Figure 3. Detection of lipid peroxidation product malondialdehyde by HPLC analysis.

The chromatogram of DNPH-MDA complex in U937 cells (A) and DNPH-MDA standard (B). In A, chromatogram of DNPH-MDA complex was measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (b) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (c) for 30 min. After the treatment, lipids were separated from proteins and DNPH was added to lipids. In B, the chromatogram of DNPH-MDA standard shows the retention time of 3 min 50 s. The insert shows the dependence of average peak area on the concentration of DNPH-MDA standard. Based on the calibration curve, the concentrations of DNPH-MDA complex determined from calibration curve were as following: 0.029±0.003 nmol ml⁻¹ (control), 0.030±0.003 (H₂O₂) and 0.09±0.02 nmol ml⁻¹ (Fenton reagent). The coefficient of determination R² was determined as 0.9997. Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.