Q orbitrap hrms

HDW segments were successively extracted (H-Total extract) and dissolved in water, then fractionated with ethyl acetate (EtOAc) and n-butanol (n-BuOH) to obtain H-H₂O, H-EtOAc and H-n-BuOH fractions. Six components, including rutin, quercetin, 6,7-dihydroxycoumarin, DMQ, geniposidic acid and methyl hexadecanoate in H-EtOAc fraction, were isolated by column chromatography on silica gel in our experimental group. The HPLC-Q-Orbitrap method was applied to identify the composition of ethyl acetate extract of Hedyotis diffusa. Ultimate 3000 RSLCnano high performance liquid chromatograph (Thermo Fisher Scientific, MA) with hyper Gold C18 chromatographic column (100 mm × 2.1 mm, 1.9 μm) was used for separation of compounds. The mobile phase for HPLC analysis consisted of 0.1% formic acid in water (solvent A) / acetonitrile (solvent B) with a flow rate of 0.3mL/min. High-resolution mass spectrometer Q-Orbitrap HRMS (Thermo Fisher Scientific, MA) with HESI-II ESI ion source were used for identification of six compounds. With the help of XCalibur and Mass Frontier 7.0 software, we speculated the possible fragmentation pathway of every composition, matching them with the database in Mass Frontier.

The UPLC-Q-Orbitrap included a UPLC system equipped with an autoinjector and quaternary UPLC pump (Waters Acquity, Milford, MA, United States), and quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS; Thermo Scientific, Waltham, MA). The chromatograph was equipped with a BEH C18 column (100 mm × 2.1 mm, 1.7 μm) and operated at a column temperature of 40°C. Samples were injected and eluted using a mobile solvent containing mobile phase A, which was an aqueous solution containing 0.1% formic acid and 5 mmol/L ammonium formate, and mobile phase B, a methanol solution containing 0.1% formic acid and 5 mmol/L ammonium formate. The mass spectrometry parameters were set similar to that in previous study (Lai et al., 2023 (link)).

Liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) analysis was performed using a UPLC Ultimate 3000 (Thermo Fisher-Dionex San Jose, CA, USA) system equipped with a HESI-II electrospray source to a Q-Exactive Orbitrap™-based mass spectrometer (all from Thermo Scientific, San Jose, CA, USA). Chromatographic separation was performed on Phenomenex Gemini C18 (100 × 2 mm), 3 µm particle size, the column was held at 37 °C. Peaks were obtained at a flow rate of 0.4 mL min⁻¹ with a sample injection volume of 5 µL.
Q-Orbitrap HRMS (Thermo Scientific, San Jose, CA) with HESI-II electrospray source was operated in positive mode. The Xcalibur™ 3.1.66 software (Thermo Scientific, Bremen, Germany) was used to control the instruments and to process the data [36 (link)].