Method of detecting 2-dimethylamino-1,3-bis-(phenylsulphonylthio)propane in biological material

FIELD: chemistry.

SUBSTANCE: analysed sample is infused with an organic insulating agent. The obtained extract is purified through chromatography on a column of silica gel L 40/100 µ, while performing elution with a mixture of organic solvents. The analysed substance is determined using a chromatographic technique using a mobile phase which contains hexane, dioxane and propanol-2. The organic insulating agent is toluene. The toluene extract is dehydrated with anhydrous sodium sulphate. During the purification process, hexane is first passed through the column and elution is then carried out with a hexane-dioxane-propanol-2 solvent mixture (8:3:0.6 by volume). Eluate fractions containing the analysed substance are merged. The eluent is evaporated. The residue is dissolved in the hexane-dioxane-propanol-2 solvent mixture (15:5:1 by volume) and detection is carried out using high-performance liquid chromatography (HPLC) in a 64×2 mm column filled with Silasorb-600 sorbent using a hexane-dioxane-propanol-2 mobile phase (15:5:1 by volume) and a UV detector.

EFFECT: high accuracy and sensitivity of analysis.

3 ex, 4 tbl

 

The invention relates to the biology, ecology, sanitary and Toxicological chemistry, and in particular to methods for determining 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in a biological material, and can be used in the practice of epidemiological stations, chemical-Toxicological, clinical, veterinary and environmental laboratories. The method refers to the mass number.

The known method for the determination of organic substances of the main character, which is 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane, namely, that the biological object is crushed, repeatedly insist with acidified ethanol (every time during the day), the extracts combined combined extract is concentrated and precipitated proteins in it and some other endogenous substances biomaterial absolute ethanol precipitated precipitate is filtered off, the concentration process of the United extraction, precipitation in him endogenous substances biomaterial and the precipitate was filtered repeat repeatedly until the termination of the appearance of the precipitate, the filtrate is concentrated to a thick syrup, diluted with water and extracted at the beginning of the analyzed compounds from acidic, and from the alkaline solution (Svickova PPM Toxicological chemistry. - M.: Medicine, 1975. - 119-123).

The method is characterized by low selectivity, lack of the enough high sensitivity and accuracy.

A known method for determining 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in biological material (tubers) by grinding the biological object, a three-time processing mode shaking portions of hexane, the mass of each of which is twice the mass of the biomaterial branch checkouts, their associations, dehydration with anhydrous sodium sulfate, evaporation to a small volume determination by TLC thin layer of broad porous silica gel plates "Silufol using the mobile phase hexane-acetone (3:2) (Methods for the determination of trace pesticides in food, feed and the environment. Vol.1 / Magliano, Ann, Craneville, Hahahahha. - M.: Kolos, 1992. - S-192).

The method has relatively low sensitivity and low accuracy.

The closest way to detect 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in a biological material, which consists in the fact that the analyzed substance isolated from a biological sample with chloroform, the resulting extract was subjected to purification in a column of silica gel L 40/100 µ, using eluent hexane-acetone (9:1) and carry out the determination by TLC thin layer of broad porous silica gel using the mobile phase hexane-dioxane-propanol-2 (80:30:6) (Sormano the VK, Baranov, YU. Application of TLC method for determination of bankol in extracts from biological material // Scientific-practical conference with international participation "the State and prospects of forensic toxicology service and research" (Kharkiv. 9-10 November 2005). - Kharkov, 2005. - S.43).

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, twice for 45 minutes to process portions of toluene, the mass of each of which exceeds 2 times the mass of a biological object, resulting extract combine, dehydrated with anhydrous sodium sulfate, the solvent of the combined extract is evaporated, the residue is dissolved in an organic solvent, purified in a column of silica gel L 40/100 µ, first passing through her hexane, and then elwira mixture solvent of hexane-dioxane-propanol-2 (8:3:0,6 volume), fractions of the eluate containing an analyte, unite, eluent is evaporated, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2 (15:5:1 by volume) and carry out the determination by HPLC column of dimensions 64×2 mm, filling the Noah sorbent "Selasar 600", using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and UV-detector.

