Method for quantitation of blood penzapyrene by liquid chromatography

FIELD: medicine.

SUBSTANCE: each blood sample is analysed twice. A fresh blood sample is centrifuged at 2,000 rpm for 5 min. The samples are separated in plasma fractions and formed elements. A solid-phase plasma extraction is performed by sequential passing of 100% acetonitrile, plasma, distilled water, 50% acetonitrile solution under vacuum through a cartridge with Oasis HLB 3 cc sorbent. The cartridge with the sorbent is dried under vacuum, and 100% methylene chloride is passed through the sorbent. An aliquot portion of the produced extract is chromatographed. Producing the extract of formed elements is ensured by dispersed solid-phase extraction: by adding 100% acetonitrile thereto and agitating intensively. That is followed by adding a number of QuECHeRS salts for extraction, agitating, centrifuging for 10 minutes at 2,000 rpm; that is accompanying by forming 3 layers; an upper layer is transferred to another test tube, which contains a number of QuECHeRS salts for purification; the layers are centrifuged at 2,000 rpm; the upper layer is sampled. Plasma and formed elements extracts are analysed by Agilent 1200 liquid chromatograph with a fluorimetric detector on Zorbax column 50 mm long and having an inner diameter of 4.6 mm with Eclipse PAH C18 sorbent at column temperature 27°C; a movable phase is presented by mixed acetonitrile and water at flow rate 1.5 cm3/min and optimising elution in the gradient mode (supplying the movable phase of 60 vl % to 68 vl % of acetonitrile for 1 min, increasing 60 vl % to 68 vl % of acetonitrile for 3 min, increasing 68 vl % to 70 vl % for 0.5 min, increasing acetonitrile 70 vl % to 90 vl % for 1.5 min, increasing acetonitrile 90 vl % to 100 vl % for 4.5 min, supplying 100% acetonitrile for 1.5 min, reducing acetonitrile to 60 vl % and supplying 60% acetonitrile for 4 min to balance the column). An excitation wavelength of the fluorimetric detector makes 265 nm, and an emission wavelength makes 412 nm. A calibration chart is used to quantify benz(a)pyrene in plasma and formed elements separately, while the results are summed up.

EFFECT: invention provides high sensitivity of the method and ensures selectivity in a combination with its accessibility for routine analyses.

3 cl, 6 tbl, 1 ex

 

The invention relates to medical Toxicological studies, in particular to sanitary toxicology, and can be used for the quantitative determination of benzo(a)pyrene in the blood, to assess the risk to human health and the development of measures to ensure chemical safety.

Benzo(a)pyrene is one of the most potent carcinogens among the polycyclic aromatic hydrocarbons (hereinafter PAHs). The concentration of benzo(a)pyrene in the air at the level of 3-6 ng/m3prolonged exposure may lead to increased frequency of lung cancer in the population. Therefore, in Russia and many other countries, legislation introduced maximum limits for benzo(a)pyrene, which, in turn, necessitates the development and application of appropriate methods of control.

For the determination of benzo(a)pyrene and other PAHs in various environments using methods of gas and liquid chromatography, mass spectroscopy, etc. the Most high identification reliability and sensitivity methods based on the use of high performance liquid chromatography with reversed phase. Determination of benzo(a)pyrene in various environmental objects is greatly complicated by the fact that it is usually present in very small concentrations. To quantify the possible loss of a substance at various �tapah analysis in particular as a result of volatilization in the process of distilling off the solvent.

Analysis of literature showed that known chemical analytical methods relate primarily to the determination of metabolites of benzo(a)pyrene in biological media at his exhibition in different ways. This is due, probably, to the fact that benzo(a)pyrene is a "latent" chemical substance. When ingested, it is metabolized with the formation of more toxic compounds that cause cancer and other dangerous for the body effects.

Various methods for the determination of benzo(a)pyrene in foods in biological systems (fat, muscles and tissues).

In accordance with SanPiN 2.3.2.560-96 acceptable level of content of benzo(a)pyrene in food is 0.001 mg/kg. Contamination of food products of benzo(a)pyrene comes from air, water and food contamination. The use of open flame or braziers, for which use charcoal, can cause pollution of PAHs not only air, but also food that is prepared in this way.

For example, the known method of determination of benzo(a)pyrene in foodstuffs of animal origin (smoked meat and fish products) [1]. Sample preparation includes the following procedures: homogenized sample was extracted with chlorodimethyl, the extract was filtered through filter paper and a layer of anhydrous sodium sulfate. The filtrate is evaporated on a rotary evaporator, pererastayut 100 cm3hexane, transferred to a separatory funnel with a capacity of 1000 cm3and with two portions of 50 cm3a mixture of dimethylformamide and water perestrelyayut. Separate the lower layer and the upper hexane extracted with PAH again. Hexane layer is cast away, and the United dimethylformamide diluted with 100 cm3and PAHs extracted with hexane three times at 40 cm3. Hexane extracts were pooled and washed with two portions of 50 cm3. Filtered through a filter paper and a layer of anhydrous sodium sulfate is placed in a pear-shaped flask at 200 cm3. The dehydrated extract was evaporated to a volume of 1-1,5 cm3and the residual solvent is removed by a current of nitrogen or air. The dried extract was dissolved in 200 mm3acetonitrile and analyzed by thin-layer chromatography. The method allows to determine 0.5-1 ág benzo(a)pyrene in 1 kg of the mass of the biomaterial.

