Development and Validation of an LC–MS/MS Method for the Quantitative Estimation of Bexicaserin in Human Cerebrospinal Fluid

ABSTRACT Bexicaserin is an investigational molecule that is a highly selective superagonist of the 5‐hydroxytryptamine 2C (5HT 2c ) serotonin receptor subtype and in Phase 3 clinical studies for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We have validated a simple LC–MS/MS method for the quantitative estimation of bexicaserin in human cerebrospinal fluid (CSF). The chromatographic separation of bexicaserin and the IS was achieved on an Acquity HSS T3, C18 column using a 5.5‐min gradient program. The sample preparation involves the dilution extraction of bexicaserin and the isotope labeled internal standard ( 13 CD 2 ‐bexicaserin; IS) from 25 μL of human CSF. The calibration curve was linear from a range of 0.1–100 ng/mL. The method was fully validated as per the regulatory guidelines. The intraday and interday precision for bexicaserin was in the range of 6.3%–8.7% and 6.0%–8.8%. Bexicaserin intraday and interday bias was in the range of −15.1% (LLOQ) to 12.0% and −10.0% to 1.2%. Stability studies showed that bexicaserin was stable on bench for 24 h, in autosampler for over 50 h, and extract stability at 6 ° C for 76 h. Bexicaserin was stable after five freeze–thaw cycles and long‐term storage at −20°C for 108 days and −80 ° C for 102 days.