Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electro spray mass spectrometry
Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electro spray mass spectrometry
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DOI：
10.1016/s0378-4347(01)00081-0
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发表时间：
2001-06-05
影响因子：
3
通讯作者：
Fanali, S
中科院分区：
文献类型：
--
作者：
Camera, E;Rinaldi, M;Fanali, S
建立了一种灵敏、特异的高效液相色谱-电喷雾-质谱法同时测定外周血单个核细胞(PBMC)中还原型谷胱甘肽(GSH)和氧化谷胱甘肽(GSSG)的方法。样品经N-乙基马来酰亚胺衍生化、硫代水杨酸为内标、冷乙腈沉淀蛋白质后，用乙腈-1%冰醋酸(25：75)洗脱，电喷雾质谱(ESI-MS)系统检测。优化后的方法具有良好的检出限(GSH和GSSG分别为0.01um和0.05um)。GSH和GSSG的线性范围分别为0.01-20um和0.05-20um。谷胱甘肽和谷胱甘肽的平均回收率分别为98.5-100.6%和105.8-111.5%。保留时间和峰面积的批间重复性GSH分别为0.06%和1.75，GSSG分别为0.18和2.50。将优化后的方法应用于20例健康人外周血中GSH和GSSG的测定。(C)2001 Elsevier Science B.V.保留所有权利。
We developed a sensitive and specific liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile-1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 muM for GSH and GSSG, respectively). Good linearity was reached in the 0.01-20 muM range for GSH and 0.05-20 muM for GSSG. The mean recoveries of GSH and GSSG were 98.5-100.6% and 105.8-111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals. (C) 2001 Elsevier Science B.V. All rights reserved.