The ACQUITY UPLC HSS T3 1.8 μm, 2.1 × 100 mm columns (Waters, Dublin, Ireland) were adopted into the present study. Ultra-performance Liquid Chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to Quadrupole-TOF MS (5600 Triple TOF Plus, AB SCIEX, Singapore) was applied to acquire metabolome data. The MS parameters for detection were: ESI source voltages were + 5.5 and -4.5 kV, respectively; vaporizer temperature, 500°C; drying gas (N2) pressure, 50 psi; nebulizer gas (N2) pressure, 50 psi; curtain gas (N2) pressure, 35 psi; The scan range was m/z 60–800. Information-dependent acquisition mode was used for MS/MS analyses of the metabolites. The collision energy was set at 35 ± 15 eV. Data acquisition and processing were performed using Analyst® TF 1.7.1 Software (AB Sciex, Concord, ON, Canada). The internal standard includes phenylalanine D8, tryptophan D8, isoleucine D10, asparagine 13C4, methionine D3, valine D8, proline D7, alanine D4, glycine D2, serine D3, glutamate D5, aspartate D3, arginine D7, glutamine D5, lysine D9, histidine D5, and taurine D2. All samples were measured at LipidALL Technologies Company (Changzhou, China).
Agilent 1290 2
Manufactured by Agilent Technologies
Sourced in Germany, United States
The Agilent 1290 II is a high-performance liquid chromatograph (HPLC) system designed for analytical applications. It features a modular design, allowing for the integration of various components such as pumps, autosampler, and detectors. The system is capable of delivering accurate and precise solvent flow rates for efficient separation and analysis of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof5600plus, by AB Sciex (5 mentions)
Accela 1250 uhplc, by Thermo Fisher Scientific (4 mentions)
Lcq fleet, by Phenomenex (4 mentions)
Kinetex 2.6μ xb c18 100a, by Phenomenex (4 mentions)
Markerview 1, by AB Sciex (4 mentions)
Peakview 2, by AB Sciex (4 mentions)
Analyst tf 1, by AB Sciex (3 mentions)
6545 qtof ms, by Agilent Technologies (2 mentions)
Acquity uplc hss t3 column, by Agilent Technologies (2 mentions)
Quadrupole tof ms 5600 triple tof plus, by AB Sciex (2 mentions)
Metabolites database, by AB Sciex (2 mentions)
Acquity uplc hss t3, by Waters Corporation (1 mentions)
Hplc ms grade, by Merck Group (1 mentions)
Infinitylab poroshell 120 hilic z column, by Agilent Technologies (1 mentions)
Acquity uplc hss t3 1.8 m, by Waters Corporation (1 mentions)
Quadrupole tof ms, by AB Sciex (1 mentions)
Acquity uplc hss t3 1.8 μm 2 1 100 mm column, by Waters Corporation (1 mentions)
14 protocols using agilent 1290 2
1
Metabolomic Profiling Using UPLC-QTOF-MS
2
Metabolite Analysis via UPLC-QTOF/MS
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The metabolites were analyzed using Agilent 1290 II (Agilent, Santa Clara, CA, USA) with ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm) with mobile phase A: ultra-pure water (0.1% formic acid) and mobile phase B: acetonitrile (0.1% formic acid). The elution gradient was set as: 0 min, 95% A; 0–11 min, 95–10% A; 11–12 min, 10% A; 12–12.1 min, 10–95% A; and 12.1–14 min, 95% A. The flow rate was 0.4 mL/min.
MS was performed using QTOF/MS-6545 (Agilent, Santa Clara, CA, USA). All analyses were performed in electrospray ionization (ESI±) mode under the following conditions: The electrospray voltage was 2.5 kV (positive ion mode) and 1.5 kV (negative ion mode); the heater temperature was 325 °C and the capillary temperature was 350 °C; the sheath gas flow rate was 45 arb; and the auxiliary gas flow rate was 8 arb.
MS was performed using QTOF/MS-6545 (Agilent, Santa Clara, CA, USA). All analyses were performed in electrospray ionization (ESI±) mode under the following conditions: The electrospray voltage was 2.5 kV (positive ion mode) and 1.5 kV (negative ion mode); the heater temperature was 325 °C and the capillary temperature was 350 °C; the sheath gas flow rate was 45 arb; and the auxiliary gas flow rate was 8 arb.
