Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification...

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.