A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-μL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0-220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.