RU2810518C1 - Method for determining cadmium in biological material - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for determining cadmium in biological material, including grinding a sample of biological material and transferring it to a Kjeldahl flask; adding in a ratio of 0.75:1 by weight of the biomaterial an oxidizing mixture containing sulfuric, nitric acids and purified water in a ratio of 1:1:1 for mineralization; an aliquot of the mineralizate is transferred to a chromatographic column with KU-2-8 cation exchanger, a 0.002% solution of dithizone in chloroform is added, the eluate is discarded, and a 20% solution of Rochelle salt, a 1% solution of dimethylglyoxime, and a 10% ammonia solution are passed successively in a given ratio; cadmium is eluted with 20% sodium hydroxide solution; a uranine solution with a concentration of 1 mg/ml is added to the eluate, transferred to a 25 ml volumetric flask and adjusted to the mark with a 20% sodium hydroxide solution, photometered at 230-240 nm; and calculations are carried out according to the given equation of the calibration graph.

EFFECT: expansion of the scope of methods for determining cadmium in biological material in a wide range of cadmium concentrations.

1 cl, 3 tbl, 2 ex

Description

The invention relates to ecology, biology and toxicological chemistry, namely to methods for the quantitative determination of cadmium in biological material and food products, and can be used in the practice of environmental, chemical-toxicological laboratories and sanitary and epidemiological stations.

There is a known method for determining cadmium in biological material (blood) by drying it, charring and ashing it, dissolving the sample in hydrochloric acid, transferring it to a separatory funnel, adding an aqueous solution of potassium iodide, bringing it to a pH close to 7 units, extracting cadmium with a mixture of benzene and tributyl phosphate , followed by discarding the aqueous phase, adding solutions of sodium bromide, rhodamine C and sulfuric acid to the organic phase, separating the extract with its subsequent photometry (Author’s certificate No. 664924 A1 USSR, IPC C01G 11/00, B01D 11/04. Extraction-photometric method determination of cadmium: No. 2328663: application 02.26.1976: publ. 05.30.1979 / P. P. Kish, I. S. Balog, P. G. Mizun; applicant UZHGOROD STATE UNIVERSITY - EDN WUCXDT).

The disadvantages of this method are the possibility of extracting only up to 95% of cadmium in the sample, the linearity zone during photometry ranges from 0.1 to 5 μg/ml, which makes it possible to determine cadmium only in a small range of content in the sample, as well as the complexity of the analysis.

The closest way to determine cadmium in food products is by mineralizing them, bringing them to a pH of about 14 units in the presence of complexing agents, adding glycerol and sodium potassium tartrate, subsequent extraction of cadmium and copper with a solution of sodium diethyldithiocarbamate, separation of the organic extract, washing it with distilled water, re-extraction of cadmium solution of hydrochloric acid, followed by its quantitative determination in the aqueous phase by extraction-photometric method with dipyridyl, by adjusting the pH to 6.5 units, adding a solution of potassium iodide prepared in a solution of sodium acetate and adding a solution of dipyridyl with a solution of Mohr's salt, extraction of cadmium complexes is carried out twice 5 ml of dichloroethane followed by photometry of the extract (Author's certificate No. 1278705 A1 USSR, IPC G01N 33/12. Method for determining the concentration of cadmium in food products: No. 3793912: application 08/14/1984: publ. 12/23/1986 / A.N. Krylova, V.N. Zhulenko, M.A. Malyarova; applicant MOSCOW ORDER OF THE RED BANNER OF LABOR TECHNOLOGICAL INSTITUTE OF MEAT AND DAIRY INDUSTRY. – EDN BMGZIY.).

The disadvantages of this method are its labor intensity, the possibility of extraction and re-extraction of cadmium only when its content is less than 0.1 mg in the sample.

The technical result of the present invention is the development of a non-labor-intensive, relatively fast method for determining cadmium in biological material with a wide range of concentrations for determining cadmium in the sample.

The technical result is achieved by the complete destruction of a biological object during acid mineralization, followed by treatment with reagents that eliminate the interfering effects of mineralizate cations. An aliquot of the mineralizate is transferred to a semi-preparative chromatographic column 1.0 cm x 20 cm filled with KU-2-8 cation exchanger, and 5 ml of a 0.002% solution of dithizone in chloroform is added, the eluate is discarded. Next, 1.0 ml of a 20% solution of Rochelle salt, 1.0 ml of a 1% solution of dimethylglyoxime and 5 ml of a 10% ammonia solution are passed through. Cadmium is eluted in 5 ml portions of 20% sodium hydroxide solution. 2.5 ml of uranine solution with a concentration of 1 mg/ml is added to the eluate, transferred to a 25 ml volumetric flask and adjusted to the mark with a 20% sodium hydroxide solution, photometered using a spectrophotometer at a wavelength of 230-240 nm. Selectivity is achieved by removing the interfering influences of cations and the ability of cadmium to elute with highly alkaline solutions. The dependence is observed in an increase in the light absorption intensity of cadmium complexes with fluorescein compared to its sodium salt (uranine).

