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Stable Isotope and Chemical Compositions of European and Australasian Ciders as a Guide to Authenticity James F. Carter,*,† Hans S. A. Yates,† and Ujang Tinggi† †

Queensland Health Forensic and Scientific Services, P.O. Box 594, Archerfield, Queensland 4108, Australia S Supporting Information *

ABSTRACT: This paper presents a data set derived from the analysis of bottled and canned ciders that may be used for comparison with suspected counterfeit or substitute products. Isotopic analysis of the solid residues from ciders (predominantly sugar) provided a means to determine the addition of C4 plant sugars. The added sugars were found to comprise cane sugar, high-fructose corn syrup, glucose, or combinations. The majority of ciders from Australia and New Zealand were found to contain significant amounts of added sugar, which provided a limited means to distinguish these ciders from European ciders. The hydrogen and oxygen isotopic compositions of the whole ciders (predominantly water) were shown to be controlled by two factors, the water available to the parent plant and evaporation. Analysis of data derived from both isotopic and chemical analysis of ciders provided a means to discriminate between regions and countries of manufacture. KEYWORDS: alcoholic beverages, authentication, chemical profile, cider, country of origin, isotope ratio

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INTRODUCTION Traditional cider, cyder, or hard cider, as it is known in the United States, is the fermented juice of apples or pears (only alcoholic beverages made from special pear cultivars should be described as perry), although the beverage is sometimes flavored with other ingredients. To make cider, the ripe fruit is crushed in a mill to form pulp or pommace, which is then pressed to release the juice or must. Fermentation occurs as a result of yeasts naturally present on the fruit or added by the brewer. The initial fermentation is typically carried out at relatively low temperatures (14−16 °C) in open vats or barrels. At the end of this stage, the cider is siphoned into new vats, leaving the sediment behind. The final fermentation is anaerobic, which creates the carbonation, and sugar may be added specifically for this purpose. In large-scale production, ciders made from different varieties of apple may be blended to accord with market taste.1 Cider is an ancient drink, and both the Greeks and Hebrews are known to have consumed fermented apple juice named Shekar or Sikera, the derivations of the word cider. After thousands of years, cider has only recently experienced a significant rise in popularity with the United States and Australia sales growing by >50% in 2011−2012.2 Although cider occupies only 1% of the market for beer, it is sold mainly to the high end of the market, and there have been reports of stylish U.S. restaurants offering English Cider priced at $26 a bottle, compared to the UK retail price of £2.59.3 Unfortunately, the demand for and high prices commanded by these fashionable drinks lead unscrupulous merchants to pass cheaper, inferior cider as a premium product. Producers are naturally protective of the status that the region of origin confers on their produce and, to defend the interests of both producers and consumers, food analysts are increasingly called upon to determine the geographical origin4,5 of suspected counterfeit goods. Published 2014 by the American Chemical Society

Very few studies have examined the chemical or isotopic compositions of ciders as a means to determine the provenance of the products. In contrast, numerous publications have described the authentication of wine through studies of its chemical composition6−10 and the stable isotopic composition of the light and heavy elements present in wine,11−13 its alcohol,14,15 and even the carbon dioxide in sparkling wines.16,17 Studies have also demonstrated correlations between isotopic composition, climatic data, and viticulture practices13,18 as another facet of authentication. Studies of the δ2H and δ18O compositions of beers have concluded that there is a correlation between the isotopic composition of drinking water and of beers produced or purchased at the same location.19,20 Studies of the δ13C composition of the dry residues of beers (composed largely of sugars) have demonstrated a means to identify the addition of C4 plant material, typically in the form of cane sugar or highfructose corn syrup (HFCS).21 Researchers have considered the polyphenolic composition of ciders as a potential guide to origin, but attributed variations more to apple variety and maturity.22,23 One study has concluded that a combination of multielement and strontium isotope ratio analysis provided a means to distinguish between ciders from England, France, Spain, and Switzerland.24 Isotopic analysis of the light elements present in ciders has, to date, been limited to distinguishing endogenous carbon dioxide from carbonation using industrial gases.17,25 In this small, initial study the authors determined the δ2H and δ18O compositions of whole ciders (predominantly water), the δ13C and δ18O compositions of the dry residues (predominantly sugar), and the concentrations of a suite of Received: Revised: Accepted: Published: 975

June 25, 2014 December 22, 2014 December 24, 2014 December 24, 2014 DOI: 10.1021/jf5030054 J. Agric. Food Chem. 2015, 63, 975−982

