Currently, the measure of the oxidative stress, from oxidized and reduced glutathione (GSSG and GSH respectively), for large cohorts of samples, is generally limited to spectrometric methods. In this study, a high-throughput assay for GSH after derivatization with N-ethylmaleimide and GSSG in blood sample was developed with an analysis time of 1.5 min. The method combines protein precipitation and a short LC (10-mm length) column where compounds were trapped in front-flush mode and eluted in back-flush mode. This setup is combined with modifier-assisted differential ion mobility spectrometry (DMS, SelexIon) and detection is performed in the selected reaction monitoring mode using positive electrospray ionization. In DMS, various modifiers were investigated including N₂, methanol, toluene, ethanol, acetonitrile, and isopropanol to improve assay selectivity. Using EtOH as modifier, the limit of quantification (LOQ) was found to be 0.4 μM for GSSG and 3.2 μM for GS-N-ethylmaleimide (NEM) using a blood volume of 60 μL. The method is linear over a wide dynamic concentration range of 0.4 to 400 μM for GSSG and from 3.2 to 3200 μM for GS-NEM. The inter-assay precision of QC samples were ≤ 6.7%, with accuracy values between 98.3 and 103%. The method was further cross-validated with a LC Hypercarb-DMS-MS/MS method by the analysis of human blood samples. The bias between both assays ranged from − 0.3 to 0.2%.

Open image in new window

目前，对于大批样品，氧化和还原型谷胱甘肽（分别为GSSG和GSH）产生的氧化应激的测量通常仅限于光谱法。在这项研究中，开发了高通量的血样中N-乙基马来酰亚胺和GSSG衍生化后的GSH，分析时间为1.5分钟。该方法结合了蛋白质沉淀和较短的LC（10毫米长）色谱柱，其中化合物以前吹扫模式捕集并以反吹扫洗模式洗脱。该设置与改性剂辅助的差分离子迁移谱法（DMS，SelexIon）相结合，并使用正电喷雾电离在选定的反应监测模式下进行检测。在DMS中，研究了各种改性剂，包括N ₂甲醇，甲苯，乙醇，乙腈和异丙醇，以提高测定的选择性。使用EtOH作为改性剂，使用60μL的血量，发现定量限（LOQ）对于GSSG为0.4μM，对于GS- N-乙基马来酰亚胺（NEM）为3.2μM。对于GSSG，该方法在0.4至400μM的宽动态浓度范围内是线性的；对于GS-NEM，该方法在3.2至3200μM的宽动态浓度范围内是线性的。QC样品的批间准确度≤6.7％，准确度值在98.3至103％之间。通过分析人类血液样本，该方法进一步与LC Hypercarb-DMS-MS / MS方法进行了交叉验证。两种测定之间的偏差范围为-0.3％至0.2％。

在新窗口中打开图像