Trace analysis of ethosuximide in human plasma with a chemically removable derivatizing reagent and high-performance liquid chromatography

Abstract

A simple and sensitive liquid chromatographic method is described for the determination of ethosuximide in human plasma, as a highly sensitive derivative. Ethosuximide spiked in plasma was extracted with toluene and derivatized with a chemically removable derivatizing reagent, 2-(2-naphthoxy)ethyl 2-[1-(4-benzyl)piperazyl]ethanesulfonate, in a homogeneous system, using magnesium oxide as base catalyst. The resulting derivative was separated on a LiChrospher diol column with 1.2% isopropanol in n-hexane as the mobile phase and using coumarin as the internal standard. Several parameters affecting the extraction/derivatization of ethosuximide from spiked plasma were investigated. The linear range for the determination of ethosuximide in spiked plasma was over 30–700 nmol/ml. For ethosuximide in plasma, the detection limit (signal-to-noise ratio=3; sample size, 10 μl) was about 9 pmol; the relative standard deviation was 6.4% for intra-day assay ( n=6) and 9.2% for inter-day assay ( n=6) and the relative recovery was found greater than 94%.