Journal of Agricultural and Food Chemistry

Article

Table 1. Descriptions of the Ciders Analyzed in This Study sample

a

brand

location

country code

regiona

ingredients

declared ABV %

calcd C4 sugarb (%)

1 2 3 4 5 6 7 8 9 10 11

Wild Magners Henry Westons Aspall Loic Raison Verano Apple Head Stassen Rekorderlig +46 Kopparberg

Tipperary, Ireland Tipperary, Ireland Herfordshire, UK Suffolk, UK Domagné, France San Sebastain, Spain Belgium Aubel, Belgium Vimmery, Sweden Herrljunga, Sweden Kopparberg, Sweden

IRL IRL GB GB F E BY BY S S S

EU EU EU EU EU EU EU EU EU EU EU

apple/strawberry pear apple apple apple apple apple apple apple apple apple

4.0 4.5 8.2 4.0 4.5 4.5 4.5 4.5 4.5 4.5 4.5

0 0 0 0 0 68 0 0 0 3 0

12 13 14 15 16 17 18 19 20 21

Mercury Cheeky Rascal The Barossa Cider Co. Matsos Bulmers Miracle Kirin Monteiths Newton’s Old Mout

TAS, Australia VIC, Australia SA, Australia WA, Australia NSW, Australia SA, Australia NSW, Australia Greymouth, New Zealand New Zealand New Zealand

AU* AU AU AU AU AU AU NZ NZ NZ

ANZ ANZ ANZ ANZ ANZ ANZ ANZ ANZ ANZ ANZ

apple apple/pomegranate apple apple/lime/ginger pear apple apple apple/pear apple apple

5.2 8.0 5.0 4.0 4.7 5.0 8.0 4.5 4.0 5.5

85 78 15 79 52 7 3 83 32 56

sugars identifed

HFCS

cane HFCS cane/HFCS cane

glucose cane HFCS

Ciders were categorized as European (EU) or Australia/New Zealand (ANZ). bCalculated according to the method of Brooks et al.21 Solid samples intended for δ18O analysis were measured into 4 × 3.2 mm silver capsules (IVA Analysentechnik, Meerbusch, Germany) and dried under vacuum, over phosphorus pentoxide for a minimum of 5 days. The 18O composition was determined using the TC/EA with the same operating conditions used for liquid samples. Capsules were crimped closed prior to analysis and introduced into the reactor by a Zero-Blank autosampler (Costech Analytical Technologies Inc., Valencia, CA, USA). Each sample was analyzed in triplicate with a median standard deviations of 0.2‰. Data were normalized to the international isotope scales (VPDBLSVEC or VSMOW-SLAP) by two-point calibration; a list of the reference materials and quality control (QC) materials used is given in the Supporting Information (cider supp 2). No attempt was made to measure the δ2H compositions of the solid residues as these samples contained exchangeable hydrogen and were highly deliquescent. In the face of ongoing controversy regarding the measurement of δ2H values for materials with exchangeable hydrogen,26 the authors considered that δ2HVSMOW data for these samples should not be compared to published data. Cation Analysis. Aliquots of approximately 2 g of sample were accurately weighed into PTFE vessels and digested with 4 mL of highpurity nitric acid (69%, Seastar Chemicals, Sidney, BC, Canada) using a MarXpress microwave digestion system (CEM, Matthews, NC, USA). Digested samples were analyzed using a Vista Pro inductively coupled plasma−optical emission spectrometer (ICP-OES) (Varian Australia Inc., VIC, Australia) and a 7700 ICP-MS (Agilent Technologies Australia Pty Ltd., VIC, Australia) as previously described.20 Reference materials and recovery spikes were used for quality control and assurance. Recoveries ranged from 77 to 119% and were deemed satisfactory (Supporting Information, cider supp 3). Anion Analysis. Samples were diluted 500-fold and transferred to autosampler tubes. Anion concentrations were measured using a Compact ion chromatograph (Metrohm, Gladesvile, Australia) as previously described.20 The recoveries for anions from spiked sample and aqueous solutions were satisfactory and >90% (Supporting Information, cider supp 3). Sugar Analysis. Degassed, filtered samples were diluted 10-fold and transferred to autosampler vials. Sugars were separated and quantified using a Shimadzu UFLC-20A High-performance liquid

cations and anions. The primary aim of this survey was to determine whether the techniques already established for beers20 could be applied to identify the geographical origin of ciders and/or the addition of C4 sugars. A further outcome of the study was to provide background data against which suspected substitute or counterfeit products can be compared.

