Thermo-Fisher Dionex UltiMate 3000 UPLC-Q Exactive Orbitrap-MS and Xcalibur workstation were used to obtain LC-MS raw data. For the chromatographic separation of the cerebral cortex, the sample was analyzed on an ACQUITY UPLC HSS T3 column. The column temperature was maintained at 35°C, the flow rate was set at 0.2 ml/min, and the injection volume was 5 μl; the mobile phase and gradient are shown in Supplementary Table S1 . Mass spectrometry conditions: samples were analyzed under positive and negative ionization modes via a heated electrospray ionization (HESI) source; the scan mode was “Full Scan 35000 FWHM/dd-MS2” (Resolution 17500, NCE 25, Stepped NCE 50%); the spray voltage was set to 3.6 kV for the positive mode and 2.5 kV for the negative mode; capillary temperature, 320°C; sheath gas flow, 35 arbitrary units; aux gas flow, 10 arbitrary units; scan range, m/z 50–1500.
Xcalibur workstation
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Xcalibur workstation is a comprehensive data system designed for mass spectrometry applications. It provides a platform for instrument control, data acquisition, and data analysis across a range of Thermo Fisher Scientific mass spectrometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Lqt orbitrap xl mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Cd software, by Thermo Fisher Scientific (1 mentions)
Mestrenova, by Mestrelab Research (1 mentions)
Syncronis hilic column, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Phoenix winnonlin software, by Certara (1 mentions)
Lipidsearch software, by Thermo Fisher Scientific (1 mentions)
Hplc grade formic acid, by Thermo Fisher Scientific (1 mentions)
Q exactive orbitrap ms, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Agilent 1290 infinity 2 uplc system, by Agilent Technologies (1 mentions)
Q exactive focus, by Thermo Fisher Scientific (1 mentions)
Eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Accela 600 pump, by Thermo Fisher Scientific (1 mentions)
Multiskan go full wavelength microplate reader, by Thermo Fisher Scientific (1 mentions)
Forma 3111 co2 incubator, by Thermo Fisher Scientific (1 mentions)
Aeg 220, by Shimadzu (1 mentions)
Tc c18, by Agilent Technologies (1 mentions)
Compound discoverer, by Thermo Fisher Scientific (1 mentions)
15 protocols using xcalibur workstation
1
Cerebral Cortex Metabolite Profiling
2
UHPLC-QE HRMS and NMR Data Processing
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The UHPLC-QE HRMS raw data were automatically processed using an Xcalibur workstation (Thermo Fisher Scientific, Waltham, MA, USA). Then, the processed data were imported into CD software (Thermo Fisher Scientific, Waltham, MA, USA) for peak extraction and normalization. The peak width at 5% height and the peak-to-peak baseline noise were automatically determined, and the processing parameters were set as follows: retention time range, 0–38 min; mass range, 100–1500 Da; mass tolerance, 5 ppm; retention time tolerance, 0.05 min; S/N threshold, 1.5; negative adducts, [M−H]−, [M + HCOO]−; and positive adducts, [M+H]+ and [M+Na]+. The compounds characterized by CD software were identified by searching the mz Cloud, Masslist, Chemspider, and PubChem databases.
The NMR spectra were processed using MestReNova (version 6.1.1, Mestrelab Research, Santiago de Compostella, Spain) to manually correct the baseline and phase and calibrated to TMS at δ 0.00 for CDCl3. All spectra were referenced to the residual signal of CD3OD at δ 3.31.
The NMR spectra were processed using MestReNova (version 6.1.1, Mestrelab Research, Santiago de Compostella, Spain) to manually correct the baseline and phase and calibrated to TMS at δ 0.00 for CDCl3. All spectra were referenced to the residual signal of CD3OD at δ 3.31.
3
GC-MS Analysis of Organic Compounds
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Chromatography was performed using the TRACE GC Ultra-DSQ II GC/MS system (Thermo Fisher Scientific, Massachusetts, USA) using an RTx-5 capillary column (30 m × 0.250 mm × 0.25 μm), the Xcalibur workstation and the NIST spectral library
[7 (link)]. The injection temperature was 270°C, and the helium gas flow rate through the column was 1 ml/min. The column temperature was isothermally maintained at 80°C for 5 min and raised to 300°C at a rate of 10°C/min. The temperature was then isothermally maintained at 300°C for 5 min. The temperatures of the transfer line and the ion source were 280°C and 230°C, respectively. The ions were generated at an electron impact (EI) energy of 70 kV, and 20 scans/s were recorded over the mass range of 45–550 m/z. The column temperature program was in Table
1 .
