Subpicogram determination of (+)-anti-benzo[a]pyrene diol-epoxide adducts in fish albumin and globin by high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive high-performance liquid chromatography/fluorescence method was developed for the determination of benzo[a]pyrene (BaP)-tetrol I-1 released after acid hydrolysis of (+)-anti-BaP diol-epoxide (BPDE)-blood protein adducts. This method does not require extensive tetrol isolation such as solvent extraction, multiple solid-phase extraction columns and/or immunoaffinity chromatography. Instead, a single cleanup step using an inexpensive, disposable C18 solid-phase extraction cartridge is performed, which yields 97% recovery of BaP-tetrol I-1. The method included a number of quality assurance measures and was validated using fish albumin (Alb) and globin (Gb) samples fortified with BaP-tetrol I-1. The absolute detection limit (S/N=3) was 0.3pg (0.9fmol) per injection, corresponding to 450fg (1.4fmol)mg-1 Alb and 90fg (0.3fmol)mg-1 Gb for 1ml of blood. The operating linear range was at least 1-100pg (r2=0.99). Coefficients of variation for within-run and betweenrun assays were <5.5 and <9.3%, respectively. The method was applied to the determination of (+)-anti-BPDE-Alb and Gb adducts in fish intraperitoneally injected with a single (10mgkg-1) or two doses (25mgkg-1 each) of BaP.