An LC–MS/MS Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Cerebrospinal Fluid

The investigational molecule bexicaserin is a highly selective 5-HT_2C superagonist currently in Phase 3 clinical studies for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). To facilitate its clinical development, a simple LC-MS/MS method was developed and validated for the quantitative estimation of its metabolites, M9, M12, and M20, in human cerebrospinal fluid (CSF). The sample preparation involves sample dilution extraction of metabolites and SIL-IS internal standards from 25 μL of human CSF. The chromatographic separation of three metabolites was achieved on an Acquity HSS T3, C18 column using an 8.0-min gradient program. The intraday and interday precision and accuracy for all three metabolites were well within the range of acceptable limits. Stability studies in CSF showed that metabolites were stable in the presence of bexicaserin on the bench for 24 h, in the auto-sampler over 92 h, and extracted stability at 6°C for 48 h. Metabolites were stable in the presence of bexicaserin after three freeze-thaw cycles and long-term storage at -20°C and -80°C for 41 days. This method has since been successfully applied in a clinical study for the quantitative estimation of metabolite concentrations in CSF from a clinical study.