Method of determining n-(4-nitro-2-phenoxyphenyl)-methanesulphonamide in biological material

FIELD: chemistry.

SUBSTANCE: invention relates to biology and toxicological chemistry and can be used in chemical-toxicology, expert forensic and clinical laboratories. The method includes: crushing a biological object containing N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide, settling twice for 45 minutes with portions of an organic isolating agent which is methyl acetate, combining the obtained extracts, evaporating the solvent from the resultant extract, treating the residue with acetone, separating the acetone extract, evaporating the solvent from resultant extract, dissolving the residue in diethyl ether, extracting the ether solution with a buffer solution at pH 9-10, acidifying the aqueous alkaline extract with 24% hydrochloric acid to pH 2-3, saturating the obtained solution with sodium bromide, extracting with ethyl acetate, evaporating the obtained extract in an air current at 20-22°C until a dry residue is obtained, dissolving the residue in a mixture of hexane and acetone taken in volume ratio of 8:2, performing chromatography on a macrocolumn with silica gel L 40/100 mcm using a hexane-acetone mobile phase in volume ratio of 8:2, combining eluate fractions containing the analysed substance, evaporating the eluent in an air current at 20-22°C until complete removal of the solvent, dissolving the residue in methanol and performing determination via a combined physical-chemical method in the form of chromatography-mass spectrometry, using a DB-5 MS EVIDEX capillary column with a mobile phase which is 5% phenyl-95% methylpolysiloxane, using a mass-selective detector operating in electron impact mode, the initial thermostat temperature of the column is 70°C, maintaining said temperature for 3 minutes, further raising the temperature from 70°C to 290°C at a rate of 20°C per minute, maintaining the final temperature of the column for 16 minutes, the temperature of the injector is 250°C, the temperature of the quadrupole is 150°C, the temperature of the detector interface is 300°C, detecting strength of the signal resulting from charged particles formed when bombarding the analysed substance coming from the capillary column and falling into an ion source with an ionising electron beam with energy of 70 eV, recording the mass spectrum on the full ion current, while calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide from the area of the chromatographic peak.

EFFECT: high sensitivity of analysis.

2 ex, 3 tbl

 

The invention relates to biology, chemistry and toxicology, and in particular to methods of determining N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in biological material, and can be used in practice in practice, chemical-Toxicological, forensic and clinical laboratories. The method refers to the mass number.

A known method for determining N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in biological material (liver tissue) on the basis of the isolation of the Stas-Otto, namely, that the biological object is crushed, insist 3-4 times in 24 hours with an organic insulating agent, which is ethanol, acidified with oxalic acid, the extracts unite, concentrate, the concentrate was added ethanol, and the resulting precipitate protein structures is filtered, the process of concentration and addition of ethanol repeat until while the addition of ethanol to the resulting once again concentrate will not be observed in the absence of precipitate protein structures, the solution was diluted with water, the pH of the resulting solution adjusted to pH 2, extracted with chloroform, the chloroform extract is evaporated to the dry residue, the residue is dissolved in ethanol, the resulting solution was centrifuged, injected and determine an analyte by HPLC in Colo is ke with sorbent Prontosil-120-5-With-18" dimensions 75×2 mm, conducting the process in gradient mode using as eluents 0.1% solution triperoxonane acid and acetonitrile, changing the content of acetonitrile in the mobile phase from 0% to 100% for up to 25 minutes (With N. A., Statsenko I. C., L. Voronkov, Isolation and identification of nimesulide in biological material // Actual problems of legal medicine and forensic experience. - 2008. No. 13. - S. 75-78).

The way is long run, labor is not high enough sensitivity and selectivity of detection.

A known method for determining N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in biological material (tissues, organs), which consists in the fact that the biological object is crushed, insist with a 1% sodium hydroxide solution once daily for 30 minutes with occasional stirring, the obtained extract was separated by filtering through cheesecloth, acidified with 6 N. solution chloroethanol acid to pH 1.0 to 2.0, extracted twice with portions of chloroform, the chloroform extracts are combined, the solvent from the combined extracts evaporated, the residue is dissolved in chloroform, the resulting solution contribute in a column of aluminum oxide, elute first with chloroform and then ethanol, ethanol eluate is collected, and the solvent of ethanol in the eluate is evaporated, the residue dissolve Aut in 0.1 N. the sodium hydroxide solution, investigate the absorption of the resulting colored solution in the interval of wavelengths 300-400 nm, an analyte identify the position of the maxima of the absorption bands and the shape of the spectral curve and calculate the quantitative content of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide the optical density measured at a wavelength of 392 nm (Dobroriz A. M. Detection of nimesulide in biological material // Judicial-medical expertise. - 2009. - T. 52, No. 4. - S. 32-34).

