RU2729620C1 - Method for simultaneous detection of a complex of antibiotics (tetracycline group, levomycetin, bacitracin) in meat and meat products using high-performance liquid chromatography - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to analytical chemistry and represents a method for simultaneous detection of a complex of tetracycline antibiotics: levomycetin, bacitracin, in meat and meat products using HPLC. Method for simultaneous detection of a complex of antibiotics, such as tetracycline hydrochloride, oxytetracycline, chlorotetracycline, chloramphenicol, bacitracin in meat and meat products using high-performance liquid chromatography, involving taking a weighed portion of tissues or animal organs weighing 5 g, extraction, stirring for 20 minutes, centrifuging, filtration, extract cleaning by three-phase extraction on Oasis HLB cartridge 3 cm×60 mg with pre-condensation of cartridge, passing the obtained extract through the cartridge at rate of 1 ml/min, washing with bidistilled water, eluting with methanol, evaporation of the obtained eluate under a weak nitrogen flow at t=25 °C, adding the dissolved residue to a chromatograph using an eluent, processing the analysis results, wherein the chromatograph is Agilent 1260 Infiniti with a UV detector G 1315D and Reprosil ODS-AC 18 column, 250×4 mm, with sorbent particles size of 5 mcm, extraction is carried out with 20 cmof a mixture of methanol, acetonitrile and McILvaine buffer in volume ratio of 37.5:37.5:25, respectively, with adding to mixture 0.6 cm 1.5 mM aqueous solution of disodium salt of ethylenediaminetetraacetic acid (EDTA), centrifuged at 4,500 rpm at pre-cooled to 4 °C centrifuge for 20 minutes, then the extraction and centrifugation procedure is repeated successively once more and the obtained extracts are combined, filtered and the obtained extract is purified on a cartridge which is preliminarily condensed with 6 cmof methanol and 6 cmof bidistilled water, eluted with 6 cm of methanol at rate of 1 ml/min, the obtained eluate is evaporated to dryness and the obtained residue is dissolved in 5 cmof a solution of mixture – 0.07 M ammonium acetate, acetonitrile and methanol in ratio of 40:15:45 respectively with addition of 1M EDTA.EFFECT: method enables simultaneous determination of a complex of said antibiotics, reduced analysis time by 3–3½ times with preservation of high sensitivity of the method.1 cl, 1 tbl, 2 dwg

Description

The invention relates to analytical chemistry, namely to methods for the simultaneous determination of a complex of antibiotics (tetracycline hydrochloride, oxytetracycline, chlortetracycline, chloramphenicol, bacitracin) by reverse phase high performance chromatography.

The problem of contamination of food products of animal origin with residual amounts of antibiotics and the increasing resistance of microorganisms to antibacterial drugs remains relevant in the world. Most experts associate the ingestion of antibiotics in food with the widespread use of therapeutic, therapeutic and prophylactic and growth-stimulating agents for farm animals and poultry.

Antibiotics of the tetracycline group (tetracycline hydrochloride, oxytetracycline, chlortetracycline), amphenicols (chloramphenicol) and the feed antibiotic bacitracin are often used in veterinary medicine, so their residual amounts can be found in food products of animal origin.

Antibiotics of the tetracycline series are drugs with a wide spectrum of action and, due to their high biological activity and relative cheapness, are still actively used in veterinary medicine for the treatment and prevention of certain infectious diseases of livestock, as well as growth stimulators of young animals.

Chloramphenicol is a broad-spectrum antibiotic with high activity against bacteria and rickettsia. The antibiotic is used for acute gastrointestinal diseases of microbial etiology of animals, as well as for surgical infections, gynecological diseases.

Bacitracin is used as an antimicrobial prophylactic and growth-stimulating agent for raising and fattening animals and poultry.

Due to the fact that the groups of antibiotics used in humans and animals in agriculture are the same, residual amounts of antibiotics in food contribute to the emergence of resistant strains in humans. Accordingly, people who consume such products develop immunity to antibiotics, and more and more powerful drugs are required to obtain the expected effect during treatment.

Under the influence of antibiotics, the body loses its ability to independently resist various infections. And, in addition, their widespread use has led to the emergence of strains of bacteria that are resistant to these drugs, and, in the end, a person may be unprotected against infections and microorganisms. The presence of antibiotics in the body can cause severe allergic reactions, accompanied by severe itching, rashes, and in rare cases - edema.

