*4.6. Determination of Catecholamines by HPLC Method*

Adrenalin, NA, DA and 5-HT were assayed in the serum using high performance liquid chromatography (HPLC, Gynkotek, Copenhagen, Denmark) with electrochemical detection using Coulochem III model 520 (ESSA, Copenhagen, Denmark).

Serum samples were mixed with 0.1 M perchloric acid containing 22.5 ng/mL ascorbic acid (ASC, Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 15,000 g, 10 min, at 4 ◦C, supernatant was filtered through a nylon syringe filter (Millipore, 0.22 μm, Merck KGaA, Darmstadt, Germany). Samples of 20 μL filtrate were injected into a high performance liquid chromatography system (Gynkotek, Copenhagen, Denmark) equipped with a Hypersil Gold (15 cm × 4.6 mm) column (Thermo Electron Corporation, Kleinostheim, Germany). The samples were eluted by a mobile phase made of 107 mM of Na2HPO4 × 2H2O, 107 mM citric acid, 0.3 mM octane-1 sulfonic acid sodium salt (OSA), 0.2 μM of EDTA, pH, 4.6, 1.5% methanol and 1.5% acetonitrile at a flow rate of 0.8 mL/min. The column temperature was set at 25 ◦C. Peaks were detected by electrochemical detection (Coulochem III, ESSA, Copenhagen, Denmark) at potentials of E1 = −50 mV and E2 = +400 mV. Data were collected and analyzed using Chromeleon software run on a PC (Gynkotek, Copenhagen, Denmark). DA and 5-HT contents in the sample were calculated by extrapolating the peak area from a standard cure.
