Certificate No. 30-08 dated 03/04/2008
FR.1.31.2008.01033

1. Objects of research

This measurement procedure applies to smoked meat, smoked fish and fatty products and establishes the determination of the mass concentration of benzo (a) pyrene by high performance liquid chromatography with fluorometric detection.

2. Measurement range

MPC of benzo (a) pyrene in fatty, smoked meat, smoked fish products is 1 μg / kg.

The method provides the results of measurements of the mass concentration of benzo (a) pyrene in the ranges presented in Table 1.

Table 1. Ranges for measuring the mass concentration of benzo (a) pyrene

Product type	Mass range concentration, μg / kg	MPC, μg / kg
fatty foods	0,5 – 2,0	1,0
smoked meat products	0,5 – 2,0	1,0
smoked fish products	0,5 – 2,0	1,0

3. Sample preparation

Sampling, conservation and storage of product samples is carried out in accordance with GOST 7631, GOST 9792, TU and other regulatory documents governing sampling for specific types of products.

Sample preparation consists of the stages of sampling (the sample is pre-cooled at a temperature of minus 12-18 ° C for 30 min), grinding, homogenization with anhydrous sodium sulfate, extraction of a homogenized sample with hexane in a flask using an ultrasonic bath, spontaneous precipitation of a solid precipitate (in for 1-2 minutes), defatting the extract in the freezer (a sample of silica gel is introduced into the cylinder, the extract is poured in, the contents of the cylinder are placed in the freezer), the supernatant hexane layer is purified by non-holding solid-phase extraction (on Strata Silica Si-1 cartridges) *, blow-off eluate in a stream of air or inert gas.

* Note. If the concentration of fats of the initial analyzed product is less than 5%, a small amount of the eluting reagent - ethyl acetate (0.1 ml to 6 ml of the purified extract) must be added to the initial extract of the sample after freezing.

4. Carrying out chromatographic analysis

4.1. Equipment and conditions for HPLC analysis of calibration solutions of benzo (a) pyrene, a prepared product sample.

For the chromatographic analysis of benzo (a) pyrene, it is necessary to use an isocratic high performance liquid chromatographic system with fluorometric detection.

For the analysis, calibration solutions are preliminarily prepared from the GSO solution of benz (a) pyrene in hexane or from the GSO solution of benz (a) pyrene in acetonitrile (the solvent is blown off, the standard sample is redissolved in hexane); carry out sample preparation; prepare the device for operation.

Equipment:

liquid chromatograph "Stayer" with a fluorometric detector;

personal computer with installed software "MultiChrom for Windows XP" version 1.5 or 2x.

isocratic regime;

column: Luna C18 (2) 150x3.0 mm 3 μm (Phenomenex, USA);

protective column: C18 4x3.0 mm (Phenomenex, USA);

mobile phase: acetonitrile / water solution (75:25);

flow rate: 0.3 ml / min;

loop volume: 10 μl;

temperature: 50 ° C;

RFU range: 0.01;

detection: fluorometric (λex: 365 ± 2 nm; λem: 400-460 nm).

Calibration is carried out using calibration solutions (in the entire range of determined concentrations) at least once every two weeks, as well as when using a new batch of reagents, replacing columns and after repairing the chromatograph.

4.2. Determination of the quantitative content of benzo (a) pyrene in a product sample.

To determine the quantitative content of a component of a product sample (benzo (a) pyrene), chromatographic analysis of one of the calibration solutions is carried out, followed by chromatographic analysis of the prepared sample. For reliability of measurements, chromatographic analysis of both the calibration solution and the prepared sample is carried out at least 2 times in a row.

Using the installed software - "MultiChrome for Windows XP" in the report or above the peak (depending on the settings of the "VIEW" options) at the end of the measurement, the result is automatically determined in the form of concentration in the sample introduced into the chromatograph (but not in the initial sample taken for research!).

To obtain a result, it is necessary to carry out at least two parallel measurements (obtain two chromatograms). The arithmetic mean of the content of benzo (a) pyrene in the concentrate of the analyzed sample C xp, μg / l (calculated from two values ​​of the mass concentration of benzo (a) pyrene in the concentrate of the analyzed sample C 1 and C 2) is taken as the measurement result.
Mass fraction of benzo (a) pyrene in the analyzed sample (in the original sample) NS, μg / kg is calculated by the formula:

where:
C xp - the average value of the concentration of benzo (a) pyrene, obtained as a result of chromatography in two parallel measurements [ng / ml];
V 1 - the volume of the original extract, actually equal to the volume of hexane taken for primary extraction (50 ml);
V 2 - the volume of the extract (part of the original) taken for freezing (30 ml);
V 3 - the volume of the extract (part of the extract after freezing) taken for SPE (6 ml) [ml];
V 5 - the volume of the final extract, a part of which is introduced into the chromatograph (about 3 ml) [ml];
K sampling - sampling factor, which takes into account the fraction of the mass of the product sample (mixed with sodium sulfate) taken for extraction, from the total mass of the sample taken for analysis. In all cases it is equal to 0.736;
K freezing Is the loss factor of benzo (a) pyrene during freezing. For all categories of the studied products, this coefficient is the same and equal to 0.95;
K ext. 1 - coefficient of primary liquid extraction with hexane. It is the same for all categories of the studied products and is equal to 0.95;
K TFExtr.2- the coefficient of solid-phase extraction of benzo (a) pyrene is 0.95;
m pr. - soil or soil taken for analysis [g].

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GOST 24104.

Rotary evaporator IR-1M.

GOST 25336.

Bath water.

Magnetic stirrer MM-ZM type with electric heating.

An ultraviolet illuminator of the "Chromatoscope" type with a spectral range of 250 - 700 nm and a BUV-15 lamp as a source of UV radiation.

Glass chromatographic chamber 40 x 40 x 40 cm.

Glass plates for thin layer chromatography 5 x 20 and 20 x 20 cm.

Glass chromatographic column 500 mm long and 20 mm in diameter with a drawn-out end at the bottom and a reservoir with a capacity of 50 - 60 cm 3 PSh 14/23.

Refrigerator ХПТ-2-400-29 / 32 ХС or ХШ-1-400-29 / 32 ХС in accordance with GOST 25336.

Along type AIO-14 / 23-50 TS or AIO-14 / 23-14 / 23-65 TS in accordance with GOST 25336.

Measuring ruler with a graduation of 0.1 cm in accordance with GOST 427.

Dephlegmator 250-19 / 26-29 / 32 ТС or 300-19 / 26-29 / 32 according to GOST 25336.

Nozzle P-1-19 / 26-14 / 23-14 / 23 ТС or Н-2-19 / 26-14 / 23 ТС in accordance with GOST 25336.

GOST 25336.

GOST 25336. Measuring cylinders 1-100, 1-250 or 3-100, 3-250 in accordance with GOST 25336.

Chemical glass V-1-100 or V-1-150 in accordance with GOST 25336.

Flasks К-1-100-29 / 32 ТХС, К-1-25Р-29/32 ТХС, К-1-500-29 / 32 ТХС or П-1-500-29 / 32 ТХС in accordance with GOST 25336.

Buchner funnel 1 or 2, or 3 in accordance with GOST 9147.

Weighing cups (weighing bottles) SV-14/8 or SV-19/9, or SV-24/10, or SV-34/12 in accordance with GOST 25336.

Microsyringes of the MSh-10 types, glass capillaries.

Universal indicator paper.

Laboratory filter paper in accordance with GOST 12026.

Scalpel or thin spatula.

Rectified ethyl alcohol according to GOST R 51652 or technical rectified ethyl alcohol according to GOST 18300.

Acetonitrile according to the normative document.

Microcrystalline powder cellulose according to the normative document.

GOST R 51650-2000

Benz (c) chrysene, the content of the main substance is not less than 98%.

Sephadex LH-20.

Silica gel of the ASKG brand according to the normative document.

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

5.2 Test preparation

5.2.1 Solvent preparation

Solvents (n-hexane, ethyl alcohol, acetone, benzene) are distilled in a conventional manner with a reflux condenser.

Dimethylformamide is distilled by adding 120 cm 3 of benzene and 36 cm 3 of water per 1 dm 3 of solvent to a distillation flask.

5.2.2 Preparation of acetylated cellulose

(50.0 ± 2.0) g of microcrystalline cellulose is placed in a flat-bottomed flask with a capacity of 500 cm 3, a mixture of 150 cm 3 of benzene or toluene, 70 cm 3 of acetic anhydride and 0.3 cm 3 of sulfuric acid prepared in a separate flask is added. The reaction mixture is stirred with a magnetic stirrer for 6 - 8 hours, left without stirring for another 18 hours, after which the liquid phase is decanted, and the residue is poured with 300 cm 3 of ethyl alcohol, stirred, left in alcohol for 24 hours, then the cellulose is filtered on a Buchner funnel , washed with 100 cm 3 of ethyl alcohol and distilled water until the wash water is neutral (according to indicator paper).

Then check the chromatographic activity of the acetylated cellulose. For this, 3-4 hours before the analysis, a mixture of ethyl alcohol, acetone and water, taken in a volume ratio of 60:25:15, is prepared and poured into a chromatographic chamber lined with strips of filter paper. The height of the solvent layer should be 1.5 - 2 cm. 1.5 g of acetylated cellulose is suspended in 7 cm 3 of ethyl alcohol and the suspension is poured into an even layer on a 5 x 20 cm glass plate, the solvent is allowed to completely evaporate in air and applied to the plate with a microsyringe or glass with a capillary to the point of 5 μl of a solution of benz (a) pyrene of a mass concentration of 1 μg / cm 3. The plate is placed in the chromatographic chamber and left in the chamber until the solvent level rises at least 100 mm from the starting line. At the end of chromatography, the plate is removed, dried in air, and a blue fluorescent spot of benzo (a) pyrene is noted under an ultraviolet irradiator lamp. Measure the distance from the starting line to the solvent front and to the middle of the benz (a) pyrene spot; calculate the value of Rj, which estimates the speed of movement of benzo (a) pyrene along the plate, according to the formula:

where X BP is the distance from the starting line to the middle of the benzo (a) pyrene spot, mm;

L is the distance from the starting line to the solvent front, mm.

The Rj value of benzo (a) pyrene should be 0.1.

To prepare a working plate, 5 g of acetylated cellulose is suspended in 20 cm 3 of ethyl alcohol and poured into an even layer on a 20x20 cm plate.

5.2.3 Preparation of benzo (a) pyrene and benzo (c) chrysene standard solutions

Weigh (10.0 + 0.2) mg of benzo (a) pyrene and benzo (c) chrysene into weighing cups (weighing bottles). The weighed portions are quantitatively transferred into volumetric flasks with a capacity of 100 cm 3: benz (a) pyrene-benzene, benz (c) chrysene-acetonitrile, then the volume of the benz (a) pyrene solution is adjusted to the mark with benzene, the volume of the benz (c) chrysene-acetonitrile solution. The resulting solutions have a mass concentration of 100 μg / cm 3. The solutions are stored in a cool, dark place for no more than three months.

