Concentration of ochratoxin A in the sample, mg/kg

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

Standard deviation repeatability (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-aqueous weighing 1.15 g, a portion of sodium disubstituted 2-aqueous 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 in retention time with ochratoxin A, calculate the mass of ochratoxin A in a poke 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 ( FROM ¢ (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.

Carrying out algorithm operational control error 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 ( FROM ¢ (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.

INTERSTATE COUNCIL FOR STANDARDIZATION, METROLOGY AND CERTIFICATION

INTERSTATE COUNCIL FOR STANDARDIZATION, METROLOGY AND CERTIFICATION

INTERSTATE

STANDARD

WINE AND WINE MATERIALS

Determination of ochratoxin A content by high performance liquid chromatography

Official edition

Standartinform

Foreword

The goals, basic principles and basic procedure for carrying out work on interstate standardization are established by GOST 1.0-92 “Interstate standardization system. Basic Provisions” and GOST 1.2-2009 “Interstate Standardization System. Interstate standards, rules and recommendations for interstate standardization. Rules for the development, adoption, application, updating and cancellation "

About the standard

1 DEVELOPED by the Limited Liability Company "Lumex-Marketing" (LLC "Lumex Marketing")

2 INTRODUCED by the Federal Agency for Technical Regulation and Metrology (Rosstaidart)

3 ADOPTED by the Interstate Council for Standardization, Metrology and Certification (Minutes No. 47 dated June 18, 2015)

4 Order federal agency on technical regulation and metrology dated July 21, 2015 No. 948-st interstate standard GOST 33287-2015 was put into effect as a national standard Russian Federation from January 1, 2017

5 INTRODUCED FOR THE FIRST TIME

Information about changes to this standard is published in the annual information index "National Standards", and the text of changes and amendments - in the monthly information index "National Standards". In case of revision (replacement) or cancellation of this standard, a corresponding notice will be published in the monthly information index to National Standards. Relevant information, notification and texts are also placed in information system general use - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet

© Standartinform. 2016

In the Russian Federation, this standard cannot be fully or partially reproduced, replicated and distributed as an official publication without the permission of the Federal Agency for Technical Regulation and Metrology

INTERSTATE STANDARD

WINE AND WINE MATERIALS

Determination of ochratoxic A content by high performance liquid chromatography

Wine and wine materials.

Determination of ochratoxin A content by high performance liquid chromatography

Introduction date - 2017-01-01

1 area of ​​use

This International Standard applies to wine and wine materials and establishes a method for determining the mass concentration of ochratoxin A using high performance liquid chromatography (HPLC).

The measurement range of the mass concentration of ochratoxin A is from 0.001 to 0.1 mg/dm 3 .

8 of this standard uses normative references to the following standards:

GOST 12.1.004-91 Occupational safety standards system. Fire safety. General requirements

GOST 12.1.007-76 Occupational safety standards system. Classification and general safety requirements

GOST 12.1.010-76 Occupational safety standards system. Explosion-proof. General requirements

GOST 12.1.019-79 Occupational safety standards system. Electrical safety. General requirements and nomenclature of types of protection

GOST 61-75 Acetic acid. Specifications

GOST 1770-74 (ISO 1042-63. ISO 4788-60) Measuring laboratory glassware. Cylinders, beakers, flasks, test tubes. General specifications

GOST ISO 3696-2013 Water for laboratory analysis. Technical requirements and control methods

GOST 4233-77 Reagents. Sodium chloride. Specifications GOST 4204-77 Reagents. Sulfuric acid. Specifications

GOST ISO 5725*6-2003° Accuracy (correctness and precision) of measurement methods and results. Part 6. Use of accuracy values ​​in practice GOST 6709-72 Distilled water. Specifications GOST 9293-74 (ISO 2435-73) Gaseous and liquid nitrogen. Specifications GOST 16317-87 Household electric refrigeration appliances. General specifications GOST ISO/IEC 17025-2009 General requirements for the competence of testing and calibration laboratories

GOST 25336-82 Glassware and laboratory equipment. Types. Basic parameters and dimensions GOST 29227-91 (ISO 835*1-61) Laboratory glassware. Pipettes graduated. Part 1. General requirements

GOST 31730-2012 Wine products. Acceptance rules and sampling methods GOST OIML R 76-1-2011 State system ensuring the uniformity of measurements. Non-automatic scales. Part 1. Metrological and technical requirements. Tests

Note - When using this standard, it is advisable to check the validity of reference standards in the public information system - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet or according to the annual information

"" In the Russian Federation there is GOST R ISO 5725-6-2002 "Accuracy (correctness and precision) of measurement methods and results. Part 6. Using precision values ​​in practice.

index "National Standards", which was published as of January 1 of the current year, and according to the issues of the monthly information index "National Standards)" for the current year. If the reference standard is replaced (modified), then when using this standard, you should be guided by the replacing (modified) standard. If the referenced standard is canceled without replacement, the provision in which the reference to it is given applies to the extent that this reference is not affected.

3 Sampling and preparation of a sample for testing

Sampling according to GOST 31730.

Wines with a high content of carbon dioxide are degassed beforehand. To do this, 50 ml of the product is placed in a flask with a 100 ml tube (see 6.22), shaken and connected to a vacuum pump (see 6.6). Degas for 10-15 minutes until the foam disappears and large bubbles appear on the surface of the liquid.

4 Safety requirements

When carrying out measurements, the following requirements must be observed:

Electrical safety in accordance with GOST 12.1.019 and technical documentation to the chromatograph:

Eeryesafety in accordance with GOST 12.1.010;

Fire safety in accordance with GOST 12.1.004;

Safety when working with harmful substances in accordance with GOST 12.1.007.

WARNING - Ochratoxin A causes kidney and liver damage and

presumably a carcinogen. All work related to sample preparation and preparation of ochratoxin A solutions should be carried out in a fume hood using protective clothing, gloves and goggles. Decontamination of glassware that has been in contact with ochratoxin A is carried out with a 4% solution of sodium hypochlorite.

5 Essence of the method

The method is based on the extraction of ochratoxin A from the sample with acidified methylene chloride, concentration of the obtained extract, screening of samples and determination of the mass concentration of ochratoxin A using HPLC on a reverse column with fluorimetric detection.

6 Measuring instruments, auxiliary equipment, reference materials, reagents, glassware and materials

6.1 Liquid chromatograph with a fluorimetric or spsktrofluorimstrich detector providing excitation of fluorescence in the spectral region (330 ± 20) nm and registration of fluorescence intensity in the spectral region (465 ± 20) nm. The applied detector should provide the limit of detection of ochratoxin A not more than 5 ng/cm 3 .

6.2 Non-automatic scales according to GOST OIML R 76-1 with limits of permissible absolute error not more than ± 0.01 g.

6.3 Analytical chromatographic column, filled with a reversed phase sorbent with a particle size of 5 µm. having an efficiency of at least 5000 theoretical plates for the peak of ochratoxin A "\

6.4 Guard column of the same internal diameter and filled with the same reversed phase sorbent as the analytical column.

6.5 Rotary evaporator, equipped with a water bath with a temperature regulator in the range from 20 °C to 50 °C.

6.6 Laboratory vacuum, diaphragm or water jet pump according to GOST 25336, providing vacuum from 2.5 to 10 kPa.

0 An example of a commercial product that meets the specified requirements. - chromatographic column with an internal diameter of 2.1 mm and a length of 120 mm. filled with reverse-faed sorbent Kromasil S-18. Alltfcna C18 and others with a particle size of 5 μm. equipped with a 25 mm long pre-cup. This information is given for the convenience of users of this International Standard and does not constitute an endorsement of the specified product.

6.7 Drying cabinet, providing temperature up to 200 C.

6.8 Household refrigerator according to GOST 16317.

6.9 Laboratory centrifuge with a speed of at least 5000 rpm.

6.10 Interstate or metrologically provided in the national measurement system of the state that adopted the standard, state standard sample 1 "of the composition of a solution of ochratoxin A in acetonitrile, mass concentration of 50 μg / cm 3 with an error of the certified value of not more than ± 2.5 μg / cm 3. The use of standard samples of the composition is allowed solution of ochratoxin A in other solvents, which must be taken into account when preparing the initial solution according to 7.3.1.

6.11 distilled water in accordance with GOST 6709 or water for laboratory analysis of purity 1 in accordance with GOST ISO 3696.

6.12 Acetic acid according to GOST 61. glacial.

6.13 Acetonitrile for liquid chromatography, optical density relative to distilled water at 200 nm, not more than 0.025, mass fraction of water, not more than 0.03%.

6.14 Methylene chloride for high performance liquid chromatography according to regulatory documents operating in the territory of the state that adopted the standard.

6.15 Sulfuric acid according to GOST 4204, x. h or h.

8.16 Sodium chloride according to GOST 4233. x. h.

6.17 Pipettes, graduated 1-2-2-1. 1-2-2-2. 1-2*2*5, 1-2-2-10 or other types and designs according to GOST 29227.

6.18 Measuring cylinders 1-25-2.1-50-2.1-250-2 or other designs according to GOST 1770.

6.19 Volumetric flasks 2-25-2. 2-50-2.2-100-2. 2-500-2 according to GOST 1770.

6.20 Pointed-bottom flasks 0-10-14/23 and 0-50-14/23 according to GOST 25336.

6.21 Flat-bottom flasks P-1-50-29/32, P-1-10O-29/32. P-1-20O-29/32. P-1-250-29/32 or Kn-1-50-29/32. Kn-1-100-29/32. Kn-1 -250-29/32 according to GOST 25336.

6.22 Flasks with a tube 2-100-19/26.2-250-29/32 according to GOST 25336.

6.23 Dividing funnels, type VD, versions 1 or 3, with a capacity of 50 cm 3 according to GOST 25336.

6.24 Laboratory funnels type B according to GOST 25336.

6.25 Paper filters "red tape" according to the regulatory documents in force in the territory of the state that adopted the standard.

