Validated LC–MS (ESI) assay for the simultaneous determination of amitriptyline and its metabolite nortriptyline in rat plasma: Application to a pharmacokinetic comparison

A rapid, sensitive and specific method based on high performance liquid chromatography with electrospray ionization mass spectrometry (HPLC–MS/ESI) has been developed for the simultaneous determination of amitriptyline and nortriptyline in rat plasma. Sample preparation involved liquid–liquid extraction with methyl t-butyl ether after alkalified with 0.5 mol/l NaOH. Chromatographic separation was performed on a XB-C4 column (4.6 mm × 250 mm, 5 μm, Welch Materials) with a mobile phase consisting of 10 mM ammonium acetate (0.6‰ formic acid)–acetonitrile (60:40, v/v) at a flow rate of 1.0 ml/min. Calibration curves were linear within the ranges of 10–3200 ng/ml for amitriptyline and 10–1000 ng/ml for nortriptyline. This method was successfully applied to the pharmacokinetic study in rats after intravenous injection of amitriptyline hydrochloride.