RU2613303C1 - Method for quantitative determination of n-nitrosamines in baby food - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention may be used for quantitative determination of N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) in baby foods, both dairy-free and dairy-based. The sample preparation requires as follows: sodium sulphate and aluminium sulphate are added to the product sample at a mass ratio of 1:1, sulphamic and sulphuric acids are added to the resulting mix to achieve pH 3. The sample is recovered by distillation with steam. The recovered distillate is subjected to solid phase extraction (SPE) using carbon cartridge, prewashed with methylene chloride/ethyl acetate/water. Sample is applied on the cartridge, dried for 20 minutes, and eluted by methylene chloride. Volume ratio of methylene chloride for cartridge washing, ethyl acetate, water, sample distillate and methylene chloride for elution is 1:1.25:1:35:2 accordingly. The resulting extract is tested by chromato-mass-spectrometry for the following nitrosamines: N-nitrosodimethylamine and N-nitrosodiethylamine.

EFFECT: invention provides selectivity, accuracy and precision of quantitative determination of N-nitrosamines and lowering detection limit for N-nitrosamines.

6 dwg, 7 tbl

Description

The invention relates to medical toxicological studies, in particular to sanitary toxicology, and can be used for the quantitative determination of N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) in food products, in particular in cereals, both milk and simple.

Nitrosamines have a wide range of toxic effects on the human body. First of all, their toxicity is directed to the liver and kidneys, resulting in a violation of their function and necrosis. When storing food, there is a significant increase in the concentration of certain amines, which can be a sign of spoilage. In connection with the foregoing, in order to solve the problem of carcinogenic food safety, it is necessary to improve food technologies that prevent the formation of carcinogenic nitrosamines in foods, especially for children, and also ensure their reliable control.

A known method for the determination of N-nitrosamines in a liquid and air medium (Auth. Certificate of the USSR No. 1343316) by treating them with chemical reagents in an acetic acid medium followed by measuring the optical density of the obtained colored solution, the treatment is carried out sequentially with zinc amalgam containing 1.5 -2% zinc, a solution of sodium hydroxide and an alcoholic solution of an aldehyde of benzene or nitrogen-containing heterocyclic series.

The disadvantages of this known method is the following:

- spectrophotometric method is non-selective;

- other nitrogen-containing compounds will interfere with the determination of nitrosamines, and therefore it is impossible to achieve the necessary accuracy of determination for a real assessment of the content of nitrosamines in the samples.

The prior art also known a method for the chromatographic determination of volatile amines and nitrosamines in food products (Auth. USSR certificate No. 1259187), including extracting them, distilling off the solvent, dissolving the residue in hydrochloric acid, dividing into several aliquots, while processing them, in addition to one, with alkali to pH 8-14, it is evaporated to dryness and dissolved, followed by the use of a solution of the untreated part as an analyte, and solutions of alkaline parts as standard after adding standard compounds to them inertia.

However, upon evaporation of the sample, losses of nitrosamines are possible, since they are highly volatile compounds, which will significantly affect the reliability and accuracy of the result.

In the article (http://www.medved.kiev.ua/arh_nutr/art_2004/n04_2_12.htm) "On the determination of n-nitrosamines in food products", authors: OS Zulfigarov, V.V. Yurchenko et al., Institute of Environmental Hygiene and Toxicology. L.I. Medveda, Kiev, Ukraine, it is also indicated that the first step in the determination of nitrosamines is their isolation from the analyzed products. Low boiling points (less than 200 ° С) and sufficient thermal stability of nitrosamines have made it possible to use steam distillation for their isolation. Simplicity in execution, versatility in relation to a wide variety of food products, completeness of extraction determined the widest application of this approach. Then, nitrosamines are isolated from aqueous distillates by extraction, the best extractant for this is methylene chloride, which is most used. This isolation scheme is effective for both liquid and solid samples. It is further recommended that the corresponding secondary amines be derivatized with 4-nitrophenyldiazonium. The derivatives thus obtained are sufficiently hydrophobic and can be easily concentrated and then separated by reverse phase high performance liquid chromatography. The essence of this known method is that the stationary phase is a non-polar (hydrophobic silica gel with grafted groups C4, C8, C18, etc.) phase; the mobile phase is polar (a mixture of water and polar solvents: acetonitrile, methanol, tetrahydrofuran, etc.). The retention of substances increases with their hydrophobicity (non-polarity). The higher the content of the organic solvent, the higher the eluting ability of the mobile phase). Since nitrophenyltriazenes are well colored, the sensitivity of their detection on conventional, most common photometric detectors is quite high and amounts to 2⋅10 ^-7 mol / L. This makes it possible to determine nitrosamines in food products when they are an order of magnitude lower than the MPC. An important circumstance is the possibility of applying this technique to determine the corresponding secondary amines. However, this method is not without drawbacks, because

