Method of determining substituted 2-methoxyhydroxybenzene in biological material

FIELD: chemistry.

SUBSTANCE: invention relates to biology and toxicological chemistry and can be used in practice of sanitary and epidemiological stations, chemical-toxicological, forensic and veterinary laboratories. A biological material, containing substituted 2-methoxyhydroxybenzene, is two times (each time for 30 minutes) infused with ethylacetate with mixing, separate extracts are separated from solid particles of the biological material, combined, ethylacetate is evaporated in an air flow at 18-22°C, residue is repeatedly processed with acetone, acetone extracts are separated, combined, dehydrated, evaporated in an air flow at 18-22°C, and then in a nitrogen flow until a solvent is completely removed, residue is dissolved in hexane, extracted with a buffer solution with pH 12-13, a water-alkaline extraction is separated, acidified to pH 2-3, saturated with sodium sulphate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in an air flow at 18-22°C, and then - in a nitrogen flow until the solvent is completely removed, residue is dissolved in a mixture of solvents hexane-dioxane-propanol-2, taken in a ratio of 20:5:1 by volume, chromatographed in macrocolumn with silicagel KSS No 3 80/120 mcm with the application of a mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume, eluate fractions, containing the analysed substance, are combined, the eluent is evaporated first in an air flow at a temperature of 18-22°C, and then in a nitrogen flow until the solvent is completely removed, residue is dissolved in dichloromethane, processed for 20 minutes with N-tert-butyl-dimethylsilyl-N-methyltrifluoroacetamide under conditions of heating at a temperature of 60°C with carrying out determination by a chromatography-mass spectrometry method with the application of a capillary column 25 m long with an internal diameter of 0.2 mm with an immobile phase (5%-phenyl)-methylpolysiloxane, with the application of a mass-selective detector, working in an electron impact mode, an initial temperature of the column thermostat constitutes 70°C, the said temperature is kept for 3 minutes, further the temperature is programmed from 70°C to 290°C at a rate of 20°C per minute, the final column temperature is kept for 10 minutes, the injector temperature constitutes 250°C, the quadrupole temperature is 150°C, the temperature of a ion source is 230°C, the temperature of the detector interface is 300°C, intensity of a signal, conditioned by charged particles, formed in the bombardment of the analysed substance, leaving the capillary column and getting into the ion source, with a ionising beam of electrons with the energy of 70 eV, is registered, the mass-spectrum by the complete ion flow is registered and an amount of substituted 2-methoxyhydroxybenzene is calculated by the area of a chromatographic peak of its trimethylsilyl derivative.

EFFECT: achievement of an increased analysis sensitivity.

4 tbl, 3 ex

 

The invention relates to biology, chemistry and toxicology, and in particular to methods of determining the substituted 2-methoxyhydroquinone in biological material, and can be used in the practice of epidemiological stations, chemical-Toxicological, forensic and veterinary laboratories. The method refers to the mass number.

A known method for determining 2-methoxy-4-arylhydroxylamine belonging to the group of substituted 2-methoxyhydroquinone, in biological material (blood) by two 30 minute infusion with ethyl acetate, separating an ethyl acetate extraction, their associations, evaporation combined extract the first in a stream of air and then in a stream of nitrogen until complete evaporation of the solvent, dissolving the residue in a solvent mixture of acetone-water, taken in the ratio of 5:5 by volume, chromatography was carried out in macrobalance with sorbent Silsor C-18 using mobile phase acetone - water in the ratio 5:5 by volume, combining fractions of the eluate containing 2-methoxy-4-arylhydrazines, dilution buffer solution with a pH of 2-3, repeated extraction with ethyl acetate, the unification of the different extracts, the first evaporation in a stream of air, then in a stream of nitrogen to evaporate the solvent, dissolve the residue in hexane, followed by chromatographytandem by HPLC in to what the PMC with the sorbent Silsor 600" using the mobile phase hexane-dioxane-propanol-2 in a ratio of 15:5:1 by volume (Sukhomlinov E. A., Shormanov C. K. Determination of eugenol in biological fluids // pharmacy. - 2008. - T. 57, No. 1. - S. 7-9).

The method is characterized not high enough sensitivity.

