3200 q trap lc ms ms system

For LPO, MDA, GSH, and GSSG assay, SN-VTA or Hip tissue block was weighed and homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer, PH 7.4. The homogenates were used to assess LPO, MDA, GSH, and GSSG spectrophotometrically using detection kits following the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, China).
For dopamine (DA) and metabolites assay, CPu or Hip tissue block was weighed and homogenized in 80% acetonitrile containing 0.1% formic acid (5 μL/mg). The homogenates were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatants were collected and stored at −80°C. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by LC-MS/MS. The LC separation was performed on Agilent 1200 LC system (Agilent, Santa Clara, USA) using a Synergi Fusion-RP C18 column (50 mm × 3.0 mm, 4 μm) provided by Phenomenex. MS/MS detection was carried out using a 3200 QTRAP™ LC-MS/MS System (Applied Biosystems, Foster City, CA, USA). The multiple-reaction monitoring mode was used for the quantification. The principal validation parameters of the LC-MS/MS were set up as shown in Table 1, based on the previous study [33 (link)].

For column chromatography, silica gel G60-230 (Merck, Germany) and Sephadex LH-20 (Sigma–Aldrich, USA) were used. Analytical thin layer chromatography (TLC) was performed on a pre-coated silica gel 60 GF₂₅₄ (Merck or Machery-Nagel, Germany). The 1D and 2D NMR spectra were performed on Bruker-400 Ascend^TM spectrometer using CDCl₃ or dimethyl sulfoxide deuterated (DMSO-d₆) as solvents. The electrospray mass spectrometry (ESMS) experiments were conducted with the 3200 Q-trap LC/MS/MS system (Applied Biosystems, Foster City, CA, USA) Analyst version 1.4.1 software (MDS Sciex; Toronto, CA, USA).
The TLC chromatogram of the obtained EtOAc extract [CH₂Cl₂–MeOH (95: 5 v/v)] revealed the presence of three major spots on visualization with 10% H₂SO₄ spray reagent and heating at 110°C for 1 min. The first spot (R_f 0.65) had no response both under UV₂₅₄ and under UV₃₆₆ lights; on visualization, however, it gave a pale orange color. The other two spots (R_f 0.25 and 0.39) quenched UV₂₅₄ light and gave a brown color.
The EtOAc extract (700 mg) was applied to a silica gel chromatographic column (35 g), packed in CH₂Cl₂ 100% and eluted with CH₂Cl₂–MeOH mixtures with different polarities to afford compounds 1 (R_f 0.65, 18 mg), 2 (R_f 0.39, 7 mg), and 3 (R_f 0.25, 10 mg). A detailed isolation procedure is presented in Supplementary Materials.