Emerging Applications of HILIC-MS/MS in Metabolomics and Drug Discovery

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) is a complementary analytical approach for the separation and sensitive detection of polar, ionic and hydrophilic compounds that are often inadequately retained by conventional reversed-phase chromatography. The technique uses a polar stationary phase and a mobile phase with a high proportion of organic solvent, commonly acetonitrile, with a small aqueous buffer component. This arrangement supports the retention of analytes such as polar metabolites, carbohydrates, peptides, nucleotides, nucleosides, amino acids and selected pharmaceutical compounds. When HILIC is coupled with tandem mass spectrometry, particularly through electrospray ionisation, the high organic content of the mobile phase can support efficient desolvation and sensitive ion detection. Tandem mass spectrometry provides additional selectivity through precursor-ion selection, fragmentation and product-ion monitoring, enabling targeted and untargeted analysis in complex matrices. This review summarises the principles, instrumentation and major ionisation interfaces used in HILIC-MS/MS, including electrospray ionisation, atmospheric pressure chemical ionisation, fast atom bombardment and matrix-assisted laser desorption/ionisation. It also describes tandem mass analyser configurations, including quadrupole, ion-trap and Orbitrap systems, and outlines detector options such as electron multipliers, Faraday cups and array detectors. The manuscript further discusses applications in metabolomics, pharmaceutical analysis, clinical studies, proteomics and polar lipid profiling through selected case studies. Overall, HILIC-MS/MS is presented as a useful analytical platform for polar analyte retention, structural confirmation and quantitative measurement, while recognising that method optimisation, retention-time reproducibility and matrix effects remain important practical considerations for reliable analytical performance across diverse sample types and laboratory workflows in contemporary biomedical research.