The method is characterized not high enough sensitivity.

The closest is the method for determining N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in biological material (tissues, organs), which consists in the fact that the biological object is crushed, twice for 45 minutes insist with portions of the organic insulating agent, which is acetone, resulting extract combine, the solvent of the combined extract is evaporated, the residue is treated with acetone, the acetone extract is separated, chromatographic in macrobalance silica gel KCC No. 3 80/120 μm using a mobile phase of hexane-acetone in the ratio of 75:25 by volume fractions of the eluate, containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 20-22°C to complete the removal process is Italia, the residue is dissolved in chloroform and spend the definition of a combined physical-chemical method, which uses gas chromatography-spectrophotometry, while chromatographic by TLC on plate "Sorbfil" brand PTSH-AF-B-UV, using mobile phase toluene-acetonitrile in the ratio of 7:3 by volume, the resulting chromatogram is shown in UV-light, elute an analyte from the sorbent of dimethylformamide, the eluate spectrophotometric, calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide the optical density of the eluate, measured at a wavelength of 439,4 nm (Shormanov C. K., Gerasimov, D. A., Siliva L. E., Galushkin, S., Simon R. Y. Distribution of nimesulide in the body of warm-blooded animals // pharmacy. - 2013. - So 62, No. 1. - S. 39-42).

The method is characterized not high enough sensitivity.

The technical result of the present invention is to increase the detection sensitivity.

The technical result is achieved by the fact that the biological object is crushed, twice for 45 minutes insist with portions of the organic insulating agent, which is the acetate, the obtained extract combine, the solvent of the combined extract is evaporated, the residue is treated with acetone, the acetone extract is separated, the solvent of the combined extract is of evaporated, the residue is dissolved in diethyl ether, the ethereal solution is extracted with a buffer solution with a pH of 9-10, aqueous alkaline extract is acidified 24% solution chloroethanol acid to pH 2-3, the resulting solution is saturated with sodium bromide, extracted with ethyl acetate, the obtained extract was evaporated in a stream of air at a temperature of 20-22°C to obtain a dry residue, the residue is dissolved in a mixture of hexane and acetone, taken in the ratio of 8:2 by volume, chromatographic in macrobalance silica gel L 40/100 μm using a mobile phase of hexane-acetone in the ratio of 8:2 by volume fractions of the eluate, containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 20-22°C until complete removal of the solvent, the residue is dissolved in methanol and spend the definition of a combined physical-chemical method, which uses gas chromatography-mass spectrometry, using a capillary column DB-5 MS EVIDEX with stationary phase, representing 5%-phenyl-95%-methylpolysiloxanes using mass-selective detector operating in electron impact mode, the initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature increases from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained within 16 minutes, those who injector temperature is 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, the ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide on the chromatographic peak area.

The method is as follows: the biological object containing N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide, crushed, twice for 45 minutes insist with portions of the organic insulating agent, which is the acetate, the obtained extract combine, the solvent of the combined extract is evaporated, the residue is treated with acetone, the acetone extract is separated, the solvent of the combined extract is evaporated, the residue is dissolved in diethyl ether, the ethereal solution is extracted with a buffer solution with a pH of 9-10, aqueous alkaline extract is acidified 24% solution chloroethanol acid to pH 2-3, the resulting solution is saturated with sodium bromide, extracted with ethyl acetate, the obtained extract was evaporated in a stream of air at a temperature of 20-22°C to obtain a dry residue, the residue is dissolved in a mixture of hexane and acetone, took the s in the ratio of 8:2 by volume, chromatographic in macrobalance silica gel L 40/100 μm using a mobile phase of hexane-acetone in the ratio of 8:2 by volume fractions of the eluate containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 20-22°C until complete removal of the solvent, the residue is dissolved in methanol and spend the definition of a combined physical-chemical method, which uses gas chromatography-mass spectrometry using capillary column DB-5 MS EVIDEX with stationary phase, representing 5%-phenyl-95%-methylpolysiloxanes using mass-selective detector, operating in electron impact mode, the initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, subsequently the temperature is increased from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained within 16 minutes, the temperature of the injector 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, calculating the amount of N-(4-n the tro-2-phenoxyphenyl)-methanesulfonamide on the chromatographic peak area.

