SNIF-NMR和IRMS技术在原产地葡萄酒鉴定中的应用

利用点特异性天然同位素分馏核磁共振技术(SNIF-NMR)和同位素比质谱仪(IRMS)技术测定葡萄酒中稳定性同位素D/H、18O/16O和13C/12C的比值,结果表明(D/H)Ⅱ、R、δ18O‰对产地的鉴别提供着主要信息,而(D/H)Ⅰ与δ13C‰提供了较次要的信息,单独使用SNIF-NMR结果只能区别开环境差异很大的地区,加上δ13C‰后,可将温差大的地区区别开,但对环境相似的地区仍无法有效鉴别,最后将(D/H)Ⅱ、R、δ13C‰、δ18O‰同时考虑,对于环境相似的地区也可以有效鉴别。研究结果为中国葡萄酒原产地鉴别提供了一种新的技术手段。     

Correlation between isotopic and meteorological parameters in Italian wines: a local-scale approach

BACKGROUND: Since the beginning of the 1980s deuterium nuclear magnetic resonance and carbon‐13 mass spectrometry have proved to be reliable techniques for detecting adulteration and for classifying natural products by their geographic origin. Scientific literature has so far mainly focused on data acquired at regional level where isotopic parameters are correlated to climatic mean data relative to large territories. RESULTS: Nebbiolo and Barbera wine samples of various vintages and from different areas within the Piedmont region (northern Italy) were analysed using SNIF‐NMR and GC‐C‐IRMS and a large set of meteorological parameters were recorded by means of weather stations placed in fields where the grapes were grown. Correlations between isotopic (²H and ¹³C) data and several climatic parameters at a local level (mean temperature, total rainfall, mean humidity and thermal sums) were attempted and some linear correlations were found. CONCLUSIONS: Mean temperature and total rainfall were found to be correlated to isotopic (²H and ¹³C) abundance in linear direct and inverse proportions respectively. Lower or no correlations between deuterium and carbon‐13 abundances and other meteorological parameters such as mean humidity and thermal sums were found. Moreover, wines produced from different grape varieties in the same grape field showed significantly different isotopic values. Copyright © 2011 Society of Chemical Industry     

Isotopic and Elemental Authenticity Markers: a Case Study on Cypriot Wines

This study monitors variations in isotopes and elements in relation to grape variety, environmental factors and provenance in order to address the wine authenticity issue. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) assessed the wines’ elemental content. Site-specific nuclear isotope fractionation-nuclear magnetic resonance and isotope ratio mass spectrometry methodologies determined in authentic and commercial wines the distribution of the naturally occurring stable isotopes of the deuterium/hydrogen (D/H) ratios and carbon (¹³C/¹²C) in ethanol of wine and oxygen ratio (¹⁸O/¹⁶O) in wine water. Chemometrics delineated the elements and isotopes responsible for wine classification. Specifically, unsupervised principal component analysis (PCA) framed the importance of grape variety and provenance, while supervised analysis pinpointed the vineyard effect and highlighted the contribution of the vintage year. Validation steps ensured that the extracted models do not predict randomly and their results are reliable. In fact, the acquired results can be incorporated to the EU Wine Isotopic Databank database providing both a guide and a tool for eventual candidatures for denomination of origin and support both Cypriot wine and winemakers. In this context, this research contributes to authenticity assurance of wines and adds value to final products, while it helps controlling hazards arising from environmental changes.     

δ18O of Ethanol in Wine and Spirits for Authentication Purposes

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

C、H、O同位素分析在葡萄酒产区鉴别中的应用

为有效鉴别产区葡萄酒,利用点特异性天然同位素分馏核磁共振技术和同位素比质谱仪技术对葡萄酒中C、H和O同位素进行研究。通过收集2010-2012年河北昌黎、山东烟台蓬莱、宁夏贺兰山东麓和河北沙城四大产区的60 个样品,发现（D/H）i、（D/H）ii、R、δ13C、δ18O、酒精体积分数的范围分别为95.68×10－6～99.15×10－6、115.99×10－6～126.39×10－6、2.38～2.59、－28.35‰～－23.00‰、－1.94‰～－0.05‰和11.3%～12.9%,总体上含量范围分布广泛,但同一地区较为集中。研究发现,单一使用任何一种元素都只能区分环境差异很大的地区,当结合3 种元素,采用线性判别分析能100%有效鉴别四大产区葡萄酒。基于沙城产区的数据库和酒精体积分数,结合t检验法,分3 步可判定出产区酒的真实性。待测的5 个盲样中只有1 个样品属于沙城产区的真实葡萄酒。     

Progress in wine authentication: GC–C/P–IRMS measurements of glycerol and GC analysis of 2,3-butanediol stereoisomers

