2.4. Analysis of Fatty Acid Methyl Esters

Plasma free fatty acids were measured as methyl esters by GC‐MS using internal standard methodology as previously described (15). Briefly, 20 µl of plasma was enriched with extraction surrogates including 20:3n3, and extracted with isopropanol/cyclohexane/ammonium acetate, solvent removed, and residues reconstituted in 1:1 methanol/toluene. Samples were enriched with 15:1n5 and fatty acids methylated with trimethylsilyl‐diazomethane in hexane (Sigma‐Aldrich, St. Louis, MO), dried under vacuum and reconstituted in hexane containing 23:0 for analysis. FAMEs were separated on an HP6890 GC equipped with a 30 m × 0.25 id × 0.25 µm DB‐225ms column (Agilent Technologies) and detected with a 5973N MSD with electron impact ionization. Analytes were quantified with ChemStation vE.02.14 software (Agilent) using internal standard methodologies against a 5‐ to 7‐point calibration curve bracketing all reported concentrations.