Protein precipitation method for determination of oxcarbazepine and its 10-monohydroxy derivative in human plasma by LC-MS/MS

Protein precipitation method for the determination of oxcarbazepine and its major active metabolite 10-monohydroxy derivative in human plasma by liquid chromatography tandem mass spectrometry was established. Analytes were extracted from human plasma samples by protein precipitation with acetonitrile.
Analyte separation was done using Phenomenex Kinetex™ Biphenyl (2.6 µm, 100×2.1 mm) column using isocratic elution with a mobile phase of 5 mM ammonium bicarbonate (42.5%) and methanol (57.5%) at a flow rate of 0.3 mL/min and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 253.1 → 180.0, 257.2 → 184.1 for oxcarbazepine and oxcarbazepine-D4, and 255.1 → 194.2, 259.2 → 198.2 for 10-monohydroxy derivative and 10,11-Dihydro-10-hydroxy carbamazepine-D4 in positive electrospray ionization mode, correspondingly. The method was validated over a concentration range
of 20-8000 ng/mL for oxcarbazepine and 37.5-15000 ng/mL for 10-monohydroxy derivative on SCIEX Triple Quad 4500 MS System. Total run time was 4 min. Sample preparation was conducted in ice water bath because of instability of oxcarbazepine in plasma at room temperature. The method was validated in accordance with U.S Food and Drug Administration and European Medicines Agency guidelines. Method validation has been proved by a variety of tests for matrix effects, extraction efficiency, selectivity, linearity, sensitivity, precision, recovery and stability and can be successfully applied for the bioequivalence/pharmacokinetic studies of oxcarbazepine and its 10-monohydroxy derivative. This method has been designed for bioequivalence study for formulations containing 600 mg of oxcarbazepine.