25) of each solution at 540 nm.
Prepare a blank using 2.0 ml of a solution containing 5 per
cent V/V each of glacial acetic acid R and water R in
acetone R. Prepare a standard curve using the absorbances
obtained with the reference solutions. From the standard
curve and the absorbance of the test solution, determine the
mass (a) of glycollic acid in the substance to be examined,
in milligrams, and calculate the content of sodium glycollate
using the following expression :
1.29 = the factor converting glycollic acid to sodium
glycollate ;
b = loss on drying as a percentage ;
m = mass of the substance to be examined, in grams.
Water-soluble substances : maximum 10.0 per cent.
Disperse 10.00 g in 800.0 ml of water R and stir for 1 min
every 10 min during the first 30 min. Allow to stand for
1 h and centrifuge if necessary. Decant 200.0 ml of the
supernatant liquid onto a fast filter paper in a vacuum
filtration funnel, apply vacuum and collect 150.0 ml of
the filtrate. Evaporate to dryness and dry the residue at
100-105 °C for 4 h.
Heavy metals (2.4.8) : maximum 20 ppm.
To the residue obtained in the determination of the sulphated
ash add 1 ml of hydrochloric acid R and evaporate on a
water-bath. Take up the residue in 20 ml of water R. 12 ml
of the solution complies with test A. Prepare the reference
solution using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 6 h.
Sulphated ash(2.4.14) : 14.0 per cent to 28.0 per cent
(dried substance), determined on 1.0 g, using a mixture of
equal volumes of sulphuric acid R and water R.
4118 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for
croscarmellose sodium used as disintegrant.
Settling volume. Place 75 ml of water R in a 100 ml
graduated cylinder and add 1.5 g of the substance to be
examined in 0.5 g portions, shaking vigorously after each
addition. Dilute to 100.0 ml with water R and shake again
until the substance is homogeneously distributed. Allow to
stand for 4 h. The settling volume is between 10.0 ml and
30.0 ml.
Degree of substitution : 0.60 to 0.85 (dried substance).
Place 1.000 g in a 500 ml conical flask, add 300 ml of a
100 g/l solution of sodium chloride R and 25.0 ml of 0.1 M
sodium hydroxide, stopper the flask and allow to stand for
5 min, shaking occasionally. Add 0.05 ml of m-cresol purple
solution R and about 15 ml of 0.1 M hydrochloric acid from
a burette. Insert the stopper and shake. If the solution is
violet, add 0.1 M hydrochloric acid in 1 ml portions until the
solution becomes yellow, shaking after each addition. Titrate
with 0.1 M sodium hydroxide until the colour turns to violet.
Calculate the number of milliequivalents (M) of base required
to neutralise the equivalent of 1 g of dried substance.
Calculate the degree of acid carboxymethyl substitution (A)
using the following expression :
C = sulphated ash as a percentage.
Calculate the degree of sodium carboxymethyl
substitution (S) using the following expression :
The degree of substitution is the sum of A and S.
Particle size distribution (2.9.31 or 2.9.38).
Hausner ratio (2.9.36).
EUROPEAN PHARMACOPOEIA 6.3 Crospovidone

01/2009:0892 TESTS
Peroxides. Type A : maximum 400 ppm expressed as H2O2 ;
CROSPOVIDONE type B : maximum 1000 ppm expressed as H2O2.
Suspend 2.0 g in 50 ml of water R. To 25 ml of this
suspension add 2 ml of titanium trichloride-sulphuric
Crospovidonum acid reagent R. Allow to stand for 30 min and filter. The
absorbance (2.2.25) of the filtrate, measured at 405 nm
using a mixture of 25 ml of a filtered 40 g/l suspension of
the substance to be examined and 2 ml of a 13 per cent V/V
solution of sulphuric acid R as the compensation liquid, has
a maximum of 0.35.
For type B use 10 ml of the suspension and dilute to 25 ml
with water R for the test.
(C6H9NO)n Mr (111.1)n Water-soluble substances: maximum 1.0 per cent.
[9003-39-8] Place 25.0 g in a 400 ml beaker, add 200 ml of water R and
stir for 1 h using a magnetic stirrer. Transfer the suspension
DEFINITION to a 250.0 ml volumetric flask, rinsing with water R, and
Cross-linked homopolymer of 1-ethenylpyrrolidin-2-one. dilute to volume with the same solvent. Allow the bulk of
the solids to settle. Filter about 100 ml of the almost clear
Content : 11.0 per cent to 12.8 per cent of N (Ar 14.01) (dried supernatant liquid through a membrane filter (nominal pore
substance). size 0.45 μm), protected by superimposing a membrane
filter (nominal pore size 3 μm). While filtering, stir the
CHARACTERS liquid above the membrane filter manually or by means of a
Appearance : hygroscopic, white or yellowish-white powder mechanical stirrer, taking care not to damage the membrane
or flakes. filter. Transfer 50.0 ml of the clear filtrate to a tared 100 ml
2 types of crospovidone are available, depending on the beaker, evaporate to dryness and dry at 105-110 °C for 3 h.
particle size : type A and type B. The residue weighs a maximum of 50 mg.
Solubility : practically insoluble in water, in ethanol 96 per Impurity A. Liquid chromatography (2.2.29).
cent and in methylene chloride. Test solution. Suspend 1.250 g in 50.0 ml of methanol R
and shake for 60 min. Leave the bulk to settle and filter
IDENTIFICATION through a filter membrane (nominal pore size 0.2 μm).
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (a). Dissolve 50 mg of
1-vinylpyrrolidin-2-one R (impurity A) in methanol R and
Comparison : crospovidone CRS. dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of
B. Suspend 1 g in 10 ml of water R, add 0.1 ml of 0.05 M this solution to 100.0 ml with methanol R. Dilute 5.0 ml of
iodine and shake for 30 s. Add 1 ml of starch solution R this solution to 100.0 ml with the mobile phase.
and shake. No blue colour develops within 30 s. Reference solution (b). Dissolve 10 mg of
C. To 10 ml of water R, add 0.1 g and shake. A suspension is 1-vinylpyrrolidin-2-one R (impurity A) and 0.50 g of
formed and no clear solution is obtained within 15 min. vinyl acetate R in methanol R and dilute to 100 ml with the
same solvent. Dilute 1.0 ml of this solution to 100.0 ml with
D. The analytical screens must be clean and dry. For the mobile phase.
this purpose the screens are washed in hot water and
allowed to dry overnight in a drying cabinet at 105 °C. Precolumn:
— size: l = 0.025 m, Ø = 4 mm ;
Place 20 g in a 1000 ml conical flask, add 500 ml of
water R and shake the suspension for 30 min. Pour the — stationary phase : octadecylsilyl silica gel for
suspension through a 63 μm analytical screen, previously chromatography R (5 μm).
tared, and rinse the screen with water R until the filtrate Column :
is clear. Dry the screen and sample residue at 105 °C for — size: l = 0.25 m, Ø = 4 mm ;
5 h in a drying cabinet without circulating air. Cool in a
desiccator for 30 min and weigh. — stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
Calculate the percentage screening residue (fraction of — temperature : 40 °C.
sample particles having a diameter of more than 63 μm),
using the following expression : Mobile phase : acetonitrile R, water R (10:90 V/V).
Flow rate : adjusted so that the retention time of the peak
due to impurity A is about 10 min.
Detection : spectrophotometer at 235 nm.
m1 = mass of the screen and sample residue, after Injection : 50 μl. After each injection of the test solution,
drying for 5 h, in grams ; wash the precolumn by passing the mobile phase backwards,
at the same flow rate as applied in the test, for 30 min.
m2 = mass of the sample, in grams ;
System suitability :
m3 = mass of the screen, in grams.
— resolution : minimum 2.0 between the peaks due to
If the screening residue fraction is more than 15 per cent, impurity A and vinyl acetate in the chromatogram
the substance is classified as type A ; if the screening obtained with reference solution (b) ;
residue fraction is less than or equal to 15 per cent, the — repeatability : maximum relative standard deviation of
substance is classified as type B. 2.0 per cent after 5 injections of reference solution (a).

General Notices (1) apply to all monographs and other texts 4119
Crospovidone
Limits :
— impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(10 ppm).
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 0.500 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Place 100.0 mg of the substance to be examined (m mg)
in a combustion flask and add 5 g of a mixture of 1 g of
copper sulphate R, 1 g of titanium dioxide R and 33 g
of dipotassium sulphate R, and 3 glass beads. Wash any
adhering particles from the neck into the flask with a
small quantity of water R. Add 7 ml of sulphuric acid R,
allowing it to run down the insides of the flask, and mix the
contents by rotation. Close the mouth of the flask loosely,
for example by means of a glass bulb with a short stem, to
avoid excessive loss of sulphuric acid. Heat gradually at
first, then increase the temperature until there is vigorous
boiling with condensation of sulphuric acid in the neck of
the flask ; precautions are to be taken to prevent the upper
part of the flask from becoming overheated. Continue the
heating for 45 min. Cool, dissolve the solid material by
cautiously adding 20 ml of water R to the mixture, cool
again and place in a steam-distillation apparatus. Add 30 ml
of strong sodium hydroxide solution R through the funnel,
rinse the funnel cautiously with 10 ml of water R and
distil immediately by passing steam through the mixture.
Collect 80-100 ml of distillate in a mixture of 30 ml of a
40 g/l solution of boric acid R and 0.05 ml of bromocresol
green-methyl red solution R and enough water R to cover
the tip of the condenser. Towards the end of the distillation
lower the receiver so that the tip of the condenser is above
the surface of the acid solution and rinse the end part of
the condenser with a small quantity of water R. Titrate the
distillate with 0.025 M sulphuric acid until the colour of the
solution changes from green through pale greyish-blue to
pale greyish-red-purple (n1 ml of 0.025 M sulphuric acid).
Repeat the test using about 100 mg of glucose R in place of
the substance to be examined (n2 ml of 0.025 M sulphuric
acid).
Percentage content of nitrogen :
4120 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
STORAGE
In an airtight container.
LABELLING
The label states the type (type A or type B).
IMPURITIES
A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for
crospovidone used as disintegrant.
Hydration capacity. Introduce 2.0 g into a 100 ml centrifuge
tube and add 40 ml of water R. Shake vigorously until a
suspension is obtained. Shake again 5 min and 10 min later,
then centrifuge for 15 min at 750 g. Decant the supernatant
liquid and weigh the residue. The hydration capacity is the
ratio of the mass of the residue to the initial mass of the
sample. It is typically 3 to 9.
Particle-size distribution (2.9.31).
Powder flow (2.9.36).
The following characteristic may be relevant for
crospovidone used as suspension stabiliser.
Settling volume. Introduce 10 g into a 100 ml graduated
cylinder and add 90 ml of water R. Shake vigorously. Dilute
to 100 ml with water R, washing the powder residues from
the walls of the cylinder. Allow to stand for 24 h, then read
the volume of the sediment. It is typically greater than 60 ml.
EUROPEAN PHARMACOPOEIA 6.3

D
Dexamethasone acetate.......................................................... 4123 Dextran 70 for injection.. ....................................................... 4127
Dextran 1 for injection............................................................ 4124 Disodium phosphate, anhydrous.......................................... 4128
Dextran 40 for injection.. ....................................................... 4125 Dydrogesterone.. ...................................................................... 4128
Dextran 60 for injection.. ....................................................... 4126

General Notices (1) apply to all monographs and other texts 4121
 EUROPEAN PHARMACOPOEIA 6.3

4122 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2008:0548
corrected 6.3
DEXAMETHASONE ACETATE
Dexamethasoni acetas
C24H31FO6 Mr 434.5
[1177-87-3]
DEFINITION
9-Fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-1,4-
dien-21-yl acetate.
Content : 97.0 per cent to 103.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone and in ethanol (96 per cent), slightly soluble in
methylene chloride.
It shows polymorphism (5.9).
IDENTIFICATION
First identification : B, C.
Second identification : A, C, D, E, F.
A. Dissolve 10.0 mg in anhydrous ethanol R and dilute
to 100.0 ml with the same solvent. Place 2.0 ml of this
solution in a ground-glass-stoppered tube, add 10.0 ml of
phenylhydrazine-sulphuric acid solution R, mix and heat
in a water-bath at 60 °C for 20 min. Cool immediately.
The absorbance (2.2.25) measured at the absorption
maximum at 419 nm is not less than 0.35.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : dexamethasone acetate CRS.
If the spectra obtained in the solid state show differences,
record new spectra using saturated solutions (about
30 g/l) in chloroform R in a 0.2 mm cell.
C. Thin-layer chromatography (2.2.27).
Solvent mixture : methanol R, methylene chloride R
(1:9 V/V).
Test solution. Dissolve 10 mg of the substance to be
examined in the solvent mixture and dilute to 10 ml with
the solvent mixture.
Reference solution (a). Dissolve 20 mg of dexamethasone
acetate CRS in the solvent and dilute to 20 ml with the
solvent mixture.
Reference solution (b). Dissolve 10 mg of cortisone
acetate R in reference solution (a) and dilute to 10 ml
with reference solution (a).
Plate : TLC silica gel F254 plate R.
Mobile phase : add a mixture of 1.2 volumes of water R
and 8 volumes of methanol R to a mixture of 15 volumes
of ether R and 77 volumes of methylene chloride R.
Application : 5 μl.
Development : over a path of 15 cm.
General Notices (1) apply to all monographs and other texts
Dexamethasone acetate
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : 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).
Detection B : spray with alcoholic solution of sulphuric
acid R. Heat at 120 °C for 10 min or until the spots
appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm.
Results B : the principal spot in the chromatogram
obtained with the test solution is similar in position,
colour in daylight, fluorescence in ultraviolet light at
365 nm and size to the principal spot in the chromatogram
obtained with reference solution (a).
System suitability : reference solution (b) :
— the chromatogram shows 2 clearly separated spots.
D. Add about 2 mg to 2 ml of sulphuric acid R and shake
to dissolve. Within 5 min, a faint reddish-brown colour
develops. Add this solution to 10 ml of water R and mix.
The colour is discharged and a clear solution remains.
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
and ignite in a crucible until an almost white residue is
obtained (usually less than 5 min). Allow to cool, add 1 ml
of water R, 0.05 ml of phenolphthalein solution R1 and
about 1 ml of dilute hydrochloric acid R to render the
solution colourless. Filter. To a freshly prepared mixture
of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl
nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to
stand for 5 min and compare the colour of the solution
with that of a blank prepared in the same manner. The
test solution is yellow and the blank is red.
F. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
Specific optical rotation (2.2.7) : + 84 to + 90 (dried
substance).
Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in about 4 ml of acetonitrile R and dilute to
10.0 ml with water R.
Reference solution (a). Dissolve 2 mg of dexamethasone
acetate CRS and 2 mg of betamethasone acetate CRS in the
mobile phase and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to
100.0 ml with the mobile phase.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : in a 1000 ml volumetric flask mix 380 ml
of acetonitrile R with 550 ml of water R and allow to
equilibrate ; dilute to 1000 ml with water R and mix again.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 254 nm.
Equilibration: with the mobile phase for about 30 min.
Injection : 20 μl.
Run time : 1.5 times the retention time of dexamethasone
acetate.
Retention time : betamethasone acetate = about 19 min ;
dexamethasone acetate = about 22 min.
4123
Dextran 1 for injection
System suitability : reference solution (a) :
— resolution : minimum 3.3 between the peaks due to
betamethasone acetate and dexamethasone acetate ; if
necessary, adjust the concentration of acetonitrile in the
mobile phase.
Limits :
— any impurity : for each impurity, not more than 0.5 times
the area of the principal in the chromatogram obtained
with reference solution (b) (0.5 per cent) ;
— total : not more than the area of the principal in the
chromatogram obtained with reference solution (b)
(1.0 per cent) ;
— disregard limit: 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 0.500 g by drying in vacuo in an oven at 105 °C.
ASSAY
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
100.0 ml with the same solvent. Dilute 2.0 ml of this solution
to 100.0 ml with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 238.5 nm.
Calculate the content of C24H31FO6 taking the specific
absorbance to be 357.
STORAGE
Protected from light.
01/2009:1506
DEXTRAN 1 FOR INJECTION
Dextranum 1 ad iniectabile
DEFINITION
Low-molecular-weight fraction of dextran, consisting of a
mixture of isomaltooligosaccharides.
Average relative molecular mass : about 1000.
PRODUCTION
It is obtained by hydrolysis and fractionation of dextrans
produced by fermentation of sucrose using Leuconostoc
mesenteroides strain NRRL B-512 = CIP 78.59 or
substrains thereof (for example L. mesenteroides
B-512 F = NCTC 10817).
It is prepared in conditions designed to minimise the risk
of microbial contamination.
CHARACTERS
Appearance : white or almost white hygroscopic powder.
Solubility : very soluble in water, very slightly soluble in
ethanol (96 per cent).
IDENTIFICATION
A. Dissolve 3.000 g in water R, heat on a water-bath and
dilute to 100.0 ml with the same solvent. The specific
optical rotation (2.2.7) is + 148 to + 164, calculated with
reference to the dried substance. Dry an aliquot of the
solution first on a water-bath and then to constant weight
in vacuo at 70 °C. Calculate the dextran content after
correction for the content of sodium chloride.
4124 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : to 1-2 mg add 1 or a few drops of water R.
Grind in an agate mortar for 1-2 min. Add about 300 mg
of potassium bromide R and mix to a slurry but do not
grind. Dry in vacuo at 40 °C for 15 min. Crush the
residue. If it is not dry, dry for another 15 min. Prepare a
disc using potassium bromide R.
Comparison : repeat the operations using dextran 1 CRS.
Blank : run the infrared spectrum with a blank disc using
potassium bromide R in the reference beam.
C. Molecular-mass distribution (see Tests).
TESTS
Solution S. Dissolve 7.5 g in carbon dioxide-free water R,
heat on a water-bath and dilute to 50 ml with the same
solvent.
Absorbance (2.2.25) : maximum 0.12, determined at 375 nm
on solution S.
Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of
phenolphthalein solution R. The solution is colourless.
Add 0.2 ml of 0.01 M sodium hydroxide. The solution is
pink. Add 0.4 ml of 0.01 M hydrochloric acid. The solution
is colourless. Add 0.1 ml of methyl red solution R. The
solution is red or orange.
Nitrogen-containing substances : maximum 110 ppm of N.
Carry out the determination of nitrogen by sulphuric acid
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
the distillate in a mixture of 0.5 ml of bromocresol green
solution R, 0.5 ml of methyl red solution R and 20 ml of
water R. Titrate with 0.01 M hydrochloric acid. Not more
than 0.15 ml of 0.01 M hydrochloric acid is required to
change the colour of the indicator.
Sodium chloride : maximum 1.5 per cent.
Accurately weigh 3-5 g and dissolve in 100 ml of water R.
Add 0.3 ml of potassium chromate solution R and titrate
with 0.1 M silver nitrate until the yellowish-white colour
changes to reddish-brown.
1 ml of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl.
Molecular-mass distribution. Size-exclusion
chromatography (2.2.30).
Test solution. Dissolve 6.0-6.5 mg of the substance to be
examined in 1.0 ml of the mobile phase.
Reference solution (a). Dissolve 6.0-6.5 mg of dextran 1 CRS
in 1.0 ml of the mobile phase.
Reference solution (b). Dissolve the content of an ampoule
of isomaltooligosaccharide CRS in 1 ml of the mobile
phase, and mix. This corresponds to approximately 45 μg
of isomaltotriose (3 glucose units), approximately 45 μg of
isomaltononaose (9 glucose units), and approximately 60 μg
of sodium chloride per 100 μl.
Column : 2 columns coupled in series :
— size: l = 0.30 m, Ø = 10 mm ;
— stationary phase : dextran covalently bound to highly
cross-linked porous agarose beads, allowing resolution
of oligosaccharides in the molecular mass range of 180
to 3000 ;
— temperature : 20-25 °C.
Mobile phase : 2.92 g/l solution of sodium chloride R.
Flow rate : 0.07-0.08 ml/min maintained constant to ± 1 per
cent.
Detection : differential refractometer.
Injection : 100 μl.
EUROPEAN PHARMACOPOEIA 6.3 Dextran 40 for injection

Identification of peaks : use the chromatogram obtained 01/2009:0999

with reference solution (b) to identify the peaks due to
isomaltotriose, isomaltononaose and sodium chloride. DEXTRAN 40 FOR INJECTION
Determine the peak areas. Disregard any peak due to sodium
chloride. Calculate the average relative molecular mass Mw Dextranum 40 ad iniectabile
and the amount of the fraction with less than 3 and more
than 9 glucose units, of dextran 1 CRS and of the substance DEFINITION
to be examined, using the following expression : Mixture of polysaccharides, principally of the α-1,6-glucan
type.
Average relative molecular mass: about 40 000.
PRODUCTION
Mw = average molecular mass of the dextran ; It is obtained by hydrolysis and fractionation of dextrans
mi = molecular mass of oligosaccharide i ; produced by fermentation of sucrose using Leuconostoc
wi = weight proportion of oligosaccharide i. mesenteroides strain NRRL B-512 = CIP 78.59 or
substrains thereof (for example L. mesenteroides
Use the following mi values for the calculation : B-512F = NCTC 10817).
It is prepared in conditions designed to minimise the risk
Oligosaccharide i mi
of microbial contamination.
glucose 180
CHARACTERS
isomaltose 342
Appearance : white or almost white powder.
isomaltotriose 504 Solubility : very soluble in water, very slightly soluble in
isomaltotetraose 666 ethanol (96 per cent).
isomaltopentaose 828 IDENTIFICATION
isomaltohexaose 990 A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried
substance).
isomaltoheptaose 1152
Dissolve 1.0 g in water R, heating on a water-bath, and
isomaltooctaose 1314 dilute to 50.0 ml with the same solvent.
isomaltononaose 1476 B. Infrared absorption spectrophotometry (2.2.24).
isomaltodecaose 1638 Comparison : dextran CRS.
isomaltoundecaose 1800
C. Molecular-mass distribution (see Tests).
isomaltododecaose 1962 TESTS
isomaltotridecaose 2124 Solution S. Dissolve 5.0 g in distilled water R, heating on a
water-bath, and dilute to 50 ml with the same solvent.
isomaltotetradecaose 2286
Appearance of solution. Solution S is clear (2.2.1) and
isomaltopentadecaose 2448 colourless (2.2.2, Method II).
isomaltohexadecaose 2610 Acidity or alkalinity. To 10 ml of solution S add 0.1 ml
isomaltoheptadecaose 2772 of phenolphthalein solution R. The solution remains
colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The
isomaltooctadecaose 2934 solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The
isomaltononadecaose 3096 solution is colourless. Add 0.1 ml of methyl red solution R.
The solution is red or orange.
System suitability : the values obtained for dextran 1 CRS Nitrogen-containing substances: maximum 110 ppm N.
are within the values stated on the label. Carry out the determination of nitrogen by sulphuric acid
Limits : digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
the distillate in a mixture of 0.5 ml of bromocresol green
— average molecular mass (Mw) : 850 to 1150 ; solution R, 0.5 ml of methyl red solution R and 20 ml of
— fraction with less than 3 glucose units : less than 15 per water R. Titrate with 0.01 M hydrochloric acid. Not more
cent ; than 0.15 ml of 0.01 M hydrochloric acid is required to
change the colour of the indicator.
— fraction with more than 9 glucose units : less than 20 per
cent . Residual solvents. Gas chromatography (2.2.28).
Internal standard : propanol R.
Heavy metals (2.4.8) : maximum 10 ppm.
Test solution. Dissolve 5 g of the substance to be examined
Dilute 20 ml of solution S to 30 ml with water R. 12 ml of in 100 ml of water R and distil. Collect the first 45 ml of the
solution complies with test A. Prepare the reference solution distillate, add 1 ml of a 25 g/l solution of propanol R and
using lead standard solution (1 ppm Pb) R. dilute to 50 ml with water R.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined Reference solution. Mix 0.5 ml of a 25 g/l solution of
on 5.000 g by drying in an oven at 105 °C for 5 h. anhydrous ethanol R, 0.5 ml of a 25 g/l solution of
propanol R and 0.5 ml of a 2.5 g/l solution of methanol R
Bacterial endotoxins (2.6.14) : less than 25 IU/g.
and dilute to 25.0 ml with water R.
Microbial contamination Column :
TAMC : acceptance criterion 102 CFU/g (2.6.12). — material: stainless steel ;

General Notices (1) apply to all monographs and other texts 4125
Dextran 60 for injection EUROPEAN PHARMACOPOEIA 6.3

— size : l = 1.8 m, Ø = 2 mm ; IDENTIFICATION

— stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (125-150 μm).
Carrier gas: nitrogen for chromatography R.
Flow rate : 25 ml/min.
Temperature :
— column : 190 °C ;
— injection port : 240 °C ;
— detector : 210 °C.
Detection : flame ionisation.
Injection : the chosen volume of each solution.
Limits :
— ethanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.5 per cent) ;
— methanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.05 per cent) ;
— sum of solvents other than ethanol, methanol and
propanol : not more than the area of the peak due to the
internal standard (0.5 per cent, calculated as propanol).Nitrogen-containing substances : maximum 110 ppm of N.
Molecular-mass distribution (2.2.39). The average molecular Carry out the determination of nitrogen by sulphuric acid
mass (Mw) is 35 000 to 45 000. The average molecular mass
of the 10 per cent high fraction is not greater than 110 000.the distillate in a mixture of 0.5 ml of bromocresol green
The average molecular mass of the 10 per cent low fraction
is not less than 7000.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined
on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h.
Sulphated ash (2.4.14) : maximum 0.3 per cent, determined
on 0.50 g.
Bacterial endotoxins (2.6.14) : less than 10 IU/g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
01/2009:1000
DEXTRAN 60 FOR INJECTION
Dextranum 60 ad iniectabile
DEFINITION
Mixture of polysaccharides, principally of the α-1,6-glucan — column : 190 °C ;
type.
Average relative molecular mass : about 60 000.
PRODUCTION
It is obtained by hydrolysis and fractionation of dextrans
produced by fermentation of sucrose using Leuconostoc
mesenteroides strain NRRL B-512 = CIP 78.59 or substrains Limits :
thereof (for example L. mesenteroides B-512F = NCTC
10817).
It is prepared in conditions designed to minimise the risk
of microbial contamination.
CHARACTERS
Appearance : white or almost white powder.
Solubility : very soluble in water, very slightly soluble in
ethanol (96 per cent).
A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried
substance).
Dissolve 1.0 g in water R, heating on a water-bath, and
dilute to 50.0 ml with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : dextran CRS.
C. Molecular-mass distribution (see Tests).
TESTS
Solution S. Dissolve 5.0 g in distilled water R, heating on a
water-bath, and dilute to 50 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.1 ml
of phenolphthalein solution R. The solution remains
colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The
solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The
solution is colourless. Add 0.1 ml of methyl red solution R.
The solution is red or orange.
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
solution R, 0.5 ml of methyl red solution R and 20 ml of
water R. Titrate with 0.01 M hydrochloric acid. Not more
than 0.15 ml of 0.01 M hydrochloric acid is required to
change the colour of the indicator.
Residual solvents. Gas chromatography (2.2.28).
Internal standard : propanol R.
Test solution. Dissolve 5 g of the substance to be examined
in 100 ml of water R and distil. Collect the first 45 ml of the
distillate, add 1 ml of a 25 g/l solution of propanol R and
dilute to 50 ml with water R.
Reference solution. Mix 0.5 ml of a 25 g/l solution of
anhydrous ethanol R, 0.5 ml of a 25 g/l solution of
propanol R and 0.5 ml of a 2.5 g/l solution of methanol R
and dilute to 25.0 ml with water R.
Column :
— material: stainless steel ;
— size : l = 1.8 m, Ø = 2 mm ;
— stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (125-150 μm).
Carrier gas: nitrogen for chromatography R.
Flow rate : 25 ml/min.
Temperature :
— injection port : 240 °C ;
— detector : 210 °C.
Detection : flame ionisation.
Injection : the chosen volume of each solution.
— ethanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.5 per cent) ;
— methanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.05 per cent) ;
— sum of solvents other than ethanol, methanol and
propanol: not more than the area of the peak due to the
internal standard (0.5 per cent, calculated as propanol).

4126 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Molecular-mass distribution (2.2.39). The average molecular
mass (Mw) is 54 000 to 66 000. The average molecular mass
of the 10 per cent high fraction is not greater than 180 000. The solution is red or orange.
The average molecular mass of the 10 per cent low fraction
is not less than 14 000.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined
on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h.
Sulphated ash (2.4.14) : maximum 0.3 per cent, determined
on 0.50 g.
Bacterial endotoxins (2.6.14) : less than 16 IU/g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
01/2009:1001 and dilute to 25.0 ml with water R.
DEXTRAN 70 FOR INJECTION
Dextranum 70 ad iniectabile
DEFINITION
Mixture of polysaccharides, principally of the α-1,6-glucan
type.
Average relative molecular mass : about 70 000.
PRODUCTION
It is obtained by hydrolysis and fractionation of dextrans
produced by fermentation of sucrose using Leuconostoc
mesenteroides strain NRRL B-512 = CIP 78.59 or substrains
thereof (for example L. mesenteroides B-512F = NCTC
10817).
It is prepared in conditions designed to minimise the risk
of microbial contamination.
CHARACTERS
Appearance : white or almost white powder.
Solubility : very soluble in water, very slightly soluble in
ethanol (96 per cent).
IDENTIFICATION
A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried Molecular-mass
substance).
Dissolve 1.0 g in water R, heating on a water-bath, and
dilute to 50.0 ml with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : dextran CRS.
C. Molecular-mass distribution (see Tests).
TESTS
Solution S. Dissolve 5.0 g in distilled water R, heating on a on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h.
water-bath, and dilute to 50 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.1 ml
of phenolphthalein solution R. The solution remains
colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The
General Notices (1) apply to all monographs and other texts
Dextran 70 for injection
solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The
solution is colourless. Add 0.1 ml of methyl red solution R.
Nitrogen-containing substances : maximum 110 ppm of N.
Carry out the determination of nitrogen by sulphuric acid
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
the distillate in a mixture of 0.5 ml of bromocresol green
solution R, 0.5 ml of methyl red solution R and 20 ml of
water R. Titrate with 0.01 M hydrochloric acid. Not more
than 0.15 ml of 0.01 M hydrochloric acid is required to
change the colour of the indicator.
Residual solvents. Gas chromatography (2.2.28).
Internal standard : propanol R.
Test solution. Dissolve 5 g of the substance to be examined
in 100 ml of water R and distil. Collect the first 45 ml of the
distillate, add 1 ml of a 25 g/l solution of propanol R and
dilute to 50 ml with water R.
Reference solution. Mix 0.5 ml of a 25 g/l solution of
anhydrous ethanol R, 0.5 ml of a 25 g/l solution of
propanol R and 0.5 ml of a 2.5 g/l solution of methanol R
Column :
— material: stainless steel ;
— size: l = 1.8 m, Ø = 2 mm ;
— stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (125-150 μm).
Carrier gas: nitrogen for chromatography R.
Flow rate : 25 ml/min.
Temperature :
— column : 190 °C ;
— injection port : 240 °C ;
— detector : 210 °C.
Detection : flame ionisation.
Injection : the chosen volume of each solution.
Limits :
— ethanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.5 per cent) ;
— methanol : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.05 per cent) ;
— sum of solvents other than ethanol, methanol and
propanol: not more than the area of the peak due to the
internal standard (0.5 per cent, calculated as propanol).
distribution (2.2.39). The average molecular
mass (Mw) is 64 000 to 76 000. The average molecular mass
of the 10 per cent high fraction is not greater than 185 000.
The average molecular mass of the 10 per cent low fraction
is not less than 15 000.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined
Sulphated ash (2.4.14) : maximum 0.3 per cent, determined
on 0.50 g.
Bacterial endotoxins (2.6.14) : less than 16 IU/g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
4127
Disodium phosphate, anhydrous
01/2008:1509 Calculate the percentage content of Na2HPO4 from the
corrected 6.3 following expression :
DISODIUM PHOSPHATE, ANHYDROUS
Dinatrii phosphas anhydricus
Na2HPO4 Mr 142.0 STORAGE
[7558-79-4]
DEFINITION
Content : 98.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder, hygroscopic.
Solubility : soluble in water, practically insoluble in ethanol
(96 per cent).
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. Loss on drying (see Tests).
C. Solution S gives reaction (b) of phosphates (2.3.1).
D. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in distilled water R and dilute to
100.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Reducing substances. To 10 ml of solution S add 5 ml of
dilute sulphuric acid R and 0.25 ml of 0.02 M potassium
permanganate and heat on a water-bath for 5 min. The
solution retains a slight red colour.
Monosodium phosphate : maximum 2.5 per cent.
From the volume of 1 M hydrochloric acid (25 ml) and of
1 M sodium hydroxide (n1 ml and n2 ml) used in the assay,
calculate the following ratio :
This ratio is not greater than 0.025.
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 5 ml of solution S to 15 ml with dilute nitric acid R.
Sulphates (2.4.13) : maximum 500 ppm.
To 6 ml of solution S add 2 ml of dilute hydrochloric acid R
and dilute to 15 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined
on 10 ml of solution S.
Iron (2.4.9) : maximum 20 ppm, determined on solution S.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using 5 ml of lead standard solution
(1 ppm Pb) R and 5 ml of water R.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
ASSAY
Dissolve 1.600 g (m) in 25.0 ml of carbon dioxide-free
water R and add 25.0 ml of 1 M hydrochloric acid. Carry
out a potentiometric titration (2.2.20) using 1 M sodium
hydroxide. Read the volume added at the 1st inflexion point
(n1 ml). Continue the titration to the 2nd inflexion point
(total volume of 1 M sodium hydroxide required, n2 ml).
4128 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
d = percentage loss on drying.
In an airtight container.
01/2009:2357
DYDROGESTERONE
Dydrogesteronum
C21H28O2 Mr 312.5
[152-62-5]
DEFINITION
9β,10α-Pregna-4,6-diene-3,20-dione.
Content : 98.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone,
sparingly soluble in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : dydrogesterone CRS.
TESTS
Specific optical rotation (2.2.7) : − 469 to − 485 (dried
substance), measured at 25 °C.
Dissolve 0.100 g in methylene chloride R and dilute to
20.0 ml with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Test solution (b). Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dissolve 3.0 mg of dydrogesterone
impurity A CRS in the mobile phase and dilute to 20.0 ml
with the mobile phase. Dilute 1.0 ml of this solution to
100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of test solution (a) to
100.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 10.0 ml with the mobile phase.
Reference solution (c). Dissolve 10 mg of the substance to
be examined in 10 ml of reference solution (a).
Reference solution (d). Dissolve 10 mg of the substance to
be examined in 30 ml of ethanol (96 per cent) R. Add 1 ml of
a 8.4 g/l solution of sodium hydroxide R and heat at 85 °C
EUROPEAN PHARMACOPOEIA 6.3 Dydrogesterone

for 10 min. Cool to room temperature, add 1 ml of a 20.6 g/l — total at 280 nm : not more than 5 times the area of
solution of hydrochloric acid R, add 20 ml of acetonitrile R, the principal peak in the chromatogram obtained with
2 mg of dydrogesterone impurity B CRS, dilute to 100 ml reference solution (b) (0.5 per cent) ;
with water R and mix. This solution contains dydrogesterone — disregard limit at 280 nm : 0.5 times the area of the
and impurities B and C. principal peak in the chromatogram obtained with
Reference solution (e). Dissolve 20.0 mg of reference solution (b) (0.05 per cent).
dydrogesterone CRS in the mobile phase and dilute Loss on drying (2.2.32) : maximum 0.5 per cent, determined
to 100.0 ml with the mobile phase. on 1.000 g by drying in an oven at 105 °C for 3 h.
Column:
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
— size : l = 0.15 m, Ø = 4.6 mm ; on 1.0 g.
— stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (3 μm) ; ASSAY
— temperature : 40 °C. Liquid chromatography (2.2.29) as described in the test for
Mobile phase : acetonitrile R, ethanol (96 per cent) R, related substances with the following modifications.
water R (21:25:54 V/V/V). Detection : spectrophotometer at 280 nm.
Flow rate : 1.0 ml/min. Injection : test solution (b) and reference solution (e).
Detection : spectrophotometer at 280 nm and at 385 nm. Calculate the percentage content of C21H28O2 from the
Injection : 10 μl of test solution (a) and reference declared content of dydrogesterone CRS.
solutions (a), (b), (c) and (d).
Run time : twice the retention time of dydrogesterone. IMPURITIES
Relative retention at 385 nm with reference to Specified impurities : A, B, C.
dydrogesterone (retention time = about 13 min) :
impurity A = about 0.9.
Relative retention at 280 nm with reference to
dydrogesterone (retention time = about 13 min) :
impurity B = about 1.1 ; impurity C = about 1.2.
System suitability :
— resolution at 385 nm: minimum 1.1 between the
peaks due to impurity A and dydrogesterone in the
chromatogram obtained with reference solution (c) ; A. 9β,10α-pregna-4,6,8(14)-triene-3,20-dione,
— resolution at 280 nm : minimum 4.5 between the peaks
due to dydrogesterone and impurity B and minimum 1.5
between the peaks due to impurity B and impurity C in
the chromatogram obtained with reference solution (d).
Limits :
— impurity A at 385 nm : not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
— impurity B at 280 nm : not more than 1.5 times the area B. pregna-4,6-diene-3,20-dione,
of the principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
— impurity C at 280 nm : not more than 3 times the area of
the principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
— unspecified impurities at 280 nm : for each impurity,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ; C. 9β,10α,17α-pregna-4,6-diene-3,20-dione.

General Notices (1) apply to all monographs and other texts 4129
 EUROPEAN PHARMACOPOEIA 6.3

4130 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

E
Ergocalciferol............................................................................ 4133 Esomeprazole magnesium trihydrate.................................. 4136
Erythritol.. ................................................................................. 4134 Ethacridine lactate monohydrate......................................... 4138

General Notices (1) apply to all monographs and other texts 4131
 EUROPEAN PHARMACOPOEIA 6.3

4132 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Ergocalciferol

01/2008:0082 Ergosterol. Examine by thin-layer chromatography (2.2.27),

corrected 6.3 using a TLC silica gel G plate R.
Test solution. Dissolve 0.25 g of the substance to be
ERGOCALCIFEROL examined in ethylene chloride R containing 10 g/l of
squalane R and 0.1 g/l of butylhydroxytoluene R and dilute
to 5 ml with the same solvent. Prepare immediately before
Ergocalciferolum use.
Reference solution (a). Dissolve 0.10 g of ergocalciferol CRS
in ethylene chloride R containing 10 g/l of squalane R and
0.1 g/l of butylhydroxytoluene R and dilute to 2 ml with the
same solvent. Prepare immediately before use.
Reference solution (b). Dissolve 5 mg of ergosterol CRS in
ethylene chloride R containing 10 g/l of squalane R and
0.1 g/l of butylhydroxytoluene R and dilute to 50 ml with
the same solvent. Prepare immediately before use.
Reference solution (c). Mix equal volumes of reference
solution (a) and reference solution (b). Prepare immediately
before use.
Apply to the plate 10 μl of the test solution, 10 μl of reference
solution (a), 10 μl of reference solution (b) and 20 μl of
reference solution (c). Develop immediately, protected from
C28H44O Mr 396.7
light, over a path of 15 cm using a mixture of equal volumes
[50-14-6]
of cyclohexane R and peroxide-free ether R, the mixture
DEFINITION containing 0.1 g/l of butylhydroxytoluene R. Allow the plate
to dry in air and spray three times with antimony trichloride
Ergocalciferol contains not less than 97.0 per cent solution R1. Examine the chromatograms for 3 min to 4 min
and not more than the equivalent of 103.0 per cent of after spraying. The principal spot in the chromatogram
(5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3β-ol. obtained with the test solution is initially orange-yellow and
1 mg of ergocalciferol is equivalent to 40 000 IU of then becomes brown. In the chromatogram obtained with the
antirachitic activity (vitamin D) in rats. test solution, any slowly appearing violet spot (corresponding
to ergosterol) immediately below the principal spot is not
CHARACTERS more intense than the spot in the chromatogram obtained
A white or slightly yellowish, crystalline powder or white or with reference solution (b) (0.2 per cent). There is no spot in
almost white crystals, practically insoluble in water, freely the chromatogram obtained with the test solution that does
soluble in alcohol, soluble in fatty oils. It is sensitive to air, not correspond to one of the spots in the chromatograms
heat and light. Solutions in volatile solvents are unstable obtained with reference solutions (a) and (b). The test is
and are to be used immediately. not valid unless the chromatogram obtained with reference
A reversible isomerisation to pre-ergocalciferol takes place in solution (c) shows two clearly separated spots.
solution, depending on temperature and time. The activity ASSAY
is due to both compounds.
Carry out the operations as rapidly as possible, avoiding
IDENTIFICATION exposure to actinic light and air.
Examine by infrared absorption spectrophotometry Examine by liquid chromatography (2.2.29).
(2.2.24), comparing with the spectrum obtained with Test solution. Dissolve 10.0 mg of the substance to be
ergocalciferol CRS. Examine the substances prepared as examined without heating in 10.0 ml of toluene R and dilute
discs. to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of
TESTS ergocalciferol CRS without heating in 10.0 ml of
Specific optical rotation (2.2.7). Dissolve 0.200 g rapidly toluene R and dilute to 100.0 ml with the mobile phase.
and without heating in aldehyde-free alcohol R and dilute to Reference solution (b). Dilute 1.0 ml of cholecalciferol for
25.0 ml with the same solvent. The specific optical rotation, system suitability CRS to 5.0 ml with the mobile phase.
determined within 30 min of preparing the solution, is + 103 Heat in a water-bath at 90 °C under a reflux condenser for
to + 107. 45 min and cool.
Reducing substances. Dissolve 0.1 g in aldehyde-free The chromatographic procedure may be carried out using :
alcohol R and dilute to 10.0 ml with the same solvent. — a stainless steel column 0.25 m long and 4.6 mm in
Add 0.5 ml of a 5 g/l solution of tetrazolium blue R internal diameter packed with a suitable silica gel (5 μm),
in aldehyde-free alcohol R and 0.5 ml of dilute
— as mobile phase at a flow rate of 2 ml/min a mixture of
tetramethylammonium hydroxide solution R. Allow to
3 volumes of pentanol R and 997 volumes of hexane R,
stand for exactly 5 min and add 1.0 ml of glacial acetic
acid R. Prepare a reference solution at the same time and — as detector a spectrophotometer set at 254 nm.
in the same manner using 10.0 ml of a solution containing An automatic injection device or a sample loop is
0.2 μg/ml of hydroquinone R in aldehyde-free alcohol R. recommended. Inject a suitable volume of reference
Measure the absorbance (2.2.25) of the two solutions solution (b). Adjust the sensitivity of the system so that the
at 525 nm using as the compensation liquid 10.0 ml of height of the principal peak is at least 50 per cent of the full
aldehyde-free alcohol R treated in the same manner. The scale of the recorder. Inject reference solution (b) 6 times.
absorbance of the test solution is not greater than that of the When the chromatograms are recorded in the prescribed
reference solution (20 ppm). conditions, the approximate relative retention times with

General Notices (1) apply to all monographs and other texts 4133
Erythritol EUROPEAN PHARMACOPOEIA 6.3

reference to cholecalciferol are 0.4 for pre-cholecalciferol

and 0.5 for trans-cholecalciferol. The relative standard
deviation of the response for cholecalciferol is not greater
than 1 per cent and the resolution between the peaks
corresponding to pre-cholecalciferol and trans-cholecalciferol
is not less than 1.0. If necessary adjust the proportions of
the constituents and the flow rate of the mobile phase to
obtain this resolution.
Inject a suitable volume of reference solution (a). Adjust the
sensitivity of the system so that the height of the principal C. (9β,10α,22E)-ergosta-5,7,22-trien-3β-ol (lumisterol2),
peak is at least 50 per cent of the full scale of the recorder.
Inject the same volume of the test solution and record the
chromatogram in the same manner.
Calculate the percentage content of ergocalciferol from the
expression :

m = mass of the substance to be examined in the test

solution, in milligrams ;
m′ = mass of ergocalciferol CRS in reference
solution (a), in milligrams ;
SD = area (or height) of the peak due to ergocalciferol D. (6E,22E)-9,10-secoergosta-5(10),6,8(14),22-tetraen-3β-ol
in the chromatogram obtained with the test (iso-tachysterol2),
solution ;
S′D = area (or height) of the peak due to ergocalciferol
in the chromatogram obtained with reference
solution (a).

STORAGE
Store in an airtight container, under nitrogen, protected
from light, at a temperature between 2 °C and 8 °C.
The contents of an opened container are to be used
immediately.

IMPURITIES
E. (6E,22E)-9,10-secoergosta-5(10),6,8,22-tetraen-3β-ol
(tachysterol2).

01/2009:1803

ERYTHRITOL
Erythritolum

A. (5E,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3β-ol
(trans-vitamin D2), C4H10O4 Mr 122.1
[149-32-6]
DEFINITION
(2R,3S)-Butane1,2,3,4-tetrol (meso-erythritol).
Content : 96.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
free-flowing granules.
Solubility : freely soluble in water, very slightly soluble in
B. (22E)-ergosta-5,7,22-trien-3β-ol (ergosterol), ethanol (96 per cent).

4134 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
IDENTIFICATION
A. Melting point (2.2.14) : 119 °C to 122 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : erythritol CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 5.0 g in water R and dilute to 50 ml with the same
solvent.
Conductivity (2.2.38) : maximum 20 μS·cm− 1.
Dissolve 20.0 g in carbon dioxide-free water R prepared
from distilled water R and dilute to 100.0 ml with the same
solvent. Measure the conductivity of the solution, while
gently stirring with a magnetic stirrer.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.50 g of the substance to be
examined in water R and dilute to 10.0 ml with the same
solvent.
Reference solution (a). Dissolve 0.50 g of erythritol CRS in
water R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 2.0 ml of the test solution to
100.0 ml with water R.
Reference solution (c). Dilute 5.0 ml of reference solution (b)
to 100.0 ml with water R.
Reference solution (d). Dissolve 1.0 g of erythritol R and
1.0 g of glycerol R in water R and dilute to 20.0 ml with the
same solvent.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : cation-exchange resin R (9 μm) ;
— temperature : 70 °C.
Mobile phase : 0.01 per cent V/V solution of sulphuric
acid R.
Flow rate : 0.8 ml/min.
Detection : refractometer maintained at a constant
temperature.
Injection : 20 μl ; inject the test solution and reference
solutions (b), (c) and (d).
Run time : 3 times the retention time of erythritol.
Relative retention with reference to erythritol (retention
time = about 11 min) : impurity A = about 0.77 ;
impurity B = about 0.90 ; impurity C = about 0.94 ;
impurity D = about 1.10.
System suitability : reference solution (d) :
— resolution : minimum 2 between the peaks due to
erythritol and impurity D.
Limits :
— any impurity : not more than the area of the principal
peak in the chromatogram obtained with reference
solution (b) (2.0 per cent) ;
General Notices (1) apply to all monographs and other texts
Erythritol
— total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (b)
(2.0 per cent) ;
— disregard limit : area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.1 per cent).
Lead (2.4.10) : maximum 0.5 ppm.
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
Microbial contamination
If intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 102 CFU/g (2.6.12).
If not intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
— absence of Escherichia coli (2.6.13) ;
— absence of Salmonella (2.6.13).
Bacterial endotoxins (2.6.14). If intended for use in
the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial
endotoxins :
— less than 4 IU/g for parenteral preparations having a
concentration of 100 g/l or less of erythritol ;
— less than 2.5 IU/g for parenteral preparations having a
concentration of more than 100 g/l of erythritol.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Calculate the percentage content of erythritol using the
chromatogram obtained with reference solution (a) and the
declared content of erythritol CRS.
LABELLING
The label states where applicable, that the substance
is suitable for use in the manufacture of parenteral
preparations.
IMPURITIES
A. maltitol,
B. sorbitol,
C. (2R,3s,4S)-pentane-1,2,3,4,5-pentol (meso-ribitol),
D. glycerol.
4135
Esomeprazole magnesium trihydrate EUROPEAN PHARMACOPOEIA 6.3

01/2009:2372 Reference solution (c). Dilute 1.0 ml of the test solution

to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
ESOMEPRAZOLE MAGNESIUM solution to 10.0 ml with the mobile phase.
TRIHYDRATE Column :
— size: l = 0.125 m, Ø = 4.6 mm ;
Esomeprazolum magnesicum trihydricum — stationary phase : octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 27 volumes of acetonitrile R and
73 volumes of a 1.4 g/l solution of disodium hydrogen
phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 280 nm.
Injection : 40 μl.
C34H36MgN6O6S2,3H2O Mr 767.2 Run time : 5 times the retention time of esomeprazole.
[217087-09-7] Relative retention with reference to esomeprazole
(retention time = about 9 min) : impurity E = about 0.6 ;
DEFINITION impurity D = about 0.8.
Magnesium bis[5-methoxy-2-[(S)-[(4-methoxy-3,5- System suitability : reference solution (a) :
dimethylpyridin-2-yl)methyl]sulphinyl]-1H-benzimidazol-1- — resolution : minimum 3.0 between the peaks due to
ide] trihydrate. impurity D and omeprazole. If necessary, adjust the pH
Content : 98.0 per cent to 102.0 per cent (anhydrous of the aqueous part of the mobile phase or its proportion
substance). of acetonitrile ; an increase in the pH will improve the
resolution.
CHARACTERS
Limits :
Appearance : white or slightly coloured powder, slightly
hygroscopic. — impurity D : maximum 0.2 per cent ;
Solubility : slightly soluble in water, soluble in methanol, — impurity E : maximum 0.1 per cent ;
practically insoluble in heptane. — unspecified impurities : for each impurity, maximum
0.10 per cent ;
IDENTIFICATION — total : maximum 0.5 per cent ;
Carry out either tests A, B, C or A, B, E or B, C, D or B, D, E. — disregard limit : 0.5 times the area of the principal peak
A. Specific optical rotation (2.2.7) : − 137 to − 155. in the chromatogram obtained with reference solution (c)
Dissolve 0.250 g in methanol R and dilute to 25.0 ml (0.05 per cent).
with the same solvent. Enantiomeric purity. Liquid chromatography (2.2.29).
B. Infrared absorption spectrophotometry (2.2.24). Buffer solution pH 6.0. Mix 70 ml of a 156.0 g/l solution of
Comparison : esomeprazole magnesium trihydrate CRS. sodium dihydrogen phosphate R with 20 ml of a 179.1 g/l
C. Atomic absorption spectrometry (2.2.23) as described in solution of disodium hydrogen phosphate R. Dilute to
the test for magnesium. 1000 ml with water R, then dilute 250 ml of this solution
to 1000.0 ml with water R.
The test solution shows the absorption maximum at
285.2 nm. Buffer solution pH 11.0. Mix 11 ml of a 95.0 g/l solution
of trisodium phosphate dodecahydrate R with 22 ml of a
D. Enantiomeric purity (see Tests). 179.1 g/l solution of disodium hydrogen phosphate R, then
E. Ignite about 0.5 g of the substance to be examined dilute to 1000.0 ml with water R.
according to the procedure for the sulphated ash test Test solution. Dissolve 40 mg of the substance to be
(2.4.14). Dissolve the residue in 10 ml of water R. 2 ml of examined in 5 ml of methanol R and dilute to 25 ml with
this solution gives the reaction of magnesium (2.3.1). buffer solution pH 11.0. Dilute 1.0 ml of this solution to
TESTS 50.0 ml with buffer solution pH 11.0.
Reference solution (a). Dissolve 2 mg of omeprazole CRS in
Absorbance (2.2.25) : maximum 0.20 at 440 nm. buffer solution pH 11.0 and dilute to 10.0 ml with the same
Dissolve 0.500 g in methanol R and dilute to 25.0 ml with buffer solution. Dilute 1.0 ml of this solution to 50.0 ml with
the same solvent. Filter the solution through a membrane buffer solution pH 11.0.
filter (nominal pore size 0.45 μm). Reference solution (b). Dilute 1.0 ml of reference solution (a)
Related substances. Liquid chromatography (2.2.29). to 50.0 ml with buffer solution pH 11.0.
Use the normalisation procedure. Use freshly prepared Column :
solutions. — size : l = 0.1 m, Ø = 4.0 mm ;
Test solution. Dissolve 3.5 mg of the substance to be — stationary phase : silica gel AGP for chiral
examined in the mobile phase and dilute to 25.0 ml with the chromatography R (5 μm).
mobile phase.
Mobile phase : acetonitrile R, buffer solution pH 6.0
Reference solution (a). Dissolve 1 mg of omeprazole CRS (65:435 V/V).
and 1 mg of omeprazole impurity D CRS in the mobile
phase and dilute to 10.0 ml with the mobile phase. Flow rate : 0.6 ml/min.
Reference solution (b). Dissolve 3 mg of the omeprazole Detection : spectrophotometer at 302 nm.
for peak identification CRS (containing impurity E) in the Injection : 20 μl.
mobile phase and dilute to 20.0 ml with the mobile phase. Elution order : impurity F, esomeprazole.

4136 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Esomeprazole magnesium trihydrate

Retention time : esomeprazole = about 4 min. Injection : 20 μl.

System suitability : Run time : 1.5 times the retention time of esomeprazole.
— resolution : minimum 3.0 between the peaks due to Retention time : esomeprazole = about 4 min.
impurity F and esomeprazole in the chromatogram Calculate the percentage content of C34H36MgN6O6S2 from
obtained with reference solution (a) ; the declared content of omeprazole CRS.
— signal-to-noise ratio : minimum 10 for the peak due to 1 g of omeprazole is equivalent to 1.032 g of esomeprazole
impurity F in the chromatogram obtained with reference magnesium.
solution (b).
Calculate the percentage content of impurity F using the STORAGE
following expression : In an airtight container, protected from light.
IMPURITIES
Specified impurities: D, E, F.
Other detectable impurities (the following substances
ri = area of the peak due to impurity F in the would, if present at a sufficient level, be detected by one
chromatogram obtained with the test solution ; or other of the tests in the monograph. They are limited
rs = sum of the areas of the peaks due to esomeprazole by the general acceptance criterion for other/unspecified
and impurity F in the chromatogram obtained impurities and/or by the general monograph Substances for
with the test solution. pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
Limits :
See also 5.10. Control of impurities in substances for
— impurity F : maximum 0.2 per cent. pharmaceutical use) : A, B, C.
Magnesium : 3.30 per cent to 3.55 per cent (anhydrous
substance).
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 0.250 g in 20 ml of a 103 g/l solution
of hydrochloric acid R, adding the acid slowly, and dilute
to 100.0 ml with water R. Dilute 10.0 ml of this solution to A. 5-methoxy-1H-benzimidazole-2-thiol,
200.0 ml with water R. To 10.0 ml of the solution obtained
add 4 ml of lanthanum chloride solution R and dilute to
100.0 ml with water R.
Reference solutions. Prepare the reference solutions using
magnesium standard solution (1000 ppm Mg) R, diluted as
necessary with a mixture of 1 ml of a 103 g/l solution of
hydrochloric acid R in 1000.0 ml of water R.
Wavelength : 285.2 nm. B. R = H, X = SO : 2-[(RS)-[(3,5-dimethylpyridin-2-
Water (2.5.12) : 6.0 per cent to 8.0 per cent, determined on yl)methyl]sulphinyl]-5-methoxy-1H-benzimidazole,
0.200 g.
C. R = OCH3, X = S : 5-methoxy-2-[[(4-methoxy-3,5-
ASSAY dimethylpyridin-2-yl)methyl]sulphanyl]-1H-benzimidazole
(ufiprazole),
Liquid chromatography (2.2.29).
Buffer solution pH 11.0. Mix 11 ml of a 95.0 g/l solution D. R = OCH3, X = SO2 : 5-methoxy-2-[[(4-methoxy-3,5-
of trisodium phosphate dodecahydrate R with 22 ml of a dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole
179.1 g/l solution of disodium hydrogen phosphate R, and (omeprazole sulphone),
dilute to 100.0 ml with water R.
Test solution. Dissolve 10.0 mg of the substance to be
examined in about 10 ml of methanol R, add 10 ml of buffer
solution pH 11.0 and dilute to 200.0 ml with water R.
Reference solution. Dissolve 10.0 mg of omeprazole CRS
in about 10 ml of methanol R, add 10 ml of buffer solution
pH 11.0 and dilute to 200.0 ml with water R.
Column: E. 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2-
yl)sulphinyl]methyl]-3,5-dimethylpyridine 1-oxide.
— size : l = 0.125 m, Ø = 4 mm ;
— stationary phase : octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 35 volumes of acetonitrile R with
65 volumes of a 1.4 g/l solution of disodium hydrogen
phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
Flow rate : 1 ml/min. F. 5-methoxy-2-[(R)-[(4-methoxy-3,5-dimethylpyridin-2-
Detection : spectrophotometer at 280 nm. yl)methyl]sulphinyl]-1H-benzimidazole((R)-omeprazole).

General Notices (1) apply to all monographs and other texts 4137
Ethacridine lactate monohydrate EUROPEAN PHARMACOPOEIA 6.3

01/2008:1591 Column :
corrected 6.3 — size: l = 0.25 m, Ø = 4.6 mm,
— stationary phase : octadecylsilyl silica gel for
ETHACRIDINE LACTATE chromatography R (5 μm).
MONOHYDRATE Mobile phase : dissolve 1.0 g of sodium octanesulphonate R
in a mixture of 300 ml of acetonitrile R and 700 ml of
phosphate buffer solution pH 2.8 R.
Ethacridini lactas monohydricus Flow rate : 1 ml/min.
Detection : spectrophotometer at 268 nm.
Injection : 10 μl.
Run time : 3 times the retention time of ethacridine.
Retention time : ethacridine = about 15 min.
Limits :
C18H21N3O4,H2O Mr 361.4 — any impurity : not more than 3 times the area of the
[6402-23-9] principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent),
DEFINITION — total : not more than the area of the principal peak in the
7-Ethoxyacridine-3,9-diamine (2RS)-2-hydroxypropanoate chromatogram obtained with reference solution (a) (1 per
monohydrate. cent),
Content : 99.0 per cent to 101.0 per cent (dried substance). — disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
CHARACTERS (0.05 per cent).
Appearance : yellow crystalline powder. Heavy metals (2.4.8) : maximum 50 ppm.
Solubility : sparingly soluble in water, very slightly soluble 1.0 g complies with test F. Prepare the reference solution
in ethanol (96 per cent), practically insoluble in methylene using 5.0 ml of lead standard solution (10 ppm Pb) R.
chloride. Loss on drying (2.2.32) : 4.5 per cent to 5.5 per cent,
determined on 1.000 g by drying in an oven in vacuo at
IDENTIFICATION 105 °C.
First identification : A. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Second identification : B, C, D. on 1.0 g.
A. Infrared absorption spectrophotometry (2.2.24). ASSAY
Comparison : ethacridine lactate monohydrate CRS.
Dissolve 0.270 g in 5.0 ml of anhydrous formic acid R.
B. Mix 0.1 ml of solution S (see Tests) and 100 ml of water R. Add 60.0 ml of acetic anhydride R and titrate with
The solution is greenish-yellow and shows a strong green 0.1 M perchloric acid, determining the end-point
fluorescence in ultraviolet light at 365 nm. Add 5 ml of potentiometrically (2.2.20).
1 M hydrochloric acid. The fluorescence remains. 1 ml of 0.1 M perchloric acid is equivalent to 34.34 mg of
C. To 0.5 ml of solution S add 1.0 ml of water R, 0.1 ml of a C18H21N3O4.
10 g/l solution of cobalt chloride R and 0.1 ml of a 50 g/l
solution of potassium ferrocyanide R. The solution is STORAGE
green. Protected from light.
D. To 50 ml of solution S add 10 ml of dilute sodium
hydroxide solution R. Filter. To 5 ml of the filtrate, add IMPURITIES
1 ml of dilute sulphuric acid R. 5 ml of the solution
obtained gives the reaction of lactates (2.3.1).
TESTS
Solution S. Dissolve 2.0 g in carbon dioxide-free water R
and dilute to 100.0 ml with the same solvent.
A. 6-amino-2-ethoxyacridin-9(10H)-one,
pH (2.2.3) : 5.5 to 7.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of test solution to
100.0 ml with the mobile phase. B. R = Cl : 6-chloro-2-ethoxyacridin-9-amine,
Reference solution (b). Dilute 1.0 ml of reference solution (a) C. R = O-CH2-CH2-OH : 2-[(9-amino-7-ethoxyacridin-3-
to 10.0 ml with the mobile phase. yl)oxy]ethanol.

4138 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

F
Ferrous gluconate.....................................................................4141 Fluvoxamine maleate.. ............................................................ 4144
Filgrastim concentrated solution.. ....................................... 4142 Frangula bark dry extract, standardised.. .......................... 4146

General Notices (1) apply to all monographs and other texts 4139
 EUROPEAN PHARMACOPOEIA 6.3

4140 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Ferrous gluconate

01/2009:0493 hydrochloric acid R and add 2 ml in excess. Boil until the

vapour no longer darkens lead acetate paper R and continue
FERROUS GLUCONATE boiling, if necessary, until the volume is reduced to about
10 ml. Cool, add 15 ml of sodium carbonate solution R,
allow to stand for 5 min and filter. Dilute the filtrate to
Ferrosi gluconas 100 ml with water R. To 5 ml of this solution add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 1 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 0.06 per cent.
Dilute 0.8 ml of solution S to 15 ml with water R.
Oxalates. Dissolve 5.0 g in a mixture of 10 ml of dilute
sulphuric acid R and 40 ml of water R. Shake the solution
C12H22FeO14,xH2O Mr 446.1 (anhydrous substance) with 50 ml of ether R for 5 min. Separate the aqueous layer
and shake it with 20 ml of ether R for 5 min. Combine the
DEFINITION ether layers, evaporate to dryness and dissolve the residue
Iron(II) di(D-gluconate). in 15 ml of water R. Filter, boil the filtrate until the volume
Content : 11.8 per cent to 12.5 per cent of iron(II) (dried is reduced to 5 ml and add 1 ml of dilute acetic acid R and
substance). It contains a variable amount of water. 1.5 ml of calcium chloride solution R. Allow to stand for
30 min. No precipitate is formed.
CHARACTERS
Sulphates (2.4.13) : maximum 500 ppm.
Appearance : greenish-yellow or grey powder or granules.
To 3.0 ml of solution S add 3 ml of acetic acid R and dilute to
Solubility : freely but slowly soluble in water giving a 15 ml with distilled water R. Examine the solutions against
greenish-brown solution, more readily soluble in hot water, the light.
practically insoluble in ethanol (96 per cent).
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined
IDENTIFICATION on 0.5 g.
A. Thin-layer chromatography (2.2.27). Barium. Dilute 10 ml of solution S to 50 ml with distilled
Test solution. Dissolve 20 mg of the substance to be water R and add 5 ml of dilute sulphuric acid R. Allow to
examined in 2 ml of water R, heating if necessary in a stand for 5 min. Any opalescence in the solution is not more
water-bath at 60 °C. intense than that in a mixture of 10 ml of solution S and
Reference solution. Dissolve 20 mg of ferrous 45 ml of distilled water R.
gluconate CRS in 2 ml of water R, heating if necessary in Ferric ion : maximum 1.0 per cent.
a water-bath at 60 °C. In a ground-glass-stoppered flask, dissolve 5.00 g in a
Plate : TLC silica gel G plate R. mixture of 10 ml of hydrochloric acid R and 100 ml of
Mobile phase : concentrated ammonia R, ethyl acetate R, carbon dioxide-free water R. Add 3 g of potassium iodide R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V). close the flask and allow to stand protected from light for
Application : 5 μl. 5 min. Titrate with 0.1 M sodium thiosulphate, using 0.5 ml
of starch solution R, added towards the end of the titration,
Development : over a path of 10 cm. as indicator. Carry out a blank titration. Not more than
Drying : at 100-105 °C for 20 min. 9.0 ml of 0.1 M sodium thiosulphate is used.
Detection : allow to cool and spray with a 50 g/l solution Heavy metals (2.4.8) : maximum 20 ppm.
of potassium dichromate R in a 40 per cent m/m solution
of sulphuric acid R. Thoroughly mix 2.5 g with 0.5 g of magnesium oxide R1 in
a silica crucible. Ignite to dull redness until a homogeneous
Results : after 5 min, the principal spot in the mass is obtained. Heat at 800 ± 50 °C for about 1 h, allow
chromatogram obtained with the test solution is similar to cool and take up the residue in 20 ml of hot hydrochloric
in position, colour and size to the principal spot in the acid R. Allow to cool. Transfer the liquid to a separating
chromatogram obtained with the reference solution. funnel and shake for 3 min with 3 quantities, each of 20 ml,
B. 1 ml of solution S (see Tests) gives reaction (a) of iron of methyl isobutyl ketone saturated with hydrochloric acid
(2.3.1). (prepared by shaking 100 ml of freshly distilled methyl
isobutyl ketone R with 1 ml of hydrochloric acid R). Allow to
TESTS
stand, separate the aqueous layer, reduce to half its volume
Solution S. Dissolve 5.0 g in carbon dioxide-free water R by boiling, allow to cool and dilute to 25 ml with water R.
prepared from distilled water R and heated to about 60 °C, Neutralise 10 ml of this solution to red litmus paper R using
allow to cool and dilute to 50 ml with carbon dioxide-free dilute ammonia R1 and dilute to 20 ml with water R. 12 ml
water R prepared from distilled water R. of the solution complies with test A. Prepare the reference
Appearance of solution. The solution is clear (2.2.1). solution using lead standard solution (1 ppm Pb) R.
Dilute 2 ml of solution S to 10 ml with water R. Examine Loss on drying (2.2.32) : 5.0 per cent to 10.5 per cent,
the solution against the light. determined on 0.500 g by drying in an oven at 105 °C for 5 h.
pH (2.2.3) : 4.0 to 5.5 for solution S, measured 3-4 h after Microbial contamination
preparation. TAMC : acceptance criterion 103 CFU/g (2.6.12).
Sucrose and reducing sugars. Dissolve 0.5 g in 10 ml of TYMC : acceptance criterion 102 CFU/g (2.6.12).
warm water R and add 1 ml of dilute ammonia R1. Pass
hydrogen sulphide R through the solution and allow to ASSAY
stand for 30 min. Filter and wash the precipitate with Dissolve 0.5 g of sodium hydrogen carbonate R in a
2 quantities, each of 5 ml, of water R. Acidify the combined mixture of 30 ml of dilute sulphuric acid R and 70 ml of
filtrate and washings to blue litmus paper R with dilute water R. When the effervescence stops, dissolve 1.00 g of the

General Notices (1) apply to all monographs and other texts 4141
Filgrastim concentrated solution
substance to be examined with gentle shaking. Using 0.1 ml
of ferroin R as indicator, titrate with 0.1 M ammonium and
cerium nitrate until the red colour disappears.
1 ml of 0.1 M ammonium and cerium nitrate is equivalent
to 5.585 mg of iron(II). D. Examine the electropherograms obtained under both
STORAGE
Protected from light.
01/2009:2206
E. Peptide mapping (2.2.55).
FILGRASTIM CONCENTRATED
SOLUTION
Filgrastimi solutio concentrata
C845H1339N223O243S9 Mr 18 799
DEFINITION
Solution of a protein having the primary structure of the
granulocyte colony-stimulating factor plus 1 additional
amino acid, an N-terminal methionine (r-met HU G-CSF).
In contrast to its natural counterpart, the protein is not
glycosylated. Human G-CSF is produced and secreted by
endothelium, monocytes and other immune cells. The
protein stimulates the differentiation and proliferation of
leucocyte stem cells into mature granulocytes.
Content : minimum 0.9 mg of protein per millilitre.
Potency : minimum 1.0 × 108 IU per milligram of protein.
PRODUCTION
Filgrastim concentrated solution is produced by a method
based on recombinant DNA (rDNA) technology, using
bacteria as host cells.
Prior to release, the following tests are carried out on each
batch of the final bulk product, unless exemption has been
granted by the competent authority.
Host-cell-derived proteins : the limit is approved by the
competent authority.
Host-cell- or vector-derived DNA : the limit is approved by
the competent authority.
CHARACTERS
Appearance : clear, colourless or slightly yellowish liquid.
IDENTIFICATION
A. It complies with the requirements described under Assay.
B. Examine the electropherograms obtained in the test for
impurities with charges differing from that of filgrastim.
Results : the principal band in the electropherogram
obtained with the test solution is similar in position to
the principal band in the electropherogram obtained with
reference solution (a).
C. Examine the chromatograms obtained in the test for
impurities with molecular masses higher than that of
filgrastim.
4142 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to the
principal peak in the chromatogram obtained with the
reference solution.
reducing and non-reducing conditions in the test for
impurities with molecular masses differing from that of
filgrastim.
Results : the principal band in the electropherogram
obtained with test solution (a) is similar in position to the
principal band obtained with the reference solution.
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS
Test solution. Introduce 50 μl of a 0.05 M sodium
phosphate buffer solution pH 8.0 into a polypropylene
tube and add a volume of the substance to be examined
corresponding to 25 μg of protein. Add 25 μl of a
0.1 mg/ml solution of glutamyl endopeptidase for
peptide mapping R, dilute to 1 ml with water R, stopper
the tube and incubate at about 37 °C for 18 h. Add 125 μl
of a 764 g/l (8 M) solution of guanidine hydrochloride R
and mix well ; add 10 μl of a 154.2 g/l (1 M) solution of
dithiothreitol R and mix well. Place the capped tube
in boiling water for 1 min, then allow to cool to room
temperature.
Reference solution. Prepare at the same time and in
the same manner as for the test solution but using
filgrastim CRS instead of the preparation to be examined.
CHROMATOGRAPHIC SEPARATION. Liquid chromatography
(2.2.29).
Column :
— size: l = 0.10 m, Ø = 2.0 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) with a pore size of 20 nm ;
— temperature : 60 °C.
Mobile phase :
— mobile phase A : dilute 0.5 ml of trifluoroacetic acid R
in 950 ml of water R, add 50 ml of acetonitrile for
chromatography R and mix ;
— mobile phase B : dilute 0.5 ml of trifluoroacetic acid R
in 50 ml of water R, add 950 ml of acetonitrile for
chromatography R and mix ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-8 97 → 94 3→6
8 - 25 94 → 66 6 → 34
25 - 40 66 → 10 34 → 90
40 - 45 10 90
45 - 46 10 → 97 90 → 3
46 - 65 97 3
Flow rate : 0.2 ml/min.
Detection : spectrophotometer at 215 nm.
Equilibration: at initial conditions for at least 30 min.
Injection : 10 μl.
System suitability : the chromatogram obtained with
the reference solution is similar to the chromatogram of
filgrastim digest supplied with filgrastim CRS.
Results : the profile of the chromatogram obtained with
the test solution corresponds to that of the chromatogram
obtained with the reference solution.
EUROPEAN PHARMACOPOEIA 6.3
TESTS
Impurities with molecular masses higher than that of
filgrastim. Size-exclusion chromatography (2.2.30) : use the
normalisation procedure.
Solution A. Dissolve 4.1 g of sodium acetate R in 400 ml of
water R, adjust to pH 4.0 with acetic acid R and dilute to
500 ml with water R.
Test solution. Dilute the preparation to be examined with
solution A to obtain a concentration of 0.4 mg/ml.
Reference solution. Dilute filgrastim CRS with solution A
to obtain a concentration of 0.4 mg/ml.
Resolution solution. Mix a sample of the reference solution
for about 30 s using a vortex mixer.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : hydrophilic silica gel for
chromatography R (5 μm), of a grade suitable for
fractionation of globular proteins in the relative molecular
mass range of 10 000 to 500 000 ;
— temperature : 30 °C.
Mobile phase. Dissolve 7.9 g of ammonium hydrogen
carbonate R in 1000 ml of water R and adjust to pH 7.0 with
phosphoric acid R ; dilute to 2000 ml with water R.
Flow rate : 0.5 ml/min.
Detection : spectrophotometer at 215 nm.
Injection : 20 μl.
Relative retention with reference to the filgrastim monomer
(retention time = about 19 min) : aggregates = about 0.60 ;
filgrastim oligomer 1 = about 0.75 ; filgrastim
oligomer 2 = about 0.80 ; filgrastim dimer = about 0.85.
System suitability : resolution solution :
— retention time : filgrastim monomer = 17 min to 20 min ;
— resolution : minimum 3 between the peaks due to the
filgrastim dimer and the filgrastim monomer.
Calculate the percentage content of the dimer, oligomers
and aggregates.
Limit :
— total of the peaks with retention times less than that of
the principal peak : maximum 2 per cent.
Impurities with molecular masses differing from that of
filgrastim. Polyacrylamide gel electrophoresis (2.2.31) under
both reducing and non-reducing conditions.
Gel dimensions : 1 mm thick.
Resolving gel : 13 per cent acrylamide.
Sample buffer (non-reducing conditions). Mix equal
volumes of water R and concentrated SDS-PAGE sample
buffer R.
Sample buffer (reducing conditions). Mix equal volumes
of water R and concentrated SDS-PAGE sample buffer for
reducing conditions R containing 2-mercaptoethanol as the
reducing agent.
Test solution (a). Dilute the preparation to be examined with
sample buffer to obtain a protein concentration of 100 μg/ml.
Test solution (b). To 0.20 ml of test solution (a) add 0.20 ml
of sample buffer.
Test solution (c). Dilute 0.20 ml of test solution (b) to 1 ml
with sample buffer.
Test solution (d). Dilute 0.20 ml of test solution (c) to 1 ml
with sample buffer.
General Notices (1) apply to all monographs and other texts
Filgrastim concentrated solution
Test solution (e). To 0.20 ml of test solution (d) add 0.20 ml
of sample buffer.
Reference solution. Solution of molecular mass markers
suitable for calibrating SDS-polyacrylamide gels in the range
of 14.4-94 kDa.
Sample treatment : boil for 5 min.
Application : 20 μl.
Detection : by silver staining.
System suitability :
— reference solution : the validation criteria are met ;
— a band is seen in the electropherogram obtained with test
solution (e) ;
— a gradation of intensity of staining is seen in the
electropherograms obtained with test solutions (a) to (e).
Limit : test solution (a) :
— impurities with molecular masses lower or higher than
that of filgrastim : no band is more intense than the
principal band in the electropherogram obtained with test
solution (d) (2.0 per cent).
Impurities with charges differing from that of filgrastim.
Isoelectric focusing (2.2.54).
Test solution. Dilute the preparation to be examined with
water R to obtain a concentration of 0.3 mg/ml.
Reference solution (a). Dilute filgrastim CRS with water R
to obtain a concentration of 0.3 mg/ml.
Reference solution (b). Dilute filgrastim CRS with water R
to obtain a concentration of 0.03 mg/ml.
Reference solution (c). Use an isoelectric point (pI)
calibration solution, in the pI range of 2.5-6.5, prepared
according to the manufacturer’s instructions.
Focusing :
— pH gradient : 4.5-8.0 ;
— catholyte : 1 M solution of sodium hydroxide R ;
— anolyte : 0.04 M solution of glutamic acid R in a
0.0025 per cent V/V solution of phosphoric acid R ;
— application : 20 μl.
Detection : as described in 2.2.54.
System suitability :
— in the electropherogram obtained with reference
solution (c), the relevant isoelectric point markers are
distributed along the entire length of the gel ;
— in the electropherogram obtained with reference
solution (a), the pI of the principal band is 5.7 to 6.3.
Limit :
— any impurity : no band is more intense than the principal
band in the electropherogram obtained with reference
solution (b) (10 per cent).
Related proteins. Liquid chromatography (2.2.29) : use the
normalisation procedure.
Solution A. A 100 mM sodium acetate buffer solution pH 4.0,
containing 0.1 mg/ml of polysorbate 80 R and 50 mg/ml
of sorbitol CRS.
Test solution. Dilute the preparation to be examined with
solution A to obtain a concentration of 0.3 mg/ml.
Reference solution (a). Dilute filgrastim CRS with solution A
to obtain a concentration of 0.3 mg/ml.
Reference solution (b). To 570 μl of reference solution (a)
add 6.8 μl of a 0.45 per cent V/V solution of hydrogen
peroxide and mix ; incubate at 25 °C for 1 h, then add 2.5 mg
of methionine CRS.
4143
Fluvoxamine maleate
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm) with a pore size of 20 nm ;
— temperature : 65 °C.
Mobile phase :
— mobile phase A : mix 499 ml of acetonitrile for
chromatography R and 500 ml of water R and add 1 ml
of trifluoroacetic acid R ;
— mobile phase B : mix 49 ml of water R and 950 ml of
acetonitrile for chromatography R and add 1 ml of
trifluoroacetic acid R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 92 8 series of 10 twofold dilutions to obtain a standard curve).
4 - 19 92 → 72 8 → 28
19 - 19.1 72 → 0 28 → 100
19.1 - 21 0 100
21 - 21.1 0 → 92 100 → 8
21.1 - 25 92 8 incubator using 6 ± 1 per cent CO2. Add 20 μl of a 5.0 g/l
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 215 nm.
Injection : 50 μl of the test solution and reference
solution (b).
Relative retention with reference to filgrastim (retention
time = about 12 min) : oxidised filgrastim 1 = about 0.90 ;
oxidised filgrastim 2 = about 0.95.
System suitability : reference solution (b) :
— the chromatogram shows 2 peaks corresponding to
oxidised filgrastim 1 and oxidised filgrastim 2 that
elute before the principal peak, the 2nd peak not being
completely separated from the principal peak.
Limits :
— any impurity : for each impurity, maximum 2.0 per cent;
— total : maximum 3.5 per cent.
Bacterial endotoxins (2.6.14) : less than 2 IU in the volume
that contains 1.0 mg of protein.
ASSAY
Protein. Liquid chromatography (2.2.29) as described in the
test for related proteins with the following modification.
Injection : test solution and reference solution (a).
Calculate the content of filgrastim (C845H1339N223O243S9) from
the declared content of C845H1339N223O243S9 in filgrastim CRS.
Potency. The potency of the preparation to be examined
is determined by comparison of the dilutions of the test
preparation with the dilutions of the International Standard
of filgrastim or with a reference preparation calibrated in
International Units.
The International Unit is the activity contained in a stated
amount of the appropriate International Standard. The
equivalence in International Units of the International
Standard is stated by the World Health Organisation.
Carry out the assay using a suitable method such as the
following, which uses the conversion of a tetrazolium
salt (MTS) as a staining method. Alternative methods of
quantifying cell proliferation, such as measurement of
intracellular ATP by luciferase bioluminescence, have also
4144 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
been found suitable, and may be used as the assay readout,
subject to appropriate validation. The assay conditions (for
example, cell concentration, incubation time and dilution
steps) are then adapted accordingly.
Use an established cell line responsive to filgrastim. NFS-60
cells (ATCC No. CRL-1838) have been found suitable.
Incubate with varying dilutions of test and reference
preparations of filgrastim. Then incubate with a solution of
tetrazolium salt R. This cytochemical stain is converted by
cellular dehydrogenases to a coloured formazan product.
The formazan is then measured spectrophotometrically.
Add 50 μl of dilution medium to all wells of a 96-well
microtitre plate. Add an additional 50 μl of this solution to
the wells designed for the blanks. Add 50 μl of each solution
to be tested in triplicate (test preparation and reference
preparation at a concentration of about 800 IU/ml, plus a
Prepare a suspension of NFS-60 cells containing 7 × 105 cells
per millilitre. Immediately before use, add 2-mercaptoethanol
to a final concentration of 0.1 mM, and add 50 μl of the
prepared cell suspension to each well, maintaining the cells
in a uniform suspension during addition.
Incubate the plate at 36.0-38.0 °C for 44-48 h in a humidified
sterile solution of tetrazolium salt R to each well and
reincubate for 4 h. Estimate the quantity of formazan
produced using a microtitre well plate reader at 490 nm.
Calculate the potency of the preparation to be examined
using a suitable statistical method, for example the parallel
line assay (5.3).
The estimated potency is not less than 80 per cent and
not more than 125 per cent of the stated potency. The
confidence limits (P = 0.95) are not less than 74 per cent and
not more than 136 per cent of the estimated potency.
LABELLING
The label states :
— the content, in milligrams of protein per millilitre ;
— the potency, in International Units per milligram of
protein.
07/2008:1977
corrected 6.3
FLUVOXAMINE MALEATE
Fluvoxamini maleas
C19H25F3N2O6 Mr 434.4
[61718-82-9]
DEFINITION
2-[[[(1E)-5-Methoxy-1-[4-(trifluoromethyl)phenyl]pentyl-
idene]amino]oxy]ethanamine (Z)-butenedioate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
EUROPEAN PHARMACOPOEIA 6.3
PRODUCTION
The production method must be evaluated to determine
the potential for formation of aziridine. Where necessary,
a validated test for the substance is carried out or the
production method is validated to demonstrate acceptable
clearance.
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : sparingly soluble in water, freely soluble in
ethanol (96 per cent) and in methanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : fluvoxamine maleate CRS.
TESTS
Related substances. Liquid chromatography (2.2.29).
Prepare the test solution immediately before use.
Test solution. Dissolve 50 mg of the substance to be
examined in the mobile phase and dilute to 25 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to
10.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve the contents of a vial
of fluvoxamine for system suitability CRS (containing
impurities A, B, C and F) in 1.0 ml of the mobile phase.
Reference solution (c). Dissolve 3.0 mg of fluvoxamine
impurity D CRS in 5 ml of the mobile phase and dilute to
10.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 370 volumes of acetonitrile R1 and
630 volumes of a buffer solution containing 1.1 g/l of
potassium dihydrogen phosphate R and 1.9 g/l of sodium
pentanesulphonate R in water R, previously adjusted to
pH 3.0 with phosphoric acid R.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 234 nm.
Injection : 20 μl.
Run time : 6 times the retention time of fluvoxamine.
Identification of impurities: use the chromatogram
supplied with fluvoxamine for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, B, C and F.
Relative retention with reference to fluvoxamine
(retention time = about 15 min) : maleic acid = about 0.15 ;
impurities F and G = about 0.5 ; impurity C = about 0.6 ;
impurity B = about 0.8 ; impurity A = about 2.5 ;
impurity D = about 5.4.
System suitability : reference solution (b) :
— resolution : minimum 1.5 between the peaks due to
impurities F and C.
Limits :
— impurity B : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
General Notices (1) apply to all monographs and other texts
Fluvoxamine maleate
— impurity C : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
— impurity D : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.15 per cent) ;
— sum of impurities F and G : not more than 3 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.3 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 : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard the peak due to maleic acid.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test B. 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 vacuo at 80 °C for 2 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g in a platinum crucible.
ASSAY
Dissolve 0.350 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 43.44 mg
of C19H25F3N2O6.
IMPURITIES
Specified impurities : A, B, C, D, F, 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. Control of impurities in substances for
pharmaceutical use) : E, I, J.
A. R1 = R2 = H : 2-[[[(1E)-1-[4-(trifluoromethyl)phenyl]pentyl-
idene]amino]oxy]ethanamine,
F. R1 = CH2-CH2-NH2, R2 = OCH3 : N-[2-[[[(1E)-5-methoxy-1-
[4-(trifluoromethyl)phenyl]pentylidene]amino]oxy]ethyl]-
ethane-1,2-diamine,
G. R1 = H, R2 = OH : (5E)-5-[(2-aminoethoxy)imino]-5-[4-
(trifluoromethyl)phenyl]pentan-1-ol,
4145
Frangula bark dry extract, standardised
B. 2-[[[(1Z)-5-methoxy-1-[4-(trifluoromethyl)phenyl]pentyl-
idene]amino]oxy]ethanamine,
C. (2RS)-2-[[2-[[[(1E)-5-methoxy-1-[4-(trifluoromethyl)-
phenyl]pentylidene]amino]oxy]ethyl]amino]butanedioic
acid,
D. 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one,
E. 2-[[[(1E)-1-[4-(difluoromethyl)phenyl]-5-methoxypentyl-
idene]amino]oxy]ethanamine,
I. (E)-N-[5-methoxy-1-[4-(trifluoromethyl)phenyl]pentyl-
idene]hydroxylamine,
J. 2-[[[(1E)-2-phenyl-1-[4-(trifluoromethyl)phenyl]-
ethylidene]amino]oxy]ethanamine.
01/2009:1214 In a tared round-bottomed flask with a ground-glass neck,
FRANGULA BARK DRY EXTRACT,
STANDARDISED
Frangulae corticis extractum siccum
normatum
DEFINITION
Standardised dry extract obtained from Frangula
bark (0025).
4146
EUROPEAN PHARMACOPOEIA 6.3
Content : 15.0 per cent to 30.0 per cent of glucofrangulins,
expressed as glucofrangulin A (C27H30O14 ; Mr 578.5) (dried
extract) ; the measured content does not deviate from that
stated on the label by more than ± 10 per cent.
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-80 per cent V/V).
CHARACTERS
Appearance : yellowish-brown, fine powder.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. To 0.05 g of the extract to be examined add
5 ml of ethanol (70 per cent V/V) R and heat to boiling.
Cool and centrifuge. Decant the supernatant solution
immediately and use within 30 min.
Reference solution. Dissolve 20 mg of barbaloin R in
ethanol (70 per cent V/V) R and dilute to 10 ml with the
same solvent.
Plate : TLC silica gel plate R.
Mobile phase : water R, methanol R, ethyl acetate R
(13:17:100 V/V/V).
Application : 10 μl as bands.
Development : over a path of 10 cm.
Drying : in air for 5 min.
Detection : spray with a 50 g/l solution of potassium
hydroxide R in ethanol (50 per cent V/V) R and heat
at 100-105 °C for 15 min ; examine immediately after
heating.
Results : the chromatogram obtained with the reference
solution shows in the median third a reddish-brown
zone due to barbaloin. The chromatogram obtained
with the test solution shows 2 orange-brown zones
(glucofrangulins) in the lower third and 2-4 red zones
(frangulins, not always clearly separated, and above them
frangula-emodin) in the upper third.
B. To about 25 mg add 25 ml of dilute hydrochloric acid R
and heat the mixture on a water-bath for 15 min. Allow
to cool, shake with 20 ml of ether R and discard the
aqueous layer. Shake the ether layer with 10 ml of dilute
ammonia R1. The aqueous layer becomes reddish-violet.
TESTS
Loss on drying (2.8.17) : maximum 5.0 per cent.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Carry out the assay protected from bright light.
weigh 0.100 g. Add 25.0 ml of a 70 per cent V/V solution
of methanol R, mix and weigh again. Heat the flask in a
water-bath under a reflux condenser at 70 °C for 15 min.
Allow to cool, weigh and adjust to the original mass with a
70 per cent V/V solution of methanol R. Filter and transfer
5.0 ml of the filtrate to a separating funnel. Add 50 ml of
water R and 0.1 ml of hydrochloric acid R. Shake with
5 quantities, each of 20 ml, of light petroleum R1. Allow the
layers to separate and transfer the aqueous layer to a 100 ml
volumetric flask. Combine the light petroleum layers and
wash with 2 quantities, each of 15 ml, of water R. Use this
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 Frangula bark dry extract, standardised

water for washing the separating funnel and add it to the Calculate the percentage content of glucofrangulins,
aqueous solution in the volumetric flask. Add 5 ml of a 50 g/l expressed as glucofrangulin A from the following expression :
solution of sodium carbonate R and dilute to 100.0 ml with
water R. Discard the light petroleum layer. Transfer 40.0 ml
of the aqueous solution to a 200 ml round-bottomed flask
with a ground-glass neck. Add 20 ml of a 200 g/l solution
of ferric chloride R and heat under a reflux condenser for
20 min in a water-bath with the water level above that of i.e. taking the specific absorbance of glucofrangulin A to
the liquid in the flask. Add 2 ml of hydrochloric acid R and be 204, calculated on the basis of the specific absorbance
continue heating for 20 min, shaking frequently, until the of barbaloin,
precipitate is dissolved. Allow to cool, transfer the mixture
to a separating funnel and shake with 3 quantities, each of A = absorbance at 515 nm ;
25 ml, of ether R, previously used to rinse the flask. Combine m = mass of the preparation to be examined, in grams.
the ether extracts and wash with 2 quantities, each of 15 ml,
of water R. Transfer the ether layer to a volumetric flask and
dilute to 100.0 ml with ether R. Evaporate 20.0 ml carefully
to dryness and dissolve the residue in 10.0 ml of a 5 g/l
solution of magnesium acetate R in methanol R. Measure LABELLING
the absorbance (2.2.25) at 515 nm using methanol R as the
compensation liquid. The label states the content of glucofrangulins.

General Notices (1) apply to all monographs and other texts 4147
 EUROPEAN PHARMACOPOEIA 6.3

4148 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
G
Galactose.................................................................................... 4151
Gelatin.. ...................................................................................... 4151
Glucose, anhydrous.. ............................................................... 4153
Glucose, liquid, spray-dried.................................................... 4154
Glucose monohydrate.. ........................................................... 4154
General Notices (1) apply to all monographs and other texts
Glycerol mono-oleate............................................................... 4155
Granisetron hydrochloride..................................................... 4156
Guar............................................................................................. 4158
Guar galactomannan.. ............................................................. 4159
4149
 EUROPEAN PHARMACOPOEIA 6.3

4150 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Gelatin

01/2009:1215 TESTS
Solution S. Dissolve, with heating in a water-bath at 50 °C,
GALACTOSE 10.0 g in carbon dioxide-free water R prepared from distilled
water R and dilute to 50 ml with the same solvent.
Galactosum Appearance of solution. Solution S is clear (2.2.1) and
not more intensely coloured than reference solution B8
(2.2.2, Method II).
Acidity or alkalinity. To 30 ml of solution S add 0.3 ml of
phenolphthalein solution R. The solution is colourless. Not
more than 1.5 ml of 0.01 M sodium hydroxide is required to
change the colour of the indicator to pink.
Specific optical rotation (2.2.7) : + 78.0 to + 81.5 (anhydrous
substance).
C6H12O6 Mr 180.2 Dissolve 10.00 g in 80 ml of water R and add 0.2 ml of
[59-23-4] dilute ammonia R1. Allow to stand for 30 min and dilute to
100.0 ml with water R.
DEFINITION
D-Galactopyranose.
Barium. Dilute 5 ml of solution S to 10 ml with distilled
water R. Add 1 ml of dilute sulphuric acid R. When
CHARACTERS examined immediately and after 1 h, any opalescence in the
Appearance : white or almost white, crystalline or finely solution is not more intense than that in a mixture of 5 ml of
granulated powder. solution S and 6 ml of distilled water R.
Solubility : freely soluble or soluble in water, very slightly Lead (2.4.10) : maximum 0.5 ppm.
soluble in ethanol (96 per cent). Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g.
IDENTIFICATION Sulphated ash: maximum 0.1 per cent.
First identification : A. To 5 ml of solution S add 2 ml of sulphuric acid R, evaporate
to dryness on a water-bath and ignite to constant mass. The
Second identification : B, C. residue weighs a maximum of 1 mg.
A. Infrared absorption spectrophotometry (2.2.24). Microbial contamination
Preparation : discs. TAMC : acceptance criterion 102 CFU/g (2.6.12).
Comparison : galactose CRS.
B. Thin-layer chromatography (2.2.27).
01/2009:0330
Test solution. Dissolve 10 mg of the substance to be
examined in a mixture of 2 volumes of water R and
3 volumes of methanol R and dilute to 20 ml with the GELATIN
same mixture of solvents.
Reference solution (a). Dissolve 10 mg of galactose CRS Gelatina
in a mixture of 2 volumes of water R and 3 volumes of
methanol R and dilute to 20 ml with the same mixture of DEFINITION
solvents. Purified protein obtained either by partial acid hydrolysis
(type A), partial alkaline hydrolysis (type B) or enzymatic
Reference solution (b). Dissolve 10 mg of galactose CRS,
hydrolysis of collagen from animals (including fish and
10 mg of glucose CRS and 10 mg of lactose CRS in
poultry) ; it may also be a mixture of different types.
a mixture of 2 volumes of water R and 3 volumes of
methanol R and dilute to 20 ml with the same mixture of The hydrolysis leads to gelling or non-gelling product grades.
solvents. Both product grades are covered by this monograph.
Plate : suitable silica gel as the coating substance. Gelatin described in this monograph is not suitable for
parenteral use or for other special purposes.
Mobile phase : water R, propanol R (15:85 V/V).
Application : 2 μl ; thoroughly dry the starting points. CHARACTERS
Development : in an unsaturated tank over a path of Appearance : faintly yellow or light yellowish-brown, solid,
15 cm. usually occurring as translucent sheets, shreds, granules
Drying : in a current of warm air. or powder.
Detection : spray with a solution of 0.5 g of thymol R in a Solubility : practically insoluble in common organic solvents ;
mixture of 5 ml of sulphuric acid R and 95 ml of ethanol gelling grades swell in cold water and give on heating a
(96 per cent) R. Heat in an oven at 130 °C for 10 min. colloidal solution which on cooling forms a more or less firm
gel.
System suitability : reference solution (b) :
The isoelectric point is a relevant quality parameter for use
— the chromatogram shows 3 clearly separated spots. of gelatin in different applications : for type A gelatin it is
Results : the principal spot in the chromatogram obtained typically between pH 6.0 and pH 9.5 and for type B gelatin
with the test solution is similar in position, colour and is typically between pH 4.7 and pH 5.6. These ranges cover
size to the principal spot in the chromatogram obtained a variety of different gelatins and for specific applications a
with reference solution (a). narrower tolerance is usually applied.
C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of Different gelatins form aqueous solutions that vary in
cupri-tartaric solution R and heat. An orange or red clarity and colour. For a particular application, a suitable
precipitate is formed. specification for clarity and colour is usually applied.

General Notices (1) apply to all monographs and other texts 4151
Gelatin
IDENTIFICATION
A. To 2 ml of solution S (see Tests) add 0.05 ml of copper
sulphate solution R. Mix and add 0.5 ml of dilute sodium 1-4 h. Heat in a water-bath at 65 ± 2 °C for 15 min. While
hydroxide solution R. A violet colour is produced.
B. To 0.5 g in a test-tube add 10 ml of water R. Allow to
stand for 10 min, heat at 60 °C for 15 min and keep the
tube upright at 0 °C for 6 h. Invert the tube ; the contents for 15 min and transfer the bottles to a thermostatically
immediately flow out for non-gelling grades and do not
flow out immediately for gelling grades.
TESTS
Solution S. Dissolve 1.00 g in carbon dioxide-free water R
at about 55 °C, dilute to 100 ml with the same solvent and
keep the solution at this temperature to carry out the tests. Report the result as the average of the 2 measurements.
pH (2.2.3) : 3.8 to 7.6 for solution S.
Conductivity (2.2.38) : maximum 1 mS·cm− 1, determined on
a 1.0 per cent solution at 30 ± 1.0 °C.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
Peroxides: maximum 10 ppm, determined using peroxide
test strips R.
Peroxidase transfers oxygen from peroxides to an organic
redox indicator which is converted to a blue oxidation
product. The intensity of the colour obtained is proportional
to the quantity of peroxide and can be compared with a
colour scale provided with the test strips, to determine the
peroxide concentration.
Suitability test. Dip a test strip for 1 s into hydrogen
peroxide standard solution (10 ppm H2O2) R, such that the
reaction zone is properly wetted. Remove the test strip,
shake off excess liquid and compare the reaction zone after
15 s with the colour scale provided with the test strips used.
The colour must match that of the 10 ppm concentration,
otherwise the test is invalid.
Test. Weigh 20.0 ± 0.1 g of the substance to be tested in a
beaker and add 80.0 ± 0.2 ml of water R. Stir to moisten all
gelatin and allow the sample to stand at room temperature
for 1-3 h. Cover the beaker with a watch-glass. Place the
beaker for 20 ± 5 min in a water bath at 65 ± 2 °C to dissolve
the sample. Stir the contents of the beaker with a glass
rod to achieve a homogeneous solution. Dip a test strip
for 1 s into the test solution, such that the reaction zone
is properly wetted. Remove the test strip, shake off excess
liquid and compare the reaction zone after 15 s with the
colour scale provided with the test strips used. Multiply the
concentration read from the colour scale by a factor of 5 to
calculate the concentration in parts per million of peroxide
in the test substance.
Gel strength (Bloom value): 80 to 120 per cent of the
labelled nominal value.
The gel strength is expressed as the mass in grams necessary TYMC : acceptance criterion 10 CFU/g (2.6.12).
to produce the force which, applied to a plunger 12.7 mm in Absence of Escherichia coli (2.6.13).
diameter, makes a depression 4 mm deep in a gel having a
concentration of 6.67 per cent m/m and matured at 10 °C. Absence of Salmonella (2.6.13).
Apparatus. Texture analyser or gelometer with :
— a cylindrical piston 12.7 ± 0.1 mm in diameter with a
plane pressure surface with a sharp bottom edge,
— a bottle 59 ± 1 mm in internal diameter and 85 mm high.
Adjust the apparatus according to the manufacturer’s
manual. Settings are : distance 4 mm, test speed 0.5 mm/s.
4152 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Method. Perform the test in duplicate. Place 7.5 g of the
substance to be tested in each bottle. Add 105 ml of water R,
place a watch-glass over each bottle and allow to stand for
heating, gently stir with a glass rod. Ensure that the solution
is uniform and that any condensed water on the inner walls of
the bottle is incorporated. Allow to cool at room temperature
controlled bath at 10.0 ± 0.1 °C, and fitted with a device
to ensure that the platform on which the bottles stand is
perfectly horizontal. Close the bottles with a rubber stopper
and allow to stand for 17 ± 1 h. Remove the sample bottles
from the bath and quickly wipe the water from the exterior of
the bottle. Centre consecutively the 2 bottles on the platform
of the apparatus so that the plunger contacts the sample as
nearly at its midpoint as possible and start the measurement.
Iron : maximum 30.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. To 5.00 g of the substance to be examined, in
a conical flask, add 10 ml of hydrochloric acid R. Close the
flask and place in a water-bath at 75-80 °C for 2 h. Allow
to cool and adjust the content of the flask to 100.0 g with
water R.
Reference solutions. Prepare the reference solutions using
iron standard solution (8 ppm Fe) R, diluted as necessary
with water R.
Wavelength : 248.3 nm.
Chromium : maximum 10.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Test solution described in the test for iron.
Reference solutions. Prepare the reference solutions using
chromium standard solution (100 ppm Cr) R, diluted if
necessary with water R.
Wavelength : 357.9 nm.
Zinc : maximum 30.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Test solution described in the test for iron.
Reference solutions. Prepare the reference solutions using
zinc standard solution (10 ppm Zn) R, diluted if necessary
with water R.
Wavelength : 213.9 nm.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.000 g, by drying in an oven at 105 °C.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
2
STORAGE
Protect from heat and moisture.
LABELLING
The label states the gel strength (Bloom value) or that it is a
non-gelling grade.
EUROPEAN PHARMACOPOEIA 6.3
01/2008:0177 C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of
corrected 6.0 cupri-tartaric solution R and heat. A red precipitate is
GLUCOSE, ANHYDROUS
Glucosum anhydricum
C6H12O6 Mr 180.2 of 0.1 M sodium hydroxide is required to change the colour
[50-99-7]
DEFINITION
(+)-D-Glucopyranose.
CHARACTERS
Appearance : white or almost white, crystalline powder.
It has a sweet taste.
Solubility : freely soluble in water, sparingly soluble in
ethanol (96 per cent).
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Thin-layer chromatography (2.2.27).
Solvent mixture : water R, methanol R (2:3 V/V).
Test solution. Dissolve 10 mg of the substance to be
examined in the solvent mixture and dilute to 20 ml with
the solvent mixture.
Reference solution (a). Dissolve 10 mg of glucose CRS in
the solvent mixture and dilute to 20 ml with the solvent
mixture.
Reference solution (b). Dissolve 10 mg each of
fructose CRS, glucose CRS, lactose CRS and
sucrose CRS in the solvent mixture and dilute to 20 ml
with the solvent mixture.
Plate : TLC silica gel G plate R.
Mobile phase : water R, methanol R, anhydrous acetic
acid R, ethylene chloride R (10:15:25:50 V/V/V/V) ;
measure the volumes accurately since a slight excess of
water produces cloudiness.
Application : 2 μl ; thoroughly dry the starting points.
Development A : over a path of 15 cm.
Drying A: in a current of warm air.
Development B : immediately, over a path of 15 cm, after
renewing the mobile phase.
Drying B: in a current of warm air.
Detection : spray with a solution of 0.5 g of thymol R in a
mixture of 5 ml of sulphuric acid R and 95 ml of ethanol
(96 per cent) R. Heat at 130 °C for 10 min.
System suitability : reference solution (b) :
— the chromatogram shows 4 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
General Notices (1) apply to all monographs and other texts
Glucose, anhydrous
formed.
TESTS
Solution S. Dissolve 10.0 g in distilled water R and dilute to
100 ml with the same solvent.
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY7
(2.2.2, Method II).
Dissolve 10.0 g in 15 ml of water R.
Acidity or alkalinity. Dissolve 6.0 g in 25 ml of carbon
dioxide-free water R and add 0.3 ml of phenolphthalein
solution R. The solution is colourless. Not more than 0.15 ml
of the indicator to pink.
Specific optical rotation (2.2.7) : + 52.5 to + 53.3 (anhydrous
substance).
Dissolve 10.0 g in 80 ml of water R, add 0.2 ml of dilute
ammonia R1, allow to stand for 30 min and dilute to
100.0 ml with water R.
Foreign sugars, soluble starch, dextrins. Dissolve 1.0 g by
boiling in 30 ml of ethanol (90 per cent V/V) R. Cool ; the
appearance of the solution shows no change.
Sulphites : maximum 15 ppm, expressed as SO2.
Test solution. Dissolve 5.0 g in 40 ml of water R, add 2.0 ml
of 0.1 M sodium hydroxide and dilute to 50.0 ml with
water R. To 10.0 ml of the solution, add 1 ml of a 310 g/l
solution of hydrochloric acid R, 2.0 ml of decolorised
fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution
of formaldehyde R. Allow to stand for 30 min.
Reference solution. Dissolve 76 mg of sodium
metabisulphite R in water R and dilute to 50.0 ml with the
same solvent. Dilute 5.0 ml of this solution to 100.0 ml
with water R. To 3.0 ml of this solution add 4.0 ml of 0.1 M
sodium hydroxide and dilute to 100.0 ml with water R.
Immediately add to 10.0 ml of this solution 1 ml of a 310 g/l
solution of hydrochloric acid R, 2.0 ml of decolorised
fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution
of formaldehyde R. Allow to stand for 30 min.
Measure the absorbance (2.2.25) of the 2 solutions
at the absorption maximum at 583 nm using for both
measurements a solution prepared in the same manner
using 10.0 ml of water R as the compensation liquid. The
absorbance of the test solution is not greater than that of
the reference solution.
Chlorides (2.4.4) : maximum 125 ppm.
Dilute 4 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 200 ppm.
Dilute 7.5 ml of solution S to 15 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 1 ppm, determined
on 1.0 g.
Barium. To 10 ml of solution S add 1 ml of dilute sulphuric
acid R. When examined immediately and after 1 h, any
opalescence in the solution is not more intense than that in a
mixture of 1 ml of distilled water R and 10 ml of solution S.
Calcium (2.4.3) : maximum 200 ppm.
Dilute 5 ml of solution S to 15 ml with distilled water R.
Lead (2.4.10) : maximum 0.5 ppm.
Water (2.5.12) : maximum 1.0 per cent, determined on 0.50 g.
4153
Glucose, liquid, spray-dried EUROPEAN PHARMACOPOEIA 6.3

Sulphated ash : maximum 0.1 per cent.
Dissolve 5.0 g in 5 ml of water R, add 2 ml of sulphuric
acid R, evaporate to dryness on a water-bath and ignite
to constant mass. If necessary, repeat the heating with
sulphuric acid R.
Pyrogens (2.6.8). If intended for use in the manufacture
of large-volume parental preparations without a further
appropriate procedure for the removal of pyrogens, the
competent authority may require that it comply with the test
for pyrogens. Inject per kilogram of the rabbit’s mass 10 ml
of a solution in water for injections R containing 50 mg of
the substance to be examined per millilitre.
Pipette 25.0 ml of cupri-tartaric solution R into a 250 ml
flask and add 18.5 ml of the test solution from the burette,
mix and add a few glass beads. Place the flask on a hot plate,
previously adjusted so that the solution begins to boil after
2 min ± 15 s. Allow to boil for exactly 120 s, add 1 ml of a
1 g/l solution of methylene blue R and titrate with the test
solution (V1) until the blue colour disappears. Maintain the
solution at boiling throughout the titration.
Standardise the cupri-tartaric solution using a 6.00 g/l
solution of glucose R (V0).
Calculate the dextrose equivalent using the following
expression :

01/2009:1525

GLUCOSE, LIQUID, SPRAY-DRIED V0 = total volume of glucose standard solution, in

millilitres ;
Glucosum liquidum dispersione desiccatum V1 = total volume of test solution, in millilitres ;
M = mass of the sample, in grams ;
DEFINITION
Mixture of glucose, oligosaccharides and polysaccharides, D = percentage content of dry matter in the substance.
obtained by the partial hydrolysis of starch. Microbial contamination
The degree of hydrolysis, expressed as dextrose TAMC : acceptance criterion 103 CFU/g (2.6.12).
equivalent (DE), is not less than 20 (nominal value).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
CHARACTERS Absence of Escherichia coli (2.6.13).
Appearance : white or almost white, slightly hygroscopic Absence of Salmonella (2.6.13).
powder or granules.
Solubility : freely soluble in water. LABELLING
The label states the dextrose equivalent (DE) (= nominal
IDENTIFICATION value).
A. Dissolve 0.1 g in 2.5 ml of water R and heat with 2.5 ml
of cupri-tartaric solution R. A red precipitate is formed.
01/2008:0178
B. Dip, for 1 s, a suitable stick with a reactive pad containing corrected 6.0
glucose-oxidase, peroxidase and a hydrogen-donating
substance, such as tetramethylbenzidine, in a 5 g/l
solution of the substance to be examined. Observe the GLUCOSE MONOHYDRATE
colour of the reactive pad ; within 60 s the colour changes
from yellow to green or blue. Glucosum monohydricum
C. It is a powder or granules.
D. Dextrose equivalent (see Tests).
TESTS
Solution S. Dissolve 12.5 g in carbon dioxide-free water R
and dilute to 50.0 ml with the same solvent.
pH (2.2.3) : 4.0 to 7.0.
Mix 1 ml of a 223.6 g/l solution of potassium chloride R C6H12O6,H2O Mr 198.2
and 30 ml of solution S. [5996-10-1]
Sulphur dioxide (2.5.29) : maximum 20 ppm.
DEFINITION
Heavy metals (2.4.8) : maximum 10 ppm.
(+)-D-Glucopyranose monohydrate.
Dilute 4 ml of solution S to 30 ml with water R. The solution
complies with test E. Prepare the reference solution using CHARACTERS
10 ml of lead standard solution (1 ppm Pb) R. Appearance : white or almost white, crystalline powder.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined It has a sweet taste.
on 10.00 g by drying in an oven at 105 °C. Solubility : freely soluble in water, sparingly soluble in
Sulphated ash (2.4.14) : maximum 0.5 per cent, determined ethanol (96 per cent).
on 1.0 g.
IDENTIFICATION
Dextrose equivalent (DE) : within 10 per cent of the nominal
value. A. Specific optical rotation (see Tests).
Weigh an amount of the substance to be examined equivalent B. Thin-layer chromatography (2.2.27).
to 2.85-3.15 g of reducing carbohydrates, calculated as Solvent mixture : water R, methanol R (2:3 V/V).
dextrose equivalent, into a 500 ml volumetric flask. Dissolve Test solution. Dissolve 10 mg of the substance to be
in water R and dilute to 500.0 ml with the same solvent. examined in the solvent mixture and dilute to 20 ml with
Transfer the solution to a 50 ml burette. the solvent mixture.

4154 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Reference solution (a). Dissolve 10 mg of glucose CRS in
the solvent mixture and dilute to 20 ml with the solvent
mixture.
Reference solution (b). Dissolve 10 mg each of
fructose CRS, glucose CRS, lactose CRS and
sucrose CRS in the solvent mixture and dilute to 20 ml
with the solvent mixture.
Plate : TLC silica gel G plate R.
Mobile phase : water R, methanol R, anhydrous acetic
acid R, ethylene chloride R (10:15:25:50 V/V/V/V) ;
measure the volumes accurately since a slight excess of
water produces cloudiness.
Application : 2 μl ; thoroughly dry the starting points.
Development A : over a path of 15 cm.
Drying A: in a current of warm air.
Development B : immediately, over a path of 15 cm, after
renewing the mobile phase.
Drying B: in a current of warm air.
Detection : spray with a solution of 0.5 g of thymol R in a mixture of 1 ml of distilled water R and 10 ml of solution S.
mixture of 5 ml of sulphuric acid R and 95 ml of ethanol
(96 per cent) R ; heat at 130 °C for 10 min.
System suitability : reference solution (b) :
— the chromatogram shows 4 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of
cupri-tartaric solution R and heat. A red precipitate is
formed.
TESTS
Solution S. Dissolve 10.0 g in distilled water R and dilute to of large-volume parenteral preparations without a further
100 ml with the same solvent.
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY7
(2.2.2, Method II).
Dissolve 10.0 g in 15 ml of water R.
Acidity or alkalinity. Dissolve 6.0 g in 25 ml of carbon
dioxide-free water R and add 0.3 ml of phenolphthalein
solution R. The solution is colourless. Not more than 0.15 ml
of 0.1 M sodium hydroxide is required to change the colour
of the indicator to pink.
Specific optical rotation (2.2.7) : + 52.5 to + 53.3 (anhydrous
substance).
Dissolve 10.0 g in 80 ml of water R, add 0.2 ml of dilute
ammonia R1, allow to stand for 30 min and dilute to
100.0 ml with water R.
Foreign sugars, soluble starch, dextrins. Dissolve 1.0 g by together with variable quantities of di- and triacylglycerols.
boiling in 30 ml of ethanol (90 per cent V/V) R. Cool ; the
appearance of the solution shows no change.
Sulphites : maximum 15 ppm, expressed as SO2.
Test solution. Dissolve 5.0 g in 40 ml of water R, add 2.0 ml or by esterification of glycerol by oleic acid, this fatty acid
of 0.1 M sodium hydroxide and dilute to 50.0 ml with
water R. To 10.0 ml of the solution, add 1 ml of a 310 g/l
solution of hydrochloric acid R, 2.0 ml of decolorised
fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution
of formaldehyde R. Allow to stand for 30 min.
Reference solution. Dissolve 76 mg of sodium
metabisulphite R in water R and dilute to 50.0 ml with the
same solvent. Dilute 5.0 ml of this solution to 100.0 ml
with water R. To 3.0 ml of this solution add 4.0 ml of 0.1 M
sodium hydroxide and dilute to 100.0 ml with water R.
Immediately add to 10.0 ml of this solution 1 ml of a 310 g/l
General Notices (1) apply to all monographs and other texts
Glycerol mono-oleate
solution of hydrochloric acid R, 2.0 ml of decolorised
fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution
of formaldehyde R. Allow to stand for 30 min.
Measure the absorbance (2.2.25) of the 2 solutions
at the absorption maximum at 583 nm using for both
measurements a solution prepared in the same manner
using 10.0 ml of water R as the compensation liquid. The
absorbance of the test solution is not greater than that of
the reference solution.
Chlorides (2.4.4) : maximum 125 ppm.
Dilute 4 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 200 ppm.
Dilute 7.5 ml of solution S to 15 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 1 ppm, determined
on 1.0 g.
Barium. To 10 ml of solution S add 1 ml of dilute sulphuric
acid R. When examined immediately and after 1 h, any
opalescence in the solution is not more intense than that in a
Calcium (2.4.3) : maximum 200 ppm.
Dilute 5 ml of solution S to 15 ml with distilled water R.
Lead (2.4.10) : maximum 0.5 ppm.
Water (2.5.12) : 7.0 per cent to 9.5 per cent, determined on
0.50 g.
Sulphated ash: maximum 0.1 per cent.
Dissolve 5.0 g in 5 ml of water R, add 2 ml of sulphuric
acid R, evaporate to dryness on a water-bath and ignite
to constant mass. If necessary, repeat the heating with
sulphuric acid R.
Pyrogens (2.6.8). If intended for use in the manufacture
appropriate procedure for the removal of pyrogens, the
competent authority may require that it comply with the test
for pyrogens. Inject per kilogram of the rabbit’s mass 10 ml
of a solution in water for injections R containing 55 mg of
the substance to be examined per millilitre.
01/2008:1430
corrected 6.3
GLYCEROL MONO-OLEATE
Glyceroli mono-oleas
DEFINITION
Mixture of monoacylglycerols, mainly mono-oleoylglycerol,
It is defined by the nominal content of monoacylglycerols
and obtained by partial glycerolysis of vegetable oils mainly
containing triacylglycerols of oleic (cis-9-octadecenoic) acid
being of vegetable or animal origin. A suitable antioxidant
may be added.
Content :
Nominal content of acylglycerol
(per cent)
40 60 90
Monoacylglycerols 32.0 - 52.0 55.0 - 65.0 90.0 - 101.0
Diacylglycerols 30.0 - 50.0 15.0 - 35.0 < 10.0
5.0 - 20.0 2.0 - 10.0 < 2.0
Triacylglycerols
4155
Granisetron hydrochloride EUROPEAN PHARMACOPOEIA 6.3

CHARACTERS Reference solutions. Into four 15 ml flasks, respectively

Appearance : amber, oily liquid which may be partially weigh, to the nearest 0.1 mg, about 2.5 mg, 5 mg, 10 mg and
solidified at room temperature. 20 mg of glycerol R. Add 5 ml of tetrahydrofuran R and
shake until well mixed. Weigh the flasks again and calculate
Solubility : practically insoluble in water, freely soluble inthe concentration of glycerol in milligrams per gram for each
methylene chloride. reference solution.
IDENTIFICATION Column :
A. Iodine value (see Tests). — size: l = 0.6 m, Ø = 7 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
B. Thin-layer chromatography (2.2.27).
(5 μm) with a pore size of 10 nm.
Test solution. Dissolve 1.0 g of the substance to be Mobile phase : tetrahydrofuran R.
examined in methylene chloride R and dilute to 20 ml
with the same solvent. Flow rate : 1 ml/min.
Reference solution. Dissolve 1.0 g of glycerol Detection : differential refractometer.
mono-oleate CRS in methylene chloride R and dilute to Injection : 40 μl.
20 ml with the same solvent. Relative retention with reference to glycerol (retention
Plate : TLC silica gel plate R. time = about 15.6 min) : triacylglycerols = about 0.76 ;
diacylglycerols = about 0.79 ; monoacylglycerols = about 0.85.
Mobile phase : hexane R, ether R (30:70 V/V).
Calculations :
Application : 10 μl.
— free glycerol : from the calibration curve obtained with
Development : over a path of 15 cm. the reference solutions determine the concentration (C)
Drying : in air. of glycerol in milligrams per gram in the test solution and
Detection : spray with a 0.1 g/l solution of rhodamine calculate the percentage content of free glycerol in the
B R in ethanol (96 per cent) R and examine in ultraviolet substance to be examined using the following expression :
light at 365 nm.
Results : the spots in the chromatogram obtained with
the test solution are similar in position to those in the
chromatogram obtained with the reference solution. — mono-, di- and triacylglycerols : calculate the percentage
content of mono-, di- and triacylglycerols using the
C. It complies with the limits of the assay (monoacylglycerol normalisation procedure.
content).
STORAGE
TESTS In an airtight container, protected from light.
Acid value (2.5.1) : maximum 6.0, determined on 1.0 g.
LABELLING
Iodine value (2.5.4, Method A) : 65.0 to 95.0.
The label states the nominal content of monoacylglycerol.
Peroxide value (2.5.5, Method A) : maximum 12.0,
determined on 2.0 g.
Saponification value (2.5.6) : 150 to 175, determined on
2.0 g. 01/2008:1695
corrected 6.3
Free glycerol : maximum 6.0 per cent, determined as
described in the assay.
Composition of fatty acids (2.4.22, Method C).
GRANISETRON HYDROCHLORIDE
Composition of the fatty acid fraction of the substance :
Granisetroni hydrochloridum
— palmitic acid : maximum 12.0 per cent,
— stearic acid : maximum 6.0 per cent,
— oleic acid : minimum 60.0 per cent,
— linoleic acid : maximum 35.0 per cent,
— linolenic acid : maximum 2.0 per cent,
— arachidic acid : maximum 2.0 per cent,
— eicosenoic acid : maximum 2.0 per cent.
C18H25ClN4O Mr 348.9
Water (2.5.12) : maximum 1.0 per cent, determined on [107007-99-8]
1.00 g. Use as the solvent a mixture of equal volumes of
anhydrous methanol R and methylene chloride R. DEFINITION
Total ash (2.4.16) : maximum 0.1 per cent. 1-Methyl-N-[(1R,3r,5S)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-
1H-indazole-3-carboxamide hydrochloride.
ASSAY Content : 97.0 per cent to 102.0 per cent (dried substance).
Size-exclusion chromatography (2.2.30).
CHARACTERS
Test solution. Into a 15 ml flask, weigh about 0.2 g (m),
to the nearest 0.1 mg. Add 5 ml of tetrahydrofuran R and Appearance : white or almost white powder.
shake to dissolve. Reweigh the flask and calculate the total Solubility : freely soluble in water, sparingly soluble in
mass of solvent and substance (M). methylene chloride, slightly soluble in methanol.

4156 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : granisetron hydrochloride CRS.
B. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 0.2 g in carbon dioxide-free water R
and dilute to 20 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3) : 4.0 to 6.5 for solution S.
Impurity E. Thin-layer chromatography (2.2.27).
Solvent mixture : water R, acetonitrile R (20:80 V/V).
Test solution. Dissolve 0.25 g of the substance to be
examined in the solvent mixture and dilute to 5 ml with the
solvent mixture.
Reference solution. Dissolve 5.0 mg of granisetron
impurity E CRS in the solvent mixture and dilute to 20.0 ml
with the solvent mixture.
Plate : TLC silica gel F254 plate R.
Mobile phase : concentrated ammonia R, 2-propanol R,
ethyl acetate R (6.5:30:50 V/V/V).
Application : 2 μl.
Development : over half of the plate.
Drying : in air.
Detection : expose to iodine vapour for 30 min.
Limit :
— impurity E : any spot due to impurity E is not more
intense than the principal spot in the chromatogram
obtained with the reference solution (0.5 per cent).
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to
50.0 ml with the mobile phase. Dilute 5.0 ml of this solution
to 20.0 ml with the mobile phase.
Reference solution (b). Transfer 2 ml of the test solution
to a colourless glass vial, stopper and expose the solution
either to sunlight for 4 h or under a UV lamp for 16 h (partial
degradation of granisetron to impurity C). A degradation of
at least about 0.3 per cent of granisetron to impurity C must
be obtained as shown by appearance of a corresponding
peak in the chromatogram. If not, expose the solution once
again to sunlight or under a UV lamp.
Reference solution (c). Dissolve 50.0 mg of granisetron
hydrochloride CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase.
Reference solution (d). Dissolve the contents of a vial of
granisetron impurity A CRS in 1 ml of the mobile phase.
Reference solution (e). Dissolve the contents of a vial of
granisetron impurity B CRS in 1 ml of the mobile phase.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : spherical base-deactivated end-capped
octadecylsilyl silica gel for chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase : dilute 1.6 ml of phosphoric acid R to 800 ml
with water R, add 200 ml of acetonitrile R and mix. Add
1.0 ml of hexylamine R and mix. Adjust to pH 7.5 ± 0.05
with freshly distilled triethylamine R (about 4 ml).
General Notices (1) apply to all monographs and other texts
Granisetron hydrochloride
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 305 nm.
Injection : 10 μl of the test solution and reference
solutions (a), (b), (d) and (e).
Run time : twice the retention time of granisetron.
Relative retention with reference to granisetron
(retention time = about 7 min) : impurity D = about 0.4 ;
impurity B = about 0.5 ; impurity A = about 0.7 ;
impurity C = about 0.8.
System suitability :
— resolution : minimum 3.5 between the peaks due to
impurity C and granisetron in the chromatogram obtained
with reference solution (b) ;
— symmetry factor : maximum 2.0 for the peak due to
granisetron.
Limits :
— correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.7 ;
— impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
— impurity C : not more than 0.4 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
— impurity D : not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent) ;
— any other impurity: for each impurity, not more
than 0.2 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.1 per cent) ;
— total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
— disregard limit : 0.1 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard any peak due to the blank.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
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 and reference solution (c).
Calculate the percentage content of C18H25ClN4O using the
declared content of granisetron hydrochloride CRS.
IMPURITIES
Specified impurities: A, B, C, D, E.
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) : F, G, H, I.
4157
Guar
A. 2-methyl-N-[(1R,3r,5S)-9-methyl-9-azabicyclo[3.3.1]non-3-
yl]-2H-indazole-3-carboxamide,
B. R = H, R′ = CH3 : N-[(1R,3r,5S)-9-methyl-9-
azabicyclo[3.3.1]non-3-yl]-1H-indazole-3-carboxamide,
C. R = CH3, R′ = H : N-[(1R,3r,5S)-9-azabicyclo[3.3.1]non-3-
yl]-1-methyl-1H-indazole-3-carboxamide,
D. R = CH3 : 1-methyl-1H-indazole-3-carboxylic acid,
H. R = H : 1H-indazole-3-carboxylic acid,
E. (1R,3r,5S)-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine,
F. 1-methyl-N-[(1R,3s,5S)-9-methyl-9-azabicyclo[3.3.1]non-3-
yl]-1H-indazole-3-carboxamide (exo-granisetron),
G. 2-methyl-2H-indazole-3-carboxylic acid,
I. 1-methyl-1H-indazole-3-carboxylic anhydride.
4158 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:1218
GUAR
Cyamopsidis seminis pulvis
DEFINITION
Guar is obtained by grinding the endosperms of seeds of
Cyamopsis tetragonolobus (L.) Taub. It consists mainly of
guar galactomannan.
CHARACTERS
Appearance : white or almost white powder.
Solubility : it yields a mucilage of variable viscosity when
dissolved in water, practically insoluble in ethanol (96 per
cent).
IDENTIFICATION
A. Examined under a microscope in glycerol R, the
substance to be examined (125) (2.9.12) shows pyriform
or ovoid cells, usually isolated, having very thick walls
around a central somewhat elongated lumen with
granular contents, and smaller polyhedral cells, isolated
or in clusters, with thinner walls.
B. In a conical flask place 2 g, add rapidly 45 ml of water R
and stir vigorously for 30 s. After 5-10 min a stiff gel
forms which does not flow when the flask is inverted.
C. Mix a suspension of 0.1 g in 10 ml of water R with 1 ml of
a 10 g/l solution of disodium tetraborate R ; the mixture
soon gels.
D. Thin-layer chromatography (2.2.27).
Test solution. To 10 mg in a thick-walled centrifuge test
tube add 2 ml of a 100 g/l solution of trifluoroacetic
acid R, shake vigorously to dissolve the forming gel,
stopper the test tube and heat the mixture at 120 °C
for 1 h. Centrifuge the hydrolysate, transfer the clear
supernatant liquid carefully into a 50 ml flask, add 10 ml
of water R and evaporate the solution to dryness under
reduced pressure. To the resulting clear film add 0.1 ml of
water R and 0.9 ml of methanol R. Centrifuge to separate
the amorphous precipitate. Dilute the supernatant liquid,
if necessary, to 1 ml with methanol R.
Reference solution. Dissolve 10 mg of galactose R and
10 mg of mannose R in 2 ml of water R, then dilute to
20 ml with methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, acetonitrile R (15:85 V/V).
Application : 5 μl, as bands.
Development : over a path of 15 cm.
Detection : spray with aminohippuric acid reagent R and
dry at 120 °C for 5 min.
Results : the chromatogram obtained with the reference
solution shows, in the lower part 2 clearly separated
brownish zones due to galactose and mannose in order
of increasing RF value. The chromatogram obtained with
the test solution shows 2 zones due to galactose and
mannose.
TESTS
Tragacanth, sterculia gum, agar, alginates, carrageenan.
To a small amount of the substance to be examined add
0.2 ml of freshly prepared ruthenium red solution R.
Examined under a microscope the cell walls do not stain red.
EUROPEAN PHARMACOPOEIA 6.3 Guar galactomannan

Protein : maximum 8.0 per cent. of methanol R. Centrifuge to separate the amorphous
Carry out the determination of nitrogen by the method of precipitate. Dilute the supernatant liquid, if necessary,
sulphuric acid digestion (2.5.9) using 0.170 g. Multiply the to 1 ml with methanol R.
result by 6.25. Reference solution. Dissolve 10 mg of galactose R and
Apparent viscosity (2.2.10) : 85 per cent to 115 per cent of 10 mg of mannose R in 2 ml of water R and dilute to
the value stated on the label. 10 ml with methanol R.
Moisten a quantity equivalent to 1.00 g of the dried substance Plate : TLC silica gel G plate R.
with 2.5 ml of 2-propanol R. While stirring, dilute to 100.0 ml Mobile phase : water R, acetonitrile R (15:85 V/V).
with water R. After 1 h, determine the viscosity at 20 °C Application : 5 μl, as bands of 20 mm by 3 mm.
using a rotating viscometer and a shear rate of 100 s−1. Development : over a path of 15 cm.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined Detection : spray with aminohippuric acid reagent R and
on 1.000 g by drying in an oven at 105 °C for 5 h. heat at 120 °C for 5 min.
Total ash (2.4.16) : maximum 1.8 per cent. Results : the chromatogram obtained with the reference
Microbial contamination solution shows in the lower part 2 clearly separated
4 brownish zones (galactose and mannose in order of
TAMC : acceptance criterion 10 CFU/g (2.6.12). increasing RF value). The chromatogram obtained with
TYMC : acceptance criterion 102 CFU/g (2.6.12). the test solution shows 2 zones due to galactose and
Absence of Escherichia coli (2.6.13). mannose.
Absence of Salmonella (2.6.13). TESTS
LABELLING Solution S. Moisten 1.0 g with 2 ml of 2-propanol R. While
The label states the apparent viscosity in millipascal seconds stirring, dilute to 100 g with water R and stir until the
for a 10 g/l solution. substance is uniformly dispersed. Allow to stand for at least
1 h. If the apparent viscosity is below 200 mPa·s, use 3.0 g
of substance instead of 1.0 g.
01/2009:0908 pH (2.2.3) : 5.5 to 7.5 for solution S.
Apparent viscosity (2.2.10) : 75 per cent to 140 per cent of
GUAR GALACTOMANNAN the value stated on the label.
Moisten a quantity of the substance to be examined
equivalent to 2.00 g of the dried substance with 2.5 ml of
Guar galactomannanum 2-propanol R and, while stirring, dilute to 100.0 ml with
DEFINITION water R. After 1 h, determine the viscosity at 20 °C using a
rotating viscometer and a shear rate of 100 s–1.
Guar galactomannan is obtained from the seeds of
Cyamopsis tetragonolobus (L.) Taub. by grinding of the Insoluble matter : maximum 7.0 per cent.
endosperms and subsequent partial hydrolysis. The main In a 250 ml flask disperse, while stirring, 1.50 g in a mixture
components are polysaccharides composed of D-galactose and of 1.6 ml of sulphuric acid R and 150 ml of water R and
D-mannose at molecular ratios of 1:1.4 to 1:2. The molecules weigh. Immerse the flask in a water-bath and heat under
consist of a linear main chain of β-(1→4)-glycosidically a reflux condenser for 6 h. Adjust to the original mass
linked mannopyranoses and single α-(1→6)-glycosidically with water R. Filter the hot solution through a tared,
linked galactopyranoses. sintered-glass filter (160) (2.1.2). Rinse the filter with
hot water R and dry at 100-105 °C. The residue weighs a
CHARACTERS maximum of 105 mg.
Appearance : yellowish-white powder. Protein : maximum 5.0 per cent.
Solubility : soluble in cold water and in hot water, practically Carry out the determination of nitrogen by sulphuric acid
insoluble in organic solvents. digestion (2.5.9), using 0.400 g. Multiply the result by 6.25.
IDENTIFICATION Tragacanth, sterculia gum, agar, alginates and
A. Mix 5 g of solution S (see Tests) with 0.5 ml of a 10 g/l carrageenan. To a small amount of the substance to be
solution of disodium tetraborate R. A gel forms within examined, add 0.2 ml of freshly prepared ruthenium red
a short time. solution R. Examined under a microscope, none of the
structures are red.
B. Heat 20 g of solution S in a water-bath for 10 min. Allow
to cool and adjust to the original mass with water R. The Loss on drying (2.2.32) : maximum 15.0 per cent, determined
solution does not gel. on 1.000 g by drying in an oven at 105 °C for 5 h.
C. Thin-layer chromatography (2.2.27). Total ash (2.4.16) : maximum 1.8 per cent, determined on
Test solution. To 10 mg of the substance to be examined 1.00 g after wetting with 10 ml of water R.
in a thick-walled centrifuge tube add 2 ml of a 230 g/l Microbial contamination
solution of trifluoroacetic acid R, shake vigorously to TAMC : acceptance criterion 103 CFU/g (2.6.12).
dissolve the forming gel, stopper the tube and heat the
TYMC : acceptance criterion 102 CFU/g (2.6.12).
mixture at 120 °C for 1 h. Centrifuge the hydrolysate,
transfer the clear supernatant liquid carefully into a 50 ml Absence of Escherichia coli (2.6.13).
flask, add 10 ml of water R and evaporate the solution to Absence of Salmonella (2.6.13).
dryness under reduced pressure. Take up the residue in
10 ml of water R and evaporate again to dryness under LABELLING
reduced pressure. To the resulting clear film, which has The label states the apparent viscosity in millipascal seconds
no odour of acetic acid, add 0.1 ml of water R and 1 ml for a 20 g/l solution.

General Notices (1) apply to all monographs and other texts 4159
 EUROPEAN PHARMACOPOEIA 6.3

4160 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
H
Haemodialysis solutions, concentrated, water for
diluting..................................................................................... 4163
Hard fat.. .................................................................................... 4164
Human haematopoietic stem cells.. ..................................... 4165
Human normal immunoglobulin for intravenous
administration.. ...................................................................... 4166
General Notices (1) apply to all monographs and other texts
Human plasma (pooled and treated for virus
inactivation).. .......................................................................... 4168
Hydroxypropylbetadex............................................................ 4170
Hypromellose.. .......................................................................... 4171
Hypromellose phthalate.. ........................................................4174
4161
 EUROPEAN PHARMACOPOEIA 6.3

4162 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Haemodialysis solutions, concentrated, water for diluting

01/2009:1167 Chlorides (2.4.4) : maximum 50 ppm.

Dilute 1 ml of the water to be examined to 15 ml with water R.
HAEMODIALYSIS SOLUTIONS, The solution complies with the limit test for chlorides.
CONCENTRATED, WATER FOR Fluorides : maximum 0.2 ppm.
Potentiometry (2.2.36, Method I) : use as indicator electrode
DILUTING a fluoride-selective solid-membrane electrode and as
reference electrode a silver-silver chloride electrode.
Aqua ad dilutionem solutionum Test solution. The water to be examined.
concentratarum ad haemodialysim Reference solutions. Dilute 2.0 ml, 4.0 ml and 10.0 ml of
The following monograph is given for information. fluoride standard solution (1 ppm F) R respectively to
20.0 ml with total-ionic-strength-adjustment buffer R1.
The analytical methods described and the limits proposed
are intended to be used for validating the procedure for Carry out the measurement of each solution.
obtaining the water. Nitrates: maximum 2 ppm.
Dilute 2 ml of the water to be examined to 100 ml with
DEFINITION nitrate-free water R. Place 5 ml of the dilution in a test-tube
Water for diluting concentrated haemodialysis solutions immersed in iced water, add 0.4 ml of a 100 g/l solution
is obtained from potable water by distillation, by reverse of potassium chloride R and 0.1 ml of diphenylamine
osmosis, by ion exchange or by any other suitable method. solution R and then, dropwise and with shaking, 5 ml of
The conditions of preparation, transfer and storage are sulphuric acid R. Transfer the tube to a water-bath at 50 °C.
designed to minimise the risk of chemical and microbial Allow to stand for 15 min. Any blue colour in the solution
contamination. is not more intense than that in a standard prepared at the
When water obtained by one of the methods described above same time and in the same manner using a mixture of 0.1 ml
is not available, potable water may be used for home dialysis. of nitrate standard solution (2 ppm NO3) R and 4.9 ml of
Because the chemical composition of potable water varies nitrate-free water R.
considerably from one locality to another, consideration must Sulphates (2.4.13) : maximum 50 ppm.
be given to its chemical composition to enable adjustments Dilute 3 ml of the water to be examined to 15 ml with
to be made to the content of ions so that the concentrations distilled water R. The solution complies with the limit test
in the diluted solution correspond to the intended use. for sulphates.
Attention has also to be paid to the possible presence of Aluminium (2.4.17) : maximum 10 μg/l.
residues from water treatment (for example, chloramines)
and volatile halogenated hydrocarbons. Prescribed solution. To 400 ml of the water to be examined
add 10 ml of acetate buffer solution pH 6.0 R and 100 ml
For the surveillance of the quality of water for diluting of water R.
concentrated haemodialysis solutions, the following methods
may be used to determine the chemical composition and/or Reference solution. Mix 2 ml of aluminium standard
to detect the presence of possible contaminants together solution (2 ppm Al) R, 10 ml of acetate buffer solution
with suggested limits to be obtained. pH 6.0 R and 98 ml of water R.
Blank solution. Mix 10 ml of acetate buffer solution
CHARACTERS pH 6.0 R and 100 ml of water R.
Clear, colourless, liquid. Ammonium : maximum 0.2 ppm.
To 20 ml of the water to be examined in a flat-bottomed
TESTS and transparent tube, add 1 ml of alkaline potassium
Acidity or alkalinity. To 10 ml of the water to be examined, tetraiodomercurate solution R. Allow to stand for 5 min.
freshly boiled and cooled in a borosilicate glass flask, add The solution is not more intensely coloured than a standard
0.05 ml of methyl red solution R. The solution is not prepared at the same time and in the same manner
red. To 10 ml of the water to be examined add 0.1 ml of using a mixture of 4 ml of ammonium standard solution
bromothymol blue solution R1. The solution is not blue. (1 ppm NH4) R and 16 ml of ammonium-free water R.
Oxidisable substances. To 100 ml of the water to be Examine the solutions along the vertical axis of the tube.
examined add 10 ml of dilute sulphuric acid R and 0.1 ml Calcium : maximum 2.0 ppm.
of 0.02 M potassium permanganate and boil for 5 min. The Atomic absorption spectrometry (2.2.23, Method I).
solution remains faintly pink. Test solution. The water to be examined.
Total available chlorine : maximum 0.1 ppm. Reference solutions. Prepare reference solutions (1 ppm to
In a 125 ml test-tube (A), place successively 5 ml of buffer 5 ppm) using calcium standard solution (400 ppm Ca) R.
solution pH 6.5 R, 5 ml of diethylphenylenediamine Source : calcium hollow-cathode lamp.
sulphate solution R and 1 g of potassium iodide R. In a Wavelength : 422.7 nm.
second 125 ml test-tube (B), place successively 5 ml of buffer
solution pH 6.5 R and 5 ml of diethylphenylenediamine Atomisation device : oxidising air-acetylene flame.
sulphate solution R. Add as simultaneously as possible to Magnesium : maximum 2.0 ppm.
tube A 100 ml of the water to be examined and to tube B a Atomic absorption spectrometry (2.2.23, Method I).
reference solution prepared as follows : to 1 ml of a 10 mg/l
Test solution. Dilute 10 ml of the water to be examined to
solution of potassium iodate R, add 1 g of potassium
100 ml with distilled water R.
iodide R and 1 ml of dilute sulphuric acid R ; allow to stand
for 1 min, add 1 ml of dilute sodium hydroxide solution R Reference solutions. Prepare reference solutions
and dilute to 100 ml with water R. Any colour in the mixture (0.1 ppm to 0.5 ppm) using magnesium standard solution
obtained with the water to be examined is not more intense (100 ppm Mg) R.
than that in the mixture obtained with the reference solution. Source : magnesium hollow-cathode lamp.

