Study of the influence of alcoholic fermentation and distillation on the oxygen-18/oxygen-16 isotope ratio of ethanol

A laboratory procedure for the analysis of the oxygen-18/oxygen-16 isotope ratios of ethanol derived from sugars and fruit juices by pyrolysis-isotope ratio mass spectrometry (IRMS) has been applied to the study of isotopic fractionation induced by the isotope effects of fermentation and distillation. For both processes, an experimental model has been established to describe and explain the observed fractionation phenomena. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. Moreover, the ability of ethanol to act as a reliable indicator of the (18)O/(16)O ratio of sugars in orange juice (and therefore to be used as an internal reference for detecting water addition) is demonstrated both in theory and in practice.     

Improved detection of added water in orange juice by simultaneous determination of the oxygen-18/oxygen-16 isotope ratios of water and ethanol derived from sugars

A procedure for the analysis of the oxygen-18/oxygen-16 isotope ratio of ethanol derived from the sugars of orange juice using the preparation steps of the SNIF-NMR method followed by pyrolysis-isotope ratio mass spectrometry is presented. The isotopic fractionation induced by the isotope effects of fermentation and distillation have been investigated, and it is shown that reproducible results can be obtained when appropriate analytical conditions are used. It is also shown that the oxygen isotope distribution in the water and organic matter pools of fruits remains quite stable during the harvest period and is not altered by the precipitation rate within the last few days before the fruits are picked. Due to the robustness of the method and the fact that most of the oxygen-18 enrichment from the initial sugars is still present in the end-product, ethanol appears as a convenient internal reference to circumvent the spatial and temporal variability observed for the oxygen-18/oxygen-16 isotope ratio of water. A very strong correlation is observed between the isotopic deviations of ethanol and water, which is altered in the event of a water addition, even at a low level. Combining the information brought by these two parameters leads to a more efficient authenticity testing tool, which avoids false positive cases and provides a lower detection limit for added water in juices not made from concentrate, whatever the origin of the sample tested.     

Determination of authenticity, regional origin, and vintage of Slovenian wines using a combination of IRMS and SNIF-NMR analyses

The authenticity and geographical origin of wines produced in Slovenia were investigated by a combination of IRMS and SNIF-NMR methods. A total of 102 grape samples of selected wines were carefully collected in three different wine-growing regions of Slovenia in 1996, 1997, and 1998. The stable isotope data were evaluated using principal component analysis (PCA) and linear discriminant analysis (LDA). The isotopic ratios to discriminate between coastal and continental regions are the deuterium/hydrogen isotopic ratio of the methylene site in the ethanol molecule (D/H)(II) and delta(13)C values; including also delta(18)O values in the PCA and LDA made possible separation between the two continental regions Drava and Sava. It was found that delta(18)O values are modified by the meteorological events during grape ripening and harvest. The usefulness of isotopic parameters for detecting adulteration or watering and to assess the geographical origin of wines is improved only when they are used concurrently.     

有机物对红外光谱技术测定植物叶片和茎秆水δ18O和δD的影响

植物叶片、茎秆和土壤水δ18O和δD是研究土壤植被大气系统生态水文循环过程的重要示踪剂。与传统的稳定同位素质谱(IRMS)技术相比,稳定同位素红外光谱(IRIS)技术具有测量速度快、运行成本低等优势,将促进稳定同位素生态学的发展。但是利用低温真空蒸馏抽提技术获得的植物叶片和茎秆水中含有甲醇和乙醇类有机污染物,造成δ18O和δD的IRIS测量值偏离IRMS测量值(2.64±0.43)‰和(3.6±0.8)‰,超过了仪器精度。本研究利用纯水混入不同浓度的色谱纯甲醇或乙醇,结合Los Gatos公司的光谱分析软件确定甲醇(NB)和乙醇(BB)类物质污染程度的光谱度量值,建立了δ18O和δD的光谱污染校正方法。研究表明,同一台分析仪建立的校正曲线无明显的时间漂移;不同分析仪建立的校正曲线存在显著差异;IRIS校正值与IRMS测量值的交叉验证表明,IRIS测定冬小麦和夏玉米叶片和茎秆水的δ18O和δD可以被准确地校正,与IRMS的差值分别为(0.11±0.12)‰和(0.7±0.4)‰。     

