Method for detecting succinate dehydrogenase bactericide by using solid-phase extraction technology in combination with liquid chromatography-tandem mass spectrometry

Abstract

According to the method for detecting the succinate dehydrogenase bactericide by using the solid-phase extraction technology in combination with the liquid chromatography-tandem mass spectrometry, after a sample to be detected is extracted, HLB solid-phase purification is adopted, the optimal solvent composition of a sample loading solution and a leacheate in the solid-phase extraction process is optimized, and interference of impurities is effectively reduced; detecting by combining an ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method, especially the ultra-high performance liquid chromatography of a biphenyl chromatographic column, so as to obtain the content of the succinate dehydrogenase bactericide in the to-be-detected sample solution. The detection method can comprehensively cover all fruit categories in GB 2763-2021, and under the condition that the detection limit of the fruit matrix corresponding to the lowest sensitivity of nine SDHIs bactericides is taken as the detection limit of the method, the technology shows more excellent sensitivity than the existing research.

Description

Technical Field

[0001] The present application belongs to the technical field of agricultural product quality and safety detection, and in particular, relates to a method for detecting succinate dehydrogenase fungicide by using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry. Background Art

[0002] In agricultural production, succinate dehydrogenase inhibitors (SDHIs) are widely used as a new type of amide broad-spectrum fungicide to prevent and control diseases caused by plant pathogenic fungi. Although the toxicity of SDHIs is relatively low, due to its large and widespread use, residues are prone to accumulation in fruits and environmental water. This not only increases the exposure risk of humans and non-target organisms, but also causes a series of toxicity problems, and therefore has received widespread attention from the society. For example, from 2014 to 2019, the global market sales share of SDHIs fungicides increased significantly to 12.7%, with a compound growth rate of 8.5%. The substantial increase in its use has made the residue status in crops and the potential threat to the ecological environment and human health a focus issue. Commonly used SDHIs fungicides include 9 compounds such as fluopyram, fluopyram, fluoxazolidinone, fluopyram, boscalid, penthiopyrad, bixafen, benzovintriazole and pyraclostrobin.

[0003] In terms of relevant technological development, common methods for the pretreatment of SDHIs fungicides include solid phase extraction (SPE) and dispersed solid phase extraction (QuEChERS). The QuEChERS method is simple to operate, fast and convenient, but the SPE method is more outstanding in terms of enrichment efficiency, impurity removal ability and stability, and is especially suitable for the detection of trace target compounds. In terms of determination methods, immunoassay, gas chromatography, liquid chromatography, gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry all have application examples. However, immunoassay, gas chromatography and liquid chromatography have the disadvantages of complex pretreatment, low sensitivity and weak qualitative ability, and are prone to false positive results under the interference of impurities. In contrast, liquid chromatography-tandem mass spectrometry has broad prospects for application in the field of SDHIs fungicide analysis due to its high sensitivity and strong qualitative and quantitative capabilities. Despite this, there is currently a lack of analytical methods for SDHIs fungicides in all major fruit categories in GB2763-2021. Therefore, developing an accurate, sensitive and stable analytical method that can simultaneously determine SDHIs fungicides in multiple fruits has important practical significance and urgent market demand. Summary of the invention

[0004] The present application provides a method for detecting succinate dehydrogenase fungicide by using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry to solve the problems existing in the related technology. The technical solution is as follows:

[0005] In a first aspect, the present application provides a method for detecting succinate dehydrogenase fungicide using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry, comprising the following steps:

[0006] The solution to be tested is separated and purified by an HLB solid phase extraction column, and then detected by ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry to obtain the content of multiple succinate dehydrogenase bactericides in the sample solution to be tested;

[0007] Among them, the chromatographic column of ultra-high performance liquid chromatography is a biphenyl chromatographic column.

[0008] Furthermore, the test solution is obtained by extracting the test sample by liquid-liquid extraction and salting-out method; the test sample includes all major fruit categories in GB 2763-2021.

[0009] Furthermore, the sample to be tested is any one of tangerine, pear, apple, fresh jujube, tree tomato, mango, banana or honey melon.

[0010] Further, the succinate dehydrogenase fungicide is one or a combination of two or more of fluopyram, fluopyram, fluoxazolidinone, fluopyram, boscalid, penthiopyrad, bixafen, benzovintriazole or pyraclostrobin.

[0011] Furthermore, after the sample to be tested is dispersed in water, it is extracted using an extractant, salt is added for salting out, and separation is performed to obtain the solution to be tested.

[0012] Furthermore, the extractant is acetonitrile; and the added salt is sodium chloride.

[0013] Further, the method of separation and purification using the HLB solid phase extraction column is:

[0014] The solution to be tested is adsorbed and analyzed by an HLB solid phase extraction column to obtain a purified sample; the eluent used for the analysis is acetonitrile.

