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Extraction for LC-q-TOF-MS to detect secon … Extraction for LC-q-TOF-MS to detect secondary metabolites<br />
Each frozen sample was extracted with 5 fold amount of solvent (methanol/water [8:2 v/v]) containing a reference compound (2.5 µM of 10-camphorsulfonic acid ([M-H]-, m/z 231.0691)) using a mixer mill MM301 (Retsch) at a frequency of 18 Hz for 7 min at 4°C. After centrifugation for 10 min at 17,000 × g, the supernatant was filtered using an Oasis® HLB µElusion plate (30 µm, Waters Co., Massachusetts, US).<br /><br />
LC-q-TOF-MS conditions to detect secondary metabolites<br />
After preparation of the extracts, the sample extracts (1 µl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions were as follows. LC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 µm, length 2.1× 100 mm, Waters); solvent system,acetonitrile(0.1% formic acid):water (0.1% formic acid); gradient program, 0.5 : 99.5 v/v at 0 min, 0.5 : 99.5 v/v at 0.1 min, 80 : 20 v/v at 10 min, 99.5 : 0.5 v/v at 10.1 min, 99.5 : 0.5 v/v at 12 min and 0.5 : 99.5 v/v at 12.1 min, 0.5 : 99.5 v/v at 15 min; flow rate, 0.3 ml/min; temperature, 40 °C; MS detection: capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature, 120°C, desolvation temperature, 450 °C, cone gas flow, 50 l/ h; desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, m/z 50‒1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; mode, centroid; polarity, negative; Lockspray (Leucineenkephalin): scan duration, 1.0 sec; interscan delay, 0.1 sec. The data were recorded using Progenesis CoMet (Nonlinear Dynamics). ing Progenesis CoMet (Nonlinear Dynamics).
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