改良的高效液相色谱法测定酵母细胞中谷胱甘肽含量

A Modified Method for Determining Glutathione of Extracts from Yeast

针对以往高效液相色谱法测定谷胱甘肽保留时间过短、易干扰的问题,探索了缓冲盐pH值对还原型和氧化型谷胱甘肽保留时间的影响,建立了一种改良的酵母细胞中谷胱甘肽高效液相色谱分析方法.采用HPLC对酵母粗提物分析,色谱柱为Venusil XBP C18(5μm×10×250mm),甲醇/水为洗脱体系(其中水相中添加庚烷磺酸钠、磷酸-二氢钾,并用磷酸调节pH值),流速1mL/min,柱温25℃,检测波长为203nm.结果表明,还原型谷胱甘肽在0.25~4.00mg/mL质量浓度范围内线性关系良好(R=0.999 7),平均回收率100.49%,RSD为0.52%;氧化型谷胱甘肽在0.25~4.00mg/mL质量浓度范围内线性关系良好(R=0.999 5),平均回收率97.49%,RSD为1.2%.本方法快速简便、灵敏度高,特别是还原型谷胱甘肽保留时间延长、实现与杂峰有效分离.

A modified method for determining glutathione of extracts from yeast using high performance liquid chromatography（HPLC）was established to increase retention time and avoid overlap with other metabolites through adjusting the pH value of water phase of elution system.The extracts of yeast was analyzed by HPLC on a Venusil XBP C-18 column（5μm×10×250 mm）using methanol-water（adding sodium heptanesulfonate,potassium dihydrogen phosphate to the water phase and adjusting the pH with phosphoric acid）as the mobile phase at a flow rate of 1.0 mL/min.The detection wavelength was 203 nm and the temperauture was 25℃.The results show that the reduced glutathione exhibits a good linear relationship in the range of 0.25-4.00 mg/mL;the correlation coefficient is 0.9997;the average recovery is 100.49% and the relative standard deviation（RSD）is 0.52%.Moreover,the oxidized glutathione also exhibits a good linear relationship in the range of 0.25-4.00 mg/mL;the correlation coefficient is 0.9995,the average recovery is 97.49%;and the RSD is 1.2%.The proposed method has the characteristics of rapidity,simpleness and accurateness,especially lengthened retention time and non-interfering separation of glutathione.