Evolution, Validation and Current Challenges in Bioanalytical Methods for Praziquantel: From Fluorometry to LC–MS/MS

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript entitled: 

Evolution, validation and current challenges in bioanalytical methods for praziquantel: From fluorometry to LC–MS/MS is a well-written review of bioanalytical methods with its clinical application. There are a few issues which authors should reconsider during revision.   1) Text from lines 826-832 should be removed (part of the MDPI template)  2) Please evaluate the entire text according to the typos mentioned in point 1.  3) In the discussion section, there are no references to literature. Please correct.  4) Please highlight the necessity of TDM of praziquantel in the introduction. 5) Please add ''best practices'' for good determination of praziquantel in conclusion or at the end of the discussion section. 

Author Response

We would like to thank the reviewer for the time and effort devoted to evaluating our manuscript. We sincerely appreciate the constructive comments and suggestions, which have significantly improved the quality and clarity of our work

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This work by Valladares Chávez et al. presents an overview of PZQ properties, pharmacokinetics and the evolution of analytical methodologies for the analysis and quantification of PZQ. The manuscript provides a thorough analysis, is very well written, with minor grammatical and/or spelling errors throughout the text.

Overall, this work and the content of the manuscript is applicable to the aims of the Scientia Pharmaceutica journal, thus, based on the above, I strongly recommend the publication of this manuscript, after considering some minor revisions, shown below:

 

1. The sentence in lines 79-81 has also been repeated earlier in the introduction.

 

2. Line 118: Authors may add “low detection limit” among with the high precision, as advantages of HPLC-MS/MS.

 

 

3. Table 1: The legend of the table should not include comments about the properties of PZQ presented in the table, or references. Thus, the second sentence should be removed (could be incorporated in the section’s text). The same applies for the legend of Figure 1 (lines 197-201), Figure 2 (lines 282-288), Figure 3 (lines 293-301), Figure 4 (lines 376-379),

 

4. Lines 435-436: HPLC and LC-MS should not be mentioned as two different analytical methods.

 

5. Lines 484-491: This paragraph describes the findings of works that used chromatographic techniques, thus it does not fit in the radiometric assay section.

 

6. Lines 542-543: HPLC and GC can both be regarded as specialized equipment that require trained professionals. The main advantage of HPLC is that can directly analyze PZQ samples, while for GC, due to the very high boiling point, derivatization is required, which costs in time and reagents and increases the complexity of the analytical protocol.

 

7. This should be changed in the whole manuscript (no comparison of HPLC vs HPLC-MS, or MS/MS). E.g. line 694 could refer to liquid chromatography techniques, rather than HPLC and HPLC-MS.MS.

 

8. I think that the sections that describe the analytical techniques should be changed. The distinction of GC as traditional and HPLC as modern is not valid. GC and HPLC method should be fall in the same section, e.g. named chromatographic techniques. Also, the distinction HPLC vs HPLC-MS is not valid. It is the same technique, using different detectors. So authors could create subsections like HPLC-UV, HPLC-MS, HPLC-MS/MS.

 

9. Table 2: There is no such thing as “optimal” chromatographic conditions, as many effective analytical protocols can be developed, depending on the instrument, column, mobile phase selected etc. I think this table should be removed.

 

10. Table 3: This table is also not valid and should be removed. LOD and LOQ can not be described in terms of percentages, but an absolute value of concentration.

 

11. Table 4: Fix “sensitive” to “sensitivity” in the GC column.

 

12. Table5: Fix in line 1 “mone” as “none”. Also, as equipment is referred for HPLC methods, the same should apply for GC. 3^rd column is confusing, it should be LOD, if only LOD values are referred.

