Supplementary information

Supplementary information

Annotated references
1. Procedure for the selection of recommended international nonproprietary
names for pharmaceutical substances and General principles for guidance in
devising international nonproprietary names for pharmaceutical substances.
In: International nonproprietary names (INN) for pharmaceutical substances. Cumu-
lative list No. 10. Geneva, World Health Organization, 2002, pp. ix–xii, avail-
able on CD-ROM.

This information is also accessible on the Internet at

http://www.who.int/medicines/

These two texts are based on World Health Assembly resolution WHA3.11.
The procedure for the selection of recommended international nonpropri-
etary names for pharmaceutical substances, and the general principles for
selecting international nonproprietary names for pharmaceutical substances,
have been updated regularly since the INN programme began in 1950.

2. Guidelines for the graphic representation of chemical formulae. In: WHO

Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-fourth
report. Geneva, World Health Organization, 1996, Annex 1 (WHO Techni-
cal Report Series, No. 863, pp. 16–49).

These guidelines are also available in The graphic representation of chemical for-
mulae in the publications of international nonproprietary names (INN) for pharma-
ceutical substances. Geneva, World Health Organization, 1995 (document
WHO/PHARM/95.579).

These unique guidelines are intended to help scientists to portray chemical

names and structures correctly and unambiguously in pharmacopoeias and
other compendia. For details of chemical nomenclature conventions, readers
are referred to the recommendations of the International Union of Pure and
Applied Chemistry.1

The guidelines should be followed as closely as possible, although it should

be noted that unwavering adherence to these principles is not always
practicable. Therefore, they may be adapted, with certain exceptions, where
necessary to produce accurately drawn structural formulae. Details of the
formulae, such as bond lengths, the position of subscripts and superscripts,

1
International Union of Pure and Applied Chemistry, Organic Chemistry Division, Commission
on the Nomenclature of Organic Chemistry. Nomenclature of organic chemistry, sections, A, B, C, D,
E, F, and H, 4th ed. Oxford, Pergamon, 1979.
Leigh GJ, ed. Nomenclature of inorganic chemistry: recommendations 1990. Oxford, Blackwell
Scientific, 1990.

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The International Pharmacopoeia

and the closeness of apposition of the individual atomic symbols, will

depend on the drawing method used, whether computer-based or manual.

The guidelines cover acyclic, cyclic, and ionic structures, isotopically

modified and coordination compounds, stereochemistry, carbohydrates,
steroids, terpenoids, prostanoids, alkaloids, antibiotics, polypeptides, and
polymers.

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 Supplementary information

List of available International Chemical Reference

Substances1
International Chemical Reference Substances (ICRS) are established on the
advice of the WHO Expert Committee on Specifications for Pharmaceutical
Preparations. They are supplied primarily for use in physical and chemical tests
and assays described in the specifications for quality control of drugs published
in The International Pharmacopoeia or proposed in draft monographs. The ICRS
are mainly intended to be used as primary standards to calibrate secondary
standards.
Directions for use, and analytical data required for the use described in the
relevant specifications of The International Pharmacopoeia, are given in the cer-
tificates enclosed with the substances when distributed. More detailed analyt-
ical reports on the substances may be obtained from the WHO Collaborating
Centre for Chemical Reference Substances.
ICRS may also be used in tests and assays not described in The International
Pharmacopoeia. However, the responsibility for assessing the suitability of the
substances then rests with the user or with the pharmacopoeia commission or
other authority that has prescribed this use.
It is generally recommended that the substances should be stored protected
from light and moisture and preferably at a temperature of about 5 °C. When
special storage conditions are required, this is stated on the label or in the
accompanying leaflet. It is recommended that the user purchase only an amount
sufficient for immediate use.
The stability of the ICRS kept at the Collaborating Centre is monitored by
regular re-examination, and any material that has deteriorated is replaced by
new batches as necessary. Lists giving control numbers for the current batches
are issued in the annual reports from the Centre and new yearly lists may be
obtained on request.
Orders for the ICRS should be sent to:

WHO Collaborating Centre for Chemical Reference Substances

Apoteket AB
Produktion & Laboratorier Centrallaboratoriet, ACL
Prismavägen 2
S-141 75 Kungens Kurva
Sweden
(Fax: +46 8 740 6040; email: [email protected])

1
As updated at the thirty-seventh meeting of the WHO Expert Committee on Specifications for
Pharmaceutical Preparations, 22–26 October 2001.

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The ICRS are supplied only in the standard packages indicated in the follow-
ing list:

Reference substance Package size Control number

p-acetamidobenzalazine 25 mg 290042
acetazolamide 100 mg 186128
allopurinol 100 mg 287049
amidotrizoic acid 100 mg 196205
2-amino-5-nitrothiazole 25 mg 186131
3-aminopyrazole-4-carboxamide 100 mg 172050
hemisulfate
3-amino-2,4,6-triiodobenzoic acid 100 mg 196206
amitriptyline hydrochloride 100 mg 181101
amodiaquine hydrochloride 200 mg 192160
amphotericin B 400 mg 191153
ampicillin (anhydrous) 200 mg 390001
ampicillin sodium 200 mg 388002
ampicillin trihydrate 200 mg 274003
anhydrotetracycline hydrochloride 25 mg 180096
atropine sulfate 100 mg 183111
azathioprine 100 mg 172060

bacitracin zinc 200 mg 192174

beclometasone dipropionate 200 mg 192175
bendazol hydrochloride 100 mg 173066
benzobarbital 100 mg 172051
benzylamine sulfate 100 mg 172052
benzylpenicillin potassium 200 mg 180099
benzylpenicillin sodium 200 mg 280047
bephenium hydroxynaphthoate 100 mg 183112
betamethasone 100 mg 183113
betamethasone sodium phosphate 100 mg 196203
betamethasone valerate 100 mg 190145
betanidine sulfate 100 mg 172053
bupivacaine hydrochloride 100 mg 289054

caffeine 100 mg 181102

calcium folinate (leucovorin calcium) 100 mg 194188
captopril 100 mg 197214
captopril disulfide 25 mg 198216
carbamazepine 100 mg 189143
carbenicillin monosodium 200 mg 383043
chloramphenicol 200 mg 486004
chloramphenicol palmitate 1g 286072
chloramphenicol palmitate (polymorph A) 200 mg 175073
5-chloro-2-methylaminobenzophenone 100 mg 172061

