SE227:/S101/M01

Sample Set Information

ID	SE227
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 (http://metabolites.in/things).
Authors	Nozomu Sakurai (National Institute of Genetics, Kazusa DNA Research Institute, email: sakurai (at) kazusa.or.jp)
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
Comment

Sample Information

ID	S101
Title	Grapefruit, Peel (Essential oil) / グレープフルーツ, 果皮（精油）
Organism - Scientific Name	Citrus x paradisi
Organism - ID	NCBI taxonomy: 37656
Compound - ID
Compound - Source
Preparation	The sample was purchased from an online store (June 12, 2019, Japan).
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
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.
Comment_of_details

Analytical Method Information

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

Analytical Method Details Information

ID	MS01
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 liquid samples that do not mix with 75% methanol, such as essential oils, 4 times volume (v/v) of 75% methanol containing 1 uM 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, or if separated into two liquid layers, the methanol layer, 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