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


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 TBA(2)-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 TBA(2)-MDA peak was 1.8 min. Reference intervals for TBARS in serum from 250 individuals were 0.53 and 2.1 micromol/l using our HPLC method and 0.07 and 0.24 micromol/l using the Chromsystems assay. Linear regression with log converted values revealed weak relationship between the two methods (r(2) = 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.
Seljeskog
Esben
E
Division of Medical Biochemistry, PO Box 8100, N-4068 Stavanger, Norway. sees@ext.sir.no <sees@ext.sir.no>
Hervig
Tor
T
Mansoor
Mohammad Azam
MA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008315:Malondialdehyde / Q000032:analysis*; D008875:Middle Aged; D011933:Reagent Kits, Diagnostic; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis*
D000925:Anticoagulants; D011933:Reagent Kits, Diagnostic; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde