Prominence diode array detector

HPLC purification of described compounds were carried out on a Shimadzu Prominence Nexera XR HPLC coupled to LC-6AD pumps, SPD-M20A Prominence Diode Array Detector, CTO-20A Prominence Column Oven, CBM-20A Communication Bus Module, and Class VP software. 1D and 2D NMR spectra were acquired on Bruker (R) – DRX500-Ultra Shield (R) (¹H: 500.13 MHz, ¹³C: 125.77 MHz) and Bruker Avance III HD 600-MHz spectrometers.

The HPLC-DAD analysis of specialized metabolites was performed using a reversed-phase system (Shimadzu LC-20AB, Prominence Diode Array Detector, Shimadzu Corporation, Japan), equipped with a binary pump. The column was a Macherey-Nagel C18 column (4.6 mm × 250 mm × 5 μm). The wavelength was set at 254 nm, and the analysis time was 70 min. Two different solvents were used as a mobile phase: A (0.05% formic acid aqueous solution) and B (methanol) programmed in a gradient as follows: 0 min (5% B); 5 min (15% B); 18 min (25% B); 35 min (35% B); 50–60 min (100% B); 65–70 min (5% B). Each mobile phase was filtered through a 0.20 μm membrane filter before use. The samples were dried by a rotavapor an solubilized in water at a concentration of 5 or 10 mg/100 μL. Samples (20 μL) were injected at a flow rate of 0.60 mL min⁻¹. The system was allowed to equilibrate for approximately 10 min for the subsequent injection. The identification of each compound was obtained according the retention time and the UV-Visible spectra of the corresponding standard compounds. The linearity of the standard was calculated by taking the peak area as a function of the concentration and expressed as mg of standard/kg of dry extract and was converted into mg of standard/100 g FW. The experiments were repeated three times and the results expressed as mean ± standard deviation.