6.26 Glass containers with a capacity of 25.50.250, 1000 cm 3 with ground glass, fluoroplastic or polyethylene stoppers according to the regulatory documents in force on the territory of the state that adopted the standard.

6.27 Hourglass or timer according to the regulations in force in the territory of the state that adopted the standard.

It is allowed to use other measuring instruments with metrological characteristics not worse than the above and auxiliary equipment, reagents and materials with technical specifications no worse than the above.

7 Preparation for the test

7.1 Preparing glassware

Vessels for the preparation and storage of the mobile phase are treated only with sulfuric acid according to 6.15 without the use of other detergents, washed thoroughly with tap water and rinsed with distilled water.

The rest of the glassware is treated with hot water and detergent, rinsed thoroughly with distilled water and dried in an oven at a temperature of 105°C.

7.2 Preparation of auxiliary solutions

7.2.1 Mobile phase preparation

8 a pre-prepared glass container with a capacity of 1000 cm 3 with a tightly closed ground glass, fluoroplastic or polyethylene stopper is placed 5 cm 3 of glacial acetic acid (see 6.12), 215 cm 3 of acetonitrile (see 6.13) and 280 cm 3 of distilled water. The mixture is thoroughly mixed. When storing the mobile phase, the use of rubber or cork stoppers is unacceptable.

Shelf life of the mixture at room temperature - no more than 1 month.

Before use, the mobile phase is degassed and filtered according to the chromatograph manufacturer's recommendations.

"In the Russian Federation - a standard sample of an approved type.

7.2.2 Preparation of a mixture of methylene chloride and acetic acid in a volume ratio of 200:1

Place 200 ml of methylene chloride (6.14) and 1.0 ml of glacial acetic acid (6.12) in a 250 ml flat-bottomed flask.

The shelf life of the mixture at room temperature in a glass container with a ground glass, fluoroplastic or polyethylene stopper is not more than 1 month.

7.2.3 Preparation of a solution of sodium chloride with a mass fraction of 20%.

Place 20 g of sodium chloride (see 6.16) into a flat-bottomed flask with a capacity of 200 cm 3, add 80 cm 3 of distilled water, mix thoroughly.

Shelf life of the solution at room temperature - no more than 3 months.

7.3 Preparation of ochratoxin A solutions

7.3.1 Preparation of a stock solution of ochratoxin A with a nominal mass concentration of 1 µg/cm 3

Pipette 1 cm 3 of a standard sample of the composition of a solution of ochratoxin A in acetonitrile with a mass concentration of 50 µg/cm 3 (see 6.10). place in a 50 ml volumetric flask and dilute to the mark with acetonitrile (6.13).

The shelf life of the prepared solution in the refrigerator at a temperature of 2 ° C to 6 * C - no more than 6 months.

The actual value of the mass concentration of ochratoxin A in the initial solution (Cm, μg / cm 3) is calculated by the formula

where Cco is the certified value of the mass concentration of ochratoxin A in the standard sample according to the passport, µg/cm 3 ;

Vco - the volume of the standard sample of the composition of the solution of ochratoxin A. selected for the preparation of the initial solution, cm 3 (1 cm 3);

V^, - the volume of the volumetric flask used to prepare the initial solution, cm 3 (50 cm 3).

Note - When using a standard sample of the composition of a solution of ochratoxin A in other solvents, an aliquot (1 cm 3) is evaporated to a dry residue in vacuum at a water bath temperature from 40 "C to 45 * C or in a stream of nitrogen. The dry residue is dissolved in 2 cm 1 of acetonitrile and stirred for 1 minute Then the resulting solution is quantitatively transferred into a volumetric flask with a capacity of 50 cm 3 and diluted to the mark with acetonitrile.

7.3.2 Preparation of a mobile phase solution of ochratoxin A with a nominal mass concentration of 50 ng/cm

In a volumetric flask with a capacity of 50 cm 3, place 2.5 cm 3 of the initial solution of ochratoxin A according to 7.3.1 and bring the volume to the mark with the mobile phase according to 7.2.1.

The shelf life of the resulting solution in a refrigerator at a temperature of 2 to 6 and C - no more than 3 months.

The actual value of the mass concentration of ochratoxin A in solution (Co, ng / cm 3) is calculated by the formula

С„ = r ~, ; v ~ -10QQ. (2)

where Cm is the actual value of the mass concentration of ochratoxin A in the initial solution (see 7.3.1), µg/cm 3 ;

The volume of the initial solution according to 7.3.1. selected for the preparation of this solution. cm 3 (2.5 cm 3);

Vo is the volume of the volumetric flask used to prepare the initial solution, cm 3 (50 cm 3);

1000 - the coefficient of coordination of the dimensions of units of mass.

7.3.3 Preparation of ochratoxin A calibration solutions

The initial solution of ochratoxin A in acetonitrile according to 7.3.1 is placed in a volumetric flask with a capacity of 50 cm 3 . The volume of ochratoxin A solution must meet the requirements of Table 1.

Table 1

The contents of the flask are diluted to the mark with the mobile phase according to 7.2.1 in accordance with Table 1.

The shelf life of calibration solutions in a refrigerator at a temperature of 2 °C to 6 °C is no more than seven days.

NOTE If necessary, for example to add ochragoxin A to the sample (see 8.2). it is allowed to prepare solutions of ochratoxin A of other concentrations in a similar way.

The actual value of the mass concentration of ochratoxin A in the calibration solutions is calculated by formula (2). based on the values ​​of the volumes of the initial solution according to table 1.

Before use, the solutions are kept until room temperature is reached.

7.4 Preparing the chromatograph

Preparation of the chromatograph for measurements is carried out in accordance with the manual (instruction) for operation.

Set the operating wavelengths for excitation and detection of fluorescence (see 6.1). The volumetric flow rate of the mobile phase and the dosing volume of the sample are set depending on the column sizes, guided by the instructions of the chromatograph and column manufacturer. For example, for the chromatographic column given in 6.3. the value of the space velocity of 200 mm 3 / min and the volume of the loop of the dosing valve from 10 to 20 mm are recommended. If there is a column thermostat, the temperature is set to (25 ± 1) ° С.

7.5 Chromatograph calibration

The linearity range of the calibration characteristic is from 5 to 100 ng/cm*. As samples for calibration of the chromatograph, calibration solutions of ochratoxin A no 7.3.3 are used.

Two chromatograms of each calibration solution are registered and, using the software for the chromatograph, the chromatograph is calibrated by setting the parameters of the calibration characteristic and the retention time of ochratoxin A.

Calculate the correlation coefficient and the deviation of the calculated values ​​of the mass concentration of ochratoxin A at each calibration point from the actual value in accordance with the procedure for preparing calibration solutions (see 7.3.3).

Graduation is considered acceptable if:

Correlation coefficient not less than 0.998:

The relative deviation of the calculated value of the mass concentration of ochratoxin A from the actual value is no more than ± 10%.

7.6 Checking the stability of the calibration characteristic

Control of the stability of the calibration characteristics is carried out daily before starting work.

8 as a control solution, use a solution of ochratoxin A in the mobile phase, prepared in the same way as 7.3.3. The mass concentration of ochratoxin A in the control solution is selected based on the expected content of ochratoxin A in the test samples: it is recommended to use a solution of ochratoxin A with a mass concentration of 20 ng/cm.

Record at least two control solution chromatograms and identify the ochratoxin A peak by retention time at a 5% identification window width. introducing, if necessary, software correction of the peak retention time, and using the calibration characteristic, the mass concentration of ochratoxin A is calculated for each input.

The repeatability of retention times and mass concentrations of ochratoxin A is checked using the formulas

l^Z^lisO.05, (3)

where h and Tr are the retention times of the ochratoxin A peak in the first and second chromatograms, respectively, min:

f is the arithmetic mean /, and t 2 , min.

i, _ "" * 0L7"

With.

where Cl and Cl2 - mass concentrations of ochratoxin A in the control solution according to the first and second chromatograms, respectively, ng/cm 3 ;

C to - the arithmetic mean of the values ​​of Cxi and C«. ng / cm 3.

The calibration dependence is recognized as stable if the condition is met where C is the actual value of the mass concentration of ochratoxin A in the control solution, ng / cm 3 -

If condition (5) is not met, then the control procedure is repeated. The results of the repeated control are considered final, and the calibration of the chromatograph according to 7.5 is carried out again.

7.7 Blank control

A blank test is carried out before testing the test samples.

15 cm * mixture of methylene chloride and acetic acid is placed in a flask for evaporation.

7.2.2 and evaporate in vacuo to dryness by placing the flask in a water bath at 40 to 45°C.

The dry residue is dissolved in 0.5 ml* of the mobile phase according to 7.2.1. stand for at least 5 minutes and carry out a chromatographic analysis of the resulting concentrate according to 8.3. If the chromatogram contains peaks that are close in retention time to the peak of ochratoxin A, then the causes of contamination of the blank sample are found and eliminated.

NOTE The most common reason for poor blank control results is insufficient purity of methylene chloride, which can be contaminated with impurities that have retention times close to those of ochratoxin A. Such methylene chloride must be replaced or subjected to thorough distillation, collecting the middle fraction with a boiling point of 39 * C to 40 * C.

8 Testing

8.1 Extraction of ochratoxin A from the sample

Pipette 5 cm 3 of the sample into a separating funnel, add 5 cm 3 of sodium chloride solution according to 7.2.3, add 5 cm 3 of a mixture of methylene chloride and acetic acid according to 7.2.2 and shake for 1 min. After phase separation, the lower organic layer is filtered through a red tape filter pre-moistened with acidified methylene chloride into an evaporating flask.

Note - When a stable emulsion is formed, it is recommended to centrifuge the mixture for 2 minutes at a speed of 5000 rpm.

