Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry (HILIC-MS/MS).

Dynamic modeling of metabolic reaction networks requires absolute quantification of intracellular and extracellular metabolite concentrations with high precision and accuracy. This chapter presents a robust HILIC-ESI-MS/MS procedure for targeted quantitative profiling of more than 50 polar key metabolites in multicomponent endogenous extracts. Without using ion-pairing-agents or prior derivatization protocols, organic acids, amino acids, sugar phosphates, coenzymes, and nucleotides are measured on a triple quadrupole platform in positive and negative electrospray ionization modes with preoptimized MRM transitions. Robust polymer-based zwitterionic stationary phases (ZIC®-pHILIC) support alkaline mobile phase conditions (pH 9.2) for enhancing retention and chromatographic performance of polar analytes in bicratic elution mode without unfavourable column bleed. The quality of the method was extensively validated and demonstrated by absolute metabolite quantification in endogenous Escherichia coli extracts by comparative use of standard-based external calibration, isotope dilution, and standard addition as quantification strategies. In sum, alkaline ZIC®-pHILIC chromatography emerged as an efficient approach providing high selectivity and sensitivity for comprehensive metabolic studies.