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

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))

Endophytes from African Rice (Oryza glaberrima L.) Efficiently Colonize Asian Rice (Oryza sativa L.) Stimulating the Activity of Its Antioxidant Enzymes and Increasing the Content of Nitrogen, Carbon, and Chlorophyll.

Bacterial endophytes support the adaptation of host plants to harsh environments. In this study, culturable bacterial endophytes were isolated from the African rice Oryza glaberrima L., which is well-adapted to grow with poor external inputs in the tropical region of Mali. Among these, six N-fixer strains were used to inoculate O. glaberrima RAM133 and the Asian rice O. sativa L. cv. Baldo, selected for growth in temperate climates. The colonization efficiency and the N-fixing activity were evaluated and compared for the two rice varieties. Oryza sativa-inoculated plants showed a fairly good colonization efficiency and nitrogenase activity. The inoculation of Oryza sativa with the strains Klebsiella pasteurii BDA134-6 and Phytobacter diazotrophicus BDA59-3 led to the highest nitrogenase activity. In addition, the inoculation of ‘Baldo’ plants with the strain P. diazotrophicus BDA59-3 led to a significant increase in nitrogen, carbon and chlorophyll content. Finally, ‘Baldo’ plants inoculated with Kl. pasteurii BDA134-6 showed the induction of antioxidant enzymes activity and the maintenance of nitrogen-fixation under salt stress as compared to the unstressed controls. As these endophytes efficiently colonize high-yielding crop varieties grown in cold temperate climates, they become good candidates to promote their growth under unfavorable conditions

Stable isotope ratio analysis to assess pharmaceuticals, cosmetics and dietary supplements authenticity

The stable isotope ratio analysis of the mayor bio-elements (hydrogen, carbon, nitrogen, oxygen and sulphur) makes it possible to authenticate pharmaceuticals, cosmetics and dietary supplements. This technique, applied to bulk samples and/or to specific compounds, can be used to detect the origin of an ingredient (synthetic or natural), the substitution of one ingredient with another, as well as the geographical and/or botanical origin of the products. The δ 13C and δ 2H values of vanillin can determine whether this product is natural (deriving from the expensive CAM plant Vanilla), biotechnologically produced or synthetic [1]. Moreover, the δ 13C values of specific components of Rosa damascene mill., one of the most expensive essential oils in the global market, can indicate the fraudulent addition of cheaper oil from C4 plants (e.g., Cymbopogon martinii, palmarosa) [2]. Finally, the δ 13C analysis is a suitable tool to discriminate between Monacolin K (contained in red yeast rice-based dietary supplements) and the marketed statin [3] and between natural L-theanine (extracted from Camellia Sinensis) and the biosynthetically produced one [4]. These examples show that the isotopic fingerprint represents an effective tool for the authenticity assessment of economically relevant pharmaceuticals, cosmetics and dietary supplement

C and H stable isotope ratio analysis using GC-IRMS for vanillin authentication

Vanilla extracts are widely used as flavouring ingredients in foods and beverages and aromatic compounds in perfumes and pharmaceuticals. Due to the high cost of producing high-quality natural extracts from Vanilla planifolia, synthetic or natural identical biosynthetic vanillin (from natural precursors such as guaiacol, ferulic acid, eugenol and lignin) are often used as a substitute [1]. To verify the authenticity of vanilla extracts from Vanilla planifolia, one of the most commonly used methods is stable isotope ratio analysis (SIRA) of 13C/12C (expressed as δ13C), since it has been found that different plants discriminate differently against 13C and differently from the synthetic source. Today this analysis is no longer enough to discover vanillin adulteration, due to the practice of adding 13C to the methylic site of synthetic vanillin [2]. In this study, we combined analysis of 13C/12C with that of 2H/1H (expressed as δ2H) using GCIRMS [3]. 16 authentic samples of Vanilla planifolia, 16 natural identical, 5 synthetic vanillin and 20 commercial extracts were considered. Authentic natural vanillin from Vanilla planifolia and natural identical vanillin are characterised by δ2H values much lower than those of synthetic vanillin. The isotopic values of all the commercial extracts declared to be from Vanilla planifolia (N=20), had δ13C within the typical range for natural vanillin, but δ2H outside the range and more similar to that of synthetic vanillin. The combination of δ13C with δ2H GC-IRMS analysis of vanillin can therefore be proposed as a suitable tool for improving the detection of vanilla extract adulteratio