Reference standard

What is the difference between a primary and a secondary reference standard?

ICH Q7 defines a primary reference standard as a substance that has been shown by an extensive set of analytical tests to be authentic material that should be of high purity. It may be obtained from an officially recognized source, prepared by independent synthesis, obtained from existing production material of high purity, or prepared by further purification of existing production material.^[2]

The same guidance defines a secondary reference standard as a substance of established quality and purity, as shown by comparison to a primary reference standard, used as a reference standard for routine laboratory analysis.^[2]

ICH Q6B, written for biotechnological and biological products, uses parallel terms. An in-house primary reference material is an appropriately characterized material prepared by the manufacturer from a representative lot or lots, and an in-house working reference material is always calibrated against that primary material. The Q6B glossary uses the term reference standards for international or national standards.^[3]

Where do reference standards come from?

FDA guidance on analytical procedures and methods validation states that reference standards can often be obtained from USP and may also be available through the European Pharmacopoeia, the Japanese Pharmacopoeia, the World Health Organization or the National Institute of Standards and Technology.^[4]

ICH Q7 states that the source of each primary reference standard should be documented, and that primary reference standards obtained from an officially recognized source are normally used without testing if stored under conditions consistent with the supplier's recommendations.^[2]

Where a primary reference standard is not available from an officially recognized source, ICH Q7 states that an in-house primary standard should be established, that testing should be performed to establish fully its identity and purity, and that documentation of this testing should be maintained. ICH Q6B notes that for new molecular entities an international or national standard is unlikely to be available.^[2],[3]

The FDA guidance states that reference materials from other sources should be characterized by procedures including routine and beyond-routine release testing as described in ICH Q6B, and names orthogonal methods among the approaches to consider for that characterization.^[4]

How is a new batch of a reference standard linked to the one before it?

ICH Q7 states that the suitability of each batch of secondary reference standard should be determined prior to first use by comparing it against a primary reference standard, and that each batch of secondary reference standard should be periodically requalified.^[2]

The FDA guidance states that a new batch of reference standard material, official or in-house, should be qualified or calibrated against the current reference standard. For biological reference standards and materials it describes a two-tiered approach, in which each new reference standard is compared with a primary reference standard, as a way to prevent drift in the quality attributes.^[4]

ICH Q6B states that in-house working reference materials used in testing production lots should be calibrated against the in-house primary reference material, and that where an international or national standard is available and appropriate, reference materials should be calibrated against it.^[3]

How is a reference standard used in an analysis?

ICH Q2(R2) states that the capability of an analytical procedure to identify an analyte can be confirmed by obtaining positive results comparable to a reference material using samples containing the analyte, along with negative results from samples that do not contain it.^[1]

ICH Q2(R2) defines accuracy as the closeness of agreement between a value accepted as a conventional true value or an accepted reference value and the value measured. One approach it describes applies the procedure to an analyte of known purity, such as a reference material, and compares the measured values with the theoretically expected values.^[1]

Where an analyte gives a different response from the reference material, for example a different specific UV absorbance, ICH Q2(R2) states that relative response factors should be calculated from the appropriate ratio of responses.^[1]

Which reference standards are used in endotoxin testing?

FDA guidance on pyrogen and endotoxins testing describes control standard endotoxins (CSEs) as endotoxin preparations other than the international or national reference standards that are traceable in their calibration to the international reference endotoxins standard. CSEs may be secondary or tertiary standards and are usually manufactured and certified by a reagent manufacturer for use with a specific lot of reagent under defined assay conditions.^[5]

The same guidance states that FDA encourages the continued use of CSEs that are suitably calibrated to the international reference endotoxins standard, and that manufacturers may be able to use another endotoxin test after demonstrating a reproducible correlation between methods and the USP reference standard.^[5]

Limitations

A comparison with a reference standard is only as informative as the standard itself. The FDA guidance notes that departures from the storage and usage conditions for a reference standard could cause modifications and contamination, resulting in additional impurities and inaccurate analysis.^[4]

One standard does not necessarily cover every attribute tested. ICH Q6B notes that a separate reference material may be necessary for biological and physicochemical testing, and that distinct reference materials for product-related substances, product-related impurities and process-related impurities may need to be established.^[3]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Compound profiles whose identity section records the sequence and molecular formula that an identity result is compared against.

• Sermorelin research profile
• Ipamorelin research profile
• TB-500 research profile

References

1. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
2. ICH Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients. U.S. FDA guidance for industry, September 2016. FDA docket FDA-1995-D-0288
3. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
4. U.S. FDA. Analytical Procedures and Methods Validation for Drugs and Biologics. Guidance for industry, July 2015. FDA docket FDA-2015-N-0007
5. U.S. FDA. Pyrogen and Endotoxins Testing: Questions and Answers. Guidance for industry. FDA docket FDA-2013-S-0610