Institute of Human Nutrition and Food Science, Christian-Albrechts-University , Hermann Rodewald Strasse 6, 24098 Kiel, Germany.
J Agric Food Chem. 2014 Jan 15;62(2):402-8. doi: 10.1021/jf403857h. Epub 2013 Dec 30.
A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R(2)) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood.
建立了一种快速、灵敏、直接的高效液相色谱电化学检测法（HPLC-ECD），用于检测培养的肝细胞（HepG2 细胞）中的还原型谷胱甘肽（GSH）。该方法按照美国食品和药物管理局的指南进行了验证，包括线性、定量下限（LOQ）、检测下限（LOD）、精密度、准确度、回收率和 GSH 标准品及质控样品的稳定性。总分析时间为 5 分钟，GSH 的保留时间为 1.78 分钟。采用等度洗脱，以 50mM 磷酸钠（pH3.0）作为流动相，使用核壳柱进行分离。GSH 的检测响应在 0.0180μmol/L 之间呈线性，相关系数（R²）＞0.99。GSH 的 LOD 为 15fmol，日内和日间回收率在 100.7%104.6%之间。该方法还能够快速检测（4 分钟内）其他与 GSH 代谢有关的化合物，如尿酸、抗坏血酸和谷胱甘肽二硫化物。优化和验证后的 HPLC-ECD 方法成功地应用于测定经丁硫氨酸亚砜（BSO，一种 GSH 合成抑制剂）和α-硫辛酸（α-LA，一种 GSH 合成诱导剂）处理的 HepG2 细胞中的 GSH 水平。结果显示，GSH 浓度依赖性地减少与 BSO 处理，增加与 α-LA 处理。该方法也可用于组织匀浆和血液等其他生物基质中 GSH、尿酸、抗坏血酸和谷胱甘肽二硫化物的定量。
