| MS Description
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Frozen leaves and roots were homogenized i … Frozen leaves and roots were homogenized in 5 μl extraction solvent (methanol:acetate:H2O = 9:1:10) per 1 mg fresh weight of tissues by mixer mill (MM300; Retsch Gmbl & Co. KG, Haan, Germany) at 30 Hz. After centrifugation at 12000 g, cell debris was discarded and extracts were centrifuged again. Fifty microliters of supernatant was applied to HPLC/PDA/ESI‐MS system comprising a Finnigan LCQ‐DECA mass spectrometer (ThermoQuest, San Jose, CA, USA) and an Agilent HPLC 1100 series (Agilent Technologies, Palo Alto, CA, USA) as described previously (Jones et al., 2003; Yamazaki et al., 2003). HPLC was carried out on a TSK‐GEL RP‐18 (φ4.6 mm × 150 mm; TOSOH, Tokyo, Japan) at a flow rate of 0.5 ml min−1. Elution gradient with solvent A [CH3CN‐H2O‐TFA (10:90:0.1)] and solvent B [CH3CN‐H2O‐TFA (90:10:0.1)] and the following elution profile (0 min 100% A, 40 min 60% A, 40.1 min 100% B, 45 min 100% B, 45.1 min 100% A, 52 min 100% A) using linear gradients in between the time points. PDA was used for detection of UV‐visible absorption in the range of 250–650 nm. Nitrogen was used as sheath gas for the positive‐ion ESI‐MS performed at capillary temperature and voltage of 350°C and 5.0 kV, respectively. The tube lens offset was set at 10.0 V. Full scan mass spectra were acquired from 200–1500 m/z at 2 scans sec−1. Tandem MS analysis was carried out with helium gas as the collision gas. The normalized collision energy was set to 30%. ormalized collision energy was set to 30%.
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