Targeted Submetabolome Profiling of Bufadienolides in Venenum Bufonis Using Scheduled Diagnostic Product Ion-Pair...

Area of Science:

• Natural Product Chemistry
• Analytical Chemistry
• Pharmacology

Background:

• Venenum bufonis (VB) is a traditional medicine with significant antitumor, immunomodulatory, and analgesic properties.
• These activities are primarily attributed to its bufadienolide content.

Purpose of the Study:

• To develop and apply a novel targeted submetabolomics strategy for comprehensive bufadienolide profiling in VB.
• To enhance the discovery and characterization of bufadienolides using advanced mass spectrometry techniques.

Main Methods:

• Utilized a targeted submetabolome strategy integrating scheduled diagnostic product ion (DPI) pair filtering and substructure recognition on an LC-MS platform.
• Employed stepwise multiple ion monitoring (MIM) with dynamic exclusion for minor peak detection and automated annotation of unconjugated bufadienolides.
• Established an extended database for conjugated bufadienolides, enabling untargeted screening and characterization via MS/MS fragmentation patterns.

Main Results:

• Identified and characterized 162 bufadienolides in VB, achieving a 96% annotation rate.
• Discovered 81 potentially novel bufadienolides, including 11 new structural subclasses.
• Demonstrated the strategy's effectiveness in profiling complex natural product mixtures.

Conclusions:

• The developed targeted submetabolome strategy significantly expanded the known diversity of bufadienolides in Venenum bufonis.
• This approach advances the application of mass spectrometry in natural product discovery and characterization.