[0015] Furthermore, before adsorption, the HLB solid phase extraction column is activated using methanol and water in sequence.

[0016] Furthermore, the mass volume ratio of the sample to be tested to water is 1 g: 0.5-1.5 mL; the mass volume ratio of the sample to be tested to the extractant is 1 g: 1-2 mL; and the amount of salt added is 0.5-0.7 times the mass of the sample to be tested.

[0017] Furthermore, the separation method is centrifugation at 3500-4500r / min for 1-3min.

[0018] Further, after adsorption, 18-23% acetonitrile was used for elution; and then acetonitrile was used for elution.

[0019] Furthermore, the conditions of the ultra-high performance liquid chromatography are:

[0020] Chromatographic column 2.1mm×100mm, 2.6μm;

[0021] Mobile phase: A is 0.1% formic acid water, B is 0.1% formic acid acetonitrile; gradient elution;

[0022] Flow rate: 0.25 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0023] Further, the gradient elution program was: 0-2.0 min: 70%-40% A, 2.0-7.0 min: 40%-30% A, 7.0-11.0 min: 30% A; 11.0-11.1 min: 30%-70% A; 11.1-15.0 min: 70% A.

[0024] Furthermore, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method include:

[0025] Electrospray ion source; curtain gas: 35 psi; collision gas: 9 psi; spray voltage: 4500 V; evaporation temperature: 400°C; evaporation gas: 65 psi; auxiliary gas: 60 psi; scanning mode: ESI+, multiple reaction detection.

[0026] The advantages or beneficial effects of the above technical solution include at least:

[0027] The present application discloses a method for detecting succinate dehydrogenase fungicides using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry, wherein a variety of succinate dehydrogenase fungicides are detected by ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry using HLB solid phase extraction column separation and biphenyl chromatography column, and the detection samples can cover all major fruit categories in GB 2763-2021, establishing a detection method with a wide range of applicable matrices; the interference of impurities can be effectively reduced, and the separation and detection effects of various succinate dehydrogenase fungicides are better; the detection of 9 succinate dehydrogenase inhibitors has higher sensitivity and accuracy, and the detection limits (LODs) are 0.10-0.90 μg / kg.

[0028] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of 9 SDHIs fungicide compounds in C 18Quantitative ion diagram of the chromatographic column and biphenyl column (10 μg / L); (a) is C 18 ; (b) Biphenyl;

[0030] Figure 2 is the average recovery rate of 9 SDHIs fungicide compounds in citrus matrix in different extraction solvents (n=6);

[0031] Figure 3 The average recovery rates of 9 SDHIs fungicides in citrus matrix at different extractant dosages (n=6)

[0032] Figure 4 The average recoveries of 9 SDHIs fungicide compounds in citrus matrix at different extraction times (n=6). DETAILED DESCRIPTION

[0033] Hereinafter, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the following description is considered to be exemplary and non-restrictive in nature.

[0034] There are few existing studies on the detection of succinate dehydrogenase inhibitors in fruits using solid phase extraction purification technology combined with liquid chromatography tandem mass spectrometry, and there are problems such as failure to cover all major fruit categories in GB 2763-2021, failure to remove impurities well during the purification process, or loss of target compounds; and low sensitivity of liquid chromatography detection. Therefore, the present application provides a method for detecting succinate dehydrogenase fungicides using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry.

[0035] A method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry comprises the following steps:

[0036] The solution to be tested is separated and purified by an HLB solid phase extraction column, and then detected by ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry to obtain the content of multiple succinate dehydrogenase bactericides in the sample solution to be tested;

[0037] Among them, the chromatographic column of ultra-high performance liquid chromatography is a biphenyl chromatographic column.

[0038] This technology uses HLB solid phase extraction to achieve the adsorption and removal of target compounds and impurities based on the principle of the difference in hydrophilic and hydrophobic interaction forces. Since the desorption capacity of the target compound on the solid phase extraction column increases with the decrease in solvent polarity, the solvent composition of the loading solution and the eluent has a significant effect on the retention of the target compound. Adjusting the organic phase content in the loading solution and the eluent can improve the recovery rate through HLB extraction, maximize the removal of interferences to achieve the best purification effect, and effectively reduce the interference of impurities in subsequent ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry detection.

[0039] The ultra-high performance liquid chromatography selected a biphenyl column, based on the fact that all nine compounds contain aromatic ring structures, and the phenyl filler can retain the target compound through π-π interactions. 18 Chromatographic column, the biphenyl chromatographic column has a stronger retention ability for the target compound, prolongs the retention time of the 9 compounds, can better separate the interfering matrix from the target, avoid peak broadening and tailing, and improve the separation degree. Therefore, the HLB solid phase extraction column is used for separation and purification and ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry is used for detection in this application, which can comprehensively cover all major fruit categories in GB 2763-2021; under the condition that the detection limit of the fruit matrix corresponding to the lowest sensitivity of each compound is used as the method detection limit, it shows a better sensitivity than the existing research. Preferably, the biphenyl chromatographic column selects the Phenomenon Kinetex Biphenyl chromatographic column.

