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Handling of Reference Standards_GMP_Dr. A. Amsavel


Definition for Reference standard Requirements Guidelines Pharmacopiea Types of Reference Standards GMP- ICH Q7 SOP for handling of Reference Standards Qualification of Secondary Standards Assigning Potency, Storage and Use Documents & Records – PowerPoint PPT presentation

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Title: Handling of Reference Standards_GMP_Dr. A. Amsavel

Handling of Reference Standards
  • Dr. A. Amsavel

  • Definition
  • Requirements
  • Guidelines
  • Pharmacopiea
  • Types of Reference Standards
  • SOP for handling of Reference Standards
  • Qualification of Secondary Standards
  • Assigning Potency, Storage and Use
  • Documents Records

Definition (ISO-Guides 30-35)
  • Reference Material (RM) 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 (CRM) Reference
    material characterised by a metrologically valid
    procedure for one or more specified properties,
    accompanied by a certificate that provides the
    value of the specified property, its associated
    uncertainty, and a statement of metrological

Definition (ISO- Guides 30-35)
  • Primary Standard Standard that is designated or
    widely acknowledged as having the highest
    metrological qualities and whose value is
    accepted without reference to other standards of
    the same quantity, within a specified context.
  • Secondary Standard Standard whose value is
    assigned by comparison with a primary standard of
    the same quantity.

Definition (ICH Q7)
  • Reference Standard, Primary A substance that has
    been shown by an extensive set of analytical
    tests to be authentic material that should be of
    high purity. This standard can be
  • obtained from an officially recognised source, or
  • prepared by independent synthesis, or
  • obtained from existing production material of
    high purity, or
  • prepared by further purification of existing
    production material.
  • Reference Standard, Secondary A substance of
    established quality and purity, as shown by
    comparison to a primary reference standard, used
    as a reference standard for routine laboratory

ICH Q7 PRS Requirement
  • 11.17. Primary reference standards (PRS) should
    be obtained as appropriate for the manufacture of
  • The source of each primary reference standard
    should be documented and Records should be
  • Storage and use as per suppliers
  • PRS obtained from an officially recognised source
    are used without testing if stored as per
    recommended conditions .
  • 11.18 Where a PRS is not available from an
    officially recognized source, an in-house
    primary standard should be established.

ICH Q7PRS Requirement
  • Appropriate testing should be performed to
    establish fully the identity and purity.
    Documentation of this testing should be
  • 11.19. Secondary reference standards should be
    appropriately prepared, identified, tested,
    approved, and stored.
  • The suitability of secondary reference standard
    should be determined prior to first use by
    comparing against a primary reference standard.
  • Each batch of secondary reference standard should
    be periodically re-qualified in accordance with a
    written protocol.

EU GMP Requirement
  • EUGMP EudraLex Volume-2, Part-1 Chapter-6 QC-
  • Reference standards should be established as
    suitable for their intended use. Their
    qualification and certification as such should be
    clearly stated and documented.
  • Whenever compendial reference standards from
    an official source exist, these should preferably
    be used as primary reference standards unless
    fully justified (the use of secondary standards
    is permitted once their traceability to primary
    standards has been demonstrated and is documented.

European Pharmacopoeia
  • 5.12. REFERENCE STANDARDS (General Chapter)
  • A Primary standard is characterised by a variety
    of analytical techniques chosen to demonstrate
    its suitability for use .
  • A Reference standard corresponding to an impurity
    is characterised for identity and purity with
    minimum content of 95.0 , The content being
    considered as 100.0 since there will be no
    appreciable effect on the determination of
  • EP Reference standards are mostly stored in
    temperature at 5 3 C, otherwise specified in
  • If the standard is presented in multi-use
    containers then re-testing may be more frequent
    for hygroscopic or oxygen-sensitive substances.
    Eg water/LOD content

European Pharmacopoeia
  • Secondary standard. A standard whose property
    value is assigned by comparison with a primary
    standard of the same property or quantity.
  • A secondary standard should exhibit the same
    properties as the primary standard, relevant
    test(s) shall be performed. The extent of testing
    may be scientifically justified. Quantitative
    purpose required comprehensive test to establish
  • The secondary standard is established by
    comparison with the primary standard to which it
    is traceable.

