Method for determining MeIQx generation level by using ultrahigh pressure liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for determining the MeIQx generation level by using ultrahigh pressure liquid chromatography-mass spectrometry. The method comprises the step of detecting the content of MeIQx, the content of precursor substances and the content of intermediate substances generated in an oil-containing system and an oil-free system of a MeIQx chemical simulation system by adopting a high performance liquid chromatograph and a triple quadrupole mass spectrometer connected in series. According to the method, the MeIQx content, precursor substances and intermediate substances in a MeIQx chemical simulation system can be quantitatively detected, the detection speed is high, the precision is high, and through the detection result, the MeIQx inhibition effect and mechanism of the exogenous inhibitor can be scientifically and reasonably analyzed when the exogenous inhibitor is used for frying aquatic products.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a method for measuring the level of MeIQx generation by using ultra-high pressure liquid chromatography-mass spectrometry. Background Art

[0002] 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a class 2B mutagenic and carcinogenic compound produced during the frying process of protein-rich aquatic products. MeIQx is mainly produced during the high-temperature processing of protein-rich aquatic products. After glucose and amino acids (glycine) undergo Strecker degradation reactions, Strecker degradation products undergo a series of complex chemical reactions such as Amadori rearrangement to form pyrazine and pyridine compounds, which then react with creatinine through the Aldol condensation mechanism. MeIQx has been detected in 38 fried aquatic products and is a common hazard factor in fried aquatic products. Therefore, studying the reduction and control methods of MeIQx is important for improving the edible safety of aquatic products.

[0003] In the prior art, inhibitors for MeIQx generation have been screened and studied, and some inhibitors (aldehyde inhibitors, pyrazine inhibitors, etc.) have been chemically synthesized, but these inhibitors are prone to cause side effects such as intestinal flora disorders and diarrhea. Therefore, turning to the search for natural antioxidants that inhibit MeIQx generation has become a hot topic, and in recent years, natural active substances such as allicin, black pepper extract and curcumin have been screened as MeIQx inhibitors.

[0004] For natural inhibitors, most of the existing technologies use free radical pathways to detect and analyze the inhibitory effects of exogenous inhibitors. The generation of MeIQx during the frying of aquatic products includes the generation of precursor substances, intermediate substances, and final MeIQx, but it is difficult to effectively and quantitatively detect and analyze the generation of these substances during the frying process in the existing technology, and it is difficult to conduct a scientific and reasonable analysis of the various pathways for the generation of MeIQx during the frying process. Summary of the invention

[0005] The object of the present invention is to provide a method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry to solve the technical problems raised in the above background technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for determining the level of MeIQx generation by ultra-high pressure liquid chromatography-mass spectrometry comprises using a high performance liquid chromatograph in series with a triple quadrupole mass spectrometer to detect the MeIQx content, precursor substance content, and intermediate substance content generated in an oil-containing system and an oil-free system of a MeIQx chemical simulation system.

[0008] Furthermore, the method for detecting the content of MeIQx includes: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding 2 ml of ethyl acetate and vortexing for 5 minutes, then ultrasonically extracting for 15 minutes, repeating the extraction step 3 times, combining the extracts and drying them with nitrogen, re-dissolving the dried extract with 1 ml of methanol, filtering with a 0.22 μm microporous filter membrane, and loading the sample into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

[0009] Furthermore, the conditions of the high performance liquid chromatograph connected to a triple quadrupole mass spectrometer are as follows: the chromatographic column uses Agilent C18, 2.1 mm*100 mm, 1.8 μm, column temperature: 30°C, flow rate: 0.3 mL / min, acquisition mode: ESI+, injection volume: 5 μL, mobile phase A: 5 mmol / L ammonium formate aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile; the mobile phase gradient is as follows: 0 min, 90% A, 10% B; 1 min 90% A, 10% B; 4 min, 70% A, 30% B; 5 min, 10% A, 90% B; 9 min, 10% A, 90% B; 9.1 min, 90% A, 10% B; 11 min, 90% A, 10% B.

