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. 2020 Jan;34(1):e4710.
doi: 10.1002/bmc.4710. Epub 2019 Nov 19.

Protein precipitation method for determination of clobazam and N-desmethylclobazam in human plasma by LC-MS/MS

Astghik Mikayelyan  1 , Armine Aleksanyan  1 , Mariam Sargsyan  1 , Arpine Gevorgyan  1 , Hasmik Zakaryan  1 , Armen Harutyunyan  1 , Lusine Zhamharyan  1 , Yeghig Armoudjian  1 , Tigran Margaryan  1   2
Affiliations

Protein precipitation method for determination of clobazam and N-desmethylclobazam in human plasma by LC-MS/MS

Astghik Mikayelyan et al. Biomed Chromatogr. 2020 Jan.

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 mm 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.

Keywords: LC-MS/MS; N-desmethylclobazam; PPT; clobazam; plasma.

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References

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