Profiling Croatian Whisky Using GC/MS-MS and UHPLC-QTOF

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes a detailed and thorough investigation of different compounds that have been detected in two varieties of Croatian whiskies, and their influence on the taste and flavor of these beverages. The introduction contains the necessary information. The used methodology is adequate for this kind of analysis. The discussions are somewhat long and very descriptive.

One important aspect, considering that four scotch type whiskies were also analyzed, is that there are no comparative results or discussion with the Croatian ones. Some data was included in the supplementary material, but was not discussed. This discussion is what I believe to be very important to this study, and should be added to the manuscript.

There are also a few other aspects which should be addressed before publication:

1. Throughout the text, the plural of whisky should be corrected as “whiskies” instead of “whisky’s”

 

2. Results and discussion should contain the number of replicates for each analysis.

3. Quality of figures 2, 3, 4, 5 and 6 is much too low. This aspect must be improved. 

4. Table 2. It would be easier to follow if “expected m/z” column was added to the table, just before the “measured m/z” column. It should contain the theoretical m/z expected for that chemical formula.

5. Table 2. A more appropriate header for "m/z [ppm]" column is needed. "Detection error" or "mass deviation" could be some options. Would be interesting if the direction of deviation would be added as “+” or “–“ (i.e. +1.24 ppm or -0.90 ppm).

6. Table 2. It is not clear what is the compound intensity measuring unit. Is it peak area? Or peak height?

7. Table 2: For multiple compounds in the "m/z [M-H]-" column the first figure is missing (i.e. for arabinose it should be 149.04 instead of 49.04).

8. Table 2: All masses and m/z should have the same number of significant figures.

9. How did the authors differentiate between isobaric compounds that elute at similar retention times? For example fumaric acid and malic acid. The annotation of isobaric compounds should be explained in the Materials and Methods section.

10. The authors should explain why some compounds have been identified at multiple retention times (such as succinic acid or gallic acid).

11. Figures 3 and 4 should be moved to supplementary materials. Discussion that follows covers this subject well enough.

12. Figures 5 and 6: The sample name is too long and does not provide much information. It should be removed and a legend should be added describing the association between colors and samples. Moreover, it would be interesting to know which of the 6 whiskies are the Croatian ones.

13. Supplementary materials are not cited and discussed anywhere in the manuscript.

Author Response

Respond to the reviewer 1

This manuscript describes a detailed and thorough investigation of different compounds that have been detected in two varieties of Croatian whiskies, and their influence on the taste and flavor of these beverages. The introduction contains the necessary information. The used methodology is adequate for this kind of analysis. The discussions are somewhat long and very descriptive.

One important aspect, considering that four scotch type whiskies were also analyzed, is that there are no comparative results or discussion with the Croatian ones. Some data was included in the supplementary material, but was not discussed. This discussion is what I believe to be very important to this study, and should be added to the manuscript.

There are also a few other aspects which should be addressed before publication:

1. Throughout the text, the plural of whisky should be corrected as “whiskies” instead of “whisky’s”

 RESPOND: Thank you for the comment all the “whisky’s” are corrected in the plural “whiskies”.

2. Results and discussion should contain the number of replicates for each analysis.

RESPOND: Thank you for noticing the missing inforamtion, we have add this information intp Material and method part:

Line 150-152. The SPME and GC-MS analysis were carried out in duplicate to ensure accuracy and reproducibility of the results.

Line 169-170. The UHPLC-QTOF analysis were carried out in duplicate to ensure accuracy and reproducibility of the results.

3. Quality of figures 2, 3, 4, 5 and 6 is much too low. This aspect must be improved. 

RESPOND: The quality of the figures are improved and it will be attached with manuscript as seperate documents.

4. Table 2. It would be easier to follow if “expected m/z” column was added to the table, just before the “measured m/z” column. It should contain the theoretical m/z expected for that chemical formula.

RESPOND: Thank you for the comment, we added the new column containg the theoretical m/z expected given chemical formula.

5. Table 2. A more appropriate header for "m/z [ppm]" column is needed. "Detection error" or "mass deviation" could be some options. Would be interesting if the direction of deviation would be added as “+” or “–“ (i.e. +1.24 ppm or -0.90 ppm).

