4.5. Lipid Extraction

Plasma lipids were extracted using the Matyash method, with slight modifications [40,41]. In brief, human plasma (10 μL) was aliquoted into microcentrifuge tubes containing ice-cold 75% methanol (400 μL) with 0.1% butylated hydroxytoluene. MTBE (1 mL) was then added, and the mixture was shaken for 1 h at room temperature. For phase separation, 250 μL water was added, and tubes were centrifuged. Aliquots of the upper phase (110 μL) and lower phase (55 μL) were transferred to new tubes, dried under a nitrogen stream, and reconstituted in 100 μL chloroform/methanol (1:9, v/v), containing the internal lipid standard (IS) mixture (40–400 ng/mL).