Determination of reduced glutathione, cystein and total thiols in pine pollen powder by in situ derivatization

Glutathione (GSH), cystein (CySH) and total thiols in pine pollen powder were determined by spectrometric, fluorometric and high performance liquid chromatographic (HPLC) assays utilizing 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) and o-phthaldehyde (OPA) as derivatizing agents. The pollen samples were extracted with phosphate buffer solutions (PBS) containing excess derivatizing agents concomitant with ultrasonic cell rupture. In this way, thiol groups in the samples could be effectively blocked by in situ formation of relatively stable derivatizing products just after the pollen cell ruptured, thereby effectively preventing thiol groups from oxidation during sample preparation and making it possible to perform sensitive and accurate detections. Using a precolumn derivatization method, DTNB, GSH and CySH, as well as other thiol compounds can be determined or estimated by the HPLC method. Total thiols in the samples were determined by the spectrometric method using DTNB as the derivatizing agent. GSH was also selectively determined by the fluorometric method using OPA as the derivatizing agent. The detection results indicated that abundant thiol compounds, such as GSH, CySH and thiol protein, existed in pine pollen powder with measured amounts of 0.50-0.53 μmol∙g− 1 of GSH, 0.41 μmol∙g− 1 of CySH and 4.15 μmol∙g− 1 of total thiols.