Enantiomeric separation of tramadol and its active metabolite in human plasma by chiral high-performance liquid chromatography: application to pharmacokinetic studies.
A sensitive and stereoselective high-performance liquid chromatographic assay for the quantitative determination of the analgesic tramadol and O-demethyltramadol, an active metabolite, is described in this work. Ketamine was used as internal standard. The assay involved a single tert-butymethylether extraction and liquid chromatography analysis with fluorescence detection. Chromatography was performed at 20 degrees C on a Chiracel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, preceded by an achiral end-capped C18 column. The mobile phase was a mixture of phosphate buffer (containing sodium perchlorate (0.2 M) and triethylamine (0.09 M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol and its active metabolite by this method was 0.5 ng/mL; only 0.5 mL of the plasma sample was required for the determination. The calibration curve was linear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 ng/mL for O-demethyltramadol enantiomers. Intra and interday precision [coefficient of variation (CV)] did not exceed 10%. Mean recoveries of 95.95 and 97.87% for (+)R,R- and (-)S,S-tramadol and 97.70 and 98.79% for (+)R,R- and (-)S,S-O-demethyltramadol with CVs < 2.15% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received 100 mg of tramadol by the intravenous route.
Campanero
M A
MA
Servicio de Farmacología Clínica, Clínica Universitaria de Navarra, Universidad de Navarra, Pamplona, Spain. macampaner@urav.es
Calahorra
B
B
Valle
M
M
Troconiz
I F
IF
Honorato
J
J
D000701:Analgesics, Opioid / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D001711:Biotransformation; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D015203:Reproducibility of Results; D013237:Stereoisomerism; D014147:Tramadol / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics*
D000701:Analgesics, Opioid; D007202:Indicators and Reagents; D014147:Tramadol