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

A simple and highly sensitive high-performance liquid chromatography/fluore scence method was developed for the determination of benzo[a]pyrene (BaP)-t etrol I-1 released after acid hydrolysis of (+)-anti-BaP diol-epoxide (BPDE )-blood protein adducts. This method does not require extensive tetrol isol ation such as solvent extraction, multiple solid-phase extraction columns a nd/or immunoaffinity chromatography. Instead, a single cleanup step using a n inexpensive, disposable Clx 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) a nd globin (Gb) samples fortified with BaP-tetrol I-1. The absolute detectio n limit (S/N = 3) was 0.3 pg (0.9 fmol) per injection, corresponding to 450 fg (1.4 fmol) mg(-1) Alb and 90 fg (0.3 fmol) mg(-1) Gb for 1 ml of blood. The operating linear range was at least 1-100 pg (r(2) = 0.99). Coefficien ts of variation for within-run and between-run 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 (10 mg kg(-1)) or two doses (25 mg kg(-1) each) of BaP. (C) 2001 Elsevier Science B.V. All rights reserved.