Application of HPLC with silica-phase and reversed-phase eluents for the determination of clomipramine and demethylated and 8-hydroxylated metabolites

A rapid and efficient high-performance liquid chromatographic method using a reversed-phase eluent of methanol-water with butylamine on a silica column was developed for the separation and quantitation of clomipramine (CMI), its demethylated metabolites (desmethyl-clomipramine and didesmethyl-clomipramine [DDCMI]), and its hydroxylated metabolites (8-hydroxy-clomipramine and 8- hydroxy-desmethylclomipramine). A liquid-liquid extraction procedure, which involved an alkaline extraction with heptane-isoamyl alcohol, first from a 1- mL serum sample and then from the acidic back extract, was used. The chromatographic separation was rapid (20 min), and the ultraviolet detection (at 254 nm) was sufficiently sensitive (limit of detection, 5-10 ng/mL). Except for DDCMI, the calibration was linear (r > 0.99, n = 5), and within- day and day-to-day coefficients of variation remained less than 11%. The method was used to analyze CMI and its metabolites in the serum of depressive patients and to determine their hydroxylation and demethylation capacities.