Validated HPLC Method for the Analysis of Clozapine in Rat Plasma and its Application to Pharmacokinetics

As a prerequisite to the determination of pharmacokinetic parameters of clozapine in rats, an HPLC method using UV detection was developed and validated. Clozapine and the internal standard, ambroxol were extracted from plasma samples using hexane and isoamyl alcohol (98.5:1.5 v/v) solutions. Chromatographic separation was achieved on a phenomenex C18 column (5 µ; 25 cm x 4.6 mm id) using a mobile phase of ACN/0.5% triethylamine solution in Ammonium acetate buffer of pH 5 in the ratio of 40:60 at a flow rate of 1.5 ml/min. Detection was at 263 nm. The calibration curve was linear from 50-500 ng/ml. The Intra-day accuracy ranged between 99.99% and 100.05% with precision of 0.006% and 0.03%. The Inter-day accuracy ranged between 99.98% and 100.01% with precision of 0.010% and 0.058%. The main pharmacokinetic parameters for rats were determined after a single intravenous administration of 100 mg/kg clozapine are t1/2, 0.0614 h; AUC0-, 83.368 ng.hr/ml; Cmax, 36.16 ng/ml; Tmax, 0.5 hrs; Keli, 11.2847 hrs-1.