尿中毒死蜱及其代谢物的分析

Simultaneous determination of chlorpyrifos and its metabolite in human urine by HPLC-DAD

目的建立尿样中毒死蜱及其代谢物3,5,6-三氯-2-吡啶醇(TCPyr)的高效液相色谱分析方法。方法采用乙酸乙酯-正己烷混合溶剂提取,Inertsil ODS-3反相分离柱,以乙腈-甲醇-水-0．05％三氟乙酸为流动相梯度洗脱,毒死蜱和TCPyr的紫外检测波长分别为290 nm与298 nm,并利用二极管阵列检测器对毒死蜱和TCPyr的紫外图谱进行鉴定。结果毒死蜱和TCPyr的线性范围分别为0．2616-13．08μg／mL和0．137 4-6．870μg／mL,相关系数良好;最低检测限分别为105．0ng／mL和62．5 ng/mL;日内及日间精密度RSD均小于10．0％,平均加标回收率分别为67．0％和85．0％。结论此方法灵敏准确,简便实用,可用于尿样中毒死蜱与TCPyr的含量分析。

Purpose To develop a simple and sensitive HPLC method for simultaneous determination of the insecticide chlorpyrifos （O, O-diethyl-O-[-3, 5, 6-trichloro-2-pyridinyl]phosphorothioate）, and its specific metabolite TCPyr （3, 5, 6-trichloro-2-pyridinol） in human urine samples by HPLC. Methods The method was based on using solvent extraction and HPLC with reversed-phase ODS-3 column. The mobile phase was gradient mixture of acetonitrile-methanol-water containing 0.05 % （V/ V） trifluoroacetic acid （TFA）. The compounds were identified by diode array detector （DAD） at 290 nm for chlorpyrifos and at 298 nm for TCP, respectively. Chlorpyrifos and TCPyr were extracted by a mixed solvent of ethyl acetate-n-hexane （3： 1, V/V）. After purification and centrifugation, the organic layer was evaporated with a stream of nitrogen at 45 ℃, and the residue was dissolved in mixed solvent acetonitrile-methanol of （1： 1, V/V） and injected into the HPLC system Results Precise separation of the chlorpyrifos and TCPyr was achieved within15 minutes. The relationship between peak areas and concentration showed a good linearity over a range from 0. 261 6 to 13. 080 μg /mL for chlorpyrifos, and from 0. 137 4 to 6.87 0 μg/mL for TCPyr, respectively. The limits of detection were 105.0 ng/ mL for chlorpyrifos and 62.5 ng/mL for TCPyr, respectively. Percentage recoveries of this procedure for fortified samples in different matrix were between 65.2% - 68.9% for chlorpyrifos, and between 71.5% - 96.8% for TCPyr. The RSD of the intraday and the inter-day variation were both lower than 10.0%. Conclusions These results demonstrated that the proposed method is simple, reliable and sensitive for simultaneous determination of chlorpyrifos and its metabolite TCPyr in human urine samples with satisfactory results.