title: Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC-MS/MS.
abstract: A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography-MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-(13)C(2),(15)N) was established using H(2)O(2) as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25-30 % (coverage factor=2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.
authors.affiliation: Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. christina.haberhauer@boku.ac.at
authors.forename: Christina
authors.initials: C
authors.lastname: Haberhauer-Troyer
authors.forename: Marizela
authors.initials: M
authors.lastname: Delic
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
mesh_terms: D002138:Calibration; D002247:Carbon Isotopes; D002845:Chromatography; D005978:Glutathione / Q000302:isolation & purification*; D019803:Glutathione Disulfide / Q000302:isolation & purification*; D006861:Hydrogen Peroxide / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D059625:Liquid-Liquid Extraction / Q000379:methods; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D010843:Pichia / Q000737:chemistry* / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D012974:Sodium Iodide / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D006861:Hydrogen Peroxide; D012974:Sodium Iodide; D005978:Glutathione; D019803:Glutathione Disulfide