间接荧光法测定水产品中L-谷胱甘肽的含量

Determination of L-glutathione in marine products by the method of indirect fluorescence

建立了间接荧光法测定水产品中L-谷胱甘肽（GSH）含量的方法。样品经三氯乙酸（TCA）沉淀蛋白，调上清液pH至10．5同时二次沉淀蛋白，提取液稀释一定倍数后以邻苯二甲醛（OPA）为荧光衍生剂，等体积（1：1，V：V）暗处混合反应1-2min，在Ex325nm和Em400nm条件下测得总荧光值A。同时以N-乙基马来酰亚胺（NEM）屏蔽待测液中舍巯基的化合物，测得杂质产生的荧光值B，以总荧光值A减去荧光值B，得出GSH所产生的荧光值，间接测得GSH含量。在0．125～1．250mg／L浓度范围内GSH线性关系良好．R2=0．9963，回归方程为Y=57．012X。该方法的相对标准偏差（RSD）为3．83％-5．25％，平均回收率为91．37％～102．20％。改进后的方法弥补了荧光法本身特异性不高的缺点，使得测定更精确．适用于水产品中L-谷胱甘肽含量的测定。

The method of indirect fluorescence was developed to the determination of L-glutathione（GSH） in marine products. The protein in products was precipitated by TCA,then the pH of supernatant was adjusted to 10.5,during which the protein was precipitated again. OPA was used as derivatization agent to be mixed with extraction liquid （1：1, v：v）,which had been diluted, and then total fluorescence intensity （FIA） was determined at Ex 325nm and Em 400nm after reacting for 1-2min in the dark. Meanwhile,FIB emitted by impurity was determined after using NEM to screen all the compounds with sulfydryl. FI emitted by GSH was known by deducting FIB from FIA,then GSH was determined indirectly. Its fluorescence intensity was linear to GSH concentration in the range of 0.125-1.250mg/L,R2=0.9963,regression equation was Y=57.012X. The GSH in three samples was determined with RSD ranged from 3.83%-5.25% as well as average recovery ranged from 91.37%-102.20%. The poor specificity of fluorescence method was made up by the method of indirect fluorescence with higher precision which could be applied to the determination of GSH in marine products.