1) many operations of preparing a sample for chemical analysis - steam distillation followed by extraction with an organic solvent and obtaining derivatives of nitrosamines (derivatization). There may be large losses of target analytes at the stages of sample preparation if nitrosamines in the sample are contained in ultramicroconcentrations. As a result, it is rather difficult to obtain reliable data on the quantitative content of nitrosamines in the sample.

2) the duration of serial studies, coextraction of a large number of interfering substances, low extraction efficiency.

The State Committee for Sanitary and Epidemiological Supervision of Russia in 1993 approved the methodology of MUK 4.4.1.011-93 “Determination of volatile N-nitrosamines in food raw materials and food products. Guidelines for control methods. - M.: Information and Publishing Center of the State Committee for Sanitary and Epidemiological Supervision of Russia, 1993. - 16 p., Which is selected as the closest analogue to the proposed invention. According to this Methodology, sample preparation is carried out, according to which sodium sulfate and aluminum sulfate are added to the sample in a mass ratio of 1: 1, then acidified to pH 3 with sulfamic acid and sulfuric acid, a hexane solution of nitrosodipropylamine is added, then distillation is carried out by distillation using water vapor to obtain distillate samples. The distillate is transferred to a separatory funnel and the nitrosamines are extracted with freshly distilled methylene chloride 4-5 times, 15 ml each. The combined chloromethylene extracts are dried with calcined magnesium sulfate, filtered off, the desiccant is washed with a small volume of methylene chloride, the combined filtrates are placed in a 100-150 ml round bottom flask, equipped with a reflux condenser (or air cooler), placed in a water bath and the methylene chloride is evaporated to 5-7 ml keeping at a bath temperature of 55-65 ° C. Determine N-nitrosamines by gas chromatography analysis.

However, the specified Methodology of MUK 4.4.1.011-93. It allows either semi-quantitative analysis (fluorimetric method) or quantitative determination of modified nitrosamines using a chemiluminescent (thermoenergetic) detector. During the derivatization procedure, loss of target analytes is possible if the matrix has sorption properties, which will significantly affect the reliability and accuracy of determination. In addition, this Methodology is distinguished by the multistage nature of their chromatographic separation and only semi-quantitative determination of nitrosamines is ensured, the lower limit of determination is 1 μg / kg of product.

The technical result achieved by the proposed method is to ensure the accuracy and reliability of the quantitative determination of N-nitrosamines due to the high completeness of their extraction from the sample, while reducing the lower limit of determination of N-nitrosamines.

The technical result is achieved by the proposed method for the quantitative determination of N-nitrosamines in cereals, including sample preparation, according to which sodium sulfate and aluminum sulfate are added to the sample in a mass ratio of 1: 1, then acidified to pH 3 with sulfamic and sulfuric acid, and distillation is carried out by distillation with using water vapor to obtain a distillate sample, the determination of N-nitrosamines using gas chromatographic analysis, while new is that before gas chromatography Of an aphic analysis, the obtained distillate is subjected to solid-phase extraction of TFE on a TFE device containing a carbon cartridge, in which first the methylene chloride is washed with a TFE cartridge, ethyl acetate is passed through it with a delay of 30 seconds, then it is washed with water; then the previously obtained sample distillate is loaded, the cartridge is dried for 20 minutes, the sample distillate is eluted from the cartridge with methylene chloride and the obtained eluents are analyzed by a chromatography-mass spectrometric method, determining the quantitative content of N-nitrosamines: N-nitrosodiamethylamine and N-nitrosodiethylamine from the chromatograms while TFE, the volume ratio of methylene chloride for washing the cartridge, ethyl acetate, water, sample distillate and methylene chloride for elution is 1: 1.25: 1: 35 : 2 respectively.

