Metabolic profiling of chrysotoxine: structural elucidation of phase I metabolites and reactive intermediate identification by liquid chromatography tandem mass spectrometry

Chrysotoxine, a bioactive bibenzyl derivative isolated from Dendrobium chrysotoxum Lindal, has recently emerged as a promising therapeutic candidate for cervical cancer. This study described an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of chrysotoxine in liver microsomes. The developed method showed excellent linearity over the concentration range of 5-1500 nM, which is suitable for in vitro high-throughput screening. Chrysotoxine showed poor stability in monkey liver microsomes, with an in vitro elimination half-life (t_1/2) of 9.7 min, contrasting with moderate stability in rat liver microsomes (t_1/2: 31.5 min) and excellent stability in dog (t_1/2: 56.5 min) and human (t_1/2: 60.1 min) liver microsomes. Metabolite identification was achieved using liquid chromatography coupled with hybrid quadrupole/Orbitrap high-resolution mass spectrometry (LC-Orbitrap-HRMS) operated in full MS/dd-MS² scan mode, enabling accurate mass measurement, formula assignment, and MS² fragmentation analysis. Seven phase I metabolites were identified, revealing three primary metabolic pathways: hydroxylation (M3 and M6), demethylation (M7), and dehydrogenation (M1 and M4). In glutathione (GSH)-supplemented liver microsomes, seven GSH conjugates were detected and identified, providing evidence for chrysotoxine bioactivation through reactive intermediate formation, particularly ortho quinone and quinone methide species. This systematic investigation elucidates the metabolic fate of chrysotoxine, offering critical insights into its biotransformation pathways and potential reactive metabolite formation. The findings provide a foundation for understanding both therapeutic efficacy and safety considerations during preclinical development of this anticancer candidate.