RU2741368C1 - Method of sample preparation for gas chromatographic determination of organochlorine compounds in biomaterial - Google Patents

Abstract

FIELD: biology; ecology; medicine.

SUBSTANCE: method of preparing a sample for gas chromatographic determination of organochlorine compounds in a biomaterial involves sampling a biomaterial in form of biological fluids or skin appendages, from which milled homogenized samples are prepared, moistened with bidistilled water to moisture content of not less than 80% and twice cryo-denatured, sample is sampled in an amount sufficient to obtain not more than 0.5 g of dry lipophilic extract, wherein sample weight of sample taken in 5 parts by weight is added with 3 cm³ of 1% sodium bicarbonate solution, and after intense shaking, extractant is added, in form of a mixture of trichloromethane: methyl alcohol: hexane, wherein in 5 parts of sample weighed portion sample is taken 6 parts of trichloromethane in cm³, 3 parts of methyl alcohol in cm³ and 5 parts of n-hexane in cm³, vigorously shaking for 5 minutes and extraction for 25 minutes, lipophilic extract of organochloride compound is settled until separation of phases, after which the water-alcohol layer is removed, and the extract is filtered through anhydrous sodium sulphate, evaporated and then purified with concentrated sulfuric acid.

EFFECT: invention provides an increase in the "output" of organochloride compounds into the concentrate of the n-hexane extract due to higher completeness of its extraction from the material of the samples and elimination of multi-stage purification by concentrated sulfuric acid.

1 cl, 1 tbl, 1 dwg

Description

The invention relates to methods for the quantitative determination of persistent organochlorine compounds - organochlorine pesticides (α-, β-, γ-, δ-hexachlorocyclohexane, 2,4- and 4,4-dichlorodiphenyltrichloroethane, 2,4- and 4,4-dichlorodiphenyldichloroethane, 2, 4- and 4,4-dichlorodiphenylethylene, dieldrin, endrin) and polychlorinated biphenyls - by gas chromatographic methods in biological fluids and human skin appendages and can be used in biology, ecology, medicine.

A known method of sample preparation for gas chromatographic determination of organochlorine pesticides in blood, including the extraction of pesticides from the blood with n-hexane, purification of coextractive substances with concentrated sulfuric acid, concentration of n-hexane extract (see RU No. 2414711, IPC G01N 33/50, G01N 33 / 48, 2011).

The disadvantage of this solution is the limited scope of application due to the impossibility of studying the entire spectrum of human biological fluids, as well as the complete impossibility of working with skin appendages - hair or nail plate.

Known guidelines for the selective gas chromatographic determination of organochlorine pesticides in biological media (urine, blood, adipose tissue and breast milk), differing in the processing of samples with a solution of potassium oxalate and then ethanol, diethyl ether and hexane (MU No. 3151 dated November 27, 1984 and No. 4362 06/08/87).

The disadvantage of this solution is the relatively incomplete yield of organochlorine compounds (OCs) in the n-hexane extract, as well as the impossibility of working with the hair or nail plate.

There is also known a method of sample preparation for gas chromatographic determination of organochlorine compounds in biomaterial, including sampling, grinding and subsequent extraction of organochlorine compounds with n-hexane, and subsequent purification from co-extractive substances with concentrated sulfuric acid and obtaining a concentrate of n-hexane extract of organochlorine compounds (see patent RF No. 2543360, IPC G01N 33/483, 2015).

The disadvantage of this method is the low efficiency and accuracy of the study due to the multiple stages of extraction and purification of the extract with sulfuric acid and, in addition, the relatively low "yield" of organochlorine compounds in the concentrate of n-hexane extract.

The problem to be solved by the claimed invention is to increase the "yield" of organochlorine compounds in the concentrate of n-hexane extract.

The technical result, which manifests itself in solving the problem, is expressed in an increase in the "yield" of organochlorine compounds in the concentrate of n-hexane extract by increasing the completeness of its extraction from the sample material and eliminating the multistage purification process with concentrated sulfuric acid.

