A method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging

Abstract

The present invention is applicable to the technical field of pesticide residue detection, and provides a method for identifying the distribution of pesticide residues in plant tissues based on in-situ mass spectrometry imaging. Based on normal pressure mass spectrometry imaging, thiram is used as a non-systemic pesticide representative, and cymoxanil, imidacloprid, and pyraclostrobin are used as systemic pesticide representatives to establish a normal pressure mass spectrometry imaging in-situ detection method for non-systemic and systemic pesticides in fresh bananas. Non-systemic pesticide thiram does not enter the inside of the banana, and systemic pesticides cymoxanil, imidacloprid, and pyraclostrobin enter the inside of the banana in a lateral penetration and longitudinal migration manner over time; the smaller the water-octanol ratio distribution coefficient and the relative molecular mass of the pesticide, the faster the migration speed and the more content, and the migration speed and content are: cymoxanil>imidacloprid>pyraclostrobin; the faster the speed of pesticide migration in the more mature bananas, the more residual content, and it is not easy to digest. The present invention helps to guide the rational application of pesticides and provide a reference for pesticide safety evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide residue detection, and in particular relates to a method for identifying the distribution of pesticide residues in plant tissues based on in-situ mass spectrometry imaging. Background Art

[0002] The use of pesticides is a common measure to ensure stable and high yields of crops, but it also has many negative effects. The correct, green, safe and reasonable use of pesticides is an urgent problem to be solved. Scientific, reasonable and targeted application of pesticides is a necessary measure to improve the prevention and control effect, ensure the safety of pesticide use and ensure agricultural harvest. The pesticides used will be transferred between plant tissues. Affected by various factors, their accumulation sites and migration patterns are different. This is closely related to the different properties of pesticides. Among them, different pesticides are affected by different octanol-water partition coefficients (Kow) and different relative molecular masses, and their migration rates will also be different. Bananas are the fruit with the largest fresh fruit trading volume in the world. The behavior of pesticide residues in bananas is the key and difficult point in the research of banana product quality and safety. Because bananas belong to "respiratory climacteric" fruits, during the ripening process of bananas, ethylene is released in banana fruits to stimulate the fruit to enhance respiration, and starch is converted into sucrose in large quantities to achieve ripening. When a certain degree of maturity is reached, a "respiratory burst" will occur, causing it to age and soften rapidly, reduce resistance, and be easily bruised or infected by fungi, resulting in blackening and rotting. Therefore, it is common to apply pesticides to bananas. However, the sugars continuously produced by bananas during their growth also affect the migration and accumulation of pesticides. Studies have found that sugars can promote the movement of compounds between plant tissues. Therefore, the migration patterns of the same pesticide in bananas of different maturity also vary. Existing studies have all used offline methods to measure and evaluate pesticide residues in banana plants or fruits, which cannot reflect their residues, metabolism, migration, and temporal and spatial distribution in real time. Traditional pesticide detection includes chromatography and mass spectrometry, which are performed on machines after homogenization, extraction, purification, centrifugation, and filtration of samples. Because it can accurately perform qualitative and quantitative analysis of pesticide residues in the test object, it is widely used, but traditional detection methods require complex pre-treatment, cannot perform in-situ detection of the test sample, and cannot intuitively clarify the distribution of pesticide residues. The introduction of mass spectrometry imaging technology has solved this problem.

[0003] Mass spectrometry imaging is to separate the analyte from the sample surface by desorption and ionization, and then detect its gas phase ions. It can visualize and analyze multiple molecules without the need for particularly complex pre-treatment or labeling, and is widely used in imaging analysis of biological tissues. At present, this technology is also widely used in plant research, mainly matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and desorption electrospray ionization mass spectrometry imaging (DESI-MSI). The MALDI-MSI sample preparation process is relatively complex and has strict requirements on the matrix, so there are still obvious deficiencies in the applicability of plant source samples. DESI-MSI overcomes the limitation of MALDI ion source to implement ionization under vacuum conditions, but this method has certain limitations on the spatial size of the sample. These two detection technologies are often used in plant research to study endogenous substances in plants, but there is no in-depth research on exogenous pesticides in plants.

