RU2521711C1 - Method for quantitative determination of n-nitrosodimethylamine and n-nitrosodiethylamine in urine by gas chromatographic analysis - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method for quantitative determination of N-nitrosamines in urine involves collecting a urine sample; preparing the urine sample for analysis by adding sodium sulphate to the sample in ratio of 1 part by volume sample to 3.2 parts by weight sodium sulphate; heating the mixture on a water bath until phase equilibrium is established; collecting the formed gas-vapour sample and feeding said sample into the capillary column of a gas chromatograph; performing gas chromatographic separation of N-nitrosodimethylamine and N-nitrosodiethylamine and the amount of each said substance is determined from a calibration curve.

EFFECT: wider range of determining nitrosamines in urine, high sensitivity and accuracy, while simplifying the sample preparation step.

4 cl, 4 tbl

Description

The invention relates to medical toxicological studies, in particular to sanitary toxicology, and can be used for the quantitative determination of N-nitrosamines in biological fluids, in particular in urine.

Methods for determining N-nitrosamines in water are known from the patent literature (RF Patent No. 2090864 and Auth. Certificate. USSR No. 1259187). According to the first patent, colorimetric analysis of natural and wastewater contaminated with highly toxic chemicals such as dimethylhydrazine and its oxidation product nitrosodimethylamine (NDMA) is performed. When analyzing in a known manner, an aqueous solution of NDMA is mixed with zinc in the presence of sodium hydroxide for 1-3 minutes at a ratio of water to sodium hydroxide and zinc equal to 1: (0.013-0.047) :( 0.005-0.15), respectively, in the reaction the solution after separation of zinc is added with hydroxylamine hydrochloric acid at a mass ratio of solution: hydroxylamine hydrochloric acid 1: 0.02 and sodium hydroxide at a mass ratio of water to the total sodium hydroxide content of 1: (0.23-0.33), followed by blowing off of the asymmetric dimethylhydrazine in a mixture of p aranitrobenzaldehyde, ethylene glycol and acetic acid, followed by its colorimetry.

And according to the technical solution for Avt.svid. USSR No. 1259187 conduct gas chromatographic determination of volatile amines and nitrosoamines in food products and in other environmental objects. When implementing this method, a sample is prepared by extraction and subsequent treatment with hydrochloric acid and alkali, and subsequent gas chromatographic analysis.

The disadvantage of this known method for the determination of N-nitrosamines in water (RF Patent No. 2090864 and Autosvid. USSR No. 1259187) is the non-specificity and non-selectivity of the colorimetric method. This is a physical method of chemical analysis based on determining the concentration of a substance by the intensity of the color of the solutions. It is not possible to compare the color intensity of a solution in the presence of other colored interfering substances. Interfering substances reduce the sensitivity of the method.

A number of methods for determining nitrosoamines in biological media are also known in the art. For example, the gas method is used to determine N-nitrosodimethylamine in biological media (Pylypiw NM Jr., Zimmerman F, Harrington GW, et al. 1985. Apparatus for trace determination of volatile N-nitrosamines in small samples. Anal Chem 57: 2996-2997) chromatography with thermal energy analysis. For this, sulfuric acid and antifoam are added to a volume of 2 cm of blood or urine samples. Then, blood or urine samples are extracted with methylene chloride in an extractor. Biological samples are concentrated and analyzed by gas chromatography with a thermoenergy analyzer. The detection limit of N-nitrosodimethylamine in the biological medium by this known method was 0.1 μg / L.

The disadvantage of this known method is the high complexity of the allocation of N-nitrosodimethylamine from the biological environment and the inability to determine N-nitrosodiethylamine. Moreover, to implement this process, expensive equipment is used - a thermoenergy analyzer.

In a known method [Preussmann R. 1984. Occurrence and exposure to N-nitroso compounds and precursors. IARC Sci Publ. 57: 3-15. Spiegelhalder B, Eisenbrand G, Preussmann R. 1982. Urinary excretion of N-nitrosamines in rats and humans. IARC Sci Publ 41: 443-449] performed a qualitative determination of N-dimethylnitrosamine in urine by thin layer chromatography. For research, silica gel was applied onto glass plates. The solvent used was a mixture of N-hexane-ether and dichloromethane in ratios of 4: 3 and 2: 2. A positive violet color on the plates confirmed the presence of nitrosamines in the urine.

