A method for detecting dimethyl fumarate in food

Abstract

The application discloses a kind of detection methods of dimethyl fumarate in food, it is related to chemical detection analysis technical field, comprising the following steps: S1, based on standard solution sample configuration standard curve;S2, the formula of purifying tube is selected and three representative samples, and based on purification scheme, sample is purified;S3, set two standard curve concentration points, carry out multiple sample data acquisition;S4, to three samples are detected in parallel double sample, and the detection limit level of sample, 1 times quantification limit level, 2 times quantification limit level and 5 times quantification limit level are 6 parallel standard addition test, based on experimental data is verified.The application makes that the dosage of organic reagent is saved about 80%, operation time is saved about 75%, the addition of internal standard DMM effectively calibrates the influence that target DMF changes with time and matrix interference, guarantees the result accuracy, while operation process is simple, to reduce the requirement of laboratory to personnel operation skill.

Description

Technical Field

[0001] This invention relates to the field of chemical testing and analysis technology, specifically to a method for detecting dimethyl fumarate in food. Background Technology

[0002] Dimethyl fumarate (DMF, CAS No. 624-49-7) is a mildew inhibitor discovered in the 1980s with broad-spectrum and highly effective antibacterial and bactericidal effects against microorganisms. Studies by Guo Ailian et al. have found that concentrations of 600 mg / L-800 mg / L have significant inhibitory effects on microorganisms such as Aspergillus, Penicillium, Mucor, Staphylococcus aureus, and Bacillus subtilis. It can significantly inhibit the growth of molds and yeasts, and greatly extend the shelf life of pastries, soy products, and other similar products. However, some manufacturers illegally add it to reduce costs and conceal raw material spoilage. Dimethyl fumarate is a non-edible substance, and relevant departments have long included it in the blacklist of illegally added non-edible substances in food, strictly prohibiting its intentional addition to food and making it a key item for food safety inspections.

[0003] DMF is currently listed as a non-edible substance that may be illegally added to food. While there are numerous studies on the analysis of DMF in food, these methods suffer from cumbersome pretreatment processes, long processing times, and unstable results. Existing methods are complex, requiring multiple extractions and are time-consuming; they also use a large amount of organic solvents, increasing costs; and the purification via solid-phase extraction (SPE) and nitrogen blowing concentration further increase time costs. SPE purification requires keeping the column moist, which increases the difficulty of operation for batch samples and also increases the use of organic solvents, raising experimental costs. Nitrogen blowing requires controlling the gas flow rate; a high flow rate leads to unstable experimental results, and this process is also time-consuming.

[0004] Meanwhile, most DMF detection currently uses the external standard quantification method. However, experimental verification shows that the results of external standard quantification have a significant downward trend over time, while the results of internal standard quantification are basically stable and not significantly affected by time changes. This indicates that DMM can not only reduce matrix interference in the sample, but also correct the changes in DMF response values ​​in real time.

[0005] Therefore, the test data of this invention are all obtained quantitatively using the internal standard method. At the same time, it provides a simple, efficient, economical, fast, environmentally friendly and stable detection method, which reduces the requirements for personnel's operating skills in the laboratory while ensuring the accuracy of the test results. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for detecting dimethyl fumarate in food, thereby solving the problems mentioned in the background section.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for detecting dimethyl fumarate in food, comprising the following steps:

[0009] S1. Configure the standard curve;

[0010] The standard was diluted and quantified to obtain a standard stock solution. Then, it was diluted to prepare standard solutions with different concentration gradients. A standard curve was prepared based on the standard solutions.

[0011] S2. Select and implement a purification solution;

[0012] Select the formulation of the purification tube and three representative samples, and purify the samples based on the purification plan;

[0013] S3. Select a quantitative method;

[0014] Two standard curve concentration points were set, and data was collected from multiple injections.

