Strontium isotopic ratio 87 Sr/ 86 Sr as a marker of terroir and its analytical determination in Portuguese wines Francisca Castro a ; Maria de Pinho a ; Sofia Catarino b a Chemical Engineering Department, Instituto Superior Técnico (IST),Universidade Técnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal b LEAF, Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Tapada da Ajuda,1349-017 Lisboa, Portugal ABSTRACT The evaluation of the geographical origin of wines based on their chemical composition for authenticity purposes is a concern of the wine sector for many years. The multielement composition, rare earth elements patterns and in particular, strontium isotopic ratio 87 Sr/ 86 Sr have shown high potential for the evaluation of wines according to their origin. The present work aims to contribute to the evaluation of strontium isotopic ratio 87 Sr/ 86 Sr as a marker of geographical origin of wines. To this end, this analytical parameter was assessed in 22 wines from the Douro Region, produced under experimental conditions. As a complement multielement analysis of the wines was performed. A previously optimized analytical procedure for determining the ratio 87 Sr/ 86 Sr in wines by ICP-MS was used. Rb and Sr were previously separated by cation exchange chromatography with EDTA. The values of the isotopic ratios 87 Sr/ 86 Sr of wines showed statistically significant differences, the values ranged between 0.7130 and 0.7175. This range is considered very small which showed a close proximity of the ratios 87 Sr/ 86 Sr of the wines from this region. As regards the multielement composition, the rare earth elements allowed a good discrimination between wines according to the geographical origin, showing its potential importance as indicators of geographical origin. Keywords: Wine, authenticity, geographical origin, multielement composition, 87 Sr/ 86 Sr ratio 1. Introduction Determination of the geographical origin of food and beverages has been a growing issue over the past decade for many countries around the world, mostly because of the concern of consumers about the authenticity of the food that they eat. The indicators commonly used are stable-isotope ratios of hydrogen, oxygen, nitrogen, carbon and sulfur, combined with isotope ratios of strontium and lead and elemental concentrations. 1
The geographical origin of wine products has a key role in ensuring its authenticity, in particular on products under Designation of Origin (DO) and Geographical Indication (GI). Portugal is the eleventh largest producer of wine (6 238 439 hl of wine, 2014 vintage) ranges of natural abundance are as follow: 84 Sr, 0.55-0.58%; 86 Sr, 9.75-9.99%; 87 Sr, 6.94-7.14%; and 88 Sr, 82.29-82.77% (Berglund and Wieser, 2011). The 84 Sr, 86 Sr and 88 Sr isotopes occur in constant relative proportions, while 87 Sr gradually increases in minerals due to the radioactive β-decay of the 87 Rb isotope. The concentration of 87 Sr in minerals depends on the age of the rock and on the Rb/Sr ratio. This makes the 87 Sr/ 86 Sr isotope ratio an important parameter in geochronology. Indeed, the 87 Sr/ 86 Sr ratio in granitic (older) rocks (typically higher than 0.710) is distinctly higher than in basaltic (younger) rocks (typically between 0.702 and 0.705). The relative abundance of 87 Sr varies with geological ages and consequently with geographical locations, providing a fingerprint for different rock types (Capo et al., 1998; Vanhaecke et al., 1999; Almeida and Vasconcelos, 2001). The present work aims to contribute to the evaluation of the strontium isotopic ratio 87 Sr/ 86 Sr as a marker of geographical origin of wines. To this end, this analytical parameter was assessed in 22 wines from the Douro region, produced under controlled experimental conditions, as part of a R&D project for the development of a biosensor for traceability of wines from this region. An analytical procedure involving the prior sample preparation for chromatographic separation of ion exchange and subsequent determination of strontium isotopic ratio 87 Sr/ 86 Sr by ICP MS was applied. As a complement, the multielement composition of the wine was evaluated in order to discriminate according to their origin. 