6540 qtof mass spectrometer

UHPLC-QTOF/MS analysis was performed using a 1290 ultra-high performance liquid chromatography system (Agilent, California, United States) and a 6540 Q-TOF mass spectrometer (Agilent, California, United States), operated in positive and negative ion modes. MS parameter settings are shown in Supplementary Table S2. Analysis were separated on a Waters BEH C18 column (2.1 × 150 mm, 1.7 μm, Milford, MA, United States) at a constant flow rate of 0.4 ml/min at a column temperature of 50°C. LC conditions were as follows: 0.1% formic acid in deionized water (solvent A) and 0.1% formic acid in acetonitrile (solvent B); gradient elution method is shown in Supplementary Table S3.

Optical rotations were
recorded at the sodium D-line (589.3 nm) on a Unipol L1000 polarimeter
with a 10 cm cell at 20 °C (Schmidt+Haensch, Berlin, Germany).
UV and ECD data were recorded in water on a Chirascan V100 with a
1.0 cm quartz cuvette (Applied Photophysics, Leatherhead, U.K.). IR
data was recorded on a PerkinElmer spectrum 100 FT-IR spectrometer
(Massachusetts, U.S.A.). NMR experiments were performed on a 500 MHz
Varian Inova spectrometer with a 5 mm OneNMR probe and a 600 MHz Agilent
Premium Compact spectrometer with a 5 mm CryoProbe (Agilent, Santa
Clara, U.S.A.). The chemical shifts (δ in ppm) are referenced
to the carbon (δ_C 49.00) and proton (δ_H 3.31) signals of residual MeOD-d₄ within the NMR solvent. High-resolution mass spectra (HRESIMS) were
obtained using an Agilent 6540 Q-Tof mass spectrometer equipped with
an Agilent 1290 UPLC and autosampler (Agilent). Large scale RP-SPE
fractionation was performed using polygoprep C₁₈-bonded
silica 35–60 μm, 120 Å (Labquip, Ireland). Semipreparative
HPLC was carried out on an Agilent 1260 HPLC system equipped with
a DAD detector. All solvents used for extraction and separations were
HPLC grade, and H₂O was milli-Q filtered. Trifluoroacetic
acid (TFA) was used for HPLC separation and was spectroscopy grade
from Alfa Aesar.

Terpene trilactones were quantified by an ultra-high-performance liquid chromatography ion-trap time-of-flight mass spectrometry (UHPLC-QTOF-MS) as described before [32 (link)] with some changes. For further analysis, authentic substrates (Ginkgolide A, B, C, J and bilobalide) were injected as positive controls. The mobile phase (delivered at 0.2 mL/min) comprised 0.1% formic acid water (A) and 100% acetonitrile (B). Gradient elution conditions were set as follows: 0–7 min, 5–30% B; 7–10 min, 30–50% B; and 10–12 min, 50–100% B. The total run time was 12 min. An Agilent 6540 Q-TOF mass spectrometer was operated for further determination. A negative mode electrospray ionization (ESI^-) source was used with the follow conditions: gas temperature = 320 °C; sheath gas temperature = 350 °C; sheath gas flow = 11 L/min, nebulizer at 35 psi; and Vcap = 2500 V, nozzle voltage (Expt) = 1000 V. Qualitative Analysis B.07.00 was used for the data analysis.

Extracts were dissolved in acetonitrile or aqueous acetonitrile (1:1) for lipophilic and polar extracts respectively, to a final concentration of 0.5 mg/mL. The samples were filtered through a 0.2 µm filter prior to analysis. A 5 µL aliquot was injected onto a diphenol column (Fortis, 150 × 2.1 mm, 1.7 µm) and eluted using a mixture of acetonitrile/water (1% formic acid) at a gradient of 5% MeCN to 100% over 12 min, with the final solvent concentration held for 3 min. Solvent was delivered at a constant flow rate of 400 µL/min. HRESIMS and HRESIMSMS data were obtained using an Agilent 6540 Q-Tof mass spectrometer. Untargeted MSMS data was obtained using a collision energy of 10, 20 and 40 eV in positive mode. These data were then imported into the GNPS website for further analysis [41 (link)].