Development and Validation of the Multi-Residue Method for Identification and Quantitation of Six Macrolide Antiparasitic Drugs

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic of this manuscript is highly relevant, and the obtained results are original and scientifically significant. However, the manuscript suffers from several major issues that need to be addressed before it can be considered for publication.

·      The manuscript is poorly written, primarily due to weak English language usage, improper writing style, and insufficient references. The authors should thoroughly revise the text for clarity, coherence, and grammatical correctness. Seeking professional language editing or assistance from a native English speaker would be beneficial. Additionally, more references should be included to support the claims made in the study.

·      The developed method lacks an adequate robustness evaluation. For a newly developed analytical method, robustness testing is essential to demonstrate its reliability under varying conditions. The authors should include an appropriate robustness study and discuss its implications for the validity of the method.

·     The discussion section is underdeveloped, lacking a comparison with previously published studies on similar topics. The authors should provide a more comprehensive discussion, contextualizing their findings within the existing body of literature. Citing relevant studies that have investigated similar methods or approaches would strengthen the manuscript and highlight its contributions.

·   In the introduction, the authors should specify the exact compounds being analyzed instead of using the terms 'avermectin' and 'ivermectin.' Given that avermectins are a group of structurally related compounds, and ivermectin itself is a mixture of B1a and B1b components, it is essential to clearly define which analytes are being investigated.

 

Comments on the Quality of English Language

Quality of English language need serious improvement.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The purpose of the study (applsci-3437200-peer-review-v1.pdf) entitled "Determination of six kinds of macrolides antiparasitic drugs in feed by high performance liquid chromatography-tandem mass spectrometry" was to develop a stable and sensitive detection method by HPLC-MS/MS, for the determination of six macrolide antiparasitic drugs in diversification feed matrices (including premix, concentrate, and complete feed) commonly used in the aquaculture industry.

First of all, I am surprised that the manuscript was submitted to the journal in a form that does not meet the criteria given in the instructions for authors. Furthermore, the title of the manuscript is not consistent with the content of the study, the keywords are identical to the title, the methods are not written in an academic style, the results are not adequately presented, without discussion of obtained results and how they can be interpreted from the perspective of previous studies, and the conclusions are too general.

However, some suggestions and recommendations to the authors I specified in the below:

Before submitting, format the manuscript according to the instructions for authors.

1.The title should be changed in way that reflected the contain of the manuscript (developtment and validation of the multi-residue method for identification and quantitation of six macrolide antiparasitic drugs)

2. Keywords- Please list the name of six macrolide antiparasitic drugs

3. ʺForewordʺplease replace with ʺIntroductionʺ

4. Figures 1 , 2, 3, 4, 5 and 6 are not listed in the manuscript.

5. The methods are not written in an academic style. The authors should improve the methods section.

6. Please,  indicate the regulations/guidelines on which you are basing your validation protocol of the developed LC-MS/MS method.

7. Authors should provide the city and state of the equipment manufacturer for LS-MS/MS.

8. The retention time of six macrolide antiparasitic drugs is missing. Please provide the retention time for each analyte.

9. The method validation subsection is not adequately presented. Authors should delete Fig. 7.  My recommendation is that authors present the method performance in a table. The table should contain data for recovery and matrix effect for each macrolide antiparasitic drug in each matrix at each concentration level.

10. The Discussion section is missing. Authors should discuss the results and how they can be interpreted from the per-spective of previous studies.

11. ʺ The dosage levels of formulated feed and vitamin were 0.05 μg/g/g, 0.5 μg/g/g and 5.0 μg/g/g.ʺ vitamin???

12. ʺIn previous studies, the purification of macrolides was mainly performed on a C18

purification column.ʺ References are missing.

13. ʺIn the previous study, acetonitrile, methanol and ethyl acetate were respectively

used for drug elution on the HLB purification column.ʺ Reference is missing.

14. The Conclusion section is too general. Please, highlight the main findings.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This research paper examines the use of high-performance liquid chromatography-tandem mass spectrometry to identify six types of macrolide antiparasitic medications in animal feed. The researchers spent considerable time and effort to produce this comprehensive scientific study. Herein, I would like to mention my reservations.

1) The reviewer believes that the manuscript lacks proper scientific organization. With few exceptions, the reviewer feels that potential readers would struggle to comprehend the main data presented in the current version.

2) The reviewer feels that the authors hastily submitted this version without adequately focusing on professional data presentation, language improvement, and organization. Despite its novelty, the manuscript's quality needs enhancement through English editing by a professional or native speaker. The entire document is full of linguistic errors, typos, and syntax issues, lacking a professional writing style. The authors should consider revising the whole manuscript, as numerous grammatical mistakes hinder the narrative and smooth flow.

