Reversed-phase high-performance liquid chromatography with electrochemical detection of anthocyanins

Anthocyanins, flavonoid compounds present in grapes and wines, were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (RP-HPLC-ED). The method developed consists of RP-HPLC gradient elution with voltammetric detection using a glassy carbon electrode after separation in an Inertsil ODS-3V analytical column. Good peak resolution was obtained following direct injection of a 50 mu L sample of anthocyanins in a mobile phase of pH 2.20. The results show that six different anthocyanins: cyanidin-3-O-glucoside chloride (kuromanin chloride), cyanidin-3,5-di-O-glucoside chloride (cyanin chloride), malvidin-3-O-glucoside chloride (oenin chloride), malvidin-3,5-di-O-glucoside chloride (malvin chloride), delphinidin-3-O-glucoside chloride (myrtillin chloride), and peonidine-3-O-glucoside chloride, all with antioxidant properties, can be separated in a single run by direct injection of solution. The limit of detection (LOD) for these compounds was lower than 0.3 mM. The method can also be applied to the analysis of these compounds in red wines and in skins and pulp extracts of red grapes, since all these antioxidants are electroactive.