What Should a Peptide Certificate of Analysis Contain?

The items a peptide COA should carry

A certificate of analysis is a release document, not a brochure. Its job is to let an independent reviewer connect one specific vial to one specific synthesis batch, and then judge that batch against limits that were set before the result was known. For a peptide, the content list is well established.

A document that states only "purity 99%" and a molecular weight has omitted every item that makes those two figures interpretable.

Why a COA without a chromatogram cannot be verified

Every number on a COA is the output of a method, and methods differ in how much they can see. A purity of 98.2% means one thing on a 60-minute shallow gradient with a 0.05% integration threshold, and something different on a 12-minute fast gradient that discards everything below 0.5%. The chromatogram shows which case applies: whether the main peak is symmetrical, whether there is a shoulder that was integrated as part of the main peak, and whether late-eluting material was still on the column when the run ended.

The mass spectrum has the same limitation. A stated mass of 1347.7 Da may come from a deconvoluted electrospray envelope with an error of 5 ppm, or from a single low-resolution MALDI peak rounded to the nearest Dalton. Those are not equivalent evidence. When a supplier sends a one-page PDF and declines to attach the underlying chromatogram and spectrum, treat the document as a declaration of intent. A buyer who cannot inspect the raw data cannot audit the claim, and a supplier who cannot produce raw data probably did not generate it for that lot.

Purity, peptide content and mass balance are three different numbers

HPLC purity is a ratio of peak areas. It answers a narrow question: of everything that absorbed at the detection wavelength and eluted from the column, what fraction was the target peptide? It says nothing about how much of the vial, by mass, is peptide.

Peptide content is a mass fraction, and it must account for water, counter-ion, residual solvent and inorganic salts. A batch can be 97% pure by HPLC and still be 84% peptide, because the balance of the mass is water and trifluoroacetate. Buyers who compare suppliers on the HPLC figure alone are comparing a ratio with a mass fraction, which is the most common analytical mistake in peptide procurement.

Mass balance is the third number: the sum of peptide, water, counter-ion, residual solvent and inorganics should close to 100%. If it does not, something was not measured. The worked arithmetic is set out in the purity testing guide.

Identity data: theoretical mass versus observed mass

Mass spectrometry confirms that the dominant species in the vial has the mass the sequence predicts. The COA should state which mass convention was used. Monoisotopic mass, calculated from the most abundant isotope of each element, is the useful reference below roughly 2 kDa. Average mass, calculated from atomic weights, is the practical reference for larger peptides where isotope envelopes merge.

For a 1.2 kDa peptide, a tolerance of 100 ppm corresponds to about 0.12 Da, and most modern instruments reach 5 to 20 ppm on a clean sample. A reported error of 0.4 Da is not instrument noise; it is a structural question. The report should also state the ionisation mode and the charge states observed, because the deconvoluted mass is only as good as the envelope it came from. Reading that envelope is covered in how to read a peptide mass spectrum.

Water, TFA and residual solvents: the part buyers ignore

Lyophilised peptides are hygroscopic. A powder that looks dry can hold 5 to 10% water by mass, and a vial opened repeatedly in a humid room will gain more. Karl Fischer titration is the reference method and should be reported as a percentage with the sample weight. Water matters twice: it lowers peptide content per milligram, and it accelerates hydrolysis and aggregation during storage.

Trifluoroacetate is the standard ion-pairing agent in reversed-phase purification, and it stays associated with basic residues after lyophilisation. Levels of 5 to 15% w/w are common, and the counter-ion is not a contaminant in the toxicological sense, but it is mass that is not peptide. Where a sequence is intended for cell work, acetate or hydrochloride exchange may be requested. Residual solvents are governed by class limits in ICH Q3C, and acetonitrile, methanol and dichloromethane are the usual suspects in solid-phase synthesis.

Consistency checks that expose a copied COA

• Entity consistency. The company name and address on the COA, the invoice and the website should match exactly. A different address for each document is a reseller pattern.
• Lot numbering logic. Lot codes should follow a rule that can be explained. Sequential numbers shared by unrelated products suggest templated documents.
• Arithmetic. Purity plus impurities, or peptide plus water plus counter-ion, should close. Numbers copied from another batch often do not.
• Dates. A release date before the synthesis date, or a retest date generated by a fixed offset for every product, is a warning sign.
• Spectrum plausibility. An observed mass that does not match the stated sequence, or a mass reported without decimals for a 3 kDa peptide, indicates the data was not measured on that lot.

None of these checks proves fraud on its own. Together they tell you whether a follow-up question will be answered with a record or with a delay.

What MOL Changes puts on a peptide COA

MOL Changes manufactures in Changsha, China, and ships every batch with the HPLC chromatogram, the mass spectrum and the COA, so the three documents can be checked against each other. Custom synthesis runs from 5 mg to kilogram scale, with HPLC and LC-MS/MS used for release testing and HPLC purity available up to 98%+ depending on sequence length and complexity.

Our COA format states the lot, sequence, formula, theoretical and observed mass, HPLC method and area percent, water content and counter-ion where measured, storage condition and retest date. We align our documentation with ISO 9001 principles; we do not claim ISO certification, GMP certification or FDA approval, and any supplier who implies those without a certificate number and scope is worth questioning. For document requests, use contact us and quote the lot number.