Orbitrap exploris 120

LC–MS/MS analyses were conducted using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo). The mobile phase included 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was 4 °C, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was used to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo). In this mode, the acquisition software evaluates the full scan MS spectrum continuously. The ESI source conditions were set as follows: sheath gas flow rate as 50 Arb, Aux gas flow rate as 15 Arb, capillary temperature 320 °C, full MS resolution as 60,000, MS/MS resolution as 15,000 collision energy as 10/30/60 in NCE mode, spray Voltage as 3.8 kV (positive) or -3.4 kV (negative), respectively. The raw data were converted to the mzXML format for peak detection, extraction, alignment, and integration, using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS. Then an in-house MS2 database was applied to metabolite annotation, for which the cutoff was set at 0.3.

LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo, Waltham, MA, USA). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The auto-sampler temperature was 4 °C, and the injection volume was 4 μL. The Orbitrap Exploris 120 mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo, Waltham, MA, USA). In this mode, the acquisition software continuously evaluates the full-scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 50 Arb, Aux gas flow rate as 15 Arb, capillary temperature as 320 °C, full MS resolution as 60,000, MS/MS resolution as 15,000, collision energy as 10/30/60 in NCE mode, spray voltage as 3.8 kV (positive) or −3.4 Kv (negative), respectively.

In both positive and negative-ion modes, all oropharyngeal samples were subjected to metabolite separation using a UHPLC system (Vanquish, Thermo Fisher Scientific). The target compounds were separated using a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 µm) liquid chromatography column. Mobile phase A was an aqueous phase with a pH of 9.75. It contained 25 mmol/L of ammonium acetate and 25 mmol/L of ammonia hydroxide, whereas mobile phase B comprised acetonitrile. The temperature of the sample tray was 4°C, and the injection volume was 2μL.
The organic phase was injected into the column at 30°C. The elution gradients were set to 95% B, 0–0.5 min; 95–65% B, 0.5–7.0 min; 65–40% B, 7.0–8.0 min; 40% B, 8.0–9.0 min; 40–95% B, 9.0–9.1 min; and 95% B, 9.1–12.0 min. The data was gathered using Xcalibur (Thermo Fisher Scientific) on the Orbitrap Exploris 120 mass spectrometer, which can collect primary and secondary mass spectrometry data in the information-dependent acquisition mode. Other conditions for the electrospray ionization source were established as follows: capillary temperature, 320°C; collision energy, 10/30/60 in NCE mode; MS/MS resolution, 15,000; full MS resolution, 60,000; auxiliary gas flow rate, 15 Arb; and sheath gas flow rate, 50 Arb. The spray voltage was set to 3.8 and -3.4 kV for the positive-ion and negative-ion modes, respectively.

Plasma samples were vortexed with methanol and centrifuged, and the supernatant was dried and resuspended in 2-chloro-l-phenylalanine with 80% methanol. Subsequent metabolite measurements were performed on a Vanquish UHPLC System (Thermo Fisher Scientific, Waltham, MA, United States). The mass spectrometric detection of metabolites was performed on Orbitrap Exploris 120 (Thermo Fisher Scientific, Waltham, MA, United States) with an ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition was used. All multivariate data analyses were mainly performed using R packages (v3.2.0), and differential metabolites were subjected to pathway analysis by MetaboAnalyst, which combines results from powerful pathway enrichment analysis with the pathway topology analysis. The identified metabolites in metabolomics were then mapped to the KEGG pathway for biological interpretation of higher-level systemic functions.

LC-MS/MS analyses were performed using an UHPLC system (Vanquish; Thermo Fisher Scientific, Jiading District, Shanghai, China) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Orbitrap Exploris 120 mass spectrometer (Orbitrap MS; Thermo). The mobile phase consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was 4 ℃, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition mode in the control of the acquisition software (Xcalibur; Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as following: sheath gas flow rate as 50 Arb, Aux gas flow rate as 15 Arb, capillary temperature 320 ℃, full MS resolution as 60,000, MS/MS resolution as 15,000 collision energy as 10/30/60 in NCE mode, spray Voltage as 3.8 kV (positive) or −3.4 kV (negative), respectively.^{[11 (link)]}

Acquisition of the phytochemical profile of fractions was carried out on a Vanquish™ Flex UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) connected to an Orbitrap Exploris 120 mass spectrometer (Thermo Scientific, Waltham, MA, USA). Separation of fractions was achieved on a Phenomenex Luna C18 column, (2.1 mm × 100 mm, 1.7 μm) using a mobile phase of A: 0.1% formic acid in water and B: methanol at a flow rate of 0.6 mL/min. The gradient program was as follows: 0 min, 10% B; 5 min, 10% B; 15 min, 100% B; 20 min, 10% B. The injection volume was 5 µL, and the column temperature was set at 35 °C.
Mass spectrometry data were recorded on an Orbitrap Exploris 140 mass spectrometer equipped with a heated ESI source and operated in the positive-ion mode with the following settings: ion spray voltage: 2.5 kV, sheath gas: 5.08 L min⁻¹, auxiliary gas: 9.37 L min⁻¹, ion transfer tube temperature: 320 °C, vaporizer temperature: 350 °C, scan range (m/z): 150–2000, and collision-energy voltage: 35 V. The full scan was operated at a mass resolution of 60,000 and MS² scan at 15,000. Data were acquired using Thermo Xcalibur software and analyzed with Compound Discoverer 3.3.