Comparison of Three Gas Chromatographic Methods—Identification of Terpenes and Terpenoids in Cannabis sativa L.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe article presents a comparison of three gas chromatography/mass spectrometry (GC/MS) methods for the identification of volatile compounds in four different chemotypes of Cannabis sativa L. The methods compared were liquid extraction with hexane (Liq), static headspace extraction (HS), and solid-phase microextraction headspace (SPME). The primary compounds analyzed were monoterpenes/monoterpenoids and sesquiterpenes/sesquiterpenoids.
- The introduction highlights the importance of terpenes and terpenoids in Cannabis sativa L. from both biogenetic and medical perspectives. The text mentions the lack of knowledge about the significance and relationship of these compounds with other bioactive compounds, mainly cannabinoids, and the need for further research to understand the medicinal effects of the plant in the context of terpenoids. It is described in the literature that cannabinoid concentrations are detected in essential oils, even in low amounts.
- Sample preparation methods and experimental conditions were described in detail. The methodology includes the use of commercial standards for compound identification and comparison with mass spectral libraries. Three different chemotypes were analyzed using each extraction method.
- The results showed that hexane extraction provided comparable results, while HS and SPME methods allowed for the identification of more substances. The tables presented the identified compounds and their relative quantities in each chemotype, highlighting the differences in the effectiveness of the methods for identifying various volatile compounds.
- The discussion addressed the relevance of the compared methods, highlighting that hexane extraction was the most effective for the quantitative analysis of compounds. However, HS and SPME methods were better for identifying a wider range of compounds. It was emphasized that the consistent use of a specific method is crucial for the comparability of results between different laboratories.
- The conclusion reaffirms that hexane extraction is advantageous for quantitative analyses, while HS and SPME are more effective for identifying a greater variety of compounds. The article suggests that standardization of the analytical method is essential for objective comparisons between laboratories.
Strengths:
1) The comparison of three different methods provides a comprehensive insight into the effectiveness of each for the analysis of volatile compounds in cannabis.
2) The research addresses an area of growing scientific and medical interest, contributing to the knowledge about the components of cannabis.
Weaknesses:
1) The limitation in the quantification of all terpenes/terpenoids due to the lack of commercial standards for some compounds may compromise the accuracy of the results.
2) The analysis relies on commercial standards that may not be available or accessible to all laboratories, limiting reproducibility.
3) The variability of the compounds identified between chemotypes and methods suggests the need for more studies to better understand these differences and their implications.
Suggestions for Improvement:
1) Investing in the acquisition of commercial standards for a broader range of compounds can improve quantification accuracy.
2) Conducting complementary studies to evaluate the stability of terpenes/terpenoids during storage and sample processing.
3) Encouraging the standardization of analytical methods among laboratories to improve comparability of results and reduce variability.
Rejection Factors and Areas for Reevaluation:
1) The article does not explicitly mention the performance of calibration curves for the solid-phase microextraction headspace (SPME) method. The methodology describes the use of commercial standards for compound identification, but there are no details about specific quantitative calibration for SPME.
2) Confidence analysis in the results was not directly discussed in the article. However, the methodology section mentions that the validation of the analytical method, including selectivity, specificity, accuracy, linearity, range, detection limit, quantification limit, robustness, and ruggedness, was performed, but these details are beyond the scope of the manuscript and will be published in another publication. This implies that a detailed confidence analysis may be available in another source.
3) The species name needs to be italicized. Throughout the text, a lack of dedication to maintaining this quality was noticeable.
4) Review the tables to present them in a better order. 1) Retention time; Compound name; Calculated IR; Percentage;
5) Add a legend in the table to describe whether the data is in elution order and which column was used for the calculation. Refer to previous works from the journal.
6) What does "cpd" mean? It needs to be defined.
7) The chemical classes were not explored in the text. It is necessary to discuss the differences and the rationale for the separation.
8)The chromatograms were not discussed. I suggest including them in the supplementary material.
9) I believe the results should be presented in table format rather than in the frame format chosen by the authors.
Conclusion:
- The article offers a valuable contribution to the understanding of methods for analyzing volatile compounds in Cannabis sativa L. The detailed comparison of GC/MS methods highlights the importance of choosing the appropriate method for different analytical objectives. However, the research could benefit from improvements in quantification and methodological standardization to maximize the reproducibility and accuracy of the results.
Author Response
Dear Reviewer, Thank you for your efforts in evaluating my manuscript. I have tried my best to make the adjustments as recommended by you and the other two reviewers. I apologize for not listing all the points, but corrections can be found in my edited manuscript. Once again, thank you very much and I wish you a nice day.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors:
I have read your paper carefully and I think that it is interesting although few modifications are required before it could be published.
