RU2795470C1 - Method for qualitative and quantitative colorimetric determination of formaldehyde in milk - Google Patents

Abstract

FIELD: dairy industry.

SUBSTANCE: invention concerns the qualitative and quantitative colorimetric determination of formaldehyde in milk. Preliminarily, denaturation and sedimentation of milk sample proteins are carried out by acidification with hydrochloric acid, heating the resulting mixture and centrifugation. The pH required for the reaction is adjusted with a sodium hydroxide solution, a mixed reagent solution is added: an aqueous solution of acetylacetone, acetic acid and ammonium acetate, and heated. Microextraction of the resulting derivative is carried out in a microvolume of molten thymol. The separation of the aqueous and organic phases is achieved by centrifugation; after that, the upper phase of the extract is taken, applied in a thin layer on a black paper substrate, and visual and/or instrumental spectrofluorimetric determination is carried out. The quantitative determination of the analyte in the sample is carried out on the basis of a pre-built calibration graph using analyte calibration solutions.

EFFECT: invention makes it possible to completely exclude the use of toxic solvents, to ensure low consumption of reagents and safe solvents, as well as high productivity and sensitivity of the determination.

2 cl, 3 tbl, 3 ex

Description

The invention relates to the field of analytical chemistry and can be used for the qualitative and quantitative colorimetric determination of formaldehyde in milk. Formaldehyde is a carcinogen and an allergen, so the determination of its content in food products is an important task for quality control.

A known method for the determination of formaldehyde in food products [1], according to which formaldehyde is derivatized with 2,4-dinitrophenylhydrazine and the reaction product is isolated into acetonitrile, which belongs to toxic organic solvents. To purify samples with a high fat content, extract into n-hexane saturated with acetonitrile (10 cm ³ per sample). At the next stage, the resulting solution is filtered and the formaldehyde derivative content is determined by high performance liquid chromatography with ultraviolet detection (HPLC-UV), using a mixture of water and acetonitrile in a ratio of 40:60, respectively, as a mobile phase.

A known method for the determination of formaldehyde in liquids [2], according to which centrifugation of the sample is performed in a high-speed centrifuge, ultrafiltration of the obtained supernatant and determination of the formaldehyde content by the spectrophotometric method. To do this, a sodium hydroxide solution, a photometric phenolic reagent, and a sample supernatant are placed in a spectral cuvette. The method involves long-term thermostating of the reaction mixture (for 30 min) to form a colored solution of the derivative, the optical density of which is proportional to the concentration of formaldehyde in the sample.

To determine the concentration of formaldehyde by the fluorimetric method, in accordance with [3], a preliminary distillation of the analyte with water vapor is performed. Next, a solution of 1,3-cyclohexanedione and ammonium acetate is added to the distillate, after which the mixture is subjected to prolonged thermostating (45 min at 60°C). Measure the fluorescence intensity of the derivative solution, which is proportional to the concentration of formaldehyde in the sample.

A known method for the qualitative determination of formaldehyde in milk [4], based on the ability of formaldehyde to reduce AgNO ₃ to metallic silver in an alkaline environment. To conduct a qualitative analysis, a milk sample is transferred to a colorimetric cylinder, solutions of silver nitrate and sodium hydroxide are added, shaken and thermostated, after which the color change of the reaction mixture is observed. The formaldehyde test is considered positive when a metallic silver color appears.

There is also a known method for the qualitative determination of formaldehyde in milk [5], which involves the introduction of concentrated sulfuric acid with traces of nitric acid into the sample. The resulting mixture is stirred and the presence of formaldehyde in milk is judged by the appearance of a colored reaction product.

Considering the fact that formaldehyde is produced in small amounts by most living organisms as part of metabolic processes and, therefore, can be naturally present in milk, the disadvantage of qualitative determination methods is the inability to quantify the formaldehyde content, on the basis of which it is possible to judge the suitability of the product for consumption. .

