HILIC-MS/MS method development for targeted quantitation of metabolites: practical considerations from a clinical...

The development of targeted assays for polar molecules is a persistent challenge in quantitative metabolite measurement. In addressing these challenges, hydrophilic interaction liquid chromatography (HILIC) has proved to be a valuable, though under-used and poorly understood chromatographic technique. This work has addressed a number of components that are intrinsic in development of a high-throughput, specific and sensitive assay for metabolites using HILIC-MS/MS. Generally accepted HILIC doctrine, such as addition of water to all mobile phases and re-equilibration time, was shown to be flawed under gradient HILIC mode. The effect of non-classical mobile phase additives on HILIC-MS/MS specificity, sensitivity and assay throughput was shown for endogenous metabolites. A broad evaluation of columns and mobile phases for the retention of varied molecular classes was performed, elucidating the empirical nature of HILIC method development. Application of the empirical evaluations performed in the paper was demonstrated by detailing a method development cycle for methylmalonic acid to achieve a highly selective and sensitive HILIC-MS/MS quantitation capable of high-throughput analysis for clinical utility.