The 'LSVEC problem' for the Vienna Peedee belemnite carbon isotope-delta scale.

Since 2016 there have been intense discussions within several established communities and networks regarding the consequences of the withdrawal of the LSVEC lithium carbonate reference material for carbon isotope-delta measurement calibration. These include the Isotope Ratio Working Group (IRWG) of the Consultative Committee for the Amount of Substance (CCQM, French: Comité Consultatif pour la Quantité de Matière) of the International Committee of Weights and Measures (CIPM, French: Comité International des Poids et Mesures); participants within the 2016, 2021 and 2024 Technical Meetings on International Atomic Energy Agency (IAEA) stable isotope ratio reference products; and the International Union of Pure and Applied Chemistry's Commission on Isotopic Abundances and Atomic Weights (IUPAC CIAAW) amongst others. The reasons for these intensive discussions relate to the privileged position that LSVEC has occupied for traceability of carbon isotope-delta measurements relative to Vienna Peedee belemnite (VPDB) and the potential implications of its withdrawal for all current and future measurements of carbon isotope-delta values. This perspective summarises the background information regarding the LSVEC material in the context of the VPDB carbon isotope-delta scale. The various proposals to address the LSVEC problem that have been discussed within those committees and networks are also presented. The information herein was used as a foundation for discussions at the 2024 IAEA Consultancy Meetings on the Development of IAEA Stable Isotope Reference Materials and Related Products and on the Definition and Realisation of the Isotopic Delta Scales for Light Elements (22-26 January 2024, Vienna, Austria). A summary of those 2024 meetings and associated outcomes can be found elsewhere.

Fatty Acid and Multi-Isotopic Analysis (C, H, N, O) as a Tool to Differentiate and Valorise the Djebel Lamb from the Mountainous Region of Tunisia.

The objective of this study was to distinguish between the Tunisian Djebel lamb meat and meat from typical Tunisian production systems (PSs) through the fatty acids (FAs) profile and the stable isotope ratio analysis (SIRA). Thirty-five lambs from three different regions and PSs (D = Djebel, B = Bou-Rebiaa, and O = Ouesslatia) were considered for this purpose. The results demonstrated that the PS and the geographic origin strongly influenced the FA profile of lamb meat. It was possible to discriminate between the Djebel lamb meat and the rest of the dataset thanks to the quantification of the conjugated linoleic acids (CLA) and the branched chain FAs. Moreover, statistically different concentrations of saturated, monounsaturated and polyunsaturated FAs and a different n-6/n-3 ratio were found for grazing (D and BR) and indoor (O) lambs, making it possible to discriminate between them. As for the stable isotope ratio analysis, all parameters made it possible to distinguish among the three groups, primarily on the basis of the dietary regimen (δ(13C) and δ(15N)) and breeding area (δ(18O) and δ(2H)).

Characterization of Beef Coming from Different European Countries through Stable Isotope (H, C, N, and S) Ratio Analysis.

The need to guarantee the geographical origin of food samples has become imperative in recent years due to the increasing amount of food fraud. Stable isotope ratio analysis permits the characterization and origin control of foodstuffs, thanks to its capability to discriminate between products having different geographical origins and derived from different production systems. The Framework 6 EU-project "TRACE" generated hydrogen (2H/1H), carbon (13C/12C), nitrogen (15N/14N), and sulphur (34S/32S) isotope ratio data from 227 authentic beef samples. These samples were collected from a total of 13 sites in eight countries. The stable isotope analysis was completed by combining IRMS with a thermal conversion elemental analyzer (TC/EA) for the analysis of δ(2H) and an elemental analyzer (EA) for the determination of δ(13C), δ(15N), and δ(34S). The results show the potential of this technique to detect clustering of samples due to specific environmental conditions in the areas where the beef cattle were reared. Stable isotope measurements highlighted statistical differences between coastal and inland regions, production sites at different latitudes, regions with different geology, and different farming systems related to the diet the animals were consuming (primarily C3- or C4-based or a mixed one).

