From Vine to Wine: Non-Colored Flavonoids as Fingerprints

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

“From Vine to Wine: Non - Colored Flavonoids as Fingerprints”

The work was based on an HPLC study of different wines, but the identification and separation of the flavonoids present in the wines is poor and poorly explained. Additional standards and an HPLC-MS assay are required to confirm the identification of the peaks..

Graphical Abstract

Abstract

The novelty of the research work should be highlighted.

Supplementary data

Improve the quality of Figure S1 and make the identification of peaks clearer. An explanation is needed as to how peaks 1, 3 and mainly 8 were considered despite not achieving good peak separation.

1. Introduction

The introduction is generally well written, however more information is needed on ‘’Non-Coloured Flavonoids” and why these flavonoids are colourless.

It is well known that flavanols are often present as part of oligomeric compounds, obviously coloured, and that this polymerisation reaction could be enhanced by the influence of pH or temperature, how could this be considered to maintain the viability of the developed models?

2. Materials and Methods

 “Tentative identification of other compounds was based on spectral data and relative retention times reported in the literature, with additional confirmation through spiking experiments. Chromatographic details from non-visible flavonoid are presented in Figure  S1 and Table S2.”

Retention times will depend on the analysis methodology used, so it is necessary to explain how this identification has been done.

 “The commercial standards used included catechin, epicatechin, quercetin, and rutin, all with purities exceeding 98% (Sigma-Aldrich).”

 The entire work is based on an HPLC assay of different wines, and only 4 standards were used. A better explanation of the standards used and their purity is needed.

A better explanation of how the identification of the compounds was performed is needed.

 

3 RESULTS AND DISCUSSION

 -It is necessary to explain how the different compounds in Table 1 and 2 were identified.

-In the chromatogram in Figure S1 and Table S2 14 compounds were identified, however in Table 1 more than 20 compounds were identified. How was this possible?

 

-The high standard deviation obtained for the quantification of the compounds in Table 1 needs to be explained.

 4 CONCLUSIONS

 It is necessary to summarise the conclusion by focusing only on the relevant progress achieved. 

Author Response

1.  Abstract. The novelty of the research work should be highlighted. We appreciate the reviewer’s valuable suggestion. The abstract has been revised to better emphasize the novelty and original contributions of our research work.

2. Supplementary data. Improve the quality of Figure S1 and make the identification of peaks clearer. An explanation is needed as to how peaks 1, 3 and mainly 8 were considered despite not achieving good peak separation. We sincerely appreciate the reviewer’s insightful comments and suggestions regarding the supplementary material. Concerning Figure S1, we included it as supplementary data primarily to provide additional transparency and support for our findings. Unfortunately, due to limitations inherent to the original experimental conditions, the quality of the chromatogram in Figure S1 cannot be substantially improved. If the reviewer considers that the current supplementary figure does not clearly support the manuscript, we are willing to remove it from the supplementary material. Regarding the identification of peaks 1, 3, and particularly peak 8 their identification was based on the retention times comparison with literature, spectral matching and UV-visible absorption characteristics and consistent peak behavior and elution order verified through repeated analyse. Thank you again for helping enhance the clarity and rigor of our work

