Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

Abstract

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C 18 column (3 μm) with a mobile phase of acetonitrile—sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (λ ex = 227 nm, λ em = 310 nm) was used. When 100 μl of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.