Using reverse‐phase high‐performance liquid chromatography (HPLC) and electrochemical detection (ECD‐300, EICOM), 5‐HT levels in the microdialysis samples were measured. We used a reverse‐phase column (EICOMPAK CAX, 2.0 × 200 mm, EICOM). The mobile phase comprised 0.1 M ammonium acetate buffer solution with 50 mg/mL EDTA‐2Na and 0.05 M sodium sulfate in methanol in water (7:3, v/v) adjusted to pH 6.0. An HPLC pump system (EP‐300, EICOM) was used, and the flow rate was set at 0.25 mL/min. The column temperature was set at 35°C, and the applied potential was set at +450 mV (ATC‐300, EICOM). Quantification was performed using standard curves.
Atc 300
Manufactured by Eicom
Sourced in Japan
The ATC-300 is a laboratory equipment product designed for temperature control applications. It features a built-in temperature control system that can be used to maintain precise temperatures in various experimental and testing environments.
Automatically generated - may contain errors
3 protocols using atc 300
1
HPLC-ECD Quantification of 5-HT
2
Quantitative Neurochemical Analysis
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The brain tissues were homogenised in 0.2 M perchloric acid (HClO4) containing 100 µM EDTA-Na and 100 ng isoproterenol as an internal standard. The homogenates were centrifuged at 20 000 × g and 4 °C for 15 min. The pH of the supernatant was adjusted to 3.0 with 1 M sodium acetate. The samples were passed through a 0.45-µm filter (UFC40HV; EMD Millipore, Billerica, MA, USA). The filtrate (10 µL) was injected into a high-performance liquid chromatography-electrochemical detection (HPLC-ECD) system (Eicom, Kyoto, Japan) consisting of a 150 mm × 3 mm octadecylsilane column (EICOMPAK SC-5ODS; Eicom, Kyoto, Japan), a pump (EP-300; Eicom, Kyoto, Japan), a column oven (ATC-300; Eicom, Kyoto, Japan), and an electrochemical detector (ECD-300; Eicom, Kyoto, Japan). The mobile phase consisted of aceto–citric acid buffer (0.1 M; pH 3.5), methanol, sodium-1-octane sulfonate (0.46 M), and disodium ethylenediaminetetraacetic acid (0.015 mM) [830:170:1.9:1]. The flow rate was 0.5 mL min−l. The levels of DA, Epi, NE, 5-HT, DOPAC, 5-HIAA, HVA, MHPG, 3-MT, and NM were calculated using PowerChrom v. 2.6.11 (eDAQ Inc., Colorado Springs, CO, USA).
3
Hydroxyl Radical Production Analysis
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To clarify the hydroxyl radical production, we introduced the DMSO trapping method of Tai et al., 28) (link) with a slight modification. The reaction mixture contained 100 mM DMSO, 1.6 mM Fe 2+ , 8 mM artesunate, and 0.24 mM 2,4-dinitrophenylhydrazine in 10 mM phosphate buffer (pH 4.0), which was maintained at room temperature for 150 min and analyzed by HPLC. HPLC was performed by using an EiCOM EP-300 pump (Eicom Co., Kyoto, Japan) and a manual injector equipped with a 50-µL loop. The separation was performed on a GL Sciences Inertsil ODS-SP (4.6×150 mm) column. The mobile phase consisted of 27.7 mM acetate buffer containing 30 mM sodium citrate (pH 4.75) and methanol (1 : 3), filtered through a 0.45-µm filter (Millipore, Bedford, MA, U.S.A.). The HPLC analysis was performed at 40°C by using a column oven (EiCOM ATC-300) under isocratic conditions with a flow rate of 1.0 mL/min and then monitored on an ECL detector (EiCOM ECD-300) at +800 mV with the full-scale current set at 0.5 nA AUFS. 29) (link) Statistical Analysis The statistical significance of differences was determined by Dunnett's or Tukey-Kramer's test, independent-sample t-test, paired-sample t test, or Wilcoxon's matched-pairs test. p<0.05 was defined as indicating statistical significance.
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