Example 1

Determination of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in liver tissue

To 10 g of liver tissue was added 5 mg of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide, carefully mix the biological object with the substance and leave for 1.5 hours at a temperature of 20-22°C. after this time the mixture is poured 20 g organic insulating agent, which is the acetate, and insist 45 minutes with stirring. The extract is separated, the operation processing is repeated in the above described conditions. The obtained extract combine, the combined organic extract evaporated to remove the solvent in a stream of air at 20-22°C, the residue repeatedly (three times for 3 minutes) is treated with acetone (portions 15 g each) with vigorous stirring, the acetone extract is separated, unite, the solvent of the combined extract is evaporated in a stream of air at 20-22°C until complete removal of the solvent, the residue is dissolved in 10 ml of diethyl ether, the resulting solution is extracted twice with portions of buffer solution with pH 9-10 10 ml each. Separate the aqueous alkaline extracts are combined acidified with 24% solution chloroethanol acid to pH 2-3, the resulting solution is saturated with sodium bromide and extracted twice with portions of ethyl acetate and 20 ml each. Separate an ethyl acetate extracts of the volume of inaut, evaporated in a stream of air at a temperature of 20-22°C to obtain a dry residue.

The residue is dissolved in 2-3 ml of a mixture of hexane and acetone, taken in the ratio of 8:2 by volume, and contribute to the chromatographic microcolony dimensions 490×11 mm, filled with 10 g of silica gel L 40/100 μm. Chromatographic, using a mobile phase of hexane-acetone in the ratio of 8:2 by volume. The eluate is collected in separate fractions of 2 ml each.

Fractions 9 to 17 inclusive unite, evaporated in a stream of air at a temperature of 20-22°C until complete solvent removal. The residue is dissolved in 10 ml of methanol (solution A). 1.0 ml of the solution And contribute in a volumetric flask with a capacity of 50 ml and adjusted with methanol to the mark (solution B).

4 ál of a solution injected into a gas chromatography-mass spectrometer.

The definition is done using a gas chromatograph company Agilent Technologies (USA) model 6850 Network GC System with a quadrupole mass-selective detector model 5973 Network the same company.

The chromatography was carried out is carried out in a capillary column DB-5 MS EVIDEX length 25 m, inner diameter of 0.2 mm with a stationary phase thickness of 0.33 μm, representing 5%-phenyl-95%-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, subsequently the temperature is increased from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained within 16 minutes the injector temperature is 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, the ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, and calculates the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide on the chromatographic peak area.

As the carrier gas helium is used. The flow of carrier gas is about 0.6 ml/min Mode with the division of the flow of 1:2. Mass-selective detector is operating in electron impact mode (70 eV). Check the mass spectrum performed on total ion current with a delay of 3.5 minutes. The scanning range is 40-500 m/z.

The peak on the chromatogram with retention time 17,14 min corresponds to N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide. In the mass spectrum of this compound, obtained by total ion current, detected signals of some characteristic of charged particles with mass numbers 51, 77, 78, 128, 154, 169, 183, 199, 229, 308. The most intensive is a particle with mass number 154, the intensity of which is taken as 100%.

N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide identify the combination of BP the seed retention in the stationary phase of the column and a particular set of signals characteristic of charged particles in its mass spectrum.

On the chromatographic peak area obtained when registering the intensity of total ion current, determine the quantitative content of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide using the equation of the calibration curve, and count on a portion of the analyte introduced into the biological material.

The construction of the calibration curve

In a series of volumetric flasks with a capacity of 25 ml contribute to 0.05, and 0.2, and 0.8, 2,0, 5,0 ml 0,00125% solution and 0.8, 2,0, 8,0, 12,0, 20,0 ml of 0.0125% solution of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in methanol and the volume was adjusted contents of each flask to the mark with methanol.

4 μl of each of the obtained solutions injected into the gas chromatography-mass spectrometer.

The definition is done using a gas chromatograph company Agilent Technologies (USA) model 6850 Network GC System with a quadrupole mass-selective detector model 5973 Network the same company.

