Two different synchronous spectrofluorimetric approaches for simultaneous determination of febuxostat and ibuprofen

Two green, simple and sensitive synchronous spectrofluorimetric methods were developed for the first time for the simultaneous estimation of febuxostat (FEB) and ibuprofen (IBU). Method I is constant-wavelength synchronous spectrofluorimetry where FEB and IBU were recorded at 329 and 258 nm, respectively, using Δλ of 40 nm. Method II is constant-energy synchronous spectrofluorimetry using a wavenumber interval of −4000 cm−1. All measurements were carried out in a borate buffer of pH 7 and distilled water for dilution which increased the methods' greenness. The two methods were rectilinear over concentration ranges of 30.0–700.0 ng ml−1 and 0.5–9.0 µg ml−1 in the first method and 20.0–500.0 ng ml−1 and 0.1–8.0 µg ml−1 in the second method for FEB and IBU, respectively. High sensitivity was attained for the two drugs with limits of quantitations (LODs) down to 0.41 and 5.51 ng ml−1 in the first method and 0.25 and 3.32 ng ml−1 in the second method for FEB and IBU, respectively. Recovery percentages were in the range of 97.3–101.9% after extraction from spiked human plasma samples, demonstrating high bioanalytical applicability. The two methods were further applied to tablet dosage forms with good recovery results. The methods' greenness was assessed according to the analytical Eco-Scale and Green Analytical Procedure Index guidelines.


2.
In the abstract; "the method greenness" in lines no. 17 and 36 should be corrected into "the methods' greenness". 3.
In the Introduction section; the first sentence "Gout is a type of inflammatory arthritis that is triggered by the deposition of monosodium urate crystals in the bones, joints, and parenchymal organs such as the kidney and is frequently associated with hyperuricemia" should be shorter. 4. Section 2.1; the second sentence, change "FEB" into "Febuxostat" so that it doesn't start with an abbreviation. 5. Section 2.2; please add other instruments used for centrifugation, vortex, and filtration. 6. Section 2.4.3; please explain why 10 min sonication is necessary. 7. Section 3.7; line no. 16, "direct methods", remove "methods" as it is a redundant word. 8.
Please revise the significant figures all over the manuscript and they should be unified. 9.
Please revise the manuscript for grammatical and spelling mistakes. 10.
Tables 2-5 should be simplified, the "Amount found" column can be removed. 11.
The authors should also consider moving some figures into the electronic supplementary material file.

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

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
Comments: 1. In the title the authors mentioned that the method was applied to human plasma. However, only spiked plasma appeared in the manuscript and no real plasma was analyzed. Analysis of spiked plasma is not a guarantee for the successful application in real samples. So the authors should itry to analyze real plasma, otherwise the plasma should be removed from the title.

2.
Page 5, line 23: the authors said "However, its direct application is inadequate for the assay of multi-component samples; " This statement is not true because there are many mixtures can be analyzed using direct measurement. So this statement should be modified. 3.
Page 5, line 39 -53: The authors explain the difference between the two modes of synchronous specrofluorimetry. Actually there is no difference between the two mode. In the first one use constant wavelength difference while the second use constant energy difference in cm [-1]. Bothe the wavelength and wave number are inter-related as the wave number is the reciprocal of wavelength (or 1/wavelength). So both type are the same. In all synchronous modes only the wavelength difference mode is used. There is mean for using the energy difference mode. It is wasting the time and efforts for repeating the same work and obtaining the same results as constant wavelength difference mode. In addition, the energy difference has no advantage over the other mode. In contrast to the claimed advantage for CESS method (mentioned in page 15, line 33), the sensitivity of both methods are almost the same LODs for FEB is 0.40 and 0.25 ng mL-1 while for IBU the values are 5.6 and 3.4 ng mL-1 for the first method and second methods, respectively. 4.
Page 7, line 51-56: The authors said "Afterwards, SF spectra were recorded at 329 nm for FEB and 258 nm for IBU, in the range of 200-400 nm keeping a constant Δλ of 40 nm between excitation and emission monochromators ". This sentence is confusing and should be modified. The phrase ", in the range of 200-400 nm " can be deleted .

