Method for enriching detection of mycotic acid in food by MOF dispersed solid phase extraction material

Abstract

The application discloses a kind of MOF dispersion solid-phase extraction material enrichment detection rice fermentation acid in food, it is related to the technology in the field of food detection.The new nitro functionalized zirconium-based organic framework material, i.e., functionalized MOF material, is prepared, and the dispersion solid-phase extraction method is used.Combining with the experiment test, the functionalized MOF material prepared by the application is used as an adsorbent, can react with rice fermentation acid in food extract, and rice fermentation acid can be desorbed from the adsorbent by using a suitable eluent, so as to realize the enrichment detection of rice fermentation acid, and the minimum detection mass of rice fermentation acid can reach 0.04 pg.The extraction liquid enrichment purification process does not use organic reagent, is environmentally friendly, reduces environmental pollution to a minimum limit.The time is shortened to 6 min from the extraction liquid enrichment purification to sample determination, which greatly improves the detection efficiency.

Description

TECHNICAL FIELD

[0001] The application relates to a kind of MOF dispersion solid phase extraction material enrichment detection methods of rice fermentation acid in food in the technical field of food detection. BACKGROUND

[0002] Rice fermentation acid is a long-chain fatty acid biological toxin produced by Pseudomonas cocovenenans, which can inhibit the activity of adenine nucleotide translocase on mitochondria in cells, causing damage to organs such as brain, heart, liver and kidney. Rice fermentation acid is easily soluble in organic solvents such as petroleum ether, n-hexane, diethyl ether, chloroform and methanol, and alkaline aqueous solution, and is stable to heat and alkaline conditions, but unstable to acidic conditions, oxidizing agents and sunlight. It is difficult to remove in the conventional food processing process. After eating food contaminated by rice fermentation acid, symptoms such as nausea, vomiting, abdominal pain, abdominal distension, dizziness and general weakness may occur within 0.5-12 hours after eating, and severe cases may develop jaundice, ascites, subcutaneous hemorrhage, convulsions, convulsions, hematuria and even liver failure and kidney failure. There is no specific antidote, and 1-1.5 mg of intake can even cause death, with a mortality rate of more than 40%. Pseudomonas cocovenenans is a common foodborne pathogen in the environment, and foods such as cereals, fermented rice and flour products, tremella fuciformis and black fungus are susceptible to the pathogen. Therefore, it is necessary to detect rice fermentation acid in food.

[0003] At present, the detection method of rice fermentation acid is high performance liquid chromatography-diode array method. This method has weak selectivity, low sensitivity, large sample sampling amount and complex sample pretreatment, especially for low concentration samples, and has insufficient qualitative and quantitative ability. High performance liquid chromatography-mass spectrometry has high sensitivity and high specificity, and can be used for the detection of rice fermentation acid in food (tremella fuciformis, black fungus, river powder), traditional Chinese medicine (Jianqu, Liushenqu), urine and plasma. The sample pretreatment method uses simple solvent extraction or selects solid phase extraction column purification. No matter which of the above detection methods involves instrument analysis, the key link in the analysis and detection is the sample pretreatment. Because sample pretreatment can remove matrix interference, increase selectivity, improve analysis accuracy and detection sensitivity, in order to achieve the highest extraction efficiency of the measured substance and the smallest interference effect of the co-extracted substance on the measured substance. Solid phase extraction is a classic sample pretreatment method, in which the target substance in the sample flows through the surface of the adsorbent and is retained by interaction. When the size of the adsorbent is smaller, the back pressure generated by the sample flowing through is larger, which affects the extraction effect and increases the operation difficulty.