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 Supplementary information

Reference substance Package size Control number

chloroquine sulfate 200 mg 195201
2-(4-chloro-3-sulfamoylbenzoyl)benzoic 50 mg 181106
acid
chlorphenamine hydrogen maleate 100 mg 182109
chlorpromazine hydrochloride 100 mg 178080
chlortalidone 100 mg 183114
chlortetracycline hydrochloride 200 mg 187138
cimetidine 100 mg 190150
ciprofloxacin hydrochloride 400 mg 197210
ciprofloxacin by-compound A 20 mg 198220
ciprofloxacin desfluoro-compound 20 mg 198219
ciprofloxacin ethylenediamine-compound 20 mg 198218
cisplatin 100 mg 197207
clomifene citrate 100 mg 187136
clomifene citrate Z-isomer see
zuclomifene
cloxacillin sodium 200 mg 274005
colecalciferol (vitamin D3) 500 mg 190146
cortisone acetate 100 mg 167006

dapsone 100 mg 183115

desoxycortone acetate 100 mg 167007
dexamethasone 100 mg 388008
dexamethasone acetate 100 mg 288009
dexamethasone phosphoric acid 100 mg 192161
dexamethasone sodium phosphate 100 mg 192158
diazepam 100 mg 172062
diazoxide 100 mg 181103
dicloxacillin sodium 200 mg 174071
dicolinium iodide 100 mg 172055
dicoumarol 100 mg 178077
diethylcarbamazine dihydrogen citrate 100 mg 181100
digitoxin 100 mg 277010
digoxin 100 mg 587011
dopamine hydrochloride 100 mg 192159
doxorubicin hydrochloride 100 mg 196202

emetine hydrochloride 100 mg 187134

4-epianhydrotetracycline hydrochloride 25 mg 288097
4-epitetracycline hydrochloride 25 mg 293098
ergocalciferol (vitamin D2) 500 mg 190147
ergometrine hydrogen maleate 50 mg 277012
ergotamine tartrate 50 mg 385013
erythromycin 250 mg 191154
erythromycin B 150 mg 194186

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Reference substance Package size Control number

erythromycin C 25 mg 194187
estradiol benzoate 100 mg 167014
estrone 100 mg 279015
etacrynic acid 100 mg 281056
ethambutol hydrochloride 100 mg 179081
ethinylestradiol 100 mg 301016
ethisterone 100 mg 167017
ethosuximide 100 mg 179088
etocarlide 100 mg 172057

flucloxacillin sodium 200 mg 195194

flucytosine 100 mg 184121
fludrocortisone acetate 200 mg 195199
fluoroqui-nolonic acid 20 mg 198217
fluorouracil 100 mg 184122
fluphenazine decanoate dihydrochloride 100 mg 182107
fluphenazine enantate dihydrochloride 100 mg 182108
fluphenazine hydrochloride 100 mg 176076
folic acid 100 mg 388019
3-formylrifamycin 200 mg 190149
framycetin sulfate (neomycin B sulfate) 200 mg 193178
furosemide 100 mg 171044

gentamicin sulfate 100 mg 194183

griseofulvin 200 mg 280040

haloperidol 100 mg 172063

hydrochlorothiazide 100 mg 179087
hydrocortisone 100 mg 283020
hydrocortisone acetate 100 mg 280021
hydrocortisone sodium succinate 200 mg 194184
(-)-3-(4-hydroxy-3-methoxyphenyl)-2-
hydrazino-2-methylalanine
(3-O-methylcarbidopa) 25 mg 193180
(-)-3-(4-hydroxy-3-methoxyphenyl)-
2-methylalanine (3-O-methylmethyldopa) 25 mg 179085

ibuprofen 100 mg 183117

imipramine hydrochloride 100 mg 172064
indometacin 100 mg 178078
o-iodohippuric acid 100 mg 171045
isoniazid 100 mg 185124

kanamycin monosulfate 12 mg 197211

322
 Supplementary information

Reference substance Package size Control number

lanatoside C 100 mg 281022
levodopa 100 mg 295065
levonorgestrel 200 mg 194182
levothyroxine sodium 100 mg 189144
lidocaine 100 mg 181104
lidocaine hydrochloride 100 mg 181105
liothyronine sodium 50 mg 193179
loperamide hydrochloride 100 mg 194185

mebendazole 200 mg 195195

melting point reference substances
azobenzene (69 °C) 1g 192168
vanillin (83 °C) 1g 299169
benzil (96 °C) 4g 294170
acetanilide (116 °C) 1g 297171
phenacetin (136 °C) 1g 297172
benzanilide (165 °C) 4g 192173
sulfanilamide (166 °C) 1g 192162
sulfapyridine (193 °C) 4g 192163
dicyanodiamide (210 °C) 1g 192164
saccharin (229 °C) 1g 192165
caffeine (237 °C) 1g 299166
phenolphthalein (263 °C) 1g 299167
metazide 100 mg 172058
methaqualone 100 mg 173069
methotrexate 100 mg 194193
methyldopa 100 mg 179084
methyltestosterone 100 mg 167023
meticillin sodium 200 mg 274024
metronidazole 100 mg 183118

nafcillin sodium 200 mg 272025

neamine hydrochloride (neomycin
A hydrochloride) 0.5 mg 193177
neomycin B sulfate see framycetin sulfate
neostigmine metilsulfate 100 mg 187135
nicotinamide 100 mg 200090
nicotinic acid 100 mg 179091
nifurtimox 100 mg 194189
niridazole 200 mg 186129
niridazole-chlorethylcarboxamide 25 mg 186130
norethisterone 100 mg 186132
norethisterone acetate 100 mg 185123
nystatin 200 mg 300152