5.
Page 8, line 35: under the title "Analysis of FEB and IBU in tablets " and page 14, line 20 -33, the authors described the analysis of separate dosage forms but not simultaneous determination . This is not matched with the title of the manuscript. 6.
According to Figure 2, it very easy to determine FEB in the presence of IBU as there is not interference of IBU spectra with that of FEB. So there is no need for the synchronous mode for FEB analysis. 7.
Page 12, line 21 -39: There is no need to present the equations as all the constants in these equations are mentioned in Table 1. 9.
Page 13, lines 39 -54 : The title section " 3.5.5. Selectivity " do not contain a new data, all results are previously mentioned and there is no additional experiment was carried to test selectivity of the method. 10.
The number of figure should be reduced and the layout of the figure should be improved.

11.
Table are presented in a very bad formats, the data should be reduced by deleting the individual numbers. In tables 2,3 and 4, the mean and SD should be calculated for each concentration but not for the data obtained from different concentrations.

12.
About the greenness of the methods: All spectrofluorimetric methods are green specially if the used solvent is water and therefore, there is no need to apply GAPI or analytical Eco-Scale.

Decision letter (RSOS-210354.R0)
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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) In this study, the authors developed two green and sensitive synchronous spectrofluorometric methods (CWSS and CESS) for simultaneous determination of febuxostat and ibuprofen. They applied the proposed methods to pharmaceuticals and human plasma. It is shown how green the methods are using two different greenness assessment tools; GAPI and analytical Eco-Scale. I think the developed methods are useful and have advantages over the published ones. The manuscript is well written and the experimental strategies were explained in detail. The context of the subject in the literature is demonstrated and the paper is easy to understand. I see this manuscript is acceptable for publication after few minor changes. The suggestions were listed below. 1. The manuscript should be rechecked and any typos have to be corrected. 2. In abstract: line number 33, "Furthermore" may be removed. 3. In section 2.2, the used cell should be written. 4. In section 3.2: line number 18, change "The intensities of SF" into "The intensities of SF spectra". 5. Reference number 60, authors' names need corrections and the title should be consistent with that in Dryad Digital Repository. 6. Table 1: add superscript letters on LOD and LOQ in the table and its footnotes. 7. Table 4: change "pharmaceutical preparations" into "tablet dosage forms" in the caption.
Reviewer: 2 Comments to the Author(s) The manuscript presents two synchronous spectrofluorimetric approaches for the simultaneous analysis of combined drugs; febuxostat and ibuprofen. The developed methods were applied to pharmaceutical tablets and human plasma. I recommend that the proposed methods are green and sensitive enough for simultaneous estimation of such a mixture. The manuscript is wellwritten and the topic is interesting. It can be accepted for publication in Royal Society Open Science after performing the required revision. Comments: 1. Please revise the adjustment of the text within the manuscript to be uniform. 2. In the abstract; "the method greenness" in lines no. 17 and 36 should be corrected into "the methods' greenness". 3. In the Introduction section; the first sentence "Gout is a type of inflammatory arthritis that is triggered by the deposition of monosodium urate crystals in the bones, joints, and parenchymal organs such as the kidney and is frequently associated with hyperuricemia" should be shorter. 4. Section 2.1; the second sentence, change "FEB" into "Febuxostat" so that it doesn't start with an abbreviation. 5. Section 2.2; please add other instruments used for centrifugation, vortex, and filtration. 6. Section 2.4.3; please explain why 10 min sonication is necessary. 7. Section 3.7; line no. 16, "direct methods", remove "methods" as it is a redundant word. 8. Please revise the significant figures all over the manuscript and they should be unified. 9. Please revise the manuscript for grammatical and spelling mistakes. 10. Tables 2-5 should be simplified, the "Amount found" column can be removed. 11. The authors should also consider moving some figures into the electronic supplementary material file.
Reviewer: 3 Comments to the Author(s) Comments: 1. In the title the authors mentioned that the method was applied to human plasma. However, only spiked plasma appeared in the manuscript and no real plasma was analyzed. Analysis of spiked plasma is not a guarantee for the successful application in real samples. So the authors should itry to analyze real plasma, otherwise the plasma should be removed from the title. 2. Page 5, line 23: the authors said "However, its direct application is inadequate for the assay of multi-component samples; " This statement is not true because there are many mixtures can be analyzed using direct measurement. So this statement should be modified. 3. Page 5, line 39 -53: The authors explain the difference between the two modes of synchronous specrofluorimetry. Actually there is no difference between the two mode. In the first one use constant wavelength difference while the second use constant energy difference in cm[-1]. Bothe the wavelength and wave number are inter-related as the wave number is the reciprocal of wavelength (or 1/wavelength). So both type are the same. In all synchronous modes only the wavelength difference mode is used. There is mean for using the energy difference mode. It is wasting the time and efforts for repeating the same work and obtaining the same results as constant wavelength difference mode. In addition, the energy difference has no advantage over the other mode. In contrast to the claimed advantage for CESS method (mentioned in page 15, line 33), the sensitivity of both methods are almost the same LODs for FEB is 0.40 and 0.25 ng mL-1 while for IBU the values are 5.6 and 3.4 ng mL-1 for the first method and second methods, respectively. 4. Page 7, line 51-56: The authors said "Afterwards, SF spectra were recorded at 329 nm for FEB and 258 nm for IBU, in the range of 200-400 nm keeping a constant Δλ of 40 nm between excitation and emission monochromators ". This sentence is confusing and should be modified. The phrase ", in the range of 200-400 nm " can be deleted . 5. Page 8, line 35: under the title "Analysis of FEB and IBU in tablets " and page 14, line 20 -33, the authors described the analysis of separate dosage forms but not simultaneous determination . This is not matched with the title of the manuscript. 6. According to Figure 2, it very easy to determine FEB in the presence of IBU as there is not interference of IBU spectra with that of FEB. So there is no need for the synchronous mode for FEB analysis. 7. Page 11, line 50-53: The authors said "The studied surfactants include β-cyclodextrin, cetrimide, sodium dodecyl sulphate, tween-80, and carboxy methyl cellulose. ". Actually βcyclodextrin and carboxy methyl cellulose are not surfactants. 8. Page 12, line 21 -39: There is no need to present the equations as all the constants in these equations are mentioned in Table 1. 9. Page 13, lines 39 -54 : The title section " 3.5.5. Selectivity " do not contain a new data, all results are previously mentioned and there is no additional experiment was carried to test selectivity of the method. 10. The number of figure should be reduced and the layout of the figure should be improved. 11. Table are presented in a very bad formats, the data should be reduced by deleting the individual numbers. In tables 2,3 and 4, the mean and SD should be calculated for each concentration but not for the data obtained from different concentrations. 12. About the greenness of the methods: All spectrofluorimetric methods are green specially if the used solvent is water and therefore, there is no need to apply GAPI or analytical Eco-Scale.