General Notices (1) apply to all monographs and other texts 4163
Hard fat EUROPEAN PHARMACOPOEIA 6.3

Wavelength : 285.2 nm. Reference solutions. Prepare reference solutions (0 ppm ;

2.5 ppm ; 5.0 ppm ; 7.5 ppm ; 10 ppm) using sodium standard
Atomisation device : oxidising air-acetylene flame.
solution (200 ppm Na) R.
Mercury : maximum 0.001 ppm. Wavelength : 589 nm.
Atomic absorption spectrometry (2.2.23, Method I). Zinc : maximum 0.1 ppm.
Test solution. Add 5 ml of nitric acid R per litre of the Atomic absorption spectrometry (2.2.23, Method I) : use
water to be examined. In a 50 ml borosilicate glass flask sampling and analytical equipment free from zinc or not
with a ground-glass-stopper, place 20 ml of the water to be liable to yield zinc under the conditions of use.
examined and add 1 ml of dilute nitric acid R and shake. Test solution. The water to be examined.
Add 0.3 ml of bromine water R1. Stopper the flask, shake Reference solutions. Prepare reference solutions (0.05 ppm
and heat the stoppered flask at 45 °C for 4 h. Allow to to 0.15 ppm) using zinc standard solution (100 ppm Zn) R.
cool. If the solution does not become yellow, add 0.3 ml of
bromine water R1 and re-heat at 45 °C for 4 h. Add 0.5 ml Source : zinc hollow-cathode lamp.
of a freshly prepared 10 g/l solution of hydroxylamine Wavelength : 213.9 nm.
hydrochloride R. Shake. Allow to stand for 20 min. Atomisation device : oxidising air-acetylene flame.
Reference solutions. Use freshly prepared reference Heavy metals (2.4.8) : maximum 0.1 ppm.
solutions (0.0005 ppm to 0.002 ppm) obtained by diluting Heat 200 ml of the water to be examined in a glass
mercury standard solution (1000 ppm Hg) R with a 5 per evaporating dish on a water-bath until the volume is
cent V/V solution of dilute nitric acid R and treat as reduced to 20 ml. 12 ml of the solution complies with limit
described for the test solution. test A. Prepare the standard using lead standard solution
To a volume of solution suitable for the instrument to be (1 ppm Pb) R.
used, add stannous chloride solution R2 equal to 1/5 of this Microbial contamination
volume. Fit immediately the device for the entrainment of TAMC : acceptance criterion 102 CFU/g (2.6.12).
the mercury vapour. Wait 20 s and pass through the device
a stream of nitrogen R as the carrier gas. Bacterial endotoxins (2.6.14) : less than 0.25 IU/ml.
Source : mercury hollow-cathode tube or a discharge lamp.
Wavelength : 253.7 nm. 01/2009:0462
Atomisation device : flameless system whereby the mercury
can be entrained in the form of cold vapour. HARD FAT
Potassium : maximum 2.0 ppm.
Adeps solidus
Atomic emission spectrometry (2.2.22, Method I).
DEFINITION
Test solution (a). Dilute 50.0 ml of the water to be examined
to 100 ml with distilled water R. Carry out a determination Mixture of triglycerides, diglycerides and monoglycerides,
using this solution. If the potassium content is more than which may be obtained either by esterification of fatty acids
0.75 mg/l, further dilute the water to be examined with of natural origin with glycerol or by transesterification of
distilled water R. natural fats.
Each type of hard fat is characterised by its melting point, its
Test solution (b). Take 50.0 ml of the water to be examined hydroxyl value and its saponification value.
or, if necessary, the water to be examined diluted as
described in the preparation of test solution (a). Add 1.25 ml It contains no added substances.
of potassium standard solution (20 ppm K) R and dilute to CHARACTERS
100.0 ml with distilled water R.
Appearance : white or almost white, waxy, brittle mass.
Reference solutions. Prepare reference solutions (0 ppm ; Solubility : practically insoluble in water, slightly soluble
0.25 ppm ; 0.50 ppm ; 0.75 ppm ; 1 ppm) using potassium in anhydrous ethanol.
standard solution (20 ppm K) R.
When heated to 50 °C, it melts giving a colourless or slightly
Wavelength : 766.5 nm. yellowish liquid.
Calculate the potassium content of the water to be examined IDENTIFICATION
in parts per million from the expression :
Thin-layer chromatography (2.2.27).
Test solution. Dissolve 1.0 g of the substance to be examined
in ethylene chloride R and dilute to 10 ml with the same
solvent.
p = dilution factor used for the preparation of test Plate : TLC silica gel G plate R.
solution (a) ; Mobile phase : ether R, ethylene chloride R (10:90 V/V).
n1 = measured value of test solution (a) ; Application : 2 μl.
n2 = measured value of test solution (b). Development : over a path of 12 cm.
Drying : in air.
Sodium : maximum 50.0 ppm.
Detection : expose to iodine vapour until the spots appear
Atomic emission spectrometry (2.2.22, Method I). and examine in daylight.
Test solution. The water to be examined. If the sodium Results : the chromatogram shows a spot with an RF value
content is more than 10 mg/l, dilute with distilled water R of about 0.6 due to triglycerides (Rst 1) and may show spots
to obtain a concentration suitable for the apparatus used. due to 1,3-diglycerides (Rst 0.5), to 1,2-diglycerides (Rst 0.3)

4164 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Human haematopoietic stem cells

and to 1-monoglycerides (Rst 0.05). If spots due to partial preparation also contains haematopoietic progenitor cells,
glycerides are not detectable the tests for melting point and which are capable of differentiation but not self-renewal. The
for hydroxyl value (see Tests) are carried out in addition to numbers of haematopoietic stem cells and haematopoietic
confirm identification. progenitor cells are correlated.
This monograph applies to haematopoietic stem cells that
TESTS
have not undergone expansion or genetic modification,
Alkaline impurities. Dissolve 2.00 g in a mixture of 1.5 ml of and that are intended to provide a successful engraftment
ethanol (96 per cent) R and 3.0 ml of ether R. Add 0.05 ml leading to a permanent restoration of all lineages of blood
of bromophenol blue solution R. Not more than 0.15 ml of cell production to a sufficient level and function in a
0.01 M hydrochloric acid is required to change the colour of recipient whose haematopoiesis has been compromised
the indicator to yellow. by, for example, disease or high doses of chemotherapy
Melting point (2.2.15) : 30 °C to 45 °C, and within 2 °C of and/or radiation therapy, or has to be replaced in certain
the nominal value. congenital diseases. The infused haematopoietic stem
cells can originate from the recipient (autologous) or from
Introduce the melted substance into the capillary tube and another individual (allogeneic).
allow to stand at a temperature below 10 °C for 24 h.
Haematopoietic stem cells are recognised by their ability
Acid value (2.5.1) : maximum 0.5. to reconstitute human haematopoiesis in vivo. They also
Dissolve 5.0 g in 50 ml of the prescribed mixture of solvents. have the capacity to differentiate into colony-forming cells,
Hydroxyl value (2.5.3, Method A) : maximum 50, and within which are able to give rise to colonies in the presence of
5 units of the nominal value ; maximum 5 if the nominal various growth factors. The membrane marker CD34 is
value is less than 5. commonly used for the successful isolation/purification of
haematopoietic stem cells from crude preparations and as
Iodine value (2.5.4, Method A) : maximum 3. an indicator of haematopoietic stem cell content in routine
Peroxide value (2.5.5, Method A) : maximum 3. quality control.
Saponification value (2.5.6) : 210 to 260, and within 5 per PRODUCTION
cent of the nominal value, determined on 2.0 g.
DONORS
Unsaponifiable matter (2.5.7) : maximum 0.6 per cent,
Where allogeneic cells are used, they are derived from
determined on 5.0 g.
carefully selected donors in accordance with donor selection
Heavy metals (2.4.8) : maximum 10 ppm. criteria. Directive 2004/23/EC of the European Union deals
2.0 g complies with test D. Prepare the reference solution with the criteria for donor selection.
using 2 ml of lead standard solution (10 ppm Pb) R. COLLECTION
Total ash (2.4.16) : maximum 0.05 per cent, determined on Peripheral blood stem cells. These are collected by
2.00 g. cytapheresis after mobilisation from the bone marrow
by administration of growth factors and/or treatment of
STORAGE autologous donors with cytotoxic substances. The cells may
Protected from light and heat. be processed to select a population of interest and may be
cryopreserved.
LABELLING Bone marrow. Bone marrow is harvested by aspirating the
The label states : cells from the cavities of hollow bones, then removing bone
— the nominal melting point ; fragments by filtration and, if necessary, separating the buffy
coat cells after centrifugation or with commercial kits based
— the nominal hydroxyl value ; on the cytapheresis principle. The cells may be processed to
— the nominal saponification value. select a population of interest and may be cryopreserved.
Umbilical cord blood. Placental blood haematopoietic cells
are collected from placentae via the vein of the umbilical
cord. The cells are then cryopreserved.
01/2008:2323
corrected 6.3 CRYOPRESERVATION
Cryopreservation allows storage for long periods. The cells
are suspended in a validated medium containing a suitable
HUMAN HAEMATOPOIETIC STEM cryoprotectant (for example, dimethyl sulphoxide) and
CELLS macromolecules (for example, autologous plasma/albumin)
and are frozen in cryobags in a manner designed to maintain
Cellulae stirpes haematopoieticae humanae viability of the cells by controlled cooling according to
a validated method. They are stored at a temperature
This monograph provides a standard for the preparation of − 140 °C or lower. Where cryobags are stored under other
and control of human haematopoietic stem cells for use conditions of temperature and duration, the functionality of
in therapy. It does not exclude the use of alternative the preparation must be validated. Cryobags from donors
preparation and control methods that are acceptable to the that test positive for any infectious disease marker must be
competent authority. stored in such a way as to avoid cross-contamination.
DEFINITION SUBSTANCES USED IN PRODUCTION
Human haematopoietic stem cells are primitive multipotent The quality of substances used in production may be critical
cells capable of self-renewal as well as differentiation and with respect to the quality, safety and efficacy of the final
maturation into all haematopoietic lineages. They are found product, particularly for substances of biological origin. This
in small numbers in bone marrow, in the mononuclear cell is of particular importance for :
fraction of circulating blood and in umbilical cord blood. The — proteins, including enzymes and antibodies ;

General Notices (1) apply to all monographs and other texts 4165
Human normal immunoglobulin for intravenous administration
— cryopreservation reagents ;
— purification reagents.
Quality assurance. All substances must be produced
within a recognised quality assurance system using suitable
production facilities.
Quality specifications. A suitable quality specification must
be presented for each substance, including notably :
— identity ;
— potency (where applicable) ;
— purity ;
— determination of bacterial endotoxins (2.6.14) (where
applicable) ;
— microbiological quality (total viable count, tests for
specified micro-organisms) ;
— sterility (2.6.1) (where applicable).
Viral safety. The requirements of chapter 5.1.7. apply.
Transmissible spongiform encephalopathies. A risk
assessment of the product with respect to transmissible
spongiform encephalopathies is carried out, and suitable
measures are taken to minimise any such risk (5.2.8).
Water. Water used in the preparation of cellular products
complies with the relevant monograph (Water for injections
(0169), Water, highly purified (1927), Purified water
(0008)). Water incorporated into the final product complies
with the section on Water for injections in bulk in the
monograph Water for injections (0169), and in addition is
sterile.
TESTS
Target specifications are established for the different tests,
but these are not used as rigid acceptance criteria.
Tests carried out include the following (further tests, such
as purging, cell depletion, allogeneic application, may be
necessary depending on any treatment applied to the cells
and on the intended recipient) :
Nucleated cell count (2.7.29).
Viability (2.7.29). Viability is assessed for products that are
not infused within 24 h of collection.
CD34+ cell count. For peripheral blood stem cells,
CD34+ cell count is determined using a validated automated
apparatus to analyse cells labelled with anti-CD34 antibodies.
The apparatus and method employed must be able to
determine the number of CD34+ cells with a sensitivity,
accuracy and reproducibility comparable with those of
immunophenotyping (2.7.23), where cells are labelled
using anti-CD34 and anti-CD45 antibodies conjugated to a
fluorochrome and analysed by flow cytometry (2.7.24).
Colony-forming cell (CFC) assay (2.7.28). Proliferative
capacity is established by a suitable assay. The test is
not necessarily carried out on each unit. The correlation
between the dose of CD34 and the number of CFCs in a given
situation (pathology, packaging, mobilisation) is determined.
The CFC assay is carried out periodically ; whenever a change
that could affect the quality of CD34+ cells is made to the
protocol for packaging or mobilisation, it is carried out on
a suitable number of units.
Microbiological control. Examine as prescribed in general
method 2.6.27. Microbiological control of cellular products.
Where justified, the product may be released before
completion of the test.
4166 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:0918
HUMAN NORMAL IMMUNOGLOBULIN
FOR INTRAVENOUS
ADMINISTRATION
Immunoglobulinum humanum normale
ad usum intravenosum
DEFINITION
Human normal immunoglobulin for intravenous adminis-
tration is a liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G (IgG). Other
proteins may be present. Human normal immunoglobulin for
intravenous administration contains the IgG antibodies of
normal subjects. This monograph does not apply to products
intentionally prepared to contain fragments or chemically
modified IgG.
Human normal immunoglobulin for intravenous
administration is obtained from plasma that complies with
the requirements of the monograph on Human plasma for
fractionation (0853). No antibiotic is added to the plasma
used.
PRODUCTION
The method of preparation includes a step or steps that have
been shown to remove or to inactivate known agents of
infection ; if substances are used for inactivation of viruses, it
shall have been shown that any residues present in the final
product have no adverse effects on the patients treated with
the immunoglobulin.
The product shall have been shown, by suitable tests in
animals and evaluation during clinical trials, to be well
tolerated when administered intravenously.
Human normal immunoglobulin for intravenous
administration is prepared from pooled material from not
fewer than 1000 donors by a method that has been shown
to yield a product that :
— does not transmit infection ;
— at an immunoglobulin concentration of 50 g/l, contains
antibodies for at least 2 of which (1 viral and 1 bacterial)
an International Standard or Reference Preparation is
available, the concentration of such antibodies being at
least 3 times that in the initial pooled material ;
— has a defined distribution of immunoglobulin G
subclasses ;
— complies with the test for Fc function of immunoglobulin
(2.7.9).
Human normal immunoglobulin for intravenous
administration is prepared as a stabilised solution or as a
freeze-dried preparation. A stabiliser may be added. In both
cases the preparation is passed through a bacteria-retentive
filter. The preparation may subsequently be freeze-dried
and the containers closed under vacuum or under an inert
gas. No antimicrobial preservative is added either during
fractionation or at the stage of the final bulk solution.
The stability of the preparation is demonstrated by suitable
tests carried out during development studies.
CHARACTERS
The liquid preparation is clear or slightly opalescent and
colourless or pale yellow. The freeze-dried preparation is a
hygroscopic, white or slightly yellow powder or solid friable
mass.
EUROPEAN PHARMACOPOEIA 6.3 Human normal immunoglobulin for intravenous administration

For the freeze-dried preparation, reconstitute as stated on System suitability : in the electropherogram obtained with
the label immediately before carrying out the identification the reference solution on cellulose acetate or on agarose
and the tests, except those for solubility and water. gels, the proportion of protein in the principal band is within
the limits stated in the leaflet accompanying the reference
IDENTIFICATION preparation.
Examine by a suitable immunoelectrophoresis technique. Results : in the electropherogram obtained with the test
Using antiserum to normal human serum, compare normal solution on cellulose acetate or on agarose gels, not more
human serum and the preparation to be examined, both than 5 per cent of protein has a mobility different from that
diluted to contain 10 g/l of protein. The main component of the principal band. This limit is not applicable if albumin
of the preparation to be examined corresponds to the IgG has been added to the preparation as a stabiliser ; for such
component of normal human serum. The preparation to preparations, a test for protein composition is carried out
be examined may show the presence of small quantities of during manufacture before addition of the stabiliser.
other plasma proteins ; if human albumin has been added as
a stabiliser, it may be seen as a major component. Distribution of molecular size. Liquid chromatography
(2.2.29).
TESTS Test solution. Dilute the preparation to be examined with
Solubility. For the freeze-dried preparation, add the volume a 9 g/l solution of sodium chloride R to a concentration
of the liquid stated on the label. The preparation dissolves suitable for the chromatographic system used. A
completely within 30 min at 20-25 °C. concentration in the range of 4-12 g/l and injection of
50-600 μg of protein are usually suitable.
pH (2.2.3) : 4.0 to 7.4.
Reference solution. Dilute human immunoglobulin
Dilute the preparation to be examined with a 9 g/l solution (molecular size) BRP with a 9 g/l solution of sodium
of sodium chloride R to obtain a solution containing 10 g/l chloride R to the same protein concentration as the test
of protein. solution.
Osmolality (2.2.35) : minimum 240 mosmol/kg. Column :
Total protein : minimum 30 g/l and between 90 per cent and — size: l = 0.6 m, Ø = 7.5 mm, or l = 0.3 m, Ø = 7.8 mm ;
110 per cent of the quantity of protein stated on the label. — stationary phase : hydrophilic silica gel for
Dilute the preparation to be examined with a 9 g/l solution chromatography R of a grade suitable for fractionation of
of sodium chloride R to obtain a solution containing about globular proteins with relative molecular masses in the
15 mg of protein in 2 ml. To 2.0 ml of this solution in a range 10 000 to 500 000.
round-bottomed centrifuge tube add 2 ml of a 75 g/l solution Mobile phase : dissolve 4.873 g of disodium hydrogen
of sodium molybdate R and 2 ml of a mixture of 1 volume phosphate dihydrate R, 1.741 g of sodium dihydrogen
of nitrogen-free sulphuric acid R and 30 volumes of phosphate monohydrate R, 11.688 g of sodium chloride R
water R. Shake, centrifuge for 5 min, decant the supernatant and 50 mg of sodium azide R in 1 litre of water R.
liquid and allow the inverted tube to drain on filter paper.
Determine the nitrogen in the centrifugation residue by the Flow rate : 0.5 ml/min.
method of sulphuric acid digestion (2.5.9) and calculate the Detection : spectrophotometer at 280 nm.
content of protein by multiplying the result by 6.25. In the chromatogram obtained with the reference solution,
Protein composition. Zone electrophoresis (2.2.31). the principal peak corresponds to the IgG monomer and
Use strips of suitable cellulose acetate gel or suitable agarose there is a peak corresponding to the dimer with a relative
gel as the supporting medium and barbital buffer solution retention to the principal peak of about 0.85. Identify
pH 8.6 R1 as the electrolyte solution. the peaks in the chromatogram obtained with the test
solution by comparison with the chromatogram obtained
If cellulose acetate is the supporting material, the method with the reference solution ; any peak with a retention time
described below can be used. If agarose gels are used, and shorter than that of the dimer corresponds to polymers and
because they are normally part of an automated system, the aggregates.
manufacturer’s instructions are followed instead.
Results : in the chromatogram obtained with the test
Test solution. Dilute the preparation to be examined with a solution :
9 g/l solution of sodium chloride R to an immunoglobulin
concentration of 30 g/l. — relative retention : for the monomer and for the dimer,
the relative retention to the corresponding peak in the
Reference solution. Reconstitute human immunoglobulin chromatogram obtained with the reference solution is
for electrophoresis BRP and dilute with a 9 g/l solution of 1 ± 0.02 ;
sodium chloride R to a protein concentration of 30 g/l.
— peak area : the sum of the peak areas of the monomer and
To a strip apply 4.0 μl of the test solution as a 10 mm band the dimer represent not less than 90 per cent of the total
or apply 0.4 μl per millimetre if a narrower strip is used. To area of the chromatogram and the sum of the peak areas
another strip apply in the same manner the same volume of of polymers and aggregates represents not more than
the reference solution. Apply a suitable electric field such 3 per cent of the total area of the chromatogram. This
that the albumin band of normal human serum applied on requirement does not apply to products where albumin
a control strip migrates at least 30 mm. Stain the strips has been added as a stabiliser ; for products stabilised with
with amido black 10B solution R for 5 min. Decolourise albumin, a test for distribution of molecular size is carried
with a mixture of 10 volumes of glacial acetic acid R and out during manufacture before addition of the stabiliser.
90 volumes of methanol R so that the background is just
free of colour. Develop the transparency of the strips Anticomplementary activity (2.6.17). The consumption of
with a mixture of 19 volumes of glacial acetic acid R and complement is not greater than 50 per cent (1 CH50 per
81 volumes of methanol R. Measure the absorbance of the milligram of immunoglobulin).
bands at 600 nm in an instrument having a linear response Prekallikrein activator (2.6.15) : maximum 35 IU/ml,
over the range of measurement. Calculate the result as the calculated with reference to a dilution of the preparation to
mean of 3 measurements of each strip. be examined containing 30 g/l of immunoglobulin.

General Notices (1) apply to all monographs and other texts 4167
Human plasma (pooled and treated for virus inactivation)
Anti-A and anti-B haemagglutinins (2.6.20). Carry out
the tests for anti-A and anti-B haemagglutinins. If the
preparation to be examined contains more than 30 g/l
of immunoglobulin, dilute to this concentration before
preparing the dilutions to be used in the test. The 1 to 64
dilutions do not show agglutination.
Anti-D antibodies (2.6.26). It complies with the test for
anti-D antibodies in human immunoglobulin for intravenous
administration.
Antibody to hepatitis B surface antigen : minimum
0.5 IU/g of immunoglobulin, determined by a suitable
immunochemical method (2.7.1).
Immunoglobulin A. As determined by a suitable
immunochemical method (2.7.1), the content of
immunoglobulin A is not greater than the maximum content
stated on the label.
Water. Determined by a suitable method, such as the
semi-micro determination of water (2.5.12), loss on drying
(2.2.32) or near infrared spectrophotometry (2.2.40), the
water content is within the limits approved by the competent
authority.
Sterility (2.6.1). It complies with the test for sterility.
Pyrogens (2.6.8). It complies with the test for pyrogens.
Inject per kilogram of the rabbit’s mass a volume equivalent
to 0.5 g of immunoglobulin but not more than 10 ml per
kilogram of body mass.
STORAGE
For the liquid preparation, store in a colourless glass
container, protected from light, at the temperature stated
on the label. For the freeze-dried preparation, store in an
airtight colourless glass container, protected from light, at a
temperature not exceeding 25 °C.
LABELLING
The label states :
— for liquid preparations, the volume of the preparation in
the container and the protein content expressed in grams
per litre ;
— for freeze-dried preparations, the quantity of protein in
the container ;
— the amount of immunoglobulin in the container ;
— the route of administration ;
— for freeze-dried preparations, the name or composition
and the volume of the reconstituting liquid to be added ;
— the distribution of subclasses of immunoglobulin G
present in the preparation ;
— where applicable, the amount of albumin added as a
stabiliser ;
— the maximum content of immunoglobulin A.
01/2009:1646
HUMAN PLASMA (POOLED AND
TREATED FOR VIRUS INACTIVATION)
Plasma humanum coagmentatum
conditumque ad exstinguendum virum
DEFINITION
Human plasma (pooled and treated for virus inactivation) is
a frozen or freeze-dried, sterile, non-pyrogenic preparation
obtained from human plasma derived from donors belonging
to the same ABO blood group. The preparation is thawed or
reconstituted before use to give a solution for infusion.
4168 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
The human plasma used complies with the monograph on
Human plasma for fractionation (0853).
PRODUCTION
The units of plasma to be used are cooled to − 30 °C or
lower within 6 h of separation of cells and always within
24 h of collection.
The pool is prepared by mixing units of plasma belonging to
the same ABO blood group.
The pool of plasma is tested for hepatitis B surface antigen
(HBsAg) and for HIV antibodies using test methods of
suitable sensitivity and specificity ; the pool must give
negative results in these tests.
Hepatitis A virus RNA. The plasma pool is tested using a
validated nucleic acid amplification technique (2.6.21). A
positive control with 1.0 × 102 IU of hepatitis A virus RNA
per millilitre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if
the positive control is non-reactive or if the result obtained
with the internal control indicates the presence of inhibitors.
The pool complies with the test if it is found non-reactive for
hepatitis A virus RNA.
Hepatitis C virus RNA. The plasma pool is tested using a
validated nucleic acid amplification technique (2.6.21). A
positive control with 1.0 × 102 IU of hepatitis C virus RNA
per millilitre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if
the positive control is non-reactive or if the result obtained
with the internal control indicates the presence of inhibitors.
The pool complies with the test if it is found non-reactive for
hepatitis C virus RNA.
Hepatitis C virus RNA for NAT testing BRP is suitable for
use as a positive control.
To limit the potential burden of B19 virus in plasma pools,
the plasma pool is also tested for B19 virus using a validated
nucleic acid amplification technique (2.6.21).
B19 virus DNA. The plasma pool contains not more than
10.0 IU/μl.
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.
The method of preparation is designed to minimise
activation of any coagulation factor (to minimise potential
thrombogenicity) and includes a step or steps that have been
shown to inactivate known agents of infection ; if substances
are used for the inactivation of viruses during production,
the subsequent purification procedure must be validated to
demonstrate that the concentration of these substances is
reduced to a suitable level and that any residues are such as
not to compromise the safety of the preparation for patients.
Inactivation process. The solvent-detergent process, which
is one of the methods used to inactivate enveloped viruses,
uses treatment with a combination of tributyl phosphate and
octoxinol 10 ; these reagents are subsequently removed by oil
extraction or by solid phase extraction so that the amount in
the final product is less than 2 μg/ml for tributyl phosphate
and less than 5 μg/ml for octoxinol 10.
No antimicrobial preservative is added.
EUROPEAN PHARMACOPOEIA 6.3
The solution is passed through a bacteria-retentive filter,
distributed aseptically into the final containers and
immediately frozen ; it may subsequently be freeze-dried.
Plastic containers comply with the requirements for sterile
plastic containers for human blood and blood components
(3.2.3).
Glass containers comply with the requirements for glass
containers for pharmaceutical use (3.2.1).
CHARACTERS
The frozen preparation, after thawing, is a clear or slightly
opalescent liquid free from solid and gelatinous particles.
The freeze-dried preparation is an almost white or slightly
yellow powder or friable solid.
Thaw or reconstitute the preparation to be examined as
stated on the label immediately before carrying out the
identification, tests and assay.
IDENTIFICATION
A. Examine by electrophoresis (2.2.31) comparing with
normal human plasma. The electropherograms show the
same bands.
B. It complies with the test for anti-A and anti-B
haemagglutinins (see Tests).
TESTS
pH (2.2.3) : 6.5 to 7.6.
Osmolality (2.2.35) : minimum 240 mosmol/kg.
Total protein : minimum 45 g/l.
Dilute with a 9 g/l solution of sodium chloride R to obtain
a solution containing about 15 mg of protein in 2 ml. Place
2.0 ml of this solution in a round-bottomed centrifuge tube
and 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 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 quantity of protein by multiplying the
result by 6.25.
Activated coagulation factors (2.6.22). It complies with the
test for activated coagulation factors. Carry out the test
with 0.1 ml of the preparation to be examined instead of
10-fold and 100-fold dilutions. The coagulation time for the
preparation to be examined is not less than 150 s.
Anti-A and anti-B haemagglutinins (2.6.20). The presence
of haemagglutinins (anti-A or anti-B) corresponds to the
blood group stated on the label.
Hepatitis A virus antibodies : minimum 1.0 IU/ml,
determined by a suitable immunochemical method (2.7.1).
Human hepatitis A immunoglobulin BRP is suitable for use
as a reference preparation.
Irregular erythrocyte antibodies. The preparation to
be examined does not show the presence of irregular
erythrocyte antibodies when examined without dilution by
an indirect antiglobulin test.
Citrate. Liquid chromatography (2.2.29).
Test solution. Dilute the preparation to be examined with an
equal volume of a 9 g/l solution of sodium chloride R. Filter
the solution using a filter with 0.45 μm pores.
Reference solution. Dissolve 0.300 g of sodium citrate R in
water R and dilute to 100.0 ml with the same solvent.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : cation exchange resin R (9 μm).
General Notices (1) apply to all monographs and other texts
Human plasma (pooled and treated for virus inactivation)
Mobile phase : 0.51 g/l solution of sulphuric acid R.
Flow rate : 0.5 ml/min.
Detection : spectrophotometer at 215 nm.
Equilibration: 15 min.
Injection : 10 μl.
Retention time : citrate = about 10 min.
Limit :
— citrate : maximum 25 mmol/l.
Calcium : maximum 5.0 mmol/l.
Atomic absorption spectrometry (2.2.23, Method I).
Source : calcium hollow-cathode lamp using a transmission
band preferably of 0.5 nm.
Wavelength : 622 nm.
Atomisation device : air-acetylene or acetylene-propane
flame.
Potassium : maximum 5.0 mmol/l.
Atomic emission spectrometry (2.2.22, Method I).
Wavelength : 766.5 nm.
Sodium : maximum 2.00 × 102 mmol/l.
Atomic emission spectrometry (2.2.22, Method I).
Wavelength : 589 nm.
Water : determined by a suitable method, such as the
semi-micro determination of water (2.5.12), loss on drying
(2.2.32) or near-infrared spectrometry (2.2.40), the water
content is within the limits approved by the competent
authority (freeze-dried product).
Sterility (2.6.1). It complies with the test for sterility.
Pyrogens (2.6.8). It complies with the test for pyrogens.
Inject 3 ml per kilogram of the rabbit’s mass.
ASSAY
Factor VIII. Carry out the assay of human coagulation
factor VIII (2.7.4) using a reference plasma calibrated against
the International Standard for blood coagulation factor VIII
in plasma.
The estimated potency is not less than 0.5 IU/ml. The
confidence limits (P = 0.95) are not less than 80 per cent and
not more than 120 per cent of the estimated potency.
Factor V. Carry out the assay of human coagulation factor V
described below using a reference plasma calibrated against
the International Standard for blood coagulation factor V
in plasma.
Using imidazole buffer solution pH 7.3 R, prepare at least
3 two-fold dilutions of the preparation to be examined,
preferably in duplicate, from 1 in 10 to 1 in 40. Test each
dilution as follows : mix 1 volume of plasma substrate
deficient in factor V R, 1 volume of the dilution to be
examined, 1 volume of thromboplastin R and 1 volume
of a 3.5 g/l solution of calcium chloride R ; measure the
coagulation times, i.e. the interval between the moment at
which the calcium chloride solution is added and the 1st
indication of the formation of fibrin, which may be observed
visually or by means of a suitable apparatus.
In the same manner, determine the coagulation time of
4 twofold dilutions (1 in 10 to 1 in 80) of human normal
plasma in imidazole buffer solution pH 7.3 R.
Check the validity of the assay and calculate the potency
of the test preparation by the usual statistical methods (for
example, 5.3).
The estimated potency is not less than 0.5 IU/ml. The
confidence limits (P = 0.95) are not less than 80 per cent and
not more than 120 per cent of the estimated potency.
4169
Hydroxypropylbetadex EUROPEAN PHARMACOPOEIA 6.3

Factor XI. Carry out the assay of human coagulation DEFINITION

factor XI (2.7.22) using a reference plasma calibrated against Hydroxypropylbetadex (β-cyclodextrin, 2-hydroxypropyl
the International Standard for blood coagulation factor XI ether) is a partially substituted poly(hydroxypropyl) ether
in plasma. of betadex. The number of hydroxypropyl groups per
The estimated potency is not less than 0.5 IU/ml. The anhydroglucose unit, expressed as molar substitution (MS),
confidence limits (P = 0.95) are not less than 80 per cent and is not less than 0.40 and not more than 1.50 and is within
not more than 125 per cent of the estimated potency. 10 per cent of the value stated on the label.
Protein C. Carry out the assay of human protein C (2.7.30) CHARACTERS
using a reference plasma calibrated against the International
Standard for human protein C in plasma. Appearance : white or almost white, amorphous or
crystalline powder.
The estimated potency is not less than 0.7 IU/ml. The
confidence limits (P = 0.95) are not less than 80 per cent and Solubility : freely soluble in water and in propylene glycol.
not more than 120 per cent of the estimated potency. IDENTIFICATION
Protein S. Carry out the assay of human protein S (2.7.31) A. Infrared absorption spectrophotometry (2.2.24).
using a reference plasma calibrated against the International
Standard for human protein S in plasma. Comparison : hydroxypropylbetadex CRS.
The estimated potency is within the limits approved for the Results : the spectrum obtained with the substance to
particular product. The confidence limits (P = 0.95) are not be examined shows the same absorption bands as the
less than 80 per cent and not more than 120 per cent of the spectrum obtained with hydroxypropylbetadex CRS. Due
estimated potency. to the difference in the substitution of the substance, the
intensity of some absorption bands can vary.
Plasmin inhibitor (α2-antiplasmin). Carry out the assay of B. Appearance of solution (see Tests).
human plasmin inhibitor (2.7.25) using a reference plasma
calibrated against human normal plasma. TESTS
1 unit of human plasmin inhibitor is equal to the activity
Solution S. Dissolve 5.0 g in carbon dioxide-free water R
of 1 ml of human normal plasma. Human normal plasma prepared from distilled water R and dilute to 50.0 ml with
is prepared by pooling plasma units from not fewer thanthe same solvent.
30 donors and storing at − 30 °C or lower.
Appearance of solution. The solution is clear (2.2.1) and
The estimated potency is not less than 0.2 units/ml. The
colourless (2.2.2, Method II), and remains so after cooling
confidence limits (P = 0.95) are not less than 80 per cent and
to room temperature.
not more than 120 per cent of the estimated potency.
Dissolve 1.0 g in 2.0 ml of water R, with heating.
LABELLING Conductivity (2.2.38) : maximum 200 μS·cm-1.
The label states : Measure the conductivity of solution S, while gently stirring
— the ABO blood group ; with a magnetic stirrer.
— the method used for virus inactivation. Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 2.50 g of the substance to be
examined in water R with heating, cool, and dilute to 25.0 ml
01/2009:1804 with the same solvent.
Reference solution (a). Dissolve 0.15 g of betadex CRS and
0.25 g of propylene glycol R in water R and dilute to 10.0 ml
HYDROXYPROPYLBETADEX with the same solvent.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
Hydroxypropylbetadexum to 50.0 ml with water R.
Precolumn:
— stationary phase : phenylsilyl silica gel for
chromatography R.
Column :
— size: l = 0.30 m, Ø = 3.9 mm ;
— stationary phase : phenylsilyl silica gel for
chromatography R ;
— temperature : 40 °C.
Mobile phase : water for chromatography R.
Flow rate : 1.5 ml/min.
Detection : differential refractometer, at 40 °C.
Injection : 20 μl.
Run time : 6 times the retention time of impurity A.
Relative retention with reference to impurity B
(retention time = about 2.5 min) : impurity A = about 4.2 ;
hydroxypropylbetadex = about 6 for the beginning of the
elution.
Hydroxypropylbetadex elutes as a very wide peak or several
C42H70O35(C3H6O)x with x = 7 MS peaks.

4170 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Hypromellose

System suitability : reference solution (a) :
— resolution : minimum 4 between the peaks due to
impurities A and B.
Limits :
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (1.5 per cent) ;
— impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (2.5 per cent) ;
— any other impurity : for each impurity, not more than
0.04 times the area of the peak due to impurity B in
the chromatogram obtained with reference solution (b)
(0.1 per cent) ;
— sum of impurities other than A and B : not more than
0.4 times the area of the peak due to impurity B in
the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
— disregard limit : 0.02 times the area of the peak due to
impurity B in the chromatogram obtained with reference If intended for use in the manufacture of parenteral
solution (b) (0.05 per cent) ; disregard any peak eluting
before impurity B or after impurity A.
Heavy metals (2.4.8) : maximum 20 ppm.
12 ml of solution S complies with test A. Prepare the reference
solution using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g by drying in an oven at 120 °C for 2 h.
Molar substitution. Nuclear magnetic resonance
spectrometry (2.2.33).
The molar substitution (MS) is calculated from the ratio
between the signal from the 3 protons of the methyl group
that is part of the hydroxypropyl group and the signal from
the proton attached to the C1 carbon (glycosidic proton)
of the anhydroglucose units.
Use a Fourier transform nuclear magnetic resonance
spectrometer of minimum frequency 250 MHz, suited to
record a proton spectrum and to carry out quantitative
analysis, at a temperature of at least 25 °C.
Introduce not less than the equivalent of 10.0 mg of the
substance to be examined (dried substance) into a 5 mm
NMR tube, equipped with a spinner in order to record
the spectrum in rotation. Add approximately 0.75 ml of
deuterium oxide R1. Cap the tube, mix thoroughly and
adapt the spinner.
Make the appropriate instrument settings (frequency, gain,
digital resolution, sample rotation, shims, probe tuning,
resolution/data point, receiver gain etc.) so as to obtain a
suitable spectrum for quantitative analysis (good FID (Free
Induction Decay), no distortion of the spectrum after Fourier
transform and phase corrections). The relaxation delay must
be adapted to the pulse angle in order to have sufficient
relaxation of the protons concerned between 2 pulses (for
example : 10 s for a 90° pulse).
Record the FID, with at least 8 scans, so as to obtain a
spectral window comprised, at least, between 0 ppm and
6.2 ppm, referring to the signal of exchangeable protons
(solvent) at 4.8 ppm (25 °C).
Make a zero filling of at least 3-fold in size relative to the
acquisition data file and transform the FID to the spectrum
without any correction of Gaussian broadening factor
(GB = 0) and with a line broadening factor not greater than
0.2 (LB ≤ 0.2). Call the integration sub-routine after phase
corrections and baseline correction between 0.5 ppm and
6.2 ppm.
Measure the peak areas of the doublet from the methyl
groups at 1.2 ppm (A1), and of the signals of the glycosidic
protons between 5 ppm and 5.4 ppm (A2).
The molar substitution is obtained using the following
equation :
A1 = area of the signal due to the 3 protons of the
methyl groups that are part of the hydroxypropyl
groups ;
A2 area of the signals due to the glycosidic protons.
=
The degree of substitution is the number of hydroxypropyl
groups per molecule of β-cyclodextrin and is obtained by
multiplying the MS by 7.
Microbial contamination
preparations :
— TAMC : acceptance criterion 102 CFU/g (2.6.12).
If not intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
— absence of Escherichia coli (2.6.13) ;
— absence of Salmonella (2.6.13).
Bacterial endotoxins (2.6.14) : less than 10 IU/g, if intended
for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.
LABELLING
The label states :
— the molar substitution (MS) ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
A. betadex,
B. propylene glycol.
01/2008:0348
corrected 6.3
HYPROMELLOSE
Hypromellosum
[9004-65-3]
DEFINITION
Hydroxypropylmethylcellulose.
Partly O-methylated and O-(2-hydroxypropylated) cellulose.
CHARACTERS
Appearance : white, yellowish-white or greyish-white powder
or granules, hygroscopic after drying.

General Notices (1) apply to all monographs and other texts 4171
Hypromellose EUROPEAN PHARMACOPOEIA 6.3

Solubility : practically insoluble in hot water, in acetone, in as an excipient (see chapter 5.15). This section is a
anhydrous ethanol and in toluene. It dissolves in cold water non-mandatory part of the monograph and it is not
giving a colloidal solution. necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
IDENTIFICATION contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
A. Evenly distribute 1.0 g on the surface of 100 ml of water R and the performance of the medicinal product during use.
in a beaker, tapping the top of the beaker, gently if Where control methods are cited, they are recognised as
necessary to ensure a uniform layer on the surface. Allow being suitable for the purpose, but other methods can also
to stand for 1-2 min : the powdered material aggregates be used. Wherever results for a particular characteristic are
on the surface. reported, the control method must be indicated.
B. Evenly distribute 1.0 g into 100 ml of boiling water R,
and stir the mixture using a magnetic stirrer with a bar The following characteristics may be relevant for
25 mm long : a slurry is formed and the particles do not hypromellose used as binder, viscosity-increasing agent or
dissolve. Allow the slurry to cool to 10 °C and stir using a film former.
magnetic stirrer : a clear or slightly turbid solution occurs
with its thickness dependent on the viscosity grade. Apparent viscosity : minimum 80 per cent and maximum
120 per cent of the nominal value for samples with a viscosity
C. To 0.1 ml of the solution obtained in identification B less than 600 mPa·s (Method 1) ; minimum 75 per cent and
add 9 ml of a 90 per cent V/V solution of sulphuric maximum 140 per cent of the nominal value for samples with
acid R, shake, heat on a water-bath for exactly 3 min, a viscosity of 600 mPa·s or higher (Method 2).
immediately cool in an ice-bath, carefully add 0.6 ml of a
20 g/l solution of ninhydrin R, shake and allow to stand Method 1, to be applied to samples with a viscosity of
at 25 °C : a red colour develops at first and changes to less than 600 mPa·s. Weigh accurately a quantity of the
purple within 100 min. substance to be examined equivalent to 4.000 g of the dried
D. Place 2-3 ml of the solution obtained in identification B substance. Transfer into a wide-mouthed bottle, and adjust
onto a glass slide as a thin film and allow the water to the mass to 200.0 g with hot water R. Capping the bottle, stir
evaporate : a coherent, clear film forms on the glass slide. by mechanical means at 400 ± 50 r/min for 10-20 min until
the particles are thoroughly dispersed and wetted. Scrape
E. Add exactly 50 ml of the solution obtained in down the insides of the bottle with a spatula if necessary, to
identification B to exactly 50 ml of water R in a beaker. ensure that there is no undissolved material on the sides of
Insert a thermometer into the solution. Stir the solution the bottle, and continue the stirring in a cooling water-bath
on a magnetic stirrer/hot plate and begin heating, maintained at a temperature below 10 °C for another
increasing the temperature at a rate of 2-5 °C per minute. 20-40 min. Adjust the solution mass if necessary to 200.0 g
Determine the temperature at which a turbidity increase using cold water R. Centrifuge the solution if necessary to
begins to occur and designate the temperature as the expel any entrapped air bubbles. Using a spatula, remove
flocculation temperature : the flocculation temperature any foam, if present. Determine the viscosity of this solution
is higher than 50 °C. using the capillary viscometer method (2.2.9) to obtain
the kinematic viscosity (ν). Separately, determine the
TESTS density (ρ) (2.2.5) of the solution and calculate the dynamic
Solution S. While stirring, introduce a quantity of the viscosity (η), as η = ρν.
substance to be examined equivalent to 1.0 g of the dried
substance into 50 g of carbon dioxide-free water R heated to Method 2, to be applied to samples with a viscosity of
90 °C. Allow to cool, adjust the mass of the solution to 100 g 600 mPa·s or higher. Weigh accurately a quantity of the
with carbon dioxide-free water R and stir until dissolution is substance to be examined equivalent to 10.00 g of the dried
complete. substance. Transfer into a wide-mouthed bottle, and adjust
Appearance of solution. Solution S is not more opalescent the mass to 500.0 g with hot water R. Capping the bottle, stir
than reference suspension III (2.2.1) and not more intensely by mechanical means at 400 ± 50 r/min for 10-20 min until
coloured than reference solution Y6 (2.2.2, Method II). the particles are thoroughly dispersed and wetted. Scrape
down the insides of the bottle with a spatula if necessary, to
pH (2.2.3) : 5.0 to 8.0 for the solution prepared as described ensure that there is no undissolved material on the sides of
under Apparent viscosity. the bottle, and continue the stirring in a cooling water-bath
Carry out the test at 20 ± 2 °C and read the indicated pH maintained at a temperature below 10 °C for another
value after the probe has been immersed for 5 ± 0.5 min. 20-40 min. Adjust the solution mass if necessary to 500.0 g
using cold water R. Centrifuge the solution if necessary to
Heavy metals (2.4.8) : maximum 20 ppm. expel any entrapped air bubbles. Using a spatula, remove
1.0 g complies with test F. Prepare the reference solution any foam, if present. Determine the viscosity (2.2.10) of this
using 2 ml of lead standard solution (10 ppm Pb) R. solution at 20 ± 0.1 °C using a rotating viscometer.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 1 h. Apparatus : single-cylinder type spindle viscometer.
Sulphated ash (2.4.14) : maximum 1.5 per cent, determined
Rotor number, revolution and calculation multiplier : apply
on 1.0 g.
the conditions specified in Table 0348.-1.
FUNCTIONALITY-RELATED CHARACTERISTICS
Allow the spindle to rotate for 2 min before taking the
This section provides information on characteristics measurement. Allow a rest period of 2 min between
that are recognised as being relevant control parameters subsequent measurements. Repeat the measurement twice
for one or more functions of the substance when used and determine the mean of the 3 readings.

4172 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Hypromellose

Table 0348.-1. Carrier gas: helium for chromatography R (thermal

conductivity) ; helium for chromatography R or nitrogen for
Labelled Rotor Revolution Calculation chromatography R (flame ionisation).
viscosity* number (r/min) multiplier
(mPa·s) Flow rate : adjusted so that the retention time of the internal
600 to less 3 60 20
standard is about 10 min.
than 1400
Detection : flame ionisation or thermal conductivity.
1400 to less 3 12 100
than 3500 Injection : 1-2 μl.
3500 to less 4 60 100 System suitability : reference solution :
than 9500

9500 to less 4 6 1000 — resolution : well resolved peaks of methyl iodide (1st peak),
than 99 500 isopropyl iodide (2nd peak) and internal standard
(3rd peak).
99 500 or more 4 3 2000
Calculation :
* the nominal viscosity is based on the manufacturer’s specifications.
— methoxy and hydroxypropoxy groups : calculate the
Degree of substitution. Gas chromatography (2.2.28). ratios (Q1 and Q2) of the areas of the peaks due to methyl
iodide and isopropyl iodide to the area of the peak due to
Apparatus : the internal standard in the chromatogram obtained with
— reaction vial: a 5 ml pressure-tight vial, 50 mm in the test solution, and the ratios (Q3 and Q4) of the areas
height, 20 mm in external diameter and 13 mm in of the peaks due to methyl iodide and isopropyl iodide to
internal diameter at the mouth, equipped with a the area of the peak due to the internal standard in the
pressure-tight butyl rubber membrane stopper coated chromatogram obtained with the reference solution.
with polytetrafluoroethylene and secured with an
aluminium crimped cap or another sealing system Calculate the percentage content of methoxy groups using
providing a sufficient air-tightness ; the following expression :
— heater : a heating module with a square aluminium block
having holes 20 mm in diameter and 32 mm in depth,
so that the reaction vials fit ; mixing of the contents of
the vial is effected using a magnetic stirrer equipped in
the heating module or using a reciprocal shaker that Calculate the percentage content of hydroxypropoxy groups
performs approximately 100 cycles/min. using the following expression :
Internal standard solution: 30 g/l solution of octane R in
xylene R.
Test solution. Weigh 65.0 mg of the substance to be
examined, place in a reaction vial, add 0.06-0.10 g of adipic m1 = mass of methyl iodide in the reference solution,
acid R, 2.0 ml of the internal standard solution and 2.0 ml in milligrams ;
of hydriodic acid R, immediately cap and seal the vial, and m2
weigh accurately. Mix the contents of the vial continuously = mass of isopropyl iodide in the reference solution,
for 60 min while heating the block so that the temperature in milligrams ;
of the contents is maintained at 130 ± 2 °C. If a reciprocal m = mass of the sample (dried substance), in
shaker or magnetic stirrer cannot be used, shake the vial milligrams.
well by hand at 5-minute intervals during the initial 30 min
of the heating time. Allow the vial to cool, and again weigh
Substitution Methoxy Hydroxypropoxy
accurately. If the loss of mass is less than 0.50 per cent of the
type (per cent) (per cent)
contents and there is no evidence of a leak, use the upper
layer of the mixture as the test solution. 1828 16.5 to 20.0 23.0 to 32.0
Reference solution. Place 0.06-0.10 g of adipic acid R, 2.0 ml
2208 19.0 to 24.0 4.0 to 12.0
of the internal standard solution and 2.0 ml of hydriodic
acid R in another reaction vial, cap and seal the vial, and 2906 27.0 to 30.0 4.0 to 7.5
weigh accurately. Add 15-22 μl of isopropyl iodide R
through the septum with a syringe, weigh accurately, add 2910 28.0 to 30.0 7.0 to 12.0
45 μl of methyl iodide R in the same manner, and weigh
accurately. Shake the reaction vial well, and use the upper The following characteristics may be relevant for
layer as the reference solution. hypromellose used as matrix former in prolonged-release
Column: tablets.
— size : l = 1.8-3 m, Ø = 3-4 mm ; Apparent viscosity : see test above.
— stationary phase : diatomaceous earth for gas Degree of substitution: see test above.
chromatography R impregnated with 10-20 per cent Molecular mass distribution (2.2.30).
of poly(dimethyl)(75)(diphenyl)(25)siloxane R (film
thickness 125-150 μm) ; Particle-size distribution (2.9.31 or 2.9.38).
— temperature : 100 °C. Powder flow (2.9.36).

General Notices (1) apply to all monographs and other texts 4173
Hypromellose phthalate
04/2008:0347 with 3 quantities, each of 20 ml, of water R, separating the
corrected 6.3 washings by centrifugation. Combine the liquid phases,
HYPROMELLOSE PHTHALATE
Hypromellosi phthalas
DEFINITION
Hydroxypropylmethylcellulose phthalate.
Monophthalic acid ester of hypromellose, containing
methoxy (-OCH3), 2-hydroxypropoxy (-OCH2CHOHCH3) and
phthaloyl (o-carboxybenzoyl C8H5O3) groups.
CHARACTERS
Appearance : white or almost white, free-flowing flakes or
granular powder.
Solubility : practically insoluble in water, soluble in a mixture STORAGE
of equal volumes of acetone and methanol and in a mixture In an airtight container.
of equal volumes of methanol and methylene chloride,
very slightly soluble in acetone and in toluene, practically
insoluble in anhydrous ethanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Preparation : dissolve 40 mg in 1 ml of a mixture of equal
volumes of methanol R and methylene chloride R ; spread
2 drops of this solution between 2 sodium chloride plates,
then remove one of the plates to evaporate the solvent.
Comparison : hypromellose phthalate CRS.
TESTS
Free phthalic acid. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.20 g of the substance to be
examined in about 50 ml of acetonitrile R with the aid of
ultrasound. Add 10 ml of water R, cool to room temperature, The following characteristics may be relevant for
dilute to 100.0 ml with acetonitrile R and mix.
Reference solution. Dissolve 12.5 mg of phthalic acid R in
125 ml of acetonitrile R. Add 25 ml of water R, dilute to
250.0 ml with acetonitrile R and mix.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm).
Mobile phase : acetonitrile R, 1 g/l solution of trifluoroacetic phosphate buffer solution pH 6.8 R with stirring.
acid R (1:9 V/V).
Flow rate : 2.0 ml/min.
Detection : spectrophotometer at 235 nm.
Injection : 10 μl.
System suitability : reference solution :
— repeatability : maximum relative standard deviation of
1.0 per cent after 2 injections.
Limit :
— phthalic acid : not more than 0.4 times the area of the
corresponding peak in the chromatogram obtained with
the reference solution (1.0 per cent).
Chlorides: maximum 0.07 per cent.
Dissolve 1.0 g in 40 ml of 0.2 M sodium hydroxide, add
0.05 ml of phenolphthalein solution R and add dilute
nitric acid R dropwise, with stirring, until the red colour
disappears. Add an additional 20 ml of dilute nitric acid R
with stirring. Heat on a water-bath with stirring until the
gel-like precipitate formed becomes granular. Cool and
centrifuge. Separate the liquid phase and wash the residue
4174 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
dilute to 200 ml with water R, mix and filter. To 50 ml of this
solution, add 1 ml of 0.1 M silver nitrate. The solution is not
more opalescent than a standard prepared by mixing 0.5 ml
of 0.01 M hydrochloric acid with 10 ml of 0.2 M sodium
hydroxide, adding 7 ml of dilute nitric acid R and 1 ml of
0.1 M silver nitrate, and diluting to 50 ml with water R.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 5.0 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
on 1.0 g.
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
hypromellose phthalate used as a gastro-resistant coating
agent.
Apparent viscosity (2.2.9) : 80 per cent to 120 per cent of
the nominal value.
Dissolve 10 g, previously dried at 105 °C for 1 h, in 90 g of
a mixture of equal masses of methanol R and methylene
chloride R by mixing and shaking.
Solubility. 0.2 g does not dissolve in 0.1 M hydrochloric
acid but dissolves quickly and completely in 100 ml of
Phthaloyl groups : typically 21.0 per cent to 35.0 per cent
(anhydrous substance).
Dissolve 1.000 g in 50 ml of a mixture of 1 volume of
water R, 2 volumes of acetone R and 2 volumes of ethanol
(96 per cent) R. Add 0.1 ml of phenolphthalein solution R
and titrate with 0.1 M sodium hydroxide until a faint pink
colour is obtained. Carry out a blank titration.
Calculate the percentage content of phthaloyl groups using
the following expression :
a = percentage content of water ;
m = mass of the substance to be examined, in grams ;
n
= volume of 0.1 M sodium hydroxide used, in
millilitres ;
S = percentage content of free phthalic acid (see
Tests).
EUROPEAN PHARMACOPOEIA 6.3

I
Ichthammol.. ............................................................................. 4177 Interferon beta-1a concentrated solution........................... 4177

General Notices (1) apply to all monographs and other texts 4175
 EUROPEAN PHARMACOPOEIA 6.3

4176 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2008:0917 formaldehyde solution R, neutralised to phenolphthalein
corrected 6.3 solution R1. Titrate with 0.1 M sodium hydroxide until a
ICHTHAMMOL
Ichthammolum
DEFINITION
Ichthammol is obtained by distillation from certain
bituminous schists, sulphonation of the distillate and
neutralisation of the product with ammonia.
Content :
— dry matter : 50.0 per cent m/m to 56.0 per cent m/m ;
— total ammonia (NH3 ; Mr 17.03) : 4.5 per cent m/m to
7.0 per cent m/m (dried substance) ;
— organically combined sulphur : minimum 10.5 per
cent m/m (dried substance) ;
— sulphur in the form of sulphate : maximum 20.0 per
cent m/m of the total sulphur.
CHARACTERS
Appearance : dense, blackish-brown liquid.
Solubility : miscible with water and with glycerol, slightly
soluble in ethanol (96 per cent), in fatty oils and in liquid
paraffin. It forms homogeneous mixtures with wool fat and
soft paraffin.
IDENTIFICATION
A. Dissolve 1.5 g in 15 ml of water R (solution A). To 2 ml of filter. Heat 100.0 ml of the filtrate almost to boiling, add
solution A add 2 ml of hydrochloric acid R. A resinous
precipitate is formed. Decant the supernatant liquid. The solution R1 dropwise and heat on a water-bath. Filter,
precipitate is partly soluble in ether R.
B. 2 ml of solution A, obtained in identification test A, gives more than 0.2 per cent of the mass of the residue.
the reaction of ammonium salts and salts of volatile bases
(2.3.1).
C. Evaporate and ignite the mixture of solution A and
dilute sodium hydroxide solution R obtained in
identification test B. Take up the residue with 5 ml of
dilute hydrochloric acid R. Gas is evolved which turns
lead acetate paper R brown or black. Filter the solution.
The filtrate gives reaction (a) of sulphates (2.3.1).
TESTS
Acidity or alkalinity. To 10.0 ml of the clear filtrate obtained
in the assay of total ammonia add 0.05 ml of methyl red
solution R. Not more than 0.2 ml of 0.02 M hydrochloric
acid or 0.02 M sodium hydroxide is required to change the
colour of the indicator.
Relative density (2.2.5) : 1.040 to 1.085, determined on a
mixture of equal volumes of the substance to be examined
and water R.
Sulphated ash (2.4.14) : maximum 0.3 per cent, determined
on 1.00 g.
ASSAY
Dry matter. Weigh 1.000 g in a tared flask containing 2 g of
sand R, previously dried to constant mass, and a small glass
rod. Heat on a water-bath for 2 h with frequent stirring and
dry in an oven at 100-105 °C until 2 consecutive weighings
do not differ by more than 2.0 mg ; the 2nd weighing is carried
out after drying again for 1 h.
Total ammonia. Dissolve 2.50 g in 25 ml of warm water R.
Rinse the solution into a 250 ml volumetric flask, add
200 ml of sodium chloride solution R and dilute to 250.0 ml
with water R. Filter the solution, discarding the first 20 ml
of filtrate. To 100.0 ml of the clear filtrate add 25 ml of
General Notices (1) apply to all monographs and other texts
Interferon beta-1a concentrated solution
faint pink colour is obtained.
1 ml of 0.1 M sodium hydroxide is equivalent to 1.703 mg
of NH3.
Organically combined sulphur. Mix 0.500 g with 4 g of
anhydrous sodium carbonate R and 3 ml of methylene
chloride R in a porcelain crucible of about 50 ml capacity,
warm and stir until all the methylene chloride has evaporated.
Add 10 g of coarsely powdered copper nitrate R, mix
thoroughly and heat the mixture very gently using a small
flame. When the initial reaction has subsided, increase the
temperature slightly until most of the material has blackened.
Cool, place the crucible in a large beaker, add 20 ml of
hydrochloric acid R and, when the reaction has ceased, add
100 ml of water R and boil until all the copper oxide has
dissolved. Filter the solution, add 400 ml of water R, heat to
boiling and add 20 ml of barium chloride solution R1. Allow
to stand for 2 h, filter, wash with water R, dry and ignite at
about 600 ± 50 °C until 2 successive weighings do not differ
by more than 0.2 per cent of the mass of the residue.
1 g of residue is equivalent to 0.1374 g of total sulphur.
Calculate the percentage content of total sulphur and
subtract the percentage content of sulphur in the form of
sulphate.
Sulphur in the form of sulphate. Dissolve 2.000 g in 100 ml
of water R, add 2 g of cupric chloride R dissolved in 80 ml
of water R and dilute to 200.0 ml with water R. Shake and
1 ml of hydrochloric acid R and 5 ml of barium chloride
wash the precipitate with water R, dry and ignite at about
600 ± 50 °C until 2 successive weighings do not differ by
1 g of residue is equivalent to 0.1374 g of sulphur present in
the form of sulphate.
Calculate the percentage content of sulphur in the form of
sulphate.
01/2009:1639
INTERFERON BETA-1a
CONCENTRATED SOLUTION
Interferoni beta-1a solutio concentrata
C908H14O6N246O252S7 Mr approx. 22 500
DEFINITION
Solution of a glycosylated protein having the same
amino acid sequence and disulphide bridge and a similar
glycosylation pattern as interferon beta produced by human
diploid fibroblasts in response to viral infections and various
other inducers. It exerts antiviral, antiproliferative and
immunomodulatory activity.
Content : minimum 0.20 mg of protein per millilitre.
Potency : minimum 1.5 × 108 IU per milligram of protein.
It may contain buffer salts.
4177
Interferon beta-1a concentrated solution EUROPEAN PHARMACOPOEIA 6.3

PRODUCTION C. Peptide mapping (2.2.55) and liquid chromatography

Interferon beta-1a concentrated solution is produced by a (2.2.29).
method based on recombinant DNA (rDNA) technology, Test solution. Add 5 μl of a 242 g/l solution of
using mammalian cells in culture. tris(hydroxymethyl)aminomethane R and a volume
Prior to release, the following tests are carried out on each of the preparation to be examined containing 20 μg of
batch of the final bulk product, unless exemption has been protein to a polypropylene tube of 0.5 ml capacity. Add
granted by the competent authority. 4 μl of a 1 mg/ml solution of endoprotease LysC R in
0.05 M tris-hydrochloride buffer solution pH 9.0 R. Mix
Host-cell-derived proteins. The limit is approved by the gently and incubate at 30 °C for 2 h. Add 10 μl of a
competent authority. 15.4 g/l solution of dithiothreitol R. Dilute the solution
Host-cell or vector-derived DNA. The limit is approved by with the same volume of a 573 g/l solution of guanidine
the competent authority. hydrochloride R. Incubate at 4 °C for 3-4 h.
N-terminal truncated forms. Examination for specific Reference solution. Prepare at the same time and in the
N-terminal truncated forms should be performed using same manner as for the test solution but using interferon
a suitable technique such as N-terminal sequence beta-1a CRS instead of the preparation to be examined.
determination. The limits are approved by the competent Precolumn:
authority.
— size: l = 0.02 m, Ø = 2.1 mm ;
Dimer and related substances of higher molecular — stationary phase : spherical octadecylsilyl silica gel
mass : not more than the amount approved by the for chromatography R (5 μm) with a pore size of
competent authority, using an appropriate validated liquid 30 nm.
chromatography method.
Column :
CHARACTERS — size: l = 0.25 m, Ø = 2.1 mm ;
Appearance : clear or slightly opalescent, colourless or — stationary phase : spherical octadecylsilyl silica gel
slightly yellowish liquid. for chromatography R (5 μm) with a pore size of
IDENTIFICATION 30 nm.
A. It shows the expected biological activity (see Assay). Mobile phase :
B. Isoform distribution. Mass spectrometry (2.2.43). — mobile phase A : dilute 1 ml of trifluoroacetic acid R
to 1000 ml with water R ;
Introduction of the sample : direct inflow of a
desalted preparation to be examined or liquid — mobile phase B : dilute 1 ml of trifluoroacetic acid R
chromatography-mass spectrometry combination. in 700 ml of acetonitrile for chromatography R, then
dilute to 1000 ml with water R ;
Mode of ionisation : electrospray.
Signal acquisition : complete spectrum mode from Time Mobile phase A Mobile phase B
1100 to 2400. (min) (per cent V/V) (per cent V/V)
0 - 30 100 → 64 0 → 36
Calibration: use myoglobin in the m/z range of 600-2400 ;
set the instrument within validated instrumental settings 30 - 45 64 → 55 36 → 45
and analyse the sample ; the deviation of the measured
45 - 50 55 → 40 45 → 60
mass does not exceed 0.02 per cent of the reported mass.
Interpretation of results : a typical spectrum consists 50 - 70 40 → 0 60 → 100
of 6 major glycoforms (A to F), which differ in their 70 - 83 0 100
degree of sialylation and/or antennarity type as shown
83 - 85 0 → 100 100 → 0
in Table 1639.-1.
Table 1639.-1. Flow rate : 0.2 ml/min.
MS peak Glycoform* Expected Mr Sialylation Detection : spectrophotometer at 214 nm.
level Injection : volume that contains 20 μg of digested protein.
A 2A2S1F 22 375 Disialylated System suitability : the chromatogram obtained with
B 2A1S1F 22 084 Monosialylated the reference solution is qualitatively similar to the
chromatogram of interferon beta-1a digest supplied with
C 3A2S1F and/or 22 739 Disialylated
2A2S1F
interferon beta-1a CRS.
+ 1 HexNacHex Results : the profile of the chromatogram obtained with
repeat the test solution corresponds to that of the chromatogram
D 3A3S1F 23 031 Trisialylated obtained with the reference solution.
E 4A3S1F and/or 23 400 Trisialylated
3A3S1F TESTS
+ 1 HexNacHex Impurities of molecular masses differing from that of
repeat
interferon beta-1a. Polyacrylamide gel electrophoresis
F 2A0S1F 21 793 Non-sialylated
(2.2.31) under reducing conditions.
* 2A = biantennary complex type oligosaccharide ; 3A = triantennary Resolving gel : 12 per cent acrylamide.
complex type oligosaccharide ; 4A = tetraantennary complex
type oligosaccharide ; 0S = non-sialylated ; 1S = monosialylated ; Concentrated sample buffer : concentrated SDS-PAGE
2S = disialylated ; 3S = trisialylated ; 1F = fucosylated. sample buffer for reducing conditions R containing
Results : the mass spectrum obtained with the preparation 2-mercaptoethanol as the reducing agent.
to be examined corresponds, with respect to the 6 major Sample buffer : mixture of equal volumes of concentrated
peaks, to the mass spectrum obtained with interferon SDS-PAGE sample buffer for reducing conditions R and
beta-1a CRS. water R.

4178 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Interferon beta-1a concentrated solution

Test solution (a). Concentrate the preparation to be Calculate the percentage of oxidation of interferon beta-1a
examined using a suitable method to obtain a protein using the following expression :
concentration of 1.5 mg/ml.
Test solution (b) : mixture of equal volumes of test
solution (a) and the concentrated sample buffer.
Test solution (c). Dilute test solution (a) to obtain a protein = area of the peak corresponding to the oxidised
A34-45ox
concentration of 0.6 mg/ml. Mix equal volumes of this
peptide fragment 34-45 ;
solution and the concentrated sample buffer.
A34-45 = area of the peak corresponding to the peptide
Test solution (d). Mix 8 μl of test solution (c) and 40 μl of
the sample buffer. fragment 34-45.
Test solution (e). Mix 15 μl of test solution (d) and 35 μl of Bacterial endotoxins (2.6.14) : less than 0.7 IU in the volume
the sample buffer. that contains 1 × 106 IU of interferon beta-1a, if intended for
Test solution (f). Mix 18 μl of test solution (e) and 18 μl of use in the manufacture of parenteral preparations without
the sample buffer. a further appropriate procedure for removal of bacterial
endotoxins.
Test solution (g). Mix 12 μl of test solution (f) and 12 μl of
the sample buffer. ASSAY
Reference solution (a). Solution of relative molecular mass Protein. Liquid chromatography (2.2.29). Prepare
markers suitable for calibrating SDS-PAGE gels in the range 3 independent dilutions for each solution.
of 15-67 kDa. Dissolve in the sample buffer.
Test solution. Dilute the preparation to be examined to
Reference solution (b) : 0.75 mg/ml solution of interferon obtain a concentration of 100 μg/ml.
beta-1a CRS in sample buffer.
Reference solution. Dissolve the contents of a vial of
Sample treatment: boil for 3 min. interferon beta-1a CRS to obtain a concentration of
Application : 20 μl of test solutions (b) to (g) and reference 100 μg/ml.
solutions (a) and (b). Precolumn:
Detection : Coomassie staining, carried out as follows : — size: l = 0.02 m, Ø = 2.1 mm ;
immerse the gel in Coomassie staining solution R1 at
33-37 °C for 90 min with gentle shaking, then remove the — stationary phase : butylsilyl silica gel for
staining solution ; destain the gel with a large excess of a chromatography R (5 μm) with a pore size of
mixture of 1 volume of glacial acetic acid R, 1 volume of 30 nm.
2-propanol R and 8 volumes of water R. Column :
Apparent molecular masses: interferon beta-1a = — size: l = 0.25 m, Ø = 2.1 mm ;
about 23 000 ; underglycosylated interferon — stationary phase : butylsilyl silica gel for
beta-1a = about 21 000 ; deglycosylated interferon chromatography R (5 μm) with a pore size of
beta-1a = about 20 000 ; interferon beta-1a dimer = about 30 nm.
46 000. Mobile phase :
Identification of bands: use the electropherogram provided — mobile phase A : 0.1 per cent V/V solution of
with interferon beta-1a CRS. trifluoroacetic acid R ;
System suitability : — mobile phase B : to 300 ml of water R, add 1 ml of
— the validation criteria are met (2.2.31) ; trifluoroacetic acid R and dilute to 1000 ml with
— a band is seen in the electropherogram obtained with test acetonitrile for chromatography R ;
solution (g) ; Time Mobile phase A Mobile phase B
— a gradation of intensity of staining is seen in the (min) (per cent V/V) (per cent V/V)
electropherograms obtained with test solutions (b) to (g). 0 - 20 100 → 0 0 → 100
Limits : 20 - 25 0 100
— in the electropherogram obtained with test solution (c),
25 - 26 0 → 100 100 → 0
the band corresponding to underglycosylated interferon
beta-1a is not more intense than the principal band in the 26 - 40 100 0
electropherogram obtained with test solution (e) (5 per
cent) ; Flow rate : 0.2 ml/min.
— in the electropherogram obtained with test solution (b), Detection : spectrophotometer at 214 nm.
the band corresponding to deglycosylated interferon Injection : 50 μl.
beta-1a is not more intense than the principal band in the Retention time : interferon beta-1a = about 20 min.
electropherogram obtained with test solution (e) (2 per System suitability : reference solution :
cent) ; any other band corresponding to an impurity of
a molecular mass lower than that of interferon beta-1a, — symmetry factor : 0.8 to 2.0 for the peak due to interferon
apart from the band corresponding to underglycosylated beta-1a ;
interferon beta-1a is not more intense than the principal — repeatability : maximum relative standard deviation
band in the electropherogram obtained with test of 3.0 per cent between the peak areas obtained after
solution (f) (1 per cent). injection of the 3 independent dilutions.
Oxidised interferon beta-1a: maximum 6 per cent. Calculate the content of interferon beta-1a
(C908H1408N246O252S7) from the declared content of
Use the chromatogram obtained with the test solution C908H1408N246O252S7 in interferon beta-1a CRS.
in identification C. Locate the peaks due to the peptide
fragment comprising amino acids 34-45 and its oxidised Potency
form using the chromatogram of oxidised interferon beta-1a The potency of interferon beta-1a is estimated by comparing
digest supplied with interferon beta-1a CRS. its ability to protect cells against a viral cytopathic effect with

General Notices (1) apply to all monographs and other texts 4179
Interferon beta-1a concentrated solution
the same ability of the appropriate International Standard
of human recombinant interferon beta-1a or of a reference
preparation calibrated in International Units.
The International Unit is the activity contained in a stated
amount of the appropriate International Standard. The
equivalence in International Units of the International
Standard is stated by the World Health Organisation.
Carry out the assay using a suitable method, based on the
following design.
Use, in standard culture conditions, an established cell line
sensitive to the cytopathic effect of a suitable virus and
responsive to interferon. The cell cultures and viruses that
have been shown to be suitable include the following :
— WISH cells (ATCC No. CCL-25) and vesicular stomatitis
virus VSV, Indiana strain (ATCC No. VR-158) as infective
agent ;
— A549 cells (ATCC No. CCL-185) and encephalomyocarditis
virus EMC (ATCC No. VR-129B) as infective agent.
Incubate in at least 4 series, cells with 3 or more different
concentrations of the preparation to be examined and the
reference preparation in a microtitre plate and include in
each series appropriate controls of untreated cells. Choose
the concentrations of the preparations such that the lowest
concentration produces some protection and the largest
4180 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
concentration produces less than maximal protection
against the viral cytopathic effect. Add at a suitable time the
cytopathic virus to all wells with the exception of a sufficient
number of wells in all series, which are left with uninfected
control cells. Determine the cytopathic effect of the virus
quantitatively with a suitable method. Calculate the potency
of the preparation to be examined by the usual statistical
methods (for example, 5.3).
The estimated potency is not less than 80 per cent and
not more than 125 per cent of the stated potency. The
confidence limits (P = 0.95) are not less than 64 per cent and
not more than 156 per cent of the estimated potency.
STORAGE
In an airtight container, protected from light, at a
temperature below − 70 °C. If the substance is sterile, store
in a sterile, airtight, tamper-proof container.
LABELLING
The label states :
— the interferon beta-1a content, in milligrams per millilitre ;
— the antiviral activity, in International Units per millilitre ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
EUROPEAN PHARMACOPOEIA 6.3

K
Kaolin, heavy............................................................................. 4183

General Notices (1) apply to all monographs and other texts 4181
 EUROPEAN PHARMACOPOEIA 6.3

4182 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Kaolin, heavy

01/2009:0503 dilute the solution 1 to 100 with water R. The solution is

not more intensely coloured than a 0.03 g/l solution of
methylene blue R.
KAOLIN, HEAVY
Swelling power. Triturate 2 g with 2 ml of water R. The
mixture does not flow.
Kaolinum ponderosum Substances soluble in mineral acids : maximum 1 per cent.
DEFINITION To 5.0 g add 7.5 ml of dilute hydrochloric acid R and 27.5 ml
Purified, natural, hydrated aluminium silicate of variable of water R and boil for 5 min. Filter, wash the residue on
composition. the filter with water R and dilute the combined filtrate and
washings to 50.0 ml with water R. To 10.0 ml of the solution
CHARACTERS add 1.5 ml of dilute sulphuric acid R, evaporate to dryness
on a water-bath and ignite. The residue weighs a maximum
Appearance : fine, white or greyish-white, unctuous powder. of 10 mg.
Solubility : practically insoluble in water and in organic
Chlorides (2.4.4) : maximum 250 ppm.
solvents.
Dilute 2 ml of solution S to 15 ml with water R.
IDENTIFICATION Sulphates (2.4.13) : maximum 0.1 per cent.
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R Dilute 1.5 ml of solution S to 15 ml with distilled water R.
and 3 g of sodium carbonate R and heat until the mixture Calcium (2.4.3) : maximum 250 ppm.
melts. Allow to cool. To the residue add 20 ml of boiling
water R, mix and filter. Wash the residue with 50 ml of Dilute 4 ml of solution S to 15 ml with distilled water R.
water R. To the residue add 1 ml of hydrochloric acid R Heavy metals (2.4.8) : maximum 50 ppm.
and 5 ml of water R. Filter. To the filtrate add 1 ml of To 5 ml of the solution prepared for the test for substances
strong sodium hydroxide solution R and filter. To the soluble in mineral acids add 5 ml of water R, 10 ml of
filtrate add 3 ml of ammonium chloride solution R. hydrochloric acid R and 25 ml of methyl isobutyl ketone R.
A gelatinous white precipitate is formed. Shake for 2 min. Separate the layers. Evaporate the aqueous
B. Add 2.0 g in 20 portions to 100 ml of a 10 g/l solution of layer to dryness on a water-bath. Dissolve the residue in
sodium laurilsulfate R in a 100 ml graduated cylinder 1 ml of acetic acid R and dilute to 25 ml with water R.
about 30 mm in diameter. Allow 2 min between additions Filter. 12 ml of the solution complies with test A. Prepare
for each portion to settle. Allow to stand for 2 h. The the reference solution using lead standard solution (1 ppm
apparent volume of the sediment is not greater than 5 ml. Pb) R.
C. 0.25 g gives the reaction of silicates (2.3.1). If intended for internal use, the above test is replaced by the
following test for heavy metals (2.4.8) : maximum 25 ppm.
TESTS To 10 ml of the solution prepared for the test for substances
Solution S. To 4 g add a mixture of 6 ml of acetic acid R and soluble in mineral acids add 10 ml of water R, 20 ml of
34 ml of distilled water R, shake for 1 min and filter. hydrochloric acid R and 25 ml of methyl isobutyl ketone R.
Shake for 2 min. Separate the layers. Evaporate the aqueous
Acidity or alkalinity. To 1.0 g add 20 ml of carbon layer to dryness on a water-bath. Dissolve the residue in
dioxide-free water R, shake for 2 min and filter. To 10 ml 1 ml of acetic acid R and dilute to 25 ml with water R.
of the filtrate add 0.1 ml of phenolphthalein solution R. Filter. 12 ml of the solution complies with test A. Prepare
The solution is colourless. Not more than 0.25 ml of 0.01 M the reference solution using lead standard solution (1 ppm
sodium hydroxide is required to change the colour of the Pb) R.
indicator to pink.
Microbial contamination
Organic impurities. Heat 0.3 g to redness in a calcination TAMC : acceptance criterion 103 CFU/g (2.6.12).
tube. The residue is only slightly more coloured than the
original substance. TYMC : acceptance criterion 102 CFU/g (2.6.12).
Adsorption power. To 1.0 g in a ground-glass-stoppered LABELLING
test-tube add 10.0 ml of a 3.7 g/l solution of methylene The label states, where applicable, that the substance is
blue R and shake for 2 min. Allow to settle. Centrifuge and suitable for internal use.

General Notices (1) apply to all monographs and other texts 4183
 EUROPEAN PHARMACOPOEIA 6.3

4184 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
L
Lactitol monohydrate.............................................................. 4187
Lactose, anhydrous.................................................................. 4188
Lactose monohydrate.............................................................. 4190
Lactulose.................................................................................... 4191
Lactulose, liquid.. ..................................................................... 4193
General Notices (1) apply to all monographs and other texts
Lamotrigine............................................................................... 4195
Lauromacrogol 400.. ............................................................... 4196
Lemon verbena leaf.. ............................................................... 4199
Levodropropizine.....................................................................4200
Lynestrenol................................................................................4202
4185
 EUROPEAN PHARMACOPOEIA 6.3

4186 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:1337 Appearance of solution. Solution S is clear (2.2.1) and not
LACTITOL MONOHYDRATE
Lactitolum monohydricum
C12H24O11,H2O Mr 362.3 examined in water R and dilute to 10.0 ml with the same
[81025-04-9]
DEFINITION
4-O-(β-D-Galactopyranosyl)-D-glucitol monohydrate.
Content : 96.5 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : very soluble in water, slightly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride
IDENTIFICATION
First identification : B.
Second identification : A, C.
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : lactitol monohydrate CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 50 mg of the substance to be
examined in methanol R and dilute to 20 ml with the
same solvent.
Reference solution (a). Dissolve 5 mg of lactitol
monohydrate CRS in methanol R and dilute to 2 ml with Run time : 2.5 times the retention time of lactitol.
the same solvent.
Reference solution (b). Dissolve 5 mg of sorbitol CRS
(impurity E) in 2 ml of reference solution (a) and dilute
to 20 ml with methanol R.
Plate : TLC silica gel G plate R.
Mobile phase : water R, acetonitrile R (25 :75 V/V).
Application : 2 μl.
Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with 4-aminobenzoic acid solution R
and dry in a current of cold air until the solvent is
removed ; heat at 100 °C for 15 min and allow to cool ;
spray with a 2 g/l solution of sodium periodate R and
dry in a current of cold air ; heat at 100 °C for 15 min.
System suitability : the chromatogram obtained with
reference solution (b) shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained Reducing sugars : maximum 0.2 per cent.
with reference solution (a).
TESTS
Solution S. Dissolve 5.000 g in carbon dioxide-free water R boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per
and dilute to 50.0 ml with the same solvent.
General Notices (1) apply to all monographs and other texts
Lactitol monohydrate
more intensely coloured than reference solution BY7 (2.2.2,
Method II).
Acidity or alkalinity. To 10 ml of solution S add 10 ml of
carbon dioxide-free water R. To 10 ml of this solution add
0.05 ml of phenolphthalein solution R. Not more than 0.2 ml
of 0.01 M sodium hydroxide is required to change the colour
of the indicator to pink. To a further 10 ml of the solution
add 0.05 ml of methyl red solution R. Not more than 0.3 ml
of 0.01 M hydrochloric acid is required to change the colour
of the indicator to red.
Specific optical rotation(2.2.7) : + 13.5 to + 15.5 (anhydrous
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
solvent.
Test solution (b). Dilute 2.0 ml of test solution (a) to 50.0 ml
with water R.
Reference solution (a). Dissolve 5.0 mg of lactitol
monohydrate CRS and 5 mg of glycerol R in water R and
dilute to 25.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of test solution (a)
to 100.0 ml with water R. Dilute 5.0 ml of this solution to
100.0 ml with water R.
Reference solution (c). Dilute 2.5 ml of reference solution (a)
to 10.0 ml with water R.
Column :
— size: l = 0.30 m, Ø = 7.8 mm ;
— stationary phase : strong cation exchange resin (calcium
form) R ;
— temperature : 60 °C.
Mobile phase : water R.
Flow rate : 0.6 ml/min.
Detection : refractive index detector maintained at a constant
temperature.
Injection : 100 μl ; inject test solution (a) and reference
solutions (b) and (c).
Relative retention with reference to lactitol (retention
time = about 13 min) : impurity A = about 0.7 ;
impurity B = about 0.8 ; glycerol = about 1.3 ;
impurity C = about 1.5 ; impurity D = about 1.8 ;
impurity E = about 1.9.
System suitability : reference solution (c) :
— resolution : minimum 5 between the peaks due to lactitol
and glycerol.
Limits :
— impurity B : not more than the area of the peak due to
lactitol in the chromatogram obtained with reference
solution (c) (1.0 per cent) ;
— total of other impurities : not more than the area of the
peak due to lactitol in the chromatogram obtained with
reference solution (c) (1.0 per cent) ;
— disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard any peak due to the solvent.
Dissolve 5.0 g in 3 ml of water R with gentle heating. Cool
and add 20 ml of cupri-citric solution R and a few glass
beads. Heat so that boiling begins after 4 min and maintain
cent V/V solution of glacial acetic acid R and 20.0 ml of
4187
Lactose, anhydrous
0.025 M iodine. With continuous shaking, add 25 ml of a
mixture of 6 volumes of hydrochloric acid R and 94 volumes
of water R. When the precipitate has dissolved, titrate the
excess of iodine with 0.05 M sodium thiosulphate using 1 ml
of starch solution R added towards the end of the titration,
as indicator. Not less than 12.8 ml of 0.05 M sodium
thiosulphate is required.
Lead (2.4.10) : maximum 0.5 ppm.
Nickel (2.4.15) : maximum 1 ppm.
Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on
0.30 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
Absence of Pseudomonas aeruginosa (2.6.13).
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 (a).
Calculate the percentage content of C12H24O11 using
the chromatograms obtained with test solution (b) and
reference solution (a) and the declared content of lactitol
monohydrate CRS.
IMPURITIES
Specified impurities : A, B, C, D, E.
A. lactose,
B. 3-O-(β-D-galactopyranosyl)-D-glucitol (lactulitol),
C. mannitol,
D. galactitol (dulcitol),
E. sorbitol.
4188
EUROPEAN PHARMACOPOEIA 6.3
01/2009:1061
LACTOSE, ANHYDROUS
Lactosum anhydricum
C12H22O11 Mr 342.3
DEFINITION
O-β-D-Galactopyranosyl-(1→4)-β-D-glucopyranose or mixture
of O-β-D-galactopyranosyl-(1→4)-α-D-glucopyranose and
O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranose.
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely but slowly soluble in water, practically
insoluble in ethanol (96 per cent).
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : anhydrous lactose CRS.
B. Thin-layer chromatography (2.2.27).
Solvent mixture : water R, methanol R (2:3 V/V).
Test solution. Dissolve 10 mg of the substance to be
examined in the solvent mixture and dilute to 20 ml with
the solvent mixture.
Reference solution (a). Dissolve 10 mg of anhydrous
lactose CRS in the solvent mixture and dilute to 20 ml
with the solvent mixture.
Reference solution (b). Dissolve 10 mg of anhydrous
lactose CRS, 10 mg of fructose CRS, 10 mg of
glucose CRS and 10 mg of sucrose CRS in the solvent
mixture and dilute to 20 ml with the solvent mixture.
Plate : TLC silica gel G plate R.
Mobile phase : water R, methanol R, glacial acetic
acid R, ethylene chloride R (10:15:25:50 V/V/V/V) ;
measure the volumes accurately, as a slight excess of
water produces cloudiness.
Application : 2 μl ; thoroughly dry the starting points.
Development A : over a path of 15 cm.
Drying A : in a current of warm air.
Development B : immediately, over a path of 15 cm, after
renewing the mobile phase.
Drying B : in a current of warm air.
Detection : spray with a solution of 0.5 g of thymol R in a
mixture of 5 ml of sulphuric acid R and 95 ml of ethanol
(96 per cent) R ; heat at 130 °C for 10 min.
System suitability : reference solution (b) :
— the chromatogram shows 4 clearly separated spots.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
C. Dissolve 0.25 g in 5 ml of water R. Add 5 ml of ammonia R
and heat in a water-bath at 80 °C for 10 min. A red colour
develops.
D. Water (see Tests).
TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY7
(2.2.2, Method II).
Dissolve 1.0 g in boiling water R and dilute to 10 ml with
the same solvent.
Acidity or alkalinity. Dissolve 6.0 g by heating in 25 ml
of carbon dioxide-free water R, cool and add 0.3 ml of
phenolphthalein solution R. The solution is colourless. Not
more than 0.4 ml of 0.1 M sodium hydroxide is required to
change the colour of the indicator to pink.
Specific optical rotation (2.2.7) : + 54.4 to + 55.9 (anhydrous
substance).
Dissolve 10.0 g in 80 ml of water R, heating to 50 °C. Allow
to cool and add 0.2 ml of dilute ammonia R1. Allow to stand
for 30 min and dilute to 100.0 ml with water R.
Absorbance (2.2.25).
Test solution (a). Dissolve 1.0 g in boiling water R and dilute
to 10.0 ml with the same solvent.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml
with water R.
Spectral range : 400 nm for test solution (a) and 210-300 nm
for test solution (b).
Results :
— at 400 nm : maximum 0.04 for test solution (a) ;
— from 210 nm to 220 nm : maximum 0.25 for test
solution (b) ;
— from 270 nm to 300 nm : maximum 0.07 for test
solution (b).
Heavy metals (2.4.8) : maximum 5 ppm.
2.0 g complies with test C. Prepare the reference solution
using 1.0 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 1.0 per cent, determined on
0.50 g, using a mixture of 1 volume of formamide R and
2 volumes of methanol R as the solvent.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
General Notices (1) apply to all monographs and other texts
Lactose, anhydrous
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for anhydrous
lactose used as a filler/diluent in solid dosage forms
(compressed and powder).
Particle size distribution (2.9.31 or 2.9.38).
Bulk and tapped density (2.9.34). Determine the bulk
density and the tapped density. Calculate the Hausner index
using the following expression :
V0 = volume of bulk substance ;
Vf = volume of tapped substance.
α-Lactose and β-lactose. Gas chromatography (2.2.28).
Silylation reagent. Mix 28 volumes of N-trimethylsilyl-
imidazole R and 72 volumes of pyridine R.
Test solution. Dissolve about 1 mg of the substance to be
examined in 0.45 ml of dimethyl sulphoxide R. Add 1.8 ml
of the silylation reagent. Mix gently and allow to stand for
20 min.
Reference solution. Prepare a mixture of α-lactose
monohydrate R and β-lactose R having an anomeric ratio of
about 1:1 based on the labelled anomeric contents of the
α-lactose monohydrate and β-lactose. Dissolve about 1 mg
of this mixture in 0.45 ml of dimethyl sulphoxide R. Add
1.8 ml of the silylation reagent. Mix gently and allow to
stand for 20 min.
Column :
— material: glass ;
— size: l = 0.9 m, Ø = 4 mm ;
— stationary phase : silanised diatomaceous earth for gas
chromatography R impregnated with 3 per cent m/m
of poly[(cyanopropyl)(methyl)][(phenyl)(methyl)]
siloxane R.
Carrier gas : helium for chromatography R.
Flow rate : 40 ml/min.
Temperature :
— column : 215 °C ;
— injection port and detector: 275 °C.
Detection : flame ionisation.
Injection : 2 μl.
System suitability : reference solution :
— relative retention with reference to β-lactose :
α-lactose = about 0.7 ;
— resolution : minimum 3.0 between the peaks due to
α-lactose and β-lactose.
Calculate the percentage content of α-lactose from the
following expression :
Calculate the percentage content of β-lactose from the
following expression :
Sa = area of the peak due to α-lactose ;
Sb = area of the peak due to β-lactose.
4189
Lactose monohydrate EUROPEAN PHARMACOPOEIA 6.3

Loss on drying (2.2.32). Determine on 1.000 g by drying

Results : the principal spot in the chromatogram obtained
in an oven at 80 °C for 2 h. with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
01/2009:0187 C. Dissolve 0.25 g in 5 ml of water R. Add 5 ml of ammonia R
and heat in a water-bath at 80 °C for 10 min. A red colour
LACTOSE MONOHYDRATE develops.
D. Water (see Tests).
Lactosum monohydricum TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY7
(2.2.2, Method II).
Dissolve 1.0 g in boiling water R and dilute to 10 ml with
the same solvent.
Acidity or alkalinity. Dissolve 6.0 g by heating in 25 ml
of carbon dioxide-free water R, cool and add 0.3 ml of
phenolphthalein solution R. The solution is colourless. Not
more than 0.4 ml of 0.1 M sodium hydroxide is required to
change the colour of the indicator to pink.
C12H22O11,H2O Mr 360.3 Specific optical rotation (2.2.7) : + 54.4 to + 55.9 (anhydrous
DEFINITION substance).
O-β-D-Galactopyranosyl-(1→4)-α-D-glucopyranose Dissolve 10.0 g in 80 ml of water R, heating to 50 °C. Allow
monohydrate. to cool and add 0.2 ml of dilute ammonia R1. Allow to stand
for 30 min and dilute to 100.0 ml with water R.
CHARACTERS Absorbance (2.2.25).
Appearance : white or almost white, crystalline powder. Test solution (a). Dissolve 1.0 g in boiling water R and dilute
Solubility : freely but slowly soluble in water, practically to 10.0 ml with the same solvent.
insoluble in ethanol (96 per cent). Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml
with water R.
IDENTIFICATION
Spectral range : 400 nm for test solution (a) and 210-300 nm
First identification : A, D. for test solution (b).
Second identification : B, C, D. Results :
A. Infrared absorption spectrophotometry (2.2.24). — at 400 nm : maximum 0.04 for test solution (a) ;
Comparison : lactose CRS. — from 210 nm to 220 nm : maximum 0.25 for test
B. Thin-layer chromatography (2.2.27). solution (b) ;
Solvent mixture : water R, methanol R (2:3 V/V). — from 270 nm to 300 nm : maximum 0.07 for test
Test solution. Dissolve 10 mg of the substance to be solution (b).
examined in the solvent mixture and dilute to 20 ml with Heavy metals (2.4.8) : maximum 5 ppm.
the solvent mixture. Dissolve 4.0 g in water R with warming, add 1 ml of 0.1 M
Reference solution (a). Dissolve 10 mg of lactose CRS in hydrochloric acid and dilute to 20 ml with water R. 12 ml
the solvent mixture and dilute to 20 ml with the solvent of the solution complies with test A. Prepare the reference
mixture. solution using lead standard solution (1 ppm Pb) R.
Reference solution (b). Dissolve 10 mg of fructose CRS, Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on
10 mg of glucose CRS, 10 mg of lactose CRS and 10 mg 0.50 g, using a mixture of 1 volume of formamide R and
of sucrose CRS in the solvent mixture and dilute to 20 ml 2 volumes of methanol R as the solvent.
with the solvent mixture.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Plate : TLC silica gel G plate R. on 1.0 g.
Mobile phase : water R, methanol R, glacial acetic
acid R, ethylene chloride R (10:15:25:50 V/V/V/V) ; Microbial contamination
measure the volumes accurately, as a slight excess of TAMC : acceptance criterion 102 CFU/g (2.6.12).
water produces cloudiness. Absence of Escherichia coli (2.6.13).
Application : 2 μl ; thoroughly dry the starting points. STORAGE
Development A : over a path of 15 cm. In an airtight container.
Drying A: in a current of warm air.
Development B : immediately, over a path of 15 cm, after FUNCTIONALITY-RELATED CHARACTERISTICS
renewing the mobile phase. This section provides information on characteristics
Drying B: in a current of warm air. that are recognised as being relevant control parameters
for one or more functions of the substance when used
Detection : spray with a solution of 0.5 g of thymol R in a
as an excipient (see chapter 5.15). This section is a
mixture of 5 ml of sulphuric acid R and 95 ml of ethanol
non-mandatory part of the monograph and it is not
(96 per cent) R ; heat at 130 °C for 10 min.
necessary to verify the characteristics to demonstrate
System suitability : reference solution (b) : compliance. Control of these characteristics can however
— the chromatogram shows 4 clearly separated spots. contribute to the quality of a medicinal product by

4190 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for lactose
monohydrate used as a filler/diluent in solid dosage forms
(compressed and powder).
Particle size distribution (2.9.31 or 2.9.38).
Bulk and tapped density (2.9.34). Determine the bulk
density and the tapped density. Calculate the Hausner Index
using the following expression :
V0 = volume of bulk substance ;
Vf = volume of tapped substance.
01/2009:1230
LACTULOSE
Lactulosum
C12H22O11 Mr 342.3
[4618-18-2]
DEFINITION
4-O-(β-D-Galactopyranosyl)-D-arabino-hex-2-ulofuranose.
Content : 95.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, sparingly soluble in
methanol, practically insoluble in toluene.
mp : about 168 °C.
IDENTIFICATION
First identification : B, C, D, E.
Second identification : A, C, D, E.
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 50.0 mg of the substance to be
examined in water R and dilute to 10.0 ml with the same
solvent.
Reference solution. Dissolve 50.0 mg of lactulose CRS in — size: l = 0.15 m, Ø = 4.6 mm ;
water R and dilute to 10.0 ml with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : glacial acetic acid R, 50 g/l solution
of boric acid R, methanol R, ethyl acetate R
(10:15:20:55 V/V/V/V).
Application : 2 μl.
General Notices (1) apply to all monographs and other texts
Lactulose
Development : over a path of 15 cm.
Drying : at 100-105 °C for 5 min and allow to cool.
Detection : spray with a 1.0 g/l solution of
1,3-dihydroxynaphthalene R in a mixture of 10 volumes
of sulphuric acid R and 90 volumes of methanol R ; heat
at 110 °C for 5 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (b).
C. Dissolve 50 mg in 10 ml of water R. Add 3 ml of
cupri-tartaric solution R and heat. A red precipitate is
formed.
D. Dissolve 0.125 g in 5 ml of water R. Add 5 ml of
ammonia R. Heat on a water-bath at 80 °C for 10 min. A
red colour develops.
E. Specific optical rotation (see Tests).
TESTS
Solution S. Dissolve 3.0 g in carbon dioxide-free water R
and dilute to 50 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY5 (2.2.2,
Method II).
pH (2.2.3) : 3.0 to 7.0.
To 10 ml of solution S add 0.1 ml of a saturated solution of
potassium chloride R.
Specific optical rotation (2.2.7) : − 46.0 to − 50.0 (anhydrous
substance).
Dissolve 1.25 g in water R, add 0.2 ml of concentrated
ammonia R and dilute to 25.0 ml with water R.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be
examined in 10 ml of water R. Add 12.5 ml of acetonitrile R
with gentle heating and dilute to 25.0 ml with water R.
Reference solution (a). To 3 ml of the test solution add
47.5 ml of acetonitrile R with gentle heating and dilute to
100.0 ml with water R.
Reference solution (b). Dissolve 1.00 g of lactulose CRS in
10 ml of water R. Add 12.5 ml of acetonitrile R with gentle
heating and dilute to 25.0 ml with water R.
Reference solution (c). Dissolve the contents of a vial of
lactulose for system suitability CRS in 1 ml of a mixture of
equal volumes of acetonitrile R and water R.
Precolumn:
— size: l = 0.05 m, Ø = 4.6 mm ;
— stationary phase : aminopropylsilyl silica gel for
chromatography R (3 μm) ;
— temperature : 38 ± 1 °C.
Column :
— stationary phase : aminopropylsilyl silica gel for
chromatography R (3 μm) ;
— temperature : 38 ± 1 °C.
Mobile phase : dissolve 0.253 g of sodium dihydrogen
phosphate R in 220 ml of water R and add 780 ml of
acetonitrile R.
4191
Lactulose
Flow rate : 1.0 ml/min.
Detection : refractometer maintained at a constant
temperature.
Injection : 20 μl of the test solution and reference
solutions (a) and (c).
Run time : 2.5 times the retention time of lactulose.
Relative retention with reference to lactulose (retention
time = about 18.3 min) : impurity E = about 0.38 ;
impurity D = about 0.42 ; impurity B = about 0.57 ;
impurity A = about 0.90 ; impurity C = about 1.17.
System suitability : reference solution (c) :
— resolution : minimum 1.3 between the peaks due to
lactulose and impurity A ; if necessary, adjust the
concentration of acetonitrile in the mobile phase to
between 75.0 per cent V/V and 82.0 per cent V/V ;
— the chromatogram obtained is similar to the
chromatogram supplied with lactulose for system
suitability CRS.
Limit :
— sum of impurities A, B, C, D and E : not more than the
area of the peak due to lactulose in the chromatogram
obtained with reference solution (a) (3 per cent).
Methanol. Head-space gas chromatography (2.2.28).
Internal standard solution. Mix 0.5 ml of propanol R with
100.0 ml of water R. Dilute 1.0 ml of this solution to 100.0 ml
with water R. Dilute 5.0 ml of the solution to 50.0 ml with
water R.
Test solution. To 79 mg of the substance to be examined in
a 20 ml vial add 1.0 ml of the internal standard solution and
5 μl of a 0.1 per cent V/V solution of methanol R.
Reference solution. To 1.0 ml of the internal standard
solution in a 20 ml vial add 5 μl of a 0.1 per cent V/V solution
of methanol R.
Column:
— size : l = 2 m, Ø = 2 mm ;
— stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (180 μm).
Carrier gas: helium for chromatography R.
Flow rate : 30 ml/min.
Static head-space conditions which may be used :
— equilibration temperature : 60 °C ;
— equilibration time : 1 h ;
— pressurisation time: 1 min.
Temperature :
— column : 140 °C ;
— injection port : 200 °C ;
— detector : 220 °C.
Detection : flame ionisation.
Injection : 1 ml of the gaseous phase.
Calculate the content of methanol, taking its density (2.2.5)
at 20 °C to be 0.79 g/ml.
Limit :
— methanol : calculate the ratio (R) of the area of the
peak due to methanol to the area of the peak due to the
internal standard in the chromatogram obtained with the
reference solution ; calculate the ratio of the area of the
peak due to methanol to the area of the peak due to the
internal standard in the chromatogram obtained with the
test solution : this ratio is not greater than 2R (50 ppm).
4192 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Boron : maximum 9 ppm.
Avoid where possible the use of glassware.
Reference solution. Dissolve 50.0 mg of boric acid R in
water R and dilute to 100.0 ml with the same solvent. Dilute
5.0 ml of this solution to 100.0 ml with water R. Keep in a
well-closed polyethylene container.
In 4 polyethylene 25 ml flasks, place separately :
— 0.50 g of the substance to be examined dissolved in 2.0 ml
of water R (solution A) ;
— 0.50 g of the substance to be examined dissolved in
1.0 ml of the reference solution and 1.0 ml of water R
(solution B) ;
— 1.0 ml of the reference solution and 1.0 ml of water R
(solution C) ;
— 2.0 ml of water R (solution D).
To each flask, add 4.0 ml of acetate-edetate buffer
solution pH 5.5 R. Mix and add 4.0 ml of freshly prepared
azomethine H solution R. Mix and allow to stand for 1 h.
Measure the absorbance (2.2.25) of solutions A, B and C at
420 nm, using solution D as the compensation liquid. The
test is not valid unless the absorbance of solution C is at
least 0.25. The absorbance of solution B is not less than
twice that of solution A.
Lead (2.4.10) : maximum 0.5 ppm.
Water (2.5.12) : maximum 2.5 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (b).
Calculate the percentage content of C12H22O11 from the
declared content of lactulose CRS.
IMPURITIES
A. 4-O-(β-D-galactopyranosyl)-D-mannopyranose (epilactose),
B. galactose,
C. lactose,
D. fructose,
E. D-lyxo-hex-2-ulopyranose (tagatose).
EUROPEAN PHARMACOPOEIA 6.3 Lactulose, liquid

01/2009:0924 pH (2.2.3) : 3.0 to 7.0.

To 10 ml of solution S, add 0.1 ml of a saturated solution of
LACTULOSE, LIQUID potassium chloride R.
Related substances. Liquid chromatography (2.2.29).
Lactulosum liquidum Test solution. Mix 4.00 g of the substance to be examined
DEFINITION with 20 ml of water R. Add 25.0 ml of acetonitrile R with
gentle heating and dilute to 50.0 ml with water R.
Aqueous solution of 4-O-(β-D-galactopyranosyl)-D-
arabino-hex-2-ulofuranose normally prepared by alkaline Reference solution (a). To 5 ml of the test solution, add
isomerisation of lactose. It may contain lesser amounts 47.5 ml of acetonitrile R with gentle heating and dilute to
of other sugars including lactose, epilactose, galactose, 100.0 ml with water R.
tagatose and fructose. Reference solution (b). Dissolve 2.00 g of lactulose CRS in
Content : minimum 620 g/l of lactulose (C12H22O11 ; Mr 342.3) 20 ml of water R. Add 25.0 ml of acetonitrile R with gentle
and 95.0 per cent to 105.0 per cent of the content of lactulose heating and dilute to 50.0 ml with water R.
stated on the label. Reference solution (c). Dissolve the contents of a vial of
It may contain a suitable antimicrobial preservative. lactulose for system suitability CRS in 1 ml of a mixture of
equal volumes of acetonitrile R and water R.
CHARACTERS
Precolumn:
Appearance : clear, viscous liquid, colourless or pale
brownish-yellow. — size: l = 0.05 m, Ø = 4.6 mm ;
Solubility : miscible with water. It may be a supersaturated — stationary phase : aminopropylsilyl silica gel for
solution or may contain crystals that disappear on heating. chromatography R (3 μm) ;
A 10 per cent V/V solution is laevorotatory. — temperature : 38 ± 1 °C.
Column :
IDENTIFICATION
First identification : B, C, D. — size: l = 0.15 m, Ø = 4.6 mm ;
Second identification : A, C, D. — stationary phase : aminopropylsilyl silica gel for
chromatography R (3 μm) ;
A. Thin-layer chromatography (2.2.27).
Test solution. Dilute 0.50 g of the substance to be — temperature : 38 ± 1 °C.
examined to 50 ml with water R. Mobile phase : dissolve 0.253 g of sodium dihydrogen
Reference solution. Dissolve 60 mg of lactulose CRS in phosphate R in 220 ml of water R and add 780 ml of
water R and dilute to 10 ml with the same solvent. acetonitrile R.
Plate : TLC silica gel G plate R. Flow rate : 1.0 ml/min.
Mobile phase : glacial acetic acid R, 50 g/l solution Detection : refractometer maintained at a constant
of boric acid R, methanol R, ethyl acetate R temperature.
(10:15:20:55 V/V/V/V). Injection : 20 μl of the test solution and reference
Application : 2 μl. solutions (a) and (c).
Development : over a path of 15 cm. Run time : 2.5 times the retention time of lactulose.
Drying : at 100-105 °C for 5 min and allow to cool. Relative retention with reference to lactulose (retention
Detection : spray with a 1.0 g/l solution of time = about 18 min) : impurity E = about 0.38 ;
1,3-dihydroxynaphthalene R in a mixture of 10 volumes impurity D = about 0.42 ; impurity B = about 0.57 ;
of sulphuric acid R and 90 volumes of methanol R ; heat impurity A = about 0.90 ; impurity C = about 1.17.
at 110 °C for 5 min. System suitability : reference solution (c) :
Results : the principal spot in the chromatogram obtained — resolution : minimum 1.3 between the peaks due to
with the test solution is similar in position, colour and lactulose and impurity A ; if necessary, adjust the
size to the principal spot in the chromatogram obtained concentration of acetonitrile in the mobile phase to
with the reference solution. between 75.0 per cent V/V and 82.0 per cent V/V ;
B. Examine the chromatograms obtained in the assay. — the chromatogram obtained is similar to the
Results : the principal peak in the chromatogram obtained chromatogram supplied with lactulose for system
with the test solution is similar in retention time to suitability CRS.
the principal peak in the chromatogram obtained with
reference solution (b). Limits :
C. To 0.1 g add 10 ml of water R and 3 ml of cupri-tartaric — impurity B : not more than 3 times the area of the peak
solution R and heat. A red precipitate is formed. due to lactulose in the chromatogram obtained with
reference solution (a) (15 per cent) ;
D. To 0.25 g add 5 ml of water R and 5 ml of ammonia R.
Heat in a water-bath at 80 °C for 10 min. A red colour — impurities A, C : for each impurity, not more than twice
develops. the area of the peak due to lactulose in the chromatogram
obtained with reference solution (a) (10 per cent) ;
TESTS — impurity E : not more than 0.8 times the area of the peak
Solution S. Mix 10 g with carbon dioxide-free water R and due to lactulose in the chromatogram obtained with
dilute to 100 ml with the same solvent. reference solution (a) (4 per cent) ;
Appearance of solution. Solution S is clear (2.2.1) and not — impurity D : not more than 0.2 times the area of the peak
more intensely coloured than reference solution BY5 (2.2.2, due to lactulose in the chromatogram obtained with
Method II). reference solution (a) (1 per cent).

General Notices (1) apply to all monographs and other texts 4193
Lactulose, liquid EUROPEAN PHARMACOPOEIA 6.3

Methanol. Head-space gas chromatography (2.2.28). In 4 polyethylene 25 ml flasks, place separately :

Internal standard solution. Mix 0.5 ml of propanol R with — 1.00 g of the substance to be examined and 1 ml of
100.0 ml of water R. Dilute 1.0 ml of this solution to 100.0 ml water R (solution A) ;
with water R. Dilute 5.0 ml of the solution to 50.0 ml with — 1.00 g of the substance to be examined and 1 ml of the
water R. reference solution (solution B) ;
Test solution. To 0.13 g of the substance to be examined in a — 1 ml of the reference solution and 1 ml of water R
20 ml vial add 1.0 ml of the internal standard solution and (solution C) ;
5 μl of a 0.1 per cent V/V solution of methanol R. — 2 ml of water R (solution D).
Reference solution. To 1.0 ml of the internal standard To each flask, add 4.0 ml of acetate-edetate buffer
solution in a 20 ml vial add 5 μl of a 0.1 per cent V/V solution solution pH 5.5 R. Mix and add 4.0 ml of freshly prepared
of methanol R. azomethine H solution R. Mix and allow to stand for 1 h.
Column: Measure the absorbance (2.2.25) of solutions A, B and C at
420 nm, using solution D as the compensation liquid. The
— size : l = 2 m, Ø = 2 mm ; test is not valid unless the absorbance of solution C is at
— stationary phase : ethylvinylbenzene-divinylbenzene least 0.25. The absorbance of solution B is not less than
copolymer R (180 μm). twice that of solution A.
Carrier gas: helium for chromatography R. Lead (2.4.10) : maximum 0.5 ppm, calculated with reference
to the declared content of lactulose.
Flow rate : 30 ml/min.
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
Static head-space conditions which may be used : on 1.5 g and calculated with reference to the declared
— equilibration temperature : 60 °C ; content of lactulose.
Microbial contamination
— equilibration time : 1 h ;
TAMC : acceptance criterion 102 CFU/g (2.6.12).
— pressurisation time: 1 min.
TYMC : acceptance criterion 101 CFU/g (2.6.12).
Temperature :
Absence of Escherichia coli (2.6.13).
— column : 140 °C ;
ASSAY
— injection port : 200 °C ;
Liquid chromatography (2.2.29) as described in the test for
— detector : 220 °C. related substances with the following modification.
Detection : flame ionisation. Injection : test solution and reference solution (b).
Injection : 1 ml of the gaseous phase. Calculate the percentage content of C12H22O11 from the
declared content of lactulose CRS.
Calculate the content of methanol, taking its density (2.2.5)
at 20 °C to be 0.79 g/ml. LABELLING
Limit : The label states the declared content of lactulose.
— methanol : calculate the ratio (R) of the area of the
peak due to methanol to the area of the peak due to the IMPURITIES
internal standard in the chromatogram obtained with the Specified impurities: A, B, C, D, E.
reference solution ; calculate the ratio of the area of the
peak due to methanol to the area of the peak due to the
internal standard in the chromatogram obtained with the
test solution : this ratio is not greater than 2R (30 ppm).
Sulphites : maximum 30 ppm.
Mix 5.0 g with 40 ml of water R, add 2.0 ml of 0.1 M sodium
hydroxide and dilute to 100 ml with water R. To 10.0 ml of
this solution, add 1.0 ml of hydrochloric acid R1, 2.0 ml
of decolorised fuchsin solution R1 and 2.0 ml of a 0.5 per
cent V/V solution of formaldehyde R. Allow to stand for
30 min and measure the absorbance (2.2.25) at 583 nm
using as the compensation liquid a solution prepared at the A. 4-O-(β-D-galactopyranosyl)-D-mannopyranose (epilactose),
same time and in the same manner with 10.0 ml of water R
instead of the solution of the substance to be examined. The B. galactose,
absorbance is not greater than that of a reference solution
prepared at the same time and in the same manner using C. lactose,
10.0 ml of sulphite standard solution (1.5 ppm SO2) R
instead of the solution of the substance to be examined. D. fructose,
Boron : maximum 5 ppm.
Avoid where possible the use of glassware.
Reference solution. Dissolve 56.0 mg of boric acid R in
water R and dilute to 100.0 ml with the same solvent. Dilute
5.0 ml of this solution to 100.0 ml with water R. Keep in a
well-closed polyethylene container. E. D-lyxo-hex-2-ulopyranose (tagatose).

4194 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:1756 Mobile phase :
— mobile phase A : mix 1 volume of triethylamine R
LAMOTRIGINE
Lamotriginum — mobile phase B : acetonitrile R ;
C9H7Cl2N5 Mr 256.1
[84057-84-1]
DEFINITION
6-(2,3-Dichlorophenyl)-1,2,4-triazine-3,5-diamine.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : very slightly soluble in water, slightly soluble
in anhydrous ethanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : lamotrigine CRS.
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20 mg of the substance to be
examined in 5 ml of methanol R and dilute to 100.0 ml with
a 10.3 g/l solution of hydrochloric acid R.
Reference solution (a). Dissolve 5 mg of lamotrigine for
system suitability CRS (containing impurity G) in 2.5 ml of
methanol R and dilute to 50.0 ml with a 10.3 g/l solution of
hydrochloric acid R. Dilute 1.0 ml of this solution to 10.0 ml
with a 10.3 g/l solution of hydrochloric acid R.
Reference solution (b). Dilute 1.0 ml of the test solution to
100.0 ml with a 10.3 g/l solution of hydrochloric acid R.
Dilute 2.0 ml of this solution to 10.0 ml with a 10.3 g/l
solution of hydrochloric acid R.
Reference solution (c). Dissolve 5.0 mg of lamotrigine
impurity E CRS in a mixture of 0.25 ml of hydrochloric
acid R and 45 ml of methanol R and dilute to 50.0 ml with
methanol R. Dilute 5.0 ml of the solution to 100.0 ml with a
10.3 g/l solution of hydrochloric acid R. To 4.0 ml of this
solution add 5 ml of methanol R and dilute to 100.0 ml with
a 10.3 g/l solution of hydrochloric acid R.
Reference solution (d). Dissolve 10 mg of lamotrigine for
peak identification CRS (containing impurities A, E and F)
in 2.5 ml of methanol R. Add 2.0 ml of reference solution (a)
and dilute to 50.0 ml with a 10.3 g/l solution of hydrochloric
acid R.
Blank solution. Mix 5 volumes of methanol R and
95 volumes of a 10.3 g/l solution of hydrochloric acid R.
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : base-deactivated end-capped
octadecylsilyl silica gel for chromatography R (5 μm) ;
— temperature : 35 °C.
General Notices (1) apply to all monographs and other texts
Lamotrigine
and 150 volumes of a 2.7 g/l solution of potassium
dihydrogen phosphate R ; adjust to pH 2.0 with
phosphoric acid R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 85 15
4 - 14 85 → 20 15 → 80
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 270 nm.
Injection : 10 μl of the test solution, reference solutions (a), (b)
and (d) and the blank solution.
Identification of impurities : use the chromatogram
supplied with lamotrigine for peak identification CRS and
the chromatogram obtained with reference solution (d) to
identify the peaks due to impurities A, E, F and G.
Relative retention with reference to lamotrigine
(retention time = about 7 min) : impurity G = about 1.1 ;
impurity A = about 1.3 ; impurity E = about 1.7 ;
impurity F = about 1.8.
System suitability: reference solution (a) :
— peak-to-valley ratio : minimum 1.2, where Hp = height
above the baseline 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 lamotrigine.
Limits :
— correction factor : for the calculation of content, multiply
the peak area of impurity F by 1.3 ;
— impurity F : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.2 per cent) ;
— impurities A, G : for each impurity, not more than 0.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent) ;
— unspecified impurities : for each impurity, not more
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ;
— total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.2 per cent) ;
— disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard any peak due to the blank and
any peak due to impurity E.
Impurity E. Liquid chromatography (2.2.29) as described
in the test for related substances with the following
modifications.
Mobile phase : acetonitrile for chromatography R, mobile
phase A (35:65 V/V).
Detection : spectrophotometer at 210 nm.
Injection : test solution and reference solutions (d) and (c).
Run time : 10 min.
Retention time : impurity E = about 5.5 min ;
impurity F = about 8.5 min.
System suitability : reference solution (d) :
— the chromatogram obtained is similar to the
chromatogram supplied with lamotrigine for peak
identification CRS.
4195
Lauromacrogol 400 EUROPEAN PHARMACOPOEIA 6.3

Limit :
— impurity E : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.1 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
To the residue obtained in the test for sulphated ash add 2 ml
of hydrochloric acid R and evaporate slowly to dryness on a
water-bath. Moisten the residue with 0.05 ml of hydrochloric C. (2Z)-[2-(diaminomethylidene)diazanylidene](2,3-
acid R, add 10 ml of boiling water R and heat the mixture for dichlorophenyl)acetonitrile,
10 min on a water-bath. Allow to cool to room temperature,
filter if necessary and adjust the volume of the filtrate and
washings to 20 ml with water R. 12 ml of the solution
complies with test A. Prepare the reference solution using
10 ml of lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 2.000 g by drying in an oven at 105 °C at a pressure not
exceeding 0.7 kPa for 3 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 2.0 g. D. 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5(2H,4H)-dione,

ASSAY
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). Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 25.61 mg
of C9H7Cl2N5. E. 2,3-dichlorobenzoic acid,

STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, E, F, 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 F. N-[5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl]-2,3-
by the general acceptance criterion for other/unspecified dichlorobenzamide,
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) : B, C, D.

G. 6-(2,4-dichlorophenyl)-1,2,4-triazine-3,5-diamine.

01/2009:2046

A. 3-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one, LAUROMACROGOL 400

B. (2E)-[2-(diaminomethylidene)diazanylidene](2,3-
dichlorophenyl)acetonitrile,
Lauromacrogolum 400
DEFINITION
Mixture of lauryl alcohol (dodecanol) monoethers of mixed
macrogols. It may contain some free macrogols and it
contains various amounts of free lauryl alcohol. The number
of moles of ethylene oxide reacted per mole of lauryl alcohol
is 9. The name of the substance is followed by a number
(400) corresponding approximately to the average molecular
mass of the macrogol portion.
This monograph applies to lauromacrogol 400 used as active
substance.

4196 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Lauromacrogol 400

CHARACTERS n1 = volume of 0.01 M sodium thiosulphate required

for the substance to be examined, in millilitres ;
Appearance : white or almost white, unctuous and n2 =
hygroscopic mass, melting at 24 °C into a colourless or volume of 0.01 M sodium thiosulphate required
yellowish, viscous liquid. for the blank, in millilitres ;
M = molarity of the sodium thiosulphate solution, in
Solubility : freely soluble in water, very soluble in acetone moles per litre ;
and in ethanol (96 per cent). m = mass of the substance to be examined, in grams.
Saponification value (2.5.6) : maximum 3.0.
IDENTIFICATION Free lauryl alcohol (dodecanol). Gas chromatography
(2.2.28).
A. Hydroxyl value (see Tests). Test solution. Dissolve 0.200 g of the substance to be
examined in acetone R and dilute to 10.0 ml with the same
B. Saponification value (see Tests). solvent.
C. Warm the substance to be examined in an incubator Reference solution. Dissolve 2.00 g of lauryl alcohol R in
at 50 °C for 1 h until fully molten and clear. Transfer acetone R and dilute to 100.0 ml with the same solvent.
50 ml to a warmed cloud-point tube (flat-bottomed glass Dilute 1.0 ml of this solution to 50.0 ml with acetone R.
tube 30-33.5 mm in internal diameter and 115-125 mm Column :
high). Insert the tube into a cooling bath that allows the
outer surface of the tube to be in contact with chilled air, — material: fused silica ;
contained within a cylindrical metal container (internal — size: l = 30 m, Ø = 0.25 mm ;
diameter 9.5-12.5 mm greater than the external diameter — stationary phase : poly(dimethyl)(diphenyl)siloxane R
of the sample tube, 115 mm high) that is surrounded by (film thickness 0.1 μm).
iced water. The base of the glass tube rests on a 6 mm
thick cork disc, which prevents direct thermal contact Carrier gas : helium for chromatography R.
with the cooled metal cylinder. Stir the substance to Flow rate : 1 ml/min.
be examined continuously with a thermometer so that Split ratio : 50:1.
the temperature is constant throughout the substance.
Periodically lift the tube out of the cooling bath to Temperature :
check for signs of cloudiness at the bottom of the tube. Time Temperature
Examine the tube against a bright light source. When (min) (°C)
cloudiness is first observed, check more frequently Column 0-1 120
until the substance becomes completely cloudy and the
thermometer, suspended in the centre of the substance, 1 - 23 120 → 350
is only just visible when viewed horizontally. Record the 23 - 33 350
temperature. It is 20 °C to 25 °C.
Injection port 300
Detector 350
TESTS
Detection : flame ionisation.
Appearance. The molten substance to be examined is clear Injection : 1.0 μl.
(2.2.1) and not more intensely coloured than reference
solution GY6 (2.2.2, Method I). Retention time : lauryl alcohol = about 5 min.
Alkalinity. Dissolve 2.0 g in a hot mixture of 10 ml of carbon Limit :
dioxide-free water R and 10 ml of ethanol (96 per cent) R. — free lauryl alcohol : not more than the area of the
Add 0.1 ml of bromothymol blue solution R1. Not more than corresponding peak in the chromatogram obtained with
0.5 ml of 0.1 M hydrochloric acid is required to change the the reference solution (2.0 per cent).
colour of the indicator to yellow. Free macrogols. Size-exclusion chromatography (2.2.30).
Acid value (2.5.1) : maximum 1.0, determined on 5.0 g. Test solution. Dissolve 5.0 g of the substance to be examined
Hydroxyl value (2.5.3, Method A) : 90 to 105, determined in the mobile phase and dilute to 250.0 ml with the mobile
on 0.35 g. phase.
Reference solution (a). Dissolve about 0.4 g of macrogol
Iodine value (2.5.4, Method A) : maximum 2.0.
1000 R in the mobile phase and dilute to 250.0 ml with the
Peroxide value (2.5.5, Method A) : maximum 5.0. mobile phase.
Reference solution (b). Dilute 50.0 ml of reference
Introduce 10.0 g into a 100 ml beaker, dissolve with glacial
solution (a) to 100.0 ml with the mobile phase.
acetic acid R and dilute to 20 ml with the same solvent. Add
1 ml of saturated potassium iodide solution R and allow to Precolumns (2) :
stand for 1 min. Add 50 ml of carbon dioxide-free water R. — size: l = 0.125 m, Ø = 4 mm ;
Titrate with 0.01 M sodium thiosulphate, determining the
— stationary phase : spherical octadecylsilyl silica gel for
end-point potentiometrically (2.2.20). Carry out a blank
chromatography R (5 μm) with a pore size of 10 nm.
titration.
Column :
Determine the peroxide value using the following expression: — size: l = 0.30 m, Ø = 7.8 mm ;
— stationary phase : hydroxylated polymethacrylate gel R
(6 μm) with a pore size of 12 nm.

General Notices (1) apply to all monographs and other texts 4197
Lauromacrogol 400 EUROPEAN PHARMACOPOEIA 6.3

Connect both precolumns to the column using a 3-way valve
and switch the mobile phase flow according to the following
programme :
— 0-114 s : precolumn 1 and column ;
— 115 s to the end : precolumn 2 and column ;
— 115 s to 8 min : flow back of precolumn 1.
Mobile phase : water R, methanol R (2:8 V/V).
Flow rate : 1.1 ml/min.
Detection : refractometer.
Injection : 20 μl.
Calculate the percentage content of free macrogols using the
following expression :
m1 = mass of the substance to be examined in the test
solution, in grams ;
m2 = mass of macrogol 1000 R in reference solution (a),
in grams ;
A1 = area of the peak due to free macrogols in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to macrogol 1000 in
the chromatogram obtained with reference
solution (a) ;
A3 = area of the peak due to macrogol 1000 in
the chromatogram obtained with reference
solution (b).
of deuterated chloroform R, containing 0.1 mol/l of
chromium(III) acetylacetonate R as a relaxation aid.
Apparatus : high resolution FT-NMR spectrometer operating
at minimum 300 MHz.
Acquisition of 13C NMR spectra. The following parameters
may be used:
— sweep width : 250 ppm (− 15 ppm to 235 ppm) ;
— irradiation frequency offset : 110 ppm ;
— time domain : 64 K ;
— pulse delay : 3 s ;
— pulse program : zgig 30 (inverse gated, 30° excitation
pulse) ;
— dummy scans : 4 ;
— number of scans : 2048.
Processing and plotting. The following parameters may be
used :
— size: 64 K (zero-filling) ;
— window multiplication : exponential ;
— Lorentzian broadening factor: 1 Hz.
Use the CD3OD signal for shift referencing. The shift of the
central peak of the multiplet is set to 49.0 ppm.
Plot the spectral region δ 0.0-80.0 ppm. Compare the
spectrum with the spectrum in Figure 2046.-1. The shift
values lie near the values given in Table 2046.-1.
Table 2046.-1. – Shift values
Signal Shift (ppm) Normalised integrals

Limit : CH3 14.4 0.989

— free macrogols : maximum 3.0 per cent. CH2 (alkyl chain) 23.2 1.000
Average chain length of the fatty alcohol and average CH2 (alkyl chain) 25.5 1.001
number of moles of ethylene oxide. Nuclear magnetic
CH2’s (alkyl chain) 30 7.410
resonance spectrometry (2.2.33).
Test solution. If the substance is in the solid state at room CH2 (alkyl chain) 32.5 0.963
temperature, heat gently before sampling. Dissolve 0.4 ml CH2 (-CH2-OH) 61.6 1.001
of the substance to be examined in 0.3 ml of a mixture (end CH2-group of
of 1 volume of deuterated methanol R and 2 volumes macrogol)

Figure 2046.-1. – 13C NMR spectrum of lauromacrogol 400

4198 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Lemon verbena leaf

CH2’s (macrogol) 70.7 16.25 IDENTIFICATION

CH2 (R-CH-O- 72.6 0.998
macrogol) (CH2 in
alpha position)
CH2 (macrogol) 73.1 0.929
System suitability :
— signal-to-noise ratio : minimum 150, for the smallest
relevant peak (CH2 at 73.1 ppm) ;
— peak width at half-height: maximum 0.05 ppm, for the
central CDCl3 signal (at δ 78.6 ppm).
Calculation of the average chain length of the fatty alcohol
and the average number of moles of ethylene oxide : define
the signal at 23.2 ppm as 1.000 and normalise the integrals
of the other signals listed in Table 2046.-1.
The average chain length of the fatty alcohol is calculated
using the following expression :
∑14-33 In,i = sum of the normalised integrals of the
signals from 14 ppm to 33 ppm ;
In,72.6 = normalised integral of the signal at
72.6 ppm.
The average number of moles of ethylene oxide is calculated
using the following expression :
A. The leaves are simple with short petioles. They are
narrow, lanceolate, and about 4 times longer than they
are wide. The entire, slightly undulating margins are
curled towards the upper surface. The upper surface is
dark green and rough to the touch ; the lower surface is
paler green and shows a prominent midrib with secondary
veins running to the margins.
B. Reduce to a powder (355) (2.9.12). The powder is light
green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters : fragments of the lamina in surface view, those
of the upper epidermis being composed of polygonal cells
with numerous short, unicellular, thick-walled cystolithic
trichomes, each arising from a rosette of cells at the base,
stomata are absent ; the cells of the lower epidermis are
more irregular and somewhat sinuous in outline and
anomocytic stomata (2.8.3) are abundant, also present are
numerous subsessile glandular trichomes with a globular
head ; fragments of parenchyma of the mesophyll and
groups of lignified tissue from the veins.

In,62, In,71, In,73

= normalised integral of the signals at
62 ppm, 71 ppm and 73 ppm respectively.
The sum of the normalised integrals of the signals at 62 ppm,
71 ppm and 73 ppm corresponds to the average number of
methylene groups in the macrogol part of lauromacrogol 400.
Limits :
— average chain length of the fatty alcohol: 10.0 to 14.0 ;
— average number of moles of ethylene oxide : 7.0 to 11.0.
Ethylene oxide and dioxan (2.4.25, Method A) : maximum
1 ppm of ethylene oxide and maximum 10 ppm of dioxan.
Water (2.5.12) : maximum 2.0 per cent, determined on
0.500 g.
Total ash (2.4.16) : maximum 0.2 per cent, determined on
2.0 g.

01/2008:1834
corrected 6.3

LEMON VERBENA LEAF

Verbenae citriodoratae folium
DEFINITION
Whole or fragmented, dried leaves of Aloysia citriodora
Palau (syn. Aloysia triphylla (L’Hér.) Kuntze ; Verbena
triphylla L’Hér. ; Lippia citriodora Kunth.).
Content :
— acteoside (C29H36O15 ; Mr 625) : minimum 2.5 per cent,
expressed as ferulic acid (dried drug) ;
— essential oil : minimum 3.0 ml/kg for the whole drug and C. Examine the chromatograms obtained in the test for
minimum 2.0 ml/kg for the fragmented drug (dried drug).
CHARACTERS
After grinding, it has a characteristic odour reminiscent of the test solution. Furthermore, other faint zones may
lemon.
A. Upper epidermis with cystolithic
trichome, with subsidiary cells
F. Surface view of lower epidermis
with striated cuticule, anomocytic
containing calcium concretions stomata and glandular trichome
B-C. Surface view of upper G. Transverse section of the lamina
epidermis, with palisade parenchyma H. Parenchyma of the mesophyll
and glandular trichome (C) with a lignified vein
D-E. Transverse section of glandular
trichomes
Figure 1834.-1. – Illustration of powdered herbal drug of
lemon verbena leaf (see Identification B)
Verbena officinalis.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and
be present in the chromatogram obtained with the test

General Notices (1) apply to all monographs and other texts 4199
Levodropropizine EUROPEAN PHARMACOPOEIA 6.3

solution. Zones may be present in the chromatogram Mobile phase :

obtained with the test solution below the zone due to — mobile phase A : 0.3 per cent V/V solution of phosphoric
rutin in the chromatogram obtained with the reference acid R ;
solution. — mobile phase B : acetonitrile R ;
Top of the plate
Time Mobile phase A Mobile phase B
_______ _______ (min) (per cent V/V) (per cent V/V)
An intense greyish-green zone 0 - 20 93 → 83 7 → 17

Arbutin : a blue or brown zone 20 - 30 83 17

30 - 35 83 → 75 17 → 25

Rutin : a dark brownish-yellow A blue or violet zone 35 - 40 75 → 20 25 → 80

zone 40 - 45 20 → 93 80 → 7
_______ _______

Reference solution Test solution

Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 330 nm.
TESTS Injection : 20 μl.
Verbena officinalis. Thin-layer chromatography (2.2.27). System suitability : test solution :
Test solution. To 0.50 g of the powdered drug (710) (2.9.12) — resolution : minimum 3.5 between the peaks due to
add 5 ml of methanol R. Heat in a water-bath at 60 °C for ferulic acid and acteoside.
10 min. Cool and filter. Calculate the percentage content of acteoside, expressed as
Reference solution. Dissolve 10 mg of arbutin R and 10 mg ferulic acid, using the following expression :
of rutin R in methanol R and dilute to 10 ml with the same
solvent.
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
plate R (2-10 μm)]. A1 = area of the peak due to acteoside in the
Mobile phase : anhydrous formic acid R, glacial acetic chromatogram obtained with the test solution ;
acid R, water R, ethyl acetate R (11:11:27:100 V/V/V/V). A2 = area of the peak due to ferulic acid in the
Application : 20 μl [or 5 μl] as bands. chromatogram obtained with the reference
Development : over a path of about 12 cm [or 6 cm]. solution ;
Drying : in air. m1 = mass of the drug in the test solution, in grams ;
Detection : spray with anisaldehyde solution R and dry at m2 = mass of ferulic acid CRS in the reference solution,
100-105 °C for about 10 min ; examine in daylight. in grams ;
Results : the chromatogram obtained with the test solution p = percentage content of ferulic acid in ferulic
shows no brownish-grey zone at a position between that of acid CRS ;
arbutin and rutin in the chromatogram obtained with the 3.1 = correlation factor between acteoside and ferulic
reference solution. acid.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered drug (710) (2.9.12) by drying Essential oil (2.8.12). Introduce 25.0 g of the freshly
in an oven at 105 °C for 2 h. crushed drug into a 1000 ml flask and add 500 ml of a 10 g/l
solution of sodium chloride R as the distillation liquid. Use
Total ash (2.4.16) : maximum 13.0 per cent. 0.50 ml of xylene R in the graduated tube. Distil at a rate of
Ash insoluble in hydrochloric acid (2.8.1) : maximum 3.0-3.5 ml/min for 3 h.
3.5 per cent.
ASSAY 01/2009:1535
Acteoside. Liquid chromatography (2.2.29).
Test solution. To 1.00 g of the powdered drug (710) (2.9.12)
LEVODROPROPIZINE
add 50.0 ml of the reference solution and stir for 2 h with
a magnetic stirrer. Centrifuge for 15 min and pass the Levodropropizinum
supernatant through a membrane filter (nominal pore size
0.45 μm).
Reference solution. Dissolve 10.0 mg of ferulic acid CRS
in ethanol (60 per cent V/V) R and dilute to 100.0 ml with
the same solvent.
Precolumn :
— size : l = 0.01 m, Ø = 4.0 mm ; C13H20N2O2 Mr 236.3
— stationary phase : octadecylsilyl silica gel for [99291-25-5]
chromatography R (5 μm).
DEFINITION
Column:
(2S)-3-(4-Phenylpiperazin-1-yl)propane-1,2-diol.
— size : l = 0.25 m, Ø = 4.0 mm ;
Content : 98.5 per cent to 101.0 per cent (dried substance).
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ; CHARACTERS
— temperature : 20 °C. Appearance : white or almost white powder.

4200 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Solubility : slightly soluble in water, freely soluble in dilute
acetic acid and in methanol, slightly soluble in ethanol
(96 per cent).
IDENTIFICATION
Carry out either tests A, B or tests B, C.
A. Specific optical rotation (2.2.7) : − 30.0 to − 33.5 (dried
substance).
Dissolve 1.50 g in a 21 g/l solution of hydrochloric acid R
and dilute to 50.0 ml with the same acid.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : levodropropizine CRS.
C. Enantiomeric purity (see Tests).
TESTS
pH (2.2.3) : 9.2 to 10.2.
Suspend 2.5 g in carbon dioxide-free water R, heat to
dissolve, cool to room temperature and dilute to 100 ml with
the same solvent.
Impurity B and related substances. Liquid chromatography
(2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the
mobile phase.
Reference solution (a). Dissolve 25.0 mg of levodropropizine
impurity B CRS in methanol R and dilute to 100.0 ml with
the same solvent. Dilute 1.0 ml of this solution to 100.0 ml
with the mobile phase.
Reference solution (b). Mix 1.0 ml of the test solution with
1.0 ml of reference solution (a).
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 μm).
Mobile phase : mix 12 volumes of methanol R and
88 volumes of a 6.81 g/l solution of potassium dihydrogen
phosphate R adjusted to pH 3.0 with phosphoric acid R.
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl.
Run time : twice the retention time of levodropropizine.
Relative retention with reference to levodropropizine
(retention time = about 7 min) : impurity B = about 1.2.
System suitability : reference solution (b) :
— resolution : minimum 2.0 between the peaks due to
levodropropizine and impurity B.
Limits :
— impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) ;
— unspecified impurities: for each impurity, not more
than 0.2 times the area of the peak due to impurity B in
the chromatogram obtained with reference solution (a)
(0.10 per cent) ;
— total : not more than 1.2 times the area of the peak due to
impurity B in the chromatogram obtained with reference
solution (a) (0.6 per cent) ;
— disregard limit: 0.1 times the area of the peak due to
impurity B in the chromatogram obtained with reference
solution (a) (0.05 per cent).
General Notices (1) apply to all monographs and other texts
Levodropropizine
Impurity C. Gas chromatography (2.2.28). Prepare the
solutions immediately before use.
Test solution. Dissolve 0.50 g of the substance to be
examined in methylene chloride R and dilute to 2.5 ml with
the same solvent.
Reference solution (a). Dissolve 0.20 g of levodropropizine
impurity C CRS in methylene chloride R and dilute to
100.0 ml with the same solvent. Dilute 0.5 ml of this solution
to 100.0 ml with methylene chloride R.
Reference solution (b). Dissolve 0.50 g of the substance to be
examined in methylene chloride R, add 0.5 ml of reference
solution (a) and dilute to 2.5 ml with methylene chloride R.
Column :
— material: fused silica ;
— size: l = 30 m, Ø = 0.53 mm ;
— stationary phase : poly[(cyanopropyl)(phenyl)][dimeth-
yl]siloxane R (film thickness 3 μm).
Carrier gas : helium for chromatography R.
Flow rate : 2.5 ml/min.
Split ratio : 1:8.
Temperature :
— column : 140 °C ;
— injection port : 170 °C ;
— detector : 250 °C.
Detection : flame ionisation.
Injection : 1 μl of the test solution and reference solution (b).
Use an appropriate split-liner, e.g. consisting of a column
about 1 cm long packed with glass wool.
At the end of a series of tests, heat the column at 250 °C
for 4-6 h.
Limit :
— impurity C : not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (10 ppm).
Enantiomeric purity. Liquid chromatography (2.2.29).
Solvent mixture : anhydrous ethanol R, hexane R
(40:60 V/V).
Test solution. Dissolve 10.0 mg of the substance to be
examined in 10.0 ml of the solvent mixture. Dilute 1.0 ml of
this solution to 50.0 ml with the solvent mixture.
Reference solution (a). Dissolve 10 mg of
levodropropizine CRS in 10.0 ml of the solvent
mixture. Dilute 1.0 ml of this solution to 50.0 ml with the
solvent mixture.
Reference solution (b). Dissolve 10.0 mg of levodropropizine
impurity A CRS in 10.0 ml of the solvent mixture. Dilute
1.0 ml of this solution to 50.0 ml with the solvent mixture.
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 50.0 ml with the solvent mixture.
Reference solution (d). Dilute 0.5 ml of reference solution (b)
to 25 ml with reference solution (a).
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : silica gel OD for chiral separations R.
Mobile phase : diethylamine R, anhydrous ethanol R,
hexane R (0.2:5:95 V/V/V).
Flow rate : 0.8 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl of the test solution and reference
solutions (a), (c) and (d).
Elution order : impurity A, levodropropizine.
4201
Lynestrenol EUROPEAN PHARMACOPOEIA 6.3

System suitability : 01/2009:0558

— retention times : the retention times of the principal
peaks in the chromatograms obtained with the test LYNESTRENOL
solution and reference solution (a) are similar ;
— resolution : minimum 1.3 between the peaks due to Lynestrenolum
impurity A and levodropropizine in the chromatogram
obtained with reference solution (d).
Limit :
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (2 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 0.500 g by drying in vacuo at 60 °C over diphosphorus
pentoxide R at a pressure of 0.15-0.25 kPa for 4 h. C20H28O Mr 284.4
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined [52-76-6]
on 1.0 g.
DEFINITION
19-Nor-17α-pregn-4-en-20-yn-17-ol.
ASSAY
Content : 98.0 per cent to 102.0 per cent (dried substance).
Dissolve 0.100 g in 50 ml of anhydrous acetic acid R.
Carry out a potentiometric titration (2.2.20), using 0.1 M CHARACTERS
perchloric acid. Read the volume added at the 2nd point of
inflexion. Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone
1 ml of 0.1 M perchloric acid is equivalent to 11.82 mg of and in ethanol (96 per cent).
C13H20N2O2.
IDENTIFICATION
STORAGE
Infrared absorption spectrophotometry (2.2.24).
Protected from light.
Comparison : lynestrenol CRS.

IMPURITIES
TESTS
Specified impurities : A, B, C.
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.2 g in ethanol (96 per cent) R and dilute to 10 ml
with the same solvent.
Specific optical rotation (2.2.7) : − 9.5 to − 11 (dried
substance).
Dissolve 0.900 g in ethanol (96 per cent) R and dilute to
25.0 ml with the same solvent.
Related substances. Gas chromatography (2.2.28).
A. (2R)-3-(4-phenylpiperazin-1-yl)propane-1,2-diol
(dextrodropropizine), Test solution. Dissolve 0.250 g of the substance to be
examined in ethyl acetate R and dilute to 25.0 ml with the
same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with ethyl acetate R. Dilute 1.0 ml of this solution
to 10.0 ml with ethyl acetate R.
Reference solution (b). Dissolve 10 mg of lynestrenol for
peak identification CRS (containing impurities A, B and C)
in 1.0 ml of ethyl acetate R.
Column :
B. 1-phenylpiperazine, — material: fused silica ;
— size: l = 50 m, Ø = 0.32 mm ;
— stationary phase : poly(dimethyl)(diphenyl)siloxane R
(film thickness 1.0 μm).
Carrier gas : helium for chromatography R.
Flow rate : 3.0 ml/min.
C. [(2RS)-oxiran-2-yl]methanol (glycidol). Split ratio : 1:34.

