Unifi software

RPLC-MS experiments were performed on an Acquity UPLC I-Class PLUS system, coupled to an FL detector (λ_excitation = 280 nm and λ_emission = 350 nm) and a high-resolution BioAccord ToF mass spectrometer from Waters. A prototype BioResolve RP mAb Polyphenyl column (2.7 μm, 10 × 2.1 mm, 450 Å) from Waters was used. The column temperature was set at 70 °C. Mobile phase A was 0.05% (v/v) DFA in water, and mobile phase B was 0.05% (v/v) DFA in ACN. The injection volume was 0.5 µL. Optimized linear gradients were run in 1 min (without re-equilibration) from 30% to 36%, 26% to 54%, 29% to 37% and 25% to 38% of mobile phase B, for bsAb1, bsAb2, bsAb3 and bsAb4, respectively. The LC flow rate was set at 1000 µL/min and split with a PEEK T-junction so that the flow rate entering the MS was equal to 275 µL/min. The MS was used in ESI-positive mode with an acquisition range of 400 to 7000 m/z. A 200 pg/µL sodium iodide solution diluted in a mixture of 50/50 water/IPA (v/v) with 0.1% formic acid was used as a mass spectrometer calibrant. The desolvation temperature was set at 550 °C, the source temperature at 120 °C, the cone voltage at 30 V, and the capillary voltage at 1.5 kV. Data acquisition and instrument control were performed with Unifi software (Waters, Milford, MA, USA) and mass spectra data treatment was performed with MassLynx software (Waters, Milford, MA, USA).

The methanol extract from the branch of A. hirsuta (Spach) Rupr. (AH), which belongs to the Betulaceae family, was collected from Gangwon Province (Gangwon-do, South Korea) and obtained from The Korea Plant Extract Bank of Korea Research Institute of Bioscience and Biotechnology (KRIBB, PB2357.6). The tentative identification of compounds in AH was analyzed by ACQUITY UPLC system coupled with Vion IMS QToF mass spectrometer (Waters Corp., Milford, MA, United States) using BEH C18 column (2.1 × 100 mm, 1.7 μm) and two mobile phases, 0.1% formic acid in water (A) and acetonitrile (B). The column and sample tray temperature were maintained at 35°C. The flow rate was 0.4 ml/min and elution conditions were optimized as follows: 0–1 min, 5% B; 1–20 min, 5–100% B; 20–20.30 min, 100% B; 20.30–22.40 min, 100–5% B; 22.40–25 min, 5% B. The mass spectrometer operated in negative mode from 100 to 1,500 Da with a 0.2 s scan time using a desolvation temperature of 350°C with desolvation gas flow of 800 L/h (N₂), source temperature of 110°C, and cone voltage of 40 V. Leucine-enkephalin was used as the lock mass ([M–H]– m/z 554.2615). The full scan data and MS/MS spectra were acquired using a UNIFI software (Waters Corporation, Milford, MA).

The Waters H-Class UPLC was utilized for the Method development and validation study (Waters Corporation, Milford, MA). The entire study was conducted using empower software to acquire, process, and report chromatographic data (Waters Corporation, Milford, MA). A statistical tool, Design-Expert-13, was employed to screen and optimize the CMPs (Stat-Ease Inc, Minneapolis, USA). Various Acquity UPLC columns (100 mm Length × 2.1 mm ID), such as BEH C₈, BEH C₁₈, BEH Phenyl, HSS T₃, and Protein BEH C₄ were assessed for the separation of components (Waters Corporation, Milford, MA). The Waters Xevo G2-XS Quadrupole time-of-flight (Q-ToF) MS instrument with step wave ion optics and XS collision cell coupled with Waters I-Class UPLC was used to separate and identify unknown impurities (Waters Corporation, Milford, MA). UNIFI software was used to identify molecular and fragment ions and their molecular structures (Waters Corporation, Milford, MA).

The LC-ESI-MS system consisted of an ultra-performance liquid chromatography (UPLC) system (ACQUITY UPLC I-Class, Waters, Milford Massachusetts, USA) and an ESI/APCI source of a 4 kDa quadrupole time-of-flight (TOF) mass spectrometer (Waters VION, Waters). The flow rate was set to 0.2 mL/min with the column temperature at 35 °C. Separation was performed with reversed-phase liquid chromatography (RPLC) on a BEH C18 column (2.1 × 100 mm, Waters, Milford Massachusetts, USA) with 7.5 μL sample injection. The elution started from 99% mobile phase A (ultrapure water + 0.1% formic acid) and 99% mobile phase B (100% methanol + 0.1% formic acid), held at 1% B for 0.5 min, raised to 90% B in 5.5 min, held at 90% B for 1 min, and then lowered to 1% B in 1 min. The column was equilibrated by pumping 1% B for 4 min. LC-ESI-MS chromatograms were acquired by positive or negative ion mode under the following conditions: capillary voltage of 2.5 kV, source temperature of 100 °C, desolvation temperature at 250 °C, cone gas maintained at 10 L/h, desolvation gas maintained at 600 L/h, and acquisition by MS^E mode with a range of m/z 100-1000 and 0.5 s scan time. The acquired data were processed by UNIFI software (Waters, Milford Massachusetts, USA) with illustrated chromatograms and summarized in an integrated area of signals.

Compound 4 metabolite discovery and identification experiments were performed on a Waters I-Class Plus UPLC connected to a Synapt G2-Si QTOF mass spectrometer with ion mobility. The method used a Waters Acquity UPLC CSH C18 (2.1 × 100 mm, 1.7 µm) column with a gradient of 25–81% B (A: water + 0.2% formic acid, B: 4:1 methanol/isopropanol). Traveling wave IMS was used with a variable wave velocity from 800 to 400 m/s. Metabolite discovery was carried out using ESI+ in HDMSe mode with a collision energy ramp from 55 to 75 eV in the high energy function. The data were processed using the metabolite discovery workflow in Waters’ UNIFI software. This was supplemented with directed LC-MS/MS experiments to confirm metabolite structures and sites of oxidation.

For the CC extract used in the experiment, fresh CC was purchased in the market of South Korea, freeze-dried, and powdered. Powdered CC was suspended in 70% ethanol in a ratio of 1:4 (w/v) and then extracted at room temperature overnight. This solution was filtered, concentrated, and freeze-dried at −80 °C, and the remainder was dissolved in DMSO used as the CC extract. Major compounds of the Chrysanthemum coronarium L. extract were analyzed using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) (Waters, Milford, MA, USA). For this procedure, the extract was injected into an Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters) equilibrated with mobile phase A (0.1% formic acid in water) and eluted using a linear gradient with mobile phase B (acetonitrile containing 0.1% formic acid). The eluted compounds ionized by negative electrospray ionization (ESI) were detected using Q-TOF MS under the following conditions: capillary voltage of 2 kV, sampling cone voltage of 40 V, desolvation temperature of 400 °C, source temperature of 100 °C, a scan range of 50–1500 m/z. Leucine-enkephalin ([M + H] = 556.2771) was used as lock mass, and the MS/MS data were collected using collision energy ramps of 10–40 eV. Compounds were tentatively identified using the online databases connected to UNIFI software (Waters).