Gas chromatography-stable isotope ratio mass spectrometry prior solid phase microextraction and gas chromatography-tandem mass spectrometry: development and optimization of analytical methods to analyse garlic (Allium sativum L.) volatile fraction.

Garlic (Allium sativum L.) is not only appreciated for its flavour and taste, but it is also recognized for various health properties. The European Commission, through the attribution of the Protected Designation of Origin (PDO) certification mark, has officially recognized some specific varieties of garlic. To protect not only the commercial value but also the reputation of this appreciated product, effective tools are therefore required. For the first time, a new compound specific isotope analysis method based on carbon stable isotopic ratio measurement of the three major volatile garlic compounds allyl alcohol (AA), diallyl disulphide (DD) and diallyl trisulphide (DT) through head-space solid phase microextraction (HS-SPME) followed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was developed. A within-day standard deviation (Srwithin-day) of 0.3 ‰, 0.4 ‰ and 0.2 ‰ for δ(13C) and a between-day standard deviation (Srbetween-day) of 0.8 ‰, 1.0 ‰ and 0.6 ‰ of AA, DT and DD was estimated. For the first time, the ranges of isotopic variability for the three volatile compounds of red garlic from two neighbouring Italian regions (Abruzzo and Lazio) were defined analysing 30 samples. The same dataset was also considered in analysing the percentage composition of the previously mentioned three volatile compounds through HS-SPME followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The two analytical approaches were combined in this explorative study, aiming to provide potential parameters to discriminate garlic samples based on their geographical origin.

Influence of flash heating and aspergillopepsin I supplementation on must and wine attributes of aromatic varieties.

The protein instability with haze formation represents one of the main faults occurring in white and rosé wines. Among the various solutions industrially proposed, aspergillopepsin I (AP-I) supplementation coupled with must heating (60-75 °C) has been recently approved by OIV and the European Commission for ensuring protein stability of wines. This study investigates the impact of AP-I either applied independently or in combination with flash pasteurization on the chemical composition of grape must and wines derived from Sauvignon Blanc and Gewürztraminer. The efficacy on protein stability of a complete treatment combining heat (70 °C) and AP-I (HP) was confirmed through heat test and bentonite requirement, although no differences were observed between must heating and HP treatments. However, high-performance liquid chromatography analysis of unstable pathogenesis-related proteins revealed that AP-I supplementation reduced chitinases and thaumatin-like proteins compared to the non-enzymed samples, with and without must heating. Amino acid increase was reported only in HP musts, particularly in Sauvignon Blanc. The concentration of yeast-derived aroma compounds in Gewürztraminer wines was increased by must heating; compared to controls, flash pasteurization rose the overall acetate esters content of 85 % and HP of 43 %, mostly due to isoamyl acetate. However, heat treatments -with or without AP-I- reduced terpenes up to 68 %. Despite the different aroma profiles, no differences were observed for any descriptor for both varieties in wine tasting, and only a slight decrease trend was observed for the floral intensity and the typicality descriptors in heated wines.

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.

Myo-Inositol, Scyllo-Inositol, and Other Minor Carbohydrates as Authenticity Markers for the Control of Italian Bulk, Concentrate, and Rectified Grape Must.

Myo-inositol polyalcohol is a characteristic component of natural and concentrated grape musts (CMs), and Regulation (EU) no. 1308/2013 prescribes its presence as a marker of the authenticity of rectified concentrated must (RCM). Other polyalcohols besides myo-inositol, such as scyllo-inositol or minor sugars, could be considered authenticity markers, but an extensive search in the literature yielded no exhaustively investigated study of their concentration variability in genuine products. The aim of this study was to create an extensive national data bank of minor carbohydrates profiles and investigate the impact of the geographical origin and the different vintages on the concentration of these compounds; to this end, 450 authentic Italian grape musts of different varieties were sampled and analyzed during the harvest season in 2019, 2020, and 2021. The grape musts from the Italian wine-growing areas CII and CIIIb had myo- and scyllo-inositol contents always higher than 756 and 39 mg/kg of sugar, respectively. Conversely, also considering other mono- and disaccharides, sucrose, sorbitol, lactose, maltose, and isomaltose showed contents always lower than 534, 1207, 390, 2222, and 1639 mg/kg of sugar, respectively. The general applicability to the CM and RCM of the proposed authenticity thresholds, established in the must, was demonstrated by studying the influence of must concentration on the myo- and scyllo-inositol content. Inter-laboratory comparison experiments were also conducted to harmonize and characterize laboratory methods and validate the analytical dataset. Based on the obtained results, the text of the EU legislation (Reg. (EU) 1308/2013), which defines the characteristics of the must and the CRM product, should be revised.

Fatty acids stable carbon isotope fractionation in the bovine organism. A compound-specific isotope analysis through gas chromatography combustion isotope ratio mass spectrometry.

The contribution of dietary fatty acids to the quality of the meat and their path through the bovine organism is currently the subject of a lot of research. Stable isotope ratio analysis represents a powerful tool for this aim, one that has not been studied in depth yet. In this work, for the first time, the carbon isotopic ratios of six fatty acids (myristic 14:0, palmitic 16:0, stearic 18:0, oleic 18:1n-9, linoleic 18:2n-6 and linolenic 18:3n-3 acids) in different matrixes (diet, rumen, duodenal content, liver and loin) were analysed through gas chromatography combustion isotope ratio mass spectrometry. Moreover, the quantification of the single fatty acids was carried out, providing important information supporting the carbon isotopic ratio results. The variation in the concentration of the fatty acids in the different matrices depends on the chemical modifications they undergo in the sequential steps of the metabolic path. GC-C-IRMS turned out to be a powerful tool to investigate the fate of dietary fatty acids, providing information about the processes they undergo inside the bovine organism.

