Ethyl propiolate as a post-column derivatization reagent for thiols: Development of a green liquid chromatographic method for the determination of glutathione in vegetables Original Research Article

The present study reports the development, validation and application of a new green liquid chromatographic method for the determination of glutathione (GSH) in vegetable samples. In this work we introduce—for the first time—ethyl propiolate (EP) as an advantageous post-column derivatization reagent for thiolic compounds. GSH (tR = 6.60 min) and N-acetylcysteine (NAC, internal standard) (tR = 11.80 min) were separated efficiently from matrix endogenous compounds by using a 100% aqueous mobile phase (0.1%, v/v CH3COOH in 1 mmol L−1 EDTA, QV = 0.5 mL min−1) and a Prevail® reversed phase column that offers the advantage of stable packing material in aqueous mobile phases. The parameters of the post-column reaction (pH, amount concentration of the reagent, flow rates, length of the reaction coil and temperature) were studied. The linear determination range for GSH was 1–200 μmol L−1 and the LOD was 0.1 μmol L−1 (S/N = 3). Total endogenous GSH was determined in broccoli, potato, asparagus and Brussels sprouts using the standards addition approach. The accuracy was evaluated by both recovery experiments (R = 91–110%) and comparison to an o-phthalaldehyde/glycine corroborative post-column derivatization fluorimetric method.