RU1786426C - Method of alkyl-dinitrophenols assay in biological material - Google Patents

Abstract

Usage: in analytical chemistry. The inventive biological sample is crushed, repeatedly treated with acetone, acetone extracts are combined and evaporated to a dry residue, the residue is dissolved in a hydrophobic solvent, treated with an aqueous alkaline solution, the alkaline solution is separated, acidified to pH 2-3, extracted with diethyl with ethyl ether, the extractant is evaporated, the residue is dissolved in acetone and chromatographed in a thin layer of silica gel in a solvent system of chloroform-hexane (7: 4). 4 tab. SB with

Description

The invention relates to biology and toxicological chemistry, in particular to methods for the determination of alkyl dinitrophenols in biological material, and can be used in the practice of sanitary epidemiological stations, chemical-toxicological and veterinary laboratories. The method is one of mass.

A known method for the determination of organic substances in biological material by grinding the test biological object, repeated infusion with ethanol (each time for 1 day), combining the extracts, condensing the combined extract, precipitating the proteins with absolute ethanol in it, filtering, evaporating the filtrate of the analyzed compounds with an organic solvent first from an acidic, and then from an alkaline solution.

The method is time-consuming, requires a considerable investment of time for its implementation, is characterized by a low degree of recovery of alkyl dinitrophenols and small select. tivnosti.

A known method for the determination of acidic organic substances (which include alkyl dinitrophenols) in a biological material consists in grinding a biological object, treating with an aqueous solution of sodium hydroxide, centrifuging, treating a centrifugate with sodium tungstate in an acidic medium while boiling in a boiled bath, and separating the precipitate by centrifugation for half an hour, extraction isolation of the analyzed compounds from the centrifugate with ether, followed by alkalization of the centrifugate and repeated ether traction.

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The method is poorly selective, characterized by a low degree of recovery of alkyl dinitrophenols.

The closest to the invention in technical essence and the achieved results is a method for determination of alkyldinitrophenols (karatana) in biological objects by grinding biological tissue, repeatedly treating it with portions of hexane every 30 minutes, separating the hexane extracts, combining them and evaporated to a dry residue, followed by dissolution of the residue in a volatile organic solvent, thin layer chromatography of silica gel in a hexane-acetone solvent system (4: 1).

The method is characterized by insufficiently high sensitivity and selectivity.

The goal is achieved by the proposed method, which is that the biological tissue (for example, liver tissue) is crushed, repeatedly treated with acetone, the acetone extracts are combined and evaporated to a dry residue, the residue is dissolved in a hydrophobic solvent, treated with an aqueous alkaline solution, the alkaline solution is separated, acidified to pH 2-3, extracted with diethyl ether, the extractant is distilled off, the residue is dissolved in acetone and chromatographed in a thin layer of silica gel in a solvent system of chloroform-hexane (7: 4).

A comparative analysis of the proposed solution and the prototype shows that the proposed method differs from the known one in that the biological tissue is treated with acetone, the dry residue is dissolved in a hydrophobic solvent, treated with an alkaline solution, the alkaline solution is separated, acidified to pH 2-3, extracted diethyl ether, the extractant is evaporated, the residue is dissolved in acetone and chromatography is carried out in a solvent system of chloroform-hexane in a volume ratio of 7: 4.

Comparison of the proposed solution not only with the prototype, but also with other technical solutions in this technical field does not allow us to identify features that distinguish the proposed technical solution from the prototype, which allows us to conclude that the proposed technical solution meets the criterion of significant differences .

The method is carried out as follows. The biological tissue containing alkyl dinitrophenols is ground, insisted three times with acetone (each time for half an hour), the acetone extracts are separated from the solid particles of the biomaterial i / are combined, the acetone is evaporated, the dry solution is dissolved in a hydrophobic solvent, extracted with an aqueous alkaline solution, the alkaline solution is separated, acidified to pH 2-3, extracted with diethyl ether, the extractant is distilled off, the residue is dissolved in acetone and chromatographed in a thin layer of silica gel with solvent system x. uroform-hexane (7: 4).

PRI me R 1. Determination of 2-methi, 1-4,6-dinitrophenol in the liver tissue.