[0040] As one implementation mode, the test solution is obtained by extracting the test sample through liquid-liquid extraction and salting-out method.

[0041] As one embodiment, the sample to be tested is dispersed in water, extracted with an extractant, salt is added for salting out, and separated to obtain the solution to be tested.

[0042] The sample to be tested is extracted by liquid-liquid extraction, so that the SDHIs fungicide is transferred from the sample matrix to the liquid phase. Salt is added for salting out. Adding salt can promote the separation of the aqueous phase and the organic phase, reduce emulsification, and ensure the smooth progress of the extraction process; at the same time, salt can change the ionic strength of the solution, which helps the target to dissolve better in the organic phase.

[0043] As one embodiment, the extractant is acetonitrile; and the added salt is sodium chloride.

[0044] Acetonitrile is a good solvent for SDHIs fungicides. Sodium chloride can change the ionic strength of the solution, which helps the target compound to dissolve better in the acetonitrile phase.

[0045] As one embodiment, the mass volume ratio of the sample to be tested to water is 1 g: 0.5-1.5 mL; the amount of salt added is 0.5-0.7 times the mass of the sample to be tested.

[0046] As one embodiment, the mass volume ratio of the sample to be tested to the extractant is 1 g: 1-2 mL.

[0047] As one embodiment, the separation method is centrifugation at 3500-4500 r / min for 1-3 min.

[0048] As one embodiment, the solid after centrifugal separation is repeatedly extracted, salted out and separated, and the separated liquids are combined.

[0049] After two extractions, the extraction effect of SDHIs fungicides can be significantly improved.

[0050] As one embodiment, the separation liquid is used to remove the acetonitrile solution, and the solution to be tested is obtained by re-dissolving with 18-23% acetonitrile solution. In this embodiment, the acetonitrile solution in the separation liquid is blown dry with a nitrogen blower to remove the acetonitrile to avoid changes in the target compound therein. The sample is re-dissolved with 18-23% acetonitrile to ensure that the 9 SDHIs fungicides are effectively re-dissolved and not eluted in the solid phase extraction column, while the interfering matrix is ​​appropriately removed.

[0051] The test solution is then re-dissolved in 20% acetonitrile solution as the loading solution for HLB solid phase extraction. If the organic phase content of the loading solution is too low, the SDHIs compound cannot be effectively dissolved, while if the organic phase content is too high, these compounds may be eluted during the column loading process, thereby reducing the recovery rate. Therefore, the degree of purification and the recovery rate can be improved.

[0052] As one embodiment, the method for separation and purification by HLB solid phase extraction column is:

[0053] The solution to be tested is adsorbed and analyzed by an HLB solid phase extraction column to obtain a purified sample; the eluent used for the analysis is acetonitrile.

[0054] HLB solid phase extraction column was used for purification, and HLB column has the characteristics of hydrophilic-lipophilic balance. Since the oil-water distribution coefficient of the target compounds of the 9 SDHIs compounds is 3.11-4.69 (pH 7.4), they have a certain lipophilicity and can form an adsorption effect with the non-polar stationary phase in the HLB column, thereby effectively removing impurities in the fruit matrix, such as fiber, organic acid, sugar and pigment.

[0055] As one embodiment, before adsorption, the HLB solid phase extraction column is activated using methanol and water in sequence.

[0056] Methanol and water were used to activate the HLB solid phase extraction column to put the filler in a suitable adsorption state.

[0057] As one embodiment, after adsorption, 18-23% acetonitrile is used for elution; and then acetonitrile is used for elution.

[0058] For the eluent, when the organic phase content is low, most of the interferences will be retained on the solid phase extraction column; only by selecting the appropriate organic phase content can the interferences be removed to the maximum extent while ensuring the recovery rate of the target, thereby achieving the best purification effect. This application optimizes the optimal solvent composition of the sample solution and the eluent during the HLB solid phase extraction process, effectively reducing the interference of impurities.

[0059] As one embodiment, after the acetonitrile is removed from the eluent, the eluent is re-dissolved in a 50% acetonitrile aqueous solution.

[0060] As one embodiment, the reconstituted solution is passed through a needle-type nylon filter membrane for separation and detection.

[0061] The present application realizes the adsorption and removal of target substances and impurities through the HLB solid phase extraction column based on the principle of difference in hydrophilic and hydrophobic interaction forces; optimizes the optimal solvent composition of the loading liquid and the eluent during the solid phase extraction process, and effectively removes impurities such as fiber, organic acids, sugars and pigments in fruits.

[0062] As one embodiment, the samples to be tested include all major fruit categories in GB 2763-2021.