5.2.4 Preparation of working solutions of benz (a) pyrene and benz (c) chrysene

Working solutions are prepared by diluting standard solutions using pipettes with a capacity of 1, 5 and 10 cm 3 and volumetric flasks with a capacity of 100 cm 3, the volume of the solution is brought to the mark with the appropriate solvent, stirred and stored in a cold dark place for no more than one month.

Preparation of a solution of benz (a) pyrene with a mass concentration of 1.0 μg / cm 3 (for determination by spectrofluorimetry): 1.0 cm 3 is taken from the standard solution and transferred to a volumetric flask with a capacity of 100 cm 3; the volume of the solution is brought to the mark with benzene.

Preparation of a solution of benz (a) pyrene of mass concentration 0.25: 1.0 and 5.0 μg / cm 3 (for determination by high-performance liquid chromatography): 0.25 is taken from the standard solution; 1.0; 5.0 cm 3, respectively, and transferred into volumetric flasks with a capacity of 100 cm 3; the volume of solutions is brought up to the mark with acetonitrile.

Preparation of benz (c) chrysene solutions of mass concentration of 0.5 and 10 μg / cm 3: 0.5 and 10 cm 3 are taken from the standard solution, respectively, and transferred to volumetric flasks with a capacity of 100 cm 3; the volume of each solution is brought up to the mark with acetonitrile.

5.2.5 Preparation of calibration solutions

To prepare calibration solutions of a mixture of benzo (a) pyrene and benzo (c) chrysene, the volumes of a standard solution of benzo (a) pyrene with a mass concentration of 100 μg / cm 3 and a working solution of benz (c) chrysene mass concentration of 10 μg / cm 3, bring the volume to the mark with acetonitrile. The resulting solutions are mixed and stored in a dark, cold place for no more than one month.

table 2

solution The volume of the initial solution, cm 3 Mass concentration in the calibration solution, μg / cm 3 Benz (a) pyrene mass concentration 100 μg / cm 3 Benz (c) chrysene mass concentration 10 μg / cm 3 Benz (a) pyrene Benz (v) chrysena

5.3 Test procedure

5.3.1 Isolation of benzo (a) pyrene from the product

In a round-bottomed flask or a flat-bottomed flask with a capacity of 100 cm 3, place a sample of the product weighing 10 g, add a solution consisting of 4 g of potassium hydroxide in 50 cm 3 of 92% ethyl alcohol. The contents of the flask are mixed by shaking. The flask is connected to a reflux condenser and heated in a water bath or on a magnetic stirrer while the reaction mixture is boiling for 3 hours. Then, 100 cm 3 of distilled water is added to the flask through a condenser. The reaction mass is cooled to room temperature. After cooling, the reaction mass is transferred into a separating funnel with a capacity of 500 cm 3. If, after hydrolysis, a solid residue remains in the reaction mass, it is separated on a Buchner funnel, washing the residue on the filter with 30 cm 3 of hot ethyl alcohol. The liquid phase of the reaction mass is used for extraction. Add 30 cm 3 N hexane to a separatory funnel. The contents of the funnel are shaken and left to separate the liquids. If an emulsion is formed, 20 cm 3 of ethyl alcohol is added to the mixture in a separating funnel. After stratification, the lower aqueous-alcoholic phase is poured into a flask, and the hexane extract is poured into another separatory funnel. This treatment of the reaction mass is carried out two more times, using 30 cm 3 n-hexane for extraction and ethyl alcohol to delaminate the emulsion in 20 cm 3 portions.

At the end of the extraction, the combined hexane extract is washed in a separating funnel with distilled water three times 30 cm 3 each, the extract is transferred into a round-bottom flask with a capacity of 100 cm 3, filtering through a layer of anhydrous sodium sulfate on a funnel with a porous filter. The solution is evaporated on a rotary evaporator to a volume of 50 cm 3 at a water bath temperature not higher than 60 ° C.

The one stripped off extract is transferred into a separating funnel with a capacity of 500 cm 3 and 50 cm 3 of a mixture of dimethylformamide and water taken in a volume ratio of 9: 1 is added to it. The mixture is vigorously shaken for 1 min, after phase separation, the lower one is poured into a flat-bottom flask with a capacity of 200 cm 3, and from the upper hexane layer, 50 cm 3 of a mixture of dimethylformamide and water is extracted again. The hexane layer is discarded, the dimethylformamide extract combined in a flat-bottomed flask is transferred to a separatory funnel, 100 cm 3 of distilled water is added and extraction is carried out from the aqueous phase with hexane three times 50 cm 3 each. The aqueous phase is discarded, and the hexane extract is washed with water three times 30 cm 3 each, transferred to a flat-bottomed flask, 10 g of anhydrous sodium sulfate is added and kept for one hour, n hexane is evaporated on a rotary evaporator to a volume of 1.5-2.0 cm 3, the remaining solvent is removed

GOST R 51650-2000

by a stream of air through a vacuum tube connected to a water-jet pump, the residue in the flask is dissolved in 0.5 cm 3 of ethyl alcohol.

Weigh (2.5 ± 0.2) g of Sephadex LH-20 into a glass with a capacity of 100 cm 3, add 20 cm 3 of ethyl alcohol and leave to swell for 3 - 4 hours. column, allow the solvent to drain so that the alcohol layer above the sorbent layer remains at least 2 mm. The remainder of the extract from the flask is pipetted onto the prepared column, washing it out of the flask three times with ethyl alcohol in portions of 0.5 cm 3. Elution from the column of polycyclic aromatic hydrocarbons, including benz (a) pyrene, is carried out with 40 cm 3 of ethyl alcohol, the first fraction with a volume of 12 cm 3 is discarded, the second fraction with a volume of 25 cm 3 is collected. A solvent elution rate of 0.5 cm 3 / min is provided by creating a slight overpressure with a stream of air or nitrogen through a nozzle connected to a blower or gas cylinder. The gas should be fed through a glass tube filled with silica gel.

The Sephadex LH-20 column is reusable. For this, without allowing the sorbent to dry out after fractionation, the column is washed with 25 cm 3 of ethyl alcohol and the next sample is applied.

The solution of the second fraction is transferred into a pear-shaped flask with a capacity of 50 cm 3, the solvent is evaporated to a volume of 0.5 - 1.0 cm 3, its remainder is removed in a stream of air or nitrogen.

The resulting fraction containing benzo (a) pyrene is then analyzed using high performance liquid chromatography or spectrofluorimetric method.

At the same time, a control experiment is carried out, carrying out all stages of the analysis using reagents according to the methodology, but without a sample of the product.

5.3.2 Determination of benzo (a) pyrene content by high performance liquid chromatography

5.3.2.1 Chromatographic conditions

Chromatography conditions are selected depending on the type of liquid chromatograph and chromatographic column used.

As an example, the following conditions for the chromatographic determination of benzo (a) pyrene can be given.

Liquid chromatograph "АИех-334" with fluorescent detector "Kratos FS-970".

Column Supelcosil LC-PAM with a grain of 5 microns, a length of 150 mm, a diameter of 4.6 mm.

Fluorometric detector: excitation wavelength 300 nm, emission filter 418 nm.

Mobile phase: acetonitrile and water in a volume ratio of 8: 2.

The elution rate is 2.0 cm 3 / min.

The volume of the injected sample is 20 μl.

The sensitivity of the amplifier is selected so that the intensity of the signals of benzo (a) pyrene and the internal standard, benzo (c) chrysene, does not exceed 95% of the scale.

Analysis time - 15 min; retention time of benz (a) pyrene - 5 min, benz (c) chrysene - 13 min.

Analyzed solutions are chromatographed twice under the same conditions. Peak areas are measured using an integrator or manually as the product of the peak height and peak width at half height.

Determination of benzo (a) pyrene content is carried out by the method of internal standard or by the method of additions.

5.3.2.2 Determination of benzo (a) pyrene content in the solution (extract) obtained according to 5.3.1 using the internal standard method

When using this method of quantitative assessment, the chromatograph is pre-calibrated using the calibration solutions prepared according to 5.2.5.

Under the conditions specified in 5.3.2.1, record three chromatograms for each of the prepared solutions and measure the peak areas of benzo (a) pyrene and benzo (c) chrysene. Determine the arithmetic mean of the area of ​​the peaks of benzo (a) pyrene and benzo (c) chrysene, calculated from three chromatograms.

Calibration coefficient K is calculated by the formula

where and and 2 are the masses of benz (a) pyrene (/ l]) and benz (c) chrysene (m 2) introduced into the chromatograph, μg; 5) and ^ 2 are the areas of the peaks of benzo (a) pyrene (.9]) and benzo (c) chrysene (A ^), cm 3.

Calibration coefficient K is calculated for each solution.

Its values ​​should not differ from the arithmetic mean of the calibration coefficient from all results by more than 10%.

With an exciting light wavelength of 300 nm and an emission filter of 418 nm, the value of the calibration factor is 9.5.

Before starting the analysis at the stage of preparing samples for alkaline hydrolysis, 50 μl of a solution of benzo (c) chrysene with a mass concentration of 0.5 μg / cm 3 are added to the product sample and the control sample. Both samples are passed through all the test steps specified in 5.3.1. The dry residue is dissolved in 200 μl of acetonitrile.

Under the conditions specified in 5.3.2.1, record the chromatograms of a solution of benzo (a) pyrene with a mass concentration of 100 μg / cm 3 and a solution of benz (c) chrysene with a mass concentration of 100 μg / cm 3, note the release time of benzo (a) pyrene and benz ( c) chrysena. Then, the chromatograms of the control experiment sample with the addition of benzo (c) chrysene and a sample of the product with the same addition of benzo (c) chrysene are recorded. Measure the areas of the peaks of benzo (a) pyrene and benzo (c) chrysene on the chromatograms of the product sample and the control sample.

Two chromatograms are recorded for each sample. The arithmetic mean of the peak areas of benzo (a) pyrene and benzo (c) chrysene is calculated from two chromatograms.

Based on the data obtained, the mass of benzo (a) pyrene, μg, is determined in the sample of the product m \ and the sample of the control experiment m 2.

“72 is the mass of benzo (a) pyrene in the control sample, μg; t st is the mass of benzo (c) chrysene introduced into the product sample and the control sample, μg;

S "] and S 2 - the areas of peaks of benzo (a) pyrene on the chromatograms of the product sample (.S"]) and the control sample (.S /), cm 2;

L /, L / - areas of peaks of benzo (s) chrysene on the chromatograms of the product sample (.SS) and the sample of the control experiment (L /), cm 2;

К - calibration coefficient established in accordance with 5.3.2.2.

