Rellán-Alvarez Rubén, Hernández Luis E, Abadía Javier, Alvarez-Fernández Ana
Plant Physiology Laboratory, Department of Biology, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain.
Anal Biochem. 2006 Sep 15;356(2):254-64. doi: 10.1016/j.ab.2006.05.032. Epub 2006 Jun 12.
A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues.
已开发出一种简单、高选择性、灵敏且可重现的液相色谱 - 电喷雾电离/质谱（飞行时间）方法，用于直接同时测定不同植物组织中的谷胱甘肽及相关化合物，如高半胱氨酸。这些化合物是参与植物细胞氧化还原稳态的低分子量抗氧化剂，目前正在努力开发方法来测定这些化合物氧化态和还原态的浓度及其比例。然而，迄今为止开发的许多方法既耗时又复杂；因此，分析物在分析过程中可能会分解，其氧化还原状态也可能会改变。我们开发的方法能够同时测定这些化合物的还原形式（谷胱甘肽[GSH]和高半胱氨酸[hGSH]）和氧化形式（谷胱甘肽二硫化物[GSSG]），并且还适用于测定抗坏血酸（ASA）和S - 亚硝基谷胱甘肽（GSNO）。使用同位素标记的GSH和ASA作为内标进行定量。所有化合物在不到6分钟内实现基峰分离，GSH的检测限为60皮摩尔，hGSH为30皮摩尔，GSSG为20皮摩尔，ASA为100皮摩尔，GSNO为30皮摩尔。保留时间和峰面积的日内重复性值分别约为0.4%和7%，而日间重复性值分别约为0.6%和9%。分析物回收率在92%至105%之间。该方法用于测定几种植物组织提取物中GSH、GSSG、hGSH和ASA的浓度。
