title: Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry.
abstract: The characterization of conjugated metabolites of benzo[a]pyrene (BP) in the urine of male germ-free rats given a single intraperitoneal dose of [(14)C]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pak C(18) cartridge and separated by lipophilic ion-exchange chromatography into neutral and acidic fractions (fractions 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 metabolites were estimated from the distribution of radioactivity. Some coeluting compounds were semiquantified from the ion current chromatograms obtained in the capillary column LC/ESMS analyses. The major conjugated metabolites in fraction II, containing about 50% of the urinary radioactivity, consisted of three tetrahydrotrihydroxy-BP-S-N-acetylcysteines, the major isomer 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-acetylcysteine. Fraction II also contained three apparently unconjugated compounds whose structures will be described elsewhere. Metabolites characterized in fractions III and IV, containing about 30% of the urinary radioactivity, 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-glutathione conjugate were detected in fraction V.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: W J
authors.initials: WJ
authors.lastname: Griffiths
authors.forename: T
authors.initials: T
authors.lastname: Midtvedt
authors.forename: J
authors.initials: J
authors.lastname: Sjövall
authors.forename: J
authors.initials: J
authors.lastname: Rafter
authors.forename: J A
authors.initials: JA
authors.lastname: Gustafsson
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005856:Germ-Free Life; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051381:Rats; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods; D016482:Urinalysis
chemical_list: D001564:Benzo(a)pyrene