Comprehensive Determination of Lipid Peroxidation Biomarkers in Ovine Tissues and Plant Oils by C18-UHPLC-DAD and GC-FID.

BACKGROUND

An original pre-column derivatisation strategy combining liquid chromatography, supported by gas chromatography, was developed for the determination of malondialdehyde (MDA), formaldehyde (FA), and 4-hydroxynonenal (4-HNE) in selected plant oils and model edible animal tissues (i.e., muscle, adipose tissue, liver, and brain).

METHODS

In oils, direct derivatisation with 2,4-dinitrophenylhydrazine (DNPH) was applied to quantify the target aldehydes (as hydrazones) without prior saponification. In the analysed animal tissue samples, MDA and FA were released by saponification and subsequently derivatised with DNPH, whereas 4-HNE was extracted from these samples and subsequently derivatised with DNPH. Derivatised aldehydes were quantified using C18 ultra-high performance liquid chromatography (C18-UHPLC) with photodiode array detection (DAD) under binary-gradient elution conditions, supported by gas chromatography (GC) with flame ionisation detection (FID).

RESULTS

The combination of the original binary gradient elution programme, selective DAD, and a high-performance C18 column (150 mm, 1.6 µm particle size) resulted in excellent baseline stability, good linearity, and satisfactory repeatability and specificity in the determination of MDA, FA, and 4-HNE. C18-UHPLC-DAD enabled satisfactory separation of MDA, FA and 4-HNE hydrazones from endogenous matrix components in solutions of processed oils and animal tissues, while the addition of acetonitrile to these sample solutions further reduced background interference. C18-UPLC-DAD provided satisfactory symmetrical peak shapes, peak purities, and recoveries of MDA, FA, and 4-HNE in analysed plant oils and ovine tissues, compared with GC-FID. Compared with GC-FID, C18-UHPLC-DAD provided superior resolution of derivatised aldehydes in matrices of analysed biological samples.

CONCLUSIONS

The determination of lipid peroxidation biomarkers in oils and animal tissues using our novel C18-UHPLC-DAD method may contribute to the optimisation of breeding practices, helping to minimise animal stress and enhance the health-promoting properties of food products.