LC-MS neg-3

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.

称取10 mg冷冻脑组织置于艾本德管（Eppendorf tube）中，加入200 µL甲醇-水混合液（体积比1:1）进行匀浆。充分研磨后，添加200 µL三氯甲烷，将混合物涡旋振荡15秒，冰浴孵育15分钟，随后于4 °C、10,000 × g条件下离心15分钟。待两相分离完成后，分别将上层和下层溶液转移至新离心管（上层用于极性代谢物分析，下层用于脂质分析）。重复上述萃取流程3次，合并各相的萃取液，经氮气吹干后于−80 °C低温保存，以待后续分析。
将冻干后的极性组分复溶于100 µL乙腈-水混合液（体积比1:1），脂质组分则用125 µL氯仿-甲醇混合液（体积比1:1）溶解。将溶液置于4 °C、15,000 × g条件下离心15分钟，收集上清液用于液相色谱-质谱（LC-MS）分析。
采用AB X500R QTOF高分辨飞行时间质谱仪（AB Sciex，美国），通过LockSpray离子源与Waters Acquity超高效液相色谱（UPLC）系统（美国马萨诸塞州米尔福德，Waters公司）联用完成UPLC-QTOF-MS分析。色谱分离采用Eclipse Plus C18色谱柱（1.8 μm，4.6 × 100 mm；安捷伦科技，美国加利福尼亚州圣克拉拉），流动相流速为0.3 mL/min，其中流动相A为水/甲醇/乙腈（体积比3:1:1，含5 mM乙酸铵），流动相B为异丙醇，用于LC-MS/MS分析。采用18分钟的液相色谱梯度洗脱程序：0–0.5 min时流动相B占比25%；0.5–1.5 min时，B相占比从25%升至40%；1.5–3 min时，B相占比从40%升至60%；3–13 min时，B相占比从60%升至98%；13–13.1 min时，B相占比从98%降至25%；13.1–18 min时，B相占比维持25%。质谱分析采用分辨率模式，分辨率约为20,000，电喷雾离子源采用正离子模式。数据以信息依赖性采集（Information-Dependent Acquisition，IDA）模式采集，采集质荷比范围为80–1,200的质心数据，低碰撞能量设置为4 V（阱碰撞能量），高碰撞能量采用30–50 V的梯度模式，单次扫描时长为0.3秒。色谱柱温度维持在40 °C，进样体积为1 μL。