Analyzing Plant Low-Molecular-Weight Polar Metabolites: A GC-MS Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article discusses multiple aspects of the GC-MS-based workflow for the analysis of low molecular weight plant metabolites, providing insights into the critical steps of workflow, targeted and non-targeted analysis of plant metabolomes and the progress achieved in translational applications for understanding plant metabolism.

Some suggestions and queries for improvement are discussed:

Abstract, line 22-25: Ongoing advancements in all stages…………………problems are needed.

There has been key progress in the advancement of GC-MS approaches for studying plant secondary metabolism, however, challenges in artifacts presence during derivatization, defines further investigations. What are the feasible solutions achieved to overcome the limitations in GC-MS studies? Please provide key examples.

 

It is also very important to discuss the studies carried out in key plant species employing GC-MS approaches. A discussion on the progress in unravelling plant metabolome, identification and quantification of new metabolites and the challenges encountered during study, should be emphasized.

Line39-41: These pioneering studies highlighted…………..molecular networks. Discussing the concept, it is suggested to briefly describe how GC-MS corelates plant genotype with phenotype, highlighting key studies.

 

Plant metabolome provides a final representation of the metabolic network, specialized metabolites and their functions together with the metabolic crosstalk. However, applications of GC-MS technique for plant metabolome study is not sufficient considering the metabolic complexity of plants. What are the key areas of plant metabolome study and objectives when GC-MS is employed? Explain.

Line 93-95: Untargeted metabolomics (metabolite profiling)……………….. additional mass selectivity. The following discussion requires citation. Please include a reference study.

The article provides an in-depth discussion on the application of GC-MS in plants. In the introduction, it is necessary to discuss the history of GC-MS development and refinement briefly to provide a complete overview to the readers.

Line 113-115: Thus, shock-freezing provides ………….. “metabolic snapshot”. In the present context, what is the meaning of  plant metabolic snapshot and why is it important in terms of plant metabolism? Discuss briefly.

In the article, there is no discussion on how the literature survey was performed, what is the methodology followed, literature searched and screened, inclusion and exclusion criteria and the rational for writing this review.

Reference sections: Very few recent works are cited in the paper. It is better to include the recent research on plant metabolome study employing GC-MS.

 

Author Response

Reviewer 1

Comments and Suggestions for Authors

The article discusses multiple aspects of the GC-MS-based workflow for the analysis of low molecular weight plant metabolites, providing insights into the critical steps of workflow, targeted and non-targeted analysis of plant metabolomes and the progress achieved in translational applications for understanding plant metabolism.

Some suggestions and queries for improvement are discussed:

Comment 1

Abstract, line 22-25: Ongoing advancements in all stages…………………problems are needed.

There has been key progress in the advancement of GC-MS approaches for studying plant secondary metabolism, however, challenges in artifacts presence during derivatization, defines further investigations. What are the feasible solutions achieved to overcome the limitations in GC-MS studies? Please provide key examples.

Answer: We thank the Reviewer for this suggestion and highlighting the subject to discuss in our review. We agree that the indicated sentences in the end of Abstract were not sufficiently clear. Thus, to improve clarity and in accordance with the Reviewer’s suggestion we have change the test as follows:

Lines 24-28: “There has been key progress in the advancement of GC-MS approaches for studying plant metabolism. However, the presence of artifacts during GC-MS analysis, particularly during derivatization, is a challenge that requires careful validations, which frequently necessitate additional investigations. The feasible solutions that were achieved to overcome the limitations in GC-MS-based studies are of particular focus in the presented discussion here. “  

As to the “feasible solutions achieved to overcome the limitations in GC-MS-based studies”, several of them already have been intensively discussed in Sample preparation and Derivatization sections. Since our review also aimed to discuss difficulties that potentially can occur at every step of the GC-MS workflow, we indicate the aim in the following text (marked with bold font) in the end of Introduction (lines129-133):

„Since the overall performance and the final output of the metabolomics analysis critically depend on each of these three steps, we address them in more detail below, highlighting the challenges and achievable solutions. However, the importance of the sample preparation, including derivatization methods, in GC-MS-based metabolomics is given special attention because inaccuracy made at this step may distort the final results of the analysis. “

For a convenient view, the key examples of those limitations and related solutions are indicated in the following table:

