Occurrence and Exposure Assessment of Mycotoxins from Beers Commercially Traded in Brazil
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDetailed comments are included in the review file attached below.
The manuscript requires minor revision before publication according to all the suggested comments.
Comments for author File: 
Comments.pdf
Detailed comments are included in the review file attached below.
Author Response
Reviewer #1:
Detailed comments are included in the review file attached below.
The manuscript requires minor revision before publication according to all the suggested comments.
Summary of the presented manuscript and general concept comments:
This study examined the presence and concentration levels of mycotoxins in commercial beers available in Săo Paulo, Brazil, and evaluated the estimated daily intake (EDI) of those mycotoxins found in quantifiable amounts. The results of this study confirm the presence of mycotoxins in commercial Brazilian beers, underscoring that their occurrence is a real and ongoing concern, even if detected levels remain below established regulatory limits. The simultaneous presence of multiple mycotoxins highlights the need for continuous and rigorous monitoring of raw materials used in beer production and the final products. Moreover, the estimated daily intake (EDI) values indicate that, despite low concentrations, chronic exposure may still pose significant health risks and cannot be disregarded. The authors also emphasize the existing regulatory gap in their country, underscoring the urgent need for updated and comprehensive legislation to ensure that all relevant mycotoxins are effectively monitored and controlled in beer production.
The manuscript's scientific content is at an average level, as it contains market analyses of mycotoxin contamination of selected beers. Still, they were performed using modern, sensitive, and reliable instruments, namely the UPLC-MS/MS method. The main strength of the presented study is the method used for the mycotoxin analysis. Based on the reviewer's assessment of the study's overall merit and relevance, as well as the importance of the results and their potential applications, it can be predicted that this research will interest readers, especially those who consume this type of alcoholic beverages.
Answer: Thanks for the comments.
The reviewer believes the manuscript’s results are generally well presented. However, the discussion of the results should be extended to other research in this field. In this case, the selection of literature is also quite limited and, consequently, should be expanded.
Answer: We sincerely hope that all concerns regarding the discussion of the results and the selection of literature were addressed properly in the revised manuscript.
Detailed comments for the manuscript, which should be considered when reviewing the article, are provided below.
Specific comments:
- Line 24 - In Keywords, I recommend using UPLC-MS/MS to emphasize the method used for the qualitative-quantitative analysis.
Answer: Done (please see L.25).
- Lines 91-92 – It would be good to add the information on why the bottom of the column was removed and what it contained in this case.
Answer: The information requested was amended in the revised manuscript (please see L.85-86).
- Line 94 - Where did this amount of acetonitrile come from in which the extract was reconstituted? It can be explained. Was it exactly 340.9 µL?
Answer: Yes, it was exactly 340.9 µL. This volume was used to keep the proportion of the mixture (extract and acetonitrile) as recommended by manufacturer of MycoSpin™ 400 colums (Romer Labs, Getzersdorf, Austria).
- Lines 96 – 96 – The Authors wrote: ‘Subsequently, 40 μL of the sample and 10 μL of an internal standard (IS) solution containing [13C17]-AFB1, [13C20]-OTA, [13C34]-FB1, [13C15]–DON, [13C18]-ZEN and [13C24]–T2 in water/acetonitrile (1:1, v/v) were introduced into an insert placed in another 2 mL vial ‘ - Was this standard a purchased ready-made mixture of selected mycotoxin standards? What was the concentration level of individual mycotoxins in 10 μl of this IS solution? I suggest specifying this in the sentence mentioned above or in another sentence.
Answer: The information requested was included in the revised manuscript, please see L91-93.
- Line 103 – UPLC should replace the abbreviation LC.
Answer: Done (please see L.96).
- Lines 105-107 - Capillary tensions, source and desolvation temperatures, and gas flow should be mentioned here. Providing them here rather than referring to another article would be better. These are just a few key data that, when given below the instrument description, provide a simpler basis for other researchers to reproduce these conditions.
Answer: The required information was amended in the revied manuscript (please see L.107-11).
- Lines 110-112 - Please explain in more detail how this residual plot was made and for what purpose.
Answer: The residual plot was constructed by the software (MassLynx software version 4.1), as mentioned in the manuscript, and its purpose was amended in the revised manuscript (please see L.117-118).
- Table 1 – below line 160 – there is a typo in the word mycotoxin –‘micotoxin’
Answer: Corrected (please see L.162).
- Table 2 – for DON, the mean content and concentration range (ng/mL) are shifted to 2 rows, which looks confusing.
Answer: Corrected.
- Table 3 - The legend under Table 3 (line 224) contains an error in the reference to Table 3 when it should be to Table 1 because the LOQs for each mycotoxin are given there.
Answer: Corrected (please see L.217).
- Lines 237-238 – The Authors wrote: ‘The EDI of mycotoxins ingested in contaminated beers by the Brazilian population varied between minimum and maximum values.’ This sentence should be deleted as it does not add anythingbeyond what is already known, since the lowest and highest mycotoxin levels found in the studies were used for the EDI calculation.
Answer: This sentence was excluded, as suggested.
- Lines 243-244 - Please provide brief information on why slightly higher EDI values were found for women.
Answer: This was possibly due to the relatively lower women’s mean body weight, as indicated in the revised manuscript (please see L.134-135).
- The discussion of the results obtained in the work is based on their comparison with only 3 scientific works in this field, which are literature items 16-18. For this reason, there is a need to extend this discussion to other data on the levels of mycotoxins in beers from different regions of the world. In this case, the list of references will be extended with new original scientific articles in this field, since the selection of literature is limited. Moreover, in this version of the manuscript, many items from references are strictly concerned with legislative requirements, standards, and statistical data regarding consumer consumption.
Answer: The Discussion was much improved, as requested (please see L.237-244, L.247-266, 276-306, and 314-331), and the list of references was extended accordingly.
Level of English:
-Some linguistic errors, even including grammatical errors, were made in the manuscript, e.g., lines 44-45: ‘…Brazilian regulations does not establish specific limits...’ or line 237 – ‘mycotoxins ingested in contaminated beers’. Therefore, the manuscript requires linguistic improvement in terms of grammar, language fluency, and clarity
Answer: The English was thoroughly revised in the new version of the manuscript manuscript.
Reviewer 2 Report
Comments and Suggestions for Authors- The main objective of the study was to detect the presence of mycotoxins in beers sold in the state of São Paulo, Brazil.
- The topic of the research undertaken by the authors is interesting. The authors used 60 beer samples purchased in cities from the state of São Paulo, Brazil. They assessed the presence of 10 mycotoxins in commercial beers and assessed the health risk associated with exposure to mycotoxins through beer consumption.
- The manuscript brings new knowledge to the subject compared to other published articles by comparing the content of 10 mycotoxins in beers produced from different raw materials: barley malt, malt and corn, and malt and rice and sold in Brazil.
- Note in the methods section:
- Lines 61-69, section 2.1. Sampling procedures: Missing information: Were all beers produced in Brazil? How many samples of beer were produced in Brazil in the state of São Paulo, and how many were imported from other countries? Were the beers used in the study pasteurized or unpasteurized?
- Note in the research results section:
- Lines 168-169 and 179-180: The authors did not intend to identify a city that sells beers with specific mycotoxins in their research. It is possible that it is not necessary to mention the cities in these sentences.
- The discussion is complete and properly discussed with the results of other authors.
- The conclusion is complete. In the summary, the authors indicated the strengths and weaknesses of the conducted study.
- References are adequate. 25 articles were used, 68% of which are from the last 10 years and 32% from last 5 years.
- Tables are properly prepared and legible.
Author Response
Reviewer #2:
- The main objective of the study was to detect the presence of mycotoxins in beers sold in the state of São Paulo, Brazil.
