GC-MS Analysis of Fatty Acids

Total lipids were extracted from serum aliquots with a mixture of chloroform-methanol (2:1, v/v), as described by Folch et al. [13]. The chloroform phase was collected, dried under a nitrogen stream, and hydrolyzed with 1 mL of 0.5 M KOH in methanol at 90 °C. After 3 h of incubation, the mixture was acidified with 6 M HCl, 1 mL H₂O was added, and free FAs were extracted thrice with 1 mL of n-hexane. Fatty acid methyl esters (FAMEs) were prepared by derivatization with 10% BF₃-methanol solution (55 °C, 1.5 h). Water was added to the mixture, and FAMEs were extracted thrice with n-hexane, as described above. The samples were dried under a nitrogen stream and stored at – 20 °C until analysis.

The analysis of FAMEs was conducted using a GC-EI-MS QP-2010SE spectrometer (Shimadzu, Kyoto, Japan) on a Zebron ZB-5MSi capillary column, 30 m × 0.25 mm i.d. × 0.25-μm film thickness (Phenomenex, Torrance, CA, USA). The analysis run time was 60 min with the column oven set at 60–300 °C (4 °C/min). The carrier gas was helium with the column head pressure at 100 kPa. The electron impact source for mass spectrometry detection operated at 70 eV, with the mass scan range set at m/z 45–700 in the full scan mode. The identification of FAs was aided by the use of reference standards (37 FAME Mix, Sigma Aldrich, St. Louis, MO, USA), and the reference library NIST 11. 19-methylarachidic served as an internal standard.