An accurately weighed amount of NBD Cl (25 mg) was quantitatively transferred to a 50 ml volumetric flask and dissolved in 10 ml methanol; the contents of the flask were diluted to volume with the same solvent to produce a stock solution of 0.05% (w/v).
The contents of the flask were diluted with 150 mL CH2Cl2 and washed with water (3 × 150 mL), 2.5 N HCl solution (3 × 150 mL), 4% aqueous sodium bicarbonate (3 × 150 mL), and finally saturated NaCl brine (3 × 150 mL).
The contents of the flasks were diluted to about 8 mL with the mobile phase, to prevent plasma protein precipitation with methanol (solvent of LEV), 1 mL of human plasma was added to each flask, and the volumes were completed to the mark with the mobile phase and mixed well.
Each portion was collected in a 5-mL volumetric flask and was diluted to the mark (if necessary) with distilled water.
10.0 mL of hydrolyzed sample was mixed with 20.0 mL of standard salicylate solution, then pH was adjusted to 6.2, and then the solution was transferred to a 50.0 mL volumetric flask, which was diluted to the mark with doubly distilled water.
The appropriate concentrations of HSA, NaCl, and patulin 15 μmol ·L−1, 1.0 mol ·L−1, and 1.0 mmol ·L−1, respectively were added to a 10 mL volumetric flask; next, the mixture was diluted to graduation with Tris-HCl buffer solution.
Rutin of 10 mg (dried at 105°C, purchased from Sinopharm Chemical Reagent Co. Ltd ,China) was dissolved in 5 ml ethanol (95% (v/v)) in a 50-ml volumetric flask, and then the solution was diluted to 50 ml using deionized water for linear assay to establish the calibration line.
A loop of E. coli cell colonies was inoculated into 20 mL LB medium (5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCl) in a 250-mL flask. 2 mL overnight seed culture was diluted into 50 mL TB medium (yeast extract 24 g/L, tryptone 12 g/L, glycerol 5 g/L, KH2PO4 2.31 g/L, K2HPO4 16.43 g/L) in a 250-mL flask and incubated for 36 h.
The following morning, the overnight culture was diluted 1∶100 in fresh TSB (flask to volume ratio 5∶1).
