Review Reports - Simultaneous Quantification of Fumonisins and Their Hydrolyzed Metabolites in Donkey Matrices: A Tool for Exposure Assessment and Toxicokinetic Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present study reports the development and full validation of a matrix-tailored LC–MS/MS method for the simultaneous quantification of fumonisins B₁, B₂, B₃ and their hydrolysed metabolites (HFB₁, HFB₂, HFB₃) in donkey plasma, urine, and feces. The method fulfils international validation criteria and, to the best of current knowledge, represents the first comprehensive analytical tool enabling multi-matrix fumonisin toxicokinetic and exposure studies in donkeys. Thus, the present study constitutes a solid methodological contribution and provides an essential foundation for future fumonisin toxicokinetic research in donkeys and potentially other species. Furthermore, the work is clear, transparent, and robustly validated, with higher applicability. Figures and tables are well–designed, and the discussion section is solid too. In my opinion, regarding the limitations, it would be desirable for the authors to include an application to real exposure or toxicokinetic samples to demonstrate performance under biological variability, although I understand that this is outside the scope of the study or relegated to future work (the objective of the work was methodological, not biological).

I have only some suggestions regarding the wording:

-Line 14: Please, replace the vague expression “satisfactory accuracy” with a more objective term such as “acceptable accuracy” or “accuracy within validation criteria”.

-Lines 16–17: Please, soften the absolute claim “This is the first reported method…” by adding a qualifying phrase such as “To our knowledge”.

-Line 45: Please, correct the grammatical inconsistency (Its primary toxicity”… by replacing “Its” with “Their”).

-Line 69: Please, correct the grammatical error “FBs and its metabolites” to “FBs and their metabolites”.

-Line 73: Please, avoid the absolute statement “are absent” and replace it with a more cautious formulation such as “are currently lacking”.

-Line 216: Please, replace the promotional wording “is highly advantageous” with a more neutral term such as “is advantageous” or “is desirable”.

-Line 218: Please, correct the sentence. It should be: “is often not required” to avoid overgeneralization.

-Line 233: Please, replace the strong wording “rendering the method ineffective” with a more precise expression, such as “rendering the method unsuitable under these conditions”.

-Line 386: Please, soften the statement “are not species-specific” to “are not inherently species-specific” to better reflect analytical variability across species.

For all the reasons mentioned previously, I consider that the current version of the manuscript must address these suggested changes (minor revisions) before publication in Toxins.

Author Response

Reviewer 1

Response:

Thank you very much for your careful review and positive assessment of our work. We are sincerely encouraged by your recognition of the methodological value and novelty of this study, as well as your favorable comments on the experimental design, presentation of figures and tables, and the overall clarity of the discussion.

We fully agree with your insightful suggestion regarding the application of this method to real exposure or toxicokinetic samples in future research to further demonstrate its performance under conditions of biological variability. This is indeed a key direction for our subsequent work.

We have thoroughly studied and addressed each of the points raised in your review. All suggested revisions will be diligently incorporated into the manuscript to enhance its accuracy, objectivity, and completeness. Your comments have significantly improved the quality and rigor of our paper, for which we are truly grateful.

I have only some suggestions regarding the wording:

Comments 1: -Line 14: Please, replace the vague expression “satisfactory accuracy” with a more objective term such as “acceptable accuracy” or “accuracy within validation criteria”.

Response 1: “satisfactory accuracy” has been revised to “acceptable accuracy” on line 15 of the revised manuscript.

Comments 2: -Lines 16–17: Please, soften the absolute claim “This is the first reported method…” by adding a qualifying phrase such as “To our knowledge”.

Response 2: “This is the first reported method…” has been modified to “To our knowledge, this is the first reported method…” on line 17 of the revised manuscript.

Comments 3: -Line 45: Please, correct the grammatical inconsistency (Its primary toxicity”… by replacing “Its” with “Their”).

Response 3: “Its primary toxicity” has been corrected to “Their primary toxicity” on line 48 of the revised manuscript.

Comments 4: -Line 69: Please, correct the grammatical error “FBs and its metabolites” to “FBs and their metabolites”.

Response 4: “FBs and its metabolites” has been corrected to “FBs and their metabolites” on line 76 of the revised manuscript.

Comments 5: -Line 73: Please, avoid the absolute statement “are absent” and replace it with a more cautious formulation such as “are currently lacking”.

