HILIC‐MS/MS method development for targeted quantitation of metabolites: Practical considerations from a clinical diagnostic perspective

Abstract

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.

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

“…So far, the majority of related LC-MS studies use reversed phase [41][42][43][44][45][46][47][48] or hydrophilic interaction stationary phases [12,13,15,16,[49][50][51][52][53][54]. Despite multiple advantages such as robustness and straightforward implementation, reversed phase liquid chromatography (RPLC) typically lacks adequate retention for nonderivatized hydrophilic analytes on silica-based reversed phase stationary phases [33].…”

A high-performance liquid chromatography – tandem mass spectrometry – based targeted metabolomics kidney dysfunction marker panel in human urine

“…Although GC/MS has become the gold standard in targeted metabolite analysis due to its superior chromatographic resolution, this approach often involves extensive sample preparation combined with derivatization, extended analysis times, and/or lacks proper bioanalytical method validation following current guidelines [18]. A primary obstacle in LC-MS based metabolite analysis has been the ability to achieve chromatographic retention and separation, which allow for the separation of small polar analytes that are often not properly retained by standard reverse phase chromatography (RP) [18,19]. As an alternative, hydrophilic interaction chromatography (HILIC) offers a unique method for retention, separation and detection of polar metabolites [19,20].…”

“…A primary obstacle in LC-MS based metabolite analysis has been the ability to achieve chromatographic retention and separation, which allow for the separation of small polar analytes that are often not properly retained by standard reverse phase chromatography (RP) [18,19]. As an alternative, hydrophilic interaction chromatography (HILIC) offers a unique method for retention, separation and detection of polar metabolites [19,20]. Moreover, as previously described by Jandera et al [21], many HILIC columns demonstrate a dual HILIC-RP retention mechanism, contingent upon the concentration of the organic solvent in the mobile phase.…”

Sample‐directed pseudotargeted method for the metabolic profiling analysis of rice seeds based on liquid chromatography with mass spectrometry

“…Metabolomics strategies can be divided into targeted and non‐targeted metabolomics . Non‐targeted method aims to cover of all the measurable known and unknown metabolites in a biological sample .…”

“…Pseudotargeted metabolomics method was first developed by our group in 2012 , a non‐targeted GC–MS metabolic profiling method was transformed to a pseudo‐targeted method using the retention time locking GC–MS‐selected ion monitoring . Then, the pseudotargeted methods using LC–MS MRM for serum and urinary metabolomics were developed .…”