The determination of glycerol and 2,3-butanediol by photometric or enzymatic methods is well established. This paper reports on the direct assessment of glycerol and stereoselective analysis of 2,3-butanediol isomers in wine using capillary GC without any derivatisation. A “model wine” and commercially available wines, as well as wines of definite origin were investigated. The contents of glycerol and 2,3-butanediol and the ratio of (R,R)- and meso-2,3-butanediol were determined. Capillary GC has proved to be a reliable alternative in glycerol assessment from wine, thus a GC-IRMS method for authenticity assessment of glycerol was developed.δ¹³C_V‐PDB and δ¹⁸O_V‐SMOW multi-element IRMS-analysis of glycerol, an important by-product of wine fermentation, is reported. For that reason glycerol, extracted from a self-prepared “model wine”, from wines of definite origin, as well as commercially available wines, was investigated. Furthermore, the ¹⁸O/¹⁶O and ¹³C/¹²C isotope ratios of commercial glycerols from different origins were determined. In addition fermentation experiments with beet and cane sugar, and with grape juices were carried out. In order to check the influence of water on the oxygen isotope ratios, water from different places in Germany was used in the fermentation experiments with cane and beet sugar.     

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

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

Analysis of isotopic ratios for the detection of illegal watering of beverages

The applications of the stable isotope ratio analyses (D/H, ¹³C and ¹⁸O) in the assessment of watering in beverages such as wine and fruit juices are discussed in this study. The measurement of ¹⁸O/¹⁶O ratio in combination with D/H and ¹³C/¹²C ratio is mainly focused and the international official methods implementing stable isotope techniques will be mentioned. Furthermore, the ongoing study on a possible use of ¹³C/¹²C ratio of CO₂ to detect the addition of technical produced CO₂ in mineral sparkling waters is also presented.     

SNIF-NMR和IRMS技术在葡萄酒质量评价中的初步研究

利用SNIF-NMR和IRMS技术测定葡萄酒中稳定性同位素D/H和18O/16O的比值。结果表明,通过测定乙醇分子中甲基位(D/H)Ⅰ和次甲基位点(D/H)Ⅱ的含量,可判断葡萄酒在酿造前是否进行了加糖操作,而通过测定葡萄酒水分中18O/16O的比值,可鉴别全汁葡萄酒和半汁葡萄酒。研究结果为中国葡萄酒的质量评价提供了一种新的技术手段。     

Correlations of carbon isotope ratios of wine ingredients for the improved detection of adulterations I. Organic acids and ethanol

The δ¹³C-values of organic acids and their correlations to those of the sugar and ethanol, respectively, from 57 EU data bank wines of the Rheinpfalz area (years 1991?–?1993) and from some of their corresponding musts have been determined. In addition to the well established difference between fermented sugar and ethanol (Δδ¹³C = –1.7±0.2‰), a new constant correlation was found in wine for ethanol and citric acid (Δδ¹³C = +2.4±0.4‰). From this result a fixed δ-value difference for citric acid in wine to the fermented sugar of +0.7±0.6‰ can be deduced. The δ¹³C-values of L-malic acid and L-tartaric acid in must were not altered by the alcoholic fermentation; they should therefore directly provide access to the δ¹³C-value of the natural sugar in must. However, in non-adulterated wines the expected δ¹³C-value differences between these acids and ethanol showed unsatisfactory correlation coefficients. For L-malate this is attributed to the secondary (partial) degradation of this acid by the malolactic fermentation; a corresponding correction is envisaged in order to make L-malate available as an internal standard. As a reason for the unsatisfactory correlation between L-tartaric acid and ethanol, it is supposed that the time of its maximum biosynthesis period does not coincide with that of glucose in the grape ripening period.     

Stable Isotope Ratios and Aroma Profile Changes Induced Due to Innovative Wine Dealcoholisation Approaches

The high ethanol level in wine has become an important issue for all the main wine producing countries. Several techniques are available to the wine industry to reduce the ethanol content; among them, the membrane contactors are certainly one of the newest. Very few studies on the effect of this practice on the wine quality and aroma profile and on the stable isotopes composition are available. A pilot and industrial plant equipped with the membrane contactor system were used in the study in the dealcoholisation on several white and red wines. Significant changes for several classes of aroma compounds in both pilot- and industrial-scale experiments were observed, even though these changes were not always in perfect agreement with the sensory evaluation carried out. Finally, modifications on the δ¹⁸O of up to 1?‰ for 2 %?v/v and of up to 4?‰ for 8 %?v/v ethanol removal were encountered. An increase of δ¹³C of ethanol of up to 1.1?‰ for 2 % and of up to 2.3?‰ for 4 % of dealcoholisation rate was also observed. Dealcoholisation via membrane contactor seemed to affect the overall wine composition (aroma and flavour), even though the main concern resided on the alteration of the isotopic composition which could be linked to product authenticity issues.     

