ICP-MS analysis for the characterization of the origins of wines MASSIMO DI MARTINO1, CIAVARDELLI DOMENICO2, FRANCO DI GIACOMO1, CARLO CIVITARESE1, ANGELO CICHELLI1* Angelo Cichelli *Corresponding Author 1. Università Chieti-Pescara, DEc. Laboratorio di Merceologia, Viale Pindaro 42, Pescara, 2. School of Engineering, Architecture/Motor Science Kore University, Enna, Italy/University Foundation, Centre of Study on Aging (Ce.S.I.), Chieti, Italy

KEYWORDS: Wine; metals; inductively coupled plasma mass spectrometry; traceability; origins; Italy; ; strontium; rubidium; barium.

ABSTRACT: Element profiling might represent a valid too for the determination of the origins of foods. In the present study, we have investigated this issue within the oenological sector on 32 wines produced in Abruzzo, Italy. In order to assess wine element composition, we used inductively coupled plasma mass spectrometry, which can be used to determine the levels of a wide range of metals in a short time and classified wine samples on the basis of their origins and element profiles using discriminant analysis. We determined the levels of the following elements: Li, Be, Na, Mg, Al, Ca, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Se, Rb, Sr, Cs, Ba and Pb. Our analytical approach provided suitable sensitivity for accurate quantification of these elements in red and white wines. The limit of quantification were between between 0.1 mg L-1 (Co, m/z 59) and 42 mg L-1 (Fe, m/z 57). Precision expressed as relative standard deviation and calculated under repeatability conditions was between 3% (Fe, m/z 57) and 22.53% (Co, m/z 59). Recovery values obtained by spike experiments were between 77±5 (Pb, m/z 206) and 110±6 (Li, m/z 7). As specific markers, we evaluated the concentrations of Sr, Ba and Rb. The classification of wines were 100% accurate in linear discriminant analysis. Overall, this study investigates wines produced in central Italy, and it contributes to their traceability and assessment of their origin. Furthermore, our data are in agreement with previously published data, and will help to contribute towards the creation of a national database to protect the consumer against Food analysis commercial fraud.

INTRODUCTION than 300 million euro, which constitutes 28% of the regional gross saleable productionand 7% of the national gross saleable Recently, grapevine (Vitis vinifera) cultivation has spread into production. The most cultivated grapevines are: Montepulciano geographical areas and continents where there was not d’Abruzzo, with an area of about 15,000 Ha; , with previously any tradition in this sector. Therefore, in a more an area of about 13,000 Ha; international grapevines (e.g., globalised market, there is a large presence of wines with , Pinot, Cabernet Sauvignon); and indigenous standardised and homogeneous characteristics of their grapevines (e.g., , Cococciola, Passerina, Montonico, composition, which often represent imitations of products coming ). from local supply chains. The differences in wine composition In 2009, approximately 75,000 hL of Montepulciano D’Abruzzo depends on the cultivars, environment, technologies used and DOP wines were bottled, with 62% of this exported, which operative techniques. All of these factors contribute to the demonstrates its commercial success worldwide (4). It is essential quality (physicochemical, sensorial, hygienically safe) and to the for the customers to know the origin of these products, in order excellence of origin according to the introduction of the DOP to keep the customers satisfied and provide the necessary and IGP classifications (1). guarantees of transparency in an ever-more-demanding market. The European Community (EC) understands the importance of the protection of local products. To achieve this, the EC issued Regulation EC 1169/2011(2),

OOD Industry Hi Tech - January/February 2013 Vol. 24(1) according to which it is mandatory to indicate the origin F of many products, including wines, extra virgin olive oil, gro

A honey, fruit, tomato sauce, fresh milk, and sheep and pork. The traceability of the origins of these products can be important in order to protect the origins of national and local foods, as well as to confirm that a certain product fulfils the necessary conditions to allow it to be linked to its own territory. This produces two main advantages: first, it gives value to the ‘made in Italy’ brand; and second, it helps small local companies that produce excellent products, but oftentimes are imitated by others that are sold as if they were the originals(3). Vineyard cultivation in Abruzzo (Italy, Figure 1), is spread overan area of approximately 33,000 Ha on the hills of the Provinces of Chieti, Pescara, and L’Aquila.

