HPLC-analysis of polyphenolic compounds in Spanish white wines and determination of their antioxidant activity by radical scavenging assay

Abstract

A high-performance liquid chromatographic method coupled with UV and fluorescence detectors for the determination of polyphenols in white wines samples is reported. The proposed method was validated regarding linearity, repeatabilities within day and between days, recoveries and polyphenol stabilities.

Eleven phenolic compounds including, flavanols and flavanols dimmers (procyanidins), hydroxycinnamates, flavonols and stilbene derivatives were identified and quantified. Samples were injected into the chromatograph directly without previous treatment.

The in-house method validation provided good results with respect to repeatabilities (within day (RSD ⩽ 4%) and between days (RSD ⩽ 5%)) and recoveries (⩾97.3%).

In the second part of the work the antioxidant activity of the wine samples was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The correlation between specific polyphenols and the antioxidant activity in samples was also investigated.

Introduction

In the past years the analysis of polyphenols in different foodstuffs; such as wine (Bravo et al., 2006, Careri et al., 2003, Castellari et al., 2002) fruits (Ehala et al., 2005, Lyons et al., 2003), and tea (Yanagida et al., 2006) has attracted the interest of the researchers due to their beneficial effects on health. Several studies reported in the literature have described their biological activities as vasodilators, anti-inflammatory and anti-microbial (Burns et al., 2000, Golberg et al., 1999, Kwang-Seok et al., 1999).

Numerous phenolic compounds including, flavanols, flavonols, stilbene derivatives, hydroxycinnamates and anthocyanins have been found in wine samples.

In addition polyphenols play an essential role in sensory properties since they contribute to colour, flavour and bitterness of wines.

High-performance liquid chromatography with diode array (DAD) (Bravo et al., 2006, Castellari et al., 2002, Malovaná et al., 2001, Peña-Neira et al., 2000), fluorescence (Bravo et al., 2006, Rodríguez-Delgado et al., 2001), and electrochemical detection (Bravo et al., 2006, Kolouchová-Hanzlíková et al., 2004), is the most commonly technique used to analyse polyphenols. Recently, LC–MS has also been successfully employed for the identification of phenolic compounds in food samples (Nicoletti et al., 2007, Puessa et al., 2006), however this equipment is very expensive and is not common in control laboratories. The use of UV–Vis and fluorescence detector in series could be an excellent alternative when a LC–MS is not available. The high selectivity of the fluorescence detector could help us to distinguish between different substances in complex matrix such as wine.

The study of the antioxidant activity in natural products has received increasing attention in recent years. Several “in vitro” methods have been used to assess the antioxidant activity in wines, including, oxygen radical absorbance capacity (ORAC) (Fernández-Pachón et al., 2004, Fernández-Pachón et al., 2006, Villaño et al., 2005), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Fernández-Pachón et al., 2004, Fernández-Pachón et al., 2006, Villaño et al., 2005; Fernández-Pachón, Villaño, Troncoso, & García-Parrilla, 2004), 1,1-diphenyl-2-picrylhydrazyl (DPPH), (Fernández-Pachón et al., 2004, Fernández-Pachón et al., 2006, Villaño et al., 2005; Que, Mao, & Pan, 2006) dimethyl-p-phenylene diamine (DMPD), (Fernández-Pachón et al., 2004), ferric reducing/antioxidant power assay (FRAP) (Katalinić, Milos, Modun, Musić, & Boban, 2004), and β-carotene bleaching (BCB) (Katalinić et al., 2004).

In this work, we report the analysis of polyphenols in white wine samples from Galicia (NW Spain) by on line HPLC coupled with UV and fluorescence detectors. The fluorescence detection was used as a confirmatory technique. The proposed method was validated with respect to linearity, repeatabilities within day and between days, recoveries and polyphenol stabilities. In the second part of the work the antioxidant activity of the wine samples was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The correlation between specific polyphenols and the antioxidant activity in samples was also investigated.