Hypersil gold analytical column

Animals were sacrificed by decapitation 3 h after cessation of treatment with drugs. Brains were separated, and several brain regions (striatum, frontal cortex) were dissected in anatomical borders. The tissue levels of DA, 5-HT, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using a HPLC with electrochemical detection. Briefly, tissue samples of brain structures were homogenized in ice-cold 0.1 M HClO₄ and were centrifuged at 10000×g for 10 min at 4 °C. The supernatant (3–5 μl) was injected into the HPLC system. The chromatography system consisted of an LC-4C amperometric detector with a cross-flow detector cell (BAS, IN, USA), an Ultimate 3000 pump (Thermo Scientific, USA), and a Hypersil Gold analytical column (3 μm, 100 × 3 mm, Thermo Scientific, USA). The mobile phase consisted of 0.1 M KH₂PO₄, 0.5 mM Na₂EDTA, 80 mg/l sodium 1-octanesulfonate, and a 4% methanol, adjusted to pH 3.7 with an 85% H₃PO₄. The flow rate was 1 ml/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V with sensitivity of 5 nA/V. The temperature of the column was maintained at 30 °C. The Chromax 2007 program (Pol-Lab, Warszawa, Poland) was used for data collection and analysis.

Animals were sacrificed by decapitation 3 h after intraperitoneal drugs administration. Brains were separated and several brain regions (striatum, nucleus accumbens septi, frontal cortex) were dissected in anatomical borders. The tissue levels of DA, 5-HT, DOPAC, HVA, and 5-HIAA were measured using a high-performance liquid chromatography (HPLC) with electrochemical detection. Briefly, tissue samples of brain structures were homogenized in ice-cold 0.1 M HClO₄ and were centrifuged at 10,000g for 10 min at 4 °C. The supernatant (3–5 µL) was injected into the HPLC system. The chromatography system consisted of an LC-4C amperometric detector with a cross-flow detector cell (BAS, IN, USA), a Ultimate 3000 pump (Thermo Scientific, USA), and a Hypersil Gold analytical column (3 μm, 100 × 3 mm, Thermo Scientific, USA). The mobile phase consisted of 0.1 M KH₂PO₄, 0.5 mM Na₂EDTA, 80 mg/L sodium 1-octanesulfonate, and a 4 % methanol, adjusted to pH 3.7 with an 85 % H₃PO₄. The flow rate was 1 mL/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V with sensitivity of 5 nA/V. The temperature of the column was maintained at 30 °C. The Chromax 2007 program (Pol-Lab, Warszawa, Poland) was used for data collection and analysis.

The LC-ESI/MS/MS experiments were performed on an Applied Biosystems/MDS Sciex (Concord, ON, Canada) API 2000 triple quadrupole mass spectrometer equipped with an electrospray ionization interface. This instrument was coupled to an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) LC system. Data acquisition and processing were accomplished using the ABSciex Analyst 1.4.2 data collection and integration software. The chromatographic separation was performed on a Hypersil GOLD analytical column (100 mm × 3 mm i.d., 5 μm, Thermo Scientific, Waltham, MA, USA) with the column temperature set at 30 °C.