Liu ChangCai, Liang LongHui, Xiang Yu, Yu HuiLan, Zhou ShiKun, Xi HaiLing, Liu ShiLei, Liu JingQuan
Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing, 102205, China.
Anal Bioanal Chem. 2015 Sep;407(23):7037-46. doi: 10.1007/s00216-015-8842-8. Epub 2015 Jul 12.
Sulfur mustard (HD) adduct to human serum albumin (ALB) at Cys-34 residue has become an important and long-term retrospective biomarker of HD exposure. Here, a novel, sensitive, and convenient approach for retrospective quantification of HD concentration exposed to plasma was established by detection of the HD-ALB adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with a novel non-isotope internal standard (IS). The HD-ALB adduct was isolated from HD-exposed plasma with blue Sepharose. The adduct was digested with proteinase K to form sulfur-hydroxyethylthioethyl ([S-HETE])-Cys-Pro-Phe tripeptide biomarker. The tripeptide adduct could be directly analyzed by UHPLC-MS/MS without an additional solid phase extraction (SPE), which was considered as a critical procedure in previous methods. The easily available 2-chloroethyl ethylsulfide (2-CEES) as HD surrogate was first reported to be used as IS in place of traditional d8-HD for quantification of HD exposure. Furthermore, 2-CEES was also confirmed to be a good IS alternative for quantification of HD exposure by investigation of product ion spectra for their corresponding tripeptide adducts which exhibited identical MS/MS fragmentation behaviors. The method was found to be linear between 1.00 and 250 ng•mL(-1) HD exposure (R(2)>0.9989) with precision of <4.50% relative standard deviation (%RSD), accuracy range between 96.5% and 114%, and a calculated limit of detection (LOD) of 0.532 ng•mL(-1). The lowest reportable limit (LRL) is 1.00 ng•mL(-1), over seven times lower than that of the previous method. The entire method required only 0.1 mL of plasma sample and took under 7 h without special sample preparation equipment. It is proven to be a sensitive, simple, and rugged method, which is easily applied in international laboratories to improve the capabilities for the analysis of biomedical samples related to verification of the Chemical Weapon Convention (CWC).
硫芥（HD）与人类血清白蛋白（ALB）在半胱氨酸-34残基处形成的加合物已成为HD暴露的一种重要且长期的回顾性生物标志物。在此，通过使用超高压液相色谱-串联质谱（UHPLC-MS/MS）并结合一种新型非同位素内标（IS）检测HD-ALB加合物，建立了一种新颖、灵敏且便捷的回顾性定量血浆中HD浓度的方法。HD-ALB加合物用蓝色琼脂糖从暴露于HD的血浆中分离出来。该加合物用蛋白酶K消化形成硫代羟乙基硫代乙基（[S-HETE]）-半胱氨酸-脯氨酸-苯丙氨酸三肽生物标志物。该三肽加合物无需额外的固相萃取（SPE）即可直接通过UHPLC-MS/MS进行分析，而SPE在以前的方法中被视为关键步骤。首次报道使用易于获得的2-氯乙基乙硫醚（2-CEES）作为HD替代物作为内标，以取代传统的d8-HD来定量HD暴露。此外，通过研究它们相应三肽加合物的产物离子光谱，证实2-CEES也是定量HD暴露的良好内标替代物，这些加合物表现出相同的MS/MS裂解行为。该方法在HD暴露浓度为1.00至250 ng•mL(-1)之间呈线性（R(2)>0.9989），相对标准偏差（%RSD）精度<4.50%，准确度范围在96.5%至114%之间，计算出的检测限（LOD）为0.532 ng•mL(-1)。最低报告限（LRL）为1.00 ng•mL(-1)，比以前的方法低七倍以上。整个方法仅需0.1 mL血浆样本，无需特殊样本制备设备，耗时不到7小时。事实证明，这是一种灵敏、简单且耐用的方法，易于应用于国际实验室，以提高与《化学武器公约》（CWC）核查相关的生物医学样本分析能力。