Also known method for the determination of benzo(a)pyrene in smoked meat by high performance liquid chromatography (HPLC) with fluorimetric and UV detection [2]. Hitch weight 5 g pulverized, triturated with 0.2 g of anhydrous sodium sulfate and placed in a conical flask. There is added 50 ml of�propanol and the extraction is carried out for 15 minutes. The extract was filtered into a round bottom flask, add 6 ml of water and 0.2 g of potassium hydroxide. Carry out the hydrolysis of the extract for 30 minutes in a flask with reflux. The hydrolysate was filtered into a beaker, add 60 ml of water and extracted with 3×10 ml of chloroform (bottom layer). The organic extracts were combined, dried over calcium chloride and evaporated on a rotary evaporator to a volume of 3-5 ml. Evaporated the extract is transferred to a glass column with a diameter of 1-2 cm, filled with 5 cm of silica gel with a perlage 60-140 μm, and polyaromatic hydrocarbons eluted in 30 ml of a mixture hexane-chloroform" 80:20. The eluate is evaporated on a rotary evaporator to a volume of 3-5 ml, transferred into a test tube to 5 ml and the solvent is distilled off by a current of air. The dry residue pererastayut in 500 µl of acetonitrile (mixture of acetonitrile / water 95:5) and analyzed on a column of Dearbom C16using UV and fluorimetric detection.

Also known is the Method used to determine PAHs in biological objects [3], and a Method for the quantitative determination of hydrocarbons in biological objects [4]. However, their disadvantage is that the definition is only the total PAH, without separation into components, making known methods nonselective.

The closest to the proposed invention is a method for determining benzo(a)pyrene in biological object - bat�Noah fabric, by HPLC with fluorimetric detector [5]. For the determination of benzo(a)pyrene 3 g of muscle homogenized with liquid nitrogen and extracted from the matrix 50 cm3hexane under the action of ultrasound for 30 minutes. The homogenates were filtered through anhydrous sodium sulfate and concentrated to approximately 1 cm3at 45°C in a rotary evaporator. The concentrated solution was purified using activated Florisil cartridge. Benzo(a)pyrene was suirable with cartridge 18 cm3solution of hexane and dichloromethane in the ratio of 3:1, the eluate was dried at 45°C, then dissolved in 1 cm3of acetonitrile and treated with ultrasound. Then 20 mm3the filtered solution was analyzed using liquid chromatography with fluorescence detector. The disadvantage of this known method is the low detection sensitivity (lower limit of detection of 1 µg/kg), so the method used in Toxicological studies.

The technical result achieved by the invention is to provide a high sensitivity of the method while ensuring selectivity and availability for serial analysis.

Said technical result is achieved by the proposed method for the quantitative determination of benzo(a)pyrene in blood by liquid chromatography, VK�uchusen sampling blood its solid-phase extraction and study of the extract by liquid chromatography, what is new is that after sampling the blood, it undergoes conservation the addition of heparin, expose the blood sample is centrifuged at a speed of 2000 rpm for 5 min with the separation of the plasma and formed elements, then separate these sample preparation of plasma and formed elements of blood, while for sample preparation of plasma carry out solid-phase extraction of the plasma with the sorbent Oasis HLB by sequentially passing through said sorbent of acetonitrile, distilled water, plasma, distilled water, 50% aqueous solution of acetonitrile, then the sorbent cartridge was dried under vacuum and then passed through the extractant - 100% methylene chloride, the extract was dried in a stream of air and the dry residue pererastayut in 100% acetonitrile, getting extract plasma and sample preparation of formed elements of the blood sample is carried out dispersive solid phase extraction by adding 100% acetonitrile, with stirring, add a set of salts QuECHeRS method for extraction, mixing, further centrifugation, the selection of the upper phase and adding to it the set of salts QuECHeRS method for cleaning, mixing, re tsentrifugirovanie� this purified the top and again the selection of the upper phase - extract of the formed elements of the blood sample for analysis, then the resulting extracts of plasma and formed elements of the blood samples analyzed separately by liquid chromatography, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 60:40 vol.% to 90:10 vol.% respectively, and 100% acetonitrile in gradient mode, which is carried out by chromatography by filing first for 1 min mobile phase consisting of a mixture of acetonitrile and water in the ratio of 60:40 vol.%, then by increasing for 3 min in the mobile phase of acetonitrile to 68 vol.%, a further increase in its concentration within 1.5 min to 70 vol.%, then increasing the concentration of acetonitrile for 1.5 min to 90 vol.%, as well as increasing the acetonitrile to 100% over the next 4.5 min and transmission of such mobile phase for 1.5 min, followed by a sharp decrease in volume of the quantity of acetonitrile to 60 vol.% and by deletion of this mobile phase through the column for 4 min prior to equilibration of the column, and the quantity of benzo(a)pyrene in blood establish on a calibration schedule, and the results obtained in chromatographicaliy extracts of plasma and formed elements, summarize.

The flow rate of the mobile phase is 1.5 ml/min.

Liquid chromatography Prov�is changed to liquid chromatograph "Agilent 1200" with fluorimetric detector at an excitation wavelength of 265 nm and the wavelength of emission 412 nm at a column temperature of 27°C.

Said technical result is achieved due to the following.

Due to the fact that the selected blood sample is subjected to preservation by the addition of heparin, provides stabilization of whole blood for further separation into plasma and corpuscles.

Due to the fact that sample collection blood was subjected to centrifugation at a speed of 2000 rpm for 5 min, there is a separation of the sample into fractions, which provides a separate analysis of plasma and formed elements in the determination of benzo(a)pyrene.

Experimentally it was found that high sensitivity and accurate determination of benzo(a)pyrene in the blood is achieved only when a specific sequence of sample preparation of plasma and formed elements derived from the sample.

Passing through said sorbent Oasis HLB of acetonitrile and distilled water provides cleaning of the sorbent from the possible presence of impurities and moisture of the sorbent prior to passing plasma (in General is the preparation of the sorbent for analysis). And washing of the sorbent after passing the plasma with distilled water and 50% aqueous solution of acetonitrile needed for the purpose of removal with sorbent labodegueta components of the biomaterial and improve the selectivity of the extraction. The use of an aqueous solution of acetonitril�namely this concentration provides good removal of unnecessary components, and at the same time is not performed extraction of the target component.

Drying of the sorbent cartridge under vacuum is required to remove water residues that prevent the further analysis (the water goes into the extract, methylene chloride, evaporation of the drop of water of unknown volume does not evaporate and remains in the test tube in which pererastayut dried extract. Thus it remains unknown total volume of the analyzed fluid, which affects the accuracy of the calculation of the concentration of benzo(a)pyrene in the blood plasma.