Response 5: We have softened the absolute statement as suggested, changing “are absent from” to “are currently lacking in” on line 79 of the revised manuscript.

Comments 6: -Line 216: Please, replace the promotional wording “is highly advantageous” with a more neutral term such as “is advantageous” or “is desirable”.

Response 6: “is highly advantageous” has been changed to “is advantageous” on line 217 of the revised manuscript.

Comments 7: -Line 218: Please, correct the sentence. It should be: “is often not required” to avoid over generalization.

Response 7: “is generally not required” has been revised to “is often not required” on line 221 of the revised manuscript.

Comments 8: -Line 233: Please, replace the strong wording “rendering the method ineffective” with a more precise expression, such as “rendering the method unsuitable under these conditions”.

Response 8: “rendering the method ineffective” has been replaced with “rendering the method unsuitable under these conditions” on lines 234-235 of the revised manuscript.

Comments 9: -Line 386: Please, soften the statement “are not species-specific” to “are not inherently species-specific” to better reflect analytical variability across species.

Response 9: We thank the reviewer for the constructive feedback regarding the phrasing of our method's broader applicability. The reviewer is correct in suggesting a more cautious tone to acknowledge potential analytical variability across different species.

we have replaced the original phrasing stating that principles "are not species-specific" with a more qualified and informative statement. The revised text now indicates that the core analytical strategies "could provide a valuable starting point for method adaptation in other species" and frames the work as offering a "methodological framework and reference." (Please see lines 404-406 of the revised manuscript.)

For all the reasons mentioned previously, I consider that the current version of the manuscript must address these suggested changes (minor revisions) before publication in Toxins.

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract

Line 15 and 16: The reported LOQ range is potentially confusing, since it spans different matrices and units (µg/L for plasma and µg/kg for feces) without a clear distinction.

Introduction

In the introduction, the authors could briefly mention whether there are any comparative studies with horses, given that they are the closest species and are known to be highly sensitive to fumonisins. This would reinforce the hypothesis as to why it is critical to study donkeys specifically. Furthermore, it is not clearly justified why donkeys are an interesting model for fumonisins, beyond their economic importance. It would be interesting to see impact or food safety studies.

Line 59: Please check figure 1. I cannot see the resolution of Figure 1

Line 61: “Equus asinus)” should be written in italics

Line 100: In the sentence “This clear research gap underscores the necessity for a novel analytical approach that is comprehensively validated for the simultaneous analysis of multiple matrices in donkeys.” the type of matrices is not specified. It would be helpful to indicate already at this point which biological matrices are targeted (e.g., plasma, urine, feces), in order to better define the scope of the study. Are there any previous review articles addressing fumonisins and/or their hydrolyzed metabolites in these types of biological matrices?

Results and discussion

Line 197: The carry-over problem is common with fumonisins due to their polar and acidic nature. Although the proposed washing protocol reduces carry-over to <0.5%, it would be advisable for the authors to mention whether they performed a solvent blank injection after the higher concentration samples during validation to ensure that there is no long-term residual accumulation in the column.

Further on, line 348 states: blank faecal sample was unavailable, but I think it is necessary to refer to this in this point: Line 264 Faeces sample.

Some recoveries slightly exceeding 100% were observed. A brief discussion on potential matrix-induced signal enhancement or internal standard correction effects would strengthen the validation discussion.

Line 360: I don´t understand this:” 0.1, 1.0, and 60 μg/L (or μg/kg),”

Line 361: In the sentence “The sensitivity achieved with the present method is comparable to that of other reported methods using similar cleanup approaches,” the authors may consider further elaborating on the advantages and limitations of the proposed method compared with those studies.

Conclusions

Line 388: he conclusions are well-supported by the experimental data provided. However, the statement regarding the applicability of the method to “other species” should be phrased more cautiously. Given that different species have distinct diets, gastrointestinal microbiotas, and varying urine/feces compositions.

Materials and methods

Line 425: “For plasma samples, a 1 mL aliquot was spiked with 20 μL of the mixed internal standard working solution, which contained each internal standard at 0.1 μg/L. Please specify”.

Line 461: “The injection volume was 5 μL” but before is written 1 μL

Line 480: Please specify the number of replicates used for matrix effect evaluation.

