Preparation of Deuterium‐Labeled Tadalafil and Omeprazole by Carbon Hydrogen–Deuterium Exchange: Hydrogen Scrambling, Acid‐Induced Transformation, and LC‐MS Applications

ABSTRACT Rationale Stable isotope‐labeled compounds are indispensable internal standards for reliable liquid chromatography–mass spectrometry (LC‐MS) analysis, but their preparation frequently requires multistep synthesis. Base‐catalyzed hydrogen–deuterium exchange at carbon centers provides a simple alternative for generating stable isotopologues. Beyond quantitative applications, carbon‐bound deuterium may serve as a mechanistic probe for investigating gas‐phase fragmentation and chemically induced molecular transformations. In this work, tadalafil and omeprazole were selected as complementary model compounds to evaluate both the analytical applicability of deuterium‐labeled standards and the behavior of carbon‐bound deuterium during tandem mass spectrometry and acid‐induced transformation. Methods Deuterium‐labeled tadalafil and omeprazole were prepared by base‐catalyzed hydrogen–deuterium exchange under optimized reaction conditions. The obtained isotopologue mixtures were characterized using high‐resolution ESI‐MS, multistage tandem mass spectrometry, 1 H NMR spectroscopy, and density functional theory (DFT) calculations. Their chromatographic behavior, short‐term isotope stability, and proof‐of‐concept applicability as LC‐MS internal standards were evaluated using chromatographic comparison and analysis of post‐dose human urine samples. Results Base‐catalyzed hydrogen–deuterium exchange produced isotopologue mixtures dominated by tadalafil‐d 3 and omeprazole‐d 2 . High‐resolution mass spectrometry and 1 H NMR spectroscopy consistently identified selective incorporation of carbon‐bound deuterium, whereas DFT calculations explained the observed regioselectivity of the exchange. Tandem mass spectrometry of deuterated tadalafil revealed product‐ion mass shifts consistent with hydrogen scrambling, demonstrating migration of carbon‐bound deuterium during collision‐induced dissociation and providing additional insight into the dissociation mechanism. In contrast, deuterium‐labeled omeprazole retained its isotope label during acid‐induced transformation. Both labeled compounds exhibited chromatographic behavior suitable for proof‐of‐concept LC‐MS applications. Conclusions Base‐catalyzed carbon hydrogen–deuterium exchange provides a rapid and cost‐effective approach for preparing stable isotope‐labeled tadalafil and omeprazole suitable for LC‐MS applications. The study further demonstrates that carbon‐bound deuterium constitutes a valuable mechanistic probe, enabling investigation of hydrogen scrambling during tandem mass spectrometry and evaluation of isotope‐label stability during acid‐induced molecular transformation. The combined HRMS, tandem MS, NMR, and DFT approach establishes a comprehensive framework for characterization of deuterium‐labeled compounds and supports their future application in quantitative analysis as well as structural elucidation by mass spectrometry.