Drying of the extract after passing through the sorbent extractant 100% methylene chloride is required to transfer the analyte to be compatible with the mobile-phase solvent (acetonitrile). And perenashivanie solids in 100% acetonitrile provides a full transition of the analyte (benzo(a)pyrene) in the solution to be analyzed.

The proposed sequence of sample preparation of formed elements of blood samples is also due to the above chemical composition.

Conduct further analysis of the extract of plasma and formed elements by liquid chromatography with special modes, namely, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 60:40 vol.% to 90:10 vol.% respectively, and 100% acetonitrile in gradient mode, which is p�control in chromatography by filing first for 1 min the mobile phase, consisting of a mixture of acetonitrile and water in the ratio of 60:40 vol.%, then by increasing for 3 min in the mobile phase of acetonitrile to 68 vol.%, a further increase in its concentration within 1.5 min to 70 vol.%, then increasing the concentration of acetonitrile for 1.5 min to 90 vol.%, as well as increasing the acetonitrile to 100% over the next 4.5 seconds and transmission of such mobile phase for 1.5 min, followed by a sharp decrease in volume of the quantity of acetonitrile to 60 vol.% and by deletion of this mobile phase through the column for 4 min prior to equilibration of the column corresponds to the optimization of elution, and thus provides high sensitivity of the proposed method. It should be clear that the change in the volume ratio of acetonitrile and water as mobile phase, at the expense of work gradient pump Agilent 1200, mixing from 2 to 4 components of the mobile phase.

The study of the extracts of a liquid chromatograph "Agilent 1200" with fluorimetric detector at an excitation wavelength of 265 nm and the wavelength of emission 412 nm at a column temperature of 27°C is due to the achievement of the maximum signal fluorimetric detector, which affects the sensitivity and accuracy of determination.

The flow rate of the mobile phase of 1.5 ml/min is optimal for research.

The number �jnt(a)pyrene in blood establish on a calibration schedule, moreover, the results obtained in chromatographicaliy extracts of plasma and formed elements, summarize. This improves the accuracy of the determination, because the sample preparation of plasma and formed elements should be done differently.

For the implementation of the proposed method is carried out the following operations in the following sequence:

- produce a selection of blood samples at 2 cm3;

- carry the specified sample in the centrifuge tube and centrifuged at a speed of 2000 rpm for 5 min;

- the upper part - the plasma is separated from the lower part of the shaped element;

- carried out separately for extraction.

The extraction algorithm

Solid-phase extraction of plasma is carried out according to the following scheme: air cartridge Oasis HLB 1 CC (30 mg), 1 cm3acetonitrile, 1 cm3distilled water, further loading of 0.5 cm3plasma on the cartridge Oasis HLB 1 CC (30 mg), flushing the cartridge caused the breakdown of 1 cm3distilled water, 0.2 cm350% aqueous solution of acetonitrile, drying cartridge in a stream of air through the vacuum pump, elution 1 cm3100% methylene chloride. Then the methylene chloride was dried in flowing air, the dry residue pererastayut in 0.5 cm3100% acetonitrile. The resulting extract plasma in further anal�evaluate 20 mm 3a liquid chromatograph.

Dispersive solid-phase extraction of formed elements: 0.5 cm3formed elements add 2 cm3100-%acetonitrile, intensely shaking 1 min, the blood proteins to fold, add 250 mg of salts set QuECHeRS method for the extraction of (ingredients: magnesium sulfate, sodium chloride, sodium citrate, disodium salt citric acid), shake intensely for 1 minute, centrifuged for 10 minutes at a speed of 2000 rpm to form 3 layers, 1.5 cm3the upper layer is transferred into another test tube, which contains 120 mg of the set of salts QuECHeRS method for the purification of (ingredients: magnesium sulfate, sorbent C18), shake 1 min, centrifuged again for 10 minutes at a speed of 2000 rpm, take away the upper layer of the extract of formed elements and analyze 20 mm3a liquid chromatograph.

The conditions of the analysis liquid chromatography for both extracts has the following characteristics:

ColumnZorbax Eclipse RAS C18and 4.6×50 mm; particle size 3.5 microns
Fluorimetric detector:
the excitation wavelength
wavelength emission
/td>
265 nm
412 nm
Mobile phaseA: water; b: acetonitrile
The gradient change in the composition of the mobile phase (water mixture)0-1 min - 60 vol.% acetonitrile
1-4 min - 68 vol.% acetonitrile
4-4,5 min - 70 vol.% acetonitrile
4,5-6,0 min - 90 vol.% acetonitrile
6-10,5 min to 100% acetonitrile
10,5-12 min - 100% acetonitrile
in 12 minutes - a sharp decline to 60 vol.% acetonitrile
12-16 min - 60 vol.% acetonitrile
Velocity1.5 ml/min
Column temperature27°C
The injection volume20 µl

The amount of benzo(a)pyrene in blood establish on a calibration schedule, and the results obtained in chromatographicaliy extracts of plasma and formed elements, summarize.

For construction of the calibration curve volumetric flask with a capacity of 100 cm3make a 1 cm3standard�Togo sample (SSS) benzo(a)pyrene in acetonitrile with a concentration of 100 μg/cm 3bring the contents of the flask to the mark with acetonitrile and alter. The concentration of benzo(a)pyrene in the initial solution (solution No. 1) is 1 g/cm3. Then 2.5 cm3solution No. 1 make a volumetric flask with a capacity of 100 cm3and bring acetonitrile to the mark. The concentration of benzo(a)pyrene in dilute solution (solution No. 2) is 25 ng/cm3. Use freshly prepared solution.

Calibration characteristic set method of absolute calibration. It expresses the dependence of peak area on the chromatogram (mm2) from the mass concentration of benzo(a)pyrene in the blood (mg/DM3) and is based on the 5 series of working standard solutions. Each series consisting of 3 standard solutions prepared in volumetric tubes with a capacity of 5 cm3. For this in each test tube add solutions of benzo(a)pyrene for grading No. 1 and 2 in accordance with table 1, bring the contents of the tube to the mark 5 cm3whole blood and mix.