A known method for determining 2-methoxy-4-arylhydroxylamine belonging to the group of substituted 2-methoxyhydroquinone, in biological material, which consists in the fact that the biological object twice for 30 minutes, treated with ethyl acetate, an ethyl acetate extract combine, dehydrate, the ethyl acetate is evaporated first in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane, extracted with a buffer solution with a pH of 12-13, aqueous alkaline extract is separated, acidified to pH 2-3, saturated with sodium sulfate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated at 18-22°C in a stream of air and then in a stream of nitrogen to remove the solvent, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 40:5:1 by volume, chromatographic in macrobalance silica gel L 40/100µ using the mobile phase hexane - dioxane-propanol-2 in a ratio of 40:5:1 by volume), fractions of the eluate containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 18-22°C to insignificant volume, then the current I is that to remove the solvent, the residue is dissolved in hexane and carry out the determination by gas chromatography-mass spectrometry using capillary column, length 30 m, internal diameter 0.25 mm with a stationary phase containing polyethylene glycol and siloxane in a mass ratio of 95:5, by using the carrier gas, helium, is supplied at a rate of 1 ml per minute, and mass-selective detector operating in electron impact mode, the initial temperature of the column thermostat 50°C (delay 1 minute), temperature programmed from 50°C to 150°C at 50°C per minute, from 150°C to 290°C at a rate of 20°C / minute with a dwell time at final temperature for 8 min, the injector temperature is 280°C, the temperature of the interface 260°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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 number of 2-methoxy-4-arylhydroxylamine on the chromatographic peak area (patent RU 2395081, IPC G01N 33/15; G01N 33/50; G01N 30/00 / Method for determining 2-methoxy-4-arylhydroxylamine in biological material / Shormanov C. K., Sukhomlinov E. A., Elizarova M. K., With whom Pliva L. E., Siplify, C.; applicants and patentees: C. K. Shormanov, E. A. Sukhomlinov, M. K. Elizarova. No. 2008138004; Statements. 23.09.2008; Published.20.07.2010 // the Invention (Claims and patents). - 2010. - №20. - 9 C.).

The method is characterized not high enough sensitivity and selectivity of detection.

The closest way to detect 2-methoxy-4-arylhydroxylamine related to substituted 2-methoxyhydroquinone, in biological material, which consists in the fact that the biological object twice for 30 minutes, treated with ethyl acetate, an ethyl acetate extract combine the ethyl acetate is evaporated, the residue is repeatedly treated with acetone, the acetone extract combine, evaporated first in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane, extracted with a buffer solution with a pH of 12-13, aqueous alkaline extract is separated, acidified to pH 2-3, saturated sodium sulfate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in a stream of air at 18-22°C, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in a solvent mixture of hexane - diethyl ether, taken in the ratio of 6:4 by volume, chromatographic in macrobalance silica gel L 40/100µ using the mobile phase hexane - diethyl ether in which the rate of 6:4 by volume, fractions of the eluate containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 18 - 22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane and carry out the determination by gas chromatography-mass spectrometry using capillary column, length 30 m, internal diameter 0.25 mm with stationary phase a (5%-phenyl)-methylpolysiloxanes using the carrier gas, helium, is supplied at a rate of 1 ml per minute, and mass-selective detector operating in electron impact mode, the initial temperature of the column thermostat 50°C, temperature programmed from 50°C to 200°C at a rate of 10°C / min, injector temperature is 280°C, the temperature of the interface 260°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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 number of 2-methoxy-4-arylhydroxylamine on the chromatographic peak area (patent RU 2456597 Russian Federation, IPC G01N 33/48; G01N 30/02 / Method for determining 2-methoxy-4-arylhydroxylamine in biological material / shore the ANOVA C. K., Astashkina A. P., Elizarova M. K., Kirichek A. C.; applicant and patentee: State educational institution of higher professional education "Kursk state medical University" of the Ministry of health and social development. No. 2011113156; Statements. 05.04.2011; Published.20.07.2012 // Invention (Claims and patents). - 2012. - №20. - 11 C.).

The method is characterized not sufficiently high detection 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 twice for 30 minutes, treated with ethyl acetate, an ethyl acetate extract combine the ethyl acetate is evaporated, the residue is repeatedly treated with acetone, the acetone extract combine, evaporated first in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane, extracted with a buffer solution with a pH of 12-13, aqueous alkaline extract is separated, acidified to pH 2-3, saturated with sodium sulfate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in a stream of air at 18-22°C, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 volume, chromatographic in macrobalance silica gel KCC No. 3 80/120 μm using the mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume), fractions of the eluate containing an analyte, unite, eluent is evaporated in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in dichloromethane, process for 20 minutes, N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C and carry out the determination by gas chromatography-mass spectrometry using capillary column length of 25 m and an inner diameter of 0.2 mm with stationary phase a (5%-phenyl)-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 programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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 papavero in the ion source, ionizing electron beam with electron energy of 70 eV, record the mass spectrum of total ion current, and calculates the number of substituted 2-methoxyhydroquinone on the chromatographic peak area of his trimethylsilyl derived.

The method is as follows: biological material containing substituted 2-methoxyhydroquinone, twice (each time for 30 minutes) insist with ethyl acetate under stirring, the extracts are separated from the solid particles of the biological material, unite, the ethyl acetate is evaporated in a stream of air at 18-22°C, the residue is repeatedly treated with acetone, the acetone extract is separated, unite, dehydrated, evaporated in a stream of air at 18-22°C, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane, extracted with a buffer solution with a pH of 12-13, aqueous alkaline extract is separated, acidified to pH 2-3, saturated with sodium sulfate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in a stream of air at 18-22°C, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume, chromatographic in macrobalance silica gel KCC No. 3 80/120 μm using the mobile phase hexane-dioxane-propanol-2 in which the compared 20:5:1 by volume, fractions of the eluate containing an analyte, unite, eluent is evaporated first in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in dichloromethane, treated for 20 minutes, N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C and carry out the determination by gas chromatography-mass spectrometry using capillary column length of 25 m and an inner diameter of 0.2 mm with stationary phase a (5%-phenyl)-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 programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C the temperature of the ion source is 230°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of the 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 number is the number of substituted 2-methoxyhydroquinone on the chromatographic peak area of his trimethylsilyl derived.