Repeat the extraction of ochratoxin A from the upper layer once again with 5 cm 3 of a mixture of acetic acid and methylene chloride according to 7.2.2. The resulting extract is filtered into the same evaporation flask.

The filter is washed with acidified methylene chloride with a volume of 5 to 10 cm 3 . The extract is evaporated to dryness in vacuo by placing the flask in a water bath at a temperature of 40°C to 45°C. The dry residue is dissolved in 0.5 cm* of the mobile phase, thus obtaining a sample concentrate.

Note -On the stage mastering the method, when changing the batch of the extractant, as well as when there is doubt about the reliability of the results obtained, find the transmission coefficient of ochratoxin A in the control sample in accordance with Appendix A and check the acceptability of the obtained value.

8.2 Screening Samples

Wine sample concentrate obtained according to 8.1. hold for at least 5 minutes. and then carry out its chromatographic analysis according to 8.3.

If there is no peak on the chromatogram identified as the peak of ochratoxin A, it is concluded that there is no ochratoxin A in the sample at the level of the lower limit of the measurement range (0.001 mg / dm 3) and the sample with the addition of ochratoxin A is not prepared.

If the sample chromatogram shows a peak. identified by the chromatograph software as an ochratoxin A peak (see 8.3). that is, the analyzed sample is introduced

an additive in the form of a solution of ochratoxin A in the mobile phase (see 7.3.2). The recommended volume of addition of ochratoxin A solution (V 4P . cm 3) is calculated by the formula

(6) where o is a coefficient, the value of which is chosen in the range from 0.5 to 2.0:

C* is the mass concentration of ochratoxin A in the sample concentrate (see 8.3). ng / cm 3;

Us is the volume of the sample concentrate, cm 3 (0.5 cm 3);

Set - mass concentration of ochratoxin A in the solution used to add the additive. ng / cm 3.

The volume of the added additive should not exceed 5% of the sample volume (U pr. cm 3). If this requirement is not consistent with the values ​​calculated by formula (6). then, to add the additive of ochratoxin A, a solution with a different mass concentration value is used.

Analyze the spiked sample according to 8.1.

8.3 Making chromatographic measurements

Record at least two chromatograms of the test sample concentrate (see 8.1) and the sample with additive (see 8.2), under the same conditions under which the chromatograph was calibrated. Identification of ochratoxin A is carried out by matching the retention time of ochratoxin A in the sample extract with its retention time obtained by monitoring the stability of the calibration characteristic, setting the width of the identification window to 5%.

An example of a chromatogram is shown in Figure B.1 (Appendix B).

Note - If it is necessary to confirm the correct identification of the ochratoxin A peak, it is recommended to add the solution of ochratoxin A in the mobile phase to the sample concentrate. The reliability of identification can be judged by the increase in the height of the expected peak of ochratoxin A. The amount of added ochratoxin A solution is determined based on the fact that the mass concentration of ochratoxin A in the sample should increase by (50 - 150)% compared to the initial value.

If there is a peak on the chromatogram of the sample concentrate, identified as the peak of ochratoxin A. calculate the mass concentration of ochratoxin A in the sample concentrate for each registered chromatogram using the calibration characteristic established in 7.5. and check the acceptability of the obtained values ​​using condition (4). If condition (4) is met, then as a result of measuring the mass concentration of ochratoxin A in the concentrate of the test sample, the arithmetic mean of the obtained concentrations (Cx. ng / cm 3) is taken. If condition (4) is not met, then the causes of instability are found and eliminated, after which the introduction of the sample concentrate is repeated.

When analyzing the sample concentrate with the addition of ochratoxin A (see 8.2), also record two chromatograms, identify the peak of ochratoxin A and calculate the mass concentration of ochratoxin in the concentrate for each chromatogram, check the acceptability of the obtained values ​​and calculate the mass concentration of ochratoxin A in the concentrate of the sample with the addition ( C X 4 D. ng / dm 3) as the arithmetic mean of the obtained values.

If the mass concentration of ochratoxin A in the sample concentrate exceeds 100 ng/cm3, then the wine sample is diluted with water according to 6.11 and the diluted sample is reanalyzed according to 8.1. The dilution factor O is calculated by the formula

where U p is the volume of the diluted sample, cm 3;

Y 4 - the volume of an aliquot of the test sample taken for dilution, cm 3 .

9 Processing of test results

The mass concentration of ochratoxin A in a wine sample (X. mg / dm) is calculated by the formula

V---ts--:--o-yu ’

where Cst is the mass concentration of the ochratoxin A solution used as an additive (see 8.2). ng/cm3:

Volume of addition of ochratoxin A solution to the sample (see 8.2), cm 3:

Vpp is the volume of the sample taken for testing according to 8.1. cm 3 (5cm 3);

Cx is the mass concentration of ochratoxin A in the sample concentrate (see 8.3), ng/cm3:

From x. c = mass concentration of ochratoxin A in the spiked sample concentrate (see 8.3). ng/cm3:

O is the dilution factor of the wine sample (see 8.3). If the sample was not diluted, then O g 1;

10" 3 - the coefficient of coordination of the dimensions of the units of mass and volume.

10 Metrological characteristics

The method provides measurement results with metrological characteristics not exceeding the values ​​given in Table 2.

table 2

Measuring range. mg/dm 1	Repeatability limit (the relative value of the allowable discrepancy between two measurement results obtained under repeatability conditions at P * 0.95) g 0, „. %	Critical difference (relative value of the allowable discrepancy between two measurement results obtained under reproducibility conditions at P = 0.951 C0% V. kg / dm 1	Accuracy indicator (boundaries* of relative error at confidence level Р = 0.95).
From 0.001 to 0.005 inclusive
Se. 0.005 » 0.1 »
* The established numerical values ​​of the relative error limits correspond to the numerical values ​​of the relative expanded uncertainty with a coverage factor k = 2.

Discrepancy between two measurement results (X| and X 2 , mg/dm - *). obtained in the same laboratory under repeatability conditions, must meet the condition

where X is the arithmetic mean of X, and X 2 . mg/dm:

L>p. - repeatability limit (table 2), %.

When condition (9) is met, the arithmetic mean of the obtained measurement results (X, and X 2 . mg / dm 3) is taken as the measurement result.

The discrepancy between two measurement results obtained in two laboratories (X 1gaC and Hahn, mg / dm 3) on identical samples must correspond to the condition

where Хпов is the arithmetic mean of Х 1лв0 and Х^. mg/DM 3: CO 095 - critical difference (table 2). %.

11 Quality control of measurement results

Control of quality indicators of measurement results in the laboratory provides for monitoring the stability of measurement results, taking into account the requirements of GOST ISO 5725 * 6 (section 6).

12 Presentation of test results

The test results are recorded in a test report, which is drawn up in accordance with the requirements of GOST ISO / IEC 17025, while the test report must contain a reference to this standard.

The results of measurements of the content of ochratoxin A (with laboratory-confirmed compliance of the analytical procedure with the requirements of this standard) are presented in the form

X±L or X±U. (11)

where X is the measurement result obtained in accordance with section 10. mg / dm 3:

A - the limits of the absolute error in measuring the content of ochratoxin A (P - 0.95), mg / dm 3, which are calculated by the formula

A = 0.0!bG: (12)

U is the expanded uncertainty at a coverage factor of k-2. mg / dm 3, which is calculated by formula 8

U = 0.CM (/ w x. (13)

The values ​​of S (U„J are given in Table 2.

The numerical values ​​of the boundaries of the absolute error (uncertainty) are expressed as a number containing no more than two significant figures, while the smallest digit of the numerical value of the final measurement result is taken to be the same. as well as the smallest digit of the numerical value of the boundaries of the absolute error (uncertainty).

Determination of the transmission coefficient of ochratoxin A

To determine the transmission coefficient of ochratoxin A, a control sample is used, for the preparation of which 5 ml of distilled water is placed in a separating funnel, 5 ml of sodium chloride solution according to 7.2. mobile phase (see 7.3.2).

Carry out the extraction of ochratoxin A according to 6.1. prepare the control sample concentrate according to 8.2 and carry out its chromatographic analysis according to 8.3 and. using the calibration characteristic according to 7.5. allocate the mass concentration of ochratoxin A in the control sample concentrate.

Then the transmission coefficient of ochratoxin A (p) is calculated by the formula

where V\ is the volume of the control sample concentrate, cm 9 (0.5 cm *):

Cx is the measured value of the mass concentration of ochratoxin A in the control sample concentrate, ng/cm*:

C. - the value of the mass concentration of ochratoxin A in solution according to 7.3.2. ng/cm*:

V. - volume of ochratoxin A solution according to 7.3.2. selected for the preparation of a control sample, cm* (0.5

The transmission coefficient of ochratoxin A is determined at least three times under repeatability conditions. The values ​​obtained must meet the following requirements:

Each of the obtained values ​​is not less than 0.8:

The relative value of the range of the obtained values ​​corresponds to the condition

I P||F Pchii I

where iv "t and tv". - the largest and smallest of the obtained values ​​of the transmission coefficient of ochratoxin A;

If both of these conditions are met, then the operations according to 8.1 are considered satisfactory. Otherwise, the reasons for the loss of ochratoxin A are found and the determination of the transmission coefficient is repeated.

Annex B (informative)

Chromatogram Example

semi-sweet

Figure B.1 shows an example of a chromatogram of a red wine sample concentrate (mass concentration of ochratoxin A in the sample is 0.0010 mg/dm 1).

concentration

Figure B.1 - Chromatogram example

The peak of ochratoxin A (marked as OTA in the figure) corresponds to the mass value of ochratoxin A in the concentrate of 7.7 ng/cm*.

UDC 543.544.5.068.7:663.2:006.354 MKS 67.160.10

Key words: wine, wine materials. test methods, high performance liquid chromatography, ochratoxin A. extraction of ochratoxin A. determination of the mass concentration of ochratoxin A. concentration of the extract, screening of samples, fluorimetric detection

Editor K.V. Dudko Proofreader P.M. Smirnov Computer history E.K. Kuzina

Signed for publication on February 8, 2016. Format 60x84V*.

