Plant diterpene glycosides are essential for diverse physiological processes. Comprehensive structural characterization proved to be a challenge due to variations in glycosylation patterns, diverse aglycone structures, and the absence of comprehensive reference databases. In this study, a method for fine-scale characterization was proposed based on energy-resolved (ER) untargeted LC-MS/MS metabolomics analysis using steviol glycosides as a demonstration. Energy-dependent fragmentation patterns were unveiled by a series of model compounds. Distinct glycosylation sites were discerned by leveraging varying fragmentation energies for the precursor ions. The sugar moiety linkage at C19OOH (R1) exhibited facile and intact cleavage at low collision energies, while the sugar moiety at C13-OH (R2) demonstrated consecutive cleavage with increasing energy. Aglycone ions exhibited a higher relative intensity at NCE 50, with relative intensities ranging from 95% to 100%. Subsequently, aglycone candidates, R1 sugar composition, and R2 sugar sequence were deduced through ER-MS/MS analysis. The developed method was applied to Stevia rebaudiana leaves. A total of 91 diterpene glycosides were unambiguously identified, including 16 steviol glycosides with novel acetylglycosylation patterns. This method offers a rapid alternative for glycan analysis and the structural differentiation of isomers. The developed method enhances the understanding of diterpene glycosides in plants, providing a reliable tool for the in-depth characterization of complex metabolite profiles.
Keywords: UPLC-HRMS/MS; diterpene glycosides; energy-resolved MS/MS; glycans; structure annotation.