Abstract

We proposed a method for determining the presence of trace amounts of adducts of isopropylmethylphosphonic acid (IMPA) and cyclohexylmethylphosphonic acid (CMPA), biomarkers of exposure to sarin and cyclosarin in a body, with a tripeptide (Tyr-Thr-Lys). The method is based on plasma trypsinolysis with the formation of stable adducts IMPA–Tyr-Thr-Lys and СMPA–Tyr-Thr-Lys and their detection by high-performance liquid chromatography (HPLC) with high-resolution tandem mass spectrometry detection. The following conditions for the sample preparation of human blood plasma exposed to sarin and cyclosarin were optimized: aliquot portion volume, enzymatic hydrolysis conditions, mass spectrometric detection (selection of optimal pairs of ion reactions and detection modes), and gradient elution programs in the HPLC separation of the test mixture. The limit of detection for sarin and cyclosarin in human blood plasma was 5 ng mL^–1. The proposed approach was tested in the analysis of blood plasma samples submitted to analysis in the framework of the Fourth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons; the results showed good determination specificity.

中文翻译：

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液相色谱-质谱法鉴定人血浆中三肽（Tyr-Thr-Lys）与O-异丙基甲基膦酸和O-环己基甲基膦酸的加合物

摘要

我们提出了一种确定人体中是否存在痕量异丙基甲基膦酸（IMPA）和环己基甲基膦酸（CMPA）的痕量加合物的方法，该生物标志物是在人体中与沙林和环沙林接触的生物标志物，带有三肽（Tyr-Thr-Lys）。该方法基于血浆胰蛋白酶解并形成稳定的加合物IMPA– Tyr-Thr-Lys和СMPA– Tyr-Thr-Lys并通过高效液相色谱（HPLC）和高分辨率串联质谱检测进行检测。优化了以下条件来制备人血中暴露于沙林和环沙林的血浆样品：等分试样体积，酶水解条件，质谱检测（选择最佳的离子反应对和检测模式对）以及HPLC中的梯度洗脱程序分离测试混合物。人体血浆中沙林和环沙林的检出限为5 ng mL ^–1。在对血浆样品进行分析的过程中对提议的方法进行了测试，该样品在禁止化学武器组织第四次官方生物医学测试的框架内进行了分析；结果显示出良好的测定特异性。