Structural feature-based comprehensive characterization of diterpenoid alkaloids of fermented Aconitum carmichaelii...

The identification of metabolites of Traditional Chinese Medicine (TCM) in vivo is challenging. The complexity of TCM components and matrix interference make it difficult to selectively identify metabolites in vivo. Fermented Aconitum carmichaelii Debx (AC) is a component of some important TCM preparations (such as Huafeng dan), but its composition and metabolites in vivo are still unclear. This study established a comprehensive characterization method based on ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry to identify diterpene alkaloids in rat plasma, urine and brain tissue after oral administration of fermented AC. Leveraging the structural features of diterpene alkaloids, the developed method integrates mass defect filtering, ring double bonds, nitrogen rule filtering, and feature mass fragment filtering to efficiently screen and identify diterpene alkaloids and their metabolites in biological samples with high selectivity. A total of 253 diterpene alkaloids were detected in rats, including 118 prototype compounds and 135 metabolites. The main biotransformation pathways of diterpene alkaloids in rats include ester hydrolysis, demethylation, oxidation, hydroxylation, and dehydration. This study provides a highly selective and high-coverage analytical strategy for identifying active ingredients that may play a pharmacological role in vivo.