6500 q tof mass spectrometer

Cell culture (300 μl) was mixed with an equal volume of ethanol by incubating at 700 r.p.m. at 30 °C for 5 min, then centrifuging at 2,500g for 30 min before loading the supernants into a 96-well sample plate for LC–MS analysis as previously described^{61 (link),62 (link)}. An Agilent 1290 Infinity system was used to analyse these prepared samples with an online diode array detector in combination with an Agilent 6500 Q-ToF mass spectrometer. An Agilent Eclipse Plus C18 2.1 × 50 mm (1.8 μm particle size) column was used at a temperature of 25 °C, with a solvent flow rate of 0.2 ml min⁻¹. LC was performed with a linear gradient of buffer A (0.1% formic acid) and buffer B (0.1% formic acid in acetonitrile) from 2% to 98% buffer B over 2.5 min, which was held at 98% buffer B for 1 min. Injection volume was 1 μl and spectra were recorded between a mass range of 90–1,000 m/z at a rate of 3 spectra per second. The prepared calibration curves of standards included p-coumaric acid and resveratrol. Quantitation was based on the MS peak area of precursor or fragment ions in comparison with the analytical standards. Negative ion detection mode was used for resveratrol samples. Error bars represent standard deviations from three independent biological samples.

Plant parts harvested was dried in oven at 37 °C. These samples were grinded to fine powder with the help of mortar and pestles. 500 mg of each sample was extracted with 10 ml absolute ethanol and kept on shaker for 48 hr, after which extract were filtered by Whatmann No. 1 filter paper and filter sterile through 0.25 micron filters. The LC-MS and LC-MS/MS were performed on a Waters TQD triple quadrupole mass spectrometer (USA). It was equipped with waters, H-Class Acquity UPLC system and electrospray ionization source. The UPLC column used was water BEH C-18 100 × 2.1 mm, 1.7 μm and dual mode (±) LC-ESI-MS experiments performed after injecting 1 μl samples by the autosampler. Similarly, LC-ESI-HRMS analysis was performed on Agilent 6500 Q-Tof Mass Spectrometer (USA). It was equipped with electrospray ionization (ESI) interface using the following operation parameters: capillary voltage 3.5 kV, nebuliser 40 psi, drying gas (nitrogen) flow rate 11.0 L/min, drying gas temperature 300 °C, fragmentor 150 V, skimmer voltage 65 V. Mass spectra were recorded across the range m/z 150–1000 Th. The accurate masses of selected metabolites were observed and compared with their exact mass (theoretical mass) values manually. Analyses were performed as per our previously established method. m/z values and retention time of different compounds identified are shown in Table 2.