Method for determination of o-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-n-methylcarbamate in biological sample

FIELD: biology, toxicological and sanitary chemistry, in particular method for determination of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in biological sample, useful in chemical, toxicological and veterinary laboratories, etc.

SUBSTANCE: claimed method includes grinding of biological tissue, double treatment thereof with ethyl acetate and acetone mixture in volume ratio of 1:1, for 30 min in each case, extractant evaporation, and residue dissolution in diethyl ether, introducing of obtained solution into silica gel column, solvent evaporation and chromatography. For chromatography hexane/acetone in ratio of 9:1 is used as mobile phase. Then effluent fractions containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate are conjugated, eluent is evaporated, and residue is dissolved in acetonitrile/water (5:5) solvent mixture and subjected to chromatography by HPLC using acetonitrile/water (5:5) as mobile phase and sensor based on photodiode matrix.

EFFECT: method of increased sensitivity and accuracy.

3 tbl, 2 ex

 

The invention relates to biology, sanitary and Toxicological chemistry, and in particular to methods of determining O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in biological material, and can be used in the practice of sanitary and epidemiological stations, chemical-Toxicological and veterinary laboratories. The method refers to the mass number.

The known method for the determination of organic substances in biological material, which consists in grinding the biological object, repeated infusion of ethanol (every time during the day), combining the extracts, thickening of the joint extraction, precipitation of proteins in it absolute ethanol, filtration, evaporation of the filtrate to a thick syrup and diluting it with water, followed by extraction of the analyzed compounds first of sour, and from the alkaline solution (Svickova PPM Toxicological chemistry. - M.: Medicine, 1975. - 119-123).

The method is time consuming, requires considerable time to implement, characterized by a low degree of extraction of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate.

There is a method of determining the derivatives carbamino acid (1-naphthyl-N-methylcarbamate) in biological objects by grinding the biological tissue, it is processed with hexane in the presence of anhydrous sodium sulfate in 2-3 hours, about the division of the hexane extract, evaporation to a dry residue, dissolving the residue in a mixture of water-methanol, taken in a volumetric ratio of 3:2, by addition of aqueous-methanolic solution of sodium chloride, extraction of the solution with chloroform, separating the chloroform extract, evaporation to a dry residue, dissolving the residue in hexane, followed by chromatographytandem in a thin layer of silica gel on the plate "Silufol using the mobile phase hexane-acetone (3:1) and the manifestation of the chromatograms by sequential processing of aqueous-ethanolic potassium hydroxide solution and a solution of diazonium salts (Laboratory tests in veterinary medicine. Chemical-Toxicological methods / edited Bientina. - M.: Agropromizdat, 1989. - P.160-162).

The method is characterized by a low degree of extraction of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-methylcarbamate, relatively low precision and sensitivity definition.

The closest way to detect O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in biological material, which consists in the fact that the biological Rob crush, process, once a portion of chloroform for 20 minutes, the chloroform extract was separated, filtered through anhydrous sodium sulfate, the chloroform from the filtrate evaporated to a dry residue, the residue is dissolved in water, the resulting solution of extras is Giroud twice with chloroform, the extracts are combined filtered through a layer of anhydrous sodium sulfate, the filtrate is evaporated at 80°With minor amount, chromatographic in a thin layer silica gel plates "Silufol" using the mobile phase hexane-acetone (3:2), and the resulting chromatogram shown by sequential processing of aqueous-ethanolic potassium hydroxide solution and a solution of diazonium salts (Methods for the determination of trace pesticides in food, feed and the environment. Vol. 1, - M.: Kolos, 1992. - S-406).

The method is characterized not high enough sensitivity and accuracy.

The present invention is to increase the sensitivity and accuracy of definition.

This object is achieved by using the proposed method, which consists in the fact that biological tissue is crushed, the twice treated with a mixture of ethyl acetate and acetone, taken in the ratio 1:1 (by volume), each time for 30 minutes. The obtained extract combine, the solvent is evaporated, the residue is dissolved in diethyl ether, the resulting solution is make a column of silica gel, the solvent is evaporated, the process of chromatography was carried out carried out using a mobile phase of hexane-acetone (9:1), fractions of the eluate containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, unite, eluent is evaporated, OST the current dissolved in a solvent mixture of acetonitrile-water (5:5) and chromatographic the HPLC column with sorbent Novapak-18" with application mobile phase acetonitrile-water (5:5) and detector-based photodiode array.

