Improvement method of fast and isocratic RP-HPLC analysis of major diterpene glycoside from Stevia rebaudiana leaves

Stevioside and rebaudioside A are major diterpene glycoside from Stevia rebaudiana. These compounds have similar structure and make both compounds have close polarity. The aim of this study was to improve isocratic RP-HPLC analysis to achieve a complete separation of stevioside and rebaudioside A in baseline peak. The method improved was conducted on Eurosphere C-18 (250 × 4.6 mm, 5 µm) as the stationary phase. Mobile phase was made by a mixture of water-methanol (90: 10, v / v, pH = 3.0); acetonitrile, and trifluoroacetic acid (TFA) in the ratio 65: 35: 0.01 (v/v/v). Flow rate of mobile phase was 0.6 mL/min. The detection was performed in UV-210 nm. The improved method has complete separation by R > 2.00 in baseline peak. The linearity of the standard curve was obtained with a correlation coefficient of 0.9998 for rebaudioside A and 0.9994 for stevioside. The percent recovery was determined by standard addition method with a mean recovery of 100.82% for rebaudioside A and 101.04% for stevioside. The relative standard deviation (RSD) of intraday precision varies from 0.34 – 1.79% for rebaudioside A and 0.31 – 1.81% for stevioside, and interday precision showed RSD 1.47% for rebaudioside A and 0.37% for stevioside. While the sensitivity reported in the limit of detection (LOD) value of method improvement was 1.00 µg/mL for both rebaudioside A and stevioside. The limit of quantitation (LOQ) of the method was 3.00 µg/mL for both respected compounds. The small changes of the mobile phase composition, the mobile phase is reused after 1 week, flow rate, and column temperature did not alter the method performance significantly. The reliable and robust of the developed and validated method was applied to both simple and complex matrices of samples including S. rebaudiana leaves for major diterpene glycoside analysis.