Microwave-assisted derivatization combined with coacervative extraction for determining glutathione in biomatrix samples, followed by capillary liquid chromatography

Abstract

In this study, an ecofriendly analytical method was developed for determining glutathione (GSH) levels in biomatrix samples. 9-(bromomethyl)acridine was used for the first time as a derivatization reagent in GSH analysis. Microwave-assisted derivatization reduced the reaction time to 1 min. After derivatization, coacervative extraction was employed to extract GSH derivative from the complex biomatrix and to increase sensitivity. Because the negatively charged group of the GSH derivative was neutralized by the extracting agent Aliquat 336, aggregates formed without any coacervating agents. Furthermore, capillary liquid chromatography coupled with ultraviolet detection was applied to decrease waste generation and increase selectivity. This method successfully quantified GSH levels in various biomatrices, including erythrocytes, HaCaT cells, BALB/3T3 cells, and 3T3-L1 fibroblasts. This method only required a low sample volume (≤10 μL). A standard addition method was utilized to spike the biomatrix samples with 0–4.8 nmol GSH to construct calibration curves. The proposed method performed well, with a determination coefficient of 0.999 and relative standard deviations of less than 6.59% for the slope and the intercept, as determined by linear regression analysis. The limit of detection of GSH in the standard solution was 800 nM or 0.4 pmol. Compared to non-derivatized GSH, the proposed method for detecting derivatized GSH provides 750-fold greater sensitivity.