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

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 H-1 and C-13 NMR. The structures of the metabolites were found to be 2 beta-hydroxy-tiamulin, 8 alpha-hydroxy-tiamulin and N-deethyl-tiamulin. In addition, the LC-MS chromatograms revealed two other minor metabolites. From their chromatography and from MS2 analysis the structures were estimated to be 2 beta-hydroxy-N-deethyl-tiamulin and 8 alpha-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 2 beta-position and 7% was hydroxylated in the 8 alpha-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 8a-hydroxy-tiamulin was considerably reduced (40% loss after 1 week). (c) 2006 Elsevier B.V. All rights reserved.