2. Materials and Methods 2.1. Wine samples In this study were used 22 varietal wines, 15 red wines and 7 white wines, from six vineyards of the Douro Region. Table 1 shows which is an important contribution to the Portuguese economy. About 50% of the total volume corresponds to wines with potential for the DO designation (IVV, 2014). Strontium is a alkaline earth metal with four naturally occurring stable isotopes which the identification of vineyards and varieties/ wines used. The wines were produced in the 2014 vintage (microvinification conditions). 2.2. Analytical procedures Reagents Trace Select 30 % (v/v) hydrogen peroxide (H 2 O 2 ) (Fluka, Sigma-Aldrich) were used for wine digestion. Ammonia (NH 3 ) 25 % (v/v) and EDTA Titriplex III of analytical grade (Merck) were used for ion-exchange chromatography. Monoelement standard solutions of Be, Co, In (1000 mg/l; Merck) and a multielement solution with Mg, Cu, Rh, Cd, In, Ba, Ce, Pb and U (10 mg/l; Perkin-Elmer) were used for ICP-MS optimization procedures. Ultrapure concentrated HNO 3 (J.T. Baker) was used for wash, blank, and standard solutions. ICP-MS semi-quantitative calibration (for Rb and Sr determinations and multielement determinations) was established with a multielement standard solution with 30 elements: Li, Be, Na, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Ag, Cd, In, Cs, Ba, Hg, Tl, Pb, Bi and U (10 mg/l ; Perkin-Elmer). ICP-MS internal standardization was performed with standard solutions of Rh and Re (1000 mg/l; Merck). The standard reference material (SRM) 987 (SrCO 3 ) from the National Institute of Standards and Technology (NIST; Gaithersburg, USA) was used as isotopic standard (external correction of mass bias). De-ionized water (conductivity <0.05 μs/cm) prepared by using a Seralpur Pro 90CN system (Seral, Ransbach-Baumbach, Germany) was used for all solutions. To avoid contamination of the samples, all the material (polypropylene and Teflon PFA) was soaked in 20% HNO3 (v/v) for at least 24 hours and then rinsed thoroughly with de-ionized water before use. For decontamination solution preparation, HNO3 reagent grade was double-distilled using an infra-red sub-boiling distillatory system (model BSB-939-IR, Berghof, Germany). 2
Table 1 Identification of the vineyards and wines Vineyards White wines Red Wines Aragonez, Rufete, Tinta Barroca, Quinta do Seixo (41 10 N, 7 33 W) Quinta da Boavista (41 10 N, 7 34 W) Quinta do Cidrô (41 08 N, 7 23 W) Quinta do Casal da Granja (41 15 N, 7 28 W) Quinta do Ventozelo (41 10 N, 7 30 W) Quinta do Cavernelho (41 17 N, 7 43 W) Côdega do Larinho, Viosinho Chardonnay Blanc Fernão Pires, Moscatel Galego Gouveio, Malvasia Fina Tinta Francisca, Tinto Cão, Touriga Fêmea, Touriga Franca, Touriga Nacional, Trincadeira, Vinhão Alicante Bouschet, Donzelinho Tinto Cabernet Sauvignon, Pinot Noir Merlot Noir HPMW mineralization Prior to ion-exchange chromatography, the wine samples were digested using a high pressure microwave (HPMW) system (Milestone MLS 1200 mega Microwave Labstation). 