- The topic of the research undertaken by the authors is interesting. The authors used 60 beer samples purchased in cities from the state of São Paulo, Brazil. They assessed the presence of 10 mycotoxins in commercial beers and assessed the health risk associated with exposure to mycotoxins through beer consumption.
- The manuscript brings new knowledge to the subject compared to other published articles by comparing the content of 10 mycotoxins in beers produced from different raw materials: barley malt, malt and corn, and malt and rice and sold in Brazil.
Answer: Thanks for the comments.
- Note in the methods section:
Lines 61-69, section 2.1. Sampling procedures: Missing information: Were all beers produced in Brazil? How many samples of beer were produced in Brazil in the state of São Paulo, and how many were imported from other countries? Were the beers used in the study pasteurized or unpasteurized?
Answer: All beers evaluated in this study were manufactured and pasteurized by companies located in the state of São Paulo, as amended in the revised manuscript (please see L.63-64).
- Note in the research results section:
Lines 168-169 and 179-180: The authors did not intend to identify a city that sells beers with specific mycotoxins in their research. It is possible that it is not necessary to mention the cities in these sentences.
Answer: We have excluded the identification of cities where beers samples were collected, as suggested.
- The discussion is complete and properly discussed with the results of other authors.
- The conclusion is complete. In the summary, the authors indicated the strengths and weaknesses of the conducted study.
- References are adequate. 25 articles were used, 68% of which are from the last 10 years and 32% from last 5 years.
- Tables are properly prepared and legible.
Answer: Thanks for the comments.
Reviewer 3 Report
Comments and Suggestions for Authors1. Compared with general survey and analysis research, this study is extremely important. Unfortunately, this study failed to show whether the presence of mycotoxins in the body with alcohol has a multiplying or reducing effect on the health risks to consumers compared to the risk of mycotoxins alone in food. The author is advised to consider combining scientific data for discussion and comparison.
2. (Lines 286–289) Another shortcoming of this study is that the authors themselves point out that the sample source limitations and seasonal changes were not included in the analysis. The authors should describe the impact of source limitations and seasonal changes on this study. And explain the impact of this limitation and why there was no plan to resolve the problem of sample source and season at the beginning of the study.
3. (Lines 281–282) mention the health risks of long-term low-dose exposure, but this study did not simulate long-term cumulative effects, and the authors fail to explain how this study further simulated long-term exposure to quantify the risks. It is suggested that the author add this description in the text.
4. (Lines 125–127) Consumption data and average weight and alcohol consumption for Brazil were used to estimate the EDI, but the authors did not include variations or differences in drinking behavior by age group or genders in the calculations.
5. In the table headers of lines 233–234, please correct Table 1 to Table 4.
6. As shown in Table 3 and lines 211–214, DON and FB1 co-occurred in 20% of the samples. The authors are requested to further evaluate their possible synergistic toxic effects rather than conduct risk assessment based on a single TDI.
7. (Lines 63–69) The sample was concentrated in three cities in São Paulo State. Can this sample size and geographic scope represent the national beer contamination trend? Are there plans to expand the study to improve national representativeness? In addition (lines 179–188), the authors state that the FB1 detection rate is 100%. What is the reason for this high detection rate? Could it be due to the fact that the sample sources are concentrated in a specific region? Are there plans to expand the sample source to improve research representativeness? It is recommended that the author address this issue in the discussion section.
8. The results in Table 1 show that the RSDs of AFG1 and ZEN are 27.8% and 26.6%, respectively, which are higher than those of other toxins. Does this mean that the quantitative accuracy of the toxin is low? Is this uncertainty considered when assessing risk? Authors should first provide a statistical explanation of the variance or impact of the data in the RSD column of Table 1 (lines 160–164).
