A comprehensive two-dimensional liquid chromatography method for the simultaneous separation of lipid species and their oxidation products

•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.