RU2416798C1 - Method of blood dichlorobromomethane measurement - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method starts with blood sample alkalisation with 10% sodium hydroxide to pH 8-10; dichlorobromomethane is recovered from the sample by hexane extraction; the extract is separated centrifugally at 7000-7500 rpm and analysed with gas chromatography in a partition gas chromatograph with an electron capture detector, while dichlorobromomethane is measured by a calibration diagram.

EFFECT: high sensitivity and accuracy of the method for blood dichlorobromomethane measurement.

1 ex, 5 tbl

Description

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

Known microdiffusion method suitable for determining chlorinated hydrocarbons in various biosubstrates - blood, urine in the range of 5-35 mg%. Quantitative determination is carried out spectrophotometrically by color intensity. The error of determination is ± 5% [1].

For the concentration and extraction of chlorinated hydrocarbons from biological fluids, the method of gas chromatographic analysis of the equilibrium vapor phase of 1,2-dichloroethane is used [2]. The disadvantage of this method is its low sensitivity: the detection limit of 10 μg. The error of determination is 5-10%.

Also known is a method of gas chromatographic determination of halogen-containing substances in water, in particular dichlorobromomethane [3]. Measurement of its concentration is carried out by gas chromatography using an electron-capture detector. The concentration of dichlorobromomethane from water is carried out by gas extraction while heating to a temperature of 80 ° C in a closed volume. Subsequently, the collected gas is introduced into the evaporator of the chromatograph, and the amount of dichlorobromomethane is set according to the calibration schedule. However, this method is not applicable for the determination of dichlorobromomethane in the blood, because in some operations, heating to a temperature at which blood coagulation will occur is required.

Another well-known method for gas chromatographic determination of halogen-substituted methane in water is a method according to which vapor-phase extraction of volatile components present in water is carried out, their concentration on a sorbent, thermal desorption and separation on a column with a sorbent at the entrance of which a layer of sulfocationionite is placed, followed by detection of separated components electron capture detector [4].

However, it is impossible to determine the halogen-substituted methane, in particular dichlorobromomethane, in the blood in the known manner it contains an operation such as thermal desorption.

The technical result achieved by the invention is to provide both high sensitivity and accuracy of the method for determining dichlorobromomethane in the blood.

The technical result is achieved by the proposed method for the quantitative determination of dichlorobromomethane in the blood, characterized in that at first they alkalize the blood sample with a 10% sodium hydroxide solution to pH 8-10, dichlorobromomethane is extracted from the sample by extraction with hexane, the extract is separated by centrifugation, it is produced research by gas chromatographic analysis, and the amount of dichlorobromomethane is set according to a calibration schedule.

The specified technical result is achieved due to the following.

The introduction of a 10% sodium hydroxide solution into the blood sample is due to the fact that this reagent does not interfere with the determination and does not cause errors.

Bringing the hydrogen index in the blood sample to pH 8-10 is explained by the fact that only at this pH a high degree of dichlorobromomethane extraction is provided, and at the same time denaturation of low molecular weight proteins is provided, which increases the accuracy of determination.

The use of hexane as an extracting solvent provides high sensitivity of the proposed method.

Thanks to the centrifugation operation, on the one hand, the proteins are removed, which determine the complex composition of the sample matrix, adversely affecting the accuracy and sensitivity of the method, and, on the other hand, the extract is separated. The centrifugation mode is recommended from 7000 rpm to 7500 rpm. When using modes of less than 7000 rpm, the extract is incompletely separated from the bioassay, while centrifugation modes of more than 7500 rpm under centrifugal acceleration occur, in addition to the precipitation of proteins necessary for analysis, precipitation of compounds whose molecular weight is greater than that of the test compound leading to overpriced results. And only when using the indicated centrifugation limits, a qualitative analysis result (high sensitivity and accuracy) is guaranteed.

The combination and sequence of these operations, their modes and allowed to achieve a high degree of accuracy and sensitivity of the proposed method.

The proposed method is implemented as follows.

Example. A blood sample in an amount of 1 cm ³ containing dichlorobromomethane is made alkaline with a 10% sodium hydroxide solution to pH 8-10. Next, 1 cm ^{3 of} hexane is extracted by shaking for 1-2 minutes. The mixture is then centrifuged at 7500 rpm for 2 minutes. After removing the proteins by centrifugation and separating the extract, the latter is poured into a test tube and chromatographed on a Crystal 2000 chromatograph with an electron capture detector. Quantitative determination of dichlorobromomethane in the prepared samples by gas chromatographic method according to the calibration schedule.

The calibration graph is constructed as follows. Blood samples taken from a control group of children with a volume of 1 cm ³ containing the desired concentration of dichlorobromomethane are made alkaline with a 10% sodium hydroxide solution to pH 8-10 and extracted with 1 cm ^{3 of} hexane. The obtained extracts — standard solutions — are chromatographed on a Crystal 2000M gas chromatograph with an electron capture detector.

The development of optimal gas chromatographic parameters for the determination of dichlorobromomethane in the blood was carried out using a hardware-software complex based on a Crystal 2000 gas chromatograph with an electron capture detector. The completeness of dichlorobromomethane separation was achieved on an Optima-5 capillary column — 25m * 0.32mm * 5.0µm at temperature conditions: column — from 50 ° C; evaporator - 200 ° C; detector - 250 ° C; carrier gas flow rate 1 (nitrogen) - 20 cm ³ / min; carrier gas consumption 30 cm ³ / min. With the same parameters of the chromatograph, the quantitative content of dichlorobromomethane in the blood is also determined.

