Metabolomics is becoming increasingly popular in livestock research, but no single analytical method can cover the entire metabolome. As such, we compared similar and complementary chromatographic methods with respect to analyte coverage and chromatographic properties of mammalian metabolites. We investigated 354 biologically relevant primary metabolites from 19 compound classes including amino acids, bile acids, biogenic amines, carboxylic acids, lipids, nucleotides and sugars. A total of 2063 selected reaction monitoring transitions were optimized on a triple quadrupole mass spectrometer. We then determined the retention profiles and peak parameters of our compounds using an anion exchange chromatography (AIC), three reversed-phase (RP) and three hydrophilic interaction liquid chromatography (HILIC) methods. On average, HILIC methods covered 54% of all metabolites with retention factors >1, while average RP coverage was 41%. In contrast to RP, HILIC methods could also retain polar metabolites such as amino acids and biogenic amines. Carboxylic acids, nucleotides, and sugar related compounds were best separated by AIC or zwitterionic pHILIC with alkaline eluents. Combining two complementary HILIC and RP methods increased the library coverage to 92%. By further including important short chain fatty acids, a combination of HILIC, RP and AIC methods achieved a coverage of 97%. The resulting dataset of LC and MS/MS parameters will facilitate the development of tailor-made quantitative targeted LC-MS/MS methods to investigate the mammalian metabolome.
Keywords: Anion-exchange chromatography; HILIC; LC-MS/MS; Metabolomics; Reversed phase chromatography.
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