https://doi.org/10.3390/cells11244110
“Gas chromatography flame ionization detection (GC-FID) was performed to identify and quantify FAs. Lipids were extracted from human milk following methodologies adapted from Folch et al. [40]. Briefly, 300 μL of human milk were homogenised and extracted in 4:2:1.75 chloroform– methanol–potassium chloride (0.88%) with 50 μg of C22:3n-3 used as an internal standard (NuChek-Prep, Elysian, MN, USA) as described previously [41]. Extracted samples were then to be dried down under nitrogen gas and then methylated by heating at 100 °C in 1:1 hexane–boron trifluoride methanol (Sigma 5 Aldrich, St. Louis, MO, USA). Deionised water was then added to stop the methylation reaction. Next, hexane was added to isolate the fatty acid methyl esters (FAMEs). FAMEs were quantified according to previously published methods using a Varian-430 gas chromatograph (Varian, Seattle, WA, USA). A quantity of 1 μL of sample was injected (splitless mode) into a capillary column (Agilent DB-23, Santa Clara, CA, USA; 30 m × 0.25 mm internal diameter, 0.25 μm film thickness) used to separate the FAMEs. A temperature of 250 °C was used for both the injector and detector. The oven temperature was initially set at 50 °C for 2 min, then increased at 20 °C/min until a temperature of 170 °C was reached. After 1 min at 170 °C, the temperature was increased by 3 °C/min until a final temperature of 212 °C was reached and held for 5 min. Helium was used as the carrier gas (0.7 mL/min). Identification of absorbance peaks was conducted using certified FAMEs standards (Nu-Chek Prep, Inc., Elysian, MN, USA). FAMEs peak analysis was conducted using Compass CDS software (version 3.0.0.68; Scion Instruments, Goes, NL) by proportional comparisons with the C22:3n-3 internal standard.”