AUTOMATED-DETERMINATION OF TRAMADOL ENANTIOMERS IN HUMAN PLASMA USINGSOLID-PHASE EXTRACTION IN COMBINATION WITH CHIRAL LIQUID-CHROMATOGRAPHY

A sensitive and automated method for the separation and individual det ermination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs ) in combination with chiral liquid chromatography (LC). The SPE opera tions were performed automatically by means of a sample processor equi pped with a robotic arm (ASPEC system). The DEC filled with ethyl sili ca (50 mg) was first conditioned with methanol and phosphate buffer, p H 7.4. A 1.0-ml volume of plasma was then applied on the DEC. The wash ing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, con taining sodium perchlorate (0.2 M) were added to the extract before in jection into the LC system. The enantiomeric separation of tramadol wa s achieved using a Chiralcel OD-R column containing cellulose tris-(3, 5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phas e was a mixture of phosphate buffer, pH 6.0, containing sodium perchlo rate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaC lO4 concentration were optimized with respect to enantiomeric resoluti on. The method developed was validated. Recoveries for both enantiomer s of tramadol were about 100%. The method was found to be linear in th e 2.5-150 ng/ml concentration range [r(2)=0.999 for (+)- and (-)-trama dol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (- )-tramadol, respectively. (C) 1997 Elsevier Science B.V.