Authenticity Analysis of Cold-Pressed Orange Essential Oils by GC/MS on Polymethoxyflavone Components

Round 1

Reviewer 1 Report

In the current study, the authors have developed a GC-MS based quality control techniques for the authenticity analysis of cold-pressed orange essential oil. This reviewer appreciates the authors’ effort in conducting the study. However, there are certain serious concerns that authors need to address.

1. While concerning the novelty of the work, this reviewer has come across few publications where similar kind of work has already carried out long before, for example:

a.       Stremple P. GC/MS analysis of polymethoxyflavones in citrus oils. Journal of High Resolution Chromatography. 1998 Nov 1;21(11):587-91.

b.      Trovato E, Arigò A, Vento F, Micalizzi G, Dugo P, Mondello L. Influence of citrus flavor addition in brewing process: Characterization of the volatile and non-volatile profile to prevent frauds and adulterations. Separations. 2021 Feb 12;8(2):18.

2. Polymethoxyflavones in orange oil are high boiling (>500°C) non volatile fraction, that are generally not suitable for GC-MS analysis. In recent years, these compounds are targeting as marker compounds for authenticity analysis of orange oil through HPLC technique, which is preferable for analyzing non-volatile compounds. For your reference see this paper

a.       Fan H, Wu Q, Simon JE, Lou SN, Ho CT. Authenticity analysis of citrus essential oils by HPLC-UV-MS on oxygenated heterocyclic components. journal of food and drug analysis. 2015 Mar 1;23(1):30-9.

Authors need to provide more details in the methodology part, how the method was developed? how the developed method can compete other non-volatile compound analyzing method such as LC-MS?, how the commercially purchased PMF was utilized in the study?

3. The figure resolution in the manuscript is very poor and not up to the mark of the journal standard.

4. In the results and discussion, section 3.2, line no. 189, the Noblientin quantity is not matching with the table 3.

5. line no. 188, “114.2~138.8ug/mL of tangeritin” should be “114.2-138.8ug/mL of tangeritin”

6. Sentence construction in the manuscript is not found good and authors are requested to focus on this. For example- In the abstract, line no. 17-19, “Therefore, the objective of this study was to develop a Gas chromatography/mass spectrometry (GC/MS) method was developed for the compositional study of total constituents in cold-pressed essential oils from major orange species.” Should be “Therefore, the objective of this study was to develop a Gas chromatography/mass spectrometry (GC/MS) method for the compositional study of total constituents in cold-pressed essential oils from major orange species.”

7. The keywords should not be chosen from the title itself.

8. In the reference section scientific names are not in italic, in some places journal abbreviation is not ended with dot, e.g. line no. 226- “Int J Food Prop.” Should be “Int. J. Food Prop.” Authors need to crosscheck the references.

Author Response

作者對審稿人意見的回應（審稿人 1）

01/04/23

Prof. Dr. Les Copeland 

Editor-in-Chief

Agriculture

Dear Editor

Dear Professor,

Thank you for considering the revised version of our manuscript: agriculture-2121319, entitled " Authenticity analysis of cold-pressed orange essential oils by GC/MS on polymethoxyflavone components", by Yang. et al. for publication in Agriculture. We are thankful to the referees and the Editor for pointing out some important modifications needed in the report. We believe the comments have been highly constructive and useful to restructure the manuscript. We have thoughtfully taken into account these comments. We have thoughtfully taken into account these comments. The explanation of what we have changed in response to the reviewers’ concerns is given point by point in the following pages.

We hope that all these changes fulfill the requirements to make the manuscript acceptable for publication in Agriculture

Looking forward to hearing from you soon.

Sincerely yours,

Chih-Wei Chen

Referees 1 Comments

• The reviewer’s comment：

1. While concerning the novelty of the work, this reviewer has come across few publications where similar kind of work has already carried out long before, for example:

The authors’ Answer:

Thanks for the reviewer’s comment, these are our references, but we emphasize the industry's applicability more. We have a comprehensive discussion in lines 63-65 and198-202.

• The reviewer’s comment：

2. Authors need to provide more details in the methodology part, how the method was developed? how the developed method can compete other non-volatile compound analyzing method such as LC-MS?, how the commercially purchased PMF was utilized in the study?

The authors’ Answer:

For the analysis of PMF by GC/MS, we have compared standard products and database ion. fragments, so there should be no problem. Tangeritin, sinensetin, and nobiletin have a high unit price (approximately NT$10,000/100mg), so 5α-Cholestane was selected as the internal standard. Section 1 (volatile matter), section 2 (blend oil), and section 3 (authenticity) in the presentation of GC/MS results are comprehensive for CP-OrEO quality monitoring, which is unprecedented in LC/MS.

• The reviewer’s comment：

3. The figure resolution in the manuscript is very poor and not up to the mark of the journal standard.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the figure resolution

Please see Figure 1.

