A rapid HPLC method for simultaneous determination of multiple antiepileptic drugs in human serum

Objective: To establish a rapid HPLC method for simultaneous determination of plasma concentrations of multiple antiepileptic drugs. Methods: The blood samples were pretreated by simple sedimentation with 5% ZnSO 1, and then analysed by Waters 2965 HPLC system on a Diamonsil C 18 column(250 mm × 4.6 mm, 5 μm) using methanol : H 2O (65 : 35) as mobile phase. Quantification was achieved with UV detection at 225 nm based on peak area with alprazolam used as internal standard. Results: The method was found to be linear over the range of 2.5-80 μg/mL (r=0.999 5) for phenobarbital, 1.25-40 μg/mL (r=0.9985) for phenytoin sodium, 0.625-20 μg/mL (r=0.9998) for carbamazepine, 0.1-3.2 μg/mL (r=0.9997) for clonazepam, 0.125-4 μg/mL (r=0.9994) for estazolam and 0.125-4 μg/mL (r=0.9987) for diazepam. The mean recovery and minimal detectable concentrations (S/N≥3) were 101.07% and 0.250 μg/mL for phenobarbital, 100.80% and 0.312 μg/mL for phenytoin sodium, 100.32% and 0.025 μg/mL for carbamazepine, 99.28% and 0.040 μg/mL for clonazepam, 99.25% and 0.031 μg/mL for estazolam, 101.05% and 0.062 μg/mL for diazepam, respectively. At three different concentrations, the intra- and inter-day precisions (RSD) were all below 10%. Conclusion: The method appeared to be a rapid, convenient and accurate method for determining the plasma concentrations of antiepileptic drugs.