Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine following consumption of a single cooked chicken meal in humans

Many studies suggest that mutagenic/carcinogenic chemicals in the diet, lik e 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), may play a role i n human cancer initiation. We have developed a method to quantify PhIP meta bolites in human urine and have applied it to samples from female volunteer s who had eaten a meal of cooked chicken. For this analysis, urine samples (5 mi) were spiked with a deuterium-labeled internal standard, adsorbed to a macroporous polymeric column and then eluted with methanol, lifter a solv ent exchange to 0.01 M HCl, the urine extracts were passed through a filter , applied to a benzenesulfonic acid column, washed with methanol/acid and e luted with ammonium acetate and concentrated on a C-18 column. The metaboli tes were eluted from the C-18 column and quantified by LC/MS/MS. In our stu dies of human PhIP metabolism, eight volunteers were fed 200 g of cooked ch icken containing a total of 27 mug PhIP, Urine samples were collected for 2 4 h after the meal, in 6 h aliquots, Although no metabolites could be found in urine collected from volunteers before eating the chicken, four major h uman PhIP metabolites, N-2-OH-PhIP-N3-glucuronide, PhIP-N-2-glucuronide 4'- PhIP-sulfate and N-2-OH-PhIP-N3-glucuronide, were found in the urine after the chicken meal, The volunteers in the study excreted 4-53% of the ingeste d PhIP dose in the urine. The rate of metabolite excretion varied among the subjects, however, in all of the subjects the majority of the metabolites were excreted in the first 12 h, Very little metabolite was detected in the urine after 18 h, In humans, N-2-OH-PhIP-N-2 glucuronide is the most abund ant urinary metabolite, followed by PhIP-N-2-glucuronide, The variation see n in the total amount, excretion time and metabolite ratios with our method suggests that individual digestion, metabolism and/or other components of the diet may influence the absorption and amounts of metabolic products pro duced from PhIP.