Quantitative Measurement of Phenytoin and Hydroxyphenytoin in Human Serum by Column Extraction-High-Performance Liquid Chromatography

Abstract

A method for the simultaneous determination of phenytoin and it's primary metabolite in humans, 5-(p-hydroxyphenyl)-5-phenylhydantoin, in human serum, was developed by column extraction-high-performance liquid chromatography. Simple and rapid extraction from the serum was carried out by using Extrelut[○!R] pre-packed column and chloroform. The chromatographic procedure utilized a reversed-phase column with 0.01% ammonium acetate in 50% methanol and monitored at 250 nm. The anticonvulsants were eluted at a flow rate of 1.0 ml/min, and their quantities were estimated from their peak height ratios to that of phenacetin added as an internal standard. The method was applied to patient serum samples and compared with a commercially available enzyme immnunoassay kit.