RO128662B1 - 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

The invention relates to a process for isotopic imprinting of aroma compounds from liquid samples, in particular wine.

At the global level, in the last period of time intense efforts are being made for the development of efficient analytical methods, with application in food authentication, the quality policy being oriented in particular towards the development of consumer protection systems, by ensuring a real quality of the materials. bonuses used to obtain food, while encouraging protection against imitations and misuse of the name of famous products, known as such to the general consumer. Consumers are supported by providing information on product characteristics and composition, as appropriate.

The present invention is in support of this goal, solving the problem of complete, global and compound-specific isotopic imprinting, of the flavors 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 on 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 mass with several mass units, due to the different number of neutrons in the nucleus, and which do not degrade through radioactive processes over time. In order to establish the authenticity of a product, taking into account stable isotopes (for example, ¹⁸ 0, ² H, ¹³ C, ¹⁵ N, etc.), determining isotopic ratios (the ratio between the heavy and the light isotope of the same element, for example, ¹⁸ O / ^1S O, ² H / ¹ H, ¹³ C / ¹² C, ¹⁵ N / ¹⁴ N, etc.) is certainly much more useful than other analytical investigations such as, for example, the composition in microelements, because C and H are constituents. of all organic compounds in liquid samples, especially wine, and O and N are also widely represented (oxygen from water to sugars, alcohols, acids, salts, flavors, polyphenols etc., and nitrogen to peptides, amino acids, proteins etc. .).

In nature, most bioelements are mixtures of two or more stable isotopes. It has been observed that, from the point of view of the isotopic content, the variation of the composition of the molecules depends on their origin. The variability is related to the isotopic content of the source and to the isotopic fractionation associated with the physical processes, chemical stages 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 takes place - the poorer isotope depletion is observed in the vapor state in relation to the liquid state. A similar isotopic fractionation occurs during the evapo-transpiration process of the plant water, with higher isotopic ratios being observed in the plant water, compared to that of the food source (for example, groundwater, precipitation).

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 occur during the synthesis.

The complementary use of analytical techniques such as gas chromatography and mass spectrometry, for isotopic reports, has gained scope in various applications of life sciences (W. Meier-Augenstein, "Applied gas chromatography coupled to isotope ratio mass spectrometry, Journal of Chromatography A"). 842,1999, pp. 351-371), the literature highlighting a variety of aspects related to isotopic analysis on a specific compound made on organic materials (PA Eakin et al., "Some instrumental effects in the determination of stable carbon isotope ratios by gas chromatography - Isotope ratio mass spectrometry, Chemical Geology. Isotope Geoscience section 101,1992, pp. 71-79), but also method optimization (K. Grob, Making and manipulating capillary

RO 128662 columns1 columns for gas chromatography, Dr. Alfred Huthig Verlag, Heidelberg, Germany, 1 1986; D. A. Merritt et al., "Performance and optimization of a combustion interface for isotope ratio monitoring gas chromatography / mass spectrometry, Analytical 3 Chemistry 67, 1995, pp. 2461-2473). Specific to the investigations related to the compositional evaluation of liquid samples, in particular wine, have been highlighted over time various 5 methods (US 005200909 A; US 006978243 B2) and compositional characterization equipment (US 20070104832 A1), for the purpose of classification and authentication. them. 7

Due to its ability to accurately and accurately measure the distribution of naturally occurring isotopes in liquid samples, GC-C-IRMS (Gas Chromatography - Combustion-lsotope 9 Ratio Mass Spectrometry) is increasingly becoming a consideration method for controlling the authenticity of food. In combination with compounds labeled with stable isotopes 11 (for example, ¹³ C), the GC-IRMS coupling was used to characterize ethyl alcohol from wine samples (FR 2673291 A1), as well as some of the majority organic compounds found in wine 13 ( AI Cabanero et al., "Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol charaterization, Rapid Communications in Mass 15 Spectrometry 22,2008, pp. 3111-3118). At the same time, the concentration of target compounds from wine samples is performed in order to optimize investigations by chromatography (Y. Kotseridis 17 et al., "Quantitative determination of β-ionone in red wines and grapes of Bordeaux using a stable isotope dillution assay, Journal of Chromatography A 848, 1999, pp. 19 317-325), one way being by using a series of liquid-liquid extractions with organic solvent, followed by the combination and concentration of the organic phases. 21

Due to the reliable information provided by stable isotopes for the classification or certification of the authenticity of organic samples, with the extension of the scope of application of the 23 GC-C-IRMS (JAJ Dungait, Variation in bulktissue, fatty acid and monosaccharide 5 ¹³ C values between autotrophic and heterotrophic plant organs, Phytochemistry 72, 25

2011, pp. 2130-2138) have also been optimized measurement systems (US 20100101304 A1).

