Novel strategy for characterizing the metabolism of GSH–enal adducts in myocardial ischemia rat model using UHPLC–MS

α,β-Unsaturated aldehydes (enals) are primarily metabolized via glutathione (GSH) conjugation, forming GSH–enal adducts, which represent the major pathway for their in vivo clearance and detoxification. These adducts undergo further metabolism to form GlyCys–enal adducts, Cys–enal adducts, and N-acetylcysteine (NAC)–enal adducts, ultimately excreted in urine. Elucidating the mechanisms by which enals undergo detoxification through GSH conjugation, metabolism, and excretion is important for understanding their roles in related diseases. However, comprehensive characterization of enal adducts remains challenging because of their structural instability and low concentrations in vivo . In this study, a novel strategy was developed to characterize the in vivo metabolism of GSH–enal adducts using liquid chromatography-mass spectrometry (LC-MS) combined with 2,4-dinitrophenylhydrazine derivatization. Characteristic fragment ions and fragmentation patterns were summarized based on 56 synthetic enal adducts analyzed by high-resolution MS. Based on the GSH–enal metabolic pathway, a database containing 4,496 predicted enal adducts was constructed to facilitate the discovery and identification of potential enal adducts. Using precursor ion scanning by LC-MS/MS integrated with the predicted database and characteristic fragment ions, 499 candidate enal adducts were initially detected in rat heart, liver, and kidney tissues. Among these, 49 novel enal adducts were further identified using dynamic multiple reaction monitoring, and 12 of the identified enal adducts were confirmed using synthetic standards. This strategy was successfully applied to dynamically profile GSH–enal adducts and their metabolites in rats with myocardial ischemia (MI), representing the first comprehensive elucidation of the detoxification and metabolic pathways of oxidative stress-induced enals. These findings provide new insights into the dynamic changes of enals and GSH–enal adducts during disease-associated oxidative damage.