PONE-D-24-56221Thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and biotin hard gelatin capsules prepared in advance and stored for the treatment of pediatric metabolic diseases: a safer alternativePLOS ONE

Dear Dr. Curti,

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PLOS ONE

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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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: N/A

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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: No

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Dear Authors,

You conducted very important and applicable study for the pharmacy practice. However, I have some comments and questions:

line 26: I don't believe that magistral practice is characterized by the absence of any quality control, it's just not mandatory. Please correct

Please merge first two paragraph in the Introduction section

Please describe how to detect excipients with known effects in Table 1.

Methods: Why you used red carmine? Is it validated procedure for your API? Mixing one components do not mean mixing another one. Please provide information about mixing duration

Why silica din not used in the volume based method, as well as in riboflavine and pyridoxine capsules?

Why exactly 15 g of MCC was added? Do you have data about bulk and tapped density?

I suggest creating a Table with the formulations contents in the method section

line 167: Which water do you used?

Whether sinkers were used in the disintegration test?

line 220: I do not understand first sentence

Which guidelines you used for forced degradation studies?

Which statistical test was applied for conclusion about stability?

line 280: I do not understand what is dosing method in the context of HPLC

Please provide data about regulatory basis for compounding in advance

Reviewer #2: The paper demonstrates adequate scientific merit and professional value. However, some sections could benefit from improvements in grammatical expression to enhance clarity. I have outlined some specific observations in this regard below:

• Some results could be further interpreted. For instance, while degradation products of riboflavin are identified, their potential pharmacological relevance or toxicity is not addressed. Similarly, the implications of biotin degradation products, such as biotin sulfoxide, for the overall system are not explored.

• The text could benefit from more concise language. Some instances of grammatical errors and awkward phrasing, such as "yielded to 22% degradation," can be improved for better readability.

• Line 49 – 50. For clarity, consider revising the sentence “Although the parenteral route is often prescribed as an emergency treatment both for thiamine3 and pyridoxine, the oral route of administration must be privileged as soon as possible.” to “While the parenteral route is commonly used for emergency treatment with thiamine and pyridoxine, the oral route should be prioritized as soon as feasible.”

• Line 105, insert the word “rate” between the words “at” and “a” in the fragment “…(VWR)) at a flow of 1 mL.min-1”

• Line 112. Replace the word “weighted with “weighed”

• Line 126. Replace the word “weighted with “weighed”

• Line 138 Replace the word “weighted with “weighed”

• Line 158 Replace the word “weighted with “weighed”

• This problem persists through the methods – correct all

• Line 354 – 356. For better clarity, rewrite the sentence “When capsules are poured in 2 mL of water, the obtained suspension must be fully administered and gently mixed when it is taken before administering to avoid any risk of underdosing.” The fragment “.. the obtained suspension must be fully administered and gently mixed when it is taken before administering to avoid any risk of underdosing” is difficult to understand. A suggested version is “When capsules are dissolved in 2 mL of water, the resulting suspension should be gently mixed and fully administered to ensure proper dosing and avoid any risk of underdosing.”

• Consider revising the sentence “However, hard gelatin capsules compounding in advance secures the adminsttration in pediatric practice.” in line 357 to read as: "However, compounding hard gelatin capsules in advance ensures proper administration in pediatric practice."

Reviewer #3: This article by M. Bonino and colleagues presents a scientific investigation into the stability of four compounded hard capsules, containing essential vitamins, either thiamine hydrochloride (50 mg), riboflavin (50 mg), pyridoxine hydrochloride (50 mg), or biotin (40 mg). This article is particularly relevant to the hospital pharmacy community as it provides valuable data on the stability profiles of these combinations when compounded into capsule form. The findings of this study could have important practical applications for pharmacists involved in preparing and dispensing compounded medications for pediatric patients with specific vitamin deficiencies or related conditions.

The manuscript is well-written, scientifically sound, and presents a methodologically rigorous examination of the stability of these compounded capsules. The authors’ approach is thorough, and the results provide useful insights that could help hospital pharmacists ensure the safe and effective use of these vitamins.

A significant flaw in the manuscript is the lack of results regarding the 'uniformity of dosage units' assay, which is a critical test as per the European and US Pharmacopeia guidelines. Uniformity of dosage units is essential to ensure that each capsule contains the correct amount of active ingredient. Although it is likely that the authors performed this assay, the manuscript does not present any data on weight uniformity or a full pharmacopeial assay on drug content.

