Development and Validation of High-Performance Liquid Chromatography–Diode Array Detector Method for the Determination of Tramadol in Human Saliva

Abstract

A high-performance liquid chromatography-diode array detector method (HPLC-DAD) was developed and validated for the determination of tramadol in human saliva. Samples of saliva were prepared utilizing liquid-liquid extraction with hexane-ethyl acetate (4:1, v/v). Propranolol was used as an internal standard (IS). The linear range and average recovery of tramadol were 0.25-4.00 µg/mL and 94.70%, respectively. The intra-and inter-assay precisions at low, intermediate and high concentrations were within 1.40-9.79 % and 0.77-3.98%, respectively. Intra-and inter-assay accuracies were within 98.50-107.13% and 100.44-103.89%, respectively. The proposed method was successfully applied to determine tramadol in saliva concentrations in three healthy volunteers for 24 hours after administration of 100 mg oral doses of tramadol.