RU2392616C1 - Method to reveal and determine origin of unknown substances in alcoholic drinks - Google Patents

Abstract

FIELD: test equipment.

SUBSTANCE: proposed invention relates to gas chromatographic analysis and can be used in alcohol quality tests. Proposed method consists in that, additionally, water-spirit mix in the ratio of 60/40% by volume is prepared to a series of model samples based thereon to prepared by adding every component of analysed drink separately in said mix. Then model sample are analysed at the inlet assembly temperature of 180°C, 250°C and 310°C. Note that unknown substance detected in model sample is qualified as artifact formed in analysis, while is it is absent from model samples, check sample is prepared by combining the entire series of model samples to be analysed at aforesaid 180°C, 250°C and 310°C. Then mass spectra and chromatograms related therewith are analyzed and, if there is no revealed unknown substance in check sample, it is qualified as marker of nonfoods origin.

EFFECT: unambiguous identification of chemical compounds and fragments thereof as well as their origin, higher accuracy and faster identification.

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Description

The invention relates to gas chromatographic analysis of various chemical compounds and can be used in the control of alcoholic beverages.

A known method of analyzing liquid preparations based on plant materials by chromatography, which involves the extraction of volatiles by stripping the initial sample with steam, the concentration of volatiles by extraction with a low boiling solvent followed by distillation of the solvent, and finally the determination of the components by gas or liquid chromatography [1].

The disadvantage of this technical solution is the low information content of the obtained data, in particular chromatographic spectra, which prevents the unambiguous identification of chemical compounds and their fragments in arbitrary combinations.

There is also a method for chromatographic identification of components of complex mixtures of organic compounds, including passing a substance through a system of series-connected columns filled with a sorbent of different polarity, sampling after each column, detecting on detectors of various types and identifying the substance to be determined by calculating the sensitivity coefficient and relative retention according to the data of two detectors [2].

The disadvantages of this method include the complexity of the analysis procedure, as well as the probability of obtaining false results in calculating the retention values of different columns.

There is also a method of identifying unknown substances by gas chromatography in combination with mass spectrometry. According to the indicated method, the chromatogram is recorded as a function of the retention time and the mass spectrum is recorded in a period of time corresponding to the exit of the substance from the column, and compared with the mass spectra of known substances in the database, then the retention index is determined and compared with those from the database and identify the substance by two parameters - the mass spectrum and the retention index [3].

The disadvantage of this method, despite the attractiveness of using the retention index, is the high probability of obtaining inaccurate analysis results, since the retention indices are initially tied to a specific column with certain parameters and often either are not reproduced or are reproduced on other equipment with distortion, which can lead to an incorrect interpretation of the analysis results.

The closest analogue to the claimed technical solution is a method of mass spectral analysis of a multicomponent system, which consists in recording the mass spectra of the sample, deconvoluting the mass spectra and associated chromatograms of one substance and recognizing them by comparison with the reference spectrum of the substance, comparing the intensities of the peaks and the retention time recognized substances with predefined parameters of the desired substance with recognition confirmation [4].

The disadvantage of this method is the low threshold of detection sensitivity, which consists in the fact that it does not allow to determine the test substances at low concentrations, and the resulting high probability of obtaining false results in such a two-stage recognition.

The technical result, the achievement of which the creation of this invention is directed, is to enable the unambiguous identification of chemical compounds and their fragments and to establish the origin of these chemical compounds while improving the accuracy and efficiency of determination.

The technical result is achieved by the fact that an aqueous-alcoholic mixture of 60/40% volumetric composition is additionally prepared, a number of model samples are prepared on its basis by introducing each component of the test drink separately into the aqueous-alcoholic mixture, and model samples are analyzed at an input node temperature of 180 ° C, 250 ° C and 310 ° C, and if an unknown substance is detected in a model sample, it is qualified as an artifact formed during analysis, and if it is absent in model samples, a control sample is prepared by combining all of a number of model samples, it is also analyzed at an input node temperature of 180 ° C, 250 ° C, and 310 ° C, mass spectra and associated chromatograms are recorded, and in the absence of an unknown substance found in the control sample, it is qualified as a marker of non-food origin.