3. Introduction. The introduction is generally well written, however more information is needed on ‘’Non-Coloured Flavonoids” and why these flavonoids are colourless. Thank you for your valuable suggestion. Additional information regarding 'Non-Coloured Flavonoids' and an explanation of why these flavonoids are colourless have now been included in the revised introduction.
4. Introduction. Tt is well known that flavanols are often present as part of oligomeric compounds, obviously coloured, and that this polymerisation reaction could be enhanced by the influence of pH or temperature, how could this be considered to maintain the viability of the developed models? It is indeed well-established that flavanols can participate in oligomeric or polymeric compounds, which are visibly colored. However, in this study, visible oligomeric compounds were not quantified. Instead, only non-colored flavanols, specifically catechin and epicatechin in their free monomeric forms, were analyzed. Coloured phenolics have been considered in our previous work cited in the scientific paper, even though polymeric pigments were not considered, as their separation by HPLC requires different working conditions. Although monomeric and oligomeric forms may exist in dynamic equilibria, these equilibria likely do not significantly impact the viability of the developed classification models. The only aspect potentially influenced could be wine aging, as long-term storage conditions might shift the equilibria toward oligomerization, potentially affecting the stability and concentration of the monomeric flavanol forms used in this study. Nevertheless, considering the robustness of the analytical approach and the specific focus on monomeric flavanol forms, to our mind the viability and applicability of the presented models remain valid.
5. Material & Methods. Retention times will depend on the analysis methodology used, so it is necessary to explain how this identification has been done. Additional standards and an HPLC-MS assay are required to confirm the identification of the peaks. A better explanation of the standards used and their purity is needed. A better explanation of how the identification of the compounds was performed is needed. We fully agree with the reviewer that an HPLC-MS assay would allow a more precise identification of chromatographic peaks than the analytical methodology employed in our study. Unfortunately, our laboratory currently lacks the necessary instrumentation for performing HPLC-MS analyses. Therefore, we relied on HPLC coupled with a Diode Array Detector (HPLC-DAD) to identify and quantify 14 non-colored flavonoids and other phenolic compounds previously reported in wine samples.

   It is important to emphasize that our study is not strictly analytical in orientation, but rather aims at providing a deeper understanding and characterization of Canary wines. While precise analytical identification is unquestionably valuable, our principal objective was to generate reliable data supporting the broader chemical characterization and appreciation of these unique wines. In this context, HPLC-DAD represents a robust and widely recognized analytical technique capable of producing trustworthy and reproducible results. Although we acknowledge that coupling with mass spectrometry would undoubtedly enhance the analytical precision, our methodology remains valid and rigorous, utilizing retention times and UV-visible spectra (200–700 nm) for peak identification. Whenever possible, direct comparison with available commercial standards was performed, including recovery assays in spiked red wine samples, consistently yielding recoveries above 90%. Compounds lacking commercial standards were identified through comparison with spectral and retention time data available in the scientific literature, demonstrating good agreement with previously reported findings.

   It is also important to highlight that our approach offers a rapid and cost-effective methodology for achieving accurate classification, despite the availability of technically more sophisticated analytical alternatives. Advanced detection systems such as HPLC-MS significantly increase operational costs and reduce the likelihood of their widespread implementation in routine wineries and enological laboratories. We have added all these considerations to the section 2 of the manuscript in order to clarify it also to the readers.

   In summary, while acknowledging the limitations highlighted by the reviewer, we reaffirm that our analytical approach is robust and appropriate for the intended purpose and scope of our research, which primarily seeks a better understanding of the distinctive properties of Canary wines rather than purely analytical validation.

6. Results & Discussion. It is necessary to explain how the different compounds in Table 1 and 2 were identified. In the chromatogram in Figure S1 and Table S2 14 compounds were identified, however in Table 1 more than 20 compounds were identified. How was this possible? The high standard deviation obtained for the quantification of the compounds in Table 1 needs to be explained. We appreciate the reviewer’s thoughtful comments. In response to the inquiry regarding the identification of compounds, we have added further details in the manuscript to clarify how the compounds listed in Tables 1 and 2 were identified. Specifically, the 14 compounds identified in Figure S1 and Table S2 were analytically determined, and the remaining flavonoid parameters included in Table 1 (more than 20 compounds) were obtained by calculation, based on those 14 analytically determined compounds. Regarding the high standard deviation observed for the quantification of the compounds in Table 1, we have also included a detailed explanation in the revised manuscript to address the potential sources of variability and to justify the observed results.

7. Conclusions.  It is necessary to summarise the conclusion by focusing only on the relevant progress achieved. Thank you for your suggestion. We have shortened the conclusion maintaining only on the most relevant considerations.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors report on the Non - Colored Flavonoids as Fingerprints to authenticate and characterize red wine from authenticate. The results have a guiding role in determining the authenticity of wine and helping consumers to make better choices. However, before this manuscript can be published, some improvements should be performed:

 

General remarks:

1. How do key markers specifically identify different samples? Taking the influence factor of Grape Cultivar as an example, the key markers screened out in the study are M3gl, Isor, Myri and Q3gl. Then whata trend do these substances present, the sample is classified as Listan Negro?
2. For the analysis in section 3.2, I don’t think correlation analysis is very relevant to the main research purpose of this study.

