New HPLC method to detect amitriptyline in the blood of rats on combination treatment

A rapid, sensitive and specific method based on high performance liquid chromatography (HPLC) has been developed for the simultaneous determination of amitriptyline hydrochloride and Sildenafil citrate in the blood of rats. Sample preparation involved liquid-liquid extraction with n-hexane after alkalified with 0.5 M NaOH. Chromatographic separation was performed on XDB C-18 column with particle size of 5 μm (150 × 4.6 mm) with mobile phase acetonitrile and 20 mM KH2PO4 buffer, pH = 3 using flow rate 1.5 ml/min. Calibration curves were linear within the ranges of 0.25-50 and 0.1-50 μg/ml for amitriptyline hydrochloride and Sildenafil citrate, respectively. This method was successfully applied to the pharmacokinetic study in rats after oral administration of amitriptyline hydrochloride and Sildenafil citrate for 24 h.