Stable Isotope Ratio Analysis for Authentication of Natural Antioxidant Cannabidiol (CBD) from Cannabis sativa.

Cannabidiol (CBD) is a non-psychoactive cannabinoid of Cannabis sativa that exhibits several beneficial pharmacological effects, including anti-inflammatory and antioxidant properties. The molecule can be obtained via extraction from the plant or through a biosynthetic route. The two products have both advantages and disadvantages, thus necessitating the development of methods capable of distinguishing between the two products. In this study, for the first time, the analysis of the stable isotope ratios of oxygen and hydrogen demonstrated high efficiency in the discrimination of CBD of a totally natural origin from that obtained through chemical synthesis. Considering a probability level of 95%, it was possible to identify threshold values for δ2H and δ18O of the totally natural CBD of -215‱ and +23.4‱, respectively. Higher values may indicate a non-entirely natural origin of CBD (i.e., a biosynthetic molecule).

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)).

Stable Isotope Ratio Analysis for the Authentication of Natural Antioxidant Curcuminoids from Curcuma longa (Turmeric).

Curcuminoid complex, a mixture of curcumin, demethoxycurcumin and didemethoxycurcumin, is one of the most popular antioxidants of natural origin, and it has a multitude of other health benefits. It is threatened by the proliferation of counterfeit products on the market containing synthetic curcuminoids whose addition is difficult to identify as they present the three curcuminoid forms in the correct ratios. Consequently, the necessity to detect this fraudulent practice is escalating. Carbon-14 analysis is the most effective available method, but it is also expensive and difficult to implement. This paper describes the first attempt to characterize natural curcuminoids and their synthetic form, used as an adulterant, through the analysis of stable isotope ratios of carbon and hydrogen (expressed as δ13C and δ2H). Carbon values greater than -28.6‱ and hydrogen values greater than -71‱ may indicate the addition of synthetic curcuminoids to the natural ones.

Evaluation of honey authenticity in Lebanon by analysis of carbon stable isotope ratio using elemental analyzer and liquid chromatography coupled to isotope ratio mass spectrometry.

Honey is one of the most valuable sweeteners consumed by humans all over the world. Consequently, it is often a target for adulteration through the addition of different sugar syrups during or after honey production, resulting in a reduction in its nutritive value. For the first time, this study analyzes honey samples of various botanical species collected from different Lebanese regions using element analyzer (EA) and liquid chromatography (LC) coupled with isotope ratio mass spectrometry (IRMS). The δ13 C of bulk honey, its protein fraction, and the main individual sugars (glucose, fructose, disaccharides, and trisaccharide) were determined, in order to characterize and evaluate the authenticity of honey consumed in Lebanon. The results showed that the δ13 C values for bulk honey and its protein range from -26.5‰ to -24.5‰ and from -26.4‰ to -24.7‰, respectively, for authentic samples. δ13 C values for samples adulterated with sugar syrups range from -11.2‰ to -25.1‰ for bulk honey and from -26.6‰ to -23.7‰ for its proteins, with a difference between bulk and protein values between -1 and -8.7‰. Using LC-C-IRMS techniques, the δ13 C of individual sugars provides additional information on the presence of undeclared sugars. We found that all authentic samples had Δδ13 Cf-g and Δδ13 C max values within the naturally occurring range of ±1‰ and ±2.1‰, respectively, while the adulterated samples fall outside the Δδ13 C ranges. The oligosaccharide peak was detected in most adulterated samples.

Stable Isotope Ratios of Herbs and Spices Commonly Used as Herbal Infusions in the Italian Market.

Stable isotope ratio analysis has been widely used for traceability and authenticity purposes in relation to various food commodities, but only in a limited number of herb and spice species. This study explored the stable isotope ratios of carbon, nitrogen, sulfur, oxygen, and hydrogen (δ13C, δ15N, δ34S, δ18O, and δ2H) of 119 herbs and spices belonging to 116 plant species and 57 plant families collected from the Italian market for the first time. The characteristic value ranges of δ13C, δ15N, δ34S, δ18O, and δ2H of the herbs and spices went from -31.0 to -11.6, -4.7 to 12.0, -5.0 to 22.0, 14.7 to 46.0, and -158 to -12‰, respectively. The isotopic profiles within and between common botanical families and their similarity/dissimilarity between herbs and spices belonging to the common botanical families are also discussed here. The results of this exploratory work highlight the possibility of characterizing herbs and spices and suggest widening the scope of the survey through more extensive sampling and focusing on specific plant species.

Characterization of L-theanine in tea extracts and synthetic products using stable isotope ratio analysis.