The method is as follows: biological tissue containing 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane, crushed, twice for 45 minutes to process portions of toluene, the mass of each of which is twice the mass of a biological object, resulting extract is separated from the solid particles of the biomaterial are pooled, filtered through a layer of anhydrous sodium sulfate, the solvent of the combined extract is evaporated, the residue is dissolved in chloroform, introducing sorbent and, after evaporation of the chloroform, chromatographic column with silica gel L 40/100µ, first passing through her hexane, and then elwira a mixture solvent of hexane-dioxane-propanol-2 (8:3:0,6 volume), fractions of the eluate containing an analyte, unite, eluent is evaporated, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2 (15:5:1 by volume) and carry out the determination by HPLC column of dimensions 64×2 mm, filled with sorbent Silsor 600", using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and UV-detector.

Example 1

Definition 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in liver tissue

To 10 g of finely ground liver tissue was added 5 mg of 2-dimethylamino-1,3-bis-(phenylsulfonyl the IO)propane, carefully mix the biological tissue with a substance and leave for half an hour at a temperature of 18-20°C. after this time the biological object containing an analyte, pour 20 g of toluene and incubated for 45 minutes with occasional stirring. The extract is separated from the solid particles of the biomaterial, the operation processing is repeated in these conditions. Separate hoods combine, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate height of 1-1,5 cm Filter additionally washed with 10 g of toluene. The filtrate and wash liquid are combined, the solvent is evaporated in a stream of air at room temperature. The residue is dissolved in 4 ml of chloroform, 2 ml of the obtained solution is mixed with 1 g of silica gel L 40/100 µ and evaporated residues of chloroform from the sorbent in a stream of air.

In column size 490×10 mm make first 9 g of silica gel type L 40/100 µ, and then, on top of the formed layer, 1 g of silica gel L 40/100 µ containing an analyte, previously entered in the form of a chloroform solution. Through the column first pass hexane, after the expiration of the column with 20 ml of the eluate solvent over the surface of the column sorbent, delete and start eluted with the solvent system hexane-dioxane-propanol-2 (8:3:0,6 - by volume). Since the filing system is s solvents hexane-dioxane-propanol-2 (8:3:0,6 - by volume) coming out of the column eluate is collected in separate fractions of 2 ml each. Fractions 11 to 14 inclusive are combined into varicellae Cup, eluent is evaporated in a stream of air at room temperature. The residue is dissolved in a mixture of 5 ml of dioxane and 1 ml of propanol-2, quantitatively transferred into a volumetric flask with a capacity of 25 ml, there make 15 ml of hexane and bring the contents of the flask with a mixture solvent of hexane-dioxane-propanol-2 (15:5:1) up to the mark. 8 μl of the obtained solution are injected type "milikhrom".

Chromatographic by HPLC. The process of chromatography was carried out is carried out in a column of size 64×2 mm, filled with normal-phase sorbent Silsor 600" using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and UV-detector.

The feed rate of eluent is 100 ál/min Scale registration of 0.8 eop, the measurement time of 0.6 sec. The optical density recorded at a wavelength of 256 nm.

The peak on the chromatogram with retention time of 6.35 min (retention 635 μl) corresponds to 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane.

The quantitative content of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane determine, based on the chromatographic peak area, the equation of the calibration graph and count on a portion of the substance made in the liver tissue.

The building is kalibrovochnogo graph.

In a series of volumetric flasks with a capacity of 25 ml make 0,25; 0,5; 1,0, 2,0; 5,0, 10,0 ml of 0.05% solution of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in a mixture solvent of hexane-dioxan-propanol-2 (15:5:1 by volume) and bring to the mark with a mixture solvent of hexane-dioxane-propanol-2 (15:5:1 by volume). 8 μl of each of the obtained solutions are injected. The chromatography was carried out is carried out in a column of size 64×2 ml, filled with sorbent Silsor 600"using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and a UV detector. The feed rate of eluent is 100 ál/min

The scale registration of 0.8 eop, the measurement time of 0.6 sec. The optical density recorded at a wavelength of 256 nm.

According to the results of measurements on the chromatograph build a graph of the dependence of chromatographic peak area against the concentration of the detected substance. Linear within the concentration range of 0.04 to 1.6 mcg.

By the method of least squares to calculate the equation of the calibration graph, which in this case is:

S=2,58489·C+0,02872,

where S is the area of the chromatographic peak, C is the concentration of analyte in khromatograficheskoi sample, ág.

The results of quantitative determination of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in liver tissue are shown in table 1.