In the article “Continuous Solid Phase Extraction Method for the Determination of Amines in Human Urine with Acid Hydrolysis in the Microwave” [Beatriz Jurado-Sanchez, Evaristo Ballesteros and Mercedes Gallego. Continuous solid-phase extraction method for the determination of amines in human urine following on-line microwave-assisted acid hydrolysis. ANALYTICAL AND BIOANALYTICAL CHEMISTRY Volume 396, Number 5, 1929-1937, DOI: 10.1007 / s00216-009-3395-3] a method for the determination of N-nitrosamines, anilines and chloralines in human urine using acid hydrolysis in a microwave oven. After that, the amines were concentrated using the solid-phase extraction method. Separation and determination were carried out by gas chromatography and mass spectrometry, operating in the control mode of target ions. The method has a low detection limit (2-26 ng / l), good accuracy (relative standard deviation less than 7%).

However, all of the above methods are characterized by the following disadvantages:

- time-consuming research, due to the complexity of the sample preparation;

- the high complexity of the allocation of N-nitrosodimethylamine from the biological environment and the inability to determine N-nitrosodiethylamine;

- the use of expensive equipment;

- non-specificity and non-selectivity of the method.

Also known is the method of gas chromatography and mass spectrometry for the quantitative determination of nitrosodimethylamine and nitrosoproline in human urine, described in the article “Isolation of nitrosodimethylamine and nitrosoprolin in the urine in humans: interpersonal and intraindividual differences, the effect of the appointment of ascorbic acid and alpha-tocopherol” [William A. Garland , 1 Wolfgang Kuenzig, Felix Rubio, Halyna Kornychuk, Edward P. Norkus, 2 and Allan H. Conney. Urinary Excretion of Nitrosodimethylamine and Nitrosoproline in Humans: Interindividual and Intraindividual Differences and the Effect of Administered Ascorbic Acid and a-Tocopherol. CANCER RESEARCH 46, 5392-5400, October 1986]. According to a known method, urine was sampled, sample prepared, and gas chromatographic analysis performed. The essence of the specified known method is as follows: in a urine sample with a volume of 20 ml or a control aqueous sample was added 40 μl of NDMA labeled with ^I5 N _2-. The sample was then poured into 50 ml centrifuge tubes, centrifuged on a vortex and left to stand for 30 minutes. After that, 1 ml of 1 molar borate buffer was added to the sample (61.8 g of boric acid and 74.7 potassium chloride were dissolved in 1 dm ^{3 of} distilled water and this solution was titrated to pH = 10 with 1 m sodium carbonate) and extracted for 30 min 20 ml of hexane. The sample was centrifuged for 15 minutes. Hexane was removed by suction and 10 ml of dichloromethane was added. The sample was mixed and centrifuged by the method described above, while the dichloromethane layer was poured into 2 5 ml tubes. The volume of dichloromethane was reduced at room temperature to 50 μl under a stream of argon, secondly purified through a trap with concentrated sulfuric acid.

Two samples of 5 μl of a dichloromethane solution were introduced into a glass column (1.9 m × 2 mm, 10% SP-1000 per 100/120 granules of an acid-purified Chromosorb W) gas chromatograph. The column temperature is 102 ° C, the evaporator temperature is 150 ° C. Hydrogen (1.6 kg / cm) was used as the carrier gas. The retention time for NDMA and NDMA with ^l5 N ₂ labeled was 90 s. The effluent from the gas chromatographic column was sent 45 seconds after the introduction to the glass separator of a high-resolution mass spectrometer with a source of chemically ionized ions. The resolution of the mass spectrometer was M / ΔM = 10,000, ions with m / z 75.0558 (MH ⁺ from NDMA) and m / z 77.0499 (MH ⁺ from ¹⁵ N ₂ -labeled NDMA) are generated by positive chemical ionization. The temperature of the separator and ion source was 150 ° C. Isobutane was introduced into the sample through chemical ionization to provide a pressure of 20 Pa. A small amount of isobutanol (MH ⁺ = 75.0810) was added to the ion source for tuning and calibration.

