How to Read a Peptide Certificate of Analysis (COA)

A certificate of analysis answers two questions about a lot of peptide: is it the right molecule (identity, by mass spectrometry) and how much of what is in the vial is that molecule (purity, by HPLC). A good COA also tells you which lot it belongs to, which laboratory ran the tests, and by what method. What it usually does not tell you is how many milligrams of peptide are actually in the vial, whether the contents are sterile, or whether the peptide is biologically active — and knowing those limits is most of what it means to read a COA well. Ours are posted on each product page (the “Additional information” tab) and the testing program is described on the Lab Testing page.

What is on a COA

HPLC purity: what the number means

Reversed-phase HPLC separates the sample into peaks by hydrophobicity and measures each peak’s UV absorbance, usually at 214–220 nm where the peptide backbone absorbs. “Purity 99.2%” means the main peak accounted for 99.2% of the total peak area. Everything else in the chromatogram is an impurity of some kind, and in a synthetic peptide they are predictable: deletion sequences (one residue missed during synthesis), truncated sequences, peptides with a protecting group still attached, oxidized (+16 Da) and deamidated (+1 Da) variants, and diastereomers from racemization during coupling. The 2014 review by D’Hondt and colleagues catalogues these systematically.

Two limits are worth knowing. Area percent assumes every impurity absorbs UV like the parent peptide, which is not exactly true; an impurity lacking an aromatic residue, for instance, can be under-counted, which is why regulators increasingly ask for relative response factors. And a chromatogram can only show what the method separates: a diastereomer or a deamidated variant may co-elute with the main peak on a routine gradient, so two competent laboratories running different methods can legitimately report different purities for the same lot.

Mass spectrometry: identity, not purity

Electrospray mass spectrometry measures the mass of the main component. For a peptide of a few thousand daltons the instrument sees multiply charged ions — [M+2H]²⁺, [M+3H]³⁺ and so on — and the software deconvolutes them to a single molecular mass. Agreement with the theoretical mass to within about a dalton confirms that the sequence is (almost certainly) the intended one and that nothing has been added or lost. It does not measure purity: a sample that is 80% peptide can produce a perfect mass spectrum. Read the MS result and the HPLC result as answering different questions.

Purity is not the same as peptide content

This is the point most often misunderstood. HPLC purity describes the peptide fraction of the sample; it says nothing about how much of the powder’s mass is peptide at all. A lyophilized peptide typically carries several percent water and, more significantly, a counter-ion: synthetic peptides are purified by HPLC in trifluoroacetic-acid mobile phases, and the basic residues (lysine, arginine, histidine, the N-terminus) each retain a trifluoroacetate ion. TFA commonly accounts for 10% or more of the mass, and more still for lysine- or arginine-rich sequences. The result is that a “10 mg” vial at “99% purity” usually contains something like 7–9 mg of actual peptide — the net peptide content — and a COA that does not report peptide content leaves you to estimate it.

For most research this does not matter; concentrations are relative and the same lot is used throughout. It matters when absolute concentration is critical, and it matters in cell-based assays, where trifluoroacetate itself has been shown to alter cell proliferation at the concentrations that come along with a TFA-salt peptide. In those cases the options are to request peptide-content data, or to specify an acetate salt, which exchanges the counter-ion.

What is usually not on a COA

• Endotoxin and sterility. Research-grade peptides are not sterile pharmaceuticals and are not tested as such unless stated. For cell culture, sterile-filter the reconstituted solution.
• Biological activity. Chemical identity and purity do not guarantee the peptide is folded or active; that is established in an assay.
• Residual solvents and heavy metals. Part of a pharmaceutical specification, rarely of a research one.
• Stability data. The COA describes the lot on the day it was tested. What happens afterward is up to storage (see How to store peptides).

Red flags

• No lot number, or a lot number that does not match the vial.
• No method details — no column, gradient, wavelength or instrument — and no chromatogram image.
• Purity reported to two or three decimal places with no supporting data, or an identical “99.9%” on every product a vendor sells.
• A molecular weight that does not match the peptide, or a mass spectrum that does not match the stated molecular weight.
• A certificate with no laboratory name, or one issued by the seller with no independent testing behind it. “Third-party tested” should mean an identifiable independent laboratory.
• A certificate that is older than the product line — i.e., one representative COA reused for every subsequent lot.

Matching a COA to your vial

Read the lot number on the vial label, open the product’s COA, and confirm the lots agree. Then check the three things that matter: the molecular weight matches the peptide, the MS result matches that weight, and the HPLC purity is what the listing claims. If the certificate reports peptide content, use that figure — not the label mass — for any calculation where absolute concentration matters (the reconstitution guide covers the arithmetic).