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Reference substance Package size Control number

oubain 100 mg 283026
oxacillin sodium 200 mg 382027
oxytetracycline dihydrate 200 mg 189142
oxytetracycline hydrochloride 200 mg 189141

papaverine hydrochloride 100 mg 185127

paracetamol 100 mg 195198
paromomycin sulfate 75 mg 195197
pheneticillin potassium 200 mg 167028
phenoxymethylpenicillin 200 mg 179082
phenoxymethylpenicillin calcium 200 mg 179083
phenoxymethylpenicillin potassium 200 mg 176075
phenytoin 100 mg 179089
piperazine adipate 100 mg 197212
piperazine citrate 100 mg 197213
praziquantel 100 mg 194191
prednisolone 100 mg 389029
prednisolone acetate 100 mg 289030
prednisolone hemisuccinate 200 mg 195196
prednisolone sodium phosphate 200 mg 194190
prednisone 100 mg 167031
prednisone acetate 100 mg 169032
probenecid 100 mg 192156
procaine hydrochloride 100 mg 183119
procarbazine hydrochloride 100 mg 184120
progesterone 100 mg 167033
propicillin potassium 200 mg 274034
propranolol hydrochloride 100 mg 187139
propylthiouracil 100 mg 185126
pyrantel embonate (pyrantel pamoate) 500 mg 192157
pyridostigmine bromide 100 mg 182110

reserpine 100 mg 186133

retinol acetate (solution) 5 capsules1 898038
riboflavin 250 mg 382035
rifampicin 200 mg 191151
rifampicin quinone 200 mg 190148

sodium amidotrizoate 100 mg 198221

sodium cromoglicate 100 mg 188140
spectinomycin hydrochloride 200 mg 193176
streptomycin sulfate 100 mg 197215

1
Each containing about 8 mg in 230 mg of oil.

324
 Supplementary information

Reference substance Package size Control number

sulfacetamide 100 mg 196200
sulfamethoxazole 100 mg 179092
sulfamethoxypyridazine 100 mg 178079
sulfanilamide 100 mg 179094
sulfasalazine 100 mg 191155

tamoxifen citrate 100 mg 196208

tamoxifen citrate E-isomer 10 mg 196209
testosterone enantate 200 mg 194192
testosterone propionate 100 mg 167036
tetracycline hydrochloride 200 mg 180095
thioacetazone 100 mg 171046
4,4¢-thiodianiline 50 mg 183116
thyroxine sodium see levothyroxine
sodium
tolbutamide 100 mg 179086
tolnaftate 100 mg 176074
toluene-2-sulfonamide 100 mg 196204
trimethadione 200 mg 185125
trimethoprim 100 mg 179093
trimethylguanidine sulfate 100 mg 172059

vincristine sulfate 9.7 mg/vial 193181

vitamin A acetate (solution) see retinol
acetate (solution)

warfarin 100 mg 168041

zuclomifene 50 mg 187137

325
The International Pharmacopoeia

List of available International Infrared Reference Spectra1

International Infrared Reference Spectra are established on the advice of the
WHO Expert Committee on Specifications for Pharmaceutical Preparations.
Full-scale reproductions of spectra produced from authenticated material on a
suitable instrument are supplied for use in identification tests described in the
specifications for quality control of drugs, published in The International Phar-
macopoeia or proposed in draft monographs.
Precise instructions for the preparation of spectra are given on the label of
each reference spectrum. All International Infrared Reference Spectra are dis-
tributed together with a document giving further details on the use of such
spectra, entitled “General recommendations for the preparation and use of
infrared spectra in pharmaceutical analysis”.2
Orders for International Infrared Reference Spectra should be sent to:

WHO Collaborating Centre for Chemical Reference Substances

Apoteket AB
Produktion & Laboratorier Centrallaboratoriet, ACL
Prismavägen 2
S-141 75 Kungens Kurva
Sweden
(Fax: +46 8 740 6040; email [email protected])

The following International Infrared Reference Spectra are currently avail-

able from the Centre:

aceclidine salicylate caffeine (anhydrous)

acetazolamide calcium folinate
allopurinol carbidopa
amiloride hydrochloride chlorphenamine hydrogen maleate
amitriptyline hydrochloride clofazimine
ampicillin trihydrate cloxacillin sodium
colchicine
beclometasone dipropionate cytarabine
benzylpenicillin potassium
biperiden dexamethasone
biperiden hydrochloride dexamethasone acetate, mono-
bupivacaine hydrochloride hydrate

1
As updated at the thirty-seventh meeting of the WHO Expert Committee on Specifications for
Pharmaceutical Preparations, 22–26 October 2001.
2
WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-fourth report. Geneva,
World Health Organization, 1996, Annex 4 (WHO Technical Report Series, No. 863).

