How to read a certificate of analysis (COA)

A certificate of analysis (COA) is the document that records the results of laboratory tests on a specific batch of a material. For a synthetic peptide, these tests answer three different questions: which molecule is in the sample (identity), what share of the detectable material is that molecule (purity) and how much substance is present (content). The quality evaluation of synthetic peptide therapeutics likewise rests on tests for identity, purity and strength, with the help of reference standards (McCarthy et al., Pharm Res 2023, PMID 36949371).

A COA is not a label. The label states what a product is supposed to contain. The COA records what was measured in a sample of the batch, by which method and by which laboratory. More on the categories of peptides in the guide "What are peptides".

The format differs from laboratory to laboratory. A complete peptide COA usually records:

• Substance identity: name, amino-acid sequence or molecular formula and theoretical molecular mass.
• Purity, usually by HPLC, as a percentage.
• Identification, usually by mass spectrometry (MS): the measured mass next to the expected one.
• Declared amount of substance per pack and, where measured, the content that was determined.
• Batch number (batch or lot).
• Dates: the date of analysis and, often, the date the batch was manufactured.
• The laboratory that ran the analysis, with name and address, and the signature of the person responsible.
• The chromatograms and spectra, the actual graphs from which the results were derived.

High-performance liquid chromatography (HPLC) separates the components of a sample as they pass through a column. Each component appears as a peak on a graph, the chromatogram. The purity percentage comes from the area of the main peak relative to the total area of all peaks.

"98%" or "99%" is therefore a relative figure: it shows what share of the substances recorded by the detector corresponds to the main peak. In a methodological study the authors noted that estimating purity from ultraviolet (UV) absorbance peak areas assumes that absorbance is uniformly proportional to mass, which is why the result may be only semi-quantitative (Preston and Phillips, Anal Bioanal Chem 2016, PMID 26879647).

The purity percentage does not say what the other components are. In synthetic peptides they include related peptides, such as chains in which one amino acid is missing or added (EMA/CHMP/CVMP/QWP/367182/2025).

Mass spectrometry (MS) measures the mass of the molecules in the sample. In a COA, the measured mass is compared with the theoretical mass of the declared sequence. When they agree, the result supports that the main molecule is the one stated.

The European Medicines Agency (EMA) guideline on synthetic peptides, in effect from 1 June 2026, describes mass spectrometry as a powerful tool for peptide structure that can determine molecular mass and confirm the amino-acid sequence. For identification of the peptide in the specification and batch release, the same guideline recommends at least two orthogonal methods, methods based on a different measuring principle (EMA/CHMP/CVMP/QWP/367182/2025).

Mass on its own has limits. Two chains with the same amino acids in a different order have the same mass. Some features, such as amino acids of identical mass or mirror-image forms of amino acids, may need additional techniques for full characterisation of a peptide reference standard (McCarthy et al., Pharm Res 2023, PMID 36949371).

HPLC purity concerns only the components the method detects. The lyophilised powder of a peptide, however, also contains water, as well as a counter-ion. The EMA guideline states that acetate is usually used, while others, such as trifluoroacetate or chloride, are also possible. The same guideline asks for content measured by liquid chromatography to be expressed as the anhydrous substance without the counter-ion, unless otherwise justified (EMA/CHMP/CVMP/QWP/367182/2025).

That is why a material can show very high HPLC purity and a lower net peptide content. Purity answers "what else is there next to the molecule". Content answers "how much of the molecule is there". A COA that records only purity does not give the second piece of information.

First, it does not say what the remaining 1% is. The EMA guideline cites the European Pharmacopoeia thresholds for peptide-related impurities: they are reported above 0.1%, identified above 0.5% and subject to qualification, that is, documentation of their level, above 1.0% (EMA/CHMP/CVMP/QWP/367182/2025). In the pharmaceutical setting, then, impurities are treated as separate, recorded items of the specification rather than as an undifferentiated remainder.

Second, two substances can appear at the same point of the chromatogram. In a study on samples of a synthetic peptide drug, two structurally similar impurities co-eluted, that is, they were not separated by conventional reversed-phase chromatography (RP-HPLC). They were measured by liquid chromatography combined with high-resolution mass spectrometry, with detection limits as low as 0.01% and 0.02%, respectively (Kodidasu et al., J Am Soc Mass Spectrom 2025, PMID 40857144). A peak that looks clean can contain more than one molecule.

Third, the percentage says nothing about identity. A material can have high purity and be a different molecule from the one declared. That is why purity is read together with identification.

A COA shows only what was measured and recorded. If there is no line for a test, that test is not documented.

• Bacterial endotoxins and microbiological purity. Endotoxins are components of the outer wall of certain bacteria and do not show up in HPLC or mass spectrometry. The EMA guideline lists them, together with microbiological purity, as separate items of the specification.
• Residual solvents and elemental impurities, also separate tests in the EMA list.
• Other batches. The COA concerns only the batch whose number it records. It does not carry over to an earlier or later production run, even if the product has the same name.
• Each unit separately. The analysis is done on a sample of the batch, not on every unit of packaging.

A COA can be issued by the manufacturer's own quality-control laboratory or by an external laboratory with no link to the manufacturer. In the second case the result does not come from the party that produced the batch.

Details that allow a laboratory to be assessed are its full name and address, accreditation to ISO/IEC 17025, the international standard for the competence of testing and calibration laboratories, where stated, and the possibility of confirming the document directly with the laboratory, for example by a reference number. A COA without a laboratory name does not allow that confirmation.

The batch number is the link between the document and the material. The same number appears, character for character, on the COA and on the pack label. If the two numbers differ, the COA describes a different batch.

The same applies to the name of the substance and the declared amount per pack, which agree between the document and the label. The date of analysis is the same as or later than the date the batch was manufactured. Published certificates of analysis are collected in the COA library.

• Confirmation of a COA depends on whether the issuing laboratory answers queries about its documents.
• The EMA and European Pharmacopoeia requirements and thresholds concern medicinal products. They are used here as a reference point for what a full analysis includes, not as a description of what every COA records.
• A COA reflects the sample on the date of analysis.

No. It concerns only the batch with the number it records.

That the main peak of the HPLC chromatogram accounts for 99% of the total area of the peaks detected. On its own it shows neither identity nor content.

They do not replace the separate tests the EMA lists in the specification, such as endotoxins, residual solvents and elemental impurities. A COA covers them only if it records a separate test with a result.

• McCarthy D et al. Pharm Res. 2023. PMID 36949371 · DOI 10.1007/s11095-023-03493-1
• Preston GW, Phillips DH. Anal Bioanal Chem. 2016. PMID 26879647 · DOI 10.1007/s00216-016-9334-1
• Kodidasu A et al. J Am Soc Mass Spectrom. 2025. PMID 40857144 · DOI 10.1021/jasms.5c00105
• European Medicines Agency (EMA). Guideline on the Development and Manufacture of Synthetic Peptides. 2025. EMA/CHMP/CVMP/QWP/367182/2025, in effect from 1 June 2026 · https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-development-manufacture-synthetic-peptides_en.pdf

An informational text summarising published research. It is not medical advice or usage guidance.