[0010] Furthermore, the precursor substances include glycine, creatinine, and glucose; and the intermediate substances include 2,5-dimethylpyrazine and aldehyde substances.

[0011] Furthermore, the glycine detection method comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, diluting to 10 ml with water, ultrasonicating for 20 minutes, centrifuging at a speed of 6000 r / min in a centrifuge for 5 minutes, taking the supernatant for purification, and loading it into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

[0012] Further, the conditions of the high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer are as follows: chromatographic column: C18SHISEIDO, 4.6mm*250mm*5μm; injection volume: 10μL; column temperature: 40°C; wavelength: 254nm; mobile phase: A: 0.1mol / L anhydrous sodium acetate + acetonitrile, adjust pH to 6.5 after mixing; B: acetonitrile + water. The mobile phase gradient is: 0min, 100% A, 0% B; 14min, 85% A, 15% B; 29min, 66% A, 34% B; 30min, 0% A, 100% B; 37min, 0% A, 100% B; 38min, 100% A, 0% B; 45min, 100% A, 0% B.

[0013] Furthermore, the creatinine detection method includes: taking 1 ml of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding first-grade water to mix and shake, adjusting the volume to 10 ml and then mixing, ultrasonicating for 10 minutes, passing through a 0.22um membrane, and then loading the sample into a high-performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18, 4.6mm*250mm*5μm; detector: DAD; column temperature: 20°C; injection volume: 10μL; flow rate: 1.0mL / min; mobile phase: acetonitrile: 0.2% phosphoric acid: water = 4:6:90.

[0014] Furthermore, the glucose detection method includes: taking 1 ml of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, diluting to 25 ml with water, and filtering with a 0.45 μm filter membrane into a liquid vial after ultrasonic extraction for 30 minutes; using high performance liquid chromatography equipped with a differential refractometer for detection, and the chromatographic conditions are: chromatographic column: Aglient amino column, 250×4.6 mm, 5 μm; column temperature: 35°C; injection volume: 10 μL; mobile phase: acetonitrile: water = 70:30; flow rate: 1.0 mL / min.

[0015] Further, the method for detecting 2,5-dimethylpyrazine includes: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding the mobile phase to mix and shake, making the volume to 25 mL of water and mixing evenly, ultrasonicating for 10 minutes, passing through a 0.22 um membrane, and loading the sample into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18, 4.6 mm*250 mm*5 μm; detector: DAD, 280 nm; column temperature: 20°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: 0.25% trifluoroacetic acid: acetonitrile = 95:5.

[0016] Furthermore, the detection method of the aldehyde substances includes: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system, respectively, adding 25 mL of water for ultrasonic extraction for 30 minutes, taking 500 μL and adding DNPH-acetonitrile, reacting at 50° C. in the dark for 1.5 hours, adding 3 mL of chloroform for extraction twice, combining nitrogen to blow dry, re-dissolving with acetonitrile to 1 mL, and simultaneously derivatizing and testing.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention can quantitatively detect the MeIQx content, precursor substances and intermediate substances in the MeIQx chemical simulation system by using a liquid chromatography-tandem mass spectrometry method, with fast detection speed and high accuracy. The detection results can reflect the scientific and reasonable analysis of the inhibitory effect and mechanism of the exogenous inhibitor on MeIQx when the exogenous inhibitor is used for frying aquatic products.

[0019] 2. The present invention analyzes different MeIQx generation-promoting factors and inducing factors and intermediates in the reaction process, and sets different chromatographic conditions to more specifically analyze the inhibitory effects of exogenous inhibitors on various MeIQx generation pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the detection result of the content of MeIQx of the present invention;

[0021] Figure 2 It is a schematic diagram of the detection result of the glycine content of the present invention;

[0022] Figure 3 It is a schematic diagram of the detection result of the content of creatinine of the present invention;

[0023] Figure 4 It is a schematic diagram of the detection result of the glucose content of the present invention;

[0024] Figure 5 It is a schematic diagram of the detection result of the content of 2,5-dimethylpyrazine of the present invention;

[0025] Figure 6 Schematic diagram of the detection result of formaldehyde content of the present invention;

