| Description
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Extraction of Metabolites
The samples were extracted with 50 μL of 80% MeOH per mg dry weight using a mixer mill (MM300, Retsch) with zirconia beads for 10 min at 20 Hz and 4°C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters).
LC–FTICR–MS/MS Analysis
The solutions (5 μL) were analyzed using an LC–FTICR–MS instrument (LC, Agilent 1260 Infinity; MS, Bruker Daltonics SolariX 7.0 T). Analytical conditions were as follows. LC: column, Xselect CSH Phenyl-Hexyl (3.5 μm, 2.1 mm × 150 mm, Waters, Milford, MA, USA); solvent system, solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid); LC gradient program, 99.5% A/0.5% B at 0 min, 0.5% A/99.5% B at 30.0 min, 0.5% A/99.5% B at 45.0 min, 99.5% A/0.5% B at 45.1 min, and 99.5% A/0.5% B at 60.0 min; flow rate, 0.3 mL/min; column temperature, 35°C; wavelength, 200–600 nm; MS/MS detection; Acquisition Mass Control (mass range, m/z 100–600; estimated resolution power, 66,000 at m/z 400; transient length, 0.4893); Data Storage, save full profile spectrum, on; save FID file, on; Accumulation (average scan, 1; source accumulation, 0 s; ion accumulation time, 0.1 s; ion cooling time, 0 s; time of flight, 0.4 s); API Source [source type, electrospray ionization (ESI); capillary, 4500 V; end plate offset, -500 V]; Source Gas Tune (nebulizer 1.6 bar; dry gas, 7.0 L/min; dry temperature, 200°C); Source Optics (capillary exit, 220 V; deflector plate, 200 V; funnel 1, 150 V; skimmer 1, 25 V; funnel RF amplitude, 150 Vpp); Octopole (frequency, 5 MHz; RF amplitude, 350 Vpp); Collision Cell (collision voltage, -2 V; DC extract bias, 0.4 V polarity, RF frequency, 2 MHz; collision RF amplitude, 1200 Vpp); Transfer Optics (time of flight, 0.4 ms; frequency, 6 MHz, RF amplitude, 350 Vpp); Infinity Cell (transient exit lens, -20 V; analyzer entrance, -10 V; side kick, 0 V; side kick offset, -1.5 V; front trap plate, 0.6 V; back trap plate, 0.6 V; sweep excitation power, 18%); Multiple Cell Accumulations (number of ICR cell fills, 1); Gated Trapping (gated trapping mode, off); Precursor Selection (prefer list, m/z of target compound [M + H]+, mass accuracy, ±20 ppm; exclude unknown charged ions, on); Quadrupole Parameters (MS/MS isolation, on; cut off in MS, m/z 100; isolation window, m/z 5); Fragmentation Mode, quadrupole collision-induced dissociation (QCID); MS/MS Boost, on (in use); MS accumulation in MS/MS Boost, 0.1 s (in use); MS/MS accumulation in MS/MS Boost, 2 s (in use); fixed collision voltage, 30 V; polarity, positive; API high voltage, on; source quench, on.
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