Characterization of In Vitro metabolic profiles of crepidatin in human liver microsomes and hepatocytes using...

Crepidatin, a bioactive bibenzyl derivative isolated from multiple plants of Dendrobium, has recently gathered significant interest due to its activity against lung cancer. The aim of this study was to elucidate the metabolic fates of crepidatin using human liver microsomes and hepatocytes. To achieve this goal, a specific HPLC-MS/MS method for the determination of crepidatin in human liver microsomes and hepatocytes was developed and validated. Separation was achieved within 2 min on an ACQUITY BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) with a mobile phase consisting of 0.1 % formic acid in water and methanol/acetonitrile (30:70, v/v), running at 0.4 mL/min. Detection employed positive electrospray ionization in multiple reaction monitoring (MRM) mode, with the transition m/z 319.1→151.1 for both crepidatin and internal standard (erianin). The developed method displayed excellent linearity over the concentration range of 1.0-1000 nM (r > 0.999). Crepidatin showed rapid metabolism both in human liver microsomes (t_1/2: 17.2 min) and hepatocytes (t_1/2: 10.8 min). Metabolite identification was achieved using HPLC-Q-Orbitrap-HRMS operated in full MS/dd-MS² scan mode. A total of 15 metabolites across five metabolic pathways were structurally characterized through accurate mass measurement and fragmentation analysis. Hydroxylation (M5, M7) and glucuronidation (M14) emerged as the predominant metabolic pathways. Furthermore, incubation with glutathione (GSH)-supplemented human liver microsomes led to the detection of three GSH conjugates, providing direct evidence for crepidatin bioactivation via the formation of ortho-quinone and quinone-methide species. Crepidatin was primarily metabolized by CYP3A4, 2C8, 2C19, UGT1A1, UGT1A8, and UGT1A9. This study describes the first integrated HPLC-MS/MS and HPLC-Q-Orbitrap-HRMS method for its in vitro metabolic profiling, and the results facilitate prediction of in vivo pharmacokinetics.