3) Units should be consistent, for example, L/hr instead of l/Hr.

4) In Table 1, the legend and footnote require correction. Specifically, "a" should be used instead of "A".

5) The current manuscript version contains many linguistic errors. For instance, the phrase "Linearity evaluated using a matrix matching calibration curve" needs correction.

6) Experiment reproducibility using identical equipment is crucial for researchers worldwide. Therefore, it is advised to provide exact LC-MS/MS specifications (such as manufacturer and model).

7) In Section 3.1, "multiple reaction detection (MRM)" is incorrect. This should be explained and rectified.

8) Figure 2 has been added by the authors, but a relevant description is missing. Proper labeling and explanation should be included in the appropriate section. The purpose of this figure should be clarified.

9) The authors state: "Due to the high lipophilic nature of the macrolide antiparasitic drugs, two extraction methods of acetonitrile and methanol were designed based on the relevant research (Ozdemir, N. & Kahraman, T.2016; Zhan et al.2013， Gabriel Rübensam et al.，2013， Wang et al.2012， de Oliveira Ferreira, F et al.2016）." It is recommended that separate methods be added or cited for both approaches.

10) The Y-axis units in Figure 3 are not specified. Additionally, a relevant description of Figure 3 is absent. Proper labeling and explanation should be added to the appropriate section.

11) The Y-axis units in Figure 4 are not provided. Again, a relevant description of Figure 3 is missing. Proper labeling and explanation should be included in the appropriate section. It is recommended to use "room temperature" or "ambient temperature" instead of "normal temperature".

12) In Section 3.2.3, the phrase "in previous study" is used. This requires clarification.

13) The following sentence is ambiguous and needs clarification: "Using 17% methanol -17% acetonitrile for best elution was continued to be validated and guaranting recovery; Effect of 18% methanol -18% acetonitrile as eluent on the recovery of target drug and impurity interference in feed."

14) The Y-axis units in Figure 5 are not specified. Once more, a relevant description of Figure 3 is missing. Proper labeling and explanation should be added to the appropriate section. This applies to other figures as well.

Overall, the manuscript lacks professional presentation because an adequate ‎demonstration is required for a research article to catch the readership of any ‎journal.  Throughout the draft, necessary explanations and discussion have not been added. Indeed, the authors have put efforts to come up with this article. Despite ‎these efforts to develop a comprehensive article, the manuscript in its current ‎state is not recommended for publication.

 

Comments on the Quality of English Language

The reviewer feels that the authors hastily submitted this version without adequately focusing on professional data presentation, language improvement, and organization. Despite its novelty, the manuscript's quality needs enhancement through English editing by a professional or native speaker. The entire document is full of linguistic errors, typos, and syntax issues, lacking a professional writing style. The authors should consider revising the whole manuscript, as numerous grammatical mistakes hinder the narrative and smooth flow.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript entitled “Determination of six kinds of macrolides antiparasitic drugs in feed by high performance liquid chromatography-tandem mass spectrometry” by Cheng et al presents a relevant study on the development of a multi-residue detection method for six macrolide antiparasitic drugs commonly used in animal feed. The use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for precise identification and quantification of these compounds is a valuable contribution to veterinary pharmacology and food safety. The reported work addresses an interesting topic, particularly in the context of regulatory efforts to monitor veterinary drug residues and their potential risks to human health.

Moreover, the study is well-structured, with a clear research objective and well-detailed methodology. The introduction provides a comprehensive overview of macrolide antiparasitic drugs, their applications, and the associated concerns related to their presence in animal feed. However, certain areas require further clarification and refinement to improve the manuscript’s clarity, coherence, and scientific impact.

Specific comments and suggestions

The introduction presents a strong foundation on the topic, but it would benefit from a more detailed discussion on the global regulatory frameworks governing macrolide residues in animal feed, particularly regarding their legal limits in different countries. Including references to current international regulations (e.g., FDA, EMA, Codex Alimentarius) would strengthen the manuscript’s relevance.

Additionally, the methodology is well described, particularly regarding the sample preparation and purification processes. However, there are some aspects that require clarification:

• The description of the sample extraction procedure should specify whether any validation steps were taken to ensure reproducibility across different types of feed matrices.
• The choice of acetonitrile over methanol as the extraction solvent is justified, but additional explanation on why acetonitrile performed better in reducing matrix interference would improve understanding.
• More details on the selection criteria for mass spectrometer parameters (e.g., cone voltage, collision energy) would enhance transparency.

