A rapid and sensitive method for the determination of malondialdehyde as its hydralazine derivative in human plasma using high performance liquid chromatography

Abstract

Malondialdehyde (MDA) is a presumptive biomarker for lipid peroxidation in plasma, live organisms, and cultured cells. This study presents an accurate and reproducible method to measure MDA as its hydralazine (HDZ) derivative in plasma using high-performance liquid chromatography (HPLC). A reference curve was prepared by mixing a 250 μL volume of each concentration of standard MDA (0.05–10.0 nmol mL⁻¹) with 25 μL HDZ 5 mM solution in 2 M hydrochloric acid and the mixture was incubated for 60 min at room temperature in the dark. An aliquot of 50 μL of this reaction mixture was injected into the HPLC system. This method was also used for the determination of MDA in phosphate buffer and plasma. The standard addition method was used to determine MDA in plasma. In order to compare this method with another method for the determination of MDA, a HPLC method was applied using derivatization of MDA with 2,4-dinitrophenylhydrazine (DNPH). Results showed that this method was valid, linear, simple, highly sensitive and reproducible for MDA determination in plasma. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.03 and 0.1 nmol mL⁻¹, respectively. This method has a shorter run time, and a lower LOQ in comparison with other HPLC methods described for MDA determination. Also, it can serve as a cost-beneficial method for determination of MDA in plasma as a biomarker for lipid peroxidation.