Empower 2 chromatographic manager system

HPLC analysis have been carried out according to Pudziuvelyte et al. [34 (link)] method. For analysis a Waters 2695 chromatography system (Waters, Milford, CT, USA), equipped with a Waters 996 PDA detector was used. Data was collected and analyzed using the Empower-2 chromatographic manager system (Waters Corporation, Milford, MA, USA). For determination of polyphenols, an ACE 5C₁₈ 250 × 4.6 mm (Advanced Chromatography Technologies, Aberdeen, Scotland, UK) column was used. The mobile phase consisted of solvent A (phosphoric acid/acetonitrile/water) (1:19:80 v/v/v) and solvent B (phosphoric acid/methanol/acetonitrile) (1:40:59 v/v/v). The linear gradient elution profile was as follows: 100% A—at 0 min, 55% A/45% B—at 20 min, 100% B—from 25 to 26 min, 100% A—from 30 to 31 min. The flow rate was 1.2 mL/min and the injection volume was 10 μL. Absorption was measured at 330 nm. Quantification of phenolic compounds was performed using reference standards of apigenin, rosmarinic acid, and chlorogenic acid. The linear calibration curves were constructed (apigenin R² = 0.999979, rosmarinic acid R² = 0.999551, chlorogenic acid R² = 0.999914), the peak areas were used for quantification. The contents were expressed as μg/g dry weight.

High-performance liquid chromatography (HPLC) analysis was carried out using Waters 2695 chromatography system (Waters, Milford, MA, USA) equipped with Waters 996 PDA detector. Data were collected and analyzed using a PC and the Empower 2 chromatographic manager system (Waters Corporation, Milford, USA). For determination of rosmarinic acid and carvacrol, an ACE 5 C18 250 × 4.6 mm column (Advanced Chromatography Technologies, Aberdeen, Scotland) was used.