PREFACE

The basic reason for this guideline is to help those involve in the analysis of drug quality
control to set up a proper secondary reference standard. Procedures laid in this guideline
cover aspects of preparation, handling, standardisation, storage dan usage of chemical
reference standard and biological reference standard (antibiotic).

Usage of appropriate reference standard is an important characteristic in Good

Manufacturing Practice. Primary reference standards such as British Pharmacopoeia
Chemical Reference Substances (BPCRS), International Chemical Reference Substances
(ICRS), United States Pharmacopoeia Reference Standards (USPRS) are too expensive
for daily analysis use. Therefore, secondary reference standards or working standards
need to to be prepared in sufficient quantity for routine use.

Assay test results which are accurate and acceptable with high level of confidence depend
mainly on the quality and purity of the reference substance in use. Proper steps need to
be taken to ensure that the reference substances used for the analysis of drug quality
control are of quality, adequately standardised, handled, kept and used correctly.

Reference Standard Unit

Drug Analysis Division
National Pharmaceutical Control Bureau
Email : ref_std@[Link]

July 2000

First print : October 1993

ISBN 983-9870-10-6

First revision : July 2000

 GUIDELINES FOR THE ESTABLISHMENT,
HANDLING, STORAGE AND USE OF CHEMICAL
REFERENCE SUBSTANCES

CONTENTS PAGE

1. Introductio n …………………………………………………………… 1

1.1. Definition ……………………………………………………... 1

1.2. Types of Analytical Procedure ……………………………….. 1

2. Acquisition of Raw Material …………………………………………. 2

3. Chemical and Physical Methods used in the the Standardisation of

Reference Substances ……………………….………………………… 2

3.1. Methods used for verifying identity of Reference Substances 2

3.2. Methods used in determining the purity of Reference

Substances and presence of impurities ………………………. 3

4. Handling and use of Reference Substances ………………………….. 4

4.1. Packing, storage and maintenance …………………………….. 4

4.2. Stability and Re-evaluation ………………………………….. 4

4.3. Documents that must be supplied with Reference Substances 5

5. Summary …………………………………………………………….. 5

6. References …………………………………………………………… 6
 GUIDELINES FOR THE ESTABLISHMENT,
HANDLING, STORAGE AND USE OF CHEMICAL
REFERENCE SUBSTANCES

1. INTRODUCTION

1.1. Definition

A Chemical Reference Substance can be defined as a material that is intended for

use in specified chemical and physical tests, in which its properties are compared
with the properties of the samples under examination. Such material should also
possess a degree of purity adequate for its intended use.

1.2. The types of analytical procedure used in specifications of pharmaceutical

products and substances that may require a chemical reference substance are :

1.2.1. Infra-red spectrophotometry for quantitative purposes

1.2.2. U.V. absorption spectrophotometry for quantitative purposes

1.2.3. Colour development and its intensity comparison, whether using

instrumentation of visual comparison for quantitative purposes

1.2.4. Chromatographic methods for identification and also quantitative

purposes

1.2.5. Titration and also optical rotation methods for quantitative purposes

1.2.6. 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.

1.2.7. Methods that might require a chemical reference substance consisting of

a fixed ratio of known components (for example, cis/trans isomers,
spiked samples).

Apart from the above analytical procedures, there are also several laboratory
equipment where their calibrations require the use of reference substances, such
as didymium or holmium oxide filters for the calibration of wavelengths and
potassium dichromate or potassium nitrate for the calibration of absorbance of
UV spectrophotometer.

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 As quality control tests involve the above analytical procedures, the use of
reference substance is of utmost importance. It is the responsibility of the quality
control laboratory concerned to prepare working standards or secondary
reference standards.

Reference substances can be divided into two categories, i.e. primary reference
substances and secondary reference standards. A secondary chemical reference
substance is a substance whose characteristics are assigned and/or calibrated by
comparison with a primary chemical reference substance. The extent of
characterization and testing of a secondary chemical reference may be less than
for a primary chemical reference substance. This definition may apply inter alia
to some substances termed “working standards”.

Primary reference substances can be obtained from sources such as :

a) British Pharmacopoeia Commission Laboratory

b) WHO Collaborating Centre for Chemical Reference Substances

c) The United States Pharmacopeial Convention, Inc.

d) European Department for the Quality of Medicines

2. ACQUISTION OF RAW MATERIAL

The quality of raw material used for the derivation of reference substance depends upon
the purpose of its use. If it is used for quantitative purpose, then its purity should be
higher than that used for qualitative purpose.

Sufficient amount for intended use of at least 3 – 5 years, should be acquired from
reliable sources such as the main manufacturer of the raw material. Analytical data, i.e.
certificate of analysis, from the supplier should be accompanied.

3. CHEMICAL AND PHYSICAL METHODS USED IN

THE STANDARDISATION OF REFERENCE SUBSTANCES

After acquiring the suitable raw material, tests are carried out on the aspects of identity,
purity, stability and suitability for its intended use.

Only adequate sample raw material is taken from its packing for analytical tests.

3.1. Methods used for verifying identity of reference substances

Tests that are capable of demonstrating that the characteristic properties of the
two specimens are identical, are usually employed. For this purpose, a
comparison of their IR absorption spectra often suffices.

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 Other than IR, highly specific techniques, such as nuclear magnetic spectroscopy,
mass spectrophotometry and X-ray diffraction crystallography, may also be used.