RESPOND: Thank you for your comment, as there is indeed a mistake in this column, where it should be expressed as |Δ m/z| [ppm]. In relation to the sign of the error, it is expressed as an absolute value, as this is the way the software gives the deviation.

6. Table 2. It is not clear what is the compound intensity measuring unit. Is it peak area? Or peak height?

RESPOND: Thank you for the question. In Table 2, we have clarified that the compound intensity is expressed as the peak area. An asterisk has been added next to the relevant column header, and a note below the table now states: "Expressed as the peak area". We hope this resolves the confusion, and we appreciate your helpful suggestion!

7. Table 2: For multiple compounds in the "m/z [M-H]-" column the first figure is missing (i.e. for arabinose it should be 149.04 instead of 49.04).

RESPOND: Thank you for the comment, we have corrected it as indicated

8. Table 2: All masses and m/z should have the same number of significant figures.

RESPOND: Thank you for the comment, We have corrected Table 2: All masses and m/z values now have the same number of significant figures.

9. How did the authors differentiate between isobaric compounds that elute at similar retention times? For example fumaric acid and malic acid. The annotation of isobaric compounds should be explained in the Materials and Methods section.

RESPOND: Thank you for your insightful comments. We appreciate the opportunity to clarify these points and have made the necessary updates to the manuscript accordingly.

In untargeted analyses, achieving absolute confirmation of all isomers is inherently challenging due to the complexity involved, even in targeted approaches. As outlined in our manuscript, this work represents an untargeted search where identifications are suggested rather than definitive. To improve reliability, we proposed a calculation algorithm to support and strengthen the identification process.

1. Fumaric and Malic Acid:

Malic acid (C4H6O5) elutes at 1.7 minutes, while fumaric acid (C4H4O4) elutes at 1.34 minutes. These compounds are not isomers and do not share retention times. However, we acknowledge a labeling error in the table, where fumaric acid was incorrectly attributed the formula C4H2O4, which actually corresponds to fumarate (the deprotonated form of fumaric acid). This has been corrected in the revised manuscript.

2. Lyoniresinol 9-glucoside and Lyoniside:

Lyoniside (C27H36O12, mass 552.6) and Lyoniresinol 9-glucoside (C28H38O13, mass 582.6) were both reported with the same formula (C28H38O13) due to processing constraints, but their distinct masses were identified through fragmentation spectra matches in MoNA. Additionally, we detected a compound with mass 628, corresponding to Lyoniresinol 9-glucoside bound to formic acid ([M-H+HCOOH]⁻), a plausible result of formic acid in the mobile phase. After formic acid dissociates, the spectrum matches Lyoniresinol 9-glucoside. These clarifications have been explicitly noted in the updated table.

3. Syringic Acid Isomers:

Two isomers were detected at retention times 6.81 and 7.33. For 6.81, a strong match with syringic acid was supported by high confidence scores in MoNA (878/999) and MetFrag. For 7.33, lower scores suggested ethyl gallate as a better match. Based on these findings, we have tentatively assigned the latter to ethyl gallate, clearly noting the tentative nature of these identifications in the revised manuscript.

4. Gluconic Acid Isomers:

We detected Gluconic Acid at 1.65 and 1.74 minutes, representing D- and L-Gluconic Acid isomers. Differentiating between these isomers in this context is complex. To improve clarity, we have labeled them as "Gluconic Acid isomers" in the updated manuscript.

This approach has also been applied to other compounds, such as Glucogallin, Gallic Acid, and Succinic Acid, where isomers were detected with high confidence.

5. D-Glucose:

For D-Glucose (C6H12O6), we recognize that there are 16 possible isomers, including fructose, galactose, and mannose. Based on high match scores from MoNA and MetFrag, as well as contextual evidence from the sample, we have tentatively identified the compound as D-Glucose. While exact isomer determination is not feasible here, we are confident in the molecular structure assignment of C6H12O6.

We hope this response addresses your concerns and provides clarity on our approach and findings. Thank you again for your valuable feedback!

10. The authors should explain why some compounds have been identified at multiple retention times (such as succinic acid or gallic acid).

RESPOND: Thank you for your comment. We believe this is due to the presence of isomers. The QToF detects the same mass twice because the isomers share the same mass, and MoNA identifies very similar fragmentation patterns, assigning them the same name. In this initial approach of our work, it is not possible to determine which specific isomer is present.

