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

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, 1H 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-d3 and omeprazole-d2. High-resolution mass spectrometry and 1H 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.