How to Read a Peptide Certificate of Analysis

Before any result, establish what the document is about. A COA should name the compound, the lot or batch number, the date of analysis and the laboratory that performed it. If it names a product but no lot, it is not a certificate of analysis — it is a specification sheet, and it says nothing about the vial in front of you.

The lot number is the field that makes the rest of the document meaningful. It is the link between the measurements on the page and a specific quantity of material that was received, stored and shipped. A result that cannot be traced to a lot cannot be revisited later if a problem emerges.

The test date matters for the same reason. A certificate is a snapshot: it describes the material as it was on that date, and it makes no statement about storage conditions in the months since.

Identity is normally established by mass spectrometry, most often electrospray ionisation (ESI-MS) or matrix-assisted laser desorption/ionisation (MALDI-TOF). The result to look for is an observed molecular mass compared against the theoretical mass calculated from the sequence.

Two conventions coexist and are easy to conflate. Monoisotopic mass uses the mass of the most abundant isotope of each element; average mass uses the isotope-weighted average. For a peptide of a few thousand daltons the two differ by a meaningful amount, and a report that appears to be off by one or two units is often comparing one convention against the other rather than describing a real discrepancy.

Electrospray spectra typically show multiply charged species — [M+2H]²⁺, [M+3H]³⁺ — rather than a single peak at the molecular mass. A report showing an m/z value well below the expected mass is usually reporting a charge state, and the deconvoluted mass is the number to compare.

What mass spectrometry establishes is that material of the expected mass is present. It is weak evidence about how much of the sample that material accounts for, and it cannot distinguish sequence isomers, which share a mass.

Purity is normally reported from reversed-phase high-performance liquid chromatography (RP-HPLC), and the figure is almost always an area percentage: the area of the main peak divided by the total area of all integrated peaks, at a stated detection wavelength.

That last clause is doing a great deal of work. Peptides are commonly detected by ultraviolet absorbance at 214 nm, which responds to the peptide bond and therefore to essentially anything peptidic in the sample, or at 280 nm, which responds mainly to tryptophan and tyrosine. A peptide containing neither is close to invisible at 280 nm. The same sample can produce quite different purity figures at the two wavelengths, and neither is wrong — they are answering different questions.

An area percentage is also not a mass percentage. Non-peptidic material that does not absorb at the detection wavelength contributes nothing to the chromatogram, so counterions, residual salts and water are invisible to it. This is why a peptide reported at 98% by HPLC may be substantially less than 98% peptide by mass, and why peptide content — determined separately, by amino acid analysis or nitrogen determination — is a different figure that some certificates report and many do not.

Read the gradient and column conditions where they are given. Two laboratories running different gradients will resolve different impurities, and a shallow gradient that separates closely-eluting related substances will report a lower and more honest purity than a steep one that co-elutes them into the main peak.

Water content, typically by Karl Fischer titration, matters for lyophilized material because it affects both stability and the accuracy of any mass-based calculation.

Counterion content — usually trifluoroacetate or acetate from the purification step — can account for a significant fraction of the vial's mass. Where a calculation depends on knowing how much peptide is actually present, this is not a detail.

Amino acid analysis independently confirms composition. It is the most direct check that the residues present are the residues claimed, and it is comparatively rare on commercial certificates.

The absence of these fields is not evidence of a problem. It is evidence that the questions were not asked, which is a different thing and worth knowing.

That the rest of the lot matches the sample. The document describes what was submitted. Its applicability to the remaining material depends on how representative the sampling was, which the certificate does not report.

That the material is still as described. Everything on it was true on the test date. Storage since then is outside its scope.

That the material is suitable for your application. Suitability is a judgment about a specific experiment against specific requirements, and no supplier's document can make it.

That the document is genuine. A certificate is a PDF. Where it matters, check the report number with the issuing laboratory directly rather than trusting the copy you were sent.

Confirm the lot number on the document matches the lot number on the vial. Confirm the compound. Check the observed mass against the theoretical mass, watching for the monoisotopic-versus-average distinction. Read the purity figure together with its detection wavelength and method, never on its own. Note what is not reported. Record the lot number alongside whatever you measure, so the result stays traceable.