이전 사이트 이동

Plasma lipids were extracted using the Matyash method, with slight modifications. In brief, human plasma (10 µL) was aliquoted into microcentrifuge tubes containing iceꠓcold 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).

all lipids and the IS were separated on a Kinetex C18 column and analyzed using an LC-MS/MS system (LCMS 8060; Shimadzu, Kyoto, Japan). The mobile phase A consisted of a water/methanol mixture (1:9, v/v), with 10 mM ammonium acetate, and the mobile phase B consisted of an isopropanol/methanol mixture (5:5, v/v), with 10 mM ammonium acetate. The gradient elution was as follows: 0 min (30% B), 0–15 min (95% B), 15–20 min (95% B), and 20–25 min (30% B). The flow rate was 0.2 mL/min. Quantitation was performed in the selected reaction monitoring (SRM) of the [M + H]+ (or [M + NH4 + ]) ion and the related product ion for each lipid and IS.

An electrospray ionization interface was used as the ionization source in the positive mode. The optimum operating conditions were as follows: capillary temperature, 350 ◦C; vaporizer temperature, 300 ◦C; collision gas (argon) pressure, 1.5 mTorr.