[0063] As one embodiment, the sample to be tested is any one of tangerine, pear, apple, fresh date, tree tomato, mango, banana or honey melon.

[0064] The influence of the matrix effects of eight kinds of fruits, including tangerines, pears, apples, fresh dates, tree tomatoes, mangoes, bananas, and honeydew melons, was investigated, covering all major fruit categories in GB 2763-2021, including the more representative citrus fruits, pome fruits, stone fruits, berries and other small fruits, tropical and subtropical fruits, and melon fruits.

[0065] As one embodiment, the succinate dehydrogenase fungicide is one or a combination of two or more of fluopyram, fluopyram, fluoxazolidinone, fluopyram, boscalid, penthiopyrad, bixafen, benzovintriazole or pyraclostrobin. The above nine succinate dehydrogenase fungicides are broad-spectrum fungicides commonly used in fruits to prevent and treat diseases caused by plant pathogenic fungi. The residual detection of the above nine SDHIs fungicides can effectively ensure the quality and safety of agricultural products and the health rights and interests of consumers.

[0066] As one embodiment thereof, the conditions of the ultra-high performance liquid chromatography are:

[0067] Chromatographic column 2.1mm×100mm, 2.6μm;

[0068] Mobile phase: A is 0.1% formic acid water, B is 0.1% formic acid acetonitrile; gradient elution;

[0069] Flow rate: 0.25 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0070] As one embodiment, the gradient elution program is: 0-2.0 min: 70%-40% A, 2.0-7.0 min: 40%-30% A, 7.0-11.0 min: 30% A; 11.0 -11.1 min: 30%-70% A; 11.1-15.0 min: 70% A.

[0071] In the prior art, when SDHIs fungicide residue detection is performed using ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry, the chromatographic column of the ultra-high performance liquid chromatography is generally C 18 column, but using C 18 The chromatographic column was used for separation, and the retention times of the nine compounds were all short, the separation effect between the interfering matrix and the target was poor, the subsequent quantitative accuracy was low, and the overall sensitivity of the detection was not high.

[0072] Therefore, the present application uses a biphenyl chromatographic column, corresponding to 9 compounds containing aromatic ring structures, and the phenyl filler can be retained with the target compound through π-π interactions and the like; the target compound retention ability is stronger, the retention time of the 9 compounds is extended, and the interfering matrix can be better separated from the target, avoiding peak broadening and tailing, and improving the separation degree. At the same time, based on the biphenyl chromatographic column, the present application uses the above-mentioned ultra-high performance liquid chromatography conditions and gradient elution procedures, and the interfering matrix is ​​separated from the target more thoroughly, the peak shape is good and there is no tailing, which effectively improves the separation degree.

[0073] As one embodiment thereof, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method include:

[0074] Electrospray ion source; curtain gas: 35 psi; collision gas: 9 psi; spray voltage: 4500 V; evaporation temperature: 400°C; evaporation gas: 65 psi; auxiliary gas: 60 psi; scanning mode: ESI+, multiple reaction detection.

[0075] Liquid chromatography separates target compounds and multiple matrices, while mass spectrometry's multiple reaction monitoring (MRM) is a highly sensitive and selective scanning analysis technique that is particularly suitable for the quantitative analysis of target compounds in complex samples. In MRM mode, the target compounds in the sample are first ionized by an electrospray ion source (ESI) to form charged ions. After ionization, these ions are introduced into the mass spectrometer. In MRM mode, the mass spectrometer sets specific parent ion and daughter ion pairs. Parent ions refer to ions that are selected and collided in mass spectrometry analysis, while daughter ions are fragment ions produced after collision of parent ions. By selecting specific parent ion and daughter ion pairs, the MRM mode can significantly improve the detection specificity of target compounds, eliminate interference, and reduce background noise.

[0076] The following is a further description with reference to specific embodiments.

[0077] The relevant mass spectrometric information of the 9 SDHIs compounds detected in the examples of this application is shown in Table 1.

[0078] Table 1 Mass spectrometry information of 9 SDHIs compounds

[0079]

[0080]

[0081] *Quantitative ion

[0082] Calculation method of the content of 9 SDHIs fungicides in samples:

[0083] The blank content must be deducted from the calculated results.

[0084] in:

[0085]

[0086] X——the content of SDHIs fungicide in the sample, μg / kg;

[0087] C——sample concentration read from blank sample matrix curve, μg / L;

[0088] V——fixed volume, mL;

[0089] m——the mass of the sample weighed, g;

[0090] f——dilution multiple.