[0015] S4. Perform detection limit, accuracy and precision verification;

[0016] Parallel dual-sample detection of dimethyl fumarate (DMF) and dimethyl malonate (DMM) was performed on three samples. Six parallel spiked tests were conducted at the limit of detection (LOD), 1-fold LOD, 2-fold LOD, and 5-fold LOD levels of the samples. The results were validated based on the experimental data.

[0017] To further optimize this technical solution, the gas chromatography test conditions used in this detection method include:

[0018] Chromatographic column: DB-WAX capillary column (30 m × 250 μm × 0.25 μm);

[0019] Carrier gas: High-purity helium (99.999% purity);

[0020] Constant flow mode: 1 mL / min;

[0021] Inlet temperature: 230 ℃;

[0022] Injection mode: splitless pulse, pulse pressure: 20 psi, time: 1 min;

[0023] Injection volume: 1 μL;

[0024] Column oven heating program: 60 ℃ (1 min), 20 ℃ / min to 120 ℃ (3 min), 20 ℃ / min to 230 ℃ (2 min), then run at 230 ℃ (8 min);

[0025] Transmission line temperature: 235 ℃.

[0026] To further optimize this technical solution, the gas chromatography-mass spectrometry testing conditions used in this detection method include:

[0027] Ion source: Electron impact source (EI), temperature: 230℃, electron energy 70 eV;

[0028] Quadrupole temperature: 150℃; Solvent delay time: 6 min;

[0029] Scanning mode: Selective ion monitoring mode (SIM);

[0030] Monitoring ions for dimethyl fumarate: quantitative ion (m / z) 113, qualitative ions (m / z) 59, 85 and 114;

[0031] Monitoring ions for dimethyl malonate: quantitative ion (m / z) 101, qualitative ions (m / z) 59, 74 and 57.

[0032] To further optimize this technical solution, the preparation of the standard solution in step S1 includes:

[0033] Preparation of 10 mg / mL internal standard stock solution: Weigh 0.1020 g DMM into a 10 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard stock solution of the corresponding concentration, and store in a refrigerator at 4 ℃.

[0034] Preparation of 100 μg / mL internal standard intermediate solution: Accurately measure 1 mL of internal standard stock solution, place it in a 100 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard intermediate solution of the corresponding concentration, and store at 4℃.

[0035] Preparation of 1000 ng / mL internal standard working solution: Accurately measure 1.000 mL of internal standard intermediate solution, place it in a 100 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard working solution of the corresponding concentration, and prepare it fresh before use;

[0036] Preparation of 10 μg / mL standard intermediate solution: Transfer 100.00 μL of 1000 μg / mL DMF standard solution into a 10 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the standard intermediate solution of the corresponding concentration, and store in a refrigerator at 4 ℃.

[0037] To further optimize this technical solution, step S1, which involves configuring a standard curve based on the injection of the standard solution, includes:

[0038] Using a 10 μg / mL standard intermediate solution, prepare standard working solutions at concentrations of 10 ng / mL, 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL. Transfer 1.000 mL of the standard working solution to a sample vial and add 100.00 μL of internal standard working solution. Prepare solutions fresh for each use.

[0039] To further optimize this technical solution, in step S2, the purification tube is formulated with PSA, C18, GCB and anhydrous sodium sulfate; representative samples include bread, hawthorn and milk.

[0040] To further optimize this technical solution, in step S2, the purification process includes:

[0041] Weigh 2.0 g of a representative sample into a 50 mL centrifuge tube, add 0.5 mL of internal standard working solution, add 5.000 mL of water to the solid sample, and add homogenous protons to vortex for 10 min until the sample is evenly dispersed.

[0042] Add 5.00 mL of acetonitrile, vortex for 10 min, add 3 g of NaCl, vortex for 2 min, and centrifuge at 5000 r / min for 3 min.

[0043] Take 3.00 mL of the supernatant into a 10 mL purification tube, vortex for 1 min, let stand for 1 min, and then filter the supernatant through a 0.22 μm organic filter membrane for testing.

