**1. Introduction**

356 Macro to Nano Spectroscopy

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Wine, especially red wine, is a very rich source of polyphenols, such as flavanols (catechin, epicatechin, etc.), flavonols (quercetin, rutin, myricetin, etc.), anthocyanins (the most abundant is malvidin-3-o-glucoside), oligomeric and polymeric proanthocyanidins, phenolic acids (gallic acid, caffeic acid, p-coumaric acid, etc.), stilbenes (*trans*-resveratrol) and many others polyphenols. Many of these compounds (e.g. resveratrol, quercetin, rutin, catechin and their oligomers and polymers proanthocyanidins) have been reported to have multiple biological activities, including cardioprotective, anti-inflammatory, anti-carcinogenic, antiviral and antibacterial properties (King et al., 2006; Santos- Buelega Scalbert, 2000). These biological properties are attributed mainly to their powerful antioxidant and antiradical activity.

Regular, moderate consumption of red wine reduced the incidence of many diseases such as risk of coronary heart disease (CHD), atherosclerosis, cancers, etc. (Cooper et al., 2004; Opie Lecour, 2007). The most intriguing are the studies which reported the possible association between red wine consumption and decrease in risk, and some suppression and inhibition of cancers (Briviba et al., 2002). Currently, chemoprevention is being used in medicine as a new strategy to prevent cancers. Natural phytochemicals, including red wine polyphenols, appear to be very promising substances to block, reverse, retard or prevent the process of carcinogenesis (Russo, 2007). Many epidemiological studies have found that regular intake of red wine or red wine polyphenols has positive effects on human health. Therefore, determination of the chemical composition, polyphenols content and antioxidant activity of red wine could be very useful for the interpretation of epidemiological studies.

Phenolic antioxidants define total antioxidant potential of wines and have the greatest influence on it. Authors showed that in grape seeds gallic acid, catechins and epicatechins prevailed, whereas in peel ellagic acid, quercetin and trans-resveratrol were most common.

<sup>\*</sup> Corresponding Author

A Comparative Study of Analytical Methods for Determination

flavonol, quercetin was also included in our research.

potential (TAPCL) of the sample (Hipler Knight, 2001)..

nm, and that with Fe2+ shows a maximum at 484 nm (Shimomura, 2006).

using the spectrophotometric and chemiluminometric method.

modelling solution.

of Polyphenols in Wine by HPLC/UV-Vis, Spectrophotometry and Chemiluminometry 359

With use of HPLC we can reach separation of non-stabile and heavy volatile analytes on the base of different chemical interactions of the analytes with mobile phase and stationary phase. We use a non-polar stationary and polar mobile phase (reversed phase chromatography). HPLC with UV-visible detection was used for determination of antioxidant compounds content of gallic acid, (+)-catechin, (-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin in numerous wine samples. The selected phenolic compounds are the most important wine antioxidants. Gallic acid, the main hydroxybenzoic acid in red wines, is a very potent antioxidant with three free hydroxyl groups. Because of the relatively slow extraction of gallic acid from grape seeds, higher concentrations are obtained with longer maceration times, which is characteristic for red wines. As the most important

Spectrophotometric determination of total antioxidant potential (TAPSP) was performed with oxidation of phenolic compounds with Folin-Ciocalteu reagent after spectrophotometric method, described by Singelton in Rossi. Gallic acid was used as

Chemiluminometric determination of polyphenols was another possibility for evaluation of the total antioxidant potential in wine. Chemiluminescence, the emission of light as a consequence of relaxation of kind, which it is evoked between chemical reactions, has become very useful technique for studying oxidation of organic materials (Costin et al., 2003; Garcia-Campana Baeyens, 2001; Hötzer et al., 2005; Kočar et al., 2008; Kuse et al., 2008). ABEL® (analysis by emitted light) antioxidant test kit, which contains photo protein Pholasin®, was used. Photo protein Pholasin® is the protein-bound luciferin from the bivalve mollusc Pholas dactylus, which reacts with luciferase and molecular oxygen to produce light (Knight, 1997; Michelson, 1978; Roberts et al., 1987). In reaction system substratecatalyser-oxidant we can therefore inhibit occurrence of chemiluminescence with antioxidants. If there are antioxidants in the sample capable of scavenging superoxide, then these antioxidants will compete with Pholasin® for the superoxide and less light will be detected. Control samples containing no antioxidants were running with each assay. With measuring of decrease of intensity of chemiluminescence we evaluate total antioxidant

