A novel HPLC method for the measurement of thiobarbituric acid reactive substances (TBARS). A comparison with a commercially available kit

Objectives: Malondialdehyde (MDA) as a part of thiobarbituric acid reacting substances (TBARS) is frequently used as an indicator of lipid peroxidation. Most methods for the measurement of TBARS require long derivatization time and addition of antioxidants in the samples. Furthermore, comparison of these methods with commercially available HPLC kits is lacking. Design and methods: We investigated column performance of five different columns, tested eight different acids for the hydrolysis of the samples, and estimated stability of derivatized plasma samples with different anticoagulants. The samples were derivatized with TBA. The peak for the TBA2–MDA adduct was separated and detected by HPLC. Results: Performance of the Phenomenex Gemini column was best. PCA at the concentration of 0.1125 N was used in this method. Coefficient of variation (CV %) within the run and between the run was 4.1% and 6.7%, and analytical recovery was 90–94%. The retention time of the TBA2–MDA peak was 1.8 min. Reference intervals for TBARS in serum from 250 individuals were 0.53 and 2.1 μmol/l using our HPLC method and 0.07 and 0.24 μmol/l using the Chromsystems assay. Linear regression with log converted values revealed weak relationship between the two methods (r2 = 0.064). Conclusions: Our HPLC method for the analysis of TBARS in serum and plasma is fast and accurate and therefore can be used in clinical studies.