Noort D, Hulst A G, Trap H C, de Jong L P, Benschop H P
Department of Chemical Toxicology, TNO Prins Maurits Laboratory, Rijswijk, The Netherlands.
Arch Toxicol. 1997;71(3):171-8. doi: 10.1007/s002040050372.
As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.
作为开发用于验证疑似接触硫芥方法的项目的一部分，我们合成并表征了三种推测由血红蛋白烷基化形成的氨基酸加合物：4-(2-羟乙硫基乙基)-L-天冬氨酸、5-(2-羟乙硫基乙基)-L-谷氨酸以及N1-和N3-(2-羟乙硫基乙基)-L-组氨酸。针对这些加合物以及半胱氨酸和N端缬氨酸加合物，开发了适用于气相色谱/质谱分析的衍生化方法。人血在体外与[35S]硫芥孵育，随后对分离出的珠蛋白进行酸性水解并用芴甲氧羰基氯（Fmoc-Cl）衍生化，经高效液相色谱分析得到三个放射性峰，其中一个与N1/N3-(2-羟乙硫基乙基)-L-组氨酸的合成芴甲氧羰基衍生物共洗脱。珠蛋白经链霉蛋白酶消化后，可初步鉴定并定量组氨酸、谷氨酸、天冬氨酸、半胱氨酸和N端缬氨酸的加合物。最终鉴定通过气相色谱/质谱分析完成。最丰富的加合物N1/N3-(2-羟乙硫基乙基)-L-组氨酸无法通过气相色谱/质谱进行灵敏分析。为此化合物开发了一种便捷的液相色谱-串联质谱方法，能够检测人血在体外接触10微摩尔硫芥的情况。