The ratio of the ion (y) with m / z 75 to m / z 77 in the experimental sample was calculated and converted to the NDMA concentration from the formula x = (yb) / m, where the m-slope and b-point of intersection are constants from the mean square linear regression analysis of the ratio of ion (ordinate) versus concentration (abscissa). Analysis data were duplicated on aqueous samples (20 ml). In each sample, 2000 pg of ¹⁵ N ₂ -labeled NDMA or 0, 200, 400, 800, 1600 pg of NDMA were added. The amount of NDMA excreted in the urine was calculated adjusted for dilution with water, morpholine, and sodium azide solutions, which were added to the urine containers. The limit of determination in a known manner for NDMA was 5 pg / ml (1 pg = 10 ^-12 grams). This very high sensitivity is achieved as a result of using a high-resolution chromatograph-mass spectrometer, with an estimated cost of about 20 million rubles.

The disadvantages of this known method are:

 - the complexity of preparing urine samples for quantification, which is carried out by prolonged extraction and mixing;

- in the known method in the urine is determined only NDMA;

- the use of expensive equipment - a high-resolution chromatography-mass spectrometer with chemical ionization of molecules;

- the use of expensive standard samples (isotope labeled ^I5 N ₂ ).

Closest to the proposed invention is a method for the determination of N-nitrosodimethylamine in a biological medium described in the article by Raymond Koseniauskas and Eric S. Burak "Reduced Blood Clearance and Increased Urinary Excretion of Nitrosodimethylamine in Patas Monkeys Exposed to Ethanol or Isopropyl Alcohol 1". American Association for Cancer Research. 52.1463-1468, March 15, 1992. According to this method, sample preparation of the selected biological medium is performed. To do this, it is recommended to use the oxidation reaction with pentafluorobenzoic acid. Then, a buffer is added to the biological sample, at pH 10 it is extracted with a solvent, then the solvent is concentrated and the studied compound is determined on a gas chromatograph with an electron capture detector.

The disadvantage of this known method is:

- insufficient sensitivity, as an electron capture detector is less sensitive than, for example, thermoenergetic analysis and requires more thorough cleaning of a biological sample;

- the impossibility of determining N-nitrosodiethylamine;

- non-specificity and non-selectivity of the method.

The technical result achieved by the proposed method is to expand the spectrum of determination of nitrosoamines in urine, to increase sensitivity and accuracy, while simplifying the stage of sample preparation.

The specified technical result is achieved by the proposed method for the quantitative determination of N-nitrosodimethylamine and N-nitrosodiethylamine in urine by gas chromatographic analysis, including sampling urine, preparing it for analysis and quantitative determination of nitrosoamine in the sample by gas chromatographic analysis, while new is that the preparation of urine samples the analysis is carried out by adding sodium sulfate to it at a ratio of: 1 volume part of the sample to 3.2 mass parts of sodium sulfate, followed by roar mixture in a water bath to establish phase equilibrium-vapor formed subsequent selection of the sample and introduction of the sample into the capillary gas chromatography column gas chromatographic separation is carried out more N-nitrosodimethylamine and N-nitrosodiethylamine, and the amount of each set of said substance by a calibration curve.

Gas chromatographic separation of N-nitrosodimethylamine and N-nitrosodiethylamine is carried out using a gas chromatograph with a thermionic detector on a DB-624 capillary column

- 30m * 0.32mm * 0.25µm, with temperature conditions: column - from 50 ° C-200 ° C; evaporator - 200 ° C; detector - 250 ° C; the flow rate of the carrier gas - nitrogen - 20 cm ³ / min, with a division of the flow 1: 14.3.

The mixture is heated in a water bath for 5 minutes. The resulting vapor-gas sample is taken with a syringe heated to a temperature of 60 ± 5 ° C.

The technical result is achieved due to the following.

