Measurement of malondialdehyde in fish: A comparison study between HPLC methods and the traditional spectrophotometric test

Alternative methods to the traditional spectrophotometric determination of the malondialdehyde-thiobarbituric acid (MDA-TBA) complex (method A) and to the overestimation of MDA levels in the TBA reaction have been develop for the evaluation of lipid oxidation in fish. In this study, two HPLC separation methods of the MDA-TBA (method B) and MDA-dinitrophenylhydrazine (MDA-DNPH) adduct (method C) were investigated and compared to the traditional spectrophotometric TBA test (method A) in samples of chilled fish (hake, sea bream and sardine). Detection limits were 0.16, 0.10 and 0.20 μM MDA and quantification limits were 0.23, 0.17 and 0.26 μM MDA, for methods A, B and C, respectively. Recovery of method B ranged between 100% and 108% and of method C between 90% and 112%. Method A presented low recovery levels (under 71%). Overall method performance followed the order HPLC method MDA-DNPH > HPLC method MDA-TBA > traditional spectrophotometric TBA test. Though showing a better accuracy and specificity, method C had, however, some disadvantages, a relatively high limit of detection (0.20 μM MDA) and a lower reproducibility at lower MDA contents in standards and samples. Nevertheless, these are not critical drawbacks for an application in routine fish analysis, given the high MDA concentrations in oxidised fish. The application of the modified HPLC methods in fish samples with different levels of MDA, showed that these methods are useful for the samples with low amounts of oxidation products, such as chilled hake as well as in samples with high levels of oxidation, like 15 days chilled stored sardine.