2.5. Ultra-High-Performance liquid chromatography-mass spectroscopy (UPLC-MS)
The metabolites of lyophilized samples of fruit with its pedicel were measured using UPLC-Quadrupole-Time-Of-Flight MS (UPLC-QTOF-MS). Each 0.05 g ground sample was extracted with a 1 mL of ethanol: water: formic acid (80:20:1) solution and incubated for more than 12 hr at 4 °C. There were four biological replicates and the extracted samples were centrifuged at 15,000 g for 5 min. Supernatants were diluted 10-fold with 100 % methanol and analysed in both positive and negative ionization modes. The UPLC-QTOF-MS system consisted of Thermo Scientific Dionex Ultimate 3000 (Sunnyvale, CA, USA) and AB SCIEX Triple TOF 5600 system (Framingham, MA, USA). The analytical column was a U-VDSpher PUR 100 C18-E (2.1 mm x 100 mm, 1.8 μm) column (VDS optilab, Germany) and the solvent composition was A=0.1% formic acid in water, B=0.1% formic acid in acetonitrile. The solvent gradient was: 100 % A 0 % B, 0-10 min; linear gradient to 80 % A 20 % B, 10-12 min; linear gradient to 70 % A 30 % B, 12-15 min; the composition held at 5 % A 95 % B, 15-16 min; linear gradient to 100 % A 0 % B to return to the initial conditions before another sample was injected at 20 min. The flow rate was 0.2 mL/min. The Triple TOF 5600 parameters were: nebulizer N2, 50 psi; heating gas, 50 psi; curtain gas, 30 psi; temperature, 500 °C; ion spray voltage floating, -4.5 kV for negative mode and 5.5 kV for positive mode; declustering potential was -60 for negative mode and 60 for positive mode; MS scan range, 50-2000 m/z.