Improved enantioselective method for the determination of the enantiomers of reboxetine in plasma by solid-phase extraction, chiral derivatization, and column-switching high-performance liquid chromatography with fluorescence detection

Abstract

A rapid enantioselective method is described for the quantitation of the reboxetine ( R, R)- and ( S, S)-enantiomers in plasma utilizing solid-phase extraction, derivatization, normal-phase high-performance liquid chromatography, and fluorescence detection. Plasma samples (0.1 ml) with added internal standard were applied to activated solid-phase extraction discs containing a nonpolar/strong cation mixed-phase, washed, eluted, evaporated to dryness, and derivatized for 5 min with (+)-1-(9-fluorenyl)ethyl chloroformate. After termination of the derivatization reaction, the samples were analyzed by isocratic normal-phase HPLC using a silica column and ethanol–heptane (1:124, v/v) as mobile phase. The derivatized reboxetine peak was column-switched onto cyano and Chiralcel OD-H columns in series using ethanol–heptane (1:49, v/v) as mobile phase to resolve the diastereomeric derivatives of the enantiomers and separate interferences. The column effluent was monitored with fluorescence detection at 260/315 nm. The range of quantitation of each enantiomer was 2–2000 ng/ml. One sample was injected every 18 min.