Method of determination of ethanol and other metabolites content in human blood

FIELD: medicine.

SUBSTANCE: invention describes a method of determination of ethanol and other metabolites content in human blood by liquid phase chromatography, including preparation of blood distillates by vapour straight distillation and blood component analyis, characterised by the fact that it is combined with one-stage quantitative determination of ethanol, diethyl ester, acetaldehyde, acetone, methylacetate, ethylacetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol with the use of capillary chromatographic columns; the concentration of the determined blood components is calculated by formula: where a is chromatographic study results, mg/dm3; V is a distillate volume, cm3; m is a whole blood weight, g.

EFFECT: method can be used in clinical laboratory diagnostics in studies of metabolic disorders caused by alcohol poisoning, and in judicial medical activity for diagnosing of a degree of intoxication of live persons.

1 ex, 1 tbl, 2 dwg

 

The invention relates to medicine and can be used in clinical laboratory diagnostics in the study of metabolic disorders in humans caused by alcohol poisoning, and in forensic medical practice for diagnosing the degree of intoxication of living persons.

The generally accepted method for determination of ethyl alcohol in human blood is a method of color reactions. The most popular of the 60-ies of the so-called "nitrite" method and method Widmark. "Nitrite" method is very time-consuming, requires rapid processing of the material researched by double distillation with water vapor, a large number of the investigated material (200-300 g). In addition, the method is not sufficiently specific. The way Widmark is used to determine the degree of intoxication of living persons. this Method is quite time-consuming, research is needed 3-4 portions of the material researched and parallel conducting 3-4 blank experiments, indicating poor reproducibility of the method.

There is a method of quantitative determination of the content of ethyl alcohol in blood and human urine by gas chromatography [Methodological letter dated April 22, 1968, No. 10-95/14-32]. The essence of the method consists in the conversion of alcohols to alkyl nitrites, which are then subjected to chromatographic separation. In osnovatyelyei is the measurement of differences in thermal conductivity of the pure carrier gas, coming in comparative camera detector thermal conductivity - katharometer, and the mixture of the analyte with the carrier gas emerging from the chromatographic column in the measuring chamber of the detector. For the study applies the chromatograph laboratory PI-4.

The concentration of ethanol is determined by calculating the ratio of the peak heights of amylnitrite and isopropylacetate (propylitic) followed by interpolation from the calibration graph. The calibration graphs are built using standard solutions of ethyl alcohol in blood and urine.

A significant drawback of this method is the absence of the possibility of quantitative determination of ethyl alcohol in the simultaneous presence in the studied material ethyl, propyl, and isopropyl alcohol. In addition, the above method does not allow for simultaneous determination of blood ethanol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol and other metabolites.

The objective of the invention is to develop a method for simultaneous quantitative determination in human blood ethyl alcohol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl Speer is a, isoamyl alcohol and other metabolites by gas chromatography.

The problem is solved in that in the method of determination of content of ethanol and other metabolites in human blood by gas chromatography, including research blood components, while conducting quantitative determination of ethyl alcohol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol and other metabolites in the distillate blood using capillary chromatographic column, calculate the concentration of blood components by the formula:

where and is the result of chromatographic studies, mg/DM3;

V - volume of distillate, cm3;

m is the mass of a sample of whole blood,

Figure 1 shows the chromatogram of distillate whole human blood, in figure 2 - model chromatogram of the mixture.

The essence of the method is to obtain distillates blood by direct steam distillation, which is then subjected to chromatographic separation. Separated through column chromatography components of the mixture in turn come in a flame ionization detector, the signals which are recorded in the form of a series of chromatogra the practical peaks. The basis of the detection is the measurement of differences in electrical conductivity of a flame of pure carrier gas flowing in comparative camera detector, and a mixture of the analyte with the carrier gas coming from the chromatographic column in the measuring chamber of the detector. For research use gas chromatograph company Hewlett Packard (USA), model 5890, series 2, with a flame ionization detector and electronic regulation of the flow of carrier gas, column chromatographic capillary HP-FFAP (USA) 50 m × 0.32 mm × 0,52 μm, the internal standard 1,2-propylene glycol, the carrier gas is nitrogen, the temperature program of the column thermostat: initial temperature 50 ° C, 10 min isotherm, further programming of the temperature at 15 degrees per minute up to 220 degrees Celsius, 10 min isotherm, the temperature of the injector - 150 degrees Celsius, the temperature of the detector 230 degrees Celsius, the division of the flow - 1:40.

Quantitative determination of ethanol and other metabolites is carried out as follows. Weigh from 10 to 50 g of whole blood in a distillation flask with a capacity of 500 cm3then add 50 cm3of distilled water. The mixture is stirred. The flask with the mixture attached to the reflux condenser with a liquid trap and a fridge with a straight tube. With careful Naga is Ivanyi (rapid expansion) the mixture is subjected to direct distillation and collect exactly 25 cm 3of distilled water in a volumetric flask with a capacity of 25 cm3. The receiving flask should be further cooling. 25 cm3the obtained distillate add 2 mm3internal standard. The mixture was thoroughly stirred and subjected to chromatographic study. Capillary chromatographic column allows to reliably separate the components of the distillate blood and simultaneous determination of ethyl alcohol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol and other metabolites.

Chromatograph pre-calibrated by ethyl alcohol and other metabolites, as well as propylene glycol, using at least three of the calibration mixtures. The values of the retention time, the calibration coefficients and the concentration of the internal standard is recorded in the computer memory. The estimated concentration of the internal standard 83,2 mg/DM3.

The study was performed on whole human blood (nine healthy men aged 25 to 39 years).

The results are shown in the table.

Table
The content of endogenous ethanol and acetaldehyde in human blood
№ p/pThe content of ethyl alcohol, 10-3%The content of acetaldehyde, 10-3%
10,110,02
20,190,04
30,250,05
40,160,02
50,950,10
60,200,05
70,320,07
80,250,04
9107,850,17

The obtained data on the content of endogenous ethanol in blood of the person correspond to the literature, according to which normal, without the introduction of outside, in human blood may contain 0.004% alcohol. In addition, the proposed method allows to reliably diagnose the degree of intoxication of living persons (table, outcome). According to literature data, the alcohol concentration of 0.02 to 0.20% of calls depending on individual sensitivity, the condition and type of nervous activity intoxication varying degrees.

Thus, in the course of the research it is established that the proposed method for determination of content of ethanol and other metabolites in human blood allows to obtain accurate and reproducible results.

Method for determination of content of ethanol and other metabolites in human blood by gas chromatography, including the production of distillates blood by direct steam distillation and research blood components, characterized in that conduct quantitative determination of ethyl alcohol, diethyl ether, acetaldehyde, acetone, methyl acetate, ethyl acetate, propyl alcohol, isobutyl alcohol, butyl alcohol, isoamyl alcohol in one study using capillary chromatographic column, the calculation of the concentration of the detected blood components produced according to the formula:

where and is the result of chromatographic studies, mg/DM3;
V - volume of distillate, cm3;
m is the mass of a sample of whole blood,



 

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Cartridge system // 2435163

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4 cl, 5 ex, 6 dwg

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1 tbl, 3 ex

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1 ex, 1 tbl, 2 dwg

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2 tbl, 2 ex

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4 ex, 3 tbl

FIELD: chemistry.

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2 dwg, 2 tbl, 1 ex

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3 ex, 4 tbl

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1 ex

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5 cl, 6 tbl

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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.

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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: 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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