Untargeted analyses were performed using an Agilent 1290 infinity liquid chromatography (LC) system coupled to an Agilent 6545 quadrupole-time-of-flight mass spectrometer (MS) equipped with an electrospray ionization (ESI) source operating in both positive and negative ion modes. Chromatographic separation was evaluated on an Acquity UPLC HSS T3 column (Waters, Wexford, Ireland) (2.1 × 100 mm, 1.8 μm) with a flow rate of 0.4 mL/min at 50°C. The mobile phase used for ESI+ consisted of 0.1% aqueous formic acid (mobile phase A) and acetonitrile (mobile phase B). For ESI-, the mobile phase consisted of (mobile phase A) 10 mmol/L ammonium acetate aqueous solution and (mobile phase B) 10 mmol/L ammonium acetate water/acetonitrile (1:9) solution. A linear gradient elution was optimized as follows: 0–1 minute, 1% B; 1–3 minutes, 1%–15% B; 3–5 minutes, 15%–70% B; 5–9 minutes, 70%–85% B; 9–10 minutes, 85%–100% B; 100% B held for 2 minutes, and then back to the initial conditions with 3 minutes for equilibration. The injection volume was 1.5 μL. MS parameters were set as follows: drying gas temperature, 320°C; drying gas flow rate, 8 L/min; nebulizer gas, 35 psi; fragmental voltage, 120 V; and capillary voltage, 3500 V. A full scan from 50 to 1050 m/z was acquired for each sample under the high-resolution mode (extended dynamic range, 2 GHz).
6545 quadrupole time of flight mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The 6545 Quadrupole Time-of-Flight Mass Spectrometer is a high-performance laboratory instrument designed for accurate and sensitive mass analysis. It combines a quadrupole mass analyzer with a time-of-flight mass analyzer to provide precise mass measurements and high-resolution data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Masshunter software, by Agilent Technologies (2 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
1290 series pump, by Agilent Technologies (1 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
1290 infinity uhplc system, by Agilent Technologies (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Jet stream ion source, by Agilent Technologies (1 mentions)
1290 infinity 2 uhplc system, by Agilent Technologies (1 mentions)
9 protocols using 6545 quadrupole time of flight mass spectrometer
1
Untargeted Metabolomics by UPLC-QTOF-MS
2
In Vitro Polyketide Synthesis Assay
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Twenty-five micromolar of LovB was incubated with 25 μM WT or mutant of LovC, 25 μM LovG, 2 mM Malonyl-CoA, 2 mM NADPH, and 2 mM SAM in buffer (100 mM NaH2PO4, pH 7.4, 10% glycerol, 2 mM DTT, 2 mM EDTA) in a 250 μl solution at 25 °C for 24 h. Reactions were quenched and extracted twice with an equal volume of 99% ethyl acetate (EA)/1% acetic acid (AcOH). The organic phase was evaporated to dryness, and redissolved in 0.05 M NaOH in 15 μl of methanol and analyzed by LC-MS. LC-MS was conducted with an Agilent 1290 Infinity Liquid chromatography and 6545 Quadrupole Time-of-Flight Mass Spectrometer by using negative electrospray ionization and an Agilent 5μ 4.6 × 150 mm C18 reverse-phase column. Samples were separated at room temperature on a linear gradient of 5–95% CH3CN (v/v) in H2O supplemented with 0.05% (v/v) formic acid over 30 min, and held at 95% CH3CN/ 0.05% formic acid for 30 min at a flow rate of 0.4 ml/min.
3
Lipid Profiling of BAL and Plasma Samples
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Reversed phase samples from the lipid fraction were randomized in the worklist and run randomly in triplicate using an Agilent 1290 series pump with an Agilent Zorbax Rapid Resolution HD (RRHD) SB-C18, 1.8 micron (2.1 × 100 mm) analytical column and an Agilent Zorbax SB-C18, 1.8 micron (2.1 × 5 mm) guard column. The autosampler tray temperature was set at 4 °C, column temperature was set at 60 °C, and the sample injection volume was 8 µL for BAL and 4 µL for plasma. The flow rate was 0.7 mL/min with the following mobile phases: mobile phase A was water with 0.1% formic acid, and mobile phase B was 60:36:4 isopropyl alcohol:acetonitrile:water with 0.1% formic acid. Gradient elution was as follows: 0–0.5 min 30–70% B, 0.5–7.42 min 70–100% B, 7.42–10.4 min 100% B, 10.4–10.5 min 100–30% B, 10.5–15.1 min 30% B. The lipid fraction MS conditions were as follows: Agilent 6545 Quadrupole Time-of-Flight mass spectrometer (QTOF-MS) in positive ionization mode with dual AJS ESI source, mass range 50–1700 m/z, scan rate 2.00, gas temperature 300 °C, gas flow 12.0 L/min, nebulizer 35 psi, sheath gas temperature 275 °C, skimmer 65 V, capillary voltage 3500 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix).
4
Metabolite Profiling of Mouse Gastrocnemius
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Frozen mouse gastrocnemius muscle was homogenized in 80% acetonitrile with 10 ml/mg tissue. After centrifugation (18,000 g, 10 min at 4°C), the supernatant of the sample was used for spectrometry analysis immediately. Liquid chromatography–mass spectrometry (LC-MS) was performed on an Agilent 1290 UHPLC system equipped with a binary solvent delivery manager, an auto sampler, and a column oven, coupled with a 6545 Quadrupole Time of Flight Mass Spectrometer with an electrospray interface (Agilent Technologies). The samples were extracted with protein precipitation as described above and separated with reverse-phase chromatography. The raw data were acquired in full scan positive mode within a mass range of m/z 100–1,000 with real-time mass calibration. Each mass response was corrected with the response of the respective internal standard. Each peak area ratio was used for relative quantitation. The data were processed with Mass Hunter software (Agilent Technologies).
