Loss of Products in Lipid Handling Procedures Compromises Accurate Analysis of Active Lipid Oxidation Pathways and Oxidation Extent

Multiple pathways that compete with the traditional free radical chain to generate products other than hydroperoxides and aldehydes have been increasingly recognized. This presents new challenges and incentives for tracking and comparing the evolution of more products, including epoxides, to assess lipid oxidation progress accurately. Significant discrepancies in the detection of epoxides in different laboratories led to the question of whether routine procedures used in handling lipids might cause loss of some types of products and thus skew views of active lipid oxidation pathways and oxidation extent.

To assess this possibility, synthetic standards of epoxides, aldehydes, and fatty acid methyl esters were dissolved in different solvents and carried through procedures used commonly in lipid extraction (manual, pressurized solvent extraction, Soxhlet, Bligh-Dyer), sample handling (filtration or separation through different media), and storage (three temperatures). These solutions were also mixed with microcrystalline cellulose to mimic food matrices and then extracted via Soxhlet and pressurized solvent extraction. Products were analyzed before and after procedures via gas chromatography with DB-5 and CP-Sil 88 columns and both normal phase and reserve phase high-performance liquid chromatography to quantitate loss and distinguish degradation of products from binding of products to media as sources of loss.

All procedures tested resulted in significant losses of the oxidation products tested. Notably, gravity filtration caused >50% losses of all products, especially at low concentrations, due to evaporation, binding to filters, and degradation. Vacuum filtration improved the recovery of short-chain epoxides and methyl esters from cellulose to ~75% and from glass fibers to 90%; recovery of aldehydes and long-chain epoxides was much lower. Soxhlet extractions accelerated thermal degradation. These observations remind us that sample handling contributes to the loss and degradation of lipid oxidation products, thereby compromising the accurate detection of lipid oxidation levels and interpretation of active pathways.