| MS Description
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BioSource amount<br />
For RDRS and … BioSource amount<br />
For RDRS and Hoshiyutaka, 100 seeds of each variety were selected according to the average weight
and length of seeds. After separating the husks from the seeds, the brown rice seeds obtained were
bulked and crushed by using a Retsch mixer mill MM301 at a frequency of 20 Hz for 2 min at
4 °C. Successively, the obtained powder was divided into three to four pools. For external set of
samples harvested in Akita, 100 seeds of each biological replicate were selected and crushed in the
same way as RDRS.<br /><br />
Extraction for LC-MS<br />
100 mg of each sample was extracted with extraction buffer [methanol/water
(5:95, v/v)] at a concentration of of 100 mg/ml using a Retsch mixer mill MM310 at a frequency
of 20 Hz for 10 min at 4°C. After centrifugation for 10 min at 15,000 x g, 500 µl of the supernatant
was transferred into a tube and diluted in 0.1% acetic acid solution. and then it was filtered using
an Oasis® HLB µ-elusion plate (30 µm, Waters Co., Massachusetts, USA). The extracts ca. 0.1
mg of each sample) were evaporated to dryness in an SPD2010 SpeedVac® concentrator.The
extracts were dissolved by 200 µl of water containing five reference compounds as follows:<br />
0.5 mg/l of lidocaine,<br />
1.0 mg/l of ampiciline,<br />
1.0 mg/l of torperizone,<br />
0.5 mg/l of 10-camphor sulfonic acid and<br />
1.0 mg/l of 2-naphthalene-4-sodium sulfate.<br /><br />
LC-q-TOF-MS conditions<br />
After filtration of the extracts (Ultrafree-MC, 0.2 µm pore size;Millipore), 5 µl of extracts (ca. 0.1 mg each sample) was analyzed using an LC-MS system
equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system;
MS, Waters Q-Tof Premier). The analytical conditions were as follows. HPLC: column, Acquity
bridged ethyl hybrid (BEH) C18 (pore size 1.7 µ m, length 2.0 x 100 mm, Waters); solvent system,
acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program, 1:99 v/v at 0 min, 1:99 v/v at 0.1 min, 64.0 : 0.5 at 10.0 min, 99.5 : 0.5 at 11.5 min, 1:99 v/v at 11.6 min and 1:99 at 14.0 min; flow rate, 0.3 ml min−1; temperature, 38°C; MS detection: capillary voltage, +3.0
keV; cone voltage, 23 V for positive mode and 35 V for negative mode; source temperature, 120°C;
desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 800 l/ h; collision
energy, 2 V for positive mode and 5 V for negative mode ; detection mode, scan (m/z 100–2000;
dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 14.0 min in
a single run. The data were recorded using MassLynx version 4.1 software (Waters). ng MassLynx version 4.1 software (Waters).
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