Method of quantitative analysis of urine dimethyl terephthalate by liquid chromatography

FIELD: medicine.

SUBSTANCE: urine is sampled, centrifuged that is followed by solid-phase extraction with Oasis HLB sorbent with using 100% acetonitrile as an extraction fluid for dimethyl terephthalate extraction. Said solid-phase extraction is conducted by consequent passing 100% acetonitrile, distilled water, the urine sample after centrifugation, distilled water, 20% aqueous acetonitrile and 100% acetonitrile as the extraction fluid through the sorbent; then the prepared extract is analysed by liquid chromatography with using as a mobile phase mixed acetonitrile and water in the at the varying ratio 25:75 vol. % to 90:10 vol. % respectively in a gradient mode which is enabled with combining chromatography by supplying at first the mobile phase containing mixed acetonitrile and water in the ratio 25:75 vol. % for 10 minutes. Then increasing the acetonitrile concentration in the mobile phase to 90 vol. % for 5 minutes and passing such mobile phase for another 5 minutes is followed by decreasing a volume amount of acetonitrile to 25 vol. % for 5 minutes and passing such mobile phase through a column for 10 minutes, while an amount of dimethyl terephthalate is determined by a calibration chart.

EFFECT: high sensitivity of the method combined with selectivity and availability for routine analyses.

5 cl, 6 tbl

 

The invention relates to medical Toxicological research, in particular to sanitary toxicology, and can be used to quantify terephthalate (dimethyl ester of terephthalic acid) in urine to assess the risks to human health and development activities to ensure chemical safety.

The prior art several methods for determining the amount of such substances. So, from source [1] there is a method of determining the dimethyl ester of terephthalic acid (or dimethyl terephthalate) in water. According to a known method measurement performed on a gas chromatograph with a flame ionization detector. The evaporator temperature 280°C, detector temperature and columns 165°C, flow rate of carrier gas (argon) 40 cm3/min. Definition does not interfere with p-xylene, acetaldehyde, diphenyl, diphenyloxide, formic, acetic, benzoic, p-tolarova, terephthalic and isophthalic acids, and methyl esters of these acids contained in water up to 10 MPC.

The concentration of water is performed by the method of solvent extraction in a separating funnel, with a capacity of 100 cm3placed 50 cm3analyzed water, make 10 g of sodium chloride, acidified chloride-hydrogen acid to pH 3, add the internal standard is 0.1 cm3solution dipropylacetate in ethyl the PTO alcohol (0.05 mg) and the extractant - 2 cm3the chloroform. Extracted for 2 min and after 15 min the lower chloroform layer is placed in a tube. The extract was evaporated in a stream of air in a fume hood until an amount of 0.2-0.3 cm3and chromatographic 1-2 mm3the extract. The range of measured concentrations is 0.15 to 3 mg/DM3. The disadvantage of this method is its low sensitivity definition.

Also there is a method of determining in the urine 16 monoamino phthalic acid [2] using solid-phase extraction and high performance liquid chromatography coupled with tandem mass spectrometry. According to this method, urine samples treated with ultrasound for 5 minutes and mix by shaking. Then to 100 mm3urine add 25 mm3the enzymatic solution and conducting the reaction diglucuronides at 37°C for 90 minutes Then the reaction diglucuronides stop adding to the sample 50 mm3acetic acid (glacial) acid and 200 mm35%aqueous solution of acetonitrile. The analysis is performed on a liquid chromatograph Agilent 1100 series connected with a quadrupole mass spectrometer with electron-beam-sustained spray. The mobile phase of 0.1% acetic acid in acetonitrile, optimized in the gradient mode, the speed of eluent 0,35 cm3/min. measurement Error is 21.0 per cent. the thinned detection monoamino phthalic acid is 0,00011-0,0043 mg/DM 3. According to this known method analyzes only monetary o-phthalic acid. Dimethyl ester of terephthalic acid is not detected by the method described above. In addition, the long sample preparation and expensive equipment is not acceptable to use a known method for mass analysis.

