TLC–Densitometric Analysis of Selected 5-Nitroimidazoles

Round 1

Reviewer 1 Report

 The manuscript titled “TLC-Densitometric Analysis of Selected 5-Nitroimidazoles” by Pyka-Pajak describes the stability of four 5-nitroimidazoles and their solutions in different solvents against prolonged heating conditions evaluated via TLC methods. The presented results can help understanding the impact of temperature exposure on the stability of these commonly used active pharmaceutical ingredients through a simple method, without needing complex equipment as HPLC or similars. Please find below my detailed comments.

1)     In the abstract, I suggest clarifying that metronidazole, ornidazole, tinidazole, secnidazole are 5-nitroimidazoles.

2)     I think that stating that TLC-densitometric and spectrodensitometric procedures used by the Author are  “newly developed” (Line13, 85) is misguiding. The use of the presented techniques is very common (for example, TLC is daily used in every synthetic chemistry laboratory to evaluate a reaction outcome), though their application to evaluate the degradation of 5-nitroimidazoles in the present work can represent some novelty. I suggest that the Author re-adjusts the claims made on this point throughout the manuscript.

3)     How did you choose the pH values of water solutions? I guess they correspond to representative acidic (2.62), neutral (5.56), and basic (8.21) conditions. But is there a rational between these numbers?

4)     Do you have specifications on the purity of the four compounds tested? I strongly suggest including these details in the Materials section.

5)     The comparison to existing data in the literature is important to highlight the coherence of the obtained results from TLC or the identification of additional degradation products. However, I suggest limiting the discussion of existing data only to the most relevant ones, otherwise the focus on the results of the present work is lost and some parts of the paper almost seems a literature review (see stability of solutions paragraphs).

6)     To reduce the length of the article, I suggest moving tables reporting the detailed description of chromatographic peaks to Supporting Information.

7)     Line 553: check sentence grammar.

Author Response

Response to Reviewer 1 Comments

 

Dear Reviewer,

I am very grateful for all your  valuable comments. I have carefully revised the manuscript and made the corrections according to these suggestions. Please find below my responses. I hope that they will meet with your requirements.

All changes made in the manuscript and supplementary materials are  marked with yellow colour.

 

Comment 1: 

In the abstract, I suggest clarifying that metronidazole, ornidazole, tinidazole, secnidazole are 5-nitroimidazoles.

Response 1:

Information that metronidazole, ornidazole, tinidazole, secnidazole are 5-nitroimidazoles is included in the ‘Abstract’ section.

 

Comment 2: 

I think that stating that TLC-densitometric and spectrodensitometric procedures used by the Author are  “newly developed” (Line 13, 85) is misguiding. The use of the presented techniques is very common (for example, TLC is daily used in every synthetic chemistry laboratory to evaluate a reaction outcome), though their application to evaluate the degradation of 5-nitroimidazoles in the present work can represent some novelty. I suggest that the Author re-adjusts the claims made on this point throughout the manuscript.

Response 2:

The statement “newly developed” has been used for the TLC-densitometric method and not for the TLC-densitometric technique. Despite this, the statement “newly developed” as suggested by the Reviewer has been removed. The entire manuscript has been reviewed and corrected accordingly.

 

Comment 3:

How did you choose the pH values of water solutions? I guess they correspond to representative acidic (2.62), neutral (5.56), and basic (8.21) conditions. But is there a rational between these numbers?

Response 3:

According to the validation rules, the stability of the substance should be tested, e.g. in an acidic, close to neutral (pure water) and alkaline environment. Therefore, as solvents was used distilled water (solution II), which had a pH of 5.56 and acidified water (pH=2.62, solution I) and water with the addition of sodium hydroxide (pH=8.21, solution III). Aqueous solutions as solvents with pH values ​​similar to those mentioned above have already been used in the study of the stability of other drugs by me and other scientists. There are many publications in which the authors do not provide the pH value of the solutions, but only provide information that the solution is acidic, neutral or basic. I believe that providing the pH value of the solutions allows for the possibility of reproducing the research by others.

Comment 4:

Do you have specifications on the purity of the four compounds tested? I strongly suggest including these details in the Materials section.

Response 4:

Specifications on the purity of the four compounds tested are introduced in the ‘Materials and Methods’ section. Namely: Metronidazole and tinidazole were pharmaceutical primary standards with purity according  with United States Pharmacopeia and European Pharmacopoeia, respectively. Secnidazole and ornidazole were analytical standards with quality level equal 100.

 

Comment 5:

The comparison to existing data in the literature is important to highlight the coherence of the obtained results from TLC or the identification of additional degradation products. However, I suggest limiting the discussion of existing data only to the most relevant ones, otherwise the focus on the results of the present work is lost and some parts of the paper almost seems a literature review (see stability of solutions paragraphs).

