Pulsed-post column derivatization coupled to green liquid chromatography for the determination of glutathione and cysteine based on thioacrylates formation
Apostolia Tsiasioti, Paraskevas D. Tzanavaras
Abstract
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.