- determination of loss on drying, which may in certain 4-2-3 Assay
circumstances replace the determinations of water and 4-2-3-1 Chemical assay. When a CRS is to be used for
residual solvents;
- determination of inorganic impurities (e.g. sulfated
ash, atomic absorption spectrometry, inductively
coupled plasma spectrometry, X-ray fluorescence
spectrometry); the results are usually not used to
determine an assigned content, except where they
would have an appreciable impact upon it;
- determination of purity by an independent method
(e.g. quantitative nuclear magnetic resonance
spectrometry, differential scanning calorimetry or
titration where appropriate; the results of these
determinations are usually used to support and
confirm the results obtained from separation
techniques; they are not used in the calculation of the
assigned content).
For biologicals, guidance is given in the WHO
recommendations for the preparation, characterisation and
establishment of international and other biological reference
standards (WHO Technical Report Series).
4-2 EUROPEAN PHARMACOPOEIA CHEMICAL
REFERENCE SUBSTANCES
The extent of testing and the number of laboratories involved
in the§stablishment of a CRS depend on the use of the CRS
and are.tailored to ensure fitness for purpose.
Where an interlaboratory study is carried out during
establishment, a protocol is provided for each participant and
only valid results derived according to the protocol are used
to determine an assigned content or otherwise confirm
suitability ,
Relevant parts of the following programme are typically
applied.
4-2-1 Identification
In general, the candidate batch is shown to comply with the
relevant requirements of the monograph; full structural
elucidation is carried out for the first batch.
4-2-2 Related substances test
A CRS corresponding to an impurity is characterised for
identity and purity. Where a CRS is used to determine the
content of a given impurity, the preferred minimum content
is 95.0 per cent; where this is achieved the assigned content
of the CRS is not given and it is considered to be
100.0 per cent; this approximation is acceptable since there
will be no appreciable effect on the determination of
impurities. When this minimum content cannot be obtained,
an assigned content is given to the CRS.
CRSs used to determine the content of a given impurity are
normally in the same acid, base or salt form as the substance
that is the subject of the corresponding monograph. Where
this is not the case, unless otherwise justified, a
corresponding stoichiometric conversion factor is applied.
If an impurity is not available in a sufficient quantity to
establish a CRS, a number of other options exist:
- preparation of a CRS that contains a mixture of the
compound(s) and the impurity or impurities;
- preparation of a CRS containing a mixture of specified
impurities.
Where such a mixture is also used to determine the content
of a given impurity, the content of the impurity in the CRS is
determined by appropriate separation methods and a content
is assigned to the reference standard.
www.webofpharma.com
quantitative determination of a substance for pharmaceutical
use (assay standard), the extent of testing is greater than
when a CRS is used for other purposes. Unless the substance
is of high purity, several collaborating laboratories are usually
involved in testing. The results obtained are used to assign a
content. It is particularly important to quantify the impurities
if a selective assay is employed. In such a case, it is best to
characterise the candidate substance by additional analytical
procedures that are scientifically justified, including, where
possible, independent methods and methods based 011
different principles. '
Fora European Pharmacopoeia chemical reference substance
established for assay purposes, the assigned content is usually
calculated from the values obtained from the analyses
performed for the determination of impurities (organic,
inorganic, water and solvents) by applying the principle of
mass balance; other suitable methods may also be used.
When possible, the assigned content is confirmed by
comparing with the result obtained by an independent
method.
If a CRS is required for a non-chromatographic assay
method (e.g, colorimetry or ultraviolet spectrophotometry),
the relative reactivity or relative absorbance of the impurities
present in a substance must be checked to ensure that they
are not markedly different from those of the substance.
Unless otherwise stated, an assigned content is given for the
substance or preparation as presented in the container ('as
is'), and the contents are not to be dried before use.
For assay standards prepared by lyophilisation, the content of
the pure substance is indicated in milligrams or International
Units per vial.
4-2-3-2 Microbiological assay. The potency is expressed in
International Units or in European Pharmacopoeia Units if
no international standard exists. The assigned potency
together with the confidence limits are calculated from
statistically valid results of an interlaboratory study, according
to the usual statistical procedures (5.3).
4-2-3-3 Assay of components of herbal drugs and herbal drug
preparations. Reference standards used in monographs on
herbal drugs vary in the extent of testing depending on the
type of reference standard.
An active component or marker constituent used as a CRS is
usually characterised and evaluated for identity and purity;
a value for content is assigned irrespective of the purity.
4-2-4 Establishment report
A report containing the results of the establishment study as
well as information concerning the use of the CRS is
prepared by EDQM, approved by the relevant group of
experts and adopted by the European Pharmacopoeia
Commission. The report for an assay standard indicates the
content assigned to the substance with the rationale for this
assignment. The estimated uncertainty of the assigned
content is calculated, and where it is less than a predefined
value, which is considered to be negligible in relation to the
acceptance criteria for the assay, then the study is accepted.
Otherwise, the study may be repeated, in whole or in part, or
the limits defined for the pharmaceutical substance may be
widened. The uncertainty of the assigned content is usually
not given as part of the information provided with the CRS,
since the precision of the method and the uncertainty of the
content assigned to the CRS are taken into account when
setting the limit(s) in a monograph.