Uel. oven l. 1.86. Circulation 45 copies. Per*. 3872.

Based on electronic version provided by the developer of the standard

Leaflet

Kits and are test systems for enzyme immunoassay. They are commercially available in accordance with ISO 9000 complete with the necessary reagents and are designed for the quantitative determination of ochratoxin in cereals, feed, grain products, beer and blood serum. Guidelines on the use of test systems RIDASCREEN® FAST Ochratoxin A and RIDASCREEN® Ochratoxin A approved by the Department of Veterinary Medicine of the Federal Agency for agriculture Ministry of Agriculture of Russia under the number MUK 5-1-14/1001. The systems are included in the "List of normative documentation permitted for use in state veterinary laboratories in diagnosing diseases of animals, fish, bees, as well as monitoring the safety of raw materials of animal and vegetable origin." Test systems RIDASCREEN® FAST Ochratoxin A correspond GOST 34108-2017"Feed, mixed feed, mixed feed raw materials. Determination of mycotoxin content by direct solid-phase competitive enzyme immunoassay".

Determination of ochratoxin A in grain, feed, grain products, beer and blood serum

Ochratoxin is a poisonous substance formed as a result of the vital activity of mold fungi of the genus Aspergillus and Penicillium. Along with pronounced nephrotoxicity, ochratoxin has hepatotoxicity, teratogenic, carcinogenic and immunosuppressive properties. With products of plant and animal origin, ochratoxin can enter the human body. It is found not only in cereals (13% of positive samples) and animal feed, but also in pig blood (60% of positive samples) and kidneys (21% of positive samples).

Technical Regulations of the Customs Union TR CU 021/2011 "On food safety" regulate the following maximum level of ochratoxin A content: in food grains, cereals, flour - 0.005 mg / kg (5 μg / kg); in baby food, food products for preschoolers and schoolchildren, food products for pregnant and lactating women - not allowed (<0,0005 мг/кг).

Draft federal law № 349084-5 The "Technical Regulations for Wine Products" establishes requirements for the content of ochratoxin A in wine no more than 0.002 mg/l.

The draft Federal Law "On requirements for the safety of food products and processes of their production, storage, transportation, sale and disposal" also includes a requirement for mandatory control of food grains for ochratoxin A, the content of which should not exceed 0.005 mg/kg. The current legal regulations can be found on the website compact24.com.

Until recently, chromatographic methods (high performance liquid chromatography, thin layer chromatography) were predominantly used to control ochratoxin. Much more convenient method of enzyme-linked immunosorbent assay (ELISA, or ELISA), which has a very high sensitivity.

Specification:	RIDASCREEN® FAST Ochratoxin A	RIDASCREEN® Ochratoxin A 30/15
Format:	Strip plate, 48 wells (6 strips of 8 wells)	Strip plate, 96 wells (12 strips of 8 wells)
Standards:	0 / 5 / 10 / 20 / 40 µg/l	0 / 50 / 100 / 300 / 900 / 1800 ng/l
Sample preparation:	Sample grinding, extraction, filtration	extraction, centrifugation/filtering (grain, feed); extraction, centrifugation, filtration, shaking, over-extraction, centrifugation, evaporation (beer/serum)
Time spent:
Limit of detection:	0.005 mg/kg	0.001250 mg/kg (grain, feed) 0.000050 mg/kg (beer, blood serum)

Related products:

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 pose 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.



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INTERSTATE COUNCIL FOR STANDARDIZATION, METROLOGY AND CERTIFICATION

INTERSTATE COUNCIL FOR STANDARDIZATION, METROLOGY AND CERTIFICATION

INTERSTATE

STANDARD

GRAIN AND ITS PROCESSING PRODUCTS, COMPOUND FEED

Determination of ochratoxin A by high performance liquid chromatography

(ISO 15141-1:1998, NEQ)

Official edition

Standartinform

Foreword

The goals, basic principles and basic procedure for carrying out work on interstate standardization are established by GOST 1.0-92 “Interstate standardization system. Basic Provisions” and GOST 1.2-2009 “Interstate Standardization System. Interstate standards, rules and recommendations for interstate standardization. Rules for the development, adoption, application, updating and cancellation "

About the standard

1 DEVELOPED by Lumex Marketing Limited Liability Company (Lumex Marketing LLC)

2 INTRODUCED by the Federal Agency for Technical Regulation and Metrology (TC 335)

3 ADOPTED by the Interstate Council for Standardization, Metrology and Certification (Minutes of November 14, 2013 No. 44-2013)

4 This standard complies with the main provisions of the international standard ISO 15141-1:1998 Foodstuffs - Determination of ochratoxin A in cereals and cereal products - Part 1: High performance liquid chromatographic method with silica gel clean up grain products - Part 1: High performance liquid chromatography method with silica gel purification).

Degree of conformity - non-equivalent (NEQ)

5 By order of the Federal Agency for Technical Regulation and Metrology dated December 30, 2013 N2 2429-st, the interstate standard GOST 32587-2013 was put into effect as the national standard of the Russian Federation from July 1, 2015.

6 INTRODUCED FOR THE FIRST TIME

7 REVISION. March 2016

Information about changes to this standard is published in the annual information index "National Standards", and the text of changes and amendments - in the monthly information index "National Standards". In case of revision (replacement) or cancellation of this standard, a corresponding notice will be published in the monthly information index "National Standards". Relevant information, notification and texts are also posted in the public information system - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet.

© Standartinform, 2016

In the Russian Federation, this standard cannot be fully or partially reproduced, replicated and distributed as an official publication without the permission of the Federal Agency for Technical Regulation and Metrology.

NOTE The reported elution volumes should be changed in accordance with the values ​​obtained from the determination of the ochratoxin A transmission coefficient (see 5.3.10).

5.4.4 Making chromatographic measurements

Record at least two chromatograms of the test sample concentrate obtained according to 5.4.3 under the same conditions under which the system was calibrated. Identification of ochratoxin A is carried out by matching the retention time of ochratoxin A in the sample extract with its retention time obtained by monitoring the stability of the calibration characteristic, setting the width of the identification window to 5%.

An example of a chromatogram is shown in Figure A.1 (Appendix A).

If there is a peak on the chromatogram of the concentrate of the sample, identified as the peak of ochratoxin A, a conclusion is made about the presence of ochratoxin A in the sample, its content is determined for each registered chromatogram, and the discrepancy between the obtained values ​​is checked using formula (2).

If the condition of formula (2) is met, then the arithmetic mean of the obtained concentrations is taken as the result of measurements of the mass concentration of ochratoxin A in the concentrate of the test sample. If the condition of formula (2) is not met, then the causes of instability are found and eliminated, after which the introduction of the sample concentrate is repeated.

If it is necessary to confirm the correct identification of the ochratoxin A peak, it is recommended to add the solution of ochratoxin A in the mobile phase to the sample concentrate. The reliability of identification can be judged by the increase in the height of the expected peak of ochratoxin A. The amount of added ochratoxin A solution is determined based on the fact that the mass fraction of ochratoxin A in the sample should increase by 50% -150% compared to the initial value.

If the mass concentration of ochratoxin A in the final concentrate C x exceeds 100 ng/cm 3 , then it is diluted with the mobile phase (see 5.3.2.1). The dilution factor Q is calculated by the formula

(8)

where Vp is the volume of the diluted concentrate of the test sample, cm 3 ;

V a is the volume of an aliquot of the original concentrate of the test sample, taken for dilution,

5.5 Processing of test results

The mass fraction of ochratoxin A in sample X, ppm 1, is calculated by the formula

X= V 1- V 2" C X .Q.10\ (9)

where Vi is the volume of chloroform taken for extraction, cm 3 (30 cm 3);

V 2 - the volume of the final concentrate of the sample, cm 3 (0.5 cm 3);

Cx is the mass concentration of ochratoxin A in the final sample concentrate (see 5.4.4), ng/cm 3 ; V 3 - the volume of the chloroform extract taken for testing according to 5.4.3, cm 3 (20 cm 3);

/l - sample weight, g;

P is the transmission coefficient of ochratoxin A (see 5.3.10);

Q is the dilution factor of the sample concentrate (see 5.4.4);

5.6 Metrological characteristics

The method provides measurement results with metrological characteristics not exceeding the values ​​given in Table 2.

table 2

Measurement range, million" 1 Repeatability limit g, ppm 1 Reproducibility limit R, ppm 1 Accuracy indicator (limits** of absolute error at confidence probability P = 0.95), ± D, mln" 1 From 0.0025 to 0.05 inclusive St. 0.05 » 1.0 » X is the result of measurements at 5.5, ppm. ** The established numerical values ​​of the absolute error limits correspond to the numerical values ​​of the expanded uncertainty U (in absolute units) with a coverage factor k = 2.

The discrepancy between two measurement results (X| and X 2, ppm 1) obtained in the same laboratory under repeatability conditions should correspond to the condition

\X 1 -X 2 \<г, (10)

where r is the repeatability limit, million" 1 (table 2).

When condition (10) is met, the arithmetic mean of the obtained measurement results (Xj and X 2, million "1) is taken as the measurement result.

The discrepancy between two measurement results obtained in two laboratories (Xi na 6 and X 2la b, ppm 1) on identical samples should correspond to the condition

where R is the limit of reproducibility, ppm 1 (table 2).

5.7 Quality control of measurement results

Control of quality indicators of measurement results in the laboratory provides for monitoring the stability of measurement results, taking into account the requirements of GOST ISO 5725-6 (section 6).

I

5.8 Presentation of test results

The test results are recorded in a test report, which is drawn up in accordance with the requirements of GOST ISO / IEC 17025, while the test report must contain a reference to this standard.

