新型前处理技术结合超高效液相色谱法测定吸烟者尿液中的苯并[a]芘及3-羟基苯并[a]芘的含量

Determination of Benzo[a]pyrene and 3-Hydroxybenzo[a]pyrene in Urine of Smokers by Ultra High Performance Liquid Chromatography Combined with a Novel Pretreatment Technique

以索氏提取装置为基础,结合基质固相分散萃取和柱层析的实践,自行设计并制成了集萃取、净化与浓缩于一体的新型样品前处理装置,用于超高效液相色谱法测定吸烟者尿液中苯并[a]芘(B[a]P)和3-羟基苯并[a]芘(3-OHB[a]P)含量时的样品前处理。采集尿样50mL,加入乙酸盐缓冲溶液(pH 5.0)0.5g,β-葡糖苷酸-芳基硫酸酯酶0.1mL,于38℃振荡水解5.0h。从中分取试液20.00mL,浓缩至近干,并分散于2.0g硅胶中。将此分散体烘干后,按规程移入于上述前处理装置中。用60mL环己烷-乙醇(98+2)混合液于90℃水浴加热回流提取45min。继续加热蒸发溶液至近干,用1 mL乙腈溶解残留,并过滤后供超高效液相色谱分析。色谱分析中用ACQUITY UPLCTM BEH C18色谱柱为固定相,以乙腈-水(68+32)混合液为流动相进行洗脱。出峰时间在0~6.5min时用λex365nm和λem450nm,荧光检测3-OHB[a]P,在6.5~12.0min时用λex384nm和λem406nm,荧光检测B[a]P,两者的质量浓度依次在4.0~200μg·L^(-1)和10~250μg·L^(-1)内与相应的峰面积呈线性关系。检出限(3S/N)依次为0.65,3.6μg·L^(-1)。加标回收率分别在86.8%~92.8%和91.2%~95.7%之间,测定值的相对标准偏差(n=7)均小于4.0%。

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 benzo[a]pyrene（B[a]P）and 3-hydroxybenzo[a]pyrene（3-OHB[a]P）in urine of smokers by ultra high performance liquid chromatography（UPLC）.50 mL of urine sample were collected and hydrolyzed vortically with0.5 g of acetate buffer solution and 0.1 mL ofβ-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 with1 mL of acetonitrile and the solution,after filtration,was used for UPLC analysis.ACQUITY UPLCTM 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-OHB[a]P in the interval of peak-appearing time from 0 to 6.5 min atλex365 nm andλem 450 nm,and for B[a]P in the time interval from 6.5 to 12.0 min atλex384 nm andλem406 nm.Linear relationships between values of peak area and respective mass concentration were found in the range of 4.0 to 200μg·L^（-1） for 3-OHB[a]P and in the range of 10 to 250μg·L^（-1） for B[a]P,with detection limits（3 S/N）of 0.65μg·L^（-1） and 3.6μg·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%.