UHPLC-Orbitrap-HRMS-based metabolite identification and profiling of methylophiopogonone A, A bioactive constituent...

Methylophiopogonone A (MON-A), an anti-inflammatory constituent of Ophiopogon japonicus, demonstrates therapeutic potential for liver fibrosis in patients with chronic hepatitis B and SARS-CoV-2 infection. This study aimed to investigate its metabolic profile using liver microsomes and cryopreserved hepatocytes from humans and three preclinical species (rat, dog, and monkey). MON-A was incubated separately with each system, and the samples were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole/Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap-HRMS) in positive electrospray ionization mode. Data were processed using Compound Discoverer software with mass defect filtering. Metabolite structures were characterized based on accurate mass and MS² fragment ions. A total of 14 metabolites were identified. The primary metabolic pathways included hydroxylation, oxidative methylenedioxyphenyl (MDP) ring-opening, oxidation, and glucuronidation. M11 (a glucuronide conjugate) was the most abundant metabolite across all species. Additionally, four glutathione (GSH) conjugates were detected, indicating the formation of reactive metabolites, notably ortho-quinone and quinone methide species. The formation of quinone methide species revealed that C₂-C₃ double bond is essential for the metabolic activation of the A-ring. This systematic study clarifies the metabolic fate of MON-A, providing important insights into its biotransformation pathways and potential for reactive metabolite formation. The results establish a foundation for evaluating both the therapeutic efficacy and safety profile of this anti-inflammatory candidate during preclinical development.