[0091] Example 1

[0092] Weigh 5.00 g of citrus sample into a 50 mL centrifuge tube, add 5 mL of ultrapure water to disperse the sample into the centrifuge tube, vortex mix, add 3 g of sodium chloride and 10 mL of acetonitrile into the centrifuge tube, shake and extract at 300 r / min for 20 min using a shaker, preliminarily extract 9 succinate dehydrogenase inhibitors into the acetonitrile phase, centrifuge at 4000 r / min for 1 min, transfer the supernatant (acetonitrile phase) to another 50 mL centrifuge tube, repeat the above steps, use 10 mL of acetonitrile to extract once and combine the supernatants, use a nitrogen blower to dry the acetonitrile solution in the centrifuge tube, re-dissolve with 20% acetonitrile solution, and wait for purification;

[0093] Activate the HLB column with 3 mL of methanol and 5 mL of water in turn to keep the filler in a suitable adsorption state; transfer the liquid to be purified into the HLB column in batches to allow the target compound to be adsorbed on the column; elute with 5 mL of 20% acetonitrile to remove some polar impurities to the maximum extent possible while ensuring that the target compound is not eluted, drain and elute the target compound with 6 mL of acetonitrile, and finally blow it dry with nitrogen; re-dissolve with 1 mL of 50% acetonitrile aqueous solution and pass through a needle-type nylon filter membrane for testing;

[0094] Preparation of matrix standard solution (for quantification): Accurately weigh or pipette appropriate amounts of 9 SDHIs fungicides (fluopyram, fluopyram, fluoxazolidinone, fluopyram, boscalid, penthiopyrad, bixafen, benzovintriazole and pyraclostrobin), dissolve with acetone and fix to obtain 9 standard stock solutions with a concentration of 100 mg / L, and store at -20°C. Take 1000 μL of the above 9 standard stock solutions in the same 10 mL volumetric flask, fix to volume with acetone, and obtain a 10 mg / L mixed standard intermediate solution. Use negative samples to obtain blank matrix solutions after the above treatment, and dilute the standard intermediate solutions step by step to 1.5, 2.5, 5.0, 15, 25, and 50 μg / L. Take the mass concentration of the analyte as X and its corresponding peak area as Y to fit the linear equation. Calculate the content of the 9 SDHIs fungicides in the sample by measuring the peak area of ​​the sample and substituting it into the linear equation;

[0095] Chromatographic analysis conditions: chromatographic column, Phenomenex Kinetex Biphenyl (2.1mm×100mm, 2.6μm); mobile phase: A is 0.1% formic acid water, B is 0.1% formic acid acetonitrile. Gradient elution program: 0-2.0min: 70%-40% A, 2.0-7.0min: 40%-30% A, 7.0-11.0min: 30% A; 11.0-11.1min: 30%-70% A; 11.1-15.0min: 70% A; flow rate: 0.25mL / min; column temperature: 40℃; injection volume: 5μL.

[0096] Mass spectrometry analysis conditions: electrospray ion source (ESI); Curtain Gas: 35 psi; Collision Gas: 9 psi; Ion Spray Voltage: 4500 V; Evaporation temperature (Temperature): 400°C; Evaporation gas (Ion Source Gas 1): 65 psi; Auxiliary gas (Ion Source Gas 2): 60 psi; Scan mode: ESI+, multiple reaction monitoring (MRM).

[0097] Method performance analysis:

[0098] 1. Selection of chromatographic column:

[0099] This patent compares the biphenyl chromatographic columns Phenomenon Kinetex Biphenyl (2.1 mm × 100 mm, 2.6 μm) and C 18 Chromatographic column Phenomenon KinetexC 18 (2.1mm×100mm, 2.6μm) two core-shell columns with different stationary phases; the results are as follows Figure 1 As shown, the chromatographic peaks are: 1. fluopyram, 2. fluopyram, 3-1. trans-fluopyram, 4. fluopyram, 5. boscalid, 3-2. cis-fluopyram, 6. penthiopyrad, 7. bixafen, 8. benzovintriazole, 9. pyraclostrobin.

[0100] Since all nine compounds contain aromatic ring structures, phenyl fillers can retain the target compounds through forces such as π-π interactions. 18 Compared with the Biphenyl column, the elution order of some chromatographic peaks changed, the retention times of the nine compounds were shortened, and the separation effect between the interfering matrix and the target compound became worse, indicating that the biphenyl column has better separation and detection effects on the target compounds in this experimental system.

[0101] 2. Selection of extraction solvent:

[0102] Since the matrix in citrus is mainly fiber, organic acid, sugar and pigment, etc., which has a great influence on the response of the 9 compounds, in order to ensure that better pre-treatment parameters can be obtained, citrus is used as the test matrix; the method is:

[0103] Weigh 5g of negative citrus samples, add 10μg / kg of 9 SDHIs fungicides, and prepare blank matrix standard curves for quantification. The oil-water partition coefficients Log D (pH 7.4, 3.11-4.69) of the 9 compounds show that the 9 compounds have certain lipophilicity. Therefore, acetonitrile, ethyl acetate, and n-hexane are used for extraction. When the extraction solvent is 15mL, sodium chloride is 3g, and the extraction is shaken for 20min, the average recovery rate is used as a reference index (flutrifloxacin is calculated as the sum of the cis and trans peak areas) to investigate the effect of different solvents on the recovery rate; the results are shown in Figure 2. Figure 2 shown.

