Determination of cysteine and glutathione in cucumber leaves by HPLC with UV detection
Cysteine (Cys) and glutathione (GSH) have been identified as key players in redox-based defense and signaling under stress in plants. Here, we describe a new high-performance liquid chromatography assay for the determination of four different forms of Cys and GSH, based on reduction of disulfide bonds with tris(2-carboxyethyl)phosphine followed by derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate and UV detection. The presented method allows facile determination of total, reduced, free and protein bound aminothiols in cucumber leaves. Via this simple yet efficient method very good precision as well as accuracy were obtained. Linearity in detector response was observed over the range of 5–100 μmol L−1 for GSH and 1–20 μmol L−1 for Cys. The LOQs for GSH and Cys were 5 μmol L−1 and 1 μmol L−1 homogenate of cucumber leaf, respectively.