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Part 1 Dr John H McB Miller Head of DLab EDQM Council of Europe Strasbourg France Pharmaceutical Reference Standards Part 1 Definitions Guidelines Need Procurement ... – PowerPoint PPT presentation

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Title: Guidelines

Pharmaceutical Reference Standards
Part 1 Dr John H McB Miller Head of
DLab EDQM Council of Europe Strasbourg France
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
  • Secondary reference standard (in-house/working

  • 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
  • WHO/PHARM/96.590

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.
DEFINITIONSFDA 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.

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.

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

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

  • 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)

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

  • 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.

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

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

Need and Use
  • Analytical procedures currently used in
    specifications for pharmaceutical substances
    products that may require a chemical reference
    substance are
  • Infrared (IR) spectrophotometry, whether for
    identification or quantitative purposes
  • Quantitative methods based on ultraviolet (UV)
    absorption spectrophotometry
  • Quantitative methods based on the development of
    a colour the measurement of its intensity,
    whether by instrumental or visual comparison.
  • Methods based on chromatographic separation for
    identification or quantitative purposes

Need and Use
  1. 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
  2. Quantitative methods, often titrimetric but
    sometimes gravimetric, that are based on
    non-stoichiometric relationships
  3. Assay methods based on measurement of optical
    rotation and
  4. Methods that might require a chemical reference
    substance consisting of a fixed ratio of known
    components (eg cis/trans isomers, spiked samples)

  • Normal production batch usually no further
    purification of active substance (gt 99.0 per cent
  • Impurities isolated or synthesised by the
    manufacturer (gt 95.0 per cent purity)

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
  • 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

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

Reference substances required for
  • identification (of substance for pharmaceutical
  • 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).

Pharmacopoeial Reference Standards used for
identification of substances for pharmaceutical
  • 1. Identification
  • Pharmaceutical reference standards can be used
    for confirmation of identity of the substance by,
  • 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
  • identification by peptide mapping requires these
    of both a CRS its chromatogram.

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

System suitability
4 mg of abacavir sulfate for system suitability
Control No 107244 monitored at 254 nm.
Chromatogram of clazuril for system suitability
Chromatogram of loperamide hydrochloride for
system suitability CRS
Location of impurity peaks
  • Confirmation of identification may be achieved by
  • reference standards of individual specified
  • mixtures (as described above) where their
    chromatograms serve also to locate the impurities
    in the chromatogram of the test sample.

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.

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
  • 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.

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
  • In general a batch of good quality, selected from
    the normal production of the substance, is

Establishment of Reference Substances
  • 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.

Establishment of Reference Substances
  • 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
  • quantitative determination of water (eg micro or
  • determination of the content of residual
  • 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.

Establishment of Reference Substances
  • 3) Assay (for the active ingredient only) by an
    absolute method (volumetric titration) is
  • 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.

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

  • 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
  • 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.

  • Assignment of content/potency of
  • Reference Substance/Preparation

  • Pharmacopoeial reference standards with an
    assigned content required for comparative assay
  • Infrared spectrophotometry
  • Ultraviolet/visible spectrophotometry
  • Chromatography

Characterising Reference Material(ISO Guide 35)
  • Single definitive method by a single organisation
  • Two or more independent reference methods by one
  • Number of methods of known and acceptable
    accuracy and precision by a network of qualified
  • Method specific approach (inter-lab study) giving
    only a method specific assessed property value

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

European PharmacopoeiaCollaborative Trials
  • Participation by
  • Experts of the European Pharmacopoeia
  • Academia
  • Industry
  • OMCLs

Initiating Laboratory (European Pharmacopoeia
  • Monograph requirements
  • Residual Solvents
  • Determination of Response Factors of Impurities
  • Absolute Analytical Method (Volumetric titration,
  • Protocol prepared by the Ph.Eur Laboratory

  • 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

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 (gt 0.5) AC for repeatability, selectivity, symmetry, retention time
Estimation of inorganic impurities (if necessary)
Representative chromatogram is supplied for
info AC Acceptance Criteria
Content of the Reference Standard
  • The content that is used to correct the purity of
    the reference standard is generally calculated as
  • No correction of loss on drying or sum water
    and solvents is done if the value is lt0.10.
  • For a chiral chromatographic quantitative
    methods, the content of antipode is not taken in
    account in the impurities.
  • Impurities are corrected for their response
  • Impurities are determined at limit of

  • Estimated uncertainty

  • Normal limit accepted for analytical error ? 2.0
  • Uncertainty of 0.24 implies 0.1 probability of
    rejecting a good result (n3)
  • 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

  • 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
  • With in-built acceptance criteria in the protocol
    this eventuality is unlikely.

Assigned value of reference substances
  • Assigned value
  • 100 (water content organic impurities
    residual solvent )
  • or
  • 100 (loss on drying organic impurities )

(No Transcript)
  • Per cent Impurities
  • Related Substances Water
  • Mean 4.03 (n6) 4.69 (n7) 4.61(n6)
  • SD (?) 0.074 0.224 0.0758
  • Variance (?2) 0.005 0.05 0.0057

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
Extended use of Ph. Eur Reference Standards
  • 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.

Misuse of European Pharmacopoeia Reference
  • 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

CERTIFICATEProposed International Chemical
Reference SubstanceABACAVIR SULFATEControl No
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 Infrared
absorption spectrophotometry (IR) A spectrum,
1.3 mg of abacavir sulfate in 300 mg of potassium
bromide, is given in figure W106238T.
(No Transcript)
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 µg were applied. Thermogravimetric analysis
(TG) When heated to 105 C a loss of lt 0.1
(w/w) was observed.
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.
CERTIFICATEProposed International Chemical
SUITABILITYControl 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//
Analytical data High performance liquid
chromatographic system suitability test 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.
4 ?g of abacavir sulfate for system suitability
Control No 107244 monitored at 254 nm.
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.
Web-page of European Pharmacopoeia
LC column information
CRS Catalogue
Batch validity Statement