Hplc ecd system

The analyses were carried out using HPLC-ECD system (Eicom, Kyoto, Japan) which consisted of a chromatograph (EP-300, flow rate: 500 μL/min), a degasser (DG-300), a column oven (ATC-300), a precolumn (CA-ODS), a separation column (Eicompak EX-3ODS, ODS (C18) column, 4.6 mm i.d. × 100 mm with 3 µm particles, column temperature 30°C), a working glassy carbon electrode (WE-GC), an ECD (ECD-300) with an applied voltage of +600 mV vs. Ag/AgCl, and a data recording system (EPC-500 controlled by PowerChrom software 2.5). The mobile phase was a mixture of 0.1 M phosphate buffer (pH 6.0) and methanol at a ratio of 82:18 including EDTA-2Na (5 mg/L).

The brain tissues were homogenised in 0.2 M perchloric acid (HClO₄) containing 100 µM EDTA-Na and 100 ng isoproterenol as an internal standard. The homogenates were centrifuged at 20 000 × g and 4 °C for 15 min. The pH of the supernatant was adjusted to 3.0 with 1 M sodium acetate. The samples were passed through a 0.45-µm filter (UFC40HV; EMD Millipore, Billerica, MA, USA). The filtrate (10 µL) was injected into a high-performance liquid chromatography-electrochemical detection (HPLC-ECD) system (Eicom, Kyoto, Japan) consisting of a 150 mm × 3 mm octadecylsilane column (EICOMPAK SC-5ODS; Eicom, Kyoto, Japan), a pump (EP-300; Eicom, Kyoto, Japan), a column oven (ATC-300; Eicom, Kyoto, Japan), and an electrochemical detector (ECD-300; Eicom, Kyoto, Japan). The mobile phase consisted of aceto–citric acid buffer (0.1 M; pH 3.5), methanol, sodium-1-octane sulfonate (0.46 M), and disodium ethylenediaminetetraacetic acid (0.015 mM) [830:170:1.9:1]. The flow rate was 0.5 mL min^−l. The levels of DA, Epi, NE, 5-HT, DOPAC, 5-HIAA, HVA, MHPG, 3-MT, and NM were calculated using PowerChrom v. 2.6.11 (eDAQ Inc., Colorado Springs, CO, USA).