HPLC法检测酶法合成中还原型/氧化型谷胱甘肽

建立酶法反应中还原型/氧化型谷胱甘肽含量的液相分析方法。采用ZOABAX SB-C18进行分离,流动相为磷酸二氢钾辛烷磺酸钠溶液∶乙腈(920∶80,V/V),流速1.0 mL/min,检测波长210 nm,进样量20μL;采用外标法定量。还原型/氧化型谷胱甘肽在10~200μg/mL范围内线性关系良好,相关系数分别为0.9994(n=4)、0.9999(n=4),在S/N=3时最低检测限分别为0.037μg/mL、0.019μg/mL;在S/N=10时最低定量限分别为1.90μg/mL、1.12μg/mL;样品加标回收率分别为98.7%~100.6%、98.5%~101.4%;色谱系统稳定性RSD,GSH、GSSH分别为0.44%、1.16%。样品分析显示出本方法操作简便,快速,准确,灵敏度高,稳定性好,适合酶催化反应中还原型/氧化型谷胱甘肽的测定。

Measurement of Reduced Glutathione/oxidized Glutathione Enzymatic Synthesis by High Performance Liquid Chromatography

To establish a new HPLC method for determination of reduced / oxidized glutathione in enzymatic reaction. The separation was carried out on a ZOABAX SB-CI8 reversed-phase column with UV detection at 210 nm and mobile phase were both a mixture of sodium dihydrogen phosphate -I-octanesulfonic acid sodium salt in water and acetonitrile （920：80, V/V）, the flow rate was 1 mL/min, the injection volume was 20 μL and the reaction mixture was quantified by external standard method. The linear range of reduced / oxidized glutathione was 10-200 μg/mL with GSH（r=0.9994, n=4） GSSH（r=0.9999, n=4）, the best limit of determination was 0.037 lag/mL and 0.019 μg/mL （S/N=3）; the quantification limit was 1.90μ g/mL and 1.12 p.g/mL; the rate of recovery of GSH was 98.7 %-100.6 %, GSSH was 98.5 %0101.4 %; The RSD of system stability on GSH and GSSH were 0.44 % and 1.16 %. The method is simple, rapid, accurate, sensitive and stable which is suitable for determination of reduced / oxidized glutathione in enzymatic reaction.