The specified technical result is achieved due to the following.

Known techniques and methods for sample preparation, such as extraction with an organic solvent, distillation with superheated water vapor, static and dynamic analysis of the equilibrium vapor phase (ARP) do not allow to achieve high completeness of extraction, and, therefore, the necessary accuracy of determinations for a real estimate of the content of nitrosamines in food products at the level of hygiene standard specified in SanPiN 2.3.2. 560-96 for baby food.

Due to the use of the solid-phase extraction operation (hereinafter - TFE) in the proposed method under the declared modes and reagents used, the completeness of nitrosamine extraction from the sample of baby porridge was ensured.

The traditional TFE procedure consists of several successive steps and is shown by the example of the TFE device shown in FIG. 2:

1 - Sepaths system;

2 - (N2) containers for organic solvents;

3 - containers for samples;

4 - containers for elution of samples from the cartridge;

5 - working platform for cartridges;

6 - vial for collecting eluate.

In order to activate the TFE system cartridge, conditioning with an organic solvent is carried out, which comes from containers 2 (shown in the diagram), then a liquid sample is loaded onto the cartridge from container 3 with nitrogen. During this, analytes are adsorbed on the sorbent, and the matrix is discharged. At this stage, the sorbent retains the target compounds to the elution step. The interfering components of the sample are completely removed in the next stage of washing the cartridge with an organic solvent that comes from tank 2. In this case, analytes of interest are eluted with a strong solvent coming from tank 4 and the eluate is collected in a vial 6.

The proposed method is implemented as follows in three stages:

- sample preparation,

- implementation of TFE,

- analysis of the obtained eluents by chromatography-mass spectrometric method.

1. Sample preparation.

When you open the box of porridge, take a portion of porridge weighing 20-50 g, dilute with water with a volume of 100 ml and the resulting sample is placed in a round bottom flask with a volume of 250 cm ³ for distillation.

Salting out reagents (5 g of sodium sulfate and 5 g of sodium chloride) are added to it.

Acidified with 2.5 ml of a 2% solution of sulfamic acid and 2.5 ml of 1 N. sulfuric acid to pH = 3.

Using superheated water vapor at a steam generator temperature of 100 ± 5 ° C and a bulb temperature with a sample of 80 ± 5 ° C, 70 cm of the distillate are distilled off.

2. The implementation of TFE.

First, the conditioning stage is carried out - the activation of the carbon cartridge, for which it is washed with methylene chloride with a volume of 2 cm ³ .

Then, a solvent of ethyl acetate with a volume of 2.5 cm ^{3 was} passed through the cartridge with a delay of the solvent for 30 seconds.

To remove residual solvents, the carbon cartridge is washed with 2 cm ³ water.

Next, load the previously obtained sample distillate with a volume of 70 cm ³ and dry the cartridge for 20 minutes.

Carry out the elution of the distillate of the sample from the cartridge with methylene chloride in a volume of 4 cm ³ .

In TFE, the volume ratio of methylene chloride for washing the carbon cartridge, ethyl acetate, water, sample distillate and methylene chloride for elution is 1: 1.25: 1: 35: 2, respectively.

3. Analysis of the obtained eluents by chromatography-mass spectrometric method.

The obtained eluents are analyzed by a known chromatography-mass spectrometric method, determining from the chromatograms the quantitative content of N-nitrosamines: N-nitrosodiamethylamine and N-nitrosodiethylamine. This method is ours (we worked out all the parameters of the chromatography-mass spectrometric method for the analysis of nitrosamines).

