解放军医学院学报2017，Vol.38Issue(7)：619-623,5.DOI:10.3969/j.issn.2095-5227.2017.07.007
全血中谷胱甘肽与N-乙基马来酰亚胺的衍生化反应条件实验
Optimizing the derivatization conditions of glutathione with N-ethylmaleimide in whole blood
摘要
Abstract
Objective To optimize the derivatization conditions of glutathione with N-ethylmaleimide (NEM) in whole blood to achieve the complete derivatization.Methods Sixty adults with 29 males and 31 females (23-79 years) in the Department of Clinical Laboratory of Chinese PLA General Hospital from April 2016 to June 2016 were enrolled in this study.Venous blood of 2 ml was collected respectively to determine the optimal derivatization reaction condition and the normal glutathione (GSH) concentration value.The effects of temperature,pH and reaction time on the derivatization process were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Group A:Samples were allowed to react at room temperature(25℃) for 10,30,60 min,respectively.Group B:Samples were heated at 50,60,70,80,90 ℃ in a water bath for 10 min,respectively.Group C:The pH was adjusted with Tris-C1 buffer (pH=8) and heated at 25,70,80,90 ℃ in a water bath for 10 min,respectively.Group D:After derivatization,samples were heated at 25,70,80,90 ℃ in a water bath for 10 rmin and then add precipitant reagent.Results The 25 ℃ subgroup of group D showed the lowest relative rate of non-derivatized GSH response compared with other groups (P ＜ 0.05,respectively).The concentrations of GSH-NEM and oxidized glutathione (GSSH) in whole blood of 60 healthy adults were (1 471.61 ± 269.43).tmol/L and (0.84 ± 0.38) μmol/L with the detection method of group D.Conclusion When the derivatization reagent NEM and the precipitant reagent 5-sulfosalicylic acid (SSA) are added respectively,a thorough derivatization reaction can be achieved without additional adjustment of pH and temperature.关键词
谷胱甘肽/衍生化/色谱法,高效液相Key words
glutathione/derivatization/chromatography/high performance liquid分类
医药卫生引用本文复制引用
徐莹莹,王志辉,王燕一..全血中谷胱甘肽与N-乙基马来酰亚胺的衍生化反应条件实验[J].解放军医学院学报,2017,38(7):619-623,5.基金项目
“十二五”国家高技术研究发展计划(863计划)(2012AA020809) （863计划）
海南省自然科学基金(20158317) （20158317）
三亚市医疗卫生科技创新项目(2014YW34)Supported by the National 12th Five Years High Technology Research and Development Program of China (2012AA020809) （2014YW34）
Supported by the Natural Science Foundation of Hainan Province (20158317) （20158317）
Medical Science and Technology Innovation Project of Sanya (2014YW34) （2014YW34）