These grapevines represent the main agricultural sector Figure 1. The provinces of Abruzzo of this region, with a gross saleable productionof more

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Food analysis 31 , 3 , in 2% -1 ; carrier -1 ) purchased from 3 ) were purchased ; spray chamber -1 -1 (2%) in polystyrene tubes 3 , in 2% HNO ) was purchased from Agilent -1 3 ) that was purchased from Romil. -1 gas, 1.13L min temperature, 2°C; sampler and skimmer cone, Ni, 1.0mm and Agilent Technologies (Palo Alto, CA, USA) and from a magnesium (Mg) standard solution (1000 mg L Multi-element calibration standard stock solution containing Li, beryllium (Be), sodium (Na), Mg, Al, calcium (Ca), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), Co, nickel (Ni), copper (Cu), zinc (Zn), gallium (Ga), arsenic (As), selenium (Se), Rb, Sr, silver (Ag), cadmium (Cd), caesium (Cs), Ba, Tl, lead (Pb), and uranium (U) (10 mg L tuning solution was prepared from a stock solution containing lithium (Li), Y, cerium (Ce), tallium (Tl) (10 mg L Technologies. Inductively coupled plasma mass spectrometry analysis The preparation of the wine samples was performed according to withprocedures, published previously some minor modifications (11,19). Briefly, the wine was diluted ten-fold with HNO (15 mL; BD Falcon, BD Biosciences, Franklin Lakes, NJ, USA) and analysed for Li, Be, Na, Mg, Al, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Ag, Cd, Cs, Ba, Tl, Pb and U. The ICP-MS analysis was performed with a 7500A ICP mass spectrometer (Agilent Technologies, Tokyo, Japan). A Babington nebuliser with a Scott spray chamber (Agilent Technologies) and shield torch system were used. The following operating conditions were applied: forward power, 1260 W; plasma gas flow rate,15 L min HNO from Fluka (Shnelldorf, Germany). Ultrapure nitric acid (HNO from Fluka (Shnelldorf, Germany). Ultrapure Teramo (TE), as 5 red wines and 8 white wines (Table 1). wines (Table and 8 white red wines (TE), as 5 Teramo commerce ready for batches taken from were The samples and accredited to type, lot number, similar according and were to samples were mixed Subsequently, these area of origin. samples of approximately significant produce statistically were: red wines wines that were sampled 1 L each. The Merlot D’Abruzzo DOP and, IGP, Montepulciano (Sangiovese IGP, Pecorino IGP, Trebbiano wines (Malvasia IGP) and white Chardonnay IGP). Their areas of originD’Abruzzo DOP, and 200 m and 300 m above seaare at altitudes of between by a substratum constituted bylevel, which are characterised emerging soils are represented mostly bysedimentary rock. The clay (12). sandstone and marine Sample preparation Standards and reagents a specific resistance of 18.2 MW cmDeionised water with Watford, UK) was used to prepare all(Milli-Q system, Millipore, the samples. Analytical grade ethanolsolutions and to dilute solution (10 mg L and the yttrium (Y) stock 69%, UpA) was purchased from Romil (Cambridge, UK). The69%, UpA) was purchased from Romil (Cambridge, Table 1. Municipalities of origin and details of the wines analysed in this study Sampling conducted on 32 samples of wineICP-MS analyses were the producers. 19 samples came fromcollected directly from as 12 red winesmunicipalities in the Province of Chieti (CH), of the Province ofand 7 white wines, and 13 from municipalities MATERIALS AND METHODS According to the literature, some metals present in wines, present some metals literature, to the According aluminium (Rb), (Ba), rubidium (Sr), barium strontium such as the (Co), can reflect (Si), boron(Bo) and cobalt (Al), silicon useful to can therefore be of the soil of origin and composition ) of origin of wines(5-16 of the different areas give indications foods (17). and other of to define the origins the present study was The aim of areas of the Abruzzo in two different wines produced the same vine, according to metalregion, but coming from of these metals was determinedconcentrations. The presence plasma mass spectrometry (ICP-MS),using inductively coupled fingerprint of a product, so that it isa tool that allows a element traceability of its origins (18). possible to have scientific 32 Agro FOOD Industry Hi Tech - January/February 2013 - Vol. 24(1)

