DETERMINATION OF MALONDIALDEHYDE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING 4-(2-PHTHALIMIDYL)BENZOHYDRAZIDE AS A PRECOLUMN FLUORESCENT LABELING REAGENT

A high-performance liquid chromatographic method was developed for the determination of malondialdehyde after conversion into 1-[4-(2-phthal imidyl)benzoyl]pyrazole with the fluorescent labelling reagent 4-(2-ph thalimidyl)benzohydrazide. The labelling reaction was carried out at 5 0 degrees C for 60 min in the presence of phosphoric acid. The extent of conversion of malondialdehyde into the fluorescent derivative was a pproximately 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 detecti on limit (signal-to-noise ratio = 3) was 8 fmol per injection (20 mu l ). The relative standard deviations for within-day and day-to-day prec ision were 3.18 and 3.53% (0.5 mu mol l(-1), n = 8), respectively. The method was applied to the determination of malondialdehyde generated from isolated rat hepatocytes stimulated with tert-butyl hydroperoxide .