Evaluation of Chromatographic Separation, with a Focus on LC-MS/MS, for the Determination of Stereoisomeric Cypermethrin and Other Synthetic Pyrethroids in Apples

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study addresses an important analytical challenge and is fundamentally sound, but requires additional details, clearer data presentation, and structural adjustments to meet publication standards. I suggested the author supplement and revise the manuscript according to the above comments before resubmitting.

1.The “modified QuEChERS-type extraction” should briefly specify how it differs from the original protocol.

2.GC-MS/MS parameters (e.g., collision energies, MRM transitions) are not fully listed; a table similar to Table 1 for LC-MS/MS is recommended.

3.Section 3.1 (“Optimization method”) contains extensive optimization descriptions that partially overlap with the Discussion. Consider merging or streamlining to avoid repetition.

4.The failure of GC-MS/MS to fully resolve isomers is attributed to poor sensitivity and long runtimes, but a more detailed explanation (e.g., column selectivity, temperature program, injector effects) would strengthen the discussion.

5.Figures 1 and 2 lack clear peak labels (e.g., isomer identification) and their captions do not fully match the presented chromatograms.

6.Matrix effect data, mentioned in the text, are not shown in Table 2. Please include or clarify why they were omitted.

7.Inconsistent citation style for EFSA documents (e.g., sometimes with full URL, sometimes only journal reference). Please unify according to journal guidelines.

Author Response

Question 1:

1.The “modified QuEChERS-type extraction” should briefly specify how it differs from the original protocol.

We thank the reviewer for this comment. In line 137, we have added a brief sentence clarifying how the modified QuEChERS-type extraction differs from the original protocol, specifically noting the omission of the second clean-up step involving purifying salts

Question 2:

2.GC-MS/MS parameters (e.g., collision energies, MRM transitions) are not fully listed; a table similar to Table 1 for LC-MS/MS is recommended.

Response 2: Thank you for this valuable suggestion. In response, we have added a new table (Table 2) that comprehensively lists the GC–MS/MS parameters, including the MRM transitions and collision energies, analogous to Table 1 for the LC–MS/MS analysis. This addition ensures full transparency and reproducibility of the GC–MS/MS method.

Question 3:

3.Section 3.1 (“Optimization method”) contains extensive optimization descriptions that partially overlap with the Discussion. Consider merging or streamlining to avoid repetition.

Response 3: To further clarify the methodological progress achieved in this work, Table 3 has been added to the revised manuscript, providing a direct comparison between previously reported chiral LC–MS/MS methods and the present approach. This table highlights the key differences in chromatographic conditions and clearly illustrates the advances achieved in terms of stereoisomeric resolution and analytical performance.

Question 4:

 4.The failure of GC-MS/MS to fully resolve isomers is attributed to poor sensitivity and long runtimes, but a more detailed explanation (e.g., column selectivity, temperature program, injector effects) would strengthen the discussion.

Response 4:  We sincerely thank the Reviewer for this valuable comment. In response, we have added a detailed discussion on the limitations of GC–MS/MS for separating cypermethrin stereoisomers, including the effects of column selectivity, temperature programs, and the structural constraints of the trans-diastereomers. This addition can be found starting from line 464 in the revised manuscript.

Question 5:

5.Figures 1 and 2 lack clear peak labels (e.g., isomer identification) and their captions do not fully match the presented chromatograms.

Response 5: We appreciate the Reviewer's important comment. The assignment of the eight cypermethrin stereoisomers shown in Figure 1 was performed using accredited cypermethrin standards, which contain a defined mixture of stereoisomers with composition provided by the manufacturer; it was alpha, beta, theta, and zeta cypermethrin. We do not have individual standards for each stereoisomer, as the cost of acquiring separate standards for all eight isomers exceeds the budget of the current study. We would like to clarify that specific isomer assignments were made only for those compounds for which we could ensure confident identification.  For other compounds, results are reported at the level of name customary without over-interpretation, to ensure analytical reliability and scientific rigor.

