Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture

Acyl glucuronides of nonsteroidal anti-inflammatory drugs having a chiral center are known to be chemically very active and form covalently bound adducts with proteins, such as human serum albumin, which may be the cause of hypersensitive reactions. Hepatic acyl glucuronosyltransferase catalyzes the transformation of α-aryl propionates into these diastereoisomeric acyl glucuronides, and, hence, its activity needs to be characterized. From this point of view, we developed a rapid, accurate and reproducible analytical method for the separation and determination of diastereoisomeric glucuronides of flurbiprofen, one of the nonsteroidal anti-inflammatory drugs, in the incubation mixture of the hepatic microsomal preparation by high-performance liquid chromatography with a simple column-switching technique for deproteinization. The glucuronides were separated on a TSKgel ODS-80Ts column with 20 mM ammonium acetate buffer (pH 5.6)–ethanol–acetonitrile as the mobile phase and monitored with a UV detector at 246 nm. The detection limit of the proposed method was 600 fmol/injection at a signal-to-noise ratio of 10. The validation results indicated that this method would be very useful for the determination of diastereomeric acyl glucuronides formed from flurbiprofen in an incubation mixture.