[0044] To further optimize this technical solution, the quantitative method in step S3 includes:

[0045] Two standard curve concentration points were selected, with concentrations of 100 μg / L and 200 μg / L, respectively. 1.000 mL of each standard curve concentration point was added, and 100 μL of internal standard working solution was added. Data were collected for the initial injection, 7 h injection, 17 h injection, 27 h injection, and 28 h injection.

[0046] To further optimize this technical solution, in step S4, parallel dual-sample detection of DMF and DMM is performed on the three samples. After confirming that there are no response values ​​of DMF and DMM in the sample solution, six parallel spiked tests are performed on the detection limit level, 1-time limit of quantitation level, 2-time limit of quantitation level and 5-time limit of quantitation level of the three samples, respectively.

[0047] To further optimize this technical solution, in the sample:

[0048] The detection limit level is 20 μg / L, which is the theoretical value of the sample concentration, and corresponds to a sample content of 0.05 mg / kg.

[0049] The limit of quantitation (LOQ) level is equivalent to a theoretical sample concentration of 40 μg / L, which corresponds to a sample concentration of 0.1 mg / kg.

[0050] The limit of quantitation (LOQ) level twice is equivalent to a theoretical sample concentration of 80 μg / L, which corresponds to a sample concentration of 0.2 mg / kg.

[0051] The 5-fold limit of quantitation level corresponds to a theoretical sample concentration of 200 μg / L, which is equivalent to a sample content of 0.5 mg / kg.

[0052] Compared with the prior art, the present invention provides a method for detecting dimethyl fumarate in food, which has the following beneficial effects:

[0053] The new method for detecting dimethyl fumarate (DMF) in this food sample requires only about 5.5 mL of acetonitrile (ADF) as a single reagent, while existing methods require a larger amount of organic solvent (20 mL ADF and 5 mL ethyl acetate), estimated at about 25 mL. The processing time for a single sample is approximately 0.5 h using this method, compared to an estimated 2 h using the existing method. This results in approximately 80% savings in organic reagent usage and 75% reduction in processing time. The addition of the internal standard DMM effectively corrects for the effects of DMF changes over time and matrix interference, ensuring the accuracy of the results. Furthermore, the simplified operation reduces the skill requirements for laboratory personnel. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a schematic diagram of the molecular structure of dimethyl fumarate (DMF).

[0056] Figure 2 This is a schematic diagram of the molecular structure of dimethyl malonate (DMM).

[0057] Figure 3 The back-calibrated concentration plots are the concentration points of the 100 μg / L standard curve injected at different time points.

[0058] Figure 4 The back-calibrated concentration plots are the concentration points of the 200 μg / L standard curve injected at different time points.

[0059] Figure 5 This is a standard curve plot using the internal standard method.

[0060] Figure 6The total ion chromatogram is for a 100 ng / mL DMF (containing DMM internal standard) standard solution.

[0061] Figure 7 Real photos taken during experiments to test detection limits, accuracy, and precision. Detailed Implementation

[0062] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0063] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0064] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0065] Example:

[0066] In this embodiment, the following materials and reagents are used:

[0067] BePure methanol DMF standard solution: 1000 μg / mL, purchased from Beijing Manhag Biotechnology Co., Ltd.

[0068] Dimethyl malonate (DMM, CAS No. 108-59-8): 98% purity, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0069] Acetonitrile: chromatographic grade, purchased from Merck, USA;

[0070] C18 chromatographic packing material (40-60μm particle size), PSA chromatographic packing material (40-60μm particle size), GCB chromatographic packing material (40-120μm particle size), and organic microporous filter membrane (0.22 μm) were all purchased from Aikeyi Biotechnology (Shandong) Co., Ltd.

[0071] Sodium chloride and anhydrous sodium sulfate: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0072] Homoprotons: Purchased from Agilent Technologies, USA.

[0073] In this embodiment, the following instruments and equipment are used:

[0074] 7890B / 5977B Gas Chromatography-Mass Spectrometer: Agilent Technologies, USA;

[0075] AX224ZH Electronic Balance: Ohaus Instruments (Changzhou) Co., Ltd.

