A silica gel orthogonal high-performance liquid chromatography method for the analyses of steviol glycosides: novel tetra-glucopyranosyl steviol

Abstract

A silica gel orthogonal method using acetonitrile: water was developed for the analyses of fractions rich in very polar steviol glycosides and resolve regions of co-elution of these compounds in reversed-phase. Additionally, we also used this normal phase analytical method to scale up the purification process of steviol glycosides. Using these approaches, one novel minor tetra-glucopyranosyl diterpene glycosides together with three known compounds were purified from a commercial Stevia rebaudiana leaf extract. Compound 1 was unambiguously elucidated as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (rebaudioside Y) based on high-performance liquid chromatography retention times, tandem mass spectrometry dissociation pattern and 1D and 2D NMR experiments. Known compounds were isolated in gram quantities and identified as rebaudioside D, E and M.

Keywords:

• Stevia rebaudiana
• steviol glycosides
• silica gel orthogonal HPLC method
• rebaudioside E isomer
• rebaudioside Y

Acknowledgements

The authors thank Dr. Bharathi Avula and Dr. Francisco Leon from University of Mississippi for recording the HRESI–MS/MS analysis and for kindly allowing us to record specific rotation and melting points of the isolated compounds. R. Carvalho thanks to CAPES, for grants and scholarship through the Brazil Scientific Mobility Program (156/2013).