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Compound-specific stable isotope analysis (CSIA) of food products is a relatively new and novel technique used to authenticate food and detect adulteration. This paper reviews recent applications of CSIA to authenticate the origin of different foods. CSIA δ¹³C values are widely used to verify geographical origin, organic production, and adulteration. The δ¹⁵N values of individual amino acids and nitrate fertilizers have proven effective to authenticate organic foods, while δ²H and δ¹⁸O values are useful to link food products with local precipitation for geographical origin verification. CSIA has a stronger analytical advantage for the authentication of food compared to bulk stable isotope analysis, especially for honey, beverages, essential oils, and processed foods.

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Isotope ratio data are increasingly used in a variety of fields including, ecology, marine sciences, earth and geosciences, forensic science, hydrology, medicine, food (including food authenticity and origin), and climate science.
 
Over the years, there have also been changes to guidelines for measurement methods, calibration conventions and even to international measurement standards that form the base of the traceability chain for isotope delta values for H, C, N, O and S.
 
It is impossible to combine isotope ratio data from a variety of sources unless the data are accompanied by a clear description of traceability and other method details.

The UK National Measurement Laboratory at LGC was part of an international group that compiled the IUPAC Technical Report presenting minimum requirements for reporting isotope ratio data, covering analytical procedure, traceability, data processing and uncertainty evaluation.

This report will help in the standardisation of methods that involve the measurement of stable isotopes.

Read the IUPAC Technical Report on minimum requirements for publishing hydrogen, carbon, nitrogen, oxygen and sulfur stable-isotope delta results.

Traditional cheeses, with protected names (PDO/PGI) are sold at a premium and hence can be susceptible to substitution and fraud. This review reports the available authentication methods, either chemical, physical, or DNA-based methods, currently used for origin authentication, highlighting their principle, reported application to cheese geographical origin authentication, performance, and respective advantages and limits. Isotope and elemental fingerprinting showed consistent accuracy in origin authentication. Other chemical and physical methods, such as near-infrared spectroscopy and NMR, require more studies and larger sampling to assess their discriminative power. Emerging DNA-based methods, such as metabarcoding, showed good potential for origin authentication. However, metagenomics, providing a more in-depth view of the cheese microbiota (up to the strain level) is an interesting approach to authenticate the origin of cheeses, but is still in its early development.

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Organic crop production is a system requirement covering many aspects of its production. One main feature, which distinguishes organic from conventional production is the prohibition on the use of synthetic fertilisers, which are produced from atmospheric nitrogen by the Haber process, and the resulting nitrate is high in ¹⁸O and low in ¹⁵N. Isotopic measurement of nitrate extracted from plant foods can indicate which fertilisation system has been used. This requires a bacterial reduction, which is costly and can only be done by specialised laboratories. Research scientists at IAEA have developed a rapid, low-cost method to measure N and O isotope ratios using a one-step Ti(III) reduction to convert the nitrate to N₂O gas for headspace isotope analysis using IRMA (isotope-ratio mass spectrometry). The new method was developed and tested using organic and conventionally grown Spanish strawberries, where the extractable nitrate N and O isotope ratios and the chemical composition were measured followed by chemometric analysis, and which revealed that the δ¹⁸O of nitrate along with δ¹⁵N and Ca²⁺ fully differentiated organic from conventional strawberry production. 

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The name Lambrusco is based on grape variety, and Lambrusco wines have been given PDO (Protected Denomination of Origin) status based on variety and method of production. They are produced in several regions in Italy, but mainly in the north including Modena, Reggio Emilia, and Mantua. In this study, 40 PDO Lambrusco wines were collected, 24 from Modena, 10 from Mantua, and 6 from Reggio Emilia. The feasibility of using isotopic ratios of  ¹¹B/¹⁰B,  ⁸⁷Sr/⁸⁶Sr, ^20yPb/^20xPb, ¹⁸O/¹⁶O,  and the elemental concentrations of boron, lead and strontium were determined. After different chemometric analyses, the best differentiation of the PDOs was based mainly on boron and strontium, in terms of concentration and isotopic ratios, and on lead isotopic ratios.

