How to guarantee authenticity and traceability of agri-food and supplements products thanks to the application of isotopic analysis of bioelements

Stable isotope ratio analysis of bio-elements (hydrogen, carbon, nitrogen, oxygen and sulphur) has been used since the 1990s to check food authenticity and traceability of a wide variety of food commodities (Rossmann, 2001). In the last few years, examples of applications also in the pharmaceutical and cosmetic field have been reported (Pellati et al., 2013; Perini et al., 2017, 2021; Perini, Paolini, et al., 2019; Perini, Pianezze, et al., 2019). The use of stable isotope analysis for products authentication purposes is possible thanks to isotopic fractionation occurring in several processes and reactions (biological, biochemical, physical, chemical etc.) which generates unique isotopic signatures. For this reason, the application of this technique on the bulk samples as well as on specific components (e.g. aroma compounds) can be used to detect the origin of an ingredient (synthetic or natural), the substitution of one ingredient for another, as well as the geographical and/or botanical origin of the products. The widespread and well-known technique based on the coupling between elemental analyzer and mass spectrometer (EA-IRMS) is now flanked by liquid chromatography (LC-IRMS) and gas chromatography (GC-IRMS). Today it is therefore possible to analyze not only the bulk of the matrices but also their individual components. The δ13C and δ2H values of vanillin can determine whether the product is natural (deriving from the expensive CAM plant Vanilla), biotechnologically derived or synthetic (Perini, Pianezze, et al., 2019). Moreover, the δ13C values of specific components of Rosa damascene mill., one of the most expensive essential oils in the market world, can indicate the fraudulent addition of cheaper oil from a C4 plant (e.g. Cymbopogon martinii, palmarosa) (Pellati et al., 2013). In pharmaceutical and cosmetic formulations, δ13C analysis is a suitable tool to discriminate between squalene and squalane from shark (illegal) and from olive oil (expensive) (Camin et al., 2010) as well as between monacolin K (contained in the fermented dietary supplement red yeast rice) and the commercially marketed statin, lovastatin (Perini et al., 2017).The L-theanine extracted from Camellia Sinensis is easily distinguishable from that obtained biosynthetically (Perini et al., 2021). It is possible to combine different isotopic signatures to guarantee the natural origin of curcumin, caffeine (Ding et al., 2019), tartaric acid and its derivatives. These examples demonstrate that the isotopic fingerprint represent an effective tool for the authenticity assessment of economically important pharmaceutical, cosmetic and supplement products

How to guarantee the natural origin of nutraceutical and pharmaceutical products? The potential of the stable isotope ratios analysis

The analysis of the ratio between the heavy and light isotopes of the elements carbon (13C/12C), nitrogen (15N/14N), sulfur (34S/32S), oxygen (18O/16O) and hydrogen (2H/1H) is well known for its power to discriminate the geographical origin and guarantee the authenticity of many agri-food products [1]. In recent years, the field of application of this technique has expanded to include nutraceuticals and pharmaceuticals, in particular in order to guarantee their natural origin. Chemically identical molecules are significantly different from an isotopic point of view due to the isotopic fractionation that occurs in different processes and reactions (biological, biochemical, physical, chemical, etc.) which generates unique isotopic signatures in the product synthesized by plants compared to that produced in the laboratory usually starting from fossil sources. Thanks to the coupling of isotopic mass spectrometry to liquid chromatography (LC-IRMS) and gas chromatography (GC-IRMS) it is now possible to discriminate between natural and/or synthetic origin not only of the bulk product but also of its specific components. The "Compound specific analysis" makes it possible to identify much more sophisticated frauds than in the past such as, for example, the addition of a single synthetic component to a natural substrate in order to artificially increase its strength. In this context, the δ13C analysis is a suitable tool to discriminate between Monacolin K (contained in red yeast rice-based dietary supplements) and the marketed statin [2] and between natural L-theanine (extracted from Camellia Sinensis) and the biosynthetically produced one [3]. The isotope ratios of hydrogen and, in some cases, carbon exhibit significantly different ranges of variability between natural extracts (such as curcuminoids [4] and cannabidiols [5]) and their synthetic adulterant, allowing for the identification of not only the two origins, but also the fraudulent additions of synthetic products to the natural complex (spiked samples). The combination of GC-MS/MS and GC-IRMS is potentially useful for botanical classification between lavender (Lavandula angostifolia) and lavandin (Lavandula hybrida) essential oils thus representing an additional powerful tool for assessing the authenticity of commercial essential oil

High density balsamic vinegar: application of stable isotope ratio analysis to determine watering down

Aceto balsamico di Modena IGP (ABM) is an Italian worldwide appreciated PGI (Protected Geographical Indication) vinegar, obtained from cooked and/or concentrated grape must (at least 20% of the volume), with the addition of at least 10% of wine vinegar and a maximum 2% of caramel for color stability (EU Reg. 583/ 2009). The geographical origin of ABM ingredients is never specified. Since 2013, the European Committee for Standardization (CEN) has issued a method for determining the water fraudulently added to the vinegar and the balsamic vinegar product (EN16466-3 18O-IRMS). The method is based on the stable isotope ratios analysis of the bulk AMB sample (expressed as δ18O in ‰ with respect to the international standard V-SMOW2). Balsamic vinegars with very high density (higher than 1.37 g / mL of sugar) are available on the market. They are obtained by adding a high amount of concentrated must or by a long aging of the product in the barrel, which leads to an intense evaporation and concentration. Products with such high density cannot be analyzed by using the official method as reported in the EN16466-3 18O-IRMS. Indeed, in this conditions, the equilibration between CO2 and the water in the sample, being the base principle of the process, does not occur. In this work, the official method has been modified and validated, calculating repeatability (r) and reproducibility (R), by proceeding with a prior dilution of the sample and by applying a correction to the data in order to eliminate the diluent isotopic contribution. Considering the limit value of δ18O for a non-watered product reported in the literature for vinegar and for rectified concentrated must [1-2], the threshold limit of δ18O below which the ABM product can be considered as adulterated was identified. References [1] J. Agric. Food Chem. 2014, 62, 32, 8197–8203 [2] Food Control 2013, 29(1), 107–11