Fingerprints for main varieties of argentinean wines: terroir differentiation by inorganic, organic, and stable isotopic analyses coupled to chemometrics

Our main goal was to investigate if robust chemical fingerprints could be developed for three Argentinean red wines based on organic, inorganic, and isotopic patterns, in relation to the regional soil composition. Soils and wines from three regions (Mendoza, San Juan, and Co?rdoba) and three varieties (Cabernet Sauvignon, Malbec, and Syrah) were collected. The phenolic profile was determined by HPLC-MS/MS and multielemental composition by ICP-MS; (87)Sr/(86)Sr and δ(13)C were determined by TIMS and IRMS, respectively. Chemometrics allowed robust differentiation between regions, wine varieties, and the same variety from different regions. Among phenolic compounds, resveratrol concentration was the most useful marker for wine differentiation, whereas Mg, K/Rb, Ca/Sr, and (87)Sr/(86)Sr were the main inorganic and isotopic parameters selected. Generalized Procrustes analysis (GPA) using two studied matrices (wine and soil) shows consensus between them and clear differences between studied areas. Finally, we applied a canonical correlation analysis, demonstrating significant correlation (r = 0.99; p < 0.001) between soil and wine composition. To our knowledge this is the first report combining independent variables, constructing a fingerprint including elemental composition, isotopic, and polyphenol patterns to differentiate wines, matching part of this fingerprint with the soil provenance.     

Authentication of natural vanilla flavorings: isotopic characterization using degradation of vanillin into guaiacol

The isotopic investigation of vanillin has been extended to the new sources of natural precursors of vanillin recently introduced with a view of obtaining natural vanillin by biotechnological processes. To check the consistency of the isotopic composition of vanillin with that of the corresponding aromatic fragment, a selective degradation reaction into guaiacol was carried out. The reaction was shown to proceed without significant isotopic fractionation at the sites of interest, and an optimized procedure was defined from the results of an experimental design involving the quantity of reagent and the temperature and duration of the experiment. Guaiacol, which can be easily obtained in a reasonable time, is an interesting isotopic probe for carbon- and oxygen-isotope ratio mass spectrometry (IRMS) determinations. It provides (13)C information specific to the aromatic fragment and, combined with delta(13)C values measured on vanillin itself, it improves the authentication potential of carbon-IRMS. Thus, the natural status of ferulic acid may be characterized by significant (13)C depletion at the formyl site. Similarly, the oxygen-18 content of guaiacol is a better authentication tool than delta(18)O of vanillin because it does not suffer the drawback of being altered by chemical exchange of the sp(2) oxygen atom with water in industrial or laboratory procedures. Although collaborative studies are still necessary to improve the interlaboratory reproducibility of the delta(18)O parameters, consistent results can be obtained in an intralaboratory context. It is shown in particular that chemical oxidation of ferulic acid is characterized by a relative enrichment of the aromatic moiety of vanillin.     

Isotopic fingerprinting for the authenticity control of crop protection active compounds using the representative insecticide Fipronil

Isotopic fingerprinting was evaluated for its potential to generate characteristic fingerprints of crop protection products in an extensive survey, using the insecticide Fipronil. One hundred and twenty batches of Fipronil from the BASF production site in France were analyzed for the isotope ratios of δ(13)C, δ(15)N, and δ(34)S. Samples spanned a production time of four years and were analyzed by elemental analysis, coupled to isotope ratio mass spectrometry (EA/IRMS). A number of Fipronil samples from other sources were analyzed in the same manner and were compared to the samples from BASF by means of multivariate data analysis. The isotopic fingerprint was sufficiently specific to differentiate between Fipronil from BASF production and Fipronil from other producers. This suggests that isotopic fingerprinting is suitable for the authenticity control of active compounds in crop protection products. It is anticipated that this technique will deliver great benefit in the defense against counterfeits and illegal parallel imports.     

