Ref Standards

Pharmaceutical Reference
Standards

Part 1

Dr John H McB Miller
Head of DLab
EDQM
Council of Europe
Strasbourg
France
QCL Training Seminar, Tanzania
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5-7 Dec 07
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Pharmaceutical Reference Standards
Part 1
Definitions
Guidelines
Need
Procurement
Uses
Establishment
Adoption/Certification

Part 2
Production/Filling
Packaging
Re-test Programme
(Monitoring)
Difference between
reference material &
reference standard
Secondary reference
standard (in-house/working
standard)
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PRIMARY
CHEMICAL REFERENCE SUBSTANCE
l A designated primary chemical reference
substance is widely acknowledged as having
appropriate qualities within a specified context,
and whose value is accepted without reliance on
comparison to another chemical substance

l WHO/PHARM/96.590
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European Pharmacopoeia Reference Standard
A reference standard established under the aegis of and approved
by the European Pharmacopoeia Commission

European Pharmacopoeia Chemical Reference Substance
A substance or mixture of substances intended for use as stated in
a monograph or general chapter of the European Pharmacopoeia.
EPCRS are primary standards, except for those, notably antibiotics,
which are calculated in International Units. The latter are secondary
standards traceable to the international standard.
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DEFINITIONS
FDA Guideline for Submitting Documentation in Drug Applications for the
Manufacturing of Drug Substances
Reference Standard
A particular lot or batch of drug substance specifically
prepared, either by independent synthesis or by additional
purification of production material, and shown, by an extensive
set of analytical tests, to be authentic material of the highest
purity reasonably attainable. It is usually used for structural
elucidation, and is the benchmark for working standards.
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Reference Standard (ICH) Q6A
A reference standard, or reference material, is a substance
prepared for use as the standard in an assay, identification
or purity test. It has a quality appropriate to its use. For new
drug substances reference standards intended for use in
assays, the impurities should be adequately identified
and/or controlled and purity should be measured by a
quantitative procedure.
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Reference Material
Material sufficiently homogeneous and stable with respect to
one or more specified properties, which has been
established to be fit for its intended use in a measurement
process
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Certified Reference Material
An RM characterised by a metrologically valid procedure for
one or more specified properties, accompanied by a
certificate that states the value of the specified property, its
associated uncertainty and a statement of metrological
traceability
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BIOLOGICAL INTERNATIONAL STANDARD
An International Biological Standard is a biological or
synthetic preparation, the mean value of which, based on a
collaborative study, is used to define an international unit.
The assignment of potency in international units of the first
international standard is generally done arbitrarily. On the
other hand, potency in international units are assigned to
replacement international standards by calibration against
the previous standard on the basis of an international
collaborative trial. In all cases, these studies must
demonstrate that the preparations are suitable to be used as
international standards.

WHO Expert Committee on Biological Standardisation 40th Report World Health Organisation, Geneva (1990) (WHO
Technical Report Series 800)
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Biological Reference Preparations (Ph.Eur)

Biological reference preparations are either primary or secondary
standards. Secondary standards are usually calibrated in
International Units. Primary standards may have an assigned potency
in European Pharmacopoeia Units. Other assigned values may also
be used for primary standards, for example virus titre, number of
bacteria.
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Guidelines

5.12 Reference Standards
European Pharmacopoeia 6th Edition (2008)
ISO Guide 34
General Requirements for the Competence of Reference Material Producers
ISO Guide 35
Certification of reference materials.
General and statistical principles.
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GUIDELINES

WHO General Guideline for the Establishment,
Maintenance & Distribution of Chemical Reference
Substances
WHO Expert Committee for Pharmaceutical Preparations,
Annex 3, 41st Report, Technical Report to Series 943

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WHO Guideline Part A
Assessment of need
Obtaining source material
Evaluation of chemical reference substances
Chemical & physical methods used in evaluating
chemical reference substances
Assignment of content
Handling & distribution
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Need and Use
Analytical procedures currently used in specifications for
pharmaceutical substances & 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
spectrophotometry;
c) Quantitative methods based on the development of a colour &
the measurement of its intensity, whether by instrumental or
visual comparison.
d) Methods based on chromatographic separation for
identification or quantitative purposes;
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Need and Use
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 & from lab to lab;
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 (eg cis/trans
isomers, spiked samples)
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Procurement