5
UHPLC-QTOF Metabolomics Analysis
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The metabolomics measurement was performed using an Agilent 1290 Infinity UHPLC system combined with a 6545 quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) rigged with an electrospray interface. Chromatographic separation was conducted on the Agilent EclipsePlus C18 column (1.8 μm, 2.1 × 50 mm) with a flow rate of 0.25 mL/min at a temperature of 40°C. The mobile phase contained 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); gradient elution procedures were as follows: 0-1 min, 1%-20% B; 1-10 min, 20%-70% B; 10-14 min, 70%-82% B; 14-15 min, 82%-85% B; 15-16 min, 85%-99% B; 16-17.5 min, 99% B; 17.5-18.5 min, 99%-1% B; and 18.5-20 min, 1% B. The injection volume was 1.5 μL. ESI-MS/MS conditions were as follows: capillary voltage 3500 V, nebulizer pressure 40 psig, flow rate of drying gas 12 L/min, temperature 350°C, and scanning m/z range 100-1000.
6
UHPLC-QTOF Analysis of Hydrophobic and Hydrophilic Fractions
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The hydrophobic fractions were analyzed using reverse-phase chromatography on an Agilent Technologies (Santa Clara, CA) 1290 ultrahigh precision liquid chromatography (UHPLC) system on an Agilent Zorbax rapid resolution HD SB-C18 analytical column (1.8 μm; 2.1 × 100 mm) as previously described (107 (link), 108 (link)). The hydrophilic fractions were analyzed using hydrophilic interaction liquid chromatography (HILIC) on a 1290 UHPLC system using an Agilent InfinityLab Poroshell 120 HILIC-Z analytical column (2.1 × 100 mm) with gradient conditions as previously described (109 ) with MS modifications as follows: nebulizer pressure, 35 lb/in2; gas flow, 12 liters/min; sheath gas temperature, 275°C; sheath gas flow, 12 liters/min; nozzle voltage, 250 V; and fragmentor, 100 V. The hydrophobic and hydrophilic fractions were run on Agilent Technologies 6545 quadrupole time-of-flight mass spectrometer. Both fractions were run in positive electrospray ionization mode.
7
Quantification of TCF and ACF by LC-ESI/MS
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TCF and ACF were analyzed by LC-ESI/MS system. The high-performance liquid chromatography (HPLC) system was 1260 Infinity II (Agilent Technologies, Waldbronn, Germany). A reversed-phase Eclipse Plus C18 RRHP (50 × 2.1 mm, 1.8 μm particle size, from Agilent Technologies) was used for the separation process. The eluents consisted of 2% acetonitrile in H2O (Phase A) and 100% methanol (Phase B), and the following gradient mode was used: 0–1 min, 15% B isocratic; 1–24 min, linear gradient from 15% to 95% B; 24–29 min, 95% B isocratic; 29–30 min, linear gradient from 95% to 15% B; and re-equilibration 30–50 min, 15% B isocratic. The column temperature was set to 30 °C, the flow rate to 0.2 mL/min, and the injection volume of 5 µL. HPLC coupled to a 6545 quadrupole time of flight mass spectrometer (Agilent Technologies) equipped with an Agilent Jet Stream electrospray ionization interface working in the positive mode. High-purity nitrogen was used as the nebulizer and auxiliary gas, and conditions were set at drying gas temperature 320 °C, sheath gas temperature 350 °C, and a flow rate of 10 L/min. The nebulizer pressure set to 35 PSIG, capillary voltage 3.5 kV, nozzle voltage 1 kV. The fragment was set to 175 (arbitrary units). Full MS scans from m/z 100–3000 Da were acquired at a scan rate of 1 spectrum/s. Data processing was performed using mestrenova software.
8
Lipidomic Analysis by LC-QTOF MS
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LC/MS lipidomic analysis was acquired on the Agilent 6545 Quadrupole Time-of-Flight Mass Spectrometer coupled with Infinity II 1290 Liquid Chromatography Ultra-High-Pressure system (Agilent Technologies Inc., Santa Clara, CA, USA).
9
UHPLC-QTOF-MS Protocol for Detailed Metabolite Analysis
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For liquid chromatography and high-resolution accurate mass spectrometry, we coupled an Agilent Technologies 1290 Infinity II UHPLC system to an Agilent Technologies 6545 Quadrupole Time-of-Flight Mass Spectrometer (i.e., QTOF-MS). Using the LC gradient profile described above, electrospray ionization (ESI) was achieved with an Agilent Jet Stream ion source. ESI was conducted in the negative ion mode (for measurement of [M – H]- ions) with capillary and nozzle voltages of 3.5 kV and 2 kV, respectively. Drying and nebulizing gases were set to 10 L/min and 25 lb/in2 (172 kPa), respectively, and a drying-gas temperature of 300 °C was used throughout. The Jet Stream ion source employed a heated nitrogen sheath gas (at 350 °C with a gas flow of 10 L/min) to improve droplet desolvation for signal enhancement. The fragmentor, skimmer, and OCT 1 RF Vpp voltages were set to 140 V, 50 V, and 300 V, respectively. The data acquisition range was from 100-2500 m/z, and the acquisition rate was 1 spectra/s. Data acquisition (Workstation B.08.00) and processing (Qualitative Analysis B.06.00) were performed via Agilent Technologies MassHunter software.
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