Publication details
Determination of thiol compounds using high-performance liquid chromatography with electrochemical detection.
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Vitamins 2004 Targeted Nutritional Therapy. |
| MU Faculty or unit | |
| Citation | |
| Field | Analytic chemistry |
| Keywords | GSH; HPLC-ED; PC |
| Description | Thiol compounds such as cystein (Cys), reduced (GSH) and oxidized (GSSG) glutathione and phytochelatins (PCs) play an important role in heavy metal detoxification in plants. These thiols are biological active compounds whose function is elimination of oxidative stress in plant cells. The aim of our work was to optimise sensitive and rapid method of high-performance liquid chromatography coupled with electrochemical detector (HPLC-ED) for determination of abovementioned thiol compounds in biological samples. An HPLC-ED system was consisted of two delivery pumps, chromatographic column Zorbax XDB (150.0 x 2.1 mm, 5 um particle size) and CoulArray detector containing eight working electrodes made from porous graphite. The most suitable isocratic mobile phase for the determination and separation of Cys, GSH, GSSG and PC2 consisted of methanol (MET) and trifluoroacetic acid (TFA). In addition, the influence of concentrations of TFA and ratio of MET:TFA on chromatographic separation and detection of the thiol compounds were studied. The mobile phase consisting from methanol and 0.05% TFA in ratio (97:3) (%; v/v) was found the most suitable for the thiol compounds determination. Optimal flow rate of the mobile phase was 0.18 ml.min-1 and the column temperature was 35 C. For the selection of the most effective working electrodes potentials, hydrodynamic voltamograms of all studied compounds was obtained. From the results we selected two detection potentials 700 mV (for the determination of GSSG and PC2) and 680 mV (Cys and GSH). HPLC-ED full scan of studied thiols. Moreover, we were able to determine femtomole levels of studied compounds by optimised HPLC-ED method. The optimised HPLC-ED method was applied to the study of influence of different cadmium concentrations (0, 10 uM and 100 uM Cd concentration) on content of Cys, GSH, GSSG and PC2 in maize (Zea mays L.) kernels. According to the increasing time of Cd treatment, content of GSH, GSSG and PC2 in maize kernels increased but content of Cys decreased. Decreasing concentration of Cys probably relates with the increasing GSH and phytochelatins synthesis. |