GC-MS limitations and potential artifacts	Solutions overcoming the limitations	Lines in revised manuscript
This technique is not compatible with analysis of high molecular weighed non-polar compounds (lipids, chlorophyls, carotinoids), which often are coextracted with relatively high yields together with low molecular weighted metabolites from the plant material	The compounds shoud be removed from the extract prior to the GC-MS analysis, e.g. by liquid-liquid extraction using non-polar solvents such as n-hexane or n-heptane	293-320
Most popular trimethylsilylation agents (MSTFA and BSTFA) which used for derivatisation and resulted TMS derivatives of metabolites highly prone to hydrolytic degradation. Even trace of water can lead to degradation of TMS derivatives and, therefore, result in artifacts in terms of mistaken metabolite quantification. This might affect   reproducibility of GC-MS analysis	1) The extracts prior to be derivatized need to be thoroughly dried. 2) An application of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and N-methyl-bis(trifluoroacetamide) (MBTFA) for derivatisation of plant polar metabolites samples with high water sensitivity. The agents  MTBSTFA and  MBTFA are less sensitive to water than traditional  MSTFA and BSTFA.	350-387
Application of trimethylsilylation as one- step derivatisation strategy often results in obtaining  multiple spatial isomers of sugars which identification presents a real challenge.	Supplementing the derivatization procedure with an additional step such as sample treatment with O-methylhydroxylamine hydrochloride (MeOX) which has to be introduced prior  trimethylsilylation. The MeOX-based derivatisation  reduces the number of spatial isomers of sugars and thereby increases the accuracy of sugar identification.	388-403
Application of combined MeOX-trimethylsilylation derivatisation strategy also has its limitations. Required elevated temperature of incubations during MEOX derivatization and  trimethylsilylation  may introduce artefacts by causing irreversible modifications (spontaneous cyclization or degradation) in structure of several metabolites. This may lead to potential misinterpretations of the results.	Prior the GC-MS results interpretation it is imperative to gain information from literature or perform experiments with standards for the compounds which stability might potentially be altered during MeOX-trimethylsilylation derivatisation	404-410
Trimethylsilylation ofter result in appearance of various by-products which co-elute with sample metabolites. This contamination affects quality of analyte mass spectra.	Those issues need further investigations	410-412
Trimethylsilylation reaction exhibited lower reactivity towards amino acid groups in compare with carboxyl and hydroxyl groups of metabolites. This results in a few derivates of amino acids which presents products of amino acid incompetent trimethylsilylation.		414-417
Trimethylsilylation may results in converting arginine to ornitine and citrulline TMS derivatives. This case indicates that some compounds detected on chromatograms may be method-related artifacts		419-421
Trimethylsilylation is not an optimal derivatization method for fatty acid analysis, as it increases the molecular weight of fatty acid derivatives, compromising their volatility.	Application of another derivatization method such as  transesterification which converts fatty acids in a reaction  with methanol into more volatile derivatives known as fatty acid methyl esters (FAMEs)	440-447
Transesterification may result in incomplete conversion of fatty acids into their corresponding  FAMEs derivatives. The derivatization  effectivity depends on the reaction time, temperature and catalist concentration. This sensitivity of transesterification on the reaction conditions may lead to the underestimation or misidentification of certain fatty acids, and, therefore, may introduce artifacts potentially affecting the  metabolomics result interpretation.	Optimization of the  transesterification reaction conditions helps to improve the efficiency and, therefore,  ensure reproducibility of the derivatization.	448-455

 

Comment 2

It is also very important to discuss the studies carried out in key plant species employing GC-MS approaches. A discussion on the progress in unravelling plant metabolome, identification and quantification of new metabolites and the challenges encountered during study, should be emphasized.

Answer: We thank the Reviewer for highlighting this issue. As suggested, we have added information about studies in which GC-MS was employed to profile metabolites of various plants in the context of both applied and fundamental plant sciences. This information is given in a way of separated steps describing the gradual progress in unravelling plant metabolome as follows: 

Lines 84-112: “…Thus, GC-MS has been widely used in research to identify metabolites responsible for the biological properties of medicinal plants (e.g. leaves of tropical plants such as Jacaranda mimosifolia (Naz et al., 2020), Aporosa cardiosperma (Abdul et al., 2024), Senecio scandens (Wang et al., 2021)). In this type of studies crude plant extracts usually are not subjected to derivatization. Their GC-MS analysis focuses on the identification of non- or semi-polar volatile secondary compounds (sesqui-, di- and triterpenoids, saturated fatty acid alcohols, alkaloids) which may exhibit biological activities and appear to be plant specific [Deng et al., 2022]. Usually such profiles cover just several dozen metabolites (Naz et al., 2020; Abdul et al., 2024; Wang et al., 2021; Deng et al., 2022).

