2.3. HPLC Biogenic Amine Analysis

The analysis of the biogenic amines methods were based from [33]. Briefly, brains were dissected on dry ice and occipital lobes were removed using a scalpel and Zeiss stereo scope (Zeiss, Munich, Germany). A total of 3 brains were pooled together within a treatment group in 1.5 mL microcentrifuge tubes and these were placed on ice for 30 s. The brains were homogenized using a pestle in 50 µL of 0.2 M perchloric acid solution. The perchloric acid solution contained 100 pg/μL of N-methylserotonin oxalate and 50 pg/μL of synephrine, which served as internal standards. N-Methylserotonin oxalate was an indicator for dopamine and serotonin, while synephrine was used for octopamine and tyramine. Each sample was then sonicated for 5 min in ice water, and then placed on ice for another 20 min. The contents were centrifuged at 4 °C for 10 min with a speed of 15,000 g (Eppendorf centrifuge 5804 R, Hamburg, Germany). For HPLC analysis, the 50-µL supernatant was taken and transferred to a 2-mL dark amber microvial that contained a 100 µL of glass insert.

Samples were run on a Thermo Scientific Dionex Ultimate 3000 HPLC system consisting of a SR-300 solvent rack, a refrigerated ISO-3100BM pump, a WPS-3000TBSL analytical autosampler and an ECD-3000RS Electrochemical Detector (Thermo Fisher Scientific, Waltham, MA, USA). The autosampler was maintained at 5 °C, and a custom made 80 mm × 4.6 mm high-efficiency, C18 reverse-phase catecholamine, HR-80 column (3-μm particle packing) (Teknikus Kromatografi Teknolojileri, Istanbul, Turkey) was used and then maintained at 32 °C. Only 2 of the 4 channels of the ECD-3000RS electrochemical detector were used: one channel was set to 650 mV for tyramine and octopamine, while the other was set to 350 mV for serotonin and dopamine. The mobile phase consisted of 10% acetonitrile, 1.7 mM of 1-octanesulfonic acid sodium salt, 25 μM of EDTA tetrasodium tetrahydrate, and 75 mM of sodium dihydrogen phosphate monohydrate with a pH of 3, which was adjusted using concentrated phosphoric acid. The flow rate was set to 0.4 mL/min with an injection volume of 2 μL. The peaks were manually integrated based on the known standard retention times using the Chromeleon v. 7.2 software (Thermo Fisher Scientific, Waltham, MA, USA). External standards were run at the beginning and end of each batch of 48 samples. The samples were run in a randomized order. The biogenic amines were quantified on a per brain basis from standard curves. All chemicals were purchased from Sigma-Aldrich (Merck, Kenilworth, NJ, USA).