Hydrophilic Interaction Chromatography–Mass Spectrometry (HILIC–MS) Approaches for Probing the Polar Metabolome

Hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization (ESI) mass spectrometry (MS) has become one of the leading analytical technologies for polar metabolome analysis. With remarkable improvement of stationary phases, HILIC has reached the separation efficiency and reproducibility equivalent to RPLC-based methods with complementary selectivity and significantly higher sensitivity for the analysis of highly hydrophilic metabolites implicated in energy producing (glycolysis, pentose phosphate pathway, TCA cycle) and biosynthetic pathways (amino acids, nucleotides, vitamins and their precursors). For polar metabolome analysis, the HILIC applications range from the targeted quantification (with a focus on one specific pathway or class of metabolites) to broad-spectrum targeted screening (of hundreds of metabolites) and untargeted discovery profiling, including stable isotope-assisted tracing experiments. These HILIC-based approaches can be applied in many areas from model systems (i.e. cell and tissue lysate analysis) to large-scale human population studies (i.e. biofluid profiling). In this review we provide numerous examples of proof-of-principle studies which unambiguously demonstrate HILIC selectivity and reproducibility. Among different stationary phases, the polymer-based zwitterionic and amide columns show the best performance in achieving the broadest metabolite coverage. Recent developments are marked by the bloom of broad-coverage, multiple pathway targeted analysis (of the well-known cellular metabolome) to take advantage of the unprecedented scan speed, sensitivity and dynamic range of tandem MS spectrometers.