Selective ionization of oxidized lipid species using different solvent additives in flow injection mass spectrometry.

Lipid oxidation in food products is a crucial problem that causes undesirable changes in the food's flavor, texture, and nutritional value. It should be carefully monitored as it can lead to the formation of potentially toxic compounds and in that way reduce the shelf life of the product. Liquid chromatography coupled to mass spectrometry is a powerful tool to monitor the formation of oxidized lipids. However, the presence of lipid species in both their non-oxidized and oxidized forms at distinctly different concentrations can hinder the detection and identification of the less abundant oxidized species, due to coelution. In this study, a flow injection mass spectrometry approach was used to selectively ionize oxidized triacylglycerols versus their non-oxidized precursors. Three mobile phase additives were investigated (ammonium formate, sodium acetate, and sodium iodide) at three different concentrations, and ion source settings (i.e., sheath gas temperature, capillary voltage, and nozzle voltage) were optimized. A fractional factorial design was conducted to examine not only the direct effect of the operating parameters on the selectivity of ionization for the oxidized lipid species, but also to assess their combined effect. Overall, selective ionization of oxidized versus non-oxidized lipid species was favored by the use of sodium-containing solvent additives. The application of specific ion source settings resulted in an increased ionization selectivity, with sheath gas temperature and capillary voltage having the most significant influence. A selectivity factor as high as 120 could be reached by combining 0.1 mg/mL sodium-containing additives, with 250 °C sheath gas temperature and 5000 V capillary voltage. These findings will contribute to future studies on fast detection and relative quantification of low abundant oxidized triacylglycerols and their possible impact on human health.

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with different PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modified assay exhibited a fivefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify various hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, buffer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same buffer as the measured sample. The use of correction factors is suggested when financial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantification by the modified FOX assay aligned well with results obtained using the commonly used conjugated diene method, while offering a quicker and broader sample pH range assessment. Thus, the modified FOX assay can be used as a reliable high-throughput screening method for determining LOX activity. KEY POINTS: • Modifying perchloric acid level in FOX reagent expands its linear detection range • The modified FOX assay is applicable for screening LOX activity in a wide pH range • The modified FOX assay effectively assesses substrate specificity of LOX.

How Digestive Processes Can Affect the Bioavailability of PCBs Associated with Microplastics: A Modeling Study Supported by Empirical Data.

The transfer kinetics of plastic-associated chemicals during intestinal digestive processes is unknown. Here, we assessed whether digestive processes affect chemical exchange kinetics on microplastics, using an in vitro gut fluid digestive model mimicking the human upper intestinal tract. Chemical exchange kinetics of microplastics were measured for 10 polychlorinated biphenyls (PCBs) as proxies for the broad class of hydrophobic organic chemicals. Following earlier studies, olive oil was used as a proxy for digestible food, under high and low digestive enzyme activities. The micelle-water and oil-water partition coefficients of the 10 PCBs were also determined to evaluate the relative contribution of each gut component to sorb PCBs. A new biphasic and reversible chemical exchange model, which included the digestion process, fitted well to the empirical data. We demonstrate that the digestive processes that break down contaminated food can lead to a substantial increase in chemical concentration in microplastics by a factor of 10-20, thereby reducing the overall chemical bioavailability in the gastrointestinal tract when compared to a scenario without microplastics. Higher enzyme activities result in more chemicals being released by the digested food, thereby resulting in higher chemical concentrations in the microplastics. While the model-calibrated kinetic parameters are specific to the studied scenario, we argue that the mechanism of the reduced bioavailability of chemicals and the modeling tool developed have generic relevance. These digestive processes should be considered when assessing the risks of microplastics to humans and also biomagnification in aquatic food webs.

Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner.