Since the consumption of foods containing residual amounts of antibiotics negatively affects the human body, in this regard, their content is normalized.

According to Appendix 21 to SanPiN 2.3.2.1078-01, the sum of all residues of tetracycline antibiotics should not exceed 0.01 mg / kg in food products (meat, fat, liver, kidneys), chloramphenicol 0.0003 mg / kg and bacitracin 0.02 mg / kg.

Since in recent years there has been a steady tendency in the world to tighten requirements for the quality of food products, in order to exclude the possibility of antibiotics getting into livestock products, it is necessary to develop and introduce into practice new, more effective and sensitive methods for their simultaneous analysis.

In the Russian Federation, there are no regulatory documents on the simultaneous determination of residues of antibiotics of the tetracycline group, chloramphenicol and bacitracin in livestock products.

Currently, many methods have been proposed for the separate determination of tetracyclines, amphenicols and bacitracin, among which the microbiological and enzyme immunoassay methods can be distinguished.

The microbiological method is based on the sensitivity of bacteria to these antibiotics, their ability to diffuse into an agar medium with spores of test microbes, preventing their growth with the formation of transparent zones of inhibition. The presence of antibiotics in meat is determined by the diameter of the zones of inhibition.

For screening purposes, when determining the antibiotics of chloramphenicol and bacitracin, the method of immunochemical analysis is used. For control of food products, preference is given to the methods of enzyme-linked immunosorbent assay, which are highly sensitive. The systems comply with the "Guidelines for the determination of residual amounts of chloramphenicol (MUK 4.1.1912-04) and bacitracin (MUK 4.1.3379-16) in animal products"

However, these methods have several disadvantages:

- low specificity. Currently, a large number of additives (preservatives, colorings, flavor enhancers, etc.) are used in food products that can have a false positive effect;

- a long period of analysis. It takes time for growing the spores of test cultures (from 1 to 7 days) and for the analysis itself (18 hours), i.e. one study takes from 2 to 8 days. In addition, for research, it is necessary to constantly maintain the purity of test cultures.

- high cost of test plates.

There are also known methods for the determination of the tetracycline group and zincbacitracin in meat and meat products, including sampling, extraction, centrifugation, filtration, introduction of the dissolved residue into the chromatograph using the eluent (GOST 33934-2016. Interstate standard. Meat and meat products. Determination of zincbacitracin by the method MKS 67, 120.10 introduced on 2018-01-01 and GOST 31694-2012 Interstate standard. Food products, food raw materials. Determination of the residual content of the tetracycline group by high performance liquid chromatography with a mass spectrometric detector).

A significant drawback of these methods, limiting their widespread use, is the use of expensive, technically difficult to master equipment, with stringent requirements for the quality of reagents and consumables.

Closest to the proposed method is a method for determining bacitracin in meat and meat products using high performance liquid chromatography (HPLC) according to patent RU N2696010 (published on 07/30/2019), which includes sampling, extraction, centrifugation, filtration, introduction of the dissolved residue into the chromatograph with using the eluent, processing the results of the analysis, as a sample, take a sample of tissues or organs of animals weighing 5 g, extraction is carried out with 5 ml of 1.5 mM aqueous solution of Trilon B and 5 ml of 15 solution of trichloroacetic acid for 15 minutes, the solution is stirred for 20 minutes, centrifuged 15 min, filtered, after filtration, the extract is purified by three-phase extraction (SPE) on an Oasis HLB cartridge 3 cm ³ × 60 mg, which is previously condensed with 3 ml of methanol and 3 ml of an aqueous solution of trichloroacetic acid pH = 4.0 before use, the resulting extract passed at a rate of 1 ml / min through the cartridge, then dried under vacuum at a pressure of 20 mm. Hg and washed with 5 ml of bidistilled water, eluted with 2.5 ml of methanol 2 times, the resulting eluate is evaporated under a weak stream of nitrogen at t + 25 ° C to 1 ml, the resulting residue is dissolved in 4 ml of a solution of a mixture of 0.05M monosubstituted potassium phosphate, acetonitrile and methanol in a ratio of 40:15:45, respectively, and an Agilent 1260 Infmiti chromatograph with an ultraviolet detector and a Reprosil ODS-AC column of 18 mm (250 × 4) mm, with a sorbent particle size of 5 μm, is used as a chromatograph.