It should be noted that the claimed method can also be carried out using the analytical system HPLC-MS / MS, since the fundamental provisions of the proposed technical solution are sample preparation, analysis in various conditioning conditions of the parameters according to the temperature gradient, the choice of objects of analysis and the subsequent identification of the substances to be determined by comparing the results analysis with basic data of reference substances.

As the studied alcoholic beverage, the entire spectrum of available alcohol-containing liquids can be used.

As a GC-MS analytical system, for example, an Agilent-5973 quadrupole analyzer coupled to an Agilent-6890 model gas chromatograph can be used.

As auxiliary equipment can be used:

- an automatic shaker from Glas-Col®, USA for liquid-based alloys;

- a centrifuge brand Rotina 46R company Hettich, (Germany), to obtain a contrasting interface between the organic and aqueous phases;

- a vacuum concentrator from Barnstead Inc. (CLUA) for evaporating the organic extract;

- FFAP type columns for chromatographic separation.

Of course, for the implementation of the claimed invention can be used and other instruments and devices with characteristics similar to the above.

Helium, nitrogen or hydrogen may be used as the carrier gas.

As internal standards (BC), cyclohexanol or another suitable compound is used.

Ionization is carried out by electron impact in a vacuum. Detection of detected substances is carried out in the registration mode of a full scan or by selected ions.

Data processing is carried out using the AMDIS automatic processing program and databases.

In principle, the search, detection and determination of the origin of unknown substances must be carried out in order to find new, previously unknown markers that may be responsible for the quality of alcoholic beverages or may be witnesses of falsification of alcoholic beverages.

As for the procedure for implementing the proposed technical solution, the authors, based on their knowledge and experience, consider it appropriate to carry out a search in the following sequence:

- search and detection of an unknown substance (GC-MS analysis of the sample of the studied alcoholic beverage at a temperature gradient of 10 ° C / min);

- identification of an unknown substance as a degradation product of the component (s) of the studied alcoholic beverage (GC-MS analysis of a number of model samples of the water-alcohol mixture + component of the studied beverage at the input node temperature of 180 ° C, 250 ° C, and 310 ° C);

- identification of an unknown substance as an impurity or a decay product of the components of the control sample (GC-MS analysis of the control sample at the input node temperature of 180 ° C, 250 ° C and 310 ° C).

The invention can be implemented as follows.

Previously, to create a database prepare samples of famous drinks-standards. Their chromatographic and mass spectrometric characteristics are recorded and recorded (at least three characteristic ions of each component are detected, retention time, molecular weight, precursor ions, characteristic ions, and lower detection limit are determined). From the obtained data form the base. Separately form a database of background laboratory pollution and instrument background.

A solution of the internal standard is prepared, then sample preparation is carried out, in which a solution of the internal standard is introduced into the sample of the alcoholic beverage under investigation, the sample is adjusted to pH 9.0 with a solid buffer, liquid-liquid extraction is carried out with a mixture of organic solvents of different polarity, the organic layer is evaporated to dryness in a stream of nitrogen, re-dissolved in ethyl acetate and then the sample is introduced into the GC-MS system. The chromatographic and mass spectrometric characteristics of the sample are removed and recorded. The results of the analysis are compared with the database and signs of an unknown substance (HB) are detected. Then, the probability of the presence of HB in each component of the studied alcoholic beverage is checked, for which a water-alcohol mixture of 60/40 vol.% Composition is prepared, and on its basis a series of model samples of the composition of the water-alcohol mixture is a component of the studied alcoholic beverage. Each sample is introduced into the GC-MS system, the chromatographic and mass spectrometric characteristics of the sample are taken and recorded at an input node temperature of 180 ° C, 250 ° C and 310 ° C and the presence of HB is determined. Next, a whole series of model samples is mixed and the control sample obtained in this way is introduced into the GC-MS system, and the chromatographic and mass spectrometric characteristics of the control sample are taken and recorded at the input node temperature of 180 ° C, 250 ° C, and 310 ° C. If no unknown substance is found in the control sample, it is qualified as a marker of non-food origin.

