Rapid detection method for semicarbazide in food contact material

Abstract

The invention provides a method for rapidly detecting semicarbazide in a food contact material, which comprises the following steps of: (1) cutting a gasket of a bottle cap of a food contact material container into pieces; (2) weighing a sample, putting the sample into a centrifugal tube, adding an acetic acid solution, and performing ultrasonic extraction; after cooling, centrifuging the centrifugal tube, and taking supernate to another centrifugal tube; (3) adding an internal standard working solution, then adding a 2-nitrobenzaldehyde solution, carrying out uniform vortex mixing, and carrying out light-shielding oscillation in a constant-temperature water bath oscillator; (4) taking out the centrifuge tube, cooling, adding a dipotassium phosphate solution, adjusting the pH value, adding ethyl acetate, carrying out vortex oscillation and centrifugation, transferring supernatant into the glass centrifuge tube, and drying in nitrogen; (5) adding a formic acid aqueous solution to dissolve residues, filtering through a filter membrane, and carrying out mass spectrum detection; (6) making a standard solution curve; the other steps are operated according to the step (3) to the step (5). The detection method disclosed by the invention is high in sensitivity, short in analysis period and high in accuracy, and meets the requirement of rapidly determining the semicarbazide in the food contact material.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a rapid detection method for semicarbazide in food contact materials. Background Art

[0002] Food safety is one of the focal issues that has attracted much attention at home and abroad. Semicarbazide is a high-risk substance with carcinogenicity and genotoxicity. Semicarbazide residues in animal food are produced by the metabolism of animals after the use of furazolidone during animal feeding. In addition to the metabolism of furazolidone, semicarbazide may also be produced by the decomposition of disinfectant byproducts, endogenous production, and azodicarbonamide. In order to enhance the sealing of containers, packaged food manufacturers use plastic gaskets, paper gaskets, etc. in bottle caps to prevent leakage.

[0003] At present, national standards, local standards, and industry standards have promulgated multiple methods for detecting aminourea components in foods such as livestock, poultry, honey, and aquatic products. There is a lack of standard analytical methods for aminourea in food container contact materials, and a small amount of literature covers this part of the research content.

[0004] There are a few methods for studying semicarbazide in food container contact materials in the literature. One is a direct determination method: the sealing gasket material is cut into pieces and soaked in a solvent overnight, and then directly analyzed by a mass spectrometer after passing through an aqueous phase filter membrane. The results are large interference, poor sensitivity, and inaccurate results; the other is a derivatization hydrolysis method: extract semicarbazide in plastic gaskets under acidic conditions and use 2-nitrobenzaldehyde (2-NBA) as a derivatization agent for 16 hours, then adjust the pH to neutral before ethyl acetate extraction, concentration, and purification. There are problems such as cumbersome experimental steps, long analysis cycle, and low reproducibility.

[0005] Research by EU agencies has confirmed that gasket materials containing azodicarbonamide foaming agents need to undergo high-temperature treatment when sealing glass bottles and sterilizing them. Under heating conditions, the azodicarbonamide in the gasket will decompose into biurea, and further decompose to produce aminourea. There is a risk of migration to food through long-term contact, causing secondary contamination of food and posing a potential threat to consumer health and safety.

[0006] Based on the safety issue of semicarbazide produced by the decomposition of azodicarbonamide in food contact materials, on January 6, 2004, the European Union issued the directive "2004 / 1 / EU amending the directive 2002 / 72 / EC on the suspension of the use of azodicarbonamide as a foaming agent", stipulating that the use of azodicarbonamide as a foaming agent should be stopped from August 2, 2005, and requiring the development and use of substitutes for azodicarbonamide. China's food container contact material hygiene standard GB 9685-2003 controls the amount of azodicarbonamide added to plastics within 3% and the amount of azodicarbonamide added to rubber within 5%; GB 9685-2008 controls the amount of azodicarbonamide added to plastics within 2% and the amount of azodicarbonamide added to rubber within 5%.