The method is illustrated by the following examples.

Example 1

Definition 2-methoxy-4-arylhydroxylamine in liver tissue

To 10 g tissue fresh cadaveric human liver add 10 mg of 2-methoxy-4-arylhydroxylamine, thoroughly mixed biological tissue with a substance and leave for 1.5 hours at a temperature of 18-22°C. after this time the mixture is poured 20 ml of ethyl acetate and leave for 30 minutes under stirring. The extract is separated, the operation processing is repeated in the above described conditions. Hoods combine the ethyl acetate is evaporated in a stream of air at 18-22°C, the residue three times for 3 minutes to process portions of acetone and 15 ml each, with vigorous stirring, the extract is separated, combine, shake with 7 g of anhydrous sodium sulfate, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, sodium sulfate and filter additionally washed with 20 ml of acetone, the filtrate and wash liquid unite, evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in 10 ml of hexane, extracted twice with portions of buffer solution with a pH of 12-13 10 ml each. Separate the aqueous alkaline extracts are combined acidified with 24% solution chloroethanol acid to pH 2-3, saturated with sodium sulfate, and ek is tracerout double-portions of diethyl ether and 20 ml each. The ether extract combine, passed through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, a filter is additionally washed with 20 ml of diethyl ether. Separate filtrates are combined evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent. The residue is dissolved in 2-3 ml of a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume, and contribute to the chromatographic microcolony dimensions 490×11 mm, filled with 10 g of silica gel KCC No. 3 80/120 mm. Chromatographic using the mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume. The eluate is collected in separate fractions of 2 ml each. Fractions 5 to 11 inclusive unite, evaporated at 18-22°C, first in a stream of air to volume of 0.5 to 1 ml, and then in a stream of nitrogen until complete evaporation of the solvent. The residue is dissolved in 10 ml dichloromethane (solution A).

0.1 ml of the solution And contribute in a test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml derivatizing reagent, which is N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide, under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane (solution B).

4 μl of the solution injected into the gas chromatography-mass with extremer.

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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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, and calculates the number of 2-methoxy-4-arylhydroxylamine on the chromatographic peak area of his trimethylsilyl derived.

As the carrier gas helium is used. The flow of carrier gas is about 0.6 ml/min Mode on the population flow 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 10,37 min corresponds trimethylsilyl derived 2-methoxy-4-arylhydroxylamine. In the mass spectrum of this compound, obtained by total ion current, detected signals of some characteristic of charged particles with mass numbers 45, 59, 73, 89, 103, 117, 163, 179, 206, 221, 236. The most intensive is a particle with mass number 206, the intensity of which is taken as 100%.

2-methoxy-4-arylhydrazines as appropriate trimethylsilyl identify derived by a combination of retention time trimethylsilyl derived 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 2-methoxy-4-arylhydroxylamine 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 25ml contribute to 0.01, 0,025, 0,05, 0,1, 0,5, 1,0, 5,0 ml 0.125% solution and 2.5, 4,0, 5,0, 10,0 ml of a 1.25% solution of 2-methoxy-4-arylhydroxylamine and bring the volume of the contents of each flask to the mark with dichloromethane.

0.1 ml each of the solutions introduced into the test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml of N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane.

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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°C, the temperature of the interface is of lectora - 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 number of 2-methoxy-4-arylhydroxylamine on the chromatographic peak area of his trimethylsilyl derived.

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 within the concentration range of 1·10-11-2·10-7he

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

S=5005133·C+7623,

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 2-methoxy-4-arylhydroxylamine in the blood to provide the Lena in table 1.

Example 2

Definition 2-methoxy-4-propylhydroxybenzoate in liver tissue

To 10 g tissue fresh cadaveric human liver add 10 mg of 2-methoxy-4-propylhydroxybenzoate, thoroughly mixed biological tissue with a substance and leave for 1.5 hours at a temperature of 18-22°C. after this time the mixture is poured 20 ml of ethyl acetate and leave for 30 minutes under stirring. The extract is separated, the operation processing is repeated in the above described conditions. Hoods combine the ethyl acetate is evaporated in a stream of air at 18-22°C, the residue three times for 3 minutes to process portions of acetone and 15 ml each, with vigorous stirring, the extract is separated, combine, shake with 7 g of anhydrous sodium sulfate, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, sodium sulfate and filter additionally washed with 20 ml of acetone, the filtrate and wash liquid unite, evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in 10 ml of hexane, extracted twice with portions of buffer solution with a pH of 12-13 10 ml each. Separate the aqueous alkaline extracts are combined acidified with 24% solution chloroethanol acid to pH 2-3, saturated with sodium sulfate and extracted twice with portions of diethyl EPE is and 20 ml each. The ether extract combine, passed through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, a filter is additionally washed with 20 ml of diethyl ether. Separate filtrates are combined evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent. The residue is dissolved in 2-3 ml of a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume, and contribute to the chromatographic microcolony dimensions 490×11 mm, filled with 10 g of silica gel KCC No. 3 80/120 mm. Chromatographic using the mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume. The eluate is collected in separate fractions of 2 ml each. Fractions 4 to 9 inclusive unite, evaporated at 18-22°C, first in a stream of air to volume of 0.5 to 1 ml, and then in a stream of nitrogen until complete evaporation of the solvent. The residue is dissolved in 10 ml dichloromethane (solution A).