2 ml of H 2 O 2 (30 % v/v) and 4 ml of de-ionized water were added to each wine sample of 2 ml. The digestion program consisted of 6 steps: 1 min at 260 W; 2 min at 0 W; 5 min at 260 W; 5 min at 430 W; 5 min at 640 W; and the ventilation step. Sr and Rb separation by ion-exchange chromatography Ion-exchange chromatography was carried out in a HIPEX Duran chromatography column (internal diameter of 1.2 cm and bed length of 20 cm) filled with Dowex 50W-X8/400 mesh resin (Sigma Aldrich). The procedure optimized and described by Martins et al. (2014) was applied. Figure 1 shows the procedure scheme of Sr and Rb ion-exchange separation and the following steps for the 87 Sr/ 86 Sr determination by Q-ICP-MS. To prevent the precipitation of residual EDTA in the ICP-MS instrument, samples were filtered using a Millipore PVDF syringe filter (0.45-μm pore size, 2.5-cm diameter). ICP-MS analysis nebulizer, a Ryton Scott-type spray chamber, and nickel cones. A four-channel peristaltic sample delivery pump (Gilson model) and a Perkin-Elmer AS-93 Plus autosampler protected by a laminar-flow-chamber clean room class 100 (Max Petek Reinraumtechnik) were used. The ICP-MS instrument was controlled by Elan 6100 Windows NT software (version 2.4). 87 Sr/ 86 Sr ratio analysis The operating conditions of the ICP-MS equipment, optimized daily, were as follows: RF power of 1200 W; Ar gas flow rates of 15 L/min for cooling, between 0.94 and 0.98 L/min for nebulizer and 1.5 L/min for auxiliary; and solution uptake rate of 1.0 ml/min. The analytical measurements were carried out by using an ICP-MS method, as described and optimized by Martins et al. (2014). The contents of Rb and Sr in Sr chromatographic fractions were determined before isotopic measurements by using an ICP-MS semi-quantitative approach, as described by Catarino et al. (2006). Each wine sample was treated (HPMW digestion followed by chromatographic separation) and analyzed in duplicate (each analysis consisted of three replicates). Periodical calibration between samples was established in order to underscore a possible shift over time. Analytical measurements were carried out with a Perkin-Elmer SCIEX Elan 9000 ICP-MS (Norwalk, CT, USA) equipped with a cross-flow 3
Multielement analysis The multielemental composition of the wines was determined (after 1:10 dilution with de-ionized water) by using an ICP-MS semi-quantitative approach, as described by Catarino et al. (2006). 2.3. Statistical analysis The statistical treatment of wine 87 Sr/ 86 Sr ratios was performed by one-way analysis of variance and comparison of means (Fisher LSD, 95 % level) using Statistica 7.0 software (StatSoft Inc., Tulsa, USA). In an early stage, normal distribution and homogeneity of variance were verified by Normal p-p (distribution of within-cell residuals) and Cochran C tests (p < 0.05), respectively. 4
Resin pretreatment (H 2 O de-ionized ; 2 N HCl) Resin activation (10% NH 3 (v/v), 30 ml) Resin washing (H 2 O de-ionized 20 ml) Resin conditioning (0,02 M EDTA ph 5,5 20 ml) Sample elution (~0,1 M EDTA ph 5,0 25-35 ml) Ca elution (0,02 M EDTA ph 5,5 40 ml) Sr elution (0,05 M EDTA ph 7,0 50 ml) Concentrationof the Sr fraction (T= 40 C V final = 10 ml) Resin washing (H 2 O de-ionized 40 ml) Reconstitution with 1% HNO 3 (v/v) (0,2 ml HNO 3, V final = 20 ml) Rb elution (3 M HNO 3 40 ml) Resin washing (H 2 O de-ionized 40 ml) Filtration PVDF 0,45 µm, d= 2,5 cm Determination of Sr and Rb concentrations by ICP-MS 87 Sr/ 86 Sr determination by Q-ICP-MS Figure 1- Procedure scheme of Sr and Rb ion-exchange separation and the following steps for the 87 Sr/ 86 Sr determination by Q-ICP-MS 5
