Method for preparing blood sample for gas chromatography of organochlorine pesticides

G01N1/10 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

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

 

The present invention relates to medicine, namely to human ecology, and can be used for early diagnosis of toxic effects of organochlorine pesticides low intensity and control during detoxification treatment of patients with pathology of the digestive system, complicated by the influence of these substances.

Organochlorine pesticides (α-, β-, γ-HCH) are the most dangerous to human health chemical factors of anthropogenic origin, doubtless representing medical-ecological problem with high risk of receipt of these substances in the human body, biocumulative, development caused diseases and impact on the overall morbidity of agricultural regions. In the absence of a specific mechanism of action of organochlorine pesticides on the development and course of diseases of particular diagnostic value gain exposure tests in biological environments. However, most potential use of blood as a biological object to assess the level of biological carriage pesticides have not found sufficient support in methodological terms.

Known gas chromatographic method for the determination of micro-amounts of pesticides of different chemical structure (organochlorine, organophosphorus pesticides, is Arbatov and others) in biological fluids [1].

The disadvantage of this method is the multiple steps involved, the expenditure of a significant amount of time for sample preparation for gas chromatographic determination of pesticides and lack of selectivity.

The described method of quantitative determination of organochlorine pesticides in serum [2] includes the extraction of a mixture of n-hexane and acetone (9:1), purification of the extract on a column of aluminum oxide and analysis by gas-liquid chromatograph. The method is time-consuming, possible loss at the stage of purification of the extract to the column.

The main disadvantage of this method is the fact that they use a serum rather than whole blood, because a significant number of pesticides adsorbed on erythrocytes.

The closest analogue of the adopted method, which is based on the principles of determination of halogenated alicyclic hydrocarbons in biological environments [3], namely gas chromatography determination of organochlorine pesticides in blood by triple extraction portions 5 cm3n-hexane for 30 minutes, twice the processing of the extract concentrated sulfuric acid at 5 cm3within 10 min and subsequent analytical determination of gas-liquid chromatograph.

The disadvantage of this method is the small volume of sample for determination of microquantities of the research is the most pesticides incomplete extraction with n-hexane-related lipids and proteins pesticides, as well as the duration of the analysis due to multiple stages of extraction and purification of the extract with sulfuric acid.

The technical problem to which the alleged invention is directed, is to develop a more reliable and rapid method of sample preparation for gas chromatographic determination of organochlorine pesticides in blood.

The essential novelty of the invention lies in the fact that the study take 5-7 cm blood and before extraction of the sample treated with 2 cm360% sulfuric acid, the extraction was carried out 30 cm3n-hexane once and before gas chromatography study of n-hexane extract once treated with 10 cm3concentrated sulphuric acid.

The technical result of the use of this method is the increased reliability of detection results of the content of organochlorine pesticides in blood and reduce the duration of the analysis due to the fact that the study take 5-7 cm3blood and before extraction of the sample treated with 2 cm360% sulfuric acid, the extraction was carried out 30 cm3n-hexane once and before gas chromatography study of n-hexane extract once treated with 10 cm of concentrated the agreement acids.

The method is as follows: 5-7 cm3the blood is placed in bottles with a wide neck and a sealing plug, there is added 2 cm360% sulfuric acid, triturated for 10 minutes followed by extraction of pesticides by processing 30 cm3n-hexane once in a closed bottle under constant shaking apparatus for 30 min and then n-hexane extract was separated and carried out once clean coextraction substances 10 cm3concentrated sulfuric acid for 30 min with shaking. Purified n-hexane layer is separated from the acid and conduct concentration in the evaporator to 1.5 cm3. In the chromatograph injected 5 μl. Mode chromatography was carried out described in the next version.

The test method was carried out in 14 patients with chronic diseases of the stomach and duodenum (gastritis, gastroduodenitis), with the process of professional contact with organochlorine pesticides (6 men, 8 women aged from 35 to 47 years).

In parallel in a single blood sample were used to define the α - and γ-HCH proposed method and the method described in analog. The results are shown in the table.

/tr>
Table
Levels of pesticides in the blood when determining the proposed method (I) and the method described in the nearest analogue of (II)
Name of pesticideConcentration in blood, mg/DM3
III
α-HCH0,0098±0,00110,0052±0,0013p≤0,05
γ-HCH0,0199±0,00150,0128±0,0014p≤0,05

Found that when using the proposed method of preparing blood samples for gas chromatographic determination detectability content of α-HCH 1.75 times, and γ-HCH in 1.55 times higher compared to analogue. The time of sample preparation to the stage of concentration of n-hexane extract in the evaporator when using the proposed method was 70 minutes, and way in the analog - 140 min, i.e. reduced in 2 times.

Example. Patient N., age 42 years, a warehouse worker agrochemicals. Received inpatient treatment in RZQHG diagnosed with chronic gastroduodenitis, chronic hepatitis. History of contact with chlorine the content of inorganic fillers pesticides for 8 years. Assigned the determination of α - and γ-HCH in blood. What made the sampling of venous blood in a volume of 5 cm3put the sample in a bottle with a wide neck and a sealing plug, there was added 2 cm360% sulfuric acid, triturated with a glass rod for 10 minutes and then spent the extraction of pesticides from the sample by processing 30 cm3n-hexane in a closed bottle under constant shaking apparatus for 30 min, then n-hexane extract was separated and held once clearance from coextracted substances 10 cm3concentrated sulfuric acid for 30 minutes with shaking. Purified n-hexane layer was separated from the acid and spent the concentration of extract in a rotary evaporator to 1.5 cm3. In the chromatograph introduced using microspace 5 ál. Mode chromatography was carried out corresponded described in analog. The calculated concentration of the studied pesticides in the blood of the examined patient accounted for α-HCH 0,0089 mg/DM3for γ-HCH 0,0135 mg/DM3. Preparation of blood samples to the stage of concentration of n-hexane extract was 70 minutes

On the basis of the results of the content of organochlorine pesticides in the blood of the patient H. appointed detoxification treatment on the basis of pectin (citrus pectin and Apple pectin - "Medetai the t").

The use of the proposed method appears to be promising in terms of timely diagnosis of chronic intoxication organochlorine pesticides, pathogenetic substantiation, treatment and rehabilitation measures for diseases of the digestive system, complicated by the influence of these factors.

Sources of information

1. Alexandrov L.G. Determination of micro-quantities of pesticides of different chemical structure in biological fluids of humans. / Occupational health. - Kiev, 1989. - VIP. - S-131.

2. Minelli E.V. Quantitive method for the determination of organochlorine pesticides in serum / Anal. Toxicol. - 1996. No. 1. - P.23-26.

3. Lysenko M.A. Methods for the determination of micro-quantities of pesticides. - M.: Kolos. - 1977. - P.15, 19.

The method of sample preparation for gas chromatographic determination of organochlorine pesticides in blood, including extraction of pesticides from blood n-hexane, purified from coextruding substances concentrated sulfuric acid, the concentration of n-hexane extract, characterized in that the study take 5-7 cm3blood and before extraction of the sample treated with 2 cm360% sulfuric acid, the extraction was carried out 30 cm3n-hexane once and before gas chromatography study of n-hexane extract once treated with 10 cm3concentrated sulfuric acid.


 

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