Thermo ultimate 3000 lc system

STPE was analyzed on a Thermo Ultimate 3000 LC system coupled with a Thermo Q Exactive HF system (Thermo Fisher Scientific, Waltham, USA). Chromatographic analyses were carried out on Zorbax Eclipse C18 (2.1 × 100 mm,1.8 μm, Agilent Technologies). The mobile phase consisted of two solvents: 0.1% formic acid in water (A) and acetonitrile (B). Elution was performed with linear gradient elution as follows: 0–2 min, 5% B; 2–6 min, 5–30% B; 6–7 min, 30% B; 7–12 min, 30–78% B; 12–14 min, 78% B; 14–17 min, 78–95% B; 17–20 min, 95% B; 20–21 min, 95–5% B; 21–25 min, 5% B. The flow rate was 0.3 mL/min and the injection volume was 2 μL.
The mass spectrometer was operated in both positive and negative ion modes and full MS (m/z 100–1500)/ data-dependent MS² (dd-MS², TopN = 10) mode. The mass parameters were as follows: spray voltage 3.5 kV; capillary temperature 330 °C; sheath gas (N₂) flow rate 45 arb; auxiliary gas (N₂) flow rate 15 arb; probe heater temperature 325 °C; and S-Lens RF level 55%. Retention time correction, peak identification, peak extraction, etc. were performed using Compound Discoverer 3.2. The identification of individual phenolic compounds was based on their retention time, mass spectrometry, and their mass-to-charge (m/z) ratio as compared with local brown algae polyphenol database constructed based on previous literature.

The following instruments were employed: YXJ-2 high speed refrigerated centrifuge (Xiang Yi Centrifuge Instrument Co., Ltd., Changsha, China), Rotary evaporator (Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China), Freeze-dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd., Beijing, China), ASP6025 Automated Vacuum Tissue Processor (Leica Co., Solms, Germany), Intellective Biological Microscope (Olympus optical Co., Ltd., Tokyo, Japan), Leica RM2016 histotome (Leica Co., Solms, Germany), Thermo UltiMate 3000 LC system and Thermo Q-Exactive system (Thermo Fisher Scientific, Bremen, Germany), Cosmosil CN column (Nacalai Tesque Co., Ltd., Kyoto, Japan), Beckman CytoFlex S Flow cytometry (Beckman Coulter, Kraemer Boulevard Brea, United States), and ONE-DScan program (Scanalytics inc., Fairfax, Va., United States).