To ensure reliable identification, the elution order was cross-checked against the data reported by the European Union Reference Laboratory (EURL) for Pesticides (https://www.eurl-pesticides.eu/library/docs/srm/EurlSrm_Observation_Cypermethrins_with_LC-MSMS.pdf). This approach—using authentic standards of the mixture combined with reference elution order—is widely accepted in the field of chiral cypermethrin analysis and provides a high level of confidence in the stereoisomer assignments, even in the absence of absolute configuration determination.

Question 6:

6.Matrix effect data, mentioned in the text, are not shown in Table 2. Please include or clarify why they were omitted.

Response 6: We appreciate the Reviewer's helpful comment. In response, the results of the matrix effect evaluation have been added to the manuscript text (lines 292–297) to ensure that all information.

Question 7:

7.Inconsistent citation style for EFSA documents (e.g., sometimes with full URL, sometimes only journal reference). Please unify according to journal guidelines.

Response 7: We thank the Reviewer for pointing this out. All EFSA references have now been revised and unified to ensure a consistent citation style in accordance with the journal’s guidelines, with redundant or inconsistent formats removed.

Reviewer 2 Report

Comments and Suggestions for Authors

Wenio et al developed a method for the determination of stereoisomeric forms of cypermethrin and other synthetic pyrethroids in Food Matrices. It is fairly useful and important for applied analytical chemistry. However, there are a couple of things that need to be addressed before the acceptance of the manuscript. 

1. I have noticed that LC-MS/MS experiments have been designed and conducted. However, only GC-MS research status on pyrethroids was provided in the intro. I would suggest the strengthening of the LC-MS/MS part from line 73-77.
2. In the method part, the authors mentioned that they have modified other methods to establish their research. However, rarely cite what the previous methods are and why they would like to modify their steps. This would suggest to be improved. 
3. I would suggest a table of comparison between the established method and the previous or other developed methods in the literature. We can clearly see the advancement of this method.
4. In Table 2, I suggest separating the results from GC-MS and those from LC.
5. I would suggest a refinement of the title to be detailed. Like "chromatographic" methods, method is a big word.
6. I would suggest a broader validation. Here, only apples have been analyzed. 

Author Response

Comments 1. I have noticed that LC-MS/MS experiments have been designed and conducted. However, only GC-MS research status on pyrethroids was provided in the intro. I would suggest the strengthening of the LC-MS/MS part from line 73-77.

Response 1: Thank you for your comment. We have taken note of your suggestion and have strengthened the LC-MS/MS section in lines 78–88. Additional details on LC-MS/MS methodologies and applications have been added to provide a more comprehensive background.

 

Comments 2. In the method part, the authors mentioned that they have modified other methods to establish their research. However, rarely cite what the previous methods are and why they would like to modify their steps. This would suggest to be improved. 

 

Response 2: We sincerely thank the reviewer for this insightful comment. We would like to emphasize that, to the best of our knowledge, this study presents the first LC-MS/MS method for determining cypermethrin enantiomers in food matrices. In line 139, we have now provided a detailed description of the specific modifications introduced to adapt previous approaches, along with proper citations of the standardized QuEChERs method that served as references. These clarifications highlight both the rationale behind our methodological adjustments and the novelty of our work, ensuring transparency and reproducibility for future studies.

Comments 3. I would suggest a table of comparison between the established method and the previous or other developed methods in the literature. We can clearly see the advancement of this method.

Response 3: To further clarify the methodological progress achieved in this work, Table 3 has been added to the revised manuscript, providing a direct comparison between previously reported chiral LC–MS/MS methods and the present approach. This table highlights the key differences in chromatographic conditions and clearly illustrates the advances achieved in terms of stereoisomeric resolution and analytical performance.

Comments 4 In Table 2, I suggest separating the results from GC-MS and those from LC.