4202 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Lynestrenol

Temperature : potentiometrically (2.2.20), using a glass indicator electrode

Time Temperature and as comparison electrode a silver-silver chloride
(min) (°C) double-junction electrode with a saturated solution of
Column 0 - 30 80 → 230
potassium nitrate R as junction liquid. Carry out a blank
titration.
30 - 32 230 → 310
1 ml of 0.1 M sodium hydroxide is equivalent to 28.44 mg
32 - 42 310 of C20H28O.
Injection port 150
STORAGE
Detector 300 Protected from light.
Detection : flame ionisation. IMPURITIES
Injection : 1.0 μl.
Specified impurities : A, C.
Identification of impurities: use the chromatogram
supplied with lynestrenol for peak identification CRS and Other detectable impurities (the following substances
the chromatogram obtained with reference solution (b) to would, if present at a sufficient level, be detected by one
identify the peaks due to impurities A, B and C. or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Relative retention with reference to lynestrenol (retention impurities and/or by the general monograph Substances for
time = about 38 min) : artefact degradation peak = about 0.97 ; pharmaceutical use (2034). It is therefore not necessary to
impurity A = about 0.99 ; impurity B = about 1.005 ; identify these impurities for demonstration of compliance.
impurity C = about 1.01. See also 5.10. Control of impurities in substances for
System suitability : reference solution (b) : pharmaceutical use) : B.
— peak-to-valley ratio : minimum 2.5, 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
lynestrenol.
Limits :
— impurity A : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
— impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference A. 19-nor-5α,17α-pregn-3-en-20-yn-17-ol,
solution (a) (0.2 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 : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
— disregard limit : 0.5 times the area of the principal peak B. 19-norpregn-4-en-20-yn-17-ol,
in the chromatogram obtained with reference solution (a)
(0.05 per cent). Disregard the artefact peak, which may
be generated in the injection system.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 0.500 g by drying in an oven at 105 °C.
ASSAY
Dissolve 0.150 g in 40 ml of tetrahydrofuran R and add
5.0 ml of a 100 g/l solution of silver nitrate R. Titrate
with 0.1 M sodium hydroxide. Determine the end-point C. 19-nor-17α-pregna-4,20-dien-17-ol.

General Notices (1) apply to all monographs and other texts 4203
 EUROPEAN PHARMACOPOEIA 6.3

4204 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
M
Macrogol 40 sorbitol heptaoleate.. ......................................4207
Magaldrate.................................................................................4207
Magnesium carbonate, light.. ................................................4208
Magnesium oxide, heavy.........................................................4209
Magnesium oxide, light...........................................................4209
Magnesium stearate................................................................. 4210
Maize starch.. ............................................................................ 4212
Mallow leaf................................................................................. 4212
Maltitol.. ..................................................................................... 4213
Maltodextrin.............................................................................. 4214
Mannitol.. ................................................................................... 4215
General Notices (1) apply to all monographs and other texts
Mefenamic acid......................................................................... 4217
Meloxicam.................................................................................. 4218
Methacrylic acid - ethyl acrylate copolymer (1:1) dispersion
30 per cent.. ............................................................................4220
Methotrexate.. ...........................................................................4220
Methylcellulose.........................................................................4223
Methylphenidate hydrochloride............................................4224
Methyltestosterone.. ................................................................4226
Mianserin hydrochloride.. ......................................................4227
Moxidectin for veterinary use.. .............................................4228
4205
 EUROPEAN PHARMACOPOEIA 6.3

4206 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2008:2396 Composition of fatty acids (2.4.22, Method C). Use the
corrected 6.3 mixture of calibrating substances in Table 2.4.22.-3.
MACROGOL 40 SORBITOL
HEPTAOLEATE
Macrogol 40 sorbitoli heptaoleas
DEFINITION
Mixture of esters of fatty acids, mainly Oleic acid (0799),
and sorbitol ethoxylated with approximately 40 moles of
ethylene oxide for each mole of sorbitol. 7 moles of oleic acid Ethylene oxide and dioxan (2.4.25, Method A) : maximum
are used for each mole of sorbitol. It also contains macrogol 1 ppm of ethylene oxide and maximum 10 ppm of dioxan.
fatty acid esters.
CHARACTERS
Appearance : clear or slightly opalescent, yellowish, viscous, in a desiccator and weigh. Evenly distribute 1.0 g of the
hygroscopic liquid.
Solubility : dispersible in water, soluble in isopropyl
myristate, in isopropyl palmitate, in mineral oils and in
vegetable fatty oils.
Relative density : about 1.0.
Viscosity (2.2.9) : about 175 mPa·s at 25 °C.
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : macrogol 40 sorbitol heptaoleate CRS.
B. Hydroxyl value (see Tests).
C. Saponification value (see Tests).
D. Composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1) : maximum 12.0, determined on 3.0 g.
Hydroxyl value (2.5.3, Method A) : 22 to 55.
Peroxide value : maximum 10.0.
Introduce 10.0 g into a 100 ml beaker and dissolve with 20 ml
of glacial acetic acid R. Add 1 ml of saturated potassium
iodide solution R and allow to stand for 1 min. Add 50 ml
of carbon dioxide-free water R and a magnetic stirring bar.
Titrate with 0.01 M sodium thiosulphate, determining the
end-point potentiometrically (2.2.20). Carry out a blank
titration.
Determine the peroxide value using the following expression :
n1 = volume of 0.01 M sodium thiosulphate required
for the titration of the substance to be examined,
in millilitres ;
n2 = volume of 0.01 M sodium thiosulphate required
for the blank titration, in millilitres ;
M = molarity of the sodium thiosulphate solution ;
m phosphate solution R ; a white, crystalline precipitate is
= mass of the substance to be examined, in grams.
Saponification value (2.5.6) : 90 to 110, determined on 4.0 g. B. The precipitate retained in identification test A gives the
Use 30.0 ml of 0.5 M alcoholic potassium hydroxide,
heat under reflux for 60 min and add 50 ml of anhydrous
ethanol R before carrying out the titration.
General Notices (1) apply to all monographs and other texts
Magaldrate
Composition of the fatty-acid fraction of the substance:
— myristic acid : maximum 5.0 per cent ;
— palmitic acid : maximum 16.0 per cent ;
— palmitoleic acid : maximum 8.0 per cent ;
— stearic acid : maximum 6.0 per cent ;
— oleic acid : minimum 58.0 per cent ;
— linoleic acid : maximum 18.0 per cent ;
— linolenic acid : maximum 4.0 per cent.
Water (2.5.12) : maximum 0.5 per cent, determined on 0.50 g.
Sulphated ash : maximum 0.25 per cent.
Heat a silica crucible to redness for 30 min, allow to cool
substance to be examined in the crucible and weigh. Dry
at 100-105 °C for 1 h and ignite in a muffle furnace at
600 ± 25 °C, until the substance is thoroughly charred.
Carry out the test for sulphated ash (2.4.14) on the residue
obtained, starting from “Moisten the substance to be
examined...”.
STORAGE
In an airtight container, protected from light.
01/2008:1539
corrected 6.3
MAGALDRATE
Magaldratum
Al5Mg10(OH)31(SO4)2,xH2O Mr 1097 (anhydrous substance)
DEFINITION
Magaldrate is composed of aluminium and magnesium
hydroxides and sulphates. Its composition corresponds
approximately to the formula Al5Mg10(OH)31(SO4)2,xH2O.
Content : 90.0 per cent to 105.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water and in ethanol
(96 per cent). It is soluble in dilute mineral acids.
IDENTIFICATION
A. Dissolve 0.6 g in 20 ml of 3 M hydrochloric acid R, add
about 30 ml of water R and heat to boiling. Adjust to
pH 6.2 with dilute ammonia R1, continue boiling for a
further 2 min, filter and retain the precipitate and the
filtrate. To 2 ml of the filtrate add 2 ml of ammonium
chloride solution R and neutralise with a solution
prepared by dissolving 2 g of ammonium carbonate R
and 2 ml of dilute ammonia R1 in 20 ml of water R ;
no precipitate is produced. Add disodium hydrogen
produced which does not dissolve in dilute ammonia R1.
reaction of aluminium (2.3.1).
C. The filtrate retained in identification test A gives
reaction (a) of sulphates (2.3.1).
4207
Magnesium carbonate, light
TESTS
Soluble chlorides : maximum 3.5 per cent.
Disperse 1 g in 50 ml of water R, boil for 5 min, cool, dilute
again to 50.0 ml, mix and filter. To 25.0 ml of the filtrate
add 0.2 ml of potassium chromate solution R and titrate
with 0.1 M silver nitrate until a persistent violet-red colour
is obtained. Not more than 5.0 ml of 0.1 M silver nitrate is
required.
Soluble sulphates: maximum 1.9 per cent.
To 2.5 ml of the filtrate obtained in the test for soluble
chlorides, add 30 ml of water R, neutralise to blue litmus
paper R with hydrochloric acid R, add 3 ml of 1 M
hydrochloric acid, 3 ml of a 120 g/l solution of barium
chloride R and dilute to 50 ml with water R. Mix and allow to
stand for 10 min. Any opalescence in the test solution is not
more intense than that of a standard prepared at the same
time in the same manner using 1 ml of 0.01 M sulphuric
acid instead of 2.5 ml of filtrate.
Sulphates : 16.0 per cent to 21.0 per cent (dried substance).
Dissolve 0.875 g in a mixture of 5 ml of glacial acetic
acid R and 10 ml of water R and dilute to 25.0 ml with
water R. Prepare a chromatographic column of 1 cm in
internal diameter containing 15 ml of cation exchange
resin R (150-300 μm), previously washed with 30 ml of
water R. Transfer 5.0 ml of the solution to be examined to
the column and elute with 15 ml of water R. To the eluate
add 5 ml of a 53.6 g/l solution of magnesium acetate R,
32 ml of methanol R and 0.2 ml of alizarin S solution R.
Add from a burette about 4.0 ml of 0.05 M barium chloride,
add a further 0.2 ml of alizarin S solution R and slowly
complete the titration until the yellow colour disappears and
a violet-red tinge is visible.
1 ml of 0.05 M barium chloride is equivalent to 4.803 mg
of SO4.
Aluminium hydroxide : 32.1 per cent to 45.9 per cent (dried
substance).
Dissolve 0.800 g in 10 ml of dilute hydrochloric acid R,
heating on a water-bath. Cool and dilute to 50.0 ml with
water R. To 10.0 ml of this solution, add dilute ammonia R1
until a precipitate begins to appear. Add the smallest
quantity of dilute hydrochloric acid R needed to dissolve
the precipitate and dilute to 20 ml with water R. Carry out
the complexometric titration of aluminium (2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 7.80 mg
of Al(OH)3.
Magnesium hydroxide : 49.2 per cent to 66.6 per cent (dried
substance).
Dissolve 0.100 g in 2 ml of dilute hydrochloric acid R
and carry out the complexometric titration of magnesium
(2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 5.832 mg
of Mg(OH)2.
Sodium : maximum 0.10 per cent.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Weigh 2.00 g into a 100 ml volumetric flask,
place in an ice-bath, add 5 ml of nitric acid R and swirl to
mix. Allow to warm to room temperature and dilute to 100 ml
with water R. Filter, if necessary, to obtain a clear solution.
Dilute 10.0 ml of the filtrate to 100.0 ml with water R.
Reference solutions. Prepare the reference solutions using
sodium standard solution (200 ppm Na) R, diluted as
necessary with dilute nitric acid R.
Source : sodium hollow-cathode lamp.
Wavelength : 589 nm.
4208 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Atomisation device : air-acetylene flame.
Heavy metals (2.4.8) : maximum 30 ppm.
Dissolve 2.0 g in 30 ml of hydrochloric acid R1 and shake
with 50 ml of methyl isobutyl ketone R for 2 min. Allow to
stand, then separate and evaporate the aqueous layer to
dryness. Dissolve the residue in 30 ml of water R. 12 ml
of the solution complies with test A. Prepare the reference
solution using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : 10.0 per cent to 20.0 per cent,
determined on 1.000 g by drying in an oven at 200 °C for 4 h.
ASSAY
To 1.500 g add 50.0 ml of 1 M hydrochloric acid. Titrate
the excess hydrochloric acid with 1 M sodium hydroxide
to pH 3.0, determining the end-point potentiometrically
(2.2.20). Carry out a blank titration.
1 ml of 1 M hydrochloric acid is equivalent to 35.40 mg
of Al5Mg10(OH)31(SO4)2.
01/2009:0042
MAGNESIUM CARBONATE, LIGHT
Magnesii subcarbonas levis
[546-93-0]
DEFINITION
Hydrated basic magnesium carbonate.
Content : 40.0 per cent to 45.0 per cent, calculated as MgO
(Mr 40.30).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water. It dissolves in
dilute acids with effervescence.
IDENTIFICATION
A. Bulk density (2.9.34) : maximum 0.15 g/ml.
B. It gives the reaction of carbonates (2.3.1).
C. Dissolve about 15 mg in 2 ml of dilute nitric acid R and
neutralise with dilute sodium hydroxide solution R. The
solution gives the reaction of magnesium (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in 100 ml of dilute acetic acid R.
When the effervescence has ceased, boil for 2 min, allow to
cool and dilute to 100 ml with dilute acetic acid R. Filter, if
necessary, through a previously ignited and tared porcelain
or silica filter crucible of suitable porosity to give a clear
filtrate.
Appearance of solution. Solution S is not more intensely
coloured than reference solution B4 (2.2.2, Method II).
Soluble substances: maximum 1.0 per cent.
Mix 2.00 g with 100 ml of water R and boil for 5 min. Filter
whilst hot through a sintered-glass filter (40) (2.1.2), allow
to cool and dilute to 100 ml with water R. Evaporate 50 ml
of the filtrate to dryness and dry at 100-105 °C. The residue
weighs a maximum of 10 mg.
Substances insoluble in acetic acid : maximum 0.05 per
cent.
Any residue obtained during the preparation of solution S,
washed, dried and ignited at 600 ± 50 °C, weighs a maximum
of 2.5 mg.
EUROPEAN PHARMACOPOEIA 6.3 Magnesium oxide, light

Chlorides (2.4.4) : maximum 700 ppm. Soluble substances: maximum 2.0 per cent.
Dilute 1.5 ml of solution S to 15 ml with water R. To 2.00 g add 100 ml of water R and boil for 5 min. Filter
Sulphates (2.4.13) : maximum 0.3 per cent. whilst hot through a sintered-glass filter (40) (2.1.2), allow
to cool and dilute to 100 ml with water R. Evaporate 50 ml
Dilute 1 ml of solution S to 15 ml with distilled water R. of the filtrate to dryness and dry at 100-105 °C. The residue
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined weighs a maximum of 20 mg.
on 10 ml of solution S. Substances insoluble in acetic acid : maximum 0.1 per cent.
Calcium (2.4.3) : maximum 0.75 per cent. Any residue obtained during the preparation of solution S,
Dilute 2.6 ml of solution S to 150 ml with distilled water R.washed, dried and ignited at 600 ± 50 °C, weighs a maximum
15 ml of the solution complies with the test. of 5 mg.
Iron (2.4.9) : maximum 400 ppm. Chlorides (2.4.4) : maximum 0.1 per cent.
Dissolve 0.1 g in 3 ml of dilute hydrochloric acid R and Dilute 1 ml of solution S to 15 ml with water R.
dilute to 10 ml with water R. Dilute 2.5 ml of this solution Sulphates (2.4.13) : maximum 1.0 per cent.
to 10 ml with water R. Dilute 0.3 ml of solution S to 15 ml with distilled water R.
Heavy metals (2.4.8) : maximum 20 ppm. Arsenic (2.4.2, Method A) : maximum 4 ppm, determined
To 20 ml of solution S add 15 ml of hydrochloric acid R1 on 5 ml of solution S.
and shake with 25 ml of methyl isobutyl ketone R for
Calcium (2.4.3) : maximum 1.5 per cent.
2 min. Allow to stand, separate the aqueous lower layer
and evaporate to dryness. Dissolve the residue in 1 ml of Dilute 1.3 ml of solution S to 150 ml with distilled water R.
acetic acid R and dilute to 20 ml with water R. 12 ml of the 15 ml of the solution complies with the test.
solution complies with test A. Prepare the reference solutionIron (2.4.9) : maximum 0.07 per cent.
using lead standard solution (1 ppm Pb) R. Dissolve 0.15 g in 5 ml of dilute hydrochloric acid R and
ASSAY dilute to 10 ml with water R. Dilute 1 ml of the solution to
10 ml with water R.
Dissolve 0.150 g in a mixture of 2 ml of dilute hydrochloric
acid R and 20 ml of water R. Carry out the complexometric Heavy metals (2.4.8) : maximum 30 ppm.
titration of magnesium (2.5.11). To 20 ml of solution S add 15 ml of hydrochloric acid R1
1 ml of 0.1 M sodium edetate is equivalent to 4.030 mg and shake with 25 ml of methyl isobutyl ketone R for 2 min.
of MgO. Allow to stand, then separate and evaporate the aqueous
layer to dryness. Dissolve the residue in 1 ml of acetic acid R
and dilute to 30 ml with water R. 12 ml of the solution
complies with test A. Prepare the reference solution using
01/2009:0041 lead standard solution (1 ppm Pb) R.
Loss on ignition : maximum 8.0 per cent, determined on
MAGNESIUM OXIDE, HEAVY 1.00 g at 900 ± 25 °C.
ASSAY
Magnesii oxidum ponderosum
Dissolve 0.320 g in 20 ml of dilute hydrochloric acid R
and dilute to 100.0 ml with water R. Using 20.0 ml of
MgO Mr 40.30
the solution, carry out the complexometric titration of
[1309-48-4]
magnesium (2.5.11).
DEFINITION 1 ml of 0.1 M sodium edetate is equivalent to 4.030 mg
Content : 98.0 per cent to 100.5 per cent of MgO (ignited of MgO.
substance).
CHARACTERS 01/2009:0040
Appearance : fine, white or almost white powder.
MAGNESIUM OXIDE, LIGHT
Solubility : practically insoluble in water. It dissolves in
dilute acids with at most slight effervescence.
Magnesii oxidum leve
IDENTIFICATION
A. Bulk density (2.9.34) : minimum 0.25 g/ml. MgO Mr 40.30
[1309-48-4]
B. Dissolve about 15 mg in 2 ml of dilute nitric acid R and
neutralise with dilute sodium hydroxide solution R. The DEFINITION
solution gives the reaction of magnesium (2.3.1).
Content : 98.0 per cent to 100.5 per cent of MgO (ignited
C. Loss on ignition (see Tests). substance).
TESTS CHARACTERS
Solution S. Dissolve 5.0 g in a mixture of 30 ml of distilled Appearance : fine, white or almost white, amorphous
water R and 70 ml of acetic acid R, boil for 2 min, cool and powder.
dilute to 100 ml with dilute acetic acid R. Filter, if necessary, Solubility : practically insoluble in water. It dissolves in
through a previously ignited and tared porcelain or silica dilute acids with at most slight effervescence.
filter crucible of suitable porosity to give a clear filtrate.
Appearance of solution. Solution S is not more intensely IDENTIFICATION
coloured than reference solution B3 (2.2.2, Method II). A. Bulk density (2.9.34) : maximum 0.15 g/ml.

General Notices (1) apply to all monographs and other texts 4209
Magnesium stearate
B. Dissolve about 15 mg in 2 ml of dilute nitric acid R and
neutralise with dilute sodium hydroxide solution R. The
solution gives the reaction of magnesium (2.3.1).
C. Loss on ignition (see Tests).
TESTS
Solution S. Dissolve 5.0 g in a mixture of 30 ml of distilled
water R and 70 ml of acetic acid R, boil for 2 min, allow to
cool and dilute to 100 ml with dilute acetic acid R. Filter, if
necessary, through a previously ignited and tared porcelain
or silica filter crucible of a suitable porosity to give a clear
filtrate.
Appearance of solution. Solution S is not more intensely
coloured than reference solution B2 (2.2.2, Method II).
Soluble substances : maximum 2.0 per cent.
To 2.00 g add 100 ml of water R and boil for 5 min. Filter
whilst hot through a sintered-glass filter (40) (2.1.2), allow
to cool and dilute to 100 ml with water R. Evaporate 50 ml
of the filtrate to dryness and dry at 100-105 °C. The residue
weighs a maximum of 20 mg.
Substances insoluble in acetic acid : maximum 0.1 per cent.
Any residue obtained during the preparation of solution S,
washed, dried, and ignited at 600 ± 50 °C, weighs a maximum
of 5 mg.
Chlorides (2.4.4) : maximum 0.15 per cent.
Dilute 0.7 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 1.0 per cent.
Dilute 0.3 ml of solution S to 15 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined
on 5 ml of solution S.
Calcium (2.4.3) : maximum 1.5 per cent.
Dilute 1.3 ml of solution S to 150 ml with distilled water R.
15 ml of this solution complies with the test.
Iron (2.4.9) : maximum 0.1 per cent.
Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and
dilute to 10 ml with water R. Dilute 2 ml of this solution to
10 ml with water R.
Heavy metals (2.4.8) : maximum 30 ppm.
To 20 ml of solution S add 15 ml of hydrochloric acid R1
and shake with 25 ml of methyl isobutyl ketone R for 2 min.
Allow to stand, then separate and evaporate the aqueous
layer to dryness. Dissolve the residue in 1.5 ml of acetic
acid R and dilute to 30 ml with water R. 12 ml of the solution
complies with test A. Prepare the reference solution using
lead standard solution (1 ppm Pb) R.
Loss on ignition : maximum 8.0 per cent, determined on
1.00 g at 900 ± 25 °C.
ASSAY
Dissolve 0.320 g in 20 ml of dilute hydrochloric acid R
and dilute to 100.0 ml with water R. Using 20.0 ml of
this solution, carry out the complexometric titration of
magnesium (2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 4.030 mg
of MgO.
4210 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:0229
MAGNESIUM STEARATE
Magnesii stearas
DEFINITION
Magnesium stearate is a mixture of magnesium salts
of different fatty acids consisting mainly of stearic
(octadecanoic) acid [(C17H35COO)2Mg ; Mr 591.3] and palmitic
(hexadecanoic) acid [(C15H31COO)2 Mg ; Mr 535.1] with minor
proportions of other fatty acids. It contains not less than
4.0 per cent and not more than 5.0 per cent of Mg (Ar 24.30),
calculated with reference to the dried substance. The fatty
acid fraction contains not less than 40.0 per cent of stearic
acid and the sum of stearic acid and palmitic acid is not less
than 90.0 per cent.
CHARACTERS
A white or almost white, very fine, light powder, greasy to
the touch, practically insoluble in water and in ethanol.
IDENTIFICATION
First identification : C, D.
Second identification : A, B, D.
A. The residue obtained in the preparation of solution S (see
Tests) has a freezing point (2.2.18) not lower than 53 °C.
B. The acid value of the fatty acids (2.5.1) is 195 to 210,
determined on 0.200 g of the residue obtained in the
preparation of solution S dissolved in 25 ml of the
prescribed mixture of solvents.
C. Examine the chromatograms obtained in the test for fatty
acid composition. The retention times of the principal
peaks in the chromatogram obtained with the test
solution are approximately the same as those of the
principal peaks in the chromatogram obtained with the
reference solution.
D. 1 ml of solution S gives the reaction of magnesium (2.3.1).
TESTS
Solution S. To 5.0 g add 50 ml of peroxide-free ether R,
20 ml of dilute nitric acid R and 20 ml of distilled water R
and heat under a reflux condenser until dissolution is
complete. Allow to cool. In a separating funnel, separate the
aqueous layer and shake the ether layer with 2 quantities,
each of 4 ml, of distilled water R. Combine the aqueous
layers, wash with 15 ml of peroxide-free ether R and dilute
to 50 ml with distilled water R (solution S). Evaporate the
organic layer to dryness and dry the residue at 100-105 °C.
Keep the residue for identification tests A and B.
Acidity or alkalinity. To 1.0 g add 20 ml of carbon
dioxide-free water R and boil for 1 min with continuous
stirring. Cool and filter. To 10 ml of the filtrate add 0.05 ml
of bromothymol blue solution R1. Not more than 0.5 ml of
0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
Chlorides (2.4.4). 0.5 ml of solution S diluted to 15 ml with
water R complies with the limit test for chlorides (0.1 per
cent).
Sulphates (2.4.13). 0.3 ml of solution S diluted to 15 ml with
distilled water R complies with the limit test for sulphates
(0.5 per cent).
EUROPEAN PHARMACOPOEIA 6.3 Magnesium stearate

Cadmium. Not more than 3.0 ppm of Cd, determined by reflux condenser for 10 min. Add 4 ml of heptane R through
atomic absorption spectrometry (2.2.23, Method II). the condenser and boil again under a reflux condenser for
10 min. Allow to cool. Add 20 ml of a saturated sodium
Test solution. Place 50.0 mg of the substance to be examined
chloride solution R. Shake and allow the layers to separate.
in a polytetrafluoroethylene digestion bomb and add
Remove about 2 ml of the organic layer and dry over 0.2 g of
0.5 ml of a mixture of 1 volume of hydrochloric acid R and
anhydrous sodium sulphate R. Dilute 1.0 ml of the solution
5 volumes of cadmium- and lead-free nitric acid R. Allow to
to 10.0 ml with heptane R.
digest at 170 °C for 5 h. Allow to cool. Dissolve the residue
in water R and dilute to 5.0 ml with the same solvent. Reference solution. Prepare the reference solution in the
same manner as the test solution using 50.0 mg of palmitic
Reference solutions. Prepare the reference solutions using
acid CRS and 50.0 mg of stearic acid CRS instead of
cadmium standard solution (10 ppm Cd) R, diluted as
magnesium stearate.
necessary with a 1 per cent V/V solution of hydrochloric
acid R. The chromatographic procedure may be carried out using :
Measure the absorbance at 228.8 nm, using a cadmium — a fused-silica column 30 m long and 0.32 mm in internal
hollow-cathode lamp as a source of radiation and a graphite diameter coated with macrogol 20 000 R (film thickness
furnace as atomic generator. 0.5 μm),
Lead. Not more than 10.0 ppm of Pb, determined by atomic — helium for chromatography R as the carrier gas at a flow
absorption spectrometry (2.2.23, Method II). rate of 2.4 ml/min,
Test solution. Use the solution described in the test for — a flame-ionisation detector,
cadmium.
with the following temperature programme :
Reference solutions. Prepare the reference solutions using
lead standard solution (10 ppm Pb) R, diluted as necessary Time Temperature Rate Comment
with water R. (min) (°C) (°C/min)
Column 0-2 70 – isothermal
Measure the absorbance at 283.3 nm, using a lead
hollow-cathode lamp as a source of radiation and a graphite 2 - 36 70 → 240 5 linear gradient
furnace as atomic generator, depending on the apparatus 36 - 41 240 – isothermal
the line at 217.0 nm may be used.
Injection port 220
Nickel. Not more than 5.0 ppm of Ni, determined by atomic
absorption spectrometry (2.2.23, Method II). Detector 260

Test solution. Use the solution described in the test for Inject 1 μl of the reference solution. When the chromatogram
cadmium. is recorded in the prescribed conditions, the relative retention
Reference solutions. Prepare the reference solutions using of methyl palmitate to that of methyl stearate is about 0.88.
nickel standard solution (10 ppm Ni) R, diluted as necessary The test is not valid unless, in the chromatogram obtained
with water R. with the reference solution, the resolution between the peaks
corresponding to methyl stearate and methyl palmitate is at
Measure the absorbance at 232.0 nm, using a nickel least 5.0.
hollow-cathode lamp as a source of radiation and a graphite
furnace as atomic generator. Inject 1 μl of the test solution. Calculate the percentage
content of stearic acid and palmitic acid from the areas of the
Loss on drying (2.2.32). Not more than 6.0 per cent, peaks in the chromatogram obtained with the test solution
determined on 1.000 g by drying in an oven at 105 °C. by the normalisation procedure, disregarding the peak due
Microbial contamination to the solvent.
TAMC : acceptance criterion 103 CFU/g (2.6.12).
FUNCTIONALITY-RELATED CHARACTERISTICS
TYMC : acceptance criterion 102 CFU/g (2.6.12).
This section provides information on characteristics
Absence of Escherichia coli (2.6.13). that are recognised as being relevant control parameters
Absence of Salmonella (2.6.13). for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
ASSAY necessary to verify the characteristics to demonstrate
Magnesium. To 0.500 g in a 250 ml conical flask add 50 ml compliance. Control of these characteristics can however
of a mixture of equal volumes of butanol R and ethanol R, contribute to the quality of a medicinal product by
5 ml of concentrated ammonia R, 3 ml of ammonium improving the consistency of the manufacturing process
chloride buffer solution pH 10.0 R, 30.0 ml of 0.1 M sodium and the performance of the medicinal product during use.
edetate and 15 mg of mordant black 11 triturate R. Heat to Where control methods are cited, they are recognised as
45-50 °C until the solution is clear and titrate with 0.1 M being suitable for the purpose, but other methods can also
zinc sulphate until the colour changes from blue to violet. be used. Wherever results for a particular characteristic are
Carry out a blank titration. reported, the control method must be indicated.
1 ml of 0.1 M sodium edetate is equivalent to 2.431 mg of Mg. The following characteristic may be relevant for
Fatty acid composition. Examine by gas chromatography magnesium stearate used as a lubricant in solid dosage
(2.2.28). forms (compressed and powder).
Specific surface area (2.9.26, Method I). Determine the
Test solution. In a conical flask fitted with a reflux
specific surface area in the P/Po range of 0.05 to 0.15.
condenser, dissolve 0.10 g of the substance to be examined in
5 ml of boron trifluoride-methanol solution R. Boil under a Sample outgassing : 2 h at 40 °C.

General Notices (1) apply to all monographs and other texts 4211
Maize starch
01/2009:0344
MAIZE STARCH
Maydis amylum
DEFINITION
Maize starch is obtained from the caryopsis of Zea mays L.
CHARACTERS
Appearance : matt, white to slightly yellowish, very fine
powder that creaks when pressed between the fingers.
Solubility : practically insoluble in cold water and in ethanol
(96 per cent).
The presence of granules with cracks or irregularities on
the edge is exceptional.
IDENTIFICATION
A. Examined under a microscope, using not less than
20 × magnification and using equal volumes of glycerol R
and water R, it appears as either angular polyhedral
granules of irregular sizes with diameters ranging from
about 2 μm to about 23 μm or as rounded or spheroidal
granules of irregular sizes with diameters ranging from
about 25 μm to about 35 μm. The central hilum consists
of a distinct cavity or 2- to 5-rayed cleft and there are no
concentric striations. Between orthogonally orientated
polarising plates or prisms, the starch granules show a
distinct black cross intersecting at the hilum.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool.
A thin, cloudy mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B
add 0.05 ml of iodine solution R1. An orange-red to dark
blue colour is produced, which disappears on heating.
TESTS
pH (2.2.3) : 4.0 to 7.0.
Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
Foreign matter. Examined under a microscope using a
mixture of equal volumes of glycerol R and water R, not
more than traces of matter other than starch granules are
present. No starch grains of any other origin are present.
Oxidising substances (2.5.30) : maximum 20 ppm, calculated
as H2O2.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
Iron (2.4.9) : maximum 10 ppm.
Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter.
The filtrate complies with the test.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.000 g by drying in an oven at 130 °C for 90 min.
Sulphated ash (2.4.14) : maximum 0.6 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
4212 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:2391
MALLOW LEAF
Malvae folium
DEFINITION
Whole or fragmented, dried leaf of Malva sylvestris L., Malva
neglecta Wallr. or a mixture of both species.
IDENTIFICATION
A. The leaves of M. sylvestris are up to 12 cm long and up to
15 cm wide with 3, 5 or 7 lobes and sinuate at the base ;
the leaves of M. neglecta are up to 9 cm long and wide,
round or kidney-shaped with 5-7 indistinct lobes. The
leaves of both species have irregular dentate margins and
are green or brownish-green. The abaxial surface of the
lamina bears more hairs and shows a more prominent
venation than the adaxial surface. The major veins on
the upper surface of the leaves and the petioles may be
violet. The petioles are as long as the leaves, up to 2 mm
wide, rounded and somewhat flattened, longitudinally
slightly grooved, green or brownish-green or violet. The
fragmented drug consists of occasionally agglomerated
crumpled pieces of leaves showing prominent veins.
B. Reduce to a powder (710) (2.9.12). The powder is green
or yellowish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the
following diagnostic characters : fragments of the upper
and lower epidermises of the lamina, in surface view,
with straight or more or less sinuous anticlinal walls ;
stomata, mostly anisocytic (2.8.3), on both surfaces ;
fragments of long covering trichomes with thickened
walls and tapering to a point at the apex, usually
unicellular but in M. sylvestris they may be stellate
with 2-8 components, each strongly pitted at the base ;
club-shaped glandular trichomes occur in both species,
composed of 2-4 cells ; fragments of the mesophyll
consisting of palisade parenchyma and spongy mesophyll
cells containing mucilage and cluster crystals of calcium
oxalate ; occasional spherical pollen grains, 130-170 μm in
diameter, with a spiny exine.
C. Thin-layer chromatography (2.2.27).
Test solution. To 2.0 g of the powdered drug (710)
(2.9.12), add 20 ml of an 80 per cent V/V solution of
tetrahydrofuran R ; extract for 10 min using sonication
and filter.
Reference solution. Dissolve 3 mg of rutin R and 3 mg of
hyperoside R in 20 ml of methanol R.
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
plate R (2-10 μm)].
Mobile phase : anhydrous formic acid R, anhydrous
acetic acid R, water R, ethyl formate R, 3-pentanone R
(4:11:14:20:50 V/V/V/V/V).
Application : 10 μl [4 μl] as bands of 10 mm [or 8 mm].
Development : over a path of 10-12 cm [or 6 cm].
Drying : in air.
Detection : heat at 100 °C for 10 min ; spray or dip the
warm plate in a 10 g/l solution of diphenylboric acid
aminoethyl ester R in methanol R. Remove the solvent
with cold air. Spray or dip the plate in a 50 g/l solution of
macrogol 400 R in methanol R. Dry in air and examine
after 15 min in ultraviolet light at 365 nm.
EUROPEAN PHARMACOPOEIA 6.3 Maltitol

Results : see below the sequence of fluorescent zones Solubility : very soluble in water, practically insoluble in
present in the chromatograms obtained with the reference anhydrous ethanol.
solution and the test solution. Furthermore, other faint
fluorescent zones may be present in the chromatogram IDENTIFICATION
obtained with the test solution. First identification : A.
Top of the plate Second identification : B, C, D.
_______ _______ A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Hyperoside : a yellow fluorescent
zone Comparison : maltitol CRS.
A yellow fluorescent zone B. Melting point (2.2.14) : 148 °C to 151 °C.
_______ _______ C. Specific optical rotation (2.2.7) : + 105.5 to + 108.5
(anhydrous substance).
Rutin : a yellow fluorescent zone
Dissolve 5.00 g in water R and dilute to 100.0 ml with
A yellow fluorescent zone the same solvent.
A light blue fluorescent zone D. Thin-layer chromatography (2.2.27).
An orange fluorescent zone Test solution. Dissolve 25 mg of the substance to be
examined in water R and dilute to 10 ml with the same
An orange fluorescent zone
solvent.
Reference solution Test solution Reference solution (a). Dissolve 25 mg of maltitol CRS
in water R and dilute to 10 ml with the same solvent.
TESTS Reference solution (b). Dissolve 25 mg of maltitol CRS
Foreign matter (2.8.2) : maximum 5 per cent of foreign and 25 mg of sorbitol CRS in water R and dilute to 10 ml
organs, maximum 5 per cent of leaves with blisters of spores with the same solvent.
of Puccinia malvacearum and maximum 2 per cent of Plate : TLC silica gel G plate R.
foreign elements. Mobile phase : water R, ethyl acetate R, propanol R
Foreign organs can be flowers, fruits and parts of the stem. (10:20:70 V/V/V).
The blisters of spores on the leaves are mostly 1 mm wide,
Application : 2 μl.
red or brown. Examine under a microscope using chloral
hydrate solution R. The spores of Puccinia malvacearum are Development : over a path of 17 cm.
oblong or oval with brownish walls and a small appendage. Drying : in air.
Loss on drying (2.2.32) : maximum 12.0 per cent, determined Detection : spray with 4-aminobenzoic acid solution R.
on 1.000 g of the powdered drug (710) (2.9.12) by drying Dry in a current of cold air until the acetone is removed.
in an oven at 105 °C for 2 h. Heat at 100-105 °C for 15 min. Allow to cool and spray
Total ash (2.4.16) : maximum 17.0 per cent. with a 2 g/l solution of sodium periodate R. Dry in a
current of cold air. Heat at 100 °C for 15 min.
Ash insoluble in hydrochloric acid (2.8.1) : maximum System suitability : test solution (b) :
3.0 per cent.
— the chromatogram shows 2 clearly separated spots.
Swelling index (2.8.4) : minimum 7, determined on 1.0 g of
the powdered drug (710) (2.9.12). Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
01/2009:1235 with reference solution (a).

MALTITOL TESTS
Appearance of solution. The solution is clear (2.2.1) and
Maltitolum colourless (2.2.2, Method II).
Dissolve 5.0 g in water R and dilute to 50 ml with the same
solvent.
Conductivity (2.2.38) : maximum 20 μS·cm− 1.
Dissolve 20.0 g in carbon dioxide-free water R prepared
from distilled water R and dilute to 100.0 ml with the same
solvent. Measure the conductivity of the solution, while
gently stirring with a magnetic stirrer.
Reducing sugars: maximum 0.2 per cent, expressed as
glucose equivalent.
C12H24O11 Mr 344.3 Dissolve 5.0 g in 6 ml of water R with the aid of gentle heat.
[585-88-6] Cool and add 20 ml of cupri-citric solution R and a few glass
beads. Heat so that boiling begins after 4 min and maintain
DEFINITION boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per
4-O-α-D-Glucopyranosyl-D-glucitol (D-maltitol). cent V/V solution of glacial acetic acid R and 20.0 ml of
Content : 98.0 per cent to 102.0 per cent (anhydrous 0.025 M iodine. With continuous shaking, add 25 ml of a
substance). mixture of 6 volumes of hydrochloric acid R and 94 volumes
of water R and, when the precipitate has dissolved, titrate
CHARACTERS the excess of iodine with 0.05 M sodium thiosulphate using
Appearance : white or almost white, crystalline powder. 1 ml of starch solution R, added towards the end of the

General Notices (1) apply to all monographs and other texts 4213
Maltodextrin
titration as indicator. Not less than 12.8 ml of 0.05 M sodium
thiosulphate is required.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 5.0 g of the substance to be examined
in 20 ml of water R and dilute to 100.0 ml with the same
solvent.
Reference solution (a). Dissolve 0.50 g of maltitol CRS in
2.0 ml of water R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to
100.0 ml with water R.
Reference solution (c). Dilute 10.0 ml of reference
solution (b) to 100.0 ml with water R.
Reference solution (d). Dissolve 0.5 g of maltitol R and 0.5 g
of sorbitol R in 5 ml of water R and dilute to 10.0 ml with
the same solvent.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : strong cation exchange resin (calcium
form) R (9 μm) ;
— temperature : 85 ± 1 °C.
Mobile phase : degassed water R.
Flow rate : 0.5 ml/min.
Detection : refractometer maintained at a constant
temperature.
Injection : 20 μl of the test solution and reference
solutions (b), (c) and (d).
Run time : 3 times the retention time of maltitol.
Relative retention with reference to maltitol (retention
time = about 16 min) : impurity B = about 0.8 ;
impurity A = about 1.8.
System suitability : reference solution (d) :
— resolution : minimum 2 between the peaks due to maltitol
and impurity A.
Limits :
— any impurity : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent) ;
— total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
(2.0 per cent) ;
— disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.1 per cent).
Lead (2.4.10) : maximum 0.5 ppm.
Nickel (2.4.15) : maximum 1 ppm.
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g.
Microbial contamination
If intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion : 102 CFU/g (2.6.12).
If not intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
— absence of Escherichia coli (2.6.13) ;
— absence of Salmonella (2.6.13).
Bacterial endotoxins (2.6.14). If intended for use in
the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial
endotoxins :
4214 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
— less than 4 IU/g for parenteral preparations having a
concentration of less than 100 g/l of maltitol ;
— less than 2.5 IU/g for parenteral preparations having a
concentration of 100 g/l or more of maltitol.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Calculate the percentage content of D-maltitol from the
declared content of maltitol CRS.
LABELLING
The label states :
— where applicable, the maximum concentration of bacterial
endotoxins,
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
A. sorbitol,
B. O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-
D-glucitol (maltotriitol).
01/2009:1542
MALTODEXTRIN
Maltodextrinum
DEFINITION
Mixture of glucose, disaccharides and polysaccharides,
obtained by the partial hydrolysis of starch.
The degree of hydrolysis, expressed as dextrose
equivalent (DE), is not greater than 20 (nominal value).
CHARACTERS
Appearance : white or almost white, slightly hygroscopic
powder or granules.
Solubility : freely soluble in water.
IDENTIFICATION
A. Dissolve 0.1 g in 2.5 ml of water R and heat with 2.5 ml
of cupri-tartaric solution R. A red precipitate is formed.
B. Dip, for 1 s, a suitable stick with a reactive pad containing
glucose-oxidase, peroxidase and a hydrogen-donating
substance, such as tetramethylbenzidine, in a 100 g/l
solution of the substance to be examined. Observe the
colour of the reactive pad ; within 60 s the colour changes
from yellow to green or blue.
C. It is a powder or granules.
D. Dextrose equivalent (see Tests).
TESTS
Solution S. Dissolve 12.5 g in carbon dioxide-free water R
and dilute to 50.0 ml with the same solvent.
EUROPEAN PHARMACOPOEIA 6.3 Mannitol

pH (2.2.3) : 4.0 to 7.0. 01/2009:0559

Mix 1 ml of a 223.6 g/l solution of potassium chloride R MANNITOL

and 30 ml of solution S.
Sulphur dioxide (2.5.29) : maximum 20 ppm. Mannitolum
Heavy metals (2.4.8) : maximum 10 ppm.

Dilute 4 ml of solution S to 30 ml with water R. The solution

complies with test E. Prepare the reference solution using
10 ml of lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined
on 10.00 g by drying in an oven at 105 °C. C6H14O6 Mr 182.2
[69-65-8]
Sulphated ash (2.4.14) : maximum 0.5 per cent, determined
on 1.0 g. DEFINITION
D-Mannitol.
Dextrose equivalent (DE) : within 2 DE units of the nominal
value. Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
Weigh an amount of the substance to be examined equivalent CHARACTERS
to 2.85-3.15 g of reducing carbohydrates, calculated as Appearance : white or almost white, crystalline powder or
dextrose equivalent, into a 500 ml volumetric flask. Dissolve free-flowing granules.
in water R and dilute to 500.0 ml with the same solvent.
Transfer the solution to a 50 ml burette. Solubility : freely soluble in water, very slightly soluble in
ethanol (96 per cent).
Pipette 25.0 ml of cupri-tartaric solution R into a 250 ml It shows polymorphism (5.9).
flask and add 18.5 ml of the test solution from the burette, IDENTIFICATION
mix and add a few glass beads. Place the flask on a hot plate,
First identification : C.
previously adjusted so that the solution begins to boil within
2 min ± 15 s. Allow to boil for exactly 120 s, add 1 ml of a Second identification : A, B, D.
1 g/l solution of methylene blue R and titrate with the test A. Specific optical rotation (2.2.7) : + 23 to + 25 (anhydrous
solution (V1) until the blue colour disappears. Maintain the substance).
solution at boiling throughout the titration. Dissolve 2.00 g of the substance to be examined and 2.6 g
of disodium tetraborate R in about 20 ml of water R at
Standardise the cupri-tartaric solution using a 6.00 g/l 30 °C ; shake continuously for 15-30 min without further
solution of glucose R (V0). heating. Dilute the resulting clear solution to 25.0 ml
with water R.
Calculate the dextrose equivalent using the following B. Melting point (2.2.14) : 165 °C to 170 °C.
expression : C. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison : mannitol CRS.
If the spectra obtained in the solid state show differences,
dissolve separately in 2 glass vials 25 mg of the substance
V0 = total volume of glucose standard solution, in to be examined and 25 mg of the reference substance
millilitres ; in 0.25 ml of distilled water R without heating. The
V1 = total volume of test solution, in millilitres ; solutions obtained are clear. Evaporate to dryness by
heating in a microwave oven with a power range of
M = sample mass, in grams ; 1000-1300 W for 15-30 min or by heating in an oven in
D = percentage content of dry matter in the substance. vacuo at 100 °C. Non-sticky, white or slightly yellowish
powders are obtained. Record new spectra using the
Microbial contamination residues.
D. Thin-layer chromatography (2.2.27).
TAMC : acceptance criterion 103 CFU/g (2.6.12). Test solution. Dissolve 25 mg of the substance to be
examined in water R and dilute to 10 ml with the same
TYMC : acceptance criterion 102 CFU/g (2.6.12). solvent.
Reference solution (a). Dissolve 25 mg of mannitol CRS
Absence of Escherichia coli (2.6.13). in water R and dilute to 10 ml with the same solvent.
Reference solution (b). Dissolve 25 mg of mannitol R
Absence of Salmonella (2.6.13). and 25 mg of sorbitol R in water R and dilute to 10 ml
with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : water R, ethyl acetate R, propanol R
LABELLING (10:20:70 V/V/V).
The label states the dextrose equivalent (DE) (= nominal Application : 2 μl.
value). Development : over 2/3 of the plate.

General Notices (1) apply to all monographs and other texts 4215
Mannitol
Drying : in air.
Detection : spray with 4-aminobenzoic acid solution R.
Dry in a current of cold air until the acetone is removed.
Heat at 100 °C for 15 min. Allow to cool and spray with a
2 g/l solution of sodium periodate R. Dry in a current of
cold air. Heat at 100 °C for 15 min.
System suitability: reference solution (b) :
— the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 5.0 g in water R and dilute to 50 ml with the same
solvent.
Conductivity (2.2.38) : maximum 20 μS·cm− 1.
Dissolve 20.0 g in carbon dioxide-free water R prepared
from distilled water R by heating at 40-50 °C and dilute
to 100.0 ml with the same solvent. After cooling, measure
the conductivity of the solution while gently stirring with a
magnetic stirrer.
Reducing sugars: maximum 0.2 per cent (calculated as
glucose equivalent).
Dissolve 5.0 g in 25 ml of water R with the aid of gentle
heating. Cool and add 20 ml of cupri-citric solution R and
a few glass beads. Heat so that boiling begins after 4 min
and maintain boiling for 3 min. Cool rapidly and add 100 ml
of a 2.4 per cent V/V solution of glacial acetic acid R and
20.0 ml of 0.025 M iodine. With continuous shaking, add
25 ml of a mixture of 6 volumes of hydrochloric acid R
and 94 volumes of water R and, when the precipitate has
dissolved, titrate the excess of iodine with 0.05 M sodium
thiosulphate using 1 ml of starch solution R, added towards
the end of the titration, as indicator. Not less than 12.8 ml of
0.05 M sodium thiosulphate is required.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 5.0 g of the substance to be examined
in 25 ml of water R and dilute to 100.0 ml with the same
solvent.
Reference solution (a). Dissolve 0.50 g of mannitol CRS in
2.5 ml of water R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 2.0 ml of the test solution to
100.0 ml with water R.
Reference solution (c). Dilute 0.5 ml of reference solution (b)
to 20.0 ml with water R.
Reference solution (d). Dissolve 0.5 g of mannitol R and
0.5 g of sorbitol R (impurity A) in 5 ml of water R and dilute
to 10.0 ml with the same solvent.
Reference solution (e). Dissolve 0.1 g of maltitol R
(impurity B) and 0.1 g of isomalt R (impurity C) in 5 ml of
water R and dilute to 100 ml with the same solvent.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : strong cation-exchange resin (calcium
form) R (9 μm) ;
— temperature : 85 ± 1 °C.
Mobile phase : degassed water R.
Flow rate : 0.5 ml/min.
Detection : refractometer maintained at a constant
temperature.
4216 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Injection : 20 μl of the test solution and reference
solutions (b), (c), (d) and (e).
Run time : twice the retention time of mannitol.
Relative retention with reference to mannitol
(retention time = about 22 min) : impurity C (eluted
in 2 peaks) = about 0.7 ; impurity B = about 0.8 ;
impurity A = about 1.2.
System suitability : reference solution (d) :
— resolution : minimum 2 between the peaks due to
mannitol and impurity A.
Limits :
— impurities A, B : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (2.0 per cent) ;
— impurity C : for the sum of the areas of the 2 peaks,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (b)
(2.0 per cent) ;
— unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.10 per cent) ;
— total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (b)
(2.0 per cent) ;
— disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.05 per cent).
Lead (2.4.10) : maximum 0.5 ppm.
Dissolve the substance to be examined in 150.0 ml of the
prescribed mixture of solvents.
Nickel (2.4.15) : maximum 1 ppm.
Dissolve the substance to be examined in 150.0 ml of the
prescribed mixture of solvents.
Water (2.5.12) : maximum 0.5 per cent, determined on
1.00 g. Use as solvent 40 ml of a mixture of equal volumes of
anhydrous methanol R and formamide R at about 50 °C.
Microbial contamination
If intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 102 CFU/g (2.6.12).
If not intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
— absence of Escherichia coli (2.6.13) ;
— absence of Salmonella (2.6.13).
Bacterial endotoxins (2.6.14). If intended for use in
the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial
endotoxins :
— less than 4 IU/g for parenteral preparations having a
concentration of 100 g/l or less of mannitol ;
— less than 2.5 IU/g for parenteral preparations having a
concentration of more than 100 g/l of mannitol.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Calculate the percentage content of D-mannitol from the
areas of the peaks and the declared content of mannitol CRS.
EUROPEAN PHARMACOPOEIA 6.3
LABELLING
The label states :
— where applicable, the maximum concentration of bacterial
endotoxins ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
Specified impurities : A, B, C.
A. sorbitol,
B. maltitol,
C. isomalt.
01/2009:1240 Injection : 10 μl.
MEFENAMIC ACID
Acidum mefenamicum
C15H15NO2 Mr 241.3 — signal-to-noise ratio: minimum 10 for the principal peak
[61-68-7]
DEFINITION
2-[(2,3-Dimethylphenyl)amino]benzoic acid.
Content : 99.0 per cent to 100.5 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, microcrystalline powder.
Solubility : practically insoluble in water, slightly soluble in
ethanol (96 per cent) and in methylene chloride. It dissolves — impurity A : not more than the area of the corresponding
in dilute solutions of alkali hydroxides.
It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : mefenamic acid CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in ethanol (96 per cent) R, evaporate
to dryness and record new spectra using the residues.
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml with the Atomic absorption spectrometry (2.2.23, Method I).
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of mefenamic acid for with the same acid.
system suitability CRS (containing impurities C and D) in
the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solutions. Prepare the reference solutions using
Reference solution (c). Dissolve 10.0 mg of mefenamic
acid impurity A CRS in the mobile phase and dilute to
10.0 ml with the mobile phase. Dilute 1.0 ml of the solution Source : copper hollow-cathode lamp.
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 100.0 ml with the mobile phase.
General Notices (1) apply to all monographs and other texts
Mefenamic acid
Reference solution (d). Dissolve 20.0 mg of benzoic acid R
in the mobile phase and dilute to 1000.0 ml with the mobile
phase. Dilute 1.0 ml of this solution to 100.0 ml with the
mobile phase.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 14 volumes of tetrahydrofuran R,
40 volumes of a 5.75 g/l solution of ammonium dihydrogen
phosphate R adjusted to pH 5.0 with dilute ammonia R2,
and 46 volumes of acetonitrile R1.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 254 nm.
Run time : 4 times the retention time of mefenamic acid.
Identification of impurities : use the chromatogram supplied
with mefenamic acid for system suitability CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities C and D.
Relative retention with reference to mefenamic acid
(retention time = about 8 min) : impurity C = about 0.3 ;
impurity D = about 0.35 ; impurity A = about 0.5.
System suitability :
— resolution : minimum 3.0 between the peaks due to
impurities C and D in the chromatogram obtained with
reference solution (b) ;
in the chromatogram obtained with reference solution (d).
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity C = 5.9 ;
impurity D = 4.0 ;
— impurities C, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
peak in the chromatogram obtained with reference
solution (c) (100 ppm) ;
— 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 : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard the peak due to impurity A.
Copper : maximum 10.0 ppm.
Test solution. Place 1.00 g in a silica crucible, moisten with
sulphuric acid R, heat cautiously on a flame for 30 min and
then progressively to 650 °C. Continue ignition until all
black particles have disappeared. Allow to cool, dissolve the
residue in 0.1 M hydrochloric acid and dilute to 25.0 ml
copper standard solution (0.1 per cent Cu) R, diluting with
0.1 M nitric acid.
Wavelength : 324.8 nm.
Atomisation device : air-acetylene flame.
4217
Meloxicam EUROPEAN PHARMACOPOEIA 6.3

Loss on drying (2.2.32) : maximum 0.5 per cent, determined 01/2009:2373

on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
MELOXICAM
ASSAY Meloxicamum
Dissolve with the aid of ultrasound 0.200 g in 100 ml of
warm anhydrous ethanol R, previously neutralised to
phenol red solution R. Add 0.1 ml of phenol red solution R
and titrate with 0.1 M sodium hydroxide.
1 ml of 0.1 M sodium hydroxide is equivalent to 24.13 mg
of C15H15NO2.

IMPURITIES
Specified impurities : A, C, D.
Other detectable impurities (the following substances C14H13N3O4S2 Mr 351.4
would, if present at a sufficient level, be detected by one [71125-38-7]
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified DEFINITION
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to 4-Hydroxy-2-methyl-N-(5-methylthiazol-2-yl)-2H-1,2-
identify these impurities for demonstration of compliance. benzothiazine-3-carboxamide 1,1-dioxide.
See also 5.10. Control of impurities in substances for Content : 99.0 per cent to 101.0 per cent (dried substance).
pharmaceutical use) : B, E.
CHARACTERS
Appearance : pale yellow powder.
Solubility : practically insoluble in water, soluble in
dimethylformamide, very slightly soluble in ethanol (96 per
cent).
A. 2,3-dimethylaniline, It shows polymorphism (5.9).

IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : meloxicam CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.

TESTS
Related substances. Liquid chromatography (2.2.29).
B. N-(2,3-dimethylphenyl)-2-[(2,3-dimethylphenyl)amino]- Test solution. Dissolve 40 mg of the substance to be
benzamide, examined in a mixture of 5 ml of methanol R and 0.3 ml
of 1 M sodium hydroxide and dilute to 20.0 ml with
methanol R.
Reference solution (a). Dilute 2.0 ml of the test solution to
100.0 ml with methanol R. Dilute 5.0 ml of this solution to
100.0 ml with methanol R.
C. 2-chlorobenzoic acid,
Reference solution (b). Dissolve 2 mg of the substance
D. benzoic acid, to be examined, 2 mg of meloxicam impurity A CRS,
2 mg of meloxicam impurity B CRS, 2 mg of meloxicam
impurity C CRS and 2 mg of meloxicam impurity D CRS in
a mixture of 5 ml of methanol R and 0.3 ml of 1 M sodium
hydroxide and dilute to 25 ml with methanol R.
Column :
— size: l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 μm) ;
E. 2,3-dimethyl-N-phenylaniline. — temperature : 45 °C.

4218 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Meloxicam

Mobile phase : 1 ml of 0.1 M perchloric acid is equivalent to 35.14 mg

— mobile phase A : 1 g/l solution of potassium dihydrogen of C14H13N3O4S2.
phosphate R adjusted to pH 6.0 with 1 M sodium
hydroxide ; STORAGE
— mobile phase B : methanol R ; Protected from light.
Time Mobile phase A Mobile phase B IMPURITIES
(min) (per cent V/V) (per cent V/V)
0-2 60 40 Specified impurities : A, B, C, D.
2 - 10 60 → 30 40 → 70
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
10 - 15 30 70 or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Flow rate : 1.0 ml/min. impurities and/or by the general monograph Substances for
Detection : spectrophotometer at 260 nm and 350 nm. pharmaceutical use (2034). It is therefore not necessary to
Injection : 10 μl. identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
Relative retention with reference to meloxicam pharmaceutical use) : E, F.
(retention time = about 7 min) : impurity B = about 0.5 ;
impurity A = about 1.4 ; impurity C = about 1.7 ;
impurity D = about 1.9.
System suitability : reference solution (b) :
— resolution : minimum 3.0 between the peaks due to
meloxicam and impurity A at 350 nm ; minimum 3.0
between the peaks due to impurity B and meloxicam at
260 nm.
Limits : A. ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-
— correction factor : for the calculation of content, multiply carboxylate 1,1-dioxide,
the peak area of impurity A by 2.0 ;
— impurity A at 350 nm : not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) at 350 nm (0.1 per cent) ;
— impurity B at 260 nm : not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) at 350 nm (0.1 per cent) ; B. 5-methylthiazol-2-amine,
— impurities C, D at 350 nm : for each impurity, not more
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) at
350 nm (0.05 per cent) ;
— unspecified impurities : for each impurity, at the
wavelength giving the higher value for the impurity,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) at the
same wavelength (0.10 per cent) ;
— total : not more than 0.3 per cent ;
C. R = CH3 : N-[(2Z)-3,5-dimethylthiazol-2(3H)-ylidene]-4-
— disregard limit : 0.3 times the area of the principal peak hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxamide
in the chromatogram obtained with reference solution (a) 1,1-dioxide,
at the same wavelength (0.03 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
D. R = C2H5 : N-[(2Z)-3-ethyl-5-methylthiazol-2(3H)-ylidene]-
1.0 g complies with test F. Prepare the reference solution 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxamide
using 2 ml of lead standard solution (10 ppm Pb) R. 1,1-dioxide,
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
In order to avoid overheating during the titration, mix
thoroughly throughout and stop the titration immediately
after the end-point has been reached. E. R = CH3 : methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-
3-carboxylate 1,1-dioxide,
Dissolve 0.250 g in a mixture of 5 ml of anhydrous formic
acid R and 50 ml of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point F. R = CH(CH3)2 : isopropyl 4-hydroxy-2-methyl-2H-1,2-
potentiometrically (2.2.20). benzothiazine-3-carboxylate 1,1-dioxide.

General Notices (1) apply to all monographs and other texts 4219
Methacrylic acid - ethyl acrylate copolymer (1:1) dispersion EUROPEAN PHARMACOPOEIA 6.3

01/2009:1129 Flow rate : 2.5 ml/min.

METHACRYLIC ACID - ETHYL
ACRYLATE COPOLYMER (1:1)
DISPERSION 30 PER CENT
Acidi methacrylici et ethylis acrylatis
polymerisati 1:1 dispersio 30 per centum
DEFINITION
Dispersion in water of a copolymer of methacrylic acid
and ethyl acrylate having a mean relative molecular mass
of about 250 000. The ratio of carboxylic groups to ester
groups is about 1:1.
Content : 46.0 per cent to 50.6 per cent of methacrylic
acid units (residue on evaporation).
It may contain suitable surface-active agents such as sodium Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
dodecyl sulphate and polysorbate 80.
CHARACTERS
Appearance : opaque, white or almost white, slightly viscous TAMC : acceptance criterion 10 CFU/g (2.6.12).
liquid.
Solubility : miscible with water. On addition of solvents such
as acetone, anhydrous ethanol or 2-propanol, a precipitate is ASSAY
formed which dissolves on addition of excess solvent. It is
miscible with a 40 g/l solution of sodium hydroxide.
It is sensitive to spoilage by microbial contaminants.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of
methacrylic acid - ethyl acrylate copolymer (1:1)
dispersion 30 per cent.
B. It complies with the limits of the assay.
TESTS
Apparent viscosity (2.2.10) : maximum 15 mPa·s, determined concentration of any surface-active agents.
using a rotating viscometer at 20 °C and at a shear rate of
50 s− l.
Appearance of a film. Place 1 ml on a glass plate and allow
to dry. A clear, brittle film is formed.
Particulate matter. Filter 100.0 g through a tared stainless
steel sieve (90). Rinse with water R until a clear filtrate is
obtained and dry at 100-105 °C. The residue weighs not
more than 1.00 g.
Ethyl acrylate and methacrylic acid. Liquid chromatography
(2.2.29).
Blank solution. To 50.0 ml of methanol R add 25.0 ml of
the mobile phase.
Test solution. Dissolve 40 mg of the dispersion to be
examined in 50.0 ml of methanol R and add 25.0 ml of the
mobile phase.
Reference solution. Dissolve 10 mg of ethyl acrylate R and C20H22N8O5
10 mg of methacrylic acid R in methanol R, then dilute to
50.0 ml with the same solvent. Dilute 0.1 ml of this solution DEFINITION
to 50.0 ml with methanol R and add 25.0 ml of the mobile
phase.
Column:
— size : l = 0.10 m, Ø = 4 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : methanol R, phosphate buffer solution
pH 2.0 R (30:70 V/V).
Detection : spectrophotometer at 202 nm.
Injection : 50 μl.
System suitability :
— resolution : minimum 2.0 between the peaks due to
ethyl acrylate and methacrylic acid in the chromatogram
obtained with the reference solution ;
— the chromatogram obtained with the blank solution does
not show peaks with the same retention times as ethyl
acrylate or methacrylic acid.
Limit :
— sum of the contents of ethyl acrylate and methacrylic
acid : maximum 0.1 per cent.
Residue on evaporation : 28.5 per cent to 31.5 per cent.
Dry 1.000 g at 110 °C for 5 h. The residue weighs not less
than 0.285 g and not more than 0.315 g.
on 1.0 g.
Microbial contamination
3
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Dissolve 1.500 g in a mixture of 40 ml of water R and 60 ml
of 2-propanol R. Titrate slowly while stirring with 0.5 M
sodium hydroxide, using phenolphthalein solution R as
indicator.
1 ml of 0.5 M sodium hydroxide is equivalent to 43.05 mg
of C4H6O2 (methacrylic acid units).
STORAGE
Protected from freezing. Handle the substance so as to
minimise microbial contamination.
LABELLING
The label states, where applicable, the name and
01/2009:0560
METHOTREXATE
Methotrexatum
Mr 454.4
[59-05-2]
(2S)-2-[[4-[[(2,4-Diaminopteridin-6-yl)methyl]methyl-
amino]benzoyl]amino]pentanedioic acid.
Content : 97.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : yellow or orange, crystalline, hygroscopic
powder.

4220 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Solubility : practically insoluble in water, in ethanol (96 per
cent) and in methylene chloride. It dissolves in dilute
mineral acids and in dilute solutions of alkali hydroxides
and carbonates.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : methotrexate CRS.
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 40.0 mg of the substance to be
examined in a mixture of 0.5 ml of dilute ammonia R1 and
5 ml of mobile phase A and dilute to 100.0 ml with mobile
phase A.
Test solution (b). Dissolve 25.0 mg of the substance to be
examined in a mixture of 0.5 ml of dilute ammonia R1 and
5 ml of mobile phase A and dilute to 50.0 ml with mobile
phase A. Dilute 5.0 ml of this solution to 50.0 ml with mobile
phase A.
Reference solution (a). Dissolve 25.0 mg of
methotrexate CRS in a mixture of 0.5 ml of dilute
ammonia R1 and 5 ml of mobile phase A and dilute to
50.0 ml with mobile phase A. Dilute 5.0 ml of this solution to
50.0 ml with mobile phase A.
Reference solution (b). Dilute 5.0 ml of test solution (a) to
100.0 ml with mobile phase A. Dilute 5.0 ml of this solution
to 50.0 ml with mobile phase A.
Reference solution (c). Dilute 5.0 ml of reference solution (b)
to 25.0 ml with mobile phase A.
Reference solution (d). Dissolve 5 mg of the substance to be
examined, 5 mg of 4-aminofolic acid R (impurity B), 5 mg
of methotrexate impurity C CRS, 5 mg of methotrexate
impurity D CRS and 5 mg of methotrexate impurity E CRS
in a mixture of 0.5 ml of dilute ammonia R1 and 5 ml of
mobile phase A and dilute to 100 ml with mobile phase A.
Reference solution (e). Dissolve 8 mg of methotrexate for
peak identification CRS (containing impurities H and I) in a
mixture of 0.1 ml of dilute ammonia R1 and 1 ml of mobile
phase A and dilute to 20 ml with mobile phase A.
Column:
— size : l = 0.25 m, Ø = 4.0 mm ;
— stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 μm).
Mobile phase :
— mobile phase A : mix 5 volumes of acetonitrile for
chromatography R and 95 volumes of a 3.4 g/l solution of
anhydrous sodium dihydrogen phosphate R previously
adjusted to pH 6.0 with a 42 g/l solution of sodium
hydroxide R ;
— mobile phase B : mix 50 volumes of acetonitrile for
chromatography R and 50 volumes of a 3.4 g/l solution of
anhydrous sodium dihydrogen phosphate R previously
adjusted to pH 6.0 with a 42 g/l solution of sodium
hydroxide R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 10 100 0
10 - 20 100 → 95 0→5
20 - 28 95 → 50 5 → 50
28 - 37 50 50
37 - 38 50 → 100 50 → 0
38 - 45 100 0
General Notices (1) apply to all monographs and other texts
Methotrexate
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 280 nm.
Injection : 20 μl of test solution (a) and reference
solutions (b), (c), (d) and (e).
Identification of impurities : use the chromatogram
supplied with methotrexate for peak identification CRS and
the chromatogram obtained with reference solution (e) to
identify the peaks due to impurities H and I.
Relative retention with reference to methotrexate
(retention time = about 18 min) : impurity B = about 0.3 ;
impurity C = about 0.4 ; impurity E = about 1.4 ;
impurity I = about 1.5 ; impurity H = about 1.6.
System suitability :
— resolution : minimum 2.0 between the peaks due to
impurities B and C and minimum 1.5 between the peaks
due to impurity D and methotrexate, in the chromatogram
obtained with reference solution (d) ; minimum 1.5
between the peaks due to impurities I and H in the
chromatogram obtained with reference solution (e) ; if the
resolution between impurity D and methotrexate does not
comply, increase the flow rate to meet the requirement.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity E = 0.8 ;
impurity I = 1.4 ;
— impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
— impurities B, E : for each impurity, not more than 0.6 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ;
— impurities H, I : for each impurity, not more than twice
the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ;
— unspecified impurities : for each impurity, not more
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.05 per cent) ;
— sum of impurities other than B, C and E : not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
— disregard limit : 0.3 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.03 per cent).
Enantiomeric purity. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with the mobile phase.
Reference solution (b). Dissolve 4.0 mg of
(RS)-methotrexate R in the mobile phase and dilute
to 100.0 ml with the mobile phase.
Column :
— size: l = 0.15 m, Ø = 4.0 mm ;
— stationary phase : bovine albumin R bound to silica gel
for chromatography R (7 μm) with a pore size of 30 nm.
Mobile phase : add 500 ml of a 7.1 g/l solution of anhydrous
disodium hydrogen phosphate R to 600 ml of a 6.9 g/l
solution of sodium dihydrogen phosphate monohydrate R,
mix, and adjust to pH 6.9 with dilute sodium hydroxide
solution R ; to 920 ml of this mixture add 80 ml of
propanol R.
4221
Methotrexate EUROPEAN PHARMACOPOEIA 6.3

Flow rate : 1.5 ml/min. I. R1 = NH2, R2 = R3 = CH3, R4 = H : (4S)-4-[[4-[[(2,4-di-

Detection : spectrophotometer at 302 nm. aminopteridin-6-yl)methyl]methylamino]benzoyl]ami-
no]-5-methoxy-5-oxopentanoic acid (methotrexate
Injection : 20 μl. 1-methyl ester),
System suitability : reference solution (b) :
— resolution : minimum 2.0 between the peaks due to J. R1 = NH2, R2 = R3 = R4 = CH3 : dimethyl (2S)-2-[[4-[[(2,4-di-
methotrexate and impurity F. aminopteridin-6-yl)methyl]methylamino]benzoyl]ami-
Limit : no]pentanedioate (methotrexate dimethyl ester),
— impurity F : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (3.0 per cent).
Heavy metals (2.4.8) : maximum 50 ppm.
1.0 g complies with test C. Prepare the reference solution
using 5 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 13.0 per cent, determined on
0.10 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined C. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
on 1.0 g. yl)methyl]methylamino]benzoyl]amino]pentanedioic acid
(N-methylfolic acid, methopterin),
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 (a).
Calculate the percentage content of C20H22N8O5 from the
declared content of methotrexate CRS.
STORAGE
In an airtight container, protected from light. D. 4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]methyl-
amino]benzoic acid (N10-methylpteroic acid),
IMPURITIES
Specified impurities : B, C, 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 E. 4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoic
impurities for demonstration of compliance. See also 5.10. acid (4-amino-N10-methylpteroic acid, APA),
Control of impurities in substances for pharmaceutical
use) : A, D, G, J, K, L.

A. (2,4-diaminopteridin-6-yl)methanol,
F. (2R)-2-[[4-[[(2,4-diaminopteridin-6-yl)methyl]methyl-
amino]benzoyl]amino]pentanedioic acid
((R)-methotrexate),

B. R1 = NH2, R2 = R3 = R4 = H : (2S)-2-[[4-[[(2,4-diamino-
pteridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic
acid (4-aminofolic acid, aminopterin),

H. R1 = NH2, R2 = R4 = CH3, R3 = H : (2S)-2-[[4-[[(2,4-di-

aminopteridin-6-yl)methyl]methylamino]benzoyl]ami- G. (2S)-2-[[4-[[4-[[(2,4-diaminopteridin-6-
no]-5-methoxy-5-oxopentanoic acid (methotrexate yl)methyl]methylamino]benzoyl]methyl-
5-methyl ester), amino]benzoyl]amino]pentanedioic acid,

4222 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Methylcellulose

with carbon dioxide-free water R and stir until dissolution

is complete. Allow to stand at 2-8 °C for 1 h before carrying
out the test for appearance of solution.
Appearance of solution. Solution S is not more opalescent
than reference suspension III (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
K. R = H : (2S)-2-[(4-aminobenzoyl)amino]pentanedioic acid, pH (2.2.3) : 5.0 to 8.0 for the solution prepared as described
L. R = CH3 : (2S)-2-[[4-(methylamino)benzoyl]amino]pen- under Apparent viscosity.
tanedioic acid. Read the pH after the probe has been immersed for
5 ± 0.5 min.
01/2008:0345 Heavy metals (2.4.8) : maximum 20 ppm.
corrected 6.3 1.0 g complies with test F. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
METHYLCELLULOSE Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 1 h.
Methylcellulosum
Sulphated ash (2.4.14) : maximum 1.5 per cent, determined
[9004-67-5] on 1.0 g.
DEFINITION FUNCTIONALITY-RELATED CHARACTERISTICS
Partly O-methylated cellulose. This section provides information on characteristics
that are recognised as being relevant control parameters
CHARACTERS for one or more functions of the substance when used
Appearance : white, yellowish-white or greyish-white powder as an excipient (see chapter 5.15). This section is a
or granules, hygroscopic after drying. non-mandatory part of the monograph and it is not
Solubility : practically insoluble in hot water, in acetone, in necessary to verify the characteristics to demonstrate
anhydrous ethanol and in toluene. It dissolves in cold water compliance. Control of these characteristics can however
giving a colloidal solution. contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
IDENTIFICATION and the performance of the medicinal product during use.
A. Evenly distribute 1.0 g onto the surface of 100 ml of Where control methods are cited, they are recognised as
water R in a beaker, tapping the top of the beaker gently if being suitable for the purpose, but other methods can also
necessary to ensure a uniform layer on the surface. Allow be used. Wherever results for a particular characteristic are
to stand for 1-2 min : the powdered material aggregates reported, the control method must be indicated.
on the surface. The following characteristics may be relevant for
B. Evenly distribute 1.0 g into 100 ml of boiling water R, methylcellulose used as binder, viscosity-enhancing agent
and stir the mixture using a magnetic stirrer with a bar or film former.
25 mm long : a slurry is formed and the particles do not Apparent viscosity : minimum 80 per cent and maximum
dissolve. Allow the slurry to cool to 5 °C and stir using a 120 per cent of the nominal value for samples with a viscosity
magnetic stirrer : a clear or slightly turbid solution occurs of less than 600 mPa·s (Method 1) ; minimum 75 per cent
with its thickness dependent on the viscosity grade. and maximum 140 per cent of the nominal value for samples
C. To 0.1 ml of the solution obtained in identification B with a viscosity of 600 mPa·s or higher (Method 2).
add 9 ml of a 90 per cent V/V solution of sulphuric Method 1, to be applied to samples with a viscosity of
acid R, shake, heat on a water-bath for exactly 3 min, less than 600 mPa·s. Weigh accurately a quantity of the
immediately cool in an ice-bath, carefully add 0.6 ml of a substance to be examined equivalent to 4.000 g of the dried
20 g/l solution of ninhydrin R, shake and allow to stand substance. Transfer into a wide-mouthed bottle, and adjust
at 25 °C : a red colour develops and does not change to the mass to 200.0 g with water R. Capping the bottle, stir
purple within 100 min. by mechanical means at 400 ± 50 r/min for 10-20 min until
D. Place 2-3 ml of the solution obtained in identification B the particles are thoroughly dispersed and wetted. Scrape
on a glass slide as a thin film and allow the water to down the insides of the bottle with a spatula if necessary,
evaporate : a coherent, clear film forms on the glass slide. to ensure that there is no undissolved material on the
E. Add exactly 50 ml of the solution obtained in sides of the bottle, and continue the stirring in a cooling
identification B to exactly 50 ml of water R in a beaker. water-bath maintained at a temperature below 5 °C for
Insert a thermometer into the solution. Stir the solution another 20-40 min. Adjust the solution mass if necessary
on a magnetic stirrer/hot plate and begin heating, to 200.0 g using cold water R. Centrifuge the solution
increasing the temperature at a rate of 2-5 °C per minute. if necessary to expel any entrapped air bubbles. Using a
Determine the temperature at which a turbidity increase spatula remove any foam, if present. Determine the viscosity
begins to occur and designate the temperature as the of this solution using the capillary viscometer method (2.2.9)
flocculation temperature : the flocculation temperature to obtain the kinematic viscosity (ν). Separately, determine
is higher than 50 °C. the density (ρ) (2.2.5) of the solution and calculate the
dynamic viscosity (η), as η = ρν.
TESTS Method 2, to be applied to samples with a viscosity of
Solution S. While stirring, introduce a quantity of the 600 mPa·s or higher. Weigh accurately a quantity of the
substance to be examined equivalent to 1.0 g of the dried substance to be examined equivalent to 10.00 g of the dried
substance into 50 g of carbon dioxide-free water R heated to substance. Transfer into a wide-mouthed bottle, and adjust
90 °C. Allow to cool, adjust the mass of the solution to 100 g the mass to 500.0 g with water R. Capping the bottle, stir

General Notices (1) apply to all monographs and other texts 4223
Methylphenidate hydrochloride
by mechanical means at 400 ± 50 r/min for 10-20 min until
the particles are thoroughly dispersed and wetted. Scrape
down the insides of the bottle with a spatula if necessary, to
ensure that there is no undissolved material on the sides of
the bottle, and continue the stirring in a cooling water-bath
maintained at a temperature below 5 °C for another
20-40 min. Adjust the solution mass if necessary to 500.0 g
using cold water R. Centrifuge the solution if necessary to
expel any entrapped air bubbles. Using a spatula, remove
any foam, if present. Determine the viscosity (2.2.10) of this
solution at 20 ± 0.1 °C using a rotating viscometer.
Apparatus : single-cylinder type spindle viscometer.
Rotor number, revolution and calculation multiplier: apply
the conditions specified in Table 0345.-1.
Table 0345.-1.
Nominal Rotor Revolution Calculation
viscosity* number (r/min) multiplier
(mPa·s)
600 to less 3 60
than 1400
1400 to less 3 12
than 3500
3500 to less 4 60
than 9500
9500 to less 4 6
than 99 500
99 500 or more 4 3
*the nominal viscosity is based on the manufacturer’s specifications.
Allow the spindle to rotate for 2 min before taking the
measurement. Allow a rest period of 2 min between
subsequent measurements. Repeat the measurement twice
and determine the mean of the 3 readings.
Degree of substitution : 26.0 per cent to 33.0 per cent of
methoxy groups (dried substance).
Gas chromatography (2.2.28).
Apparatus :
— reaction vial: a 5 ml pressure-tight vial, 50 mm in
height, 20 mm in external diameter and 13 mm in
internal diameter at the mouth, equipped with a
pressure-tight butyl rubber membrane stopper coated
with polytetrafluoroethylene and secured with an
aluminium crimped cap or another sealing system
providing a sufficient air-tightness ;
— heater : a heating module with a square aluminium block
having holes 20 mm in diameter and 32 mm in depth,
so that the reaction vials fit ; mixing of the contents of
the vial is effected using a magnetic stirrer equipped in
the heating module or using a reciprocal shaker that
performs approximately 100 cycles/min.
Internal standard solution: 30 g/l solution of octane R in
xylene R.
Test solution. Weigh 65.0 mg of the substance to be
examined, place in a reaction vial, add 0.06-0.10 g of adipic
acid R, 2.0 ml of the internal standard solution and 2.0 ml
of hydriodic acid R, immediately cap and seal the vial, and
weigh accurately. Using a magnetic stirrer, mix the contents
of the vial continuously for 60 min while heating the block
so that the temperature of the contents is maintained at
130 ± 2 °C. If a reciprocal shaker or magnetic stirrer cannot
be used, shake the vial well by hand at 5-minute intervals
during the initial 30 min of the heating time. Allow the vial
4224
EUROPEAN PHARMACOPOEIA 6.3
to cool, and again weigh accurately. If the loss of mass is less
than 0.50 per cent of the contents and there is no evidence of
a leak, use the upper layer of the mixture as the test solution.
Reference solution. Place 0.06-0.10 g of adipic acid R,
2.0 ml of the internal standard solution and 2.0 ml of
hydriodic acid R in another reaction vial, cap and seal the
vial, and weigh accurately. Add 45 μl of methyl iodide R
through the septum with a syringe, and weigh accurately.
Shake the reaction vial well, and use the upper layer as the
reference solution.
Column :
— size: l = 1.8-3 m, Ø = 3-4 mm ;
— stationary phase : diatomaceous earth for gas
chromatography R impregnated with 10-20 per cent
of poly(dimethyl)(75)(diphenyl)(25)siloxane R (film
thickness 125-150 μm) ;
— temperature : 100 °C.
Carrier gas: helium for chromatography R (thermal
conductivity) ; helium for chromatography R or nitrogen for
20 chromatography R (flame ionisation).
Flow rate : adjusted so that the retention time of the internal
standard is about 10 min.
100
Detection : flame ionisation or thermal conductivity.
Injection : 1-2 μl.
100
System suitability : reference solution :
— resolution : well-resolved peaks of methyl iodide (1st peak)
1000 and internal standard (2nd peak).
Calculation :
2000 — methoxy groups : calculate the ratio (Q) of the area of the
peak due to methyl iodide to the area of the peak due
to the internal standard in the chromatogram obtained
with the test solution, and the ratio (Q1) of the area of the
peak due to methyl iodide to the area of the peak due
to the internal standard in the chromatogram obtained
with the reference solution.
Calculate the percentage content of methoxy groups
using the following expression :
m1 = mass of methyl iodide in the reference solution,
in milligrams ;
m
= mass of the sample (dried substance), in
milligrams.
01/2009:2235
METHYLPHENIDATE
HYDROCHLORIDE
Methylphenidati hydrochloridum
C14H20ClNO2 Mr 269.8
[298-59-9]
DEFINITION
Methyl (2RS)-phenyl[(2RS)-piperidin-2-yl]acetate
hydrochloride.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, fine crystalline powder.
Solubility : freely soluble in water, soluble in ethanol (96 per
cent), slightly soluble in methylene chloride.
IDENTIFICATION
First identification : A, C.
Second identification : B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : methylphenidate hydrochloride CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 5 mg of the substance to be
examined in 1.0 ml of methanol R.
Reference solution. Dissolve 5 mg of methylphenidate
hydrochloride CRS in 1.0 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, methanol R,
methylene chloride R (1:4:95 V/V/V).
Application : 5 μl.
Development : over 2/3 of the plate.
Drying : at 60 °C for 5 min.
Detection : spray with a freshly prepared 5 g/l solution of
fast blue B salt R. Heat to 60 °C for 1 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot obtained with the reference solution.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Dissolve 25 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the inflexion.
mobile phase.
Reference solution (a). Dissolve the contents of a vial
of methylphenidate impurity mixture CRS (containing
impurities A and B) in 1.0 ml of the test solution.
Reference solution (b). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase : mix 7 volumes of methanol R2 with
18 volumes of a 1.82 g/l solution of potassium dihydrogen
phosphate R.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 209 nm.
Injection : 10 μl.
Run time : 1.5 times the retention time of methylphenidate.
Identification of impurities: use the chromatogram
supplied with methylphenidate impurity mixture CRS and
the chromatogram obtained with reference solution (a) to
identify the peaks due to impurities A and B.
General Notices (1) apply to all monographs and other texts
Methylphenidate hydrochloride
Relative retention with reference to methylphenidate
(retention time = about 11 min) : impurity B = about 0.6 ;
impurity A = about 0.9.
System suitability : reference solution (a) :
— peak-to-valley ratio : minimum 2.5, where Hp = height
above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
methylphenidate.
Limits :
— impurities A, B : for each impurity, not more than 5 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
— unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
— total : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (1.0 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test A. 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 in vacuo at 60 °C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R
and add 5.0 ml of 0.01 M hydrochloric acid. Carry out
a potentiometric titration (2.2.20) using 0.1 M sodium
hydroxide and an electrode for non-aqueous acid-base
titrations. Read the volume added between the 2 points of
1 ml of 0.1 M sodium hydroxide is equivalent to 26.98 mg of
C14H20ClNO2.
STORAGE
Protected from light.
IMPURITIES
Specified impurities: A, B.
A. (2RS)-phenyl[(2RS)-piperidin-2-yl]acetic acid,
B. methyl (2RS)-phenyl[(2SR)-piperidin-2-yl]acetate.
4225
Methyltestosterone EUROPEAN PHARMACOPOEIA 6.3

07/2008:0410 Test solution. Dissolve 50 mg of the substance to be

corrected 6.3 examined in methanol R and dilute to 100.0 ml with the
same solvent.
METHYLTESTOSTERONE Reference solution (a). Dilute 0.5 ml of the test solution to
100.0 ml with methanol R.
Methyltestosteronum Reference solution (b). Dissolve 5 mg of methyltestosterone
for system suitability CRS (containing impurity A) in
methanol R and dilute to 10 ml with the same solvent.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 μm).
Mobile phase :
C20H30O2 Mr 302.5 — mobile phase A : water R ;
[58-18-4] — mobile phase B : methanol R ;
Time Mobile phase A Mobile phase B
DEFINITION
(min) (per cent V/V) (per cent V/V)
17β-Hydroxy-17-methylandrost-4-en-3-one. 0 - 15 30 70
Content : 97.0 per cent to 103.0 per cent (dried substance).
15 - 45 30 → 0 70 → 100
CHARACTERS 45 - 50 0 100
Appearance : white or slightly yellowish-white, crystalline
powder. Flow rate : 1.5 ml/min.
Solubility : practically insoluble in water, freely soluble in Detection : spectrophotometer at 254 nm.
ethanol (96 per cent). Injection : 20 μl.
IDENTIFICATION Identification of impurities : use the chromatogram supplied
with methyltestosterone for system suitability CRS and
First identification : B. the chromatogram obtained with reference solution (b) to
Second identification : A, C. identify the peak due to impurity A.
A. Melting point (2.2.14) : 162 °C to 168 °C. Relative retention with reference to methyltestosterone
B. Infrared absorption spectrophotometry (2.2.24). (retention time = about 8 min) : impurity A = about 1.5.
Comparison : methyltestosterone CRS. System suitability : reference solution (b):
C. Thin-layer chromatography (2.2.27). — resolution : minimum 5 between the peaks due to
Test solution. Dissolve 0.2 g of the substance to be methyltestosterone and impurity A.
examined in a mixture of 1 volume of methanol R and Limits :
9 volumes of chloroform R and dilute to 10 ml with the — impurity A : not more than the area of the principal peak
same mixture of solvents. in the chromatogram obtained with reference solution (a)
Reference solution. Dissolve 20 mg of methyl- (0.5 per cent) ;
testosterone CRS in 1 ml of a mixture of 1 volume of — unspecified impurities : for each impurity, not more
methanol R and 9 volumes of chloroform R. than 0.2 times the area of the principal peak in the
Plate : TLC silica gel F254 plate R. chromatogram obtained with reference solution (a)
Mobile phase : anhydrous acetic acid R, light (0.10 per cent) ;
petroleum R, butyl acetate R (1:30:70 V/V/V). — total : not more than twice the area of the principal peak
Application : 5 μl. in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
Development : over 2/3 of the plate.
— disregard limit : 0.1 times the area of the principal peak
Drying : in air.
in the chromatogram obtained with reference solution (a)
Detection : examine in ultraviolet light at 254 nm (0.05 per cent).
and spray with a saturated solution of potassium
dichromate R in a mixture of 30 volumes of water R and Loss on drying (2.2.32) : maximum 2.0 per cent, determined
70 volumes of sulphuric acid R. Examine immediately in on 0.500 g by drying in an oven at 105 °C for 2 h.
daylight. ASSAY
Results : the principal spot in the chromatogram obtained Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
with the test solution is similar in position, colour and 50.0 ml with the same solvent. Dilute 10.0 ml of the solution
size to the principal spot in the chromatogram obtained to 100.0 ml with ethanol (96 per cent) R. Dilute 10.0 ml
with the reference solution. of this solution to 100.0 ml with ethanol (96 per cent) R.
TESTS Measure the absorbance (2.2.25) at the absorption maximum
at 241 nm.
Specific optical rotation (2.2.7) : + 79 to + 85 (dried
substance). Calculate the content of C20H30O2 , taking the specific
absorbance to be 540.
Dissolve 0.250 g in ethanol (96 per cent) R and dilute to
25.0 ml with the same solvent. STORAGE
Related substances. Liquid chromatography (2.2.29). Protected from light.

4226 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Mianserin hydrochloride

IMPURITIES Reference solution (a). Dissolve 10 mg of mianserin

Specified impurities : A. hydrochloride CRS in methylene chloride R and dilute
to 5 ml with the same solvent.
Reference solution (b). Dissolve 10 mg of mianserin
hydrochloride CRS and 10 mg of cyproheptadine
hydrochloride CRS in methylene chloride R and dilute
to 5 ml with the same solvent.
Plate : TLC silica gel GF254 plate R.
Mobile phase : diethylamine R, ether R, cyclohexane R
(5:20:75 V/V/V).
A. 17α-hydroxy-17-methylandrost-4-en-3-one.
Application : 2 μl.
Development : over 2/3 of the plate.
Detection : examine in ultraviolet light at 254 nm.
01/2009:0846
System suitability : reference solution (b) :
— the chromatogram shows 2 clearly separated principal
MIANSERIN HYDROCHLORIDE spots.
Results : the principal spot in the chromatogram obtained
Mianserini hydrochloridum with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (a).
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
pH (2.2.3) : 4.0 to 5.5.
Dissolve 0.10 g in carbon dioxide-free water R and dilute to
10 ml with the same solvent.
C18H21ClN2 Mr 300.8 Related substances. Liquid chromatography (2.2.29).
[21535-47-7] Buffer solution pH 3.0. Dissolve 5.0 g of sodium
octanesulphonate R in water R and dilute to 350 ml with
DEFINITION the same solvent. Stir until complete dissolution. Adjust to
(14bRS)-2-Methyl-1,2,3,4,10,14b-hexahydrodibenzo- pH 3.0 with a mixture of 1 volume of phosphoric acid R and
[c,f]pyrazino[1,2-a]azepine hydrochloride. 3 volumes of water R. Dilute to 400 ml with water R.
Content : 98.5 per cent to 101.0 per cent (dried substance). Test solution. Dissolve 25 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml with the
CHARACTERS mobile phase.
Appearance : white or almost white, crystalline powder or Reference solution (a). Dilute 1.0 ml of the test solution
crystals. to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
Solubility : sparingly soluble in water, soluble in methylene solution to 10.0 ml with the mobile phase.
chloride, slightly soluble in ethanol (96 per cent). Reference solution (b). Dissolve the contents of a vial
of mianserin for system suitability CRS (containing
IDENTIFICATION impurities A, D and E) in 1.0 ml of the mobile phase.
First identification : B, D. Reference solution (c). Dissolve 5.0 mg of mianserin
Second identification : A, C, D. impurity B CRS in the mobile phase and dilute to 50.0 ml
A. Ultraviolet and visible absorption spectrophotometry with the mobile phase. Dilute 1.0 ml of this solution to
(2.2.25). 100.0 ml with the mobile phase.
Test solution. Dissolve 50.0 mg in water R and dilute Column :
to 50.0 ml with the same solvent. Dilute 5.0 ml of the — size: l = 0.15 m, Ø = 3.9 mm ;
solution to 50.0 ml with water R. — stationary phase : end-capped octylsilyl silica gel for
Spectral range : 230-350 nm. chromatography R (5 μm).
Absorption maximum : at 279 nm. Mobile phase : buffer solution pH 3.0, methanol R
Specific absorbance at the absorption maximum : 64 (37:63 V/V).
to 72. Flow rate : 0.5 ml/min.
B. Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 250 nm.
Injection : 10 μl.
Comparison : mianserin hydrochloride CRS. Run time : twice the retention time of mianserin.
If the spectra obtained in the solid state show differences, Identification of impurities : use the chromatogram
dissolve the substance to be examined and the reference supplied with mianserin for system suitability CRS and
substance separately in methanol R, evaporate to dryness the chromatogram obtained with reference solution (b) to
and record new spectra using the residues. identify the peaks due to impurities A, D and E.
C. Thin-layer chromatography (2.2.27). Relative retention with reference to mianserin (retention
Test solution. Dissolve 10 mg of the substance to be time = about 18 min) : impurity B = about 0.2 ;
examined in methylene chloride R and dilute to 5 ml impurity A = about 0.5 ; impurity D = about 0.7 ;
with the same solvent. impurity E = about 1.1.

General Notices (1) apply to all monographs and other texts 4227
Moxidectin for veterinary use EUROPEAN PHARMACOPOEIA 6.3

System suitability : reference solution (b) : D. R = CH2-C6H5 : [2-[(2RS)-4-benzyl-2-phenylpiperazin-1-

— peak-to-valley ratio : minimum 4.0, where Hp = height yl]phenyl]methanol,
above the baseline of the peak due to impurity E and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
mianserin.
Limits :
— correction factor : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 2.4 ;
impurity D = 2.1 ; B. (14bRS)-2-methyl-1,2,3,4,10,14b-hexahydrodibenzo-
— impurity B : not more than 3 times the area of the [c,f]pyrazino[1,2-a]azepine-8-sulfonic acid,
principal peak in the chromatogram obtained with
reference solution (c) (0.3 per cent) ;
— impurities A, D, E : for each impurity, not more
than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.15 per cent) ;
— unspecified impurities: for each impurity, not more C. (2-aminophenyl)methanol,
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
— total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined E. (14bRS)-1,2,3,4,10,14b-hexahydrodibenzo-
on 1.000 g by drying over diphosphorus pentoxide R at [c,f]pyrazino[1,2-a]azepine,
65 °C at a pressure not exceeding 0.7 kPa for 3 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.200 g in a mixture of 5.0 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 F. (14bRS)-2-benzyl-1,2,3,4,10,14b-hexahydrodibenzo-
of inflexion. [c,f]pyrazino[1,2-a]azepine.
l ml of 0.1 M sodium hydroxide is equivalent to 30.08 mg
of C18H21ClN2.
STORAGE 01/2008:1656
Protected from light. corrected 6.3
IMPURITIES
Specified impurities : A, B, D, E.
MOXIDECTIN FOR VETERINARY USE
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one Moxidectinum ad usum veterinarium
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) : C, F.

A. R = CH3 : [2-[(2RS)-4-methyl-2-phenylpiperazin-1- C37H53NO8 Mr 640

yl]phenyl]methanol, [113507-06-5]

4228 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
DEFINITION
(2aE,2′R,4E,4′E,5′S,6R,6′S,8E,11R,15S,17aR,20R,20aR,
20bS)-6′-[(1E)-1,3-Dimethylbut-1-enyl]-20,20b-dihydroxy-
4′-(methoxyimino)-5′,6,8,19-tetramethyl-3′,4′,5′,6,6′,7,10,-
11,14,15,17a,20,20a,20b-tetradecahydrospiro[2H,17H-11,-
15-methanofuro[4,3,2-pq][2,6]benzodioxacyclooctadecine-
13,2′-pyran]-17-one ((6R,23E,25S)-5O-demethyl-28-
deoxy-25-[(1E)-1,3-dimethylbut-1-enyl]-6,28-epoxy-23-
(methoxyimino)milbemycin B).
Semi-synthetic product derived from a fermentation product.
It may contain suitable stabilisers such as antioxidants.
Content : 92.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or pale yellow, amorphous powder.
Solubility : practically insoluble in water, very soluble in
ethanol (96 per cent), slightly soluble in hexane.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : moxidectin CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution GY5
(2.2.2, Method II).
Dissolve 0.40 g in benzyl alcohol R and dilute to 20 ml with
the same solvent.
Related substances. Liquid chromatography (2.2.29).
A. Test solution. Dissolve 25.0 mg of the substance to be
examined in acetonitrile R and dilute to 25.0 ml with the
same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml acetonitrile R.
Reference solution (b). Dissolve 5 mg of moxidectin for
system suitability CRS (containing impurities A, B, C, D,
E, F, G, H, I, J and K) 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.
Column:
— size : l = 0.15 m, Ø = 3.9 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (4 μm) ;
— 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.
Flow rate : 2.5 ml/min.
Detection : spectrophotometer at 242 nm.
Injection : 10 μl of the test solution and reference
solutions (a) and (b).
Run time : 2 times the retention time of moxidectin.
Identification of impurities : use the chromatogram
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
(retention time = about 12 min) : impurity A = about 0.5 ;
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.
General Notices (1) apply to all monographs and other texts
Moxidectin for veterinary use
System suitability : reference solution (b) :
— peak-to-valley ratio : minimum 3.0, where Hp = height
above the baseline of the peak due to impurity D and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
moxidectin.
Limits :
— impurity D : not more than 2.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (2.5 per cent) ;
— sum of impurities E and F : not more than 1.7 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.7 per cent) ;
— impurities A, C, G : for each impurity, not more
than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(1.5 per cent) ;
— impurity B : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
— any other impurity eluting before impurity G : for
each impurity, not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
— disregard limit : 0.1 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.1 per cent) ; disregard the peak due to
the stabiliser (identify this peak, where applicable, by
injecting a suitable reference solution).
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.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with acetonitrile R.
Reference solution (b). Dissolve 5 mg of moxidectin for
system suitability CRS (containing impurities A, B, C, D,
E, F, G, H, I, J and K) in 5 ml of acetonitrile R.
Column :
— size: l = 0.15 m, Ø = 3.9 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (4 μm) ;
— 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.
Flow rate : 2.0 ml/min.
Detection : spectrophotometer at 242 nm.
Injection : 10 μl.
Run time : 10 times the retention time of moxidectin.
Identification of impurities: use the chromatogram
supplied with moxidectin for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities H + I, J and K.
Relative retention with reference to moxidectin
(retention time = about 4 min) : impurity G = about 1.4 ;
impurities H + I = about 2.0 ; impurity J = about 2.2 ;
impurity K = about 3.4.
System suitability : reference solution (b) :
— resolution : baseline separation between the peaks due
to impurities H + I and J.
4229
Moxidectin for veterinary use EUROPEAN PHARMACOPOEIA 6.3

Limits :

— sum of impurities H and I : not more than the area of

the principal peak in the chromatogram obtained with
reference solution (a) (1.0 per cent) ;

— impurities J, K : for each impurity, not more than

0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.5 per cent) ;

— any other impurity eluting after impurity G : for each

impurity, not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
A. R1 = R2 = R3 = R4 = CH3, R5 = R6 = H :
25-des[(1E)-1,3-dimethylbut-1-enyl]-25-[(1E)-1-
— disregard limit : 0.1 times the area of the principal methylprop-1-enyl]moxidectin,
peak in the chromatogram obtained with reference
solution (a) (0.1 per cent) ; disregard the peak due to
the stabiliser (identify this peak, where applicable, by
injecting a suitable reference solution). B. R1 = R2 = R3 = R5 = R6 = CH3, R4 = H :
24-desmethylmoxidectin,
Total of all impurities. Calculate the sum 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 C. R1 = R2 = R3 = R4 = R5 = CH3, R6 = H :
total of all impurities is not more than 7.0 per cent. 25-des[(1E)-1,3-dimethylbut-1-enyl]-25-[(1E)-1-
methylbut-1-enyl]moxidectin,
Heavy metals (2.4.8) : maximum 20 ppm.

Prescribed solution. Dissolve 0.50 g in 20 ml of ethanol F. one of groups R1 to R6 is C2H5, the others are CH3 :
(96 per cent) R. x-desmethyl-x-ethylmoxidectin,
Reference solution. Mix 6 ml of a 1 ppm Pb standard solution
with 2 ml of the prescribed solution and 4 ml of water R.

To each solution add 2 ml of buffer solution pH 3.5 R. Mix.

Add 1.2 ml of thioacetamide reagent R. Mix immediately.
Filter the solutions through a membrane filter (pore size
0.45 μm). Carry out the filtration slowly and uniformly,
applying moderate and constant pressure to the piston.
Compare the spots on the filters obtained with the different
solutions. The test is invalid if the reference solution does not
show a slight brown colour compared to the blank solution.
The substance to be examined complies if the test solution is
not more intense than that in the reference solution.
Water (2.5.12) : maximum 1.3 per cent, determined on 0.50 g.
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
on 1.0 g.
D. 2-epi-moxidectin,

ASSAY

Liquid chromatography (2.2.29) as described in test A for

related substances with the following modification.

Injection : test solution and reference solution (c).

Calculate the percentage content of C37H53NO8 using the

declared content of moxidectin CRS.

IMPURITIES

Specified impurities : A, B, C, D, E, F, G, H, I, J, K. E. (4S)-2-dehydro-4-hydromoxidectin,

4230 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Moxidectin for veterinary use

G. (23E,25S)-5O-desmethyl-28-deoxy-25-[(1E)-1,3-
dimethylbut-1-enyl]-23-(methoxyimino)milbemycin B, J. R = CH2-S-CH3, R′ = H : 7-O-[(methylsulphanyl)methyl]-
moxidectin,

K. R = H, R′ = CO-C6H4-pNO2 : 5-O-(4-nitrobenzoyl)moxidectin,

H. 2,5-didehydro-5-deoxymoxidectin,

I. (23S)-23-des(methoxyimino)-23-[(methylsulphanyl)meth-
oxy]moxidectin, L. (23Z)-moxidectin.

General Notices (1) apply to all monographs and other texts 4231
 EUROPEAN PHARMACOPOEIA 6.3

4232 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

N
Naphazoline hydrochloride....................................................4235 Nicotine resinate.. ....................................................................4237
Nicotine.. ....................................................................................4236

General Notices (1) apply to all monographs and other texts 4233
 EUROPEAN PHARMACOPOEIA 6.3

4234 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Naphazoline hydrochloride

01/2009:0730 Reference solution (b). Dissolve 5.0 mg of naphazoline

impurity A CRS in the mobile phase and dilute to 100.0 ml
with the mobile phase. Dilute 1.0 ml of this solution to
NAPHAZOLINE HYDROCHLORIDE 100.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of the test solution to
Naphazolini hydrochloridum 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
Column :
— size: l = 0.25 m, Ø = 4.0 mm ;
— stationary phase : base-deactivated end-capped octylsilyl
silica gel for chromatography R (4 μm) with a pore size
of 6 nm.
C14H15ClN2 Mr 246.7 Mobile phase : dissolve 1.1 g of sodium octanesulphonate R
[550-99-2] in a mixture of 5 ml of glacial acetic acid R, 300 ml of
acetonitrile R and 700 ml of water R.
DEFINITION Flow rate : 1 ml/min.
2-(Naphthalen-1-ylmethyl)-4,5-dihydro-1H-imidazole Detection : spectrophotometer at 280 nm.
hydrochloride.
Injection : 20 μl.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Run time : 3 times the retention time of naphazoline.
CHARACTERS Retention time : naphazoline = about 14 min.
Appearance : white or almost white, crystalline powder. System suitability : reference solution (a) :
Solubility : freely soluble in water, soluble in ethanol (96 per
cent). — resolution : minimum 5.0 between the peaks due to
naphazoline and impurity B.
mp : about 259 °C, with decomposition.
Limits :
IDENTIFICATION — impurity A : not more than the area of the principal peak
First identification : B. in the chromatogram obtained with reference solution (b)
(0.1 per cent) ;
Second identification : A, C.
— unspecified impurities : not more than the area of the
A. Dissolve 50.0 mg in 0.01 M hydrochloric acid and dilute principal peak in the chromatogram obtained with
to 250.0 ml with the same acid. Dilute 25.0 ml of the reference solution (c) (0.10 per cent) ;
solution to 100.0 ml with 0.01 M hydrochloric acid.
Examined between 230 nm and 350 nm (2.2.25), the — total : not more than 5 times the area of the principal peak
solution shows 4 absorption maxima, at 270 nm, 280 nm, in the chromatogram obtained with reference solution (c)
287 nm and 291 nm. The ratios of the absorbances (0.5 per cent) ;
measured at the maxima at 270 nm, 287 nm and — disregard limit : 0.5 times the area of the principal peak
291 nm to that measured at the maximum at 280 nm are in the chromatogram obtained with reference solution (c)
0.82 to 0.86, 0.67 to 0.70 and 0.65 to 0.69, respectively. (0.05 per cent).
B. Infrared absorption spectrophotometry (2.2.24). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Comparison : naphazoline hydrochloride CRS. on 1.000 g by drying in an oven at 105 °C.
C. It gives reaction (a) of chlorides (2.3.1). Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
TESTS
Solution S. Dissolve 0.5 g in carbon dioxide-free water R ASSAY
and dilute to 50 ml with the same solvent. Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric
Appearance of solution. Solution S is clear (2.2.1) and acid and 50 ml of ethanol (96 per cent) R. Carry out
colourless (2.2.2, Method II). a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
Acidity or alkalinity. To 20 ml of solution S add 0.2 ml of inflexion.
of 0.01 M sodium hydroxide and 0.1 ml of methyl red
solution R. The solution is yellow. Not more than 0.6 ml of 1 ml of 0.1 M sodium hydroxide is equivalent to 24.67 mg
0.01 M hydrochloric acid is required to change the colour of C14H15ClN2.
of the solution to red.
Related substances. Liquid chromatography (2.2.29). STORAGE
Test solution. Dissolve 50.0 mg of the substance to be Protected from light.
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
IMPURITIES
Reference solution (a). Dissolve 5 mg of 1-naphthylacetic
Specified impurities : A.
acid R in the mobile phase, add 5 ml of the test solution and
dilute to 100 ml with the mobile phase. Other detectable impurities : B, C, D.

General Notices (1) apply to all monographs and other texts 4235
Nicotine
A. R = CO-NH-[CH2]2-NH2 : N-(2-aminoethyl)-2-(naphthalen-1-
yl)acetamide (naphthylacetylethylenediamine),
B. R = CO2H : (naphthalen-1-yl)acetic acid (1-naphthylacetic
acid),
C. R = CN : (naphthalen-1-yl)acetonitrile (1-naphthylace-
tonitrile),
D. 2-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-imidazole
(β-naphazoline).
01/2009:1452
NICOTINE
Nicotinum
C10H14N2 Mr 162.2 identify the peaks due to impurities A, B, C, D, E, F and G.
[54-11-5]
DEFINITION
3-[(2S)-1-Methylpyrrolidin-2-yl]pyridine.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : colourless or brownish viscous liquid, volatile,
hygroscopic.
Solubility : soluble in water, miscible with anhydrous
ethanol.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of nicotine.
TESTS
Appearance of solution. Dissolve 1.0 g in water R and dilute
to 10 ml with the same solvent. The solution is clear (2.2.1) — disregard limit : 0.5 times the area of the principal peak
and not more intensely coloured than reference solution Y5,
BY5 or R5 (2.2.2, Method II).
Specific optical rotation (2.2.7) : − 140 to − 152.
Dissolve 1.00 g in anhydrous ethanol R and dilute to 50.0 ml
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Dissolve 20.0 mg of the substance to be
examined in water R and dilute to 25.0 ml with the same
solvent.
4236 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Reference solution (a). Dissolve the contents of a vial of
nicotine for system suitability CRS (containing impurities
A, B, C, D, E, F and G) in 1.0 ml of water R.
Reference solution (b). Dilute 1.0 ml of the test solution
to 10.0 ml with water R. Dilute 1.0 ml of this solution to
100.0 ml with water R.
Column :
— size: l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R
(5 μm).
Mobile phase :
— mobile phase A : to 900 ml of water R, add 25 ml of
a 60 g/l solution of acetic acid R, then add 6 ml of
concentrated ammonia R1. Adjust to pH 10.0 with
dilute ammonia R2 or dilute acetic acid R and dilute to
1000 ml with water R ;
— mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-3 100 0
3 - 3.01 100 → 95 0→5
3.01 - 28 95 → 74 5 → 26
28 - 32 74 → 60 26 → 40
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl.
Identification of impurities : use the chromatogram
supplied with nicotine for system suitability CRS and
the chromatogram obtained with reference solution (a) to
Relative retention with reference to nicotine (retention
time = about 17.8 min) : impurity E = about 0.3 ;
impurity C = about 0.55 ; impurity F = about 0.7 ;
impurity A = about 0.8 ; impurity D = about 0.86 ;
impurity G = about 0.9 ; impurity B = about 1.6.
System suitability : reference solution (a) :
— resolution : minimum 3 between the peaks due to
impurity G and nicotine.
Limits :
— impurities A, B, C, D, E, F, G : for each impurity, not
more than 3 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.3 per cent) ;
— unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
— total : not more than 8 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.8 per cent) ;
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
ASSAY
Dissolve 60.0 mg in 30 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 8.11 mg of
C10H14N2.
EUROPEAN PHARMACOPOEIA 6.3
STORAGE
Under nitrogen, in an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G.
A. (2S)-1,2,3,6-tetrahydro-2,3′-bipyridyl (anatabine),
B. 3-(1-methyl-1H-pyrrol-2-yl)pyridine (β-nicotyrine),
C. (5S)-1-methyl-5-(pyridin-3-yl)pyrrolidin-2-one (cotinine),
D. 3-(4,5-dihydro-3H-pyrrol-2-yl)pyridine (myosmine),
E. (1RS,2S)-1-methyl-2-(pyridin-3-yl)pyrrolidine 1-oxide
(nicotine N′-oxide),
F. 3-[(2S)-pyrrolidin-2-yl]pyridine (nornicotine),
G. 3-[(2S)-piperidin-2-yl]pyridine (anabasine).
01/2009:1792
NICOTINE RESINATE
Nicotini resinas
DEFINITION
Complex of nicotine (3-[(2S)-1-methylpyrrolidin-2-yl]pyridine)
with a weak cationic exchange resin.
Content : 95.0 per cent to 115.0 per cent of the declared
content of nicotine (C10H14N2) stated on the label (anhydrous
susbtance).
It may contain glycerol.
CHARACTERS
Appearance : white or slightly yellowish powder.
General Notices (1) apply to all monographs and other texts
Nicotine resinate
Solubility : practically insoluble in water.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : shake a quantity of the substance to be
examined equivalent to 100 mg of nicotine with a mixture
of 10 ml of dilute ammonia R2, 10 ml of water R, 5 ml
of strong sodium hydroxide solution R and 20 ml of
hexane R for 5 min. Transfer the upper layer to a beaker
and evaporate to produce an oily residue. Record the
spectrum of the oily residue as a thin film between
sodium chloride R plates.
Comparison : Ph. Eur. reference spectrum of nicotine.
B. It complies with the test for nicotine release (see Tests).
TESTS
Nicotine release : minimum 70 per cent of the content
determined under Assay in 10 min.
Transfer an accurately weighed quantity of the substance
to be examined equivalent to about 4 mg of nicotine, to a
glass-stoppered test-tube, add 10.0 ml of a 9 g/l solution of
sodium chloride R previously heated to 37 °C and shake
vigorously for 10 min. Immediately filter the liquid through
a dry filter paper discarding the 1st millilitre of filtrate.
Transfer 1.0 ml of the filtrate to a 20 ml volumetric flask,
dilute to 20 ml with 0.1 M hydrochloric acid and mix.
Determine the absorbance (2.2.25) at the minima at about
236 nm and 282 nm and at the maximum at 259 nm using
1.0 ml of a 9 g/l solution of sodium chloride R diluted to
20 ml with 0.1 M hydrochloric acid as compensation liquid.
Calculate the percentage of nicotine release using the
following expression :
323 specific absorbance of nicotine at
=
259 nm ;
C = percentage of nicotine in the
substance to be examined
determined in the assay ;
m = mass of the substance to be
examined, in milligrams ;
A236, A259, A282 = absorbances of the solution at
the wavelength indicated by the
subscript.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Weigh a quantity of the substance to
be examined equivalent to 30.0 mg of nicotine into a
glass-stoppered test-tube, add 10.0 ml of dilute ammonia R2
solution and shake vigorously for 10 min. Centrifuge for
20 min at about 3000 r/min. To 5.0 ml of the clear solution,
add 5 ml of a 60 g/l solution of acetic acid R and dilute to
25.0 ml with water R.
Reference solution (a). Dissolve the contents of a vial of
nicotine for system suitability CRS (containing impurities
A, B, C, D, E, F and G) in 1.0 ml of water R.
Reference solution (b). Dilute 1.0 ml of the test solution
to 10.0 ml with water R. Dilute 1.0 ml of this solution to
100.0 ml with water R.
Reference solution (c). Dissolve 46.0 mg of nicotine
ditartrate CRS in water R and dilute to 25.0 ml with the
same solvent.
Column :
— size: l = 0.15 m, Ø = 4.6 mm ;
4237
Nicotine resinate EUROPEAN PHARMACOPOEIA 6.3

— stationary phase : end-capped polar-embedded Calculate the percentage content of nicotine (C10H14N2)
octadecylsilyl amorphous organosilica polymer R (anhydrous substance) from the declared content of C10H14N2
(5 μm). in nicotine ditartrate CRS.
Mobile phase : STORAGE
— mobile phase A : to 900 ml of water R, add 25 ml of In an airtight container, protected from light.
a 60 g/l solution of acetic acid R, then add 6 ml of
concentrated ammonia R1 ; adjust to pH 10.0 with dilute LABELLING
ammonia R2 or dilute acetic acid R and dilute to 1000 ml
with water R ; The label states the content of nicotine.
— mobile phase B : acetonitrile R ; IMPURITIES
Time Mobile phase A Mobile phase B Specified impurities : A, B, C, D, E, F, G.
(min) (per cent V/V) (per cent V/V)
0-3 100 0
3 - 3.01 100 → 95 0→5
3.01 - 28 95 → 74 5 → 26
28 - 32 74 → 60 26 → 40
A. (2S)-1,2,3,6-tetrahydro-2,3′-bipyridyl (anatabine),
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl.
Identification of impurities: use the chromatogram
supplied with nicotine for system suitability CRS and
the chromatogram obtained with reference solution (a) to B. 3-(1-methyl-1H-pyrrol-2-yl)pyridine (β-nicotyrine),
identify the peaks due to impurities A, B, C, D, E, F and G.
Relative retention with reference to nicotine (retention
time = about 17.8 min) : impurity E = about 0.3 ;
impurity C = about 0.55 ; impurity F = about 0.7 ;
impurity A = about 0.8 ; impurity D = about 0.86 ;
impurity G = about 0.9 ; impurity B = about 1.6. C. (5S)-1-methyl-5-(pyridin-3-yl)pyrrolidin-2-one (cotinine),
System suitability : reference solution (a) :
— resolution : minimum 3 between the peaks due to
impurity G and nicotine.
Limits :
— impurities A, B, C, D, E, F, G : for each impurity, not
more than 3 times the area of the principal peak in D. 3-(4,5-dihydro-3H-pyrrol-2-yl)pyridine (myosmine),
the chromatogram obtained with reference solution (b)
(0.3 per cent) ;
— unspecified impurities: for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
— total : not more than 8 times the area of the principal peak E. (1RS,2S)-1-methyl-2-(pyridin-3-yl)pyrrolidine 1-oxide
in the chromatogram obtained with reference solution (b) (nicotine N′-oxide),
(0.8 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : maximum 5.0 per cent.
Suspend 1.0 g in 20.0 ml of methanol R, shake for 30 min F. 3-[(2S)-pyrrolidin-2-yl]pyridine (nornicotine),
and allow to stand for 30 min. Use 10 ml of the methanol
layer for the titration. Carry out a blank titration.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (c). G. 3-[(2S)-piperidin-2-yl]pyridine (anabasine).

4238 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

O
Olive leaf.. .................................................................................. 4241 Omeprazole magnesium.........................................................4248
Omega-3-acid ethyl esters 60.................................................4242 Oxaliplatin.. ...............................................................................4249
Omega-3-acid ethyl esters 90.................................................4244 Oxymetazoline hydrochloride.. .............................................4252
Omega-3-acid triglycerides.. ...................................................4246

General Notices (1) apply to all monographs and other texts 4239
 EUROPEAN PHARMACOPOEIA 6.3

4240 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Olive leaf

01/2009:1878 Top of the plate

A dark violet-blue zone (solvent
front)
OLIVE LEAF A dark violet-blue zone
_______ _______

Oleuropein : a brownish-green A brownish-green zone

Oleae folium zone (oleuropein)
_______ _______

Rutin : a brownish-yellow zone

DEFINITION
Reference solution Test solution
Dried leaf of Olea europaea L.
Content : minimum 5.0 per cent of oleuropein (C25H32O13 ;
Mr 540.5) (dried drug).

IDENTIFICATION
A. The leaf is simple, thick and coriaceous, lanceolate to
obovate, 30-50 mm long and 10-15 mm wide, with a
mucronate apex and tapering at the base to a short
petiole ; the margins are entire and reflexed abaxially. The
upper surface is greyish-green, smooth and shiny, the
lower surface paler and pubescent, particularly along the
midrib and main lateral veins.
B. Reduce to a powder (355) (2.9.12). The powder is
yellowish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the
following diagnostic characters : fragments of the
epidermis in surface view with small, thick-walled
polygonal cells and, in the lower epidermis only, small
anomocytic stomata (2.8.3) ; fragments of the lamina
in sectional view showing a thick cuticle, a palisade
composed of 3 layers of cells and a small-celled spongy
parenchyma ; numerous sclereids, very thick-walled
and mostly fibre-like with blunt or, occasionally, forked
ends, isolated or associated with the parenchyma of
the mesophyll ; abundant, very large peltate trichomes,
with a central unicellular stalk from which radiate
some 10-30 thin-walled cells that become free from the
adjoining cells at the margin of the shield, giving an
uneven, jagged appearance.
C. Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the powdered drug (355) A. Peltate trichome, seen from F. Fragment of the lamina, in
(2.9.12) add 10 ml of methanol R. Boil under a reflux above transverse section, showing a thick
B. Peltate trichome, seen from cuticle (Fa), palisade parenchyma
condenser for 15 min. Cool and filter. composed of 3 layers of cells (Fb),
below
and spongy parenchyma (Fc)
Reference solution. Dissolve 10 mg of oleuropein R and C. Palisade parenchyma
J. Fragment of lower epidermis
1 mg of rutin R in 1 ml of methanol R. D, G, H and L. Fibre-like with anomocytic stomata (Ja) and
sclereids, some accompanied cicatrix of peltate trichome (Jb)
Plate : TLC silica gel plate R. by parenchymatous fragments of
the spongy mesophyll K. Fragment of upper epidermis, in
surface view, with underlying
Mobile phase : water R, methanol R, methylene E. Spongy parenchyma palisade parenchyma (Ka)
chloride R (1.5:15:85 V/V/V). and sclereids of the spongy
mesophyll (Kb)
Application : 10 μl, as bands.
Figure 1878.-1. — Illustration of powdered herbal drug of
Development : over a path of 10 cm. olive leaf (see Identification B)
Drying : in air. TESTS
Detection : spray with vanillin reagent R and heat at Loss on drying (2.2.32) : maximum 10.0 per cent, determined
100-105 °C for 5 min ; examine in daylight. on 1.000 g of the powdered drug (355) (2.9.12) by drying
in an oven at 105 °C for 2 h.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and Total ash (2.4.16) : maximum 9.0 per cent.
the test solution. Furthermore, other faint zones may
be present in the chromatogram obtained with the test ASSAY
solution. Liquid chromatography (2.2.29).

General Notices (1) apply to all monographs and other texts 4241
Omega-3-acid ethyl esters 60 EUROPEAN PHARMACOPOEIA 6.3

Test solution. In a flask, place 1.000 g of the powdered A1
drug (355) (2.9.12) and add 50 ml of methanol R. Heat in a
water-bath at 60 °C for 30 min with shaking. Allow to cool A2
and filter into a 100 ml volumetric flask. Rinse the flask and
the filter with methanol R and dilute to 100.0 ml with the
same solvent. Dilute 2.5 ml of this solution to 25.0 ml with
water R. m1
Reference solution. Dissolve 5.0 mg of oleuropein CRS m2
in 5.0 ml of methanol R. Dilute 1.0 ml of this solution to
25.0 ml with water R. p =
Column:
— size : l = 0.15 m, Ø = 3.9 mm ;
= area of the peak due to oleuropein in the
chromatogram obtained with the test solution ;
= area of the peak due to oleuropein in the
chromatogram obtained with the reference
solution ;
= mass of the drug to be examined in the test
solution, in grams ;
= mass of oleuropein CRS in the reference solution,
in grams ;
percentage content of oleuropein in
oleuropein CRS.
01/2009:2063

— stationary phase : octadecylsilyl silica gel for OMEGA-3-ACID ETHYL ESTERS 60

chromatography R (5 μm) ;
Omega-3 acidorum esteri ethylici 60
— temperature : 25 °C.
DEFINITION
Mobile phase : Ethyl esters of alpha-linolenic acid (C18:3 n-3), moroctic
acid (C18:4 n-3), eicosatetraenoic acid (C20:4 n-3),
— mobile phase A : dilute 1.0 ml of glacial acetic acid R to timnodonic (eicosapentaenoic) acid (C20:5 n-3 ; EPA),
100 ml with water R ; heneicosapentaenoic acid (C21:5 n-3), clupanodonic acid
(C22:5 n-3) and cervonic (docosahexaenoic) acid (C22:6 n-3 ;
— mobile phase B : methanol R ; DHA). Omega-3-acid ethyl esters 60 are obtained by
transesterification of the body oil obtained from fish of
Time Mobile phase A Mobile phase B families such as Engraulidae, Carangidae, Clupeidae,
(min) (per cent V/V) (per cent V/V)
Osmeridae, Salmonidae and Scombridae and subsequent
0-5 85 → 40 15 → 60
physico-chemical purification processes, including molecular
distillation. The minimum content of total omega-3-acid ethyl
5 - 12 40 → 20 60 → 80 esters and the minimum content of the omega-3-acids EPA
12 - 15 20 → 85 80 → 15 and DHA ethyl esters are indicated in Table 2063.-1.
Table 2063.-1
Flow rate : 1 ml/min. Total omega-3- EPA and DHA EPA ethyl DHA ethyl
acid ethyl esters ethyl esters esters esters
Detection : spectrophotometer at 254 nm. Minimum content (per cent)
65 50 25 20
Injection : 20 μl. –
60 50 40
Retention time : oleuropein = about 9 min. 55 50 40 –

Calculate the percentage content of oleuropein using the Authorised antioxidants in concentrations not exceeding the
following expression : levels specified by the competent authority may be added.
CHARACTERS
Appearance : light yellow liquid.

1. oligomers 2. monoglycerides 3. fatty acid ethyl esters

Figure 2063.-1. – Chromatogram for the test for oligomers and partial glycerides

4242 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Omega-3-acid ethyl esters 60

Slight fish-like odour. Peroxide value (2.5.5, Method A) : maximum 10.0.

Solubility : practically insoluble in water, very soluble in
acetone, in ethanol (96 per cent), in heptane and in methanol. chromatography (2.2.30).
IDENTIFICATION
A. Examine the chromatograms obtained in the assay
for EPA and DHA ethyl esters.
Results : the peaks due to eicosapentaenoic acid ethyl
ester and docosahexaenoic acid ethyl ester in the
chromatogram obtained with the test solution are similar Column 1 :
in retention time to the corresponding peaks in the
chromatogram obtained with the reference solution.
B. It complies with the limits of the assay for total
omega-3-acid ethyl esters.
TESTS
Absorbance (2.2.25) : maximum 0.60 at 233 nm.
Dilute 0.300 g to 50.0 ml with trimethylpentane R. Dilute
2.0 ml of this solution to 50.0 ml with trimethylpentane R.
Acid value (2.5.1) : maximum 2.0, determined on 10 g in
50 ml of the prescribed mixture of solvents.
Anisidine value (2.5.36) : maximum 20.0.
Oligomers and partial glycerides. Size-exclusion
Test solution. Dilute 50.0 mg of the substance to be
examined to 10.0 ml with tetrahydrofuran R.
Reference solution. Dissolve 50 mg of monodocosa-
hexaenoin R, 30 mg of didocosahexaenoin R and 20 mg of
tridocosahexaenoin R in tetrahydrofuran R and dilute to
100.0 ml with the same solvent.
— dimensions: l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
(7 μm), with a pore size of 10 nm.
Columns 2 and 3 placed closest to the injector :
— dimensions: l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
(7 μm), with a pore size of 50 nm.
Mobile phase : tetrahydrofuran R.
Flow rate : 0.8 ml/min.
Detection : differential refractometer.
Injection : 40 μl.

1. C16:0 5. C18:2 n-6 9. C20:1 n-9 12. C20:4 n-3 16. C21:5 n-3
2. C18:0 6. C18:3 n-3 9a. C20:1 n-11 13. EPA 17. C22:5 n-6
3. C18:1 n-9 7. C18:4 n-3 10. C20:1 n-7 14. C22:1 n-11 18. C22:5 n-3
4. C18:1 n-7 8. C20:0 11. C20:4 n-6 15. C22:1 n-9 19. DHA

Figure 2063.-2. – Chromatogram for the assays

General Notices (1) apply to all monographs and other texts 4243
Omega-3-acid ethyl esters 90 EUROPEAN PHARMACOPOEIA 6.3

System suitability : reference solution : CHARACTERS

— elution order: tridocosahexaenoin, didocosahexaenoin,
monodocosahexaenoin ;
— resolution : minimum 2.0 between the peaks due
to monodocosahexaenoin and didocosahexaenoin ;
minimum 1.0 between the peaks due to didocosahexaenoin
and tridocosahexaenoin.
Calculate the percentage content of oligomers plus partial
glycerides using the following expression :
A = sum of the areas of all the peaks in the
chromatogram ;
B = sum of the areas of the peaks with a retention
time less than the retention time of the peaks due
to ethyl esters.
The ethyl ester peaks, which may be present in the form of
an unresolved double peak, are identified as the major peaks
in the chromatogram (see Figure 2063.-1).
Limit :
— sum of oligomers and partial glycerides: maximum
7.0 per cent.
ASSAY
EPA and DHA ethyl esters (2.4.29). For identification of
the peaks, see Figure 2063.-2.
Total omega-3-acids ethyl esters (2.4.29). See
Figure 2063.-2.
STORAGE
Under an inert gas, in an airtight container, protected from
light.
LABELLING
The label states :
— the content of total omega-3-acid ethyl esters ;
— the content of EPA ethyl ester and DHA ethyl ester.
Appearance : light yellow liquid.
Solubility : practically insoluble in water, very soluble in
acetone, in ethanol (96 per cent), in heptane and in methanol.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay for
EPA and DHA ethyl esters.
Results : the peaks due to eicosapentaenoic acid ethyl
ester and docosahexaenoic acid ethyl ester in the
chromatogram obtained with the test solution are similar
in retention time to the corresponding peaks in the
chromatogram obtained with the reference solution.
B. It complies with the limits of the assay for total
omega-3-acid ethyl esters.
TESTS
Absorbance (2.2.25) : maximum 0.55 at 233 nm.
Dilute 0.300 g to 50.0 ml with trimethylpentane R. Dilute
2.0 ml of this solution to 50.0 ml with trimethylpentane R.
Acid value (2.5.1) : maximum 2.0, determined on 10 g in
50 ml of the prescribed mixture of solvents.
Anisidine value (2.5.36) : maximum 20.0.
Peroxide value (2.5.5, Method A) : maximum 10.0.
Oligomers. Size-exclusion chromatography (2.2.30).

01/2009:1250

OMEGA-3-ACID ETHYL ESTERS 90

Omega-3 acidorum esteri ethylici 90
DEFINITION
Ethyl esters of alpha-linolenic acid (C18:3 n-3), moroctic
acid (C18:4 n-3), eicosatetraenoic acid (C20:4 n-3),
timnodonic (eicosapentaenoic) acid (C20:5 n-3 ; EPA),
heneicosapentaenoic acid (C21:5 n-3), clupanodonic
acid (C22:5 n-3) and cervonic (docosahexaenoic) acid
(C22:6 n-3 ; DHA). Omega-3-acid ethyl esters are obtained
by transesterification of the body oil obtained from fish
of families such as Engraulidae, Carangidae, Clupeidae,
Osmeridae, Salmonidae and Scombridae and subsequent
physico-chemical purification processes, including urea
fractionation followed by molecular distillation.
Content :
1. oligomers 2. ethyl esters
— EPA and DHA ethyl esters : minimum 80 per cent, with
minimum 40 per cent of EPA ethyl esters and minimum Figure 1250.-1. – Chromatogram for the test for oligomers :
34 per cent of DHA ethyl esters ; spiked sample
— total omega-3-acid ethyl esters: minimum 90 per cent. Test solution. Dilute 50.0 mg of the substance to be
Tocopherol may be added as an antioxidant. examined to 10.0 ml with tetrahydrofuran R.

4244 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Reference solution. Dissolve 50 mg of monodocosa-
hexaenoin R, 30 mg of didocosahexaenoin R and 20 mg of
tridocosahexaenoin R in tetrahydrofuran R and dilute to
100.0 ml with the same solvent.
Column 1 :
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
(7 μm) with a pore size of 10 nm.
Columns 2 and 3 placed closest to the injector :
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
(7 μm) with a pore size of 50 nm.
Mobile phase : tetrahydrofuran R.
Flow rate : 0.8 ml/min.
Detection : differential refractometer.
Injection : 40 μl.
System suitability : reference solution :
— elution order: tridocosahexaenoin, didocosahexaenoin,
monodocosahexaenoin ;
— resolution : minimum 2.0 between the peaks due to
monodocosahexaenoin and didocosahexaenoin and
minimum 1.0 between the peaks due to didocosahexaenoin
and tridocosahexaenoin.
Calculate the percentage content of oligomers using the
following expression :
1. phytanic acid 7. C18:4 n-3
2. C16:3 n-4 8. C18:4 n-1
3. C16:4 n-1 9. furan acid 5
4. C18:3 n-6 10. C19:5
5. C18:3 n-4 11. C20:3 n-6
6. C18:3 n-3 12. C20:4 n-6
Figure 1250.-2. – Chromatogram for the assays
General Notices (1) apply to all monographs and other texts
Omega-3-acid ethyl esters 90
A = sum of the areas of all the peaks in the
chromatogram ;
B = sum of the areas of the peaks with a retention
time less than the retention time of the peaks due
to ethyl esters.
The ethyl ester peaks, which may be present in the form of
an unresolved double peak, are identified as the major peaks
in the chromatogram (see Figure 1250.-1).
Where the result obtained exceeds the limit due to the
presence of monoglycerides, the following procedure is
carried out.
Test solution. Weigh 10.0 mg of the substance to be
examined into a quartz tube. Add 1.5 ml of a 20 g/l
solution of sodium hydroxide R in methanol R, cover with
nitrogen R, cap tightly with a polytetrafluoroethylene-lined
cap, mix and heat on a water-bath for 7 min. Allow to cool.
Add 2 ml of boron trichloride-methanol solution R, cover
with nitrogen R, cap tightly, mix and heat on a water-bath for
30 min. Cool to 40-50 °C, add 1 ml of trimethylpentane R,
cap and shake vigorously for at least 30 s. Immediately
add 5 ml of saturated sodium chloride solution R, cover
with nitrogen R, cap and shake thoroughly for at least 15 s.
Transfer the upper layer to a separate tube. Shake the
13. furan acid 7 19. C22:4
14. C20:4 n-3 20. furan acid 10
15. furan acid 8 21. C22:5 n-6
16. EPA 22. furan acid 11
17. furan acid 9 23. C22:5 n-3
18. C21:5 n-3 24. DHA
4245
Omega-3-acid triglycerides EUROPEAN PHARMACOPOEIA 6.3

methanol layer once more with 1 ml of trimethylpentane R. acids with glycerol or by transesterification of the omega-3
Carefully evaporate the solvent under a current of nitrogen R acid ethyl esters with glycerol. The origin of the omega-3
then add 10.0 ml of tetrahydrofuran R to the residue. Add a acids is the body oil obtained from fish of families such
small amount of anhydrous sodium sulphate R and filter. as Engraulidae, Carangidae, Clupeidae, Osmeridae,
Calculate the percentage content of oligomers using the Salmonidae and Scombridae. The omega-3 acids are
following expression : identified as the following acids : alpha-linolenic acid (C18:3
n-3), moroctic acid (C18:4 n-3), eicosatetraenoic acid (C20:4
n-3), timnodonic (eicosapentaenoic) acid (C20:5 n-3 ; EPA),
heneicosapentaenoic acid (C21:5 n-3), clupanodonic acid
(C22:5 n-3) and cervonic (docosahexaenoic) acid (C22:6 n-3 ;
B′ = sum of the areas of the peaks with a retention DHA).
time less than the retention time of the peaks due Content :
to methyl esters.
— sum of the contents of the omega-3 acids EPA and DHA,
Limit : expressed as triglycerides : minimum 45 per cent ;
— oligomers : maximum 1.0 per cent.
— total omega-3 acids, expressed as triglycerides : minimum
ASSAY 60 per cent.
EPA and DHA ethyl esters (2.4.29). For identification of Authorised antioxidants in concentrations not exceeding the
the peaks, see Figure 1250.-2. levels specified by the competent authority may be added.
Total omega-3-acid ethyl esters (2.4.29). See Figure 1250.-2.
CHARACTERS
STORAGE Appearance : pale yellow liquid.
Under an inert gas, in an airtight container, protected from
light. Solubility : practically insoluble in water, very soluble
in acetone and in heptane, slightly soluble in anhydrous
01/2009:1352 ethanol.
IDENTIFICATION
OMEGA-3-ACID TRIGLYCERIDES
Examine the chromatograms obtained in the assay for EPA
and DHA.
Omega-3 acidorum triglycerida
Results : the peaks due to eicosapentaenoic acid methyl ester
DEFINITION and docosahexaenoic acid methyl ester in the chromatogram
Mixture of mono-, di- and triesters of omega-3 acids with obtained with test solution (b) are similar in retention time
glycerol containing mainly triesters and obtained either to the corresponding peaks in the chromatogram obtained
by esterification of concentrated and purified omega-3 with reference solution (a).

1. oligomers 2. triglycerides 3. diglycerides 4. monoglycerides

Figure 1352.-1. – Chromatogram for the test for oligomers and partial glycerides

4246 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Omega-3-acid triglycerides

1. C14:0 5. C18:0 9. C18:3 n-3 13. C20:1 n-9 17. C20:4 n-3 21. C22:1 n-9 25. DHA
2. C16:0 6. C18:1 n-9 10. C18:4 n-3 14. C20:1 n-7 18. EPA 22. C21:5 n-3 26. C24:1 n-9
3. C16:1 n-7 7. C18:1 n-7 11. C20:0 15. C20:2 n-6 19. C22:0 23. C22:5 n-6
4. C16:4 n-1 8. C18:2 n-6 12. C20:1 n-11 16. C20:4 n-6 20. C22:1 n-11 24. C22:5 n-3

Figure 1352.-2. – Chromatogram for the assays

TESTS — stationary phase : styrene-divinylbenzene copolymer R

Absorbance (2.2.25) : maximum 0.73 at 233 nm.
Dilute 0.300 g to 50.0 ml with trimethylpentane R. Dilute
2.0 ml of this solution to 50.0 ml with trimethylpentane R.
Acid value (2.5.1) : maximum 3.0, determined on 10.0 g in
50 ml of the prescribed mixture of solvents.
Anisidine value (2.5.36) : maximum 30.0.
Peroxide value (2.5.5, Method A) : maximum 10.0.
Oligomers and partial glycerides. Size-exclusion
chromatography (2.2.30).
Test solution. Dilute 50.0 mg of the substance to be
examined to 10.0 ml with tetrahydrofuran R.
Reference solution. Dissolve 50 mg of monodocosa-
hexaenoin R, 30 mg of didocosahexaenoin R and 20 mg of
tridocosahexaenoin R in tetrahydrofuran R and dilute to
100.0 ml with the same solvent.
Column 1 :
— dimensions : l = 0.3 m, Ø = 7.8 mm ;
(7 μm) with a pore size of 10 nm.
Columns 2 and 3 placed closest to the injector :
— dimensions: l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : styrene-divinylbenzene copolymer R
(7 μm) with a pore size of 50 nm.
Mobile phase : tetrahydrofuran R.
Flow rate : 0.8 ml/min.
Detection : differential refractometer.
Injection : 40 μl.
System suitability : reference solution :
— elution order : tridocosahexaenoin, didocosahexaenoin,
monodocosahexaenoin ;
— resolution : minimum 2.0 between the peaks due to
monodocosahexaenoin and didocosahexaenoin ; minimum
1.0 between the peaks due to didocosahexaenoin and
tridocosahexaenoin.

