Liquid chromatography with tandem mass spectrometry experiments were performed on an Agilent 1290 Infinity UPLC system (Agilent) coupled to a QTrap5500 mass spectrometer (Sciex), equipped with an ESI TurboIonSpray source. The flow rate was 0.22 ml/min, autosampler temperature was set at 6 °C, and the column compartment was set to 35 °C. Separations were performed on an Asahipak NH2P-40 (250 × 2 mm, 4-μm particle size; Showa Denko). The mobile phase was composed of 20 mM ammonium carbonate (10361-29-2; Merck) in H2O/5% acetonitrile (ACN; HN40.2; Roth), pH 10, and ACN. After an initial 3.5-min isocratic elution of 99.9% ACN, the percentage of ACN was decreased to 85% at 3.6 min, to 75% at 8.1 min, to 0% at 14 min, and back to 99.9% at 34 min. The composition was maintained at 99.9% ACN until 42 min. The QTrap5500-MS system was operated in triple quadrupole mode with positive/negative ion switching. Ion spray voltages of 5,500/−4,500 V were applied, respectively. Curtain gas was set to 30 psi, collision gas to medium, source temperature to 500 °C, ion source gas 1 to 35 psi, and ion source gas 2 to 35 psi. Analyst 1.6.2 and MultiQuant 3.0 (both from Sciex) were used for data acquisition and analysis, respectively. Data were analyzed with Metaboanalyst 4.0.
Agilent 1290 infinity uplc system
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Agilent 1290 Infinity UPLC system is a high-performance liquid chromatography (HPLC) instrument designed for ultra-high performance liquid chromatography (UPLC) applications. It features an advanced pump design, rapid and precise sample handling, and a sensitive and selective detector. The system is capable of delivering stable and reliable performance for a wide range of chromatographic separations.
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Lab products found in correlation
Masshunter qualitative analysis software, by Agilent Technologies (3 mentions)
Multiquant 3, by AB Sciex (2 mentions)
Xdb c18 column, by Agilent Technologies (2 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Q tof mass spectrometer, by Agilent Technologies (1 mentions)
Silymarin, by Merck Group (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
Uplc esi qtof ms system, by Agilent Technologies (1 mentions)
Esi l low concentration tuning mix, by Agilent Technologies (1 mentions)
6550 accurate mass quadrupole time of flight mass spectrometer, by Agilent Technologies (1 mentions)
Agilent 1290 diode array detector, by Agilent Technologies (1 mentions)
Zorbax extend c18 column, by Agilent Technologies (1 mentions)
Zorbax rrhd sb c18 column, by Agilent Technologies (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
Agilent masshunter workstation data acquisition software, by Agilent Technologies (1 mentions)
6550accurate mass quadrupole time of flight, by Agilent Technologies (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Rosmarinic acid, by Merck Group (1 mentions)
15 protocols using agilent 1290 infinity uplc system
1
Rapid Metabolite Analysis by LC-MS/MS
2
UPLC-QTOF Analysis of RSV Metabolites
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A previously validated method
(linearity, precision, accuracy, limits of detection, and quantification)
was used to analyze RSV and derived metabolites.30 (link) Briefly, the analyses were performed on an Agilent 1290
Infinity UPLC system coupled to a 6550 accurate-mass quadrupole-time-of-flight
(QTOF) mass spectrometer (Agilent Technologies, Waldbronn, Germany)
using an electrospray interface (Jet Stream Technology), using chrysin
as an internal control of the ionization signal. Spectra were acquired
in the m/z range of 100 to 1100
in a negative polarity mode and at an acquisition rate of 1.5 spectra/s.
Data were processed using Mass Hunter Qualitative Analysis software
(version B.06.00, Agilent), which lists and rates possible molecular
formulas consistent with the accurate mass measurement and the actual
isotopic pattern. A target screening strategy was applied to qualitatively
analyze possible metabolites that could be present after RSV consumption.
