Localization of malonyl and acetyl on substituted saikosaponins according to the full‐scan mass spectra and the fragmentation of sodium‐adduct ions in the positive mode

Abstract

Rationale:Discriminating between aglycone-substituted and saccharide-substituted saikosaponins by liquid chromatography/tandem mass spectrometry (LC/MS n ) is a long-standing issue that is still to be resolved. It is necessary to characterize the two types of substituted saikosaponins taking into consideration the potential significant difference in their bioactivity. Methods:Taking the substituents malonyl and acetyl as examples, we developed a MS strategy to discriminate between the aglycone-substituted and saccharidesubstituted saikosaponins through comparing their Y 0 − nH 2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and their B ions in the MS 2 spectra of sodium-adduct molecules in the positive mode. Results:The deprotonated molecules of the aglycone-substituted saikosaponins presented similar fragmentation patterns to those of saccharide-substituted ones in the negative mode, which could not discriminate whether the substitutes were located on the aglycone or the saccharide. In contrast, the Y 0 − nH 2 O (n = 1-2) ions containing or no substituent were observed respectively in the mass fragmentation of the protonated molecules of aglycone-substituted or saccharide-substituted saikosaponins in the positive mode. In addition, the B ions containing or no substituent were observed respectively in the mass fragmentation of the sodiumadduct molecules of the saccharide-substituted or aglycone-substituted saikosaponins in the positive mode. Two aglycone-malonylated saikosaponins were reported for the first time. Conclusions:Whether the substituents were located on the aglycone or the saccharide could be determined according to the Y 0 − nH 2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and the B ions in the MS 2 spectra of sodium-adduct molecules in the positive mode. Our results have updated the mass fragmentation patterns of substituted saikosaponins, which is helpful for the quality control of pharmaceutical preparations containing saikosaponins. More importantly, this MS strategy should be able to be extended to characterize other substituted saponins of bioactive significance in future studies.

Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS

“…In the ESI – mode, the fragmentation behavior of the two epimers was similar to that of SSa/SSd, and they also produced the following fragment ions: m/z 617 ([M−Glu−H] – ), m/z 541 ([M−Glu−C 3 H 8 O 2 −H] – ), m/z 471 ([M−Glu−Fuc−H] – , corresponding to [aglycone−H] − ), and m/z 439 ([aglycone−CH 3 OH−H] − ). Compared with SSa/SSd (type I), SSb 1 /SSb 2 lost a CH 2 OH group at position C 17 and a hydroxyl (OH) group at position C 16 in the aglycone, simultaneously, and generated the m/z 423 ([aglycone−48−H] − ) fragment ion, which was consistent with the fragmentation behavior found by Liu et al ( Liu et al, 2019 ). Because SSf presented a single double bond of aglycone and did not produce a m/z 409 ([aglycone−48−H] − ) fragment ion, it can be inferred that this fragmentation pathway only occurred when two double bonds were conjugated.…”

“…As a representative example, the fragmentation pathway of 6″- O -acetyl-SSa is shown in Figure 7 , while that of 6″- O -acetyl-SSb 3 in the ESI + mode is shown in Supplementary Figure S10 . These rules and findings were also applicable to malonylated saikosaponins (the malonyl group is substituted by the saccharide chain) according to the related literature ( Liu et al, 2019 ) and also produce [aglycone-nH 2 O + H] + (n = 1 or 2 or 3) in the ESI + mode, while the fragment ions m/z 497 ([aglycone+42–H 2 O + H] + ) and m/z 479 ([aglycone+42–2H 2 O + H] + indicate that the acetyl group is substituted by the aglycone. For example, the fragmentation pathway in the ESI + mode of 23- O -acetyl-SSa (the malonyl group is substituted by the aglycone) ( Liu et al, 2019 ) is shown in Supplementary Figure S11 .…”

“…Thus, we combined the DDA and DIA (MS E ) modes to complement each other to scan and analyze parent ions and product fragment ions of saikosaponins accurately and comprehensively. The ESI – mode provides more abundant structural information of saikosaponin compounds, while the ESI + spectra of saikosaponins provide less information and compounds are subjected to breakage in the low CE scanning channel, which results in more interference with the analysis but can also document the characteristic fragment ions in ESI + and contribute to the determination of the specific position of substituent groups in acetylated/malonylated saikosaponins ( Liu et al, 2019 ). Thus, this approach was used to identify saikosaponins in the ESI – , whereas for the identified acetylated/malonylated saikosaponins in ESI – , their fragment ions in ESI + were combined to speculate on the specific position of the respective acetyl/malonyl groups.…”

Z0 ion from saikosaponins with 16 α‐OH and Y0‐H2O from saikosaponins with 16 β‐OH may underlie their different dissociation patterns of [aglycone − H2O + H]+

“…Based on the previous method reported by our group, saikosaponins c, a, b 2 , b 1 and d were detected in the MeOH extract of vinegar‐processed Radix Bupleuri , as shown in Figure . Their t R , UV absorption and MS information is summarized in Table and their accurate mass data with errors (ppm) for ions are listed in Table S1 (supporting information).…”

“…To improve the quality control of Radix Bupleuri and its pharmaceutical preparations, the characterization and identification of saikosaponins in herbs have been reported based on their fragmentation patterns using liquid chromatography/mass spectrometry (LC/MS) in combination with their UV absorption and retention time (t R ). [2][3][4][5][6][7] In these reports, the types and linkage sequences of sugars in the glycosyl part on the saikosaponins were clarified clearly by multi-stage mass spectrometry (MS n ) in the negative ion mode. However, it is the dissociation patterns in the positive ion mode which provide the direct structural information aglycones of SSa and SSd by comparing their mass spectra based on our previous work.…”