A method for detecting succinate dehydrogenase inhibitor fungicides by using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry

Abstract

The application provides a method for detecting succinate dehydrogenase bactericides by using solid phase extraction technology combined with liquid chromatography tandem mass spectrometry. After the sample to be detected is extracted, HLB solid phase purification is adopted, the optimal solvent composition of the sample solution and the elution solution in the solid phase extraction process is optimized, and the interference of impurities is effectively reduced. The sample to be detected is detected by combining ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry, in particular, the ultra-high performance liquid chromatography of a biphenyl column, so that the content of the succinate dehydrogenase bactericide in the sample solution to be detected is obtained. 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 the nine SDHI bactericides is used as the method detection limit, the technology exhibits more excellent sensitivity than the existing research.

Description

Technical Field

[0001] This application belongs to the field of agricultural product quality and safety testing technology, and in particular relates to a method for detecting succinate dehydrogenase bactericides using solid phase extraction technology combined with liquid chromatography-tandem mass spectrometry. Background Technology

[0002] In agricultural production, succinate dehydrogenase inhibitors (SDHIs) are widely used as a new type of broad-spectrum amide fungicide to control diseases caused by plant pathogenic fungi. Although SDHIs have relatively low toxicity, their large-scale and widespread use easily leads to residue accumulation in fruits and environmental water. This not only increases the exposure risk to humans and non-target organisms but also raises a series of toxicity issues, thus attracting widespread social attention. For example, between 2014 and 2019, the global market share of SDHI fungicides increased significantly to 12.7%, with a compound annual growth rate of 8.5%. This substantial increase in usage has made residue status in crops and the potential threats to the ecological environment and human health a focal point. Commonly used SDHI fungicides include nine compounds: fluopyram, fluopyram, fluopyram, fluopyram, fluopyram, boscalid, pyraclostrobin, bifenthiophanate-methyl, benzalkonium chloride, and pyraclostrobin.

[0003] Regarding the development of related technologies, common pretreatment methods for SDHIs fungicides include solid-phase extraction (SPE) and dispersive solid-phase extraction (QuEChERS). QuEChERS is simple, rapid, and convenient, but SPE excels in enrichment efficiency, impurity removal, and stability, making it particularly suitable for the detection of trace target compounds. For analytical methods, immunoassay, gas chromatography, liquid chromatography, gas chromatography-tandem mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS) all have application examples. However, immunoassay, GC, and LC suffer from complex pretreatment, low sensitivity, and weak qualitative capabilities, making them prone to false positives due to impurities. In contrast, LC-MS, with its high sensitivity and powerful qualitative and quantitative capabilities, has broad prospects for application in the analysis of SDHIs fungicides. Nevertheless, analytical methods for SDHIs fungicides in all major fruit categories specified in GB2763-2021 are still relatively lacking. Therefore, developing an accurate, sensitive, and stable analytical method capable of simultaneously determining SDHI fungicides in multiple fruits is of significant practical importance and has an urgent market demand. Summary of the Invention

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

[0005] In a first aspect, embodiments of this application provide a method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry, comprising the following steps:

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

[0007] The ultra-high performance liquid chromatography column is a biphenyl column.

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

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

[0010] Furthermore, the succinate dehydrogenase bactericide is one or a combination of two or more of the following: fluopyram, fluopyram, fluoxastrobin, fluopyram, cyproconazole, pyrimethanil, bifenthiophanate-methyl, benzo[a]fluoroquinolones, or pyraclostrobin.

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

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

[0013] Furthermore, the HLB solid-phase extraction column separation and purification method is as follows:

[0014] The test solution was purified by adsorption and elution using an HLB solid-phase extraction column; the eluent used for elution was acetonitrile.

[0015] Furthermore, prior to adsorption, the HLB solid-phase extraction column is activated sequentially with methanol and water.

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

[0017] Further separation is achieved by centrifugation at 3500-4500 r / min for 1-3 min.

[0018] Further, after adsorption, the sample was rinsed with 18-23% acetonitrile; then eluted with acetonitrile.