Starting from the state of the art and existing research, the technical problem that this invention solves is related to obtaining a global and specific imprint of the aroma compounds from liquid samples, in particular wine, by isotopic mass spectrometry, 29 coupled with chromatography. of gas.

The process of isotopic imprinting of aroma compounds from liquid samples, produced 31 liquids of natural origin, in particular wine, according to the invention, comprises the following steps:

(a) preparation of a sample represented by an organic extract of the liquid sample to be analyzed by liquid-liquid extraction in an organic solvent, and concentration of the aroma compounds; 35 (b) identification of the target aroma compounds in the extract organic obtained in step (a), by gas chromatography coupled with mass spectrometry (GC-MS), 37 (c) global isotopic imprinting, by combustion on an elemental analyzer (EA-IRMS) of the organic solvents used in step (a) ) and pure flavor compounds, used as reference materials 39 for GC-MS analysis of step (b) and GC-C-IRMS from (d), and (d) specific isotopic imprinting of the flavor compounds from the organic extract obtained 41 in step (a) by (GC-C-IRMS), for the purpose of isotopic imprinting of the aroma compounds that define the typicality of the variety, respectively, of the minority ones, in order to determine the origin of the liquid samples 43.

The present invention comes to support the activities of certifying the authenticity of a product, 45 solving the problem of complete isotopic imprinting, globally and specifically on the compound, of the aromas of a liquid, especially wine. 47

RO 128662 Β1

Evaluation of the natural composition in stable isotopes of a liquid sample, in particular, wine, is one of the best performing applications of analytical chemistry, through which we can establish relationships between the finished product (for example, wine) and the raw materials (for example, water, plant) from their natural environment.

Using this isotopic imprinting process, an easily understandable, quantitative assessment of the characteristics of a liquid, for example, the wine, in which the specific aroma compounds of the liquid are evaluated, can be provided.

The following is a brief description of the figures:

FIG. 1, the conceptual model of the specific isotopic imprint on the compound for a liquid sample, in particular wine;

FIG. 2, matrix containing an induced mixture of 4 volatile organic compounds (aromas): 1-hexaneol, ethyl octanoate, ethyl decanoate and diethyl succinate (TG-WAXMS column 30 mx 0.25 mm x 0.25 pm, splitless injection mode, split flow 50 ml / min, helium eluent 1 bar, reference gas CO ₂ 1.1 bar, starting temperature 40 ° C, isothermal 3 min, heating rate 70 ° C / min up to 220 ° C);

FIG. 3, real wine matrix (TG-WAXMS column 30 mx 0.25 mm x 0.25 pm, splitless injection mode, split flow 50 ml / min, helium eluent 1 bar, CO ₂ reference gas 1.1 bar, temperature start 40 ° C, isotherm 3 min, heating rate 70 ° C / min to 220 ° C).

The present invention describes a process for isotopic imprinting of aroma compounds from liquid samples, in particular wine, based on a succession of steps, according to FIG. 1, starting from the preparation of the sample by extraction of the target compounds (step I), identifying their qualitative preliminary (step II), determining the retention times and establishing the working parameters for the specific isotopic imprinting on the compound (step III), and ending with their imprinting. isotopic global (step IV) and specific (step V).

An essential first step in the isotopic imprinting of flavor compounds is the extraction of 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, minimum three, with an individual duration of 10 ... 60 minutes, having the sample volumetric ratios: solvent in a range of values of 5: 1 ... 25: 1. The organic extracts are combined and then dried with a desiccant (for example, sodium sulfate or silica gel). The dry organic extract is concentrated to a final volume of 50 ... 2000 µl, which constitutes the sample for gas chromatography 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, no sophisticated laboratory equipment required, nor any technical personnel having a special training.

From the extract thus obtained, the aroma compounds (step II) are thus identified, using gas chromatography coupled with mass spectrometry. The operating parameters of the gas chromatograph used for this type of determination are: dilution of the injected sample: no dilution - 50: 1 dilution, especially 10: 1 dilution; chromatographic column type: capillary column with medium or high polarity stationary phase, especially from polyethylene glycol; the sample volume injected between 0.1 ... 10 μΙ, in particular 1 μΙ; injector temperature in the range

150 .. .250 ° C, especially 190 ... 200 ° C; the initial oven temperature chromatography between 5 ° C above ambient temperature and 50 ° C; final temperature of the oven chromatography of

70 .., 250 ° C, especially 150 .., 200 ° C; speed of increase of oven temperature chromatography