The chromatography was carried out is carried out in a capillary column DB-5 MS EVIDEX length 25 m, inner diameter of 0.2 mm with a stationary phase thickness of 0.33 μm, representing 5%-phenyl-95%-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, subsequently the temperature is increased from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained within 16 minutes, the temperature inject the RA is 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, the ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, and calculates the amount of N-[-(4-nitro-2-phenoxyphenyl)-methanesulfonamide on the chromatographic peak area.

As the carrier gas helium is used. The flow of carrier gas is about 0.6 ml/min Mode with the division of the flow of 1:2. Mass-selective detector is operating in electron impact mode (70 eV). Check the mass spectrum performed on total ion current with a delay of 3.5 minutes. The scanning range is 40-500 m/z.

According to the results of measurements on gas chromatography-mass spectrometer build a graph of peak area against the concentration of the detected substance. Linear in the concentration range 5·10-11-2,0·10-7he

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

S=429918·-7874,

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

The results of quantitative determination of N-(4-nitro-2-f is noxi-phenyl)-methanesulfonamide in liver tissue are shown in table 1.

Example 2

Determination of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in the tissue of the lungs

To 10 g of lung tissue is added 5 mg of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide, carefully mix the biological object with the substance and leave for 1.5 hours at a temperature of 20-22°C. after this time the mixture is poured 20 g organic insulating agent, which is the acetate, and insist 45 minutes with stirring. The extract is separated, the operation processing is repeated in the above described conditions. The obtained extract combine, the combined organic extract evaporated to remove the solvent in a stream of air at 20-22°C, the residue repeatedly (three times for 3 minutes) is treated with acetone (portions 15 g each) with vigorous stirring, the acetone extract is separated, unite, the solvent of the combined extract is evaporated in a stream of air at 20-22°C until complete removal of the solvent, the residue is dissolved in 10 ml of diethyl ether, the resulting solution is extracted twice with portions of buffer solution with pH 9-10 10 ml each. Separate the aqueous alkaline extracts are combined acidified with 24% solution chloroethanol acid to pH 2-3, the resulting solution is saturated with sodium bromide and extracted twice with portions of ethyl acetate and 20 ml each. Separate an ethyl acetate extracts of the volume of inaut, evaporated in a stream of air at a temperature of 20-22°C to obtain a dry residue.

The residue is dissolved in 2-3 ml of a mixture of hexane and acetone, taken in the ratio of 8:2 by volume, and contribute to the chromatographic microcolony dimensions 490×11 mm, filled with 10 g of silica gel L 40/100 μm. Chromatographic, using a mobile phase of hexane-acetone in the ratio of 8:2 by volume. The eluate is collected in separate fractions of 2 ml each.

Fractions 9 to 17 inclusive unite, evaporated in a stream of air at a temperature of 20-22°C until complete removal of solvent.

The residue is dissolved in 10 ml of methanol (solution A). 1.0 ml of the solution And contribute in a volumetric flask with a capacity of 50 ml and adjusted with methanol to the mark (solution B).

4 ál of a solution injected into a gas chromatography-mass spectrometer.

The definition is done using a gas chromatograph company Agilent Technologies (USA) model 6850 Network GC System with a quadrupole mass-selective detector model 5973 Network the same company.

The chromatography was carried out is carried out in a capillary column DB-5 MS EVIDEX length 25 m, inner diameter of 0.2 mm with a stationary phase thickness of 0.33 μm, representing 5%-phenyl-95%-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, subsequently the temperature is increased from 70°C to 290°C at a rate of 20°C / minute, final temperature to which once is aged for 16 minutes the injector temperature is 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, the ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, and calculates the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide on the chromatographic peak area.

As the carrier gas helium is used. The flow of carrier gas is about 0.6 ml/min Mode with the division of the flow of 1:2. Mass-selective detector is operating in electron impact mode (70 eV). Check the mass spectrum performed on total ion current with a delay of 3.5 minutes. The scanning range is 40-500 m/z.

The peak on the chromatogram with retention time 17,14 min corresponds to N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide. In the mass spectrum of this compound, obtained by total ion current, detected signals of some characteristic of charged particles with mass numbers 51, 77, 78, 128, 154, 169, 183, 199, 229, 308. The most intensive is a particle with mass number 154, the intensity of which is taken as 100%.

N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide identify the combination of BP the seed retention in the stationary phase of the column and a particular set of signals characteristic of charged particles in its mass spectrum.

