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

Abstract

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 to 5.0 ng/μL, and limits of detection (LODs) 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.

Evaluation of 3-hydroxy-benzo[a]pyrene levels in Nile tilapia (Oreochromis niloticus) after waterborne exposure to Benzo[a]pyrene

“…Upon exposure, fish have a well-developed system that rapidly metabolizes BaP in two phases (Varanasi, Stein, and Nishimoto 1989), in a complex mixture of quinones, phenols, dihydrodiols, triols, and tretols (Kennedy and Tierney 2008;Willett et al 2000;Zhu et al 2008). In phase I, BaP is metabolized into hydroxylated derivatives (OH-BaP) by the multienzymatic system cytochrome P450 (CYP450), CYP 1A1 isozyme being the predominant, although 1A2, 2A1, and 1B1 can also be active to a minor extent (Kim et al 1998).…”

Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea)

“…As a consequence, PAHs are only detected at trace levels in the tissues, that is the case in fish. In order to assess the level of PAH exposure, several analytical protocols were developed these last years to detect and quantify several PAH metabolites in biological fluids such as plasma and bile (Zhu et al, 2008).…”

Analysis of pyrene metabolites in marine snails by liquid chromatography using fluorescence and mass spectrometry detection

“…In GC-MS, only parent PAH can be sensitively analyzed without derivatization, and in most studies polar phase II metabolites must also be hydrolyzed to phase I oxidation products before analysis [39]. LC-MS with electrospray ionization (LC-ESI-MS) detection has recently been used to quantify phase I and phase II metabolites of PY [40] and other PAHs [41]. The strategies used for the detection of common phase II bioconjugates of pharmaceuticals and contaminants have been recently reviewed [42] and are promising for the analysis of a large number of PAH metabolites in complex samples.…”