[0076] Arium Pro ultrapure water system: Sartorius, Germany;

[0077] Multi Reax universal vortex mixer: Heidolph GmbH, Germany;

[0078] 3-18K high-speed refrigerated centrifuge: Sigma, Germany.

[0079] In this embodiment, the preparation of the standard curve includes:

[0080] Preparation of 10 mg / mL internal standard stock solution: Weigh 0.1020 g DMM into a 10 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard stock solution of the corresponding concentration, and store in a refrigerator at 4 ℃.

[0081] Preparation of 100 μg / mL internal standard intermediate solution: Accurately measure 1 mL of internal standard stock solution and place it in a 100 mL brown volumetric flask. Dilute to the mark with acetonitrile, shake well, and obtain the internal standard intermediate solution of the corresponding concentration. Store at 4 °C.

[0082] Preparation of 1000 ng / mL internal standard working solution: Accurately measure 1,000 mL of internal standard intermediate solution and place it in a 100 mL brown volumetric flask. Dilute to the mark with acetonitrile, shake well, and obtain the internal standard working solution of the corresponding concentration. Prepare fresh before use.

[0083] Preparation of 10 μg / mL standard intermediate solution: Transfer 100.00 μL of 1000 μg / mL DMF standard solution into a 10mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the standard intermediate solution of the corresponding concentration, and store in a refrigerator at 4 ℃.

[0084] Preparation of standard curves: Using a 10 μg / mL standard intermediate solution, prepare standard working solutions of 10 ng / mL, 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL; transfer 1.000 mL of standard working solution into a sample vial and add 100.00 μL of internal standard working solution. Prepare fresh solutions before use.

[0085] The gas chromatography test conditions used in this method include:

[0086] Chromatographic column: DB-WAX capillary column (30 m × 250 μm × 0.25 μm);

[0087] Carrier gas: High-purity helium (99.999% purity);

[0088] Constant flow mode: 1 mL / min;

[0089] Inlet temperature: 230 ℃;

[0090] Injection mode: splitless pulse, pulse pressure: 20 psi, time: 1 min;

[0091] Injection volume: 1 μL;

[0092] Column oven heating program: 60 ℃ (1 min), 20 ℃ / min to 120 ℃ (3 min), 20 ℃ / min to 230 ℃ (2 min), then run at 230 ℃ (8 min);

[0093] Transmission line temperature: 235 ℃.

[0094] The gas chromatography-mass spectrometry (GC-MS) testing conditions used in this method include:

[0095] Ion source: Electron impact source (EI), temperature: 230℃, electron energy 70 eV;

[0096] Quadrupole temperature: 150℃; Solvent delay time: 6 min;

[0097] Scanning mode: Selective ion monitoring mode (SIM);

[0098] Monitoring ions for dimethyl fumarate: quantitative ion (m / z) 113, qualitative ions (m / z) 59, 85 and 114;

[0099] Monitoring ions for dimethyl malonate: quantitative ion (m / z) 101, qualitative ions (m / z) 59, 74 and 57.

[0100] Using these chromatographic and mass spectrometric conditions, the total ion chromatogram of a 100 ng / mL DMF (containing DMM internal standard) standard solution is detailed in [link to chromatographic and mass spectrometric data]. Figure 6 .

[0101] To verify the purification effect of different samples and different mass ratios, the purification scheme was selected as follows: DMF and DMM have simple molecular structures and belong to linear small molecule compounds, such as... Figure 1 and Figure 2 As shown, it is not easily adsorbed by PSA, C18, and GCB. This method used four formulations (see Table 1 below) to purify the limit-of-quantitation (LOQ) level spiked solutions of three representative samples (bread, hawthorn, and milk). The purification process was as follows:

[0102] Weigh 2.0 g (0.0001 g accurate) of sample into a 50 mL centrifuge tube, add 0.5 mL of internal standard working solution, add 5.000 mL of water to the solid sample, add homogenizer and vortex for 10 min until the sample is evenly dispersed (add 3.00 mL of water to the liquid sample and vortex for 1 min until the sample is mixed); add 5.00 mL of acetonitrile, vortex for 10 min, add 3 g of NaCl, vortex for 2 min, centrifuge at 5000 r / min for 3 min, take 3.00 mL of supernatant into a 10 mL purification tube, vortex for 1 min, let stand for 1 min, take the supernatant and filter it through a 0.22 μm organic filter membrane for analysis.