[0026] Figure 7 It is a schematic diagram of the detection result of acetaldehyde content of the present invention;

[0027] Figure 8 It is a schematic diagram of the detection result of the content of acrolein of the present invention;

[0028] Fig. 9 It is a schematic diagram of the detection result of the content of glyoxal of the present invention;

[0029] Fig.10 It is a schematic diagram of the detection result of the content of methylglyoxal in the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0031] Example 1

[0032] A method for determining the level of MeIQx generation by ultra-high pressure liquid chromatography-mass spectrometry comprises using a high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer to detect the MeIQx content, precursor substance content, and intermediate substance content generated in an oil-containing system and an oil-free system of a MeIQx chemical simulation system; the precursor substance comprises glycine, creatinine, and glucose; and the intermediate substance comprises 2,5-dimethylpyrazine and aldehyde substances.

[0033] The construction method of the MeIQx chemical simulation system is as follows: 10 mL of diethylene glycol and 10 mL of water are added to a reaction tube, and then 1 mmol of glycine, 1 mmol of creatinine, and 0.05 mmol of glucose are added to a polytetrafluoroethylene tube and mixed to obtain a MeIQx generation simulation system, and the mixture is mixed evenly. Two portions of the MeIQx generation simulation system are prepared, one portion is added with soybean oil to obtain an oil-containing MeIQx simulation system, and the other portion is not added to obtain an oil-free MeIQx simulation system; gallic acid is added to the polytetrafluoroethylene tubes of the two systems respectively, and the mixture is mixed evenly; and the polytetrafluoroethylene tube is subjected to a simulated reaction: heating at 180° C. for 40 minutes, and cooling after heating, thereby simulating the generation of MeIQx during frying of aquatic products.

[0034] The method for detecting the content of MeIQx comprises: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, respectively, adding 2 ml of ethyl acetate and vortexing for 5 minutes, then ultrasonically extracting for 15 minutes, repeating the extraction steps 3 times, combining the extracts and drying them with nitrogen, re-dissolving the dried extract with 1 ml of methanol, filtering with a 0.22 μm microporous filter membrane, and loading the sample into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

[0035] The conditions of the high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer are as follows: the chromatographic column adopts Agilent C18, 2.1mm*100mm, 1.8μm, column temperature: 30°C, flow rate: 0.3mL / min, acquisition mode: ESI+, injection volume: 5μL, mobile phase A: 5mmol / L ammonium formate aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile; the mobile phase gradient is as follows: 0min, 90% A, 10% B; 1min90% A, 10% B; 4min, 70% A, 30% B; 5min, 10% A, 90% B; 9min, 10% A, 90% B; 9.1min, 90% A, 10% B; 11min, 90% A, 10% B.

[0036] The glycine detection method comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, respectively, diluting to 10 ml with water, ultrasonicating for 20 minutes, centrifuging at a speed of 6000 r / min in a centrifuge for 5 minutes, taking the supernatant for purification, and loading it into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

[0037] The conditions of the high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer are as follows: chromatographic column: C18SHISEIDO, 4.6mm*250mm*5μm; injection volume: 10μL; column temperature: 40°C; wavelength: 254nm; mobile phase: A: 0.1mol / L anhydrous sodium acetate + acetonitrile, adjust pH to 6.5 after mixing; B: acetonitrile + water. The mobile phase gradient is: 0min, 100% A, 0% B; 14min, 85% A, 15% B; 29min, 66% A, 34% B; 30min, 0% A, 100% B; 37min, 0% A, 100% B; 38min, 100% A, 0% B; 45min, 100% A, 0% B.

[0038] The creatinine detection method comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, adding first-grade water for mixing and shaking, fixing the volume to 10 ml and then mixing, ultrasonicating for 10 minutes, passing through a 0.22 um membrane, and then loading the sample into a high performance liquid chromatograph connected to a triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18, 4.6 mm*250 mm*5 μm; detector: DAD; column temperature: 20°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: acetonitrile: 0.2% phosphoric acid: water = 4:6:90.