L-theanine involves a great number of health benefits and dietary supplements containing this molecule are becoming increasingly popular. There is, therefore, a growing need to find ways to discriminate between natural L-theanine extracted from tea leaves and the cheaper, synthetic one obtained using specific bacterial enzymes. A first attempt of stable isotope ratio analysis characterization of the possible synthetic adulterant L-theanine (δ13C of -14.3 ± 1.5‰), obtained from vegetable substrates with C4 photosynthetic cycle, and of the more expensive natural L-theanine (δ13C of -24.4 ± 1.3‰), extracted from Camellia sinensis plants with C3 photosynthetic cycle, is reported here.

Isotopic and elemental characterisation of Italian white truffle: A first exploratory study.

White truffle (Tuber magnatum Pico) is a typical product of some Italian regions, and due to its organoleptic properties it is of high economic value. To date, there is no available information on the elemental and isotopic composition of this product. For the first time the characteristic value ranges of the stable isotope ratios of the bio-elements as a whole (δ13C, δ15N, δ18O and δ34S) and of the concentration of some macro- and micro-elements (Ag, Al, B, Ba, Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Sr, Zn) are now available, determined in white truffles produced in different areas of Italy (Toscana, Molise, Marche, Piemonte - Langhe, Monferrato-) and in Croatia. All the samples analysed showed values for the toxic, potentially toxic or allergenic elements lower than the legal limits. Furthermore, the ranges of variability of some elements or elements ratios (i.e. Na, B, Mg/Ca, Ba/Ca, Sr/Ca, Li/Fe, B/Al, Ba/Al) and stable isotope ratios (δ34S and δ18O) seem to be different according to the geographical origin. Therefore, the results of this first exploratory work highlight the possibility of characterising white truffles of different provenance, and suggest widening the scope of the survey throughout Italy and foreign regions.

Stable isotope ratio analysis combined with inductively coupled plasma-mass spectrometry for geographical discrimination between Italian and foreign saffron.

Seventy-six samples of saffron were analysed through inductively coupled plasma-mass spectrometry (ICP-MS) and stable isotope ratio analysis. The dataset was formed by 67 samples harvested in different areas of Italy, Morocco and Iran, and nine samples purchased in the Italian market. For the first time, 42 elements and five stable isotopes (δ13 C, δ15 N, δ34 S, δ2 H and δ18 O) were considered to carry out the discrimination of the samples on the basis of their geographical origin. Combined ICP-MS and isotopic composition data turned out to be a useful tool for the geographical discrimination of saffron among predefined cultivation sites. K, Cr, Mn, Ni, Zn, Rb, Sr, Mo, Cs, Nd, Eu, Pb, δ13 C, δ15 N, δ34 S and δ2 H were identified as the significant variables in geographical discrimination. Moreover, the class models generated for saffron cultivated in two specific areas of Central Italy exhibited 100% specificity for Moroccan, Iranian and commercial samples and a high specificity (83% and 84%) for the saffron samples cultivated in other, although close, Italian sites.

δ34 S for tracing the origin of cheese and detecting its authenticity.

Casein δ34 S of 725 samples of cheese from all over the world were measured using IRMS. δ34 S alone made it possible to establish characteristic ranges of values for two types of Italian cheese (Grana Padano PDO and Parmigiano Reggiano PDO) and for the different regions and provinces of both the Grana Padano PDO and the Parmigiano Reggiano PDO zones. Moreover, δ34 S of PDO Italian samples were compared to both Italian (not PDO) and foreign competitive cheese samples. In all the cases, sulfur isotopic ratio analysis was a powerful tool to fix characteristic ranges of values for cheeses coming from different countries and to improve the information given by other isotopic parameters.

Geographical discrimination of garlic (Allium Sativum L.) based on Stable isotope ratio analysis coupled with statistical methods: The Italian case study.

Stable isotope ratio analysis of the major bioelements (δ2H, δ13C, δ15N, δ18O, δ34S), considered for the first time as a whole, was used to attempt a geographical characterization of red garlic (Allium sativum L.) cultivated throughout different Italian territories (Abruzzo, Lazio and Sicily). Up to now, no official methods are available to determine the geographical origin of this type of product. In this context, the Isotope Ratio Mass Spectrometry method (IRMS) represents a powerful analytical technique. The characteristic ranges of variability of the five isotope ratios in 56 red Italian garlic samples are here presented as well as their relationships. The geographical origin has some influence over the different ratios, although their data distribution shows some overlap when Principal Component Analysis (PCA) is applied. In spite of the relative closeness of the sampling sites, a model with very good predictive performance of the geographical classification was achieved byLinear Discriminant Analysis (LDA) and k-nearest neighbours (k-NN) method. Moreover, preliminary class modelling based on Soft Independent Modelling of Class Analogy (SIMCA) supports the ability of stable isotope ratios analysis for the geographical traceability of garlic.

