An Agilent 1260 HPLC coupled to an Agilent 6540B QTOF was used. An Agilent extended LC C18 column, 5 cm × 2.1 mm, 1.7 µm, was used. Gradient elution was performed with 95%/5% water, 0.1% formic acid:acetonitrile, 0.1% formic acid to 75% water, 0.1% formic acid: 25% acetonitrile, 0.1% formic acid from 0–16 min, then back to 5% acetonitrile, 0.1% formic acid from 16.1 to 19 min at a flow rate of 0.4 mL/min. Samples or standards were injected at 5 µL. The QTOF MS was equipped with a dual JetStream ESI probe at positive mode to provide a continuous injection of API-TOF Mass Reference (Agilent, Santa Clara, CA). It was tuned prior to HPLC-MS analysis to ensure mass error <10 ppm, and continuously calibrated with the reference to maintain mass accuracy during HPLC-MS analysis. The QTOF was run with the following settings: gas temperature 300 °C; drying gas flow rate 12 L/min; nebulizer pressure 35 psig; sheath gas temperature 325 °C; sheath gas flow rate 10 L/min; Vcap 4 kV; fragmentor 135 V; skimmer 65 V; OCT RF Vapp 750 V; mass scan range m/z 100–1000. High accuracy QTOF MS data were analyzed using MassHunter Qualitative Analysis B.06.00 software. Compound identification was processed by extracting MS ions with intensity above 20,000 counts and comparing with a user-defined database of reported plant flavonoids in literature for mass match with a mass defect tolerance of 0.05 Da.
Jetstream esi probe
Manufactured by Agilent Technologies
The JetStream ESI probe is a compact, high-performance electrospray ionization source designed for use with Agilent mass spectrometry systems. It provides efficient sample ionization and introduction into the mass spectrometer for accurate and sensitive analyte detection.
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2 protocols using jetstream esi probe
1
HPLC-QTOF Analysis of Plant Flavonoids
2
UPLC-QTOF-MS for Metabolite Profiling
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The UPLC-QTOF-MS system used in this work consisted of an Agilent 1290 Infinity UPLC system coupled with an Agilent 6540 QTOF mass spectrometer. The UPLC system included a solvent reservoir, a degasser, a G4220A binary pump, a G1330B thermostat, a G4226A autosampler, a G1316C thermostatted column compartment, and a G4212A diode array detector. The mass spectrometer was equipped with an Agilent jet stream ESI probe.
Liquid chromatographic separation was achieved using gradient elution on a Waters ACQUITY UPLC BEH C18 (
GHz). The MS scan range was 50-1000 m/z at a scan rate 5 spectra/s. The MS/MS scan range was 50-1000 m/z at a scan rate of 3 spectra/s with an isolation width at narrow (~1.3 m/z). To maintain the mass accuracy, the mass spectrometer was calibrated and tuned before analysis, and internal reference masses from the reference mass solution were used for real-time mass correction at m/z 121.0508 and 922.0098 for the positive ion mode, and m/z 112.9885 and 1033.9881 for the negative ion mode throughout the acquisition process. The reference mass solution was prepared using an Agilent API-TOF reference mass solution kit (Part No: G1969-85001).
Liquid chromatographic separation was achieved using gradient elution on a Waters ACQUITY UPLC BEH C18 (
GHz). The MS scan range was 50-1000 m/z at a scan rate 5 spectra/s. The MS/MS scan range was 50-1000 m/z at a scan rate of 3 spectra/s with an isolation width at narrow (~1.3 m/z). To maintain the mass accuracy, the mass spectrometer was calibrated and tuned before analysis, and internal reference masses from the reference mass solution were used for real-time mass correction at m/z 121.0508 and 922.0098 for the positive ion mode, and m/z 112.9885 and 1033.9881 for the negative ion mode throughout the acquisition process. The reference mass solution was prepared using an Agilent API-TOF reference mass solution kit (Part No: G1969-85001).
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