ELISA and UPLC/FLD as Screening and Confirmatory Techniques for T-2/HT-2 Mycotoxin Determination in Cereals

Round 1

Reviewer 1 Report

The study presented in this paper addressed the screening and confirmatory methods for the detection of toxic T-2 and HT-2 toxins which could adversely affects human and animal health. Authors used ELISA as the screening while UPLC/FLD as the confirmatory approach for the detection of these toxins in cereal samples. The use of ELISA as a screening method reduces cost and analysis time while UPLC/FLD as a confirmatory approach provides a high-resolution column with better sensitivity and validation. Authors found that both these methods met the performance parameters including selectivity, precision, sensitivity, linearity, and ruggedness. Furthermore, they recommended that these two techniques should be considered as complementary techniques for T-2 and HT-2 detection in cereals.

 

I have few suggestions:

• Paragraph 2.3.2 Immunoassay procedure, line 95-96 “50 µL of 96 standard/sample, of enzyme conjugate and antibody were added into the wells.” I would suggest double checking the accuracy of the method described in this sentence. Does the author mean - 50 µL of standard/sample, T-2 toxin enzyme conjugate and anti-T2/HT-2 toxin antibodies were added into the wells??

 

• Line 18 and line 53, be consistent on how you use the full form of ELISA -Enzyme linked immunosorbent assay, i.e sorbent is missing on line 18.

 

• Fig 1D – Fig 1D shows the corn sample naturally contaminated with HT-2 toxins of 315.9 µg kg-1 (as described in the Fig legend)  also shows the presence of T-2-toxin at the retention time ~2 min. Because the y-axis goes to only 600, the peak of T-2 toxin is cut off. I would suggest increasing the y-axis such that the peak of T-2 toxin is shown and/or maybe put as an inset?? Also, I would suggest discussing Fig1D within the body of the text.

 

• Be consistent on using HT2 or HT-2 throughout the paper.

 

 

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 1

The study presented in this paper addressed the screening and confirmatory methods for the detection of toxic T-2 and HT-2 toxins which could adversely affects human and animal health. Authors used ELISA as the screening while UPLC/FLD as the confirmatory approach for the detection of these toxins in cereal samples. The use of ELISA as a screening method reduces cost and analysis time while UPLC/FLD as a confirmatory approach provides a high-resolution column with better sensitivity and validation. Authors found that both these methods met the performance parameters including selectivity, precision, sensitivity, linearity, and ruggedness. Furthermore, they recommended that these two techniques should be considered as complementary techniques for T-2 and HT-2 detection in cereals.

I have few suggestions:

    Paragraph 2.3.2 Immunoassay procedure, line 95-96 “50 µL of 96 standard/sample, of enzyme conjugate and antibody were added into the wells.” I would suggest double checking the accuracy of the method described in this sentence. Does the author mean - 50 µL of standard/sample, T-2 toxin enzyme conjugate and anti-T2/HT-2 toxin antibodies were added into the wells??

Response: The sentence was modified, according to the referee’s comment (lines 100-102).

    Line 18 and line 53, be consistent on how you use the full form of ELISA -Enzyme linked immunosorbent assay, i.e sorbent is missing on line 18.

Response: Following the reviewer’s suggestion, the definition has been corrected at lines 19 and 90.

    Fig 1D – Fig 1D shows the corn sample naturally contaminated with HT-2 toxins of 315.9 µg kg^-1 (as described in the Fig legend)  also shows the presence of T-2-toxin at the retention time ~2 min. Because the y-axis goes to only 600, the peak of T-2 toxin is cut off. I would suggest increasing the y-axis such that the peak of T-2 toxin is shown and/or maybe put as an inset?? Also, I would suggest discussing Fig1D within the body of the text.

Response: The peak obtained at 2.083 min, out of scale, is not related to T-2 toxin, since it is out of the time-window of interest, corresponding to 1.912 ± 2.5% (as specified at line 236). Other retention time repeatability tests, carried out by injecting both the T-2 toxin standard solution and spiked samples, gave a good value of precision, obviously higher for the first (1.912 ± 0.002, n=10) in respect to the second (1.925 ± 0.01, n=10) due to the matrix effect. Thus, the peak out of scale is an interfering compound, so it was not considered during figure elaboration. Moreover, following the reviewer’s suggestion, a brief comment about Figure 1D has been added in the text (lines 364-367).