Isotopes as tracers of the Hawaiian coffee-producing regions

Green coffee bean isotopes have been used to trace the effects of different climatic and geological characteristics associated with the Hawaii islands. Isotope ratio mass spectrometry (IRMS) and inductively coupled plasma mass spectrometry ((MC)-ICP-SFMS and ICP-QMS) were applied to determine the isotopic composition of carbon (δ13C), nitrogen (δ15N), sulfur (δ34S), and oxygen (δ18O), the isotope abundance of strontium (87Sr/86Sr), and the concentrations of 30 different elements in 47 green coffees. The coffees were produced in five Hawaii regions: Hawaii, Kauai, Maui, Molokai, and Oahu. Results indicate that coffee plant seed isotopes reflect interactions between the coffee plant and the local environment. Accordingly, the obtained analytical fingerprinting could be used to discriminate between the different Hawaii regions studied.     

Compound specific plant amino acid δ15N values differ with functional plant strategies in temperate grassland

The distribution and natural abundance isotopic (δ¹⁵N) content of whole tissue and individual amino acids in plants in a temperate grassland were determined using ion chromatography (IC), continuous flow‐isotope ratio mass spectrometry (CF‐IRMS), and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS). The results showed that the selected plants (Lolium perenne, Juncus effusus, and Brachythecium rutabulum) differed in their amino acid content and distribution from the parent grassland soil. Bulk and individual amino acid δ¹⁵N isotope signatures were different between the plants, which concurred with their functional strategy in relation to the relative acquisition of available N sources. The individual amino acid δ¹⁵N values of histidine and phenylalanine could be used to differentiate between the three plant species.     

Application of multielement stable isotope ratio analysis to the characterization of French, italian, and spanish cheeses

The stable isotope ratios (delta13C, delta15N, and delta34S of casein and delta13C and delta18O of glycerol) measured by IRMS of French, Italian, and Spanish cheeses are presented and discussed. Variability factors such as animal-feeding regimen, geographical origin, and climatic and seasonal conditions were studied to check the possibilities of cheese characterization offered by each isotopic parameter. Delta13C values of both casein and glycerol appeared to be strongly correlated to the amount of maize in the animal diet. Delta15N and delta34S of casein proved to be mostly influenced by the geoclimatic conditions of the area (aridity, closeness to the sea, altitude). Delta18O of glycerol was more dependent on the geographical origin of the cheeses and on climatic/seasonal parameters. By applying a multivariate stepwise canonical discriminant analysis, good discrimination possibilities for the different European cheeses were obtained, confirmed by the classification analysis, when >90% of the samples were correctly reclassified.     

花生油生产过程中稳定同位素变化规律及影响因素研究

花生油的稳定同位素特征研究是该技术应用于花生油掺假检测的基础,为探究花生油生产过程中碳、氢、氧的稳定同位素比值变化规律及其受产地、年份的影响,本试验收集了3个年份6个省份的花生样品,利用稳定同位素比值质谱仪(IRMS)测定油脂中3种元素的稳定同位素比值(δ¹³C、δ²H和δ¹⁸O)。结果表明,不同产地花生样品中,油脂的δ¹³C、δ²H和δ¹⁸O值均不相同。以δ¹³C为例,平均值由大到小依次为湖北>辽宁>广东>山东>河南>江苏,部分省份之间差异显著(P<0.05);因年份不同油脂稳定同位素特征也存在差异。另外,研究结果表明不同生产阶段的油脂中稳定同位素特征几乎一致,加工工艺的差异未改变油脂的稳定同位素分馏。可见,油脂中保留了花生的稳定同位素特征。本研究结果为花生油掺假检测技术研究提供了理论基础与应用可行性。     

Detection of exogenous citric acid in fruit juices by stable isotope ratio analysis

A new method has been developed for measuring the D/H ratio of the nonexchangeable sites of citric acid by isotope ratio mass spectrometry (IRMS). Pure citric acid is transformed into its calcium salt and subsequently analyzed by pyrolysis-IRMS. The citric acid isolated from authentic fruit juices (citrus, pineapple, and red fruits) systematically shows higher D/H values than its nonfruit counterpart produced by fermentation of various sugar sources. The discrimination obtained with this simplified method is similar to that obtained previously by applying site specific isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) to an ester derivative of citric acid. The combination of carbon 13 and deuterium measurements of extracted citric acid is proposed as a routine method for an optimum detection of exogenous citric acid in all kinds of fruit juices.     

