Multiple-Stage Tandem Mass Spectrometry for Structural Characterization of Saponins

Abstract

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 developed 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 monosaccharide residue facilitated the determination of the substitution pattern, due to the induction of ring-cross fragmentation. Deuteriomethylation 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 interpretation. The applied method is attractive as a pre-NMR analysis, especially because it resulted rapidly in an overall idea of the structure even when starting from scratch. The multiple-stage tandem approach enabled structural determination of saponins to a more detailed level than achievable with current single-stage tandem mass spectrometry.

Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa

“…To determine whether this difference is hardware-specific, we acquired additional MS 2 spectra of Compound 19 on the LCQ Duo ITMS. The [M ϩ Na] ϩ ions generated undergo fragmentation similar to results reported for triterpene and steroid saponins [21,24] but do not show any product ion series specific for aglycones and the ϩ ion, we think this is due to a different waveform design used in the Agilent LC/MSD XCTPlus ITMS to ramp the amplitude of the CID fragmentation voltage. As a consequence, stable product ions or product ions with low-energy fragmentation pathways can also be fragmented.…”

“…The resulting signals at [M ϩ H Ϫ 162] ϩ correspond to the neutral loss of a hexose residue (Hex; Figure 4; m/z 811, 957, 767, 633 for B, G, I, K, respectively). Quantum chemical calculations using the Gaussian 03 suite of programs [32] show that the ␥-H-rearrangement [21] followed by bond breaking between the C-28 carboxylic acid and the monosaccharide is energetically more favored (⌬E ϭ 2.7 kcal/mol) compared to fragmentation at the C-3 bound saccharide. Furthermore, the carboxyl group is the place of initial ionization and therefore the starting point for fragmentation.…”

“…The coupling of ESI-MS n with nano-HPLC (nLC) allows studying saponin molecular species, aglycones, the saccharide sequence, mod-ification, branching [17][18][19][20][21][22][23][24][25][26][27][28][29][30], and deals with relative low amounts of sample, respectively. Due to the short analysis time (LC)-MS n analysis was recommended prior the common way of natural product identification [20].…”

Structural analysis of saponins from medicinal herbs using electrospray ionization tandem mass spectrometry

“…More recently, electrospray ionization (ESI) has become one of the most effective analytical tools for the structural characterization of a variety of polar and thermally labile molecules, e.g., polypeptides, carbohydrates and natural glycosides [19 -22]. Recent studies have reported the use of ESI-MS for determination of saponins with higher sensitivity and better reproducibility than the other types of ionization [23][24][25][26][27][28]. Moreover multistage mass spectrometry has great advantages for characterization of compounds by providing structural information [24,25,[27][28][29][30][31].…”

Structural Determination of Saponins Extracted from Starfish by Fast Atom Bombardment Collision-Induced Dissociation Mass Spectrometry

“…1,2 Some saponins have properties of hemolysis, bitterness, complex formation with cholesterol, and fish poisons. [3][4][5][6] Saponins consist of sugar moieties linked to aglycone, which are classified into triterpenoid and steroid types, sugars are linked to the aglycone generally through an ester or an ether linkage at one or two sites. [7][8][9] Saponins are found predominantly in the secondary metabolites of starfish and exist as complex mixtures in minute amounts.…”