  Be consistent on using HT2 or HT-2 throughout the paper.

Response: The toxin acronyms have been standardized throughout the Paper. Corrections made at lines 83, 158, 223, 233 and 351.

Author Response File: Author Response.doc

Reviewer 2 Report

The manuscript submitted by D’Agnello and colleagues questions the comparability of two analytical approaches to determine the concentration of T-2/HT-2 trichothecenes in cereals in a low to medium throughput laboratory setting. In fact, this article describes and justifies the steps undertaken to implement a new analytical procedure in an analytical laboratory.

T-2/HT-2 trichothecene mycotoxins can occur in many cereals, and are particularly abundant in oats. Suspected cancerogenic and other chronic consequences in humans and animals, T-2/HT-2 mycotoxins are a major concern in food and feed safety and for regulators worldwide.

Today, we dispose of a series of chemical analytical methods to detect and quantify these mycotoxins in cereal matrices. Yet, unlike many other mycotoxins, the EU has not yet established limit values of T-2/HT-2 mycotoxins in unprocessed and processed food and feed. However, the regulation is expected in the near future. Meanwhile, the EU commission recommends to collect information of occurrence and severity of T-2/HT-2 in the member states.

In the present manuscript, the authors test and compare a classical Uplc/Fld analytical method with a much faster and simpler antibody-based ELISA method. Arguably, the ELISA method would simplify and speed up the large-scale screening of cereal samples, with the option to use the more precise Uplc/Fld on samples with unclear outcome.

 

Overall, the manuscript has been written in a clear English. A series of minor to medium errors in spelling and wording shall be corrected by a proficient English writer.

 

The objective of the work is clearly stated in the introduction.

 

Unfortunately, the content of the manuscript does not allow me to recommend its publication.

• What is the originality of the article?
  • There is no new method, but a comparison of methods.

Certainly, this journal invites articles that implement new applications in sciences. Yet, the authors have to clearly state and justify that this contribution is new, original and can be implemented generally. What is its use once it is implemented. who shall use it then?

 

• The origin and the type of cereal samples remain obscure as well as the effective analytical results.

 

More details are needed to understand the topic. It is not satisfactory when a large part of the samples are below the detection limit. Please provide data of samples from other regions with a higher incidence of contaminated samples. Arguably, 100 samples have been analysed, but only a part of them can finally be used in this study.

• There are 4different cereal matrices, but the extraction method seems to be the same. This topic is not discussed. 

Please consider that oats contain more fat than durum wheat or maize. It is well known that fats, anthocyanins, soluble and non-soluble fibres as well as other grain constituents may interfere with the extraction of myctoxins.

• Is it correct to simply rely on “manufacturers recommendations” instead of improving the extraction methods for ELISA

Composition of the manuscript

• A large part of the RESULTS are Materials and Methods.
• There is no DISCUSSION but everything is somehow stuffed in the RESULTS and culminates finally in a CONCLUSION, that is not clear and, I daresay, superficial.
• Authors cite insufficiently and hardly compare their findings with other work of other authors. Also, the cited work is quite old (most recent work is from 2008).

A lot of articles on T-2/HT-2 analytics has been published in recent years. In a new version, this work has to be intectated.

To my esteem, the article has to be re-organised and completed to be scientifically acceptable way. The proof of originality must be provided.

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 2

The manuscript submitted by D’Agnello and colleagues questions the comparability of two analytical approaches to determine the concentration of T-2/HT-2 trichothecenes in cereals in a low to medium throughput laboratory setting. In fact, this article describes and justifies the steps undertaken to implement a new analytical procedure in an analytical laboratory.

T-2/HT-2 trichothecene mycotoxins can occur in many cereals, and are particularly abundant in oats. Suspected cancerogenic and other chronic consequences in humans and animals, T-2/HT-2 mycotoxins are a major concern in food and feed safety and for regulators worldwide.

Today, we dispose of a series of chemical analytical methods to detect and quantify these mycotoxins in cereal matrices. Yet, unlike many other mycotoxins, the EU has not yet established limit values of T-2/HT-2 mycotoxins in unprocessed and processed food and feed. However, the regulation is expected in the near future. Meanwhile, the EU commission recommends to collect information of occurrence and severity of T-2/HT-2 in the member states.

