How to read a Certificate of Analysis

What a COA actually certifies

A Certificate of Analysis is a lot-specific record: it reports what was actually measured on the batch you are being sent, not a general spec sheet for the compound. A real COA carries a lot or batch number that ties it to one specific production run, and every figure on it — purity, identity, water content — should be the result of an assay run against that lot, not a number copied from a product description.

Three things a COA needs to establish are identity (is this molecule what the label says it is), purity (how much of the material is the target compound versus impurities), and quantity or concentration (how much is actually in the vial). A document missing any of the three is not doing the job of a COA, regardless of how official it looks.

Identity confirmation: mass spectrometry

Identity is normally confirmed by ESI-MS (electrospray ionization mass spectrometry), which measures the molecule's mass-to-charge ratio and should match the compound's known molecular weight within the instrument's expected tolerance. For a well-characterized peptide this is a straightforward match; for larger or more complex molecules, a COA may report multiple charge-state peaks that resolve to the same underlying mass.

The number to actually check is the measured mass against the compound's known/theoretical molecular weight — not just that the document contains the word 'confirmed.' A COA that reports a mass thousands of daltons off from the expected value, with no explanation, is describing a different molecule.

Purity: reading the RP-HPLC chromatogram

Purity on a peptide COA is almost always reported by reversed-phase HPLC (RP-HPLC): the sample is run through a column, and different components separate by hydrophobicity, eluting at different retention times. The area under the main peak, as a percentage of the total area under all peaks, is the reported purity — for example, purity of 98% or higher means the main compound accounts for at least 98% of the integrated peak area at 210 to 220 nm UV detection.

A COA worth trusting includes the actual chromatogram image, not just the number. Without the chromatogram, there is no way to see whether the 98% figure came from a clean single dominant peak with minor shoulders, or from more generous integration around a messier separation.

• Look for: a visible chromatogram, a clearly resolved main peak, and a stated wavelength (commonly 210 or 214 nm).
• Be cautious of: a purity percentage with no chromatogram attached, or a chromatogram that doesn't match the lot number on the same page.

Water content and counter-ions

RP-HPLC purity describes the compound's chromatographic purity — the ratio of target peak to total peak area — not what fraction of the vial's total weight is peptide. Lyophilized powders retain residual water (commonly measured by Karl Fischer titration) and, depending on the synthesis and purification route, may carry a counter-ion such as acetate or trifluoroacetate (TFA) bound to the peptide. Both count toward the powder's gross mass without being an impurity in the purity-percentage sense.

A COA that reports water content and counter-ion/salt form alongside HPLC purity is giving a more complete picture of what's actually in the vial. See our companion article on net peptide content vs. gross powder mass for how these figures combine into a real cost-per-milligram comparison.

Red flags

A few patterns are worth treating as a reason to ask more questions before ordering, rather than as automatic disqualifiers: a purity or identity claim with no attached chromatogram or spectrum; a document that looks identical across multiple different product listings (a template, not a lot-specific record); a lot number that doesn't appear anywhere on the actual vial label; or a molecular weight that doesn't match the compound's known value without an explanation.

None of these alone proves a problem, but a document that has several of them together is not functioning as a real Certificate of Analysis — it is marketing collateral wearing a COA's format.