*2.10. Analysis of Phenolic Compounds*

The HPLC-DAD Shimadzu system (Shimadzu, Kioto, Japan), which consisted of a CBM-20A controller, DGU-20A5R degassing unit, two LC-30AD pumps, SIL-30AC autosampler, SPD-M30A diode array detector (DAD), and CTO-20AC oven, was used to analyze the phenolic compound of the amaranth extracts. Extracts dissolved in 80% (*v*/*v*) methanol were injected (10 μL) into a Luna C8(2) (4.6 × 150 mm, particle size 3 μm, Phenomenex, Torrance, CA, USA) column [34]. The mobile phase consisted of solvents A (acetonitrile-water-trifluoroacetic acid, 5:95:0.1, *v*/*v*/*v*) and B (acetonitrile-trifluoroacetic acid, 100:0.1, *v*/*v*): From 0–16 min, the eluent composition was changed from 0–24% B. The flow rate was 1 mL/min. The oven temperature was 25 ◦C and detector wavelength ranged from 200 to 600 nm. The content of individual phenolic compounds in the extracts was expressed on the basis of a calibration curve of the corresponding standards or structurally related substances.

For identification of extract compounds, HPLC-MS/MS analysis was carried out using a liquid chromatography (LC) system coupled with a quadrupole ion trap mass spectrometer (QTRAP® 5500 LC-MS/MS System, AB Sciex, Framingham, MA, USA). Operating MS/MS conditions were the following: Nitrogen curtain gas flow rate of 25 L/min, collision gas glow rate of 9 L/min, ion spray source voltage of −4.5 kV, temperature of 350 ◦C, nebulizer gas flow rate of 35 L/min, and turbo gas flow rate of 30 L/min. Negative-mode of electrospray ionization (ESI–) was used. Qualification was based on multiple reaction monitoring (MRM) of selected ion pairs in the first quadrupole (Q1) and third quadrupole (Q3). The presence of compounds previously identified in aerial parts of amaranth [10,18–20,35] were verified.
