Investigation into the use of carbon isotope ratios (13C/12C) of Scotch whisky congeners to establish brand authenticity using gas chromatography-combustion-isotope ratio mass spectrometry

Volatile congeners of whisky have been analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Acetaldehyde, ethyl acetate, n-propanol, isobutanol and amyl alcohol from a popular blended whisky have been separated using established GC conditions and their δ¹³C%. values determined. Eight samples of the whisky blend taken over the last 2 years of production have been analysed to allow the authentic range to be determined. A radar diagram has been used to graphically represent the δ¹³C%. data. This was used as a simple means of comparing the carbon isotope profiles of whiskies. Two other whisky samples were analysed and shown to differ from the whisky blend on the basis of the δ¹³C values.     

Gehalte an stabilen Sauerstoff-Isotopen in Wasser von Apfelsaftkonzentrat als Kriterium für den Nachweis einer Zuckerung

Using a modified method for the determination of the¹⁸O/¹⁶O ratio in water the δ¹⁸O values for authentic apple juice concentrates (70 °Brix) of known origin, for industrially produced invert sugar syrups, for commercial apple juice concentrates, and for spiked apple juice concentrates have been measured. The δ¹⁸O values for the authentic apple juice concentrates were correlated to the δ¹⁸O values of the related pressed (single strenght) apple juices and to the sugar content of the concentrates in °Brix. The results for the samples were compared to and evaluated together with the δ¹³C values for the sugars. Concluding from the results, a minimum value for authentic apple juice concentrate of 0 to 1%., depending on the regional origin of the material, is suggested, while the determination of the δ¹⁸O value of apple juice concentrate is preferably used in addition to the already well known methods on the basis of stable isotope measurement.     

B-HIVE: Beeswax hydrogen isotopes as validation of environment. Part I: Bulk honey and honeycomb stable isotope analysis

Stable isotope analysis is an established method for detecting honey adulteration. We extend its application to include honey and honeycomb region-of-origin assignment using hydrogen (δ²H) and oxygen (δ¹⁸O) isotopes. We observed that liquid honey δ²H and δ¹⁸O values had the potential to change because of water absorption and H atom exchange between sugars and water vapour. This suggested that liquid honey has limited use for geo-location, because specimens will record analysis location humidity. Paired liquid honey and honeycomb δ²H values were significantly correlated; therefore, we propose using wax δ²H values for region-of-origin assignment. We observed significant correlations between beeswax δ²H values and both mean annual precipitation and tap water δ²H values predicted for hive locations, suggesting that geographical variation in water δ²H values are recorded by beeswax δ²H values. This survey demonstrates the promise of stable isotopes for region-of-origin assignment, using honeycomb wax δ²H values, which complements carbon isotope analysis to detect adulteration.     

Detection of the illicit extension of potable spirituous liquors using 13C: 12C ratios

The stable carbon isotope ratios of 122 samples of common liquors and nine ferment able substrates have been determined. The δ¹³C values of the liquors analysed reflected those in the plant from which the fermentable substrate was derived. These values ranged from —23.4 to– -26.0% relative to Pee Dee Belemnite (PDB) for substrates derived from C₃ plants while those from C₄ plants ranged from —10.3 to —11.8% PDB. The application of this technique to detect the illicit extension of whisky has been investigated. As little as 2.9% grape spirit or 5.7% molasses spirit can be detected in whisky although the lower limit is dependent on the δ¹³C value of both the authentic whisky and the diluent. Measurement of ¹³C: ¹²C ratios could also be used to check that imported brandies conform with local requirements.     

