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Acrylamide (AA) is reported present in high-temperature-processed food and classified as a possible human carcinogen. In vivo metabolic activation of AA by CYP 2E1 to glycidamide (GA) may play an important role on AA carcinogenicity. AA and GA can be detoxified by glutathione-S-transferase to form AA and isomeric GA glutathione conjugates (AA-, GA2- and GA3-GSH, respectively), which can be further metabolized to mercapturic acids (MAs). Although many studies analyzed MAs in urine of rodents and humans, few studies have characterized and analyzed the GSH conjugates. The objectives of this study were to synthesize, purify, and characterize AA-GSH, GA2-GSH, GA3-GSH, (¹³C₃)-AA-GSH, (¹³C₃)-GA2-GSH, and (¹³C₃)-GA3-GSH to develop an isotope-dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method to analyze AA- and GA-GSHs in blood of rats treated with AA. After …