The results of measurements of the content of ochratoxin A (with laboratory-confirmed compliance of the analytical procedure with the requirements of this standard) are presented in the form

X ± D, million "1 or X ± U, million" 1, (12)

where X is the measurement result obtained in accordance with 5.6, ppm 1 ;

A - the limits of the absolute error in measuring the content of ochratoxin A (P = 0.95) according to

table 2, million" 1;

U is the expanded uncertainty with a coverage factor k = 2, million" 1 .

The numerical value of the absolute error (uncertainty) boundaries is expressed as a number containing no more than two significant digits, while the smallest digit of the numerical value of the final measurement result is taken to be the same as the smallest digit of the numerical value of the absolute error (uncertainty) boundaries.

6 Method B

6.1 Essence of the method

The method is based on the extraction of ochratoxin A with toluene after acidifying the test sample with hydrochloric acid and adding magnesium chloride to increase the ionic strength. The extract was purified using solid phase extraction columns filled with silica gel, and the content of ochratoxin A was determined by HPLC on a reverse phase column using fluorimetric detection. If necessary, confirm the identification of ochratoxin A by derivatization with a solution of boron trifluoride in methanol.

6.2 Reagents

When carrying out tests, use water according to 5.2.13, reagents according to 5.2.14 - 5.2.17, as well as the following.

6.2.7 Mixed solvent No. 1: toluene (6.2.3) and glacial acetic acid (5.2.15) in a volume ratio of 99:1.

6.2.8 Mixed solvent No. 2: acetone (6.2.5) and toluene (6.2.3) in a volume ratio of 5:95.

6.2.9 Mixed solvent No. 3: toluene (6.2.3) and glacial acetic acid (5.2.15) in a 90:10 volume ratio.

6.2.10 Mobile phase

Mix 99 parts by volume of acetonitrile (5.2.16) with 99 parts by volume of water (5.2.13) and two parts by volume of glacial acetic acid (5.2.15). The mobile phase is degassed before use.

6.2.11 Boron trifluoride, mass fraction of the main substance, not less than

6.2.12 Solution of boron trifluoride in methanol, p (BF 3) = 14 g/100 cm 3 .

ATTENTION! It is necessary to work in a well-functioning fume hood. Avoid contact with skin, eyes and respiratory organs.

6.2.13 Ochratoxin A in crystalline form or in the form of an ampoule preparation in film form with a mass fraction of the main substance of at least 95%

6.2.14 Ochratoxin A stock solution

Dissolve 1 mg of crystalline ochratoxin A (6.2.13) or the contents of the film ampoule containing ochratoxin A in such a volume of mixed solvent No. 1 (6.2.7) to obtain a solution of 20 to 30 µg of ochratoxin A, by mass. /cm 3 .

To determine the exact value of the mass concentration of ochratoxin A in the main solution, record its optical density in the wavelength range from 300 to 370 nm in 5 nm steps in a quartz cuvette with an optical layer thickness of 1 cm according to 6.3.2 using a spectrophotometer (see 6.3.1 ). Mixed solvent No. 1 according to 6.2.7 is used as a reference solution. The exact position of the absorption maximum is identified by recording the optical density near it in 1 nm increments. Calculate the value of the mass concentration of ochratoxin A, C ref, µg/cm 3 , according to the formula

where A max is the optical density at the absorption maximum (in this case at 333 nm);

M is the molar mass of ochratoxin A, g/mol (M=403.8 g/mol);

100 - coefficient of agreement between units of length and mass; e - molar absorption coefficient of ochratoxin A in mixed solvent No. 1, m 2 / mol, (e = 544 m 2 / mol);

/ is the thickness of the absorbing layer of the cuvette, see

6.2.15 Ochratoxin A, 1 µg/cm3 intermediate solution Evaporate to dryness in a stream of nitrogen an aliquot of the stock solution (6.2.14) containing 100 µg of ochratoxin A and dissolve in 100 ml of the mobile phase (6.2. ten). The volume of the aliquot is calculated by the formula:

\/ a - aliquot volume, cm 3 ;

100 - mass of ochratoxin A, mcg;

Cie* - mass concentration of the initial solution of ochratoxin A according to 6.2.14, µg/cm 3 .

The shelf life of the solution in a refrigerator at a temperature of 2°C to 6°C and regular monitoring of its stability is no more than 6 months.

6.2.16 Ochratoxin A calibration solutions

In volumetric flasks with a capacity of 100 cm 3 place 1; 2.5; 4 and 5 ml of ochratoxin A intermediate solution (6.2.15) and diluted to the mark with mobile phase (6.2.10). The mass concentration of ochratoxin A in the prepared calibration solutions is 10; 25; 40 and 50 ng/cm 3 respectively, which corresponds to the mass of ochratoxin And 0.2; 0.5; 0.8 and 1.0 ng in dosing volume 20

6.3 Measuring instruments, auxiliary equipment, materials and utensils

When conducting tests, use measuring instruments, auxiliary equipment, materials and utensils according to 5.2.1 - 5.2.2, 5.2.6 - 5.2.11, 5.2.21 -5.2.34, as well as the following.

6.3.1 Spectrophotometer, suitable for measurements in the wavelength range from 300 to 370 nm and having a spectral bandwidth of not more than 2 nm.

6.3.2 Quartz cuvettes with an absorbing layer thickness of 1 cm, not absorbing light in the wavelength range from 300 to 370 nm.

6.3.3 Centrifuge tubes, for example, 250 ml capacity, made of high-density polyethylene with screw caps.

6.3.4 Refrigerated centrifuge, preferably refrigerated, for centrifuge tubes (see 6.3.3), capable of generating an acceleration of at least 3 500 in.

NOTE The centrifuge speed to achieve the specified acceleration is selected according to the centrifuge manufacturer's recommendations.

6.3.5 SPE column, disposable, filled with silica gel.

After opening the package, the column is conditioned for two hours at 105°C and stored over activated silica gel with a moisture indicator. Pass 10 ml of toluene through the column before use (see 6.2.3). With each new batch of columns, the yield of ochratoxin A is checked. For example, columns made from a 3 cm3 polypropylene tube with the following characteristics are suitable:

The average weight of silica gel is 690 mg;

Pore ​​size - 12.5 nm;

Particle size - from 55 to 105 microns.

6.3.6 Reservoirs for solvents, such as syringes, for example 50 ml, with center hole and stopper.

6.3.7 Separating funnel with a capacity of 50 cm 3 according to GOST 25336.

6.3.8 Membrane filters for aqueous solutions, made of polytetrafluoroethylene, with a pore size of 0.45 µm.

6.3.9 Crimp or screw cap vials.

6.3.10 Microspirits, 500 mm 3 .

6.3.11 HPLC apparatus

6.3.11.1 Liquid chromatograph according to 5.2.1.

Length................................................. ..250 mm;

Inner Diameter...................................4 mm;

The size of spherical particles .......................... 5 µm.

6.3.11.2 Analytical chromatographic column, filled with 5 µm reversed-phase sorbent, providing separation of the ochratoxin A peak and the remaining peaks to baseline 1 with the following characteristics:

Note - It is allowed to use analytical columns of other standard sizes (for example, from 120 to 150 mm long).

6.3.11.3 Guard column of the same internal diameter and filled with the same reverse

phase sorbent, which is the same as the analytical column with a length

6.3.11.4 Guard column of the same internal diameter and filled with the same reverse phase sorbent as the analytical column, for example, with the following characteristics:

Length................................................. ...40 mm;

Inner Diameter...................................4mm;

The size of spherical particles .......................... 5 µm.

Note - It is allowed to use pre-columns of other standard sizes.

It is allowed to use other measuring instruments with metrological characteristics and auxiliary equipment with technical characteristics not worse, as well as materials and reagents in quality not lower than the above.

6.4 Testing

6.4.1 General

Tests must be completed within one working day.

6.4.2 Extraction of ochratoxin A from the sample

Place (20,0 ± 0,1) g of the sample prepared in accordance with Section 3 in the centrifuge tube (6.3.3). If an ochratoxin content of more than 0.005 ppm is expected, the sample weight is reduced to 10 g; otherwise, the possible risk of reduced recovery of ochratoxin A from the sample should be taken into account.

Add successively 30 ml of hydrochloric acid solution (see 6.2.1), 50 ml of magnesium chloride solution (see 6.2.2), mix with a glass rod and add 100 ml of toluene

Stir for 60 minutes and then centrifuge the suspension. The centrifugation time depends on the efficiency of the centrifuge. Centrifugation is carried out under cooling to prevent loss of toluene. Remove a 50 ml aliquot of the top (toluene) layer (at 2) and pass it through the SPE column prepared in 6.3.5 with a reservoir connected to it (see 6.3.6).

NOTE Measures should be taken to avoid overloading the dispenser.

Wash the column with two 10 ml portions of hexane (5.2.17) and then with two portions of mixed solvent No. 2 (6.2.8). Then washed with 5 cm 3 of toluene, discarding all eluates.

Elute the ochratoxin A with two 15 ml portions of mixed solvent No. 3 (6.2.9) into a 50 ml pointed-bottomed flask (5.2.25). The combined eluate is carefully evaporated to dry residue under reduced pressure and at a temperature not exceeding 40 °C. The residue is dissolved in 1 ml (1/3) of the mobile phase (see 6.2.10) and transferred to a vial (see 6.3.9).

Notes

1 Conditions for elution of ochratoxin A and subsequent operations may depend on the type of columns used for solid phase extraction. For example, check if the eluent volume indicated above matches the type of column being used.

2 The size and/or shape of the flask may have a negative effect on the yield of ochratoxin A.

6.4.3 Conditions for chromatographic analysis

lysis is carried out under the following conditions:

Mobile phase flow rate .................1 cm 3 /min;

Fluorescence excitation wavelength.........330 nm;

Wavelength of registration of fluorescence ......... 460 nm;

Dosing volume ........................................20 mm 3 .

