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Sample Set Information

ID SE225
Title LC-MS based untargeted metabolome analysis of various samples
Description Compounds in various samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). The same analytical conditions are applied to all samples. Therefore, the compound peaks can be compared to each other by the mass values, the retention time of the LC, and the CID mass spectrum. The data were obtained for the construction of the “Thing Metabolome Repository” website (
Authors Nozomu Sakurai (National Institute of Genetics, Kazusa DNA Research Institute, email: sakurai (at)
Reference The Thing Metabolome Repository family (XMRs): comparable untargeted metabolome databases for analyzing sample-specific unknown metabolites. Sakurai N, Yamazaki S, Suda K, Hosoki A, Akimoto N, Takahashi H, Shibata D and Aoki Y, Nucleic Acids Research Database Issue) 51 (D1): D660-D677 (2023), DOI: 10.1093/nar/gkac1058

Sample Information

ID S001
Title Hama-giku (a kind of chrysanthemum, NBRP YPK35), Leaf / ハマギク (NBRP YPK35), 葉
Organism - Scientific Name Nipponanthemum nipponicum (Franch. ex Maxim.) Kitamura
Organism - ID NCBI taxonomy: 99084
Compound - ID
Compound - Source
Preparation The sample was kindly provided by Makoto Kusaba (Hiroshima Univ, NBRP) (Jan 18, 2022, Japan).
Sample Preparation Details ID SS1

Sample Preparation Details Information

Title Homogenated and stored at -80 C
Description After harvesting, the sample was immediately frozen in liquid nitrogen and stored at -80 degree C. The sample was ground to a fine powder under liquid nitrogen using mortar and pestle and stored at -80 degree C until use.

Analytical Method Information

ID M01
Title LC-QTOF-MS, ESI, Positive
Method Details ID MS01
Sample Amount 0.5 mg / 2 ul injection

Analytical Method Details Information

Title LC-Q-Tof-MS, ESI, Positive
Instrument Nexera X2 (Shimadzu), Compact (Bruker Daltonics)
Instrument Type LC-QTOF-MS
Ionization ESI
Ion Mode Positive
Description - 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, 40 or 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).

Comment_of_details [column] InertSustain AQ-C18 (2.1 x 150 mm, 3 micrometer; GL Sciences)

[gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile; Gradient 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)

[total separation time] 42 min

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