[0004] The in-situ detection of exogenous pollutants in plants by atmospheric pressure mass spectrometry imaging is an issue that has not been deeply studied and analyzed. Due to simple pretreatment, in-situ visualization, and precise positioning of the analyte, the instrument detection platform based on atmospheric pressure mass spectrometry (MS) provides a unique opportunity for detecting small molecules in plants. Therefore, this experiment selected banana, a typical tropical fruit with the characteristic of "respiratory jump", as the research object. By further optimizing and adjusting the atmospheric pressure MSI system to make it more suitable for imaging of fresh plant tissues, an in-situ visualization research method for the residual behavior of four pesticides, thiram, cymoxanil, imidacloprid, and pyraclostrobin, in banana fruits based on mass spectrometry imaging technology was established. The factors that lead to the differences between pesticides were clarified through the lateral penetration and longitudinal conduction of pesticides in bananas. At the same time, the migration and accumulation of pesticides in bananas of different maturity levels under the influence of sugar were further intuitively explored. The study of the migration and accumulation of pesticides provides a reference for the rational application of pesticides and the formulation of pesticide safety evaluation. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging, aiming to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging comprises the following steps:

[0008] Step 1: Sample preparation

[0009] Four pesticides, namely, thiram, propamocarb, pyraclostrobin and imidacloprid, were weighed respectively, and prepared into equal concentration solutions with ultrapure water. After being diluted 1000 times with ultrapure water, the solutions were uniformly mixed to prepare a pesticide mixture. Banana samples of different maturity were obtained from a banana base, and bananas of the same maturity were ensured to be from the same batch. The dirt on the surface of the banana samples was cleaned with ultrapure water, and the cycle was repeated three times.

[0010] Take a completely unripe banana sample, fully immerse it in the mixed pesticide solution and take it out after 30 minutes, place it in a room at 25℃, and freeze it in a refrigerator at -80℃ every 2 hours. After freezing, use an oscillating slicer to slice the banana tissue into slices of 25mm×20mm×5mm in length×width×height in both horizontal and vertical ways, adhere and fix them on a positively charged anti-shedding slide, and store them in a refrigerator at -80℃ for imaging analysis;

[0011] Take a completely unripe banana sample, immerse the bottom stalk of the whole banana fruit in the prepared mixed solution, take it out after 30 minutes, place it at 25℃ for 12 hours, and then put it in a refrigerator at -80℃. Use a slicer to cut tissue sections horizontally from the top of the banana, place them on a slide and freeze them in a refrigerator at -80℃ until analysis;

[0012] Take banana samples of 20%, 60%, and 100% different degrees of maturity, immerse them in the mixed solution, take them out after 30 minutes, place them at 25°C, freeze the fresh samples in a -80°C refrigerator once every 2 hours, use a slicer to make longitudinal slices of 25mm×20mm×5mm in length×width×height, adhere the longitudinal slices to a glass slide, seal and freeze them until detection and analysis;

[0013] Step 2: Mass spectrometry imaging

[0014] First, install and fix the spray needle, then connect the gas passage and liquid passage of the electrospray needle, let N2 into the gas passage, and the liquid solvent in the liquid passage is methanol water containing 0.1% formic acid, turn on the connected vacuum pump, fix the ion transfer tube to the interface of the electrostatic field orbital trap high-resolution mass spectrometer, and be identified by the mass spectrometer through the ion source identifier;

[0015] Adjust the movable loading platform to place the glass slide with the banana sample attached in an appropriate position, and adjust the position of the electrospray needle;

[0016] By applying high voltage to the liquid path, the spray solvent is charged. Under the action of high-speed spray gas N2, the micro-flow rate liquid is atomized to form electrospray droplets. The charged droplets bombard the sample surface. Under the extraction action of methanol and water solvent, the sample is desorbed and ionized. After Coulomb rupture occurs, the sample solution is desolvated and enters the mass spectrometer interface through the ion transfer tube. Under the action of vacuum, it enters the mass spectrometer cone port for collection.

[0017] Step 3: Data Analysis

[0018] Imaging data was acquired by continuously capturing mass spectra with a given number of scans, and Xcalibur software was used for mass spectrometry processing. The file format was converted into CDF format, and Massimager was used for visualization of mass spectrometry images and ion image data. The actual length and width of the scanned sample slices and the number of scan lines were set, and the scanned images were imported. The intensity normalization was set to a global image, and the corresponding analyte images were obtained by setting the mass-to-charge ratios of thiram, propamocarb, pyraclostrobin, and imidacloprid with a mass-to-charge ratio tolerance of 0.05 Da.

[0019] Furthermore, in the step 2, a high voltage of 7000V is applied to the liquid path to charge the spray solvent, and under the action of 0.6Mpa high-speed spray gas N2, the micro-flow rate liquid is atomized to form electrospray droplets.

[0020] Furthermore, in step 2, the volume ratio of methanol to water in the methanol water is 80:20.

[0021] Furthermore, in the step 2, the spray solvent flow rate of the electrospray needle is 5 μL / min.

[0022] Furthermore, in step 2, the suction flow rate of the vacuum pump is 50 L / min.

[0023] Furthermore, in step 2, the length of the ion transfer tube is 50 cm, and the distance between the ion transfer tube and the conical collection port is 2.5 mm.

[0024] Furthermore, in step 2, the scanning speed of the loading platform is 0.5 mm / s, and the line spacing is 0.5 mm.

