An HPTLC method was developed and validated for simultaneous determination of stevioside (STE) and rebaudioside-A (REB-A) in Stevia rebaudiana leaves. The HPTLC separation was performed on precoated silica gel 60F254 HPTLC plates with the mobile phase ethyl acetate-ethanol-acetone-water (15 + 3 + 6 + 6, v/v/v/v). The densitometric quantification of steviol glycosides was carried out at Amax 580 nm in the reflection-absorption mode after spraying with anisaldehyde-sulfuric acid reagent. Rf values were 0.34 and 0.28 for STE and REB-A, respectively. The content of STE and REB-A in leaf extract was found to be 6.94 and 6.35%, respectively. The method was validated in terms of precision, accuracy, specificity, and robustness. The method can be useful for routine analysis of the sweeteners.
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