Determination of atomoxetine metabolites in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

4-Hydroxyatomoxetine (4-HAT) and N-desmethylatomoxetine (N-DAT) are major metabolites of atomoxetine, a potent and selective inhibitor of the presynaptic norepinephrine transporter that is used for the treatment of attention deficit/hyperactivity disorder. The pharmacological activity of 4-HAT is similar to that of atomoxetine. We have developed and validated a simple, rapid and sensitive liquid chromatography analytical method with tandem mass spectrometry (LC–MS/MS) for the determination of 4-HAT and N-DAT in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of analytes was performed using a reversed-phase Luna C18 column (2.0 mm × 100 mm, 3 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)–methanol (10:90, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 250 μL/min and the retention times of 4-HAT, N-DAT and internal standard (IS, metoprolol) were 0.9, 1.0 and 1.0 min, respectively. The calibration curves were linear over the range of 0.05–20 ng/mL for 4-HAT and 0.1–20 ng/mL for N-DAT. The lower limits of quantification, using 200 μL human plasma, were 0.05 and 0.1 ng/mL for 4-HAT and N-DAT, respectively. The mean accuracy and precision for intra- and inter-day validation of 4-HAT and N-DAT were both within the acceptable limits. This LC–MS/MS method showed improved sensitivity for quantification of the two main metabolites of atomoxetine in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.