3. Results and discussion 3.1. Effectiveness of the strontium and rubidium separation by ion-exchange chromatography In order to monitor the presence of Rb in the Sr elution fraction, directly related to the effectiveness of the chromatographic separation, the contents of Rb and Sr were determined in this solution. This evaluation must be carried out prior to the 87 Sr/ 86 Sr determination by ICP-MS. According to the results of previous studies (Martins et al., 2014), a ratio value of 4% between Rb and Sr concentrations can contribute to the overestimation of 87 Sr/ 86 Sr ratio. Rb was successfully removed in all samples, except in Touriga Franca (both replicates) where this ratio was of 3%. Still, to occur, its influence on the measurement of the 87 Sr/ 86 Sr ratio will be minimal. In addition, other elements from alkaline earth metals group (Ca, Mg and Ba) were quantified in high concentrations in the Sr elution fraction as expected, as EDTA forms complexes with other cations with behavior and properties similar to Sr. 3.2. 87 Sr/ 86 Sr in wines from the Douro region Recently, the existence of a good correlation between the values of the 87 Sr/ 86 Sr ratio and the ratio between the concentration of Rb and Sr ([Rb]/[Sr]) in the soils of different Portuguese DO vineyards was verified (Martins et al., 2014). In order to investigate a possible correlation between these parameters in wines was performed a graphical representation of the 87 Sr/ 86 Sr ratio and the [Rb]/[Sr] ratio (Figure 2). It was found that the values of 87 Sr/ 86 Sr isotope ratio are not related to the ratio of Rb and Sr concentrations ([Rb]/[Sr]) of the wine. 3.3. Multielement composition Regarding the multielemental characterization of the wines (35 elements), the 13 rare earth elements seemed to be the most appropriate for discrimination purposes. These results were consistent with the results of a study previously developed by Catarino et al. (2011), confirming the potential of rare earth elements profiles for traceability and evaluation of geographical origin. Finally, the results indicated the potential application of this technique, by itself or in conjunction with other techniques, for provenance establishment purposes (Catarino et al., 2014). The values of the total concentration of Rb and Sr, the ratio between these concentrations, Sr levels in Sr chromatographic fraction and isotope ratio 87 Sr/ 86 Sr in wines are shown in Table 2. 6
Table 2 Total Rb and Sr concentrations and Rb/Sr ratio in wines. Rb and Sr concentration in Sr chromatographic fraction and 87 Sr/ 86 Sr ratio in wines. Sr fraction after ionexchange separation Wines Rb (µg/l) Sr (µg/l) Rb/Sr Sr (µg/l) Rb (µg/l) Wines 87 Sr/ 86 Sr Chardonnay 874 527 1,66 137 0,078 0,715±0,001 a,b,c Côdega de Larinho 203 532 0,38 121 0,050 0,7155±0,0005 b,c,d Fernão Pires 1944 1355 1,44 220 0,043 0,7175±0,0005 d Gouveio 1132 390 2,90 105 0,215 0,7135±0,0005 a,b Malvasia Fina 944 459 2,05 121 0,075 0,713±0,001 a Moscatel Galego 1457 1017 1,43 156 0,076 0,7155±0,0005 b,c,d Viosinho 1443 500 2,88 126 0,176 0,714±0,002 a,b Alicante Bouschet 1107 1295 0,85 204 0,231 0,7145±0,0005 a,b,c Aragonês 2097 764 2,75 166 0,113 0,713 a Cabernet Sauvignot 2862 1316 2,18 257 0,111 0,716 c,d Donzelinho Tinto 1677 1814 0,92 248 0,091 0,716±0,001 c,d Merlot 2173 1207 1,80 186 0,224 0,7145±0,0005 a,b,c Pinot Noir 2801 1120 2,50 152 0,244 0,713 a Rufete 4230 2737 1,55 324 0,463 0,713 a Tinta Barroca 2369 962 2,46 130 0,146 0,7135±0,0005 a,b Tinta Francisca 3053 1819 1,68 195 0,274 0,7155±0,0005 b,c,d Tinto Cão 3068 2042 1,50 247 0,140 0,715 a,b,c Touriga Fêmea 1844 1392 1,33 195 0,780 0,713±0,002 a Touriga Franca 3218 1554 2,07 183 3,945 0,714 a,b,c Touriga Nacional 2706 1482 1,83 213 0,180 0,713±0,001 a Trincadeira 2245 1001 2,24 143 0,045 0,7130 a Vinhão 1721 1918 0,90 214 0,440 0,7135±0,0005 a,b Means followed by the same letter are not significantly different at the 0.05 level of significance 7
Figure 2 87 Sr/ 86 Sr ratios and [Rb]/[Sr] ratios of wines 8
