DLLμE/GC-MS as a Powerful Analytical Approach to Establish the Volatilomic Composition of Different Whiskeys
, Michael Caldeira 1, Freddy Rodrigues 1, Jorge A. M. Pereira 1 and José S. Câmara 1,2,*
Round 1
Reviewer 1 Report
The volatilomic fingerprint of nine different whiskeys was established using a dispersive liquid-liquid microextraction (DLLµE) followed by gas chromatography mass spectrometry detection (GC-MS) and gas chromatography with flame ionization detection (GC-FID). The influence of extractor solvent on the extraction efficiency of volatile compounds (VOCs) was evaluated. Used method showed no matrix effect, good linearity in the assessed concentration range, recovery, precision and sensitivity. A total of 37 VOCs including alcohols, esters, acids, carbonyl compounds, furanic compounds and volatile phenols. Alcohols (3-methylbutan-1-ol, propan-1-ol), esters (ethyl decanoate, ethyl octanoate, ethyl hexanoate) and acids (decanoic acid, octanoic acid, hexanoic acid) were the most abundant. The multivariate statistical analysis allowed the discrimination of whiskeys based on their volatilomic fingerprint, namely octanoic acid, 2-furfural, ethyl octanoate, ethyl hexanoate, acetic acid, ethyl dodecanoate, butan-1-ol and ethyl decanoate. The manuscript is interesting but needs major revision according to further reamarks.
lines 93-113: Among the methods used for the research of volatiles from alcoholic beverages, important references are missing such as: Angioni, A.; Pintore, G.A.M.; Caboni, P. Determination of wine aroma compounds by dehydration followed by GC/MS. J. AOAC Int. 2012, 95, 813–819 and new one Kus, P.M.; Czabaj, S.; Jerkovic, I. Comparison of Volatile Profiles of Meads and Related Unifloral Honeys: Traceability Markers. Molecules 2022, 27, 4558. The methods used in these researches should me mentioned in the introduction part. In addition, it is not clear if dispersive liquid-liquid microextraction (DLLµE) was previously used for the extraction of beverages or not and it should be stated clearly. Is this new method?
lines 101-102: “…may be necessary to there is the need of…” – it is not clear, should be corrected
At the end of introduction there is need to explain what is the novelty of performed research. What was done for the first time? In addition, the research hypothesis is missing and how it will be tested.
The nomenclature of the compounds should be rechecked and corrected throughout overall manuscript with respect to IUPAC rules, octan-3-ol instead of 3-octanol, 3-methylbutan-1-ol instead of 3-methyl-1-butanol, hexan-1-ol instead of 1-hexanol; and similar should be used and C7 to C30 should be corrected with 7 and 30 in subscript.
Paragraph 2.3. Dispersive liquid-liquid microextraction procedure – the procedure was explained good. There is need to explain the extractor solvent and acetone as disperser solvent. What about solubility of hexane as the dispenser solvent in the matrix? The addition of Na2SO4 to whisky should cause water removal from whiskey, but it is not described. Why one mL of acetone containing 50 µL of dichloromethane was added after the addition of Na2SO4 and not before? Why only dichloromethane was mentioned as added in acetone, and not other solvents mentioned (ethyl ether (E), and hexane (H), and mixture of these extractor solvents (D:E (3:1 v/v), D:E (1:3 v/v), D:H (3:1 v/v), D:H (1:3 v/v), E:H (1:3 v/v)). Selection of extractor solvent using DLLµE/GC-MS described at the beginning of discussion should be mentioned in the experimental part.
Paragraph 2.4.1. Gas chromatography-mass spectrometry (GC-MS) conditions - The emission current and the maximum ionization time were 10 μA and 15 μs, respectively. Why these data are presented and not energy of ionisation (EI mode)?
Line 165 How the comparison of the compounds retention index (RI) was done? The literature used for comparison should me mentioned. The same remark is valid for identification of the compounds by gas chromatography-flame ionization detector (GC-FID). If the retention indices were compared that there is no need to mention that the retention times were compared.
Figure 2. is not clear. What the circles present?
Figure 4 a) – what are total concentrations (the values) presented?
Author Response
REVIEWER #1
Reviewer#1 “The volatilomic fingerprint of nine different whiskeys was established using a dispersive liquid-liquid microextraction (DLLµE) followed by gas chromatography mass spectrometry detection (GC-MS) and gas chromatography with flame ionization detection (GC-FID). The influence of extractor solvent on the extraction efficiency of volatile compounds (VOCs) was evaluated. Used method showed no matrix effect, good linearity in the assessed concentration range, recovery, precision and sensitivity. A total of 37 VOCs including alcohols, esters, acids, carbonyl compounds, furanic compounds and volatile phenols. Alcohols (3-methylbutan-1-ol, propan-1-ol), esters (ethyl decanoate, ethyl octanoate, ethyl hexanoate) and acids (decanoic acid, octanoic acid, hexanoic acid) were the most abundant. The multivariate statistical analysis allowed the discrimination of whiskeys based on their volatilomic fingerprint, namely octanoic acid, 2-furfural, ethyl octanoate, ethyl hexanoate, acetic acid, ethyl dodecanoate, butan-1-ol and ethyl decanoate. The manuscript is interesting but needs major revision according to further remarks.”
