2.2. Fatty Acids Analysis

Total lipids were extracted with hexane:isopropanol 3:2 (v/v) [27] with appropriate adaptations for each food matrix [10]. Fatty acids were methylated through base-catalyzed transesterification [28] in order to avoid CLA isomerization. Briefly, lipid extracts were dissolved in n-hexane followed by addition of a NaOCH₃ solution in methanol (0.3 mol/L) and heating at 50 °C in a water bath for 10 min. Then, 10% HCl (w/v) in methanol was added and heated at 80 °C in a water bath for 10 min. The resulting fatty acid methyl esters (FAME) were extracted with hexane by centrifugation after addition of 28% NaCl solution (w/v in water). The upper hexane layer was evaporated, and the FAME were suspended in hexane and stored at −20 °C until analysis by gas-chromatography (GC). All samples were analyzed in triplicate.

FAME were analyzed in a GC-2010 gas chromatograph (Shimadzu, Tokyo, Japan) equipped with a flame ionization detector (FID), a split/splitless injector (with a 1:30 split ratio), and a polyethylene glycol capillary column (30 m, 0.32 mm i.d., 0.25 μm film thickness; Omegawax-320, Supelco Co., Bellefonte, PA, USA). In order to confirm CLA isomer distribution in dairy fat, two samples of butter were analyzed in a highly polar (88%-cyanopropyl) polysiloxane capillary column (60 m, 0.25 mm i.d., 0.25 μm film thickness; Agilent, Santa Clara, CA, USA). Detailed gas-chromatographic operational conditions were previously reported [29]. Relative retention times of commercial standards (37 FAME mix and CLA methyl esters; Sigma-Aldrich, São Paulo, Brazil) were used for identification of gas-chromatographic FAME peaks. The method described by Torres et al. (2002) [30] and the equivalent chain lengths system [31] were used as complementary tools for the identification of FAME peaks. The areas of the peaks were corrected by the theoretical response factors of Ackman and Sipos for the FAME responses of the flame ionization detector as described by Wolff et al. (1995) [32]. Content for an individual fatty acid was expressed as g/100 g total fatty acids. The AI of the dairy products was calculated based on the contents of lauric (C12:0), myristic (C14:0), and palmitic (C16:0) acids and the sum of unsaturated fatty acids (Equation (1)) [11]. All the materials used for analysis of fatty acids were rinsed with alcoholic KOH (2.5%, w/v) and soaked in HNO₃ solution (13%, w/v) for 2 h. All solvents were of chromatographic grade from Tedia (Fairfield, OH, USA). HPLC grade water (Milli-Q System, Millipore, Bedford, MA, USA) was used throughout the experiments.

where, AI: atherogenicity index; C12:0, C14:0, and C16:0 are contents (g/100 g fatty acids) of these saturated fatty acids; ΣUFA: sum of contents of all unsaturated fatty acids.