The midbrain and SN samples were weighed and then homogenized using Tissue Lyzer (QIAGEN, Hilden, Germany) with 400 μl of methanol. The homogenate was incubated for 15 min at 4 °C. 200 μl of 1 μM dopamine-1,1,2,2-d4 hydrochloride, internal standard, was added into the sample after incubation, and mixed well. The sample was then centrifuged at 14,000 r.p.m. for 15 min. The supernatant was collected and equal volume of 1% formic acid was added. The sample was mixed well and ready for solid phase extraction. Oasis wax 3cc cartridge was conditioned with 1 ml of methanol and 0.5% formic acid, sequentially. The sample solution was loaded to the cartridge and incubated for 10 min. Then the cartridge was completely dried under vacuum. Finally, 1 ml of methanol was added for sample elution, and the eluant was dried under vacuum. The dried sample was stored at −20 °C until analysis and reconstituted with 20 μl of 50% methanol before Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis.
