Protein precipitation method for determination of c c lobazam and N N N ‐desmethylclobazam in human plasma by LC–MS/MS LC–MS/MS

摘要

Abstract A protein precipitation method for the determination of clobazam (CLB) and its major active metabolite N ‐desmethylclobazam ( N‐ CLB) in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS) was established. CLB and N‐ CLB were extracted from human plasma samples by protein precipitation with methanol. Analyte separation was done using a Phenomenex Kinetex? Biphenyl (50?×?2.1?mm, 1.7?μm) column using isocratic elution with a mobile phase of 5?m m ammonium formate with 0.01% ammonium hydroxide (40%) and methanol (60%) at a flow rate of 0.4?mL/min and an injection volume of 10?μL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor the precursor‐to‐product ion transitions of m / z 301.1?→?259.0, 306.0?→?263.9 for CLB and CLB‐D5 and 287.0?→?245.0, 292.0?→?250.0 for N‐ CLB and N‐ CLB‐D5 in positive electrospray ionization mode, respectively. The method was validated over a concentration range of 2.0–750?ng/mL for CLB and 0.7–200?ng/mL for N‐ CLB on SCIEX Triple Quad 4500 MS System. Total run time was 5?min. This method has been designed for bioequivalence study for formulations containing 20?mg of CLB.