Testing methods, and what they actually prove
Each analytical method answers a different question. Confusing them is how bad batches pass "QC".
Reverse-phase HPLC — how much of the main thing is there?
RP-HPLC separates components by hydrophobicity. For peptides, purity is usually reported as the area percentage of the main peak at a defined wavelength. It answers: what fraction of the UV-absorbing material is the target peak? It does not, by itself, prove identity, and it can overstate purity if the compound has low UV absorbance or if impurities co-elute. Area percent is also not a concentration: determining how much peptide is actually in a solution is a separate measurement, covered in measuring peptide concentration. The chromatogram behind the number carries the rest of the story — peak shape, integration and the units that were excluded — which is covered in how to read a peptide chromatogram. The choices that produce that chromatogram in the first place — column chemistry, ion pairing and gradient shape — are set out in designing an HPLC method for peptides.
Mass spectrometry — is it the right molecule?
ESI-MS or MALDI-TOF measures the mass-to-charge ratio of ions. It answers: does the material contain a species with the expected molecular mass? It is the identity check. It does not measure purity, and a match at the expected mass does not exclude isomers or modified variants with the same nominal mass.
Karl Fischer — how much water is in the powder?
Residual moisture affects stability, weighing accuracy and apparent purity. A lyophilized powder with high moisture can degrade faster and weigh inaccurately. The method answers: what is the water content? It measures a process outcome as much as a product attribute — a high result usually traces back to the freeze-drying cycle, which is why the number belongs next to the lyophilization record rather than on its own. How that number tracks over time — and what a shelf-life claim needs — is set out in stability studies.
Peptide content (assay) — how much is peptide vs counter-ion/salt?
Net peptide content differs from HPLC purity: a sample can be 98% pure by HPLC and still contain a significant fraction of counter-ions, water or excipients. For quantitative work, ask for assay or net-peptide content, not just purity. The arithmetic — and how it changes a price comparison — is worked through in net peptide content and cost per mg.
What a combined package proves
| Question | Method | Limited by |
|---|---|---|
| Identity | MS | Resolution; isomers |
| Purity profile | HPLC | UV response; co-elution |
| Moisture | Karl Fischer | Sample handling |
| Net peptide content | Assay / amino-acid analysis | Method availability |
| Sterility / endotoxin | Microbial / LAL | Only if actually performed |
The last row is the one buyers most often assume rather than verify. HPLC and MS describe the peptide; they say nothing about viable organisms or pyrogenic residue, and a 0.22 µm filter does not remove endotoxin. See endotoxin and bioburden for what each test measures and what to request. For the identity check itself — mass conventions, adducts and the limits of a single intact mass — see peptide identity by mass spectrometry; for what fragmentation adds, see MS/MS sequence confirmation.
None of these methods measures what happens after the powder is dissolved. A lot can pass identity, purity and moisture and still lose soluble material through adsorption or aggregation and self-assembly in the customer's buffer — which is why the release panel and the end-use preparation are two different questions.
One common gap in a release panel is solvent residue: HPLC and Karl Fischer say nothing about acetonitrile, DMF or trapped TFA, and “loss on drying” is not a substitute. Headspace GC and the fields to ask for are covered in residual solvents in peptides. Non-volatile contamination is a separate panel: metals from catalysts, reactors and reagents are covered in elemental impurities.
Each of these methods also has a capability limit that is established separately from the result: whether the method is specific for the target, what range it was validated over, and what its limit of quantification is. A number from an unvalidated method is still a number — see analytical method validation. The value that method is calibrated against needs its own qualification, covered in peptide reference standards.
A panel is only as good as the sample it was run on. How many units were drawn, whether they were pooled, and what to do when your own receipt result disagrees with the certificate are separate questions from which tests are listed — see sampling and batch release.