The method is carried out as follows: a sample (precise sample) of biological material is crushed in a suitable manner and quantitatively transferred to a Kjeldahl flask. An oxidizing mixture containing sulfuric and nitric acids and purified water in a ratio of 1 to 1 to 1 is added to the flask in a ratio of 0.75 to 1 by weight of the biomaterial and mineralization is carried out, if necessary, by adding a mixture of nitric acid and water (1 to 1). Mineralization is carried out until a clear solution is obtained. Upon completion of mineralization and complete destruction of organic substances, the samples are denitrated by adding formalin drop by drop until the emission of brown smoke stops. The mineralizate is transferred to a volumetric flask of the appropriate volume and diluted with water to the mark. Further, the amounts of reagents are indicated per 10.0 grams of starting biological material. An aliquot of the mineralizate is transferred to a semi-preparative chromatographic column 1.0 cm x 20 cm filled with KU-2-8 cation exchanger, and 5 ml of a 0.002% solution of dithizone in chloroform is added, the eluate is discarded. Next, 1.0 ml of a 20% solution of Rochelle salt, 1.0 ml of a 1% solution of dimethylglyoxime and 5 ml of a 10% ammonia solution are passed through. Cadmium is eluted in 5 ml portions of 20% sodium hydroxide solution. 2.5 ml of uranine solution with a concentration of 1 mg/ml is added to the eluate, transferred to a 25 ml volumetric flask and adjusted to the mark with a 20% sodium hydroxide solution, photometered using a spectrophotometer at a wavelength of 230-240 nm. Calculations are carried out according to the equation of the calibration graph, taking into account the taken flasks, pipettes, samples and dilutions.

Example 1. Determination of cadmium in cadaveric liver

10.0 g of liver was crushed to 0.5 cm pieces and treated with 0.8 ml of cadmium chloride solution containing 1000 μg/ml. The amount added corresponds to 1/50 of the half lethal dose of cadmium for a laboratory animal (subchronic poisoning). The model mixture was infused for two hours to form cadmium bonds with the components of the biomatrix. The resulting model mixture was transferred to a Kjeldahl flask and 7.5 ml of the oxidizing mixture was added. Mineralization was carried out at low boiling of the model mixture, periodically adding an aqueous solution of nitric acid. After the mineralizate had cleared, formaldehyde was added drop by drop until the emission of brown smoke stopped. The mineralizate was transferred quantitatively into a 100 ml volumetric flask and brought to the mark with purified water. A 2.0 ml aliquot of the mineralizate was transferred to a semipreparative chromatographic column 1.0 cm x 20 cm filled with KU-2-8 cation exchanger, and 5 ml of a 0.002% solution of dithizone in chloroform was added, the eluate was discarded. Next, 1.0 ml of a 20% solution of Rochelle salt, 1.0 ml of a 1% solution of dimethylglyoxime and 5 ml of a 10% ammonia solution were passed through. Cadmium was eluted with two 5 ml portions of 20% sodium hydroxide solution. 2.5 ml of uranine solution with a concentration of 1 mg/ml was added to the eluate, transferred to a 25 ml volumetric flask and diluted to the mark with 20% sodium hydroxide solution. Samples were photometered at 230-240 nm on a BioRad SmartSpecPlus spectrophotometer (USA), calculations were carried out using the equation of the calibration curve, in parallel, a control experiment was carried out without adding cadmium and its result was subtracted in the calculations (Table 1).

Construction of a calibration graph.

The calibration curve was constructed in the range of 0 – 25 μg of cadmium in 25 ml of solution. Standard solutions of cadmium containing 0; 5.0; 20.0; 25.0 μg of cadmium was placed in separatory funnels and equalized with 20% sodium hydroxide solution to 22.5 ml. Then a solution of uranine with a concentration of 1.0 mg/ml was added to 2.5 ml and shaken well. Colored solutions were photometered at 230-240 nm on a BioRad SmartSpecPlus spectrophotometer (USA). The obtained data were used to calculate the calibration graph equation. The equation looked like:

A = 0.0319x + 0.0227,

where A is the optical density, x is the concentration of the analyte in the sample.

