Oxidation of PUFA ethyl esters with NMBHA. Adapted from ref ; approximately 50 mg (≈17 µmol) of esterified fatty acid was diluted in 2 mL of anhydrous CH3CN, mixed with 7 mg of V70 (≈24 µmol), followed by addition of 25 mg of N-methyl benzohydroxamic acid (NMBHA) (≈17 µmol), and incubated for 24 hours opened to room air (i.e. 21% O2) at 37 °C in sand bath. The oxidation product was purified to eliminate NMBHA, the unreacted PUFA and other byproducts with Flash-LC employing a Reveleris X2 system from Buchi (Eastern Switzerland) and a puriflash 30SI-JP/12g 30 µm column from Interchim (Montluçon, France). Solvent was eliminated with rotavap, resuspended in 1 mL of cyclohexane with a few drops of ethyl acetate until soluble material could be injected into the Flash-LC system. The separation gradient was cyclohexane (0.1% acetic acid) (A) and ethyl acetate (0.1% acetic acid) (B) as eluents, and the flow rate was set at 30 mL/min, (25mL per tube). The proportion of B was gradually increased from 0% to 10% over 6.2 minutes, then to 23% over 2.3 minutes, then maintained isocratic for 4.7 minutes and increased to 100% over 0.7 minutes which was held 5 minutes for a total run time of 18.9 minutes. The elution of the unreacted PUFA, oxPUFA, and the NMBHA were controlled by TLC and only the fractions of oxPUFA were finally collected. It should be noted that the amount of NMBHA employed could be very problematic if it was injected concentrated into the LC-MS system so this cleaning step is mandatory. The oxPUFA fractions were concentrated with rotavap collecting approximately 25 mg of oxPUFA of which 5 mg were diluted in 2 mL of water to reach the same concentration as in the H2O2 oxidation. Then, it was proceeded to saponification, SPE, P(OMe)3 reduction and IS dilution such as in the H2O2 oxidation.