Example 2

Definition 2-dimethylamino-1,3-bis-(peninsul is phenylthio)propane in the tissue of the stomach

To 10 g of finely ground tissue of the stomach is added 5 mg of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane, thoroughly mixed biological tissue with a substance and leave for half an hour at a temperature of 18-20°C. after this time the biological object containing an analyte, pour 20 g of toluene and incubated for 45 minutes with occasional stirring. The extract is separated from the solid particles of the biomaterial, the operation processing is repeated in these conditions. Separate hoods combine, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate height of 1-1,5 cm Filter additionally washed with 10 g of toluene. The filtrate and wash liquid are combined, the solvent is evaporated in a stream of air at room temperature. The residue is dissolved in 4 ml of chloroform, 2 ml of the obtained solution is mixed with 1 g of silica gel L 40/100 µ and evaporated residues of chloroform from the sorbent in a stream of air.

In column size 490×10 mm make first 9 g of silica gel type L 40/100 µ, and then, on top of the formed layer, 1 g of silica gel L 40/100 µ containing an analyte, previously entered in the form of a chloroform solution. Through the column first pass hexane, after the expiration of the column with 20 ml of the eluate solvent over the surface of the column sorbent, remove and begin ale is encoded by the solvent system hexane-dioxane-propanol-2 (8:3:0,6 - by volume). Since the filing of the solvent system hexane-dioxane-propanol-2 (8:3:0,6 vol.) emerging from the column eluate is collected in separate fractions of 2 ml each. Fractions 11 to 14 inclusive are combined into varicellae Cup, eluent is evaporated in a stream of air at room temperature. The residue is dissolved in a mixture of 5 ml of dioxane and 1 ml of propanol-2, quantitatively transferred into a volumetric flask with a capacity of 25 ml, there make 15 ml of hexane and bring the contents of the flask with a mixture solvent of hexane-dioxane-propanol-2 (15:5:1) up to the mark. 8 μl of the obtained solution are injected type "milikhrom".

Chromatographic by HPLC. The process of chromatography was carried out is carried out in a column of size 64×2 mm, filled with normal-phase sorbent Silsor 600" using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and UV-detector.

The feed rate of eluent is 100 ál/min Scale registration of 0.8 eop, the measurement time of 0.6 sec. The optical density recorded at a wavelength of 256 nm.

The peak on the chromatogram with retention time of 6.35 min (retention 635 μl) corresponds to 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane.

The quantitative content of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane determine, based on the chromatographic peak area, using equation kalibrovochnoj the graph and count on a portion of the substance, made in the tissue of the stomach.

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

The results of quantitative determination of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in the tissue of the stomach presented in table 2.

Example 3

Definition 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in the kidneys

To 10 g of finely ground tissue of the kidneys is added 5 mg of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane, thoroughly mixed biological tissue with a substance and leave for half an hour at a temperature of 18-20°C. after this time the biological object containing an analyte, pour 20 g of toluene and incubated for 45 minutes with occasional stirring. The extract is separated from the solid particles of the biomaterial, the operation processing is repeated in these conditions. Separate hoods combine, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate height of 1-1,5 cm Filter additionally washed with 10 g of toluene. The filtrate and wash liquid are combined, the solvent is evaporated in a stream of air at room temperature. The residue is dissolved in 4 ml of chloroform, 2 ml of the obtained solution is mixed with 1 g of silica gel L 40/100 µ, and evaporated residues of chloroform from the sorbent in a stream of air.

In column size 490×10 mm make first 9 g C is images type L 40/100 µ, and then, on top of the formed layer, 1 g of silica gel L 40/100 µ containing an analyte, previously entered in the form of a chloroform solution. Through the column first pass hexane, after the expiration of the column with 20 ml of the eluate solvent over the surface of the column sorbent, delete and start eluted with the solvent system hexane-dioxane-propanol-2 (8:3:0,6 vol.). Since the filing of the solvent system hexane-dioxane-propanol-2 (8:3:0,6 vol.) emerging from the column eluate is collected in separate fractions of 2 ml each. Fractions 11 to 14 inclusive are combined into varicellae Cup, eluent is evaporated in a stream of air at room temperature. The residue is dissolved in a mixture of 5 ml of dioxane and 1 ml of propanol-2, quantitatively transferred into a volumetric flask with a capacity of 25 ml, there make 15 ml of hexane and bring the contents of the flask with a mixture solvent of hexane-dioxane-propanol-2 (15:5:1) up to the mark. 8 μl of the obtained solution are injected type "milikhrom".

Chromatographic by HPLC. The process of chromatography was carried out is carried out in a column of size 64×2 mm, filled normalnotorowym sorbent "Selasar 600" using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and UV-detector.