In the present manuscript, the authors test and compare a classical Uplc/Fld analytical method with a much faster and simpler antibody-based ELISA method. Arguably, the ELISA method would simplify and speed up the large-scale screening of cereal samples, with the option to use the more precise Uplc/Fld on samples with unclear outcome.

Overall, the manuscript has been written in a clear English. A series of minor to medium errors in spelling and wording shall be corrected by a proficient English writer.

Response: Following the referee’s remark, the manuscript has been checked and grammar typos have been corrected. The spell-check of the main text was also performed.

 

The objective of the work is clearly stated in the introduction.

Unfortunately, the content of the manuscript does not allow me to recommend its publication.

    What is the originality of the article?

        There is no new method, but a comparison of methods.

Certainly, this journal invites articles that implement new applications in sciences. Yet, the authors have to clearly state and justify that this contribution is new, original and can be implemented generally. What is its use once it is implemented. who shall use it then?

Response: The originality of this MS can be resumed in two main points, both interesting especially for  personnel involved in food safety official control activity.

The first is the implementation of an effective laboratory approach which allows the execution of large amounts of analysis in brief time, maintaining high level of reliability. This approach, developed by describing how the validation parameters should be calculated and evaluated, can be applied to most of food contaminants, other than T-2 and HT-2 toxins, for which both ELISA and chromatographic approach are available.

The second is the “point-to-point” comparison among different analytical approaches available for the same purpose. These types of studies results very useful, especially for laboratory managers, since they report an essential report of “pros and cons” of different approaches, to take into account when the laboratory has to implement its own protocol of analysis. Usually, this choice is based on specific need, expertise, instrument availability, economic capabilities of laboratory, so, these studies may be considered as particularly useful, since they resume all these aspects.

 

More details are needed to understand the topic. It is not satisfactory when a large part of the samples are below the detection limit. Please provide data of samples from other regions with a higher incidence of contaminated samples. Arguably, 100 samples have been analysed, but only a part of them can finally be used in this study.

Response: According to the reviewer’s suggestion, results and discussion has been improved. A new section related to the results obtained from the monitoring has been added. In this section, the results have been evaluated, commented and compared to other similar studies developed in Italy (lines 373-389). Moreover, two new references have been added.

  

 There are 4 different cereal matrices, but the extraction method seems to be the same. This topic is not discussed. Please consider that oats contain more fat than durum wheat or maize. It is well known that fats, anthocyanins, soluble and non-soluble fibres as well as other grain constituents may interfere with the extraction of myctoxins. Is it correct to simply rely on “manufacturers recommendations” instead of improving the extraction methods for ELISA

Response: Following the referee’s suggestion, a comment about the versatility of extraction procedure has been added at lines 174-178.

 

Composition of the manuscript

    A large part of the RESULTS are Materials and Methods.

Response: Results and discussion section reports brief indications about the methods used for evaluating each validation parameter. These parts have been reduced to essential, but the authors think that these sentences are useful for the reader to simplify understanding of experimental design and appraising of most significant results.

   

There is no DISCUSSION but everything is somehow stuffed in the RESULTS and culminates finally in a CONCLUSION, that is not clear and, I daresay, superficial.

Response: According to the referee’s comment, the “results” paragraph has been changed to “Results and discussion”, since other than results, it included a series of comments and the final discussion.

   

Authors cite insufficiently and hardly compare their findings with other work of other authors. Also, the cited work is quite old (most recent work is from 2008). A lot of articles on T-2/HT-2 analytics has been published in recent years. In a new version, this work has to be intectated.

Response: Following the reviewer’s suggestion, the discussion section has been improved adding other updated references concerning both the presence of T-2 and HT-2 toxins in cereals (lines 384 and 387) and the analytical parameters of other published methods (lines 210, 212, 251 and 253). New comments have been added about both aspects (lines 209-212 and 250-257). 

 

To my esteem, the article has to be re-organised and completed to be scientifically acceptable way. The proof of originality must be provided

Response: We appreciate the reviewer’s comments, very useful for improving the scientific level of this Paper. We hope our comments and manuscript improvement can satisfy the referee’s requests. 

