RO128662A2 - Process for isotopic fingerprinting of flavour compounds in liquid samples, in particular wine - Google Patents

Abstract

The invention relates to a method for identifying the wine flavour compounds which define the wine flavour profile. According to the invention, the method consists in preparing the sample by solvent extraction in a target compounds ratio of 5:1...20:1, concentrating the extracts to a volume of 50...2000 μL, qualitative identification by gas chromatography coupled with mass spectrometry, evaluating the global isotopic ratios for each component and establishing the working parameters for the compound-specific isotopic fingerprint, isotopic labeling with C13, wherefrom, on the basis of the retention time established by injecting each compound separatedly and on the basis of the measured global isotopic fingerprint, there is created a measuring sequence, then the identified solvents are removed from the system and on the obtained chromatogram only the peaks of the compounds of interest remain, the values of these peaks being quantified.

Description

Deposit date

Process for isotopic printing of flavor compounds in liquid samples, in particular wine

Globally, in recent times intense efforts are being made to develop research aimed at finding high-performance analytical methods with application in food authentication, quality policy being focused mainly on the development of consumer protection systems by ensuring quality of raw materials used to obtain food, while encouraging protection against imitations and misuse of famous products known as such to the general consumer. Consumers are supported by providing information on the characteristics and composition of the products, as appropriate.

The present invention supports this desideratum, solving the problem of complete isotopic imprinting, globally and specifically on the compound, of a liquid, especially wine. Its principle is based on the fact that the natural ratio of stable isotopes in plant organic matter is not constant, but depends on the original isotopic content of the molecules used by the plant for photosynthesis (water and carbon dioxide) and the isotopic fractionation that takes place. along biosynthetic pathways. Both precursor molecules are characterized by specific isotopic ratios. Stable isotopes are atoms of the same element, with a different atomic weight by several units of mass, due to the different number of neutrons in the nucleus and which do not degrade by radioactive processes over time. To determine the authenticity of a product, taking into account stable isotopes (eg ¹⁸ O / ¹⁶ O, ² H / * H, ¹³ C / ¹² C, ¹⁵ N / ⁱ⁴ N, etc.), the determination of isotopic ratios (isotope ratio heavy and light of the same element) are certainly much more useful than other analytical investigations such as composition in microelements, because C and H are constituents of all organic compounds in liquid samples, especially wine, and O and N are also they are widely represented (oxygen from water to sugars, alcohols, acids, salts, flavors, polyphenols, etc. and nitrogen to peptides, amino acids, proteins, etc.).

The evaluation of the natural composition in stable isotopes of a liquid sample, in particular wine, is one of the best applications of analytical chemistry, through which we can establish the relationship between the finished product (eg wine) and raw materials (eg water and CO2). ) from their natural environment.

In nature, most bio-elements are mixtures of two or more stable isotopes. It was observed that in terms of isotopic content the variation

CX-2 Ο 1 1 - ο 1 3 - Ο 7 -12- 2011 of the starting tank and the isotopic fractionation associated with the physical processes, chemical steps and / or biochemical pathways involved during the formation of the molecule. For example, in the water cycle, during the evaporation of water from the oceans, the phenomenon of isotopic fractionation occurs, the impoverishment in the heavier isotope is observed in the vapor state in relation to the liquid one. A similar isotopic fractionation occurs during the evapo-transpiration process of plant water, with higher isotopic ratios being observed in plant water compared to groundwater.

We emphasize that the products obtained by chemical synthesis have an isotopic content dependent on that of the raw material used and on the thermodynamic and kinetic effects of the isotopic fractionation that take place during the synthesis.

The literature [1-4] presents a variety of methods for investigating liquids, especially wine, from general analytical methods [5, 6] to specific methods [7].

The present invention describes a process for isotopic impression of flavor compounds from liquid samples, especially wine, based on a sequence of steps, according to Figure 1, starting from sample preparation and extraction of target compounds (step I), their preliminary qualitative identification ( step II), determining the retention times and establishing the working parameters for the specific isotopic impression on the compound (step III) and ending with their global (step IV) and specific (step V) isotopic footprint.