326
 Supplementary information

dextromethorphan hydrobromide niclosamide

diazepam nicotinamide
dicolinium iodide noscapine
dicoumarol
diethylcarbamazine dihydrogen oxamniquine
citrate
diphenoxylate hydrochloride papaverine hydrochloride
phenobarbital
erythromycin ethylsuccinate phenoxymethylpenicillin calcium
erythromycin stearate phenytoin
etacrynic acid primaquine phosphate
ethionamide propylthiouracil
ethosuximide protionamide
pyrimethamine
furosemide
salbutamol
gallamine triethiodide salbutamol sulfate
glibenclamide sulfadimidine
sulfadoxine
haloperidol sulfamethoxazole
hydrochlorothiazide sulfamethoxypyridazine

ibuprofen tiabendazole
imipramine hydrochloride trihexyphenidyl hydrochloride
indometacin trimethoprim
isoniazid
valproic acid
lidocaine verapamil hydrochloride
lidocaine hydrochloride
lindane

metronidazole
miconazole nitrate

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General guidelines for the establishment, maintenance, and

distribution of chemical reference substances
Introduction 329

Part A. Primary chemical reference substances 330

1. Assessment of need for the establishment of chemical reference
substances 330
2. Obtaining source material 331
3. Evaluation of chemical reference substances 332
3.1 Use in identification tests 332
3.2 Use in purity tests 332
3.3 Use in assays 333
3.4 Use in the calibration of an instrument 333
4. Chemical and physical methods used in evaluating chemical
reference substances 333
4.1 Methods used to verify the identity of chemical reference
substances 334
4.2 Methods used to determine the purity of chemical reference
substances 334
4.2.1 Separation techniques 334
Chromatographic methods 334
Capillary electrophoresis 335
4.2.2 Methods based on intrinsic thermodynamic properties 335
Differential scanning calorimetry 335
Phase solubility analysis 336
4.2.3 Other methods 336
Spectrophotometric methods 336
Titrimetric methods 336
Optical rotation methods 336
Determination of water and organic volatiles 336
5. Assignment of content 337
6. Handling and distribution of chemical reference substances 337
6.1 Packaging operations 337
6.2 Storage 338
6.3 Stability 338
6.4 Information to be supplied with chemical reference substances 339
6.5 Distribution and supply 340
6.6 Period of use 340

Part B. Secondary chemical reference substances 341

References 341

328
 Supplementary information

Introduction
In 1975, the WHO Expert Committee on Specifications for Pharmaceutical
Preparations recommended “General guidelines for the establishment, mainte-
nance and distribution of chemical reference substances” (1).1 At that time these
general guidelines were aimed at fostering greater collaboration and har-
monization among various national and regional authorities responsible for
collections of chemical reference substances. This aim is still relevant. The
guidelines were initially drawn up for particular use by the WHO Collaborat-
ing Centre for Chemical Reference Substances in Sweden, which provides
International Chemical Reference Substances (ICRS). These substances are pri-
marily intended for use with pharmacopoeial monographs included in The Inter-
national Pharmacopoeia (2).
It became evident that in order to meet particular national or regional phar-
macopoeial requirements, it was necessary to establish chemical reference sub-
stances external to the WHO Collaborating Centre for Chemical Reference
Substances. Another difficulty was to ensure prompt dispatch of the substances.
Since the meticulous work of the WHO Collaborating Centre establishing the
international collection would have to be duplicated in local or regional labo-
ratories, guidelines were necessary to ensure the integrity of national or regional
collections. In order to clarify the need for national and regional collections, the
1975 guidelines were reviewed and modified in 1982 (3). In view of refinements
in pharmaceutical and analytical methods since then, the present revision was
considered essential.
The purpose of having chemical reference substances is to achieve accuracy
and reproducibility of the analytical results required by pharmacopoeial testing
and pharmaceutical control in general. These substances are normally prepared
and issued by the regional/national pharmacopoeial commission or the
regional/national quality control laboratory on behalf of the drug regulatory
authority. In the context of these guidelines, the general use of a chemical ref-
erence substance should be considered an integral part of a compliance-oriented
monograph or test procedure used to demonstrate the identity, purity and
content of pharmaceutical substances and preparations.
The establishment of chemical reference substances should be based on
reports in which the results of analytical testing have been evaluated. These
reports should subsequently be approved and adopted by a certifying body,
normally the relevant pharmacopoeial committee or the drug regulatory
authority. Such establishment can be on an international, national or regional
basis. Each substance is generally established for a specific analytical purpose,
defined by the issuing body. Its use for any other purpose becomes the respon-
sibility of the user and a suitable caution is included in the information sheet
accompanying a reference substance. The present guidelines are concerned

1
The term chemical reference substances, as used in this text, refers to an authenticated uniform mat-
erial that is intended for use in specified chemical and physical tests, in which its properties are
compared with the properties of a product under examination, and which possesses a degree of
purity adequate for its intended use.

329
The International Pharmacopoeia

with both primary and secondary chemical reference substances as defined

below.
The preparation of a chemical reference substance should comply with
the requirements for quality assurance systems, including principles of good
manufacturing practices (GMP) and good control laboratory practices (4–6).
Adequate training programmes are also required. Both the WHO Collabo-
rating Centre and other laboratories concerned with the evaluation and estab-
lishment of chemical reference substances give assistance in training, subject to
the availability of resources.

Primary chemical reference substance

A designated primary chemical reference substance is one that is widely
acknowledged to have the appropriate qualities within a specified context, and
whose value is accepted without requiring comparison to another chemical
substance.

Secondary chemical reference substance

A secondary chemical reference substance is a substance whose characteristics
are assigned and/or calibrated by comparison with a primary chemical refer-
ence substance. The extent of characterization and testing of a secondary chem-
ical reference substance may be less than for a primary chemical reference
substance. This definition may apply inter alia to some substances termed
“working standards”.