Table 1
Calibration solutions for the determination of benzo(a)pyrene in the blood in the range of mass concentrations of 0.0001-3,002 mg/DM3
Room solution1 2345
The volume of solution No. 1, mm3-2,55,07,510,0
The volume of solution No. 2, mm320,0----
The amount of benzo(a)pyrene, 5 cm3calibration solution, ng0,52,55,07,510,0
Mass concentration of benzo(a)pyrene in the blood, mg/DM30,00010,00050,0010,00150,002

Table 2
Calibration solutions for the determination of benzo(a)pyrene in the blood in the range of mass concentrations 0,00001-0,0001 mg/DM3
Room solution 12345
The volume of solution No. 2, mm32,04,010,016,020,0
The amount of benzo(a)pyrene, 5 cm3calibration solution, ng0,050,10,250,40,5
Mass concentration of benzo(a)pyrene in the blood, mg/DM30,00001of 0.000020,000050.00008 inch0,0001

Practicing optimal liquid-chromatographic parameters for the determination of benzo(a)pyrene in the blood by the proposed method was carried out using a liquid chromatograph "Agilent 1200", equipped with a thermostat column, gradient pump and a fluorimetric detector.

The completeness of the separation of benzo(a)pyrene in the presence of matrix components was achieved on a column Zorbax length of 50 mm and an inner diameter of 4.6 mm with a sorbent Eclipse RAS (C18) a perlage 3.5 µm at a temperature of �of column 27°C, when using the modes specified above, the gradient changes in the composition of the mobile phase. Maximum signal fluorimetric detector obtained at an excitation wavelength of 265 nm and the wavelength of emission 412 nm. In the case that was not applied in a gradient elution mode the sensitivity of the method was drastically changed.

In the process of investigating the selection of optimum conditions of carrying out solid phase extraction (SPE) benzo(a)pyrene from blood plasma, comprising the conditioning of the sorbent Oasis HLB solvent - 100% acetonitrile and distilled water rinsing of the cartridge from interfering matrix components with distilled water and 50% aqueous solution of acetonitrile after drawing a test sample of plasma to the specified sorbent and subsequent desorption of benzo(a)pyrene 100% methylene chloride, were tested 2 options for sample preparation: without prior diglucuronides and diglucuronide by adding in the plasma of a solution of β-glucuronidase in acetate buffer (2500 ME), mixing and incubation in a water bath at 37°C for 1 hour. The data obtained in the tests are shown in table 3.

Table 3
The efficiency of extraction of benzo(a)Pyrenees blood plasma by solid-phase extraction
No. modeModes of sample preparationThe degree of extraction, %
1Elution with methylene chloride without diglucuronidesof 38.7
2Pre diglucuronide and elution
the methylene chloride
13,2

The data in table 3 show that the maximum degree of extraction of benzo(a)pyrene from plasma was achieved by using mode # 1, which was used in further studies.

Experimentally, were also installed, along with the need to use as the extractant 100% methylene chloride, and other features and modes of the proposed method, providing for efficient and selective recovery of benzo(a)pyrene from blood samples and increase the sensitivity and accuracy of the definition, namely: centrifuging blood samples at 2000 rpm, when using solid-phase extraction of 1 cm3100% acetonitrile and 1 cm3distilled water to contentioncount adsorbent, washing the cartridge with 1 cm3distilled water and 0.2 cm350% aqueous solution of acetonitrile after proposin�I fraction of blood plasma prior to extraction 1.0 cm 3100% methylene chloride.

In the study the choice of optimal conditions of extraction of benzo(a)pyrene from shaped elements using dispersive solid-phase extraction, comprising adding to blood cells 100% acetonitrile and salts set QuECHeRS method for the extraction and the separation and purification of the extract bath salts QuECHeRS method "to clean", it was found that the degree of extraction is 64%.

An example of a specific implementation of the proposed method

Analyze blood samples (surveyed population). Each blood sample is analyzed twice. Vegetariano the blood sample with a volume of 2 cm3centrifuged at a speed of 2000 rpm for 5 min, fractionated plasma and formed elements. Take 0.5 cm3plasma and carry out solid-phase extraction, consistently flowing under vacuum through the cartridge sorbent Oasis HLB 3 CC 1 cm3100% acetonitrile, 1 cm3distilled water, 0.5 cm3plasma, 1 cm3distilled water, 0.2 cm350% aqueous solution of acetonitrile. Then the sorbent cartridge was dried under vacuum, transferred to a cumulative vessel (BUCs) and passed through the sorbent 1.0 cm3100% of methylene chloride. The extraction speed (flow rate) of not more than cm3/min aliquot of the extract obtained from�playing and chromatographic a liquid chromatograph Agilent 1200 series with fluorimetric detector column (Zorbax length of 50 mm and an inner diameter of 4.6 mm with a sorbent Eclipse RAS C 18when the column temperature 27°C, using as mobile phase a mixture of acetonitrile and water at a flow rate of 1.5 cm3/min and optimization in gradient elution mode (1 min flow mobile phase: a mixture of 60 vol.% of acetonitrile and 40 vol.% water, the increase of acetonitrile with 60 vol.% to 68 vol.% within 3 min, the increase of acetonitrile with 68 vol.% up to 70 vol.% for 0.5 min, increase of acetonitrile with 70 vol.% up to 90 vol.% for 1.5 min, increase of acetonitrile to 90 vol.% to 100 vol.% over 4.5 min, flow 100% acetonitrile for 1.5 min, then a sharp decrease of acetonitrile to 60 vol.% and filing 60 vol.% acetonitrile for 4 min to trim columns). In this case, the excitation wavelength fluorimetric detector was 265 nm and the wavelength of emission 412 nm.

On the calibration chart to determine the content of benzo(a)pyrene in the plasma (samples 1-5). The results are shown in table 4.