Author Response

Reviewer 2

Comments 1: Abstract

Line 15 and 16: The reported LOQ range is potentially confusing, since it spans different matrices and units (µg/L for plasma and µg/kg for feces) without a clear distinction.

Response 1: Thank you for pointing this out. Accordingly, the LOQ range has been clarified to explicitly state the units for each matrix: “0.1–0.15 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces.” This revision can be found on lines 16–17 of the revised manuscript.

Comments 2: Introduction

Response 2: We sincerely thank the reviewer for this insightful and constructive suggestion. We fully agree that placing our study within the context of the sensitivity of equids, particularly horses, would significantly strengthen the rationale for investigating fumonisins in donkeys.

As suggested, we have revised the introduction (in the third paragraph) to explicitly address this point. We now state:

“Notably, equids are known to be highly sensitive to fumonisin toxicosis, with horses (Equus caballus), the closest relative to donkeys, developing the characteristic and fatal equine leukoencephalomalacia (ELEM) upon FB1 exposure [2]. … Despite the known sensitivity of the equine family and the economic importance of donkeys, scientific research on the absorption, distribution, metabolism, excretion, and toxicological effects of FBs in donkeys is extremely limited.”

Comments 3: Line 59: Please check figure 1. I cannot see the resolution of Figure 1

Response 3: Thank you for pointing out this issue. I have redrawn the molecular structure diagram in an appropriate format to ensure sufficient clarity and resolution.

Comments 4: Line 61: “Equus asinus)” should be written in italics

Response 4: “Equus asinus” has been italicized on lines 64 of the revised manuscript.

Comments 5: Line 100: In the sentence “This clear research gap underscores the necessity for a novel analytical approach that is comprehensively validated for the simultaneous analysis of multiple matrices in donkeys.” the type of matrices is not specified. It would be helpful to indicate already at this point which biological matrices are targeted (e.g., plasma, urine, feces), in order to better define the scope of the study. Are there any previous review articles addressing fumonisins and/or their hydrolyzed metabolites in these types of biological matrices?

Response 5: Thank you for your valuable comments. The issues you pointed out are indeed pertinent, as we did not specify the types of biological matrices targeted in the study in the original manuscript, which might have led to an unclear definition of the research scope.

Based on your suggestion, we have revised the original text to explicitly state the three key biological matrices targeted in this study: plasma, urine, and feces. Citing insights from review articles, we note that these matrices are commonly used samples for assessing mycotoxin exposure, toxicokinetics, and excretion behaviors in animals, and are crucial for comprehensively understanding the fate of fumonisins in donkeys. The specific revisions can be found in lines 107-112 of the revised version.

Comments 6: Results and discussion

Response 6: We sincerely thank the reviewer for raising this critical point regarding the assessment of long-term system carry-over, which is indeed essential for validating the robustness of an analytical method for continuous runs. The reviewer is absolutely correct.

In fact, this was indeed our standard practice during the validation process. In our method validation experiments, a solvent blank was routinely injected immediately after the high-concentration calibration standards to check for any potential carry-over of FBs. At the spiked concentrations used in this validation, no significant carry-over of FBs was observed. As suggested by the reviewer, we have now explicitly added the following statement in the revised manuscript: "To confirm the absence of long-term carry-over, solvent blanks injected after the highest calibration standard showed no detectable analyte peaks (S/N < 3), validating the effectiveness of the optimized wash protocol." (Please see lines 203-206 of the revised manuscript.)

Comments 7: Further on, line 348 states: blank faecal sample was unavailable, but I think it is necessary to refer to this in this point: Line 264 Faeces sample.

Response 7: We thank the reviewer for the valuable suggestion to improve the clarity of our method description. As recommended, we have revised the beginning of the "2.3.2. Faeces sample" section to explicitly state the unavailability of blank feces and the selection of the sample with the lowest FBs background for our experiments.

The revised text now reads: “Donkey feces contain undigested forage, feed components, and endogenous metabolites. Importantly, true blank fecal samples (free of fumonisin contamination) were unavailable, as preliminary analysis detected FBs in all samples collected. For method development and validation, a fecal sample with the lowest measured FB₁ concentration (375 μg/kg dry matter) was selected as the representative matrix for optimization and standard addition experiments. FBs originate from...” (Please see lines 268-272 of the revised manuscript.)

This revision enhances the logical flow of the manuscript.