AUTHORS ANSWER: The authors thank the reviewer comment and acknowledge the reviewer for helping us to significantly improve our manuscript.
Reviewer#1 “lines 93-113: Among the methods used for the research of volatiles from alcoholic beverages, important references are missing such as: Angioni, A.; Pintore, G.A.M.; Caboni, P. Determination of wine aroma compounds by dehydration followed by GC/MS. J. AOAC Int. 2012, 95, 813–819 and new one Kus, P.M.; Czabaj, S.; Jerkovic, I. Comparison of Volatile Profiles of Meads and Related Unifloral Honeys: Traceability Markers. Molecules 2022, 27, 4558. The methods used in these researches should me mentioned in the introduction part. In addition, it is not clear if dispersive liquid-liquid microextraction (DLLµE) was previously used for the extraction of beverages or not and it should be stated clearly. Is this new method?
lines 101-102: “…may be necessary to there is the need of…” – it is not clear, should be corrected
At the end of introduction there is need to explain what is the novelty of performed research. What was done for the first time? In addition, the research hypothesis is missing and how it will be tested.”
AUTHORS ANSWER: As suggested by the reviewer, the important references were added to the introduction section (Lines 93-113). The DLLµE has been previously used in the extraction of volatile organic compounds from beverages, and this information was added in the introduction (Lines 109-117). The sentence was corrected to avoid misunderstanding. A statement was added to the end of the introduction to highlight the novelty of the work.
Reviewer#1 “The nomenclature of the compounds should be rechecked and corrected throughout overall manuscript with respect to IUPAC rules, octan-3-ol instead of 3-octanol, 3-methylbutan-1-ol instead of 3-methyl-1-butanol, hexan-1-ol instead of 1-hexanol; and similar should be used and C7 to C30 should be corrected with 7 and 30 in subscript.”
AUTHORS ANSWER: Thanks for your suggestion. The correction was done in all manuscript as suggested by the reviewer.
Reviewer#1 “Paragraph 2.3. Dispersive liquid-liquid microextraction procedure – the procedure was explained good. There is need to explain the extractor solvent and acetone as disperser solvent. What about solubility of hexane as the dispenser solvent in the matrix? The addition of Na2SO4 to whisky should cause water removal from whiskey, but it is not described. Why one mL of acetone containing 50 µL of dichloromethane was added after the addition of Na2SO4 and not before? Why only dichloromethane was mentioned as added in acetone, and not other solvents mentioned (ethyl ether (E), and hexane (H), and mixture of these extractor solvents (D:E (3:1 v/v), D:E (1:3 v/v), D:H (3:1 v/v), D:H (1:3 v/v), E:H (1:3 v/v)). Selection of extractor solvent using DLLµE/GC-MS described at the beginning of discussion should be mentioned in the experimental part.”
AUTHORS ANSWER: According to reviewer comment, the information related to the addition of Na2SO4 was added in the section 2.3. Regarding to extractor solvent, different extractor solvents were tested with different density and polarity with the purpose to achieve the best extraction efficiency. On the other hand, acetone was used as disperser solvent since it is soluble in extraction solvent (dichloromethane) and immiscible in sample, that enabling the extraction solvent to be dispersed as fine particles in aqueous phase to form a cloudy solution, Lines 222-225.
Dichloromethane was added in acetone, since the results demonstrated that this extractor solvent presented the best extraction efficiency. Nevertheless, to avoid misunderstanding, dichloromethane was removed. The information related to the selection of extractor solvent was added to the manuscript, Lines 149 and 150.
Reviewer#1 “Paragraph 2.4.1. Gas chromatography-mass spectrometry (GC-MS) conditions - The emission current and the maximum ionization time were 10 μA and 15 μs, respectively. Why these data are presented and not energy of ionisation (EI mode)?”
AUTHORS ANSWER: The authors agree with the reviewer, and the values of energy of ionization (70 eV) was added to the manuscript.
Reviewer#1 “Line 165 How the comparison of the compounds retention index (RI) was done? The literature used for comparison should me mentioned. The same remark is valid for identification of the compounds by gas chromatography-flame ionization detector (GC-FID). If the retention indices were compared that there is no need to mention that the retention times were compared.”
AUTHORS ANSWER: The RI was compared with the values reported in the literature (LRI) for similar columns, and the literature used for comparison was added to the manuscript, Lines 170-171.
Reviewer#1 “Figure 2. is not clear. What the circles present?”
AUTHORS ANSWER: In Fig 2(a) the circles represent the extraction efficiency of different mixtures of solvents assayed expressed as total relative peak area: Fig. 2(b) represent the distribution of different chemical families according to used solvent mixture.
