Analysis of sweet diterpene glycosides from Stevia rebaudiana: Improved HPLC method

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevi a rebaudiana before processing; in a selective sampling program searching f or plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure de veloped involves two steps: solvent extraction followed by an isocratic HPL C analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degreesC water bath. After the extract was cooled, it was fi ltered and analyzed by HPLC using an NH2 column (250 x 4.6 mm) and a mixtur e of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with ac etic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantit ation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevio side and rebaudioside A. Working under these conditions there were no obser ved interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having lit tle loss of precision [coefficient of variation (CV%) between 1.8% and 3.0% ] and recovery [between 98.5% and 100.5%]. The method was applied to 30 sam ples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevi oside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudio side A content ranged between 1.62 and 7.27% (weight).