Source of information [3] a known method of determining diethylhexylphthalate in urine samples by gas chromatography/mass spectrometry. The urine sample in the amount of 0.5 cm3was padillas to pH=2-3 and extracted twice with 1.5 cm3ethyl acetate before and after enzymatic hydrolysis with beta-glucuronidase. The combined extracts were dried under a stream of nitrogen and the reaction was performed derivatization with diazomethane to translate the investigated substances into the corresponding methyl esters. Quantitative analysis of diethylhexylphthalate and products of its hydrolysis performed by gas chromatography/mass spectrometry on a capillary column with a mass spectrometer (Germany). The detector temperature 280°C, the temperature of the column thermostat was programmed as follows: 40°C (1 min), increased to 80°C at a rate of 20°C/min, increased to 320°C at a rate of 10°C/min, exposure (5 minutes). The carrier gas is helium. The limit of detection of 0.01 mg/DM3.

In the source of information [4] described a method for determining the end of the ation of phthalic acid and its isomers (isophthalic and terephthalic acids), and their derivatives, including exporter in microbiological substrate used for biodegradation, using high-performance liquid chromatographie (HPLC) with UV detector. Samples of the substrate centrifuged for 3 minutes with a speed of 10,000 rpm, diluted to concentrations less than 50 mg/DM3and analyze aliquots of 10 µl. Separation of the mixture is achieved by using a column (100×3 mm)filled with phase Chromospher 5C18. As an eluting liquid is used a mixture of methanol and 1%aqueous solution of acetic acid in the ratio of 40:60, the flow rate of the mobile phase, 0.3 ml/min Detection of the individual components of the mixture is carried out with ultraviolet detector (Spectroflow 773) at a wavelength of 230 nm. A disadvantage of the known method is its low sensitivity definition.

Also known a method of quantitative determination of phthalates (diethylphthalate, dibutyl phthalate, dihexylfluorene and dioctylphthalate) in surface and drinking waters by high performance liquid chromatography with pre-procedure solid phase extraction [5]. According to a known method preconcentration of water samples by the method of solid phase extraction is carried out by passing 250 cm3analyzed water samples speeds of 10-20 cm3/min through the cartridge brand Diapak P, which tentatively is satisfactory conditionyou 12 cm 3methanol and washed with 18 cm3of distilled water. After the deposition of the sorbent Diapak P the analyzed water sample cartridge was washed with 6 cm3distilled water and dried under vacuum for 7 minutes Then elute phthalates 1.5 cm3methanol, evaporated the solvent in a stream of nitrogen and dissolve the residue in 100 mm3of methanol. Aliquot part of the extract analyzed by liquid chromatograph Agilent 1100 series with diode-matrix detector at a wavelength of detection 220 nm. Lower limit of detection of these phthalates in water is 0,0005-0.024 mg/DM3. This method is not defined dimethyl ester of terephthalic acid.

Specified known method cannot be used to determine terephthalate in the urine with a lower limit of detection of 0.0005 mg/DM3as in the analysis of urine must be 2 parallel detection of a single sample and therefore will require a volume of the urine sample in a quantity of 500 ml, which is not always feasible in non-invasive sampling. In addition, due to the interfering influence of the matrix, reducing the analytical signal, the lower limit of detection of dimethyl terephthalate in the urine is above the specified lower limit of detection for water, as in this known method.

The technical result provided by the invention, is to provide the highest Chu the reality of the way to determine the dimethyl ester of terephthalic acid (or dimethyl terephthalate) in the urine, including multicomponent samples, while maintaining selectivity and availability for serial analysis.

The specified technical result is achieved by the proposed method for quantitative determination of terephthalate in urine by liquid chromatography, whereby to produce a sample of urine, subjecting it to centrifugation, carry out solid-phase extraction on Oasis HLB sorbent using to retrieve terephthalate extractant - 100%acetonitrile, with a specified solid-phase extraction is carried out by sequential passage through the sorbent 100%acetonitrile, distilled water, urine samples after centrifugation, distilled water, 20%aqueous solution of acetonitrile and extractant - 100%acetonitrile, and then the resulting extract was analyzed by liquid chromatography, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 25:75 vol.% to 90:10 vol.% accordingly, in the gradient mode, which is carried out by chromatography by filing first for 10 min mobile phase consisting of a mixture of acetonitrile and water in the ratio 25:75 vol.%, then by increasing for 5 min in a mobile phase of acetonitrile to 90% vol. and transmission of such mobile phase for another min, with the subsequent reduction of volume quantities of acetonitrile for 5 min to 25 vol.% and deletion of such mobile phase through the column for 10 min, and the number of terephthalate set on the calibration curve.