5.3.2.3 Determination of benzo (a) pyrene content in solution (extract) obtained according to 5.3.1 by the method of additions

For quantitative assessment when using the addition method, a sample of the control experiment is analyzed simultaneously with the product sample. The fractions isolated from the product samples and the control experiment according to 5.3.1 are dissolved in 400 μl of acetonitrile. The resulting solutions are divided into two parts, taking a smaller part (40 μl) into a test tube or pear-shaped flask.

The chromatograms of the product sample, the control sample and the chromatogram of the benz (a) pyrene solution of 0.25 μg / cm 3 mass concentration are recorded. The time of release of benzo (a) pyrene is noted.

In the remaining parts of the product sample and control experiment (360 μl), add 10 - 20 μl of benzo (a) pyrene solution with a mass concentration of 5 μg / cm 3. The resulting solutions are reintroduced into the chromatograph.

All chromatograms are recorded twice. Measure the areas of the peaks of benzo (a) pyrene. The arithmetic mean of the peak area of ​​benzo (a) pyrene is calculated from the two chromatograms.

Based on the data obtained, determine the mass of benzo (a) pyrene, μg, in the product sample / 77] and in the sample of the control experiment t 2:

t op ■ S 1. _ t to ■ S 3 (9)

S 2 - 0.95) '2 5 4 - 0.95 3'

where / 77 op and / 77 k is the mass of benzo (a) pyrene added to the part of the extract from the product sample (t op) and the control sample (/%), μg;

S "] and S 2 are the areas of the peaks of benzo (a) pyrene on the chromatograms of the product sample (.S"]) and the product sample with the addition of benzo (a) pyrene (L /), cm 2;

L / i S) - the area of ​​the peaks of benz (a) pyrene on the chromatograms of the sample of the control experiment (L /) and the sample of the control experiment with the addition of benz (a) pyrene (L /), cm 2;

0.9 is the proportion of the sample to which benzo (a) pyrene is added.

5.3.3 Determination of benzo (a) pyrene content by spectrofluorimetry at room temperature

When determining the content of benzo (a) pyrene by spectrofluorimetry, simultaneously with the product sample, a control sample is analyzed, to which 50 μl of a solution of benzo (a) pyrene of a mass concentration of 1 μg / cm 3 is added.

The fractions obtained according to 5.3.1, containing benzo (a) pyrene, from a sample of the product and a sample of the control experiment with the addition, are dissolved in 0.5 cm 3 of benzene and then subjected to additional purification in a thin layer of acetylated cellulose.

For this, a 20 x 20 cm plate, prepared as indicated in 5.2.2, is divided into two fields: lateral - 1.5 - 2 cm wide and the main one, passing a dividing strip along the sorbent layer with a scalpel or a thin spatula. On the main field in a continuous strip, 2 cm from the bottom edge of the plate and 1 cm from the side edges, apply a solution of the fraction isolated in 5.3.1. The solution is applied using a thinly drawn capillary or microsyringe, the size of the spots should not exceed 5 mm. For quantitative transfer of the substance, it is washed off twice from the walls of the flask with a small amount of benzene (0.4 - 0.6 cm 3). On the starting line of the lateral field, 5 μl of a solution of benzo (a) pyrene of a mass concentration of 1 μg / cm 3 is applied at the point. After complete evaporation of the solvent, the plate is placed in a pre-saturated chromatographic chamber at an angle of 70 ° - 85 ° and elution is carried out in a mixture of ethyl alcohol, acetone and water taken in a ratio of 60:25:15. When the front of the solvent reaches 2 cm from the upper edge of the plate, it is taken out of the chamber, dried in air, and the chromatographic zone of benzo (a) pyrene is developed under an ultraviolet irradiator lamp. The sorbent from the benz (a) pyrene zone from the main field is scraped off the plate with a scalpel or thin spatula and transferred to a glass filter, from which the substance is eluted in several steps 50 cm 3 of benzene into flasks with a capacity of 100 cm 3, then the solvent is evaporated to a small volume , the remainder of the solvent is removed with an air stream and 1 cm 3 of benzene is added to the flask.

On a spectrofluorometer at a wavelength of 386 nm exciting light in the range 400 - 440 nm at a scanning speed of 60 nm / min, the fluorescence spectra of the product sample and the control sample with the addition of benzo (a) pyrene are recorded.

The spectra of solutions are recorded in one amplification mode, adjusting the slit and the gain in the solution of the control sample so that the signal of benzo (a) pyrene at 406 nm was 0.4 - 0.6 of the instrument scale. For each solution, the spectrum is recorded twice to achieve good reproducibility. On the obtained spectrograms at a maximum at 406 nm, measure in millimeters the height of the spectral line of benzo (a) pyrene for a sample of the product and a sample of the control experiment. Calculate the average value of benzo (a) pyrene heights from the data of two spectrograms. At high levels of benzo (a) pyrene in the product, the samples are diluted with benzene and the spectrum is re-recorded in the same amplification mode as for the control sample.

Two parallel determinations are made.

5.4 Expression of results

5.4.1 The mass fraction of benzo (a) pyrene in the product X \,%, or X 2, mg / kg, when using the high-performance liquid chromatography method, is calculated by the formulas:

(t 1 - t 2) ■ 100 (t g - t 2) t ■ 1000 1000 “t 10

where mi is the mass of benzo (a) pyrene in the product sample, μg;

m2 — mass of benzo (a) pyrene in the control sample, μg; t is the mass of the product taken for analysis, g.

5.4.2 The mass fraction of benzo (a) pyrene in product A),%, or X 2, mg / kg, when using the spectrofluorimetry method, is calculated by the formulas:

S st NU-100 s ST // V t ■ 1000 ■ 1000 ■

where c st is the mass concentration of benzo (a) pyrene in the working solution prepared according to 5.2.4 and added to the control experiment sample, μg / cm 3;

the height of the spectral line of benzo (a) pyrene on the spectrogram of the product sample, mm; the height of the spectral line of benzo (a) pyrene on the spectrogram of the control experiment sample, mm;

V is the volume of the working solution of benzo (a) pyrene added to the control sample, cm 3; t is the mass of the sample of the product taken for testing, g.

The arithmetic mean of two parallel determinations with the same number of significant digits is taken as the final test result.

If the discrepancy between the results of parallel determinations does not exceed \ X-y - YG 2 |<

< 0,01 dX, где, Х 2 и X- результаты параллельных определений и их среднее арифметическое, а d - норматив контроля сходимости, то среднее арифметическое X принимают за результат анализа. В противном случае анализ повторяют. Значение норматива d приведено в таблице 3.

Based on the obtained analysis result X and the value of the relative error q given in Table 3, calculate the absolute error A = 0, (ШХ

The analysis result is presented as (X ± A), mg / kg or% at P = 0.95.

5.5 Controlling the accuracy of analysis results

5.5.1 The repeatability of parallel determinations is controlled for each analyzed sample in accordance with 5.3.

5.5.2 Use working samples to control reproducibility. The sample is divided into two equal parts and analyzed in accordance with the methodology in different laboratories or in one laboratory, varying the conditions of the analysis as much as possible, that is, different sets of volumetric glassware are used, analyzes are performed on different days or by two different analysts.

The reproducibility of control analyzes is considered satisfactory if \ X-y - X 2 \<

< 0,01 DX, где Л), Х 2 и X- результаты анализа одной и той же пробы, полученные в разных лабораториях или при варьирующих условиях в одной лаборатории и их среднее арифметическое значение, D - значение норматива внутреннего оперативного контроля воспроизводимости. Значение норматива D приведено в таблице 3.

Frequency of reproducibility control - at least once every two weeks

Table 3 - The range of measurements, the value of the characteristic of the relative error and the standards of operational control of the random component of the relative error (convergence and reproducibility) at a confidence level of P = 0.95

5.5.3 To control the accuracy, use working samples with a known addition of benzo (a) pyrene. The sample is divided into two equal parts, the first of which is analyzed in accordance with the procedure, and in the second, a known additive of benzo (a) pyrene is introduced and then also analyzed in accordance with the procedure. The amount of the additive should be 50 - 150% of the benzopyrene content in the analyzed sample.

The accuracy of control analyzes is considered satisfactory if | A) - X- c \< К, где Л), X и с - результаты контрольных анализов пробы с добавкой бенз(а)пирена, реальной пробы и величина добавки бенз(а)пирена соответственно; К - норматив оперативного контроля точности.

Typed in FSUE "STANDARTINFORM" on a PC.

Printed at the branch of FSUE STANDARTINFORM - type. "Moscow printer", 105062 Moscow, Lyalin per., 6

GOST R 51650-2000

1 area of ​​use............................................... ......... 1

3 Sampling ............................................... ................ 2

4 Low-temperature spectrofluorimetry method ................................. 2

4.1 Apparatus, materials and reagents .......................................... 2

4.2 Preparation for testing .............................................. ... 3

4.3 Carrying out the test ............................................... .... 3

4.4 Processing of results ............................................... .... 6

4.5 Controlling the accuracy of the analysis results ...................................... 6

5 Methods of high performance liquid chromatography and spectrofluorimetry at

room temperature ................................................ ...... 7

5.1 Apparatus, materials and reagents .......................................... 8

5.2 Preparation for testing .............................................. ... nine

5.3 Testing ............................................... ....ten

5.4 Processing of results ............................................... ....13

5.5 Controlling the accuracy of the analysis results ...................................... 14

6 Safety requirements .............................................. 15

7 Operator qualification requirements .......................................... 15

Appendix A Bibliography

STATE STANDARD OF THE RUSSIAN FEDERATION

FOOD PRODUCTS Methods for determination of the mass fraction of benzo (a) pyrene

Methods for determination of benz (a) pyren fraction of total mass

Introduction date 2001-07-01

1 area of ​​use

This standard applies to food raw materials, food products, food and flavor additives and specifies methods for the determination of the mass fraction of benzo (a) pyrene using spectrofluorimetry at low and room temperature and high performance liquid chromatography.

General purpose laboratory scales of the 2nd class of accuracy with the maximum weighing limit of 500 g in accordance with GOST 24104.

Rotary evaporator IR-1M.

Bath water.

Household electric stove with a closed spiral and a heating regulator in accordance with GOST 14919.

Dewar vessel for liquid nitrogen of any capacity

Baths for chromatography (enameled photo cuvettes).

Glass plates measuring 15 x 30 and 20 x 40 cm.

Flasks К-1-250-29 / 32 ТХС, К-1-100-29 / 32 ТХС, К-1-500-29 / 32 ТХС or P-1-500-29 / 32 ТХС in accordance with GOST 25336.

Refrigerators HIT-1-300-14 / 23 XC or HIT-1-400-14 / 23 XC in accordance with GOST 25336.

Refrigerators HPT-2-400-29 / 32 XC and HPT-1-300-29 / 32 or HPT-400-29 / 32 XC in accordance with GOST 25336.

Reflux condenser 250-19 / 26-29 / 32 ТС or reflux condenser 300-19 / 26-29 / 32 ТС in accordance with GOST 25336.

