Quantitation of Steviol Glycosides from Stevia Species by High-Performance Liquid Chromatography

Extracts and plant material of Stevia rebaudiana, a plant native to South America, are widely used as sweeteners by indigenous populations and by the food industry. The active sweet components, steviol glycosides, all share the same steviol backbone. Steviol glycosides are generally separated by normal phase liquid chromatography using amino columns or by reverse phase chromatography using octadecyl silica columns. In this study, steviol glycosides are extracted by sonicating the plant material at 60°C in 70:30 water/acetonitrile (v/v) for 60min. Active components are separated by HPLC using a polyamine column (YMC-Pack PA, 100×3.0mm, Allentown, PA), or a mixed mode weak ion exchange/reversed phase column (Acclaim® Mixed-Mode Wax-1, 3.0×150mm, Dionex, Sunnyvale, CA) with a linear gradient of water (pH 3 adjusted with phosphoric acid) and acetonitrile. Both separations were monitored by UV detection at 210nm. Quantitation of stevioside, rebaudioside A, dulcoside A, rebaudioside B, rebaudioside C, rebaudioside D and steviolbioside was achieved using both HPLC columns. Steviol, which is not retained by the polyamine column because of its higher hydrophobicity, was separated and quantified using the mixed mode column.