Centrifugation should be performed within 5 min with a speed of 2000 rpm/min

The flow rate of the mobile phase is 1.0 cm3/min

The volumetric ratio of the urine sample and extractant is 3:1, respectively.

Chromatography is performed on a liquid chromatograph Agilent 1200 with fluorimetric detector at a wavelength of 246 nm excitation and wavelength emission 330 nm.

This technical result is achieved due to the following.

Due to the fact that the collected urine sample is subjected to centrifugation, provided the denaturation of the protein, preventing its influence on the accuracy of determination.

Experimentally it was found that high sensitivity and accuracy exporter in the urine sample is achieved only under strictly defined sequence of implementation of solid-phase extraction on Oasis HLB sorbent using as extractant - 100%acetonitrile. When this transmission (konwencjonalna) sorbent 100%acetonitrile and 1 cm3distilled water is used for cleaning sorbent from possible prisutstvie the impurities and moisture sorbent before passing urine sample (in General - this preparation of the sorbent to the analysis), and washing of the sorbent after the urine sample with distilled water and 20%aqueous solution of acetonitrile necessary for the purpose of removal with sorbent slobodianik components of the matrix and increasing the selectivity of the extraction. The use of an aqueous solution of acetonitrile precisely this concentration provides good removal of unnecessary components, and at the same time is not possible extraction.

Conduct further analysis of the extract by liquid chromatography with special modes, namely, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 25:75 vol.% to 90:10 vol.% accordingly, in a gradient mode (gradient elution), which is carried out by first filing within 10 min of the mobile phase consisting of a mixture of acetonitrile and water in the ratio 25:75 vol%, and then by increasing for 5 min in a mobile phase of acetonitrile to 90% vol. and transmission of such mobile phase for another 5 min, followed by a decline bulk amounts of acetonitrile for 5 min to 25 vol.% and deletion of such mobile phase through the column for 10 min, corresponds to the optimization of the elution, and thus provides a high sensitivity of the method. It should be clear that changing the volume ratio of acetonitrile and water in the mobile f the se is due to the 4-channel gradient pump Agilent 1200, mixing up to 4 components of the mobile phase.

The flow rate of the mobile phase 1.0 cm3rpm is optimal for research.

The volumetric ratio of the urine sample and extractant 3:1 respectively selected based on the results of experimental studies on the completeness of the extraction of terephthalate with Oasis HLB cartridges SS in the range defined concentrations of 0.001-1.0 mg/DM3in the urine. For the degree of extraction, equal 99,3% of the dose introduced in a urine sample that does not contain terephthalate, it's enough to pass through the cartridge 1 cm3100%of acetonitrile, and the first pass of 0.5 cm3100%of acetonitrile in a first vial, and then following 0.5 cm3acetonitrile in the second vial. The contents of the vial analysed separately, and the final result is given by summing the detected amount of terephthalate in the first and second vials.

The study of the extract on a liquid chromatograph Agilent 1200 with fluorimetric detector at a wavelength of 246 nm excitation and wavelength emission 330 nm is due to the maximum signal fluorimetric detector, which affects the sensitivity and precision of definition.

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

- produce a sample of the eyes in the amount of 10 cm 3;

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

- select 6 cm3the top layer of urine and conduct solid-phase extraction, sequentially passing under vacuum through cartridge sorbent Oasis HLB 1 cm3100%acetonitrile, 1 cm3distilled water, 3 cm3urine (miss only 3 cm3because spend 2 parallel detection of one sample, the result is presented as the average of the two parallel definitions), 3 cm3distilled water, 2 cm320%aqueous solution of acetonitrile with a flow rate of 1 cm3/min. and Then transferred to the cartridge with a sorbent in the accumulation vessel (tube) and is passed through the sorbent 1.0 cm3extractant - 100%acetonitrile;

