Concentration of ochratoxin A in the sample, mg/kg

Relative error limits (accuracy index) (±d), %, R = 0,95

Repeatability standard deviation (s r), %

Repeatability limit ( r), %

Completeness of extraction of substances, %

4. Measuring instruments, auxiliary devices, glassware, reagents and materials

4.1. Measuring instruments

4.2. Auxiliary equipment


Apparatus for shaking samples type АВУ-6С or similar	TU 64-1-2451
Rotary evaporator IR-1M with a trap or similar	TU 25-11917
Laboratory drying cabinet with temperature maintenance error ±2.5 in the range from 50 to 350 °C	TU 16-531.639
Refrigerator household
Electric laboratory mill EM-3A or similar pH meter	TU 46-22-236-79
Magnetic stirrer type MM 5 with stirrer bar	TU 25-11.834-80
Flat-bottomed conical flasks 250 cm 3 with NSh 29, type KnKSh 250-29/32	GOST 10394-74
Dark glass screw bottles (vile), volume 7 cm 3
Volumetric flasks, capacity 100, 500, 1000 cm 3 type 2-100-2,2-500-2
Funnels laboratory
Pear-shaped flasks, 10 cm 3 with NSh 14.5, type GrKSH-10-14/23	GOST 10394-72

4.3. Reagents and materials


Sodium phosphate monosubstituted, 2-aqueous, chda
Sodium chloride, chemically pure
Acetonitrile, high purity grade 0
Methanol, osch
Phosphoric acid, high purity	TU 2612-014-00203677-97
Glacial acetic acid, chemically pure
Toluene, chda
Ochraprep immunoaffinity columns (R-Biopharm, UK)

. Preparing to take measurements

5.1. Preparation of standard solutions of ochratoxin A

To prepare a standard storage solution (concentration of ochratoxin A - 10 ng / μl), a sample of crystalline ochratoxin A weighing 5 mg is placed in a volumetric flask with a volume of 500 cm 3, 50 cm 3 of a mixture of toluene-acetic acid (98:2% vol.) is poured, carefully stir until complete dissolution of the substance and bring the same mixture of solvents to the mark. To establish the exact concentration of the storage solution, its optical density is measured at a wavelength of 333 nm (D 333). The concentration of the solution is calculated by the formula:

Further, 5 cm 3 of a standard solution of ochratoxin A with a concentration of 10 ng/μl is diluted with a mixture of toluene-acetic acid (98: 2% vol.) to a volume of 100 cm 3, obtaining a working solution with a concentration of 0.5 ng/μl.

For the preparation of working solutions of ochratoxin A with a concentration of 0.005; 0.05 and 0.1 ng/µl, respectively, 50, 500 and 1000 µl of a solution with a concentration of 0.5 ng/µl are taken, evaporated to dryness and dissolved in 5 cm 3 of the mobile phase.

The storage solution of ochratoxin A is kept in a glass container with a ground stopper in a dark, cool place (at a temperature of about 0 ° C) for up to one year and is used to prepare working standard solutions. Working standard solutions are stored in dark glass vials in a dark, cool place (at a temperature of about 0 °C) for 1 month.

Before using working standard solutions, they should be brought to room temperature and only then should the stoppers be opened.

5.2. Preparation of phosphate buffer solution, pH = 7.4

A portion of sodium phosphate disubstituted 12-water weighing 1.15 g, a portion of sodium monosubstituted 2-water weighing 0.124 g and a portion of sodium chloride weighing 1.74 g are transferred to a volumetric flask with a capacity of 100 cm 3, 10 - 20 cm 3 of distilled water are added. Stir and bring the volume of the solution in the flask to the mark. Shelf life - 1 month in the refrigerator.

5.3. Preparation of solvent mixtures

Toluene-acetic acid (98:2 % about.).

In a 1000 cm 3 volumetric flask, add 20 cm 3 of acetic acid and, while stirring, bring to the mark with toluene. Shelf life - 1 month in a dark cool place.