[0019] Furthermore, the conditions for the ultra-high performance liquid chromatography are as follows:

[0020] The chromatographic column is 2.1 mm × 100 mm, with a diameter of 2.6 μm.

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

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

[0023] Furthermore, the gradient elution program is as follows: 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 for the ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method include:

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

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

[0027] This application discloses a method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method employs ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry (HPLC-MS / MS) with an HLB solid-phase extraction column and a biphenyl column for separation and detection of multiple succinate dehydrogenase bactericides. The method covers all major fruit categories specified in GB 2763-2021, establishing a detection method applicable to a wide range of matrices. It effectively reduces interference from impurities, resulting in superior separation and detection of multiple succinate dehydrogenase bactericides. The method also exhibits higher sensitivity and accuracy in detecting nine succinate dehydrogenase inhibitors, with limits of detection (LODs) ranging from 0.10 to 0.90 μg / kg.

[0028] The above overview 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 this application will become readily apparent from the following detailed description. Attached Figure Description

[0029] Figure 1 Nine SDHI bactericidal compounds in C 18Quantitative ion chromatograms (10 μg / L) for chromatographic columns and biphenyl columns; where (a) represents C 18 (b) is Biphenyl;

[0030] Figure 2 The average recovery rates of nine SDHI bactericide compounds in citrus matrix in different extraction solvents (n=6);

[0031] Figure 3 The average recovery rate of nine SDHI fungicides in citrus matrix at different extractant dosages (n=6) was calculated.

[0032] Figure 4 The average recovery rate of nine SDHI fungicides in citrus matrix at different extraction times (n=6). Detailed Implementation

[0033] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the following description is considered to be exemplary in nature and not restrictive.

[0034] Existing research reports on the detection of succinate dehydrogenase inhibitors in fruits using solid-phase extraction purification technology combined with liquid chromatography-tandem mass spectrometry are limited, and these methods suffer from drawbacks such as failing to cover all major fruit categories in GB 2763-2021, inadequate removal of impurities during purification, or loss of target compounds; and low sensitivity of liquid chromatography detection. Therefore, this application provides a method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry.

[0035] A method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry includes the following steps:

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

[0037] The ultra-high performance liquid chromatography column is a biphenyl column.

[0038] This technique employs HLB solid-phase extraction, leveraging the difference in hydrophilic and hydrophobic interactions to adsorb and remove target analytes and impurities. Since the desorption capacity of target compounds on the solid-phase extraction column increases with decreasing solvent polarity, the solvent composition of the loading and eluents significantly affects the retention of target compounds. Adjusting the organic phase content in the loading and eluents can improve the recovery rate through HLB solid-phase extraction, maximizing the removal of interfering substances and achieving optimal purification, effectively reducing interference from impurities in subsequent ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry detection.

[0039] Ultra-high performance liquid chromatography (UHPLC) used a biphenyl column. This is because all nine compounds contain aromatic ring structures, and the phenyl packing material can retain the target compounds through π-π interactions. Compared to C... 18 The chromatographic column, specifically the biphenyl column, exhibits stronger retention capacity for the target compounds, extending the retention time of nine compounds and better separating interfering matrices from the target analytes, avoiding peak broadening and tailing, and improving resolution. Therefore, this application employs HLB solid-phase extraction column separation and purification followed by ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry for detection, comprehensively covering all major fruit categories in GB 2763-2021; under the condition that the detection limit of the fruit matrix corresponding to the lowest sensitivity for each compound is used as the method detection limit, it demonstrates superior sensitivity compared to existing studies. Preferably, the biphenyl column is a Phenomenon Kinetex Biphenyl column.

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

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

[0042] The sample to be tested was extracted using liquid-liquid extraction, which transferred the SDHI-type bactericide from the sample matrix to the liquid phase. Salt was added for salting out, which promotes the separation of the aqueous and organic phases, reduces emulsification, and ensures the smooth progress of the extraction process. At the same time, the salt can change the ionic strength of the solution, which helps the target analyte to dissolve better in the organic phase.

