Understanding peptide purity percentages — what worked for you?

Dr.NateNeph said:

They are not contradictory, they measure different things, and this is the single most common misreading of a certificate.

Mass spectrometry confirmation for certificates of analysis: beyond HPLC purity, mass spec tells you the molecule's exact molecular weight. This confirms identity — not just purity.

Why this matters: HPLC can show you a "pure" compound, but if it's pure of the WRONG compound, purity is meaningless. Mass spec at m/z 4113.58 Da confirms it's actually semaglutide. My Janoshik report includes both HPLC and mass spec.

If your testing provider only offers HPLC, consider upgrading to include mass spec for at least your first batch from a new vendor.

A narrower follow-up, since the general answer is now clear:

Whether a purity figure and a content figure can disagree without one of them being wrong?

labquiet_amy said:

Mass spectrometry confirmation for certificates of analysis: beyond HPLC purity, mass spec tells you the molecule's exact molecular weight.

Reconstituted peptide stability data for certificates of analysis: I had Janoshik test my reconstituted vial at day 1, day 14, and day 28 stored in the refrigerator.

Day 1: 100.5% purity
Day 14: 99.3% purity
Day 28: 98.1% purity

Minimal degradation over 28 days when properly refrigerated. This confirms the standard 28-day beyond-use date is appropriate. Beyond 28 days, I'd reconstitute a fresh vial to be safe.

Closing the loop on my own question.

Purity and content being different measurements was the piece I was missing. The vial was overfilled and pure, which is a very different problem from the one I thought I had.

Dr.SurgeonPGH said:

Reconstituted peptide stability data for certificates of analysis: I had Janoshik test my reconstituted vial at day 1, day 14, and day 28 stored in…

Agreed, and mass spec answers a question purity does not: identity. High purity tells you one thing dominates the chromatogram, not that it is the molecule you ordered. A mass within about a dalton of expected is the identity check.