Comparative study of ¹³C composition in ethanol and bulk dry wine using isotope ratio monitoring by mass spectrometry and by nuclear magnetic resonance as an indicator of vine water status.

The potential of wine (13)C isotope composition (δ(13)C) is presented to assess vine water status during grape ripening. Measurements of δ(13)C have been performed on a set of 32 authentic wines and their ethanol recovered after distillation. The data, obtained by isotope ratio monitoring by mass spectrometry coupled to an elemental analyser (irm-EA/MS), show a high correlation between δ(13)C of the bulk wine and its ethanol, indicating that the distillation step is not necessary when the wine has not been submitted to any oenological treatment. Therefore, the ethanol/wine δ(13)C correlation can be used as an indicator of possible enrichment of the grape must or the wine with exogenous organic compounds. Wine ethanol δ(13)C is correlated to predawn leaf water potential (R(2) = 0.69), indicating that this parameter can be used as an indicator of vine water status. Position-specific (13)C analysis (PSIA) of ethanol extracted from wine, performed by isotope ratio monitoring by nuclear magnetic resonance (irm-(13)C NMR), confirmed the non-homogenous repartition of (13)C on ethanol skeleton. It is the δ(13)C of the methylene group of ethanol, compared to the methyl moiety, which is the most correlated to predawn leaf water potential, indicating that a phase of photorespiration of the vine during water stress period is most probably occurring due to stomata closure. However, position-specific (13)C analysis by irm-(13)C NMR does not offer a greater precision in the assessment of vine water status compared to direct measurement of δ(13)C on bulk wine by irm-EA/MS.

(13)C/(12)C isotope ratios of organic acids, glucose and fructose determined by HPLC-co-IRMS for lemon juices authenticity.

High performance liquid chromatography linked to isotope ratio mass spectrometry via an interface allowing the chemical oxidation of organic matter (HPLC-co-IRMS) was used to simultaneously determine carbon 13 isotope ratio (δ(13)C) of organic acids, glucose and fructose in lime and lemon juices. Because of the significant difference between organic acids and sugars concentrations, the experimental protocol was optimised by applying a "current jump" to the IRMS device. The filament current is increased of 300µA during elution in order to enhance IRMS sensitivity. Then, analysis were performed on 35 lemon and lime fruits from various geographical origins and squeezed in the laboratory. An overall average δ(13)C values of -25.40±1.62‰, -23.83±1.82‰ and -25.67±1.72‰ is found for organic acids mixture mainly made up of citric acid, glucose and fructose, respectively. These authentic samples allowed the definition of a confidence domain to which have been confronted 30 commercial juices (24 "pure juices" and 6 coming from concentrate). Among these 30 samples, 10 present δ(13)C values outside the defined range revealing an added "C4" type organic acids or sugars, addition not specified on the label that is not in agreement with EU regulation.

Potential of ion chromatography coupled to isotope ratio mass spectrometry via a liquid interface for beverages authentication.

New tools for the determination of characteristic parameters for food authentication are requested to prevent food adulteration from which health concerns, unfair competition could follow. A new coupling in the area of compound-specific carbon 13 isotope ratio (δ(13)C) analysis was developed to simultaneously quantify δ(13)C values of sugars and organic acids. The coupling of ion chromatography (IC) together with isotope ratio mass spectrometry (IRMS) can be achieved using a liquid interface allowing a chemical oxidation (co) of organic matter. Synthetic solutions containing 1 polyol (glycerol), 3 carbohydrates (sucrose, glucose and fructose) and 12 organic acids (gluconic, lactic, malic, tartaric, oxalic, fumaric, citric and isocitric) were used to optimize chromatographic conditions (concentration gradient and 3 types of column) and the studied isotopic range (-32.28 to -10.65‰) corresponds to the values found in food products. Optimum chromatographic conditions are found using an IonPac AS15, an elution flow rate of 0.3mLmin(-1) and a linear concentration gradient from 2 to 76mM (rate 21mMmin(-1)). Comparison between δ(13)C value individually obtained for each compound with the coupling IRMS and elemental analyzer, EA-IRMS, and the ones measured on the mixture of compounds by IC-co-IRMS does not reveal any isotope fractionation. Thus, under these experimental conditions, IC-co-IRMS results are accurate and reproducible. This new coupling was tested on two food matrices, an orange juice and a sweet wine. Some optimization is necessary as the concentration range between sugars and organic acids is too large: an increase in the filament intensity of the IRMS is necessary to simultaneously detect the two compound families. These first attempts confirm the good results obtained on synthetic solutions and the strong potential of the coupling IC-co-IRMS in food authentication area.

