Using Hydrogen/Deuterium Exchange in Untargeted LC-MS Analysis of Small Molecules

Liquid chromatography–hydrogen/deuterium exchange–mass spectrometry (LC-HDX-MS) provides orthogonal structural information that complements conventional LC-MS and improves confidence in small-molecule structural elucidation. Although HDX-MS is well established in protein research, its application to small molecules remains less developed, and practical guidance for experimental implementation and data interpretation is limited. Here, we evaluate major LC-HDX-MS strategies, including post-column co-infusion, partial HDX, and full HDX approaches, and assess their analytical performance using reference standards and NIST SRM 1950 human plasma. The effects of deuterated mobile phases on background contamination, labeled/unlabeled signal ratios, retention-time shifts, adduct chemistry, and mass spectral interpretation are examined, and currently available software tools for LC-HDX-MS data processing are discussed. Full HDX provided the most complete deuterium incorporation, whereas deuterated mobile phases increased background signals and generally reduced analyte responses. Retention-time shifts were typically small but should be considered when matching features. HDX-derived information provides an additional experimental structural descriptor by defining the number of exchangeable hydrogen atoms and refining adduct assignments, thereby reducing ambiguity during structural annotation. These findings provide practical recommendations for implementing LC-HDX-MS in untargeted LC-MS workflows and demonstrate its value as an orthogonal tool for small-molecule structural elucidation.