9. For the target mycotoxins in this study, sample preparation included drying and re-dissolution during analysis. Could such steps result in the loss of some easily degradable or volatile mycotoxins? (Lines 93–94) It can be said that OTA seems to be stable up to 180 °C; however aflatoxin B1 was almost completely degraded at heating temperatures of 160 °C and above. So please briefly explain the temperature control part in the text.
10. In order to improve research insights and results, it is recommended that the author discuss the importance of selecting specific brands of materials. Avoid trapping your hard research into a specific framework. In lines 88–90, the authors used a custom-made MycoSpin™ 400 column for sample cleanup. Please explain in the text whether this adsorption column has the same recovery efficiency for each mycotoxin. Are there any specific mycotoxins whose recovery rates are low and thus affect the overall test results? If there are similar products from different brands that can be used, please state this in the discussion section of this article.
11. The analytical method used by the author is "in-house validated method" (line 15). Please explain whether this method has been validated before this study? If so, please explain its source and add them in reference section. (line 78-79) If analytical validation was part of the study, authors should state the validation criteria used to validate the analytical method. And state whether this analytical method has been cross-validated with other international standard methods (such as AOAC, ISO) to enhance its reliability and comparability?
Author Response
Reviewer #3:
- Compared with general survey and analysis research, this study is extremely important. Unfortunately, this study failed to show whether the presence of mycotoxins in the body with alcohol has a multiplying or reducing effect on the health risks to consumers compared to the risk of mycotoxins alone in food. The author is advised to consider combining scientific data for discussion and comparison.
Answer: This statement “Unfortunately, this study failed to show whether the presence of mycotoxins in the body with alcohol has a multiplying or reducing effect on the health risks to consumers compared to the risk of mycotoxins alone in food” cannot be accepted, because this was not the focus of the study. Our study aimed to determine multiple mycotoxins in commercially available beers in Brazil and assess their dietary exposure through the consumption of Brazilian beers, not to identify the effect of mycotoxins and alcohol combinations on the human health.
- (Lines 286–289) Another shortcoming of this study is that the authors themselves point out that the sample source limitations and seasonal changes were not included in the analysis. The authors should describe the impact of source limitations and seasonal changes on this study. And explain the impact of this limitation and why there was no plan to resolve the problem of sample source and season at the beginning of the study.
Answer: The limitations of the study and their impacts were improved (please see L.318-323).
- (Lines 281–282) mention the health risks of long-term low-dose exposure, but this study did not simulate long-term cumulative effects, and the authors fail to explain how this study further simulated long-term exposure to quantify the risks. It is suggested that the author add this description in the text.
Answer: This sentence excluded in the revised manuscript, to avoid misinterpretation.
- (Lines 125–127) Consumption data and average weight and alcohol consumption for Brazil were used to estimate the EDI, but the authors did not include variations or differences in drinking behavior by age group or genders in the calculations.
Answer: While we acknowledge that drinking behavior may vary by age group and gender, detailed and updated public data on beer consumption specifically, stratified by these demographic variables, are not available for Brazil.
- In the table headers of lines 233–234, please correct Table 1 to Table 4.
Answer: Done (please see L.225).
- As shown in Table 3 and lines 211–214, DON and FB1 co-occurred in 20% of the samples. The authors are requested to further evaluate their possible synergistic toxic effects rather than conduct risk assessment based on a single TDI.
Answer: This issue was addressed in the Discussion section of the revised manuscript, as requested (please see L.286-306).
- (Lines 63–69) The sample was concentrated in three cities in São Paulo State. Can this sample size and geographic scope represent the national beer contamination trend? Are there plans to expand the study to improve national representativeness? In addition (lines 179–188), the authors state that the FB1 detection rate is 100%. What is the reason for this high detection rate? Could it be due to the fact that the sample sources are concentrated in a specific region? Are there plans to expand the sample source to improve research representativeness? It is recommended that the author address this issue in the discussion section.