Assessing the authenticity of animal rennet using δ15N analysis of chymosin.

Chymosin is a protease that curdles the milk casein. Animal rennet was the first discovered source of chymosin and its use is mandatory for the production of PDO cheeses such as Parmigiano Reggiano and Grana Padano. Of the alternatives, fermentation-produced chymosin is the most competitive because it functions in a similar way, but is much cheaper. Analytical tools are necessary in order to distinguish the 2 types of chymosin and verify the compulsory use of animal rennet in the production of PDO cheeses. In this work, a method to analyse 15N/14N in chymosin after extraction was developed. The δ15N values of animal rennet range from 5.7‰ to 8‰, whereas the δ15N values of fermentation-produced chymosin are significantly lower, ranging from -5.3‰ to 2.2‰. A threshold value of 5.7‰ was defined for authentic animal rennet. Addition of fermentation-produced chymosin to animal rennet, or its complete substitution, can be therefore detected.

Characterisation and attempted differentiation of European and extra-European olive oils using stable isotope ratio analysis.

European law requires a designation of origin for virgin and extra virgin olive oils (at least in terms of EU/non-EU provenance). Stable isotope ratios have been successfully applied to determine the geographical origin of olive oils, but never to distinguish EU and non-EU oils. In this study 2H/1H, 13C/12C and 18O/16O ratios were analysed in bulk olive oils using Isotope Ratio Mass Spectrometry (IRMS) as well as 13C/12C and 2H/1H in the four main fatty acids (linoleic, oleic, palmitic and stearic acids) using IRMS coupled with GC. The isotopic composition of olive oils was successfully used to distinguish samples originating in the two areas. Specifically, when bulk data were combined with fatty acid isotopic data the differentiation power of the method improved clearly. This separation is due to the specific isotopic fingerprint of the individual countries making up the EU and non-EU samples.

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.

Stable isotope composition of cocoa beans of different geographical origin.

The isotopic profile (δ(13) C, δ(15) N, δ(18) O, δ(2) H, δ(34) S) was used to characterise a wide selection of cocoa beans from different renowned production areas (Africa, Asia, Central and South America). The factors most influencing the isotopic signatures of cocoa beans were climate and altitude for δ(13) C and the isotopic composition of precipitation water for δ(18) O and δ(2) H, whereas δ(15) N and δ(34) S were primarily affected by geology and fertilisation practises. Multi-isotopic analysis was shown to be sufficiently effective in determining the geographical origin of cocoa beans, and combining it with Canonical Discriminant Analysis led to more than 80% of samples being correctly reclassified. Copyright © 2016 John Wiley & Sons, Ltd.

δ(15) N from soil to wine in bulk samples and proline.

The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd.

Food web of a confined and anthropogenically affected coastal basin (the Mar Piccolo of Taranto) revealed by carbon and nitrogen stable isotopes analyses.

Carbon and nitrogen stable isotope analysis was used to examine the food web of the Mar Piccolo of Taranto, a coastal basin experiencing several anthropogenic impacts. Main food sources (algal detritus, seaweeds, particulate organic matter (POM) and sediment organic matter (SOM)) and benthic and pelagic consumers were collected during two contrasting seasons (June and April), at four sites distributed over two inlets, and characterized by different level of confinements, anthropogenic inputs and the presence of mussels farming. δ(13)C values of organic sources revealed an important contribution of POM to both planktonic and benthic pathways, as well as the influence of terrigenous inputs within both inlets, probably due to high seasonal land runoff. Although δ(13)C of both sources and consumers varied little between sampling sites and dates, δ(15)N spatial variability was higher and clearly reflected the organic enrichment in the second inlet as well as the uptake of anthropogenically derived material by benthic consumers. On the other hand, within the first inlet, the isotopic composition of consumers did not change in response to chemical contamination. However, the impact of polluted sediments near the Navy Arsenal in the first inlet was detectable at the level of the macrobenthic trophic structure, showing high dominance of motile, upper level consumers capable to face transient conditions and the reduction of the more resident deposit feeders. We therefore underline the great potential of matching stable isotope analysis with quantitative studies of community structure to assess the effects of multiple anthropogenic stressors.

Validation of methods for H, C, N and S stable isotopes and elemental analysis of cheese: results of an international collaborative study.

