Abstract
In a previous paper, we reported the availability of mass spectrometry as a useful technique for the identification of drug metabolites. In the present study, the metabolites of 7-hydroxy-4-methylcoumarin in rat were investigated by mass spectrometric technique as shown in Chart 1. A small amount of the administrated urine was evaporated under reduced pressure and the mass spectrum of the residue was recorded(MS-1). From this spectrum, we estimated the presence of various metabolites by observing the shift of mass numbers in high mass region. The remained urine was added with saturated aqueous solution of basic lead acetate and the precipitate was filtrated. After evaporation of the filtrate under reduced pressure various metabolites in the residue were separated by thin-layer chromatography. The mass spectra of extracts of each spot on the thin-layer chromatography were recorded(MS-3). From these spectra, structures of major metabolites were identified by aid of various physical methods(UV, IR, NMR etc.)and of some chemical techniques. Futhermore, the precipitate. was suspended in 50% aqueous MeOH and treated with H2S gas. The filtrate thus obtained was evaporated under reduced pressure and the mass spectrum of the residue was recorded after silylation(MS-4). From this spectrum, we can confirm the presence of various glucuronides.By using these techniques, four metabolites and their glucuronides of the substrate, 7-hydroxy-4-methylcoumarin, were easily identified(Fig. 10).