11. Figures 3 and 4 should be moved to supplementary materials. Discussion that follows covers this subject well enough.

RESPOND: Thank you for the comment the figure 3  and 4 will be moved to supplementary materials

12. Figures 5 and 6: The sample name is too long and does not provide much information. It should be removed and a legend should be added describing the association between colors and samples. Moreover, it would be interesting to know which of the 6 whiskies are the Croatian ones.

RESPOND: Thank you for your valuable feedback regarding Figures 5 and 6. We appreciate your suggestions and have made the requested changes in the revised manuscript.

The sample names have been removed from the figures, and a clear legend has been added to describe the association between colors and the respective samples. Additionally, we have indicated which of the six whiskies are Croatian to provide clarity and context. We believe these changes enhance the readability and presentation of the figures.

Thank you again for pointing this out!

13. Supplementary materials are not cited and discussed anywhere in the manuscript.

RESPOND: Thank you for the comment, We have now included references to the supplementary materials within the manuscript. Specifically, we cite the supplementary figures and tables that present the detailed data analyses (PCA, PLS-DA, sPLS-DA, Scores Heat Map, and Dendrogram) showing the distinction between Croatian and Scotch whiskies based on untargeted QToF analysis of non-volatile compounds

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I revised the paper “Profiling Croatian whisky using GC/MS-MS and UHPLC- QTOF” submitted to Applied Sciences.

The paper matches the aim and scope of the Journal. However, in my opinion, the paper in its current state is not suitable for publication.

The quality of the presentation should be amended. Moreover, results should be properly discussed.

Please, see the following comments.

Abstract

The abstract should include the main results of the study. Please, amend.

Introduction

The Introduction should set the background of the study and the novelty of the study. Are studies on Croatian whisky already available? Please, amend.

Results and discussion

Table 1: please, add Retention Index of each identified compounds.

Lines 365-374: please, mind that this paragraph is not clear. Are there any differences between single mail and four grain whiskey? Please, amend.

Figure 3 and Figure 4: They should be discussed. Please, amend the text. Moreover, they are not clear. Please, replace.

Figure 5: Please, identify “whysky_1, whisky_2…., etc. Please, add the variance (%) explained by PC1 and PC2. Moreover, the Figure should be properly discussed in the text. Please, amend.

Figure 6: It is not possible to read the text in the Figure. Please, replace. 

Author Response

Dear Authors,

I revised the paper “Profiling Croatian whisky using GC/MS-MS and UHPLC- QTOF” submitted to Applied Sciences.

The paper matches the aim and scope of the Journal. However, in my opinion, the paper in its current state is not suitable for publication.

The quality of the presentation should be amended. Moreover, results should be properly discussed.

Please, see the following comments.

Abstract

The abstract should include the main results of the study. Please, amend.

RESPOND: Thank you for the comment we have include the main findings of the study in the abstract.

“…The results revealed a diverse range of compounds, including esters, alcohols, phenols, and acids, which are integral to the whisky's sensory characteristics. Furthermore, key volatile and non-volatile compounds were identified in Croatian whiskies, revealing a complex aromatic profile influenced by compounds like D-limonene, linalool, and β-damascenone, which contribute citrus, floral, and fruity notes. The findings also highlighted compounds associated with aging, such as esculetin and ellagic acid, which enhance flavor complexity, and emphasized the role of maturation, grain type, and production methods in shaping the unique sensory characteristics of Croatian whiskies…“

Introduction

The Introduction should set the background of the study and the novelty of the study. Are studies on Croatian whisky already available? Please, amend.

RESPOND: Thank you for the comment. We have add the following sentence

“….To our knowledge, there are no studies on profiling Croatian whiskies to date. Therfore, the primary objective is to identify and quantify the volatile and non-volatile compounds contributing to the flavor and aroma profile of Croatian whisky.….“

Results and discussion

Table 1: please, add Retention Index of each identified compounds.

RESPOND: Thank you for the comment the retention indexes are added into the Table 1.

Lines 365-374: please, mind that this paragraph is not clear. Are there any differences between single mail and four grain whiskey? Please, amend.