To solve the problem, a method for preparing a sample for gas chromatographic determination of organochlorine compounds in biomaterial is proposed, including sampling, grinding it and subsequent extraction of organochlorine compounds with n-hexane, and subsequent purification from co-extractive substances with concentrated sulfuric acid and obtaining a concentrate of n-hexane extract of organochlorine compounds, characterized in that biological fluids or skin appendages are used as a biomaterial, from which crushed homogenized samples are obtained, moistened with bidistilled water to a moisture content of at least 80% and cryodenatured twice, a sample is taken in an amount sufficient to obtain not more than 0.5 g of dry lipophilic extract, while in the weighed sample of the sample, which is taken as 5 parts by weight, add 3 cm ^{3 of a} 1% sodium bicarbonate solution, and after vigorous shaking, add the extractant, which is used as a mixture of tr ichloromethane: methyl alcohol: hexane, and 6 parts of trichloromethane in cm ³ , 3 parts of methyl alcohol in cm ³ and 5 parts of n-hexane in cm ³ are taken for 5 parts of the weighed portion of the sample in g, shaking vigorously for 5 minutes and extraction is carried out for 25 min, the lipophilic extract of the organochlorine compound is defended until the phases are separated, after which the aqueous-alcoholic layer is removed, and the extract is filtered through anhydrous sodium sulfate, evaporated and then purified with concentrated sulfuric acid.

Comparative analysis of the features of the claimed solution with the features of the prototype and analogs indicates the compliance of the declared solution with the "novelty" criterion.

The set of features of the claims provides the possibility of increasing the "yield" of organochlorine compounds in the concentrate of n-hexane extract by increasing the completeness of its extraction from the sample material and eliminating the multistage process of purification with concentrated sulfuric acid from coextractive substances during the reaction of the lipophilic extract with it. For example, when using a sample of breast milk, as a result of the extraction of which less than 0.5 g of lipophilic extract is formed, the "yield" of all declared COS may not be complete, and the compounds that are in minimal concentrations may "not come out" at all. At the same time, if we take a sample, as a result of the extraction of which more than 0.5 g of lipophilic extract is formed, then several stages of washing with sulfuric acid will be required, which leads to a “loss” of concentrations of the desired compounds and an increase in the time of the general analysis.

In this case, the features of the characterizing part of the claims solve the following functional problems.

The sign indicating that "biological fluids or skin appendages are used as a biomaterial" expands the scope of research of the entire spectrum of human biological fluids, as well as the ability to work with skin appendages - hair or nail plate.

The signs "... use crushed homogenized samples, moistened with bidistilled water to a moisture content of at least 80% ..." indicate the sample preparation parameters required for a more efficient cryodenaturation process.

Signs indicating that homogenized samples "double cryodenatured" provide a significant increase in the "yield" of extracted substances due to the processes of repeated low-temperature denaturation of protein-lipid complexes of the sample.

Signs indicating that a sample is taken "in an amount sufficient to obtain no more than 0.5 g of dry lipophilic extract" provide the maximum yield of the analyte from the sample.

A sign indicating that "3 cm ^{3 of a} 1% sodium bicarbonate solution is added to the weighed portion of the sample, which is taken as 5 parts by weight," provides a high degree of caseinogen denaturation, binding of calcium ions, and improved sample wettability.

Signs indicating that "after vigorous shaking, an extractant is added, which is a mixture of trichloromethane: methyl alcohol: hexane" are necessary for effective destruction of lipoprotein complexes of the sample and more complete extraction of organochlorine compounds.

Signs indicating that "6 parts of trichloromethane in cm ³ , 3 parts of methyl alcohol in cm ³ and 5 parts of n-hexane in cm ³ are taken for 5 parts of the sample in g" are necessary to clarify the best ratio of reagents that effectively destroy lipoglycoprotein complexes samples and, as a result, increasing the "yield" of the investigated organochlorine substances.

The sign indicating that "vigorously shake for 5 minutes and carry out the extraction for 25 minutes" provides acceleration of work due to an increase in the area of the interface between the phases of trichloromethane-hexane and water-alcohol layers.

Signs indicating that "the lipophilic extract of the organochlorine compound is defended before phase separation, after which the aqueous-alcoholic layer is removed" describe the technological process of separating the lipophilic extract of organochlorine compounds from the aqueous-alcoholic layer.

