2.5. High performance liquid chromatography-electrochemical detection (HPLC-ED)

HPLC-ED was used to detect the levels of sympathetic neurotransmitter NE in the STAD tissues and the adjacent tissues [18]. To prepare the NE standard solution for HPLC chromatograms, NE powder (Sigma, USA) was dissolved in ultrapure water to achieve a NE stock solution of 10⁻³ mol/L, and 0.1 mol/L acetic acid (10%) was added to the solution and then stored at −20 °C to prevent NE oxidative denaturation. The NE solution was diluted to a working concentration (10⁻⁷ mol/L) before HPLC-ED. Freshly harvested STAD tissues (0.183 g) and adjacent tissues (0.183 g) were respectively homogenized in 200 μL buffer containing (in mmol/L) 147 NaCl, 4 KCl, 2.3 CaCl₂ and 1.2 MgCl₂ (pH 7.4), and then the homogenates were centrifuged at 26,916 g for 15 min at 4 °C to remove any residues. The supernatant of each sample was diluted with 600 μL diluent buffer, then was treated with 20 μL of 10% acetic acid (to stabilize NE) and filtered with 0.2-μm Millipore. HPLC-ED was performed using a LC-20A liquid chromatograph equipped with a LC-20AT solvent delivery unit and an electrochemical detector (ED723, GL-Sciences, Kyoto, Japan). A laboratory solution workstation software (Shimadzu, Kyoto, Japan) was used for data acquisition. Chromatographic separations were carried out on a Shim-pack GIST C18 column (250 mm × 4.6 mm, 5 μm particle size, Shimadzu, Kyoto, Japan). The mobile phase consisted of (in mmol/L) 30 KH₂PO₄, 0.05 EDTA, and 0.32 sodium 1-octane sulfonate (SOS) in 400 mL ultrapure water (pH adjusted to 4.0 with phosphoric acid). Then the mobile phase solution was mixed with 44 mL of 10% methanol and filtered with a 0.22-μm filter membrane and was degassed. The detection voltage of the ED723 detector was set to 750 mV (Diamond electrode). The flow rate was 1 mL/min and the injection volume were 5 μL. The column temperature was kept at 30 °C. The HPLC-ED analyses were performed on the STAD tissues and adjacent tissues from three patients.