Answer: Thanks for pointing out these questions. Based on the data presented in the manuscript, which described the occurrence of mycotoxins in beers commercially available in the state of São Paulo, it’s not possible to confirm if they represent a national beer contamination trend. So, of course further studies are needed for including samples from different states and regions to assess national representativeness and to identify possible regional variations in the occurrence of mycotoxins in beers in Brazil, as amended in the revised manuscript (please see L.323-331). The 100% detection of FB1 observed in our study may be explained by the widespread use of corn as an adjunct in beer production in São Paulo, as well as the region’s climatic conditions, which favor contamination by Fusarium fungi. This information was included in the revised manuscript (please see L.47-249).
- The results in Table 1 show that the RSDs of AFG1 and ZEN are 27.8% and 26.6%, respectively, which are higher than those of other toxins. Does this mean that the quantitative accuracy of the toxin is low? Is this uncertainty considered when assessing risk? Authors should first provide a statistical explanation of the variance or impact of the data in the RSD column of Table 1 (lines 160–164).
Answer: The RSD values observed for ZEN (26.6%) and AFG₁ (27.8%) may be associated with variations in the adsorption ability of these compounds by MycoSpin™ 400 columns during clean-up procedures of beer samples, or losses during other steps of sample preparation. In our study, there is no statistical inference that could be used to explain this variance. Although these values were higher than those obtained for the other mycotoxins, all the obtained RSD values were within the acceptable values according to the Eurachem Guide for compounds analyzed in complex matrices at parts per billion (ppb) levels. This information was amended in the revised manuscript, for clarity (please see L.242-244). In addition, this variability was considered when evaluating the risk in the present study, but the exposure estimates remained well below established TDI values.
- For the target mycotoxins in this study, sample preparation included drying and re-dissolution during analysis. Could such steps result in the loss of some easily degradable or volatile mycotoxins? (Lines 93–94) It can be said that OTA seems to be stable up to 180 °C; however aflatoxin B1 was almost completely degraded at heating temperatures of 160 °C and above. So please briefly explain the temperature control part in the text.
Answer: Yes, as mentioned in our response to your previous comment no. 8 (above), extraction and clean-up procedures can cause losses of the analyte in a sample. That’s why we evaluated the performance of the analytical method, and the results clearly indicated that all the obtained RSD and recovery values were within the acceptable values according to the Eurachem Guide for compounds analyzed in complex matrices at parts per billion (ppb) levels. As the aflatoxins are well known for their strong stability and high heat resistance, we cannot corroborate your comment “however aflatoxin B1 was almost completely degraded at heating temperatures of 160 °C and above”, unless an appropriate reference is provided for this statement.
- In order to improve research insights and results, it is recommended that the author discuss the importance of selecting specific brands of materials. Avoid trapping your hard research into a specific framework. In lines 88–90, the authors used a custom-made MycoSpin™ 400 column for sample cleanup. Please explain in the text whether this adsorption column has the same recovery efficiency for each mycotoxin. Are there any specific mycotoxins whose recovery rates are low and thus affect the overall test results? If there are similar products from different brands that can be used, please state this in the discussion section of this article.
Answer: In fact, the column used for clean-up (MycoSpin™ 400) was not custom-made, it’s a commercially available product for mycotoxin analysis of foods – among many other types of columns intended for mycotoxin analysis. It’s not possible to explain whether the adsorption column has the same recovery efficiency for each mycotoxin, neither the recovery rates for each mycotoxin eluted in this column, because this was not accessed in the study. The experimental design was focused on the occurrence of mycotoxins in beers commercially available in the state of São Paulo, not on specific attributes of the analytical method.
- The analytical method used by the author is "in-house validated method" (line 15). Please explain whether this method has been validated before this study? If so, please explain its source and add them in reference section. (line 78-79) If analytical validation was part of the study, authors should state the validation criteria used to validate the analytical method. And state whether this analytical method has been cross-validated with other international standard methods (such as AOAC, ISO) to enhance its reliability and comparability?