Author Response File: Author Response.doc

Reviewer 3 Report

The manuscript submitted by Paola D’Agnello et al. refers to the comparison of ELISA and HPLC methods for determination of T-2 and HT-2 in cereals.  ELISA method was proposed as screening test whereas HPLC-FLD for confirmatory analysis.  The manuscript reports an accurate validation approach, however there are issue to be addressed.

In particular, this work is not an original research and no scientific novelty  was reported. The ELISA method test is based on commercially available test kits (R-Biopharm AG RIDASCREEN® T-2 and HT-2 (Enzymatic) Kit) already used and validated in Pleadin et al, 2018 for the analysis of T -2 and HT- 2 in cereals. The HPLC-FLD method was described and validated by Pascale 2012 for the analysis of the same mycotoxins in oats and wheat.

Given the lack of novelty, I would suggest proposing a workflow optimized in terms of time and validation parameters more comprehensive, which takes into account in the comparison both the other existing screening and confirmation methods.

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 3

The manuscript submitted by Paola D’Agnello et al. refers to the comparison of ELISA and HPLC methods for determination of T-2 and HT-2 in cereals.  ELISA method was proposed as screening test whereas HPLC-FLD for confirmatory analysis.  The manuscript reports an accurate validation approach, however there are issue to be addressed.

In particular, this work is not an original research and no scientific novelty  was reported. The ELISA method test is based on commercially available test kits (R-Biopharm AG RIDASCREEN® T-2 and HT-2 (Enzymatic) Kit) already used and validated in Pleadin et al, 2018 for the analysis of T -2 and HT- 2 in cereals. The HPLC-FLD method was described and validated by Pascale 2012 for the analysis of the same mycotoxins in oats and wheat.

Response: Two references cited by the referee have been added in the text and used for comparison. More comments related to methods validation have been added, according to the reviewer’s suggestion (lines 209-212 and 250-257).

 

Given the lack of novelty, I would suggest proposing a workflow optimized in terms of time and validation parameters more comprehensive, which takes into account in the comparison both the other existing screening and confirmation methods.

Response: A new figure that describes two workflows related to methods validation has been added (Figure 2), and cited/commented at lines 161-164. Other comments regarding the comparison to other available screening and confirmatory methods have been added (lines 209-212 and 250-257). 

Author Response File: Author Response.doc

Reviewer 4 Report

In subjected paper, the Authors presented studies upon the the validation and then the comparison between the ELISA, as screening method, and UPLC/FLD, as confirmatory method, for the quantitative determination of T-2 and HT-2 toxins in cereals.

Authors presented this issue in a clear way by describing it correctly in a chapter “Materials and Methods”. They also presented and discussed those results in chapter “Results”.

Minor notes:

Use uppercase letters in the title correctly.

The year of origin of the cereal samples must be completed.

Chapter 3 should be "Results and Discussion"

Tables 1-4 and Figures 1-2 should be included in the manuscript.

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 4

In subjected paper, the Authors presented studies upon the validation and then the comparison between the ELISA, as screening method, and UPLC/FLD, as confirmatory method, for the quantitative determination of T-2 and HT-2 toxins in cereals.

Authors presented this issue in a clear way by describing it correctly in a chapter “Materials and Methods”. They also presented and discussed those results in chapter “Results”.

Minor notes:

Use uppercase letters in the title correctly.

Response: The title has been checked and verified.

 

The year of origin of the cereal samples must be completed.

Response: As requested by the referee, the time interval dedicated to samples collecting has been specified at lines 73 and 349.

 

Chapter 3 should be "Results and Discussion"

Response: The paragraph title has been completed, as suggested by the reviewer (line 160).

 

Tables 1-4 and Figures 1-2 should be included in the manuscript.

Response: Due to possible resolution loss, the figures have been attached as supplementary files, using high resolution, together with tables. So, in order to satisfy the referee’s comment, the position of tables and figures has been suggested, adding the indication in the text (lines 49, 165, 197, 216, 343, 357 and 391).

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

The revision version looks good! Author have made the changes as suggested and I will support the paper for publication.

Author Response

The revision version looks good! Author have made the changes as suggested and I will support the paper for publication.