General Notices (1) apply to all monographs and other texts 4247
Omeprazole magnesium
Identify the peaks using the chromatogram shown in
figure 1352.-1. Calculate the percentage content of oligomers
using the following expression :
A = sum of the areas of all the peaks in the
chromatogram ;
B = area of the peak with a retention time less than the
retention time of the peak due to the triglyceride.
Calculate the percentage content of partial glycerides using
the following expression :
C = (sum of the) area(s) of the peak(s) due to the
mono- and diglycerides.
Limits :
— oligomers : maximum 3.0 per cent ;
— partial glycerides : maximum 50.0 per cent.
ASSAY
EPA and DHA (2.4.29). For identification of the peaks, see and 1 mg of omeprazole impurity D CRS in the mobile
Figure 1352.-2.
Total omega-3-acids (2.4.29). See Figure 1352.-2.
STORAGE
In an airtight, well-filled container, protected from light,
under an inert gas.
01/2009:2374
OMEPRAZOLE MAGNESIUM
Omeprazolum magnesicum
C34H36MgN6O6S2 Mr 713 (retention time = about 9 min) : impurity E = about 0.6,
[95382-33-5]
DEFINITION
Magnesium bis[5-methoxy-2-[(RS)-[(4-methoxy-3,5-
dimethylpyridin-2-yl)methyl]sulphinyl]-1H-benzimidazol-1-
ide]. It contains a variable quantity of water.
Content : 97.5 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, hygroscopic powder.
Solubility : very slightly soluble in water, sparingly soluble
in methanol, practically insoluble in heptane.
IDENTIFICATION
Carry out either tests A, B, C or tests A, B, D.
A. Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 0.250 g in methanol R and dilute to 25.0 ml
with the same solvent.
4248 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : omeprazole magnesium CRS.
C. Atomic absorption spectrometry (2.2.23) as described in
the test for magnesium.
The test solution shows the absorption maximum at
285.2 nm.
D. Ignite about 0.5 g of the substance to be examined
according to the procedure for the sulphated ash test
(2.4.14). Dissolve the residue in 10 ml of water R. 2 ml of
this solution gives the reaction of magnesium (2.3.1).
TESTS
Absorbance (2.2.25) : maximum 0.10 at 440 nm.
Dissolve 0.500 g in methanol R and dilute to 25.0 ml with
the same solvent. Filter the solution through a membrane
filter (nominal pore size 0.45 μm).
Related substances. Liquid chromatography (2.2.29) :
use the normalisation procedure. Prepare the solutions
immediately before use.
Test solution. Dissolve 3.5 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml with the
mobile phase.
Reference solution (a). Dissolve 1 mg of omeprazole CRS
phase and dilute to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 3 mg of omeprazole for
peak identification CRS (containing impurity E) in the
mobile phase and dilute to 20.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Column :
— size: l = 0.125 m, Ø = 4.6 mm ;
— stationary phase : octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 27 volumes of acetonitrile R and
73 volumes of a 1.4 g/l solution of disodium hydrogen
phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 280 nm.
Injection : 40 μl.
Run time : 5 times the retention time of omeprazole.
Relative retention with reference to omeprazole
impurity D = about 0.8.
System suitability : reference solution (a) :
— resolution : minimum 3.0 between the peaks due to
impurity D and omeprazole ; if necessary, adjust the pH
of the aqueous part of the mobile phase or its proportion
of acetonitrile ; an increase in the pH will improve the
resolution.
Limits :
— impurities D, E : for each impurity, maximum 0.1 per cent ;
— unspecified impurities : for each impurity, maximum
0.10 per cent ;
— total : maximum 0.5 per cent ;
— disregard limit : half the area of the principal peak in
the chromatogram obtained with reference solution (c)
(0.05 per cent).
Magnesium : 3.30 per cent to 3.55 per cent (anhydrous
substance).
Atomic absorption spectrometry (2.2.23, Method I).
EUROPEAN PHARMACOPOEIA 6.3 Oxaliplatin

Test solution. Dissolve 0.250 g in 20.0 ml of a 103 g/l

solution of hydrochloric acid R by slow addition of the acid
and dilute to 100.0 ml with water R. Dilute 10.0 ml of the
solution to 200.0 ml with water R. To 10.0 ml of this solution
add 4 ml of lanthanum chloride solution R and dilute to
100.0 ml with water R.
Reference solutions. Prepare the reference solutions using
magnesium standard solution (1000 ppm Mg) R, diluting B. R = H, X = SO : 2-[(RS)-[(3,5-dimethylpyridin-2-
with a mixture of 1 ml of a 103 g/l solution of hydrochloric yl)methyl]sulphinyl]-5-methoxy-1H-benzimidazole,
acid R and 1000.0 ml of water R. C. R = OCH3, X = S : 5-methoxy-2-[[(4-methoxy-3,5-
Wavelength : 285.2 nm. dimethylpyridin-2-yl)methyl]sulphanyl]-1H-benzimidazole,
Water (2.5.12) : 7.0 per cent to 10.0 per cent, determined D. R = OCH3, X = SO2 : 5-methoxy-2-[[(4-methoxy-3,5-
on 0.200 g. dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole,
ASSAY
Liquid chromagraphy (2.2.29).
Buffer pH 11.0. Mix 11 ml of a 95.0 g/l solution of trisodium
phosphate dodecahydrate R and 22 ml of a 179.1 g/l
solution of disodium hydrogen phosphate R. Dilute to
100.0 ml with water R.
Test solution. Dissolve 10.0 mg of the substance to be E. 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2-
examined in about 10 ml of methanol R. Add 10 ml of buffer yl)sulphinyl]methyl]-3,5-dimethylpyridine 1-oxide.
pH 11.0 and dilute to 200.0 ml with water R.
Reference solution. Dissolve 10.0 mg of omeprazole CRS in
about 10 ml of methanol R. Add 10 ml of buffer pH 11.0 and
01/2009:2017
dilute to 200.0 ml with water R.
Column:
— size : l = 0.125 m, Ø = 4 mm;
OXALIPLATIN
— stationary phase : octylsilyl silica gel for
chromatography R (5 μm).
Oxaliplatinum
Mobile phase : mix 35 volumes of acetonitrile R and
65 volumes of a 1.4 g/l solution of disodium hydrogen
phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 280 nm. C8H14N2O4Pt Mr 397.3
Injection : 20 μl. [61825-94-3]
Run time : 1.5 times the retention time of omeprazole.
DEFINITION
Retention time : omeprazole = about 4 min.
(SP-4-2)-[(1R,2R)-Cyclohexane-1,2-diamine-κN,κN′]
Calculate the percentage content of C34H36MgN6O6S2 from [ethanedioato(2-)-κO1,κO2]platinum.
the declared content of omeprazole CRS.
Content : 98.0 per cent to 102.0 per cent (dried substance).
1 g of omeprazole is equivalent to 1.032 g of omeprazole
magnesium. CHARACTERS
Appearance : white or almost white, crystalline powder.
STORAGE
Solubility : slightly soluble in water, very slightly soluble in
In an airtight container, protected from light. methanol, practically insoluble in anhydrous ethanol.
IMPURITIES IDENTIFICATION
Specified impurities : D, E. A. Infrared absorption spectrophotometry (2.2.24).
Other detectable impurities (the following substances Comparison : oxaliplatin CRS.
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited B. It complies with the test for specific optical rotation (see
by the general acceptance criterion for other/unspecified Tests).
impurities and/or by the general monograph Substances for TESTS
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance. Appearance of solution. The solution is clear (2.2.1) and
See also 5.10. Control of impurities in substances for colourless (2.2.2, Method II).
pharmaceutical use) : A, B, C. Dissolve 0.10 g in water R and dilute to 50 ml with the same
solvent.
Acidity. Dissolve 0.10 g in carbon dioxide-free water R,
dilute to 50 ml with the same solvent and add 0.5 ml of
phenolphthalein solution R1. The solution is colourless.
Not more than 0.60 ml of 0.01 M sodium hydroxide is
A. 5-methoxy-1H-benzimidazole-2-thiol, required to change the colour of the indicator to pink.

General Notices (1) apply to all monographs and other texts 4249
Oxaliplatin
Specific optical rotation (2.2.7) : + 74.5 to + 78.0 (dried
substance).
Dissolve 0.250 g in water R and dilute to 50.0 ml with the
same solvent.
Related substances
A. Impurity A. Liquid chromatography (2.2.29). Use
vigorous shaking and very brief sonication to dissolve
the substance to be examined. Inject the test solution
within 20 min of preparation.
Test solution. Dissolve 0.100 g of the substance to be
examined in water R and dilute to 50.0 ml with the same
solvent.
Reference solution (a). Dissolve 14.0 mg of oxalic acid R
(impurity A) in water R and dilute to 250.0 ml with the
same solvent.
Reference solution (b). Dilute 5.0 ml of reference
solution (a) to 200.0 ml with water R.
Reference solution (c). Dissolve 12.5 mg of sodium
nitrate R in water R and dilute to 250.0 ml with the same
solvent. Dilute a mixture of 2.0 ml of this solution and
25.0 ml of reference solution (a) to 100.0 ml with water R.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : base-deactivated octadecylsilyl
silica gel for chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase : mix 20 volumes of acetonitrile R with
80 volumes of a solution prepared as follows : to 10 ml of
a 320 g/l solution of tetrabutylammonium hydroxide R
add 1.36 g of potassium dihydrogen phosphate R, dilute
to 1000 ml with water R and adjust to pH 6.0 with
phosphoric acid R.
Flow rate : 2 ml/min.
Detection : spectrophotometer at 205 nm.
Injection : 20 μl of the test solution and reference
solutions (b) and (c).
Run time : twice the retention time of impurity A.
Retention times : nitrate = about 2.7 min ;
impurity A = about 4.7 min.
System suitability :
— resolution : minimum 9 between the peaks due to
nitrate and impurity A in the chromatogram obtained
with reference solution (c) ;
— signal-to-noise ratio: minimum 10 for the peak due
to impurity A in the chromatogram obtained with
reference solution (b).
Limit :
— impurity A : not more than twice the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent).
B. Impurity B. Liquid chromatography (2.2.29). Use
vigorous shaking and very brief sonication to
dissolve the substance to be examined. Inject the test
solution within 20 min of preparation. Use suitable
polypropylene containers for the preparation and
injection of all solutions. Glass pipettes may be used
for diluting solutions.
Test solution. Dissolve 0.100 g of the substance to be
examined in water R and dilute to 50.0 ml with the same
solvent.
4250 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Reference solution (a). Add 12.5 mg of oxaliplatin
impurity B CRS to 63 ml of methanol R and dilute to
250.0 ml with water R. Sonicate for about 1.5 h until
dissolved. Dilute 3.0 ml of this solution to 200.0 ml with
water R.
Reference solution (b). In order to prepare impurity E
in situ, add 12.5 mg of oxaliplatin impurity B CRS to
63 ml of methanol R and dilute to 250 ml with water R.
Sonicate for about 1.5 h until dissolved. Adjust to pH 6.0
with a 0.2 g/l solution of sodium hydroxide R. Heat at
70 °C for 4 h and allow to cool.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : base-deactivated octadecylsilyl
silica gel for chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase : mix 20 volumes of acetonitrile R with
80 volumes of a solution prepared as follows : dissolve
1.36 g of potassium dihydrogen phosphate R and 1 g of
sodium heptanesulphonate R in 1000 ml of water R and
adjust to pH 3.0 ± 0.05 with phosphoric acid R.
Flow rate : 2.0 ml/min.
Detection : spectrophotometer at 215 nm.
Injection : 20 μl.
Run time : 2.5 times the retention time of impurity B.
Retention time : impurity B = about 4.3 min ;
impurity E = about 6.4 min.
System suitability :
— resolution : minimum 7 between the peaks due to
impurities B and E in the chromatogram obtained with
reference solution (b) ;
— signal-to-noise ratio : minimum 10 for the peak due
to impurity B in the chromatogram obtained with
reference solution (a).
Limit :
— impurity B : not more than 3.3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent).
C. Impurity C and other related substances. Liquid
chromatography (2.2.29). Use vigorous shaking and
very brief sonication to dissolve the substance to be
examined. Inject the test solution within 20 min of
preparation.
Test solution (a). Dissolve 0.100 g of the substance to
be examined in water R and dilute to 50.0 ml with the
same solvent.
Test solution (b). Dissolve 50.0 mg of the substance to
be examined in water R and dilute to 500.0 ml with the
same solvent.
Reference solution (a). Dissolve 10 mg of oxaliplatin
impurity C CRS and 10 mg of oxaliplatin CRS in water R
and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of reference
solution (a) to 100.0 ml with water R.
Reference solution (c). Dissolve 5 mg of
dichlorodiaminocyclohexaneplatinum CRS in
methanol R and dilute to 50.0 ml with the same solvent.
To 10.0 ml of this solution add 10.0 ml of reference
solution (a) and dilute to 100.0 ml with water R.
Reference solution (d). Dissolve 50.0 mg of
oxaliplatin CRS in water R and dilute to 500.0 ml with
the same solvent.
EUROPEAN PHARMACOPOEIA 6.3
Reference solution (e). Dissolve 5.0 mg of
dichlorodiaminocyclohexaneplatinum CRS in reference
solution (d) and dilute to 50.0 ml with reference
solution (d).
Reference solution (f). To 0.100 g of the substance to be
examined add 1.0 ml of reference solution (a) and dilute
to 50.0 ml with water R.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 40 °C.
Mobile phase : mix 1 volume of acetonitrile R with
99 volumes of a solution prepared as follows : dilute
0.6 ml of dilute phosphoric acid R in 1000 ml of water R
and adjust to pH 3.0 with either sodium hydroxide
solution R or phosphoric acid R.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μl of test solution (a) and reference
solutions (b), (c) and (f).
Run time : 3 times the retention time of oxaliplatin.
Retention time : impurity C = about 4.4 min ;
dichlorodiaminocyclohexaneplatinum = about 6.9 min ;
oxaliplatin = about 8.0 min.
System suitability:
— resolution : minimum 2.0 between the peaks due to
dichlorodiaminocyclohexaneplatinum and oxaliplatin
in the chromatogram obtained with reference
solution (c) ;
— signal-to-noise ratio: minimum 50 for the peak due
to impurity C and minimum 10 for the peak due
to oxaliplatin in the chromatogram obtained with
reference solution (b).
Limits :
— impurity C : not more than 0.5 times the area of the
peak due to impurity C in the chromatogram obtained
with reference solution (f) (0.1 per cent) ;
— any other impurity : not more than twice the area
of the peak due to oxaliplatin in the chromatogram
obtained with reference solution (b) (0.1 per cent) ;
— total of other impurities : not more than twice the area
of the peak due to oxaliplatin in the chromatogram
obtained with reference solution (b) (0.1 per cent) ;
— disregard limit : the area of the peak due to oxaliplatin
in the chromatogram obtained with reference
solution (b) (0.05 per cent) ; disregard any peak with a
retention time less than 2 min.
D. Total of impurities : the sum of impurities A, B, C and
other related impurities is not greater than 0.30 per cent.
Impurity D. Liquid chromatography (2.2.29).
Test solution. Dissolve 30 mg of the substance to be
examined in methanol R and dilute to 50.0 ml with the same
solvent.
Reference solution (a). Dissolve 5 mg of oxaliplatin
impurity D CRS in methanol R and dilute to 100.0 ml with
the same solvent.
Reference solution (b). Dilute 15.0 ml of reference
solution (a) to 50.0 ml with methanol R.
Reference solution (c). Dissolve 150.0 mg of oxaliplatin CRS
in methanol R and dilute to 200.0 ml with the same solvent.
Reference solution (d). Dilute 5.0 ml of reference solution (c)
to 100.0 ml with methanol R.
General Notices (1) apply to all monographs and other texts
Oxaliplatin
Reference solution (e). To 40 ml of reference solution (c)
add 1.0 ml of reference solution (b) and dilute to 50.0 ml
with methanol R.
Reference solution (f). To 4.0 ml of reference solution (a)
add 5.0 ml of reference solution (d) and dilute to 50.0 ml
with methanol R.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : silica gel OC for chiral separations R ;
— temperature : 40 °C.
Mobile phase : anhydrous ethanol R, methanol R
(30:70 V/V).
Flow rate : 0.3 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl of the test solution and reference
solutions (e) and (f).
Run time : twice the retention time of oxaliplatin.
Retention time : oxaliplatin = about 14 min ;
impurity D = about 16 min.
System suitability :
— resolution : minimum 1.5 between the peaks due to
oxaliplatin and impurity D in the chromatogram obtained
with reference solution (f) ;
— signal-to-noise ratio : minimum 10 for the peak due to
impurity D in the chromatogram obtained with reference
solution (e).
Limit :
— impurity D : not more than twice the peak height of the
corresponding peak in the chromatogram obtained with
reference solution (e) (0.1 per cent).
Silver : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 0.1000 g of the substance to be
examined in water R and dilute to 50.0 ml with the same
solvent. Dilute 20 μl of this solution to 40 μl with 0.5 M
nitric acid.
Reference solution (a). Dilute a solution of silver nitrate R
containing 1000 ppm of silver in 0.5 M nitric acid with 0.5 M
nitric acid to obtain a solution which contains 10 ppb of
silver.
Reference solution (b). Mix 20 μl of the test solution and
8 μl of reference solution (a) and dilute to 40 μl with 0.5 M
nitric acid.
Reference solution (c). Mix 20 μl of the test solution and
16 μl of reference solution (a) and dilute to 40 μl with 0.5 M
nitric acid.
Source : silver hollow-cathode lamp.
Wavelength : 328.1 nm.
Atomisation device : furnace.
Measure the absorbance of the test solution and reference
solutions (b) and (c).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
Bacterial endotoxins (2.6.14) : less than 1.0 IU/mg,
if intended for use in the manufacture of parenteral
preparations without a further appropriate procedure for the
removal of bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
impurity C and other related substances with the following
modifications.
4251
Oxymetazoline hydrochloride
Injection : 20 μl of test solution (b) and reference solutions (d)
and (e).
System suitability :
— resolution : minimum 2.0 between the peaks due to
dichlorodiaminocyclohexaneplatinum and oxaliplatin in
the chromatogram obtained with reference solution (e),
— repeatability : reference solution (d).
Calculate the percentage content of oxaliplatin using the
chromatogram obtained with reference solution (d).
IMPURITIES
Specified impurities : A, B, C, D.
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) : E.
A. ethanedioic acid (oxalic acid),
B. (SP-4-2)-diaqua[(1R,2R)-cyclohexane-1,2-diamine-
κN,κN′]platinum (diaquodiaminocyclohexaneplatinum),
C. (OC-6-33)-[(1R,2R)-cyclohexane-1,2-diamine-
κN,κN′][ethanedioato(2-)-κO1,κO2]dihydroxyplatinum,
D. (SP-4-2)-[(1S,2S)-cyclohexane-1,2-diamine-
κN,κN′][ethanedioato(2-)-κO1,κO2]platinum
(S,S-enantiomer of oxaliplatin),
E. (SP-4-2)-di-μ-oxobis[(1R,2R)-cyclohexane-1,2-diamine-
κN,κN′]diplatinum (diaquodiaminocyclohexaneplatinum
dimer).
4252
EUROPEAN PHARMACOPOEIA 6.3
01/2009:0943
OXYMETAZOLINE HYDROCHLORIDE
Oxymetazolini hydrochloridum
C16H25ClN2O Mr 296.8
[2315-02-8]
DEFINITION
3-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-6-(1,1-
dimethylethyl)-2,4-dimethylphenol hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water and in ethanol (96 per
cent).
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : oxymetazoline hydrochloride CRS.
B. Thin layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in a mixture of equal volumes of ethyl acetate R
and methanol R and dilute to 5 ml with the same mixture
of solvents.
Reference solution. Dissolve 20 mg of oxymetazoline
hydrochloride CRS in a mixture of equal volumes of ethyl
acetate R and methanol R and dilute to 5 ml with the
same mixture of solvents.
Plate : TLC silica gel G plate R.
Mobile phase : diethylamine R, cyclohexane R,
anhydrous ethanol R (6:15:79 V/V/V).
Application : 5 μl.
Development : over 2/3 of the plate.
Drying : in a current of warm air for 5 min, then allow
to cool.
Detection : spray with a freshly prepared 5.0 g/l
solution of potassium ferricyanide R in ferric chloride
solution R2; examine in daylight.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
C. Dissolve about 2 mg in 1 ml of water R, then add 0.2 ml
of a 50 g/l solution of sodium nitroprusside R and 0.2 ml
of dilute sodium hydroxide solution R. Allow to stand
for 10 min. Add 2 ml of sodium hydrogen carbonate
solution R. A violet colour develops.
D. It gives reaction (a) of chlorides (2.3.1).
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 Oxymetazoline hydrochloride

TESTS — unspecified impurities : for each impurity, not more

Appearance of solution. The solution is clear (2.2.1) and than the area of the principal peak in the chromatogram
not more intensely coloured than reference solution BY7 obtained with reference solution (a) (0.10 per cent) ;
(2.2.2, Method II). — total : not more than 5 times the area of the principal peak
Dissolve 2.5 g in water R and dilute to 50 ml with the same in the chromatogram obtained with reference solution (a)
solvent. (0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
Acidity or alkalinity. Dissolve 0.25 g in carbon dioxide-free in the chromatogram obtained with reference solution (a)
water R and dilute to 25 ml with the same solvent. Add (0.05 per cent).
0.1 ml of methyl red solution R and 0.2 ml of 0.01 M
hydrochloric acid. The solution is red. Not more than 0.4 ml Loss on drying (2.2.32) : maximum 1.0 per cent, determined
of 0.01 M sodium hydroxide is required to change the colour on 1.000 g by drying in an oven at 105 °C.
of the indicator to yellow. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Related substances. Liquid chromatography (2.2.29). on 1.0 g.
Prepare the solutions immediately before use.
ASSAY
Test solution. Dissolve 50.0 mg of the substance to be Dissolve 0.200 g in a mixture of 20 ml of anhydrous
examined in water R and dilute to 50.0 ml with the same acetic acid R and 20 ml of acetic anhydride R. Titrate
solvent. with 0.1 M perchloric acid, determining the end-point
Reference solution (a). Dilute 5.0 ml of the test solution potentiometrically (2.2.20).
to 100.0 ml with water R. Dilute 2.0 ml of this solution to 1 ml of 0.1 M perchloric acid is equivalent to 29.68 mg of
100.0 ml with water R. C16H25ClN2O.
Reference solution (b). Dissolve 5.0 mg of oxymetazoline
impurity A CRS and 5 mg of the substance to be examined IMPURITIES
in water R and dilute to 50.0 ml with the same solvent. Specified impurities : A.
Dilute 10.0 ml of this solution to 50.0 ml with water R. Other detectable impurities (the following substances
Reference solution (c). Dilute 1.0 ml of reference solution (b) would, if present at a sufficient level, be detected by one
to 20.0 ml with water R. or other of the tests in the monograph. They are limited
Column: by the general acceptance criterion for other/unspecified
— size : l = 0.25 m, Ø = 4.6 mm ; impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
— stationary phase : end-capped octadecylsilyl silica gel identify these impurities for demonstration of compliance.
for chromatography with polar incorporated groups R See also 5.10. Control of impurities in substances for
(5 μm). pharmaceutical use) : B, C, D, E.
Mobile phase :
— mobile phase A : 1.36 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 3.0 with
phosphoric acid R ;
— mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 70 30 A. N-(2-aminoethyl)-2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-
dimethylphenyl]acetamide,
5 - 20 70 → 15 30 → 85
20 - 35 15 85
B. xylometazoline,

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 220 nm.
Injection : 10 μl.
Relative retention with reference to oxymetazoline (retention
time = about 5.0 min) : impurity A = about 0.9.
System suitability : reference solution (b) :
C. R=CO-NH2 : 2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-
— resolution : minimum 4.0 between the peaks due to dimethylphenyl]acetamide,
impurity A and oxymetazoline.
Limits : D. R=CO2H : [4-(1,1-dimethylethyl)-3-hydroxy-2,6-
dimethylphenyl]acetic acid,
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference E. R=CN : [4-(1,1-dimethylethyl)-3-hydroxy-2,6-
solution (c) (0.1 per cent) ; dimethylphenyl]acetonitrile.

General Notices (1) apply to all monographs and other texts 4253
 EUROPEAN PHARMACOPOEIA 6.3

4254 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
P
Paclitaxel....................................................................................4257
Pancreas powder.. ....................................................................4260
Pea starch.. ................................................................................4263
Pepsin powder...........................................................................4263
Perphenazine.. ..........................................................................4265
Phenol.. ......................................................................................4266
Pholcodine.................................................................................4266
Pilocarpine hydrochloride......................................................4268
Pilocarpine nitrate.. .................................................................4269
Polyacrylate dispersion 30 per cent.....................................4270
General Notices (1) apply to all monographs and other texts
Polysorbate 20.. ........................................................................4271
Polysorbate 40.. ........................................................................4272
Polysorbate 60.. ........................................................................4273
Polysorbate 80.. ........................................................................ 4274
Poly(vinyl acetate) dispersion 30 per cent.. .......................4275
Potassium citrate.. ................................................................... 4276
Potassium dihydrogen phosphate.. ......................................4277
Potato starch.............................................................................4277
Pravastatin sodium.. ................................................................4278
4255
 EUROPEAN PHARMACOPOEIA 6.3

4256 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:1794
PACLITAXEL
Paclitaxelum
C47H51NO14 Mr 854
[33069-62-4]
DEFINITION
5β,20-Epoxy-1,7β-dihydroxy-9-oxotax-11-ene-2α,4,10β,-
13α-tetrayl 4,10-diacetate 2-benzoate 13-[(2R,3S)-3-
(benzoylamino)-2-hydroxy-3-phenylpropanoate].
It is isolated from natural sources or produced by
fermentation or by a semi-synthetic process.
Content : 97.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in methanol
and freely soluble in methylene chloride.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : paclitaxel CRS.
If the spectra obtained in the solid state show differences,
dissolve 10 mg of the substance to be examined and the
reference substance separately in 0.4 ml of methylene
chloride R, evaporate to dryness and record new spectra
using the residues.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.1 g in 10 ml of methanol R.
Specific optical rotation (2.2.7) : − 49.0 to − 55.0 (anhydrous
substance).
Dissolve 0.250 g in methanol R and dilute to 25.0 ml with
the same solvent.
Related substances. Liquid chromatography (2.2.29).
A. Paclitaxel isolated from natural sources or produced by
fermentation.
General Notices (1) apply to all monographs and other texts
Paclitaxel
Test solution (a). Dissolve 20.0 mg of the substance to be
examined in acetonitrile R1 and dilute to 10.0 ml with
the same solvent.
Test solution (b). Dilute 1.0 ml of test solution (a) to
20.0 ml with acetonitrile R1.
Reference solution (a). Dilute 1.0 ml of test solution (a)
to 10.0 ml with acetonitrile R1. Dilute 1.0 ml of this
solution to 100.0 ml with acetonitrile R1.
Reference solution (b). Dissolve 5.0 mg of paclitaxel CRS
in acetonitrile R1 and dilute to 5.0 ml with the same
solvent. Dilute 2.0 ml of this solution to 20.0 ml with
acetonitrile R1.
Reference solution (c). Dissolve 2.0 mg of paclitaxel
impurity C CRS in acetonitrile R1 and dilute to 20.0 ml
with the same solvent.
Reference solution (d). Dilute 1.0 ml of reference
solution (c) to 50.0 ml with acetonitrile R1.
Reference solution (e). To 1 ml of reference solution (b)
add 1 ml of reference solution (c).
Reference solution (f). Dissolve 5 mg of paclitaxel natural
for peak identification CRS (containing impurities A, B,
C, D, E, F, H, O, P, Q and R) in acetonitrile R1 and dilute
to 5 ml with the same solvent.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : diisopropylcyanopropylsilyl silica
gel for chromatography R (5 μm) with a specific
surface area of 180 m2/g and a pore size of 8 nm ;
— temperature : 20 ± 1 °C.
Mobile phase :
— mobile phase A : methanol R, water R (200:800 V/V) ;
— mobile phase B : methanol R, acetonitrile for
chromatography R (200:800 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 60 85 → 56 15 → 44
60 - 61 56 → 85 44 → 15
61 - 75 85 15
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 227 nm.
Injection : 10 μl of test solution (a) and reference
solutions (a), (d), (e) and (f).
Identification of impurities: use the chromatogram
supplied with paclitaxel natural for peak
identification CRS and the chromatogram obtained
with reference solution (f) to identify the peaks due to
impurities A, B, C, D, E, F, H, O, P, Q and R.
Relative retention with reference to paclitaxel (retention
time = about 50 min) : impurities A and B = about 0.90 ;
impurity R = about 0.93 ; impurity H = about 0.96 ;
impurities Q and P = about 1.02 ; impurity C = about 1.05 ;
impurity D = about 1.07 ; impurities O and E = about 1.15 ;
impurity F = about 1.20.
System suitability : reference solution (e) :
— resolution : minimum 3.5 between the peaks due to
paclitaxel and impurity C.
4257
Paclitaxel
Limits :
— sum of impurities E and O : not more than 5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ;
— impurity R : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
— sum of impurities A and B : not more than 4 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.4 per cent) ;
— impurity C : not more than 3 times the area of the
corresponding peak in the chromatogram obtained
with reference solution (d) (0.3 per cent) ;
— impurity D : not more than twice the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
— sum of impurities P and Q : not more than twice
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
— impurity F : not more than the area of the principal
peak in the chromatogram obtained with reference
solution (d) (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 : not more than 15 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.5 per cent) ;
— disregard limit : 0.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.05 per cent).
B. Paclitaxel produced by a semi-synthetic process.
Test solution. Dissolve 10.0 mg of the substance to be
examined in acetonitrile R1 and dilute to 10.0 ml with
the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution
to 10.0 ml with acetonitrile R1. Dilute 1.0 ml of this
solution to 100.0 ml with acetonitrile R1.
Reference solution (b). Dissolve 5.0 mg of paclitaxel CRS
in acetonitrile R1 and dilute to 5.0 ml with the same
solvent.
Reference solution (c). Dissolve 5 mg of paclitaxel
semi-synthetic for peak identification CRS (containing
impurities A, G, I and L) in acetonitrile R1 and dilute to
5 ml with the same solvent.
Reference solution (d). Dissolve the contents of a vial
of paclitaxel semi-synthetic for system suitability CRS
(containing impurities E, H and N) in 1 ml of
acetonitrile R1.
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (3 μm) with a specific surface
area of 300 m2/g and a pore size of 12 nm ;
— temperature : 35 °C.
Mobile phase :
— mobile phase A : acetonitrile for chromatography R,
water R (400:600 V/V) ;
— mobile phase B : acetonitrile for chromatography R ;
4258 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 20 100 0
20 - 60 100 → 10 0 → 90
60 - 62 10 → 100 90 → 0
62 - 70 100 0
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 227 nm.
Injection : 15 μl of the test solution and reference
solutions (a), (c) and (d).
Identification of impurities : use the chromatogram
supplied with paclitaxel semi-synthetic for peak
identification CRS and the chromatogram obtained
with reference solution (c) to identify the peaks due
to impurities A, G, I and L ; use the chromatogram
supplied with paclitaxel semi-synthetic for system
suitability CRS and the chromatogram obtained with
reference solution (d) to identify the peaks due to
impurities E, H and N.
Relative retention with reference to paclitaxel (retention
time = about 23 min) : impurity N = about 0.2 ;
impurity G = about 0.5 ; impurity A = about 0.8 ;
impurities M, J and H = about 0.9 ; impurity E = about 1.3 ;
impurity I = about 1.4 ; impurity L = about 1.5 ;
impurity K = about 2.2.
System suitability : reference solution (d) :
— resolution : minimum 1.5 between the peaks due to
impurity H and paclitaxel.
Limits :
— correction factor : for the calculation of content,
multiply the peak area of impurity N by 1.29 ;
— impurity A : not more than 7 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.7 per cent) ;
— impurity L : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
— impurities E, I : for each impurity, not more than
4 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.4 per cent) ;
— sum of impurities H, J and M : not more than 4 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.4 per cent) ;
— impurities G, K, N : for each impurity, not more
than twice the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.2 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 : not more than 12 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.2 per cent) ;
— disregard limit : 0.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 1.0 g in methanol R and dilute to 20 ml with the
same solvent. 12 ml of the solution complies with test B.
Prepare the reference solution using 10 ml of lead standard
solution (1 ppm Pb), obtained by diluting lead standard
EUROPEAN PHARMACOPOEIA 6.3 Paclitaxel

solution (100 ppm Pb) R with methanol R and 2 ml of identify these impurities for demonstration of compliance.
the test solution. To 12 ml of each solution, add 2 ml of See also 5.10. Control of impurities in substances for
buffer solution pH 3.5 R. Mix. Add 1.2 ml of thioacetamide pharmaceutical use) : H.
reagent R. The substance will precipitate. Dilute to 40 ml Test B
with methanol R ; the substance re-dissolves completely.
Filter the solution through a membrane filter (pore size Specified impurities : A, E, G, H, I, J, K ,L, M, N.
0.45 μm). Compare the spots on the filters obtained with
the different solutions. The substance to be examined
complies with the test if any brownish-black colour in the
spot obtained with the test solution is not more intense than
that of the spot obtained with the reference solution.
Water (2.5.32) : maximum 3.0 per cent, determined on
0.050 g.
Microbial contamination
TAMC : acceptance criterion 102 CFU/g (2.6.12).
Bacterial endotoxins (2.6.14) : less than 0.4 IU/mg.

ASSAY
A. Paclitaxel isolated from natural sources or produced by
fermentation.
Liquid chromatography (2.2.29) as described in test A for
related substances with the following modification.
Injection : test solution (b) and reference solution (b).
Calculate the percentage content of C47H51NO14 from the
declared content of paclitaxel CRS.
B. Paclitaxel produced by a semi-synthetic process.
Liquid chromatography (2.2.29) as described in test B for
related substances with the following modification.
Injection : 10 μl of the test solution and reference
solution (b).
Calculate the percentage content of C47H51NO14 from the
declared content of paclitaxel CRS.

STORAGE
In an airtight container, protected from light.

LABELLING A. R1 = Tg, R2 = Ac, R3 = Bz, R4 = R6 = H, R5 = OH :

2-O-debenzoyl-2-O-tigloylpaclitaxel,
The label states the origin of the substance :
— isolated from natural sources ;
B. R1 = Bz, R2 = Ac, R3 = Tg, R4 = R6 = H, R5 = OH :
— produced by fermentation ; N-debenzoyl-N-tigloylpaclitaxel (cephalomannine),
— produced by a semi-synthetic process.

C. R1 = Bz, R2 = Ac, R3 = Hx, R4 = R6 = H, R5 = OH :

IMPURITIES N-debenzoyl-N-hexanoylpaclitaxel (paclitaxel C),
Test A
Specified impurities : A, B, C, D, E, F, O, P, Q, R. D. R1 = Bz, R2 = Ac, R3 = Tg, R4 = R5 = H, R6 = OH :
N-debenzoyl-N-tigloyl-7-epi-paclitaxel (7-epi-
Other detectable impurities (the following substances
cephalomannine),
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 E. R1 = R3 = Bz, R2 = Ac, R4 = R5 = H, R6 = OH :
pharmaceutical use (2034). It is therefore not necessary to 7-epi-paclitaxel,

General Notices (1) apply to all monographs and other texts 4259
Pancreas powder
F. R1 = Bz, R2 = Ac, R3 = Hx, R4 = CH3, R5 = OH, R6 = H :
N-debenzoyl-N-hexanoyl-N-methylpaclitaxel
(N-methylpaclitaxel C),
G. R1 = R3 = Bz, R2 = R4 = R6 = H, R5 = OH :
10-O-deacetylpaclitaxel,
H. R1 = R3 = Bz, R2 = R4 = R5 = H, R6 = OH :
10-O-deacetyl-7-epi-paclitaxel,
I. R1 = R3 = Bz, R2 = Pa, R4 = R6 = H, R5 = OH :
10-O-[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-
phenylpropanoyl]-10-O-deacetylpaclitaxel,
J. R1 = R3 = Bz, R2 = Aa, R4 = R6 = H, R5 = OH :
10-O-deacetyl-10-O-(3-oxobutanoyl)paclitaxel,
K. R1 = R3 = Bz, R2 = Ac, R4 = R6 = H, R5 = O-Si(C2H5)3 :
7-O-(triethylsilanyl)paclitaxel,
L. R1 = R3 = Bz, R2 = Ac, R4 = R6 = H, R5 = O-CO-CH3 :
7-O-acetylpaclitaxel,
O. R1 = Bz, R2 = Ac, R3 = Cn, R4 = R6 = H, R5 = OH :
N-cinnamoyl-N-debenzoylpaclitaxel,
P. R1 = Bz, R2 = Ac, R3 = Ba, R4 = R6 = H, R5 = OH :
N-debenzoyl-N-(phenylacetyl)paclitaxel,
Q. R1 = Bz, R2 = Ac, R3 = He, R4 = R6 = H, R5 = OH :
N-debenzoyl-N-[(3E)-hex-3-enoyl]paclitaxel,
R. R1 = Bz, R2 = Ac, R3 = Mb, R4 = R6 = H, R5 = OH :
N-debenzoyl-N-[(2S)-2-methylbutanoyl]paclitaxel,
M. 1,2α,4,7β-dihydroxy-9-oxotax-11-ene-5β,10β,13α,20-
tetrayl 5,10-diacetate 20-benzoate 13-[(2R,3S)-3-
(benzoylamino)-2-hydroxy-3-phenylpropanoate],
N. 13-O-de[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-
phenylpropanoyl]paclitaxel (baccatin III).
4260 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:0350
PANCREAS POWDER
Pancreatis pulvis
DEFINITION
Pancreas powder is prepared from the fresh or frozen
pancreases of mammals. It contains various enzymes having
proteolytic, lipolytic and amylolytic activities.
1 mg of pancreas powder contains not less than
1.0 Ph. Eur. U. of total proteolytic activity, 15 Ph. Eur. U. of
lipolytic activity and 12 Ph. Eur. U. of amylolytic activity.
CHARACTERS
A slightly brown, amorphous powder, partly soluble in water,
practically insoluble in ethanol (96 per cent).
IDENTIFICATION
A. Triturate 0.5 g with 10 ml of water R and adjust to pH 8
with 0.1 M sodium hydroxide, using 0.1 ml of cresol
red solution R as indicator. Divide the suspension into
2 equal parts (suspension (a) and suspension (b)). Boil
suspension (a). To each suspension add 10 mg of fibrin
congo red R, heat to 38-40 °C and maintain at this
temperature for 1 h. Suspension (a) is colourless or
slightly pink and suspension (b) is distinctly more red.
B. Triturate 0.25 g with 10 ml of water R and adjust to
pH 8 with 0.1 M sodium hydroxide, using 0.1 ml of
cresol red solution R as indicator. Divide the suspension
into 2 equal parts (suspension (a) and suspension (b)).
Boil suspension (a). Dissolve 0.1 g of soluble starch R
in 100 ml of boiling water R, boil for 2 min, cool and
dilute to 150 ml with water R. To 75 ml of the starch
solution add suspension (a) and to the remaining 75 ml
add suspension (b). Heat each mixture to 38-40 °C and
maintain at this temperature for 5 min.
To 1 ml of each mixture add 10 ml of iodine solution R2.
The mixture obtained with suspension (a) has an
intense blue-violet colour ; the mixture obtained with
suspension (b) has the colour of the iodine solution.
TESTS
Fat content. In an extraction apparatus, treat 1.0 g with
light petroleum R1 for 3 h. Evaporate the solvent and dry
the residue at 100-105 °C for 2 h. The residue weighs not
more than 50 mg (5.0 per cent).
Loss on drying (2.2.32). Not more than 5.0 per cent,
determined on 0.50 g by drying at 60 °C at a pressure not
exceeding 670 Pa for 4 h.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Total proteolytic activity. The total proteolytic activity
of pancreas powder is determined by comparing the
quantity of peptides non-precipitable by a 50 g/l solution of
trichloroacetic acid R released per minute from a substrate
of casein solution with the quantity of such peptides released
by pancreas powder (protease) BRP from the same substrate
in the same conditions.
Casein solution. Suspend a quantity of casein BRP
equivalent to 1.25 g of dried substance in 5 ml of water R,
add 10 ml of 0.1 M sodium hydroxide and stir for 1 min.
EUROPEAN PHARMACOPOEIA 6.3 Pancreas powder

(Determine the water content of casein BRP prior to the Table 0350.-1
test by heating at 60 °C in vacuo for 4 h.) Add 60 ml of
Tubes
water R and stir with a magnetic stirrer until the solution
is practically clear. Adjust to pH 8.0 with 0.1 M sodium S1 S1b S2 S2b S3 S3b T Tb B
hydroxide or 0.1 M hydrochloric acid. Dilute to 100.0 ml Buffer solution 2 2 1 1 1 1 3
with water R. Use the solution on the day of preparation.
Reference suspension 1 1 2 2 3 3
Enterokinase solution. Dissolve 50 mg of enterokinase BRP
in 0.02 M calcium chloride solution R and dilute to 50.0 ml Test suspension 2 2
with the same solvent. Use the solution on the day of Trichloroacetic acid solution 5 5 5 5 5
preparation.
Mix + + + + +
For the test suspension and the reference suspension, + + + + + + + + +
Water-bath 35 °C
prepare the suspension and carry out the dilution at 0-4 °C.
Casein solution 2 2 2 2 2
Test suspension. Triturate 0.100 g of the substance to
be examined for 5 min adding gradually 25 ml of 0.02 M Mix + + + + +
calcium chloride solution R. Transfer completely to a Casein solution 2 2 2 2
volumetric flask and dilute to 100.0 ml with 0.02 M calcium
chloride solution R. To 10.0 ml of this suspension add Mix + + + +
10.0 ml of the enterokinase solution and heat on a water-bath Water-bath 35 °C 30 min + + + + + + + + +
at 35 ± 0.5 °C for 15 min. Cool and dilute with borate buffer
solution pH 7.5 R at 5 ± 3 °C to a final concentration of Trichloroacetic acid solution 5 5 5 5
about 0.065 Ph. Eur. U. of total proteolytic activity per Mix + + + +
millilitre calculated on the basis of the stated activity. + + + + + + + + +
Room temperature
Reference suspension. Prepare a suspension of pancreas 20 min
powder (protease) BRP as described for the test suspension Filter + + + + + + + + +
but without the addition of enterokinase so as to obtain a
known final concentration of about 0.065 Ph. Eur. U. per Measure the absorbance (2.2.25) of the filtrates at 275 nm
millilitre calculated on the basis of the stated activity. using the filtrate obtained from tube B as the compensation
Designate tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b ; liquid.
designate a tube B. Correct the average absorbance values for the filtrates
Add borate buffer solution pH 7.5 R to the tubes as follows : obtained from tubes S1, S2 and S3 by subtracting the average
values obtained for the filtrates from tubes S1b, S2b and S3b
B : 3.0 ml, respectively. Draw a calibration curve of the corrected values
S1 and S1b : 2.0 ml, against volume of reference suspension used.
S2, S2b, T and Tb : 1.0 ml. Determine the activity of the substance to be examined using
the corrected absorbance for the test suspension (T − Tb) and
Add the reference suspension to the tubes as follows : the calibration curve and taking into account the dilution
S1 and S1b : 1.0 ml, factors.
S2 and S2b : 2.0 ml, The test is not valid unless the corrected absorbance values
are between 0.15 and 0.60.
S3 and S3b : 3.0 ml.
Lipolytic activity. The lipolytic activity is determined by
Add 2.0 ml of the test suspension to tubes T and Tb. comparing the rate at which a suspension of pancreas
Add 5.0 ml of a 50 g/l solution of trichloroacetic acid R to powder hydrolyses a substrate of olive oil emulsion with the
tubes B, S1b, S2b, S3b and Tb. Mix by shaking. rate at which a suspension of pancreas powder (amylase
and lipase) BRP hydrolyses the same substrate under the
Place the tubes and the casein solution in a water-bath same conditions. The test is carried out under nitrogen.
at 35 ± 0.5 °C. Place a glass rod in each tube. When
temperature equilibrium is reached, add 2.0 ml of the casein Olive oil stock emulsion. In an 800 ml beaker 9 cm in
solution to tubes B, S1b, S2b, S3b and Tb. Mix. At time zero, diameter, place 40 ml of olive oil R, 330 ml of acacia
add 2.0 ml of casein solution successively and at intervals of solution R and 30 ml of water R. Place an electric mixer
30 s to tubes S1, S2, S3 and T. Mix immediately after each at the bottom of the beaker. Place the beaker in a vessel
addition. Exactly 30 min after addition of the casein solution, containing ethanol (96 per cent) R and a sufficient quantity
taking into account the regular interval adopted, add 5.0 ml of ice as a cooling mixture. Emulsify using the mixer at an
of a 50 g/l solution of trichloroacetic acid R to tubes S1, S2, average speed of 1000-2000 r/min. Cool to 5-10 °C. Increase
S3 and T. Mix. Withdraw the tubes from the water-bath and the mixing speed to 8000 r/min. Mix for 30 min keeping
allow to stand at room temperature for 20 min. the temperature below 25 °C by the continuous addition of
crushed ice into the cooling mixture. (A mixture of calcium
Filter the contents of each tube twice through the same chloride and crushed ice is also suitable). Store the stock
suitable filter paper previously washed with a 50 g/l solution emulsion in a refrigerator and use within 14 days. The
of trichloroacetic acid R, then with water R and dried. emulsion must not separate into 2 distinct layers. Check the
A suitable filter paper complies with the following test : filter diameter of the globules of the emulsion under a microscope.
5 ml of a 50 g/l solution of trichloroacetic acid R on a 7 cm At least 90 per cent have a diameter below 3 μm and none
disc of white filter paper ; the absorbance (2.2.25) of the has a diameter greater than 10 μm. Shake the emulsion
filtrate, measured at 275 nm using unfiltered trichloroacetic thoroughly before preparing the emulsion substrate.
acid solution as the compensation liquid, is less than 0.04. Olive oil emulsion. For 10 determinations, mix the following
A schematic presentation of the above operations is shown solutions in the order indicated : 100 ml of the stock
in Table 0350.-1. emulsion, 80 ml of tris(hydroxymethyl)aminomethane

General Notices (1) apply to all monographs and other texts 4261
Pancreas powder EUROPEAN PHARMACOPOEIA 6.3

solution R1, 20 ml of a freshly prepared 80 g/l of sodium

taurocholate BRP and 95 ml of water R. Use on the day of
preparation.
Apparatus. Use a reaction vessel of about 50 ml capacity n = average volume of 0.1 M sodium hydroxide
provided with : used per minute during the titration of the test
suspension, in millilitres ;
— a device that will maintain a temperature of 37 ± 0.5 °C ; n1 = average volume of 0.1 M sodium hydroxide used
— a magnetic stirrer ; per minute during the titration of the reference
suspension, in millilitres ;
— a lid with holes for the insertion of electrodes, the tip of m = mass of the substance to be examined, in
a burette, a tube for the admission of nitrogen and the milligrams ;
introduction of reagents. m1 = mass of the reference preparation, in milligrams ;
An automatic or manual titration apparatus may be used. A = activity of pancreas powder (amylase and
In the latter case, the burette is graduated in 0.005 ml and lipase) BRP, in European Pharmacopoeia Units
the pH-meter is provided with a wide reading scale and per milligram.
glass-calomel or glass-silver-silver chloride electrodes. After
each test the reaction vessel is evacuated by suction and Amylolytic activity. The amylolytic activity is determined
washed several times with water R, the washings being by comparing the rate at which a suspension of pancreas
removed each time by suction. powder hydrolyses a substrate of starch solution with the
rate at which a suspension of pancreas powder (amylase
Test suspension. In a small mortar cooled to 0-4 °C, and lipase) BRP hydrolyses the same substrate under the
triturate carefully a quantity of the substance to be examined same conditions.
equivalent to about 2500 Ph. Eur. U. of lipolytic activity with
1 ml of cooled maleate buffer solution pH 7.0 R (lipase Starch solution. To a quantity of starch BRP equivalent
solvent) until a very fine suspension is obtained. Dilute the to 2.0 g of the dried substance add 10 ml of water R and
suspension with cold maleate buffer solution pH 7.0 R, mix. (Determine the water content of starch BRP prior to
transfer quantitatively to a volumetric flask and dilute to the test by heating at 120 °C for 4 h). Add this suspension,
100.0 ml with the cold buffer solution. Keep the flask whilst stirring continuously, to 160 ml of boiling water R.
containing the test suspension in iced water during the Wash the container several times with successive quantities,
titration. each of 10 ml, of water R and add the washings to the hot
starch solution. Heat to boiling, stirring continuously. Cool
Reference suspension. To avoid absorption of water to room temperature and dilute to 200 ml with water R. Use
formed by condensation, allow the reference preparation the solution on the day of preparation.
to reach room temperature before opening the container.
Prepare a suspension of pancreas powder (amylase and For the test suspension and the reference suspension,
lipase) BRP as described for the test suspension using a prepare the suspension and carry out the dilution at 0-4 °C.
quantity equivalent to about 2500 Ph. Eur. U.
Test suspension. Triturate a quantity of the substance to be
Carry out the titrations immediately after preparation of the examined equivalent to about 1500 Ph. Eur. U. of amylolytic
test suspension and the reference suspension. Place 29.5 ml activity with 60 ml of phosphate buffer solution pH 6.8 R1
of olive oil emulsion in the reaction vessel equilibrated at for 15 min. Transfer quantitatively to a volumetric flask and
37 ± 0.5 °C. Fit the vessel with the electrodes, a stirrer and dilute to 100.0 ml with phosphate buffer solution pH 6.8 R1.
the burette (the tip being immersed in the olive oil emulsion).
Put the lid in place and switch on the apparatus. Carefully Reference suspension. Prepare a suspension of pancreas
add 0.1 M sodium hydroxide with stirring to adjust to powder (amylase and lipase) BRP as described for the
pH 9.2. Using a rapid-flow graduated pipette transfer about test suspension, using a quantity equivalent to about
0.5 ml of the previously homogenised reference suspension, 1500 Ph. Eur. U.
start the chronometer and add continuously 0.1 M sodium
hydroxide to maintain the pH at 9.0. After exactly 1 min, In a test tube 200 mm long and 22 mm in diameter, fitted
note the volume of 0.1 M sodium hydroxide used. Carry with a ground-glass stopper, place 25.0 ml of starch solution,
out the measurement a further 4 times. Discard the first 10.0 ml of phosphate buffer solution pH 6.8 R1 and 1.0 ml
reading and determine the average of the 4 others (S1). Make of an 11.7 g/l solution of sodium chloride R. Close the tube,
2 further determinations (S2 and S3). Calculate the average shake and place in a water-bath at 25.0 ± 0.1 °C. When the
of the values S1, S2 and S3. The average volume of 0.1 M temperature equilibrium has been reached, add 1.0 ml of the
sodium hydroxide used should be about 0.12 ml per minute test suspension and start the chronometer. Mix and place
with limits of 0.08 ml to 0.16 ml. the tube in the water-bath. After exactly 10 min, add 2 ml of
1 M hydrochloric acid. Transfer the mixture quantitatively
Carry out 3 determinations in the same manner for the test to a 300 ml conical flask fitted with a ground-glass stopper.
suspension (T1, T2 and T3). If the quantity of 0.1 M sodium Whilst shaking continuously, add 10.0 ml of 0.05 M iodine
hydroxide used is outside the limits of 0.08 ml to 0.16 ml immediately followed by 45 ml of 0.1 M sodium hydroxide.
per minute, the assay is repeated with a quantity of test Allow to stand in the dark at a temperature between 15 °C
suspension that is more suitable but situated between 0.4 ml and 25 °C for 15 min. Add 4 ml of a mixture of 1 volume
and 0.6 ml. Otherwise the quantity of the substance to be of sulphuric acid R and 4 volumes of water R. Titrate the
examined is adjusted to comply with the conditions of the excess of iodine with 0.1 M sodium thiosulphate using
test. Calculate the average of the values T1, T2 and T3. a microburette. Carry out a blank titration adding the
2 ml of 1 M hydrochloric acid before introducing the
Calculate the activity in European Pharmacopoeia Units per test suspension. Carry out the titration of the reference
milligram using the following expression : suspension in the same manner.

4262 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Calculate the amylolytic activity in European Pharmacopoeia Foreign matter. Examined under a microscope using a
Units per milligram using the following expression :
n = volume of 0.1 M sodium thiosulphate used in the
titration of the test suspension, in millilitres ;
n1 = volume of 0.1 M sodium thiosulphate used in the Iron (2.4.9) : maximum 50 ppm.
titration of the reference suspension, in millilitres ; Shake 1.0 g with 50 ml of dilute hydrochloric acid R. Filter.
n′ = volume of 0.1 M sodium thiosulphate used in
the blank titration of the test suspension, in
millilitres ;
n′1 = volume of 0.1 M sodium thiosulphate used in the Sulphated ash (2.4.14) : maximum 0.6 per cent, determined
blank titration of the reference suspension, in
millilitres ;
m Microbial contamination
= mass of the substance to be examined, in
milligrams ;
m1 = mass of the reference preparation, in milligrams ;
A = activity of pancreas powder (amylase and
lipase) BRP, in European Pharmacopoeia Units
per milligram.
STORAGE
Store in an airtight container.
01/2009:2403
PEA STARCH
Pisi amylum
DEFINITION
Pea starch is obtained from the seeds of Pisum sativum L.
CHARACTERS
Appearance : white or almost white, very fine powder.
Solubility : practically insoluble in cold water and in ethanol
(96 per cent).
IDENTIFICATION
A. Examined under a microscope using equal volumes of
glycerol R and water R, it presents a majority of large
elliptical granules, 25-45 μm in size, sometimes irregular,
or reniform. It also presents a minority of small rounded
granules, 5-8 μm in size. Granules can present cracks or
irregularities. Sometimes, granules show barely visible
concentric striations. Exceptionally, granules show a
slit along the main axis. Between orthogonally oriented
polarising plates or prisms, the granules show a distinct
black cross.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool.
A thin, cloudy mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B,
add 0.05 ml of iodine solution R1. A dark blue colour is
produced, which disappears on heating.
TESTS
pH (2.2.3) : 5.0 to 8.0.
Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for
60 s. Allow to stand for 15 min and shake again.
General Notices (1) apply to all monographs and other texts
Pepsin powder
mixture of equal volumes of glycerol R and water R, not
more than traces of matter other than starch granules are
present. No starch granules of any other origin are present.
Oxidising substances (2.5.30) : maximum 20 ppm, calculated
as H2O2.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
The filtrate complies with the test for iron.
Loss on drying (2.2.32) : maximum 16.0 per cent, determined
on 1.000 g by drying in an oven at 130 °C for 90 min.
on 1.0 g.
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
01/2009:0682
PEPSIN POWDER
Pepsini pulvis
[9001-75-6]
DEFINITION
Pepsin powder is prepared from the gastric mucosa of pigs,
cattle or sheep. It contains gastric proteinases, active in
acid medium (pH 1 to 5). It has an activity not less than
0.5 Ph. Eur. U./mg, calculated with reference to the dried
substance.
PRODUCTION
The animals from which pepsin powder is derived must
fulfil the requirements for the health of animals suitable for
human consumption.
CHARACTERS
A white or slightly yellow, crystalline or amorphous powder,
hygroscopic, soluble in water, practically insoluble in
ethanol (96 per cent). The solution in water may be slightly
opalescent with a weak acidic reaction.
IDENTIFICATION
In a mortar, pound 30 mg of fibrin blue R. Suspend in 20 ml
of dilute hydrochloric acid R2. Filter the suspension on a
filter paper and wash with dilute hydrochloric acid R2 until
a colourless filtrate is obtained. Perforate the filter paper
and wash the fibrin blue R through it into a conical flask
using 20 ml of dilute hydrochloric acid R2. Shake before
use. Dissolve a quantity of the substance to be examined,
equivalent to not less than 20 Ph. Eur. U., in 2 ml of dilute
hydrochloric acid R2 and adjust to pH 1.6 ± 0.1. Add 1 ml
of this solution to a test-tube containing 4 ml of the fibrin
blue suspension, mix and place in a water-bath at 25 °C with
gentle shaking. Prepare a blank solution at the same time
and in the same manner using 1 ml of water R. After 15 min
of incubation the blank solution is colourless and the test
solution is blue.
4263
Pepsin powder EUROPEAN PHARMACOPOEIA 6.3

TESTS S1 and S1b : 0.5 ml

Loss on drying (2.2.32). Not more than 5.0 per cent,
determined on 0.500 g by drying at 60 °C over diphosphorus
pentoxide R at a pressure not exceeding 670 Pa for 4 h.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
The activity of pepsin powder is determined by comparing the
quantity of peptides, non-precipitable by trichloroacetic acid
solution R and assayed using the phosphomolybdotungstic
reagent R, which are released per minute from a substrate of
haemoglobin solution R, with the quantity of such peptides
released by pepsin powder BRP from the same substrate in
the same conditions.
For the test solution and the reference solution, prepare the
solution and carry out the dilution at 0 °C to 4 °C.
Avoid shaking and foaming during preparation of the test
and reference solutions.
Test solution. Immediately before use, prepare a solution
of the substance to be examined expected to contain
0.5 Ph. Eur. U./ml in dilute hydrochloric acid R2; before
dilution to volume, adjust to pH 1.6 ± 0.1, if necessary, using
1 M hydrochloric acid.
Reference solution. Less than 15 min before use,
prepare a solution of pepsin powder BRP containing
0.5 Ph. Eur. U./ml in dilute hydrochloric acid R2; before
dilution to volume, adjust to pH 1.6 ± 0.1, if necessary, using
1 M hydrochloric acid.
Designate tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b ;
designate a tube B.
Add dilute hydrochloric acid R2 to the tubes as follows :
B : 1.0 ml
S1 and S1b : 0.5 ml
S2, S2b and T and Tb : 0.25 ml
Add the reference solution to the tubes as follows :
S2 and S2b : 0.75 ml
S3 and S3b : 1.0 ml
Add 0.75 ml of the test solution to tubes T and Tb.
Add 10.0 ml of trichloroacetic acid solution R to tubes S1b,
S2b, S3b, Tb and B. Mix by shaking.
Place the tubes and haemoglobin solution R in a water bath
at 25 ± 0.1 °C. When temperature equilibrium is reached,
add 5.0 ml of haemoglobin solution R to tubes B, S1b, S2b,
S3b and Tb. Mix.
At time zero add 5.0 ml of haemoglobin solution R
successively and at intervals of 30 s to tubes S1, S2, S3 and T.
Mix immediately after each addition.
Exactly 10 min after adding the haemoglobin solution R,
stop the reaction by adding, at intervals of 30 s, 10.0 ml of
trichloroacetic acid solution R to tubes S1, S2, S3 and T (the
use of a fast-flowing or blow-out pipette is recommended)
and mix.
Filter the contents of each tube (samples and blanks) twice
through the same suitable filter paper previously washed
with a 50 g/l solution of trichloroacetic acid R, then with
water R and dried. Discard the first 5 ml of filtrate. Place
3.0 ml of each filtrate separately in a tube containing 20 ml
of water R. Mix.
A suitable filter paper complies with the following test : filter
5 ml of a 50 g/l solution of trichloroacetic acid R through a
7 cm disc of white filter paper : the absorbance (2.2.25) of the
filtrate, measured at 275 nm using unfiltered trichloroacetic
acid R solution as the compensation liquid, is less than 0.04.
Add to each tube 1.0 ml of sodium hydroxide solution R and
1.0 ml of phosphomolybdotungstic reagent R, beginning
with the blanks and then the samples of each set, in a known
order.
A schematic presentation of the above operations is shown
in Table 0682.-1.
After 15 min measure the absorbance (2.2.25) of solutions
S1, S2, S3, S1b, S2b, S3b and T at 540 nm using the filtrate
obtained from tube B as the compensation liquid. Correct
the average absorbance values for the filtrates obtained
from tubes S1, S2 and S3 by subtracting the average values
obtained for the filtrates from tubes S1b, S2b, S3b respectively.

Table 0682.-1
Tubes
S1 S1b S2 S2b S3 S3b T Tb B
Dilute hydrochloric acid R2 (ml) 0.5 0.5 0.25 0.25 0.25 0.25 1.0
Reference solution (ml) 0.5 0.5 0.75 0.75 1.0 1.0
Test solution (ml) 0.75 0.75
Trichloroacetic acid solution R (ml) 10.0 10.0 10.0 10.0 10.0
Mix + + + + +

Water bath at 25 °C + + + + + + + + +

Haemoglobin solution R (ml) 5.0 5.0 5.0 5.0 5.0

Mix + + + + +

Haemoglobin solution R (ml) 5.0 5.0 5.0 5.0

Mix + + + +

Water bath at 25 °C, 10 min + + + + + + + + +

Trichloroacetic acid solution R (ml) 10.0 10.0 10.0 10.0

Mix + + + +

Filter + + + + + + + + +

4264 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Perphenazine

Draw a calibration curve of the corrected values — stationary phase : spherical base-deactivated octylsilyl
against volume of reference solution used. Determine the silica gel for chromatography R (4 μm) ;
activity of the substance to be examined using the corrected — temperature : 30 °C.
absorbance for the test solution (T − Tb) together with the
Mobile phase :
calibration curve and taking into account the dilution factors.
— mobile phase A : mix 35 volumes of acetonitrile R and
STORAGE 65 volumes of a 7 g/l solution of sodium dihydrogen
Store in an airtight container, protected from light, at a phosphate R ;
temperature of 2 °C to 8 °C. — mobile phase B : acetonitrile R ;
LABELLING Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
The label states the activity in European Pharmacopoeia
0-5 100 0
Units per milligram.
5 - 10 100 → 80 0 → 20
10 - 33 80 → 30 20 → 70
01/2009:0629
33 - 48 30 → 100 70 → 0

PERPHENAZINE Flow rate : 1.3 ml/min.

Perphenazinum
C21H26ClN3OS Mr 404.0 — resolution : minimum 4.0 between the peaks due to
[58-39-9]
DEFINITION
2-[4-[3-(2-Chloro-10H-phenothiazin-10-yl)propyl]piperazin-
1-yl]ethanol.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or yellowish-white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
methylene chloride, soluble in ethanol (96 per cent). It
dissolves in dilute solutions of hydrochloric acid.
IDENTIFICATION
A. Melting point (2.2.14) : 96 °C to 100 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : perphenazine CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1).
Dissolve 0.20 g in 10 ml of methanol R.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use. Carry out
the test protected from light.
Test solution. Dissolve 20 mg of the substance to be
examined in mobile phase A and dilute to 10.0 ml with
mobile phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to potentiometrically (2.2.20).
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution 1 ml of 0.1 M perchloric acid is equivalent to 20.20 mg of
to 10.0 ml with mobile phase A.
Reference solution (b). Dissolve 2 mg of perphenazine for
system suitability CRS (containing impurities A and B) in
1.0 ml of mobile phase A.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
Detection : spectrophotometer at 245 nm.
Injection : 10 μl.
Identification of impurities : use the chromatogram
supplied with perphenazine for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A and B.
Relative retention with reference to perphenazine
(retention time = about 12 min) : impurity A = about 0.3 ;
impurity B = about 0.8.
System suitability : reference solution (b) :
impurity B and perphenazine.
Limits :
— correction factor : for the calculation of content, multiply
the peak area of impurity A by 0.6 ;
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
— impurity B : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 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 : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 65 °C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.150 g in 25 ml of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid determining the end-point
C21H26ClN3OS.
STORAGE
Protected from light.
IMPURITIES
Specified impurities: A, B.

General Notices (1) apply to all monographs and other texts 4265
Phenol
A. 2-[4-[3-(2-chloro-5-oxido-10H-phenothiazin-10-
yl)propyl]piperazin-1-yl]ethanol,
B. 2-[4-[3-(10H-phenothiazin-10-yl)propyl]piperazin-1-
yl]ethanol.
01/2008:0631
corrected 6.3
PHENOL
Phenolum
C 6 H6 O Mr 94.1
[108-95-2]
DEFINITION
Content : 99.0 per cent to 100.5 per cent.
CHARACTERS
Appearance : colourless or faintly pink or faintly yellowish,
crystals or crystalline masses, deliquescent.
Solubility : soluble in water, very soluble in ethanol (96 per
cent), in glycerol and in methylene chloride.
IDENTIFICATION
A. Dissolve 0.5 g in 2 ml of concentrated ammonia R. The
substance dissolves completely. Dilute to about 100 ml
with water R. To 2 ml of this solution add 0.05 ml of
strong sodium hypochlorite solution R. A blue colour
develops and becomes progressively more intense.
B. To 1 ml of solution S (see Tests) add 10 ml of water R
and 0.1 ml of ferric chloride solution R1. A violet colour
is produced which disappears on addition of 5 ml of
2-propanol R.
C. To 1 ml of solution S add 10 ml of water R and 1 ml of
bromine water R. A white precipitate is formed.
TESTS
Solution S. Dissolve 1.0 g in water R and dilute to 15 ml
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution B6 (2.2.2,
Method II).
Acidity. To 2 ml of solution S add 0.05 ml of methyl orange
solution R. The solution is yellow.
Freezing point (2.2.18) : minimum 39.5 °C.
4266 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Residue on evaporation : maximum 0.05 per cent.
Evaporate 5.000 g to dryness on a water-bath and dry at
100-105 °C for 1 h. The residue weighs a maximum of 2.5 mg.
ASSAY
Dissolve 2.000 g in water R and dilute to 1000.0 ml with
the same solvent. Transfer 25.0 ml of the solution to a
ground-glass-stoppered flask and add 50.0 ml of 0.0167 M
bromide-bromate and 5 ml of hydrochloric acid R, close the
flask, allow to stand with occasional swirling for 30 min.
Then allow to stand for 15 min. Add 5 ml of a 200 g/l
solution of potassium iodide R, shake and titrate with 0.1 M
sodium thiosulphate until a faint yellow colour remains.
Add 0.5 ml of starch solution R and 10 ml of chloroform R
and continue the titration with vigorous shaking. Carry out
a blank titration.
1 ml of 0.0167 M bromide-bromate is equivalent to 1.569 mg
of C6H6O.
STORAGE
In an airtight container, protected from light.
01/2008:0522
corrected 6.3
PHOLCODINE
Pholcodinum
C23H30N2O4,H2O Mr 416.5
[509-67-1]
DEFINITION
7,8-Didehydro-4,5α-epoxy-17-methyl-3-[2-(morpholin-4-
yl)ethoxy]morphinan-6α-ol monohydrate.
Content : 98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : sparingly soluble in water, freely soluble in
acetone and in ethanol (96 per cent). It dissolves in dilute
mineral acids.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : pholcodine CRS.
TESTS
Specific optical rotation (2.2.7) : − 94 to − 98 (dried
substance).
Dissolve 1.000 g in ethanol (96 per cent) R and dilute to
50.0 ml with the same solvent.
Related substances. Liquid chromatography (2.2.29).
0.02 M phosphate buffer solution. To 80.0 ml of 0.2 M
sodium hydroxide add 100.0 ml of 0.2 M potassium
dihydrogen phosphate R and dilute to 1000.0 ml with
water R.
EUROPEAN PHARMACOPOEIA 6.3 Pholcodine

Solvent mixture. Dilute 80 ml of acetonitrile R to 1000 ml ASSAY

with the 0.02 M phosphate buffer solution. Dissolve 0.180 g in 50 ml of anhydrous acetic acid R,
Test solution. Dissolve 50 mg of the substance to be warming gently. Titrate with 0.1 M perchloric acid,
examined in the solvent mixture and dilute to 50 ml with the determining the end-point potentiometrically (2.2.20).
solvent mixture. 1 ml of 0.1 M perchloric acid is equivalent to 19.93 mg
Reference solution (a). Dissolve 10 mg of codeine R of C23H30N2O4.
(impurity B) in the solvent mixture and dilute to 10 ml with
the solvent mixture. To 0.5 ml of this solution add 0.5 ml of IMPURITIES
the test solution and dilute to 50 ml with the solvent mixture. Specified impurities : A, B, D.
Reference solution (b). Dilute 1.0 ml of the test solution Other detectable impurities (the following substances
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this would, if present at a sufficient level, be detected by one
solution to 10.0 ml with the solvent mixture. or other of the tests in the monograph. They are limited
Reference solution (c). Dissolve 5 mg of pholcodine for peak by the general acceptance criterion for other/unspecified
identification CRS (containing impurities A, B and D) in the impurities and/or by the general monograph Substances for
solvent mixture and dilute to 5 ml with the solvent mixture. pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
Column: See also 5.10. Control of impurities in substances for
— size : l = 0.075 m, Ø = 4.6 mm ; pharmaceutical use) : C, E, F.
— stationary phase : spherical end-capped phenylhexylsilyl
silica gel for chromatography R (3 μm) with a specific A. morphine,
surface area of 400 m2/g and a pore size of 10 nm ;
— temperature : 35 °C. B. codeine,
Mobile phase : to 50 ml of tetrahydrofuran for
chromatography R add 75 ml of acetonitrile R and dilute to
1000 ml with the 0.02 M phosphate buffer solution ; adjust
to pH 7.9 ± 0.05 with 0.2 M sodium hydroxide ; the pH must
not exceed 8.0.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 238 nm.
Injection : 20 μl.
Run time : 5 times the retention time of pholcodine. C. (17RS)-7,8-didehydro-4,5α-epoxy-17-methyl-3-[2-
Identification of impurities: use the chromatogram (morpholin-4-yl)ethoxy]morphinan-6α-ol 17-oxide
supplied with pholcodine for peak identification CRS and (pholcodine N-oxide),
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B and D.
D. unknown structure,
Relative retention with reference to pholcodine
(retention time = about 10 min) : impurity A = about 0.4 ;
impurity B = about 0.8 ; impurity D = about 2.3.
System suitability : reference solution (a) :
— resolution : minimum 3 between the peaks due to
impurity B and pholcodine.
Limits :
— impurities A, B, D : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ; E. 7,8-didehydro-4,5α-epoxy-17-methyl-3-[2-(4-
oxidomorpholin-4-io)ethoxy]morphinan-6α-ol
— unspecified impurities: for each impurity, not more (pholcodine N′-oxide),
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
— total : not more than 7 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.7 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : 3.9 per cent to 4.5 per cent,
determined on 0.500 g by drying in an oven at 105 °C. F. (17RS)-7,8-didehydro-4,5α-epoxy-17-methyl-3-[2-(4-
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined oxidomorpholin-4-io)ethoxy]morphinan-6α-ol 17-oxide
on 1.0 g. (pholcodine N,N′-dioxide).

General Notices (1) apply to all monographs and other texts 4267
Pilocarpine hydrochloride
01/2008:0633 Appearance of solution. Solution S is clear (2.2.1) and not
corrected 6.3 more intensely coloured than reference solution Y7 (2.2.2,
PILOCARPINE HYDROCHLORIDE
Pilocarpini hydrochloridum
C11H17ClN2O2 Mr 244.7 20.0 ml with water R.
[54-71-7]
DEFINITION
(3S,4R)-3-Ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]-
dihydrofuran-2(3H)-one hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals, hygroscopic.
Solubility : very soluble in water and in ethanol (96 per cent). — size: l = 0.15 m, Ø = 4.6 mm ;
mp : about 203 °C.
IDENTIFICATION
First identification : A, B, E.
Second identification : A, C, D, E.
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : pilocarpine hydrochloride CRS.
If the substances are examined as discs, prepare them
using potassium chloride R.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg in methanol R and dilute
to 2 ml with the same solvent.
Reference solution. Dissolve 10 mg of pilocarpine
hydrochloride CRS in methanol R and dilute to 2 ml
with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, methanol R,
methylene chloride R (1:14:85 V/V/V).
Application : 2 μl.
Development : over a path of 15 cm.
Drying : at 100-105 °C for 10 min, then allow to cool.
Detection : spray with dilute potassium iodobismuthate
solution R.
Results : the principal spot in the chromatogram obtained — sum of impurities other than A and B : not more than the
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
D. Dilute 0.2 ml of solution S (see Tests) to 2 ml with
water R. Add 0.05 ml of a 50 g/l solution of potassium
dichromate R, 1 ml of dilute hydrogen peroxide
solution R and 2 ml of methylene chloride R and shake. Iron (2.4.9) : maximum 10 ppm, determined on solution S.
A violet colour develops in the organic layer.
E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R
and dilute to 50.0 ml with the same solvent.
4268 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Method II).
pH (2.2.3) : 3.5 to 4.5 for solution S.
Specific optical rotation (2.2.7) : + 89 to + 93 (dried
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be
examined in water R and dilute to 100.0 ml with the same
solvent.
Reference solution (a). Dilute 5.0 ml of the test solution
to 100.0 ml with water R. Dilute 2.0 ml of this solution to
Reference solution (b). Dissolve 5.0 mg of pilocarpine
nitrate for system suitability CRS (containing impurity A) in
water R and dilute to 50.0 ml with the same solvent.
Reference solution (c). To 5 ml of the test solution, add
0.1 ml of ammonia R and heat the solution on a water-bath
for 30 min, cool and dilute to 25 ml with water R. Dilute 3 ml
of this solution to 25 ml with water R. Mainly pilocarpic acid
(impurity B) is formed.
Column :
— stationary phase : octadecylsilyl silica gel for
chromatography R1 (5 μm) with a pore size of 10 nm and
a carbon loading of 19 per cent.
Mobile phase : mix 55 volumes of methanol R, 60 volumes
of acetonitrile R and 885 volumes of a 0.679 g/l solution of
tetrabutylammonium dihydrogen phosphate R previously
adjusted to pH 7.7 with dilute ammonia R2.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 220 nm.
Injection : 20 μl.
Run time : twice the retention time of pilocarpine.
Elution order: impurity B, impurity C, impurity A,
pilocarpine.
Retention time : pilocarpine = about 20 min.
System suitability : reference solution (b) :
— resolution : minimum 1.6 between the peaks due to
impurity A and pilocarpine.
Limits :
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1 per cent) ;
— sum of impurities A and B : not more than 3 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.5 per cent) ;
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
— disregard limit : 0.4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent).
Prepare the standard using 5 ml of iron standard solution
(1 ppm Fe) R and 5 ml of water R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
EUROPEAN PHARMACOPOEIA 6.3
ASSAY
Dissolve 0.200 g in 50 ml of ethanol (96 per cent) R and add
5 ml of 0.01 M hydrochloric acid. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
(2.2.20). Read the volume added between the 2 points of
inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 24.47 mg
of C11H17ClN2O2.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B.
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) : C.
A. (3R,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-
yl)methyl]dihydrofuran-2(3H)-one (isopilocarpine),
B. R = C2H5, R′ = H : (2S,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-
methyl-1H-imidazol-5-yl)butanoic acid (pilocarpic acid),
C. R = H, R′ = C2H5 : (2R,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-
methyl-1H-imidazol-5-yl)butanoic acid (isopilocarpic acid). E. It gives the reaction of nitrates (2.3.1).
01/2008:0104 and dilute to 50.0 ml with the same solvent. Prepare
corrected 6.3 immediately before use.
PILOCARPINE NITRATE
Pilocarpini nitras
C11H17N3O5 Mr 271.3 Reference solution (a). Dissolve 5.0 ml of the test solution
[148-72-1]
DEFINITION
(3S,4R)-3-Ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]-
dihydrofuran-2(3H)-one nitrate.
General Notices (1) apply to all monographs and other texts
Pilocarpine nitrate
Content : 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals, sensitive to light.
Solubility : freely soluble in water, sparingly soluble in
ethanol (96 per cent).
mp : about 174 °C, with decomposition.
IDENTIFICATION
First identification : A, B, E.
Second identification : A, C, D, E.
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : pilocarpine nitrate CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in water R and dilute to 10 ml with the same
solvent.
Reference solution. Dissolve 10 mg of pilocarpine
nitrate CRS in water R and dilute to 10 ml with the same
solvent.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, methanol R,
methylene chloride R (1:14:85 V/V/V).
Application : 10 μl.
Development : over a path of 15 cm.
Drying : at 100-105 °C for 10 min and allow to cool.
Detection : spray with potassium iodobismuthate
solution R.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
D. Dilute 0.2 ml of solution S (see Tests) to 2 ml with
water R. Add 0.05 ml of a 50 g/l solution of potassium
dichromate R, 1 ml of dilute hydrogen peroxide
solution R and 2 ml of methylene chloride R and shake.
A violet colour develops in the organic layer.
TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
pH (2.2.3) : 3.5 to 4.5 for solution S.
Specific optical rotation (2.2.7) : + 80 to + 83 (dried
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be
examined in water R and dilute to 100.0 ml with the same
solvent.
to 100.0 ml with water R. Dilute 2.0 ml of this solution to
20.0 ml with water R.
Reference solution (b). Dissolve 5.0 mg of pilocarpine
nitrate for system suitability CRS (containing impurity A) in
water R and dilute to 50.0 ml with the same solvent.
4269
Polyacrylate dispersion 30 per cent
Reference solution (c). To 5 ml of the test solution, add
0.1 ml of ammonia R and heat the solution on a water-bath
for 30 min, cool and dilute to 25 ml with water R. Dilute 3 ml
of this solution to 25 ml with water R. Mainly pilocarpic acid
(impurity B) is formed.
Column:
— size : l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R1 (5 μm) with a pore size of 10 nm and
a carbon loading of 19 per cent.
Mobile phase : mix 55 volumes of methanol R, 60 volumes
of acetonitrile R and 885 volumes of a 0.679 g/l solution of
tetrabutylammonium dihydrogen phosphate R previously
adjusted to pH 7.7 with diluted ammonia R2.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 220 nm.
Injection : 20 μl.
Run time : twice the retention time of pilocarpine.
Elution order : impurity B, impurity C, impurity A,
pilocarpine.
Retention time : pilocarpine = about 20 min.
System suitability : reference solution (b) :
— resolution : minimum 1.6 between the peaks due to
impurity A and pilocarpine.
Limits :
— impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1 per cent) ;
— sum of impurities A and B : not more than 3 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.5 per cent) ;
— sum of impurities other than A and B : not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
— disregard limit : 0.4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ; disregard any peak due to the nitrate ion
with a relative retention time with reference to pilocarpine
of about 0.3.
Chlorides (2.4.4) : maximum 70 ppm, determined on
solution S.
Iron (2.4.9) : maximum 10 ppm, determined on solution S.
Prepare the standard using 5 ml of iron standard solution
(1 ppm Fe) R and 5 ml of water R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.250 g in 30 ml of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid determining the end-point A. Infrared absorption spectrophotometry (2.2.24).
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 27.13 mg
of C11H17N3O5.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B.
4270 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
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) : C.
A. (3R,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-
yl)methyl]dihydrofuran-2(3H)-one (isopilocarpine),
B. R = C2H5, R′ = H : (2S,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-
methyl-1H-imidazol-5-yl)butanoic acid (pilocarpic acid),
C. R = H, R′ = C2H5 : (2R,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-
methyl-1H-imidazol-5-yl)butanoic acid (isopilocarpic acid).
01/2009:0733
POLYACRYLATE DISPERSION
30 PER CENT
Polyacrylatis dispersio 30 per centum
DEFINITION
Dispersion in water of a copolymer of ethyl acrylate and
methyl methacrylate having a mean relative molecular mass
of about 800 000.
Content : 28.5 per cent to 31.5 per cent (residue on
evaporation).
It may contain a suitable emulsifier.
CHARACTERS
Appearance : opaque, white or almost white, slightly viscous
liquid.
Solubility : miscible with water, soluble in acetone, in
anhydrous ethanol and in 2-propanol.
IDENTIFICATION
First identification : A.
Second identification : B, C, D, E.
Comparison : Ph. Eur. reference spectrum of
polyacrylate.
B. To 1 g add 5 ml of water R and mix ; the mixture remains
opaque. Take 3 portions of 1 g and mix separately with
5 g of anhydrous ethanol R, 5 g of acetone R and 5 g of
2-propanol R. Transparent solutions are obtained.
C. To 1 g add 10 ml of 0.1 M sodium hydroxide. The mixture
remains opaque.
D. Appearance of a film (see Tests).
EUROPEAN PHARMACOPOEIA 6.3
E. Dry 4 g in a Petri dish at 60 °C in an oven for 4 h and
transfer the resulting clear film to a small test-tube
(100 mm × 12 mm). Heat over a flame and collect the
fumes that evolve in a 2nd test-tube held over the mouth of
st
the 1 tube. The condensate gives the reaction of esters
(2.3.1).
TESTS
Relative density (2.2.5) : 1.037 to 1.047.
Apparent viscosity (2.2.10) : maximum 50 mPa·s, determined
using a rotating viscometer at 20 °C and a shear rate of
10 s− l.
Appearance of a film. Pour 1 ml on a glass plate and allow
to dry. A clear elastic film is formed.
Particulate matter. Filter 100.0 g through a tared stainless
steel sieve (90). Rinse with water R until a clear filtrate is
obtained and dry at 80 °C to constant mass. The residue
weighs not more than 0.500 g.
Residual monomers. Liquid chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be
examined in tetrahydrofuran R and dilute to 50.0 ml
with the same solvent. To 5.0 ml of a 35 g/l solution of
sodium perchlorate R add 10.0 ml of the solution dropwise
whilst stirring continuously. Centrifuge and filter the clear
supernatant liquid. Dilute 5.0 ml of this solution to 10.0 ml
with water R.
Reference solution. Dissolve 10 mg of ethyl acrylate R and
10 mg of methyl methacrylate R in tetrahydrofuran R and
dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 100.0 ml with tetrahydrofuran R. To 10.0 ml
of the solution add 5.0 ml of a 35 g/l solution of sodium
perchlorate R and mix. Dilute 5.0 ml of the mixture to
10.0 ml with water R.
Column:
— size : l = 0.12 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm).
Mobile phase : acetonitrile R1, water for chromatography R
(15:85 V/V).
Flow rate : 2 ml/min.
Detection : spectrophotometer at 205 nm.
Injection : about 50 μl.
Limit :
— residual monomers : maximum 100 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.
Sulphated ash (2.4.14) : maximum 0.4 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
ASSAY
Dry 1.000 g at 110 °C for 3 h and weigh the residue.
STORAGE
At a temperature of 5 °C to 25 °C. Protect from freezing.
Handle the substance so as to minimise microbial
contamination.
General Notices (1) apply to all monographs and other texts
Polysorbate 20
LABELLING
The label states, where applicable, the name and
concentration of any added emulsifier.
01/2008:0426
corrected 6.3
POLYSORBATE 20
Polysorbatum 20
DEFINITION
Mixture of partial esters of fatty acids, mainly lauric
(dodecanoic) acid, with sorbitol and its anhydrides
ethoxylated with approximately 20 moles of ethylene oxide
for each mole of sorbitol and sorbitol anhydrides.
CHARACTERS
Appearance : oily, yellow or brownish-yellow, clear or slightly
opalescent liquid.
Solubility : soluble in water, in anhydrous ethanol, in ethyl
acetate and in methanol, practically insoluble in fatty oils
and in liquid paraffin.
Relative density : about 1.10.
Viscosity : about 400 mPa·s at 25 °C.
IDENTIFICATION
First identication : A, D.
Second identification : B, C, D, E.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of
polysorbate 20.
B. It complies with the test for hydroxyl value (see Tests).
C. It complies with the test for saponification value (see
Tests).
D. It complies with the test for composition of fatty acids
(see Tests).
E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g
of potassium thiocyanate R and 0.1 g of cobalt nitrate R.
Stir with a glass rod. The solution becomes blue.
TESTS
Acid value (2.5.1) : maximum 2.0.
Dissolve 5.0 g in 50 ml of the prescribed solvent mixture.
Hydroxyl value (2.5.3, Method A) : 96 to 108.
Peroxide value : maximum 10.0.
Introduce 10.0 g into a 100 ml beaker and dissolve with 20 ml
of glacial acetic acid R. Add 1 ml of saturated potassium
iodide solution R and allow to stand for 1 min. Add 50 ml
of carbon dioxide-free water R and a magnetic stirring bar.
Titrate with 0.01 M sodium thiosulphate, determining the
end-point potentiometrically (2.2.20). Carry out a blank
titration.
4271
Polysorbate 40
Determine the peroxide value using the following expression :
n1 = volume of 0.01 M sodium thiosulphate required
for the substance to be examined, in millilitres ;
n2 = volume of 0.01 M sodium thiosulphate required
for the blank, in millilitres ;
M = molarity of the sodium thiosulphate solution, in
moles per litre ;
m = mass of substance to be examined, in grams.
Saponification value (2.5.6) : 40 to 50, determined on 4.0 g.
Use 15.0 ml of 0.5 M alcoholic potassium hydroxide and
dilute with 50 ml of ethanol (96 per cent) R before carrying
out the titration. Heat under reflux for 60 min.
Composition of fatty acids (2.4.22, Method C). Prepare
reference solution (a) as indicated in Table 2.4.22.-2.
Column:
— material: fused silica ;
— size : l = 30 m, Ø = 0.32 mm ;
— stationary phase : macrogol 20 000 R (film
thickness 0.5 μm).
Carrier gas: helium for chromatography R.
Linear velocity : 50 cm/s.
Temperature :
Time Temperature
(min) (°C)
Column 0 - 14 80 → 220
14 - 54 220
Injection port 250
Detector 250
Detection : flame ionisation.
Injection : 1 μl.
Composition of the fatty-acid fraction of the substance:
— caproic acid: maximum 1.0 per cent ;
— caprylic acid : maximum 10.0 per cent ;
— capric acid : maximum 10.0 per cent ;
— lauric acid : 40.0 per cent to 60.0 per cent ;
— myristic acid: 14.0 per cent to 25.0 per cent ;
— palmitic acid : 7.0 per cent to 15.0 per cent ;
— stearic acid : maximum 7.0 per cent ;
— oleic acid : maximum 11.0 per cent ;
— linoleic acid: maximum 3.0 per cent.
Ethylene oxide and dioxan (2.4.25, Method A) : maximum
1 ppm of ethylene oxide and 10 ppm of dioxan.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 3.0 per cent, determined on 1.00 g.
Total ash (2.4.16) : maximum 0.25 per cent, determined on
2.0 g.
STORAGE
In an airtight container, protected from light.
4272 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2008:1914
corrected 6.3
POLYSORBATE 40
Polysorbatum 40
DEFINITION
Mixture of partial esters of fatty acids, mainly Palmitic
acid (1904), with sorbitol and its anhydrides ethoxylated
with approximately 20 moles of ethylene oxide for each mole
of sorbitol and sorbitol anhydrides.
CHARACTERS
Appearance : oily, viscous, yellowish or brownish-yellow
liquid.
Solubility : miscible with water, with anhydrous ethanol,
with ethyl acetate and with methanol, practically insoluble
in fatty oils and in liquid paraffin.
Relative density : about 1.10.
Viscosity : about 400 mPa·s at 30 °C.
IDENTIFICATION
First identication : A, D.
Second identification : B, C, D, E.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of
polysorbate 40.
B. It complies with the test for hydroxyl value (see Tests).
C. It complies with the test for saponification value (see
Tests).
D. It complies with the test for composition of fatty acids
(see Tests).
E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g
of potassium thiocyanate R and 0.1 g of cobalt nitrate R.
Stir with a glass rod. The solution becomes blue.
TESTS
Acid value (2.5.1) : maximum 2.0.
Dissolve 5.0 g in 50 ml of the prescribed solvent mixture.
Hydroxyl value (2.5.3, Method A) : 89 to 105.
Peroxide value : maximum 10.0.
Introduce 10.0 g into a 100 ml beaker and dissolve with 20 ml
of glacial acetic acid R. Add 1 ml of saturated potassium
iodide solution R and allow to stand for 1 min. Add 50 ml
of carbon dioxide-free water R and a magnetic stirring bar.
Titrate with 0.01 M sodium thiosulphate, determining the
end-point potentiometrically (2.2.20). Carry out a blank
titration.
Determine the peroxide value using the following expression :
n1 = volume of 0.01 M sodium thiosulphate required
for the substance to be examined, in millilitres ;
n2 = volume of 0.01 M sodium thiosulphate required
for the blank, in millilitres ;
M = molarity of the sodium thiosulphate solution, in
moles per litre ;
m = mass of substance to be examined, in grams.
EUROPEAN PHARMACOPOEIA 6.3 Polysorbate 60

Saponification value (2.5.6) : 41 to 52, determined on 4.0 g. A. Infrared absorption spectrophotometry (2.2.24).
Use 15.0 ml of 0.5 M alcoholic potassium hydroxide and Comparison : Ph. Eur. reference spectrum of
dilute with 50 ml of ethanol (96 per cent) R before carrying polysorbate 60.
out the titration. Heat under reflux for 60 min. B. It complies with the test for hydroxyl value (see Tests).
Composition of fatty acids (2.4.22, Method C). Prepare C. It complies with the test for saponification value (see
reference solution (a) as indicated in Table 2.4.22.-1. Tests).
Column: D. It complies with the test for composition of fatty acids
— material: fused silica ; (see Tests).
— size : l = 30 m, Ø = 0.32 mm ; E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g
— stationary phase : macrogol 20 000 R (film of potassium thiocyanate R and 0.1 g of cobalt nitrate R.
thickness 0.5 μm). Stir with a glass rod. The solution becomes blue.
Carrier gas: helium for chromatography R. TESTS
Linear velocity : 50 cm/s. Acid value (2.5.1) : maximum 2.0.
Temperature : Dissolve 5.0 g in 50 ml of the prescribed solvent mixture.
Time Temperature Hydroxyl value (2.5.3, Method A) : 81 to 96.
(min) (°C)
Peroxide value : maximum 10.0.
Column 0 - 14 80 → 220
14 - 54 220 Introduce 10.0 g into a 100 ml beaker and dissolve with 20 ml
Injection port 250 of glacial acetic acid R. Add 1 ml of saturated potassium
iodide solution R and allow to stand for 1 min. Add 50 ml
Detector 250 of carbon dioxide-free water R and a magnetic stirring bar.
Titrate with 0.01 M sodium thiosulphate, determining the
Detection : flame ionisation.
end-point potentiometrically (2.2.20). Carry out a blank
Injection : 1 μl. titration.
Composition of the fatty-acid fraction of the substance: Determine the peroxide value using the following expression :
— palmitic acid : minimum 92.0 per cent.
Ethylene oxide and dioxan (2.4.25, Method A) : maximum
1 ppm of ethylene oxide and maximum 10 ppm of dioxan.
Heavy metals (2.4.8) : maximum 10 ppm. n1 = volume of 0.01 M sodium thiosulphate required
2.0 g complies with test C. Prepare the reference solution for the substance to be examined, in millilitres ;
using 2 ml of lead standard solution (10 ppm Pb) R. n2 = volume of 0.01 M sodium thiosulphate required
Water (2.5.12) : maximum 3.0 per cent, determined on 1.00 g. for the blank, in millilitres ;
Total ash (2.4.16) : maximum 0.25 per cent, determined on M = molarity of the sodium thiosulphate solution, in
2.0 g. moles per litre ;
m = mass of substance to be examined, in grams.
STORAGE
In an airtight container, protected from light. Saponification value (2.5.6) : 45 to 55, determined on 4.0 g.
Use 15.0 ml of 0.5 M alcoholic potassium hydroxide and
dilute with 50 ml of ethanol (96 per cent) R before carrying
01/2008:0427 out the titration. Heat under reflux for 60 min.
01/2009
Composition of fatty acids (2.4.22, Method C). Prepare
reference solution (a) as indicated in Table 2.4.22.-1.
POLYSORBATE 60 Column :
— material: fused silica ;
Polysorbatum 60 — size: l = 30 m, Ø = 0.32 mm ;
DEFINITION — stationary phase : macrogol 20 000 R (film thickness
Mixture of partial esters of fatty acids, mainly Stearic 0.5 μm).
acid 50 (1474), with sorbitol and its anhydrides ethoxylated Carrier gas : helium for chromatography R.
with approximately 20 moles of ethylene oxide for each mole Linear velocity : 50 cm/s.
of sorbitol and sorbitol anhydrides. Temperature :
CHARACTERS Time Temperature
Appearance : yellowish-brown gelatinous mass which (min) (°C)
becomes a clear liquid at temperatures above 25 °C. Column 0 - 14 80 → 220
Solubility : soluble in water, in anhydrous ethanol, in ethyl 14 - 54 220
acetate and in methanol, practically insoluble in fatty oils
and in liquid paraffin. Injection port 250
Relative density : about 1.10. Detector 250
Viscosity : about 400 mPa·s at 30 °C. Detection : flame ionisation.
IDENTIFICATION Injection : 1 μl.
First identication : A, D. Composition of the fatty-acid fraction of the substance:
Second identification : B, C, D, E. — stearic acid : 40.0 per cent to 60.0 per cent ;

General Notices (1) apply to all monographs and other texts 4273
Polysorbate 80 EUROPEAN PHARMACOPOEIA 6.3

— sum of the contents of palmitic and stearic acids : Titrate with 0.01 M sodium thiosulphate, determining the
minimum 90.0 per cent. end-point potentiometrically (2.2.20). Carry out a blank
Ethylene oxide and dioxan (2.4.25, Method A) : maximum titration.
1 ppm of ethylene oxide and maximum 10 ppm of dioxan. Determine the peroxide value using the following expression :
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 3.0 per cent, determined on 1.00 g. n1 = volume of 0.01 M sodium thiosulphate required
for the substance to be examined, in millilitres ;
Total ash (2.4.16) : maximum 0.25 per cent, determined on n2
2.0 g. = volume of 0.01 M sodium thiosulphate required
for the blank, in millilitres ;
STORAGE M = molarity of the sodium thiosulphate solution, in
In an airtight container, protected from light. moles per litre ;
m = mass of substance to be examined, in grams.
Saponification value (2.5.6) : 45 to 55, determined on 4.0 g.
01/2008:0428 Use 30.0 ml of 0.5 M alcoholic potassium hydroxide,
corrected 6.3 heat under reflux for 60 min and add 50 ml of anhydrous
ethanol R before carrying out the titration.
POLYSORBATE 80 Composition of fatty acids. Gas chromatography (2.4.22,
Method C). Use the mixture of calibrating substances in
Table 2.4.22.-3.
Polysorbatum 80 Column :
DEFINITION — material: fused silica ;
Mixture of partial esters of fatty acids, mainly Oleic — size: l = 30 m, Ø = 0.32 mm ;
acid (0799), with sorbitol and its anhydrides ethoxylated — stationary phase : macrogol 20 000 R (film thickness
with approximately 20 moles of ethylene oxide for each mole 0.5 μm).
of sorbitol and sorbitol anhydrides. Carrier gas : helium for chromatography R.
CHARACTERS Linear velocity : 50 cm/s.
Appearance : oily, yellowish or brownish-yellow, clear or Temperature :
slightly opalescent liquid.
Time Temperature
Solubility : dispersible in water, in anhydrous ethanol, in (min) (°C)
ethyl acetate and in methanol, practically insoluble in fatty Column 0 - 14 80 → 220
oils and in liquid paraffin. 14 - 54 220
Relative density : about 1.10. Injection port 250
Viscosity : about 400 mPa·s at 25 °C. Detector 250

IDENTIFICATION Detection : flame ionisation.

First identication : A, D. Injection : 1 μl.
Second identification : B, C, D, E. Composition of the fatty-acid fraction of the substance:
A. Infrared absorption spectrophotometry (2.2.24). — myristic acid : maximum 5.0 per cent ;
Comparison : Ph. Eur. reference spectrum of — palmitic acid : maximum 16.0 per cent ;
polysorbate 80. — palmitoleic acid : maximum 8.0 per cent ;
B. Hydroxyl value (see Tests). — stearic acid : maximum 6.0 per cent ;
C. Saponification value (see Tests).
— oleic acid : minimum 58.0 per cent ;
D. Composition of fatty acids (see Tests).
— linoleic acid : maximum 18.0 per cent ;
E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g
— linolenic acid : maximum 4.0 per cent.
of potassium thiocyanate R and 0.1 g of cobalt nitrate R.
Stir with a glass rod. The solution becomes blue. Ethylene oxide and dioxan (2.4.25, Method A) : maximum
1 ppm of ethylene oxide and maximum 10 ppm of dioxan.
TESTS Heavy metals (2.4.8) : maximum 10 ppm.
Acid value (2.5.1) : maximum 2.0. 2.0 g complies with test C. Prepare the reference solution
Dissolve 5.0 g in 50 ml of the prescribed mixture of solvents. using 2 ml of lead standard solution (10 ppm Pb) R.
Hydroxyl value (2.5.3, Method A) : 65 to 80. Water (2.5.12) : maximum 3.0 per cent, determined on 1.00 g.
Peroxide value : maximum 10.0. Total ash (2.4.16) : maximum 0.25 per cent, determined on
Introduce 10.0 g into a 100 ml beaker and dissolve with 20 ml 2.0 g.
of glacial acetic acid R. Add 1 ml of saturated potassium
iodide solution R and allow to stand for 1 min. Add 50 ml STORAGE
of carbon dioxide-free water R and a magnetic stirring bar. In an airtight container, protected from light.

4274 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Poly(vinyl acetate) dispersion 30 per cent

01/2009:2152 — mobile phase B : methanol R2, acetonitrile for

chromatography R, water for chromatography R
POLY(VINYL ACETATE) DISPERSION (5:45:50 V/V/V) ;
30 PER CENT Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 100 0
Poly(vinylis acetas) dispersio 30 per centum
2 - 26 100 → 80 0 → 20
DEFINITION
26 - 27 80 → 0 20 → 100
Dispersion in water of poly(vinyl acetate) having a mean
relative molecular mass of about 450 000. It may contain 27 - 30 0 → 100 100 → 0
Povidone (0685) and a suitable surface-active agent, such as
Flow rate : 1.0 ml/min.
Sodium laurilsulphate (0098), as stabilisers.
Detection : spectrophotometer at 205 nm.
Content : 25.0 per cent to 30.0 per cent of poly(vinyl acetate).
Injection : 10 μl of the test solution and reference
CHARACTERS solutions (a) and (b).
Appearance : opaque, white or almost white, slightly viscous System suitability: reference solution (b) :
liquid. — resolution : minimum 5.0 between the peaks due to vinyl
Solubility : miscible with water and with ethanol (96 per acetate and 1-vinylpyrrolidin-2-one.
cent). Limit :
It is sensitive to spoilage by microbial contaminants. — vinyl acetate : not more than the area of the principal
IDENTIFICATION peak in the chromatogram obtained with reference
solution (a) (100 ppm).
A. Infrared absorption spectrophotometry (2.2.24).
Povidone : maximum 4.0 per cent.
Preparation : dry 1 ml in vacuo, dissolve the residue in
acetone R, and spread 1 drop of the solution between Carry out the determination of nitrogen by sulphuric acid
2 sodium chloride R plates ; remove 1 plate and allow the digestion (2.5.9) on 0.25 g. Calculate the percentage content
solvent to evaporate. of povidone using the following expression :
Comparison : repeat the operation using poly(vinyl
acetate) dispersion 30 per cent CRS.
B. Place 3 ml on a glass plate and allow to dry. A clear film
is formed. N = percentage content of nitrogen.
C. 50 mg gives the reaction of acetyl (2.3.1). Acetic acid. Liquid chromatography (2.2.29).
TESTS Test solution. Mix 0.200 g with water for chromatography R.
Agglomerates. Filter 100.0 g through a tared stainless Sonicate for about 10 min and dilute to 10.0 ml with water
steel sieve (90). Rinse with water R until a clear filtrate is for chromatography R.
obtained and dry to constant mass at 100-105 °C. The mass Reference solution. Dissolve 30.0 mg of acetic acid R and
of the residue is not greater than 0.500 g. 30 mg of citric acid R in the mobile phase. Shake gently to
dissolve and dilute to 100.0 ml with the mobile phase.
Vinyl acetate. Liquid chromatography (2.2.29).
Column :
Test solution. Introduce 0.250 g into a 10 ml volumetric flask
and add about 1 ml of methanol R2. Sonicate. Add about — size: l = 0.25 m, Ø = 4.6 mm ;
8 ml of water for chromatography R. Sonicate and dilute — stationary phase : octadecylsilyl silica gel for
to 10.0 ml with water for chromatography R. Centrifuge chromatography R (5 μm).
for about 10 min and filter. Mobile phase : 0.005 M sulphuric acid.
Reference solution (a). Dissolve 5.0 mg of vinyl acetate CRS Flow rate : 1.0 ml/min.
in methanol R2 and dilute to 10.0 ml with the same solvent. Detection : spectrophotometer at 205 nm.
Dilute 1.0 ml of the solution to 20.0 ml with mobile phase A.
Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Injection : 20 μl. After each injection, rinse the column
with a mixture of equal volumes of acetonitrile for
Reference solution (b). To 5 mg of vinyl acetate R and 5 mg chromatography R and 0.005 M sulphuric acid.
of 1-vinylpyrrolidin-2-one R, add 10 ml of methanol R2 and
sonicate. Dilute to 50 ml with mobile phase A. Dilute 1 ml of Retention time : acetic acid = about 6 min ; citric acid = about
this solution to 20 ml with mobile phase A. 8 min.
A precolumn containing octadecylsilyl silica gel for System suitability : reference solution :
chromatography R (5 μm) may be used if a matrix effect is — resolution : minimum 2.0 between the peaks due to acetic
observed. acid and citric acid.
Column: Limit :
— size : l = 0.25 m, Ø = 4.0 mm ; — acetic acid : not more than the area of the corresponding
— stationary phase : octadecylsilyl silica gel for peak in the chromatogram obtained with the reference
chromatography R (5 μm) ; solution (1.5 per cent).
— temperature : 30 °C. Residue on evaporation : 0.285 g to 0.315 g, determined
Mobile phase : on 1.000 g at 110 °C for 5 h.
— mobile phase A : acetonitrile for chromatography R, Sulphated ash : maximum 0.5 per cent, determined on 1.0 g.
methanol R2, water for chromatography R Heat a silica crucible to redness for 30 min, allow to cool
(5:5:90 V/V/V) ; in a desiccator and weigh. Evenly distribute 1.00 g of the

General Notices (1) apply to all monographs and other texts 4275
Potassium citrate
preparation to be examined in the crucible and weigh. Dry
at 100-105 °C for 1 h and ignite in a muffle furnace at
600 °C ± 25 °C, until the substance is thoroughly charred.
Carry out the test for sulphated ash (2.4.14) on the residue
obtained, starting with “Moisten the substance to be
examined...”.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
ASSAY
Determine the saponification value (2.5.6) on 1.5 g and
calculate the percentage content of poly(vinyl acetate) using
the following expression :
Is = saponification value.
STORAGE
At a temperature of 5 °C to 30 °C. Handle the substance so
as to minimise microbial contamination.
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics
that are recognised as being relevant control parameters
for one or more functions of the substance when used
as an excipient (see chapter 5.15). This section is a
non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process
and the performance of the medicinal product during use.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic are
reported, the control method must be indicated.
The following characteristics may be relevant for poly(vinyl
acetate) dispersion 30 per cent used in the manufacture of
modified-release dosage forms and to mask taste.
Solubility of a film. Place the film obtained in identification
test B in 50 ml of phosphate buffer solution pH 6.8 R whilst
stirring continuously. The film does not dissolve within
30 min.
Apparent viscosity (2.2.10) : maximum 100 mPa·s,
determined using a rotating viscometer at 20 °C and a shear
rate of 100 s-1.
01/2009:0400
POTASSIUM CITRATE
Kalii citras
C6H5K3O7,H2O Mr 324.4
[6100-05-6]
DEFINITION
Tripotassium 2-hydroxypropane-1,2,3-tricarboxylate
monohydrate.
4276 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, granular powder or
transparent crystals, hygroscopic.
Solubility : very soluble in water, practically insoluble in
ethanol (96 per cent).
IDENTIFICATION
A. To 1 ml of solution S (see Tests) add 4 ml of water R. The
solution gives the reaction of citrates (2.3.1).
B. 0.5 ml of solution S gives reaction (b) of potassium (2.3.1).
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100 ml with
the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of
phenolphthalein solution R. Not more than 0.2 ml of 0.1 M
hydrochloric acid or 0.1 M sodium hydroxide is required to
change the colour of the indicator.
Readily carbonisable substances. To 0.20 g of the powdered
substance to be examined add 10 ml of sulphuric acid R and
heat in a water-bath at 90 ± 1 °C for 60 min. Cool rapidly.
The solution is not more intensely coloured than reference
solution Y2 or GY2 (2.2.2, Method II).
Chlorides (2.4.4) : maximum 50 ppm.
Dilute 10 ml of solution S to 15 ml with water R.
Oxalates : maximum 300 ppm.
Dissolve 0.50 g in 4 ml of water R, add 3 ml of hydrochloric
acid R and 1 g of zinc R in granules and heat on a water-bath
for 1 min. Allow to stand for 2 min, decant the liquid
into a test-tube containing 0.25 ml of a 10 g/l solution of
phenylhydrazine hydrochloride R and 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 0.05 g/l solution
of oxalic acid R.
Sulphates (2.4.13) : maximum 150 ppm.
To 10 ml of solution S add 2 ml of hydrochloric acid R1 and
dilute to 15 ml with distilled water R.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Sodium : maximum 0.30 per cent.
Atomic emission spectrometry (2.2.22, Method II).
Test solution. To 10 ml of solution S add 1 ml of dilute
hydrochloric acid R and dilute to 100 ml with distilled
water R.
Reference solutions. Prepare the reference solutions using
a solution of sodium chloride R containing 1 mg of Na per
millilitre diluted as necessary with distilled water R.
Wavelength : 589 nm.
Water (2.5.12) : 4.0 per cent to 7.0 per cent, determined
on 0.250 g. Use a mixture of 1 volume of formamide R
and 2 volumes of methanol R as solvent. After adding the
substance to be examined, stir for 15 min before titrating.
EUROPEAN PHARMACOPOEIA 6.3 Potato starch

ASSAY Test solution. Dissolve 1.00 g of the substance to be

Dissolve 0.150 g in 20 ml of anhydrous acetic acid R, examined in water R and dilute to 100.0 ml with the same
heating to about 50 °C. Allow to cool. Titrate with 0.1 M solvent.
perchloric acid using 0.25 ml of naphtholbenzein solution R Reference solutions. Prepare the reference solutions using
as indicator until a green colour is obtained. the following solution, diluted as necessary with water R :
1 ml of 0.1 M perchloric acid is equivalent to 10.21 mg dissolve 0.5084 g of sodium chloride R, previously dried at
of C6H5K3O7. 100-105 °C for 3 h, in water R and dilute to 1000.0 ml with
the same solvent (200 μg of Na per millilitre).
STORAGE Wavelength : 589 nm.
In an airtight container. Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
01/2008:0920 Pb) R.
corrected 6.3 Loss on drying (2.2.32) : maximum 2.0 per cent, determined
on 1.000 g by drying in an oven at 125-130 °C.
POTASSIUM DIHYDROGEN ASSAY
PHOSPHATE Dissolve 1.000 g in 50 ml of carbon dioxide-free water R.
Titrate with carbonate-free 1 M sodium hydroxide,
Kalii dihydrogenophosphas determining the end-point potentiometrically (2.2.20).
1 ml of 1 M sodium hydroxide is equivalent to 0.1361 g
KH2PO4 Mr 136.1 of KH2PO4.
[7778-77-0]
LABELLING
DEFINITION The label states, where applicable, that the substance is
Content : 98.0 per cent to 100.5 per cent (dried substance). suitable for use in the manufacture of parenteral dosage
forms.
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
01/2009:0355
Solubility : freely soluble in water, practically insoluble in
ethanol (96 per cent).
POTATO STARCH
IDENTIFICATION
A. Solution S (see Tests) is faintly acid (2.2.4). Solani amylum
B. Solution S gives reaction (b) of phosphates (2.3.1).
C. 0.5 ml of solution S gives reaction (b) of potassium (2.3.1). DEFINITION
Potato starch is obtained from the tuber of Solanum
TESTS tuberosum L.
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100 ml with CHARACTERS
the same solvent. Appearance : very fine, white or almost white powder which
Appearance of solution. Solution S is clear (2.2.1) and creaks when pressed between the fingers.
colourless (2.2.2, Method II). Solubility : practically insoluble in cold water and in ethanol
pH (2.2.3) : 4.2 to 4.5. (96 per cent).
To 5 ml of solution S add 5 ml of carbon dioxide-free water R. Potato starch does not contain starch grains of any other
origin. It may contain a minute quantity, if any, of tissue
Reducing substances. To 5 ml of solution S add 5 ml of fragments of the original plant.
dilute sulphuric acid R and 0.25 ml of 0.02 M potassium
permanganate. Heat on a water-bath for 5 min. The colour IDENTIFICATION
of the permanganate is not completely discharged.
A. Examined under a microscope using a mixture of
Chlorides (2.4.4) : maximum 200 ppm. equal volumes of glycerol R and water R, it presents
Dilute 2.5 ml of solution S to 15 ml with water R. granules, either irregularly shaped, ovoid or pear-shaped,
usually 30-100 μm in size but occasionally exceeding
Sulphates (2.4.13) : maximum 300 ppm. 100 μm, or rounded, 10-35 μm in size. There are
To 5 ml of solution S add 0.5 ml of hydrochloric acid R and occasional compound granules having 2-4 components.
dilute to 15 ml with distilled water R. The ovoid and pear-shaped granules have an eccentric
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined hilum and the rounded granules acentric or slightly
on 0.5 g. eccentric hilum. All granules show clearly visible
concentric striations. Between orthogonally orientated
Iron (2.4.9) : maximum 10 ppm, determined on solution S. polarising plates or prisms, the granules show a distinct
Sodium : maximum 0.10 per cent, if intended for use in the black cross intersecting at the hilum.
manufacture of parenteral dosage forms. B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool.
Atomic emission spectrometry (2.2.22, Method I). A thick, opalescent mucilage is formed.

General Notices (1) apply to all monographs and other texts 4277
Pravastatin sodium
C. To 1 ml of the mucilage obtained in identification test B, B. Infrared absorption spectrophotometry (2.2.24).
add 0.05 ml of iodine solution R1. An orange-red to dark
blue colour is produced which disappears on heating.
TESTS
pH (2.2.3) : 5.0 to 8.0.
Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
Foreign matter. Examined under a microscope using a
mixture of equal volumes of glycerol R and water R, not
more than traces of matter other than starch granules are
present. No starch grains of any other origin are present.
Oxidising substances (2.5.30) : maximum 20 ppm, calculated
as H2O2.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
Iron (2.4.9) : maximum 10 ppm.
Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter.
The filtrate complies with the limit test for iron.
Loss on drying (2.2.32) : maximum 20.0 per cent, determined
on 1.000 g by drying in an oven at 130 °C for 90 min.
Sulphated ash (2.4.14) : maximum 0.6 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
01/2009:2059
PRAVASTATIN SODIUM
Pravastatinum natricum
C23H35NaO7 Mr 446.5
[81131-70-6]
DEFINITION
Sodium (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-
hydroxy-2-methyl-8-[[(2S)-2-methylbutanoyl]oxy]-1,2,6,7,8,8a- 0.3 ; impurity A = about 0.6 ; impurity D = about 1.9 ;
hexahydronaphthalen-1-yl]heptanoate.
Content : 97.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or yellowish-white powder or crystalline Limits :
powder, hygroscopic.
Solubility : freely soluble in water and in methanol, soluble
in anhydrous ethanol.
IDENTIFICATION
A. Specific optical rotation (see Tests).
4278 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Comparison : Ph. Eur. reference spectrum of pravastatin
sodium.
C. 1 ml of solution S (see Tests) gives reaction (a) of sodium
(2.3.1).
TESTS
Solution S. Dissolve 1.00 g in carbon dioxide-free water R
and dilute to 20.0 ml with the same solvent.
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY6
(2.2.2, Method II).
Dilute 2.0 ml of solution S to 10.0 ml with water R.
pH (2.2.3) : 7.2 to 9.0 for solution S.
Specific optical rotation (2.2.7) : + 153 to + 159 (anhydrous
substance).
Dilute 2.0 ml of solution S to 20.0 ml with water R.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : methanol R, water R (9:11 V/V).
Test solution (a). Dissolve 0.1000 g of the substance to be
examined in the solvent mixture and dilute to 100.0 ml with
the solvent mixture.
Test solution (b). Dilute 10.0 ml of test solution (a) to
100.0 ml with the solvent mixture.
Reference solution (a). Dissolve the contents of a vial of
pravastatin impurity A CRS in 1.0 ml of test solution (b).
Reference solution (b). Dilute 2.0 ml of test solution (a)
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 (c). Dissolve 12.4 mg of pravastatin
1,1,3,3-tetramethylbutylamine CRS in the solvent mixture
and dilute to 100.0 ml with the solvent mixture.
Column :
— size: l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 25 °C.
Mobile phase : glacial acetic acid R, triethylamine R,
methanol R, water R (1:1:450:550 V/V/V/V).
Flow rate : 1.3 ml/min.
Detection : spectrophotometer at 238 nm.
Injection : 10 μl of test solution (a) and reference solutions (a)
and (b).
Run time : 2.5 times the retention time of pravastatin.
Relative retention with reference to pravastatin
(retention time = about 21 min) : impurity F = about
0.1 ; impurity B = about 0.2 ; impurity E = about
impurity C = about 2.1.
System suitability : reference solution (a) :
— resolution : minimum 7.0 between the peaks due to
impurity A and pravastatin.
— impurity A : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
— impurities B, C, D, E : for each impurity, not more than
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
EUROPEAN PHARMACOPOEIA 6.3 Pravastatin sodium

— impurity F : not more than 0.75 times the area of the B. R1 = R4 = OH, R2 = R3 = R5 = H : (3R,5R)-3,5-
principal peak in the chromatogram obtained with dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-8-[[(2S,
reference solution (b) (0.15 per cent) ; 3R)-3-hydroxy-2-methylbutanoyl]oxy]-2-methyl-1,2,
— unspecified impurities: for each impurity, not more 6,7,8,8a-hexahydronaphthalen-1-yl]heptanoic acid
than 0.5 times the area of the principal peak in the (3″-(R)-hydroxypravastatin),
chromatogram obtained with reference solution (b)
(0.10 per cent) ;
— total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) C. R1 = OH, R2 = R3 = R4 = H, R5 = CH3 :
(0.6 per cent) ; (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-
— disregard limit: 0.25 times the area of the principal peak hydroxy-2-methyl-8-[[(2S)-2-methylpentanoyl]oxy]-1,2,6,7,
in the chromatogram obtained with reference solution (b) 8,8a-hexahydronaphthalen-1-yl]heptanoic acid,
(0.05 per cent).
Ethanol (2.4.24, System A) : maximum 3.0 per cent m/m.
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in a mixture of 15 volumes of water R and E. R1 = R3 = OH, R2 = R4 = R5 = H : (3R,5R)-3,5-
85 volumes of methanol R and dilute to 20 ml with the same dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-8-[[(2S,
mixture of solvents. 12 ml of the solution complies with 3S)-3-hydroxy-2-methylbutanoyl]oxy]-2-methyl-1,2,
test B. Prepare the reference solution using lead standard 6,7,8,8a-hexahydronaphthalen-1-yl]heptanoic acid
solution (2 ppm Pb) obtained by diluting lead standard (3″-(S)-hydroxypravastatin),
solution (100 ppm Pb) R with a mixture of 15 volumes of
water R and 85 volumes of methanol R.
Water (2.5.12) : maximum 4.0 per cent, determined on
0.500 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 C23H35NaO7 using
the chromatogram obtained with reference solution (c)
and the declared content of pravastatin in pravastatin
1,1,3,3-tetramethylbutylamine CRS.
1 mg of pravastatin is equivalent to 1.052 mg of pravastatin
sodium. D. (1S,3S,7S,8S,8aR)-3-hydroxy-8-[2-[(2R,4R)-4-hydroxy-6-
oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl-1,2,3,7,8,
STORAGE 8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate
In an airtight container. (pravastatin lactone),
IMPURITIES
Specified impurities : A, B, C, D, E, F.

A. R1 = R3 = R4 = R5 = H, R2 = OH : (3R,5R)-3,5-dihydroxy-
7-[(1S,2S,6R,8S,8aR)-6-hydroxy-2-methyl-8-[[(2S)-2- F. (3R,5R)-7-[(1S,2S,6S,8S,8aR)-6,8-dihydroxy-2-
methylbutanoyl]oxy]-1,2,6,7,8,8a-hexahydronaphthalen-1- methyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-
yl]heptanoic acid (6′-epipravastatin), dihydroxyheptanoic acid.

General Notices (1) apply to all monographs and other texts 4279
 EUROPEAN PHARMACOPOEIA 6.3

4280 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

R
Racecadotril.. ............................................................................4283 Rice starch.................................................................................4284

General Notices (1) apply to all monographs and other texts 4281
 EUROPEAN PHARMACOPOEIA 6.3

4282 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Racecadotril

07/2008:2171 Column :
corrected 6.3 — size: l = 0.25 m, Ø = 4.0 mm ;
— stationary phase : end-capped octadecylsilyl silica gel
RACECADOTRIL for chromatography R (5 μm) ;
— temperature : 30 °C.
Racecadotrilum Mobile phase :
— mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R in water R, adjust to pH 2.5 with phosphoric
acid R and dilute to 1000 ml with water R ;
— mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 60 40
5 - 25 60 → 20 40 → 80
C21H23NO4S Mr 385.5
[81110-73-8] 25 - 35 20 80

DEFINITION Flow rate : 1.0 ml/min.

Benzyl [[(2RS)-2-[(acetylsulfanyl)methyl]-3-
phenylpropanoyl]amino]acetate.
Content : 98.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, freely soluble in
methanol and in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : racecadotril CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
Dissolve 5.0 g in 10 ml of acetone R.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : mobile phase A, mobile phase B
(50:50 V/V).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 25.0 ml with
the solvent mixture.
Test solution (b). Dilute 5.0 ml of test solution (a) to 25.0 ml
with the solvent mixture.
Reference solution (a). Dilute 1.0 ml of test solution (a)
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). Dilute 500 μl of racecadotril
impurity A CRS in acetonitrile R and dilute to 250.0 ml with
the same solvent. Dilute 1.0 ml of the solution to 10.0 ml
with the solvent mixture. Dilute 1.0 ml of this solution to
100.0 ml with the solvent mixture.
Reference solution (c). Dissolve 5 mg of racecadotril
impurity G CRS in the solvent mixture and dilute to 50 ml
with the solvent mixture. To 5 ml of this solution add 1 ml
of test solution (b) and dilute to 100 ml with the solvent
mixture.
Reference solution (d). Dissolve 50.0 mg of racecadotril CRS ASSAY
in the solvent mixture and dilute to 25.0 ml with the solvent
mixture. Dilute 5.0 ml of this solution to 25.0 ml with the
solvent mixture.
Reference solution (e). Dissolve 2 mg of racecadotril for
peak identification CRS (containing impurities C, E and F) Calculate the percentage content of C21H23NO4S from the
in 1.0 ml of the solvent mixture.
Detection : spectrophotometer at 210 nm.
Injection : 10 μl of the solvent mixture, test solution (a) and
reference solutions (a), (b), (c) and (e).
Identification of impurities : use the chromatogram
supplied with racecadotril for peak identification CRS and
the chromatogram obtained with reference solution (e) to
identify the peaks due to impurities C, E and F.
Relative retention with reference to racecadotril
(retention time = about 16 min) : impurity A = about 0.2 ;
impurity C = about 0.3 ; impurity E = about 0.5 ;
impurity F = about 0.9.
System suitability: reference solution (c) :
— resolution : minimum 1.5 between the peaks due to
impurity G and racecadotril.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity C = 1.4 ;
impurity E = 0.6 ; impurity F = 0.7 ;
— impurities C, E, F : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
— impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
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 : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (b) and reference solution (d).
declared content of racecadotril CRS.

General Notices (1) apply to all monographs and other texts 4283
Rice starch
IMPURITIES
Specified impurities : A, C, E, F.
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) : B, D, G, H.
A. ethanethioic acid (thioacetic acid),
B. R = H : [[(2RS)-2-benzyl-3-sulfanylpropanoyl]amino]acetic
acid,
G. R = CH2-C6H5 : benzyl [[(2RS)-2-benzyl-3-
sulfanylpropanoyl]amino]acetate,
C. (2RS)-2-[(acetylsulfanyl)methyl]-3-phenylpropanoyl]-
amino]acetic acid,
D. R = H : 5,10-dibenzyl-4,11-dioxo-7,8-dithia-3,12-
diazatetradecanedioic acid,
H. R = CH2-C6H5 : dibenzyl 5,10-dibenzyl-4,11-dioxo-7,8-
dithia-3,12-diazatetradecanedioate,
E. R = OH : 2-benzylprop-2-enoic acid (2-benzylacrylic acid),
F. R = NH-CH2-CO-O-CH2-C6H5 : benzyl [(2-benzylprop-2-
enoyl)amino]acetate.
4284 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
01/2009:0349
RICE STARCH
Oryzae amylum
DEFINITION
Rice starch is obtained from the caryopsis of Oryza sativa L.
CHARACTERS
Appearance : very fine, white or almost white powder, which
creaks when pressed between the fingers.
Solubility : practically insoluble in cold water and in ethanol
(96 per cent).
Rice starch does not contain starch grains of any other
origin. It may contain traces of, if any, fragments of the
endosperm tissue of the fruit.
IDENTIFICATION
A. Examined under a microscope using a mixture of equal
volumes of glycerol R and water R, it presents polyhedral,
simple grains 1-10 μm, mostly 4-6 μm, in size. These
simple grains often gather in ellipsoidal, compound grains
50-100 μm in diameter. The grains have a poorly visible
central hilum and there are no concentric striations.
Between orthogonally orientated polarising plates or
prisms, the starch grains show a distinct black cross
intersecting at the hilum.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool.
A thin, cloudy mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B
add 0.05 ml of iodine solution R1. An orange-red to dark
blue colour is produced, which disappears on heating.
TESTS
pH (2.2.3) : 5.0 to 8.0.
Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
Iron (2.4.9) : maximum 10 ppm for the filtrate.
Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter.
Foreign matter. Examine under a microscope using a
mixture of equal volumes of glycerol R and water R. Not
more than traces of matter other than starch granules are
present. No starch grains of any other origin are present.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.00 g by drying in an oven at 130 °C for 90 min.
Sulphated ash (2.4.14) : maximum 0.6 per cent, determined
on 1.0 g.
Oxidising substances (2.5.30) : maximum 0.002 per cent,
calculated as H2O2.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
EUROPEAN PHARMACOPOEIA 6.3
S
Saquinavir mesilate.. ...............................................................4287
Schisandra fruit........................................................................4288
Senna leaf dry extract, standardised.. .................................4289
Sertraline hydrochloride.. ......................................................4290
Sesame oil, refined.. ................................................................4292
Sevoflurane.. .............................................................................4294
Sodium alendronate.. ..............................................................4296
Sodium alginate.. .....................................................................4297
Sodium ascorbate.. ..................................................................4298
Sodium glycerophosphate, hydrated.. .................................4299
Sodium hyaluronate.. ..............................................................4300
Sodium molybdate dihydrate.. ..............................................4302
General Notices (1) apply to all monographs and other texts
Sodium polystyrene sulphonate.. .........................................4303
Sodium stearate.. .....................................................................4304
Sorbitol.......................................................................................4305
Sorbitol, liquid, partially dehydrated...................................4307
Stanozolol..................................................................................4308
Starch, pregelatinised.. ...........................................................4308
St. John’s wort dry extract, quantified................................4309
Sucrose....................................................................................... 4311
Sugar spheres.. ......................................................................... 4312
Sultamicillin tosilate dihydrate............................................. 4313
Sumatriptan succinate............................................................ 4315
4285
 EUROPEAN PHARMACOPOEIA 6.3

4286 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Saquinavir mesilate

01/2009:2267 Reference solution (c). Dissolve 30.0 mg of saquinavir

mesilate CRS in the solvent mixture, using sonication, and
dilute to 100.0 ml with the same solvent.
SAQUINAVIR MESILATE
Column :
Saquinaviri mesilas — size: l = 0.15 m, Ø = 4.6 mm ;
— stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (3.5 μm).
Mobile phase :
— mobile phase A : to 2.5 ml of strong sodium hydroxide
solution R add 900 ml of water for chromatography R,
adjust to pH 1.8 with perchloric acid R and dilute to
1000 ml with water for chromatography R ;
— mobile phase B : mobile phase A, acetonitrile R1
(38:62 V/V) ;
Time Mobile phase A Mobile phase B
C39H54N6O8S Mr 767 (min) (per cent) (per cent)
[149845-06-7] 0-1 50 50
1 - 31 50 → 0 50 → 100
DEFINITION
(2S)-N1-[(1S,2R)-1-Benzyl-3-[(3S,4aS,8aS)-3-[(1,1-dimethyl- Flow rate : 1.0 ml/min.
ethyl)carbamoyl]octahydroisoquinolin-2(1H)-yl]-2-hydroxy- Detection : spectrophotometer at 210 nm.
propyl]-2-[(quinolin-2-ylcarbonyl)amino]butanediamide Injection : 10 μl of the test solution and reference
methanesulphonate. solutions (a) and (b).
Content : 97.5 per cent to 102.0 per cent (anhydrous Identification of impurities : use the chromatogram
substance). supplied with saquinavir for system suitability CRS and
PRODUCTION the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, B, C and D.
The production method must be evaluated to determine
the potential for formation of alkyl mesilates, which is Relative retention with reference to saquinavir
particularly likely to occur if the reaction medium contains (retention time = about 17 min) : impurity A = about 0.2 ;
lower alcohols. Where necessary, the production method impurity B = about 0.3 ; impurity C = about 0.5 ;
is validated to demonstrate that alkyl mesilates are not impurity D = about 0.9.
detectable in the final product. System suitability : reference solution (b) :
— peak-to-valley ratio : minimum 3, where Hp = height above
CHARACTERS the baseline of the peak due to impurity D and Hv = height
Appearance : white or almost white, slightly hygroscopic above the baseline of the lowest point of the curve
powder. separating this peak from the peak due to saquinavir.
Solubility : practically insoluble in water, sparingly soluble Limits :
in methanol, slightly soluble in ethanol (96 per cent). — correction factors : for the calculation of content,
IDENTIFICATION multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.5 ;
A. Specific optical rotation (see Tests). impurity B = 0.5 ; impurity C = 2.5 ;
B. Infrared absorption spectrophotometry (2.2.24). — impurities A, B, C : for each impurity, not more
Comparison : saquinavir mesilate CRS. than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
TESTS (0.15 per cent) ;
Specific optical rotation (2.2.7) : − 35.0 to − 42.0 (anhydrous — unspecified impurities : for each impurity, not more
substance). than 0.5 times the area of the principal peak in the
Dissolve 0.25 g in anhydrous methanol R and dilute to chromatogram obtained with reference solution (a)
50.0 ml with the same solvent. (0.05 per cent) ;
Related substances. Liquid chromatography (2.2.29). — total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Solvent mixture : water for chromatography R,
(0.5 per cent) ;
acetonitrile R1 (47:53 V/V).
— disregard limit : not more than 0.3 times the area of
Test solution. Dissolve 30.0 mg of the substance to be
the principal peak in the chromatogram obtained with
examined in the solvent mixture, using sonication, and dilute
reference solution (a) (0.03 per cent).
to 100.0 ml with the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution Heavy metals (2.4.8) : maximum 10 ppm.
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this 0.50 g complies with test G. Prepare the reference solution
solution to 10.0 ml with the solvent mixture. using 0.5 ml of lead standard solution (10 ppm Pb) R. The
Reference solution (b). Dissolve the contents of a vial solution may become yellow again after pH-adjustment.
of saquinavir for system suitability CRS (containing Filter the solutions through a membrane filter (0.45 μm).
impurities A, B, C and D) in 1.0 ml of the solvent mixture Water (2.5.12) : maximum 1.0 per cent, determined on
and sonicate for 2 min. 0.250 g.

General Notices (1) apply to all monographs and other texts 4287
Schisandra fruit
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 F. R1 = H, R2 = CH -CN : N-[(1S)-2-[[(1S,2R)-1-benzyl-
related substances with the following modification.
Injection : 10 μl of the test solution and reference solution (c).
Calculate the percentage content of saquinavir mesilate from
the declared content of saquinavir mesilate CRS.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C.
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) : D, E, F, G, H.
A. R = H : (2S)-4-amino-4-oxo-2-[(quinolin-2-
ylcarbonyl)amino]butanoic acid,
B. R = C2H5 : ethyl (2S)-4-amino-4-oxo-2-[(quinolin-2-
ylcarbonyl)amino]butanoate,
C. (3S,4aS,8aS)-2-[(2R,3S)-3-amino-2-hydroxy-4-phenylbutyl]-
N-(1,1-dimethylethyl)decahydroisoquinoline-3-
carboxamide,
D. R1 = CH2-CO-NH2, R2 = H : (2R)-N1-[(1S,2R)-1-benzyl-
3-[(3S,4aS,8aS)-3-[(1,1-dimethylethyl)carbamoyl]octa-
hydroisoquinolin-2(1H)-yl]-2-hydroxypropyl]-2-[(quinolin-
2-ylcarbonyl)amino]butanediamide (2-epi-saquinavir),
4288 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
E. R1 = H, R2 = CH2-CO2H : (3S)-4-[[(1S,2R)-1-benzyl-
3-[(3S,4aS,8aS)-3-[(1,1-dimethylethyl)carbamoyl]octa-
hydroisoquinolin-2(1H)-yl]-2-hydroxypropyl]amino]-4-oxo-
3-[(quinolin-2-ylcarbonyl)amino]butanoic acid,
2
3-[(3S,4aS,8aS)-3-[(1,1-dimethylethyl)carbamoyl]octa-
hydroisoquinolin-2(1H)-yl]-2-hydroxypropyl]amino]-1-
(cyanomethyl)-2-oxoethyl]quinoline-2-carboxamide,
G. R1 = H, R2 = CH2-CO-OCH3 : methyl (3S)-4-
[[(1S,2R)-1-benzyl-3-[(3S,4aS,8aS)-3-[(1,1-
dimethylethyl)carbamoyl]octahydroisoquinolin-
2(1H)-yl]-2-hydroxypropyl]amino]-4-oxo-3-[(quinolin-2-
ylcarbonyl)amino]butanoate,
H. N-[(3S)-1-[(1S,2R)-1-benzyl-3-[(3S,4aS,8aS)-3-[(1,1-
dimethylethyl)carbamoyl]octahydroisoquinolin-2(1H)-
yl]-2-hydroxypropyl]-2,5-dioxopyrrolidin-3-yl]quinoline-2-
carboxamide.
01/2009:2428
SCHISANDRA FRUIT
Schisandrae chinensis fructus
DEFINITION
Whole, dried or steamed and dried, ripe fruit of Schisandra
chinensis (Turcz.) Baill.
Content : minimum 0.40 per cent of schisandrin (C24H32O7 ;
Mr 432.5) (dried drug).
IDENTIFICATION
A. The berry is more or less spherical, up to 8 mm in
diameter ; red, reddish-brown or blackish outer surface,
sometimes covered in a whitish frost ; strongly shrivelled
pericarp ; presence of 1-2 reniform, yellowish-brown,
lustrous seeds, with thin seed-coat.
B. Reduce to a powder (355) (2.9.12). The powder is
reddish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows the
following diagnostic characters : reddish-brown fragments
of pericarp, consisting of 1 layer of thin-walled epicarp
cells, accompanied by sparse oil cells and several layers
of ovoid, more-or-less flattened mesocarp cells ; fragments
of the outer testa of the seed consisting of thick-walled,
finely channelled sclereids, polygonal in surface view
(15-50 μm in diameter) and in palisade arrangement in
side view ; fragments of the inner testa with sclereids,
isolated or in small groups, about 80 μm in diameter,
with slightly thickened and markedly channelled walls ;
fragments of endosperm consisting of polyhedral cells
containing oil droplets and aleurone grains. Examine
under a microscope using a 50 per cent V/V solution of
glycerol R : the powder shows parenchymatous cells of
the mesocarp containing numerous small, round starch
granules.
C. Examine the chromatograms obtained in the test for
Schisandra sphenanthera.
EUROPEAN PHARMACOPOEIA 6.3 Senna leaf dry extract, standardised

Results A : see below the sequence of quenching zones Test solution. Weigh 1.250 g of the powdered drug (355)
present in the chromatograms obtained with the reference (2.9.12) into a 250 ml conical flask, add 90 ml of methanol R
solution and the test solution. Furthermore, other weak and sonicate for 30 min. Filter the solution into a volumetric
quenching zones may be present in the chromatogram flask, add 10 ml of methanol R whilst rinsing the filter and
obtained with the test solution. dilute to 100.0 ml with the same solvent.
Top of the plate Reference solution. Dissolve 5.0 mg of schisandrin R in
methanol R and dilute to 100.0 ml with the same solvent.
γ-Schisandrin : a quenching zone A quenching zone (γ-schisandrin)
Column :
_______ _______
— size: l = 0.25 m, Ø = 4.6 mm ;
A weak quenching zone — stationary phase : end-capped octadecylsilyl silica gel
_______ _______ for chromatography R ;
Schisandrin : a quenching zone A quenching zone (schisandrin) — temperature : 25 °C.
Reference solution Test solution Mobile phase :
— mobile phase A : water R, methanol R (35:65 V/V) ;
Results B : see below the sequence of zones present in — mobile phase B : methanol R ;
the chromatograms obtained with the reference solution
and the test solution. Furthermore, other faint zones Time Mobile phase A Mobile phase B
may be present in the chromatogram obtained with the (min) (per cent V/V) (per cent V/V)
test solution. 0 - 10 100 0

Top of the plate 10 - 16 100 → 58 0 → 42

γ-Schisandrin : a brown zone A brown zone (γ-schisandrin) 16 - 26 58 42

_______ _______
Flow rate : 1 ml/min.
Detection : spectrophotometer at 250 nm.
_______ _______ Injection : 10 μl.
Schisandrin : an intense, An intense, brownish-green zone Retention time : schisandrin = about 8 min.
brownish-green zone (schisandrin)
System suitability :
Reference solution Test solution
— number of theoretical plates : minimum 5000, calculated
for the peak due to schisandrin in the chromatogram
TESTS obtained with the reference solution.
Schisandra sphenanthera. Thin-layer chromatography Calculate the percentage content of schisandrin using the
(2.2.27). following expression :
Test solution. To 2.5 g of the powdered drug (355) (2.9.12)
add 10 ml of methanol R. Extract at 25 °C in an ultrasonic
bath for 5 min and centrifuge.
Reference solution. Dissolve 5 mg of schisandrin R and A1 = area of the peak due to schisandrin in the
5 mg of γ-schisandrin R in 5 ml of methanol R. chromatogram obtained with the test solution ;
Plate : TLC silica gel F254 plate R (5-40 μm) [or TLC silica A2 = area of the peak due to schisandrin in the
gel F254 plate R (2-10 μm)]. chromatogram obtained with the reference
Mobile phase : acetic acid R, ethyl acetate R, toluene R solution ;
(2:22:46 V/V/V). m1 = mass of the drug to be examined used to prepare
the test solution, in grams ;
Application : 5 μl [or 2 μl] as bands of 10 mm [or 6 mm].
m2 = mass of schisandrin R used to prepare the
Development : over a path of 10 cm [or 7 cm]. reference solution, in grams ;
Drying : in air. p = percentage content of schisandrin in
Detection A : examine in ultraviolet light at 254 nm. schisandrin R.
Detection B : spray with a 100 g/l solution of sulphuric
acid R in methanol R and heat in an oven at 120 °C for
7 min ; examine in daylight.
01/2009:1261
Results B : the chromatogram obtained with the test
solution shows a zone due to schisandrin and a zone due
to γ-schisandrin ; the chromatogram shows no intense SENNA LEAF DRY EXTRACT,
violet-pink zone in the middle third. STANDARDISED
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered drug (355) (2.9.12) by drying Sennae folii extractum siccum normatum
in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 6.0 per cent. [8055-96-7]
ASSAY DEFINITION
Liquid chromatography (2.2.29). Standardised dry extract produced from Senna leaf (0206).