Example 2. Determination of cadmium in potato tuber tissue

100.0 g of potatoes were grated and treated with 0.3 ml of cadmium chloride solution containing 10 μg/ml. The applied amount corresponds to the maximum permissible concentration of cadmium in potatoes. The model mixture was infused for two hours to form cadmium bonds with the components of the biomatrix. The resulting model mixture was transferred to a Kjeldahl flask, dried, and 15 ml of the oxidizing mixture was added. Mineralization was carried out at low boiling of the model mixture, periodically adding an aqueous solution of nitric acid. After the mineralizate had cleared, formaldehyde was added drop by drop until the emission of brown smoke stopped. The mineralizate was transferred quantitatively into a 25 ml volumetric flask and brought to the mark with purified water. 25 ml of the mineralizate, a semi-preparative chromatographic column 1.0 cm x 20 cm, filled with the KU-2-8 cation, and added 5 ml of a 0.002% solution of dithizone in chloroform. Next, 10.0 ml of a 20% Rochelle salt solution, 1.0 ml of a 1% dimethylglyoxime solution and 5.0 ml of a 10% ammonia solution were eluted successively. Cadmium was eluted with two 5 ml portions of 20% sodium hydroxide solution. 2.5 ml of uranine solution with a concentration of 1 mg/ml was added to the eluate, transferred to a 25 ml volumetric flask and diluted to the mark with 20% sodium hydroxide solution. Samples were photometered at 230-240 nm on a BioRad SmartSpecPlus spectrophotometer (USA), calculations were carried out using the calibration curve equation, a control experiment was carried out in parallel without adding cadmium, and its result was subtracted in the calculations (Table 2).

The construction of the calibration graph was carried out similarly to that described in example 1.

Table 1
Results of determination of cadmium in cadaveric liver (n = 5, P = 0.95) No. Contributed, mcg per 10 g of liver Found Metrological characteristics mcg % 1. 800 784.2 98.02 X av = 97.75
S = 0.5786
S xav = 0.2588
ΔХ av = 0.72 2. 800 774.8 96.85 3. 800 787.3 98.41 4. 800 781.0 97.63 5. 800 782.6 97.83

table 2
Results of determination of cadmium in potato tubers (n = 5, P = 0.95) No. Contributed, mcg per 10 g of liver Found Metrological characteristics mcg % 1. 3.0 2.94 97.78 X av = 97.41
S = 0.6289
S xav = 0.2812
ΔХ av = 0.80 2. 3.0 2.90 96.73 3. 3.0 2.95 98.30 4. 3.0 2.92 97.26 5. 3.0 2.91 97.00

Table 3
Comparison of the proposed and known method for determining cadmium Index Suggested method Known method Example 1 Example 2 Percentage of cadmium determination, % 97.75 97.41 92 - 94 Time spent on 1 determination, hour 0.25 0.3 1.0

Thus, the proposed method in a wide range of cadmium concentrations in the sample, relatively quickly compared to the prototype, increases the percentage of determination of cadmium in samples by 4-6%.

Claims (3)

A method for determining cadmium in biological material, including grinding a sample of biological material and transferring it to a Kjeldahl flask; then add an oxidizing mixture containing sulfuric, nitric acids and purified water in a ratio of 1:1:1 in a ratio of 0.75:1 by weight of the biomaterial and carry out mineralization, if necessary adding a mixture of nitric acid with water in a ratio of 1:1 until a transparent solution; then add formalin drop by drop until the release of brown smoke stops; an aliquot of the resulting mineralizate is added to a semi-preparative chromatographic column 1.0 cm × 20 cm filled with KU-2-8 cation exchanger, 5 ml of a 0.002% solution of dithizone in chloroform is added, the eluate is discarded and 1.0 ml of a 20% solution of Rochelle salt is passed successively, 1. 0 ml of 1% dimethylglyoxime solution and 5 ml of 10% ammonia solution; then cadmium is eluted in 5 ml portions of 20% sodium hydroxide solution; 2.5 ml of uranine solution with a concentration of 1 mg/ml is added to the eluate, transferred to a 25 ml volumetric flask and adjusted to the mark with 20% sodium hydroxide solution, photometered using a spectrophotometer at a wavelength of 230-240 nm; and calculations are carried out using the equation of the calibration graph: A = 0.0319x + 0.0227, where A is the optical density, x is the concentration of the analyte in the sample.