Simultaneous Quantitative Assay of Phenobarbital, Phenytoin, and p-Hydroxydiphenylhydantoin by HPLC and a Comparison with EMIT®

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Abstract
The antiepileptic drugs, phenobarbital and phenytoin, were determined in plasma by enzyme immunoassay (EMIT) and by high-pressure liquid chromatography (HPLC). In addition, the major metabolite of phenytoin, p-hydroxydiphenylhydantoin (HPPH), was determined by HPLC. Within-day and between-day coefficients of variation for both methods were 7.6% or less for phenobarbital and 8.7% or less for phenytoin, and for HPPH, by liquid chromatography, were 7.4% or less. Both methods also showed good linearity from subtherapeutic through low toxic concentrations. Comparison of the results of the two methods for phenobarbital and for phenytoin showed good correlations r = 0.99 and 0.97 respectively, indicating that HPLC can be used rather than the more commonly used EMIT. In addition, this HPLC method simultaneously measures both antiepileptic drugs and the unconjugated form of the major metabolite of phenytoin, and with prior hydrolysis, the conjugated form of p-hydroxydiphenylhydantoin.