5 mg of 2; methyl-4,6-dinitrophenol are added to 50 g of finely divided tissue of fresh human cadaveric liver, the biological tissue is thoroughly mixed with the substance and left for 1 day at 18-20 ° C. After this time, the mixture is poured into 100 ml of acetone and incubated for 0.5 h, stirring occasionally. The extract is healed and the operation of infusion is repeated twice more. The acetone extracts are combined, evaporated to dryness in a stream of air at room temperature. The residue was dissolved in 50 ml of chloroform. The resulting solution was extracted three times with a pH 9.5 alkaline buffer solution (in 50 ml portions each). The alkaline extracts are separated, combined, acidified with a 24% hydrochloric acid solution to p H 2-3 and extracted with 100 ml portions of diethyl ether. The ether extracts are combined, the ether is evaporated. The dry residue was dissolved in acetone, the solution was transferred into a 50 ml volumetric flask and adjusted to the mark with acetone. 0.1 ml of this solution was applied to the start line of a chromatographic plate, such as Silufol UV-254 and chromium plates, which were formed in a solvent system of chloro-ormhexane (7: 4). 2-Methyl-4,6-dinitroenol appears on the chromatogram as a yellow spot, 42.

To determine the amount of 2-methyl-4,6-dinitrophenol recovered and the degree of extraction, a portion of the chromatographic plate with a stain was cut out, treated with 5 ml of dimethylformamide and the resulting colored solution was filtered into a spectrophotometric cell. The optical density of the solution was measured on an SF-26 spectrophotometer at a wavelength of 435 nm. The measurements are carried out against the background of the solution obtained in the control experiment. The quantitative content of 2-methyl-4,6-dinitrophenol is determined

by

recounted for a sample of the substance introduced into the biological material.

calibration graph equation and

Building a calibration graph

ka.

0.05, 0.10, 0.20, 0.40, 0.80, 1.20, 1.60 ml of a 0.005% solution of 2-methyl-4.6 are applied to the start line of a Silufol UV-254 chromatographic plate -dinitrophenol in acetone and chromatography in a solvent system of chloroform-hexane (7: 4) was carried out. 2-Methyl-4,6-dinitrophenol is detected in the chromatogram. As yellow spots, 42. Parts of the plate with stains of substance on them are cut out and each spot is eluted with 5 ml of dimethylformamide for 5 minutes. The extracts were filtered directly into spectrophotometric cuvettes through a paper filter, and the absorbance was measured at 435 on a spectrophotometer. The reference solution is the zlate obtained from the control experiment. Based on the measurement results, a plot is made of the dependence of the optical density of the solutions on the concentration of the analyte. The least squares method calculates the equation of the calibration graph, which in this case has the form

, 08495-0 + 0.0005, where D is the optical density;

C is the concentration of the colored solution, µg / ml.

The results of the determination of 2-methyl-4,6-dinitrophenol in liver tissue are presented in Table 1.

PRI me R 2. Determination of 2-sec-octyl-4,6-dinitrophenol in the liver tissue.

26 mg of 2-sec-octyl-4,6-dinitrophenol was added to 50 g of finely ground tissue of fresh human cadaveric liver, the biological tissue was thoroughly mixed with the substance and left for 1 day at 18-20 ° C. After this time, the mixture is poured into 100 ml of acetone and incubated for 0.5 h, stirring occasionally. The extraction is separated and the setting operation is repeated twice more. The acetone extracts are combined, evaporated to dryness in a stream of air at room temperature. The residue was dissolved in 50 ml of hexane. The resulting solution was extracted three times with an alkaline solution (universal buffer with a pH of 11.98, diluted four times with water) in 50 ml portions. The alkaline extracts are separated, combined, acidified with a 24% hydrochloric acid solution to pH 2-3 and extracted with 100 ml portions of diethyl ether. The ether extracts are combined, the ether is evaporated. The dry residue was dissolved in acetone, the solution was transferred to a 50 ml volumetric flask and adjusted to the mark with acetone. 0.1 ml of this solution was applied to the start line of a Silufol UV-254 chromatographic plate and chromatographed in a solvent system of chloroform-hexane (7: 4). 2-sec-0-octyl-4,6-dinitrophenol appears on the chromatogram as a yellow spot with

, 76.

To determine the amount of 2-sec-octyl-4,6-dinitrophenol recovered and the degree of extraction, a section of the chromatographic plate with a stain of the substance was cut out, treated with 5 ml of dimethylformamide and the resulting colored solution was filtered into a spectrophotometric cuvette. The optical density of the solution is measured on a spectrophotometer.

SF-26 at a wavelength of 445 nm. The measurements are carried out against the background of the solution obtained in the control experiment. The quantitative content of 2-sec-octyl-4,6-dinitrophenol is determined by the calibration equation

graphics and recounted for a sample of substances introduced into biological material.

