Discussion forum for Pharma Quality events, Regulatory Actions

Warning letters, 483s, Recalls, Import Alerts, Audit observations

Updated on April 14, 2025

Authored by: Srinivas Churya

This Quality Insight post presents a concise approach for selection and qualification of Inhouse Reference Standards and Secondary standards (Working standards) by USP Mass balance approach for pharmaceutical actives (APIs) and impurities.

Broadly the standard qualification program and management can be divided into following steps

  1. Policy for Selection of material
  2. Assigning Potency of the standard
  3. Dispensing of standard to make Master Vial and Usage Vials of standard.
  4. Storage of standard
  5. Assigning validity and unique identification number for the standard
  6. Issuance and usage of standard, Vial testing
  7. Requalification of standard
  8. Retesting of the working standard at the end of the shelf life
  9. Destruction of standard at the end of the shelf life

A. Policy for selection of material:

  1. Defne a policy for the selection of material for standard preparation (for both API and Impurity standards). Typically:
  • synthesized from R&D
  • a commercial batch or raw material from a supplier is selected
  • prepared by other isolation technique such as Preparative HPLC, TLC etc.

 In case of commercial batches, choose from the best available material.

Example:

  • In case of API select a batch, which has low level of impurities (including water, Loss on Drying – LOD, or Residue on ignition).
  • Often an available batch is selected and additional purifications are performed in R&D lab for removing the impurities
  • The material should be complying to the specifications
    • For APIs the applicable API specifications
    • For impurity standards, define a specification based on the Route of synthesis. Parameters to be considered (but not limited to) could be Purity / Assay, %Water or LOD, Residue on Ignition (ROI) etc.
  1. Define level of characterization required for the material
  • Inhouse reference standard – Typically UV, IR, Mass, NMR spectral characterisation and Elemental analysis (CHNO plus any other elements in the structure)
  • Secondary standard – IR spectra comparison with primary standard

B. Assigning Potency of Standard

Prepare a protocol defining what are the parameters to be analysed.

  • Parameters which impact the content or assay of the material shall be selected for analysis apart from material characteristics
    • Parameters related to Material Characteristic: Description, IR, UV, SOR etc
    • Parameters Impacting content: Related substances by HPLC and / or GC, Residual solvents by GC, Residue on ignition, Loss on Drying-LOD or Water content shall be included as applicable.
    • While qualifying secondary standards for which an Official standard (Pharmacopeia Standard) is available, perform the assay analysis and identification test by IR spectral comparison. The material shall meet the Pharmacopeia requirements. This qualifies the secondary standard against the pharmacopeia standard and establish its traceability to the official standard.
  • Arrive at Potency by USP Mass Balance approach
    • USP Mass Balance approach can be considered a most robust and comprehensive method for estimating the potency of a reference material as it accounts for all measurable components of the material. The mass balance approach integrates multiple analytical techniques, providing a holistic view of the material’s composition and assigned potency will be most reflective of the material’s true active content

Assigning potency for API standards:

mg of analyte per mg of the analyte =

 (100%-Sum of % of dry basis Impurities) X (100%-Sum of % of as is basis Impurities)                                                                             10000

 % Purity or Potency (as is basis) = mg of the analyte X 100

 ·        % dry basis impurities are related substances  and impurities, typically determined by HPLC and/ or GC, for example:Sum of %dry basis impurities =         

=          Total impurities observed by the HPLC and / or GC X 100

                                                                        [100 – %LOD or %(Water + Residual solvent)

Note: If different impurities or related substances are estimated by different HPLC and GC methods add all of these impurities to get total impurities. 

·   % as is basis impurities are parameters such as LOD, Water content, Residue on ignition etc, for example

 Sum of % of as is basis Impurities =

 = (%Residual solvents by GC+% LOD or Water content+% Residue on ignition)

Note: Either water content and residuals solvents by GC shall be considered or % LOD alone shall be considered

Exceptions & Notes:

  • Potency is assigned by USP Mass Balance approach. Analysis of assay for secondary standard is only meant for qualifying the standard against official standard, not for assigning potency
  • Where the Pharmacopeia doesn’t have a Chromatographic assay or Purity / Related Substances method and only assay by titration are defined consider following approaches with appropriate scientific justification.
    • Establish an inhouse chromatographic method for Purity / Impurities, validate the method. Use this method to determine chromatographic impurities in the material and further proceed to assign potency as per mass balance approach or
    • Perform Assay as per Pharmacopeia method to qualify standard. Anyway potency of the standard may not be consequential as it is not involved in determining assay of the sample.

Assigning potency for impurity standards:

  • Same procedure described above for API shall be applied when there is sufficient quantity of material available to establish parameters like Water Content, LOD, ROI etc.
  • If the quantity of material available is not sufficient, establish the % dry basis impurities by HPLC and / or GC to assign potency. Justify the approach in the protocol. (Example impurity standard is only used for identification, impurities in the samples are quantified against the API standard etc).
  • However, if it is critical to have the accurate potency of the impurity standard, generate sufficient quantity of the standard to establish potency by mass balance and further establish the response factor against the API standard. Use this potency factor while calculating impurities in sample. The impurity standard itself will be used only for qualitative purposes like identification of retention time, system suitability etc in routine analysis.