6.4.4 Chromatograph calibration

If a column according to 6.3.11.2 and a mobile phase according to 6.2.10 are used, then the chromatographic analysis

The calibration of the chromatograph is carried out during the development of the technique and in the future in all cases when the conditions of chromatographic analysis change.

Inject into the chromatograph at least four calibration solutions with different mass concentrations of ochratoxin A (see 6.2.16). The dosing volume of each calibration solution must be the same.

The calibration characteristic is established as the dependence of the area or height of the peak on the mass of ochratoxin A in the injected volume.

Check the linearity of the established calibration characteristic using the correlation coefficient (see 5.3.7). Every day before starting work, the stability of the calibration characteristic is checked in the same way as in 5.3.8, using a calibration solution of ochratoxin A with a mass concentration of 25 ng/cm 3 (see 6.2.16).

6.4.5 Identification of ochratoxin A

The ochratoxin A peak is identified by comparing the retention times of the peaks in the chromatogram of the sample and the calibration solutions. In some cases it may be necessary to inject a sample supplemented with a solution of ochratoxin A in the mobile phase (see 5.4.4).

6.4.6 Quantification

The sample solution prepared according to 6.4.2 is immediately injected into the chromatograph. The volume of dosing must be equal to the volume of dosing of calibration solutions when calibrating the chromatograph (see 6.4.4).

Measure the area or height of the ochratoxin A peak on the chromatogram of the sample and, using the calibration characteristic (see 6.4.4), find its mass (/n-i, ng) in the injected volume.

If the response value of ochratoxin A on the sample chromatogram is outside the calibration characteristic, then the prepared sample solution is diluted or concentrated before entering into the chromatograph, taking this fact into account when calculating the measurement results according to 6.5.

6.4.7 Confirmation

If necessary, confirm the identification of the ochratoxin A peak by its disappearance and the simultaneous appearance of a peak with a retention time coinciding with the retention time of the ochratoxin A methyl ester peak as a result of boron strifluoride derivatization.

Take 0.5 cm 3 of the solution prepared according to 6.4.2 into a pointed flask, evaporate to dryness on a rotary evaporator (see 5.2.7). Dissolve the dry residue in 1 ml of methylene chloride (6.2.4) and add 2 ml of a solution of boron trifluoride in methanol (6.2.12).

The flask is tightly stoppered and heated in a water bath at 50°C to 60°C for 15 min. After cooling, the solution is transferred to a separating funnel with a capacity of 50 cm 3 containing 30 cm 3 of water, shaken by 30 with three portions of methylene chloride, 10 cm 3 each. Combine the organic phases after each extraction in another 50 cm 3 separating funnel, add 20 cm 3 of water to wash and shake for 30 s.

Filter the organic phase through a pad of anhydrous sodium sulfate (5.2.14) into a pointed flask, evaporate to dryness, dissolve in 500 mm 3 of the mobile phase (6.2.10) and record the chromatogram of the resulting solution under the conditions of 6.4.3.

To find the retention time of ochratoxin A methyl ester and check the completeness of derivatization under the above conditions, the intermediate solution is treated according to 6.2.15.

6.5 Processing of test results

The mass fraction of ochratoxin A X, million "1 is calculated by the formula

where Vi is the volume of toluene taken for extraction according to 6.4.2, cm 3 (100 cm 3);

\/ 3 - the volume of the sample solution prepared according to 6.4.2, cm 3 (1 cm 3);

mi is the mass of ochratoxin A determined according to 6.4.6, ng;

V 2 - the volume of an aliquot of the centrifuge according to 6.4.2, cm 3 (50 cm 3);

V 4 - the volume of dosing of the prepared sample solution, cm 3;

/77 - mass of the sample taken for testing, g;

10" 3 - the coefficient of agreement of the dimensions of units of mass.

6.6 Metrological characteristics

6.6.1 The results of an interlaboratory test to determine the accuracy of the method, carried out in accordance with GOST ISO 5725-2, are given in Appendix B. The values ​​obtained from the interlaboratory test may not be applicable in the ranges of ochratoxin A concentrations and for matrices other than those specified in appendix B.

6.6.2 The absolute discrepancy between two single test results obtained under repeatability conditions may exceed the repeatability limit r in no more than 5% of cases.

Values ​​of repeatability limits r in the analysis of wheat flour from whole grains:

/=0.18 µg/kg (0.00018 ppm 1) at X=0.41 µg/kg (0.00041 ppm 1);

/=0.70 µg/kg (0.00070 ppm 1) at X=1.23 µg/kg (0.00123 ppm 1).

6.6.3 The absolute discrepancy between two single test results obtained under reproducibility conditions may exceed the reproducibility limit R in no more than 5% of cases.

The values ​​of the limits of reproducibility R in the analysis of wheat flour from whole grains: R=0.30 µg/kg (0.00030 ppm 1) at X=0.41 µg/kg (0.00041 ppm 1);

R=1.10 µg/kg (0.00110 ppm 1) with X=1.23 µg/kg (0.00123 ppm 1).

6.7 Quality control of measurement results - according to 5.7.

6.8 Registration of test results - according to 5.8.

Annex A (informative)

Chromatogram Example

A.1 Figure A.1 shows an example chromatogram (Method A) of R-Biopharm Rhone Ltd. OW-815 "Ochratoxin A in Ground Wheat" standard concentrate. with a mass fraction of ochratoxin A (0.0049 + 0.0010) million "1.

4 6 8 10 12 14 16 Time, min

Figure A.1 - Example of a chromatogram 2 3

Annex B (informative)

Precision Data

B.1 The data given in Table B.1 were obtained as a result of interlaboratory tests carried out for method B in accordance with GOST ISO 5725-2 by the Max von Pettenkofer Institute (Berlin, Germany) on samples of wheat flour.

Table B.1 - Method precision data

Name Wheat flour Wheat flour indicator Number of laboratories Number of samples Number of laboratories remaining after elimination of outliers Number of emissions Number of accepted results Average X value, µg/kg Repeatability standard deviation s r , µg/kg Relative standard deviation of repeatability RSD r Repeatability limit g, µg/kg Reproducibility standard deviation s R , µg/kg Relative standard deviation of reproducibility RSD r Reproducibility limit R, µg/kg

UDC 543.544.5.068.7:006.354 MKS 67.060 NEQ

Key words: grain, food grain, feed grain, animal feed, grain processing products, test methods, chromatography, high performance liquid chromatography, ochratoxin A, mycotoxins

Signed for publication on April 14, 2016. Format 60x84V 8 .

Uel. oven l. 2.33. Circulation 22 copies. Zach. 1070.

Prepared on the basis of the electronic version provided by the developer of the standard

FSUE "STANDARTINFORM"

123995 Moscow, Granatny per., 4. www.gostinfo.ru [email protected]

STANDARD GRAIN AND ITS PROCESSING PRODUCTS, MIXED FEED Determination of ochratoxin A by high performance liquid chromatography

INTERSTATE

Grain and products of its processing, mixed feeds.

Determination of ochratoxin A by high performance liquid chromatography

Introduction date - 2015-07-01

1 area of ​​use

This standard applies to grain and its processed products and establishes methods for determining the content of ochratoxin A using high performance liquid chromatography (hereinafter referred to as HPLC) with fluorimetric detection using:

Screening of samples and purification by column chromatography (method A) in food grains, flour and cereal products based on wheat, corn, barley, rye, oats and rice, animal feed and raw materials for their production on a grain basis (cake, meal) in the measurement range mass fraction of ochratoxin A from 0.0025 to 1.0 million "1;

Purification by solid-phase extraction (method B) in cereal grains and flour in the measurement range of the mass fraction of ochratoxin A more than 0.0004 million "1.

Note -1 ppm 1 corresponds to 1 mg/kg or 1000 µg/kg.

2 Normative references

This standard uses normative references to the following standards:

From the combined sample, a laboratory sample weighing at least 100 g is isolated, which is ground to such a state that it passes through a sieve with a hole diameter of 1 mm. The isolated ground laboratory sample is thoroughly mixed and stored until testing in a dry, dark place in a vessel under a lid.

Flour samples are tested without grinding.

4 Safety requirements

When carrying out measurements, the following requirements must be observed:

Electrical safety in accordance with GOST 12.1.019 and technical documentation for the chromatograph;

Explosion safety in accordance with GOST 12.1.010;

Fire safety in accordance with GOST 12.1.004;

Safety when working with harmful substances in accordance with GOST 12.1.007.

WARNING - Ochratoxin A causes kidney and liver damage and is suspected to be a carcinogen. All work related to sample preparation and preparation of ochratoxin A solutions should be carried out in a fume hood using protective clothing, gloves and goggles. Decontamination of glassware that has been in contact with ochratoxin A is carried out with a 4% solution of sodium hypochlorite.

5 Method A

5.1 Essence of the method

The method is based on the extraction of ochratoxin A from the analyzed sample of the product with acidified chloroform, screening of samples that may contain ochratoxin A, purification of their extracts by column chromatography on silica gel and determination of the mass fraction of ochratoxin A using HPLC with fluorimetric detection.

5.2 Measuring instruments, auxiliary equipment, reference materials, reagents, glassware and materials

5.2.1 Liquid chromatograph with a fluorimetric or spectrofluorimetric detector providing excitation of fluorescence in the spectral region (330 + 20) nm and registration

fluorescence intensity in the spectral region (465 ± 20) nm. The detector used must ensure the detection limit of ochratoxin A is not more than 5 ng/cm 4 5

5.2.2 Non-automatic scales in accordance with GOST OIML R 76-1-2011 with limits of permissible absolute error of not more than ± 0.01 g.

5.2.3 Analytical chromatographic column, filled with a reverse-phase sorbent with a grain size of 5 µm, having an efficiency of at least 5000 theoretical plates for the ochratoxin A* peak.

5.2.4 Guard column of the same internal diameter and filled with the same reverse phase sorbent as the analytical column.

