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-muL 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.
Liao
Kai H
KH
Quantitative and Computational Toxicology Group, Center for Environmental Toxicology and Technology, Colorado State University, Fort Collins, CO 80523, USA.
Mayeno
Arthur N
AN
Reardon
Kenneth F
KF
Yang
Raymond S H
RS
D001564:Benzo(a)pyrene / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*
D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1; D015032:Zinc