[0025] Furthermore, the capillary in the electrospray needle extends 0.1 mm, the electrospray needle is arranged at an angle of 60° to the sample, and the electrospray needle is arranged at a distance of 1.5 mm from the ion transfer tube.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The method for identifying the distribution of pesticide residues in plant tissues based on in-situ mass spectrometry imaging is based on atmospheric pressure mass spectrometry imaging to establish an atmospheric pressure mass spectrometry imaging in-situ detection method for thiram, propamocarb, imidacloprid, and pyraclostrobin in fresh bananas, and analyzes the factors affecting the migration of pesticides. The results show that the non-systemic pesticide thiram does not enter the banana, and the systemic pesticides propamocarb, imidacloprid, and pyraclostrobin enter the banana in a lateral infiltration and longitudinal migration manner over time; the smaller the water-octanol ratio distribution coefficient and the relative molecular mass of the pesticide, the greater the migration speed and the more content, and the migration speed and content are: propamocarb>imidacloprid>pyraclostrobin; the more mature the banana, the faster the migration speed of the pesticide, the more residual content, and it is not easy to digest. The present invention helps to guide the rational application of pesticides, provide a reference for pesticide safety evaluation, and promote the formulation of rational application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural schematic diagram of the present invention.

[0029] Figure 2 Schematic diagram of the mass spectrometry imaging system device in the present invention.

[0030] Figure 3 This is a banana slice diagram in the present invention. ((A)(E) is a 3D stereoscopic image of a banana cross-slice; (B) is a real image of a banana cross-slice; (C) is a 3D stereoscopic image of a banana longitudinal slice; (D) is a real image of a banana longitudinal slice; (F) is a real image of a banana transverse half slice)

[0031] Figure 4 This is the ion fragment diagram of the analyte in the present invention. (m / z 240.995 is thiram; pyraclostrobin is m / z 388.105; m / z 189.159 is propamocarb; m / z 256.059 is imidacloprid)

[0032] Figure 5 This is a parameter optimization diagram in the present invention. ((a) mobile phase optimization; (b) spray voltage optimization; (c) optimization of the distance between the spray needle and the ion transfer tube; (d) optimization of the distance between the transfer tube and the mass spectrometer collection cone; (e) spray pressure optimization; (f) spray needle angle optimization)

[0033] Figure 6 This is a mass spectrometry image of a transverse half-slice of a sample of the present invention in which the bottom stalk of a banana fruit is soaked in pesticides. (m / z 240.995 is thiram; m / z 189.159 is propamocarb; m / z 256.059 is imidacloprid; and pyraclostrobin is m / z 388.105)

[0034] Figure 7The mass spectrometry images of the banana peel sample soaked in pesticides in the present invention. ((A) is the mass spectrometry image of the longitudinal section of the banana sample; (B) is the mass spectrometry image of the transverse half section of the banana tissue)

[0035] Figure 8 These are bananas of different maturity in the present invention. (From left to right, they are banana samples of 20%, 60%, and 100% maturity)

[0036] Fig. 9 This is the migration process of pesticides inside bananas of different maturity in the present invention. (Each pesticide has 20%, 60%, and 100% maturity in the horizontal direction, and the migration change in the vertical direction is every 2 hours) DETAILED DESCRIPTION

[0037] 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 in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0039] An embodiment of the present invention provides a method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging, comprising the following steps:

[0040] Step 1: Sample preparation

[0041] Weigh thiram (C6H 12 N2S4), cymoxanil (C9H 20 N2O2), pyraclostrobin (C 19 H 18 ClN3O4), imidacloprid (C9HClN5O2) Figure 1 ), prepare an equal concentration solution with ultrapure water, dilute it 1000 times with ultrapure water, and evenly mix it to prepare a pesticide mixture, obtain banana samples of different maturity at a banana base, and ensure that bananas of the same maturity are from the same batch, and wash the dirt on the surface of the banana samples with ultrapure water, and repeat three times;

[0042] Take a completely unripe banana sample, immerse it fully in the mixed pesticide solution for 30 minutes, take it out, freeze it in a -80°C refrigerator at 25°C every 2 hours, and cut the banana tissue into slices of 25mm×20mm×5mm in length×width×height using an oscillating slicer in both horizontal and vertical ways, adhere and fix them on Fisher SuperfrostPlus positively charged anti-shedding slides, and store them in a -80°C refrigerator for imaging analysis;

[0043] Take a completely unripe banana sample, immerse the bottom stalk of the whole banana fruit in the prepared mixed solution for 30 minutes, then take it out, put it in a -80℃ refrigerator after an interval of 12 hours at 25℃, and then use a slicer to cut the tissue section horizontally from the top of the banana, place it on a slide and store it in a -80℃ refrigerator until analysis;

