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 mu L) 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 mu mol 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.

Automated summary

In this study, a new method called Pulsed-post column derivatization (Pulsed-PCD) was developed for the determination of thiols in yeast samples. The method involved using 100% aqueous mobile phase for chromatographic separation and on-line reaction with a derivatizing reagent. Spectrophotometric detection was carried out and the method was validated for endogenous content determination. The proposed method was found to be greener in terms of chemical usage and waste production compared to a corroborative method based on classical PCD.