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A targeted approach has been applied to quantitative analysis of eicosanoids derived from omega-6 fatty acids in serum from individuals diagnosed with coronary artery disease (CAD). The target metabolites were series-2 prostaglandins, thromboxane B2, hydroxyeicosatetraenoic acids, and hydroxyoctadecadienoic acids. The method was based on SPELC-MS/MS in selected reaction monitoring mode for highly selective and sensitive determination of the target eicosanoids. The combination of SPE and LC-MS/MS involved the benefits from both direct analysis of serum without a step for protein precipitation and fully automation of the analysis. The method allowed comparison of omega-6-derived eicosanoids in serum from patients diagnosed with CAD and from control individuals. The effect of treatment with aspirin on the profile of the target compounds was evaluated through its incidence on the different pathways. Finally, the serum levels of the target metabolites in patients diagnosed with CAD were also statistically examined according to the severity of the coronary lesion stratified as stable angina, non-ST-elevation acute coronary syndrome, and acute myocardial infarction.