The disadvantage of this method is the determination of only one antibiotic - bacitracin, whereas there are many different types of antibiotics in meat and meat products, and they also require additional analyzes and time to determine them, and for their simultaneous determination, a method with high sensitivity is currently not exist.

The objective of the present invention is the development of a reproducible, precision method for the simultaneous isolation and concentration of a complex of antibiotics [tetracycline group, chloramphenicol (chloramphenicol), bacitracin] from biological objects and their quantitative determination by HPLC-UV.

The technical result, which the invention is aimed at, consists in the simultaneous extraction and quantitative determination of the complex of antibiotics of the tetracycline group, chloramphenicol (chloramphenicol) and bacitracin, as well as in expanding the range of application of the method, in ease of use and in reducing the time of analysis while maintaining the high sensitivity of the method ...

To achieve the named technical result in the proposed method for the simultaneous determination of a complex of antibiotics (tetracycline group, chloramphenicol, bacitracin) in meat and meat products using HPLC, including taking a sample of animal tissues or organs weighing 5 g, stirring for 20 minutes, extraction, centrifugation, filtration, purification of the extract by solid-phase extraction on an Oasis HLB cartridge 3 cm ³ × 60 with its preliminary condensation, passing the obtained extract through the cartridge at a rate of 1 ml / min, washing with bidistilled water, elution with methanol, evaporation of the resulting eluent under a weak nitrogen flow at t + 25 ° C, introduction of the dissolved residue into the chromatograph using an eluent, processing of the analysis results, extraction is carried out with 20 cm ^{3 of a} mixture of methanol, acetonitrile and MclLvaine buffer in a volume ratio of 37.5 / 37.5 / 25, respectively, with the addition of 0.6 cm ³ 1 , 5 mM aqueous solution of ethylenediaminetetraacetic disodium salt th acid (EDTA), centrifuged at 4500 rpm in a centrifuge pre-cooled to 4 ° C for 20 min, then the extraction and centrifugation procedure is repeated one more time, then the obtained extracts are combined, filtered and the resulting extract is purified on a cartridge, which is condensed before use with 6 cm ^{3 of} methanol and 6 cm ^{3 of} bidistilled water, washed with 6 cm ^{3 of} bidistilled water, eluted with 6 cm ^{3 of} methanol at a rate of 1 ml / min, the resulting eluate is evaporated to dryness, and the resulting precipitate is dissolved in 5 cm ^{3 of a} solution of the mixture - 0.07M ammonium acetate, acetonitrile and methanol in a ratio of 40:15:45, respectively, with the addition of 1M EDTA. As a chromatograph, an Agilent 1260 Infiniti chromatograph with an ultraviolet detector G 1315 D and a Reprosil ODS-AC 18 mm (250 × 4) mm column with a sorbent particle size of 5 μm is used.

Distinctive features of the proposed method are the extraction of 20 cm ^{3 of a} mixture of methanol, acetonitrile and MclLvaine buffer in a volume ratio of 37.5: 37.5: 25, respectively, with the addition of a 1.5 mM aqueous solution of disodium salt of ethylenediaminetetraacetic acid to the mixture of 0.6 cm ³ ( EDTA), centrifugation at 4500 rpm, on a centrifuge pre-cooled to 4 ° C, for 20 min, repeating the extraction and centrifugation procedures one more time, combining the obtained extracts, filtering, purifying the obtained extract on a cartridge, condensing the cartridge before use 6 cm ^{3 of} methanol and 6 cm ^{3 of} bidistilled water, washing with 6 cm ^{3 of} bidistilled water, elution with 6 cm ^{3 of} methanol at a flow rate of 1 ml / min, evaporation of the resulting eluent to dryness, dissolution of the resulting residue in 5 cm ³ of a mixture solution - 0.07 M acetate ammonium, acetonitrile and methanol in a ratio of 40:15:45, respectively, with the addition of 1M EDTA. This allows you to simultaneously isolate, concentrate and quantitatively determine the complexes of such antibiotics as chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin, simplify the process of use, reduce the analysis time while maintaining high sensitivity of the method and expand the range of application of the method - at the same time, you can determine the complex of antibiotics (tetracycline group, chloramphenicol, bacitracin).

