Development of Validation RP-HPLC Method for Estimation of Stevioside and Rebaudioside a In Leaves of Stevia Rebaudiana

Abstract

Stevia rebaudiana is a plant species in the stevia genus from the family Asteraceae. It is a small seasonal plant which grows to a height of 30-60 cm. It has elongated leaves that grow along the stems and line up against each other. The leaves of the stevia plant and extracts made from them have a sweet taste. Steviol glycosides are the constituents of stevia leaves responsible for the sweet taste. It is also called the herb stevia and has been cultivated for hundreds of years and leaves are 10 to15 times sweet as sugar while its extracts are 200 to 300 times sweet as sugar. Stevia rebaudiana bioactives were determined using an RP-HPLC method that was precise, selective, and accurate. Stevioside and Rebaudioside A was in solid form and determination was simultaneously done using HPLC. The C18 column size (250 * 4.6 mm, 5 μm) was utilized for the chromatographic process, and the mobile phase was made up of acetonitrile and sodium hydrogen phosphate buffer at a 35:65% V/V ratio. The detecting wavelength was 210 nm, in hydroalcoholic extract Stevioside and Rebaudioside A was retention times of 3.870 and 4.787 minutes, respectively and 3.953 and 4.630 minutes in water extract respectively. For stevioside standard retention time was 5.887 minutes and for rebaudioside A retention time was 5.520 minutes.