Acetonitrile-water (60:40 % about.).

In a 1000 cm 3 volumetric flask, add 600 cm 3 of acetonitrile and, stirring, bring to the mark with water. Shelf life - 1 month in a dark cool place.

Acetonitrile-water (60:40 % about.; pH = 3 ,0 ).

In a 1000 cm 3 volumetric flask, add 600 cm 3 of acetonitrile and, stirring, bring to the mark with bidistilled water. By adding phosphoric acid, the pH of the mixture is adjusted to a value equal to 3.0. Shelf life - 1 month in a dark cool place.

methanol-acetic acid (98:2 % about.).

In a 1000 cm 3 volumetric flask add 20 cm 3 of acetic acid and, while stirring, bring to the mark with methanol. Shelf life - 1 month in a dark cool place.

. Sampling and preparation of samples for analysis

6.1. Sample selection

To take into account the specifics of sampling certain types of products, one should be guided by the current regulatory and technical documentation:

"Corn. Acceptance rules and sampling methods” GOST 13586.3-83;

"Krupa. Acceptance rules and sampling methods” GOST 26312.1-84;

“Flour and bran. Acceptance and sampling methods” GOST 27668-88;

“Canned food products. Sampling and preparing them for testing” GOST 8756.0-70.

Samples for analysis representative of the concentration of mycotoxins for the entire batch should be taken from a pre-homogenized average (initial) sample weighing 2 kg.

6.2. Sample preparation for analysis

The selected samples are crushed for 1 - 2 min in a laboratory mill. In this case, two parallel samples are used.

6.2.1. Extraction

A portion of 25 g of the crushed sample is placed in a 250 cm flat-bottomed conical flask, 100 cm 3 of a mixture of acetonitrile-water (60:40% vol.) are added. Extract on a shaker for 30 minutes. The resulting mixture is filtered through a blue ribbon pleated paper filter. Select 10 cm 3 of the filtrate and add 90 ml of phosphate buffer solution, pH = 7.4.

6.2.2. Purification of the extract

100 ml of the resulting mixture is applied to the immunoaffinity column at a rate of 1 - 2 drops per second, washed with 20 cm 3 of a phosphate buffer solution, pH = 7.4. Ochratoxin A is eluted with 3 cm 3 of a mixture of methanol-acetic acid (98:2 % vol.).

. Taking measurements

7.1. Test sample preparation

The eluate is evaporated to dryness. The dry residue is dissolved in 400 μl of the mobile phase (solution A).

7.2. Chromatography conditions

HPLC conditions: mobile phase - acetonitrile-water (60:40% vol.; pH = 3.0); mobile phase speed - 1.5 cm 3 /min.

The fluorimetric detector is set to the excitation wavelength of 333 nm, an emission filter with a bandwidth of 466 nm is installed on the emission line.

For sample analysis, 50 μl of the test sample (solution A) is injected into the chromatograph injector using a microsyringe. In the presence of a peak coinciding with the retention time of ochratoxin A, the mass of ochratoxin A in the injection is calculated using a calibration curve.

. Processing measurement results

8.1. Construction of a graduated dependence

To build a calibration graph, a chromatographic analysis of a series of working solutions of standards is carried out. Using a microsyringe, 50 µl of the standard working solution with a concentration of 0.005 ng/µl is injected into the injector, which corresponds to 0.25 ng of ochratoxin A. The same is done for other standard solutions with concentrations of 0.05 and 0.10 ng/µl, which in turn corresponds to 2.5 and 5.0 ng of ochratoxin A per injection. Under these conditions, the retention time for ochratoxin A is in the range of 4 to 5 minutes. Based on the data obtained, a calibration graph is built (dependence of the area of the chromatographic peak on the mass of ochratoxin A in the injection).

