全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量

摘要

建立一种测定贝类肌肉中谷胱甘肽和游离氨基酸的全自动氨基酸分析仪法,以4种常见贝类为研究对象,采用0.02 mol/L盐酸溶液作为提取剂,体积分数5％磺基水杨酸溶液为蛋白沉淀剂,同时优化了缓冲溶液的组成及洗脱程序,并对该方法的线性范围、检出限、精密度以及回收率进行测定.结果表明:贝类肌肉中的谷胱甘肽和游离氨基酸有着较好的分离效果,线性关系良好,相关系数为0.999 1～0.999 9,检出限(RSN=3)为0.07～0.27 μmol/L,加标平均回收率为86.40％～102.42％,日内和日间相对标准偏差分别为0.31％～0.73％和1.14％～2.60％.4种贝类肌肉组织中的游离氨基酸含量比较丰富,总游离氨基酸含量分别为1 396.39、1 016.04、911.15、287.01mg/100 g,文蛤＞青蛤＞缢蛏＞牡蛎,主要的游离氨基酸为牛磺酸(Tau)、谷氨酸(Glu)、丙氨酸(Ala)、精氨酸(Arg);谷胱甘肽含量分别为103.20、82.53、61.77、33.37 mg/100 g,青蛤＞文蛤＞缢蛏＞牡蛎.该方法适用于贝类肌肉中谷胱甘肽和游离氨基酸含量的测定与分析.%An analytical method was established for the simultaneous determination of glutathione and free amino acids in shellfish using a fully automatic amino acid analyzer.The extraction of the analytes was carried out using 0.02 mol/L hydrochloric acid solution as the extraction solvent followed by protein precipitation with 5％ sulfonic acid solution.The buffer composition and the elution procedure were optimized at the same time.The linear range,limit of detection (LOD),precision and recovery rate of the method were determined.The results showed that good separation of glutathione and free amino acids in shellfish was achieved by this method with a good linear relationship.The correlation coefficients of the calibration curves were between 0.999 1 and 0.999 9.The minimum LODs at a signal to noise ratio (RsN) of 3 ranged from 0.07 to 0.27 μmol/L.The average recovery rates of spiked samples were between 86.40％ and 102.42％.The relative standard deviations (RSDs) of intra-day and inter-day assays were 0.31％-).73％ and 1.14％-2.60％,respectively.Free amino acids were abundant in muscle tissue of four shellfish species,and their total free amino acid contents in decreasing order were clam (1 396.39 mg/100 g) ＞ Cyclina sinensis (1 016.04 mg/100 g) ＞ razor clam (911.15 mg/100 g) ＞ oyster (287.01 mg/100 g).The main free amino acids were taurine,glutamic acid,alanine,and arginine.The glutathione contents of Cyclina sinensis,clam,razor clam and oyster were 103.20,82.53,61.77,and 33.37 mg/100 g,respectively.This method is suitable for the determination of glutathione and free amino acids in shellfish.