SU1742713A1 - Method of determination of tetracyclines - Google Patents

Abstract

The invention relates to agriculture, in particular to veterinary medicine, and can be used to control the quality of honey. Tetracyclines are determined by adding a solvent to a sample, applying a complete sample to thin layer chromatography plates, pre-impregnated with a 0.1 M solution of Trilon B, followed by distilling the sample in a mixture of ethyl acetate and acetone. A mixture of 0.005-0.015 N hydrochloric acid and acetone with a volume ratio of a sample of hydrochloric acid and acetone 4.0 4- 4.5: 1.3 5-4.0 is used as a solvent. Trilon B solution is used with a pH of 4.5-4. , 8 The method allows to identify and determine the residual amounts of antibiotics from the tetracycline group (tetracycline oxytetracycline, metacycline and doxycycline) at the level of 0 0005% or 0.005 mg per 1 g of honey LH

Description

The invention relates to agriculture, in particular to veterinary medicine, and can be used to control the quality of honey.

There is a known method for determining tetracyclines (in particular, in serum) by adding a solvent to tetracycline sample, applying the obtained sample on a thin layer chromatography plate, pre-impregnated with a 0.1 M solution of Trilon B, followed by distillation in a mixture of ethyl acetate and acetone and water.

However, this method cannot be used to identify and determine residual tetracyclines in honey with high accuracy.

The aim of the invention is to improve the accuracy of the method for the determination of tetracycline in honey.

To achieve this goal, according to the method of determining tetracyclics by adding honey to the sample, put the obtained sample on the thin-layer chromatography plate with a 0.1 M Trilon E solution, pH 4 5 - 4.8, followed by distillation in a mixture of ethyl acetate and acetone and water as a solvent, use a mixture of 0.005 - 0.015 N hydrochloric acid and acetone in a volume ratio of a sample of hydrochloric acid and acetone 4.0 4 5.1 35-40

The method is carried out as follows.

Preparation for analysis.

For the preparation of a U.1 M solution of Trilon B. 3.76 g of Trilon B is introduced into a 100 ml volumetric flask, dissolved in a small amount of water a volume of solution VJ

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The pa in the flask was made up to the mark with water and stirred. The pH of the resulting solution should be 4.5-4.8 (checked potentiometrically); If necessary, the pH of the solution is adjusted to the desired value with a 10% sodium hydroxide solution.

Preparation of standard tetracycline solutions.

50 mg of each antibiotic (tetracycline, oxytetracycline, metacycline and doxycycline) are introduced into a 250 ml volumetric flask, 10 ml of 0.01 N is added. hydrochloric acid, shake until complete dissolution, then the volume of the solution in the flask is made up to the mark with acetone. Add 5 ml of this solution to a 50 ml volumetric flask, bring the volume of the solution in the flask to the mark with acetone (solution 1).

Preparation of a standard doxycycline solution to determine all tetracyclines.

Add 50 mg of doxycycline hydrochloride to a measuring flask with a capacity of 250 ml, add 10 ml of 0.01N hydrochloric acid, shake until complete dissolution, volume of the solution in the flask is adjusted to the mark with acetone, and stirred. Add 5 ml of this solution to a 50 ml measuring flask and volume of the solution in the flask is made up to the mark with acetone (solution 2).

Preparation of honey samples for analysis.

To 4.0–4.5 ml (4.5–5.5 g) of honey in a flask with a ground stopper add 1 ml of 0.005–0.015 n hydrochloric acid, mix thoroughly, triturate, add 3.5–4 , 0 ml of acetone, mix (solution 3).

Preparation and regeneration of chromatographic plates.

The finished plates for chromatography (TU 15/16 ESSR-7-88) of reusable size 6x10 cm are placed for 30 minutes in a 0.1 M solution of Trilon B (pH 4.5-4.8) for impregnation. The plates are removed, air dried for 2 to 3 hours and placed in an oven for 1 hour at 105-110 ° C for activation. The cooled plate is ready for analysis.

After performing the chromatographic analysis, the plates are regenerated by placing them in a solution of the chromium mixture (carefully) for 5-10 minutes, removed with forceps and transferred to a washing vessel in running water for 30 minutes. Finally, each plate is washed with distilled water (100 ml), dried in air, then impregnated with a solution of Trilon B and activated as above.

Preparation of the mobile phase (solvent system) for chromatography.

In a measuring cylinder with a ground glass stopper with a capacity of 50-100 ml, 20 ml of ethyl acetate, 19 ml of acetone and 3 ml of water are mixed, the mobile phase is poured into the chromatography chamber with a lapped cover.

Perform analysis.

The following samples were applied to the starting line of the glass plate at points at a distance of 1 cm from one another and 1.5 cm from the edge of the lower plate:

5 µl of solution 1 (0.1 µg of each antibiotic);

5 µg of solution 2 (0.1 µg dexycycline),

5, 10 and 20 μl of solution 3 (honey sample).

