δ¹³C-Ethanol as a Potential Exclusionary Criterium for the Authentication of Scotch Whiskies in Taiwan: Normal vs. 3-Parameter Lognormal Distributions of δ¹³C-Ethanol Found in Single Malt and Blended Scotch Whiskies

Round 1

Reviewer 1 Report

This paper is designed to investigate if theδ13C-Ethanol can be used as a potential exclusionary criterium for the authentication of Scotch Whiskies in Taiwan. The study is meaningful and the presentation is well organized, but the authors should pay further attention that measurement of 85 δ13C-ethanol has been used to differentiate between the raw materials of spirits from C3 plants with δ13C ‰ values of -21‰ to -34‰ and C4 plants with δ13C values of -9‰ to -87 20‰, sequentially [20-25] (Line 85-87). This means 51 single malt Scotch whiskies selected is actually an unrepresentative case and it will seriously influence the results of Fig. 4, although the obtained result is unsatisfactory (especially the addition ratio of 9:1 and 8:2). Furthermore, only one edible rectified spirit (Taiwan Sugar Corp.) was selected, what is the result if edible rectified spirit from other company is used? Such as a company in Scotch or a neighboring country.

Some specific comments:

Lines 99-109. Some references about the discrimination of products using data distribution model are needed.

Line 172-175. Where is the result?

Line 206. Figure 2c?

Line 244-245. (a), (a) and (a)?

Line 248. The Standard deviation should be incorrect.

Author Response

Response to Reviewer 1 Comments

Point 1: This paper is designed to investigate if the δ¹³C-Ethanol can be used as a potential exclusionary criterium for the authentication of Scotch Whiskies in Taiwan. The study is meaningful, and the presentation is well organized, but the authors should pay further attention that measurement of 85 δ¹³C-ethanol has been used to differentiate between the raw materials of spirits from C3 plants with δ¹³C ‰ values of -21‰ to -34‰ and C4 plants with δ¹³C values of -9‰ to -20‰, sequentially [20-25] (Line 85-87). This means 51 single malt Scotch whiskies selected is actually an unrepresentative case and it will seriously influence the results of Fig. 4, although the obtained result is unsatisfactory (especially the addition ratio of 9:1 and 8:2).

Response 1: We thank the reviewer for the comments. Single malt Scotch whisky is mainly made from barley, C3-derived spirits, as defined in the Scotch Whisky Regulations 2009. Thus, our results demonstrate a narrower range from −23.21‰ to −30.07‰, consistent with the reference δ¹³C‰ values, between −21‰ and −34‰, but more concentrated on the basis of characteristics of raw materials and statistics. The 99.7% CI boundary established in this study is for the exclusionary criterium. Therefore, more analytical methods should be used to provide more evidence of authentication for those samples that cannot be ruled out, such as the simulative adulterated single malt Scotch whiskies with adding ratio of 9:1 and 8:2 in this study.

Point 2: Furthermore, only one edible rectified spirit (Taiwan Sugar Corp.) was selected, what is the result if edible rectified spirit from other company is used? Such as a company in Scotch or a neighboring country.

Response 2: The edible rectified spirits selected in this study are based on some seized scotch whisky cases in Taiwan. While using edible rectified spirits from another company in Scotland or a neighbouring country, the relative exclusory result may be different. However, the exclusionary criteria in this study would not be affected, because it was established by the authentic Scotch whisky samples.

Point 3: Lines 99-109. Some references about the discrimination of products using data distribution model are needed.

Response 3: We thank the reviewers for the comments and understand the concerns. However, many studies discuss various analytical methods for discriminating products, but most use statistical tools such as PCA and PLS. Therefore, to discuss discriminative methods by data distribution models, we add our previous publication [19] as a reference at line 103. In addition, we also add reference labeling in line 105 to strengthen the description of the importance of involving data distribution in various scientific research.

Point 4: Line 172-175. Where is the result?

