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Incrementally Modified Drug (IMD), Yuhan Research Institute 416-1, Gyeonggi-do, KoreaReceived February 21, 2012, Accepted March 26, 2012A rapid, specific, and reliable LC-MS/MS-based bioanalytical method was developed and validated in ratplasma for the simultaneous quantitation of amitriptyline and its metabolite nortriptyline. Chromatographicseparation of these analytes was achieved on a Gemini C18 column (50 × 4.60 mm, 5 µm) using reversed-phasechromatography. The mobile phase was an isocratic solvent system consisting of 1% formic acid in water andmethanol (10:90, v/v), at a flow rate of 0.2 mL/min. The analytical range was set as 0.1-500 ng/mL foramitriptyline and 0.08-500 ng/mL for nortriptyline using a 200 µL plasma sample. The accuracy and precisionof the assay were in accordance with FDA regulations for the validation of bioanalytical methods. Thevalidated method was successfully applied to a pharmacokinetic study in six rats after oral administration ofamitriptyline (15 mg/kg). This method allows laboratory scientists to rapidly determine amitriptyline andnortriptyline concentrations in plasma.Key Words : Amitriptyline, Nortriptyline, LC-MS/MS, Quantitation, PharmacokineticsIntroductionAmitriptyline is a typical tricyclic antidepressant (TCA)used for the treatment of major depression since the 1960s. Itinduces a specific pharmacodynamic effect primarily byblocking presynaptic uptake of amines (norepinephrine,dopamine, and serotonin). Amitriptyline metabolism involveshepatic microsomal enzymes (mainly CYP2C19 and CYP3A4)that demethylate the aliphatic side chain, generating thepharmacologically active metabolite nortriptyline.

Quantitative Determination of Amitriptyline and Its Metabolite in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry