Automated determination of clomipramine and its major metabolites in human and rat serum by high-performance liquid chromatography with on-line column-switching

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for simultaneous determination of the tricyclic antidepressant clomipramine and its metabolites demethylclomipramine, 2-, 8-, and 10-hydroxyclomipramine, 2-, and 8-hydroxydemethylclomipramine and didemethylclomipramine in serum. After serum injection into the HPLC system and on-line sample clean-up on a clean-up column (Hypersil CN; 10×4.6 mm) by an eluent consisting of 35% acetonitrile and 65% deionized water, the chromatographic separation was performed on an analytical column (LiChrospher CN; 250×4.6 mm I.D.) by an eluent consisting of 38% acetonitrile and 62% aqueous sodium perchlorate (0.02 M, pH 2.5). The UV detector was set at 260 nm. The limit of quantification was about 15 ng/ml for all analytes. The coefficients of variation ranged between 3 and 12% with recovery rates between 64 and 110%. Linear regression analyses revealed coefficients of correlation between 0.98 and 0.99. The method could be applied to therapeutic drug monitoring as well as metabolism studies in man and rat.