Immediately before LC–MS analysis each sample was filtered using 0.25 μm filter discs with a low protein binding Durapore polyvinylidene fluoride (PVDF) membrane Fasudil cell line (Millex; EMD Millipore, Billerica, MA, USA) and diluted with 9 ml of HPLC-grade water. Samples were run in a random order with QC samples (Dunn, Wilson, Nicholls, & Broadhurst, 2012). An external reference standard of sinigrin hydrate was also prepared for quantification of GSL compounds, and isorhamnetin
for flavonol compounds. Preparation was as follows: A 12 mM solution was prepared in 70% methanol. A dilution series of concentrations was prepared as an external calibration curve with HPLC-grade water (200, 150, 100, 56, 42, 28, 14 and 5.6 ng μl; sinigrin correlation coefficient: y = 12.496x − 15.012; r2 = 0.993, isorhamnetin correlation coefficient: y = 0.3205x − 5.3833, r2 = 0.921). Standard response factors were used in the calculation of GSL concentration where available ( Wathelet, Iori, Leoni, Quinsac, & Palmieri, 2004). Where such data could not be found for intact GSLs, response factors were assumed to be 1.00 ( Lewis & Fenwick, 1987). LC–MS analysis was performed in the negative ion mode on an Agilent 1200 Series LC system equipped with a binary pump, degasser, autosampler, thermostat, column heater, photodiode array detector and Agilent 1100 Series LC/MSD mass trap spectrometer.
Separation of samples was achieved find more on a Zorbax SB C18 column (2.1 × 100 mm; 1.8 μm; Agilent, Santa Clara, CA, USA) with precolumn filter. Myosin Both GSLs and flavonols
were separated in the same sample during a 40-min chromatographic run. Mobile phases consisted of ammonium formate (0.1%) and acetonitrile with a gradient of 95% and 5% respectively at a flow rate of 0.3 ml/min, with a column temperature of 30 °C. 5 μl of sample was injected. MS analysis settings were as follows: ESI was carried out at atmospheric pressure in negative ion mode (scan range m/z 50–1050 Da). Nebulizer pressure was set at 50psi, gas-drying temperature at 350 °C, and capillary voltage at 20,000 V. Compounds were identified using their nominal mass and characteristic fragment ions, and by comparing data with those published in the literature (see Table 1 and Table 2). GSLs were quantified at a wavelength of 229 nm, and flavonols at 330 nm. All data were analysed using Bruker Daltronics software. The results reported are the averages of three biological replicates and three separately extracted technical replicates (n = 9). Processed GSL and flavonol data were analysed with ANOVA and Tukey’s HSD test, and principal component analysis (PCA) was performed in XL Stat (Addinsoft, New York City, New York, USA). Table 1 lists all of the GSL compounds identified across all rocket samples, including systematic names, common names and the identifying ions. Unlike previous studies, the GSL profiles of each rocket accession were markedly different in some cases.