Multielement isotope analysis of bovine muscle for determination of international geographical origin of meat

Multielemental (C, N, H, S) stable isotope ratio analysis was used as an analytical tool to verify the geographical origin of beef from several European and non-European countries. Beef samples were collected from nine different countries, and the (13)C/(12)C, (15)N/(14)N, (2)H/(1)H, and (34)S/(32)S ratios of defatted beef were measured using isotope ratio mass spectrometry (IRMS). There were highly significant differences in the mean isotopic values of the beef from different countries. The results of discriminant analysis showed that the four isotope ratios were significant for the discrimination of geographical origin and that 84.9% of the samples were correctly assigned to the country of origin (82.2% when cross-validated). Beef was also classified according to geographical origin when additional information on different feeding regimens used in Ireland was included, with 85.0% of the samples correctly allocated and 82.9% cross-validated using the isotopic signatures. All of the Irish beef samples verifiable as pasture-fed beef were correctly classified and then cross-validated.     

Improved detection of sugar addition to maple syrup using malic acid as internal standard and in 13C isotope ratio mass spectrometry (IRMS)

Stable carbon isotope ratio mass spectrometry (delta13C IRMS) was used to detect maple syrup adulteration by exogenous sugar addition (beet and cane sugar). Malic acid present in maple syrup is proposed as an isotopic internal standard to improve actual adulteration detection levels. A lead precipitation method has been modified to isolate quantitatively malic acid from maple syrup using preparative reversed-phase liquid chromatography. The stable carbon isotopic ratio of malic acid isolated from this procedure shows an excellent accuracy and repeatability of 0.01 and 0.1 per thousand respectively, confirming that the modified lead precipitation method is an isotopic fractionation-free process. A new approach is proposed to detect adulteration based on the correlation existing between the delta13Cmalic acid and the delta13Csugars-delta13Cmalic acid (r = 0.704). This technique has been tested on a set of 56 authentic maple syrup samples. Additionally, authentic samples were spiked with exogeneous sugars. The mean theoretical detection level was statistically lowered using this technique in comparison with the usual two-standard deviation approach, especially when maple syrup is adulterated with beet sugar : 24 +/- 12% of adulteration detection versus 48 +/- 20% (t-test, p = 7.3 x 10-15). The method was also applied to published data for pineapple juices and honey with the same improvement.     

基于氧同位素的玉米农田蒸散发估算和区分

农田蒸散发(evapotranspiration,ET)的估算和区分是土壤-植物-大气连续体中的重要研究内容,是农业水资源高效利用的重要基础。该研究分析了土壤水、蒸发水汽、蒸腾水汽和大气背景混合水汽氧同位素组成分布特征,并采用2种同位素的方法对玉米农田蒸散发进行估算和区分:1)结合Keeling plot和Craig-Gordon模型的同位素方法(Iso-CG);2)基于土壤水同位素守恒和水量平衡的方法(Iso-WB)。结果表明,在玉米生育期内Iso-WB方法与Iso-CG方法所计算的玉米蒸腾比例分别为0.64~0.91和0.52~0.91,平均值分别为0.80和0.78。玉米蒸散发总量在前期、中期和后期均值分别为3.95、5.30和4.98 mm/d。通过比较参数并与前人研究结果对比分析,表明采用Iso-CG方法估算区分ET相对精确,采用Iso-WB方法计算蒸散发要求的测量精度相对较高,计算误差较大。该研究成果不仅为玉米农田制定灌溉制度及提高用水效率提供了理论依据,而且对深入探索氧同位素水文学领域具有重要意义。     

陕西关中地区肉牛产地同位素溯源技术初探

为应用牛组织中C、N同位素组成的区域分布和同位素指标溯源肉牛产地,本文利用同位素比率质谱仪(IRMS)对陕西关中不同区县来源的牛尾毛样品的1δ3C和1δ5N值进行检测,通过聚类分析,研究陕西关中地区肉牛组织中同位素组成的区域分布情况。结果表明,杨凌区、眉县牛尾毛样品聚为一类;乾县、永寿县样品聚为一类;麟游县样品聚为一类;扶风县、岐山县和凤翔县分类不十分明显,分散在上述3类中。说明陕西关中不同地区肉牛组织中同位素组成存在差异,利用它们可进行肉牛地域的小范围溯源。     

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.     