8.2. Registration of results

Calculation of the concentration of ochratoxin A in the sample is carried out according to the formula:

C (protection A)- concentration of ochratoxin A in the sample, mg/kg;

M- weight of sample for analysis, g (25.0);

tis the mass of ochratoxin A, corresponding to the volume of solution A introduced into the chromatograph, ng;

V 1 - volume of solution for extraction, cm 3 (100);

D - limit of absolute error:

d - limit of the relative error of the technique (accuracy index), % (Table 1).

If the content of ochratoxin A in the sample is less than the lower limit of the range of determined concentrations, the result of the analysis is presented as:

* 0.0001 mg/kg - limit of detection.

8.3. Checking the acceptability of the results of parallel determinations

Permissible discrepancy between parallel measurements (R) is determined based on the repeatability limit (r) (Table 1):

R = 0,01 (r, %) × , mg/kg

If the discrepancy between parallel definitions does not exceed the allowable:

then the arithmetic mean is taken as the result of the analysis.

When exceeding the standardRmeasurements should be repeated using backup samples.

. Quality control of measurement results

The frequency of measurement error control depends on the number of working measurements for the controlled period and is determined by control plans.

Control samples are working samples of food raw materials and food products. A sample is taken and divided into 2 equal parts. One of them is left unchanged, and an amount of ochratoxin A standard solution is added to the other so that its mass fraction in the sample increases by 50 - 100% compared to the initial value. The additive must be introduced into the sample before the start of sample preparation.

Both samples are analyzed in strict accordance with the prescription of the methodology and the results of the analysis of the original sample are obtained ( C (neg. A)) and samples with the addition ( With ¢ (neg. A)). The determination is carried out under the same conditions, namely: the analysis is carried out by one analyst, using one set of volumetric utensils, reagents, solutions, etc.

The algorithm for conducting operational error control using the additive method consists in comparing the result of the control determination, which is equal to the difference between the result of the control measurement of the sample with the additive ( With ¢ (ocher)), samples without additive ( C (protection A)) and the value of the additive ( C ext (arm. A)) with operational control standard (K). The decision on a satisfactory error is made when the following condition is met (when R and

Ambient temperature from 15 to 25 °С.

Relative air humidity no more than 80% at 25 °С.

Atmospheric pressure 730 - 760 mm Hg.

Power supply voltage: 210 - 220 V. AC frequency: 45 - 50 Hz.

Ochratoxins are produced by certain types of fungi. Aspergillus and Penicillium. The main producers are A.ochraceus and P. viridicatum. These mushrooms are found everywhere. Aspergillus produces ochratoxins at elevated temperature and humidity, and Penicillium already at 5°C. Ochratoxins are highly toxic compounds with a pronounced teratogenic effect.

Ochratoxins A, B, and C are a group of structurally related compounds that are isocoumarins associated with L-phenylalanine peptide bond. Depending on the nature of the radicals, various types of ochratoxins are formed (Table 2.3.).

Ochratoxin A is a colorless crystalline substance, slightly soluble in water, moderately soluble in polar organic solvents (methanol, chloroform), as well as in an aqueous solution of sodium carbonate. In a chemically pure form, it is unstable and very sensitive to light and air, but in an ethanol solution it can remain unchanged for a long time. In UV light it has green fluorescence.

Ochratoxin B is a crystalline substance, an analogue of ochratoxin A, which does not contain a chlorine atom. It is about 50 times less toxic than ochratoxin A. In UV light, it has blue fluorescence.

Ochratoxin C is an amorphous substance, ethyl ester of ochratoxin A, which is close to it in toxicity, but has not been found as a natural food and feed contaminant. In Y-light it has a pale green fluorescence.

Ochratoxins belong to toxic mycotoxins, have high toxicity to the liver, kidneys, teratogenic and immunosuppressive properties, and a pronounced hemolytic effect. Of the ochratoxins, ochratoxin A is the most toxic (LD 50 = 3.4 mg/kg, (day-old chicks, oral)). It is more toxic than aflatoxins. Other mycotoxins of this group are an order of magnitude less toxic.