Normal production batch: usually no further purification of
active substance (> 99.0 per cent purity)
Impurities: isolated or synthesised by the manufacturer
(> 95.0 per cent purity)
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Supplied with:
a certificate of analysis including test methods, test results with
complete identification by appropriate physico-chemical methods,
e.g. nuclear magnetic resonance spectroscopy, infrared
spectrophotometry, mass spectroscopy etc
Stability data of the substance with an indication of the storage
conditions to be employed,
Information as to its hygroscopicity and its solid-state properties,
e.g. amorphous, crystalline, polymorphic form etc
A material safety data sheet,
A list of potential impurities (if an active substance) with response
factors

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The Uses of the Reference
Substances/Preparations
Are clearly described in the individual monographs of the
Pharmacopoeias;
Are applicable to active pharmaceutical substances,
excipients and products
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Reference substances required for:
identification (of substance for pharmaceutical use),
system suitability (to ensure adequate performance of the
applied technique),
purity (identification and estimation of specified impurities
in a substance for pharmaceutical use manufactured by a
particular route of synthesis),
assay (of content of the substance for pharmaceutical use
or its products).
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Pharmacopoeial Reference Standards used for
identification of substances for pharmaceutical use
1. Identification:
Pharmaceutical reference standards can be used for
confirmation of identity of the substance by, e.g.
spectroscopy usually, infrared spectrophotometry where the
spectrum of the substance to be examined is compared to the
spectrum of the CRS, or to the reference spectrum,
separation techniques where the retention times (or migration
distance or migration time) of both the substance to be
examined and the CRS are compared.
identification by peptide mapping requires these of both a CRS
its chromatogram.
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2. Related Substances (organic impurities)
The system suitability criteria for selectivity may be:
resolution of two closely eluting impurities or an impurity and the main
compound;
peak-to-valley ratio of an impurity which is incompletely separated to the
main compound. A mixture of the compound with a small amount of the
impurity is required;
mixtures of impurities or a mixture of impurities and the compound as
reference standard (may be supplied with a chromatogram if prescribed
in the monograph) as a system suitability test based on the unadjusted
relative retentions of the impurity peaks to the substance peak and the
resolution or peak-to-valley ratio of specified peaks.

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System suitability

4 mg of abacavir sulfate for system suitability Control No 107244 monitored at 254 nm.

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Chromatogram of clazuril for system suitability CRS
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Chromatogram of
loperamide hydrochloride for system suitability CRS
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Location of impurity peaks

Confirmation of identification may be achieved by :
reference standards of individual specified impurities;
mixtures (as described above) where their chromatograms
serve also to locate the impurities in the chromatogram of
the test sample.
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Impurity Standards
By-products & degradation products

Synthesis of by-products & degradation products is often challenging
& the preparation of samples with greater than 95.0% purity may be
impossible. Since such comparison substances are used to determine
relative response factors, errors in the purity of 5.0% are not critical for
the quantitation of impurities in the drug substance at the 0.5% level or
less. However, purity correction must be made in the calculation of
the response factor.

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Pharmacopoeial Reference Standards used for Assay
physico-chemical methods chemical reference substances are employed to
assay for content of synthetic and semi-synthetic pharmaceutical substances,
herbal substances and peptides:
microbiological assay of antibiotics to determine their potencies. These
microbiological reference standards are referred to a chemical reference
substances in the European Pharmacopoeia and are secondary reference
standards since they have been established against the International Standard;
biological assays (in vitro or in vivo) to determine the activity/potency of
biological substances. The biological reference standards are referred to as
Biological Reference Preparations in the European Pharmacopoeia and may be
primary or secondary standards.
The assigned content/potency of the reference standard is method-specific i.e. it
is to be applied only with the method described in the specification or the
monograph.
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Establishment of Reference Substances
The extent of analyses required for the establishment
of Chemical Reference Substances depends on the
purpose(s) for which it is employed.
In general a batch of good quality, selected from the
normal production of the substance, is satisfactory.
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1) to characterise the substance (proof of molecular
structure) by appropriate chemical attributes such as
structured formula, empirical formula and molecular
weight. A number of techniques may be used including:
Nuclear magnetic resonance (NMR) spectroscopy ;
Mass spectroscopy;
Infrared spectroscopy. The spectra are to be interpreted to
support the structure.
Elemental analysis to confirm the percentage composition of
the elements.

