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 μL 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.