The GABA and glutamate levels were estimated by the method described by Donzanti (Donzanti and Yamamoto, 1988 (link)) with slight modifications, which involved HPLC with an ECD. (A Waters standard system comprising a high-pressure isocratic pump, a 20 microlitres manual sample injector valve, a C18 reversed-phase column, and an ECD used in the study. The mobile phase comprised 100 mM anhydrous disodium hydrogen phosphate, 25 mM EDTA, and 22 % methanol (pH: 6.5). The electrochemical conditions for the experiment were +0.65 V, with sensitivity ranging from 5 to 50 nA. The separation was carried out at a flow rate of 1.2 mL/minutes, and the column temperature maintained at 40 °C. Samples (20 μl) injected manually through a rheodyne valve injector. On the day of the experiment, the frozen brain samples thawed and homogenized in 0.2 M perchloric acid. Then, the samples anteroposterior at 12,000×g for 15 min. The supernatant was derivatized using OPA/β-ME (o-pthalaldehyde/β-mercaptoethanol) and then filtered through the 0.22-mm nylon filters before being injected into the HPLC sample injector). Data were recorded and analyzed using Breeze software version 3.2 purchase from Waters HPLC. The amino acid concentrations calculated from the standard curve generated using a standard in the concentration range of 10–100 ng/ml. The values expressed as a percentage of the normal control group.
Standard system
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4 protocols using standard system
1
HPLC-Based Quantification of Neurotransmitters
2
HPLC-ECD Analysis of DA Levels
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DA levels in the brain were measured using high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD). A Waters standard system was used during the research. This system includes a high-pressure isocratic pump, a 20 μL manual sample injector valve, a C18 reversed-phase column and an ECD. The mobile phase was an acetonitrile-sodium citrate buffer (pH 4.5) mixture (87:13, v/v). The sodium citrate buffer was composed of 10 mM citric acid, 25 mM NaH2HPO4, 25 mM EDTA (ethylene diamine tetra-acetic acid) and 2 mM 1-heptane sulfonic acid. The electrochemical parameters of the experiment were +0.75 V, with a 5–50 nA sensitivity. A flow rate of 0.8 mL/min was applied for this separation. Each sample was injected by hand at a volume of 20 μL. The experiment’s homogenising solution contained 0.2 M perchloric acid, which was used to defrost the frozen brain samples before homogenisation. After that, the samples were centrifuged at 12,000× g for 5 min. The supernatant was filtered through 0.22-mm nylon filters before being injected into the HPLC sample injector. Breeze software was used to collect and analyse the data [67 ].
3
Quantifying Dopamine Levels in Rat Brains
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The level of dopamine in the striatum is a marker of neuronal excitability leading to mood alterations. The dopamine levels in the brain were estimated via high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD). A Waters standard system comprised of a high-pressure isocratic pump, a 20-microlitre manual sample injector valve, a C18 reversed-phase column, and an ECD was used in the study. The mobile phase is comprised of sodium citrate buffer (pH 4.5)–acetonitrile (87:13, v/v). The sodium citrate buffer contained 10 mM citric acid, 25 mM sodium hydroxide, 25 mM EDTA (ethylene diamine tetraacetic acid), and 2 mM 1-heptane sulfonic acid. The electrochemical conditions for the experiment were +0.75 V, with sensitivity ranging from 5 to 50 nA. The separation was carried out at a 0.8 mL/minute flow rate. The samples (20 microlitres) were injected manually. On the experiment day, the frozen brain samples were thawed and homogenized in a homogenizing solution containing 0.2 M perchloric acid. Then, the samples were centrifuged at 12,000× g for 5 min. The supernatant was filtered through 0.22 mm nylon filters before being injected into the HPLC sample injector. Data were recorded and analysed with Breeze software. The dopamine activity in rat brain homogenates is expressed as ng/mg protein [63 (link)].
4
Quantifying Brain Dopamine Levels via HPLC-ECD
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The levels of dopamine in the brain were estimated via high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) (Jamwal and Kumar, 2016 (link)). A Waters standard system comprising a high-pressure isocratic pump, a 20 microlitres manual sample injector valve, a C18 reversed-phase column, and an ECD used in the study. The mobile phase comprised of sodium citrate buffer (pH 4.5)–acetonitrile (87:13, v/v). The sodium citrate buffer comprised 10 mM citric acid, 25 mM NaH2HPO4, 25 mM EDTA (ethylene diamine tetra-acetic acid), and 2 mM 1-heptane sulfonic acid. The electrochemical conditions for the experiment were +0.75 V, with sensitivity ranging from 5 to 50 nA. The separation carried out at a flow rate of 0.8 mL/minutes. The samples (20 microlitres) were injected manually. On the day of the experiment, the frozen brain samples thawed and homogenized in a homogenizing solution containing 0.2 M perchloric acid. Then, the samples were centrifuged at 12,000×g for 5 min. The supernatant filtered through 0.22-mm nylon filters before being injected into the HPLC sample injector). Data were recorded and analyzed with Breeze software.
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