Quantitative determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence and GC–MS

Abstract

Two analytical methods have been evaluated for quantitative determination of de-conjugated chrysene metabolites in fish bile. High performance liquid chromatography-fluorescence (HPLC-F) and gas chromatography–mass spectrometry (GC–MS) were compared regarding instrumental and overall limits of detection (LOD) as well as recoveries for the following nine chrysene compounds: 1-, 2,- 3-, 4- and 6-hydroxychrysene (1-, 2-, 3-, 4- and 6-OH-chr), 1,2-dihydroxy-1,2-dihydrochrysene (1,2-DHD-chr), 3,4-dihydroxy-3,4-dihydrochrysene (3,4-DHD-chr), 5,6-dihydroxy-5,6-dihydrochrysene (5,6-DHD-chr) and chrysene. Instrumental LODs were comparable for the two methods whereas the overall LOD was better for HPLC-F. Recoveries varied per chrysene compound for both HPLC-F (62–107%) and GC–MS (48–124%). In vivo formed chrysene metabolites were studied in the bile of Atlantic cod ( Gadus morhua) exposed to chrysene (1 mg/kg) via intra-peritoneal (i.p.) and inter-muscular (i.m.) injection. Total amounts of chrysene metabolites were three times higher in i.p. compared to i.m. exposed cod bile, but the relative distribution of determined metabolites was very similar. 1,2-DHD-chr was the most prominent metabolite in de-conjugated bile and constituted more than 88% of the total chrysene metabolites. Additional chrysene metabolites formed were 3,4-DHD-chr and 1-, 2-, 3- and 4-OH-chr. K-region chrysene metabolites (oxidation at carbons 5 and 6) were not detected and seem to be a less favoured biotransformation route. The two methods were applied and evaluated for analysis of chrysene metabolites in two bile reference materials (BCR 720 and 721) and a limited number of field exposed cods.