Quick answer: HPLC measures purity (how much of the sample is the target compound); mass spectrometry confirms identity (whether the compound is what the label says). A complete COA reports both, because a sample can be pure and still be the wrong molecule.
Two questions, two instruments
A Certificate of Analysis answers two separate questions. Is this the right molecule, and how much of the sample is that molecule? Each question needs a different instrument.
HPLC: purity
High-performance liquid chromatography pushes the dissolved sample through a column under pressure. Components separate by how strongly they interact with the column material and elute at different times. A detector records a chromatogram, a trace with peaks. The main peak is the target peptide; smaller peaks are impurities, side products, or degradation. Purity is the area of the main peak divided by the total area, reported as a percentage.
What HPLC cannot tell you: what the main peak actually is. The wrong peptide can still show a single clean peak and a 99% purity figure.
Mass spectrometry: identity
Mass spectrometry ionizes the sample and measures the mass-to-charge ratio of the ions. The result is a molecular mass that can be compared with the theoretical mass calculated from the sequence. A match within instrument tolerance confirms the molecule is what the label claims. Modern COAs often use LC-MS, where the HPLC output feeds directly into the mass spectrometer, so both measurements come from one run.
What MS cannot tell you on its own: how much of the sample is the target. It confirms identity, not proportion.
Reading them together
- High purity, mass matches: the material is what it says and mostly clean.
- High purity, mass does not match: wrong compound or a modified version. Do not use.
- Mass matches, low purity: right compound, significant impurities. Usable only if the study can tolerate them.
- Only one test reported: the COA is incomplete. Ask for the other.
Other tests you may see
Karl Fischer titration for water content, peptide content assays (amino acid analysis or UV), and endotoxin testing for some applications. These refine the picture but do not replace HPLC and MS.
Sources
- United States Pharmacopeia, General Chapter 621, Chromatography.
- International Council for Harmonisation, ICH Q6B: Specifications for Biotechnological/Biological Products.
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