[0039] The glucose detection method comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, respectively, diluting to 25 ml with water, and filtering with a 0.45 μm filter membrane into a liquid phase vial after ultrasonic extraction for 30 minutes; using high performance liquid chromatography equipped with a differential refractometer (HPLC-RID) for detection, and the chromatographic conditions are: chromatographic column: Aglient amino column, 250×4.6 mm, 5 μm; column temperature: 35°C; injection volume: 10 μL; mobile phase: acetonitrile: water = 70:30; flow rate: 1.0 mL / min.

[0040] The method for detecting 2,5-dimethylpyrazine comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, adding the mobile phase to mix and shake, making the volume to 25 mL of water and mixing evenly, ultrasonicating for 10 minutes, passing through a 0.22 um membrane, and loading the sample into a high performance liquid chromatograph connected to a triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18 (4.6 mm*250 mm*5 μm); detector: DAD, 280 nm; column temperature: 20°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: 0.25% trifluoroacetic acid: acetonitrile = 95:5.

[0041] The detection method of the aldehyde substances comprises: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system after the simulation reaction, adding 25 ml of water for ultrasonic extraction for 30 min, taking 500 μL, adding 0.2 ml of DNPH-acetonitrile (4 mg / ml), reacting at 50° C. in the dark for 1.5 h, adding 3 ml of chloroform for extraction twice, combining nitrogen to blow dry, re-dissolving with acetonitrile to 1 ml, and simultaneously derivatizing and testing.

[0042] 1. Effects of exogenous inhibitors on MeIQx production

[0043] According to the method of Example 1, 0.25, 0.1, and 1 mmol of gallic acid were added to the oil-containing and oil-free systems of the MeIQx chemical simulation system as experimental groups, and a control group without gallic acid was set up. According to the method of Example 1, the MeIQx content, the precursor content, and the intermediate content in the system were detected respectively.

[0044] 1.1MeIQx content test results

[0045] The inhibitory effect of gallic acid on MeIQx in oil-free and oil-containing simulation systems is shown in Figure 2. Figure 1As can be seen from the figure, the MeIQx content of the oil-free control group is 6.57ng / ml, and the MeIQx content of the oil-containing system is 9.66ng / ml; compared with the control group, the experimental groups with 0.25, 0.5, and 1mmol gallic acid added have reduced MeIQx concentrations in the oil-free system by 7.24%, 16.83%, and 39.5%, and reduced MeIQx concentrations in the oil-containing system by 17.02%, 26.42%, and 31.76%, indicating that gallic acid has excellent inhibitory ability and can significantly reduce the MeIQx produced during oil processing.

[0046] 1.2 Precursor substance detection results

[0047] Effect of gallic acid on the consumption levels of glucose, glycine and creatinine in the MeIQx generation simulation system Figure 2-4 As shown. It can be seen from the figure that in the experimental group, with the increase of gallic acid addition, the concentrations of the three precursor substances in the oil-free system and the oil-containing system increased. In the oil-free system, compared with the control group, in the experimental groups with the addition of 0.25 and 1 mmol gallic acid, the glucose concentration increased by 17.08%, 29.82%, and 29.57%, the glycine concentration increased by 25.22%, 38.46%, and 37.61%, and the creatinine concentration increased by 5.4%, 15.52%, and 22.09%; in the oil-containing system, the glucose concentration increased by 29.22%, 39.65%, and 37.31%, respectively, and the creatinine concentration increased by 5.54%, 7.32%, and 10.12%, respectively. In addition, the addition of 0.5 and 1 mmol gallic acid increased the glycine concentration by 12.31% and 16.28%, respectively.

[0048] This is because in the process of MeIQx generation, glucose, glycine and creatinine are continuously consumed as precursors to generate MeIQx. When gallic acid is added, the concentration of precursors increases, indicating that these precursors are not consumed to generate MeIQx, but accumulate in the system, indicating that gallic acid inhibits them from entering the chemical reaction chain to generate MeIQx, thereby reducing the generation of MeIQx.