Insights into the stable isotope ratio variability of hybrid grape varieties: a preliminary study.

BACKGROUND: Official stable isotope databases, based on the analysis of (D/H)I ethanol , (D/H)II ethanol , δ13 Cethanol and δ18 Owater of wine, are an indispensable tool for establishing the limits beyond which the mislabeling or the addition of sugar and/or water in wine production can be detected. The present study investigates, for the first time, whether the use of hybrid varieties instead of European Vitis vinifera for wine production can have an impact on the stable isotope ratios. RESULTS: The analyses were performed by isotope ratio mass spectrometry and site-specific natural isotope fractionation by nuclear magnetic resonance, in accordance with the official methods of the International Organization of Grapes and Wine. The comparison shows the tendency of some stable isotope ratios of hybrid varieties, in particular (D/H)I , to deviate from the regional averages of the V. vinifera samples. Notably, Baron, Monarch and Regent showed significantly different values at one of the two sampling sites. Particularly high δ13 C values characterize Helios compared to other hybrid varieties. CONCLUSION: For the first time, and from an isotopic point of view, the present study investigates the wine obtained from hybrid varieties, showing that further attention should be paid to their interpretation, on the basis of the database established according to the European Regulation 2018/273. © 2022 Society of Chemical Industry.

Natural variation in stomatal dynamics drives divergence in heat stress tolerance and contributes to seasonal intrinsic water-use efficiency in Vitis vinifera (subsp. sativa and sylvestris).

Stomata control CO2 uptake for photosynthesis and water loss through transpiration, thus playing a key role in leaf thermoregulation, water-use efficiency (iWUE), and plant productivity. In this work, we investigated the relationship between several leaf traits and hypothesized that stomatal behavior to fast (i.e. minutes) environmental changes co-determines, along with steady-state traits, the physiological response of grapevine to the surrounding fluctuating environment over the growing season. No relationship between iWUE, heat stress tolerance, and stomatal traits was observed in field-grown grapevine, suggesting that other physiological mechanisms are involved in determining leaf evaporative cooling capacity and the seasonal ratio of CO2 uptake (A) to stomatal conductance (gs). Indeed, cultivars that in the field had an unexpected combination of high iWUE but low sensitivity to thermal stress displayed a quick stomatal closure to light, but a sluggish closure to increased vapor pressure deficit (VPD) levels. This strategy, aiming both at conserving water under a high to low light transition and in prioritizing evaporative cooling under a low to high VPD transition, was mainly observed in the cultivars Regina and Syrah. Moreover, cultivars with different known responses to soil moisture deficit or high air VPD (isohydric versus anisohydric) had opposite behavior under fluctuating environments, with the isohydric cultivar showing slow stomatal closure to reduced light intensity but quick temporal responses to VPD manipulation. We propose that stomatal behavior to fast environmental fluctuations can play a critical role in leaf thermoregulation and water conservation under natural field conditions in grapevine.

Simultaneous evaluation of the enantiomeric and carbon isotopic ratios of Cannabis sativa L. essential oils by multidimensional gas chromatography.

Recent times have witnessed an upsurge of interest in hemp and hemp-derived products, as driven by the scientific findings specific to the pharmacological properties of Cannabis sativa L. and its constituents. There has been evidence that the terpene profile, along with the cannabinoid content, produces in humans the effects associated with different strains, beyond fragrance perception. A great deal of effort has been put into developing analytical approaches to strengthen the scientific knowledge on cannabis essential oil composition and provide effective tools for ascertaining the authenticity of commercial cannabis samples. For this concern, enantio-selective-GC-C-IRMS has proven to be effective for assessing the ranges characteristic of the genuine samples and detecting any fraudulent additions. This research aimed at providing for the first time the enantiomeric and isotopic ratios of target terpenes in cannabis essential oils, obtained from microwave-assisted hydro-distillation from the fresh and dried inflorescences of different cannabis varieties. Implementing multidimensional gas chromatography separation was mandatory prior to detection, in order to obtain accurate δ13C values and enantiomeric data from completely separated peaks. For this purpose, a heart-cut method was developed, based on the coupling of an apolar first dimension column to a secondary chiral cyclodextrin-based stationary phase. Afterwards, the data gathered from enantio-selective-MDGC-C-IRMS/qMS analysis of a set of genuine samples were used to evaluate the quality of nineteen commercial cannabis essential oils purchased from local stores. Remarkably, the data in some cases evidenced enantiomeric ratios and δ13C values outside the typical ranges of genuine oils. Such findings suggest the usefulness of the method developed to ascertain the genuineness and quality of cannabis essential oils.

