Qtrap 5500 ms

Liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to evaluate the content of dopamine (DA) as well as the dopamine metabolites dihydroxy‐phenyl acetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. Tissue samples were ground in 500 μL of 80% methanol for 5 min and subsequently sonicated for 30 min at 4°C and let stand for an additional hour. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected and filtered through a 0.22 μm mesh prior to preparation for LC–MS/MS analysis. The various apparatuses used for LC–MS/MS analysis were as follows: liquid chromatography (Water Acuity UPLC); mass spectrography (AB SCIEX 5500 Qtrap‐MS); and chromatographic column (Acquity UPLC HSS T3; 1.8 μm, 2.1 mm × 100 mm).

For the chromatographic separation of organic acids, an Acquity BEH C18 column was used (1.7 µm, 2.1 × 100 mm; Waters, Eschborn, Germany). The flow rate was set to 0.4 mL/min, and 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) served as solvents. The gradient started with an isocratic step at 7% solvent B (2 min) and increased to 45% (7.5 min) and then to 100% solvent B (0.5 min). After isocratic elution (1 min), the starting conditions we re-established (0.1 min) followed by the equilibration of the system at 7% solvent B (2.9 min). The UPLC System (Shimadzu Nexera X2, Shimadzu, Duisburg, Germany) was coupled to a 5500 Q-Trap MS (Sciex, Darmstadt, Germany) with Analyst 1.6.2. Ionisation was performed in negative electrospray mode. The setting was 35 psi for curtain gas, −4500 V for ion spray voltage, 55 psi for heater gas, and 65 psi for turbo gas. The source temperature was set to 500 °C. MS/MS analysis was done in MRM mode, in which each transition was recorded with a dwell time of 5 ms. See Supporting Table 2 for MS/MS parameters.

All biological samples participated in LC–MS/MS measurements. All chromatographic separations were conducted on Shimadzu LC modular system (Kyoto, Japan) equipped with a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm, Milford, CT). Mobile phase consisting of 0.1% aqueous formic acid (A) and ACN (B) was delivered in gradient at a total flow rate of 0.2 mL/min by operating the program as follows: 0–4 min, 2–10% B; 4–20 min, 10–30% B; 20–25 min, 30–65% B; 25–28 min, 65–100% B; and 28–32 min, 100% B. After each analytical run, 2% B was delivered for another five minutes for the re-equilibration. Column oven and injection volume were set as 40 °C and 2 μL, respectively.
The column outlet was directly connected to ESI interface of either Qtof-MS (TripleTOF 6600⁺, SCIEX, Foster City, CA) or Qtrap-MS (5500 Qtrap-MS, SCIEX) device. Actually, the whole measurement consisted of three progressive steps: UPLC–Qtof-MS/MS for acquiring HR-m/z values of precursor and fragment ions, online ER-MS for obtaining OCEs of the targeted 1^st-generation fragment ions, and post-CID ER-MS for gaining the trajectories from selected 1^st-generation fragment ions to 2^nd-generation fragment ions.