0.1 ml of the solution And contribute in a test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml derivatizing reagent, which is N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide, under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane (solution B).

4 μl of the solution injected into the gas chromatography-mass with extremer.

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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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, and calculates the number of 2-methoxy-4-propylhydroxybenzoate on the chromatographic peak area of his trimethylsilyl derived.

As the carrier gas helium is used. The flow of carrier gas is about 0.6 ml/min By dividing flow 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 11,06 min corresponds trimethylsilyl derived 2-methoxy-4-propylhydroxybenzoate. In the mass spectrum of this compound, obtained by total ion current, detected signals of some characteristic of charged particles with mass numbers 45, 59, 73, 89, 103, 115, 161, 179, 191, 206, 221, 236. The most intensive is a particle with mass number 206, the intensity of which is taken as 100%.

2-methoxy-4-propylhydroxybenzoate as appropriate trimethylsilyl identify derived by a combination of retention time trimethylsilyl derived 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 2-methoxy-4-propylhydroxybenzoate 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 VM is the cost of 25 ml make 0,025, of 0.05, 0.1 and 0.5, and 1.0, 5.0 ml 0.125% solution and 2.5, 4,0, 5,0, 10,0, 20,00 ml of a 1.25% solution of 2-methoxy-4-propylhydroxybenzoate and bring the volume of the contents of each flask to the mark with dichloromethane.

0.1 ml each of the solutions introduced into the test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml of N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane.

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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°C, the temperature of the interface, the sa 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 number of 2-methoxy-4-propylhydroxybenzoate on the chromatographic peak area of his trimethylsilyl derived.

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 within the concentration range of 2.5·10-11-4·10-7he

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

S=290141·C-4363,

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 2-methoxy-4-propylhydroxybenzoate in tissue p is Cheney presented in table 2.

Example 3

Definition 2-methoxyhydroquinone in liver tissue

To 10 g tissue fresh cadaveric human liver add 10 mg of 2-methoxyhydroquinone, thoroughly mixed biological tissue with a substance and leave for 1.5 hours at a temperature of 18-22°C. after this time the mixture is poured 20 ml of ethyl acetate and leave for 30 minutes under stirring. The extract is separated, the operation processing is repeated in the above described conditions. Hoods combine the ethyl acetate is evaporated in a stream of air at 18-22°C, the residue three times for 3 minutes to process portions of acetone and 15 ml each, with vigorous stirring, the extract is separated, combine, shake with 7 g of anhydrous sodium sulfate, filtered through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, sodium sulfate and filter additionally washed with 20 ml of acetone, the filtrate and wash liquid unite, evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent, the residue is dissolved in 10 ml of hexane, extracted twice with portions of buffer solution with a pH of 12-13 10 ml each. Separate the aqueous alkaline extracts are combined acidified with 24% solution chloroethanol acid to pH 2-3, saturated with sodium sulfate and extracted twice with portions of diethyl ether and 20 ml each. EPE is data extraction unite, passed through a glass filter with a diameter of 4 cm with a layer of anhydrous sodium sulfate thickness of 1-1,5 cm, a filter is additionally washed with 20 ml of diethyl ether. Separate filtrates are combined evaporated in a stream of air at 18-22°C to a volume of 0.5-1.0 ml, and then in a stream of nitrogen to remove the solvent. The residue is dissolved in 2-3 ml of a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume, and contribute to the chromatographic microcolony dimensions 490×11 mm, filled with 10 g of silica gel KCC No. 3 80/120 mm. Chromatographic using the mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume. The eluate is collected in separate fractions of 2 ml each. Fractions 4 to 9 inclusive unite, evaporated at 18-22°C, first in a stream of air to volume of 0.5 to 1 ml, and then in a stream of nitrogen until complete evaporation of the solvent. The residue is dissolved in 10 ml dichloromethane (solution A).

0.1 ml of the solution And contribute in a test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml derivatizing reagent, which is N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide, under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane (solution B).

4 μl of the solution injected into the gas chromatography-mass spectrometer.

Definition about what W ill result, using 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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°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, and calculates the number of 2-methoxyhydroquinone on the chromatographic peak area of his trimethylsilyl derived.

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 behaviour the first 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 8,58 min corresponds trimethylsilyl derived 2-methoxyhydroquinone. In the mass spectrum of this compound, obtained by total ion current, detected signals of some characteristic of charged particles with mass numbers 41, 58, 73, 89, 121, 136, 151, 166, 167, 181, 196. The most intensive is a particle with mass number 166, the intensity of which is taken as 100%.

