ABSTRACT
RATIONALE: Discriminating between aglycone-substituted and saccharide-substituted saikosaponins by liquid chromatography/tandem mass spectrometry (LC/MSn ) 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 saccharide-substituted saikosaponins through comparing their Y0 - nH2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and their B ions in the MS2 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 Y0 - nH2 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 sodium-adduct 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 Y0 - nH2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and the B ions in the MS2 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.
