An HPLC-automated Derivatization for Glutathione and Related Thiols Analysis in Brassica rapa L

The high content of glucosinolates and glutathione makes the Brassicaceae an important healthy food. Thiols and especially glutathione and γ-Glu-Cys-Gly tripeptide are involved in many fundamental cellular functions such as oxidative stress protection. Although several methods for sulphur compounds analysis in biological samples are actually used, the determination of glutathione and other sulphur derivatives in plant tissues is rather problematic due to their extreme susceptibility to oxidation, which can lead to their overestimation. The aim of this work was the improvement and validation of an automated method for determination of reduced and oxidised glutathione, cysteine and γ-glutamylcysteine in plant tissues. The method consists of a fully automated pre-column derivatization of thiols based on monobromobimane reagent, a high-performance liquid chromatography derivatives separation, and a fluorimetric detection and quantification. The method was successfully applied for determination of the oxidized and reduced forms of Cys, γ-GC and GSH content in leaves, petioles, inflorescences and roots of Brassica rapa L. subsp. Sylvestris. At harvest, in freshly cut plants, the average contents of GSH/2GSSG were 840/45, 345/70 and 150/70 nmol g⁻¹ FW for the florets, leaf blades and stems, respectively; those of Cys/2Cys were 80/12, 29/12 and 24/6 nmol g-1 FW; while those of γ-GC/γ-GCCG-γ were 8.0/4.0, and 6.0/3.0, 3.0/2.0 nmol g⁻¹ FW, respectively. Such amounts were lower in low-sulphur-grown plants at harvest. The very low coefficient of variation between repeated tests (maximum 1.6%), the high recovery of internal standard (>96%) and the linear correlation coefficient of the calibration (R² > 0.99) support the efficiency of this method that allowed analysing about 50 samples/die in a totally automated manner with no operator intervention. Our results show that the reported method integrations can significantly improve thiols detection via HPLC.