Retrospective analysis of metabolite patterns of clobazam and N-desmethylclobazam in human plasma by LC-MS/MS

Clobazam is a benzodiazepine drug, used to treat Lennox-Gastaut syndrome in patients aged 2 years and older.To support patient care, our laboratory developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma or serum samples.The chromatographic separation was achieved with an Agilent Zorbax Eclipse Plus C-18 RRHD column with mobile phase consisting of 0.05% formic acid in 5 mM ammonium formate, pH 3.0 and 0.1% formic acid in acetonitrile at a flow rate of 600 µL/minute and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor precursor-to-product ion transitions in positive electrospray ionization mode.The method was validated over a concentration range of 20-2000 ng/mL for CLB and 200-10,000 ng/mL for N-CLB. The lower limit of quantification was 20 ng/mL for CLB and 200 ng/mL for N-CLB with good accuracy and precision. The method performance was successfully evaluated by comparison with two different external laboratories. Retrospective data analysis was performed to evaluate the positivity rate and metabolic patterns for clobazam from our patient population, as a reference laboratory. Among the positive samples, both parent and metabolite were detected in 96.4% of the samples.The method was developed to support therapeutic drug monitoring and the data generated from retrospective analysis could be useful for result interpretation in conjunction with clinical patient information.