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Sample Set Information

ID SE116
Title Untargeted metabolome analysis of foods using LC-MS (5) / LC-MSによる食品のノンターゲットメタボローム解析 (5)
Description Untargeted metabolome analyses of foods were performed using LC-MS. Food items were selected from the Standard Tables of Food Composition in Japan-2005 (Seventh Revised Version) published by the Ministry of Education, Culture, Sports, Science and Technology, Japan (hereafter, refered to as the standard table). Metabolites in foods were extracted with methanol, separated by reversed-phase LC and detected using a high-resolution mass spectrometry (LTQ-FT, ThermoFisher Scientific). Two different conditions (Method 1, and Method 5) were applied to ESI positive mode detections, and Method 1 was applied to ESI negative mode detection. A series of different parameter settings for PowerGetBatch software were used for peak detection, and valid peaks were selected by an alignment of the resulted peaks with those detected from several mock samples. The detailed peak information including the results from compound database search and prediction of flavonoid aglycones using FlavonoidSearch were published from the Food Metabolome Repository (

The IDs mean as follows:

Sample ID: Prefix "S" + Food Item No. in the standard table + blanch No.

∗ The basic blanch No. is 1, and larger numbers were assigned for different types of the food items, and so on.

M01: Method 1 for accurate mass measurement in ESI positive mode

M11: Method 1 for accurate mass measurement in ESI negative mode

M05: Method 5 for acquire MS3 spectrum in ESI positive mode

M90, M91: Pseudo analysis representing a set of analyses used in the alignment for each positive and negative analysis, respectively


LC-MSを用いて食品をノンターゲットメタボローム分析した。食品は、日本食品標準成分表2015年版(七訂)(文部科学省)(以後「食品成分表」)から代表的なものを選んだ。メタノール抽出後、逆相HPLCで分離し、高分解能フーリエ変換型質量分析(LTQ-FT, ThermoFisher Scientific)による分析を行った。一つの食品につき、二つの異なる条件(Method 1, Method 5)でESIポジティブ分析を行い、精密質量値とMS2、MS3スペクトルを得た。ESIネガティブ分析は、1つの条件で精密質量値とMS2スペクトルを得た。ピーク検出は、PowerGetBatchソフトの異なるパラメーター設定による複数回の検出結果をアラインメントし、同時にアラインメントした試料の代わりに水を用いたMock分析データを差し引くことで実施した。検出されたピーク情報から、データベース検索、FlavonoidSearchによるフラボノイドアグリコンの推定を行った結果は、食品メタボロームレポジトリ( )から公開している。


サンプルID: 接頭辞S + 食品成分表の食品番号 + 枝番(1, 2)


M01:Method 1(精密質量分析用)によるESIポジティブ分析

M11:Method 1(精密質量分析用)によるESIネガティブ分析

M05:Method 5(MS3スペクトル取得用)によるESIポジティブ分析

M90, M91:アラインメントに用いた分析データ(複数)を示すための疑似的な分析

Authors Sakurai N1,2, Suda K1, Akimoto N1, Osawa S1 (1 Kazusa DNA Research Institute, 2 National Institute of Genetics), Contact: sakurai AT (replace AT with @)


櫻井望1,2、須田邦裕1、秋元奈弓1、大澤祥子1(1 かずさDNA研究所, 2 国立遺伝学研究所)

Reference Sakurai N and Shibata D. Tools and databases for an integrated metabolite annotatio environment for liquid chromatography-mass spectrometry-based untargeted metabolomics. Carotenoid Science 22: 16-22 (2017)

Link icon database.png

Sample Information

ID S063011
Title 06301 よもぎ 葉 生, Japanese wormwood, leaves, raw
Organism - Scientific Name Artemisia indica var. maximowiczii
Organism - ID NCBI taxonomy: 1242858
Compound - ID
Compound - Source
Preparation Harvested from field (Kisarazu, Chiba, Japan, 11 May, 2017)


採取 (木更津市, 2017年5月11日)

Sample Preparation Details ID SS1

Sample Preparation Details Information

Title Preparation of food samples
Description Foods without 'Removed Portion' were ground into fine powder in liquid nitrogen. 'Removed Portion' described in the 'Remarks' in the Standard Tables were removed. Solid samples were ground into fine powder in liquid nitrogen with mortal and pestle. When food size is large, a representative portion is cutout and ground. For example, in the case of a water melon, a portion of comb cut where the edge is along to the line between the petiole and the tip of the fruit is used to minimize the deviation of maturity in the fruit. Liquid samples are used without grinding.




Analytical Method Information

ID M05
Title Method 5: ESI Positive, Data dependent MS2/MS3 scan (FT, IT, IT)
Method Details ID MS05
Sample Amount Approx. 5 mg FW / 20 µL injection

Analytical Method Details Information

Title LC-FT-MS, ESI, Positive (method 5)
Instrument Agilent1100 HPLC (Agilent), LTQ-FT (Thermo Fisher Scientific)
Instrument Type LC-FTICR-MS
Ionization ESI
Ion Mode Positive
Description Metabolite Extraction

Compounds in foods were extracted by approximately 75%(v/v) methanol. In the case of liquid samples and powders from foods containing abundant water (such as vegetables), 3 times volume (v/v or w/v) of 100% methanol were added. In the case of samples with low water content (such as dried seaweed), 400 times volume (w/v) of 75% methanol were added. We did not measure the moisture content of each food. A 25 µM of 7-hydroxy-5-methylflavone is contained as internal standard (IS) in the added methanol. The sample in methanol in 2 mL tubes was homogenized using Mixer Mill MM 300 (QIAGEN) at 25 Hz, for 2 min, twice. A homogenate was centrifuged under 17,400 x g, 5 min at 4 ºC. A supernatants was passed through a Polytetrafluoroethylene (PTFE) filter (pore size 0.2 µm, Millipore), and a filtrate was applied to a C18 silica column (MonoSpin C18, GL Science) to remove highly-hydrophobic contaminants. The filtrate passed through the C18 column was used for LC-MS analyzes.

