Ultra-High Performance Liquid Chromatography Method for Bioanalysis of Fampridine Using Dried Blood Spot (DBS) Methodology: Application to Pharmacokinetic Study in Albino Rats.

Abstract

Fampridine (dalfampridine) is used to improve walking in people who have multiple sclerosis (a disease in which the nerves do not function properly and may cause weakness, numbness, loss of muscle coordination and problems with vision, speech and bladder control). Measurement of fampridine plasma concentrations is not practical at sites lacking the facilities to prepare and process blood samples. A dried blood spot (DBS) sampling method, in which a few drops of blood, drawn by lancet from the finger, are applied onto specially manufactured absorbent filter paper, can be used as an alternative to plasma monitoring and would allow for simplified sample storage and transport. Using blood samples from pharmacokinetic studies, an ultra-high performance liquid chromatography assay method for quantification of fampridine in DBS is developed and validated for specificity, selectivity, accuracy, precision, reproducibility and stability. Method was specific and selective relative to endogenous compounds, with required process efficiency, and no matrix effect. Inaccuracy and precision for intra-day and inter-day analyses were tested at all concentrations. Quantification of fampridine in DBS assay was not affected by blood deposit volume and punch position within spot, and hematocrit level had a limited but acceptable effect on measurement accuracy. Fampridine was stable for at least 2 months at room temperature. The correlation between DBS and plasma concentrations with an average blood-to-plasma ratio is determined. DBS sampling is a simple and practical method for monitoring fampridine concentrations. The method is completely validated as per ICH guidelines and extended to the in vivo determination of fampridine in male albino rats.