How to read a peptide COA: HPLC, mass spec, purity

What is a certificate of analysis?

The structure: a batch identifier, a list of tests, the method behind each test, an acceptance criterion, and a result. Everything useful on the page hangs on those last three columns being filled in.

The part that carries the weight: the method. A purity figure with no method named is a number without a definition, because purity by area percent, purity by mass and purity by a different chromatographic system are three different quantities.

What it is not: a specification. In an approved product the specification comes first and the certificate reports against it. With no approved product there is no compendial monograph forcing which tests appear, so the certificate defines its own scope.

The consequence of that: a short certificate is not a failing certificate. It is a narrower one, and the tests it leaves out are the ones nobody is reporting.

The lines, one at a time

How to read this: the middle column is what the method physically measures; the right column is the question it leaves open. Both matter, and the right one gets skipped.

The identity line's real limit: mass spectrometry returns a mass. Two sequences built from the same residues in a different order have the same mass, and so do certain residue substitutions, which is why sequence confirmation is a separate exercise from a mass match.

Why regulators ask for more than one method: FDA's guidance on synthetic peptides encourages applicants to apply orthogonal analytical methods to characterise primary sequence and physicochemical properties, secondary structure, oligomer and aggregation states, and biological activity. One method characterising one property is the baseline, not the finish.

What the impurities in a synthetic peptide are

Where they come from: most peptides are made by solid-phase synthesis, a cycle of coupling and deprotection repeated once per residue. Every cycle is a chance for the reaction to go incompletely or sideways.

The catalogue, from the review that assembled it: deletion sequences from incomplete deprotection, insertion sequences from excess reagent, racemised residues producing diastereomers, side-chain protection adducts from incomplete deprotection, oxidation of side chains, dimers and larger oligomers, residual counterions such as trifluoroacetate, and, where practice was poor, contamination by unrelated peptides (D'Hondt, 2014).

The second family: degradation products that form later, through routes the same review names, including beta-elimination, diketopiperazine formation, pyroglutamate formation and succinimide formation.

Why deletion sequences are the awkward ones: a molecule missing one residue is chemically close to the intended one, so it can sit near the main peak in a single gradient and carry a similar detector response. That is a separation problem, not a detection problem.

The threshold a regulator uses for comparison: in the generic-peptide pathway, FDA treats a new specified peptide-related impurity above 0.5 percent of the drug substance as raising a question about immunogenicity risk, and expects each new impurity below that to be characterised and justified.

Which tests are usually absent?

The pattern: the tests that appear are the chemistry ones, because they run on the same instrument set. The tests that do not appear are the microbiology and trace ones, because they need different samples, different labs and different time.

• Sterility. The compendial sterility test, USP General Chapter 71, is a microbiological test with its own incubation period. No chemical result implies it.
• Bacterial endotoxin. USP General Chapter 85, the bacterial endotoxins test. Endotoxin survives conditions that kill the organism that produced it, so a sterile preparation can still carry it.
• Elemental impurities. Covered by USP General Chapter 233 and by ICH Q3D, which sets out a risk-based process for assessing and controlling them.
• Residual solvents. ICH Q3C recommends acceptable amounts of residual solvents in pharmaceuticals. Synthesis and purification both use solvents, and none of them show as a peak in a peptide purity run.
• Visible particulates. USP General Chapter 790 sets the standard for visible particulates in injections, with the how-to in chapter 1790.

The reading: a certificate covering identity, purity and water has answered three questions out of a longer list, and the unanswered ones are the ones that decide whether a preparation is safe to put into a body.

What does a peptide COA test?

Typically five things: what the solid looks like, what mass the molecule has, what share of the detected chromatogram is the main peak, how much water is in the solid, and which counterion is present. Some certificates add peptide content, which is the only line that speaks to how much peptide a weighed sample contains.

The single most useful question to ask of any certificate: which method produced this number. Purity, identity and content each have several possible methods, and the number is only interpretable once the method is named.

What purity number is normal?

High numbers are normal, which is precisely why the number discriminates so poorly. Modern solid-phase synthesis followed by preparative chromatography routinely lands in the high nineties by area percent, so a figure in that range tells you the process was ordinary rather than that the material is good.

The comparison worth making instead: the impurity profile. A regulator comparing a generic synthetic peptide against an approved one does not read the headline purity, it verifies that each shared impurity is no higher than in the reference and that each new one is characterised and justified.

Why that reframing matters: two batches at the same area percent can carry entirely different impurities, and the impurities are what the immunogenicity question is about.

Does a certificate prove the vial is sterile?