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The report gives evidence of the illegal trade of wild caught sturgeon in the lower region of the Danube specifically in Romania, Bulgaria, Serbia and Ukraine. Because sturgeon is an endangered species, the trade in both wild and aquaculture fish and its products (primarily caviar) is regulated through CITES (Convention on International Trade of Endangered Species). The project looked at official data of enforcement on illegal fishing activities. It also carried out a market survey from October 2016 to July 2020, and collected 145 samples of fresh and processed sturgeon, as well as caviar, from the retail and catering sectors in the four countries. During this period all fishing and trade in wild sturgeon was prohibited in Romania, Bulgaria and Ukraine. Serbia was permitted to have a restrictive catch of wild sterlet sturgeon above 40cm in length, but even this was only until the end of 2018, after which it became illegal as well.

All the samples were analysed by 3 DNA methods (mitochondrial DNA sequencing for species, microsatellites for identification of species and hybrids, and SNPs (single nucloeotide polymorphism) for hybrid and species identification). In addition, stable isotopes analysis (SIRA) was carried out to give information on whether the fish was wild or farmed based on feedstuffs, and geographic origin. The results indicated that 30% of the samples tested were illegal, 27 samples were from illegally caught wild sturgeon, 17 samples of caviar were in violation of CITES Regulations.

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This book highlights the use of specific physicochemical parameters, such as sugar content, moisture content, electrical conductivity, acidity, colour, and attributes in the production of honey. It also discusses the use of honey micro-constituents, including volatile compounds, polyphenols, minerals, organic acids, free amino acids and isotopic data, in the determination of the botanical and geographical origins of honey, in combination with chemometrics. It represents the ultimate research guide and reference manual for the determination of honey uniqueness. 

More information on the content here

Methods to determine the geographic origin of EVOO are a useful tool to prevent fraud for this high value oil. Portuguese researchers have applied SIRA of carbon, oxygen and hydrogen (δ¹³C, δ²H and δ¹⁸O) with chemometrics to verify whether the geographical origin of VOOs obtained from three Mediterranean countries can be determined. One hundred and thirty eight authentic VOO samples were collected from Portugal, France and Turkey from two different harvest years (2016 and 2017). The samples were analysed for the isotopic ratios using an elemental analyser coupled to an isotope ratio mass spectrometer (EA-IRMS). Using meteorological and geographical parameters, a meteoric water line for olive oil from Portugal, France and Turkey, in two harvest years, were created to assess the impact of climate change on their δ²H and δ¹⁸O values. After principal component analysis, the VOOs from Portugal and France were well separated, Turkey slightly less so, but also the 2016 and 2017 harvest years of each country were also discriminated.  

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The Canadian Food Inspection Agency (CFIA) conducted targeted surveillance between 2019 and 2020 as part of ongoing efforts to detect honey adulteration with exogenous sugars in both domestic and imported honey sold in Canada. A total of 275 samples were collected across Canada and analysed using Stable Isotope Ratio Analysis (SIRA) and Nuclear Magnetic Resonance (NMR). Two types of honey samples were collected. One group of 127 samples, consisted of single-ingredient honey products such as bulk and honey for further processing from importers and a small proportion from domestic establishments. These were collected from suppliers where the chance of non-compliance was higher, based on risk-factors such as a history of non-compliance, gaps in preventive controls, or unusual trading patterns. The other 148 samples of honey were collected by an independent third party at retailers in various cities across Canada as part of CFIA's compliance monitoring of the marketplace, to gauge overall compliance. Of the targeted samples 17(13%) were considered unsatisfactory from the analytical results of which only 1 was Canadian honey. Of the retail samples, only 3 were considered unsatisfactory, and all of these were imported. As a result of CFIA's actions, an estimated 83,461 kg of adulterated honey was prevented from being sold in the Canadian marketplace between April 1, 2019 and March 31, 2020.