It should be explained that to increase the completeness of the extraction of the studied compounds from urine, a salting out technique is used, which is a factor that reduces the solubility of a substance in urine and increases its extractability from biological fluids [Toxicological chemistry: textbook for high schools / ed. Wicker. - 2nd ed., Rev. - M .: GEOTAR-Media, 2006. - 512 s]. Salting out or isolating a substance from a biological fluid by introducing a salting-out substance, which is usually well soluble in this solvent, leads to the destruction of bonds between substances and charged groups of urine proteins [Mikhailov V.A., J. Phys. Chemistry, 1962, t. 36, No. 2, p. 306-13; Salting-salting out of substances from solutions, Kaunas, 1970]. In the process of salting out, recoverable substances can be released in the form of a new phase - a solid precipitate, a liquid or gas phase. In the proposed method, as a salting out agent, it is recommended to use sodium sulfate in the form of a powder or, in other words, in the form of a solid phase. The salting out mechanism consists in the interaction of anions (SO ₄ ^2- ) and cations (Na ⁺ ) of the salt with charged groups of urine proteins (NH ₄ ⁺ and COO ^- groups). As a result, the protein charge disappears and the hydration shell decreases sharply at the same time, because salt ions attract polarized water molecules. The amount of water interacting with the protein decreases, because at high salt concentrations, the amount of salt ions is huge compared to charged groups of proteins. And since the solubility of proteins in water depends on the formation of a hydration shell around hydrophilic ionic groups, the movement of water molecules to other ions reduces the solubility of the protein. All this leads to the "sticking together" of protein molecules, their precipitation, the release of extracted substances and their appearance in urine in higher concentrations.

When sample preparation was carried out, a mixture of urine with sodium sulfate was subsequently heated in an equilibrium vapor dispenser (mainly for 5 minutes) until phase equilibrium was established. In this case, when working out the optimal conditions for the preparation of a biological sample (urine) for vapor-phase analysis, such parameters as temperature and pressure created in a closed heterogeneous liquid-gas system were taken into account. To exclude the influence of temperature and total pressure changes on the accuracy of vapor-phase analysis in quantitative measurements, we used the pneumatic vapor-phase dosing technique for bioassay into the capillary column of a chromatograph with a DANI HSS 86.50 HEAD SPACE SAMPLER dispenser.

The dosing cycle is a mechanical operation performed by a DANI HSS 86.50 HEAD SPACE SAMPLER batcher to transfer the gas phase from the vial to the gas chromatographic column. In the process of research, the optimal parameters of the urine sample dosing cycle were worked out: thermostat temperature - 80 ° C; metering unit - 130 ° C; transition line - 135 ° C; incubation time, min - 25; carrier gas flow rate - 60 ml / min; optimal boost pressure - 0.2 bar.

In addition, the above operations, apparently, provide a lower detection limit and an increase in the sensitivity of the determinations of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) in urine due to their concentration using the selective extraction method (equilibrium vapor phase analysis method) from urine N-nitrosamines.

Experimentally, we also selected optimal conditions for the chromatographic process for a clear separation of the NDMA and NDEA peaks from the peaks of other compounds on the Crystal 5000 hardware-software complex using standard samples. In the process of research, the following capillary columns of various lengths and thicknesses of the film of the stationary phase were tested: DB-624, HP-FFAP, HP-Plot / U, HP-VOC. Qualitative separation of N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) with compounds with similar physicochemical properties was achieved on a capillary column of the DB-624 series - 30m * 0.32mm * 0.25µm, at a temperature regime: column - from 50 ° C-200 ° C; evaporator - 200 ° C; detector - 250 ° C; the flow rate of the carrier gas - nitrogen - 20 cm ³ / min, with a division of the flow 1: 14.3. This allowed us to expand the spectrum of determination of nitrosoamines in urine (two components instead of one in the prototype) and increase the sensitivity and accuracy of their determination.

Thus, due to the totality and sequence of these operations of the proposed method, their modes and provides a high degree of accuracy and sensitivity.