[7 (link)]. The injection temperature was 270°C, and the helium gas flow rate through the column was 1 ml/min. The column temperature was isothermally maintained at 80°C for 5 min and raised to 300°C at a rate of 10°C/min. The temperature was then isothermally maintained at 300°C for 5 min. The temperatures of the transfer line and the ion source were 280°C and 230°C, respectively. The ions were generated at an electron impact (EI) energy of 70 kV, and 20 scans/s were recorded over the mass range of 45–550 m/z. The column temperature program was in Table
4
Targeted Amino Acid Profiling in Osteosarcoma
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Prior to LC-MS/MS analysis, osteosarcoma cells (1 × 106 cells) and tissue (5–10 mg) were thoroughly mixed or homogenized (tissue) in 1 mL of buffer containing 60% methanol and 40% ddH2O, which was pre-cooled to −40 °C. The mixture was then centrifuged. Amino acids were the UltiMate 3000-TSQ Endura UPLC–MS/MS system from Thermo Fisher Scientific, equipped with a Syncronis HILIC Column (100 × 2.1 mm, 1.7 μm). Mass spectrometric detection was performed on a TSQ Endura triple quadruple mass spectrometer with an elect ionization source. Compound-specific parameters of the mass spectrometer were set as follows: spray voltage at 3500 V, capillary temperature at 320 °C, vaporizer temperature at 350 °C, sheath gas at 35 (Arb), and auxiliary gas at (10b). The detections were carried out in the (SRM) positive mode with transitions of m/z values as follows: Met - m/z150.2 → 104.1511; SAH - m/z385.45 → 134.111; SAM - m/z399.35 → 250.111 respectively. Instrument control and data acquisition were performed using an Xcalibur workstation from Thermo Fisher Scientific.
5
Quantitative Metabolite Profiling
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The data was acquired
and processed with Xcalibur workstation (Thermo Fisher Scientific,
Waltham, MA, USA). Compound Discoverer 3.0 and 3.1 by Thermo Fisher
Scientific (Waltham, MA, USA) were used for the data filtering of
most predominant phase I and phase II o-glucuronide-metabolites.
MassFrontier 7.0 and 8.0 (Thermo Fisher Scientific, Waltham, MA, USA)
were used for data filtering and elucidating fragmentation patterns
from the data in MS/HRMS studies. The graphs were plotted and statistical
calculations executed with GraphPad Prism 8.0 (GraphPad Software,
San Diego, CA, USA). The graphs with statistics are presented as mean
± standard deviation (SD).
and processed with Xcalibur workstation (Thermo Fisher Scientific,
Waltham, MA, USA). Compound Discoverer 3.0 and 3.1 by Thermo Fisher
Scientific (Waltham, MA, USA) were used for the data filtering of
most predominant phase I and phase II o-glucuronide-metabolites.
MassFrontier 7.0 and 8.0 (Thermo Fisher Scientific, Waltham, MA, USA)
were used for data filtering and elucidating fragmentation patterns
from the data in MS/HRMS studies. The graphs were plotted and statistical
calculations executed with GraphPad Prism 8.0 (GraphPad Software,
San Diego, CA, USA). The graphs with statistics are presented as mean
± standard deviation (SD).
6
Pharmacokinetic Analysis of Compound X
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The LC-MS data were examined using the Xcalibur workstation’s Qual Browser and Quan Browser (Thermo Scientific, United States, Version 4.1). SPSS 23.0 software (SPSS Inc., United States, Version 23) was used for statistical analysis, and sample concentrations were expressed as mean ± SD. The pharmacokinetic profile was depicted by GraphPad Prism software (Bethesda, United States, Version 6.02), and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Certara, Corp., Version 6.3).
7
UHPLC-HRMS Lipid Identification
8
Untargeted Metabolomics Analysis by UHPLC-MS
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An UltiMate 3,000 ultrahigh performance liquid chromatography (UHPLC, Thermo-Fisher, United States) in tandem with a Q Exactive Orbitrap-MS (Thermo-Fisher Scientific, United States) was used. Xcalibur workstation (Thermo Fisher Scientific, United States), IKA T10 tissue homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd, China), Neofuge13R high-speed freezing centrifuge (Likang Biotechnology Company, China), and MX-S Adjustable mixer (Scilogex, United States) were used. LC-MS grade acetonitrile and HPLC grade formic acid were obtained from Thermo-Fisher (United States).
9
UHPLC-Q-Orbitrap/MS Metabolomics Analysis
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Using Thermo-Fisher Dionex UltiMate 3000 UHPLC-Q Exactive Orbitrap-mass spectrometer (UHPLC-Q-Orbitrap/MS) and Xcalibur workstation (Thermo Fisher, Waltham, MA, USA) were used to acquire raw data. The complete details of the UHPLC-Q-Orbitrap/MS method for pharmacochemistry analysis and metabolomics analysis are provided in Supplemental Method 6 .
10
UPLC-MS/MS Analysis of PDBW Components
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Components in PDBW were analyzed with a LC-MS/MS system. Chromatographic separation was performed using an Agilent 1,290 Infinity II UPLC system (Agilent, Santa Clara, CA, USA) equipped with an Agilent Eclipse XDB-C18 column (100 mm × 2.1 mm i.d., 3.5 μm). The column temperature was set at 35°C and the UV absorption wavelengths were set as 254 and 320 nm, respectively. Elution were accomplished on a gradient of formic acid (0.1%) in water (mobile phase A) vs. formic acid (0.1%) in acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min and the injection volume was 10 μL. An optimal gradient elution program was applied to separate the components effectively: 0–15 min, 5–90% B; 15–20 min, 90% B. MS/MS analysis was operated using a high resolution mass spectrometer (Q-Exactive Focus, Thermo Fisher Scientific). The MS data were acquired from an electrospray ionization (ESI) source in positive and negative ion mode, respectively. The parameters of the source were set as follows: nebulizer gas pressure 45.00 psi; electrospray voltage 4,000 V; fragmentor 150 V; desolvation gas (nitrogen > 99.99%) flow 600 L/h; desolvation temperature 350°C and source temperature 100°C; target mass m/z 400; scan range m/z 100–1,500. Data acquisition processing was carried out using Thermo Fisher Xcalibur workstation (Xcalibur software, version 4.0).
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