The reactions of Pholasin® have been studied extensively (Dunstan et al., 2000; Müller et al., 1989; Reichl et al., 2000). It was found to have a 50- to 100-fold greater sensitivity towards superoxide than luminol. In addition, the decay of the Pholasin® chemiluminescent product was more rapid than that of the luminol product, leading to a greater accuracy in real-time kinetic studies. For these reasons, Pholasin® offers several advantages over luminol (Roberts et al., 1987). The luminescence of Pholasin® elicited with luciferase has a maximum at 490

In addition, we compared the results of the chromatographic method with those obtained

In order to evaluate their potential individual contributions to TAP, phenolic antioxidants were analysed as pure solutions in the same concentration. The order of contributions of individual phenolic antioxidants to TAP determined according to spectrophotometric method was different than those determined with chemiluminometric method. Generalised, *cis*-resveratrol has the biggest contribution to TAP, following by *trans*-resveratrol, (-)-

The high antioxidant potential of red wines can be ascribed to the synergistic effect of the mixture of natural phenolic antioxidants (Lopez-Velez et al., 2003).

Production technology is one of the main factors influencing the high antioxidant potential of red wines (Downey et al., 2006; Vršič et al., 2009). During winemaking the grape pulp is fermented, and fruit peel and seeds are very rich in phenolic antioxidants. The concentration of polyphenols in peel is higher than in the flesh (Darias-Martin et al., 2000; Fuhrman et al., 2001). During intensive pressing or during long contact of juice with pulp the content of phenolic compounds increases rapidly (Fuhrman et al., 2001). It was found that in wines fermented with peel the concentration of phenolic antioxidants was 2 times higher than in wines fermented without peel (Darias Martin et al., 2000).

Antioxidant potential and polyphenol composition were assessed in wine of Croatian origin (Katalinic et al., 2004). The concentration of total polyphenols in red wines ranged from 2200 to 3200 mg gallic acid per litter (mg GA/L).

In winemaking, phenolic antioxidants are extracted from berry skins, seeds and stems during crushing and fermentation. Due to the market demand, knowledge of the concentration of phenolic antioxidants in wine and their antioxidant potential is very important.

Wine, especially red wine, is a very rich source of flavonol quercetin and many others polyphenols. Various methods for characterisation of total antioxidant potential are presently in routine use, although some are non-stoichiometric (Alimelli et al., 2007; Campanella et al., 2004; Careri et al., 2003; Carralero Sanz et al., 2005; De Beer et al., 2005; Fernandez-Pachon et al., 2004; Giovanelli, 2005; Gomez-Alonso et al., 2007; Magalhaes et al., 2009; Makris et al., 2003; Malovana et al, 2001; Mozetič et al., 2006; Prior et al., 2005; Prosen et al., 2007; Recamales et al., 2006; Spigno De Faveri, 2007; Staško et al., 2008; Weingerl et al., 2009; Woraratphoka, 2007). Some of these methods allow for rapid characterization of wines, and allow for evaluation of synergistic effects of various wine components, e.g. transition metals (Strlič et al., 2002), which can have pro-oxidative effects in a mixture with phenolic compounds. The results of such analyses are usually given in equivalents of gallic acid or other reference compounds. Among these methods, determination of total phenolic content using the Folin-Ciocalteu reagent, as described by Singleton and Rossi (Singelton Rossi, 1965), is very common.

Considering the accumulated knowledge on the effect of phenolic antioxidants on human health and the resulting market requirements it is highly important to have well developed, robust and established methods for their determination (Minussi et al., 2003; Urbano-Cuadrado et al., 2004).

In this study we compared three analytical methods: high pressure liquid chromatography (HPLC) with UV-vis detection, UV-vis spectrophotometry and chemiluminometry.

For separation and determination of phenolic acids and flavonoids, HPLC is the established technique (Nave et al., 2007; Rodriguez-Delgado et al., 2001; Spranger et al., 2004; Vitrac et al., 2002). The chromatographic conditions include the use of, almost exclusively, a reversed phase C18 column; UV-vis diode array detector, and a binary solvent system containing acidified water and a polar organic solvent (Tsao Deng, 2004).