The method is as follows: biological tissue containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, crushed, twice insist with a mixture of ethyl acetate and acetone, taken in the ratio 1:1 (by volume) and each time for 30 minutes. The extract is separated from the solid particles of the biomaterial are combined and the combined extract evaporated to a dry residue. The resulting residue is dissolved in a solvent mixture of hexane-acetone (9:1) and chromatographic column with silica gel L 100/160 multiples μusing a mobile phase of hexane-acetone (9:1). Fractions of the eluate containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, unite, eluent is evaporated, the residue is dissolved in a solvent mixture of acetonitrile-water (5:5) and chromatographic the HPLC column with sorbent Novapak-18" using the mobile phase acetonitrile-water (5:5) and detector on the basis of the photo diode array.

Example 1

Determination of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in liver tissue

To 10 g of finely ground liver tissue was added 5 mg of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, thoroughly mixed biological tissue with a substance and leave for days at a temperature of 18-20°C. after this time the biological object containing an analyte, pour 20 ml of a mixture of e is racette and acetone, taken in the ratio 1:1 (by volume) and incubated for 30 minutes with occasional stirring. The extract is separated, the operation processing is repeated in these conditions. Separate hoods combine, extractant and extracted from the biological tissue water is evaporated in a current of warm air. The residue is dissolved in 10 ml of diethyl ether. Two portions of the resulting solution (each 1 ml) consistently make the column size 490×11 mm, filled with 10 g of silica gel type L 100/160 multiples μ. After making each portion and occurrence of the sorbent solution in diethyl ether is evaporated from the sorbent by blowing free space column above the surface of the sorbent air using a vacuum pump. The chromatography was carried out carried out using mobile phase gxen-acetone (9:1). Eluent is collected in a separate racemi 2 ml each. Fractions from 27 to 30 containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, unite, eluent is evaporated, the residue is dissolved in 12.5 ml of acetonitrile, quantitatively transferred into a volumetric flask with a capacity of 25 ml and adjusted to the mark with water. The flask contents are stirred. 8 μl of the obtained solution are injected Water Alian (USA). The process of chromatography was carried out is carried out in a column dimensions 150×3.9 mm, filled obremenennym sorbent "Novapak C-18", using a mobile phase of acetonitrile-water is (5:5) and the detector on the basis of the photodiode array. The feed rate of eluent is 1 ml/min at a column temperature of 20°C. the Optical density recorded at a wavelength of 278 nm.

The peak on the chromatogram with retention time of 2.25 min (volume retention 2250 μl) corresponds to O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate.

The quantitative content of O-(2,3-dihydro-2,2-dimethyl-7-benzo-furanyl)-N-methylcarbamate determined on the basis of the chromatographic peak area by the equation of the calibration graph and count on a portion of the analyte introduced into the liver tissue.

Preparation of calibration graph

In a series of volumetric flasks with a capacity of 50 ml make 0,19, 0,37, 1,25, 2,50, 5,00, 10,00, 20,00 ml of 0.02% solution, as well as 4,00, of 6.25 and 12.5 0.2% solution of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in acetonitrile, are added respectively 24,81, 24,63, 23,75, 22,50, 20,00, 15,00, 5,00, 21,00, 18,75 and 12,50 ml of acetonitrile and bring the volume of the contents of each flask to the mark with water. 8 μl of each of the obtained solutions are injected. The chromatography was carried out is carried out in a column size of 150×3.9 mm with sorbent Novapak C-18", using a mobile phase of acetonitrile-water (5:5) and the detector on the basis of the photodiode array. The feed rate of eluent is 1 ml/min at a column temperature of 20°C. the Optical density recorded at a wavelength of 278 nm. According to the results of measurements on which hromatography build a graph of peak area against the concentration of the detected substance. Linear within the concentration range 0,006-4.0 µg. By the method of least squares to calculate the equation of the calibration graph, which in this case is:

S=0,75547×C-0,00156,

where: S is the area of the chromatographic peak, cm

C - concentration of analyte in khromatograficheskoi sample, ág.