Part A. Primary chemical reference substances

1. Assessment of need for the establishment of chemical
reference substances
The production, validation, maintenance and distribution of chemical reference
substances is a costly and time-consuming undertaking. It is therefore of great
importance to determine critically whether a need for a given substance exists.
Requests for new chemical reference substances usually arise when a particu-
lar approach to developing a specification for a new substance or product has
been adopted. Methods may have been proposed in a specification that require
the establishment of a chemical reference substance for use as a comparative
standard. Therefore, the first matter that should be assessed is whether an alter-
native, equally satisfactory, procedure could be adopted that does not require
a comparative standard.
Analytical procedures currently used in specifications for pharmaceutical
substances and products that may require a chemical reference substance are:

(a) infrared (IR) spectrophotometry, whether for identification or quantitative

purposes;
(b) quantitative methods based on ultraviolet (UV) absorption spectro-
photometry;

330
 Supplementary information

(c) quantitative methods based on the development of a colour and the mea-
surement of its intensity, whether by instrumental or visual comparison;
(d) methods based on chromatographic separation for identification or quanti-
tative purposes;
(e) quantitative methods (including automated methods) based on other
separation techniques that depend on partition of the substance to be
determined between solvent phases, where the precise efficiency of the
extraction procedure might depend upon ambient conditions that vary from
time to time and from laboratory to laboratory;
(f) quantitative methods, often titrimetric but sometimes gravimetric, that are
based on non-stoichiometric relationships;
(g) assay methods based on measurement of optical rotation; and
(h) methods that might require a chemical reference substance consisting of
a fixed ratio of known components (for example, cis/trans isomers, spiked
samples).

2. Obtaining source material

Source material of satisfactory quality can be selected from a batch (lot) of the
substance originating from the normal production process, if the purity is
acceptable. Further purification techniques may be needed to render the
material acceptable for use as a chemical reference substance.
The purity requirements for a chemical reference substance depend upon
its intended use. A chemical reference substance proposed for an identification
test does not require meticulous purification, since the presence of a small per-
centage of impurities in the substance often has no noticeable effect on the test.
On the other hand, chemical reference substances that are to be used in
assays should possess a high degree of purity. As a guiding principle, a purity
of 99.5% or higher is desirable, calculated on the basis of the material in its
anhydrous form or free of volatile substances. However, where the selectivity
of the analytical procedure for which the chemical reference substance is
required is low, such a degree of purity may not be necessary. In making a deci-
sion about the suitability of a chemical reference substance, the most impor-
tant consideration is the influence of the impurity on the attribute measured in
the assay when used in a non-specific assay procedure. Impurities with
physicochemical characteristics similar to those of the main component will
not impair the usefulness of a chemical reference substance, whereas even
traces of impurities with significantly different properties may render a sub-
stance unsuitable as a chemical reference substance.
When source material to be used as a chemical reference substance is
obtained from a supplier, the following should be supplied with the material:
• Certificate of analysis with complete information as to test methods
employed, values found and number of replicates used, where applicable,
and relevant spectra and/or chromatograms.

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The International Pharmacopoeia

• Information on optimal storage conditions required for stability (tempera-

ture and humidity considerations).
• Results of any hygroscopicity study and/or statement of the hygroscopicity
of the source material.
• Results of any accelerated stability studies.
• Identification of detected impurities (by preference), and/or specific infor-
mation on the relative response factor as determined in compendial
methods concerning the principal component, and/or the percentage mass
of the impurity.
• Updated Material Safety Data Sheet outlining any health hazards associated
with the material.

For new drug substances, manufacturers should be aware that elaboration

of pharmacopoeial monographs will be needed and a batch of the new sub-
stance should be set aside to be used if necessary as the chemical reference sub-
stance. It is desirable for bodies that issue chemical reference substances to
provide each other with a sample of the same batch of material, even if the
substance will be employed for different test methods. This will require the
exchange of information concerning the establishment process, supplier(s),
availability and conditions of supply.

3. Evaluation of chemical reference substances

The suitability of a substance proposed for use as a chemical reference requires
careful evaluation by the issuing body. It is necessary to consider all data
obtained from testing the material by a wide variety of analytical methods.
When taken as a whole, this will ensure that the substance is suitable for its
intended use. The extent of the analyses required depends on the purpose(s)
for which the chemical reference substance is to be employed, and may involve
a number of independent laboratories.

3.1 Use in identification tests

For use in identification tests (infrared spectrophotometry and/or chromato-
graphic methods), a batch of good quality material selected from the normal
production process is satisfactory if it is of acceptable purity. Additional purifi-
cation by the supplier may be necessary. The most important check is the appli-
cation of the test(s) for which the substance is intended. It is usual for at least
one laboratory to apply all the tests described in the relevant monograph.

3.2 Use in purity tests

The characterization of a chemical reference substance used in the determina-
tion of a specific impurity is more extensive, especially when used in a limit

332
 Supplementary information

test. If the technique employed is thin-layer chromatography (TLC) an accept-

able minimum purity is recommended (normally at least 90%), but purer
material may be required for liquid chromatography (LC) or gas chromato-
graphy (GC). If the proposed reference substance is being prepared or isolated
for the first time, appropriate chemical and physicochemical tests, such as
nuclear magnetic resonance (NMR), mass spectrometry (MS) and elemental
analysis, must be applied to characterize it.

3.3 Use in assays

If the chemical reference substance is to be used in an assay (colorimetry, LC,
GC or UV spectrophotometry), the extent of testing is very much greater.
Several (a minimum of three) laboratories should collaborate in testing the
proposed substance, using a variety of established and validated techniques,
including the method used in the pharmacopoeial specification. The relative
reactivity or relative absorbance of the impurities present must be checked
when a non-specific assay method is employed, e.g. by colorimetry or UV spec-
trophotometry. When a selective assay method is employed, it is particularly
important to determine the quantity of impurities. In such a case, it is best to
examine the proposed reference substance by as many methods as practicable
including, where possible, absolute methods. For substances that are acidic or
basic a titration with alkali or acid is simple, but other reactions which are
known to be stoichiometric may be used. Phase solubility analysis and differ-
ential scanning calorimetry may also be employed in certain cases.
The total of the determinations of water content, organic solvents, mineral
impurities and organic components should amount to 100%. For most chemi-
cal reference substances intended for assays, the content may be expressed “as
is”. When establishing the chemical reference substance it is therefore essential
to determine the content of water and residual solvents for a non-specific assay,
and also to determine the content of impurities for a selective assay.

