Method for detecting n-nitrosodimethylamine in meat products

Abstract

The application discloses a kind of detection methods of N-dimethyl nitrosamine in meat product, comprising: step one, cut piece of meat product, put into soaking liquid and soak, ultrasonic oscillation, clean with clean water;Step two, the meat block is crushed, add acetonitrile, anhydrous magnesium sulfate, n-hexane, drop 0.1% glacial acetic acid, ultrasonic oscillation, stand, liquid separation, take acetonitrile layer, repeat 1-3 times, combine acetonitrile layer, add extractant again, ultrasonic oscillation, centrifugal, obtain filtrate;Step three, take solid-phase extraction column activation, sample filter liquor, elution, nitrogen blowing, redissolve, filter, obtain sample to be measured;Step four, the sample to be measured is carried out qualitative and quantitative detection using gas chromatography mass spectrometry, and the content of N-dimethyl nitrosamine is obtained.The application detects N-dimethyl nitrosamine by removing grease and residual components after pretreatment of meat product, with high accuracy and precision.

Description

Technical Field

[0001] This invention relates to the field of food analysis and testing technology. More specifically, this invention relates to a method for detecting N-dimethylnitrosamine in meat products. Background Technology

[0002] Nitrites are often added during the processing of meat products such as cured pork, braised pork, and sausages to prevent spoilage and enhance color, aroma, and flavor. However, the curing process of meat with added nitrites can lead to the formation of N-nitrosodimethylamine (NDM), which not only causes food contamination but is also a potent carcinogen. Consuming these meat products with alcohol can exponentially increase the health risks of NDM. Therefore, detecting the NDM content in meat products is of great significance. Gas chromatography-mass spectrometry (GC-MS) combines the separation capabilities of GC with the molecular weight and structure determination capabilities of mass spectrometry, making it an effective detection system for analyzing and identifying complex multi-component organic mixtures. It can be used to detect NDM in meat products. However, meat products require pretreatment. When using steam distillation for extraction, on the one hand, meat products usually contain fat, which increases the difficulty of extraction; on the other hand, steam distillation requires a long distillation time to ensure the recovery rate, which is time-consuming and poses many safety risks. Exploring a fast and safe pretreatment method suitable for meat products to facilitate the detection of N-dimethylnitrosamine content is an urgent technical problem to be solved. Summary of the Invention

[0003] This invention provides a method for detecting N-dimethylnitrosamine in meat products. The method involves pre-treating the meat products to remove grease and residual components before detecting N-dimethylnitrosamine, and it has high accuracy and precision.

[0004] To achieve these objectives and other advantages according to the present invention, a method for detecting N-dimethylnitrosamine in meat products is provided, comprising:

[0005] Step 1: Cut the meat products into pieces and soak them in a soaking solution, which includes flour, water, disodium citrate, modified oil ethoxylate, and sodium sulfate of fatty alcohol polyoxyethylene ether. Air is blown into the soaking solution to form tiny bubbles, ultrasonic vibration is applied, and the meat is then rinsed clean with water.

[0006] Step 2: Crush the meat pieces, add acetonitrile, anhydrous magnesium sulfate, and n-hexane, add 0.1% glacial acetic acid, ultrasonically vibrate, let stand, separate the liquids, take the acetonitrile layer, repeat 1 to 3 times, combine the acetonitrile layers, add the extraction agent, which includes sodium alginate and anhydrous magnesium sulfate, ultrasonically vibrate, centrifuge, and obtain the filtrate.

[0007] Step 3: Activate the solid phase extraction column, load the filtrate onto the sample, elute with 5-10% (w / w) ammoniated methanol, collect the eluent, purge with nitrogen, redissolve, and filter with a 0.22 μm organic filter membrane to obtain the sample to be tested;

[0008] Step four: The sample to be tested is subjected to qualitative and quantitative detection using gas chromatography-mass spectrometry to obtain the content of N-dimethylnitrosamine.

[0009] Preferably, in step one, the soaking solution comprises flour, water, disodium citrate, modified oil ethoxylate, and sodium sulfate of fatty alcohol polyoxyethylene ether in a weight ratio of 15-20:100:15-20:5-10:5-10.

[0010] Preferably, in step one, the meat product is cut into pieces, soaked in the soaking solution for 30-60 minutes, the ultrasonic temperature is room temperature, the ultrasonic frequency is 60 kHz, and the ultrasonic extraction time is 15 minutes.

