| MS Description
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- Compound Extraction
Compounds in the s … - Compound Extraction
Compounds in the sample were extracted by approximately 75%(v/v) methanol. In the case of liquid samples and samples containing abundant water such as plant leaves, 3 times volume (v/v or w/v) of 100% methanol containing 1 uM 7-hydroxy-5-methylflavone as an internal control (IS) was added. In the case of samples with low water content such as granules of herbal medicine, 100 times volume (w/v) of 75% methanol containing 1 uM IS was added. The sample in methanol in a 2 mL tube was homogenized using a zirconia bead (5 mm diameter) and Mixer Mill MM 400 (Verder Scientific, Co., Ltd.) at 25 Hz for 2 min, twice. A homogenate was centrifuged at 17,400 x g, 5 min at 4 degree C. A supernatant was applied to a C 18 silica column (MonoSpin C18, GL Sciences Inc.) to remove highly hydrophobic contaminants. The filtrate was passed through a polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore) and used for LC-MS analyses.
- Liquid chromatography (LC)-mass spectrometry (MS) analysis
Nexera X2 system (Shimadzu Corporation) and Compact system (Bruker Japan K.K.) were used. An aliquot (2 uL) of the methanol extract was applied to an InertSustain AQ-C18 column (3 um x 2.1 mm x 150 mm, GL Sciences) connected after a guard column (InertSustain AQ-C18 Cartridge Guard Column E, GL Sciences), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile (Solvent B). The gradient program was as follows: 2% B (0 min), 2% B (3 min), 98% B (30 min), 98% B (35 min), 2% B (35.01 min), and 2% B (42 min). The flow rate was set at 0.2 mL/min. The column oven temperature was set to 40 degree C.
The compounds separated by the LC were detected using the mass spectrometer under the conditions below: Ionization, Electrospray Ionization (ESI); Polarity, Positive; Scan rate, 1 Hz; Mass scan range, 50-1200; End plate offset, 500 V; Capillary voltage, 4000 V; Nebulizer gas (N2) pressure, 2.5 bar; Dry gas (N2) flow, 8.0 L/min; Dry gas temperature, 200 degree C; Transfer Funnel1 RF, 200.0 Vpp; Funnel2 RF, 200.0 Vpp; In-source CID Energy, 0.0 eV; Hexapole RF, 50.0 Vpp; Quadrupole Ion Energy, 3.0 eV; Low mass m/z, 55.00; Collision energy, 10.0 eV; Collision RF, 450.0 Vpp; Transfer time, 80.0 us; and PrePulse storage, 3.0 us. The data-dependent MS/MS spectra were obtained with the conditions below: Isolation width, 3-15 Da; Collision energy 35 eV; Precursor ion number, 5; Active Exclusion, on; Exclude, after 3 spectra; Release, after 0.3 min; Reconsider precursor, on; and if current intens. / previous intens., 2.0. For mass calibration, 1 mM sodium formate in 50% (v/v) 2-propanol was injected directly into the MS at 38.50–40.50 min of LC separation with a flow rate 0.1 mL/min. The eluent at 0-3 min was wasted. The raw data were obtained by Hystar software (ver.3.2 SR4, Bruker Daltonik, GmbH). ware (ver.3.2 SR4, Bruker Daltonik, GmbH).
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