To encompass the broad spectrum of oxylipins produced from PUFAs, various oxidation protocols were applied to DHA, ALA, AA, and EPA acids and esters. The objective was to generate precise samples for encompassing the chemical signature of NEO-PUFAS (oxPUFA data in the text). Oxidation of PUFAs with H2O2.Approximately 5 mg of free fatty acid (≈17 µmol) were dissolved in 100 µL of ethanol. Subsequently, it was mixed with a 1.8 mL Tyrode's solution and 100 µL H2O2 solution (120 mM in water) to reach a final volume of 2 mL and approximate concentrations of 8.5 mM and 6 mM for PUFA and H2O2, respectively (i.e., 1.4 PUFA/H2O2 molar ratio). The oxidation proceeded for 30 minutes, then the reaction mixture was divided in two (1 mL each) aliquots. In one aliquot, 0.9 mL of 1M KOH in water was added and incubated for 30 minutes at 40 °C with mid rotation in tube rotator. After this KOH saponification, 100 µL of formic acid was added to obtain an acidic pH of 4. In the other aliquot, only 1 mL of acidified water with formic acid (pH = 4) was added. Of note: this alkaline hydrolysis step was included as a control experiment since best practice for NEO-PUFAs profiling requires such step in the extraction procedure of plasma/tissues (to the opposite of oxylipins), and that it was reported to transform or degrade a few NEO-PUFA/oxylipin structures . Other oxylipin metabolites possessing 3,5-disubstituted 1,2dioxolane units (endoperoxides) are also known to rearrange under basic conditions via the Kornblum-DeLaMare reaction onto ketohydroxyl derivatives (i.e; PGG2 into PGE2), however the fate of endoperoxide metabolites (derived from initial 5-exo-trig cyclization followed by oxygenation of peroxyl radical precursor) remains uncertain. On model studies , linear 1,2dioxolanes do not rearrange under our alkaline conditions (see Supplementary Information Figure ). Other endoperoxide derivatives (like PGGs) were not investigated. Each aliquot (i.e., saponified and non-saponified) was purified by automated solid phase extraction (SPE) process employing Extrahera system from Biotage (Uppsala, Sweden) and Oasis Max cartridge as stationary phase (60 mg, 30 µm particle size) from Waters (Mildford, MA, USA). The SPE procedure was optimized for the extraction of oxylipins in our previous works ; briefly, after the conditioning and sample loading, the cartridge was washed with 2 mL each of 2% NH3 solution, MeOH/formic acid (20 mM, 30:70), pure hexane, and hexane/ethyl acetate (70:30). Subsequently, elution is carried out using 1.5 mL of hexane/EtOH/acetic acid (70:29.4:0.6). Finally, each aliquot elution was divided in two again (750 µL each) and 50 µL of P(OMe)3 was added in one of the aliquots and incubated 30 min at room temperature in order to reduce hydroperoxide derivatives and bicyclic endoperoxides, keeping intact linear endoperoxides (1,2-dioxolanes) . The robustness of the linear endoperoxides was demonstrated through reduction tests on an isolated compound (Supplementary Information Figure ).