  However, by implementing a derivatization strategy into the GC-MS workflow, the coverage of metabolites can be significantly expanded (up to several hundreds metabolites (Xu et al., 2023; Osmolovskaya et al., 2025)) as it transforms most non-volatile semi-polar and polar molecules dominating in plant extracts into volatile targets for GC-MS. Most metabolites of the groups are participants of major energy and biosynthetic metabolite pathways. Therefore, even though derivatization introduces complexity (potential incompliteness of reactions, formation of several derivatives) leading to appearing artifacts (Bekele et al., 2014; Abadie et al., 2018; Domergue et al., 2022; Tarakhovskaya et al., 2023), this strategy has made GC-MS to be an apropriate method for analysing plant primary metabolome, significantly increasing the method’s versatility. Moreover, this progress in unravelling plant primary metabolome using GC-MS has been proven to be a significant contribution to multi-omics research (Frolova et al. Gas chromatography-mass spectrometry (GC-MS) in the plant metabolomics toolbox: GC-MS in multi-platform metabolomics and integrated multi-omics research. IJMS. Under review). This can be demonstrated by multiple studies in which GC-MS-based metabolite profiles helped in linking genomic and proteomic data to phenotyic traits of interest enabling metabolomics-assisted beeding for crop improvement (Acharjee et al., 2016); revealing mechanisms of development (Yun et al., 2019), growth protectors (Wang et al., 2021), or responses to a changing enviromental (Strenkert et al., 2019). Thus, by used of GC-MS Acharjee and co-workers (Acharjee et al., 2016) identified four metabolites assotiated with carotenoid biosynthesis contributing to the potato tuber flesh color. In another work, Wang and co-authors (Wang et al., 2021) reaviled that growth promoting effect of zaxinone on rice seedlings was accompanied by marked increase in contents of many sugars and stimulating central energy pathways. And in the third example, Yun and colleagues (Yun et al., 2019) found 36 GC-MS-identified primary metabolites and 81 volatiles that contributed to the ripening of the peel in harvested bananas.“

New literature references

Naz R, Roberts TH, Bano A, Nosheen A, Yasmin H, Hassan MN, Keyani R, Ullah S, Khan W, Anwar Z. GC-MS analysis, antimicrobial, antioxidant, antilipoxygenase and cytotoxic activities of Jacaranda mimosifolia methanol leaf extracts and fractions. PLoS One. 2020 Jul 29;15(7):e0236319. doi: 10.1371/journal.pone.0236319. PMID: 32726328; PMCID: PMC7390342.

Abdul, U., Manikandan, D.B., Arumugam, M. et al. GC–MS based metabolomic profiling of Aporosa cardiosperma (Gaertn.) Merr. leaf extracts and evaluating its therapeutic potential. Sci Rep 14, 16010 (2024). https://doi.org/10.1038/s41598-024-66491-2

Wang L, Liang J, Xie X, Liu J, Shen Q, Li L, Wang Q. Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens. Plant Mol Biol. 2021 Jan;105(1-2):55-64. doi: 10.1007/s11103-020-01068-x. Epub 2020 Sep 11. PMID: 32915351

Comment 3

Line 39-41: These pioneering studies highlighted…………..molecular networks. Discussing the concept, it is suggested to briefly describe how GC-MS corelates plant genotype with phenotype, highlighting key studies.

Plant metabolome provides a final representation of the metabolic network, specialized metabolites and their functions together with the metabolic crosstalk. However, applications of GC-MS technique for plant metabolome study is not sufficient considering the metabolic complexity of plants. What are the key areas of plant metabolome study and objectives when GC-MS is employed? Explain.

Answer: We appreciate the Reviewer’s suggestion. We have revised the indicated text and significantly expanded it by including discussion on “areas of plant metabolome study and objectives” of GC-MS-based studies as it has been suggested. The revised text can be seen in lines 46-56.

