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Harvested sample is frozen by liquid N2 an … Harvested sample is frozen by liquid N2 and resulting powder (100mg) are solved in 300uL 80% methanol solution. 5uL sample is injected into HPLC after 0.2um membrane filter treatment. HPLC conditions: Shimadzu Nexera (Shimadzu), Column: TSKgel ODS-100V (4.6 x 250 mm, 5 micrometer; TOSOH), Solvent: A; 0.1% formic acid aq. B; ACN (addition 0.1% formic acid fc.), Gradient: (B) 0 to 3% for 15 min, 3 to 97% for 20 min, 97 to 3% for 0.1 min, and 3% for 25 min., Column temp.: 40 degree C, Flow rate=0.4mL/min, PDA: 190-800 nm (2 nm step). Orbitrap-MS conditions: Filter 1: FTMS + c norm !corona !pi res=60000 o(100.0-1500.0); 2: ITMS + c norm !corona !pi Dep MS/MS Most intense ion from (1); 3: ITMS + c norm !corona !pi Dep MS/MS 2nd most intense ion from (1); 4: ITMS + c norm !corona !pi Dep MS/MS 3rd most intense ion from (1); 5: ITMS + c norm !corona !pi Dep MS/MS 4th most intense ion from (1). i Dep MS/MS 4th most intense ion from (1).
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