Validated high-performance thin-layer chromatography method for steviol glycosides in Stevia rebaudiana

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate–ethanol–water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at λ = 510 nm in reflection–absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160–960 ng/spot for steviolbioside, 1–6 μg/spot for stevioside and 0.5–3 μg/spot for rebaudioside-A with good correlation coefficients (0.998–0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to monitor the commercial production of stevioside and its allied molecules during different stages of its processing.