Development and Validation of a High Performance Liquid Chromatographic (HPLC) Method for the Determination of Phenytoin Prodrug (Fosphenytoin) in Solutions, Parenteral Formulations, and Active Drug Substance

A rapid, stability indicating high performance liquid chromatographic (HPLC) procedure for the determination of phenytoin prodrug (fosphenytoin) content in solutions, parenteral formulations and the active drug substance was developed. Reverse-phase liquid chromatography was performed using a 15 cm microparticulate (5 μm) NOVA-PAK phenyl packed column, a variable wavelength detector (214 nm), and a mobile phase that consisted of a buffer that was 0.025 M in potassium phosphate monobasic and 0.05 M in 1-heptanesulfonic acid sodium salt (pH = 4.1 with phosphoric acid) and methanol (65:35) pumped at 1.0 mL/minute. The method is precise and accurate, with three separate analysts achieving an accuracy greater than 99.0% and a relative standard deviation of less than 0.8%. A limit of detection determined by visual examination was found to be approximately 0.1 μg/mL. Accuracy of this method decreases at concentrations of less than 10 μg/mL fosphenytoin. Either peak height or peak area ratios at 214 nm can be used for quantitation.