[0007] Regarding the detection of semicarbazide residues of nitrofuran metabolites in animal-derived foods, national and industry standards have promulgated various detection and analysis methods. However, there are few studies on the detection of semicarbazide components in food contact materials. It is urgent to establish a quantitative method with high accuracy, good repeatability and short analysis cycle to analyze the semicarbazide content in daily food contact materials and provide data analysis reference for relevant companies and regulators. Summary of the invention

[0008] In order to understand the content of semicarbazide components in food container contact materials, it is urgent to develop a rapid, sensitive, easy-to-operate and available detection and analysis method. The present invention provides a rapid detection method for semicarbazide in food contact materials, comprising the following steps:

[0009] (1) Cut the gaskets of food contact material containers and bottle caps into pieces;

[0010] (2) Weigh the sample into a centrifuge tube, add acetic acid solution, and perform ultrasonic extraction; after cooling, centrifuge the centrifuge tube and take the supernatant into another centrifuge tube;

[0011] (3) Add the internal standard working solution and then the 2-nitrobenzaldehyde solution, vortex mix, and place in a constant temperature water bath oscillator in the dark for oscillation;

[0012] (4) Take out the centrifuge tube and cool it, add potassium hydrogen phosphate solution to adjust the pH value, add ethyl acetate, vortex and oscillate, centrifuge, transfer the supernatant to a glass centrifuge tube, and dry it in nitrogen;

[0013] (5) adding formic acid aqueous solution to dissolve the residue, filtering through a filter membrane, and performing mass spectrometry;

[0014] (6) Prepare a standard solution curve; the other steps are carried out according to steps (3) to (5).

[0015] Preferably, in step (1), the size of the fragments is controlled within the specification of 5 mm×5 mm.

[0016] Preferably, in step (2), the volume concentration of the acetic acid solution is 4%; and the centrifugal speed of the centrifuge tube is 9000-9500 r / min.

[0017] Preferably, in step (3), the concentration of the internal standard working solution is 100 ng / mL; the concentration of the 2-nitrobenzaldehyde solution is 0.1 to 0.15 mol / L.

[0018] Preferably, in step (3), the temperature in the constant temperature water bath oscillator is 60°C±1°C.

[0019] Preferably, in step (4), the pH value is 7.0-7.5, the centrifugal speed is 9000-9500 r / min, and the nitrogen is nearly dry at 40°C±1°C.

[0020] Preferably, in step (5), the residue is dissolved in 0.500 mL of 0.1% formic acid aqueous solution and filtered through a 0.22-0.45 μm filter membrane.

[0021] Preferably, in step (6), a standard solution curve is prepared with 0, 0.500, 1.00, 2.00, 5.00, 10.0, and 20.0 ng.

[0022] Preferably, the instrument is a liquid chromatography-tandem mass spectrometer (LC-MS / MS);

[0023] Chromatographic column: ACQUITY UPLC BEH C18 1.7μm 2.1×100mm Column;

[0024] Column temperature: 35°C;

[0025] Injection volume: 4 μL;

[0026] Gradient elution: see Table 1

[0027] Table 1 Gradient elution program

[0028]

[0029] For multiple ion reaction monitoring (MRM), the mass spectrometer parameters are shown in Table 2, and the ion source parameters are shown in Table 3.

[0030] Table 2 Mass spectrometry parameters

[0031]

[0032] Table 3 Ion source parameter conditions

[0033] Ionization method ESI+ Curtain Gas(CUR) 40.0psi Collision Gas(CAD) 8psi IonSpray Voltage(IS) 4500.0V Temperature(TEM) 550.0℃ Ion Source Gas 1(GS1) 55.0psi Ion Source Gas 1(GS2) 55.0psi

[0034] The detection method of the invention has high sensitivity, short analysis period and high accuracy, and meets the requirements for rapid determination of semicarbazide in food contact materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result of the experimental conditions using 0.2 mol / L hydrochloric acid in the examples of the present invention.

[0036] Figure 2 This is the result of the experimental conditions using 0.5 mol / L hydrochloric acid in the examples of the present invention.

