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 ass ay for the quantitative determination of the analgesic tramadol and O-demet hyltramadol, 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 con taining cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, p receded by an achiral end-capped C-18 column. The mobile phase was a mixtur e of phosphate buffer (containing sodium perchlorate (0.2M) and triethylami ne (0.09M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol an d its active metabolite by this method was 0.5 ng/mL; only 0.5 mt of the pl asma sample was required for the determination. The calibration curve was l inear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 n g/mL for O-demethyltramadol enantiomers. Intra and interday precision [coef ficient of variation (CV)I 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- an d (-) S,S-O-demethyltramadol with CVs <2.15% were obtained. Applicability o f the method was demonstrated by a pharmacokinetic study in normal voluntee rs who received 100 mg of tramadol by the intravenous route. Chirality 11:2 72-279, 1999. (C) 1999 Wiley-Liss, Inc.