Determination of malondialdehyde by high-performance liquid chromatography using 4-(2-phthalimidyl)benzohydrazide as a pre-column fluorescent labelling reagent

Abstract

A high-performance liquid chromatographic method was developed for the determination of malondialdehyde after conversion into 1-[4-(2-phthalimidyl)-benzoyl]pyrazole with the fluorescent labelling reagent 4-(2-phthalimidyl)benzohydrazide. The labelling reaction was carried out at 50 °C for 60 min in the presence of phosphoric acid. The extent of conversion of malondialdehyde into the fluorescent derivative was approximately 100%. The fluorescent derivative was separated on an ODP-50 column with elution using 40% aqueous acetonitrile and detected by fluorescence at 320 nm (excitation) and 385 nm (emission). The detection limit (signal-to-noise ratio = 3) was 8 fmol per injection (20 µl). The relative standard deviations for within-day and day-to-day precision were 3.18 and 3.53%(0.5 µmol l^–1, n= 8), respectively. The method was applied to the determination of malondialdehyde generated from isolated rat hepatocytes stimulated with tert-butyl hydroperoxide.