Response 4: Thank you very much for your suggestion regarding Table 2. We would like to clarify that validation in our study was performed only for compounds analyzed by LC‑MS/MS. The decision not to perform a full validation of the GC‑MS method was based on practical and analytical considerations. A complete GC-MS/MS validation would require approximately 40 injections due to the long run time (≈approximately 157 minutes per analysis), resulting in a validation process spanning more than four days. Moreover, our laboratory does not have a cooled tray for the autosampler. It is well-documented that temperature, solvent exposure, and analysis time can significantly influence the stereoisomerization of cypermethrin and the stability of its enantiomers during GC analysis, leading to potential artifacts in chiral measurements and undermining the reliability of the results. Under our current conditions, performing a full validated GC-MS method would risk generating non-reproducible and non-representative data. Therefore, we focused on LC‑MS/MS validation for the analytes.

 

Comments 5 I would suggest a refinement of the title to be detailed. Like "chromatographic" methods, method is a big word.

Response 5: We sincerely thank the reviewer for the suggestion. We have refined the title to better reflect the study's focus and novelty. The revised title emphasizes the use of LC-MS/MS for determining the stereoisomeric forms of cypermethrin and other synthetic pyrethroids in apples, while subtly indicating the method development and evaluation aspects. Title is:” Evaluation of Chromatographic Separation, with a Focus on LC-MS/MS, for the Determination of Stereoisomeric Cypermethrin and Other Synthetic Pyrethroids in Apples”.

Comments 6. I would suggest a broader validation. Here, only apples have been analyzed. 

 Response 6: We sincerely thank the Reviewer for this valuable and constructive comment. We acknowledge that chiral separation and method validation were performed using apples as a representative high-water-content matrix, and that broader validation across matrices of differing composition (e.g., high-fat or dry commodities) would be required to demonstrate universal applicability. The primary aim of this study was not to establish a fully multi-matrix residue method, but to develop and critically evaluate an enantioselective LC–MS/MS approach for resolving and quantifying stereoisomeric forms of cypermethrin and selected synthetic pyrethroids under conditions relevant to regulatory analysis. Apples were deliberately chosen as a commonly used reference matrix in pesticide method validation, in line with SANTE guidelines. To avoid overinterpretation of the method’s scope, the manuscript title has been refined to clearly reflect its focus on apples and on chromatographic separation. Importantly, matrices with high lipid content pose specific analytical challenges for stereoisomeric cypermethrin analysis, as matrix effects and extraction conditions may promote isomerization, making direct extrapolation from high-water matrices inappropriate. In future studies, we plan to extend the validation to a broader range of food matrices and other pyrethroid compounds to systematically investigate matrix-dependent effects on stereoisomer stability and method performance.

Reviewer 3 Report

Comments and Suggestions for Authors

The present study aimed to develop, optimize, and validate a robust analytical method for quantifying cypermethrin and other pyrethroid residues in complex food matrices. The study is well performed, analytical methods are valid and the conclusions are mainly supported by the results. However, revision is needed before the manuscript can be accepted for publication.

Introduction

There are surprisingly too few references cited in the Introduction, and many of them are from EFSA. I suggest including more relevant references, especially regarding analytical methods.

Material and methods

Instead of GC-MS/MS condition and LC-MS/MS condition I suggest writing “GC-MS/MS operating conditions” and  “LC-MS/MS operating conditions”.

Additionally, the method section lacks a clear description of the method validation procedure. The validation part currently appears in the Results section but should be moved to the Methods. This section should specify the number of samples analyzed and the levels or ranges evaluated for each validation parameter.

Results

Much of the content under Result section describes method development and validation procedures rather than reporting experimental findings. This material should be moved to the Materials and Methods section (for example under Method optimization and Validation subsections). The Results section should focus on the analytical outcomes (actual residue levels, isomeric profiles, comparison among matrices, or implications for MRL determination).

Discussion

The discussion section frequently begins sentences with phrases such as  “In a study by...” or “In a study conducted by...”. This repetitive structure affects the readability and narrative flow. Consider varying sentence openings and integrating references more smoothly into the discussion. For example, instead of “In the study by Yao et al. (2015)…”, you could write “Yao et al. (2015) demonstrated that…”

Some references are not formatted according to the journal citation style (ex. lines 329, 358, 374…) Please ensure consistency in reference formatting.

Conclusion

Some statements in Conclusion section extend beyond the experimental evidence presented in the manuscript. The results primarily describe method optimization and validation, with no applied residue data or toxicological comparisons presented. The conclusion should therefore focus on the method analytical performance. The authors should avoid broader claims about toxicological risk assessment or regulatory applications unless supported by data.