On the chromatographic peak area obtained when registering the intensity of total ion current, determine the quantitative content of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide using the equation of the calibration curve, and count on a portion of the analyte introduced into the biological material.

The construction of the calibration curve

The construction of the calibration curve and its equation is given in example 1.

The results of quantitative determination of N-(4-nitro-2-phenoxy-phenyl)-methanesulfonamide in lung tissue is presented in table 2.

The proposed method is compared with the prototype 5.7·104time increases the detection sensitivity for the detected sample and 12 times in biological material.

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

Table 1
The results of the determination of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in liver tissue (n=5; p=0.95)
No.Made N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide, mg in 10 g of liver tissueFound
the peak area on the chromatogram, the river. unitsmg khromatograficheskoi samplemg in terms of the sample introduced into the biomaterial% of the paid in biomaterial sampleMetrological characteristics,
%
15,0075913561,768·10-54,41988,38x-=84,57
25,0073420041,710·10-54,27485,48S=2,50
35,0070470801,641·10-54,10382,05Sx-=1,12
45,00711008 1,656·10-54,14082,79Δx-=3,11
55,0072267861,683·10-54,20784,14ε=3,68

Table 2
The results of the determination of N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in the lung tissue (n=5; p=0,95)
No.Made N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide, mg in 10 g of lung tissueFoundMetrological characteristics, %
the peak area on the chromatogram, the river. unitsmg khromatograficheskoi samplemg in terms of the linkage made in the bio is the material % of the paid in biomaterial sample
15,0076240301,775·10-54,43888,76x-=85,72
25,0070281641,637·10-54,09281,83S=2,72
35,0072465621,687·10-54,21984,37Sx-=1,21
45,0075148311,750·10-54,37587,49Δx-=3,38
55,0073996131,723·10-54,30886,15ε=4,94

Table 3
Comparative characteristics of the proposed and known methods (for example, the study of the liver tissue)
IndicatorsThe proposed methodThe known method
Sensitivity (open low)
a) 100 g of a biomaterial5,0·10-6g6,0·10-5g
b) the detected sample3,5·10-11g2,0·10-6g
The interval of linearity of the calibration curve (g detectable in the sample)
5,0·10-11-2,0·10-7g2,0·10-6sphere-6,4·10-5g

The method for determining N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide in biological material (tissues, organs), which consists in the fact that the biological object is crushed, twice for 45 minutes insist with portions of the organic insulating agent, resulting extract combine, the solvent of the combined extract is evaporated, the residue is treated with acetone, the acetone extract is separated, chromatographic in macrobalance silica gel using the mobile phase hexane-acetone fractions of the eluate containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 20-22°C until complete removal of the solvent, the residue is dissolved and spend the definition of a combined physical-chemical method, characterized in that the organic insulating agent is acetate, before chromatographytandem in macrobalance with silica gel, the solvent of the acetone extract is evaporated, the residue is dissolved in diethyl ether, the ethereal solution is extracted with a buffer solution with a pH of 9-10, aqueous alkaline extract is acidified 24% solution chloroethanol acid to pH 2-3, the resulting solution is saturated with sodium bromide, extracted with ethyl acetate, who received the extract is evaporated in a stream of air at a temperature of 20-22°C to obtain a dry residue, the residue is dissolved in a mixture of hexane and acetone, taken in the ratio of 8:2 by volume, when chromatographicaliy in macrobalance use silica gel L 40/100 μm, the ratio of hexane and acetone in the mobile phase is 8:2 by volume, the balance before the combined physico-chemical method is dissolved in methanol, as a combined physical-chemical method uses gas chromatography-mass spectrometry using capillary column DB-5 MS EVIDEX with stationary phase, representing 5%-phenyl-95%-methylpolysiloxanes, using mass-selective detector, operating in electron impact mode, the initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, subsequently the temperature is increased from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained within 16 minutes, the temperature of the injector 250°C, the temperature of the quadrupole 150°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of analyte released from the capillary column and trapped in the ion source, ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, calculating the amount of N-[-(4-nitro-2-Fenox is phenyl)-methanesulfonamide on the chromatographic peak area.