Authentication and geographical characterisation of Italian grape musts through glucose and fructose carbon isotopic ratios determined by LC IRM

The authenticity of grape musts is normally checked through the stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. Unfortunately, it presents some issues that are difficult to over come. Grape must samples can only be analysed after they have been fermented to obtain ethanol. The process must be carried out under careful control of the fermentation to avoid the presence of unwanted by-products arising from a premature fermentation interruption. Moreover, if the musts have been preserved by the addition of sulphur dioxide (SO2 ), they must undergo an additional step to eliminate the SO2 , which would affect the fermentation. Once the product has been fermented, the ethanol must be separated using specific distillation columns (such as the Cadiot ones) making it possible to obtain ethanol free of isotopic fractionation with a minimum alcohol degree of 95% vol. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. In LC–IRMS, analytes are separated on an LC system and consecutively oxidized in an online reactor to CO2 , which is required for the determination of compound-specific carbon isotopic ratios. This technique has been already used in the study of matrices such as wine [1], ethanol [1,2], glycerol [2], and honey [3] to detect fraudulent alterations of their natural composition such as the addition of exogenous sugars to the products. The LC-IRMS allows a single separation of the individual components of a sample and makes it possible to determine their δ13C values online, avoiding both the disadvantages of off-line methods and the disadvantages of methods requiring a derivatization step (such as GC-C-IRMS), causing the addition of extra carbons. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic Italian must samples from 16 different sampling regions were analysed. In addition, the δ13C variability in authentic and fake must (added with increasing percentages of exogenous sugars) has been explored and tested to verify their validity as fraud detectors. The two analysed parameters, ranging from −29.8‰ to −21.9‰, are well correlated (R2 = 0.7802) and the northern Italian regions showed significantly more negative δ13C values for both sugars than the rest of the dataset (Figure 1). By using the LC-IRMS technique, the addition of exogenous sugars, such as fructose and glucose from C4 photosynthetic cycle plants, is easily detectable as it modifies the δ13C of the individual sugar

High-density balsamic vinegar: application of stable isotope ratio analysis to determine watering down

Balsamic vinegar of Modena (ABM) is a product obtained from concentrated grape must with the addition of wine vinegar. It can be adulterated with the addition of exogenous water. The official method EN16466-3, based on the analysis of the stable isotope ratio δ18O of the water, is not applicable to ABM with high density (above 1.20 at 20 °C). In this work, for the first time, the official method was modified, providing for a prior dilution of the sample and applying a correction of the data in order to eliminate the isotopic contribution of the diluent, whereupon the within- and between-day standard deviations of repeatability (Sr) were estimated. Considering the limit values of δ18O for vinegar and concentrated must, the threshold limit of δ18O, below which the ABM product can be considered adulterated, has been identified

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

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)

Authentication and geographical characterisation of Italian grape musts through glucose and fructose carbon isotopic ratios determined by LC-IRMS

The authenticity of grape musts is normally checked through a time-consuming stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. It allows not only the detection of the fraudulent addition to grape must of exogenous glucose and fructose deriving from C4 plants but also the characterisation of it based on its geographical origin. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic samples were analysed. The two analysed parameters, ranging from -29.8‱ to -21.9‱, are well correlated (R2 = 0.7802) and the northern regions showed significantly more negative δ13C values for both sugars than the rest of the datase

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

5openopenPerini, Matteo; Nfor, Mohamadou Bawe; Camin, Federica; Pianezze, Silvia; Piasentier, EdiPerini, Matteo; Nfor, Mohamadou Bawe; Camin, Federica; Pianezze, Silvia; Piasentier, Ed

Improving honey identification: stable isotope ratios variability of mono and polyfloral honeys from the citrus growing area of Salto/Concordia and from Uruguayan coastal areas

Concordia in Argentina and Salto in Uruguay are two neighbouring regions famous for the production of a precious citrus honey, whose valorisation passes through the ability to guarantee its geographical origin. In this study the influence of the different botanical origins (monofloral and polyfloral) within the same region of origin on the different stable isotope ratios was evaluated. Moreover, the effectiveness of the stable isotope ratio parameters δ13Choney, δ13Cprotein, δ15Nprotein, δ34Sprotein, δ2 Hprotein and δ18Oprotein to discriminate between honeys produced in the internal border regions between Argentina and Uruguay (namely, Concordia and Salto) and honeys sampled in the two different Uruguayan coastal regions (Canelones and Maldonado) was tested on 82 monofloral and polyfloral honey samples. The results show that the different stable isotope ratios, except that of nitrogen, are not significantly influenced by the botanical origin of the samples. The sulphur isotopic ratio of proteins and carbon of honey are the most significant parameters for discriminating the geographical origin of the honeys considered. Applied a Principal Component Analysis, the first two factors overall explain 63.5% of the total variance, while the Discriminant Analysis provided optimal discrimination between the three origins, reaching a minimum of 96.7% correct reclassification