Plasma Fatty Acid Extraction Protocol

FAs were extracted as described by Yang et al. [40 (link)]. Waters Oasis-HLB cartridges (30 mg/30 μm) were treated with ethyl acetate (1 mL), methanol (2 × 1 mL), and 95:5 v/v water/methanol containing 0.1% acetic acid (1 mL). We mixed 100 μL of plasma with 7 μL of internal standard solution (stock concentration: 500 nM), 10 μL of butylated hydroxytoluene (BHT; stock concentration: 2 mg/mL), and 120 μL of H₂O:methanol (MeOH) (95:5) containing 0.1% acetic acid. The resulting samples (240 μL) were then loaded onto pre-treated cartridges and washed twice with 750 μL of H₂O/MeOH (95:5) containing 0.1% acetic acid. The aqueous plug was pulled from the cartridges using a high vacuum, and the cartridges were then dried further under a low vacuum for about 20 min. Waters Oasis-HLB cartridges were eluted into tubes with 250 μL of methanol followed by 1 mL of ethyl acetate into 2 mL tubes containing 6 μL of 30% glycerol in MeOH, a trap solution. The samples were dried under nitrogen and then dissolved in 70 μL of methanol containing 20 nM of 1-cyclohexyl-dodecanoic acid urea. The samples were then vortexed for 5 min, transferred to autosampler vials with low-volume inserts, and stored at −20 °C until further analysis.