Construction of a calibration graph. On-line chromatographic start

plates of the Silufol type UV-254 are applied t 0.025, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00 ml of a 0.005% solution of 2-sec-octyl-4,6-dinitrophenol in acetone and chromatography is carried out in

solvent system chloroform-hexane (7: 4). 2-sec-Octyl-4,6-dinitrophenol is detected on the chromatogram as yellow spots, 76. Parts of the plate with stains of substance on them are cut out and each spot is eluted with 5 ml of dimethylformamide for 5 minutes. Extracts are filtered directly into spectrophotometric cuvettes through a paper filter, absorbance is measured

at 445 gm on a spectrophotometer. Comparison solution - eluent obtained from a control experiment. Based on the measurement results, a plot is made of the dependence of the optical density of solutions on the concentration of the analyte. The least squares method calculates the equation of the calibration graph, which in this case has the form, 05545-C + 0.00504,

where D is the optical density;

C is the concentration of the colored solution, µg / ml.

The results of the determination of 2-sec-octyl-4,6-dinitrophenol in the liver tissue are shown in Table 2.

Example 3. Determination of 4-sec-octyl-2,6-dinitrophenol in liver tissue.

25 mg of 4-sec-octyl-2,6-dinitrophenol is added to 50 g of finely ground tissue of fresh human cadaveric liver, the biological tissue is thoroughly mixed with the substance and left for 1 day at 18-20 ° C. After this time, the mixture is poured into 100 ml of acetone and incubated for 0.5 h, stirring occasionally. The extract was separated and the infusion operation was repeated twice more. The acetone extracts are combined, evaporated to dryness in a stream of air at room temperature. The residue was dissolved in 50 ml of hexane. The resulting solution was extracted three times with an alkaline solution (universal buffer with a pH of 11.98, diluted four times with water) in 50 ml portions. The alkaline extracts are separated, combined, acidified with a 24% hydrochloric acid solution to pH 2-3 and extracted with 100 ml portions of diethyl ether. The ether extracts are combined, the ether is evaporated. The dry residue was dissolved in acetone, the solution was transferred to a 50 ml volumetric flask and adjusted to the mark with acetone. 0.1 ml of this solution is applied to the start line of a Silufol UV-254 chromatographic plate and chromatographed in a solvent system of chloroform-hexane (7: 4). 4-sec-0-octyl-2,6-dinitrophenol appears on the chromatogram as a yellow spot with

,thirty.

To determine the amount of 4-sec-octyl-2,6-dinitrophenol recovered and the degree of extraction ... a portion of the chromatographic plate with a stain of the substance was cut out, treated with 5 ml of dimethylformamide and the resulting colored solution was filtered in an SF-26 spectrophotometer with wavelength 490 nm. Measurements were carried out against the background of a solution obtained in a control experiment. The quantitative content of 4-sec-octyl-2,6-dinitrophenol is determined by the equation of the calibration graph and converted to a sample of the substance introduced into the biological material.

Construction of a calibration graph. 0.02, 0.04, 0.08, 0.16, 0.32, 0.48, 0.64, 0.80 ml of a 0.005% solution 4 are applied to the start line of a Silufol UV-254 chromatographic plate 2-octyl-2,6-dinitrophenol in acetone and chromatography was performed on a solvent system of chloroform-hexane (7: 4). 4-sec-0-octyl-2,6-dinitrophenol is detected on the chromatogram as yellow spots, 70. Parts of the plate with stains of substance on them are cut out and each spot is eluted with 5 ml of dimethylformamide for 5 minutes. The extracts were filtered directly into spectrophotometric cuvettes through a paper filter, the optical density was measured at 590 nm on a spectrophotometer, and the reference solution was the eluate obtained from the control experiment. Based on the measurement results, a plot is made of the dependence of the optical density of the solutions on the concentration of the analyte. The least squares method calculates the equation of the calibration graph, which in this case has the form

, 02576-C + 0.00482,

where D is the optical density;

C is the concentration of the colored solution, µg / ml.

The results of the determination of 4-sec-ok IL-2, 6-dinitrophenol in the liver tissue are shown in Table 3.

