Liquid Chromatography Methods for Separation of Polar and Charged Intracellular Metabolites for C Metabolic Flux Analysis.

Accurate quantification of mass isotopolog distribution (MID) of intracellular metabolites is a key requirement for C metabolic flux analysis (C-MFA). Liquid chromatography coupled with mass spectrometry (LC/MS) has emerged as a frontrunner technique that combines two orthogonal separation strategies. While metabolomics requires separation of monoisotopic peaks, C-MFA imposes additional demands for chromatographic separation as isotopologs of metabolites significantly add to the number of analytes. In this protocol chapter, we discuss two liquid chromatography methods, namely, reverse phase ion-pairing and hydrophilic interaction chromatography (HILIC) that together can separate a wide variety of metabolites that are typically used for C metabolic flux analysis.