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MATERIALS AND METHODS

Sample Collection and Processing. Twenty bottled and one canned cider were purchased from licensed retail outlets in the Brisbane metropolitan area (Queensland, Australia). The packaging of each sample was examined to verify the integrity and authenticity of the product and to confirm the location of manufacture. As with many studies of food authenticity, it was difficult to obtain truly authentic samples. In obtaining the samples for this study our strategy was to seek out reputable vendors. Prior to analysis, the samples were stored at room temperature. Once opened, samples were filtered through hardened ashless paper to remove the majority of the dissolved carbon dioxide and then through 0.45 μm Durapore filters (Merck Millipore, Billerica, MA, USA) into two 50 mL sterile containers for cation and anion determination. Degassed samples intended for δ2H and δ18O analysis were dispensed into 2 mL autosampler vials. To obtain a solid residue, approximately 5 mL aliquots of unfiltered ciders were heated in wide beakers at 102 °C until visibly dry. Stable Isotopic Analysis. All isotopic measurements were performed using a Thermo Scientific (Bremen, Germany) Delta VPLUS isotope ratio mass spectrometer (IRMS) coupled to a ConFlo IV interface for working gas introduction and sample dilution. The carbon isotopic analysis of solid samples and hydrogen and oxygen isotopic analyses of liquid samples were performed using a Thermo Scientific Flash 2000 HT operated in Elemental Analyzer (EA) or Thermal Conversion EA (TC/EA) mode, respectively, as previously described.20 The median standard deviations for replicate measurements of samples were 0.03‰ for solid 13C measurements (n = 3) and 0.3 and 0.1‰, respectively, for aqueous 2H and 18O measurements (n = 5). 976

DOI: 10.1021/jf5030054 J. Agric. Food Chem. 2015, 63, 975−982


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Ba), four anions (Cl, NO3, S, and P), the δ2H and δ18O compositions of the whole ciders (predominantly water), and the δ13C and δ18O compositions of the dry residues (predominantly sugar). A previous study24 has identified the concentrations of a similar combination of cations (Na, Ca, K, Mg, Si, V, Mn, Sr, Ba, Al, Ti, Zn, As, Rb, and Mo) were sufficient to classify ciders according to country of origin, and the findings of this study were broadly in agreement with that conclusion. A Pearson correlation matrix was determined for all of the measured parameters (Supporting Information, cider supp 1) and, in contrast to results previously reported for beers,20 few strong correlations were observed. Correlations observed within the data for beer were attributed to the addition of brewing salts, such as calcium chloride and calcium sulfate, used to control the character of the brewing water and of the finished beer. There is no documented evidence for the addition of brewing salts to ciders, which may account for the general lack of correlations between anion and cation concentrations. In common with the data obtained for beer, significant correlations were observed between the concentrations of potassium, magnesium, and manganese. There was no apparent explanation for these relationships other than all three elements being important nutritional elements for plants. PCA was applied to the combined anion and cation concentrations to determine which parameters accounted for the largest differences between samples. The loading factors for PC1 were dominated by potassium concentration, as was the case for an earlier study of beer, and the loading factors for PC2 were dominated by sulfur concentration. The latter finding could be explained because all but two of the ciders (samples 1 and 7) declared the preservative sulfur dioxide (or sulfite) as an ingredient. As confirmation, samples 1 and 7 contained the lowest concentrations of sulfur of the samples tested, 30 and 11 mg/kg, respectively, compared to a median concentration of 173 mg/kg. The loading factors for PC3 were predominantly the concentrations of phosphorus, sodium, and calcium. Overall, these finding were in broad agreement with an earlier study of beer,31 which identified the concentrations of potassium and phosphate as important factors in distinguishing between styles of beers and the region of production. Isotopic Composition. A Pearson correlation matrix (Supporting Information, cider supp 1) showed that a strong correlation existed between the δ2H and δ18O compositions of the liquid component of ciders. Both the δ2H and δ18O compositions of the liquid component correlated with the δ18O composition of the solid residue and with the concentrations of potassium, magnesium, manganese, and silicon. A correlation was also observed between the δ13C and δ18O compositions of the solid residue. Sugar in Cider. Figure 1 shows the distribution of the δ13C compositions determined for the dry residues of the ciders (predominantly sugar). The majority of samples had δ13CVPDB values of approximately −26‰, which is typical of C3 plant material, that is, unadulterated apple or pear sugars, consistent with AIJN guidelines. Other samples were, however, significantly enriched in 13C, suggesting the inclusion of C4 sugar. An estimate of the proportion of C4 sugar present in the samples was calculated using the equation adapted from Brooks et al.28

