Interconversion from N‐Desmethyl Clomipramine to Clomipramine and its impact on a bioequivalence study

Interconversion or back conversion from a parent compound to its metabolites or vice versa has never been clearly understood and established by using mass spectrometry. This makes it a daunting challenge for which the technique of liquid chromatography with tandem mass spectrometry supports it while an other hyphenated technique liquid chromatography with ultraviolet detection employed for investigation lacks it. Herein, a selective and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous determination of Clomipramine and its active metabolite N‐Desmethyl Clomipramine in human plasma was developed and validated. A suitable method for investigating interconversion between Clomipramine and its active metabolite to each other by two analytical tools was achieved and its impact on a bioequivalence study was evaluated. Stable labeled internal standards i.e. Clomipramine D3 and N‐Desmethyl Clomipramine D3 were used as internal standard to track and compensate the parent compound during processing and extraction from plasma. The method involves a rapid liquid‐liquid extraction (followed by flash freezing under dry ice bath) from plasma followed by reversed gradient chromatography condition and mass spectrometry detection. The mean recovery for Clomipramine and N‐Desmethyl Clomipramine were 62.1 and 63.2%, respectively.