Monoamine analysis

To evaluate the levels of dopamine and its metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] in the brain, tissue was homogenized in 0.2-M perchloric acid (Wako Pure Chemicals) containing 100-μM EDTA-Na₂ (MilliporeSigma). After centrifugation, the supernatant was analyzed by HPLC using an Eicompak SC-5ODS column and PrePak column (Eicom, Kyoto, Japan) with electrochemical detection (ECD). The mobile phase consisted of 83% 0.1-M acetic acid in citric acid buffer (pH 3.5), 17% methanol (Wako Pure Chemicals), 190 mg/ml of sodium 1-octanesulfonate sodium (Wako Pure Chemicals), and 5-mg/ml EDTA-Na₂. For ECD, the applied voltage was 750 mV vs. an Ag/AgCl reference electrode.