Determination of glutathione and glutathione disulfide using zone fluidics and fluorimetric detection

Abstract In the present study we report the simultaneous determination of glutathione (GSH) and glutathione disulfide (GSSG) by an automated flow method based on the concept of zone fluidics. GSH is quantified selectively in a first run by reaction with o-phthalaldehyde at a mildly basic pH = 8, without interference from GSSG. The latter was also found to react with o-phthalaldehyde but in highly basic medium (0.2 mol L−1 NaOH) and was determined after masking of GSH with N-ethyl-maleimide. Detection was carried out fluorimetrically at 340/425 nm. The flow procedure was optimized and validated, paying special attention to its selectivity. The LOD was 60 nmol L−1 for GSH and for 53 nmol L−1 for GSSG, while the within-day and day-to-day precisions were better than 1.5% and 3.7% respectively. Real yeast samples were successfully analyzed without matrix effect (−2.0 to +4.1%) and with percent recoveries being in the range of 87.0 and 103.3%.