Test methods using UV spectrophotometry and other supplementary tests as

necessary, may also be required to verify the identity of the intended substance.

3.2. Methods used in determining the purity of reference substance and presence
of impurities

The purity requirement for reference substance depends upon its intended use.
Reference substances that are used in assays should possess a high degree of
purity : preferably a purity of 100% + 0.5% is desirable calculated on the basis of
the material in its anhydrous form or free of volatile substances.

3.2.1. Chromatographic Methods

These methods are especially useful for determining whether there are
impurities in reference substances. TLC, GC and HPLC are methods
often used. TLC is more useful for qualitative purposes. The use of GC
and HPLC have more advantages over TLC because of their applicability
for quantitative purposes.

3.2.2. Spectrophotometric Methods

UV Spectrophotometry is widely used for determination of purity of a

reference substance.

3.2.3. Titration Methods

These methods are also used for determining the purity of a reference
substances. The type of titration often used is non aqueous titration.

3.2.4. Optical Rotation Methods

For reference substances that are optically active, the optical rotation
methods are useful for quantitative purposes.

3.2.5. Differential Scanning Colorimetry Methods

The use of these methods is based on the determination of the heat of

fusion of the intended substance. From the difference in melting point,
the presence of impurities can be determined.

3.2.6. Other Methods

Other Methods such as gravimetric analysis, electrophoresis, and atomic

absorption spectroscopy, are also used in determination of purity.

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 Whatever methods are used to standarised reference substances, it is essential
that the moisture content and the content of the volatile contaminants, are also
assessed. Moisture content can be determinded by total drying or by Karl Fischer
titration. The content of the volatile contaminates may be determined by GC
method.

4. HANDLING AND USE OF REFERENCE SUBSTANCES

The measures employed in handling and use of reference substances must provide for
assurance that their integrity will be safeguarded and maintained throughout their period
of use.

4.1. Packing, Storage and Maintenance

Containers used should afford protection to reference substances from moisture,

light and oxygen. From the point of stability, sealed glass ampoules are
recommended. An important matter to be considered when choosing suitable
containers, is their permeability to moisture.. Additional measures may be
necessary to ensure long term integrity and stability.

The filling of reference substances into containers can be carried out in a glove
box with facility for nitrogen supply, where humidity is controlled.

Theoretically, the stability of the substances should be enhanced by keeping by

keeping them at low temperatures but, for substances that contain water, storage
below 00 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 +50 C, with precautions to prevent such absorption, has proved
satisfactory for most chemical reference substances.

The storage and maintenance of unopened containers of the chemical reference

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 substances
necessary for short-term need and obtain fresh stocks (held under controlled and
known conditions) when needed. Long-term storage of substances in opened
containers is to be avoided. Similarly, efforts should be made to avoid possible
degradation, contamination and/or introduction of moisture during the repeated
use of a substance.

4.2. Stability and Re-evaluation

It is important that the stability of reference substances be maintained by carrying

out regular re-examination so that action can be taken should there be a change
from their original properties.

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 Any change in their properties, such as formation of degraded products in small
amounts may not impair the use of the reference standards for IR identification,
but may affect assay tests using HPLC or GC methods. Re-standardisation is
recommended within a period of three years.

4.3. Documents that must be supplied with Reference Substances

Two important documents that are required to be supplied together with

reference substances are labels and certificate of analysis.

4.3.1. Information that should be indicated on the labels of a reference

substances are :-

a) Name of reference substance :

b) Batch or Control Number :

c) Approximate quantity of reference substance in container :

d) Purity in percentage :

4.3.2. The certificate of analysis should contain information such as :-

a) Name of reference substance :

b) Batch or Control Number:

c) Description of the physical nature of the reference substance, test

carried out together with their results, particularly those tests
where their information are needed for calculation of the results
of tests :

d) Intended use of the reference substance :

e) Direction for use (e.g. instruction about drying before use) :

f) Storage conditions required for stability:

5. SUMMARY

All reference substances used in a quality control laboratory should be maintained by a

responsible person who shall keep a central record of the materials. Among the
information that should be recorded includes :-

a) Name of reference substance :

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 b) Identification number (code number) :

c) Source acquired :

d) Batch Number :

e) Intended use :

f) Storage location :

f) Storage condition :

The identification number should be labelled on every vial of the reference substance and
this number should be noted on the analytical work sheets whenever the material is used.

The personnel responsible for the maintenance of reference substances is also advised to
keep a file for every reference substances available. The information that may be filed
includes a summary of all tests and standardisation carried out to prepare the reference
substance, as well as test results or certificate of analysis, and the name of the analyst
who evaluates the reference substances.

The personnel responsible should also regularly check the storage condition to ensure that
reference substances are properly kept to avoid deterioration.

6. REFERENCES

6.1 British Pharmacopoeia 1988, A 146.

6.2 WHO Expert Committee on Biological Standardisation, 37th report. Technical
Report Series 760, WHO Geneva, 1987.
6.3 Draft Guidelines for Collaborative Assays. National Biological Standards
Laboratory, Australia.
6.4 Int. biological standards and reference preparations. Journal of Biol. Standardisation
1974, 2, 249 – 258.
6.5 World Health Organisation. WHO Technical Report Series, No. 885, 1999.

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