2.3. Fatty Acid Analysis

Human milk samples were stored at −80 °C until analysis of fatty acids between 2015 and 2018 by high-resolution capillary gas-liquid chromatography by using the method of Bligh and Dyer [18] and Beermann et al. [19]. The lipid content was analyzed using the method outlined by Lucas et al. [20] and Jones et al. [21]. Data are reported for 45 fatty acids which include saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), trans-fatty acids (TFAs), branched-chain fatty acids (BFAs), and polyunsaturated fatty acids (PUFAs). Peak identification of SFAs, MUFAs, TFAs, and both n-3 and n-6 PUFA methyl esters were verified and calibrated by comparison with authentic standards (NuChek Prep; Elysian, MN, USA: GLC-463, GLC-473, GLC-642, GLC-643, GLC-674, and Sigma-Aldrich Ltd., St. Louis, MO, USA: Supelco PUFA3). The standard mix of BFA methyl esters was originally from Danone Nutricia Research, Utrecht, the Netherlands. Fatty acid concentrations were recorded as %weight of total fatty acids.