The optimal chromatographic-mass-spectrometric parameters for determining the volatile N-nitrosamines in porridge samples were tested using gas chromatography and mass spectrometry (GC / MS): an Agilent 7890A gas chromatograph (USA) with a 5975C mass-selective detector and a quadrupole mass analyzer. Electron impact ionization mode at 70 eV.

In the process of experimental studies, the following capillary columns of various lengths and thicknesses of the film of the stationary phase were studied: DB-624, HP-FFAP, HP-Plot / U, HP-VOC. Qualitative separation of N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) with close physicochemical properties was achieved on a capillary column of the HP-FFAP series 30 m ⋅ 0.250 mm ⋅ 0.250 μm with a length of 30 meters, an inner diameter of 0.250 mm and a film thickness of the stationary phase 0.250 μm. Modes of chromatography-mass spectrometric parameters are presented in table 1.

Chromatogram of a standard solution of N-nitrosamines under optimally selected conditions of chromatography-mass spectrometric analysis, recorded by the total ion current, with the content of N-nitrosamines (With _NDMA = 0.02 μg / cm ³ , With _NDEA = 0.02 μg / cm ³ ) is presented in FIG. one.

In modes 2 and 3 (Table 1), a sufficiently effective separation of volatile N-nitrosamines was not observed. Qualitative separation was achieved in mode 1, which was chosen for further work.

To select the optimal modes and reagents of the proposed method, laboratory studies were conducted to establish the necessary brands of cartridges in a TFE device, conditions for TFE elution and distillation schemes on standard samples of nitrosamines obtained by constructing a calibration dependence were worked out. To build the calibration characteristics prepared a series of solutions. For this, standard samples were used.

Preparation of solutions for analysis

Preparation of a solution of sulfuric acid. 60 cm ^{3 of} double-distilled water was placed in a heat-resistant chemical beaker; with continuous stirring, 29 cm ^{3 of} concentrated sulfuric acid was poured. The solution was cooled. Shelf life is unlimited.

Preparation of a solution of 2% sulfamic acid. Dissolve 2 g of sulfamic acid in 100 cm ³ bidistilled water. Shelf life 1 month.

Preparation of stock solution. Bidistilled water in a volume of 10 cm ³ is added to a 10 cm ³ volumetric tube, and 2 mm ^{3 of} N-nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine) are injected with a microsyringe. The weight content of the components in the initial solution is (taking into account the density and content of the main substance): N-nitrosodimethylamine 0.2 mg / cm ³ , N-nitrosodiethylamine 0.2 mg / cm ³ . The shelf life of the solution is 30 days.

Preparation of working solution. Into a volumetric flask with a volume of 50 cm ³ containing bidistilled water in a volume of 50 cm ³ , a 2 mm ³ dispenser of the initial solution of N-nitrosamines is introduced. The mass concentration of N-nitrosodimethylamine in the working solution is 0.008 mg / cm ³ and N-nitrosodiethylamine 0.008 mg / cm ³ . The shelf life of the solution is 5 hours.

Preparation of calibration solutions. Calibration solutions of N-nitrosamines are prepared in volumetric flasks with a volume of 100 cm ³ . To do this, add 50 cm ³ bidistilled water to each flask and add the working solution for graduation in accordance with Table 2, the contents of the flask are adjusted to the mark with bidistilled water and mix thoroughly.

It should be emphasized that the modes and reagents for the sample preparation stage were the same as in the prototype described in the Methodology of MUK 4.4.1.011-93.

To obtain extracts of the required purity for chromatography-mass spectrometric analysis and to establish a sufficient degree of concentration, replaceable TFE cartridge cartridges filled with various sorbents, organic solvents for elution of nitrosamines from cartridges were tested and the conditions of the optimal elution scheme were studied. The selectivity of the TFE process was achieved by selecting a cartridge with the appropriate phase.

For this, the following cartridge types were tested: coal Coconut 6 ml; filled with octadecyl Chromabond C18 100 and 500 mg; 200 mg Strata polymer-based cartridges.

