How to Read a Peptide Certificate of Analysis (COA)

A peptide certificate of analysis (COA) is the testing laboratory's record of what was measured on one specific batch: usually identity by mass spectrometry and purity by HPLC, and sometimes net peptide content, counter-ion, water and endotoxin. Read it in this order: does the identity match the compound named, what does the purity figure actually measure, how much of the powder is peptide, and who did the testing?

A COA is only as useful as your ability to check it. This guide walks through each section, what it can and cannot tell you, and the red flags that should make you stop and ask questions.

What a peptide certificate of analysis should contain

The peptide manufacturer Bachem frames quality control around four questions: is it the desired product, how pure is it, what by-products does it contain, and how high is the product content?¹ A useful certificate answers them with data for the specific batch:

Identity: does the COA match the compound named?

This is the first check and the easiest to fail. The sequence, molecular formula and mass on the certificate must belong to the compound on the vial. A certificate that shows a different sequence, a different formula or a mass belonging to another peptide is a red flag whatever its purity figure says, because high purity of the wrong molecule is worthless.

You can check the theoretical values yourself on PubChem. For example:

Two details trip people up. First, the average molar mass (used for weighing) and the monoisotopic mass (the lightest isotopic form, which high-resolution instruments report) are different numbers; for BPC-157 on PubChem the gap is about 0.8. Second, electrospray mass spectrometry produces a series of multiply charged ions⁶, so a spectrum may show its main peaks at m/z values well below the molecular mass. For BPC-157, the doubly protonated ion ([M+2H]2+) would appear near m/z 710.4. The certificate should either report the deconvoluted mass or say which charge state it is quoting. Our guide to HPLC and mass spectrometry explains these readouts in more detail.

An observed mass that differs from the expected value by the mass of one amino acid residue, one oxygen atom or a protecting group points to a specific synthesis or degradation impurity, such as a deletion sequence, an oxidised residue or incomplete deprotection, rather than the target peptide.⁷

Purity: what the HPLC percentage does and does not tell you

For peptides, analytical HPLC with UV detection at 210–220 nm is the standard purity method, and "the area of the main peak in relation to the total area of all peaks reflects the peptide purity".¹ A figure such as 98.6% therefore means that 98.6% of the UV-absorbing peak area, under those conditions, belongs to the main peak. It does not mean:

• That 98.6% of the powder is peptide. Water and counter-ions are not measured by the purity method.¹
• That every impurity is counted equally. At 214 nm, tryptophan absorbs about 30 times more strongly than a peptide bond, and phenylalanine, tyrosine and histidine about six times more, so impurities of different composition give different signals per mole.⁸
• That nothing is hiding under the main peak. An impurity that co-elutes with the target inflates the figure; mass spectrometry across the peak helps catch it.
• That no unrelated peptide is present. In one vaccine-research case, an HIV peptide from a commercial supplier contained about 1% by weight of a cytomegalovirus peptide, enough to produce false-positive T-cell responses.⁹

Ask for the chromatogram, not just the number. A certificate that states a purity but shows no trace, method, column or wavelength gives you nothing to check.

What purity grade does an experiment need?

Higher is not automatically necessary, but it has to fit the use. Bachem's own grade guide recommends more than 95% purity for NMR and crystallography and for peptides used as references in quantitative enzyme–substrate, receptor–ligand and competition studies; 90–95% for quantitative assays, monoclonal antibody production and in vitro or in vivo studies; and more than 80% for qualitative work such as western blotting.¹ It also notes that purity varies from batch to batch and that peptidic by-products in lower-purity batches can have biological activity of their own.¹

Peptide content vs purity

Bachem defines net peptide content (NPC) as "the percentage of peptides relative to non-peptidic material (mostly counterions and moisture)", and stresses that "NPC and purity are not equivalent, because the former includes peptidic contaminants". It adds that basic peptides can have a low NPC even when extremely pure, because of salt formation, and that hydrophilic peptides can absorb considerable moisture.¹ NPC is often estimated from nitrogen content by elemental analysis. Amino acid analysis, which breaks the peptide down with strong acid and quantifies the released amino acids, confirms composition but gives no sequence information.¹

