PONE-D-20-26989

A validated LC-MS/MS method for simultaneous determination of azithromycin, hydroxychloroquine, and its metabolites desethylhydroxychloroquine and bisdesethylchoroquine in human plasma

PLOS ONE

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Reviewers' comments:

Reviewer #1: Comments to Author:

A validated LC-MS/MS method for simultaneous determination of azithromycin, hydroxychloroquine, and its metabolites desethylhydroxychloroquine and

bisdesethylchoroquine in human plasma

The paper presented in this study is a validated LC-MS/MS method for simultaneous determination of Hydroxychloroquine (HCQ) and azithromycin (AZM) are antimalarial drugs recently reported to be active against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and quantitation method to assess the pharmacokinetics of AZM, HCQ, and its metabolites desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ) in patients’ plasma. The work is well written and organized and the results are satisfactorily supported by the reported data. Figures and tables are comprehensive and helpful. However, some minor changes are required as discussed in the following:

1. Abstract should be limited to 200 words

2. Keywords should not be the repetitions of the title words, please find such words which are not in the title, this way search engines of the web will find your manuscript with higher probability.

3. Suggestions for improvements in the Title

4. The structure of scientific publication should include the general chapters (Introduction, Materials and Methods, Results, Discussion, Conclusion). Please follow instructions on journal webpage. Conclusion chapter is missing.

5. Recommendations for future studies are needed in the conclusion section. Kindly provide strong recommendations for future researches.

6. Some citations are missing from the References section.

7. Some references are not cited in the text.

8. Formatting does not match journal criteria. (e.g. References section; section title spacing; paragraph indents)

The paper generates the following kinds of data.

1. Only 20 µL plasma sample by volume is needed for simultaneous quantitation of AZM, HCQ, DHCQ, and BDCQ.

2. The run time is 3.5 min which is fast turnaround time.

3. Statistical measurements are missing in whole of the data which should be added in the manuscript.

However, before I can recommend its publication, the authors should address the following questions

Some questions to author

1. The %differences from controls are 3.8% and -2.9% for AZM, 1.3% and 2.7% for HCQ, -1.9% and -0.9% for DHCQ, and -2.0% and -5.7% for BDCQ. Can author explain what would be the structural changes?

2. Why the study is important, what benefits readers can get from this study?

Reviewer #2: This is an interesting manuscript describing a method to analyze the compounds of interest. The authors follow the FDA and CPQA methodology. The quality of the data and results are outstanding. There are several minor points that the authors should address prior to acceptance of the manuscript. These points are listed below in order of appearance in the paper.

1. Page 5, line 3: There are two periods after [21].

2. Page 6, Figure 1: I recommend having the same relative geometry of the rings in the structure for the last 6 compounds to aid in visualizing the differences between structures.

3. Page 6, Section 2.2: PFP should be defined for the more general reader.

4. Page 7, section 2.4: HLB should be defined for the more general reader.

5. Page 5 and 22-23: These equations should be reformatted and numbered using the recommended standards of the journal.

6. Page 19, end of Room Temperature Stability in Plasma section: (Table] should be replaced with (Table 5).

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Development and validation of an LC-MS/MS method for determination of hydroxychloroquine, its two metabolites, and azithromycin in EDTA-treated human plasma

PONE-D-20-26989R1

Dear Dr. Huang,

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.

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Kind regards,

Pasquale Avino, Ph.D.

Academic Editor

PLOS ONE