Glass test tubes P2-10-180 XC in accordance with GOST 25336.

Nozzle P-1-19 / 26-14 / 23 ТС or Н2-19 / 23 in accordance with GOST 25336.

Laboratory water-jet pump in accordance with GOST 25336.

Pipettes with a capacity of 1, 2, 5, 10 cm 3 in accordance with GOST 29228 and GOST 29229.

Funnel VFO-32-POR 100-14 / 23 XC or VFO-32-POR 160-14 / 23 XC in accordance with GOST 25336.

Measured tubes P-2-15-14 / 23 XC in accordance with GOST 1770.

Separating funnel VD-1-500 or VD-3-500 in accordance with GOST 25336.

Measuring cylinders 1-100, 1-250 or 3-100, 3-250 in accordance with GOST 25336.

Weighing cups (weighing bottles) SV-14/8, or SV-19/9, or SV-24/10, or SV-34/12 in accordance with GOST 25336.

A thermometer with a temperature measurement range of 0-250 ° C with a graduation of 1 ° C in accordance with GOST 29224.

Glass capillaries, glass rods.

n octane, h., according to the normative document.

n.hexane, h., according to the normative document.

Rectified technical ethyl alcohol according to GOST 18300 or rectified ethyl alcohol according to GOST R 51652.

Petroleum ether fraction 40 - 70 ° С according to the normative document.

Aluminum oxide for chromatography of the 2nd degree of activity according to the normative document.

Benz (a) pyrene, the basic substance content is not less than 98%.

1,12-Benzperylene, the content of the basic substance is not less than 98%.

It is allowed to use other measuring instruments with metrological characteristics and equipment with technical characteristics, as well as reagents and materials of quality not lower than those indicated.

4.2 Test preparation

4.2.1 Cleaning of solvents

Solvents (n-octane, ethyl alcohol, petroleum ether, chloroform, and n-hexane) are distilled in a conventional manner with a reflux condenser.

4.2.2 Preparation of alumina

Alumina is dried in an oven at a temperature of (250 + 4) ° C for 4 hours and stored in a vessel with a ground stopper.

4.2.3 Preparation of benzo (a) pyrene solution for thin layer chromatography (witness solution).

About 10 mg of benzo (a) pyrene is weighed into a weighing bottle, several milliliters of petroleum ether are added until the sample is completely dissolved.

The resulting solution is quantitatively transferred into a volumetric flask with a capacity of 100 cm 3 and the volume of the solution is brought to the mark with petroleum ether. The shelf life of the solution is no more than three months in the refrigerator.

4.2.4 Preparation of benzo (a) pyrene standard solution

Weigh (10.0 ± 0.2) mg of benzo (a) pyrene into a weighing bottle, add several milliliters of n-octane until the sample is completely dissolved. The resulting solution is quantitatively transferred into a volumetric flask with a ground stopper with a capacity of 100 cm 3 and brought to the mark with n-octane. The mass concentration of benz (a) pyrene in the resulting solution is 100 μg / cm 3. Store the solution in a refrigerator. The shelf life of the solution is no more than three months.

4.2.5 Preparation of working solutions of benz (a) pyrene

Working solutions of benz (a) pyrene of mass concentration 0.1; 0.04 and 0.02 μg / cm 3 in n-octane are prepared by successive dilution of the initial standard solution of benzo (a) pyrene, prepared according to 4.2.4, in volumetric flasks with a ground-in stopper with a capacity of 100 cm 3. The solutions are stored in a refrigerator. The shelf life of the solutions is not more than one month.

4.2.6 Preparation of 1,12-benzperylene standard solution (internal standard)

To prepare the initial solution, (10.0 + 0.2) mg of 1,12-benzperylene are weighed in a weighing bottle, a few milliliters of n-octane are added until the sample is completely dissolved. The resulting solution is quantitatively transferred into a volumetric flask with a ground stopper with a capacity of 100 cm 3 and brought to the mark with n-octane. The mass concentration of 1,12-benzperylene in the resulting solution is 100 μg / cm 3. Store the solution in a refrigerator. The shelf life of the solution is no more than three months.

4.2.7 Preparation of working solutions of 1,12-benzperylene (internal standard solutions)

Working solutions of 1,12-benzperylene of mass concentration 0.01; 0.005; 0.002 and 0.001 μg / cm 3

prepare in n-octane by successive dilution of the stock standard solution prepared according to 4.2.6 in volumetric flasks with a ground-in stopper with a capacity of 100 cm 3. The solutions are stored in a refrigerator. The shelf life of the solutions is not more than one month.

4.3 Carrying out the test

4.3.1 Isolation of benzo (a) pyrene from the product

In a round-bottomed flask with a capacity of 500 cm 3, place a sample of the product weighing 25 g, add 20 cm 3 of distilled water, 200 cm 3 of ethyl alcohol and 20 g of potassium hydroxide to the flask.

The contents of the flask are mixed by shaking. The flask is connected to a reflux condenser and heated in a water bath while the reaction mixture is boiling for 3 hours. Then 150 cm 3 of water is added to the flask through a condenser; the flask is removed from the bath and cooled to room temperature.

After cooling, the liquid phase of the reaction mixture is transferred by decantation into a separatory funnel, leaving the remainder of the product in the flask. 150 cm 3 of n hexane is added to the flask with the residue, the contents of the flask are vigorously stirred and the n hexane is decanted into a separatory funnel.

The funnel is capped and shaken vigorously, then secured in a tripod and left to separate the liquids. To separate the resulting emulsion, 20 cm 3 of ethyl alcohol is added to the mixture in a separating funnel. After delamination, the lower aqueous-alcoholic phase is poured back into the flask with the sediment, and the hexane extract is poured into a flask with a capacity of 500 cm 3.

This treatment of the reaction mixture is carried out two more times, using for extraction n hexane, 100 cm 3 and ethyl alcohol for exfoliation of the emulsion, in portions of 20 cm 3.

At the end of the extraction, the residue in the flask and the hydrolyzate are discarded, and the extract is washed in a separating funnel with distilled water three times 50 cm 3 each and evaporated in portions in a round-bottomed flask previously weighed to the second decimal place with a capacity of 250 cm 3 on a rotary evaporator at a water bath temperature no more than 60 ° C. The flask with the extract is left in a fume hood to remove traces of solvent, and then weighed again. The difference between weighings is used to determine the mass of the isolated extract.

From the extract in the flask, take 1/5 part into a weighing bottle without weighing. The flask with the remainder of the extract is weighed. Add 0.1-0.2 cm 3 of the "witness" benz (a) pyrene solution prepared according to 4.2.3 to the bottle with a part of the extract. The contents of the bottle and the remainder of the flask are dissolved in a small volume of petroleum ether.

For chromatographic separation of the extract, alumina is evenly poured onto a 20x40 cm glass plate. Then, using a glass rod, divided into three parts (14, 1 and 3 cm) with rubber rings 1 mm thick and 3 mm wide, the alumina is carefully leveled.

The resulting solutions are quantitatively applied to the prepared plate with glass capillaries: on the narrow part - the solution from the bottle ("witness"), on the wide part - the extract of the product from the flask. The solutions are applied evenly in a continuous strip, departing from the lower edge of the plate 7 - 8 cm.

The plate is placed in a chromatographic bath at a slight 20 ° - 25 ° angle, petroleum ether is poured so that it does not reach the sample line. The bath is covered with glass and chromatography is carried out, bringing the solvent front to the upper edge of the plate.

Without drying the plate, irradiate it with ultraviolet light and determine the location of benzo (a) pyrene in the test sample by the luminous band of the "witness". Mark the boundaries of the benz (a) pyrene band on the chromatogram of the test sample. The plate is air dried in a fume hood.

The strip of aluminum oxide marked on the chromatogram of the test sample is removed from the plate using a glass slide and quantitatively transferred to the porous plate of the filter funnel. The funnel is connected to a round-bottom flask with a capacity of 100 cm 3 and benzo (a) pyrene is eluted from aluminum oxide with 50 cm 3 of benzene, adding benzene in small portions and stirring the alumina on the funnel with a stick. Benzene is evaporated to dryness on a rotary evaporator at a water bath temperature of not more than 60 ° C. The remainder of the flask is quantitatively transferred with octane into a test tube. The volume of the solution in the test tube should not exceed 5 cm 3.

In the analysis of some products, there is no complete and clear separation of the fluorescent components of the sample during the primary chromatography of the extract isolated from the product. In this case, a wider strip of alumina is isolated on the plate at the "witness" level; benz (a) pyrene is eluted from alumina with benzene as described above, and the evaporation residue is dissolved in ethyl alcohol and the resulting alcohol extract is rechromatographed.

For the chromatographic separation of the alcoholic extract, a 15 x 30 cm plate with a 0.3 mm alumina layer thickness is used. Two strips 10 and 3 cm wide are separated on the plate. An alcoholic extract of the analyzed product is applied to the wide part of the plate using a glass capillary, and a solution of benz (a) pyrene in petroleum ether (“witness” solution) is applied to the narrow part. The plate is placed in the bath at an angle 20 - 25 ° and chromatography is carried out in chloroform, bringing the front of the solvent to the upper edge of the plate. In ultraviolet light, a "witness" band of alumina with benzo (a) pyrene of the test product is marked. Then benz (a) pyrene is eluted from alumina with benzene and all further operations are carried out as described above.

GOST R 51650-2000

A solution of benzo (a) pyrene in n-octane is transferred into a test tube. The volume of the solution should not exceed 5 cm 3 with an initial sample of the product of 25 g.

In the resulting solution (extract), the content of benzo (a) pyrene is determined by low-temperature spectrofluorimetry, using the addition method or the internal standard method for quantitative assessment.

4.3.2 Determination of benzo (a) pyrene content in the solution (extract) obtained in 4.3.1 by the method of additions.

Pour 1 cm 3 of the resulting solution of benzo (a) pyrene in n-octane into three test tubes with a pipette. Then, 2 cm 3 of N. octane are poured into the first test tube. Into the second test tube, pour 1.5 cm 3 of N. octane and 0.5 cm 3 of a working solution of benzo (a) pyrene with a mass concentration of 0.1 μg / cm 3, prepared according to 4.2.5. Into the third test tube add 1 cm 3 of N octane and 1 cm 3 of the same working solution of benzo (a) pyrene as in the second test tube.

Spectrofluorometric analysis begins with the third tube. To do this, the third test tube is placed in a Dewar flask with liquid nitrogen in front of the entrance slit of the spectrophotometer; set the analytical line of benzo (a) pyrene fluorescence 403 nm at a wavelength of the exciting light 367 nm. By adjusting the gain and opening the slit, as well as by simultaneously adjusting the test tube in the Dewar flask, the maximum signal is achieved by the recording device of the spectrophotometer (up to 50 - 80%), after which the spectrogram of benzo (a) pyrene is recorded in the region of 401 - 404 nm, fixing the value of the recording device spectrophotometer at a wavelength of 401 nm. The spectrum recording is repeated twice.

