How to read a certificate of analysis

A certificate of analysis is not marketing. It is a record of what a laboratory measured on a specific batch of material on a specific day, and almost everything worth knowing about a supplier is visible in how much of it they are willing to publish.

Purity is not one number

Purity by RP-HPLC is the percentage of the material that came off the column as your peak rather than as something else. It is the figure everybody quotes. It is also the easiest one to make look good, because it says nothing about what the other percent is, and nothing at all about how much of the powder in the vial is peptide.

A chromatogram at 214 nm is reading the peptide bond. Salts, counter-ions and water are close to invisible to it. You can have a 99% pure peptide that is two-thirds salt by weight, and both numbers are true.

Net peptide content: how much of the solid is peptide

Net peptide content is the fraction of the lyophilized solid that is actually peptide. The rest is trifluoroacetate from the purification, residual water, and counter-ions. It typically runs between 75 and 90 per cent.

It separates the mass of peptide from the mass of solid: a lot at 82 per cent net peptide content holds 8.2 mg of peptide in every 10 mg of powder, and the other 1.8 mg is counter-ion and water. Most suppliers do not publish it. The certificates in our testing library report the measured content of the vial in milligrams, which is a related but different figure.

Identity is a separate question from purity

Mass spectrometry answers a different question: is this the molecule you ordered. A pure sample of the wrong peptide passes an HPLC purity test perfectly. The observed mass should sit within a fraction of a unit of the calculated molecular weight, and any certificate that reports purity without reporting identity has answered the easier half.

Water, and why it is on there

Karl Fischer titration measures residual moisture. It matters for two reasons: water is part of the solid's mass, and a lyophilized peptide that came out of the dryer wet degrades faster in storage than one that did not.

What a weak certificate leaves out

• No lot number, or a lot number that appears nowhere else — on the vial, on the packing slip, in any index you can search.
• Purity without identity. One assay, usually HPLC, and no mass spectrometry.
• No net peptide content, which leaves the peptide fraction of the solid unknown.
• No date of analysis and no retest date, so you cannot tell whether the document describes material tested this year or three years ago.

Who tested it, and for whom

A manufacturer tests its own material at release, which is normal and not sufficient: it is the one party with an interest in the answer. A certificate from an independent laboratory is worth more, and it names both the laboratory and the party that submitted the sample. The certificates in our testing library come from a named third-party laboratory, on samples submitted by our supplier, and each can be checked by number on the laboratory's own site.

The ways a genuine certificate can still describe the wrong material are set out in seven ways a certificate of analysis misleads.

Common questions

What does the purity figure on a certificate of analysis actually measure?

It is the percentage of the material that came off the RP-HPLC column as the intended peak instead of as something else. It says nothing about what the remaining percentage is, and nothing about how much of the powder in the vial is peptide, because salts, counter-ions and water are close to invisible at 214 nm.

What is net peptide content on a COA?

It is the fraction of the lyophilized solid that is actually peptide. The remainder is trifluoroacetate from the purification, residual water and counter-ions. It typically runs between 75 and 90 per cent, so a lot at 82 per cent net peptide content holds 8.2 mg of peptide in every 10 mg of powder.

Does a high HPLC purity prove the peptide is the right one?

No. A pure sample of the wrong peptide passes an HPLC purity test perfectly. Identity is a separate question answered by mass spectrometry, where the observed mass should sit within a fraction of a unit of the calculated molecular weight. A certificate that reports purity without identity has answered the easier half.

What is missing from a weak certificate of analysis?

Four things are commonly missing. A weak certificate has no lot number, or one that appears nowhere else, reports purity without identity, leaves out net peptide content, and carries no date of analysis or retest date. Without a matching lot number, it is a document about some other material.