Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry

The characterization of conjugated metabolites of benzo[a]pyrene (BP) in th e urine of male germ-free rats given a single intraperitoneal dose of [C-14 ]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pair Cls cartridge and separated by lipophilic ion-exchange chromatography into neutral and acidic fractions (f ractions I-V). Metabolites in the latter fractions, constituting more than 80% of the urinary radioactivity, were characterized by reversed-phase HPLC and capillary column liquid chromatography/electrospray mass spectrometry (LC/ESMS) and tandem mass spectrometry (MS/MS). Relative quantities of BP m etabolites were estimated from the distribution of radioactivity. Some coel uting compounds were semiquantified from the ion current chromatograms obta ined in the capillary column LC/ESMS analyses. The major conjugated metabol ites in fraction II, containing about 50% of the urinary radioactivity, con sisted of three tetrahydrotrihydroxy-BP-S-N-acetylcysteines, the major isom er being 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine, two dihydrotrihydroxy-BP-S-N-acetylcysteines and a tetrahydrotetrahydroxy-BP-S -N-acetylcysteines Fraction II also contained three apparently unconjugated compounds whose structures will be described elsewhere. Metabolites charac terized in fractions III and IV, containing about 30% of the urinary radioa ctivity, included three BP-O,O'-disulfates, two monohydroxy-BP-O- sulfates, three dihydrodihydroxy-BP-O-sulfates, three BP-O,O'-diglucuronides, and a BP-O-sulfate-O'-glucuronide. Trace levels of a tetrahydrotrihydroxy-BP-S-gl utathione conjugate were detected in fraction V.