[0044] Take banana samples of 20%, 60%, and 100% different degrees of maturity, immerse them in the mixed solution for 30 minutes and then take them out, freeze the fresh samples in a -80°C refrigerator at 25°C every 2 hours, and use a slicer to make longitudinal slices of 25mm×20mm×5mm in length×width×height, adhere the longitudinal slices to a glass slide and seal and freeze them until detection and analysis (due to the respiratory transition of bananas, they soften rapidly after maturity, and the moisture and viscosity increase. Directly using fresh samples to prepare slices will cause sample damage during the experiment. On the other hand, due to the increase in sugar content, the frozen ripe bananas melt too quickly and produce a large amount of moisture, resulting in changes in morphology. Therefore, the longitudinal slices prepared are freeze-dried at -80°C using a freeze dryer (Beijing Sihuan Technology Instrument Factory Co., Ltd., Beijing). In order to avoid moisture affecting the experimental results, the freeze-dried samples are sealed and frozen until detection and analysis);

[0045] Step 2: Mass spectrometry imaging

[0046] First, install and fix the spray needle, then connect the electrospray needle gas passage (Sprayl gas) and liquid passage (Spray the solvent), let N2 into the gas passage, and the liquid solvent in the liquid passage is methanol water containing 0.1% formic acid. Turn on the connected vacuum pump, fix the ion transfer tube (Ion transfer tube) to the interface of Q Exactive plus electrostatic field orbital trap high-resolution mass spectrometer (Thermo, Germany), and be identified by the mass spectrometer through the ion source identifier;

[0047] Adjust the movable loading platform to place the glass slide with the banana sample attached in an appropriate position, and adjust the position of the electrospray needle;

[0048] By applying high voltage to the liquid path, the spray solvent is charged (Charged solvent spray). Under the action of high-speed spray gas N2, the micro-flow rate liquid is atomized to form electrospray droplets. The charged droplets bombard the sample surface. Under the extraction action of methanol and water solvent, the sample undergoes desorption ionization (Molecular desorption ionization). After Coulomb rupture occurs, the sample solution is desolvated and enters the mass spectrometer interface through the ion transfer tube. Under the action of vacuum, it enters the mass spectrometer cone port for collection.

[0049] Step 3: Data Analysis

[0050] The imaging data was acquired by continuously capturing the mass spectrum (Mass spectrum acquisition) with a given number of scans. The mass spectrometry was processed using Xcalibur software. The file format was converted into CDF format. Massimager was used to visualize the mass spectrometry image and ion image data. The actual length and width of the scanned sample slice and the number of scan lines were set. The scanned image was imported, and the intensity normalization was set to the global image. The mass-to-charge ratio of thiram, propamocarb, pyraclostrobin, and imidacloprid was set with a mass-to-charge ratio tolerance of 0.05 Da to obtain the corresponding analyte image.

[0051] In the embodiment of the present invention, thiram (C6H 12 N2S4), cymoxanil (C9H 20 N2O2), pyraclostrobin (C 19 H 18 The molecular structures of the four pesticides are as follows:

[0052]

[0053] Preferably, a positive ion tuning solution is used to perform mass calibration on the positive ion detection mode of the Q Exactive plus high-resolution mass spectrometer under the data processing system of Xcalibur to ensure the accuracy of instrument data acquisition. The atmospheric pressure ion source is coupled to the Q Exactive plus high-resolution mass spectrometer (High voltage potential coupling), the various components are installed, the syringe, vacuum pump, and N2 gas line are turned on, and the ion intensities of m / z 149.023 and m / z 279.158 are the highest in the positive ion detection mode, the TCI coefficient of variation is within 10%, and the instrument is stable. By adjusting the spray voltage, spray pressure, the distance between the spray needle and the transmission port, the spray needle angle, the distance between the transmission tube collection cone, and the mobile phase, the electrospray ionization effect, the instrument detection sensitivity, and the imaging accuracy are optimized (specific optimization conditions such as Figure 5 A blank banana sample was placed on a glass slide and the total ion intensity was compared. In step 3, a 500 μm step size was used between pixels for all images.

[0054] As a preferred embodiment of the present invention, in step 2, a high voltage of 7000V is applied to the liquid path to charge the spray solvent, and under the action of 0.6Mpa high-speed spray gas N2, the micro-flow rate liquid is atomized to form electrospray droplets.

