Mauro Paolini

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.

The use of IRMS, (1)H NMR and chemical analysis to characterise Italian and imported Tunisian olive oils.

Isotope Ratio Mass Spectrometry (IRMS), (1)H Nuclear Magnetic Resonance ((1)H NMR), conventional chemical analysis and chemometric elaboration were used to assess quality and to define and confirm the geographical origin of 177 Italian PDO (Protected Denomination of Origin) olive oils and 86 samples imported from Tunisia. Italian olive oils were richer in squalene and unsaturated fatty acids, whereas Tunisian olive oils showed higher δ(18)O, δ(2)H, linoleic acid, saturated fatty acids β-sitosterol, sn-1 and 3 diglyceride values. Furthermore, all the Tunisian samples imported were of poor quality, with a K232 and/or acidity values above the limits established for extra virgin olive oils. By combining isotopic composition with (1)H NMR data using a multivariate statistical approach, a statistical model able to discriminate olive oil from Italy and those imported from Tunisia was obtained, with an optimal differentiation ability arriving at around 98%.

Gas chromatography combined with mass spectrometry, flame ionization detection and elemental analyzer/isotope ratio mass spectrometry for characterizing and detecting the authenticity of commercial essential oils of Rosa damascena Mill.

RATIONALE The essential oil of Rosa damascena Mill. is known for its fine perfumery application, use in cosmetic preparations and for several pharmacological activities. Due to its high value, it can be easily adulterated with flavors or cheaper oils. This study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil and at their authenticity assessment. METHODS Nineteen commercial samples of R. damascena essential oil of different geographic origin and an additional authentic one, directly extracted by hydro-distillation from fresh flowers, were considered. GC/MS and GC/FID techniques were applied for the phytochemical analysis of the samples. EA/IRMS (Elemental Analyzer/Isotope Ratio Mass Spectrometry) and GC/C (Combustion)/IRMS were used to determine the δ(13)C composition of bulk samples and of some specific components. RESULTS Citronellol (28.7-55.3%), geraniol (13.5-27.3%) and nonadecane (2.6-18.9%) were the main constituents of Bulgarian and Turkish essential oils, while those from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.7-23.2%). The δ(13)C values of bulk samples were between -28.1 and -26.9‰, typical for C3 plants. The δ(13)C values of specific components were in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate, which displayed higher values (up to -18‰). These unusual δ(13)C values were explained by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa), which was found to occur in most of the essential oils. CONCLUSION GC/C/IRMS, in combination with GC/MS and GC/FID, can be considered as an effective and reliable tool for the authenticity control of R. damascena essential oil.

Botanical traceability of commercial tannins using the mineral profile and stable isotopes

Commercial tannins are natural polyphenolic compounds extracted from different plant tissues such as gall, the wood of different species and fruit. In the food industry they are mainly used as flavourings and food ingredients, whereas in winemaking they are classified as clarification agents for wine protein stabilisation, although colour stabilisation, metal removal, unpleasant thiol removal and rheological correction are also well-known and desired effects. Due to their particular technical properties and very different costs, the possibility of correct identification of the real botanical origin of tannins can be considered a primary target in oenology research and in fulfilling the technical and economic requirements of the wine industry. For some categories of tannins encouraging results have already been achieved by considering sugar or polyphenolic composition. For the first time this work verifies the possibility of determining the botanical origin of tannins on the basis of the mineral element profile and analysis of the (13) C/(12) C isotopic ratio. One hundred two commercial tannins originating from 10 different botanical sources (grapes, oak, gall, chestnut, fruit trees, quebracho, tea, acacia, officinal plants and tara) were analysed to determine 57 elements and the (13) C/(12) C isotopic ratio, using inductively coupled plasma mass spectrometry and isotope-ratio mass spectrometry, respectively. Forward stepwise discriminant analysis provided good discrimination between the 8 most abundant groups, with 100% correct re-classification. The model was then validated five times on subsets of 10% of the overall samples, randomly extracted, achieving satisfactory results. With a similar approach it was also possible to distinguish toasted and untoasted oak tannins as well as tannins from grape skin and grape seeds.

