Sensitive quantitation of reboxetine enantiomers in rat plasma and brain, using an optimised reverse phase chiral LC–MS/MS method

Abstract

A sensitive liquid chromatography–mass spectrometry (LC–MS) method has been developed for stereoselective determination of reboxetine in rat plasma and brain homogenate (LLOQ, 50 pg/ml). The method optimised ionisation efficiency with an electro-ionspray source, by adjusting the composite flow conditions (rate, pH, organic content) from column eluent and post-column organic modifier. LC conditions utilized a chiral AGP column (5 μm) with 12.5 mM ammonium carbonate buffer adjusted with formic acid (pH 6.7) and included a wash step (0.05% acetic acid in water) to maintain assay robustness and chromatographic performance. The total method cycle time was 23 min. Imprecision (R.S.D.) was below 10% and inaccuracy (% error) below 7% for both enantiomers in plasma and brain homogenate, over a 2000-fold dynamic range (0.05–100 ng/ml). An automated liquid–liquid extraction technique was used (borate buffer, pH 10/ tert-butyl methyl ether) and the matrix type used produced no difference in the assay performance. The method was successfully applied to determine the pharmacokinetic profiles of S,S- and R,R-reboxetine in rats, following subcutaneous administration of racemate drug.