基于UHPLC-MS/MS分析痕量芥子气染毒血样中血红蛋白加合物

芥子气可以与血红蛋白的组氨酸侧链发生反应,生成芥子气暴露染毒的生物标志物。本研究建立了定性、定量检测痕量芥子气暴露染毒人血红蛋白加合物的方法。采用2-氯乙基乙基硫醚(2-CEES)染毒的血红蛋白溶液作为内标,用链霉蛋白酶对芥子气-珠蛋白加合物进行酶解,经PPL柱固相萃取纯化以及苄氧羰基氯(Cbz-Cl)衍生,使用超高效液相色谱-串联质谱(UHPLC-MS/MS)进行分析检测。经过优化实验条件,最终选择链霉蛋白酶的酶解温度55℃,酶解时间10.0 h,使用3.0 mL 5%甲醇水溶液淋洗,1.0 mL 50%甲醇水溶液洗脱,Cbz-Cl衍生时间30 min。结果表明,在10.0~1 000μg/L线性范围内,线性关系良好(R^2>0.997),准确度为89.8%~113%,精密度(RSD)不高于12.4%,检出限(LOD)为10.0μg/L(S/N>5)。该方法采用2对离子对进行确证,有助于痕量芥子气暴露染毒的溯源性分析,与传统酸解法相比,该方法样品处理时间短,反应条件温和、操作简单,且灵敏度可提高约150倍。

Analysis of the Sulfur Mustard Adduct to Human Hemoglobin in Blood Samples Exposed to Trace Sulfur Mustard by UHPLC-MS/MS

Sulfur mustard (2, 2’-dichloroethyl-sulfide, HD) is an alkylating agent and a powerful vesicant, which was widely imposed and caused the bulk of injuries and deaths in World War Ⅰ and Iran-Iraq War. Since World War Ⅱ, many casualties have been caused by the chemical weapons containing HD abandoned by Japanese in China. It still represents potential threat to military and civilian population. It’s very important to study reliable bioanalytical methods for detecting HD exposure. HD reacts with histidine residues of hemoglobin producing the HD adduct which has been used as a biomarker for retrospective detection of HD exposure because of its long half-life (about 120 days). A novel method based on the HD-hemoglobin adduct for qualitative and quantitative detection of trace HD exposure was developed. The HD-hemoglobin was isolated from blood through centrifugation and hemolyzation in water. Then the heme was removed with acidic acetone to obtain the HD-globin. HD-globin adduct was digested with pronase, purified by PPL solid phase extraction following benzyl chloroformate (Cbz-Cl) derivatization, and analyzed with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Upon optimization, the digestion was performed at 55 ℃ for 10.0 h. The PPL loading samples was washed with 3.0 mL 5% methanol solution and eluted with 1.0 mL 50% methanol solution. The derivatization time and temperature were 30 min and 4 ℃, respectively. With 2-chloroethyl ethyl sulfide spiked hemoglobin solution serving as internal standard (ISTD), the method was found to be linear between 10.0 and 1 000 μg/L HD exposure (R²>0.997) with precision of ≤12.4% and accuracy ranged between 89.8% and 113%. In spite of a shorter sample treatment time (compared with the acid hydrolysis, which needed reflux in hydrochloric acid at 110 ℃ for 24 h), the limit of detection (LOD) was 10.0 μg/L (S/N>5) which was 150 times lower than that by the acid hydrolysis in a previous article. Furthermore, two transitions were more beneficial to retrospective detection for trace HD exposure than only one transition used in the previous article. This method showed great specificity with absent interference, stable ratio of confirmation transition, quantitative transition, mild sample preparation conditions and simplified operations. This study provides a method for the retrospective detection of trace-HD exposure, which is beneficial to the official Organization for the Prohibition of Chemical Weapons (OPCW) proficiency tests. Furthermore, this method can provide evidence for allegations of the use of HD, treatments of the persons exposed to HD and the study of toxication mechanism.