Retention-Guided LC-MS/MS Enables Discovery and Structural Assignment of Trace Steviol Glycosides from in Vitro...

Stevia rebaudiana produces diverse steviol glycosides (SVGs), but many positional isomers remain difficult to distinguish by MS/MS alone because of similar fragmentation patterns and low abundance. Here, we established a retention-guided LC-MS/MS workflow for discovery and structural characterization of trace SVGs generated by in vitro glycosylation using crude enzyme extracts from Stevia leaves. Reversed-phase retention trends associated with sugar identity and linkage patterns enabled chromatographic discrimination of isomeric SVGs, including rebaudiosides D and I, which exhibit indistinguishable MS/MS and MS3 spectra. Enzymatic reactions using UDP-13C6-glucose and UDP-xylose generated rare sugar-chain elongated derivatives, including rebaudioside FX1 and the previously unreported compound Reb FX0 carrying Glcβ(1-2)Glcβ1- at C-13 and Xylβ(1-2)Glcβ1- at C-19. Structural assignments were confirmed by comparison with semisynthetic standards. This integrated analytical-biochemical approach provides a practical platform for discovery-oriented characterization of trace SVGs and supports compositional analysis of stevia-derived ingredients and bioconversion products relevant to food applications.