Then, the second and first tubes are sequentially frozen in liquid nitrogen and the fluorescence spectra are recorded in the wavelength range of 401 - 404 nm, making sure to set the pen of the recorder at a wavelength of 401 nm in the same position as when scanning the sample in the third tube.

The mass concentration of benzo (a) pyrene in the analyzed extract is determined according to the graph, on which the value of benzo (a) pyrene addition (μg) is plotted along the abscissa axis, and the height of the maximum of the characteristic line of benzo (a) pyrene at 403 nm, along the ordinate axis, measured from the obtained spectrograms in millimeters.

If the mass concentration of benzo (a) pyrene in the test solution falls into the range suitable for measurements, then the obtained experimental points lie on one straight line. Extrapolation of this straight line to the intersection with the abscissa axis gives on it a segment corresponding to the content of benzo (a) pyrene in the solution without additive, that is, in 1 cm 3 of the test solution. If the mass concentration of benz (a) pyrene in the analyzed solution is higher than the upper limit of the range of concentrations measured by the device, then the analyzed solution is diluted with n-octane.

4.3.3 Determination of benzo (a) pyrene content in solution (extract) obtained according to 4.3.1 by internal standard method

1,12-benzperylene was used as an internal standard. 3 cm 3 of a solution of benzo (a) pyrene in n-octane, obtained according to 4.3.1, is poured into a test tube, and placed in a Dewar flask with liquid nitrogen in front of the entrance slit of the spectrophotometer, the analytical line is set at 403 nm at an exciting light wavelength of 367 nm and carried out recording the spectrum of the solution in the wavelength range 401 - 409 nm. According to the intensity of the line (according to the height of the peak of the maximum of the characteristic line of benzo (a) pyrene at 403 nm), the approximate content of benzo (a) pyrene in the sample is estimated. In accordance with this estimate, a solution of 1,12-benzperylene is added to a test tube with 3 cm 3 of a solution of benz (a) pyrene in n-octane in such an amount that the intensity of 1,12-benzperylene in the spectrum of the sample at

406.3 nm was 3 - 5 times the intensity of the benz (a) pyrene line at a wavelength of 403 nm.

The spectrum is recorded in the wavelength interval 401 - 409 nm twice.

Intensities of characteristic lines of benzo (a) pyrene at 403 nm and 1,12-benzperylene at

406.3 nm (H | and H 2, respectively) is determined from spectrograms, measuring the peak heights in the maxima of the characteristic lines of these compounds in millimeters. In the calculations, the average value is taken. Calculate the ratio coefficient (K) of the intensity of the benz (a) pyrene line (EHD to the intensity of the 1,12-benzperylene (EG 2) line, K = //] /// 2.

Further, this coefficient is determined for standard solutions of benzo (a) pyrene (X st). For this, 3 cm 3 of standard solutions of benzo (a) pyrene with a mass concentration of 0.02 and 0.04 μg / cm 3 are poured into two test tubes. The same amount of 1,12-benzperylene is poured into each of the test tubes as into the test tube with the sample. The spectra of each solution are recorded twice in the wavelength range of 401 - 409 nm.

In this case, it is imperative that the position of the pen of the recorder at a wavelength of 401 nm is fixed at the same level in all cases.

Next, the spectrograms determine the intensity of the characteristic lines of benzo (a) pyrene at 403 nm and 1,12-benzperylene at 406.3 nm (H and H 2, respectively). In the calculations, the average value is taken. Calculate K st = H ^ H 2 for each concentration of benzo (a) pyrene.

The mass concentration of benz (a) pyrene in the analyzed solution c, μg / cm 3, is calculated by the formula:

s s st * K / K st, (1)

where c st is the concentration of benzo (a) pyrene in a standard solution, μg / cm 3;

K is the coefficient found from the spectrogram of the analyzed solution with the addition of 1,12-benzperylene;

K C1 is the coefficient found from the spectrograms of a standard solution of benzo (a) pyrene with the addition of 1,12-benzperylene, the value of which is closer in value to the coefficient of the analyzed solution with the corresponding addition of 1,12-benzperylene.

Two parallel determinations are carried out and at the same time a control experiment is carried out through all stages of the analysis using all reagents according to the methodology, but without a sample of the product.

4.4 Expression of results

The mass fraction of benzo (a) pyrene L),%, X and X 2, mg / kg, is calculated by the formulas:

= (s - ep) ■ m l V ■ 100 = (s - ep) ■ t 1 ■ V (2)

3 t 2 ■ t ■ 1000 ■ 1000 t 2 ■ t '

_ (s - s 0) ■ V ■ t 1 (3)

where c is the concentration of benzo (a) pyrene, established according to 4.3.2 or 4.3.3 in the solution (extract) of the analyzed product obtained according to 4.3.1, μg / cm 3; с 0 - the concentration of benzo (a) pyrene in the solution of the control experiment obtained in 4.3.1, μg / cm 3; V is the volume of benzo (a) pyrene solution isolated from the analyzed product sample, cm 3;

/ "I is the mass of the extract isolated from the analyzed product, g; t 2 is the mass of the extract applied to a wide strip of the plate, g; t is the mass of the sample of the product, g.

The result is rounded to the second significant digit.

The arithmetic mean of two parallel determinations with the same number of significant digits is taken as the final result of the determination.

If the discrepancy between the results of parallel determinations does not exceed | A) - X 2 \<

< 0,01яЖ, где Xi, Х 2 и X- результаты первого и второго параллельных определений и их среднеарифметическое, a d- норматив контроля сходимости, то среднеарифметическое X принимают за результат анализа. В противном случае анализ повторяют. Значение норматива контроля сходимости d приведено в таблице 1.

Based on the obtained analysis result X and the value of the relative error q given in Table 1, calculate the absolute error A = 0, (SD mg / kg or%.

The analysis result is presented as (X ± A), mg / kg or% at P = 0.95.

4.5 Controlling the accuracy of analysis results

Internal operational control (EQA) of the quality of the analysis results includes the control of the convergence, reproducibility and accuracy of the analysis results.

4.5.1 The repeatability of parallel determinations is controlled for each analyzed sample in accordance with 4.4.

4.5.2 Use working samples for internal control of reproducibility. The sample is divided into two equal parts and analyzed in accordance with the methodology in different laboratories or in one laboratory, varying the conditions of the analysis as much as possible, that is, different sets of volumetric glassware are used, analyzes are performed on different days or by two different analysts.

The reproducibility of control analyzes is considered satisfactory if \ X ^ - X 2 \<

< 0,01 DX, где X/, Х 2 и X- результаты анализа одной и той же пробы, полученные в разных лабораториях или при варьирующих условиях в одной лаборатории и их среднеарифметическое значение, D - значение норматива внутреннего оперативного контроля воспроизводимости. Значение норматива D приведено в таблице 1.

GOST R 51650-2000

The frequency of the reproducibility control is at least once every two weeks.

Table 1- Measurement range, value of the characteristic of the relative error and standards of operational control of the random component of the relative error (convergence and reproducibility) at a confidence level of P = 0.95

4.5.3 To control the accuracy, use working samples with a known addition of benzo (a) pyrene. The sample is divided into two equal parts, one of which is analyzed in accordance with the procedure; in the second, a known additive of benzo (a) pyrene is introduced and then also analyzed in accordance with the procedure. The amount of the additive should be 50 - 150% of the benzopyrene content in the analyzed sample.

The accuracy of control analyzes is considered satisfactory if \ Xy-X- c \< 0,01 К, где Ху, Xи с - результаты контрольных анализов пробы с добавкой бенз(а)пирена, реальной пробы и величина добавки бенз(а)пирена, соответственно; К- норматив оперативного контроля точности. Норматив оперативного контроля точности рассчитывают по формулам: при проведении внутрилабораторного контроля (Р = 0,90)

K = 0.84 V (A X]) 2 + (A x) 2; (4)

during external control (P = 0.95)

K = V (A ^) 2 + (A z) 2, (5)

where A ^ + A x are the values ​​of the error characteristic corresponding to the mass concentration

benzo (a) pyrene in the additive sample and in the real sample;

Ay, = 0.01 Xy and A x = 0.01d x X, where Xy and X are the mass fraction of benzo (a) pyrene in the sample with the addition

and in a real sample,% or mg / kg.

The value of the relative error dx (8y) is given in table 1.

The analysis accuracy is monitored at least once a month, as well as when changing reagents or after a long break in work.

If the standards for operational control of accuracy are exceeded, repeated analyzes are carried out. If the specified standards are exceeded again, the analyzes are suspended, the reasons leading to unsatisfactory results are found out, and they are eliminated.

EQA results are recorded in a special journal.

5 Methods of high performance liquid chromatography and spectrofluorimetry at room temperature

The essence of the method consists in the extraction of hydrocarbons, including benz (a) pyrene, with hexane from the product pretreated with an alcoholic solution of potassium hydroxide, the isolation of a fraction of polycyclic aromatic hydrocarbons containing benzo (a) pyrene, and the purification of the resulting fraction from interfering impurities on a Sephadex column. and in a thin layer of acetylated cellulose, followed by quantitative determination of the isolated benz (a) pyrene by high performance liquid chromatography or spectrofluorimetry at room temperature.

The range of the determined values ​​of the mass fraction of benz (a) pyrene in the analyzed products using the method of high-performance liquid chromatography and the method of spectrofluorimetry at room temperature 0.0001-0.002 mg / kg or 0.1 x 10 -7 - 2.0 x 10 -7% ... The optimal range of determined mass concentrations of benzo (a) pyrene in solution when using the high-performance liquid chromatography method is 0.01-0.02 μg / cm 3, when using the spectrofluorimetry method - 0.02-0.2 μg / cm 3.

Benzapyrene belongs to the class of polycyclic aromatic hydrocarbons - PAHs. This is a group of organic compounds, the chemical structure of which contains benzene rings - groups of three or more rings. Chemical definition of benzopyrene: an organic substance containing carbon belonging to the group of polycyclic hydrocarbons, with a molar mass of 252.31 g / mol.

What is benzopyrene

Benzapirene, like all PAHs, is mainly the result of technical progress, a consequence of human activity. The main sources of anthropogenic pollution with PAHs are the combustion of solid and liquid organic substances, including oil and oil products, wood, and anthropogenic waste. Among the natural sources of benzopyrene, it is worth noting forest fires, volcanic eruptions.

However, the formation of benzopyrene can also occur without combustion processes - during pyrolysis, smoldering, polymerization.

Benzapirene is emitted during smoking: the content of benzopyrene in the smoke of one cigarette averages 0.025 μg, which is many times higher than the MPC (on average 10,000 -15,000 times). It has been calculated that smoking one cigarette in terms of benzopyrene content is equivalent to sixteen hours of inhalation of exhaust gases.

