Hydrogen–Deuterium Back-Exchange for High-Resolution Mass-Spectrometry-Based Metabolite Characterization

Methods for unknown metabolite characterization rely heavily on one or more of the following: spectroscopic approaches; tandem mass spectrometry (MS/MS); ion mobility or liquid chromatography; and/or guided intervention through reverse metabolomics, derivatization, etc. The limitations of these methodologies vary widely. For example, spectroscopic approaches are slow and limited in throughput as they necessitate metabolite purification. Additionally, tandem mass spectrometry is often insufficient for unambiguous identification, and guided interventions can be limited in scope. A complementary approach to high-resolution mass spectrometry that is high-throughput, is robust, and provides nondiscrete data (i.e., unique reaction rate) that can rationally inform metabolite structure is desirable. Here, we present an optimized method utilizing sodium bicarbonate for hydrogen-deuterium exchange (HDX) to label slowly exchanging and labile sites over time, after which back-exchange of all labile sites leaves only slowly exchanging sites labeled for subsequent MS analysis. The exchange kinetics and extent of deuterium incorporation aid in metabolite structure identification. The myriads of different metabolite structures impart differences in resonance, inductive, and stearic effects at the labeling site and are resolvable kinetically. This methodology is used to identify different functional groups and provide kinetic resolution of structural isomers (methyl xanthine and methyl 2-oxovalerate as well as ortho, para, and meta isomers of hydroxyhippurate), complemented with density functional theory (DFT) calculations.