Nontargeted SWATH acquisition mode for metabolites identification of osthole in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, anti-apoptosis and neuroprotection. In this study, a rapid and reliable method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and MetabolitePilot2.0™ software with principal component variable grouping (PCVG) filtering was developed to observe probable metabolites of OST firstly. The high resolution mass data were acquired by data-independent acquisition mode (DIA), i.e., sequential window acquisition of all theoretical fragmentation spectra (SWATH), which could significantly improved the hit rate of low-level and trace metabolites. A novel data processing method ‘key product ions (KPIs)’ were employed for metabolites rapid hunting and identification as an assistant tool. A total of 72 metabolites of OST were detected in vitro and in vivo, including 39 metabolites in rat liver microsomes (RLMs), 20 metabolites in plasma, 32 metabolites in bile, 32 metabolites in urine and 37 metabolites in feces. The results showed that mono-oxidation, demethylation, dehydrogenation, sulfate conjugation and glucuronide conjugation were major metabolic reactions of OST. More significant, oxydrolysis, 3,4-epoxide-aldehylation, phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation were considered as unique metabolic pathways of OST, and phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation reactions were characterized in rat biological samples for the first time. Preparation of active metabolites will be greatly helpful in elucidating the potential biological mechanism of OST, and the proposed metabolic pathways of it might provide further understanding of the safety and efficacy of simple coumarins.