[0037] Figure 3 This is the result of the experimental conditions using 4% volume concentration of acetic acid in the examples of the present invention.

[0038] Figure 4 The food contact material immersion test is adopted in the embodiment of the present invention.

[0039] Figure 5 It is a diagram of the derived results of the embodiment of the present invention. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings:

[0041] Sample preparation

[0042] (1) Cut the gaskets of food contact material containers and bottle caps into pieces, and control the size of the pieces to be within the specification of 5 mm × 5 mm.

[0043] Experimental operation

[0044] (2) Weigh 1.00 g of the sample into a 50 mL centrifuge tube, add 5 mL of 4% acetic acid solution, and perform ultrasonic extraction for 30 minutes; cool the centrifuge tube and centrifuge it at 9000-9500 r / min for 4 minutes, and take 2.5 mL of the supernatant into another 50 mL centrifuge tube.

[0045] (3) Add 0.05 mL of 100 ng / mL internal standard working solution and 0.15 mL of 0.1-0.15 mol / L 2-nitrobenzaldehyde solution. Vortex mix for 1 min and place in a constant temperature water bath oscillator at 60°C ± 1°C in the dark for 2 h.

[0046] (4) Remove the centrifuge tube and cool it to room temperature. Add 4 mL of potassium dihydrogen phosphate solution to adjust the pH to 7.0-7.5. Add 10 mL of ethyl acetate and vortex for 10 minutes. Centrifuge at 9000-9500 r / min for 4 minutes. Transfer the supernatant to a 10 mL glass centrifuge tube and dry it under nitrogen at 40°C±1°C.

[0047] (5) Accurately add 0.500 mL of 0.1% formic acid aqueous solution to dissolve the residue, filter through a 0.22-0.45 μm filter membrane, and then measure the mass spectrum.

[0048] (6) Prepare a standard solution curve. Take 0, 0.500, 1.00, 2.00, 5.00, 10.0, and 20.0 (ng) and add them to 5 mL of 4% acetic acid solution. The other steps are carried out according to steps (2) to (4).

[0049] Instrument reference conditions

[0050] Instrument: Liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0051] Chromatographic column: ACQUITY UPLC BEH C18 1.7μm 2.1×100mm Column

[0052] Column temperature: 35°C

[0053] Injection volume: 4 μL

[0054] Gradient elution: see Table 1

[0055] Table 1 Gradient elution program

[0056]

[0057] For multiple ion reaction monitoring (MRM), the mass spectrometer parameters are shown in Table 2, and the ion source parameters are shown in Table 3.

[0058] Table 2 Mass spectrometry parameters

[0059]

[0060] Table 3 Ion source parameter conditions

[0061] Ionization method ESI+ Curtain Gas(CUR) 40.0psi Collision Gas(CAD) 8psi IonSpray Voltage(IS) 4500.0V Temperature(TEM) 550.0℃ Ion Source Gas 1(GS1) 55.0psi Ion Source Gas 1(GS2) 55.0psi

[0062] Results Analysis

[0063] Selecting extraction reagents

[0064] Regarding the screening of extraction reagents for semicarbazide (SEM), according to the relevant standard methods for detecting semicarbazide, 0.2mol / L hydrochloric acid and 0.5mol / L hydrochloric acid are mainly used. Organic experiments on food contact materials mostly use 4% acetic acid solution to extract the target. The experimental plan first compared the experimental operation effects of 0.2mol / L hydrochloric acid, 0.5mol / L hydrochloric acid, and 4% acetic acid solution. The results showed that under the experimental conditions of 0.2mol / L hydrochloric acid, there were many interfering peaks near the target peak of SEM quantitative ion 209.1-166.0 ( Figure 1), the peak area response intensity is second; under the experimental conditions of 0.5mol / L hydrochloric acid, there is an obvious interference peak near the target peak of SEM quantitative ion 209.1-166.0, and the peak area response intensity is the lowest; under the experimental conditions of volume concentration 4% acetic acid solution, the SEM quantitative ion 209.1-166.0 target peak separation and response are the highest, and there is no obvious interference peak nearby (see Figure 3 ), so 4% acetic acid solution was finally selected as the target extraction reagent.

