HPLC and mass spectrometry on a peptide CoA, explained

Definitions

• HPLC purity: the area of the main peak as a percentage of the total peak area detected by the instrument, usually by UV absorbance at about 214–220 nm.
• Mass spectrometry (MS) identity: a measured molecular mass compared with the theoretical mass calculated from the peptide's sequence.
• LC-MS: liquid chromatography coupled to mass spectrometry, so each peak can be given a mass.

How reversed-phase HPLC separates a peptide

In reversed-phase HPLC the sample passes through a column packed with a hydrophobic stationary phase. An increasing proportion of organic solvent (commonly acetonitrile with a little trifluoroacetic or formic acid) elutes peptides in order of hydrophobicity. A UV detector records a peak for each separated component; the chromatogram is that record.

A purity figure is only as good as the separation. Two components that elute together appear as one peak, so a single symmetrical peak supports, but does not prove, high purity. Published work on pharmaceutical peptides uses orthogonal methods such as two-dimensional LC to test whether a main peak hides co-eluting impurities.

Source: Reversed-phase liquid chromatography of peptides: a tutorial, J Proteome Res 2022 (PubMed 36355445); Peak purity of pharmaceutical peptides by 2D-LC, J Chromatogr A 2023 (PubMed 36871316)

What mass spectrometry adds

MS measures mass-to-charge ratio. Electrospray ionisation often produces several charge states of the same peptide, which software converts back to one molecular mass. If the measured and theoretical masses agree within the instrument's tolerance, the main component is the expected molecule.

MS on its own does not give purity, and a correct mass does not rule out impurities of similar mass, such as a sequence with two amino acids swapped. LC-MS characterisation of synthetic peptides is reviewed in detail in the literature, including typical impurities: deletions, truncations, incomplete deprotection, oxidation and deamidation.

Source: Characterization of synthetic peptide therapeutics using LC-MS, J Am Soc Mass Spectrom 2021 (PubMed 34110145)

Reading the two results together

• HPLC says how much of what the detector saw was the main peak.
• MS says whether the main peak is the right molecule.
• Neither states how much peptide is in the vial; that is net peptide content (see purity vs net peptide content).
• Both should refer to the same lot number as the one printed on your vial.

Questions to put to a supplier

• Which lab ran the tests, and is there a report number that lab will confirm?
• What wavelength and gradient were used for HPLC?
• Is the chromatogram and mass spectrum available, not just the summary figures?
• Which lot does the certificate cover?

How Sable documents this

Sable releases lots at ≥99% HPLC purity with identity confirmed by mass spectrometry. There is a CoA for every product and every lot, and every vial has a printed lot number that matches its certificate. Request a CoA and it is emailed within 2 hours.

Frequently asked questions