To implement the proposed method carry out the following operations in the following sequence:

- conduct sampling of urine in an amount of about 10 cm ³ ;

- 5 cm ³ of a urine sample and 16 g of sodium sulfate are placed in vials (i.e., at a ratio of 1 volume part of the sample to 3.2 mass parts of sodium sulfate);

- vials are placed in a dispenser of equilibrium steam (pneumatic vapor-phase dispenser bioassay);

- after establishing phase equilibrium with optimal parameters of the urine sample dosing cycle (thermostat temperature is 80 ° C, the dosing unit is 130 ° C, the transition line is 135 ° C, the incubation time is min 25, and the carrier gas flow rate is 60 ml / min, optimal boost pressure is 0.2 bar) a gas-vapor sample is introduced into the capillary column of the gas chromatograph through an evaporator and analyzed under the conditions: thermionic detector (TID), capillary column DB-624 -30m * 0.32mm * 0.25µm, temperature: column - from 50 ° C-200 ° C; evaporator - 200 ° C; detector - 250 ° C; carrier gas flow rate 1 (nitrogen) - 1.4 cm ³ / min; carrier gas consumption 2 (nitrogen) - 20 cm ³ / min; the quantitative content of N-nitrosodimethylamine and N-nitrosodiethylamine is established according to calibration graphs, which are constructed by using urine samples taken from the control group of children (children live in an ecologically safe area and their urine samples do not contain nitrosamines) and standard solutions of N-nitrosodimethylamine and N- nitrosodiethylamine.

The calibration schedule is constructed as follows.

First, the initial solution is prepared. For this purpose a measuring tube of 10 cm ³ of bidistilled water dispenser added in a volume of 10 cm ^3, is introduced by a microsyringe 0.2 mm ³ N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) (administered separately in a volumetric flask. Build separately schedule for the first and second). The weight content of the components in the initial standard solution is (taking into account the density and content of the main substance): N-nitrosodimethylamine - 0.2 μg / cm ³ , N-nitrosodiethylamine - 0.19 μg / cm ³ . The shelf life of the solution is 12 hours.

Calibration characteristics are established on calibration solutions of N-nitrosoamines by absolute calibration. The prepared standard solutions are chromatographed on a capillary column at least 5 times. On the obtained chromatogram, the peak areas of the determined components are determined, and according to average results from 5 series of calibration solutions, a calibration characteristic is constructed. It expresses the dependence of the peak area of the analyte on the chromatogram (mV - with automatic calculation using the hardware-software complex) on the concentration (μg / cm ³ ). Each series consists of 6 solutions. Calibration solutions of N-nitrosamines are prepared in 10 cm ³ volumetric tubes. For this, 10 cm of bidistilled water are added to each tube with a dispenser and the initial standard solution for calibration is added in accordance with Table 1 and mixed thoroughly.

Table 1 Solutions for establishing calibration characteristics when determining the concentrations of N-nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine) Calibration solution one 2 3 four 5 6 The volume of the original standard solution, mm ³ one 2 3 5 10 fifteen The concentration of N-nitrosodimethylamine, μg / cm ³ 0.04 0.08 0.12 0.2 0.4 0.6 The concentration of N-nitrosodiethylamine, μg / cm ³ 0,038 0,076 0.114 0.19 0.38 0.57

To build a calibration graph using urine that does not contain the studied components. 5 cm ³ urine samples with a standard mixture (table 1) and 16 g of sodium sulfate are placed in vials and placed in a pneumatic dispenser of equilibrium steam. After the phase equilibrium is established at the optimal parameters of the urine sample dosing cycle (thermostat temperature is 80 ° C, the dosing unit is 130 ° C, the transition line is 135 ° C, the incubation time is 25 minutes, the carrier gas flow rate is 60 ml / min, the optimal boost pressure - 0.2 bar) a gas-vapor sample is introduced into the capillary column of a gas chromatograph through an evaporator and analyzed under the conditions: column thermostat temperature is 50-200 ° C at a heating rate of 10 ° C / min; carrier gas flow rate (nitrogen) - 1.4 cm ³ / min; dividing the stream 1: 14.3 (part of the vapor sample enters the column, but the main part of the sample is removed from the system. The use of a flow divider ensures that narrow sample zones are received at the column inlet).