[0065] Extraction method

[0066] Method 1: Weigh 1.00 g of sample into a 50 mL centrifuge tube, add 5 mL of 4% acetic acid solution, and perform ultrasonic extraction for 30 minutes. Add internal standard substances and derivatives directly to the extraction solution in the 50 mL centrifuge tube for subsequent operations.

[0067] Method 2: Weigh 1.00 g of sample into a 50 mL centrifuge tube, add 5 mL of 4% acetic acid solution, and extract by ultrasonic for 30 minutes. Cool and centrifuge the centrifuge tube at 9000-9500 r / min for 4 minutes, take 2.5 mL of supernatant into another 50 mL centrifuge tube, add internal standard substances and derivatives to the extraction solution, and proceed with subsequent operations.

[0068] After comparing the results of instrumental analysis, the results of method 1 were not ideal, the derivatization effect was not good, and the reproducibility was poor; the invention first separated the food contact material and the extraction and soaking solution before conducting subsequent experimental operations. The results showed that the recovery rate met the requirements of the analysis method and had good reproducibility. Reasons for the improvement of the results: The food contact material samples were soaked in the solution, and it was easy to absorb water and swell in the later stage (see Figure 4 ); During the detection process, the internal standard substances and derivative substances measured at the ng level are directly added to the solution containing the food contact material sample, and are indirectly adsorbed by the food contact material sample. The derivatization reaction fails, resulting in the inability of ethyl acetate to extract the derivative substances of semicarbazide, and the instrument cannot detect the target substance.

[0069] Derivative Condition Selection

[0070] The molecular weight of semicarbazide (SEM) is only 76, and the secondary fragment ions are 30.9 and 60.0. Direct mass spectrometry detection of semicarbazide is easily interfered by fragments of other substances and has low sensitivity. Therefore, we choose derivative substances with mature technology to derive and synthesize new substances to increase the molecular weight and improve the detection sensitivity. The experimental conditions of the derivatization experiment are 60℃±1℃. Figure 5 The results showed that shaking in a water bath at 60℃±1℃ for 2 hours also achieved the results of deriving at 37℃ for 16 hours. The experimental operating conditions of shaking in a water bath at 60℃±1℃ for 2 hours significantly shortened the detection cycle of semicarbazide, providing strong technical support for handling emergency events. Figure 5 shown.

[0071] Methodological research

[0072] 1.00g of blank food contact material without target drug was taken and placed in a 50mL polypropylene centrifuge tube. Standard solution was added to make low-level 0.200μg / kg, 0.500μg / kg, and 5.00μg / kg positive test samples. The samples were analyzed according to the 3 technical schemes. Each addition level was tested in parallel 6 times, and the average recovery and precision were calculated. The standard working curve was configured without adding blank samples. The linear equation correlation coefficient (r) was 0.9996 in the range of 0.200-100μg / L, which was greater than 0.995. The linearity of the regression equation was good, and the recovery was between 95.1% and 105%, which met the requirements of the analytical method. The detailed data are shown in Table 4.

[0073] Table 4 Limit of quantification, correlation coefficient (r), spike recovery and precision of semicarbazide (n = 6)

[0074]

[0075] Actual sample testing

[0076] The rapid detection method of semicarbazide in food contact materials of the present invention is used for actual sample detection. The sample types mainly involve sealing materials of cans, honey, health foods, etc. Two parallel experiments are set for each sample, and the experiments are carried out according to the operating procedures of the present invention. The analysis results are shown in Table 5. After testing 23 batches of samples, it was found that semicarbazide components were detected in 14 batches of food contact materials, with a detection rate of about 60%, and a SEM content of 0.883-4.10×10 3 (μg / kg), indicating that such food testing materials still have certain risks, and production companies and regulatory authorities need to strengthen risk prevention and control, adjust and improve the composition of food contact materials, and further reduce the risk of food contamination.