Author Response

Comments 1. Introduction

There are surprisingly too few references cited in the Introduction, and many of them are from EFSA. I suggest including more relevant references, especially regarding analytical methods.

Response 1: We appreciate the Reviewer's valuable comment. In response, the Introduction has been expanded and supplemented with additional relevant references, particularly focusing on analytical methods for pyrethroid determination. This ensures a more comprehensive and up-to-date contextualization of the study, better situating our work within the existing literature.

Comments 2. Material and methods

Instead of GC-MS/MS condition and LC-MS/MS condition I suggest writing “GC-MS/MS operating conditions” and  “LC-MS/MS operating conditions”.

Additionally, the method section lacks a clear description of the method validation procedure. The validation part currently appears in the Results section but should be moved to the Methods. This section should specify the number of samples analyzed and the levels or ranges evaluated for each validation parameter.

Response 2: We appreciate the Reviewer's suggestions. The section titles have been updated to “GC–MS/MS operating conditions” and “LC–MS/MS operating conditions” for clarity. Additionally, the method validation details have been moved from the Results to the Materials and Methods sectionand the subsection name is Validation parameters

Comments 3. Results

Much of the content under Result section describes method development and validation procedures rather than reporting experimental findings. This material should be moved to the Materials and Methods section (for example under Method optimization and Validation subsections). The Results section should focus on the analytical outcomes (actual residue levels, isomeric profiles, comparison among matrices, or implications for MRL determination).

Response 3: We appreciate the Reviewer's constructive comment. In response, the manuscript has been reorganized so that detailed descriptions of method development and validation procedures have been moved from the Results section to the Materials and Methods, specifically under the Method Optimization and Validation parameter subsections.

Comments 4. Discussion

The discussion section frequently begins sentences with phrases such as  “In a study by...” or “In a study conducted by...”. This repetitive structure affects the readability and narrative flow. Consider varying sentence openings and integrating references more smoothly into the discussion. For example, instead of “In the study by Yao et al. (2015)…”, you could write “Yao et al. (2015) demonstrated that…”

Some references are not formatted according to the journal citation style (ex. lines 329, 358, 374…) Please ensure consistency in reference formatting.

Response 4: We appreciate the Reviewer's helpful suggestions. The Discussion section has been revised to improve readability and narrative flow by varying sentence openings and integrating references more smoothly. Additionally, all references have been carefully checked and formatted consistently in accordance with the journal’s citation style.

Comments 5. Conclusion

Some statements in Conclusion section extend beyond the experimental evidence presented in the manuscript. The results primarily describe method optimization and validation, with no applied residue data or toxicological comparisons presented. The conclusion should therefore focus on the method analytical performance. The authors should avoid broader claims about toxicological risk assessment or regulatory applications unless supported by data.

Response 5: We appreciate the Reviewer's comment. The Conclusions section has been substantially revised to focus specifically on the analytical performance of the optimized LC–MS/MS method, including enantiomeric resolution, sensitivity, accuracy, precision, and linearity. Statements regarding toxicological risk assessment. These revisions ensure that the Conclusions are now fully aligned with the Reviewer’s suggestions and accurately summarize the method validation results without extending beyond the presented evidence.

Reviewer 4 Report

Comments and Suggestions for Authors

The study develops an LC-MS/MS-based enantioselective method for quantifying stereoisomers of cypermethrin and other pyrethroids in food matrices. The motivation is well-justified (i.e., current MRLs do not differentiate high-toxicity isomers), method validation complies with SANTE guidelines, and systematic optimization identifies column temperature as the critical parameter. However, gaps remain regarding method generality, depth of GC-MS/MS comparison, details on isomerization control, and regulatory applicability.  

Comments to the Author:

Question 1：The chiral separation and method validation were performed only in apple (a high-water matrix). Evidence for applicability across diverse food matrices (e.g., fatty, dry, or acidic commodities) is lacking, which limits the claimed “robustness” for general residue monitoring.

