title: Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans.
abstract: This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.
authors.affiliation: Servicio de Farmacología Clínica, Clínica Universitaria de Navarra, C/Pio XII s/n, 31080 Pamplona, Spain. macampaner@unav.es
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Campanero
authors.forename: Emilio
authors.initials: E
authors.lastname: García-Quetglas
authors.forename: Belén
authors.initials: B
authors.lastname: Sádaba
authors.forename: Jose Ramón
authors.initials: JR
authors.lastname: Azanza
mesh_terms: D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003640:Dealkylation; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008297:Male; D009294:Narcotics / Q000097:blood* / Q000493:pharmacokinetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D013237:Stereoisomerism; D013696:Temperature; D014147:Tramadol / Q000097:blood* / Q000493:pharmacokinetics*
chemical_list: D007202:Indicators and Reagents; D009294:Narcotics; D012996:Solutions; D014147:Tramadol