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

[0044] Acetonitrile is a good solvent for SDHI-type bactericides. Sodium chloride can change the ionic strength of the solution, which helps the target substance to dissolve better in the acetonitrile phase.

[0045] In one implementation method, the mass-to-volume ratio of the sample 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.

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

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

[0048] As one implementation method, the solids after centrifugation are repeatedly extracted, salted out, and separated, and the separated liquids are combined.

[0049] Two extractions can significantly improve the extraction efficiency of SDHI-type bactericides.

[0050] In one embodiment, the separation solution uses an acetonitrile-removed solution, which is redissolved in 18-23% acetonitrile solution to obtain the test solution. In this embodiment, the acetonitrile solution in the separation solution is dried using a nitrogen evaporator to remove acetonitrile and prevent changes in the target compounds. Redissolving the sample in 18-23% acetonitrile ensures that the nine SDHI-type bactericides are effectively redissolved and not eluted in the solid-phase extraction column, while also appropriately removing interfering matrices.

[0051] Furthermore, the test solution is redissolved in 20% acetonitrile solution and used as the loading solution for HLB solid-phase extraction. If the organic phase content of the loading solution is too low, SDHI compounds will not dissolve effectively, while if the organic phase content is too high, these compounds may be eluted during column loading, reducing the recovery rate. Therefore, this method can improve the purification level and increase the recovery rate.

[0052] As one implementation method, the HLB solid-phase extraction column separation and purification method is as follows:

[0053] The test solution was purified by adsorption and elution using an HLB solid-phase extraction column; the eluent used for elution was acetonitrile.

[0054] Purification was performed using an HLB solid-phase extraction column, which possesses a hydrophilic-lipophilic balance. Since the oil-water distribution coefficients of the nine SDHI target compounds ranged from 3.11 to 4.69 (pH 7.4), they exhibited a certain degree of lipophilicity and could adsorb onto the non-polar stationary phase within the HLB column, thereby effectively removing impurities from the fruit matrix, such as fiber, organic acids, sugars, and pigments.

[0055] As one implementation method, the HLB solid-phase extraction column is activated sequentially with methanol and water before adsorption.

[0056] The HLB solid-phase extraction column is activated with methanol and water to bring the packing material into a suitable adsorption state.

[0057] As one implementation method, after adsorption, the adsorption is performed by rinsing with 18-23% acetonitrile; then elution is performed using acetonitrile.

[0058] For the eluent, when the organic phase content is low, most interfering substances will be retained on the solid-phase extraction column. Only by selecting an appropriate organic phase content can the target analyte recovery rate be guaranteed while maximizing the removal of interfering substances, thereby achieving the best purification effect. This application optimizes the optimal solvent composition of the loading solution and eluent during HLB solid-phase extraction, effectively reducing interference from impurities.

[0059] As one implementation method, after the acetonitrile is removed from the eluent, it is reconstituted using a 50% aqueous solution of acetonitrile.

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

[0061] This application utilizes an HLB solid-phase extraction column to achieve the adsorption and removal of target substances and impurities based on the difference in hydrophilic and hydrophobic interactions. The optimal solvent composition of the loading solution and eluent during solid-phase extraction is optimized, effectively removing impurities such as fiber, organic acids, sugars, and pigments from fruits.

[0062] As one implementation method, the samples to be tested include all major categories of fruits according to GB 2763-2021.

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

[0064] The study investigated the matrix effects of eight fruits: citrus, pear, apple, fresh jujube, tree tomato, mango, banana, and honeydew melon. These fruits covered all major fruit categories in GB 2763-2021, including representative citrus fruits, pome fruits, stone fruits, berries and other small fruits, tropical and subtropical fruits, and melon fruits.

[0065] In one embodiment, the succinate dehydrogenase fungicide is one or a combination of two or more of the following: fluopyram, fluopyram, fluoxastrobin, fluopyram, boscalid, pyraclostrobin, bifenthiophanate-methyl, benzalkonium chloride, or pyraclostrobin. These nine succinate dehydrogenase fungicides are commonly used broad-spectrum fungicides for controlling diseases caused by plant pathogenic fungi in fruits. Residue detection of these nine SDHI fungicides can effectively ensure the quality and safety of agricultural products and protect the health rights of consumers.