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2) determination the purity
determination of the content of organic impurities by an appropriate separation technique
(eg gas chromatography (GC), liquid chromatography (LC) or capillary electrophoresis
(CE));
quantitative determination of water (eg micro or semi-microdetermination);
determination of the content of residual solvents;
determination of loss on drying may in certain circumstances replace the determinations of
water and residual solvents;
determination of the purity by an absolute method (eg differential scanning calorimetry or
phase solubility analysis where appropriate. The results of these determinations are to
support and confirm the results obtained from separation techniques. They are not
normally included in the calculation of the assigned value);
determination of inorganic impurities (test for heavy metals, sulphated ash, atomic
absorption spectrophotometry, ICP, X-ray fluorescence) often the values obtained will
have no consequence on the assignment of the purity of the standard.
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3) Assay (for the active ingredient only) by an absolute
method (volumetric titration) is recommended.
The assigned content of the primary chemical reference
standard is calculated from the values obtained from the
analysis performed for the determination of purity and are
verified by a calculation of the mass balance.
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Purity of Impurities

at least 90.0 per cent when used only to locate a peak in the
chromatogram of the substance for pharmaceutical use;
at least 90.0 per cent when used in the system suitability test for
resolution;
at least 95.0 per cent when used to estimate the content of a
specified impurity (for the purpose of the estimation it is considered
to be 100 per cent). When the minimum purity cannot be obtained,
then a purity value is assigned to the standard.

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Most chemical reference substances are fully
characterised & are traceable to the molecular weight.
They are considered to be Primary Reference
Substances - no comparison to existing standards.
Chemical Reference Substances (some antibiotics) used
for the microbiological assay of potency & many of the
Biological Reference Preparations are secondary
reference substances since they are calibrated against
the international standard established by WHO.

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Assignment of content/potency of
Reference Substance/Preparation
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Assay
Pharmacopoeial reference standards with an assigned
content required for comparative assay techniques:
Infrared spectrophotometry
Ultraviolet/visible spectrophotometry
Chromatography

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Characterising Reference Material
(ISO Guide 35)
Single definitive method by a single organisation
Two or more independent reference methods by one
organisation
Number of methods of known and acceptable accuracy
and precision by a network of qualified organisations
Method specific approach (inter-lab study) giving only a
method specific assessed property value
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Assignment of Content
Ph.Eur. Policy
The value assigned is method specific and is
estimated from the results obtained by a
collaborative trial (min. 6 participants). Other
techniques may be employed to determine the
content but these results are not used for
assigning the value but give complementary
supporting information
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European Pharmacopoeia
Collaborative Trials

Participation by:
Experts of the European Pharmacopoeia
Academia
Industry
OMCLs
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Initiating Laboratory
(European Pharmacopoeia Laboratory)
Monograph requirements
Residual Solvents
Determination of Response Factors of Impurities
Absolute Analytical Method (Volumetric titration, DSC)

Protocol prepared by the Ph.Eur Laboratory
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Protocol
the determination of water (or loss on drying);
the estimation of the organic impurities (including residual
solvents when appropriate) using the prescribed
separation techniques;
the determination of the content of the substance by an
absolute method (usually volumetric titration) may be
included. These are confirmatory determinations and the
results are not used in the calculation of the assigned
value and are not necessarily performed by all
participants.
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Protocol
Loss on drying or
Semi-micro determination of water
AC for repeatability
AC for repeatability
Estimation of organic impurities by the
assay method
AC for repeatability, selectivity,
sensitivity, symmetry, retention
time
Estimation of Residual Solvents (> 0.5%) AC for repeatability, selectivity,
symmetry, retention time
Estimation of inorganic impurities (if
necessary)
Representative chromatogram is supplied for info
AC = Acceptance Criteria
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Content of the Reference Standard
The content that is used to correct the purity of the reference
standard is generally calculated as follows:

No correction of % loss on drying or sum water and solvents is
done if the value is <0.10%.
For a chiral chromatographic quantitative methods, the content
of antipode is not taken in account in the % impurities.
Impurities are corrected for their response factors.
Impurities are determined at limit of quantification.

100
purity %
x solvents) & water sum or drying on (loss - % 100
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CHEMICAL REFERENCE SUBSTANCES
UNCERTAINTY OF THE ASSIGNED VALUE
Estimated uncertainty =

o
i
2
+o
w
2
+o
s
2
n
t
n1
( )

Where
n = number of participating laboratories
o
i
2
= is the variance for the estimation of impurities
o
w
2
= is the variance for the estimatin of water
o
s
2
= is the variance for the estimation of residual solvents
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ASSAY STANDARD FOR LIQUID
CHROMATOGRAPHY
Normal limit accepted for analytical error 2.0%
Uncertainty of 0.24% implies 0.1% probability of rejecting
a good result (n=3)
If this is considered to be insignificant then there is no
justification for giving uncertainty values with the assigned
value
Maximum permitted uncertainty varies proportionally to
the limits set e.g. maximum permitted uncertainty for
limits of 1.0% would 0.12
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Uncertainty
The estimated uncertainty of the result of a collaborative
trial is employed to assess the acceptability of the trial.
If the estimated uncertainty is greater than a pre-defined
criterion then the trial is considered to be unsatisfactory &
after investigation & identification of the causes of failure
the monograph may be revised & the trial repeated.
With in-built acceptance criteria in the protocol this
eventuality is unlikely.

