Peonidin 3 o glucoside chloride

Qualitative analysis of the extract was performed by high-performance liquid chromatography (HPLC) using an HP 1100/1200 instrument (Agilent Technologies, Palo Alto, CA, USA), equipped with an autosampler (100 µL sample loop), and a diode array detector [26 (link)]. About 225 μg of raw extract was loaded in 100 µL of HPLC starting conditions (6% of eluent A, composed by water/acetonitrile/formic acid, 87:3:10 v/v/v) on a C12 column (Synergi 4 µm Max-RP 80 A, 250 × 4.6 mm ID, Phenomenex, Torrance, California, USA), preceded by a Synergi guard column (4 × 3.00 mm, Phenomenex) with the same stationary phase, at room temperature. The flow rate was set at 0.5 mL/min with a multistep gradient of eluent B (water/acetonitrile/formic acid, 40:50:10 v/v/v in A from 6% to 90%, according to the scheme provided in Table 1. Elution was followed by recording the absorbance between 190 and 700 nm. As a reference, a standard pool composed by 60 µL of a 2.5 ng/µL solution of malvidin-3-O-glucoside chloride, 60 µL of a 5 ng/µL solution of cyanidin-3-O-glucoside chloride, and 60 µL of a 2.5 ng/µL solution of peonidin-3-O-glucoside chloride (Sigma-Aldrich) was run on the same gradient.