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• Comprehensive LC×LC is a promising tool for lipid oxidation analysis in foods. • Combining SEC (1D) and NPLC (2D) can separate various lipid classes and their oxidation products. • Diagnosis of peak displacement needs to be considered in NPLC. Lipid oxidation is one of the major causes of food spoilage for lipid-rich foods. In particular, oil-in-water emulsions, like mayonnaises and spreads, are prone to oxidation due to the increased interfacial area that facilitates contact between the lipids and hydrophilic pro-oxidants present in the water phase. Polar, amphiphilic lipid species present at the oil/water interface, like the mono- (MAGs) and di-acylglycerols (DAGs), act as oxidation starters that initiate subsequent oxidation reactions of the non-polar lipids in the oil droplets. A comprehensive two-dimensional liquid chromatography (LC×LC) method with evaporative light-scattering detection (ELSD) was set up to study the composition of the complex mixture of oxidized polar and non-polar lipids. The LC×LC-ELSD method employs size exclusion chromatography (SEC) in the 1D (1st dimension) to separate the various lipid species according to size. In the 2D (2nd dimension), normal-phase liquid chromatography (NPLC) is used to separate the fractions according to their degree of oxidation. The coupling of SEC with NPLC yields a good separation of the oxidized triacylglycerols (TAGs) from the large excess of non-oxidized TAGs. In addition, it allows the isolation of non-oxidized DAGs and MAGs that usually interfere with the detection of a variety of oxidized products that have similar polarities. This method facilitates elucidating how lipid composition affects oxidation kinetics in emulsified foods and will aid in the development of more oxidation-stable products. [ABSTRACT FROM AUTHOR]