Although I have include few comments throughout the text I think that you should include few explanation about:
1.- I think that you should use the same amounts of samples for the different methods to compare them. The different results correspond to the extraction method. I think it would be interesting if you could compare each method under the same conditions. Did you consider using an extraction method that allows the ingestion of the product? If you were told about the effect of different chemotypes it would be interesting to know which compounds will be in the product to be used for medicinal purposes.
2.- The methods that you have used are not very reproductive, particularly SPME. I think it is important you include the number of repetitions you have done and give the results with the standard deviation.
3.- yo can not use this: "We chose the biggest peak (by space) as 100% and calculated the relative ratios of the other ones (in Tables as. % norm)." You should recalculate all the compounds so the sum of all of them is 100. Neither compound of the sample can have 100%.
4.- Please, considering the above comments, try to give the information of table 5 with statistical analysis.
Best wishes.
Comments for author File: Comments.pdf
Author Response
Dear reviewer,
I corrected my manuscript according to your suggestions.
Thank you
Lumír Hanuš
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIt is not significant original scientific research.
There are no novel methodologies or concepts at work.
The application of the method is limited since it is known that the chemical composition of plants changes depending on the place and date of collection.
There are already many studies on this matter, and in the following review there is a lot of information.
Terpenes in Cannabis sativa - From plant genome to humans, Booth, Judith K.; Bohlmann, Jorg, Plant Science (2019), 284, 67-72.
Author Response
Dear Reviewer, Thank you for your efforts in evaluating my manuscript. I have tried my best to make the adjustments as recommended by you and the other two reviewers. I apologize for not listing all the points, but corrections can be found in my edited manuscript. Once again, thank you very much and I wish you a nice day.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear,
The author fulfilled most of the requests.
I just ask you to write a caption for "cpd" in the text.
Author Response
Dear,
The author fulfilled most of the requests.
I just ask you to write a caption for "cpd" in the text.
Abbreviation cpd was added to the text to Table 1. Explanation is also at Abbreviations.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsWith the improvements to the manuscript, it can provide relevant information about the chemistry of Cannabis sativa, it was recommended to review the following to consider it for publication.
· Propose selection criteria for chemotypes and not select them at random.
· Discuss the differences found in each extraction method, because one method detects curcumene and the other two do not.
· Check if all the compounds are of biosynthetic origin, for example, 4-methyloctane or 2,4-dimethylheptane.
· I suggest that compounds with a % less than 0.09 be grouped and identified as minor components (tables 1-4).
· In the conclusions, the authors indicate that extraction with hexane gives balanced results (which is advantageous for quantitative analysis), but they must clarify the purity of the hexane to be used to avoid artifacts in the analysis.
Author Response
- Propose selection criteria for chemotypes and not select them at random.
These four varieties that are used to treat patients were chosen so that we could compare the effectiveness of the given variety in treatment in relation to their content substances.
- 2. Discuss the differences found in each extraction method, because one method detects curcumene and the other two do not.
The determination of some substances in the sample depends on the sensitivity of the method used. The volatility of the given substance and the amount of sample used are also important. Therefore, some substances were not identified by all three methods
- 3. Check if all the compounds are of biosynthetic origin, for example, 4-methyloctane or 2,4-dimethylheptane.
4-Methyloctane and 2,4-dimethylheptane are natural product found for instance in Nicotiana tabacum (published information). Thes compounds were found in cannabis also by others – for instance Lorenzo Calvi, Daniela Pentimalli, Sara Panseri, Luca Giupponi, Fabrizio Gelmini, Giangiacomo Beretta, Davide Vitali, Massimo Bruno, Emanuela Zilio, Radmila Pavlovic, Annamaria Giorgi: Comprehensive quality evaluation of medical Cannabis sativa L.inflorescence and macerated oils based on HS-SPME coupled toGC–MS and LC-HRMS (q-exactive orbitrap®) approach. Journal of Pharmaceutical and Biomedical Analysis 150, 208–219 (2018)
- I suggest that compounds with a % less than 0.09 be grouped and identified as minor components (tables 1-4).
The content of substances with a content lower than 0.09% was marked as traces.
- 5. In the conclusions, the authors indicate that extraction with hexane gives balanced results (which is advantageous for quantitative analysis), but they must clarify the purity of the hexane to be used to avoid artifacts in the analysis.
n-Hexane for gas chromatography ≥ 98.0% (Merck) was used in sample processing.
Dear reviewer, I found that I did not correct Table one. As I did not succeed to attach it here I sent it to the Journal (to Ms Gwen Ma). Thank you for understanding and have a nice day.
Author Response File: Author Response.pdf