Type of EP CRS
  • Type of Reference Standards available in EDQM
  • Identification
  • Assay
  • Impurities Quantitative
  • Impurities Qualitative
  • Mixtures for System Suitability/ Peak
  • Purpose/Usage
  • Batch testing release
  • System suitability test
  • Evaluation of a measurement system

Type of EP CRS
  • Availability of EP CRS other than API
  • Reference spectrum
  • Elemental impurity eg,Lead solution CRS (1.00
  • Instrument calibration / qualification
  • Caffeine CRS for Calibration standard for MR,
    WL accuracy for LC
  • Sodium Aminosalicylate dihydrate CRS for
    equipment qualification / Calibration. Loss on
    drying(2.2.32 ) Value 169.6 ( 2.4) mg/g
    Uncertainty 0.4 mg/g
  • Refer Leaflet Contains Scientific information,
    purpose, use, potency, uncertainty of assigned
    value, Instruction for use, validity, Storage

USP RS General Chapter lt11gt
  • Official applications of USP RS are specified in
    the monographs and general chapters.
  • Pharmacopeial Reference Standards are highly
    characterized specimens
  • Where USP states in the monograph that use
    USPRS, in the Assays and/or the Identification
    tests. It is legal requirement.
  • Secondary standard can be used, but it should be
    qualified using the USPRS.
  • Potency or content of USP RS is assumed as100.0
    pure, unless otherwise directed or stated in the

USP RS General Chapter lt11gt
  • In case of dispute in the test results, value
    obtained using the specified USP RS is
  • If any USP RS is not yet available, that portion
    of the standard containing the requirement shall
    not be official until USP RS is available
  • USP Reference Standards for Assay and
    Identification for Qualitative or Quantitative
  • Refer for more details
  • Technical Data Sheets or Typical Chromatograms.
  • Weights and Balances lt41gt Volumetric Apparatus
  • Adjustments to Solutions in the General

Types of Reference Standards
  • USP Reference Standards for Impurities
  • Organic impurities Starting materials,
    intermediates, by-products, reagents, catalysts,
    and/or degradation products.
  • Inorganic impurities Reagents, catalysts, heavy
    metals, or inorganic salts
  • Residual solvents that may be either inorganic or
    organic liquids.
  • Authentic Visual References (AVR) are used for
    visual images to compare certain test that they
    meet compendial requirements.
  • Performance Verification Test Standards-
    Instruments qualification and calibration.
    Method-specific uses, (e.g., performance
    verification standards, AVR, melting point
    standards, and the particle count set)

Information USP RS
  • USP RS are provided with Technical Data Sheets or
    Typical Chromatograms.
  • USP RS do not carry an expiration date on the
    label. It may be used as long as it is listed as
    "Current Lot" in the Catalog or has not reached
    its Valid Use Date.
  • Catalogs published USP website (check quarterly
    for validity)
  • Upon depletion, the lot is designated in the
    catalog as "Previous Lot" and a "Valid Use Date"
    is assigned.
  • Refer for more details_at_ https//www.usp.org/refere

How to Use USP RS
  • Use USPRS as per Technical data sheet or as per
  • As Is Use without any prior treatment or
    correction for volatiles. (confirm from valid
    data that the volatiles are constant over time)
  • Dry Before Use Use immediately after drying
    under stated conditions. Drying should not be
    performed in the original container. A portion of
    the material should be transferred to a separate
    drying vessel.
  • Determine Water Content Titrimetrically At Time
    of Use Use with a correction for the water
    content or the loss on drying, determined on a
    separate portion of material.
  • Test Water Determination as per Method I (921).
  • Instrumental or microanalytical methods are
  • When about 50 mg of RS is used for water test,
    use dilute reagent with 2 to 5 fold

How to Use USP RS
  • Some of the USP RS provided in single-use
    containers. Labeled as reconstituted in its
    entirety without any additional weighing, where
    given in the monographs.
  • Intended use and that value assignment and
    uncertainty information are considered.
  • USP RS should be stored in the packaging
    configuration provided by USP (e.g., vials
    packaged in hermetically sealed).
  • When special storage conditions are specified,
    label directions should be followed.

SOP for Handling of RS/CRS
  • Establish the procedure for handling of Reference
    standards to address the following, but not
    limited to .
  • Purpose Scope
  • Responsibility Assign co-ordinator for handling
    reference standards/ working standards
  • obtained from an officially recognized source
    like Pharmacopoeia reference substances /
    certified RS
  • Ordering and receipt of RS/CRS
  • Label with details

SOP for Handling of RS/CRS
  • Maintain the records such as PO, date of receipt,
    COA or leaf let,
  • Storage store as per storage condition in
    secured area
  • Use appropriate RS for testing, calibration,
    qualification of equipment, instruments or other
  • Usage log with purpose and reconciliation
  • Periodic verification of validity or availability
    of new standard
  • Include the qualification of secondary reference
    standard or Prepare separate SOP.

