**3. Results and discussion**

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 UV/VIS detection.

A Comparative Study of Analytical Methods for Determination

chemiluminometry.

methods.

results (TAPCL).

of Polyphenols in Wine by HPLC/UV-Vis, Spectrophotometry and Chemiluminometry 363

determined with HPLC/UV-vis and total antioxidant potential TAPCL determined with

Sum of phenolic compounds, determined with HPLC method, correlate well with total antioxidant potential TAPCL, determined with chemiluminometric method (r2 = 0.80) (Fig 1). With use of HPLC method we have the information about single phenolic antioxidants content, which doesn't consider synergistic influences between phenolic compounds in wine. Those effects considered only methods for determining total antioxidant potential. On the other hand, matrix effects may lead to different results obtained using other methods. In order to obtain a better insight into the extent of these effects, we compared all used

To estimate the quantification limits, weighted tolerance intervals were used. The limit of quantification (in response units) is defined as 10 times the standard deviation at the lowest detectable signal (*LC*) plus the weighted intercept. The corresponding concentration LQ can be obtained by *LQ = (yQ – a)/b*, where *a* is the intercept, *b* is the slope of correlation curve (Zorn et al. 1997). The quantification limit for TAPSP was calculated from data represented in

Fig. 2. Comparison of spectrophotometric determinations of TAPSP and chemiluminometric

For comparison, Table 1 shows further limits of quantification for determination of sum of

HPLC determined phenolic antioxidants, TAPCL and TAPSP in wine.

Fig. 2, and amounts to 844 µmol/L gallic acid (Weingerl et al., 2011).

Sum of phenolic compounds, determined using HPLC with UV-vis detection, summarized six manly phenolic compounds (gallic acid, (+)-catechin, (-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin). Regarding the knowledge about the influence of phenolic antioxidants on human health, selected phenolic compounds are the most important antioxidants of red wines. Gallic acid, the main hydroxybenzoic acid in red wines, is with three free hydroxyl groups a very strong antioxidant. Because of relative slow extraction of gallic acid from grape seeds, we obtain higher concentrations with longer maceration time, which is characteristic for red wines. In the group of non-flavonoid phenols we analysed also the main representative of stilbenes – resveratrol. Resveratrol (3,5,4'-trihydroxystilbene) is present in wine in four forms; we ware determining *trans*-resveratrol and *cis*-resveratrol. In the group of flavonoid phenols we choose isomers (+)-catechin and (-)-epicatechin, more specific, flavan-3-ols. As most important flavonol we include in our research quercetin. Chemically are flavonols 3-glycosides.

Relative good correlation is result between sum of phenolic compounds determined with HPLC/UV-vis and spectrophotometric determinations of total antioxidant potential TAPSP (r2 = 0.91) (Weingerl et al., 2009).

Light emitted by chemiluminescent substrate was expressed in relative light units (RLU). The time dependence of light intensity was measured and the peak intensity was converted into percent of inhibition relative to control. We expressed the results as total antioxidant potential, expressed as percent inhibition: TAPCL = ((max RLUcontrol) – (max RLUsample)) 100 / (max RLUcontrol). Figure 1 shows the correlation between sum of phenolic compounds

Fig. 1. Comparison between natural logarithm of sum of gallic acid, (+)-catechin, (-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin, determined using HPLC and TAPCL. All wine samples included.

Sum of phenolic compounds, determined using HPLC with UV-vis detection, summarized six manly phenolic compounds (gallic acid, (+)-catechin, (-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin). Regarding the knowledge about the influence of phenolic antioxidants on human health, selected phenolic compounds are the most important antioxidants of red wines. Gallic acid, the main hydroxybenzoic acid in red wines, is with three free hydroxyl groups a very strong antioxidant. Because of relative slow extraction of gallic acid from grape seeds, we obtain higher concentrations with longer maceration time, which is characteristic for red wines. In the group of non-flavonoid phenols we analysed also the main representative of stilbenes – resveratrol. Resveratrol (3,5,4'-trihydroxystilbene) is present in wine in four forms; we ware determining *trans*-resveratrol and *cis*-resveratrol. In the group of flavonoid phenols we choose isomers (+)-catechin and (-)-epicatechin, more specific, flavan-3-ols. As most important flavonol we include in our research quercetin.

