Development of an HPLC method for the determination of fampridine (dalfampridine) in human plasma by fluorimetric derivatization and application to a prototype pharmacokinetic study

Fampridine (dalfampridine) is used to improve walking in people who have multiple sclerosis. In this study, a new, fast and sensitive HPLC method with fluorometric detection was developed for the determination of fampridine in human plasma and applied to a pharmacokinetic study. Fampridine was precolumn derivatized with 7-chloro-4-nitrobenzofurazan (NBD-Cl) and the fluorescent derivative was separated on a C18 (150 × 4.6 mm × 2.6 μm) analytical column at 30 ºC using a mobile phase composed of acetonitrile – 0.1% o-phosphoric acid in water (80:20, v/v) by isocratic elution with flow rate of 1.0 mL min–1 . The method was based on the measurement of the derivative using fluorescence detection (λex= 310 nm, λem = 365 nm). The retention time of fampridine is 4.10 ± 0.02 min. This currently developed method was validated according to EMA criteria by evaluating the specificity, linearity, precision, accuracy and robustness. The method was determined to be linear in a concentration range of 0.01–20 ng mL–1 with the correlation coefficient of 0.9996. LOD and LOQ were found to be 0.003 and 0.01 ng mL–1 , respectively. Intraday and interday RSD values were less than 2.45%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC0–t, AUC0–∞, Cmax, tmax, t1/2, were calculated according to the assays. The presented method can certainly be used for bioequivalence and bioavailability investigations and routine analysis of the drug in plasma.