Biochemical, molecular, cellular mechanisms of action of ochratoxins are not well understood. It is known that ochratoxin A inhibits protein synthesis and carbohydrate metabolism, in particular glycogenosis, by inhibiting the activity of phenylalanine, a tRNA, a specific enzyme that plays a key role in the initial stage of protein synthesis.

Ochratoxin A is found in corn, barley, wheat, oats, and barley. It is important and dangerous that ochratoxin A is found in livestock products (ham, bacon, sausages) at high contamination of feed grains and animal feed. Ochratoxin B is rare. Ochratoxins also affect all fruits of horticultural crops. Apples are especially affected: up to 50% of the crop can be contaminated with mycotoxins.

It should be noted that ochratoxins are stable compounds. So, for example, during prolonged heating of wheat contaminated with ochratoxin A, its content decreased only by 32% (at a temperature of 250–300ºС). Thus, the prevalence in food products, toxicity and persistence of ochratoxins create a real danger to human health.

Analysis Methods

Ochratoxin A is found in oxidized foods. It readily dissolves in many organic solvents, which is used for extraction. The most commonly used is extraction with chloroform and an aqueous solution of phosphoric acid, followed by purification on a column and quantitative determination using the TLC method.

An HPLC method has also been developed. Before HPLC analysis, the sample is prepared as follows. The crushed sample is treated with a mixture of 2 M hydrochloric acid and 0.4 M magnesium chloride solution. After homogenization, extract with toluene for 60 minutes. The mixture is centrifuged. The centrifuge is passed through a column of silica gel and washed with a mixture of toluene and acetone (mobile phase). Ochratoxin A is eluted with a mixture of toluene and acetic acid (9:1) and dried at 40°C. The residue is dissolved and filtered. The analysis is carried out using HPLC.

In addition, a number of bioassays on shrimp and bacteria have been developed, but the results obtained did not allow the use of these methods for the determination of ochratoxins.

Concentration of ochratoxin A in the sample, mg/kg

Relative error limits (accuracy index) (±d), %, R = 0,95

Repeatability standard deviation (s r), %

Repeatability limit ( r), %

Completeness of extraction of substances, %

4.2. Auxiliary equipment


Apparatus for shaking samples type АВУ-6С or similar
Rotary evaporator IR-1M with a trap or similar
Laboratory drying cabinet with temperature maintenance error ±2.5 in the range from 50 to 350 °C
Refrigerator household
Electric laboratory mill EM-3A or similar pH meter	TU 46-22-236-79
Magnetic stirrer type MM 5 with stirrer bar	TU 25-11.834-80
Flat-bottomed conical flasks 250 cm3 with NSh 29, type KnKSh 250-29/32	GOST 10394-74
Dark glass screw bottles (vile), 7 cm3
Volumetric flasks, capacity 100, 500, 1000 cm3 type 2-100-2,2-500-2
Funnels laboratory
Pear-shaped flasks, 10 cm3, with NSh 14.5, type GrKSh-10-14/23	GOST 10394-72

4.3. Reagents and materials

. Preparing to take measurements

5.1. Preparation of standard solutions of ochratoxin A

To prepare a standard storage solution (the concentration of ochratoxin A is 10 ng/µl), a 5 mg sample of crystalline ochratoxin A is placed in a volumetric flask with a volume of 500 cm3, 50 cm3 of a mixture of toluene-acetic acid (98:2% vol.) is added, thoroughly mixed until complete dissolution of the substance and bring the same mixture of solvents to the mark. To establish the exact concentration of the storage solution, its optical density is measured at a wavelength of 333 nm (D333). The concentration of the solution is calculated by the formula:

Before using working standard solutions, they should be brought to room temperature and only then should the stoppers be opened.

5.2. Preparation of phosphate buffer solution, pH = 7.4

A weighing of 1.15 g of sodium phosphate disubstituted 12-aqueous with a mass of 1.15 g, a weighing of sodium of disubstituted 2-aqueous sodium with a mass of 0.124 g and a weighing of sodium chloride with a mass of 1.74 g are transferred to a volumetric flask with a capacity of 100 cm3, 10 - 20 cm3 of distilled water are added. Stir and bring the volume of the solution in the flask to the mark. Shelf life - 1 month in the refrigerator.