• The reviewer’s comment：

4. In the results and discussion, section 3.2, line no. 189, the Noblientin quantity is not matching with the table 3.
5. line no. 188, “114.2~138.8ug/mL of tangeritin” should be “114.2-138.8ug/mL of tangeritin”

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the errors

The error has been revised. Please see line 195

14, line 257.

• The reviewer’s comment：

6. Sentence construction in the manuscript is not found good and authors are requested to focus on this. For example- In the abstract, line no. 17-19, “Therefore, the objective of this study was to develop a Gas chromatography/mass spectrometry (GC/MS) method was developed for the compositional study of total constituents in cold-pressed essential oils from major orange species.” Should be “Therefore, the objective of this study was to develop a Gas chromatography/mass spectrometry (GC/MS) method for the compositional study of total constituents in cold-pressed essential oils from major orange species.”

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the errors

The error has been revised. Please see line 17-19

• The reviewer’s comment：

7. The keywords should not be chosen from the title itself.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the keywords

The error has been revised. Please see keywords

• The reviewer’s comment：

8. In the reference section scientific names are not in italic, in some places journal abbreviation is not ended with dot, e.g. line no. 226- “Int J Food Prop.” Should be “Int. J. Food Prop.” Authors need to crosscheck the references.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the errors

The error has been revised. Please see line 231-232

Author Response File: Author Response.docx

Reviewer 2 Report

The authors of this manuscript have done valuable research on the authenticity of orange essential oils by GC/MS analysis. Checking the authenticity of oils is a very urgent task, due to the presence on the market of a large amount of low-quality oil material. I consider this work worthy of publication after making the following rectification:

Lines 44-46 Please change the symbols (i), (ii) and (iii) into capital letters.

Lines 64-69 Please clearly state the purpose and objectives of the study.

Lines 86-88 These components should be calculated and entered in relation to the internal standard or in relation to the built calibration programs.

Lines 93-95 The volume of analyzed samples is not specified. The dilution rate of the samples with 100% isooctane should be indicated.

Line 92 The feasibility of using this external standard is not clear. Either a literary reference to the method should be given, or generally accepted standards such as hexadecane should be used.

Line 93 The expediency of using maximum concentrations, in particular 1000 μg / ml, is not clear.

Please specify sample application mode (split or splitless) and sample dilution.

Lines 95-96 The analysis of GC/MS does not depend on the day of determination, and if artifact peaks appear, control injections of the solvent used should be carried out. Explain what do you mean.

Line 98 A reference of the signal-to-noise peak selection should be added, as well as the difference from the classical signal-to-noise 2:1 peak selection should be explained.

Lines 96-97 What do you mean when you use "Triplicate injections"? Clarify please.

Lines 109-110 The column should be described in detail, as well as a comparative analysis of the performance of the above column with the commonly used HP-5MS one (30 m × 0.25 mm, phase film thickness - 0.25 mm, (5% - phenyl-, 95% - methylpolysiloxane).

Lines 183-184 Give a reference or research reasons for the presence of 5α-cholesterol in the sample.

Line 191 The table 3 does not contain the retention indices of tangeritin, sinensetin, nobiletin. Difficulties in identifying oil components by the classic NIST20 mass spectrum library are not described. And also with the possible difficulty in identifying the components of the essential oil, it is necessary to determine the molecular ions of the highest intensity.

Line 198-203 Please indicate the prospects for using this method in the analysis of the authenticity of essential oils and whether this method is suitable for other citrus crops.

Author Response

01/04/23

Prof. Dr. Les Copeland 

Editor-in-Chief

Agriculture

Dear Editor

Dear Professor,

Thank you for considering the revised version of our manuscript: agriculture-2121319, entitled " Authenticity analysis of cold-pressed orange essential oils by GC/MS on polymethoxyflavone components", by Yang. et al. for publication in Agriculture. We are thankful to the referees and the Editor for pointing out some important modifications needed in the report. We believe the comments have been highly constructive and useful to restructure the manuscript. We have thoughtfully taken into account these comments. We have thoughtfully taken into account these comments. The explanation of what we have changed in response to the reviewers’ concerns is given point by point in the following pages.

We hope that all these changes fulfill the requirements to make the manuscript acceptable for publication in Agriculture

Looking forward to hearing from you soon.

Sincerely yours,

Chih-Wei Chen

Referees 2 Comments

• The reviewer’s comment：

1. Lines 44-46 Please change the symbols (i), (ii) and (iii) into capital letters.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised the symbols .

The error has been revised. Please see line 43-45.

• The reviewer’s comment：

2. Lines 64-69 Please clearly state the purpose and objectives of the study.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised it. Please see line 63-71.

