Monoamine levels in the NAc were quantified by HPLC with electrochemical detection as described in our previous publications [5] (link).
Briefly, NAc was homogenized in 0.01 M HClO4 and centrifuged at 14, 000×g for 15 min. DA, DOPAC, HVA, 5-HT, and 5-HIAA levels were measured by HPLC with electrochemical detection. The analytical column was SunFire C18 (5 µm particle size, 4.6×150.0 mm) from Waters (Waters Corp., Millford, MA). The mobile phase was 0.01 M sodium dihydrogenphosphate, 0.01 M citric acid, 2 mM sodium EDTA, 1 mM sodium octylsulfate, 10% methanol, pH 3.5 at flow rate 1.0 ml/min and temperature 35°C. The installation consisted of Waters 1525 Binary HPLC pump and Esa Coulochem III electrochemical detector (Thermo Fisher Scientific, Sunnyvale, CA). The glassy carbon electrode was used at a potential of 0.75 V. Peak areas and sample concentrations were calculated with the proprietary software program, Breezes (Waters Corp.). The program was used to calculate peak height and to integrate known standards for the HPLC data. Contents of DA, DOPAC and HVA were calculated as pg/mg of tissue weight.
Briefly, NAc was homogenized in 0.01 M HClO4 and centrifuged at 14, 000×g for 15 min. DA, DOPAC, HVA, 5-HT, and 5-HIAA levels were measured by HPLC with electrochemical detection. The analytical column was SunFire C18 (5 µm particle size, 4.6×150.0 mm) from Waters (Waters Corp., Millford, MA). The mobile phase was 0.01 M sodium dihydrogenphosphate, 0.01 M citric acid, 2 mM sodium EDTA, 1 mM sodium octylsulfate, 10% methanol, pH 3.5 at flow rate 1.0 ml/min and temperature 35°C. The installation consisted of Waters 1525 Binary HPLC pump and Esa Coulochem III electrochemical detector (Thermo Fisher Scientific, Sunnyvale, CA). The glassy carbon electrode was used at a potential of 0.75 V. Peak areas and sample concentrations were calculated with the proprietary software program, Breezes (Waters Corp.). The program was used to calculate peak height and to integrate known standards for the HPLC data. Contents of DA, DOPAC and HVA were calculated as pg/mg of tissue weight.
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