A Sensitive and Selective LC–MS/MS-ESI Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Plasma and Urine Matrices

Bexicaserin is a highly selective 5HT_2c receptor agonist being developed for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC–MS/MS method for the quantitative estimation of three pharmacologically inactive metabolites (M9, M12, and M20) of bexicaserin in human plasma/urine. Sample preparation involves the extraction of M9, M12, M20, and internal standards (ISs) from 25-μL plasma and 50-μL urine following protein precipitation. The chromatographic separation of analytes was achieved on a HSS T3-C18 column. The calibration curves ranged from 0.1 to 100 ng/mL for M9, 0.5–500 ng/mL for M12, and 1.0–1000 ng/mL for M20 in plasma and 2.0–2000 ng/mL for M9 and M12 and 10–10,000 ng/mL for M20 in urine. Intraday/interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, stability studies, and incurred sample reanalysis were performed in both plasma and urine. The intraday and interday accuracy and precision for metabolites met the stipulated regulatory guidelines. Stability studies in plasma and urine showed that analytes were stable at bench-top for > 23.5 h and in autosampler for > 69 h. Analytes were stable after five freeze–thaw cycles and > 552 days of long-term storage at −20°C and −80°C.