RESPOND: Thank you for the comment.  “Fig. 2, illustrate a ˝topography view˝ presenting the intensities of the compounds plotted 369 against their retention time and m/z values.“ between single mail and four grain whiskey. The distinct differences between single mail and four grain whiskey, as determined by the untargeted compound analysis using UHPLC-QTOF, are shown in Table 2.  Single malt whisky is produced by a single distillery using a single malted grain, typically barley. On the other hand, four grain whiskey includes four distinct grains.

Figure 3 and Figure 4: They should be discussed. Please, amend the text. Moreover, they are not clear. Please, replace.

RESPOND: The figures have been replaced with better quality ones and moved in supplementary materials as requested by other reviwer (Figures 3 and 4 should be moved to supplementary materials. Discussion that follows covers this subject well enough.)

Figure 5: Please, identify “whysky_1, whisky_2…., etc. Please, add the variance (%) explained by PC1 and PC2. Moreover, the Figure should be properly discussed in the text. Please, amend.

Figure 6: It is not possible to read the text in the Figure. Please, replace. 

RESPOND: Thank you for your valuable feedback regarding Figures 5 and 6. We appreciate your suggestions and have made the requested changes in the revised manuscript.

The sample names have been removed from the figures, and a clear legend has been added to describe the association between colors and the respective samples. Additionally, we have indicated which of the six whiskies are Croatian to provide clarity and context. We believe these changes enhance the readability and presentation of the figures.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper “Profiling Croatian whisky using GC/MS-MS and UHPLC- QTOF” contributes to the growth of the literature for research and alcoholic beverage producers, especially whisky.

Before  the manuscript acceptation for publication in “Applied Sciences”, the following items should be revised:

 

Introduction

Lines 52-55

The presented content should be supported by literature.

 

 

 

In Table 1 and Table 2, I suggest standardizing the precision of the results after the decimal point.

Figure 6 - I suggest an enlargement of the data description.

line 634

Is such several numbers after the decimal point necessary?

 

 

Conclusions

Line 669-671

Did the authors perform a sensory analysis? Isn't this an overinterpretation of the results?

I suggest adding the summary conclusion -  the strengths and weaknesses of the experience.

Author Response

The paper “Profiling Croatian whisky using GC/MS-MS and UHPLC- QTOF” contributes to the growth of the literature for research and alcoholic beverage producers, especially whisky.

Before  the manuscript acceptation for publication in “Applied Sciences”, the following items should be revised:

 Introduction

Lines 52-55

The presented content should be supported by literature.

RESPOND: Thank you for the comment. The lines 52-55 are supported with literature (reference 1) ˝ Despite these standards, factors such as cereal variety, fermentation conditions, distillation process/design, malting regime, aging time, cask selection, maturation environment and blending significantly influence the final character of the whisky. These factors result in whisky with distinct flavors, shaped by numerous active flavor compounds known as congeners. The main congeners in whisky include alcohols, aldehydes, acids, esters, phenols, terpenes, tannins and lactones which are produced at different stages of the production process and may be more prominent depending on the specific process involved [1]. ˝

In Table 1 and Table 2, I suggest standardizing the precision of the results after the decimal point.

RESPOND: Thank you for the comment. We appreciate your suggestion but the results will be presented as they are due to different opinions of reviewers.

Figure 6 - I suggest an enlargement of the data description.

RESPOND: Thank you for the suggestion. We have enlarged the description for Figure 6 ˝…Dendrogram of clustering based on the similarity matrix of analyzed whisky´s. Red: Croatian single malt whisky, light green: Croatian four grain whisky, dark blue: blended Scotch whisky, pink: single malt Scotch whisky, light blue: blended Scotch whisky, dark green: single malt Scotch whisky, olive: blended Scotch whisky, purple: blended Scotch whisky, brown: methanol..˝

line 634 Is such several numbers after the decimal point necessary?

RESPOND: Thank you for the question. Several numbers after the decimal point is necessary due to different opinions of reviewers

 

Conclusions

Line 669-671

Did the authors perform a sensory analysis? Isn't this an overinterpretation of the results?

I suggest adding the summary conclusion -  the strengths and weaknesses of the experience.

RESPOND: Thank you for the suggestion, but we didin´t perform the sensory analysis because we don´t have trained senzor analysts. Due to, we can´t express our strengths and weaknesses of the experience.