Benzapyrene formula

There are two isomers of benzopyrene. The first is 1,2-Benzapirene (3,4-benzpyrene), which is contained in all combustion products - oil, tar, coal, smoke of various origins, including. In their pure form, these are needle-shaped crystals or plates of light yellow color, with a melting point of about 177 ° C.

4,5-Benzopyrene - crystals in the form of needles and light yellow plates, with a melting point of 179 ° C. Contained in coal tar, found in soils (especially near enterprises and highways). Does not possess mutagenic, carcinogenic properties.

The chemical formula of benzopyrenes is C20H12.

Benzapyrene in soil and air

Benzapyrene practically does not occur in a free state, but is always deposited on particles contained in the air. Together with the moving air masses, benzopyrene is spread over a large area, and falling out together with solid particles from the air (for example, during precipitation) falls into the soil layers, water bodies, on the surface of buildings.

Its source, road transport, also plays a role in the migration and accumulation of benzopyrene. On the one hand, when traveling long distances, cars contribute to the even distribution of benzopyrene. On the other hand, settled benzopyrene accumulates in large quantities along highways and at facilities near them (the so-called "secondary sources").

Benzapirene is easily “included” in the cycle of substances in nature: with atmospheric precipitation, which always contains solid particles, it is introduced even in areas far from the main source of PAHs, enters water bodies, from where, during evaporation, it rises again into the air. It is this ability of benzopyrene to migrate that leads to the fact that its content can be high in places where there is no powerful source of this substance.

Getting into the environment and accumulating in it, benzopyrene penetrates into plants, which later serve as fodder for livestock or are used in human nutrition. The concentration of benzopyrene in plants is higher than its content in soil, and in food (or feed) it is higher than in the raw materials for their manufacture. This effect of increasing the concentration of chemicals, including benzopyrene, is called bioaccumulation.

Thus, benzopyrene is dangerous not only as a background pollution of the environment, but also as a substance that enters the body through the food chain.

MPC benzopyrene

The main method for the determination and control of benzopyrene is liquid chromatography.

According to the Hygienic Standards 2.1.6.695-98 and 2.1.6.1338-03, the maximum permissible average daily amount of benzopyrene in the air (MPCss) is 0.1 μg / 100 m3 or 10-9 g / m3, and its MPC in the soil is according to Hygienic Standards 2.1. 7.2041-06 - 0.02 mg / kg in total, taking into account the background level. In the air at workplaces, the average shift maximum permissible concentration is not more than 0.00015 mg / m3. (from item 1 and item 2. GN 2.2.5. 1313-03).

MPC of benzopyrene in water is not more than 0.000001 mg / l, in drinking water with a centralized water supply system - not more than 0.000005 mg / l. In bottled drinking water - from no more than 0.001 μg / l (high quality water) to no more than 0.005 μg / l in bottled water of the first quality category.

In food products in which the presence of benzopyrene is permissible due to technological features, the permissible level of benzopyrene is not more than 0.001 mg / kg. These include: sausages and products with the use of by-products, including smoked ones; smoked bacon; sausages and smoked products from meat and poultry offal; smoked canned food and fish preserves, smoked fish; food grain.

When using smoke flavorings, the benzopyrene content is not more than 2 μg / kg (l), and after their use, the benzopyrene content in finished products should not exceed 0.03 μg / kg (l).

Benzopyrene is not allowed in other food products.

However, according to the monitoring results, the benzopyrene content standards have been exceeded many times. On average, the level of air pollution in cities is 5-12 times higher than the MPC, in soils - 3-7 times, in food - from 1.5 to 11 times.

The effect of benzopyrene on the human body

Benzapiren is classified as a class 1 hazardous substance. The first class of hazard is substances with an extremely high hazardous impact on the environment, while the changes caused by them are irreversible and cannot be restored.

Benzapirene is one of the most potent and widespread carcinogens. Being chemically and thermally stable, possessing bioaccumulation properties, it gets into and accumulates in the body and acts constantly and powerfully. In addition to carcinogenic, benzopyrene has mutagenic, embryotoxic, hematotoxic effects.

The pathways for benzopyrene to enter the body are varied: with food and water, through the skin and by inhalation. The degree of danger is regardless of how the benzopyrene enters the body. In experiments, as well as according to monitoring data from ecologically unfavorable areas, benzopyrene is introduced into the DNA complex, causing irreversible mutations that are passed on to subsequent generations. Of particular concern is the fact of bioaccumulation of benzopyrene: the likelihood of mutations in the next generations of offspring increases due to bioaccumulation many times over.

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4.1.2. Technique for measuring the mass fraction of benzo (a) pyrene in food raw materials, food products and soil by high performance liquid chromatography

Purpose and scope

The technique is intended for the quantitative determination of benzo (a) pyrene (BP) in food raw materials, food products and soil at its mass fraction indicated in table. 1. The lower limit of the measurement range corresponds to 1/2 of the permissible level (content) of the toxin in products and raw materials, the upper limit to five times the permissible level.

Benz (a) pyrene is a highly toxic carcinogenic compound, and its permissible content in food products and food raw materials is established by the Sanitary Rules and Norms SanPiN 2.3.2.560-96.

The technique can be applied by institutions of the State Sanitary and Epidemiological Supervision of the Russian Federation, laboratories of other organizations and enterprises related to research, chemical analysis and certification of food products. The methodology is not arbitration.

Rice. 11.14.

• 1. Naphthalene 9. Chrysene (0.17 *)
• 2. Acenaphthene (1.40 *) 10. Benz (a) pyrene
• 3. Fluorene (2.60 *) 11. Benz (b) fluoranthene (0.26 *)
• 4. Phenanthrene * 2.40 *) 12. Benz (c) fluoranthene (0.10 *)
• 5. Anthracene (0.13 *) 13. Benz (a) pyrene (0.2 *)
• 6. Fluoranthene (0.74 *) 14. Dibenz (a, b) anthracene
• 7. Pyrene (0.67 *) 15. BeH3 (g, h, i) nepHaen (0.21 *)
• 8. Benz (a) anthracene (0.07 *) 16. Indeno (1,2,3-cc1) pyrene (0.26 *)

Analysis conditions:

Column: Supelcosil® LC RAS ​​(250 mm x 2.1 mm; 5 μm);

Gradient: Acetonitrile (A) 50-100%, water (B) 50-0%; 200 μl / min Temperature: 25 ° С Sample volume: 10 μl Detection: СFlu, according to the program

Measurement uncertainty characteristics

The boundaries of the relative error (± 5) of the measurement result of the mass fraction of BP (with a confidence level of 0.95) are shown in table. 1.

Table 1. Analyzed objects, ranges and characteristics of measurements

Product group (analyzed object)	Valid content ( IV X), mg / kg	BP mass fraction measurement range, mg / kg	Coefficient extraction,	Limits of relative error (± 8), %
Smoked meat, fish and fat products

Measuring instruments, auxiliary devices, materials and reagents

Measuring instruments

Liquid chromatographs:

• - Microcolumn "Milichrom-5", TU 25-7405.0009-89, version 3 with a fluorometric (FLD) detector (Option 1).
• - Isocratic or gradient liquid chromatograph, for example "Kpayer" (Germany), State Register of Measuring Instruments of the Russian Federation 16848-97 or Chromatographic attachment "HPZhKh-3", LLP "Lumex" (St. Petersburg), equipped with FLD "Fluorat-02" -2M ", LLP" Lumex ", State Register of Measuring Instruments of the Russian Federation 14093-99 (Option 2).
• - Isocratic or gradient liquid chromatograph with FLD of any type, for example "Kpayer" (Germany), State Register of Measuring Instruments of the Russian Federation 16848-97 (Option 3).

Chromatographic columns "Diasfer-110-S 16", TU 4215-001-05451931-94, CJSC "BioKhimMak ST" (Moscow), with standard sizes corresponding to the variant of the chromatographic system:

• - 2 x 80 mm, dp = 5 - 7 microns (Option 1)
• - 2 x 150 mm, dp = 5 - 7 microns (Option 2)
• - 4 x 150 mm, dp = 5 - 7 microns (Option 3)

Hardware and software complex "MultiChrom-Spectrum", TU AZHRTs

3.036.001, JSC "Ampersand" (Moscow), or any other software that allows for calibration and quantitative determination by the method of an external standard.

GSO 7515-98 composition of benz (a) pyrene solution in acetonitrile with benz (a) pyrene mass concentration of 100 μg / cm 3, AOZT Ekros (St. Petersburg).

GSO 7064-93 composition of benz (a) pyrene solution in hexane with benz (a) pyrene mass concentration of 100 μg / cm 3, AOZT "Ekros" (St. Petersburg).

Electronic laboratory scales 4 cl. accuracy model VLE 134, GOST 24104-88 or others.

Microsyringes, 100 μL Hamilton, Model Microliter # 1710 or equivalent.

Micropipettes 0.5; 0.2; 0.1 μl; GOST 20292-74.

Measuring cylinders 2-25,2-50, 2-100 and 2-500, GOST 1770-74.

Volumetric flasks 2-10-2, 2-100-2, GOST 1770-74.

Graduated pipettes 1, 2, 5, 10 cm 3, GOST 29227-91.

Reagents and materials

Acetonitrile for liquid chromatography, OP-3 high purity grade, TU 6-09-14-2167-84, rectified.

Double-distilled water, TU 6-09-2502-77.

Hexane, chemically pure grade, TU 6-09-3375-78, dried over Na 2 S 04 rectified.

Benzene, chemically pure, GOST 5955-75, dried over Na 2 S 04 rectified.

Anhydrous sodium sulfate, chemically pure, GOST 4166-76.

Concentrating cartridges "Diapak": A-3, P-3, S; TU 4215-002-05451931-94, CJSC BioChimMak ST (Moscow).

Assistive devices

System for filtration and degassing of eluents ZAO BioKhimMak ST (Moscow) or others.

Glass vials for calibration and analysis solutions with a capacity of 1.8 and 5.0 cm 3 with screw caps and Teflon gaskets from Supelco, catalog numbers 2-6951, 2-7037 and

2-7039, or similar.

Micro mixer PPE-3, Ekros (St. Petersburg).

Rotary evaporator IR-1M2, TU 25-1173.102-84 or others.

Pointed-bottom flasks with stoppers with a capacity of 25, 10 and 5 cm 3, GOST 25336.

A device for blowing off solutions in a stream of nitrogen, equipped with a thermostatted aluminum block (dry air bath) JV "BioMark" (Lvov) or others.

Membrane filters with dp = 0.4-0.5 microns.

A device for creating a vacuum of about 7 mm Hg. Art. (water jet pump, GOST 25336; water ring vacuum pump "Begemot", UVK-RK2 / 1, JSC "BioKhimMak ST", Moscow).

A vacuum device for sample preparation (vacuum manifold) or others with sample receivers with a capacity of at least 10 cm 3.

Buchner flask, Bunsen funnel with a capacity of at least 500 and 200 cm 3, respectively, GOST 1770.