Comments 8: Some recoveries slightly exceeding 100% were observed. A brief discussion on potential matrix-induced signal enhancement or internal standard correction effects would strengthen the validation discussion.

Response 8: We thank the reviewer for this insightful observation. The reviewer correctly notes that the recoveries presented in Table 2 are already corrected using isotope-labeled internal standards, making the simple explanation of “signal enhancement corrected by IS” insufficient.

In response, we have revised the added discussion in the method validation section (2.3.5) to provide a more precise explanation. We now attribute the recoveries slightly above 100% to the inherent complexity of the fecal matrix and potential trace-level background interference. The text clarifies that despite internal standard correction, residual matrix effects—potentially from components co-eluting with the analytes—could lead to this observation.

The revised text reads: “The occasional recoveries slightly exceeding 100% (Table 2), particularly for the FBs in feces, are likely a combined result of inherent matrix complexity and background interference. Despite the application of isotope-labeled internal standard correction for FBs, residual matrix-induced signal enhancement, possibly co-eluting with the analytes, could contribute to this observation.” (Please see lines 366-370 of the revised manuscript.)

Comments 9: Line 360: I don´t understand this:” 0.1, 1.0, and 60 μg/L (or μg/kg),”

Response 9: We appreciate the reviewer for pointing out the ambiguity in the presentation of the Limit of Quantification (LOQ) values. The confusion is understandable, and we apologize for the lack of clarity.

We have revised the sentence in Section 2.3.5 (Method validation) to explicitly state each LOQ value along with its corresponding unit and matrix. The revised text reads: “Specifically, the LOQs for FBs were 0.1 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces, respectively. Correspondingly, the LOQs for HFBs were 0.15 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces.” (Please see lines 373-375 of the revised manuscript.)

Comments 10: Line 361: In the sentence “The sensitivity achieved with the present method is comparable to that of other reported methods using similar cleanup approaches,” the authors may consider further elaborating on the advantages and limitations of the proposed method compared with those studies.

Response 10: We thank the reviewer for the valuable suggestion to deepen the discussion regarding method comparison. Following the reviewer's advice, we have expanded the discussion in the final paragraph of Section 2.3.5 (Method validation). Immediately after comparing the sensitivity of our method to others, we have added a brief statement highlighting the key practical advantage (throughput and cost-effectiveness due to simplified cleanup) and acknowledging a potential trade-off (possible need for further optimization for highly complex matrices) of our strategy compared to methods employing more rigorous purification.

The added text reads: “The key advantage of our method is its speed and cost-effectiveness, as it avoids the need for specialized purification columns. However, for samples with highly complex matrices, this simplified preparation may need further optimization to manage stronger matrix interference.” (Please see lines 378-381 of the revised manuscript.)

Comments 11: Conclusions

Response 11: We thank the reviewer for the constructive comment regarding the phrasing of the method's applicability to other species. As suggested, we have revised the concluding statement in Section 3. Conclusions to reflect a more careful and qualified perspective.

The revised text reads: “Importantly, the core analytical principles and matrix-tailored sample preparation strategies (e.g., SALLE for complex fluids, DES for plasma) could provide a valuable starting point for method adaptation in other species. Therefore, this work not only addresses an immediate need in donkey research but also offers a methodological framework and reference for investigating fumonisin metabolism and exposure in other livestock and animal species.” (Please see lines 402-408 of the revised manuscript.)

Comments 12: Materials and methods

Line 425: “For plasma samples, a 1 mL aliquot was spiked with 20 μL of the mixed internal standard working solution, which contained each internal standard at 0.1 μg/L. Please specify”.

Response 12: We sincerely thank the reviewer for raising this critical point regarding the internal standard spiking procedure. The reviewer correctly identified an unclear description and a typographical error in the initial manuscript.

In response, we have thoroughly revised the relevant sections to ensure absolute clarity and accuracy.

① We have corrected the typo in the concentration unit and clearly stated that two distinct internal standard working solutions were prepared: one at 1 mg/L (for urine and feces) and another at 0.1 mg/L (for plasma), each containing all labeled analytes.

② Detailed specification of the spiking procedure (Section 4.3): For each matrix—plasma, urine, and feces—we now explicitly specify:

a) The volume added.
b) The concentration of the working solution used.
c) The resulting nominal concentration of the internal standard in the sample after spiking.

