Identification and characterization of the metabolites of sinomenine using liquid chromatography combined with benchtop Orbitrap mass spectrometry and nuclear magnetic resonance spectroscopy

Abstract

RationaleSinomenine, a major bioactive compound isolated from Sinomenium acutum, has been used for the treatment of rheumatoid arthritis and other cardio‐cerebrovacular diseases. However, the metabolism of this drug has not been fully investigated. The current work was carried out to investigate the in vitro metabolism of sinomenine in liver microsomes.MethodsThe metabolites were generated by incubating sinomenine (3 μM) with the liver microsomes in the presence of NADPH at 37°C. The structure of the metabolites was characterized using liquid chromatography coupled to high‐resolution mass spectrometry (HRMS). Two major metabolites synthesized and their structures were further confirmed using nuclear magnetic resonance spectroscopy.ResultsUnder the current conditions, 12 metabolites were found and structurally identified using high resolution MS and MS2 spectra. Among these metabolites, M1, M2, M3, M4, M5, M6, M7, M9, M11, and M12 were first reported. The metabolites M8 and M10 were synthesized and unambiguously identified as N‐desmethyl‐sinomenine and sinomenine N‐oxide, respectively. The phenotyping study revealed that the formation of M8 was catalyzed by CYP2C8, 2C19, 2D6, and 3A4, whereas the formation of M3, M6, and M10 were exclusively catalyzed by CYP3A4. The metabolic pathways of sinomenine include N‐demethylation, O‐demethylation, dehydrogenation, oxygenation, and N‐oxygenation.ConclusionsN‐Demethylation and N‐oxygenation were the primary metabolic pathways of sinomenine. This study provides new insight into the in vitro metabolism of sinomenine, which would help prospects of sinomenine disposition and safety assessments.