The method for the simultaneous determination of a complex of antibiotics (tetracycline group, chloramphenicol, bacitracin) in meat and meat products using high performance liquid chromatography is as follows. Take a sample of meat, grind it in a laboratory homogenizer MPW-302 in an amount of 5 g, weighed with the recording of the result to the second decimal place on a laboratory scale VLTE-500, place it in a centrifuge tube and add to 20 cm ^{3 of a} mixture of methanol, acetonitrile and MclLvaine buffer in bulk a ratio of 37.5: 37.5: 25, respectively, add 0.6 cm ^{3 of} 1.5 mM aqueous solution of disodium salt of ethylenediamine tetraacetic acid (EDTA), stir for 20 minutes on a shaker, extract on an ultrasonic bath at 20 ° C for 15 minutes. The solution is centrifuged at 4500 rpm in a SORVALL RC-6 centrifuge, pre-cooled to 4 ° C for 20 min. The extraction and centrifugation procedure is repeated one more time. The combined extracts are filtered through a 0.45 micron membrane filter.

Purification of the extract is carried out by solid-phase extraction on a cartridge Oasis HLB 3 cm ³ × 60 mg. The cartridge is preliminarily condensed with 6 cm ^{3 of} methanol and equilibrated with 6 cm ^{3 of} bidistilled water. Then the combined extract is passed through the cartridge at a flow rate of 1 ml / min and washed with 6 cm ^{3 of} bidistilled water, eluted with 6 cm ^{3 of} methanol at a flow rate of 1 ml / min, the resulting eluate is evaporated under a weak flow of nitrogen on a thermostatted heating module with a system for stripping solvents with inert gas at a temperature of + 25 ° C to dryness, the resulting residue is dissolved in 5 cm ^{3 of a} solution of a mixture of 0.07M ammonium acetate, acetonitrile and methanol taken in a ratio of 40:15:45 with the addition of 1M EDTA.

If necessary, the resulting solution is passed through a membrane filter with a pore diameter of 0.45 microns.

The final volume is introduced into an Agilent 1260 Inflniti type G 1315 D liquid chromatograph with a G 1315 D UV detector.

The conditions for the analysis of liquid chromatography correspond to the following parameters:

The processing of the results is carried out by calculating the indicator, the area of the chromatographic peak, the sample under study and the standard solution according to the formula:

S = hW _h ;

where h is the height of the peak, that is, the distance from the maximum of the peak to its base, measured along the axis of the detector response, W _h is the width of the peak at the base, that is, a line segment cut off by the peak, drawn parallel to the base of the peak in the middle of its height.

The analysis results are processed using the absolute calibration method, the antibiotic content in biological objects (X, mg / kg) is calculated by the formula:

Where

X — antibiotic content in the sample, mg / kg;

C is the concentration of a standard antibiotic solution, μg / ml;

S ₁ - the area of the peak of the antibiotic in a standard solution, mm ² ;

S ₂ - the area of the peak of the antibiotic in the analyzed sample, mm ² ;

V is the volume of the sample prepared for chromatographic analysis, ml;

P - weight of the analyzed sample, g.

Analysis of the metrological characteristics of the method for the simultaneous determination of a complex of antibiotics (tetracycline hydrochloride, oxytetracycline, chlortetracycline, chloramphenicol, bacitracin) in meat and meat products by high performance liquid chromatography shows high sensitivity and selectivity and the availability of the method corresponds to the developed regulatory documentation due to the precise selection of the extractant mixture of methanol, acetone and buffer MclLvaine in a volume ratio of 37.5: 37.5: 25, respectively, containing a 1.5 mM aqueous solution of EDTA, using solid-phase extraction on Oasis HLB 3 cm ³ × 60 mg cartridges and a column (250 × 4) mm Reprosil ODS - AC 18 with a particle size of 5 microns, as well as a mobile phase of a mixture of 0.07M ammonium acetate, acetonitrile and methanol in a ratio of 40:15:45 allows and 1 M aqueous EDTA solution allows you to simultaneously extract and determine the tetracycline group, chloramphenicol and polypeptide antibiotic - bacitracin in meat by HPLC -UF at the level of the established norms of SanPiN 2.3.2.1078-01, use fast and safe sample preparation and maintain high sensitivity of the analysis and reduce the time of simultaneous determination of antibiotics to 3-3.5 times.