Question 2：The introduction states both LC-MS/MS and GC-MS/MS are used, yet GC-MS/MS was discontinued due to poor resolution and sensitivity. This contradiction should be addressed upfront to avoid misleading readers about the dual-platform strategy.

Question 3：The QuEChERS protocol omits primary secondary amine (PSA) cleanup. Given that acetonitrile without PSA leads to 12-15% isomerization (line 442), this choice risks artifact formation in pigmented or fatty foods, undermining accuracy.

Question 4：While the focus on cypermethrin’s isomerization is justified, the paper must clarify how its temperature-driven optimization offers a substantive advance over existing chiral LC-MS/MS methods (e.g., Zhao et al., 2019), beyond being analyte-specific.

Question 5：The claim that non-enantioselective methods “underestimate toxicological risk” lacks quantitative context. A brief risk scenario (e.g., if α-cypermethrin constitutes 20% of total but is 10× more toxic) would strengthen the regulatory argument.

Question 6：The conclusion asserts suitability for “routine analysis,” yet the 30-min isocratic LC run and expensive chiral columns may hinder high-throughput labs. This practical limitation should be acknowledged.

Question 7：The assignment of eight cypermethrin stereoisomers (Fig. 1) lacks confirmation of absolute configuration. Were authentic isomer standards used? Or based solely on retention order from literature?

Question 8：The manuscript treats EFSA’s proposed dual MRL framework as established policy (lines 83-91), though it remains provisional. This could mislead readers about current regulatory requirements.

Question 9：The terms “enantiomer” and “stereoisomer” are used interchangeably. Cypermethrin has diastereomers (due to multiple chiral centers), not just enantiomers. Precise stereochemical language is essential.

Question 10：The conclusions state that “the developed LC-MS/MS method... demonstrates high sensitivity, accuracy, precision, and linearity, which confirms its suitability for routine residue analysis.” This is supported for LC-MS/MS. However, the concluding phrase “enabling accurate stereochemical characterization” is too broad, as the failed GC-MS/MS arm suggests significant challenges in characterizing all stereoisomers by this complementary technique. The conclusions should be refined to reflect that accurate characterization was achieved specifically by the optimized LC-MS/MS method, acknowledging the limitations encountered with GC-MS/MS.

Question 11：The introduction poses the challenge of accurate stereoisomer determination for refined risk assessment and MRL setting. While the developed LC-MS/MS method is a step forward, the conclusions do not explicitly circle back to how this method directly addresses the regulatory problem of setting isomer-specific MRLs (e.g., for alpha-cypermethrin) mentioned in the introduction. A stronger concluding statement linking the methodological success to this primary question is needed.

   This work provides valuable technical insights into chiral pyrethroid analysis, particularly regarding temperature effects on isomer resolution. Major revisions are recommended: (1) include multi-matrix validation; (2) clarify method limitations and regulatory status; (3) correct terminology and strengthen stereochemical assignment. Acceptable after major revision.

Author Response

Question 1：The chiral separation and method validation were performed only in apple (a high-water matrix). Evidence for applicability across diverse food matrices (e.g., fatty, dry, or acidic commodities) is lacking, which limits the claimed “robustness” for general residue monitoring.

Response 1: We sincerely thank the Reviewer for this valuable and constructive comment. We acknowledge that chiral separation and method validation were performed using apples as a representative high-water-content matrix, and that broader validation across matrices of differing composition (e.g., high-fat or dry commodities) would be required to demonstrate universal applicability. The primary aim of this study was not to establish a fully multi-matrix residue method, but to develop and critically evaluate an enantioselective LC–MS/MS approach for resolving and quantifying stereoisomeric forms of cypermethrin and selected synthetic pyrethroids under conditions relevant to regulatory analysis. Apples were deliberately chosen as a commonly used reference matrix in pesticide method validation, in line with SANTE guidelines. To avoid overinterpretation of the method’s scope, the manuscript title has been refined to clearly reflect its focus on apples and on chromatographic separation. Importantly, matrices with high lipid content pose specific analytical challenges for stereoisomeric cypermethrin analysis, as matrix effects and extraction conditions may promote isomerization, making direct extrapolation from high-water matrices inappropriate. In future studies, we plan to extend the validation to a broader range of food matrices and other pyrethroid compounds to systematically investigate matrix-dependent effects on stereoisomer stability and method performance.

