Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.
Department of Bioengineering Sciences, Weihenstephan-Triesdorf University of Applied Sciences, Freising, Germany.
Drug Test Anal. 2022 Jun;14(6):1140-1148. doi: 10.1002/dta.3236. Epub 2022 Feb 17.
Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re-dissolution analysis were performed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, μLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE] at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis, the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The eightfold deuterated SM analog (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C), and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at -30°C for at least 33 days and for three freeze-and-thaw cycles. At the body temperature of chicken (+40°C), Glu(-HETE) was degraded in serum (period of half-change 3 days), whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.
硫芥（SM，双[2-氯乙基]硫醚）是一种被禁止的化学战剂，曾在中东的暴力冲突中被使用，毒害了人类和动物。出于法律原因，必须采用生物分析方法来证明 SM 的暴露。SM 与内源性蛋白质（例如血清白蛋白（SA））的反应产物（加合物）是分析的有价值的长寿命靶标。尽管迄今为止几乎所有方法都集中在人类蛋白质上，但我们首次针对纯净的鸡 SA 和来自鸡、鸭和鸵鸟的禽血清进行了研究。经过蛋白酶解、蛋白质沉淀、上清液蒸发和重新溶解分析后，采用微液相色谱-电喷雾电离串联质谱法（μLC-ESI MS/MS）在选择反应监测模式（SRM）下进行分析，μLC-ESI MS/MS（SRM）用于检测 m/z 105.0 处的羟乙基硫乙基产物离子[HETE]。在与 SM 体外孵育并经蛋白酶催化的蛋白水解作用后，检测到了烷基化氨基酸 Glu（-HETE）和 His（-HETE）。两者都带有结合在其侧链上的 SM 特征性 HETE 部分。还应用了八重氘代 SM 类似物（d8-SM）来支持加合物鉴定。针对 pH 值（8.0）、温度（50°C）和时间优化了蛋白水解条件，以最大程度地提高 Glu（-HETE）（30 分钟）和 His（-HETE）（180 分钟）的产量。在自动进样器中，氨基酸加合物至少稳定 24 小时。在-30°C 下，血清中的蛋白质加合物至少稳定 33 天，并可耐受三个冻融循环。在鸡的体温（+40°C）下，血清中的 Glu（-HETE）降解（半衰期 3 天），而 His（-HETE）保持稳定。所提出的方法扩展了用于记录 SM 中毒的程序工具箱。