The proposed method for the simultaneous determination of a complex of antibiotics (tetracycline group, chloramphenicol, bacitracin) in meat and meat products using high performance liquid chromatography on a UV detector was tested in the chemical analysis laboratory of the Kazan Federal Center for Toxicological, Radiation and Biological Safety.

The experimental data are shown in FIG. 1 and 2.

FIG. 1 shows the identification of a complex of antibiotics (chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin) in a standard solution containing 90% of the active ingredients (European Pharmacopeia Reference Standard) by HPLC.

FIG. 1 shows a chromatogram of antibiotics chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin in a standard solution using an Agilent 1260 Infiniti liquid chromatograph with a G 1315 D UV detector at an excitation wavelength of 235 nm. Under these conditions, the retention time of the peaks of antibiotics was established: chloramphenicol - 3.267 min, tetracycline hydrochloride - 3.884 min, oxytetracycline - 4.114 min, chlortetracycline - 4.388 min, bacitracin A - 16.488 min, the area of the chromatographic peak of chloramphenicol - 15.99555 mm ² , tetracycline hydrochloride - 149.58323 mm ² , oxytetracycline - 224.65234 mm ² , chlortetracycline - 153.58354 mm ² , bacitracin A - 442.79001 mm ² .

FIG. 2 presents the identification of chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin major peak A in a test meat sample. Artificial seeding of meat with a complex of antibiotics was carried out at the MRL level for chloramphenicol at a dose of 0.0003 mg / kg, for tetracycline hydrochloride, oxytetracycline and chlortetracycline - 0.01 mg / kg, and bacitracin A - 0.02 mg / kg.

FIG. 2 shows the chromatogram of the antibiotic complex chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline. bacitracin in a test sample using an Agilent 1260 Infiniti liquid chromatograph with a G 1315 D UV detector at an excitation wavelength of 235 nm. Under these conditions, the retention time of the peaks of chloramphenicol antibiotics was 3.259 min, tetracycline hydrochloride - 3.890 min, oxytetracycline - 4.121 min, chlortetracycline - 4.395 min, bacitracin A - 16.507 min, the area of the chromatographic peak of chloramphenicol - 12.63648 mm ² , tetracycline hydrochloride - 121 , 16242 mm ² , oxytetracycline - 181.96839 mm ^2; chlortetracycline - 122.86683 mm ² , bacitracin A - 354.23201 mm ² .

Here is the calculation of the peak areas of chloramphenicol in the standard solution and in the analyzed sample.

S ₁ = 15.99555 mm ² (peak area of chloramphenicol in standard solution)

S ₂ = 12.6364845 mm (area of the chloramphenicol peak in the analyzed sample)

(chloramphenicol content in the analyzed sample)

Recoverability percentage is 79%.

We present the calculation of the peak areas of tetracycline hydrochloride in the standard solution and in the analyzed sample.

S ₁ = 149.58323 mm ² (peak area of tetracycline hydrochloride in standard solution)

S ₂ = 121.16242 mm ² (peak area of tetracycline hydrochloride in the analyzed sample)

(content of tetracycline hydrochloride in the analyzed sample)

Recoverability percentage is 81%.

Here is the calculation of the areas of the peaks of oxytetracycline in the standard solution and in the analyzed sample.

S ₁ = 224.65234 mm (peak area of oxytetracycline in standard solution)

S ₂ = 181.96839 mm (the area of the peak of oxytetracycline in the analyzed sample)

(content of oxytetracycline in the analyzed sample)

Recoverability percentage is 80%.

We present the calculation of the peak areas of chlortetracycline in the standard solution and in the analyzed sample.

S ₁ = 153.58354 mm ² (peak area of chlortetracycline in standard solution)

S ₂ = 122.86683 mm ² (peak area of chlortetracycline in the analyzed sample)

(content of chlortetracycline in the analyzed sample)

Recoverability percentage is 80%.

We present the calculation of the peak areas of bacitracin A in the standard solution and in the analyzed sample.

S ₁ = 442.79001 mm ² (peak area of bacitracin A in standard solution)

S ₂ = 354.23201 mm ² (area of the peak of bacitracin A in the analyzed sample)

(content of bacitracin A in the analyzed sample) Recoverability in percentage is 80%.

The development of the optimal liquid chromatographic parameters for the determination of chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin in meat and meat products by the proposed method was carried out using an Agilent 1260 Infiniti liquid chromatograph equipped with an ultraviolet column thermostat, a 13-gradient pump and a detector.