 

Comment 4

Line 93-95: Untargeted metabolomics (metabolite profiling)……………….. additional mass selectivity. The following discussion requires citation. Please include a reference study.

The article provides an in-depth discussion on the application of GC-MS in plants. In the introduction, it is necessary to discuss the history of GC-MS development and refinement briefly to provide a complete overview to the readers.

Answer: As suggested, we added the following reference to the indicated sentence (Line 173):

Fiehn O. Metabolomics by Gas Chromatography-Mass Spectrometry: Combined Targeted and Untargeted Profiling. Curr Protoc Mol Biol. 2016;114:30.4.1-30.4.32. doi: 10.1002/0471142727.mb3004s114. PMID: 27038389; PMCID: PMC4829120.

And we agree with the Reviewer and thank for raising this point that information indicating the history of GC-MS development and refinement was not presented enough in Introduction section to provide a complete overview to the readers. In accordance to the Reviewer’s suggestion, we have revised the Introduction section and supplemented it with the information indicating the major steps in GC-MS development. The corresponding text can be seen in lines 57-79.

Comment 5

Line 113-115: Thus, shock-freezing provides ………….. “metabolic snapshot”. In the present context, what is the meaning of  plant metabolic snapshot and why is it important in terms of plant metabolism? Discuss briefly.

Answer: We thank the Reviewer for highlighting this point. We have revised the text and supplemented it with the following explanation:

Lines 191-197: “Thus, shock-freezing provides a footprint of plant metabolism in the form of a “metabolic snapshot”, preventing any further modification of the sample, such as decomposition of metabolites or changes in their concentration, chemical or physical properties. Since metabolites are highly dynamic (in time and space), such a snapshot helps to clearly assess the current physiological state of the plant at a given time and helps to minimize errors associated with sample preparation (Rodrigues et al., 2019).“

 

Comment 6

In the article, there is no discussion on how the literature survey was performed, what is the methodology followed, literature searched and screened, inclusion and exclusion criteria and the rational for writing this review.

Answer: We thank and agree with the Reviewer’s remark. It should be clarified that this article is a part of a series of three articles which we dedicated with aim to highlight the role of GC-MS in plant metabolomics. The first part is devoted to the sample preparation and instrumental analysis, and has been recently published:

N Frolova, A Orlova, V Popova, T Bilova, A Frolov Gas Chromatography–Mass Spectrometry (GC-MS) in the Plant Metabolomics Toolbox: Sample Preparation and Instrumental Analysis Biomolecules 16 (1), 16. https://www.mdpi.com/2218-273X/16/1/16/pdf

The other part is devoted to the role of GC-MS in multi-platform metabolomics and integrated multi-omic studies: Gas Chromatography-Mass Spectrometry (GC-MS) in the Plant Metabolomics Toolbox: GC-MS in Multi-Platform Metabolomics and Integrated Multi-Omics Research (currently it is under revision process in IJMS).

This manuscript is devoted to the analysis of the primary metabolome of plants using GC-MS, where we address the critical aspects of GC-MS analytical workflow with a special emphasis on the selection of the analytical platform, data acquisition, processing and data interpretation as well as description of databases and tools for GC-MS data analysis.

Accordingly, the criteria for selecting literary sources were their relevance, mainly in terms of the universality of the method and its application to plant objects. We have not consciously sought to select publications only in recent years.

Comment 7

Reference sections: Very few recent works are cited in the paper. It is better to include the recent research on plant metabolome study employing GC-MS.

Answer:

We thank the Reviewer for this comment. Indeed, we did not aim to select publications from only recent years, but rather to show the development of methodological approaches over time. Nevertheless, in the revised version of the manuscript we have updated the list of references according to the Reviewer's comment, adding publications from the last 5 years.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is a comprehensive review of GC-MS analysis of polar metabolites in plants, covering sample preparation, analytical acquisition, and data interpretation. Although the essential information is well covered, the following points were difficult for non-specialists in analytical chemistry to understand.

The authors should revise those points as follows.

Line 28. The introduction demonstrates the achievements and trends of GC-MS metabolomics over the past 20 years. However, in the introduction section, the overview of this review manuscript is not presented. Especially, the sample preparation, including derivatization technique, is one of the essential points in GC-MS metabolomics, which is less mentioned. The authors should add that information in introduction section.

