Freddy Thomas

Isotopic and Elemental Data for Tracing the Origin of European Olive Oils

H, C, and O stable isotope ratios and the elemental profile of 267 olive oils and 314 surface waters collected from 8 European sites are presented and discussed. The aim of the study was to investigate if olive oils produced in areas with different climatic and geological characteristics could be discriminated on the basis of isotopic and elemental data. The stable isotope ratios of H, C, and O of olive oils and the ratios of H and O of the relevant surface waters correlated to the climatic (mainly temperature) and geographical (mainly latitude and distance from the coast) characteristics of the provenance sites. It was possible to characterize the geological origin of the olive oils by using the content of 14 elements (Mg, K, Ca, V, Mn, Zn, Rb, Sr, Cs, La, Ce, Sm, Eu, U). By combining the 3 isotopic ratios with the 14 elements and applying a multivariate discriminant analysis, a good discrimination between olive oils from 8 European sites was achieved, with 95% of the samples correctly classified into the production site.

1H NMR-based protocol for the detection of adulterations of refined olive oil with refined hazelnut oil.

A (1)H NMR analytical protocol for the detection of refined hazelnut oils in admixtures with refined olive oils is reported according to ISO format. The main purpose of this research activity is to suggest a novel analytical methodology easily usable by operators with a basic knowledge of NMR spectroscopy. The protocol, developed on 92 oil samples of different origins within the European MEDEO project, is based on (1)H NMR measurements combined with a suitable statistical analysis. It was developed using a 600 MHz instrument and was tested by two independent laboratories on 600 MHz spectrometers, allowing detection down to 10% adulteration of olive oils with refined hazelnut oils. Finally, the potential and limitations of the protocol applied on spectrometers operating at different magnetic fields, that is, at the proton frequencies of 500 and 400 MHz, were investigated.

Determination of origin of Atlantic salmon (Salmo salar): the use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin.

Variability within the stable isotope ratios in various lipidic fractions and the fatty acid composition of muscle oil has been analyzed for a large sample (171 fish) of wild and farmed Atlantic salmon ( Salmo salar) from 32 origins within Europe, North America, and Tasmania. Sampling was extended over all seasons in 2 consecutive years and included fish raised by different practices, in order to maximize the range of variation present. It is shown that two readily measured parameters, delta 15N measured on choline and delta18 O measured on total oil, can be successfully used to discriminate between fish of authentic wild and farmed origin. However, the certainty of identification of mislabeling in market-derived fish is strengthened by including the percentage of linoleic acid C18:2n-6 in the lipidic fraction. Thus, several apparent misidentifications were found. The combination of these three analytical parameters and the size of the database generated makes the method practical for implementation in official laboratories as a tool of labeling verification.

Isotopic finger-printing of active pharmaceutical ingredients by 13C NMR and polarization transfer techniques as a tool to fight against counterfeiting.

The robustness of adiabatic polarization transfer methods has been evaluated for determining the carbon isotopic finger-printing of active pharmaceutical ingredients. The short time stabilities of the adiabatic DEPT and INEPT sequences are very close to that observed with the one pulse sequence, but the DEPT long time stability is not sufficient for isotopic measurements at natural abundance or low enrichment. Using the INEPT sequence for (13)C isotopic measurements induces a dramatic reduction in the experimental time without deterioration in short time or long time stability. It appears, therefore, to be a method of choice for obtaining the isotopic finger-print of different ibuprofen samples in a minimum time. The results obtained on 13 commercial ibuprofen samples from different origins show that this strategy can be used effectively to determine (13)C distribution within a given molecule and to compare accurately differences in the isotopic distribution between different samples of the given molecule. The present methodology is proposed as a suitable tool to fight against counterfeiting.

Rapid approach to identify the presence of Arabica and Robusta species in coffee using 1H NMR spectroscopy

NMR spectroscopy was used to verify the presence of Arabica and Robusta species in coffee. Lipophilic extracts of authentic roasted and green coffees showed the presence of established markers for Robusta (16-O-methylcafestol (16-OMC)) and for Arabica (kahweol). The integration of the 16-OMC signal (δ 3.165 ppm) was used to estimate the amount of Robusta in coffee blends with an approximate limit of detection of 1-3%. The method was successfully applied for the analysis of 77 commercial coffee samples (coffee pods, coffee capsules, and coffee beans). Furthermore, principal component analysis (PCA) was applied to the spectra of lipophilic and aqueous extracts of 20 monovarietal authentic samples. Clusters of the two species were observed. NMR spectroscopy can be used as a rapid prescreening tool to discriminate Arabica and Robusta coffee species before the confirmation applying the official method.

Fast and global authenticity screening of honey using 1H-NMR profiling

An innovative analytical approach was developed to tackle the most common adulterations and quality deviations in honey. Using proton-NMR profiling coupled to suitable quantification procedures and statistical models, analytical criteria were defined to check the authenticity of both mono- and multi-floral honey. The reference data set used was a worldwide collection of more than 800 honeys, covering most of the economically significant botanical and geographical origins. Typical plant nectar markers can be used to check monofloral honey labeling. Spectral patterns and natural variability were established for multifloral honeys, and marker signals for sugar syrups were identified by statistical comparison with a commercial dataset of ca. 200 honeys. Although the results are qualitative, spiking experiments have confirmed the ability of the method to detect sugar addition down to 10% levels in favorable cases. Within the same NMR experiments, quantification of glucose, fructose, sucrose and 5-HMF (regulated parameters) was performed. Finally markers showing the onset of fermentation are described.

2H NMR and 13C-IRMS analyses of acetic acid from vinegar, 18O-IRMS analysis of water in vinegar: international collaborative study report.