Question 2：The introduction states both LC-MS/MS and GC-MS/MS are used, yet GC-MS/MS was discontinued due to poor resolution and sensitivity. This contradiction should be addressed upfront to avoid misleading readers about the dual-platform strategy.

Response 2: We thank the Reviewer for this important and constructive comment. We agree that the initial wording of the Introduction could have suggested a dual-platform analytical strategy, which required clarification. To address this and avoid any potential misunderstanding, the Introduction has been revised to clearly explain that both LC–MS/MS and GC–MS/MS were initially evaluated during the method development stage; however, only LC–MS/MS was ultimately validated and applied.

These revisions ensure that the analytical strategy and rationale for method selection are clearly communicated upfront and that readers are not misled regarding the role of GC–MS/MS in the study.

Question 3：The QuEChERS protocol omits primary secondary amine (PSA) cleanup. Given that acetonitrile without PSA leads to 12-15% isomerization (line 442), this choice risks artifact formation in pigmented or fatty foods, undermining accuracy.

Response 3: We fully agree with the Reviewer that omission of PSA cleanup in the QuEChERS protocol can promote isomerization, particularly in challenging matrices. Our preliminary studies indicated that the extent of stereoisomer formation is strongly matrix-dependent. In high-water-content matrices, such as apples, isomerization was largely minimized under the applied conditions. However, complex or lipid-rich matrices can substantially enhance isomerization; for example, repeated injection of oil-rich matrices in GC–MS/MS analysis resulted in approximately a 30% formation of stereoisomeric isomers. These observations highlight that fatty or highly pigmented commodities require tailored sample preparation to preserve enantiomeric integrity, which was beyond the scope of the present study focused on apples.

Question 4：While the focus on cypermethrin’s isomerization is justified, the paper must clarify how its temperature-driven optimization offers a substantive advance over existing chiral LC-MS/MS methods (e.g., Zhao et al., 2019), beyond being analyte-specific.

Response 4: We thank the Reviewer for this important comment and for pointing out the need to better contextualize the methodological advance relative to existing chiral LC–MS/MS approaches, including Zhao et al. (2019). We would like to clarify that the method reported in Zhao et al. primarily focused on the chiral separation of terramethrin and α-cypermethrin and did not demonstrate complete chromatographic resolution of all eight stereoisomers of cypermethrin. In the present study, we initially evaluated the chromatographic conditions described by Zhao et al., including the use of the same chiral column. However, under these conditions, satisfactory separation of all cypermethrin stereoisomers could not be achieved in our laboratory. This limitation prompted a systematic optimization effort involving alternative chiral stationary phases and chromatographic conditions. Through this approach, we identified a distinct column chemistry and optimized temperature-controlled separation parameters, enabling, for the first time, complete and reproducible resolution of all eight cypermethrin stereoisomers in an apple matrix. To further clarify the methodological progress achieved in this work, Table 3 has been added to the revised manuscript, providing a direct comparison between previously reported chiral LC–MS/MS methods and the present approach. This table highlights the key differences in chromatographic conditions and clearly illustrates the advances achieved in terms of stereoisomeric resolution and analytical performance.

Question 5：The claim that non-enantioselective methods “underestimate toxicological risk” lacks quantitative context. A brief risk scenario (e.g., if α-cypermethrin constitutes 20% of total but is 10× more toxic) would strengthen the regulatory argument.

