Metabolic Stability and Comprehensive Metabolite Profiling of Chrysotobibenzyl in Human Liver Microsomes and Hepatocytes Employing UPLC-MS/MS and UPLC-Orbitrap-HRMS Analysis

Chrysotobibenzyl, a bioactive ingredient from Dendrobium chrysotoxum, exhibits potent anti-tumor activity. However, its metabolic profiles remain unelucidated. This study aimed to disclose the metabolic fates of chrysotobibenzyl using human liver fractions. In vitro metabolism of chrysotobibenzyl was assessed using human liver microsomes and hepatocytes. The concentration of unchanged parent compound was quantified using a validated UPLC-MS/MS method. Metabolite profiling was achieved by Q-Exactive Orbitrap HRMS combined with Compound Discoverer software using a mass defect filtering approach, with structures characterized by accurate mass measurement and fragmentation pattern interpretation. Chrysotobibenzyl exhibited poor metabolic stability in human liver microsomes (t_1/2: 16.24 min) and hepatocytes (t_1/2: 44.35 min). Totally, 36 metabolites were identified, comprising 19 phase I metabolites, 10 glucuronide conjugates, and seven GSH adducts. Using reference standards, M22, M25, M27, and M28 were unambiguously identified as moscatilin, chrysotoxine, erianin, and crepidatin, respectively. The detection of GSH conjugates indicated the formation of reactive metabolites, including ortho-quinone and quinone-methide intermediates. This study demonstrates the effectiveness of UPLC-MS/MS and UPLC-Orbitrap-HRMS platforms for metabolic profiling. Metabolic pathways include demethylation, hydroxylation, dehydrogenation, glucuronidation, and GSH conjugation. These findings provide critical insights into the metabolism of chrysotobibenzyl in humans, enhancing our understanding of its biological activity.