Lc 10advp hplc pump

The levels of serotonin (5HT) and 5-hydroxyindole acetic acid (5-HIAA) were measured in frontal cortex tissue dissected from the brain hemisphere contralateral to that used for protein extraction (see above). Thus, a total of 12 samples per group (6 per replicate, chosen as described for protein extraction), were subjected to HPLC analysis as previously described (Nguyen et al., 2010 ), with some modifications. Fragments of cortical tissue were homogenized in 0.06 M perchloric acid (1:10 w/v) and then centrifuged (14,000g, 5 min) to remove any tissue debris. All samples were analysed using HPLC with electrochemical detection and separated with an ACE column (C18, 3 μm, 125 × 3 mm + ACE C18 guard, 10 × 3 mm run at 35 °C). The eluent was as previously described (Nguyen et al., 2010 ) (9% methanol, 50 mM citric acid, 50 mM H₃PO₄, 0.1 mM Na₂EDTA, 2 mM 1-octanesulphonic acid, pH 3.2) pumped with a flow rate of 0.6 ml/min (Shimadzu LC10 ADVP HPLC Pump). Monoamines were detected using a porous graphitic electrode held at +0.40 V (Coulochem 5100A). The sample content was determined with reference to daily-calibrated standard solutions in 0.06 M perchloric acid (5 pmol 5-HT and 5-HIAA). Chromatograms were displayed and analysed using Shimadzu LC solution software.

HPLC analysis was performed according to the method by Amakura et al., with slight modification.^{12 (link)} An LC-10AD VP HPLC pump connected to a SPD-10A UV/VIS detector (254 nm) and C-R8A chromatopac, all of which were Shimadzu products (Kyoto, Japan), were used in the analysis. After the samples (20 μL) were subjected to a reversed-phase column (Cosmosil-packed 5C₁₈ AR II 4.6 mm × 150 mm, 5 μm particle size, Nacalai Tesque, Kyoto, Japan), equilibrated with a mobile phase comprising 20% (v/v) acetonitrile with 0.5% (v/v) acetic acid solution, the eluates were analyzed for 20 min. AG was detected at the retention time of ∼17.0 min when the flow rate was 1.0 mL min⁻¹. The calibration curve for AG was drawn in the concentration range of 0.049–12.5 μg mL⁻¹ with a LOD of 0.23 μg mL⁻¹. All sample analyses were performed in triplicate.