The feed rate of eluent is 100 ál/min Scale registration of 0.8 eop, the maintenance time is 0.6 seconds. The optical density recorded at a wavelength of 256 nm.

The peak on the chromatogram with retention time of 6.35 min (retention 635 μl) corresponds to 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane.

The quantitative content of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane determine, based on the chromatographic peak area, the equation of the calibration graph and count on a portion of the substance entered the tissue of the kidneys.

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

The results of quantitative determination of 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in the tissue of the kidney are presented in table 3.

The proposed method is compared with the prototype 6 times increases the sensitivity of the determination in biological material, and in 30-40 times in khromatograficheskoi the sample, and also characterized by higher accuracy (half-width of the confidence interval decreases by 2.5 times). Comparative characteristics of the proposed and known methods are presented in table 4.

Table 4
Comparative characteristic featur is imago and known methods (for example, the study of the liver tissue)
IndexThe proposed methodThe known method
1. Sensitivity (open minimum):
in khromatograficheskoi sampleof 0.01 mcg0.3 to 0.4 mcg
b) 100 g of a biomaterial0.3 mg2 mg
2. The degree of extraction (provided content 50 mg analyte per 100 g of biomaterial)75,63% (isolate toluene)is 55.74% (isolate chloroform)
3. Half-width confidence interval (accuracy rate) (n=5; P=0.95)2,90%8,68%

The method for determining 2-dimethylamino-1,3-bis-(phenylsulfonyl)propane in a biological material, which consists in the fact that the analyzed sample insist organic insulating agent, the obtained extract purified by chromatography was carried out in a column of silica gel L 40/100 µ, by elution with a mixture of organic solvents, and carry out the determination of the analyte XP is motographics method using a mobile phase comprising hexane, dioxane and propanol-2, characterized in that the organic insulating agent is toluene, the toluene extract dehydrated with anhydrous sodium sulfate, in the clearing process through the column first pass hexane, and then elute with a mixture solvent of hexane-dioxane-propanol-2 (8:3:0,6 volume), fractions of the eluate containing an analyte, unite, eluent is evaporated, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2 (15:5:1 by volume) and spend the definition method in VIEH column dimensions 64×2 mm, filled with sorbent Silsor-600" using the mobile phase hexane-dioxane-propanol-2 (15:5:1 by volume) and a UV detector.



 

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5 cl, 1 ex, 4 tbl

FIELD: chemistry.

SUBSTANCE: disclosed is a method of detecting unknown substances in body fluids of patients taking narcotic or psychoactive substances. The method involves preparation of three body fluid samples - the first through extraction with re-solution, the second through acid hydrolysis and the third through enzymatic hydrolysis. The first sample undergoes GC/MS analysis at temperature gradient of 15°C/min and data are analysed by comparing with a data base from which features of the unknown substance are detected, specifically spectra with m/z values which coincide with basic ions of the narcotic or psychoactive substance or metabolites and content of the unknown substance in the sample. The second sample undergoes GC/MS analysis at temperature gradient of 25°C/min and the third sample undergoes GC/MS analysis also at temperature gradient of 15°C/min and, if content of the unknown substances in the last two samples is higher than the in the first, the narcotic or psychoactive substance undergoes GC/MS analysis for presence of the unknown substance also at temperature gradient of 15°C/min, and if also not present in the basic substance. Presence of the unknown substance in intact body fluid is also checked, for which a sample of the intact body fluid is prepared via acid hydrolysis and undergoes GC/MS analysis at temperature gradient of 15°C/min and 25°C/min, and if the unknown substance is detected in the intact body fluid, the substance is classified as endogenous, and in the absence of features, an aliquot of the first sample is mixed with the sample of intact body fluid. The sample is prepared via acid hydrolysis of the mixture. The sample undergoes GC/MS analysis at temperature gradient of 15°C/min and 25°C/min. Further, content of the unknown substance is determined from results of both analysis modes and then compared with content of the known substance in the first sample. If content values of the unknown substance in the said three samples coincide, the unknown substance is classified as a new, previously unknown product of metabolism of the basic narcotic or psychoactive substance.

EFFECT: possibility of unique identification of chemical compounds and their fragments in arbitrary combinations while increasing accuracy and rapidness of detection.

4 tbl

FIELD: chemistry.

