Reference Standards and Certified Reference Materials: How Labs Anchor a Measurement

In plain English

Every measurement compares something unknown with something known. A balance is checked with calibrated masses. In chemistry, the “something known” is often a reference standard: a carefully tested sample of the substance being measured ^{[1, 4]}.

To measure how much peptide is in a sample, a laboratory compares it with a standard whose content is already known. If the standard’s value is wrong, every result calculated from it is wrong by the same proportion.

Some standards come from official bodies, such as pharmacopoeias (official books of standards for medicines). A certified reference material, or CRM, comes with a certificate that states its value, how uncertain that value is, and how it links back to international units of measurement ^{[3, 6]}.

The short version

• A result is only as good as the standard behind it.
• Primary standards anchor secondary (working) standards ^{[2, 4]}.
• A CRM certificate gives a value, an uncertainty and traceability ^[3].
• Purity by area % needs no standard; peptide content does ^[7].

Why a measurement needs a reference

The international vocabulary of metrology (the science of measurement) defines metrological traceability as the property of a result that lets it be related to a reference through a documented, unbroken chain of calibrations, each of which adds to the measurement uncertainty ^[1]. Traceability needs an established calibration hierarchy ^[1].

A reference material is material that is sufficiently homogeneous and stable for specified properties and has been shown fit for its intended use ^[1]. Only reference materials with assigned values can be used for calibration ^[1].

In a chromatographic assay, the amount of a compound is read from a calibration made with an authentic specimen, either directly (an absolute calibration curve) or relative to an internal standard ^[7]. USP’s peptide reference standard content, for example, is usually determined by HPLC assay against an external standard whose purity was assigned by mass balance ^[5]. The standard’s assigned value is built into every result.

Primary, secondary and working standards

A primary standard from an officially recognised source is normally used without further testing, provided it is stored as the supplier recommends ^[2]. The WHO guidelines on chemical reference substances make the same distinction: a primary substance’s assigned content is accepted without comparison with another substance, while a secondary substance’s value is calibrated against a primary one ^[4].

Chains of standards can drift. FDA guidance says each new batch of a reference standard, official or in-house, should be qualified against the current one, and recommends a two-tiered approach for biological reference standards to prevent drift ^[8]. European guidance on synthetic peptides asks manufacturers using a primary and working standard to explain how they will prevent drift in peptide content, and to take precautions because peptides are often very hygroscopic ^[9].

Pharmacopoeial reference standards

FDA guidance notes that reference standards can often be obtained from USP, and may also be available through the European Pharmacopoeia, Japanese Pharmacopoeia, WHO or the US National Institute of Standards and Technology ^[8]. European Pharmacopoeia reference standards, known as CRS, are distributed through an online catalogue run by the EDQM ^[10].

How a pharmacopoeial peptide standard is valued. USP uses a two-step approach. First, the purity of a bulk material is assigned by mass balance: all detectable impurities, including related peptides, counter-ion, water, residual solvents and non-combustible residue, are measured and subtracted from 100%. Second, that bulk is used as the standard to assay the peptide content of the final lyophilised vials ^[11]. Several laboratories take part, statistical outliers are removed, and the value is cross-checked against the current lot and the European Pharmacopoeia standard ^[11]. Released standards stay on a real-time stability monitoring programme with alert and action limits, and most peptide standards are stored at −20 °C or colder ^[11].

What comes with them. Labels should give the substance name, the issuing body, the approximate quantity and the batch number; accompanying documents give storage conditions, intended use and the assigned value needed for calculations ^[4]. Pharmacopoeial reference substances usually do not carry a conventional expiry date. Instead, the issuing body monitors each batch and tells users when it is no longer valid ^[4]. See Pharmacopoeias Explained.

Certified reference materials and ISO 17034

A certified reference material is a reference material accompanied by documentation from an authoritative body giving one or more property values with their uncertainties and traceabilities ^[1]. NATA describes the certificate as stating the value of the specified property, its associated uncertainty and a statement of metrological traceability ^[3].

Who makes them. ISO 17034 sets requirements for the competent operation of reference material producers. Accreditation to it covers assignment of property values, characterisation, homogeneity, stability, labelling and distribution ^[3]. The 2012 metrology vocabulary pointed to ISO Guides 34 and 35 for CRM production and certification ^[1]. A 2025 CRM report, for example, cites ISO 17034:2016 for producer competence and ISO 33405:2024 for characterisation and the assessment of homogeneity and stability ^[12].

