1Emergency Response Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, NE, Atlanta, GA 30341, USA.
J Anal Toxicol. 2014 Jan-Feb;38(1):8-15. doi: 10.1093/jat/bkt088. Epub 2013 Nov 7.
Here, we report an enhanced throughput method for the diagnosis of human exposure to sulfur mustard. A hydroxyethylthioethyl (HETE) ester-adducted tripeptide, produced by pronase digestion of human serum albumin, was selected as the quantitative exposure biomarker. Cibacron Blue enrichment was developed from an established cartridge method into a 96-well plate format, increasing throughput and ruggedness. This new method decreased sample volume 2.5-fold. Addition of a precipitation and solid-phase extraction concentration step increased the sensitivity of the method. With the conversion to a 96-well plate and optimization of chromatography, the method resulted in a 3-fold decrease in analysis time. Inclusion of a confirmation ion has increased specificity. The method was found to be linear between 0.050 and 50 µM sulfur mustard exposure with a precision for both quality control samples of ≤6.5% relative standard deviation and an accuracy of >96%. The limit of detection (3So) was calculated to be ∼0.0048 µM, an exposure value similar to that of the HETE-albumin adduct method first described by Noort and co-workers (Noort et al., 1999; Noort el al., 2004) which used protein precipitation to isolate albumin. A convenience set of 124 plasma samples from healthy unexposed individuals was analyzed using this method to assess background levels of exposure to sulfur mustard; no positive results were detected.
在这里，我们报告了一种用于诊断人体接触芥子气的高通量方法。一种羟乙基硫乙基（HETE）酯加合物三肽，通过人血清白蛋白的胰蛋白酶消化产生，被选为定量暴露生物标志物。Cibacron Blue 从已建立的试剂盒方法中开发出来，成为 96 孔板格式，提高了通量和坚固性。这种新方法将样品体积减少了 2.5 倍。加入沉淀和固相萃取浓缩步骤增加了方法的灵敏度。通过转换为 96 孔板并优化色谱，分析时间缩短了 3 倍。加入确认离子增加了特异性。该方法在 0.050 至 50 μM 芥子气暴露范围内呈线性，两个质控样品的精密度均≤6.5%相对标准偏差，准确性>96%。检测限（3So）计算为约 0.0048 μM，暴露值与 Noort 及其同事首次描述的 HETE-白蛋白加合物方法相似（Noort 等人，1999 年；Noort 等人，2004 年），该方法使用蛋白质沉淀来分离白蛋白。使用该方法分析了 124 份来自健康未暴露个体的便利血浆样本，以评估接触芥子气的背景水平；未检测到阳性结果。