chromatograph (HPLC) system coupled to an RID-10A refractive index detector. Separation was achieved using a 150 mm × 4.6 mm Alltima NH2 3 μm column (Grace, Archerfield, QLD, Australia) maintained at 30 °C with an isocratic mobile phase of water/ acetonitrile (30:70). Standard solutions were prepared at concentrations of 10, 5.0, and 2.5 g/L from high-purity fructose, glucose, and sucrose (Sigma-Aldrich, Castle Hill, NSW, Australia). Statistical Analysis. Data were analyzed and plotted using R 3.0.1 software environment for statistical computing and graphics.27 Least absolute deviation (LAD) regression was applied to plotted data to determine the equation of robust fit and the correlation R2 (observed vs predicted). Principal component analysis (PCA) was performed on the chemical data to determine which parameters accounted for the largest differences between samples. This was performed using the L1norm to increase robustness to outliers compared to traditional PCA.28 Canonical discriminant analysis (CDA) was applied to both chemical and isotopic data to determine to what extent these parameters distinguish between the different regions of origin. These results represent the recognition ability based on the samples used to generate the model.

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RESULTS Overview of Samples. Although the number of samples in this study was limited, it was considered to be representative of the brands readily available within Australia at the time of the survey (October 2013). The analytical data are presented in the Supporting Information (cider supp 1). The ciders obtained for this study are detailed in Table 1, categorized by country and region of origin and ingredients. Seven of the samples were produced in Australia, three in New Zealand (NZ), and 12 in European Union (EU) countries (Belgium, France, Ireland, Spain, Sweden, and the United Kingdom). Within Australia and New Zealand the composition of cider is controlled by the Food Standards Australia and New Zealand (FSANZ) code. There is no common EU legislation governing cider, but the majority of cider-producing countries adhere to the AVIC (L’Association des Industries des Cidres et Vins de fruit de l’U.E.) code of practice. The declared alcoholic content of the ciders (reported in Table 1) ranged from 4.0 to 8.2% alcohol by volume (ABV) with a median value of 4.5% ABV. (For a product to be described as cider, FSANZ and AVIC codes require an alcoholic strength between 1.5 and 8.5% ABV.) Two of the ciders claimed to be manufactured from pears (samples 2 and 16) and one from apples and pears (sample 19). Both FSANZ and AVIC codes allow apple cider to contain up to 25% pear juice and vice versa. Three samples (1, 13, and 15) contained novel ingredients: strawberry, pomegranate, lime, or ginger. The authors assumed that these ingredients would be added in small amounts, principally for flavor, and, consequently, contribute little to the chemical or isotopic composition of the finished product. None of the analytical data acquired suggested differences between beverages produced from apples or pears or due to the inclusion of novel flavoring ingredients. This finding was in agreement with AIJN (The European Fruit Juice Assocaition) reference guides for apples and pears,29,30 which indicate no significant differences between the juices of the two fruits. The sole sample of canned cider was not distinct from the bottled varieties other than containing the lowest concentration of sulfur. It was assumed that a canned product would be pasteurized, negating the need for added preservative. Chemical Composition. All measured parameters that were below the limit of detection for the majority of the samples were removed from the overall data. Complete data are reported for nine cations (Na, Ca, K, Mg, Si, V, Mn, Sr, and

%C4 carbon = 977

(δ13Ccider − δ13C3) (δ13C4 − δ13C3) DOI: 10.1021/jf5030054 J. Agric. Food Chem. 2015, 63, 975−982


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would not be possible to distinguish them from a Spanish cider sweetened with HFCS. Figure 2a shows a scatter plot of the δ18O composition of the solid residues against that of the corresponding whole ciders defined by δ18Osolid = δ18Oliquid × 0.59 + 33 (R2 = 0.87)

Figure 1. Histogram showing the distribution of δ13C compositions for the solid residues from ciders.