2-methoxyhydroquinone as appropriate trimethylsilyl identify derived by a combination of retention time trimethylsilyl derived 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 the 2-methoxyhydroquinone 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 make 0,01, 0,025, 0,05, 0,1, 0,5, 1,0, 5,0 ml 0.125% solution and 2.5, 4,0, 5,0, 10,0 ml of a 1.25% solution of 2-methoxyphenoxy is Anzola and bring the volume of the contents of each flask to the mark with dichloromethane.

0.1 ml each of the solutions introduced into the test tube with a capacity of 0.2 ml, treated for 20 minutes to 0.016 ml of N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C, the reaction product together with the tube content was quantitatively transferred into a volumetric flask with a capacity of 10 ml and make up to the mark with dichloromethane.

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 a (5%-phenyl)-methylpolysiloxanes.

The initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the quadrupole - 150°C, the temperature of the ion source is 230°C, the temperature of the interface detector 300°C, record the intensity of the signal due to charged particles produced by the bombardment of the analyte, vyshedshih is of 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, and calculates the number of 2-methoxyhydroquinone on the chromatographic peak area of his trimethylsilyl derived.

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 within the concentration range of 1·10-11-2·10-7he

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

S=4065703·C-14791,

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 2-methoxyhydroquinone in liver tissue are presented in table 3.

The proposed method is compared with the prototype 100-fold increases detection sensitivity in khromatograficheskoi sample and 5 times in a biological material.

Comparative characteristics the tick proposed and known methods are presented in table 4.

Table 1
The results of the determination of 2-methoxy-4-arylhydroxylamine in liver tissue (n=5; P=0.95)
No.Made 2-methoxy-4-arylhydroxylamine, mg in 10 g liverFoundMetrological characteristics, %
the peak area on the chromatogram, the river.ed.mg khromatograficheskoi samplemg in terms of the sample introduced into the biomaterial% of the paid in biomaterial sample
110,08435412416,852·10-68,42684,26x=84,54
210,08261233816,504·10-68,25282,52 S=3,59
310,08947938117,876·10-68,93889,38Sx=1,60
410,08656639317,294·10-68,64786,47Δx=4,46
510,08018484916,019·10-68,00580,05ε=5,28

Table 2
The results of the determination of 2-methoxy-4-propylhydroxybenzoate is in the liver tissue (n=5; P=0.95)
No.Made 2-methoxy-4-propenyl-hydroxy-benzene, mg in 10 g liverFoundMetrological characteristics, %
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 sample
110,0507542617,508·10-68,75487,54x=85,24
210,0517233317,842·10-68,92189,21S=3,33
310,0482474416,644·10-68,32283,22 Sx=1,49
410,0468721716,170·10-6br8.085to 80.85Δx=4,14
510,0494892417,072·10-68,53685,36ε=4,86

Table 3
The results of the 2-methoxyhydroquinone in liver tissue (n=5; P=0.95)
No.Made of 2-methoxyhydroquinone, mg in 10 g liverFoundMetrol the environmental characteristics, %
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 sample
110,06432089315,824·10-67,91279,12x=83,69
210,06744335316,592·10-68,29682,96S=2,89
310,06932983917,056·10-68,52885,28Sx=1,29
410,0 7044384117,330·10-68,66586,65Δx=3,59
510,06870372116,902·10-68,45184,51ε=4,29

Table 4
Comparative characteristics of the proposed and known methods (for example, determination of 2-methoxy-4-arylhydroxylamine in liver tissue)
IndicatorsThe proposed methodThe known method
Sensitivity (open minimum):
a) 100 g of a biomaterial3·10-6g1,5·0 -5g
b) in khromatograficheskoi sample5·10-12g5·10-10g
The interval of linearity of the calibration curve (g khromatograficheskoi sample)1·10-11-2·10-7g1·10-9-1·10-7g

The method of determination of substituted 2-methoxyhydroquinone in biological material, which consists in the fact that the biological object twice for 30 minutes, treated with ethyl acetate, an ethyl acetate extract combine the ethyl acetate is evaporated, the residue is repeatedly treated with acetone, the acetone extract combine, evaporated first in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved in hexane, extracted with a buffer solution with a pH of 12-13, aqueous alkaline extract is separated, acidified to pH 2-3, saturated with sodium sulfate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated at 18-22°C in a stream of air and then in a stream of nitrogen to remove the solvent, the residue is dissolved in a mixture of solvents, chromatographic in macrobalance silica gel using the mobile phase fractions of the eluate containing an analyte, combine the, eluent is evaporated in a stream of air at a temperature of 18-22°C, then in a stream of nitrogen to remove the solvent, the residue is dissolved and carried out the determination by gas chromatography-mass spectrometry using capillary column with a stationary phase (5%-phenyl)-methylpolysiloxanes using the carrier gas is helium and mass-selective detector operating in electron impact mode, the temperature of the quadrupole is 150°C, the temperature of the ion source is 230°C, record the intensity of the signal due to charged particles produced by the bombardment of the 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 number of substituted 2-methoxyhydroquinone on the chromatographic peak area, characterized in that, before chromatographytandem in macrobalance, the residue is dissolved in a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume, when chromatographicaliy in macrobalance use silica gel KCC No. 3 80/120 μm, mobile phase for chromatography was carried out in macrobalance is a solvent mixture of hexane-dioxane-propanol-2, taken in the ratio of 20:5:1 by volume) prior to gas chromatography-mass spectrophotometric determination of the STATCOM is dissolved in dichloromethane and treated for 20 minutes, N-tert-butyl-dimethylsilane-N-methyltrifluoroacetamide under conditions of heating at 60°C, for gas chromatography-mass spectrometric determination applies capillary column length of 25 m and an inner diameter of 0.2 mm, the initial temperature of the column thermostat 70°C, this temperature is maintained for 3 minutes, further temperature programmed from 70°C to 290°C at a rate of 20°C / minute, final temperature of the column is maintained during 10 min, the injector temperature is 250°C, the temperature of the interface detector 300°C, calculate the number of substituted 2-methoxyhydroquinone carry out the chromatographic peak area of his trimethylsilyl derived.