Take 0.5 cm3formed elements and spend a dispersive solid-phase extraction: 0.5 cm3formed elements add 2 cm3100% of acetonitrile, shaken vigorously for 1 min, add 250 mg of salts set QuECHeRS method for extraction", shake intensely for 1 minute, centrifuged for 10 minutes at a speed of 2000 rpm to form 3 layers, 1.5 cm3the top layer Perrin�asked in another test tube, which contains 120 mg of the set of salts QuECHeRS method "to clean", shake 1 min, centrifuged again for 10 minutes at a speed of 2000 rpm, select the top layer and analyze 20 mm3a liquid chromatograph Agilent 1200 series similar to plasma.

On the calibration chart to determine the content of benzo(a)pyrene in structural elements (samples 1-5). The results are shown in table 4.

Table 4
The results of the study blood samples for the content of benzo(a)pyrene in the range of mass concentrations 0,00001-0.002 mg/DM3
No.
sample
Blood plasmaBlood corpusclesWhole blood
Results
parallel
definitions
mg/DM3
The result of the measurement,
mg/DM3
Results
parallel
definitions
mg/DM3
The result of the measurement,
mg/DM3
The measurement result (summarized),
mg/DM3
Relative�I error,
%
10,0000100,000011±0,00000260,0000280,000031±0,00000740,000042±0,000005a 20.7
0,0000120,000034
2000,0000170,000018±0,00000430,000018±0,000002117,6
00,000020
30,0000110,000012±0,00000290,0000110,000010±0,00000240,000022±0,000002620,1
0,0000130,000009
40,0000100,000010±0,00000240,0000110,000010±0,00000240,000021±0,000002418,5
0,000008
50,0000230,000021±0,0000050,0000240,000046±0,0000110,000067±0,00000815,0
0,0000190,000022

The sensitivity of detection of benzo(a)pyrene in the analyzed volume of blood (10 mm3) was 0.2 PG (or 0,00001 mg/DM3). The determination error does not exceed 23.6 per cent.

The measurement technique provides the results of measurements with an error not exceeding the values given in tables 5 and 6.

Table 5
Values of repeatability, reproducibility, accuracy
Name of the analyte and the measurement range, mg/DM3The rate of recurrence, σr, %Indicator of reproducibility, σR, %Indicator accuracy ±δ % (P=0,95)
Benzo(a)pyrene from 0.00001 to 0.002 on.2, 7,723,6

Table 6
Values of the limits of repeatability and reproducibility at a confidence level p=0,95
Name of the analyte and the measurement range, mg/DM3The limit of repeatability, rn, %Limit intralaboratory reproducibility,
RC, %
Benzo(a)pyrene from 0.00001 to 0.002 on.6,2a 21.4

The application of the proposed method allows to increase the sensitivity of detection of benzo(a)pyrene at least 10 times, for example, in comparison with the prototype.

The inventive method is simple and can be recommended for serial analysis.

Sources of information

1. RF patent №2153167.

2. Sychev S. N. Methods to improve chromatographic systems and mechanisms of retention in HPLC. Orel, Orel state technical University, 2000, p. 211.

3. RF patent №2187106.

4. RF patent №1337764.

5. Kang N., Jeong S., Cho., Cho J. Changes of biomarkers with oral exposure to benzo(a)pyrene, phenanthrene and pyrene in rats // J. Vet. Sci. 2007. - 8(4). - p.361-368.

1. Method of quantitative determination of benzo(a)pyrene in blood by liquid chromatography, comprising a sample of blood, its solid-phase extraction and study of the extract by liquid chromatography, characterized in that after sampling the blood, it undergoes conservation the addition of heparin, expose the blood sample is centrifuged at a speed of 2000 rpm for 5 min with the separation of the plasma and formed elements, then separate these sample preparation of plasma and formed elements of blood, while for sample preparation of plasma carry out solid-phase extraction of the plasma with the sorbent Oasis HLB by sequentially passing through said sorbent acetonitrile, distilled water, plasma, distilled water, 50% aqueous solution of acetonitrile, then the sorbent cartridge was dried under vacuum and then passed through the extractant - 100% methylene chloride, the extract was dried in a stream of air and the dry residue pererastayut in 100% acetonitrile, getting extract plasma and sample preparation of formed elements of the blood sample is carried out dispersive solid phase extraction by adding 100% acetonitrile, with stirring, add a set of salts on m�Todd QuECHeRS extraction, mixing, further centrifugation, the selection of the upper phase and adding to it the set of salts QuECHeRS method for cleaning, mixing, re-centrifugation of this refined top and again the selection of the upper phase extract of the formed elements of the blood sample for analysis, then the resulting extracts of plasma and formed elements of the blood samples analyzed separately by liquid chromatography, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 60:40 vol.% to 90:10 vol.% respectively, and 100% acetonitrile in gradient mode, which is carried out by chromatography by filing first for 1 min mobile phase consisting of a mixture of acetonitrile and water in the ratio of 60:40 vol.%, then by increasing for 3 min in the mobile phase of acetonitrile to 68 vol.%, a further increase in its concentration within 1.5 min to 70 vol.%, then increasing the concentration of acetonitrile for 1.5 min to 90 vol.%, as well as increasing the acetonitrile to 100% over the next 4.5 seconds and transmission of such mobile phase for 1.5 min, followed by a sharp decrease in volume of the quantity of acetonitrile to 60 vol.% and by deletion of this mobile phase through the column for 4 min prior to equilibration of the column, and the quantity of benzo(a)pyrene in the blood set on grad�eremochloa schedule moreover, the results obtained in chromatographicaliy extracts of plasma and formed elements, summarize.

2. A method according to claim 1, characterized in that the flow rate of the mobile phase is 1.5 ml/min.

3. A method according to claim 1, characterized in that the liquid chromatography was performed on liquid chromatograph "Agilent 1200" with fluorimetric detector at an excitation wavelength of 265 nm and the wavelength of emission 412 nm at a column temperature of 27°C.



 

Same patents:

FIELD: medicine.