5.2.5 Chromatographic glass column with a ground stopper, 200 mm long and 10 mm in inner diameter.

5.2.6 Sample grinder capable of grinding to particles smaller than 1 mm, such as a laboratory mill.

5.2.7 Rotary evaporator type IR-1 M2 or similar, equipped with a water bath with a temperature controller in the range from 20 °C to 50 °C.

5.2.8 A device for mixing samples (shaker), providing a shaking frequency of up to 120 min" 1 .

5.2.9 Laboratory vacuum, diaphragm or water jet pump according to GOST 25336, providing a vacuum from 2.5 to 10 kPa.

5.2.10 Drying cabinet, providing temperature up to 200 °С.

5.2.12 Standard sample of the composition of a solution of ochratoxin A in acetonitrile with a mass concentration of 50 µg/cm 5 with a tolerance of ± 2.5 µg/cm 5 . Applicable

standard samples of the composition of the solution of ochratoxin A in other solvents, which should be taken into account when preparing the initial solution according to 5.3.3.1.

5.2.13 Distilled water according to GOST 6709 or water for laboratory analysis, purity 1 according to GOST ISO 3696.

5.2.22 Graduated pipettes of types 1-3, versions 1(1 a) or 2(2a), 2nd accuracy class, with a capacity of 1.2, 5 cm 5 according to GOST 29227.

5.2.23 Dimensional cylinders, versions 1-4, 2nd accuracy class, with a capacity of 25, 50, 250 cm 5 according to GOST 1770.

5.2.24 Volumetric flasks 2-25-2, 2-50-2, 2-100-2, 2-500-2 according to GOST 1770.

5.2.25 Pointed-bottom flasks 0-10-14/23 and 0-50-14/23 according to GOST 25336.

5.2.26 Flat-bottomed flasks of type P-1 or Kn-1 with a capacity of 50, 100, 250 cm 5 according to GOST 25336.

5.2.27 Single-channel pipettors of variable volume from 100 to 1000 mm 5 with metrological characteristics in accordance with GOST 28311.

5.2.28 Laboratory funnels type B in accordance with GOST 25336.

5.2.30 Single use microsample tube (Eppendorf type)

5.2.31 Glass containers with a capacity of 50, 250, 1000 cm 5 with ground glass, fluoroplastic or polyethylene stoppers.

5.2.32 Sieve with a hole diameter of 1.0 mm.

5.2.33 Glass rods.

5.2.34 Hourglass or timer.

5.2.35 Paper filters "red tape".

5.2.37 Silica gel for column chromatography, particle size 100 to 200 µm. It is allowed to use other measuring instruments with metrological characteristics not

worse than the above and auxiliary equipment and materials with technical characteristics not worse than the above.

5.3 Preparation for testing

5.3.1 Preparing glassware

Vessels for the preparation and storage of the mobile phase are washed only with sulfuric acid according to 5.2.20 (without the use of other detergents), washed thoroughly with tap water and rinsed with distilled water.

The rest of the glassware is washed with hot water and detergent, rinsed thoroughly with distilled water and dried in an oven at a temperature of 105 °C.

5.3.2 Preparation of auxiliary solutions

5.3.2.1 Mobile phase preparation

In a volumetric flask with a capacity of 500 cm 3 with a tightly closed ground glass, fluoroplastic or polyethylene stopper, place 5 cm 3 of glacial acetic acid (see 5.2.15), 215 cm 3 of acetonitrile (see 5.2.16) and dilute to the mark with distilled water. The mixture is thoroughly mixed. Contact of the mobile phase with rubber is not allowed.

Shelf life of the mixture at room temperature - no more than 1 month.

5.3.2.2 Preparation of a 1% acetic acid solution

Place 200 ml of distilled water and 2 ml of glacial acetic acid (5.2.15) into a 250 ml flat-bottomed flask and mix thoroughly.

The shelf life of the solution at room temperature in a glass container with a tightly closed ground glass, fluoroplastic or polyethylene stopper is no more than 1 month.

5.3.2.3 Preparation of a mixture of chloroform and formic acid in a volume ratio

Place 200 ml of chloroform (5.2.18) and 4 ml of formic acid (5.2.19) in a 250 ml flat-bottomed flask. Add 10 g of anhydrous sodium sulfate (5.2.14). The mixture is thoroughly mixed, filtered through a "red tape" filter.

The shelf life of the mixture at room temperature in a glass container with a ground glass, fluoroplastic or polyethylene stopper is no more than 1 month.

5.3.3 Preparation of ochratoxin A solutions

5.3.3.1 Preparation of a stock solution of ochratoxin A with a mass concentration of 1

With a pipette, 1 cm 3 of a standard sample of the composition of a solution of ochratoxin A in acetonitrile with a mass concentration of 50 µg/cm 3 (see 5.2.12) is taken, placed in a volumetric flask with a capacity of 50 cm 3 and the volume is adjusted to the mark with acetonitrile (see 5.2.16).

The shelf life of the prepared solution in the refrigerator at a temperature of 2°C to 6°C - no more than 6 months.

When using a standard sample of the composition of a solution of ochratoxin A in other solvents, an aliquot (1 cm 3) is evaporated to a dry residue in vacuum at a water bath temperature from 40 °C to 45 °C or in a stream of nitrogen. The dry residue is dissolved in 2 cm 3 of acetonitrile and stirred for 1 min. Then the resulting solution was quantitatively transferred into a volumetric flask with a capacity of 50 cm 3 and diluted to the mark with acetonitrile.

Note - It is allowed to prepare the stock solution of ochratoxin A according to 6.2.14-6.2.15 using acetonitrile instead of the mobile phase according to 6.2.10.

5.3.3.2 Preparation of a solution of ochratoxin A in acetonitrile with a mass concentration of 100

In a volumetric flask with a capacity of 50 cm 3, place 5 cm 3 of a solution of ochratoxin A with a mass concentration of 1 μg/cm 3 according to 5.3.3.1, which corresponds to 1000 ng/cm 3 , and bring the volume to the mark with the mobile phase according to 5.3.2.1.

The shelf life of the resulting solution in a refrigerator at a temperature of 2°C to 6°C - no more than 3 months.

5.3.3.3 Preparation of ochratoxin A calibration solutions

The initial solution of ochratoxin A in acetonitrile according to 5.3.3.1 is placed in a volumetric flask with a capacity of 50 cm 3 . The volume of ochratoxin A solution must comply with the requirements of Table 1.

Table 1

solution	The volume of the initial solution according to 5.3.3.1, cm 3	Nominal value of mass concentration, ng / cm 3

The contents of the flask are diluted to the mark with the mobile phase according to 5.3.2.1 in accordance with Table 1.

Shelf life of calibration solutions - no more than seven days.

Note - If necessary, it is allowed to prepare solutions of ochratoxin A in a similar way with other concentrations in the range of linearity of the calibration characteristic (see 5.3.7).

5.3.3.4 All solutions of ochratoxin A are stored in a refrigerator at a temperature not exceeding 6 °C or in a freezer at a temperature not exceeding minus 18 °C in glass or fluoroplastic vessels.

The actual value of the mass concentration of ochratoxin A in solutions according to 5.3.3.1 - 5.3.3.3 is calculated based on the values ​​of the mass concentration of ochratoxin A according to the standard sample certificate.

Before use, the solutions are kept until room temperature is reached.

5.3.4 Preparing the glass chromatographic column

The glass chromatographic column is prepared immediately before the test.

Place (1,0 ± 0,1) g of silica gel (5.2.37) in a 50 ml beaker, add 10 to 15 ml of chloroform (5.2.18) and mix thoroughly. The resulting suspension is kept in a beaker for 4-5 minutes and then, in several stages, the silica gel suspension is transferred into the column through a funnel with a diameter of 25 or 36 mm, after placing a piece of cotton wool on its bottom.

After the silica gel has settled in the column, a layer of about 2 g of anhydrous sodium sulfate is placed on top of it (see 5.2.14).

In the process of working with the column, the sorbent should not be allowed to dry out, for which it is necessary to maintain a solvent layer above the desiccant. When ready, the column must be filled with chloroform above the level of the sorbent, closed with a ground glass stopper, and the column outlet must be immersed in chloroform.

The prepared column is used once.

5.3.5 Preparing the chromatograph

Preparation of the chromatograph for measurements is carried out in accordance with the manual (instruction) for operation.

Set the operating wavelengths for excitation and fluorescence detection (see 5.2.1). The mobile phase feed rate is set depending on the column size, guided by the instructions of the chromatograph and column manufacturer. For example, for the column specified in 5.2.3, the recommended mobile phase space velocity is 200 mm 3 /min. If there is a column thermostat, the temperature is set to (25 ± 1) °C.

5.3.6 Conditions for chromatographic analysis

If a chromatographic column according to 5.2.3 and a mobile phase according to 6.2.10 are used, then the chromatographic analysis is carried out under the following conditions:

Fluorescence Excitation Wavelength .................................330 nm;

Wavelength of registration of fluorescence...................465 nm;

Mobile phase flow rate ...............................200 mm 3 /min;

Dosing volume .................................................................. 20 mm 3;

The temperature of the column thermostat (if any) ... (25 ± 1) °С.

When using columns of other standard sizes, the flow rate of the mobile phase and the volume of dosing are selected in accordance with the instructions of the manufacturer of the chromatograph and column.

5.3.7 Chromatograph calibration

The linearity range of the calibration characteristic is from 10 to 100 ng/cm 3 . Ochratoxin A calibration solutions according to 5.3.3.3 are used as samples for chromatograph calibration.

At least two chromatograms of each calibration solution are recorded and the chromatograph is calibrated by setting the parameters of the calibration characteristic and the retention time of ochratoxin A using the software for the chromatograph.

Calculate the correlation coefficient and the deviation of the calculated values ​​of the mass concentration of ochratoxin A at each calibration point from the actual value in accordance with the procedure for preparing calibration solutions (see 5.3.3.3).

