Salting‐out assisted liquid–liquid extraction method optimized by design of experiments for the simultaneous high‐performance liquid chromatography analysis of perampanel and stiripentol in mouse matrices

Abstract

We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART® Purospher Star column (C18 , 55×4mm; 3μm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372nm and perampanel at 275/430nm. Good linearity was demonstrated for perampanel at 1-500ng/mL range in brain, 2-2000ng/mL in liver and 1-2000ng/mL in plasma and kidney (r2 ≥0.9922), and for stiripentol between 10 and 2000ng/mL in brain and 10 and 20000ng/mL in the remaining matrices (r2 ≥0.9917). Precision (CV ≤15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.