Multi-isotopic signatures of organic and conventional Italian pasta along the production chain.

The variability of stable isotope ratios (δ(2) H, δ(13) C, δ(15) N, δ(18) O and δ(34) S) along the production chain of pasta (durum wheat, flour and pasta) produced by using both conventional and organic farming systems in four Italian regions in 2 years was investigated. The aim was to evaluate if and how the farming system and geographical origin affect stable isotope ratios determined along the production chain. Irrespective of the processing technology, 65% of the samples were correctly classified according to the farming system and 98% were correctly classified regarding the geographical region. When considering both farming system and geographical region simultaneously, 80% of the samples were correctly classified. The measured isotope parameters were thus primarily affected by the geographical origin. In conclusion, it is expected that the use of these parameters will allow the development of analytical control procedures that can be used to check the geographical origin of Italian organic and conventional pasta and its raw materials. Copyright

δ15N 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

Compound-specific δ 13 C and δ 2 H analysis of olive oil fatty acids

Fatty acids are the most abundant biomolecules in olive oil. In this study, we describe the analytical method developed to measure the δ13C, and for the first time, the δ2H values of fatty acid methyl esters (FAMEs) extracted from olive oil triglycerides, after base-catalysed transesterification with methanol. The extracted FAMEs are analysed using Gas Chromatography Combustion/Pyrolysis Isotope Ratio Mass Spectrometry (GC-C/Py-IRMS). The method made it possible to obtain baseline separation between the peaks of the main FAMEs (C16, C18, C18:1, C18:2). The accuracy, precision and uncertainty of the whole method (from preparation to measurement) were determined. Furthermore, the impact of methylation on δ13C and δ2H values was investigated. The obtained performance of the method is satisfying and allow the determination of reliable data. As δ13C and δ2H determined in bulk olive oils already demonstrated to be able on a some extent in distinguishing products according to their geographical origins, what it is expected applying this new method, is the improvement of the capacity of provenance differentiation.

Combined use of isotopic fingerprint and metabolomics analysis for the authentication of saw palmetto (Serenoa repens) extracts

Saw palmetto (Serenoa repens, SP) is the most expensive oil source of the pharmaceutical and healthfood market, and its high cost and recurrent shortages have spurred the development of designer blends of fatty acids to mimic its phytochemical profile and fraudulently comply with the current authentication assays. To detect this adulteration, the combined use of isotopic fingerprint and omic analysis has been investigated, using Principal Component Analysis (PCA) to handle the complex databases generated by these techniques and to identify the possible source of the adulterants. Surprisingly, the presence of fatty acids of animal origin turned out to be widespread in commercial samples of saw palmetto oil.

The use of stable isotope ratio analysis to characterise saw palmetto (Serenoa Repens) extract

Saw palmetto extract (SPE) has many pharmacological effects. Thus, its demand and value has increased steadily, along with the presence of counterfeit SPEs on the market. In this work bulk δ13C, δ2H, δ18O and fatty acid δ13C, δ2H analysis was performed in 20 authentic and 9 commercial SPEs, 12 meat fats and 4 pure fatty acids. Authentic SPEs are characterised by bulk values from -31.0‰ to -29.7‰ for δ13C, -176‰ to -165‰ for δ2H, 27.2‰ to 40.7‰ for δ18O, and values of capric, caprylic, lauric, myristic, palmitic and oleic acids from -37.4‰ to -30.5‰ for δ13C and -187‰ to -136‰ for δ2H. The isotopic values of all the commercial SPEs were out of these ranges and more similar to those of meat fat and pure fatty acids. Stable isotope ratio analysis can therefore be proposed as a suitable tool for detecting adulteration in SPEs.

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.