An essential first step in the isotopic imprinting of flavor compounds is the extraction of the target compounds from the sample matrix so that the solvents used interfere as little as possible with the sample and can be removed as easily as possible in the final step.

Sample preparation (step I) involves the separation of volatile compounds from the liquid sample, in particular wine, by liquid-liquid extraction using organic solvents, for example pentane, hexane, petroleum ether and especially dichloromethane. A number of extractions are performed, at least three, with an individual duration between 10-60 min, having the volumetric ratios of the sample: solvent in a range of values of 5: 1 - 25: 1. The organic extracts are combined and dried with a desiccant (eg sodium sulphate or silica gel). The dry organic extract is concentrated to a final volume of 50-2000 μΐ, which is the sample for gas chromatographic determinations. The advantages of this method are the reduction of extraction time, the use of indigenous solvents, better reproducibility, simplicity and speed and low costs, not requiring sophisticated laboratory equipment or technical staff with special training.

gas chromatography coupled with spectrometry

From the extract thus obtained, the dextrin compounds (step II) are identified by the use of the a? '

Ο 1 1 - Ο 1 - Ο 7 -12- 2011

gas chromatograph used for this type of determination are: split (dilution of the injected sample): without split - split 50: 1, especially split of 10: 1; chromatographic column type: capillary column with stationary phase of medium or high polarity, in particular of polyethylene glycol; the volume of sample injected between 0.1 - 1 Ομί, especially Ιμΐ; injector temperature in the range 150 - 250 ° C, especially 190-200 ° C; the initial temperature of the chromatographic furnace between 5 ° C above ambient temperature and 50 ° C; the final temperature of the chromatographic furnace between 70 250 ° C, especially 150-200 ° C; the rate of increase of the temperature of the chromatographic furnace between 1 - 30 ° C / min, in particular 3 - 5 ° C / min. The operating parameters set for the mass spectrometer are: ionization mode - electronic impact with reading of the whole mass spectrum (Full EI); and mass reading range in the range 10 - 400 m / z, especially 30-150 m / z.

Flavor compounds shall be identified by a) comparing their mass spectra with a bank of mass spectra existing in the internal memory of the analysis instrument, b) comparing the mass spectra of flavor compounds with those of the reference compounds and by c ) comparing the retention times on the chromatographic column of the analyzes with the retention times of the reference compounds. Flavor compounds identified by this technique will be considered as target compounds in gas chromatographic analysis coupled with isotopic ratio mass spectrometry (GC-C-IRMS) - step V.

Determination of retention times and establishment of working parameters for the specific isotopic imprinting on the compound of volatile organic flavors / compounds in liquid samples, especially wine is characterized by the fact that it includes two steps: determination of retention times of solvents involved in sample preparation, materials certified reference marks, globally isotopically imprinted in step IV and setting the working parameters for both gas chromatograph, oxidation / reduction furnaces, regulation of reference gas flows, etc. The identification of the target compounds in step II allowed the choice of a stationary phase capillary column with the polarity of the sample constituents (polar eg TG-WAXMS) and their specific reference materials. The type of injection used is splitless, and to avoid widening the peak, the split is opened 10 seconds after injection, splitting flow 50 ml / min. Temperature gradients have been chosen to exploit the maximum column length: the longer the column length, the slower the temperature per minute (eg min. 3 ° C / min). Column pressure and eluent velocity (eg helium purity 99.9999%) is set to fit the capillary column diameter (min. 1 bar). To help focus the injected sample and obtain sharper peaks, a retention gap known as a pre-column or guard column is used, one piece

