RU2431829C1 - Method for determination of chloramphenicol content in food products and sorbent for its implementation - Google Patents

Abstract

FIELD: food industry. ^ SUBSTANCE: chloramphenicol is extracted from specimen with acetonitrile, a solution containing the antibiotic is separated in a centrifuge, a sorbent represented by vermiculite is added to the centrifugate at a ratio of vermiculite taken in an amount of 0.1 g of the sorbent per 1 g of lipid in the specimen and shaking is done for 30 minutes, then the solution is separated and passed through the sorbents mixture represented by activated carbon and vermiculite taken in an amount of 1:1 and washed with distilled water; chloramphenicol is eluated with ethanol, the eluate is evaporated up to dryness in water bath and cooled to a room temperature, then the remainder is dissolved in a mobile phase (acetonitrile:water, 70:30 volume per volume), the chloramphenicol solution is removed with a doser into dry vials and chloramphenicol is quantified by RP HELC method with UV detection at wave length 278 nm; the antibiotic content is calculated using an analytical curve. ^ EFFECT: enhancement of analysis accuracy and safety. ^ 2 cl, 1 tbl, 4 dwg

Description

The invention relates to analytical chemistry, and in particular to methods for determining the antibiotic chloramphenicol in food products by the method of reverse phase high performance liquid chromatography.

Antibiotics, in particular chloramphenicol (chloramphenicol), are used as effective anti-infective agents in veterinary medicine and in the prevention of diseases of poultry and livestock. In this case, the antibiotic is able to pass into meat, animal milk, bird eggs and other food products. Since in recent years there has been a steady tendency in the world to tighten food quality requirements, it is necessary to develop and put into practice new, more effective and sensitive analysis methods.

There are various methods for determining chloramphenicol in foods (enzyme-linked immunosorbent assay, chemical methods (qualitative reactions to this antibiotic with various specially selected reagents), physico-chemical methods (voltammetry, polarography, spectrophotometry), thin-layer chromatography, gas chromatography). When using these methods of analysis, they often encounter certain difficulties, for example, the need to use specific, rare and quite expensive reagents; the impossibility of determining several drugs at once with the joint presence and unsatisfactory lower detection limit of the antibiotic; the need to use a pre-column (for gas chromatography).

One of the most promising methods for determining the antibiotic levomycetin is the method of reverse phase high performance liquid chromatography (RP HPLC), which makes it possible to extract, separate, identify and quantify drugs in biological fluids.

A known method for the quantitative determination of chloramphenicol in food products by measuring the mass fraction of the antibiotic by RP HPLC with photometric detection using a Lumahrom liquid chromatograph [1]. The method consists in extracting chloramphenicol from a sample with distilled water, further purifying the extract with a DIAPAC-C ₁₆ concentrating cartridge and determining chloramphenicol by RP-HPLC using a photometric detector (254 nm). Diapak-S ₁₆ cartridges are chemically stable polymer cases filled with high-quality chemically modified sorbents based on silica gel with grafted hexadecyl groups. The method has several disadvantages: chloramphenicol is extracted from the samples with water, which complicates further analysis, because extracts are obtained with sufficiently contaminated extraneous substances that interfere with the determination of the antibiotic; the method involves the use of volatile and toxic solvents - chloroform and hexane; determination of chloramphenicol by RP-HPLC with an ultraviolet detector is carried out at a wavelength of 254 nm. However, in this spectral region, intense absorption is characteristic of almost all substances containing an aromatic cycle; therefore, the known method for determining chloramphenicol does not guarantee accurate results.

The possibility of DIAPAC-C ₁₆ cartridges for the concentration of cefazolin and chloramphenicol antibiotics from dilute solutions with their subsequent determination by RP-HPLC was studied [2]. Sorption of the drugs is carried out in static and dynamic modes, antibiotics are desorbed with acetonitrile, the resulting solutions are analyzed by RP HPLC using the mobile phase (acetonitrile-water, 50:50 v / v); Zorbax ODS C ₁₈ columns (15 × 0.46 cm), working wavelengths - 210 and 254 nm. However, this technique allows the isolation and analysis of antibiotics from dilute aqueous solutions of pharmaceuticals and is not adapted for the analysis of foods with a high fat content.

A known method for the determination of chloramphenicol in crab meat and shrimp using HPLC with mass detection [3]. The antibiotic is extracted with ethyl acetate, methanol is used as the mobile phase. The disadvantages of this method are: the use of highly toxic methanol as a mobile phase; the use of an expensive and difficult to master liquid chromatograph with a mass detector, which complicates the widespread use of the known method of analysis.