[0004] The adsorbent of the dispersive solid phase extraction (DSPE) is not directly filled into the adsorption column, but is dispersed into the sample solution to adsorb the target analyte to the surface of the adsorbent, under the action of centrifugal force, the target analyte migrates with the adsorbent, and finally the measured substance is eluted by a suitable solvent, so as to separate from the sample matrix. This process reduces the use of harmful organic solvents, simplifies the cumbersome sample elution step, and can enrich trace compounds in the sample by a high multiple. Metal organic framework materials (MOF) can be used as adsorbents in dispersive solid phase extraction due to their regular periodic arrangement structure, rich active center, high specific surface area, high porosity and diversity, and exhibit excellent performance in chemical and biological sensing, sample pretreatment, chemical separation, chromatographic stationary phase, etc. SUMMARY

[0005] The present application aims at the defects of the existing mycotoxin detection and pretreatment method, such as complicated and time-consuming steps, large reagent consumption, low sensitivity and low detection accuracy, and proposes a method for enriching and detecting mycotoxin in food by using dispersive solid phase extraction material, which can quickly separate and enrich mycotoxin. The selective adsorption of the target substance is realized by the electrostatic interaction, π-π stacking effect, hydrogen bond and other intermolecular weak interactions between the functionalized MOF material and the mycotoxin molecules, and the main factors affecting the dispersive solid phase extraction are optimized, including solution pH value, adsorbent dispersion method, adsorbent dosage, extraction time, desorption solvent type, time and method, etc., so as to realize the enrichment and purification of mycotoxin. The sample solution after elution and purification is determined by liquid chromatography-mass spectrometer, and the content of mycotoxin in food is obtained. The method for enriching and detecting mycotoxin in food based on the dispersive solid phase extraction material of the functionalized MOF material has the advantages of less time consumption, greatly reduced consumption of organic reagents, high detection sensitivity, detection limit of pg level, high detection accuracy, which is superior to the existing detection technology, and provides more reliable technical support for the determination of trace mycotoxin in food.

[0006] The present application is realized by the following technical solutions:

[0007] The present application provides a MOF dispersive solid phase extraction material for detecting mycotoxin in food, the precursor is zirconium chloride, the ligand is 2-nitro-p-terephthalic acid, and the MOF dispersive solid phase extraction material is prepared by a solvothermal self-assembly reaction of the precursor and the ligand.

[0008] Preferably, the specific surface area is 300-500 cm 2 / g. The specific surface area of the MOF dispersive solid phase extraction material prepared in the embodiment 1 is 315 m 2 / g.

[0009] Specifically, the precursor and the ligand are dissolved in an organic solvent, mixed, and the MOF dispersive solid phase extraction material is prepared by a solvothermal self-assembly reaction.

[0010] Preferably, the molar ratio of the precursor and the ligand is 1-1.17:1.

[0011] The organic solvent is DMF; the molar volume ratio of the precursor and the organic solvent is 0.015-0.025 mmol:1 L.

[0012] Preferably, the temperature of the solvothermal self-assembly reaction is 120-125 DEG C, and the reaction time is 16-24 h.

[0013] The application further provides the MOF dispersed solid-phase extraction material in the detection of the nigeric acid in food.

[0014] The application further provides a method for enriching and detecting the nigeric acid in food by using the MOF dispersed solid-phase extraction material, comprising the following steps:

[0015] (1) soaking the sample to be detected by using an extraction reagent to obtain an extraction solution;

[0016] (2) dispersing the MOF dispersed solid-phase extraction material as an adsorbent for dispersing solid-phase extraction in the extraction solution obtained in step (1) to adsorb the nigeric acid, discarding the upper solution, collecting the precipitate, adding a buffer solution to the precipitate for elution, and collecting the upper solution;

[0017] (3) filtering the upper solution obtained in step (2) and then analyzing by using a liquid chromatography-tandem mass spectrometer.

[0018] The buffer solution is a phosphate buffer solution, and the components are: 6.45 g of disodium hydrogen phosphate dodecahydrate, 1.09 g of sodium dihydrogen phosphate dihydrate, and 4.25 g of sodium chloride, which are dissolved in water and then diluted to 500 mL.

[0019] Specifically, in step (1), 10-20 mL of methanol aqueous solution with a volume concentration of 80% is added to every 2-5 g of the sample to be detected, the sample is soaked for 1-2 h, and then 5000-8000 rpm centrifugation is performed for 2-3 min to obtain the extraction solution.

[0020] In step (2), 10-50 mg of the functionalized MOF material is added to every 2-10 mL of the extraction solution.