[0011] Preferably, in step two, the meat chunks are ground into powder, and 0.5 mL of acetonitrile, 0.5 g of anhydrous magnesium sulfate, and 2 mL of n-hexane are added per gram of powder. The ultrasonic temperature is room temperature, the ultrasonic frequency is 60 kHz, and the ultrasonic extraction time is 5 min.

[0012] Preferably, in step two, the amount of extractant added to each milliliter of acetonitrile layer is 8g, and by weight, the extractant includes sodium alginate and anhydrous magnesium sulfate in a ratio of 1:0.5 to 2. The ultrasonic temperature is room temperature, the ultrasonic frequency is 60kHz, and the ultrasonic extraction time is 15min.

[0013] Preferably, in step three, the packing material of the solid-phase extraction column is graphitized carbon with an average particle size of 120–400 mesh and a specific surface area of ​​100 m². 2 / g, the sample loading amount is 2-10% of the packing mass, and the elution solvent volume is 10-15 times the volume of the packing in the column.

[0014] Preferably, step three includes:

[0015] S1: Prepare N-dimethylnitrosamine standard solutions of different concentrations, inject them into a gas chromatograph-tandem mass spectrometer, and plot a standard curve based on the detection results;

[0016] S2: Inject the sample solution to be tested into a gas chromatograph-tandem mass spectrometer to obtain the chromatographic peak of the quantitative ion of N-dimethylnitrosamine. Combined with the standard curve, the concentration of N-dimethylnitrosamine in the sample to be tested is calculated.

[0017] Preferably, the gas chromatography conditions are as follows: the column is an INNOWAX quartz capillary column; the injection port temperature is 220℃; the temperature program is as follows: initial column temperature 40℃, increased to 80℃ at a rate of 10℃ / min, increased to 100℃ at a rate of 1℃ / min, then increased to 240℃ at a rate of 20℃ / min, and held for 2 min; carrier gas is helium; flow rate is 1.0 mL / min; injection volume is 1.0 μL.

[0018] Mass spectrometry conditions were as follows: ion detection, scanning of N-dimethylnitrosamine starting at 9.9 min, selected ions 15.0, 42.0, 43.0, 44.0, and 74.0; electron impact ionization (EI) source, voltage 70 eV, ionization current 300 μA, ion source temperature 230 °C, interface temperature 230 °C, and ion source vacuum 1.33 × 10⁻⁶. -4 Pa.

[0019] The present invention has at least the following beneficial effects:

[0020] First, after the meat products were pretreated using the method of this invention, the detection of N-nitrosodimethylamine showed linearity in the concentration range of 0.01 μg / mL to 0.5 μg / mL, with a linear correlation coefficient of 0.9998 and a precision RSD of 4.6%. The recovery rate was 71.25% to 86.17% in the concentration range of 10 μg / kg to 80 μg / kg, with an average recovery rate of 78.29% and an RSD of 6.6%. Therefore, this invention has high accuracy and precision.

[0021] Secondly, the pretreatment method of this invention introduces disodium citrate into the soaking solution to precipitate metal ions. The introduction of SOE and AES provides good biodegradability and non-toxicity. Furthermore, the combination of SOE and AES has a thickening effect, strong emulsifying ability for oils, and adheres to bubbles, enhancing the oil removal capacity. Adding glacial acetic acid improves the extraction of N-dimethylnitrosamine by acetonitrile. Hexane is used to remove oil, and the extraction agent is introduced for further adsorption and removal of oil. Solid-phase extraction at room temperature avoids the oxidation of unsaturated fatty acids caused by heating, further removing oil and residual components.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0024] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0025] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0026] 1. Reagents and Instruments

[0027] Chromatographic column: INNOWAX quartz capillary column (30m long, 0.25mm inner diameter, 0.25μm membrane thickness), centrifuge, ultrasonic oscillator, bubbler, balance, 0.22μm organic filter membrane, N-dimethylnitrosamine (chromatographic grade), cooked meat products (purchased from supermarket).

[0028] 2. Preparation of standard products

[0029] Standard stock solution: Weigh N-dimethylnitrosamine standard and prepare a 1 μg / mL standard stock solution with acetonitrile;

[0030] Standard solutions: Dilute the standard stock solution with acetonitrile to obtain standard solutions with concentrations of 0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL, respectively.

[0031] 3. Preparation of the test sample

[0032] Step 1: Cut 100g of meat products (cured meat) into pieces and soak them in the soaking solution for 30 minutes. The soaking solution contains flour, water, disodium citrate, modified oil ethoxylate (SOE), and sodium fatty alcohol polyoxyethylene ether sulfate (AES) in a weight ratio of 20:100:15:10:10. Air is bubbled into the soaking solution to form tiny bubbles. Ultrasonic oscillation is performed at room temperature and 60kHz for 15 minutes. The product is then rinsed with clean water.

