Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS.

We have developed a new hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method with mobile phases optimized for high metabolite coverage in individual polarity modes. This dual mobile phase strategy expands the range of annotated metabolites and improves identification confidence, providing broader and more accurate metabolic profiles. The incorporation of a bioinert chromatographic system further enhances sensitivity. The bridged ethyl hybrid (BEH) amide column yields the best results for metabolomic analysis among the three chromatographic columns in this comparison. The method development involves investigating the effects of mobile phase composition, pH, and a medronic acid additive on the MS response under bioinert chromatographic conditions. The results highlight the important role of alkaline pH for the sensitive detection of polyphosphorylated metabolites, while demonstrating the redundancy of chelating additives in a fully bioinert system. Finally, the optimized method is applied to mouse plasma, pancreas, and liver samples to demonstrate its versatility and reliable performance in complex biological matrices, establishing it as a powerful tool for confident and reproducible metabolomics studies.