SUBSTANCE: cognitive disorders are stated according to the clinical recognition and neuropsychological test results in a child. Child's blood is analysed to measure the manganese content, and if its content tends to increase more than the reference, the levels are stated: lipid hydroperoxide in blood serum, malondialdehyde MDA in blood plasma and 8-hydroxy-2-deoxyguanosine 8-OHdG in urine; glutathione peroxidase GlPO, Cu/Zn-superoxide dismutase Cu/Zn-SOD, antioxidant activity AOA of blood serum; glutamate and γ-aminobutyric acid in blood serum; hormones of the pituitary-adrenal axis: adrenocorticotropic hormone ACTH, cortisol and serotonine in blood serum, cyclic adenosine monophosphate cAMP and cyclic guanosine monophosphate cGMP. If the blood manganese content is more than 0.029 mcg/cm3 with at least 50% of the above clinical-laboratory values having the following characteristics: lipid hydroperoxide, MDA, 8-OHdG, glutamate, ACTH, cortisol, cGMP increased as compared to the age physiological norms; GlPO, Cu/Zn-SOD, AOA, γ-aminobutyric acid and cAMP reduced as compared to the age physiological norms, cognitive disorders associated with the exposure to manganese in the child.

EFFECT: using the invention provides high diagnostic accuracy.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: blood is sampled, acidified to pH 2-3 with aqueous oxalic acid, extracted in toluene for 5 min; the prepared extract is centrifuged for 60 min at 7,000 rpm, added with sodium sulphate to dewater and acetylated for 3 hours by introducing trifluoroacetic anhydride while stirring continuously in the pyridine medium. The blood sample, toluene, trifluoroacetic anhydride and pyridine are taken in volume ratio 5:2.5:0.2:0.1 respectively.

EFFECT: simplifying the stage of sample preparation and increasing the sensitivity of pentachlorophenol test.

3 cl, 4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention describes the diagnostic technique for oxidative stress in children exposed to nickel from the external environment and involves measuring nickel in blood samples and determining the laboratory values: in blood serum - lipid hydroperoxide, in urine - 8-hydroxy-2-deoxyguanosine; that is follows by a correlation analysis of the above laboratory values and blood nickel, if establishing the significant dependencies: high blood nickel more than 0.083 mg/dm3 and blood serum lipid hydroperoxide increased as compared to the reference and urine 8-hydroxy-2-deoxyguanosine increased as compared to the reference enables diagnosing the oxidative stress in the body at the level of cell DNA and membrane.

EFFECT: invention provides high diagnostic accuracy for the oxidative stress at the early stage in the exposure to nickel from the external environment by the complex use with interpreting the results along with the laboratory values describing the oxidative damages of cell DNA and membrane, as well as the values of blood chemical contaminant - blood nickel.

2 tbl, 2 dwg, 1 ex

FIELD: biotechnology.

SUBSTANCE: method is implemented as follows: the biological material containing novocaine is crushed, treated three times with acetone, containing 0.2-0.4% water, the liquid extract is separated, the acetone from the liquid extract is evaporated together with water in the air stream at a temperature of 18-22°C until complete removal of the solvent, the residue obtained as a result of evaporation is repeatedly treated with acetone containing 0.2-0.4% water, the acetone extract is separated, combined, and the solvent from the combined extract is evaporated, the residue is dissolved in diethyl ether, the resulting solution is diluted in hexane in a ratio of 1:1 by volume, extracted with buffer solution with pH 1-2, the acid-water extract is separated, neutralized with 10% ammonia solution, alkalized with ammonium buffer solution to pH 9.0-9.5, the resulting aqueous alkaline solution is saturated with ammonium sulphate, extracted twice with portions of the organic extractant which is used as a 30% solution of camphor in methyl acetate, at a ratio of aqueous and organic phases as 1:1 by volume, the organic extracts are separated, combined, and the solvent from the combined extract is evaporated in a stream of air at a temperature of 18-22°C to dry residue, the residue is dissolved in a mixture of dichloromethane and ethanol taken in a volume ratio of 1:1, the resulting solution is applied to a column of silica gel CRA No. 80/120 mcm, chromatographed using two-phase movable phase dichloromethane-ethanol in a ratio of 1:1 by volume, the fractions of eluate containing the analyte, combined, the eluent is evaporated, the residue is dissolved in methanol and determining is performed by gas-liquid chromatography coupled with mass spectrometric detection in a capillary column DB-5 MS EVIDEX with the length of 25 m and an inner diameter of 0.2 mm with the stationary phase, which is 5%-phenyl-95%-methylpolysiloxane using helium carrier gas supplied at a rate of 0.6 ml/min and a mass selective detector operating in electron impact mode, the initial temperature of the column oven is 80°C, this temperature is maintained for 1 minute, then the temperature is raised from 80°C to 200°C with the rate of 40°C per minute, then from 200°C to 300°C with the rate of 12.5°C per minute, the final column temperature is maintained for 16 minutes, the temperature of the injector is 200°C, the temperature of quadrupole is 150°C, the temperature of the detector interface is 300°C, the intensity of the signal is recorded, due to charged particles produced by bombarding of the analyte emerged from the capillary column and entered into the ion source, with ionizing electron beam with energy of 70 eV, a mass spectrum is recorded on total ion current and the amount of novocaine is calculated based on the chromatogram peak area.

EFFECT: increased sensitivity of determining.

2 ex, 3 tbl

FIELD: chemistry.

