Mass spectrometry

A mass spectrometer turns molecules into charged particles (ions), separates them by their mass-to-charge ratio (m/z) and counts them. The output is a spectrum: peaks at particular m/z values. From those, the instrument's software works out the mass of the molecule.

Peptides are usually ionised by electrospray ionisation (ESI), which sprays the dissolved sample into a fine mist and leaves the molecules carrying several extra protons each. The method, described for large biomolecules by Fenn and colleagues in 1989, earned a share of the 2002 Nobel Prize in Chemistry. MALDI, which uses a laser and a crystal matrix, is the other common method.

What the numbers look like

Because a peptide picks up several charges, the same molecule shows up as a series of peaks. Semaglutide, with a molecular weight of 4113.58 g/mol on the Wegovy label, carrying three extra protons, appears at an m/z of about 1,372; carrying four, at about 1,029. The software combines the series back into one mass.

A test report should state the expected mass, the observed mass and the difference. A match within the instrument's stated accuracy supports identity. A mass that is off by about 18 suggests a lost or gained water molecule; off by 16, an oxidation; off by the weight of a single amino acid, a missing or extra residue.

Reading the report

Check that the expected mass is for the peptide itself. Counter-ions such as acetate or TFA from a salt form separate from the peptide in solution and are not part of the measured mass, so the observed value should match the peptide, not the salt. For semaglutide and tirzepatide the fatty acid side chain is part of the molecule and is included. A spectrum dominated by one charge series is reassuring. Extra series at other masses point to related substances, which HPLC should then quantify. And the batch number on the report should match the vial.

What it does not show

• How much. Mass spectrometry as usually reported confirms identity. It does not say how many milligrams are in the vial.
• Order of amino acids. A scrambled sequence with the same building blocks has the same mass. Tandem mass spectrometry (MS/MS), which breaks the peptide into fragments and weighs those, is needed to confirm order.
• Leucine versus isoleucine. They have identical mass, so a standard spectrum cannot tell them apart.
• Purity. That is HPLC's job. LC-MS joins the two: chromatography separates the peaks and the mass spectrometer weighs each one.
• Endotoxin or sterility. Separate tests. See endotoxin.

A complete certificate of analysis for an injectable peptide usually pairs identity by mass spectrometry with purity by HPLC, and adds content, endotoxin and sterility where they apply.

The app's vial tracker keeps the batch number and source with each vial, so a test report can be matched to the doses that came from it.

Related terms: HPLC, amino acid sequence, certificate of analysis, purity testing. Back to the glossary.