Response 5: We agree that, in its original form, the statement regarding potential underestimation of toxicological risk by non-enantioselective methods lacked sufficient quantitative context and could be misleading. Therefore, this wording has been removed from the revised manuscript. Under the current European Union regulatory framework, this consideration is not directly applicable, as the residue definition for enforcement purposes is “cypermethrin (cypermethrin including other mixtures of constituent isomers (sum of isomers)) (F)”, and compliance with MRLs is assessed based on the total sum of cypermethrin isomers rather than individual stereoisomers. Consequently, from a present regulatory perspective, enantiomer-specific toxicity does not affect MRL compliance. The development of an enantioselective analytical method, including the capability to distinguish α-cypermethrin from other cypermethrin stereoisomers, was motivated by the anticipated regulatory evolution toward a separate residue definition and MRL for α-cypermethrin, which has already been established as a distinct active substance. The original wording was intended to highlight this forward-looking analytical relevance rather than to imply immediate regulatory consequences. We fully agree with the Reviewer that quantitative toxicological risk scenarios would only be scientifically and regulatory justified once enantiomer-specific residue definitions and MRLs are formally implemented. This clarification has now been incorporated into the manuscript to ensure accurate interpretation and regulatory consistency.

Question 6：The conclusion asserts suitability for “routine analysis,” yet the 30-min isocratic LC run and expensive chiral columns may hinder high-throughput labs. This practical limitation should be acknowledged.

Response 6: We thank the Reviewer for this important and constructive comment. In response, the term “routine” has been removed from the Conclusion to avoid any potential overstatement of the method’s applicability. Additionally, the following sentence has been added to acknowledge the practical limitations: “However, due to the 30-minute isocratic LC run and the requirement for costly chiral columns, its application in high-throughput laboratories may be limited. This practical consideration should be taken into account when planning routine residue monitoring.” These revisions ensure that the Conclusion accurately reflects both the analytical performance and the practical considerations relevant to the method.

Question 7：The assignment of eight cypermethrin stereoisomers (Fig. 1) lacks confirmation of absolute configuration. Were authentic isomer standards used? Or based solely on retention order from literature?

Response 7:  We thank the Reviewer for this important comment. The assignment of the eight cypermethrin stereoisomers shown in Figure 1 was performed using accredited cypermethrin standards, which contain a defined mixture of stereoisomers with composition provided by the manufacturer; it was alpha, beta, theta, and zeta cypermethrin. We do not have individual standards for each stereoisomer, as the cost of acquiring separate standards for all eight isomers exceeds the budget of the current study.

To ensure reliable identification, the elution order was cross-checked against the data reported by the European Union Reference Laboratory (EURL) for Pesticides (https://www.eurl-pesticides.eu/library/docs/srm/EurlSrm_Observation_Cypermethrins_with_LC-MSMS.pdf). This approach—using authentic standards of the mixture combined with reference elution order—is widely accepted in the field of chiral cypermethrin analysis and provides a high level of confidence in the stereoisomer assignments, even in the absence of absolute configuration determination.

Question 8：The manuscript treats EFSA’s proposed dual MRL framework as established policy (lines 83-91), though it remains provisional. This could mislead readers about current regulatory requirements.

Response 8: We thank the reviewer for highlighting this important regulatory nuance. The text has been revised to clarify that EFSA’s proposal regarding a dual MRL framework for cypermethrin and α-cypermethrin is not yet established policy, but rather a provisional recommendation intended to support a refined risk assessment.

Question 9：The terms “enantiomer” and “stereoisomer” are used interchangeably. Cypermethrin has diastereomers (due to multiple chiral centers), not just enantiomers. Precise stereochemical language is essential.

Response 9: We appreciate the Reviewer's important comment. We acknowledge that cypermethrin contains multiple chiral centers and therefore forms both diastereomers; therefore, precise stereochemical terminology is essential. Accordingly, the manuscript has been revised throughout to use appropriate terms. In the revised manuscript, the term “stereoisomer” is consistently used throughout the text wherever appropriate, ensuring accurate representation of cypermethrin’s stereochemistry.

Question 10：The conclusions state that “the developed LC-MS/MS method... demonstrates high sensitivity, accuracy, precision, and linearity, which confirms its suitability for routine residue analysis.” This is supported for LC-MS/MS. However, the concluding phrase “enabling accurate stereochemical characterization” is too broad, as the failed GC-MS/MS arm suggests significant challenges in characterizing all stereoisomers by this complementary technique. The conclusions should be refined to reflect that accurate characterization was achieved specifically by the optimized LC-MS/MS method, acknowledging the limitations encountered with GC-MS/MS.