Furthermore, the results are well presented with appropriate tables and figures. However, the discussion section could be improved in the following ways:

• The comparison of recovery rates across different macrolide drugs should be expanded to explain why some compounds exhibit higher variability in recovery.
• The limitations of the method should be explicitly discussed, particularly regarding the detection of low-concentration residues in complex feed matrices.
• A comparative analysis with existing methods from the literature should be included to highlight the advantages of the proposed approach.
• Figure captions should be revised for clarity. For example, "Mean recovery of macrolide anthelmintic from premix feed extracted with methanol and acetonitrile" could be reworded to "Comparison of macrolide anthelmintic recovery from premix feed using methanol and acetonitrile as extraction solvents."
• If available, a graphical abstract summarizing the workflow could improve the manuscript’s accessibility.

Finally, the conclusion summarizes the study but should emphasize the broader implications of the findings. Discussing how this method could be applied in routine monitoring programs and potential future improvements (e.g., adaptation for other veterinary drugs) would enhance the manuscript’s impact.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The work is improved after revision.

Comments on the Quality of English Language

English should be polished.

Author Response

Dear reviewer, thank you for your valuable comments. We have checked the content of the article again, corrected the grammatical errors in the content of the article, and refined the language of the article again, hoping to help readers better understand the content and meaning of the article.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did their best to revise the manuscript according to the reviewers' comments to improve its quality. However, the manuscript still requires some minor corrections, as detailed below:

The authors did not correct the manuscript title.

The title should be changed in a way that reflects the content of the manuscript (e.g., “Development and validation of the multi-residue method for identification and quantitation of six macrolide antiparasitic drugs”).

Page 17, Line 398: Figures 7–13 are not listed in the text of the manuscript.

Page 8, Lines 288-289: The authors should provide the manufacturer, city, and state both for C18 and HLB (Hydrophilic-Lipophilic Balance) purification columns.

Technical errors

In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation.

The list of references should be corrected by following the Instructions for Authors.

Author Response

• The manuscript titlehavs been corrected as your advise to be “Development and validation of the multi-residue method for identification and quantitation of six macrolide antiparasitic drugs”.
• Page 17, Line 398: Figures 7–13 are not listed in the text of the manuscript.
• Figures 7-13: The corresponding descriptions have been added to Page 17, Line 420of the revised manuscript.

3、 Page 8, Lines 288-289: The authors should provide the manufacturer, city, and state both for C18 and HLB (Hydrophilic-Lipophilic Balance) purification columns.

• SPE columns (C18 & HLB):

While the C18 and HLB purification columns were not explicitly described in Page 8, Lines 288-289, their manufacturers were previously stated in Page 3, Line 124. C18 was added in here too. Both SPE columns were bought from Waters Co.

4、In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation.

• Yes, we have edited these and four references were deleted because of no quoted in the text. All references have been verified and adjusted to comply with the journal’s style guidelines.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The author has not adequately addressed my comments provided in the previous review. 1)-It is recommended that the author submit a version of the manuscript with modifications highlighted in different colors for each reviewer. It is hard to find the modifications.

2)-Simply stating that modifications have been made is insufficient. Specific details regarding the modifications, including their exact location, line number, and nature, are necessary.

3)-Currently, the entire draft is highlighted, making it difficult to discern the specific changes.

4)-Additionally, many responses to previous comments appear to be missing.

 

Comments on the Quality of English Language

The authors should think about revising the entire manuscript one more time, as the numerous grammatical errors disrupt the narrative and impede its smooth flow.

Author Response

Dear reviewer:

Hello, based on your review comments this time, we have first checked the full text of the article again and corrected the grammatical errors. We will make a specific response to the last review comments, and we have sent the revised article together with the reply comments to you this time(Please see the attachment). The following is our response to the last review comments:

1. For the data in the article, we have given specific explanations to some of them;
2. Unified revision of language errors, misspellings and grammatical problems in the article, as well as unified revision of the writing style of the article, striving to present it in a more professional language;
3. The problem of unit consistency has also been modified;
4. For the a and A problems in Table 1, the article refers to solution A in Table 1, rather than the legend or annotation in Table 1;
5. We have checked and corrected the language errors in the manuscript version;
6. We have accurately provided the manufacturer of LC-MS/MS in the article;
7. We have explained the multiple response detection (MRM) in 3.1;
8. We added a description of Figure 2 to the article;
9. For the extraction methods of acetonitrile and methanol, we described them separately from the cited literatures;
10. The description of Y-axis in Figure 3, Figure 4 and Figure 5 has been added in the article.
11. For the word "in previous study" in 3.2.3, we have added the reference literature;
12. For ambiguous statements, "Using 17% methanol -17% acetonitrile for best elution was continued to be validated and guaranting. recovery;  Effect of 18% methanol -18% acetonitrile as eluent on the recovery of target drug and impurity interference in  "feed" for further explanation;

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

File attached

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Hello, and thank you for your invaluable suggestions regarding our manuscript. We have carefully revised the manuscript in accordance with your comments, and the specific changes include the following:

1. The reviewer believes that the manuscript lacks proper scientific organization. With few exceptions, the reviewer feels that potential readers would struggle to comprehend the main data presented in the current version.