Detection of counterfeit scotch whisky by H and O stable isotope analysis

Neat Scottish whiskies and the local source water used for and during whisky production have been analysed for their ²H and ¹⁸O isotopic composition by High Temperature Conversion–Isotope Ratio Mass Spectrometry (TC/EA–IRMS). With coefficients of determination R² for δ²H and δ¹⁸O of 0.71 and 0.88, respectively, measured δ²H and δ¹⁸O values for water used in the production of Scottish whisky were found to be well correlated with the δ²H and δ¹⁸O values observed for the corresponding whiskies. By plotting bulk ²H against bulk ¹⁸O isotope abundance data of authentic whisky and counterfeit whisky samples it was possible to discriminate between samples of authentic whisky and samples of counterfeit whisky.     

酿造食醋中碳、氧同位素比值的测定及应用研究

以酿造食醋为研究对象,建立元素分析/连续流-稳定同位素比质谱法（EA/GasBench Ⅱ-IRMS）测定食醋总碳、水中氧同位素比值（δ13C和δ18O）的方法。通过优化稀释倍数与进样体积,得到δ13C值测定最佳条件为食醋稀释2倍,进样体积1.0 μL;通过优化平衡时间和样品体积,得到δ18O值测定时平衡时间为24 h,样品体积为500 μL。结果表明,在最佳条件下,食品样品碳同位素比测定值标准偏差（SD）值均＜0.30‰,氧的同位素比测定值的SD值均＜0.10‰,表明该测定方法的稳定性较好。山西食醋总碳δ13C值分布在-23.26‰～-20.80‰,水中氧的δ18O值在-5.66‰～-4.49‰;镇江食醋总碳δ13C值在-25.93‰～-20.70‰,水中氧的δ18O值在-8.35‰～-5.61‰;结合碳氧同位素比值分析,可以将山西老陈醋、镇江香醋和镇江陈醋区分开（P＜0.01）。     

Characterization of Swiss vineyards using isotopic data in combination with trace elements and classical parameters

The objective of this work was to study methods to check the geographical origin of Swiss wines. Authentic grape samples from the vintages 2000 and 2001 were collected in representative locations, and the wines produced from these samples were subjected to isotopic (²H/¹H_ethanol; ¹⁸O/¹⁶O_{wine water}), elemental (Mn, Al, B, Ba, Ca, Fe, Mg, Na, Rb, Sr, Zn) and FT-IR (ethanol, pH, total acidity, volatile acidity, malic acid, fructose, tartaric acid, lactic acid, succinate, citric acid, glycerol, 2,3-butandiol, dry matter and relative density (d₂₀)) analyses. The results showed that the variables ¹⁸O/¹⁶O_{wine water}, strontium, rubidium, and ethanol%, when considered in a monovariate approach, allowed the best discrimination of the geographical origins of Swiss wines. By using a combination of all variables (multivariate approach), Switzerland was divided in four main production zones with a separation power that has never before been achieved. These zones were defined as Tessin, Valais, Romandie and Alemanique. A fifth zone (Graubünden) could be evidenced in vintage 2001.     

Comparison between isotope ratio mass spectrometry (IRMS) and cavity ring-down spectroscopy (CRDS) for analysing the carbon isotope ratio and detection of adulteration in coconut water

This study investigated the carbon isotope ratio (¹³C/¹²C, δ¹³C in unit of parts per thousand or per mill, ‰) of different coconut water samples using elemental analysis isotope ratio mass spectrometry (EA-IRMS) and combustion module cavity ring-down spectroscopy (CM-CRDS). Natural coconut water from young coconuts from 12 provinces in Thailand (n = 94), adulterated coconut water containing three sweeteners (sucrose, glucose and fructose) at different concentrations and eight brands of commercial coconut water were analysed. The δ¹³C of all samples were not significantly different (P > 0.05) between the EA-IRMS and CM-CRDS analyses. The natural coconut water (C3 plant) had δ¹³C ranging from −21.58‰ to −27.79‰ (mean −24.64 ± 0.91‰). Three sweeteners (C4 plant) contained δ¹³C between −11.46‰ to −13.16‰. The use of δ¹³C determination can detect adulteration of a singular extraneous sweetener (either glucose or sucrose) down to a level of 2% of adulteration. For the detected δ¹³C values of commercial products labelled as ‘no added sugar’, about 50% of products were adulterated. This study demonstrates that CM-CRDS can be used as an alternative analytical platform to EA-IRMS for detecting adulterated products, especially coconut water.     

