Identification of metabolites of oridonin in rats with a single run on UPLC-Triple-TOF-MS/MS system based on multiple mass defect filter data acquisition and multiple data processing techniques

Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.