Source of Liquid chromatography-mass spectrometry

{{Infobox method | name = Liquid chromatography-mass spectrometry | subtitle = Hyphenated technique | Abbreviation = LC-MS | Combines = Chromatographic separation and mass detection | Interface = Usually [[Electrospray ionisation|electrospray]] | Adds = Identity to a retention-based separation }} '''Liquid chromatography-mass spectrometry''' couples a chromatographic separation to a mass spectrometer, so that each eluting peak is characterised by both its [[Retention time|retention time]] and its mass. It converts a weak identification into a strong one.{{r|aebersold2003}} The interface is almost always [[Electrospray ionisation|electrospray]], which works directly from flowing solution. This compatibility is what made the technique routine, and it constrains the chromatography: mobile phases must be volatile and free of non-volatile buffers.{{r|fenn1989}} For peptide work its principal use is identity confirmation — establishing that the peak dominating a chromatogram has the expected mass — and characterisation of impurities, since each minor peak carries a mass that often identifies what it is.{{r|usp1503}} == What it adds over either alone == Chromatography alone establishes that one species dominates; mass spectrometry alone establishes that a species of the expected mass is present. Together they establish that the dominant species has the expected mass, which is a materially stronger statement than either.{{r|aebersold2003}} Impurity identification is where the combination is most valuable. A deletion sequence appears as a peak of lower mass by one residue; an oxidised species by 16 Da more; an incompletely deprotected species by the mass of the retained group. See [[Resin cleavage]] and [[Solid-phase peptide synthesis]].{{r|usp1503}} Tandem operation — selecting a precursor, fragmenting it, and measuring the fragments — adds sequence information and can locate a modification to a residue rather than merely detecting it.{{r|aebersold2003}} == Quantification == LC-MS is not inherently quantitative: ionisation efficiency varies by orders of magnitude between species and with what else is eluting. Quantification requires calibration against a standard of the same substance, ideally with an isotopically labelled internal standard.{{r|aebersold2003}} This is why a purity figure is normally reported from ultraviolet detection rather than from mass detection: ultraviolet response at 214 nm is roughly proportional to the number of peptide bonds and is therefore a reasonable proxy for mass across related peptides, whereas mass-spectrometric response is not.{{r|usp1503}} A certificate reporting purity "by LC-MS" without saying which detector produced the figure is ambiguous on this point.{{r|reports}} == Reading an LC-MS result == The fields that make an LC-MS determination interpretable are the chromatographic conditions, the ionisation mode, the mass analyser and its resolution, and whether the reported mass is monoisotopic or average.{{r|aebersold2003}} Resolution determines what could have been distinguished: a 0.984 Da [[Deamidation|deamidation]] shift is invisible on a unit-resolution instrument, while a 16 Da oxidation is not. A report omitting the instrument omits the answer to what it could have seen.{{r|usp1503}} As with every determination discussed on this wiki, these are observations about which fields make a result checkable rather than assessments of any laboratory or supplier.{{r|reports}} == References == {{reflist}} <ref name="aebersold2003">Aebersold R, Mann M. "Mass spectrometry-based proteomics." ''Nature'' 422(6928):198–207 (2003). PMID 12634793.</ref> <ref name="fenn1989">Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. "Electrospray ionization for mass spectrometry of large biomolecules." ''Science'' 246(4926):64–71 (1989). PMID 2675315.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="reports">PeptidePedia Wiki community test-report tally, 2024–2026 (self-reported; see [[Project:Sourcing_guidelines]]).</ref> == See also == * [[Mass spectrometry]] * [[Electrospray ionisation]] * [[High-performance liquid chromatography]] * [[Retention time]] * [[Deamidation]] {{DEFAULTSORT:Liquid chromatography-mass spectrometry}} [[Category:Mass spectrometry]] [[Category:Chromatography]] [[Category:Analytical methods]]