δ18O of Ethanol in Wine and Spirits for Authentication Purposes

Since 1986 the European Union has established official isotopic analysis methods for detecting the illegal addition of sugar and water to wine and to enable geographical traceability. In this paper we investigate the possibility of using analysis of the ¹⁸O/¹⁶O stable isotope ratio (expressed as δ¹⁸O) of ethanol to improve detection of the watering of wine and to determine the origin of ethanol. Sixty‐nine authentic wine samples from all over Italy, 59 spirits from fruit and cereals, 5 chemically synthesized ethanols, one concentrated and rectified must, one beet and one cane sugar, one fresh must, and 6 waters with increasing δ¹⁸O values were considered. Ethanol was recovered by distillation, using a Cadiot spinning band column, following the official OIV methods. The residual water was trapped by storing the distillate for at least 24 h on a molecular sieve. The ¹⁸O/¹⁶O ratio was measured using a pyrolyser interfaced with an isotope ratio mass spectrometer. The δ^‐18O of ethanol is significantly related to the δ¹⁸O of the fermentation water and can be considered as a reliable internal reference. The values ranged from +24‰ to +36‰ in wine (years 2008 to 2012), +10‰ to +26‰ in fruit and cereal distillates, and from –2‰ to +12‰ in synthetic ethanol. The method was shown to be effective in improving detection of the watering of wine and determining the origin of ethanol (from grapes, other fruit, or synthesis), but not in detecting the addition of cane or beet sugar to wine.     

Gas chromatography carbon isotope ratio mass spectrometry applied to the detection of neutral alcohol in Scotch whisky: an internal reference approach

Ethanol and the volatile congeners of Scotch whisky have been analysed by GC–combustion stable isotope ratio mass spectrometry (GC–C–IRMS). Ethanol, propan-1-ol, 2-methylpropan-1-ol and methylbutan-1-ol (2- and 3-methylbutan-1-ol) have been separated using gas chromatography and their ratios of stable isotopes of carbon (¹³C/¹²C) determined by combustion isotope ratio mass spectrometry. The internal isotopic correlations for the ethanol and the congeners from the same sample were determined. A close and reproducible correlation was found for ¹³C/¹²C ratios of propan-1-ol and ethanol in authentic whisky samples and is the basis for determining the addition of neutral alcohol to whisky. Initial studies suggest that the method has the ability to detect the addition of neutral alcohol, depending on the cereal source of the whisky and neutral alcohol concerned, without the need for reference to databases.     

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ¹³C, δ¹⁵N, and δ¹⁸O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n = 1), Japan (n = 12), and USA (n = 1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2–40.0% (C content), 0.8–1.4% (N content), −27.1 to −25.4% (δ¹³C), +0.4 to +9.0% (δ¹⁵N), and +18.8 to +22.9% (δ¹⁸O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ¹³C, δ¹⁵N, and δ¹⁸O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.     

The application of SNIF‐NMR and IRMS combined with C,H and O isotopes for detecting the geographical origin of Chinese wines

The regional origin of Chinese wines was investigated using two important complementary techniques, site‐specific natural isotopic fractionation nuclear magnetic resonance (SNIF‐NMR) and isotope ratio mass spectrometry (IRMS). Twenty samples from five different grape varieties were collected from north Xinjiang in 2009, along with 100 wine samples from five different regions during 2010–2013. The (D/H)_I and (D/H)_II in wine ethanol ranged from 95.10 to 102.86 ppm and from 115.99 to 126.39 ppm using SNIF‐NMR, respectively. The ¹³C/¹²C of wine ethanol and ¹⁸O/¹⁶O of wine water were detected using IRMS. The δ¹³C value (?23.36‰) in coastal regions was higher than that in continental regions (?27.75‰). The temperature is the key for δ¹³C value. The δ¹⁸O ranged from ?1.94 to 4.57‰. The δ¹⁸O values were only positive in north Xinjiang which had the arid climate and strong sunshine. No difference was found for isotope ratios for wines made from five different grape varieties in north Xinjiang. All data evaluated by principal component analysis and linear discriminant analysis showed that the best method to distinguish the regional wine origin correctly is a combination of (D/H)_I, (D/H)_II, R, δ¹³C and δ¹⁸O. Therefore, natural multi‐elemental isotope ratios are effective in contributing to wine quality control in the Chinese market.     