Author's Response to Decision Letter for (RSOS-210354.R0)
See Appendix A.

Decision letter (RSOS-210354.R1)
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Dear Prof. Editor of Royal Society Open Science,
On the behalf of all authors, I would like to thank you for the opportunity that we have been given to further revise our manuscript entitled: ''Two Different Synchronous Spectrofluorimetric

Approaches for Simultaneous Determination of Febuxostat and Ibuprofen: Application to
Human Plasma''. We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback and insightful comments on the manuscript.
We have carefully revised the manuscript taking into account your recommendations and the reviewers' comments. Please, find enclosed the revised version of the manuscript where the areas containing the major changes were highlighted and the font color was changed into red.
Here is a point-by-point response to the reviewers' comments and concerns.

Comments from Reviewer #1
In this study, the authors developed two green and sensitive synchronous spectrofluorometric methods (CWSS and CESS) for simultaneous determination of febuxostat and ibuprofen. They applied the proposed methods to pharmaceuticals and human plasma. It is shown how green the methods are using two different greenness assessment tools; GAPI and analytical Eco-Scale. I think the developed methods are useful and have advantages over the published ones. The manuscript is well written and the experimental strategies were explained in detail. The context of the subject in the literature is demonstrated and the paper is easy to understand. I see this manuscript is acceptable for publication after few minor changes. The suggestions were listed below.

The manuscript should be rechecked and any typos have to be corrected
Author's reply: The manuscript was carefully revised and any grammatical or spelling mistakes were corrected as recommended by the reviewer.
Author's reply: For analysis of FEB and IBU in commercial tablets, 10 min. sonication was necessary after the addition of 40 mL methanol to ensure complete extraction and separation of the cited drugs from the tablet excipients which are insoluble in methanol. This in turn guarantees the method selectivity for the determination of the drugs in presence of tablet excipients with high recoveries and without any interference; which is in compliance with the pharmacopeial methods for assay of drugs in pharmaceutical preparations. This method was reported in many published articles [1][2][3][4].

