Analysis of oligosaccharide sequences of trace Caulophyllum robustum saponins by direct infusion multiple-stage tandem mass spectrometry

• Some fragmentation approaches were proposed through analysis of [M+Na]+ ions.
• Three [M+Na]+ ions could produce non-complementary [Y0α+Na]+ and [Bα+Na]+.
• Isomeric β-sugar chains Glc→3Ara and Glc→2Ara were perfectly differentiated.
• An α-oligosaccharide was identified as Glc→4Glc→6Glc→4Rha→4Glc→6Glc.
• A nonlinear β-oligosaccharide was identified as Rha→Glc→Glc (Glc)→2,3Ara.

The saponins in Caulophyllum robustum have not yet been fully characterized. Furthermore these saponins are often present in trace amounts and are structurally complex. Here, a simple direct infusion electrospray ion trap multiple-stage tandem mass spectrometry (DI-ESI-IT-MSn) method was described for the characterization of trace C. robustum saponins. Eight reference saponins from the C. robustum hairy root were investigated by DI-ESI-IT-MSn in positive ion mode. Some fragmentation approaches were proposed through analysis of the [M+Na]+ ions: (1) preferential cleavage of the C-28 ester glycosidic bond to provide complementary [Y0α+Na]+ and [Bα+Na]+ ions for bidesmosidic saponins; (2) diagnostically neutral loss of CO2 from free carboxyl groups at C-28 for monodesmosidic saponins; and (3) the ion intensity ratio between [C2β+Na]+ and [B2β+Na]+, which is sensitive to the structural differences between the two isomeric β-sugar chains (Glc→2Ara and Glc→3Ara). The DI-ESI-IT-MSn method was successfully used for the analysis of trace C. robustum saponins with [M+Na]+ ions at m/z 1745.6, 1729.5, 1583.7, 1567.7, 1421.7 and 1405.7. This article highlights the discovery and identification of complex α- and β-oligosaccharide moieties in Caulophyllum saponins by glycosidic product ions along with cross ring cleavage product ions. Five oligosaccharide moieties were unambiguously or tentatively identified as Rha→4Glc→6Glc→4Rha→4Glc→6Glc, Glc→4Glc→6Glc→4Rha→4Glc→6Glc, Rha→Glc→Glc (Glc)→2,3Ara, Glc→Glc (Glc) →2,3Ara and Glc (Glc)→2,3Ara. Accuracy of the analytical procedure was demonstrated by structural identification of two saponins isolated using 1D and 2D-NMR spectroscopy. The DI-ESI-IT-MSn method facilitates rapid discovery and analysis of trace Caulophyllum saponins and is a powerful and practical tool for structural characterization of complex oligosaccharide moieties in triterpene saponins.

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