The same extraction procedure were performed using 75%(v/v) methanol without sample to prepare mock samples and used as negative controls.

Liquid chromatography (LC)-mass spectrometry (MS) analysis

Agilent 1100 system (Agilent) and Finnigan LTQ-FT (Thermo Fisher Scientific) were used. Aliquots (20 µL) of the methanol extract were applied to a TSK-gel ODS-100V column (4.6 x 250 mm, 5 µm, TOSO), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile containing 0.1%(v/v) formic acid (Solvent B). The gradient program was as follows: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min). The flow rate was set at 0.25 mL/min for 0-100 min, and 0.5 mL/min for 100.1-107 min. The column oven temperature was set to 40 ºC. To monitor the HPLC eluate, a photodiode array detector was used with a wavelength range of 200-650 nm.

Electrospray ionization (ESI)-positive mode with a spray voltage of 4.0 kV and capillary temperature of 300 ºC was used for compound detection. The nitrogen sheath gas and auxiliary gas were set at 40 and 15 arbitrary units, respectively. The MS scan setting was as below:

- Method 5

A setting for obtaining MS3 spectra.

Full scan: Resolution = 12,500 by FT

MS2 scan: Data dependent scan by IT for the most intense 5 ions in the full scan

MS3 scan: Data dependent scan by IT for the most intense 2 ions in the MS2 scan

The dynamic exclusion settings for MS2 scan was follows: repeat count, 3; repeat duration, 30s; exclusion list size, 500; margin, 10 ppm, exclusion duration, 20s. A m/z range for Full scan was set to 100-1500. Raw data were obtained by Xcalibur software (ver. 2.0.7, Thermo Fisher Scientific).



終濃度約75%(v/v)のメタノールで抽出した。液体や含水率の高い葉野菜などの試料は、3倍容または3倍量の100%メタノールを添加した。のりなどの乾燥した試料は、75%メタノールを加えた。なお、正確な含水率は測定していない。また、用いたメタノールには、質量分析での内部標準として、25μMの7-ヒドロキシ-5-メチル フラボンを添加した。メタノールを添加した試料を、Mixer Mill MM 300 (QIAGEN社)にて、25 Hz, 2 min, 2回の条件で激しく撹拌し、その後、 17,400×g, 5 min, 4℃で遠心分離した。遠心上清を、ポアサイズ0.2μmのポリテトラフルオロエチレン(PTFE) フィルター(Millipore社)で濾過して微粒子を除去したあと、濾液をC18シリカカラム(MonoSpin C18、 GLサイエンス社)に通して疎水性の強い成分を吸着除去した。C18カラムを通過した試料を、液体クロマトグラフィー- 質量分析に供試した。

試料を含まない75%メタノール抽出液で、全く同じ処理を行ったもの(mockサンプル)を準備し、 ネガティブコントロールとして用いた。


液体クロマトグラフィーとしてAgilent 1100システム(Agilent社)、その下流に接続した質量分析器として Finnigan LTQ-FT(Thermo Fisher Scientific社)を用いた。上記までに調製したメタノール抽出試料20μLを、LC装置に接続したTSK-gel ODS-100Vカラム(4.6×250 mm, 5μm, 東ソー社)にロードし、溶媒A(0.1%(v/v)ギ酸を含む HPLCグレードの水)と溶媒B(0.1%(v/v)ギ酸を含むHPLCグレードのアセトニトリル)によるリニアグラジエントにより分離した。グラジエントプログラムは、3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), 3% B (107 min)とした。流速は、0-100 minの分離時間が0.25 mL/min、100.1-107 minのウォッシュ時間が0.5 mL/minとした。流速は0.25 mL/min (0-100 min)、0.5 mL/min (100.1-107 min)とした。カラムオーブン温度は40℃とした。HPLCによる溶出をモニタするため、UV/Vizフォトダイオードアレイ検出器により200-650 nmの吸収波長を測定した。

エレクトロスプレーイオン化法(ESI)ポジティブモードにより化合物を検出した。スプレイ電圧は4.0 kV、キャピラリー温度は300℃とした。窒素シースガスおよび補助ガスは、それぞれ40および15ユニットに設定した。以下のスキャン条件を設定した。

- Method 5


フルスキャン: 分解能12,500 (FT)

MS2スキャン: フルスキャンで得た強度の強い上位5つのイオン (IT)

MS3スキャン: MS2スキャンで得た強度の強い上位2つのイオン (IT)

MS2取得時のダイナミックエクスクルージョン設定は以下とした: repeat count, 3; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 20 s。フルスキャンの検出レンジはm/z 100-1500とした。データの取得はXcaliburソフト(バージョン2.0.7、Thermo Fisher Scientific)にて行った。

Comment_of_details [column] TSK-gel ODS-100V (4.6 x 250 mm, 5 micrometer; TOSOH)

[gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile containing 0.1% v/v formic acid; Gradient: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min)

[total separation time] 107 min

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