General Notices (1) apply to all monographs and other texts 4289
Sertraline hydrochloride
Content : 5.5 per cent to 8.0 per cent of hydroxyanthracene
glycosides, expressed as sennoside B (C42H38O20 ; Mr 863)
(dried extract). The measured content does not deviate from
the value stated on the label by more than ± 10 per cent.
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-80 per cent V/V).
CHARACTERS
Appearance : brownish or brown powder.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Solvent mixture : ethanol (96 per cent) R, water R
(50:50 V/V).
Test solution. To 0.1 g of the extract to be examined add layers to a volumetric flask and dilute to 100.0 ml with
5 ml of the solvent mixture and heat to boiling. Cool and ether R. Evaporate 10.0 ml carefully to dryness and dissolve
centrifuge. Use the supernatant liquid.
Reference solution. Dissolve 10 mg of senna extract CRS acetate R in methanol R. Measure the absorbance (2.2.25)
in 1 ml of the solvent mixture (a slight residue remains). at 515 nm using methanol R as the compensation liquid.
Plate : TLC silica gel plate R.
Mobile phase : glacial acetic acid R, water R, ethyl
acetate R, 1-propanol R (1:30:40:40 V/V/V/V).
Application : 10 μl as bands.
Development : over a path of 10 cm.
Drying : in air.
Detection : spray with a 20 per cent V/V solution of nitric A
acid R and heat at 120 °C for 10 min ; allow to cool and
spray with a 50 g/l solution of potassium hydroxide R in
ethanol (50 per cent V/V) R until the zones appear.
Results : the principal zones in the chromatogram
obtained with the test solution are similar in position,
colour and size to the principal zones in the chromatogram
obtained with the reference solution. The chromatograms
show in the lower third a prominent brown zone due
to sennoside B and above it a yellow zone followed by
another prominent brown zone due to sennoside A. In the
upper half of the chromatograms are visible, in order of
increasing RF value, a prominent reddish-brown zone and
an orange-brown zone followed by a faint pink zone and
2 yellow zones. Close to the solvent front a dark pink zone
appears, which may be followed by several faint zones.
B. Place about 25 mg of the extract to be examined in
a conical flask and add 50 ml of water R and 2 ml of
hydrochloric acid R. Heat in a water-bath for 15 min,
cool and shake with 40 ml of ether R. Separate the ether
layer, dry over anhydrous sodium sulphate R, evaporate
5 ml to dryness and to the cooled residue add 5 ml of
dilute ammonia R1. A yellow or orange colour develops.
Heat on a water-bath for 2 min. A reddish-violet colour
develops.
TESTS
Loss on drying (2.8.17) : maximum 5.0 per cent.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
ASSAY
Carry out the assay protected from bright light.
Place 0.150 g of the extract to be examined in a 100 ml
flask, dissolve in water R and dilute to 100.0 ml with the
same solvent. Filter the solution, discard the first 10 ml
4290 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
of the filtrate. Transfer 20.0 ml of the filtrate to a 150 ml
separating funnel. Add 0.1 ml of dilute hydrochloric acid R
and shake with 3 quantities, each of 15 ml, of ether R. Allow
the layers to separate and discard the ether layer. Add 0.10 g
of sodium hydrogen carbonate R to the aqueous layer and
shake for 3 min. Centrifuge and transfer 10.0 ml of the
supernatant liquid to a 100 ml round-bottomed flask with a
ground-glass neck. Add 20 ml of ferric chloride solution R1
and mix. Heat for 20 min under a reflux condenser in a
water-bath with the water level above that of the liquid in the
flask ; add 3 ml of hydrochloric acid R and heat for a further
30 min with frequent shaking to dissolve the precipitate.
Cool, transfer the mixture to a separating funnel and shake
with 3 quantities, each of 25 ml, of ether R previously used
to rinse the flask. Combine the ether layers and wash with
2 quantities, each of 15 ml, of water R. Transfer the ether
the residue in 10.0 ml of a 5.0 g/l solution of magnesium
Calculate the percentage content of hydroxyanthracene
glycosides expressed as sennoside B using the following
expression :
i.e. taking the specific absorbance of sennoside B to be 240.
= absorbance at 515 nm ;
m
= mass of the herbal drug to be examined, in grams.
LABELLING
The label states the content of hydroxyanthracene glycosides.
04/2008:1705
corrected 6.3
SERTRALINE HYDROCHLORIDE
Sertralini hydrochloridum
C17H18Cl3N Mr 342.7
[79559-97-0]
DEFINITION
(1S,4S)-4-(3,4-Dichlorophenyl)-N-methyl-1,2,3,4-
tetrahydronaphthalen-1-amine hydrochloride.
Content : 97.5 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water, freely soluble in
anhydrous ethanol, slightly soluble in acetone and in
isopropanol.
It shows polymorphism (5.9).
EUROPEAN PHARMACOPOEIA 6.3
IDENTIFICATION
A. Specific optical rotation (2.2.7) : + 38.8 to + 43.0
(anhydrous substance), measured at 25 °C.
Solvent mixture. Dilute 1 volume of a 103 g/l solution of
hydrochloric acid R to 20 volumes with methanol R.
Dissolve 0.250 g in the solvent mixture and dilute to
25.0 ml with the solvent mixture.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : sertraline hydrochloride CRS.
If the spectra obtained in the solid state show differences,
record new spectra using 10 g/l solutions in methylene
chloride R.
C. Dissolve 10 mg in 5 ml of anhydrous ethanol R and
add 5 ml of water R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
Enantiomeric purity. Liquid chromatography (2.2.29).
Solvent mixture : diethylamine R, hexane R, 2-propanol R Related substances. Gas chromatography (2.2.28) : use the
(1:40:60 V/V/V).
Test solution. Dissolve 60.0 mg of the substance to be
examined in the solvent mixture and dilute to 10.0 ml with
the solvent mixture.
Reference solution (a). Dissolve the contents of a vial of
sertraline for system suitability CRS (containing impurity G) Add 8.0 ml of methylene chloride R, stopper the tube and
in 1.0 ml of solvent mixture.
Reference solution (b). Dilute 0.5 ml of the test solution to
100.0 ml with the solvent mixture.
Column:
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : silica gel AD for chiral separation R
(5 μm).
Mobile phase : mix 30 volumes of hexane R and 70 volumes Column :
of a mixture of 1 volume of diethylamine R, 25 volumes of
2-propanol R and 975 volumes of hexane R.
Flow rate : 0.4 ml/min.
Detection : spectrophotometer at 275 nm.
Injection : 20 μl.
Run time : 30 min.
Elution order : sertraline, impurity G.
System suitability:
— resolution : minimum 1.5 between the peaks due to
sertraline and impurity G in the chromatogram obtained
with reference solution (a) ;
— signal-to-noise ratio : minimum 10 for the peak due to
sertraline in the chromatogram obtained with reference
solution (b).
Limit :
— impurity G : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (1.5 per cent).
Impurity E. Thin-layer chromatography (2.2.27).
Solvent mixture : methanol R, methylene chloride R
(50:50 V/V).
Test solution. Dissolve 0.500 g of the substance to be
examined in the solvent mixture and dilute to 10.0 ml with
the solvent mixture.
Reference solution (a). Dissolve 5.0 mg of mandelic acid R time = about 24 min) : impurity B = about 0.5 ;
(impurity E) in the solvent mixture and dilute to 50.0 ml
with the solvent mixture.
General Notices (1) apply to all monographs and other texts
Sertraline hydrochloride
Reference solution (b). Dissolve 5 mg of mandelic acid R
(impurity E) and 5 mg of the substance to be examined with
the solvent mixture and dilute to 50.0 ml with the solvent
mixture.
Plate : TLC silica gel F254 plate R.
Mobile phase : dilute ammonia R2, methanol R, methylene
chloride R (15:50:120 V/V/V).
Application : 50 μl as bands of about 4 cm. Allow to dry.
Development : over 2/3 of the plate.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
System suitability: reference solution (b) :
— the chromatogram shows 2 clearly separated zones.
Limit :
— impurity E : any zone due to impurity E is not more
intense than the zone in the chromatogram obtained with
reference solution (a) (0.2 per cent).
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.
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.
Reference solution (a). Dissolve the contents of a vial
of sertraline for peak identification CRS (containing
impurities A, B, C and F) in 0.2 ml of methylene chloride R.
Reference solution (b). Dilute 1.0 ml of the test solution to
100.0 ml with methylene chloride R. Dilute 1.0 ml of this
solution to 20.0 ml with methylene chloride R.
— material: fused silica ;
— size: l = 30 m, Ø = 0.53 mm ;
— stationary phase : polymethylphenylsiloxane R (film
thickness 1.0 μm).
Carrier gas : helium for chromatography R.
Flow rate : 9 ml/min.
Split ratio : 1:10.
Temperature :
Time Temperature
(min) (°C)
Column 0-1 200
1 - 31 200 → 260
31 - 39 260
Injection port 250
Detector 280
Detection : flame ionisation.
Injection : 1 μl.
Identification of impurities : use the chromatogram
supplied with sertraline for peak identification CRS and
the chromatogram obtained with reference solution (a) to
identify the peaks due to impurities A, B, C and F.
Relative retention with reference to sertraline (retention
impurities C and D = about 0.7 ; impurity A = about 1.05 ;
impurity F = about 1.1.
4291
Sesame oil, refined EUROPEAN PHARMACOPOEIA 6.3

System suitability : reference solution (a) :

— peak-to-valley ratio : minimum 15, where Hp = height
above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to sertraline.
Limits :
— impurities A, B, F : for each impurity, maximum 0.2 per
cent ;
— sum of impurities C and D : maximum 0.8 per cent ; A. R1 = NH-CH3, R2 = H, R3 = R4 = Cl : (1RS,4SR)-4-(3,4-
— unspecified impurities: for each impurity, maximum dichlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-
0.10 per cent ; 1-amine,
— total : maximum 1.5 per cent ; B. R1 = R3 = R4 = H, R2 = NH-CH3 : (1RS,4RS)-N-methyl-4-
— disregard limit: the area of the principal peak in the phenyl-1,2,3,4-tetrahydronaphthalen-1-amine,
chromatogram obtained with reference solution (b) C. R1 = R3 = H, R2 = NH-CH3, R4 = Cl : (1RS,4RS)-4-(4-
(0.05 per cent). chlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-1-
Heavy metals (2.4.8) : maximum 20 ppm. amine,
Dissolve 2.0 g in ethanol (96 per cent) R and dilute to 20.0 ml D. R1 = R4 = H, R2 = NH-CH3, R3 = Cl : (1RS,4RS)-4-(3-
with the same solvent. 12 ml of the solution complies with chlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-1-
test B. Prepare the reference solution using lead standard amine,
solution (2 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R with ethanol (96 per cent) R.
Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
on 1.0 g. E. (2R)-hydroxyphenylacetic acid ((R)-mandelic acid),
ASSAY
Liquid chromatography (2.2.29).
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.
Test solution. Dissolve 55.0 mg of the substance to be
examined 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. F. (4R)-4-(3,4-dichlorophenyl)-3,4-dihydronaphthalen-1(2H)-
one,
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, Ø = 3.9 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (4 μm) ;
— temperature : 30 °C.
Mobile phase : methanol R, buffer solution, acetonitrile R G. (1R,4R)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-
(15:40:45 V/V/V). tetrahydronaphthalen-1-amine (sertraline enantiomer).
Flow rate : 1.8 ml/min. 01/2008:0433
Detection : spectrophotometer at 254 nm. corrected 6.3
Injection : 20 μl.
Run time : twice the retention time of sertraline. SESAME OIL, REFINED
Retention time : sertraline = about 1.9 min. Sesami oleum raffinatum
Calculate the percentage content of C17H18Cl3N from the
declared content of sertraline hydrochloride CRS. DEFINITION
Fatty oil obtained from the ripe seeds of Sesamum
STORAGE indicum L. by expression or extraction. It is then refined.
Improved colour and odour may be obtained by further
Protected from light.
refining. It may contain a suitable antioxidant.
IMPURITIES CHARACTERS
Specified impurities : A, B, C, D, E, F, G. Appearance : clear, light yellow liquid, almost colourless.

4292 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Solubility : practically insoluble in ethanol (96 per cent),
miscible with light petroleum.
Relative density : about 0.919.
Refractive index : about 1.473.
It solidifies to a butter-like mass at about − 4 °C.
IDENTIFICATION
First identification : A.
Second identification : B.
A. Composition of triglycerides (see Tests).
B. Identification of fatty oils by thin-layer chromatography
(2.3.2).
Results : the chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2-1.
TESTS
Acid value (2.5.1) : maximum 0.5, determined on 10.0 g ;
maximum 0.3 if intended for use in the manufacture of
parenteral preparations.
Peroxide value (2.5.5) : maximum 10.0 ; maximum 5.0
if intended for use in the manufacture of parenteral
preparations.
Unsaponifiable matter (2.5.7) : maximum 2.0 per cent,
determined on 5.0 g.
Alkaline impurities (2.4.19). It complies with the test for
alkaline impurities in fatty oils.
Cottonseed oil. Mix 5 ml in a test-tube with 5 ml of a
mixture of equal volumes of pentanol R and a 10 g/l
solution of sulphur R in carbon disulphide R. Warm the
mixture carefully until the carbon disulphide is expelled, and
immerse the tube to 1/3 of its depth in boiling saturated
sodium chloride solution R. No reddish colour develops
within 15 min.
Composition of triglycerides. Liquid chromatography
(2.2.29).
Test solution. Dilute 50.0 mg of the substance to be
examined to 10.0 ml with a mixture of equal volumes of
acetone R and methylene chloride R.
Reference solutions. Dissolve 80.0 mg of triolein R in a
mixture of equal volumes of acetone R and methylene
chloride R and dilute to 50.0 ml with the same mixture of
solvents. Prepare 5 reference solutions by dilution of this
solution so as to cover concentrations ranging from the
disregard limit (0.5 per cent) to the upper limit for OLL
(30.0 per cent).
Plot the logarithm of the area of the peak due to triolein
against the logarithm of the concentration of triolein in the In an airtight, well-filled container, protected from light ;
reference solution.
Column: 2 columns coupled in series :
— size of each column : l = 0.25 m, Ø = 4 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (4 μm).
Mobile phase :
— mobile phase A : acetone R, methylene chloride R,
acetonitrile R (5:15:80 V/V/V) ;
— mobile phase B : acetone R, acetonitrile R, methylene
chloride R (20:20:60 V/V/V) ;
General Notices (1) apply to all monographs and other texts
Sesame oil, refined
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 100 → 75 0 → 25
15 - 25 75 25
25 - 70 75 → 0 25 → 100
70 - 75 0 → 100 100 → 0
75 - 80 100 0
Flow rate : 1.0 ml/min.
Detection : evaporative light-scattering detector ; the
following settings have been found to be suitable ; if the
detector has different setting parameters, adjust the detector
settings so as to comply with the system suitability criterion :
— carrier gas : nitrogen R ;
— flow rate : 0.7 litre/min ;
— evaporator temperature : 85 °C ;
— nebuliser temperature : 45 °C.
Injection : 20 μl.
Identification of peaks: use the chromatograms obtained
with the reference solutions to identify the peak due to
triolein ; identify the other peaks using the chromatogram
shown in Figure 0433.-1. The fatty acids are designated
as linolenic (Ln), linoleic (L), oleic (O), palmitic (P) and
stearic (S).
System suitability : test solution :
— resolution : minimum 1.5 between the peaks due to OOO
(triolein) and SOL.
Using the calibration curve obtained with the reference
solutions, determine the percentage content of each peak
with an area greater than that of the peak corresponding to
the disregard limit (0.5 per cent). Assuming that the sum of
these peak areas is 100 per cent, normalise the percentage
content of each of the 8 triglycerides specified below.
Composition of triglycerides:
— LLL : 7.0 per cent to 19.0 per cent ;
— OLL : 13.0 per cent to 30.0 per cent ;
— PLL : 5.0 per cent to 9.0 per cent ;
— OOL : 12.0 per cent to 23.0 per cent ;
— POL : 6.0 per cent to 14.0 per cent ;
— OOO : 5.0 per cent to 14.0 per cent ;
— SOL : 2.0 per cent to 8.0 per cent ;
— POO : 2.0 per cent to 10.0 per cent.
Water (2.5.12) : maximum 0.05 per cent, determined on
5.0 g, if intended for use in the manufacture of parenteral
preparations.
STORAGE
if intended for use in the manufacture of parenteral
preparations store under an inert gas in an airtight container.
When the container has been opened, its contents are to be
used as soon as possible. Any part of the contents not used
at once is protected by an atmosphere of an inert gas.
LABELLING
The label states :
— whether the oil is obtained by expression or extraction ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations ;
— where applicable, the name of the inert gas used.
4293
Sevoflurane EUROPEAN PHARMACOPOEIA 6.3

1. LLLn 4. OLLn 7. PLL 10. POL 13. SOL 16. PPO 19. SSL
2. OLnLn 5. OLL 8. OOL 11. PPL 14. POO 17. SOO 20. PPS
3. LLL 6. OOLn 9. SLL 12. OOO 15. PSL 18. PSO 21. SSO

Figure 0433.-1. – Chromatogram for the composition of triglycerides in refined sesame oil
01/2009:2269 Preparation : examine the substance in the gaseous state
or in the liquid state.
SEVOFLURANE Comparison : sevoflurane CRS.
TESTS
Sevofluranum Acidity or alkalinity. Introduce 20.0 ml of the substance to
be examined and 20 ml of carbon dioxide-free water R into a
separating funnel, shake for 3 min and allow to stand. Collect
the aqueous upper layer and add 0.2 ml of bromocresol
purple solution R. Not more than 0.10 ml of 0.01 M sodium
C 4 H3 F 7 O Mr 200.1 hydroxide or not more than 0.60 ml of 0.01 M hydrochloric
[28523-86-6] acid is required to change the colour of the indicator.
DEFINITION Refractive index (2.2.6) : 1.2745 to 1.2760.
1,1,1,3,3,3-Hexafluoro-2-(fluoromethoxy)propane. Related substances. Gas chromatography (2.2.28).
Internal standard : methylal R.
CHARACTERS
Test solution. Introduce 20.0 ml of the substance to be
Appearance : clear, colourless, volatile liquid. examined into a vial and seal with a cap and septum. Using
Solubility : slightly soluble in water, miscible with ethanol a microsyringe, add 5 μl of the internal standard and mix
(96 per cent). thoroughly.
Relative density : about 1.52. Reference solution (a). Introduce 2.0 ml of ethylene
bp : about 59 °C. chloride R into a screw-cap vial and immediately seal with
a cap and septum. Using a microsyringe, add about 20 μl
It is non-flammable. of the substance to be examined. Record the quantity
It decomposes in the presence of Lewis acids ; this added, in milligrams, of the substance to be examined (M2).
decomposition is inhibited by water in sufficient quantity. Then, using a microsyringe, add about 20 μl of the internal
standard. Record the quantity added, in milligrams, of the
IDENTIFICATION internal standard (M1).
Infrared absorption spectrophotometry (2.2.24).

4294 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Sevoflurane

Reference solution (b): sevoflurane CRS (containing 0.859 = relative density of the internal standard ;
impurities A and B).
1.52 = relative density of sevoflurane ;
Reference solution (c). Introduce 20.0 ml of ethylene
chloride R into a vial and seal with a cap and septum. Using R1 = ratio of the area of the peak due to the impurity to
a microsyringe, add 20 μl of the substance to be examined the area of the peak due to the internal standard
and mix thoroughly. Dilute 0.5 ml of this solution to 100.0 ml from the chromatogram obtained with the test
with ethylene chloride R. solution ;
F1 = relative response factor for reference solution (a).
Column:
— material: fused silica ; Limits :
— impurity A : maximum 25 ppm ;
— size : l = 30 m, Ø = 0.32 mm ;
— impurity B : maximum 100 ppm ;
— stationary phase : poly[(cyanopropyl)(phenyl)]-
[dimethyl]siloxane R (film thickness 3 μm). — unspecified impurities : for each impurity, maximum
100 ppm ;
Carrier gas: helium for chromatography R.
— total : maximum 300 ppm ;
Flow rate : 1.0 ml/min. — disregard limit : the area of the peak due to sevoflurane
Split ratio : 1:20. in the chromatogram obtained with reference solution (c)
Temperature : (5 ppm).
Fluorides : maximum 2 μg/ml.
Time Temperature
(min) (°C)
Potentiometry (2.2.36, Method I). Use plastic utensils
throughout this test.
Column 0 - 10 40
Buffer solution. Dissolve 0.5 g of sodium citrate R and 55 g
10 - 26 40 → 200 of sodium chloride R in 350 ml of water R. Carefully add
26 - 40 200 75 g of sodium hydroxide R and shake to dissolve. Cool to
room temperature and carefully add 225 ml of glacial acetic
Injection port 200
acid R while stirring. Cool and add 300 ml of isopropyl
Detector 225 alcohol R. Dilute with water R to 1000.0 ml. The apparent
pH of this solution is between 5.0 and 5.5.
Detection : flame ionisation. Test solution. Introduce 50.0 ml of the substance to be
Injection : 2 μl. examined and 50.0 ml of water R into a separating funnel,
Rinse the syringe with a solution containing ethylene shake vigorously for 3 min and allow the layers to separate
chloride R before the injection of the reference solutions. completely. Dilute 25.0 ml of the aqueous upper layer to
Rinse the syringe with the substance to be examined before 50.0 ml with the buffer solution.
the injection of the test solution. Fluoride standard solution (1000 ppm F). Dissolve 221.0 mg
of sodium fluoride R, previously dried at 150 °C for 4 h, in
Identification of impurities: use the chromatogram supplied water R. Add 1.0 ml of 0.01 M sodium hydroxide and dilute
with sevoflurane CRS and the chromatogram obtained to 100.0 ml with water R.
with reference solution (b) to identify the peaks due to
impurities A and B. Reference stock solutions. Dilute the fluoride standard
solution (1000 ppm F) diluted with water R to obtain
Relative retention with reference to sevoflurane (retention solutions having known concentrations of about 5 μg, 2 μg,
time = about 6.6 min) : impurity A = about 0.78 ; 0.5 μg, and 0.2 μg of fluoride per millilitre.
impurity B = about 0.83 ; internal standard = about 1.35.
Reference solutions. Dilute 25.0 ml of each reference stock
System suitability : reference solution (b) : solution to 50.0 ml with the buffer solution.
— resolution : minimum 2.0 between the peaks due to Indicator electrode : fluoride-selective.
impurities A and B. Reference electrode : glass-sleeved calomel.
Calculate the relative response factor (F1) for reference Apparatus : voltmeter capable of a minimum reproducibility
solution (a), using the following expression : of ± 0.2 mV.
Carry out the measurements on the reference solutions
and test solution. To take measurements, transfer the
solution under test to a 100 ml beaker containing a
polytetrafluoroethylene-coated magnetic stirring bar, and
M1 = mass of the internal standard in reference immerse the electrodes. Allow to stir on a magnetic stirrer
solution (a), in milligrams ; with an insulated top until equilibrium is attained (about
M2 = mass of the substance to be examined in reference 2-3 min), and record the potential. Rinse the electrodes with
solution (a), in milligrams ; the buffer solution and dry, taking care to avoid damaging
R = ratio of the area of the peak due to sevoflurane to the crystal of the specific-ion electrode.
the area of the peak due to the internal standard Calculate the concentration of fluorides using the calibration
from the chromatogram obtained with reference curve.
solution (a).
Non-volatile residue : maximum 100 mg/l.
Calculate the quantity of each impurity in the substance Transfer 10.0 ml to a tared evaporating dish, evaporate to
to be examined, in parts per million, using the following dryness on a water-bath and dry the residue at 105 °C for
expression : 2 h. The residue weighs a maximum of 1.0 mg.
Water (2.5.12) : maximum 0.050 per cent m/m, determined
on 10.0 ml.

General Notices (1) apply to all monographs and other texts 4295
Sodium alendronate EUROPEAN PHARMACOPOEIA 6.3

STORAGE B. It gives reaction (a) of sodium (2.3.1).

In an airtight, stainless-steel container, protected from light.
TESTS
IMPURITIES Solution S. Dissolve 0.5 g in carbon dioxide-free water R
Specified impurities : A, B. prepared from distilled water R and dilute to 50 ml with the
Other detectable impurities (the following substances same solvent.
would, if present at a sufficient level, be detected by one Appearance of solution. Solution S is clear (2.2.1) and not
or other of the tests in the monograph. They are limited more intensely coloured than reference solution B7 or BY7
by the general acceptance criterion for other/unspecified (2.2.2, Method II).
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to pH (2.2.3). The pH of solution S is 4.0 to 5.0.
identify these impurities for demonstration of compliance. 4-aminobutanoic acid. Examine by thin-layer
See also 5.10. Control of impurities in substances for chromatography (2.2.27), using a TLC silica gel plate R.
pharmaceutical use) : C. Test solution. Dissolve 0.10 g of the substance to be
examined in water R and dilute to 10 ml with the same
solvent.
Reference solution (a). Dissolve 0.10 g of 4-aminobutanoic
acid R in water R and dilute to 200 ml with the same solvent.
A. 1,1,3,3,3-pentafluoro-2-(fluoromethoxy)prop-1-ene,
Reference solution (b). Dilute 1 ml of reference solution (a)
to 10 ml with water R.
Apply to the plate 5 μl of the test solution and 5 μl of
reference solution (b). Allow the plate to dry in air. Develop
over a path of 15 cm using a mixture of 20 volumes of
B. 1,1,1,3,3,3-hexafluoro-2-methoxypropane, water R, 20 volumes of glacial acetic acid R and 60 volumes
of butanol R. Dry the plate in a current of warm air. Spray
with ninhydrin solution R and heat at 100 °C to 105 °C for
15 min. Any spots corresponding to 4-aminobutanoic acid
in the chromatogram obtained with the test solution are not
C. 1,1,1,3,3,3-hexafluoropropan-2-ol. more intense than the spot in the chromatogram obtained
with reference solution (b) (0.5 per cent).
Phosphate and phosphite. Examine the chromatograms
obtained in the assay. In the chromatogram obtained with
01/2008:1564 the test solution : the area of any peak corresponding to
corrected 6.3 phosphate is not greater than that of the peak due to
phosphate in the chromatogram obtained with reference
SODIUM ALENDRONATE solution (d) (0.5 per cent) ; the area of any peak corresponding
to phosphite is not greater than that of the peak due to
phosphite in the chromatogram obtained with reference
Natrii alendronas solution (d) (0.5 per cent).
Heavy metals (2.4.8). 1.0 g complies with limit test F for
heavy metals (20 ppm). Prepare the reference solution using
2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : 16.1 per cent to 17.1 per cent,
determined on 1.000 g by drying in an oven at 140 °C to
145 °C.
C4H12NNaO7P2,3H2O Mr 325.1
[121268-17-5] ASSAY
Examine by liquid chromatography (2.2.29).
DEFINITION
Sodium alendronate contains not less than 98.0 per Test solution. Dissolve 50.0 mg of the substance to be
cent and not more than the equivalent of 102.0 per cent examined in water R and dilute to 25.0 ml with the same
of (4-amino-1-hydroxybutylidene)bisphosphonic acid solvent.
monosodium salt, calculated with reference to the dried Reference solution (a). Dissolve 50.0 mg of sodium
substance. alendronate CRS in water R and dilute to 25.0 ml with the
same solvent.
CHARACTERS
Reference solution (b). Dissolve 3.0 g of phosphoric acid R
A white or almost white, crystalline powder, soluble in water, in water R and dilute to 100.0 ml with the same solvent.
very slightly soluble in methanol, practically insoluble in Dilute 1.0 ml of the solution to 100.0 ml with water R.
methylene chloride.
Reference solution (c). Dissolve 2.5 g of phosphorous acid R
IDENTIFICATION in water R and dilute to 100.0 ml with the same solvent.
A. Examine by infrared absorption spectrophotometry Dilute 1.0 ml of the solution to 100.0 ml with water R.
(2.2.24), comparing with the spectrum obtained with Reference solution (d). Mix 2.0 ml of reference solution (b)
sodium alendronate CRS. Examine the substances and 2.0 ml of reference solution (c) and dilute to 50.0 ml
prepared as discs. with water R.

4296 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Sodium alginate

The chromatographic procedure may be carried out using : C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared
— a column 0.15 m long and 4.6 mm in internal diameter 10 g/l solution of 1,3-dihydroxynaphthalene R in
packed with anion exchange resin R1 (7 μm), ethanol (96 per cent) R and 5 ml of hydrochloric acid R.
Boil for 3 min, cool, add 5 ml of water R, and shake with
— as mobile phase at a flow rate of 1.2 ml/min a solution of 15 ml of di-isopropyl ether R. Carry out a blank test. The
0.2 ml of anhydrous formic acid R in 1000 ml of water R, upper layer obtained with the substance to be examined
adjusted to pH 3.5 with 2 M sodium hydroxide R, exhibits a deeper bluish-red colour than that obtained
— as detector a refractometer, with the blank.
— a 100 μl loop injector, D. It complies with the test for sulphated ash. The residue
maintaining the temperature of the column at 35 °C. obtained, dissolved in 2 ml of water R, gives reaction (a)
of sodium (2.3.1).
Inject reference solution (a) six times. The assay is not valid
unless the relative standard deviation of the peak area of
sodium alendronate is at most 1.0 per cent. Inject the test TESTS
solution, reference solution (a) and reference solution (d). Solution S. Dissolve 0.10 g in water R, with constant stirring,
The retention time of sodium alendronate is about 16 min dilute to 30 ml with the same solvent and allow to stand for
and the relative retention times are : phosphate about 1.3 and 1 h.
phosphite about 1.6. Record the chromatograms for twice
the retention time of the principal peak in the chromatogram Appearance of solution. The solution is not more opalescent
obtained with the test solution. than reference suspension II (2.2.1) and not more intensely
coloured than intensity 6 of the range of reference solutions
Calculate the percentage content of C4H12NNaO7P2 from of the most appropriate colour (2.2.2, Method II).
the peak areas and the declared content of sodium
alendronate CRS. Dilute 1 ml of solution S to 10 ml with water R.
Chlorides : maximum 1.0 per cent.
IMPURITIES
To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and
dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml
of the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of
toluene R. Titrate with 0.1 M ammonium thiocyanate, using
A. 4-aminobutanoic acid, 2 ml of ferric ammonium sulphate solution R2 as indicator
and shaking vigorously towards the end point.
B. phosphate, 1 ml of 0.1 M silver nitrate is equivalent to 3.545 mg of Cl.
Calcium : maximum 1.50 per cent.
C. phosphite. Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 0.10 g in 50 ml of dilute ammonia R2,
heating on a water-bath. Allow to cool and dilute to 100.0 ml
with distilled water R (solution (a)). Dilute 3.0 ml of
01/2009:0625 solution (a) to 100.0 ml with distilled water R.
Reference solutions. Prepare 3 reference solutions in the
SODIUM ALGINATE same manner as the test solution but add 0.75 ml, 1.0 ml
and 1.5 ml respectively of calcium standard solution
(100 ppm Ca) R to the 3.0 ml of solution (a).
Natrii alginas
Set the zero of the instrument using a mixture of 1.5 volumes
DEFINITION of dilute ammonia R2 and 98.5 volumes of distilled water R.
Sodium alginate consists mainly of the sodium salt of alginic Source : calcium hollow-cathode lamp.
acid, which is a mixture of polyuronic acids [C6H8O6)n]
Wavelength : 422.7 nm.
composed of residues of D-mannuronic acid and L-guluronic
acid. Sodium alginate is obtained mainly from algae Atomisation device : air-acetylene flame.
belonging to the Phaeophyceae.
Heavy metals (2.4.8) : maximum 20 ppm.
CHARACTERS 1.0 g complies with test F. Prepare the reference solution
Appearance : white or pale yellowish-brown powder. using 2 ml of lead standard solution (10 ppm Pb) R.
Solubility : slowly soluble in water forming a viscous, Loss on drying (2.2.32) : maximum 15.0 per cent, determined
colloidal solution, practically insoluble in ethanol (96 per on 0.1000 g by drying in an oven at 105 °C for 4 h.
cent). Sulphated ash (2.4.14) : 30.0 per cent to 36.0 per cent (dried
substance), determined on 0.1000 g.
IDENTIFICATION Microbial contamination
A. Dissolve 0.2 g with shaking in 20 ml of water R. To 5 ml
of this solution add 1 ml of calcium chloride solution R. TAMC : acceptance criterion 103 CFU/g (2.6.12).
A voluminous gelatinous mass is formed. TYMC : acceptance criterion 102 CFU/g (2.6.12).
B. To 10 ml of the solution prepared in identification test A Absence of Escherichia coli (2.6.13).
add 1 ml of dilute sulphuric acid R. A gelatinous mass
is formed. Absence of Salmonella (2.6.13).

General Notices (1) apply to all monographs and other texts 4297
Sodium ascorbate EUROPEAN PHARMACOPOEIA 6.3

01/2009:1791 Phosphate buffer solution. Dissolve 6.8 g of potassium

SODIUM ASCORBATE
Natrii ascorbas
C6H7NaO6 Mr 198.1 200.0 ml with the mobile phase. Mix 1.0 ml of this solution
[134-03-2]
DEFINITION
Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2,5- — size: l = 0.25 m, Ø = 4.6 mm ;
dihydrofuran-3-olate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or yellowish, crystalline powder or
crystals.
Solubility : freely soluble in water, sparingly soluble in
ethanol (96 per cent), practically insoluble in methylene
chloride.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
A. Specific optical rotation (2.2.7) (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : sodium ascorbate CRS.
C. To 1 ml of solution S (see Tests) add 0.2 ml of dilute nitric — impurity C : not more than the area of the corresponding
acid R and 0.2 ml of silver nitrate solution R2. A grey
precipitate is formed.
D. 1 ml of solution S gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100.0 ml with — total : not more than twice the area of the peak due to
the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 or BY6
(2.2.2, Method II) ; examine the colour immediately after
preparation of the solution.
pH (2.2.3) : 7.0 to 8.0 for solution S.
Specific optical rotation (2.2.7) : + 103 to + 108 (dried
substance), determined on freshly prepared solution S.
Impurity E : maximum 0.3 per cent.
Test solution. Dissolve 0.25 g in 5 ml of water R. Add 1 ml
of dilute acetic acid R and 0.5 ml of calcium chloride
solution R.
Reference solution. Dissolve 70 mg of oxalic acid R in
water R and dilute to 500 ml with the same solvent ; to 5 ml (0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting copper standard
of the solution add 1 ml of dilute acetic acid R and 0.5 ml of solution (10 ppm Cu) R with 0.1 M nitric acid.
calcium chloride solution R.
Allow the solutions to stand for 1 h. Any opalescence in the
test solution is not more intense than that in the reference
solution.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
dihydrogen phosphate R in water R and dilute to about
175 ml with the same solvent. Filter (porosity 0.45 μm) and
dilute to 1000 ml with water R.
Test solution. Dissolve 0.500 g of the substance to be
examined in the phosphate buffer solution and dilute to
10.0 ml with the phosphate buffer solution.
Reference solution (a). Dissolve 10.0 mg of ascorbic acid
impurity C CRS in the mobile phase and dilute to 5.0 ml
with the mobile phase.
Reference solution (b). Dilute 2.5 ml of reference solution (a)
to 100.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of the test solution to
with 1.0 ml of reference solution (a).
Column :
— stationary phase : aminopropylsilyl silica gel for
chromatography R (5 μm) ;
— temperature : 45 °C.
Mobile phase : phosphate buffer solution, acetonitrile R1
(30:70 V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 210 nm.
Injection : 20 μl of the test solution and reference
solutions (b) and (c).
Run time : twice the retention time of sodium ascorbate.
Relative retention with reference to sodium ascorbate
(retention time = about 8 min) ; impurity C = about 1.4.
System suitability : reference solution (c) :
— resolution : minimum 3.0 between the peaks due to
ascorbic acid and impurity C.
Limits :
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent) ;
— unspecified impurities : for each impurity, not more
than the area of the peak due to impurity C in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ;
impurity C in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
— disregard limit : 0.5 times the area of the peak due to
impurity C in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Sulphates (2.4.13) : maximum 150 ppm.
To 10 ml of solution S add 2 ml of hydrochloric acid R1 and
dilute to 15 ml with distilled water R.
Copper : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 2.0 g in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutions. Prepare the reference solutions
Source : copper hollow-cathode lamp.
Wavelength : 324.8 nm.
Atomisation device : air-acetylene flame.
Iron : maximum 2.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).

4298 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Test solution. Dissolve 5.0 g in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutions. Prepare the reference solutions
(0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting iron standard
solution (20 ppm Fe) R with 0.1 M nitric acid.
Source : iron hollow-cathode lamp.
Wavelength : 248.3 nm.
Atomisation device : air-acetylene flame.
Nickel : maximum 1.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 10.0 g in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutions. Prepare the reference solutions
(0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting nickel standard
solution (10 ppm Ni) R with 0.1 M nitric acid.
Source : nickel hollow-cathode lamp.
Wavelength : 232.0 nm.
Atomisation device : air-acetylene flame.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in water R and dilute to 20 ml with the same
solvent. 12 ml of the solution complies with test A. Prepare
the reference solution using lead standard solution (1 ppm
Pb) R.
Loss on drying (2.2.32) : maximum 0.25 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
ASSAY
Dissolve 80 mg in a mixture of 10 ml of dilute sulphuric
acid R and 80 ml of carbon dioxide-free water R. Add 1 ml
of starch solution R. Titrate with 0.05 M iodine until a
persistent violet-blue colour is obtained.
1 ml of 0.05 M iodine is equivalent to 9.91 mg of C6H7NaO6.
STORAGE
In a non-metallic container, protected from light.
IMPURITIES
Specified impurities : C, E.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one C. In a test-tube fitted with a glass tube, mix 0.1 g with 5 g
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, B, D.
A. 2-furaldehyde,
B. R = [CH2]3-CH3 : butyl D-sorbosonate,
C. R = H : D-sorbosonic acid,
D. R = CH3 : methyl D-sorbosonate,
General Notices (1) apply to all monographs and other texts
Sodium glycerophosphate, hydrated
E. oxalic acid.
01/2009:1995
SODIUM GLYCEROPHOSPHATE,
HYDRATED
Natrii glycerophosphas hydricus
C3H7Na2O6P, xH2O Mr 216.0 (anhydrous substance)
DEFINITION
Mixture of variable proportions of sodium
(2RS)-2,3-dihydroxypropyl phosphate and sodium
2-hydroxy-1-(hydroxymethyl)ethyl phosphate. The mixture
may contain various amounts of sodium glycerol diphosphate
isomers and sodium glycerol triphosphate. The degree of
hydration is 4 to 6.
Content : 98.0 per cent to 105.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
crystals.
Solubility : freely soluble in water, practically insoluble in
acetone and in ethanol (96 per cent).
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of sodium (2.3.1).
B. To 0.1 g add 5 ml of dilute nitric acid R. Heat to boiling
and boil for 1 min. Cool. The solution gives reaction (b)
of phosphates (2.3.1).
of potassium hydrogen sulphate R. Heat strongly and
direct the white vapour into 5 ml of decolorised fuchsin
solution R. A violet-red colour develops which becomes
violet upon heating for 30 min on a water-bath.
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100 ml with
the same solvent.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
Alkalinity. To 10 ml of solution S add 0.2 ml of
phenolphthalein solution R. Not more than 1.0 ml of 0.1 M
hydrochloric acid is required to change the colour of the
indicator, (n2).
Glycerol and ethanol (96 per cent)-soluble substances :
maximum 1.0 per cent.
Shake 1.000 g with 25 ml of ethanol (96 per cent) R for
10 min. Filter. Evaporate the filtrate on a water-bath and dry
the residue at 70 °C for 1 h. The residue weighs not more
than 10 mg.
4299
Sodium hyaluronate
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 2.5 ml of solution S to 15 ml with water R.
Phosphates (2.4.11) : maximum 0.1 per cent.
Dilute 1 ml of solution S to 10 ml with water R. Dilute 1 ml
of this solution to 100 ml with water R.
Sulphates (2.4.13) : maximum 500 ppm.
Dilute 3 ml of solution S to 15 ml with water R.
Iron (2.4.9) : maximum 20 ppm.
Dilute 5 ml of solution S to 10 ml with water R.
Heavy metals (2.4.8) : maximum 20 ppm.
Dilute 10 ml of solution S to 20 ml with water R. 12 ml of
the solution complies with limit test A. Prepare the standard
using 10 ml of lead standard solution (1 ppm Pb) R.
Water (2.5.12) : 25.0 per cent to 35.0 per cent, determined
on 0.100 g.
ASSAY
Dissolve 0.250 g in 30 ml of water R. Titrate with 0.05 M
sulphuric acid, determining the end-point potentiometrically
(2.2.20), (n1).
Calculate the percentage content of sodium glycerophosphate
(anhydrous substance) from the expression :
a = percentage content of water,
n1 = volume of 0.05 M sulphuric acid used in the
assay, in millilitres,
n2 = volume of 0.1 M hydrochloric acid used in the
test for alkalinity, in millilitres,
m = mass of the substance to be examined, in grams.
01/2009:1472 Mix solutions A and B until a pH of 7.0 is reached. Filter
SODIUM HYALURONATE
Natrii hyaluronas
(C14H20NNaO11)n
[9067-32-7]
DEFINITION
Sodium salt of hyaluronic acid, a glycosaminoglycan
consisting of D-glucuronic acid and N-acetyl-D-glucosamine
disaccharide units.
Content : 95.0 per cent to 105.0 per cent (dried substance). (t ) and for the 4 test solutions (t , t , t and t ), at
Intrinsic viscosity : 90 per cent to 120 per cent of the value 25.00 ± 0.03 °C. Use an appropriate suspended level
stated on the label.
4300 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
PRODUCTION
It is extracted from cocks’ combs or obtained by fermentation
from Streptococci, Lancefield Groups A and C. When
produced by fermentation of gram-positive bacteria, the
process must be shown to reduce or eliminate pyrogenic or
inflammatory components of the cell wall.
CHARACTERS
Appearance : white or almost white, very hygroscopic
powder or fibrous aggregate.
Solubility : sparingly soluble or soluble in water, practically
insoluble in acetone and in anhydrous ethanol.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of sodium
hyaluronate.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Weigh a quantity of the substance to be examined
equivalent to 0.10 g of the dried substance and add 30.0 ml
of a 9 g/l solution of sodium chloride R. Mix gently on a
shaker until dissolved (about 12 h).
Appearance of solution. Solution S is clear (2.2.1) and its
absorbance (2.2.25) at 600 nm is not greater than 0.01.
pH (2.2.3) : 5.0 to 8.5.
Dissolve the substance to be examined in carbon dioxide-free
water R to obtain a solution containing a quantity equivalent
to 5 mg of the dried substance per millilitre.
Intrinsic viscosity. Sodium hyaluronate is very hygroscopic
and must be protected from moisture during weighing.
Buffer solution (0.15 M sodium chloride in 0.01 M
phosphate buffer solution pH 7.0). Dissolve 0.78 g of
sodium dihydrogen phosphate R and 4.50 g of sodium
chloride R in water R and dilute to 500.0 ml with the same
solvent (solution A). Dissolve 1.79 g of disodium hydrogen
phosphate R and 4.50 g of sodium chloride R in water R
and dilute to 500.0 ml with the same solvent (solution B).
through a sintered-glass filter (4) (2.1.2).
Test solution (a). Weigh 0.200 g (m0p) (NOTE : this value
is only indicative and should be adjusted after an initial
measurement of the viscosity of test solution (a)) of the
substance to be examined and dilute with 50.0 g (m0s) of
buffer solution at 4 °C. Mix the solution by shaking at 4 °C
during 24 h. Weigh 5.00 g (m1p) of the solution and dilute
with 100.0 g (m1s) of buffer solution at 25 °C. Mix this
solution by shaking for 20 min. Filter the solution through a
sintered-glass filter (100) (2.1.2), and discard the first 10 ml.
Test solution (b). Weigh 30.0 g (m2p) of test solution (a) and
dilute with 10.0 g (m2s) of buffer solution at 25 °C. Mix this
solution by shaking for 20 min. Filter the solution through a
sintered-glass filter (100) (2.1.2) and discard the first 10 ml.
Test solution (c). Weigh 20.0 g (m3p) of test solution (a) and
dilute with 20.0 g (m3s) of buffer solution at 25 °C. Mix this
solution by shaking for 20 min. Filter the solution through a
sintered-glass filter (100) (2.1.2) and discard the first 10 ml.
Test solution (d). Weigh 10.0 g (m4p) of test solution (a) and
dilute with 30.0 g (m4s) of buffer solution at 25 °C. Mix this
solution by shaking for 20 min. Filter the solution through a
sintered-glass filter (100) (2.1.2) and discard the first 10 ml.
Determine the flow-times (2.2.9) for the buffer solution
0 1 2 3 4
viscometer (specifications : viscometer constant about
EUROPEAN PHARMACOPOEIA 6.3
0.005 mm2/s2, kinematic viscosity of 1-5 mm2/s, internal
diameter of tube R 0.53 mm, volume of bulb C 5.6 ml,
internal diameter of tube N 2.8-3.2 mm) with a funnel-shaped
lower capillary end. Use the same viscometer for all
measurements ; measure all outflow times in triplicate. The
test is not valid unless the results do not differ by more than
0.35 per cent from the mean and if the flow time t1 is not less
than 1.6 and not more than 1.8 times t0. If this is not the
case, adjust the value of m0p and repeat the procedure.
Calculation of the relative viscosities
Since the densities of the sodium hyaluronate solutions and
of the solvent are almost equal, the relative viscosities ηri
(being ηr1, ηr2, ηr3 and ηr4) can be calculated from the ratio
of the flow times for the respective solutions ti (being t1,
t2, t3and t4) to the flow time of the solvent t0, but taking
into account the kinetic energy correction factor for the
capillary (B = 30 800 s3), using the following expression :
Calculation of the concentrations
Calculate the concentration c1 (expressed in kg/m3) of
sodium hyaluronate in test solution (a) using the following
expression:
x = percentage content of sodium hyaluronate as
determined under Assay ;
h = percentage loss on drying ;
ρ25
= 1005 kg/m3 (density of the test solution at 25 °C).
Calculate the concentration c2 (expressed in kg/m3) of
sodium hyaluronate in test solution (b) using the following
expression :
Calculate the concentration c3 (expressed in kg/m3) of
sodium hyaluronate in test solution (c) using the following
expression :
Calculate the concentration c4 (expressed in kg/m3) of
sodium hyaluronate in test solution (d) using the following
expression :
Calculation of the intrinsic viscosity
Calculate the intrinsic viscosity [η] by linear least-squares
regression analysis using the Martin equation :
General Notices (1) apply to all monographs and other texts
Sodium hyaluronate
The decimal antilogarithm of the intercept is the intrinsic
viscosity expressed in m3/kg.
Sulphated glycosaminoglycans : maximum 1 per cent, if the
product is extracted from cocks’ combs.
Appropriate safety precautions are to be taken when
handling perchloric acid at elevated temperature.
Test solution. Introduce a quantity of the substance to be
examined equivalent to 50.0 mg of the dried substance into
a test-tube 150 mm long and 16 mm in internal diameter and
dissolve in 1.0 ml of perchloric acid R.
Reference solution. Dissolve 0.149 g of anhydrous sodium
sulphate R in water R and dilute to 100.0 ml with the same
solvent. Dilute 10.0 ml of this solution to 100.0 ml with
water R. Evaporate 1.0 ml in a test-tube 150 mm long and
16 mm in internal diameter in a heating block at 90-95 °C,
and dissolve the residue in 1.0 ml of perchloric acid R.
Plug each test-tube with a piece of glass wool. Place the
test-tubes in a heating block or a silicone oil bath maintained
at 180 °C and heat until clear, colourless solutions are
obtained (about 12 h). Remove the test-tubes and cool to
room temperature. Add to each test-tube 3.0 ml of a 33.3 g/l
solution of barium chloride R, cap and shake vigorously.
Allow the test-tubes to stand for 30 min. Shake each
test-tube once again, and determine the absorbance (2.2.25)
at 660 nm, using water R as a blank.
The absorbance obtained with the test solution is not greater
than the absorbance obtained with the reference solution.
Nucleic acids. The absorbance (2.2.25) of solution S at
260 nm is maximum 0.5.
Protein : maximum 0.3 per cent ; maximum 0.1 per cent,
if intended for use in the manufacture of parenteral
preparations.
Test solution (a). Dissolve the substance to be examined in
water R to obtain a solution containing a quantity equivalent
to about 10 mg of the dried substance per millilitre.
Test solution (b). Mix equal volumes of test solution (a) and
water R.
Reference solutions. Prepare a 0.5 mg/ml stock solution
of bovine albumin R in water R. Prepare 5 dilutions of the
stock solution containing between 5 μg/ml and 50 μg/ml
of bovine albumin R.
Add 2.5 ml of freshly prepared cupri-tartaric solution R3
to test-tubes containing 2.5 ml of water R (blank), 2.5 ml
of the test solutions (a) or (b) or 2.5 ml of the reference
solutions. Mix after each addition. After about 10 min, add
to each test-tube 0.50 ml of a mixture of equal volumes of
phosphomolybdotungstic reagent R and water R prepared
immediately before use. Mix after each addition. After
30 min, measure the absorbance (2.2.25) of each solution
at 750 nm against the blank. From the calibration curve
obtained with the 5 reference solutions determine the
content of protein in the test solutions.
Chlorides (2.4.4) : maximum 0.5 per cent.
Dissolve 67 mg in 100 ml of water R.
Iron : maximum 80.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve a quantity of the substance to be
examined equivalent to 0.25 g of the dried substance in 1 ml
of nitric acid R by heating on a water-bath. Cool and dilute
to 10.0 ml with water R.
Reference solutions. Prepare 2 reference solutions in the
same manner as the test solution, adding 1.0 ml and 2.0 ml
respectively of iron standard solution (10 ppm Fe) R to the
dissolved substance to be examined.
4301
Sodium molybdate dihydrate EUROPEAN PHARMACOPOEIA 6.3

Source : iron hollow-cathode lamp using a transmission cg = mean of concentrations of D-glucuronic acid in
band of 0.2 nm. the test solutions, in milligrams per gram ;
Wavelength : 248.3 nm. cs = mean of concentrations of the substance to be
Atomisation device : air-acetylene flame. examined in the test solutions, in milligrams per
gram ;
Heavy metals (2.4.8) : maximum 20 ppm ; maximum
10 ppm if intended for use in the manufacture of parenteral Z = determined percentage content of C6H10O7 in
D-glucuronic acid R ;
preparations.
1.0 g complies with test F. Prepare the reference solution h = percentage loss on drying ;
using 2.0 ml of lead standard solution (10 ppm Pb) R. 401.3 = relative molecular mass of the disaccharide
Loss on drying (2.2.32) : maximum 20.0 per cent, determined fragment ;
on 0.500 g by drying at 100-110 °C over diphosphorus 194.1 = relative molecular mass of glucuronic acid.
pentoxide R for 6 h.
STORAGE
Microbial contamination
In an airtight container, protected from light and humidity.
TAMC : acceptance criterion 102 CFU/g (2.6.12). Use 1 g of If the substance is sterile, store in a sterile, airtight,
the substance to be examined. tamper-proof container.
Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg,
if intended for use in the manufacture of parenteral LABELLING
preparations without a further appropriate procedure for The label states :
the removal of bacterial endotoxins ; less than 0.05 IU/mg, — the intrinsic viscosity ;
if intended for use in the manufacture of intra-ocular — the origin of the substance ;
preparations or intra-articular preparations without a
further appropriate procedure for the removal of bacterial — the intended use of the substance ;
endotoxins. — where applicable, that the substance is suitable for
parenteral administration other than intra-articular
ASSAY administration ;
Determine the glucuronic acid content by reaction with — where applicable, that the substance is suitable for
carbazole as described below. parenteral administration, including intra-articular
administration ;
Reagent A. Dissolve 0.95 g of disodium tetraborate R in
100.0 ml of sulphuric acid R. — where applicable that the material is suitable for
intra-ocular use.
Reagent B. Dissolve 0.125 g of carbazole R in 100.0 ml of
anhydrous ethanol R.
Test solution. Prepare this solution in triplicate. Dissolve 01/2008:1565
0.170 g of the substance to be examined in water R and corrected 6.3
dilute to 100.0 g with the same solvent. Dilute 10.0 g of this
solution to 200.0 g with water R. SODIUM MOLYBDATE DIHYDRATE
Reference stock solution. Dissolve 0.100 g of D-glucuronic
acid R, previously dried to constant mass in vacuum over Natrii molybdas dihydricus
diphosphorus pentoxide R (2.2.32), in water R and dilute to
100.0 g with the same solvent. MoNa2O4,2H2O Mr 241.9
Reference solutions. Prepare 5 dilutions of the reference [10102-40-6]
stock solution containing between 6.5 μg/g and 65 μg/g of DEFINITION
D-glucuronic acid R.
Content : 98.0 per cent to 100.5 per cent (dried substance).
Place 25 test-tubes, numbered 1 to 25, in iced water. Add
1.0 ml of the 5 reference solutions in triplicate to the CHARACTERS
test-tubes 1 to 15 (reference tubes), 1.0 ml of the 3 test Appearance : white or almost white powder or colourless
solutions in triplicate to the test-tubes 16 to 24 (sample crystals.
tubes), and 1.0 ml of water R to test-tube 25 (blank). Add Solubility : freely soluble in water.
to each test-tube 5.0 ml of freshly prepared reagent A,
previously cooled in iced water. Tightly close the test-tubes IDENTIFICATION
with plastic caps, shake the contents, and place on a water A. Loss on drying (see Tests).
bath for exactly 15 min. Cool in iced water, and add to each
test tube 0.20 ml of reagent B. Recap the tubes, shake, and B. Dissolve 0.2 g in 5 ml of a mixture of equal volumes of
put them again on a water-bath for exactly 15 min. Cool to nitric acid R and water R and add 0.1 g of ammonium
room temperature and measure the absorbance (2.2.25) of chloride R. Add 0.3 ml of disodium hydrogen phosphate
the solutions at 530 nm, against the blank. solution R and heat slowly at 50-60 °C. A yellow
precipitate is formed.
From the calibration curve obtained with the mean C. Dissolve 0.15 g in 2 ml of water R, the solution gives
absorbances read for each reference solution, determine reaction (a) of sodium (2.3.1).
the mean concentrations of D-glucuronic acid in the test
solutions. TESTS
Calculate the percentage content of sodium hyaluronate Solution S. Dissolve 10.0 g in water R and dilute to 50 ml
using the following expression : with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).

4302 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Chlorides : maximum 50 ppm.
To 10 ml of a mixture of equal volumes of nitric acid R and
water R add 10 ml of solution S with shaking. Add 1 ml of
0.1 M silver nitrate. Any opalescence in the solution is not
more intense after 5 min than that of a standard solution
prepared at the same time in the same manner with 2 ml of
chloride standard solution (50 ppm Cl) R.
Phosphates: maximum 200 ppm.
Dissolve 2.0 g by heating in 13 ml of water R. In the still-hot
solution, dissolve 8.0 g of ammonium nitrate R1. Add this
solution to 27 ml of a mixture of equal volumes of nitric
acid R and water R. Any yellow colour or opalescence in
the solution is not more intense within 3 h than that in a
standard solution prepared at the same time in the same
manner as follows : dissolve 1.0 g in 12 ml of water R and
add 1 ml of phosphate standard solution (200 ppm PO4) R.
Ammonium (2.4.1, Method B) : maximum 10 ppm,
determined on 0.10 g.
Prepare the standard using 1 ml of ammonium standard
solution (1 ppm NH4) R.
Heavy metals: maximum 10 ppm.
To 10 ml of solution S, add 2 ml of water R, 6 ml of a 168 g/l
solution of sodium hydroxide R and 2 ml of concentrated
ammonia R (solution A). To 0.5 ml of thioacetamide
reagent R add a mixture of 15 ml of solution A and 5 ml of
water R. Any coloration of the solution is not more intense
after 2 min than that of a reference solution prepared at the
same time as follows : to 0.5 ml of thioacetamide reagent R
add a mixture of 5 ml of solution A, 1 ml of lead standard
solution (10 ppm Pb) R and 14 ml of water R.
Loss on drying (2.2.32) : 14.0 per cent to 16.0 per cent,
determined on 1.000 g by drying in an oven at 140 °C for 3 h.
ASSAY
Dissolve 0.100 g in 30 ml of water R, add 0.5 g of
hexamethylenetetramine R and 0.1 ml of a 250 g/l solution
of nitric acid R. Heat to 60 °C. Titrate with 0.05 M lead
nitrate using 4-(2-pyridylazo)resorcinol monosodium salt R
as indicator.
1 ml of 0.05 M lead nitrate is equivalent to 10.30 mg
of Na2MoO4.
01/2009:1909
SODIUM POLYSTYRENE
SULPHONATE
Natrii polystyrenesulfonas
DEFINITION
Polystyrene sulphonate resin prepared in the sodium form.
Exchange capacity : 2.8 mmol to 3.4 mmol of potassium per
gram (dried substance).
Content : 9.4 per cent to 11.0 per cent of Na (dried substance).
CHARACTERS
Appearance : almost white or light brown powder.
Solubility : practically insoluble in water, in ethanol (96 per
cent) and in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
General Notices (1) apply to all monographs and other texts
Sodium polystyrene sulphonate
Preparation : discs using finely ground substance.
Comparison : Ph. Eur. reference spectrum of sodium
polystyrene sulphonate.
B. Suspend 0.1 g in water R, add 2 ml of a 150 g/l solution
of potassium carbonate R, and heat to boiling. Allow
to cool and filter. To the filtrate add 4 ml of potassium
pyroantimonate solution R and heat to boiling. Allow
to cool in iced water and if necessary rub the inside of
the test-tube with a glass rod. A dense white precipitate
is formed.
TESTS
Styrene. Liquid chromatography (2.2.29).
Test solution. Shake 10.0 g of the substance to be examined
with 10 ml of acetone R for 30 min, centrifuge and use the
supernatant liquid.
Reference solution. Dissolve 10 mg of styrene R in acetone R
and dilute to 100 ml with the same solvent. Dilute 1 ml of
this solution to 100 ml with acetone R.
Column :
— size: l = 0.25 m, Ø = 4 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : acetonitrile R, water R (1 :1 V/V).
Flow rate : 2 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μl.
Limit :
— styrene : not more than the area of the principal peak in
the chromatogram obtained with the reference solution
(1 ppm).
Calcium : maximum 0.10 per cent.
Atomic emission spectrometry (2.2.22, Method I).
Test solution. To 1.10 g add 5 ml of hydrochloric acid R,
heat to boiling, cool and add 10 ml of water R. Filter, wash
the filter and residue with water R and dilute the filtrate and
washing to 25.0 ml with water R.
Reference solutions. Prepare the reference solutions using
calcium standard solution (400 ppm Ca) R, diluted as
necessary with water R.
Wavelength : 422.7 nm.
Potassium : maximum 0.10 per cent.
Atomic emission spectrometry (2.2.22, Method I).
Test solution. To 1.10 g add 5 ml of hydrochloric acid R,
heat to boiling, cool and add 10 ml of water R. Filter, wash
the filter and residue with water R and dilute the filtrate and
washings to 25.0 ml with water R.
Reference solutions. Prepare the reference solutions using
potassium standard solution (100 ppm K) R, diluted as
necessary with water R.
Wavelength : 766.5 nm.
Heavy metals (2.4.8) : maximum 10 ppm.
Treat 1.0 g as described in limit test F. After the addition
of the buffer solution pH 3.5 R and of the thioacetamide
reagent R, dilute to 50 ml with water R and continue as
described in limit test E, beginning at the words “mix and
allow to stand for 10 min...”.
Prepare the standard using 10 ml of lead standard solution
(1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
4303
Sodium stearate EUROPEAN PHARMACOPOEIA 6.3

Microbial contamination (2.6.13) CHARACTERS

Bile-tolerant gram-negative bacteria : acceptance criterion Appearance : white or yellowish, fine powder, greasy to the
less than 102 CFU/g. touch.
ASSAY Solubility : slightly soluble in water and in ethanol (96 per
cent).
Sodium. Atomic emission spectrometry (2.2.22, Method I).
Test solution. In a platinum crucible moisten 0.90 g with a IDENTIFICATION
few drops of sulphuric acid R, ignite very gently and allow First identification : C, D.
to cool. Moisten with a few drops of sulphuric acid R again,
ignite at 800 ± 50 °C until a carbon-free ash is obtained and Second identification : A, B, D.
allow to cool. A. Freezing point (2.2.18) : minimum 53 °C for the residue
Add 20 ml of water R to the crucible, warm gently on a obtained in the preparation of solution S (see Tests).
water-bath until dissolution, cool, transfer quantitatively to a B. Acid value (2.5.1) : 195 to 210, determined on 0.200 g
100 ml graduated flask and dilute to 100.0 ml with water R. of the residue obtained in the preparation of solution S
Dilute 5 ml of this solution to 1000.0 ml with water R. dissolved in 25 ml of the prescribed mixture of solvents.
Reference solutions. Prepare the reference solutions using C. Examine the chromatograms obtained in the assay of
sodium standard solution (200 ppm Na) R, diluted as stearic acid and palmitic acid.
necessary with water R. Results : the 2 principal peaks in the chromatogram
Wavelength : 589 nm. obtained with the test solution are similar in retention
Exchange capacity. Atomic emission spectrometry (2.2.22, time and size to the 2 principal peaks in the chromatogram
Method I). obtained with the reference solution.
Solution A. 9.533 g/l solution of potassium chloride R. D. Solution S gives reaction (b) of sodium (2.3.1).
Test solution. To 1.6 g of the substance to be examined in TESTS
a dry 250 ml ground-glass-stoppered flask add 100 ml of
Solution S. To 10.0 g add 100 ml of peroxide-free ether R
solution A, stopper and shake for 15 min. Filter, discard the
and 80 ml of acetic acid R. Boil under a reflux condenser
first 20 ml of the filtrate and dilute 4 ml of the filtrate to
1000 ml with water R. until dissolution is complete. Allow to cool. In a separating
funnel, separate the aqueous layer and shake the ether layer
Reference solutions. Prepare the reference solutions by with 2 quantities, each of 8 ml, of acetic acid R. Combine
diluting 0, 1, 2, 3 and 4 ml of solution A respectively and 4,
the aqueous layers, wash with 30 ml of peroxide-free ether R
3, 2, 1 and 0 ml of a 7.63 g/l solution of sodium chloride Rand dilute to 100 ml with distilled water R (solution S).
to 1000 ml with water R. Evaporate the ether layers to dryness on a water-bath and
Wavelength : 766.5 nm. dry the residue at 100-105 °C.
Prepare a calibration curve using the reference solutions Acidity or alkalinity. Suspend 2.0 g in 50 ml of previously
and calculate the potassium exchange capacity of the neutralised ethanol (96 per cent) R. Heat under reflux to
substance to be examined in millimoles per gram taking the dissolve and add 3 drops of phenolphthalein solution R ;
concentration of potassium in solution A as 128 mmoles the solution is colourless. Not less than 0.60 ml and not
of K per litre. more than 0.85 ml of 0.1 M sodium hydroxide is required
STORAGE to change the colour of the indicator.
In an airtight container. Chlorides (2.4.4) : maximum 0.2 per cent.
Dilute 0.25 ml of solution S to 15 ml with water R.
IMPURITIES
Sulphates (2.4.13) : maximum 0.3 per cent.
Specified impurities : A.
Dilute 0.5 ml of solution S to 15 ml with distilled water R.
A. styrene. Nickel : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
01/2009:2058 Test solution. Place 50.0 mg of the substance to be examined
in a polytetrafluoroethylene digestion flask and add 0.5 ml
SODIUM STEARATE of a mixture of 1 volume of heavy metal-free hydrochloric
acid R and 5 volumes of heavy metal-free nitric acid R.
Allow to digest at 170 °C for 5 h. Allow to cool. Dissolve the
Natrii stearas residue in water R and dilute to 5.0 ml with the same solvent.
DEFINITION Reference solutions. Prepare the reference solutions using
nickel standard solution (10 ppm Ni) R, diluted as necessary
Mixture of sodium salts of different fatty acids consisting with water R.
mainly of stearic (octadecanoic) acid [C17H35COONa ;
Mr 306.5] and palmitic (hexadecanoic) acid [C15H31COONa ; Source : nickel hollow-cathode lamp.
Mr 278.4]. Wavelength : 232.0 nm.
Content : Atomisation device : air-acetylene flame.
— sodium : 7.4 per cent to 8.5 per cent (Ar 22.99) (dried Loss on drying (2.2.32) : maximum 5.0 per cent.
substance) ;
In a weighing glass introduce 1.0 g of previously washed
— stearic acid in the fatty acid fraction : minimum 40 per sand R, dry at 105 °C and weigh. Add 0.500 g of the
cent ; substance to be examined and 10 ml of ethanol (96 per
— sum of stearic acid and palmitic acid in the fatty acid cent) R. Evaporate at 80 °C and dry the residue at 105 °C
fraction : minimum 90 per cent. for 4 h.

4304 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Sorbitol

Microbial contamination 01/2009:0435

3
TAMC : acceptance criterion 10 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
SORBITOL
Absence of Escherichia coli (2.6.13).
Sorbitolum
Absence of Salmonella (2.6.13).

ASSAY
Sodium. Dissolve 0.250 g with gentle heating in a mixture
of 5 ml of acetic anhydride R and 20 ml of anhydrous
acetic acid R. Cool and add 20 ml of dioxan R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). C6H14O6 Mr 182.2
[50-70-4]
1 ml of 0.1 M perchloric acid is equivalent to 2.299 mg of Na.
Stearic acid and palmitic acid. Gas chromatography DEFINITION
(2.2.28) : use the normalisation procedure. D-Glucitol (D-sorbitol).
Test solution. In a conical flask fitted with a reflux Content : 97.0 per cent to 102.0 per cent (anhydrous
condenser, dissolve 0.10 g of the substance to be examined in substance).
5 ml of boron trifluoride-methanol solution R. Boil under a
reflux condenser for 10 min. Add 4 ml of heptane R through CHARACTERS
the condenser and boil again under a reflux condenser Appearance : white or almost white, crystalline powder.
for 10 min. Allow to cool. Add 20 ml of saturated sodium Solubility : very soluble in water, practically insoluble in
chloride solution R. Shake and allow the layers to separate. ethanol (96 per cent).
Remove about 2 ml of the organic layer and dry over 0.2 g of
anhydrous sodium sulphate R. Dilute 1.0 ml of the solution It shows polymorphism (5.9).
to 100.0 ml with heptane R. IDENTIFICATION
Reference solution. Prepare the reference solution in the First identification : A.
same manner as the test solution using 50.0 mg of palmitic
Second identification : B, C, D.
acid CRS and 50.0 mg of stearic acid CRS instead of the
substance to be examined. A. Examine the chromatograms obtained in the assay.
Column: Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
— material: fused silica ; to the principal peak in the chromatogram obtained with
— size : l = 30 m, Ø = 0.32 mm ; reference solution (a).
B. Dissolve 0.5 g with heating in a mixture of 0.5 ml of
— stationary phase : macrogol 20 000 R (film thickness
pyridine R and 5 ml of acetic anhydride R. After 10 min,
0.5 μm).
pour the solution into 25 ml of water R and allow to
Carrier gas: helium for chromatography R. stand in iced water for 2 h. The precipitate, recrystallised
from a small volume of ethanol (96 per cent) R and dried
Flow rate : 2.4 ml/min.
in vacuo, melts (2.2.14) at 98 °C to 104 °C.
Temperature : C. Thin-layer chromatography (2.2.27).
Time Temperature Test solution. Dissolve 25 mg of the substance to be
(min) (°C) examined in water R and dilute to 10 ml with the same
Column 0-2 70 solvent.
2 - 36 70 → 240 Reference solution (a). Dissolve 25 mg of sorbitol CRS in
water R and dilute to 10 ml with the same solvent.
36 - 41 240
Reference solution (b). Dissolve 25 mg of mannitol CRS
Injection port 220 and 25 mg of sorbitol CRS in water R and dilute to 10 ml
Detector 260 with the same solvent.
Plate : TLC silica gel G plate R.
Detection : flame ionisation. Mobile phase : water R, ethyl acetate R, propanol R
Injection : 1 μl. (10:20:70 V/V/V).
Relative retention with reference to methyl stearate (retention Application : 2 μl.
time = about 40 min) : methyl palmitate = about 0.88. Development : over a path of 17 cm.
System suitability : reference solution : Drying : in air.
— resolution : minimum 5.0 between the peaks due to Detection : spray with 4-aminobenzoic acid solution R ;
methyl stearate and methyl palmitate. dry in a current of cold air until the acetone is removed ;
heat at 100 °C for 15 min ; allow to cool and spray with
Calculate the content of stearic acid and palmitic acid. a 2 g/l solution of sodium periodate R ; dry in a current
of cold air ; heat at 100 °C for 15 min.
STORAGE System suitability : reference solution (b) :
In an airtight container, protected from light. — the chromatogram shows 2 clearly separated spots.

General Notices (1) apply to all monographs and other texts 4305
Sorbitol
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
D. Specific optical rotation (2.2.7) : + 4.0 to + 7.0 (anhydrous
substance).
Dissolve 5.00 g of the substance to be examined and 6.4 g
of disodium tetraborate R in 40 ml of water R. Allow to
stand for 1 h, shaking occasionally, and dilute to 50.0 ml
with water R. Filter if necessary.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 5 g in water R and dilute to 50 ml with the same
solvent.
Conductivity (2.2.38) : maximum 20 μS·cm− 1.
Dissolve 20.0 g in carbon dioxide-free water R prepared
from distilled water R and dilute to 100.0 ml with the same
solvent. Measure the conductivity of the solution while
gently stirring with a magnetic stirrer.
Reducing sugars : maximum 0.2 per cent, expressed as
glucose equivalent.
Dissolve 5.0 g in 6 ml of water R with the aid of gentle
heat. Cool and add 20 ml of cupri-citric solution R and a
few glass beads. Heat so that boiling begins after 4 min and
maintain boiling for 3 min. Cool rapidly and add 100 ml
of a 2.4 per cent V/V solution of glacial acetic acid R and
20.0 ml of 0.025 M iodine. With continuous shaking, add
25 ml of a mixture of 6 volumes of hydrochloric acid R
and 94 volumes of water R and, when the precipitate has
dissolved, titrate the excess of iodine with 0.05 M sodium
thiosulphate using 1 ml of starch solution R, added towards
the end of the titration, as indicator. Not less than 12.8 ml of
0.05 M sodium thiosulphate is required.
Related products. Liquid chromatography (2.2.29).
Test solution. Dissolve 5.0 g of the substance to be examined
in 20 ml of water R and dilute to 100.0 ml with the same
solvent.
Reference solution (a). Dissolve 0.50 g of sorbitol CRS in
2 ml of water R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 2.0 ml of the test solution to
100.0 ml with water R.
Reference solution (c). Dilute 5.0 ml of reference solution (b) ASSAY
to 100.0 ml with water R.
Reference solution (d). Dissolve 0.5 g of sorbitol R and 0.5 g related products with the following modification.
of mannitol R (impurity A) in 5 ml of water R and dilute to
10.0 ml with the same solvent.
Column:
— size : l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : strong cation exchange resin (calcium LABELLING
form) R (9 μm) ;
— temperature : 85 ± 1 °C.
Mobile phase : degassed water R.
Flow rate : 0.5 ml/min.
Detection : refractometer maintained at a constant
temperature.
Injection : 20 μl of the test solution and reference
solutions (b), (c) and (d).
4306 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Run time : 3 times the retention time of sorbitol.
Relative retention with reference to sorbitol (retention
time = about 27 min) : impurity C = about 0.6 ;
impurity A = about 0.8 ; impurity B = about 1.1.
System suitability : reference solution (d) :
— resolution : minimum 2 between the peaks due to
impurity A and sorbitol.
Limits :
— any impurity : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (2 per cent) ;
— total : not more than 1.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (3 per cent) ;
— disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.1 per cent).
Lead (2.4.10) : maximum 0.5 ppm.
Nickel (2.4.15) : maximum 1 ppm.
Dissolve the substance to be examined in 150.0 ml of the
prescribed mixture of solvents.
Water (2.5.12) : maximum 1.5 per cent, determined on 1.00 g.
Microbial contamination
If intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 102 CFU/g (2.6.12).
If not intended for use in the manufacture of parenteral
preparations :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
— absence of Escherichia coli (2.6.13) ;
— absence of Salmonella (2.6.13).
Bacterial endotoxins (2.6.14). If intended for use in
the manufacture of parenteral preparations without a
further appropriate procedure for the removal of bacterial
endotoxins :
— less than 4 IU/g for parenteral preparations having a
concentration of less than 100 g/l of sorbitol ;
— less than 2.5 IU/g for parenteral preparations having a
concentration of 100 g/l or more of sorbitol.
Liquid chromatography (2.2.29) as described in the test for
Injection : test solution and reference solution (a).
Calculate the percentage content of D-sorbitol from the
declared content of sorbitol CRS.
The label states :
— where applicable, the maximum concentration of bacterial
endotoxins ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
A. mannitol,
EUROPEAN PHARMACOPOEIA 6.3 Sorbitol, liquid, partially dehydrated

Reducing sugars : maximum 0.3 per cent, calculated as

glucose (anhydrous substance).
To an amount of the substance to be examined equivalent to
3.3 g of anhydrous substance, add 3 ml of water R, 20.0 ml
of cupri-citric solution R and a few glass beads. Heat so
that boiling begins after 4 min. Maintain boiling for 3 min.
B. iditol, Cool rapidly and add 100 ml of a 2.4 per cent V/V solution
of glacial acetic acid R and 20.0 ml of 0.025 M iodine.
With continuous shaking, add 25 ml of a mixture of 6 ml
of hydrochloric acid R and 94 ml of water R. When the
C. maltitol. precipitate has dissolved, titrate the excess of iodine with
0.05 M sodium thiosulphate using 2 ml of starch solution R,
added towards the end of the titration, as indicator. Not less
than 12.8 ml of 0.05 M sodium thiosulphate is required.
01/2009:2048 Nickel (2.4.15) : maximum 1 ppm (anhydrous substance).
Water (2.5.12) : 15.0 per cent to 32.0 per cent, determined
on 0.10 g.
SORBITOL, LIQUID, PARTIALLY Microbial contamination
DEHYDRATED
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Sorbitolum liquidum partim deshydricum
Absence of Escherichia coli (2.6.13).
DEFINITION Absence of Salmonella (2.6.13).
Partially dehydrated liquid sorbitol is obtained by
acid-catalysed partial internal dehydration of liquid sorbitol. ASSAY
It contains not less than 68.0 per cent m/m and not more Liquid chromatography (2.2.29).
than 85.0 per cent m/m of anhydrous substances, composed
Test solution. Dissolve 0.400 g of the substance to be
of a mixture of mainly D-sorbitol and 1,4-sorbitan, with
examined in water R and dilute to 20.0 ml with the same
mannitol, hydrogenated oligo- and disaccharides, and
solvent.
sorbitans.
Reference solution (a). Dissolve 50.0 mg of sorbitol CRS
Content (nominal value) : and 20.0 mg of 1,4-sorbitan CRS in water R and dilute to
— 1,4-sorbitan (C6H12O5) : minimum 15.0 per cent 5.0 ml with the same solvent.
(anhydrous substance) ; Reference solution (b). Dissolve 0.100 g of mannitol R and
— D-sorbitol
(C6H14O6) : minimum 25.0 per cent (anhydrous 0.100 g of sorbitol R in water R and dilute to 10.0 ml with
substance). the same solvent.
The contents of 1,4-sorbitan and D-sorbitol are within Column :
95.0 per cent to 105.0 per cent of the nominal values. — size: l = 0.3 m, Ø = 7.8 mm ;
— stationary phase : strong cation exchange resin (calcium
CHARACTERS
form) R (9 μm) ;
Appearance : clear, colourless, syrupy liquid.
— temperature : 80 ± 5 °C.
Solubility : miscible with water, practically insoluble in Mobile phase : degassed water R.
mineral oils and vegetable oils.
Flow rate : 0.5 ml/min.
IDENTIFICATION Detection : refractometer maintained at a constant
Examine the chromatograms obtained in the assay. temperature of about 30-35 °C.
Results : the 2 principal peaks in the chromatogram obtained Injection : 40 μl.
with the test solution are similar in retention time and size Relative retention with reference to D-sorbitol (retention
to the peaks in the chromatogram obtained with reference time = about 25 min) : 1,4-sorbitan = about 0.5 ;
solution (a). mannitol = about 0.8.
System suitability : reference solution (b) :
TESTS
— resolution : minimum 2.0 between the peaks due to
Solution S. Dilute the substance to be examined with carbon mannitol and D-sorbitol.
dioxide-free water R prepared from distilled water R to
obtain a solution containing 50.0 per cent m/m of anhydrous Calculate the percentage contents of 1,4-sorbitan and
substance. D-sorbitol using the chromatogram obtained with reference
solution (a) and the declared contents of 1,4-sorbitan CRS
Appearance of solution. Solution S is clear (2.2.1) and and of sorbitol CRS.
colourless (2.2.2, Method II).
Conductivity (2.2.38) : maximum 20 μS·cm− 1. LABELLING
Measure the conductivity of solution S, while gently stirring The label states the content of D-sorbitol and the content of
with a magnetic stirrer. 1,4-sorbitan (= nominal values).

General Notices (1) apply to all monographs and other texts 4307
Stanozolol EUROPEAN PHARMACOPOEIA 6.3

01/2008:1568 Reference solution (c). Dissolve 10 mg of stanozolol CRS

in a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R and dilute to 5 ml with the same
STANOZOLOL mixture of solvents.
Apply to the plate 5 μl of each solution. Develop over a
Stanozololum path corresponding to two thirds of the plate height using
a mixture of 10 volumes of methanol R and 90 volumes of
methylene chloride R. Allow the plate to dry in air, spray
with alcoholic solution of sulphuric acid R, heat at 105 °C
for 15 min and examine under ultraviolet light at 365 nm.
Any secondary spot in the chromatogram obtained with
test solution (a) is not more intense than the spot in the
chromatogram obtained with reference solution (a) (0.5 per
cent). The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated spots.
C21H32N2O Mr 328.5 Loss on drying (2.2.32). Not more than 1.0 per cent,
[10418-03-8] determined on 1.000 g by drying at 100 °C at a pressure
not exceeding 0.7 kPa.
DEFINITION
Stanozolol contains not less than 98.5 per cent and ASSAY
not more than the equivalent of 101.0 per cent of Dissolve 0.250 g in 50 ml of anhydrous acetic acid R. Titrate
17-methyl-2′H-5α-androst-2-eno[3,2-c]pyrazol-17β-ol, with 0.1 M perchloric acid, determining the end-point
calculated with reference to the dried substance. potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 32.85 mg of
CHARACTERS C21H32N2O.
A white or almost white crystalline powder, hygroscopic,
practically insoluble in water, soluble in dimethylformamide, STORAGE
slightly soluble in ethanol (96 per cent), very slightly soluble Store in an airtight container, protected from light.
in methylene chloride.
IMPURITIES
It shows polymorphism (5.9).
IDENTIFICATION
A. Examine by infrared spectrophotometry (2.2.24),
comparing with the spectrum obtained with
stanozolol CRS. If the spectra obtained in the solid
state show differences, dissolve the substance to be
examined and the reference substance separately in the
minimum volume of methylene chloride, evaporate to
dryness at room temperature under an air-stream and A. R = H2 : 17β-hydroxy-17-methyl-5α-androstan-3-one
record new spectra using the residues. (mestanolone),
B. Examine the chromatograms obtained in the test B. R = CH-OH : 17β-hydroxy-2-(hydroxymethylene)-17-methyl-
for related substances. The principal spot in the 5α-androstan-3-one (oxymetholone).
chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (c). 01/2009:1267
TESTS
STARCH, PREGELATINISED
Specific optical rotation (2.2.7). Dissolve 0.10 g in
chloroform R and dilute to 10.0 ml with the same solvent.
The specific optical rotation is + 34 to + 40, calculated with Amylum pregelificatum
reference to the dried substance. DEFINITION
Related substances. Examine by thin-layer chromatography Pregelatinised starch is prepared from Maize starch (0344),
(2.2.27), using a TLC silica gel F254 plate R. Potato starch (0355) or Rice starch (0349) by mechanical
Test solution (a). Dissolve 0.10 g of the substance to be processing in the presence of water, with or without heat, to
examined in a mixture of 1 volume of methanol R and rupture all or part of the starch granules, and subsequent
9 volumes of methylene chloride R and dilute to 5 ml with drying. It contains no added substances but it may be
the same mixture of solvents. modified to render it compressible and to improve its flow
characteristics.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml
with a mixture of 1 volume of methanol R and 9 volumes of CHARACTERS
methylene chloride R. Appearance : white or yellowish-white powder.
Reference solution (a). Dilute 1.0 ml of test solution (a) It swells in cold water.
to 200 ml with a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R. IDENTIFICATION
Reference solution (b). Dissolve 5 mg of stanozolol A. Examined under a microscope using a mixture of equal
impurity A CRS in reference solution (a) and dilute to 50 ml volumes of glycerol R and water R it presents irregular,
with the same solution. translucent, white or yellowish-white flakes or pieces

4308 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 St. John’s wort dry extract, quantified

with an uneven surface. Under polarised light (between IDENTIFICATION

crossed nicol prisms), starch granules with a distinct Thin-layer chromatography (2.2.27).
black cross intersecting at the hilum may be seen.
Test solution. Disperse 0.25 g of the extract to be examined
B. Disperse 0.5 g in 2 ml of water R without heating and in 5 ml of methanol R.
add 0.05 ml of iodine solution R1. A reddish-violet to
blue colour is produced. Reference solution. Dissolve 5 mg of rutin R and 5 mg of
hyperoside R in methanol R and dilute to 10 ml with the
TESTS same solvent.
pH (2.2.3) : 4.5 to 7.0. Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
Progressively add 3.0 g to 100.0 ml of carbon dioxide-free plate R (2-10 μm)].
water R, stirring continuously. Determine the pH when a Mobile phase : anhydrous formic acid R, water R, ethyl
homogeneous solution is obtained. acetate R (6:9:90 V/V/V).
Oxidising substances (2.5.30). It complies with the test for Application : 10 μl [or 5 μl] as bands of 10 mm [or 8 mm].
oxidising substances. Use a mixture of equal volumes of Development : over a path of 10 cm [or 7.5 cm] .
water R and methanol R as solvent. Drying : at 100-105 °C for 10 min.
Sulphur dioxide (2.5.29) : maximum 50 ppm. Detection : spray with a 10 g/l solution of diphenylboric
Iron (2.4.9) : maximum 20 ppm. acid aminoethyl ester R in methanol R and then with a
Dissolve the residue obtained in the test for sulphated ash 50 g/l solution of macrogol 400 R in methanol R. Examine
in 20 ml of dilute hydrochloric acid R. Filter. The filtrate after about 30 min in ultraviolet light at 365 nm.
complies with the test. Results : see below the sequence of the zones present in the
Foreign matter. Examined under a microscope using a chromatograms obtained with the reference solution and the
mixture of equal volumes of glycerol R and water R, not more test solution. Furthermore, other fluorescent zones may be
than traces of matter other than starch granules are present. present in the chromatogram obtained with the test solution.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined Top of the plate
on 1.000 g by drying in an oven at 130 °C for 90 min. A yellowish-orange fluorescent
Sulphated ash (2.4.14) : maximum 0.6 per cent, determined zone
on 1.0 g. 2 red fluorescent zones (hypericin
and pseudohypericin)
Microbial contamination _______ _______
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
3 yellowish-orange fluorescent
Absence of Escherichia coli (2.6.13). zones
Absence of Salmonella (2.6.13).
LABELLING _______ _______

The type of starch used as starting material is stated. Hyperoside : a yellowish-orange
fluorescent zone
07/2008:1874
corrected 6.3 Rutin : a yellowish-orange
fluorescent zone
ST. JOHN’S WORT DRY EXTRACT, Reference solution
A yellowish-orange fluorescent
zone (hyperoside)
Yellow and blue possibly
superimposed fluorescent zones
A yellowish-orange fluorescent
zone (rutin)
Test solution

QUANTIFIED
ASSAY
Hyperici herbae Total hypericins. Liquid chromatography (2.2.29).
extractum siccum quantificatum Test solution. Dissolve 70.0 mg of the extract to be examined
in 25.0 ml of methanol R. Sonicate and centrifuge the
DEFINITION solution. Expose the solution to a xenon lamp at about
2
Quantified dry extract obtained from St. John’s wort (1438). 765 W/m for 8 min.
Content : Reference solution. Dissolve a quantity of St. John’s wort
— total hypericins, expressed as hypericin (C30H16O8 ; standardised dry extract CRS corresponding to 0.15 mg of
Mr 504.5) : 0.10 per cent to 0.30 per cent (dried extract) ; hypericin in 25.0 ml of methanol R. Sonicate and centrifuge.
Expose the solution to a xenon lamp at about 765 W/m2
— flavonoids, expressed as rutin (C27H30O16 ; Mr 610.5) : for 8 min.
minimum 6.0 per cent (dried extract) ;
Column :
— hyperforin (C35H52O4 ; Mr 536.8) : maximum 6.0 per cent
(dried extract) and not more than the content stated on — size: l = 0.15 m, Ø = 4.6 mm ;
the label. — stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
PRODUCTION
— temperature : 40 °C.
The extract is produced from the herbal drug by a suitable
procedure using ethanol (50-80 per cent V/V) or methanol Mobile phase : mix 39 volumes of ethyl acetate R, 41 volumes
(50-80 per cent V/V). of a 15.6 g/l solution of sodium dihydrogen phosphate R
adjusted to pH 2 with phosphoric acid R and 160 volumes
CHARACTERS of methanol R.
Appearance : brownish-grey powder. Flow rate : 1.0 ml/min.

General Notices (1) apply to all monographs and other texts 4309
St. John’s wort dry extract, quantified EUROPEAN PHARMACOPOEIA 6.3

Detection : spectrophotometer at 590 nm.
Injection : 20 μl.
Run time : 15 min.
Identification of peaks : use the chromatogram supplied
with St. John’s wort standardised dry extract CRS and
the chromatogram obtained with the reference solution to
identify the peaks due to pseudohypericin and hypericin.
System suitability : reference solution :
— the chromatogram obtained is similar to the
chromatogram supplied with St. John’s wort
standardised dry extract CRS ;
— resolution : minimum 2 between the peaks due to
pseudohypericin and hypericin.
Calculate the percentage content of total hypericins,
expressed as hypericin, using the following expression :
Detection : spectrophotometer at 360 nm, then at 275 nm
after the elution of biapigenin (about 22 min).
Injection : 10 μl.
Identification of peaks : use the chromatogram supplied
with St. John’s wort standardised dry extract CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to rutin, hyperoside, isoquercitroside,
quercitroside, quercetin, biapigenin, hyperforin and
adhyperforin.
System suitability : reference solution (b) :
— the chromatogram obtained is similar to the
chromatogram supplied with St. John’s wort
standardised dry extract CRS ;
— resolution : minimum 2.0 between the peaks due to rutin
and hyperoside, and minimum 2.0 between the peaks due
to hyperforin and adhyperforin.
Calculate the percentage content of hyperforin using the
following expression :

A1 = area of the peak due to pseudohypericin in the

chromatogram obtained with the test solution ;
A2 = area of the peak due to hypericin in the
A4 = area of the peak due to hyperforin in the
chromatogram obtained with the test solution ;
chromatogram obtained with the test solution ;
A3 = area of the peak due to hypericin in the
A5 = area of the peak due to rutin in the chromatogram
chromatogram obtained with the reference
obtained with reference solution (a) ;
solution ;
m3 = mass of the extract to be examined used to
m1 = mass of the extract to be examined used to
prepare the test solution, in grams ;
prepare the test solution, in grams ;
m4 = mass of rutoside trihydrate CRS used to prepare
m2 = mass of St. John’s wort standardised dry
the reference solution, in grams ;
extract CRS used to prepare the reference
solution, in grams ; 2.3 = correction factor for hyperforin with respect to
p = percentage content of hypericin in St. John’s rutin ;
wort standardised dry extract CRS. p = percentage content of rutin in rutoside
trihydrate CRS.
Hyperforin and flavonoids. Liquid chromatography (2.2.29).
Carry out the assay protected from light. Calculate the percentage content of flavonoids, expressed as
rutin, using the following expression :
Solvent mixture : water R, methanol R (20:80 V/V).
Test solution. Dissolve 75.0 mg of the extract to be examined
in 20.0 ml of the solvent mixture. Sonicate and centrifuge.
Reference solution (a). Dissolve 20.0 mg of rutoside
trihydrate CRS in 200.0 ml of the solvent mixture. A5 = area of the peak due to rutin in the chromatogram
obtained with reference solution (a) ;
Reference solution (b). Dissolve 75.0 mg of St. John’s wort
standardised dry extract CRS in 20.0 ml of the solvent A6 = area of the peak due to rutin in the chromatogram
mixture. Sonicate and centrifuge. obtained with the test solution ;
A7 = area of the peak due to hyperoside in the
Column:
chromatogram obtained with the test solution ;
— size : l = 0.15 m, Ø = 4.6 mm ; =
A8 area of the peak due to isoquercitroside in the
— stationary phase : octadecylsilyl silica gel for chromatogram obtained with the test solution ;
chromatography R (3 μm). =
A9 area of the peak due to quercitroside in the
Mobile phase : chromatogram obtained with the test solution ;
— mobile phase A : phosphoric acid R, water R (3:1000 V/V) ; A10 = area of the peak due to quercetin in the
— mobile phase B : phosphoric acid R, acetonitrile R chromatogram obtained with the test solution ;
(3:1000 V/V) ; A11 = area of the peak due to biapigenin in the
Time Mobile phase A Mobile phase B Flow rate
chromatogram obtained with the test solution ;
(min) (per cent V/V) (per cent V/V) (ml/min) m3 = mass of the extract to be examined used to
0-8 82 18 0.8 prepare the test solution, in grams ;
m4 = mass of rutoside trihydrate CRS used to prepare
8 - 18 82 → 47 18 → 53 0.8
the reference solution, in grams ;
18 - 18.1 47 → 3 53 → 97 0.8 p = percentage content of rutin in rutoside
18.1 - 19 3 97 0.8 → 1.2 trihydrate CRS.
19 - 29 3 97 1.2
LABELLING
29 - 30 3 → 82 97 → 18 1.2
The label states the content of hyperforin.

4310 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
01/2009:0204
SUCROSE
Saccharum
C12H22O11 Mr 342.3
[57-50-1]
DEFINITION
β-D-Fructofuranosyl α-D-glucopyranoside.
It contains no additives.
CHARACTERS
Appearance : white or almost white, crystalline powder, or
lustrous, colourless or white or almost white crystals.
Solubility : very soluble in water, slightly soluble in ethanol
(96 per cent), practically insoluble in anhydrous ethanol.
IDENTIFICATION
First identification : A.
Second identification : B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : sucrose CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in a mixture of 2 volumes of water R and
3 volumes of methanol R and dilute to 20 ml with the
same mixture of solvents.
Reference solution (a). Dissolve 10 mg of sucrose CRS
in a mixture of 2 volumes of water R and 3 volumes of
methanol R and dilute to 20 ml with the same mixture of b
solvents.
Reference solution (b). Dissolve 10 mg each of
fructose CRS, glucose CRS, lactose CRS and
sucrose CRS in a mixture of 2 volumes of water R and
3 volumes of methanol R and dilute to 20 ml with the
same mixture of solvents.
Plate : TLC silica gel G plate R.
Mobile phase : cold saturated boric acid solution R,
60 per cent V/V solution of glacial acetic acid R,
ethanol R, acetone R, ethyl acetate R (10:15:20:60:60
V/V/V/V/V).
Application : 2 μl.
Development : in an unsaturated tank over a path of
15 cm.
Drying : in a current of warm air.
Detection : spray with a solution of 0.5 g of thymol R
in a mixture of 5 ml of sulphuric acid R and 95 ml of
alcohol R. Heat the plate at 130 °C for 10 min.
System suitability : the chromatogram obtained with
reference solution (b) shows 4 clearly separated spots.
General Notices (1) apply to all monographs and other texts
Sucrose
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
C. Dilute 1 ml of solution S (see Tests) to 100 ml with
water R. To 5 ml of the solution add 0.15 ml of freshly
prepared copper sulphate solution R and 2 ml of freshly
prepared dilute sodium hydroxide solution R. The
solution is blue and clear and remains so after boiling. To
the hot solution add 4 ml of dilute hydrochloric acid R
and boil for 1 min. Add 4 ml of dilute sodium hydroxide
solution R. An orange precipitate is formed immediately.
TESTS
Solution S. Dissolve 50.0 g in water R and dilute to 100 ml
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1).
Conductivity (2.2.38) : maximum 35 μS·cm− 1 at 20 °C.
Dissolve 31.3 g in carbon dioxide-free water R prepared
from distilled water R and dilute to 100 ml with the same
solvent. Measure the conductivity of the solution (C1), while
gently stirring with a magnetic stirrer, and that of the water
used for preparing the solution (C2). The readings must be
stable within 1 per cent over a period of 30 s. Calculate the
conductivity of the solution of the substance to be examined
from the following expression :
Specific optical rotation (2.2.7) : + 66.3 to + 67.0.
Dissolve 26.0 g in water R and dilute to 100.0 ml with the
same solvent.
Colour value : maximum 45.
Dissolve 50.0 g in 50.0 ml of water R. Mix, filter (diameter of
pores 0.45 μm) and degas. Measure the absorbance (2.2.25)
at 420 nm, using a cell of minimum 4 cm (a cell length of
10 cm or more is preferred).
Calculate the colour value using the following expression :
A = absorbance measured at 420 nm ;
= path length in centimetres ;
c = concentration of the solution, in grams per
millilitre, calculated from the refractive index
(2.2.6) of the solution ; use Table 0204.-1 and
interpolate the values if necessary.
Table 0204.-1
c
(g/ml)
1.4138 0.570
1.4159 0.585
1.4179 0.600
1.4200 0.615
1.4221 0.630
1.4243 0.645
1.4264 0.661
System suitability :
— repeatability : the absolute difference between 2 results
is not greater than 3.
4311
Sugar spheres
Dextrins. If intended for use in the manufacture of
large-volume parenteral preparations, it complies with the
test for dextrins. To 2 ml of solution S add 8 ml of water R,
0.05 ml of dilute hydrochloric acid R and 0.05 ml of 0.05 M
iodine. The solution remains yellow.
Reducing sugars. To 5 ml of solution S in a test-tube about
150 mm long and 16 mm in diameter add 5 ml of water R,
1.0 ml of 1 M sodium hydroxide and 1.0 ml of a 1 g/l
solution of methylene blue R. Mix and place in a water-bath.
After exactly 2 min, take the tube out of the bath and
examine the solution immediately. The blue colour does
not disappear completely. Ignore any blue colour at the
air/solution interface.
Sulphites : maximum 10 ppm, calculated as SO2.
Determine the sulphites content by a suitable enzymatic
method based on the following reactions. Sulphite is
oxidised by sulphite oxidase to sulphate and hydrogen
peroxide which in turn is reduced by nicotinamide-adenine
dinucleotide-peroxidase in the presence of reduced
nicotinamide-adenine dinucleotide (NADH). The amount of
NADH oxidised is proportional to the amount of sulphite.
Test solution. Dissolve 4.0 g of the substance to be examined
in freshly prepared distilled water R and dilute to 10.0 ml
with the same solvent.
Reference solution. Dissolve 4.0 g of the substance to be
examined in freshly prepared distilled water R, add 0.5 ml
of sulphite standard solution (80 ppm SO2) R and dilute to
10.0 ml with freshly prepared distilled water R.
Blank solution. Freshly prepared distilled water R.
Separately introduce 2.0 ml each of the test solution, the
reference solution and the blank in 10 mm cuvettes and add
the reagents as described in the instructions in the kit for
sulphite determination. Measure the absorbance (2.2.25)
at the absorption maximum at about 340 nm before and at
the end of the reaction time and subtract the value obtained
with the blank.
The absorbance difference of the test solution is not greater
than half the absorbance difference of the reference solution.
Loss on drying (2.2.32) : maximum 0.1 per cent, determined
on 2.000 g by drying in an oven at 105 °C for 3 h.
Bacterial endotoxins (2.6.14) : less than 0.25 IU/mg,
if intended for use in the manufacture of large-volume
parenteral preparations.
LABELLING
The label states, where applicable, that the substance
is suitable for use in the manufacture of large-volume
parenteral preparations.
01/2009:1570
SUGAR SPHERES
Sacchari sphaerae
DEFINITION
Sugar spheres contain not more than 92 per cent of sucrose, Microbial contamination
calculated on the dried basis. The remainder consists of
maize starch and may also contain starch hydrolysates and
colour additives. The diameter of sugar spheres varies
usually from 200 μm to 2000 μm and the upper and lower
limits of the size of the sugar spheres are stated on the label. Absence of Salmonella (2.6.13).
4312 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using
a TLC silica gel G plate R.
Solvent mixture: water R, methanol R (2:3 V/V).
Test solution. Mix 2 ml of solution S (see Tests) with
3 ml of methanol R and dilute to 20 ml with the solvent
mixture.
Reference solution (a). Dissolve 10 mg of sucrose CRS in
the solvent mixture and dilute to 20 ml with the solvent
mixture.
Reference solution (b). Dissolve 10 mg of fructose CRS,
10 mg of glucose CRS, 10 mg of lactose CRS and 10 mg
of sucrose CRS in the solvent mixture and dilute to 20 ml
with the solvent mixture.
Apply to the plate 2 μl of each solution and thoroughly
dry the starting points. Develop over a path of 15 cm
using a mixture of 10 volumes of water R, 15 volumes of
methanol R, 25 volumes of anhydrous acetic acid R and
50 volumes of ethylene chloride R, measured accurately
as a slight excess of water causes cloudiness of the
solution. Dry the plate in a current of warm air. Repeat
the development immediately after renewing the mobile
phase. Dry the plate in a current of warm air and spray
evenly with a 5 g/l solution of thymol R in a mixture of
5 volumes of sulphuric acid R and 95 volumes of ethanol
(96 per cent) R. Heat at 130 °C for 10 min. The principal
spot in the chromatogram obtained with the test solution
is similar in position, colour 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 4 clearly separated
spots.
B. To a water slurry of the insoluble portion obtained in the
assay, add 0.05 ml of iodine solution R1. A dark-blue
colour is produced, which disappears on heating.
C. To 5 ml of solution S add 0.15 ml of freshly prepared
copper sulphate solution R and 2 ml of freshly prepared
dilute sodium hydroxide solution R. The solution is blue
and clear and remains so after boiling. To the hot solution
add 4 ml of dilute hydrochloric acid R and boil for 1 min.
Add 4 ml of dilute sodium hydroxide solution R. An
orange precipitate is formed immediately.
TESTS
Solution S. To 0.5 g in a 100 ml volumetric flask add 80 ml
of water R and shake until the sucrose is dissolved. Dilute
to 100.0 ml with water R. Filter under vacuum to obtain a
clear solution.
Fineness (2.9.35): not less than 90 per cent m/m of the
sugar spheres are between the lower and the upper limits of
the size of the sugar spheres stated on the label.
Heavy metals (2.4.8): maximum 5 ppm.
2.0 g complies with test C. Prepare the reference solution
using 1.0 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32): maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Sulphated ash (2.4.14): maximum 0.2 per cent, determined
on 2 g.
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
EUROPEAN PHARMACOPOEIA 6.3
ASSAY
Sucrose content
Weigh 10.000 g of ground sugar spheres in a 100 ml flask
and make up to 100.0 ml with water R. Stir and decant.
Filter under vacuum to obtain a clear solution (the insoluble
portion is used for identification test B). Measure the angle ofphosphate R in 900 ml of water R. Add 7.0 ml of phosphoric
optical rotation (2.2.7) and calculate the sucrose percentage
content using the following expression :
α = angle of rotation;
l = length of the polarimeter tube, in decimetres;
m tosilate CRS in 35 ml of solution A and sonicate for about
= exact mass of the sample, in grams;
H = loss on drying.
LABELLING
The label states the upper and the lower limits of the size
of the sugar spheres.
01/2008:2212
corrected 6.3 Reference solution (c). Dissolve 0.200 g of the substance to
SULTAMICILLIN TOSILATE
DIHYDRATE
Sultamicillini tosilas dihydricus
C32H38N4O12S3,2H2O Mr 803
DEFINITION
4-Methylbenzenesulphonate of methylene
(2S,5R,6R)-6-[[(2R)-aminophenylacetyl]amino]-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate
6
(2S,5R)-3,3-dimethyl-4,4,7-trioxo-4λ -thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate dihydrate.
Semi-synthetic product derived from a fermentation product.
Content : 95.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, sparingly soluble reference solutions (b), (c) and (d).
in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : sultamicillin tosilate CRS.
TESTS
Specific optical rotation (2.2.7) : + 178 to + 195 (anhydrous
substance).
Dissolve 1.000 g in dimethylformamide R and dilute to
50.0 ml with the same solvent.
General Notices (1) apply to all monographs and other texts
Sultamicillin tosilate dihydrate
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use or keep at
2-8 °C for not more than 6 h.
Solution A : methanol R1, acetonitrile R1 (20:80 V/V).
Solution B. Dissolve 1.56 g of sodium dihydrogen
acid R and dilute to 1000 ml with water R.
Blank solution : solution B, solution A (30:70 V/V).
Test solution. Dissolve 70.0 mg of the substance to be
examined in 35 ml of solution A and sonicate for about
1 min. Add 13 ml of solution B, mix and sonicate for about
1 min. Dilute to 50.0 ml with solution B and mix.
Reference solution (a). Dissolve 70.0 mg of sultamicillin
1 min. Add 13 ml of solution B, mix and sonicate for about
1 min. Dilute to 50.0 ml with solution B and mix.
Reference solution (b). Suspend 15 mg of the substance
to be examined in 20 ml of a 0.4 g/l solution of sodium
hydroxide R and sonicate in an ultrasonic bath for about
5 min. Add 20 ml of a 0.36 g/l solution of hydrochloric
acid R and dilute to 100.0 ml with water R.
be examined in 70.0 ml of solution A and sonicate for about
1 min. Add 25.0 ml of solution B, mix and sonicate for about
1 min. Dilute to 100.0 ml with solution B and mix. Dilute
1.0 ml of this solution to 100.0 ml with the blank solution.
Reference solution (d). Dissolve 32.3 mg of ampicillin
trihydrate CRS (impurity B) and 7.0 mg of sulbactam CRS
(impurity A) in water R and dilute to 1000 ml with the same
solvent.
Column :
— size: l = 0.10 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (3.5 μm) ;
— temperature : 25 °C.
Mobile phase :
— mobile phase A : 4.68 g/l solution of sodium dihydrogen
phosphate R adjusted to pH 3.0 with phosphoric acid R ;
— mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 95 → 30 5 → 70
15 - 16 30 70
16 - 16.5 30 → 95 70 → 5
16.5 - 20 95 5
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 215 nm.
Injection : 5 μl of the blank solution, the test solution and
Relative retention with reference to sultamicillin (retention
time = about 9.3 min) : impurity A = about 0.41 ; ampicillin
penicilloic acid = about 0.47 ; tosilate = about 0.50 ;
impurity B = about 0.55 ; impurity C = about 0.94 ;
impurity D = about 1.09 ; impurity F = about 1.23 ;
impurity E = about 1.26 ; impurity G = about 1.42.
System suitability : reference solution (b) :
— resolution : minimum 2.5 between the peaks due to
ampicillin penicilloic acid and tosilate and minimum 2.5
between the peaks due to tosilate and impurity B.
4313
Sultamicillin tosilate dihydrate EUROPEAN PHARMACOPOEIA 6.3

Limits : Injection : 1 ml.

— impurity B : not more than the area of the corresponding Relative retention with reference to dimethylformamide
peak in the chromatogram obtained with reference (retention time = about 14 min) : ethyl acetate = about 0.7.
solution (d) (2.0 per cent) ; Limit :
— impurity A : not more than the area of the corresponding — ethyl acetate : maximum 2.0 per cent.
peak in the chromatogram obtained with reference
solution (d) (0.5 per cent) ; Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in a mixture of 40 volumes of methanol R and
— impurities C, D, E, F, G : for each impurity, not more
60 volumes of acetonitrile R and dilute to 20.0 ml with the
than 0.5 times the area of the peak due to sultamicillin
same mixture of solvents. 12 ml of the solution complies with
in the chromatogram obtained with reference solution (c)
test B. Prepare the reference solution using lead standard
(0.5 per cent) ;
solution (2 ppm Pb) obtained by diluting lead standard
— any other impurity : for each impurity, not more than solution (100 ppm Pb) R with a mixture of 40 volumes of
0.5 times the area of the peak due to sultamicillin in methanol R and 60 volumes of acetonitrile R.
the chromatogram obtained with reference solution (c) Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on
(0.5 per cent) ; 0.200 g.
— total : not more than 4 times the area of the peak due Sulphated ash (2.4.14) : maximum 0.2 per cent, determined
to sultamicillin in the chromatogram obtained with on 1.0 g.
reference solution (c) (4.0 per cent) ;
— disregard limit: 0.1 times the area of the peak due ASSAY
to sultamicillin in the chromatogram obtained with Liquid chromatography (2.2.29) as described in the test for
reference solution (c) (0.1 per cent). related substances with the following modification.
Ethyl acetate. Head space gas chromatography (2.2.28). Injection : test solution and reference solution (a).
Test solution. Dissolve 0.200 g in 7.0 ml of a mixture of Calculate the percentage content of sultamicillin tosilate
1 volume of water R and 99 volumes of dimethylformamide R. (C32H38N4O12S3) from the declared content of sultamicillin
Reference solution. Dissolve 0.200 g of ethyl acetate R in tosilate CRS.
240 ml of a mixture of 1 volume of water R and 99 volumes
of dimethylformamide R and dilute to 250.0 ml with the STORAGE
same mixture of solvents. Dilute 5.0 ml of this solution to In an airtight container.
7.0 ml with a mixture of 1 volume of water R and 99 volumes
of dimethylformamide R. IMPURITIES
Immediately close the vials with a tight rubber membrane Specified impurities : A, B, C, D, E, F, G.
stopper coated with polytetrafluoroethylene and secure
with an aluminium crimped cap. Shake to obtain a A. sulbactam,
homogeneous solution.
Column: B. ampicillin,
— material: fused silica ;
— size : l = 50 m, Ø = 0.32 mm ;
— stationary phase : poly(dimethyl)siloxane R (film
thickness : 1.8 μm or 3 μm).
Carrier gas: helium for chromatography R.
Linear velocity : 35 cm/s.
Split ratio : 1:5.
C. [[(2R)-aminophenylacetyl]amino][(4S)-4-[[[[[(2S,5R)-
Static head-space conditions that may be used : 3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]hept-
— equilibration temperature: 105 °C; 2-yl]carbonyl]oxy]methoxy]carbonyl]-5,5-
dimethylthiazolidin-2-yl]acetic acid (penicilloic
— equilibration time : 45 min ;
acids of sultamicillin),
— transfer-line temperature : 110 °C ;
— pressurisation time : 30 s.
Temperature :
Time Temperature
(min) (°C)
Column 0-6 70
6 - 16 70 → 220
16 - 18 220
D. methylene (2S,5R,6R)-3,3-dimethyl-6-[[(2R)-[(1-methyl-
Injection port 140
4-oxopentylidene)amino]phenylacetyl]amino]-7-oxo-4-
Detector 250 thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)-
3,3-dimethyl-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-
Detection : flame ionisation. carboxylate,

4314 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
E. methylene bis[(2S,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate] (sulbactam
methylene ester),
F. methylene (2S,5R,6R)-6-[[(2R)-[[[(2S,5R,6R)-6-[[(2R)-
aminophenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-
phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)-3,3-
dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane- Test solution. Dissolve 30.0 mg of the substance to be
2-carboxylate (ampicillin sultamicillin amide),
G. methylene (2S,5R,6R)-6-[[(2R)-[[[[(2R)-amino-
phenylacetyl]amino][(4S)-4-[[[[[(2S,5R)-3,3-dimethyl-
4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]hept-2-yl]-
carbonyl]oxy]methoxy]carbonyl]-5,5-dimethylthiazolidin- Detection : spectrophotometer at 282 nm.
2-yl]acetyl]amino]phenylacetyl]amino]-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate
6
(2S,5R)-3,3-dimethyl-4,4,7-trioxo-4λ -thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylate (sultamicillin dimer). Relative retention with reference to sumatriptan
01/2009:1573 impurity H = about 3.0.
SUMATRIPTAN SUCCINATE
Sumatriptani succinas
C18H27N3O6S Mr 413.5
[103628-48-4]
General Notices (1) apply to all monographs and other texts
Sumatriptan succinate
DEFINITION
[3-[2-(Dimethylamino)ethyl]-1H-indol-5-yl]-N-
methylmethanesulphonamide hydrogen butanedioate.
Content : 97.5 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : freely soluble in water, sparingly soluble in
methanol, practically insoluble in methylene chloride.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison : sumatriptan succinate CRS.
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R
and dilute to 25.0 ml with the same solvent.
pH (2.2.3) : 4.5 to 5.3.
Dilute 2.5 ml of solution S to 10 ml with carbon dioxide-free
water R.
Absorbance (2.2.25) : maximum 0.10, measured at 440 nm
on solution S.
Impurities A and H. Liquid chromatography (2.2.29).
examined in the mobile phase and dilute to 10.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve the contents of a vial
of sumatriptan for system suitability CRS (containing
impurities A and H) in the mobile phase and dilute to 1 ml
with the mobile phase.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : silica gel for chromatography R
(5 μm).
Mobile phase : mix 10 volumes of a 771 g/l solution of
ammonium acetate R and 90 volumes of methanol R.
Flow rate : 2.0 ml/min.
Injection : 20 μl.
Run time : 5 times the retention time of sumatriptan.
(retention time = about 2.5 min) : impurity A = about 2.2 ;
System suitability : reference solution (b) :
— the chromatogram is similar to the chromatogram
supplied with sumatriptan for system suitability CRS ;
— resolution : minimum 3.0 between the peaks due to
impurities A and H.
Limits :
— correction factor : for the calculation of content, multiply
the peak area of impurity A by 0.6 ;
— impurity A : not more than 6 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.6 per cent) ;
— impurity H : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent).
4315
Sumatriptan succinate EUROPEAN PHARMACOPOEIA 6.3

Related substances. Liquid chromatography (2.2.29). — total : not more than 6 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Solution A. Dissolve 2.925 g of sodium dihydrogen
(0.6 per cent) ;
phosphate R in 600 ml of water R, adjust to pH 6.5 with
strong sodium hydroxide solution R, dilute to 750 ml with — disregard limit : 0.5 times the area of the principal peak
water R, add 250 ml of acetonitrile R and mix. in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Test solution (a). Dissolve 30.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 ml with the Water (2.5.12) : maximum 1.0 per cent, determined on
mobile phase. 0.500 g.
Test solution (b). Dissolve 15.0 mg of the substance to Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
be examined in solution A and dilute to 100.0 ml with on 1.0 g.
solution A.
ASSAY
Reference solution (a). Dilute 1.0 ml of test solution (a) to
100.0 ml with the mobile phase. Dilute 1.0 ml of this solution Liquid chromatography (2.2.29) as described in the test for
to 10.0 ml with the mobile phase. related substances with the following modification.
Reference solution (b). Dissolve the contents of a vial of Injection : test solution (b) and reference solution (c).
sumatriptan impurity mixture CRS (containing impurities B, Calculate the percentage content of C18H27N3O6S from the
C, D and E) in the mobile phase and dilute to 1 ml with the declared content of sumatriptan succinate CRS.
mobile phase.
STORAGE
Reference solution (c). Dissolve 15.0 mg of sumatriptan
succinate CRS in solution A and dilute to 100.0 ml with Protected from light.
solution A.
IMPURITIES
Column:
Specified impurities: A, B, C, D, E, H.
— size : l = 0.25 m, Ø = 4 mm ;
Other detectable impurities (the following substances
— stationary phase : octadecylsilyl silica gel for would, if present at a sufficient level, be detected by one
chromatography R (5 μm). or other of the tests in the monograph. They are limited
Mobile phase : mix 25 volumes of acetonitrile R with by the general acceptance criterion for other/unspecified
75 volumes of a solution prepared as follows : dissolve impurities and/or by the general monograph Substances for
0.970 g of dibutylamine R, 0.735 g of phosphoric acid R pharmaceutical use (2034). It is therefore not necessary to
and 2.93 g of sodium dihydrogen phosphate R in 750 ml identify these impurities for demonstration of compliance.
of water R, adjust to pH 6.5 with strong sodium hydroxide See also 5.10. Control of impurities in substances for
solution R and dilute to 1000 ml with water R. pharmaceutical use) : F, G.
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 282 nm.
Injection : 10 μl of test solution (a) and reference solutions (a)
and (b).
Run time : 4 times the retention time of sumatriptan.
Identification of impurities: use the chromatogram
obtained with reference solution (b) and the chromatogram
supplied with sumatriptan impurity mixture CRS to identify
the peaks due to impurities B, C, D and E.
Relative retention with reference to sumatriptan
(retention time = about 7 min) : impurity E = about 0.5 ; A. [3-[2-(dimethylamino)ethyl]-2-[[3-[2-(dimethylamino)eth-
impurity B = about 0.6 ; impurity D = about 0.7 ; yl]-1H-indol-5-yl]methyl]-1H-indol-5-yl]-N-methylmethane-
impurity C = about 0.8. sulphonamide,
System suitability : reference solution (b) :
— resolution : minimum 1.5 between the peaks due to
sumatriptan and impurity C ;
— the chromatogram shows 5 clearly separated peaks.
Limits :
— impurities B, C, D : for each impurity, not more than
5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ;
— impurity E : not more than the area of the principal peak B. R1 = R2 = H : N-methyl[3-[2-(methylamino)ethyl]-1H-indol-
in the chromatogram obtained with reference solution (a) 5-yl]methanesulphonamide,
(0.1 per cent) ;
— unspecified impurities: for each impurity, not more C. R1 = CH2-OH, R2 = CH3 : [3-[2-(dimethylamino)eth-
than the area of the principal peak in the chromatogram yl]-1-(hydroxymethyl)-1H-indol-5-yl]-N-methylmethanesul-
obtained with reference solution (a) (0.10 per cent) ; phonamide,

4316 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Sumatriptan succinate

F. R = H : N-methyl(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-
6-yl)methanesulphonamide,
G. R = CH3 : N-methyl(2-methyl-2,3,4,9-tetrahydro-1H-
D. N,N-dimethyl-2-[5-[(methylsulphamoyl)methyl]-1H-indol-3- pyrido[3,4-b]indol-6-yl)methanesulphonamide,
yl]ethanamine N-oxide,

H. [3-[2-(dimethylamino)ethyl]-1-[[3-[2-(dimethylamino)eth-
E. [3-(2-aminoethyl)-1H-indol-5-yl]-N-methylmethanesulphon- yl]-1H-indol-5-yl]methyl]-1H-indol-5-yl]-N-methylmethane-
amide, sulphonamide.

General Notices (1) apply to all monographs and other texts 4317
 EUROPEAN PHARMACOPOEIA 6.3

4318 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
T
Talc.............................................................................................. 4321
Teicoplanin.. ..............................................................................4323
Telmisartan................................................................................4325
Tetracosactide...........................................................................4326
Tragacanth.. ..............................................................................4328
General Notices (1) apply to all monographs and other texts
Triamterene.. .............................................................................4329
Tributyl acetylcitrate.. .............................................................4330
Trypsin.. ..................................................................................... 4331
Tryptophan................................................................................4333
4319
 EUROPEAN PHARMACOPOEIA 6.3

4320 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Talc

01/2009:0438 — fibre bundles displaying frayed ends,

— fibres in the form of thin needles,
TALC — matted masses of individual fibres and/or fibres showing
curvature.
Talcum
[14807-96-6]
DEFINITION
Powdered, selected, natural, hydrated magnesium silicate.
Pure talc has the formula Mg3Si4O10(OH)2 (Mr 379.3). It may
contain variable amounts of associated minerals among
which chlorites (hydrated aluminium and magnesium
silicates), magnesite (magnesium carbonate), calcite (calcium
carbonate) and dolomite (calcium and magnesium carbonate)
are predominant.
PRODUCTION
Talc derived from deposits that are known to contain
associated asbestos is not suitable for pharmaceutical use.
The manufacturer is responsible for demonstrating by the
test for amphiboles and serpentines that the product is
free from asbestos. The presence of amphiboles and of
serpentines is revealed by X-ray diffraction or by infrared
spectrophotometry (see A and B). If detected, the specific
morphological criteria of asbestos are investigated by a
suitable method of optical microscopy to determine whether
tremolite asbestos or chrysotile is present, as described
below.
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of potassium bromide R.
In the range 740 cm− 1 to 760 cm− 1 using scale expansion,
any absorption band at 758 ± 1 cm− 1 may indicate the
presence of tremolite or of chlorite. If the absorption band
remains after ignition of the substance to be examined at TESTS
850 ± 50 °C for at least 30 min, it indicates the presence
of the tremolite. In the range 600 cm− 1 to 650 cm− 1 using Solution S1. Weigh 10.0 g into a conical flask fitted with
scale expansion, any absorption band or shoulder may
indicate the presence of serpentines.
B. X-ray diffraction.
Preparation : place the sample on the sample holder ;
pack and smooth its surface with a polished glass
microscope slide.
Radiation : Cu Kα monochromatic, 40 kV, 24-30 mA.
Incident slit : 1°.
Detection slit : 0.2°.
Goniometer speed : 1/10° 2θ/min.
Scanning range: 10-13° 2θ and 24-26° 2θ.
Sample : not oriented.
Results : the presence of amphiboles is detected by
a diffraction peak at 10.5 ± 0.1° 2θ, the presence
of serpentines is detected by diffraction peaks at
24.3 ± 0.1° 2θ and at 12.1 ± 0.1° 2θ.
If, by one of the 2 methods, amphiboles and/or serpentine the dish with water R. Transfer into a volumetric flask, rinse
are detected, examine by a suitable method of optical
microscopy to determine the asbestos character.
The presence of asbestos is shown if the following 2 criteria Acidity or alkalinity. Boil 2.5 g with 50 ml of carbon
are met :
— a range of length to width ratios of 20:1 to 100:1, or
higher for fibres longer than 5 μm,
— capability of splitting into very thin fibrils,
and if at least 2 of the following 4 criteria are met :
— parallel fibres occurring in bundles,
CHARACTERS
Appearance : light, homogeneous, white or almost white
powder, greasy to the touch (non abrasive).
Solubility : practically insoluble in water, in ethanol (96 per
cent) and in dilute solutions of acids and alkali hydroxides.
IDENTIFICATION
First identification : A.
Second identification : B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of potassium bromide R.
Absorption bands: at 3677 ± 2 cm− 1, 1018 ± 2 cm− 1 and
669 ± 2 cm− 1.
B. In a platinum crucible, melt a mixture of 0.2 g of
anhydrous sodium carbonate R and 2.0 g of potassium
carbonate R. To the melted mass add 0.1 g of the
substance to be examined and heat until the mixture is
completely melted. Allow to cool and transfer the melted
mass into an evaporating dish with 50 ml of hot water R.
Add hydrochloric acid R until effervescence ceases. Add
10 ml of hydrochloric acid R and evaporate to dryness on
a water-bath. Allow to cool. Add 20 ml of water R, heat
to boiling and filter (the residue is used for identification
test C). To 5 ml of the filtrate add 1 ml of ammonia R
and 1 ml of ammonium chloride solution R and filter. To
the filtrate add 1 ml of disodium hydrogen phosphate
solution R. A white, crystalline precipitate is formed.
C. The residue obtained in identification test B gives the
reaction of silicates (2.3.1).
a reflux condenser, add 50 ml of 0.5 M hydrochloric acid
gradually while stirring and heat on a water-bath for 30 min.
Allow to cool. Transfer the mixture to a beaker and allow
the undissolved material to settle. Filter the supernatant
through medium-speed filter paper into a 100 ml volumetric
flask, retaining as much as possible of the insoluble material
in the beaker. Wash the residue and the beaker with
3 quantities, each of 10 ml, of hot water R. Wash the filter
with 15 ml of hot water R, allow the filtrate to cool and
dilute to 100.0 ml with the same solvent.
Solution S2. Perchlorates mixed with heavy metals are
known to be explosive. Take proper precautions while
performing this procedure. Weigh 0.5 g in a 100 ml
polytetrafluoroethylene dish, add 5 ml of hydrochloric
acid R, 5 ml of lead-free nitric acid R and 5 ml of perchloric
acid R. Stir gently then add 35 ml of hydrofluoric acid R and
evaporate slowly to dryness on a hot plate. To the residue,
add 5 ml of hydrochloric acid R, cover with a watch-glass,
heat to boiling and allow to cool. Rinse the watch-glass and
the dish with water R and dilute to 50.0 ml with the same
solvent.
dioxide-free water R under reflux. Filter in vacuo. To 10 ml
of the filtrate add 0.1 ml of bromothymol blue solution R1 ;
not more than 0.4 ml of 0.01 M hydrochloric acid is required
to change the colour of the indicator to green. To 10 ml of
the filtrate add 0.1 ml of phenolphthalein solution R1 ; not
more than 0.3 ml of 0.01 M sodium hydroxide is required to
change the colour of the indicator to pink.

General Notices (1) apply to all monographs and other texts 4321
Talc
Water-soluble substances : maximum 0.2 per cent.
To 10.0 g add 50 ml of carbon dioxide-free water R, heat to
boiling and maintain boiling under a reflux condenser for
30 min. Allow to cool, filter through a medium-speed filter
paper and dilute to 50.0 ml with carbon dioxide-free water R.
Take 25.0 ml of the filtrate, evaporate to dryness and heat at
105 °C for 1 h. The residue weighs a maximum of 10 mg.
Aluminium : maximum 2.0 per cent.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. To 5.0 ml of solution S2 add 10 ml of a
25.34 g/l solution of caesium chloride R, 10.0 ml of
hydrochloric acid R and dilute to 100.0 ml with water R.
Reference solutions. Into 4 identical volumetric flasks,
each containing 10.0 ml of hydrochloric acid R and 10 ml
of a 25.34 g/l solution of caesium chloride R, introduce
respectively 5.0 ml, 10.0 ml, 15.0 ml and 20.0 ml of
aluminium standard solution (100 ppm Al) R and dilute to
100.0 ml with water R.
Source : aluminium hollow-cathode lamp.
Wavelength : 309.3 nm.
Atomisation device : nitrous oxide-acetylene flame.
Calcium : maximum 0.90 per cent.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. To 5.0 ml of solution S2 add 10.0 ml of
hydrochloric acid R, 10 ml of lanthanum chloride solution R
and dilute to 100.0 ml with water R.
Reference solutions. Into 4 identical volumetric flasks,
each containing 10.0 ml of hydrochloric acid R and 10 ml
of lanthanum chloride solution R, introduce respectively
1.0 ml, 2.0 ml, 3.0 ml and 4.0 ml of calcium standard solution
(100 ppm Ca) R1 and dilute to 100.0 ml with water R.
Source : calcium hollow-cathode lamp.
Wavelength : 422.7 nm.
Atomisation device : nitrous oxide-acetylene flame.
Iron : maximum 0.25 per cent.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. To 2.5 ml of solution S1, add 50.0 ml of 0.5 M
hydrochloric acid and dilute to 100.0 ml with water R.
Reference solutions. Into 4 identical volumetric flasks, each
containing 50.0 ml of 0.5 M hydrochloric acid, introduce
respectively 2.0 ml, 2.5 ml, 3.0 ml and 4.0 ml of iron standard
solution (250 ppm Fe) R and dilute to 100.0 ml with water R.
4322 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Source : iron hollow-cathode lamp.
Wavelength : 248.3 nm.
Atomisation device : air-acetylene flame.
Correction : deuterium lamp.
Lead : maximum 1.0 × 101 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Use solution S1.
Reference solutions. Into 4 identical volumetric flasks, each
containing 50.0 ml of 0.5 M hydrochloric acid, introduce
respectively 5.0 ml, 7.5 ml, 10.0 ml and 12.5 ml of lead
standard solution (10 ppm Pb) R1 and dilute to 100.0 ml
with water R.
Source : lead hollow-cathode lamp.
Wavelength : 217.0 nm.
Atomisation device : air-acetylene flame.
Magnesium : 17.0 per cent to 19.5 per cent.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dilute 0.5 ml of solution S2 to 100.0 ml
with water R. To 4.0 ml of the solution, add 10.0 ml
of hydrochloric acid R, 10 ml of lanthanum chloride
solution R and dilute to 100.0 ml with water R.
Reference solutions. Into 4 identical volumetric flasks,
each containing 10.0 ml of hydrochloric acid R and 10 ml
of lanthanum chloride solution R, introduce respectively
2.5 ml, 3.0 ml, 4.0 ml and 5.0 ml of magnesium standard
solution (10 ppm Mg) R1 and dilute to 100.0 ml with water R.
Source : magnesium hollow-cathode lamp.
Wavelength : 285.2 nm.
Atomisation device : air-acetylene flame.
Loss on ignition : maximum 7.0 per cent, determined on
1.00 g by ignition to constant weight at 1050-1100 °C.
Microbial contamination
If intended for cutaneous administration :
— TAMC : acceptance criterion 102 CFU/g (2.6.12).
If intended for oral administration :
— TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
— TYMC : acceptance criterion 102 CFU/g (2.6.12).
LABELLING
The label states, where applicable, that the substance is
suitable for oral or cutaneous administration.
EUROPEAN PHARMACOPOEIA 6.3 Teicoplanin

01/2009:2358 IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
TEICOPLANIN Comparison : teicoplanin for identification CRS.
B. Examine the chromatograms obtained in the test for
Teicoplaninum composition and related substances.
Results : the principal peaks (teicoplanins A3-1, A2-1, A2-2,
A2-3, A2-4 and A2-5) in the chromatogram obtained with
the test solution are similar in retention time and size to
the principal peaks in the chromatogram obtained with
reference solution (a).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY3 or B4
(2.2.2, Method I).
Dissolve 0.8 g in 10 ml of water R.
pH (2.2.3) : 6.5 to 7.5.
Dissolve 0.50 g in carbon dioxide-free water R and dilute to
10 ml with the same solvent.
Composition and related substances. Liquid
chromatography (2.2.29) : use the normalisation procedure.
Test solution. Dissolve 0.100 g of the substance to be
examined in water R and dilute to 50.0 ml with the same
solvent.
Reference solution (a). Dissolve 20 mg of teicoplanin for
identification CRS in water R and dilute to 10.0 ml with
the same solvent.
Reference solution (b). Dilute 1.0 ml of reference solution (a)
to 10.0 ml with water R. Dilute 1.0 ml of this solution to
20.0 ml with water R.
Reference solution (c). Dissolve 50.0 mg of mesityl
oxide CRS in water R and dilute to 25.0 ml with the same
solvent. Dilute 1.0 ml of the solution to 10.0 ml with water R.
Dilute 1.0 ml of this solution to 100.0 ml with water R.
Column :
— size: l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 μm).
Mobile phase :
— mobile phase A : mix 900 ml of a 3.0 g/l solution of
anhydrous sodium dihydrogen phosphate R, adjusted
to pH 6.0 with 1 M sodium hydroxide, and 100 ml of
acetonitrile R ;
— mobile phase B : mix 300 ml of a 3.0 g/l solution of
anhydrous sodium dihydrogen phosphate R, adjusted
to pH 6.0 with 1 M sodium hydroxide, and 700 ml of
acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 30 100 → 50 0 → 50
DEFINITION
30 - 31 50 → 10 50 → 90
Mixture of glycopeptides produced by certain strains
of Actinoplanes teichomyceticus sp.; the 6 principal 31 - 35 10 90
components of the mixture are teicoplanin A2-1 to A2-5 and
teicoplanin A3-1. Flow rate : 2.3 ml/min.
Detection : spectrophotometer at 254 nm.
Fermentation product.
Injection : 20 μl.
Potency : minimum 900 IU/mg (anhydrous and sodium
chloride-free substance). Identification : use the chromatogram supplied with
teicoplanin for identification CRS and the chromatogram
CHARACTERS obtained with reference solution (a) to identify the groups
Appearance : yellowish, amorphous powder. and impurities.
Solubility : freely soluble in water, sparingly soluble in Relative retention of groups and impurities with reference
dimethylformamide, practically insoluble in ethanol (96 per to teicoplanin A2-2 :
cent V/V). — teicoplanin A3 group ≤ 0.70 ;

General Notices (1) apply to all monographs and other texts 4323
Teicoplanin EUROPEAN PHARMACOPOEIA 6.3

— teicoplanin A2 group > 0.70 and ≤ 1.25 and within this S3 = sum of the areas of the peaks due to teicoplanin A2-3
group : group in the chromatogram obtained with the
test solution ;
— teicoplanin A2-1 group < 1 ;
S4 = area of the peak due to teicoplanin A2-4 in the
— teicoplanin A2-2 = 1 ; chromatogram obtained with the test solution ;
S5 = sum of the areas of the peaks due to teicoplanin A2-5
— teicoplanin A2-3 group > 1 and < 1.12 ; group in the chromatogram obtained with the
test solution.
— teicoplanin A2-4 = about 1.12 ;
Limits :
— teicoplanin A2-5 group > 1.12 and ≤ 1.25 ; — teicoplanin A2 group : minimum 80.0 per cent ;
— impurities > 1.25. — teicoplanin A2-1 group : maximum 20.0 per cent ;
— teicoplanin A2-2 : 35.0 to 55.0 per cent ;
Relative retention of principal peaks of the groups with — teicoplanin A2-3 group : maximum 20.0 per cent ;
reference to teicoplanin A2-2 (retention time = about 18 min) :
teicoplanin A3-1 = about 0.43 ; teicoplanin A2-1 = about 0.93 ; — teicoplanin A2-4 : maximum 20.0 per cent ;
teicoplanin A2-3 = about 1.04 ; teicoplanin A2-4 = about 1.12 ; — teicoplanin A2-5 group : maximum 20.0 per cent ;
teicoplanin A2-5 = about 1.14. — teicoplanin A3 group : maximum 15.0 per cent ;
System suitability : reference solution (a) : — total of impurities other than mesityl oxide with a
retention time more than 1.25 : maximum 5.0 per cent ;
— the chromatogram obtained is similar to the — disregard limit : the area of the peak due to teicoplanin A2-2
chromatogram supplied with teicoplanin for in the chromatogram obtained with reference solution (b)
identification CRS ; (0.25 per cent).
— resolution : minimum 1.0 between the peaks due to Chlorides : maximum 5.0 per cent, expressed as sodium
teicoplanin A2-4 and teicoplanin A2-5. chloride (anhydrous substance).
Dissolve 1.000 g in 300 ml of water R, stir and acidify with
Calculate the percentage content of the different components 2 ml of nitric acid R. Titrate with 0.1 M silver nitrate,
using the following equations : determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl.
teicoplanin A2 group = Heavy metals (2.4.8) : maximum 20 ppm.
0.50 g complies with test G. Prepare the reference solution
teicoplanin A2-1 group = using 100 μl of lead standard solution (100 ppm Pb) R.
Filter the solutions through a membrane filter (nominal pore
size 0.45 μm).
teicoplanin A2-2 = Impurity A. Liquid chromatography (2.2.29) as described
under the test for composition and related substances with
the following modifications.
teicoplanin A2-3 group = Injection : 20 μl of the test solution and reference solution (c).
Relative retention with reference to teicoplanin A2-2
(retention time = about 18 min) : impurity A = about 0.6.
teicoplanin A2-4 =
Limits :
— impurity A : maximum twice the area of the principal peak
teicoplanin A2-5 group = in the chromatogram obtained with reference solution (c)
(0.2 per cent).
Water (2.5.12) : maximum 15.0 per cent, determined on
teicoplanin A3 group = 0.300 g.
Bacterial endotoxins (2.6.14) : less than 0.31 IU/mg.
impurities = ASSAY
Carry out the microbiological assay of antibiotics (2.7.2),
Sa = sum of the areas of the peaks due to teicoplanin using the diffusion method. Use teicoplanin CRS as the
A2 group in the chromatogram obtained with the reference substance.
test solution ;
Sb = sum of the areas of the peaks due to teicoplanin A3 STORAGE
group in the chromatogram obtained with the test Protected from light, at a temperature of 2 °C to 8 °C.
solution ; disregard any peak due to mesityl oxide ;
IMPURITIES
Sc = sum of the areas of the peaks with a relative
retention more than 1.25 ; Specified impurities : A.
S1 = sum of the areas of the peaks due to teicoplanin A2-1
group in the chromatogram obtained with the
test solution ;
S2 = area of the peak due to teicoplanin A2-2 in the
chromatogram obtained with the test solution ; A. 4-methylpent-3-en-2-one (mesityl oxide).

4324 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
07/2008:2154 Column :
corrected 6.3 — size: l = 0.125 m, Ø = 4.0 mm ;
TELMISARTAN
Telmisartanum
C33H30N4O2 Mr 514.6 Detection : spectrophotometer at 230 nm.
[144701-48-4]
DEFINITION
4′-[[4-Methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-
benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or slightly yellowish, crystalline powder. Relative retention with reference to telmisartan
Solubility : practically insoluble in water, slightly soluble
in methanol, sparingly soluble in methylene chloride. It
dissolves in 1 M sodium hydroxide.
It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : telmisartan CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in hot anhydrous ethanol R, evaporate
to dryness and record new spectra using the residues.
TESTS
Appearance of solution. The solution is not more intensely
coloured than reference solution Y4 (2.2.2, Method II).
Dissolve 0.5 g in 1 M sodium hydroxide and dilute to 10 ml
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. To 25 mg of the substance to be examined add — total : not more than 10 times the area of the principal
about 5 ml of methanol R and 100 μl of a 40 g/l solution of
sodium hydroxide R. Dissolve with the aid of ultrasound
and dilute to 50 ml with methanol R.
Reference solution (a). Dilute 1.0 ml of the test solution to
10.0 ml with methanol R. Dilute 1.0 ml of this solution to
100.0 ml with methanol R.
Reference solution (b). Dissolve the contents of a vial
of telmisartan for system suitability CRS (containing
impurities A, B, C, E and F) in 2 ml of methanol R.
Reference solution (c). To 5 mg of telmisartan for peak
identification CRS (containing impurity D) add about 5 ml ASSAY
of methanol R and 100 μl of a 40 g/l solution of sodium
hydroxide R. Dissolve with the aid of ultrasound and dilute 75 ml of acetic anhydride R. Titrate with 0.1 M perchloric
to 10 ml with methanol R.
General Notices (1) apply to all monographs and other texts
Telmisartan
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) with a pore size of 10 nm ;
— temperature : 40 °C.
Mobile phase :
— mobile phase A : dissolve 2.0 g of potassium dihydrogen
phosphate R and 3.8 g of sodium pentanesulphonate
monohydrate R1 in water R, adjust to pH 3.0 with dilute
phosphoric acid R and dilute to 1000 ml with water R ;
— mobile phase B : methanol R2, acetonitrile R1
(20:80 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-3 70 30
3 - 28 70 → 20 30 → 80
Flow rate : 1 ml/min.
Injection : 10 μl.
Identification of impurities : use the chromatogram
supplied with telmisartan for system suitability CRS and
the chromatogram obtained with reference solution (b)
to identify the peaks due to impurities A, B, C, E and F ;
use the chromatogram supplied with telmisartan for peak
identification CRS and the chromatogram obtained with
reference solution (c) to identify the peak due to impurity D.
(retention time = about 15 min) : impurity A = about 0.2 ;
impurity E = about 0.6 ; impurity F = about 0.7 ;
impurity B = about 0.9 ; impurity C = about 1.5 ;
impurity D = about 1.6.
System suitability : reference solution (b) :
— the chromatogram obtained with reference solution (b) is
similar to the chromatogram supplied with telmisartan
for system suitability CRS ;
— resolution : minimum 3.0 between the peaks due to
impurity B and telmisartan.
Limits :
— impurities C, D : for each impurity, not more than twice
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
— impurities A, B : for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.15 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) ;
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
— disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Dissolve 0.190 g in 5 ml of anhydrous formic acid R. Add
acid, determining the end-point potentiometrically (2.2.20).
4325
Tetracosactide
1 ml of 0.1 M perchloric acid is equivalent to 25.73 mg
of C33H30N4O2.
IMPURITIES
Specified impurities : A, B, C, D.
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 F. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-
pharmaceutical use) : E, F, G, H.
A. 4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-
benzimidazole,
B. 4′-[[7-methyl-5-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-
1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid,
C. 1,1-dimethylethyl 4′-[[4-methyl-6-(1-methyl-1H-
benzimidazol-2-yl)-2-propyl-1H-benzimidazol-1-
yl]methyl]biphenyl-2-carboxylate,
D. unidentified impurity,
E. 1-[(2′-carboxybiphenyl-4-yl)methyl]-4-methyl-2-propyl-1H-
benzimidazol-6-carboxylic acid,
4326 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxamide,
G. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-
1H-benzimidazol-1-yl]methyl]biphenyl-2-carbonitrile,
H. 1,1-dimethylethyl 4′-(bromomethyl)biphenyl-2-carboxylate.
01/2009:0644
TETRACOSACTIDE
Tetracosactidum
C136H210N40O31S Mr 2933
[16960-16-0]
DEFINITION
Synthetic tetracosapeptide, in which the sequence of amino
acids is the same as that of the first 24 residues of human
corticotropin. It increases the rate at which corticoid
hormones are secreted by the adrenal glands. It is available
as an acetate.
Content : 90 per cent to 102 per cent (anhydrous and acetic
acid-free substance). By convention, 1 μg of tetracosactide
is equivalent to 1 IU of tetracosactide.
CHARACTERS
Appearance : white or yellow, amorphous powder.
Solubility : sparingly soluble in water.
IDENTIFICATION
A. Liquid chromatography (2.2.29) as described in the test
for related peptides.
EUROPEAN PHARMACOPOEIA 6.3 Tetracosactide

Results : the principal peak in the chromatogram obtained Flow rate : 0.8 ml/min.
with the test solution is similar in retention time and size
Detection : spectrophotometer at 275 nm.
to the principal peak in the chromatogram obtained with
the reference solution. Injection : 20 μl.
B. Amino acid analysis (2.2.56). For hydrolysis use Method 1 Identification of impurities : use the chromatogram supplied
and for analysis use Method 1. with tetracosactide CRS and the chromatogram obtained
Express the content of each amino acid in moles. with reference solution (a) to identify the peak due to
Calculate the relative proportions of the amino acids, impurity B ; use the chromatogram obtained with reference
taking that of valine to be equivalent to 3. The values fall solution (b) to identify the peak due to impurity A.
within the following limits : lysine 3.5 to 4.7 ; histidine Relative retention with reference to tetracosactide
0.9 to 1.1 ; arginine 2.7 to 3.3 ; serine 1.1 to 2.2 ; glutamic (retention time = about 26 min) : impurity A = about 0.3 ;
acid 0.9 to 1.1 ; proline 2.5 to 3.5 ; glycine 1.8 to 2.2 ; impurity B = about 0.95.
methionine 0.9 to 1.1 ; tyrosine 1.7 to 2.2 ; phenylalanine
0.9 to 1.1. Not more than traces of other amino acids System suitability : reference solution (a) :
are present. — peak-to-valley ratio : minimum 3, where Hp = height above
TESTS the baseline of the peak due to impurity B and Hv = height
above the baseline of the lowest point of the curve
Specific optical rotation (2.2.7) : − 99 to − 109 (anhydrous separating this peak from the peak due to tetracosactide.
and acetic acid-free substance).
Limits :
Dissolve 10.0 mg in 1.0 ml of a mixture of 1 volume of glacial
acetic acid R and 99 volumes of water R. — impurity A : maximum 3 per cent ;
Absorbance (2.2.25) : 0.51 to 0.61 (anhydrous and acetic — impurity B : maximum 4 per cent ;
acid-free substance), determined at the absorption maximum — unspecified impurities : for each impurity, maximum
between 240 nm and 280 nm, at 276 nm. The ratio of the 2.5 per cent ;
absorbance at the maximum at 276 nm to the absorbance
at 248 nm is 2.4 to 2.9. — sum of impurities other than A : maximum 9 per cent.
Dissolve 1.0 mg in 0.1 M hydrochloric acid and dilute to Acetic acid (2.5.34) : 8.0 per cent to 13.0 per cent.
5.0 ml with the same acid.
Test solution. Dissolve 10.0 mg of the substance to be
Related peptides. Liquid chromatography (2.2.29) : use the examined in a mixture of 5 volumes of mobile phase B and
normalisation procedure. 95 volumes of mobile phase A and dilute to 10.0 ml with the
Test solution. Dissolve 1.0 mg of the substance to be same mixture of mobile phases.
examined in 1 ml of water R. Water (2.5.32) : maximum 14.0 per cent, determined on
Reference solution (a). Dissolve the contents of a vial of 20.0-50.0 mg.
tetracosactide CRS in water R to obtain a concentration of Bacterial endotoxins (2.6.14) : less than 10 IU/mg,
1.0 mg/ml. if intended for use in the manufacture of parenteral
Reference solution (b). In order to prepare impurity A in preparations without a further appropriate procedure for the
situ, dissolve 1.0 mg of the substance to be examined in 1 ml removal of bacterial endotoxins.
of a 1 per cent V/V solution of glacial acetic acid R, add
50 μl of a mixture of 1 volume of strong hydrogen peroxide ASSAY
solution R and 999 volumes of water R, and allow to stand
for 2 h. Liquid chromatography (2.2.29) as described in the test for
related peptides.
Column:
— size : l = 0.15 m, Ø = 4.6 mm ; Calculate the content of C136H210N40O31S using the declared
content of tetracosactide CRS.
— stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm) ;
STORAGE
— temperature : 25 °C.
Mobile phase : Protected from light, at a temperature of 2 °C to 8 °C.
— mobile phase A : mix 5.0 ml of glacial acetic acid R, 60 ml
of acetonitrile R and 5.0 g of ammonium sulphate R and LABELLING
dilute to 1000 ml with water R ; The label states :
— mobile phase B : mix 5.0 ml of glacial acetic acid R, — the mass of peptide in the container ;
310 ml of acetonitrile R and 5.0 g of ammonium
sulphate R and dilute to 1000 ml with water R ; — where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
— mobile phase C : acetonitrile R.
Time Mobile phase A Mobile phase B Mobile phase C IMPURITIES
(min) (per cent V/V) (per cent V/V) (per cent V/V)
0 - 50 55 → 40 45 → 60 0 Specified impurities : A, B.
50 - 50.1 40 → 0 60 → 15 0 → 85
50.1 - 55 0 15 85 A. tetracosactide sulphoxide,
55 - 55.1 0 → 55 15 → 45 85 → 0
55.1 - 60 55 45 0
B. unidentified impurity.