Signs indicating that "the extract is filtered through anhydrous sodium sulfate" describes the process of drying and removal of solid fragments of coagulated protein from the trichloromethane-hexane extract.

Signs indicating that the extract is “evaporated and then purified with concentrated sulfuric acid” describe a sequence of steps that significantly speeds up the process.

FIG. shows the distribution of lipids in biological fluids and such appendages of human skin as hair and nail plates to determine the volume of the sample depending on the type of material.

The sample volume is determined based on the concentration of the analytes in the sample, the sensitivity of the analytical method and the reproducibility of the results when the quantitative analysis is repeated.

The detection limit was determined on the basis of three standard deviations per eight to ten samples analyzed on COS. For the analysis of trace concentrations of COS, samples from 1 to 10 g are usually used. However, for a more efficient determination of COS with a high octanol-water coefficient, it is necessary to take into account the different lipid content in biological fluids and human skin appendages, as well as the degree of their chemical participation in phospholipid complexes. membranes or keratin matrix of skin appendages, since this approach allows unification of further steps, including extraction.

It was found that the most effective purification of the studied compounds from co-extractive substances with concentrated sulfuric acid occurs during the reaction of a lipophilic extract dissolved in 20 cm ^{3 of} n-hexane and concentrated sulfuric acid with a volume of 10 cm ³ with stirring on a laboratory shaker.

The largest mass of the lipophilic extract that can be purified with the indicated volume of sulfuric acid is 0.5 g. FIG. shows the distribution of lipids in biological fluids and in such appendages of human skin as hair or nail plate.

Table 1 shows the maximum sample volume that can be optimally cleaned with a single acid extraction.

Table 1

Distribution of lipids in human organs and tissues

Biomaterial % lipids Maximum sample weight, g Nail plate ten five Hair 4 12.5 Breast milk 4 12.5 Saliva 0.5 100 Urine 0.5 100 Sperm 0.3 167 Exudate 0.5-4 12.5-100 Transudate 0.5 100 Sweat departments 4 12.5 Bile one 50 Cerebrospinal fluid 0.01 5000 Lymph 0.5-2 25-100 Menstrual blood 0.05 1000

To ensure repeatability of studies, the following sampling rules are mandatory.

For urine, it is recommended to use a daily urine sample, pre-stripped to a volume of 100 ml.

At a low lipid concentration in the sample, the samples are pre-concentrated by evaporation and then transferred to the extraction process chain.

Hair: samples are collected from the occipital region of the head; the hair should be cut as close to the root as possible.

The nail plate: the part of the nail plate, at least 3 mm long, protruding above the pad of the I, II or III finger of the left hand, is cut off repeating the anatomical outlines of the finger pad.

For saliva, it is recommended to collect a sample in a calm state no earlier than 3 hours after eating or drinking.

For bile, it is recommended to study the portion due to the highest stable concentration of the lipid complex - 7.69 g / liter.

A disposable instrument is used to collect samples to avoid contamination with analytes.

An example of the implementation of the method

Samples of biological fluids or skin appendages are taken. All collected samples are placed in glass containers of sufficient volume and frozen to a temperature of -20 ° C or below. An empty test tube is required to control the cleanliness of the container.

Samples are crushed mechanically in an ultrahomogenizer, if necessary, moistened with bidistilled water to 80%. Further, double cryodenaturation is performed at a rate of more than 5 K / minute.

Weigh such an amount of the homogenized sample to obtain up to 0.5 g of dry lipophilic extract (Table 1). For example, for breast milk with a fat content of 4%, a sample of 12.5 g is taken, which will correspond to 0.5 g of dry lipophilic extract (Table 1). Into a cylindrical separating funnel with a capacity of 50 cm ^3, add 3 cm ^{3 of} 1% sodium bicarbonate and shake vigorously for 1 minute, add a mixture of trichloromethane-methyl alcohol in a calculated amount according to the proportion of the mass-volume ratio "sample sample (12.5 g): trichloromethane (15 cm ³ ): methyl alcohol (7.5 cm ³ ): n-hexane (12.5 cm ³ ): as 5: 6: 3: 5, and shake vigorously, after which the volume of n-hexane required for extraction is added (at using high-lipid samples with a fat content above 5%, for example, a nail plate, the volumes of solvents should be 9 cm ^{3 of a} mixture of "trichloromethane-methyl alcohol" and 5 cm ^{3 of} p-hexane, respectively, to prevent losses), cover the flask with a Parafilm paraffin film and shake vigorously for 5 minutes and leave to extract for 25 minutes. After phase separation, the aqueous-alcoholic layer is removed.

