How to read a peptide certificate of analysis

A certificate of analysis is meant to be the bridge between a vial and the data behind it. In practice, a large number of the certificates circulating in this industry are decorative — a table of favourable numbers with nothing underneath them. This is a guide to telling the two apart.

Start with the lot number

Everything else on the document is meaningless without it. A certificate identifies a single production and release batch, and the number on it has to match the number on the vial in front of you. A certificate that carries a product name but no lot number is not a certificate of analysis. It is a specification sheet, which is a statement of intent rather than a record of measurement.

Purity: the number is the least interesting part

Purity is normally reported by area normalisation from an RP-HPLC run at 220 nm, where the peptide bond absorbs. The figure is the main peak's area as a percentage of total integrated area. Two things follow from that definition, and both are routinely glossed over.

• It assumes everything in the sample absorbs at 220 nm roughly like the peptide does. Non-absorbing contaminants — salts, residual water, some solvents — are invisible to the method and simply do not appear in the denominator.
• It depends entirely on the integration window and the gradient. A fast gradient that fails to resolve a close-eluting deletion sequence will report that impurity as part of the main peak, and the number goes up.

This is why the chromatogram matters more than the percentage. With the trace in front of you, you can see peak symmetry, baseline stability, whether the main peak has a shoulder, and whether the run was long enough for anything late-eluting to appear. Without it, you are taking the integration on trust.

Identity: purity's necessary partner

A clean chromatogram tells you the sample contains one thing. It does not tell you that thing is the compound you ordered. Retention time supports identity but does not establish it — plenty of unrelated sequences elute at similar times under the same method.

Mass spectrometry closes the gap. Look for an observed mass reported against a theoretical one, ideally with the spectrum shown. Peptides ionise into several charge states in electrospray, so a genuine spectrum has a family of peaks rather than a single line. A certificate that lists only the theoretical mass has shown you an arithmetic result, not a measurement.

The supporting assays, and why they exist

• Water content by Karl Fischer. Typically 1–8%. Water drives hydrolysis in the solid state, so this is a stability indicator as well as a composition one.
• Acetate content by ion chromatography. Typically 4–12%. Peptides purified with TFA are usually exchanged to the acetate salt because residual TFA is cytotoxic in cell culture at concentrations easily reached by accident.
• Peptide content by amino acid analysis. Often 70–90%. This is the honest answer to how much of the vial's mass is actually peptide rather than salt and water, and it is the figure to use when you calculate a concentration that has to be right.
• Endotoxin by LAL. Matters for any cell-based or in-vivo work, because lipopolysaccharide triggers immune responses at vanishingly low concentrations.

Three omissions worth walking away from

• No chromatogram. The purity figure cannot be checked, so it is an assertion. This is the most common and most consequential omission.
• No lot number, or a lot number that does not match the vial. The document does not describe your material.
• No named analyst, laboratory or date. Nobody has put their name to it, and you cannot tell whether it was measured last month or three years ago.

None of this requires you to be an analytical chemist. It requires the supplier to show their working — and a supplier who will not is telling you something useful about themselves.