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Not AvailableBackground: Fish oils, which are rich in health promoting polyunsaturated fatty acids (PUFA), have emerged as promising functional foods in the global health and wellness food market. Their source regarding the fish type, season, and location of harvesting might influence the nutritional value of such bioactive oils and determine their market price. The differences in price among such oils often lead to economically motivated mislabeling and adulteration. Objective: In this study, our objective was to demonstrate how a qualitative targeted shotgun lipid profile workflow using an electrospray ionization–quadrupole-linear ion trap MS could differentiate fish oils originating from two different species. Methods: Five samples each of sardine oil and shark liver oil were diluted to a concentration of 80 microgram per millilitre in chloroform–methanol with 5 mM ammonium acetate. These samples were directly infused into a QTrap MS. The data were acquired for 23 precursor ion and 4 neutral loss scan experiments in the positive ionization mode and compared. Results: We identifed the following major lipid classes: cholesteryl ester, diacyl glycerol, triacylglycerol, monoalkyldiacylglycerol, and phophatydyl choline. The relative peak areas of the identifed lipid species, when subjected to supervised multivariate analysis, could effectively distinguish the sardine oil and shark liver oil. Conclusions: The approach will be useful in establishing authenticity of fish oil and to support the regulatory agencies in dispute resolution. It can also be extended to establish authenticity in other agricultural and food commodities. Highlights: This paper reports a proof of concept for authenticating PUFA-rich fish supplements. A shotgun targeted lipidomics profile and chemometrics modeling successfully discriminated sardine oil and shark liver oil.Not Availabl