高效液相色谱法测定L02细胞中还原型及氧化型谷胱甘肽  

Determination of the Reduced and Oxidized Glutathione in L02 cells by HPLC

作　　者：王晓娜[1] 徐晓敏[1] 许波[2] 马成俊[2] 李刚[2] 王振华[1] 

机构地区：[1]石河子大学药学院/新疆特种植物药资源教育部重点实验室,石河子832002 [2]烟台大学生命科学学院,烟台264005

出　　处：《石河子大学学报（自然科学版）》2013年第4期484-488,共5页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(11175222);教育部新世纪优秀人才支持计划项目(NCET-10-0967);山东省优秀中青年科学家奖励基金项目(2006BS01226);山东省自然科学基金项目(ZR2009BM027)

摘　　要：为建立准确测定L02人胎肝细胞中还原型谷胱甘肽（oxidized glutathione，GSH）与氧化型谷胱甘肽（oxidized glutathione，GSSG）的高效液相色谱法。采用5，5r_二硫代双硝基苯甲酸（5，5＇-dithiobis（2-nitrobenzoicacid），DT—NB）作为衍生剂，二硫苏糖醇（dithiothreitol，DTT）作为还原剂，利用C18色谱柱，pH5．6的0．05mol／L醋酸钠缓冲液-甲醇（93：7，V：V）为流动相，流速为0．8mL／min，检测波长为320Dm，柱温为40℃的高效液相色谱法测定细胞内的GSH和GSSG的浓度。结果表明，GSH在0．1～2mmol／L范围内线性关系良好，最低定量限为0．1mmol／L，批内，批间精密度均小于10％，样品的回收率均为95％～105％。由此可知，本方法准确，精密，重复性好，稳定性高。可用于细胞中还原型和氧化型谷胱甘肽的测定。To establish an exact and specific HPLC method for measuring the reduced and oxidative glutathione in L02 cells. The 5,5＇-dithiohis（2-nitrobenzoic acid）（DTNB）was used as derivating agent, and the dithiothreitol（DTT）was used as reducing agent. The derivative of the reduced and oxidative glutathione was analyzed by HPLC with the RP18 column at 40 ℃. And the mobile phase was chosen as the 0.05 mol/L sodium acetate buffer（pH 5.6）and methanol （93 ： 7,V ： V）at a flow rate of 0.8 mL/min. The detection wavelength was at 320 nm. The results showed that the standard eurve was linear over the range of 0.1 to 2 mmol/L. The minimum quantitative limit was 0.1 mmol/L. The precision discrepancy of inter and intra-batch was below 10%. The recoveries of the low, medium and high concentrations were within the range of 95 % to 105 %. Therefore, this method is accurate,quick,precise and suitable for the study of glutathione in cells.

关 键 词：还原型谷胱甘肽 高效液相色谱法 准确测定 胎肝细胞 氧化型 C18色谱柱 硝基苯甲酸 二硫苏糖醇 

分 类 号：R284[医药卫生—中药学]