Closest to the claimed invention and selected as a prototype is a method for determining formaldehyde in milk [6], including precipitation of proteins, formaldehyde derivatization using 2,4-dinitrophenylhydrazine, extraction of the obtained derivative into acetonitrile with freezing of the aqueous phase at -4°C for several hours and HPLC-UV analysis of the liquid organic phase with the derivative isolated into it. The disadvantage of the proposed method is the use of toxic organic solvent acetonitrile, which is also a precursor of prohibited substances. In addition, the extraction stage involves a long-term freezing of the aqueous phase, which negatively affects the performance of the proposed method.

The technical result of the claimed invention in comparison with the prototype is a productive and environmentally friendly method for preparing milk samples, which allows qualitatively and quantitatively determine formaldehyde in milk.

The technical task of the claimed invention is to reduce the amount of extractant used, use an environmentally friendly solvent, and increase productivity.

This technical result is achieved by the fact that at the stage of sample preparation for protein precipitation, the milk sample is acidified with hydrochloric acid and the mixture is heated at 80 ± 5 ° C for 5 minutes, after which it is centrifuged for 5 minutes and the pH of the resulting supernatant is adjusted by adding a concentrated sodium hydroxide solution to a value of 5.0-5.5, necessary for the next stage of derivatization, which is carried out by adding a mixed reagent (an aqueous solution of acetylacetone, acetic acid and ammonium acetate in a molar ratio of 1:1:30, respectively) to the selected supernatant. The formation and extraction of the derivative into a natural environmentally friendly solvent - thymol, occurs simultaneously with heating (80±5°C) and stirring for 8-10 minutes. Separation of the aqueous and organic phases is achieved by centrifugation at 5000 rpm for 5 min, after which 0.015 cm ³ of the upper phase of the extract is taken with a dispenser and applied in a thin layer on a paper substrate for solidification. Black paper is used as the substrate material, since it does not give a background signal in the fluorescence spectrum. The qualitative determination of formaldehyde is carried out by comparing the initial color of the extractant and the color of the obtained extract on the substrate, the appearance of a yellow color indicates a formaldehyde content of ≥300 µg/dm ³ (the natural content of formaldehyde in milk varies in the range of 100-300 µg/dm ³ ). Quantification is carried out by placing the substrate with the extract in a solid sample holder in a spectrofluorimeter and measuring the fluorescence intensity of the derivative on the substrate at excitation and emission wavelengths of 420 and 494 nm, respectively. Quantitative determination of the analyte in the sample is carried out using the method of absolute calibration. The proposed method provides a degree of formaldehyde extraction from 83 to 100%. The limit of detection is 15 μg/dm ³ , the range of determined concentrations is 45-500 μg/dm ³ , the time for one analysis is 35 minutes.

Distinctive features of the proposed method from the closest analogue, taken as a prototype, are:

- the use of thymol as an extractant - a natural solvent with a low melting point (50.5°C);

- using microvolumes of organic extractant (thymol);

- carrying out liquid extraction during heating (80±5), which significantly reduces the limiting stage of sample preparation;

- qualitative determination of formaldehyde due to visual fixation of changes in the intensity of the color of the extract.

The claimed distinguishing features allow for the qualitative and quantitative determination of formaldehyde in milk without the use of toxic organic extractants and high time costs while maintaining the sensitivity of the method in the case of quantitative determination.

The claimed method was tested in laboratory conditions at St. Petersburg State University, the results of testing are presented in the form of specific examples of implementation. The studies were carried out using the equipment of the resource center "Methods of analysis of the composition of matter" of the Science Park of St. Petersburg State University and instrumental support of the Department of Analytical Chemistry.

Example 1

Sampling

Milk sample No. 1 was purchased from a local supermarket (St. Petersburg). The safety of the product was ensured by compliance with the conditions specified by the manufacturer on the packaging.