Response 4: We understand your concern and summarized the distribution fitting results from the software as follows. Based on the p value≥0.05, a fitting distribution demonstrated that the δ¹³C-ethanol data of single malt Scotch whiskies not only conformed to the normal distribution but also to the 3-parameter lognormal distribution.

 

Distribution	Threshold	AD	p Value	AIC
Normal	--	0.150	0.960	161.2
LogNormal - Three Parameter	-49.97	0.152	0.958	163.0

Also, the results shown in Table 1 and Figures 2c/2d in the manuscript can also demonstrate the fitting consequence for the 3-parameter lognormal distribution.

Point 5: Line 206. Figure 2c?

Response 5: Thanks for noticing. The number has been corrected.

Point 6: Line 244-245. (a), (a) and (a)?

Response 6: Thanks for noticing. The labels have been corrected.

Point 7: Line 248. The Standard deviation should be incorrect.

Response 7: We realize your concern and explain as follows. The standard deviations in Line 248 are calculated on log scale and then transformed to original scale. After rechecking data, the standard deviations in Line 248 are correct.

 

Reviewer 2 Report

 

In their study, the authors used stable isotope ratio between 13C/12C of ethanol, i.e., δ13C-ethanol, that was a base for the analysis of Scotch whiskies made from different raw materials. They analyzed 51 authentic single malt and 34 authentic blended Scotch whiskies by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) and then statistically analyzed the obtained data by a series of fitting distribution processes. The obtained results showed that δ13C-ethanol distribution of single malt Scotch whiskies fitted both normal and 3-parameter lognormal distribution. In the case of blended Scotch whiskies, the data distribution of δ13C-ethanol fitted with a 3-parameter lognormal distribution. Moreover, they established the confidence intervals (CI) of δ13C-ethanol. CI were for single malt Scotch whiskies between -23.21‰ to -30.07‰ for 3-parameter lognormal distribution, and between -11.19‰ to -28.93‰ for blended Scotch whiskies. The authors proposed to use this combination of analytical and statistical methods for the authentication of Scotch whiskies in an effort to reduce adulteration of these beverages.

 

This study is important because it is a good combination of analytical and statistical methods that could be used in practice to reduce the adulteration of Scotch whiskies

This study was well designed and performed and the obtained data is presented in a clear manner.

The methods are adequately described.

The conclusions are appropriate and based on the data from the study.

The cited references are sufficient and relevant.

The manuscript is well written and easy to read.

 

Author Response

Response to Reviewer 2 Comments

We would like to thank the reviewer for the review and kind comments. We appreciate inspiring remarks.

Reviewer 3 Report

The article is well designed and focused on achieving the assumed goal. The research is well conducted and described. The scientific value of the article consists primarily in the development of an interesting methodology for identifying counterfeit alcohols, with particular emphasis on Whikey. From this point of view, the article is also of great application importance. In the introduction, the authors should synthetically explain the concept of "δ13C for ethanol", which may not be familiar to many readers. Although this concept is generally described in Wikipedia, placing an explanation in the article will help readers better understand the research methodology.

In the text of the article, I found a few typographical errors that should have been removed in the editing process.

Authors should supplement the bibliography with an article published in 2010 - Madeleine E. Spitzke · Carsten Fauhl-Hassek, “Determination of the 13C/12C ratios of ethanol and higher alcohols in wine by GC-C-IRMS analysis”, Eur Food Res Technol ( 2010) 231:247–257; DOI 10.1007/s00217-010-1267-x. They should also include this article in the discussion of the results obtained.

Author Response

Response to Reviewer 3 Comments

Point 1: The article is well designed and focused on achieving the assumed goal. The research is well conducted and described. The scientific value of the article consists primarily in the development of an interesting methodology for identifying counterfeit alcohols, with particular emphasis on Whiskey. From this point of view, the article is also of great application importance. In the introduction, the authors should synthetically explain the concept of "δ¹³C for ethanol", which may not be familiar to many readers. Although this concept is generally described in Wikipedia, placing an explanation in the article will help readers better understand the research methodology.