A round-bottom flask K-1-100-29 / 32, previously dried in an oven for 30-40 minutes until the weight stabilizes, cooled to room temperature in a desiccator with indicator silica gel, is weighed on an analytical balance (4th class of accuracy). The data are entered into the laboratory log.

The layer remaining in the funnel is filtered through anhydrous sodium sulfate, soaked in n-hexane, into a round-bottom flask K-1-100-29 / 32 and placed on a rotary evaporator (water bath - 35 ° C + vacuum) until n-hexane is completely evaporated. Next, the round bottom flask is placed in an oven at 102 ° C until the weight is stabilized.

After stabilizing the weight of the lipophilic extract, the round bottom flask is reweighed. The data are entered into a laboratory log and the weight of the sample and the percentage of total fats obtained during the extraction are calculated for the subsequent conversion of COS concentrations as ng / g lipids (generally accepted measurements of the concentration of organic pollutants in living organisms).

To purify the obtained lipophilic extract from coextractive substances in a fume hood, 20-25 cm ^{3 of} n-hexane are introduced into a round-bottom flask with a lipophilic extract, mix thoroughly and add 10 cm ³ H ₂ SO _{4 conc} .

The contents are mixed again and left for 24 hours. Part of the acid is separated by an automatic acid-resistant dispenser with a disposable polypropylene tip and the n-hexane layer is transferred to a separating funnel VD-3-250-29 / 32 with a PTFE tap.

The solution of the extracted test substances in n-hexane is washed with a 20 cm ³ portion ^of 1% sodium bicarbonate, and then with 20-30 cm ³ portions of bidistilled water until the neutral reaction of the universal paper indicator.

A solution of the test substances in n-hexane is filtered through anhydrous sodium sulfate into a conical flask KO-30-14 / 23; KO-30-29 / 32 and put on a rotary evaporator (water bath - 35 ° C) until n-hexane is completely evaporated.

The determination of the concentrations of organochlorine pesticides and polychlorinated biphenyls in the samples is carried out on a gas chromatograph with a mass-selective detector and on a gas chromatograph with an electron capture detector.

Thus, the proposed method for preparing a sample for gas chromatographic determination of organochlorine compounds in biological fluids and human skin appendages can reduce the cost of sulfuric acid, n-hexane and bidistilled water, and reduce labor costs for producing one sample by 48-72 hours.

Claims (1)

A method for preparing a sample for gas chromatographic determination of organochlorine compounds in a biomaterial, including sampling, crushing it and subsequent extraction of organochlorine compounds with n-hexane, and subsequent purification from co-extractive substances with concentrated sulfuric acid and obtaining a concentrate of n-hexane extract of organochlorine compounds, characterized in that in biological fluids or skin appendages are used as a biomaterial, from which crushed homogenized samples are obtained, moistened with bidistilled water to a moisture content of at least 80% and twice cryodenatured, a sample is taken in an amount sufficient to obtain not more than 0.5 g of dry lipophilic extract, while 3 cm ^{3 of} 1% sodium bicarbonate solution is added to the weighed portion of the sample, which is taken as 5 parts by weight, and after vigorous shaking, the extractant is added, which is used as a mixture of trichloromethane: methyl alcohol: hexane, with than 5 parts of the weighed portion of the sample in g take 6 parts of trichloromethane in cm ³ , 3 parts of methyl alcohol in cm ³ and 5 parts of n-hexane in cm ³ , shake vigorously for 5 minutes and extract for 25 minutes, lipophilic extract of organochlorine compound settle until phase separation, after which the aqueous-alcoholic layer is removed, and the extract is filtered through anhydrous sodium sulfate, evaporated and then purified with concentrated sulfuric acid.

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