Biomonitoring of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and Its Carcinogenic Metabolites in Urine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine that is produced in cooked meats. The simultaneous analysis of PhIP and its metabolites in human urine is a challenge, because these biomarkers only occur in urine at parts per billion or lower concentrations and must be selectively purifed from thousands of other urinary constituents. We have developed a facile solid-phase extraction method, employing a mixed-mode reverse-phase cation exchange resin, to simultaneously isolate PhIP, its glucuronide conjugates, and the glucuronide conjugates of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine from the urine of meat eaters. PhIP and its metabolites were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS), using a triple stage quadrupole mass spectrometer in the selected reaction monitoring scan mode. The lower limit of quantification (LOQ) of PhIP is 5 parts per trillion (ppt), and the LOQ values for the glucuronide conjugates are 50 ppt, when 25 mu L of urine is employed for assay. The extraction scheme is versatile and has been employed to isolate other ring-hydroxylated and glucuronidated metabolites of PhIP, for characterization by LC-ESI/MS/MS.