[0103] The four materials serve the following functions: PSA removes polar interfering substances such as organic acids, sugars, and pigments from the sample through weak anion exchange and hydrogen bonding; C18 removes non-polar interfering substances such as oils and fats from the sample through hydrophobic adsorption; GCB's graphite planar structure selectively adsorbs substances with aromatic rings, removing interfering substances such as chlorophyll and carotenoids from the sample. Anhydrous sodium sulfate provides gentle dehydration and salting-out separation, while also protecting the target analyte, ensuring stable recovery, and protecting the instrument.

[0104] Table 1 Test Results of Purification Pipe Formulation

[0105]

[0106] As shown in Table 1, the theoretical concentration of DMF is 40 μg / L. The test results of the purified sample solutions ranged from 40.0778 μg / L to 41.1227 μg / L. Compared with the backcalibrated result of 40.0445 μg / L for the 40 μg / L standard solution, the matrix enhancement effect still exists, but the deviation of the detection results is between 1.3% and 2.6%, indicating that the matrix enhancement effect of the purified sample solution is weak and basically does not affect the accurate quantification of the detection results. The weak enhancement of the matrix effect also indicates that the adsorption of DMF by PSA, C18, and GCB is not significant and will not cause the detection results to be too low. Since the No. 4 formulation uses less material and has a relatively low cost, the No. 4 formulation was used for subsequent experimental purification.

[0107] Quantitative Method Selection: Since DMM and DMF have similar structures and retention times, and DMM was undetectable in the sample, using DMM as an internal standard effectively reduces matrix interference. Experiments showed that the standard curve correction point concentration for monitoring instrument and standard substance stability decreased with increasing injection time. Data was collected at initial injection, 7 h injection, 17 h injection, 27 h injection, and 28 h injection by adding 100 μL of internal standard working solution to each of the two standard curve concentration points (100 μg / L and 200 μg / L). The results are as follows: Figure 3 and Figure 4 As shown, the results of external standard quantification show a significant downward trend over time, while the results of internal standard quantification remain relatively stable and are not significantly affected by time changes. This indicates that DMM can not only reduce matrix interference in the sample, but also provide real-time feedback on changes in the response value of DMF.

[0108] This method refers to the standard curve range of BJS202407. Following the above operations, the standard curve prepared is as follows: Figure 5 As shown, Y = 1.268472 × x + 0.043252; R 2 =0.99977181, indicating a good linear relationship. The recalibration concentrations of the 100 μg / L and 200 μg / L standard solutions were essentially unaffected by time changes.

[0109] Validation of detection limit, accuracy, and precision: This method was used to perform parallel dual-sample detection of DMF and DMM on three samples. After confirming that there were no response values ​​of DMF and DMM in the sample solution, six parallel spiked tests were conducted on the detection limit level (theoretical sample solution concentration of 20 μg / L, corresponding to a sample content of approximately 0.05 mg / kg), the 1st limit of quantitation level (theoretical sample solution concentration of 40 μg / L, corresponding to a sample content of approximately 0.1 mg / kg), the 2nd limit of quantitation level (theoretical sample solution concentration of 80 μg / L, corresponding to a sample content of approximately 0.2 mg / kg), and the 5th limit of quantitation level (theoretical sample solution concentration of 200 μg / L, corresponding to a sample content of approximately 0.5 mg / kg). The results are detailed in Table 2.