2.
Page 5, line 23: the authors said "However, its direct application is inadequate for the assay of multi-component samples; " This statement is not true because there are many mixtures can be analyzed using direct measurement. So this statement should be modified.
Author's reply: Thank you for pointing this out. It is true that the direct emission spectrofluorimetric technique could be applied for the determination of some mixtures, but this can occur if there is an adequate resolution between the mixture components and well-resolved peaks which allow the determination of each component in presence of the other without interference.
Selectivity problems can arise if the components have broad bands and overlapped emission spectra so that resolution between mixture components is insufficient. The limited selectivity of direct emission spectrofluorimetry can be improved by applying total luminescence, synchronous spectrofluorimetric, or derivative synchronous spectrofluorimetric techniques [1,2]. This is what we meant by the word ''inadequate''. and matrix isopotential synchronous spectrofluorimetry (MISS). The differences between these modes, their principles, and applications have been detailed in the literature [1][2][3].
It is true that CWSS is the most used technique as the major advantage of CWSS is that it can be facilely conducted using the existing capabilities of data acquisition software programs of most commercial fluorescence spectrometers [1]. Therefore, most published reports use CWSS but it is not the only mode to be applied. Synchronous scanning could also be carried out in CESS mode since 1982, when Inman and Winefordner [4] developed this mode, which has demonstrated great utility in the resolution of multi-component mixtures [5]. Since this date, CESS was applied and reported in some published articles [6][7][8][9][10].
It was reported that, CESS could offer improvement in sensitivity and selectivity and overcome the limitations of CWSS technique [11,12]. This is because CESS can greatly reduce the effect of solvent Raman scatter and Rayleigh scatter interferences with the fluorescence spectra of the analytes [12]. Conventional CESS does not entirely eliminate Rayleigh and Raman interference especially when the difference between the excitation and emission wavelengths exceeds the difference between the wavelengths of the Rayleigh and Raman peaks [12,13]. This advantage of CESS could be useful especially in the case of weakly fluorescent analytes or low concentrations of strongly fluorescent compounds [12,13].
Some published articles used both CWSS and CESS in the same study and reported an improvement of sensitivity in the case of CESS over CWSS [9,10,13]. Similarly, the current study applied both techniques to obtain sensitivity as high as possible. It was found that CESS improved the sensitivity of the developed method to some extent where LODs decreased to almost half which could be important for the determination of very low concentrations of the studied drugs.
Hopefully, the point is clarified.

5.
Page 8, line 35: under the title "Analysis of FEB and IBU in tablets " and page 14, line 20 -33, the authors described the analysis of separate dosage forms but not simultaneous determination. This is not matched with the title of the manuscript.
Author's reply: Thank you for your comment. The proposed methods were applied for the simultaneous determination of FEB and IBU in synthetic mixtures of pure forms and spiked human plasma samples which is consistent with the title of the manuscript. We want to clarify that, FEB and IBU are co-administered drugs for the treatment of gout as mentioned in the ''Introduction'' section and not co-formulated so that we applied the developed methods for the determination of each drug in its tablet dosage form as reported in many published articles [1][2][3]. Author's reply: Thank you for pointing this out. Although the emission spectra of FEB and IBU are not overlapped as shown in Figure 2, we preferred to use synchronous spectrofluorimetry to be able to determine both drugs simultaneously in a single scan. The use of conventional emission spectrofluorimetry for analysis of such mixture will be tedious and time-consuming where each mixture will need to be measured two times; one at λ excitation of FEB and the other at λ excitation of IBU. This reason in addition to the well-known advantages of the synchronous spectrofluorimetric technique which was discussed in detail under the ''3.1'' section made the synchronous spectrofluorimetric technique the best choice for the analysis of such a binary mixture.
Author's reply: Thank you for your valuable comment. It is true that β-cyclodextrin and carboxy methyl cellulose are not surfactants. They are macromolecules and their mechanism as organized media is similar to surfactants. They provide a viscous and very rigid microenvironment that can inhibit quenching by molecular oxygen and restrict the freedom of fluorophores and consequently diminish the probabilities of non-radiative processes. These factors can increase the fluorescence quantum yield and enhance the fluorescence intensity [1,2]. Therefore, the word ''surfactants'' was replaced by ''organized media'' in the revised manuscript according to the reviewer's comment. In addition, the sentence was corrected into ''The studied organized media include surfactants such as cetrimide, sodium dodecyl sulphate, and tween-80, as well as macromolecules such as carboxy methyl cellulose, and β-cyclodextrin'' in ''3.4.3.'' section. We used the mentioned examples for organized media in the study as reported in most of the published articles [1][2][3][4][5][6]. References: