6545 q tof

Sample analyses were performed using an Agilent QTOF 6545 with Jetstream ESI spray source coupled to an Agilent 1260 Infinity II Quaternary Pump UHPLC (Agilent Technologies, Santa Clara, USA) with 1260 autosampler, column oven compartment and variable wavelength detector. The MS was operated in all-ions mode with scan range 50–1700 m/z, 20 000 : 1 resolution and was calibrated using a reference introduced from the independent ESI reference sprayer.
Samples from the in vitro experiments were injected in negative ionisation mode onto a reverse-phase InfinityLab Poroshell 120 EC-C18 (3.0 × 50 mm, 2.7 μm) column (Agilent Technologies, Santa Clara, USA) using 0.1% (v/v) formic acid in water (mobile phase A) and in methanol (mobile phase B). Accurate mass measurements are reported to four decimal places. For improved retention of amino acids, samples from the experiments in fructo were subjected to further LC-MS analyses using a HILIC column (Agilent InfinityLab Poroshell 120 HILIC-Z; dimensions, 2.1 × 100 mm, 2.7 μm). Details of the gradient schedules are given in ESI Table S1.† LC-MS operational conditions are in ESI Table S2.†

The purified compounds were individually subjected to spectrum analysis for structure identification. The high-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum of the compound was recorded with an Agilent QTOF 6545 instrument operated in a positive-ion mode at capillary and cone voltages of 3.6 kV and 40 to 150 V, respectively. The collision energy was optimized from 15 to 50 V. For accurate measurement of metabolite mass, the instrument was calibrated each time using a standard calibration mix (Agilent) in the range of m/z 150 to 1,900. The 1D and 2D NMR (nuclear magnetic resonance) spectrum data for each compound (including ¹H, ¹³C, heteronuclear single quantum coherence [HSQC], correlation spectroscopy [COSY], nuclear Overhauser effect spectroscopy distortionless enhancement by polarization transfer [NOESY DEPT], and/or heteronuclear multiple-bond correlation [HMBC]) were recorded at 25°C using a Bruker Avance III-500 spectrometer equipped with a 5-mm Pabbo BB-¹H/D probe (60 (link)). The chemical shift values (δ) are given in parts per million, and the coupling constants (J values) are shown in hertz. Chemical shifts were referenced to the residual solvent peaks of CD₃OD-d₄. All spectra were processed with the MestRe Nova program (version 9.0.1; Metrelab Research, Santiago de Compostela, Spain).

Two microliters of metabolic extract were analyzed according to a previously described method [23 (link)]. A reversed-phase column (Zorbax SB-Aq 2.1 × 50 mm, 1.8 µm particle size, Agilent Technologies, CA, USA) with a pre-column (Zorbax SB-C8 Rapid Resolution Cartridge, 2.1 × 30 mm, 3.5 µm particle size, Agilent Technologies, CA, USA) set at 60 °C was used for chromatographic separation [23 (link)]. Afterwards, electrospray ionization was made in positive and negative ion modes using N₂ at a pressure of 50 psi for the nebulizer with a flow of 12 L/min and a temperature of 325 °C.
The analysis was performed using a UHPLC 1290 model coupled to a Q-TOF 6545 instrument (Agilent Technologies, CA, USA) as previously described [23 (link)], and data were collected using the MassHunter Data Analysis Software (Agilent Technologies, CA, USA).

All reagents were commercially available and used as supplied without further purification. Deuterated solvents were purchased from Cambridge Isotope Laboratory (Andover, MA). Compound 1^{39 (link)}, 2^{40 (link)}, 4^{39 (link)}, 5^{40 (link)} was prepared according to the published procedures. ¹H NMR, ³¹P{¹H} NMR spectra and 2D COSY NMR spectra were recorded on Bruker AVANCE III HD 400 MHz spectrometer and Bruker AVANCE III HD 600 MHz spectrometer. ¹H NMR chemical shifts are reported relative to residual solvent signals. Mass spectra were recorded on the Micromass Quattro II triple-quadrupole mass spectrometer using electrospray ionization, Thermo Scientific Q Exactive mass spectrometer using electrospray ionization and Agilent 6545 Q-TOF using electrospray ionization. The single crystals data were collected on a Nonius KappaCCD diffractometer equipped with Mo K-alpha radiation (λ = 0.71073 Å) and a BRUKER APEXII CCD.

Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was realized on Agilent 1290 Infinity II UHPLC (Agilent Technologies) with diode array detector (DAD) coupled to an Agilent 6545 QTOF with an electrospray ionization source. Analyses were performed in negative and positive ion mode. Compound 2 was prepared at 250 uM in ethanol and 1 μL was used for injection. The analyses were performed on a reversed-phase column Agilent Poroshell 120 Phenyl Hexyl column (150 × 2.1 mm, 1.9 µm), using water/acetonitrile mobile phase, both containing formic acid at 20 mM (phase A/B respectively). Phase B increased from 10% to 100% in 10 min, then held at 100% B for 2 min, returned to 10% in 0.1 min, and equilibrated for 2 min at a flow rate of 350 µL/min, and column temperature of 40 °C.
The raw data were processed by MassHunter workstation software (Agilent Technologies), Qualitative Analysis (version B.07.00).