<λ ~ 2 Ο 1 1 - o 1 3 - o 7 -12- 2111 simple requires the use of a combustion interface between gas chromatograph and mass spectrometer, where the effluent (Helium as carrier and sample gas) is fed into a combustion reactor (oxidation). This reactor is a ceramic tube filled with CuO / NiO / Pt and maintained at a temperature of approx. 960 ° C. To remove water vapor generated during combustion, the effluent passes through a nylon hatch. Naphion is a fluorinated polymer that reacts as a semi-permanent membrane through which water is retained, while all other combustion products are retained in the carrier gas stream (Helium). Quantitative removal of water before the introduction of flue gases into the ion source is essential, as any water residue would lead to the “protonation” of CO2 gas producing HCO2 ⁺ , which interferes with CO2 analysis (isobaric interference). For each analysis sequence of a sample, it must be verified that the SD Standard Deviation at three successive measurements of the same sample is less than 0.6% o. The final result for the sample is given by the average value for three measurements. If the deviation is greater than 0.6% o, the measurement must be repeated. The C value of the working standard must not differ by more than 0.5% from the permitted (certified) value. otherwise, the spectrometer settings must be checked and adjusted if necessary.

Prior to the specific isotopic impression of the flavor compounds in the sample, an evaluation of the overall isotopic ratios (step IV) is performed on both the solvents involved in the preparation and extraction of the target compounds from the sample matrix and on the pure, undiluted flavor compounds / organic compounds. , used as reference materials, compounds which are subsequently used in the step of determining the retention times on the capillary column and establishing the working parameters for the specific isotopic impression on the compound.

The reference materials used are chemical compounds of purity for gas chromatography or equivalent, being divided into the following categories:

pure flavor compounds, which may include alcohols, acids, esters, terpenes, carbonyl compounds, etc. which can be found in liquid samples, especially wine;

organic solvents, which are represented by extraction solvents and solvents in which flavor compounds are dissolved, for example ethanol, methanol, acetone, ethyl ether; especially ethanol;

solutions of flavor compounds in organic solvents. The solubility of the flavor compound in the selected organic solvent must be as high as possible. Prepare solutions in the concentration range of ± 20% of the concentration values of the flavor compound in the liquid sample, in particular wine.

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For the compound-specific isotopic impression, the same extract obtained in step I and investigated in step II is introduced into the separation system of a GC-C / TC chromatograph coupled to an IRMS mass spectrometer. Gas chromatograph allows in a first phase the separation of the compounds from the extract on the chromatographic column and then the identification of the retention times on the column and the creation of a working method so that only the target compounds pass through the combustion or pyrolysis furnace, the rest being eliminated by the vacuum pump. preliminary. Measurements by isotopic mass spectrometry are performed by analyzing gaseous compounds (eg H2, CO2 or N2) obtained by combustion or pyrolysis of the sample to determine the stable isotope content of the component elements (eg hydrogen, carbon, oxygen or nitrogen).

The intensities of the ionic currents m / z corresponding to the isotopomers of the analyzed elements are measured by mass spectrometry both for the sample and for the standard used. The isotopic ratio is expressed as the deviation% ₀ from an international reference standard:

R - R

ΔΧ% ₀ = _x i 00 reference where X = ² Η / * Η, ^l3 C / ^l2 C, ¹⁵ N / ¹⁴ N and ¹⁸ O / ¹⁶ O, respectively, and R _pro ba and Rreference are the isotopic ratios for the sample and the standard of international reference. All the values of the isotopic ratios obtained will make an isotopic footprint specific to each compound, contributing to the creation of an aromatic profile typical of an authentic wine.

Figures 2 and 3 show experimental results on the specific imprint of flavor compounds.

The problem solved by the present invention is to provide a complementary method of authentication of food, liquid samples, especially wine, after specific flavor compounds, based on parameters that do not change over time or following technological processes applied to the product. .