Authenticity of carbon dioxide bubbles in French ciders through multiflow-isotope ratio mass spectrometry measurements.

A procedure to detect whether carbon dioxide was added to French ciders has been developed. For this purpose, an optimised and simplified method is proposed to determine (13)C/(12)C isotope ratio of carbon dioxide (δ(13)C) in ciders. Three critical steps were checked: (1) influence of atmospheric CO2 remaining in the loaded vial, (2) impact of helium flush, (3) sampling speed. This study showed that atmospheric CO2 does not impact the measurement, that helium flush can lead to isotopic fractionation and finally, that a fractionation occurs only 5h after bottle opening. The method, without any other preparation, consists in sampling 0.2 mL of cold (4 °C) cider in a vial that is passed in an ultrasonic bath for 10 min at room temperature to enhance cider de-carbonation. The headspace CO2 is then analysed using the link Multiflow®-isotope ratio mass spectrometer. Each year, a data bank is developed by fermenting authentic apples juices in order to control cider authenticity. Over a four year span (2008-2011), the CO2 produced during the fermentation step was studied. This set of 61 authentic ciders, from various French production areas, was used to determine a δ(13)C value range of -22.59±0.92‰ for authentic ciders CO2 bubbles. 75 commercial ciders were analysed with this method. Most of the samples analysed present a gas δ(13)C value in the expected range. Nevertheless, some ciders have δ(13)C values outside the 3σ limit, revealing carbonation by technical CO2. This practice is not allowed for organic, "Controlled Appellation of Origin" ciders and ciders specifying natural carbonation on the label.

Intrinsic ratios of glucose, fructose, glycerol and ethanol 13C/12C isotopic ratio determined by HPLC-co-IRMS: toward determining constants for wine authentication.

High-performance liquid chromatography linked to isotope ratio mass spectrometry (HPLC-co-IRMS) via a Liquiface© interface has been used to simultaneously determine (13)C isotope ratios of glucose (G), fructose (F), glycerol (Gly) and ethanol (Eth) in sweet and semi-sweet wines. The data has been used the study of wine authenticity. For this purpose, 20 authentic wines from various French production areas and various vintages have been analyzed after dilution in pure water from 20 to 200 times according to sugar content. If the (13)C isotope ratios vary according to the production area and the vintage, it appears that internal ratios of (13)C isotope ratios (R((13)C)) of the four compounds studied can be considered as a constant. Thus, ratios of isotope ratios are found to be 1.00 ± 0.04 and 1.02 ± 0.08 for R((13)C(G/F)) and R((13)C(Gly/Eth)), respectively. Moreover, R((13)C(Eth/Sugar)) is found to be 1.15 ± 0.10 and 1.16 ± 0.08 for R((13)C(Gly/Sugar)). Additions of glucose, fructose and glycerol to a reference wine show a variation of the R((13)C) value for a single product addition as low as 2.5 g/L(-1). Eighteen commercial wines and 17 concentrated musts have been analyzed. Three wine samples are suspicious as the R((13)C) values are out of range indicating a sweetening treatment. Moreover, concentrated must analysis shows that (13)C isotope ratio can be also used directly to determine the authenticity of the matrix.

Quantification method and organoleptic impact of added carboxymethyl cellulose to dry white wine.

