| MS Description
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The authentic samples of withanolides were … The authentic samples of withanolides were isolated from plants as described in our previous paper (Ozawa et al., 2013). The leaves of P. alkekengi and P. peruviana were collected at five different developmental stages of the growing season before bearing fruit, because our pilot study showed to detect withanolides of P. alkekengi and P. peruviana at these stages (data not shown). Fresh samples of leaves at five different developmental stages were extracted with 5 μl of 80% MeOH containing 2.5 μM lidocaine (internal standard) per mg fresh weight using a mixer mill with zirconia beads (7 min at 18 Hz and 4°C). After 10-min centrifugation, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μl) were analyzed with LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions for metabolite profiling were as described in elsewhere (Tamura et al., 2014). The polarity of electrospray ionization was applied in positive ionization mode. n was applied in positive ionization mode.
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