[0055] In the embodiment of the present invention, preferably, the spray voltage is directly related to the spray ionization effect, such as Figure 5b, starting from 3000V, the ionic strength is 8.13E 5 , gradually increase the voltage until 7000V, the ion strength reaches 1E 8 When the voltage is further increased to 8000V, the ion intensity of the instrument drops to 8.21E 6 This proves that under too low voltage, spray ionization cannot achieve the ideal detection effect, and when the spray voltage is too high, the excessive voltage causes discharge breakdown, turning it into a path, generating current, and preventing the ions from being fully excited. Therefore, the spray voltage is set to 7000V. On the other hand, spray pressure is also an important factor affecting ionization. First, set the spray pressure to 0.2Mpa, and the ion strength is displayed as 1.29E 6 , the spray pressure is continuously increased, and the ion strength of the instrument continues to increase. When the spray pressure reaches 0.6Mpa, the ion strength reaches 1E 8 However, considering that fresh banana slices have high moisture content and are easily damaged, excessive spray pressure will accelerate the drying of banana slices or directly cause damage to banana slices. When the spray pressure is set to 0.7Mpa, the drying degree of banana samples is accelerated ( Figure 5 e). Therefore, the spray pressure is set to 0.6Mpa.

[0056] As a preferred embodiment of the present invention, in step 2, the volume ratio of methanol to water in the methanol water is 80:20.

[0057] In the embodiment of the present invention, preferably, during the detection and analysis, the process of the main component ions and the exchange groups forming ion pairs is affected by the mobile phase, and the ionic strength is changed by adjusting the proportion of the mobile phase, so that the separation effect and selectivity are improved. In order to improve the sensitivity, 0.1% formic acid is added to methanol water. First, methanol water containing 50% methanol is selected as the mobile phase, and its ionic strength is 1.04E 5 , and continue to increase the proportion of methanol. Figure 5 a shows that when the methanol content in methanol-water reaches 70%, the response is greatly improved by continuing to increase the organic phase ratio. When methanol:water=80:20 (v / v), the instrument response intensity reaches 1E 8 , the instrument response value dropped significantly when the proportion of methanol was further increased. This may be due to the high voltage effect. The excessively high organic phase is more likely to volatilize, resulting in a decrease in the sensitivity of the detection. Therefore, the mobile phase was determined to be methanol: water = 80:20 (v / v).

[0058] As a preferred embodiment of the present invention, in the step 2, the spray solvent flow rate of the electrospray needle is 5 μL / min.

[0059] As a preferred embodiment of the present invention, in step 2, the suction flow rate of the vacuum pump is 50 L / min.

[0060] As a preferred embodiment of the present invention, in step 2, the length of the ion transfer tube is 50 cm, and the distance between the ion transfer tube and the conical collection port is 2.5 mm.

[0061] In the embodiment of the present invention, preferably, when the ions to be detected pass through the ion transfer tube to reach the mass spectrometer collection port, changing the length of the transfer tube from the mass spectrometer collection cone port can also improve the instrument detection sensitivity ( Figure 5 d). When the interval distance is set to the range of 1mm to 2.5mm, the detected ion intensity will continue to increase. When the interval distance reaches 2.5mm, the ion intensity reaches 7.61E 7 , continue to increase the spacing distance. When the spacing exceeds 2.5mm, the instrument response intensity begins to gradually decrease. The reason for this is that the length of the transmission tube from the mass spectrometer collection cone is too long, resulting in the inability of the analyte ions to fully enter the ion transmission tube, and the detected ion intensity is low.

[0062] As a preferred embodiment of the present invention, in step 2, the scanning speed of the loading platform is 0.5 mm / s, and the line spacing is 0.5 mm.

[0063] As a preferred embodiment of the present invention, the capillary in the electrospray needle extends 0.1 mm, the electrospray needle is arranged at an angle of 60° to the sample, and the electrospray needle is arranged at a distance of 1.5 mm from the ion transfer tube.

[0064] In the embodiment of the present invention, preferably, during the process from excitation to collection, the sample will pass through various parts, and the charged droplets sprayed by the spray needle will bombard the sample surface. In this part, the angle between the spray needle and the sample determines the ion intensity. When the angle between the electrospray needle and the sample is 30°, the detected ion intensity is only 1.12E 6 , gradually increasing the angle, when the angle between the electrospray needle and the sample is 60°, the ion intensity reaches 2.75E 7 , continue to increase the angle, and find that the response value begins to decrease ( Figure 5 f), this may be because too high or too low an injection angle will cause incomplete ion sputtering into the ion transfer tube. When the charged droplets bombard the sample, another factor that affects the ion entry into the transfer tube is the distance between the spray needle and the transfer port. Figure 5 c It can be seen that when the spacing is set between 0.5mm and 1.5mm, the detected ion intensity continues to increase. When the distance between the spray needle and the inlet of the ion transmission tube is adjusted to exceed 1.5mm, the ion intensity begins to decrease and reaches the maximum response value of 3.21E when the spacing is 1.5mm. 7This is because if the distance is too close, the ions cannot fully enter the transmission tube, and if the distance is too far, the ions cannot enter the transmission tube.

