A Highly Sensitive Triple Quad LC–MS/MS Method Development and Validation for the Determination of Bexicaserin (LP352) in Human Plasma and Urine Matrices

Abstract

Bexicaserin is a highly selective agonist at the 5-HT_2c receptor in clinical development for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC–MS/MS method for the quantitative estimation of bexicaserin in human plasma and urine. The sample preparation involves the extraction of bexicaserin and internal standard (¹³CD₂-bexicaserin; IS) from 150 μL plasma and 50 μL urine using a solid phase extraction method. The chromatographic separation of bexicaserin and IS was achieved on a Poroshell EC-C18 column using 5 min gradient program. The calibration curve ranged from 0.1 to 100 ng/mL in plasma and 1.0 to 1000 ng/mL in urine. Intraday and interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, a battery of stability studies, and incurred sample reanalysis were performed for bexicaserin in both plasma and urine. The intraday and interday accuracy and precision were well within the acceptable limits in both plasma and urine matrices. Stability studies in plasma and urine showed that bexicaserin was stable on bench for 24 h, in autosampler over 54 h, five freeze–thaw cycles, and long-term storage at −20°C and −80°C for > 368 days. The validated methods were successfully applied in clinical study.