 

Same patents:

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: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical, laboratory diagnostics, microbiological methods of research, and is aimed at standardisation of saliva analysis by method of wedge dehydration/crystallography. Claimed is method of obtaining standard, quality sample of mixed saliva facia for crystallography with application of portable laboratory device with inbuilt levels on axes X and Y, legs, regulated by height and isolating cover. From 20 to 40 microscope slides are simultaneously placed on the surface of portable laboratory device after obtaining smooth horizontal without inclination angle surface. After that, one sample of mixed saliva in amount 0.02 ml, preliminarily centrifuged for 20 min at 3000 rev/min, is applied on each microscope slide by means of micropipette. As a result a round 1.0 mm high drop of mixed saliva with diameter from 4.0 to 5.0 mm, corresponding to standard parameters, is obtained. Then, standard in dimensions drop on microscope slide placed on the surface of portable laboratory device, adjusted by levels, is dried at room temperature +18…+25°C under isolating cover for 5 hours, with further performance of microscopy of standard quality sample of saliva facia.

EFFECT: obtained sample makes it possible to interpret indices of crystallography without distortions, as standard in volume, dimensions and shape drop of mixed saliva is obtained, and there is no displacement of crystallisation centre and impairment of figures of saliva facia (crystallography pattern) in drop, dried on ideal horizontal surface of the device, ratio of central and peripheral zones of facia exactly correspond to organism's condition, which reduces percentage of false results of crystallography.

3 ex, 1 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention aims at asserting the maximum allowable blood concentrations (MAC) of heavy metals in the children living in the dirty environment as shown by health risk criteria after the chronic integrated exposure. An environmentally neglected zone is selected; a representative sampling of the children for the examination is drawn that is a basic group with using biological, social and hygienic criteria; the same criteria are used to draw a representative sampling of the children to a reference group living in the environmentally friendly zone. In the territory of the above zones, the chronic exposure of the analysed heavy metal is qualitatively assessed by establishing its average daily concentration in the ambient environment; the derived value is used to calculate a total average daily doses of a heavy metal supplied from various sources into a child's body averaged over the annual exposure for the children of both groups. Blood is sampled from the children every three months for one year to determine the content of the analysed heavy metal and also to measure the biochemical values of blood plasma and serum characterizing body responses presented by actual or potential health problems that are response markers. That is followed by calculating the average blood concentration of the analysed heavy metal and comparing it to the reference for the same heavy metal with using a Student two-sample test, thereby stating whether the children were sampled from the main and reference groups adequately. A mathematical modelling procedure is used to establish a relation between the exposure that is the total average daily doses of the analysed metal, and the exposure marker that is the average blood metal concentration. A sliding window technology is used to assert the response markers selected. The maximum allowable concentration of the exposure marker and respective marker is determined by a technique based on ratio analysis.

EFFECT: enabled measurement of the blood MAC of the heavy metals in the children after the integrated exposure with using sparing techniques making it possible to avoid a health risk.

4 tbl, 2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention represents an instant diagnostic technique for acute intestinal infections (AIIs), involving detecting indication markers of the AII aetiology with the use of laboratory immunology tests, differing by the fact that the AII aetiology is stated in children of an early age category, preferentially in the newborn children; that is accompanied by measuring the concentration of cytokine, interleukin IL-10 in coprofiltrate and diagnosing chronic placental insufficiency (CPI); a probability (P) of the bacterial AII aetiology is calculated; the value P of more than 50% testifies to the bacterial AII aetiology, while the value P being less than 50% shows the absence of the bacterial AII aetiology, and enables considering the diagnostics second stage to be necessary, which implies measuring the concentration of cytokine, interleukin IL-4 in coprofiltrate; the time of latching the newborn child to the breast is established with considering the type of feeding; that is combined with calculating a probability (P) of the viral or viral-bacterial AII aetiology, with the value P of more than 50% testifying to the viral AII aetiology, while the value being less than 50% makes it possible to state the viral-bacterial AII aetiology.

EFFECT: more accurate diagnosing of the aetiology of acute intestinal infection and simplifying the diagnostic procedure.

2 tbl

FIELD: medicine.

