A Novel HPLC Method for Determination of Phenytoin in Human Plasma
Jesse Flores
Department of Biomedical and Pharmaceutical Sciences, Touro College of Pharmacy, New York, USA.
Sheril Alexander
Department of Biomedical and Pharmaceutical Sciences, Touro College of Pharmacy, New York, USA.
Mariana Babayeva *
Department of Biomedical and Pharmaceutical Sciences, Touro College of Pharmacy, New York, USA.
*Author to whom correspondence should be addressed.
Abstract
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.
Keywords: Phenytoin, HPLC, validation, human plasma.