Mass frontier 7

LC-MS was performed with a Thermo Fisher Scientific LTQ XL mass spectrometer coupled with a Thermo Scientific UltiMate 3000 HPLC system. The MS detector was set with a +3.5 kV spray voltage, a 275 °C capillary temperature, a 300 °C heater temperature, a sheath gas flow of 25 L/min, auxiliary gas flow of 10 L/min, and a mass scan range of 80–900 m/z.
The gradient mobile phases were (A) 10 mM ammonium formate in 95:5 acetonitrile/water + 0.1% formic acid and (B) 10 mM ammonium formate in 50:50 acetonitrile/water + 0.1% formic acid for 30 min at a flow rate of 300 µL/min. Aliquots of serum extract (5 μL) were injected into a Thermo Fisher Scientific Accurore C₁₈ Column (100 × 2.1 mm, 2.6 μm particle size), with a column temperature of 40 °C. MS data acquisition was performed using Thermo Fisher Scientific Xcalibur™ software v 2.2 SP1.48. Metabolites were determined through comparison of the ion features in the experimental samples to those of control samples based on retention time and molecular weight (m/z) using Thermo-Fisher Scientific Mass Frontier 7.0 software [30 (link),31 ].

Instrument: Ultimate 3000 UPLC instrument, LTQ-Orbitrap velos pro mass spectrometer; column: ACQUITY UPLC BEH Shield RP18 (2.1 × 100 mm, 1.7 μm); flow rate: 0.3 mL/min; column temperature: 40 °C; mobile phase: phase A—0.1% formic acid water, phase B—acetonitrile; injection volume: 2 μL; gradient elution procedure: 0–1.0 min, 90–80% A; 1–5.5 min, 80–60% A; 5.5–6 min, 60–20% A; 6–8 min, 20–5% A; 8–8.5 min, 5% A; 8.5–9 min, 5–90% A; 9–10 min, 90–90% A.
The analysis was performed in positive and negative ion modes. Heater temperature: 350 °C; capillary temperature: 350 °C; capillary voltage: 35 V; spray voltage: 3.4 kV; sheath gas (N₂) flow rate: 35 arb; auxiliary gas (N₂) flow rate: 10 arb; mass standard calibration using external standards (mass error is less than 5 ppm). The primary mass spectrum was scanned in FT mode (resolution R is 30,000, and the m/z scan range is from 50 to 1500). The MS² and MS³ spectra were obtained using a data-dependent scan. Dynamic ion exclusion mode was used to obtain additional compound information. Data acquisition and analysis were performed using Xcalibur, Metaworks, Mass Frontier 7.0 software (Thermo Fisher Scientific, San Jose, CA, USA).

Mass Frontier 7.0 (Thermo Fisher Scientific software, San Jose, CA, USA) was used for the management, evaluation, and interpretation of mass spectra. For statistical analyses, one-way ANOVA and SigmaPlot 13.0 software (Systat Software, Inc., San Jose, CA, USA) were used, and the results are presented as mean ± standard deviation (SD). Student’s two-tailed t test was used for independent comparisons of the heights of common peaks between the groups of Zucker rats. A p-value < 0.05 was considered a significant difference.

The LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was coupled to the UPLC by a HESI interface. The specific parameters were set as aforementioned. The mass spectrometer calibration was conducted before each experiment. In the MSⁿ experiments, data-dependent scanning was adopted to trigger multistage fragmentation, which was to select the strongest several parent ions in each scanning point as targeted precursor ions for the further fragmentation: four ions for MS² fragmentation and one ion for MS³ fragmentation, respectively. The dynamic exclusion function was utilized to prevent the repetitive ion scans and save the analysis time. The software Xcalibur 4.1 (Thermo Fisher Scientific, Waltham, MA, USA) and Mass Frontier 7.0 (Thermo Fisher Scientific, Waltham, MA, USA) were employed to process the UPLC-MS data. To ensure the reliability of the identification results, those peaks with intensity over 10⁵ in TIC were selected for identification. The formulas of all parent and fragment ions in selected peaks were generated according to their accurate mass using a formula predictor. The maximal mass accuracy error was confined to ±3 ppm. In consideration of the possible elemental compositions of existed compounds in B. paniculatum, the number of four types of atoms were limited as follows: C ≤ 100, H ≤ 150, O ≤ 50 and N ≤ 10.

High-resolution tandem mass spectrometry was performed on GsONC201 and ONC201 (Oncoceutics) using direct infusion into an accurate mass Thermo Scientific Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Samples were resuspended in 1 mL of dimethyl sulfoxide (DMSO), diluted 1 in 100 in 50:50 methanol:acetonitrile/0.1% formic acid, and analyzed using a resolution of 140 000 at 200 m/z for both MS1 and MS2. Data analysis was performed using Mass Frontier 7.0 (Thermo Fisher Scientific).

An ultimate 3000 hyperbaric liquid chromatography (LC) system coupled with a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used in this study. An UPLC reverse-phase C18 analytical column (2.1 × 100 mm, 1.8 μm, ACQUITY UPLC^® T3, Waters, Milford, MA, USA) was used to analyze the samples. The mobile phase consisted of A (water containing 0.1% formic acid) and B (acetonitrile). The linear gradient conditions were optimized as follows: 0–6.0 min, 3% B; 6.0–8.0 min, 3–10% B; 8.0–9.0 min, 10–20% B; 9.0–22.0 min, 20–50% B; and 22.0–30.0 min, 50–95% B. The flow rate was 0.3 mL/min, and the column temperature was maintained at 35 °C. The injection volume was 1 μL. Mass detection was performed in positive ion mode. The ESI source parameters were as follows: ion spray voltage, +5.0 kV; sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb; capillary temperature, 350 °C; S-lens RF level, 60%. The mass resolution of Fourier transform (FT) was 30,000 with a full scan in the range of m/z 50–1000. The MS/MS and MS3 experiments were set as data-dependent scans. Data processing was performed using Xcalibur 3.0 (Thermo Fisher Scientific, San Jose, CA, USA), Metworks 1.3 (Thermo Fisher Scientific, San Jose, CA, USA) and Mass Frontier 7.0 software packages (Thermo Fisher Scientific, San Jose, CA, USA).