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

[0067] The chromatographic column is 2.1 mm × 100 mm, with a diameter of 2.6 μm.

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

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

[0070] As one implementation method, the gradient elution program is as follows: 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 existing technologies for detecting SDHI (diethyltoluene-based) bactericide residues, when using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), the UHPLC column is typically a C12C ... 18 Column, but using C 18 When the chromatographic column was used for separation, the retention times of all nine compounds were short, the separation effect between the interfering matrix and the target analytes was poor, the subsequent quantitative accuracy was low, and the overall sensitivity of the detection was not high.

[0072] Therefore, this application uses a biphenyl column, corresponding to the fact that all nine compounds contain aromatic ring structures. The phenyl packing material can retain the target compounds through π-π interactions, resulting in stronger retention of the target compounds and extending the retention time of the nine compounds. This allows for better separation of the interfering matrix from the target analytes, avoiding peak broadening and tailing, and improving resolution. Furthermore, based on the biphenyl column, this application uses the aforementioned ultra-high performance liquid chromatography conditions and gradient elution program, resulting in more thorough separation of the interfering matrix from the target analytes, good peak shape, and no tailing, effectively improving resolution.

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

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

[0075] Liquid chromatography separates target compounds from various matrices, while multiple reaction monitoring (MRM) in mass spectrometry is a highly sensitive and selective scanning analytical technique, particularly suitable for the quantitative analysis of target compounds in complex samples. In MRM mode, the target compound in the sample is first ionized using an electrospray ionization (ESI) source, forming charged ions. After ionization, these ions are introduced into the mass spectrometer. In MRM mode, the mass spectrometer is programmed with specific precursor and daughter ion pairs. The precursor ion is the ion selected for collision during mass spectrometry analysis, while the daughter ion is the fragment ion produced after the precursor ion collision. By selecting specific precursor and daughter ion pairs, MRM mode can significantly improve the detection specificity of target compounds, eliminate interference, and reduce background noise.

[0076] The following is a further explanation using specific embodiments.

[0077] The relevant mass spectrometry information of the nine SDHIs compounds detected in the embodiments of this application is shown in Table 1.

[0078] Table 1. Mass spectrometry information of 9 SDHI compounds

[0079]

[0080]

[0081] *Quantitative Ions

[0082] Methods for calculating the content of nine SDHI bactericides in the sample:

[0083] The blank content needs to be deducted from the calculation results.

[0084] in:

[0085]

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

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

[0088] V—Volume at constant volume, mL;

[0089] m — the mass of the sample taken, in grams;

[0090] f – Dilution factor.

[0091] Example 1

[0092] Weigh 5.00g of citrus sample into a 50mL centrifuge tube, add 5mL of ultrapure water to disperse the sample, vortex mix, add 3g of sodium chloride and 10mL of acetonitrile to the centrifuge tube, and extract by shaking at 300r / min for 20min. The nine succinate dehydrogenase inhibitors are initially extracted into the acetonitrile phase. Centrifuge at 4000r / min for 1min, and transfer the supernatant (acetonitrile phase) to another 50mL centrifuge tube. Repeat the above steps, extract once more with 10mL of acetonitrile and combine the supernatants. Dry the acetonitrile solution in the centrifuge tube with a nitrogen blower, redissolve with 20% acetonitrile solution, and wait for purification.

[0093] The HLB column was activated sequentially with 3 mL of methanol and 5 mL of water to bring the packing material to a suitable adsorption state. The solution to be purified was then transferred into the HLB column in portions to allow the target analyte to be adsorbed onto the column. The column was then eluted with 5 mL of 20% acetonitrile to remove as much polar impurity as possible without eluting the target compound. After drying, the target analyte was eluted with 6 mL of acetonitrile and finally dried with nitrogen. The solution was then reconstituted with 1 mL of 50% acetonitrile aqueous solution and filtered through a needle-type nylon filter membrane for analysis.