RO 128662 Β1 of 1 ... 30 ° C / min, especially 3 ... 5 ° C / min. The operating parameters set for the mass spectrometer 1 are: ionization mode - electronic impact with the reading of the entire mass spectrum, and mass reading range in the range 10 ... 400 m / z, in particular 30 ... 150 m / z. 3

The flavor compounds will be identified by:

a) comparing their mass spectra with a mass spectra bank 5 existing in the internal memory of the analysis instrument,

b) comparing the mass spectra of the aroma compounds with those of the reference compounds 7, and by

c) comparing the retention times on the chromatographic column of the analytes with the 9 retention times of the reference compounds. The aroma compounds identified by this technique will be considered target compounds in gas chromatography analysis coupled with mass spectrometry 11 isotopic ratios (GC-C-IRMS) - step V.

Determining the retention times and establishing the working parameters for the specific isotopic induction on the compound of the volatile organic aromas / compounds in liquid samples, in particular wine, is characterized by the fact that it includes two stages: determining the 15 retention times of the solvents involved in preparation of the sample, of the certified reference materials, isotopically imprinted globally in step IV, and establishing the working parameters for the gas chromatograph 17, for the oxidation / reduction furnaces, regulating the reference gas flow rates, etc. The identification of the target compounds in step II allowed the selection of a stationary phase capillary column 19 with the polarity of the sample constituents (polar, for example, TG-WAXMS) and of the reference materials specific to them. The type of injection used is of splitless type, and for 21 avoiding the enlargement of the envelope, the split is opened 10 s after the injection, splitting flow 50 ml / min. The temperature gradients were chosen to maximize the column length: the 23 the greater the column length, the slower the temperature per minute (for example, minimum 3 ° C / min). The column pressure and the eluent velocity (for example, Helium, purity 99.9999%) are set to match the diameter of the capillary column (minimum 1 bar).

To help focus the injected sample and obtain sharper peaks, a retention gap known as a pre-column or guard column, a deactivated piece of a silicon capillary, at least 1 m long, is used. The conversion of the sample into simple gases 29 requires the use of a combustion interface between the gas chromatograph and the mass spectrometer, where the effluent (Helium as carrier gas and sample) is fed into a combustion (oxidation) reactor. This reactor is a ceramic tube filled with CuO / NiO / Pt and maintained at a temperature of approximately 960 ° C. In order to eliminate the 33 water vapors generated during combustion, the effluent passes through a suction trap. Nafion 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 prior to the introduction of flue gas into the 37-ion source is essential, as any water residue would lead to the protonation of CO ₂ gas, producing HCO ₂ ⁺ , which interferes with CO ₂ (isobaric interference) analyzes. For 39 each sequence of analysis of a sample must be checked that the standard deviation of three successive measurements of the same sample is less than 0.6% o. The final result for 41 samples is given by the average value for three measurements. If the deviation is greater than 0.6% o, the measurement must be repeated. The value ¹³ C of the working standard should not differ by more than 0.5% compared to the admitted (certified) value. otherwise, the spectrometer settings should be checked and, if necessary, adjusted. 45

Prior to the specific isotopic imprinting of the aroma compounds in the sample, an evaluation of the global isotopic ratios (step IV) is performed for both the solvents involved 47 in the preparation and extraction of the target compounds from the sample matrix, as well as on the test compounds.

RO 128662 Β1 pure, undiluted volatile organic compounds / compounds, used as reference materials, compounds which are subsequently used in the determination of retention times on the capillary column, and setting the working parameters for the specific isotopic imprinting on the compound. Knowing the retention times of the above mentioned compounds of interest, in order to obtain a chromatogram only with the peaks of these compounds and to eliminate the solvents used in the preparation step, dilution with helium (100: 1) is introduced for the time intervals outside the field. of interest t, ± 100 s (z = l, z? where n = number of target compounds).

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

- solutions of aroma compounds in organic solvents. The solubility of the aroma compound in the selected organic solvent should be as high as possible. Solutions are prepared in the concentration range of ± 20% compared to the concentration values of the aroma compound in the liquid sample, especially wine.

