Analysis of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Its Phase I and Phase II Metabolites in Mouse Urine Using LC?CUV?CMS?CMS

2-Amino-1-methyl-6-phenylimidazo4,5-b]pyridine (PhIP) is an abundantly present heterocyclic aromatic amine which is found to be carcinogenic in rodents, mice and rats. The biotransformation of PhIP is extensive and involves both the formation of bioactivated as well as detoxification metabolites. In order to understand its carcinogenicity, the metabolism of PhIP needs to be studied. Numerous metabolites of PhIP have been described but, so far, assays for their quantitative determination in biological matrices are scarce. We present the development and application of an assay, using reversed phase liquid chromatography coupled to ultraviolet and mass spectrometry detectors for the quantification of PhIP, three phase I and nine phase II metabolites in urine. Additionally, the identification of two PhIP-sulfates by the use of NMR is presented. Sample pretreatment consisted of straightforward dilution of urine. PhIP and its metabolites were shown to be stable in diluted urine for at least 22 h when stored at 2?C8 °C. Precision of the analysis was within 15%. The assay has been successfully applied for the quantification of PhIP and 12 of its metabolites in urine from mice that received 200 mg kg^?1 PhIP via oral gavage.