The completeness of the separation of the antibiotic complex (chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin) in the presence of matrix components was achieved on a Reprosil ODS - AC 18 column 250 mm long and 4 mm in inner diameter with an Eclipse RAS (AC 18) sorbent with a grain size of 5 μm at a column temperature + 25 ° С, when using the modes of the above gradient composition of the mobile phase. The maximum UV detector signal was obtained at an excitation wavelength of 235 nm.

Thus, the proposed method for the determination of chloramphenicol, tetracycline hydrochloride, oxytetracycline, chlortetracycline, bacitracin in meat and meat products using HPLC is highly sensitive, simple and economical to implement and can be applied in any chemical laboratory where there is a liquid chromatograph with a UV detector. ... The standard deviation of the measurement is 0.45% for chloramphenicol concentrations up to 0.0003 mg / kg, tetracycline hydrochloride, oxytetracycline and chlortetracycline up to 0.01 mg / kg, and bacitracin A up to 0.02 mg / kg.

Sources of information

1. GOST 33934-2016 MEAT AND MEAT PRODUCTS. Determination of zincbacitracin by high performance liquid chromatography with a mass spectrometric detector. Interstate standard. - M. Standartinform, 2017 .-- 12 p.

2. GOST 31694-2012 FOOD PRODUCTS, FOOD RAW MATERIALS. Method for determination of the residual content of antibiotics of the tetracycline group using high-performance liquid chromatography with a mass spectrometric detector. - M. Standartinform, 2012 .-- 21 p.

3. MUK 4.1.1912-04 CONTROL METHODS, CHEMICAL FACTORS. Determination of residual amounts of chloramphenicol, chloromycetin in animal products by high performance liquid chromatography and enzyme immunoassay. Approved. Ch. state dignity doctor of the Russian Federation, Per. deputy. Minister of Health of the Russian Federation G.G. Onishchenko, 06.03.2004, 7 p.

4. MU 3049-84 Determination of residual amounts of antibiotics in animal products. Approved. Deputy Ch. state dignity doctor A.I. Zaichenko, 1984 .-- 25 p.

5. MUK 4.1.3379-16 Determination of residual amounts of bacitracin in animal products by enzyme immunoassay: Methodological guidelines. - M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2016 .-- 10 p.

Claims (1)

A method for the simultaneous determination of a complex of antibiotics, such as tetracycline hydrochloride, oxytetracycline, chlortetracycline, chloramphenicol, bacitracin in meat and meat products using high performance liquid chromatography, including taking a sample of tissues or organs of animals weighing 5 g, extraction, stirring for 20 minutes, centrifugation, filtration, purification of the extract by three-phase extraction on an Oasis HLB cartridge 3 cm ³ × 60 mg with preliminary condensation of the cartridge, passing the obtained extract through the cartridge at a rate of 1 ml / min, washing with bidistilled water, elution with methanol, evaporation of the resulting eluate under a weak stream of nitrogen at t = 25 ° C, introduction of the dissolved residue into the chromatograph using the eluent, processing of the analysis results, characterized in that the chromatograph is an Agilent 1260 Infiniti chromatograph with an ultraviolet detector G 1315D and a Reprosil ODS-AC 18 column, 250 × 4 mm, with a size sorb particles enta 5 μm, extraction is carried out with 20 cm ^{3 of a} mixture of methanol, acetonitrile and McILvaine buffer in a volume ratio of 37.5: 37.5: 25, respectively, with the addition of a 1.5 mM aqueous solution of ethylenediamine tetraacetic acid disodium salt (EDTA) to the mixture of 0.6 cm , centrifuged at 4500 rpm on a centrifuge pre-cooled to 4 ° C for 20 min, then the extraction and centrifugation procedure is repeated sequentially once more and the resulting extracts are combined, filtered and the resulting extract is purified on a cartridge, which is previously condensed with 6 cm ³ methanol and 6 cm ^{3 of} bidistilled water, eluted with 6 cm of methanol at a rate of 1 ml / min, the resulting eluant is evaporated to dryness and the resulting residue is dissolved in 5 cm ^{3 of a} solution of a mixture - 0.07 M ammonium acetate, acetonitrile and methanol in a ratio of 40:15 : 45 respectively with the addition of 1M EDTA.

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