Mass Spectrometry Identity Confirmation

Mass spectrometry confirms identity by ionising the sample, measuring mass-to-charge ratio, and comparing the observed mass with the value calculated from the claimed sequence. Electrospray ionisation produces a series of multiply charged ions that are deconvoluted to a single mass; MALDI-TOF usually yields singly charged ions directly. Coupling the source to a liquid chromatograph, as LC-MS, separates the sample first so that each chromatographic peak gets its own mass assignment.

Resolution decides what the test can see. A truncation missing several residues shifts the mass by hundreds of daltons and is unmissable. Deamidation of an asparagine adds roughly 1 dalton and oxidation of a methionine about 16, so separating those from the intact molecule needs a high-resolution instrument rather than a nominal-mass one. Semaglutide, at roughly 4,114 daltons, sits comfortably inside the working range of routine electrospray analysis.

What the method returns is a mass consistent with the sequence, which is a weaker statement than the sequence itself. Establishing the order of residues requires tandem mass spectrometry, in which the molecule is fragmented and the resulting ladder of fragment masses is read. On a certificate of analysis, a matching parent mass rules out gross substitution and most chain-length errors, and rules out very little else.

The error worth naming is treating a matching mass as proof of the right compound. Diastereomers carrying a D-amino acid in place of an L, scrambled disulfide isomers, isoaspartate rearrangements and reversed sequences all weigh exactly what the target weighs. A spectrum also says nothing about quantity: identity and content are separate measurements, and a vial can contain the correct molecule in an amount nobody has determined.