Supercritical fluid extraction and HPLC determination of relevant polyphenolic compounds in grape skin

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Abstract
The polyphenols determined are: (+)-catechin, (–)-epicatechin, rutin, quercetin and trans-resveratrol. Suitable conditions of supercritical fluid extraction were established using ethanol as a modifier of the polarity solvent (supercritical carbon dioxide). Final extraction conditions were: 20% v/v ethanol, 60°C, 250 bars and flow rate 2 mL/min. Static step time and dynamic step time were established using a selected grape skin sample. The extract was collected in water; the more polar polyphenols ((+)-catechin and (–)-epicatechin) remain in solution but rutin, quercetin and trans-resveratrol precipitate in this medium, thereby the solution of the extracted polyphenols was filtered. (+)-Catechin and (–)-epicatechin were determined in the liquid fraction, while the solid fraction, containing rutin, quercetin and trans-resveratrol, was solved with ethanol/H2O (40:60). HPLC determination was carried out at C18 stationary phase, with ethanol/water/acetic acid as mobile phases and UV-visible diode array detection. Due to the significant differences between the polarity of the polyphenols, two different mobile phases were used. An ethanol/water/acetic acid (5:93:2) mobile phase was used to determine (+)-catechin (280 nm) and (–)-epicatechin (280 nm). On the other hand, rutin (254 nm), quercetin (254 nm) and trans-resveratrol (306 nm) were resolved using ethanol/water/acetic acid (40:58:2) as mobile phase. Instrumental parameters were optimised and analytical parameters obtained. The analytical method was validated and applied to five different varieties of Vitis vinifera from the geographical area of Valencia.

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