Qualification of Secondary Standard
  • Qualification of Secondary /Working standard
  • Standard for Quantitative Use
  • Extensive physico-chemical testing is required
    for the standards used for quantitative purpose.
  • The level of characterization depends on the
    intended use
  • Choose the tests method to confirm the mass
  • The secondary /working standard can be procured
    external source or synthesis or select from
    commercial batch.
  • Assay Select one of the batches which have
    minimum 99.0 of assay /purity and confirm the
  • Purify the material further if purity is less.

Qualification of Secondary Standard
  • Qualify against official Reference Standard
  • Test the assay / chromatographic purity at least
    in triplicate
  • Test other parameters, which are related to
    potency like Loss on drying and / or water by
    TGA or Karl Fischer, Residual solvents, Residue
    on Ignition etc.
  • Perform Inter-laboratory study to assign a
    content (if required)
  • Calculate the uncertainty of measurement
  • Recommended to assign the content on an as is
  • Assign the potency / content based on mass
    balance considering LOD / water content, Residual
    solvent or ash content etc
  • Potency (100 LOD/water/solvents/inorganics)
    x chromatographic purity()/100.

Qualification of Secondary Standard
  • Assigned value has to be checked by Orthogonal
    techniques like non-aqueous titration or other
    suitable techniques, such as CHN-analysis.
  • Perform characterization against official
    reference standard Mass, FTIR, NMR, UV
    spectroscopy DSC, XRD polymorph as required.
  • In case of impurity standard select the
    assay/purity of min 95
  • Record all the details like source batch, tests,
    primary reference used, and potency and keep the
  • Maintain the test record with raw data, ref
    standard used.
  • Pack and Label the vials with potency, use,
    validity and storage .

Qualification of Secondary Standard
  • Standard for Quantitative Use (RT
    Identification or system suitability)
  • Procure or synthesis or select from in-house,
    ensure that content is at least 95 If t is less
    justify scientifically for its suitability.
  • Test against reference standard (if available)
    otherwise perform complete test for
  • Test the following physico-chemical properties to
    elucidate the structure.
  • Chromatographic purity, FTIR- UV spectrum, mass,
    NMR and CHN-analysis and prepare the COA with
  • Maintain the test record with raw data, spectrum,
  • Pack and Label the vials with potency, validity,
    storage and pre-defined date for use and
    numbering the vials
  • Retest and confirm to extend the validity / usage

Instruction for Usage
  • Take a reference standard vial from storage area
    and allow to controlled room temperature prior to
  • Open the reference standard vial carefully and
    keep the inner cap and outer cap in the inverted
    position on a clean butter paper.
  • Use a clean stainless steel spatula or paper for
    one time use for transfer of RS.
  • Dont return unused standard to the vial.
  • After the weighing / transfer close the vial
  • Dont expose the reference standard for too long
    to avoid pick up of moisture.
  • Return the closed vial to their respective
    places, securely.

Label Reference Standards
  • Reference standards labelled with information
  • In-house ref. No
  • Name
  • Batch, lot No
  • Date of opening
  • Shelf-life (if applicable)
  • Potency
  • Storage conditions
  • Usage condition
  • Vial No.

Records for Reference standards
  • Record for reference standard
  • Ref No /identification No. of the material
  • Description of the material
  • Source date of receipt
  • batch designation or other identification code
  • Potency , LOD any other details as appropriate
  • Intended use of the material (e.g. as an infrared
    reference material, as an impurity reference
    material for thin-layer chromatography, etc.)
  • Location of storage in the laboratory, and any
    special storage conditions

Packing and Use
  • Pack the working standard in 2ml/5ml/10ml vials
    or as appropriate.
  • Pack required number of vials based on the
    requirement and frequency of use, eg. one vial
    for each month / three months
  • Assign the validity / expiry based on the
  • Pack required number of vials and assign validity
    of each vial. Record the details in the working
    standard usage login register
  • If molecule is stable assign 2 years and the
    extent validity period after retest. Specify
    clearly in the SOP extend the validity period for
    6 months.
  • Label the vials and store at recommended storage
  • Record the opening date use until the valid date
    of vial
  • Dispose the excess sample as per validity/ expiry

  • Contact Dr. A. Amsavel
  • aamsavel_at_gmail.com

Thank You
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