Flow rate was 1.0 mL min−1 during separation, and the injection volume was 10 L.
The mobile phase consisted of a combination of solvent A
(0.1% [v/v] phosphoric acid buffer with 0.1% [v/v] acetonitrile) and
solvent B (acetonitrile). The conditions for HPLC analysis were a
linear gradient from 75:25 A:B to 55:45 A:B over 8 min, and then,
solvent B was increased to 100% in 5 min. Absorbance at 345 nm
was used for real-time monitoring of peak intensities. Rutin and
scopoletin standards were obtained from Fluka and Sigma-Aldrich,
respectively.