[0033] Step 2: Crush the meat chunks into powder. Add 0.5 mL of acetonitrile, 0.5 g of anhydrous magnesium sulfate, and 2 mL of n-hexane to each gram of powder. Add 0.1% glacial acetic acid dropwise. Perform ultrasonic oscillation at room temperature and 60 kHz for 5 min. Allow to stand, separate the layers, and take the acetonitrile layer. Repeat this process 3 times. Combine the acetonitrile layers and add the extractant. The amount of extractant added to each milliliter of acetonitrile layer is 8 g. The extractant includes sodium alginate and anhydrous magnesium sulfate in a 1:1 ratio. Perform ultrasonic oscillation at room temperature and 60 kHz for 15 min. Centrifuge to obtain the filtrate.

[0034] Step 3: The solid-phase extraction column is packed with graphitized carbon, with an average particle size of 120–400 mesh and a specific surface area of ​​100 m². 2 / g, the sample loading amount is 5% of the packing mass, the elution solvent volume is 15 times the volume of the packing in the column, the solid phase extraction column is activated, the filtrate is loaded onto the sample, and eluted with 5% ammoniated methanol by mass, the eluent is collected, nitrogen is blown out, redissolved, and the volume is adjusted to 5mL. The sample is then filtered through a 0.22μm organic filter membrane to obtain the sample to be tested.

[0035] Qualitative and quantitative detection of 4N-dimethylnitrosamine

[0036] Qualitative and quantitative detection were performed using gas chromatography-mass spectrometry.

[0037] The gas chromatography conditions were as follows: INNOWAX quartz capillary column, injection port temperature 220℃, and programmed temperature rise: initial column temperature 40℃, increased to 80℃ at a rate of 10℃ / min, increased to 100℃ at a rate of 1℃ / min, then increased to 240℃ at a rate of 20℃ / min and held for 2 min; helium as carrier gas; flow rate 1.0 mL / min; injection volume 1.0 μL.

[0038] Mass spectrometry conditions were as follows: ion detection, scanning of N-dimethylnitrosamine starting at 9.9 min, selected ions 15.0, 42.0, 43.0, 44.0, and 74.0; electron impact ionization (EI) source, voltage 70 eV, ionization current 300 μA, ion source temperature 230 °C, interface temperature 230 °C, and ion source vacuum 1.33 × 10⁻⁶. -4 Pa.

[0039] The standard solution was injected for analysis, and the peak area was used to perform linear regression on the concentration. A standard curve was plotted based on the detection results.

[0040] The sample solution to be tested was injected for analysis to obtain the chromatographic peak of the quantitative ion of N-dimethylnitrosamine. Combined with the standard curve, the concentration of N-dimethylnitrosamine in the sample to be tested was calculated.

[0041] 5. Method Evaluation

[0042] 5.1 Standard Curve

[0043] N-Dimethylnitrosamine showed good linearity in the concentration range of 0.01 μg / mL to 0.5 μg / mL. y represents the peak area, x represents the concentration (ng / mL), and the regression equation is y = 8179.7x + 18255, with a linear correlation coefficient r = 0.9998.

[0044] 5.2 Precision

[0045] Five samples were weighed and analyzed. The average content of N-dimethylnitrosamine was 2.17 μg / kg, and the RSD was 4.6%.

[0046] 5.3 Accuracy and Recovery Rate

[0047] Fresh pork was used to simulate meat products free of N-nitrosodimethylamine. Five portions of fresh pork were weighed and spiked with N-nitrosodimethylamine at concentrations of 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 4 μg / kg, and 6 μg / kg, respectively, injected as solutions. After pretreatment, the spiked samples were analyzed. The average recovery rate of N-nitrosodimethylamine was 78.29%, with an RSD of 6.6%.

[0048] In summary, the detection of N-nitrosodimethylamine in meat products after pretreatment using the method of this invention shows linearity in the concentration range of 0.01 μg / mL to 0.5 μg / mL, with a linear correlation coefficient of 0.9998 and a precision RSD of 4.6%. The recoveries range from 71.25% to 86.17% with a spiked amount of 0.5 μg / kg to 6 μg / kg, with an average recovery rate of 78.29% and an RSD of 6.6%. Therefore, this invention exhibits high accuracy and precision.

