Determination of selenium species in plant leaves by HPLC–UV–HG-AFS

The purpose of this work was the development of a method for the determination of Se compounds in leaves of plants. Water-soluble Se compounds were extracted from samples by water. Enzymatic hydrolysis with the non-specific enzyme protease XIV was used for the release of Se compounds bound to proteins. Separation of Se species was made by ion exchange chromatography, using an anion exchange column for SeIV, SeVI and selenomethionine (SeMet), and a cation exchange column for selenomethylselenocysteine (SeMeSeCys) and selenocystine (SeCys2). Columns were connected “on line” to a hydride generation atomic fluorescence spectrometer (HG-AFS) using a UV lamp between the separation and detection system. The repeatability of the results obtained by the developed method was under 15% (R.S.D.) for all Se species; the detection limit was 2–10 ng Se/g of supernatant. The accuracy was checked by comparison with some literature data for reference materials since there were no suitable certified reference materials available. The method was used for the determination of Se compounds in chicory (Cichorium intybus L.) leaves from plants which were cultivated aeroponically with elevated concentrations of Na2SeO4 for different periods. Se accumulated efficiently in chicory leaves; up to 480 μg/g after 41 days of exposure, mostly (64%) as SeVI, i.e. in the form of Se added. Beside inorganic Se, in the extracts from enzyme hydrolysis we also found SeMet (4.2–8.4%) and SeMeSeCys (