The Influence of the Hydrogen Isotope Effect on the Kinetics of Amoxicillin and Essential Elements Interaction
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript presents an interesting and fairly coherent optical-kinetic study of amoxicillin-zinc complex formation in H₂O vs D₂O. Experiments on Zn²⁺ vs Ca²⁺/Mg²⁺ with polarimetry, fluorimetry, and UV–vis data support a two-stage binding model.
Quantitative KIE analysis across temperatures and Arrhenius treatment with distinct Ea values for H₂O vs D₂O provide a solid physico-chemical contribution. The use of AMX’s chirality and optical rotation as a kinetic observable is conceptually appealing and reasonably executed.
However, the manuscript overstates clinical implications and needs clarification and tightening in several methodological and interpretational areas. The step from in vitro chiral/complexation changes to “potential complete loss of bactericidal activity” is speculative and currently under-supported by data; no microbiological assay or in vivo-relevant system is presented.
The rationale and realism of some concentration choices (e.g., 4 mmol/L AMX and 40 mmol/L ZnCl₂, or 1:10 molar ratio) relative to intestinal or systemic conditions should be better justified and contextualized.
Some mechanistic claims (e.g., exact 2AMX:Zn²⁺ stoichiometry, “rigid intermolecular structure” leading to chirality cancellation) would benefit from additional or clearer evidence, or more cautious wording.
Minor comments
Use consistent symbols for rate constants (k₁, k₂) across text, figures, and table 1.
Check uniform formatting of units (e.g., 10⁻⁴ s⁻¹) and decimal separators in Table 1.
Ensure that all figure axes have units and that the kinetic curves in semilog coordinates are readable at the journal’s final size (especially Figure 7 inset).
For Figures 2–7, it would be helpful to indicate H₂O vs D₂O directly in legends, not only in the main text.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe interaction between commonly prescribed antibiotics and other medicines is an object of intensive investigation, but there is not sufficient information on how the complementary medicines, herbal remedies, and supplements are safe to take with antibiotics. From this point of view, the presented manuscript, dedicated to the chemical interactions between amoxicillin trihydrate and some essential element ions, taken as usual supplements (zinc, magnesium, and calcium) in solutions, is important. The study was conducted with varying water isotopic compositions, focusing on the kinetic isotope effect (KIE) and the impact of replacing hydrogen with deuterium in water on the reaction rates and mechanisms of antibiotic-metal ion interactions. The research underscores the significance of understanding drug-mineral supplement interactions, particularly in the context of polypharmacy and prolonged supplement use during antibiotic treatments. It also emphasizes the role of water's isotopic composition in pharmaceutical processes and its potential implications for drug efficacy and safety. I recommend publishing the manuscript with some minor revisions (additional clarification) concerning the following points:
- It was pointed out that the interaction between amoxicillin and zinc is more pronounced than interactions with magnesium or calcium due to the differences in electron configuration. Although I want to ask the Authors to comment in more detail on the differences between the s-block elements (magnesium and calcium) interactions with amoxicillin.
- Could polarimetric monitoring be used as a routine analytical method in pharmaceutical quality control or drug–drug interaction studies?
- How might the obtained results influence guidelines or recommendations for taking antibiotics and mineral supplements simultaneously? Should the Authors specify the correction of daily doses of zinc and/or amoxicillin?
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
presented work is interesting and add value in clinical practice. However, I have some suggestions.
INTRODUCTION
The statement that drug–drug interactions account for 21% of all hospitalizations is not supported by reference [1]. The cited source reports that 21% refers specifically to adverse drug event–related hospital admissions, not total hospitalizations, and therefore the percentage has been misinterpreted. Please revise this sentence to accurately reflect the data in the original reference. Additionally, please revise all others percentages
FTIR is a widely used and highly informative technique for detecting chemical interactions and is often considered a gold standard for assessing chemical incompatibility. It is notable that this method is not mentioned in the introduction. While FTIR alone is not sufficient to fully evaluate compatibility, since physical incompatibilities such as changes in crystallinity or polymorphism require complementary techniques like DSC or X-ray diffraction, it could provide valuable additional insight. The authors may consider citing relevant studies, such as https://www.mdpi.com/2227-9040/13/12/417 , which demonstrate the use of FTIR in combination with other methods for a comprehensive assessment of substance compatibility.
METHODS
The methodological descriptions provided for polarimetry, fluorimetry, and UV–Vis spectroscopy are very brief. For reproducibility and clarity, the manuscript should include details such as:
-
the type and number of samples analyzed,
-
sample preparation procedures,
-
solvents or media used,
-
path length of the polarimetric cell,
-
excitation and emission parameters for fluorimetry (if applicable),
-
and any other relevant experimental conditions
It is recommended that the authors justify the choice of buffer systems used in the study. In particular, the manuscript does not explain why a medium with pH 1.2 was not considered, which is relevant for simulating gastric conditions and assessing the behavior of the substances under acidic conditions. Providing a rationale for buffer selection would strengthen the experimental design and improve the relevance of the results.
CONCLUSION
The current conclusion is quite long and contains detailed experimental data that may be more appropriate for the Results section. I suggest condensing the Conclusion to focus on the main findings and their significance.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors of the manuscript investigated the chemical incompatibility between amoxicillin trihydrate and essential element salts using different methods across different pH values and in water isotopologues. They proposed a two-stage interaction mechanism: rapid ionic binding via the deprotonated carboxylate (common to Zn²⁺/Ca²⁺/Mg²⁺), followed by donor–acceptor coordination unique to Zn²⁺ that progressively reduces optical activity to near zero, interpreted as racemization or chirality cancellation in a putative 2AMX:Zn²⁺ complex. The authors quantified a solvent kinetic isotope effect (KIE) on the AMX–Zn²⁺ interaction, reporting slower kinetics and higher activation energy in D₂O (KIE ~2.0 at 20 °C). The work aims to highlight potential clinical implications for coadministration of amoxicillin and zinc supplements and suggests polarimetry as a sensitive tool to monitor chiral API–metal interactions. The clinical meaning, workload and creativity of the manuscript are enough for the acceptance by the journal Hydrogen. Here are some concerns for the authors to improve the quality of the manuscript:
1) In the Keywords section, the essential elements should be deleted.
2) For the Introduction section, the authors may need to rewrite this section. The authors may need to delete some information about drug-drug interactions as the work of the manuscript present none association with DDI. What is more, the Introduction section reads no logically enough. Therefore, the author may need to rewrite this section.
3) For Figure 1 and Figure 4, these two pictures are not read clearly enough. The authors may need to redraw these pictures using ChemDraw.
4) For Section 2, the authors provide detailed information about the materials and the equipment. However, the authors almost provide nothing about the methods. Therefore, the authors may need to provide such information.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for your response. The manuscript is now ready for publication in its current form.
Author Response
Thank you for your response
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors have revised the manuscript according to the concerns. The present version is recommended to be accepted by the journal Hydrogen. Congratulations to the auhtors.
Author Response
Thank you for your response
