Chemometric analysis of minerals and trace elements in Sicilian wines from two

Transcription

1 SUPPLEMENTARY MATERIAL Chemometric analysis of minerals and trace elements in Sicilian wines from two different grape cultivars Angela Giorgia Potortί, Vincenzo Lo Turco*, Marcello Saitta, Giuseppe Daniel Bua, Alessia Tropea, Giacomo Dugo, Giuseppa Di Bella. Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF), Università di Messina, Viale Annunziata Polo Universitario Messina, Italy. *Address correspondence to Vincenzo Lo Turco; Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF), Università di Messina, Viale Annunziata Polo Universitario Messina, Italy; phone: ; vloturco@unime.it. Abstract Chemometric analysis are used for food authenticity evaluation, correlating botanical and geographical origins with food chemical composition. This research was carried out in order to proved that it is possible linked red wines to Nero d'avola and Syrah cultivars of Vitis vinifera according to their mineral content, while the values of the physical and chemical parameters do not affect relevantly this discrimination. The levels of mineral elements were determined by ICP-OES and ICP-MS. Samples from cv Nero d Avola had the highest content of Zn, Cr, Ni, As and Cd, whereas the highest mineral concentration in cv Syrah samples was represented by K, Mg, Cu, and Sb. The research highlights that it is possible linked red wines to Nero d'avola and Syrah cultivars of Vitis vinifera according to their mineral contents, adding knowledge to the determination studies of the wine botanical origin. Keywords: Red wines; Minerals; Trace elements; Chemometric analysis; Botanical discrimination. 1

2 Experimental Reagents and materials High purity water with resistivity of 10 MΩ cm (J.T. Baker, Milan, Italy), was used throughout. Concentrated (65%, v/v) nitric acid trace metal analysis grade (J.T. Baker, Milan, Italy) was used together with concentrated (30%, v/v) hydrogen peroxide (J.T. Baker, Milan, Italy) for samples digestion. The first one was also employed for cleaning glassware. Single element standards (1000 mg L 1 in 2% nitric acid) were purchased from Fluka (Milan, Italy) and from Merck (Darmstadt, Germany) and were mixed to prepare a multi-element standard solution that has been subsequently diluted for calibration analysis. To correct instrumental drift and variations due to the matrix, standard solutions of 45 Sc, 103 Rh and 209 Bi (1000 mg L 1 in 2% nitric acid) were purchased from Fluka (Milan, Italy) and were used as on-line internal standards (at level of 1.5 mg L -1 ). To verify the digestion of sample and to correct the volumetric changes, standard solution of Re at 1000 mg L -1 in 2% nitric acid was acquired by Fluka (Milan, Italy) and was used as preparation standard (at level of 0.5 mg L -1 ). A solution containing 1 g L -1 of 7 Li, 59 Co, 80 Y and 205 Tl in 2% HNO 3 was obtained from Agilent (Santa Clara, CA) and was used to tune the ICP-MS instrument. A diagnostic standard solution containing 1000 mg L -1 of Ba, Mg and Zn in 5% HNO 3 (JYICP- DIAG) was obtained from Horiba Jobin Yvon (Longjumeau, France) and used for the periodic check of the ICP-OES instrument. Argon (N 5.0) of % purity and helium (N 5.5) of % purity were supplied by Rivoira gases (Milan, Italy). All reagents used for enological parameters determination, provided by Sigma Aldrich, were analytical grade. Wine samples The analyses were carried out on 39 sicilian red wines, obtained from grapes of 100 % cv Nero d Avola and 34 from 100% cv Syrah, and cultivated in the same geographic areas in province of Syracuse. All samples were obtained directly from producers and were from 2015 vintage. The wine glass bottles (750 ml) were stored in the dark at 2 C and opened before analysis. 2