 

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2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: instrument comprises an oral sample collection vessel, a detector able to detect a marker in this sample, an indicator actuated by a detector signal. The above vessel is detachably connected to an oral cavity instrument. The vessel comprises a sample collection element, a sample storage container, and a passage connecting the collection element and the container to supply the sample to the container by capillary action. The indicator is integrated into the container. The declared instrument is used to diagnose oral diseases by collecting the oral sample, detecting one or more markers in this sample and indicating the presence of one of the disease markers.

EFFECT: inventions enables establishing an accurate and fast diagnosis of the oral pathologies accompanying the daily oral care by placing the detector inside the container able to accumulate a required amount of the sample to be diagnosed.

25 cl, 1 dwg

FIELD: medicine.

SUBSTANCE: technique involves the clinical-laboratory examination of a sportsman who completed heavy physical activity 12-16 hours ago. The examination extent is determined taking into account the organs and systems most vulnerable to the physical activity while deriving the prognostically significant criteria of the morphofunctional body state. The examination involves measuring and analyzing the biochemical, haematological, immunological and functional values, as well as vitamin-mineral saturation. And if the above values are stably unchanged, reliably different from the norm, nonspecific changes of the sportsman's organs and systems are diagnosed.

EFFECT: technique provides the early diagnosis of the significant changes of the organs and systems during trainings and competitions that enables taking further timely measures to prevent the further progression of pathological conditions and maintaining thereby occupational performance and achieving stable high sport results.

FIELD: biotechnology.

SUBSTANCE: efficiency of treatment of patients with high grade non-Hodgkin malignant lymphoma is determined by the likelihood of achieving remission, and 5-year total and relapse-free survival. The method comprises the study of polymorphism G13494A 6th intron of gene TP53 of the patient. In case of revealing in the patient of homozygous genotype G/G in a given locus the low efficiency of treatment is predicted, namely, low likelihood of 5-year survival of the patient and low likelihood of absence of relapse. In the case of revealing in the patient of the genotype A/A or G/A in a given locus, the high efficiency of treatment is predicted, namely the high likelihood of remission and 5-year survival of patient.

EFFECT: invention enables to assess the efficiency of treatment of patients with high grade non-Hodgkin malignant lymphoma on the degree of polymorphism G13494A of 6th intron of gene TP53.

5 dwg, 10 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention represents a diagnostic technique for the disturbed thrombocyte aggregation accompanying mucoviscidosis in children involving a thrombocyte aggregation test using the Multiplate aggregometer inducers. Trays with a magnetic mixer and electrodes are added with NaCl 400 mcl at 37°C and immediately added with whole blood 400 mcl from a hirudin test tube, incubated in the chamber for two minutes; the tray is added with 30 mcl of an aggregation inducer specified in a group: soluble thrombin receptor - peptid-6, adenosine diphosphate, arachidonic acid. The thrombocyte aggregation rate is displayed on the screen in the form of a curve, and the sub-curve area U is automatically calculated; the sub-curve area U shows the thrombocyte aggregation state as compared to reference values in the group of healthy children; if the threshold area U has appeared to exceed the reference, the thrombocyte hyperaggregation, while the threshold area U being less than the reference, the thrombocyte hypoaggregation is stated.

EFFECT: invention provides the timely diagnosis of microcirculatory disorders accompanying mucoviscidosis.

2 ex, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to the field of microbiology, namely to a method of microorganism characteristic. The essence of the method consists in the following: (a) obtaining a sample to be tested, about which it is known that it contains or can contain microorganisms; (b) layering the tested sample on a density buffer in a container, where the said density buffer possesses a uniform density from approximately 1.025 to approximately 1.120 g/ml; (c) addition of an identifier into the said tested sample and/or into the said density buffer; (d) centrifugation of the said container in order to separate microorganisms from other components of the said tested sample and to form a deposit of microorganisms; (e) spectroscopic analysis of the deposit and/or the said one or more than one identifier with obtaining measurements, which characterise the microorganisms, where the said spectroscopic analysis is carried out when the said deposit is located in the said container; and (f) characteristic of the microorganisms in the deposit on the basis of the obtained measurements and/or the presence or absence of the said identifier or a metabolised form of the said identifier in the deposit, where the said microorganisms are characterised by one or more classification models, selected from the group, consisting of Gram groups, clinical Gram groups, therapeutic and functional groups.

EFFECT: application of the claimed invention makes it possible to increase the accuracy of the microorganism characteristic.

15 cl, 5 dwg, 1 tbl, 4 ex

FIELD: physics.