SUBSTANCE: polymerase chain reaction method is used to recognise polymorphous variants of IL6 and TGFb1 genes. Recognising the homozygous genotype CC in -174 position of IL6 gene in males and females, as well as the heterozygous genotype GC in -915 position of TGFb1 gene in females enables predicting the high risk of the complicated clinical course of the urogenital Chlamydial infection.

EFFECT: invention enables deciding on reasonable grounds on selecting a therapeutic approach to a specific patient suffering from urogenital Chlamydial infection in order to prevent complications of the urogenital Chlamydial infection and reproductive dysfunctions.

4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention concerns diagnosing undifferentiated connective tissue dysplasia (UCTD) in females with a personal history of miscarriage. The technique involves determining the fibrinolytic activity in an endometrial biopsy sample. If the value is less than 23.55 mm2, undifferentiated connective tissue dysplasia is diagnosed.

EFFECT: invention provides the high diagnostic accuracy and enables diagnosing UCTD in the females with the personal history of early miscarriage.

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.

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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.

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2 ex, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention is used to identify unknown components of complex mixtures of natural and man-made substances in different industries: chemical, gas, oil, medicine, environmental, food, perfume etc. In the method, the analysed mixture is fed into a two-phase system consisting of immiscible liquids - hexane and acetonitrile. Gas chromatography is then used to determine percentage content of components in each phase and the logarithms of the distribution constant thereof, expressed in form of indices, as well as retention indices of non-polar phase components with linear programming of column temperature, and the difference between retention indices and indices of the distribution constant logarithm is used to determine indices of molecular weight and boiling point.

EFFECT: high accuracy of determining molecular weight and boiling point.

2 tbl

FIELD: medicine.

SUBSTANCE: method for acquiring samples for the spectral biochemical blood analysis involving preparing and drying blood serum and extracting for the chromatographic examination differs by the fact that a process of a dry matter of blood serum obtaining is performed with stirring constantly at a temperature of 50-60°C for 21-27 hours to produce the dry matter in the form of a plug compacted in the centre and coated with a superficial film; the plug is perforated with a sterile and chemically intact object; 85% methanol is added to a test tube containing the dry matter. The prepared mixture is placed into the stirring device again at a temperature of 48-52°C for 21-27 hours and compacted in a centrifuge at an acceleration of 11500-12500 g. The prepared sample is added to an auto-sampler test tube of a liquid chromatograph in an amount of 3/4 - 2/3 volumes of the test tube.

EFFECT: using the present invention enables producing chromatograms with a low-error reproducibility within one sample that is adequate to provide the analysis result reliability with the use of t liquid chromatography.

FIELD: chemistry.

SUBSTANCE: method includes the following stages: interaction of eluate 68Ge/68Ga generator with a cation-exchange resin, washing the cation-exchange resin with a mixture of hydrochloric acid and ethanol, eluting 68Ga from the cation-exchange resin with the mixture of hydrochloric acid and ethanol, interaction of the obtained eluate with an anion-exchange resin, washing the anion-exchange resin with ethyl alcohol, drying the anion-exchange resin with air or inert gas and eluting 68Ga from the anion-exchange resin with a water solution of hydrochloric acid.

EFFECT: increased output of the process.

2 tbl, 2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: method involves blood sampling, 2,4-dichlorophenol extraction by an organic extractant from the above sample and measurement by gas chromatography analysis with using a calibration curve; before extraction, the blood sample is acidified with an oxalic acid solution to pH 2-3; the organic extractant for the extraction is presented by toluene; further, the prepared extract is added with a bromating agent and a water-thinned sulphuric acid in volume ratio of water : concentrated sulphuric acid as 3:1 respectively; the extract is brominated for 5 minutes that is followed by neutralising a bromine excess with sodium sulphite; the prepared brominated extract is centrifuged to separate toluene, and acetylated with trifluoroacetic anhydride in the pyridine medium for 5 minutes; the blood sample, the organic extractant toluene, the bromating agent, trifluoroacetic anhydride and pyridine are taken in the following volume ratio of 1:0.5:0.4:0.02:0.02 respectively.

EFFECT: simplifying the sample preparation stage in a combination with higher sensitivity.

5 cl, 6 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an express, safe and cheap method of determining mycotoxins in animal and vegetable products. Determination is carried out from 2 g of a sample, the QuEChERS purified extract is divided into three portions of 2 ml each and 300 mcl of chloroform is used as a dispersant in dispersion liquid-liquid micro-extraction. The obtained extracts are collected in micro-vials. The solvent is evaporated. The residue in the first and third micro-vials is dissolved in 50 mcl acetonitrile and the residue in the second micro-vial is dissolved in 50 mcl hexane. Aflatoxins (B1, B2, G1, G2), zearalenone and ochratoxin A are determined in the first micro-vial by HPLC with a fluorimetric detector. Trichothecene mycotoxins (deoxynivalenol, nivalenol, HT-2, T-2, diacetoxyscirpenol, 13-, 15-acetyl deoxynivalenol), penicidin, ochratoxin A and zearalenone are determined in the second micro-vial by gas-liquid chromatography with an electron capture detector. Penicidin and zearalenone are determined in the third micro-vial by HPLC with a donor-matrix detector. The duration of determining mycotoxins is 1.5-2 hours when operating with three chromatographs simultaneously. Sample preparation requires 10.1 ml aceonitrile, 0.9 ml chloroform and 0.05 ml hexane.