3 Analytical procedure for minerals and trace elements determination From the freshly opened bottles, about 1 ml of each wine was transferred and accurately weighed into acid-prewashed PTFE vessels; it was added with internal Re standard and then digested with 6 ml of HNO 3 (69%, v/v) and 2 ml of H 2 O 2 (30%, v/v) in a microwave digestion system Ethos 1 (Milestone, Bergamo, Italy) equipped with sensors for temperature and pressure control. Instrumental parameters and settings were: 10 min for 1000W up to 200 C, 10 min for 1000W at 200 C. Allowed to cool, each sample was made up to volume of 10 ml with HNO 3 (2%, v/v). Each sample was digested in triplicate. The K, Ca, Mg, Na, Zn, Fe, Mn and Cu determination was carried out by Horiba Jobin Yvon ULTIMA 2 (HORIBA Scientific, Longjumeau, France) ICP-OES spectrometer, equipped with a glass concentric pneumatic nebulizer (i.d. 0.3mm) coupling with a quartz cyclonic type spray chamber (50mL). For Cr, Pb, Ni, Co, Se, As, Cd and Sb measurements an Agilent 7500cx (Agilent Technologies, Santa Clara, CA) ICP-MS spectrometer, equipped with a MicroMist glass concentric pneumatic nebulizer coupling with a cooled Scott double pass type spray chamber made of quartz, was used. To minimize polyatomic interferences resulting from plasma and matrix, an octopole collision system with 4 ml min -1 helium as collision gas and kinetic energy discrimination mode was used (collision mode) for almost all the elements. The instrument operating parameters for ICP-OES and ICP-MS analyses are presented in Table S1. According to literature, ethanol may affects the quantification of the different elements in wine samples since its content could influence the transport properties towards atomization devices of the instrument as well as the viscosity and the density of the samples (Aceto et al. 2002). Thus, the possible interference of alcohol on elemental measurements was corrected by addiction of 1.3% ethanol to all standard solutions used for calibration since wine samples were diluted 10 times before analysis (Rodriguez et al. 2011). The evaluation of the linearity was based on the 6 standard solutions injections. Each solution was injected three times (n=3). The instrumental detection limits (LODs) and quantification (LOQs) were experimentally calculated as 3.3σ/S and 10σ/S, respectively, where σ is the standard deviation of the response of six blanks and S is the slope of the calibration curve (EURACHEM 2000). A lab-made wine containing 5 g L -1 tartaric acid and 13% ethanol in water was used as blank solution. It was digested as describe above and it was run with each batch of wines. Moreover, for recovery studies, 18 spiked lab-made wine solutions were prepared: 9 were used for ICP-OES analysis (3 at level of 50 mg L -1, 3 at level of 100 mg L -1 and 3 at level of 300 mg L -1 ) and 9 were 3

4 used for ICP-MS analysis (3 at level of 10 µg L -1, 3 at level of 20 µg L -1 and 3 at level of 50 µg L -1 ). Each solution was analyzed in triplicate. For repeatability estimation and intermediate precision, each spiked level was prepared and analyzed in 12 replicates in the same batch and in 24 replicates in different days. Enological parameters determination The main enological parameters, among which alcohol content, ph, total acidity, volatile acidity, malic acid, SO 2 and total SO 2 contents, were determined following the procedures specified in detail in EC Regulation 2676/90 (1990). Finally, according to Ribéreau Gayon and Stonestreet (1965), anthocyanins quantification was carried out in wine samples. Validation of ICP-MS and ICP-OES analysis Method linearity, sensitivity, accuracy, precision and repeatability are reported in Table S2. Results showed that the adopted procedures were suitable for the research. Indeed, good linearity was observed in each investigate concentration range with R Instrumental LOD values ranged from to mg L -1 for ICP-OES analysis and from to µg L -1 for ICP- MS analysis, while instrumental LOQ values ranged from to 2.50 mg L -1 and from to µg L -1, respectively. Thus the analytical limit of quantification for elements analyzed by ICP- OES were between 0.1 to 25 mg L -1 (Cu and K, respectively), while for elements analyzed by ICP- MS varied from (value determined for Sb) to µg L -1 (value determined for Se and As). The recovery for all elements was always within the interval of %. The repeatability RSD% was lower or equal to 5.1%, while for intermediate precision it was lower or equal to 9.5%. Statistical analysis The SPSS 13.0 statistical software package for Windows (SPSS Inc., Chicago, IL, USA) was used for all statistical calculations. Statistical methods were conducted on starting multivariate matrix where variables were the concentrations of 24 detected parameters (8 were the enological parameters and 16 were the concentrations of minerals and trace elements) and the cases were the 73 analyzed wine samples. Data below LOQ were replaced with the LOD/2 values and all concentrations were log e - transformed to reduce the effect of outliers on skewing the data distribution and to bring the concentrations of element within the same range (Škrbić et al. 2010). The data were subdivided in two groups, according to the cultivar of Vitis vinifera: the first one (39 samples) consisting of wines from Nero d Avola grapes, and the second one (34 samples) represented by wines from Syrah grapes. 4

