The simultaneous determination of five anti-epileptic drugs in plasma by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method is described for the simultaneous determination in plasma of carbamazepine, ethosuximide, phenobarbitone, phenytoin and primidone. The procedure involved the preliminary extraction of the drugs and the internal standard (hexobarbitone) into a mixture of organic solvents at pH 2. The dried extract was dissolved in methanol and 25 microliter of the concentrate was injected into a liquid chromatograph linked to a reverse-phase column. The drugs and internal standard were eluted from the column by a mixture of methanol and water, as the mobile phase, and detected with a UV spectrophotometer at 204 nm. This HPLC assay, which required 0.5 ml of plasma, was used to determine anti-epileptic drugs levels in 136 mentally-handicapped children suffering from epilepsy. Comparison between different batches of assays showed that recovery of the drugs from plasma varied from 60 to 98% and with a coefficient of variance between 3.8 to 9.8%. Detection limit of the method ranged from 2 micrograms ml-1 for primidone, to 1 microgram ml-1 for the remainder of the anti-epileptic drugs.