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

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.