A Novel HPLC Method for Determination of Phenytoin in Human Plasma

Aim: Aim of this research was to develop and validate a simple, efficient and reproducible high-performance liquid chromatography method to measure phenytoin concentrations in human plasma

Study Design: Linearity, selectivity, sensitivity, accuracy and precision of the analytical methods were validated according to ICH guidelines.

Methodology: The method employed a Phenomenex C18 column kept at 25ºC. The mobile phase consisted of a 0.05 M potassium dihydrogen phosphate buffer solution (pH 2.8) and methanol in a ratio of 60:40, respectively. The flow rate of the mobile phase was 0.7 mL/min. Phenytoin was detected at a wavelength of 250 nm.

Results: Phenytoin was eluted at 7.4 minutes with no interference with the other components of the human plasma. The method was linear in the range of 1-25 μg/mL (R2 = 0.9998). The LOD and LOQ were calculated as 0.07 μg/mL and 0.20μg/mL, respectively. Recovery, tested in the range of 5-20 μg/mL, was found to be 99.21 - 102.09%. Intraday and interday precision RSDs were 0.65 and 0.29%, respectively.

Conclusion: The proposed method is fast, reliable and reproducible, and can be recommended to measure free phenytoin levels in the blood.