Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana

A simple reversed-phase high-performance liquid chromatographic method has been developed for the determination of the major steviol glycosides, the diterpene sweeteners derived from Stevia rebaudiana. The method is based on a water extraction step and a solid-phase extraction (SPE) clean-up. Different SPE cartridges and two HPLC columns were tested in the separation of the main steviol glycosides stevioside and rebaudioside A. A good separation was performed on a Luna HILIC analytical column with a mobile phase consisting of acetonitrile/water (85:15 v/v). The calibration curves were linear from 10 to 800 µg/mL and the recoveries for stevioside and rebaudioside A were 99 ± 4.4 and 100 ± 5.0%, respectively. The applicability of this method was demonstrated in the analysis of stevioside and rebaudioside A from Stevia plants grown in two different areas in Germany. Stevioside and rebaudioside A contents showed statistically significant differences (F- and t-test) between the two harvests. Nevertheless, the total concentrations (>12%) and the ratio of stevioside to rebaudioside A (6:4) were similar to those found in the countries in which Stevia rebaudiana originates. Based on a comparison of yields from different harvests, we discussed whether Stevia rebaudiana can be economically grown in the temperate zones of the northern European hemisphere.