Tissue preparation and high-pressure liquid chromatogra­phy and electrochemical detection (HPLC-ECD)

DA and its major metabolites, HVA and DOPAC, were measured in the striatum by HPLC-ECD. Brain tissue blocks were rapidly sacrificed on ice and homogenized with ice-cold 0.4 M perchloric acid with 1× PBS, then incubated at –80°C overnight. After centrifugation at 15,000 rpm for 30 min at 4°C, the supernatant was filtered using an appropriate column (#SC1000-1Kt, Sigma Prep spin column, Sigma-Aldrich). Filtered supernatants were directly injected into the X-bridge column (XBridge^TM C18 5 µm, Waters Corp., MA, USA). The mobile phase consisted of 85 mM citric acid, 100 mM sodium acetate, 0.9 mM sodium-octyl sulfate, 0.2 mM ethylenediaminetetraacetic acid (EDTA), and 14% methanol at pH 3.8 (Tianzhu et al., 2014). The flow rate was kept constant at 1 mL/min. Chromatographic peak analysis was accompanied by identification of unknown peaks in a sample matched according to retention times.