Method for determining monohydroxybenzo[a]pyrene isomers using column-switching high-performance liquid chromatography
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- 作者：Toriba ; Akira ; Nakamura ; Hiroaki ; Chetiyanukornkul ; Thaneeya ; Kizu ; Ryoichi ; Makino ; Tsunehisa ; et. al.
- 关键词：Hydroxybenzo[a]pyrene ; Hydroxypyrene ; PAH exposure ; Urine ; Cytochrome P450
- 刊名：Analytical Biochemistry
- 出版年：2003
- 出版时间：January 1, 2003
- 年：2003
- 卷：312
- 期：1
- 页码：14-22
- 全文大小：157 K
文摘
A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a β-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3–8pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant P450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by β-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91–103 % , which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200ml of urine were 95.5 and 100.9 % , and those for 1-OHP were 96.4 and 103.6 % , respectively. The intra- and interday precision values were 3.9 and 2.4 % for 3-OHBaP and 2.4 and 3.2 % for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.