Tissue turnover in ovine muscles and lipids as recorded by multiple (H,C, O,S) stable isotope ratios

Multiple stable isotope ratios (δ²H, δ¹³C, δ¹⁸O and δ³⁴S) were measured in muscle, muscle lipids and lipid fractions collected from 28 lambs, subjected to a diet-switch and raised on two energy allowances (EAs), to determine tissue turnover and diet-tissue fractionation. The diet-muscle fractionations prior to the diet-switch were estimated to be −44.0‰, +1.9‰ and 0‰ for H, C and S, respectively, while the drinking water was demonstrated to be the main source of muscle O and thus δ¹⁸O variation. The diet-intra-muscular lipid fractionations prior to the diet-switch were estimated to be −172.7‰, −1.3‰ and −11.5‰ for H, C and O, respectively. The C half-lives of muscle were determined to be 75.7 and 91.6 days for animals receiving the high and low EA, respectively. Extracting temporally resolved pre-slaughter dietary information from meat by analysing bulk muscle, muscle lipids and muscle lipid fractions appeared to be not practicable due to possible incomplete turnover of lipids.     

Principles and Limitations of Stable Isotopes in Differentiating Organic and Conventional Foodstuffs: 1. Plant Products

Among the lighter elements having two or more stable isotopes (H, C, N, O, S), δ¹⁵N appears to be the most promising isotopic marker to differentiate plant products from conventional and organic farms. Organic plant products vary within a range of δ¹⁵N values of +0.3 to +14.6%, while conventional plant products range from negative to positive values, i.e. ?4.0 to +8.7%. The main factors affecting δ¹⁵N signatures of plants are N fertilizers, biological N₂ fixation, plant organs and plant age. Correlations between mode of production and δ¹³C (except greenhouse tomatoes warmed with natural gas) or δ³⁴S signatures have not been established, and δ²H and δ¹⁸O are unsuitable markers due to the overriding effect of climate on the isotopic composition of plant-available water. Because there is potential overlap between the δ¹⁵N signatures of organic and conventionally produced plant products, δ¹⁵N has seldom been used successfully as the sole criterion for differentiation, but when combined with complementary analytical techniques and appropriate statistical tools, the probability of a correct identification increases. The use of organic fertilizers by conventional farmers or the marketing of organic produce as conventional due to market pressures are additional factors confounding correct identification. The robustness of using δ¹⁵N to differentiate mode of production will depend on the establishment of databases that have been verified for individual plant products.     

Measurement of stable isotope abundances in milk and milk ingredients — a possible tool for origin assignment and quality control

Relative carbon and nitrogen stable isotope abundances in total milk reflect the isotopic composition of the diet fed to the dairy cows; this diet and its δ-values depend on geographical and climatic factors. Milk from regions dominated by grassland typically shows relatively negative δ¹³C-values, while in regions dominated by crop cultivation the δ¹³C-values are more positive. The δ¹⁵N-values are influenced by factors such as soil conditions, the intensity of agricultural use and the climate. Casein in authentic milk samples is enriched in both ¹³C and ¹⁵N as compared to total milk, while the whey fraction is slightly enriched in ¹³C and depleted of ¹⁵N. The isotopic content of milk, casein and whey from one location have been measured throughout a period of longer than 1 year; variations are usually not greater than 1‰. In milk water, the ¹⁸O content was increased by between 2 and 6‰ as compared to ground water.     

Reliability of stable isotope values from meat juice for the determination of the meat origin

For the determination of the origin of foods, analysis of the oxygen and hydrogen isotope composition of the food water often is of great importance, because of the differences in the isotope signature of water between different regions. However, it was reported previously that the changes in δ¹⁸O during storage of meat are of such magnitude that existing variations between regions and countries are obscured. We have investigated the δ²H and δ¹⁸O changes in meat juice after slaughtering during storage under real cold storage conditions. Our results disagree with a previous publication as we did not observe heavy isotope enrichment of meat juice during industrial cold storages. Additionally we investigated possible changes in δ¹⁸O of the meat juice during the roasting process.     