EFFECT: using different types of chromatography to determine penicidin, zearalenone and ochratoxin A enables to obtain more reliable analysis results.

1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of determining content, in a gaseous medium, of semi-volatile organic compounds such as polyaromatic hydrocarbons, carboxylic acids, alcohols, esters, n-alkanes- C15-30. The method involves passing the gaseous medium through a sorbent containing at least one compound selected from MgO, CaO, CaCO3, MgCO3. The sorbent is then dissolved in a first aqueous solution with pH less than 7 to obtain a second aqueous solution. The semi-volatile compound in the second aqueous solution is then extracted with an organic solvent to obtain an extract. Content of the semi-volatile compound in the extract is determined using a suitable physical and chemical analysis method.

EFFECT: invention enables to determine content of organic compounds while reducing duration of sample preparation.

23 cl, 6 dwg, 5 tbl

Gas flow controller // 2509334

FIELD: power engineering.

SUBSTANCE: gas flow controller is proposed for a gas chromatograph, comprising a pressure stabiliser, the inlet of which is connected to a gas manifold, a gas flow sensor, a programmer - a gas flow setter. It contains an electronic controller containing a low-frequency filter with proportionate-integral-differential (PID) law of regulation, one of inlets of which "setting action" is connected with the "control" outlet of the programmer-setter of gas flow, and the second "signal" inlet is connected with the signal outlet of the gas flow sensor, which represents a converter of mass gas flow - voltage, at the same time the flow sensor is connected by its gas outlet with the gas inlet of the analytic part of the chromatograph, and the gas inlet is connected with the gas outlet of the pressure stabiliser, which represents an electronic pressure controller "downstream" and including a pneumatic proportionate valve with electronic control, connected pneumatically between the inlet of the gas manifold and the outlet of the pressure stabiliser. The electromagnet of the drive is connected with the outlet of the comparison circuit, one of inlets of which is connected with the control outlet of the controller with the proportionate-integral-differential law of control, and the second inlet with the signal outlet of the converter pressure-voltage, connected pneumatically to the gas outlet of the pressure stabiliser.

EFFECT: increased absolute and relative accuracy to maintain gas flow and analysis accuracy in chromatographs.

4 cl, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to chromatography. An anionite suspension in 20-25% aqueous glycerol solution is prepared, followed by centrifuging and decantation to obtain an anionite base residue with particle size of 12-16 mcm. A cationite modifier nanosuspension is prepared by converting cationite functional groups to a hydrogen form, washing with water, drying, grinding the particles in a ball mill, followed by centrifuging and decantation to obtain a cationite suspension in water with particle size of 50-300 nm. A column is filled with the anionite suspension in aqueous glycerol solution; the anionite layer is packed under pressure; the anionite is converted to a hydroxyl form. The cationite nanoparticles are laid into macropores of the anionite base. Nanoparticles are removed from the outer surface of the anionite by passing the cationite modifier nanosuspension through the column until breakthrough, and excess modifier is then removed by passing an acid, water and a solvent until equilibrium is established.

EFFECT: obtaining chromatography columns with variable selectivity, having longevity, chemical resistance and capable of being regenerated.

7 cl, 2 tbl, 4 dwg

FIELD: physics.

SUBSTANCE: method of analysing optical and structural isomers by separating the analysed mixture on a binary sorbent, containing chiral macrocyclic methylated β- cyclodextrin, followed by determination of the composition of the analysed components of the mixture from results of measuring chromatographic signals from the obtained chromatograms. The methylated β- cyclodextrin is deposited on a flat uniform surface of a carbon adsorbent/support Carbopack Y in an amount sufficient to completely cover the surface with a dense layer.

EFFECT: high selectivity of separating optical and structural isomers in one chromatographic cycle.

FIELD: chemistry.

SUBSTANCE: method wherein separation of a sample into separate components takes place in a capillary column with a sorbent under the effect of an ascending stream of a liquid eluent, the flow rate of which is programmed by exponential raising of pressure at the input of the column. The apparatus has a fused silica column with a sorbent, a sealed container with a liquid mobile phase, a video densitometric sensor, an inert gas preparation unit, a controlled fluidic resistor and a hollow container connected to the gas space of the sealed container with liquid eluent.

EFFECT: stabilising linear ascending speed of the liquid mobile phase in the sorbent layer and shorter analysis time.

2 cl, 1 dwg, 1 tbl

FIELD: chemical engineering; medical engineering.

SUBSTANCE: method involves plotting two chromatograms one of which is based on radioactivity (No 1) and the other one on ultraviolet absorption (No 2) or on radioactivity (No 1) and on fluorescence (No 2) and chromatogram specific relative to ultraviolet absorption (No 3) or relative to fluorescence (No 3). Material quality is estimated to be the more high the more close studied labeled compound peak shape is to trapezoid shape on the third chromatogram.

EFFECT: high accuracy of the method.

8 dwg

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