Simultaneous estimation of troxerutin and calcium dobesilate in presence of the carcinogenic hydroquinone using green spectrofluorimetric method

In the present study, we conducted two facile and highly sensitive spectrofluorimetric approaches in order to quantify the vasoprotective agents; troxerutin (TROX) and calcium dobesilate (DOB) in the presence of hydroquinone (HQ) (as a highly toxic impurity and potential degradation product of DOB) in commercial formulations and human plasma. The first approach relies simply on using ethanol as an eco-friendly solvent for the estimation of DOB at 345 nm after being excited at 305 nm. The linearity was carefully investigated between DOB concentration and the relative fluorescence intensity in the range of 0.05–0.8 µg ml−1. Due to the high method simplicity and sensitivity, applying the first approach to quality control analysis and spiked human plasma samples with mean % recoveries 100.74 ± 3.71 adds another merit. The second approach involved rapid conventional fluorimetric estimation of ethanolic TROX solution in TROX/DOB combined dosage forms at 455/350 nm (emission/excitation) with a linear calibration chart covering the range of 0.1–1.2 µg ml−1. Moreover, the second approach involved a comprehensive study in a trial to solve the problem of superposition of DOB and HQ graph adopting the first derivative synchronous fluorimetric mechanism in ethanol at Δλ = 60 nm. Therefore, DOB was measured at 286 and 323 nm, while HQ could be quantitated at 301 nm. The Beer–Lambert Law has complied over the ranges of 0.1–1.0 and 0.02–0.4 µg ml−1 for DOB and HQ, respectively. Guidelines adopted by the International Council of Harmonization (ICH) were used to validate the target approaches. The developed methods are more convenient for routine quality control laboratory instead of the time-consuming and sophisticated reported techniques. Moreover, different aspects of evaluating the greenness of the proposed approaches were conducted to have a complete image of their environmental impact.


Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The manuscript presents two spectrofluorimetric methods for the determination of calcium dobesilate alone or simultaneously with troxerutin and its potential degradation product; hydroquinone. The current study is the first report for the spectrofluorimetric assay of this mixture. The work described is valuable and suitable for publication in Royal Society Open Science (RSOS) after considering the following revisions: (1) The title can be better rewritten in a clearer way as follows: Simultaneous estimation of troxerutin and calcium dobesilate in presence of the carcinogenic degradation product hydroquinone using green spectrofluorimetric method. (2) The manuscript includes some minor English mistakes and should be generally revised regarding grammatical or spelling mistakes.
(3) P2, L25: Replace "Due to the method's high simplicity and sensitivity" by "Due to the high method simplicity and sensitivity".
Avoid writing details about the results at the introduction section.
Did the authors study the effect of surfactants in water or in ethanol? (9) P7, L3: Replace "Results and the discussion" by "Results and discussion". (10) In "Section 3. Results and discussion", please give a highlight to the relation of the chemical structures of the studied analytes and the presence of their native fluorescence. (11) P8, L4: Please re-phrase this sentence: "All the mentioned attributes forced us to continue using ethanol as diluent". (12) P11, L34: The sentence starting with "Considering the proposed …." should be rewritten in a clear way.
The access dates in references 23, 26 and 27 should be updated and the URL addresses in reference 26 and 27 should be checked. Also, it is preferable to include reference to royal society open science journal: Ex: 1-Abdelhameed AS, Attwa MW, Kadi AA. Liquid chromatography-tandem mass spectrometry metabolic profiling of nazartinib reveals the formation of unexpected reactive metabolites. Royal Society Open Science. 2019;6(8):190852. 2-Abdelhameed AS, Attwa MW, Al-Shaklia NS, Kadi AA. A highly sensitive LC-MS/MS method to determine novel Bruton's tyrosine kinase inhibitor spebrutinib: application to metabolic stability evaluation. Royal Society Open Science. 2019;6(6):190434.
References corresponding to the methods used for evaluation of greenness should be added to Table 6. (15) The manuscript lacks the figure numbers on the presented figures.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments) Comments to the Author(s) I read the paper many times, it sounds accepetable.
One remark, the authors should give the values of the optical densities at the excication and the emission wavelengths, used, to obtain excitation and emission spectra.
Second remark: They should write Fluorescence excitation and emission spectra.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The manuscript# RSOS-201888 entitled ''Green spectrofluorimetric estimation of troxerutin and dobesilate simultaneously in the presence of the carcinogenic degradation product hydroquinone'' is of interest and suitable for the scope of Royal Society Open Science. This paper may be published after a revision considering the following points: 1.
The language needs some revision and editing.

