Development and Bio-Analytical Validation of Chromatographic Determination Method of Rufinamide in Presence of its Metabolite in Human Plasma.

Abstract

Rufinamide (RF), antiepileptic drug, is biotransformed to inactive metabolite. Frequent plasma monitoring is required for dose adjustment. This work is concerned with the development and validation of a sensitive and selective RP-HPLC method for the quantitative determination of RF in spiked human plasma in the presence of its main metabolite. Lacosamide was selected as internal standard. Preparation of plasma samples involved precipitation of plasma proteins using methanol. Isocratic elution mode was applied and the chromatographic separation was performed on Prontosil CN column (5μm, 250×4.6mm). Good resolution was achieved using acetonitrile: water (10:90, v/v, adjusted with 0.01N aqueous solution of o-phosphoric acid to pH=3) as a mobile phase at a flow rate of 1mL/min, and UV detection was carried out at 210nm. Linearity was observed over the concentration range of 0.5-50μg/mL of RF in plasma. Bio-analytical validation of the developed method was carried out in accordance to the European Medicines Agency guidelines. The accuracy ranged from 95.97 to 114.13%, and the coefficient of variation of the assay intra-day and inter-day precision did not exceed 10%. The samples were stable under the employed experimental conditions. In conclusion, the findings of the present study revealed its usefulness for therapeutic drug monitoring, assessment of drug pharmacokinetics and application for bioequivalence study.