Response 5:

According to the Reviewer's suggestion, the discussion has been shortened. New subsection (3.3.) entitled 'Comparison of obtained degradation products of metronidazole, secnidazole, ornidazole and tinidazole with literature data' has been incorporated into the manuscript. A new Table 2 is presented. Table 2 compares the maximum number of degradation products of metronidazole, secnidazole, ornidazole and tinidazole that have been detected in this work and by other researchers. The data presented in Table 2 showed the importance of TLC in the study of 5-nitroimidazoles, without the need application for e.g. HPLC.

 

Comment 6:

To reduce the length of the article, I suggest moving tables reporting the detailed description of chromatographic peaks to Supporting Information.

Response 6:

Tables 2, 3, 4, 5 and 6 have been moved to Supplementary Materials.

 

Comment 7:

Line 553: check sentence grammar.

Response 7:

Sentence from the line 553 ". Whereas HPLC detected only two ornidazole degradation products” was corrected to: Meanwhile only two ornidazole degradation products were detected in neutral solution of ornidazole analyzed by HPLC.

 

Sincerely yours,

Alina Pyka-Pająk

Reviewer 2 Report

Manuscript appears incomplete.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 

Dear Reviewer,

I am very grateful for all your  valuable comments. I have carefully revised the manuscript and made the corrections according to these suggestions. Please find below my responses. I hope that they will meet with your requirements.

All changes made in the manuscript and supplementary materials are  marked with yellow colour.

 

Comment 1: 

The manuscript presents some potentially interesting results but appears incomplete. It is not clear from the presentation of the data if the degradation studies were replicated or if the data being presented are single replicates. The densitograms in Figures 3,4, 7, 9, 11 and 13 depict multiple traces which should be identified with legends.  The method was validated quantitatively and results for the degradation studies are not presented quantitatively. It is unclear why the author chose to do this as more robust comparisons to the cited literature could be made. The use of percent area to show degradation results as seen in Figures 2, 6, 8 10 and 12 seem less important when the author can quantitate the parent compound for each degradation study. The identity of many of the degradation products is known and referenced by the author. Why standards for these were not obtained and prepared to use to quantitate the degradation products is unclear. Measures for dispersion for results should be included in all figures.

 

Response 1:

All studies were replicated six times, which was supplemented in the revised manuscript.

Traces are visible in densitograms 3, 4, 7, 9, 11 and 13. It is possible that after even longer heating, there would be enough of them to be measurable with a densitometer. According to the reviewer's suggestion, traces were described in densitograms 3, 4, 7, 9, 11 and 13 using the abbreviation TR for all traces.

The method was validated using methanol solutions of metronidazole, secnidazole, ornidazole and tinidazole. Such validation may be used in the future, for example, to determine these substances in pharmaceutical preparations after their methanol extraction. For stress tests, solutions of the tested 5-nitroimidazoles were prepared in various solvents. In thin-layer chromatography, the solvent used affects the size of the spot of the test substance applied to the chromatographic plate, and this in turn affects the blurring of the chromatographic spot under the influence of the mobile phase used. The size of the chromatographic spots after development on silica gel will be different depending on the solvent used in which 5-nitroimidazole is dissolved. Therefore, the obtained calibration curves (validation) cannot be used for quantitative calculations of 5-nitroimidazoles in aqueous solutions (at different pH), in saline solutions, in hydrogen peroxide. For each tested 5-nitroimidazole in solvents from I to V, calibration curves should be determined separately. The calibration curves shown in Table 3 can only be used for 5-nitroimidazoles dissolved in methanol (solutions marked VI). In drug stability studies, it is common in the literature to use the percentage area of ​​the chromatographic band and not the absolute content of the substance subjected to stress conditions. Therefore, I consider the manuscript to be complete.

In most publications, the authors only state what percentage of 5-nitroimidazoles remains after exposure to stress conditions. Only in a few publications the authors give the amount of degradation products of 5-nitroimidazoles, and they identified one or two degradation products. Introduction of a new subsection entitled ‘Comparison of the obtained degradation products of metronidazole, secnidazole, ornidazole and tinidazole with literature data’’ should dispel all doubts. Table 2 compares the maximum number of degradation products of metronidazole, secnidazole, ornidazole and tinidazole that have been detected in this work and by other researchers. Table 2 also contains information on the identification of the resulting 5-nitroimidazole degradation products.The data presented in Table 2 showed the importance of TLC in the study of 5-nitroimidazoles, without the need application for e.g. HPLC.

In my manuscript, I used only one standard - a degradation product, namely 2-methyl-5-nitroimidazole, which was commercially available at the time of the study. In thin-layer chromatography, there is a rule that to consider a method as specific, it must be demonstrated that degradation products can be separated under stress conditions, but without the need to identify them.

As suggested by the Reviewer, Figures 2, 6, 8, 10 and 12 show the dispersion of the results.