Relative good correlation is result between sum of phenolic compounds determined with HPLC/UV-vis and spectrophotometric determinations of total antioxidant potential TAPSP

Light emitted by chemiluminescent substrate was expressed in relative light units (RLU). The time dependence of light intensity was measured and the peak intensity was converted into percent of inhibition relative to control. We expressed the results as total antioxidant potential, expressed as percent inhibition: TAPCL = ((max RLUcontrol) – (max RLUsample)) 100 / (max RLUcontrol). Figure 1 shows the correlation between sum of phenolic compounds

Fig. 1. Comparison between natural logarithm of sum of gallic acid, (+)-catechin,

(-)-epicatechin, *trans*-resveratrol, *cis*-resveratrol and quercetin, determined using HPLC and

Chemically are flavonols 3-glycosides.

(r2 = 0.91) (Weingerl et al., 2009).

TAPCL. All wine samples included.

determined with HPLC/UV-vis and total antioxidant potential TAPCL determined with chemiluminometry.

Sum of phenolic compounds, determined with HPLC method, correlate well with total antioxidant potential TAPCL, determined with chemiluminometric method (r2 = 0.80) (Fig 1).

With use of HPLC method we have the information about single phenolic antioxidants content, which doesn't consider synergistic influences between phenolic compounds in wine. Those effects considered only methods for determining total antioxidant potential. On the other hand, matrix effects may lead to different results obtained using other methods. In order to obtain a better insight into the extent of these effects, we compared all used methods.

To estimate the quantification limits, weighted tolerance intervals were used. The limit of quantification (in response units) is defined as 10 times the standard deviation at the lowest detectable signal (*LC*) plus the weighted intercept. The corresponding concentration LQ can be obtained by *LQ = (yQ – a)/b*, where *a* is the intercept, *b* is the slope of correlation curve (Zorn et al. 1997). The quantification limit for TAPSP was calculated from data represented in Fig. 2, and amounts to 844 µmol/L gallic acid (Weingerl et al., 2011).

Fig. 2. Comparison of spectrophotometric determinations of TAPSP and chemiluminometric results (TAPCL).

For comparison, Table 1 shows further limits of quantification for determination of sum of HPLC determined phenolic antioxidants, TAPCL and TAPSP in wine.

A Comparative Study of Analytical Methods for Determination

Fig. 4. TAPCL of individual phenolic antioxidant solutions (50 µmol/L).

following by *trans*-resveratrol, (-)-epicatechin, (+)-catechin and quercetin.

are very strongly co-correlated and most strongly affected by quercetin content.

resveratrol.

Regarding their antioxidant potential, *cis*-resveratrol has been found to be the most powerful scavenger among the analysed phenolic antioxidants. *cis*-resveratrol, (+)-catechin, (-)-epicatechin, quercetin, *trans*-resveratrol and gallic acid was the order of contributions to TAPSP, determined according the spectrophotometric method. *trans*-resveratrol, *cis*-resveratrol, (-)-epicatechin, quercetin, (+)-catechin and gallic acid was the order of contributions to TAPCL, determined according to chemiluminometric method. Gallic acid has, interesting, the lowest contribution to TAP. Generalised, *cis*-resveratrol has the biggest contribution to TAP,

To reduce the number of variables and to investigate the extent of correlation between the six individual phenolic antioxidants and total antioxidant potentials, determined with spectrophotometric and chemiluminometric method, principal component analysis (PCA) was performed. While 57.5% of the variation is explained by PC1 and another 11 % by PC2, we compared the loading factors in Figure 4 to investigate how the different variables might be co-correlated. TAPSP and amount of gallic acid, determined with HPLC/UV-vis method

As is evident from Figure 4, there is a very strong co-correlation between TAPCL and content of *cis*-resveratrol. Referred to this co-correlation TAPCL is also strongly correlated to further antioxidants present especially in red grape varieties: (+)-catechin, (-)-epicatechin and *trans*-

of Polyphenols in Wine by HPLC/UV-Vis, Spectrophotometry and Chemiluminometry 365


Table 1. Limits of quantification (LOQ) for determination of polyphenols in wine.

As it is evident from Table 1, the chemiluminometric method exhibits similar quantification limits to the HPLC method for the determination of the sum of gallic acid, (+)-catechin, (-) epicatechin, *trans*-resveratrol, *cis-*resveratrol and quercetin in wines (32 µmol/L). This is much lower quantification limit comparable with the spectrophotometric method, for the determination of total polyphenols, expressed in µmol/L of gallic acid.

Like total antioxidant potential determined with spectrophotometric or chemiluminometric method, like sum of six determined phenolic compounds are higher by red wines than white wines, rosé wines are giving intermediate results.

Evaluation of individual contributions of selected phenolic antioxidants to total antioxidant potential shows different results for spectrophotometric and chemiluminometric method (Fig.3 and Fig.4).

Fig. 3. TAPSP of individual phenolic antioxidant solutions (50 µmol/L).

As it is evident from Table 1, the chemiluminometric method exhibits similar quantification limits to the HPLC method for the determination of the sum of gallic acid, (+)-catechin, (-) epicatechin, *trans*-resveratrol, *cis-*resveratrol and quercetin in wines (32 µmol/L). This is much lower quantification limit comparable with the spectrophotometric method, for the

Like total antioxidant potential determined with spectrophotometric or chemiluminometric method, like sum of six determined phenolic compounds are higher by red wines than

Evaluation of individual contributions of selected phenolic antioxidants to total antioxidant potential shows different results for spectrophotometric and chemiluminometric method

Chemiluminometric method 32 µmol/L ascorbic acid

Spectrophotometric method 844 µmol/L gallic acid

Table 1. Limits of quantification (LOQ) for determination of polyphenols in wine.

Method LOQ Unit

Sum HPLC/UV-vis 27 µmol/L

determination of total polyphenols, expressed in µmol/L of gallic acid.

Fig. 3. TAPSP of individual phenolic antioxidant solutions (50 µmol/L).

white wines, rosé wines are giving intermediate results.

(Fig.3 and Fig.4).

Fig. 4. TAPCL of individual phenolic antioxidant solutions (50 µmol/L).

Regarding their antioxidant potential, *cis*-resveratrol has been found to be the most powerful scavenger among the analysed phenolic antioxidants. *cis*-resveratrol, (+)-catechin, (-)-epicatechin, quercetin, *trans*-resveratrol and gallic acid was the order of contributions to TAPSP, determined according the spectrophotometric method. *trans*-resveratrol, *cis*-resveratrol, (-)-epicatechin, quercetin, (+)-catechin and gallic acid was the order of contributions to TAPCL, determined according to chemiluminometric method. Gallic acid has, interesting, the lowest contribution to TAP. Generalised, *cis*-resveratrol has the biggest contribution to TAP, following by *trans*-resveratrol, (-)-epicatechin, (+)-catechin and quercetin.

To reduce the number of variables and to investigate the extent of correlation between the six individual phenolic antioxidants and total antioxidant potentials, determined with spectrophotometric and chemiluminometric method, principal component analysis (PCA) was performed. While 57.5% of the variation is explained by PC1 and another 11 % by PC2, we compared the loading factors in Figure 4 to investigate how the different variables might be co-correlated. TAPSP and amount of gallic acid, determined with HPLC/UV-vis method are very strongly co-correlated and most strongly affected by quercetin content.

As is evident from Figure 4, there is a very strong co-correlation between TAPCL and content of *cis*-resveratrol. Referred to this co-correlation TAPCL is also strongly correlated to further antioxidants present especially in red grape varieties: (+)-catechin, (-)-epicatechin and *trans*resveratrol.

A Comparative Study of Analytical Methods for Determination

methods.

**5. Acknowledgements** 

**6. References** 

56.

respect during my academic growth.

597, 103-112.

*Bioelectr*. 19, 641-651.

of Polyphenols in Wine by HPLC/UV-Vis, Spectrophotometry and Chemiluminometry 367

considered both described methods for determining total antioxidant potential (TAPSP and TAPCL), on which indicates quality of correlation determining according to these two


If we consider content of individually determined phenolic antioxidants and not only antioxidative capacity of wine, we may use only HPLC method with UV-vis detection.

I offer my regards and blessings to all of those precious people who supported me in any

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Fig. 5. Loading plot for PCA performed with all measured variables: single phenolic antioxidants, TAPSP and TAPCL.