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Grass-based milk production is a major contributor to Irish agricultural output, and the terms "grass-fed" and "pasture-raised" are appreciated by many consumers as a more sustainable and welfare friendly means of producing milk. This study characterised the Irish raw milk pool using SIRA of carbon, nitrogen, oxygen and sulphur. Authentic raw milk samples were collected from 50 farms on five occasions over 13 months. δ¹³C values reflected a high level of grass input, and values increased with increasing cereal concentrate feed input (P < 0.001). δ¹⁸O values were most negative in spring. There was a significant interaction between feed and season for δ¹³C and δ¹⁵N values (P < 0.05), with the impact of concentrate feeding most evident in spring. The isotopic ratio values of the Irish milk pool may serve as authenticity markers with the potential to discriminate Irish milk and dairy products from similar commodities from other countries.

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This study has determined the isotopic ratios, i.e. of oxygen and hydrogen isotopes, of Slovenian milk and its major constituents: water, casein, and lactose. In addition, the oxygen isotope ratios of cow, sheep, and goat’s milk were compared. Oxygen isotope ratios in milk show seasonal variability, and are also 18O enriched in relation to animal drinking water. The δ18O water values were higher in sheep and goat’s milk when compared to cow milk, reflecting the isotopic composition of the drinking water source, and the effect of differences in the animal’s thermoregulatory physiologies. The relationship between δ18O milk and δ18O lactose is an indication that even lower amounts (>7%) of added water to milk, can be determined. This approach could be a possible confirmatory method to determining added water in milk to the freezing point method.

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Vanilla is a high value flavouring used in ice cream, desserts and confectionery, and mainly grown in Madagascar. Synthetic vanillin and biovanillin (produced by fermentation) are cheaper. By using SIRA (stable isotope ratio analysis) to look at the δ13C, it is possible to distinguish between the three types of vanilla flavouring. The method was tested on market samples of yoghurt and ic cream. Also FTIR (Fourier Transform Infrared) spectoscopy with chemometric analysis was calibrated with authentic samples of black pepper so that it was possible to detect the adulteration of ground black pepper with black pepper husk and defatted spent material. The research was undertaken as a Ph.D registered at the Technical University of Denmark, but carried out at the Danish National Food Institute and IGFS - Queens University Belfast.  

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Stable Isotope Ratio (SIRA) analysis is widely used to investigate different authenticity issues from exogenous sugar adulteration to geographic origin. An international project  has developed, quality-tested, and measured isotope–delta values of 10 new food matrix reference materials (RMs) for hydrogen, carbon, nitrogen, oxygen, and sulfur SIRA measurements. The RMs include (i) two honeys from Canada and tropical Vietnam, (ii) two flours from C3 (rice) and C4 (millet) plants, (iii) four vegetable oils from C3 (olive, peanut) and C4 (corn) plants, and (iv) two collagen powders from marine fish and terrestrial mammal origins. The RMs were collaboratively tested by 8 laboratories to obtain consensus values and measurement uncertainties. These new RMs should facilitate mutual compatibility of stable isotope data if accepted normalisation procedures are applied and documented.

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The δ¹³C and δ¹⁵N isotopic ratios  are important in the investigation of food authenticity and fraud. Previous studies in Brazil on targeted foods revealed  that many of them were adulterated; mislabelled or even fraudulent. Hence to improve future authenticity studies, Brazilian researchers have determined baseline values of δ¹³C and δ¹⁵N in 1245 food items and 374 beverages; most of them made in Brazil. The average δ¹³C and δ¹⁵N values of C3 plants, C4 plants, plant-based processed foods, meat (including beef, poultry, pork and lamb), meat products, and beverages (including beer and wine) are all given in the paper. Because cattle are grass fed, and chickens and pigs fed on soya and maize, C-C₄ constitutes a large proportion of fresh meat, dairy products, as well as meat products. Also cane sugar and maize predominate as ingedients, and hence there is a large proportion of C-C4 in plant-based processed foods.

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This pilot study used SIRA (stable isotope ration analysis) and trace element analysis to verify the organic status of pork purchased in the markets from four different regions of China. Four stable isotope ratios (δ¹³C, δ¹⁵N, δ²H and δ¹⁸O) and the concentrations of seven elements (K, Na, Mg, Ca, Fe, Cu and Se) were determined in organic and conventional pork samples from the four locations of China. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyse stable isotope ratios and multi-element concentrations in pork. Discrimination between the organic and conventionally reared pigs was based mainly on δ¹⁵N as the marker for organic feedstuffs. Using the limited database of analytical values, the methodology would potentially be able to confirm whether a sample of pork came from the region and organic status it claimed. 