Reviewer 4 Report

Comments and Suggestions for Authors

A very interesting and highly informative manuscript on the chemical compounds present in Croatian whisky. The manuscript is very well written; however, attention is needed to the following:

1. l. 38. It would be better to cite the respective EU regulations. References 1 and 2 may contain such references, but it would be better for the manuscript (and the reader) to cite directly these EU regulations. 

2. The usefulness of the study is obvious and adequately described. However, the novelty of the study should also be highlighted.

3. The discussion is extensive but very well written and very comprehensive and informative. However, only the effect of the specific compounds on the quality of the whisky is discussed and only limited comparison is made with other similar studies. Therefore, the question 'what distinguishes Croatian whisky from other products available in the market?' can be partially addressed and only by scientists familiarized with the chemical composition of these products. A paragraph highlighting the key differences would add value to the manuscript.  

 

Author Response

A very interesting and highly informative manuscript on the chemical compounds present in Croatian whisky. The manuscript is very well written; however, attention is needed to the following:

1. 38. It would be better to cite the respective EU regulations. References 1 and 2 may contain such references, but it would be better for the manuscript (and the reader) to cite directly these EU regulations. 

RESPOND: Thank you for the valuable suggestion, the reference 2 is changed with the reference for EU regulations.

2. The usefulness of the study is obvious and adequately described. However, the novelty of the study should also be highlighted.

RESPOND: Thank you for the comment, the novelty of the study is highlighted in the conclusion. ˝… the aromatic and non-volatile compounds identified in Croatian whiskies not only differentiate them from other whiskies but also underline the potential for further exploration in the field of whisky chemistry and flavor science. This study provides a foundation for future research aimed at understanding and enhancing the sensory qualities of whisky through detailed chemical analysis..˝

3. The discussion is extensive but very well written and very comprehensive and informative. However, only the effect of the specific compounds on the quality of the whisky is discussed and only limited comparison is made with other similar studies. Therefore, the question 'what distinguishes Croatian whisky from other products available in the market?' can be partially addressed and only by scientists familiarized with the chemical composition of these products. A paragraph highlighting the key differences would add value to the manuscript.  

RESPOND: Thank you for your insightful comment. In this work we only discussed about the effect of the specific compounds on the quality of the whisky because we didn´t performe the sensory analysis. Therefore, we cannot compare Croatian whisky with others when it comes to aroma, taste, etc. However, in conclusion we summerise that Croatian whiskies are distinguished by their unique aromatic and non-volatile compound profiles, which set them apart from other whiskies available on the market. Key compounds such as D-limonene, linalool, and β-damascenone contribute citrus, floral, and fruity notes, providing a distinctive sensory character. The presence of aging-related compounds like esculetin, ellagic acid, and lyoniresinol adds depth and complexity to the flavor while showcasing the influence of maturation, grain type, and oak barrels. Unique fruity notes such as red apple and pineapple (from ethyl hexanoate and ethyl heptoate) and the toasted, fatty aromas of (E)-2-nonenal further highlight their characteristic profile. These findings underline the craftsmanship and chemical intricacies that make Croatian whiskies stand out in the global whisky market.

Reviewer 5 Report

Comments and Suggestions for Authors

All my suggestions are given as comments in the pdf document.

Comments for author File: Comments.pdf

Author Response

1. I think that at this point the most important compounds on the basis of which the types of whiskey are distinguished should be listed.

 

RESPOND: Thank you for your insightful comment. We listed the most important compounds on the basis of which the types of whiskey are distinguished. ˝Furthermore, key volatile and non-volatile compounds were identified in Croatian whiskies, revealing a complex aromatic profile influenced by compounds like D-limonene, linalool, and β-damascenone, which contribute citrus, floral, and fruity notes. The findings also highlighted compounds associated with aging, such as esculetin and ellagic acid, which enhance flavor complexity, and emphasized the role of maturation, grain type, and production methods in shaping the unique sensory characteristics of Croatian whiskies.˝

 

2. What about collision energy? Is these any MS2 data? Add MS2 data with relative abundance, if MS2 was recorded at all.

 

RESPOND: Thank you for pointing this out, we belive that here is all necessery dana for the method used:

„…The injector temperature was set at 260 °C and splitless mode was used (1 min, 75 mL min^-1). The mass spectrometer detector (MSD) was operated in the electron ionization (EI) positive mode (+70 eV), and the temperatures of the transfer line, the quadrupole and the ion source were set at 290 °C, 150 °C and 230 °C, respectively. Full Scan (FS) acquisition mode was employed monitoring mass/charge (m/z) fragments between 30 and 800. The system was operated by Agilent MSD ChemStation E.02.00.493 software….“

Regarding MS2 data with relative abundance, unfortinulay in this moment we are unable to provide the above information due to the short time frame for submitting revised work and our current busy schedule. However, if there is an opportunity to provide the information at a later date, we will do our best to organize and provide it.