During laboratory tests, the dependence of the degree of extraction of dichlorobromomethane on the pH of the medium and the nature of organic solvents was studied. The results are shown in table 1.

Table 1 The dependence of the degree of extraction of dichlorobromomethane from a blood sample on the pH of the medium and the nature of organic solvents Detectable ingredient Organic solvent PH range of the start of extraction The degree of extraction,% Dichlorobromomethane in the blood heptane 2-3 42 4-6 37.7 8-10 39 diethyl ether 2-3 58 4-6 70 8-10 85 hexane 2-3 86 4-6 89 8-10 97

The data in table 1 show that the most effective extractant for the extraction of dichlorobromomethane is hexane at pH 8-10.

To denature low molecular weight proteins and increase the degree of extraction, the analyzed blood samples are made alkaline with a 10% sodium hydroxide solution to pH 8-10.

Another proof of the above is the results presented in table 2, on the extraction of dichlorobromomethane from the blood with organic solvents under the chosen optimal extraction conditions (pH = 8-10).

table 2 The average values of the degree of extraction of dichlorobromomethane from a blood sample with organic solvents at n = 5 and p = 0.95 Solvent The content of dichlorobromomethane, mcg Introduced Found The degree of extraction,% Heptane 0.012 0.0047 39 Diethyl ether 0.012 0.0102 85 Hexane 0.012 0.0116 97 Note: n is the number of definitions; p is the probability

As a result of the experiment, it was once again confirmed that the most complete extraction of dichlorobromomethane from the blood occurs upon extraction with hexane in an alkaline medium (pH 8-10).

To determine the sensitivity, we further analyzed blood samples in children by the method of additives with different dichlorobromomethane contents in them. Blood samples containing the specified concentration of dichlorobromomethane are made alkaline with a 10% sodium hydroxide solution to pH 8-10 and extracted with 1 cm ^{3 of} hexane for 1-2 minutes. After extract separation and protein removal by centrifugation at 7000-7500 rpm for 2 min, the analyzed extracts are chromatographed at least 5 times on a Crystal 2000M chromatograph with an electron capture detector.

The calibration graph determines the content of the component in the samples. The results are shown in table 3.

Table 3 Blood sample no. The content of the determined dichlorobromomethane, μg / cm ³ one 0.002 2 0.004 3 0.008 four 0.012 5 0.016

The sensitivity of the determination for dichlorobromomethane was 0.002 μg / cm ³ . It is possible to determine dichlorobromomethane 0.004 μg in the analyzed sample volume.

Also during the tests it was found that the determination error for dichlorobromomethane of the proposed method is 24.01%. The data are shown in table 4.

Table 4 Range of measurements of indicators of accuracy, repeatability, reproducibility The name of the determined component and the measurement range, μg / cm ³ Repeatability index (relative standard deviation of repeatability), σ _r ,% Reproducibility index (relative standard deviation of reproducibility) σ _R ,% Accuracy indicator (limits of relative error with probability Р = 0.95), ± δ,% Dichlorobromomethane, from 0.002 to 0.01 incl. 2.91 9.92

Also, in the course of laboratory tests, the limits of repeatability and reproducibility of the determination of dichlorobromomethane by the proposed method were determined, which characterize the accuracy of the measurement results. Data on these indicators are shown in table 5.

Table 5 The values of the limits of repeatability and reproducibility of the determination of dichlorobromomethane by the proposed method with a confidence probability of p = 0.95 The name of the determined component and the measurement range, μg / cm ³ Repeatability limit (relative value of the permissible discrepancy between two results of parallel determinations), r _n ,%

, %The limit of internal laboratory reproducibility (the relative value of the permissible discrepancy between the two measurement results obtained in the same laboratory, but under different conditions),

% Dichlorobromomethane, from 0.002 to 0.01 incl. 8.06 2.75

The results of laboratory studies showed that the claimed method is really characterized by high sensitivity (from 0.002 μg / cm ³ ), high accuracy of determination (24.01%) and provides high limits of repeatability and reproducibility. This allows you to recommend it for use in specialized medical laboratories.

Information sources

1. Gadaskina I.D., Filov V.A. The conversion and determination of organic poisons in the body. "Medicine", 1971

2. Guidelines for the detection and determination of 1,2-dichloroethane in biological material by gas-liquid chromatography. M .: Ministry of Health of the USSR, 1978

3. Guidelines for gas chromatographic determination of halogen-containing substances in water. MUK 4.1.646-96. Approved by the State Committee for Sanitary and Epidemiological Supervision of Russia on October 31, 1996

Claims (1)

A method for the quantitative determination of dichlorobromomethane in the blood, characterized in that the blood sample is first alkalized with a 10% sodium hydroxide solution to pH 8-10, dichlorobromomethane is extracted from the sample by extraction with hexane, the extract is separated by centrifugation at 7000-7500 rpm, its study is carried out by gas chromatographic analysis on a gas chromatograph with an electron capture detector, and the amount of dichlorobromomethane is set according to a calibration graph.