Method for determining monohydroxybenzo[a]pyrene isomers using column-switching high-performance liquid chromatography.
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 beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/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 beta-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 200 ml 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 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.
Toriba
Akira
A
Graduate School of Natural Science and Technology, Kanazawa University, 13-1, Takara-machi, Kanazawa City, Ishikawa 920-0934, Japan.
Nakamura
Hiroaki
H
Chetiyanukornkul
Thaneeya
T
Kizu
Ryoichi
R
Makino
Tsunehisa
T
Nakazawa
Hiroyuki
H
Yokoi
Tsuyoshi
T
Hayakawa
Kazuichi
K
D001564:Benzo(a)pyrene / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D007536:Isomerism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results
D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1