 

Response: The authors would like to thank again the reviewer for his effort in improving the scientific level of the Paper.

 

Reviewer 2 Report

Dear authors,

I appreciate you replying in detail to my previous comments. 

Indeed, I see an improvement, but the manuscript requires some addional revisions.

Please explain in the manuscript, why this work is important and original. Please understand me, I suppose that other laboratories did the same comparison. You will agree that such methodological comparisons are very common. If nobody else published it, then it is an original contribution.

Refer in you introduction on the necessity for this type of comparison and the and the changing EU legislation / ordinances concerning mycotoxins. You expect a lot more analysis in your laboratory (...).

Correlation coefficients are designed with "r" (minor r), while regression coefficients are designed "R" (capital R). Check what you did in line 25/26, please.

Line 54 "Recently, Pascale et al (2003)". 17 year old articles are not recent, but quite old, considering the evolution of analytical technology. 

Figure 1: what is the purpose of this figure? Explain it in the text and in the legend, please.

Formular (1) and following: why don't you put these in the Material and Methods?

all the descriptions of Ruggedness (234) and other validation schemes should be described in Material and Methods. You may remember the audience of methods in the results section, but not be described there.

Please prove that this comparison has not been done by other authors and refer to it in the text.

 

 

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 2

Dear authors,

I appreciate you replying in detail to my previous comments.

Indeed, I see an improvement, but the manuscript requires some additional revisions.

Please explain in the manuscript, why this work is important and original. Please understand me, I suppose that other laboratories did the same comparison. You will agree that such methodological comparisons are very common. If nobody else published it, then it is an original contribution.

Refer in you introduction on the necessity for this type of comparison and the and the changing EU legislation / ordinances concerning mycotoxins. You expect a lot more analysis in your laboratory (...).

Response: Following the referee’s remark, some other comments have been added, regarding EU Recommendation and possible future trends in the field (lines 47-50).

 

Correlation coefficients are designed with "r" (minor r), while regression coefficients are designed "R" (capital R). Check what you did in line 25/26, please.

Response: The indication has been standardized as r² throughout the text (lines 27 and 269).

 

Line 54 "Recently, Pascale et al (2003)". 17 year old articles are not recent, but quite old, considering the evolution of analytical technology.

Response: Following the referee’s remark, the adverb “Recently” has been removed (line 58).

 

Figure 1: what is the purpose of this figure? Explain it in the text and in the legend, please.

Response: Figure 1 was added as requested by the referee n.3, who suggested the addition of a comprehensive workflow, optimized in terms of time and validation parameters. According to the referee’s suggestion, a new sentence has been added at lines 188-189, and the legend has been improved. Moreover, the figures numbering in the text has been checked and corrected.

 

Formular (1) and following: why don't you put these in the Material and Methods?

Response: Following the referee’s suggestion, the equation used for calculating the measurement uncertainty, the trust semi-interval and the limits of detection and quantification have been moved in Materials and Methods, at lines 157, 165 and 171, respectively. The equation used for the calculation of the uncertainty function has been confirmed at line 313 for simplifying the reading of this part.

 

all the descriptions of Ruggedness (234) and other validation schemes should be described in Material and Methods. You may remember the audience of methods in the results section, but not be described there.

Response: According to the reviewer’s comment the procedures adopted for evaluating method selectivity and ruggedness have been moved in Materials and Methods (lines 174-181).

 

Please prove that this comparison has not been done by other authors and refer to it in the text.

Response: Following the referee’s remark, another sentence regarding the need and usefulness of such comparisons between different techniques has been added at lines 338-340.

Author Response File: Author Response.pdf

Reviewer 3 Report

The aim of this work was the implementation of an effective laboratory approach for the analysis of T-2 and HT-2 in cereal samples.

Two main analytical methods were considered for the analysis of T-2 and HT-2 toxins: an instrumental method (liquid chromatography with spectrofluorometric detection) and ELISA (enzyme-linked immunosorbent assay). The validation of the method was carried out according to Commission Decision 2002/657 / EC establishing the performance criteria and procedures for the validation of the screening and confirmatory methods for residues in products of animal origin.