Separating funnel with a capacity of 100 and 500 cm 3, GOST 25336.

Flat-bottomed conical flasks with stoppers with a capacity of 50, 100 and 250 cm 3, GOST 25336.

Pear-shaped flasks with stoppers with a capacity of 50 and 100 cm 3, GOST 25336.

Conical funnel with a diameter of at least 10 cm, GOST 1770.

Filter paper of the "blue ribbon" type.

Non-sterile medical cotton wool, cotton.

Measurement method

The technique includes the following basic procedures:

• - primary extraction with hexane and re-extraction into acetonitrile BP from a sample of the smoked product;
• - primary extraction of BP with an acetonitrile-water mixture from a grain or soil sample;
• - concentration and purification of the primary extract by solid-phase extraction;
• - dilution of the prepared sample extract with an acetonitrile-water mixture;
• - calibration of the chromatograph by solutions with a known value of the mass concentration of BP;
• - analysis of the solution of the prepared sample extract by high performance liquid chromatography (HPLC) with registration of the fluorescence signal;
• - identification of the determined BP by retention parameters;
• - calculation of BP mass concentration based on the registered analytical signal and calibration characteristics;
• - calculation of the mass fraction of BP, based on the mass concentration of BP, the mass of the food sample and the volume of the prepared extract solution.

Safety requirements

When working with the chemical compounds used, it is necessary to comply with the safety requirements established for working with toxic, caustic and flammable substances, GOST 12.1.018-86 and GOST 12.1.004-76, fire safety requirements, GOST 12.1.004-76.

If the BP solutions get on the skin "or the surface of objects, it is necessary to treat them with water and detergent, and then with ethyl alcohol. The solutions should be stored in a refrigerator in a sealed package.

When operating the system for HPLC and carrying out the corresponding measurements, it is necessary to observe the rules of electrical safety, GOST

12.1.019-79 and instructions for use of the device.

Operator qualification requirements

Persons are allowed to work:

• - qualified as a chemical engineer or chemical technician;
• - with experience in a chemical laboratory;
• - passed the relevant training courses and internships in laboratories accredited to perform analyzes using HPLC;
• - those who received positive results when performing control operations.

Measurement conditions

Sample preparation, preparation of solutions, preparation and measurements are carried out at an ambient temperature of 18-25 ° C, atmospheric pressure 84.0-100.7 kPa (630-800 mm Hg), air humidity not more than 80% (at temperature 25 ° C).

When making measurements in the laboratory, the following conditions must be met: voltage in the network 220 ± 10 V, frequency of current in the network 50zh 1 Hz. Measurements are carried out under the conditions recommended by the description and instructions for use of the device.

Preparing to take measurements

Glassware

Before further use, the used glassware is rinsed with the last of the solvents used and thoroughly washed with hot water with any detergent powder, rinsed sequentially with distilled and bidistilled water and dried. Store clean dishes with a cork or cotton swab.

Sampling, storage and handling

Sampling and averaging of samples is carried out in accordance with the regulatory documents for each type of product (GOST 13586.3-83, GOST 27668-88, GOST 9792-73, GOST 7631-85). The determined BP is extracted from the samples of smoked products by extraction with dry hexane, after evaporation of which it is re-extracted into acetonitrile. When extracting BP from grain or soil samples, a mixture of water-acetonitrile is used (16:84). Subsequent concentration and purification of the primary extract of the sample containing BP, regardless of the nature of the initial product, is performed in accordance with the complex scheme of solid-phase extraction using three diapak A-3, P-3, C concentrating cartridges.

Prepared samples (sample extracts) are dissolved in an acetonitrile-water mixture (70:30).

Each measurement of the mass fraction of BP includes preparation and chromatographic analysis of at least two samples.

Preparation of solvent mixtures

Mixtures of solvents are prepared by the volumetric method in graduated cylinders. The required volumes of acetonitrile and water are measured with separate graduated cylinders and then mixed. Extractant A: acetonitrile-water mixture (84:16).

Preparation of extractants

To prepare mutually saturated acetonitrile and hexane, about 300 cm 3 of acetonitrile and 100 cm 3 of hexane are shaken in a separating funnel with a capacity of 500 cm 3. After separation of the solvents, the layers are separated separately: the lower (acetonitrile saturated with hexane - extractant B) and upper (hexane saturated with acetonitrile - extractant C), the interphase is discarded.

Preparation of eluents

For HPLC measurements, acetonitrile-water mixtures are prepared in the following ratios: (90:10) - eluent 90, (84:16) - eluent 84, (80:20) - eluent 80, (70:30) - eluent 70. Ready eluents are filtered through a membrane filter and vacuum or thermal degassing is carried out.

Preparation of calibration solutions

The state standard sample of the composition of the BP solution in acetonitrile (see above) is diluted with a mixture of acetonitrile-water (7: 3) before use. Take with a pipette

1.0 cm 3 of the stock solution, place in a volumetric flask with a capacity of 100 cm 3 and add the solvent to the mark. Then, using a pipette, take certain volumes of the resulting solution, place them in a volumetric flask with a capacity of 10 cm 3 and add the solvent to the mark. The corresponding volumes of solutions used for dilution and the concentration of calibration solutions 2-5 (Option 1) and 3-7 (Options 2, 3) are shown in table. 2. When using GSO of the composition of BP solution in hexane (see below), after evaporation of the solvent under the conditions given below, the dry residue is redissolved in acetonitrile and diluted as described above, taking into account the value of the certified concentration of GSO.

Table 2. Calibration solutions for benzopyrene (BP) analysis

BP mass concentration (certified value of GSO), μg / cm 3	Stock solution for dilution		Calibration
				BP mass concentration, μg / cm 3

* Not used for direct calibration of the chromatographic system.

Preparing the chromatographic system

The chromatograph is switched on and prepared for operation in accordance with its description and operation manual. Set the column "Diasfer-110-C 16" with standard sizes in accordance with the chromatograph option (see above). The eluent with the highest concentration of acetonitrile is pumped through the chromatographic system until the baseline of the detector is stabilized, and then it is conditioned under the initial conditions of the gradient according to the corresponding sections "Analysis conditions".

Preparation of concentrating cartridges

Diapak A-3 and P-3 concentrating cartridges are prepared for operation as follows:

• 1. In a polypropylene case with a volume of 10 cm 3 with a replaceable filter in the lower part, 3 cm 3 of dry diapak A or P sorbent are poured. The upper empty part of the case is used as a funnel for applying a sample or eluent.
• 2. Mount the cartridge vertically in a suitable evacuation device and tap into an even horizontal top layer of sorbent. For the final preparation of the Diapak A-3 cartridge, it is enough to fix the sorbent layer with a small cotton swab.
• 3. To prepare the cartridge Diapak P-3, the sorbent is washed sequentially with 10 cm 3 of benzene, acetone and extractant A with weak evacuation (dripping rate no more than 1-2 drops per second), preventing air from entering the sorbent. After filling the cartridge with acetone, the sorbent is allowed to settle, the upper polymer filter is introduced, compacted over the upper layer of the sorbent and rinsing is continued. Upon reaching extractant A at a level of 2-3 cm above the filter, flushing is stopped and the cartridge is sealed with a lower plug and an upper cap (for storage). In case of accidental drying, the cartridge is washed extractant A. Before applying the sample, the plugs are removed and, under weak evacuation, the remains of the water-acetonitrile mixture are passed to the level of the upper filter, then the test solution is immediately poured. Regeneration of the reusable cartridge Diapak P-3 is carried out according to a similar scheme, excluding the removal of the upper filter.

The diapak C concentrating cartridge is prepared for work as follows:

• 1. A finished polypropylene capsule with 1 cm 3 of Diapak C sorbent is sealed with plugs. After removing the plugs, the cartridge is prepared for work by passing 5 cm 3 of hexane through it with a syringe at a dripping rate of 1-2 drops per second.
• 2. The sample is applied by gravity, using an empty polypropylene case with a volume of 10 cm 3 as a funnel, tightly fixed in the upper fitting of the Diapak C capsule.

Sample preparation for measurements

Extraction of benz (a) pyrene with hexane from a sample of smoked product A weighed portion of 10.0 g of a sample of the smoked product is ground in a mortar with 30 g of anhydrous sodium sulfate. The mixture is quantitatively transferred into a flat-bottom flask with a capacity of 100 cm 3 and extracted with hexane in a volume of 40 cm 3 for at least 30 minutes with stirring. The primary hexane extract of the total fat of the sample is decanted and passed through 10 g of anhydrous sodium sulfate into a stripping flask. The extraction procedure is repeated twice with two volumes of 20 cm 3 of hexane and portions of the extract are passed through a desiccant into the same stripping flask. Hexane is evaporated on a rotary evaporator at a temperature not higher than 35 ° C until the smell disappears.

The total fat extract is dissolved in 20 cm 3 extractant B, quantitatively transferred into a measuring cylinder with a capacity of 50 cm 3 and bring the volume of the solution to 40.0 cm 3 with the same solvent.

20.0 cm 3 of the resulting solution is transferred into a separating funnel with a capacity of 100 cm 3 and re-extraction of BP into acetonitrile is carried out in three volumes of 20 cm 3 extractant B. Each time, achieving the most complete phase separation possible, the lower layer (acetonitrile extract of BP) is taken and evaporated on a rotary evaporator at a temperature not exceeding 50 ° C to a volume of 10-15 cm 3. Quantitatively (using acetonitrile) transfer the solution into a measuring cylinder with a capacity of 25 cm 3, bring the volume to 21.0 cm 3 with acetonitrile, add 4.0 cm 3 of bidistilled water and thoroughly mix the resulting water-acetonitrile extract of BP.

Extraction of benzo (a) pyrene from a grain or soil sample with an acetonitrile-water mixture

A weighed portion of 10-25 g of the sample is transferred into a flat-bottomed flask, add 50-125 cm 3 extractant A, strictly observing the ratio 1: 5 of the weighed portion of the product to the volume of the extractant, and stirring for 1 hour. Filter water-acetonitrile extract of BP through a paper filter on a Buchner funnel under vacuum and squeeze the cake out on the filter.

Pre-purification and concentration of the primary extract of grain or smoked product

Pass through the cartridge Diapak A-3 25.0 cm 3 and then 3 cm 3 extractant A at a dripping rate of 2-3 drops per second into a receiving flask.

The collected eluate is passed through the prepared cartridge Diapak P-3, and then 5 cm 3 of acetonitrile at a dripping rate of 1-2 drops per second, discarding the washings. The target fraction containing BP is eluted from the cartridge with a mixture of benzene-acetonitrile (1: 1) in a volume of 7 cm 3 at a rate of 1-2 drops per second into a stripping flask. The eluate is evaporated on a rotary evaporator at a temperature not higher than 50 ° С, 0.5 cm 3 of hexane is added to the flask and thoroughly shaken on a micro mixer until the dry residue is completely dissolved.

