Simultaneous determination of plasma phenytoin and its primary hydroxylated metabolites in carbon tetrachloride-intoxicated rats by high-performance liquid chromatography

We have developed a simple and sensitive method for the simultaneous determination of phenytoin (PHT), 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in rat plasma by high-performance liquid chromatography. The three substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 250 mm x 4.6 mm I.D.) using acetonitrile-0.008 M NaH2PO4 (pH 6) (35:65, v/v) as a mobile phase at a flow-rate of 0.8 ml/min. Absorbance was monitored at 215 nm. The quantification limit was 50 ng/ml for each of PHT, m-HPPH and p-HPPH. The mean recoveries for DPH, m-HPPH and p-HPPH from plasma were 95.6 +/- 3.6, 94.5 +/- 4.2 and 98.6 +/- 2.9%, respectively.