Response 1: We thank the reviewer for the comments. The sentence about the concept of δ¹³C for ethanol has been added in Line 88-90.

Point 2: In the text of the article, I found a few typographical errors that should have been removed in the editing process.

Response 2: We thank the reviewer for the comments. The typographical errors has been corrected.

Point 3: Authors should supplement the bibliography with an article published in 2010 - Madeleine E. Spitzke · Carsten Fauhl-Hassek, “Determination of the ¹³C/¹²C ratios of ethanol and higher alcohols in wine by GC-C-IRMS analysis”, Eur Food Res Technol ( 2010) 231:247–257; DOI 10.1007/s00217-010-1267-x. They should also include this article in the discussion of the results obtained.

Response 3: We thank the reviewer for the comments and recommendation. The sentence have been included in the section of result and discussion in Line 300-304. Also, the bibliography has been added as suggested.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The results of a narrower range from −23.21‰ to −30.07‰ (CI 99.7%) is not good enough because the simulative samples of the addition ratio of 9:1 and 8:2 were in this range, furthermore the addition ratio of 7:3 is really closed to 99.7% Upper bound. If more analytical methods will be considered to establish more authentication criteria, the results obtained in this manuscript made little sense. When there is more than 30% exogenous rectified spirit, the fake Scotch whiskies can be probably discriminated by experienced tasters according to the sensory properties.

 

The CI range from −23.21‰ to −30.07‰ (99.7%) was obtained based on the 51 samples selected (Table 2, between -24.21‰ and -29.19‰?). If the selected samples are more comprehensive or samples size is enlarged, the range of CI 99.7% should be much narrower. Thus, it may be a good choice to enlarge the samples size. Besides, edible rectified spirit from at least 3 company is necessary to prepare simulative samples (the fake Scotch whiskies).

 

The original standard deviations should be showed in Table 2. Please check the whole text, if the exhibited data is calculated on log scale, the detailed explanation is necessary.

Author Response

Response to Reviewer 1 Comments

We would like to thank the reviewer for the review and for providing valuable comments.

The responses to comments were listed point-by-point as follows.

Point 1: The results of a narrower range from −23.21‰ to −30.07‰ (CI 99.7%) is not good enough because the simulative samples of the addition ratio of 9:1 and 8:2 were in this range, furthermore the addition ratio of 7:3 is really closed to 99.7% Upper bound. If more analytical methods will be considered to establish more authentication criteria, the results obtained in this manuscript made little sense. When there is more than 30% exogenous rectified spirit, the fake Scotch whiskies can be probably discriminated by experienced tasters according to the sensory properties.

 

Response 1: We thank the reviewer for the comment and apologize for not expressing ourselves clearly. Based on the reports from seized cases in Taiwan, most forgers adulterated or counterfeit Scotch whiskies with more than 30% dilution in order to gain high profits. The CI of δ¹³C-ethanol we establish in this study is used as a preliminary exclusionary criterium for the seized Scotch Whiskies in Taiwan and can be seen as a complementary method with other discriminating methods, such as pH measurement, Methanol concentration determination [19], GC-MS with qualitative/quantitative analysis, or other spectra measurements [9]. For those samples that cannot be ruled out, more analytical methods should be included as evidence. We have revised the corresponding part of the manuscript by adding more examples of analytical methods to make the paragraph clear. The paragraph in the conclusion from Lines 346-352 is rewriting as: “It should be emphasized that the CI boundaries established in this work for exclusive authentication are based on the δ¹³C-ethanol value, which is highly associated with the characteristics of raw materials used in the process of Scotch whiskies. For those adulterated Scotch whiskies whose δ¹³C-ethanol values fell into the CI range of authentic Scotch whiskies, more analytical methods, such as pH measurement, the gas chromatography-mass spectrometry analysis of methanol or other Scotch whisky congeners, or other spectra measurements, should be considered to establish more authentication criteria.”