3
Purification of Diastereomeric Compound
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Example 96
Second Eluting Diastereomer: 1H NMR (400 MHz, methanol-d4) δ 7.82 (s, 1H), 7.30 (dd, J=8.6, 7.2 Hz, 2H), 7.23-7.12 (m, 3H), 6.83 (d, J=4.5 Hz, 1H), 6.74 (d, J=4.5 Hz, 1H), 5.87 (d, J=6.0 Hz, 1H), 5.79 (dd, J=5.9, 4.9 Hz, 1H), 5.68 (d, J=4.9 Hz, 1H), 4.43 (t, J=5.6 Hz, 2H), 4.01 (dd, J=10.9, 6.7 Hz, 1H), 3.90 (ddd, J=10.1, 8.7, 6.8 Hz, 2H), 2.55 (hept, J=7.4 Hz, 1H), 2.12 (d, J=19.7 Hz, 6H), 2.06-1.66 (m, 6H), 1.27 (dd, J=7.1, 1.1 Hz, 3H). 31P NMR (162 MHz, methanol-d4) δ 3.02. LCMS: MS m/z=671.30 [M+1], tR=1.03 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ XB-C18 100A, 50×3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN at 1800 μL/min. HPLC: tR=5.376 min; Agilent 1290 II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6 mm; Solvents: A: Water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; Gradient: 2-98% B with 8.5 min gradient at 1.5 mL/min.
4
Purification and Characterization of Diastereomeric Compound
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Example 109
Second Eluting Diastereomer: 1H NMR (400 MHz, methanol-d4) δ 7.83 (s, 1H), 7.31 (t, J=7.9 Hz, 2H), 7.24-7.13 (m, 3H), 6.84 (d, J=4.6 Hz, 1H), 6.75 (d, J=4.5 Hz, 1H), 5.89 (d, J=5.9 Hz, 1H), 5.80 (dd, J=5.9, 4.8 Hz, 1H), 5.68 (d, J=4.9 Hz, 1H), 4.50-4.37 (m, 2H), 3.97-3.78 (m, 5H), 3.34 (m, 2H), 2.53-2.35 (m, 4H), 1.95-1.68 (m, 1H), 1.55 (d, J=13.3 Hz, 2H), 1.34-1.22 (m, 5H), 1.15 (dt, J=14.3, 7.5 Hz, 6H). 31P NMR (162 MHz, methanol-d4) δ 2.98. LCMS: MS m/z=729.27 [M+1], tR=1.01 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ XB-C18 100A, 50×3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN at 1800 μL/min. HPLC: tR=5.214 min; Agilent 1290 II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6 mm; Solvents: A: Water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; Gradient: 2-98% B with 8.5 min gradient at 1.5 mL/min.
5
Synthesis and Characterization of Diastereomeric Compound
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Example 108
First Eluting Diastereomer: 1H NMR (400 MHz, methanol-d4) δ 7.81 (s, 1H), 7.35-7.26 (m, 2H), 7.20-7.12 (m, 3H), 6.85 (d, J=4.5 Hz, 1H), 6.78 (d, J=4.5 Hz, 1H), 5.97 (d, J=5.9 Hz, 1H), 5.86 (dd, J=5.9, 4.6 Hz, 1H), 5.69 (d, J=4.6 Hz, 1H), 4.46 (ddd, J=36.1, 11.1, 5.5 Hz, 2H), 3.94-3.79 (m, 5H), 3.3 (m, 2H), 2.54-2.36 (m, 4H), 1.86 (dp, J=10.6, 3.8 Hz, 1H), 1.64-1.46 (m, 2H), 1.35-1.09 (m, 11H). 31P NMR (162 MHz, methanol-d4) δ 3.03. LCMS: MS m/z=729.30 [M+1], tR=1.00 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ XB-C18 100A, 50×3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN at 1800 μL/min. HPLC: tR=5.151 min; HPLC system: Agilent 1290 II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6 mm; Solvents: A: Water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; Gradient: 2-98% B with 8.5 min gradient at 1.5 mL/min.
6
Purification and Characterization of Diastereomer
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Example 117
Second Eluting Diastereomer: 1H NMR (400 MHz, methanol-d4) δ 7.83 (s, 1H), 7.31 (dd, J=8.6, 7.1 Hz, 2H), 7.24-7.12 (m, 3H), 6.84 (d, J=4.5 Hz, 1H), 6.74 (d, J=4.6 Hz, 1H), 5.89 (d, J=5.9 Hz, 1H), 5.80 (dd, J=5.9, 4.8 Hz, 1H), 5.68 (d, J=4.8 Hz, 1H), 4.50-4.37 (m, 2H), 3.97-3.78 (m, 3H), 3.04-2.88 (m, 4H), 2.53-2.35 (m, 4H), 2.26 (t, J=11.7 Hz, 2H), 1.65-1.51 (m, 3H), 1.34-1.08 (m, 11H). 19F NMR (376 MHz, methanol-d4) δ −71.22 (t, J=9.9 Hz). 31P NMR (162 MHz, methanol-d4) δ 2.99. LCMS: MS m/z=405.88 [M+1], tR=1.04 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ XB-C18 100A, 50×3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN at 1800 μL/min. HPLC: tR=4.673 min; Agilent 1290 II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6 mm; Solvents: A: Water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; Gradient: 2-98% B with 8.5 min gradient at 1.5 mL/min.
