Development and Validation of an LC-MS/MS Method for the Quantitative Estimation of Bexicaserin in Human...

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 (¹³CD₂-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.