Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography–tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazole

Thiago José Dionísio; Gabriela Moraes Oliveira; Marina Morettin; Flavio Cardoso Faria; Carlos Ferreira Santos; Adriana Maria Calvo

doi:10.1371/journal.pone.0236297

Peer Review History

Original SubmissionApril 5, 2020
14 May 2020 Decision Letter - Joseph Banoub, Editor PONE-D-20-08997 Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by LC MS / MS: PK study of naproxen associated or not with esomeprazole PLOS ONE Dear Dr Calvo, 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. We would appreciate receiving your revised manuscript by Jun 28 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ******** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript does not appear complete, as there are no analyses, results, or discussion sections. It appears to only be a partly-developed protocol as there is no mention of any planned statistical analyses for the data collected from 17 time points. This needs to be amended, and results should be shown if it is to be a true P/K study. Additionally, it is mentioned that time point 0 for each drug were analyzed to ensure no interference, but there is no mention of how this analysis was performed. Reviewer #2: The submission was not complete as only the abstract, introduction and methods were provided. Despite this, the following major issues were identified which must be addressed in the complete version: • The method is intended for a clinical application among patients. Method validation is a must- it is not optional when dealing with patient samples. There was no mention of method validation. The authors must completely validate the method as per regulatory guidelines, the FDA, EMA or ICH. • Though the authors indicate that this is the first time a method is reported for Saliva, did they attempt the conditions of the reported methods measuring the same compounds. Is there any similarities or improvements form an analytical stand point? • Patient sample preparation [containing the drug] was not adequately described. • PK studies are usually done in plasma- what information we can get from a Saliva for a medication intended for pain. Clarification and justification are needed to justify publication. • What is the structures of the drugs and their monitored product ions? MSMS data should be shown along with the proposed structures. Why these specific product ions were chosen? • Did the authors monitor the ratios of the quantifier/qualifier ions? A must when doing quantification with LC-MS/MS to ensure peak purity. • In fact, it was not clear which product ion was the quantifier and which one is the qualifier • The authors mention molecular ions. I think the authors mean protonated ions as molecular ions are the radical cations and it is not commonly seen in ESI. • Why a structural analogue was used for internal standard instead of isotopically labelled IS. ****** 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 #2: No [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0236297.r001
Revision 1
29 Jun 2020 Author Response Answer to reviewers Reviewer #1: This manuscript does not appear complete, as there are no analyses, results, or discussion sections. It appears to only be a partly-developed protocol as there is no mention of any planned statistical analyses for the data collected from 17 time points. This needs to be amended, and results should be shown if it is to be a true P/K study. Additionally, it is mentioned that time point 0 for each drug were analyzed to ensure no interference, but there is no mention of how this analysis was performed. Answer: Thank you for your observation. The statistical analyses, which we intend to use, were added in the method section. Our article type is Registered Report Protocol, therefore, the results will be presented in the second manuscript as well as the discussion. The time point 0 refers to the collection of saliva from the volunteers before taking the medications. In fact, this collection is important to ensure that the pre-determined methods of detecting the drugs under study do not detect other molecules in the volunteers' saliva. Reviewer #2: The submission was not complete as only the abstract, introduction and methods were provided. Despite this, the following major issues were identified which must be addressed in the complete version: • The method is intended for a clinical application among patients. Method validation is a must- it is not optional when dealing with patient samples. There was no mention of method validation. The authors must completely validate the method as per regulatory guidelines, the FDA, EMA or ICH. Answer: Thank you for your observation. Our article type is Registered Report Protocol, therefore, the results will be presented in the second manuscript as well as the discussion (if this manuscript is able to be accepted). We will we will execute the validations in all analyzes carried out in accordance with the FDA Please find the description of the experiments for validation in the methods section. • Though the authors indicate that this is the first time a