General Notices (1) apply to all monographs and other texts 4327
Tragacanth EUROPEAN PHARMACOPOEIA 6.3

01/2009:0532 Plate : TLC silica gel plate R.

Mobile phase : 16 g/l solution of sodium dihydrogen
TRAGACANTH phosphate R, butanol R, acetone R (10:40:50 V/V/V).
Application : 10 μl as bands.
Tragacantha Development A : over a path of 10 cm.
Drying A : in a current of warm air for a few minutes.
[9000-65-1] Development B : over a path of 15 cm using the same mobile
phase.
DEFINITION Drying B : at 110 °C for 10 min.
Air-hardened, gummy exudate, flowing naturally or obtained Detection : spray with anisaldehyde solution R and dry at
by incision from the trunk and branches of Astragalus 110 °C for 10 min.
gummifer Labill. and certain other species of Astragalus Results : the chromatogram obtained with the reference
from western Asia. solution shows 4 clearly separated coloured zones
IDENTIFICATION due to galactose (greyish-green or green), arabinose
(yellowish-green), xylose (greenish-grey or yellowish-grey)
A. Tragacanth occurs in thin, flattened, ribbon-like, white and rhamnose (yellowish-green), in order of increasing R
or pale yellow, translucent strips, about 30 mm long and value ; the chromatogram obtained with the test solutionF
10 mm wide and up to 1 mm thick, more or less curved, does not show a yellowish-green zone corresponding to the
horny, with a short fracture ; the surface is marked by zone of rhamnose in the chromatogram obtained with the
fine longitudinal striae and concentric transverse ridges. reference solution.
It may also contain pieces similar in shape but somewhat
thicker, more opaque and more difficult to fracture. Methylcellulose. Examine the chromatograms obtained in
the test for acacia.
B. Reduce to a powder (355) (2.9.12). The powder is white
or almost white and forms a mucilaginous gel with about Results : the chromatogram obtained with the test solution
10 times its mass of water R. Examine under a microscope does not show a red zone near the solvent front.
using a 50 per cent V/V solution of glycerol R. The Sterculia gum
powder shows in the gummy mass numerous stratified A. Place 0.2 g of the powdered drug (355) (2.9.12) in a 10 ml
cellular membranes that turn slowly violet when treated ground-glass-stoppered cylinder graduated in 0.1 ml. Add
with iodinated zinc chloride solution R. The gummy 10 ml of ethanol (60 per cent V/V) R and shake. Any gel
mass includes starch grains, isolated or in small groups, formed occupies not more than 1.5 ml.
usually rounded in shape and sometimes deformed, with B. To 1.0 g of the powdered drug (355) (2.9.12) add 100 ml of
diameters varying between 4 μm and 10 μm, occasionally water R and shake. Add 0.1 ml of methyl red solution R.
up to 20 μm, and a central hilum visible between crossed Not more than 5.0 ml of 0.01 M sodium hydroxide is
nicol prisms. required to change the colour of the indicator.
C. Examine the chromatograms obtained in the test for
Foreign matter : maximum 1.0 per cent.
acacia.
Place 2.0 g of the powdered drug (355) (2.9.12) in a 250 ml
Results : the chromatogram obtained with the test round-bottomed flask and add 95 ml of methanol R. Swirl to
solution shows 3 zones due to galactose, arabinose and moisten the powder and add 60 ml of hydrochloric acid R1.
xylose. A faint yellowish zone at the solvent front and a Add a few glass beads about 4 mm in diameter and heat
greyish-green zone between the zones due to galactose on a water-bath under a reflux condenser for 3 h, shaking
and arabinose may be present. occasionally. Remove the glass beads and filter the hot
D. Moisten 0.5 g of the powdered drug (355) (2.9.12) with suspension in vacuo through a sintered-glass filter (160)
1 ml of ethanol (96 per cent) R and add gradually, while (2.1.2). Rinse the flask with a small quantity of water R and
shaking, 50 ml of water R until a homogeneous mucilage pass the rinsings through the filter. Wash the residue on the
is obtained. To 5 ml of the mucilage add 5 ml of water R filter with about 40 ml of methanol R and dry to constant
and 2 ml of barium hydroxide solution R. A slight mass at 110 °C (about 1 h). Allow to cool in a desiccator and
flocculent precipitate is formed. Heat on a water-bath for weigh. The residue weighs a maximum of 20 mg.
10 min. An intense yellow colour develops.
Flow time : minimum 10 s, or minimum 50 s if the substance
TESTS to be examined is to be used for the preparation of emulsions.
Acacia. Thin-layer chromatography (2.2.27). Place 1.0 g of the powdered drug (125-250) (2.9.12) in a
1000 ml round-bottomed flask with a ground-glass stopper,
Test solution. To 100 mg of the powdered drug (355) add 8.0 ml of ethanol (96 per cent) R and close the flask.
(2.9.12) in a thick-walled centrifuge test-tube, add 2 ml of a Disperse the suspension over the inner surface of the flask by
100 g/l solution of trifluoroacetic acid R, shake vigorously shaking, taking care not to wet the stopper. Open the flask
to dissolve the forming gel, stopper the test-tube and heat and add as a single portion 72.0 ml of water R. Stopper the
the mixture at 120 °C for 1 h. Centrifuge the resulting flask and shake vigorously for 3 min. Allow to stand for 24 h
hydrolysate, transfer the clear supernatant carefully into and shake vigorously again for 3 min. Eliminate air bubbles
a 50 ml flask, add 10 ml of water R and evaporate the by applying vacuum above the mucilage for 5 min. Transfer
solution to dryness under reduced pressure. To the resulting the mucilage to a 50 ml cylinder. Dip in the mucilage a piece
clear film add 0.1 ml of water R and 0.9 ml of methanol R. of glass tubing 200 mm long and 6.0 mm in internal diameter
Centrifuge to separate the amorphous precipitate, collect and graduated at 20 mm and 120 mm from the lower end ;
the supernatant and, if necessary, dilute to 1 ml with the tubing must not be rinsed with surface-active substances.
methanol R. When the mucilage has reached the upper mark, close the
Reference solution. Dissolve 10 mg of arabinose R, 10 mg tube with a finger. Withdraw the closed tube, remove the
of galactose R, 10 mg of rhamnose R and 10 mg of xylose R finger and measure with a stop-watch the time needed for
in 1 ml of water R and dilute to 10 ml with methanol R. the meniscus to reach the lower graduation. Carry out this

4328 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
operation 4 times and determine the average value of the
last 3 determinations.
Total ash (2.4.16) : maximum 4.0 per cent.
Microbial contamination
TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
LABELLING
The label states whether or not the contents are suitable for
preparing emulsions.
04/2008:0058
corrected 6.3
TRIAMTERENE
Triamterenum
C12H11N7 Mr 253.3
[396-01-0]
DEFINITION
6-Phenylpteridine-2,4,7-triamine.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : yellow, crystalline powder.
Solubility : very slightly soluble in water and in ethanol
(96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : triamterene CRS.
TESTS
Acidity. Boil 1.0 g with 20 ml of water R for 5 min, cool,
filter and wash the filter with 3 quantities, each of 10 ml, of
water R. Combine the filtrate and washings and add 0.3 ml
of phenolphthalein solution R. Not more than 1.5 ml of
0.01 M sodium hydroxide is required to change the colour
of the indicator.
Impurity D. Gas chromatography (2.2.28).
Internal standard solution. Dilute 0.1 ml of nitrobenzene R
to 100 ml with methanol R. Dilute 1 ml of this solution to
50 ml with methanol R.
Test solution. Introduce 0.800 g of the substance to
be examined into a suitable vial, add 5 ml of dimethyl
sulphoxide R and heat until the sample is dissolved (do not
heat to boiling). Allow to cool. Add 5 ml of cold methanol R
to enhance the precipitation of triamterene. Filter and wash
the filter with 5 ml of methanol R. Combine the filtrate and
washing, add 2.0 ml of the internal standard solution and
dilute to 20.0 ml with methanol R.
Reference solution. Dissolve 20.0 mg of benzyl cyanide R
(impurity D) in methanol R and dilute to 100.0 ml with the
same solvent. Dilute 5.0 ml of the solution to 50.0 ml with
methanol R. To 2.0 ml of this solution add 2.0 ml of the
internal standard solution and 5 ml of dimethyl sulphoxide R
and dilute to 20.0 ml with methanol R.
General Notices (1) apply to all monographs and other texts
Triamterene
Blank solution. Dilute 5 ml of dimethyl sulphoxide R to
20 ml with methanol R.
Column :
— material: fused silica ;
— size: l = 30 m, Ø = 0.25 mm ;
— stationary phase : macrogol 20 000 R (0.5 μm).
Carrier gas : helium for chromatography R.
Flow rate : 1.5 ml/min.
Split ratio : 1:15.
Temperature :
— column : 170 °C ;
— injection port : 210 °C ;
— detector : 230 °C.
Detection : flame ionisation.
Injection : 1 μl.
Run time : twice the retention time of the internal standard.
Relative retention with reference to the internal standard
(retention time = about 6 min) : impurity D = about 1.6.
System suitability : reference solution :
— resolution : minimum 2.0 between the peak due to
impurity D and the nearest peak due to the solvent (blank
solution) ;
— signal-to-noise ratio : minimum 10 for the peak due to
impurity D.
Limit :
— impurity D : calculate the ratio (R) of the area of the peak
due to impurity D to the area of the peak due to the
internal standard from the chromatogram obtained with
the reference solution ; from the chromatogram obtained
with the test solution, calculate the ratio of the area of
the peak due to impurity D to the area of the peak due
to the internal standard : this ratio is not greater than R
(50 ppm).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 5.0 mg of
nitrosotriaminopyrimidine CRS (impurity A) in the
mobile phase and dilute to 100.0 ml with the mobile phase.
Dilute 1.0 ml of the solution to 100.0 ml with the mobile
phase. Dilute 1.0 ml of this solution to 10.0 ml with the
mobile phase.
Reference solution (c). Dissolve the contents of a vial
of triamterene impurity B CRS in 200 μl of dimethyl
sulphoxide R. Add 5.0 ml of the test solution and dilute to
50.0 ml with the mobile phase. Filter the solution through a
0.45 μm membrane filter before injection.
Column :
— size: l = 0.25 m, Ø = 4.0 mm ;
— stationary phase : spherical end-capped octylsilyl silica
gel for chromatography R (5 μm).
Mobile phase : butylamine R, acetonitrile R, methanol R,
water R (2:200:200:600 V/V/V/V), adjusted to pH 5.3 with
acetic acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 320 nm and at 355 nm.
Injection : 50 μl.
4329
Tributyl acetylcitrate
Relative retention with reference to triamterene
(retention time = about 5 min) : impurity A = about 0.6 ;
impurity B = about 0.8 ; impurity C = about 1.7.
System suitability :
— resolution : minimum 1.5 between the peaks due to
impurity B and triamterene in the chromatogram obtained
with reference solution (c) at 355 nm ; if necessary,
increase the quantity of water R in the mobile phase ;
— signal-to-noise ratio : minimum 10 for the principal peak
in the chromatogram obtained with reference solution (b)
at 320 nm.
Limits :
— correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity B = 1.8 ;
impurity C = 1.5 ;
— impurity A at 320 nm : not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (50 ppm) ;
— impurities B, C at 355 nm : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent) ;
— unspecified impurities at 355 nm : 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 at 355 nm: not more than twice the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
— disregard limit at 355 nm : 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.150 g in 5 ml of anhydrous formic acid R
and add 100 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 25.33 mg
of C12H11N7.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D.
A. 5-nitrosopyrimidine-2,4,6-triamine (nitrosotriamino-
pyrimidine),
B. R = OH, R′ = NH2 : 2,7-diamino-6-phenylpteridin-4-ol,
C. R = NH2, R′ = OH : 2,4-diamino-6-phenylpteridin-7-ol,
4330
EUROPEAN PHARMACOPOEIA 6.3
D. phenylacetonitrile (benzyl cyanide).
01/2009:1770
TRIBUTYL ACETYLCITRATE
Tributylis acetylcitras
C20H34O8 Mr 402.5
[77-90-7]
DEFINITION
Tributyl 2-(acetyloxy)propane-1,2,3-tricarboxylate.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : clear, oily liquid.
Solubility : not miscible with water, miscible with ethanol
(96 per cent) and with methylene chloride.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Preparation : thin films between 2 sodium chloride plates.
Comparison : tributyl acetylcitrate CRS.
TESTS
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution BY6
(2.2.2, Method II).
Acidity. Dilute 10 g with 10 ml of previously neutralised
ethanol (96 per cent) R and add 0.5 ml of bromothymol
blue solution R2. Not more than 0.3 ml of 0.1 M sodium
hydroxide is required to change the colour of the indicator
to blue.
Refractive index (2.2.6) : 1.442 to 1.445.
Related substances. Gas chromatography (2.2.28).
Test solution. Dissolve 0.5 g of the substance to be examined
in methylene chloride R and dilute to 20 ml with the same
solvent.
Reference solution (a). Dissolve 50 mg of the substance to
be examined and 50 mg of tributyl citrate R (impurity A)
in methylene chloride R and dilute to 20 ml with the same
solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to
20.0 ml with methylene chloride R. Dilute 1.0 ml of this
solution to 25.0 ml with methylene chloride R.
Reference solution (c). Dissolve the contents of a vial of
tributyl acetylcitrate for peak identification CRS (containing
impurities B and C) in 1 ml of methylene chloride R.
Column :
— material: fused silica ;
— size: l = 30 m, Ø = 0.53 mm ;
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3 Trypsin

— stationary phase : poly[(cyanopropyl)(methyl)]- ASSAY

[(phenyl)(methyl)]siloxane R (film thickness 1.0 μm). Introduce 1.500 g into a 250 ml borosilicate glass flask.
Carrier gas: helium for chromatography R. Add 25 ml of 2-propanol R, 50 ml of water R, 25.0 ml
of 1 M sodium hydroxide and a few glass beads. Heat
Linear velocity : 36 cm/s. under a reflux condenser for 3 h. Allow to cool. Add
Split ratio : 1:20. 1 ml of phenolphthalein solution R1 and titrate with 1 M
hydrochloric acid. Carry out a blank titration.
Temperature :
1 ml of 1 M sodium hydroxide is equivalent to 100.6 mg
Time Temperature of C20H34O8.
(min) (°C)
Column 0-7 70 → 210 IMPURITIES
Specified impurities: A, B, C.
7 - 50 210
Other detectable impurities (the following substances
Injection port 250 would, if present at a sufficient level, be detected by one
Detector 250 or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Detection : flame ionisation. impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
Injection : 1 μl ; inject via an inert, glass-lined injection port identify these impurities for demonstration of compliance.
using an automatic injection device. See also 5.10. Control of impurities in substances for
Identification of impurities: use the chromatogram supplied pharmaceutical use) : D, E.
with tributyl acetylcitrate for peak identification CRS and
the chromatogram obtained with reference solution (c)
to identify the peaks due to impurities B and C ; use the
chromatogram obtained with reference solution (a) to
identify the peak due to impurity A.
Relative retention with reference to tributyl acetylcitrate
(retention time = about 24 min) : impurity B = about 0.70 ;
impurity C = about 0.83 ; impurity A = about 0.87. A. tributyl 2-hydroxypropane-1,2,3-tricarboxylate (tributyl
citrate),
System suitability :
— resolution : minimum 2.0 between the peaks due to
impurity A and tributyl acetylcitrate in the chromatogram
obtained with reference solution (a) ;
— repeatability : maximum relative standard deviation of
5.0 per cent after 6 injections of reference solution (b).
Limits : B. tributyl propene-1,2,3-tricarboxylate (tributyl aconitate),
— impurity A : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.6 per cent) ;
— impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.4 per cent) ;
C. 1,2-dibutyl 3-(2-methylpropyl) 2-(acetyloxy)propane-1,2,
— impurity B : not more than the area of the principal peak 3-tricarboxylate,
in the chromatogram obtained with reference solution (b)
(0.2 per cent) ;
— unspecified impurities: for each impurity, not more
than 0.5 times the area of the principal peak in the D. R = H : butan-1-ol,
chromatogram obtained with reference solution (b) E. R = CO-CH3 : butyl acetate.
(0.10 per cent) ;
— total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b) 01/2009:0694
(1.0 per cent) ;
— disregard limit: 0.25 times the area of the principal peak TRYPSIN
in the chromatogram obtained with reference solution (b)
(0.05 per cent). Trypsinum
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test F. Prepare the reference solution [9002-07-7]
using 2 ml of lead standard solution (10 ppm Pb) R.
DEFINITION
Water (2.5.12) : maximum 0.25 per cent, determined on Trypsin is a proteolytic enzyme obtained by the activation
2.00 g. of trypsinogen extracted from the pancreas of healthy
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined mammals. It has an activity of not less than 0.5 microkatal
on 1.0 g. per milligram, calculated with reference to the dried

General Notices (1) apply to all monographs and other texts 4331
Trypsin EUROPEAN PHARMACOPOEIA 6.3

substance. In solution, it has maximum enzymic activity at Absence of Escherichia coli (2.6.13).
pH 8 ; the activity is reversibly inhibited at pH 3, at which pH Absence of Salmonella (2.6.13).
it is most stable.
ASSAY
PRODUCTION The activity of trypsin is determined by comparing the
The animals from which trypsin is derived must fulfil the rate at which it hydrolyses benzoylarginine ethyl ester
requirements for the health of animals suitable for human hydrochloride R with the rate at which trypsin BRP
consumption. hydrolyses the same substrate in the same conditions.
The method of manufacture is validated to demonstrate that Apparatus. Use a reaction vessel of about 30 ml capacity
the product, if tested, would comply with the following test. provided with :
Histamine (2.6.10). Not more than 1 μg of histamine base — a device that will maintain a temperature of 25.0 ± 0.1 °C ;
per 0.2 microkatal of trypsin activity. Use a 10 g/l solution — a stirring device (for example, a magnetic stirrer) ;
of the substance to be examined in 0.0015 M borate buffer — a lid with holes for the insertion of electrodes, the tip of
solution pH 8.0 R inactivated by heating on a water-bath for a burette, a tube for the admission of nitrogen and the
30 min. Carry out dilutions with a 9 g/l solution of sodium introduction of reagents.
chloride R.
An automatic or manual titration device may be used. For
the latter, the burette is graduated in 0.005 ml and the pH
CHARACTERS meter is provided with a wide-range scale and glass-calomel
A white or almost white, crystalline or amorphous powder, or glass-silver-silver chloride electrodes.
sparingly soluble in water. The amorphous form is Test solution. Dissolve sufficient of the substance to be
hygroscopic. examined in 0.001 M hydrochloric acid and dilute to 25.0 ml
with the same acid in order to obtain a solution containing
IDENTIFICATION approximately 700 nanokatals per millilitre.
A. Dilute 1 ml of solution S (see Tests) to 100 ml with Reference solution. Dissolve 25.0 mg of trypsin BRP in
water R. In a depression in a white spot-plate, mix 0.1 ml 0.001 M hydrochloric acid and dilute to 25.0 ml with the
of this solution with 0.2 ml of tosylarginine methyl same acid.
ester hydrochloride solution R. A reddish-violet colour Store the solutions at 0-5 °C. Warm 1 ml of each solution
develops within 3 min. to about 25 °C over 15 min and use 50 μl of each solution
B. Dilute 0.5 ml of solution S to 5 ml with water R. Add for each titration. Carry out the titration in an atmosphere
0.1 ml of a 20 g/l solution of tosyl-lysyl-chloromethane of nitrogen. Transfer 10.0 ml of 0.0015 M borate buffer
hydrochloride R. Adjust to pH 7.0, shake for 2 h and solution pH 8.0 R to the reaction vessel and, while stirring,
dilute to 50 ml with water R. In one of the depressions of add 1.0 ml of a freshly prepared 6.86 g/l solution of
a white spot-plate, mix 0.1 ml of this solution with 0.2 ml benzoylarginine ethyl ester hydrochloride R. When the
of tosylarginine methyl ester hydrochloride solution R. temperature is steady at 25.0 ± 0.1 °C (after about 5 min)
No reddish-violet colour develops within 3 min. adjust the pH to exactly 8.0 with 0.1 M sodium hydroxide.
Add 50 μl of the test solution and start a timer. Maintain the
TESTS pH at 8.0 by the addition of 0.1 M sodium hydroxide, the tip
of the microburette being immersed in the solution ; note
Solution S. Dissolve 0.10 g in carbon dioxide-free water R the volume added every 30 s. Follow the reaction for 8 min.
and dilute to 10.0 ml with the same solvent. Calculate the volume of 0.1 M sodium hydroxide used per
Appearance of solution. Solution S is not more opalescent second. Carry out a titration in the same manner using the
than reference suspension III (2.2.1). reference solution and calculate the volume of 0.1 M sodium
pH (2.2.3). The pH of solution S is 3.0 to 6.0. hydroxide used per second.
Calculate the activity in microkatals per milligram using the
Absorbance (2.2.25). Dissolve 30.0 mg in 0.001 M
following expression :
hydrochloric acid and dilute to 100.0 ml with the same
acid. The solution shows an absorption maximum at 280 nm
and a minimum at 250 nm. The specific absorbance at the
absorption maximum is 13.5 to 16.5 and at the absorption
minimum is not greater than 7.0. m = mass of the substance to be examined, in
Chymotrypsin. To 1.8 ml of buffer solution pH 8.0 R add milligrams ;
7.4 ml of water R and 0.5 ml of 0.2 M acetyltyrosine ethyl m′ = mass of trypsin BRP, in milligrams ;
ester R. While shaking the solution, add 0.3 ml of solution S
and start a timer. After exactly 5 min, measure the pH (2.2.3) V = volume of 0.1 M sodium hydroxide used per
(test solution). Prepare a reference solution in the same second by the test solution ;
manner, replacing solution S by 0.3 ml of a 0.5 g/l solution V′ = volume of 0.1 M sodium hydroxide used per
of chymotrypsin BRP and measure the pH (2.2.3) exactly second by the reference solution ;
5 min after adding the chymotrypsin. The pH of the test A = activity of trypsin BRP, in microkatals per
solution is higher than that of the reference solution. milligram.
Loss on drying (2.2.32). Not more than 5.0 per cent,
determined on 0.500 g by drying at 60 °C at a pressure not STORAGE
exceeding 670 Pa for 2 h. In an airtight container, protected from light, at a
Microbial contamination temperature of 2 °C to 8 °C.
TAMC : acceptance criterion 104 CFU/g (2.6.12). LABELLING
TYMC : acceptance criterion 102 CFU/g (2.6.12). The label states the activity in microkatals per milligram.

4332 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Tryptophan

01/2009:1272 Reference solution (a). Dissolve 10 mg of tryptophan CRS

in the solvent mixture and dilute to 50 ml with the solvent
mixture.
TRYPTOPHAN
Reference solution (b). Dilute 5 ml of test solution (b) to
20 ml with the solvent mixture.
Tryptophanum Reference solution (c). Dissolve 10 mg of tryptophan CRS
and 10 mg of tyrosine CRS in the solvent mixture and dilute
to 25 ml with the solvent mixture.
Plate : TLC silica gel plate R.
Mobile phase : glacial acetic acid R, water R, butanol R
(20:20:60 V/V/V).
C11H12N2O2 Mr 204.2 Application : 5 μl.
[73-22-3]
Development : over a path of 15 cm.
DEFINITION Drying : in air.
(S)-2-Amino-3-(1H-indol-3-yl)propanoic acid. Detection : spray with ninhydrin solution R and heat at
Content : 98.5 per cent to 101.0 per cent (dried substance). 100-105 °C for 15 min.
System suitability : reference solution (c) :
CHARACTERS
— the chromatogram shows 2 clearly separated spots.
Appearance : white or almost white, crystalline or
amorphous powder. Limit : test solution (a) :
Solubility : sparingly soluble in water, slightly soluble in — any impurity : any spot, apart from the principal spot,
ethanol (96 per cent). It dissolves in dilute solutions of is not more intense than the principal spot in the
mineral acids and alkali hydroxides. chromatogram obtained with reference solution (b)
(0.5 per cent).
IDENTIFICATION Impurity A and other related substances. Liquid
First identification : A, B. chromatography (2.2.29). Prepare the standard, test and
reference solutions immediately before use.
Second identification : A, C, D.
Buffer solution pH 2.3. Dissolve 3.90 g of sodium
A. Specific optical rotation (see Tests).
dihydrogen phosphate R in 1000 ml of water R. Add about
B. Infrared absorption spectrophotometry (2.2.24). 700 ml of a 2.9 g/l solution of phosphoric acid R and adjust
Preparation : discs. to pH 2.3 with the same acid solution.
Comparison : tryptophan CRS. Solvent mixture : acetonitrile R, water R (10:90 V/V).
C. Examine the chromatograms obtained in the test for Standard solution. Dissolve 10.0 mg of N-acetyltryptophan R
ninhydrin-positive substances. in the solvent mixture and dilute to 100.0 ml with the solvent
Results : the principal spot in the chromatogram obtained mixture. Dilute 2.0 ml of this solution to 100.0 ml with the
with test solution (b) is similar in position, colour and size solvent mixture.
to the principal spot in the chromatogram obtained with Test solution (a). Dissolve 0.10 g of the substance to be
reference solution (a). examined in the solvent mixture and dilute to 10.0 ml with
D. Dissolve about 20 mg in 10 ml of water R. Add 5 ml the solvent mixture.
of dimethylaminobenzaldehyde solution R6 and 2 ml Test solution (b). Dissolve 0.10 g of the substance to be
of hydrochloric acid R1. Heat on a water-bath. A examined in the standard solution and dilute to 10.0 ml with
purple-blue colour develops. the standard solution.
Reference solution (a). Dissolve the contents of a vial of
TESTS 1,1′-ethylidenebistryptophan CRS (impurity A) in 1.0 ml
Appearance of solution. The solution is clear (2.2.1) and of the solvent mixture.
not more intensely coloured than reference solution BY6 Reference solution (b). Dissolve the contents of a vial of
(2.2.2, Method II). 1,1′-ethylidenebistryptophan CRS (impurity A) in 1.0 ml of
Dissolve 0.1 g in 1 M hydrochloric acid and dilute to 10 ml the standard solution.
with the same acid.
Reference solution (c). Dilute 0.5 ml of reference solution (a)
Specific optical rotation (2.2.7) : − 30.0 to − 33.0 (dried to 5.0 ml with the solvent mixture.
substance).
Column :
Dissolve 0.25 g in water R, heating on a water-bath if
— size: l = 0.25 m, Ø = 4.6 mm ;
necessary, and dilute to 25.0 ml with the same solvent.
— stationary phase : octadecylsilyl silica gel for
Ninhydrin-positive substances. Thin-layer chromatography
chromatography R (5 μm) ;
(2.2.27).
Solvent mixture : glacial acetic acid R, water R (50:50 V/V). — temperature : 40 °C.
Test solution (a). Dissolve 0.10 g of the substance to be Mobile phase :
examined in the solvent mixture and dilute to 10 ml with the — mobile phase A : acetonitrile R, buffer solution pH 2.3
solvent mixture. (115:885 V/V) ;
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml — mobile phase B : acetonitrile R, buffer solution pH 2.3
with the solvent mixture. (350:650 V/V) ;

General Notices (1) apply to all monographs and other texts 4333
Tryptophan EUROPEAN PHARMACOPOEIA 6.3

Time Mobile phase A Mobile phase B of methyl isobutyl ketone R1, shaking for 3 min each time.
(min) (per cent V/V) (per cent V/V) To the combined organic layers add 10 ml of water R and
0 - 10 100 0 shake for 3 min. Examine the aqueous layer.
10 - 45 100 → 0 0 → 100 Heavy metals (2.4.8) : maximum 10 ppm.
45 - 65 0 100 2.0 g complies with test D. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
65 - 66 0 → 100 100 → 0
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
66 - 80 100 0 on 1.000 g by drying in an oven at 105 °C.
Flow rate : 0.7 ml/min. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Detection : spectrophotometer at 220 nm. on 1.0 g.
Injection : 20 μl of test solutions (a) and (b) and reference ASSAY
solutions (b) and (c).
Retention time : tryptophan = about 8 min ; Dissolve 0.150 g in 3 ml of anhydrous formic acid R.
N-acetyltryptophan = about 29 min ; impurity A = about Add 30 ml of anhydrous acetic acid R. Titrate with 0.1 M
34 min. perchloric acid, using 0.1 ml of naphtholbenzein solution R
as indicator.
System suitability :
1 ml of 0.1 M perchloric acid is equivalent to 20.42 mg
— resolution : minimum 8.0 between the peaks due to of C11H12N2O2.
N-acetyltryptophan and impurity A in the chromatogram
obtained with reference solution (b) ; if necessary, adjust STORAGE
the time programme for the elution gradient (an increase
in the duration of elution with mobile phase A produces Protected from light.
longer retention times and a better resolution) ;
IMPURITIES
— signal-to-noise ratio : minimum 15 for the principal peak
in the chromatogram obtained with reference solution (c) ;
— symmetry factor : maximum 3.5 for the peak due to
impurity A in the chromatogram obtained with reference
solution (b).
— in the chromatogram obtained with test solution (a)
there is no peak with the same retention time as
N-acetyltryptophan (in such case correct the area of the
N-acetyltryptophan peak).
Limits : test solution (b) :
— impurity A : not more than 0.5 times the area of the A. 3,3′-[ethylidenebis(1H-indole-1,3-diyl)]bis[(2S)-2-
principal peak in the chromatogram obtained with aminopropanoic] acid (1,1′-ethylidenebistryptophan),
reference solution (c) (10 ppm) ;
— sum of impurities with a retention time less than that of
tryptophan : not more than 0.6 times the area of the peak
due to N-acetyltryptophan in the chromatogram obtained
with reference solution (b) (100 ppm) ;
— sum of impurities with a retention time greater than that
of tryptophan and up to 1.8 times the retention time of
N-acetyltryptophan : not more than 1.9 times the area of B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-
the peak due to N-acetyltryptophan in the chromatogram 3-yl]propanoic acid (dioxyindolylalanine),
obtained with reference solution (b) (300 ppm) ;
— disregard limit : 0.02 times the area of the peak due
to N-acetyltryptophan in the chromatogram obtained
with reference solution (b) ; disregard the peak due to
N-acetyltryptophan.
Chlorides (2.4.4) : maximum 200 ppm.
Dissolve 0.25 g in 3 ml of dilute nitric acid R and dilute
to 15 ml with water R. The solution, without any further C. R = H : (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid
addition of nitric acid, complies with the test. (kynurenine),
Sulphates (2.4.13) : maximum 300 ppm. E. R = CHO : (S)-2-amino-4-[2-(formylamino)phenyl]-4-
Dissolve 0.5 g in a mixture of 5 volumes of dilute oxobutanoic acid (N-formylkynurenine),
hydrochloric acid R and 25 volumes of distilled water R,
and dilute to 15 ml with the same mixture of solvents.
Ammonium (2.4.1, Method B) : maximum 200 ppm,
determined on 0.10 g.
Prepare the standard using 0.2 ml of ammonium standard
solution (100 ppm NH4) R.
Iron (2.4.9) : maximum 20 ppm.
In a separating funnel, dissolve 0.50 g in 10 ml of dilute D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
hydrochloric acid R. Shake with 3 quantities, each of 10 ml, (5-hydroxytryptophan),

4334 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Tryptophan

F. (S)-2-amino-3-(phenylamino)propanoic acid
(3-phenylaminoalanine),

J. R = CHOH-CH2-OH : (S)-2-amino-3-[2-[2,3-dihydroxy-1-(1H-
indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid,
K. R = H : (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-3-
yl]propanoic acid,
G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid
(2-hydroxytryptophan),

H. R = H : (3RS)-1,2,3,4-tetrahydro-9H-β-carboline-3-
carboxylic acid,
I. R = CH3 : 1-methyl-1,2,3,4-tetrahydro-9H-β-carboline-3- L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H-β-carboline-
carboxylic acid, 3-carboxylic acid.

General Notices (1) apply to all monographs and other texts 4335
 EUROPEAN PHARMACOPOEIA 6.3

4336 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

W
Water for injections.. ...............................................................4339 Water, purified..........................................................................4344
Water, highly purified.. ...........................................................4342 Wheat starch.. ...........................................................................4346

General Notices (1) apply to all monographs and other texts 4337
 EUROPEAN PHARMACOPOEIA 6.3

4338 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Water for injections

01/2009:0169 Growth promotion of R2A agar

— Preparation of test strains. Use standardised stable
suspensions of test strains or prepare them as stated
WATER FOR INJECTIONS in Table 0169.-1. Seed lot culture maintenance
techniques (seed-lot systems) are used so that the viable
Aqua ad iniectabilia micro-organisms used for inoculation are not more than
5 passages removed from the original master seed-lot.
Grow each of the bacterial strains separately as described
H 2O Mr 18.02 in Table 0169.-1. Use buffered sodium chloride-peptone
solution pH 7.0 or phosphate buffer solution pH 7.2
DEFINITION to make test suspensions. Use the suspensions within
Water for the preparation of medicines for parenteral 2 h, or within 24 h if stored at 2-8 °C. As an alternative
administration when water is used as vehicle (water for to preparing and then diluting a fresh suspension
injections in bulk) and for dissolving or diluting substances of vegetative cells of Bacillus subtilis, a stable spore
or preparations for parenteral administration (sterilised suspension is prepared and then an appropriate volume
water for injections). of the spore suspension is used for test inoculation. The
stable spore suspension may be maintained at 2-8 °C for
a validated period of time.
Water for injections in bulk — Growth promotion. Test each batch of ready-prepared
medium and each batch of medium, prepared either from
PRODUCTION dehydrated medium or from the ingredients described.
Water for injections in bulk is obtained from water that Inoculate plates of R2A agar separately with a small
complies with the regulations on water intended for human number (not more than 100 CFU) of the micro-organisms
consumption laid down by the competent authority or from indicated in Table 0169.-1. Incubate under the conditions
purified water by distillation in an apparatus of which the described in the table. Growth obtained must not differ
parts in contact with the water are of neutral glass, quartz by a factor greater than 2 from the calculated value for a
or a suitable metal and which is fitted with an effective standardised inoculum. For a freshly prepared inoculum,
device to prevent the entrainment of droplets. The correct growth of the micro-organisms must be comparable to
maintenance of the apparatus is essential. The first portion that obtained with a previously tested and approved batch
of the distillate obtained when the apparatus begins to of medium.
function is discarded and the distillate is collected. Table 0169.-1. – Growth promotion of R2A agar
In order to ensure the appropriate quality of the water, Micro-organism Preparation of the test
Growth promotion
validated procedures and in-process-monitoring of the strain
electrical conductivity and regular microbial monitoring are Pseudomonas Casein soyabean digest R2A agar
aeruginosa agar or casein soyabean ≤ 100 CFU
applied. digest broth
such as : 30-35 °C
Water for injections in bulk is stored and distributed in ATCC 9027 30-35 °C
≤ 3 days
conditions designed to prevent growth of micro-organisms NCIMB 8626 18-24 h
and to avoid any other contamination. CIP 82.118
Microbiological monitoring. During production and NBRC 13275
subsequent storage, appropriate measures are taken to Bacillus subtilis Casein soyabean digest R2A agar
ensure that the microbial count is adequately controlled such as : agar or casein soyabean ≤ 100 CFU
digest broth
and monitored. Appropriate alert and action levels are set ATCC 6633 30-35 °C
30-35 °C
so as to detect adverse trends. Under normal conditions, an NCIMB 8054 ≤ 3 days
18-24 h
appropriate action level is a microbial count of 10 CFU per CIP 52.62
100 ml when determined by filtration through a membrane NBRC 3134
with a nominal pore size not greater than 0.45 μm, using R2A
Total organic carbon (2.2.44) : maximum 0.5 mg/l.
agar, using at least 200 ml of water for injections in bulk and
incubating at 30-35 °C for not less than 5 days. For aseptic Conductivity. Determine the conductivity off-line or in-line
processing, stricter alert levels may need to be applied. under the following conditions.
R2A agar EQUIPMENT
Yeast extract 0.5 g Conductivity cell :
— electrodes of a suitable material such as stainless steel ;
Proteose peptone 0.5 g
— cell constant : the cell constant is generally certified
Casein hydrolysate 0.5 g by the supplier and is subsequently verified at suitable
Glucose 0.5 g intervals using a certified reference solution with a
conductivity less than 1500 μS·cm− 1 or by comparison
Starch 0.5 g with a cell having a certified cell constant. The cell
Dipotassium hydrogen phosphate 0.3 g constant is confirmed if the value found is within 2 per
cent of the certified value, otherwise re-calibration must
Magnesium sulphate, anhydrous 0.024 g
be performed.
Sodium pyruvate 0.3 g Conductometer : accuracy of 0.1 μS·cm− 1 or better at the
Agar 15.0 g lowest range.
Purified water to 1000 ml
System calibration (conductivity cell and conductometer) :
— against one or more suitable certified reference solutions ;
Adjust the pH so that after sterilisation it is 7.2 ± 0.2. — accuracy : within 3 per cent of the measured conductivity
Sterilise by heating in an autoclave at 121 °C for 15 min. plus 0.1 μS·cm− 1.

General Notices (1) apply to all monographs and other texts 4339
Water for injections EUROPEAN PHARMACOPOEIA 6.3

Conductometer calibration: calibration is carried out for When the change in conductivity (due to uptake of
each range of measurement to be used, after disconnection atmospheric carbon dioxide) is less than 0.1 μS.cm− 1 per
of the conductivity cell, using certified precision resistors 5 min, note the conductivity.
or equivalent devices with an uncertainty not greater than 5. If the conductivity is not greater than 2.1 μS.cm− 1, the
0.1 per cent of the certified value. water to be examined meets the requirements of the
If in-line conductivity cells cannot be dismantled, system test for conductivity. If the conductivity is greater than
calibration may be performed against a calibrated 2.1 μS.cm− 1, proceed with stage 3.
conductivity-measuring instrument with a conductivity cell Stage 3
placed close to the cell to be calibrated in the water flow. 6. Perform this test within approximately 5 min of the
Temperature measurement : tolerance ± 2 °C. conductivity determination in step 5 under stage 2, while
PROCEDURE maintaining the sample temperature at 25 ± 1 °C. Add
Stage 1 a recently prepared saturated solution of potassium
1. Measure the conductivity without temperature chloride R to the test sample (0.3 ml per 100 ml of the
compensation, recording simultaneously the temperature. test sample), and determine the pH (2.2.3) to the nearest
Temperature-compensated measurement may be 0.1 pH unit.
performed after suitable validation. 7. Using Table 0169.-3, determine the conductivity limit
2. Using Table 0169.-2, find the closest temperature value at the measured pH value in step 6. If the measured
that is not greater than the measured temperature. The conductivity in step 4 under stage 2 is not greater than
corresponding conductivity value is the limit at that the conductivity requirements for the pH determined,
temperature. the water to be examined meets the requirements of the
test for conductivity. If either the measured conductivity
3. If the measured conductivity is not greater than the is greater than this value or the pH is outside the range
value in Table 0169.-2, the water to be examined meets of 5.0-7.0, the water to be examined does not meet the
the requirements of the test for conductivity. If the requirements of the test for conductivity.
conductivity is higher than the value in Table 0169.-2,
proceed with stage 2. Table 0169.-3. – Stage 3
pH and conductivity requirements (for atmosphere-
Table 0169.-2. – Stage 1 and temperature-equilibrated samples)
Temperature and conductivity requirements pH Conductivity
(for non-temperature-compensated conductivity (μS·cm− 1)
measurements) 5.0 4.7
Temperature Conductivity
(°C) (μS·cm− 1) 5.1 4.1
0 0.6 5.2 3.6
5 0.8 5.3 3.3
10 0.9 5.4 3.0
15 1.0 5.5 2.8
20 1.1 5.6 2.6
25 1.3 5.7 2.5
30 1.4 5.8 2.4
35 1.5 5.9 2.4
40 1.7 6.0 2.4
45 1.8 6.1 2.4
50 1.9 6.2 2.5
55 2.1 6.3 2.4
60 2.2 6.4 2.3
65 2.4 6.5 2.2
70 2.5 6.6 2.1
75 2.7 6.7 2.6
80 2.7 6.8 3.1
85 2.7 6.9 3.8
90 2.7 7.0 4.6
95 2.9
100 3.1 CHARACTERS
Appearance : clear and colourless liquid.
Stage 2
TESTS
4. Transfer a sufficient amount of the water to be examined
(100 ml or more) to a suitable container, and stir the test Nitrates: maximum 0.2 ppm.
sample. Adjust the temperature, if necessary, and while Place 5 ml in a test-tube immersed in iced water, add 0.4 ml
maintaining it at 25 ± 1 °C, begin vigorously agitating the of a 100 g/l solution of potassium chloride R, 0.1 ml of
test sample while periodically observing the conductivity. diphenylamine solution R and, dropwise with shaking,

4340 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
5 ml of nitrogen-free sulphuric acid R. Transfer the tube
to a water-bath at 50 °C. After 15 min, any blue colour in
the solution is not more intense than that in a reference
solution prepared at the same time in the same manner
using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml
of nitrate standard solution (2 ppm NO3) R.
Aluminium (2.4.17) : maximum 10 ppb, if intended for use in of a 100 g/l solution of potassium chloride R, 0.1 ml of
the manufacture of dialysis solutions.
Prescribed solution. To 400 ml of the water to be examined 5 ml of nitrogen-free sulphuric acid R. Transfer the tube
add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of to a water-bath at 50 °C. After 15 min, any blue colour in
distilled water R.
Reference solution. Mix 2 ml of aluminium standard
solution (2 ppm Al) R, 10 ml of acetate buffer solution
pH 6.0 R and 98 ml of distilled water R.
Blank solution. Mix 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of distilled water R.
Bacterial endotoxins (2.6.14) : less than 0.25 IU/ml.
Sterilised water for injections
DEFINITION
Water for injections in bulk that has been distributed
into suitable containers, closed and sterilised by heat in
conditions which ensure that the product still complies
with the test for bacterial endotoxins. Sterilised water for
injections is free from any added substances.
Examined in suitable conditions of visibility, it is clear and
colourless.
Each container contains a sufficient quantity of water for
injections to permit the nominal volume to be withdrawn.
TESTS
Acidity or alkalinity. To 20 ml add 0.05 ml of phenol
red solution R. If the solution is yellow, it becomes red
on the addition of 0.1 ml of 0.01 M sodium hydroxide ;
if red, it becomes yellow on the addition of 0.15 ml of
0.01 M hydrochloric acid.
Conductivity : maximum 25 μS·cm− 1 for containers with a
nominal volume of 10 ml or less ; maximum 5 μS·cm− 1 for
containers with a nominal volume greater than 10 ml.
Use equipment and the calibration procedure as defined
under Water for injections in bulk, maintaining the sample
temperature at 25 ± 1 °C.
Oxidisable substances. For containers with a nominal
volume less than 50 ml : heat 100 ml to boiling with 10 ml
of dilute sulphuric acid R, add 0.4 ml of 0.02 M potassium
permanganate and boil for 5 min ; the solution remains
faintly pink.
For containers with a nominal volume equal to or greater
than 50 ml : heat 100 ml to boiling with 10 ml of dilute
sulphuric acid R, add 0.2 ml of 0.02 M potassium
permanganate and boil for 5 min ; the solution remains
faintly pink.
Chlorides (2.4.4) : maximum 0.5 ppm for containers with a
nominal volume of 100 ml or less.
15 ml complies with the limit test for chlorides. Prepare
the standard using a mixture of 1.5 ml of chloride standard
solution (5 ppm Cl) R and 13.5 ml of water R. Examine the
solutions down the vertical axes of the tubes.
General Notices (1) apply to all monographs and other texts
Water for injections
For containers with a nominal volume greater than 100 ml,
use the following test : to 10 ml add 1 ml of dilute nitric
acid R and 0.2 ml of silver nitrate solution R2. The solution
shows no change in appearance for at least 15 min.
Nitrates: maximum 0.2 ppm.
Place 5 ml in a test-tube immersed in iced water, add 0.4 ml
diphenylamine solution R and, dropwise with shaking,
the solution is not more intense than that in a reference
solution prepared at the same time in the same manner
using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml
of nitrate standard solution (2 ppm NO3) R.
Sulphates. To 10 ml add 0.1 ml of dilute hydrochloric acid R
and 0.1 ml of barium chloride solution R1. The solution
shows no change in appearance for at least 1 h.
Aluminium (2.4.17) : maximum 10 ppb, if intended for use in
the manufacture of dialysis solutions.
Prescribed solution. To 400 ml of the water to be examined
add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
distilled water R.
Reference solution. Mix 2 ml of aluminium standard
solution (2 ppm Al) R, 10 ml of acetate buffer solution
pH 6.0 R and 98 ml of distilled water R.
Blank solution. Mix 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of distilled water R.
Ammonium : for containers with a nominal volume less than
50 ml : maximum 0.6 ppm ; for containers with a nominal
volume equal to or greater than 50 ml : maximum 0.2 ppm.
Containers with a nominal volume less than 50 ml : to
20 ml add 1 ml of alkaline potassium tetraiodomercurate
solution R ; after 5 min, examine the solution down the
vertical axis of the tube ; the solution is not more intensely
coloured than a standard prepared at the same time by
adding 1 ml of alkaline potassium tetraiodomercurate
solution R to a mixture of 4 ml of ammonium standard
solution (3 ppm NH4) R and 16 ml of ammonium-free
water R.
Containers with a nominal volume equal to or greater
than 50 ml : to 20 ml add 1 ml of alkaline potassium
tetraiodomercurate solution R ; after 5 min, examine the
solution down the vertical axis of the tube ; the solution
is not more intensely coloured than a standard prepared
at the same time by adding 1 ml of alkaline potassium
tetraiodomercurate solution R to a mixture of 4 ml of
ammonium standard solution (1 ppm NH4) R and 16 ml
of ammonium-free water R.
Calcium and magnesium. To 100 ml add 2 ml of ammonium
chloride buffer solution pH 10.0 R, 50 mg of mordant
black 11 triturate R and 0.5 ml of 0.01 M sodium edetate. A
pure blue colour is produced.
Residue on evaporation : maximum 4 mg (0.004 per cent) for
containers with a nominal volume of 10 ml or less ; maximum
3 mg (0.003 per cent ) for containers with a nominal volume
greater than 10 ml.
Evaporate 100 ml to dryness on a water-bath and dry in an
oven at 100-105 °C.
Particulate contamination : sub-visible particles (2.9.19). It
complies with test A or test B, as appropriate.
Sterility (2.6.1). It complies with the test for sterility.
Bacterial endotoxins (2.6.14) : less than 0.25 IU/ml.
4341
Water, highly purified EUROPEAN PHARMACOPOEIA 6.3

01/2009:1927 2 h, or within 24 h if stored at 2-8 °C. As an alternative

to preparing and then diluting a fresh suspension
WATER, HIGHLY PURIFIED of vegetative cells of Bacillus subtilis, a stable spore
suspension is prepared and then an appropriate volume
of the spore suspension is used for test inoculation. The
Aqua valde purificata stable spore suspension may be maintained at 2-8 °C for
a validated period of time.
H 2O Mr 18.02 — Growth promotion. Test each batch of ready-prepared
DEFINITION medium and each batch of medium, prepared either from
dehydrated medium or from the ingredients described.
Water intended for use in the preparation of medicinal Inoculate plates of R2A agar separately with a small
products where water of high biological quality is needed, number (not more than 100 CFU) of the micro-organisms
except where Water for injections (0169) is required. indicated in Table 1927.-1. Incubate under the conditions
PRODUCTION described in the table. Growth obtained must not differ
by a factor greater than 2 from the calculated value for a
Highly purified water is obtained from water that complies standardised inoculum. For a freshly prepared inoculum,
with the regulations on water intended for human growth of the micro-organisms must be comparable to
consumption laid down by the competent authority. that obtained with a previously tested and approved batch
Current production methods include, for example, of medium.
double-pass reverse osmosis coupled with other suitable
techniques such as ultrafiltration and deionisation. Correct Table 1927.-1. – Growth promotion of R2A agar
operation and maintenance of the system is essential. Micro-organism Preparation of the test
Growth promotion
strain
In order to ensure the appropriate quality of the water,
Pseudomonas Casein soyabean digest R2A agar
validated procedures and in-process monitoring of the aeruginosa agar or casein soyabean ≤ 100 CFU
electrical conductivity and regular microbial monitoring are such as : digest broth
30-35 °C
applied. ATCC 9027 30-35 °C
≤ 3 days
Highly purified water is stored in bulk and distributed in NCIMB 8626 18-24 h
conditions designed to prevent growth of micro-organisms CIP 82.118
and to avoid any other contamination. NBRC 13275
Microbiological monitoring. During production and Bacillus subtilis Casein soyabean digest R2A agar
subsequent storage, appropriate measures are taken to such as : agar or casein soyabean ≤ 100 CFU
digest broth
ensure that the microbial count is adequately controlled ATCC 6633 30-35 °C
30-35 °C
and monitored. Appropriate alert and action levels are set NCIMB 8054 ≤ 3 days
18-24 h
so as to detect adverse trends. Under normal conditions, an CIP 52.62
appropriate action level is a microbial count of 10 CFU per NBRC 3134
100 ml when determined by filtration through a membrane
with a nominal pore size not greater than 0.45 μm, using Total organic carbon (2.2.44) : maximum 0.5 mg/l.
R2A agar, at least 200 ml of highly purified water and Conductivity. Determine the conductivity off-line or in-line
incubating at 30-35 °C for not less than 5 days. under the following conditions.
R2A agar EQUIPMENT
Yeast extract 0.5 g Conductivity cell :
Proteose peptone 0.5 g — electrodes of a suitable material such as stainless steel ;
Casein hydrolysate 0.5 g
— cell constant : the cell constant is generally certified
by the supplier and is subsequently verified at suitable
Glucose 0.5 g intervals using a certified reference solution with a
Starch 0.5 g conductivity less than 1500 μS·cm− 1 or by comparison
with a cell having a certified cell constant ; the cell
Dipotassium hydrogen phosphate 0.3 g constant is confirmed if the value found is within 2 per
Magnesium sulphate, anhydrous 0.024 g cent of the certified value, otherwise re-calibration must
be performed.
Sodium pyruvate 0.3 g
Conductometer : accuracy of 0.1 μS·cm− 1 or better at the
Agar 15.0 g lowest range.
Purified water to 1000 ml System calibration (conductivity cell and conductometer) :

Adjust the pH so that after sterilisation it is 7.2 ± 0.2. — against one or more suitable certified reference solutions ;
Sterilise by heating in an autoclave at 121 °C for 15 min. — accuracy : within 3 per cent of the measured conductivity
Growth promotion of R2A agar plus 0.1 μS·cm− 1.
— Preparation of test strains. Use standardised stable Conductometer calibration : calibration is carried out for
suspensions of test strains or prepare them as stated each range of measurement to be used, after disconnection
in Table 1927.-1. Seed lot culture maintenance of the conductivity cell, using certified precision resistors
techniques (seed-lot systems) are used so that the viable or equivalent devices with an uncertainty not greater than
micro-organisms used for inoculation are not more than 0.1 per cent of the certified value.
5 passages removed from the original master seed-lot. If in-line conductivity cells cannot be dismantled, system
Grow each of the bacterial strains separately as described calibration may be performed against a calibrated
in Table 1927.-1. Use buffered sodium chloride-peptone conductivity-measuring instrument with a conductivity cell
solution pH 7.0 or phosphate buffer solution pH 7.2 placed close to the cell to be calibrated in the water flow.
to make test suspensions. Use the suspensions within Temperature measurement: tolerance ± 2 °C.

4342 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Water, highly purified

PROCEDURE
Stage 1
1. Measure the conductivity without temperature
compensation, recording simultaneously the temperature.
Temperature-compensated measurement may be
performed after suitable validation.
2. Using Table 1927.-2, find the closest temperature value
that is not greater than the measured temperature. The 7. Using Table 1927.-3, determine the conductivity limit
corresponding conductivity value is the limit at that
temperature.
3. If the measured conductivity is not greater than the
value in Table 1927.-2, the water to be examined meets
the requirements of the test for conductivity. If the
conductivity is higher than the value in Table 1927.-2,
proceed with stage 2.
Table 1927.-2. – Stage 1
Temperature and conductivity requirements
(for non-temperature-compensated conductivity
measurements)
Temperature Conductivity
(°C) (μS·cm− 1)
0 0.6
Stage 3
6. Perform this test within approximately 5 min of the
conductivity determination in step 5 under stage 2, while
maintaining the sample temperature at 25 ± 1 °C. Add
a recently prepared saturated solution of potassium
chloride R to the test sample (0.3 ml per 100 ml of the
test sample), and determine the pH (2.2.3) to the nearest
0.1 pH unit.
at the measured pH value in step 6. If the measured
conductivity in step 4 under stage 2 is not greater than
the conductivity requirements for the pH determined,
the water to be examined meets the requirements of the
test for conductivity. If either the measured conductivity
is greater than this value or the pH is outside the range
of 5.0-7.0, the water to be examined does not meet the
requirements of the test for conductivity.
Table 1927.-3. – Stage 3 - pH and conductivity requirements
(for atmosphere and temperature equilibrated samples)
pH Conductivity
(μS·cm− 1)
5.0 4.7
5.1 4.1

5 0.8 5.2 3.6

10 0.9 5.3 3.3

15 1.0 5.4 3.0

20 1.1 5.5 2.8

25 1.3 5.6 2.6

30 1.4 5.7 2.5

35 1.5 5.8 2.4

40 1.7 5.9 2.4

45 1.8 6.0 2.4

50 1.9 6.1 2.4

55 2.1 6.2 2.5

60 2.2 6.3 2.4

65 2.4 6.4 2.3

70 2.5 6.5 2.2

75 2.7 6.6 2.1

80 2.7 6.7 2.6

85 2.7 6.8 3.1

90 2.7 6.9 3.8

95 2.9 7.0 4.6

100 3.1
Stage 2
4. Transfer a sufficient amount of the water to be examined
(100 ml or more) to a suitable container, and stir the test
sample. Adjust the temperature, if necessary, and while
maintaining it at 25 ± 1 °C, begin vigorously agitating the
test sample while periodically observing the conductivity.
When the change in conductivity (due to uptake of
atmospheric carbon dioxide) is less than 0.1 μS·cm− 1 per
5 min, note the conductivity.
5. If the conductivity is not greater than 2.1 μS·cm− 1, the
water to be examined meets the requirements of the
test for conductivity. If the conductivity is greater than
2.1 μS·cm− 1, proceed with stage 3.
CHARACTERS
Appearance : clear and colourless liquid.
TESTS
Nitrates: maximum 0.2 ppm.
Place 5 ml in a test-tube immersed in iced water, add 0.4 ml
of a 100 g/l solution of potassium chloride R, 0.1 ml of
diphenylamine solution R and, dropwise with shaking,
5 ml of nitrogen-free sulphuric acid R. Transfer the tube
to a water-bath at 50 °C. After 15 min, any blue colour in
the solution is not more intense than that in a reference
solution prepared at the same time in the same manner
using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml
of nitrate standard solution (2 ppm NO3) R.

General Notices (1) apply to all monographs and other texts 4343
Water, purified EUROPEAN PHARMACOPOEIA 6.3

Aluminium (2.4.17) : maximum 10 ppb, if intended for use in Adjust the pH so that after sterilisation it is 7.2 ± 0.2.
the manufacture of dialysis solutions. Sterilise by heating in an autoclave at 121 °C for 15 min.
Prescribed solution. To 400 ml of the water to be examined Growth promotion of R2A agar
add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of — Preparation of test strains. Use standardised stable
distilled water R. suspensions of test strains or prepare them as stated
Reference solution. Mix 2 ml of aluminium standard in Table 0008.-1. Seed lot culture maintenance
solution (2 ppm Al) R, 10 ml of acetate buffer solution techniques (seed-lot systems) are used so that the viable
pH 6.0 R and 98 ml of distilled water R. micro-organisms used for inoculation are not more than
Blank solution. Mix 10 ml of acetate buffer solution 5 passages removed from the original master seed-lot.
pH 6.0 R and 100 ml of distilled water R. Grow each of the bacterial strains separately as described
in Table 0008.-1. Use buffered sodium chloride-peptone
Bacterial endotoxins (2.6.14) : less than 0.25 IU/ml. solution pH 7.0 or phosphate buffer solution pH 7.2
to make test suspensions. Use the suspensions within
LABELLING 2 h, or within 24 h if stored at 2-8 °C. As an alternative
The label states, where applicable, that the substance is to preparing and then diluting a fresh suspension
suitable for use in the manufacture of dialysis solutions. of vegetative cells of Bacillus subtilis, a stable spore
suspension is prepared and then an appropriate volume
of the spore suspension is used for test inoculation. The
01/2009:0008 stable spore suspension may be maintained at 2-8 °C for
a validated period of time.
WATER, PURIFIED — Growth promotion. Test each batch of ready-prepared
medium and each batch of medium, prepared either from
Aqua purificata dehydrated medium or from the ingredients described.
Inoculate plates of R2A agar separately with a small
H 2O Mr 18.02 number (not more than 100 CFU) of the micro-organisms
indicated in Table 0008.-1. Incubate under the conditions
DEFINITION described in the table. Growth obtained must not differ
Water for the preparation of medicines other than those by a factor greater than 2 from the calculated value for a
that are required to be both sterile and apyrogenic, unless standardised inoculum. For a freshly prepared inoculum,
otherwise justified and authorised. growth of the micro-organisms must be comparable to
that obtained with a previously tested and approved batch
of medium.
Purified water in bulk
Table 0008.-1. – Growth promotion of R2A agar
PRODUCTION
Micro-organism Preparation of the test strain Growth promotion
Purified water in bulk is prepared by distillation, by ion
Pseudomonas Casein soyabean digest agar or R2A agar
exchange, by reverse osmosis or by any other suitable aeruginosa casein soyabean digest broth ≤ 100 CFU
method from water that complies with the regulations on such as : 30-35 °C 30-35 °C
water intended for human consumption laid down by the ATCC 9027 18-24 h
competent authority. ≤ 3 days
NCIMB 8626
Purified water in bulk is stored and distributed in conditions CIP 82.118
designed to prevent growth of micro-organisms and to avoid NBRC 13275
any other contamination. Bacillus subtilis Casein soyabean digest agar or R2A agar
Microbiological monitoring. During production and such as : casein soyabean digest broth ≤ 100 CFU
subsequent storage, appropriate measures are taken to ATCC 6633 30-35 °C 30-35 °C
ensure that the microbial count is adequately controlled NCIMB 8054 18-24 h ≤ 3 days
and monitored. Appropriate alert and action levels are set CIP 52.62
so as to detect adverse trends. Under normal conditions, an NBRC 3134
appropriate action level is a microbial count of 100 CFU/ml,
determined by filtration through a membrane with a nominal Total organic carbon or oxidisable substances. Carry out
pore size not greater than 0.45 μm, using R2A agar and the test for total organic carbon (2.2.44) with a limit of
incubating at 30-35 °C for not less than 5 days. The size of 0.5 mg/l or alternatively the following test for oxidisable
the sample is to be chosen in relation to the expected result. substances : to 100 ml add 10 ml of dilute sulphuric acid R
and 0.1 ml of 0.02 M potassium permanganate and boil for
R2A agar 5 min ; the solution remains faintly pink.
Yeast extract 0.5 g
Conductivity. Determine the conductivity off-line or in-line
Proteose peptone 0.5 g under the following conditions.
Casein hydrolysate 0.5 g EQUIPMENT
Glucose 0.5 g Conductivity cell :
— electrodes of a suitable material such as stainless steel ;
Starch 0.5 g
— cell constant : the cell constant is generally certified
Dipotassium hydrogen phosphate 0.3 g by the supplier and is subsequently verified at suitable
Magnesium sulphate, anhydrous 0.024 g intervals using a certified reference solution with a
conductivity less than 1500 μS·cm− 1 or by comparison
Sodium pyruvate 0.3 g
with a cell having a certified cell constant ; the cell
Agar 15.0 g constant is confirmed if the value found is within 2 per
cent of the certified value, otherwise re-calibration must
Purified water to 1000 ml
be performed.

4344 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Water, purified

Conductometer : accuracy of 0.1 μS·cm− 1 or better at the solution prepared at the same time in the same manner
lowest range. using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml
System calibration (conductivity cell and conductometer) : of nitrate standard solution (2 ppm NO3) R.
— against one or more suitable certified reference solutions ; Aluminium (2.4.17) : maximum 10 ppb, if intended for use in
— accuracy : within 3 per cent of the measured conductivity the manufacture of dialysis solutions.
plus 0.1 μS·cm− 1. Prescribed solution. To 400 ml of the water to be examined
Conductometer calibration: calibration is carried out for add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
each range of measurement to be used, after disconnection distilled water R.
of the conductivity cell, using certified precision resistors Reference solution. Mix 2 ml of aluminium standard
or equivalent devices with an uncertainty not greater than solution (2 ppm Al) R, 10 ml of acetate buffer solution
0.1 per cent of the certified value. pH 6.0 R and 98 ml of distilled water R.
If in-line conductivity cells cannot be dismantled, system Blank solution. Mix 10 ml of acetate buffer solution
calibration may be performed against a calibrated pH 6.0 R and 100 ml of distilled water R.
conductivity-measuring instrument with a conductivity cell Heavy metals (2.4.8) : maximum 0.1 ppm.
placed close to the cell to be calibrated in the water flow. To 200 ml add 0.15 ml of 0.1 M nitric acid and heat in a
Temperature measurement : tolerance ± 2 °C. glass evaporating dish on a water-bath until the volume
PROCEDURE is reduced to 20 ml. 12 ml of the concentrated solution
Measure the conductivity without temperature complies with test A. Prepare the reference solution using
compensation, recording simultaneously the temperature. 10 ml of lead standard solution (1 ppm Pb) R and adding
Temperature-compensated measurement may be performed 0.075 ml of 0.1 M nitric acid. Prepare the blank solution
after suitable validation. adding 0.075 ml of 0.1 M nitric acid.
The water to be examined meets the requirements if the Bacterial endotoxins (2.6.14) : less than 0.25 IU/ml, if
measured conductivity at the recorded temperature is not intended for use in the manufacture of dialysis solutions
greater than the value in Table 0008.-2. without a further appropriate procedure for removal of
bacterial endotoxins.
Table 0008.-2. – Temperature and conductivity
requirements LABELLING
Temperature Conductivity The label states, where applicable, that the substance is
(°C) (μS·cm− 1) suitable for use in the manufacture of dialysis solutions.
0 2.4
10 3.6 Purified water in containers
20 4.3
DEFINITION
25 5.1 Purified water in bulk that has been filled and stored in
30 5.4 conditions designed to assure the required microbiological
quality. It is free from any added substances.
40 6.5
50 7.1 CHARACTERS
60
Appearance : clear and colourless liquid.
8.1
70 9.1 TESTS
75 9.7 It complies with the tests prescribed in the section on
Purified water in bulk and with the following additional tests.
80 9.7
Acidity or alkalinity. To 10 ml, freshly boiled and cooled in a
90 9.7 borosilicate glass flask, add 0.05 ml of methyl red solution R.
100 10.2 The solution is not coloured red.
To 10 ml add 0.1 ml of bromothymol blue solution R1. The
For temperatures not listed in Table 0008.-2, calculate the solution is not coloured blue.
maximal permitted conductivity by interpolation between Oxidisable substances. To 100 ml add 10 ml of dilute
the next lower and next higher data points in the table. sulphuric acid R and 0.1 ml of 0.02 M potassium
Heavy metals. If purified water in bulk complies with permanganate and boil for 5 min. The solution remains
the requirement for conductivity prescribed for Water for faintly pink.
injections (0169) in bulk , it is not necessary to carry out the Chlorides. To 10 ml add 1 ml of dilute nitric acid R and
test for heavy metals prescribed below. 0.2 ml of silver nitrate solution R2. The solution shows no
CHARACTERS change in appearance for at least 15 min.
Appearance : clear and colourless liquid. Sulphates. To 10 ml add 0.1 ml of dilute hydrochloric acid R
and 0.1 ml of barium chloride solution R1. The solution
TESTS shows no change in appearance for at least 1 h.
Nitrates : maximum 0.2 ppm. Ammonium : maximum 0.2 ppm.
Place 5 ml in a test-tube immersed in iced water, add 0.4 ml To 20 ml add 1 ml of alkaline potassium tetraiodomercurate
of a 100 g/l solution of potassium chloride R, 0.1 ml of solution R. After 5 min, examine the solution down the
diphenylamine solution R and, dropwise with shaking, vertical axis of the tube. The solution is not more intensely
5 ml of nitrogen-free sulphuric acid R. Transfer the tube coloured than a standard prepared at the same time by
to a water-bath at 50 °C. After 15 min, any blue colour in adding 1 ml of alkaline potassium tetraiodomercurate
the solution is not more intense than that in a reference solution R to a mixture of 4 ml of ammonium standard

General Notices (1) apply to all monographs and other texts 4345
Wheat starch
solution (1 ppm NH4) R and 16 ml of ammonium-free
water R.
Calcium and magnesium. To 100 ml add 2 ml of ammonium
chloride buffer solution pH 10.0 R, 50 mg of mordant
black 11 triturate R and 0.5 ml of 0.01 M sodium edetate. A
pure blue colour is produced.
Residue on evaporation : maximum 0.001 per cent.
Evaporate 100 ml to dryness on a water-bath and dry in an
oven at 100-105 °C. The residue weighs a maximum of 1 mg.
Microbial contamination
TAMC : acceptance criterion 102 CFU/ml (2.6.12). Use casein
soya bean digest agar.
LABELLING
The label states, where applicable, that the substance is
suitable for use in the manufacture of dialysis solutions.
01/2009:0359
WHEAT STARCH
Tritici amylum
DEFINITION
Wheat starch is obtained from the caryopsis of Triticum
aestivum L. (T. vulgare Vill.).
CHARACTERS
Appearance : very fine, white or almost white powder that
creaks when pressed between the fingers.
Solubility : practically insoluble in cold water and in ethanol Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter.
(96 per cent).
Wheat starch does not contain starch grains of any other
origin. It may contain a minute quantity, if any, of tissue
fragments of the original plant.
IDENTIFICATION
A. Examined under a microscope using equal volumes
of glycerol R and water R, it presents large and small
granules, and, very rarely, intermediate sizes. The large
granules, 10-60 μm in diameter, are discoid or, more
rarely, reniform when seen face-on. The central hilum and Absence of Escherichia coli (2.6.13).
striations are invisible or barely visible and the granules Absence of Salmonella (2.6.13).
4346 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
sometimes show cracks on the edges. Seen in profile,
the granules are elliptical and fusiform and the hilum
appears as a slit along the main axis. The small granules,
rounded or polyhedral, are 2-10 μm in diameter. Between
orthogonally orientated polarising plates or prisms, the
granules show a distinct black cross intersecting at the
hilum.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool.
A thin, cloudy mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B
add 0.05 ml of iodine solution R1. A dark blue colour is
produced, which disappears on heating.
TESTS
pH (2.2.3) : 4.5 to 7.0.
Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
Foreign matter. Examined under a microscope using a
mixture of equal volumes of glycerol R and water R, not
more than traces of matter other than starch granules are
present. No starch grains of any other origin are present.
Total protein : maximum 0.3 per cent of total protein
(corresponding to 0.048 per cent N2, conversion factor :
6.25), determined on 6.0 g by sulphuric acid digestion (2.5.9)
modified as follows : wash any adhering particles from the
neck into the flask with 25 ml of sulphuric acid R ; continue
the heating until a clear solution is obtained ; add 45 ml of
strong sodium hydroxide solution R.
Oxidising substances (2.5.30) : maximum 20 ppm, calculated
as H2O2.
Sulphur dioxide (2.5.29) : maximum 50 ppm.
Iron (2.4.9) : maximum 10 ppm.
The filtrate complies with the test.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.000 g by drying in an oven at 130 °C for 90 min.
Sulphated ash (2.4.14) : maximum 0.6 per cent, determined
on 1.0 g.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
EUROPEAN PHARMACOPOEIA 6.3

X
Xanthan gum.. ..........................................................................4349 Xylitol..........................................................................................4350

General Notices (1) apply to all monographs and other texts 4347
 EUROPEAN PHARMACOPOEIA 6.3

4348 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Xanthan gum

01/2009:1277 Test solution. To 200 ml of water R in a 1000 ml

round-bottomed flask, add 5.0 g of the substance to be
XANTHAN GUM examined and 1 ml of a 10 g/l emulsion of dimeticone R in
liquid paraffin R, stopper the flask and shake for 1 h. Distil
about 90.0 ml, mix the distillate with 4.0 ml of the internal
Xanthani gummi standard solution and dilute to 100.0 ml with water R.
Reference solution. Dilute a suitable quantity of
[11138-66-2] 2-propanol R, accurately weighed, with water R to obtain
a solution having a known concentration of 2-propanol of
DEFINITION about 1 mg/ml. To 4.0 ml of this solution add 4.0 ml of
High-molecular-mass anionic polysaccharide produced the internal standard solution and dilute to 100.0 ml with
by fermentation of carbohydrates with Xanthomonas water R.
campestris. It consists of a principal chain of β(1→4)-linked Column :
D-glucose units with trisaccharide side chains, on alternating
anhydroglucose units, consisting of 1 glucuronic acid unit — size: l = 1.8 m, Ø = 4.0 mm ;
included between 2 mannose units. Most of the terminal units — stationary phase : styrene-divinylbenzene copolymer R
contain a pyruvate moiety and the mannose unit adjacent to (149-177 μm).
the principal chain may be acetylated at C-6. Carrier gas : helium for chromatography R.
Xanthan gum has a relative molecular mass of approximately Flow rate : 30 ml/min.
1 × 106. It exists as the sodium, potassium or calcium salt. Temperature :
Content : minimum 1.5 per cent of pyruvoyl groups (C3H3O2 ; — column : 165 °C ;
Mr 71.1) (dried substance).
— injection port and detector: 200 °C.
CHARACTERS Detection : flame ionisation.
Appearance : white or yellowish-white, free-flowing powder. Injection : 5 μl.
Solubility : soluble in water giving a highly viscous solution, Relative retention with reference to 2-propanol :
practically insoluble in organic solvents. 2-methyl-2-propanol = about 1.5.
IDENTIFICATION Limit :
A. In a flask, suspend 1 g in 15 ml of 0.1 M hydrochloric — 2-propanol: maximum 750 ppm.
acid. Close the flask with a fermentation bulb containing Other polysaccharides. Thin-layer chromatography (2.2.27).
barium hydroxide solution R and heat carefully for 5 min. Test solution. To 10 mg of the substance to be examined
The barium hydroxide solution shows a white turbidity. in a thick-walled centrifuge test tube add 2 ml of a 230 g/l
B. To 300 ml of water R, previously heated to 80 °C and solution of trifluoroacetic acid R, shake vigorously to
stirred rapidly with a mechanical stirrer in a 400 ml dissolve the forming gel, stopper the test tube, and heat
beaker, add, at the point of maximum agitation, a dry the mixture at 120 °C for 1 h. Centrifuge the hydrolysate,
blend of 1.5 g of carob bean gum R and 1.5 g of the transfer the clear supernatant liquid carefully into a 50 ml
substance to be examined. Stir until the mixture forms flask, add 10 ml of water R and evaporate the solution to
a solution, and then continue stirring for 30 min or dryness under reduced pressure. Take up the residue thus
longer. Do not allow the water temperature to drop below obtained in 10 ml of water R and evaporate to dryness under
60 °C during stirring. Discontinue stirring and allow the reduced pressure. Wash 3 times with 20 ml of methanol R
mixture to stand for at least 2 h. A firm rubbery gel forms and evaporate under reduced pressure. To the resulting
after the temperature drops below 40 °C but no such clear film which has no odour of acetic acid, add 0.1 ml of
gel forms in a 1 per cent control solution of the sample water R and 1 ml of methanol R. Centrifuge to separate
prepared in the same manner but omitting the carob the amorphous precipitate. Dilute the supernatant liquid, if
bean gum. necessary, to 1 ml with methanol R.
TESTS Reference solution. Dissolve 10 mg of glucose R and 10 mg
of mannose R in 2 ml of water R and dilute to 10 ml with
pH (2.2.3) : 6.0 to 8.0 for a 10.0 g/l solution. methanol R.
Viscosity (2.2.10) : minimum 600 mPa·s. Plate : TLC silica gel plate R.
Add 3.0 g within 45-90 s into 250 ml of a 12 g/l solution Mobile phase : 16 g/l solution of sodium dihydrogen
of potassium chloride R in a 500 ml beaker stirring with phosphate R, butanol R, acetone R (10:40:50 V/V/V).
a low-pitch propeller-type stirrer rotating at 800 r/min. Application : 5 μl, as bands.
When adding the substance take care that agglomerates are
destroyed. Add an additional quantity of 44 ml of water R, Development : over a path of 15 cm.
to rinse any adhering residue from the walls of the beaker. Detection : spray with a solution of 0.5 g of diphenylamine R
Stir the preparation at 800 r/min for 2 h whilst maintaining in 25 ml of methanol R to which 0.5 ml of aniline R and
the temperature at 24 ± 1 °C. Determine the viscosity within 2.5 ml of phosphoric acid R have been added. Heat for
15 min at 24 ± 1 °C using a rotating viscosimeter set at 5 min at 120 °C and examine in daylight.
60 r/min and equipped with a rotating spindle 12.7 mm System suitability : reference solution:
in diameter and 1.6 mm high which is attached to a shaft — the chromatogram shows 2 clearly separated
3.2 mm in diameter. The distance from the top of the greyish-brown zones due to glucose and mannose in the
cylinder to the lower tip of the shaft being 25.4 mm, and the middle third.
immersion depth being 50.0 mm.
Results : the chromatogram obtained with the test solution
2-Propanol. Gas chromatography (2.2.28). shows 2 zones corresponding to the zones due to glucose
Internal standard solution. Dilute 0.50 g of and mannose in the chromatogram obtained with the
2-methyl-2-propanol R to 500 ml with water R. reference solution. In addition, 1 weak reddish and 2 faint

General Notices (1) apply to all monographs and other texts 4349
Xylitol
bluish-grey bands may be visible just above the starting
line. 1 or 2 bluish-grey bands may also be seen in the upper B. Infrared absorption spectrophotometry (2.2.24).
quarter of the chromatogram. No other bands are visible.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2.5 h.
Total ash (2.4.16) : 6.5 per cent to 16.0 per cent.
Microbial contamination
TAMC : acceptance criterion 103 CFU/g (2.6.12).
2
TYMC : acceptance criterion 10 CFU/g (2.6.12).
ASSAY
Test solution. Dissolve a quantity of the substance to be
examined corresponding to 120.0 mg of the dried substance
in water R and dilute to 20.0 ml with the same solvent.
Reference solution. Dissolve 45.0 mg of pyruvic acid R in
water R and dilute to 500.0 ml with the same solvent.
Place 10.0 ml of the test solution in a 50 ml round-bottomed
flask, add 20.0 ml of 0.1 M hydrochloric acid and weigh.
Boil on a water-bath under a reflux condenser for 3 h.
Weigh and adjust to the initial mass with water R. In a
separating funnel mix 2.0 ml of the solution with 1.0 ml of
dinitrophenylhydrazine-hydrochloric solution R. Allow to
stand for 5 min and add 5.0 ml of ethyl acetate R. Shake
and allow the solids to settle. Collect the upper layer and
shake with 3 quantities, each of 5.0 ml, of sodium carbonate
solution R. Combine the aqueous layers and dilute to 50.0 ml
with sodium carbonate solution R. Mix. Treat 10.0 ml of the
reference solution at the same time and in the same manner
as for the test solution.
Immediately measure the absorbance (2.2.25) of the
2 solutions at 375 nm, using sodium carbonate solution R
as the compensation liquid.
The absorbance of the test solution is not less than that of
the reference solution, which corresponds to a content of
pyruvic acid of not less than 1.5 per cent.
01/2009:1381 same solvent.
XYLITOL
Xylitolum
C5H12O5 Mr 152.1 maintain boiling for 3 min. Cool rapidly and add 100 ml
[87-99-0]
DEFINITION
Meso-xylitol.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
crystals.
Solubility : very soluble in water, sparingly soluble in
ethanol (96 per cent).
IDENTIFICATION
First identification : B.
Second identification : A, C.
4350 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
A. Melting point (2.2.14) : 92 °C to 96 °C.
Preparation : mulls in liquid paraffin R.
Comparison : xylitol CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
examined in water R and dilute to 5 ml with the same
solvent.
Reference solution (a). Dissolve 25 mg of xylitol CRS in
water R and dilute to 5 ml with the same solvent.
Reference solution (b). Dissolve 25 mg of mannitol CRS
and 25 mg of xylitol CRS in water R and dilute to 5 ml
with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : water R, ethyl acetate R, propanol R
(10:20:70 V/V/V).
Application : 2 μl.
Development : over 3/4 of the plate.
Drying : in air.
Detection : spray with 4-aminobenzoic acid solution R,
dry in a current of cold air until the acetone is removed,
then heat at 100 °C for 15 min ; allow to cool, spray with
a 2 g/l solution of sodium periodate R, dry in a current
of cold air, then heat at 100 °C for 15 min.
System suitability : reference solution (b) :
— the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
TESTS
Appearance of solution. The solution is not more opalescent
than reference suspension IV (2.2.1) and not more intensely
coloured than reference solution BY7 (2.2.2, Method II).
Dissolve 2.5 g in water R and dilute to 50.0 ml with the
Conductivity (2.2.38) : maximum 20 μS·cm− 1.
Dissolve 20.0 g in carbon dioxide-free water R prepared
from distilled water R and dilute to 100.0 ml with the same
solvent. Measure the conductivity of the solution while
gently stirring with a magnetic stirrer.
Reducing sugars : maximum 0.2 per cent, calculated as
glucose equivalent.
Dissolve 5.0 g in 6 ml of water R with the aid of gentle
heat. Cool and add 20 ml of cupri-citric solution R and a
few glass beads. Heat so that boiling begins after 4 min and
of a 2.4 per cent V/V solution of glacial acetic acid R and
20.0 ml of 0.025 M iodine. With continuous shaking, add
25 ml of a mixture of 6 volumes of hydrochloric acid R
and 94 volumes of water R and, when the precipitate has
dissolved, titrate the excess of iodine with 0.05 M sodium
thiosulphate using 1 ml of starch solution R, added towards
the end of the titration, as indicator. Not less than 12.8 ml of
0.05 M sodium thiosulphate is required.
Related substances. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 5 mg of erythritol R in
water R and dilute to 25.0 ml with the same solvent.
Test solution (a). Dissolve 5.000 g of the substance to be
examined in water R and dilute to 100.0 ml with the same
solvent.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml
with water R.
EUROPEAN PHARMACOPOEIA 6.3
Reference solution (a). Dissolve 5.0 mg each of
L-arabinitol CRS (impurity A), galactitol CRS (impurity B),
mannitol CRS (impurity C) and sorbitol CRS (impurity D) in
water R and dilute to 20.0 ml with the same solvent.
Reference solution (b). Dissolve 50.0 mg of xylitol CRS in
water R and dilute to 10.0 ml with the same solvent.
Pipette 1.0 ml of test solutions (a) and (b) and reference
solutions (a) and (b) into 4 separate 100 ml round-bottomed
flasks. Add 1.0 ml of the internal standard solution to
each of the flasks containing test solution (a) or reference
solution (a), and 5.0 ml of the internal standard solution to
each of the flasks containing test solution (b) or reference
solution (b). Evaporate each mixture to dryness in a
water-bath at 60 °C with the aid of a rotary evaporator.
Dissolve each dry residue in 1 ml of anhydrous pyridine R,
add 1 ml of acetic anhydride R to each flask and boil each
solution under reflux for 1 h to complete acetylation.
Column:
— size : l = 30 m, Ø = 0.25 mm ;
— stationary phase : poly(cyanopropylphenyl)(14)(methyl)-
(86)siloxane R (0.25 μm).
Carrier gas : nitrogen R.
Flow rate : 1 ml/min.
Split ratio : 1:50 to 1:100.
Temperature :
Time Temperature
(min) (°C)
Column 0-1 170
1-6 170 → 230
6 - 30 230
Injection port 250
Detector 250
Detection : flame-ionisation.
Injection : 1 μl of test solution (a) and reference solution (a)
(solutions obtained after derivatisation).
Relative retention with reference to xylitol (retention
time = about 15 min) : internal standard = about 0.6 ;
impurity A = about 0.9 ; impurity C = about 1.4 ;
impurity B = about 1.45 ; impurity D = about 1.5.
System suitability : reference solution (a) :
— resolution : minimum 2.0 between the peaks due to
impurities B and D.
Calculate the percentage content of each related substance in
the substance to be examined using the following expression :
ms = mass of the particular component in 1 ml of
reference solution (a), in milligrams ;
mu = mass of the substance to be examined in 1 ml of
test solution (a), in milligrams ;
Rs = ratio of the area of the peak due to the particular
derivatised component to the area of the peak
due to the derivatised internal standard in
the chromatogram obtained with reference
solution (a) ;
Ru = ratio of the area of the peak due to the particular
derivatised component to the area of the peak
due to the derivatised internal standard in the
chromatogram obtained with test solution (a).
General Notices (1) apply to all monographs and other texts
Xylitol
The sum of the percentage contents of the related substances
in the chromatogram obtained with test solution (a) is not
greater than 2.0 per cent. Disregard any peak with an area
corresponding to a percentage content of 0.05 per cent or
less.
Lead (2.4.10) : maximum 0.5 ppm.
Dissolve the substance to be examined in 150.0 ml of the
prescribed mixture of solvents.
Nickel (2.4.15) : maximum 1 ppm.
Dissolve the substance to be examined in 150.0 ml of the
prescribed mixture of solvents.
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g.
Bacterial endotoxins (2.6.14) : less than 4 IU/g if the
concentration is less than 100 g/l of xylitol and less than
2.5 IU/g if the concentration is 100 g/l or more of xylitol,
when intended for use in the manufacture of parenteral
preparations without a further appropriate procedure for the
removal of bacterial endotoxins.
ASSAY
Gas chromatography (2.2.28) as described in the test for
related substances with the following modifications.
Injection : 1 μl of test solution (b) and reference solution (b)
(solutions obtained after derivatisation).
Calculate the percentage content of C5H12O5 using the
following expression :
T = declared percentage content of xylitol CRS ;
mt = mass of xylitol CRS in 1 ml of reference
solution (b), in milligrams ;
mv = mass of the substance to be examined in 1 ml of
test solution (b), in milligrams ;
Rt = ratio of the area of the peak due to derivatised
xylitol to the area of the peak due to the derivatised
internal standard in the chromatogram obtained
with reference solution (b) ;
Rv = ratio of the area of the peak due to derivatised
xylitol to the area of the peak due to the derivatised
internal standard in the chromatogram obtained
with test solution (b).
LABELLING
The label states :
— where applicable, the maximum concentration of bacterial
endotoxins ;
— where applicable, that the substance is suitable for use in
the manufacture of parenteral preparations.
IMPURITIES
A. L-arabinitol,
4351
Xylitol EUROPEAN PHARMACOPOEIA 6.3

C. mannitol,

B. meso-galactitol, D. sorbitol.

4352 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3

INDEX
To aid users the index includes a reference to the supplement where the latest version of a text can be found.
For example: Amikacin...............................................6.1-3396
means the monograph Amikacin can be found on page 3396 of Supplement 6.1.
Note that where no reference to a supplement is made, the text can be found in the principal volume.

Monographs deleted from the 6th Edition are not included in the index; a list of deleted texts is found in the Contents of
this supplement, page xxxix.

English index ........................................................................ 4355 Latin index ................................................................................. 4387

General Notices (1) apply to all monographs and other texts 4353
 EUROPEAN PHARMACOPOEIA 6.3

