Liquid-chromatographic method for simultaneous determination of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in plasma and urine.

We describe a liquid-chromatographic assay for phenytoin and its para-hydroxylated metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in plasma and urine. Unlike previously reported methods, this procedure is sensitive enough to allow quantitation of the unconjugated metabolite in plasma at concentrations observed clinically (50-500 microgram/L). Simultaneous measurements of the drug and its metabolite in plasma and urine may be helpful in explaining changes in the apparent clearance of phenytoin, assumed to occur through its metabolite. The method may also be used, without modification, for analysis for phenobarbital and carbamazepine in plasma. The accuracy of our method for these three drugs was assessed for 25 months in quality-assurance program for determining drug concentrations. Regression slopes for measured vs reported values for phenytoin, phenobarbital, and carbamazepine were 1.02, 0.99, and 0.99, respectively.