SUBSTANCE: sodium fluoride is added to an analysed sample in amount of 10% of the mass of the biological object and infused twice in 45 minutes with portions of ethyl acetate, the mass of each of which is twice higher than the mass of the biological material. Separate extractions are combined, filtered through anhydrous sodium sulphate. The solvent from the filtrate is evaporated at temperature 50-60°C. The residue is dissolved in a mixture of hexane-dioxane-propanol-2 solvents. Chromatography is performed in a column with silica gel L 40/100 µ using a hexane-dioxane-propanol-2 mobile phase. The eluate fractions which contain the analysed substance are merged. The eluate is evaporated. The residue is dissolved in a mixture of hexane-dioxane-propanol-2 solvents and the analysed substance is determined via high performance liquid chromatography (HPLC) in a 64x2 mm column filled with Silasorb 600 sorbent using a hexane-dioxane-propanol-2 mobile phase and a UV detector.

EFFECT: invention shortens the duration of detecting tetraethyl thiuram disulphide in blood and increases its sensitivity.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: biological tissue is crushed, processed twice for 30 minutes with portions of ethyl acetate, weight of each twice exceeding weight of a biological object; prepared extractions are combined, filtered through anhydrous sodium sulphate; a solvent is evaporated from the filtrate; the residue is dissolved in acetonitrile; the prepared solution is watered down in the volume ratio 1:4, extracted twice in portions of chloroform, volume of each being equal to volume of a hydrophilic layer; the chloroform extracts are combined, steamed to a dry residue; the residue is dissolved in mixed solvents hexane-dioxane-propanol-2, cleaned in a silica gel column L 40/100µ with using a mobile phase hexane-dioxane-propanol-2; eluate fractions containing an analysed substance are combined; the eluent is evaporated; the residue is dissolved in mixed solvents hexane-dioxane-propanol-2 and analysed by a HELC method in a column of dimensions 64×2 mm filled with the sorbent Silasorb 600 with using a mobile phase hexane-dioxane-propanol-2 and a UV detector.

EFFECT: invention allows higher selectivity, sensitivity and accuracy of biological material analysis for tetramethylthiuramdisulfide.

4 ex, 5 tbl

FIELD: chemistry.

SUBSTANCE: in the method of detecting phenol in aqueous solution via reverse-phase micro-column high-performance liquid chromatography with a preliminary sample preparation step through liquid-liquid extraction with acetonitrile, extraction is carried out at temperature 263±2 K for 30 minutes with ratio of equilibrium volume of water to the organic phase equal to 1:1.

EFFECT: simple and cheap method, high degree of extracting phenol, low detection limit.

1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: for an assay, 5-7 cm3 of blood is taken, and before extraction the sample is pre-treated with 2 cm3 of 60% sulphuric acid; the extraction process is executed with 30 cm3 of n-hexane once, and gas chromatography is preceded with single treatment of n-hexane extract with 10 cm3 of concentrated sulphuric acid.

EFFECT: invention provides higher reliability of α-HCCH, γ-HCCH test results and twofold reduced time of sample preparation.

1 ex, 1 tbl

Gas analysis method // 2410679

FIELD: test equipment.

SUBSTANCE: invention refers to analysis of the quantity of impurities in carbon dioxide during manufacturing and/or cleaning process. Measurement method of concentration of impurities during gas cleaning consists in the fact that, first, gas flow containing impurities passes through gas absorbing device during the time period at ambient temperature or higher so that impurities can be absorbed. Than gas flow movement is stopped. Then, desorption and analysis of impurities in stopped gas flow movement is performed by means of detector. At that, impurities have been chosen from the group consisting of H2S, COS, dimethyl sulphide, benzene, aldehydes, spirits with low length of carbon chain and hydrocarbons. Also, in the proposed method, gas absorbing device includes column with absorbent layer in gas chromatograph, and gas is desorbed from column with absorbent layer through gas-distributing column.

EFFECT: improving accuracy and reducing costs for measurement of concentration of impurities during gas cleaning.

9 cl, 1 dwg

FIELD: industrial organic synthesis.

SUBSTANCE: thiolsulfonates of general formula RSO2SR (I), wherein R represents C1-C20-alkyl or benzyl, are prepared by oxidation of thiols RSH (II) or disulfides RSSR (III), wherein R is as above, oxidation being performed with chlorine dioxide at thiol (disulfide)-to-oxidant molar ratio 1:(2-4) at 10-30°C. Products obtained can be used as biologically active substances possessing bactericidal and fungicidal activities.

EFFECT: increased yield of thiolsulfonate (to 92%) owing to use of new oxidant for organosulfur compounds.

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