Sample preparation

At the first stage, to eliminate the interfering effect of the sample components, denaturation and precipitation of proteins were performed. To do this, 1.4 cm ^{3 of} hydrochloric acid with a concentration of 4.0 mol/dm ³ was added to 7.0 cm ³ of milk samples. The resulting mixture was heated for 5 min at 75°C, stirred and centrifuged at 5000 rpm for 5 min. Next, 290 μl of an aqueous solution of sodium hydroxide with a concentration of 10 mol/DM ³ was added to 5.0 ^{cm 3} of the supernatant solution to establish pH=5.0. At the second stage, formaldehyde was derivatized to obtain a yellow-colored form, including one capable of fluorescence, and the reaction product was concentrated by microextraction to reduce the limits of detection. For formaldehyde derivatization in a narrow eppendorf, to an aliquot of 4.0 cm ³ of the resulting solution was added 0.4 cm ³ of a mixed reagent solution, which is an aqueous solution of acetylacetone, acetic acid and ammonium acetate in a molar ratio of 1:1:30, respectively, and cm ³ thymol melt. Eppendorf was placed in a thermostat at 80°C and the mixture was stirred for 8 min. Next, centrifugation was performed at 5000 rpm for 5 min, 0.015 cm ³ of the extract phase was taken with a dispenser and applied in a thin layer on a black paper substrate (oval 6×12 mm). The extract was left on the substrate at room temperature until solidified.

Analysis

Qualitative determination of formaldehyde was carried out visually. No color change was observed.

Quantitative determination of formaldehyde was carried out using a spectrofluorimeter. The substrate with the frozen phase of the extract was placed in a holder for solid samples in a spectrofluorimeter to measure the fluorescence intensity at excitation and emission wavelengths of 420 and 494 nm, respectively. Quantitative determination of the analyte in the sample was carried out using the absolute calibration method. The results of the analysis are presented in table 1. The content of formaldehyde in the sample was 105 μg/DM ³ that does not exceed the norms of the natural content of formaldehyde in milk.

Example 2

Sampling

Milk sample No. 2 was purchased from a local supermarket (St. Petersburg). The safety of the product was ensured by compliance with the conditions specified by the manufacturer on the packaging.

Sample preparation

At the first stage, to eliminate the interfering effect of the sample components, denaturation and precipitation of proteins were performed. To do this, 0.5 cm ^{3 of} concentrated hydrochloric acid was added to 7.0 cm ³ of milk samples. The resulting mixture was heated for 5 min at 80°C, stirred and centrifuged at 5000 rpm for 5 min. Next, 300 μl of an aqueous solution of sodium hydroxide with a concentration of 10 mol/DM ³ was added to 5.0 ^{cm 3} of the supernatant solution to establish pH=5.3. At the second stage, formaldehyde was derivatized to obtain a yellow-colored form, including one capable of fluorescence, and the reaction product was concentrated by microextraction to reduce the limits of detection. For formaldehyde derivatization in a narrow eppendorf, to an aliquot of 4.0 cm ³ of the resulting solution was added 0.4 cm ³ of a mixed reagent solution, which is an aqueous solution of acetylacetone, acetic acid and ammonium acetate in a molar ratio of 1:1:30, respectively, and cm ³ thymol melt. Eppendorf was placed in a thermostat at 80°C and the mixture was stirred for 9 min. Next, centrifugation was performed at 5000 rpm for 5 min, 0.015 cm ³ of the extract phase was taken with a dispenser and applied in a thin layer on a black paper substrate (oval 6×12 mm). The extract was left on the substrate at room temperature until solidified.

Analysis

Qualitative determination of formaldehyde was carried out visually. No color change was observed.

Quantitative determination of formaldehyde was carried out using a spectrofluorimeter.

The substrate with the frozen phase of the extract was placed in a holder for solid samples in a spectrofluorimeter to measure the fluorescence intensity at excitation and emission wavelengths of 420 and 494 nm, respectively. Quantitative determination of the analyte in the sample was carried out using the absolute calibration method. The results of the analysis are presented in table 2. The content of formaldehyde in the sample was 117 μg/DM ³ that does not exceed the norms of the natural content of formaldehyde in milk.

Example 3

Sampling

Milk sample No. 2 was purchased from a local supermarket (St. Petersburg). The safety of the product was ensured by compliance with the conditions specified by the manufacturer on the packaging.