For a better understanding, the invention can be illustrated, but not exhausted by the following examples of its specific implementation.

Example 1

Identification of unknown substances in cognac.

A. Preparation and analysis of samples of the reference and test samples for the presence of volatile compounds.

A solution of cyclohexanol in ethanol with a concentration of 0.1 vol.% Is used as an internal standard. To 1 ml of reference cognac add 30 μl of the internal standard to a concentration of 28 mg / l and the sample is introduced into the GC-MS system with an FFAP column.

Similarly prepare and analyze a sample of the investigated cognac.

The analysis is carried out on an Agilent 6890 / 5973N gas chromatography-mass spectrometric system with a mass-selective detector (quadrupole mass spectrum and “ion trap” mass spectrum are recorded). The process is carried out with the following parameters: temperature of the sample inlet unit - 180 ° C, analytical interface 190 ° C. The separation is carried out on a quartz capillary column with a stationary phase (NF) HP-FFAP with a length of 50 m, an inner diameter of 0.32 mm, a film thickness of the stationary phase of 0.5 μm. The carrier gas flow rate of 1.2 ml / min, the average linear velocity of the carrier gas 32 cm / sec. The volume of the injected sample is 1 μl when dividing the flow 1/10. Temperature program: 60 ° C (4 min), 10 ° C / min, 190 ° C, (40 min). The signal is recorded by the total ion current (SCAN) in the mass range m / z 29 - 300 amu The retention time of the internal standard is 4.52 minutes. When using the GC-DIP method, the analysis is carried out under similar conditions. The retention time of the internal standard is 9.27 minutes. The carrier gas is nitrogen. Quantitative analysis is carried out on selected ions.

B. Preparation and analysis of samples of the reference and test samples for the presence of age markers.

To a sample of reference cognac (3 ml) add 100 μl of the internal standard (solution of orthovaniline in ethanol, 2 mg / l), extract 3 ml of a mixture of hexane / diethyl ether 1: 1. The extract was evaporated, 100 μl of ethyl acetate was added and 1 μl of the sample was added to the GC-MS system with an HP-5MS column.

Similarly prepare and analyze a sample of the investigated cognac. The chromatographic and mass spectrometric characteristics of the samples are removed and recorded (at least three characteristic ions of each reference substance are detected, retention time, molecular weight, precursor ions, characteristic ions, and lower detection limit are determined).

According to the results of the analysis, two substances were identified in the investigated cognac (see Table 1) that were identified as not characteristic of cognac.

Table 1 Retention time Mass spectrum Content Estimated Substance * 24.56 min 103, 116, 145 8 ng / μl triacetin 26.75 min 31, 43, 60, 172 6 ng / μl * - found in the database

Next, prepare a water-alcohol (cognac alcohol of the appropriate exposure) mixture of 60/40 vol.% And on the basis of this mixture prepare a number of samples of the individual components of the reference cognac, including:

water-alcohol mixture + ethylhexenal

water-alcohol mixture + diethylcrotonal

water-alcohol mixture + ethyl lactate

water-alcohol mixture + vanillin

water-alcohol mixture + sucrose

water-alcohol mixture + eugenol

Each sample is introduced into the GC-MS system, and the chromatographic and mass spectrometric characteristics of the sample are removed and recorded at an input node temperature of 180 ° C, 250 ° C, and 310 ° C.

