Development and validation of an ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry bioanalytical method for quantifying clonazepam in human plasma.

Journal of AOAC International

PubMedID: 24000746

Favreto WA, Pinto AM, Manfio JL, Hoss I, Pristch MC, Bordignon SF. Development and validation of an ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry bioanalytical method for quantifying clonazepam in human plasma. J AOAC Int. 2013;96(4):745-50.
A sensitive, selective, and rapid ultra-performance LC (UPLC)/MSIMS method was validated for the confirmation and quantification of clonazepam in human plasma. The analyte was extracted from human plasma with diethyl ether, reaching an average recovery of 64.02 and 66.48% for clonazepam and the internal standard, respectively. The separation was performed on a Waters ACQUITY UPLC BEH C18 column (50 x 2.1 mm id, 1.7 microm particle size) with gradient elution at a flow rate of 0.25 mL/min using a 0.5% formic acid solution (mobile phase A) and acetonitrile-methanol-formic acid (75+25 + 0.5, v/v/v; mobile phase B). Detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode via electrospray ionization. Linear calibration curves were obtained in the concentration range of 0.3-50.0 ng/mL, with an LOQ of 0.3 ng/mL. The intraday and interday precision (CV) values were below 10%, and accuracy (relative error) ranged from -2.6 to 6.6% at all QC levels. The suggested method was successfully applied for the determination of clonazepam in human plasma in a bioequivalence study.