in which δ13C3 and δ13C4 are the typical δ13CVPDB values for C3 and C4 plant material, respectively. (In this study δ13C3 was taken to be the average value for the ciders that did not appear to contain C4 sugar (−26.1‰): δ13C4 was taken as the value reported by Brooks et al. (−12.5‰).) Applying this equation to the δ13C compositions of the dry residues gave the results presented in Table 1 and revealed that seven of the nine ANZ samples contained C4 sugar, in contrast to a single EU sample. The EU sample (sample 6) was produced in Spain and claimed to be made from “100% local ingredients”. In France, the UNICID (L’Union Natioale Interprofessionnelle Cidricole) code distinguishes between products branded with the English or French spellings, cider or Cidre, and the addition of water or sugar to the latter is strictly prohibited. Reassuringly, the δ13C composition of Normandy Cidre (sample 5) showed no evidence of adulteration with C4 sugar. Ciders that were considered to contain >25% C4 sugar were analyzed by HPLC to identify the sugars present (Supporting Information, cider supp 1). It was assumed that most of the sugars originally present in the fruit juice would be consumed during primary fermentation and that the profile of the sugars present in the final product would reflect those added after the primary fermentation. The AIJN guidelines suggest that the glucose/fructose ratio of natural apple and pear juice should not exceed approximately 0.5 and, therefore, samples with ratios of approximately unity were deemed to contain HFCS. A single sample (sample 19) was found to have a glucose/fructose ratio >3 and was assumed to contain added glucose syrup. The AIJN guidelines also suggest that the sucrose/fructose ratio of natural apple and pear juice should not exceed approximately 0.4. Ciders with a sucrose concentration exceeding 0.5 (in combination with 13C enrichment) were deemed to contain added cane sugar. On the basis of these guidelines the nature of the added sugar is reported in Table 1. In this study ANZ and Spanish cider producers were found to use a variety of sugar types (cane sucrose, HFCS, and glucose) or combinations to sweeten their products. In contrast, manufacturers in other EU countries appeared to rely on natural fruit sugars. FSANZ, AVIC, and local Spanish codes (BOE-A-1979-21034) allow for the addition of sugar (and water) to ciders and do not restrict the source of added sugar. This practice makes it difficult to distinguish the origin of ciders based on δ 13 C measurements and, although C 4 sweetened products may be indicative of an ANZ origin, it

Figure 2. (a) Plot of δ18O for solid residue against water content; (b) plot of δ2H against δ 18O for the water content of cider samples (Global Meteoric Water Line shown dotted). Open symbols indicate samples for which δ13C results suggest the inclusion of C4 sugars.

The enrichment in 18O of the residues with respect to the whole ciders was consistent with biosynthesis.32 The robust trend line shown was derived from the samples considered not to contain C4 sugar (shown as solid symbols, ◆). All of the solid residues that were deemed to contain C4 sugar (shown as open symbols, ◇) were observed to be enriched in 18O with respect to the robust trend. Water in Cider. Applying LAD regression to the δ2H and 18 δ O data for the whole ciders produced, the results shown in Figure 3a are defined by the relationship δ 2 H = δ18O × 7.6 + 4.3 (R2 = 0.98)

with a slope that approximated to that of the Global Meteoric Water Line,33 which defines all natural waters according to δ 2 H = δ18O × 8 + 10

The liquid components of all ciders were found to be enriched in 18O with respect to meteoric waters. A recent study has demonstrated that an offset from the GWML develops during the manufacture of beer,34 which was attributed largely to the boiling of brewery water. Although boiling should not occur during cider making, it is proposed that slower 978



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Table 2. Percentage Correct Classification by CDA country codea isotopic data chemical data all data

AU

BY

E

F

GB

IRL

NZ

S

100 100

100 100

100 100

100 100

50 100

100 100

67 100

100 100

100

100

100

100

100 100 regional code

100

100

isotopic data chemical data all data

Figure 3. Least absolute deviation (LAD) regression plots of δ2H against δ18O for the water content of (a) all cider samples and (b) samples identified as outliers. Robust fit lines are shown dashed.

AU

NZ

EU

100 100 100

100 100 100

91 100 100

a

Australia (AU), Belgium (BY), Spain (E), France (F), UK (GB), Ireland (IRL), New Zealand (NZ), Sweden (S).

evaporation, from open vats, during initial fermentation may have a similar effect. Another possibility is that the observed offset results from the presence of alcohol and sugar in the analyte, of which cider typically contains more than beer.35 Four ciders produced in Australia (samples 13, 14, 17, and 18) deviated significantly from the robust trend line shown in Figure 3a. Applying LAD regression to these data alone produced the result shown in Figure 3b with a relationship defined by