An international collaborative study of isotopic methods applied to control the authenticity of vinegar was organized in order to support the recognition of these procedures as official methods. The determination of the 2H/1H ratio of the methyl site of acetic acid by SNIF-NMR (site-specific natural isotopic fractionation-nuclear magnetic resonance) and the determination of the 13C/12C ratio, by IRMS (isotope ratio mass spectrometry) provide complementary information to characterize the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic acetic acid. Both methods use the same initial steps to recover pure acetic acid from vinegar. In the case of wine vinegar, the determination of the 18O/16O ratio of water by IRMS allows to differentiate wine vinegar from vinegars made from dried grapes. The same set of vinegar samples was used to validate these three determinations. The precision parameters of the method for measuring delta13C (carbon isotopic deviation) were found to be similar to the values previously obtained for similar methods applied to wine ethanol or sugars extracted from fruit juices: the average repeatability (r) was 0.45 per thousand, and the average reproducibility (R) was 0.91 per thousand. As expected from previous in-house study of the uncertainties, the precision parameters of the method for measuring the 2H/1H ratio of the methyl site were found to be slightly higher than the values previously obtained for similar methods applied to wine ethanol or fermentation ethanol in fruit juices: the average repeatability was 1.34 ppm, and the average reproducibility was 1.62 ppm. This precision is still significantly smaller than the differences between various acetic acid sources (delta13C and delta18O) and allows a satisfactory discrimination of vinegar types. The precision parameters of the method for measuring delta18O were found to be similar to the values previously obtained for other methods applied to wine and fruit juices: the average repeatability was 0.15 per thousand, and the average reproducibility was 0.59 per thousand. The above values are proposed as repeatability and reproducibility limits in the current state of the art. On the basis of this satisfactory inter-laboratory precision and on the accuracy demonstrated by a spiking experiment, the authors recommend the adoption of the three isotopic determinations included in this study as official methods for controlling the authenticity of vinegar.

Improved Characterization of the Botanical Origin of Sugar by Carbon-13 SNIF-NMR Applied to Ethanol

Until now, no analytical method, not even isotopic ones, had been able to differentiate between sugars coming from C4-metabolism plants (cane, maize, etc.) and some crassulacean acid metabolism plants (e.g., pineapple, agave) because in both cases the isotope distributions of the overall carbon-13/carbon-12 and site-specific deuterium/hydrogen isotope ratios are very similar. Following recent advances in the field of quantitative isotopic carbon-13 NMR measurements, a procedure for the analysis of the positional carbon-13/carbon-12 isotope ratios of ethanol derived from the sugars of pineapples and agave using the site-specific natural isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) method is presented. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. When applied to pineapple juice, this new method demonstrates a unique ability to detect cane and maize sugar, which are major potential adulterants, with a detection limit in the order of 15% of the total sugars, which provides an efficient mean of controlling the authenticity of juices made from this specific fruit. When applied to tequila products, this new method demonstrates a unique ability to unambiguously differentiate authentic 100% agave tequila, as well as misto tequila (made from at least 51% agave), from products made from a larger proportion of cane or maize sugar and therefore not complying with the legal definition of tequila.

NMR spectrometry isotopic fingerprinting: A tool for the manufacturer for tracking Active Pharmaceutical Ingredients from starting materials to final medicines

In the frame of increasingly stringent quality assessment required by the regulators, the pharmaceutical industry has to face increasingly sophisticated counterfeiting practices. Counterfeits based on deliberate copying of processes or on the infringement of current patents for generic medicines are not straightforward to detect, unless the molecular probe is the active molecule itself. In this context, impurity profiling is limited. A tool based on the determination of intramolecular isotopic profiles has been developed to provide manufacturers of APIs (Active Pharmaceutical Ingredients) with a new solution to meet this double requirement. Stable isotope analyses by NMR gives direct access to site-specific isotope content at natural abundance. In this report, it is shown how both ²H and ¹³C NMR spectrometry can provide complementary and valuable information that could be applied to link APIs to their manufacturing source. Isotopic ¹³C NMR offers additional benefits over ²H NMR in using robust adiabatic polarization transfer methods, leading to a tremendous reduction in experimental time. Two approaches are illustrated. Firstly, the use of ²H and single pulse ¹³C NMR spectra obtained on 20 commercial ibuprofen samples from different origins show that this combined strategy leads to (i) a unique intramolecular isotope identification and (ii) a preliminary classification of the samples according to the synthetic pathways of the main industrial processes. An approach employing polarization transfer methods applied to 11 commercial naproxen samples, for which ²H and single pulse ¹³C NMR spectra are not exploitable and/or are not accessible in reasonable time. The relative and partial intramolecular ¹³C distribution obtained on naproxen by applying this methodology is sufficiently informative to allow the same conclusions as for ibuprofen. The additional benefits that these approaches should bring to API manufacturers are discussed.

Combination of 1H NMR and chemometrics to discriminate manuka honey from other floral honey types from Oceania

Manuka honey is a product produced essentially in New Zealand, and has been widely recognised for its antibacterial properties and specific taste. In this study, 264 honeys from New Zealand and Australia were analysed using proton NMR spectroscopy coupled with chemometrics. Known manuka markers, methylglyoxal and dihydroxyacetone, have been characterised and quantified, together with a new NMR marker, identified as being leptosperin. Manuka honey profiling using 1H NMR is shown to be a possible alternative to chromatography with the added advantage that it can measure methylglyoxal (MGO), dihydroxyacetone (DHA) and leptosperin simultaneously. By combining the information from these three markers, we established a model to estimate the proportion of manuka in a given honey. Markers of other botanical origins were also identified, which makes 1H NMR a convenient and efficient tool, complementary to pollen analysis, to control the botanical origin of Oceania honeys.