Development, Validation, and Application of a Simple and Rugged HPLC Method for Boswellic Acids for a Comparative Study of Their Abundance in Different Species of Boswellia Gum Resins

Round 1

Reviewer 1 Report

A. Figure 2 on Page 6 should be correctly labeled as Figure 1.

B. Lines 203-204 = Authors mention that they have acquired additional data from peer reviewed publications. They should mention these references here. Next, authors mention in table 3, Line 214 that all quantitation data was obtained from one single manuscript (Reference 22). Both of above comments are misleading. .

C, Authors have pointed to Reference 22 observation on Line 221. Considering the importance, authors could detail how the present study differs from theirs in methodology, experimental plan and present work add upon the literature knowledge.

D. The resolution of Figures 1 and 2 is low and legends are unreadable even after zooming in up to 200%. This made interpreting results difficult.

E. The results in section 3.3 Multivariate analysis are not explained and only figure is presented. It will help to know how PCA analysis can confirm or enhance results already presented in Table 2.

F. Line 121 - Authors could mention which HPLC method was used to generate method validation parameters. 

G. Line 254 - It is not clear where the second HPLC method was previously used and by whom. Authors prepare a table with retention times for boswellic acids for the third HPLC method. This will help others in the filed to validate their results.

H. It will be interesting to see if retention times could be used as variables for PCA analysis.

I. Table 3 = Supposed specie is in 2nd column and not 4th.

 

Author Response

General changes

The authors names and surnames were inverted in the form “name surname”

We added a supplementary section for the chromatograms

 

 

1. Figure 2 on Page 6 should be correctly labeled as Figure 1.

We corrected the figures numbering

1. Lines 203-204 = Authors mention that they have acquired additional data from peer reviewed publications. They should mention these references here. Next, authors mention in table 3, Line 214 that all quantitation data was obtained from one single manuscript (Reference 22). Both of above comments are misleading.

The observation is correct. We specified that only one peer-reviewed work was used to build the model (now rows 253-254) we also specified the reference. Furthermore, we cited the article in M&M. (now line 81). We changed the reference also in table 3.

C, Authors have pointed to Reference 22 observation on Line 221. Considering the importance, authors could detail how the present study differs from theirs in methodology, experimental plan and present work add upon the literature knowledge.

Thank you very much for the suggestion. We added the discussion about the different methodology proposed. We also discussed about other literature methods and in what our instrumental method adds to literature. The discussion may be found now at rows 347-352.

A discussion about what our meta-analysis work adds to the scientific literature knowledge is presented at rows 398-402.

1. The resolution of Figures 1 and 2 is low and legends are unreadable even after zooming in up to 200%. This made interpreting results difficult.

The reviewer is right. Figures 1 and 2 and their legends are now more readable.

3. The results in section 3.3 Multivariate analysis are not explained and only figure is presented. It will help to know how PCA analysis can confirm or enhance results already presented in Table 2.

We added the explanation of the results obtained by the PCA analysis. A new paragraph can be found at lines 236-250

1. Line 121 - Authors could mention which HPLC method was used to generate method validation parameters. 

Thanks, now the method used is specified (see row 146)

1. Line 254 - It is not clear where the second HPLC method was previously used and by whom. Authors prepare a table with retention times for boswellic acids for the third HPLC method. This will help others in the field to validate their results.

Thank you for the observation. The second method was previously used in our analytical laboratory as we specified it in the discussion (row 333). We added the Rt data in table 1.

1. It will be interesting to see if retention times could be used as variables for PCA analysis.

Thank you for your comment, however retention times cannot be used as variables since in chromatography the retention time do not vary.

1. Table 3 = Supposed specie is in 2nd column and not 4th.

Thank you, we corrected the error

Author Response File: Author Response.docx

Reviewer 2 Report

1.      Abstract. The introductory part was complete ignored. The first line (14) is repeated again with similar meaning in line 18. Common RP C18 column is not an appropriate sentence. The methods shall be clearly explain, the results as well, the results shall be quantitative. Then draw the conclusion in connection with different varieties of boswellia plants, also no need to use capitalization for plant name

2.      Keywords. The uncommon abbreviation shall not be given, also the words must be different from title of manuscript

3.      First use the full name then abbreviate throughout the manuscript.

4.      Line 41, gum-resins contain up to 30%..it must be 30 %, and follow throughout the manuscript

5.      Line 41-53, the sentence is too long, split in small sentences to understand the meaning clearly

6.      The research work is related to analysis by HPLC of plant material, and then identified the different origins of plant species. The rational of the study is very weak, the literature reported methods for analysis of same type of plant species were not incorporated for the comparison. What is novelty of this study, no explanations, and the advantages of present study over the reported study need to explain?

7.      Samples, the market name from where the samples were purchased

8.      Samples for meta-multivariate analysis. Explain clearly how the data was obtained, methodology, and the time span for which the data was collected.

9.      Samples preparation. Clearly mention the species name for sample preparation, each species name must be mentioned

10.    The LC gradient program as well as mobile phase ratios were not clear mentioned, make it easy for understanding

11.    Line 118. Acquisition bandwidth was set 118 at ±4nm, shall be ±4 nm

12.    The accuracy was expressed in terms of BIAS% using a low (n=3), a medium (n=3) and a high concentration level, (n=3). Explain the concentration of all three levels.

