Isocratic separation of phenytoin metabolites by high-performance liquid chromatography from human and animal microsomes and urine

A rapid and simple high-performance liquid chromatography analytical method is described for the quantitative determination of phenytoin and five of its metabolites—phenytoin dihydrodiol, catechol, methoxycatechol, para-hydroxyphenytoin, and meta-hydroxyphenytoin—in biological materials. Following ethyl acetate extraction and incubation with betaglucuronidase, samples were passed through a C₁₈ Sep-Pak cartridge. The eluate was chromatographed on a reverse-phase 10 cm C₁₈ Radial Nova-Pak (5 µm) column using a mobile phase of isopropanol: Water (20:80) at a flow rate of 1.4 ml/min and monitored at 235 nm. Total chromatographic analysis time was 23 min, with complete baseline resolution of all metabolites. Five different columns and 10 different mobile phase conditions were studied to determine the best system. The 10-cm C₁₈ Radial Nova-Pak (5 µm) gave the best resolution, reproducibility, and durability. This method should prove applicable for clinical use as well as for research purposes.