Response 10: We thank the Editor for this constructive suggestion. In response, we have clarified the Conclusions by adding the following sentence: “It should be noted that accurate stereochemical characterization was achieved specifically by the optimized LC–MS/MS method, while the GC–MS/MS approach did not provide complete stereoisomer resolution under the tested conditions.” This revision ensures that the manuscript accurately reflects the capabilities of the LC–MS/MS method, while acknowledging the limitations of GC–MS/MS, thereby addressing concerns regarding the overgeneralization of stereochemical characterization.

Question 11：The introduction poses the challenge of accurate stereoisomer determination for refined risk assessment and MRL setting. While the developed LC-MS/MS method is a step forward, the conclusions do not explicitly circle back to how this method directly addresses the regulatory problem of setting isomer-specific MRLs (e.g., for alpha-cypermethrin) mentioned in the introduction. A stronger concluding statement linking the methodological success to this primary question is needed.

Response 11: We thank the reviewer for this important comment. To explicitly link the analytical development to the regulatory challenge highlighted in the Introduction, the Conclusions section has been revised, and an additional sentence has been added at the end of this paragraph.

   This work provides valuable technical insights into chiral pyrethroid analysis, particularly regarding temperature effects on isomer resolution. Major revisions are recommended: (1) include multi-matrix validation; (2) clarify method limitations and regulatory status; (3) correct terminology and strengthen stereochemical assignment. Acceptable after major revision.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

There are two tables both numbered “Table 2”. The first Table 2 (pp. 7-8) lists MRM parameters for GC-MS/MS, while the second Table 2 (p. 10) presents method validation data (recoveries and precision). This duplication of table numbers seriously undermines the manuscript's rigor and readability and must be corrected. Beyond this issue, the authors have comprehensively and effectively addressed the previous round of review comments, including enhancing methodological details, optimizing the discussion structure, and adding comparative data, leading to a significant improvement in the scientific quality of the work. Therefore, it is recommended that the manuscript be accepted after minor revisions, pending the resolution of the table numbering conflict (renumbering and updating all in-text citations) and the completion of the other minor adjustments.

Author Response

Comment 1: There are two tables both numbered “Table 2”. The first Table 2 (pp. 7-8) lists MRM parameters for GC-MS/MS, while the second Table 2 (p. 10) presents method validation data (recoveries and precision). This duplication of table numbers seriously undermines the manuscript's rigor and readability and must be corrected. Beyond this issue, the authors have comprehensively and effectively addressed the previous round of review comments, including enhancing methodological details, optimizing the discussion structure, and adding comparative data, leading to a significant improvement in the scientific quality of the work. Therefore, it is recommended that the manuscript be accepted after minor revisions, pending the resolution of the table numbering conflict (renumbering and updating all in-text citations) and the completion of the other minor adjustments. 

Response 1: We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments. We thank the Reviewer for pointing out the duplication of table numbering in the manuscript. The error has been corrected by renumbering the tables appropriately. All corresponding in-text references have also been carefully revised and updated to ensure consistency and clarity throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, the quality of this paper has been improved. The only remaining suggestion is to strengthen the connection to apples throughout the manuscript, following the change in the title. At present, the link to apples is not sufficiently clear, particularly in the Introduction.

Author Response

Comments 1: Overall, the quality of this paper has been improved. The only remaining suggestion is to strengthen the connection to apples throughout the manuscript, following the change in the title. At present, the link to apples is not sufficiently clear, particularly in the Introduction.

Response 1: 

We thank the Reviewer for this valuable suggestion. In response, the Introduction has been revised to more clearly and explicitly emphasize the relevance of apples as the target matrix, in line with the revised title. The following text has been added to the Introduction:

Apples were selected as the target matrix due to their widespread consumption and frequent exposure to pyrethroid insecticides. Moreover, the relatively simple composition of the apple matrix allows for the straightforward determination of pyrethroid residues with minimal matrix interferences.

We sincerely thank the Reviewer for the constructive feedback, which has helped further to improve the clarity and focus of the manuscript.