title: Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols.
abstract: The development of drugs with the ability to increase the level of the antioxidant glutathione and related metabolites has become an important research area for many age-related diseases. Here we describe a high-performance liquid chromatography (HPLC) method that uses the thiol-specific, fluorogenic reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) for the simultaneous determination of total glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), and homocysteine (Hcys) in cell culture medium. ABD-F-labeled thiols were separated using an isocratic mobile phase consisting of 14% methanol and 86% 0.1M acetate buffer at pH4.0. The method was validated for linearity, accuracy, and intra- and interday precision, and the lower and upper limits of quantitation (LLOQ and ULOQ, respectively) were determined using a Dionex RF-2000 detector set to medium sensitivity. In addition, the suitability of N-acetylcysteine (NAC) as an internal standard was evaluated by external and internal standard calibration methods. Although both calibration methods showed acceptable linearity (correlation coefficients>0.99) and intra- and interday precision (relative standard deviations=10.2 and 6.6%, respectively), the external standard calibration method performed better in terms of accuracy (recovery=93.7-125%) and also had lower LLOQ values for all analytes (Cys=6.3μM, CysGly=0.8μM, Hcys=0.8μM, and GSH=1.6μM).
authors: Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia.
authors: Megan L
authors: ML
authors: Steele
authors: Lezanne
authors: L
authors: Ooi
authors: Gerald
authors: G
authors: Münch
mesh_terms: D001253:Astrocytes / Q000166:cytology; D002138:Calibration; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004220:Disulfides / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D010084:Oxidation-Reduction; D012015:Reference Standards; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds; D005978:Glutathione