[0021] Specifically, in step (3), when the liquid chromatography-tandem mass spectrometer is analyzed,

[0022] The liquid chromatography conditions are as follows:

[0023] Mobile phase A is water, mobile phase B is acetonitrile containing 0.1% formic acid by volume concentration, elution gradient: 0→1.00 min, 30%-60% B; 1.00→5.00 min, 60%-95.0% B; 5.00→7.10 min, 95%-30.0% B; 7.10→9.10 min 30% B;

[0024] Mass spectrometry conditions:

[0025] Ion source: electrospray ion source; scanning mode: negative ion scanning; quantitative detection mode: multiple reaction monitoring mode; electrospray voltage: -4500V; atomization gas pressure: 50.0 psi; auxiliary gas flow rate: 50.0 psi; gas curtain pressure: 40.0 psi; collision gas: 6.0 psi; ion source temperature: 550 DEG C; scanning time: 50 ms; collision chamber outlet voltage: 11.0V; collision chamber inlet voltage: 10.0V; qualitative ion pair 485>441 m / z or 485>397 m / z, quantitative ion pair 485>441 m / z, collision gas energy -20 eV or -30 eV, de-clustering voltage 80V.

[0026] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0027] The novel nitro functionalized zirconium-based organic framework material (NO2-MOF) is prepared, and the dispersion solid phase extraction method is combined. Through experimental testing, the metal organic framework material (NO2-MOF) prepared by the method can react with rice koji mycotoxin in food extract, and the rice koji mycotoxin can be desorbed from the adsorbent by using a suitable eluent, so that the enrichment and detection of the rice koji mycotoxin are realized, and the minimum detection mass of the rice koji mycotoxin can reach 0.04 pg.

[0028] Compared with the conventional extraction and detection method, the dispersion solid phase extraction combined with the functionalized metal organic framework material as the adsorbent can effectively extract and enrich the rice koji mycotoxin in the food extract. No organic reagent is used in the extraction and purification process, which is environmentally friendly and minimizes environmental pollution. From the extraction and purification to the sample loading and determination, the time is shortened to 6 minutes, which cannot be achieved by the general solid phase extraction purification method, and the detection efficiency is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a scanning electron microscope graph of the nitro functionalized zirconium-based organic framework material (NO2-MOF).

[0030] Figure 2 It is an infrared spectrum graph of the NO2-MOF.

[0031] Figure 3X-ray photoelectron spectroscopy (XPS) pattern of NO2-MOF.

[0032] Figure 4 Thermogravimetric pattern of NO2-MOF.

[0033] Figure 5 X-ray diffraction (XRD) pattern of NO2-MOF.

[0034] Figure 6 Nitrogen adsorption isotherm of NO2-MOF.

[0035] Figure 7 Pore size distribution of NO2-MOF.

[0036] Figure 8 MRM chromatogram-mass spectrum of mycophenolic acid standard (0.19 ng / mL).

[0037] Figure 9 Standard curve.

[0038] Figure 10 MRM chromatogram-mass spectrum of mycophenolic acid in positive sample (Auricularia auricula).

[0039] Figure 11 Mass spectrum signal change of toxoflavin (a) and mycophenolic acid (b) before (curve 1), after (curve 2) adsorption and after elution with buffer (curve 3) of NO2-MOF material.

[0040] Figure 12 Comparison of tetrodotoxin before (curve 1) and after (curve 2) adsorption. DETAILED DESCRIPTION

[0041] Example 1: Preparation of nitro-functionalized zirconium-based organic framework material (NO2-MOF)

[0042] 0.6 millimoles of zirconium chloride was added to 20 mL of DMF and stirred to dissolve to obtain solution A. Then 0.6 millimoles of nitro-p-terephthalic acid and 10 mL of DMF were added to solution A, and stirring and ultrasonic dissolution were continued. Then it was transferred to a 100 mL stainless steel high-pressure reaction kettle, and reacted in a 120°C constant temperature oven for 24 h. After the reaction was completed, the product was washed with water and ethanol three times, respectively. The centrifugal speed during washing was 6000-8000 rpm, and the time was 5-7 min. Finally, the product was dried in a 75°C vacuum drying oven.