• The reviewer’s comment：

3. Lines 86-88 These components should be calculated and entered in relation to the internal standard or in relation to the built calibration programs.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised it. Please see line 89-92

• The reviewer’s comment：

4. Line 92 The feasibility of using this external standard is not clear. Either a literary reference to the method should be given, or generally accepted standards such as hexadecane should be used.
5. Lines 183-184 Give a reference or research reasons for the presence of 5α-cholesterol in the sample.

The authors’ Answer:

At line 169 =>We analyzed with GC/MS to identify phytosterol and polymethoxyflavones in section 3. that cannot be removed with determination.

We changed the text 5α-cholesterol to 5α-Cholestane for a more general and convenient query.

The following literature references show that phytosterol assays will use 5α-Cholestane as an internal. standard.

Jun-Hua, H. A. N., Yue-Xin, Y. A. N. G., & Mei-Yuan, F. E. N. G. (2008). Contents of phytosterols in vegetables and fruits commonly consumed in China. Biomedical and Environmental Sciences, 21(6), 449-453.

Ciou, J. Y., Chen, H. C., Chen, C. W., & Yang, K. M. (2021). Relationship between antioxidant. components and oxidative stability of peanut oils as affected by roasting temperatures. Agriculture, 11(4), 300.

• The reviewer’s comment：

6. Line 93 The expediency of using maximum concentrations, in particular 1000 μg / ml, is not clear. (Please specify sample application mode (split or splitless) and sample dilution.)

The authors’ Answer:

In the experiment, it was not detected at 1 μg/ml, so the calibration line was set at 10 μg/ml as the lowest. concentration, and the amount of internal standard added in the experiment was 100 μg/ml, so 1000 μg/ml was used as the highest concentration of the calibration line.

• The reviewer’s comment：

7. The analysis of GC/MS does not depend on the day of determination, and if artifact peaks appear, control injections of the solvent used should be carried out. Explain what do you mean.
8. Line 98 A reference of the signal-to-noise peak selection should be added, as well as the difference from the classical signal-to-noise 2:1 peak selection should be explained.
9. Lines 96-97 What do you mean when you use "Triplicate injections"? Clarify please.

The authors’ Answer:

In line 93, there is a reference to the method validation, and we refer to the presentation of the following. documents for the convenience of readers.

Chen, W. C., Lai, Y. S., Lu, K. H., Lin, S. H., Liao, L. Y., Ho, C. T., & Sheen, L. Y. (2015). Method. development and validation for the high-performance liquid chromatography assay of gastrodin in water   extracts from different sources of Gastrodia elata Blume. journal of food and drug analysis, 23(4), 803-810.

Shao, X., Lv, L., Parks, T., Wu, H., Ho, C. T., & Sang, S. (2010). Quantitative analysis of ginger. components in commercial products using liquid chromatography with electrochemical array detection. Journal of agricultural and food chemistry, 58(24), 12608-12614.

Chan, K. H., Hsu, M. C., & Chu, W. L. (2007). Determination of pentazocine in urine by gas. chromatography-mass spectrometry. Journal of Food and Drug Analysis, 15(3), 4.

• The reviewer’s comment：

10. Lines 109-110 The column should be described in detail, as well as a comparative analysis of the performance of the above column with the commonly used HP-5MS one (30 m × 0.25 mm, phase film thickness - 0.25 mm, (5% - phenyl-, 95% - methylpolysiloxane).

The authors’ Answer:

We have completed the establishment of essential oil and phytosterol methods in the DB-1 column. and published many SCI papers, so we chose the DB-1 column for analysis.

Lin, L. Y., Chuang, C. H., Chen, H. C., & Yang, K. M. (2019). Lime (Citrus aurantifolia (Christm.) Swingle) essential oils: Volatile compounds, antioxidant capacity, and hypolipidemic effect. Foods, 8(9), 398.

Ciou, J. Y., Chen, H. C., Chen, C. W., & Yang, K. M. (2021). Relationship between antioxidant. components and oxidative stability of peanut oils as affected by roasting temperatures. Agriculture, 11(4), 300.

Yeh, M. Y., Ko, W. C., & Lin, L. Y. (2014). Hypolipidemic and antioxidant activity of enoki mushrooms. (Flammulina velutipes). BioMed research international, 2014.

• The reviewer’s comment：

11. Line 191 The table 3 does not contain the retention indices of tangeritin, sinensetin, nobiletin. Difficulties in identifying oil components by the classic NIST20 mass spectrum library are not described. And also with the possible difficulty in identifying the components of the essential oil, it is necessary to determine the molecular ions of the highest intensity.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised table 3. Please see table 3

• The reviewer’s comment：

12. Line 198-203 Please indicate the prospects for using this method in the analysis of the authenticity of essential oils and whether this method is suitable for other citrus crops.

The authors’ Answer:

Thanks for the reviewer’s comment, I’ve carefully checked again and revised it. Please see line 198-202

Author Response File: Author Response.docx