Pulsed-post column derivatization coupled to green liquid chromatography for the determination of glutathione and cysteine based on thioacrylates formation

In the present work, we developed a method for the determination of thiols (cysteine and glutathione) in yeast samples under the new concept of Pulsed-post column derivatization (Pulsed-PCD). For the chromatographic separation of the analytes, 100% aqueous mobile phase was used and the eluted compounds reacted on-line with the injected pulses (100 μL) of the derivatizing reagent (ethyl propiolate + Britton-Robinson buffer). Spectrophotometric detection of the derivatives was carried out at 285 nm. The Pulsed-PCD configuration, the selection of the analytical column and the pulsed-PCD reaction conditions were investigated. The method was validated for the determination of endogenous content of the analytes in dry and fresh yeasts, with LODs of 3.0 μmol L-1. The percent recovery ranged between 85.2 and 114.4% in all cases and the results were compared with a corroborative method based on classical PCD. The analytical greenness of the proposed method was evaluated using two tools; Analytical Eco-Scale and Green Analytical Procedure Index (GAPI). The greenness score of the HPLC-Pulsed-PCD method (score = 77) was compared with that of the corroborative HILIC-PCD method (score = 71) and was found to be greener in terms of the amount of chemicals used and the produced wastes.