RATIONALE: PDO cheeses, such as Parmigiano Reggiano and Grana Padano, which cost more than double generic similar cheeses, must be protected against mislabelling. The aim of this study was to validate the methods for the isotopic and elemental analysis of cheese, in order to support official recognition of their use in authenticity assessment. METHODS: An international collaborative study based on blind duplicates of seven hard cheeses was performed according to the IUPAC protocol and ISO Standards 5725/2004 and 13528/2005. The H, C, N and S stable isotope ratios of defatted cheese determined using Isotope Ratio Mass Spectrometry (IRMS) and the content of Li, Na, Mn, Fe, Cu, Se, Rb, Sr, Mo, Ba, Re, Bi, U in cheese after acid microwave digestion using Inductively Coupled Plasma Mass Spectrometry or Optical Emission Spectrometry (ICP-MS or -OES) were measured in 13 different laboratories. RESULTS: The average standard deviations of repeatability (sr) and reproducibility (sR) were 0.1 and 0.2 ‰ for δ(13)C values, 0.1 and 0.3 ‰ for δ(15)N values, 2 and 3 ‰ for δ(2)H values, and 0.4 and 0.6 ‰ for δ(34)S values, thus comparable with results of official methods and the literature for other food matrices. For elemental data, the average RSDr and RSDR values ranged between 2 and 11% and between 9 and 28%, respectively, consistent with methods reported by the FDA and in the literature for cheese. CONCLUSIONS: The validation data obtained here can be submitted to the standardisation agencies to obtain official recognition for the methods, which is fundamental when they are used in commercial disputes and legal debates.

Compound-Specific δ¹5N and δ¹³C Analyses of Amino Acids for Potential Discrimination between Organically and Conventionally Grown Wheat.

We present a study deploying compound-specific nitrogen and carbon isotope analysis of amino acids to discriminate between organically and conventionally grown plants. We focused on grain samples of common wheat and durum wheat grown using synthetic nitrogen fertilizers, animal manures, or green manures from nitrogen-fixing legumes. The measurement of amino acid δ(15)N and δ(13)C values, after protein hydrolysis and derivatization, was carried out using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Our results demonstrated that δ(13)C of glutamic acid and glutamine in particular, but also the combination of δ(15)N and δ(13)C of 10 amino acids, can improve the discrimination between conventional and organic wheat compared to stable isotope bulk tissue analysis. We concluded that compound-specific stable isotope analysis of amino acids represents a novel analytical tool with the potential to support and improve the certification and control procedures in the organic sector.

Studying trophic interactions between a plant pathogen and two different antagonistic microorganisms using a 13C-labeled compound and isotope ratio mass spectrometry.

RATIONALE: The study of the interactions among microorganisms, especially between pathogens and other microorganisms, is a very useful way to identify possible biocontrol agents (BCAs). In this study we verified the capability of δ(13)C analysis using isotope ratio mass spectrometry (IRMS) to detect active parasitism or metabolic assimilation of (13)C-labeled Armillaria mellea (plant pathogen) by Trichoderma atroviride and Pseudomonas fluorescens (two BCAs). METHODS: The three microorganisms were labeled in pure-culture using a specific medium to which D-glucose (13)C was added. The δ(13)C analysis of mycelia/cells and DNA was undertaken using IRMS at different times, to study the uptake kinetics of (13)C. The mechanisms of interaction were studied by implementing dual-culture tests and measuring the δ(13)C values of the two BCAs after 29 days of contact with the labeled pathogen. RESULTS: A. mellea absorbed (13)C more slowly (plateau at 21 days) than T. atroviride and P. fluorescens (3 and 1 day, respectively) in pure-culture. The maximum δ(13)C values were higher in A. mellea and T. atroviride mycelia (8,019.9‰ and 10,383.7‰, respectively) than in P. fluorescens (953.4‰ in cells). In dual-culture the mycelia of T. atroviride which remained in direct contact with labeled A. mellea showed an increased δ(13)C value with respect to the unlabeled treatment (66.4‰ and -26.6‰, respectively), due to active interaction. Lower assimilation of (13)C was detected in P. fluorescens. CONCLUSIONS: This work demonstrates that IRMS can be used for the in-depth study of direct parasitism and interaction process between biocontrol agents and labeled pathogens, allowing the screening of potential new BCAs.

H, C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses.

In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors. This paper presents two statistical models, based on isotopic and elemental composition, able to trace the origin of cheese also in grated and shredded forms, for which it is not possible to check the logo fire-marked on the rind. One model is able to predict the origin of seven types of European hard cheeses (in a validation step, 236 samples out of 240 are correctly recognised) and the other specifically to discriminate the PDO Parmigiano Reggiano cheese from 9 European and 2 extra-European imitators (260 out of 264 correct classifications). Both models are based on Random Forests. The most significant variables for cheese traceability common in both models are δ(13)C, δ(2)H, δ(15)N, δ(34)S and Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, and Li. These variables are linked not only to geography, but also to cow diet and cheese making processes.