Triple quad 5500 lc ms ms system

Kiwifruit pericarp tissues from three independent biological/pooled samples were lyophilized and ground into fine powders. 0.1 g freeze-dried powders were extracted with 1mL 80% methanol/water (v/v) containing 0.1% formic acid. The mixture was vortexed for 30 sec, sonicated at 4°C for 20 min, vortexed again for 30 sec, centrifuged (14,000g, 10 min, 4°C), and filtered through a 0.2-mm polytetrafluoroethyle ne Reduced AsA and total AsA contents were measured as previously described (Liu et al., 2016) . In brief, 0.5g freeze-dried sample powders were extracted with 10 mL 1% oxalic acid at 4 °C for 12 hours and centrifuged (14,000g, 10 min, 4 °C). To determine the total AsA level, 1 mL of the supernatant was transferred to a 2-ml Eppendorf tube containing 10 uL of 5 mmol dithiothreitol (DTT) and incubated at 4 °C for 1 hour to convert the oxidized ascorbate into the reduced form. Supernatants were collected and filtered before the LC-MS analysis was performed using the SCIEX Triple Quad 5500 LC-MS/MS System. To assay reduced AsA, DTT was replaced by the same volume of potassium phosphate buffer (pH 7.4), while the rest of the procedure was the same as for the total AsA assay. All samples were analyzed in three independent biologic a l replicates.