[0104] from Figure 2 It can be seen that the extraction efficiency of acetonitrile is better than that of ethyl acetate and n-hexane, with a recovery rate of 81.3% to 104%. When ethyl acetate and n-hexane are used for extraction, the recovery rates are relatively low. The reason is that the polarity is quite different from that of the target, and it is difficult to effectively extract the target from the aqueous phase. At the same time, some impurities with lower polarity are extracted, which interfere with the analysis results. Acetonitrile is used as the extraction solvent.

[0105] 3. Selection of extraction solvent dosage:

[0106] Weigh 5g of negative citrus samples, add 10μg / kg of 9 kinds of SDHIs fungicides, and investigate the effect of different acetonitrile addition amounts (2.5, 5.0, 7.5, 10.0, 12.5mL) on the recovery rate. In order to extract more fully, the number of extractions was set to 2. The results are shown in Figure 3 shown.

[0107] from Figure 3 It can be seen that when the amount of extraction solvent is 10mL, the compounds except penthiopyrad and bifenthiopyrad all obtain the best recovery rate, and the average recovery rate is 86.7%-99.7%. When the amount of extraction solvent is 5mL, the recovery rate of penthiopyrad and bifenthiopyrad is the best, but the recovery rate deviation is large and the stability is poor. When the amount of extraction solvent is 12.5mL, the recovery rate of 9 compounds drops sharply, and the recovery rate is 40.0%-57.5%. It may be because the increase in the amount of solvent, more matrix interference components are extracted, and after concentration, they are combined with the target, so that the final re-dissolution sample step cannot dissolve all the compounds. Therefore, it is appropriate to use an extraction solvent with a volume of 1-2 times the sample mass. Considering all factors, 10mL of acetonitrile with a volume of 2 times the mass of the citrus sample is the optimal solvent usage.

[0108] 4. Determination of extraction times:

[0109] Weigh 5g of negative citrus samples, add 10μg / kg of 9 SDHIs fungicides, and use 10.0mL acetonitrile to extract once, twice, and three times. The effect of extraction times on the extraction rate of target compounds was evaluated by the average recovery rate of 9 SDHIs compounds. The results are shown in Figure 4 shown.

[0110] In addition to the amount of extraction solvent, the number of extractions also affects the extraction efficiency of the target compound. When the number of extractions is too small, the target component may not be fully extracted. When the number of extractions is too large, in addition to greatly reducing the experimental efficiency, more interfering substances will be extracted. Therefore, it is necessary to find a suitable number of extractions. Figure 4 In the test, when the number of extractions was 1, the target was not fully extracted and the recovery rate was low, ranging from 45.7% to 76.5%. When the number of extractions was 3, the recovery rate was 33.0% to 63.2%. The main reason was that more matrix interference components were extracted, which in turn affected the re-dissolution of the target compound, resulting in a lower recovery rate. When the number of extractions was 2, the recovery rates of the 9 SDHIs compounds were between 90.2% and 105%, which was high and met the detection requirements.

[0111] 5. Determination of solid phase extraction column type:

[0112] Accurately weigh 5g of homogenized citrus sample into a 50mL stoppered centrifuge tube, add 10mL of water, vortex to disperse the matrix, add 10mL of acetonitrile, 4g of anhydrous magnesium sulfate and 1g of sodium chloride in sequence, vortex and shake for 15min. Centrifuge at 4000r / min for 5min. Accurately transfer 5mL of acetonitrile phase to a 50mL centrifuge tube pre-filled with 1g of anhydrous magnesium sulfate, 30mg of PSA and 30mg of C 18 The solid phase extractant was placed in a 15 mL centrifuge tube, vortexed for 1 min, and the upper layer solution was placed in a 2 mL centrifuge tube and centrifuged at 10000 r / min for 5 min. 1 mL of the upper acetonitrile phase was concentrated to near dryness in a nitrogen blowpipe, and then re-dissolved with 0.5 mL of a 1:1 acetonitrile aqueous solution, and the same instrument conditions as in Example 1 were selected for detection.

[0113] Since the oil-water distribution coefficients of the nine compounds were between 3.11 and 4.69 (pH 7.4), they were lipophilic and could form a certain adsorption effect with the non-polar stationary phase. Therefore, the non-polar filler C was compared in the initial stage of the experiment. 18 The effect of solid phase extraction cartridge and HLB solid phase extraction cartridge. 18 Both the HLB solid phase extraction cartridges and the C 18 The recovery rates were 85.3%-104%, and the HLB recovery rates were 86.1%-106%. The recovery rates of the compounds were similar, but considering the product performance, HLB was better than C 18The solid phase extraction column has a larger adsorption upper limit and better dryness resistance. It can tolerate a wider pH range and has a higher operating error tolerance.