Answer: The analytical method was not fully validated in this study, only its performance parameters were evaluated. This section was modified in the revised manuscript, to clearly describe the analytical procedures conducted in the experiment (please see L.73-78).
Reviewer 4 Report
Comments and Suggestions for AuthorsReview of the manuscript entitled "Occurrence and Exposure Assessment of Mycotoxins in Beers Commercially Traded in Brazil".
The authors present a LC-IDMS method for the analysis of selected mycotoxins in beer. The method is based on a fast sample purification, and subsequently, isotope dilution and LC-MS/MS separation. The results are reliable and strengthened by an isotope dilution approach that is not commonly used unfortunately. The manuscript is in the scope of the journal, it provides important data for future risk assessment on mycotoxins.
Real sample analysis and result comparison to other data are presented. I did not find any major issues to be addressed, however the comments below need to be clarified.
My comments:
I did not find the description of HPLC separation.
Why did the authors not analyse the Alternaria toxins that are also important in beer. I suggest to cite these papers and mention that a future aspect could be to extend the method for other toxins, e.g. Alternarias.
- Quantification of the Alternaria mycotoxin tenuazonic acid in beer, Food Chemistry, Volume 120, Issue 3, 1 June 2010, Pages 902-906
- A Dilute and Shoot Strategy for Determining Alternaria Toxins in Tomato-Based Samples and in Different Flours Using LC-IDMS Separation, Molecules 2021, 26(4), 1017; https://doi.org/10.3390/molecules26041017
Author Response
Reviewer # 4:
Review of the manuscript entitled "Occurrence and Exposure Assessment of Mycotoxins in Beers Commercially Traded in Brazil".
The authors present a LC-IDMS method for the analysis of selected mycotoxins in beer. The method is based on a fast sample purification, and subsequently, isotope dilution and LC-MS/MS separation. The results are reliable and strengthened by an isotope dilution approach that is not commonly used unfortunately. The manuscript is in the scope of the journal, it provides important data for future risk assessment on mycotoxins.
Real sample analysis and result comparison to other data are presented. I did not find any major issues to be addressed, however the comments below need to be clarified.
Answer: Thanks for the comments.
My comments:
I did not find the description of HPLC separation.
Answer: The chromatographic run conditions was included in the revised manuscript (please see L.98-105).
Why did the authors not analyse the Alternaria toxins that are also important in beer. I suggest to cite these papers and mention that a future aspect could be to extend the method for other toxins, e.g. Alternarias.
Quantification of the Alternaria mycotoxin tenuazonic acid in beer, Food Chemistry, Volume 120, Issue 3, 1 June 2010, Pages 902-906
A Dilute and Shoot Strategy for Determining Alternaria Toxins in Tomato-Based Samples and in Different Flours Using LC-IDMS Separation, Molecules 2021, 26(4), 1017; https://doi.org/10.3390/molecules26041017
Answer: Yes, including Alternaria’s mycotoxins would substantially improve the experiment. However, there were no available standards including isotopic standards of alternariol or tenuazoic acid in the laboratory when the analyses were conducted. We have included the suggested articles in the revised manuscript, also addressing the importance of future studies on the occurrence of these emerging mycotoxins in Brazilian beers (please see L.325-328).
Round 2
Reviewer 3 Report
Comments and Suggestions for Authors- The title of this study, "Occurrence and Exposure Assessment of Mycotoxins in Beers Commercially Traded in Brazil", implies that the assessment includes mycotoxins in Beers. But what the author wants to express and show is the mycotoxins brought by beer, not the beer itself. Therefore, it is recommended to revise it to "Occurrence and Exposure Assessment of Mycotoxins from Beers Commercially Traded in Brazil", which means that the assessment is for mycotoxins from Beers. I am not an expert in English writing and grammar, and my suggestions are only for authors to think about and refer to in order to avoid misunderstandings among readers.
