How to Read a Peptide Certificate of Analysis
Almost every peptide supplier publishes certificates. Far fewer publish certificates that answer the question you are actually asking, which is whether the vial in your hand contains what the label says in the amount the label says. Here is how to tell the difference.
Purity is a percentage of what, exactly?
An HPLC purity figure is an area-percent measurement: the area under the main peak divided by the total area of all peaks the detector saw, usually at 214 nm. That is a statement about the relative proportions of UV-absorbing species in the sample. It is not a statement about how much peptide is in the vial.
This matters more than it sounds. A lot can be 99.5 % pure and still be half salt and water by mass, because salt and water do not absorb at 214 nm and therefore contribute no peak area at all. Purity and content are different questions, and a certificate that answers only the first is answering the easier one.
Net peptide content is the number that governs your concentration
Net peptide content, determined by amino acid analysis or nitrogen determination, tells you what fraction of the cake mass is actually peptide. Typical values for a lyophilised TFA salt run 80–90 %; the remainder is counter-ion and residual water.
If you weigh out 10 mg of an 85 % net-content lot and calculate your molarity as though you had 10 mg of peptide, every concentration in your experiment is 15 % high. Suppliers who report net peptide content are giving you the number you need to avoid that. Suppliers who omit it are leaving you to assume 100 %, which is never true.
At Certus we label vials by net peptide content rather than gross cake weight, so a 10 mg vial contains 10 mg of peptide. Read the certificate to confirm this on any supplier you use — including us.
One method is not identity confirmation
HPLC tells you how many distinct species are present and in what proportion. It does not tell you what they are. Retention time is suggestive, but it is only meaningful against a qualified reference standard run on the same column, same gradient, same day.
Mass spectrometry tells you the molecular weight, which strongly constrains identity but does not fix it. Diastereomers have identical masses. Aspartimide isomers have identical masses. A cyclised peptide and its linear precursor differ by 18 daltons, which is inside the tolerance a careless operator might use on a 4,000 Da molecule.
Identity confirmation means two orthogonal methods that fail in different ways. Purity by HPLC, mass by LC-MS, and — where the compound has a specific known failure mode — a third assay chosen to catch it.
The four ways a real certificate still misleads
First, the lot mismatch: the certificate is real, the testing was real, and it describes a different lot than the one in your box. If the lot number on the certificate does not match the vial label character for character, you have no certificate.
Second, the stale certificate: a document from eighteen months ago attached to material synthesised last week, on the theory that the process is the same. Check the test date against the manufacture date.
Third, the in-house certificate presented as third-party: a report on the manufacturer's own letterhead, or from a laboratory that is a subsidiary of the manufacturer. Look for the testing laboratory's name and its accreditation number, and check that the accreditation is real.
Fourth, the selective panel: a certificate reporting only the assays the lot passed. If a supplier tests for endotoxin sometimes and publishes it sometimes, the absence of that line is information.
What to check, in order
Lot number matches the vial. Test date is after the manufacture date and recent. Testing laboratory is named, independent, and accredited to ISO/IEC 17025 with a number you can verify. Purity is reported with the method and wavelength. Net peptide content is present. Mass is reported against a stated theoretical value. Endotoxin and sterility are present for anything intended for cell culture.
If all seven are there, you are reading a real certificate. If more than one is missing, ask why — a supplier who runs the tests will happily send you the missing page, and a supplier who does not will change the subject.
Put it into practice
Every Certus lot has a public certificate you can audit against everything above, before you spend anything.