4. Conclusions This study extends the limited knowledge available on the isotope ratios of strontium 87 Sr/ 86 Sr in Portuguese wines, in particular wines of the Douro wine region. Regarding the analytical procedure, the ion exchange separation method used, proved to be very effective in the separation of Sr and Rb, being a crucial step in order to avoid the overestimation of the 87 Sr/ 86 Sr ratio. In most cases the ratio between the concentration of Rb and Sr concentration of Sr in the eluted fraction was less than 0.5%. The analytical method by ICP-MS applied to the determination of the 87 Sr/ 86 Sr of wines presented enough precision to allow distinguishing between the isotopic ratios of the samples. This is essential since the natural range of variation of the isotopic ratio 87 Sr/ 86 Sr is very narrow. Thus, for comparison purposes a high precision is required. Precision (%RSD) values ranged from 0.02 to 0.42%, in the limit of Q-ICP-MS technique. The wines from 6 vineyards of the Douro Region showed isotope ratio values 87 Sr/ 86 Sr between 0.7130 and 0.7175. Statistical analysis revealed highly significant differences between the wines, which could probably be less evident if wines from other region/soils had been included in this study. Still, the range is too narrow, revealing a close proximity of the wines with respect to this analytical parameter/indicator. Moreover, it was found that the values of 87 Sr/ 86 Sr isotope ratio are not related to the ratio of Rb and Sr concentrations ([Rb]/[Sr]) of the wine. The results of the multielement composition (analyzed by multivariate statistical methods), obtained in addition to isotopic data, showed the importance of the rare earth elements as potential indicators of geographical origin, corroborating previous results. The results showed that the ratio 87 Sr/ 86 Sr could be a reliable tool for geographic identification and extended the existing information on 87 Sr/ 86 Sr values for Portuguese wines from the Douro region. However, it is necessary to gather more information about the strontium isotopic ratio 87 Sr/ 86 Sr of Portuguese wines in connection with their origin soils. It is also necessary to evaluate the evolution of the 87 Sr/ 86 Sr ratio over the biotechnological sequence (soil, grapes, must and wine) to ascertain how it is affected by the technological processes. In future, this information may be included in the European Wine Database, along with other parameters, in order to be used as a tool for geographical identification and authentication of wines. Acknowledgments: The authors would like to acknowledge Otília Cerveira for help in Mineral Analysis activities (INIAV, I.P., Dois Portos); Ilda Caldeira for help with the multivariate statistical analysis data; and Laboratório de Análises do Instituto Superior Técnico (LAIST, Lisboa) for wine mineralization equipment and support. References Almeida C.M., Vasconcelos M.T.S.D., 2001. ICP-MS determination of strontium isotope ratio in wine in order to be used as fingerprint of its regional origin. J. Anal. Atom. Spectrom., 16, 607-611. Almeida C.M., Vasconcelos M.T.S.D., 2003. Multi-element composition and 87 Sr/ 86 Sr of wines and their potentialities as fingerprints of wine provenance. Ciência Téc. Vitiv., 18 (1), 15-27. Almeida C.M., Vasconcelos M.T.S.D., 2004. Does the winemaking process influence the wine 87 Sr/ 86 Sr? A case study. Food Chem., 85, 7-12. Berglund M., Wieser M. E., 2011. Isotopic compositions of the elements 2009 (IUPAC Technical Report). Pure Appl. Chem., 83, 397-410. Catarino S., Curvelo-Garcia A.S., Bruno de Sousa R., 2006. Measurements of contaminant elements of wines by inductively coupled plasma mass spectrometry: A comparison of two calibration approaches. Talanta, 70, 1073-1080. Catarino S., Madeira M., Monteiro M., Caldeira I., Rosa T., Martins P., Bruno de Sousa R., Curvelo-Garcia A.S., 2014. Multielemental analysis throughout soil-wine system as a generator of information on geographic 9
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