C. Dispensing of standard to make a Master Vials and Usage Vials of standard. 

  • Dispense the standards into vials under laminar air flow unit and under dehumidifier (for hygroscopic material) to make Master Vials of the standard and Usage vials. Record the weight of each vial and label the vials. Extra care should be taken for the hygroscopic material during dispensing.
  • Usage vials are prepared from Master vials which will be used in routine analysis, For e.g monthly usage vials, weekly usage vials etc. The periodicity shall depend on number of times the usage vials will be opened during its usage time validity, stability, hygroscopicity of the material etc.
  • Master Vials are opened and used only rarely; for e.g. preparing usage vials for customers / other laboratories, retesting the standard etc.

D. Storage of standard

  • For storage conditions of the standard check the reference standard recommendation in pharmacopeia ( USP and EP) and follow the same.
  • For compounds not having pharmacopeial reference, check other appropriate references (e.g. scientific literature, material safety data sheets, supplier recommendations etc.).
  • Create a system for the monitoring storage temperature of the standard {Refrigerated storage (2-8°C), Controlled room temperature, Room temperature etc}.
  • Any excursions of storage temperature shall be handled through the deviation management system.

E. Assigning validity and unique identification number for the standard

  • Define the validity period of the standard (e.g. 1 year, 6 months etc) with due consideration for characteristic of the material (hygroscopicity, stability). The validity shall not exceed the expiry period of the material from which the standard is prepared.
  • Assign a unique identification number for each standard certified.

F. Issuance and usage of standard, Vial testing

  • Create a system for the subdivision of standard material into smaller portions as Usage Vials (for example monthly vials). Prepare the required number of usage vials along with Master vial. For example, if the validity of the standard is one year and usage vials will have validity of 1 month (monthly vials), prepare 12+1 or 2 vials.
  • For impurity standards where quantities available are very small, it may not be practical to prepare monthly usage vials. In such cases take adequate precautions shall be taken to ensure integrity of the standard is not compromised by frequent opening and closing.
  • Create a system to document and monitor the usage of the Usage vials (e.g. monthly vials).
  • Create a system to document and trace usage of Master vials.
  • Define a system for Vial testing of the Usage vials (monthly vials) at the end the defined usage period ( example at the end of the month). Typically Chromatographic purity by HPLC or GC and any other critical parameter which can potentially be impacted by repeated opening and closing of vial during usage shall be evaluated. For other parameters initial analysis values can be used. Assess the potency of the standard. The potency shall be 0.98 – 1.02.
  • Vial testing is performed to confirm the stability and validity of the usage vial at the end of the defined usage time (e.g. end of the month). If the results of vial testing are not complying, handle it as a deviation. Evaluate the impact and actions to be taken, for e.g.
    • The usage vial validity may need to be reduced (for example from monthly vial to fortnightly or weekly vial).
    • Replace the standard, requalify a new standard.
    • Define shorter validity period for the standard (for e.g revise validity to six months from 1 year).
    • Evaluate impact on the batches analysed and released.
  • Define a system to record vial usage of standard. This data will be helpful during failure investigations.

G. Requalification of standard 

  • Create a system for requalification of the standard if the same material to be continued as a standard beyond its defined validity.
  • Follow the same procedure as defined under Section B for assigning the potency of the requalified standard
  • Requalification of standard shall be an exception than rule. For example, in the case of difficult to synthesize impurities, rarely manufactured APIs.
  • The validity for the requalified standard shall not exceed the expiry of the material from which the standard was selected.

H. Retesting of the standard at the end of the shelf life

  • Create a system for retesting the potency of standard (from Master vial) at the end of the defined validity. This data is useful for the future working standard qualification and defining validity period of the standard.
  • Evaluate the same parameters that was tested for assigning the potency of the standard under Section B and calculate the potency.
  • Typical acceptance criteria for Potency shall be 0.98 to 1.02.
  • If the potency if not meeting acceptance criteria during retesting, handle it as a deviation. Evaluate the impact and actions to be taken, as defined in Section F.

I. Destruction of standard at the end of the shelf life

  • Create a system for destruction and disposal of the standard at the end of the shelf life.
  • After the validity period over for the vial, discard the left over material following the procedures for disposal of chemical waste
  • Remove the vial label. Send both vial and material for destruction.

Definitions

Primary Standard

:

Primary standards are obtained from certified sources and come with a Certificate of Analysis (COA) that verifies their purity and quality. They are used without requiring comparison to another substance, ensuring reliability in analytical procedures

Official Standard

:

Refers to a reference material or substance that is officially recognized by regulatory authorities, such as the United States Pharmacopeia (USP), European Pharmacopoeia (EP), or other pharmacopeial bodies

Secondary Standard / Working Standard

:

A reference material that is established and validated against a primary standard. It is used for routine analysis and quality control testing when the use of a primary standard is not practical or necessary

Inhouse Reference Standard

:

A reference material developed and characterized internally by a pharmaceutical company when an official pharmacopeial standard is unavailable or unsuitable for specific testing purposes

Potency of Standard

:

Refers to the exact quantitative content of the active ingredient in a reference material. It is expressed as a percentage and accounts for all impurities, such as organic and inorganic impurities, residual solvents, counter ions, loss on drying, or water content.

LOD

:

Loss on Drying

ROI

:

Residue on Ignition

References:  Pharmacopeial Forum Vol. 33(6) [Nov-Dec 2007], The Application of Uncertainty to USP’s Compendial Reference Standards Program: Certified Reference Materials

Tags :

Leave a Comment