Direct determination of protein binding of phenytoin in serum by high-performance liquid chromatography

A reliable, routine method has been developed which speeds and simplifies the determination of free phenytoin levels in serum. The free fraction of the drug was determined by a single direct injection of serum (350 microL) into a high-performance liquid chromatography (HPLC) system. The mobile phase was a mixture of isopropanol (0.5%) and pH 7.4 phosphate buffer. An internal surface reversed-phase silica column, the Pinkerton column, was used with a flow rate of 1.0 mL/min at ambient temperature, and the wavelength was set at 254 nm. The resolution of free and bound phenytoin peaks was affected by the percentage of isopropanol, the serum volume injected, and the protein concentration in the sample. The concentrations of free and bound drug agreed well with those determined by the conventional ultrafiltration method (MPS-1). The determination of protein binding by HPLC appears to be a satisfactory method for routine therapeutic drug monitoring. The factors affecting the binding and chromatographic separation and the application of this method for clinical use are discussed.