Glycosylated platycosides: Identification by enzymatic hydrolysis and structural determination by LC–MS/MS

In this study, enzymatic hydrolysis and chemometric methods were utilized to discriminate glycosylated platycosides in the extract of Platycodi Radix by LC–MS. Laminarinase, whose enzymatic activity was evaluated using gentiobiose and laminaritriose, was a suitable enzyme to identify the glycosylated platycosides. The laminarinase produced deapi‐platycodin D and platycodin D from the isolated deapi‐platycoside E and platycoside E through the loss of two glucose units by enzymatic reaction, respectively. After hydrolyzing a crude extract by laminarinase, the reconstructed total ion chromatogram generated by a chemometric technique sorted peaks of deglycosylated platycosides easily. Structural information of the glycosylated isomers was revealed through fragment ions generated by the sodiated C₀ᵦ ion corresponding to reduced disaccharides in the positive MS⁴ spectra. Characteristic fragment ions of Glc‐(1→6)‐Glc moieties were observed through ring cleavages of ⁰,²A₀ᵦ, ⁰,³A₀ᵦ, and ⁰,⁴A₀ᵦ, whereas Glc‐(1→3)‐Glc moieties produced only ⁰,³A₀ᵦ ions. Lithium‐adducted platycosides allowed more detailed structural analysis of glycosidic bond cleavage corresponding to Y₁ᵦ and B₁ᵦ in addition to ring cleavage.