Food analysis The external standard method was used for quantification The externalstandardmethodwasusedforquantification was washedwith5%HNO ( best sensitivityof1µgL replicates, 3.Theoperatingconditionswereoptimisedforthe by spikingsamplesat1000,1500and2000µgL the standardadditionmethod(n=3),whichwasperformed major elements,suchasNa,MgandCa,werequantifiedby 235 82 43 0.4 mminsidediameter;sampleuptakerate,mLmin of ceriumoxide( sample depth,7mm;measuredm/z, 140 Se, Ca, of unspikedsample,Si=concentrationcorrespondingtothei-thspikelevel,i=1,2,3. dR%, meanpercentrecovery(n=5)calculatedasR%i=(xs,i-xe)/Si*100,wherexs,I=concentrationofthei-thspikedsample,xe=concentration cRSD, relativestandarddeviationcalculatedasRSD=SD/x*100,wherexismeanconcentration. bSD,standard deviation,n=5 blank signal(n=10)andSDBk=standarddeviationofexperimental(n=10). aLOQ, Limitofquantificationcalculatedastheconcentrationcorrespondingtosignaly=yBk+10SDBk,whereyBk=meanexperimental ten-fold dilutedconcentrationsofelementsfoundintheun-spikedandspikedsamplematrix. Cs; 10,50,100µgL-1forLi,Al,Mn,Ni,Cu,Se,Rb,Sr,Ba,andPb;100,200FeZn.Dataaremeanvalues(n=5)showthe the analysedwinesamplesandspikedwithknownconcentrationsoftargetanalytes.Spikelevels:1,10,50µgL-1forBe,V,Co,Ga, Table 2.AnalyticalfiguresofmeritICP-MSanalysiswine.Arepresentativesamplematrixwasobtainedbypoolingequivalentvolumes of U; sampling period per mass, 0.3 s; number of instrumental U; samplingperiodpermass,0.3s;numberofinstrumental Ce 85 51 2+ Rb, V, / 140 53 86 Ce Cr, Sr, calculated Table 4.Rb,Srand Baconcentrationsinspecificsampled winetypes.SD:standarddeviation. NC:not standard deviation. Table 3.minimum,maximumandaveragevaluesofconcentrationRb,SrBrinthesampledwines.SD: + 89 55 ) ion ratios. The sample introduction system ) ionratios.Thesampleintroductionsystem 140 Y, Mn, 107 Ce 57 Ag, 16 -1 Fe, withMg, Y and Tl, and for the reduction withMg, YandTl,forthereduction O 111 3 + 59 and water between analyses. The andwaterbetweenanalyses.The / 140 Co, Cd, Ce 60 133 + Ni, ) and doubly charged ) anddoublycharged Cs, 7 63 Li, 137 Cu, 9 Be, Ba, 66 23 Zn, 203 -1 Na, , respectively. , respectively. Tl, 69 Ga, 24 206 Mg, Pb and Pb and 75 -1 As, As, 27 ; Al, Al, 2% ethanol/2%HNO concentration, by analysis of the element standards prepared in functions werecalculatedovertwoordersofmagnitude of thetraceelements(n=3).Matrix-matchedcalibration Excel software. Excel software. using ICP-MSChemstation(AgilentTechnologies)andMicrosoft previously reported(24-25).Thedataanalysiswascarriedout percentages ofrecovery(R%),respectivelyandevaluatedas are expressedasrelativestandarddeviations(RSDs)and analysis of10experimentalblanks.Repeatabilityandtrueness standard deviations(SD)ofthemeanblanksignalsfrom µg L addition inaT-pieceofaninternalstandardsolutionY(50 was performedforbothquantificationmethodsbyonline -1 ). The limit of quantification (LOQ) was calculated from ). Thelimitofquantification(LOQ)wascalculatedfrom 3 . Furthermore, internal standard correction . Furthermore,internalstandardcorrection Figure 2. Score plot from LDA of the wines produced in each municipality of the Provinces of Teramo and Chieti.

Statistical analysis Linear discriminant analysis (LDA) was performed in order to classify wine samples on the basis of their provenience in relation to their element profile. After the assessment of normality of data, it was applied to raw data by using stepwise regression mode. Variable importance to the model was evaluated by Wilks’ lambda selection criterion. p values lower than 0.050 were considered for statistical significance. Statistical analysis was performed using SPSS statistical package.