3.4 Use in the calibration of an instrument

Where the chemical reference substance is to be employed as calibration
material, the extent of testing is similar to that for a chemical reference substance
used in assays. Several laboratories should collaborate in testing the proposed
substance using a variety of techniques to check that its purity is adequate. An
appropriate number of collaborating laboratories should also participate, after
the reference substance has been deemed suitable, to establish a value for the
essential property of the substance using an appropriate instrument.

4. Chemical and physical methods used in evaluating chemical

reference substances
It is important to establish by individual testing that a substance proposed for
use as a chemical reference is suitable.

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The methods used to establish the suitability of such a substance fall into
two broad groups: those intended primarily to identify the substance and those
used to establish its purity. With most methods, the percentage purity of a
chemical reference substance cannot be expressed as an absolute value if the
impurities have not been identified. The quoted purity is then an estimate
based upon the data obtained by the various analytical methods.

4.1 Methods used to verify the identity of chemical reference

substances
Where a proposed substance consists of a compound whose structure has been
satisfactorily defined, its identity may be confirmed by matching the IR spectra
of the substance to that of an authentic compound. Particular care should be
taken when polymorphism exists (7). Other highly specific techniques, such as
NMR spectroscopy, MS, or X-ray diffraction crystallography, may also be used
for such comparisons. The identity of a substance that is intended to replace
an established chemical reference substance of the same molecular constitution
must be verified, to determine that the characteristic properties of the two
specimens are identical. For this purpose it is often sufficient to compare their
IR absorption spectra.
However, where no authentic specimen of the proposed substance is avail-
able for comparison, and definitive data about its properties are lacking, it may
be necessary to verify its identity by applying several analytical techniques
currently used to characterize new compounds. Such analytical methods may
include elemental analyses, crystallographic studies, MS, NMR spectroscopy,
functional group analyses, and IR or UV spectrophotometry, as well as other
supplementary tests as required to establish that the proposed substance is fully
characterized.

4.2 Methods used to determine the purity of chemical reference

substances
The analytical methods to be employed in examining a substance should be
considered in relation to its intended use. These analytical methods may be
divided into three broad categories: those that require comparison with an
external chemical reference substance (e.g. chromatographic or spectrophoto-
metric methods), those that depend solely on an intrinsic dynamic property
(e.g. phase solubility analysis and differential scanning calorimetry) and other
methods.

4.2.1 Separation techniques

The methods used for the determination of purity should be established and
validated with system suitability requirements as appropriate.

Chromatographic methods. Methods of analysis based on chromatographic

separation are especially useful for detecting and determining impurities in

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chemical reference substances. High-performance liquid chromatography

(HPLC) is the most widely used chromatographic method, but TLC and GC
are also used. The individual components separated by chromatographic
methods may sometimes be recovered for characterization.
The selectivity of HPLC and of GC usually exceeds that of TLC. Both the
first two methods also have the advantage of being readily applicable on a
quantitative basis, but they require more complex equipment. HPLC, employ-
ing a spectrophotometric method of detection, is of particular value in the
examination of chemical reference substances intended for use in UV spec-
trophotometric assays. The UV wavelength of detection employed for deter-
mining the impurity content of the chemical reference substance should be
chosen so that the detection responses of the substance and its known impu-
rities are similar. When the response factors are significantly different at the
optimal wavelength of detection, appropriate corrections must be made to esti-
mate the content of impurities. LC with diode-array detection is very useful for
recording the UV spectra of both the main peak and the impurities. LC with
MS detection is used for identification of separated impurities as well as for
the main component, and is particularly important for chemical reference
substances where no other reference standards or IR reference spectra are
available.
In a GC method used for an assay, as with LC, the detection responses of
the known impurities are determined. Generally, GC monograph methods are
of particular value in detecting and determining volatile impurities, including
solvent residues, in chemical reference substances.
TLC uses apparatus that is simple and cheap; the technique is easy to carry
out and is readily applicable even in the microgram range. It can separate closely
related compounds, such as geometric isomers and the members of a homolo-
gous series. All the constituents of a substance submitted to chromatography
appear somewhere on the chromatogram. However, some constituents may
remain on the starting line, some may move with the solvent front, some may
migrate at the same rate as the main component, and some may remain un-
detected. For this reason, the usefulness of the method may be greatly enhanced
by means of two-dimensional chromatography and by using a number of
different solvent systems and a variety of detection methods. In some cases
the method may be used quantitatively with acceptable accuracy by using a
densitometer.

Capillary electrophoresis. Capillary electrophoresis is an increasingly common

method. It may be considered as complementary to LC for detecting impurities.

4.2.2 Methods based on intrinsic thermodynamic properties

Methods in this group measure total impurity levels in absolute terms.

Differential scanning calorimetry. This technique is used to check the presence

of different polymorphic forms and to determine the total amount of solid
impurities. Purity estimation is based on determination of the heat of fusion

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of the sample and of the change in its melting point caused by the presence of
impurities. This analytical method can be performed rapidly and with high
precision. However, it is not applicable if the substance decomposes on melting.
This limits its value as a general procedure for purity estimation of chemical
reference substances. It is also inapplicable if solid solutions are formed.

Phase solubility analysis. The method has occasionally been used, but its value
is limited and the procedure is time consuming. It may be employed to detect
contaminating substances, including isomeric species, and to estimate their con-
centration. Some factors that may make the method inapplicable are degrada-
tion of the substance during the course of analysis, formation of a solid solution,
and polymorphism in the main component.

4.2.3 Other methods

Spectrophotometric methods. UV spectrophotometry is occasionally used to
determine purity. Since it depends upon the presence of a characteristic chro-
mophore, it can detect impurities that contribute excessively to the absorbance
value and may indicate the presence of impurities that have a negligible or dis-
tinctive absorbance.
However, the utility of the method is limited by the small number of absorp-
tion maxima in the UV range, the large numbers of compounds containing
similar characteristic chromophores, and the need for an external chemical
reference substance.
IR spectrophotometry may be used to identify and determine the propor-
tions of geometric isomers. NMR spectroscopy, a powerful spectroscopic iden-
tification tool, is also occasionally useful in the determination of purity.

