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The contents of GSLs and OAS were measured … The contents of GSLs and OAS were measured by LC-MS using ultra performance liquid chromatography–tandem quadrupole mass spectrometry (UPLC-TQMS) according to Sawada et al. (2009a, 2009b).<br />
Approximately 10–50 mg of rosette leaves was sampled from 3-week-old plants and transferred to a pre-frozen, 2 ml tube containing 5 mm zirconia beads. After chilling in liquid nitrogen, samples were stored at −80°C until use. Plant samples were lyophilized using a freeze dryer (FDU-2100, EYELA) in a vacuum, homogenized using a Shake Master (Bio Medical Science) at a speed of 1,000 r.p.m. and homogenized again following the addition of 800 µl of the extraction buffer (methanol : milliQ water = 4 : 1, 0.1% formic acid, 5 µM sinigrin as the AGSL internal standard, 1.68 µM 10-camphorsulfonic acid as the internal standard for negative ion mode analysis and 0.0336 µM lidocaine as the internal standard for positive ion mode analysis). A 200 µl homogenate was transferred to a new tube, concentrated to dryness using a Speedvac (Thermo Fisher Scientific) vacuum centrifuge, dissolved with 200 µl of ultrapure water (LC-MS grade) and filtered using Amicon Ultrafree-MC centrifugal filter devices (0.2 µm, 100 PK). For seed analysis, five seeds were extracted with 500 µl of extraction buffer without sinigrin, and diluted to 1/6 with ultrapure water.<br />
For UPLC-TQMS analysis, samples were diluted to 1/500 with ultrapure water (LC-MS grade) using an automated liquid handling system (Microlab STARplus, Hamilton). Compounds were separated using a HSS T3 1.8 µm column (1.0 × 50 mm, Waters), and the UPLC gradient program: 0–0.25 min, 99.9% of solvent A; 0.4 min, 91% of solvent A; 0.8 min, 83% of solvent A; 1.9 min, 0.1% of solvent A; 1.9–2.1 min, 0.1% of solvent A; 2.11–2.7 min, 99.9% of solvent A; and a flow rate of 0.24 ml min−1. Conditions for the TQMS (Waters) detection were capillary voltage of −0.80 kV (negative ion mode) or 0.50 kV (positive ion mode), cone voltage of 50 V (negative ion mode) or 30 V (positive ion mode), source temperature of 150°C, and desolvation gas of 1,000 l h−1 at 600°C.<br /> n gas of 1,000 l h−1 at 600°C.<br />
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