The high antioxidant potential of red wines can be ascribed to the synergistic effect of the

Production technology is one of the main factors influencing the high antioxidant potential of red wines (Downey et al., 2006; Vršič et al., 2009). During winemaking the grape pulp is fermented, and fruit peel and seeds are very rich in phenolic antioxidants. The concentration of polyphenols in peel is higher than in the flesh (Darias-Martin et al., 2000; Fuhrman et al., 2001). During intensive pressing or during long contact of juice with pulp the content of phenolic compounds increases rapidly (Fuhrman et al., 2001). It was found that in wines fermented with peel the concentration of phenolic antioxidants was 2 times higher than in

Antioxidant potential and polyphenol composition were assessed in wine of Croatian origin (Katalinic et al., 2004). The concentration of total polyphenols in red wines ranged from 2200

In winemaking, phenolic antioxidants are extracted from berry skins, seeds and stems during crushing and fermentation. Due to the market demand, knowledge of the concentration of phenolic antioxidants in wine and their antioxidant potential is very

Wine, especially red wine, is a very rich source of flavonol quercetin and many others polyphenols. Various methods for characterisation of total antioxidant potential are presently in routine use, although some are non-stoichiometric (Alimelli et al., 2007; Campanella et al., 2004; Careri et al., 2003; Carralero Sanz et al., 2005; De Beer et al., 2005; Fernandez-Pachon et al., 2004; Giovanelli, 2005; Gomez-Alonso et al., 2007; Magalhaes et al., 2009; Makris et al., 2003; Malovana et al, 2001; Mozetič et al., 2006; Prior et al., 2005; Prosen et al., 2007; Recamales et al., 2006; Spigno De Faveri, 2007; Staško et al., 2008; Weingerl et al., 2009; Woraratphoka, 2007). Some of these methods allow for rapid characterization of wines, and allow for evaluation of synergistic effects of various wine components, e.g. transition metals (Strlič et al., 2002), which can have pro-oxidative effects in a mixture with phenolic compounds. The results of such analyses are usually given in equivalents of gallic acid or other reference compounds. Among these methods, determination of total phenolic content using the Folin-Ciocalteu reagent, as described by Singleton and Rossi (Singelton

Considering the accumulated knowledge on the effect of phenolic antioxidants on human health and the resulting market requirements it is highly important to have well developed, robust and established methods for their determination (Minussi et al., 2003; Urbano-

In this study we compared three analytical methods: high pressure liquid chromatography

For separation and determination of phenolic acids and flavonoids, HPLC is the established technique (Nave et al., 2007; Rodriguez-Delgado et al., 2001; Spranger et al., 2004; Vitrac et al., 2002). The chromatographic conditions include the use of, almost exclusively, a reversed phase C18 column; UV-vis diode array detector, and a binary solvent system containing

(HPLC) with UV-vis detection, UV-vis spectrophotometry and chemiluminometry.

acidified water and a polar organic solvent (Tsao Deng, 2004).

mixture of natural phenolic antioxidants (Lopez-Velez et al., 2003).

wines fermented without peel (Darias Martin et al., 2000).

to 3200 mg gallic acid per litter (mg GA/L).

important.

Rossi, 1965), is very common.

Cuadrado et al., 2004).

With use of HPLC we can reach separation of non-stabile and heavy volatile analytes on the base of different chemical interactions of the analytes with mobile phase and stationary phase. We use a non-polar stationary and polar mobile phase (reversed phase chromatography). HPLC with UV-visible detection was used for determination of antioxidant compounds content of gallic acid, (+)-catechin, (-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin in numerous wine samples. The selected phenolic compounds are the most important wine antioxidants. Gallic acid, the main hydroxybenzoic acid in red wines, is a very potent antioxidant with three free hydroxyl groups. Because of the relatively slow extraction of gallic acid from grape seeds, higher concentrations are obtained with longer maceration times, which is characteristic for red wines. As the most important flavonol, quercetin was also included in our research.

Spectrophotometric determination of total antioxidant potential (TAPSP) was performed with oxidation of phenolic compounds with Folin-Ciocalteu reagent after spectrophotometric method, described by Singelton in Rossi. Gallic acid was used as modelling solution.

Chemiluminometric determination of polyphenols was another possibility for evaluation of the total antioxidant potential in wine. Chemiluminescence, the emission of light as a consequence of relaxation of kind, which it is evoked between chemical reactions, has become very useful technique for studying oxidation of organic materials (Costin et al., 2003; Garcia-Campana Baeyens, 2001; Hötzer et al., 2005; Kočar et al., 2008; Kuse et al., 2008). ABEL® (analysis by emitted light) antioxidant test kit, which contains photo protein Pholasin®, was used. Photo protein Pholasin® is the protein-bound luciferin from the bivalve mollusc Pholas dactylus, which reacts with luciferase and molecular oxygen to produce light (Knight, 1997; Michelson, 1978; Roberts et al., 1987). In reaction system substratecatalyser-oxidant we can therefore inhibit occurrence of chemiluminescence with antioxidants. If there are antioxidants in the sample capable of scavenging superoxide, then these antioxidants will compete with Pholasin® for the superoxide and less light will be detected. Control samples containing no antioxidants were running with each assay. With measuring of decrease of intensity of chemiluminescence we evaluate total antioxidant potential (TAPCL) of the sample (Hipler Knight, 2001)..