The plate with the applied samples is dried in air for 1–2 min, placed in a chamber with a mixture of solvents, ethylacetate: acetone: water (20: 19: 3), and chromatographed using an ascending method.

When the solvent front rises 7–8 cm from the start line, the plate is removed from the chamber, dried in air for 10 minutes, in a drying cabinet for 5 minutes (at 100 ° C). After cooling, the plate is placed for 3–5 minutes in an ammonia vapor chamber and then viewed in the light of a UV lamp at a wavelength of 366 nm. On the chromatogram, 4 zones of antibiotics should be visible, which were obtained by chromatographing solution 1: metacycline with Rsno doxycyc - 0.47 ± 0.05; tetracycline with Rsno doxycic 0.76 ± 0.05; doxycycline with Rsno doxycycl 1.0 ± 0.05 and oxytetracycline with Rsno doxycyc 1.18 ± 0.05. The intensity of luminescence of spots of substances obtained by chromatography of solution 3 (honey sample) applied in different quantities (from 5 to 20 µl) using a microsyringe or pipette with intensity of areas of substances obtained by chromatographic solution 1 is compared. The intensity of the luminescence zones of antibiotics obtained by chromatographing solution 3 should be no more than the intensity of the luminescence zones of antibiotics obtained by chromatographing solution 1.

The detected amount of each antibiotic in the honey sample of the proposed method is 0.0005% or 0.005 mg per 1 g of honey.

PRI me R 1. Place 4.0 ml of honey (sample 1) in a flask with a ground stopper, add 1 ml of 0.005 N salt of acid, mix, triturate, c.) 5 South.

3.5 ml of acetone. Chromatographic plates are prepared as described above, but the pH of a 0.1 M solution of Trilon B is 4.5; air-dried for 2 hours and activated at 105 ° C. The following samples are applied to the plates at a distance of 1 cm from one another:

5 μl of solution 1 (preparation of a standard tetracycline solution is described above);

5 µl of Solution 2 (preparation of a standard for doxycycline for the determination of tetracycline is described above);

5, 10 and 20 μl of solution 3 (honey sample).

When viewing the plates after chromatography in the light of a UV lamp, oxytetracycline was detected, the intensity of the luminescence zone of which corresponds to 0.01 mg of antibiotic per 1 g of honey.

In the example, 3 samples of honey were used. When used to determine residual amounts of tetracyclines in a known manner, no tetracyclines were detected in any of the samples.

EXAMPLE 2 The determination was carried out simultaneously on 3 samples of honey (sample 2). To 4.5 ml of honey in a flask with a ground stopper, add 1 ml of 0.015 n hydrochloric acid, mix thoroughly, triturate, add 4.0 ml of acetone.

The following samples were applied to the points on the start line of the chromatographic plate prepared as indicated above, but impregnated with a 0.1 M solution of Trilon B at pH 4.0 and dried in air for 3 hours when activated in a cabinet at 110 ° C.

5 μl of solution 1 (standard - a mixture of tetracyclines);

5 μl of solution 2 (doxycycline standard);

5, 10 and 20 μl of honey solution.

When viewing the waffle plate with a lamp after the chromatographic process

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No zones of tetracycline emission were detected.

EXAMPLE 3 3 samples of honey (sample 3) were taken at 4.25 ml, 1 ml of 0.01 n hydrochloric acid and 3.75 ml of acetone was added to each sample, then all operations were carried out as in examples 1 and 2. The plates are impregnated with the same solution of Trilon B, but pH 4.65, dried in air for 2.5 hours, activated at 107 ° C. Samples are applied to the plate in the same sequence and quantities as in Examples 1 and 2.

After carrying out the process of chromatography and viewing the plate in the light of a UV lamp, the luminescence zone of oxytetracycline was detected, the intensity of which corresponds to 0.005 mg of antibiotic per 1 g of honey.

The method allows identification and determination of residual amounts of antibiotics from the tetracycline group (tetracycline, oxytetracycline, methacycline and doxycycline) at the level of 0.0005% or 0.005 mg per 1 g of honey.

Claims (1)

Invention Formula The method for determining tetracyclines by adding a solvent to a sample containing tetracycline, applying the obtained sample to thin layer chromatography plates, is pre-impregnated with a 0.1 M solution of trilon 6, followed by distillation of the sample in a mixture of ethyl acetate and acetone, characterized in that increase the accuracy of the method for determining tetracycline in honey; as a solvent, use a mixture of 0.005 - 0.015 n hydrochloric acid with acetone, taken in a volume ratio of the sample: salt: acid: acetone, 4.0 - 4.5: 1: 3.5 - 4.0, and Trylon B solution is used with a pH of 4.5-4.8.