In adult rats, omega-6 linoleic acid (LA, 18:2n-6) serves as a precursor to oxidized LA metabolites (OXLAMs) known to regulate multiple signaling processes in the brain. However, little is known regarding the levels or role(s) of LA and its metabolites during brain development. To address this gap, fatty acids within various brain lipid pools, and their oxidized metabolites (oxylipins) were quantified in brains from 1-day-old male and female pups using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. Primary neuron-glia co-cultures derived from postnatal day 0-1 male and female rat neocortex were exposed to vehicle (0.1% ethanol), LA, the OXLAM 13-hydroxyoctadecadienoic acid (13-HODE), or prostaglandin E2 at 10-1000 nM for 48 h to test their effects on neuronal morphology. In both male and female pups, LA accounted for 1-3% of fatty acids detected in brain phospholipids and cholesteryl esters. It was not detected in triacylglycerols, and free fatty acids. Unesterified OXLAMs constituted 47-53% of measured unesterified oxylipins in males and females (vs. ~5-7% reported in adult rat brain). Of these, 13-HODE was the most abundant, accounting for 30-33% of measured OXLAMs. Brain fatty acid and OXLAM concentrations did not differ between sexes. LA and 13-HODE significantly increased axonal outgrowth. Separate analyses of cultures derived from male versus female pups revealed that LA at 1, 50, and 1000 nM, significantly increased axonal outgrowth in female but not male cortical neurons, whereas 13-HODE at 100 nM significantly increased axonal outgrowth in male but not female cortical neurons. prostaglandin E2 did not alter neuronal outgrowth in either sex. This study demonstrates that OXLAMs constitute the majority of unesterified oxylipins in the developing rat brain despite low relative abundance of their LA precursor, and highlights a novel role of LA and 13-HODE in differentially influencing neuronal morphogenesis in the developing male and female brain.

Brain oxylipin concentrations following hypercapnia/ischemia: effects of brain dissection and dissection time.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

31 P NMR assessment of the phosvitin-iron complex in mayonnaise.

Lipid oxidation is the main reason for the limited shelf life of mayonnaise. One of the main catalysts of this process is iron, which is introduced in its ferric (Fe(III)) form via phosvitin, an egg yolk phosphoprotein rich in phosphoserines. The binding of Fe(III) to phosvitin and its ability to establish a redox couple with Fe(II) is believed to determine the oxidation rate of unsaturated lipids. In this work, a 31 P NMR based method was developed to quantify loading of phosvitin with Fe(III) and its reductive release. Both features could be quantified in model phosvitin solutions by exploiting the paramagnetic broadening of 31 P NMR signal of phosphoserine residues by Fe(III). This method was then successfully applied to quantify the phosvitin-Fe(III) loading in mayonnaise water phase by liquid NMR, whereas 31 P NMR MAS could only provide a qualitative measure. The 31 P NMR method showed a direct relation between loading of the Fe(III)-phosvitin complex and lipid oxidation.

Oxidized linoleic acid metabolites induce liver mitochondrial dysfunction, apoptosis, and NLRP3 activation in mice.

Circulating oxidized linoleic acid (LA) metabolites (OXLAMs) are increased in patients with nonalcoholic steatohepatitis (NASH) and their levels correlate with disease severity. However, the mechanisms by which OXLAMs contribute to NASH development are incompletely understood. We tested the hypothesis that LA or OXLAMs provided directly through the diet are involved in the development of hepatic injury. C57BL/6 mice were fed an isocaloric high-fat diet containing low LA, high LA, or OXLAMs for 8 weeks. The livers of OXLAM-fed mice showed lower triglyceride concentrations, but higher FA oxidation and lipid peroxidation in association with increased oxidative stress. OXLAM-induced mitochondrial dysfunction was associated with reduced Complex I protein and hepatic ATP levels, as well as increased mitochondrial biogenesis and cytoplasmic mitochondrial DNA. Oxidative stress increased thioredoxin-interacting protein (TXNIP) in the liver and stimulated the activation of mitochondrial apoptosis signal-regulating kinase 1 (ASK1) leading to apoptosis. We also found increased levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and Caspase-1 activation in the livers of OXLAM-fed mice. In vitro, OXLAMs induced hepatocyte cell death, which was partly dependent on Caspase-1 activation. This study identified key mechanisms by which dietary OXLAMs contribute to NASH development, including mitochondrial dysfunction, hepatocyte cell death, and NLRP3 inflammasome activation.

Caffeine intake increases plasma ketones: an acute metabolic study in humans.

Brain glucose uptake declines during aging and is significantly impaired in Alzheimer's disease. Ketones are the main alternative brain fuel to glucose so they represent a potential approach to compensate for the brain glucose reduction. Caffeine is of interest as a potential ketogenic agent owing to its actions on lipolysis and lipid oxidation but whether it is ketogenic in humans is unknown. This study aimed to evaluate the acute ketogenic effect of 2 doses of caffeine (2.5; 5.0 mg/kg) in 10 healthy adults. Caffeine given at breakfast significantly stimulated ketone production in a dose-dependent manner (+88%; +116%) and also raised plasma free fatty acids. Whether caffeine has long-term ketogenic effects or could enhance the ketogenic effect of medium chain triglycerides remains to be determined.

