Method of preparing sample for gas-chromatographic identification of pesticides in biomaterial

FIELD: chemistry.

SUBSTANCE: method includes selection, and crushing of biomaterial, two-stage extraction of pesticides with n-hexane, purification of biomaterial from coextractive substances with concentrated sulphuric acid, formation of concentrate of n-hexane extract of pesticides, drying, sample formation by dissolution in 0.5-1 ml of n-hexane and carrying out gaschromatographic identification.

EFFECT: invention is characterised by higher effectiveness and accuracy of research and can be used in biology, ecology, medicine for gaschromatographic identification of organochlorine pesticides, namely α-HCCH, β-HCCH, γ-HCCH, DDT, DDD, DDE, in various biomaterials, such as lipids of internal organs and tissues, blood, milk, bird feathers.

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The invention relates to methods for sample preparation and can be used in biology, ecology, medicine for gas chromatographic determination of organochlorine pesticides, namely α-HCH, β-HCH, γ-HCH, DDT, DDD, DDE in various biomaterial containing lipids, internal organs and tissues, blood, milk. In addition, as a biomaterial, you can use the feathers of birds.

The known 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 (see RF patent №2414711, IPC G01N 33/50, G01N 33/48, publication date 20.03.2011).

The lack of technical solutions is a limited scope due to the inability of studies of internal organs and tissues.

As the closest analogue of the adopted method of sample preparation for gas chromatographic determination of organochlorine pesticides in the biomaterial, including screening, grinding and subsequent extraction of pesticides n-hexane, after which the resulting material is purified from coextruding substances concentrated sulfuric acid, to form a concentrate of n-hexane extract of pesticides (see Klimenko M. A. Methods for the determination of microco is icest pesticides. - M.: Kolos. - 1977. - S. 15, 19).

The disadvantage of the closest analogue is the low efficiency and accuracy of the study because of incomplete extraction with n-hexane-related lipids pesticides, as well as the duration of the analysis due to multiple stages of extraction and purification of the extract with sulfuric acid.

The problem to which the invention is directed, is to develop an efficient method of sample preparation for gas chromatographic determination of pesticides in various biomaterial.

Technical result achieved when solving a task, expressed in increasing the efficiency and accuracy of research by conducting a more complete extraction with n-hexane-related lipids of pesticides and reduce the number of stages of extraction and purification from coextruding substances concentrated sulfuric acid.

The problem is solved in that in the method of sample preparation for gas chromatographic determination of pesticides in the biomaterial, including the selection, grinding and subsequent extraction of pesticides n-hexane, after which the resulting material is purified from coextruding substances concentrated sulfuric acid, to form a concentrate of n-hexane extract of pesticides, use crushed the biomaterial in the representative volume containing lipids to the th is subjected to at least twice the extraction of lipids by treatment of biomaterial n-hexane with the combined filtrates, received at various stages of extraction, and for the first time extracted at least 20 minutes, then for at least 10 minutes, then combined filtrate is evaporated at a temperature 69-70°, the resulting fat is weighed and dissolved in n-hexane, after which the named material is poured concentrated sulfuric acid to the destruction of coextruding substances and select hexanamide layer, then the material hexenstieg layer selected and washed from concentrated sulfuric acid to achieve pH=6, then evaporated n-hexane at a temperature lower temperature of its boiling point, and then form the sample, dissolving the resulting material in a fixed volume of n-hexane, preferably 0.5 to 1 ml.

In addition, as a biomaterial use internal organs, mainly the liver, weighing not less than 10 g, and/or tissue, for example muscle mass not less than 10 g, and/or blood, milk, not less than 20 ml in Addition, as a biomaterial, you can use the feathers of birds weighing not less than 5,

Comparative analysis of the essential features of the proposed solution with essential features analogues demonstrates its compliance with the criterion of "novelty".

This distinctive features in the claims solves the following functional tasks.

The sign "use crushed b is material in the representative volume, containing lipids provides research and allows us to expand the scope of application due to the use as a biomaterial is not only blood, but also of organs, tissues. In addition, as a biomaterial, you can use the feathers of birds.

Topic "... biomaterial is subjected to at least twice the extraction of lipids by treatment of biomaterial n-hexane with the combined filtrates obtained at different stages of extraction, and for the first time extracted at least 20 minutes, and subsequently at least 10 minutes" provides the possibility of separating the lipids from the crushed biomaterial.

The sign of "the combined filtrate is evaporated at a temperature 69-70°" allows you to remove excess n-hexane while maintaining lipids.