- aliquot of the resulting extract in the amount of 10 mm3analyzed by liquid chromatograph Agilent 1200 with fluorimetric detector, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 25:75 vol.% to 90:10 vol.% accordingly, in the gradient mode, which is carried out by chromatography by filing first for 10 min mobile phase consisting of a mixture of acetonitrile and water in the ratio 25:75 vol.% (i.e. about 25. hours of acetonitrile and 75 rpm. including water), then led by the program for 5 min in a mobile phase of acetonitrile to 90% vol. (i.e. about 90. hours of acetonitrile and about 10. including water) and transmission of such mobile phase for another 5 min, followed by a decline bulk amounts of acetonitrile for 5 min to 25 vol.% and deletion of such mobile phase through the column for 10 min;

- quantitative content exporter install calibration curve, which is constructed through the use of selected control group of children urine samples and standard solutions exporter.

To construct the calibration curve in a volumetric flask with a capacity of 100 cm3make 50,0 mg terephthalate (dimethyl ester of terephthalic acid) and adjusted with methanol to the mark. The concentration of dimethyl terephthalate in the original solution (solution No. 1) is 0.5 mg/cm3. Then 0.1 cm3solution No. 1 contribute in a volumetric flask with a capacity of 100 cm3and bring with methanol to the mark. The concentration of dimethyl terephthalate in dilute solution (solution No. 2) is 0.5 mg/DM3. Use fresh solution.

Calibration characteristic set method for the absolute calibration. It expresses the dependence of peak area on the chromatogram (mm2from the concentration of dimethyl terephthalate in the urine (mg/DM3) and is based on the 6 series of working standard solutions. Each series consisting of 3 standard solutions prepared in volumetric flasks 25 cm 3. For this purpose, in each flask contribute solutions for calibration No. 1 and 2 in accordance with tables 1 and 2, bring the volume of the flask to the mark with urine and mix. 10 cm3freshly prepared standard solutions are transferred into centrifuge tubes and centrifuged with a speed of 2000 rpm for 5 minutes Select 6 cm3the top layer of urine and conduct solid-phase extraction on Oasis HLB sorbent SS as in the proposed method. The obtained extracts chromatographic on a liquid chromatograph Agilent 1200 with fluorimetric detector.

Table 1
Standard solutions for establishing calibration characteristics when determining terephthalate in the urine in the concentration range of 0.001-0,020 mg/DM3
Room solution123456
The volume of solution No. 2, cm30,050,100,150,250,501,00
The amount of dimethyl terephthalate in the calibration solution, ág0,0250,0500,0750,1250,2500,500
Mass concentration of dimethyl terephthalate in the urine, mg/DM30,0010,0020,0030,0050,0100,020

Table 2
Standard solutions for establishing calibration characteristics when determining terephthalate in the urine in the concentration range 0,020-1.0 mg/DM3
Room solution123456
The volume of solution No. 2, cm31,02,55,0---
the volume of solution No. 1, cm3---0,0050,0120,025
The amount of dimethyl terephthalate in the calibration solution, ág0,501,252,55,0to 12.025,0
Mass concentration of dimethyl terephthalate in the urine, mg/DM30,0200,050,10,20,481,0

Optimization of liquid-chromatographic parameters for the determination of dimethyl terephthalate in the urine of the proposed method was carried out using a liquid chromatograph Agilent 1200 equipped with a thermostat column, gradient pump and fluorimetric detector.

The complete separation of the dimethyl terephthalate in the presence of dimethyl esters of phthalic and isophthalic acids, monometallic esters of phthalic, isophthalic and terephthalic acid, and phthalic, isophthalic and terephthalic acids was achieved on a column of 4.6×150 mm with a sorbent Eclipse XDB-C 18when the column temperature is 27°C, when using as the mobile phase at the beginning of a mixture of acetonitrile and water in a volume ratio of 25:75 a flow rate of 1.0 cm3/min and optimization of elution in a gradient mode (10 min the flow of the mobile phase of 25% vol. acetonitrile and 75 vol.% water, increasing acetonitrile with 25 vol.% up to 90% vol. within 5 min, 5 min supply 90%acetonitrile, then the reduction of acetonitrile to 25 vol.% within 5 min and feed of 25%acetonitrile for 10 min prior to the equilibration of the column). The maximum signal fluorimetric detector obtained at a wavelength of 246 nm excitation and wavelength emission 330 nm. If you don't apply a gradient elution mode the sensitivity of the method was dramatically changed.