2.
Page#2, Line#32: Remove the space at the end of the paragraph and make the abstract one paragraph.
The purity of the studied drugs [troxerutin, dobesilate, and hydroquinine] should be written.

5.
Under the procedure section, the statement "completing to the volume" must be changed to be "completing to the mark." 6.
Page#7 Line #10: We used the higher sensitivity of the first approach………… Please rewrite the sentences in an acceptable scientific writing way. 9.
The resolution and the shape of figure #1 are poor. Please, try to improve. 12.
The number of figures is a large number and some of them can be moved to the supplementary material.

Decision letter (RSOS-201888.R0)
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Dear Dr Salim: Title: Green spectrofluorimetric estimation of troxerutin and dobesilate simultaneously in the presence of carcinogenic degradation product hydroquinone Manuscript ID: RSOS-201888 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: (There are no comments.) RSC Subject Editor: Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript presents two spectrofluorimetric methods for the determination of calcium dobesilate alone or simultaneously with troxerutin and its potential degradation product; hydroquinone. The current study is the first report for the spectrofluorimetric assay of this mixture. The work described is valuable and suitable for publication in Royal Society Open Science (RSOS) after considering the following revisions: (1) The title can be better rewritten in a clearer way as follows: Simultaneous estimation of troxerutin and calcium dobesilate in presence of the carcinogenic degradation product hydroquinone using green spectrofluorimetric method.
(2) The manuscript includes some minor English mistakes and should be generally revised regarding grammatical or spelling mistakes.
(3) P2, L25: Replace "Due to the method's high simplicity and sensitivity" by "Due to the high method simplicity and sensitivity". (4) Unify the use of the term "fluorimetry" instead of "fluorometry" all over the text. (5) P2, L30: Replace "TROX ethanolic solution" by "ethanolic TROX solution". (6) P2, L44: Replace "They were" by "The developed methods are". (7) Avoid writing details about the results at the introduction section. (8) Did the authors study the effect of surfactants in water or in ethanol? (9) P7, L3: Replace "Results and the discussion" by "Results and discussion". (10) In "Section 3. Results and discussion", please give a highlight to the relation of the chemical structures of the studied analytes and the presence of their native fluorescence. (11) P8, L4: Please re-phrase this sentence: "All the mentioned attributes forced us to continue using ethanol as diluent". (14) References corresponding to the methods used for evaluation of greenness should be added to Table 6. (15) The manuscript lacks the figure numbers on the presented figures.

Reviewer: 2
Comments to the Author(s) I read the paper many times, it sounds accepetable.
One remark, the authors should give the values of the optical densities at the excication and the emission wavelengths, used, to obtain excitation and emission spectra.
Second remark: They should write Fluorescence excitation and emission spectra.

Reviewer: 3
Comments to the Author(s) The manuscript# RSOS-201888 entitled ''Green spectrofluorimetric estimation of troxerutin and dobesilate simultaneously in the presence of the carcinogenic degradation product hydroquinone'' is of interest and suitable for the scope of Royal Society Open Science. This paper may be published after a revision considering the following points: 1. The language needs some revision and editing. 2. Page#2, Line#32: Remove the space at the end of the paragraph and make the abstract one paragraph.
3. Page#2, Line#46: Replace "evaluating the proposed approaches' greenness" by "evaluating the greenness of the proposed approaches" 4. The purity of the studied drugs [troxerutin, dobesilate, and hydroquinine] should be written. 5. Under the procedure section, the statement "completing to the volume" must be changed to be "completing to the mark." 6. Page #6 Line # 2: concentration in an ascending way…….should be deleted. 7. In section # 2.4.3.2.: The dosage form's nominal content was determined…….., the obtained statistical data should be added in Table #4. 8. Page#7 Line #10: We used the higher sensitivity of the first approach………… Please rewrite the sentences in an acceptable scientific writing way. 9. Page#7, Line#42: Replace "Experimental condition optimization" by "Optimization of experimental conditions". 10. Reference #14…………………please, adhere to the journal guidelines for reference formatting. 11. The resolution and the shape of figure #1 are poor. Please, try to improve. 12. The number of figures is a large number and some of them can be moved to the supplementary material.

Author's Response to Decision Letter for (RSOS-201888.R0)
See Appendix A.

Decision letter (RSOS-201888.R1)
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Dear Dr Salim:
Title: Simultaneous estimation of troxerutin and calcium dobesilate in presence of the carcinogenic hydroquinone using green spectrofluorimetric method Manuscript ID: RSOS-201888.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.