Effect of irrigation and variety on oxygen (δ18O) and carbon (δ13C) stable isotope composition of grapes cultivated in a warm climate

Background and Aims: δ¹³C values from Vitis vinifera leaves, whole grape, seed, pulp, skin and/or grape must sugars have been investigated as an integrated marker of vine water status or intrinsic water-use efficiency during berry growth and across region of origin, vintage and variety. The use of ¹⁸O/¹⁶O isotopic ratio as a marker of water addition, vintage and geographical origin has also been studied. This paper examines the effect of irrigation and grapevine variety on δ¹⁸O and δ¹³C of grape must from eight varieties, all cultivated in the same vineyard to reduce the effects from other variables. Methods and Results: Stable isotope compositions of grape must water and sugar were determined by isotope ratio mass spectrometry. The result of the study showed statistically significant effects of irrigation and vine variety on both δ¹⁸O and δ¹³C. The effect of vintage on δ¹⁸O was only significant for non-irrigated vines. Conclusion: This research highlights the effect of variety and irrigation on δ¹³C and δ¹⁸O of grape. Significance of the Study: This is the first report to demonstrate that the varietal effect on δ¹³C and δ¹⁸O of grape is not due only to differences in the vegetative cycle of each variety. It further suggests that water exhibits a lower isotopic discrimination in the indigenous Spanish varieties studied than in non-indigenous varieties.     

Characterisation of authentic Italian extra-virgin olive oils by stable isotope ratios of C,O and H and mineral composition

The paper shows the isotopic ratios (¹³C/¹²C, D/H, ¹⁸O/¹⁶O) in oil and extracted glycerol and the mineral composition of authentic PDO and PGI Italian extra-virgin olive oils, officially collected from 2000 to 2005 (N=539$N=539$) to establish a national databank. ¹³C/¹²C and ¹⁸O/¹⁶O increased from Trentino to Sicily, each year distinguishing Northern Italy from Sicily and Calabria. Significant differences were found among the years and in some cases also between PDOs from the same region. ¹³C/¹²C and ¹⁸O/¹⁶O in bulk oil were significantly correlated with those in glycerol. D/H, measured in 2005 for the first time in oil, showed promising geographical discrimination capability. The content of 26 elements – Li, Rb, Cs, La, Ce and Yb rarely reported in the literature – was measured in well settled 2005 oils after ultrasound acid extraction.     

Verifying the geographical origin of beef: The application of multi-element isotope and trace element analysis

Beef samples originating from the major cattle producing regions of the world (Europe, USA, South America, Australia and New Zealand) have been analysed using IRMS and ICP-MS. C and N isotope composition of the beef defatted dry mass and H and O isotope composition of the corresponding lipid fractions were determined. It was observed that intensive maize and/or C₄ pasture feeding, during cattle production, gave rise to significant differences in the ¹³C content of beef produced in Brazil and the USA versus British beef fed predominantly on C₃ pasture and fodder. The mean δ²H‰ and δ¹⁸O‰ values of beef lipid correlated well with the latitude of production regions and the relationship between the H and O isotopic contents were found to parallel the Meteoric Water Line. These findings support the hypothesis that the systematic global variations in the ²H and ¹⁸O content of precipitation are transferred through drinking water and feed into beef lipid. Multi-element concentrations determined in the beef were combined with the stable isotope data and submitted to multivariate analysis. Six key variables (δ¹³C‰ (defatted dry mass), Sr, Fe, δ²H‰ (lipid), Rb and Se) were identified by canonical discriminant analysis as providing the maximum discrimination between beef samples on the basis of the broad geographical areas (Europe, South America and Australasia). It was concluded that the methodology in its current state can be used to provide reliable origin information, but this is dependent upon the countries under investigation.     

