Jayatilleke E, Shaw S
Department of Medicine, Mount Sinai School of Medicine, Bronx, New York 10468.
Anal Biochem. 1993 Nov 1;214(2):452-7. doi: 10.1006/abio.1993.1522.
A high-performance liquid chromatographic method was developed for the measurement of oxidized and reduced glutathione in biological samples. The method allowed the separation of glutathione (oxidized and reduced) from related thiols (homocysteine, cysteine, cystine, methionine) and other intermediates of glutathione pathways without derivatization or enzymatic reduction. Quantitation was achieved with uv detection. Biological samples were prepared by rapid homogenization in iced KCl followed immediately by deproteinization and acidification with sulfosalicylic acid. Samples were eluted isocratically at 1 ml/min using 0.0025 M sodium phosphate buffer, pH 3.50, containing 0.005 M tetrabutylammonium phosphate (Waters, Milford, MA) and 13% methanol and analyzed on a 30-cm x 3.9-mm C-18 mu Bondapak column and detected with a uv detector at 190 nm. The determination of nanomole levels of glutathione and glutathione disulfide and their separation from other thiols are described.
开发了一种高效液相色谱法用于测定生物样品中的氧化型和还原型谷胱甘肽。该方法无需衍生化或酶促还原，就能将谷胱甘肽（氧化型和还原型）与相关硫醇（同型半胱氨酸、半胱氨酸、胱氨酸、蛋氨酸）以及谷胱甘肽途径的其他中间体分离。通过紫外检测进行定量。生物样品通过在冰KCl中快速匀浆制备，随后立即用磺基水杨酸进行脱蛋白和酸化。使用含0.005 M磷酸四丁铵（沃特世公司，马萨诸塞州米尔福德）和13%甲醇的0.0025 M磷酸钠缓冲液（pH 3.50），以1 ml/min的等度洗脱速率对样品进行洗脱，在30 cm×3.9 mm的C-18 μ Bondapak柱上进行分析，并用紫外检测器在190 nm波长处进行检测。描述了谷胱甘肽和谷胱甘肽二硫化物纳摩尔水平的测定及其与其他硫醇的分离。