[0110] Table 2. Experimental data on detection limit, accuracy, and precision.

[0111]

[0112] As shown in Table 2, the recoveries of the three samples at the detection limit level ranged from 76.3% to 82.3%, indicating that this detection method can achieve accurate quantification at the detection limit. The relative range of the six parallel samples in each group ranged from 4.8% to 6.8%, meeting the requirement of the relevant standard that the absolute difference between parallel samples should not exceed 10% of the arithmetic mean. The recoveries of the three samples at the 1st, 2nd, and 5th limits of quantitation levels ranged from 88.0% to 97.7%, and the recoveries showed an upward trend towards 100% with increasing spiking amount. The relative range of the six parallel samples in each group ranged from 1.1% to 4.1%, meeting the precision requirements of the relevant standard. The relative range of the milk and hawthorn slice spiked samples showed a downward trend with increasing spiking amount, possibly because DMF is slightly soluble in water, and a small amount of DMF remains in the aqueous phase of the extraction step, resulting in samples with smaller spiking amounts being relatively more affected, while samples with larger spiking amounts were relatively less affected. Unlike other samples, the relative range in bread showed no trend with the amount of spiking, which may be related to the porous structure of the bread sample. Before extraction with acetonitrile, 5.00 mL of water and homogenants were added to the sample for re-homogenization. The porous structure of the bread may not have been completely destroyed and there may have been differences, resulting in different amounts of DMF being adsorbed. This also shows that the operation of re-homogenization with homogenants during the extraction process is beneficial to improving the precision of the experimental results.

[0113] A detailed comparison of the detection method of this invention with the operation steps of BJS202407 is shown in Table 3. When processing the same solid sample using the two methods, this method requires approximately 5.5 mL of organic reagent, while the BJS 202407 method requires approximately 26 mL of organic reagent. The reagent usage is approximately 20% of that of the BJS 202407 method, saving approximately 20.5 mL of organic reagent. The time required using this method is approximately 0.5 h, while the time required using the BJS202407 method is approximately 2 h, which is approximately 25% of the time required by the BJS 202407 method, thus saving approximately 1.5 h of operation time.

[0114] Table 3 Comparison of the operation steps of this method with those of BJS202407

[0115]

[0116] Compared with the BJS 202407 method, this invention reduces the amount of organic reagents by about 80%, the operation time by about 75%, and the addition of internal standard DMM effectively corrects the effects of the target analyte DMF changing over time and matrix interference.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting dimethyl fumarate in food, characterized in that, Includes the following steps: S1. Configure the standard curve; The standard was diluted and quantified to obtain a standard stock solution. Then, it was diluted to prepare standard solutions with different concentration gradients. A standard curve was prepared based on the standard solutions. S2. Select and implement a purification solution; Select the formulation of the purification tube and three representative samples, and purify the samples based on the purification plan; S3. Select a quantitative method; Two standard curve concentration points were set, and data was collected from multiple injections. S4. Perform detection limit, accuracy and precision verification; Parallel dual-sample detection of dimethyl fumarate (DMF) and dimethyl malonate (DMM) was performed on three samples. Six parallel spiked tests were conducted at the limit of detection (LOD), 1-fold LOD, 2-fold LOD, and 5-fold LOD levels of the samples. The results were validated based on the experimental data.

2. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, The gas chromatography test conditions used in this detection method include: Chromatographic column: DB-WAX capillary column (30 m × 250 μm × 0.25 μm); Carrier gas: High-purity helium (99.999% purity); Constant flow mode: 1 mL / min; Inlet temperature: 230 ℃; Injection mode: splitless pulse, pulse pressure: 20 psi, time: 1 min; Injection volume: 1 μL; Column oven heating program: 60 ℃ (1 min), 20 ℃ / min to 120 ℃ (3 min), 20 ℃ / min to 230 ℃ (2 min), then run at 230 ℃ (8 min); Transmission line temperature: 235 ℃.

3. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, The gas chromatography-mass spectrometry (GC-MS) testing conditions used in this detection method include: Ion source: Electron impact source (EI), temperature: 230℃, electron energy 70 eV; Quadrupole temperature: 150℃; Solvent delay time: 6 min; Scanning mode: Selective ion monitoring mode (SIM); Monitoring ions for dimethyl fumarate: quantitative ion (m / z) 113, qualitative ions (m / z) 59, 85 and 114; Monitoring ions for dimethyl malonate: quantitative ion (m / z) 101, qualitative ions (m / z) 59, 74 and 57.

4. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S1, the preparation of the standard solution includes: Preparation of 10 mg / mL internal standard stock solution: Weigh 0.1020 g DMM into a 10 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard stock solution of the corresponding concentration, and store in a refrigerator at 4 ℃. Preparation of 100 μg / mL internal standard intermediate solution: Accurately measure 1 mL of internal standard stock solution, place it in a 100 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard intermediate solution of the corresponding concentration, and store at 4℃. Preparation of 1000 ng / mL internal standard working solution: Accurately measure 1.000 mL of internal standard intermediate solution, place it in a 100 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the internal standard working solution of the corresponding concentration, and prepare it fresh before use; Preparation of 10 μg / mL standard intermediate solution: Transfer 100.00 μL of 1000 μg / mL DMF standard solution into a 10 mL brown volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the standard intermediate solution of the corresponding concentration, and store in a refrigerator at 4 ℃.

5. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S1, configuring a standard curve based on the injection of the standard solution includes: Using a 10 μg / mL standard intermediate solution, prepare standard working solutions at concentrations of 10 ng / mL, 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL. Transfer 1.000 mL of the standard working solution to a sample vial and add 100.00 μL of internal standard working solution. Prepare solutions fresh for each use.

6. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S2, the purification tube is formulated with PSA, C18, GCB and anhydrous sodium sulfate; representative samples include bread, hawthorn and milk.

7. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S2, the purification process includes: Weigh 2.0 g of a representative sample into a 50 mL centrifuge tube, add 0.5 mL of internal standard working solution, add 5.000 mL of water to the solid sample, and add homogenous protons to vortex for 10 min until the sample is evenly dispersed. Add 5.00 mL of acetonitrile, vortex for 10 min, add 3 g of NaCl, vortex for 2 min, and centrifuge at 5000 r / min for 3 min. Take 3.00 mL of the supernatant into a 10 mL purification tube, vortex for 1 min, let stand for 1 min, and then filter the supernatant through a 0.22 μm organic filter membrane for testing.

8. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S3, the quantitative method includes: Two standard curve concentration points were selected, with concentrations of 100 μg / L and 200 μg / L, respectively. 1.000 mL of each standard curve concentration point was added, and 100 μL of internal standard working solution was added. Data were collected for the initial injection, 7 h injection, 17 h injection, 27 h injection, and 28 h injection.

9. The method for detecting dimethyl fumarate in food according to claim 1, characterized in that, In step S4, parallel dual-sample detection of DMF and DMM is performed on the three samples. After confirming that there are no response values ​​of DMF and DMM in the sample solution, six parallel spiked tests are performed on the detection limit level, 1-time limit of quantitation level, 2-time limit of quantitation level and 5-time limit of quantitation level of the three samples, respectively.

10. The method for detecting dimethyl fumarate in food according to claim 9, characterized in that, In the sample: The detection limit level is 20 μg / L, which is the theoretical value of the sample concentration, and corresponds to a sample content of 0.05 mg / kg. The limit of quantitation (LOQ) level is equivalent to a theoretical sample concentration of 40 μg / L, which corresponds to a sample content of 0.1 mg / kg. The limit of quantitation level twice is equivalent to a theoretical sample concentration of 80 μg / L, corresponding to a sample content of 0.2 mg / kg. The 5-fold limit of quantitation level corresponds to a theoretical sample concentration of 200 μg / L, which is equivalent to a sample content of 0.5 mg / kg.

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