The reactions of Pholasin® have been studied extensively (Dunstan et al., 2000; Müller et al., 1989; Reichl et al., 2000). It was found to have a 50- to 100-fold greater sensitivity towards superoxide than luminol. In addition, the decay of the Pholasin® chemiluminescent product was more rapid than that of the luminol product, leading to a greater accuracy in real-time kinetic studies. For these reasons, Pholasin® offers several advantages over luminol (Roberts et al., 1987). The luminescence of Pholasin® elicited with luciferase has a maximum at 490 nm, and that with Fe2+ shows a maximum at 484 nm (Shimomura, 2006).

In addition, we compared the results of the chromatographic method with those obtained using the spectrophotometric and chemiluminometric method.

In order to evaluate their potential individual contributions to TAP, phenolic antioxidants were analysed as pure solutions in the same concentration. The order of contributions of individual phenolic antioxidants to TAP determined according to spectrophotometric method was different than those determined with chemiluminometric method. Generalised, *cis*-resveratrol has the biggest contribution to TAP, following by *trans*-resveratrol, (-)-

A Comparative Study of Analytical Methods for Determination

mmol gallic acid per litter (gallic acid equivalents - GAE).

(Folin Ciocalteu, 1927).

**2.3 Chemiluminometry** 

(1:10) while white wines were not.

component analysis (PCA) was used.

**3. Results and discussion** 

UV/VIS detection.

white wines.

**2.4 Statistical analysis** 

of Polyphenols in Wine by HPLC/UV-Vis, Spectrophotometry and Chemiluminometry 361

ortophosphoric acid, hydrochloric acid, lithium sulphate, bromine, hydrogen peroxide

Briefly, 25 µL of a red and rosé wine sample or 250 µL of a white wine sample, 15 mL of distilled water, 1.25 mL of the diluted (1:2) Folin–Ciocalteu reagent, 3.75 mL of a sodium carbonate solution (20%) were mixed and distilled water was added to make up the total volume of 25 mL. The solution was agitated and left to stand for 120 min at room temperature for the reaction to take place. The calibration curve was prepared with gallic acid solutions in the concentration range from 0 to 1000 mg/L. The results are expressed as

The results for standards were highly reproducible (calibration curve squared regression coefficient >0.9993). All determinations were performed in triplicates. Typical standard deviation for spectrophotometric determinations of total phenolic content (TAPSP) was 0.10

The Abel®-21 M2 antioxidant test kit (Knight Scientific Limited, Plymouth, UK) was used for chemiluminometric determination of total antioxidant potential (TAPCL). Superoxide, generated in a tube containing Pholasin® with and without a sample of wine with unknown antioxidant potential leads to appearance of chemiluminescence, which was measured using a micro plate luminometer model Lucy (Anthos Labtec Instruments, Wals, Austria). Pholasin® is a bioluminescent photo protein of *Pholas dactylus*, which is a marine, rock-

The Antioxidant Test procedure for Superoxide is provided by the supplier of the test kit (Hipler and Knight, 2001). The amount of sample was optimised to obtain not more than 90% and typically 50% signal inhibition. This signal was then corrected for sample dilution: 10 µL of sample was used, however, red wines and rosé wines were first diluted with water

The results are calculated as TAPCL, expressed as % signal inhibition. Typical measurement uncertainty was 0.024 mmol/L for red wines, 0.016 mmol/L for rosé and 0.002 mmol/L for

Measurements are expressed as means ± standard deviations (SD) for three replicate determinations. Multivariate analysis was performed using SPSS 17.0 for Windows (SPSS Inc., Chicago, USA). As a typical data reduction and visualisation technique, principal

Comparison of determinations of total antioxidant potential in different wines was performed with spectrophotometric and chemiluminometric method, while comparison of antioxidant compounds content in the same samples was performed using HPLC with

mmol/L for red wines, 0.09 mmol/L for rosé and 0.02 mmol/L for white wines.

boring, bivalve mollusc. Antioxidant test kit used contains assay buffer (pH 7.2).

epicatechin, (+)-catechin and quercetin. Interesting, gallic acid, as modelling solution by spectrophotometric determination of TAPSP, shows the lowest contribution to TAP.