RESULTS AND DISCUSSION

In this study, we applied a previously published method for ICP-MS element profiling of wine with some minor modification of the sample preparation method (11). Therefore, we decided to evaluate and verify the analytical figures of merit of this procedure by assessing the limit of quantification (LOQ), linearity of matrix matched calibration functions, precision under repeatability condition, and trueness. Repeatability and trueness were evaluated by spiking a representative sample matrix obtained by pooling equivalent volumes of the analysed wine samples. The determination coefficients (r2) of matrix matched calibrations were higher than 0.980 for all analytes (data not shown). Furthermore, the concentrations of Li, Be, Na, Mg, Al, Ca, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Se, Rb, Sr, Cs, Ba and Pb were higher than the LOQ (Table 2). In contrast, the Cr, As, Ag, Cd, Tl and U concentrations were below the LOQ. Acceptable levels of repeatability and trueness were calculated for all target elements (Table 2). In fact, the relative standard deviations of mean concentrations measured in the unspiked matrix sample were between 3% (Fe, m/z 57) and 22.53% (Co, m/z 59). Furthermore, the mean recovery calculated from analysis of matrix sample spiked at three concentration levels were between 77±5 (Pb, m/z 206) and 110±6 (Li, m/z 7). Previous study showed that whenever wines have to be classified according to their area of origin, there is the need to identify one or more chemical elements that are linked with the territory of origin. Statistical analysis of differential element profiles allows the classification of wines into specific groups (20). In the present study, we carried out an analysis that was aimed at the attribution of the samples of wines to one or more groups (i.e., municipalities). LDA based on element composition of wine was aimed at classifying wine samples according to their origin. The Wilk’s lambda criterion indicated that Na (p=0.02), Al (p=0.014), Ca (p=0.017), Mn (p<0.001), Co (p=0.001), Cu (p=0.010), Ga (p=0.001), Rb (p=0.050), Sr (p=0.013), and Pb (p<0.001) were capable to discriminate wine samples on the basis of their origin. Six significant discriminant functions were calculated as linear combinations of these variables (element concentrations). The scatter plot of function 1 vs function 2 is shown in Figure 2. The first two functions guarantee an optimal interpretation The discriminant analysis of these metals confirms that through the because they retain 88% of the global information (Figure 2). We concentrations of Sr, Ba and Rb in wines that are specific in certain also found that the classification of the municipalities based on areas, it is possible to verify some ‘anomalies’. This should allow the the estimated discriminant variables is 100% accurate. identification of any commercial fraud. It is also possible to identify These results indicate that it is possible to distinguish the wines elements in common in different areas of production and in the produced in each municipality, independently of whether these area of origin of the wines, considering that the production control are white, red, or white vinified red wines (Figure 2). allows us to narrow down the sampling. Our findings are in line with previously published data.In particular, The analytical method of ICP-MS and the diversified statistical Martin et al. (7) was able to distinguish wines from different regions analysis are here proposed as useful protocols for the typifying of of the Bordeaux area (France) from analysis of Ba, Sr, Mg, Al and wines and for the verification of their chemical traceability and Rb,. Frias (21) distinguished Spanish wines from the Canary Islands origins. The current study will be further developed by increasing through the analysis of Rb. La Torre (5) classified Spanish wines the number of samples and by the creation of a database from the Galicia area through a study of the levels of Liand Rb. that is similar to those in each Italian region, as per Regulation Galgano(22)classified some wines from southern Italy through EC: 2676/90 (23), with the collaboration of the consortium for the analysis of the elements that were present in small and large protection. The connections between the mineral content of a quantities. In the present study, the elements Sr, Ba and Rb were wine and its soil of origin can also be investigated further, which present at significant levels, such that these can be used to might allow the creation of an identity map of each product (2). typify the white and red wines of the different municipalities in It would be desirable to validate any model of analysis on an the Provinces of Teramo and Chieti. The concentrations of these international level by bearing in mind other innovative techniques, elements are not influenced by the production cycle, but are such as nuclear magnetic resonance. These techniques can be determined by the contributions of the minerals in the soil and by used to introduce an effective system for a barcode system on the retention of these minerals by the grapevines (10). the labels of wines that can contain these analytical data (e.g., The mean levels of Rb in the sampled wines varied from 1815.36 the content of Sr in a specific batch of wine, for a particular year), μgL-1 in the Province of Chieti, to 1599.7 μgL-1in the Province of which can then be used as a reference in cases of antifraud Teramo. Those are values that are typical of areas that have controls. mostly clayey soils. The mean levels of Sr (an alkaline earth-soiled metal) varied from 395.58 μgL-1in the Province of Chieti, to 458.36 μgL-1in the . These are typical values for REFERENCES AND NOTES the Adriatic ridge in central Italy. As far as Ba is concerned, the highest concentrations were in the wines of the Province of Chieti 1. Regolamento CE 607/2009 della Commissione del 14 luglio 2009 (97.33μgL-1),as compared to those of the Province of Teramo recante modalità di applicazione del regolamento CE n. 479/2008 del -1 Consiglio, L 193/60 del 24/07/2009.