We also provide our GA to decribe this concept. Please see the attachment.

Point 2: The CI range from −23.21‰ to −30.07‰ (99.7%) was obtained based on the 51 samples selected (Table 2, between -24.21‰ and -29.19‰?).

Response 2: We apologize for not expressing this clearly. The values of -24.21‰ and -29.19‰ shown in Table 2 describe the observed data of δ¹³C-ethanol value with minimum and maximum, not the 99.7% CI range. The explanation is added from Lines 205 to 206 as “The data descriptions of δ¹³C-ethanol data for authentic Scotch whiskies are summarized in Table 2, including the min and max of the observed values.” Also, we change the title of Table 2 to make the expression clearer and more accurate. The title of Table 2 is rewriting as “Data descriptions of δ¹³C-ethanol data for authentic Scotch whiskies”.

Point 3: If the selected samples are more comprehensive or samples size is enlarged, the range of CI 99.7% should be much narrower. Thus, it may be a good choice to enlarge the samples size.

Response 3: We thank the reviewer's suggestion. Enlarging the sample size is a reasonable method to narrow the CI range further. However, the 51 reference materials, prepared to establish the discrimination criteria of seized Scotch whiskies in Taiwan, have covered nearly all common brands imported from Scotland into the Taiwan market. Therefore, it is not easy to cover more legal brands of Scotch Whiskies found in Taiwan in a short time. Meanwhile, we think that the statistical model built by 51 samples should be representative enough for the preliminary exclusory purpose to conduct significant statistics based on the statistical definition(N = 30). Thus, we will try to collect more legal brands of Scotch whiskies from the markets and apply that additional experiments be included in follow-up papers.

Point 4: Besides, edible rectified spirit from at least 3 company is necessary to prepare simulative samples (the fake Scotch whiskies).

Response 4: We thank the reviewer's suggestion and add the δ¹³C-ethanol of three commonly-used edible rectified spirits by forgers in Table 4. However, two samples were belonged to C4-derived spirits with δ¹³C=-13.5‰ and -14.63‰, and one was a C3-derived spirit with δ¹³C=-24.76‰. Since the single malt Scotch whisky belongs to C3-derived spirits, C3-derived edible distilled spirits do not apply to this study's exclusivity criteria for Scotch whisky seized by stable isotope analysis. Based on the types of rectified spirits found in some seized cases of Scotch whiskies in Taiwan, the C4-derived rectified spirit was selected to prepare the simulative Scotch whiskies in this study. Because of the closeness of the δ¹³C values ​​of these two samples, we selected one for simulating the adulteration of single malt Scotch whisky. The relative explanation paragraph is added from lines 290 to 299 as follows: “To make adulterated or counterfeit Scotch whiskies, three commonly-used edible rectified spirits by forgers, which comply with regulations of "The Tobacco and Alcohol Administration Act" in Taiwan [44], were selected to examine the δ¹³C-ethanol for reference. Two samples were belonged to C4-derived spirits, and one was a C3-derived spirit, as shown in Table 4. Since the single malt Scotch whisky belongs to C3-derived spirits, C3-derived edible rectified spirit is not suitable for using the exclusory criterium for seized Scotch whiskies by stable isotope analysis in this study. On the basis of the types of rectified spirits found in some seized cases of Scotch whiskies in Taiwan, the C4-derived rectified spirit was selected to prepare the simulative Scotch whiskies in this study. The simulative results are shown in Figure 4.”

Table 4. δ¹³C-ethanol data of commonly-used edible rectified spirit in Taiwan

Supplier	Taiwan Sugar Corp.	Taiwan Tobacco & Liquor Corp.	Echo Chemical Co., Ltd.
δ13C-ethanol	-13.50(‰)	-14.63(‰)	-24.76(‰)
Type	C4-derived spirit	C4-derived spirit	C3-derived spirit

Point 5: The original standard deviations should be showed in Table 2. Please check the whole text, if the exhibited data is calculated on log scale, the detailed explanation is necessary.