Claims (4)

CLAIMS: 1. The method of extracting flavoring compounds from liquid samples, in particular wine, characterized in that it includes a) a number of extractions with an organic solvent of at least three, b) pooling and drying of the extracts, followed by c) their concentration at a final volume of 50-2000 μΐ. 2. The method of identifying target flavor compounds by gas chromatography coupled with mass spectrometry characterized in that the identification is performed in a sequence of steps a) by comparing the mass spectra of the compounds with the existing mass spectrum bank in memory internal of the analysis instrument, b) comparison of the mass spectra of the flavor compounds with those of the reference compounds and by c) comparison of the retention times on the chromatographic column of the analyzes with the retention times of the reference compounds. 3. Method of global isotopic imprinting by combustion on an elemental analyzer characterized in that the identification of global isotopic ratios for solvents involved in the preparation and extraction of target compounds from the sample matrix and pure, undiluted flavor compounds / volatile organic compounds used as reference, compounds that will be used later to determine the retention times on the capillary column and to establish the working parameters for the specific isotopic impression on the compound is done by combustion of the sample and reduction on the reduction column of the gas mixture obtained at CO ₂ where the mass spectrometer IRMS provides the global isotopic footprint of the compound (eg Carbon 13) and by ionizing the molecules by electronically bombarding the ion source of the spectrometer, a fragmentation process occurs, the molecules are "broken", dissociated into their component parts, forming fragments, free radicals , even atoms) characteristic of each mole resulting from an identity card in the form of a "fragmentation spectrum" where the fragments that form (ions) are accelerated by an electric or magnetic field, which separates them according to the mass / electric charge ratio (m / z ), then the detector converts the separated ions into electrical signals proportional to the number of ions, thus allowing the recording of the mass spectrum characteristic of each organic substance and the injection of the liquid sample (e.g. solvents, pure certified reference materials) is made using a microliter syringe, the amount injected being of the order of μΐ and where the eluent flow (Helium purity 99.9999%) through the elemental analyzer is at least 90 fgfS ^^^ oxygen through the reference ^x furnace (CO ₂ purity combustion of 250 ml / min for 1 second, presj ^ -2011-0 lj ^ 3 - 0 7 -12- 2011 99.998%) is set to at least 1 bar at the interface of the elemental analyzer with the mass spectrometer and the CO gas? will be separated from the gas mixture obtained by combustion by passing it over a 1.5 m PoraPlot chromatographic column at 50 ° C. 4. Method for determining the specific isotopic footprint of real matrix flavor compounds characterized in that based on the retention times determined by injection of each individual compound (eg solvents, certified reference materials), the measured overall isotopic footprint is created. an acquisition / measurement sequence that also contains backflush steps - the removal out of the system only of the solvents used at the sample matrix extraction stage and which are identified on the chromatogram, so as not to reach the ionization source of the mass spectrometer so that , on the chromatogram obtained will remain only the peaks of the compounds of interest, whose values will be quantified and where the small variations that may occur during the online measurement are due to changes in instrumental conditions and in determining the values of the isotopic ratio 6 ¹³ C for the target compounds in the sample are first corrected for the values obtained for working standards and reference materials, namely: by the difference between the measured value 6 ^I3 C and their value obtained when measuring with the elemental analyzer and between two measurements of the same sample, the variation, and therefore the correction, must be linear and the samples with working standards and materials reference are measured at the beginning and end of all series of samples then, using linear interpolation, a correction is determined for each sample so that for each of them the SD Standard Deviation at three successive measurements of the same sample is verified to be smaller of 0.6% o the final result for the test is given by the average value for three measurements and if the deviation is greater than 0.6% o, the measurement must be repeated taking into account that the ¹³ C value of the working standard does not differ by more than 0.5% of the permissible value (certified) and otherwise adjust and check the spectrometer settings ensuring that verification a correct measurement is made by measuring the ion current for the mass m / z = 44, which is proportional to the amount of carbon injected into the system where under standard conditions the ion current is approximately constant for the analyzed samples and a significant deviation indicates that there is either an imperfect separation in the capillary column and the oxidation of the sample or an instability of the mass spectrometer.