Following the resolution Oeno 2-2008 of the OIV, "Organisation Internationale de la Vigne et du Vin", the addition of carboxymethyl cellulose (CMC) in wines at a concentration lower than 100 mg L-1 is authorized in the EU (RCE 606-2009). In order to control this level, a method has been elaborated to quantify CMC addition at the maximum limit authorized. It is based on the absorbance level of a coloured complex obtained by the addition of a solution of 2,7-dihydronaphthalene in concentrated sulfuric acid to a dry white wine that has been dialyzed; the proposed method has a quantification limit of 60 mg L-1 with a 40 mg L-1 uncertainty. Despite these relatively high values, the method is, nevertheless, suitable for its purpose. Some wines, elaborated and CMC supplemented, allowed one to test the CMC quantification method, in real conditions of wine production. All the measurements provide a CMC quantification at the expected level, within the uncertainty. These samples, sets of treated and untreated wines, were submitted to sensory evaluation to assess the impact of CMC addition to wine. At the maximum authorized level, no significant difference was detected by the 20 expert panel under quality control requirements. Moreover, some experiments, performed to determine the concentration threshold at which the CMC effect is noticeable, showed that even at a level 15 times higher than the maximum amount authorized, CMC has no impact on the taste or the nose of the wines.

Characterization and quantification of grape variety by means of shikimic acid concentration and protein fingerprint in still white wines.

Protein profiles, obtained by high-performance capillary electrophoresis (HPCE) on white wines previously dialyzed, combined with shikimic acid concentration and multivariate analysis, were used for the determination of grape variety composition of a still white wine. Six varieties were studied through monovarietal wines elaborated in the laboratory: Chardonnay (24 samples), Chenin (24), Petit Manseng (7), Sauvignon (37), Semillon (24), and Ugni Blanc (9). Homemade mixtures were elaborated from authentic monovarietal wines according to a Plackett-Burman sampling plan. After protein peak area normalization, a matrix was elaborated containing protein results of wines (mixtures and monovarietal). Partial least-squares processing was applied to this matrix allowing the elaboration of a model that provided a varietal quantification precision of around 20% for most of the grape varieties studied. The model was applied to commercial samples from various geographical origins, providing encouraging results for control purposes.

Effects of must concentration techniques on wine isotopic parameters.

Despite the robustness of isotopic methods applied in the field of wine control, isotopic values can be slightly influenced by enological practices. For this reason, must concentration technique effects on wine isotopic parameters were studied. The two studied concentration techniques were reverse osmosis (RO) and high-vacuum evaporation (HVE). Samples (must and extracted water) have been collected in various French vineyards. Musts were microfermented at the laboratory, and isotope parameters were determined on the obtained wine. Deuterium and carbon-13 isotope ratios were studied on distilled ethanol by nuclear magnetic resonance (NMR) and isotope ratio mass spectrometry (IRMS), respectively. The oxygen-18 ratio was determined on extracted and wine water using IRMS apparatus. The study showed that the RO technique has a very low effect on isotopic parameters, indicating that this concentration technique does not create any isotopic fractionation, neither at sugar level nor at water level. The effect is notable for must submitted to HVE concentration: water evaporation leads to a modification of the oxygen-18 ratio of the must and, as a consequence, ethanol deuterium concentration is also modified.

Alternative method for the quantification by gas chromatography triacylglycerol class analysis of cocoa butter equivalent added to chocolate bars.

Directive 2000/36/EC allows chocolate makers to add up to 5% of only six specific cocoa butter equivalents (CBEs) to cocoa butter (CB). A quantification method based on triacylglycerol (TAG) class analysis by gas chromatography with an unpolar column was set up for routine control purposes of chocolate bars. Mixtures of CBEs/CB were elaborated according to a Placket-Burman experiment design and analyzed by gas chromatography. A matrix was built with the normalized values of TAG classes (C50, C52, C54, and C56) of pure CBs of various origins, homemade CB/CBE mixtures (1 CB type), and mixtures containing CBE with CBs of various origins. A multivariate calibration equation was computed from this matrix using a partial least-squares regression technique. CBE addition can be detected at a minimum level of 2%, and the mathematical model allows its quantification with an uncertainty of 2% with respect to the cocoa butter fats. The model has also been applied for deconvolution and quantification of each CBE of a CBE mixture in chocolate bars.