Simultaneous separation and determination of zonisamide,phenytoin carbamazepine and carbamazepine 10,11-epoxide in human plasma using isocratic reversed-phase HPLC

Purpose To develop a simple reversed-phase high-performance liquid chromatography(HPLC) method for the simultaneous determination of the antiepileptic drugs(AEDs) zonisamide(ZNS),phenytoin(PHT),carbamazepine(CBZ) and carbamazepine 10,11-epoxide(CBZE) in human plasma. Methods Plasma sample(100 μL) pre-treatment was taken based on simple deproteinization by 300 μL methanol containing 20 μg/mL chlorzoxazone as internal standard.The HPLC separation was carried out using a Phenomenex SynergiTM Fusion-RP C18 column(250 mm×4.6 mm,particle size 4 μm).The mobile phase was consisted of methanol-acetonitrile-0.1% triflouroacetic acid(27∶15∶58,V/V/V),and delivered at a 1.2 mL/min flow rate.PHT and CBZE were detected at the wavelength of 215 nm, and ZNS and CBZ were detected at 235 nm. Results The lowest limits of quantification for ZNS,PHT,CBZ and CBZE were all 1.0 μg/mL,The method was found to be linear over the concentration ranges investigated,i.e.,1-80 μg/mL for ZNS,1-50 μg/mL for PHT,1-25 μg/mL for CBZ and 1-10 μg/mL for CBZE,respectively.The intra-and inter-day reproducibility was adequate with the coefficients of variation of 7.21% or below and observed recovery was ranged from 94.77% to 113.41% for all the analytes. Conclusions This method was proved to be accurate,sensitive and convenient.It is suitable for therapeutic drug monitoring of these AEDs' concentrations in human plasma.