[0065] Results and discussion

[0066] (1) Confirmation of in situ mass spectrometry imaging detection

[0067] Bananas that have been soaked in the pesticide mixture and then frozen were sliced ​​longitudinally every 2 hours using a slicer to make banana tissue longitudinal slices with a length × width × height of 25 mm × 20 mm × 5 mm, so that the slices included both the banana peel and the banana flesh ( Figure 3 C. Figure 3 D) is used to observe the vertical migration of pesticides inside bananas. To further prove that pesticides can migrate inside bananas, we selected samples of the bottom of the banana stalk and soaked them in pesticides. After one day, we selected the top of the banana to ensure that it contains both the peel and the flesh, and made cross-sections ( Figure 3 A. Figure 3 B). While proving its longitudinal migration, in order to facilitate the observation of the distribution and accumulation areas of pesticides at different times, banana samples were selected to be cut horizontally to make cross-sections. In order to ensure the rapid detection of fresh samples and avoid the influence of too long detection time on accuracy, half of the banana sample cross-section was selected for scanning to ensure that the half-section included the peel and the flesh ( Figure 3 F). The prepared tissue slices are frozen and stored for testing. The prepared tissue slices are placed on the instrument loading platform, fixed, and the optimal parameters for debugging are set. During the detection process, the ion fragmentation diagram of the object to be tested can be seen in the mass spectrum ( Figure 4 ), m / z 240.995 is thiram, m / z 388.105 is pyraclostrobin, m / z 189.159 is propamocarb, and m / z 256.059 is imidacloprid. Through Massimager software processing, a visualized image of the banana sample was obtained.

[0068] (2) Effect of systemicity on pesticide migration

[0069] After application, systemic pesticides will produce systemic and conduction behaviors in plants. As a property of systemic pesticides, it is not possible to see it intuitively. Therefore, we compared the typical non-systemic dithiocarbamate fungicide thiram with the systemic carbamate fungicide cymoxanil, the nitromethylene insecticide imidacloprid, and the strobilurin fungicide pyraclostrobin to visualize the impact of systemic pesticide migration patterns.

[0070] First, the sample of the bottom end of the banana stalk soaked in pesticide was selected for scanning imaging. According to the mass spectrometry imaging of the top slice ( Figure 6), and found that after one day, even if the peel was not soaked in pesticides and the top part had no contact with pesticides, pesticides were still detected. This proves that the pesticides enter through the bottom handle and gradually migrate to the top. The three systemic pesticides have residues in the peel and flesh, while a small amount of non-systemic thiram can be observed in the peel. This may be because the fiber tubular structure of the banana peel has a conductive effect, and a small part of the flowing pesticide solution will be transferred to the top through the fiber tube. This further proves that pesticides can migrate vertically inside the banana.

[0071] In order to further demonstrate the migration and distribution of different pesticides in bananas, longitudinal sections of banana tissues were selected when the peel was soaked in pesticides ( Figure 7 A) Scanning, the three systemic pesticides all began to migrate inwards at 2 hours, and the longitudinal migration of pesticides to the banana core was clearly observed at 6 hours. Pesticide residues were observed in the banana core at 8 hours. After 2 hours, thiram still only stayed in the skin and did not enter the banana. After 4-8 hours, the pesticides in the skin gradually decreased. It may be that some pesticides evaporated with water in the air, and a small amount of pesticides degraded. Then, we took a transverse half-slice and scanned it according to Figure 7 B shows that after 2-8 hours, no thiram was detected inside the banana, while the three systemic pesticides continued to diffuse from the banana peel to the inside with the increase of time. This shows that the pesticides that can enter the banana will continue to penetrate horizontally as time goes by while migrating vertically, while non-systemic pesticides will not remain in other tissue parts except for spraying in any way. Therefore, systemic pesticides enter the banana through vertical migration and horizontal penetration.

[0072] (3) Effect of octanol ratio on pesticide migration

[0073] Since non-systemic pesticides do not enter the plant body, systemic pesticides will have regular vertical migration in the plant body, so further analysis of the migration of different systemic pesticides in bananas is carried out to explore the law of pesticide transmission. The conduction of systemic pesticides in plants is affected by the structural type of the target compound, which is often divided into hydrophilic compounds and lipophilic compounds.