The results of the study of the completeness of extraction of nitrosamines from a standard sample using various brands of cartridges are presented in table 3.

An analysis of the results shown in Table 3 showed that on a Coconut carbon cartridge of 6 ml, the N-nitrosamine extraction rate from a standard sample was 97.5% complete.

Studies have shown an insufficient degree of extraction of nitrosamines from standard samples. Therefore, additional studies were conducted to test the optimal conditions for the elution of nitrosamines from a Coconut 6 ml carbon cartridge using various solvents. For this, we varied:

- volumes of solvents at the stage of conditioning - activation of the cartridge;

- sample volumes at the stage of loading into the cartridge;

- cartridge drying time;

- the volume of the drain when eluting the sample from the cartridge with methylene chloride.

Experimentally developed schemes for the elution of nitrosamines of a standard sample from a Coconut 6 ml carbon cartridge by solid-phase extraction are presented in table 4.

The results of the study of the completeness of the extraction of nitrosamines from a standard sample by the introduced-found method using various schemes (see table 4) for TFE elution and a 6 ml Coconut carbon cartridge are shown in table 5.

In the process of experimental studies, it was found that the most optimal scheme for the elution of nitrosamines by TFE using a 6 ml Coconut carbon cartridge in the proposed method is scheme 7, which includes several stages:

1. The conditioning step — activation of the cartridge: the cartridge was washed with 2 cm ³ methylene chloride, then 2.5 cm ³ ethyl acetate was passed through the cartridge with a solvent delay of 30 seconds. To remove residual solvents, the cartridge was washed with 2 cm ³ water;

2. The stage of adsorption of the target components of the standard sample on the cartridge when loading samples with a volume of 70 cm ³ .

3. Dry the cartridge for 20 minutes to remove residual sample.

4. The final stage of TFE is the elution of target analytes from a cartridge of methylene chloride with a volume of 4 cm ³ .

Thus, it was found that when implementing the proposed method at the TFE stage, the volume ratio of methylene chloride for washing the cartridge, ethyl acetate, water, sample distillate and methylene chloride for elution should be 1: 1.25: 1: 35: 2, respectively.

Under these optimally established conditions of TFE, then with subsequent chromatography-mass spectrometric analysis, when implementing the proposed method, high separation efficiency of nitrosamines of a standard sample was achieved, which is clearly illustrated by the chromatogram shown in FIG. 3. This chromatogram shows that the following N-nitrosamines are contained in the standard solution: N-NDMA - N-nitrosodimethylamine = 0.02 μg / cm ³ , N-NDEA - N-nitrosodiethylamine = 0.02 μg / cm ³ .

The completeness of extraction of nitrosamines from the standard sample for NDMA - 97.5%, for NDEA - 100% was established using a Coconut carbon cartridge 6 ml and elution scheme 7 (table 5).

To increase the completeness of extraction extraction of analytes, especially nitrosodimethylamine from a standard sample, distillation with superheated steam was used (it is known that nitrosamines are volatile compounds and are decomposed with water vapor without decomposition).

Therefore, the next stage of research was to develop the conditions for the distillation of nitrosamines of a standard sample, followed by sorption on a 6 ml Coconut carbon cartridge. To do this, the investigated standard sample in a volume of 25 cm was placed in a round bottom flask with a volume of 250 cm ³ for distillation, salting out reagents (5 g of sodium sulfate and 5 g of sodium chloride) were added. A standard sample of nitrosamines was acidified with 2.5 ml of 2% sulfamic acid and 2.5 ml of 1 N. sulfuric acid to pH = 3. Using superheated water vapor at t ° steam generator = 100 ± 5 ° C and t ° flasks with a standard sample = 80 ± 5 ° C, 25 cm ³ and 70 cm ^{3 of} distillate were distilled off. The resulting distillate (volume 25 cm ³ and 70 cm ³ ) was passed through a 6 ml Coconut cartridge in a solid phase extraction system according to the elution scheme 7.

Then, the obtained eluents were analyzed by chromatography-mass spectrometric method.