The closest in technical essence and the achieved result to the method for determining chloramphenicol in food products is the method of extracting antibiotics from food products with organic solvents - methanol and acetonitrile, followed by purification of the extract with Carreza solutions (aqueous solution of Zn (CH ₃ COO) ₂ and K ₄ [Fe ( CN) ₆ ] × 3H ₂ O), after which the extract is treated with diethyl ether to remove lipids and analyzed by RP-HPLC using the mobile phase (acetonitrile: water, 70:30 v / v) (Zorbax ODS C ₁₈ column (15 × 0.46 cm), with UV detection tion at 220, 254 and 273 nm) [4]. The amount of antibiotic in the sample is determined using a calibration graph. The detection limit of the antibiotic chloramphenicol in the prototype method is 0.9 ± 0.2 ng in one portion of the solution introduced into the chromatograph for analysis. The disadvantages of this method are the need to use a highly toxic reagent - methanol, as well as the lack of accuracy in determining chloramphenicol due to partial deposition of the antibiotic on a flocculent precipitate of zinc hexacyanoferrate.

The task of the invention is to develop a reproducible, precision method for the isolation and concentration of chloramphenicol from food products, as well as its quantitative determination.

The task is achieved by the fact that chloramphenicol is extracted from the sample with acetonitrile, the solution containing the antibiotic is separated in a centrifuge, sorbent - vermiculite is added to the centrifuge (mass ratio of which is 0.1 g of sorbent per 1 g of fat in the sample) and shaken for 30 minutes , then the solution is separated and passed through a mixture of sorbents - activated carbon and vermiculite, taken in a mass ratio of 1: 1, and washed with distilled water, eluted with chloramphenicol ethanol, the eluate is evaporated to dryness in a water bath and about cooled to room temperature, then the residue was dissolved in the mobile phase (acetonitrile: water, 70:30 vol./about.), the chloramphenicol solution was transferred to dry vials with a dispenser and quantitatively determined chloramphenicol by RP-HPLC with UV detection at a wavelength of 278 nm; the antibiotic content is calculated using a calibration graph.

The problem is best solved by new conditions of sample preparation, not previously known from the prior art, namely:

- consistent use of sorbents:

- first - vermiculite, the mass ratio of which is 0.1 g of sorbent per 1 g of fat in the sample;

- then - a mixture of activated carbon and vermiculite with their mass ratio equal to 1: 1;

- subsequent re-extraction of the antibiotic with ethanol.

In the inventive method, vermiculite (natural zeolite) of the Koksharovskoye deposit of the Primorsky Territory is used.

These significant distinguishing features of the claimed invention provide the concentration of the antibiotic, purification of the extract from the components that interfere with the analysis, which is especially important when analyzing foods with a high (> 30%) fat content and make it possible to determine levomycetin by the known RP HPLC method with a sufficient degree of accuracy.

The technical result of the proposed method for determining the content of chloramphenicol in foods is to increase the accuracy of quantitative determination of the antibiotic in foods with different fat contents, as well as reducing the toxicity of analytical studies.

The method for determining the content of chloramphenicol in food products is as follows. Chloramphenicol is extracted from the sample with acetonitrile and the solution containing the antibiotic with impurities is separated from the precipitate (coagulated protein). Vermiculite is added to the obtained antibiotic solution in acetonitrile, the mass ratio of which is 0.1 g of sorbent per 1 g of fat in the sample, the flask with the mixture is shaken for 30 minutes on a shaker and the solution is separated from the sorbent. The filtered solution is passed through a mixture of sorbents (activated carbon and vermiculite taken at a mass ratio of 1: 1), washed with distilled water and the antibiotic is eluted with ethanol. The eluate in round-bottom flasks was evaporated to dryness in a water bath, cooled to room temperature and dissolved in the mobile phase (acetonitrile: water, 70:30 v / v). The solution of chloramphenicol is transferred by the dispenser to dry vials and the antibiotic is quantified by RP-HPLC with UV detection at a wavelength of 278 nm; the antibiotic content is calculated using a calibration graph.

The need for consistent use of sorbents has been experimentally established: first, vermiculite for additional purification of the extract from impurities. The need to use vermiculite with its mass ratio equal to 0.1 g of sorbent per 1 g of fat in the sample has been experimentally confirmed and is due to the fact that during sorption from the solution on the vermiculite sorption of impurities (lipids, proteins, etc.) occurs in the extract. Thus, a preliminary purification of the extract from ballast substances is achieved.