Food analysis (87.86μgL ) (Table 3). Through the analysis of these values obtained for the most 2. Reg. Ce 1169/2011 della Commissione del Consiglio del 25 Ottobre 2011, L 304 del 22/11/2011. important types of these wines on a commercial scale (Table 3. C. Tsolakis et al., Proceedings of 34th World Congress of vine and wine, 4), it can be seen that the concentrations of Rb and Ba in the OIV 2011 Porto Portogallo, (2011). Montepulciano d’Abruzzo DOP are higher in the Province 4. Sintesi della viticoltura Regione Abruzzo Vinitaly 2012; http://www. of Chieti, while the concentration of Sr is higher in the wines vinitaly.com/pdf/italia/abruzzo/Vini_Abruzzo_Vinitaly2012.pdf produced in the Province of Teramo. The element that 5. M.L. Latorre et al., J. Agric. Food Chem., 42: 1451-1455, (1994). differentiates the Pecorino IGP the most is Sr, which is higher in 6. J.D.Greenough et al., Australian Journal of Grape and Wine Research, the Province of Teramo. The concentration of Sr is higher in the 3: 75-83, (1997). Province of Teramo also with the Trebbiano d’Abruzzo DOP, 7. G.J. Martin et al., Am. J. Enol. Vitic., 50, 4: 409-417, (1999). as well as for the Chardonnay IGP, while Rb and Ba are mostly 8. V.F. Taylor. et al., J. Agric. Food Chem., 51: 856-860, (2003). present in the wines of the Province of Chieti. These data are 9. A. Del Signore, Journal of Trace Elements in Medicine and Biology, Volume 17, Issue SUPPL. 1, 57-63, (2003). similar to those reported in recent studies (12). 10. M.d.M. Castineira Gomez et al., J. Agric. Food Chem., 52: 2962-2974, From the study by Polo et al.(12), it can be seen that wine (2004). produced in the hills of the Province of Treviso, is characterised 11. P.P. Coetzee et al., F. J. Agric. Food Chem., 53: 5060-5066, (2005). -1 by an average content of Sr of 200μgL , which is a typical value 12. M. Polo et al., Proceedings of 31th Congress of Vine and Wine, OIV for wines produced in areas with a low clayey soil. The wines of 2008 Verona Italia, (2008). Abruzzo contain high concentrations of Sr compared to a wine 13. C. M. R. Almeida et al., J. Agric. Food Chem., 51, 4788e4798(2003). of northern Italy, this data could be used to discriminate wines 14. N.Jakubowski et al., J. Anal. Chem. 364, 424 e 428 (1999). with different origin. The values of the metals could be included in 15. E. Marengo et al., Food Chemistry, 81, 621e630 (2003). production rules. 16. G. Thiel et al., Analytical and Bioanalytical Chemistry, 378, 1630e1636. (2004). OOD Industry Hi Tech - January/February 2013 Vol. 24(1)

F 17. F. Di Giacomo et al., J. Agric. Food Chem. 55, 860-866, (2007). 18. M. Giaccio et al., Journal commodity technology quality, 47 (I-IV), 267- gro

A CONCLUSIONS 284, (2008). 19. G. Van der Linde et al., S. Afr. J. Enol. Vitic., 31 (2): 143-153, (2010). From the point of view of the traceability of wines, there is no 20. A. Cichelli et al., American Journal of Enology and Viticulture Volume doubt that the most promising variables are the chemical 51, Issue 2, 108-114, (2000). elements of natural origin, because these can highlight 21. S. Frias et al., Talanta, 59(2) 335-344, (2003). the relationships to the characteristics of the soils, as widely 22. F. Galgano et al., LWT, Food Science and Technology, 41 1808-1815, documented in the literature(10). 2008. On the premises of geographical origin and the comparison of 23. Reg. CE 2676/90 della Commissione del 17 settembre 1990, L 272 del these numerous areas, the distributions of the metals in the wines 3/10/1990. 24. The Fitness for Purpose of Analytical Methods: A Laboratory Guide to analysed in the present study show that the concentrations of Method Validation and Related Topics, Eurachem Guide, LGC: some of these metals are connected with determined areas and Teddington Ltd, 1st English edn, 1998 specific characteristics of the soil. The statistical analysis indicates 25. W. Funk, V. Dammann and G. Donnovert, Quality Assurance in that the wines produced in each of these municipalities can be Analytical Chemistry: Application in Environmental, Food, and distinguished one from the other, whether these are white, red or Material Analysis, Biotechnology and Medical Engineering, Wiley- white vinified red wines. VCH, Weinheim, 2007

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