4354 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3
Numerics
1. General notices .......................................................................... 3
2.1.1. Droppers..............................................................................15
2.1.2. Comparative table of porosity of sintered-glass
filters..............................................................................................15
2.1.3. Ultraviolet ray lamps for analytical purposes..............15
2.1.4. Sieves ...................................................................................16
2.1.5. Tubes for comparative tests ............................................17
2.1.6. Gas detector tubes.............................................................17
2.1. Apparatus ...............................................................................15
2.2.10. Viscosity - Rotating viscometer method .................... 28
2.2.11. Distillation range ............................................................ 30
2.2.12. Boiling point ....................................................................31
2.2.13. Determination of water by distillation........................31
2.2.14. Melting point - capillary method................................. 32
2.2.15. Melting point - open capillary method ...................... 32
2.2.16. Melting point - instantaneous method ...................... 33
2.2.17. Drop point ........................................................................ 33
2.2.18. Freezing point................................................................. 35
2.2.19. Amperometric titration ................................................. 35
2.2.1. Clarity and degree of opalescence of liquids...............21
2.2.20. Potentiometric titration ................................................ 35
2.2.21. Fluorimetry...................................................................... 36
2.2.22. Atomic emission spectrometry.................................... 36
2.2.23. Atomic absorption spectrometry ................................ 37
2.2.24. Absorption spectrophotometry, infrared .................. 39
2.2.25. Absorption spectrophotometry, ultraviolet and
visible.............................................................................................41
2.2.26. Paper chromatography ................................................. 43
2.2.27. Thin-layer chromatography .......................................... 43
2.2.28. Gas chromatography ..................................................... 45
2.2.29. Liquid chromatography ................................................ 46
2.2.2. Degree of coloration of liquids...................................... 22
2.2.30. Size-exclusion chromatography .................................. 47
2.2.31. Electrophoresis ............................................................... 48
2.2.32. Loss on drying ................................................................ 53
2.2.33. Nuclear magnetic resonance spectrometry ...6.3-3909
2.2.34. Thermal analysis.................................................. 6.1-3311
2.2.35. Osmolality ........................................................................ 57
2.2.36. Potentiometric determination of ionic
concentration using ion-selective electrodes....................... 58
2.2.37. X-ray fluorescence spectrometry................................. 59
2.2.38. Conductivity .................................................................... 59
2.2.39. Molecular mass distribution in dextrans .................. 60
2.2.3. Potentiometric determination of pH ............................ 24
2.2.40. Near-infrared spectrophotometry ............................... 62
2.2.41. Circular dichroism.......................................................... 66
2.2.42. Density of solids ..................................................6.3-3912
2.2.43. Mass spectrometry ......................................................... 68
2.2.44. Total organic carbon in water for pharmaceutical
use..................................................................................................71
2.2.45. Supercritical fluid chromatography............................71
2.2.46. Chromatographic separation techniques.................. 72
2.2.47. Capillary electrophoresis .............................................. 77
2.2.48. Raman spectrometry ..................................................... 82
2.2.49. Falling ball viscometer method................................... 84
2.2.4. Relationship between reaction of solution,
approximate pH and colour of certain indicators .............. 25
2.2.54. Isoelectric focusing........................................................ 84
2.2.55. Peptide mapping............................................................. 86
2.2.56. Amino acid analysis ....................................................... 89
2.2.57. Inductively coupled plasma-atomic emission
spectrometry ............................................................................... 96
2.2.58. Inductively coupled plasma-mass spectrometry ...... 98
2.2.5. Relative density................................................................. 25
2.2.60. Melting point - instrumental method..............6.1-3313
2.2.6. Refractive index ................................................................ 26
2.2.7. Optical rotation ................................................................. 26
2.2.8. Viscosity.............................................................................. 27
General Notices (1) apply to all monographs and other texts
Index
2.2.9. Capillary viscometer method ......................................... 27
2.2. Physical and physicochemical methods...........................21
2.3.1. Identification reactions of ions and functional
groups ........................................................................................ 103
2.3.2. Identification of fatty oils by thin-layer
chromatography....................................................................... 106
2.3.3. Identification of phenothiazines by thin-layer
chromatography....................................................................... 107
2.3.4. Odour ................................................................................ 107
2.3. Identification....................................................................... 103
2.4.10. Lead in sugars............................................................... 115
2.4.11. Phosphates......................................................................116
2.4.12. Potassium........................................................................116
2.4.13. Sulphates ........................................................................116
2.4.14. Sulphated ash ................................................................116
2.4.15. Nickel in polyols ............................................................116
2.4.16. Total ash..........................................................................116
2.4.17. Aluminium.......................................................................117
2.4.18. Free formaldehyde ........................................................117
2.4.19. Alkaline impurities in fatty oils ..................................117
2.4.1. Ammonium........................................................................111
2.4.21. Foreign oils in fatty oils by thin-layer
chromatography........................................................................117
2.4.22. Composition of fatty acids by gas chromato-
graphy .........................................................................................118
2.4.23. Sterols in fatty oils....................................................... 120
2.4.24. Identification and control of residual solvents...... 121
2.4.25. Ethylene oxide and dioxan......................................... 126
2.4.26. N,N-Dimethylaniline .................................................... 127
2.4.27. Heavy metals in herbal drugs and fatty oils........... 128
2.4.28. 2-Ethylhexanoic acid ................................................... 129
2.4.29. Composition of fatty acids in oils rich in omega-3
acids...................................................................................6.2-3623
2.4.2. Arsenic ...............................................................................111
2.4.30. Ethylene glycol and diethylene glycol in ethoxylated
substances ..................................................................................131
2.4.31. Nickel in hydrogenated vegetable oils ......................131
2.4.32. Total cholesterol in oils rich in omega-3 acids ...... 132
2.4.3. Calcium..............................................................................111
2.4.4. Chlorides .......................................................................... 112
2.4.5. Fluorides .......................................................................... 112
2.4.6. Magnesium....................................................................... 112
2.4.7. Magnesium and alkaline-earth metals ....................... 112
2.4.8. Heavy metals ................................................................... 112
2.4.9. Iron .................................................................................... 115
2.4. Limit tests.............................................................................111
2.5.10. Oxygen-flask method................................................... 140
2.5.11. Complexometric titrations.......................................... 140
2.5.12. Water : semi-micro determination..............................141
2.5.13. Aluminium in adsorbed vaccines...............................141
2.5.14. Calcium in adsorbed vaccines ................................... 142
2.5.15. Phenol in immunosera and vaccines ....................... 142
2.5.16. Protein in polysaccharide vaccines .......................... 142
2.5.17. Nucleic acids in polysaccharide vaccines ................ 142
2.5.18. Phosphorus in polysaccharide vaccines.................. 142
2.5.19. O-Acetyl in polysaccharide vaccines......................... 143
2.5.1. Acid value......................................................................... 137
2.5.20. Hexosamines in polysaccharide vaccines................ 143
2.5.21. Methylpentoses in polysaccharide vaccines ........... 143
2.5.22. Uronic acids in polysaccharide vaccines ................. 144
2.5.23. Sialic acid in polysaccharide vaccines ..................... 144
2.5.24. Carbon dioxide in gases.....................................6.3-3915
2.5.25. Carbon monoxide in gases................................6.3-3915
2.5.26. Nitrogen monoxide and nitrogen dioxide in
gases ........................................................................................... 146
2.5.27. Oxygen in gases ................................................... 6.3-3916
2.5.28. Water in gases............................................................... 146
2.5.29. Sulphur dioxide............................................................ 146
2.5.2. Ester value ....................................................................... 137
4355
Index
2.5.30. Oxidising substances................................................... 147
2.5.31. Ribose in polysaccharide vaccines............................ 147
2.5.32. Water : micro determination ...................................... 147
2.5.33. Total protein.................................................................. 148
2.5.34. Acetic acid in synthetic peptides .............................. 151
2.5.35. Nitrous oxide in gases................................................. 152
2.5.36. Anisidine value ............................................................. 152
2.5.3. Hydroxyl value ................................................................ 137
2.5.4. Iodine value ..................................................................... 137
2.5.5. Peroxide value................................................................. 138
2.5.6. Saponification value ...................................................... 139
2.5.7. Unsaponifiable matter ................................................... 139
2.5.8. Determination of primary aromatic
amino-nitrogen ......................................................................... 139
2.5.9. Determination of nitrogen by sulphuric acid
digestion .................................................................................... 139
2.5. Assays ................................................................................... 137
2.6.10. Histamine ....................................................................... 165
2.6.11. Depressor substances.................................................. 166
2.6.12. Microbiological examination of non-sterile products :
microbial enumeration tests ........................................6.3-3923
2.6.13. Microbiological examination of non-sterile products :
test for specified micro-organisms ..............................6.3-3927
2.6.14. Bacterial endotoxins .................................................... 182
2.6.15. Prekallikrein activator................................................. 189
2.6.16. Tests for extraneous agents in viral vaccines for
human use................................................................................. 190
2.6.17. Test for anticomplementary activity of
immunoglobulin........................................................................191
2.6.18. Test for neurovirulence of live virus vaccines........ 193
2.6.19. Test for neurovirulence of poliomyelitis vaccine
(oral) ........................................................................................... 193
2.6.1. Sterility .................................................................... 6.3-3919
2.6.20. Anti-A and anti-B haemagglutinins
(indirect method) ..................................................................... 195
2.6.21. Nucleic acid amplification techniques ..................... 195
2.6.22. Activated coagulation factors.................................... 198
2.6.24. Avian viral vaccines : tests for extraneous agents in
seed lots ..................................................................................... 198
2.6.25. Avian live virus vaccines : tests for extraneous agents
in batches of finished product .............................................. 202
2.6.26. Test for anti-D antibodies in human immunoglobulin
for intravenous administration ....................................6.2-3627
2.6.27. Microbiological control of cellular products .......... 205
2.6.2. Mycobacteria ................................................................... 159
2.6.7. Mycoplasmas........................................................... 6.1-3317
2.6.8. Pyrogens........................................................................... 164
2.6.9. Abnormal toxicity ........................................................... 165
2.6. Biological tests ................................................................... 155
2.7.10. Assay of human coagulation factor VII ................... 228
2.7.11. Assay of human coagulation factor IX ..................... 229
2.7.12. Assay of heparin in coagulation factors .................. 230
2.7.13. Assay of human anti-D immunoglobulin................. 230
2.7.14. Assay of hepatitis A vaccine ....................................... 232
2.7.15. Assay of hepatitis B vaccine (rDNA)......................... 233
2.7.16. Assay of pertussis vaccine (acellular)....................... 233
2.7.17. Assay of human antithrombin III .............................. 234
2.7.18. Assay of human coagulation factor II ...................... 234
2.7.19. Assay of human coagulation factor X ...................... 235
2.7.19. Assay of human coagulation factor X (2.7.19.)....... 235
2.7.1. Immunochemical methods ........................................... 209
2.7.20. In vivo assay of poliomyelitis vaccine
(inactivated) .............................................................................. 235
2.7.21. Assay of human von Willebrand factor.................... 237
2.7.22. Assay of human coagulation factor XI..................... 238
2.7.23. Numeration of CD34/CD45+ cells in
haematopoietic products........................................................ 238
2.7.24. Flow cytometry ............................................................. 240
2.7.25. Assay of human plasmin inhibitor...................6.2-3631
4356 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
2.7.27. Flocculation value (Lf) of diphtheria and tetanus
toxins and toxoids (Ramon assay)........................................ 241
2.7.28. Colony-forming cell assay for human
haematopoietic progenitor cells ........................................... 242
2.7.29. Nucleated cell count and viability............................. 243
2.7.2. Microbiological assay of antibiotics...................6.3-3935
2.7.30. Assay of human protein C .................................6.2-3631
2.7.31. Assay of human protein S..................................6.2-3632
2.7.32. Assay of human α-1-proteinase inhibitor .......6.2-3633
2.7.4. Assay of human coagulation factor VIII .....................216
2.7.5. Assay of heparin...............................................................217
2.7.6. Assay of diphtheria vaccine (adsorbed) ......................217
2.7.7. Assay of pertussis vaccine............................................. 222
2.7.8. Assay of tetanus vaccine (adsorbed)........................... 223
2.7.9. Test for Fc function of immunoglobulin ................... 227
2.7. Biological assays ................................................................ 209
2.8.10. Solubility in alcohol of essential oils ....................... 250
2.8.11. Assay of 1,8-cineole in essential oils ........................ 250
2.8.12. Determination of essential oils in herbal drugs .... 251
2.8.13. Pesticide residues................................................6.2-3637
2.8.14. Determination of tannins in herbal drugs.............. 255
2.8.15. Bitterness value ............................................................ 255
2.8.16. Dry residue of extracts................................................ 256
2.8.17. Loss on drying of extracts .......................................... 256
2.8.18. Determination of aflatoxin B1 in herbal drugs ...... 256
2.8.1. Ash insoluble in hydrochloric acid ............................. 249
2.8.20. Herbal drugs : sampling and sample preparation.. 258
2.8.2. Foreign matter ................................................................ 249
2.8.3. Stomata and stomatal index ........................................ 249
2.8.4. Swelling index................................................................. 249
2.8.5. Water in essential oils.................................................... 249
2.8.6. Foreign esters in essential oils .................................... 250
2.8.7. Fatty oils and resinified essential oils in essential
oils............................................................................................... 250
2.8.8. Odour and taste of essential oils................................. 250
2.8.9. Residue on evaporation of essential oils................... 250
2.8. Methods in pharmacognosy ............................................ 249
2.9.10. Ethanol content and alcoholimetric tables ............ 281
2.9.11. Test for methanol and 2-propanol ............................ 282
2.9.12. Sieve test ........................................................................ 283
2.9.14. Specific surface area by air permeability ................ 283
2.9.15. Apparent volume .......................................................... 285
2.9.16. Flowability...................................................................... 286
2.9.17. Test for extractable volume of parenteral
preparations.............................................................................. 287
2.9.18. Preparations for inhalation : aerodynamic assessment
of fine particles ........................................................................ 287
2.9.19. Particulate contamination : sub-visible particles... 300
2.9.1. Disintegration of tablets and capsules..............6.3-3943
2.9.20. Particulate contamination : visible particles.......... 302
2.9.22. Softening time determination of lipophilic
suppositories............................................................................. 302
2.9.23. Gas pycnometric density of solids ...................6.2-3642
2.9.25. Dissolution test for medicated chewing gums....... 304
2.9.26. Specific surface area by gas adsorption.................. 306
2.9.27. Uniformity of mass of delivered doses from multidose
containers.................................................................................. 309
2.9.29. Intrinsic dissolution..................................................... 309
2.9.2. Disintegration of suppositories and pessaries ......... 265
2.9.31. Particle size analysis by laser light diffraction .......311
2.9.32. Porosity and pore-size distribution of solids by
mercury porosimetry .....................................................6.2-3643
2.9.33. Characterisation of crystalline and partially crystalline
solids by X-ray powder diffraction (XRPD)................6.3-3945
2.9.34. Bulk density and tapped density of powders ..6.2-3646
2.9.35. Powder fineness ..................................................6.2-3648
2.9.36. Powder flow................................................................... 320
2.9.37. Optical microscopy....................................................... 323
EUROPEAN PHARMACOPOEIA 6.3
2.9.38. Particle-size distribution estimation by analytical
sieving ...............................................................................6.2-3649
2.9.3. Dissolution test for solid dosage forms ..................... 266
2.9.40. Uniformity of dosage units................................6.1-3325
2.9.41. Friability of granules and spheroids ........................ 330
2.9.42. Dissolution test for lipophilic solid dosage forms.. 332
2.9.43. Apparent dissolution ..........................................6.1-3327
2.9.4. Dissolution test for transdermal patches .................. 275
2.9.5. Uniformity of mass of single-dose preparations....... 278
2.9.6. Uniformity of content of single-dose preparations.. 278
2.9.7. Friability of uncoated tablets ....................................... 278
2.9.8. Resistance to crushing of tablets................................ 279
2.9.9. Measurement of consistency by penetrometry ........6.2-
3641
2.9. Pharmaceutical technical procedures ........................... 263
3.1.10. Materials based on non-plasticised poly(vinyl chloride)
for containers for non-injectable, aqueous solutions ...... 360
3.1.11. Materials based on non-plasticised poly(vinyl
chloride) for containers for dry dosage forms for oral
administration .......................................................................... 362
3.1.1.1. Materials based on plasticised poly(vinyl chloride) for
containers for human blood and blood components....... 339
3.1.1.2. Materials based on plasticised poly(vinyl chloride)
for tubing used in sets for the transfusion of blood and
blood components ................................................................... 342
3.1.13. Plastic additives ...................................................6.2-3655
3.1.14. Materials based on plasticised poly(vinyl chloride)
for containers for aqueous solutions for intravenous
infusion ...................................................................................... 366
3.1.15. Polyethylene terephthalate for containers for
preparations not for parenteral use..................................... 369
3.1.1. Materials for containers for human blood and blood
components............................................................................... 339
3.1.3. Polyolefines...................................................................... 344
3.1.4. Polyethylene without additives for containers
for parenteral preparations and for ophthalmic
preparations.............................................................................. 348
3.1.5. Polyethylene with additives for containers
for parenteral preparations and for ophthalmic
preparations.............................................................................. 349
3.1.6. Polypropylene for containers and closures for
parenteral preparations and ophthalmic preparations ... 352
3.1.7. Poly(ethylene - vinyl acetate) for containers and tubing
for total parenteral nutrition preparations ........................ 356
3.1.8. Silicone oil used as a lubricant ................................... 358
3.1.9. Silicone elastomer for closures and tubing .............. 358
3.1. Materials used for the manufacture of containers ..... 339
3.2.1. Glass containers for pharmaceutical use .................. 373
3.2.2.1. Plastic containers for aqueous solutions for
infusion ...................................................................................... 379
3.2.2. Plastic containers and closures for pharmaceutical
use............................................................................................... 378
3.2.3. Sterile plastic containers for human blood and
blood components ................................................................... 379
3.2.4. Empty sterile containers of plasticised poly(vinyl
chloride) for human blood and blood components.......... 381
3.2.5. Sterile containers of plasticised poly(vinyl chloride) for
human blood containing anticoagulant solution ............. 382
3.2.6. Sets for the transfusion of blood and blood
components............................................................................... 383
3.2.8. Sterile single-use plastic syringes ............................... 384
3.2.9. Rubber closures for containers for aqueous
parenteral preparations, for powders and for
freeze-dried powders ............................................................... 386
3.2. Containers ........................................................................... 373
4.1.1. Reagents ........................................................................... 391
4.1.1. Reagents ..................................................................6.1-3331
4.1.1. Reagents ..................................................................6.2-3661
4.1.1. Reagents ..................................................................6.3-3953
General Notices (1) apply to all monographs and other texts
Index
4.1.2. Standard solutions for limit tests................................ 504
4.1.2. Standard solutions for limit tests.......................6.3-3954
4.1.3. Buffer solutions .............................................................. 508
4.1.3. Buffer solutions .....................................................6.1-3331
4.1.3. Buffer solutions .....................................................6.3-3954
4.1. Reagents, standard solutions, buffer solutions ........... 391
4.2.1. Primary standards for volumetric solutions..............514
4.2.2. Volumetric solutions.......................................................514
4.2.2. Volumetric solutions.............................................6.3-3954
4.2. Volumetric analysis.............................................................514
4-Aminobenzoic acid ............................................................... 1164
4. Reagents.................................................................................. 391
5.10. Control of impurities in substances for pharmaceutical
use............................................................................................... 653
5.11. Characters section in monographs .............................. 659
5.1.1. Methods of preparation of sterile products .............. 525
5.1.2. Biological indicators of sterilisation........................... 527
5.12. Reference standards........................................................ 663
5.1.3. Efficacy of antimicrobial preservation ....................... 528
5.14. Gene transfer medicinal products for human use .... 669
5.1.4. Microbiological quality of non-sterile pharmaceutical
preparations and substances for pharmaceutical
use......................................................................................6.3-3957
5.1.5. Application of the F0 concept to steam sterilisation of
aqueous preparations ....................................................6.3-3958
5.15. Functionality-related characteristics of
excipients..........................................................................6.1-3339
5.1.6. Alternative methods for control of microbiological
quality......................................................................................... 532
5.1.7. Viral safety........................................................................ 543
5.1.9. Guidelines for using the test for sterility .........6.3-3958
5.1. General texts on microbiology ........................................ 525
5.2.1. Terminology used in monographs on biological
products ..................................................................................... 547
5.2.2. Chicken flocks free from specified pathogens for the
production and quality control of vaccines........................ 547
5.2.3. Cell substrates for the production of vaccines for
human use........................................................................6.3-3963
5.2.4. Cell cultures for the production of veterinary
vaccines...................................................................................... 553
5.2.5. Substances of animal origin for the production of
veterinary vaccines.................................................................. 555
5.2.6. Evaluation of safety of veterinary vaccines and
immunosera ............................................................................. 556
5.2.7. Evaluation of efficacy of veterinary vaccines and
immunosera .....................................................................6.1-3335
5.2.8. Minimising the risk of transmitting animal spongiform
encephalopathy agents via human and veterinary medicinal
products ..................................................................................... 558
5.2.9. Evaluation of safety of each batch of veterinary
vaccines and immunosera...................................................... 567
5.2. General texts on biological products............................. 547
5.3. Statistical analysis of results of biological assays and
tests............................................................................................. 571
5.4. Residual solvents ............................................................... 603
5.5. Alcoholimetric tables ........................................................ 613
5.6. Assay of interferons........................................................... 627
5.7. Table of physical characteristics of radionuclides
mentioned in the European Pharmacopoeia..................... 633
5.8. Pharmacopoeial harmonisation ..................................... 645
5.9. Polymorphism..................................................................... 649
A
Abbreviations and symbols (1.) ................................................... 3
Abnormal toxicity (2.6.9.)......................................................... 165
Absorption spectrophotometry, infrared (2.2.24.)................ 39
Absorption spectrophotometry, ultraviolet and visible
(2.2.25.) .........................................................................................41
Acacia...................................................................................6.3-4013
4357
Index
Acacia, spray-dried ............................................................ 6.3-4014
Acamprosate calcium .............................................................. 1088
Acarbose..................................................................................... 1089
Acebutolol hydrochloride....................................................... 1091
Aceclofenac.........................................................................6.2-3685
Acemetacin .........................................................................6.3-4015
Acesulfame potassium ............................................................ 1095
Acetazolamide........................................................................... 1096
Acetic acid, glacial ................................................................... 1097
Acetic acid in synthetic peptides (2.5.34.) ............................ 151
Acetone....................................................................................... 1098
Acetylcholine chloride ............................................................ 1099
Acetylcysteine ........................................................................... 1100
β-Acetyldigoxin ..........................................................................1101
Acetylsalicylic acid ................................................................... 1103
Acetyltryptophan, N- ........................................................ 6.3-4016
Acetyltyrosine, N- ..................................................................... 1106
Aciclovir ..................................................................................... 1107
Acid value (2.5.1.)....................................................................... 137
Acitretin...................................................................................... 1109
Actinobacillosis vaccine (inactivated), porcine .................... 943
Activated charcoal.............................................................6.3-4088
Activated coagulation factors (2.6.22.).................................. 198
Additives, plastic (3.1.13.)................................................6.2-3655
Adenine ...................................................................................... 1110
Adenosine ........................................................................... 6.3-4018
Adenovirus vectors for human use ........................................ 670
Adipic acid ................................................................................. 1113
Adrenaline ..........................................................................6.2-3686
Adrenaline tartrate ...................................................................1114
Aerodynamic assessment of fine particles in preparations for
inhalation (2.9.18.) .................................................................. 287
Aflatoxin B1 in herbal drugs, determination of (2.8.18.)... 256
Agar...................................................................................... 6.3-4019
Agnus castus fruit.............................................................6.2-3688
Agrimony ....................................................................................1117
Air, medicinal .....................................................................6.3-4020
Air, synthetic medicinal .......................................................... 1121
Alanine ....................................................................................... 1121
Albendazole............................................................................... 1122
Albumin solution, human.......................................................2057
Alchemilla .................................................................................. 1123
Alcoholimetric tables (2.9.10.) ................................................ 281
Alcoholimetric tables (5.5.) ...................................................... 613
Alcuronium chloride................................................................ 1124
Alendronate sodium .........................................................6.3-4296
Alexandrian senna pods .........................................................2870
Alfacalcidol ................................................................................ 1126
Alfadex........................................................................................ 1127
Alfentanil hydrochloride......................................................... 1128
Alfuzosin hydrochloride ..................................................6.1-3394
Alginic acid.........................................................................6.3-4022
Alkaline-earth metals and magnesium (2.4.7.) .................... 112
Alkaline impurities in fatty oils (2.4.19.)................................117
Allantoin..................................................................................... 1131
Allergen products....................................................................... 679
Allopurinol................................................................................. 1132
all-rac-α-Tocopherol.................................................................3086
all-rac-α-Tocopheryl acetate ..................................................3089
Almagate .............................................................................6.3-4023
Almond oil, refined .................................................................. 1136
Almond oil, virgin .................................................................... 1136
Aloes, Barbados........................................................................ 1137
Aloes, Cape ................................................................................ 1138
Aloes dry extract, standardised......................................6.2-3690
Alphacyclodextrin .................................................................... 1127
Alprazolam ................................................................................ 1139
Alprenolol hydrochloride ........................................................1141
Alprostadil ................................................................................. 1143
4358 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Alteplase for injection ............................................................. 1145
Alternative methods for control of microbiological quality
(5.1.6.)......................................................................................... 532
Altizide ................................................................................6.2-3691
Alum............................................................................................ 1149
Aluminium (2.4.17.) ....................................................................117
Aluminium chloride hexahydrate......................................... 1149
Aluminium hydroxide, hydrated, for adsorption........6.1-3395
Aluminium in adsorbed vaccines (2.5.13.).............................141
Aluminium magnesium silicate......................................6.3-4024
Aluminium oxide, hydrated.............................................6.3-4025
Aluminium phosphate gel ...............................................6.3-4026
Aluminium phosphate, hydrated .......................................... 1153
Aluminium sodium silicate .............................................6.3-4026
Aluminium sulphate ................................................................ 1154
Alverine citrate ......................................................................... 1154
Amantadine hydrochloride .................................................... 1156
Ambroxol hydrochloride......................................................... 1156
Amfetamine sulphate .............................................................. 1158
Amidotrizoic acid dihydrate................................................... 1158
Amikacin .............................................................................6.1-3396
Amikacin sulphate ............................................................6.1-3398
Amiloride hydrochloride......................................................... 1163
Amino acid analysis (2.2.56.)..................................................... 89
Aminobenzoic acid, 4- ............................................................. 1164
Aminocaproic acid ................................................................... 1166
Aminoglutethimide.................................................................. 1167
Amiodarone hydrochloride .............................................6.3-4028
Amisulpride ............................................................................... 1170
Amitriptyline hydrochloride ...........................................6.3-4029
Amlodipine besilate ................................................................. 1173
Ammonia (13N) injection ........................................................... 981
Ammonia solution, concentrated ......................................... 1175
Ammonio methacrylate copolymer (type A) ...................... 1175
Ammonio methacrylate copolymer (type B) .......................1176
Ammonium (2.4.1.) .....................................................................111
Ammonium bromide................................................................ 1177
Ammonium chloride................................................................ 1178
Ammonium glycyrrhizate....................................................... 1179
Ammonium hydrogen carbonate .......................................... 1180
Amobarbital............................................................................... 1180
Amobarbital sodium ................................................................ 1181
Amoxicillin sodium .................................................................. 1182
Amoxicillin trihydrate ............................................................. 1184
Amperometric titration (2.2.19.) ............................................... 35
Amphotericin B .................................................................6.3-4031
Ampicillin, anhydrous ............................................................ 1188
Ampicillin sodium .................................................................... 1190
Ampicillin trihydrate ............................................................... 1193
Anaesthetic ether ..................................................................... 1834
Analysis, thermal (2.2.34.)............................................... 6.1-3311
Analytical sieving, particle-size distribution estimation by
(2.9.38.) .............................................................................6.2-3649
Angelica root............................................................................. 1196
Animal anti-T lymphocyte immunoglobulin for human
use.............................................................................................1203
Animal immunosera for human use....................................... 685
Animal spongiform encephalopathies, products with risk of
transmitting agents of............................................................. 694
Animal spongiform encephalopathy agents, minimising the
risk of transmitting via human and veterinary medicinal
products (5.2.8.) ....................................................................... 558
Aniseed....................................................................................... 1199
Anise oil...................................................................................... 1197
Anisidine value (2.5.36.) ........................................................... 152
Antazoline hydrochloride....................................................... 1199
Anthrax spore vaccine (live) for veterinary use................... 859
Anthrax vaccine for human use (adsorbed, prepared from
culture filtrates) ....................................................................... 757
EUROPEAN PHARMACOPOEIA 6.3
Anti-A and anti-B haemagglutinins (indirect method)
(2.6.20.) ...................................................................................... 195
Antibiotics, microbiological assay of (2.7.2.) ...............6.3-3935
Antibodies (anti-D) in human immunoglobulin for
intravenous administration, test for (2.6.26.) ...........6.2-3627
Antibodies for human use, monoclonal ................................ 690
Anticoagulant and preservative solutions for human blood
...................................................................................................1200
Anticomplementary activity of immunoglobulin (2.6.17.) ..191
Anti-D antibodies in human immunoglobulin for intravenous
administration, test for (2.6.26.)..................................6.2-3627
Anti-D immunoglobulin for intravenous administration,
human ......................................................................................2059
Anti-D immunoglobulin, human ....................................6.2-3757
Anti-D immunoglobulin, human, assay of (2.7.13.)............. 230
Antimicrobial preservation, efficacy of (5.1.3.).................... 528
Antiserum, European viper venom ........................................ 970
Antithrombin III concentrate, human .................................2060
Antithrombin III, human, assay of (2.7.17.) .......................... 234
Anti-T lymphocyte immunoglobulin for human use,
animal.......................................................................................1203
Apomorphine hydrochloride .................................................1207
Apparatus (2.1.) .............................................................................15
Apparent dissolution (2.9.43.)........................................6.1-3327
Apparent volume (2.9.15.)........................................................ 285
Application of the F0 concept to steam sterilisation of
aqueous preparations (5.1.5.).......................................6.3-3958
Aprotinin .............................................................................6.3-4033
Aprotinin concentrated solution....................................6.3-4035
Arachis oil, hydrogenated ...............................................6.2-3694
Arachis oil, refined................................................................... 1211
Arginine...................................................................................... 1212
Arginine aspartate ................................................................... 1213
Arginine hydrochloride........................................................... 1214
Arnica flower......................................................................6.3-4038
Arnica tincture...................................................................6.3-4040
Arsenic (2.4.2.).............................................................................111
Arsenious trioxide for homoeopathic preparations.......... 1073
Articaine hydrochloride.......................................................... 1217
Artichoke leaf............................................................................ 1219
Artichoke leaf dry extract ...............................................6.3-4041
Ascorbic acid......................................................................6.3-4042
Ascorbyl palmitate ...................................................................1222
Ash insoluble in hydrochloric acid (2.8.1.)........................... 249
Ash leaf.......................................................................................1222
Asparagine monohydrate .......................................................1223
Aspartame..................................................................................1224
Aspartic acid..............................................................................1225
Assay of 1,8-cineole in essential oils (2.8.11.) ...................... 250
Assay of diphtheria vaccine (adsorbed) (2.7.6.) ....................217
Assay of heparin (2.7.5.) ............................................................217
Assay of heparin in coagulation factors (2.7.12.)................ 230
Assay of hepatitis A vaccine (2.7.14.) ..................................... 232
Assay of hepatitis B vaccine (rDNA) (2.7.15.) ...................... 233
Assay of human anti-D immunoglobulin (2.7.13.)............... 230
Assay of human antithrombin III (2.7.17.) ............................ 234
Assay of human coagulation factor II (2.7.18.).................... 234
Assay of human coagulation factor IX (2.7.11.)................... 229
Assay of human coagulation factor VII (2.7.10.) ................. 228
Assay of human coagulation factor VIII (2.7.4.)...................216
Assay of human coagulation factor X (2.7.19.) .................... 235
Assay of human coagulation factor XI (2.7.22.) .................. 238
Assay of human plasmin inhibitor (2.7.25.).................6.2-3631
Assay of human protein C (2.7.30.) ...............................6.2-3631
Assay of human protein S (2.7.31.)................................6.2-3632
Assay of human von Willebrand factor (2.7.21.) ................. 237
Assay of interferons (5.6.)......................................................... 627
Assay of pertussis vaccine (2.7.7.)........................................... 222
Assay of pertussis vaccine (acellular) (2.7.16.) .................... 233
General Notices (1) apply to all monographs and other texts
Index
Assay of poliomyelitis vaccine (inactivated), in vivo
(2.7.20.) ...................................................................................... 235
Assay of tetanus vaccine (adsorbed) (2.7.8.) ........................ 223
Assays (2.5.)................................................................................. 137
Astemizole .................................................................................1226
Atenolol......................................................................................1228
Atomic absorption spectrometry (2.2.23.) .............................. 37
Atomic emission spectrometry (2.2.22.).................................. 36
Atomic emission spectrometry, inductively coupled plasma-
(2.2.57.) ........................................................................................ 96
Atracurium besilate .................................................................1230
Atropine ..............................................................................6.3-4044
Atropine sulphate..............................................................6.3-4045
Aujeszky’s disease vaccine (inactivated) for pigs................ 859
Aujeszky’s disease vaccine (live) for pigs for parenteral
administration .......................................................................... 861
Avian infectious bronchitis vaccine (inactivated)................ 864
Avian infectious bronchitis vaccine (live) ....................6.1-3371
Avian infectious bursal disease vaccine (inactivated) ........ 867
Avian infectious bursal disease vaccine (live) ...................... 869
Avian infectious encephalomyelitis vaccine (live) ............... 871
Avian infectious laryngotracheitis vaccine (live)................. 872
Avian live virus vaccines : tests for extraneous agents in
batches of finished product (2.6.25.)................................... 202
Avian paramyxovirus 1 (Newcastle disease) vaccine
(inactivated) .............................................................................. 937
Avian paramyxovirus 3 vaccine (inactivated)....................... 874
Avian tuberculin purified protein derivative...................... 3146
Avian viral tenosynovitis vaccine (live).................................. 875
Avian viral vaccines : tests for extraneous agents in seed lots
(2.6.24.) ...................................................................................... 198
Azaperone for veterinary use ................................................1234
Azathioprine..............................................................................1236
Azelastine hydrochloride........................................................1236
Azithromycin......................................................................6.3-4047
B
Bacampicillin hydrochloride...........................................6.1-3409
Bacitracin...................................................................................1245
Bacitracin zinc ..........................................................................1247
Baclofen .....................................................................................1250
Bacterial cells used for the manufacture of plasmid vectors
for human use .......................................................................... 676
Bacterial endotoxins (2.6.14.).................................................. 182
Bambuterol hydrochloride..................................................... 1251
Barbados aloes ......................................................................... 1137
Barbital.......................................................................................1252
Barium chloride dihydrate for homoeopathic
preparations............................................................................ 1073
Barium sulphate.......................................................................1253
Basic butylated methacrylate copolymer............................1254
BCG for immunotherapy .................................................6.3-4053
BCG vaccine, freeze-dried ........................................................ 759
Bearberry leaf ....................................................................6.1-3410
Beclometasone dipropionate, anhydrous ....................6.3-4054
Beclometasone dipropionate monohydrate ................6.3-4056
Bee for homoeopathic preparations, honey....................... 1079
Beeswax, white .........................................................................1260
Beeswax, yellow........................................................................ 1261
Belladonna leaf......................................................................... 1261
Belladonna leaf dry extract, standardised ...................6.3-4059
Belladonna leaf tincture, standardised................................1264
Belladonna, prepared .......................................................6.2-3698
Benazepril hydrochloride................................................6.3-4060
Bendroflumethiazide ..............................................................1266
Benfluorex hydrochloride ......................................................1267
Benperidol .................................................................................1269
Benserazide hydrochloride ....................................................1270
Bentonite ............................................................................6.3-4062
4359
Index
Benzalkonium chloride...........................................................1272
Benzalkonium chloride solution ..........................................1273
Benzathine benzylpenicillin ..................................................1283
Benzbromarone........................................................................1273
Benzethonium chloride ..........................................................1275
Benzocaine ................................................................................ 1276
Benzoic acid .............................................................................. 1276
Benzoin, Siam...........................................................................1277
Benzoin, Sumatra ....................................................................1278
Benzoin tincture, Siam ...........................................................1278
Benzoin tincture, Sumatra.....................................................1279
Benzoyl peroxide, hydrous ....................................................1280
Benzyl alcohol .......................................................................... 1281
Benzyl benzoate .......................................................................1283
Benzylpenicillin, benzathine .................................................1283
Benzylpenicillin potassium....................................................1285
Benzylpenicillin, procaine......................................................1287
Benzylpenicillin sodium .........................................................1288
Betacarotene .............................................................................1290
Betacyclodextrin ...................................................................... 1291
Betacyclodextrin, poly(hydroxypropyl) ether ............. 6.3-4170
Betadex ...................................................................................... 1291
Betahistine dihydrochloride ..................................................1292
Betahistine mesilate ................................................................1293
Betamethasone.........................................................................1295
Betamethasone acetate ..........................................................1297
Betamethasone dipropionate ................................................1298
Betamethasone sodium phosphate......................................1300
Betamethasone valerate ..................................................6.3-4062
Betaxolol hydrochloride .........................................................1303
Bezafibrate ................................................................................1304
Bifonazole..................................................................................1306
Bilberry fruit, dried .................................................................1307
Bilberry fruit dry extract, fresh, refined and
standardised.....................................................................6.2-3745
Bilberry fruit, fresh...........................................................6.1-3412
Biological assays (2.7.) .............................................................. 209
Biological assays and tests, statistical analysis of results of
(5.3.)............................................................................................ 571
Biological indicators of sterilisation (5.1.2.) ........................ 527
Biological products, general texts on (5.2.).......................... 547
Biological products, terminology used in monographs on
(5.2.1.)......................................................................................... 547
Biological tests (2.6.)................................................................. 155
Biotin ..........................................................................................1308
Biperiden hydrochloride.........................................................1309
Biphasic insulin injection....................................................... 2140
Biphasic isophane insulin injection ..................................... 2140
Birch leaf.............................................................................6.2-3699
Bisacodyl.................................................................................... 1312
Bismuth subcarbonate............................................................ 1313
Bismuth subgallate.................................................................. 1314
Bismuth subnitrate, heavy ..................................................... 1315
Bismuth subsalicylate ............................................................. 1316
Bisoprolol fumarate..........................................................6.1-3412
Bistort rhizome ........................................................................ 1317
Bitter fennel .............................................................................. 1873
Bitter-fennel fruit oil................................................................ 1318
Bitterness value (2.8.15.).......................................................... 255
Bitter-orange epicarp and mesocarp.............................6.3-4064
Bitter-orange-epicarp and mesocarp tincture ....................1320
Bitter-orange flower .........................................................6.3-4065
Bitter-orange-flower oil...........................................................2490
Black horehound ..................................................................... 1321
Bleomycin sulphate .................................................................1322
Blood and blood components, empty sterile containers of
plasticised poly(vinyl chloride) for (3.2.4.) ......................... 381
Blood and blood components, materials for containers for
(3.1.1.)......................................................................................... 339
4360 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Blood and blood components, sets for the transfusion of
(3.2.6.) ........................................................................................ 383
Blood and blood components, sterile plastic containers for
(3.2.3.) ........................................................................................ 379
Blood, anticoagulant and preservative solutions for .......1200
Blood, sterile containers of plasticised poly(vinyl chloride)
containing anticoagulant solution (3.2.5.) ......................... 382
Bogbean leaf .............................................................................1323
Boiling point (2.2.12.) ..................................................................31
Boldo leaf...................................................................................1324
Boldo leaf dry extract.......................................................6.1-3415
Borage (starflower) oil, refined.............................................1326
Borax ..........................................................................................1326
Boric acid...................................................................................1327
Botulinum antitoxin .................................................................. 965
Botulinum toxin type A for injection...................................1327
Bovine infectious rhinotracheitis vaccine (live) .................. 924
Bovine insulin........................................................................... 2135
Bovine leptospirosis vaccine (inactivated)............................ 876
Bovine parainfluenza virus vaccine (live)............................. 878
Bovine respiratory syncytial virus vaccine (live)................. 879
Bovine serum ............................................................................1329
Bovine tuberculin purified protein derivative ................... 3147
Bovine viral diarrhoea vaccine (inactivated)........................ 880
Bromazepam ............................................................................. 1331
Bromhexine hydrochloride ....................................................1332
Bromocriptine mesilate ..........................................................1333
Bromperidol ..............................................................................1335
Bromperidol decanoate ..........................................................1337
Brompheniramine maleate.....................................................1339
Brotizolam .................................................................................1340
Brucellosis vaccine (live) (Brucella melitensis Rev. 1 strain)
for veterinary use..................................................................... 881
Buccal tablets and sublingual tablets.................................... 734
Buckwheat herb ....................................................................... 1341
Budesonide................................................................................1342
Bufexamac .................................................................................1344
Buffer solutions (4.1.3.) ............................................................ 508
Buffer solutions (4.1.3.) ...................................................6.1-3331
Buffer solutions (4.1.3.) ...................................................6.3-3954
Buflomedil hydrochloride ......................................................1345
Bulk density and tapped density of powders
(2.9.34.) .............................................................................6.2-3646
Bumetanide ...............................................................................1346
Bupivacaine hydrochloride ....................................................1347
Buprenorphine .........................................................................1349
Buprenorphine hydrochloride ..............................................1350
Buserelin.............................................................................6.3-4067
Buspirone hydrochloride........................................................1353
Busulfan.....................................................................................1355
Butcher’s broom................................................................6.1-3416
Butylated methacrylate copolymer, basic...........................1254
Butylhydroxyanisole................................................................1357
Butylhydroxytoluene...............................................................1357
Butyl parahydroxybenzoate...................................................1358
C
Cabergoline ...............................................................................1363
Cachets ......................................................................................... 719
Cadmium sulphate hydrate for homoeopathic
preparations............................................................................ 1074
Caffeine ...............................................................................6.1-3421
Caffeine monohydrate.............................................................1365
Calcifediol ..................................................................................1366
Calcipotriol, anhydrous ..........................................................1367
Calcipotriol monohydrate ......................................................1370
Calcitonin (salmon)..................................................................1372
Calcitriol.....................................................................................1375
Calcium (2.4.3.)............................................................................111
EUROPEAN PHARMACOPOEIA 6.3
Calcium acetate ........................................................................ 1376
Calcium ascorbate....................................................................1377
Calcium carbonate ............................................................6.2-3703
Calcium carboxymethylcellulose .......................................... 1422
Calcium chloride dihydrate....................................................1378
Calcium chloride hexahydrate ..............................................1379
Calcium dobesilate monohydrate ..................................6.2-3703
Calcium folinate ................................................................6.3-4071
Calcium glucoheptonate.........................................................1383
Calcium gluconate ............................................................6.3-4073
Calcium gluconate, anhydrous ......................................6.3-4074
Calcium gluconate for injection.....................................6.3-4074
Calcium glycerophosphate.....................................................1386
Calcium hydrogen phosphate, anhydrous ..........................1387
Calcium hydrogen phosphate dihydrate .............................1388
Calcium hydroxide ...................................................................1389
Calcium in adsorbed vaccines (2.5.14.) ................................. 142
Calcium iodide tetrahydrate for homoeopathic prepara-
tions .......................................................................................... 1074
Calcium lactate, anhydrous ...................................................1389
Calcium lactate monohydrate ...............................................1390
Calcium lactate pentahydrate ...............................................1390
Calcium lactate trihydrate...................................................... 1391
Calcium levofolinate pentahydrate ......................................1392
Calcium levulinate dihydrate.................................................1394
Calcium pantothenate.............................................................1395
Calcium pentetate (sodium) for radiopharmaceutical
preparations.....................................................................6.3-4001
Calcium phosphate ..................................................................1396
Calcium stearate................................................................6.3-4076
Calcium sulphate dihydrate...................................................1398
Calendula flower ......................................................................1398
Calf coronavirus diarrhoea vaccine (inactivated)................ 882
Calf rotavirus diarrhoea vaccine (inactivated)..................... 884
Calicivirosis vaccine (inactivated), feline............................... 909
Calicivirosis vaccine (live), feline .............................................910
Camphor, D- ............................................................................... 1400
Camphor, racemic .................................................................... 1401
Canine adenovirus vaccine (inactivated) .............................. 885
Canine adenovirus vaccine (live) ............................................ 886
Canine distemper vaccine (live) .............................................. 887
Canine leptospirosis vaccine (inactivated)............................ 888
Canine parainfluenza virus vaccine (live)............................. 890
Canine parvovirosis vaccine (inactivated)............................. 891
Canine parvovirosis vaccine (live) .......................................... 892
Cape aloes.................................................................................. 1138
Capillary electrophoresis (2.2.47.)............................................ 77
Capillary viscometer method (2.2.9.)....................................... 27
Caprylic acid.............................................................................. 1402
Caprylocaproyl macrogolglycerides..................................... 1403
Capsicum.............................................................................6.2-3704
Capsicum oleoresin, refined and quantified ...................... 1405
Capsicum tincture, standardised .......................................... 1406
Capsules ........................................................................................717
Capsules and tablets, disintegration of (2.9.1.) ..........6.3-3943
Capsules, gastro-resistant......................................................... 718
Capsules, hard ............................................................................ 718
Capsules, intrauterine......................................................6.3-3977
Capsules, modified-release ....................................................... 718
Capsules, oromucosal ............................................................... 734
Capsules, rectal........................................................................... 745
Capsules, soft .............................................................................. 718
Capsules, vaginal........................................................................ 752
Captopril .................................................................................... 1407
Caraway fruit............................................................................. 1408
Caraway oil ................................................................................ 1408
Carbachol................................................................................... 1410
Carbamazepine ..........................................................................1411
Carbasalate calcium................................................................. 1412
General Notices (1) apply to all monographs and other texts
Index
Carbidopa .................................................................................. 1413
Carbimazole ...............................................................................1414
Carbocisteine ............................................................................ 1415
Carbomers ..........................................................................6.1-3422
Carbon dioxide ..........................................................................1417
Carbon dioxide in gases (2.5.24.) ..................................6.3-3915
Carbon monoxide (15O) ............................................................. 982
Carbon monoxide in gases (2.5.25.)..............................6.3-3915
Carboplatin................................................................................ 1419
Carboprost trometamol .......................................................... 1420
Carboxymethylcellulose calcium .......................................... 1422
Carboxymethylcellulose sodium........................................... 1423
Carboxymethylcellulose sodium, cross-linked ............ 6.3-4117
Carboxymethylcellulose sodium, low-substituted............. 1424
Carisoprodol.............................................................................. 1421
Carmellose calcium.................................................................. 1422
Carmellose sodium .................................................................. 1423
Carmellose sodium and microcrystalline cellulose ..........2422
Carmellose sodium, low-substituted .................................... 1424
Carmustine ................................................................................ 1425
Carnauba wax ........................................................................... 1425
Carprofen for veterinary use ..........................................6.3-4077
Carteolol hydrochloride.......................................................... 1426
Carvedilol................................................................................... 1427
Cascara ....................................................................................... 1429
Cascara dry extract, standardised ........................................ 1430
Cassia oil .............................................................................6.2-3707
Castor oil, hydrogenated ........................................................ 1432
Castor oil, polyoxyl ..................................................................2304
Castor oil, polyoxyl hydrogenated........................................2303
Castor oil, refined .................................................................... 1433
Castor oil, virgin....................................................................... 1434
Catgut, sterile............................................................................ 1045
Catgut, sterile, in distributor for veterinary use ............... 1057
CD34/CD45+ cells in haematopoietic products, numeration
of (2.7.23.).................................................................................. 238
Cefaclor ...................................................................................... 1435
Cefadroxil monohydrate ..................................................6.1-3423
Cefalexin monohydrate....................................................6.1-3425
Cefalotin sodium ...................................................................... 1440
Cefamandole nafate................................................................. 1441
Cefapirin sodium...................................................................... 1443
Cefatrizine propylene glycol.................................................. 1444
Cefazolin sodium...................................................................... 1445
Cefepime dihydrochloride monohydrate ............................ 1448
Cefixime ..................................................................................... 1450
Cefoperazone sodium ............................................................. 1451
Cefotaxime sodium .................................................................. 1453
Cefoxitin sodium ...................................................................... 1455
Cefradine.................................................................................... 1457
Ceftazidime................................................................................ 1459
Ceftriaxone sodium.................................................................. 1461
Cefuroxime axetil ..................................................................... 1462
Cefuroxime sodium.................................................................. 1464
Celiprolol hydrochloride......................................................... 1465
Cell count and viability, nucleated (2.7.29.)......................... 243
Cell cultures for the production of veterinary vaccines
(5.2.4.) ........................................................................................ 553
Cell substrates for the production of vaccines for human use
(5.2.3.) ...............................................................................6.3-3963
Cellular products, microbiological control of (2.6.27.) ...... 205
Cellulose acetate ...............................................................6.3-4078
Cellulose acetate butyrate...................................................... 1468
Cellulose acetate phthalate.............................................6.3-4079
Cellulose, microcrystalline..............................................6.3-4080
Cellulose (microcrystalline) and carmellose sodium........2422
Cellulose, powdered .........................................................6.3-4084
Centaury .................................................................................... 1477
Centella ...................................................................................... 1477
4361
Index
Cetirizine dihydrochloride ..............................................6.2-3715
Cetostearyl alcohol .................................................................. 1480
Cetostearyl alcohol (type A), emulsifying .................... 6.2-3717
Cetostearyl alcohol (type B), emulsifying....................6.2-3718
Cetostearyl isononanoate....................................................... 1484
Cetrimide ................................................................................... 1484
Cetyl alcohol ............................................................................. 1485
Cetyl palmitate.......................................................................... 1486
Cetylpyridinium chloride........................................................ 1486
Ceylon cinnamon bark oil ...............................................6.2-3721
Ceylon cinnamon leaf oil........................................................1544
CFC assay for human haematopoietic progenitor cells
(2.7.28.) ...................................................................................... 242
Chamomile flower, Roman..................................................... 1487
Characterisation of crystalline and partially crystalline solids
by X-ray powder diffraction (XRPD) (2.9.33.) ...........6.3-3945
Characters section in monographs (5.11.)............................ 659
Charcoal, activated ...........................................................6.3-4088
Chenodeoxycholic acid .......................................................... 1489
Chewing gum, medicated (2.9.25.)......................................... 304
Chewing gums, medicated ....................................................... 719
Chicken flocks free from specified pathogens for the
production and quality control of vaccines (5.2.2.).......... 547
Chicken infectious anaemia vaccine (live)............................ 925
Chitosan hydrochloride .......................................................... 1490
Chlamydiosis vaccine (inactivated), feline ............................911
Chloral hydrate......................................................................... 1491
Chlorambucil............................................................................. 1492
Chloramine ................................................................................ 3103
Chloramphenicol...................................................................... 1492
Chloramphenicol palmitate ................................................... 1493
Chloramphenicol sodium succinate..................................... 1495
Chlorcyclizine hydrochloride ................................................ 1496
Chlordiazepoxide ..................................................................... 1497
Chlordiazepoxide hydrochloride .......................................... 1498
Chlorhexidine diacetate.......................................................... 1499
Chlorhexidine digluconate solution ....................................1500
Chlorhexidine dihydrochloride .............................................1502
Chlorides (2.4.4.) ........................................................................ 112
Chlorobutanol, anhydrous .....................................................1503
Chlorobutanol hemihydrate ..................................................1504
Chlorocresol ..............................................................................1504
Chloroquine phosphate ..........................................................1505
Chloroquine sulphate..............................................................1506
Chlorothiazide ..........................................................................1507
Chlorphenamine maleate ................................................6.1-3427
Chlorpromazine hydrochloride.............................................1509
Chlorpropamide........................................................................ 1510
Chlorprothixene hydrochloride ............................................ 1511
Chlortalidone ............................................................................ 1513
Chlortetracycline hydrochloride........................................... 1514
Cholecalciferol .......................................................................... 1516
Cholecalciferol concentrate (oily form)........................6.3-4089
Cholecalciferol concentrate (powder form).................6.3-4091
Cholecalciferol concentrate (water-dispersible
form) ..................................................................................6.3-4093
Cholera vaccine ...........................................................................761
Cholera vaccine, freeze-dried ...................................................761
Cholera vaccine (inactivated, oral)......................................... 762
Cholesterol ................................................................................1524
Cholesterol in oils rich in omega-3 acids, total (2.4.32.) ... 132
Chondroitin sulphate sodium.........................................6.3-4095
Chromatographic separation techniques (2.2.46.) ............... 72
Chromatography, gas (2.2.28.).................................................. 45
Chromatography, liquid (2.2.29.) ............................................. 46
Chromatography, paper (2.2.26.).............................................. 43
Chromatography, size-exclusion (2.2.30.)............................... 47
Chromatography, supercritical fluid (2.2.45.) ........................71
Chromatography, thin-layer (2.2.27.)....................................... 43
4362 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Chromium (51Cr) edetate injection ................................6.2-3677
Chymotrypsin............................................................................1527
Ciclopirox...................................................................................1528
Ciclopirox olamine ...................................................................1530
Ciclosporin ................................................................................ 1531
Cilastatin sodium ..............................................................6.1-3428
Cilazapril....................................................................................1534
Cimetidine..................................................................................1536
Cimetidine hydrochloride.......................................................1537
Cinchocaine hydrochloride....................................................1538
Cinchona bark ...................................................................6.2-3720
Cinchona liquid extract, standardised.................................1540
Cineole........................................................................................ 1541
Cineole in essential oils, 1,8-, assay of (2.8.11.)................... 250
Cinnamon ..................................................................................1542
Cinnamon bark oil, Ceylon .............................................6.2-3721
Cinnamon leaf oil, Ceylon ......................................................1544
Cinnamon tincture...................................................................1545
Cinnarizine ................................................................................1545
Ciprofibrate ...............................................................................1547
Ciprofloxacin.............................................................................1548
Ciprofloxacin hydrochloride..................................................1550
Circular dichroism (2.2.41.) ....................................................... 66
Cisapride monohydrate........................................................... 1551
Cisapride tartrate .....................................................................1552
Cisplatin ..............................................................................6.3-4097
Citalopram hydrobromide ...............................................6.3-4099
Citalopram hydrochloride ............................................... 6.3-4101
Citric acid, anhydrous .............................................................1554
Citric acid monohydrate .........................................................1555
Citronella oil..............................................................................1556
Cladribine ..................................................................................1557
Clarithromycin..........................................................................1559
Clarity and degree of opalescence of liquids (2.2.1.).............21
Clary sage oil............................................................................. 1561
Classical swine-fever vaccine (live, prepared in cell
cultures)............................................................................6.2-3669
Clazuril for veterinary use .....................................................1562
Clebopride malate....................................................................1564
Clemastine fumarate ........................................................6.1-3430
Clenbuterol hydrochloride.....................................................1567
Clindamycin hydrochloride....................................................1568
Clindamycin phosphate ..........................................................1570
Clioquinol .................................................................................. 1571
Clobazam ...................................................................................1572
Clobetasol propionate.............................................................1573
Clobetasone butyrate ..............................................................1575
Clodronate disodium tetrahydrate ................................6.2-3722
Clofazimine................................................................................1577
Clofibrate ...................................................................................1578
Clomifene citrate ......................................................................1579
Clomipramine hydrochloride.................................................1580
Clonazepam...............................................................................1582
Clonidine hydrochloride..................................................6.3-4102
Clopamide...........................................................................6.1-3431
Closantel sodium dihydrate for veterinary use .................1584
Clostridium botulinum vaccine for veterinary use ............. 894
Clostridium chauvoei vaccine for veterinary use.......6.3-3997
Clostridium novyi alpha antitoxin for veterinary use ........ 973
Clostridium novyi (type b) vaccine for veterinary use ....... 895
Clostridium perfringens beta antitoxin for veterinary use
..................................................................................................... 974
Clostridium perfringens epsilon antitoxin for veterinary use
..................................................................................................... 975
Clostridium perfringens vaccine for veterinary use ........... 897
Clostridium septicum vaccine for veterinary use................ 899
Closures and containers for parenteral preparations and
ophthalmic preparations, polypropylene for (3.1.6.)........ 352
EUROPEAN PHARMACOPOEIA 6.3
Closures and containers for pharmaceutical use, plastic
(3.2.2.) ........................................................................................ 378
Closures and tubing, silicone elastomer for (3.1.9.)........... 358
Closures for containers for aqueous parenteral preparations,
for powders and for freeze-dried powders, rubber
(3.2.9.) ........................................................................................ 386
Clotrimazole.......................................................................6.1-3433
Clove ...........................................................................................1587
Clove oil .....................................................................................1588
Cloxacillin sodium....................................................................1589
Clozapine ...................................................................................1590
Coagulation factor II, assay of (2.7.18.)................................. 234
Coagulation factor IX, human...............................................2064
Coagulation factor IX, human, assay of (2.7.11.)................. 229
Coagulation factors, activated (2.6.22.)................................. 198
Coagulation factors, assay of heparin (2.7.12.) ................... 230
Coagulation factor VII, human ............................................. 2061
Coagulation factor VII, human, assay of (2.7.10.)............... 228
Coagulation factor VIII, human............................................2062
Coagulation factor VIII, human, assay of (2.7.4.).................216
Coagulation factor VIII (rDNA), human .............................2063
Coagulation factor X, assay of (2.7.19.)................................. 235
Coagulation factor XI, human...............................................2065
Coagulation factor XI, human, assay of (2.7.22.) ................ 238
Coated granules ......................................................................... 724
Coated tablets ............................................................................. 749
Cocaine hydrochloride............................................................1592
Coccidiosis vaccine (live) for chickens .........................6.2-3665
Coconut oil, refined..........................................................6.2-3723
Cocoyl caprylocaprate.............................................................1594
Codeine ...............................................................................6.1-3434
Codeine hydrochloride dihydrate.........................................1596
Codeine phosphate hemihydrate..........................................1598
Codeine phosphate sesquihydrate .......................................1599
Codergocrine mesilate ..................................................... 6.3-4103
Cod-liver oil, farmed ......................................................... 6.3-4105
Cod-liver oil (type A)......................................................... 6.3-4109
Cod-liver oil (type B)......................................................... 6.3-4113
Cola ............................................................................................. 1611
Colchicine .................................................................................. 1612
Cold-water vibriosis vaccine (inactivated) for
salmonids..........................................................................6.2-3671
Colestyramine ........................................................................... 1613
Colibacillosis vaccine (inactivated), neonatal piglet........... 934
Colibacillosis vaccine (inactivated), neonatal ruminant .... 936
Colistimethate sodium ............................................................ 1614
Colistin sulphate ...................................................................... 1615
Colloidal anhydrous silica ......................................................2877
Colloidal hydrated silica .........................................................2877
Colloidal silica, hydrophobic .................................................2878
Colloidal silver, for external use ...........................................2879
Colony-forming cell assay for human haematopoietic
progenitor cells (2.7.28.) ........................................................ 242
Colophony ..................................................................................1617
Coloration of liquids (2.2.2.)...................................................... 22
Common stinging nettle for homoeopathic
preparations............................................................................ 1075
Comparative table of porosity of sintered-glass filters
(2.1.2.)............................................................................................15
Complexometric titrations (2.5.11.)........................................ 140
Composition of fatty acids by gas chromatography
(2.4.22.) .......................................................................................118
Composition of fatty acids in oils rich in omega-3 acids
(2.4.29.) .............................................................................6.2-3623
Compressed lozenges................................................................ 734
Concentrated solutions for haemodialysis .........................2022
Concentrates for injections or infusions............................... 736
Concentrates for intrauterine solutions.......................6.3-3977
Conductivity (2.2.38.).................................................................. 59
General Notices (1) apply to all monographs and other texts
Index
Coneflower herb, purple ........................................................2785
Coneflower root, narrow-leaved............................................2483
Coneflower root, pale..............................................................2602
Coneflower root, purple .........................................................2787
Conjugated estrogens ............................................................. 1824
Consistency by penetrometry, measurement of
(2.9.9.) ...............................................................................6.2-3641
Containers (3.2.)......................................................................... 373
Containers and closures for parenteral preparations and
ophthalmic preparations, polypropylene for (3.1.6.)........ 352
Containers and closures for pharmaceutical use, plastic
(3.2.2.) ........................................................................................ 378
Containers and tubing for total parenteral nutrition
preparations, poly(ethylene - vinyl acetate) for (3.1.7.) ... 356
Containers for aqueous solutions for infusion, plastic
(3.2.2.1.) ..................................................................................... 379
Containers for aqueous solutions for intravenous infusion,
materials based on plasticised poly(vinyl chloride) for
(3.1.14.) ...................................................................................... 366
Containers for dry dosage forms for oral administration,
materials based on non-plasticised poly(vinyl chloride) for
(3.1.11.)....................................................................................... 362
Containers for human blood and blood components,
materials based on plasticised poly(vinyl chloride) for
(3.1.1.1.) ..................................................................................... 339
Containers for human blood and blood components,
materials for (3.1.1.) ................................................................ 339
Containers for human blood and blood components, plastic,
sterile (3.2.3.) ............................................................................ 379
Containers for non-injectable aqueous solutions, materials
based on non-plasticised poly(vinyl chloride) for
(3.1.10.) ...................................................................................... 360
Containers for parenteral preparations and for ophthalmic
preparations, polyethylene with additives for (3.1.5.) ..... 349
Containers for parenteral preparations and for ophthalmic
preparations, polyethylene without additives for
(3.1.4.)......................................................................................... 348
Containers for pharmaceutical use, glass (3.2.1.)............... 373
Containers for preparations not for parenteral use,
polyethylene terephthalate for (3.1.15) .............................. 369
Containers of plasticised poly(vinyl chloride) for human
blood and blood components, empty sterile (3.2.4.)........ 381
Containers of plasticised poly(vinyl chloride) for human blood
containing anticoagulant solution, sterile (3.2.5.)............ 382
Contamination, microbial : microbial enumeration tests
(2.6.12.) .............................................................................6.3-3923
Contamination, microbial : test for specified micro-organisms
(2.6.13.) .............................................................................6.3-3927
Content uniformity of single-dose preparations (2.9.6.).... 278
Control of impurities in substances for pharmaceutical use
(5.10.).......................................................................................... 653
Control of microbiological quality, alternative methods for
(5.1.6.)......................................................................................... 532
Copolymer, basic butylated methacrylate ..........................1254
Copolymer, methacrylic acid - ethyl acrylate (1:1) ....6.2-3781
Copolymer, methacrylic acid - ethyl acrylate (1:1) dispersion
30 per cent .......................................................................6.3-4220
Copolymer (type A), ammonio methacrylate ..................... 1175
Copolymer (type B), ammonio methacrylate ......................1176
Copovidone.................................................................................1617
Copper acetate monohydrate for homoeopathic prepara-
tions .......................................................................................... 1075
Copper for homoeopathic preparations.............................. 1076
Copper sulphate, anhydrous.................................................. 1619
Copper sulphate pentahydrate.............................................. 1620
Coriander ................................................................................... 1620
Coriander oil ............................................................................. 1621
Cortisone acetate ..................................................................... 1622
Cotton, absorbent .................................................................... 1624
4363
Index
Cottonseed oil, hydrogenated .......................................6.2-3724
Couch grass rhizome .............................................................. 1625
Creams.................................................................................6.3-3980
Cresol, crude ............................................................................. 1626
Croscarmellose sodium.................................................... 6.3-4117
Crospovidone ..................................................................... 6.3-4119
Crotamiton ................................................................................ 1629
Crystalline and partially crystalline solids, characterisation
by X-ray powder diffraction (XRPD) of (2.9.33.) ......6.3-3945
Cutaneous application, liquid preparations for................... 728
Cutaneous application, powders for .............................6.3-3978
Cutaneous application, semi-solid preparations for ..6.3-3979
Cutaneous application, veterinary liquid preparations
for ................................................................................................ 752
Cutaneous foams........................................................................ 728
Cyanocobalamin ....................................................................... 1630
Cyanocobalamin (57Co) capsules ............................................ 983
Cyanocobalamin (57Co) solution ............................................. 984
Cyanocobalamin (58Co) capsules ............................................ 985
Cyanocobalamin (58Co) solution ............................................. 986
Cyclizine hydrochloride...................................................6.2-3725
Cyclopentolate hydrochloride ............................................... 1632
Cyclophosphamide................................................................... 1633
Cyproheptadine hydrochloride ............................................. 1634
Cyproterone acetate ................................................................ 1635
Cysteine hydrochloride monohydrate ................................. 1636
Cystine........................................................................................ 1637
Cytarabine ................................................................................. 1638
D Diethylene glycol and ethylene glycol in ethoxylated
Dacarbazine............................................................................... 1641
Dalteparin sodium ................................................................... 1642
Danaparoid sodium ................................................................. 1644
Dapsone ..................................................................................... 1646
Daunorubicin hydrochloride ................................................. 1647
D-Camphor ................................................................................. 1400
Decyl oleate ............................................................................... 1648
Deferoxamine mesilate............................................................ 1649
Degree of coloration of liquids (2.2.2.).................................... 22
Dembrexine hydrochloride monohydrate for veteri-
nary use ................................................................................... 1650
Demeclocycline hydrochloride.............................................. 1651
Density of powders, bulk density and tapped
(2.9.34.) .............................................................................6.2-3646
Density of solids (2.2.42.)................................................6.3-3912
Density of solids, gas pycnometric (2.9.23.)................6.2-3642
Density, relative (2.2.5.) .............................................................. 25
Dental type silica......................................................................2878
Depressor substances (2.6.11.)................................................ 166
Deptropine citrate.................................................................... 1653
Dequalinium chloride.............................................................. 1654
Desflurane ..........................................................................6.1-3439
Desipramine hydrochloride ................................................... 1655
Deslanoside ............................................................................... 1656
Desmopressin............................................................................ 1657
Desogestrel ................................................................................ 1658
Desoxycortone acetate............................................................ 1659
Detector tubes, gas (2.1.6.) .........................................................17
Determination of aflatoxin B1 in herbal drugs (2.8.18.).... 256
Determination of essential oils in herbal drugs (2.8.12.).. 251
Determination of nitrogen by sulphuric acid digestion
(2.5.9.) ........................................................................................ 139
Determination of primary aromatic amino-nitrogen
(2.5.8.) ........................................................................................ 139
Determination of tannins in herbal drugs (2.8.14.)............ 255
Determination of water by distillation (2.2.13.) .....................31
Detomidine hydrochloride for veterinary use ................... 1660
Devil’s claw dry extract........................................................... 1662
Devil’s claw root ................................................................6.2-3729
4364 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Dexamethasone ........................................................................ 1663
Dexamethasone acetate...................................................6.3-4123
Dexamethasone isonicotinate ............................................... 1666
Dexamethasone sodium phosphate ..................................... 1667
Dexchlorpheniramine maleate .............................................. 1669
Dexpanthenol............................................................................ 1670
Dextran 1 for injection.....................................................6.3-4124
Dextran 40 for injection ..................................................6.3-4125
Dextran 60 for injection ..................................................6.3-4126
Dextran 70 for injection ..................................................6.3-4127
Dextranomer ............................................................................. 1675
Dextrans, molecular mass distribution in (2.2.39.) .............. 60
Dextrin........................................................................................ 1675
Dextromethorphan hydrobromide ....................................... 1676
Dextromoramide tartrate ....................................................... 1677
Dextropropoxyphene hydrochloride.................................... 1678
Diazepam ................................................................................... 1679
Diazoxide ................................................................................... 1680
Dibrompropamidine diisetionate .......................................... 1681
Dibutyl phthalate ..................................................................... 1682
Dichloromethane......................................................................2387
Diclazuril for veterinary use.................................................. 1683
Diclofenac potassium .............................................................. 1685
Diclofenac sodium ................................................................... 1686
Dicloxacillin sodium ................................................................ 1687
Dicycloverine hydrochloride.................................................. 1689
Didanosine................................................................................. 1689
Dienestrol .................................................................................. 1691
Diethylcarbamazine citrate.................................................... 1693
substances (2.4.30.)..................................................................131
Diethylene glycol monoethyl ether...................................... 1694
Diethylene glycol palmitostearate........................................ 1695
Diethyl phthalate ..............................................................6.1-3441
Diethylstilbestrol ...................................................................... 1696
Diffraction, laser light, particle size analysis by (2.9.31.) ..311
Diflunisal.................................................................................... 1697
Digitalis leaf .............................................................................. 1698
Digitoxin..................................................................................... 1700
Digoxin ....................................................................................... 1701
Dihydralazine sulphate, hydrated .................................6.1-3442
Dihydrocodeine hydrogen tartrate....................................... 1704
Dihydroergocristine mesilate ................................................ 1705
Dihydroergotamine mesilate ..........................................6.1-3444
Dihydroergotamine tartrate .................................................. 1709
Dihydrostreptomycin sulphate for veterinary use .....6.2-3730
Dihydrotachysterol .................................................................. 1712
Diltiazem hydrochloride ..................................................6.1-3446
Dimenhydrinate........................................................................ 1715
Dimercaprol............................................................................... 1716
Dimethylacetamide ...................................................................1717
Dimethylaniline, N,N- (2.4.26.)................................................ 127
Dimethyl sulfoxide ................................................................... 1716
Dimeticone .........................................................................6.2-3732
Dimetindene maleate .............................................................. 1719
Dinoprostone ............................................................................ 1722
Dinoprost trometamol............................................................. 1720
Diosmin ...................................................................................... 1723
Dioxan and ethylene oxide (2.4.25.) ...................................... 126
Dip concentrates ........................................................................ 753
Diphenhydramine hydrochloride.......................................... 1725
Diphenoxylate hydrochloride ................................................ 1726
Diphtheria and tetanus toxins and toxoids, flocculation value
(Lf) of, (Ramon assay) (2.7.27.) ............................................. 241
Diphtheria and tetanus vaccine (adsorbed) ......................... 763
Diphtheria and tetanus vaccine (adsorbed, reduced antigen(s)
content)...................................................................................... 764
Diphtheria antitoxin .................................................................. 965
EUROPEAN PHARMACOPOEIA 6.3
Diphtheria, tetanus and hepatitis B (rDNA) vaccine
(adsorbed).................................................................................. 765
Diphtheria, tetanus and pertussis (acellular, component)
vaccine (adsorbed)................................................................... 767
Diphtheria, tetanus and pertussis vaccine (adsorbed) ..... 768
Diphtheria, tetanus and poliomyelitis (inactivated) vaccine
(adsorbed, reduced antigen(s) content) .............................. 770
Diphtheria, tetanus, pertussis (acellular, component) and
haemophilus type b conjugate vaccine (adsorbed) .......... 771
Diphtheria, tetanus, pertussis (acellular, component) and
hepatitis B (rDNA) vaccine (adsorbed) ............................... 774
Diphtheria, tetanus, pertussis (acellular, component) and
poliomyelitis (inactivated) vaccine (adsorbed) .................. 775
Diphtheria, tetanus, pertussis (acellular, component) and
poliomyelitis (inactivated) vaccine (adsorbed, reduced
antigen(s) content) .................................................................. 778
Diphtheria, tetanus, pertussis (acellular, component),
hepatitis B (rDNA), poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 780
Diphtheria, tetanus, pertussis (acellular, component),
poliomyelitis (inactivated) and haemophilus type b
conjugate vaccine (adsorbed).......................................6.3-3983
Diphtheria, tetanus, pertussis and poliomyelitis (inactivated)
vaccine (adsorbed)................................................................... 785
Diphtheria, tetanus, pertussis, poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 787
Diphtheria vaccine (adsorbed) ................................................ 789
Diphtheria vaccine (adsorbed), assay of (2.7.6.)...................217
Diphtheria vaccine (adsorbed, reduced antigen content).. 791
Dipivefrine hydrochloride ...................................................... 1727
Dipotassium clorazepate ........................................................ 1728
Dipotassium phosphate .......................................................... 1729
Diprophylline ............................................................................ 1730
Dipyridamole............................................................................. 1731
Dirithromycin.....................................................................6.1-3447
Disintegration of suppositories and pessaries (2.9.2.)....... 265
Disintegration of tablets and capsules (2.9.1.) ...........6.3-3943
Disodium edetate ..................................................................... 1734
Disodium phosphate, anhydrous...................................6.3-4128
Disodium phosphate dihydrate............................................. 1735
Disodium phosphate dodecahydrate ............................6.1-3449
Disopyramide ............................................................................ 1737
Disopyramide phosphate........................................................ 1738
Dispersible tablets ..................................................................... 750
Dissolution, apparent (2.9.43.).......................................6.1-3327
Dissolution, intrinsic (2.9.29.) ................................................. 309
Dissolution test for lipophilic solid dosage forms
(2.9.42.) ...................................................................................... 332
Dissolution test for solid dosage forms (2.9.3.)................... 266
Dissolution test for transdermal patches (2.9.4.)................ 275
Distemper vaccine (live), canine ............................................. 887
Distemper vaccine (live) for mustelids .................................. 900
Distillation range (2.2.11.).......................................................... 30
Distribution estimation by analytical sieving, particle-size
(2.9.38.) .............................................................................6.2-3649
Disulfiram .................................................................................. 1739
Dithranol.................................................................................... 1740
DL-Methionine ...........................................................................2380
DL-α-Tocopheryl hydrogen succinate...................................3093
Dobutamine hydrochloride.....................................................1741
Docusate sodium...................................................................... 1743
Dodecyl gallate ......................................................................... 1744
Dog rose..................................................................................... 1744
Domperidone ............................................................................ 1745
Domperidone maleate............................................................. 1747
Dopamine hydrochloride........................................................ 1749
Dopexamine dihydrochloride ................................................ 1750
Dorzolamide hydrochloride................................................... 1752
Dosage units, uniformity of (2.9.40.) ............................6.1-3325
General Notices (1) apply to all monographs and other texts
Index
Dosulepin hydrochloride........................................................ 1753
Doxapram hydrochloride........................................................ 1754
Doxazosin mesilate .................................................................. 1756
Doxepin hydrochloride ....................................................6.1-3449
Doxorubicin hydrochloride.................................................... 1759
Doxycycline hyclate ................................................................. 1760
Doxycycline monohydrate...................................................... 1762
Doxylamine hydrogen succinate....................................6.1-3451
Droperidol.................................................................................. 1765
Droppers (2.1.1.)............................................................................15
Drop point (2.2.17.)...................................................................... 33
Drops (nasal) and sprays (liquid nasal) ................................. 731
Drops, oral ................................................................................... 730
Dry extracts ........................................................................6.1-3344
Dry residue of extracts (2.8.16.).............................................. 256
Duck plague vaccine (live) ....................................................... 901
Duck viral hepatitis type I vaccine (live)............................... 902
Dwarf pine oil ........................................................................... 1766
Dydrogesterone .................................................................6.3-4128
E
Ear drops and ear sprays.......................................................... 720
Ear powders ................................................................................ 720
Ear preparations......................................................................... 719
Ear preparations, semi-solid .................................................... 720
Ear sprays and ear drops.......................................................... 720
Ear tampons................................................................................ 720
Ear washes................................................................................... 720
Ebastine ..................................................................................... 1771
Econazole .................................................................................. 1772
Econazole nitrate..................................................................... 1773
Edetic acid ................................................................................. 1774
Edrophonium chloride............................................................ 1775
Effervescent granules................................................................ 724
Effervescent powders ................................................................ 739
Effervescent tablets ................................................................... 749
Efficacy of antimicrobial preservation (5.1.3.)..................... 528
Efficacy of veterinary vaccines and immunosera, evaluation
of (5.2.7.) ...........................................................................6.1-3335
Egg drop syndrome ′76 vaccine (inactivated)...................... 904
Elder flower............................................................................... 1776
Electrophoresis (2.2.31.)............................................................. 48
Electrophoresis, capillary (2.2.47.)........................................... 77
Eleutherococcus....................................................................... 1777
Emedastine difumarate........................................................... 1779
Emetine hydrochloride heptahydrate.................................. 1780
Emetine hydrochloride pentahydrate.................................. 1781
Empty sterile containers of plasticised poly(vinyl chloride)
for human blood and blood components (3.2.4.) ............. 381
Emulsifying cetostearyl alcohol (type A) .....................6.2-3717
Emulsifying cetostearyl alcohol (type B).....................6.2-3718
Emulsions, solutions and suspensions, oral ........................ 729
Enalaprilat dihydrate .............................................................. 1784
Enalapril maleate ..................................................................... 1782
Encephalitis vaccine (inactivated), tick-borne ..................... 845
Endotoxins, bacterial (2.6.14.)................................................. 182
Enilconazole for veterinary use............................................ 1785
Enoxaparin sodium.................................................................. 1787
Enoxolone.................................................................................. 1788
Ephedrine, anhydrous............................................................. 1789
Ephedrine hemihydrate .......................................................... 1790
Ephedrine hydrochloride ....................................................... 1791
Ephedrine hydrochloride, racemic....................................... 1792
Epinephrine........................................................................6.2-3686
Epinephrine tartrate ................................................................1114
Epirubicin hydrochloride ....................................................... 1793
Equine herpesvirus vaccine (inactivated) ............................. 905
Equine influenza vaccine (inactivated) ................................. 907
Equisetum stem........................................................................ 1794
4365
Index
Ergocalciferol.....................................................................6.3-4133
Ergometrine maleate............................................................... 1797
Ergotamine tartrate................................................................. 1798
Erysipelas vaccine (inactivated), swine ................................. 955
Erythritol ............................................................................6.3-4134
Erythromycin ............................................................................ 1801
Erythromycin estolate............................................................. 1803
Erythromycin ethylsuccinate................................................. 1806
Erythromycin lactobionate .................................................... 1808
Erythromycin stearate ............................................................ 1810
Erythropoietin concentrated solution................................. 1813
Eserine salicylate .....................................................................2677
Eserine sulphate.......................................................................2678
Esketamine hydrochloride ......................................................1817
Esomeprazole magnesium trihydrate...........................6.3-4136
Essential oils ............................................................................... 680
Essential oils, assay of 1,8-cineole in (2.8.11.) ..................... 250
Essential oils, fatty oils and resinified essential oils in
(2.8.7.)......................................................................................... 250
Essential oils, foreign esters in (2.8.6.) ................................. 250
Essential oils in herbal drugs, determination of (2.8.12.).. 251
Essential oils, odour and taste (2.8.8.) .................................. 250
Essential oils, residue on evaporation (2.8.9.)..................... 250
Essential oils, solubility in alcohol (2.8.10.)......................... 250
Essential oils, water in (2.8.5.) ................................................ 249
Ester value (2.5.2.)..................................................................... 137
Estradiol benzoate............................................................6.1-3455
Estradiol hemihydrate............................................................. 1819
Estradiol valerate ..................................................................... 1821
Estriol ......................................................................................... 1822
Estrogens, conjugated ............................................................ 1824
Etacrynic acid ........................................................................... 1826
Etamsylate ..........................................................................6.2-3737
Ethacridine lactate monohydrate..................................6.3-4138
Ethambutol hydrochloride..............................................6.1-3456
Ethanol (96 per cent) .............................................................. 1829
Ethanol, anhydrous ................................................................. 1831
Ethanol content and alcoholimetric tables (2.9.10.) .......... 281
Ether ........................................................................................... 1833
Ether, anaesthetic.................................................................... 1834
Ethinylestradiol ........................................................................ 1834
Ethionamide.............................................................................. 1835
Ethosuximide ............................................................................ 1836
Ethoxylated substances, ethylene glycol and diethylene
glycol in (2.4.30.) ......................................................................131
Ethyl acetate ............................................................................. 1838
Ethyl acrylate - methacrylic acid copolymer (1:1) .....6.2-3781
Ethyl acrylate - methacrylic acid copolymer (1:1) dispersion
30 per cent .......................................................................6.3-4220
Ethylcellulose ........................................................................... 1841
Ethylenediamine ...................................................................... 1843
Ethylene glycol and diethylene glycol in ethoxylated
substances (2.4.30.)..................................................................131
Ethylene glycol monopalmitostearate................................. 1842
Ethylene glycol monostearate............................................... 1842
Ethylene oxide and dioxan (2.4.25.) ...................................... 126
Ethylhexanoic acid, 2- (2.4.28.)............................................... 129
Ethylmorphine hydrochloride............................................... 1843
Ethyl oleate ............................................................................... 1838
Ethyl parahydroxybenzoate................................................... 1839
Ethyl parahydroxybenzoate sodium .................................... 1840
Etidronate disodium................................................................ 1844
Etilefrine hydrochloride ......................................................... 1845
Etodolac ..................................................................................... 1847
Etofenamate .............................................................................. 1849
Etofylline.................................................................................... 1850
Etomidate .................................................................................. 1851
Etoposide ................................................................................... 1852
Eucalyptus leaf ......................................................................... 1857
4366 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Eucalyptus oil ....................................................................6.2-3738
Eugenol ...................................................................................... 1859
European goldenrod ...............................................................2000
European viper venom antiserum.......................................... 970
Evaluation of efficacy of veterinary vaccines and immunosera
(5.2.7.)................................................................................6.1-3335
Evaluation of safety of each batch of veterinary vaccines and
immunosera (5.2.9.) ................................................................ 567
Evaluation of safety of veterinary vaccines and immunosera
(5.2.6.) ........................................................................................ 556
Evening primrose oil, refined................................................ 1860
Extractable volume of parenteral preparations, test for
(2.9.17.)....................................................................................... 287
Extracts ...............................................................................6.1-3343
Extracts, dry.......................................................................6.1-3344
Extracts, dry residue of (2.8.16.)............................................. 256
Extracts, liquid...................................................................6.1-3343
Extracts, loss on drying of (2.8.17.)........................................ 256
Extracts, soft ......................................................................6.1-3344
Extraneous agents in viral vaccines for human use, tests for
(2.6.16.) ...................................................................................... 190
Extraneous agents : tests in batches of finished product of
avian live virus vaccines (2.6.25.)......................................... 202
Extraneous agents : tests in seed lots of avian viral vaccines
(2.6.24.) ...................................................................................... 198
Eye drops ..................................................................................... 721
Eye lotions................................................................................... 721
Eye preparations ........................................................................ 721
Eye preparations, semi-solid .................................................... 722
F
F0 concept to steam sterilisation of aqueous preparations,
application of (5.1.5.) .....................................................6.3-3958
Factor II, human coagulation, assay of (2.7.18.) ................. 234
Factor IX, human coagulation ..............................................2064
Factor IX, human coagulation, assay of (2.7.11.) ................ 229
Factor VII, human coagulation............................................. 2061
Factor VII, human coagulation, assay of (2.7.10.) .............. 228
Factor VIII, human coagulation ...........................................2062
Factor VIII, human coagulation, assay of (2.7.4.) ................216
Factor VIII (rDNA), human coagulation .............................2063
Factor X, human coagulation, assay of (2.7.19.) ................. 235
Factor XI, human coagulation ..............................................2065
Factor XI, human coagulation, assay of (2.7.22.)................ 238
Falling ball viscometer method (2.2.49.) ................................ 84
Famotidine................................................................................. 1865
Fat, hard..............................................................................6.3-4164
Fatty acids, composition by gas chromatography
(2.4.22.) .......................................................................................118
Fatty acids in oils rich in omega-3 acids, composition of
(2.4.29.) .............................................................................6.2-3623
Fatty oils, alkaline impurities in (2.4.19.) ..............................117
Fatty oils and herbal drugs, heavy metals in (2.4.27.) ....... 128
Fatty oils and resinified essential oils in essential oils
(2.8.7.)......................................................................................... 250
Fatty oils, foreign oils in, by thin-layer chromatography
(2.4.21.) .......................................................................................117
Fatty oils, identification by thin-layer chromatography
(2.3.2.) ........................................................................................ 106
Fatty oils, sterols in (2.4.23.) ................................................... 120
Fatty oils, vegetable................................................................... 712
Fc function of immunoglobulin, test for (2.7.9.) ................. 227
Febantel for veterinary use.................................................... 1870
Felbinac ...................................................................................... 1866
Feline calicivirosis vaccine (inactivated) ............................... 909
Feline calicivirosis vaccine (live)..............................................910
Feline chlamydiosis vaccine (inactivated)..............................911
Feline infectious enteritis (feline panleucopenia) vaccine
(inactivated) .............................................................................. 912
EUROPEAN PHARMACOPOEIA 6.3
Feline infectious enteritis (feline panleucopenia) vaccine
(live) .............................................................................................913
Feline leukaemia vaccine (inactivated)...................................914
Feline panleucopenia vaccine (inactivated).......................... 912
Feline panleucopenia vaccine (live) ........................................913
Feline viral rhinotracheitis vaccine (inactivated) .................916
Feline viral rhinotracheitis vaccine (live)...............................917
Felodipine .................................................................................. 1867
Felypressin................................................................................. 1869
Fenbendazole for veterinary use .......................................... 1871
Fenbufen .................................................................................... 1872
Fennel, bitter............................................................................. 1873
Fennel, sweet............................................................................. 1874
Fenofibrate ................................................................................ 1875
Fenoterol hydrobromide......................................................... 1876
Fentanyl ..................................................................................... 1878
Fentanyl citrate......................................................................... 1879
Fenticonazole nitrate .............................................................. 1880
Fenugreek.................................................................................. 1882
Fermentation, products of ....................................................... 693
Ferric chloride hexahydrate .................................................. 1882
Ferrous fumarate ..................................................................... 1883
Ferrous gluconate............................................................. 6.3-4141
Ferrous sulphate, dried........................................................... 1885
Ferrous sulphate heptahydrate............................................. 1886
Feverfew ..................................................................................... 1887
Fexofenadine hydrochloride .................................................. 1888
Fibrinogen, human ..................................................................2066
Fibrin sealant kit ...................................................................... 1890
Filgrastim concentrated solution .................................. 6.3-4142
Finasteride................................................................................. 1891
Fineness, powder (2.9.35.) ..............................................6.2-3648
Fish oil, rich in omega-3 acids............................................... 1893
Flavoxate hydrochloride ......................................................... 1895
Flecainide acetate .................................................................... 1896
Flocculation value (Lf) of diphtheria and tetanus toxins and
toxoids (Ramon assay) (2.7.27.) ............................................ 241
Flowability (2.9.16.) ................................................................... 286
Flow cytometry (2.7.24.)........................................................... 240
Flubendazole ............................................................................ 1898
Flucloxacillin magnesium octahydrate ........................ 6.2-3741
Flucloxacillin sodium .............................................................. 1899
Fluconazole...............................................................................1900
Flucytosine ................................................................................1902
Fludarabine phosphate...........................................................1903
Fludeoxyglucose (18F) injection .....................................6.2-3678
Fludrocortisone acetate..........................................................1906
Flumazenil.................................................................................1908
Flumazenil (N-[11C]methyl) injection ..................................... 989
Flumequine ...............................................................................1909
Flumetasone pivalate .............................................................. 1910
Flunarizine dihydrochloride.................................................. 1911
Flunitrazepam........................................................................... 1913
Flunixin meglumine for veterinary use............................... 1914
Fluocinolone acetonide .......................................................... 1915
Fluocortolone pivalate............................................................ 1916
Fluorescein................................................................................ 1918
Fluorescein sodium ................................................................. 1919
Fluorides (2.4.5.) ........................................................................ 112
Fluorimetry (2.2.21.) ................................................................... 36
Fluorodopa (18F) (prepared by electrophilic substitution)
injection ..................................................................................... 990
Fluorouracil...............................................................................1920
Fluoxetine hydrochloride .......................................................1922
Flupentixol dihydrochloride ..................................................1924
Fluphenazine decanoate ........................................................1926
Fluphenazine dihydrochloride..............................................1928
Fluphenazine enantate ...........................................................1927
Flurazepam monohydrochloride ..........................................1930
General Notices (1) apply to all monographs and other texts
Index
Flurbiprofen .............................................................................. 1931
Fluspirilene ...............................................................................1932
Flutamide...................................................................................1933
Fluticasone propionate...........................................................1934
Flutrimazole..............................................................................1936
Fluvoxamine maleate ....................................................... 6.3-4144
Foams, cutaneous ...................................................................... 728
Foams, intrauterine ..........................................................6.3-3977
Foams, medicated ...................................................................... 723
Foams, rectal............................................................................... 746
Foams, vaginal ............................................................................ 752
Folic acid....................................................................................1938
Foot-and-mouth disease (ruminants) vaccine
(inactivated) ...............................................................................918
Foreign esters in essential oils (2.8.6.) .................................. 250
Foreign matter (2.8.2.) .............................................................. 249
Foreign oils in fatty oils by thin-layer chromatography
(2.4.21.) .......................................................................................117
Formaldehyde, free (2.4.18.) .....................................................117
Formaldehyde solution (35 per cent) ..................................1939
Formoterol fumarate dihydrate ............................................1940
Foscarnet sodium hexahydrate.............................................1942
Fosfomycin calcium .................................................................1943
Fosfomycin sodium..................................................................1945
Fosfomycin trometamol ..........................................................1946
Fowl cholera vaccine (inactivated) ......................................... 920
Fowl-pox vaccine (live) .............................................................. 921
Framycetin sulphate................................................................1947
Frangula bark ...........................................................................1949
Frangula bark dry extract, standardised ..................... 6.3-4146
Frankincense, Indian .............................................................. 2128
Free formaldehyde (2.4.18.)......................................................117
Freezing point (2.2.18.)............................................................... 35
Fresh bilberry fruit dry extract, refined and
standardised.....................................................................6.2-3745
Friability of granules and spheroids (2.9.41.) ...................... 330
Friability of uncoated tablets (2.9.7.)..................................... 278
Fructose ..................................................................................... 1951
Fucus .......................................................................................... 2213
Fumitory ....................................................................................1952
Functional groups and ions, identification reactions of
(2.3.1.)......................................................................................... 103
Furosemide................................................................................1953
Furunculosis vaccine (inactivated, oil-adjuvanted, injectable)
for salmonids ...................................................................6.2-3668
Fusidic acid ...............................................................................1954
G
Galactose............................................................................. 6.3-4151
Gallamine triethiodide ............................................................1959
Gallium (67Ga) citrate injection ............................................... 992
Gargles.......................................................................................... 733
Garlic for homoeopathic preparations ................................ 1077
Garlic powder............................................................................ 1961
Gas chromatography (2.2.28.) ................................................... 45
Gas detector tubes (2.1.6.) ..........................................................17
Gases, carbon dioxide in (2.5.24.)..................................6.3-3915
Gases, carbon monoxide in (2.5.25.).............................6.3-3915
Gases, nitrogen monoxide and nitrogen dioxide in
(2.5.26.) ...................................................................................... 146
Gases, nitrous oxide in (2.5.35.).............................................. 152
Gases, oxygen in (2.5.27.) ................................................ 6.3-3916
Gases, water in (2.5.28.)............................................................ 146
Gas-gangrene antitoxin, mixed ................................................ 966
Gas-gangrene antitoxin (novyi) ............................................... 966
Gas-gangrene antitoxin (perfringens) .................................... 967
Gas-gangrene antitoxin (septicum)......................................... 968
Gas pycnometric density of solids (2.9.23.).................6.2-3642
Gastro-resistant capsules.......................................................... 718
4367
Index
Gastro-resistant granules.......................................................... 724
Gastro-resistant tablets ............................................................. 750
Gelatin ................................................................................. 6.3-4151
Gels.......................................................................................6.3-3980
Gels for injections ...................................................................... 737
Gemcitabine hydrochloride....................................................1963
Gemfibrozil ................................................................................1964
General notices (1.)........................................................................ 3
General texts on biological products (5.2.) .......................... 547
General texts on microbiology (5.1.)...................................... 525
Gene transfer medicinal products for human use (5.14.).. 669
Gentamicin sulphate................................................................1965
Gentian root ..............................................................................1967
Gentian tincture .......................................................................1968
Ginger ..................................................................................6.2-3751
Gingival solutions ...................................................................... 733
Ginkgo dry extract, refined and quantified.................6.1-3461
Ginkgo leaf ................................................................................1969
Ginseng....................................................................................... 1971
Glass containers for pharmaceutical use (3.2.1.) ................ 373
Glibenclamide ...........................................................................1972
Gliclazide.................................................................................... 1974
Glimepiride ................................................................................1975
Glipizide .....................................................................................1977
Glossary.........................................................................................717
Glossary (dosage forms) ............................................................717
Glucagon, human.....................................................................1979
Glucose, anhydrous ..........................................................6.3-4153
Glucose, liquid ...................................................................6.2-3752
Glucose, liquid, spray-dried.............................................6.3-4154
Glucose monohydrate ......................................................6.3-4154
Glutamic acid ............................................................................1984
Glutathione.........................................................................6.1-3463
Glycerol ......................................................................................1987
Glycerol (85 per cent)..............................................................1988
Glycerol dibehenate.................................................................1990
Glycerol distearate ................................................................... 1991
Glycerol monocaprylate..........................................................1992
Glycerol monocaprylocaprate................................................1993
Glycerol monolinoleate...........................................................1994
Glycerol mono-oleate........................................................6.3-4155
Glycerol monostearate 40-55.................................................1996
Glycerol triacetate.................................................................... 3112
Glyceryl trinitrate solution..............................................6.1-3465
Glycine........................................................................................1998
Glycyrrhizate ammonium ....................................................... 1179
Goldenrod ..................................................................................1999
Goldenrod, European..............................................................2000
Goldenseal rhizome..........................................................6.1-3467
Gonadorelin acetate ................................................................2003
Gonadotrophin, chorionic ......................................................2004
Gonadotrophin, equine serum, for veterinary use............2005
Goserelin ....................................................................................2005
Gramicidin .................................................................................2007
Granisetron hydrochloride..............................................6.3-4156
Granules ....................................................................................... 723
Granules and powders for oral solutions and
suspensions............................................................................... 729
Granules and powders for syrups........................................... 730
Granules and spheroids, friability of (2.9.41.)...................... 330
Granules, coated......................................................................... 724
Granules, effervescent............................................................... 724
Granules, gastro-resistant......................................................... 724
Granules, modified-release....................................................... 724
Greater celandine..................................................................... 2010
Griseofulvin ............................................................................... 2011
Guaiacol ..................................................................................... 2012
Guaifenesin................................................................................ 2014
Guanethidine monosulphate ................................................. 2015
4368 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Guar .....................................................................................6.3-4158
Guar galactomannan ........................................................6.3-4159
Guidelines for using the test for sterility (5.1.9.) .......6.3-3958
H
Haematopoietic products, numeration of CD34/CD45+ cells
in (2.7.23.).................................................................................. 238
Haematopoietic progenitor cells, human, colony-forming cell
assay for (2.7.28.) ..................................................................... 242
Haematopoietic stem cells, human ...............................6.3-4165
Haemodiafiltration and for haemofiltration, solutions
for ..............................................................................................2025
Haemodialysis, concentrated solutions for ........................2022
Haemodialysis solutions, concentrated, water for
diluting..............................................................................6.3-4163
Haemodialysis, solutions for..................................................2022
Haemofiltration and for haemodiafiltration,
solutions for ............................................................................2025
Haemophilus type b (conjugate), diphtheria, tetanus and
pertussis (acellular, component) vaccine (adsorbed)....... 771
Haemophilus type b (conjugate), diphtheria, tetanus,
pertussis (acellular, component) and poliomyelitis
(inactivated) vaccine (adsorbed) ..................................6.3-3983
Haemophilus type b (conjugate), diphtheria, tetanus,
pertussis (acellular, component), hepatitis B (rDNA) and
poliomyelitis (inactivated) vaccine (adsorbed) .................. 780
Haemophilus type b (conjugate), diphtheria, tetanus,
pertussis and poliomyelitis (inactivated) vaccine
(adsorbed).................................................................................. 787
Haemophilus type b conjugate vaccine........................6.3-3985
Haemorrhagic disease vaccine (inactivated), rabbit........... 949
Halofantrine hydrochloride ...................................................2027
Haloperidol................................................................................2028
Haloperidol decanoate............................................................2030
Halothane .................................................................................. 2031
Hamamelis leaf ..................................................................6.1-3471
Hard capsules.............................................................................. 718
Hard fat ...............................................................................6.3-4164
Hard paraffin............................................................................. 2612
Hawthorn berries.....................................................................2034
Hawthorn leaf and flower ......................................................2035
Hawthorn leaf and flower dry extract .................................2036
Hawthorn leaf and flower liquid extract, quantified........2037
Heavy bismuth subnitrate ...................................................... 1315
Heavy kaolin.......................................................................6.3-4183
Heavy magnesium carbonate .........................................6.2-3779
Heavy magnesium oxide..................................................6.3-4209
Heavy metals (2.4.8.) ................................................................. 112
Heavy metals in herbal drugs and fatty oils (2.4.27.)......... 128
Hedera helix for homoeopathic preparations.................... 1078
Helium ........................................................................................2038
Heparin, assay of (2.7.5.) ...........................................................217
Heparin calcium .......................................................................2039
Heparin in coagulation factors, assay of (2.7.12.)............... 230
Heparins, low-molecular-mass ............................................... 2041
Heparin sodium........................................................................2040
Hepatitis A immunoglobulin, human ..................................2068
Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine
(adsorbed).................................................................................. 794
Hepatitis A vaccine, assay of (2.7.14.).................................... 232
Hepatitis A vaccine (inactivated, adsorbed) ......................... 795
Hepatitis A vaccine (inactivated, virosome) ......................... 797
Hepatitis B immunoglobulin for intravenous administration,
human ......................................................................................2069
Hepatitis B immunoglobulin, human ..................................2069
Hepatitis B (rDNA), diphtheria and tetanus vaccine
(adsorbed).................................................................................. 765
Hepatitis B (rDNA), diphtheria, tetanus and pertussis
(acellular, component) vaccine (adsorbed) ........................ 774
EUROPEAN PHARMACOPOEIA 6.3
Hepatitis B (rDNA), diphtheria, tetanus, pertussis (acellular,
component), poliomyelitis (inactivated) and haemophilus
type b conjugate vaccine (adsorbed) ................................... 780
Hepatitis B vaccine (rDNA)...................................................... 800
Hepatitis B vaccine (rDNA), assay of (2.7.15.) ..................... 233
Hepatitis C virus (HCV), validation of nucleic acid
amplification techniques for the detection of HCV RNA in
plasma pools : Guidelines....................................................... 195
Heptaminol hydrochloride.....................................................2043
Herbal drug preparations......................................................... 684
Herbal drugs ............................................................................... 684
Herbal drugs and fatty oils, heavy metals in (2.4.27.)........ 128
Herbal drugs, determination of aflatoxin B1 in (2.8.18.)... 256
Herbal drugs, determination of essential oils in herbal drugs
(2.8.12.) ...................................................................................... 251
Herbal drugs, determination of tannins (2.8.14.) ............... 255
Herbal drugs for homoeopathic preparations ................... 1065
Herbal teas................................................................................... 685
Herpes zoster (shingles) vaccine (live) .........................6.3-3991
Hexamidine diisetionate .........................................................2044
Hexetidine..................................................................................2045
Hexobarbital..............................................................................2047
Hexosamines in polysaccharide vaccines (2.5.20.) ............. 143
Hexylresorcinol.........................................................................2047
Highly purified water .......................................................6.3-4342
Histamine (2.6.10.)..................................................................... 165
Histamine dihydrochloride ....................................................2049
Histamine phosphate ..............................................................2049
Histidine.....................................................................................2050
Histidine hydrochloride monohydrate ................................ 2051
Homatropine hydrobromide ..................................................2052
Homatropine methylbromide ................................................2053
Homoeopathic preparations .................................................. 1065
Homoeopathic preparations, arsenious trioxide for ........ 1073
Homoeopathic preparations, calcium iodide tetrahydrate
for .............................................................................................. 1074
Homoeopathic preparations, common stinging nettle
for .............................................................................................. 1075
Homoeopathic preparations, copper acetate monohydrate
for .............................................................................................. 1075
Homoeopathic preparations, copper for............................. 1076
Homoeopathic preparations, garlic for ............................... 1077
Homoeopathic preparations, hedera helix for................... 1078
Homoeopathic preparations, herbal drugs for .................. 1065
Homoeopathic preparations, honey bee for....................... 1079
Homoeopathic preparations, hyoscyamus for ................... 1079
Homoeopathic preparations, hypericum for ...................... 1080
Homoeopathic preparations, iron for .................................. 1081
Homoeopathic preparations, mother tinctures for........... 1072
Homoeopathic preparations, oriental cashew for............. 1082
Homoeopathic preparations, saffron for............................. 1084
Homoeopathic stocks (methods of preparation of) and
potentisation....................................................................6.1-3385
Honey .........................................................................................2055
Honey bee for homoeopathic preparations........................ 1079
Hop strobile........................................................................6.1-3472
Human α-1-proteinase inhibitor ....................................6.2-3762
Human albumin injection, iodinated (125I)............................ 993
Human albumin solution .......................................................2057
Human anti-D immunoglobulin .....................................6.2-3757
Human anti-D immunoglobulin, assay of (2.7.13.).............. 230
Human anti-D immunoglobulin for intravenous
administration ........................................................................2059
Human antithrombin III, assay of (2.7.17.)........................... 234
Human antithrombin III concentrate ..................................2060
Human coagulation factor II, assay of (2.7.18.)................... 234
Human coagulation factor IX................................................2064
Human coagulation factor IX, assay of (2.7.11.).................. 229
Human coagulation factor VII .............................................. 2061
General Notices (1) apply to all monographs and other texts
Index
Human coagulation factor VII, assay of (2.7.10.) ................ 228
Human coagulation factor VIII.............................................2062
Human coagulation factor VIII, assay of (2.7.4.)..................216
Human coagulation factor VIII (rDNA)...............................2063
Human coagulation factor X, assay of (2.7.19.) ................... 235
Human coagulation factor XI................................................2065
Human coagulation factor XI, assay of (2.7.22.) ................. 238
Human fibrinogen....................................................................2066
Human haematopoietic progenitor cells, colony-forming cell
assay for (2.7.28.) ..................................................................... 242
Human haematopoietic stem cells ................................6.3-4165
Human hepatitis A immunoglobulin ...................................2068
Human hepatitis B immunoglobulin ...................................2069
Human hepatitis B immunoglobulin for intravenous
administration ........................................................................2069
Human insulin .......................................................................... 2137
Human measles immunoglobulin.........................................2069
Human normal immunoglobulin...................................6.2-3757
Human normal immunoglobulin for intravenous
administration .................................................................6.3-4166
Human plasma for fractionation....................................6.2-3759
Human plasma (pooled and treated for virus
inactivation) .....................................................................6.3-4168
Human plasmine inhibitor, assay of (2.7.25.)..............6.2-3631
Human protein C, assay of (2.7.30.)..............................6.2-3631
Human protein S, assay of (2.7.31.) ..............................6.2-3632
Human prothrombin complex............................................... 2076
Human rabies immunoglobulin............................................2078
Human rubella immunoglobulin ..........................................2079
Human tetanus immunoglobulin .........................................2079
Human varicella immunoglobulin........................................2080
Human varicella immunoglobulin for intravenous
administration ........................................................................ 2081
Human von Willebrand factor............................................... 2081
Human von Willebrand factor, assay of (2.7.21.) ................ 237
Hyaluronidase ..........................................................................2082
Hydralazine hydrochloride ....................................................2083
Hydrochloric acid, concentrated...........................................2085
Hydrochloric acid, dilute ........................................................2085
Hydrochlorothiazide................................................................2086
Hydrocodone hydrogen tartrate 2.5-hydrate .....................2087
Hydrocortisone.........................................................................2089
Hydrocortisone acetate........................................................... 2091
Hydrocortisone hydrogen succinate....................................2092
Hydrogenated arachis oil ................................................6.2-3694
Hydrogenated castor oil ......................................................... 1432
Hydrogenated cottonseed oil .........................................6.2-3724
Hydrogenated soya-bean oil............................................6.2-3837
Hydrogenated vegetable oils, nickel in (2.4.31.)...................131
Hydrogenated wool fat............................................................3226
Hydrogen peroxide solution (30 per cent) .........................2094
Hydrogen peroxide solution (3 per cent)............................2094
Hydromorphone hydrochloride ............................................2095
Hydrophobic colloidal silica ..................................................2878
Hydrous wool fat......................................................................3227
Hydroxocobalamin acetate.....................................................2096
Hydroxocobalamin chloride...................................................2098
Hydroxocobalamin sulphate ..................................................2099
Hydroxycarbamide ................................................................... 2100
Hydroxyethylcellulose............................................................. 2102
Hydroxyethylmethylcellulose ................................................2390
Hydroxyethyl salicylate........................................................... 2101
Hydroxyl value (2.5.3.) .............................................................. 137
Hydroxypropylbetadex..................................................... 6.3-4170
Hydroxypropylcellulose .......................................................... 2105
Hydroxypropylmethylcellulose....................................... 6.3-4171
Hydroxypropylmethylcellulose phthalate.................... 6.3-4174
Hydroxyzine hydrochloride ................................................... 2106
Hymecromone........................................................................... 2107
4369
Index
Hyoscine..................................................................................... 2108
Hyoscine butylbromide........................................................... 2109
Hyoscine hydrobromide.......................................................... 2110
Hyoscyamine sulphate ............................................................ 2112
Hyoscyamus for homoeopathic preparations .................... 1079
Hypericum ..........................................................................6.2-3839
Hypericum for homoeopathic preparations....................... 1080
Hypromellose ..................................................................... 6.3-4171
Hypromellose phthalate .................................................. 6.3-4174
I Infectious bursal disease vaccine (live), avian ..................... 869
Ibuprofen ............................................................................6.1-3479
Iceland moss.............................................................................. 2121
ICH (5.8.)...................................................................................... 645
Ichthammol ........................................................................ 6.3-4177
Identification (2.3.) .................................................................... 103
Identification and control of residual solvents (2.4.24.).... 121
Identification of fatty oils by thin-layer chromatography
(2.3.2.) ........................................................................................ 106
Identification of phenothiazines by thin-layer
chromatography (2.3.3.) ......................................................... 107
Identification reactions of ions and functional groups
(2.3.1.)......................................................................................... 103
Idoxuridine ................................................................................ 2122
Ifosfamide .................................................................................. 2123
Imipenem ................................................................................... 2125
Imipramine hydrochloride ..............................................6.2-3769
Immunochemical methods (2.7.1.) ......................................... 209
Immunoglobulin for human use, anti-T lymphocyte,
animal.......................................................................................1203
Immunoglobulin for intravenous administration, human
anti-D ........................................................................................2059
Immunoglobulin for intravenous administration, human
hepatitis B ...............................................................................2069
Immunoglobulin for intravenous administration, human
normal ............................................................................... 6.3-4166
Immunoglobulin for intravenous administration, human
varicella.................................................................................... 2081
Immunoglobulin, human anti-D ....................................6.2-3757
Immunoglobulin, human anti-D, assay of (2.7.13.)............. 230
Immunoglobulin, human hepatitis A...................................2068
Immunoglobulin, human hepatitis B ..................................2069
Immunoglobulin, human measles ........................................2069
Immunoglobulin, human normal ..................................6.2-3757
Immunoglobulin, human rabies ...........................................2078
Immunoglobulin, human rubella .........................................2079
Immunoglobulin, human tetanus.........................................2079
Immunoglobulin, human varicella .......................................2080
Immunoglobulin, test for anticomplementary activity of
(2.6.17.)........................................................................................191
Immunoglobulin, test for Fc function of (2.7.9.)................. 227
Immunosera and vaccines, phenol in (2.5.15.) .................... 142
Immunosera and vaccines, veterinary, evaluation of efficacy
of (5.2.7.) ...........................................................................6.1-3335
Immunosera and vaccines, veterinary, evaluation of safety
(5.2.6.) ........................................................................................ 556
Immunosera and vaccines, veterinary, evaluation of the
safety of each batch (5.2.9.)................................................... 567
Immunosera for human use, animal...................................... 685
Immunosera for veterinary use............................................... 687
Implants ....................................................................................... 737
Impurities in substances for pharmaceutical use, control of
(5.10.).......................................................................................... 653
Indapamide................................................................................ 2127
Indian frankincense................................................................. 2128
Indicators, relationship between approximate pH and colour
(2.2.4.) .......................................................................................... 25
Indinavir sulphate.................................................................... 2130
Indium (111In) chloride solution .............................................. 994
4370 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Indium (111In) oxine solution ................................................... 995
Indium (111In) pentetate injection........................................... 996
Indometacin .............................................................................. 2132
Inductively coupled plasma-atomic emission spectrometry
(2.2.57.) ........................................................................................ 96
Inductively coupled plasma-mass spectrometry (2.2.58.).... 98
Infectious bovine rhinotracheitis vaccine (live)................... 924
Infectious bronchitis vaccine (inactivated), avian ............... 864
Infectious bronchitis vaccine (live), avian....................6.1-3371
Infectious bursal disease vaccine (inactivated), avian........ 867
Infectious chicken anaemia vaccine (live) ............................ 925
Infectious encephalomyelitis vaccine (live), avian .............. 871
Infectious laryngotracheitis vaccine (live), avian ................ 872
Influenza vaccine (split virion, inactivated) ......................... 801
Influenza vaccine (surface antigen, inactivated)................. 803
Influenza vaccine (surface antigen, inactivated, prepared in
cell cultures) ............................................................................. 804
Influenza vaccine (surface antigen, inactivated,
virosome) ................................................................................... 806
Influenza vaccine (whole virion, inactivated) ...................... 808
Influenza vaccine (whole virion, inactivated, prepared in cell
cultures)......................................................................................810
Infrared absorption spectrophotometry (2.2.24.) ................. 39
Infusions ...................................................................................... 736
Inhalation gas, krypton (81MKr) ............................................. 1000
Inhalation, preparations for..................................................... 739
Inhalation, preparations for : aerodynamic assessment of fine
particles (2.9.18.) ..................................................................... 287
Injectable insulin preparations ............................................. 2146
Injections ..................................................................................... 736
Injections, gels for...................................................................... 737
Injections or infusions, concentrates for .............................. 736
Injections or infusions, powders for ...................................... 736
Inositol, myo- ............................................................................2460
Inserts, ophthalmic.................................................................... 722
Insulin aspart ............................................................................ 2133
Insulin, bovine .......................................................................... 2135
Insulin, human.......................................................................... 2137
Insulin injection, biphasic ...................................................... 2140
Insulin injection, biphasic isophane .................................... 2140
Insulin injection, isophane......................................................2141
Insulin injection, soluble .........................................................2141
Insulin lispro ..............................................................................2141
Insulin, porcine......................................................................... 2144
Insulin preparations, injectable ............................................ 2146
Insulin zinc injectable suspension ....................................... 2148
Insulin zinc injectable suspension (amorphous) .............. 2149
Insulin zinc injectable suspension (crystalline) ................ 2149
Interferon alfa-2 concentrated solution .............................. 2150
Interferon beta-1a concentrated solution.................... 6.3-4177
Interferon gamma-1b concentrated solution ..................... 2153
Interferons, assay of (5.6.)........................................................ 627
International System (SI) units (1.) ........................................... 3
Intramammary preparations for veterinary use.................. 725
Intraruminal devices ................................................................. 725
Intrauterine capsules .......................................................6.3-3977
Intrauterine foams ............................................................6.3-3977
Intrauterine preparations for veterinary use ..............6.3-3977
Intrauterine solutions, suspensions..............................6.3-3977
Intrauterine sticks.............................................................6.3-3977
Intrauterine tablets...........................................................6.3-3977
Intrinsic dissolution (2.9.29.) .................................................. 309
In vivo assay of poliomyelitis vaccine (inactivated)
(2.7.20.) ...................................................................................... 235
Iobenguane (123I) injection....................................................... 997
Iobenguane (131I) injection for diagnostic use ..................... 998
Iobenguane (131I) injection for therapeutic use................... 999
EUROPEAN PHARMACOPOEIA 6.3
Iobenguane sulphate for radiopharmaceutical
preparations.....................................................................6.1-3381
Iodinated (125I) human albumin injection ............................. 993
Iodinated povidone..................................................................2734
Iodine.......................................................................................... 2156
Iodine value (2.5.4.) ................................................................... 137
Iohexol........................................................................................ 2157
Ionic concentration, potentiometric determination of using
ion-selective electrodes (2.2.36.)............................................. 58
Ions and functional groups, identification reactions of
(2.3.1.)......................................................................................... 103
Ion-selective electrodes, potentiometric determination of
ionic concentration (2.2.36.) ................................................... 58
Iopamidol................................................................................... 2160
Iopanoic acid............................................................................. 2162
Iotalamic acid............................................................................ 2163
Iotrolan....................................................................................... 2164
Ioxaglic acid .............................................................................. 2167
Ipecacuanha liquid extract, standardised ........................... 2168
Ipecacuanha, prepared.....................................................6.2-3770
Ipecacuanha root ..................................................................... 2170
Ipecacuanha tincture, standardised..................................... 2171
Ipratropium bromide........................................................6.2-3771
Iron (2.4.9.).................................................................................. 115
Iron for homoeopathic preparations ................................... 1081
Irrigation, preparations for ...................................................... 743
Isoconazole................................................................................ 2173
Isoconazole nitrate .................................................................. 2175
Isoelectric focusing (2.2.54.)...................................................... 84
Isoflurane....................................................................................2176
Isoleucine................................................................................... 2177
Isomalt........................................................................................ 2178
Isoniazid..................................................................................... 2180
Isophane insulin injection.......................................................2141
Isoprenaline hydrochloride.................................................... 2181
Isoprenaline sulphate.............................................................. 2182
Isopropyl alcohol...................................................................... 2182
Isopropyl myristate.................................................................. 2183
Isopropyl palmitate.................................................................. 2184
Isosorbide dinitrate, diluted ................................................. 2185
Isosorbide mononitrate, diluted .......................................... 2186
Isotretinoin ................................................................................ 2188
Isoxsuprine hydrochloride ..................................................... 2189
Ispaghula husk ......................................................................... 2191
Ispaghula seed .......................................................................... 2192
Isradipine ................................................................................... 2192
Itraconazole .............................................................................. 2194
Ivermectin.................................................................................. 2196
Ivy leaf ........................................................................................ 2198
J Limit tests, standard solutions for (4.1.2.)............................ 504
Javanese turmeric .................................................................... 3150
Java tea .......................................................................................2203
Josamycin...................................................................................2204
Josamycin propionate..............................................................2205
Juniper........................................................................................2206
Juniper oil ..................................................................................2207
K Liothyronine sodium........................................................6.1-3486
Kanamycin acid sulphate ....................................................... 2211
Kanamycin monosulphate...................................................... 2212
Kaolin, heavy......................................................................6.3-4183
Kelp ............................................................................................. 2213
Ketamine hydrochloride ......................................................... 2214
Ketobemidone hydrochloride................................................ 2215
Ketoconazole ............................................................................ 2216
Ketoprofen................................................................................. 2218
Ketorolac trometamol .............................................................2220
General Notices (1) apply to all monographs and other texts
Index
Ketotifen hydrogen fumarate ................................................ 2221
Knotgrass...................................................................................2223
Krypton (81mKr) inhalation gas.............................................. 1000
L
Labetalol hydrochloride .........................................................2227
Lactic acid..................................................................................2228
Lactic acid, (S)- .........................................................................2229
Lactitol monohydrate.......................................................6.3-4187
Lactobionic acid ....................................................................... 2231
Lactose, anhydrous...........................................................6.3-4188
Lactose monohydrate.......................................................6.3-4190
Lactulose............................................................................. 6.3-4191
Lactulose, liquid ................................................................6.3-4193
Lamivudine................................................................................2238
Lamotrigine........................................................................6.3-4195
Lansoprazole.............................................................................2240
Laser light diffraction, particle size analysis by (2.9.31.) ..311
Lauromacrogol 400 ..........................................................6.3-4196
Lauroyl macrogolglycerides ..................................................2242
Lavender flower........................................................................2243
Lavender oil...............................................................................2244
Lead in sugars (2.4.10.) ............................................................ 115
Leflunomide ..............................................................................2245
Lemon oil...................................................................................2246
Lemon verbena leaf ..........................................................6.3-4199
Leptospirosis vaccine (inactivated), bovine.......................... 876
Leptospirosis vaccine (inactivated), canine .......................... 888
Letrozole....................................................................................2249
Leucine.......................................................................................2250
Leuprorelin................................................................................ 2251
Levamisole for veterinary use ...............................................2253
Levamisole hydrochloride......................................................2254
Levocabastine hydrochloride ................................................2255
Levocarnitine ............................................................................2257
Levodopa....................................................................................2258
Levodropropizine ..............................................................6.3-4200
Levomenthol ............................................................................. 2261
Levomepromazine hydrochloride.........................................2262
Levomepromazine maleate ....................................................2263
Levomethadone hydrochloride .............................................2264
Levonorgestrel..........................................................................2266
Levothyroxine sodium ............................................................2267
Lidocaine ............................................................................6.1-3485
Lidocaine hydrochloride.........................................................2269
Light liquid paraffin ................................................................ 2612
Light magnesium carbonate...........................................6.3-4208
Light magnesium oxide ...................................................6.3-4209
Lime flower ...............................................................................2270
Limit tests (2.4.)...........................................................................111
Limit tests, standard solutions for (4.1.2.)...................6.3-3954
Lincomycin hydrochloride .....................................................2271
Lindane ......................................................................................2272
Linen thread, sterile, in distributor for veterinary use ... 1058
Linoleoyl macrogolglycerides................................................2273
Linseed .......................................................................................2273
Linseed oil, virgin .................................................................... 2274
Lipophilic solid dosage forms, dissolution test for
(2.9.42.) ...................................................................................... 332
Liquid chromatography (2.2.29.).............................................. 46
Liquid extracts...................................................................6.1-3343
Liquid glucose ...................................................................6.2-3752
Liquid glucose, spray-dried.............................................6.3-4154
Liquid lactulose .................................................................6.3-4193
Liquid maltitol ..........................................................................2332
Liquid paraffin .......................................................................... 2613
Liquid preparations for cutaneous application ................... 728
4371
Index
Liquid preparations for cutaneous application,
veterinary .................................................................................. 752
Liquid preparations for inhalation ......................................... 740
Liquid preparations for oral use............................................. 728
Liquids, clarity and degree of opalescence of (2.2.1.)...........21
Liquid sorbitol (crystallising) ................................................2942
Liquid sorbitol (non-crystallising) ........................................2943
Liquid sorbitol, partially dehydrated ............................6.3-4307
Liquorice dry extract for flavouring purposes ...........6.1-3488
Liquorice ethanolic liquid extract, standardised .......6.2-3775
Liquorice root ........................................................................... 2276
Lisinopril dihydrate .................................................................2277
Lithium carbonate ...................................................................2279
Lithium citrate..........................................................................2279
L-Methionine ([11C]methyl) injection.................................... 1001
Lobeline hydrochloride...........................................................2280
Lomustine.................................................................................. 2281
Loosestrife .................................................................................2283
Loperamide hydrochloride.....................................................2283
Loperamide oxide monohydrate...........................................2285
Loratadine .................................................................................2286
Lorazepam.................................................................................2288
Loss on drying (2.2.32.).............................................................. 53
Loss on drying of extracts (2.8.17.)........................................ 256
Lovage root................................................................................2290
Lovastatin .................................................................................. 2291
Low-molecular-mass heparins ............................................... 2041
Lozenges and pastilles.............................................................. 734
Lozenges, compressed .............................................................. 734
Lubricant, silicone oil (3.1.8.).................................................. 358
Lymecycline........................................................................6.1-3489
Lynestrenol.........................................................................6.3-4202
Lyophilisates, oral...................................................................... 748
Lysine acetate ...........................................................................2295
Lysine hydrochloride...............................................................2296
M for containers for non-injectable, aqueous solutions
Macrogol 15 hydroxystearate ................................................2305
Macrogol 20 glycerol monostearate ....................................2304
Macrogol 40 sorbitol heptaoleate .................................6.3-4207
Macrogol 6 glycerol caprylocaprate.....................................2302
Macrogol cetostearyl ether .................................................... 2301
Macrogolglycerol cocoates.....................................................2302
Macrogolglycerol hydroxystearate .......................................2303
Macrogolglycerol ricinoleate .................................................2304
Macrogol lauryl ether .............................................................2306
Macrogol oleate ........................................................................2307
Macrogol oleyl ether ...............................................................2308
Macrogols...................................................................................2308
Macrogol stearate..................................................................... 2311
Macrogol stearyl ether............................................................ 2312
Magaldrate..........................................................................6.3-4207
Magnesium (2.4.6.) .................................................................... 112
Magnesium acetate tetrahydrate .......................................... 2313
Magnesium aluminium silicate ......................................6.3-4024
Magnesium and alkaline-earth metals (2.4.7.) ..................... 112
Magnesium aspartate dihydrate ........................................... 2314
Magnesium carbonate, heavy .........................................6.2-3779
Magnesium carbonate, light ...........................................6.3-4208
Magnesium chloride 4.5-hydrate .......................................... 2317
Magnesium chloride hexahydrate ........................................ 2316
Magnesium citrate, anhydrous.............................................. 2318
Magnesium gluconate......................................................6.1-3495
Magnesium glycerophosphate............................................... 2318
Magnesium hydroxide............................................................. 2319
Magnesium lactate dihydrate ................................................2320
Magnesium oxide, heavy..................................................6.3-4209
Magnesium oxide, light....................................................6.3-4209
Magnesium peroxide ............................................................... 2321
4372 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Magnesium pidolate ................................................................2322
Magnesium stearate..........................................................6.3-4210
Magnesium sulphate heptahydrate......................................2325
Magnesium trisilicate ..............................................................2325
Maize oil, refined...............................................................6.2-3779
Maize starch .......................................................................6.3-4212
Malathion...................................................................................2327
Maleic acid.................................................................................2328
Malic acid ...................................................................................2329
Mallow flower............................................................................2330
Mallow leaf..........................................................................6.3-4212
Maltitol ................................................................................6.3-4213
Maltitol, liquid...........................................................................2332
Maltodextrin....................................................................... 6.3-4214
Mandarin oil ..............................................................................2333
Manganese gluconate ......................................................6.1-3495
Manganese glycerophosphate, hydrated.............................2334
Manganese sulphate monohydrate ......................................2335
Mannheimia vaccine (inactivated) for cattle ........................ 927
Mannheimia vaccine (inactivated) for sheep........................ 928
Mannitol ..............................................................................6.3-4215
Maprotiline hydrochloride .....................................................2337
Marbofloxacin for veterinary use ..................................6.1-3496
Marek’s disease vaccine (live).................................................. 930
Marshmallow leaf .....................................................................2338
Marshmallow root ....................................................................2339
Mass spectrometry (2.2.43.)....................................................... 68
Mass spectrometry, inductively coupled plasma- (2.2.58.).. 98
Mass uniformity of delivered doses from multidose containers
(2.9.27.) ...................................................................................... 309
Mass uniformity of single-dose preparations (2.9.5.) ......... 278
Mastic..........................................................................................2340
Materials based on non-plasticised poly(vinyl chloride) for
containers for dry dosage forms for oral administration
(3.1.11.)....................................................................................... 362
Materials based on non-plasticised poly(vinyl chloride)
(3.1.10.) ...................................................................................... 360
Materials based on plasticised poly(vinyl chloride) for
containers for aqueous solutions for intravenous infusion
(3.1.14.) ...................................................................................... 366
Materials based on plasticised poly(vinyl chloride) for
containers for human blood and blood components
(3.1.1.1.) ..................................................................................... 339
Materials based on plasticised poly(vinyl chloride) for
tubing used in sets for the transfusion of blood and blood
components (3.1.1.2.).............................................................. 342
Materials for containers for human blood and blood
components (3.1.1.) ................................................................. 339
Materials used for the manufacture of containers (3.1.) ... 339
Matricaria flower......................................................................2340
Matricaria liquid extract ..................................................6.2-3780
Matricaria oil.............................................................................2342
Meadowsweet ............................................................................2344
Measles immunoglobulin, human ........................................2069
Measles, mumps and rubella vaccine (live) .................6.1-3347
Measles vaccine (live) .......................................................6.1-3348
Measurement of consistency by penetrometry
(2.9.9.) ...............................................................................6.2-3641
Mebendazole .............................................................................2345
Meclozine hydrochloride........................................................2346
Medicated chewing gum (2.9.25.)........................................... 304
Medicated chewing gums ......................................................... 719
Medicated feeding stuffs for veterinary use, premixes for.. 739
Medicated foams......................................................................... 723
Medicated plasters ............................................................6.3-3980
Medicated tampons.................................................................... 751
Medicated vaginal tampons ..................................................... 752
Medicinal air.......................................................................6.3-4020
EUROPEAN PHARMACOPOEIA 6.3
Medicinal air, synthetic........................................................... 1121
Medium-chain triglycerides.................................................... 3122
Medroxyprogesterone acetate ...............................................2347
Mefenamic acid.................................................................. 6.3-4217
Mefloquine hydrochloride......................................................2350
Megestrol acetate .....................................................................2352
Meglumine.................................................................................2353
Melilot.........................................................................................2354
Melissa leaf ................................................................................2355
Meloxicam...........................................................................6.3-4218
Melting point - capillary method (2.2.14.)............................... 32
Melting point - instantaneous method (2.2.16.) .................... 33
Melting point - open capillary method (2.2.15.) .................... 32
Menadione .................................................................................2356
Meningococcal group C conjugate vaccine...........................814
Meningococcal polysaccharide vaccine..................................816
Menthol, racemic......................................................................2356
Mepivacaine hydrochloride....................................................2357
Meprobamate ............................................................................2359
Mepyramine maleate ...............................................................2360
Mercaptopurine ........................................................................ 2361
Mercuric chloride..................................................................... 2361
Mercury porosimetry, porosity and pore-size distribution of
solids by (2.9.32.) ............................................................6.2-3643
Mesalazine .................................................................................2362
Mesna..........................................................................................2364
Mesterolone...............................................................................2366
Mestranol ...................................................................................2367
Metacresol .................................................................................2368
Metamizole sodium .................................................................2369
Metformin hydrochloride .......................................................2370
Methacrylate copolymer, basic butylated ...........................1254
Methacrylic acid - ethyl acrylate copolymer (1:1) ......6.2-3781
Methacrylic acid - ethyl acrylate copolymer (1:1) dispersion
30 per cent .......................................................................6.3-4220
Methacrylic acid - methyl methacrylate copolymer (1:1) ..2373
Methacrylic acid - methyl methacrylate copolymer (1:2).. 2374
Methadone hydrochloride...................................................... 2374
Methanol .................................................................................... 2376
Methanol and 2-propanol, test for (2.9.11.) .......................... 282
Methaqualone ...........................................................................2377
Methenamine ............................................................................2378
Methionine ................................................................................2379
Methionine ([11C]methyl) injection, L-.................................. 1001
Methionine, DL-.........................................................................2380
Methods in pharmacognosy (2.8.) .......................................... 249
Methods of preparation of homoeopathic stocks and
potentisation....................................................................6.1-3385
Methods of preparation of sterile products (5.1.1.)............ 525
Methotrexate ......................................................................6.3-4220
Methylatropine bromide .........................................................2383
Methylatropine nitrate ............................................................2383
Methylcellulose..................................................................6.3-4223
Methyldopa................................................................................2386
Methylene blue .........................................................................2402
Methylene chloride ..................................................................2387
Methylergometrine maleate...................................................2388
Methylhydroxyethylcellulose.................................................2390
Methyl nicotinate .....................................................................2390
Methyl parahydroxybenzoate ................................................ 2391
Methylpentoses in polysaccharide vaccines (2.5.21.) ......... 143
Methylphenidate hydrochloride.....................................6.3-4224
Methylphenobarbital ...............................................................2392
Methylprednisolone.................................................................2393
Methylprednisolone acetate...................................................2395
Methylprednisolone hydrogen succinate ............................2397
Methylpyrrolidone, N- .............................................................2399
Methylrosanilinium chloride .................................................2400
Methyl salicylate....................................................................... 2401
General Notices (1) apply to all monographs and other texts
Index
Methyltestosterone ...........................................................6.3-4226
Methylthioninium chloride ....................................................2402
Metixene hydrochloride..........................................................2404
Metoclopramide.................................................................6.2-3783
Metoclopramide hydrochloride.............................................2407
Metolazone ................................................................................2407
Metoprolol succinate...............................................................2409
Metoprolol tartrate .................................................................. 2410
Metrifonate ................................................................................ 2412
Metronidazole ........................................................................... 2414
Metronidazole benzoate ......................................................... 2415
Mexiletine hydrochloride........................................................ 2416
Mianserin hydrochloride .................................................6.3-4227
Miconazole ................................................................................ 2418
Miconazole nitrate ...................................................................2420
Microbial enumeration tests (microbiological examination of
non-sterile products) (2.6.12.) ......................................6.3-3923
Microbiological assay of antibiotics (2.7.2.).................6.3-3935
Microbiological control of cellular products (2.6.27.)........ 205
Microbiological examination of non-sterile products :
microbial enumeration tests (2.6.12.).........................6.3-3923
Microbiological examination of non-sterile products : test for
specified micro-organisms (2.6.13.) ............................6.3-3927
Microbiological quality, alternative methods for control of
(5.1.6.)......................................................................................... 532
Microbiological quality of non-sterile pharmaceutical
preparations and substances for pharmaceutical use
(5.1.4.)................................................................................6.3-3957
Microbiology, general texts on (5.1.) ..................................... 525
Microcrystalline cellulose................................................6.3-4080
Microcrystalline cellulose and carmellose sodium ...........2422
Micro determination of water (2.5.32.).................................. 147
Microscopy, optical (2.9.37.) .................................................... 323
Midazolam .................................................................................2422
Milk thistle dry extract, refined and standardised............2426
Milk-thistle fruit........................................................................2425
Minimising the risk of transmitting animal spongiform
encephalopathy agents via human and veterinary medicinal
products (5.2.8.) ....................................................................... 558
Minocycline hydrochloride dihydrate..................................2427
Minoxidil ....................................................................................2429
Mint oil, partly dementholised ..............................................2430
Mirtazapine ............................................................................... 2431
Misoprostol................................................................................2433
Mitomycin ..................................................................................2434
Mitoxantrone hydrochloride..................................................2436
Modafinil ....................................................................................2437
Modified-release capsules......................................................... 718
Modified-release granules ........................................................ 724
Modified-release tablets ............................................................ 750
Molecular mass distribution in dextrans (2.2.39.) ................ 60
Molgramostim concentrated solution .................................2438
Molsidomine.......................................................................6.1-3499
Mometasone furoate................................................................ 2441
Monoclonal antibodies for human use.................................. 690
Morantel hydrogen tartrate for veterinary use .................2443
Morphine hydrochloride..................................................6.1-3501
Morphine sulphate............................................................6.2-3785
Moss, Iceland ............................................................................ 2121
Mother tinctures for homoeopathic preparations ............ 1072
Motherwort ...............................................................................2447
Mouthwashes .............................................................................. 733
Moxidectin for veterinary use ........................................6.3-4228
Moxifloxacin hydrochloride ............................................6.2-3786
Moxonidine................................................................................2453
Mucoadhesive preparations ..................................................... 735
Mullein flower...........................................................................2454
Multidose containers, uniformity of mass of delivered doses
(2.9.27.) ...................................................................................... 309
4373
Index
Mumps, measles and rubella vaccine (live) .................6.1-3347
Mumps vaccine (live) ........................................................6.1-3349
Mupirocin...................................................................................2454
Mupirocin calcium ...................................................................2456
Mycobacteria (2.6.2.) ................................................................. 159
Mycophenolate mofetil............................................................2458
Mycoplasma gallisepticum vaccine (inactivated)................. 932
Mycoplasmas (2.6.7.)......................................................... 6.1-3317
myo-Inositol ..............................................................................2460
Myrrh .......................................................................................... 2461
Myrrh tincture .......................................................................... 2461
Myxomatosis vaccine (live) for rabbits .................................. 933
N Nitrous oxide............................................................................. 2515
Nabumetone ..............................................................................2465
N-Acetyltryptophan........................................................... 6.3-4016
N-Acetyltyrosine ....................................................................... 1106
Nadolol .......................................................................................2466
Nadroparin calcium .................................................................2467
Naftidrofuryl hydrogen oxalate.............................................2470
Nalidixic acid.............................................................................2472
Naloxone hydrochloride dihydrate.......................................2473
Naltrexone hydrochloride....................................................... 2474
Nandrolone decanoate ............................................................ 2476
Naphazoline hydrochloride.............................................6.3-4235
Naphazoline nitrate .................................................................2479
Naproxen.............................................................................6.2-3791
Naproxen sodium ..............................................................6.1-3507
Narrow-leaved coneflower root .............................................2483
Nasal drops and liquid nasal sprays....................................... 731
Nasal powders............................................................................. 732
Nasal preparations ..................................................................... 730
Nasal preparations, semi-solid................................................. 732
Nasal sprays (liquid) and nasal drops .................................... 730
Nasal sticks.................................................................................. 732
Nasal washes ............................................................................... 732
Near-infrared spectrophotometry (2.2.40.)............................. 62
Neohesperidin-dihydrochalcone ...........................................2485
Neomycin sulphate ..................................................................2487
Neonatal piglet colibacillosis vaccine (inactivated) ............ 934
Neonatal ruminant colibacillosis vaccine (inactivated) ..... 936
Neostigmine bromide ..............................................................2489
Neostigmine metilsulfate........................................................2490
Neroli oil ....................................................................................2490
Netilmicin sulphate..................................................................2492
Nettle leaf...................................................................................2493
Neurovirulence test for poliomyelitis vaccine (oral)
(2.6.19.) ...................................................................................... 193
Neurovirulence test of live viral vaccines (2.6.18.) ............. 193
Nevirapine, anhydrous............................................................2495
Newcastle disease vaccine (inactivated) ................................ 937
Newcastle disease vaccine (live).............................................. 939
Nicergoline ................................................................................2496
Nickel in hydrogenated vegetable oils (2.4.31.)....................131
Nickel in polyols (2.4.15.)..........................................................116
Niclosamide, anhydrous .........................................................2497
Niclosamide monohydrate .....................................................2498
Nicotinamide .............................................................................2499
Nicotine ...............................................................................6.3-4236
Nicotine resinate ...............................................................6.3-4237
Nicotinic acid ............................................................................2502
Nifedipine...................................................................................2503
Niflumic acid ......................................................................6.1-3508
Nifuroxazide ....................................................................... 6.1-3510
Nikethamide ..............................................................................2505
Nilutamide ..........................................................................6.2-3792
Nimesulide.................................................................................2506
Nimodipine ................................................................................2507
Nitrazepam ................................................................................2508
4374 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Nitrendipine ..............................................................................2509
Nitric acid .................................................................................. 2510
Nitric oxide .........................................................................6.2-3794
Nitrofural ................................................................................... 2512
Nitrofurantoin........................................................................... 2513
Nitrogen ..............................................................................6.2-3795
Nitrogen determination by sulphuric acid digestion
(2.5.9.) ........................................................................................ 139
Nitrogen determination, primary aromatic amino
(2.5.8.) ........................................................................................ 139
Nitrogen, low-oxygen............................................................... 2514
Nitrogen monoxide and nitrogen dioxide in gases
(2.5.26.) ...................................................................................... 146
Nitrous oxide in gases (2.5.35.)............................................... 152
Nizatidine................................................................................... 2516
N-Methylpyrrolidone................................................................2399
NMR spectrometry (2.2.33.)............................................6.3-3909
N,N-Dimethylaniline (2.4.26.) .................................................. 127
Nomegestrol acetate................................................................ 2518
Nonoxinol 9............................................................................... 2519
Non-sterile products, microbiological examination of
(microbial enumeration tests) (2.6.12.)......................6.3-3923
Non-sterile products, microbiological examination of (test for
specified micro-organisms) (2.6.13.) ...........................6.3-3927
Noradrenaline hydrochloride ................................................2520
Noradrenaline tartrate............................................................ 2521
Norcholesterol injection, iodinated (131I) ............................ 1003
Norepinephrine hydrochloride..............................................2520
Norepinephrine tartrate ......................................................... 2521
Norethisterone..........................................................................2523
Norethisterone acetate ...........................................................2524
Norfloxacin.........................................................................6.2-3796
Norgestimate .............................................................................2526
Norgestrel ..................................................................................2527
Normal immunoglobulin for intravenous administration,
human ...............................................................................6.3-4166
Normal immunoglobulin, human ..................................6.2-3757
Nortriptyline hydrochloride...................................................2528
Noscapine ..................................................................................2529
Noscapine hydrochloride........................................................2530
Notoginseng root ..................................................................... 2531
Nuclear magnetic resonance spectrometry (2.2.33.) ..6.3-3909
Nucleated cell count and viability (2.7.29.) .......................... 243
Nucleic acid amplification techniques (2.6.21.)................... 195
Nucleic acids in polysaccharide vaccines (2.5.17.) .............. 142
Numeration of CD34/CD45+ cells in haematopoietic
products (2.7.23.) ..................................................................... 238
Nutmeg oil ..........................................................................6.2-3797
Nystatin ......................................................................................2534
O
O-Acetyl in polysaccharide vaccines (2.5.19.) ...................... 143
Oak bark ....................................................................................2539
Octoxinol 10 ..............................................................................2539
Octyldodecanol.........................................................................2540
Octyl gallate ..............................................................................2539
Odour (2.3.4.).............................................................................. 107
Odour and taste of essential oils (2.8.8.) .............................. 250
Ofloxacin.............................................................................6.2-3801
Oils, essential .............................................................................. 680
Oils, fatty, vegetable .................................................................. 712
Oils rich in omega-3 acids, composition of fatty acids in
(2.4.29.) .............................................................................6.2-3623
Oils rich in omega-3 acids, total cholesterol in (2.4.32.) ... 132
Ointments ...........................................................................6.3-3980
Oleic acid ...................................................................................2543
Oleoresins ...........................................................................6.1-3344
Oleoyl macrogolglycerides.....................................................2543
EUROPEAN PHARMACOPOEIA 6.3
Oleyl alcohol .............................................................................2544
Olive leaf .............................................................................6.3-4241
Olive oil, refined ................................................................6.2-3802
Olive oil, virgin ..................................................................6.2-3803
Olsalazine sodium....................................................................2548
Omega-3-acid ethyl esters 60..........................................6.3-4242
Omega-3-acid ethyl esters 90..........................................6.3-4244
Omega-3 acids, composition of fatty acids in oils rich in
(2.4.29.) .............................................................................6.2-3623
Omega-3 acids, fish oil rich in ............................................... 1893
Omega-3 acids, total cholesterol in oils rich in (2.4.32.) ... 132
Omega-3-acid triglycerides ..............................................6.3-4246
Omeprazole ...............................................................................2557
Omeprazole magnesium..................................................6.3-4248
Omeprazole sodium.................................................................2558
Ondansetron hydrochloride dihydrate ................................2560
Opalescence of liquids, clarity and degree of (2.2.1.)............21
Ophthalmic inserts .................................................................... 722
Opium dry extract, standardised ..........................................2562
Opium, prepared.......................................................................2563
Opium, raw ................................................................................2564
Opium tincture, standardised................................................2565
Optical microscopy (2.9.37.) .................................................... 323
Optical rotation (2.2.7.)............................................................... 26
Oral drops .................................................................................... 730
Oral lyophilisates........................................................................ 748
Oral powders............................................................................... 738
Oral solutions, emulsions and suspensions ......................... 729
Oral use, liquid preparations for............................................. 728
Orciprenaline sulphate.....................................................6.2-3804
Oregano......................................................................................2568
Organ preservation, solutions for.........................................2929
Oriental cashew for homoeopathic preparations.............. 1082
Orodispersible tablets ............................................................... 750
Oromucosal capsules ................................................................ 734
Oromucosal drops, oromucosal sprays and sublingual
sprays.......................................................................................... 733
Oromucosal preparations......................................................... 732
Oromucosal preparations, semi-solid..................................... 733
Oromucosal solutions and oromucosal suspensions ......... 733
Oromucosal sprays, oromucosal drops and sublingual
sprays.......................................................................................... 732
Oromucosal suspensions and oromucosal solutions ......... 732
Orphenadrine citrate...............................................................2569
Orphenadrine hydrochloride.................................................2570
Osmolality (2.2.35.)...................................................................... 57
Ouabain......................................................................................2571
Oxacillin sodium monohydrate ......................................6.2-3806
Oxaliplatin ..........................................................................6.3-4249
Oxazepam ..................................................................................2577
Oxeladin hydrogen citrate......................................................2578
Oxfendazole for veterinary use......................................6.2-3808
Oxidising substances (2.5.30.)................................................. 147
Oxitropium bromide ................................................................ 2581
Oxolinic acid..............................................................................2582
Oxprenolol hydrochloride ......................................................2583
Oxybuprocaine hydrochloride...............................................2584
Oxybutynin hydrochloride .....................................................2585
Oxycodone hydrochloride ......................................................2587
Oxygen (15O) .............................................................................. 1004
Oxygen.................................................................................6.2-3809
Oxygen-flask method (2.5.10.)................................................. 140
Oxygen in gases (2.5.27.)................................................. 6.3-3916
Oxymetazoline hydrochloride ........................................6.3-4252
Oxytetracycline dihydrate ......................................................2590
Oxytetracycline hydrochloride .............................................. 2591
Oxytocin .....................................................................................2593
Oxytocin concentrated solution............................................2594
General Notices (1) apply to all monographs and other texts
Index
P
Paclitaxel.............................................................................6.3-4257
Pale coneflower root ...............................................................2602
Palmitic acid..............................................................................2604
Pamidronate disodium pentahydrate ..................................2604
Pancreas powder ...............................................................6.3-4260
Pancuronium bromide ............................................................2608
Pansy, wild (flowering aerial parts) ..................................... 3217
Pantoprazole sodium sesquihydrate............................. 6.1-3518
Papaverine hydrochloride ......................................................2609
Paper chromatography (2.2.26.)............................................... 43
Paracetamol .............................................................................. 2611
Paraffin, hard ............................................................................ 2612
Paraffin, light liquid ................................................................ 2612
Paraffin, liquid .......................................................................... 2613
Paraffin, white soft ...........................................................6.2-3815
Paraffin, yellow soft..........................................................6.2-3816
Parainfluenza virus vaccine (live), bovine............................ 878
Parainfluenza virus vaccine (live), canine ............................ 890
Paraldehyde............................................................................... 2615
Paramyxovirus 1 (Newcastle disease) vaccine (inactivated),
avian ........................................................................................... 937
Parenteral preparations............................................................ 735
Parenteral preparations, test for extractable volume of
(2.9.17.)....................................................................................... 287
Parnaparin sodium .................................................................. 2616
Paroxetine hydrochloride, anhydrous ................................. 2616
Paroxetine hydrochloride hemihydrate............................... 2619
Particles, fine, aerodynamic assessment of in preparations
for inhalation (2.9.18.) ............................................................ 287
Particle size analysis by laser light diffraction (2.9.31.) .....311
Particle-size distribution estimation by analytical sieving
(2.9.38.) .............................................................................6.2-3649
Particulate contamination : sub-visible particles
(2.9.19.) ...................................................................................... 300
Particulate contamination : visible particles (2.9.20.)........ 302
Parvovirosis vaccine (inactivated), canine ............................ 891
Parvovirosis vaccine (inactivated), porcine .......................... 946
Parvovirosis vaccine (live), canine.......................................... 892
Passion flower .......................................................................... 2621
Passion flower dry extract .....................................................2622
Pastes...................................................................................6.3-3980
Pasteurella vaccine (inactivated) for sheep .......................... 941
Pastilles and lozenges............................................................... 734
Patches, transdermal................................................................. 737
Patches, transdermal, dissolution test for (2.9.4.) .............. 275
Pea starch ...........................................................................6.3-4263
Pefloxacin mesilate dihydrate ...............................................2623
Pelargonium root .....................................................................2625
Penbutolol sulphate ................................................................2625
Penetrometry, measurement of consistency by
(2.9.9.) ...............................................................................6.2-3641
Penicillamine.............................................................................2626
Pentaerythrityl tetranitrate, diluted ....................................2628
Pentamidine diisetionate........................................................2630
Pentazocine............................................................................... 2631
Pentazocine hydrochloride ....................................................2632
Pentazocine lactate .................................................................2632
Pentetate sodium calcium for radiopharmaceutical
preparations.....................................................................6.3-4001
Pentobarbital ............................................................................2633
Pentobarbital sodium..............................................................2634
Pentoxifylline ............................................................................2635
Pentoxyverine hydrogen citrate............................................2637
Peppermint leaf ........................................................................2638
Peppermint oil ..........................................................................2639
Pepsin powder ...................................................................6.3-4263
Peptide mapping (2.2.55.) .......................................................... 86
Peptides, synthetic, acetic acid in (2.5.34.)........................... 151
4375
Index
Pergolide mesilate.................................................................... 2641
Perindopril tert-butylamine....................................................2643
Peritoneal dialysis, solutions for...........................................2646
Peroxide value (2.5.5.)............................................................... 138
Perphenazine .....................................................................6.3-4265
Pertussis (acellular, component), diphtheria and tetanus
vaccine (adsorbed)................................................................... 767
Pertussis (acellular, component), diphtheria, tetanus and
haemophilus type b conjugate vaccine (adsorbed) .......... 771
Pertussis (acellular, component), diphtheria, tetanus and
hepatitis B (rDNA) vaccine (adsorbed) ............................... 774
Pertussis (acellular, component), diphtheria, tetanus and
poliomyelitis (inactivated) vaccine (adsorbed) .................. 775
Pertussis (acellular, component), diphtheria, tetanus and
poliomyelitis (inactivated) vaccine (adsorbed, reduced
antigen(s) content) .................................................................. 778
Pertussis (acellular, component), diphtheria, tetanus,
hepatitis B (rDNA), poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 780
Pertussis (acellular, component), diphtheria, tetanus,
poliomyelitis (inactivated) and haemophilus type b
conjugate vaccine (adsorbed).......................................6.3-3983
Pertussis, diphtheria, tetanus and poliomyelitis (inactivated)
vaccine (adsorbed)................................................................... 785
Pertussis, diphtheria, tetanus, poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 787
Pertussis vaccine (acellular), assay of (2.7.16.).................... 233
Pertussis vaccine (acellular, component, adsorbed) .......... 820
Pertussis vaccine (acellular, co-purified, adsorbed) ........... 822
Pertussis vaccine (adsorbed) ................................................... 824
Pertussis vaccine, assay of (2.7.7.) .......................................... 222
Peru balsam........................................................................ 6.2-3817
Pessaries....................................................................................... 751
Pessaries and suppositories, disintegration of (2.9.2.) ...... 265
Pesticide residues (2.8.13.)..............................................6.2-3637
Pethidine hydrochloride.........................................................2650
Pharmaceutical technical procedures (2.9.)......................... 263
Pharmacognosy, methods in (2.8.)......................................... 249
Pharmacopoeial harmonisation (5.8.) ................................... 645
Phenazone................................................................................. 2651
Pheniramine maleate ..............................................................2652
Phenobarbital ...........................................................................2653
Phenobarbital sodium.............................................................2654
Phenol .................................................................................6.3-4266
Phenol in immunosera and vaccines (2.5.15.)..................... 142
Phenolphthalein.......................................................................2656
Phenolsulfonphthalein ...........................................................2657
Phenothiazines, identification by thin-layer chromatography
(2.3.3.) ........................................................................................ 107
Phenoxyethanol........................................................................2657
Phenoxymethylpenicillin .................................................6.1-3520
Phenoxymethylpenicillin potassium.............................6.1-3521
Phentolamine mesilate ...........................................................2662
Phenylalanine ...........................................................................2663
Phenylbutazone .......................................................................2664
Phenylephrine...........................................................................2665
Phenylephrine hydrochloride................................................2667
Phenylmercuric acetate ..........................................................2668
Phenylmercuric borate ...........................................................2669
Phenylmercuric nitrate ...........................................................2669
Phenylpropanolamine hydrochloride ..................................2670
Phenytoin...................................................................................2671
Phenytoin sodium....................................................................2672
Phloroglucinol, anhydrous ....................................................2672
Phloroglucinol dihydrate .......................................................2673
Pholcodine..........................................................................6.3-4266
Phosphates (2.4.11.) ...................................................................116
Phosphoric acid, concentrated .............................................2675
Phosphoric acid, dilute........................................................... 2676
4376 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Phosphorus in polysaccharide vaccines (2.5.18.) ............... 142
pH, potentiometric determination of (2.2.3.) ......................... 24
Phthalylsulfathiazole .............................................................. 2676
Physical and physicochemical methods (2.2.) ........................21
Physostigmine salicylate.........................................................2677
Physostigmine sulphate..........................................................2678
Phytomenadione ......................................................................2679
Phytosterol ................................................................................2680
Picotamide monohydrate .......................................................2682
Pilocarpine hydrochloride...............................................6.3-4268
Pilocarpine nitrate ............................................................6.3-4269
Pimobendan ..............................................................................2685
Pimozide ....................................................................................2686
Pindolol......................................................................................2688
Pine (dwarf) oil ......................................................................... 1766
Pine sylvestris oil .....................................................................2689
Pinus pinaster type turpentine oil ....................................... 3151
Pipemidic acid trihydrate .......................................................2690
Piperacillin ................................................................................ 2691
Piperacillin sodium..................................................................2692
Piperazine adipate ...................................................................2694
Piperazine citrate.....................................................................2695
Piperazine hydrate...................................................................2696
Piracetam...................................................................................2697
Pirenzepine dihydrochloride monohydrate .......................2698
Piretanide ..................................................................................2699
Piroxicam ...................................................................................2700
Pivampicillin..............................................................................2702
Pivmecillinam hydrochloride.................................................2704
Plasma for fractionation, human...................................6.2-3759
Plasma (pooled and treated for virus inactivation),
human ...............................................................................6.3-4168
Plasmid vectors for human use............................................... 674
Plasmid vectors for human use, bacterial cells used for the
manufacture of ......................................................................... 676
Plasmin inhibitor, assay of human (2.7.25.)................6.2-3631
Plasters, medicated...........................................................6.3-3979
Plastic additives (3.1.13.).................................................6.2-3655
Plastic containers and closures for pharmaceutical use
(3.2.2.) ........................................................................................ 378
Plastic containers for aqueous solutions for infusion
(3.2.2.1.) ..................................................................................... 379
Plastic containers for human blood and blood components,
sterile (3.2.3.) ............................................................................ 379
Plastic syringes, single-use, sterile (3.2.8.) ........................... 384
Pneumococcal polysaccharide conjugate vaccine
(adsorbed).................................................................................. 825
Pneumococcal polysaccharide vaccine ................................. 827
Poliomyelitis (inactivated), diphtheria and tetanus vaccine
(adsorbed, reduced antigen(s) content) .............................. 770
Poliomyelitis (inactivated), diphtheria, tetanus and pertussis
(acellular, component) vaccine (adsorbed) ........................ 775
Poliomyelitis (inactivated), diphtheria, tetanus and pertussis
(acellular, component) vaccine (adsorbed, reduced
antigen(s) content) .................................................................. 778
Poliomyelitis (inactivated), diphtheria, tetanus and pertussis
vaccine (adsorbed)................................................................... 785
Poliomyelitis (inactivated), diphtheria, tetanus, pertussis
(acellular, component) and haemophilus type b conjugate
vaccine (adsorbed)..........................................................6.3-3983
Poliomyelitis (inactivated), diphtheria, tetanus, pertussis
(acellular, component), hepatitis B (rDNA) and haemophilus
type b conjugate vaccine (adsorbed) ................................... 780
Poliomyelitis (inactivated), diphtheria, tetanus, pertussis and
haemophilus type b conjugate vaccine (adsorbed) .......... 787
Poliomyelitis vaccine (inactivated) ................................6.3-3988
Poliomyelitis vaccine (inactivated), in vivo assay of
(2.7.20.) ...................................................................................... 235
Poliomyelitis vaccine (oral) .............................................6.1-3351
EUROPEAN PHARMACOPOEIA 6.3
Poliomyelitis vaccine (oral), test for neurovirulence
(2.6.19.) ...................................................................................... 193
Poloxamers ................................................................................2705
Polyacrylate dispersion 30 per cent..............................6.3-4270
Polyamide 6/6 suture, sterile, in distributor for veterinary
use ............................................................................................ 1059
Polyamide 6 suture, sterile, in distributor for veterinary use
................................................................................................... 1058
Polyethyleneglycols .................................................................2308
Polyethylene terephthalate for containers for preparations
not for parenteral use (3.1.15.) ............................................. 369
Poly(ethylene terephthalate) suture, sterile, in distributor for
veterinary use ........................................................................ 1059
Poly(ethylene - vinyl acetate) for containers and tubing for
total parenteral nutrition preparations (3.1.7.) ................. 356
Polyethylene with additives for containers for parenteral
preparations and for ophthalmic preparations (3.1.5.) ... 349
Polyethylene without additives for containers for parenteral
preparations and for ophthalmic preparations (3.1.4.) ... 348
Polymorphism (5.9.) .................................................................. 649
Polymyxin B sulphate .............................................................2707
Polyolefines (3.1.3.) ................................................................... 344
Polyoxyl castor oil....................................................................2304
Polyoxyl hydrogenated castor oil .........................................2303
Polypropylene for containers and closures for parenteral
preparations and ophthalmic preparations (3.1.6.).......... 352
Polysaccharide vaccines, hexosamines in (2.5.20.)............. 143
Polysaccharide vaccines, methylpentoses in (2.5.21.)........ 143
Polysaccharide vaccines, nucleic acids in (2.5.17.) ............. 142
Polysaccharide vaccines, O-acetyl in (2.5.19.)...................... 143
Polysaccharide vaccines, phosphorus in (2.5.18.)............... 142
Polysaccharide vaccines, protein in (2.5.16.) ....................... 142
Polysaccharide vaccines, ribose in (2.5.31.) ......................... 147
Polysaccharide vaccines, sialic acid in (2.5.23.) .................. 144
Polysaccharide vaccines, uronic acids in (2.5.22.).............. 144
Polysorbate 20 ...................................................................6.3-4271
Polysorbate 40 ...................................................................6.3-4272
Polysorbate 60 ...................................................................6.3-4273
Polysorbate 80 ...................................................................6.3-4274
Poly(vinyl acetate).................................................................... 2712
Poly(vinyl acetate) dispersion 30 per cent ..................6.3-4275
Poly(vinyl alcohol) ................................................................... 2715
Poly(vinyl chloride), non-plasticised, materials based on for
containers for dry dosage forms for oral administration
(3.1.11.)....................................................................................... 362
Poly(vinyl chloride), non-plasticised, materials based on
for containers for non-injectable aqueous solutions
(3.1.10.) ...................................................................................... 360
Poly(vinyl chloride), plasticised, empty sterile containers of
for human blood and blood components (3.2.4.) ............. 381
Poly(vinyl chloride), plasticised, materials based on for
containers for aqueous solutions for intravenous infusion
(3.1.14.) ...................................................................................... 366
Poly(vinyl chloride), plasticised, materials based on for
containers for human blood and blood components
(3.1.1.1.) ..................................................................................... 339
Poly(vinyl chloride), plasticised, materials based on for
tubing used in sets for the transfusion of blood and blood
components (3.1.1.2.).............................................................. 342
Poly(vinyl chloride), plasticised, sterile containers of
for human blood containing anticoagulant solution
(3.2.5.) ........................................................................................ 382
Poppy petals, red...................................................................... 2811
Porcine actinobacillosis vaccine (inactivated) ..................... 943
Porcine influenza vaccine (inactivated) ................................ 944
Porcine insulin.......................................................................... 2144
Porcine parvovirosis vaccine (inactivated) ........................... 946
Porcine progressive atrophic rhinitis vaccine
(inactivated) .....................................................................6.1-3373
General Notices (1) apply to all monographs and other texts
Index
Pore-size distribution of solids by mercury porosimetry,
porosity and (2.9.32.) .....................................................6.2-3643
Porosimetry, mercury, porosity and pore-size distribution of
solids by (2.9.32.) ............................................................6.2-3643
Porosity and pore-size distribution of solids by mercury
porosimetry (2.9.32.)......................................................6.2-3643
Porosity of sintered-glass filters (2.1.2.)...................................15
Potassium (2.4.12.) .....................................................................116
Potassium acetate .................................................................... 2716
Potassium bromide .................................................................. 2716
Potassium carbonate............................................................... 2717
Potassium chloride ...........................................................6.2-3819
Potassium citrate ..............................................................6.3-4276
Potassium clavulanate ............................................................ 2719
Potassium clavulanate, diluted ............................................. 2721
Potassium dihydrogen phosphate .................................6.3-4277
Potassium hydrogen aspartate hemihydrate .....................2723
Potassium hydrogen carbonate ............................................2724
Potassium hydrogen tartrate.................................................2725
Potassium hydroxide ...............................................................2726
Potassium iodide......................................................................2726
Potassium metabisulphite......................................................2727
Potassium nitrate .....................................................................2728
Potassium perchlorate ............................................................2728
Potassium permanganate.......................................................2729
Potassium sodium tartrate tetrahydrate.............................2729
Potassium sorbate....................................................................2730
Potassium sulphate ................................................................. 2731
Potato starch......................................................................6.3-4277
Potentiometric determination of ionic concentration using
ion-selective electrodes (2.2.36.)............................................. 58
Potentiometric determination of pH (2.2.3.).......................... 24
Potentiometric titration (2.2.20.).............................................. 35
Potentisation, methods of preparation of homoeopathic
stocks and.........................................................................6.1-3385
Poultices..............................................................................6.3-3980
Pour-on preparations ................................................................ 753
Povidone .............................................................................6.1-3523
Povidone, iodinated.................................................................2734
Powdered cellulose ...........................................................6.3-4084
Powder fineness (2.9.35.) ................................................6.2-3648
Powder flow (2.9.36.) ................................................................ 320
Powders and granules for oral solutions and
suspensions............................................................................... 729
Powders and granules for syrups ........................................... 730
Powders and tablets for rectal solutions and suspensions.. 746
Powders, bulk density and tapped density of
(2.9.34.) .............................................................................6.2-3646
Powders, ear................................................................................ 720
Powders, effervescent................................................................ 739
Powders for cutaneous application...............................6.3-3978
Powders for eye drops and powders for eye lotions........... 722
Powders for inhalation.............................................................. 742
Powders for injections or infusions ....................................... 736
Powders for oral drops.............................................................. 730
Powders, nasal ............................................................................ 732
Powders, oral .............................................................................. 738
Poxvirus vectors for human use ............................................. 672
Pravastatin sodium ...........................................................6.3-4278
Prazepam ...................................................................................2736
Praziquantel..............................................................................2737
Prazosin hydrochloride ..........................................................2738
Prednicarbate............................................................................ 2740
Prednisolone ............................................................................. 2741
Prednisolone acetate............................................................... 2742
Prednisolone pivalate.............................................................. 2744
Prednisolone sodium phosphate .......................................... 2745
Prednisone................................................................................. 2746
Prekallikrein activator (2.6.15.) .............................................. 189
4377
Index
Premixes for medicated feeding stuffs for veterinary use.. 739
Preparations for inhalation...................................................... 739
Preparations for inhalation : aerodynamic assessment of fine
particles (2.9.18.) ..................................................................... 287
Preparations for irrigation....................................................... 743
Pressurised pharmaceutical preparations ............................ 744
Prilocaine................................................................................... 2748
Prilocaine hydrochloride........................................................2750
Primaquine diphosphate ........................................................ 2751
Primary aromatic amino-nitrogen, determination of
(2.5.8.) ........................................................................................ 139
Primary standards for volumetric solutions (4.2.1.)............514
Primidone ..................................................................................2752
Primula root ..............................................................................2753
Probenecid.................................................................................2754
Procainamide hydrochloride .................................................2755
Procaine benzylpenicillin .......................................................1287
Procaine hydrochloride ..........................................................2756
Prochlorperazine maleate ......................................................2756
Products of fermentation ......................................................... 693
Products of recombinant DNA technology .......................... 701
Products with risk of transmitting agents of animal
spongiform encephalopathies............................................... 694
Progenitor cells, human haematopoietic, colony-forming cell
assay for (2.7.28.) ..................................................................... 242
Progesterone.............................................................................2757
Progressive atrophic rhinitis vaccine (inactivated),
porcine ..............................................................................6.1-3373
Proguanil hydrochloride ........................................................2758
Proline ........................................................................................ 2760
Promazine hydrochloride....................................................... 2761
Promethazine hydrochloride................................................. 2761
Propacetamol hydrochloride ................................................. 2763
Propafenone hydrochloride ................................................... 2764
Propanol..................................................................................... 2766
Propanol and methanol, 2-, test for (2.9.11.) ....................... 282
Propantheline bromide........................................................... 2767
Propofol...................................................................................... 2768
Propranolol hydrochloride.....................................................2770
Propylene glycol.......................................................................2773
Propylene glycol dicaprylocaprate....................................... 2774
Propylene glycol dilaurate ..................................................... 2774
Propylene glycol monolaurate ..............................................2775
Propylene glycol monopalmitostearate............................... 2776
Propylene glycol monostearate............................................. 2776
Propyl gallate............................................................................2771
Propyl parahydroxybenzoate.................................................2772
Propylthiouracil .......................................................................2777
Propyphenazone ......................................................................2778
Protamine hydrochloride .......................................................2779
Protamine sulphate .................................................................2780
Protein C, human, assay of (2.7.30.) .............................6.2-3631
Protein in polysaccharide vaccines (2.5.16.) ........................ 142
Protein S, human, assay of (2.7.31.)..............................6.2-3632
Protein, total (2.5.33.) ............................................................... 148
Prothrombin complex, human .............................................. 2076
Protirelin.................................................................................... 2781
Proxyphylline ............................................................................2783
Pseudoephedrine hydrochloride ...................................6.2-3820
Psyllium seed ............................................................................2785
Purified water ....................................................................6.3-4344
Purified water, highly ......................................................6.3-4342
Purple coneflower herb..........................................................2785
Purple coneflower root...........................................................2787
Pycnometric density of solids, gas (2.9.23.) ................6.2-3642
Pygeum africanum bark .........................................................2789
Pyrantel embonate...................................................................2790
Pyrazinamide ............................................................................ 2791
Pyridostigmine bromide .........................................................2792
4378 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Pyridoxine hydrochloride.......................................................2793
Pyrimethamine .........................................................................2794
Pyrogens (2.6.8.)......................................................................... 164
Pyrrolidone................................................................................2794
Q
Quality of non-sterile pharmaceutical preparations and
substances for pharmaceutical use, microbiological
(5.1.4.)................................................................................6.3-3957
Quantified hawthorn leaf and flower liquid extract.........2037
Quinidine sulphate ..................................................................2799
Quinine hydrochloride............................................................2800
Quinine sulphate......................................................................2802
R
Rabbit haemorrhagic disease vaccine (inactivated) ........... 949
Rabies immunoglobulin, human ..........................................2078
Rabies vaccine for human use prepared in cell
cultures .............................................................................6.1-3355
Rabies vaccine (inactivated) for veterinary use..........6.1-3375
Rabies vaccine (live, oral) for foxes ........................................ 952
Racecadotril .......................................................................6.3-4283
Racemic camphor..................................................................... 1401
Racemic ephedrine hydrochloride ....................................... 1792
Racemic menthol .....................................................................2356
Raclopride ([11C]methoxy) injection..................................... 1005
Radionuclides, table of physical characteristics (5.7.) ....... 633
Radiopharmaceutical preparations ........................................ 695
Radiopharmaceutical preparations, iobenguane sulphate
for .......................................................................................6.1-3381
Radiopharmaceutical preparations, pentetate sodium
calcium for........................................................................6.3-4001
Raman spectrometry (2.2.48.) ................................................... 82
Ramipril...............................................................................6.2-3826
Ramon assay, flocculation value (Lf) of diphtheria and
tetanus toxins and toxoids (2.7.27.) ..................................... 241
Ranitidine hydrochloride........................................................2809
Rapeseed oil, refined........................................................6.2-3829
Reagents (4.) ............................................................................... 391
Reagents (4.1.1.)......................................................................... 391
Reagents (4.1.1.)................................................................6.1-3331
Reagents (4.1.1.)................................................................6.2-3661
Reagents (4.1.1.)................................................................6.3-3953
Reagents, standard solutions, buffer solutions (4.1.)......... 391
Recombinant DNA technology, products of......................... 701
Rectal capsules ........................................................................... 745
Rectal foams................................................................................ 746
Rectal preparations.................................................................... 744
Rectal preparations, semi-solid ............................................... 746
Rectal solutions and suspensions, powders and tablets
for ................................................................................................ 744
Rectal solutions, emulsions and suspensions...................... 745
Rectal tampons........................................................................... 746
Red poppy petals...................................................................... 2811
Reference standards (5.12.) ..................................................... 663
Refractive index (2.2.6.) .............................................................. 26
Relationship between reaction of solution, approximate pH
and colour of certain indicators (2.2.4.) ............................... 25
Relative density (2.2.5.)............................................................... 25
Repaglinide................................................................................ 2812
Reserpine ................................................................................... 2814
Residual solvents (5.4.) ............................................................. 603
Residual solvents, identification and control (2.4.24.) ...... 121
Residue on evaporation of essential oils (2.8.9.)................. 250
Resistance to crushing of tablets (2.9.8.) ............................. 279
Resorcinol.................................................................................. 2815
Restharrow root ....................................................................... 2815
Rhatany root ............................................................................. 2816
EUROPEAN PHARMACOPOEIA 6.3
Rhatany tincture ...................................................................... 2817
Rhinotracheitis vaccine (inactivated), viral, feline ...............916
Rhinotracheitis vaccine (live), viral, feline.............................917
Rhubarb ..................................................................................... 2817
Ribavirin..................................................................................... 2818
Riboflavin...................................................................................2820
Riboflavin sodium phosphate................................................ 2821
Ribose in polysaccharide vaccines (2.5.31.) ......................... 147
Ribwort plantain ......................................................................2823
Rice starch..........................................................................6.3-4284
Rifabutin ....................................................................................2825
Rifampicin..................................................................................2826
Rifamycin sodium.....................................................................2827
Rilmenidine dihydrogen phosphate.....................................2829
Risperidone ...............................................................................2830
Ritonavir ....................................................................................2832
Rocuronium bromide ..............................................................2835
Roman chamomile flower ...................................................... 1487
Ropivacaine hydrochloride monohydrate...........................2837
Roselle .................................................................................6.1-3529
Rosemary leaf ...........................................................................2839
Rosemary oil .............................................................................2840
Rotating viscometer method - viscosity (2.2.10.) .................. 28
Rotation, optical (2.2.7.) ............................................................. 26
Roxithromycin...........................................................................2842
RRR-α-Tocopherol ...................................................................3088
RRR-α-Tocopheryl acetate.....................................................3090
RRR-α-Tocopheryl hydrogen succinate ..............................3095
Rubber closures for containers for aqueous parenteral
preparations, for powders and for freeze-dried powders
(3.2.9.) ........................................................................................ 386
Rubella immunoglobulin, human.........................................2079
Rubella, measles and mumps vaccine (live) ................6.1-3347
Rubella vaccine (live) .......................................................6.1-3358
Rutoside trihydrate..................................................................2844
S Silicone oil used as a lubricant (3.1.8.) ................................. 358
Saccharin ...................................................................................2849
Saccharin sodium ....................................................................2850
Safety, viral (5.1.7.) .................................................................... 543
Safflower flower ....................................................................... 2851
Safflower oil, refined...............................................................2852
Saffron for homoeopathic preparations.............................. 1084
Sage leaf (salvia officinalis)....................................................2853
Sage leaf, three-lobed..............................................................2854
Sage oil, Spanish...............................................................6.2-3838
Sage tincture.............................................................................2854
Salbutamol ................................................................................2855
Salbutamol sulphate ...............................................................2857
Salicylic acid..............................................................................2859
Salmeterol xinafoate................................................................2860
Salmonella Enteritidis vaccine (inactivated) for chickens.. 953
Salmonella Typhimurium vaccine (inactivated) for
chickens ..................................................................................... 954
Salmon oil, farmed...................................................................2862
Sanguisorba root...............................................................6.1-3533
Saponification value (2.5.6.).................................................... 139
Saquinavir mesilate ..........................................................6.3-4287
Saw palmetto fruit ...................................................................2864
Schisandra fruit.................................................................6.3-4288
Scopolamine.............................................................................. 2108
Scopolamine butylbromide .................................................... 2109
Scopolamine hydrobromide................................................... 2110
Selamectin for veterinary use ........................................6.1-3534
Selegiline hydrochloride ........................................................2866
Selenium disulphide................................................................2867
Semi-micro determination of water (2.5.12.) ........................141
Semi-solid ear preparations ..................................................... 720
Semi-solid eye preparations ..................................................... 722
General Notices (1) apply to all monographs and other texts
Index
Semi-solid intrauterine preparations ............................6.3-3977
Semi-solid nasal preparations.................................................. 732
Semi-solid oromucosal preparations...................................... 733
Semi-solid preparations for cutaneous application ...6.3-3979
Semi-solid rectal preparations................................................. 746
Semi-solid vaginal preparations.............................................. 752
Senega root ...............................................................................2867
Senna leaf ..................................................................................2868
Senna leaf dry extract, standardised ............................6.3-4289
Senna pods, Alexandrian........................................................2870
Senna pods, Tinnevelly...........................................................2871
Separation techniques, chromatographic (2.2.46.) .............. 72
Serine..........................................................................................2872
Sertaconazole nitrate.......................................................6.1-3535
Sertraline hydrochloride .................................................6.3-4290
Sesame oil, refined ...........................................................6.3-4292
Sets for the transfusion of blood and blood components
(3.2.6.) ........................................................................................ 383
Sevoflurane ........................................................................6.3-4294
Shampoos .................................................................................... 728
Shellac ................................................................................6.2-3833
Shingles (herpes zoster) vaccine (live).........................6.3-3991
Sialic acid in polysaccharide vaccines (2.5.23.)................... 144
Siam benzoin tincture.............................................................1278
Sieves (2.1.4.) .................................................................................16
Sieve test (2.9.12.)...................................................................... 283
Sieving, analytical, particle-size distribution estimation by
(2.9.38.) .............................................................................6.2-3649
SI (International System) units (1.) ........................................... 3
Silica, colloidal anhydrous .....................................................2877
Silica, colloidal hydrated ........................................................2877
Silica, dental type.....................................................................2878
Silica, hydrophobic colloidal .................................................2878
Silicate, aluminium magnesium.....................................6.3-4024
Silicate, aluminium sodium ............................................6.3-4026
Silicone elastomer for closures and tubing (3.1.9.)............ 358
Silk suture, sterile, braided, in distributor for veterinary use
................................................................................................... 1059
Silver, colloidal, for external use ..........................................2879
Silver nitrate .............................................................................2880
Simeticone.................................................................................2880
Simvastatin................................................................................ 2881
Single-dose preparations, uniformity of content (2.9.6.)... 278
Single-dose preparations, uniformity of mass (2.9.5.)........ 278
Sintered-glass filters (2.1.2.) .......................................................15
Size-exclusion chromatography (2.2.30.)................................ 47
(S)-Lactic acid............................................................................2229
Smallpox vaccine (live) ....................................................6.1-3359
Sodium acetate ([1-11C]) injection......................................... 1006
Sodium acetate trihydrate .....................................................2883
Sodium alendronate .........................................................6.3-4296
Sodium alginate ................................................................6.3-4297
Sodium aluminium silicate .............................................6.3-4026
Sodium amidotrizoate.............................................................2886
Sodium aminosalicylate dihydrate .......................................2887
Sodium ascorbate .............................................................6.3-4298
Sodium aurothiomalate..........................................................2889
Sodium benzoate .....................................................................2890
Sodium bromide....................................................................... 2891
Sodium calcium edetate .........................................................2892
Sodium calcium pentetate for radiopharmaceutical
preparations.....................................................................6.3-4001
Sodium caprylate .....................................................................2893
Sodium carbonate, anhydrous ..............................................2894
Sodium carbonate decahydrate ............................................2894
Sodium carbonate monohydrate ..........................................2895
Sodium carboxymethylcellulose........................................... 1423
Sodium carboxymethylcellulose, cross-linked ............ 6.3-4117
4379
Index
Sodium carboxymethylcellulose, low-substituted............. 1424
Sodium cetostearyl sulphate .................................................2895
Sodium chloride.......................................................................2897
Sodium chromate (51Cr) sterile solution ............................. 1007
Sodium citrate ..........................................................................2898
Sodium cromoglicate ..............................................................2899
Sodium cyclamate....................................................................2900
Sodium dihydrogen phosphate dihydrate .......................... 2901
Sodium fluoride .......................................................................2902
Sodium fluoride (18F) injection ............................................. 1008
Sodium fusidate .......................................................................2902
Sodium glycerophosphate, hydrated ............................6.3-4299
Sodium hyaluronate .........................................................6.3-4300
Sodium hydrogen carbonate .................................................2906
Sodium hydroxide....................................................................2907
Sodium iodide...........................................................................2907
Sodium iodide (123I) injection ................................................ 1009
Sodium iodide (123I) solution for radiolabelling ................ 1010
Sodium iodide (131I) capsules for diagnostic use................1011
Sodium iodide (131I) capsules for therapeutic use ............ 1012
Sodium iodide (131I) solution ................................................. 1013
Sodium iodide (131I) solution for radiolabelling .................1014
Sodium iodohippurate (123I) injection ..................................1014
Sodium iodohippurate (131I) injection ................................. 1015
Sodium lactate solution..........................................................2908
Sodium laurilsulfate ................................................................ 2910
Sodium metabisulphite........................................................... 2911
Sodium methyl parahydroxybenzoate................................. 2911
Sodium molybdate (99Mo) solution (fission) ...................... 1016
Sodium molybdate dihydrate .........................................6.3-4302
Sodium nitrite........................................................................... 2913
Sodium nitroprusside ............................................................. 2913
Sodium perborate, hydrated.................................................. 2914
Sodium pertechnetate (99mTc) injection (fission) .............. 1018
Sodium pertechnetate (99mTc) injection (non-fission) ...... 1020
Sodium phenylbutyrate ...................................................6.1-3539
Sodium phosphate (32P) injection ........................................ 1020
Sodium picosulfate ................................................................. 2915
Sodium polystyrene sulphonate ....................................6.3-4303
Sodium propionate .................................................................. 2917
Sodium propyl parahydroxybenzoate.................................. 2918
Sodium salicylate ..................................................................... 2919
Sodium selenite pentahydrate .............................................. 2919
Sodium (S)-lactate solution ...................................................2909
Sodium starch glycolate (type A) .........................................2920
Sodium starch glycolate (type B) ......................................... 2921
Sodium starch glycolate (type C) .........................................2922
Sodium stearate ................................................................6.3-4304
Sodium stearyl fumarate ........................................................2924
Sodium sulphate, anhydrous.................................................2924
Sodium sulphate decahydrate...............................................2925
Sodium sulphite, anhydrous..................................................2926
Sodium sulphite heptahydrate..............................................2926
Sodium thiosulphate...............................................................2927
Sodium valproate .....................................................................2927
Soft capsules ............................................................................... 718
Softening time determination of lipophilic suppositories
(2.9.22.) ...................................................................................... 302
Soft extracts .......................................................................6.1-3344
Solid dosage forms, dissolution test for (2.9.3.).................. 266
Solids by mercury porosimetry, porosity and pore-size
distribution of (2.9.32.)..................................................6.2-3643
Solids, density of (2.2.42.)...............................................6.3-3912
Solids, gas pycnometric density of (2.9.23.)................6.2-3642
Solubility in alcohol of essential oils (2.8.10.) ..................... 250
Soluble tablets............................................................................ 750
Solutions, emulsions and suspensions, oral ........................ 729
Solutions for haemodialysis...................................................2022
4380 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Solutions for haemodialysis, concentrated, water for
diluting..............................................................................6.3-4163
Solutions for haemofiltration and for haemodiafiltra-
tion............................................................................................2025
Solutions for organ preservation..........................................2929
Solutions for peritoneal dialysis ...........................................2646
Solutions, suspensions, intrauterine ............................6.3-3977
Solvents, residual (5.4.) ............................................................ 603
Solvents, residual, identification and control (2.4.24.)...... 121
Somatostatin .............................................................................2930
Somatropin................................................................................ 2931
Somatropin concentrated solution ......................................2933
Somatropin for injection ........................................................2935
Sorbic acid.................................................................................2937
Sorbitan laurate .......................................................................2938
Sorbitan oleate .........................................................................2938
Sorbitan palmitate ...................................................................2939
Sorbitan sesquioleate..............................................................2939
Sorbitan stearate......................................................................2940
Sorbitan trioleate.....................................................................2940
Sorbitol................................................................................6.3-4305
Sorbitol, liquid (crystallising)................................................2942
Sorbitol, liquid (non-crystallising)........................................2943
Sorbitol, liquid, partially dehydrated............................6.3-4307
Sotalol hydrochloride .............................................................2944
Soya-bean oil, hydrogenated...........................................6.2-3837
Soya-bean oil, refined.......................................................6.2-3838
Spanish sage oil.................................................................6.2-3838
Specific surface area by air permeability (2.9.14.).............. 283
Specific surface area by gas adsorption (2.9.26.) ............... 306
Spectinomycin dihydrochloride pentahydrate ..................2947
Spectinomycin sulphate tetrahydrate for veterinary use ..2949
Spectrometry, atomic absorption (2.2.23.)............................. 37
Spectrometry, atomic emission (2.2.22.)................................. 36
Spectrometry, mass (2.2.43.) ..................................................... 68
Spectrometry, nuclear magnetic resonance
(2.2.33.) .............................................................................6.3-3909
Spectrometry, Raman (2.2.48.) ................................................. 82
Spectrometry, X-ray fluorescence (2.2.37.)............................. 59
Spectrophotometry, infrared absorption (2.2.24.)................ 39
Spectrophotometry, near-infrared (2.2.40.)............................ 62
Spectrophotometry, ultraviolet and visible absorption
(2.2.25.) .........................................................................................41
SPF chicken flocks for the production and quality control of
vaccines (5.2.2.)........................................................................ 547
Spheroids and granules, friability of (2.9.41.)...................... 330
Spiramycin..........................................................................6.1-3540
Spirapril hydrochloride monohydrate.................................2954
Spironolactone .........................................................................2955
Spot-on preparations................................................................. 753
Sprays ........................................................................................... 753
Sprays (liquid nasal) and drops (nasal) ................................. 731
Squalane ....................................................................................2956
Standard solutions for limit tests (4.1.2.) ............................. 504
Standard solutions for limit tests (4.1.2.) ....................6.3-3954
Standards, reference (5.12.)..................................................... 663
Stannous chloride dihydrate .................................................2959
Stanozolol...........................................................................6.3-4308
Star anise...................................................................................2960
Star anise oil .............................................................................2962
Starch glycolate (type A), sodium ........................................2920
Starch glycolate (type B), sodium ........................................ 2921
Starch glycolate (type C), sodium ........................................2922
Starch, maize .....................................................................6.3-4212
Starch, potato ....................................................................6.3-4277
Starch, pregelatinised ......................................................6.3-4308
Starch, rice .........................................................................6.3-4284
Starch, wheat .....................................................................6.3-4346
Starflower (borage) oil, refined.............................................1326
EUROPEAN PHARMACOPOEIA 6.3
Statistical analysis of results of biological assays and tests
(5.3.)............................................................................................ 571
Stavudine...................................................................................2964
Steam sterilisation of aqueous preparations, application of
the F0 concept (5.1.5.)....................................................6.3-3958
Stearic acid................................................................................2966
Stearoyl macrogolglycerides .................................................2967
Stearyl alcohol..........................................................................2968
Stem cells, human haematopoietic ............................... 6.3-4165
Sterile braided silk suture in distributor for veterinary
use............................................................................................. 1059
Sterile catgut............................................................................. 1045
Sterile catgut in distributor for veterinary use ................. 1057
Sterile containers of plasticised poly(vinyl chloride)
for human blood containing anticoagulant solution
(3.2.5.) ........................................................................................ 382
Sterile linen thread in distributor for veterinary use....... 1058
Sterile non-absorbable strands in distributor for veterinary
use............................................................................................. 1060
Sterile non-absorbable sutures ............................................. 1046
Sterile plastic containers for human blood and blood
components (3.2.3.)................................................................. 379
Sterile polyamide 6/6 suture in distributor for veterinary
use............................................................................................. 1059
Sterile polyamide 6 suture in distributor for veterinary
use............................................................................................. 1058
Sterile poly(ethylene terephthalate) suture in distributor for
veterinary use ......................................................................... 1059
Sterile products, methods of preparation (5.1.1.)............... 525
Sterile single-use plastic syringes (3.2.8.)............................. 384
Sterile synthetic absorbable braided sutures .................... 1050
Sterile synthetic absorbable monofilament sutures......... 1052
Sterilisation procedures, biological indicators (5.1.2.) ...... 527
Sterility (2.6.1.) .................................................................. 6.3-3919
Sterility, guidelines for using the test for (5.1.9.) ......6.3-3958
Sterols in fatty oils (2.4.23.)..................................................... 120
Sticks ............................................................................................ 748
Sticks, intrauterine ...........................................................6.3-3977
Sticks, nasal................................................................................. 732
St. John’s wort...................................................................6.2-3839
St. John’s wort dry extract, quantified.........................6.3-4309
Stomata and stomatal index (2.8.3.) ...................................... 249
Stramonium leaf.......................................................................2968
Stramonium, prepared.....................................................6.2-3842
Strands, sterile non-absorbable, in distributor for veterinary
use ............................................................................................ 1060
Streptokinase concentrated solution ...........................6.2-3843
Streptomycin sulphate ............................................................2972
Strontium (89Sr) chloride injection ...................................... 1021
Subdivision of tablets................................................................ 748
Sublingual sprays, oromucosal drops and oromucosal
sprays.......................................................................................... 732
Sublingual tablets and buccal tablets ................................... 734
Substances for pharmaceutical use ..............................6.3-3969
Substances for pharmaceutical use, control of impurities in
(5.10.).......................................................................................... 653
Substances of animal origin for the production of veterinary
vaccines (5.2.5.)........................................................................ 555
Sub-visible particles, particulate contamination (2.9.19.).. 300
Succinylsulfathiazole .............................................................. 2974
Sucrose................................................................................ 6.3-4311
Sucrose monopalmitate...................................................6.1-3543
Sucrose stearate ................................................................6.1-3544
Sufentanil ..................................................................................2977
Sufentanil citrate .....................................................................2978
Sugars, lead in (2.4.10.) ............................................................ 115
Sugar spheres ....................................................................6.3-4312
Sulbactam sodium ............................................................6.2-3845
Sulfacetamide sodium......................................................6.2-3847
General Notices (1) apply to all monographs and other texts
Index
Sulfadiazine...............................................................................2983
Sulfadimidine............................................................................2984
Sulfadoxine................................................................................2984
Sulfafurazole.............................................................................2985
Sulfaguanidine..........................................................................2986
Sulfamerazine...........................................................................2987
Sulfamethizole..........................................................................2988
Sulfamethoxazole ....................................................................2989
Sulfamethoxypyridazine for veterinary use .......................2990
Sulfanilamide ............................................................................ 2991
Sulfasalazine .............................................................................2992
Sulfathiazole .............................................................................2994
Sulfinpyrazone .........................................................................2995
Sulfisomidine ............................................................................2996
Sulindac .....................................................................................2996
Sulphated ash (2.4.14.) ..............................................................116
Sulphates (2.4.13.) ......................................................................116
Sulphur dioxide (2.5.29.).......................................................... 146
Sulphur for external use ........................................................2998
Sulphuric acid...........................................................................2998
Sulpiride.....................................................................................2999
Sultamicillin .......................................................................6.1-3545
Sultamicillin tosilate dihydrate......................................6.3-4313
Sumatra benzoin......................................................................1278
Sumatra benzoin tincture ......................................................1279
Sumatriptan succinate.....................................................6.3-4315
Sunflower oil, refined ......................................................6.2-3848
Supercritical fluid chromatography (2.2.45.) .........................71
Suppositories .............................................................................. 745
Suppositories and pessaries, disintegration of (2.9.2.)...... 265
Suppositories, lipophilic, softening time determination
(2.9.22.) ...................................................................................... 302
Suspensions, solutions and emulsions, oral ........................ 729
Suspensions, solutions, intrauterine ............................6.3-3977
Sutures, sterile non-absorbable ............................................ 1046
Sutures, sterile synthetic absorbable braided .................. 1050
Sutures, sterile synthetic absorbable monofilament ...... 1052
Suxamethonium chloride.......................................................3007
Suxibuzone................................................................................3008
Sweet fennel.............................................................................. 1874
Sweet orange oil.......................................................................3009
Swelling index (2.8.4.)............................................................... 249
Swine erysipelas vaccine (inactivated) .................................. 955
Swine-fever vaccine (live, prepared in cell cultures),
classical .............................................................................6.2-3669
Symbols and abbreviations (1.)................................................... 3
Synthetic absorbable braided sutures, sterile ................... 1050
Synthetic absorbable monofilament sutures, sterile........ 1052
Syringes, plastic, sterile single-use (3.2.8.)........................... 384
Syrups........................................................................................... 730
T
Table of physical characteristics of radionuclides mentioned
in the European Pharmacopoeia (5.7.) ............................... 633
Tablets .......................................................................................... 748
Tablets and capsules, disintegration of (2.9.1.) ..........6.3-3943
Tablets, buccal ............................................................................ 734
Tablets, coated............................................................................ 749
Tablets, dispersible .................................................................... 750
Tablets, effervescent .................................................................. 749
Tablets for intrauterine solutions and suspensions ..6.3-3977
Tablets for use in the mouth ................................................... 750
Tablets for vaginal solutions and suspensions .................... 752
Tablets, gastro-resistant............................................................ 750
Tablets, intrauterine .........................................................6.3-3977
Tablets, modified-release .......................................................... 750
Tablets, orodispersible .............................................................. 750
Tablets, resistance to crushing (2.9.8.) ................................. 279
Tablets, soluble........................................................................... 750
4381
Index
Tablets, subdivision of .............................................................. 748
Tablets, sublingual..................................................................... 734
Tablets, uncoated ....................................................................... 749
Tablets, uncoated, friability of (2.9.7.) ................................... 278
Tablets, vaginal........................................................................... 752
Talc.......................................................................................6.3-4321
Tamoxifen citrate ..................................................................... 3014
Tampons, ear............................................................................... 720
Tampons, medicated ................................................................. 751
Tampons, rectal .......................................................................... 746
Tampons, vaginal, medicated .................................................. 752
Tamsulosin hydrochloride ..................................................... 3016
Tannic acid ................................................................................ 3018
Tannins in herbal drugs, determination of (2.8.14.) .......... 255
Tapped density of powders, bulk density and
(2.9.34.) .............................................................................6.2-3646
Tartaric acid .............................................................................. 3018
Teat dips....................................................................................... 753
Tea tree oil................................................................................. 3019
Teat sprays................................................................................... 753
Technetium (99mTc) bicisate injection .................................. 1022
Technetium (99mTc) colloidal rhenium sulphide injection
............................................................................................6.3-4002
Technetium (99mTc) colloidal sulphur injection ................. 1024
Technetium (99mTc) colloidal tin injection .......................... 1025
Technetium (99mTc) etifenin injection .................................. 1026
Technetium (99mTc) exametazime injection ........................ 1027
Technetium (99mTc) gluconate injection .............................. 1028
Technetium (99mTc) human albumin injection ................... 1029
Technetium (99mTc) macrosalb injection.......................6.3-4003
Technetium (99mTc) mebrofenin injection ....................6.3-4004
Technetium (99mTc) medronate injection............................. 1031
Technetium (99mTc) mertiatide injection ............................. 1033
Technetium (99mTc) microspheres injection.................6.3-4005
Technetium (99mTc) pentetate injection............................... 1035
Technetium (99mTc) sestamibi injection ............................... 1036
Technetium (99mTc) succimer injection................................ 1037
Technetium (99mTc) tin pyrophosphate injection........6.3-4006
Teicoplanin .........................................................................6.3-4323
Telmisartan.........................................................................6.3-4325
Temazepam................................................................................3020
Tenosynovitis avian viral vaccine (live) ................................. 875
Tenoxicam.................................................................................. 3021
Terazosin hydrochloride dihydrate ......................................3022
Terbinafine hydrochloride......................................................3024
Terbutaline sulphate ...............................................................3025
Terconazole ........................................................................6.1-3553
Terfenadine.........................................................................6.1-3554
Terminology used in monographs on biological products
(5.2.1.)......................................................................................... 547
Test for anticomplementary activity of immunoglobulin
(2.6.17.)........................................................................................191
Test for anti-D antibodies in human immunoglobulin for
intravenous administration (2.6.26.) ..........................6.2-3627
Test for extractable volume of parenteral preparations
(2.9.17.)....................................................................................... 287
Test for Fc function of immunoglobulin (2.7.9.) ................. 227
Test for methanol and 2-propanol (2.9.11.) .......................... 282
Test for neurovirulence of live virus vaccines (2.6.18.) ..... 193
Test for neurovirulence of poliomyelitis vaccine (oral)
(2.6.19.) ...................................................................................... 193
Test for specified micro-organisms (microbiological
examination of non-sterile products) (2.6.13.) .........6.3-3927
Testosterone ..............................................................................3030
Testosterone decanoate .......................................................... 3031
Testosterone enantate.............................................................3033
Testosterone isocaproate........................................................3034
Testosterone propionate.........................................................3035
4382 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Tests for extraneous agents in viral vaccines for human use
(2.6.16.) ...................................................................................... 190
Tetanus and diphtheria toxins and toxoids, flocculation value
(Lf) of, (Ramon assay) (2.7.27.) ............................................. 241
Tetanus and diphtheria vaccine (adsorbed, reduced
antigen(s) content) .................................................................. 764
Tetanus antitoxin for human use ........................................... 969
Tetanus antitoxin for veterinary use...................................... 976
Tetanus, diphtheria and hepatitis B (rDNA) vaccine
(adsorbed).................................................................................. 765
Tetanus, diphtheria and pertussis (acellular, component)
vaccine (adsorbed)................................................................... 767
Tetanus, diphtheria and poliomyelitis (inactivated) vaccine
(adsorbed, reduced antigen(s) content) .............................. 770
Tetanus, diphtheria, pertussis (acellular, component) and
haemophilus type b conjugate vaccine (adsorbed) .......... 771
Tetanus, diphtheria, pertussis (acellular, component) and
hepatitis B (rDNA) vaccine (adsorbed) ............................... 774
Tetanus, diphtheria, pertussis (acellular, component) and
poliomyelitis (inactivated) vaccine (adsorbed) .................. 775
Tetanus, diphtheria, pertussis (acellular, component) and
poliomyelitis (inactivated) vaccine (adsorbed, reduced
antigen(s) content) .................................................................. 778
Tetanus, diphtheria, pertussis (acellular, component),
hepatitis B (rDNA), poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 780
Tetanus, diphtheria, pertussis (acellular, component),
poliomyelitis (inactivated) and haemophilus type b
conjugate vaccine (adsorbed).......................................6.3-3983
Tetanus, diphtheria, pertussis and poliomyelitis (inactivated)
vaccine (adsorbed)................................................................... 785
Tetanus, diphtheria, pertussis, poliomyelitis (inactivated) and
haemophilus type b conjugate vaccine (adsorbed) .......... 787
Tetanus immunoglobulin, human ........................................2079
Tetanus vaccine (adsorbed) ..................................................... 844
Tetanus vaccine (adsorbed), assay of (2.7.8.) ....................... 223
Tetanus vaccine for veterinary use ........................................ 957
Tetracaine hydrochloride ................................................6.1-3556
Tetracosactide....................................................................6.3-4326
Tetracycline ...............................................................................3040
Tetracycline hydrochloride .................................................... 3041
Tetra-O-acetyl-mannose triflate for radiopharmaceutical
preparations.....................................................................6.3-4008
Tetrazepam ................................................................................3043
Tetryzoline hydrochloride......................................................3044
Thallous (201Tl) chloride injection......................................... 1039
Theobromine.............................................................................3045
Theophylline .............................................................................3046
Theophylline-ethylenediamine ..............................................3048
Theophylline-ethylenediamine hydrate ...............................3049
Theophylline monohydrate....................................................3047
Thermal analysis (2.2.34.) ............................................... 6.1-3311
Thermogravimetry (2.2.34.)............................................ 6.1-3311
Thiamazole ................................................................................3050
Thiamine hydrochloride ......................................................... 3051
Thiamine nitrate.......................................................................3053
Thiamphenicol ..........................................................................3054
Thin-layer chromatography (2.2.27.)........................................ 43
Thioctic acid ..............................................................................3055
Thiomersal.................................................................................3056
Thiopental sodium and sodium carbonate.........................3057
Thioridazine ..............................................................................3058
Thioridazine hydrochloride ...................................................3059
Three-lobed sage leaf...............................................................2854
Threonine...................................................................................3060
Thyme ......................................................................................... 3061
Thyme oil ..................................................................................3063
Thyme, wild ............................................................................... 3219
Thymol........................................................................................3064
EUROPEAN PHARMACOPOEIA 6.3
Tiabendazole .............................................................................3064
Tiamulin for veterinary use ...................................................3065
Tiamulin hydrogen fumarate for veterinary use ...............3068
Tianeptine sodium ...................................................................3070
Tiapride hydrochloride ...........................................................3071
Tiaprofenic acid ........................................................................3072
Tibolone ..................................................................................... 3074
Ticarcillin sodium.....................................................................3075
Tick-borne encephalitis vaccine (inactivated) ...................... 845
Ticlopidine hydrochloride ......................................................3077
Tilidine hydrochloride hemihydrate ....................................3079
Timolol maleate ........................................................................3080
Tinctures .............................................................................6.1-3344
Tinidazole ...........................................................................6.2-3852
Tinnevelly senna pods.............................................................2871
Tinzaparin sodium ...................................................................3082
Tioconazole ...............................................................................3083
Titanium dioxide ......................................................................3084
Titration, amperometric (2.2.19.).............................................. 35
Titration, potentiometric (2.2.20.)............................................ 35
Titrations, complexometric (2.5.11.) ...................................... 140
Tobramycin.........................................................................6.2-3854
Tocopherol, all-rac-α- ..............................................................3086
Tocopherol, RRR-α- .................................................................3088
Tocopheryl acetate, all-rac-α- ................................................3089
α-Tocopheryl acetate concentrate (powder form) ............ 3091
Tocopheryl acetate, RRR-α-...................................................3090
Tocopheryl hydrogen succinate, DL-α- ................................3093
Tocopheryl hydrogen succinate, RRR-α- ............................3095
Tolbutamide ..............................................................................3097
Tolfenamic acid.........................................................................3097
Tolnaftate ..................................................................................3099
Tolu balsam ...............................................................................3099
Torasemide, anhydrous........................................................... 3100
Tormentil ....................................................................................3101
Tormentil tincture.................................................................... 3102
Tosylchloramide sodium......................................................... 3103
Total ash (2.4.16.)........................................................................116
Total cholesterol in oils rich in omega-3 acids (2.4.32.) .... 132
Total organic carbon in water for pharmaceutical use
(2.2.44.) .........................................................................................71
Total protein (2.5.33.) ............................................................... 148
Toxicity, abnormal (2.6.9.)........................................................ 165
Toxin, botulinum type A for injection..................................1327
Tragacanth .........................................................................6.3-4328
Tramadol hydrochloride ......................................................... 3104
Tramazoline hydrochloride monohydrate .......................... 3106
Trandolapril............................................................................... 3107
Tranexamic acid ....................................................................... 3108
Transdermal patches ................................................................. 737
Transdermal patches, dissolution test for (2.9.4.) .............. 275
Trapidil ....................................................................................... 3110
Tretinoin .....................................................................................3111
Triacetin ..................................................................................... 3112
Triamcinolone........................................................................... 3112
Triamcinolone acetonide.........................................................3114
Triamcinolone hexacetonide ................................................. 3115
Triamterene ........................................................................6.3-4329
Tribenoside.................................................................................3117
Tributyl acetylcitrate ........................................................6.3-4330
Trichloroacetic acid ................................................................. 3119
Triethanolamine ....................................................................... 3133
Triethyl citrate .......................................................................... 3120
Trifluoperazine hydrochloride .............................................. 3121
Triflusal ...................................................................................... 3121
Triglycerides, medium-chain.................................................. 3122
Triglycerides, omega-3-acid.............................................6.3-4246
Triglycerol diisostearate ..................................................6.1-3558
Trihexyphenidyl hydrochloride............................................. 3125
General Notices (1) apply to all monographs and other texts
Index
Trimetazidine dihydrochloride.............................................. 3126
Trimethadione .......................................................................... 3127
Trimethoprim............................................................................ 3128
Trimipramine maleate............................................................. 3130
Tri-n-butyl phosphate .............................................................. 3132
Tritiated (3H) water injection................................................. 1040
Trolamine................................................................................... 3133
Trometamol ............................................................................... 3135
Tropicamide............................................................................... 3135
Tropisetron hydrochloride ..................................................... 3136
Trospium chloride.................................................................... 3138
Troxerutin.................................................................................. 3139
Trypsin ................................................................................6.3-4331
Tryptophan.........................................................................6.3-4333
TSE, animal, minimising the risk of transmitting via human
and veterinary medicinal products (5.2.8.) ........................ 558
TSE, animal, products with risk of transmitting agents
of.................................................................................................. 694
Tuberculin for human use, old.............................................. 3144
Tuberculin purified protein derivative, avian .................... 3146
Tuberculin purified protein derivative, bovine.................. 3147
Tuberculin purified protein derivative for human use .... 3147
Tubes for comparative tests (2.1.5.) ..........................................17
Tubing and closures, silicone elastomer for (3.1.9.)........... 358
Tubing and containers for total parenteral nutrition
preparations, poly(ethylene - vinyl acetate) for (3.1.7.) ... 356
Tubing used in sets for the transfusion of blood and blood
components, materials based on plasticised poly(vinyl
chloride) for (3.1.1.2.) ............................................................. 342
Tubocurarine chloride ............................................................ 3150
Turmeric, Javanese .................................................................. 3150
Turpentine oil, Pinus pinaster type ..................................... 3151
Tylosin for veterinary use ...................................................... 3152
Tylosin phosphate bulk solution for veterinary use ........ 3154
Tylosin tartrate for veterinary use ....................................... 3156
Typhoid polysaccharide vaccine ............................................. 847
Typhoid vaccine.......................................................................... 849
Typhoid vaccine, freeze-dried.................................................. 849
Typhoid vaccine (live, oral, strain Ty 21a)............................ 849
Tyrosine...................................................................................... 3157
Tyrothricin................................................................................. 3158
U
Ubidecarenone.......................................................................... 3163
Udder-washes .............................................................................. 753
Ultraviolet and visible absorption spectrophotometry
(2.2.25.) .........................................................................................41
Ultraviolet ray lamps for analytical purposes (2.1.3.)............15
Uncoated tablets......................................................................... 749
Undecylenic acid ...................................................................... 3164
Uniformity of content of single-dose preparations
(2.9.6.) ........................................................................................ 278
Uniformity of dosage units (2.9.40.) .............................6.1-3325
Uniformity of mass of delivered doses from multidose
containers (2.9.27.).................................................................. 309
Uniformity of mass of single-dose preparations (2.9.5.) .... 278
Units of the International System (SI) used in the
Pharmacopoeia and equivalence with other units (1.)........ 3
Unsaponifiable matter (2.5.7.) ................................................. 139
Urea............................................................................................. 3165
Urofollitropin ............................................................................ 3166
Urokinase................................................................................... 3167
Uronic acids in polysaccharide vaccines (2.5.22.)............... 144
Ursodeoxycholic acid ............................................................. 3168
V
Vaccines, adsorbed, aluminium in (2.5.13.)...........................141
Vaccines, adsorbed, calcium in (2.5.14.)................................ 142
4383
Index
Vaccines and immunosera, phenol in (2.5.15.).................... 142
Vaccines and immunosera, veterinary, evaluation of efficacy
of (5.2.7.) ...........................................................................6.1-3335
Vaccines and immunosera, veterinary, evaluation of safety
(5.2.6.) ........................................................................................ 556
Vaccines and immunosera, veterinary, evaluation of the
safety of each batch (5.2.9.)................................................... 567
Vaccines for human use...................................................6.3-3971
Vaccines for human use, cell substrates for the production of
(5.2.3.) ...............................................................................6.3-3963
Vaccines for human use, viral, extraneous agents in
(2.6.16.) ...................................................................................... 190
Vaccines for veterinary use...................................................... 707
Vaccines, polysaccharide, hexosamines in (2.5.20.)............ 143
Vaccines, polysaccharide, methylpentoses in (2.5.21.)....... 143
Vaccines, polysaccharide, nucleic acids in (2.5.17.) ............ 142
Vaccines, polysaccharide, O-acetyl in (2.5.19.)..................... 143
Vaccines, polysaccharide, phosphorus in (2.5.18.) ............. 142
Vaccines, polysaccharide, protein in (2.5.16.) ...................... 142
Vaccines, polysaccharide, ribose in (2.5.31.) ........................ 147
Vaccines, polysaccharide, sialic acid in (2.5.23.) ................. 144
Vaccines, polysaccharide, uronic acids in (2.5.22.)............. 144
Vaccines, SPF chicken flocks for the production and quality
control of (5.2.2.) .................................................................... 547
Vaccines, veterinary, cell cultures for the production of
(5.2.4.) ........................................................................................ 553
Vaccines, veterinary, substances of animal origin for the
production of (5.2.5.) .............................................................. 555
Vaccines, viral live, test for neurovirulence (2.6.18.).......... 193
Vaginal capsules ......................................................................... 752
Vaginal foams.............................................................................. 752
Vaginal preparations ................................................................. 751
Vaginal preparations, semi-solid ............................................. 752
Vaginal solutions and suspensions, tablets for.................... 752
Vaginal solutions, emulsions and suspensions.................... 752
Vaginal tablets ............................................................................ 752
Vaginal tampons, medicated.................................................... 752
Valerian dry hydroalcoholic extract..................................... 3173
Valerian root...............................................................................3174
Valerian tincture....................................................................... 3175
Valine ...........................................................................................3176
Valnemulin hydrochloride for veterinary use ................... 3177
Valproic acid.............................................................................. 3178
Vancomycin hydrochloride .................................................... 3180
Vanillin ....................................................................................... 3182
Varicella immunoglobulin for intravenous administration,
human ...................................................................................... 2081
Varicella immunoglobulin, human.......................................2080
Varicella vaccine (live)......................................................6.3-3992
Vectors for human use, adenovirus ....................................... 670
Vectors for human use, plasmid ............................................. 674
Vectors for human use, plasmid, bacterial cells used for the
manufacture of ......................................................................... 676
Vectors for human use, poxvirus............................................ 672
Vecuronium bromide............................................................... 3183
Vegetable fatty oils..................................................................... 712
Venlafaxine hydrochloride ..................................................... 3184
Verapamil hydrochloride ........................................................ 3186
Verbena herb............................................................................. 3188
Veterinary liquid preparations for cutaneous application.. 752
Veterinary vaccines and immunosera, evaluation of efficacy
of (5.2.7.) ...........................................................................6.1-3335
Viability, nucleated cell count and (2.7.29.) ......................... 243
Vibriosis (cold-water) vaccine (inactivated) for
salmonids..........................................................................6.2-3671
Vibriosis vaccine (inactivated) for salmonids..............6.2-3672
VICH (5.8.)................................................................................... 645
Vinblastine sulphate................................................................ 3189
Vincristine sulphate................................................................. 3190
4384 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Vindesine sulphate .................................................................. 3192
Vinorelbine tartrate ................................................................. 3194
Vinpocetine................................................................................ 3196
Viper venom antiserum, European ........................................ 970
Viral rhinotracheitis vaccine (inactivated), feline.................916
Viral rhinotracheitis vaccine (live), feline ..............................917
Viral safety (5.1.7.) ..................................................................... 543
Viscometer method, capillary (2.2.9.)...................................... 27
Viscometer method, falling ball (2.2.49.)................................ 84
Viscose wadding, absorbent .................................................. 3197
Viscosity (2.2.8.) ........................................................................... 27
Viscosity - rotating viscometer method (2.2.10.)................... 28
Visible and ultraviolet absorption spectrophotometry
(2.2.25.) .........................................................................................41
Visible particles, particulate contamination (2.9.20.) ........ 302
Vitamin A ................................................................................... 3199
Vitamin A concentrate (oily form), synthetic.....................3200
Vitamin A concentrate (powder form), synthetic.............. 3201
Vitamin A concentrate (solubilisate/emulsion),
synthetic ..................................................................................3203
Volumetric analysis (4.2.) ..........................................................514
Volumetric solutions (4.2.2.).....................................................514
Volumetric solutions (4.2.2.)...........................................6.3-3954
Volumetric solutions, primary standards for (4.2.1.) ..........514
von Willebrand factor, human .............................................. 2081
von Willebrand factor, human, assay of (2.7.21.) ................ 237
W
Warfarin sodium.......................................................................3207
Warfarin sodium clathrate .....................................................3208
Washes, nasal.............................................................................. 732
Water (15O) injection................................................................ 1040
Water, determination by distillation (2.2.13.) .........................31
Water for diluting concentrated haemodialysis
solutions ...........................................................................6.3-4163
Water for injections ..........................................................6.3-4339
Water for pharmaceutical use, total organic carbon in
(2.2.44.) .........................................................................................71
Water, highly purified ......................................................6.3-4342
Water in essential oils (2.8.5.) ................................................. 249
Water in gases (2.5.28.) ............................................................ 146
Water : micro determination (2.5.32.).................................... 147
Water, purified...................................................................6.3-4344
Water : semi-micro determination (2.5.12.) ...........................141
Wheat-germ oil, refined .......................................................... 3215
Wheat-germ oil, virgin............................................................. 3216
Wheat starch ......................................................................6.3-4346
White beeswax ..........................................................................1260
White horehound..................................................................... 3216
White soft paraffin............................................................6.2-3815
Wild pansy (flowering aerial parts)...................................... 3217
Wild thyme ................................................................................ 3219
Willow bark ........................................................................6.1-3563
Willow bark dry extract ...................................................6.1-3564
Wool alcohols............................................................................ 3221
Wool fat ......................................................................................3222
Wool fat, hydrogenated...........................................................3226
Wool fat, hydrous.....................................................................3227
Wormwood ................................................................................3228
X
Xanthan gum .....................................................................6.3-4349
Xenon (133Xe) injection............................................................ 1042
X-ray fluorescence spectrometry (2.2.37.)............................... 59
X-ray powder diffraction (XRPD), characterisation
of crystalline and partially crystalline solids by
(2.9.33.) .............................................................................6.3-3945
Xylazine hydrochloride for veterinary use .........................3234
EUROPEAN PHARMACOPOEIA 6.3 Index