Geography and vintage predicted by a novel GIS model of wine delta18O

Wine hydrogen and oxygen stable isotopes record the climatic conditions experienced by the grape vine and the isotopic composition of the vine's source water during berry development. As such, stable isotopes have been explored extensively for use in detecting wine adulteration or for independently verifying claims of origin. We present the results of a study designed to evaluate the relationships between wine water delta18O and spatial climate and precipitation delta18O patterns across the winegrape-growing regions of Washington, Oregon, and California. Retail wines produced from typically small vineyards across these regions were obtained from the 2002 vintage, and the delta18O of wine water was analyzed using a CO2 equilibration method. Significant correlations were observed between the measured wine water delta18O from 2002 and the long-term average precipitation delta18O and late season 2002 climate, based on a spatial join with continuous geographic information system (GIS) maps of these drivers. We then developed a regression model that was implemented spatially in a GIS. The GIS model is the first of its kind and allows spatially explicit predictions of wine delta18O across the region. Because high spatial resolution monthly climate layers are now available for many years, wine delta18O could be modeled for previous years. We therefore tested the model by executing it for specific years and comparing the model predictions with previously published results for wine delta18O from seven vintages from Napa and Livermore Valleys, California. With the exception of one year, an anomaly potentially related to the effects of El Ni?o on precipitation isotopic composition, the model predicted well the wine delta18O for both locations for all vintages and generally reflected the consistent enrichment of wine from Napa relative to Livermore. Our results suggest that wine water delta18O records both source water delta18O and climate during the late stages of winegrape ripening and that GIS models of wine water delta18O are useful tools for independently verifying claims of regional origin and vintage.     

蒸发与降水入渗过程中不同水体氢氧同位素变化规律

为了研究土壤水蒸发与降水入渗非饱和带过程中不同水体氢氧同位素的变化规律,该文选用2种不同性质的土壤-砂土和黄土,设计了土壤水蒸发和降水入渗室内试验。结果表明:对于砂土,土壤水蒸发过程中剩余水体氢氧同位素分馏遵从瑞利模式;对于黄土,随着蒸发时间的延长,剩余土壤水氢氧同位素值越来越远离瑞利分馏关系线;在降水入渗非饱和带的初期,相对于风干砂土,风干的黄土颗粒对土柱出流水的氢氧同位素值产生了影响,并且出流水的氢氧同位素亦受到土壤原水同位素值的影响,只有入渗的降水达到了一定数量,土柱出流水才能与降水的同位素值相同。该研究可为运用氢氧同位素研究降水能否通过非饱和带补给地下水提供数据分析的依据。     

Authentication of farmed and wild turbot (Psetta maxima) by fatty acid and isotopic analyses combined with chemometrics

Fatty acid composition and stable isotope ratios of carbon (delta(13)C) and nitrogen (delta(15)N) were determined in muscle tissue of turbot (Psetta maxima). The multivariate analysis of the data was performed to evaluate their utility in discriminating wild and farmed fish. Wild (n=30) and farmed (n=30) turbot of different geographical origins (Denmark, The Netherlands, and Spain) were sampled from March 2006 to February 2007. The application of linear discriminant analysis (LDA) and soft independent modeling of class analogy (SIMCA) to analytical data demonstrated the combination of fatty acids and isotopic measurements to be a promising method to discriminate between wild and farmed fish and between wild fish of different geographical origin. In particular, IRMS (Isotope Ratio Mass Spectrometry) alone did not permit us to separate completely farmed from wild samples, resulting in some overlaps between Danish wild and Spanish farmed turbot. On the other hand, fatty acids alone differentiated between farmed and wild samples by 18:2n-6 but were not able to distinguish between the two groups of wild turbot. When applying LDA isotope ratios, 18:2n-6, 18:3n-3, and 20:4n-6 fatty acids were decisive to distinguish farmed from wild turbot of different geographical origin, while delta(15)N, 18:2n-6, and 20:1n-11 were chosen to classify wild samples from different fishing zones. In both cases, 18:2n-6 and delta(15)N were determinant for classification purposes. We would like to emphasize that IRMS produces rapid results and could be the most promising technique to distinguish wild fish of different origin. Similarly, fatty acid composition could be used to easily distinguish farmed from wild samples.