Peptide identity and mass spectrometry

Purity and identity are two different questions. HPLC can tell you how much of a sample is the main peak, but it cannot, on its own, tell you what that peak is. Mass spectrometry (MS) is how identity is confirmed.

The question MS answers

A peptide has a predictable molecular mass for its amino-acid sequence. Mass spectrometry measures the mass of the ions formed from the sample and compares them to that expected value. If the observed mass matches the theoretical mass for the intended sequence, the material is consistent with being that compound. If it does not, the sample is not what it is meant to be, whatever its purity.

How the measurement works

Molecules are ionised and separated by their mass-to-charge ratio in a vacuum, then detected. Peptides ionise in predictable ways, so the pattern of signals can be read back as a mass. A single-stage measurement gives the molecular mass; a tandem (MS/MS) measurement fragments the peptide and reads the fragments, which can indicate the amino-acid sequence itself.

Observed versus theoretical mass

The certificate should show both numbers and the difference between them. A small deviation at the right resolution is expected; a large one is a problem. The resolution of the instrument matters too — a low-resolution reading can only confirm an approximate mass, while a high-resolution reading can distinguish between candidate sequences that are close in mass.

Why both tests belong together

• HPLC alone — tells you how much of the sample is the main peak, but not what the peak is.
• MS alone — tells you a mass is present, but not how much of the sample it represents.
• Both together — a single dominant peak at the expected mass is what "verified" should mean.

That pairing is why we test every batch by HPLC and mass spectrometry rather than reporting one figure. The certificate ties both results to a batch number, so the CoA you receive describes the material in your vial. For the purity side of the same picture, see HPLC purity explained, and check the lab test results page for published batches.