A novel strategy for the characterization of glaucocalyxin A metabolites in vivo and in vitro by UHPLC-Q-TOF-MS based on DDA and DIA data acquisitions

Glaucocalyxin A (GLA) belongs to the natural ent-kauranoid diterpenoids family with antitumor, antifibrotic, anticoagulative, antioxidant, and anti-AD effects. In this study, ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) system was applied to observe probable metabolites of GLA in vitro and in vivo firstly. The mass data were respectively obtained by two typical acquisition methods, ‘data-dependent acquisition’ (DDA) and ‘data-independent acquisition’ (DIA) modes. The combinations can not only guarantee sensitivity but also capture more precursor ions and MS/MS spectra. Then, multiple data processing techniques were applied to hunt metabolites rapidly. As a result, 32 phase I metabolites of different structures and 6 phase II metabolites were identified, including 25, 18, 17 and 7 in rat urine, feces, bile, and plasma, respectively. Besides, under the action of rat intestinal flora (RIF), 7 metabolites were detected. In the study, the main bio-transformations were oxidation and demethylation. Conjugation with methylation, sulfate, and glucuronide produced phase II metabolites. This study laid the foundation for the further study of the pharmacological effects of GLA and was conducive to mechanism research.