Applying CDA to data composed of a large number of variables with only a small number of samples assigned to each class might be considered disingenuous. In an initial attempt to address this concern, CDA was applied to two broader groupings, ANZ and EU countries. This approach proved unsuccessful as it transpired the NZ ciders had many characteristics intermediate between those of the Australian and EU samples. CDA was, therefore, recalculated using NZ as a separate grouping, which gave the results shown in Table 2. Figure 4a shows the results of CDA applied to the isotopic data. Although Australian and EU samples formed discrete groupings, the NZ grouping overlapped both and encompassed the sample from Spain, clearly demonstrating why a single ANZ grouping was ineffective. All of the isotopic data contributed similar weighting to the discriminant functions. A similar pattern was observed for the chemical data (Figure 4b), the three classes forming discrete grouping with the Spanish sample correctly classified. In contrast to PCA results, the discriminant functions were dominated by the concentrations of strontium and barium. When the isotopic and chemical data were combined (Figure 4c), there was a clear distinction between Australian and EU samples. From this figure it was also apparent that the NZ samples had more in common with the EU samples than with those from Australia. The discriminant functions were again dominated by the concentrations of strontium and barium with significant contributions from manganese and chloride. In contrast to PCA results, the concentrations of potassium and phosphorus made little contribution; that is, the discriminatory information was not aligned with the direction of maximum variance. It will be necessary to check the established discrimination functions with a new suite of samples to evaluate fully the real discriminating power. Geo-location or CyderSpace. Figure 2b shows a scatter plot of the δ2H and δ18O compositions of the whole ciders. As discussed above, these parameters appeared to be controlled by two factors, groundwater and evaporation. To reflect this, two trend lines are shown in Figure 2b; the dashed line shows the robust trend determined by LAD regression (above), and the solid line shows a polynomial fit to all data according to

δ 2 H = δ18O × 3.3 − 11 (R2 = 0.95)

This result is consistent with published δ2H and δ18O compositions of orange and grape juices,36 which have been reported to have a relationship of approximately 4. This enrichment in 18O with respect to groundwater was reported to result from evapotranspiration and to be more apparent in slow-growing fruits such as apples and pears, which are typically grown in cooler climates than grapes or oranges. From Figures 2a and 3 it was apparent that the water content of ciders reflected the meteoric water available to the parent fruit tree but, when grown in warmer climates, such as Australia, isotopic enrichment occurred as a result of evaporation. It is also possible that some of the isotopic enrichment observed in the Australian samples resulted from evaporative enrichment of the waters used for irrigation or through water loss during storage or transportation of fruit.

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DISCUSSION Classification. The results of CDA applied to the chemical, isotopic, and combined compositions of the ciders in an attempt to distinguish the country of origin are summarized in Table 2. When considering the chemical data, it was possible to assign the ciders to their country of origin with 100% correct classification. In agreement with PCA, the discriminant functions were dominated by the concentrations of potassium, phosphorus, calcium, and magnesium. Although the concentration of sulfur dominated PC2, it did not feature strongly in the discriminant functions, most likely because the majority of sulfur was derived from an additive rather than from a natural component of the fruit juice. Combining the chemical and isotopic data again provided 100% correct classification. CDA analysis of the isotopic data in isolation proved somewhat less successful in discriminating the country of origin, and some U.K. samples were variously misclassified as France or New Zealand possibly because these regions share similar geography and/or climate. All of the isotopic parameters contributed approximately equal weighting to the discriminant functions.

δ 2 H = −0.2 × δ18O2 + 3.7 × δ18O − 10 (R2 = 0.99)

Of note, the linear part of this equation was similar to the evapotranspiration line determined in Figure 3b (δ2H = δ18O × 3.3−11). 979



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Figure 4. Factor plots derived from canonical discriminant analysis of (a) isotopic data, (b) chemical data, and (c) combined data: Australian (red, ○), EU (blue, ×), and NZ (green, △).

Figure 5. Isoscape of (a) the oxygen isotopic composition of whole ciders compared to (b) the predicted isotopic composition of leaf water. Reprinted with permission from ref 38. Copyright 2008 Public Library of Science.

both were scaled to encompass the full range of data rather than presented on an absolute scale. Although limited data were available for ciders, the visual similarity between the two maps was apparent. A similar comparison was obtained when the δ18O data derived from analysis of the dry residues were mapped, which might be expected given the strong correlation observed between the solid and liquid δ18O data. The presentation of data in a geo-spatial framework provided a more refined tool to determine country of origin by addressing the following question: Is the isotopic composition of a cider consistent with the claimed country of origin? Such an approach will allow an assessment of the authenticity of ciders for which no reference samples can be obtained. Caution, however, must be applied when the isotopic composition of ciders is compared to isoscapes derived from precipitation. First, ciders have been shown to be enriched in 18O with respect to meteoric waters and, second, plants may be exposed to waters from a combination of precipitation and irrigation, which may vary with time.39 As a further caveat, it should be noted that once harvested, apples, pears, juice, or sugar may be transported considerable distances, and the composition of a finished cider may not reflect the region of production of any single ingredient. As with any comparison, it is vital to understand variability to know if differences are significant. The next goal of this study