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Sugar adulteration using carbon isotope ratio analysis by mass spectrometry is well established, but it can only be used for C4 sugar (from cane or maize) adulteration. Although a method has been developed to determine C3 sugar (from beet, most fruits or wheat) adulteration using hydrogen/deuterium isotope ratio analysis by mass spectrometry, it requires very time consuming chemical derivative preparation. The established method for C3 sugar determination has been quantitative deuterium nuclear magnetic resonance (²H NMR) measurement of ethanol derived from the sugars in the sample, which again is time consuming, and requires a normalisation process to compensate for the deuterium content of the fermentation water. Researchers at IAEA have developed a rapid method for both C3 and C4 sugar detection, which derivatises the carbohydrate's exchangeable hydroxyl-hydrogens, so that the derivative compound is sufficiently volatile to be separated and measured by a gas chromatograph coupled to an isotope ratio mass spectrometer. Feasibility of the method has been shown by measuring sugars from fruit juice and honey, but further work is required to assess the reproducibility of this method and establish its applicability for detection of undeclared addition of exogenous sugars and syrups to a range of other foods and beverages.  .

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Honey is one of the most adulterated foods by addition of sugar syrups. Most if not all commercial honeys derive from the nectar of C3 plants. Sugar syrups derived from cane sugar or maize, which are C4 plants. The accepted standard method to check C4 sugar adulteration, is to determine the δ¹³C (C13/C12 ratio) by SIRA mass spectrometry.  Honeys that are tested with a δ¹³C value of -23.5 and lower are deemed to be pure. Honeys that have a δ¹³C value between -23.5 and -21.5, fall into a grey area. Honeys that have a δ¹³C of -21.5 or higher are deemed to be adulterated. However, false positives of syrup adulteration may occur if the bees have been collecting nectar from C4 plants, and hence low level adulteration is difficult to detect. This review discusses the background to testing honey by SIRA, and the limitations of this method.

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Analytical methods for authenticity testing of organically grown vegetables are urgently needed. Danish researchers have developed a novel assay for organic authentication based on stable isotope ratio analysis (SIRA) of oxygen in plant-derived sulphate. This method was compared with SIRA of bulk plant tissue and plant-derived nitrate to discriminate organic and conventionally grown potato, carrot, and cabbage from rigidly controlled long-term field trials and from a case study using retail potatoes. The results indicated that oxygen isotope ratios of sulphate from organic vegetables were significantly lower compared to their conventionally grown counterparts, and the values were directly linked to the fertilisation strategy. The classification power of sulphate isotope analysis was superior compared to known bulk tissue isotope markers and nitrate isotope values, and hence represents a promising new method for authentication of organic vegetables.

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Mozzarella di Bufala Campana (MBC) is a PDO cheese produced from whole buffalo milk in specific regions of southern Italy. Due to the high price and the limited amount of buffalo milk, MBC is potentially subject to mislabelling, substitution or fraud. Italian researchers have used elemental and isotopic profiles of authentic samples of buffalo milk and the corresponding MBC samples collected in the reference area in winter and summer in an initial exploratory study. A model was developed to classify product categories for this cheese by merging MBC-PDO samples with non-PDO samples of buffalo mozzarella produced both inside and outside the reference area. Despite differences caused during processing, along with differences in the season and production area, the model was effective in distinguishing PDO and non-PDO mozzarella, particularly when non-PDO cheeses were made outside the MBC reference area.

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As the European Union has committed to only accept authenticated “sustainably sourced” palm oils, it is important to ensure that such imported oils are really from the declared source, preferably via proven analytical methods. This full review looks at the legal requirements for the traceability and authentication of palm oil, and assesses some new and emerging chemically-based technologies that should contribute to improving the monitoring of palm oil and other vegetable oil supply chains in Europe and elsewhere. 

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