Luna 5 m c18 2 250x4.6 mm column

The content of individual anthocyanins in the obtained juices was determined by gradient HPLC using a DAD detector based on the method proposed by Mieszczakowska-Frąc et al. [28] with own modifications. The flow rate of the mobile phase consisting of 5 % HCOOH (reagent A) and 100 % ACN (reagent B), was 1 mL/min. The gradient programme was as follows: 0–5 min 3 % B, 5–20 min 9 % B, 20–30 min 9–12 % B, 30–35 min 12 % B, 35–38 min 12–40 % B, 38–40 min 40 % B, 40–41 min 3 % B, 41–56 min 3 % B. The gradient varied linearly. The separation was carried out on a Luna 5 µm C18(2) 250x4.6 mm column (Phenomenex, Torrance, California, USA) precolumn at 520 nm and an oven temperature of 25 °C. Anthocyanin content was expressed in mg/100 mL of juice. The concentration of compounds was determined from a cyanidin-3-glucoside standard (Sigma-Aldrich). Analyses were repeated thrice for each material. The total anthocyanins content (TA) was calculated as a sum of individual anthocyanins.

The content of iridoid compounds was determined in three repetitions according to the method of Duan et al. [11] (link). with own modifications. The mobile phase of the flow rate of 1 mL/min was: H₃PO₄ (0.1 %) - reagent A and methanol (100 %) - reagent B. The results were recorded at a wavelength of 210 nm. Luna 5 µm C18(2), 250x4.6 mm column (Phenomenex, Torrance, California, USA) and PDA detector were used for chromatographic separation. The gradient program varied linearly: 0–4 min 0–25 % B, 4–10 min 25–35 % B, 10–22 min 35–40 % B, 22–27 min 40–0 % B, 27–42 min 0 % B. The amounts of iridoids were calculated based on standards of loganic acid (ChromaDex), loganin (Sigma-Aldrich), secologanin (ChromaDex) and sweroside (ChromaDex) and the total iridoids (TI) was calculated (mg/100 mL) as a sum of identified iridoids.