5 Initially, the non-parametric Mann Whitney U test was applied to study the significances of differences. Successively, the data set was normalized and Principal Component Analysis (PCA) was performed to differentiate samples belonging to the two red wine types based on the enological parameters values and the concentrations of minerals and trace elements. In addition to PCA, Linear Discriminant Analysis (LDA) in the stepwise mode was carried out to discriminate among wine cultivars in according to F-value. References Aceto M, Abollino O, Bruzzoniti MC, Mentasti E, Sarzanini C and Malandrino M Determination of metals in wine with atomic spectroscopy (flame-aas, GF-AAS and ICP-AES): a review. Food Addit Contam 19: D.M. 29 December Decree of the Minister of Agriculture and Forests, published on Official Gazette, No. 13, January 17th EC Regulation 2676/90 (17 Sept. 1990). Determining Community methods for the analysis of wines. Off J Eur Communities 1990, No. 272 (Oct 3), EURACHEM 2000, Guide. (2 nd Ed.). Editors: S L R Ellison (LGC, UK), M Rosslein (EMPA, Switzerland), A Williams (UK). European Directive EC/1881/2006/. Commission Regulation, No. 1881, December 19 th OIV (Organisation Internationale de la Vigne et du Vin) Compendium of international methods of wine and must analysis. Ribéreau GP and Stonestreet E Le dosage des anthocyanes dans le vin rouge. Bull Soc Chim Fr 9: Rodriguez SM, Otero M, Alves AA, Coimbra J, Coimbra MA, Pereira E and Duarte AC Elemental analysis for categorization of wines and authentication of their certified brand of origin. J Food Comp Anal 24: Škrbić B, Szyrwińska K, Đurišić-Mladenović N, Nowicki P and Lulek J Principal component analysis of indicator PCB profiles in breast milk from Poland. Environ Int 36:

6 Table S1 Instrument operating parameters for ICP-OES and ICP-MS analyses. ICP-OES analysis ICP-MS analysis Rf power 1000W Auxiliary/nebulizer/plasma Argon flow rate 0.2/1/12 L min -1 Nebulization pressure 2.98 bar Nebulizer pump 20 rpm Sample introduction flow rate 1 ml min -1 Acquisition mode maxima Integration time 2 sec for K, Ca, Mg and Na; 4 sec for Zn, Fe, Mn and Ca Monitored isotopes and wavelengths (nm) K, ; Ca, ; Mg, ; Na, ; Zn, ; Fe, ; Mn, ; Cu, RF power 1500W Plasma/auxiliary/carrier gas flow rate 15/0.9/1.1 L min -1 Helium collision gas flow rate 4 ml min -1 Spray chamber temperature 2 C Sample depth 9 mm Sample introduction flow rate 1 ml min -1 Nebulizer pump 0.1rps Extract lens V Octopole collision system setting He mode for Cr, Ni, Co, Se, As and Cd; No-gas mode for Pb and Sb Monitored isotopes 52 Cr, 59 Co, 60 Ni, 75 As, 80 Se, 114 Cd, 121 Sb and 208 Pb On-line internal standards 45 Sc for Cr, Co, Ni, As and Se; 103 Rh for Cd and Sb; 209 Bi for Pb Integration times 0.8 s/point and for Se; 0.5 s/point for As; 0.2 s/point for Cr, Co and Ni; 0.1 s/point for Cd, Sb and Pb Point for mass 3 (3 replicates acquisitions) 6

7 Table S2 Validation parameters for ICP-OES and ICP-MS analyses. ICP-OES analysis Element R 2 LODi (mg/l) LOQi (mg/l) LOQa (mg/l) Accuracy (% ± RSD%, n=9) Repeatability (RSD%, n=12) Intermediate precision (RSD%, n=24) Level I (50 mg L -1 ) Level II (100 mg L -1 ) Level III (300 mg L -1 ) Level I (50 mg L -1 ) Level II (100 mg L -1 ) Level III (300 mg L -1 ) Level I (50 mg L -1 ) Level II (100 mg L -1 ) K ± ± ± Ca ± ± ± Mg ± ± ± Na ± ± ± Zn ± ± ± Fe ± ± ± Mn ± ± ± Cu ± ± ± Level III (300 mg L -1 ) ICP-MS analysis Element R 2 LODi (mg/l) LOQi (mg/l) LOQa (mg/l) Accuracy (% ± RSD%, n=9) Repeatability (RSD%, n=12) Intermediate precision (RSD%, n=24) Level I (10 µg L -1 ) Level II (20 µg L -1 ) Level III (50 µg L -1 ) Level I (10 µg L -1 ) Level II (20 µg L -1 ) Level III (50 µg L -1 ) Level I (10 µg L -1 ) Level II (20 µg L -1 ) Cr ± ± ± Pb ± ± ± Ni ± ± ± Co ± ± ± Se ± ± ± As ± ± ± Cd ± ± ± Sb ± ± ± R 2, determination coefficient; LODi, instrumental detection limit; LOQi, instrumental quantification limit; LOQa, analytical quantification limit. Level III (50 µg L -1 ) 7