Studies on the optimum conditions for solid phase extraction exporter of urine, including konwencjonalna Oasis HLB sorbent solvent a to 100%acetonitrile and distilled water, rinsing of the cartridge from the interfering components of urine distilled water and 20%aqueous solution of acetonitrile after application of the sample of urine for a specified sorbent and subsequent desorption of the terephthalate of 100%acetonitrile as a solvent were tested following solvents: methanol, ethanol, acetonitrile and water RA the works. The data obtained in the tests are shown in table 3.

Table 3
The average values of the degree of extraction exporter of urine depending on the nature of solvent
no experienceThe solvent (extractant)The degree of extraction, %
15%aqueous solution of methanol0
2100%methanol20,5
3100%ethanol6,3
460%aqueous solution of acetonitrile51,0
5100%acetonitrile99,3

The data in table 3 show that the maximum degree of extraction exporter of urine samples was achieved using as extractant 100%acetonitrile, which was used in further studies.

Experimentally were also install the Lena, along with the necessity of using as extractant 100%acetonitrile, and other characteristics and modes of the proposed method for enabling the efficient and selective extraction of terephthalate and increase the sensitivity and accuracy of definition, namely: centrifugation of urine samples at 2000 rpm, when using solid phase extraction 1 cm3100%acetonitrile and 1 cm3distilled water for kondensirovannie adsorbent, washing cartridge 3 cm3distilled water and 2 cm3a 20%aqueous solution of acetonitrile, after transmission of the analyzed urine samples and extraction of 1.0 cm3100%acetonitrile.

A specific example of the proposed method.

Analyze urine sample (examined group of children). Each urine sample analyzed twice. Vegetariano a urine sample volume of 10 cm3centrifuged with a speed of 2000 rpm for 5 minutes Select 3 cm of the upper layer and conducting solid-phase extraction, sequentially passing under vacuum through cartridge with Oasis HLB sorbent SS 1 cm3100%acetonitrile, 1 cm3distilled water, 3 cm3urine, 3 cm3distilled water, 2 cm320%aqueous solution of acetonitrile. Then transfer the cartridge with a sorbent in the accumulation vessel (tube) and passed through orbent 1.0 cm 3100%acetonitrile. The extraction speed (flow rate) of 1 cm3/min Aliquot of the resulting extract select and chromatographic on a liquid chromatograph Agilent 1200 series" with fluorimetric detector column of 4.6×150 mm with a sorbent Eclipse XDB-C18when the column temperature is 27°C, using as mobile phase a mixture of acetonitrile and water with a flow rate of 1.0 cm3/min and optimization of elution in a gradient mode (10 min the flow of the mobile phase of 25% vol. acetonitrile and 75 vol.% water, increasing acetonitrile with 25 vol.% up to 90% vol. within 5 min, 5 min supply 90%acetonitrile, then the reduction of acetonitrile to 25 vol.% within 5 min and feed of 25%acetonitrile for 10 min prior to the equilibration of the column). At this wavelength excitation fluorimetric detector was 246 nm and the wavelength of emission 330 nm.

For calibration curve and determine the content exporter in samples 1-5. The results are shown in table 4.

td align="center"> The result of measurement g/cm3
Table 4
The results of the study samples of urine on the content exporter
No. sampleThe results of parallel measurements, mg/cm3Relative error, %
10,00350,00325±0,000824,6
0,0030
20,00520,0050±0,001224,0
0,0048
30,00620,0061±0,001524,6
0,0060
40,01000,0105±0,002624,7
0,0110
50,00740,0071±0,001825,7
0,0068

The detection sensitivity of terephthalate in the analyzed volume of the urine sample (10 mm3) was 0.01 ng (or 0.001 mg/DM3). The measurement error does not exceed 26%.

The method of measurement provides the results of measurements with pogress the stew, not exceeding the values given in tables 5 and 6.

