A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in the urine of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed to isolate MeIQx and its oxidized metabolites, 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f] quinoxaline (8-CH2OH-IQx) and 2-amino-3-methylimidazo[4,5-f] quinoxaline-8-carboxylic acid (IQx-8-COOH), which are produced by cytochrome P450 1A2 (P450 1A2). The phase II conjugates N2-(β-1- glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and N 2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)-sulfamic acid were measured indirectly, following acid hydrolysis to form MeIQx. The enrichment procedure permitted the simultaneous analysis of PhIP, N2-(β-1- glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, N3-(β-1-glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1-methyl-6-(4′-hydroxy)-phenylimidazo[4,5-b]pyridine (4′-HO-PhIP), and the isomeric N2- and N3-glucuronide conjugates of the carcinogenic metabolite, 2-hydroxyamino-1-methyl-6- phenylimidazo[4,5-b]pyridine (HONH-PhIP), which is formed by P450 1A2. The limit of quantification (LOQ) for MeIQx, PhIP, and 4′-HO-PhIP was ∼ 5 pg/mL; the LOQ values for 8-CH2OH-IQx and IQx-8-COOH were, respectively, <15 and <25 pg/mL, and the LOQ values for the glucuronide conjugates of PhIP and HONH-PhIP were 50 pg/mL. The metabolism was extensive; less than 9% of the dose was eliminated in urine as unaltered MeIQx, and <1% was eliminated as unaltered PhIP. Phase II conjugates of the parent amines accounted for up to 12% of the dose of MeIQx and up to 2% of the dose of PhIP. 8-CH2OH-IQx and IQx-8-COOH accounted for up to 76% of the dose of MeIQx, and the isomeric glucuronide conjugates of HONH-PhIP accounted for up to 33% of the dose of PhIP that were eliminated in urine within 10 h of meat consumption. P450 1A2 significantly contributes to the metabolism of both HAAs but with marked differences in substrate specificity. P450 1A2 primarily catalyzes the detoxification of MeIQx by oxidation of the 8-methyl group, whereas it catalyzes the bioactivation of PhIP by oxidation of the exocyclic amine group.
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