Mass Spectrometry

Mass Spectrometry (MS) is an analytical tool that ionizes chemical species and sorts the ions based on their mass-to-charge ratio (m/z). For peptides, Electrospray Ionization (ESI-MS) or Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF) is utilized to measure exact molecular mass down to fractional Dalton accuracy without fragmenting the intact peptide backbone, confirming that the synthesized molecule possesses the correct chemical formula.

HPLC can tell you if a sample is pure, but only Mass Spectrometry can confirm what the molecule actually is. A sample could be 99% pure on HPLC while being the completely wrong peptide without MS confirmation.

The most basic quality question about any peptide product is whether it contains the right molecule. Mass spectrometry answers this more directly than any other routine test, because a peptide’s mass follows from its amino acid composition. Reviews of falsified biotechnology drugs describe products containing the wrong active ingredient, the wrong amount or no active ingredient at all, problems that identity testing is designed to catch.

Mass spectrometry also helps identify impurities. A peptide missing one amino acid, carrying an extra one or oxidised during storage has a different mass from the intended molecule. When liquid chromatography is connected to a mass spectrometer (LC-MS), each separated peak can be characterised. Researchers used this approach in 2024 to quantify semaglutide in vials bought from illegal online pharmacies.

A mass spectrum on a certificate is only meaningful if it can be traced to a named laboratory and a specific batch, and if it shows the observed mass alongside the expected one. A generic or reused spectrum, or one without a lab identity, offers little assurance. Agencies that analyse seized products often combine mass spectrometry with other techniques to be confident of what a sample contains.

“A matching mass proves the exact sequence”

Intact mass confirms composition, not order. Two peptides with the same amino acids in a different sequence have identical masses, and swapping an L-amino acid for its D-form does not change mass at all. Confirming sequence requires fragmentation methods such as tandem mass spectrometry (MS/MS), and some isomers need additional techniques.

“Mass spectrometry tells you the purity”

Intact-mass analysis is mainly an identity test. Different molecules ionise with different efficiencies, so peak heights in a mass spectrum do not translate directly into percentages. Purity is usually measured by HPLC with UV detection, and quantification by LC-MS needs calibrated reference standards. A good quality report includes both identity and purity data.

“If a lab report includes mass spectrometry, the product is safe”

Mass spectrometry does not test for bacteria, endotoxin or particulate contamination. In the 2024 online semaglutide study, endotoxin was detected in every vial tested by a separate method. A correct mass shows that the intended molecule is present; it says nothing about sterility or whether the product is safe to use.

What does mass spectrometry show on a peptide certificate of analysis?

It typically shows the measured molecular mass of the main component compared with the theoretical mass calculated from the peptide’s formula. A close match supports the claim that the correct peptide is present. A useful report also states the instrument method, the laboratory, the batch tested and the date of analysis.

What is LC-MS?

LC-MS stands for liquid chromatography-mass spectrometry. The liquid chromatography step separates the components of a sample, and the mass spectrometer then measures the mass of each separated component. Combining the two allows analysts to identify the main peptide and characterise impurities in a single run, which is why it is widely used for synthetic peptides.

What is the difference between HPLC and mass spectrometry?

HPLC separates a mixture and, with UV detection, estimates the relative amount of each component, which is the basis of most purity figures. Mass spectrometry measures molecular mass, which is the basis of identity confirmation. HPLC answers how clean a sample is; mass spectrometry answers what the main molecule is.

Can mass spectrometry detect counterfeit peptides?

Yes, it is one of the main tools for this. Laboratories analysing falsified biotechnology products use mass spectrometry, often alongside other methods, to identify the active ingredient, detect when the wrong substance is present, and find unauthorised or unexpected peptides. It cannot, however, detect microbial or endotoxin contamination.