IMPROVED RP-HPLC METHOD FOR QUANTITATIVE ESTIMATION OF STEVIOSIDE IN STEVIA REBAUDIANA BERTONI BURM
An RP-HPLC method with UV array detection was established for the determination of stevioside, an extract of herbal S. rebaudiana plant. The stevioside was separated using isocratic solvent system consisting of methanol and 0.1% orthophosphoric acid (v/v) in water (70:30) at flow rate of 1.0 ml/min and the detection wavelength of 219 nm. The method was validated for linearity, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ). The linearity of the proposed method was obtained in the range of 5.0-75 Î¼g/ml with regression coefficient of 0.9999. Intraday and interday precision studies showed the relative standard deviation less than 2.5%. The accuracy of the proposed method was determined by a recovery study conducted at 3 different levels. The average recovery was 97-99%. The LOD and LOQ were 0.02 and 0.05 Âµg/ml, respectively. The content of stevioside obtained in the dried leaves powder was within the ranges of 6.83 â€“ 7.91% and 1.7 â€“ 2.9 % w/w, respectively. The proposed method is simple, sensitive, yet reproducible. It is therefore suitable for routine analysis of stevioside in S. rebaudiana Bertoni.
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