Development, validation, and clinical application of a UPLC-MS/MS method for clobazam and N-desmethylclobazam in...

Clobazam (CLB) and its active metabolite N-desmethylclobazam (N-CLB) exhibit substantial interindividual variability in paediatric patients with refractory epilepsy, highlighting the value of therapeutic drug monitoring (TDM). This study aimed to develop and validate a rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of CLB and N-CLB and to investigate clinical factors associated with their concentrations in plasma. The method required 50 μL of plasma and achieved chromatographic separation within 3 min. The linear ranges were 25-800 ng/mL for CLB and 237.5-7600 ng/mL for N-CLB, and the method was validated according to the core elements of the ICH M10 guidelines. The validated method was applied to 61 paediatric patients with refractory epilepsy, whose concomitant antiseizure medications and CYP3A4 and CYP2C19 genotypes were evaluated. Multivariate analysis revealed that the daily CLB dose was positively associated with CLB concentration (β = 5.75; 95% CI: 3.01-8.49), whereas the use of VPA was associated with lower CLB concentration (β = -88.37; 95% CI: -134.43 to -42.31) and the use of STP was associated with higher CLB concentrations (β = 206.29; 95% CI: 137.11-275.47). For N-CLB, poor CYP2C19 metabolism (β = 4354.34, 95% CI: 3259.61-5449.07) and STP use (β = 3005.10, 95% CI: 1740.35-4269.84) were associated with higher concentrations. The method is rapid, requires a small plasma volume, and is suitable for routine simultaneous TDM of CLB and N-CLB. Integrating TDM with CYP2C19 genotyping may support individualized CLB therapy and safety monitoring in paediatric patients with refractory epilepsy.