Xylitol...................................................................................6.3-4350 Zinc acetate dihydrate.............................................................3250

Xylometazoline hydrochloride ..............................................3237 Zinc acexamate ......................................................................... 3251
Xylose..........................................................................................3238 Zinc chloride .............................................................................3253
Zinc oxide...................................................................................3253
Y Zinc stearate..............................................................................3254
Yarrow ........................................................................................3243 Zinc sulphate heptahydrate ...................................................3254
Yellow beeswax ......................................................................... 1261 Zinc sulphate hexahydrate.....................................................3255
Yellow fever vaccine (live) ...............................................6.1-3365 Zinc sulphate monohydrate ...................................................3255
Yellow soft paraffin........................................................... 6.2-3816 Zinc undecylenate ....................................................................3256
Yohimbine hydrochloride .......................................................3244 Zolpidem tartrate .....................................................................3256
Zopiclone ...................................................................................3257
Zoster (shingles) vaccine (live), herpes ........................6.3-3991
Z
Zuclopenthixol decanoate ......................................................3259
Zidovudine.................................................................................3249

General Notices (1) apply to all monographs and other texts 4385
 EUROPEAN PHARMACOPOEIA 6.3

4386 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.3 Index

Numerics Acidum pipemidicum trihydricum.....................................2690

α-1-Proteinasi inhibitor humanum ............................6.2-3762 Acidum salicylicum ................................................................2859
Acidum (S)-lacticum ...............................................................2229
A Acidum sorbicum ....................................................................2937
Acidum stearicum ...................................................................2966
Absinthii herba ........................................................................3228
Acidum sulfuricum .................................................................2998
Acaciae gummi .................................................................6.3-4013
Acidum tartaricum ................................................................. 3018
Acaciae gummi dispersione desiccatum.................... 6.3-4014
Acidum thiocticum..................................................................3055
Acamprosatum calcicum....................................................... 1088
Acidum tiaprofenicum...........................................................3072
Acarbosum ................................................................................ 1089
Acidum tolfenamicum............................................................3097
Acebutololi hydrochloridum ................................................ 1091
Acidum tranexamicum .......................................................... 3108
Aceclofenacum..................................................................6.2-3685
Acidum trichloraceticum ...................................................... 3119
Acemetacinum ..................................................................6.3-4015
Acidum undecylenicum ........................................................ 3164
Acesulfamum kalicum ........................................................... 1095
Acidum ursodeoxycholicum................................................. 3168
Acetazolamidum...................................................................... 1096
Acidum valproicum ................................................................ 3178
Acetonum .................................................................................. 1098
Acitretinum............................................................................... 1109
Acetylcholini chloridum........................................................ 1099
Adeninum.................................................................................. 1110
Acetylcysteinum ...................................................................... 1100
Adenosinum ...................................................................... 6.3-4018
β-Acetyldigoxinum...................................................................1101 Adeps lanae ..............................................................................3222
Aciclovirum .............................................................................. 1107
Adeps lanae cum aqua...........................................................3227
Acidi methacrylici et ethylis acrylatis polymerisati 1:1
Adeps lanae hydrogenatus ...................................................3226
dispersio 30 per centum .....................................................2372
Adeps solidus.....................................................................6.3-4164
Acidi methacrylici et ethylis acrylatis polymerisati 1:1
Adrenalini tartras.....................................................................1114
dispersio 30 per centum ..............................................6.3-4220
Adrenalinum .....................................................................6.2-3686
Acidi methacrylici et ethylis acrylatis polymerisatum
Aer medicinalis.................................................................6.3-4020
1:1 ......................................................................................6.2-3781
Aer medicinalis artificiosus ................................................. 1121
Acidi methacrylici et methylis methacrylatis polymerisatum
Aether ......................................................................................... 1833
1:1 .............................................................................................2373
Aether anaestheticus .............................................................. 1834
Acidi methacrylici et methylis methacrylatis polymerisatum
Aetherolea ................................................................................... 680
1:2 ............................................................................................. 2374
Agar ..................................................................................... 6.3-4019
Acidum 4-aminobenzoicum ................................................. 1164
Agni casti fructus .............................................................6.2-3688
Acidum aceticum glaciale..................................................... 1097
Agrimoniae herba ....................................................................1117
Acidum acetylsalicylicum ..................................................... 1103
Alaninum................................................................................... 1121
Acidum adipicum.................................................................... 1113
Albendazolum .......................................................................... 1122
Acidum alginicum............................................................6.3-4022
Albumini humani solutio......................................................2057
Acidum amidotrizoicum dihydricum ................................ 1158
Alchemillae herba ................................................................... 1123
Acidum aminocaproicum ..................................................... 1166
Alcohol benzylicus.................................................................. 1281
Acidum ascorbicum.........................................................6.3-4042
Alcohol cetylicus...................................................................... 1485
Acidum asparticum ................................................................1225
Alcohol cetylicus et stearylicus ........................................... 1480
Acidum benzoicum................................................................. 1276
Alcohol cetylicus et stearylicus emulsificans A ....... 6.2-3717
Acidum boricum......................................................................1327
Alcohol cetylicus et stearylicus emulsificans B .......6.2-3718
Acidum caprylicum ................................................................ 1402
Alcoholes adipis lanae ........................................................... 3221
Acidum chenodeoxycholicum.............................................. 1489
Alcohol isopropylicus............................................................. 2182
Acidum citricum anhydricum .............................................1554
Alcohol oleicus.........................................................................2544
Acidum citricum monohydricum .......................................1555
Alcohol stearylicus..................................................................2968
Acidum edeticum .................................................................... 1774
Alcuronii chloridum............................................................... 1124
Acidum etacrynicum.............................................................. 1826
Alfacalcidolum ......................................................................... 1126
Acidum folicum .......................................................................1938
Alfadexum ................................................................................. 1127
Acidum fusidicum...................................................................1954
Alfentanili hydrochloridum.................................................. 1128
Acidum glutamicum ...............................................................1984
Alfuzosini hydrochloridum ...........................................6.1-3394
Acidum hydrochloridum concentratum............................2085
Allantoinum.............................................................................. 1131
Acidum hydrochloridum dilutum .......................................2085
Allii sativi bulbi pulvis ........................................................... 1961
Acidum iopanoicum............................................................... 2162
Allium sativum ad praeparationes homoeopathicas ..... 1077
Acidum iotalamicum.............................................................. 2163
Allopurinolum.......................................................................... 1132
Acidum ioxaglicum................................................................. 2167
Almagatum.........................................................................6.3-4023
Acidum lacticum .....................................................................2228
Aloe barbadensis ..................................................................... 1137
Acidum lactobionicum........................................................... 2231
Aloe capensis............................................................................ 1138
Acidum maleicum ...................................................................2328
Aloes extractum siccum normatum.............................6.2-3690
Acidum malicum .....................................................................2329
Alprazolamum ......................................................................... 1139
Acidum mefenamicum.................................................... 6.3-4217
Alprenololi hydrochloridum..................................................1141
Acidum nalidixicum...............................................................2472
Alprostadilum........................................................................... 1143
Acidum nicotinicum...............................................................2502
Alteplasum ad iniectabile...................................................... 1145
Acidum niflumicum ........................................................6.1-3508
Althaeae folium........................................................................2338
Acidum nitricum ..................................................................... 2510
Althaeae radix ..........................................................................2339
Acidum oleicum.......................................................................2543
Altizidum............................................................................6.2-3691
Acidum oxolinicum ................................................................2582
Alumen....................................................................................... 1149
Acidum palmiticum ................................................................2604
Aluminii chloridum hexahydricum ................................... 1149
Acidum phosphoricum concentratum...............................2675
Aluminii hydroxidum hydricum ad adsorptionem ..6.1-3395
Acidum phosphoricum dilutum .......................................... 2676
Aluminii magnesii silicas ..............................................6.3-4024

General Notices (1) apply to all monographs and other texts 4387
Index
Aluminii natrii silicas.....................................................6.3-4026
Aluminii oxidum hydricum...........................................6.3-4025
Aluminii phosphas hydricus ................................................ 1153
Aluminii phosphatis liquamen.....................................6.3-4026
Aluminii sulfas......................................................................... 1154
Alverini citras........................................................................... 1154
Amantadini hydrochloridum ............................................... 1156
Ambroxoli hydrochloridum .................................................. 1156
Amfetamini sulfas ................................................................... 1158
Amikacini sulfas ...............................................................6.1-3398
Amikacinum ......................................................................6.1-3396
Amiloridi hydrochloridum.................................................... 1163
Aminoglutethimidum............................................................. 1167
Amiodaroni hydrochloridum ........................................6.3-4028
Amisulpridum .......................................................................... 1170
Amitriptylini hydrochloridum ......................................6.3-4029
Amlodipini besilas .................................................................. 1173
Ammoniae (13N) solutio iniectabilis ..................................... 981
Ammoniae solutio concentrata ........................................... 1175
Ammonii bromidum ............................................................... 1177
Ammonii chloridum ............................................................... 1178
Ammonii glycyrrhizas ........................................................... 1179
Ammonii hydrogenocarbonas ............................................. 1180
Ammonio methacrylatis copolymerum A......................... 1175
Ammonio methacrylatis copolymerum B .........................1176
Amobarbitalum ........................................................................ 1180
Amobarbitalum natricum ..................................................... 1181
Amoxicillinum natricum....................................................... 1182
Amoxicillinum trihydricum.................................................. 1184
Amphotericinum B ..........................................................6.3-4031
Ampicillinum anhydricum ................................................... 1188
Ampicillinum natricum......................................................... 1190
Ampicillinum trihydricum.................................................... 1193
Amygdalae oleum raffinatum .............................................. 1136
Amygdalae oleum virginale ................................................. 1136
Amylum pregelificatum..................................................6.3-4308
Angelicae radix........................................................................ 1196
Anisi aetheroleum................................................................... 1197
Anisi fructus ............................................................................. 1199
Anisi stellati aetheroleum .....................................................2962
Anisi stellati fructus................................................................2960
Antazolini hydrochloridum.................................................. 1199
Anticorpora monoclonalia ad usum humanum ............... 690
Antithrombinum III humanum densatum .......................2060
Apis mellifera ad praeparationes homoeopathicas........ 1079
Apomorphini hydrochloridum ............................................1207
Aprotinini solutio concentrata .....................................6.3-4035
Aprotininum......................................................................6.3-4033
Aqua ad dilutionem solutionum concentratarum ad
haemodialysim ............................................................... 6.3-4163
Aqua ad iniectabilia ........................................................6.3-4339
Aquae (15O) solutio iniectabilis............................................ 1040
Aquae tritiatae (3H) solutio iniectabilis............................. 1040
Aqua purificata.................................................................6.3-4344
Aqua valde purificata......................................................6.3-4342
Arachidis oleum hydrogenatum...................................6.2-3694
Arachidis oleum raffinatum ................................................. 1211
Argenti nitras ...........................................................................2880
Argentum colloidale ad usum externum ..........................2879
Arginini aspartas .................................................................... 1213
Arginini hydrochloridum...................................................... 1214
Argininum................................................................................. 1212
Arnicae flos........................................................................6.3-4038
Arnicae tinctura ...............................................................6.3-4040
Arsenii trioxidum ad praeparationes homoeopathicas .. 1073
Articaini hydrochloridum..................................................... 1217
Ascorbylis palmitas.................................................................1222
Asparaginum monohydricum..............................................1223
Aspartamum .............................................................................1224
4388 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Astemizolum.............................................................................1226
Atenololum................................................................................1228
Atracurii besilas.......................................................................1230
Atropini sulfas...................................................................6.3-4045
Atropinum..........................................................................6.3-4044
Aurantii amari epicarpii et mesocarpii tinctura ............1320
Aurantii amari epicarpium et mesocarpium............6.3-4064
Aurantii amari flos ..........................................................6.3-4065
Aurantii dulcis aetheroleum.................................................3009
Auricularia.................................................................................. 719
Azaperonum ad usum veterinarium..................................1234
Azathioprinum.........................................................................1236
Azelastini hydrochloridum...................................................1236
Azithromycinum...............................................................6.3-4047
B
Bacampicillini hydrochloridum...................................6.1-3409
Bacitracinum............................................................................1245
Bacitracinum zincum ............................................................1247
Baclofenum...............................................................................1250
Ballotae nigrae herba ............................................................ 1321
Balsamum peruvianum.................................................. 6.2-3817
Balsamum tolutanum ............................................................3099
Bambuteroli hydrochloridum .............................................. 1251
Barbitalum ................................................................................1252
Barii chloridum dihydricum ad praeparationes
homoeopathicas .................................................................... 1073
Barii sulfas................................................................................1253
BCG ad immunocurationem.........................................6.3-4053
Beclometasoni dipropionas anhydricus ....................6.3-4054
Beclometasoni dipropionas monohydricus ..............6.3-4056
Belladonnae folii extractum siccum normatum ......6.3-4059
Belladonnae folii tinctura normata ...................................1264
Belladonnae folium ................................................................ 1261
Belladonnae pulvis normatus.......................................6.2-3698
Benazeprili hydrochloridum ........................................6.3-4060
Bendroflumethiazidum .........................................................1266
Benfluorexi hydrochloridum ...............................................1267
Benperidolum ..........................................................................1269
Benserazidi hydrochloridum ...............................................1270
Bentonitum........................................................................6.3-4062
Benzalkonii chloridi solutio ................................................1273
Benzalkonii chloridum..........................................................1272
Benzbromaronum...................................................................1273
Benzethonii chloridum .........................................................1275
Benzocainum ........................................................................... 1276
Benzoe sumatranus................................................................1278
Benzoe tonkinensis ................................................................1277
Benzois sumatrani tinctura..................................................1279
Benzois tonkinensis tinctura...............................................1278
Benzoylis peroxidum cum aqua .........................................1280
Benzylis benzoas.....................................................................1283
Benzylpenicillinum benzathinum......................................1283
Benzylpenicillinum kalicum................................................1285
Benzylpenicillinum natricum .............................................1288
Benzylpenicillinum procainum..........................................1287
Betacarotenum ........................................................................1290
Betadexum ................................................................................ 1291
Betahistini dihydrochloridum .............................................1292
Betahistini mesilas .................................................................1293
Betamethasoni acetas ............................................................1297
Betamethasoni dipropionas .................................................1298
Betamethasoni natrii phosphas ..........................................1300
Betamethasoni valeras....................................................6.3-4062
Betamethasonum ....................................................................1295
Betaxololi hydrochloridum...................................................1303
Betulae folium...................................................................6.2-3699
Bezafibratum............................................................................1304
Bifonazolum.............................................................................1306
EUROPEAN PHARMACOPOEIA 6.3
Biotinum ...................................................................................1308
Biperideni hydrochloridum .................................................1309
Bisacodylum............................................................................. 1312
Bismuthi subcarbonas ........................................................... 1313
Bismuthi subgallas ................................................................. 1314
Bismuthi subnitras ponderosus........................................... 1315
Bismuthi subsalicylas ............................................................ 1316
Bisoprololi fumaras .........................................................6.1-3412
Bistortae rhizoma ................................................................... 1317
Bleomycini sulfas....................................................................1322
Boldi folii extractum siccum .........................................6.1-3415
Boldi folium..............................................................................1324
Boragonis officinalis oleum raffinatum............................1326
Borax ..........................................................................................1326
Bromazepamum...................................................................... 1331
Bromhexini hydrochloridum ...............................................1332
Bromocriptini mesilas ...........................................................1333
Bromperidoli decanoas .........................................................1337
Bromperidolum .......................................................................1335
Brompheniramini maleas ....................................................1339
Brotizolamum ..........................................................................1340
Budesonidum ...........................................................................1342
Bufexamacum ..........................................................................1344
Buflomedili hydrochloridum ...............................................1345
Bumetanidum ..........................................................................1346
Bupivacaini hydrochloridum...............................................1347
Buprenorphini hydrochloridum .........................................1350
Buprenorphinum ...................................................................1349
Buserelinum ......................................................................6.3-4067
Buspironi hydrochloridum...................................................1353
Busulfanum ..............................................................................1355
Butylhydroxyanisolum ..........................................................1357
Butylhydroxytoluenum..........................................................1357
Butylis parahydroxybenzoas................................................1358
C Carvi fructus............................................................................. 1408
Cabergolinum ..........................................................................1363
Cadmii sulfas hydricus
ad praeparationes homoeopathicas................................. 1074
Calcifediolum ...........................................................................1366
Calcii acetas ............................................................................. 1376
Calcii ascorbas.........................................................................1377
Calcii carbonas .................................................................6.2-3703
Calcii chloridum dihydricum ..............................................1378
Calcii chloridum hexahydricum .........................................1379
Calcii dobesilas monohydricus ....................................6.2-3703
Calcii folinas .....................................................................6.3-4071
Calcii glucoheptonas..............................................................1383
Calcii gluconas .................................................................6.3-4073
Calcii gluconas ad iniectabile ......................................6.3-4074
Calcii gluconas anhydricus...........................................6.3-4074
Calcii glycerophosphas..........................................................1386
Calcii hydrogenophosphas anhydricus.............................1387
Calcii hydrogenophosphas dihydricus..............................1388
Calcii hydroxidum ..................................................................1389
Calcii iodidum tetrahydricum ad praeparationes
homoeopathicas .................................................................... 1074
Calcii lactas anhydricus........................................................1389
Calcii lactas monohydricus..................................................1390
Calcii lactas pentahydricus ..................................................1390
Calcii lactas trihydricus ........................................................ 1391
Calcii laevulinas dihydricus.................................................1394
Calcii levofolinas pentahydricus ........................................1392
Calcii pantothenas..................................................................1395
Calcii stearas .....................................................................6.3-4076
Calcii sulfas dihydricus .........................................................1398
Calcipotriolum anhydricum.................................................1367
Calcipotriolum monohydricum...........................................1370
Calcitoninum salmonis .........................................................1372
General Notices (1) apply to all monographs and other texts
Index
Calcitriolum..............................................................................1375
Calendulae flos........................................................................1398
Camphora racemica ............................................................... 1401
Capsici fructus ..................................................................6.2-3704
Capsici oleoresina raffinata et quantificata .................... 1405
Capsici tinctura normata...................................................... 1406
Capsulae .......................................................................................717
Captoprilum ............................................................................. 1407
Carbacholum............................................................................ 1410
Carbamazepinum ....................................................................1411
Carbasalatum calcicum......................................................... 1412
Carbidopum.............................................................................. 1413
Carbimazolum ..........................................................................1414
Carbo activatus .................................................................6.3-4088
Carbocisteinum ....................................................................... 1415
Carbomera .........................................................................6.1-3422
Carbonei dioxidum..................................................................1417
Carbonei monoxidum (15O) .................................................... 982
Carboplatinum......................................................................... 1419
Carboprostum trometamolum ............................................. 1420
Carboxymethylamylum natricum A...................................2920
Carboxymethylamylum natricum B................................... 2921
Carboxymethylamylum natricum C...................................2922
Carisoprodolum....................................................................... 1421
Carmellosum calcicum .......................................................... 1422
Carmellosum natricum ......................................................... 1423
Carmellosum natricum conexum ................................ 6.3-4117
Carmellosum natricum, substitutum humile................... 1424
Carmustinum ........................................................................... 1425
Carprofenum ad usum veterinarium..........................6.3-4077
Carteololi hydrochloridum ................................................... 1426
Carthami flos ........................................................................... 2851
Carthami oleum raffinatum .................................................2852
Carvedilolum............................................................................ 1427
Carvi aetheroleum .................................................................. 1408
Caryophylli floris aetheroleum ...........................................1588
Caryophylli flos .......................................................................1587
Cefaclorum ............................................................................... 1435
Cefadroxilum monohydricum ......................................6.1-3423
Cefalexinum monohydricum ........................................6.1-3425
Cefalotinum natricum ........................................................... 1440
Cefamandoli nafas.................................................................. 1441
Cefapirinum natricum........................................................... 1443
Cefatrizinum propylen glycolum........................................ 1444
Cefazolinum natricum........................................................... 1445
Cefepimi dihydrochloridum monohydricum................... 1448
Cefiximum................................................................................. 1450
Cefoperazonum natricum..................................................... 1451
Cefotaximum natricum ......................................................... 1453
Cefoxitinum natricum ........................................................... 1455
Cefradinum............................................................................... 1457
Ceftazidimum........................................................................... 1459
Ceftriaxonum natricum......................................................... 1461
Cefuroximum axetili............................................................... 1462
Cefuroximum natricum......................................................... 1464
Celiprololi hydrochloridum.................................................. 1465
Cellulae stirpes haematopoieticae humanae............6.3-4165
Cellulosi acetas .................................................................6.3-4078
Cellulosi acetas butyras......................................................... 1468
Cellulosi acetas phthalas................................................6.3-4079
Cellulosi pulvis .................................................................6.3-4084
Cellulosum microcristallinum......................................6.3-4080
Cellulosum microcristallinum et carmellosum
natricum..................................................................................2422
Centaurii herba ....................................................................... 1477
Centellae asiaticae herba...................................................... 1477
Cera alba ...................................................................................1260
Cera carnauba ......................................................................... 1425
4389
Index
Cera flava .................................................................................. 1261
Cetirizini dihydrochloridum.........................................6.2-3715
Cetobemidoni hydrochloridum ........................................... 2215
Cetostearylis isononanoas.................................................... 1484
Cetrimidum............................................................................... 1484
Cetylis palmitas ....................................................................... 1486
Cetylpyridinii chloridum ...................................................... 1486
Chamomillae romanae flos .................................................. 1487
Chelidonii herba...................................................................... 2010
Chinidini sulfas .......................................................................2799
Chinini hydrochloridum .......................................................2800
Chinini sulfas...........................................................................2802
Chitosani hydrochloridum ................................................... 1490
Chlorali hydras ........................................................................ 1491
Chlorambucilum...................................................................... 1492
Chloramphenicoli natrii succinas ...................................... 1495
Chloramphenicoli palmitas.................................................. 1493
Chloramphenicolum............................................................... 1492
Chlorcyclizini hydrochloridum........................................... 1496
Chlordiazepoxidi hydrochloridum ..................................... 1498
Chlordiazepoxidum ................................................................ 1497
Chlorhexidini diacetas........................................................... 1499
Chlorhexidini digluconatis solutio.....................................1500
Chlorhexidini dihydrochloridum........................................1502
Chlorobutanolum anhydricum............................................1503
Chlorobutanolum hemihydricum .......................................1504
Chlorocresolum .......................................................................1504
Chloroquini phosphas ...........................................................1505
Chloroquini sulfas...................................................................1506
Chlorothiazidum .....................................................................1507
Chlorphenamini maleas.................................................6.1-3427
Chlorpromazini hydrochloridum .......................................1509
Chlorpropamidum .................................................................. 1510
Chlorprothixeni hydrochloridum ....................................... 1511
Chlortalidonum ....................................................................... 1513
Chlortetracyclini hydrochloridum...................................... 1514
Cholecalciferoli pulvis ....................................................6.3-4091
Cholecalciferolum ................................................................... 1516
Cholecalciferolum densatum oleosum .......................6.3-4089
Cholecalciferolum in aqua dispergibile .....................6.3-4093
Cholesterolum ..........................................................................1524
Chondroitini natrii sulfas ..............................................6.3-4095
Chorda resorbilis sterilis ....................................................... 1045
Chorda resorbilis sterilis in fuso
ad usum veterinarium......................................................... 1057
Chromii (51Cr) edetatis solutio iniectabilis................6.2-3677
Chymotrypsinum ....................................................................1527
Ciclopirox olaminum .............................................................1530
Ciclopiroxum............................................................................1528
Ciclosporinum ......................................................................... 1531
Cilastatinum natricum ...................................................6.1-3428
Cilazaprilum ............................................................................1534
Cimetidini hydrochloridum .................................................1537
Cimetidinum ............................................................................1536
Cinchocaini hydrochloridum ..............................................1538
Cinchonae cortex .............................................................6.2-3720
Cinchonae extractum fluidum normatum .......................1540
Cineolum................................................................................... 1541
Cinnamomi cassiae aetheroleum ................................6.2-3707
Cinnamomi cortex ..................................................................1542
Cinnamomi corticis tinctura................................................1545
Cinnamomi zeylanici folii aetheroleum...........................1544
Cinnamomi zeylanicii corticis aetheroleum ............6.2-3721
Cinnarizinum ..........................................................................1545
Ciprofibratum...........................................................................1547
Ciprofloxacini hydrochloridum ..........................................1550
Ciprofloxacinum .....................................................................1548
Cisapridi tartras ......................................................................1552
Cisapridum monohydricum................................................. 1551
4390 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Cisplatinum .......................................................................6.3-4097
Citaloprami hydrobromidum ........................................6.3-4099
Citaloprami hydrochloridum ........................................ 6.3-4101
Citri reticulatae aetheroleum...............................................2333
Citronellae aetheroleum........................................................1556
Cladribinum .............................................................................1557
Clarithromycinum ..................................................................1559
Clazurilum ad usum veterinarium.....................................1562
Clebopridi malas .....................................................................1564
Clemastini fumaras .........................................................6.1-3430
Clenbuteroli hydrochloridum ..............................................1567
Clindamycini hydrochloridum............................................1568
Clindamycini phosphas.........................................................1570
Clioquinolum ........................................................................... 1571
Clobazamum ............................................................................1572
Clobetasoli propionas ............................................................1573
Clobetasoni butyras ................................................................1575
Clofaziminum ..........................................................................1577
Clofibratum...............................................................................1578
Clomifeni citras .......................................................................1579
Clomipramini hydrochloridum ...........................................1580
Clonazepamum .......................................................................1582
Clonidini hydrochloridum.............................................6.3-4102
Clopamidum......................................................................6.1-3431
Closantelum natricum dihydricum
ad usum veterinarium.........................................................1584
Clotrimazolum..................................................................6.1-3433
Cloxacillinum natricum ........................................................1589
Clozapinum ..............................................................................1590
Cocaini hydrochloridum.......................................................1592
Cocois oleum raffinatum................................................6.2-3723
Cocoylis caprylocapras..........................................................1594
Codeini hydrochloridum dihydricum................................1596
Codeini phosphas hemihydricus ........................................1598
Codeini phosphas sesquihydricus ......................................1599
Codeinum...........................................................................6.1-3434
Codergocrini mesilas ......................................................6.3-4103
Coffeinum...........................................................................6.1-3421
Coffeinum monohydricum ...................................................1365
Colae semen ............................................................................. 1611
Colchicinum ............................................................................. 1612
Colestyraminum ...................................................................... 1613
Colistimethatum natricum ................................................... 1614
Colistini sulfas ......................................................................... 1615
Colophonium ............................................................................1617
Compressi.................................................................................... 748
Copolymerum methacrylatis butylati basicum ...............1254
Copovidonum............................................................................1617
Coriandri aetheroleum.......................................................... 1621
Coriandri fructus .................................................................... 1620
Corpora ad usum pharmaceuticum ............................6.3-3969
Cortisoni acetas....................................................................... 1622
Crataegi folii cum flore
extractum fluidum quantificatum ....................................2037
Crataegi folii cum flore extractum siccum.......................2036
Crataegi folium cum flore.....................................................2035
Crataegi fructus .......................................................................2034
Cresolum crudum ................................................................... 1626
Croci stigma ad praeparationes homoeopathicas.......... 1084
Crospovidonum ................................................................ 6.3-4119
Crotamitonum.......................................................................... 1629
Cupri acetas monohydricus ad praeparationes
homoeopathicas .................................................................... 1075
Cupri sulfas anhydricus ........................................................ 1619
Cupri sulfas pentahydricus .................................................. 1620
Cuprum ad praeparationes homoeopathicas .................. 1076
Curcumae xanthorrhizae rhizoma..................................... 3150
Cyamopsidis seminis pulvis..........................................6.3-4158
Cyanocobalamini (57Co) capsulae ........................................ 983
EUROPEAN PHARMACOPOEIA 6.3
Cyanocobalamini (57Co) solutio ............................................ 984
Cyanocobalamini (58Co) capsulae ........................................ 985
Cyanocobalamini (58Co) solutio ............................................ 986
Cyanocobalaminum ............................................................... 1630
Cyclizini hydrochloridum..............................................6.2-3725
Cyclopentolati hydrochloridum .......................................... 1632
Cyclophosphamidum ............................................................. 1633
Cynarae folii extractum siccum ...................................6.3-4041
Cynarae folium ........................................................................ 1219
Cyproheptadini hydrochloridum........................................ 1634
Cyproteroni acetas.................................................................. 1635
Cysteini hydrochloridum monohydricum........................ 1636
Cystinum ................................................................................... 1637
Cytarabinum............................................................................. 1638
D Dimercaprolum........................................................................ 1716
Dacarbazinum ......................................................................... 1641
Dalteparinum natricum ........................................................ 1642
Danaparoidum natricum ...................................................... 1644
Dapsonum................................................................................. 1646
Daunorubicini hydrochloridum.......................................... 1647
D-Camphora .............................................................................. 1400
Decylis oleas............................................................................. 1648
Deferoxamini mesilas ............................................................ 1649
Dembrexini hydrochloridum monohydricum ad usum
veterinarium .......................................................................... 1650
Demeclocyclini hydrochloridum ........................................ 1651
Deptropini citras ..................................................................... 1653
Dequalinii chloridum............................................................. 1654
Desfluranum......................................................................6.1-3439
Desipramini hydrochloridum .............................................. 1655
Deslanosidum .......................................................................... 1656
Desmopressinum..................................................................... 1657
Desogestrelum.......................................................................... 1658
Desoxycortoni acetas ............................................................. 1659
Detomidini hydrochloridum ad usum veterinarium..... 1660
Dexamethasoni acetas ....................................................6.3-4123
Dexamethasoni isonicotinas................................................ 1666
Dexamethasoni natrii phosphas ......................................... 1667
Dexamethasonum ................................................................... 1663
Dexchlorpheniramini maleas .............................................. 1669
Dexpanthenolum..................................................................... 1670
Dextranomerum ...................................................................... 1675
Dextranum 1 ad iniectabile ..........................................6.3-4124
Dextranum 40 ad iniectabile ........................................6.3-4125
Dextranum 60 ad iniectabile ........................................6.3-4126
Dextranum 70 ad iniectabile ........................................6.3-4127
Dextrinum................................................................................. 1675
Dextromethorphani hydrobromidum ................................ 1676
Dextromoramidi tartras......................................................... 1677
Dextropropoxypheni hydrochloridum............................... 1678
Diazepamum ............................................................................ 1679
Diazoxidum .............................................................................. 1680
Dibrompropamidini diisetionas.......................................... 1681
Dibutylis phthalas ................................................................... 1682
Diclazurilum ad usum veterinarium................................. 1683
Diclofenacum kalicum........................................................... 1685
Diclofenacum natricum ........................................................ 1686
Dicloxacillinum natricum..................................................... 1687
Dicycloverini hydrochloridum ............................................ 1689
Didanosinum............................................................................ 1689
Dienestrolum............................................................................ 1691
Diethylcarbamazini citras .................................................... 1693
Diethylenglycoli aether monoethilicus ............................. 1694
Diethylenglycoli palmitostearas.......................................... 1695
Diethylis phthalas ............................................................6.1-3441
Diethylstilbestrolum ............................................................... 1696
Diflunisalum ............................................................................ 1697
Digitalis purpureae folium ................................................... 1698
General Notices (1) apply to all monographs and other texts
Index
Digitoxinum.............................................................................. 1700
Digoxinum ................................................................................ 1701
Dihydralazini sulfas hydricus.......................................6.1-3442
Dihydrocodeini hydrogenotartras ...................................... 1704
Dihydroergocristini mesilas................................................. 1705
Dihydroergotamini mesilas...........................................6.1-3444
Dihydroergotamini tartras.................................................... 1709
Dihydrostreptomycini sulfas ad usum veterinarium..... 1710
Dihydrostreptomycini sulfas ad usum veterinarium.......6.2-
3730
Dihydrotachysterolum ........................................................... 1712
Dikalii clorazepas ................................................................... 1728
Dikalii phosphas...................................................................... 1729
Diltiazemi hydrochloridum...........................................6.1-3446
Dimenhydrinatum .................................................................. 1715
Dimethylacetamidum..............................................................1717
Dimethylis sulfoxidum........................................................... 1716
Dimeticonum.....................................................................6.2-3732
Dimetindeni maleas ............................................................... 1719
Dinatrii clodronas tetrahydricus .................................6.2-3722
Dinatrii edetas ......................................................................... 1734
Dinatrii etidronas ................................................................... 1844
Dinatrii pamidronas pentahydricus ..................................2604
Dinatrii phosphas anhydricus......................................6.3-4128
Dinatrii phosphas dihydricus .............................................. 1735
Dinatrii phosphas dodecahydricus .............................6.1-3449
Dinitrogenii oxidum............................................................... 2515
Dinoprostonum........................................................................ 1722
Dinoprostum trometamolum ............................................... 1720
Diosminum ............................................................................... 1723
Diphenhydramini hydrochloridum.................................... 1725
Diphenoxylati hydrochloridum........................................... 1726
Dipivefrini hydrochloridum ................................................. 1727
Diprophyllinum ....................................................................... 1730
Dipyridamolum ....................................................................... 1731
Dirithromycinum .............................................................6.1-3447
Disopyramidi phosphas......................................................... 1738
Disopyramidum....................................................................... 1737
Disulfiramum ........................................................................... 1739
Dithranolum ............................................................................. 1740
DL-Methioninum ......................................................................2380
DL-α-Tocopherylis hydrogenosuccinas..............................3093
Dobutamini hydrochloridum ................................................1741
Dodecylis gallas....................................................................... 1744
Domperidoni maleas.............................................................. 1747
Domperidonum ....................................................................... 1745
Dopamini hydrochloridum................................................... 1749
Dopexamini dihydrochloridum........................................... 1750
Dorzolamidi hydrochloridum.............................................. 1752
Dosulepini hydrochloridum................................................. 1753
Doxaprami hydrochloridum ................................................ 1754
Doxazosini mesilas................................................................. 1756
Doxepini hydrochloridum .............................................6.1-3449
Doxorubicini hydrochloridum............................................. 1759
Doxycyclini hyclas.................................................................. 1760
Doxycyclinum monohydricum............................................ 1762
Doxylamini hydrogenosuccinas ..................................6.1-3451
Droperidolum........................................................................... 1765
Dydrogesteronum ............................................................6.3-4128
E
Ebastinum ................................................................................. 1771
Echinaceae angustifoliae radix...........................................2483
Echinaceae pallidae radix ....................................................2602
Echinaceae purpureae herba...............................................2785
Echinaceae purpureae radix................................................2787
Econazoli nitras ...................................................................... 1773
Econazolum.............................................................................. 1772
4391
Index
Edrophonii chloridum ........................................................... 1775
Eleutherococci radix .............................................................. 1777
Emedastini difumaras............................................................ 1779
Emetini hydrochloridum heptahydricum......................... 1780
Emetini hydrochloridum pentahydricum ........................ 1781
Emplastra transcutanea .......................................................... 737
Enalaprilatum dihydricum................................................... 1784
Enalaprili maleas.................................................................... 1782
Enilconazolum ad usum veterinarium ............................. 1785
Enoxaparinum natricum ...................................................... 1787
Enoxolonum ............................................................................. 1788
Ephedrini hydrochloridum .................................................. 1791
Ephedrini racemici hydrochloridum................................. 1792
Ephedrinum anhydricum ..................................................... 1789
Ephedrinum hemihydricum ................................................ 1790
Epirubicini hydrochloridum................................................ 1793
Equiseti herba .......................................................................... 1794
Ergocalciferolum..............................................................6.3-4133
Ergometrini maleas................................................................ 1797
Ergotamini tartras .................................................................. 1798
Erythritolum......................................................................6.3-4134
Erythromycini estolas............................................................ 1803
Erythromycini ethylsuccinas............................................... 1806
Erythromycini lactobionas................................................... 1808
Erythromycini stearas ........................................................... 1810
Erythromycinum..................................................................... 1801
Erythropoietini solutio concentrata................................... 1813
Eserini salicylas ......................................................................2677
Eserini sulfas............................................................................2678
Esketamini hydrochloridum .................................................1817
Esomeprazolum magnesicum trihydricum ..............6.3-4136
Estradioli benzoas............................................................6.1-3455
Estradioli valeras..................................................................... 1821
Estradiolum hemihydricum ................................................. 1819
Estriolum................................................................................... 1822
Estrogeni coniuncti ................................................................ 1824
Etamsylatum .....................................................................6.2-3737
Ethacridini lactas monohydricus ................................6.3-4138
Ethambutoli hydrochloridum .......................................6.1-3456
Ethanolum (96 per centum)................................................. 1829
Ethanolum anhydricum ........................................................ 1831
Ethinylestradiolum ................................................................. 1834
Ethionamidum......................................................................... 1835
Ethosuximidum ....................................................................... 1836
Ethylcellulosum ....................................................................... 1841
Ethylendiaminum ................................................................... 1843
Ethylenglycoli monopalmitostearas................................... 1842
Ethylis acetas ........................................................................... 1838
Ethylis oleas.............................................................................. 1838
Ethylis parahydroxybenzoas................................................ 1839
Ethylis parahydroxybenzoas natricus ............................... 1840
Ethylmorphini hydrochloridum.......................................... 1843
Etilefrini hydrochloridum..................................................... 1845
Etodolacum............................................................................... 1847
Etofenamatum ......................................................................... 1849
Etofyllinum ............................................................................... 1850
Etomidatum .............................................................................. 1851
Etoposidum............................................................................... 1852
Eucalypti aetheroleum ...................................................6.2-3738
Eucalypti folium...................................................................... 1857
Eugenolum................................................................................ 1859
Extracta...............................................................................6.1-3343
F Fluorouracilum........................................................................1920
Factor humanus von Willebrandi....................................... 2081
Factor IX coagulationis humanus ......................................2064
Factor VII coagulationis humanus .................................... 2061
Factor VIII coagulationis humanus...................................2062
Factor VIII coagulationis humanus (ADNr) ....................2063
4392 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Factor XI coagulationis humanus ......................................2065
Fagopyri herba ........................................................................ 1341
Famotidinum............................................................................ 1865
Febantelum ad usum veterinarium.................................... 1870
Felbinacum ............................................................................... 1866
Felodipinum ............................................................................. 1867
Felypressinum.......................................................................... 1869
Fenbendazolum ad usum veterinarium............................ 1871
Fenbufenum.............................................................................. 1872
Fenofibratum............................................................................ 1875
Fenoteroli hydrobromidum .................................................. 1876
Fentanyli citras........................................................................ 1879
Fentanylum............................................................................... 1878
Fenticonazoli nitras ............................................................... 1880
Ferri chloridum hexahydricum........................................... 1882
Ferrosi fumaras ....................................................................... 1883
Ferrosi gluconas............................................................... 6.3-4141
Ferrosi sulfas desiccatus ....................................................... 1885
Ferrosi sulfas heptahydricus................................................ 1886
Ferrum ad praeparationes homoeopathicas ................... 1081
Fexofenadini hydrochloridum............................................. 1888
Fibrini glutinum...................................................................... 1890
Fibrinogenum humanum .....................................................2066
Fila non resorbilia sterilia.................................................... 1046
Fila non resorbilia sterilia in fuso ad usum
veterinarium .......................................................................... 1060
Fila resorbilia synthetica monofilamenta sterilia.......... 1052
Fila resorbilia synthetica torta sterilia.............................. 1050
Filgrastimi solutio concentrata .................................... 6.3-4142
Filipendulae ulmariae herba ...............................................2344
Filum bombycis tortum sterile in fuso ad usum
veterinarium .......................................................................... 1059
Filum ethyleni polyterephthalici sterile in fuso ad usum
veterinarium .......................................................................... 1059
Filum lini sterile in fuso ad usum veterinarium ............ 1058
Filum polyamidicum-6/6 sterile in fuso ad usum
veterinarium .......................................................................... 1059
Filum polyamidicum-6 sterile in fuso ad usum
veterinarium .......................................................................... 1058
Finasteridum............................................................................ 1891
Flavoxati hydrochloridum .................................................... 1895
Flecainidi acetas ..................................................................... 1896
Flubendazolum........................................................................ 1898
Flucloxacillinum magnesicum octahydricum.......... 6.2-3741
Flucloxacillinum natricum................................................... 1899
Fluconazolum ..........................................................................1900
Flucytosinum ...........................................................................1902
Fludarabini phosphas ............................................................1903
Fludeoxyglucosi (18F) solutio iniectabilis ..................6.2-3678
Fludrocortisoni acetas ...........................................................1906
Flumazenili (N-[11C]methyl) solutio iniectabilis ............... 989
Flumazenilum..........................................................................1908
Flumequinum...........................................................................1909
Flumetasoni pivalas ............................................................... 1910
Flunarizini dihydrochloridum ............................................ 1911
Flunitrazepamum ................................................................... 1913
Flunixini megluminum ad usum veterinarium.............. 1914
Fluocinoloni acetonidum ..................................................... 1915
Fluocortoloni pivalas ............................................................. 1916
Fluoresceinum ......................................................................... 1918
Fluoresceinum natricum ...................................................... 1919
Fluorodopae (18F) ab electrophila substitutione solutio
iniectabilis ................................................................................ 990
Fluoxetini hydrochloridum ..................................................1922
Flupentixoli dihydrochloridum ...........................................1924
Fluphenazini decanoas.........................................................1926
Fluphenazini dihydrochloridum ........................................1928
Fluphenazini enantas............................................................1927
EUROPEAN PHARMACOPOEIA 6.3
Flurazepami monohydrochloridum...................................1930
Flurbiprofenum ....................................................................... 1931
Fluspirilenum ..........................................................................1932
Flutamidum ..............................................................................1933
Fluticasoni propionas............................................................1934
Flutrimazolum .........................................................................1936
Fluvoxamini maleas........................................................ 6.3-4144
Foeniculi amari fructus......................................................... 1873
Foeniculi amari fructus aetheroleum................................ 1318
Foeniculi dulcis fructus......................................................... 1874
Formaldehydi solutio (35 per centum)..............................1939
Formoteroli fumaras dihydricus .........................................1940
Foscarnetum natricum hexahydricum..............................1942
Fosfomycinum calcicum .......................................................1943
Fosfomycinum natricum.......................................................1945
Fosfomycinum trometamolum ............................................1946
Framycetini sulfas ..................................................................1947
Frangulae cortex .....................................................................1949
Frangulae corticis extractum siccum normatum .... 6.3-4146
Fraxini folium ..........................................................................1222
Fructosum ................................................................................. 1951
Fucus vel Ascophyllum.......................................................... 2213
Fumariae herba.......................................................................1952
Furosemidum...........................................................................1953
G Hexobarbitalum .......................................................................2047
Galactosum ........................................................................ 6.3-4151
Gallamini triethiodidum .......................................................1959
Gallii (67Ga) citratis solutio iniectabilis ............................... 992
Gelatina .............................................................................. 6.3-4151
Gemcitabini hydrochloridum...............................................1963
Gemfibrozilum .........................................................................1964
Gentamicini sulfas ..................................................................1965
Gentianae radix .......................................................................1967
Gentianae tinctura..................................................................1968
Ginkgonis extractum siccum raffinatum et
quantificatum .................................................................6.1-3461
Ginkgonis folium.....................................................................1969
Ginseng radix........................................................................... 1971
Glibenclamidum ......................................................................1972
Gliclazidum............................................................................... 1974
Glimepiridum...........................................................................1975
Glipizidum ................................................................................1977
Glucagonum humanum ........................................................1979
Glucosum anhydricum ...................................................6.3-4153
Glucosum liquidum .........................................................6.2-3752
Glucosum liquidum dispersione desiccatum............6.3-4154
Glucosum monohydricum .............................................6.3-4154
Glutathionum ....................................................................6.1-3463
Glyceroli dibehenas ................................................................1990
Glyceroli distearas .................................................................. 1991
Glyceroli monocaprylas ........................................................1992
Glyceroli monocaprylocapras..............................................1993
Glyceroli monolinoleas ........................................................1994
Glyceroli mono-oleas.......................................................6.3-4155
Glyceroli monostearas 40-55................................................1996
Glyceroli trinitratis solutio ............................................6.1-3465
Glycerolum................................................................................1987
Glycerolum (85 per centum) ................................................1988
Glycinum ...................................................................................1998
Gonadorelini acetas ...............................................................2003
Gonadotropinum chorionicum ...........................................2004
Gonadotropinum sericum equinum ad usum
veterinarium ..........................................................................2005
Goserelinum .............................................................................2005
Gossypii oleum hydrogenatum.....................................6.2-3724
Gramicidinum..........................................................................2007
Graminis rhizoma................................................................... 1625
Granisetroni hydrochloridum.......................................6.3-4156
General Notices (1) apply to all monographs and other texts
Index
Granulata .................................................................................... 723
Griseofulvinum ........................................................................ 2011
Guaiacolum .............................................................................. 2012
Guaifenesinum ........................................................................ 2014
Guanethidini monosulfas ..................................................... 2015
Guar galactomannanum................................................6.3-4159
H
Halofantrini hydrochloridum ..............................................2027
Haloperidoli decanoas...........................................................2030
Haloperidolum.........................................................................2028
Halothanum.............................................................................. 2031
Hamamelidis folium........................................................6.1-3471
Harpagophyti extractum siccum......................................... 1662
Harpagophyti radix .........................................................6.2-3729
Hederae folium ........................................................................ 2198
Hedera helix ad praeparationes homoeopathicas.......... 1078
Helianthi annui oleum raffinatum..............................6.2-3848
Helium .......................................................................................2038
Heparina massae molecularis minoris ............................. 2041
Heparinum calcicum .............................................................2039
Heparinum natricum.............................................................2040
Heptaminoli hydrochloridum..............................................2043
Hexamidini diisetionas .........................................................2044
Hexetidinum.............................................................................2045
Hexylresorcinolum .................................................................2047
Hibisci sabdariffae flos ...................................................6.1-3529
Histamini dihydrochloridum ...............................................2049
Histamini phosphas................................................................2049
Histidini hydrochloridum monohydricum....................... 2051
Histidinum ................................................................................2050
Homatropini hydrobromidum .............................................2052
Homatropini methylbromidum ...........................................2053
Hyaluronidasum .....................................................................2082
Hydralazini hydrochloridum ...............................................2083
Hydrargyri dichloridum ........................................................ 2361
Hydrastis rhizoma ...........................................................6.1-3467
Hydrochlorothiazidum ..........................................................2086
Hydrocodoni hydrogenotartras 2.5-hydricus ..................2087
Hydrocortisoni acetas............................................................ 2091
Hydrocortisoni hydrogenosuccinas ...................................2092
Hydrocortisonum ....................................................................2089
Hydrogenii peroxidum 30 per centum..............................2094
Hydrogenii peroxidum 3 per centum ................................2094
Hydromorphoni hydrochloridum .......................................2095
Hydroxocobalamini acetas ...................................................2096
Hydroxocobalamini chloridum ...........................................2098
Hydroxocobalamini sulfas ....................................................2099
Hydroxycarbamidum ............................................................. 2100
Hydroxyethylcellulosum........................................................ 2102
Hydroxyethylis salicylas........................................................ 2101
Hydroxypropylbetadexum ............................................. 6.3-4170
Hydroxypropylcellulosum .................................................... 2105
Hydroxyzini hydrochloridum .............................................. 2106
Hymecromonum...................................................................... 2107
Hyoscini butylbromidum ...................................................... 2109
Hyoscini hydrobromidum..................................................... 2110
Hyoscinum................................................................................ 2108
Hyoscyamini sulfas ................................................................ 2112
Hyoscyamus niger
ad praeparationes homoeopathicas................................. 1079
Hyperici herba ..................................................................6.2-3839
Hyperici herbae extractum siccum quantificatum ..6.3-4309
Hypericum perforatum
ad praeparationes homoeopathicas................................. 1080
Hypromellosi phthalas.................................................... 6.3-4174
Hypromellosum ................................................................ 6.3-4171
4393
Index
I Interferoni gamma-1b solutio concentrata ...................... 2153
Ibuprofenum......................................................................6.1-3479
Ichthammolum ................................................................. 6.3-4177
Idoxuridinum........................................................................... 2122
Iecoris aselli oleum A...................................................... 6.3-4109
Iecoris aselli oleum B ..................................................... 6.3-4113
Iecoris aselli oleum domestici ...................................... 6.3-4105
Ifosfamidum ............................................................................. 2123
Imipenemum............................................................................ 2125
Imipramini hydrochloridum.........................................6.2-3769
Immunoglobulinum anti-T lymphocytorum ex animale ad
usum humanum....................................................................1203
Immunoglobulinum humanum anti-D.......................6.2-3757
Immunoglobulinum humanum anti-D ad usum
intravenosum .........................................................................2059
Immunoglobulinum humanum hepatitidis A .................2068
Immunoglobulinum humanum hepatitidis B .................2069
Immunoglobulinum humanum hepatitidis B ad usum
intravenosum .........................................................................2069
Immunoglobulinum humanum morbillicum ..................2069
Immunoglobulinum humanum normale ..................6.2-3757
Immunoglobulinum humanum normale ad usum
intravenosum .................................................................. 6.3-4166
Immunoglobulinum humanum rabicum..........................2078
Immunoglobulinum humanum rubellae..........................2079
Immunoglobulinum humanum tetanicum ......................2079
Immunoglobulinum humanum varicellae.......................2080
Immunoglobulinum humanum varicellae ad usum
intravenosum ......................................................................... 2081
Immunosera ad usum veterinarium.................................... 687
Immunosera ex animali ad usum humanum.................... 685
Immunoserum botulinicum ................................................... 965
Immunoserum Clostridii novyi alpha ad usum
veterinarium ............................................................................ 973
Immunoserum Clostridii perfringentis beta ad usum
veterinarium ............................................................................ 974
Immunoserum Clostridii perfringentis epsilon ad usum
veterinarium ............................................................................ 975
Immunoserum contra venena viperarum
europaearum ........................................................................... 970
Immunoserum diphthericum ................................................ 965
Immunoserum gangraenicum (Clostridium novyi) ........ 966
Immunoserum gangraenicum
(Clostridium perfringens)..................................................... 967
Immunoserum gangraenicum (Clostridium septicum).. 968
Immunoserum gangraenicum mixtum............................... 966
Immunoserum tetanicum ad usum humanum................. 969
Immunoserum tetanicum ad usum veterinarium............ 976
Indapamidum .......................................................................... 2127
Indii (111In) chloridi solutio .................................................... 994
Indii (111In) oxini solutio ......................................................... 995
Indii (111In) pentetatis solutio iniectabilis .......................... 996
Indinaviri sulfas ...................................................................... 2130
Indometacinum ....................................................................... 2132
Inhalanda.................................................................................... 739
Insulini zinci amorphi suspensio iniectabilis................. 2149
Insulini zinci cristallini suspensio iniectabilis............... 2149
Insulini zinci suspensio iniectabilis .................................. 2148
Insulinum aspartum .............................................................. 2133
Insulinum biphasicum iniectabile ..................................... 2140
Insulinum bovinum................................................................ 2135
Insulinum humanum............................................................. 2137
Insulinum isophanum biphasicum iniectabile............... 2140
Insulinum isophanum iniectabile .......................................2141
Insulinum lisprum...................................................................2141
Insulinum porcinum.............................................................. 2144
Insulinum solubile iniectabile..............................................2141
Interferoni alfa-2 solutio concentrata................................ 2150
Interferoni beta-1a solutio concentrata...................... 6.3-4177
4394 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
int-rac-α-Tocopherolum .........................................................3086
int-rac-α-Tocopherylis acetas ...............................................3089
Iobenguani (123I) solutio iniectabilis.................................... 997
Iobenguani (131I) solutio iniectabilis ad usum
diagnosticum ........................................................................... 998
Iobenguani (131I) solutio iniectabilis ad usum
therapeuticum.......................................................................... 999
Iobenguani sulfas ad radiopharmaceutica ...............6.1-3381
Iodinati (125I) humani albumini solutio iniectabilis ........ 993
Iodum ......................................................................................... 2156
Iohexolum ................................................................................. 2157
Iopamidolum............................................................................ 2160
Iotrolanum................................................................................ 2164
Ipecacuanhae extractum fluidum normatum ................. 2168
Ipecacuanhae pulvis normatus....................................6.2-3770
Ipecacuanhae radix................................................................ 2170
Ipecacuanhae tinctura normata ......................................... 2171
Ipratropii bromidum .......................................................6.2-3771
Isoconazoli nitras ................................................................... 2175
Isoconazolum........................................................................... 2173
Isofluranum...............................................................................2176
Isoleucinum.............................................................................. 2177
Isomaltum ................................................................................. 2178
Isoniazidum ............................................................................. 2180
Isoprenalini hydrochloridum .............................................. 2181
Isoprenalini sulfas .................................................................. 2182
Isopropylis myristas ............................................................... 2183
Isopropylis palmitas............................................................... 2184
Isosorbidi dinitras dilutus..................................................... 2185
Isosorbidi mononitras dilutus ............................................. 2186
Isotretinoinum ......................................................................... 2188
Isoxsuprini hydrochloridum ................................................ 2189
Isradipinum.............................................................................. 2192
Itraconazolum.......................................................................... 2194
Iuniperi aetheroleum.............................................................2207
Iuniperi pseudo-fructus.........................................................2206
Ivermectinum........................................................................... 2196
J
Josamycini propionas............................................................2205
Josamycinum ...........................................................................2204
K
Kalii acetas ............................................................................... 2716
Kalii bromidum........................................................................ 2716
Kalii carbonas .......................................................................... 2717
Kalii chloridum.................................................................6.2-3819
Kalii citras..........................................................................6.3-4276
Kalii clavulanas ....................................................................... 2719
Kalii clavulanas dilutus......................................................... 2721
Kalii dihydrogenophosphas .................................................2723
Kalii dihydrogenophosphas ..........................................6.3-4277
Kalii hydrogenoaspartas hemihydricus............................2723
Kalii hydrogenocarbonas......................................................2724
Kalii hydrogenotartras...........................................................2725
Kalii hydroxidum ....................................................................2726
Kalii iodidum ...........................................................................2726
Kalii metabisulfis ....................................................................2727
Kalii natrii tartras tetrahydricus.........................................2729
Kalii nitras ................................................................................2728
Kalii perchloras .......................................................................2728
Kalii permanganas .................................................................2729
Kalii sorbas ...............................................................................2730
Kalii sulfas ................................................................................ 2731
Kanamycini monosulfas ....................................................... 2212
Kanamycini sulfas acidus..................................................... 2211
Kaolinum ponderosum...................................................6.3-4183
EUROPEAN PHARMACOPOEIA 6.3
Ketamini hydrochloridum .................................................... 2214
Ketoconazolum........................................................................ 2216
Ketoprofenum .......................................................................... 2218
Ketorolacum trometamolum ................................................2220
Ketotifeni hydrogenofumaras .............................................. 2221
Kryptonum (81mKr) ad inhalationem ................................ 1000
L Macrogola..................................................................................2308
Labetaloli hydrochloridum...................................................2227
Lacca ...................................................................................6.2-3833
Lactitolum monohydricum............................................6.3-4187
Lactosum anhydricum....................................................6.3-4188
Lactosum monohydricum..............................................6.3-4190
Lactulosum ........................................................................ 6.3-4191
Lactulosum liquidum......................................................6.3-4193
Lamivudinum ..........................................................................2238
Lamotriginum...................................................................6.3-4195
Lansoprazolum........................................................................2240
Lanugo cellulosi absorbens.................................................. 3197
Lanugo gossypii absorbens .................................................. 1624
Lauromacrogolum 400...................................................6.3-4196
Lavandulae aetheroleum ......................................................2244
Lavandulae flos .......................................................................2243
Leflunomidum .........................................................................2245
Leonuri cardiacae herba.......................................................2447
Letrozolum................................................................................2249
Leucinum ..................................................................................2250
Leuprorelinum......................................................................... 2251
Levamisoli hydrochloridum.................................................2254
Levamisolum ad usum veterinarium ................................2253
Levistici radix...........................................................................2290
Levocabastini hydrochloridum ...........................................2255
Levocarnitinum.......................................................................2257
Levodopum ...............................................................................2258
Levodropropizinum.........................................................6.3-4200
Levomentholum....................................................................... 2261
Levomepromazini hydrochloridum ...................................2262
Levomepromazini maleas ....................................................2263
Levomethadoni hydrochloridum ........................................2264
Levonorgestrelum ...................................................................2266
Levothyroxinum natricum ...................................................2267
Lichen islandicus.................................................................... 2121
Lidocaini hydrochloridum ...................................................2269
Lidocainum .......................................................................6.1-3485
Limonis aetheroleum.............................................................2246
Lincomycini hydrochloridum..............................................2271
Lindanum .................................................................................2272
Lini oleum virginale .............................................................. 2274
Lini semen ................................................................................2273
Liothyroninum natricum ..............................................6.1-3486
Liquiritiae extractum fluidum ethanolicum normatum ..6.2-
3775
Liquiritiae extractum siccum ad saporandum.........6.1-3488
Liquiritiae radix ...................................................................... 2276
Lisinoprilum dihydricum .....................................................2277
Lithii carbonas.........................................................................2279
Lithii citras ...............................................................................2279
L-Methionini ([11C]methyl) solutio iniectabilis ................ 1001
Lobelini hydrochloridum......................................................2280
Lomustinum ............................................................................. 2281
Loperamidi hydrochloridum................................................2283
Loperamidi oxidum monohydricum..................................2285
Loratadinum ............................................................................2286
Lorazepamum..........................................................................2288
Lovastatinum ........................................................................... 2291
Lupuli flos..........................................................................6.1-3472
Lymecyclinum ..................................................................6.1-3489
Lynestrenolum..................................................................6.3-4202
Lysini acetas.............................................................................2295
General Notices (1) apply to all monographs and other texts
Index
Lysini hydrochloridum..........................................................2296
Lythri herba..............................................................................2283
M
Macrogol 20 glyceroli monostearas ...................................2304
Macrogol 40 sorbitoli heptaoleas.................................6.3-4207
Macrogol 6 glyceroli caprylocapras...................................2302
Macrogolglyceridorum caprylocaprates ........................... 1403
Macrogolglyceridorum laurates ..........................................2242
Macrogolglyceridorum linoleates.......................................2273
Macrogolglyceridorum oleates ............................................2543
Macrogolglyceridorum stearates.........................................2967
Macrogolglyceroli cocoates ..................................................2302
Macrogolglyceroli hydroxystearas......................................2303
Macrogolglyceroli ricinoleas................................................2304
Macrogoli 15 hydroxystearas ...............................................2305
Macrogoli aether cetostearylicus ........................................ 2301
Macrogoli aether laurilicus ..................................................2306
Macrogoli aether oleicus.......................................................2308
Macrogoli aether stearylicus................................................ 2312
Macrogoli oleas........................................................................2307
Macrogoli stearas .................................................................... 2311
Magaldratum .....................................................................6.3-4207
Magnesii acetas tetrahydricus ............................................. 2313
Magnesii aspartas dihydricus.............................................. 2314
Magnesii chloridum 4.5-hydricum ..................................... 2317
Magnesii chloridum hexahydricum ................................... 2316
Magnesii citras anhydricus .................................................. 2318
Magnesii gluconas ...........................................................6.1-3495
Magnesii glycerophosphas ................................................... 2318
Magnesii hydroxidum ............................................................ 2319
Magnesii lactas dihydricus...................................................2320
Magnesii oxidum leve .....................................................6.3-4209
Magnesii oxidum ponderosum.....................................6.3-4209
Magnesii peroxidum............................................................... 2321
Magnesii pidolas .....................................................................2322
Magnesii stearas...............................................................6.3-4210
Magnesii subcarbonas levis...........................................6.3-4208
Magnesii subcarbonas ponderosus .............................6.2-3779
Magnesii sulfas heptahydricus ............................................2325
Magnesii trisilicas...................................................................2325
Malathionum ............................................................................2327
Maltitolum..........................................................................6.3-4213
Maltitolum liquidum...............................................................2332
Maltodextrinum ................................................................ 6.3-4214
Malvae folium....................................................................6.3-4212
Malvae sylvestris flos .............................................................2330
Mangani gluconas ...........................................................6.1-3495
Mangani glycerophosphas hydricus ..................................2334
Mangani sulfas monohydricus ............................................2335
Mannitolum .......................................................................6.3-4215
Maprotilini hydrochloridum ................................................2337
Marbofloxacinum ad usum veterinarium .................6.1-3496
Marrubii herba......................................................................... 3216
Masticabilia gummis medicata.............................................. 719
Mastix .........................................................................................2340
Matricariae aetheroleum.......................................................2342
Matricariae extractum fluidum ....................................6.2-3780
Matricariae flos .......................................................................2340
Maydis amylum ................................................................6.3-4212
Maydis oleum raffinatum...............................................6.2-3779
Mebendazolum.........................................................................2345
Meclozini hydrochloridum ...................................................2346
Medroxyprogesteroni acetas ................................................2347
Mefloquini hydrochloridum.................................................2350
Megestroli acetas .....................................................................2352
Megluminum ............................................................................2353
Mel...............................................................................................2055
4395
Index
Melaleucae aetheroleum ....................................................... 3019
Meliloti herba ...........................................................................2354
Melissae folium........................................................................2355
Meloxicamum....................................................................6.3-4218
Menadionum ............................................................................2356
Menthae arvensis aetheroleum partim mentholum
depletum..................................................................................2430
Menthae piperitae aetheroleum ..........................................2639
Menthae piperitae folium .....................................................2638
Mentholum racemicum .........................................................2356
Menyanthidis trifoliatae folium ..........................................1323
Mepivacaini hydrochloridum ..............................................2357
Meprobamatum........................................................................2359
Mepyramini maleas................................................................2360
Mercaptopurinum ................................................................... 2361
Mesalazinum ............................................................................2362
Mesnum .....................................................................................2364
Mesterolonum ..........................................................................2366
Mestranolum.............................................................................2367
Metacresolum ...........................................................................2368
Metamizolum natricum.........................................................2369
Metformini hydrochloridum ................................................2370
Methadoni hydrochloridum ................................................. 2374
Methanolum.............................................................................. 2376
Methaqualonum ......................................................................2377
Methenaminum .......................................................................2378
Methioninum............................................................................2379
Methotrexatum..................................................................6.3-4220
Methylatropini bromidum.....................................................2383
Methylatropini nitras .............................................................2383
Methylcellulosum .............................................................6.3-4223
Methyldopum ...........................................................................2386
Methyleni chloridum..............................................................2387
Methylergometrini maleas....................................................2388
Methylhydroxyethylcellulosum............................................2390
Methylis nicotinas...................................................................2390
Methylis parahydroxybenzoas ............................................. 2391
Methylis parahydroxybenzoas natricus ............................ 2911
Methylis salicylas .................................................................... 2401
Methylphenidati hydrochloridum................................6.3-4224
Methylphenobarbitalum ........................................................2392
Methylprednisoloni acetas....................................................2395
Methylprednisoloni hydrogenosuccinas...........................2397
Methylprednisolonum............................................................2393
Methylrosanilinii chloridum ................................................2400
Methyltestosteronum .......................................................6.3-4226
Methylthioninii chloridum ...................................................2402
Metixeni hydrochloridum .....................................................2404
Metoclopramidi hydrochloridum........................................2407
Metoclopramidum............................................................6.2-3783
Metolazonum............................................................................2407
Metoprololi succinas ..............................................................2409
Metoprololi tartras .................................................................. 2410
Metrifonatum............................................................................ 2412
Metronidazoli benzoas .......................................................... 2415
Metronidazolum ...................................................................... 2414
Mexiletini hydrochloridum................................................... 2416
Mianserini hydrochloridum..........................................6.3-4227
Miconazoli nitras ....................................................................2420
Miconazolum............................................................................ 2418
Midazolamum ..........................................................................2422
Millefolii herba.........................................................................3243
Minocyclini hydrochloridum dihydricum........................2427
Minoxidilum .............................................................................2429
Mirtazapinum .......................................................................... 2431
Misoprostolum .........................................................................2433
Mitomycinum ...........................................................................2434
Mitoxantroni hydrochloridum.............................................2436
Modafinilum .............................................................................2437
4396 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Molgramostimi solutio concentrata ...................................2438
Molsidominum..................................................................6.1-3499
Mometasoni furoas ................................................................. 2441
Moranteli hydrogenotartras ad usum veterinarium......2443
Morphini hydrochloridum.............................................6.1-3501
Morphini sulfas.................................................................6.2-3785
Moxidectinum ad usum veterinarium........................6.3-4228
Moxifloxacini hydrochloridum ....................................6.2-3786
Moxonidinum...........................................................................2453
Mupirocinum............................................................................2454
Mupirocinum calcicum .........................................................2456
Musci medicati........................................................................... 723
Mycophenolas mofetil............................................................2458
myo-Inositolum........................................................................2460
Myristicae fragrantis aetheroleum ..............................6.2-3797
Myrrha ....................................................................................... 2461
Myrrhae tinctura ..................................................................... 2461
Myrtilli fructus recens.....................................................6.1-3412
Myrtilli fructus recentis extractum siccum raffinatum et
normatum ........................................................................6.2-3745
Myrtilli fructus siccus.............................................................1307
N
Nabumetonum .........................................................................2465
N-Acetyltryptophanum.................................................... 6.3-4016
N-Acetyltyrosinum .................................................................. 1106
Nadololum.................................................................................2466
Nadroparinum calcicum .......................................................2467
Naftidrofuryli hydrogenooxalas..........................................2470
Naloxoni hydrochloridum dihydricum .............................2473
Naltrexoni hydrochloridum.................................................. 2474
Nandroloni decanoas............................................................. 2476
Naphazolini hydrochloridum .......................................6.3-4235
Naphazolini nitras..................................................................2479
Naproxenum......................................................................6.2-3791
Naproxenum natricum ...................................................6.1-3507
Nasalia ......................................................................................... 730
Natrii acetas trihydricus .......................................................2883
Natrii acetatis ([1-11C]) solutio iniectabilis ....................... 1006
Natrii alendronas .............................................................6.3-4296
Natrii alginas ....................................................................6.3-4297
Natrii amidotrizoas ................................................................2886
Natrii aminosalicylas dihydricus........................................2887
Natrii ascorbas..................................................................6.3-4298
Natrii aurothiomalas..............................................................2889
Natrii benzoas..........................................................................2890
Natrii bromidum...................................................................... 2891
Natrii calcii edetas ..................................................................2892
Natrii calcii pentetas ad radiopharmaceutica..........6.3-4001
Natrii caprylas .........................................................................2893
Natrii carbonas anhydricus..................................................2894
Natrii carbonas decahydricus..............................................2894
Natrii carbonas monohydricus............................................2895
Natrii cetylo- et stearylosulfas. ............................................2895
Natrii chloridum......................................................................2897
Natrii chromatis (51Cr) solutio sterilis ............................... 1007
Natrii citras...............................................................................2898
Natrii cromoglicas ..................................................................2899
Natrii cyclamas........................................................................2900
Natrii dihydrogenophosphas dihydricus .......................... 2901
Natrii docusas .......................................................................... 1743
Natrii fluoridi (18F) solutio iniectabilis.............................. 1008
Natrii fluoridum ......................................................................2902
Natrii fusidas............................................................................2902
Natrii glycerophosphas hydricus.................................6.3-4299
Natrii hyaluronas.............................................................6.3-4300
Natrii hydrogenocarbonas....................................................2906
Natrii hydroxidum ..................................................................2907
Natrii iodidi (123I) solutioad radio-signandum ................ 1010
EUROPEAN PHARMACOPOEIA 6.3
Natrii iodidi (123I) solutio iniectabilis ................................ 1009
Natrii iodidi (131I) capsulae ad usum diagnosticum........1011
Natrii iodidi (131I) capsulae ad usum therapeuticum..... 1012
Natrii iodidi (131I) solutio....................................................... 1013
Natrii iodidi (131I) solutio ad radio-signandum ................1014
Natrii iodidum .........................................................................2907
Natrii iodohippurati (123I) solutio iniectabilis ..................1014
Natrii iodohippurati (131I) solutio iniectabilis.................. 1015
Natrii lactatis solutio..............................................................2908
Natrii laurilsulfas .................................................................... 2910
Natrii metabisulfis .................................................................. 2911
Natrii molybdas dihydricus ...........................................6.3-4302
Natrii molybdatis (99Mo) fissione formati solutio ........... 1016
Natrii nitris ............................................................................... 2913
Natrii nitroprussias ................................................................ 2913
Natrii perboras hydricus ....................................................... 2914
Natrii pertechnetatis (99mTc) fissione formati solutio
iniectabilis .............................................................................. 1018
Natrii pertechnetatis (99mTc) sine fissione formati solutio
iniectabilis .............................................................................. 1020
Natrii phenylbutyras .......................................................6.1-3539
Natrii phosphatis (32P) solutio iniectabilis ....................... 1020
Natrii picosulfas ...................................................................... 2915
Natrii polystyrenesulfonas.............................................6.3-4303
Natrii propionas ...................................................................... 2917
Natrii salicylas ......................................................................... 2919
Natrii selenis pentahydricus ................................................ 2919
Natrii (S)-lactatis solutio .......................................................2909
Natrii stearas .....................................................................6.3-4304
Natrii stearylis fumaras.........................................................2924
Natrii sulfas anhydricus........................................................2924
Natrii sulfas decahydricus ....................................................2925
Natrii sulfis anhydricus.........................................................2926
Natrii sulfis heptahydricus ...................................................2926
Natrii thiosulfas .......................................................................2927
Natrii valproas .........................................................................2927
Neohesperidin-dihydrochalconum .....................................2485
Neomycini sulfas .....................................................................2487
Neostigmini bromidum..........................................................2489
Neostigmini metilsulfas .........................................................2490
Neroli aetheroleum .................................................................2490
Netilmicini sulfas ....................................................................2492
Nevirapinum anhydricum ....................................................2495
Nicergolinum ...........................................................................2496
Nicethamidum .........................................................................2505
Niclosamidum anhydricum..................................................2497
Niclosamidum monohydricum............................................2498
Nicotinamidum........................................................................2499
Nicotini resinas ................................................................6.3-4237
Nicotinum ..........................................................................6.3-4236
Nifedipinum..............................................................................2503
Nifuroxazidum.................................................................. 6.1-3510
Nilutamidum .....................................................................6.2-3792
Nimesulidum ............................................................................2506
Nimodipinum...........................................................................2507
Nitrazepamum .........................................................................2508
Nitrendipinum .........................................................................2509
Nitrofuralum............................................................................. 2512
Nitrofurantoinum.................................................................... 2513
Nitrogenii oxidum............................................................6.2-3794
Nitrogenium ......................................................................6.2-3795
Nitrogenium oxygenio depletum ........................................ 2514
Nizatidinum.............................................................................. 2516
N-Methylpyrrolidonum ..........................................................2399
Nomegestroli acetas................................................................ 2518
Nonoxinolum 9........................................................................ 2519
Noradrenalini hydrochloridum...........................................2520
Noradrenalini tartras ............................................................. 2521
Norcholesteroli iodinati (131I) solutio iniectabilis ........... 1003
General Notices (1) apply to all monographs and other texts
Index
Norethisteroni acetas .............................................................2524
Norethisteronum .....................................................................2523
Norfloxacinum..................................................................6.2-3796
Norgestimatum ........................................................................2526
Norgestrelum............................................................................2527
Nortriptylini hydrochloridum..............................................2528
Noscapini hydrochloridum...................................................2530
Noscapinum..............................................................................2529
Notoginseng radix................................................................... 2531
Nystatinum ...............................................................................2534
O
Octoxinolum 10 .......................................................................2539
Octyldodecanolum ..................................................................2540
Octylis gallas ............................................................................2539
Oenotherae oleum raffinatum ............................................. 1860
Ofloxacinum......................................................................6.2-3801
Oleae folium ......................................................................6.3-4241
Olea herbaria ............................................................................ 712
Olibanum indicum.................................................................. 2128
Olivae oleum raffinatum ................................................6.2-3802
Olivae oleum virginale ...................................................6.2-3803
Olsalazinum natricum...........................................................2548
Omega-3 acidorum esteri ethylici 60..........................6.3-4242
Omega-3 acidorum esteri ethylici 90..........................6.3-4244
Omega-3 acidorum triglycerida ...................................6.3-4246
Omeprazolum...........................................................................2557
Omeprazolum magnesicum ..........................................6.3-4248
Omeprazolum natricum........................................................2558
Ondansetroni hydrochloridum dihydricum ....................2560
Ononidis radix ......................................................................... 2815
Ophthalmica ............................................................................... 721
Opii extractum siccum normatum......................................2562
Opii pulvis normatus .............................................................2563
Opii tinctura normata............................................................2565
Opium crudum ........................................................................2564
Orciprenalini sulfas.........................................................6.2-3804
Origani herba...........................................................................2568
Orphenadrini citras................................................................2569
Orphenadrini hydrochloridum............................................2570
Orthosiphonis folium .............................................................2203
Oryzae amylum ................................................................6.3-4284
Ouabainum ...............................................................................2571
Oxacillinum natricum monohydricum ......................6.2-3806
Oxaliplatinum ...................................................................6.3-4249
Oxazepamum ...........................................................................2577
Oxeladini hydrogenocitras ...................................................2578
Oxfendazolum ad usum veterinarium .......................6.2-3808
Oxitropii bromidum................................................................ 2581
Oxprenololi hydrochloridum ..............................................2583
Oxybuprocaini hydrochloridum .........................................2584
Oxybutynini hydrochloridum ..............................................2585
Oxycodoni hydrochloridum .................................................2587
Oxygenium (15O)...................................................................... 1004
Oxygenium.........................................................................6.2-3809
Oxymetazolini hydrochloridum...................................6.3-4252
Oxytetracyclini hydrochloridum......................................... 2591
Oxytetracyclinum dihydricum.............................................2590
Oxytocini solutio concentrata..............................................2594
Oxytocinum ..............................................................................2593
P
Paclitaxelum......................................................................6.3-4257
Pancreatis pulvis ..............................................................6.3-4260
Pancuronii bromidum ...........................................................2608
Pantoprazolum natricum sesquihydricum ............... 6.1-3518
Papaverini hydrochloridum.................................................2609
Papaveris rhoeados flos ........................................................ 2811
4397
Index
Paracetamolum........................................................................ 2611
Paraffinum liquidum.............................................................. 2613
Paraffinum perliquidum ....................................................... 2612
Paraffinum solidum................................................................ 2612
Paraldehydum.......................................................................... 2615
Parenteralia ................................................................................ 735
Parnaparinum natricum....................................................... 2616
Paroxetini hydrochloridum anhydricum.......................... 2616
Paroxetini hydrochloridum hemihydricum ..................... 2619
Passiflorae herba..................................................................... 2621
Passiflorae herbae extractum siccum ................................2622
Pefloxacini mesilas dihydricus ...........................................2623
Pelargonii radix.......................................................................2625
Penbutololi sulfas....................................................................2625
Penicillaminum .......................................................................2626
Pentaerythrityli tetranitras dilutus.....................................2628
Pentamidini diisetionas ........................................................2630
Pentazocini hydrochloridum ...............................................2632
Pentazocini lactas...................................................................2632
Pentazocinum .......................................................................... 2631
Pentobarbitalum......................................................................2633
Pentobarbitalum natricum ...................................................2634
Pentoxifyllinum .......................................................................2635
Pentoxyverini hydrogenocitras ...........................................2637
Pepsini pulvis....................................................................6.3-4263
Pergolidi mesilas ..................................................................... 2641
Perphenazinum................................................................6.3-4265
Pethidini hydrochloridum ....................................................2650
Phenazonum ............................................................................ 2651
Pheniramini maleas...............................................................2652
Phenobarbitalum.....................................................................2653
Phenobarbitalum natricum..................................................2654
Phenolphthaleinum................................................................2656
Phenolsulfonphthaleinum ....................................................2657
Phenolum...........................................................................6.3-4266
Phenoxyethanolum ................................................................2657
Phenoxymethylpenicillinum.........................................6.1-3520
Phenoxymethylpenicillinum kalicum ........................6.1-3521
Phentolamini mesilas ............................................................2662
Phenylalaninum......................................................................2663
Phenylbutazonum...................................................................2664
Phenylephrini hydrochloridum ..........................................2667
Phenylephrinum .....................................................................2665
Phenylhydrargyri acetas .......................................................2668
Phenylhydrargyri boras.........................................................2669
Phenylhydrargyri nitras........................................................2669
Phenylpropanolamini hydrochloridum............................2670
Phenytoinum............................................................................2671
Phenytoinum natricum .........................................................2672
Phloroglucinolum anhydricum...........................................2672
Phloroglucinolum dihydricum ............................................2673
Pholcodinum .....................................................................6.3-4266
Phthalylsulfathiazolum ......................................................... 2676
Physostigmini salicylas .........................................................2677
Physostigmini sulfas ..............................................................2678
Phytomenadionum .................................................................2679
Phytosterolum..........................................................................2680
Picotamidum monohydricum..............................................2682
Pilocarpini hydrochloridum .........................................6.3-4268
Pilocarpini nitras.............................................................6.3-4269
Pimobendanum .......................................................................2685
Pimozidum ...............................................................................2686
Pindololum ...............................................................................2688
Pini pumilionis aetheroleum............................................... 1766
Pini sylvestris aetheroleum ..................................................2689
Piperacillinum ........................................................................ 2691
Piperacillinum natricum ......................................................2692
Piperazini adipas....................................................................2694
Piperazini citras ......................................................................2695
4398 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Piperazinum hydricum .........................................................2696
Piracetamum ............................................................................2697
Pirenzepini dihydrochloridum monohydricum .............2698
Piretanidum..............................................................................2699
Piroxicamum............................................................................2700
Piscis oleum omega-3 acidis abundans ............................ 1893
Pisi amylum.......................................................................6.3-4263
Pivampicillinum......................................................................2702
Pivmecillinami hydrochloridum ........................................2704
Plantae ad ptisanam ................................................................ 685
Plantae medicinales ................................................................. 684
Plantae medicinales ad praeparationes
homoeopathicas .................................................................... 1065
Plantae medicinales praeparatae ......................................... 684
Plantaginis lanceolatae folium ...........................................2823
Plantaginis ovatae semen..................................................... 2192
Plantaginis ovatae seminis tegumentum ......................... 2191
Plasma humanum ad separationem...........................6.2-3759
Plasma humanum coagmentatum conditumque ad
exstinguendum virum ..................................................6.3-4168
Poloxamera...............................................................................2705
Polyacrylatis dispersio 30 per centum.......................6.3-4270
Poly(alcohol vinylicus) .......................................................... 2715
Polygalae radix ........................................................................2867
Polygoni avicularis herba.....................................................2223
Polymyxini B sulfas................................................................2707
Polysorbatum 20 ..............................................................6.3-4271
Polysorbatum 40 ..............................................................6.3-4272
Polysorbatum 60 ..............................................................6.3-4273
Polysorbatum 80 ..............................................................6.3-4274
Poly(vinylis acetas)................................................................. 2712
Poly(vinylis acetas) dispersio 30 per centum...........6.3-4275
Povidonum.........................................................................6.1-3523
Povidonum iodinatum...........................................................2734
Praeadmixta ad alimenta medicata
ad usum veterinarium........................................................... 739
Praeparationes ad irrigationem............................................ 743
Praeparationes buccales ......................................................... 732
Praeparationes homoeopathicas ........................................ 1065
Praeparationes insulini iniectabiles.................................. 2146
Praeparationes intramammariae
ad usum veterinarium........................................................... 725
Praeparationes intraruminales ............................................. 725
Praeparationes intra-uterinae ad usum veterinarium....6.3-
3977
Praeparationes liquidae ad usum dermicum .................... 728
Praeparationes liquidae peroraliae...................................... 728
Praeparationes liquidae veterinariae ad usum dermicum
..................................................................................................... 752
Praeparationes molles ad usum dermicum ..............6.3-3979
Praeparationes pharmaceuticae in vasis cum pressu..... 744
Pravastatinum natricum ................................................6.3-4278
Prazepamum ............................................................................2736
Praziquantelum.......................................................................2737
Prazosini hydrochloridum ...................................................2738
Prednicarbatum....................................................................... 2740
Prednisoloni acetas ................................................................ 2742
Prednisoloni natrii phosphas .............................................. 2745
Prednisoloni pivalas............................................................... 2744
Prednisolonum ........................................................................ 2741
Prednisonum............................................................................ 2746
Prilocaini hydrochloridum...................................................2750
Prilocainum.............................................................................. 2748
Primaquini diphosphas......................................................... 2751
Primidonum .............................................................................2752
Primulae radix.........................................................................2753
Probenecidum..........................................................................2754
Procainamidi hydrochloridum............................................2755
Procaini hydrochloridum .....................................................2756
EUROPEAN PHARMACOPOEIA 6.3
Prochlorperazini maleas.......................................................2756
Producta ab arte ADN recombinandorum ......................... 701
Producta ab fermentatione..................................................... 693
Producta allergenica ................................................................ 679
Producta cum possibili transmissione vectorium
enkephalopathiarum spongiformium animalium ......... 694
Progesteronum ........................................................................2757
Proguanili hydrochloridum .................................................2758
Prolinum ................................................................................... 2760
Promazini hydrochloridum.................................................. 2761
Promethazini hydrochloridum............................................ 2761
Propacetamoli hydrochloridum .......................................... 2763
Propafenoni hydrochloridum .............................................. 2764
Propanolum.............................................................................. 2766
Propanthelini bromidum ...................................................... 2767
Propofolum ............................................................................... 2768
Propranololi hydrochloridum..............................................2770
Propylenglycoli dicaprylocapras ........................................ 2774
Propylenglycoli dilauras ....................................................... 2774
Propylenglycoli monolauras................................................2775
Propylenglycoli monopalmitostearas................................ 2776
Propylenglycolum...................................................................2773
Propylis gallas..........................................................................2771
Propylis parahydroxybenzoas .............................................2772
Propylis parahydroxybenzoas natricus ............................ 2918
Propylthiouracilum ................................................................2777
Propyphenazonum .................................................................2778
Protamini hydrochloridum ..................................................2779
Protamini sulfas ......................................................................2780
Prothrombinum multiplex humanum ............................... 2076
Protirelinum ............................................................................. 2781
Proxyphyllinum.......................................................................2783
Pruni africanae cortex ..........................................................2789
Pseudoephedrini hydrochloridum ..............................6.2-3820
Psyllii semen ............................................................................2785
Pulveres ad usum dermicum ........................................6.3-3978
Pulveres perorales..................................................................... 738
Pyranteli embonas..................................................................2790
Pyrazinamidum....................................................................... 2791
Pyridostigmini bromidum ....................................................2792
Pyridoxini hydrochloridum .................................................2793
Pyrimethaminum....................................................................2794
Pyrrolidonum...........................................................................2794
Q Scopolamini butylbromidum ............................................... 2109
Quercus cortex .........................................................................2539
R Selamectinum ad usum veterinarium........................6.1-3534
Racecadotrilum.................................................................6.3-4283
Raclopridi ([11C]methoxy) solutio iniectabilis ................. 1005
Radiopharmaceutica................................................................ 695
Ramiprilum .......................................................................6.2-3826
Ranitidini hydrochloridum ..................................................2809
Rapae oleum raffinatum ................................................6.2-3829
Ratanhiae radix ....................................................................... 2816
Ratanhiae tinctura.................................................................. 2817
Rectalia ........................................................................................ 744
Repaglinidum........................................................................... 2812
Reserpinum .............................................................................. 2814
Resorcinolum ........................................................................... 2815
Rhamni purshianae cortex................................................... 1429
Rhamni purshianae extractum siccum normatum........ 1430
Rhei radix.................................................................................. 2817
Rhenii sulfidi colloidalis et technetii (99mTc) solutio
iniectabilis .......................................................................6.3-4002
Ribavirinum ............................................................................. 2818
Riboflavini natrii phosphas ................................................. 2821
Riboflavinum............................................................................2820
General Notices (1) apply to all monographs and other texts
Index
Ricini oleum hydrogenatum ................................................ 1432
Ricini oleum raffinatum........................................................ 1433
Ricini oleum virginale........................................................... 1434
Rifabutinum..............................................................................2825
Rifampicinum ..........................................................................2826
Rifamycinum natricum .........................................................2827
Rilmenidini dihydrogenophosphas....................................2829
Risperidonum ..........................................................................2830
Ritonavirum .............................................................................2832
Rocuronii bromidum..............................................................2835
Ropivacaini hydrochloridum monohydricum ................2837
Rosae pseudo-fructus ............................................................. 1744
Rosmarini aetheroleum.........................................................2840
Rosmarini folium ....................................................................2839
Roxithromycinum ...................................................................2842
RRR-α-Tocopherolum ............................................................3088
RRR-α-Tocopherylis acetas ..................................................3090
RRR-α-Tocopherylis hydrogenosuccinas..........................3095
Rusci rhizoma...................................................................6.1-3416
Rutosidum trihydricum.........................................................2844
S
Sabalis serrulatae fructus .....................................................2864
Sacchari monopalmitas .................................................6.1-3543
Saccharinum............................................................................2849
Saccharinum natricum .........................................................2850
Sacchari sphaerae ...........................................................6.3-4312
Sacchari stearas ...............................................................6.1-3544
Saccharum......................................................................... 6.3-4311
Salbutamoli sulfas .................................................................2857
Salbutamolum..........................................................................2855
Salicis cortex .....................................................................6.1-3563
Salicis corticis extractum siccum ................................6.1-3564
Salmeteroli xinafoas ..............................................................2860
Salmonis domestici oleum ...................................................2862
Salviae lavandulifoliae aetheroleum ..........................6.2-3838
Salviae officinalis folium ......................................................2853
Salviae sclareae aetheroleum .............................................. 1561
Salviae tinctura .......................................................................2854
Salviae trilobae folium ..........................................................2854
Sambuci flos............................................................................. 1776
Sanguisorbae radix .........................................................6.1-3533
Saquinaviri mesilas.........................................................6.3-4287
Schisandrae chinensis fructus .....................................6.3-4288
Scopolamini hydrobromidum.............................................. 2110
Scopolaminum......................................................................... 2108
Selegilini hydrochloridum....................................................2866
Selenii disulfidum...................................................................2867
Semecarpus anacardium
ad praeparationes homoeopathicas................................. 1082
Sennae folii extractum siccum normatum................6.3-4289
Sennae folium..........................................................................2868
Sennae fructus acutifoliae....................................................2870
Sennae fructus angustifoliae ...............................................2871
Serinum.....................................................................................2872
Serpylli herba........................................................................... 3219
Sertaconazoli nitras ........................................................6.1-3535
Sertralini hydrochloridum ............................................6.3-4290
Serum bovinum .......................................................................1329
Sesami oleum raffinatum ..............................................6.3-4292
Sevofluranum ...................................................................6.3-4294
Silica ad usum dentalem.......................................................2878
Silica colloidalis anhydrica..................................................2877
Silica colloidalis hydrica.......................................................2877
Silica hydrophobica colloidalis...........................................2878
Silybi mariani extractum siccum raffinatum et
normatum ...............................................................................2426
4399
Index
Silybi mariani fructus............................................................2425
Simeticonum ............................................................................2880
Simvastatinum......................................................................... 2881
Soiae oleum hydrogenatum ..........................................6.2-3837
Soiae oleum raffinatum .................................................6.2-3838
Solani amylum .................................................................6.3-4277
Solidaginis herba ....................................................................1999
Solidaginis virgaureae herba...............................................2000
Solutiones ad conservationem partium corporis...........2929
Solutiones ad haemocolaturam
haemodiacolaturamque ......................................................2025
Solutiones ad haemodialysim .............................................2022
Solutiones ad peritonealem dialysim................................2646
Solutiones anticoagulantes et sanguinem humanum
conservantes ..........................................................................1200
Somatostatinum ......................................................................2930
Somatropini solutio concentrata ........................................2933
Somatropinum......................................................................... 2931
Somatropinum iniectabile....................................................2935
Sorbitani lauras.......................................................................2938
Sorbitani oleas.........................................................................2938
Sorbitani palmitas ..................................................................2939
Sorbitani sesquioleas .............................................................2939
Sorbitani stearas .....................................................................2940
Sorbitani trioleas ....................................................................2940
Sorbitolum .........................................................................6.3-4305
Sorbitolum liquidum cristallisabile....................................2942
Sorbitolum liquidum non cristallisabile...........................2943
Sorbitolum liquidum partim deshydricum ...............6.3-4307
Sotaloli hydrochloridum .......................................................2944
Spectinomycini dihydrochloridum pentahydricum ......2947
Spectinomycini sulfas tetrahydricus ad usum
veterinarium ..........................................................................2949
Spiramycinum ..................................................................6.1-3540
Spiraprili hydrochloridum monohydricum .....................2954
Spironolactonum ....................................................................2955
Squalanum................................................................................2956
Stanni colloidalis et technetii (99mTc)
solutio iniectabilis ................................................................ 1025
Stanni pyrophosphatis et technetii (99mTc) solutio
iniectabilis .......................................................................6.3-4006
Stannosi chloridum dihydricum.........................................2959
Stanozololum ....................................................................6.3-4308
Stavudinum ..............................................................................2964
Stramonii folium .....................................................................2968
Stramonii pulvis normatus............................................6.2-3842
Streptokinasi solutio concentrata................................6.2-3843
Streptomycini sulfas...............................................................2972
Strontii (89Sr) chloridi solutio iniectabilis........................ 1021
Styli............................................................................................... 748
Succinylsulfathiazolum......................................................... 2974
Sufentanili citras.....................................................................2978
Sufentanilum............................................................................2977
Sulbactamum natricum..................................................6.2-3845
Sulfacetamidum natricum.............................................6.2-3847
Sulfadiazinum .........................................................................2983
Sulfadimidinum.......................................................................2984
Sulfadoxinum...........................................................................2984
Sulfafurazolum ........................................................................2985
Sulfaguanidinum ....................................................................2986
Sulfamerazinum......................................................................2987
Sulfamethizolum .....................................................................2988
Sulfamethoxazolum................................................................2989
Sulfamethoxypyridazinum ad usum veterinarium .......2990
Sulfanilamidum....................................................................... 2991
Sulfasalazinum........................................................................2992
Sulfathiazolum ........................................................................2994
Sulfinpyrazonum ....................................................................2995
Sulfisomidinum.......................................................................2996
4400
EUROPEAN PHARMACOPOEIA 6.3
Sulfur ad usum externum.....................................................2998
Sulfuris colloidalis et technetii (99mTc) solutio
iniectabilis .............................................................................. 1024
Sulindacum ..............................................................................2996
Sulpiridum................................................................................2999
Sultamicillini tosilas dihydricus ..................................6.3-4313
Sultamicillinum................................................................6.1-3545
Sumatriptani succinas....................................................6.3-4315
Suxamethonii chloridum ......................................................3007
Suxibuzonum...........................................................................3008
T
Talcum ................................................................................6.3-4321
Tamoxifeni citras..................................................................... 3014
Tamponae medicatae ............................................................... 751
Tamsulosini hydrochloridum .............................................. 3016
Tanaceti parthenii herba ...................................................... 1887
Tanninum ................................................................................. 3018
Technetii (99mTc) bicisati solutio iniectabilis.................... 1022
Technetii (99mTc) et etifenini solutio iniectabilis............. 1026
Technetii (99mTc) exametazimi solutio iniectabilis ......... 1027
Technetii (99mTc) gluconatis solutio iniectabilis.............. 1028
Technetii (99mTc) humani albumini solutio iniectabilis .. 1029
Technetii (99mTc) macrosalbi suspensio iniectabilis.........6.3-
4003
Technetii (99mTc) mebrofenini solutio iniectabilis ...6.3-4004
Technetii (99mTc) medronati solutio iniectabilis.............. 1031
Technetii (99mTc) mertiatidi solutio iniectabilis............... 1033
Technetii (99mTc) microsphaerarum suspensio
iniectabilis .......................................................................6.3-4005
Technetii (99mTc) pentetatis solutio iniectabilis............... 1035
Technetii (99mTc) sestamibi solutio iniectabilis................ 1036
Technetii (99mTc) succimeri solutio iniectabilis............... 1037
Teicoplaninum..................................................................6.3-4323
Telmisartanum..................................................................6.3-4325
Temazepamum ........................................................................3020
Tenoxicamum .......................................................................... 3021
Terazosini hydrochloridum dihydricum...........................3022
Terbinafini hydrochloridum ................................................3024
Terbutalini sulfas ....................................................................3025
Terconazolum ...................................................................6.1-3553
Terebinthinae aetheroleum a Pino pinastro.................... 3151
Terfenadinum....................................................................6.1-3554
tert-Butylamini perindoprilum............................................2643
Testosteroni decanoas............................................................ 3031
Testosteroni enantas ..............................................................3033
Testosteroni isocaproas .........................................................3034
Testosteroni propionas ..........................................................3035
Testosteronum..........................................................................3030
Tetracaini hydrochloridum ...........................................6.1-3556
Tetracosactidum ...............................................................6.3-4326
Tetracyclini hydrochloridum ............................................... 3041
Tetracyclinum ..........................................................................3040
Tetra-O-acetylmannosi triflas ad radiopharmaceutica ...6.3-
4008
Tetrazepamum .........................................................................3043
Tetryzolini hydrochloridum.................................................3044
Thallosi (201Tl) chloridi solutio iniectabilis....................... 1039
Theobrominum ........................................................................3045
Theophyllinum.........................................................................3046
Theophyllinum et ethylenediaminum ...............................3048
Theophyllinum et ethylenediaminum hydricum............3049
Theophyllinum monohydricum ..........................................3047
Thiamazolum ...........................................................................3050
Thiamini hydrochloridum .................................................... 3051
Thiamini nitras........................................................................3053
Thiamphenicolum...................................................................3054
Thiomersalum..........................................................................3056
Thiopentalum natricum et natrii carbonas .....................3057
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Thioridazini hydrochloridum..............................................3059
Thioridazinum.........................................................................3058
Threoninum..............................................................................3060
Thymi aetheroleum ................................................................3063
Thymi herba ............................................................................. 3061
Thymolum .................................................................................3064
Tiabendazolum ........................................................................3064
Tiamulini hydrogenofumaras ad usum veterinarium ..3068
Tiamulinum ad usum veterinarium ..................................3065
Tianeptinum natricum ..........................................................3070
Tiapridi hydrochloridum ......................................................3071
Tibolonum................................................................................. 3074
Ticarcillinum natricum .........................................................3075
Ticlopidini hydrochloridum.................................................3077
Tiliae flos...................................................................................2270
Tilidini hydrochloridum hemihydricum...........................3079
Timololi maleas .......................................................................3080
Tincturae maternae
ad praeparationes homoeopathicas................................. 1072
Tinidazolum ......................................................................6.2-3852
Tinzaparinum natricum .......................................................3082
Tioconazolum ..........................................................................3083
Titanii dioxidum......................................................................3084
Tobramycinum..................................................................6.2-3854
α-Tocopherylis acetatis pulvis ............................................. 3091
Tolbutamidum..........................................................................3097
Tolnaftatum ..............................................................................3099
Torasemidum anhydricum ................................................... 3100
Tormentillae rhizoma .............................................................3101
Tormentillae tinctura ............................................................. 3102
Tosylchloramidum natricum ............................................... 3103
Toxinum botulinicum typum A ad iniectabile ................1327
Tragacantha.......................................................................6.3-4328
Tramadoli hydrochloridum .................................................. 3104
Tramazolini hydrochloridum monohydricum ................ 3106
Trandolaprilum ....................................................................... 3107
Trapidilum ................................................................................ 3110
Tretinoinum...............................................................................3111
Triacetinum .............................................................................. 3112
Triamcinoloni acetonidum....................................................3114
Triamcinoloni hexacetonidum............................................ 3115
Triamcinolonum ..................................................................... 3112
Triamterenum ...................................................................6.3-4329
Tribenosidum ............................................................................3117
Tributylis acetylcitras......................................................6.3-4330
Tricalcii phosphas...................................................................1396
Triethylis citras ........................................................................ 3120
Trifluoperazini hydrochloridum......................................... 3121
Triflusalum ............................................................................... 3121
Triglycerida saturata media................................................. 3122
Triglyceroli diisostearas .................................................6.1-3558
Trigonellae foenugraeci semen........................................... 1882
Trihexyphenidyli hydrochloridum ..................................... 3125
Trimetazidini dihydrochloridum........................................ 3126
Trimethadionum ..................................................................... 3127
Trimethoprimum..................................................................... 3128
Trimipramini maleas ............................................................. 3130
Tri-n-butylis phosphas............................................................ 3132
Tritici aestivi oleum raffinatum .......................................... 3215
Tritici aestivi oleum virginale.............................................. 3216
Tritici amylum ..................................................................6.3-4346
Trolaminum.............................................................................. 3133
Trometamolum ........................................................................ 3135
Tropicamidum ......................................................................... 3135
Tropisetroni hydrochloridum .............................................. 3136
Trospii chloridum ................................................................... 3138
Troxerutinum ........................................................................... 3139
Trypsinum..........................................................................6.3-4331
Tryptophanum ..................................................................6.3-4333
General Notices (1) apply to all monographs and other texts
Index
Tuberculini aviarii derivatum proteinosum
purificatum............................................................................. 3146
Tuberculini bovini derivatum proteinosum
purificatum............................................................................. 3147
Tuberculini derivatum proteinosum purificatum ad usum
humanum ............................................................................... 3147
Tuberculinum pristinum ad usum humanum ................ 3144
Tubocurarini chloridum........................................................ 3150
Tylosini phosphatis solutio ad usum veterinarium ....... 3154
Tylosini tartras ad usum veterinarium ............................. 3156
Tylosinum ad usum veterinarium...................................... 3152
Tyrosinum................................................................................. 3157
Tyrothricinum.......................................................................... 3158
U
Ubidecarenonum..................................................................... 3163
Ureum......................................................................................... 3165
Urofollitropinum ..................................................................... 3166
Urokinasum .............................................................................. 3167
Urtica dioica ad praeparationes homoeopathicas ......... 1075
Urticae folium ..........................................................................2493
Uvae ursi folium ...............................................................6.1-3410
V
Vaccina ad usum humanum .........................................6.3-3971
Vaccina ad usum veterinarium ............................................. 707
Vaccinum actinobacillosidis inactivatum ad suem.......... 943
Vaccinum adenovirosidis caninae vivum........................... 886
Vaccinum adenovirosis caninae inactivatum ................... 885
Vaccinum anaemiae infectivae pulli vivum....................... 925
Vaccinum anthracis adsorbatum ab colato culturarum ad
usum humanum...................................................................... 757
Vaccinum anthracis vivum ad usum veterinarium.......... 859
Vaccinum aphtharum epizooticarum inactivatum ad
ruminantes ................................................................................918
Vaccinum bronchitidis infectivae aviariae inactivatum.. 864
Vaccinum bronchitidis infectivae aviariae vivum...6.1-3371
Vaccinum brucellosis (Brucella melitensis stirpe Rev. 1)
vivum ad usum veterinarium .............................................. 881
Vaccinum bursitidis infectivae aviariae inactivatum...... 867
Vaccinum bursitidis infectivae aviariae vivum................. 869
Vaccinum calicivirosis felinae inactivatum ....................... 909
Vaccinum calicivirosis felinae vivum ...................................910
Vaccinum chlamydiosidis felinae inactivatum ..................911
Vaccinum cholerae ....................................................................761
Vaccinum cholerae aviariae inactivatum........................... 920
Vaccinum cholerae cryodesiccatum......................................761
Vaccinum cholerae perorale inactivatum........................... 762
Vaccinum Clostridii botulini ad usum veterinarium ...... 894
Vaccinum Clostridii chauvoei ad usum veterinarium ....6.3-
3997
Vaccinum Clostridii novyi B ad usum veterinarium....... 895
Vaccinum Clostridii perfringentis
ad usum veterinarium........................................................... 897
Vaccinum Clostridii septici ad usum veterinarium ......... 899
Vaccinum coccidiosidis vivum ad pullum .................6.2-3665
Vaccinum colibacillosis fetus a partu recentis inactivatum
ad ruminantes ......................................................................... 936
Vaccinum colibacillosis fetus a partu recentis inactivatum
ad suem ..................................................................................... 934
Vaccinum diarrhoeae viralis bovinae inactivatum .......... 880
Vaccinum diphtheriae adsorbatum ...................................... 789
Vaccinum diphtheriae, antigeniis minutum,
adsorbatum............................................................................... 791
Vaccinum diphtheriae et tetani adsorbatum ..................... 763
Vaccinum diphtheriae et tetani, antigeni-o(-is) minutum,
adsorbatum............................................................................... 764
4401
Index
Vaccinum diphtheriae, tetani et hepatitidis B (ADNr)
adsorbatum............................................................................... 765
Vaccinum diphtheriae, tetani et pertussis adsorbatum .. 768
Vaccinum diphtheriae, tetani et pertussis sine cellulis ex
elementis praeparatum adsorbatum.................................. 767
Vaccinum diphtheriae, tetani et poliomyelitidis
inactivatum, antigeni-o(-is) minutum, adsorbatum....... 770
Vaccinum diphtheriae, tetani, pertussis et poliomyelitidis
inactivatum adsorbatum....................................................... 785
Vaccinum diphtheriae, tetani, pertussis, poliomyelitidis
inactivatum et haemophili stirpi b coniugatum
adsorbatum............................................................................... 787
Vaccinum diphtheriae, tetani, pertussis sine cellulis ex
elementis praeparatum cumque haemophili stirpi b
coniugatum adsorbatum....................................................... 771
Vaccinum diphtheriae, tetani, pertussis sine cellulis
ex elementis praeparatum et hepatitidis B (ADNr)
adsorbatum............................................................................... 774
Vaccinum diphtheriae, tetani, pertussis sine cellulis ex
elementis praeparatum et poliomyelitidis inactivatum
adsorbatum............................................................................... 775
Vaccinum diphtheriae, tetani, pertussis sine cellulis ex
elementis praeparatum et poliomyelitidis inactivatum,
antigeni-o(-is) minutum, adsorbatum................................ 778
Vaccinum diphtheriae, tetani, pertussis sine cellulis
ex elementis praeparatum, hepatitidis B (ADNr),
poliomyelitidis inactivatum et haemophili stirpi b
coniugatum adsorbatum....................................................... 780
Vaccinum diphtheriae, tetani, pertussis sine cellulis ex
elementis praeparatum, poliomyelitidis inactivatum et
haemophili stirpi b coniugatum adsorbatum.........6.3-3983
Vaccinum encephalitidis ixodibus advectae
inactivatum .............................................................................. 845
Vaccinum encephalomyelitidis infectivae aviariae
vivum.......................................................................................... 871
Vaccinum erysipelatis suillae inactivatum ........................ 955
Vaccinum febris flavae vivum.......................................6.1-3365
Vaccinum febris typhoidi ........................................................ 849
Vaccinum febris typhoidi cryodesiccatum ......................... 849
Vaccinum febris typhoidis polysaccharidicum ................. 847
Vaccinum febris typhoidis vivum perorale (stirpe
Ty 21a) ....................................................................................... 849
Vaccinum furunculosidis inactivatum ad salmonidas cum
adiuvatione oleosa ad iniectionem...........................6.2-3668
Vaccinum haemophili stirpi b coniugatum...............6.3-3985
Vaccinum hepatitidis A inactivatum adsorbatum ............ 795
Vaccinum hepatitidis A inactivatum et hepatitidis B (ADNr)
adsorbatum............................................................................... 794
Vaccinum hepatitidis A inactivatum virosomale.............. 797
Vaccinum hepatitidis B (ADNr)............................................. 800
Vaccinum hepatitidis viralis anatis stirpe I vivum .......... 902
Vaccinum herpesviris equini inactivatum ......................... 905
Vaccinum inactivatum diarrhoeae vituli coronaviro
illatae ......................................................................................... 882
Vaccinum inactivatum diarrhoeae vituli rotaviro
illatae ......................................................................................... 884
Vaccinum influenzae equi inactivatum.............................. 907
Vaccinum influenzae inactivatum ad suem ...................... 944
Vaccinum influenzae inactivatum ex cellulis corticisque
antigeniis praeparatum......................................................... 804
Vaccinum influenzae inactivatum ex cellulis virisque
integris praeparatum ..............................................................810
Vaccinum influenzae inactivatum ex corticis antigeniis
praeparatum............................................................................. 803
Vaccinum influenzae inactivatum ex corticis antigeniis
praeparatum virosomale....................................................... 806
Vaccinum influenzae inactivatum ex viris integris
praeparatum............................................................................. 808
4402 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.3
Vaccinum influenzae inactivatum ex virorum fragmentis
praeparatum............................................................................. 801
Vaccinum laryngotracheitidis infectivae aviariae
vivum.......................................................................................... 872
Vaccinum leptospirosis bovinae inactivatum.................... 876
Vaccinum leptospirosis caninae inactivatum ................... 888
Vaccinum leucosis felinae inactivatum................................914
Vaccinum mannheimiae inactivatum ad bovinas............ 927
Vaccinum mannheimiae inactivatum ad ovem ................ 928
Vaccinum meningococcale classis C coniugatum ............814
Vaccinum meningococcale polysaccharidicum.................816
Vaccinum morbi Aujeszkyi ad suem inactivatum ............ 859
Vaccinum morbi Aujeszkyi ad suem vivum ad usum
parenteralem............................................................................ 861
Vaccinum morbi Carrei vivum ad canem........................... 887
Vaccinum morbi Carrei vivum ad mustelidas ................... 900
Vaccinum morbi haemorrhagici cuniculi inactivatum .. 949
Vaccinum morbillorum, parotitidis et rubellae
vivum.................................................................................6.1-3347
Vaccinum morbillorum vivum......................................6.1-3348
Vaccinum morbi Marek vivum .............................................. 930
Vaccinum morbi partus diminutionis MCMLXXVI
inactivatum ad pullum .......................................................... 904
Vaccinum Mycoplasmatis galliseptici inactivatum.......... 932
Vaccinum myxomatosidis vivum ad cuniculum ............... 933
Vaccinum panleucopeniae felinae infectivae
inactivatum .............................................................................. 912
Vaccinum panleucopeniae felinae infectivae vivum........913
Vaccinum parainfluenzae viri canini vivum..................... 890
Vaccinum paramyxoviris 3 aviarii inactivatum ............... 874
Vaccinum parotitidis vivum ..........................................6.1-3349
Vaccinum parvovirosis caninae inactivatum .................... 891
Vaccinum parvovirosis caninae vivum ............................... 892
Vaccinum parvovirosis inactivatum ad suem ................... 946
Vaccinum pasteurellae inactivatum ad ovem.................... 941
Vaccinum pertussis adsorbatum ........................................... 824
Vaccinum pertussis sine cellulis copurificatum
adsorbatum............................................................................... 822
Vaccinum pertussis sine cellulis ex elementis praeparatum
adsorbatum............................................................................... 820
Vaccinum pestis anatis vivum ............................................... 901
Vaccinum pestis classicae suillae vivum ex cellulis.........6.2-
3669
Vaccinum pneumococcale polysaccharidicum................. 827
Vaccinum pneumococcale polysaccharidicum coniugatum
adsorbatum............................................................................... 825
Vaccinum poliomyelitidis inactivatum ......................6.3-3988
Vaccinum poliomyelitidis perorale .............................6.1-3351
Vaccinum pseudopestis aviariae inactivatum................... 937
Vaccinum pseudopestis aviariae vivum.............................. 939
Vaccinum rabiei ex cellulis ad usum humanum .....6.1-3355
Vaccinum rabiei inactivatum ad usum veterinarium .....6.1-
3375
Vaccinum rabiei perorale vivum ad vulpem ...................... 952
Vaccinum rhinitidis atrophicantis ingravescentis suillae
inactivatum .....................................................................6.1-3373
Vaccinum rhinotracheitidis infectivae bovinae vivum ... 924
Vaccinum rhinotracheitidis viralis felinae inactivatum ..916
Vaccinum rhinotracheitidis viralis felinae vivum .............917
Vaccinum rubellae vivum ..............................................6.1-3358
Vaccinum Salmonellae Enteritidis inactivatum ad
pullum........................................................................................ 953
Vaccinum Salmonellae Typhimurium inactivatum ad
pullum........................................................................................ 954
Vaccinum tenosynovitidis viralis aviariae vivum ............ 875
Vaccinum tetani adsorbatum ................................................. 844
Vaccinum tetani ad usum veterinarium ............................. 957
Vaccinum tuberculosis (BCG) cryodesiccatum ................. 759
Vaccinum varicellae vivum ...........................................6.3-3992
EUROPEAN PHARMACOPOEIA 6.3
Vaccinum variolae gallinaceae vivum ............................... 921
Vaccinum variolae vivum ..............................................6.1-3359
Vaccinum vibriosidis aquae frigidae inactivatum ad
salmonidas.......................................................................6.2-3671
Vaccinum vibriosidis inactivatum ad salmonidas ..6.2-3672
Vaccinum viri parainfluenzae bovini vivum..................... 878
Vaccinum viri syncytialis meatus spiritus bovini
vivum.......................................................................................... 879
Vaccinum zonae vivum ..................................................6.3-3991
Vaginalia ..................................................................................... 751
Valerianae extractum hydroalcoholicum siccum ........... 3173
Valerianae radix .......................................................................3174
Valerianae tinctura................................................................. 3175
Valinum ......................................................................................3176
Valnemulini hydrochloridum ad usum veterinarium... 3177
Vancomycini hydrochloridum............................................. 3180
Vanillinum ................................................................................ 3182
Vaselinum album..............................................................6.2-3815
Vaselinum flavum ............................................................ 6.2-3816
Vecuronii bromidum .............................................................. 3183
Venlafaxini hydrochloridum ................................................ 3184
Verapamili hydrochloridum ................................................. 3186
Verbasci flos..............................................................................2454
Verbenae citriodoratae folium ......................................6.3-4199
Verbenae herba ........................................................................ 3188
Via praeparandi stirpes homoeopathicas et
potentificandi..................................................................6.1-3385
Vinblastini sulfas..................................................................... 3189
Vincristini sulfas ..................................................................... 3190
Vindesini sulfas ....................................................................... 3192
Vinorelbini tartras................................................................... 3194
Vinpocetinum........................................................................... 3196
Violae herba cum flore .......................................................... 3217
Vitamini synthetici densati A pulvis .................................. 3201
Vitaminum A ............................................................................ 3199
Vitaminum A syntheticum densatum oleosum ...............3200
General Notices (1) apply to all monographs and other texts
Index
Vitaminum A syntheticum, solubilisatum densatum in
aqua dispergibile...................................................................3203
W
Warfarinum natricum............................................................3207
Warfarinum natricum clathratum......................................3208
X
Xanthani gummi ..............................................................6.3-4349
Xenoni (133Xe) solutio iniectabilis ....................................... 1042
Xylazini hydrochloridum ad usum veterinarium ..........3234
Xylitolum ............................................................................6.3-4350
Xylometazolini hydrochloridum .........................................3237
Xylosum .....................................................................................3238
Y
Yohimbini hydrochloridum ..................................................3244
Z
Zidovudinum............................................................................3249
Zinci acetas dihydricus .........................................................3250
Zinci acexamas........................................................................ 3251
Zinci chloridum.......................................................................3253
Zinci oxidum ............................................................................3253
Zinci stearas .............................................................................3254
Zinci sulfas heptahydricus....................................................3254
Zinci sulfas hexahydricus .....................................................3255
Zinci sulfas monohydricus ...................................................3255
Zinci undecylenas...................................................................3256
Zingiberis rhizoma ..........................................................6.2-3751
Zolpidemi tartras.....................................................................3256
Zopiclonum...............................................................................3257
Zuclopenthixoli decanoas.....................................................3259
4403
 KEY TO MONOGRAPHS

Carbimazole EUROPEAN PHARMACOPOEIA 6.3

Version date of the text 01/2008:0884 of this solution to 10.0 ml with a mixture of 20 volumes
corrected 6.3 of acetonitrile R and 80 volumes of water R.
CARBIMAZOLE Reference solution (b). Dissolve 5.0 mg of thiamazole R
Text reference in a mixture of 20 volumes of acetonitrile R and
number 80 volumes of water R and dilute to 10.0 ml with
Carbimazolum the same mixture of solvents. Dilute 1.0 ml of this
Modification to be taken solution to 100.0 ml with a mixture of 20 volumes of
into account from the acetonitrile R and 80 volumes of water R.
publication date of Column:
Supplement 6.3
– size: l = 0.15 m, Ø = 3.9 mm,
C7H10N2O2S – stationary phase: octadecylsilyl silica gel for
CAS number [22232-54-8] Mr 186.2 chromatography R (5 µm).
Mobile phase: acetonitrile R, water R (10:90 V/V).
DEFINITION
Flow rate: 1 ml/min.
Chemical name Ethyl 3-methyl-2-thioxo-2,3-dihydro-1H-imidazole-1-
in accordance carboxylate. Detection: spectrophotometer at 254 nm.
with IUPAC Content: 98.0 per cent to 102.0 per cent (dried substance). Injection: 10 µl.
nomenclature CHARACTERS Run time: 1.5 times the retention time of carbimazole.
rules
Appearance: white or yellowish-white, crystalline powder. Retention time: carbimazole = about 6 min.
Solubility: slightly soluble in water, soluble in acetone and System suitability: reference solution (a):
in ethanol (96 per cent). – resolution: minimum 5.0 between the peaks due to
Application of the impurity A and carbimazole.
first and second IDENTIFICATION
identification is First identification: B. Limits:
defined in the Second identification: A, C. – impurity A: not more than 0.5 times the area of the
principal peak in the chromatogram obtained with

N
General Notices
A. Melting point (2.2.14): 122 °C to 125 °C. reference solution (b) (0.5 per cent),
(chapter 1)
B. Infrared absorption spectrophotometry (2.2.24).
E
– unspecified impurities: for each impurity, not more

M
than 0.1 times the area of the principal peak in the

I
Preparation: discs.
chromatogram obtained with reference solution (b)

C
Reference Comparison: carbimazole CRS. (0.10 per cent).

E
standard available C. Thin-layer chromatography (2.2.27). Loss on drying (2.2.32): maximum 0.5 per cent,

SP
from the Test solution. Dissolve 10 mg of the substance to be determined on 1.000 g by drying in a desiccator over
Secretariat examined in methylene chloride R and dilute to 10 ml diphosphorus pentoxide R at a pressure not exceeding
(see www.edqm.eu) with the same solvent. 0.7 kPa for 24 h.
Reference solution. Dissolve 10 mg of carbimazole CRS Sulphated ash (2.4.14): maximum 0.1 per cent,
in methylene chloride R and dilute to 10 ml with the determined on 1.0 g.
same solvent. ASSAY
Plate: TLC silica gel GF254 plate R. Dissolve 50.0 mg in water R and dilute to 500.0 ml
Reagents described Mobile phase: acetone R, methylene chloride R with the same solvent. To 10.0 ml add 10 ml of dilute
in chapter 4 (20:80 V/V). hydrochloric acid R and dilute to 100.0 ml with water R.
Measure the absorbance (2.2.25) at the absorption
Application: 10 µl. maximum at 291 nm.
Development: over a path of 15 cm. Calculate the content of C7H10N2O2S taking the specific
Further Drying: in air for 30 min. absorbance to be 557.
information
Detection: examine in ultraviolet light at 254 nm. IMPURITIES
available on
www.edqm.eu Results: the principal spot in the chromatogram Specified impurities: A.
obtained with the test solution is similar in position Other detectable impurities (the following substances
(KNOWLEDGE) and size to the principal spot in the chromatogram would, if present at a sufficient level, be detected by one
obtained with the reference solution. or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Reference to a TESTS impurities and/or by the general monograph Substances
general chapter Related substances. Liquid chromatography (2.2.29). for pharmaceutical use (2034). It is therefore not
necessary to identify these impurities for demonstration
Test solution. Dissolve 5.0 mg of the substance to be of compliance. See also 5.10. Control of impurities in
Line in the examined in 10.0 ml of a mixture of 20 volumes of substances for pharmaceutical use): B.
margin acetonitrile R and 80 volumes of water R. Use this
indicating solution within 5 min of preparation.
where part of ❚
❚ Reference solution (a). Dissolve 5 mg of thiamazole R and
the text has ❚ 0.10 g of carbimazole CRS in a mixture of 20 volumes of
❚
❚
been modified acetonitrile R and 80 volumes of water R and dilute to
(technical 100.0 ml with the same mixture of solvents. Dilute 1.0 ml A. 1-methyl-1H-imidazole-2-thiol (thiamazole),
modification)

See the information section on general monographs (cover pages)

General Notices (1) apply to all monographs and other texts
 IMPORTANT NOTICE
GENERAL MONOGRAPHS
The European Pharmacopoeia contains a number of general monographs covering classes of products. These general
monographs give requirements that are applicable to all products in the given class or, in some cases, to any product in the
given class for which there is a specific monograph in the Pharmacopoeia (see 1. General Notices, General monographs).
Where no restriction on scope of a general monograph is given in a preamble, it is applicable to all products in the class
defined, irrespective of whether there is an individual monograph for the product in the Pharmacopoeia.
Whenever a monograph is used, it is essential to ascertain whether there is a general monograph applicable to the product
in question. The general monographs listed below are published in the section General Monographs (unless otherwise
stated). This list is updated where necessary and republished in each Supplement.

Allergen products (1063)

Dosage Forms monographs
(published in the Dosage Forms section)
Essential oils (2098)
Extracts (0765)
Herbal drug preparations (1434)
Herbal drugs (1433)
Herbal drugs for homoeopathic preparations (2045)
(published in the Homoeopathic Preparations section)
Herbal teas (1435)
Homoeopathic preparations (1038)
(published in the Homoeopathic Preparations section)
Immunosera for human use, animal (0084)
Immunosera for veterinary use (0030)
Methods of preparation of homoeopathic stocks and potentisation (2371)
(published in the Homoeopathic Preparations section)
Monoclonal antibodies for human use (2031)
Mother tinctures for homoeopathic preparations (2029)
(published in the Homoeopathic Preparations section)
Products of fermentation (1468)
Products with risk of transmitting agents of animal spongiform encephalopathies (1483)
Radiopharmaceutical preparations (0125)
Recombinant DNA technology, products of (0784)
Substances for pharmaceutical use (2034)
Vaccines for human use (0153)
Vaccines for veterinary use (0062)
Vegetable fatty oils (1579)