Agilent 7890 series 2 and 5975c

Blood samples were collected after an overnight fasting period. Samples were centrifuged (3000× g, 10 min, 4 °C), and stored in plastic vials at −80 °C.
The profile of the FAs was assessed using Glaser’s method [2 (link)]. One hundred microliters of serum, 100 μL of an internal standard (146 µg PC15:0/mL methanol), and 0.6 mL of cold methanol were combined in glass tubes and shaken for 30 s. After centrifugation (2300× g, 10 min, 4 °C), the supernatant was transferred into a glass vial. FA methyl esters (FAMEs) were synthesized at room temperature by adding 25 µL sodium methoxide (25 wt % in methanol; Sigma Aldrich, Saint Louis, MO, USA). The transesterification reaction was stopped after 3 min by adding 75 µL 3 M methanolic HCl. FAMEs were extracted twice into 2× 300 µL hexane. The extracts were combined and the solvent was evaporated under nitrogen. FAMEs were redissolved in 50 µL hexane containing butylated hydroxytoluene (2 g/L) and stored at −20 °C until further analysis.
Individual FAMEs were quantified by gas chromatography with mass spectrum (Agilent 7890 series II and 5975C, Agilent Technologies, Santa Clara, CA, USA) using a BPX 70 column (BPX70, 25 m × 0.22 mm ID × 0.25 μm, SGE Analytical Science, Ringwood, Australia) as described previously [2 (link)]. Peak integration was performed using MSD ChemStation (Agilent Technologies, Santa Clara, CA, USA).

Total FAs were assessed using a modified Gotlieb's method [22 (link)]. The samples were prepared for processing by the addition of 0.3 mL of concentrated H₂SO₄ and maintaining 65°C for 15 minutes. Then, 3 mL of ethanol (99%) were added and the sample was vortexed. The extraction was performed twice with 2 mL of n-hexane and centrifugation at 2300 rpm for 10 minutes. The supernatant was dried in nitrogen at room temperature. All the samples were frozen without delay at -20°C. Ten μL of the extract were vortexed with 20 μL of 2N methanolic KOH. Then, 1 mL of n-hexane was added, the sample was vortexed and left for 5 minutes at room temperature. For the analysis, 200 μL of the extract were placed in the chromatographic vial. Gas chromatography with mass spectrometry was conducted using Agilent 7890 series II and 5975C (Agilent Technologies, Santa Clara, CA, USA) with BPX70 column (SGE Analytical Science, Ringwood, Australia). Peaks were analyzed using MSD ChemStation (Agilent Technologies).