High-performance liquid chromatographic assay for simultaneous determination of tramadol and its active metabolite in human plasma.: Application to pharmacokinetic studies

A sensitive liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its active metabolite is described. Fluconazole was used as internal standard. The assay involved a single tert-butyl methyl ether extraction and LC analysis with fluorescence detection. Chromatography was at 30 degrees C pumping an isocratic mobile phase of acetonitrile-water (19:81, v/v) containing 0.06 M NaH2PO4 and 0.05 M triethylamine, adjusted to pH 7.90, at 1 mL min(-1) through a reversed-phase, 250 x 4 mm base-stable column. The limit of quantitation of tramadol and its active metabolite was 1 ng mL(-1), only 0.5 mL plasma sample was required for the determination. The calibration curve was linear from 1-1000 ng mL(-1). Intra and inter-day precision (C.V.) did not exceed 10 %. Mean recoveries of 96.38 % for tramadol and 96.62 % for O-demethyltramadol with CVs of 0.43 % and 1.46 % were obtained. Applicability of the method was demonstrated by a pharmacokinetic study on normal volunteers who received 100 mg tramadol intravenously.