PONE-D-25-03276A SYSTEMIC APPROACH TO ESTIMATE AND VALIDATE RP-HPLC ASSAY METHOD FOR REMDESIVIR AND FAVIPIRAVIR IN CAPSULE DOSAGE FORMPLOS ONE

Dear Dr. Khan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Abdelwahab Omri, Pharm B, Ph.D, Laurentian University

Academic Editor

PLOS ONE

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2. In the online submission form, you indicated that “Data id available from corresponding author on reasonable request”

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4.  Please ensure that you refer to Figure 1-3 in your text as, if accepted, production will need this reference to link the reader to the figure.

5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 1-7 in your text; if accepted, production will need this reference to link the reader to the Table.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In Assay method for dual drug combination by HPLC method is lacking novely and new finding.

Chromatogram of blank, specificity, and LLQ is missing

Without any innovative steps and methods like QbD, greenness, and chemometrics, it seems simple experimental methods. For a research article, the author should incorporate new methodology, processes, and findings that add value to existing research.

Reviewer #2: 1 References are cited as numbers superscripts, however are listed alphabetically , revise as per journal guidelines

2. Table no.7 degree Celsius sign missing

3. Elaborate on significance of degradation studies in discussion

Reviewer #3: Correct manuscript for grammatical mistakes.

Write complete source information for instruments, materials and software used in the proposed research work.

Format references properly as per guidelines.

The proposed method can be evaluated for greenness and whiteness profile.

For greenness and whiteness profile assessment of the proposed method, the authors can cite following recently published articles;

https://doi.org/10.1093/jaoacint/qsad013, https://doi.org/10.1007/s42250-024-01007-z, https://doi.org/10.1016/j.scp.2024.101523, https://doi.org/10.1093/chromsci/bmad054, https://doi.org/10.1093/jaoacint/qsad108

Reviewer #4: Reviewer Comments for Authors

Abstract: References should not be included in the abstract.

Mobile Phase Composition: It is preferable to state the mobile phase composition in terms of ratios rather than actual volumes.

Run Time Justification: A justification is needed for the extended run time for the separation of only two drugs.

Linearity Range & LOQ: Justify the selected linearity range for both drugs. Additionally, discrepancies between the linearity range and LOQ should be clarified.

Novelty Justification: The manuscript claims novelty, but several HPLC methods have been reported in the literature for this combination. The authors should justify why their method is unique compared to existing ones (e.g., references: https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/cem.3548?af=R, https://pmc.ncbi.nlm.nih.gov/articles/PMC10203959/).

Drug Purity: The purity of the drugs used should be mentioned in the materials section.

Methodology Heading (Section 2.4): The heading "Procedure" is vague. It should clearly define the purpose of the procedure.

Target Concentration Justification: The rationale for selecting target concentrations must be provided.

Incomplete Units: Some values in the methodology section are missing units. Ensure all numerical values have appropriate units.

Precision Studies: Percentage relative standard deviation (%RSD) should be reported for precision studies.

Robustness Studies: The robustness table should be revised for clarity, showing parameter variations and their impact more effectively.

Quality by Design (QbD) Application: The abstract mentions the application of QbD, but there is no detailed discussion on risk assessment, DOE application, MODR, or model validation in the results and discussion section. This needs to be addressed.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Attachments

Attachment

Submitted filename: PONE comments.docx

PONE-D-25-03276R1A SYSTEMIC APPROACH TO ESTIMATE AND VALIDATE RP-HPLC ASSAY METHOD FOR REMDESIVIR AND FAVIPIRAVIR IN CAPSULE DOSAGE FORMPLOS ONE

Dear Dr. Khan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Apr 09 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Abdelwahab Omri, Pharm B, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear Dr. Khan,

Thank you for submitting the revised manuscript titled "A Systemic Approach to Estimate and Validate RP-HPLC Assay Method for Remdesivir and Favipiravir in Capsule Dosage Form" to PLOS ONE. We appreciate the efforts you and your co-authors have made in addressing the reviewers’ comments. After careful evaluation of your revisions, we find that the manuscript has significantly improved. However, a few minor concerns need to be addressed before we can proceed with final acceptance.

Required Revisions:

1. Explicit Comparison with Existing Methods & Justification of Uniqueness:

o While the manuscript claims novelty based on mobile phase composition and co-loading in oral capsules, it does not critically compare the proposed method with existing RP-HPLC methods.

o Please provide a detailed comparison with prior studies, highlighting the advantages of your method in terms of sensitivity, specificity, robustness, or applicability. A direct comparison table or a critical discussion in the introduction or discussion section would be beneficial.

2. Details on Risk Assessment, DOE Application, MODR, and Model Validation:

o The manuscript briefly mentions quality by design (QbD) and risk assessment but does not provide sufficient details on method operable design region (MODR) and model validation.

o Please include a structured discussion explaining the specific risk factors considered, how DOE (Design of Experiments) was applied, and how model validation was performed. Adding supporting data would enhance clarity.

3. Justification for Extended Run Time:

o The explanation provided for the extended run time due to remdesivir’s inconsistent elution pattern is acceptable. However, including chromatographic overlays or additional discussion on why shorter run times were not feasible would further strengthen the justification.

4. Explanation of LOQ and Linearity Discrepancies:

o The manuscript presents LOQ and linearity values but does not explain why any potential discrepancies might exist. If non-linearity was observed at higher concentrations, please clarify how the method ensures accuracy within the tested range.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

A SYSTEMIC APPROACH TO ESTIMATE AND VALIDATE RP-HPLC ASSAY METHOD FOR REMDESIVIR AND FAVIPIRAVIR IN CAPSULE DOSAGE FORM

PONE-D-25-03276R2

Dear Dr. Barkat Ali Khan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Abdelwahab Omri, Pharm B, Ph.D, Laurentian University, Canada

Academic Editor

PLOS ONE