Determination of malonaldehyde by coupled high-performance liquid chromatography—spectrofluorimetry after derivatization with luminarin 3

Abstract

A high-performance liquid chromatographic method with fluorescence detection is described for the specific determination of free malonaldehyde (MDA). Malonaldehyde was labelled with luminarin 3 in acetate buffer (pH 4.0) at room temperature in the dark. An aliquot of the reaction mixture was injected on to an octadecyl-bonded column using acetonitrile—imidazole buffer (30:70, v/v) as the mobile phase. The eluate was monitored with a fluorescence detector at 395 nm (excitation) and 500 nm (emission). A linear calibration graph was established between 7.2 and 90 ng/ml of MDA and the limit of quantification (LOQ) was lower than 7 ng/ml of MDA. The precision was characterized by R.S.D.s of 11% at 7.2 ng/ml and 2% at 90 ng/ml. The structure of the derivative was confirmed as the 5-hydroxy-2-pyrazoline form. UV absorbance and corrected fluorescence spectral data and quantum yields of the luminarin 3 derivative of malonaldehyde are presented.