High Performance Liquid Chromatographic Isolation and Spectroscopic Characterization of Metabolites from the Bile of Rats Receiving Rapamycin (Sirolimus) Intravenously

Abstract

Ten major metabolites of rapamycin (M2, M3, M8, M9, M10, M11, M13, M14, M15, and M16) were isolated from pooled bile of intravenously dosed rats. Metabolites were extracted from the bile with ethyl acetate prior to isolation by HPLC using a Supelcosil SPLC-18, μm, 10 × 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS analyses. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M10 is oxygenated in the southern portion of rapamycin and the macrolide ring is intact. M13 is a monohydroxylation and demethylation metabolite and both biotransformations occurred at the southern portion. M8, M9, and M11 are monohydroxylation and demethylation metabolites. M14 and M15 are di-hydroxylation metabolites. M16 is mainly a dihydroxylation metabolite.