Docosahexaenoic acid (DHA) prevents corticosterone-induced changes in astrocyte morphology and function.

The many functions of astrocytes, such as glutamate recycling and morphological plasticity, enable them to stabilize synapses environment and protect neurons. Little is known about how they adapt to glucocorticoid-induced stress, and even less about the influence of dietary factors. We previously showed that omega-3 polyunsaturated fatty acids (ω3PUFA), dietary fats which alleviate stress responses, influence the way astroglia regulate glutamatergic synapses. We have explored the role of docosahexaenoic acid (DHA), the main ω3PUFA, in the astroglial responses to corticosterone, the main stress hormone in rodents to determine whether ω3PUFA help astrocytes resist stress. Cultured rat astrocytes were enriched in DHA or arachidonic acid (AA, the main ω6PUFA) and given 100 nM corticosterone for several days. Corticosterone stimulated astrocyte glutamate recycling by increasing glutamate uptake and glutamine synthetase (GS), and altered the astrocyte cytoskeleton. DHA-enriched astrocytes no longer responded to the action of corticosterone on glutamate uptake, had decreased GS, and the cytoskeletal effect of corticosterone was delayed, while AA-enriched cells were unaffected. The DHA-dependent anti-corticosterone effect was related to fewer glucocorticoid receptors, while corticosterone increased DHA incorporation into astrocyte membranes. Thus, DHA helps astrocytes resist the influence of corticosterone, so perhaps promoting a sustainable response by the stressed brain. We show that corticosterone increases the glutamate recycling capacity of rat cortical astrocytes in culture, and alters their morphology, which may be detrimental in the long term. Increasing the membrane incorporation of docosahexaenoic acid (DHA), the main omega-3 in brain, reduces the amount of glucocorticoid receptors (GR) and prevents the effects of corticosterone. This may help the astrocytes maintain a functional phenotype in chronic stress situations.

Insights into the emulsification mechanism of the surfactant-like protein oleosin.

Oleosins are proteins with a unique central hydrophobic hairpin designed to stabilize lipid droplets (oleosomes) in plant seeds. For efficient droplet stabilization, the hydrophobic hairpin with a strong affinity for the apolar droplet core is flanked by hydrophilic arms on each side. This gives oleosins a unique surfactant-like shape making them a very interesting protein. In this study, we tested if isolated oleosins retain their ability to stabilize oil-in-water emulsions, and investigated the underlying stabilization mechanism. Due to their surfactant-like shape, oleosins when dispersed in aqueous buffers associated to micelle-like nanoparticles with a size of ∼33 nm. These micelles, in turn, clustered into larger aggregates of up to 20 µm. Micelle aggregation was more extensive when oleosins lacked charge. During emulsification, oleosin micelles and micelle aggregates dissociated and mostly individual oleosins adsorbed on the oil droplet interface. Oleosins prevented the coalescence of the oil droplets and if sufficiently charged, droplet flocculation as well.

Influence of emulsifier on lipid oxidation in spray-dried microencapsulated O/W emulsions.

Lipid oxidation limits the shelf-life of dried microencapsulated oils (DMOs), such as infant formula. However, it is poorly understood how lipid oxidation is affected by different types of emulsifiers. To improve our understanding, we prepared DMOs with different emulsifiers (whey protein isolate (WPI), pea protein isolate (PPI), and non-proteinaceous CITREM) and studied lipid oxidation in both the free and encapsulated fat. Only a small difference in oxidation rate was observed between these fat fractions for all formulations. We ascribed this to a non-discrete distribution of the fractions and the subsequent low fractionation selectivity as shown by Raman microscopy. The DMO with PPI showed hardly any oxidation during a 7-week incubation at 40 °C, whereas the DMOs with WPI and CITREM both reached significantly higher contents of oxidation products (lipid hydroperoxides, aldehydes, and epoxides). The enhanced stability of DMO-PPI could not be ascribed to the presence of phytic acid. In conclusion, we demonstrate the potential of using PPI to produce oxidatively stable DMOs.

Droplet size dependency and spatial heterogeneity of lipid oxidation in whey protein isolate-stabilized emulsions.

Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification (respectively producing mono- and polydisperse droplets) on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. We further used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.

Lipid oxidation in emulsions: New insights from the past two decades.

Lipid oxidation constitutes the main source of degradation of lipid-rich foods, including food emulsions. The complexity of the reactions at play combined with the increased demand from consumers for less processed and more natural foods result in additional challenges in controlling this phenomenon. This review provides an overview of the insights acquired over the past two decades on the understanding of lipid oxidation in oil-in-water (O/W) emulsions. After introducing the general structure of O/W emulsions and the classical mechanisms of lipid oxidation, the contribution of less studied oxidation products and the spatiotemporal resolution of these reactions will be discussed. We then highlight the impact of emulsion formulation on the mechanisms, taking into consideration the new trends in terms of emulsifiers as well as their own sensitivity to oxidation. Finally, novel antioxidant strategies that have emerged to meet the recent consumer's demand will be detailed. In an era defined by the pursuit of healthier, more natural, and sustainable food choices, a comprehensive understanding of lipid oxidation in emulsions is not only an academic quest, but also a crucial step towards meeting the evolving expectations of consumers and ensuring the quality and stability of lipid-rich food products.

A mechanistic kinetic model for lipid oxidation in Tween 20-stabilized O/W emulsions.

Models predicting lipid oxidation in oil-in-water (O/W) emulsions are a requirement for developing effective antioxidant solutions. Existing models do, however, not include explicit equations that account for composition and structural features of O/W emulsions. To bridge this gap, a mechanistic kinetic model for lipid oxidation in emulsions is presented, describing the emulsion as a one-dimensional three phase (headspace, water, and oil) system. Variation in oil droplet sizes, overall surface area of oil/water interface, oxidation of emulsifiers, and the presence of catalytic transition metals were accounted for. For adequate predictions, the overall surface area of oil/water interface needs to be determined from the droplet size distribution obtained by dynamic and static light scattering (DLS, SLS). The kinetic model predicted well the formation of oxidation products in both mono- and polydisperse emulsions, with and without presence of catalytic transition metals.

Unravelling the effect of droplet size on lipid oxidation in O/W emulsions by using microfluidics.

Lipid oxidation in emulsions is hypothesised to increase with decreasing droplet size, as this increases the specific oil-water interfacial area, where lipid oxidation is expected to be initiated. In literature, however, contradictory results have been reported, which can be caused by confounding factors such as the oil droplet polydispersity and the distribution of components between the available phases. In this work, monodisperse surfactant-stabilised emulsions with highly controlled droplet sizes of 4.7, 9.1, and 26 µm were produced by microfluidic emulsification. We show that lipid oxidation increases with decreasing droplet size, which we ascribe to the increased contact area between lipids and continuous phase prooxidants. Besides, a significant amount of oxygen was consumed by oxidation of the surfactant itself (Tween 20), an effect that also increased with decreasing droplet size. These insights substantiate the importance of controlling droplet size for improving the oxidative stability of emulsions.

Separation of flavonoid isomers by cyclic ion mobility mass spectrometry.

Analytical techniques, such as liquid chromatography coupled to mass spectrometry (LC-MS) or nuclear magnetic resonance (NMR), are widely used for characterization of complex mixtures of (isomeric) proteins, carbohydrates, lipids, and phytochemicals in food. Food can contain isomers that are challenging to separate, but can possess different reactivity and bioactivity. Catechins are the main phenolic compounds in tea; they can be present as various stereoisomers, which differ in their chemical properties. Currently, there is a lack of fast and direct methods to monitor interconversion and individual reactivity of these epimers (e.g. epicatechin (EC) and catechin (C)). In this study, cyclic ion mobility mass spectrometry (cIMS-MS) was explored as a potential tool for the separation of catechin epimers. Formation of sodium and lithium adducts enhanced IMS separation of catechin epimers, compared to deprotonation and protonation. Baseline separation of the sodium adducts of catechin epimers was achieved. Moreover, we developed a fast method for the identification and semi-quantification of cIMS-MS separated catechin epimers. With this method, it is possible to semi-quantify the ratio between EC and C (1:5 to 5:1, within 50-1200 ng mL-1) in food samples, such as tea. Finally, the newly developed approach for cIMS-MS separation of flavonoids was demonstrated to be successful in separation of two sets of positional isomers (i.e. morin, tricetin, and quercetin; and kaempferol, fisetin, luteolin, and scutellarein). To conclude, we showed that both epimers and positional isomers of flavonoids can be separated using cIMS-MS, and established the potential of this method for challenging flavonoid separations.