method is reported for Saliva, did they attempt the conditions of the reported methods measuring the same compounds. Is there any similarities or improvements form an analytical stand point? Answer: Thank you for your question. Yes, we did a research where we compared the detection and quantification of Piroxicam in saliva and plasma. These results confirmed that in saliva it is possible to perform pharmacokinetic tests as in plasma (Calvo et al. J Pharm Biomed Anal. 2016 Feb 20;120:212-20. doi: 10.1016/j.jpba.2015.12.042). • Patient sample preparation [containing the drug] was not adequately described. Answer: Thank you for your observation. We adequately described patient sample preparation. Please find this new information in method section. • PK studies are usually done in plasma- what information we can get from a Saliva for a medication intended for pain. Clarification and justification are needed to justify publication. Answer: Thank you for your question. The idea of the study is to share the experience that it is possible to perform pharmacokinetic tests, using saliva, of any drug, including anti-inflammatory drugs. Pharmacokinetic studies are important for planning treatment according to the speed of metabolism of each person. This information was added in the introduction. • What is the structures of the drugs and their monitored product ions? MSMS data should be shown along with the proposed structures. Why these specific product ions were chosen? Answer: Thank you for your observation. Our mass spectrometer, based on the reported drug mass, automatically optimizes all parameters including product ions, which can be seen in Table 1. • Did the authors monitor the ratios of the quantifier/qualifier ions? A must when doing quantification with LC-MS/MS to ensure peak purity. Answer: Thank you for your observation. These information is in the new table 2. Table 2: Quantifier and qualifier product ion to be used in analysis. O-Desmethylnaproxen Type m/z Inten Act % Idenf. Rang % T 214.900>171.250a 199246 100.00 100% Ref 1 214.900>169.250b 49605 24.90 - Ref 2 214.900>143.050 3283 1.65 - Naproxen Type m/z Inten Act % Idenf. Rang % T 228.900>170.30a 513749 100.00 100% Ref 1 228.900>169.250b 336855 65.57 - Ref 2 228.900>185.200b 288128 56.08 - Piroxicam Type m/z Inten Act % Idenf. Rang % T 331.900>95.100a 33688 100.00 100% Ref 1 331.900>121.050 15274 45.37 - Ref 2 331.900>78.100b 23870 70.90 - aQuantifier - the most intense peak bQualifier - a second peak (SRM)t have used as a qualifier for the same analysis • In fact, it was not clear which product ion was the quantifier and which one is the qualifier Answer: Thank you for your observation. Please check the answer above. • The authors mention molecular ions. I think the authors mean protonated ions as molecular ions are the radical cations and it is not commonly seen in ESI. Answer: Thank you for your observation. Sorry for the mistake. In fact we refer to the protonated ions. • Why a structural analogue was used for internal standard instead of isotopically labelled IS. Answer: Thank you for your question. Previous studies by our group have shown that Piroxicam could be used successfully as an internal standard. However, we have the conscience and the intention to start using the isotopically labeled IS. During the validation process, we will guarantee that there will be no interference from the chosen IS. Answer to editors 1. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. 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After analyzing the PK and pharmacodynamic results using the Phoenix WinNonlin software (version 8.1), the data will be organized descriptively and a paired t-test will be performed to compare naproxen absorption after volunteers are administered, at two different times, naproxen alone or naproxen in combination with esomeprazole. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Answer: Thank you for your observation. We do not wish to make changes to our data availability statement. 5. It appears that your ORCiD iD has not been validated in your Editorial Manager account and we are unable to proceed until that step is complete. Answer: Thank you for your observation. We validated the ORCiD iD in Editorial Manager. Attachments Attachment Submitted filename: Answer to reviewers 2.docx https://doi.org/10.1371/journal.pone.0236297.r002
6 Jul 2020 Decision Letter - Joseph Banoub, Editor Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by LC MS / MS: PK study of naproxen associated or not with esomeprazole PONE-D-20-08997R1 Dear Dr. Calvo, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Joseph Banoub, Ph,D., D. Sc. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0236297.r003
Formally Accepted
24 Jul 2020 Acceptance Letter - Joseph Banoub, Editor PONE-D-20-08997R1 Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography–tandem mass spectrometry: pharmacokinetic study of naproxen alone and associated with esomeprazole Dear Dr. Calvo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Joseph Banoub Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236297.r004

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