Titrimetric methods. Titrimetric methods provide a valuable means of confirming

the identity and purity of a proposed chemical reference substance and are
useful in confirming purity values obtained by other methods.

Optical rotation methods. Many chemical reference substances are optically active
and the relative proportion of optical isomers can sometimes be determined by
an optical rotation method, but generally such methods lack sensitivity.
However, the quantitative use of these techniques is well established and can
yield results of high precision, depending on the solvent and the wavelength
chosen for measurement. Chiral chromatography and NMR are becoming
increasingly important.

Determination of water and organic volatiles. It is essential that an accurate assess-

ment of the moisture content and the content of volatile contaminants be made.
These total values may often be obtained by drying under defined conditions
that are appropriate to the proposed substance. Sometimes this may not be
possible or may yield misleading results. In such cases, thermogravimetric
analysis may be used to determine the water and volatile content. Alternatively,

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the water content may be determined by Karl Fischer titration and the content
of volatile solvents by GC. Without an accurate assessment of these values at
the time that other determinations are being made, judgements of the accept-
ability of the proposed chemical reference substance will be invalid.

5. Assignment of content
If a content is to be assigned to a chemical reference substance, it should
be borne in mind that the value is based on the results of a collaborative
interlaboratory programme using different analytical methods. This experi-
mentally obtained value represents the best estimate of the true value. In
general, the assignment of content for a chemical reference substance is 100%
minus the content of water and volatiles, and when a substance is intended for
use as an assay standard based on a separation technique the impurity content,
as determined by that method, must also be subtracted. Sometimes the
chemical reference substances must be dried before use, in which case the
content is expressed on the basis of the dried material.

6. Handling and distribution of chemical reference substances

The handling, distribution and use of established chemical reference substances
must ensure that their integrity is safeguarded and maintained throughout their
period of use.

6.1 Packaging operations

Current GMP requirements (5) should be observed. The various stages in pack-
aging chemical reference substances should be clearly defined and controlled,
to avoid contamination of the sample, mislabelling of containers, or any other
event which might result in mishandling or mismanagement.
Containers for chemical reference substances should protect their contents
from moisture, light and oxygen and must be tested for moisture permeability.
Additional measures may be necessary to ensure long-term integrity and
stability. The best containers for chemical reference substances from the point
of view of stability are sealed glass ampoules, but these have certain disad-
vantages. There is the risk of contaminating the substance with glass particles
when the ampoules are opened, and reclosure is difficult. Sealable glass
ampoules are therefore principally used for substances that must be kept in an
oxygen-free atmosphere. Certain other substances may require even more elab-
orate protection. Most chemical reference substances, however, are conve-
niently supplied in reclosable containers which should be uniform in type and
size to facilitate distribution. The lack of permeability to moisture is an impor-
tant factor in determining the suitability of container closure systems.
Before undertaking any packaging operations, the health hazards of the item
to be packaged should be assessed through information sources, e.g. the
Material Safety Data Sheet. Appropriate precautions should be taken to protect
the person handling the chemical reference substance.

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The packaging of a batch of a chemical reference substance into containers

is a small-scale operation for which suitable equipment is now always avail-
able to the manufacturer of the material. Therefore, the packaging of chemical
reference substances is usually undertaken by the responsible issuing body.
Screw-type feeders have been constructed, but generally the packaging of
chemical reference substances is carried out manually. Substances which are
expensive or only available in very small quantities may have to be divided
between containers in solution and then lyophilized, or evaporated to dryness.
Some chemical reference substances must be packaged under an inert gas
or in conditions of controlled humidity. Therefore, the use of a glove-box or an
air-tight cabinet is necessary.

6.2 Storage
Information about suitable storage conditions can often be obtained from the
manufacturer of the source material and should be requested routinely when
a new chemical reference substance is established. Theoretically, the stability
of the substances should be enhanced by keeping them at low temperatures
but, for substances that contain water, storage below 0 °C may impair the
stability. It should also be remembered that the relative humidity in normal
refrigerators or cold-rooms may be high and, unless ampoules or other tightly
closed containers are used, the improvement in stability may be more than
offset by degradation due to the absorption of moisture. Storage at about
+5 °C, with precautions to prevent such absorption, has proved satisfactory
for most chemical reference substances.

6.3 Stability
A chemical reference substance is an integral part of the drug specification. Thus,
if the reference substance deteriorates, this will change the specification of the
drug. It is therefore of the utmost importance that the stability of chemical
reference substances should be monitored by regular re-examination and that
they should be replaced as soon as a significant change in a property is noted.
The definition of what is a “significant change” differs according to the
intended use of the chemical reference substance. Several per cent of degrada-
tion products found in a substance may not impair the usefulness of the mate-
rial in identification tests. For chemical reference substances that are used in
chromatographic assays, however, even small amounts of impurities may be
unacceptable. When establishing a chemical reference substance, consideration
must be given to its intended use and to the performance characteristics of the
analytical methods in which it will be used. The tolerable degree of degrada-
tion will be different from case to case.
Laboratories in charge of collections of chemical reference substances should
have a system for regular re-examination of the materials in stock. The fre-
quency of re-testing may be modified according to the need. It must be borne

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in mind that the stability of a specially prepared chemical reference substance

may not always be the same as that of commercial samples of the same
material.
The selection of suitable analytical methods for monitoring the stability of
chemical reference substances depends on the nature and intended use of the
substance. A substance used solely for identification purposes will normally
only require demonstration that it is still suitable for this use, e.g. that the IR
spectrum is identical to that obtained during establishment. If substances are
employed for other purposes, the testing must be more extensive but should
use methods which are rapid and sensitive so as not to consume too much of
the existing stock. It is important to check that there has been no significant
uptake of moisture, which could result in degradation by hydrolysis and/
or a decrease in the assigned content of the substance. Chromatography
is employed extensively, as well as absolute methods such as differential
scanning calorimetry where applicable. Changes in the impurity profile or
purity determination usually mean that the batch must be replaced. Changes
which compromise the integrity of the batch indicate it should immediately be
withdrawn from use. Sometimes a batch of a chemical reference substance will
discolour or otherwise change in appearance. Steps should be taken to replace
this substance whether or not the results of subsequent analyses indicate
significant degradation. Such changes in physical appearance reduce the confi-
dence of the user in the suitability of the chemical reference substance. Appro-
priate testing of active bulk substance should be carried out before further
dispensing into vials or ampoules.