The results of quantitative determination of O-(2,3-dihydro-2,2-di-methyl-7-benzofuranyl)-N-methylcarbamate in liver tissue are shown in table 1.

Example 2

Determination of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in sugar beet roots

To 10 g of finely ground tissue of sugar beet roots was added 5 mg of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, thoroughly mixed biological tissue with Westbam and leave for days at a temperature of 18-20°C. after this time the biological object containing an analyte, pour 20 ml of a mixture of ethyl acetate and acetone, taken in the ratio 1:1 (by volume) and incubated for 30 minutes, stirring occasionally. The extract is separated, the operation processing is repeated in these conditions. Separate hoods combine, extractant and extracted from the biological tissue water is evaporated in a current of warm air. The residue is dissolved in 10 ml of diethyl ether. Two portions of the resulting solution (each 1 ml) the placenta is therefore contribute to column size 490× 11 mm, filled with 10 g of silica gel type L 100/160 multiples μ. After making each portion and the reference solution in the sorbent, diethyl ether is evaporated from the sorbent by blowing free space column above the surface of the sorbent air using a vacuum pump. The chromatography was carried out carried out using a mobile phase of hexane-acetone (9:1). The eluate is collected in separate fractions of 2 ml each. Fractions from 27 to 30 containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, unite, eluent is evaporated, the residue is dissolved in 12.5 ml of acetonitrile, quantitatively transferred into a volumetric flask with a capacity of 25 ml and adjusted to the mark with water. The flask contents are stirred. 8 μl of the obtained solution are injected Water Alian (USA). The process of chromatography was carried out is carried out in a column size of 150×3.9 mm, filled obremenennym sorbent "Novapak C-18", using a mobile phase of acetonitrile-water (5:5) and the detector on the basis of the photo diode array. The feed rate of eluent is 1 ml/min at a column temperature of 20°C. the Optical density recorded at a wavelength of 278 nm.

The peak on the chromatogram with retention time of 2.25 min (retention 2250 μl) corresponds to O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate.

The quantitative content of O-(2,3-dihydro-2,2-dimethyl-7-benzo-furanyl)-N-methylcarbamate ODA is really based on the chromatographic peak area by the equation of the calibration graph and count on a portion of the analyte, made in the fabric of sugar beet roots.

Preparation of calibration graph and its equation is given in example 1.

The results of quantitative determination of O-(2,3-dihydro-2,2-di-methyl-7-benzofuranyl)-N-methylcarbamate in the tissue of sugar beet roots are presented in table 2.

The proposed method is compared with the prototype in 4·103time increases the sensitivity of the determination in khromatograficheskoi sample and 8·102once in a biological material, 1.15 times increases the degree of extraction of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate of biological tissues, characterized by higher accuracy (relative error of the mean of the reduced approximately 4.5 times - from ±9% to 1.79%).

Comparative performance of the proposed and known methods are presented in table 3.

The method of determination of O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate in biological material by grinding the sample, processing the extractant of organic nature, evaporation of the solvent, dissolving the residue and the chromatography was carried out, characterized in that the treatment of biological tissue is carried out with a mixture of ethyl acetate and acetone, taken in aspect] is to 1:1 by volume, twice each time for 30 min, after evaporation of the solvent the residue is dissolved in diethyl ether, making a column of silica gel, the solvent is evaporated, the process of chromatography was carried out carried out using a mobile phase of hexane: acetone (9:1), fractions of the eluate containing O-(2,3-dihydro-2,2-dimethyl-7-benzofuranyl)-N-methylcarbamate, unite, eluent is evaporated, and the residue is dissolved in a solvent mixture of acetonitrile-water (5:5) and chromatographic the HPLC column with sorbent Novapak C-18", using a mobile phase of acetonitrile-water (5:5) and the detector on the basis of the photodiode array.



 

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