SUBSTANCE: method is proposed for identifying narcotic and psychoactive substances in biological fluids, where two aliquot samples of the analysed specimen are prepared - native and derived. Each of the two aliquot samples is passed through a column in at least two modes of conditioning parametres of temperature gradient variation. Each of the said aliquots is additionally passed through the column with division of the stream in the same conditioning modes. Further, detector signals are picked up on chromatograms. Peaks with asymmetry values on 0.1; 0.5 and 0.6 the height of the peak from the base ≤1.05 are selected on the chromatograms as best corresponding to binomial distribution of probability density and undeformed by effect of background components. Further, detected substances are identified on the selected peaks by comparing with standard analytical characteristics of the detected substances.

EFFECT: increased efficiency and accuracy of detection, possibility of unique identification of chemical compounds and their fragments in arbitrary combinations.

26 dwg

FIELD: chemical technology.

SUBSTANCE: invention relates to a method for synthesis of ester perfluorinated derivative by using a chemical reaction. This reaction represents the fluorination reaction of the parent compound as a raw, the reaction of chemical conversion of fragment of ester perfluorinated derivative to yield another ester perfluorinated derivative or the interaction reaction of carboxylic acid with alcohol under condition that at least one or reagent, i. e. carboxylic acid or alcohol, represents a perfluorinated compound wherein indicated perfluorinated derivative of ester represents a compound comprising a fragment of the formula (1):

with a boiling point 400°C, not above. The reaction time for carrying out abovementioned chemical reaction is sufficient to provide the required yield of ester perfluorinated derivative and wherein this yield of ester perfluorinated compound is determined by the gas chromatography method by using a nonpolar column. Also, invention relates to a method for pyrolysis of ester perfluorinated derivative with a boiling point 400°C, not above, to yield the dissociation product wherein this product represents a derivative of acyl fluoride or ketone and wherein pyrolysis time is sufficient to provide the required degree of conversion of ester perfluorinated derivative and wherein the indicated conversion degree of ester perfluorinated derivative is determined by gas chromatography method by using a nonpolar column. Also, invention relates to a method for analysis of ester perfluorinated derivative with a boiling point 400°C, not above, that involves analysis of ester perfluorinated derivative in a sample containing ester perfluorinated derivative by gas chromatography method by using a nonpolar column wherein ester perfluorinated derivative represents compound comprising a fragment of above given formula (1).

EFFECT: improved method of synthesis.

8 cl, 1 dwg, 2 ex

The invention relates to the field of high-performance liquid chromatography

The invention relates to the field of high-performance liquid chromatography

The invention relates to chromatography and can be used to identify individual compounds or individual components of complex mixtures in various industries: chemical, oil, gas, petrochemical, oil refining, metallurgy, medicine, biology, ecology, etc

FIELD: chemical technology.

SUBSTANCE: invention relates to a method for synthesis of ester perfluorinated derivative by using a chemical reaction. This reaction represents the fluorination reaction of the parent compound as a raw, the reaction of chemical conversion of fragment of ester perfluorinated derivative to yield another ester perfluorinated derivative or the interaction reaction of carboxylic acid with alcohol under condition that at least one or reagent, i. e. carboxylic acid or alcohol, represents a perfluorinated compound wherein indicated perfluorinated derivative of ester represents a compound comprising a fragment of the formula (1):

with a boiling point 400°C, not above. The reaction time for carrying out abovementioned chemical reaction is sufficient to provide the required yield of ester perfluorinated derivative and wherein this yield of ester perfluorinated compound is determined by the gas chromatography method by using a nonpolar column. Also, invention relates to a method for pyrolysis of ester perfluorinated derivative with a boiling point 400°C, not above, to yield the dissociation product wherein this product represents a derivative of acyl fluoride or ketone and wherein pyrolysis time is sufficient to provide the required degree of conversion of ester perfluorinated derivative and wherein the indicated conversion degree of ester perfluorinated derivative is determined by gas chromatography method by using a nonpolar column. Also, invention relates to a method for analysis of ester perfluorinated derivative with a boiling point 400°C, not above, that involves analysis of ester perfluorinated derivative in a sample containing ester perfluorinated derivative by gas chromatography method by using a nonpolar column wherein ester perfluorinated derivative represents compound comprising a fragment of above given formula (1).

EFFECT: improved method of synthesis.

