LC-MS pos-2

The frozen brain tissue (10 mg) was weighed into an Eppendorf tube and homogenized with 200 µL of methanol/water (v:v, 1:1). After thorough grinding, 200 µL of chloroform was added. The mixture was vortexed for 15 seconds, incubated on ice for 15 minutes, and centrifuged at 10,000 × g for 15 minutes at 4 °C. Following phase separation, the upper and lower layers were separately transferred into new tubes (the upper phase for polar metabolite analysis and the lower phase for lipid analysis). This extraction procedure was repeated three times, and the combined extracts from each phase were dried under a stream of nitrogen and stored at −80 °C until further analysis.
The lyophilized polar fractions were reconstituted in 100 µL of acetonitrile/water (v:v, 1:1), while the lipid fractions were dissolved in 125 µL of chloroform/methanol (v:v, 1:1). The solutions were centrifuged at 15,000 × g for 15 minutes at 4 °C, and the resulting supernatants were collected for LC-MS analysis.
UPLC-QTOF-MS analysis was performed using an AB X500R QTOF UPLC-QTOF-MS analysis was performed using an AB X500R QTOF high-resolution time-of-flight mass spectrometer (AB Sciex, USA) coupled via a LockSpray ion source to a Waters Acquity UPLC system (Milford, MA, USA). The chromatographic separation was achieved on an Eclipse Plus C18 column (1.8 μm, 4.6 × 100 mm; Agilent Technologies, Santa Clara, CA, USA) with a mobile phase (delivered at 0.3 mL/min) comprising solvent A (H2O/ MeOH/ACN, 3: 1: 1, v: v: v ; containing 5 mM ammonium acetate ) and solvent B (isopropanol) for LC-MS/MS-based analysis. A 18 min LC gradient program was used, consisting of 0–0.5 min, 25% B; 0.5–1.5 min, 25–40% B; 1.5–3 min, 40–60% B; 3–13 min, 60–98% B; 13–13.1 min, 98–25% B; 13.1–18 min, 25–25% B. The mass spectrometry was achieved in resolution mode with a resolving power of approximately 20,000 and the electrospray ion source was operated in the positive ion mode. Data were acquired in IDA (Information-Dependent Acquisition) mode in centroid form over an m/z range of 80–1,200, with low collision energy (trap collision energy, 4 V) and ramped high collision energy (30–50 V), and a scan time of 0.3 seconds. The column temperature was maintained at 40℃, and the sample injection volume was 1 μL.