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

Yang Yang, William J. Griffiths, Tore Midtvedt, Jan Sjövall, Joseph Rafter, Jan-Ake Gustafsson

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The characterization of conjugated metabolites of benzo[α]pyrene (BP) in the urine of male germ-free rats given a single intraperitoneal dose of [14C]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pak C18 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.

Original languageEnglish (US)
Pages (from-to)1182-1189
Number of pages8
JournalChemical Research in Toxicology
Issue number12
StatePublished - Dec 1 1999

ASJC Scopus subject areas

  • Toxicology


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