7
UPLC-MS/MS Metabolite Profiling
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Ultrahigh performance liquid chromatography (UPLC, Agilent 1290 II, Agilent Technologies, Waldbronn, Germany) combined with tandem quadrupole time-of-flight (5600 Triple TOF Plus, AB Sciex, Singapore), and ACQUITY UPLC HSS T3 (1.8 μm, 2.1 mm × 100 mm, Waters, Dublin, Ireland) chromatographic column were used for the analysis. All analyses were performed in electrospray ionization mode. Instrument conditions were as previously reported (Song et al., 2020 (link)), including the following: curtain gas = 35; positive ion spray voltage = 5,500 V; negative ion spray voltage = -4,500 V; temperature = 450°C; ion source gas 1 = 50; and ion source gas 2 = 50. Data acquisition mode included a full scan of the primary mass spectrum and information-dependent acquisition of secondary mass spectrum data. MarkerView 1.3 (AB Sciex, Concord, ON, Canada) was used to extract the peak area, mass-to-charge ratio, and retention time of the primary mass spectrum data to generate a two-dimensional data array. Secondary mass spectrum data were extracted by PeakView 2.2 (AB Sciex), and metabolite IDs were identified after interrogation of a metabolite database, HMDB, and METLIN standards. Metabolite IDs were assigned to the corresponding ion of the two-dimensional data array.
8
Metabolic Flux Analysis of Palmitate Tracing
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For metabolic flux experiments, 13C-palmitate tracing was conducted using LC/MS at LipidALL Technologies. Cells (1 × 107) were incubated for 8 h at 37 °C in RPMI-1640 medium with 10% FBS. For 13C-palmitate incorporation in metabolites, cells were incubated in growth medium with 50 μM [U-13C] palmitate for 24 h. Then, cells were quickly washed with 1×PBS and fixed with pre-cooled methanol (HPLC-MS grade, Millipore) for 30 min at −80 °C. The extraction method was referenced to a previous paper, but with modification [52 (link)]. Samples were incubated for 30 min at 1500 rpm and 4 °C and then centrifuged for 10 min at 12,000 rpm and 4 °C. The supernatant fractions were placed into clean 1.5-ml centrifuge tubes and dried using a SpeedVac. The dried extracts were dissolved with 50% acetonitrile in water and the upper layer liquids were collected for LC–MS analysis. The InfinityLab Poroshell 120 HILIC-Z column (2.1 mm × 50 mm, 2.7 μm, Agilent Technologies, Germany) was used in this study. Ultra-performance Liquid Chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to the Quadrupole-TOF MS 5600 Triple TOF Plus, AB SCIEX, Singapore) was used to acquire metabolome data.
9
Metabolomic Analysis Using UPLC-QTOF MS
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The column ACQUITY UPLC HSS T3 1.8 µm, 2.1 × 100 mm columns (Waters, Dublin, Ireland) was adopted into the present study. Ultra-performance Liquid Chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to Quadrupole-TOF MS (5600 Triple TOF Plus, AB SCIEX, Singapore) was applied to acquire metabolome data.
10
Metabolite Analysis via UPLC-QTOF/MS
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The metabolites were analyzed using Agilent 1290 II (Agilent, Santa Clara, CA, USA) with ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm) with mobile phase A: ultra-pure water (0.1% formic acid) and mobile phase B: acetonitrile (0.1% formic acid). The elution gradient was set as: 0 min, 95% A; 0–11 min, 95–10% A; 11–12 min, 10% A; 12–12.1 min, 10–95% A; and 12.1–14 min, 95% A. The flow rate was 0.4 mL/min.
MS was performed using QTOF/MS-6545 (Agilent, Santa Clara, CA, USA). All analyses were performed in electrospray ionization (ESI±) mode under the following conditions: The electrospray voltage was 2.5 kV (positive ion mode) and 1.5 kV (negative ion mode); the heater temperature was 325 °C and the capillary temperature was 350 °C; the sheath gas flow rate was 45 arb; and the auxiliary gas flow rate was 8 arb.
MS was performed using QTOF/MS-6545 (Agilent, Santa Clara, CA, USA). All analyses were performed in electrospray ionization (ESI±) mode under the following conditions: The electrospray voltage was 2.5 kV (positive ion mode) and 1.5 kV (negative ion mode); the heater temperature was 325 °C and the capillary temperature was 350 °C; the sheath gas flow rate was 45 arb; and the auxiliary gas flow rate was 8 arb.
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