Metabolite Quantification in Infected and Non-Infected Bean Leaves

Infected (race 1 and 3) and non-infected common bean leaf extracts were subjected to analysis on a liquid chromatography-quadrupole time-of flights tandem mass spectrometry instrument (LCMS-9030 qTOF, Shimadzu Corporation, Kyoto, Japan) for quantification of metabolites at different time intervals. A Shim-pack Velox C18 column (100 mm × 2.1 mm with a 2.7 µm particle size) was used for chromatographic separation at 55°C (Shimadzu Corporation, Kyoto, Japan). An injection volume of 3 µL was used for all samples and were run on a binary mobile phase including solvent A: 0.1% formic acid in Milli-Q HPLC grade water (Merck, Darmstadt, Germany) and solvent B: UHPLC grade methanol with 0.1% formic acid (Romil Ltd., Cambridge, United Kingdom). Chromatographic analysis was done using qTOF high-definition mass spectrometer that was set to negative electrospray ionisation for data acquisition. Parameters set included nebulization, interface voltage (4.0 kV), interface temperature (300°C), dry gas flow (3 L/min), detector voltage (1.8 kV), heat block (400°C), DL (280°C) and flight tube (42°C) temperatures. Ion fragmentation was achieved using argon gas for collision with an energy of 30 eV and 5 eV spread (Ramabulana et al., 2021 (link)).