Isolation and structural elucidation of tiamulin metabolites formed in liver microsomes of pigs

Anne Kruse Lykkeberg, Claus Cornett, Bent Halling-Sørensen, Steen Honoré Hansen

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    Abstract

    Although the antimicrobial tiamulin is extensively metabolized in pigs, the metabolism is not well investigated. In this work the NADPH dependent metabolism of tiamulin in liver microsomes from pigs has been studied. The tiamulin metabolites formed in the incubations were analysed using LC-MS, and three major metabolites were isolated using solid phase extraction and preparative HPLC. The final structure elucidations were performed by tandem mass spectrometry and (1)H and (13)C NMR. The structures of the metabolites were found to be 2beta-hydroxy-tiamulin, 8alpha-hydroxy-tiamulin and N-deethyl-tiamulin. In addition, the LC-MS chromatograms revealed two other minor metabolites. From their chromatography and from MS(2) analysis the structures were estimated to be 2beta-hydroxy-N-deethyl-tiamulin and 8alpha-hydroxy-N-deethyl-tiamulin, but the structures were not confirmed by NMR. In these studies approximately 20% of tiamulin was deethylated, 10% was hydroxylated in the 2beta-position and 7% was hydroxylated in the 8alpha-position. About 40% of tiamulin was metabolized during the incubation conditions used. The protein precipitation in the incubations was performed using perchloric acid, and the preparative purification was performed under alkaline conditions. Therefore, the stability of the metabolites under these conditions was studied. The metabolites were found to be stable in the acid solution, but under alkaline conditions, particularly at room temperature, the stability of especially 8alpha-hydroxy-tiamulin was considerably reduced (40% loss after 1 week).
    Original languageEnglish
    JournalJournal of Pharmaceutical and Biomedical Analysis
    Volume42
    Issue number2
    Pages (from-to)223-31
    Number of pages8
    ISSN0731-7085
    DOIs
    Publication statusPublished - 2006

    Bibliographical note

    Keywords: Animals; Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diterpenes; Female; Magnetic Resonance Spectroscopy; Mass Spectrometry; Metabolic Detoxication, Drug; Microsomes, Liver; Molecular Structure; NADP; Swine

    Keywords

    • Former Faculty of Pharmaceutical Sciences

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