Nuclear magnetic resonance spectroscopy in extra virgin olive oil authentication.

Extra virgin olive oil (EVOO) is a high-quality product that has become one of the stars in the food fraud context in recent years. EVOO can encounter different types of fraud, from adulteration with cheaper oils to mislabeling, and for this reason, the assessment of its authenticity and traceability can be challenging. There are several officially recognized analytical methods for its authentication, but they are not able to unambiguously trace the geographical and botanical origin of EVOOs. The application of nuclear magnetic resonance (NMR) spectroscopy to EVOO is reviewed here as a reliable and rapid tool to verify different aspects of its adulteration, such as undeclared blends with cheaper oils and cultivar and geographical origin mislabeling. This technique makes it possible to use both targeted and untargeted approaches and to determine the olive oil metabolomic profile and the quantification of its constituents.

Using Bioelements Isotope Ratios and Fatty Acid Composition to Deduce Beef Origin and Zebu Feeding Regime in Cameroon.

The purpose of this study was to address the lack of knowledge regarding the stable isotopic composition of beef from zebu cattle reared in tropical Africa. Sixty beef carcasses belonging to the most common zebu breeds (Goudali, white Fulani, and red Mbororo) were selected and classified according to their subcutaneous fat color (white, cream or yellow). The stable isotope ratios of five bioelements-H, O, C, N, and S-in muscle fractions and the fatty acids composition were analyzed. Zebu meat from Cameroon shows peculiar δ13C values, related to the almost exclusive intake of grazed tropical grasses with photosynthetic cycle C4. It also shows δ2H and δ18O values higher than those reported in other areas of the world and correlated with the isotopic composition of animal drinking water. The white subcutaneous fat ("white type") zebu showed higher δ2H and lower δ13C than the "yellow type", that is correlated with a higher content of polyunsaturated fatty acid (PUFA) and a lower amount of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA). Multielement analysis seems to provide promising results for tracing the regional origin of Cameroon beef and some aspects of the livestock system, such as the nutritional status of the animals.

NMR spectroscopy in wine authentication: An official control perspective.

Wine authentication is vital in identifying malpractice and fraud, and various physical and chemical analytical techniques have been employed for this purpose. Besides wet chemistry, these include chromatography, isotopic ratio mass spectrometry, optical spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, which have been applied in recent years in combination with chemometric approaches. For many years, 2 H NMR spectroscopy was the method of choice and achieved official recognition in the detection of sugar addition to grape products. Recently, 1 H NMR spectroscopy, a simpler and faster method (in terms of sample preparation), has gathered more and more attention in wine analysis, even if it still lacks official recognition. This technique makes targeted quantitative determination of wine ingredients and nontargeted detection of the metabolomic fingerprint of a wine sample possible. This review summarizes the possibilities and limitations of 1 H NMR spectroscopy in analytical wine authentication, by reviewing its applications as reported in the literature. Examples of commercial and open-source solutions combining NMR spectroscopy and chemometrics are also examined herein, together with its opportunities of becoming an official method.

Special Issue "Isotopic Techniques for Food Science".

Today, the analytical verification of food safety and quality together with authenticity and traceability plays a central role in food analysis [...].

Influence of Fermentation Water on Stable Isotopic D/H Ratios of Alcohol Obtained from Concentrated Grape Must.

