What a certificate of analysis contains for a peptide batch

The vial arrives with a small label. On it, a name and a string of letters and digits. The certificate of analysis (CoA) is the document that ought to tie that string to measurements made on the exact material in the vial. We say “ought to” because we have seen plenty of certificates that describe a peptide in general and no batch in particular. Below we go through what we look for on the page, from top to bottom.

The header: which material this is about

First, the batch number. It has to be the same, character for character, as the one on the vial label. If no batch number appears on the certificate, the rest of the page has nothing to refer to.

Next to it sit the identifying details of the peptide: the name, the amino acid sequence, the molecular formula and the theoretical molecular mass. We need the theoretical mass later, when we compare it with the measured one. Some certificates also state the salt form, trifluoroacetate or acetate, and the quantity per vial.

Then the dates. The analysis date shows when the measurements were made, and some manufacturers add the manufacturing date and a retest date. An analysis only makes sense if it comes after the batch was made. That sounds self-evident. We check anyway.

HPLC purity and the method behind it

The result appears as a percentage, for example “98.4%”. A result is a measured value. Wording such as “≥ 98%” is a specification, that is, the limit the batch was supposed to meet. Well-made certificates have both, in separate columns.

The percentage on its own says little without the method. We look for the column type (for peptides, usually C18, reversed-phase), the mobile phases (water and acetonitrile, with trifluoroacetic acid), the gradient and the detection wavelength, which for the peptide bond lies in the 214–220 nm region. And we look for the chromatogram itself, attached, with its peak table. From the table the arithmetic can be redone: the area of the main peak relative to the total area.

On the chromatogram we check the sample name in the header printed by the instrument. If it shows a different batch from the one on the certificate, we have a documentation problem, however good the numbers look.

Mass confirmed by spectrometry

HPLC shows how clean the sample is. It does not show what is in it. For identity, the certificate needs a mass spectrometry line giving the theoretical mass, the mass found and, in the best case, the spectrum.

Under electrospray ionisation, peptides often appear as ions carrying several charges. What we read in the spectrum is not the molecular mass directly but mass-to-charge ratios: [M+H]⁺, [M+2H]²⁺ and so on. The doubly charged ion appears at roughly half the molecular mass plus one. The mass is recalculated from these signals, and it has to agree with the theoretical value within the precision of the instrument. We also check that the two values are of the same kind, both monoisotopic or both average. Otherwise a small difference looks like an error when it is not one.

A certificate that lists only the theoretical mass, with no measured value, confirms nothing. It repeats what was already known from the sequence.

Appearance and the small print

The “appearance” line looks like a formality. It usually reads “white lyophilised powder” or something close to that. We read it anyway, because it is the only result we can verify ourselves, by eye, when the parcel is opened.

More detailed certificates add net peptide content, water content and counter-ion content. Not all of them do, and their absence does not make the document false. It only means that HPLC purity cannot be read as the amount of peptide in the vial. At the bottom there should be the name of the laboratory that ran the analysis and of the person who approved it.

Signs of a generic document

Over time we have collected a few things that make us ask for clarification before we accept a certificate as belonging to the batch we received.

1. The batch number is missing or does not match the one on the vial.
2. Purity is given only as a limit (“≥ 98%”), with no measured value.
3. There is no chromatogram attached, or the same image appears, identical, for different batches. Two real runs do not give exactly the same baseline.
4. The analysis date is earlier than the manufacturing date, or missing altogether.
5. The spectrometry line shows only the theoretical mass.
6. The method is not described at all: no column, no gradient, no wavelength.

None of these signs proves, on its own, that the material is poor. It proves that the document cannot be used as evidence for that particular batch. In a laboratory notebook, that amounts to having no document at all.

What we do with the certificate once we have read it

If it does not come with the parcel, we ask the supplier for it, with the batch number written in the message. A reply that arrives with a file valid “for all batches” sends us back to the list above.

Once we have it, we keep it next to a photograph of the label, taken on receipt, and we note the date the batch entered the laboratory. A few months on, when someone repeats an experiment and wants to know which material was used the first time round, these papers are the only answer.