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Assigned value of reference substances
Assigned value =

100 (water content % + organic impurities % + residual solvent %)
or
100 (loss on drying % + organic impurities %)

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DETERMINATION OF THE UNCERTAINTY OF THE ASSIGNED
VALUE
TICARCILLIN SODIUM CRS
Per cent Impurities
Related Substances Water
Mean 4.03 (n=6) 4.69 (n=7) 4.61(n=6)
SD (o) 0.074 0.224 0.0758
Variance (o
2
) 0.005 0.05 0.0057

Approx uncertainty =
0.005
6
+
0.005
7
2.5=0.1
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Assay
USP/BP
Active pharmaceutical
ingredient + pharmaceutical
products

If no content is given for RS
assumed to be 100 per
cent for assay.
Ph.Eur.
Active pharmaceutical
ingredient

CRS with assigned content
only for assay method
described in the
corresponding monograph
for the API & pharmaceutical
product
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Extended use of Ph. Eur Reference Standards
1. Assay reference standard used for determination of content in a
monograph for a substance for pharmaceutical use can be used
for the assay of that substance in a pharmaceutical product.
Provided that:
- the assay method employed for the product is that described in
the monograph of the substance for pharmaceutical use;
- any pre-treatment of the sample (eg extraction) is validated by the
user.
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Misuse of European Pharmacopoeia Reference
Standards

If used for another purpose other than those described in
the monograph it is the responsibility of the user to
validate appropriately the reference standard
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CERTIFICATE
Proposed International Chemical Reference Substance
ABACAVIR SULFATE
Control No 106238

Intended use
The International Chemical Reference Substance for abacavir sulfate is
intended to be used in the infrared absorption spectrophotometric and
thin-layer chromatographic tests for identity according to the monograph
for Abacavir sulfate in The International Pharmacopoeia.

Analytical data
mg of 1.3 : A spectrum, nfrared absorption spectrophotometry (IR) I
abacavir sulfate in 300 mg of potassium bromide, is given in figure
W106238T.

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4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400,0
0,0
10
20
30
40
50
60
70
80
90
100,0
cm-1
%T
W106238T

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Ultraviolet spectrophotometric absorption data: = 416 at 297 nm,
calculated with reference to the dried substance. Abacavir sulfate was
dissolved in 0.1 M hydrochloric acid.

High performance liquid chromatography (HPLC): The purity was
estimated by peak area normalization to 99.8% at 254 nm.

Thin-layer chromatography (TLC): The purity was estimated to
100.0% at 254 nm, when 100 g were applied.

Thermogravimetric analysis (TG): When heated to 105 C a loss of <
0.1% (w/w) was observed.
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Storage

Abacavir sulfate should be kept in a tightly closed container, protected from
light.

Reference: This certificate is extracted from the report, which is the basis for
the adoption of this International Chemical Reference Substance by the
WHO Expert Committee on Specifications for Pharmaceutical Preparations.

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CERTIFICATE
Proposed International Chemical Reference Substance
ABACAVIR SULFATE FOR SYSTEM SUITABILITY
Control No 107244
Intended use

The International Chemical Reference Substance for abacavir sulfate for system
suitability is intended to be used in the liquid chromatographic test for related
substances according to the monograph for Abacavir sulfate in The International
Pharmacopoeia, Fourth Edition,
. http://www.who.int/medicines/areas/quality_safety/safety_efficacy/mon_arvs/en/

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Analytical data
iquid chromatographic system suitability test High performance l
The method used is described in the monograph for Abacavir sulfate.
When abacavir sulfate for system suitability was injected, the following peaks were
eluted at the following relative retention times with reference to abacavir (retention
time about 19 minutes):
Impurity B = 1.3
Impurity C = 0.7
Impurity D = 1.04
Impurity E = 1.10
Impurity F = 1.6
The resolution between abacavir and impurity D was 2.3.

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4 g of abacavir sulfate for system suitability Control No 107244 monitored at 254 nm.
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Storage

Abacavir sulfate for system suitability should be kept in a tightly closed container,
protected from light.

Reference: This certificate is extracted from the report, which is the basis for the
adoption of this International Chemical Reference Substance by the WHO
Expert Committee on Specifications for Pharmaceutical Preparations.

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Web-page of European Pharmacopoeia
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LC column information
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CRS Catalogue
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Batch validity Statement

Ref Standards

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