In addition, targeted MS/MS analysis provided additional information
to achieve a reliable compound identification. MS/MS product ion spectra
were collected at m/z 50–800
range using a retention time window of 1 min, collision energy of
20 V, and an acquisition rate of 4 spectra/s.
(linearity, precision, accuracy, limits of detection, and quantification)
was used to analyze RSV and derived metabolites.30 (link) Briefly, the analyses were performed on an Agilent 1290
Infinity UPLC system coupled to a 6550 accurate-mass quadrupole-time-of-flight
(QTOF) mass spectrometer (Agilent Technologies, Waldbronn, Germany)
using an electrospray interface (Jet Stream Technology), using chrysin
as an internal control of the ionization signal. Spectra were acquired
in the m/z range of 100 to 1100
in a negative polarity mode and at an acquisition rate of 1.5 spectra/s.
Data were processed using Mass Hunter Qualitative Analysis software
(version B.06.00, Agilent), which lists and rates possible molecular
formulas consistent with the accurate mass measurement and the actual
isotopic pattern. A target screening strategy was applied to qualitatively
analyze possible metabolites that could be present after RSV consumption.
In addition, targeted MS/MS analysis provided additional information
to achieve a reliable compound identification. MS/MS product ion spectra
were collected at m/z 50–800
range using a retention time window of 1 min, collision energy of
20 V, and an acquisition rate of 4 spectra/s.
3
Silymarin Extraction and Analysis
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Extraction solvents were of analytical grade from XiLong chemical Co. Ltd. (Guangdong, China). Silymarin was obtained from Sigma-Aldrich (SIGMA-ALDRICH, Co., China) and used as a positive control in this research. UPLC-DAD-ESI-MS experiments were performed on an Agilent 1290 infinity UPLC system (Agilent, USA). Absorbance was measured with a microplate reader (DNM-9602, Beijing Pu Long new technology Co. Ltd., Beijing, China). A wan-neng pulverizer (Zhejiang Yi Li Co. Ltd., Zhejiang, China) was used for grinding medicines.
4
Quantitative Analysis of Cellular Metabolites
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HeLa cells were seeded in 10 cm dishes and exposed to 5mM butyrate for 48 h. Cells were grown to approximately 90% confluency and then harvested. After washing with cold PBS and cold normal saline three times, the cells were dissolved in 1ml of cold 4/4/2 (v/v/v) methanol/acetonitrile/water. Then, all cell samples were collected in 1.5ml centrifuge tubes stored in a refrigerator at − 80 °C after flash freezing in liquid nitrogen. Samples were separated by the Agilent 1,290 Infinity UPLC system at Shanghai Applied Protein Technology Co.,Ltd. (Shanghai, China). Data were acquired using QTRAP5500 (ABSCIEX) mass spectrometry in negative ionization mode. Chromatographic peak area and retention time were extracted by Multiquanta software. Retention time corrected for energy metabolite standard.
5
Comprehensive Metabolite Profiling by UPLC-ESI-QTOF/MS
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The urine, feces, and microsome samples were analyzed by the UPLC-ESI-QTOF/MS system (Agilent, Santa Clara, CA, USA). Metabolites showed good separation in the Agilent 1290 infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an XDB-C18 column (2.1 mm × 100 mm, 1.8 µM). The column temperature was maintained at 45 °C, and the flow was set at 0.3 mL/min. Elution was performed using gradient elution ranging from 2% to 98% acetonitrile, containing 0.1% formic acid for 16 min. The injection volume was 5 µL, and the mass signals of ions were collected in both positive (ESI +) and negative (ESI −) modes with electrospray ionization. Nitrogen was used as the collision gas and drying gas, which was set at 350 °C and 9 L/min. Nebulizer pressure was set at 35 psi, and the capillary voltage was set at 3.5 kV. The structures of metabolites were identified by the accurate mass measurements compared to the fragmentary mode of the parent compound, and the MS/MS chromatogram of metabolites was obtained using four collision energy, 10, 15, 20, and 30 eV. The MS was calibrated using the ESI-L Low-Concentration Tuning Mix (Agilent, Santa Clara, CA, USA).