It was possible to use these data to make inferences about geographical origin. Simplistically, from Figure 2b the most isotopically depleted samples originated from Sweden (S) or Belgium (BY), whereas the most enriched samples originated from Australia (AU). Although AIJN guidelines recommend a minimum δ18OVSMOW composition for both apple and pear juice of −6.5‰, it was clear that all of the Swedish samples fell below this value, but these guidelines do note that “lower values are possible due to the effects of geographical origins...”. It was difficult to interpret data in the central region of Figure 2b because samples from similar geographical regions such as the United Kingdom (GBR), France (F), or Ireland (IRL) had similar isotopic compositions, as did samples from regions with similar climates, EU, NZ, and Tasmania (AU*), especially Spanish and NZ samples. Figure 5a shows the δ18O composition of the whole ciders, color scaled and projected onto a map, often referred to as an isoscape37 or, in this example, CyderSpace. This map was compared to a map in which records of the isotopic composition of precipitation were used to predict the δ18O composition leaf water38 (Figure 5b). The extent of isotopic enrichment of water within plant tissue is generally assumed to be leaves > fruit > stem > groundwater.36 Although the water in fruit was expected to be less enriched than in leaves, it was possible to compare fruits and leaves in these maps because 980



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(13) Dutra, S. V.; Adami, L.; Marcon, A. R.; Carnieli, G. J.; Roani, C. A.; Spinelli, F. R.; Leonardelli, S.; Vanderlinde, R. Characterization of wines according the geographical origin by analysis of isotopes and minerals and the influence of harvest on the isotope values. Food Chem. 2013, 141 (3), 2148−2153. (14) Cabañero, A. I.; Recio, J. L.; Rupérez, M. Simultaneous stable carbon isotopic analysis of wine glycerol and ethanol by liquid chromatography coupled to isotope ratio mass spectrometry. J. Agric. Food Chem. 2009, 58 (2), 722−728. (15) Perini, M.; Camin, F. δ 18O of ethanol in wine and spirits for authentication purposes. J. Food Sci. 2013, 78 (6), C839−C844. (16) Martinelli, L. A.; Moreira, M. Z.; Ometto, J. P. H. B.; Alcarde, A. R.; Rizzon, L. A.; Stange, E.; Ehleringer, J. R. Stable carbon isotopic composition of the wine and CO2 bubbles of sparkling wines: detecting C4 sugar additions. J. Agric. Food Chem. 2003, 51 (9), 2625− 2631. (17) Cabañero, A. I.; San-Hipólito, T.; Rupérez, M. GasBench/ isotope ratio mass spectrometry: a carbon isotope approach to detect exogenous CO2 in sparkling drinks. Rapid Commun. Mass Spectrom. 2007, 21 (20), 3323−3328. (18) Christoph, N.; Rossmann, A.; Schlicht, C.; Voerkelius, S. Wine authentication using stable isotope ratio analysis: significance of geographic origin, climate, and viticultural parameters. In Authentication of Food and Wine; American Chemical Society: Washington, DC, USA, 2006; Vol. 952, pp 166−179. (19) Chesson, L. A.; Valenzuela, L. O.; O’Grady, S. P.; Cerling, T. E.; Ehleringer, J. R. Links between purchase location and stable isotope ratios of bottled water, soda, and beer in the United States. J. Agric. Food Chem. 2010, 58 (12), 7311−7316. (20) Carter, J. F.; Yates, H. S. A.; Tinggi, U. A global survey of the stable isotope and chemical compositions of bottled and canned beers as a guide to authenticity. Sci. Justice 2014, DOI: /10.1016/ j.scijus.2014.05.002. (21) Brooks, J. R.; Buchmann, N.; Phillips, S.; Ehleringer, B.; Evans, R. D.; Lott, M.; Martinelli, L. A.; Pockman, W. T.; Sandquist, D.; Sparks, J. P.; Sperry, L.; Williams, D.; Ehleringer, J. R. Heavy and light beer: a carbon isotope approach to detect C4 carbon in beers of different origins, styles, and prices. J. Agric. Food Chem. 2002, 50 (22), 6413−6418. (22) Alonso-Salces, R. M.; Guyot, S.; Herrero, C.; Berrueta, L. A.; Drilleau, J.-F.; Gallo, B.; Vicente, F. Chemometric classification of Basque and French ciders based on their total polyphenol contents and CIELab parameters. Food Chem. 2005, 91 (1), 91−98. (23) Alonso-Salces, R. M.; Herrero, C.; Barranco, A.; LópezMárquez, D. M.; Berrueta, L. A.; Gallo, B.; Vicente, F. Polyphenolic compositions of Basque natural ciders: a chemometric study. Food Chem. 2006, 97 (3), 438−446. (24) García-Ruiz, S.; Moldovan, M.; Fortunato, G.; Wunderli, S.; García Alonso, J. I. Evaluation of strontium isotope abundance ratios in combination with multi-elemental analysis as a possible tool to study the geographical origin of ciders. Anal. Chim. Acta 2007, 590 (1), 55−66. (25) Cabañero, A. I.; Rupérez, M. Carbon isotopic characterization of cider CO2 by isotope ratio mass spectrometry: a tool for quality and authenticity assessment. Rapid Commun. Mass Spectrom. 2012, 26 (16), 1753−1760. (26) Coplen, T. B.; Qi, H. Recognizing the potential pitfalls of hydrogen isotopic analysis of keratins with steam equilibration to infer origins of wildlife, food, and people. Rapid Commun. Mass Spectrom. 2013, 27 (22), 2569−2569. (27) R. C. Team. R: A Language and Nnvironment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2010. (28) Brooks, J. P.; Dulá, J. H.; Boone, E. L. A pure -norm principal component analysis. Comput. Stat. Data Anal. 2013, 61 (0), 83−98. (29) Association of the Industry of Juices and Nectars from fruits and vegetables of the EEC. COP 6.3 Reference guideline for apple − revision April 2014; AIJN: Brussels, Belgium, 2004.