Please see lines 423-426, 445-447, 457-459, and 469-472 of the revised manuscript.

Comments 13: Line 461: “The injection volume was 5 μL” but before is written 1 μL

Response 13: We sincerely thank the reviewer for their meticulous attention to detail in identifying this apparent inconsistency in the injection volume.

The reviewer is absolutely correct in noting the discrepancy. This arose because Figure 2 presents chromatographic data collected during the initial method optimization phase. At that stage, a 1 μL injection volume was used for preliminary screening to conserve standards and minimize potential system carryover during the evaluation of different columns and mobile phases. The final, validated method parameters, as detailed in Section 4.4, utilize a 5 μL injection volume to achieve the required sensitivity for quantifying low-level analytes in biological matrices.

To resolve this ambiguity and prevent confusion, we have clarified the caption of Figure 2. The revised caption now explicitly states that the data were obtained during the optimization phase with a 1 μL injection and notes the different volume used in the final method.

Revised Figure 2 Caption:

“Figure 2. Extracted ion chromatograms (XICs) of the target analytes (20 ng/mL in solvent) obtained during method optimization with an injection volume of 1 μL under different mobile phase conditions... (Note: The final validated method uses an injection volume of 5 μL.)” (Please see lines 165-168 of the revised manuscript.)

Comments 14: Line 480: Please specify the number of replicates used for matrix effect evaluation.

Response 14: We thank the reviewer for pointing out the need to specify the experimental design for the matrix effect evaluation.

As suggested, we have revised the method validation section (Section 4.5) to explicitly state the number of replicates used in the matrix effect assessment. The text now clarifies that the comparison was performed using three replicate measurements (n = 3) for each matrix. (Please see lines 507-508 of the revised manuscript.)

Reviewer 3 Report

Comments and Suggestions for Authors

Simultaneous quantification of fumonisins and their hydrolysed metabolites in donkey matrices : a tool for exposure assessment and toxicokinetic studies

The authors did efforts to optimize an extraction protocol and validate a method for the analysis of fumonisins and their hydrolysed metabolites in donkey matrices, which is interesting for this particular animal species, but also for future exposure assessments and toxicokinetic studies in other animals.

The experiments for method optimization were well designed and thoroughly investigated. The choice of the final method parameters is critically motivated, which is interesting for the reader of the manuscript. The manuscript is clearly written.

The method was validated according to international guidelines and the results are shown. Although real samples have not been analysed using the optimized method, the applicability of the method is promising.

Therefore, the manuscript can be accepted for publication in Toxins following minor revision.

Some minor additional remarks:

Section 1. Introduction

1, line 40: please explain the abbreviation DDGS.
3, line 86 – 94: authors are mentioned in these paragraphs (i.e. Siegrid De Baere et al; Prathapkumar H. Shetty and Ramesch V. Bhat; Shuo Shang et al); however, it is only necessary to mention the surname in the text and not the whole name of the authors.

Section 2. Results and discussion

Sub-section 2.1. “Optimization of MS parameters”

4, table 1: In this table, it is indicated that FB2 elutes before FB3 and HFB2 before HFB3. Are the authors sure about this elution order? In other manuscripts in the literature (DOI 10.1002/jssc.201000423, DOI 10.1002/jssc.201200753; https://doi.org/10.3390/toxins14020131; …), it is reported that FB3 elutes before FB2. In Figure 2, it is indicated that FB3 and HFB3 eluted before FB2 and HFB2. Probably, a writing error occurs in Table 1? Could the authors check this ?

Sub-section 2.3. “Sample preparation”

7, Figure 4 and other figures: it is not described how the matrix-corrected extraction recoveries were determined. Can the authors add the procedure to the manuscript (in the materials and methods section) ?

Abbreviations : not all abbreviations were listed.

Author Response

Reviewer 3

Simultaneous quantification of fumonisins and their hydrolysed metabolites in donkey matrices : a tool for exposure assessment and toxicokinetic studies

Therefore, the manuscript can be accepted for publication in Toxins following minor revision.

Response: We sincerely thank you for the thorough evaluation and positive feedback on our manuscript titled "Simultaneous quantification of fumonisins and their hydrolysed metabolites in donkey matrices: a tool for exposure assessment and toxicokinetic studies." We are pleased to learn that the reviewer found the experimental design rigorous, the methodology clearly described, and acknowledged the potential applicability of the method for exposure assessment and toxicokinetic studies in donkeys and other animal species.