This omission is a major concern because without this data, it is difficult to assess whether the observed variability in the stability results stems from inconsistencies in the compounding process itself or from factors unrelated to formulation stability. In the absence of dosage uniformity data, the article lacks the necessary information to fully evaluate the consistency of the compounded capsules, which could ultimately affect their safety and efficacy in a clinical setting.

Other points to improve:

Introduction section:

1- Neonates and Hard Capsules: The authors mention neonates in the context of the study, but hard capsules are generally not considered an appropriate dosage form for this age group due to issues such as swallowing difficulties and the need for precise dosing. It would be helpful if the authors could provide further explanation or justification for why hard capsules need to be compounded in this case, particularly for neonates. Are there specific circumstances or clinical reasons that warrant this formulation, or could alternative dosage forms (e.g., liquids or powders) be more suitable for this population?

2- Table 1 - Terminology: In Table 1, the term ‘commercial drugs’ could be more accurately described as ‘available commercial formulations’ or ‘available commercial drug products’. This would provide a clearer and more precise reference. Additionally, it would be beneficial to specify which country or region this information pertains to, as pharmaceutical formulations can vary significantly by location.

3- Scope of the Article: The statement ‘In our hospital, prescribers…’ refers to a very specific context, which may limit the broader applicability of the article's findings. It would be helpful if the authors could clarify that the scope of the study extends beyond this specific setting. Ideally, the manuscript should aim to describe a problem or solution that is of general relevance to the wider hospital pharmacy community, rather than being confined to a single institutional context.

Methodology section:

4- Capsules Compounding: Please provide the exact weights of the powders used in the study, including the number of decimals that reflect the precision of the weighing scale. This will ensure greater transparency and reproducibility of the compounding process. Additionally, this section could be streamlined by describing the general compounding procedure briefly, followed by a table that presents the specific weightings for each formulation. This would make the methodology more concise and easier to follow.

5- HPLC Stability-Indicating Method: The description of the HPLC method could be more concise. This streamlined information will provide a clearer and more focused explanation without unnecessary repetition. Please include the following key details:

(i) The equipment used (e.g., HPLC system, detector, etc).

(ii) The exact duration of the analytical steps (sample analysis).

(iii) The duration of vortexing during sample preparation, as this can affect consistency.

6- Stability Studies: Please specify the reference for the climatic chamber used in the study. It would also be beneficial to use the terms ‘stability chamber’ or ‘environmental test chamber’ instead, as these are more commonly used in the pharmaceutical field to describe equipment used for stability testing. This terminology is more precise and aligns with established industry standards.

Results section:

7- Unless mistaken, this manuscript nor its supporting data does not seem to include the spectra for each compound alone and not degraded (reference spectra). This is a major concern as the readers should be able to judge how the degradation process affects the spectra.

8- Tables 3-4 : some RRT are close enough to the parent peak. Which guidelines or scientifically established data/norms did the authors use to validate the suitability of their methods in this matter ?

9- Table 7: include precise information on which criterion was accepted based on European or US pharmacopeia and why.

10- Table 8 and corresponding text: please precise which criterion/assay was used to reject the batches.

11- I would rather be able to see the standard deviations on each point, so maybe Figure 1 could be divided into 4 smaller figures to include this important dataset.

12- Figure 2: legend is not detailed enough to understand what is presented on this figure. Thin lines, thick lines? SD?

Discussion Section:

13- The discussion does not address whether the HPLC methods used are novel or adapted from existing protocols, such as those outlined in pharmacopeias or previous studies. Additionally, while degradation products are mentioned, they are only observed rather than identified.

14- For some of these products, the relative retention times (RRT) are similar to that of the parent drug, suggesting that the resolution of the method could be optimized. If improving the resolution is not feasible, this limitation should be explicitly acknowledged and discussed.

15- The discussion section is relatively lengthy and could benefit from being more concise while ensuring the relevance of each point.

16- Given that the results section requires revision, the discussion should also be adjusted to reflect these changes.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and biotin hard gelatin capsules prepared in advance and stored for the treatment of pediatric metabolic diseases: a safer alternative

PONE-D-24-56221R1

Dear Dr. Curti,

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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Kind regards,

Fırat Aşir

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. 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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. 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 #3: Yes

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5. 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: Yes

Reviewer #3: Yes

**********

6. 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: Language is clear. All is well written and explained. I do advice authors to read text one more time since some typos might be omitted.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #3: No

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