WM Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
Anal Bioanal Chem. 2012 Jan;402(1):357-66. doi: 10.1007/s00216-011-5416-2. Epub 2011 Sep 29.
A simple and robust isotope dilution mass spectrometry-based assay was developed for the determination of free cysteine and glutathione (GSH) in aquatic insects. Several experimental parameters were evaluated and optimized to provide specific and sensitive detection of both compounds by in situ derivatization with N-ethylmaleimide followed by acid alkylation quenching and reverse-phased liquid chromatography coupled with selected reaction monitoring. For both targets, the assay was evaluated over a concentration range of 0.313 to 320 μM and was demonstrated to have a quantitative dynamic range spanning nearly three orders of magnitude, with lower limits of quantification being 0.330 μM for GSH and 0.370 μM for cysteine. Additionally, measurements were observed to be highly reproducible over the course of several days. When applied to the analysis of four different species of insects, large biological variation between and within species was observed. Different feeding regimens were also tested within two species of insects but statistical comparisons revealed no significant difference in the levels of either compound.
建立了一种简单而稳健的基于同位素稀释质谱的分析方法，用于测定水生昆虫中的游离半胱氨酸和谷胱甘肽（GSH）。对多个实验参数进行了评估和优化，通过原位与 N-乙基马来酰亚胺衍生化，然后进行酸烷基化淬灭和反相液相色谱与选择反应监测相结合，对这两种化合物进行特异性和灵敏性检测。对于这两个目标物，该分析方法的浓度范围为 0.313 至 320 μM，具有定量动态范围跨越近三个数量级的特点，GSH 的定量下限为 0.330 μM，半胱氨酸的定量下限为 0.370 μM。此外，在几天的时间内观察到测量结果具有高度重现性。当应用于四种不同昆虫的分析时，观察到种间和种内存在很大的生物学变异。还在两种昆虫中测试了不同的喂养方案，但统计比较显示两种化合物的水平均无显著差异。