Sample preparation

At the first stage, to eliminate the interfering effect of the sample components, denaturation and precipitation of proteins were performed. To do this, 0.6 cm ^{3 of} concentrated hydrochloric acid was added to 8.0 cm ³ of milk samples. The resulting mixture was heated for 5 min at 85°C, stirred and centrifuged at 5000 rpm for 5 min. Next, 300 μl of an aqueous solution of sodium hydroxide with a concentration of 10 mol/DM ³ was added to 5.0 ^{cm 3} of the supernatant solution to establish pH=5.5. At the second stage, formaldehyde was derivatized to obtain a yellow-colored form, including one capable of fluorescence, and the reaction product was concentrated by microextraction to reduce the limits of detection. For formaldehyde derivatization in a narrow eppendorf, to an aliquot of 4.0 cm ³ of the resulting solution was added 0.4 cm ³ of a mixed reagent solution, which is an aqueous solution of acetylacetone, acetic acid and ammonium acetate in a molar ratio of 1:1:30, respectively, and cm ³ thymol melt. Eppendorf was placed in a thermostat at 80°С and the mixture was stirred for 10 min. Next, centrifugation was performed at 5000 rpm for 5 min, 0.015 cm ³ of the extract phase was taken with a dispenser and applied in a thin layer on a black paper substrate (oval 6×12 mm). The extract was left on the substrate at room temperature until solidified.

Analysis

Qualitative determination of formaldehyde was carried out visually. No color change was observed.

Quantitative determination of formaldehyde was carried out using a spectrofluorimeter. The substrate with the frozen phase of the extract was placed in a holder for solid samples in a spectrofluorimeter to measure the fluorescence intensity at excitation and emission wavelengths of 420 and 494 nm, respectively. Quantitative determination of the analyte in the sample was carried out using the absolute calibration method. The results of the analysis are presented in table 3. The content of formaldehyde in the sample was 108 μg/DM ³ that does not exceed the norms of the natural content of formaldehyde in milk.

The proposed method is easy to implement, provides high performance and sensitivity of determination due to microextraction preconcentration, eliminates the use of toxic solvents, which is fully consistent with modern trends in "green" analytical chemistry. The method can be used by food laboratories to control the quality of dairy products.

List of information sources used

1. CN 109342620 A "Method for rapidly determining formaldehyde in foods by derivative extraction method".

2. CN 105606603 A "Method for measuring formaldehyde content in liquid".

3. GOST P 55227-2012 "WATER. Methods for determining the content of formaldehyde".

4. CN 1266469 С "Quick detecting method for formaldehyde in fresh milk".

5. SU 1727060 A1 "Method for determining the presence of formaldehyde in milk".

6.F.B. de Freitas Rezende et al., "Determination of formaldehyde in bovine milk using a high sensitivity HPLC-UV method", Microchem. J., vol. 134, pp. 383-389, 2017, doi: 10.1016/j.microc.2017.07.003 (prototype).

Claims (2)

1. A method for the qualitative and quantitative colorimetric determination of formaldehyde in milk, including precipitation of proteins, derivatization, liquid extraction of the derivative into an extractant and analysis of the extract, characterized in that precipitation of proteins is carried out by mixing hydrochloric acid and milk samples in a volume ratio of 1:13-1:15 at 80±5°C for 5 min, after which the mixture is centrifuged and the pH of the resulting supernatant is adjusted to a value of 5.0-5.5 by adding concentrated sodium hydroxide solution, formaldehyde derivatization and extraction of the resulting derivative are carried out simultaneously by adding a reaction mixture consisting of an aqueous solution of acetylacetone, acetic acid and ammonium acetate, taken in molar ratios of 1:1:30, respectively, and pre-melted thymol extractant to the selected supernatant in volume ratios of 1:10 and 1:40, respectively, the mixture is stirred and heated at a temperature of 80 ± 5 °C for 8-10 min, after which it is centrifuged, the upper organic phase of the extract is taken, applied to the substrate and left in the open air to solidify; after which, a visual comparison of the initial color of the extractant and the color of the obtained extract on the substrate is carried out and the presence of formaldehyde in the sample is judged by the change in color intensity, the quantitative determination is carried out by measuring the fluorescence intensity of the derivative on the substrate at excitation and emission wavelengths of 420 and 494 nm, respectively. 2. The method according to p. 1, characterized in that centrifugation is carried out for 5-10 minutes at 5000 rpm.