The method used was based on a previously reported protocol (Vosse et al., 2018 (link)). Separation was performed on an Agilent Rapid Res 1200 HPLC system using a HILICON iHILIC-Fusion (2.1 × 150 mm, 3.5 μm) column. Mobile phase A was water with 35 mM NH4OH, pH 5.75 and mobile phase B was ACN. Initial conditions were 3:97 A:B, held for 0.5 minute, followed by a linear gradient to 25:75 at 26.5 min, 40:60 at 27 min, and held until 33 min. Column re-equilibration was performed by returning to 3:97 A:B at 35 min and held until 45 min. Column flow rate was 0.3 mL/min. The retention time for PG was 7.5 min. Analytes were detected by MS/MS, based on multiple reaction monitoring, utilizing an Agilent 6460 triple quadrupole mass spectrometer. Electrospray ionization in negative mode was used with a transition of 773.4 to 281.2, with a collision energy of 45 V, for both BMP and PG. A fragmentor energy of 100 V and a dwell time of 200 ms was used. Source parameters were as follows: nitrogen gas temperature = 325 °C and flow rate = 9 L/min, nebulizer pressure = 40 psi, sheath gas temperature = 250 °C, sheath gas flow rate = 7 L/min, and capillary potential = 3.8 kV. All data were collected and analyzed with Agilent MassHunter B.03 software.
Masshunter b 03
Manufactured by Agilent Technologies
MassHunter B.03 software is a data analysis software for mass spectrometry. It provides tools for data acquisition, processing, and visualization.
Automatically generated - may contain errors
Lab products found in correlation
1290 infinity, by Agilent Technologies (2 mentions)
Agilent 6410 q tof ms ms, by Agilent Technologies (2 mentions)
6460 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Agilent 1290 uplc system, by Agilent Technologies (1 mentions)
Agilent 6540 q tof mass spectrometer, by Agilent Technologies (1 mentions)
4 protocols using masshunter b 03
1
HPLC-MS/MS protocol for BMP and PG
2
UPLC-QTOF Mass Spectrometry Protocol
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Agilent 1290 UPLC system (Agilent Technologies), equipped with an auto sampler, a binary pump and a thermostatted column compartment, was used for chromatographic analysis. The samples were separated on ACQUITY UPLC BEH C18 column (2.1 mm × 100 nm, 1.7 μm) at 40 °C. The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B). Programmed gradient elution was performed as follow: 0–3 min, 2% B; 3–9 min, 2–12% B; 9–24 min, 12–32% B; 24–29 min, 32–75% B; 29–29.1 min, 75–100% B; 29.1–32 min, 100% B; 32.1–35 min, 100-2%B. The flow rate was 0.4 mL/min. Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream electrospray (ESI) ion source was utilized to acquire MS and MS/MS data in positive ion mode. Data acquisition was managed by MassHunter B.03 software (Agilent Technologies). The working parameters were as follow: nebulizing gas (N2) flow rate, 8.0 L/min; nebulizing gas temperature, 300 °C; jet stream gas flow, 9 L/min; sheath gas temperature, 350 °C; nebulizer, 45 psi; capillary, 3000 V; skimmer, 65 V; Oct RFV, 600 V; fragment voltage, 150 V. Mass spectrum was documented with mass range at 100–1700 m/z of all mass peaks.
3
Targeted LC-MS Metabolite Analysis
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Liquid chromatography‐MS analysis was carried out with a ultra high performance liquid chromatography system (Infinity 1290; Agilent Technologies) connected to a Q‐TOF/MS instrument (Agilent 6410 Q‐TOF MS/MS; Agilent Technologies). Elution with solvent A (0.1% formic acid) and solvent B (acetonitrile) in a linear gradient manner was undertaken at a flow rate of 0.4 mL/min as follows: 5% B (0‐2 minutes), 50% B (2‐10 minutes), 100% B (10‐18 minutes), 100% B (18‐18.5 minutes), 5% B (18.5‐19 minutes), and 5% B (19‐20 minutes). The injection volume was 5 μL, the flow rate was 300 μL/min, and the temperature was 40°C. Electrospray ionization in positive ionization mode was carried out with the following parameters: the capillary voltage and cone voltage were set at 4.5 and 35 kV, respectively, the drying gas temperature was set at 400°C, and the mass range was scanned as m/z 50‐1000. The LC‐MS data were collected and analyzed using MassHunter B.03.01 (Agilent Technologies).
4
Metabolite Identification by LC-MS/MS
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LC‐MS analysis was conducted by an UHPLC system (Infinity 1290; Agilent Technologies). The system was connected to a Q‐TOF/MS instrument (Agilent 6410 Q‐TOF MS/MS, Agilent Technologies). LC‐MS data were collected and analysed by MassHunter B.03.01 (Agilent Technologies). The injection of each sample was performed twice, 2 μL for negative ionization mode analysis and positive ionization mode analysis, respectively. The column compartment temperature was 40°C, the setup of flow rate was 0.4 mL/min, the total separation time of two ionization modes was 20 minutes and the autosampler temperature was kept at 4°C. The mobile phase comprised Solvents A (5 mmol/L 0.1% formic acid) and Solvents B (acetonitrile with 0.2% acetic acid). By spiking the pooled serum samples containing mixtures of five standard metabolites, the identities of metabolites were proved.
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