[0077] Table 5 Actual sample test results

[0078]

[0079]

[0080] (1) The present invention first separates the food contact material sample gasket and the extract, and takes the supernatant for subsequent experiments to improve the extraction effect;

[0081] (2) The present invention can greatly shorten the derivatization time by increasing the derivatization temperature, thereby achieving the purpose of rapid analysis of semicarbazide;

[0082] (3) The present invention provides an experimental analysis method for detecting food contact materials, which is combined with mass spectrometry and has high specificity;

[0083] Key steps of experimental operation:

[0084] 1. Cut the food container bottle cap gasket material into pieces, and control the size of the pieces within the specification of 5mm×5mm.

[0085] 2. Weigh 1.00 g of sample into a 50 mL centrifuge tube, add 5 mL of 4% acetic acid solution, and perform ultrasonic extraction for 30 minutes. Cool the centrifuge tube and centrifuge at 9000-9500 r / min for 4 minutes, and take 2.5 mL of supernatant into another 50 mL centrifuge tube.

[0086] 3. Add 0.05 mL of 100 ng / mL internal standard working solution, then add 0.15 mL of 0.1-0.15 mol / L 2-nitrobenzaldehyde solution, vortex mix for 1 min, and place in a constant temperature water bath oscillator at 60℃±1℃ in the dark for 2 h.

[0087] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A rapid detection method for semicarbazide in food contact materials, characterized in that: It includes the following steps: (1) Cut the gaskets of food contact material containers and bottle caps into pieces; (2) Weigh the sample into a centrifuge tube, add acetic acid solution, and perform ultrasonic extraction; after cooling, centrifuge the centrifuge tube and take the supernatant into another centrifuge tube; (3) Add the internal standard working solution and then the 2-nitrobenzaldehyde solution, vortex mix, and place in a constant temperature water bath oscillator in the dark for oscillation; (4) Take out the centrifuge tube and cool it, add potassium hydrogen phosphate solution to adjust the pH value, add ethyl acetate, vortex and oscillate, centrifuge, transfer the supernatant to a glass centrifuge tube, and dry it in nitrogen; (5) adding formic acid aqueous solution to dissolve the residue, filtering through a filter membrane, and performing mass spectrometry; (6) Prepare a standard solution curve; the other steps are carried out according to steps (3) to (5).

2. The method according to claim 1, characterized in that In the step (1), the fragments are within 5 mm×5 mm.

3. The method according to claim 1, characterized in that In the step (2), the concentration of the acetic acid solution is 4%; and the centrifugal speed of the centrifuge tube is 9000-9500 r / min.

4. The method according to claim 1, characterized in that In the step (3), the concentration of the internal standard working solution is 100 ng / mL; the concentration of the 2-nitrobenzaldehyde solution is 0.1-0.15 mol / L.

5. The method according to claim 1, characterized in that In the step (3), the temperature in the constant temperature water bath oscillator is 60°C±1°C.

6. The method according to claim 1, characterized in that In the step (4), the pH value is 7.0-7.5, the centrifugal speed is 9000-9500 r / min, and the nitrogen is nearly dry at 40°C±1°C.

7. The method according to claim 1, characterized in that In the step (5), the concentration of the residue dissolved by the formic acid aqueous solution is 0.500 mL.

8. The method according to claim 1, characterized in that The filter membrane has a filtration pore size of 0.22 to 0.45 μm.

9. The method according to claim 1, characterized in that In the step (6), a standard solution curve is prepared by taking 0, 0.500, 1.00, 2.00, 5.00, 10.0, and 20.0 ng.

10. The method according to claim 1, characterized in that Instruments and equipment: Liquid chromatography-tandem mass spectrometry (LC-MS / MS); Chromatographic column: ACQUITY UPLC BEH C18 1.7μm 2.1×100mm Column; Column temperature: 35°C; Injection volume: 4 μL; Gradient elution: see Table 1 Table 1 Gradient elution program For multiple ion reaction monitoring (MRM), the mass spectrometer parameters are shown in Table 2, and the ion source parameters are shown in Table 3. Table 2 Mass spectrometry parameters Table 3 Ion source parameter conditions

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