Method Development and Validation of a Novel UHPLC Coupled with MS/MS System for the Estimation of Brivaracetam in Human (K2EDTA) Plasma Samples and its Application to Pharmacokinetic Study

Abstract

Background: Brivaracetam is a novel antiepileptic drug clinically approved for the treatment of partial onset seizures in adults and adolescents. It has some abuse potential and assigns to Schedule V category under the Controlled Substance Act by the Drug Enforcement Administration. It is essential to develop a faster, simple, and highly sensitive method for the quantification of Brivaracetam in human plasma by employing simple liquid-liquid extraction. Objective: The objective of this study is to develop and validate a novel UHPLC-MS/MS method for the estimation of brivaracetam in human plasma samples and application to pharmacokinetic study. Methods: An ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current regulatory guidelines for bioanalytical methods. Sample processing (50 μL) involved only a simple liquid-liquid extraction by ethyl acetate as extraction solvent. Brivaracetam-d7 was used as an internal standard. The chromatographic analysis was performed by a Unisol C18 (4.6 X 100 mm, 5μm) column using 0.1% formic acid in water/acetonitrile (20/80 V/V) as an isocratic mobile phase, at a flow rate of 1.0 mL/min with a run time of 2.2 min. Brivaracetam and its internal standard Brivaracetam D7 were detected and quantified in positive ion mode using multiple reaction monitoring transitions at m/z 213.100→168.100 and m/z 220.000→175.100, respectively. The developed method was applied to assess pharmacokinetic parameters like Cmax, Tmax, t1/2 and AUC for Brivaracetam in healthy, male, and adult humans. Results: The method was validated over a concentration range of 20.000 ng/mL to 4000. 000 ng/mL. Both intra- and inter-assay precision and accuracy were <15% for all quality control samples. No matrix effect was observed. Pharmacokinetic results showed that test formulation is bioequivalent with reference formulation. Conclusion: The present assay is faster, highly sensitive and simpler than previously published analytical reports for brivaracetam in human plasma samples and is suitable for pharmacokinetic evaluation of any marketed formulation.