Purity and identity are two separate questions. Purity asks 'how much of the sample is a single component?' Identity asks 'is that component the molecule on the label?' Both are required to characterise a batch, and each is measured by a different technique.
Mass spectrometry
Mass spectrometry is the primary tool for identity verification of peptides. Electrospray ionisation (ESI-MS) and matrix-assisted laser desorption ionisation (MALDI-TOF) are the two most common ionisation modes. Both produce a mass-to-charge (m/z) spectrum in which the observed monoisotopic or average mass of the intact peptide is compared to the theoretical mass calculated from the sequence.
- Observed mass within ±1 Da of theoretical mass for typical peptide sizes
- For larger sequences, isotope envelope matching confirms charge state
- Fragmentation (MS/MS) can be used to confirm the amino-acid sequence itself
Amino acid analysis
Amino acid analysis (AAA) hydrolyses the peptide into free amino acids, which are then quantified. The observed ratios are compared to the expected composition of the sequence. AAA is also the reference method for absolute quantification of peptide content — a purity number alone does not tell you how much peptide is in a vial.
Sequence confirmation
For higher assurance, tandem MS or Edman degradation can be used to read the sequence directly. This is more common on characterisation batches than on routine release testing, but is expected for new reference materials.
Cross-checking against the COA
When reviewing a COA, look for an identity method (typically ESI-MS or MALDI-TOF) alongside the HPLC purity value. The observed mass and theoretical mass should both be listed. A COA that reports only purity should be treated as incomplete for identity purposes.