How to read a peptide certificate of analysis
A certificate of analysis summarizes the tests run on one lot of peptide. This guide walks through each section of a typical COA, what the numbers mean, and the red flags that suggest the document cannot be relied on.
What a COA is and is not
A certificate of analysis (COA) is a document that reports the results of tests performed on a specific production lot of a material. For a research peptide, it should tie a lot number to identity, purity and, where relevant, content and contaminant data, along with the methods used and the laboratory that performed them.
A COA is a summary, not proof. It is only as trustworthy as the laboratory behind it and the link between the tested sample and the material in hand. For that reason, the most useful habit is to read a COA for internal consistency: whether the methods, results and identifiers make sense together and match what the sequence predicts.
Header information
The top of a COA should identify the product and the lot unambiguously. Look for the compound name and full sequence, including any modifications such as C-terminal amidation, acetylation or cyclization, plus the lot or batch number, test date, and the name and address of the testing laboratory. A sequence written without modifications, for a peptide that is supposed to carry them, is a mismatch worth questioning.
Many COAs also list the salt form (for example acetate or trifluoroacetate), appearance, and the amount of material in the container. The salt form matters because it changes the mass of powder needed to supply a given amount of peptide; see counter-ions: TFA vs acetate.
- Compound name and full sequence with modifications
- Lot or batch number that matches the label
- Test date and, ideally, a retest or expiry date
- Laboratory name, address and accreditation (for example ISO/IEC 17025)
- Salt form and appearance
Identity: mass spectrometry
Identity is normally confirmed by mass spectrometry, using electrospray ionization (ESI) or MALDI-TOF. The COA should state the theoretical mass and the observed mass, and say whether the value is monoisotopic or average. With ESI, peptides appear as multiply charged ions, so the spectrum shows several peaks at different m/z values that should all deconvolute to the same neutral mass.
The expected mass for any sequence can be computed with the sequence analyzer, and each compound page on this wiki shows it. Compare that figure with the reported one. Small differences consistent with instrument accuracy are normal; differences that correspond to a whole residue, an oxidation (+16 Da), a missing amide (+1 Da versus the acid), or an incomplete protecting-group removal suggest the wrong or a modified product. See HPLC and mass spec explained.
Purity: HPLC
Purity is usually reported as the percentage of total UV absorbance area under the main peak in a reversed-phase HPLC chromatogram, typically detected around 214 to 220 nm where peptide bonds absorb. A good COA includes the chromatogram itself, the column type, the mobile phases and gradient, and the detection wavelength, along with a peak table.
HPLC purity describes how much of the UV-absorbing material is the main peptide. It does not detect water, salts or counter-ions, and it can miss impurities that co-elute with the main peak or absorb weakly. That is why HPLC purity must be paired with MS identity, and why a high purity value does not mean the powder is nearly all peptide by weight.
Content and contaminant tests
Net peptide content (NPC) reports the fraction of the powder's weight that is actually peptide, with the remainder made up of counter-ions, bound water and residual solvent. It is determined by amino acid analysis or nitrogen elemental analysis. NPC is important whenever solutions must be prepared to a known concentration; see purity vs net peptide content.
Depending on the intended experiments, a COA may also report endotoxin (in EU/mg, by LAL or recombinant factor C methods), sterility or bioburden, residual moisture by Karl Fischer titration, residual TFA, and for metal complexes such as GHK-Cu, metal content. For cell-based work, endotoxin results are often as important as purity; see endotoxin testing.
Red flags
No single item proves a COA is unreliable, but each of the following should prompt questions or independent testing before the material is used in experiments.
- No lot number, or a lot number that does not match the vial or packaging.
- Purity by HPLC only, with no MS identity: a clean peak says nothing about whether it is the right molecule.
- Identical chromatograms across different lots: real lots differ slightly in retention time, baseline and minor peaks; pixel-identical traces suggest a reused image.
- Purity reported to two decimals with no method: precision without column, gradient or wavelength details is not meaningful.
- No laboratory name or accreditation, or a laboratory that cannot be identified.
- MS mass that does not match theory, or an MS value given without stating monoisotopic or average.
- Missing net peptide content when relevant, for example when the material will be used to prepare solutions of defined concentration.
- Sequence or modifications on the COA that differ from the product description.
- A test date long before the material was produced or received, with no retest data.
Verifying and documenting
Where results matter, laboratories commonly send a retained sample to an independent accredited laboratory for MS and HPLC, and for endotoxin if the material will contact cells. Contacting the issuing laboratory to confirm that a report with that number exists is another simple check. Keeping the COA with experimental records, along with the lot number used in each experiment, makes results traceable if a problem is found later.
Finally, record how the material was stored after receipt. A COA describes the lot at the time of testing; oxidation, moisture uptake or repeated freeze-thaw afterward can change the material, as described in peptide storage and stability.
Frequently asked questions
Is 99% HPLC purity the same as 99% peptide by weight?
No. HPLC purity is the share of UV-absorbing material in the main peak. The powder also contains counter-ions such as acetate or TFA, bound water and sometimes residual solvent, none of which appear in the chromatogram. The weight fraction that is actually peptide is the net peptide content, which is usually noticeably lower than the HPLC purity.
What mass should the mass spectrum show?
It should match the theoretical mass of the exact sequence including all modifications, within the accuracy of the instrument. The COA should say whether the value is monoisotopic or average, since the two differ for larger peptides. ESI spectra show several charge states that should all correspond to the same neutral mass. Expected values can be calculated from the sequence.
Does a COA guarantee the material in my vial?
No. A COA describes the sample that was tested from a given lot. Its value depends on the competence and independence of the laboratory, a clear link between that lot number and your container, and appropriate storage since testing. Independent testing of a retained sample is the usual way to confirm critical results.
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