How to read a certificate of analysis (COA) for peptides
Sep
What a certificate of analysis is
A certificate of analysis is a document reporting the results of analytical testing performed on one specific lot of material. Each phrase in that sentence carries weight.
It reports results, so it describes what was measured rather than what was intended. It covers one lot, so it applies to material produced in a single manufacturing run and not to the product line in general. And it is analytical, so it is only as informative as the methods listed on it.
A certificate that could apply to any lot is not a certificate of analysis. It is a specification sheet, which states what a product is supposed to be. Both documents have a use. They are not interchangeable, and they are frequently presented as though they were.
Identity: the top block
Compound name and sequence
The name should be unambiguous, and for a peptide the sequence should be stated in single-letter or three-letter code. Trade names and abbreviations vary between suppliers, but a sequence does not. Where the compound has an entry in a public reference such as UniProt or PubChem, that identifier removes the remaining ambiguity.
Check the sequence against what you ordered rather than checking the name. Names collide. Sequences do not.
Molecular formula and molecular weight
These are the theoretical values calculated from the sequence, and they are what the mass spectrometry section will be compared against. Note whether the certificate reports average or monoisotopic mass, because the two differ and comparing one against the other produces a false mismatch.
If the peptide is supplied as a salt, the molecular weight of the free base and the salt form differ. A certificate that does not state which is which is ambiguous on the one number the rest of the document depends on.
CAS number
Present for many compounds, absent for many others, particularly for sequences that are not separately registered. Absence is not itself a red flag. A wrong one is.
Lot number and dates
The lot number is the link between this document and the vial in your hand. If they do not match, the certificate tells you nothing about your material, however impressive its contents.
Manufacturing date, analysis date and retest or expiry date should all appear. Analysis substantially predating manufacture is an inconsistency worth asking about.
Appearance and physical description
Usually a short line such as “white to off-white lyophilized powder”. It is easy to skip, and it is a useful cross-check: if the certificate describes a white powder and the vial contains a yellow film, something has happened between the laboratory and you, whether that is degradation, an oxidation, or a mix-up.
Purity: the HPLC section
This section should report a purity figure, a method, and ideally the chromatogram itself.
The purity figure is chromatographic area percent:
Chromatographic purity
purity % = (area of the main peak / total peak area) x 100
The method parameters matter because the number depends on them. Column chemistry and dimensions, mobile phase composition, gradient profile, flow rate, and detection wavelength should all be stated. Detection at 214 nm targets the peptide bond and is standard. Detection at 280 nm responds mainly to aromatic residues and will report a different number for the same sample.
Where the chromatogram is included, look at it. Retention time, peak symmetry and baseline quality are visible there and nowhere else. A tall symmetrical peak on a flat baseline with one small early-eluting impurity is a different material from a peak with tailing, a shoulder, and a drifting baseline, even when both are reported as 98.2%.
Identity confirmation: the mass spectrometry section
This section should give the observed mass alongside the theoretical mass, and state the ionization technique, commonly electrospray ionization.
The two masses should agree within the instrument’s tolerance. A discrepancy is informative rather than merely wrong, and the size of it often indicates the cause. A shift of around 16 mass units suggests oxidation, frequently at methionine. A shift matching the mass of a single residue suggests a deletion sequence, a peptide missing one amino acid, which is a common synthesis byproduct and one that can co-elute closely with the target.
Where the raw spectrum is included, the charge state envelope is visible and the deconvoluted mass can be checked rather than taken on trust.
Content: the section most often missing
Chromatographic purity describes the peptide fraction of the ultraviolet-absorbing material. It does not describe how much of the vial is peptide. The following entries close that gap.
- Water content. Determined by Karl Fischer titration. Lyophilizates commonly retain several percent.
- Counterion content. Most commonly trifluoroacetate from reversed-phase purification, and it can represent a substantial share of the total mass. Some applications are sensitive to residual TFA, in which case a salt exchange to acetate or hydrochloride may be specified.
- Net peptide content. Determined by amino acid analysis or nitrogen determination, and the figure that ties everything together:
Net peptide content
net peptide % = (mass of peptide / total mass of lyophilizate) x 100
A lot reporting 99% chromatographic purity and 80% net peptide content is not contradicting itself. It is telling you that four fifths of the powder is peptide, and that the peptide present is 99% homogeneous. Any calculation involving quantity needs the first number. Most certificates in circulation omit it.
Additional testing where applicable
Depending on the material and its intended laboratory application, a certificate may also report residual solvent by gas chromatography, endotoxin by LAL assay, bioburden, or specific rotation. These are not universal, and their absence is only meaningful relative to the application. Endotoxin data matters for cell culture work and is irrelevant to a binding assay.
Authorization
The foot of the document should identify the testing laboratory, name or sign for the person releasing the results, and date the release. Where the laboratory holds accreditation, the reference should be stated and should be verifiable against the accrediting body’s register.
An unsigned certificate with no laboratory named is an assertion in the format of a document.
A short checklist
- Lot number on the certificate matches the lot number on the vial.
- Sequence matches what was ordered, checked residue by residue.
- Observed mass agrees with theoretical mass, comparing like with like.
- Purity figure is accompanied by the method that produced it.
- Chromatogram is present and the baseline and peak shape look reasonable.
- Net peptide content is stated, or water and counterion content are, if quantity matters to the work.
- Dates are internally consistent.
- Testing laboratory is identified.
Items one through three take under a minute and catch the majority of real problems. The rest determine how much weight the document can bear.
Keeping the record
Certificates should be archived against the lot and retained after the material is consumed. When a result is queried months later, the question is usually what the input material was, and a certificate that was read once and discarded cannot answer it.
All compounds discussed here are supplied for laboratory research use only. They are not medicines, are not for human or veterinary use, and are not for diagnostic or therapeutic application of any kind.