[0074] like Figure 6 and Figure 7It can be seen that at the same time, the migration content of cymoxanil is significantly greater than that of imidacloprid and pyraclostrobin, and the migration speed is also faster than that of imidacloprid and pyraclostrobin. The speed and content of lateral penetration of cymoxanil in 2h are also the largest. According to the comparison, the internal pesticide concentration, content and migration speed at the same time are: cymoxanil> imidacloprid> pyraclostrobin. Therefore, there are differences in migration between different pesticides. The n-octanol-water partition coefficient of organic compounds is an important parameter reflecting the migration ability between the aqueous phase and the organic phase, which is used to characterize the distribution of pesticides between lipids and water. The migration of organic matter is largely related to its partition coefficient. The larger the value of the partition coefficient, the greater the solubility of the organic matter in the organic phase and the smaller the solubility in water. Conversely, the smaller the hydrophilic organic phase partition coefficient. Studies have shown that when pesticides are absorbed by plants, hydrophilic pesticides can be transported and migrate inside the plants. Comparing the distribution coefficients of the three systemic pesticides, propamocarb logKow = 0.0018, imidacloprid logKow = 0.57, and pyraclostrobin logKow = 3.99. According to the distribution coefficient logKow comparison, propamocarb < imidacloprid < pyraclostrobin, so the migration rate and content of propamocarb are greater than imidacloprid and greater than pyraclostrobin. Therefore, the reason for the difference in pesticide migration is the difference in the proportional distribution coefficients of pesticides in water and n-octanol.

[0075] (4) Effect of relative molecular mass on pesticide migration

[0076] After the banana peel is soaked in pesticides, the small molecules of pesticides will enter the inside through the pores on the surface of the banana peel under the action of systemic absorption. The smaller the relative molecular mass, the faster its movement speed, and the easier it is for the pesticide to pass through the aperture and enter the inside of the fruit. The relative molecular mass of propamocarb is 189.159, the relative molecular mass of imidacloprid is 256.059, and the relative molecular mass of pyraclostrobin is 388.105. Comparing the relative molecular mass, propamocarb is easier to penetrate into the banana. In comparison, the penetration ability of pyraclostrobin is poor, which is consistent with our experimental results.

[0077] Based on the above conclusions, it can be inferred that the migration of systemic pesticides in bananas is affected by the relative molecular mass of the pesticides and the water-n-octanol ratio distribution coefficient. The larger the distribution coefficient, the smaller the systemic effect, the less the pesticide migration content, and the slower the migration speed. Pesticides with smaller relative molecular mass are easier to penetrate the interior, and the migration speed and content are also higher.

[0078] (5) Effect of banana maturity on pesticide migration

[0079] In order to observe the differences in the migration patterns of pesticides in bananas of different maturity, we selected bananas of 20%, 60%, and 100% maturity ( Figure 8 ) samples, and perform mass spectrometry imaging scanning detection ( Fig. 9). At 2h, a small part of the three pesticides began to migrate in the green banana, while the pesticides in the fully ripe banana had gradually migrated to the inside. At 4h, the 100% mature banana almost completely migrated to the inside, while the 20% mature banana only migrated slowly at 4h. At 8h, the pesticides in the green banana migrated to the center of the banana and the accumulation of pesticides was observed to decrease. It is possible that some pesticides degraded over time, while the ripe bananas could still cause a large amount of residue accumulation after migrating to the center. According to the mass spectrometry imaging diagram, under the same maturity conditions, the three pesticides still migrated according to the law of systemic absorption, and for the same pesticide, the more mature the banana, the greater the accumulation of pesticide residues, the faster the coverage speed, and the slower the degradation speed. The reason for this difference is that as the banana continues to mature, starch is converted into sugar, and the internal sugar increases significantly, which can promote the flow of pesticides. Therefore, it can be inferred that the content of sugar substances produced inside the banana during the ripening process will affect the migration of internal pesticides.

[0080] Comparing different pesticides, regardless of the maturity of bananas, the migration speed of propamocarb in bananas is faster than that of imidacloprid and pyraclostrobin, and the degree of systemic absorption also determines the migration speed and accumulation content of pesticides in bananas. Comparing the maturity, regardless of the systemicity of the pesticide, the more mature the banana, the faster its migration speed, the more accumulated content, and the slower its degradation.

[0081] in conclusion

[0082] The present invention uses mass spectrometry imaging technology to visualize the residual accumulation and migration of four pesticides, namely, thiram, propamocarb, imidacloprid, and pyraclostrobin, in bananas. By debugging and optimizing the normal pressure MSI imaging system, it is more suitable for the detection of exogenous small molecules in fresh plants, and an in-situ imaging detection method for four residual pesticides in fresh bananas based on mass spectrometry imaging technology is established, which clarifies that propamocarb, imidacloprid, and pyraclostrobin mainly migrate vertically and penetrate into the interior of bananas horizontally.

[0083] The present invention further studies the factors that affect the migration of pesticides, and finds that systemicity, water-octanol ratio distribution coefficient, relative molecular mass and the maturity of bananas themselves are all important factors that change the migration and accumulation of pesticides. Pesticides can be absorbed into the banana under the influence of systemicity. The smaller the water-octanol ratio distribution coefficient and relative molecular mass of the pesticide, the greater its migration rate and content. The sugar produced by bananas during the ripening process can accelerate the migration of internal pesticides, resulting in residual accumulation. In general, the present invention provides a new model to study drug transfer and residues in fresh plants after pesticide application, provides a reference for pesticide safety evaluation, and promotes the formulation of reasonable pesticide application.

