Xcalibur system

The samples were dissolved in methanol and analyzed using a Thermo U3000-LTQ XL ion trap mass spectrometer (Thermo Scientific, Waltham, MA, United States) equipped with an electrospray ionization (ESI) mass source. Chromatographic separation of the compounds was achieved using a Waters HSS T3 C18 column (2.1 × 150 mm; 2.5 μm) at the flow rate of 0.3 ml/min. The mobile phases, A and B, contained 0.1% formic acid along with water and acetonitrile, respectively. Gradient elution was performed as follows: 5–100% B for 0–15 min with a linear gradient, followed by 5 min of 100% B. The MS/MS system was operated in ESI mode. The operating parameters were as follows: spray needle voltage, +5 kV; ion transfer capillary temperature, 275°C; nitrogen sheath gas, 35 (arbitrary units); auxiliary gas, 5 (arbitrary units). The ion trap contained helium damping gas, which was introduced in accordance with the manufacturer’s recommendations. Mass spectra were acquired in an m/z range of 100–2000, applying three microscans and a maximum ion injection time of 100 ms. Data-dependent mass spectrometry experiments were controlled using the menu-driven software provided in the Xcalibur system (version 4.0; Thermo Scientific).

The qualitative analysis was performed on a UHPLC/Q-Orbitrap-MS (Thermo Fisher Scientific, San Jose, CA, USA) with chromatographic separation achieved using an ACQUITY UPLC® BEH C18 (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA) at 40°C. The mobile phase system was composed of 0.1% formic acid aqueous solution (v/v) (A) and methanol (B) at 0.3 mL min⁻¹, which was performed according to following optimized gradient program: 0–5 min, 3%–9% B; 5–9 min, 9%–15% B; 9–11 min, 15%–17% B; 11–15 min, 17%–27% B; 15–18 min, 27%–28% B; 18–26 min, 28%–50% B; 26–31 min, 50%–66% B; 31–35 min, 66%–74% B; and 35–36 min, 74%–3% B. The mass spectrometer was carried out in both positive and negative ion modes with centroided MS and MS² spectra recorded from 100 to 1500 m/z in full MS and dd-MS² (TopN) modes at a resolution of 70000 and 17500, respectively. The maximum injection time was set at 50 and 100 ms for MS¹ and MS², respectively. The automatic gain control (AGC) target of MS¹ and MS² was 3e⁶ and 1e⁵, respectively. The optimal MS parameters were as follows: spray voltage, −2.5 kV in negative ion mode and 3.5 kV in positive ion mode; sheath gas flow rate, 35 Arb; aux gas flow rate, 10 Arb; capillary temperature, 350°C; aux gas heater temperature, 350°C; collision energy at 20, 40, and 60 eV. All the data were acquired and processed by the Thermo Scientific™ Xcalibur™ system.

The cultures were extracted using an equal volume of ethyl acetate. Ethyl acetate extracts were then dried and resuspended in methanol. Twenty microliters of the extract was injected into a J’sphere ODS-H80 column (4.6 × 150 mm i.d., 5 μm; YMC, Japan) using an UltiMate U3000 system (CH₃CN–H₂O (0.05% TFA) 20–100% acetonitrile (CH₃CN) for 20 min and 100% CH₃CN for 5 min, at a flow rate of 1 ml/min; Thermo Fisher Scientific, Unites States) equipped with a photodiode array detector. The quantification of the compounds was based on the peak areas of absorbance at 320 nm. The quantification data shown in this study were generated from independent experiments performed in triplicate. For LC/MS analysis, samples were dissolved in methanol and analyzed by electrospray ionization (ESI) using an UltiMate U3000-LTQ XL linear ion trap mass spectrometer system (Thermo Fisher Scientific, United States). A 2-μl volume of the sample was applied to an ACQUITY UPLC HSS T3 C18 column (2.1 × 150 mm; 2.5 μm particle size; Waters, United States), and the mobile phase used for the linear gradient condition (5–100% CH₃CN–H₂O containing 0.1% formic acid for 15 min, 100% CH₃CN for 5 min, at a flow rate 0.3 ml/min). The mass spectrometry experiments were controlled and analyzed using menu-driven software provided with the Xcalibur system (version 2.2 SP1.48; Thermo Fisher Scientific).

The dried C. atrati (CA) root was purchased from Daekwang Medicine Company (Chuncheon, Korea), and the extraction procedure was followed as in the previous report [22 (link)]. Briefly, CA was powdered and extracted in 70% ethanol at room temperature for 3 h. The filtered extract was evaporated to remove solvent (LABOTORA 4000eco, Heidolph Instruments GmbH&Co., Schwabach, Germany) and stored in a freezer at −20 °C until used.
For the compositional analysis of CA, sample solution was subjected to ultrahigh pressure liquid chromatography system (UHPLC) using an LTQ Orbitrap XL linear ion trap mass spectrometry (MS) system (Thermo Scientific, San Jose, CA, USA) equipped with an electrospray ionization source. Chromatographic separation was performed using an ACQUITY UPLC^® BEH C₁₈ column (2.1 × 150 mm, 1.7 μm; Waters, Milford, MA, USA) and operated using mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). Each compound was detected with photodiode array at 200~500 nm. The Orbitrap analyzer was used for high-resolution and accurate mass (HR/AM) data acquisition with 30,000 FWHM resolving power at 400 m/z. The MS/MS experiments were adjusted using the Xcalibur system (Thermo Scientific, San Jose, CA, USA) and performed under automatic gain control (AGC) conditions.