Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols

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% 0.1 M acetate buffer at pH 4.0. 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 = 6.3 μM, CysGly = 0.8 μM, Hcys = 0.8 μM, and GSH = 1.6 μM).