Table 5
The range of measurements, the values of precision, repeatability, reproducibility of the proposed method
The name of the component to be determined and the measurement range, mg/DM3(ág/cm3)The measure of repeatability (relative standard deviation repeatability),σr, %The measure of reproducibility (relative standard deviation of reproducibility) σR, %Accuracy rate (bounds the relative error with probability P=0,95), ±δ, %
The exporter from 0.001 to 0.02 on.10,311,025,0
The exporter from 0.02 to 1.0 on.6,3the 9.729,4

Table 6
The limits of repeatability and play is the necessity of the proposed method with probability P=0,95
The name of the component to be determined and the measurement range, mg/DM3The limit of repeatability (relative value allowable discrepancies between the two results of parallel measurements), rn, %Limit intralaboratory reproducibility (relative value allowable discrepancies between the two measurement results obtained in one laboratory, but in different conditions),%
The exporter from 0.001 to 0.02 on.29,031,0
The exporter from 0.02 to 1.0 on.the 17.327,0

The application of the proposed method can improve the detection sensitivity of the terephthalate (dimethyl ester of terephthalic acid) is at least 3 times, for example, compared with the method described in the source of information [4].

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

Sources of information

1. MUK 4.1.745-99. Gas chromatographic determination of dimethyl ester of terephthalic acid in water. Sat. guidelines the Determination of concentrations of chemicals in water centralized the drinking water supply systems. V.2. M: Federal center of state sanitary and epidemiological surveillance Ministry of health of Russia, 1999.

2. Kato K., M.J. Silva, L.L. Needham, A.M. Calafat Determination of 16 Phthalate Metabolites in Urine Using Automated Sample Preparation and Online Preconcentration/High-Performance Liquid Chromatography/Tandem Mass Spectrometry // J. of Analytical Chemistry, Vol.77, No.9, May 1, 2005 2985-2991.

3. Mettang So, Alscher D., Pauli-Magnus C. Dunst R., Kuhlmann U., A. Rettenmeier Phthalic Acid Is the Main Metabolite of the Plasticizer Di(2-ethylhexyl) Phthalate in Peritoneal Dialysis Patients // Adv. Perit. Dial. 1999. - 15. - p.229-33.

4. Kleerebezem R., Hulshoff P., Lettinga, G. Anaerobic biodegradability of phthalic acid isomers and related compounds // J. Biodegradation. - 10. - 1999. - 63-73.

5. Turnaev VA, Tretyakov POSTGRADUATE, Turaeva E.A. Chromatographic methods for the determination of phthalates in surface and drinking waters // Bulletin of the Tyumen state University. - 2007. No. 3. - s-146.

1. The method of quantitative determination of dimethyl terephthalate in urine by liquid chromatography, characterized by the fact that produce a sample of urine, subjecting it to centrifugation, carry out solid-phase extraction on Oasis HLB sorbent using to retrieve terephthalate extractant - 100%acetonitrile, with a specified solid-phase extraction is carried out by sequential passage through the sorbent 100%acetonitrile, distilled water, urine samples after centrifugation, distilled water, 20%aqueous solution of acetonitrile and extractant - 100%acetonitrile, and then received extras the CT analyzing by liquid chromatography, using as mobile phase a mixture of acetonitrile and water when changing the ratio from 25:75 vol.% to 90:10 vol.% accordingly, in the gradient mode, which is carried out by chromatography by filing first for 10 min mobile phase consisting of a mixture of acetonitrile and water in the ratio 25:75 vol.%, then by increasing for 5 min in a mobile phase of acetonitrile to 90% vol. and transmission of such mobile phase for another 5 min, followed by a decline bulk amounts of acetonitrile for 5 min to 25 vol.% and deletion of such mobile phase through the column for 10 min, and the number of terephthalate set on the calibration curve.

2. The method according to claim 1, characterized in that the centrifugation should be performed within 5 min with a speed of 2000 rpm/min

3. The method according to claim 1, characterized in that the flow rate of the mobile phase is 1.0 cm3/min

4. The method according to claim 1, characterized in that the volume ratio of the urine sample and extractant is 3:1, respectively.

5. The method according to claim 1, characterized in that the chromatography is performed on a liquid chromatograph Agilent 1200 with fluorimetric detector at a wavelength of 246 nm excitation and wavelength emission 330 nm.