[0094] Preparation of matrix standard solutions (for quantification): Accurately weigh or pipette appropriate amounts of nine SDHI fungicides (fluopyram, fluopyram, fluopyram, fluopyram, fluopyram, boscalid, pyraclostrobin, bifenthiophanate-methyl, benzalkonium chloride, and pyraclostrobin), dissolve in acetone, and dilute to a final concentration of 100 mg / L to obtain standard stock solutions of the nine compounds. Store at -20℃. Take 1000 μL of each of the above nine standard stock solutions into the same 10 mL volumetric flask and dilute to a final volume with acetone to obtain a mixed standard intermediate solution of 10 mg / L. Use negative samples treated as described above to obtain blank matrix solutions. Serially dilute the standard intermediate solutions to 1.5, 2.5, 5.0, 15, 25, and 50 μg / L. Using the mass concentration of the analyte as X and its corresponding peak area as Y, fit a linear equation. By measuring the peak area of ​​the sample and substituting it into the linear equation, calculate the content of the nine SDHI fungicides in the sample.

[0095] Chromatographic conditions: Column: Phenomenex Kinetex Biphenyl (2.1 mm × 100 mm, 2.6 μm); Mobile phase: A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile. 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; 11.0–11.1 min: 30%–70% A; 11.1–15.0 min: 70% A; Flow rate: 0.25 mL / min; Column temperature: 40 °C; Injection volume: 5 μL.

[0096] Mass spectrometry analysis conditions: Electrospray ionization (ESI); Curtain gas: 35 psi; Collision gas: 9 psi; Ion spray voltage: 4500 V; Evaporation temperature: 400 °C; Evaporation gas (Ion Source Gas 1): 65 psi; Auxiliary gas (Ion Source Gas 2): 60 psi; Scan mode: ESI+, multiple reaction detection (MRM).

[0097] Method performance analysis:

[0098] 1. Selection of chromatographic column:

[0099] This patent compares a Phenomenon Kinetex Biphenyl column (2.1 mm × 100 mm, 2.6 μm) and a C24 column with the same particle size under the same gradient elution program conditions. 18 Chromatographic column: Phenomenon Kinetex C 18 Two core-shell chromatographic columns with different stationary phases (2.1 mm × 100 mm, 2.6 μm); results are as follows. Figure 1 As shown, the chromatographic peaks are: 1. Fluopyram, 2. Fluopyram, 3-1. trans-fluopyram, 4. Fluopyram aniline, 5. Cyclopyram, 3-2. cis-fluopyram, 6. Pythiamethoxam, 7. Bifenpyroxime, 8. Benzofluoxime, 9. Pyraclostrobin.

[0100] Since all nine compounds contain aromatic ring structures, the phenyl filler can be retained by the target compounds through forces such as π-π interactions. 18 Compared with the biphenyl column, the elution order of some chromatographic peaks changed, and the retention times of all nine compounds were shortened, indicating that the separation effect between the interfering matrix and the target analytes was worse. This suggests that the biphenyl column is superior in the separation and detection of the target compounds in this experimental system.

[0101] 2. Selection of extraction solvent:

[0102] Since the matrix in citrus mainly consists of fiber, organic acids, sugars, and pigments, it has a significant impact on the response of the nine compounds. Therefore, to ensure good pretreatment parameters, citrus was used to test the matrix. The method was as follows:

[0103] Five g of negative-labeled citrus samples were weighed and nine different SDHI fungicides (10 μg / kg) were added. A blank matrix standard curve was prepared for quantification. The oil-water partition coefficients (Log D, pH 7.4, 3.11-4.69) of the nine compounds indicated that they possessed some lipophilicity. Therefore, three extraction solvents of different polarities—acetonitrile, ethyl acetate, and n-hexane—were used for extraction. With 15 mL of extraction solvent, 3 g of sodium chloride, and a shaking time of 20 min per extraction, the average recovery rate was used as a reference indicator (fluoxadiazon was calculated as the sum of the cis and trans peak areas). The effect of different solvents on the recovery rate was investigated. The results are as follows: Figure 2 As shown.