[0043] Figure 1 The scanning electron microscope (SEM) image of the nitro-functionalized zirconium-based organic framework material obtained in Example 1 can be seen from the scanning electron microscope image that the NO2-MOF material has a polyhedral structure, is relatively uniform in dispersion, and has a certain agglomeration phenomenon. Figure 2is the infrared spectrum of the nitro-functionalized zirconium-based organic framework material obtained in Example 1. The infrared spectrum shows that the stretching vibration peak of carboxyl group at 3390 cm -1 and 1600 cm -1 , the anti-symmetrical stretching vibration peak of N-O at 1540 cm -1 , indicating that the MOF material has carboxyl group and functional group nitro on the structure. Figure 3 is the photoelectron spectrum of the nitro-functionalized zirconium-based organic framework material obtained in Example 1. From the photoelectron spectrum, it can be seen that the material has strong signals at electron binding energies of 282 eV, 528 eV, 402 eV, 180 and 182 eV, corresponding to C, O, N, Zr four elements, respectively, indicating that the material contains C, O, N, Zr four elements. Figure 4 is the thermogravimetric graph of the nitro-functionalized zirconium-based organic framework material obtained in Example 1. From the thermogravimetric graph, it can be seen that the NO2-MOF material has good overall thermal stability. There is a 10% weight loss at about 300°C, including water and other small molecules initially attached to the surface of the material; at about 420°C, the structure of the material begins to decompose, and about 30% of the weight is lost, which is consistent with the thermogravimetric analysis results of other zirconium-based MOF materials. Even if the temperature is increased to 800°C, there are still 40% of the material remaining in the original state, which overall indicates that the MOF material has good thermal stability. Figure 5 is the X-ray diffraction graph of the nitro-functionalized zirconium-based organic framework material obtained in Example 1. From the X-ray diffraction graph, it can be seen that the NO2-MOF material has good crystallinity, and the diffraction angles 7.3 and 8.5 correspond to the crystal planes (111) and (200). The X-ray diffraction graph of the material is basically consistent with the diffraction graph of other zirconium-based MOF materials. Figure 6 and Figure 7 is the nitrogen adsorption and pore size distribution graph of the nitro-functionalized zirconium-based organic framework material obtained in Example 1. From the graph, it can be seen that the specific surface area of the prepared NO2-MOF material is the BET specific surface area of 315 m 2 / g. The total pore distribution graph of nitrogen adsorption shows that the average pore size of the NO2-MOF material is 8.8 nm, and the maximum adsorption point pore volume is 0.19 cm 3 / g.

[0044] Example 2: Preparation of nitro-functionalized zirconium-based organic framework material (NO2-MOF)

[0045] 0.7 mmol of zirconium chloride was added into 20 mL of DMF, and stirred and dissolved to obtain solution A. Then 0.6 mmol of nitro-p-terephthalic acid and 10 mL of DMF were added into solution A, and continued to be stirred and ultrasonically dissolved. Then it was transferred into a 100 mL stainless steel high-pressure reaction kettle, and reacted in a 125°C constant-temperature oven for 16 h. After the reaction was completed, the product was washed with water and ethanol for three times respectively. The centrifugal speed was 6000-8000 rpm, and the time was 5-7 min. Finally, the product was dried in a 75°C vacuum drying oven.

[0046] Example 3: Implementation of method with standard addition recovery rate

[0047] One sample of rice powder and one sample of black fungus were taken, and each sample was uniformly pulverized and then evenly divided into four portions (each portion had a mass of 5 g). One portion of each sample was taken as a blank control, and low, medium and high levels of rice mycotoxin standard solution were added into the other three portions of each sample. 20 mL of methanol aqueous solution (methanol content 80%) was added, and the samples were soaked for 1 h, and then centrifuged at 6000 rpm for 3 min to collect the supernatant. 2 mL of the supernatant was taken and added into 20 mg of NO2-MOF material, and then vortexed for 1 min, and centrifuged at 15000 rpm for 2 min, and the supernatant was discarded. 0.2 mL of phosphate buffer solution (6.45 g of disodium hydrogen phosphate dodecahydrate, 1.09 g of sodium dihydrogen phosphate dihydrate, and 4.25 g of sodium chloride were dissolved in water and diluted to 500 mL) was added into the obtained precipitate, and then vortexed for 1 min, and centrifuged at 15000 rpm for 2 min. The supernatant was filtered through a 0.22 μm filter membrane, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). The analysis results are shown in Table 1.