 

Minor

Line 16: “catechin and epicatechin” may be a more general expression.

Line 22: change to “denomination of origin (DO)”.

Line 298 and 345: wrong title number.

Line 491: 75.1%?

Line 768: This section is too long and is recommended to condense.

 

Comments for author File: Comments.pdf

Author Response

1. General Remark 1: How do key markers specifically identify different samples? Taking the influence factor of Grape Cultivar as an example, the key markers screened out in the study are M3gl, Isor, Myri and Q3gl. Then whata trend do these substances present, the sample is classified as Listan Negro? We thank the reviewer for this consideration. The compounds identified as critical for classification according to grape cultivar (M3gl, Isor, Myri, and Q3gl) exhibit distinctive trends. Specifically, for the Listán Negro samples, M3gl and Q3gl present relatively high mean concentrations compared to several other cultivars, whereas Isor and Myri show intermediate concentration levels. Such concentration patterns of these flavonoids collectively enable accurate differentiation and classification of Listán Negro samples. We have specifically added to our manuscript.
2. General remarks 2.  For the analysis in section 3.2, I don’t think correlation analysis is very relevant to the main research purpose of this study.  We would like to thank the reviewer for their thoughtful feedback. While we understand the concern regarding the relevance of the correlation analysis to the main research purpose of the study, we believe that it adds valuable insight into the relationships between the variables examined. To our mind the correlation analysis provides supporting evidence that enhances our understanding of the data, even though it is true that these relationships are not the primary focus of our study. Nevertheless, we really think this additional information contributes to a more comprehensive interpretation of the results.
3. Minor remarks. Line 16: “catechin and epicatechin” may be a more general expression. Line 22: change to “denomination of origin (DO)”. Line 298 and 345: wrong title number. Line 491: 75.1%? Line 768: This section is too long and is recommended to condense.  We would like to thank the reviewer for their helpful remarks. We have carefully addressed all of these suggested revisions

Reviewer 3 Report

Comments and Suggestions for Authors

A very interesting manuscript on the use of non-colored flavonoids as an indicator of wine origin, cultivar and aging. The manuscript is very carefully prepared, the materials and methods are suitable for such a study, the results are clearly presented and thoroughly discussed. Only a couple of minor suggestions can be offered:

1. catequine and epicatequine (l. 16) should read catechin and epicatechin (l. 112).
2. It would be easier to follow the text if the abbreviations were only kept in the Tables and Figures (along with the explanatory footnotes) but written in full in the text. Please consider this only as a suggestion.
3. Please add error bars and statistical analysis in figure 1
4. Please write scientific names in the references in italics (e.g. l. 846,847, 863 and throughout)

Author Response

1. catequine and epicatequine (l. 16) should read catechin and epicatechin (l. 112). Thank you very much, we have adressed it.
2. It would be easier to follow the text if the abbreviations were only kept in the Tables and Figures (along with the explanatory footnotes) but written in full in the text. We sincerely appreciate the reviewer’s suggestion regarding the use of abbreviations. However, we have opted to maintain the abbreviations in the text in order to preserve the consistency and structure of the manuscript. We believe this approach enhances the flow of the text while ensuring clarity. We hope this decision is acceptable.
3. Please add error bars and statistical analysis in figure 1. We really appreciate you comment that improve the version of our manuscript. We have improved figure 1 following your suggestions.
4. Please write scientific names in the references in italics. We have changed it, thank you very much.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1.  Abstract. The novelty of the research work should be highlighted. We appreciate the reviewer’s valuable suggestion. The abstract has been revised to better emphasize the novelty and original contributions of our research work.
2. Supplementary data. Improve the quality of Figure S1 and make the identification of peaks clearer. An explanation is needed as to how peaks 1, 3 and mainly 8 were considered despite not achieving good peak separation. 

