For LC-MS analysis of phenylpropanoid compounds, tomato fruit were harvested 7 days after breaker. The whole fruit pericarp was freeze dried and ground into a fine powder. An amount of 1 g of dry powder was extracted with 25 ml 80% MeOH at 4 °C under agitation, overnight. The sample was then centrifuged at 3,000 × g at 4 °C for 15 min and the supernatant was taken. The pellet was extracted again with 25 ml 80% MeOH at 4 °C for another 2 h. The supernatant was combined and further diluted 10 times with 80% MeOH. The samples were filtered through a 0.45 μm filter before injection. For each line, three biological replicates were analysed.
For flavonoid and isoflavone analysis, the samples were run on a Surveyor high-performance liquid chromatography (HPLC) system (Thermo) attached to a DecaXPplus ion trap MS (Thermo). Separation was on a 100 × 2.1 mm 2.6 μ Kinetex XB-C18 column (Phenomenex) using the following gradient of methanol versus 0.1% formic acid in water, run at 200 μl min−1 and 30 °C: 0 min, 2% MeOH; 24 min, 38% MeOH; 30 min, 70% MeOH; 33.6 min, 70% MeOH, 34.2 min, 2% MeOH and 43.2 min, 2% MeOH. Data were collected for 34 min. UV-visible absorbance was collected with spectra from 200–600 nm from which chromatograms could be extracted at any wavelength, and a specific channel of 260 nm (bandwidth 9 nm), and positive electrospray MS spectra from m/z 150–2,000. The instrument also collected data-dependent MS2 spectra at an isolation width of m/z 4.0 and 35% collision energy, using dynamic exclusion to maximize the number of ions selected for fragmentation. Spray chamber conditions were 50 units sheath gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV. Sheath gas was supplied via the aux gas line and the entire flow from the LC passed through diode array to the mass spec ionization chamber without teeing.
Resveratrols were measured using a Surveyor HPLC system attached to a DecaXPplus MS (both Thermo). Separation was on a 100 × 2 mm 3 μm Luna C18(2) column (Phenomenex) running the following gradient of acetonitrile (ACN) versus 0.1% formic acid in water at 30 °C and 250 μl min−1: 0 min, 2% ACN; 30 min, 70% ACN; 30.5 min, 2% ACN and 38 min, 2% ACN. resveratrol and polydatin were detected by UV absorbance at 306 nm (9 nm bandwidth) and by selected reaction monitoring in positive mode electrospray MS. The mass spec was set up to collect full spectra from m/z 150–1,000 and also targeted MS2 of precursor ion 229.0 at 40% collision energy and an isolation width of m/z 4.0, and precursor 391.1 at 40% collision energy and an isolation width of m/z 3.0. Spray chamber conditions were 50 units sheath gas, 5 units aux gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV using a steel needle kit.
For flavonoid and isoflavone analysis, the samples were run on a Surveyor high-performance liquid chromatography (HPLC) system (Thermo) attached to a DecaXPplus ion trap MS (Thermo). Separation was on a 100 × 2.1 mm 2.6 μ Kinetex XB-C18 column (Phenomenex) using the following gradient of methanol versus 0.1% formic acid in water, run at 200 μl min−1 and 30 °C: 0 min, 2% MeOH; 24 min, 38% MeOH; 30 min, 70% MeOH; 33.6 min, 70% MeOH, 34.2 min, 2% MeOH and 43.2 min, 2% MeOH. Data were collected for 34 min. UV-visible absorbance was collected with spectra from 200–600 nm from which chromatograms could be extracted at any wavelength, and a specific channel of 260 nm (bandwidth 9 nm), and positive electrospray MS spectra from m/z 150–2,000. The instrument also collected data-dependent MS2 spectra at an isolation width of m/z 4.0 and 35% collision energy, using dynamic exclusion to maximize the number of ions selected for fragmentation. Spray chamber conditions were 50 units sheath gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV. Sheath gas was supplied via the aux gas line and the entire flow from the LC passed through diode array to the mass spec ionization chamber without teeing.
Resveratrols were measured using a Surveyor HPLC system attached to a DecaXPplus MS (both Thermo). Separation was on a 100 × 2 mm 3 μm Luna C18(2) column (Phenomenex) running the following gradient of acetonitrile (ACN) versus 0.1% formic acid in water at 30 °C and 250 μl min−1: 0 min, 2% ACN; 30 min, 70% ACN; 30.5 min, 2% ACN and 38 min, 2% ACN. resveratrol and polydatin were detected by UV absorbance at 306 nm (9 nm bandwidth) and by selected reaction monitoring in positive mode electrospray MS. The mass spec was set up to collect full spectra from m/z 150–1,000 and also targeted MS2 of precursor ion 229.0 at 40% collision energy and an isolation width of m/z 4.0, and precursor 391.1 at 40% collision energy and an isolation width of m/z 3.0. Spray chamber conditions were 50 units sheath gas, 5 units aux gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV using a steel needle kit.
Free full text: Click here