For sorption of chloramphenicol from the solution after separation of vermiculite, a binary sorbent is used (activated carbon and vermiculite at a mass ratio of 1: 1), the use of which allows not only to purify the extract from coextracted impurities, but also to concentrate chloramphenicol in the amounts necessary for chromatographic analysis . Activated carbon adsorbs impurities from a solution of chloramphenicol in ethanol, provides a transparent and suitable for RP HPLC analysis of a solution of chloramphenicol. In order to minimize the possibility of sorption of the antibiotic on coal, the required amount of activated carbon is preliminarily calculated according to formula (1) if the fat content in the product is ≥3.9%; and according to the formula (2), if the fat content in the product is less than 3.9%:

where m _S is the mass of the sorbent;

Δm = m _R -m _{P ,,}

Where

m _P is the sample of the product;

m _R is the mass of the residue after extraction with acetonitrile;

f is the fat content in the test product.

where m _S2 is the mass of the sorbent necessary for the analysis of a food product with a fat content of less than 3.9%,

f is the fat content in the test product.

After calculating the weight of activated carbon necessary for the analysis of the product for chloramphenicol, a mixture of sorbents is prepared at a mass ratio of 1: 1; this ratio of sorbents is optimal and experimentally established.

A change in the ratio of activated carbon: vermiculite leads either to a decrease in the degree of extraction of the antibiotic, or to the formation of turbid solutions unsuitable for analysis by RP HPLC.

Elution of chloramphenicol is carried out with ethanol, since this solvent has sufficient polarity for the most complete elution of chloramphenicol from the sorbent. Less polar solvents are removed from the sorbent and non-polar impurities, which leads to a decrease in the accuracy of the analysis of the antibiotic of the claimed method. Not unimportant when choosing a solvent at the stage of elution of chloramphenicol is lower toxicity of ethanol compared to methanol used in the prototype method.

The inventive method for the first time allows you to analyze foods with different fat contents - from 1.0% to 60% with a sufficiently high degree of antibiotic extraction, namely (98.5-90)%, which is confirmed by the table.

The degree of extraction of chloramphenicol from foods with different fat content Product under investigation The fat content of the test product,% The degree of extraction,% Margarine 1 60.0 88.9 89.3 Margarine 2 40,0 91.2 90.9 Milk 3.9 96.4 97.1 Eggs 1,2 98.7 98.9

The correctness of the proposed method for determining the content of chloramphenicol in food products is confirmed by the method of "entered-found" and experimental data (examples 1-3); chromatograms are shown in figures 1-4.

Figure 1 presents the chromatogram of a standard solution of chloramphenicol with an antibiotic concentration of 0.01 mg / ml, prepared from the pharmaceutical preparation "Chloramphenicol sodium succinate", purchased in the pharmacy network. The peak of the substance with a retention time of 2.492 min was taken as the peak of chloramphenicol.

Figure 2 presents the chromatogram of a solution of chloramphenicol with a concentration of 0.01 mg / ml, introduced into a food product (vegetable margarine, 40% fat) and isolated from it by the present method. The peak of the substance with a retention time of 2.429 min was taken as the peak of chloramphenicol.

Figure 3 presents the chromatogram of a solution of chloramphenicol isolated by the present method from the food product "chicken egg". The peak of the substance with a retention time of 2.418 min was taken as the peak of chloramphenicol.

Figure 4 presents the chromatogram of a solution of chloramphenicol with a concentration of 0.01 mg / ml, introduced into a food product (vegetable margarine, fat content of 60%) and isolated from it by the present method. The peak of the substance with a retention time of 2.437 min was taken as the peak of chloramphenicol.

The possibility of implementing the proposed method is illustrated by examples.

Example 1. Determination of chloramphenicol (chloramphenicol) in chicken eggs

In a conical flask with a capacity of 100.0 ml, 50 g of chicken eggs, previously thoroughly homogenized in a glass dish, are added, 100 ml of acetonitrile (hc) are added and put on a shaker for 2 hours. The resulting extract was filtered through glass wool, placed in a glass funnel and washed with 5 ml of acetonitrile. To the resulting solution add 1 g of vermiculite. The flask with the extract and the sorbent is placed on the shaker for 30 minutes, the solution is filtered and passed through a binary sorbent - a mixture of 3.85 g of activated carbon and 3.85 g of vermiculite, after which the sorbents are washed with 10 ml of distilled water. Then, chloramphenicol was eluted with 10 ml of ethanol and the solution was evaporated in a water bath in a round bottom flask to dryness. The flask was cooled in air to room temperature and rinsed with 1 ml of the mobile phase (acetonitrile: water, 70:30 v / v). A solution of the antibiotic in the mobile phase is collected with a 1000 μl dispenser and transferred to a dry, clean vial. The amount of antibiotic is determined by RP-HPLC (Shimadzu LC-6A liquid chromatograph with UV detector, Zorbax ODS C ₁₈ column (15 × 0.46 cm), mobile phase (acetonitrile: water, 70:30 v / v) at operating wavelength of 278 nm, the content of chloramphenicol (mg / kg), calculated by the method of the calibration graph was: 0.0140 ± 0.0011 mg / kg; the degree of extraction of 98.9% (figure 3).

