MULTIPLE-STAGE TANDEM MASS-SPECTROMETRY FOR STRUCTURAL CHARACTERIZATION OF SAPONINS

Nanoelectrospray ion trap multiple-stage tandem mass spectrometry was applied to characterize saponins present in HPLC fractions from Quil A , a commercially available bark extract. An analytical strategy was de veloped based on recognition of carbohydrate sequence ions as well as glycosidic ring-cross ions formed by gamma-hydrogen rearrangements and successive retro-Diels-Alder fragmentations. These ions could be used for the determination of several glycosidic linkages, ring sizes, and positions of acyl groups. The presence of an acyl group on a monosacc haride residue facilitated the determination of the substitution patte rn, due to the induction of ring-cross fragmentation. Deuteriomethylat ion resulted in a more extended set of ring-cross ions, thus allowing determination of additional glycosidic linkages. An analysis typically consumed 200 ng of sample and a total of 1-4 h for measurement and in terpretation. The applied method is attractive as a pre-NMR analysis, especially because it resulted rapidly in an overall idea of the struc ture even when starting from scratch. The multiple-stage tandem approa ch enabled structural determination of saponins to a more detailed lev el than achievable with current single-stage tandem mass spectrometry.