Graduation is considered acceptable if:

Correlation coefficient not less than 0.998;

The relative deviation of the calculated value of the mass concentration of ochratoxin A from the actual value is no more than + 10%. Instead of a relative deviation, the acceptability of a calibration characteristic can be assessed by a relative standard deviation, which should not exceed 5%.

5.3.8 Checking the stability of the calibration characteristic

Control of the stability of the calibration characteristics is carried out daily before starting

As a control solution, use a solution of ochratoxin A in the mobile phase, prepared in the same way as 5.3.3.3. The mass concentration of ochratoxin A in the control sample is selected based on the expected content of ochratoxin A in the test samples; it is recommended to use a solution of ochratoxin A with a mass concentration of 20 ng/cm.

At least two chromatograms of the control solution are recorded and the ochratoxin A peak is identified by the retention time with an identification window width of 5%, making, if necessary, a software correction of the peak retention time, and using the calibration characteristic, the mass concentration of ochratoxin A is calculated for each input.

The convergence of the values ​​of the retention time and the values ​​of the mass concentration of ochratoxin A is checked by the formulas:

where U and t 2 - the retention time of the peak of ochratoxin A in the first and second chromatograms, respectively, min;

I - arithmetic mean value of U and t 2 , min.

Sk: and С«2 - mass concentrations of ochratoxin A in the control solution according to the first and second chromatograms, respectively, ng/cm 3 ;

C to - the arithmetic mean of the values ​​C K: and C u, ng / cm 3.

The calibration dependence is recognized as stable if the condition

where C is the mass concentration of ochratoxin A in the control solution, ng/cm.

If condition (3) is not met, then the control procedure is repeated. The results of the repeated control are considered final, and the calibration of the chromatograph according to 5.3.7 is carried out again.

Note - Recalibration of the chromatograph is usually required when the efficiency of the chromatographic system or the sensitivity of the detector changes (for example, after repairs or a long downtime of the chromatograph).

5.3.9 Inspection of a reactive blank

A reactive blank test is carried out before testing the test samples.

Place 30 cm 3 of chloroform and 2.5 cm 3 of acetic acid solution according to 5.3.2.2 into a flat-bottomed flask with a capacity of 50 cm 3, mix thoroughly and carefully take 20 cm 3 of chloroform from the bottom layer into a 50 cm 3 sharp-bottomed flask for evaporation, avoiding water getting into it.

The pointed flask is placed on a rotary evaporator and evaporated in vacuo to dryness at a water bath temperature of 40°C to 45°C. Continue with all steps of 5.4.3, thus obtaining a blank concentrate. Carry out a chromatographic study of the resulting concentrate according to 5.4.4. If the chromatogram contains peaks that are close in retention time to the peak of ochratoxin A, then the causes of contamination of the blank sample (reagents or glassware) are found and eliminated.

NOTE The most common cause of poor blank control results is insufficient purity of chloroform, which can be contaminated with impurities that have retention times close to those of ochratoxin A. Such chloroform must be replaced or subjected to thorough distillation, collecting the middle fraction with a boiling point of 60 ° C to 62 ° C.

5.3.10 Accounting for loss of ochratoxin A during sample preparation

30 cm 3 of chloroform (see 5.2.18) and 2.5 cm 3 of a solution of acetic acid (see 5.3.2.2) are placed in a flat-bottomed flask with a capacity of 50 cm 3, 0.25 cm 3 of a solution of ochratoxin A in acetonitrile, mass concentration of 100 ng /cm3 (see 5.3.3.2). Mix thoroughly and carefully remove 20 cm 3 of the chloroform (lower) layer into a 50 cm 3 pointed evaporator flask, avoiding the ingress of water into it. Evaporate to dryness in vacuo at a water bath temperature of 40°C to 45°C.

Further, all operations according to 5.4.3 are continued, the chromatographic analysis of the obtained concentrate is carried out according to 5.4.4 and, using the calibration characteristic according to 5.3.7, the mass concentration of ochratoxin A in the concentrate is calculated.

Then the transmission coefficient of ochratoxin A p is calculated by the formula

(4)

V 0 - the volume of ochratoxin A solution according to 5.3.3.2, selected for the preparation of the control

sample, cm 3 (0.25 cm 3).

The transmission coefficient of ochratoxin A is determined at least three times at the stage of mastering the technique and then when changing batches of reagents. The values ​​obtained must meet the following requirements:

Each of the obtained values ​​is not less than 0.7;

The relative value of the range of the obtained values ​​corresponds to the condition

I max Umin I q jq ^

where Dmax and t| min - the largest and smallest of the obtained values ​​of the transmission coefficient of ochratoxin A;

d) - arithmetic mean of the obtained values ​​of the ochratoxin transmission coefficient

If both of these conditions are met, then the arithmetic mean value of the transmission coefficient of ochratoxin A is used when calculating the results of the determination according to formula (9). Otherwise, the reasons for the loss of ochratoxin A are found and the determination of the transmission coefficient is repeated.

Notes

1 The reason for the unsatisfactory result of the determination of the coefficient of transmission may be an increased speed of eluent through the column.

2 Particular attention should be paid to the need to determine the transmission coefficient of ochratoxin A when changing the batch of silica gel. When switching to a new batch of silica gel, it is recommended to optimize the volumes of eluents used in column purification of samples (see 5.4.3), achieving the highest value of the transmission coefficient. The volumes found in this case are used in the purification of samples on columns made from a given batch of the reagent.

3 It is recommended to clarify the ochratoxin A transmission coefficient for specific matrices using standard samples of the product composition with a certified value of the mass fraction of ochratoxin A or by analyzing a “clean” sample (no ochratoxin A is detected in the sample), which has been supplemented with ochratoxin A. Analyze the standard sample ( ochratoxin A) sample according to 5.4.1 to 5.4.4. Calculate the result of the analysis (see 5.5) and find the ochratoxin A transmission coefficient as the ratio of the result obtained to the certified value of the mass fraction of ochratoxin A in the standard sample or to the mass fraction of ochratoxin A in the additive.

5.4 Testing

5.4.1 Extraction of ochratoxin A from the sample

A sample of the crushed product weighing (5.00 ± 0.01) g is placed in a flat-bottomed flask with a capacity of 100 cm 3 . Add 30 ml of chloroform (5.2.18) and 2.5 ml of acetic acid solution (5.3.2.2) and stir vigorously for 30 minutes. Pass the resulting extract through a paper pleated filter "red tape", selecting 20 cm 3 of the filtrate for evaporation.

If it is not possible to select 20 cm 3 of the filtrate, then select the maximum possible volume, measure it and use the obtained value in the formula (9).

Transfer the resulting filtrate to a 50 cm3 sharp-bottomed flask and evaporate to dryness in vacuo at a water bath temperature of 40°C to 45°C.

The dry residue is treated according to 5.4.2 or 5.4.3 depending on the purpose of the test - screening samples or quantifying the mass fraction of ochratoxin A in a sample in which a peak identified as an ochratoxin A peak was detected during screening.

NOTE When testing samples for which there is reason to believe that they are contaminated with ochratoxin A, it is allowed to proceed directly to purification on a column according to 5.4.3, bypassing screening.

5.4.2 Screening samples

The dry residue obtained according to 5.4.1 is dissolved in 0.5 cm 3 of the mobile phase according to 5.3.2.1 and stirred for 1 min. Then add 3 ml of hexane (see 5.2.17) and mix vigorously again. After separation of the two immiscible phases, carefully transfer approximately 0.3 cm 3 of the bottom layer into a 1.5 cm 3 single use microsample tube (Eppendorf tube) or equivalent.

Record the chromatogram of the resulting solution according to 5.4.4 on the day of the test. If a peak is found on the chromatogram, which is identified by the software for the chromatograph as an ochratoxin A peak, then the test of the sample is repeated, including mandatory purification according to 5.4.3.

If there is no peak identified as the peak of ochratoxin A, it is concluded that there is no ochratoxin A in the sample at the level of the lower limit of the measurement range (0.0025 ppm).

5.4.3 Sample cleaning and sample preparation

The dry residue according to 5.4.1 is dissolved in 1 cm 3 of chloroform.

Prepare the glass column according to 5.3.4. Allow the chloroform to drain to the level of anhydrous sodium sulfate and apply the resulting solution to the column. The pointed-bottom flask, in which the extract was evaporated, is rinsed twice with 1 cm 3 of chloroform, which is also applied to the column. After that, the column is washed with 10 cm 3 of hexane, the eluate is discarded.

Then 30 ml of a mixture of chloroform and formic acid (see 5.3.2.3) are passed through the column at a rate of not more than one drop per second, eluting ochratoxin A. The entire eluate is collected in a sharp-bottomed flask and evaporated to dryness in vacuo at water-bath temperature. from 40 °С to 45 °С.

The dry residue is dissolved in 0,5 ml of the mobile phase (see 5.3.2.1) and stirred for 1 min. Then 3 cm 3 of hexane are added and again vigorously stirred. After separation of the two immiscible phases, carefully transfer approximately 0.3 cm 3 of the bottom layer into a 1.5 cm 3 single use microsample tube (Eppendorf tube) or equivalent. The resulting concentrate is used for chromatographic measurements according to 5.4.4 on the day of the test.

For example, the Lichrospher® 100 RP 18 column is an example of a commercial product that meets these requirements. This information is given for the convenience of users of this International Standard and does not constitute an endorsement of the specified product.

The peak corresponds to an ochratoxin A content of 0.0042 ppm.

* An example of a commercial product that meets the specified requirements is a chromatographic column with an internal diameter of 2.1 mm and a length of 120 mm, filled with a reverse-phase sorbent Kromasil C-18 with a particle size of 5 μm, equipped with a pre-column 25 mm long. This information is given for the convenience of users of this International Standard and does not constitute an endorsement of the specified product.