• We have provided further clarification and explanation for the data presented in the manuscript.

Reviewers Current Comment: Where are the modifications? Please highlight the text. Insert line numbering to indicate modifications.

• We have highlighted all modifications in yellow, primarily by adding references to the selected data to help readers better understand the rationale and justification behind our data selection.

2. The reviewer feels that the authors hastily submitted this version without adequately focusing on professional data presentation, language improvement, and organization. Despite its novelty, the manuscript's quality needs enhancement through English editing by a professional or native speaker. The entire document is full of linguistic errors, typos, and syntax issues, lacking a professional writing style. The authors should consider revising the whole manuscript, as numerous grammatical mistakes hinder the narrative and smooth flow.

• The language of the manuscript has been refined to ensure a more professional and natural English expression. We have corrected language errors, typos, and grammatical issues, and revised the content using a more professional writing style to the best of our ability.

Reviewers Current Comment: Yes, some mistakes have been corrected. Nonetheless, many problems remain.

• We have thoroughly re-examined the manuscript and corrected all grammatical errors identified.

3. The current manuscript version contains many linguistic errors. For instance, the phrase "Linearity evaluated using a matrix matching calibration curve" needs correction.

• Language errors in the manuscript have been corrected.

Reviewers Current Comment: Yes, some mistakes have been corrected. Nonetheless, many problems remain.

• We have thoroughly re-examined the manuscript and corrected all grammatical errors identified.

4. Experiment reproducibility using identical equipment is crucial for researchers worldwide. Therefore, it is advised to provide exact LC-MS/MS specifications (such as manufacturer and model).

• The specifications of the LC-MS/MS equipment have been accurately provided in the manuscript.

Reviewers Current Comment: If included in the revised version, highlight the change with the specified line number. I have not seen that modification.

• The modified information (equipment manufacturer details) has been highlighted in yellow on page 4, line 136 of the manuscript.

5. Figure 2 has been added by the authors, but a relevant description is missing. Proper labeling and explanation should be included in the appropriate section. The purpose of this figure should be clarified.

• Figure 2 has been described in detail within the manuscript.

 Reviewers Current Comment: There is no description available for Figure 2. Could you clarify your reasoning behind Figure 2? Please provide an explanation for figure 2. Simply stating that the results are shown in Figure 2 is insufficient.

• The design basis has been highlighted in yellow in the article.On lines 236 to 241 of page 6 of the article

We sincerely hope that these revisions address your concerns and enhance the overall quality of the manuscript. Thank you once again for your constructive feedback.

Author Response File: Author Response.docx

Round 4

Reviewer 3 Report

Comments and Suggestions for Authors

 

The author has performed some necessary modifications. It is suggested that before further processing following minor changes should be considered.

• In Figure 2, after optimization. The author states that -These precursor and product ions were subsequently used for multiple reaction monitoring (MRM) analysis (the 242 determined mass numbers of the precursor ions are illustrated in Figure 2). The author should mention here the precursor and product ions.
• Figure 1. Displays the molecular structures of six macrolide drugs. Regarding the use of red color (functional groups) in Avermectin (AVM) and Milbemycin oxime (MIL), I believe there is no particular reason for it. Please standardize the format (uniform) of the chemical structures by redrawing them.

Author Response

1. In Figure 2, after optimization. The author states that -These precursor and product ions were subsequently used for multiple reaction monitoring (MRM) analysis (the 242 determined mass numbers of the precursor ions are illustrated in Figure 2). The author should mention here the precursor and product ions.

• Thank you for your valuable advice. We have provided further explanations of the precursor ions and sub-ions. The relevant content has been added to lines 241-268 on page 6 of the article and has been highlighted in yellow.

2. Figure 1. Displays the molecular structures of six macrolide drugs. Regarding the use of red color (functional groups) in Avermectin (AVM) and Milbemycin oxime (MIL), I believe there is no particular reason for it. Please standardize the format (uniform) of the chemical structures by redrawing them.

• Ok, Thank you. We have unified the chemical structural formulas of AVM and MIL with those of other drugs and formats, and removed the red markings of functional groups.

Author Response File: Author Response.pdf