 

13. Table 6: Add the corresponding detector for all HPLC examples stated in the table.

Author Response

We would like to thank the reviewer for the time and effort devoted to evaluating our manuscript. We sincerely appreciate the constructive comments and suggestions, which have significantly improved the quality and clarity of our work.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review manuscript provides a comprehensive overview of analytical methods developed for praziquantel (PZQ) quantification over several decades. The authors have compiled valuable information about traditional and modern techniques, along with relevant pharmacokinetic and physicochemical properties of this essential antiparasitic drug. While the manuscript contains substantial useful information and has the potential to become a valuable reference for researchers in the field, several significant issues require attention before publication. The following recommendations address structural, technical, and presentation concerns that would improve the quality and impact of this work.

1. It is recommended to restructure the “Introduction.” It should be more focused on analytical challenges rather than extensive disease epidemiology.
2. Table 3 incorrectly lists acceptance criteria for LOD and LOQ as "80%-120%"—these parameters are defined by signal-to-noise ratios or precision/accuracy at low concentrations, not percentage recovery ranges. It is recommended to revise this table with appropriate validation parameters and acceptance criteria according to ICH M10 guidelines.
3. While ICH guidelines are mentioned, the manuscript lacks detailed discussion of critical validation parameters (matrix effects, carryover, dilution integrity, bench-top stability), especially for LC-MS/MS methods. It is suggested to add a dedicated subsection addressing modern bioanalytical validation requirements.
4. The abstract mentions "green analytical chemistry, automation, and artificial intelligence-driven data processing," but these topics receive minimal coverage in the text. It is suggested to expand these sections substantially or revise the abstract to better reflect the actual content.
5. The review would benefit from a dedicated section discussing implementation challenges in resource-limited settings where PZQ is most needed. Include considerations of cost, equipment requirements, training needs, and electricity/water requirements for different analytical methods.
6. It is suggested to standardize the description of PZQ's BCS classification throughout the text.
7. It is recommended to carefully check for duplicate references (e.g., references 36 and 41 appear identical).
8. It is suggested to update references to include more recent developments (2022-2024) in PZQ analysis.
9. Tables 4, 5, and 6 would benefit from additional comparison parameters (cost, analysis time, and sample throughput).
10. It is suggested to include detection limits in consistent units across all analytical methods.
11. It is recommended to add information about sample preparation requirements for each method
12. The conclusion section needs substantial enhancement to address emerging technologies (paper-based analytical devices, smartphone-based detection), the potential for AI/ML in data processing and method optimization, point-of-care testing developments for field applications, and integration with telemedicine approaches for remote settings.
13. It is suggested to add discussion about regulatory requirements for bioanalytical methods used in different contexts (drug development quality control vs. therapeutic drug monitoring).
14. It is recommended to dedicate a section to discussing sample preparation methods (protein precipitation, SPE, LLE, etc.) and their impact on method performance, as this is critical for bioanalysis but currently underrepresented.
15. It is suggested to expand coverage of quality control procedures necessary for reliable PZQ quantification in different matrices and settings.
16. While chirality is mentioned, provide more practical guidance on implementing enantioselective methods, including column selection, mobile phase optimization, and validation considerations specific to chiral separations.

Author Response

We would like to thank the reviewer for the time and effort devoted to evaluating our manuscript. We sincerely appreciate the constructive comments and suggestions, which have significantly improved the quality and clarity of our work.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The submitted review article focused on the description of the analytical methods used for the determination of praziquantel in biological samples. 

In general terms the article is adequately written and referenced. However, there some points that need correction prior to its final publication (see the following comments):

Comments

1) Figure 4 and Table 2 are meaningless and they can be deleted.

2) Some representative chromatograms of the analysis of the drug in biological samples can be added.

3) Table 4 is also meaningless. A table with comparative data (i.e. sample, analytical technique, LOD, LOQ, recovery data) of the published methods must be added instead.

 

 

Author Response

We thank the reviewer for their thoughtful comments. Detailed responses are provided below, and all modifications have been marked in the revised manuscript using track changes.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised the manuscript according to suggestions; therefore, I recommend considering this manuscript for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been revised accordingly.