Response 5: We thank the reviewer for the comment. The standard deviations on the original scale and log scale have been added in Table 2. We also checked the whole manuscript and added the explanation as suggested. The sentences are amended as “……, where x̄* and s* is back-transformed from x̄ and s* on the log scale.” in Line 204, and “…….the mentioned evaluation and back-transformed procedure was also carried out …” in Line 237, respectively.

Table 2. Data descriptions of δ¹³C-ethanol data for authentic Scotch whiskies.

Data descriptions
	No. of samples		Min	Max	Threshold	Mean	Standard deviation*	Standard deviation**
Single malt Scotch whisky		51	-29.19‰	-24.21‰	-49.97 ‰	-26.89‰	1.050	0.021
Blended Scotch whisky	34		-28.04‰	-17.14‰	-30.35 ‰	-25.13‰	1.542	0.188
*On the original scale; ** On the log scale.

 

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Lines 305-308. Here the authors should discussed complementally about “Based on the reports from seized cases in Taiwan, most forgers adulterated or counterfeit Scotch whiskies with more than 30% dilution in order to gain high profits.”. This point also should be mentioned in the conclusion section.

 

The identification results obtained from simulative samples should be introduced emphatically in the abstract section.

Author Response

Response to Reviewer 1 Comments

We would like to thank the reviewer for taking the time to review our manuscript and provide many valuable comments. The responses to comments were listed point-by-point as follows.

Point 1: Lines 305-308. Here the authors should discussed complementally about “Based on the reports from seized cases in Taiwan, most forgers adulterated or counterfeit Scotch whiskies with more than 30% dilution in order to gain high profits.”. This point also should be mentioned in the conclusion section.

Response 1: We thank the reviewer for the important comments. We have added this part in Lines 303-308. The paragraph is amended as “…. Based on the reports from seized cases in Taiwan, most forgers adulterated or counterfeit Scotch whiskies with more than 30% dilution in order to gain high profits. Consequently, the simulation experiment of adding C4-derived spirits to authentic single malt Scotch whisky proves the importance of establishing δ¹³C-ethanol CI and provides a reference for seized Scotch whisky cases in Taiwan.”

Meanwhile, we amended the sentences in the conclusion to point out this part in Lines 340-344 as “With these CI boundaries, the simulative adulterated Scotch whiskies could be exclusively screened while the seized Scotch whisky containing more than 30% C4-derived edible distilled spirits without comparing specific authentic reference samples. This result has been proved to effectively discriminate most adulterated and counterfeit Scotch whiskies in Taiwan” and in Lines 352-356 as “For those adulterated Scotch whiskies whose δ¹³C-ethanol values fell into the CI boundaries, those results can’t be directly recognized as authentic Scotch whiskies. To obtain other evidence, some of the analytical methods, such as pH measurement, the gas chromatography-mass spectrometry analysis of methanol or other Scotch whisky congeners, or other spectra measurements, should be considered to establish more authentication criteria” as suggested.

Point 2: The identification results obtained from simulative samples should be introduced emphatically in the abstract section.

Response 2: We thank the reviewer for the important comment. We added a sentence in the abstract section in Lines 21-23 as” The simulative adulterated Scotch whiskies using more than 30% C4-derived edible distilled spirits can be effectively discriminated by means of CI of δ¹³C-ethanol.” We also rewrite the sentence to emphasize this result in Lines 116-120 as “Also, with a known modus operandi of illicit Scotch whiskies, a series of simulative experiments with adding rectified spirits to authentic Scotch whiskies may evaluate the parts of clues from seized Scotch whiskies cases and further verify the importance of establishing a CI of δ¹³C-ethanol for authentication of Scotch whiskies.”

Author Response File: Author Response.docx