High-Performance Liquid Chromatographic Measurement of Phenytoin, Phenobarbital and Their Major Metabolites in Serum, Brain Tissue and Urine

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Abstract
A rapid HPLC method for the simultaneous determination of phenytoin, phenobarbital, 5-(p-hydroxyphenyl)-5-phenylhydantoin and p-hydroxyphenobarbital in serum, brain tissue and urine is described. The chromatographic separation is carried out using a Spherisorb 5 ODS column and monitoring at 195 nm. The mobile phase is a mixture of acetonitrile and phosphate buffer (28:72) with a flow rate of 2 ml/min. Serum and brain tissue homogenate samples are extracted with tert-butyl-methyl ether at low pH in the presence of an excess of ammonium sulfate. Glucuronide conjugates in urine samples are hydrolyzed by enzymatic cleavage with ß-glucuronidase and then extracted with tert-butyl-methyl ether at low pH. Quantitation is based on peak-height ratio of analyte to internal standard (p-methylphenobarbital). The statistical analysis of the results demonstrate that the method is precise and accurate.