Analysis of clobazam and its active metabolite norclobazam in plasma and serum using HPLC/DAD

Abstract

This report describes a simple reversed-phase high-performance liquid chromatographic (HPLC) method with automated solid-phase extraction (SPE) for analysing clobazam and norclobazam concentrations in human serum or plasma. For the HPLC analysis the samples and standards are prepared with an ASPEC automatic sample preparer using 100-mg Bond-Elut C-18 solid-phase extraction columns. The HPLC method is an isocratic method with a mobile phase of acetonitrile:methanol:10 mmol l-1 dipotassium hydrogen phosphate, pH 3.7 (30:2:100), at a flow rate of 1.5 ml min-1. The benzodiazepines are detected with a diode array detector (DAD) at 240 nm and the peak purity analyses are performed at 210-365 nm. The recovery is over 97% for both analytes, and it is independent of the drug concentration. The intra-assay CVs vary between 0.7 and 2.2% and inter-assay CVs between 3.8 and 4.6% at therapeutic drug concentrations. The detection limit is 15 nmol l-1. The assay is linear from 30 to 20,000 nmol l-1 (clobazam) and from 170 to 105,000 nmol l-1 (norclobazam). This method leads to a very good separation of norclobazam from carbamazepine and phenytoin. None of the anti-epileptic or antidepressant drugs tested interfere with the assay.