The results of studies of the completeness of the extraction of nitrosamines from standard samples by the introduced-found method using various distillation schemes and TFE according to scheme 7 are presented in table 6. The data shown in table 6 show that the integrated use of distillation in combination with the optimal elution scheme 7 and concentration of the distillate in a volume of 70 cm ³ for a 6 ml Coconut carbon cartridge, it was possible to achieve high completeness of extraction of N-nitrosamines from a standard sample, which amounted to 98.5% for NDMA and 100% for NDEA.

It should be clarified that absolutely the same data (tables 3-6) as for standard samples were obtained for the samples of the studied cereals, which confirms the reliability of the data presented.

The final stage of the studies was the testing of the developed method using the Sepaths automatic solid-phase extraction system (TFE) for chromatography-mass spectrometric analysis of N-nitrosamines for various types of baby food.

Studies of standard samples and samples of cereals of various manufacturers for the content of nitrosamines were performed by chromatography-mass spectrometry: gas chromatograph Agilent 7890A (USA) with a mass-selective detector 5975C and a quadrupole mass analyzer. Electron impact ionization mode at 70 eV. For studies, we used a capillary column of the HP-VOC series 60 m ⋅ 0.2 mm ⋅ 1.12 μm 60 meters long, 0.2 mm inner diameter, and 1.8 μm stationary phase film thickness.

The data are shown in table 7, as well as on chromatograms of nitrosamines found in porridge samples. In FIG. 4 - for Winnie oat milk porridge (N-NDMA = 0.0038 mg / kg, N-NDEA = 0.0035 mg / kg); in FIG. 5 - for oat milk with Heinz banana (N-NDMA = 0.0043 mg / kg, N-NDEA = 0.0012 mg / kg); in FIG. 6 - for multi-cereal milk “FrutoNanny” (N-NDMA = 0 mg / kg, N-NDEA = 0,00066 mg / kg).

The data shown in table 7 show that the proposed method was found to have an increased content of the determined components by the sum of N-nitrosamines in cereals relative to the hygiene standard in the range from 5.3 to 10.8 times.

In multigrain and buckwheat cereals with peaches and apricots FrutoNyanya, the content of nitrosamines is set below the MPC.

Thus, the proposed method has the following advantages over the known:

- provides almost complete extraction of nitrosamines from the test sample (for NDMA - 98.5%, for NDEA - 100%), which indicates the accuracy and reliability of the proposed method;

- can significantly reduce the lower limit of determination of nitrosamines (up to 0.4 μg / kg of product), while in the prototype this figure is 1 μg / kg of product;

- provide selectivity and specificity; more effective separation of nitrosamines; high quantitative extraction of the test sample (> 75%); excellent reproducibility, reliability and accuracy of determination; ease of implementation; the possibility of optimizing the TFE process; saving expensive solvents.

Claims (1)

A method for the quantitative determination of N-nitrosamines in cereals, including the preparation, according to which sodium sulfate and aluminum sulfate are added to the sample in a mass ratio of 1: 1, then acidified to pH 3 with sulfamic and sulfuric acid, and distillation is carried out by distillation using water vapor to obtain sample distillate, determination of N-nitrosamines using gas chromatographic analysis, characterized in that before the gas chromatographic analysis, the obtained distillate is subjected to solid phase extraction ii TFE in the TFE unit, containing carbon cartridge at which produces first cartridge washing with methylene chloride TFE instrument is passed therethrough with its delayed acetate for 30 seconds, followed by washing with water produced; then the previously obtained sample distillate is loaded, the cartridge is dried for 20 minutes, the sample distillate is eluted from the cartridge with methylene chloride and the obtained eluents are analyzed by a chromatography-mass spectrometric method, determining the quantitative content of N-nitrosamines: N-nitrosodiamethylamine and N-nitrosodiethylamine from the chromatograms while TFE, the volume ratio of methylene chloride for washing the cartridge, ethyl acetate, water, sample distillate and methylene chloride for elution is 1: 1.25: 1: 35: 2 ootvetstvenno.

Images