[0104] from Figure 2 It can be seen that acetonitrile has a higher extraction efficiency than ethyl acetate and n-hexane, with a recovery rate of 81.3% to 104%. When using ethyl acetate and n-hexane for extraction, the recovery rate is relatively low because the polarity of the target analyte is significantly different from that of the target analyte, making it difficult to effectively extract the target analyte from the aqueous phase. At the same time, some impurities with low polarity are extracted, which interfere with the analytical results. Therefore, acetonitrile is used as the extraction solvent.

[0105] 3. Selection of extraction solvent dosage:

[0106] Five grams of negative-labeled citrus samples were weighed and 10 μg / kg of nine SDHI fungicides were added. The effect of different acetonitrile addition amounts (2.5, 5.0, 7.5, 10.0, and 12.5 mL) on the recovery rate was investigated. To ensure more thorough extraction, the extraction was performed twice. The results are as follows: Figure 3 As shown.

[0107] from Figure 3 The results show that when the extraction solvent volume is 10 mL, all compounds except pyraclostrobin and bifenthrin achieve optimal recovery rates, with average recoveries ranging from 86.7% to 99.7%. When the extraction solvent volume is 5 mL, pyraclostrobin and bifenthrin show the best recoveries, but their recovery rates exhibit significant deviations and poor stability. When the extraction solvent volume is 12.5 mL, the recoveries of all nine compounds decrease sharply, ranging from 40.0% to 57.5%. This may be because the increased solvent volume leads to the extraction of more matrix interference components, which bind to the target analytes after concentration, preventing complete dissolution in the final reconstitution step. Therefore, an extraction solvent volume of 1-2 times the sample mass is suitable. Considering all factors, 10 mL of acetonitrile (twice the sample mass) is the optimal solvent volume.

[0108] 4. Determining the number of extractions:

[0109] Five g of negative-labeled citrus sample was weighed and 10 μg / kg of nine SDHIs bactericides were added. Extraction was performed once, twice, and three times using 10.0 mL of acetonitrile. The effect of the number of extractions on the extraction rate of the target compounds was evaluated by the average recovery rate of the nine SDHIs compounds. The results are as follows: Figure 4 As shown.

[0110] Besides the amount of extraction solvent, the number of extractions also affects the extraction efficiency of the target compound. Too few extractions may result in insufficient extraction of the target component, while too many extractions will not only significantly reduce experimental efficiency but also extract more interfering substances. Therefore, it is necessary to find an appropriate number of extractions. Figure 4 In the analysis, when the extraction was performed once, the target analytes were not fully extracted, resulting in low recoveries ranging from 45.7% to 76.5%. When the extraction was performed three times, the recoveries ranged from 33.0% to 63.2%, mainly because more matrix interference components were extracted, affecting the redissolution of the target compounds and leading to lower recoveries. When the extraction was performed twice, the recoveries of the nine SDHI compounds ranged from 90.2% to 105%, which were high and met the detection requirements.

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

[0112] Accurately weigh 5g of homogenized citrus sample into a 50mL stoppered centrifuge tube, add 10mL of water, vortex to disperse the matrix, then add 10mL of acetonitrile, 4g of anhydrous magnesium sulfate, and 1g of sodium chloride sequentially, and vortex for 15min. Centrifuge at 4000r / min for 5min. Accurately transfer 5mL of acetonitrile to a pre-filled container containing 1g of anhydrous magnesium sulfate, 30mg of PSA, and 30mg of sodium chloride. 18 In a 15 mL centrifuge tube containing the solid-phase extractant, vortex for 1 min, then transfer the supernatant to a 2 mL centrifuge tube and centrifuge at 10000 rpm for 5 min. Take 1 mL of the supernatant acetonitrile phase and concentrate to near dryness in a nitrogen blow-off tube, then redissolve in 0.5 mL of a 1:1 acetonitrile aqueous solution. Analyze under the same instrument conditions as in Example 1.

