In vitro Metabolites of Tenvermectin in Rat and Dog Liver Microsomes Analyzed by UPLC-Q-Exactive Orbitrap MS

Tenvermectin (TVM) is a novel 16-membered macrolide antibiotic synthesized from genetically engineered bacteria that produce avermectin and milbemycin. The in vitro metabolites of the new veterinary drug Tenvermectin (TVM) in the liver microsomal model were analyzed and identified by high-resolution mass spectrometry. It directly reflects the in vitro metabolic characteristics of the drug, and at the same time provides a theoretical basis and important clues for in vivo metabolic studies.

In this study, rat and dog liver microsomes were first prepared by differential centrifugation. The in vitro metabolic activities of both were determined by incubating them with specific substrates for rat liver microsomes (7-ethoxy coumarin) and dog liver microsomes (coumarin), respectively. An in vitro liver microsome metabolic model of TVM was established. Under in vitro conditions, the metabolites in the incubation system were extracted and purified by incubating rat and dog liver microsomes with TVM. The metabolites of TVM were detected by UPLC-Q-Exactive Orbitrap MS. The existence of the metabolites was determined through software prediction and instrument verification of the metabolites. At the same time, combined with the elemental composition and structure of the original tetraviridin, each metabolite and its fragment ions were rapidly and accurately identified.

The findings indicated that nine TVM metabolites were identified in rat liver microsomes, comprising demethylated products (M2, M5, M8), oxidized products (M1, M3, M4, M9), ketone products (M6), and demonosaccharified products (M7). In contrast, ten TVM metabolites were detected in dog liver microsomes, including demethylated products (M2, M5, M8), oxidized products (M9, M10, M11, M13), ketone products (M6), demonosaccharified products (M7), and demethylated ketone products (M12). The metabolite types and contents in the liver microsomes of the two species were different.

UPLC-Q-Exactive Orbitrap MS was used to study the metabolites and metabolic pathways of TVM. The metabolic pathways of TVM in rat and dog liver microsome in vitro included demethylation, oxidation, ketonization, demonoglycation and ketonization after demethylation, among which demethylation and oxidation were the main ones. This research clarified the in vitro metabolic mode of tenvermectin and refined its metabolic pathway. The research results provide an important theoretical basis and reference for further studying the metabolic products, pharmacokinetics and pharmacodynamics of TVM in animals.