According to Organisation Internationale de la vigne et du vin (OIV) standards, when analysing the stable isotope ratio of deuterium to hydrogen D/H at the methyl (I) and methylene (II) site of ethanol from concentrated must, a dilution with tap water is needed in order to carry out the alcoholic fermentation. This dilution causes a partial transfer of water hydrogens to the sugar, and this affects the (D/H)I and (D/H)II isotopic values of ethanol, which need to be normalised through specific equations based on the analysis of water δ18O or δ2H. The aim of this study was to evaluate the effectiveness and correctness of these equations experimentally. Grape, cane, and beet sugar, as well as grape must were diluted with water with increasing H and O stable isotope ratios, fermented, and analysed. SNIF-NMR and IRMS techniques were applied following the respective OIV methods. The equations based on the δ2H analysis of the diluted sugar/must solutions proved to be reliable in all the cases, although it is not an OIV standard. When using the equations based on the values of δ18O of the diluted solution, data normalisation was reliable only in cases where the water used for dilution had not undergone isotopic fractionation due, for example, to evaporation. In these cases, δ2H should be analysed.

Milk Authentication: Stable Isotope Composition of Hydrogen and Oxygen in Milks and Their Constituents.

This paper summarises the isotopic characteristics, i.e., oxygen and hydrogen isotopes, of Slovenian milk and its major constituents: water, casein, and lactose. In parallel, the stable oxygen isotope ratios of cow, sheep, and goat's milk were compared. Oxygen stable isotope ratios in milk water show seasonal variability and are also 18O enriched in relation to animal drinking water. The δ18Owater values were higher in sheep and goat's milk when compared to cow milk, reflecting the isotopic composition of drinking water source and the effect of differences in the animal's thermoregulatory physiologies. The relationship between δ18Omilk and δ18Olactose is an indication that even at lower amounts (>7%) of added water to milk can be determined. This procedure once validated on an international scale could become a reference method for the determination of milk adulteration with water.

Validation of the 2H-SNIF NMR and IRMS Methods for Vinegar and Vinegar Analysis: An International Collaborative Study.

This paper presents the results of a collaborative study involving seven laboratories and concerning two samples of wine vinegar, one of apple vinegar and four of balsamic vinegar. The aim of the study was to define standard deviations of repeatability (sr) and reproducibility (sR) for vinegar and balsamic vinegar stable isotope ratios of H (D/H), C (δ13C) and O (δ18O), in order to establish them as fully recognized official standards. Acetic acid was extracted and subjected to (D/H)CH3 and δ13C analysis. δ18O analysis was performed on whole samples. The grape must solution remained after distillation of balsamic vinegar was fermented and the resulting ethanol was subjected to (D/H)I, (D/H)II, R and δ13C analysis. The sr and sR were 0.6 ppm and 1.1 ppm for (D/H)CH3, 0.14‱ and 0.25‱ for δ13C of acetic acid, 0.1‱ and 0.17‱ for δ18O of water, 0.19 ppm and 0.64 ppm for ethanol (D/H)I, 1.14 and 1.31 ppm for (D/H)II, 0.09 and 0.11‱ for δ13C of ethanol. These data are in line with those in the literature or reported in corresponding official methods, and sr and sR of balsamic vinegar are in line with those of vinegar and must.

Can We Discover Truffle's True Identity?

This study used elemental and stable isotope composition to characterize Slovenian truffles and used multi-variate statistical analysis to classify truffles according to species and geographical origin. Despite the fact that the Slovenian truffles shared some similar characteristics with the samples originating from other countries, differences in the element concentrations suggest that respective truffle species may respond selectively to nutrients from a certain soil type under environmental and soil conditions. Cross-validation resulted in a 77% correct classification rate for determining the geographical origin and a 74% correct classification rate to discriminate between species. The critical parameters for geographical origin discriminations were Sr, Ba, V, Pb, Ni, Cr, Ba/Ca and Sr/Ca ratios, while from stable isotopes δ18O and δ13C values are the most important. The key variables that distinguish T.magnatum from other species are the levels of V and Zn and δ15N values. Tuber aestivum can be separated based on the levels of Ni, Cr, Mn, Mg, As, and Cu. This preliminary study indicates the possibility to differentiate truffles according to their variety and geographical origin and suggests widening the scope to include stable strontium isotopes.

