A Novel HPLC Method for Determination of Phenytoin in Human Plasma
Jesse Flores, Sheril Alexander, Mariana Babayeva
Journal of Pharmaceutical Research International · pp. 1–7 · Published 5 Jul 2018
10.9734/JPRI/2018/41885Abstract
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.
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