The proposed method in comparison with the prototype increases the degree of extraction of alkyl dinitrophenols from liver tissue E 2.5 times and the sensitivity of determination by 4 times. In contrast to the prototype, the proposed method is more selective and allows the determination of alkyl dinitrophenols in the presence of various classes of organic compounds: derivatives of tropane (TROPINO ester of dl-tropic acid, scopinone ester of l-tropic acid), piperidine (1,2,5-trimethyl-4 -propionyloxy-4-phen | / 1l-piperidine), quinuclidine (3-acetoxyquinuclidine), quinoline (/ -diethylaminortylamide-2-butoxycinquinonic acid), 4- (1-methyl-4-diethylamino) -7- chloroquinoline, 2- (4-nitrostyryl} -4- (1 | -methyl-4-diethylamine-obutylamino) -6-methoxyquinoline, b -megoc - siquinolyl- (41) - 5-vinylquinuclide and l - (2) - carbinol), phenothiazine 2-chloro-10- (3-d11methylaminopropyl) -phenothiazine, 10- (3 dimethylaminopropyl) -phenothiazine, 10- (2-dimethylaminopropyl) -phenothiazine, quinolysidine (a-spartein), pyrrolisidine (platifillin), isoquinoline 6,7-dimethoxy-1- (3,4-dimethoxybenzyl) -isoquinoline, phenanthrenisinoquinoline (morphine, code ), imidazole (pilocarpine).

A comparative characteristic of the proposed and known methods is presented in Table 4.

Claims (1)

The claims A method for determining alkyl dinitrophenols in biological material by grinding a sample treated with an organic solvent, evaporating, dissolving the obtained solid residue and chromatographing the solution in a thin layer of gel forces, characterized in that, with a chain for increasing the sensitivity and selectivity of the method, as an organic solvent acetone is used, the dry residue is dissolved in a hydrophobic solvent, treated with an alkaline solution, the alkaline solution is separated, acidified to pH 2-3, and diethyls are extracted ether, the extractant is evaporated, the residue is dissolved in acetone and chromatography is carried out in a solvent system of chloroform - hexane with a volume ratio of 7: 4. Results of determination of 2-methyl-4,6-dinitrophenol in liver tissue Results of determination of 2-sec-octyl-4,6-dinitrophenol in liver tissue Results of determination of 4-sec-octyl-2,6-dinitrophenol in liver tissue Table 1 table 2 Table 3 A comparative characteristic of the proposed and known methods (for example, 2-methyl-, 6-dinitrophenol) Index The proposed method the heat of extraction,% sensitivity, mg per 0 g of liver lectivity 81, EO% Allows the selective determination of alkyl dinitrophenols in the presence of tropane derivatives (tropic ester d, 1-tropic acid, scopine ester of 1-tropic acid); piperidine (1. 3-trimethyl-propionyloxy-4-phenylpiperidine); quinucidine (3-acetoxyquinuclidine, 3-benzoyloxyquinuclidine); quinoline (/ -diethylaminoethylamide-2-butoxycinquinonic acid), k- (1-methyl-diethylaminobutylamino) -7-chloroquinoline, 2- (4-nitrostyryl) g - {11-methyl-1-diethylaminoamyl - but) -6-methoxyquinoline, b-methoxyquinolyl- (4) -5 vinylquinuclidyl) (2c-carbinol); phenothiazine 12-chloro-1 p- (3-dimethylaminopropyl) -phenothiazine J, 10- (3-dimethylaminopropyl) -phenothiazine, 10- (2-dimethylaminopropyl) -phenothiazine; quinolysidine (l-spartein); pyrrolisidine (platyphyllin); isoquinoline 6,7-Dimethoxy-1- (Z1, -dimethoxybenzyl) -isoquinoline; phenatrenisoquinoline (morphine, codeine); imidazo a (pilocarpine) Table a The known method of 28.83% It does not allow the selective determination of alkyl dinitrophenols in the presence of tropane derivatives (tropic ester d, 1-tropic acid, scopine ester of 1-tropic acid); piperidine (1,2,5-trimethyl-propionyloxy-phenylpiperidine); quinuclidine (3-acetoxyquinuclidine, 3-benzoyloxyquinuclidine); quinoline (Ј-diethylaminoethylamide-2-butoxycinquinonic acid), Ag (1-methyl- - diethylaminobutylamino) -7-chloroquinoline, 2- {V-nitrostyryl) - 4- (1-methyl-4- diethylaminobutyl amino) -6-methoxyquinoline; 6-methoxyquinolyl- () - 5-vinylquinuclidyl- (2) J-carbinol; phenothiazine 2-chloro-1 O- (3-dimethylaminopropyl) -phenothiazine, 1 O - (3-dimethylaminopropyl) -phenothiazine., 10- (2G-dimethylaminopropyl) -phenothiazine. j quinolizidine (OC-spartein); pyrrolisidine (platifillin); isoquinoline f6, 7-Dimethoxy-1 - (3, - dimethoxybenzyl) -isoquinoline |; phenanthrene isoquinoline (morphine, codeine); imidazole (pilocarpine)