13.    BIAS % 5, 20, and 100 mg/L while the RSD % 1, 20, and 100 mg/L?

14.    0.19, and 0,45, use similar decimal points throughout the manuscript

15.    Following the ICH guidelines the sample solutions spiked with known concentrations, and results must be presented in terms of % recovery for accuracy studies whereas repeatability and reproducibility explained for precision, ICH guidelines were not followed, The HPLC chromatograms were not presented, at least chromatograms of blank, standard, and samples must be given.

 

Author Response

General changes

The authors names and surnames were inverted in the form “name surname”

We added a supplementary section for the chromatograms

 

 

Reviewer 2

1. Abstract. The introductory part was completely ignored. The first line (14) is repeated again with similar meaning in line 18. Common RP C18 column is not an appropriate sentence. The methods shall be clearly explain, the results as well, the results shall be quantitative. Then draw the conclusion in connection with different varieties of boswellia plants, also no need to use capitalization for plant name

We addressed the suggested observations:

We added the introduction abstract and deleted the sentence which was repeated.

In order to avoid increasing the abstract length over 200 recommended words, we decided to briefly summarize the methodology (now rows 22-23) and the discussion of results (29), furthermore, since the journal is an open access, all data can be easily accessed.

2. Keywords. The uncommon abbreviation shall not be given, also the words must be different from title of manuscript

Thank you for your suggestion, We removed the uncommon abbreviations as well as the repeated title words

3. First use the full name then abbreviate throughout the manuscript.

Thank you for your suggestion, we addressed the issue in the introductory part as well as in the rest of the MS

4. Line 41, gum-resins contain up to 30%..it must be 30 %, and follow throughout the manuscript

Thank you for the observation, we corrected the error in the whole manuscript

5. Line 41-53, the sentence is too long, split in small sentences to understand the meaning clearly

We split the sentence in two parts, one describing the activity of Boswellia sacra, the second one for Boswellia serrata. Now at line 61

6. The research work is related to analysis by HPLC of plant material, and then identified the different origins of plant species. The rational of the study is very weak, the literature reported methods for analysis of same type of plant species were not incorporated for the comparison. What is novelty of this study, no explanations, and the advantages of present study over the reported study need to explain?

We tried to strengthen the rational of our study by focusing its potential as an approach to use those six molecules to characterize different species’ fingerprint. Consequently it’s possible avoid the misrecognition of crude resins to produce food-supplement grade extracts. The new discussion can be found in the paragraph of Discussion (rows 300-317)

Other HPLC methods reporting the analysis for boswellic acids were discussed in the first part of the discussion paragraph (now rows 347-352)

A discussion about what our meta-analysis work adds to the scientific literature knowledge is presented at rows 398-402

7. Samples, the market name from where the samples were purchased

We specified the market name of the samples of crude resin we purchased at line 76-77

8. Samples for meta-multivariate analysis. Explain clearly how the data was obtained, methodology, and the time span for which the data was collected.

The data regarding the samples for multivariate analysis were from 2019; our data are obtained in the timespan between 2020 and 2022, please find the revision at row 81-82

9. Samples preparation. Clearly mention the species name for sample preparation, each species name must be mentioned

Thank you, we mentioned the species name at row 98

10. The LC gradient program as well as mobile phase ratios were not clear mentioned, make it easy for understanding

We specified from line 119-140 the total analysis time and the single steps by a simpler notation (time expressed in minutes and % B)

11. Line 118. Acquisition bandwidth was set 118 at ±4nm, shall be ±4 nm

We corrected the refuse (now at line 144)

12. The accuracy was expressed in terms of BIAS% using a low (n=3), a medium (n=3) and a high concentration level, (n=3). Explain the concentration of all three levels.

We added the concentration value in mg/L (new rows 154-154)

13. BIAS % 5, 20, and 100 mg/L while the RSD % 1, 20, and 100 mg/L?

There was a refuse, thanks for your observation. Both BIAS% and RSD% studies were conducted on the 5 mg/L Low calibration level

14. 0.19, and 0,45, use similar decimal points throughout the manuscript

We uniformed the decimals using dots.

15. Following the ICH guidelines the sample solutions spiked with known concentrations, and results must be presented in terms of % recovery for accuracy studies whereas repeatability and reproducibility explained for precision, ICH guidelines were not followed.

Regarding your comment on the validation part, we think that the terms of BIAS and RSD% may be misleading. For BIAS% we meant the accuracy as the percentage of distance between the real value and the calculated value.  We modified the term BIAS% to “accuracy BIAS %” in the validation method discussion to clarify its meaning according the ICH definition. (Row 154)

The RSD% explains the precision as percent value of the standard deviation of three measurements: two led in the same day (repeatability) and one in a different day by a different operator (intermediate precision).

This is the ICH paragraph which explains how to report accuracy:

“Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value together with the confidence intervals”

This is the paragraph which explains how to report precision:

“The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated.”

 

 

The HPLC chromatograms were not presented, at least chromatograms of blank, standard, and samples must be given

Chromatograms were added in supplementary.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have satisfactorily answered my comments.

Reviewer 2 Report

Manuscript in its current form reached the level for final acceptance for publication