These guidelines were also used in the field of mycotoxins until the entry into force of Regulation No. 2014/519 / EUR which specifies the performance criteria for mycotoxin screening and confirmatory methods. Both validation procedures are valid, however the authors could explain why laboratories should use Commission Decision 2002/657 / EC instead of the more recent Regulation 2014/519 to procedure validation.

Although previous comments have been partially  addressed, some issue in the manuscript submitted by Paola D’Agnello et al., should  be facedto further improve the quality of manuscript.

The purpose of the validation was to demonstrate that a method is "fit for purpose".

The objective of the screening methods is to detect the presence of T-2 and HT-2 toxins at the level of interest in order to verify compliance with the maximum permitted levels (STC = maximum level) or for process management (STC predefined by the laboratory) . s no legal limits have been defined in Europe For T-2 and Ht-2 toxin, but only a recommendation is available.

Commission decision no. 2002/657 / EC defines a set of parameters to be determined in the validation of the screening method, namely, selectivity / specificity detection capability (CCbeta), decision limit (CCalfa), precision and applicability / robustness / stability.

•  Selectivity / specificity was assessed for five representative products (wheat, maize, compound feed, barley and oats) by setting the level of intere 250 μg kg-1 of HT-2 toxin. This value is greater than the value indicated for the sum of the T-2 and HT-2 toxins for the matrices considered (see table 1) therefore it is advisable to add a lower level. Alternatively, the authors should state that the method may be non-selective at lower values ​​by being validated at 250 μg kg-1 of HT-2 toxin. Furthermore, I suggest to report the result in Table 2 also in concentration to allow to calculate the detection capabilities (CCβ) and the decision limit (CCalfa).
• Precision and recovery were performed on two series of blank feed samples (mixture of compound feed and raw materials) fortified at two different concentrations of T-2 + HT-2 toxins (100 and 250 μg kg-1) Line 201 stated that the test results were reported in Table 3, however the data in Table 3 refer to durum wheat samples.

Other comment:

- Line 169: write barley instead of cereals

- Line 239: 20mg / L instead of mg / Kg

- Line 161: elisA (21 DAYS instead of 44 days)

- Line 334: this statement is not clear “The sample is considered suspect positive if its concentration is ≥ MRL (Maximum residue limit, as defined by the relevant Regulation) - 2 • repeatability SD. In this case: 250 - 24.32 = 225.68 μg kg-1. "

- According to Commission decision no. 2002/657 / EC the sample is considered positive if its concentration is ≥ average of all fortified samples at the maximum residue limit - 1.64 • repeatability SD

Author Response

The authors would like to thank the reviewer for his effort in improving the scientific impact of the Paper. The manuscript has been revised, according to reviewer’s suggestions, editing corrections and rewording the text where necessary.

 

Reply to Reviewer 3

The aim of this work was the implementation of an effective laboratory approach for the analysis of T-2 and HT-2 in cereal samples.

Two main analytical methods were considered for the analysis of T-2 and HT-2 toxins: an instrumental method (liquid chromatography with spectrofluorometric detection) and ELISA (enzyme-linked immunosorbent assay). The validation of the method was carried out according to Commission Decision 2002/657 / EC establishing the performance criteria and procedures for the validation of the screening and confirmatory methods for residues in products of animal origin.

These guidelines were also used in the field of mycotoxins until the entry into force of Regulation No. 2014/519 / EUR which specifies the performance criteria for mycotoxin screening and confirmatory methods. Both validation procedures are valid, however the authors could explain why laboratories should use Commission Decision 2002/657 / EC instead of the more recent Regulation 2014/519 to procedure validation.

Response: As stated at lines 293-294, both reference documents (Reg. No. 519/2014/EC and Dec. No. 657/2002/EC) have been taken into account for methods validation. The authors added more references in the text to Dec. No. 2002/657/EC due to its applicability to both screening and confirmatory methods, and to its complete description of all validation parameters. This is useful in comparison studies like this.

 

Although previous comments have been partially  addressed, some issue in the manuscript submitted by Paola D’Agnello et al., should  be faced to further improve the quality of manuscript.

The purpose of the validation was to demonstrate that a method is "fit for purpose".

