Enantioselective determination of tramadol and its main phase I metabolites in human plasma by high-performance liquid chromatography

A sensitive and relatively rapid reversed-phase HPLC method was applied to the enantiomeric separation of tramadol and its two main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in plasma samples. Chromatography was performed on an AGP column containing α1-acid glycoprotein as chiral selector with a mobile phase of 30 mM diammonium hydrogen phosphate buffer–acetonitrile–triethylamine (98.9:1:0.1, v/v), adjusted to pH 7 by phosphoric acid, and a flow rate of 0.5 ml/min. The fluorescence of analytes was detected at excitation and emission wavelengths of 200 and 301 nm, respectively. The sample preparation was a simple extraction with ethyl acetate using fluconazol as internal standard (IS). The enantiomers of all analytes and IS peaks eluted within 32 min, without any endogenous interference. The calibration curves were linear (r2 > 0.993) in the concentration range of 2–200, 2.5–100 and 2.5–75 ng/ml for tramadol, M1, and M2 enantiomers, respectively. The within- and between-day variation determined by the measurement of quality control samples at four tested concentrations, showed acceptable values. The lower limit of quantitation was 2 ng/ml for tramadol enantiomers and 2.5 ng/ml for M1 or M2 enantiomers. Mean recoveries of enantiomers from plasma samples were >81% for all analytes. The procedure was applied to assess the pharmacokinetics of the enantiomers of tramadol and its two main metabolites following oral administration of single 100-mg doses to healthy volunteers.