Determination of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its metabolite 2-hydroxyamino-PhIP by liquid chromatography/electrospray ionization-ion trap mass spectrometry

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. R is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N-2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography-electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N-2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N-2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C-18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C-18. liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N-2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues. (C) 2001 Academic Press.