5.3. Preparation of solvent mixtures

Toluene-acetic acid (98:2 % about.).

Add 20 cm3 of acetic acid to a 1000 cm3 volumetric flask and, while stirring, make up to the mark with toluene. Shelf life - 1 month in a dark cool place.

Acetonitrile-water (60:40 % about.).

Add 600 cm3 of acetonitrile to a 1000 cm3 volumetric flask and, while stirring, make up to the mark with water. Shelf life - 1 month in a dark cool place.

Acetonitrile-water (60:40 % about.; pH = 3 ,0 ).

Add 600 cm3 of acetonitrile to a 1000 cm3 volumetric flask and, while stirring, make up to the mark with bidistilled water. By adding phosphoric acid, the pH of the mixture is adjusted to a value equal to 3.0. Shelf life - 1 month in a dark cool place.

methanol-acetic acid (98:2 % about.).

Add 20 cm3 of acetic acid to a 1000 cm3 volumetric flask and, while stirring, make up to the mark with methanol. Shelf life - 1 month in a dark cool place.

. Sampling and preparation of samples for analysis

6.1. Sample selection

To take into account the specifics of sampling certain types of products, one should be guided by the current regulatory and technical documentation:

"Corn. Acceptance Rules and Sampling Methods" GOST 13586.3-83 ;

"Krupa. Acceptance Rules and Sampling Methods" GOST 26312.1-84 ;

“Flour and bran. Acceptance and sampling methods" GOST 27668-88 ;

“Canned food products. Sampling and preparation for testing" GOST 8756.0-70.

Samples for analysis representative of the concentration of mycotoxins for the entire batch should be taken from a pre-homogenized average (initial) sample weighing 2 kg.

6.2. Sample preparation for analysis

The selected samples are crushed for 1 - 2 min in a laboratory mill. In this case, two parallel samples are used.

6.2.1. Extraction

A portion of 25 g of the crushed sample is placed in a 250 cm flat-bottomed conical flask, 100 cm3 of an acetonitrile-water mixture (60:40% by volume) is added. Extract on a shaker for 30 minutes. The resulting mixture is filtered through a blue ribbon pleated paper filter. Withdraw 10 ml of the filtrate and add 90 ml of phosphate buffer solution, pH = 7.4.

6.2.2. Purification of the extract

100 ml of the resulting mixture is applied to the immunoaffinity column at a rate of 1-2 drops per second, washed with 20 cm3 of a phosphate buffer solution, pH = 7.4. Ochratoxin A is eluted with 3 cm3 of methanol-acetic acid (98:2% by volume).

. Taking measurements

7.1. Test sample preparation

The eluate is evaporated to dryness. The dry residue is dissolved in 400 μl of the mobile phase (solution A).

7.2. Chromatography conditions

HPLC conditions: mobile phase - acetonitrile-water (60:40% vol.; pH = 3.0); mobile phase rate - 1.5 cm3/min.

The fluorimetric detector is set to a wavelength of exciting radiation of 333 nm, an emission filter with a bandwidth of 466 nm is installed on the emission line.

. Processing measurement results

8.1. Construction of a graduated dependence

The result of the analysis is presented in the form (with a probability R = 0,95):

D - absolute error limit:

d is the limit of the relative error of the technique (accuracy index), % (Table 1).

* 0.0001 mg/kg - limit of detection.

. Performer qualification requirements

To perform the analysis of ochratoxin A in grain and grain products, persons with a special higher education or a secondary specialized education who own the technique of HPLC analysis, have received appropriate training and have experience in a chemical laboratory are allowed.

. Measurement conditions

Ambient temperature from 15 to 25 °С.

Relative air humidity no more than 80% at 25 °С.

Atmospheric pressure 730 - 760 mm Hg.

Power supply voltage: 210 - 220 V. AC frequency: 45 - 50 Hz.