Poster Presentations
Day 3, June 12（Fri.） Room P (5F 501+502)
- 3P-35
Retention-Guided LC–MS/MS Enables Structural Assignment of Trace Steviol Glycosides Generated by In Vitro Glycosylation Using Crude Stevia Leaf Extracts
(1Suntory Foundation for Life Sciences, 2SBF)
oTakehiro Watanabe1, Kohki Fujikawa1, Kazunari Iwaki2, Tadayoshi Hirai2, Jun Murata1
Steviol glycosides (SVGs) are natural sweeteners found in stevia leaves and exhibit considerable structural diversity in their glycan moieties. Based on their biosynthetic pathways, additional trace-level SVGs with even greater structural diversity are likely to exist. The detection and structural characterization of such minor components require highly sensitive analytical techniques, particularly LC–MS/MS. However, C-19 side-chain glycan isomers generate identical MS/MS fragment ions, making structural discrimination by mass spectrometry alone difficult. Moreover, their extremely low abundance limits structural confirmation by NMR spectroscopy.
In this study, we established a reversed-phase LC–MS/MS workflow and constructed a systematic retention map to clarify the effects of sugar type and linkage position on hydrophobicity (rhamnose [Rha] > xylose [Xyl] > glucose [Glc]; 3-O-glycosylation prolongs retention time, whereas 6-O-glycosylation shortens it). To further verify minor components, in vitro glycosylation assays were performed using crude stevia leaf extracts. Co-incubation of SVGs with UDP-¹³C₆-glucose and UDP-xylose generated rare glucose–xylose mixed glycosides. Their structures were deduced from retention behavior and MS³ fragmentation patterns, and previously unreported compounds were confirmed by comparison with synthetically prepared authentic standards. This retention-guided LC–MS/MS strategy provides a robust approach for the structural elucidation of trace-level steviol glycosides.