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Updated: May 20, 2026
Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro
Published on: June 28, 2024
Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and
Megan L Steele1, Lezanne Ooi, Gerald Münch
1Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia.
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).