Pre-purification and concentration of the primary soil extract

Pass through the cartridge Diapak A-3 5.0 cm 3 BP water-acetonitrile extract, then 3 cm 3 extractant A at a dripping rate of 2-3 drops per second into a receiving flask. Transfer the eluate into a measuring cylinder with a capacity of 25 cm 3, rinse the flask with two volumes of 5 cm 3 extractant A and bring the volume of the solution in the cylinder to 20 cm 3.

Pass through the prepared cartridge Diapak P-3 5.0 cm 3 of the diluted eluate, and then 5 cm 3 of acetonitrile at a dripping rate of 1-2 drops per second, discarding the washings. The target fraction containing BP is eluted from the cartridge with a mixture of benzene-acetonitrile (1: 1) in a volume of 7 cm 3 at a rate of 1-2 drops per second into a stripping flask. The eluate is evaporated on a rotary evaporator at a temperature not higher than 50 ° С, 0.5 cm 3 of hexane is added to the flask and thoroughly shaken on a micro mixer until the dry residue is completely dissolved.

Fine purification of the extract

Apply 0.5 cm 3 of a sample solution in hexane to the prepared cartridge Diapak C by gravity, then wash the flask with two portions of 0.5 cm 3 of hexane and subsequently apply them to the cartridge, discarding all washes. BP is eluted with benzene in a volume of 2.0 cm 3 at a dripping rate of 1-2 drops per second into a stripping flask and evaporated on a rotary evaporator at a temperature not exceeding 50 ° C. Dissolve the dry residue of the BP sample extract in an acetonitrile-water mixture (7: 3), the volumes of which are indicated in the “Measurement conditions” sections for each variant of the chromatographic system (see below).

Calibration and Measurement

Chromatograph calibration

The chromatograph is calibrated by sequential input (under the conditions of BP measurement) of the nominal volume of the calibration solutions (Table 2) in the order of increasing their mass concentrations. Each solution is injected into the chromatograph at least twice. With the correct adjustment of the chromatographic system, the height of the peak in the chromatogram of the calibration solution with the lowest concentration must exceed the baseline noise level by at least 10 times.

After mathematical processing of the chromatograms, the retention parameters and peak areas are recorded and calibration characteristics (GC) are plotted, reflecting the dependence of the average peak area value on the BP mass concentration in the calibration solution.

The correctness of the construction of the calibration characteristic is monitored.

The calibration characteristic is rebuilt when changing columns, after carrying out maintenance and repair work, with negative results of GC stability control (see below).

Determination of benzo (a) pyrene

Measurement conditions (Option 1)

For analysis, the prepared extract of the BP sample is dissolved in 0.1 cm 3 of an acetonitrile-water mixture.

Operating modes of FMD and UVPA are set from the computer keyboard in accordance with the User's Manual (PAK "MultiChrom-Spectrum") and controlled on monitors in the following form:

Fluorometric detector

• excitation wavelength 296 nm;
• emission wavelength - light filter No. 2 (more than 380 nm);
• measurement time 0.2 s.

Automatic dispenser

• the volume of regeneration is 0.4 cm 3;
• sample volume 0.04 cm 3;
• flow rate 0.15 cm 3 / min;
• set speed 0.3 cm 3 / min;
• retention time of BP 11 min.
• the composition of the eluents in the vessels and the scheme for drawing up the gradient of acetonitrile are presented in table. 3.

Table 3. Eluents

Measurement conditions (Option 2)

For analysis, the prepared (see above) extract of the BP sample is dissolved in 0.5 cm 3 of an acetonitrile-water mixture.

• the filter on the excitation line - "X4";
• emission line filter - "ХЗ";
• sample volume 0.02 cm 3;
• the sensitivity is average;
• smoothing - 4;
• the "background" level is selected according to the results of registration of the test chromatogram of the calibration solution No. 3.

• flow rate 0.2 cm 3 / min;
• eluent 84;
• retention time of BP is about 12 min.

Gradient separation mode:

• flow rate 0.25 cm 3 / min;
• eluent 100 in eluent 70 in 20 minutes;

Measurement conditions (Option 3)

For analysis, the prepared extract of the BP sample is dissolved in 0.5 cm 3 of an acetonitrile-water mixture.

• excitation wavelength 375 nm;
• emission wavelength 405 nm;
• flow rate 0.8 cm 3 / min;
• sample volume 0.02 cm 3;
• time constant 1.0 s.

Isocratic Separation Mode:

• eluent 84;
• retention time of BP is about 12 min;

Gradient separation mode:

• linear gradient from 30 to 70% eluent 100 in eluent 70 in 20 minutes;
• retention time of BP is about 14 min.

Obtaining and processing of chromatograms

The sample extract solution is introduced into the chromatograph twice. BP identification is carried out on the basis of comparison of the retention parameters of the peaks in the chromatograms of the sample extract and calibration solutions. Approximate retention parameters are given in the Measurement Conditions sections. Reliable identification of the compound to be determined corresponds to the difference between the values ​​of the retention parameters for the calibration solution and the sample, which does not exceed 0.2 min.

Dilution of the extract solution

Carried out if the mass concentration of the determined BP exceeds its highest mass concentration in the calibration solutions. The extract solution is diluted by half (dilution ratio, dil = 2), taking equal volumes of this solution and the acetonitrile-water mixture (70:30) and mixing the latter. If a single dilution does not eliminate the “off-scale”, the procedure is repeated (dilution ratio, dil = 4).

Processing of measurement results

Calculate the average value of the peak area (output signal of the chromatograph) for two injections of the sample extract solution into the chromatograph. Control the convergence of the output signals (see below).

The calibration dependence is used to find the value of the mass concentration of BP in the solution, which corresponds to the average value of the peak area.

The mass fraction of BP (I ^), mg / kg, in the i-th sample (the result of determination) is calculated by the formula

where Sbp is the mass concentration of the analyte in the solution of the extract of the i-th sample of BP, μg / cm 3 (calculated from the calibration dependence, based on the average value of the peak area); V p- the volume of the extract solution of the i-th sample of BP, cm 3; R- the degree of extraction of BP at the stage of sample preparation according to table. 2; Meq- the mass of a part of the sample, corresponding to the fraction of the water-acetonitrile extract of BP used for purification and subsequent chromatographic determination; - the equivalent mass of the sample, which is 5.0 g (grain, smoked products) or 0.25 g (soil).

In the case of dilution of the extract (see above), the mass fraction of BP ( W, -, mg / kg) in the i-th measurement is calculated by the formula

where Wj- the value obtained by the formula (II. 1), dil - the degree of dilution (see above).

Calculate the average value of the mass fraction of BP (analysis result) for two samples:

The convergence of the results of determining the mass fraction of BP is controlled (see below).

Registration of measurement results

The result of the analysis (measurement) of the mass fraction of BP in the determined object is presented in the form

where W - mass fraction of BP, calculated by the formula (II.3).

If the power supply unit is not found, the measurement result is presented in the form

where P "ds is the permissible BP content according to Table 1.

Error control MVI

Control of the convergence of the output signals of the chromatograph

The control is carried out during the calibration and analysis of each sample in relation to the output signals (values ​​of the BP peak areas on the chromatograms) obtained with two injections of the solution into the chromatograph. The control result is considered satisfactory if the range of the output signals, referred to the arithmetic mean, does not exceed 8%.

Control of the correctness of the construction of the calibration characteristic

Control is carried out at each calibration. The control result is considered satisfactory when the conditions are met for each "th calibration solution

where Sj is the average value of the BP peak area for the y "-th calibration solution, a.u .; S)- the value of the peak area corresponding to the calibration characteristic of the mass concentration of BP in the y "-th calibration solution, c.u.

Control of the stability of the calibration characteristic

The control is carried out daily before starting work with the analyzed samples using a control solution, which is used as a calibration solution with a BP mass concentration corresponding to its permissible content in the analyzed object.

The control result is considered satisfactory if the condition

where C ki is the value of the mass concentration of BP in the control solution, found from the calibration characteristic for the average value of the peak area, μg / cm 3; C to - the value of the mass concentration of BP in the control solution according to table. 2.

Checking the convergence of the determination results

Control is carried out at each analysis (measurement). The control result is considered satisfactory if the range of determination results, referred to the arithmetic mean (analysis result), does not exceed 10%.

Error control by the addition method

Control is carried out:

• a) before starting to apply this MVI - without fail;
• b) when dubious results of determining the mass fraction of BP appear;
• c) in accordance with the plans for internal laboratory control;
• d) at the request of organizations that control the activities of the laboratory.

The additive is formed on the basis of a calibration solution. Additive amount (D, mg / kg) is chosen such that the mass fraction of BP in the sample increases by 1.5-2.5 times. The calculation is carried out according to the formula

where C D- mass concentration of BP in the calibration solution, μg / cm 3; Vo- the volume of the BP calibration solution introduced into the sample, cm 3; M zhv is the equivalent mass of the sample taken for analysis, g.

The additive is introduced into the primary extract of a grain or soil sample in the form of a calibration solution prepared in accordance with table. 2. The additive is introduced into the primary extract of the smoked product in the form of a solution of the same concentration in hexane. For this, the GSO composition of BP in hexane (see above) is diluted (taking into account the correction for the certified concentration value) in accordance with the procedure for preparing calibration solutions (see above), using hexane as a solvent and obtaining a solution of the required concentration. When using GSO composition BP in acetonitrile, after evaporation of the solvent, the dry residue is redissolved in hexane and diluted with hexane, as described above.

Analysis of two samples with natural BP content and two samples with BP addition is carried out under the same conditions (one device, one calibration characteristic, one operator). The control result is considered satisfactory if the condition

where W D- mass fraction of BP in samples with additive, mg / kg; W- mass fraction of BP in samples without additive, mg / kg ( W D and W- mean values ​​of mass fraction for two samples with positive results of control of convergence).

An illustration of the application of this technique in practical ecoanalytics can be a chromatogram and a calibration graph for the determination of benzo (a) pyrene in food (Figures 11.15 and 11.16).

Results of monitoring the error in the determination of benzo (a) pyrene in objects by the addition method with fluorimetric detection of 375 Ex / 405 Et (Option 3).

The additive level corresponds to 0.5 permissible content of benzopyrene (0.0005 mg / kg - grain and smoked products, 0.01 mg / kg - soil).

Rice. 11.15.

Graduation for component: BaP Correlation coefficient: 0.999667 Response: Area

Reference channel 365 Ex / 405 Em

Formula Y = Ki X

The values ​​100 ( W D - W - D) / D are 10 and 16% for samples 1 and 2, respectively. The compound to be determined in the initial sample of wheat flour is present in an amount of 0.00009 kg / kg.

Sample 1 - 0.00064 mg / kg Sample 2 - 0.00067 mg / kg

• In the general case, the calibration characteristic has the following types: S = AC + B, where S is the peak area, a.u .; C is the mass concentration of the pollutant, μg / cm3; A and B are coefficients.