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 <i>Stevia rebaudiana</i> leaf extract. Compound <b>1</b> was unambiguously elucidated as 13-[(2-<i>O</i>-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]<i>ent</i>-kaur-16-en-19-oic acid-(6-<i>O</i>-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.