A Novel HPLC Method for Quantification of 10 Antiepileptic Drugs or Metabolites in Serum/Plasma Using a Monolithic Column

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important in maximizing the therapeutic response while minimizing the adverse effects. High-performance liquid chromatography (HPLC) is the most commonly used technique for this purpose. Recently, commercial monolithic columns were introduced, which consist of a single rod of fused silica or polymer. The objective of this work was to develop a simple and fast method to quantify 10 commonly measured AEDs or metabolites [carbamazepine, carbamazepine-10,11-epoxide, felbamate, lamotrigine, 10,11-dihydro-10-hydroxy-carbamazepine (active metabolite of oxcarbazepine), pentobarbital, phenobarbital, phenytoin, primidone, and zonisamide] in serum/plasma by HPLC using a reverse-phase monolithic column. Serum/heparin plasma (100 mu L) was mixed with an internal standard solution (5-ethyl-5-p-tolylbarbituric acid in methanol, 250 mu L). After centrifugation at 15,500g for 10 minutes, 15 mu L of supernatant was injected into a monolithic column. The analytes were eluted with an isocratic solution of 0.1 M, pH 6.5, phosphate buffer: methanol: acetonitrile (77:20:3), monitored at 210 nm. The chromatography time was 16 minutes. The method was linear from 0.4-4.9 to 21.2-190.9 mu g/mL depending on the analytes with analytical recovery of 80%-114%. The inter- and intra-assay coefficients of variation were < 8% in 3 levels of serum-based controls for all the analytes. No significant carryover was observed. Commercial controls containing > 100 therapeutic drugs and common endogenous substances were tested and showed no interference. Comparison studies for 6 AEDs or metabolites were performed against commercial HPLC methods. Three AEDs were compared with Food and Drug Administration-approved immunoassays. All comparisons had R > 0.96 with slope ranging from 0.86 to 1.20. This is a simple and fast HPLC method suitable for measuring the 10 AEDs or metabolites. The use of the monolithic column resulted in increased sensitivity, better resolution, and a shorter analytical time compared with a regular C18 column.