Oe T, Ohyagi T, Naganuma A
Department of Molecular and Biochemical Toxicology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
J Chromatogr B Biomed Sci Appl. 1998 Apr 24;708(1-2):285-9. doi: 10.1016/s0378-4347(97)00676-2.
A method was developed for the simultaneous determination of gamma-glutamylglutathione (gamma-GluGSH) and other low-molecular-mass thiol compounds (cysteine, cysteamine, homocysteine, cysteinylglycine, gamma-glutamylcysteine, glutathione and N-acetylcysteine) using high-performance liquid chromatography combined with precolumn fluorescence labeling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). These SBD-labeled thiol compounds were separated within 35 min on a Cosmosil 5C-18AR column with isocratic elution using 75 mM sodium citrate buffer (pH 2.90)-methanol (98:2) and detected fluorimetrically (ex. 386 nm, em. 516 nm). The calibration graphs using 2-mercaptoethanol as an internal standard showed good linearity in the range from 20 pmol to 10 nmol for all thiol compounds examined. The application of this method for the quantitative determination of thiol compounds in the urine from gamma-glutamyl transpeptidase-deficient mice was also demonstrated. This method is sufficiently simple, rapid and sensitive for the determination of gamma-GluGSH and other low-molecular-mass thiol compounds in biological samples.
建立了一种同时测定γ-谷氨酰谷胱甘肽（γ-GluGSH）和其他低分子量硫醇化合物（半胱氨酸、半胱胺、高半胱氨酸、半胱氨酰甘氨酸、γ-谷氨酰半胱氨酸、谷胱甘肽和N-乙酰半胱氨酸）的方法，该方法采用高效液相色谱结合7-氟苯并-2-恶唑-1,3-二氮杂环丁烷-4-磺酸盐（SBD-F）进行柱前荧光标记。这些SBD标记的硫醇化合物在Cosmosil 5C-18AR柱上，使用75 mM柠檬酸钠缓冲液（pH 2.90）-甲醇（98:2）等度洗脱，在35分钟内分离，并通过荧光检测（激发波长386 nm，发射波长516 nm）。以2-巯基乙醇为内标绘制的校准曲线表明，所有检测的硫醇化合物在20 pmol至10 nmol范围内具有良好的线性。还证明了该方法在定量测定γ-谷氨酰转肽酶缺陷小鼠尿液中硫醇化合物的应用。该方法对于生物样品中γ-GluGSH和其他低分子量硫醇化合物的测定足够简单、快速且灵敏。