[0048] Table 1: Analysis results of blank control and spiked samples

[0049]

[0050] As can be seen from Table 1, rice mycotoxin was not detected in rice powder and black fungus. The recovery rate of rice mycotoxin detection in spiked samples with different levels was 75-96%, and the recovery rate was good.

[0051] Example 4: Determination of method limit of quantification and sensitivity

[0052] Take 1 portion of rice flour and black fungus sample, crush uniformly. Take 2 g of the crushed sample, add 20 mL of methanol aqueous solution (methanol content 80%), soak for 1 h, centrifuge at 6000 rpm for 3 min, collect the supernatant. Take 2 mL of the supernatant, add 20 mg of NO2-MOF material, vortex for 1 min, centrifuge at 15000 rpm for 2 min, discard the supernatant, add 0.2 mL of phosphate buffer solution to the obtained precipitate, vortex for 1 min, centrifuge at 15000 rpm for 2 min, take the upper filtrate through a 0.22 μm filter membrane, and analyze by liquid chromatography-tandem mass spectrometry (LC-MS). It is analyzed that the orsellinic acid in the rice flour and black fungus is not detected. Add orsellinic acid standard solution to the above sample extract without orsellinic acid to make its concentration 0.02 ng / mL. After the above same method is handled, 10 times concentration, and the standard curve (as shown in Figure 9 The concentration of orsellinic acid in the sample is 0.19 ng / mL. The minimum detection mass of orsellinic acid in the rice flour and black fungus is 0.04 pg. Figure 8 The MRM chromatogram-mass spectrum of orsellinic acid is shown. The two pairs of qualitative ion pairs extracted are 485>441 and 485>397, and the quantitative ion pair is 485>441. When the concentration of orsellinic acid in the sample is 0.19 ng / mL, the signal-to-noise ratio of the quantitative ion pair 485>441 is 10, so the concentration of orsellinic acid in the original extract is 0.02 ng / mL, which can be used as the quantitative limit of the method, and thus the minimum detection mass of the whole method is 0.04 pg (the injection amount is 2 μL).

[0053] Example 5: Determination of positive sample

[0054] Take 1 portion of black fungus sample, crush uniformly. Take 2 g of the crushed sample, add 20 mL of methanol aqueous solution (methanol content 80%), soak for 1 h, centrifuge at 6000 rpm for 3 min, collect the supernatant. Take 2 mL of the supernatant, add 20 mg of NO2-MOF material, vortex for 1 min, centrifuge at 15000 rpm for 2 min, discard the supernatant, add 0.2 mL of phosphate buffer solution to the obtained precipitate, vortex for 1 min, centrifuge at 15000 rpm for 2 min, take the upper filtrate through a 0.22 μm filter membrane, and analyze by liquid chromatography-tandem mass spectrometry (LC-MS). The concentration of orsellinic acid in the sample is 4.9 ng / mL. The content of orsellinic acid in the black fungus sample is 4.9 μg / kg. Figure 9 The MRM chromatogram-mass spectrum of orsellinic acid measured in the black fungus of this example is shown. Figure 10

