Named, unnamed and unspecified impurities as regulatory categories — the long version

Relative response factors mean impurities are not detected in proportion to how much of them is present. A one per cent peak is not one per cent by mass unless the response factors happen to match.

This is the sort of thing the wiki should carry and currently does not.

Nothing to add, except that this is the answer I would give if asked.

Post #15 is right about the mechanism and I think understates the practical bit.

Storage-related degradation and synthesis-related impurity look different on a chromatogram. A growing oxidation peak over time is not a manufacturing finding.

Coming back to post #14, because the follow-up matters more than the original answer.

Oxidation at methionine or tryptophan adds sixteen per oxygen and typically elutes earlier. In aged material it is the modification that grows.

Deletion sequences arise from incomplete coupling and are lower by one residue mass. Where they elute depends on the hydrophobicity of the residue that is missing, so they can appear on either side of the main peak.

Worth separating two things that post #18 runs together.

The honest reading of a purity figure: it is an upper bound on how much of what the method could see was the intended species, under one integration convention, on one sample.

Post #19 describes the usual case. This is about the unusual one.

Reporting thresholds matter: below a stated threshold, peaks are usually not reported at all. A clean-looking table may reflect a high threshold rather than a clean synthesis.

Relative response factors mean impurities are not detected in proportion to how much of them is present. A one per cent peak is not one per cent by mass unless the response factors happen to match.

Genuinely open to being wrong about this one.

A supplier that can tell you what its principal impurity is has answered a harder question than one that can tell you its purity, and the answer is more useful.

Flagging that the sources on this are thinner than the confidence in the thread suggests.

On post #23 — agreed on the reasoning, with one qualification.

Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.

Scavengers and cleavage-cocktail residues can persist and appear as small early-eluting peaks. They are process-related rather than sequence-related, which is a useful distinction when reading a profile.

That distinction has done more work for me than anything else in this category.

Disulfide formation: if a peptide contains cysteine, it can form disulfide bonds with itself or with other molecules. Under oxidising conditions multiple species appear. Reducing conditions (like DTT) convert them back.

If that is already documented somewhere, ignore me and link it.

Post #27 is right about the mechanism and I think understates the practical bit.

Peptide impurities that differ by a single residue are the hardest to resolve and the most likely to be biologically relevant, which is an unfortunate combination.

Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.

That is all I can say without guessing.

Truncation products are substantially lower in mass and usually well separated chromatographically, which makes them the easiest class to see and the least likely to be missed.

I would rather say I do not know than round it up to an answer.