Example 2. Determination of chloramphenicol in margarine with a fat content of 40.0%

The antibiotic was determined as in Example 1, but before extraction, 50 g of margarine was melted in a flask at 55 ° C (sufficient temperature to melt the fat contained in margarine and not destroy the antibiotic), 1 ml of 0.1 mg chloramphenicol solution was added / ml Then, without cooling the sample, 100 ml of acetonitrile was added and the chloramphenicol was extracted on a shaker for 3 hours at a temperature of 50 ° C. To the resulting solution was added 1 g of vermiculite. The flask with the extract and sorbent was placed on a shaker for 30 minutes, the solution was filtered and passed through a binary sorbent - a mixture of 27.0 g of activated carbon and 27.0 g of vermiculite. Further, according to example 1. The content of chloramphenicol was 0.09105 mg / kg; the degree of extraction is 91% (figure 2).

Example 3. Determination of the content of chloramphenicol in margarine with a fat content of 60.0%. The amount of antibiotic added is the same as in Example 2. The extraction time with acetonitrile is 4 hours. Binary sorbent - 40.5 g of activated carbon and 45.5 g of vermiculite. The content of chloramphenicol was 0.0891 mg / kg; the degree of extraction is 89% (figure 4).

It was experimentally established that, depending on the fat content in the product, the extraction time must be changed. For products with a fat content of 1 to 10%, it is 2 hours, for products with a fat content of 10 to 40% - 3 hours, above 40% - 4 hours. An increase in extraction time of more than 4 hours does not increase the degree of extraction of the antibiotic; at an extraction time of less than 2 hours, the extraction of chloramphenicol is not complete.

The inventive method for isolation, concentration and identification of the antibiotic chloramphenicol (chloramphenicol) is simple, not laborious, does not require a large number of reagents, and can be used in any chemical laboratory where there is a liquid chromatograph with a UV detector. The measurement error is 3-4% for concentrations up to 0.01 mg / kg and 10% for large concentrations.

Bibliography

1. No. M 01-28-2002. "The methodology for measuring the mass fraction of chloramphenicol (chloramphenicol) in animal products by RP HPLC with photometric detection using a Lumahrom liquid chromatograph." - St. Petersburg: Lumex LLC, 2008.

2. Concentration of antibiotics cefazolin, cefotaxime and chloramphenicol on modified silicas. / L.I. Sokolova, I.V. Chuchalina. // Journal of analytical chemistry. - 2006. - T.61, No. 12, p.1238-1242.

3. Rupp. H.S. Liquid chromatography / tandem mass spectrometry analysis of chloramphenicol in cooked crabmeat / H.S. Rupp, J.S. Stuart, J.A. Hurlbut // Journal of AOAC International. - 2005. - Vol.88, N 4. - PP.1155-1159.

4. Determination of benzylpenicillin, chloramphenicol and tetracycline in foods by high performance liquid chromatography. / L.I. Sokolova, A.P. Chernyaev. // Journal of analytical chemistry. - 2001. - T.56, No. 11, pp. 1177-1180.

Claims (2)

1. A method for determining the content of chloramphenicol in food products, including extraction of chloramphenicol from the sample with acetonitrile, separation of the solution containing chloramphenicol from the precipitate, purification of the solution with sorbent-vermiculite in a mass ratio of 0.1 g of sorbent per 1 g of fat in the sample, tertiary treatment of the solution and concentration from of chloramphenicol when passing through a sorbent in the form of a mixture of activated carbon and vermiculite at a mass ratio of 1: 1, desorption of chloramphenicol when eluting with ethanol, evaporation of the eluate, followed by dissolved using a cooled residue in the mobile phase of acetonitrile-water at a volume ratio of 70:30, analysis of the resulting solution by reverse phase high performance liquid chromatography and calculation of the content of chloramphenicol using a calibration graph. 2. Sorbent for implementing the method according to claim 1, containing activated carbon and vermiculite in their mass ratio equal to 1: 1.

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