Ultra-high pressure liquid chromatography (UPLC) coupled with ECD was used to simultaneously measure DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT, 5-hydroxy-3-acetic acid (5-HIAA), and NE. A 10-μl aliquot was injected onto a Luna Omega 2.1 x 150 mm column (Phenomenex, Torrance, CA) coupled to a LPG-3400RS pump, WPS-3000TBRS autosampler, and a CoulArray electrochemical detector. The column was heated to 30°C. The mobile phase consisted of 50 mM sodium phosphate, 47 mM citric acid, 0.14 mM EDTA, 0.64 mM octanesulfonic acid, and 5% methanol, with a flow rate of 0.4 ml/min. The detector was set to sequentially deliver potentials of −150 mV, 150 mV, 400 mV, and 600 mV.
Standards for ECD were prepared by weighing approximately 1 mg of analytes DA (Sigma-Aldrich, Buchs, Switzerland), DOPAC (Sigma-Aldrich, Buchs, Switzerland), HVA (Sigma-Aldrich, Buchs, Switzerland), 5-HT (Sigma-Aldrich, St. Louis, MO), 5-HIAA (Sigma-Aldrich, St. Louis, MO), and NE (Sigma-Aldrich, St. Louis, MO). Each standard was transferred to a volumetric flask and diluted to volume with tissue buffer to create stock solutions. A stock solution containing approximately 10 μg/ml of analyte was then diluted to encompass a concentration range from 1000 to 0.5 ng/ml.
Standards for ECD were prepared by weighing approximately 1 mg of analytes DA (Sigma-Aldrich, Buchs, Switzerland), DOPAC (Sigma-Aldrich, Buchs, Switzerland), HVA (Sigma-Aldrich, Buchs, Switzerland), 5-HT (Sigma-Aldrich, St. Louis, MO), 5-HIAA (Sigma-Aldrich, St. Louis, MO), and NE (Sigma-Aldrich, St. Louis, MO). Each standard was transferred to a volumetric flask and diluted to volume with tissue buffer to create stock solutions. A stock solution containing approximately 10 μg/ml of analyte was then diluted to encompass a concentration range from 1000 to 0.5 ng/ml.