Clomipramine and N-desmethyl Clomipramine: Selective and Sensitive Quantification in Human Plasma by Liquid Chromatography Tandem Mass Spectroscopy

Abstract

A sensitive, rapid and reproducible liquid chromatography-positive electrospray ionization- tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of Clomipramine and its active metabolite N-desmethyl Clomipramine in human plasma using Clomipramine-d3 and N-desmethyl Clomipramine d3 as an internal standard (IS). The analytes were extracted with solid phase extraction using cation exchange cartridges and analyzed on a BDS Hypersil C18 (100 mm × 4.6 mm, 5 μm) column. The mobile phase was composed of methanol: acetonitrile: 10 mM ammonium acetate (35:35:30 v/v/v). Clomipramine, N-desmethyl Clomipramine, Clomipramine d3 and N-desmethyl Clomipramine d3 detected in the multi-reaction monitoring (MRM) mode using LC-MS/MS (API 5500) productions of transitions as Q1→Q3 m/z 315.2→86.2, m/z 301.1→72.1, m/z 318.1→89.3 and m/z 304.2→75.2 respectively. The matrix effect, recovery, sensitivity, linearity, accuracy and precision were all evaluated over the concentration range of 0.500-200.000 ng/mL for both Clomipramine and N-desmethyl Clomipramine. In summary, the method was developed and validated successfully for intended to be use for the analysis of plasma samples of Bioavailability and Bioequivalence study of Clomipramine Tablets.

GRAPHICAL ABSTRACT

Keywords:

• LC-MS/MS
• Biological fluid
• Bioavailability
• Bioequivalence