Not unless it reports a sterility test, and most do not. Sterility is established by USP General Chapter 71, a microbiological method with its own sampling plan and incubation. Bacterial endotoxin is a further, separate test under General Chapter 85, and it answers a different question again, because endotoxin is a heat-stable fragment that remains after the bacteria are gone.

The inference people make and should not: that a clean chromatogram implies a clean preparation. Chromatographic purity and microbiological quality are unrelated measurements on unrelated samples.

Can a certificate be wrong?

Labels demonstrably are. When a research group bought 44 products sold online in one unapproved category and analysed them, the labelled amount matched the analysed amount in only 18, eleven held substances the label never mentioned, and four contained no active compound at all (Van Wagoner, 2017). In a separate action, a regulator reported confirming by laboratory analysis that two consumer products contained an undeclared active ingredient.

The structural gap: a certificate describes a batch. Nothing in the document links it to the container in front of anyone, and in an unapproved supply chain there is no independent party checking that link.

What that does not mean: that certificates are worthless. A certificate naming methods, criteria and a batch is far more information than none, and it can be checked against the methods it names. It is evidence, weighted by who produced it and whether anyone can verify the chain.

What a certificate leaves unsettled

The open items on almost every one:

• Chain of custody. Which lot the vial came from, and whether the tested sample and the sold material share a history.
• Sequence. A mass match constrains composition, not order.
• Net mass of peptide. Without a peptide content line, the milligram figure on the label is a fill target, not a measurement.
• Microbiological quality. Absent unless a sterility and an endotoxin result appear.
• Method detail. Column, gradient, wavelength and system suitability are the parameters that make a purity number reproducible, and they are usually not printed.

How this page is sourced

Methods from the method documents: the analytical descriptions come from the compendial chapters named by number and from FDA's guidance on synthetic peptide applications, read directly. The impurity catalogue comes from a 2014 review of peptide-related impurities.

Seven sources: three papers with DOIs and PubMed IDs, one FDA guidance, one FDA enforcement document, one compendial entry covering the chapters named in the text, and one ICH entry covering the two impurity guidances.

Last reviewed: 27 July 2026.

1. 1

   U.S. Food and Drug Administration, Center for Drug Evaluation and Research. ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin. Guidance for Industry, May 2021. Sections III.A on active ingredient sameness and orthogonal methods, and III.B on the 0.5 percent new specified peptide-related impurity threshold. Accessed 27 July 2026. fda.gov, guidance on synthetic peptide ANDAs.

2. 2

   D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. doi:10.1016/j.jpba.2014.06.012. PMID 25044089.

3. 3

   Vergote V, Burvenich C, Van de Wiele C, De Spiegeleer B. Quality specifications for peptide drugs: a regulatory-pharmaceutical approach. J Pept Sci. 2009;15(11):697-710. doi:10.1002/psc.1167. PMID 19750489.

4. 4

   Van Wagoner RM, Eichner A, Bhasin S, Deuster PA, Eichner D. Chemical composition and labeling of substances marketed as selective androgen receptor modulators and sold via the internet. JAMA. 2017;318(20):2004-2010. doi:10.1001/jama.2017.17069. PMID 29183075.

5. 5

   U.S. Food and Drug Administration. Warning letter reference 718252, 19 December 2025, reporting that laboratory analysis of product samples confirmed an undeclared active ingredient. Accessed 27 July 2026. fda.gov warning letter 718252.

6. 6

   United States Pharmacopeia. General Chapter 621, Chromatography, Stage 4 harmonisation, official 1 December 2022; General Chapter 71, Sterility Tests; General Chapter 85, Bacterial Endotoxins Test; General Chapter 233, Elemental Impurities; General Chapter 790, Visible Particulates in Injections; General Chapter 921, Water Determination. Chapter 621 accessed 27 July 2026. usp.org, General Chapter 621.

7. 7

   International Council for Harmonisation. Q3C Impurities: Residual Solvents, and Q3D Elemental Impurities, both cited in the FDA synthetic peptide guidance above as the applicable standards for those impurity classes.

Related pages

Around this page: five neighbouring pages on what a test covers and what a vial can be shown to hold.

• What an HPLC purity number misses: the same purity line taken apart in detail, with the compendial definition.
• What a lyophilized peptide is: why water content sits on a certificate at all.
• What a vial cake means: the visual check that runs before any of these tests, and its ceiling.
• Is ostarine a steroid: the regulatory reason an unapproved substance has no specification behind it.
• The dose-math hub: the arithmetic section, which assumes the mass on the label is real.

The certificate series: every method explainer in this run ships first inside The Decadewise briefing.