[0114] Specifically, 1 g of anhydrous magnesium sulfate, 30 mg of PSA and 30 mg of C 18 Compared with the HLB solid phase extraction column of Example 1, the solid phase extraction agent column obtained more serious sample matrix interference, and the method detection limits (LODs) of the 9 compounds were 1.00-6.00 μg / kg, and the sensitivity of the 9 compounds was lower than that of the HLB solid phase extraction column. The method detection limits of the 9 compounds in the HLB solid phase extraction column were 0.10-0.90 μg / kg, and the detection method of the HLB solid phase extraction column combined with liquid chromatography tandem mass spectrometry showed better sensitivity.

[0115] 6. Determination of sample solution and eluent:

[0116] Weigh 5g of negative citrus sample, add 10μg / kg of 9 kinds of SDHIs compounds, extract the target components according to the method of Example 1, blow the solvent to dryness with nitrogen, and then use 5mL of water, 10% acetonitrile aqueous solution, 20% acetonitrile aqueous solution, 30% acetonitrile aqueous solution, and 40% acetonitrile aqueous solution to re-dissolve the sample solution. Transfer all the sample solutions to the activated solid phase extraction column for purification (using pure water in the elution step) in batches, and then test on the machine;

[0117] After optimizing the solvent composition of the loading solution, the effects of 5 mL of water, 10% acetonitrile aqueous solution, 20% acetonitrile aqueous solution, and 30% acetonitrile aqueous solution as eluents on the recovery rate were investigated.

[0118] The results showed that when 20% acetonitrile water was used for loading, the recovery rates of the nine compounds did not decrease significantly, and the recovery rates were between 85.2% and 110%. When pure water was used to re-dissolve the target compounds and load them on the column, the recovery rates were only 64.6% to 86.4% because the target compounds could not be effectively re-dissolved; and when 30% acetonitrile aqueous solution was used for elution, the recovery rates of boscalid and flupyraclostrobin decreased significantly, by 9.4% and 18.7% respectively. Therefore, 20% acetonitrile water was used for re-dissolution and loading.

[0119] When 20% acetonitrile water was used for reconstitution and loading, the eluent was 20% acetonitrile water, and the recovery rates of the 9 compounds did not decrease significantly, ranging from 84.3% to 103%. However, when 30% acetonitrile aqueous solution was used for elution, the recovery rates of boscalid and fluopicolide also decreased significantly. Therefore, using 20% ​​acetonitrile aqueous solution as the loading solution and eluent can remove interferences to the maximum extent while ensuring the recovery rates of the 9 compounds.

[0120] HLB solid phase extraction is mainly based on the principle of difference between hydrophilic interaction force and hydrophobic interaction force to complete adsorption, so as to achieve the purpose of adsorbing target substances or impurities. Since the desorption capacity of target compounds in solid phase extraction columns increases with the decrease of solvent polarity, the solvent composition of the loading solution and the eluent will affect the retention of target compounds. When the organic phase content of the loading solution is low, the 9 SDHIs compounds cannot be effectively dissolved. When the organic phase content is too high, it will be eluted during the column loading process, reducing the recovery rate of the target compound. For the eluent, when the organic phase content is low, most of the interferences will be retained in the solid phase extraction column. The appropriate organic phase content can maximize the removal of interferences while ensuring the recovery rate of the target, thereby achieving the best purification effect.

[0121] 7. The influence of the matrix effect of 8 kinds of fruits, including tangerine, pear, apple, fresh jujube, tree tomato, mango, banana, and honeydew, was explored, covering all the major fruit categories in GB 2763-2021, including the more representative citrus fruits, pome fruits, stone fruits, berries and other small fruits, tropical and subtropical fruits, and melon fruits. The pretreatment method of Example 1 was used to obtain a blank matrix solution, prepare a matrix standard curve, and then use 50% acetonitrile aqueous solution to prepare a solvent standard curve. The ratio K was obtained by the ratio of the slope of the matrix standard curve to the slope of the solvent standard curve; when K < 0.8, it indicates the presence of matrix inhibition, and when K > 1.2, it indicates the presence of matrix enhancement; the results are shown in Table 2.

[0122] Table 2

[0123]

[0124]

[0125] The K values ​​in Table 2 show that, except for pyraclostrobin in apple matrix, matrix inhibition occurred in almost all the nine compounds in all fruit matrices to varying degrees, requiring matrix-matched standard curves for quantification.

