Measurement of total and free malondialdehyde by gas–chromatography mass spectrometry – comparison with high-performance liquid chromatography methology

Abstract

Malondialdehyde (MDA) is considered to be a biomarker for enzymatic degradation and lipid peroxidation of polyunsaturated fatty acids. Usually, MDA determination from different biological materials is performed by reaction with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis and fluorometric detection. As this method lacks specificity and sensitivity, we developed a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of MDA with 2,4-dinitrophenylhydrazine. Representative ions in negative ion chemical ionization (NICI) mode were recorded at m/z 204 for MDA and at m/z 206 for the deuterated analogon (MDA-d₂) as internal standard. This stable and precise GC–MS method showed good linearity (r² = 0.999) and higher specificity and sensitivity than the HPLC method and was validated for both total MDA (t-MDA) and free MDA (f-MDA). Within-day precisions were 1.8–5.4%, between-day precisions were 4.8–9.2%; and accuracies were between 99% and 101% for the whole calibration range (0.156–5.0 μmol/L for t-MDA and 0.039–0.625 μmol/L for f-MDA). Although comparison of t-MDA levels from GC–MS and HPLC results using Passing–Bablok regression analysis as well as Bland–Altman plot showed a correlation of the data, a tendency to increased results for the HPLC values was detectable, due to possible formation of unspecific products of the TBA reaction.

Keywords::

• GC–MS
• HPLC
• malondialdehyde
• oxidative stress
• total and free MDA

Acknowledgements

We thank Prof. Gholamali Khoschsorur, from whose profound understanding of the material we greatly benefited, and Eugenia Lamont for language editing.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.