How to Read a Peptide Certificate of Analysis (Without Taking It on Faith)

A certificate of analysis is the single most important document a peptide supplier gives you, and it is also the easiest one to fake. A convincing-looking PDF costs nothing to produce. Learning to read one properly costs an afternoon and saves entire experiments.

What a real COA contains

Every legitimate COA has six things. If any are missing, treat the document as marketing rather than data.

• Product identity — the compound name, and ideally the CAS number and molecular formula
• Batch or lot number — which must match the number printed on the vial you received
• Test date — recent, and after the manufacture date
• Testing laboratory — named, independent, and contactable
• Methods used — typically RP-HPLC for purity and ESI-MS or MALDI-TOF for identity
• The raw traces — an actual chromatogram and mass spectrum, not just a number in a table

That last point is where most fraudulent COAs fall down. Anyone can type "99.2%" into a table. Producing a plausible chromatogram with the right retention time, peak shape and integration is much harder.

Reading the HPLC chromatogram

Reverse-phase HPLC separates the peptide from its impurities by hydrophobicity. What you get back is a plot of detector response against time.

Look at the main peak. It should be tall, symmetrical and sharp. A peak that leans heavily to one side — a phenomenon called tailing — suggests either column problems or a co-eluting impurity hiding inside what looks like a single peak.

Look at the baseline. A clean run has a flat, quiet baseline. A noisy or drifting baseline makes integration unreliable, which means the purity number sitting on top of it is unreliable too.

Look at the small peaks. Purity is calculated as the area of the main peak divided by the total area of all peaks. A 99% result with one 1% impurity is a very different material from a 99% result with twenty 0.05% impurities. The first suggests a specific, identifiable side product — often a deletion sequence. The second suggests general degradation.

Check the gradient is stated. A purity figure means little without knowing the mobile phase, gradient and column. A shallow gradient resolves impurities that a steep one hides.

Reading the mass spectrum

HPLC tells you the material is pure. Mass spectrometry tells you it is the right thing. These are different questions, and you need both answers.

The spectrum should show a molecular ion matching the theoretical monoisotopic or average mass, usually within a few daltons for larger peptides. For multiply charged electrospray data, you will often see a charge envelope — a series of peaks at m/z values corresponding to [M+2H]2+, [M+3H]3+ and so on — which deconvolutes to a single mass.

Two common findings worth understanding:

1. Mass 18 Da low — a dehydration product, often from aspartimide formation
2. Mass short by one residue — a deletion sequence, meaning a coupling step failed during synthesis

Neither is necessarily disqualifying at trace level, but both should appear in the impurity profile rather than the main peak.

The tests people forget to ask for

Purity and identity are the headline. For serious work, three more matter:

• Water content by Karl Fischer titration. Lyophilized peptides are hygroscopic. A vial labelled 10 mg that is 12% water contains 8.8 mg of peptide, and your concentrations are all wrong.
• Peptide content by amino acid analysis or nitrogen determination. This distinguishes net peptide from counter-ions and residual salts. Net peptide content of 80% is completely normal for a TFA salt — but you need to know.
• Residual solvents by GC. Acetonitrile and TFA carry over from purification.

A peptide can be 99% pure by HPLC and still be only 78% peptide by mass. Purity and content are not the same number, and conflating them is the most common concentration error in peptide research.

Four red flags

The batch number does not match the vial. This is the fastest check available and it catches recycled COAs immediately.

The testing lab is the manufacturer. Self-certification is not third-party testing. It may be honest, but it is not independent.

Purity is quoted to an implausible precision. "99.99%" is beyond the resolution of routine RP-HPLC integration. Real numbers look like 99.2% or 98.7%.

There is no chromatogram. A table of results without the underlying trace is an assertion, not evidence.

Verifying independently

If the material matters, send a sample out yourself. Independent analytical labs will run HPLC and MS on a peptide sample for a modest fee, and the peace of mind on a long study is worth it. Compare the retention time and impurity profile against the supplier's trace — they should look like the same material.

Every batch we ship has its COA published on the product page before you order, with the chromatogram and mass spectrum included, tested by an independent laboratory. If a batch number on a vial does not match the document, we want to hear about it.

All materials discussed here are supplied for laboratory research use only and are not for human or veterinary consumption.