8 Table S3 Contents and significant differences of the mineral and trace elements composition of wines from cv Nero d Avola and cv Syrah grapes. K (mg L -1 ) Ca (mg L -1 ) Mg (mg L -1 ) Na (mg L -1 ) Zn (mg L -1 ) Fe (mg L -1 ) Mn (mg L -1 ) Cu (mg L -1 ) Nero d Avola (n=39) Syrah (n=34) Min Max Mean S. D Min Max Mean S. D Mann Whitney U Wilcoxon W Asymp. Sign Cr (µg L -1 ) Pb (µg L -1 ) Ni (µg L -1 ) Co (µg L -1 ) Se (µg L -1 ) As (µg L -1 ) Cd (µg L -1 ) Sb (µg L -1 ) Nero d Avola (n=39) Syrah (n=34) Min n.d. n.d. Max n.d. Mean _ S. D _ Min n.d. n.d. Max Mean S. D Mann Whitney U Wilcoxon W Asymp. Sign Asymp. Sign. bold values indicate element concentrations significantly different at 95%. 8

9 Table S4 Legal limits of Zn, Cu, Pb, As and Cd, and comparison with results obtained in this study. Element Legal limits (mg/l) Concentrations in wine (mg/l) Samples exceeded legal limits (%) Nero d'avola Syrah Nero d'avola Syrah Mean value Max value Mean value Max value Pb 0.2 a b 0 0 Cu 1 c,d Zn 5 c,d As 0.2 d Cd 0.1 d a Regulation EC/1181/2006; b OIV, 2006; c Ministerial Decree of 29 December 1986; d OIV,

10 Table S5 Values and significant differences of the enological parameters of wines from cv Nero d Avola and cv Syrah grapes. Alcoholic grade (% vol) ph Total acidity (g L -1 ) Volatile acidity (g L -1 ) Malic acid (g L -1 ) SO 2 (mg L -1 ) Total SO 2 (mg L -1 ) Anthocyanins (mg L -1 ) Nero d Avola (n=39) Min Max Mean S. D Syrah (n=34) Min Max Mean S. D Mann Whitney U Wilcoxon W Asymp. Sign Asymp. Sign. bold values indicate element concentrations significantly different at 95%. 10

11 Table S6 The Recommended Dietary Allowance (RDA) of non toxic elements and RDA shares from consumption of Nero d Avola and Syrah wines. Non Toxic Elements RDA (mg day -1 ) a Mean concentrations in wine (mg/l) % of RDA estimated by mean value Nero d'avola Syrah Nero d'avola Syrah Zn Fe Se Cu Cr Mn Ca K Mg Na 1500 b a Commission Directive 2008/100/EC; b AI (Adeguate intake) EFSA

12 Table S7 Protection Limits (TDI, TWI, PTWI and BMDL 01 ) of potentially toxic elements and Protection Limits shares from consumption of Nero d Avola and Syrah wines. Potentially Toxic Elements Mean concentrations in wine (mg/l) % of Protection limit estimated by mean value Reference Nero d'avola Syrah Nero d'avola Syrah Pb PTWI (mg kg.-1 b.w week -1 ) EFSA, 2010 BMDL 01 (µg kg.-1 b.w day -1 ) As PTWI (mg kg.-1 b.w week -1 ) EFSA, 2009 BMDL 01 (µg kg.-1 b.w day -1 ) BMDL 01 (µg kg.-1 b.w day -1 ) Cd TWI (µg kg.-1 b.w week -1 ) EFSA, 2012 Ni TDI (µg kg.-1 b.w day -1 ) WHO, 2005 Sb TDI (µg kg.-1 b.w day -1 ) 6 n.d n.d WHO, 2003 n.d., not determinable. 12

13 Figure S1-2D Scatterplot for the 73 wine samples categorized by cultivars of Vitis vinifera. Insert: loading plot for PC1 and PC2. 13