Routine determination of hydroxyphenytoin in urine by high-performance liquid chromatography using an automatic...

A high-performance liquid chromatographic method for the determination of the main phenytoin metabolite, hydroxyphenytoin, in the urine of epileptic patients is described. The use of an automated column-switching technique greatly simplifies the pretreatment steps. Thereby, both time and chemicals are saved. The possibility of error arising during the several pretreatment steps is considerably reduced. Following acid hydrolysis of the hydroxyphenytoin glucuronic acid conjugate the sample is diluted with water and after centrifugation is injected onto the pre-column. After washing for a short time with water, the substances which were absorbed on the head of the pre-column were backflushed with water--acetonitrile as eluent onto the analytical column. Separation is achieved by gradient elution using an ODS reversed-phase column with a particle size of 5 microns.