8 cl, 1 dwg, 2 ex

FIELD: physics.

SUBSTANCE: method is proposed for identifying narcotic and psychoactive substances in biological fluids, where two aliquot samples of the analysed specimen are prepared - native and derived. Each of the two aliquot samples is passed through a column in at least two modes of conditioning parametres of temperature gradient variation. Each of the said aliquots is additionally passed through the column with division of the stream in the same conditioning modes. Further, detector signals are picked up on chromatograms. Peaks with asymmetry values on 0.1; 0.5 and 0.6 the height of the peak from the base ≤1.05 are selected on the chromatograms as best corresponding to binomial distribution of probability density and undeformed by effect of background components. Further, detected substances are identified on the selected peaks by comparing with standard analytical characteristics of the detected substances.

EFFECT: increased efficiency and accuracy of detection, possibility of unique identification of chemical compounds and their fragments in arbitrary combinations.

26 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to biology and toxicological chemistry and can be used in chemical-toxicology, expert forensic and clinical laboratories. The method includes: crushing a biological object containing N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide, settling twice for 45 minutes with portions of an organic isolating agent which is methyl acetate, combining the obtained extracts, evaporating the solvent from the resultant extract, treating the residue with acetone, separating the acetone extract, evaporating the solvent from resultant extract, dissolving the residue in diethyl ether, extracting the ether solution with a buffer solution at pH 9-10, acidifying the aqueous alkaline extract with 24% hydrochloric acid to pH 2-3, saturating the obtained solution with sodium bromide, extracting with ethyl acetate, evaporating the obtained extract in an air current at 20-22°C until a dry residue is obtained, dissolving the residue in a mixture of hexane and acetone taken in volume ratio of 8:2, performing chromatography on a macrocolumn with silica gel L 40/100 mcm using a hexane-acetone mobile phase in volume ratio of 8:2, combining eluate fractions containing the analysed substance, evaporating the eluent in an air current at 20-22°C until complete removal of the solvent, dissolving the residue in methanol and performing determination via a combined physical-chemical method in the form of chromatography-mass spectrometry, using a DB-5 MS EVIDEX capillary column with a mobile phase which is 5% phenyl-95% methylpolysiloxane, using a mass-selective detector operating in electron impact mode, the initial thermostat temperature of the column is 70°C, maintaining said temperature for 3 minutes, further raising the temperature from 70°C to 290°C at a rate of 20°C per minute, maintaining the final temperature of the column for 16 minutes, the temperature of the injector is 250°C, the temperature of the quadrupole is 150°C, the temperature of the detector interface is 300°C, detecting strength of the signal resulting from charged particles formed when bombarding the analysed substance coming from the capillary column and falling into an ion source with an ionising electron beam with energy of 70 eV, recording the mass spectrum on the full ion current, while calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide from the area of the chromatographic peak.

EFFECT: high sensitivity of analysis.

2 ex, 3 tbl

FIELD: chemistry.

SUBSTANCE: method includes transforming a compound with an asymmetrical structure into two compounds with a symmetrical structure relative to a selected centre of symmetry and determining the retention index as the half sum of retention indices of compounds with a symmetrical structure, characterised by that the centre of symmetry of an O-alkylalkylfluorophosphonate compound is the carbon atom in the O-alkyl radical at the branching of which two different alkyl fragments or an alkyl fragment and a hydrogen atom are located; further, the method includes structural transformation of the compound into two compounds of the same class of O-alkylalkylfluorophosphonates with a symmetrical structure relative to the selected carbon atom using only said fragments and without structural change of the rest of the molecule.

EFFECT: novel method of predicting gas-chromatographic indices with improved reliability and objectivity.

1 ex, 2 tbl, 3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to identification of unknown compounds and, in particular, but not exclusively, to methods and systems for unknown compounds identification by gas chromatography - mass spectrometry using a retention index as the second parameter for identification. The computer method for creation of a standard compounds database with associated retention indices for unknown compounds identification using gas chromatography - mass spectrometry, GC-MS, includes assessment of the predicted retention index for a standard compound from the mass spectrometric GC-MS standard library on the basis of standard compound atomic structure. Also, the computer method includes assigning of the predicted retention index to a standard compound to create a record in the database, identifying the standard compound and the predicted retention index.

EFFECT: increased reliability and efficiency of unknown compounds identification.

15 cl 3 dwg

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