Simultaneous high performance liquid-chromatographic determination of carbamazepine, carbamazepine-10,11-epoxide, ethosuximide, phenobarbital, phenytoin, primidone and phenylethylmalonamide in plasma.

A common methodology is reported for the determination of five major anticonvulsants (carbamazepine, ethosuximide, phenobarbital, phenytoin, primidone) and their active metabolites (carbamazepine-10,11-epoxide, phenylethylmalonamide) in 30 microliters of plasma. After a single step of deproteinisation and extraction with acetonitrile, leading to an almost complete recovery of all the analytes, 5 microliters is injected on a reversed-phase column (Lichrosorb RP-18, 5 microns). The anticonvulsants are eluted isocratically at a column temperature of 50 degrees C with a mobile phase consisting of acetonitrile/phosphate buffer p' 6.9 (40/60 by vol), and monitored at 208 nm. Quantitation, using peak height or peak area, is based on the ratio of analyte to internal standard (allylisobutylbarbital) referenced to a serum-based multiple drug standard. The composition and pH of the mobile phase, temperature of the column, choice of wavelength of detection and size of the column material are crucial for the optimal separation of these five drugs and their two active metabolites in a chromatographic time of only 12 min, without sacrificing high sensitivity and column life.