Line 60. This section, "2. Choosing an analytical platform," mainly demonstrated the GC-MS platform. As GC-MS is performed after the sample preparation, the authors should move this section after 3. Sample preparation. Before demonstrating 4. analytical acquisition, the description of the analytical platform makes it easy for readers to understand.

Author Response

Reviewer 2.

Comments and Suggestions for Authors

This manuscript is a comprehensive review of GC-MS analysis of polar metabolites in plants, covering sample preparation, analytical acquisition, and data interpretation. Although the essential information is well covered, the following points were difficult for non-specialists in analytical chemistry to understand.

The authors should revise those points as follows.

Comment 1:

Line 28. The introduction demonstrates the achievements and trends of GC-MS metabolomics over the past 20 years. However, in the introduction section, the overview of this review manuscript is not presented. Especially, the sample preparation, including derivatization technique, is one of the essential points in GC-MS metabolomics, which is less mentioned. The authors should add that information in introduction section.

Answer: We thank the Reviewer for the suggestion.

It should be clarified that this article is a part of a series of three papers dedicated to the role of GC-MS in plant metabolomics. In the first part of the series, we described the sample preparation in detail, the article has been recently published (N Frolova, A Orlova, V Popova, T Bilova, A Frolov Gas Chromatography–Mass Spectrometry (GC-MS) in the Plant Metabolomics Toolbox: Sample Preparation and Instrumental Analysis Biomolecules 16 (1), 16.https://www.mdpi.com/2218-273X/16/1/16/pdf).

This (i.e. presented here) manuscript we have focused on the GC-MS workflow for the analysis of polar metabolites. Therefore, we have designed the Introduction section to emphasize firstly the versatility of GC-MS methodology and secondly its applicability for the analysis of polar compounds. We also have supplemented the Introduction with historical information on the development of GC-MS technology in the revised manuscript. In that context, we have referred to the sample preparation and derivatization steps as tools that significantly enhance the GC-MS coverage of plant metabolome. However, we try not to make emphasis on variety of details of the both steps because we would like to avoid duplication in content with the first article.

However, we have decided to supplement the Introduction with the following text and the reference to our recently published article –

Lines 130-137: “However, the importance of the sample preparation, including derivatization methods, in GC-MS-based metabolomics is given special attention because inaccuracy made at this step may distort the final results of the analysis. Numerous sample preparation protocols for GC-MS provide all the necessary information concerning of harvesting, freezing, storage, homogenizing, as well as metabolite extraction, concentration, derivatization, and resuspension (Dagar et al., 2024). In general, the details of the sample preparation protocol depend on the purpose and object of the study, and the choice of the analytical platform (Frolova et al., 2026).”

New literature reference

Dagar R, Gautam A, Priscilla K, Sharma V, Gupta P, Kumar R. Sample Preparation from Plant Tissue for Gas Chromatography-Mass Spectrometry (GC-MS). Methods Mol Biol. 2024;2788:19-37. doi: 10.1007/978-1-0716-3782-1_2. PMID: 38656506.

Comment 2:

Line 60. This section, "2. Choosing an analytical platform," mainly demonstrated the GC-MS platform. As GC-MS is performed after the sample preparation, the authors should move this section after 3. Sample preparation. Before demonstrating 4. analytical acquisition, the description of the analytical platform makes it easy for readers to understand.

Answer: We thank the Reviewer for highlighting this point. However, we do not agree with the Reviewer’s suggestion that the section  2. Choosing an analytical platform, should be moved after 3. Sample preparation. but before demonstrating 4. analytical acquisition.

It is very important that Choosing an analytical platform can be done in the very beginning of the whole experiment. The selection of an analytical platform mainly depends on the experiment’s objectives, namely (i) what should be a group of target metabolites in the targeted strategy of GC-MS analysis or (ii) profiling of volatiles or non-volatile metabolites will be of interest in the untargeted GC-MS strategy. Thus, the selection of an analytical platform determines the molecular targets which can be most effectively analysed by it, and therefore the following appropriate sample preparation workflow and, if necessary, derivatization strategy should be selected.

Accordingly, to bring more clarify to the section organization we have added the following text to the end of Introduction:

Lines 135-137: “In general, the details of the sample preparation protocol depend on the purpose and object of the study, and the choice of the analytical platform”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been substantially revised and may be considered in the present form.

Congratulations to the authors.