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Research Article Open Access

Development of an Analytical Method for the Plasma Determination of Five Anti-Epileptics and their Metabolites by HPLC: Comparison with Immunoanalytical Methods


As bioanalysis emerged as a critical tool in the process of drug discovery and development, and it is desirable the availability of less expensive, faster and simpler analytical methodologies to support drug interaction screening studies with statistical validation over a wide concentration range, the aim of this work is to develop a simple, rapid and cost-effective High-Performance Liquid Chromatographic method for the simultaneous quantification of human plasma concentration of six antiepileptic drugs frequently used in clinical practice (phenobarbital, lamotrigine, oxcarbazepine, mono-hydroxycarbazepine, carbamazepine and phenytoin). Clonazepam was used as an internal standard. Sample preparation consisted of a deproteinization step with acetonitrile. After extraction, the analytes were separated on C18 reversedphase column thermostated at 25°C. The mobile phase was composed of 50% phosphate buffer (pH=7.5) and 50% of methanol/acetonitrile (80:20, v/v), pumped isocratically at 1 mL/min. The UV detector was set at 215 nm. The total run time was only 10 min. The extraction yield values were between 84% and 99%. All of these antiepileptics get separated with good peak shapes and resolution factor greater than 2. The new method proved to be simple and rapid. Calibration curves were linear with regression coefficients greater than 0.998. Intraand interday precision CV% varied between 0.91% and 5.69%. Bias value for accuracy ranged from 2.42% to 1.27%. The mean absolute extraction recoveries at low, middle and high quality control sample levels ranged from 84.17% to 99.02%. This bioanalytical method was successfully applied to real plasma samples from 26 epileptic patients. Our results were compared to those obtained by immunoassay methods in biochemistry-toxicology FATTOUMA hospital laboratory and in Tunisia Pharmacovigilance Center. The developed method seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies.

Kaouthar Louati, Sarah Mahfoudhi and Fathi Safta

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