Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2–5.0 ng/μL, and limits of detection (LOD) were lower than 0.01 ng/μL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4 ± 21.3% to 92.4 ± 3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC–MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.