Gas chromatography/mass spectroscopy (GC/MS) analysis of plasma fatty acid compositions

Plasma fatty acids were methylated following previously reported methods but with minor modifications [16–18]. In brief, 20 μL of internal standards in hexane containing 1 mg/mL of methyl heptadecanoate, 0.5 mg/mL of methyl tricosanoate, and 28 mg/mL of butylated hydroxytoluene (BHT) were added to a Pyrex tube followed by the addition of 50 μL plasma and 1 mL of methanol/hexane mixture (4:l, v/v). After cooling down the tubes in a self-made liquid nitrogen bath for 10 min, 100 μL of precooled acetyl chloride was added to the mixture and then flushed briefly with nitrogen gas. The tubes were then screw-capped and kept at room temperature in the dark for 24 h. The resultant mixture was cooled in an ice bath for 10 min followed by the gradual addition of 2.5 mL of 6% K₂CO₃ solution (with shaking) to neutralize. After standing for another 30 min, 200 μL of hexane was added to extract methylated fatty acids. The mixture was rested for 10 min, and the top layer was transferred into a glass sample vial. This extraction process was further repeated twice, and the combined supernatants were evaporated to dryness. The resultant residues were re-dissolved in 50 μL of hexane followed with GC- flame ionization detector (FID)/MS analysis.

Methylated fatty acids were measured on a Shimadzu GC/MS-QP2010Plus spectrometer (Shimadzu Scientific Instruments, USA) equipped with a MS with an electron impact (EI) ion source and an FID. An Agilent DB-225 capillary GC column (10 m, 0.1 mm ID, 0.1 μm film thickness) was employed with a sample injection volume of 1 μL and a splitter (1:60). Helium gas was used as carrier and makeup gas. The injection port and detector temperatures were both set at 230 °C. The column temperature was set to 55 °C for 1 min and then increased to 205 °C with a rate of 30 °C/min. The column temperature was then kept at 205 °C for 3 min and then increased to 230 °C (5 °C/min). The MS spectra were acquired with the EI voltage of 70 eV and the m/z range of 45 to 450. Methylated fatty acids were identified by comparing with a chromatogram from a mixture of 37 known standards and further confirmed with their mass spectral data. Each fatty acid was quantified with the FID data from its signal integrals and internal standards. The results were presented as μmol of fatty acids per liter of plasma.