Lipid Profiling and Stable Isotopic Data Analysis for Differentiation of Extra Virgin Olive Oils Based on Their Origin.

To differentiate extra virgin olive oils (EVOO) according to the origin of purchase, such as monocultivar Italian EVOO with protected denomination of origin (PDO) and commercially-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of various lipid species by liquid chromatography/mass spectrometry (LC-ESI-MS/MS, LC-ESI-IT-MS) and proton nuclear magnetic resonance analysis (1H-NMR). Many putative chemical markers were extracted as differentiators by uni- and multivariate statistical analysis. Commercially-blended EVOO contained higher concentrations of the majority of minor lipids, including free fatty acids, their alkyl (methyl and ethyl) esters, monoglycerides, and diglycerides, which may be indicative of a higher degree of triglyceride lipolysis in these than in monocultivar PDO EVOO. Triterpenoids and particular TAG species were also found in higher proportions in the samples from the commercially-blended EVOO class, suggesting a possible influence of factors such as the cultivar and geographical origin. The largest differences between the classes were determined for the concentrations of uvaol and oleanolic acid. The results of the analysis by isotopic ratio mass spectrometry (IRMS) were reasonably consistent with the information about the geographical origin declared on the labels of the investigated EVOOs, showing considerable variability, which possibly also contributed to the differences in lipid composition observed between the two investigated classes of EVOO.

Lessons learned from inter-laboratory studies of carbon isotope analysis of honey.

Forensic application of carbon isotope ratio measurements of honey and honey protein to investigate the degree of adulteration with high fructose corn syrup or other C4 plant sugars is well established. These measurements must use methods that exhibit suitable performance criteria, particularly with regard to measurement uncertainty and traceability - low levels of adulteration can only be detected by methods that result in suitably small measurement uncertainties such that differences of 1‰ or less can be reliably detected. Inter-laboratory exercises are invaluable to assess the state-of-the art of measurement capabilities of laboratories necessary to achieve such performance criteria. National and designated metrology institutes from a number of countries recently participated in an inter-laboratory assessment (CCQM-K140) of stable carbon isotope ratio determination of bulk honey. The same sample material was distributed to a number of forensic isotope analysis laboratories that could not participate directly in the metrological comparison. The results from these studies have demonstrated that the majority of participants provided isotope delta values with acceptable performance metrics; that all participants ensured traceability of their results; and that where measurement uncertainties were reported; these were fit-for-purpose. A number of the forensic laboratories only reported precision rather than full estimates of measurement uncertainty and this was the major cause of the few instances of questionable performance metrics. Reporting of standard deviations in place of measurement uncertainties is common practice outside metrology institutes and the implications for interpretations of small differences in isotopic compositions are discussed. The results have also highlighted a number of considerations that are useful for organisers of similar inter-laboratory studies in the future.

Differentiation of wood-derived vanillin from synthetic vanillin in distillates using gas chromatography/combustion/isotope ratio mass spectrometry for δ13 C analysis.

