GC-MS Analysis of Serum Free Fatty Acids

The analysis of FFAs was performed by Agilent gas chromatography-mass spectrometry (GC-MS) system composed of 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph, and 7673 autosampler, with a dedicated previously described protocol (Baldi et al., 2021 (link)). The chemicals, GC-MS conditions, and calibration parameters were reported in supporting information SX. Just before the analysis, each sample was thawed. The FFAs were extracted as follows: an aliquot of 300 μl of serum sample was added to 10 μl of ISTD mixture, 100 μl of tert-butyl methyl ether, and 20 μl of 6 M HCl + 0.5 M NaCl solution in 0.5 ml centrifuge tube. Afterward, each tube was stirred in a vortex for 2 min, centrifuged at 10,000 rpm for 5 min, and finally, the solvent layer was transferred to a vial with a microvolume insert and analyzed.