A 2024 test-purchase study shows why the distinction matters. Researchers made test purchases from illegal online pharmacies and analysed the three lyophilised semaglutide vials that were delivered, using liquid chromatography–mass spectrometry. Each chromatogram showed a single semaglutide signal with no peptide-like impurities, yet semaglutide made up only 7.7% to 14.4% of the powder's total weight, against the 99% purity claimed by the sellers. The semaglutide content per vial exceeded the labelled amount by 28.6% to 38.7%, and endotoxin was detected in all samples.¹⁰ A purity claim, a content figure and a label amount are three different numbers.

Counter-ions, water and residual solvents

Peptides purified by reverse-phase HPLC are often isolated as trifluoroacetate (TFA) salts, because trifluoroacetic acid is part of the mobile phase.¹¹ Bachem notes that TFA "cannot be completely removed due to salt formation" and suggests ordering a different salt form where residual TFA is a concern.¹ It can be a concern. In cell culture, TFA at 10 to 100 nM reduced proliferation of osteoblasts and chondrocytes, and the TFA salts of amylin and calcitonin consistently gave less proliferation than the hydrochloride salts, enough to hide a proliferative effect or suggest an antiproliferative one that was not there.¹¹ A review of counter-ions in peptides describes how the salt form also affects structure and physicochemical properties.¹²

Bachem lists the methods behind the rest of the non-peptide mass: water by Karl Fischer titration, residual solvents by gas chromatography, acetate by HPLC or ion chromatography, and residual TFA by ion chromatography.¹ Together with NPC, these results explain the gap between a purity figure and the amount of peptide you can actually weigh out. Moisture also changes after the vial is opened, which is one reason storage matters; see how to store peptides.

Endotoxin and sterility claims

An endotoxin result should name its method. The FDA's guidance on pyrogen and endotoxins testing (revised March 2026) describes the gel-clot, photometric and kinetic methods set out in USP chapter <85>, the Bacterial Endotoxins Test.¹³ Results are usually reported per milligram of material, for example in EU/mg. Sterility is a separate test: in the semaglutide study above, sterility and endotoxin were assessed under different pharmacopoeial chapters, and the samples were free of viable microorganisms yet still contained endotoxin.¹⁰ A certificate that reports one does not establish the other. For sterile handling at the bench, see reconstituting peptides aseptically.

Peptide COA example: reading one line by line

Here is how the checks come together on a hypothetical certificate for 10 mg of angiotensin II, a peptide widely used as a laboratory standard. The certificate and its figures are invented for illustration; the reference values are PubChem's.¹⁴

Change a single line and the verdict changes. A mass that does not match 1045.5, a sequence belonging to another peptide or a lot number that does not match the vial would each make the rest of the certificate irrelevant. For converting net peptide content into an accurate stock concentration, see the arithmetic in our guide to reconstituting peptides.

Who tested it?

• The laboratory's name and address, not just a logo.
• A named analyst or approver, a signature and a date.
• The methods used, with enough detail (column, wavelength, instrument type) to judge them.
• Whether the laboratory is accredited to ISO/IEC 17025, the international standard for testing laboratories, and whether that particular test falls within its accredited scope.
• A way to verify the report with the laboratory, if one is offered.

Red flags on a peptide COA

• The sequence, formula or mass belongs to a different peptide from the one named.
• No lot number, or a lot number that does not match the vial.
• A purity figure with no chromatogram, method or wavelength.
• An identity claim with no mass spectrum or observed mass.
• Identical results, dates or chromatograms across different lots or different compounds.
• No laboratory name, signatory or date.
• Purity quoted as if it were content, with no net peptide content anywhere.

For what synthesis and lyophilisation leave in the vial in the first place, see what research peptides are and how they are made.

Frequently asked questions

References