6.4 Information to be supplied with chemical

reference substances
Labels on chemical reference substances should give the following information:

— the appropriate name of the substance: the international nonproprietary

name (INN) should be used wherever possible;
— name and address of the issuing body;
— approximate quantity of material in the container; and
— batch or control number.

Where associated documents are provided they should incorporate relevant

items from the list above. The following information should be given, as
necessary, on the labels and/or in associated documents:

— recommended storage conditions (if special conditions apply);

— intended use of the chemical reference substance;
— directions for use (e.g. storage and handling);
— information about assigned analytical value of the chemical reference sub-
stance (needed for calculation of the results of tests in which the substance
will be used);

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— a disclaimer of responsibility when chemical reference substances are

misused, or stored under inappropriate conditions, or used for other pur-
poses than those intended by the issuing body; and
— health hazard information or warning in conformity with national and
regional regulations or international agreements.

If analytical data are to be supplied with the chemical reference substances,

it is recommended that the data provided be limited to what is necessary for
the proper use of the substances in the tests and assays.

6.5 Distribution and supply

Distribution of chemical reference substances within the same country usually
does not present problems. However, when samples are to be sent to other
countries, both the sender and the receiver of the goods may encounter diffi-
culties because of the vagaries of postal and customs regulations, e.g. the
application of special procedural requirements applicable to substances under
international control. Distributors of chemical reference substances waste con-
siderable resources in seeking information on different international import
regulations, and in completing the required forms. A way of reducing such
difficulties and barriers to effective distribution of chemical reference sub-
stances should be sought. There should be the minimum delay in providing the
chemical reference substances to the users, and the most speedy means of
transport should be chosen.

6.6 Period of use

Chemical reference substances do not carry an “expiry date” in the conven-
tional sense. To avoid the unnecessary discarding of satisfactory substances, a
mechanism for general control of the batch of a chemical reference substance
may be used by the issuing body. If the issuing body applies stability con-
siderations and a monitoring procedure based on its experience to its collec-
tion, this should guarantee the user of the acceptability of the chemical
reference substance for its intended use.
If it is considered necessary to specify a beyond-use date, it should be stated
on the label and/or on a document accompanying the chemical reference
substances. Adequate shipping records should exist to enable contact with the
purchaser of a batch for recall or other notification.
The storage and maintenance of unopened containers of the chemical ref-
erence substance in accordance with information provided are integral to its
suitability of use. To avoid potential doubts concerning the integrity of opened
containers, it is suggested that potential users obtain only the quantities of sub-
stances necessary for short-term need and obtain fresh stocks (held under con-
trolled and known conditions) when needed. Long-term storage of substances
in opened containers is to be avoided. Similarly, efforts should be made to avoid

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possible degradation, contamination and/or introduction of moisture during the

repeated use of a substance.

Part B. Secondary chemical reference substances

The establishment of secondary chemical reference substances calibrated
against a primary chemical reference substance may be desirable for various
practical reasons, e.g. the latter may not be available in adequate quantities to
supply all local needs. Moreover, the availability of such secondary chemical
reference substances (for example, on a regional basis) would reduce the delay
in receiving the reference material.
The body which establishes a secondary chemical reference substance for
national/regional use should be clearly defined by the competent drug
regulatory authority. Clear documentation must exist to establish the rela-
tionship between the secondary and the primary chemical reference
substance.

References
1. WHO Expert Committee on Specifications for Pharmaceutical Preparations. Twenty-fifth
report. Geneva, World Health Organization, 1975, Annex 3 (WHO Technical Report
Series, No. 567).
2. The International Pharmacopoeia, 3rd ed. Vol. 1. General methods of analysis; Vol. 2. Quality
specifications; Vol. 3. Quality specifications; Vol. 4. Tests, methods, and general requirements.
Quality specifications for pharmaceutical substances, excipients, and dosage forms. Geneva,
World Health Organization, 1979–1994.
3. WHO Expert Committee on Specifications for Pharmaceutical Preparations. Twenty-eighth
report. Geneva, World Health Organization, 1982, Annex 1 (WHO Technical Report
Series, No. 681).
4. Good laboratory practices in governmental drug control laboratories. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations. Thirtieth report. Geneva, World
Health Organization, 1987, Annex 1 (WHO Technical Report Series, No. 748).
5. Good manufacturing practices for pharmaceutical products. In: WHO Expert Com-
mittee on Specifications for Pharmaceutical Preparations. Thirty-second report. Geneva,
World Health Organization, 1992, Annex 1 (WHO Technical Report Series, No. 823).
6. Good manufacturing practices for biological products. In: WHO Expert Committee on
Specifications for Pharmaceutical Preparations. Thirty-third report. Geneva, World Health
Organization, 1993, Annex 3 (WHO Technical Report Series, No. 834).
7. General recommendations for the preparation and use of infrared spectra in phar-
maceutical analysis. In: WHO Expert Committee on Specifications for Pharmaceutical
Preparations. Thirty-fourth report. Geneva, World Health Organization, 1996, Annex 4
(WHO Technical Report Series, No. 863).

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