Identification and estimation of stevioside in the commercial samples of stevia leaf and powder by HPTLC and HPLC

Abstract

Stevioside, a major sweet component of leaves of Stevia rebaudiana (Bertoni), is about 300 times sweeter than sucrose. Highly refined stevia leaf extracts and stevioside are officially used as low calorie sweeteners in many countries. The present study describes a method for rapid identification and estimation of stevioside in commercial samples using HPTLC and HPLC. Identification of stevioside in samples was done by HPTLC. The separation was achieved on a pre-coated silica gel 60F₂₅₄ plate with mobile phase; ethyl acetate: methanol: water (75:15:10 v/v/v). Densitometric scanning was perfomed at 510 nm after visualization with a solution of acetic anhydride: sulphuric acid: ethanol (1:1:10 v/v/v). The identity of the peak corresponding to stevioside was further confirmed by spectral analysis. HPLC was used for the estimation of stevioside content. The calibration curve was linear in the concentration range from 0.1 to 1 mg/ml. The detection limit for stevioside was 0.05 mg/ml (1 µg per injection). The percentage stevioside content of the samples i.e. stevia powder and stevia leaf was found to be; 8.859 % and 3.703 % respectively. The method allows rapid identification and quantification of stevioside in different samples and can be used for routine analysis of stevioside in commercial samples.