[0049] 1.3 Test results of intermediate substances

[0050] 1.31 Effect of Gallic Acid on 2,5-Dimethylpyrazine in the Simulation System of MeIQx Generation

[0051] The inhibitory effect of gallic acid on the 2,5-dimethylpyrazine intermediate in the MeIQx simulation system is shown in Figure 2. Figure 5As shown in the figure, compared with the control group, in the oil-containing system, the experimental groups with 0.25 and 1 mmol gallic acid added, the concentration of 2,5-dimethylpyrazine decreased by 2.67% and 4.61%, respectively, and in the oil-free system, the concentration of 2,5-dimethylpyrazine decreased by 6.1%, 12.52%, and 11.99%, respectively. This is because gallic acid changes the carbonyl valence of the reaction and quenches free radicals through coordination, complexation, multinuclear complexation, and chelation with 2,5-dimethylpyrazine, thereby blocking the MeIQx generation reaction cascade chain and inhibiting the generation of 2,5-dimethylpyrazine.

[0052] 1.32 Effect of Gallic Acid on Aldehydes in MeIQx in the Simulated System of MeIQx Generation

[0053] The inhibitory effect of gallic acid on formaldehyde, acetaldehyde and acrolein in the MeIQx generation simulation system Figure 6-8 As shown. It can be seen from the figure that gallic acid has a significant inhibitory effect on formaldehyde, acetaldehyde and acrolein; in the experimental groups with 0.25, 0.5 and 1 mmol gallic acid added, the formaldehyde inhibition rate in the oil-free system was 20.15%, 34.55% and 44.57%, the inhibition rate of acetaldehyde was 18.39%, 36.2% and 53.81%, and the inhibition rate of acrolein was 15.47%, 33.63% and 45.96%; in the oil-containing system, the inhibition rate of formaldehyde was 20.22%, 36.70% and 59.8%, the inhibition rate of acetaldehyde was 32.51%, 47.87% and 66.24%, and the inhibition rate of acrolein was 15.37%, 42.21% and 60.80%, and compared with the control group, gallic acid has a significant inhibitory effect on formaldehyde, acetaldehyde and acrolein in the oil-containing system and the oil-free system.

[0054] The inhibitory effect of gallic acid on the generation of glyoxal and methylglyoxal in the MeIQx simulation system is shown in Figure 2. Figure 9-10 As shown. It can be seen from the figure that gallic acid can significantly inhibit glyoxal and methylglyoxal in oil-free and oil-containing systems. In the experimental groups with 0.25, 0.5 and 1 mmol gallic acid added, the inhibition rates of glyoxal in the oil-free system were 11.52%, 23.22% and 36.17%, respectively, and the inhibition rates of glyoxal in the oil-containing system were 6.89%, 26.34% and 38.52%, respectively; the inhibition rates of methylglyoxal in the oil-free system were 7.21%, 16.15% and 22.30%, respectively, and the inhibition rates of methylglyoxal in the oil-containing system were 4.57%, 9.67% and 17.62%, respectively. Compared with the control group, gallic acid has a significant inhibitory effect on glyoxal and methylglyoxal in the oil-containing system and the oil-free system.

[0055] By detecting the intermediate substances in the process of MeIQx generation, it is shown that gallic acid can not only remove aldehydes in the process of MeIQx generation, but also inhibit the activity of carbonyl compounds produced by fat oxidation, thereby inhibiting the generation of intermediate substances and reducing the generation of MeIQx.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the level of MeIQx production using ultra-high pressure liquid chromatography-mass spectrometry, characterized in that: The method includes using a high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer to detect the content of MeIQx, the content of precursor substances, and the content of intermediate substances produced in the oil-containing system and the oil-free system of the MeIQx chemical simulation system.

2. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 2, characterized in that: The method for detecting the content of MeIQx comprises: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding 2 ml of ethyl acetate and vortexing for 5 minutes, then ultrasonically extracting for 15 minutes, repeating the extraction steps 3 times, combining the extracts and drying them with nitrogen, re-dissolving the dried extract with 1 ml of methanol, filtering with a 0.22 μm microporous filter membrane, and loading the sample into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

3. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 2, characterized in that: The conditions of the high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer are as follows: the chromatographic column adopts Agilent C18, 2.1 mm*100 mm, 1.8 μm, column temperature: 30°C, flow rate: 0.3 mL / min, acquisition mode: ESI+, injection volume: 5 μL, mobile phase A: 5 mmol / L ammonium formate aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile; the mobile phase gradient is as follows: 0 min, 90% A, 10% B; 1 min 90% A, 10% B; 4 min, 70% A, 30% B; 5 min, 10% A, 90% B; 9 min, 10% A, 90% B; 9.1 min, 90% A, 10% B; 11 min, 90% A, 10% B.

4. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 1, characterized in that: The precursor substances include glycine, creatinine and glucose; the intermediate substances include 2,5-dimethylpyrazine and aldehyde substances.

5. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 4, characterized in that: The glycine detection method comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, diluting to 10 ml with water, ultrasonicating for 20 minutes, centrifuging at a speed of 6000 r / min for 5 minutes in a centrifuge, taking the supernatant for purification, and loading it into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection.

6. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 5, characterized in that: The conditions of the high performance liquid chromatograph connected in series with a triple quadrupole mass spectrometer are as follows: chromatographic column: C18SHISEIDO, 4.6mm*250mm*5μm; injection volume: 10μL; column temperature: 40°C; Wavelength: 254nm; Mobile phase: A: 0.1mol / L anhydrous sodium acetate + acetonitrile, mix and adjust pH to 6.5; B: acetonitrile + water. Mobile phase gradient: 0min, 100% A, 0% B; 14min, 85% A, 15% B; 29min, 66% A, 34% B; 30min, 0% A, 100% B; 37min, 0% A, 100% B; 38min, 100% A, 0% B; 45min, 100% A, 0% B.

7. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 4, characterized in that: The creatinine detection method comprises: taking 1 ml of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding first-grade water for mixing and shaking, fixing the volume to 10 ml and then mixing, ultrasonicating for 10 minutes, passing through a 0.22 um membrane, and then loading the sample into a high performance liquid chromatograph connected to a triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18, 4.6 mm*250 mm*5 μm; detector: DAD; column temperature: 20°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: acetonitrile: 0.2% phosphoric acid: water = 4:6:

90.

8. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 7, characterized in that: The glucose detection method comprises: taking 1 ml of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, diluting to 25 ml with water, and filtering to a liquid phase vial with a 0.45 μm filter membrane after ultrasonic extraction for 30 minutes; using a high performance liquid chromatography equipped with a differential refractometer for detection, and the chromatographic conditions are: chromatographic column: Aglient amino column, 250×4.6 mm, 5 μm; column temperature: 35°C; injection volume: 10 μL; mobile phase: acetonitrile: water = 70:30; flow rate: 1.0 mL / min.

9. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 5, characterized in that: The method for detecting 2,5-dimethylpyrazine comprises: taking 1 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding the mobile phase to mix and shake, making the volume to 25 ml of water and mixing evenly, ultrasonicating for 10 minutes, passing through a 0.22 um membrane, and loading the sample into a high performance liquid chromatograph tandem triple quadrupole mass spectrometer for detection, and the chromatographic conditions are: chromatographic column: Agilent C18, 4.6 mm*250 mm*5 μm; detector: DAD, 280 nm; column temperature: 20°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: 0.25% trifluoroacetic acid: acetonitrile = 95:

5.

10. The method for measuring the level of MeIQx production by ultra-high pressure liquid chromatography-mass spectrometry according to claim 9, characterized in that: The detection method of the aldehyde substances comprises: taking 2 ml of the solution of the oil-containing system and the oil-free system of the MeIQx chemical simulation system respectively, adding 25 ml of water for ultrasonic extraction for 30 min, taking 500 μL and adding DNPH-acetonitrile, reacting at 50° C. in the dark for 1.5 h, adding 3 ml of chloroform for extraction twice, combining nitrogen to blow dry, re-dissolving with acetonitrile to 1 ml, and simultaneously derivatizing and testing.

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