To obtain reliable results, the analysis of each calibration mixture is carried out at least 2 times. The procedure is repeated similarly for each calibration solution.

The development of optimal gas chromatographic parameters for the determination of N-nitrosamines in urine was carried out using a hardware-software complex based on a Crystal 5000 gas chromatograph with a thermionic detector (TID). The high efficiency of the method was achieved by selecting the optimal conditions for gas chromatographic analysis: thermionic detector (TID), capillary column DB-624 - 30m * 0.32mm * 0.25µm, temperature: column - from 50 ° C-200 ° C; evaporator - 200 ° C; detector - 250 ° C; carrier gas flow rate 1 (nitrogen) - 1.4 cm ³ / min; carrier gas flow rate 2 (nitrogen) -20 cm ³ / min.

Given that not any salting out agent is suitable for determining N-nitrosoamines in urine, in the course of research for the extraction of N-nitrosoamines from urine by the proposed method when choosing a salting out

used neutral salts of alkali and alkaline earth metals (Na ₂ S0 ₄ , K ₂ NRA ₄ , NaCl, NH ₄ HPO ₄ ), as well as a mixture of salts of potassium chloride, K ₂ NRA, Na ₂ S0 ₄ . The precision of the analysis and the efficiency of the extraction of N-nitrosamines from the urine were determined experimentally by the "introduced-found" method using standard solutions. The average values of the completeness of extraction of N-nitrosodimethylamine and N-nitrosodiethylamine from urine by the analysis of the equilibrium vapor phase using neutral salts at n = 5 (n-number of samples) and with a probability of p = 0.95 are presented in table 2.

table 2

Average values of the completeness of extraction of N-nitrosoamines from a urine sample with the addition of various salts (salting out agents)

Salting out Introduced Found The degree of extraction,% GM-nitrosodimethylamine (concentration, μg / cm ^-5 ) l.Na ₂ S0 ₄ 0.2 ± 0.015 0.180 ± 0.042 90.0 2. To ₂ NRA ₄ 0.2 ± 0.022 0.107 ± 0.039 53.5 3. NaCl 0.2 ± 0.025 0,053 ± 0,028 26.5 4. NH ₄ HPO ₄ 0.2 ± 0.019 0.120 ± 0.084 60.0 5. A mixture of salts of potassium chloride, K ₂ NRA, Na ₂ S0 ₄ . 0.2 ± 0.020 0.120 ± 0.084 60.0 N-nitrosodiethylamine (concentration, μg / cm ^- *) l.Na ₂ SO ₄ 0.2 ± 0.015 0.192 ± 0.064 96.0 2. K ₂ HPO ₄ 0.2 ± 0.022 0.168 ± 0.045 84.0 3. NaCl 0.2 ± 0.025 0.133 ± 0.014 66.5 4. NH ₄ HPO ₄ 0.2 ± 0.019 0.180 ± 0.133 90.0 5. A mixture of salts of potassium chloride, K ₂ NRA, Na ₂ SO ₄ 0.2 ± 0.020 0.170 ± 0.133 85.0

The data shown in table 2 show that the greatest

the completeness of extraction of N-nitrosodimethylamine and N-nitrosodiethylamine in urine is ensured by the use of sodium sulfate as the salting out agent in the proposed method.

The conducted studies showed, nevertheless, the completeness of extraction of N-nitrosocompounds from urine is insufficiently high with the developed parameters of sample preparation. Therefore, additional studies were conducted to study the completeness of extraction of the studied compounds from a urine sample using a different mass of sodium sulfate as a salting out reagent. The results are presented in table 3.

