Simultaneous rapid HPLC determination of anticonvulsant drugs in plasma and correlation with EMIT

A method is presented for measuring simultaneously five anti-convulsants (primidone, ethosuximide, phenobarbital, carbamazepine and diphenylhydantoin) in serum and plasma by high-performance liquid chromatography (HPLC) using alphenal (5 - allyl - 5 - phenyl - barbituric acid) as the internal standard. All five drugs are separated from each other and, in the case of primidone, from its metabolie, 2 - phenyl - 2 - ethyl - malondiamide. Total HPLC time for the separation is seven minutes. The chromatography is performed on a C-8 reverse phase column with a mobile phase consisting of acetonitrile/phosphate buffer (35/65) at 25 degrees C. The eluted drugs are detected at 220 nm and quantitated from their peak heights relative to that of the internal standard. The lower limits of detection for each drug is 200 ng per ml for primidone, 1000 ng per ml for ethosuximide, 200 ng per ml for phenobarbital, 100 ng per ml for carbamazepine and 200 ng per ml for diphenylhydantoin. Analytical recoveries for the five drugs ranged from 97 to 107 percent. Correlation of results for 187 specimens by enzyme-immunoassay (emit) was 0.981 for primidone, 0.827 for ethosuximide, 0.975 for phenobarbital, 0.889 for carbamazepine and 0.990 for diphenylhydantoin. In the HPLC assay, ethotoin was found to interfere with phenobarbital and phensuximide with carbamazepine.