2.2. Polyunsaturated Fatty Acid Laboratory Analysis

Plasma samples (n = 55 maternal and n = 55 cord) were analyzed for n-3 PUFAs including α-linolenic acid (ALA), docosahexaenoic acid (DHA), n-3 docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), and total n-3 PUFAs (sum of ALA, EPA, DPA, and DHA), as well as the n-6 PUFAs arachidonic acid (AA) and linoleic acid (LA), and total n-6 PUFAs (sum of AA, LA, n-6 docosapentaenoic acid, eicosadienoic acid, dihomo-γ-linolenic acid, and docosatetraenoic acid). Quantitation of these nutrients was conducted using gas chromatography with flame ionization detection (GC-FID) at OmegaQuant Analytics LLC (Sioux Falls, SD, USA). Plasma was transferred to a screw-cap glass vial, and boron trifluoride–methanol (BTM) solution (methanol containing 14% boron trifluoride, toluene, methanol; 35:30:35 v/v/v; Sigma-Aldrich, St. Louis, MO, USA) and an internal standard were added. The vial was briefly vortexed and heated in a bath at 100 °C for 45 min. After cooling, hexane (Merck KGaA, Darmstadt, Germany) and high-performance liquid chromatography-grade water were added. The tubes were recapped, vortexed, and centrifuged to separate layers. An aliquot of the hexane layer was transferred to a GC vial. GC was conducted using a GC-2010 Gas Chromatograph (Shimadzu Corporation, Columbia, MD, USA) equipped with an SP-2560 100 m fused silica capillary column (0.25 mm in internal diameter, 0.2 um in film thickness; Supelco, Bellefonte, PA, USA). Fatty acids were identified by comparison with a standard mixture of fatty acids (GLC OQ-A; NuChek Prep, Elysian, MN, USA), which was also used to determine individual fatty acid calibration curves.