Chemical Isotope Labeling LC-MS for High Coverage and Quantitative Profiling of the Hydroxyl Submetabolome in Metabolomics

A key step in metabolomics is to perform accurate relative quantification of the metabolomes in comparative samples with high coverage. Hydroxyl-containing metabolites are an important class of the metabolome with diverse structures and physical/chemical properties; however, many of them are difficult to detect with high sensitivity. We present a high-performance chemical isotope labeling liquid chromatography mass spectrometry (LC-MS) technique for in-depth profiling of the hydroxyl submetabolome, which involves the use of acidic liquid–liquid extraction to enrich hydroxyl metabolites into ethyl acetate from an aqueous sample. After drying and then redissolving in acetonitrile, the metabolite extract is labeled using a base-activated 12C- or 13C-dansylation reaction. A fast step-gradient LC-UV method is used to determine the total concentration of labeled metabolites. On the basis of the concentration information, a 12C-labeled individual sample is mixed with an equal mole amount of a 13C-labeled pool or...