[0049] <Example 1>

[0050] Step 1: Select 100g of fresh pork for spiked treatment and soak it in the soaking solution for 30 minutes. The soaking solution consists of flour, water, disodium citrate, modified oil ethoxylate (SOE), and sodium fatty alcohol polyoxyethylene ether sulfate (AES) in a weight ratio of 20:100:15:10:10. Air is bubbled into the soaking solution to form tiny bubbles. Ultrasonic oscillation is performed at room temperature and 60kHz for 15 minutes. The pork is then rinsed with clean water.

[0051] Step 2: Crush the meat chunks into powder. Add 0.5 mL of acetonitrile, 0.5 g of anhydrous magnesium sulfate, and 2 mL of n-hexane to each gram of powder. Add 0.1% glacial acetic acid dropwise. Perform ultrasonic oscillation at room temperature and 60 kHz for 5 min. Allow to stand, separate the layers, and take the acetonitrile layer. Repeat this process 3 times. Combine the acetonitrile layers and add the extractant. The amount of extractant added to each milliliter of acetonitrile layer is 8 g. The extractant includes sodium alginate and anhydrous magnesium sulfate in a 1:1 ratio. Perform ultrasonic oscillation at room temperature and 60 kHz for 15 min. Centrifuge to obtain the filtrate.

[0052] Step 3: The solid-phase extraction column is packed with graphitized carbon, with an average particle size of 120–400 mesh and a specific surface area of ​​100 m². 2 / g, the sample loading amount is 5% of the packing mass, the elution solvent volume is 15 times the volume of the packing in the column, the solid phase extraction column is activated, the filtrate is loaded onto the sample, and eluted with 5% ammoniated methanol by mass, the eluent is collected, nitrogen is blown out, redissolved, and the volume is adjusted to 5mL. The sample is then filtered through a 0.22μm organic filter membrane to obtain the sample to be tested.

[0053] Step four: The sample to be tested is qualitatively and quantitatively detected using gas chromatography-mass spectrometry to obtain the N-dimethylnitrosamine content in μg / mL, which is then converted to obtain the N-dimethylnitrosamine content in μg / kg.

[0054] <Comparative Example 1>

[0055] Similar to Example 1, except that in step one, the soaking solution only includes flour and water, and the ratio of flour to water by weight is 20:100.

[0056] <Comparative Example 2>

[0057] Similar to Example 1, except that in step two, glacial acetic acid is not added, no extractant is added, and the combined acetonitrile layers are directly subjected to ultrasonic oscillation and centrifugation to obtain the filtrate.

[0058] <Comparative Example 3>

[0059] Similar to Example 1, except that in step three, solid-phase extraction is not performed, and the filtrate obtained in step two is directly subjected to nitrogen blowing.

[0060] The accuracy and recovery rate of Example 1 and Comparative Examples 1-3 were calculated. The dosage of N-dimethylnitrosamine was 2 μg / kg and 4 μg / kg, and each group was repeated 3 times. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] As shown in Table 1, comparing Example 1 and Comparative Example 1, introducing disodium citrate into the soaking solution precipitates metal ions. Introducing SOE and AES not only ensures good biodegradability and non-toxicity, but also provides a thickening effect and strong emulsifying ability for oils. The SOE and AES adhere to bubbles, enhancing oil removal. Comparing Example 1 and Comparative Example 2, adding glacial acetic acid improves the extraction of N-dimethylnitrosamine by acetonitrile. Using n-hexane to remove oil further adsorbs and removes it. Comparing Example 1 and Comparative Example 3, solid-phase extraction at room temperature avoids oxidation of unsaturated fatty acids caused by heating, further removing oil and residual components.

[0064] <Example 2>

[0065] 100g of beef balls were selected and pretreated using the method in Example 1 to obtain the sample to be tested. The sample was then qualitatively and quantitatively detected using gas chromatography-mass spectrometry, and the N-dimethylnitrosamine content was calculated to be 1.61 μg / kg.

[0066] <Example 3>

[0067] 100g of sausage was selected and pretreated using the method in Example 1 to obtain the sample to be tested. The sample was qualitatively and quantitatively detected using gas chromatography-mass spectrometry, and the N-dimethylnitrosamine content was calculated to be 2.46 μg / kg.

[0068] <Example 4>

[0069] 100g of smoked meat was selected and pretreated using the method in Example 1 to obtain the sample to be tested. The sample was then qualitatively and quantitatively detected using gas chromatography-mass spectrometry, and the N-dimethylnitrosamine content was calculated to be 3.57μg / kg.

[0070] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0071] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details shown and described herein.