We will carefully address all the specific comments provided by the reviewer and make the necessary revisions to further improve the clarity, accuracy, and scientific rigor of the manuscript. Thank you once again for your time and valuable guidance throughout the review process.

Some minor additional remarks:

Section 1. Introduction

Comments 1: 1, line 40: please explain the abbreviation DDGS.

Response 1: We thank the reviewer for pointing out that the abbreviation DDGS was not defined upon its first use in the manuscript. And now we have revised the sentence in the Introduction section (Line 42) to include the full explanation of the abbreviation DDGS upon its first mention. The text now reads: “...such as wheat bran and distillers dried grains with solubles (DDGS), may contain...”

Comments 2: 3, line 86 – 94: authors are mentioned in these paragraphs (i.e. Siegrid De Baere et al; Prathapkumar H. Shetty and Ramesch V. Bhat; Shuo Shang et al); however, it is only necessary to mention the surname in the text and not the whole name of the authors.

Response 2: We thank the reviewer for the valuable comment regarding the proper formatting of author names in the text. We have removed the given names and initials, retaining only the surname(s) followed by the publication year (e.g., “De Baere et al. (2018)”, “Shetty and Bhat (1998)”, “Zhang et al. (2022)”).

Comments 3: Section 2. Results and discussion

Sub-section 2.1. “Optimization of MS parameters”

4, table 1: In this table, it is indicated that FB2 elutes before FB3 and HFB2 before HFB3. Are the authors sure about this elution order? In other manuscripts in the literature (DOI 10.1002/jssc.201000423, DOI 10.1002/jssc.201200753; https://doi.org/10.3390/toxins14020131; …), it is reported that FB3 elutes before FB2. In Figure 2, it is indicated that FB3 and HFB3 eluted before FB2 and HFB2. Probably, a writing error occurs in Table 1? Could the authors check this ?

Response 4: We sincerely thank the reviewer for their meticulous observation regarding the elution order of FB2/FB3 and HFB2/HFB3 in Table 1. The reviewer is absolutely correct. There was indeed a writing error in Table 1. The retention times (RT) for the isomeric pairs FB2/FB3 and HFB2/HFB3 were inadvertently transposed. As correctly pointed out by the reviewer and as clearly shown in Figure 2 of our manuscript, FB3 elutes before FB2, and HFB3 elutes before HFB2. This order is consistent with the literature cited by the reviewer.

We have carefully corrected Table 1 to reflect the accurate elution order and corresponding retention times. The values in the "RT (min)" column for FB2, FB3, HFB2, and HFB3 have been swapped accordingly.

Comments 4: Sub-section 2.3. “Sample preparation”

7, Figure 4 and other figures: it is not described how the matrix-corrected extraction recoveries were determined. Can the authors add the procedure to the manuscript (in the materials and methods section) ?

Response 4: We appreciate the reviewer's request for clarification regarding the determination of matrix-corrected recoveries presented in the figures.

To ensure full transparency, we have added a clear description of the recovery calculation procedure to the Materials and Methods section (4.5. Method validation). The added text states: “The matrix-corrected recovery was also calculated to evaluate the efficiency of the sample preparation procedure. It was determined by comparing the peak areas of analytes spiked into blank matrix before extraction with those spiked into the post-extracted blank matrix after extraction (matrix-matched standards), according to the following formula: Matrix-corrected recovery (%) = (Peak area of pre-spiked sample / Peak area of post-spiked sample) × 100.” (Please see lines 510-515 of the revised manuscript.)

Comments 5: Abbreviations : not all abbreviations were listed.

Response 5: We sincerely thank the reviewer for this meticulous observation. We have conducted a thorough check of all abbreviations used throughout the manuscript, including the main text, figures, tables, and footnotes. We have identified and added the missing abbreviations to the “Abbreviations” list at the revised manuscript. The added entries are as follows:

DDGS: Distillers dried grains with solubles

ELEM: Equine leukoencephalomalacia

IARC: International agency for research on cancer

PPE: Porcine pulmonary edema

pKa: Acid dissociation constant

SAX: Strong anion exchange

S1P: Sphingosine-1-phosphate

XIC: Extracted ion chromatogram