Novel bioanalytical method for the quantification of rufinamide in mouse plasma and tissues using HPLC-UV: A tool to support pharmacokinetic studies
Rufinamide is an antiepileptic drug approved for seizures treatment associated with Lennox-Gastaut syndrome. To support future pharmacokinetic studies in rodents, this work aimed to validate for the first time a fast and simple high-performance liquid chromatographic (HPLC) method for rufinamide quantification in mouse plasma and brain, liver and kidney tissues. For that, aliquots (100 μL) of plasma or tissues homogenates were spiked with known amounts of rufinamide and chloramphenicol (internal standard). Compounds were extracted using a combination of protein precipitation and liquid-liquid extraction. Their separation was accomplished using a LiChroCART® Purospher Star column (C18, 55 mm × 4 mm; 3 μm) protected by a LiChroCART® Purospher Star pre-column (C18, 4 mm × 4 mm; 5 μm) at 35 °C. Mobile phase [water/acetonitrile (82:18, v/v)] was isocratically pumped at 1.0 mL min-1 and detection was performed using a diode-array detector set at 210 nm. A preliminary in vivo pharmacokinetic study was also performed by orally administering rufinamide (10 mg kg-1) to mice. The bioanalytical method herein developed was validated according to international bioanalytical guidelines and showed to be selective and linear (r2 ≥ 0.9918) over the concentration range of 0.1-30 μg mL-1. Regarding quality control samples, overall imprecision was lower than 14.5% and inaccuracy ranged between -14.6% and 15.0%. In all tested matrices, rufinamide recoveries varied between 73.1% and 85.2%. This method was successfully applied in a preliminary pharmacokinetic study, suggesting to be a useful bioanalytical tool to support further non-clinical pharmacokinetic-based studies involving rufinamide.