An analytical method to measure malondialdehyde (MDA) was developed. MDA was derivatized with N-methylhydrazine (NMH) to 1-methylpyrazole (1-MP). 1-MP was extracted and analyzed by capillary gas chromatography with nitrogen-phosphorus detection. Analyte concentration, pH, and matrix effects of 1-MP-spiked samples were investigated to determine optimal recovery conditions. Efficiencies for solid-phase extraction ranged from 95.6 ± 0.9 to 81.6 ± 3.5% compared to 75.0 ± 6.4 to 67.5 ± 9.6% for liquid-liquid extraction for 20 to 1 nmol/ml 1-MP-spiked samples, respectively. Solid-phase extraction of 1-MP was more effective than liquid-liquid extraction over a range of pH 2-8.5 and in various aqueous matrices. Addition of methanol to the matrix decreased the solid-phase extraction efficiency. Reaction yield at pH 2-8.5 showed full conversion of MDA to 1-MP following reaction with NMH. Recovery of bound MDA was investigated by incubating bovine serum albumin (BSA) spiked with MDA at 37°C for 18 h and separating the free MDA and MDA-bound protein. The recovery of bound MDA from BSA increased by increasing the acidity and temperature. Specific applications of this method for biological samples are given for the analysis of endogenous MDA in the plasma and red blood cells of mice and the formation of MDA in ultraviolet-irradiated cells in culture.
ASJC Scopus subject areas
- Molecular Biology