Table 3 The average values of the completeness of extraction of N-nitroso compounds from a urine sample using a different mass of sodium sulfate (per 5 cm ^{3 of} urine) The mass of salting out reagent, g The ratio of the volume of urine to the mass of sodium sulfate Introduced Found The degree of extraction,% N-nitrosodimethylamine (concentration, μg / cm ³ ) 5 1: 1 0.2 ± 0.014 0.10 ± 0.009 fifty 8 1: 1.6 0.2 ± 0.019 0.18 ± 0.075 90 16 1: 3,2 0.2 ± 0.060 0.198 ± 0.075 99 N-nitrosodiethylamine (concentration, μg / cm ³ ) 5 1: 1 0.2 ± 0.06 0.140 ± 0.028 70 8 1: 1.6 0.2 ± 0.045 0.198 ± 0.085 99 16 1: 3,2 0.2 ± 0.039 0.20 ± 0.035 one hundred

In the course of the studies, it was found that the highest degree of extraction of N-nitrosoamines from urine by the analysis of the equilibrium vapor phase is achieved with the ratio: 1 volume part of the urine sample to 3.2 mass parts of sodium sulfate, and amounted to 99% for N-nitrosodimethylamine, for N -nitrosodiethylamine-100%.

Thus, the research results prove that due to the combination and sequence of operations of the proposed method, their modes, a high degree of accuracy and sensitivity for the determination of N-nitrosodimethylamine and N-nitrosodiethylamine in urine is achieved by gas chromatographic analysis.

During the studies, urine samples were also analyzed in children (examination group) living in environmental conditions with a possible effect of N-nitrosamines on the child's body. Urine samples were processed by the proposed method and the content of components in the samples was determined by a calibration schedule. The results are shown in table 4.

Table 4 The content of N-nitrosodimethylamine and N-nitrosodiethylamine in the urine of children of the survey group living in conditions of environmental distress established by the proposed method Sample number N-nitrosodimethylamine N-nitrosodiethylamine Concentration, mcg / cm ³ one 0 0,023 2 0.003 0.005 3 0.0022 0.0028 four 0 0.0015 5 0 0.0021 6 0.0026 0.002

Studies have shown that the proposed method allows the determination of N-nitrosoamines (N-nitrosodimethylamine and N-nitrosodiethylamine) in urine with a high degree of accuracy and sensitivity in the concentration range from 0.04 to 0.6 μg / cm ³ (concentration of N-nitrosodimethylamine and N-nitrosodiethylamine of Table 4 in the urine of children was determined at the level of the lower limit of determination, which is 0.002 μg / cm ³ for the method), with a method error of 23.5% with optimal parameters of gas chromatographic analysis and sample preparation. It was found that the highest degree of extraction of N-nitrosoamines from urine by the equilibrium vapor phase analysis (the proposed method) is achieved using a sodium sulfate salting out reagent, with the ratio: 1 volume part of urine sample to 3.2 mass parts of sodium sulfate, and for N- nitrosodimethylamine - 99%, for N-nitrosodiethylamine - 100%.

Claims (4)

1. A method for the quantitative determination of N-nitrosodimethylamine and N-nitrosodiethylamine in urine by gas chromatographic analysis, including sampling the urine, preparing it for analysis and quantifying nitrosoamine in the sample by gas chromatographic analysis, characterized in that the preparation of the urine sample for analysis is carried out by adding to sodium sulfate at a ratio of 1 volume part of the sample to 3.2 mass parts of sodium sulfate, followed by heating the mixture in a water bath until phase equilibrium is established, after eduyuschego selection resulting vapor-gas sample and introduction of the sample into the capillary gas chromatography column gas chromatographic separation is carried out more N-nitrosodimethylamine and N-nitrosodiethylamine, and the amount of each set of said substance by a calibration curve. 2. The method according to claim 1, characterized in that the gas chromatographic separation of N-nitrosodimethylamine and N-nitrosodiethylamine is carried out using a gas chromatograph with a thermionic detector on a DB-624 capillary column - 30m * 0.32mm * 0.25µm, at a temperature regime: column - from 50 ° C-200 ° C; evaporator - 200 ° C; detector - 250 ° C; the flow rate of the carrier gas - nitrogen - 20 cm ³ / min, with a division of the flow 1: 14.3. 3. The method according to claim 1, characterized in that the mixture is heated in a water bath for 5 minutes. 4. The method according to claim 1, characterized in that the selection of the combined vapor-gas sample is produced by a syringe heated to a temperature of 60 ± 5 ° C.