SIMULTANEOUS DETERMINATION OF METHYLPHENOBARBITAL ENANTIOMERS AND PHENOBARBITAL IN HUMAN PLASMA BY ONLINE COUPLING OF AN ACHIRAL PRECOLUMN TO A CHIRAL LIQUID-CHROMATOGRAPHIC COLUMN

A fully automated liquid chromatographic (LC) method for the simultane ous determination of methylphenobarbital enantiomers and phenobarbital in human plasma has been developed. The method is based on the use of a precolumn packed with an internal-surface reversed-phase packing ma terial (LiChrospher ADS) for sample clean-up coupled to LC analysis on a cellulose tris(4-methylbenzoate) based chiral stationary phase (Chi ralcel OJ-R). A 100-mu l plasma sample was injected directly on the pr ecolumn packed with LiChrospher RP-18 ADS using a mixture of pH 5.0 ph osphate buffer-methanal (97:3, v/v) as washing liquid. The analytes we re then eluted in the back-flush mode with the LC mobile phase. The en antiomeric separation of methylphenobarbital was achieved on Chiralcel OJ-R. The retention times were modelled using a D-optimal design with ten experimental points in order to optimise the LC mobile phase for the separation of phenobarbital from the enantiomers of mephobarbital. The factors selected were the acetonitrile content, the pH and the so dium perchlorate concentration in the mobile phase. A Derringer's desi rability function was used to find an optimal and robust solution with in the experimental domain. The mobile phase selected consisted of a m ixture of pH 7.0 phosphate buffer-acetonitrile (60:40, v/v). The eluti on profiles of phenobarbital, methylphenobarbital and blank plasma sam ples on the precolumn and the time needed for analyte transfer from th e precolumn to the analytical column were then determined. Finally, th e method developed was validated. (C) 1998 Elsevier Science B.V. All r ights reserved.