A CRM certificate typically states:

• the certified value, with units (for a pure substance, usually a mass fraction such as mg/g);
• the expanded uncertainty and its coverage factor, k ^[1];
• a statement of metrological traceability ^[3];
• the method or methods used to assign the value;
• homogeneity and stability information, and a validity period set from stability data with post-certification monitoring ^[12];
• storage and handling instructions.

Peptide examples. A candidate angiotensin II CRM was assigned 691 ± 9 mg/g (k = 2) by combining isotope dilution amino acid analysis, quantitative NMR and mass balance; trifluoroacetate counter-ion made up nearly 25% of its mass ^[13]. In another study, NIST SRM 998 (angiotensin I) and a certified C-peptide reference material were used to validate a peptide hydrolysis procedure, and the resulting method then certified an in-house peptide standard ^[14]. A mass balance for a synthetic nine-residue peptide gave an SI-traceable value of 796.5 mg/g with an expanded uncertainty of 6.5 mg/g (k = 2) ^[15]. See Amino Acid Analysis.

Why area-% purity needs no standard but an assay does

Area-% purity compares peaks within a single chromatogram: each peak’s area divided by the total ^[7]. No external standard is needed, because the answer is a ratio. Its weakness is the assumption of equal response. ICH Q2(R2) calls for relative response factors, calculated from the responses of characterised materials, and for a correction factor when they fall outside 0.8–1.2 ^[16].

An assay of content asks how many milligrams of peptide are present, which requires a known amount to compare with ^{[7, 9]}.

Worked example (illustrative). A standard solution is made at 0.500 mg/mL of powder from a reference standard with an assigned content of 0.912 mg of peptide per mg, so it contains 0.500 × 0.912 = 0.456 mg/mL of peptide. Its main peak area is 1,820. A test sample made at 0.600 mg/mL of powder gives an area of 1,905.

• Peptide in sample solution = 1,905 ÷ 1,820 × 0.456 = 0.477 mg/mL
• Peptide content = 0.477 ÷ 0.600 = 79.5%

Had the analyst assumed the standard was 100% peptide, the answer would have been 87.2%, nearly 8 percentage points too high. The assigned value, its basis (“as is” or water-corrected) and its uncertainty all carry into the result. Water uptake by a hygroscopic standard after opening has the same effect, which is why moisture content may need confirming before use ^[9]. See Karl Fischer Titration.

Qualifying an in-house standard

When no official standard exists, a laboratory establishes its own ^[2]. Good practice, drawn from the guidance cited, looks like this:

1. Confirm identity with orthogonal methods, such as mass spectrometry, NMR, peptide mapping and amino acid analysis ^[11].
2. Assign purity or content by mass balance, with water, organic solvents, inorganic residue and organic impurities together accounting for 100% ^[4], or by qNMR or AAA. For a standard used in assays, the WHO guidelines recommend that at least three laboratories collaborate, using several validated techniques ^[4].
3. Compare with a primary or official standard where one exists ^{[2, 8]}.
4. Check homogeneity and stability across vials, and set storage conditions ^{[11, 12]}.
5. Protect from moisture. Vials should not be opened until they reach room temperature, to prevent condensation ^[4]; dispensing into single-use vials helps with hygroscopic materials ^[4].
6. Requalify on a schedule and record the results ^[2].

How the methods used in these steps are shown to be reliable is covered in Analytical Method Validation.

What this means in Australia

• National Measurement Institute. Australia’s National Measurement Institute (NMI) produces certified reference materials with metrological traceability to the SI unit of mass, the kilogram, and reports purity as a mass fraction with its uncertainty on the certificate ^[6]. It holds NATA accreditation to ISO 17034:2016 and ISO/IEC 17025:2017 for this work ^[6].
• Accreditation. NATA accredits reference material producers to ISO 17034 and testing laboratories to ISO/IEC 17025 ^{[3, 17]}. See ISO/IEC 17025 and NATA Accreditation.
• Pharmacopoeias. For medicines, the Therapeutic Goods Act 1989 defines monographs in the British Pharmacopoeia, European Pharmacopoeia and United States Pharmacopeia–National Formulary as standards ^[18].
• Labels. The Therapeutic Goods Administration (TGA) has stated that a “research use only” disclaimer does not make supply lawful, change regulatory status, permit importation, or remove advertising or supply obligations ^[19]. Testing against a reference standard does not change any of this.

This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.

Frequently asked questions

What is a reference standard?

A well-characterised material, of documented identity and purity, against which test samples are compared to identify them or measure their content ^{[2, 4]}.

What is the difference between a reference standard and a certified reference material?

