Determination of Benzoapyrene and 3-Hydroxybenzoapyrene in Urine of Smokers by Ultra High Performance Liquid Chromatography Combined with a Novel Pretreatment Technique

A novel sample pretreatment device integrating extraction, purification and concentration was designed and fabricated on the base of Soxhlet extraction device, and by practice of matrix solid-phase dispersion extraction and column chromatography, and it was used for sample pretreatment in the determination of benzoapyrene (BaP) and 3-hydroxybenzoapyrene (3-OHBaP) in urine of smokers by ultra high performance liquid chromatography (UPLC). 50 mL of urine sample were collected and hydrolyzed vortically with 0.5 g of acetate buffer solution and 0.1 mL of β-glucuronidearyl sulfatase at 38℃ for 5 h. An aliquot (20.00 mL) of the hydrolyzed solution was concentrated to near dryness and dispersed with 2.0 g of silica gel. The dispersion, after drying, was introduced into the novel sample pretreatment device as prescribed, extracted and purified by refluxing at 90℃ in a water bath for 45 min using 60 mL of a mixture of cyclohexane and ethanol (98+2) as extractant. Heating was continued until the solution was evaporated to near-dryness. The residue was taken up with 1 mL of acetonitrile and the solution, after filtration, was used for UPLC analysis. ACQUITY UPLC^TM BEH C₁₈ column was used as stationary phase and a mixture of acetonitrile and water (68+32) was used as mobile phase to elute the analytes. Fluorophotometric detections were made for 3-OHBaP in the interval of peak-appearing time from 0 to 6.5 min at λ_ex 365 nm and λ_em 450 nm, and for BaP in the time interval from 6.5 to 12.0 min at λ_ex 384 nm and λ_em 406 nm. Linear relationships between values of peak area and respective mass concentration were found in the range of 4.0 to 200 μg·L^-1 for 3-OHBaP and in the range of 10 to 250 μg·L^-1 for BaP, with detection limits (3S/N) of 0.65 μg·L^-1 and 3.6 μg·L^-1 respectively. Values of recovery obtained by standard addition method were in the ranges of 86.8% to 92.8% and 91.2% to 95.7% respectively. Values of RSDs (n=7) were all less than 4.0%.