- As mentioned earlier, this research is of considerable value, and the data provided are therefore critical and important for future references and even official management. Therefore, it is recommended to make some adjustments in the presentation of the analysis method. Line 157-160 has clearly presented the recovery and precision of the method, but if you want to emphasize the international nature and effectiveness of this study, you can consider adding the following elements: First, comparison with international standards. Mentioning which international standards or guidelines this method complies with, such as relevant regulations of ISO, AOAC or USFDA, can enhance its authority. Point 2, comparison with other methods, if there is data showing that this method is more accurate or sensitive than existing analytical techniques, these comparison results can be supplemented. Point 3, scope of application and practical verification, emphasizes that this method has been successfully verified in beer samples from different countries or different brands, proving its wide applicability. Point 4, If the method has been published in an international journal, evaluated by experts, or certified, this information can be included to enhance its credibility. At the same time, Line 76 also specifies with some modifications. Although there is no complete full set of method validation/verification information, it is recommended to briefly explain the modifications to reduce the impact of the results and improve the efficiency in order to complete the robustness of this study's analysis method.
Author Response
Answers to Reviewer #3:
- The title of this study, "Occurrence and Exposure Assessment of Mycotoxins in Beers Commercially Traded in Brazil", implies that the assessment includes mycotoxins in Beers. But what the author wants to express and show is the mycotoxins brought by beer, not the beer itself. Therefore, it is recommended to revise it to "Occurrence and Exposure Assessment of Mycotoxins from Beers Commercially Traded in Brazil", which means that the assessment is for mycotoxins from Beers. I am not an expert in English writing and grammar, and my suggestions are only for authors to think about and refer to in order to avoid misunderstandings among readers.
Answer: Done.
- As mentioned earlier, this research is of considerable value, and the data provided are therefore critical and important for future references and even official management. Therefore, it is recommended to make some adjustments in the presentation of the analysis method. Line 157-160 has clearly presented the recovery and precision of the method, but if you want to emphasize the international nature and effectiveness of this study, you can consider adding the following elements: First, comparison with international standards. Mentioning which international standards or guidelines this method complies with, such as relevant regulations of ISO, AOAC or USFDA, can enhance its authority. Point 2, comparison with other methods, if there is data showing that this method is more accurate or sensitive than existing analytical techniques, these comparison results can be supplemented. Point 3, scope of application and practical verification, emphasizes that this method has been successfully verified in beer samples from different countries or different brands, proving its wide applicability. Point 4, If the method has been published in an international journal, evaluated by experts, or certified, this information can be included to enhance its credibility. At the same time, Line 76 also specifies with some modifications. Although there is no complete full set of method validation/verification information, it is recommended to briefly explain the modifications to reduce the impact of the results and improve the efficiency in order to complete the robustness of this study's analysis method.
Answer: Point 1, comparison with internationals standards: We have already made such comparison for the obtained RSD values, which were within the acceptable values according to the Eurachem Guide for compounds analyzed in complex matrices at parts per billion (ppb) levels (please see L.242-244). In fact, Eurachem is a network of organizations in Europe having the objective of establishing a system for the international traceability of chemical measurements and the promotion of good quality practices. Therefore, Eurachem is an international entity that cannot be ignored as an authority in the field of Analytical Chemistry. For more information, please check its website at https://www.eurachem.org/. Regarding Point 2, comparison of the method used in the experiment with other analytical methods is not feasible, as the experimental design was focused on the occurrence of mycotoxins in beers commercially available in the state of São Paulo, not on specific attributes of the analytical method that could be comparable with others. Thanks for the suggestion made in Point 3: We intend to apply the analytical method in future studies on the occurrence of mycotoxins in beer samples from different countries and different brands available in Brazil. As for Point 4, the answer is no: The method has not been previously published in an international journal, neither evaluated by experts or certified, other than in the present manuscript submitted to Beverages. Finally, the modifications done in the extraction procedures are already described in L.76-82.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper is ready for publication.
Author Response
Thanks for the comments.