Comment 2: 

The objective of the manuscript as identified in line 78 is not clearly achieved. The  manuscript is verbose. In the introduction starting at line 31 through line 38 a list of uses is provided for the nitroimidizoles as antibiotics but no citations are provided. It seems this kind of information could be deleted as it is not directly relevant to the manuscript or properly cited and tied into the results and discussion making it more relevant

Response 2:

All substances, i.e. metronidazole, secnidazole, ornidazole and tinidazole, were tested under exactly the same stress conditions. The concentrations of 5-nitroimidazoles in the individual solvents were also identical before being subjected to heating. In objective of the manuscript, I supplemented that the stress conditions were identical (now there will be no doubt). So the objective of the manuscript as identified in line 78 has been achieved.

As suggested by the Reviewer, the text from line 31 to line 38 was removed from the Introduction.

Comment 3:  

In the Results & Discussion section summary tables for the degradation studies done in the literature and the results from those studies might help a reader follow the importance of the results from this study.

Capturing the referenced literature in references 16-48 more succinctly as to allow a comparison of what was done in this work compared to the previous work would be extremely helpful.

 

Response 3:

New subsection (3.3.) entitled 'Comparison of the obtained degradation products of metronidazole, secnidazole, ornidazole and tinidazole with literature data' has been incorporated into the manuscript. A new Table 2 is presented. Table 2 compares the maximum number of degradation products of metronidazole, secnidazole, ornidazole and tinidazole that have been detected in this work and by other researchers. Table 2 also contains information on the identification of the resulting 5-nitroimidazole degradation products. The data presented in Table 2 showed the importance of TLC in the study of 5-nitroimidazoles, without the need application for e.g. HPLC.

 

Sincerely yours,

Alina Pyka-Pająk

 

 

Round 2

Reviewer 2 Report

For figures 3, 4, 7, 9, 11, and 13 each line should be identified as to which of these treatments it corresponds to: I – in water at pH=2.62; II – in water at pH=5.56; III- in water at pH=8.21; IV- in hydrogen 130 peroxide (3%); V – in physiological salt (0.9% NaCl); VI- in methanol.

For figures 2, 6, 8, 10 and 12 specify that the bars are means +/- 1 standard deviation or standard error.

Author Response

Response to Reviewer 2 Comments  (Round 2)

 

Dear Reviewer,

I am very grateful for all your  valuable comments. I have carefully revised the manuscript and made the corrections according to these suggestions. Please find below my responses. I hope that they will meet with your requirements.

All changes made in the manuscript and supplement materials are  marked with blue colour.

 

Comment 1: 

For figures 3, 4, 7, 9, 11, and 13 each line should be identified as to which of these treatments it corresponds to: I – in water at pH=2.62; II – in water at pH=5.56; III- in water at pH=8.21; IV- in hydrogen 130 peroxide (3%); V – in physiological salt (0.9% NaCl); VI- in methanol.

Response 1:

The lines in figures 3, 4, 7, 9, 11, and 13 do not correspond to stress conditions. Figures 3, 4, 7, 9, 11, and 13 are densitograms obtained as a result of densitometry analysis at multiple wavelengths from 200 nm to 380 nm (precisely at seven wavelengths, i.e. 200 nm, 230 nm, 260 nm , 290 nm, 320 nm, 350 nm and 380 nm). In the densitograms, each line corresponds to a different wavelength at which the densitometry analysis was performed.

Independent densitograms were obtained for each 5-nitroimidazole subjected to particular stress conditions. For example:

• Figure 7 shows the densitometric analysis of metronidazole dissolved in hydrogen peroxide. Metronidazole is stable in other solvents (see Table S2), therefore metronidazole densitograms from these solutions are not presented in the manuscript and supplementary materials.
• Figure 11 shows the densitometric analysis of ornidazole dissolved in water at pH=5.56. On the other hand, supplementary materials in Figures S4, S5, S6, S7, S8 present densitograms of ornidazole, which was dissolved in water with pH=8.21 (Fig. S4), in water with pH=2.62 (Fig. S5), in 3% hydrogen peroxide (Fig. S6), in saline (Fig. S7), in methanol (Fig. S8).

Therefore, the description of the densitometric analysis in chapter "2.6. Densitometric and Spectrodensitometric Analysis” has been precisely corrected. The description of all densitograms in the manuscript and Supplementary Materials has also been corrected (Figures 3, 4, 7, 9, 11, and 13 as well as Figures from S1 to S12). The descriptions of the densitograms were supplemented with the information that they have been made at multiple wavelengths (MLW) from 200 to 380 nm, at wavelength intervals of 30 nm at each step.

 

Comment 2: 

For figures 2, 6, 8, 10 and 12 specify that the bars are means +/- 1 standard deviation or standard error.

 Response 2:

The bars in Figures 2, 6, 8, 10 and 12 are the average of six measurements with the calculated standard deviation (±SD). This description has been added to Figures 2, 6, 8, 10 and 12.

 

Sincerely yours,

Alina Pyka-Pająk