We sincerely appreciate the reviewer’s insightful comments and suggestions regarding the supplementary material. Concerning Figure S1, we included it as supplementary data primarily to provide additional transparency and support for our findings. Unfortunately, due to limitations inherent to the original experimental conditions, the quality of the chromatogram in Figure S1 cannot be substantially improved. If the reviewer considers that the current supplementary figure does not clearly support the manuscript, we are willing to remove it from the supplementary material. Regarding the identification of peaks 1, 3, and particularly peak 8 their identification was based on the retention times comparison with literature, spectral matching and UV-visible absorption characteristics and consistent peak behavior and elution order verified through repeated analyse. Thank you again for helping enhance the clarity and rigor of our work

The quality of the figure is still unacceptable, it is impossible to read the values on the axes.

How was it possible to identify the retention time if the peaks are not well separated?

1. Introduction. The introduction is generally well written, however more information is needed on ‘’Non-Coloured Flavonoids” and why these flavonoids are colourless. Thank you for your valuable suggestion. Additional information regarding 'Non-Coloured Flavonoids' and an explanation of why these flavonoids are colourless have now been included in the revised introduction.

Its necessary to add a reference about the Non-coloured flavonoids explanation

 

1. Introduction. Tt is well known that flavanols are often present as part of oligomeric compounds, obviously coloured, and that this polymerisation reaction could be enhanced by the influence of pH or temperature, how could this be considered to maintain the viability of the developed models?

 

 It is indeed well-established that flavanols can participate in oligomeric or polymeric compounds, which are visibly colored. However, in this study, visible oligomeric compounds were not quantified. Instead, only non-colored flavanols, specifically catechin and epicatechin in their free monomeric forms, were analyzed. Coloured phenolics have been considered in our previous work cited in the scientific paper, even though polymeric pigments were not considered, as their separation by HPLC requires different working conditions. Although monomeric and oligomeric forms may exist in dynamic equilibria, these equilibria likely do not significantly impact the viability of the developed classification models. The only aspect potentially influenced could be wine aging, as long-term storage conditions might shift the equilibria toward oligomerization, potentially affecting the stability and concentration of the monomeric flavanol forms used in this study. Nevertheless, considering the robustness of the analytical approach and the specific focus on monomeric flavanol forms, to our mind the viability and applicability of the presented models remain valid.

OK

1. Material & Methods. Retention times will depend on the analysis methodology used, so it is necessary to explain how this identification has been done. Additional standards and an HPLC-MS assay are required to confirm the identification of the peaks. A better explanation of the standards used and their purity is needed. A better explanation of how the identification of the compounds was performed is needed.

 We fully agree with the reviewer that an HPLC-MS assay would allow a more precise identification of chromatographic peaks than the analytical methodology employed in our study. Unfortunately, our laboratory currently lacks the necessary instrumentation for performing HPLC-MS analyses. Therefore, we relied on HPLC coupled with a Diode Array Detector (HPLC-DAD) to identify and quantify 14 non-colored flavonoids and other phenolic compounds previously reported in wine samples.

It is important to emphasize that our study is not strictly analytical in orientation, but rather aims at providing a deeper understanding and characterization of Canary wines. While precise analytical identification is unquestionably valuable, our principal objective was to generate reliable data supporting the broader chemical characterization and appreciation of these unique wines. In this context, HPLC-DAD represents a robust and widely recognized analytical technique capable of producing trustworthy and reproducible results. Although we acknowledge that coupling with mass spectrometry would undoubtedly enhance the analytical precision, our methodology remains valid and rigorous, utilizing retention times and UV-visible spectra (200–700 nm) for peak identification. Whenever possible, direct comparison with available commercial standards was performed, including recovery assays in spiked red wine samples, consistently yielding recoveries above 90%. Compounds lacking commercial standards were identified through comparison with spectral and retention time data available in the scientific literature, demonstrating good agreement with previously reported findings.

It is also important to highlight that our approach offers a rapid and cost-effective methodology for achieving accurate classification, despite the availability of technically more sophisticated analytical alternatives. Advanced detection systems such as HPLC-MS significantly increase operational costs and reduce the likelihood of their widespread implementation in routine wineries and enological laboratories. We have added all these considerations to the section 2 of the manuscript in order to clarify it also to the readers.

In summary, while acknowledging the limitations highlighted by the reviewer, we reaffirm that our analytical approach is robust and appropriate for the intended purpose and scope of our research, which primarily seeks a better understanding of the distinctive properties of Canary wines rather than purely analytical validation.

 

It is necessary to reference the scientific literature used to identify all compounds, to really ensure that all compounds are well identified. Please add the theoretical reference times and the absorbance and wavenumber (UV-visible spectra (200-700 nm)) used to identify the compounds to really check that the analytical conditions were the same.

1. Results & Discussion. It is necessary to explain how the different compounds in Table 1 and 2 were identified. In the chromatogram in Figure S1 and Table S2 14 compounds were identified, however in Table 1 more than 20 compounds were identified. How was this possible? The high standard deviation obtained for the quantification of the compounds in Table 1 needs to be explained.

 We appreciate the reviewer’s thoughtful comments. In response to the inquiry regarding the identification of compounds, we have added further details in the manuscript to clarify how the compounds listed in Tables 1 and 2 were identified. Specifically, the 14 compounds identified in Figure S1 and Table S2 were analytically determined, and the remaining flavonoid parameters included in Table 1 (more than 20 compounds) were obtained by calculation, based on those 14 analytically determined compounds. Regarding the high standard deviation observed for the quantification of the compounds in Table 1, we have also included a detailed explanation in the revised manuscript to address the potential sources of variability and to justify the observed results.

It is necessary to explain how they were calculated the remaining flavonoid.

1. Conclusions.  It is necessary to summarise the conclusion by focusing only on the relevant progress achieved. 

Thank you for your suggestion. We have shortened the conclusion maintaining only on the most relevant considerations.

OK

Author Response

• Comment 1. The quality of the figure is still unacceptable, it is impossible to read the values on the axes.

We agree with the reviewer; the image quality of the figure is not good. So, upon careful consideration, we have decided to remove Figure S1, as we have not been able to significantly improve it. However, we have incorporated into Table S2 the employed wavelengths for detection and quantification.

Additionally, we have extended Table S2 including other valuable observations provided by the reviewer.

 

• Comment 2. How was it possible to identify the retention time if the peaks are not well separated?

We appreciate the reviewer’s comment and concern regarding the peak separation. The identification of peaks was accomplished following these steps:

1. Retention times were consistently assessed in a reliable manner comparing with data from literature. Wine samples were directly spiked with standards, when they were available, in order to confirm that the corresponding peaks were increased.
2. For each peak we analysed the UV-Vis absorption spectral in order to comparing the characteristic UV-Vis absorption of the wine samples with those characteristics of the corresponding standards.
3. Elution order of the peaks corresponding to flavonoid compounds were compared with elution order previously observed in data from scientific literature.

 

• Comment 3. It is necessary to add a reference about the non-coloured flavonoids explanation

A new reference relative to  non-colored flavonoids was added [reference 2].

 

• Comment 4. It is necessary to reference the scientific literature used to identify all compounds, to really ensure that all compounds are well identified. Please add the theoretical reference times and the absorbance and wavenumber (UV-visible spectra (200-700 nm)) used to identify the compounds to really check that the analytical conditions were the same.

We appreciate the reviewer’s comment. We have added the relevant scientific references used to identify all compounds directly in the Materials and Methods section of the manuscript and in Table S2. In addition, Table S2 has been extended to include the UV-visible characteristic absorption spectra (200-700 nm) used for compound identification.

We believe these additions provide clarity and strengthen the validity of our compound identification process.

 

• Comment 5. It is necessary to explain how they were calculated the remaining flavonoid.

The formulas used to calculate the remaining flavonoids are detailed in the Materials and Methods section, which were numbered as equations 1 to 7.

 

We sincerely appreciate the reviewer’s insightful comments, which have significantly improved our manuscript.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have clarified all the reviewers' questions. 

Author Response

We sincerely thank the reviewer for their thorough work, which has helped us improve the final outcome.