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

Area of Science:

• Analytical Chemistry
• Phytochemistry

Background:

• Stevia rebaudiana leaves are a natural source of sweet steviol glycosides.
• Accurate quantification of steviol glycosides is crucial for quality control and commercial processing.

Purpose of the Study:

• To develop and validate a high-performance thin-layer chromatographic (HPTLC) method for simultaneous quantification of key steviol glycosides.
• To establish a reliable method for quality assessment of Stevia rebaudiana leaf extracts.

Main Methods:

• Development and validation of an HPTLC method following International Conferences on Harmonization (ICH) guidelines.
• Utilized ethyl acetate-ethanol-water mobile phase on silica gel 60 F254 plates for separation.
• Densitometric quantification at 510 nm after spraying with acetic anhydride:sulphuric acid:ethanol reagent.

Main Results:

• Achieved linear calibration curves for steviolbioside (160-960 ng/spot), stevioside (1-6 µg/spot), and rebaudioside-A (0.5-3 µg/spot) with high correlation coefficients (0.998-0.999).
• Demonstrated method reproducibility across Stevia rebaudiana leaf samples from ten different locations.
• The method proved effective for simultaneous quantification of steviolbioside, stevioside, and rebaudioside-A.

Conclusions:

• The validated HPTLC method provides accurate and reproducible simultaneous quantification of major steviol glycosides.
• This method serves as a valuable quality control tool for monitoring commercial production of stevioside and related compounds.
• The developed HPTLC technique supports consistent quality assessment of Stevia rebaudiana leaf products.