Bacterial lipoxygenases: Biochemical characteristics, molecular structure and potential applications.

Lipoxygenases (LOXs) are enzymes that catalyze dioxygenation of polyunsaturated fatty acids into fatty acid hydroperoxides. The formed fatty acid hydroperoxides are of interest as they can readily be transformed to a number of value-added compounds. LOXs are widely distributed in both eukaryotic and prokaryotic organisms, including humans, animals, plants, fungi and bacteria. Compared to eukaryotic enzymes, bacterial enzymes are typically easier to produce at industrial scale in a heterologous host. However, many bacterial LOXs were only identified relatively recently and their structure and biochemical characteristics have not been extensively studied. A better understanding of bacterial LOXs' structure and characteristics will lead to the wider application of these enzymes in industrial processes. This review focuses on recent findings on the biochemical characteristics of bacterial LOXs in relation to their molecular structure. The basis of LOX catalysis as well as emerging determinants explaining the regio- and enantioselectivity of different LOXs are also summarized and critically reviewed. Clustering and phylogenetic analyses of bacterial LOX sequences were performed. Finally, the improvement of bacterial LOXs by mutagenesis approaches and their application in chemical synthesis are discussed.

Quantitative assessment of epoxide formation in oil and mayonnaise by 1H-13C HSQC NMR spectroscopy.

Lipid oxidation is detrimental for the quality of oil-based foods. Historically, lipid oxidation research focussed on hydroperoxides and aldehydes, but a third class, the epoxides, have been proposed to resolve observed mechanistic anomalies. Here, we developed a 2D 1H-13C HSQC NMR spectroscopic method to quantify epoxides in food in a reproducible (relative standard deviation ≤11.6 %) and sensitive (LoQ 0.62 mmol/kg oil) manner. Lipid hydroperoxides, aldehydes, and epoxides generated in rapeseed oil and mayonnaise were quantified over time by NMR. Epoxides accounted at most for 10-40 % of the products. They were formed after hydroperoxide accumulation, most likely primarily via alkoxyl radical intermediates, which limits their potential as an early oxidation marker. As 99 % and ∼60 % of the epoxide signal intensities were assigned in a fatty acid and sub-structure specific manner, respectively, our quantitative HSQC method will enable unravelling and quantitative modelling of lipid oxidation mechanisms.

Evaluation of oxygen partial pressure, temperature and stripping of antioxidants for accelerated shelf-life testing of oil blends using 1H NMR.

Lipid oxidation compromises the shelf-life of lipid-containing foods, leading to the generation of unpleasant off-flavours. Monitoring lipid oxidation under normal shelf-life conditions can be time-consuming (i.e. weeks or months) and therefore accelerated shelf-life conditions are often applied. However, little is known on their impact on the lipid oxidation mechanisms. In this study, different oxygen partial pressures (PO2; 10 and 21%), temperatures (20, 30 and 40 °C), and the removal of antioxidants through stripping of the oil were tested to accelerate lipid oxidation. Increasing the incubation temperature of stripped oil blends from 30 to 40 °C reduced the onset of lipid oxidation from 4 to 2 weeks, whereas the PO2 had no impact. Surprisingly, at room temperature, an increase in PO2 resulted in a longer onset time (10 weeks under 10% oxygen, 15 weeks under 21% oxygen). We hypothesize that this is due to a shift in (initiation) mechanism. In non-stripped oil, an increase in PO2 from 10 to 21% decreased the onset time from 16 to 10 weeks (40 °C). Temperature elevations and stripping led to a shift towards more trans-trans diene hydroperoxides, as compared to the cis-trans conformation. Additionally, oil stripping led to an increase in oxidized PUFAs with three or more double bonds in which the hydroperoxide group is located between the double bond pattern, instead of on the edge of it. Lastly, it was shown that small additions of LC-PUFAs (0, 0.3, 0.6, 1.2 and 2.3%, w/w) accelerate lipid oxidation, even in relatively stable stripped oils. In conclusion, increased PO2 and slightly elevated temperatures hold fair potential for accelerated shelf-life testing of non-stripped oils with a limited impact on the lipid oxidation mechanisms, whereas stripping significantly changes propagation mechanisms.

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

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.