SUBSTANCE: biological material containing 3-methoxyhydroxybenzene is repeatedly (three times) treated for 45 minutes with an alkyl acetate in the form of methyl acetate; the separate extracts are combined; the solvent from the combined alkyl acetate extract is evaporated; the residue is repeatedly treated with acetone; the acetone extracts are combined, evaporated in an air current at 18-22°C, and then in an nitrogen current until complete removal of the solvent; the residue is dissolved in ether; the obtained solution is diluted with hexane in volume ratio of 1:1, extracted with a buffer solution with pH 12-13; the water-alkaline extract 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 current at 18-22°C and then in a nitrogen current until complete removal of the solvent; the residue is dissolved in a hexane-dioxane-propanol-2 solvent mixture, taken in volume ratio of 20:5:1, subjected to chromatography in a silica gel macrocolumn L 40/100 mcm using a mobile phase of hexane-dioxane-propanol-2 in volume ratio of 20:5:1; eluate fractions containing the analyte are combined; the eluent is evaporated in an air current at 18-22°C and then in a nitrogen current until complete removal of the solvent; the residue is dissolved in dichloromethane, followed by determination using a chromatographic-mass-spectrometric technique using a capillary column with length of 25 m and internal diameter of 0.2 mm with a stationary phase with thickness (5% phenyl)-methylpolysiloxane, using a helium carrier gas, fed at a rate of 0.6 ml/min, and a mass-selective detector operating in electronic impact mode; the initial thermostat temperature of the column is 70°C; said temperature is maintained for 3 min, and the temperature is then raised from 70°C to 290°C at a rate of 20°C/min; the injector temperature is 250°C, the detector interface temperature is 300°C; the method also includes detecting the strength of the signal resulting from charged particles formed when bombarding the analyte coming from the capillary column and falling into an ion source which ionises an electron beam with energy of 70 eV; recording the mass spectrum on the full ion current and calculating the amount of 3methoxyhydroxybenzene from the area of the chromatographic peak obtained by detecting the signal from the characteristic molecular ion 124 m/Z.

EFFECT: high sensitivity.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to medical diagnostics in oncology, and describes a method for predicting metastases in the patients with skin melanoma. The method involves measuring VEGF C and VEGF A markers in the tumour tissue removed intraoperatively, calculating their relation; if the relation increases 5 times as much in relation to the values in intact skin derived intraoperatively from non-oncologic patients, lymphatic metastases are predicted, whereas decreasing this value 10 times as much enables predicting haematogenous metastases. The invention can be used in routine clinical practice of health care institutions, research and development establishments and oncologic dispensaries.

EFFECT: method is characterised by accessibility, relative simplicity and usability of assessing the vascular growth factors and enables predicting the risk of latent metastatic phase of the disease, conducting the objective control of the malignant process, particularly skin melanoma, predicting metastases before the clinical manifestations and including the patients into the risk group for intensive follow-up, taking the timely measures for prescribing the adjuvant treatment.

2 ex

FIELD: medicine.

SUBSTANCE: presented group of inventions refers to medicine. What is presented is a method for the prediction of a high probability of the therapeutic effect of high-affinity anti-VEGF antibody, particularly ranibizumab in the patient suffering age-related moist macular degeneration. What is presented is a kit comprising a first oligonucleotide and additional nucleotides specific for allele polymorphisms of matrix metalloprotease 25 (MMP25) gene corresponding to rs1064875. If the respective genotype contains AA or AG, the high probability of the effect of the above treatment is predicted.

EFFECT: presented group of inventions provides the effective agents and methods for the prediction of the high probability of the effect of the treatment with the high-affinity anti-VEGF antibody in the patient with moist AMD.

9 cl, 1 dwg, 5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for the differential diagnostics of hepatic steatosis and steatohepatitis by biochemical tests characterised by measuring blood serum phospholipase A2, nitrogen oxide and endotoxin; if phospholipase A2 is 199.7-252.5 ng/ml, nitrogen oxide is 93-94.6 mcmole/l and endotoxin is 2.2-2.6 EU/ml, hepatic steatosis is diagnosed; if phospholipase A2 is 412.5-576.5 ng/ml, nitrogen oxide is 137.5-168.5 mcmole/l and endotoxin is 3.32-4.18 EU/ml, steatohepatitis is diagnosed.

EFFECT: higher accuracy of differential diagnostics and reduced analysis injuries.

4 ex

FIELD: medicine.

SUBSTANCE: invention described a method for predicting the high risk of industrial and occupational diseases in chemical workers employed in harm involving measuring the total antioxidant status in blood serum, which is also analysed for quantitative content of lipid peroxidation products; if an increase of lipid peroxidation products of more than 4.31 mcmole/l and a decrease of total antioxidant status of less than 1.3 mcmole/l are observed, the high risk of industrial and occupational diseases is predicted.

EFFECT: using the invention simplifies and reduces the examination time, as well as labour inputs and material expenses.

3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: treating secondary mitochondrial dysfunction in the children suffering an urinogenital pathology is ensured by measuring a pre-therapeutic level of reactive oxygen intermediate (ROI) generation by whole capillary blood leukocytes by luminol-dependent chemoluminescence stimulated by barium sulphate crystals and antioxidant activity (AOA) of serum by chemiluminescence activated by rhodamine B in the presence of ferrous iron ions. If ROI is more than 2.7×105 quantum/sec × 4π, AOA is less than 29 relative units, an energotropic therapy is prescribed in the form of oral administration of 30% carnitine 30 mg/kg/day for one month; thereafter ROI and AOA is re-measured. If ROI is less than 2.7×105 quantum/sec × 4π, AOA is more than 29 relative units, the treatment is terminated; if ROI is more than 2.7×105 quantum/sec × 4π, and AOA is less than 29 relative units, continuing the carnitine therapy is indicated in the same dose for another month.

EFFECT: invention enables optimising the drug-induced treatment of secondary mitochondrial dysfunction in the children with congenital urogenital abnormalities by the individual approach, and grounds prescribing the energotropic therapy and its length.

3 tbl, 2 ex

FIELD: instrumentation.

SUBSTANCE: device for preparing control gas mixtures comprises a gas mixer, at least one channel for supplying target gas to the gas mixer, at least two channels for supplying the diluent gas to the gas mixer, and the channel for output of the gas mixture from the gas mixer. And in each channel for supplying gas to the gas mixer the mass flow controller of gas and a solenoid valve are mounted successively, at least in one channel for supplying the diluent gas to the gas mixer the gas humidifier and the solenoid valve are mounted successively. In each of the channels equipped with the gas humidifier at least one bypass pipeline with the additional solenoid valve is mounted. and the output of the gas flow controller of this channel is connected to the input of the additional pipeline which output is connected to the output of the last solenoid valve, and at the input of the target gas and the input of the diluent gas at least one filter is mounted, which outputs are connected to the inputs of manually operated valves.

EFFECT: possibility of operational automated obtaining the dry or moist gas mixture, and reliable obtaining of the given values of concentrations of gas mixtures at the output of the device.

