Method of blood dichlorobromomethane measurement

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

 

The invention relates to medical Toxicological research, in particular to sanitary toxicology, and can be used for the quantitative determination dichlorobromomethane in biological fluids such as blood.

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

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

Also there is a method of gas chromatographic determination of halogenated substances in water, in particular, dichloromethane [3]. The measurement of its concentration carried out by gas chromatography using electron-capture detector. Concentration dichlorobromomethane of water carry gas extraction at temperatures up to 80°C in a confined space. Further assembled gas is injected into the chromatograph evaporator, and the number of dicarbamate is to establish on a calibration curve. However, this method is not applicable to the determination of dichloromethane in the blood, because the number of operations required to heat up to the temperature at which happens clotting of blood.

Another known method of gas chromatographic determination halogen-substituted methane in water is the way in which produce vapor extraction present in water volatile components, their concentration on the sorbent, thermal desorption and separation on the column with sorbent, the entrance of which is placed a layer of sulfonic cation exchanger, with subsequent detection of the separated components of the detector of electron capture [4].

However, the mentioned known method, it is impossible to determine halogen-substituted methane, in particular dichloromethane, in the blood, because it has such an operation, such as thermal desorption.

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

The technical result is achieved by the proposed method for quantitative determination dichloromethane in the blood, characterized by the fact that initially carry out alkalization of the blood samples of 10%sodium hydroxide solution to a pH of 8-10, produce the removing of dihl brometane from the sample by extraction of its hexane, separate the extract by centrifugation, to produce a study of gas chromatographic analysis, and the number dichlorobromomethane set via a calibration curve.

This technical result is achieved due to the following.

Introduction in a blood sample of 10%aqueous sodium hydroxide solution due to the fact that this reagent does not interfere with the determination and does not cause error.

Bringing the pH in the blood sample to a pH of 8-10 explained by the fact that only with the high pH provides a high degree of extraction dichloromethane, and simultaneously provides the denaturation of low molecular weight proteins, which increases the accuracy of determination.

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

Thanks to the operation of centrifugation is on the one hand the destruction of proteins that define a complex matrix composition of the sample which may adversely affect the accuracy and sensitivity of the method, and on the other hand - is the separation of the extract. Mode centrifugation recommended from 7000 rpm to 7500 rpm When using modes less than 7000 rpm is incomplete separation of the extract from the assay, when the modes centrifugation over 7500 rpm the centrifugal acceleration occurs, in addition to the besieged who I proteins, for analysis, the deposition of compounds of molecular weight greater than that of the compounds that lead to overly optimistic results. And only when using the specified limits centrifugation guaranteed quality of the analysis (high sensitivity and precision).

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

The proposed method is implemented as follows.

Example. A sample of blood in quantities of 1 cm3containing dichloromethane, alkalinized with 10% sodium hydroxide solution to a pH of 8-10. Next, make the extraction of 1 cm3hexane by shaking for 1-2 minutes Then the mixture is centrifuged at 7500 rpm for 2 minutes After removal of the protein by centrifugation and separation of the extract, the latter is poured into a test tube and chromatographic on the chromatograph "Crystal 2000" with electron capture detector. Conduct quantitative determination dichloromethane in the prepared samples by gas chromatography on a calibration curve.

The calibration graph is constructed as follows. Blood samples taken from a control group of children, volume 1 cm3containing specified concentrations of dicarbamate is on, alkalinized with 10% sodium hydroxide solution to a pH of 8-10 and extracted with a 1 cm3hexane. The obtained extracts - standard solutions - chromatographic on gaschromatographie "Crystal 2000" with electron capture detector.

Optimization of gas chromatographic parameters to determine dichlorobromomethane in blood was carried out using a hardware-software complex based on gas chromatograph "Crystal 2000" with electron capture detector. Weight selection dichlorobromomethane achieved capillary column Optima-5 - 25m*0,32mm*5,0µm at temperature: column - 50°C; evaporator - 200°C, detector 250°C; flow rate of carrier gas 1 (nitrogen) - 20 cm3/min; flow rate of carrier gas was 30 cm3/min. With the same parameters chromatograph is determined and quantitative content dichloromethane in the blood.