A CRM is a reference material whose certificate, issued by an authoritative body, gives an assigned value with its uncertainty and traceability ^{[1, 3]}. Pharmacopoeial reference standards are established for specific pharmacopoeial tests and are managed by batch monitoring rather than conventional expiry dates ^[4].

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

A primary standard is accepted on the strength of its own extensive characterisation. A secondary standard’s value is set by comparison with a primary standard ^[2].

Does HPLC purity need a reference standard?

Area-% purity does not, because it is a ratio of peaks in one chromatogram. Measuring peptide content by HPLC does, because the result is calculated from a standard of known content ^[7].

What should a CRM certificate include?

At minimum, the certified value, its uncertainty and a statement of metrological traceability, plus storage instructions and a validity period ^{[3, 12]}.

References

1. Joint Committee for Guides in Metrology. JCGM 200:2012. International vocabulary of metrology: basic and general concepts and associated terms (VIM), 3rd edition. bipm.org. Accessed 25 October 2026.
2. International Council for Harmonisation. ICH Q7: Good manufacturing practice guide for active pharmaceutical ingredients. Sections 11.17–11.19 and Glossary. database.ich.org. Accessed 25 October 2026.
3. National Association of Testing Authorities, Australia (NATA). Reference materials producers (ISO 17034) accreditation. nata.com.au. Accessed 25 October 2026.
4. WHO Expert Committee on Specifications for Pharmaceutical Preparations. WHO general guidelines for the establishment, maintenance and distribution of chemical reference substances. WHO Technical Report Series 943, Annex 3. 2007. who.int. Accessed 25 October 2026.
5. Li C, Bhavaraju S, Thibeault MP, et al. Survey of peptide quantification methods and comparison of their reproducibility: a case study using oxytocin. J Pharm Biomed Anal. 2019;166:105–112. doi:10.1016/j.jpba.2018.12.028. PMID: 30640042
6. National Measurement Institute (Australia). Chemical reference materials. industry.gov.au. Accessed 25 October 2026.
7. The Japanese Pharmacopoeia, 18th edition (English version). General Tests, Processes and Apparatus: 2.01 Liquid Chromatography. mhlw.go.jp. Accessed 25 October 2026.
8. US Food and Drug Administration. Analytical procedures and methods validation for drugs and biologics: guidance for industry. July 2015. Section V, Reference standards and materials. fda.gov. Accessed 25 October 2026.
9. European Medicines Agency. Guideline on the development and manufacture of synthetic peptides. EMA/CHMP/CVMP/QWP/367182/2025. Adopted 4 December 2025; effective 1 June 2026. ema.europa.eu. Accessed 25 October 2026.
10. EDQM. European Pharmacopoeia reference standards: CRS catalogue. crs.edqm.eu. Accessed 25 October 2026.
11. McCarthy D, Han Y, Carrick K, et al. Reference standards to support quality of synthetic peptide therapeutics. Pharm Res. 2023;40(6):1317–1328. doi:10.1007/s11095-023-03493-1. PMID: 36949371
12. Sommerfeld T, Riedel J, Lisec J, et al. Development of a certified reference material for per- and polyfluoroalkyl substances (PFAS) in textiles. Anal Bioanal Chem. 2025;417(26):6007–6015. doi:10.1007/s00216-025-06098-2. PMID: 40926081
13. Melanson JE, Thibeault MP, Stocks BB, et al. Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II. Anal Bioanal Chem. 2018;410(26):6719–6731. doi:10.1007/s00216-018-1272-7. PMID: 30143839
14. Nicolás Carcelén J, Potes Rodríguez H, González-Gago A, et al. Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards. Anal Bioanal Chem. 2024;416(7):1717–1731. doi:10.1007/s00216-024-05176-1. PMID: 38363304
15. Wang S, Wu P, Li M, et al. Mass balance method for SI-traceable purity assignment of synthetic oxytocin. J Pharm Biomed Anal. 2022;207:114401. doi:10.1016/j.jpba.2021.114401. PMID: 34656934
16. International Council for Harmonisation. ICH Q2(R2): Validation of analytical procedures. Adopted 1 November 2023. database.ich.org. Accessed 25 October 2026.
17. National Association of Testing Authorities, Australia (NATA). Laboratory accreditation: ISO/IEC 17025. nata.com.au. Accessed 25 October 2026.
18. Therapeutic Goods Act 1989 (Cth), section 3: definitions of “standard” and “default standard”. Federal Register of Legislation. legislation.gov.au. Accessed 25 October 2026.
19. Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 25 October 2026.