Reviewer#1 “Figure 4 a) – what are total concentrations (the values) presented?”
AUTHORS ANSWER: The Figure 4a represent the total concentration of the free volatile fraction of assayed whiskeys. The total concentration levels were added on the figure.
Reviewer 2 Report
Below, the comments regarding the article: DLLmE/GC-MS as a powerful analytical approach to establish the volatilomic composition of different whiskeys.
1. Line 31: remove (reviewed in [1]) and replace it with [1]
2. Line 97: correct stir bar sorptive extraction
3. Rephrase the lines 101-102-103
4. Rephrase the lines 105-106-107
5. Line 108: remove “do”
6. Line 231: complete abbreviation list: D3E1, D1E3, D3H1, D1H3, and E1H3?
7. Line 235: correct “was” to “were”
8. Line 246: replace “after” by “once”
9. Line 247: remove “of whiskeys under study” and replace it with “to establish their volatilomic fingerprints.
10. Line 276: correct “was” to “were”
11. Line 279: correct “was” to “were”
12. Line 302: correct “was” to “were”
13. Line 309: correct “ageing process”
14. Rephrase the lines 331-332-333
15. Line 346: rephrase “which were not significant different” to “where statistically significant difference is barely detected”
16. Line 352: correct “The highest and lowest concentrations of esters were determined”
17. Line 353: correct “Similar concentrations of esters were determined”
18. Line 359: correct “The total concentration of the alcohols is ranged”
19. Lines “368-369: correct “the total concentration of carbonyl compounds is ranging”
20. Line 369: correct “Acetaldehyde was”
21. Line 371: correct “syringaldehyde was”
22. Line 372: correct “except for”
23. Line 373: correct “the total furanic compounds concentration is ranged”
24. Line 380: replace the sentence “is not too easy” with “is challenging”
25. References to be quoted:
· Chemical composition analysis and authentication of whisky, https://0-doi-org.brum.beds.ac.uk/10.1002/jsfa.6960
· Analysis of Barrel-Aged Kentucky Bourbon Whiskey by Ultrahigh Resolution Mass Spectrometry, https://0-doi-org.brum.beds.ac.uk/10.1007/s12161-020-01850-z
· A Rapid Sample Screening Method for Authenticity Control of Whiskey Using Capillary Electrophoresis with Online Preconcentration, https://0-doi-org.brum.beds.ac.uk/10.1021/jf202218r
· Whiskey Composition: Identification of Additional Components by Gas Chromatography-Mass Spectrometry, https://0-doi-org.brum.beds.ac.uk/10.1111/j.1365-2621.1969.tb12096.x
· Aroma Perception and Chemistry of Bitters in Whiskey Matrices: Modeling the Old-Fashioned, https://0-doi-org.brum.beds.ac.uk/10.1007/s12078-017-9229-3
· Whiskey Composition: Identification of Components by Single-Pass Gas Chromatography-Mass Spectrometry, https://0-doi-org.brum.beds.ac.uk/10.1111/j.1365-2621.1968.tb03637.x
· Determination of the chemical composition of alcoholic beverages by gas chromatography-mass spectrometry, https://0-doi-org.brum.beds.ac.uk/10.1111/jfpp.14676
· Profiling of nonvolatiles in whiskeys using ultra high pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC–QTOF MS), https://0-doi-org.brum.beds.ac.uk/10.1016/j.foodchem.2014.04.095
Author Response
AUTHORS ANSWER: The authors thank the reviewer comments/corrections. All the suggested corrections/alterations were done and included in the revised version of the manuscript
Reviewer 3 Report
The work seems to have been carried out competently and properly. The article is clear, well organized and the statistical analysis well done.
Line 92: please add some references
Line 134: please add production years for all whiskey samples
Line 133: would the authors explain what compounds were used as standards, how the standards solutions were prepared, what was the concentration range?
Author Response
REVIEWER #2 “The work seems to have been carried out competently and properly. The article is clear, well organized and the statistical analysis well done.”
AUTHORS ANSWER: The authors thank the reviewer comment.
REVIEWER #2 “Line 92: please add some references”
AUTHORS ANSWER: The references were added as suggested by the reviewer.
REVIEWER #2 “Line 134: please add production years for all whiskey samples
Line 133: would the authors explain what compounds were used as standards, how the standards solutions were prepared, what was the concentration range?”
AUTHORS ANSWER: The years of production for all whiskeys were added in the section 2.2. The standards used in the current study, as well as the concentration range tested is available in Table 1. Nevertheless, to avoid possible misunderstanding, Table 1 was referred in section 2.2. The information related to standard solutions preparation is available in section 2.2. The stock solution was prepared in ethanol, whereas the working solutions used to perform the method validation was prepared in hydroalcoholic solution of 40 % v/v.
Round 2
Reviewer 1 Report
The authors made revision according to the comments.