 

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FIELD: medicine.

SUBSTANCE: vaginal fluid is analysed. The vaginal secretion is collected by means of a common tampon placed in a vagina for 8-9 hours, further weighted; microbial metabolites are extracted in equiponderate amount of distilled water, and the extract is analysed by gas-liquid chromatography. If the vaginal discharge contain acetic acid more than 0.315 mg/g and total propionic and butyric acids ≤0.200 mg/g in an age group of 17 to 34 years, and acetic acid more than 0.210 mg/g with total propionic and butyric acids ≤0.120 mg/g in an age group of 35 to 48 years, nonspecific aerobic vaginitis is diagnosed.

EFFECT: more accurate diagnosis of nonspecific aerobic vaginitis.

4 ex

FIELD: medicine.

SUBSTANCE: invention describes a method of quantitative evaluation of blood acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids by gas chromatography analysis wherein a blood sample is acidified with 1 % sulphuric acid to pH 2-3, evaluated acids are extracted with isobutyl alcohol volume of which is related to the blood sample volume as 1:1. The protein separation is enabled by centrifugation. 2-3 drops of 0.4 % alkali is added, and the extract is evaporated dry, further the solid residue is added consistently with 1 % sulphuric acid and isobutyl alcohol that is followed with gas chromatography separation of the mixed acids in a capillary column with a flame ionisation detector, and the amount of each acid is evaluated by a calibration diagram.

EFFECT: higher sensitivity and accuracy of the method of quantitative evaluation of acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids if found in blood together.

5 cl, 1 ex, 4 tbl

FIELD: chemistry.

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

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

4 tbl

FIELD: chemistry.

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

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

3 tbl, 2 ex

FIELD: medicine.

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

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

4 ex, 5 tbl

FIELD: chemistry.

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

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

1 ex, 3 tbl

FIELD: medicine.

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

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

1 ex, 1 tbl

Gas analysis method // 2410679

FIELD: test equipment.

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

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

9 cl, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry. The method is realised as follows: a sample of ground up premix is filled with a hydrochloric acid solution and put into an opaque case which is put into an ultrasonic bath. Extraction is carried out for 15-20 minutes at 38-42°C and centrifuging is then carried out for 10 minutes at 8000 rpm. The mixture is then brought up to the mark in a measurement flask. The obtained solution undergoes chromatographic separation on a column with Purospher sorbent. Chromatography conditions: eluent A - 0.005 M lithium perchlorate solution, pH=2.5; eluent B- acetonitrile; elution gradient mode - from 0 to 26% eluent B for 14 minutes.

EFFECT: high efficiency and accuracy and possibility of detecting a wider range of vitamins independent of the premix base.

2 dwg, 5 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: method for chromatographic analysis of a substance involves exposing the separated mixture of substances carried by a carrier through a chromatographic column to acoustic oscillations. Before chromatographic analysis, a liquid nematic crystal is deposited on the wall of the chromatographic column, where the said crystal is directed across the propagation of sound oscillations.

EFFECT: high efficiency of separating an analysed mixture of substances into components using sound waves.

1 dwg

FIELD: oil and gas industry.

SUBSTANCE: gas chromatograph includes chamber for samples with piston position sensor, which is connected through sample valve to pipeline and through oil pump to reservoir for compensation of hydraulic oil pressure, electrical thermostat with temperature sensor and chromatograph tube located inside thermostat, which is in-series connected on one side through rotating sample injector, zeolite filter, the first return valve and isolating valve of chromatograph with connection line of sample valve and chamber for specimens, in-series connected on the other side to the second return valve, fraction detector, bottle with sample portion and the second pressure sensor. At that, rotating sample injector is in-series connected to pressure reducer, valve for transporting medium, bottle with compressed nitrogen and the first pressure sensor, bypass line with bypass valve is parallel connected to rotating sample injector, chromatograph tube and fraction detector, and circuit of electronic telemetry is connected to output of fraction detector. Method of downhole gas chromatography is proposed as well.

EFFECT: development of device allowing to perform gas chromatography for determining the type of well fluids in well in real time.

3 cl, 5 dwg

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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