How to Read a Peptide Certificate of Analysis (COA)

Start with report identity and traceability

Before reading any number, establish what the report describes. A certificate that cannot be tied to a specific batch is a statement about a product type, not evidence about the vial on the bench. Preserve the original file, because changing the client, manufacturer, QR code, signature, result or verification text breaks the link to the laboratory record.

• The issuing laboratory and its contact details
• A task, report or sample number, and any verification key or QR destination
• The client and manufacturer names as they appear on the laboratory record
• The product name, the batch identifier and the testing and release dates
• The signature or approval of a named person

If the supplied vial carries a different batch code, the report may still describe the same compound, but it is not batch-specific evidence for that vial. The guide to batch-specific verification explains how to document this link.

Separate identity, purity and content

Three questions are often collapsed into one number. Identity asks whether the main substance is the expected molecule. Purity asks what share of the detected signal belongs to the main peak. Content asks how much target material is in the vial. A report can show high chromatographic purity while the measured content differs from the label, so each result should be reviewed on its own.

Read the HPLC result in context

Do not stop at the printed purity percentage. Review the chromatogram itself, the detector and its wavelength, the column, the mobile phases, the gradient, the run time, the retention time of the main peak and the way peaks were integrated. Peptides are normally detected at 214 or 220 nm, where the peptide bond absorbs, and area normalization compares integrated responses at that wavelength only.

Impurities that do not absorb there are invisible, and impurities that co-elute with the main peak are counted as part of it.

• A baseline blank run shows which peaks belong to the system rather than the sample.
• A visible shoulder on the main peak suggests a closely related impurity that the integration may have merged.
• The integration threshold decides which small peaks are counted at all.
• The same sample can give different percentages with different gradients.

The guide to reading an HPLC chromatogram goes through these points with examples.

Review the mass-spectrometry evidence

Check whether the report gives the expected formula or mass, the observed ions, their charge states and a calculated or deconvoluted mass. Peptides usually carry several charges, so a raw mass-to-charge value is not the intact mass. For a 1,000 Da peptide, the singly charged ion appears near m/z 1001 and the doubly charged ion near m/z 501.

The observed result should be explained by the expected molecule, the ionization mode, the salt form and any adducts. Agreement within a few tenths of a dalton is typical for a low-resolution instrument, and within a few parts per million for a high-resolution one.

Compare the numbers with the reference values on the compound page for the product. If the report compares an observed mass with an expected value, the expected value should be stated on the report, along with whether it is monoisotopic or average.

Look for content, water and counter-ion data

A lyophilized peptide is a salt. The powder holds counter-ions such as trifluoroacetate or acetate and a few percent of water, so the peptide is only part of the weighed mass. A report that gives net peptide content, water content and counter-ion information lets a laboratory work out how much peptide a vial contains. A report that gives only a purity percentage leaves that question open.

Recognise common warning signs

Some features do not prove a problem but justify a question to the supplier before the material is accepted.

• Only a purity percentage, with no chromatogram
• No batch identifier, or an identifier that differs from the vial
• A chromatogram cropped so that the baseline and early or late peaks are not visible
• A mass listed without a charge state, an ion type or an expected value
• Identical-looking reports for different compounds, with only the name changed
• A verification key or QR code that opens a page that does not match the printed report

Use a repeatable acceptance checklist

A receiving laboratory can document five decisions: the report is authentic, the material and the report are traceably linked, the methods are appropriate, the results meet internal specifications, and any discrepancy has been resolved before release. Keep the report, the verification evidence, the purchase record, photographs of the received batch and the internal decision together.

The compound identity file template gives a layout for this record.