Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.