6
Metabolite Profiling via UPLC-MS/MS
7
Quantitative Analysis of RSV Metabolites
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A previously validated method (linearity, precision, accuracy, limits of detection, and quantification) was used to analyze RSV and its derived metabolites [36 (link)]. Briefly, the analyses were performed on an Agilent 1290 Infinity UPLC system coupled to a 6550 Accurate-Mass quadrupole-time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Waldbronn, Germany) using an electrospray interface (Jet Stream Technology). Chrysin was used as an internal control of the ionization signal. Spectra were acquired in the m/z range from 100 to 1100, in negative polarity mode (m/z−) and an acquisition rate of 1.5 spectra/s. Data were processed using the Mass Hunter Qualitative Analysis software (version B.06.00, Agilent). A targeted screening was used to identify possible phase-II metabolites (glucuronides, sulfates, and sulfoglucuronides) after RSV metabolism. In addition, MS/MS analysis provided additional information to achieve reliable compound identification. MS/MS product ion spectra were collected at m/z 50–800 range using a retention time window of 1 min, collision energy of 20 V, and acquisition rate of 4 spectra/s.
8
Quantitative Analysis of Total Fungal Glucans
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The total GA was detected using the HPLC method according to the previously described method [33 (link)]. Briefly, dried mycelia (0.05 g) were weighed and fully ground and then added to 2 mL of a 10% (w/v) KOH-75% (v/v) ethanol solution. The samples were left for 2 h for saponification and the mixture was extracted 3 times with 2 mL hexane. Samples of the hexane layer were collected and vaporized under nitrogen gas until dry. The residue was dissolved in 0.5 mL of acetonitrile. An Agilent 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA) was used with an Agilent 1290 diode array detector and an Agilent Zorbax Eclipse Pluss C18 Rapid Resolution HD 18-Micron column (2.1 ×100 mm) (Agilent, Tokyo, Japan).
9
Quantification of Kinase Metabolite in Plasma
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The preparation and quantification of KM in the plasma were performed as previously reported (Ye et al., 2020 (link)). In brief, 50 μL of plasma was spiked with GM at a final concentration of 80 ng ml−1 and evaporated to dryness. The dry sample was mixed with 400 μL of ethyl acetate by vortexing and then centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was carefully transferred into fresh tubes and evaporated to dryness. The residues were redissolved with 100 μL of 50% methanol solution, of which 5 μL was injected for ultra-performance liquid chromatography (UPLC)–tandem mass spectrometry (MS/MS) analysis. The assay was performed on an Agilent 1290 Infinity UPLC® system (Agilent Technologies, Santa Clara, CA, United States). The mobile phase comprised methanol and water with 0.1% formic acid. The flow rate was 0.4 ml min−1 at 40°C. The gradient program is shown in SupplementaryTable S1 . Only the eluent between 2.0 and 5.0 min was passed into the MS/MS system, operated in the positive ionization mode. The multiple reaction monitoring transition of KM was at m/z 307.2/180.1 and GM was at m/z 323.1/70.1.
10
Pharmaceutical Compound Separation by UPLC
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Analysis was performed on an Agilent 1290 Infinity UPLC system (Agilent Technology, Waldbronn, Germany) equipped with a binary pump, a diode-array detector, an auto sampler, and a thermostatically controlled column compartment. Samples were separated on an Agilent ZORBAX Extend-C18 column (2.1 mm × 50 mm, 1.8 μm) at 25°C using water (solvent A) and acetonitrile (solvent B). A gradient elution program was used according to the following profile: 20% B at 0–0.5 min, 20–26% B at 0.5–3 min, 26–38% B at 3–6 min, 38–55% B at 6–8 min, 55% B at 8–10 min, and 55–100% B at 10–15 min. The flow rate was kept at 0.8 mL/min and the optimum wavelength was set at 203 nm.
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