A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent

Abstract

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid–liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 × 4.6 mm) column. In this method the limit of quantification of 0.01 μg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 μg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from −3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.