Principles and limitations of stable isotopes in differentiating organic and conventional foodstuffs: 2. Animal products

In this review, we examine the variation in stable isotope signatures of the lighter elements (δ²H, δ¹³C, δ¹⁵N, δ¹⁸O, and δ³⁴S) of tissues and excreta of domesticated animals, the factors affecting the isotopic composition of animal tissues, and whether stable isotopes may be used to differentiate organic and conventional modes of animal husbandry. The main factors affecting the δ¹³C signatures of livestock are the C3/C4 composition of the diet, the relative digestibility of the diet components, metabolic turnover, tissue and compound specificity, growth rate, and animal age. δ¹⁵N signatures of sheep and cattle products have been related mainly to diet signatures, which are quite variable among farms and between years. Although few data exist, a minor influence in δ¹⁵N signatures of animal products was attributed to N losses at the farm level, whereas stocking rate showed divergent findings. Correlations between mode of production and δ²H and δ¹⁸O have not been established, and only in one case of an animal product was δ³⁴S a satisfactory marker for mode of production. While many data exist on diet–tissue isotopic discrimination values among domesticated animals, there is a paucity of data that allow a direct and statistically verifiable comparison of the differences in the isotopic signatures of organically and conventionally grown animal products. The few comparisons are confined to beef, milk, and egg yolk, with no data for swine or lamb products. δ¹³C appears to be the most promising isotopic marker to differentiate organic and conventional production systems when maize (C4) is present in the conventional animal diet. However, δ¹³C may be unsuitable under tropical conditions, where C4 grasses are abundant, and where grass-based husbandry is predominant in both conventional and organic systems. Presently, there is no universal analytical method that can be applied to differentiate organic and conventional animal products.     

Influence of feed and water on the stable isotopic composition of dairy milk

Traceability of dairy products is an important issue for the dairy industry. Empirical studies have demonstrated the utility of stable isotope measurements on milk for verification of origin. However, various drivers influence the isotopic composition of a food stuff, such as rainfall levels or food supplements. Relationships between the isotopic composition (δ²H, δ¹⁸O, δ¹³C and δ¹⁵N) of milk fatty acids, the water component of the milk, and feed and farm water from 18 New Zealand dairy farms were studied. Significant relationships between the isotopic composition of milk and both farm water and feed fatty acids were observed. Measurement of both diet and drinking water from each dairy farm enabled the underlying environmental correlations to milk fatty acids to be explored by analysis of their respective δ²H values. Development of this technology may provide a diagnostic tool for determining type and the origin of dietary feed utilised in milk production.     

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA⁺, γ-tir⁺, stx2⁺, rfbE⁺, fliC⁺, hlyA⁺) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.     

Isotopic consequences of consumer food choice: Hydrogen and oxygen stable isotope ratios in foods from fast food restaurants versus supermarkets

We investigated geographic trends in the isotopic composition of the modern American diet, purchasing paired food items from fast food restaurants and supermarkets across the USA. We observed large ranges in δ²H and δ¹⁸O values, suggesting variation in the region-of-origin for beef, wheat, and potatoes. Mean restaurant meal δ²H and δ¹⁸O values (−114 and 22.6‰, respectively) were similar to supermarket values (−111 and 22.1‰, respectively). There were no correlations between restaurant beef and local tap water isotope values but significant correlations between supermarket beef and water (δ²H_beef = δ²H_water * 0.19−115‰ and δ¹⁸O_beef = δ¹⁸O_water * 0.17 + 14.8‰) suggesting regionality in the source of beef available to supermarket patrons. We observed no correlations between the stable isotopic composition of carbohydrates and local tap water. Understanding regional differences observed in some foods but not others will help refine parameters in models used to explore human movements in anthropological, archaeological, and forensic studies.