液相色谱-质谱/质谱法测定柞蚕幼虫体内敌敌畏代谢产物磷酸三甲酯及代谢过程分析

Determination of trimethyl phosphate of dichlorvos metabolite in tussah silkworm larvae by LC-MS/MS and analysis of its metabolic process

[目的]建立液相色谱-质谱/质谱法测定柞蚕幼虫体内敌敌畏代谢产物磷酸三甲酯方法,并用此方法研究敌敌畏在柞蚕幼虫体内分解代谢情况。[方法]样品前处理用0.2%甲酸乙腈超声提取,经HLB固相萃取柱净化;色谱条件以水和甲醇为流动相梯度洗脱,在Biphenyl色谱柱上分离;质谱分析采用电喷雾离子源(ESI),正离子扫描、多反应监测(+MRM)模式下检测,基质外标法定量。采用食下毒叶法给药,设定食药后不同时间点选取活着的柞蚕幼虫进行检测。[结果]在0.01~1.00μg/L范围内,磷酸三甲酯的线性关系良好,相关系数r达0.99958;在低、中、高6种质量分数加标水平(0.05、0.10、0.25、0.50、1.00、2.50μg/kg)下,平均回收率在80.3%~110.8%之间,相对标准偏差(RSD,n=6)在3.3%~18.7%范围内;方法检出限为0.02μg/kg,定量限为0.05μg/kg。敌敌畏在柞蚕体内迅速代谢分解为磷酸三甲酯,药后0~2.0 h,磷酸三甲酯质量分数从0.062μg/kg逐渐蓄积达到峰值0.198μg/kg,之后柞蚕体内的磷酸三甲酯质量分数逐渐降低,3.0 h降至0.050μg/kg;在柞蚕幼虫体内完全检不出敌敌畏原药。[结论]该方法的准确度、灵敏度和重现性均较高,可用于柞蚕幼虫体内敌敌畏代谢产物磷酸三甲酯的测定。此研究结果为研究柞蚕幼虫敌敌畏中毒的定性确证以及在体内代谢过程提供技术依据和理论参考。

[Aims]An liquid chromatography-mass spectrometry/mass spectrometry method was established for the determination of trimethyl phosphate,a metabolite of dichlorvos in tussah silkworm larvae,and to study the catabolism of dichlorvos in tussah silkworm larvae.[Methods]Samples were ultrasonically extracted with 0.2%(V/V)formic acid acetonitrile,purified by HLB solid-phase extraction column;Biphenyl column was used for chromatographic separation,with water and methanol as mobile phase for gradient elution;electrospray ionization source(ESI)was adopted for mass spectrometry,with positive ion scanning and multiple reaction monitoring(+MRM)mode;matrix external standard method was used for quantification.The pesticide was administered by the method of ingesting poison leaves,and the live tussah silkworm larvae were selected at different time points after ingestion for detection.[Results]In the range of 0.01-1.00μg/L,the linear relationship of trimethyl phosphate was good,and the correlation coefficient was 0.99958;at the spiked levels(0.05,0.10,0.25,0.5,1.0,2.5μg/kg),the average recoveries ranged from 80.3%to 110.8%,and the relative standard deviation(RSD,n=6)ranged from 3.3%to 18.7%;the detection limit of the method was 0.02μg/kg,and the limit of quantification was 0.05μg/kg.The results showed that dichlorvos was rapidly metabolized and decomposed into trimethyl phosphate in the tussah silkworm.From 0 to 2.0 h after taking the pesticide,the concentration of trimethyl phosphate increased from 0.062μg/kg gradually to the peak value of 0.198μg/kg,and after that,the concentration of trimethyl phosphate in the body gradually decreased to 0.050μg/kg after 3.0 h;the original pesticide dichlorvos could not be detected in tussah silkworm larvae.[Conclusions]The method has high sensitivity,accuracy and reproducibility,and can be used for the determination of trimethyl phosphate as a metabolite of dichlorvos in tussah silkworm larvae.The research results provide a technical basis and theoretical reference for the qualitative confirmation of dichlorvos poisoning in tussah silkworm larvae and the metabolic process in vivo.