[0126] 8. Select 8 kinds of fruit matrices, including tangerine, pear, apple, fresh jujube, tree tomato, mango, banana, and honey melon, and process and prepare mixed standard matrix curves according to the method of Example 1 to measure the samples. Take the mass concentration of the analyte as X and its corresponding peak area as Y to fit the linear equation. The instrument detection limit of different fruits under this method was determined with about 3 times the signal-to-noise ratio (S / N≈3). Combined with the pretreatment conditions, the detection limit of the fruit matrix corresponding to the lowest sensitivity of each compound was used as the method detection limit. Then the method quantitative limit was determined with about 10 times the signal-to-noise ratio (S / N≈10); the results are shown in Table 3.

[0127] Table 3

[0128]

[0129]

[0130] The nine SDHIs compounds showed good linearity within the linear range, with correlation coefficients ranging from 0.9954 to 0.9993. The method limits of detection (LODs) of the nine analytes were 0.10-0.90 μg / kg, and the limits of quantification (LOQs) were 0.30-3.0 μg / kg. 18 The high performance liquid separation of the chromatographic column has lower detection limit and quantification limit.

[0131] 9. Select 8 different fruits (tangerine, pear, apple, fresh jujube, tree tomato, mango, banana and honeydew melon) for 3-level spike recovery test. The addition levels were 1, 2 and 5 times the quantitative limit. Each addition level was repeated 6 times, and the recovery rate and precision were calculated.

[0132] Table 4

[0133]

[0134] The recoveries of the nine target compounds in different fruits ranged from 73.4% to 108%, with relative standard deviations of 1.4% to 4.9%.

[0135] In summary, the present invention discloses a method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry. HLB solid phase extraction is used to optimize the optimal solvent composition of the sample solution and the eluent during the solid phase extraction process, effectively reducing the interference of impurities; combined with ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry for detection, especially ultra-high performance liquid chromatography with a biphenyl column, the content of succinate dehydrogenase fungicide in the sample solution to be tested is obtained. This detection method can comprehensively cover all major fruit categories in GB 2763-2021, and under the condition that the detection limit of the fruit matrix corresponding to the lowest sensitivity of each compound is used as the method detection limit, this technology shows better sensitivity than existing research.

[0136] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0137] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0138] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for detecting succinate dehydrogenase fungicide using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: The solution to be tested is separated and purified by an HLB solid phase extraction column, and then detected by ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry to obtain the content of succinate dehydrogenase bactericide in the sample solution to be tested; Among them, the chromatographic column of ultra-high performance liquid chromatography is a biphenyl chromatographic column.

2. The method for detecting succinate dehydrogenase bactericide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The test solution is obtained by extracting the test sample by liquid-liquid extraction and salting-out method; the test sample includes all major fruit categories in GB2763-2021; preferably, the test sample is any one of tangerine, pear, apple, fresh date, tree tomato, mango, banana or honeydew.

3. The method for detecting succinate dehydrogenase bactericide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The succinate dehydrogenase fungicide is one of fluopyram, fluopyram, flutoxafen, fluopyram, boscalid, penthiopyrad, bixafen, benzovintriazole or pyraclostrobin, or a combination of two or more thereof.

4. The method for detecting succinate dehydrogenase bactericide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: After the sample to be tested is dispersed in water, it is extracted using an extractant, salt is added for salting out, and separation is performed to obtain the solution to be tested; The extractant is acetonitrile; the added salt is sodium chloride.

5. The method for detecting succinate dehydrogenase bactericide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The separation and purification method of HLB solid phase extraction column is: The solution to be tested is adsorbed and analyzed by an HLB solid phase extraction column to obtain a purified sample; the eluent used for the analysis is acetonitrile; Before adsorption, the HLB solid phase extraction column is activated with methanol and water in sequence.

6. The method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 4, characterized in that: The mass volume ratio of the sample to be tested to water is 1g:0.5-1.5mL; the amount of salt added is 0.5-0.7 times the mass of the sample to be tested; The separation method is centrifugation at 3500-4500r / min for 1-3min.

7. The method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 5, characterized in that: After adsorption, 18-23% acetonitrile was used for elution; then acetonitrile was used for elution.

8. The method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The conditions of the ultra-high performance liquid chromatography are: Chromatographic column 2.1mm×100mm, 2.6μm; Mobile phase: A is 0.1% formic acid water, B is 0.1% formic acid acetonitrile; gradient elution; Flow rate: 0.25 mL / min; column temperature: 40°C; injection volume: 5 μL.

9. The method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: Gradient elution program: 0-2.0 min: 70%-40% A, 2.0-7.0 min: 40%-30% A, 7.0-11.0 min: 30% A; 11.0~11.1min: 30%~70%A; 11.1~15.0min:70%A.

10. The method for detecting succinate dehydrogenase fungicide by solid phase extraction technology combined with liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method include: Electrospray ion source; curtain gas: 35 psi; collision gas: 9 psi; spray voltage: 4500 V; evaporation temperature: 400°C; evaporation gas: 65 psi; auxiliary gas: 60 psi; scanning mode: ESI+, multiple reaction detection.

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