Chromeleon7 chromatography data system

The dried extract was re-dissolved under the acid hydrolysis method as previously reported [51 (link)]. In this condition, the glycosidic bond between sugar and phenolics is disrupted, leading to its aglycone. Briefly, the extract was re-dissolved in formic acid and 62.5% (v/v) methanol containing tert-butyl hydroquinone, then shaken at 80 °C for 2 h and filtered through a 0.22 µm polytetrafluoroethylene (PTFE) filter. The extract was then loaded to an Accucore RP-MS column (a 2.1 mm × 100 mm, 2.6 μm column (Thermo Fisher Scientific, Bremen, Germany), which was connected to the LC–ESI–MS/MS system consisting of a Dionex Ultimate 3000 series ultra-high-performance liquid chromatograph (UHPLC) system, a diode array detector, a TSQ Quantis Triple Quadrupole mass spectrometer (MS), and a Chromeleon 7 chromatography data system (version 7.2.9.11323, Thermo Fisher Scientific, Bremen, Germany).
Acetonitrile (A) and 0.1% v/v formic acid in Milli-Q water (B) were used as mobile phase. A gradient solvent system was set as follows: 0.0–0.8 min, 10–80% A; 8.0–8.1 min, 80–10% A; 8.1–10.0 min, 10% A, at a flow rate of 0.5 mL/min. Supplementary Tables S1 and S2 show a list of authentic standards with parameters and validations, respectively.

For high performance liquid chromatography (HPLC) analysis, adrenal glands were dissected and immediately stored at −70 °C. Tissue samples were homogenized in DEPROT solution (1 mg:30 μL) containing 2% ethylene glycol tetra-acetic acid, 0.1 N HClO4, and 0.2% MgCl2, sonicated and centrifuged (30 min, 18000 rpm, 4 °C). Fifty µL of collected supernatants were injected with the autosampler of a Dionex UltiMate 3000 HPLC system (Thermo Scientific, Sunnyvale, CA, USA) equipped with an Acclaim Polar Advantage II (C18, 5 µm, 4.6 mm, 150 mm) HPLC column (Thermo Scientific, Waltham, Massachusetts, United States). The Chromeleon7 Chromatography Data System (Thermo Scientific, Sunnyvale, CA, USA) was used for instrument control and data acquisition. The mobile phase consisted of 98% ammonium formate buffer (Fisher Scientific, Cambridge, UK, pH 3.6) and 2% methanol (J.T. Baker, Griesheim, Germany), with the flow rate set at 500 μL/min. The electrochemical measurement was set at +850 mV potential and the separation temperature at 25 °C. Noradrenaline (DL-noradrenaline hydrochloride, Sigma-Aldrich) and adrenaline ((±)-adrenaline hydrochloride, Sigma-Aldrich) standard solutions were created from the stock standard solution (1 mg/mL of noradrenaline in methanol) in DEPROT, with concentration range 0.5–50 μg/mL.

For high-performance liquid chromatography (HPLC) analysis, adrenal glands were dissected and immediately stored at −70 °C. Tissue samples were homogenized in DEPROT solution (1 mg:30 μL) containing 2% ethylene glycol tetra-acetic acid, 0.1 N HClO4 and 0.2% MgCl2, sonicated and centrifuged (30 min, 18,000 rpm, 4 °C). A total of 50 µL of collected supernatants were injected with the autosampler of a Dionex UltiMate 3000 HPLC system (Thermo Scientific, Sunnyvale, CA, USA) equipped with a Acclaim Polar Advantage II (C18, 5 µm, 4.6 mm, 150 mm) HPLC column (Thermo Scientific, Waltham, MA, USA). Chromeleon7 Chromatography Data System (Thermo Scientific, Sunnyvale, CA, USA) was used for instrument control and data acquisition. The mobile phase consisted of 98% ammonium formate buffer (Fisher Scientific, Cambridge, UK, pH 3.6) and 2% methanol (J.T. Baker, Griesheim, Germany), with the flow rate set at 500 μL/min. The electrochemical measurement was set at +850 mV potential and the separation temperature at 25 °C. Noradrenaline (DL-noradrenaline hydrochloride, Sigma-Aldrich) and adrenaline (±)-adrenaline hydrochloride, Sigma-Aldrich) standard solutions were made from the stock standard solution (1 mg/mL of noradrenaline in methanol) in DEPROT, with concentration range 0.5–50 μg/mL.