Aix Marseille Univ, CNRS, LCE, Marseille, France.
Cabinet conseil Santé-Environnement, 43 Rue Antoine Brun, 06150 Cannes, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 15;1113:69-76. doi: 10.1016/j.jchromb.2019.03.005. Epub 2019 Mar 6.
In this study, an accurate and robust gas chromatography/mass spectrometry method was developed for quantitative analysis of diphenylamine, tolylfluanid, propargite and phosalone in liver fractions. Different injector parameters were optimized by an experimental design technique (central composite design). An optimal combination of injector temperature (°C), splitless time (min) and overpressure (kPa) values enabled to maximize the chromatographic responses. Sample preparation was based on protein precipitation using trichloroacetic acid followed by liquid-liquid extraction (LLE) of the pesticides with hexane. All compounds and endrin as internal standard were quantified without interference in selected ion monitoring mode. The calibration curves for diphenylamine, tolylfluanid, propargite and phosalone compounds were linear over the concentration range of 0.1 to 25 μM with determination coefficients (R) higher than 0.999. A lower limit of quantification of 0.1 μM was obtained for all analytes, i.e. 422.5, 868.0, 876.2 and 919.5 μg/kg of liver fraction (hepatocytes) for diphenylamine, tolylfluanid, propargite and phosalone, respectively. All compounds showed extraction recoveries higher than 93%, with a maximum RSD of 3.4%. Intra- and inter-day accuracies varied from 88.4 to 102.9% and, imprecision varied from 1.1 to 6.7%. Stability tests demonstrated that all pesticides were stable in liver extracts during instrumental analysis (20 °C in the autosampler tray for 72 h) following three successive freeze-thaw cycles and, at -20 °C for up to 12 months. This simple and efficient analytical procedure is thus suitable for metabolism studies or for assessing mammals liver contamination.
本研究建立了一种准确、稳健的气相色谱/质谱法，用于定量分析肝组织中二苯胺、甲苯氟磺胺、丙炔螨特和磷胺。采用实验设计技术（中心复合设计）优化了不同的进样器参数。优化的进样器温度（°C）、不分流时间（min）和过压（kPa）值组合可使色谱响应最大化。样品制备基于用三氯乙酸沉淀蛋白质，然后用己烷进行农药的液-液萃取（LLE）。所有化合物和异狄氏剂内标在选择离子监测模式下均无干扰定量。二苯胺、甲苯氟磺胺、丙炔螨特和磷胺化合物的校准曲线在 0.1 至 25µM 的浓度范围内呈线性，相关系数（R）均高于 0.999。所有分析物的定量下限均为 0.1µM，即肝组织（肝细胞）中二苯胺、甲苯氟磺胺、丙炔螨特和磷胺的含量分别为 422.5、868.0、876.2 和 919.5µg/kg。所有化合物的提取回收率均高于 93%，最大相对标准偏差（RSD）为 3.4%。日内和日间准确度在 88.4%至 102.9%之间变化，精密度在 1.1%至 6.7%之间变化。稳定性测试表明，所有农药在仪器分析过程中（自动进样器托盘内 20°C 时 72 h）在经过三个连续的冻融循环后以及在-20°C 下可稳定保存长达 12 个月。因此，这种简单高效的分析程序适用于代谢研究或评估哺乳动物肝脏污染。