[0084] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging, characterized in that: The following steps are involved: Step 1: Sample preparation Four pesticides, namely, thiram, propamocarb, pyraclostrobin and imidacloprid, were weighed respectively, and prepared into equal concentration solutions with ultrapure water. After being diluted 1000 times with ultrapure water, the solutions were uniformly mixed to prepare a pesticide mixture. Banana samples of different maturity were obtained from a banana base, and bananas of the same maturity were ensured to be from the same batch. The dirt on the surface of the banana samples was cleaned with ultrapure water, and the cycle was repeated three times. Take a completely unripe banana sample, fully immerse it in the mixed pesticide solution and take it out after 30 minutes, place it in a 25℃ room, and freeze it in a -80℃ refrigerator every 2 hours. After freezing, use an oscillating slicer to cut the banana tissue into slices of 25 mm × 20 mm × 5 mm in length × width × height in both horizontal and vertical ways, adhere and fix them on a positively charged anti-shedding slide, and store them in a -80℃ refrigerator for imaging analysis; Take a completely unripe banana sample, immerse the bottom stalk of the whole banana fruit in the prepared mixed solution, take it out after 30 minutes, place it at 25℃ for 12 hours, and then put it in a refrigerator at -80℃. Use a slicer to cut tissue sections horizontally from the top of the banana, place them on a slide and freeze them in a refrigerator at -80℃ until analysis; Take banana samples of 20%, 60%, and 100% different degrees of maturity, immerse them in the mixed solution, take them out after 30 minutes, place them at 25℃, freeze the fresh samples in a -80℃ refrigerator every 2 hours, use a slicer to make longitudinal slices of 25 mm × 20 mm × 5 mm in length × width × height, adhere the longitudinal slices to a glass slide, seal and freeze them until detection and analysis; Step 2: Mass spectrometry imaging First, install and fix the spray needle, then connect the gas passage and liquid passage of the electrospray needle, let N2 into the gas passage, and the liquid solvent in the liquid passage is methanol water containing 0.1% formic acid. Turn on the connected vacuum pump, fix the ion transfer tube to the interface of the electrostatic field orbital trap high-resolution mass spectrometer, and identify it by the mass spectrometer through the ion source identifier; Adjust the movable loading platform to place the glass slide with the banana sample attached in an appropriate position, and adjust the position of the electrospray needle; By applying high voltage to the liquid path, the spray solvent is charged. Under the action of high-speed spray gas N2, the micro-flow rate liquid is atomized to form electrospray droplets. The charged droplets bombard the sample surface. Under the extraction action of methanol and water solvent, the sample is desorbed and ionized. After Coulomb rupture occurs, the sample solution is desolvated and enters the mass spectrometer interface through the ion transfer tube. Under the action of vacuum, it enters the mass spectrometer cone port for collection. Step 3: Data Analysis Imaging data was acquired by continuously capturing mass spectra with a given number of scans, and Xcalibur software was used for mass spectrometry processing. The file format was converted into CDF format, and Massimager was used for visualization of mass spectrometry images and ion image data. The actual length and width of the scanned sample slices and the number of scan lines were set, and the scanned images were imported. The intensity normalization was set to a global image, and the corresponding analyte images were obtained by setting the mass-to-charge ratios of thiram, propamocarb, pyraclostrobin, and imidacloprid with a mass-to-charge ratio tolerance of 0.05 Da.

2. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 1, characterized in that: In the step 2, a high voltage of 7000V is applied to the liquid path to charge the spray solvent, and the micro-flow rate liquid is atomized to form electrospray droplets under the action of 0.6Mpa high-speed spray gas N2.

3. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 2, characterized in that: In the step 2, the volume ratio of methanol to water in the methanol water is 80:

20.

4. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 3, characterized in that: In the step 2, the spray solvent flow rate of the electrospray needle is 5 μL / min.

5. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 4, characterized in that: In the step 2, the suction flow rate of the vacuum pump is 50 L / min.

6. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 5, characterized in that: In the step 2, the length of the ion transfer tube is 50 cm, and the distance between the ion transfer tube and the mass spectrometer cone is 2.5 mm.

7. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 6, characterized in that: In the step 2, the scanning speed of the loading platform is 0.5 mm / s, and the line spacing is 0.5 mm.

8. The method for identifying the distribution of pesticide residues in plant tissues based on in situ mass spectrometry imaging according to claim 4, characterized in that: The capillary in the electrospray needle extends 0.1 mm, the electrospray needle is arranged at an angle of 60° to the sample, and the electrospray needle is arranged at a distance of 1.5 mm from the ion transfer tube.

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