A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction
| Autor: | Kenneth F. Reardon, Kai H. Liao, Raymond S. H. Yang, Arthur N. Mayeno |
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| Rok vydání: | 2005 |
| Předmět: |
Time Factors
animal structures Clinical Biochemistry chemistry.chemical_element Zinc complex mixtures Biochemistry High-performance liquid chromatography Analytical Chemistry chemistry.chemical_compound Benzo(a)pyrene Cytochrome P-450 CYP1A1 polycyclic compounds Humans Epoxide hydrolase Derivatization Chromatography High Pressure Liquid Detection limit Chromatography organic chemicals Reproducibility of Results Cell Biology General Medicine Fluorescence Recombinant Proteins chemistry Calibration embryonic structures Pyrene Oxidation-Reduction |
| Zdroj: | Journal of Chromatography B. 824:166-174 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2005.07.021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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