 

3. This column is not important, only M-H should remain, but correct where necessary.

RESPOND: Thank you for the suggestion. We changed the column with Theoretical m/z [M-H]-

4. M-H for fumaric acid in negative mode is around 115.00368, formula of molecular ion will be C4H3O4. Formula is wrong also, C4H4O4 must be

RESPOND: Thank you for the comment, we acknowledge a labeling error in the table, where fumaric acid was incorrectly attributed the formula C4H2O4, which actually corresponds to fumarate (the deprotonated form of fumaric acid). This has been corrected in the revised manuscript.

5. Check again all M-H values, there are many error.

 

 

RESPOND: Thank you for the suggestion. All dana has been reviewed again.

 

6. This is wrong annotation. It seems to be ellagic acid dimer.

RESPOND: Thank you for pointing this out, the named is corrected in table

7. Where does salvigenin come from in whiskey?

RESPOND: Thank you for the question. The answer is given in the lines 552-558 ˝.. The presence of salvigenin in whisky  is less documented, but like other phenolic compounds, it may contribute to the overall  astringency or might provide subtle herbal nuances to the flavor. 3-O-methyl ellagic acid is likely to contribute the bitter and astringent aspects of the whisky flavor profile. Bothsalvigenin and 3-O-methyl ellagic acid are more specific derivatives of ellagic acid, related  to the oak wood tannins. Moreover, they can contribute to the overall depth of flavor  through their bittering and astringency properties, but their precise contributions to whisky´s flavor profile are not as well characterized as ellagic acid.˝

8. Where are data for these six samples?

 

RESPOND: Thank you for the question. The results for other six samples are not present in this work because the work is only focused on presenting the Croatian whiskey.

 

9. I would remove the information about sugars from the work. Where did the sugars come from, they didn't turn into alcohol? However, sugars are not analyzed with MS detection.

RESPOND: Thank you for the suggestion. The mention of sugars in our work reflects their identification as part of the untargeted analysis we conducted. All the compounds presented were determined using UHPLC- QTOF which allow for the detection of trace-level compounds. However, it is important to note that our study was focused solely on qualitative identification rather than quantitative analysis. Therefore, the presence of sugars could be attributed to trace amounts rather than significant concentrations, as we did not perform targeted quantification.

10. resolution is very low

RESPOND: Thank you for the comment.We include new figures with higher resolution.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

some comments were not addressed. The Introduction should set the background of the study and the novelty of the study. The quality of figures is too low. Please, amend.

Author Response

Review 2

Comment 1:

Response 1: Thank you very much for your comment, the introduction has been changed to accommodate your opinion and paragraphs related to the background of the research and novelty have been added. The changes are marked in the text

Comment 2: The quality of figures is too low. Please, amend.

Response 1: Unfortunately, the image exported from the device is of this quality, and we are unable to enhance it despite having tried all the tools at our disposal.

Reviewer 5 Report

Comments and Suggestions for Authors

However, try to provide MS2 data.

Author Response

Cemment 1: However, try to provide MS2 data.

Response 1: In SPME-GC-MS, a single quadrupole instrument was used, which does not allow MS/MS. In addition, the acquisition mode was Full Scan to screen and tentatively identify compounds by comparison with experimental and theoretical spectra from the NIST library, so MS/MS was not performed. The mass spectrometer detector (MSD) was operated in the electron ionization (EI) positive mode (+70 eV), as commented in section 2.4. Therefore, the collision energy is +70 eV. This is precisely what made it possible to compare the experimental spectra with the theoretical ones (also obtained at +70eV).

In the case of QTOF, although the instrument does allow MS/MS, an untarget and not a target analysis has been performed, since the aim of the work was to search for unknown compounds and not their quantification. For this reason, MS/MS was not performed.