Claims

1. A method for detecting N-dimethylnitrosamine in meat products, characterized in that, include: Step 1: Cut the meat products into pieces and soak them in a soaking solution, which includes flour, water, disodium citrate, modified oil ethoxylate, and sodium sulfate of fatty alcohol polyoxyethylene ether. Air is blown into the soaking solution to form tiny bubbles, ultrasonic vibration is applied, and then the meat is rinsed clean with water. Step 2: Crush the meat pieces, add acetonitrile, anhydrous magnesium sulfate and n-hexane, add 0.1% glacial acetic acid, ultrasonically vibrate, let stand, separate the liquids, take the acetonitrile layer, repeat 1 to 3 times, combine the acetonitrile layers, add the extraction agent, which includes sodium alginate and anhydrous magnesium sulfate, ultrasonically vibrate, centrifuge, and obtain the filtrate. Step 3: Activate the solid-phase extraction column. The solid-phase extraction column is filled with graphitized carbon. Load the filtrate onto the column and elute with 5-10% (w / w) ammoniated methanol. Collect the eluent, blow it with nitrogen, redissolve it, and filter it with a 0.22 μm organic filter membrane to obtain the sample to be tested. Step four: The sample to be tested is subjected to qualitative and quantitative detection using gas chromatography-mass spectrometry to obtain the content of N-dimethylnitrosamine.

2. The method for detecting N-dimethylnitrosamine in meat products as described in claim 1, characterized in that, In step one, the soaking solution comprises flour, water, disodium citrate, modified oil ethoxylate, and sodium sulfate of fatty alcohol polyoxyethylene ether in a weight ratio of 15~20:100:15~20:5~10:5~10.

3. The method for detecting N-dimethylnitrosamine in meat products as described in claim 2, characterized in that, In step one, the meat products are cut into pieces and soaked in the soaking solution for 30-60 minutes. The ultrasonic temperature is room temperature, the ultrasonic frequency is 60 kHz, and the ultrasonic extraction time is 15 minutes.

4. The method for detecting N-dimethylnitrosamine in meat products as described in claim 1, characterized in that, In step two, the meat chunks are ground into powder. For every gram of powder, 0.5 mL of acetonitrile, 0.5 g of anhydrous magnesium sulfate, and 2 mL of n-hexane are added. The ultrasonic temperature is room temperature, the ultrasonic frequency is 60 kHz, and the ultrasonic extraction time is 5 min.

5. The method for detecting N-dimethylnitrosamine in meat products as described in claim 4, characterized in that, In step two, the amount of extractant added to each milliliter of acetonitrile layer is 8 g. By weight, the extractant includes sodium alginate and anhydrous magnesium sulfate in a ratio of 1:0.5~2. The ultrasonic temperature is room temperature, the ultrasonic frequency is 60 kHz, and the ultrasonic extraction time is 15 min.

6. The method for detecting N-dimethylnitrosamine in meat products as described in claim 1, characterized in that, In step three, the average particle size of the graphitized carbon is 120-400 mesh, and the specific surface area is 100 m². 2 / g, the sample loading amount is 2~10% of the packing mass, and the amount of elution solvent is 10~15 times the volume of the packing in the column.

7. The method for detecting N-dimethylnitrosamine in meat products as described in claim 1, characterized in that, Step three includes: S1: Prepare N-dimethylnitrosamine standard solutions of different concentrations, inject them into a gas chromatograph-tandem mass spectrometer, and plot a standard curve based on the detection results; S2: Inject the sample solution to be tested into a gas chromatograph-tandem mass spectrometer to obtain the chromatographic peak of the quantitative ion of N-dimethylnitrosamine. Combined with the standard curve, the concentration of N-dimethylnitrosamine in the sample to be tested is calculated.

8. The method for detecting N-dimethylnitrosamine in meat products as described in claim 7, characterized in that, The gas chromatography conditions were as follows: INNOWAX quartz capillary column, injection port temperature 220℃, and programmed temperature rise: initial column temperature 40℃, increased to 80℃ at a rate of 10℃ / min, increased to 100℃ at a rate of 1℃ / min, then increased to 240℃ at a rate of 20℃ / min and held for 2 min; helium as carrier gas; flow rate 1.0 mL / min; injection volume 1.0 μL. Mass spectrometry conditions were as follows: ion detection, scanning of N-dimethylnitrosamine starting at 9.9 min, selected ions 15.0, 42.0, 43.0, 44.0, and 74.0; electron impact ionization (EI) source, voltage 70 eV, ionization current 300 μA, ion source temperature 230 °C, interface temperature 230 °C, and ion source vacuum 1.33 × 10⁻⁶. -4 Pa.

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