For the specific isotopic imprinting on the compound, the same extract obtained in step I and investigated in step II is introduced into the separation system of a GC-C / TC (Gas Chromatograph - Combustion / Total Combustion) chromatograph, coupled to an IRMS mass spectrometer. The gas chromatograph allows, in the 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, for the peaks of the compounds of interest appearing on the chromatogram, creating a working method by which dilution with helium is carried out. (100: 1) for time intervals outside the range t, ± 100 s (z = 1, / where n = number of target compounds), so that only the compounds of interest pass through the combustion or pyrolysis furnace. being removed from the preliminary vacuum pump. In the created method, the time interval in which the samples are automatically introduced into the gas chromatograph takes into account the reference gas peaks (for example, CO ₂ , H ₂ or N ₂ ) with known value of the isotopic ratio, determined by means of the standards of the appropriate reference, and which appear in the first part of the working sequence, do not overlap with both the solvent peaks and the target compounds. Measurements by isotopic mass spectrometry are carried out by analyzing gaseous compounds (eg, H ₂ , CO ₂ or N ₂ ) obtained by combustion or pyrolysis of the sample, to determine the content in stable isotopes of the component elements (for example, hydrogen, carbon, oxygen). or nitrogen).

The intensities of ionic currents m / z corresponding to the isotopomers of the analyzed elements are measured by mass spectrometry for both the sample and the standard used. The measured value is the difference between the isotopic ratio of the sample (R _x ) and the isotopic ratio of a reference compound (R _std ) expressed by the size δ (% o), defined as:

δχ = ^Κχ ~ ^Rstd xlOOO

I ^ Std where X = ² H, ¹³ C, ¹⁵ N, respectively, ¹⁸ O, and R = ² H / ¹ H, ¹³ C / ¹² C, ¹⁵ N / ¹⁴ N, respectively, ¹⁸ O / ¹⁶ O. All values the obtained isotopic reports will form an isotopic imprint specific to each compound, contributing to the composition of an aromatic profile typical of a liquid sample, especially of an authentic wine.

RO 128662 Β1 in fig. 2 and 3 are presented experimental results regarding the specific imprint 1 of the flavor compounds identified in a wine sample. The example for a wine sample is the following: 3 (a) Initially, a mixture of 4 volatile organic compounds (1-Hexanol: ethidiethylsuccinate ethdecanoate octanoate) is injected into GC-C-IRMS, each time being obtained for 5 compounds retention: t, = 755 s (1-Hexanol), t ₂ = 845 s (ethyl octanoate), t ₃ = 1051 s (ethyl decanoate) and t ₄ = 1088 s (diethylsuccinate); 7 (b) subsequently, the real matrix is introduced, in this case the wine sample (fig. 3), and the values of the isotopic ratios obtained for the peaks appeared at the retention times 9 identified previously (± 3 s) are evaluated. For this chromatogram, the dilutions with helium (100: 1) are made in the range: 250- ^ 640 s and 11701400 s. According to the created method, peaks 11 of the reference gas (for example, CO ₂ ) whose value appears in the first 200 s of the isotopic ratio is known, being determined using the appropriate reference standards. The values of the 13 isotopic ratios obtained for the target aroma compounds of each wine sample constitute an isotopic imprint specific to the respective product. 15

The problem solved by the present invention is to provide a complementary method of authentication of food products, liquid samples, especially wine, after 17 specific flavoring compounds, based on parameters that do not change in time or following technological processes applied. product. 19

Claims (3)

claims 1. Process for isotopic imprinting of aroma compounds from liquid samples, liquid products of natural origin, in particular wine, by isotopic spectrometry, coupled with gas chromatography (GC-C-IRMS), characterized in that it will comprise the following steps: a) preparation of a sample represented by an organic extract of the liquid sample to be analyzed, by liquid-liquid extraction in an organic solvent, and the concentration of the aroma compounds, b) identification of the target aroma compounds from the organic extract obtained in step (a), by gas chromatography coupled with mass spectrometry (GC-MS), c) Global isotopic combustion imprinting on an elemental analyzer (EA-IRMS) of the organic solvents used in step (a) and of the pure flavor compounds used as reference materials for the GC-MS analysis of step (b) and GC -C-IRMS from step (d), and d) specific isotopic imprinting of the aroma compounds from the organic extract obtained in step (a) by GC-C-IRMS, in order to isotope imprinting the aroma compounds that define the typicality of the variety, including the minority ones, in order to determine the authenticity of the origin of the liquid samples . Process according to claim 1, characterized in that the sample is subjected to at least three times a liquid-liquid extraction which involves the use of an organic solvent, such as pentane pentane, petroleum ether, especially dichloromethane, followed by the extraction and drying of the extract. organic result with a desiccant, in particular anhydrous sodium sulfate, and their concentration to a final volume of 502000 µL. 3. Process according to claims 1 and 2, characterized in that the specific isotopic imprinting by GC-C-IRMS of the aroma compounds in the organic extract is carried out by means of a measurement sequence containing steps of eliminating the peaks of the solvents used in the extraction step. of the target compounds in the sample, for analysis remaining only the compounds of interest, the isotopic imprints of which will be quantified.