1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry, particularly to methods of determining benzoic acid, and describes a method for quantitative determination of benzoic acid from a methyl derivative thereof - methyl ether in aqueous matrices with determination sensitivity of 5.0·10-5 mg/cm3 with determination error of not more than 25%. The method is characterised by that quantitative determination of benzoic acid is carried out using a chromatographic method with flame-ionisation detection and includes the following steps: extraction concentration of an analyte with benzene in water samples acidified with 25% sulphuric acid solution to pH 1-3 while adding sodium chloride until a saturated solution is obtained, conducting a benzoic acid methylation reaction with diazomethane to obtain a derivative - methyl ether of benzoic acid and determining the formed methyl ether of benzoic acid by a chromatographic method with flame-ionisation detection.

EFFECT: method provides high sensitivity, selectivity and easy implementation during quantitative determination of benzoic acid in aqueous media and enables use thereof in practice in factory analytical laboratories, central laboratories of chemical companies and chemical-toxicology laboratories.

1 ex, 3 tbl

FIELD: engines and pumps.

SUBSTANCE: proposed method is based on application of simplified model of intake of admixtures into cabin which allows for only oil decomposition products in gas turbine engine. Major portion of air samples, 95-97%, required for identification and quantitative determination of oil decomposition products, is sampled on surface from device simulating oil decomposition conditions including air temperature and pressure at point of sampling from engine compressor, and oil stay time in hot zone.

EFFECT: decreased time of in-flight experiments and that of surface analysis of samples.

1 cl, 1 ex, 1 tbl, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to laboratory methods of analysis and deals with method of quantitative determination of manganese, lead and nickel in bile by method of atomic-absorption analysis with atomisation in flame. Essence of method lies in the following: sampling of bile is carried out during duodenal probing, after that it is frozen, and unfrozen at room temperature, homogenisation of bile by mixing being performed already at partial soft unfreezing. After that, sampling of homogenised bile is carried out for preparation for analysis, concentrated nitric acid is introduced into it with volume ratio 1:1, mixture is kept at room temperature, then heated and further mixture is kept for not less than 2.5 hours at room temperature. In order to obtain analyte, to obtained mixture added is concentrated hydrogen peroxide in volume ratio 1:1 to volume of bile sample volume, analyte is heated, after that cooled to room temperature. After that by method of atomic-absorption spectrometry, using graduated diagram, quantitative content of particular type of metal: manganese, lead and nickel is determined in analyte.

EFFECT: invention allows increasing accuracy of quantitative determination of manganese, lead and nickel in bile.

6 tb

FIELD: physics.

SUBSTANCE: vapour-gas mixture source has a mixer which has connecting pieces for inlet and outlet of the vapour-gas mixture. The vapour-gas mixture source also has a diffusion pipe filled with working fluid and an auxiliary pipe designed for filling the diffusion pipe with working fluid. Part of the diffusion pipe is filled with substance which retains the working fluid. The level of working fluid in the auxiliary pipe is lower than the level of substance in the diffusion pipe. The substance which retains the working fluid used can be sand, granular material with particle size between 10 and 10000 mcm, porous substances, e.g. ceramic metal etc.

EFFECT: more accurate measurement and maintenance of concentration of the vapour-gas mixture coming out of the source, provision for constant diffusion flow of vapour of working fluid into the mixer.

11 cl, 2 dwg

FIELD: instrument engineering.

SUBSTANCE: invention is designed for calibrating gas analyser detectors, according to which there prepared is calibration substance solution with concentration A=By/k (%) as per Henry constant value k (mg/m %) at calibration temperature and as per the specified value of calibration substance mass concentration in calibration steam/gas mixture By (mg/m). After the solution has been introduced into the vessel in quantity enough for fully saturated equilibrium calibration steam/gas mixture to appear above the solution surface, the sensor calibration is carried out by means of mixture; at that, mixture concentration is changed by means of direct proportional change of solution concentration by diluting concentrated reference solution of calibration substance with analytical accuracy up to the specified concentration value A (%). There also proposed is the device for realising this method, which includes a solution point for preparing calibration solution with analytical accuracy, vessel with thermostatic device for obtaining steam/gas mixture with constant concentration corresponding to Henry law; at that, solution point includes graduated dose metre, graduated diluter, mixer with a reducer, capacity with solvent, and reference container with reference solution, which is stabilised with a gate valve meant for multiple use of container, and vessel with thermostatic device consists of thermometre and heat-insulating cover plate with an inlet branch pipe containing a normally closed return valve and a pusher for valve opening.

EFFECT: decrease of calibration substance losses; accuracy and reproducibility of metrological performance, and meeting requirements of industrial and ecological safety.

6 cl, 2 dwg

FIELD: instrument engineering.

SUBSTANCE: device for generating flow of vapor-gas mixture with preset concentration of vapor has vessel partially filled with fluid, second vessel provided with branch pipes for supply and removal of gas, and vapors of fluid pipeline-leak. One of vessels is connected with gas discharge forcer; fluid vapors pipeline-leak connects both vessels. Vessel, partially filled with fluid, is mounted inside second vessel. Pipeline-line, connecting both vessels, is totally placed inside second vessel. Device is also provided with additional discharge forcer for adjusting concentration of fluid vapor in second vessel. Granulated filler is introduced into vessel partially filled with fluid. Device is also provided with gas analyzer for providing gas concentration in space of second vessel.

EFFECT: higher precision of keeping of preset concentration of vapor; improved efficiency of vapor concentration control and adjustment.

FIELD: chemical technology.

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

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

EFFECT: improved method of synthesis.

8 cl, 1 dwg, 2 ex

The invention relates to the field of analytical chemistry of organic compounds, namely, the field determination of organic compounds in their joint presence using gas-liquid column chromatography, and can be used for the separate determination of phenols in liquid environments, mainly in industrial effluents, as well as the analysis of natural waters

The invention relates to the field of gas analysis and can be used for calibration of gas analysis equipment

FIELD: chemical technology.

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

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

EFFECT: improved method of synthesis.

8 cl, 1 dwg, 2 ex

Up!