The in vivo and in vitro biotransformation study of lefucoxib, 5-(3,4-dimethyl-phenyl)-1-methanesulfonyl-3-trifluoromethol-pyrazole, a new cyclooxygenase-2 (COX-2) inhibitor, was investigated in rats. To conduct the in vivo metabolism study, Wistar rats received lefucoxib in an oral dose, then their plasma and excreta were collected and analyzed. Through HPLC coupled with fluorescence detector and LC-MSn analysis, hydroxylation was found to be the primary metabolism pathway of lefucoxib in rats. The chemical structure of the di-hydroxy metabolite was identified by MSn spectra, and it was detected in rat plasma, urine and feces after an oral dose. However, the chemical structure of mono-hydroxy metabolites could not be identified by MSn analysis due to the existence of two similar methyls on the phenyl ring of rofecoxib. To solve this problem, in vitro metabolism studies with liver microsome incubation helped accumulate enough metabolites for 1H NMR analysis, which was employed and proved to be successful. Through further analysis of 1H-(1)H correlated spectroscopy (1H-(1)H COSY), chemical structures of two isomeric metabolites (mono-hydroxy metabolites) which had the same retention time in chromatograms were identified. The quantitative ratio of the two isomeric metabolites was also clarified to be 1:2 after analysis of the integrating height of 1H NMR signals. Additionally, the present study illustrated the co-application of in vivo and in vitro metabolism on drug metabolite identification.
