Simultaneous Determination of Six Anticonvulsants in Serum by High Performance Liquid Chromatography

The authors describe a simultaneous determination method of six anticonvulsants (ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, valproic acid) using 500 μl of serum. After the addition of the internal standard (5-(p-methylphenyl)-5 phenylhydantoin), the anticonvulsants are extracted in an acid medium with ethyl acetate. They are separated by reverse phase chromatography on a μ Bondapack C18 column, eluted with a water/methanol mixture (36/64 V/V) at a flow rate of 0.7 ml/min. The column effluent is first analyzed by monitoring the ultraviolet absorption at 197 nm and then at 425 nm after the addition of a color indicator: bromocresol purple. The analysis lasts 12 minutes at ambient temperature. The sensitivity obtained with the serum for the range of products investigated is of the order of 0.5 to 2 mg/l, the extraction recoveries varying from 75 to 100% depending on the drug. Reproducibility is good (cv ≤ 9%).