[0055] Example 6: Interference test

[0056] ​Both of the mycotoxins are produced by Pseudomonas cocovenenans, and it is very possible that they exist in the food contaminated by the bacteria. Tetrodotoxin is a small molecule non-protein neurotoxin with extremely high toxicity, which is widely found in dinoflagellate cysts, calcareous algae, and bivalves. In addition, tetrodotoxin is also found in sediments in marine and freshwater environments. The three toxins are all natural toxins produced in nature, and are small molecule compounds, which are difficult to be found in food. Therefore, the tetrodotoxin and the toxic yellow substance were selected as the interference experimental objects of the rice fungus acid. One sample of agaric was taken, and the sample was crushed and evenly divided into 3 parts (5g each). One part was used as a blank control, and the other two parts were added with a certain amount of mixed standard solution of rice fungus acid, toxic yellow substance and tetrodotoxin. Then 20 mL of methanol aqueous solution (80% methanol) was added, soaked for 1 h, centrifuged at 6000 rpm for 3 min, and the supernatant was collected. 2 mL of the supernatant was added to 20 mg of NO2-MOF material, vortexed for 1 min, centrifuged at 15000 rpm for 2 min, and the supernatant was discarded. 0.2 mL of phosphate buffer solution was added to the obtained precipitate, vortexed for 1 min, centrifuged at 15000 rpm for 2 min, and the upper filtrate was filtered through a 0.22 μm filter membrane and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). Figure 11 In the figure, curves 1, 2 and 3 represent the mass spectrum signal changes of the toxic yellow substance (a) and the rice fungus acid (b) before adsorption, after adsorption and after elution with buffer, respectively. Figure 12 is the comparison of tetrodotoxin before adsorption (curve 1) and after adsorption (curve 2). The experimental results show that both the rice fungus acid and the toxic yellow substance are adsorbed by the NO2-MOF material, and the tetrodotoxin is not adsorbed. In the subsequent elution experiment, the toxic yellow substance is not eluted, and only the rice fungus acid is finally detected. From the results of this interference experiment, it can be seen that the NO2-MOF material can selectively determine the target rice fungus acid. The mechanism is analyzed as follows: due to the unique pore size distribution and nitro functionalization of the NO2-MOF material, the toxic yellow substance and the slightly negatively charged rice fungus acid interact with the NO2-MOF material through weak forces such as electrostatic interaction, π-π stacking effect and hydrogen bonding and are adsorbed. In the phosphate buffer, the weak interaction between the rice fungus acid and the NO2-MOF material is destroyed, causing the rice fungus acid to be released from the NO2-MOF material. However, under the same elution conditions, because the toxic yellow substance molecule is smaller, it is captured by the relatively small micropores (<2 nm) of the NO2-MOF material, so the toxic yellow substance is not released. Tetrodotoxin is an alkaloid, its molecules generally have weak positive charge, and its molecular structure has two saturated six-membered rings, which is relatively rigid. It is repulsive with the NO2-MOF material which also has positive charge, and they also fail to form π-π stacking effect, so tetrodotoxin is not adsorbed by the NO2-MOF material.

Claims

1. Application of MOF dispersive solid-phase extraction material in detection of rice mycotoxin in food, The precursor is zirconium chloride, and the ligand is 2-nitro-p-terephthalic acid. The MOF dispersive solid-phase extraction material is prepared by a solvothermal self-assembly reaction of the precursor and the ligand. The precursor and the ligand are dissolved in an organic solvent, mixed, and subjected to a solvothermal self-assembly reaction to prepare the MOF dispersive solid-phase extraction material. The food is rice powder or black fungus. In application, the detection of rice mycotoxin in food includes the following steps: (1) soaking the sample to be detected with an extraction reagent to obtain an extract; The extraction reagent is a methanol aqueous solution with a volume ratio concentration of 80%; (2) using the MOF dispersive solid-phase extraction material as an adsorbent for dispersing solid-phase extraction to adsorb rice mycotoxin in the extract obtained in step (1), discarding the supernatant, collecting the precipitate, adding a buffer solution to the precipitate for elution, and collecting the supernatant; The buffer solution is a phosphate buffer solution; (3) filtering the supernatant obtained in step (2) and then analyzing it by liquid chromatography-tandem mass spectrometry.

2. Use according to claim 1, characterized in that, The molar ratio of the precursor to the ligand is 1-1.17:

1.

3. Use according to claim 1, characterized in that, The organic solvent is DMF; The molar volume ratio of the precursor to the organic solvent is 0.015-0.025 mmol: 1 L.

4. Use according to claim 1, characterized in that, The solvothermal self-assembly reaction temperature is 120-125°C, and the reaction time is 16-24 h.

5. The use according to claim 1, characterized in that, In step (1), 10-20 mL of a methanol aqueous solution with a volume ratio concentration of 80% is added to every 2-5 g of the sample to be detected, soaked for 1-2 h, and centrifuged at 5000-8000 rpm for 2-3 min to obtain the extract; In step (2), 10-50 mg of the functionalized MOF material is added to every 2-10 mL of the extract.

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