RATIONALE: Typical storage in oak barrels releases in distillates different degradation products such as vanillin, which play an important role in flavour and aroma. The addition of vanillin, as well as other aroma compounds, of different origin is prohibited by European laws. As vanillin samples from different sources have different δ13 C values, the δ13 C value could be used to determine whether the vanillin is authentic (lignin-derived), or if it has been added from another source (e.g. synthetic). METHODS: The δ13 C values for vanillin derived from different sources, including natural, synthetic and tannins, were measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), after diethyl ether addition and/or ethanol dilution. A method for analysing vanillin in distillates after dichloromethane extraction was developed. Tests were undertaken to prove the reliability, reproducibility and accuracy of the method with standards and samples. Distillate samples were run to measure the δ13 C values of vanillin and to compare them with values for other sources of vanillin. RESULTS: δ13 C values were determined for: natural vanillin extracts (-21.0 to -19.3‰, 16 samples); vanillin ex-lignin (-28.2‰, 1 sample); and synthetic vanillin (-32.6 to -29.3‰, 7 samples). Seventeen tannin samples were found to have δ13 C values of -29.5 to -26.7‰, which were significantly different (p < 0.05) from those of the natural and synthetic vanillins. The vanillin δ13 C values measured in distillates (-28.9 to -25.7‰) were mainly in the tannin range, although one spirit (-32.5‰) was found to contain synthetic vanillin. CONCLUSIONS: The results show that synthetic vanillin added to a distillate could be differentiated from vanillin derived from oak barrels by their respective δ13 C values. The GC/C/IRMS method could be a useful tool in the determination of adulteration of distillates.

Determination of the 13C/12C Carbon Isotope Ratio in Carbonates and Bicarbonates by 13C NMR Spectroscopy.

This paper is the first study focused on the innovative application of 13C NMR (nuclear magnetic resonance) spectroscopy to determine the bulk 13C/12C carbon isotope ratio, at natural abundance, in inorganic carbonates and bicarbonates. In the past, 13C NMR spectroscopy (irm-13C NMR) was mainly used to measure isotope ratio monitoring with the potential of conducting 13C position-specific isotope analysis of organic molecules with high precision. The reliability of the newly developed methodology for the determination of stable carbon isotope ratio was evaluated in comparison with the method chosen in the past for these measurements, i.e., isotope ratio mass spectrometry (IRMS), with very encouraging results. We determined the 13C/12C ratio of carbonates and bicarbonates (∼50-100 mg) with a precision on the order of 1‰ in the presence of a relaxation agent, such as Cr(acac)3, and CH313COONa as an internal standard. The method was first applied to soluble inorganic carbonates and bicarbonates and then extended to insoluble carbonates by converting them to Na2CO3, following a simple procedure and without observing isotopic fractionation. Here, we demonstrate that 13C NMR spectroscopy can also be successfully adopted to characterize the 13C/12C isotope ratio in inorganic carbonates and bicarbonates with applications in different fields, such as cultural heritage and geological studies.

Changes in stable isotope ratios in PDO cheese related to the area of production and green forage availability. The case study of Pecorino Siciliano.

RATIONALE: No study has investigated the variations in stable isotope ratios (SIRs) of bioelements within a Protected Designations of Origin (PDO) cheese and few studies have focused on the dietary background of animals. For traceability purposes, it is important to know how and whether these issues affect SIRs in a PDO cheese. METHODS: Thirty-six Pecorino Siciliano cheese samples were collected in three east-Sicilian areas in seasons in which green herbage was present or absent in the diet of the animals. The determination of C, N and S SIRs was performed using an isotope ratio mass spectrometer coupled with an elemental analyser. The H and O SIRs were measured using an isotope ratio mass spectrometer equipped with a thermal conversion elemental analyser pyrolysis unit. RESULTS: The C, N, H, O and S SIRs measured in defatted cheeses were subjected to a multivariate stepwise discriminant analysis to verify if cheeses could be distinguished based on their geographical origin and on the animals' feeding regimen. Sulfur and nitrogen SIRs allowed the best discrimination among the three areas (97.2% correct classification of the cheeses). The discrimination of the feeding system, to check the presence or not of fresh forage in the diet of the animals, correctly classified 86.1% of the cheeses. The C and O SIRs were the most effective parameters. CONCLUSIONS: This study demonstrates that the variability in C, H, O, N and S SIRs can allow discrimination between cheeses produced in a narrow geographical region within a PDO area. This may lead to the search for new tools in authentication studies such as the creation of specific isoscapes. Moreover, this study confirms that SIR determination can also discriminate cheeses obtained from animals fed in stalls from those produced when animals have access to green forage, with benefit in terms of environmental impact, animal welfare and product quality. Copyright © 2017 John Wiley & Sons, Ltd.