When analyzing the sample water-alcohol mixture + sucrose, an unknown substance was found with the following parameters: retention time 26.75 min; characteristic ions 31, 43, 60, 172. The specified substance, therefore, is a product of thermal decomposition of sucrose in the injector of the chromatograph. According to the database, the substance is defined as a dimer of 1,3-dihydroxyacetone. A complete analysis of the components of the reference cognac separately and in aggregate did not reveal the presence of a second unknown substance. According to the database, this substance is triacetin. Triacetin is known to be an essential component of artificial flavors. According to the results of the analysis, triacetin is classified as a marker of non-food origin.

Example 2

Determination of unknown substances in vodka.

Samples of the reference and test vodkas are prepared as in Example 1. The analysis is carried out as in part B of Example 1.

The results of the analysis are presented in Table 2.

table 2 Retention time Mass spectrum Content Estimated Substance * 33.85 122, 105, 77 13.0 40.76 212, 194, 167, 105 8.5 45.02 234, 120, 86 7.6

Next, prepare a reference water-alcohol mixture with a composition of 60/40 vol.% (In accordance with the recipe of the studied vodka) and analyze the sample, as in example 1. In the control sample, unknown substances were not found. According to the database, unknown substances found in the studied vodka are benzoic acid, benzoyl benzoate, and lidocaine, respectively, and are the products of thermal decomposition of bitrex, a denaturing additive to alcohols, and are qualified according to the results of analysis as markers of non-food origin.

Example 3

Determination of unknown substances in vodka.

Samples of the reference and test vodkas are prepared as in Example 2. The analysis is carried out as in part B of Example 1.

The results of the analysis are presented in Table 3.

Table 3 Retention time Mass spectrum Content Estimated Substance * 39.45 222, 177, 149 34.7

An unknown substance was not detected in the control sample. According to the database, an unknown substance found in the studied vodka is diethyl phthalate.

At the same time, diethyl phthalate and other phthalic acid esters are typical representatives of laboratory contaminants (injectors, dishes, etc.), and therefore, for a qualified determination of the origin of the found dimethyl phthalate, a control determination of the level of laboratory contaminants is required. The analysis is carried out at the following parameters: 100 ° C (1 min), 35 ° C / min, 300 ° C, (20 min), HP-5MS column.

Laboratory contamination analysis results are presented in Table 4.

According to the results of the analysis, to qualify an unknown substance discovered in the studied vodka - dimethyl phthalate - qualify as a marker of non-food origin, denaturing additive to alcohol.

As can be seen from the description and examples of the method, the claimed technical solution provides the ability to uniquely identify chemical compounds and their fragments, allows you to establish the origin of these chemical compounds while improving the accuracy and efficiency of determination.

Information sources

1. RU 2093822 C1, M.C. G01N 30/04, publ. 1997 year

2. RU 2069363 C1, M.C. G01N 30/02, publ. 1996 year

3. WO 2004/104571, M.C. G01N 30/00, publ. 2004 year

4. EP 1846757 A2, M.C. G01N 30/86, publ. 2007 - the closest analogue.

Claims (1)

A method for identifying and determining the origin of unknown substances in alcoholic beverages, in which a sample of the test beverage is prepared, subjected to GC / MS analysis, mass spectra and associated chromatograms are recorded and components are recognized by comparison with a database of reference analytical characteristics of the beverage, characterized in that an additional water-alcohol mixture of 60/40% volume is prepared, a number of model samples are prepared on its basis by introducing each component into the water-alcohol mixture of a drink separately and model samples are analyzed at an input node temperature of 180 ° C, 250 ° C and 310 ° C, and if an unknown substance is detected in a model sample, it is qualified as an artifact formed during analysis, and in the absence of it, a control sample is prepared in model samples by combining a whole series of model samples, it is also analyzed at an input node temperature of 180 ° С, 250 ° С and 310 ° С, mass spectra and associated chromatograms are recorded and in the absence of an unknown substance found in the control sample e go qualify as a marker of non-food origin.