4.5. Neurotransmitter Extraction and High Performance Liquid Chromatography (HPLC) Determination

Extracellular DA, 5-HT, and NE levels were analyzed through an HPLC apparatus consisting of a Jasco (Tokyo, Japan) PU-2080 chromatographic pump and an ESA (Chelmsford, MA, USA) Coulochem III coulometric detector, equipped with microdialysis cell (ESA-5014b) porous graphite working electrode and solid state palladium reference electrode. The analytical conditions for biogenic amine identification and quantification were selected as previously reported [40]. Briefly, the analytical cell was set at −0.150 V for detector 1 and at +0.300 V for detector 2, with a range of 100 nA. The chromatograms were monitored at the analytical detector 2. Integration was performed by Jasco Borwin Chromatography software, version 1.5. The chromatographic separation was performed by isocratic elution on Phenomenex Kinetex reverse phase column (C18, 150 mm × 4.6 mm i.d., 2.6 µm). The mobile phase was (10:90, v/v) acetonitrile and 75 mM pH 3.00 phosphate buffer containing octanesulfonic acid 1.8 mM, EDTA 30 µM, and triethylamine 0.015% v/v. Flow rate was 0.6 mL/min and the samples were manually injected through a 20 µL loop. Neurotransmitter peaks were identified by comparison with the retention time of pure standard. Neurotransmitter concentrations in the samples were calculated by linear regression curve (y = bx + m) obtained with standard. Neither internal nor external standard were necessary for neurotransmitter quantification in the hypothalamus homogenate, and all tests performed for method validation yielded results in accordance to limits indicated in official guidelines for applicability in laboratory trials (See Supplementary Materials). The standard stock solutions of DA, NE, 5-HT, DOPAC, and 5-HIIA at 2 mg/mL were prepared in bidistilled water containing 0.004% EDTA and 0.010% sodium bisulfite. The stock solutions were stored at 4 °C. Work solutions (1.25–20.00 ng/mL) were obtained daily, progressively diluting stock solutions in mobile phase.