Bacterial Complexity of Traditional Mountain Butter Is Affected by the Malga-Farm of Production.

Bacteria can play different roles affecting flavors and food characteristics. Few studies have described the bacterial microbiota of butter. In the present paper, next-generation sequencing was used to determine bacterial diversity, together with aromatic characteristics, in raw cow milk butter processed by traditional fermentation, in fourteen small farms called "Malga", located in the Trentino province (Alpine region, North-East of Italy). The physicochemical and aromatic characterization of traditional mountain butter (TMB) showed a low moisture level depending on the Malga producing the butter. Counts of lactic acid bacteria, Staphylococci, and coliforms, as well as diacetyl/acetoin concentrations exhibited changes according to the geographical origin of Malga and the residual humidity of butter. MiSeq Illumina data analysis revealed that the relative abundance of Lactococcus was higher in TMB samples with the highest values of acetoin (acetoin higher than 10 mg/kg). The traditional mountain butter bacterial community was characterized by a "core dominance" of psychrotrophic genera, mainly Acinetobacter and Pseudomonas, but according to ANCOM analysis, a complex bacterial population emerged and specific bacterial genera were able to characterize the TMB bacteria community, with their high abundance, based on the Malga producing the butter.

Bulk and compound-specific stable isotope ratio analysis for authenticity testing of organically grown tomatoes.

Until now, there has been a lack of analytical methods that can reliably verify the authenticity of organically grown plants and derived organic food products. In this study, stable isotope ratio analysis of hydrogen (H, δ2H), carbon (C, δ13C), nitrogen (N, δ15N), oxygen (O, δ18O) and sulfur (S, δ34S) was conducted along the tomato passata production process using organic and conventionally grown tomatoes from two Italian regions over two years. A gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) based method was developed and applied for analysis of C and N isotope ratios in amino acids derived from tomatoes. Of the bulk isotope ratios, δ15N was the most significant parameter for discriminating organic from conventional products. The classification power was improved significantly by compound-specific isotope analysis regardless of the production years and regions. We conclude that isotope analysis of amino acids is a novel analytical tool for complementing existing certification and control procedures in the organic tomato sector.

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.

Development of a fast gas chromatography-tandem mass spectrometry method for volatile aromatic compound analysis in oenological products.

Aroma plays an important role in food quality, and knowledge of volatile composition is essential to support technological choices during industrial transformation processes. In this study, an innovative GC-MS/MS method useful for fast analysis of the broad aroma profile of oenological products was developed. Fifty volatile compounds of oenological interest belonging to the main chemical classes (acetates, acids, alcohols, aldehydes, esters, ketones, phenols, terpenes, and thiols) were identified and quantified in a single chromatographic run taking only 15 minutes. The high sensitivity of the mass spectrometer, combined with accurate fast chromatography performed with a suitable capillary column, allowed us to avoid further concentration steps after solid phase extraction. Validation of the method was performed in relation to the limit of detection, limit of quantification, linearity range, repeatability, reproducibility, and recovery. Once validated, the method was successfully tested on commercial oenological samples, demonstrating its applicability to fast routine multiaroma analysis.

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.

Differentiation of wood-derived vanillin from synthetic vanillin in distillates using gas chromatography/combustion/isotope ratio mass spectrometry for δ13 C analysis.

RATIONALE: Typical storage in oak barrels releases in distillates different degradation products such as vanillin, which play an important role in flavour and aroma. The addition of vanillin, as well as other aroma compounds, of different origin is prohibited by European laws. As vanillin samples from different sources have different δ13 C values, the δ13 C value could be used to determine whether the vanillin is authentic (lignin-derived), or if it has been added from another source (e.g. synthetic). METHODS: The δ13 C values for vanillin derived from different sources, including natural, synthetic and tannins, were measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), after diethyl ether addition and/or ethanol dilution. A method for analysing vanillin in distillates after dichloromethane extraction was developed. Tests were undertaken to prove the reliability, reproducibility and accuracy of the method with standards and samples. Distillate samples were run to measure the δ13 C values of vanillin and to compare them with values for other sources of vanillin. RESULTS: δ13 C values were determined for: natural vanillin extracts (-21.0 to -19.3‰, 16 samples); vanillin ex-lignin (-28.2‰, 1 sample); and synthetic vanillin (-32.6 to -29.3‰, 7 samples). Seventeen tannin samples were found to have δ13 C values of -29.5 to -26.7‰, which were significantly different (p < 0.05) from those of the natural and synthetic vanillins. The vanillin δ13 C values measured in distillates (-28.9 to -25.7‰) were mainly in the tannin range, although one spirit (-32.5‰) was found to contain synthetic vanillin. CONCLUSIONS: The results show that synthetic vanillin added to a distillate could be differentiated from vanillin derived from oak barrels by their respective δ13 C values. The GC/C/IRMS method could be a useful tool in the determination of adulteration of distillates.

Compound-specific δ13C and δ2H 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.

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.

From soil to grape and wine: Variation of light and heavy elements isotope ratios.

In the development of a geographical traceability model, it is necessary to understand if the value of the monitored indicators in a food is correlated to its origin or if it is also influenced by 'external factors' such as those coming from its production. In this study, a deeper investigation of the trend of direct geographical traceability indicators along the winemaking process of two traditional oenological products was carried out. Different processes were monitored, sampling each step of their production (grape juice, intermediate products and wine). The results related to the determinations of δ(18)O, (D/H)I, (D/H)II, δ(13)C, δ(15)N and (87)Sr/(86)Sr have been reported. Furthermore, correspondence with the isotopic values coming from the respective soil and vine-branch samples have been investigated as well, showing the optimal traceability power of the monitored geographical tracers.

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 ß-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%.

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.

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.

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.