The characteristic of the resulting fat is weighed and dissolved in n-hexane produces a portion of fat known weight lipids for subsequent discharge from her pesticides by gas chromatography.

The characteristic material pour concentrated sulfuric acid to the destruction of coextruding substances and select hexanamide layer allows for efficient removal of lipids saving in material of pesticides.

The characteristic material hexenstieg layer selected and washed from concentrated sulfuric acid to achieve pH=6" allows jet to remove the remnants of concentrated sulfuric acid at achieving an optimal level of acidity and provide a more reliable and durable equipment, namely, the chromatographic column.

The characteristic is evaporated n-hexane at a temperature lower temperature of its boiling point" allows you to remove n-hexane while maintaining the material containing pesticides.

The characteristic shape of the sample, dissolving the resulting material in a fixed volume of n-hexane, preferably 0.5 to 1 ml makes it easy to control the amount of sample for analysis.

In Fig.1 shows a chromatogram of a mixture of standard solutions of pesticides.

The method is as follows.

Pre-produce a selection of biomaterial - internal organs, mainly the liver, weighing not less than 10 g, and/or tissue, for example muscle mass not less than 10 g, and/or blood, milk, not less than 20 ml in Addition, as a biomaterial, you can use the feathers of birds weighing not less than 5,

The selected biomaterial pulverized by grinding in a porcelain mortar with anhydrous sodium sulfate (Na2SO4betw.) to remove moisture. Then prepare the first portion, for which the crushed material is transferred into a conical flask, pour 40-50 ml of n-hexane and extracted with not less than 20 minutes After that, the liquid part is filtered in a pear-shaped flask, previously dried and weighed on an analytical balance to the fourth digit. Next, prepare the second portion, for that crushed the biomaterial from the nutrient conical flask pour 20 ml of n-hexane and extracted for 10 min, then the liquid portion connected to the first portion of the filtrate in pear-shaped flask.

Then the combined filtrate is evaporated on a water bath with temperature 69-70°C (the boiling point of n-hexane - 68,742°C), select a portion of the resulting fat and determine its weight.

After weighing the fat pear-shaped flask was dissolved in n-hexane (25 ml), then the resulting material is poured concentrated sulfuric acid (5-7 ml), gently shaken and left for a few hours (2-4 hours). After the destruction of coextruding substances, when the mixture is stratified, select hexanamide layer glass syringe into a separating funnel with a volume of 250 ml.

Next hexanamide layer in a separating funnel cleaned with concentrated sulfuric acid to obtain a colorless layer of sulfuric acid. After that hexanamide layer washed with distilled water until neutral pH=6 by universal paper indicator. Then washed hexanamide layer is dried by filtering through sodium sulfate in sharp-bottom flask with one neck with cut (volume of 25-100 ml). Forth from sharp-bottom flask is evaporated n-hexane at a temperature lower temperature of its boiling point, for example on a rotary evaporator, or leave for a few days until complete evaporation of n-hexane. Then form the sample, dissolving the resulting Mat is real in a fixed volume of n-hexane, preferably 0.5 to 1 ml, and spend gas chromatographic determination of the content of organochlorine pesticides on the equipment of known construction in the standard modes. If the peaks on the chromatogram are not too large, the material of the sample add a controlled amount of n-hexane.

1. The method of sample preparation for gas chromatographic determination of pesticides in the biomaterial, including the selection, grinding and subsequent extraction of pesticides n-hexane, after which the resulting material is purified from coextruding substances concentrated sulfuric acid, to form a concentrate of n-hexane extract of pesticides, characterized in that use crushed the biomaterial in the representative volume containing lipids, which expose at least a twofold extraction of lipids with n-hexane filtered extracts each stage with the consolidation of the obtained filtrate, and the duration of the first extraction 20 minutes, and the next 10 minutes, then combined filtrate is evaporated at a temperature 69-70°, the resulting residue is weighed and dissolved in n-hexane, after which the solution is poured concentrated sulfuric acid to the destruction of coextruding substances and selected material from hexenstieg layer, which is washed from concentrated sulfuric acid up to the achievements pH=6, then evaporated n-hexane at a temperature lower temperature of its boiling point, and then form the sample, dissolving the material obtained in 0.5-1 ml n-hexane.

2. The method of sample preparation for gas chromatographic determination of pesticides in biomaterial under item 1, characterized in that as a biomaterial using samples taken from the internal organs, mainly the liver, weighing not less than 10 g, and/or tissues, such as muscles, weighing not less than 10 g, and/or blood or milk, not less than 20 ml, and/or sample weighing not less than 5 g is formed from the feathers of birds.



 

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