[0113] Since the oil-water distribution coefficients of the nine compounds ranged from 3.11 to 4.69 (pH 7.4), indicating a certain degree of lipophilicity, they could form certain adsorption interactions with the non-polar stationary phase. Therefore, in the initial stage of the experiment, the non-polar packing material C was compared with... 18 The effects of solid-phase extraction (SPE) columns and HLB SPE columns. C 18 Both C and HLB solid-phase extraction columns achieved excellent recoveries. 18 The recovery rates were 85.3%-104%, while HLB's recovery rate was 86.1%-106%. The recoveries of the various compounds were similar, but considering product performance, HLB was superior to C. 18Solid-phase extraction columns have a higher adsorption limit, better dryness resistance, and a wider pH tolerance range, resulting in higher operational tolerance.

[0114] Specifically, the above-mentioned 1g anhydrous magnesium sulfate, 30mg PSA, and 30mg C 18 Compared to the HLB solid-phase extraction column in Example 1, the solid-phase extraction column resulted in more severe matrix interference in the samples, with method detection limits (LODs) for nine compounds ranging from 1.00 to 6.00 μg / kg. The sensitivity for all nine compounds was lower than that of the HLB solid-phase extraction column method. The method detection limits for the nine compounds in the HLB solid-phase extraction column ranged from 0.10 to 0.90 μg / kg, demonstrating that the detection method combining HLB solid-phase extraction column with liquid chromatography-tandem mass spectrometry exhibited superior sensitivity.

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

[0116] Weigh 5g of negative citrus sample, add 10μg / kg of 9 SDHI compounds, extract the target components according to the method in Example 1, and after nitrogen blowing to dryness, reconstitute the sample solution with 5mL of water, 10% acetonitrile aqueous solution, 20% acetonitrile aqueous solution, 30% acetonitrile aqueous solution, and 40% acetonitrile aqueous solution, respectively. Transfer the entire sample solution into an activated solid-phase extraction column for purification in several portions (using pure water for the rinsing step), and then perform analysis.

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

[0118] The results showed that when using 20% ​​acetonitrile-water for loading, the recoveries of the nine compounds did not decrease significantly, ranging from 85.2% to 110%. When the target compounds were reconstituted in pure water for column loading, the recoveries were only 64.6% to 86.4% because the target compounds could not be effectively reconstituted. However, when eluted with 30% acetonitrile-water, the recoveries of boscalid and fluopyram decreased significantly, by 9.4% and 18.7%, respectively. Therefore, reconstitution with 20% acetonitrile-water was recommended for loading.

[0119] When reconstituted with 20% acetonitrile-water and used for sample loading, the eluent was also 20% acetonitrile-water. The recoveries of the nine compounds showed no significant decrease, ranging from 84.3% to 103%. However, when eluted with 30% acetonitrile-water, the recoveries of boscalid and fluopyram also showed a significant decrease. Therefore, using 20% ​​acetonitrile-water as both the sample loading and eluent can maximize the removal of interfering substances while ensuring the recovery rates of the nine compounds.

[0120] HLB solid-phase extraction primarily utilizes the difference between hydrophilic and hydrophobic interactions to adsorb target analytes or impurities. Since the desorption capacity of target compounds in the solid-phase extraction column increases with decreasing solvent polarity, the solvent composition of the loading and eluents affects the retention of these compounds. A low organic phase content in the loading solution is insufficient to effectively dissolve the nine SDHI compounds, while an excessively high organic phase content results in elution during loading, reducing the recovery rate. For the eluent, a low organic phase content leads to the retention of most interfering substances in the solid-phase extraction column. A suitable organic phase content maximizes the removal of interfering substances while ensuring target analyte recovery, achieving optimal purification.

[0121] 7. The effects of matrix on eight fruits—citrus, pear, apple, fresh jujube, tree tomato, mango, banana, and honeydew melon—were investigated, covering all major fruit categories in GB 2763-2021, including 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, and a matrix standard curve was prepared. A solvent standard curve was then prepared using a 50% acetonitrile aqueous solution. The ratio K was obtained by comparing the slope of the matrix standard curve with the slope of the solvent standard curve. A K < 0.8 indicated matrix inhibition, while a K > 1.2 indicated matrix enhancement. The results are shown in Table 2.

