Reference Standards, Types, Uses, Preparation & Qualification

Reference standard material is commonly defined as a highly purified compound that is well characterized, the quality and purity of reference standards are very crucial in determining scientifically reasonable results for the analytical methods in the pharmaceutical industry and are also very well designated by FDA (US Food and Drug Administration) or a company or laboratory.

In the pharmaceutical industry, active pharmaceutical ingredient APIs and drug products about their quality reference standards are used to identify, purity, quality, and potency.  

Reference standards are used to determine quantitative data (such as assay and impurity), qualitative data (such as identification test), and calibration (such as melting point standard). Therefore, the quality and purity of the reference standards are critical in achieving scientifically valid results.  

Drug reference standards in the pharmaceutical industry

A drug reference standard or the pharmaceutical reference standard is the most characterized material suitable to test the strength, identification, quality, and purity of substances for pharmaceutical uses and medicinal products.

A drug reference standard or the pharmaceutical reference standard is the most characterized material suitable to test the strength, identification, quality, and purity of substances for pharmaceutical use and medicinal products.

USFDA: The U.S. Food and Drug Administration defines the reference standard material as a highly purified compound that is well characterized, specifically prepared drug substance batch and an authentic material of high purity used for structural elucidation and working standards.

ICH (Q6A): A reference standard from new drug substances intended for assays, its impurities should be adequately identified and/or controlled and purity should be measured by a quantitative procedure.

USP: US Pharmacopeia (USP) international organization defines reference standard materials as highly characterized specimens of drug substances, excipients, reportable impurities, degradation products, compendial reagents, and performance calibrators”.

The USP reference standard certificates of the previous and current lots can be viewed on the website on the product description of the RS on the online USP store and can be accessed through the catalog number of the RS in the USP reference standards catalog.

The USP reference standards are the integral/basic components of monographs and documentary standards set by the USP to help to ensure the quality, strength, identity, and purity of medicines and foods and are provided essentially for quality control used in conducting the tests and assays in these documentary standards.

Further, United States Pharmacopeia (USP) – NF(National Formulary) includes over 5000 quality standards for medicines, both biological and chemical, active pharmaceutical ingredients and excipients (inactive ingredients), and is considered the widest source in the world for medicine quality standards.

USP-NF is the combination of two compendia the United States Pharmacopeia (USP) and National Formulary (NF) includes monographs for the dosage forms, drug substance, and compounded preparations as featured in USP.     

The pharmacopoeial reference standards are established for the uses described in pharmacopeial monographs and general chapters. The Pharmacopeial reference standards are accessible in various pharmacopeias like United States Pharmacopoeia and European pharmacopeia.   

EDQM (European Directorate for the Quality of Medicines & Healthcare) also supplies the chemical reference substance (CRS), biological reference preparations (BRP) and herbal reference standards (HRS) and also the reference spectra for the assays and tests to be carried out as the per the official methods as specified in the European pharmacopeia. 

Types of reference standards

• Laboratory-made Reference Standards
• Company Reference Standards
• Official Reference Standards
• Primary Reference Standards
• Secondary Reference Standards
• Supplementary Reference Standards

The reference standards are used as the basis for quantitative and qualitative testing. Impurity Reference standards: Not much guidance from authorities here as per ICH Q3A. “The reference standards used in the analytical procedures to control impurities should be evaluated and characterized according to their intended uses.”

Different types of reference standard materials and their uses

Type of Reference STD	Uses
Assays	To determine the potency of active pharmaceutical ingredients (APIs) and salts
Degradation Products	To identify and possibly quantitate degradation products
Process Impurities	To identify and possibly quantitate process-related compounds
Resolution Standard	To determine assay performance or impurity method
Metabolites Standard	To identify and possibly quantitate substances generated through a metabolic process.

Reference Standards are primarily used for the following types of analytical studies:

• Essay
• Identification tests
• Limit tests for related substances
• Analytical method validation
• System suitability for analysis techniques and in particular spectroscopic and chromatographic analysis

When a pharmacopeial reference standard is not available, then it is prepared by synthesizing as per below:

Reference material is selected from a particular batch. After assigning its batch number, its characteristic properties are documented for reference purposes and comparison with future production lots. The material selected as a primary standard should be highly stable, free from the water of hydration, and bear traceability to a national or international standards body.

Besides the qualification results, the batch-specific certificate of analysis also includes details on the recommended storage conditions as well as sample spectra and chromatograms.

Storage conditions: The conditions of storage for reference standards and impurity standards need definite precautions like storage at room temperature, protection from light, moisture, freezing, and excessive heat, and also as per the storage condition as given or proposed. Further precautions need to be taken to store the reference standard and impurity standards in clean vials or suitable vials for storage, regarding storage requirements for the USP-NF refer to the general chapters <659> for online publication for storage and handling terms. 

The reference standard materials were prepared as per the below procedure:

• Highest purity grade material with completely characterized materials
• Reliable chromatographic methods for purity determination.
• Residual solvent impurities include organic, inorganic, and an aqueous solvent.
• Validated methods used to confirm the consistent and precise results
• Qualified instruments are used for the qualification of the reference standard.

Reference Standard identity and purity standards:

As a part of the qualification of identity and purity standards, minimal required tests for initial characterization were typically performed by using the following tests:

• Purity & structure confirmation by 1H-​NMR and 13C-​NMR spectroscopy,
• Recording of coupled spectra as required Mass spectroscopy
• Metals impurity by inductively coupled plasma mass spectrometry (ICP-MS)
• Identification & structure confirmation by UV and/or IR spectroscopy
• Chromatographic purity by using HPLC
• Residual solvents by Headspace gas chromatography (HSGC-FID)
• Water content by Karl Fischer titration or Thermogravimetric analysis.
• Non-combustible impurities by residue on ignition/sulfated ash test

Scientists and Experts at Veeprho continuously develop and manufacture the highest purity grade reference standard with complete characterization using various analytical techniques like HPLC, ICP-MS, GC-FID, NMR, LC-MS, TGA, and FTIR.

FAQs

References:

• http://www.usp.org/reference-standards
• http://www.fda.gov/downloads/Drugs/Guidances/ucm122858.pdf
• Hand Book Of Isolation And Characterization Of Impurities In Pharmaceuticals, By Satinder Ahuja, Elsevier Publications, Volume 5, Pg No: 15, 119-143.
• www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM122858.pdf
• ICH Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances
• ICH Q6B Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Biological Products.
• https://database.ich.org/sites/default/files/Q6A%20Guideline.pdf
• ICH Q7 Good Manufacturing Practice
• https://www.usp.org › how-read-listings
• Culbert, Paul A.; Johnson, Bruce D. (2004). “Reference standards”. Handbook of Isolation and Characterization of Impurities in Pharmaceuticals. Separation Science and Technology. Vol. 5. pp. 119–143. 
• https://www.edqm.eu/en/ph-eur-reference-standards-purpose-and-use https://www.usp.org/about/public-policy/overview-of-monographs
• https://www.usp.org/frequently-asked-questions/excipents