will be to examine intraproduct heterogeneity as a function of time because growing conditions will vary from year-to-year and suppliers of fruit and sugar may change.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary data (cider supp 1, cider supp 2, and cider supp 3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.F.C.) Phone: +61 (07) 3274 9228. Fax: +61 (07) 3274 9229. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank a number of reviewers who have provided insightful feedback and additional references that have greatly improved this paper. We also extend thanks to Stefan Petry and Elizabeth Harrison for their technical assistance with anion analysis, and we thank Jason B. West of Texas A&M University for permission to reproduce part of Figure 1 from ref 36 as Figure 5b.

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REFERENCES

(1) The Apple Journal; http://www.applejournal.com/fr05.htm (accessed June 12, 2014). (2) Blenkinsop P. Insight: cider, the golden apple of brewer’s eyes. Rueters Dec 20, 2012. (3) Boffey D. Cider’s cool new image to lead the way in British export push. Observer Oct 20, 2013. (4) Asche, S.; Michaud, A. L.; Brenna, J. T. Sourcing organic compounds based on natural variations measured by high precision isotope ratio mass spectrometry. Curr. Org. Chem. 2003, 7, 1527− 1543. (5) LeBot, B.; Oulhote, Y.; Deguen, S.; Glorennec, P. Using and interpreting isotope data for source identification. Trends Anal. Chem. 2011, 30 (2), 302−312. (6) Martin, A. E.; Watling, R. J.; Lee, G. S. The multi-element determination and regional discrimination of Australian wines. Food Chem. 2012, 133 (3), 1081−1089. (7) Baxter, M. J.; Crews, H. M.; John Dennis, M.; Goodall, I.; Anderson, D. The determination of the authenticity of wine from its trace element composition. Food Chem. 1997, 60 (3), 443−450. (8) Geana, I.; Iordache, A.; Ionete, R.; Marinescu, A.; Ranca, A.; Culea, M. Geographical origin identification of Romanian wines by ICP-MS elemental analysis. Food Chem. 2013, 138 (2−3), 1125−1134. (9) Fabani, M. P.; Arrúa, R. C.; Vázquez, F.; Diaz, M. P.; Baroni, M. V.; Wunderlin, D. A. Evaluation of elemental profile coupled to chemometrics to assess the geographical origin of Argentinean wines. Food Chem. 2010, 119 (1), 372−379. (10) Gonzálvez, A.; Llorens, A.; Cervera, M. L.; Armenta, S.; de la Guardia, M. Elemental fingerprint of wines from the protected designation of origin Valencia. Food Chem. 2009, 112 (1), 26−34. (11) Ogrinc, N.; Košir, I. J.; Kocjančič, M.; Kidrič, J. Determination of authenticy, regional origin, and vintage of Slovenian wines using a combination of IRMS and SNIF-NMR analyses. J. Agric. Food Chem. 2001, 49 (3), 1432−1440. (12) Marchionni, S.; Braschi, E.; Tommasini, S.; Bollati, A.; Cifelli, F.; Mulinacci, N.; Mattei, M.; Conticelli, S. High-precision 87Sr/86Sr analyses in wines and their use as a geological fingerprint for tracing geographic provenance. J. Agric. Food Chem. 2013, 61 (28), 6822− 6831. 981



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