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

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

Stable Isotope Ratio Analysis for Assessing the Authenticity of Food of Animal Origin

The main elemental constituents (H, C, N, O, and S) of bio‐organic material have different stable isotopes (²H, ¹H; ¹³C,¹²C; ¹⁵N,¹⁴N; ¹⁸O,¹⁷O,¹⁶O; ³⁶S, ³⁴S, ³³S, and ³²S). Isotopic ratios can be measured precisely and accurately using dedicated analytical techniques such as isotope ratio mass spectrometry (IRMS). Analysis of these ratios shows potential for assessing the authenticity of food of animal origin. In this review, IRMS analysis of food of animal origin and variability factors related to stable isotope ratios in animals are described. The study also lists examples of application of stable isotope ratio analysis to meat, dairy products, fish, and shellfish and emphasizes the strengths and weaknesses of the technique. Geographical, climatic, pedological, geological, botanical, and agricultural factors affect the stable isotope ratios (SIR) of bio‐elements, and SIR variations are ultimately incorporated into animal tissue through eating, drinking, breathing, and exchange with the environment, being recorded in the resulting foods. SIR analysis was capable of determining geographical origin, animal diet, and the production system (such as organic/conventional or wild/farmed) for pork, beef, lamb, poultry, milk, butter, cheese, fish, and shellfish. In the case of the hard PDO (protected designations of origin) cheeses Grana Padano and Parmigiano Reggiano it is also used in real‐life situations to assess the authenticity of grated and shredded cheese on the market.     

New isotopic criteria for the short-term dating of brandies and spirits

As a result of the atmospheric atomic experiments carried out in the 1960s a method for short-term dating of wines and spirits could be based on the determination of the ¹⁴C radioactivity of ethanol. However, due to the turnover of atmospheric ¹⁴CO₂ this method is no longer applicable to the most recent samples. The aptitude of stable isotopes of carbon and hydrogen to provide new probes for dating brandies such as cognacs and armagnacs is evaluated. The overall carbon isotope ratio of ethanol is measured by isotope ratio mass spectrometry and the site-specific hydrogen parameters of the methyl, (D/H)_I, and methylene, (D/H)_II, sites are determined by the SNIF-NMR method. The isotopic results can be interpreted in terms of fractionation phenomena associated with thermo-kinetic evaporation undergone in the casks during the ageing period. A theoretical estimation of the vintage founded on this isotopic evolution requires the knowledge of the isotope ratios of the starting product. In principle, these values can be estimated on the basis of the meteorological parameters associated with the growing period of the corresponding vine. However, although the combined contributions of climatic effects and of evaporation phenomena conveniently explain the general isotopic behaviour, the influence of oenological practices such as ullage (replacement of the amount of a liquid lost in a cask by evaporation) must also be considered. Further progress towards an unequivocal dating procedure using stable isotope ratios is expected from the access to isotope databanks of the wine precursors. © 1998 SCI.     

Free radical scavenging capacity of selected red,rosé and white wines

The free radical scavenging capacity of selected red, rosé and white Spanish wines from different grape varieties was determined by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^·) method using a new methodology developed at our laboratory. The amount of sample necessary to decrease by 50% the initial DPPH^· concentration (EC₅₀), the time needed to reach the steady state at EC₅₀ concentration (T_EC50) and the antiradical efficiency (AE = 1/EC₅₀T_EC50) were determined in the wine samples. Some differences between rosé wines made with Garnacha and Tempranillo grape varieties were observed in the UV‐vis spectra and in the free radical scavenging parameters, those from Garnacha variety having the highest antioxidant activity. The antiradical efficiency of the wines followed a decreasing order: red wines (22.44 × 10⁻⁶) > rosé wines (4.90 × 10⁻⁶) > white wines (1.88 × 10⁻⁶). There was a correlation between antiradical efficiency and total polyphenol (TP): AE = −3.33135 + 0.0180535TP; the correlation coefficient was r = 0.951454. © 1999 Society of Chemical Industry     

Authentication of tequila by gas chromatography and stable isotope ratio analyses

Gas chromatographic (GC) determination of volatile constituents and isotope ratio mass spectrometry (IRMS) analysis of ¹³C/¹²C isotope ratios as well as SNIF-NMR analysis of (D/H)-ratios of ethanol in authentic (n=12) and commercial tequila samples (n=13) were used to differentiate analytically between tequila derived from 100% agave (Agave tequilana Weber var. Azul) and tequila produced with other fermentable sugars ('mixed' tequila). Evaluating the correlation of methanol and 2-/3-methyl-1-butanol concentrations, GC analysis was found to be a suitable method for the authenticity assessment of '100% agave' and 'mixed' tequilas. Additional determinations of ¹³C_VPDB and (D/H) ratios of ethanol were used to show the perspectives and limits of the methods.