METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF STEVIOSIDE, REBAUDIOSIDE-A, REBAUDIOSIDE C AND DULCOSIDE A CONTAINED IN STEVIA REBAUDIANA BERTONI USING HPLC-ELSD

Objective: To develop and validate a selective HPLC-ELSD method for determination of steviol glycosides contained in Stevia rebaudiana, mainly stevioside, rebauside A, rebaudioside C, and dulcoside A. Methods: The chromatographic separation of stevioside, rebaudioside A, rebaudioside C, and dulcoside A was achieved using Phenomenex Luna column 250 mm x 4.6 mm i.d. in isocratic system mode with a mobile phase of acetonitrile-water (35: 65). The temperature of nebulization and evaporization of the ELS detector was set at 50 oC and 70 oC, respectively. Results: The good separation of stevioside, rebaudioside A, rebaudioside C, and dulcoside A was obtained, yielding the resolution of all the analytes more than 1.5. All the validation parameters like specificity, linearity, range, accuracy and precision met the acceptance criteria according to ICH guidelines. Conclusion: The proposed HPLC-ELSD method is simple and sensitive for the simultaneously detection and determination of stevioside, rebaudioside A, rebaudioside C and dulcoside A contained in Stevia rebaudiana. The method was successfully applied for the determination of the samples product of Stevia rebaudiana. Keywords: Stevioside, Rebaudioside A, Rebaudioside C, Dulcoside A, HPLC-ELSD