In laboratory tests the dependence of the degree of extraction dichloromethane from pH and nature of organic solvents. The results are shown in table 1.

Table 1
The degree of extraction dichlorobromomethane of blood samples from the pH and nature of organic solvents
Defined ingredient Organic solventThe range of pH start extractionThe degree of extraction, %
Dichloromethane in the bloodheptane2-342
4-637,7
8-1039
diethyl ether2-358
4-670
8-1085
hexane2-386
4-689
8-1097

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

For denaturation of low molecular weight proteins and increase the degree of extraction of the sample of blood is alkalinized with 10% sodium hydroxide solution to a pH of 8-10.

Another proof of the above results are presented in t the blitz 2, extraction dichlorobromomethane of blood organic solvents when the selected optimal extraction conditions (pH=8-10).

Table 2
The average values of the degree of extraction dichloromethane from blood samples of organic solvents for n=5 and p=0,95
SolventContent dichlorobromomethane, mcg
IntroducedFoundThe degree of extraction, %
Heptane0,0120,004739
Diethyl ether0,0120,010285
Hexane0,0120,011697
Note: n - number of definitions; p - probability

The result of this experiment, it is confirmed once again that the most complete extraction of dichloromethane from the blood takes place by extraction with hexane in SEL is offered by the environment (pH 8-10).

To determine the sensitivity, then analyzed blood samples from children by the method of supplementation with different content in them dichloromethane. Blood samples containing defined concentrations dichlorobromomethane, alkalinized with 10% sodium hydroxide solution to a pH of 8-10 and extracted with a 1 cm3hexane within 1-2 minutes After separation of the extract and removal of proteins by centrifugation at 7000-7500 rpm for 2 min analyzed extracts chromatographic at least 5 times on the chromatograph "Crystal 2000M with electron capture detector.

Via a calibration curve and determine the content of the component in the samples. The results are shown in table 3.

Table 3
No. blood samplesThe content defined dichlorobromomethane, mg/cm3
10,002
20,004
30,008
40,012
50,016

Thus the detection sensitivity for dichlorobromomethane were 0.002 mg/cm3. Prob is tenderly definition dichlorobromomethane of 0.004 µg in the analyzed sample volume.

During testing it was found that the measurement error for dichlorobromomethane the proposed method is 24,01%. The data are given in table 4.

Table 4
The range of values of precision, repeatability, reproducibility
The name of the component to be determined and the measurement range, µg/cm3The 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, %
Dichlorobromomethane, from 0.002 to 0.01 on.2,919,92

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

Table 5
The limits of repeatability and reproducibility of the determination dichlorobromomethane the proposed method with a confidence level of p=0,95
The name of the component to be determined and the measurement range, µg/cm3The 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),, %
Dichlorobromomethane, from 0.002 to 0.01 on.of 8.062,75

Laboratory results showed that the proposed method indeed characterized by a high sensitivity (from 0.002 ág/cm3), high accuracy (24,01%) and provides high limits of repeatability and reproducibility. This allows to recommend it for use in medical laboratories.

Sources of information

1. Galuskina I.D., Filov VA Pre the treatment and definition of organic poisons in the body. "Medicine", 1971

2. Guidelines on the detection and determination of 1,2-dichloroethane in a biological material by gas chromatography. M: the Ministry of health of the USSR, 1978

3. Guidelines for gas chromatographic determination of halogenated compounds in water. MUK 4.1.646-96. Approved by the state Committee of sanitary and Russia 31.10.1996,

The method of quantitative determination of dichloromethane in the blood, characterized by the fact that initially carry out alkalization of the blood samples of 10%sodium hydroxide solution to a pH of 8-10, produce removing dichloromethane from the sample by extraction of its hexane, separating the extract by centrifugation at 7000-7500 rpm and produce his study of gas chromatographic analysis on a gas chromatograph with electron capture detector, and the number dichlorobromomethane set via a calibration curve.



 

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