Dopamine determination by HPLC

Cells were lysed in 200 μl of 0.1 M perchloric acid, sonicated and spun in a microcentrifuge at 10 000 × g for 10 min at 4 °C. The supernatant was transferred in ultra-free microcentrifuge tubes (Millipore) and spun at 10 000 × g for 5 min at 4 °C. Samples (11 μl) were injected into the HPLC apparatus. Measurements of dopamine were made by HPLC with an electrochemical detection system (ALEXYS LC-EC, Antec Leyden BV, Zoeterwoude, The Netherlands) equipped with a reverse-phase column (3-μm particles, ALB-215 C18, 1 × 150 mm, Antec Leyden BV) at a flow rate of 50 μl/min and electrochemically detected by a 0.7 mm glass carbon electrode (VT-03, Antec Leyden BV). The mobile phase contained 50 mM H₃PO₄, 50 mM citric acid, 8 mM KCl, 0.1 mM EDTA, 400 mg/l octanesulfonic acid sodium salt and 8% (vol/vol) acetonitrile (pH 3.0).