Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts

11 pages, 5 figures, 4 tables.-- PMID: 16828049 [PubMed].-- Printed version published Sep 15, 2006.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.This work was funded by projects from the Spanish Ministry of Education and Science (MEC) (REN2001-113-C01-02 and REN2002-04229-C02-01 to L. E. Hernández and AGL2004-00194 [cofinanced with FEDER] to J. Abadía). R. Rellán-Álvarez was supported by the Universidad Autónoma de Madrid through a grant of the Ph.D. Student Exchange Program and an FPI fellowship of the Spanish MEC. A. Álvarez-Fernández was supported by a “Ramón y Cajal” research contract from the Spanish MEC. Acquisition of the HPLC/MS(TOF) apparatus was cofinanced with FEDER.Peer reviewe