[0122] Table 2

[0123]

[0124]

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

[0126] 8. Eight fruit matrices—citrus, pear, apple, fresh jujube, tree tomato, mango, banana, and honeydew melon—were selected and processed according to the method in Example 1. A mixed standard matrix curve was prepared to analyze the samples. The mass concentration of the analyte was defined as X, and its corresponding peak area as Y. A linear equation was fitted. The instrument detection limit for different fruits was determined using this method at approximately a signal-to-noise ratio of 3 (S / N≈3). Based on the pretreatment conditions, the detection limit of the fruit matrix corresponding to the lowest sensitivity for each compound was taken as the method detection limit. Then, the method quantitation limit was determined at approximately a signal-to-noise ratio of 10 (S / N≈10). The results are shown in Table 3.

[0127] Table 3

[0128]

[0129]

[0130] Nine SDHI compounds exhibited good linearity within the linear range, with correlation coefficients ranging from 0.9954 to 0.9993. The limits of detection (LODs) for the nine analytes ranged from 0.10 to 0.90 μg / kg, and the limits of quantitation (LOQs) ranged from 0.30 to 3.0 μg / kg. Compared to C... 18 High-performance liquid chromatography (HPLC) columns offer lower limits of detection and quantitation.

[0131] 9. Eight different fruits (tangerine, pear, apple, fresh jujube, tree tomato, mango, banana and honeydew melon) were selected for a three-level spiked recovery test. The spiked levels were 1, 2 and 5 times the limit of quantitation. Each spiked level was repeated 6 times. 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, this application presents a method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method employs HLB solid-phase extraction, optimizing the solvent composition of the sample and eluent during the extraction process to effectively reduce interference from impurities. Detection is then performed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), particularly UHPLC with a biphenyl column, to obtain the content of succinate dehydrogenase bactericides in the test sample solution. This detection method comprehensively covers all major fruit categories in GB 2763-2021. Furthermore, under the condition that the detection limit of the fruit matrix corresponding to the lowest sensitivity for each compound is used as the method detection limit, this technique exhibits superior sensitivity compared to existing studies.

[0136] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0137] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0138] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for detecting succinate dehydrogenase bactericides using solid-phase extraction combined with liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: The test solution was separated and purified by 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 test solution; Among them, the chromatographic column for ultra-high performance liquid chromatography is a biphenyl column; The test solution was obtained by extracting the test sample using liquid-liquid extraction and salting-out methods; the test sample included all major categories of fruits according to GB2763-2021; The succinate dehydrogenase bactericide is fluopyram, fluopyram, fluopyram, fluopyram, cyproconazole, pyraclostrobin, bifenthiophanate-methyl, benzalkonium chloride, or pyraclostrobin. The sample to be tested is dispersed in water, extracted with an extractant, and then salted out to obtain the test solution; the extractant is acetonitrile; the added salt is sodium chloride. The HLB solid-phase extraction column separation and purification method is as follows: the test solution is adsorbed and desorbed by the HLB solid-phase extraction column to obtain a purified sample; the eluent used for desorption is acetonitrile; before adsorption, the HLB solid-phase extraction column is activated sequentially with methanol and water; After adsorption, the sample was rinsed with 18-23% acetonitrile; then eluted with acetonitrile. The conditions for the ultra-high performance liquid chromatography are as follows: Column: 2.1 mm × 100 mm, 2.6 μm; Mobile phase: A is 0.1% formic acid aqueous solution, B is 0.1% formic acid acetonitrile solution; gradient elution is performed; Flow rate: 0.25 mL / min; Column temperature: 40℃; Injection volume: 5 μL; 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; 11.0–11.1 min: 30%–70% A; 11.1–15.0 min: 70% A; The mass spectrometry conditions for the ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry method include: Electrospray ionization source; curtain gas: 35psi; collision gas: 9psi; spray voltage: 4500V; evaporation temperature: 400℃; evaporation gas: 65psi; auxiliary gas: 60psi; scanning mode: ESI+, multiple reaction detection.

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

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