The objective of the screening methods is to detect the presence of T-2 and HT-2 toxins at the level of interest in order to verify compliance with the maximum permitted levels (STC = maximum level) or for process management (STC predefined by the laboratory) . s no legal limits have been defined in Europe For T-2 and Ht-2 toxin, but only a recommendation is available.

Commission decision no. 2002/657 / EC defines a set of parameters to be determined in the validation of the screening method, namely, selectivity / specificity detection capability (CCbeta), decision limit (CCalfa), precision and applicability / robustness / stability.

Selectivity / specificity was assessed for five representative products (wheat, maize, compound feed, barley and oats) by setting the level of intere 250 μg kg^-1 of HT-2 toxin. This value is greater than the value indicated for the sum of the T-2 and HT-2 toxins for the matrices considered (see table 1) therefore it is advisable to add a lower level. Alternatively, the authors should state that the method may be non-selective at lower values ​​by being validated at 250 μg kg^-1 of HT-2 toxin. Furthermore, I suggest to report the result in Table 2 also in concentration to allow to calculate the detection capabilities (CCβ) and the decision limit (CCalfa).

Response: As reported at lines 232-233, according to the reviewer’s remark, the ELISA was validated taking into consideration two spiking levels, 100 and 250 μg kg^-1, obtaining satisfactory results. Following the reviewer’s suggestion, table 2 has been integrated, adding the concentrations measured on spiked samples.

 

Precision and recovery were performed on two series of blank feed samples (mixture of compound feed and raw materials) fortified at two different concentrations of T-2 + HT-2 toxins (100 and 250 μg kg^-1) Line 201 stated that the test results were reported in Table 3, however the data in Table 3 refer to durum wheat samples.

Response: We are sorry for the mistake. The values reported in table 3 are related to UPLC/FLD determinations, which were carried out using spiked durum wheat. The results obtained from ELISA accuracy tests (100 and 250 μg kg^-1) were just commented at lines 234-241, and not reported in detail, since these data were only used for comparison, and then added in table 4. Some corrections have been made in the text for improving the readability (lines 233-234 and 255). The heading of table 3 was also integrated.

 

Other comment:

 

- Line 169: write barley instead of cereals

Response: Following the reviewer’s comment the word “cereals” has been replaced by “barley” (line 199).

 

- Line 239: 20mg / L instead of mg / Kg

Response: According to the reviewer’s remark, the mistake has been corrected. (line 250).

 

- Line 161: ELISA (21 DAYS instead of 44 days)

Response: The number of days required for the completion of each validation has been switched (line 191).

 

- Line 334: this statement is not clear “The sample is considered suspect positive if its concentration is ≥ MRL (Maximum residue limit, as defined by the relevant Regulation) - 2 • repeatability SD. In this case: 250 - 24.32 = 225.68 μg kg-1. "

- According to Commission decision no. 2002/657/EC the sample is considered positive if its concentration is ≥ average of all fortified samples at the maximum residue limit - 1.64 • repeatability SD

Response: The approach used for identifying a “suspect positive” sample was developed taking into account the main focus of this study, that is the comparison between two analytical methods. So, the coefficient 1.64 was rounded up to 2, in order to increase the number of samples useful for comparison. In this sense, this approach was not linked to method validation (par. 3.1), but only used for sample selection within methods comparison (par. 3.3.). In order to improve the readability, this section was re-arranged, adding some brief sentences. (lines 351-363).

Author Response File: Author Response.pdf

Reviewer 4 Report

The authors corrected the work in line with the reviewer's comments.

Author Response

The authors corrected the work in line with the reviewer's comments.

 

Response: The authors would like to thank again the reviewer for his effort in improving the scientific level of the Paper.

Round 3

Reviewer 2 Report

The manuscript has been modified according to my suggestions. 

Please check the meaning of R/ r (coefficient of correlation) and R square rooted (coefficient of determination) and correct your r / R in your manuscript.

 

Author Response

The manuscript has been modified according to my suggestions. 

Please check the meaning of R/ r (coefficient of correlation) and R square rooted (coefficient of determination) and correct your r / R in your manuscript.

Response: we are sorry for the mistake. The correct indications (r-R²) were reported when the correlation between results obtained by applying two different techniques and the determination coefficient of calibration curve, respectively, are discussed. Please see lines 26-27, 256 and 373.