A method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods.

Abstract

This invention belongs to the technical field of bactericide residue analysis and determination in plant-derived foods, specifically relating to a method for detecting isobutylethoxyquinoline and its metabolites in plant-derived foods. The method includes extraction with acetonitrile under citrate conditions, followed by purification with C18 (25 mg), PAX (25 mg), and XFM64 (nitrogen-doped multi-walled carbon nanotubes, 10 mg) adsorbents. Chromatographic separation is performed using a C18 column with methanol-2 mmol / L ammonium acetate (containing 0.1% formic acid) as the mobile phase, isocratic elution at a flow rate of 0.40 mL / min, electrospray ionization (ESI+), and detection in multiple reaction monitoring (MRM) mode. This invention is simple to operate, has high pretreatment efficiency, and its precision and recovery rate meet the regulatory residue limit monitoring requirements. It is suitable for the simultaneous determination of isobutylethoxyquinoline and its metabolites in plant-derived foods.

Description

Technical Field

[0001] This invention belongs to the technical field of bactericide residue analysis and determination in plant-derived foods, specifically relating to a method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods. Background Technology

[0002] Tebufloquin is a new fungicide containing a fluoroquinoline structure, developed and launched by Meiji Seika Co., Ltd. in 2012. Its chemical name is (6-tert-butyl-8-fluoro-2,3-dimethylquinoline-4-yl)acetic acid ester. This compound exhibits broad-spectrum fungicidal activity and demonstrates excellent control effects against various plant pathogenic fungi. However, despite its high bioactivity against the target pathogens, isobutylethoxyquinoline showed a 50% contact kill rate against Tetranychus carmine eggs at a concentration of 500 mg / L. Even at higher doses (500 mg / L), it still maintained approximately a 50% contact kill rate against Tetranychus carmine eggs, a non-target arthropod. High concentrations applied in the field may cause non-selective toxicity to some arthropods. In vivo, isobutylethoxyquinoline can be enzymatically hydrolyzed to produce its metabolite, tebufloquin-deacetyl. This metabolite is persistent in the environment, easily accumulates in soil, and may migrate into aquatic systems via surface runoff. Once in water, the metabolite can bind to suspended particulate matter and remain in sediments for extended periods, posing a potential risk to aquatic ecosystems and disrupting their ecological balance. Given the potential environmental and food safety issues that its residues may pose, my country currently lacks effective detection methods for monitoring the residue status of isobutylethoxyquinoline and its metabolites. This, to some extent, restricts the effective monitoring of its residue status. In order to overcome the technical barriers imposed by foreign countries and prevent the importation of food contaminated by this type of pesticide into my country, establishing sensitive, accurate, and efficient detection methods for isobutylethoxyquinoline and its metabolites is of significant scientific importance and urgent practical need.

[0003] In existing technologies, isobutylethoxyquinoline and its metabolites are non-volatile substances and are not suitable for gas chromatography-mass spectrometry (GC-MS, GC-MS / MS). HPLC-MS / MS, with its high sensitivity, low detection limit, and ability to perform accurate qualitative and quantitative analysis, is therefore chosen as the preferred detection method. Furthermore, pretreatment methods for pesticide residue analysis mainly include gel permeation chromatography (GPC), solid-phase extraction (SPE), and QuEChERS. GPC requires specialized equipment and consumes a large amount of reagents; SPE involves complex steps such as activation, sample loading, washing, and elution; while QuEChERS reduces solvent consumption and has advantages such as simplicity, speed, and low cost. This paper improves the QuEChERS pretreatment technique using C18 (25 mg), PAX (25 mg), and XFM64 (nitrogen-doped multi-walled carbon nanotubes, 10 mg) as adsorbents, and establishes an efficient, rapid, and accurate analytical method for isobutylethoxyquinoline and tebufloquin-defacetyl residues from plant sources, combined with HPLC-MS / MS. This method is simple to operate, has high pretreatment efficiency, and its precision and recovery rate meet the regulatory residue limit monitoring requirements. It is suitable for the simultaneous determination of isobutylethoxyquinoline and its metabolites in plant-derived foods. Summary of the Invention

[0004] To address the above problems, the purpose of this invention is to provide a method for detecting isobutylethoxyquinoline and its metabolites in plant-derived foods that is simple to operate, has high pretreatment efficiency, and whose precision and recovery rate meet the regulatory residue limit monitoring requirements. This method is suitable for the simultaneous determination of isobutylethoxyquinoline and its metabolites in plant-derived foods.

[0005] The objective of this invention is achieved through the following technical solution: a method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods, comprising the following steps:

[0006] Step 1, Preparation of standard solutions: Dissolve the solid standard isobutylethoxyquinoline and its metabolite in acetonitrile to prepare a standard stock solution. Dilute the standard stock solution with acetonitrile to prepare a mixed standard intermediate solution.

[0007] Step 2, Sample pretreatment: Add acetonitrile to the homogenized sample for extraction. The optimal extraction environment is pH≈5.5.

[0008] Step 3: The supernatant after step 2 is purified by PAX, C18 and XFM64 adsorbents and filtered through a membrane. The mass ratio of the adsorbent combination is: C18 20-25 mg + PAX 20-25 mg + XFM64 8-10 mg.

[0009] Step 4: HPLC-MS / MS detection. The filtrate after membrane filtration in step 3 is collected in a dedicated HPLC-MS / MS sample vial and separated by chromatography using a C18 column. The mobile phase is methanol-2 mmol / L ammonium acetate (containing 0.1% formic acid), and the elution is isocratic at a flow rate of 0.40 mL / min. Electrospray positive ionization (ESI+) is performed, and the detection is performed in multiple reaction monitoring mode (MRM).

[0010] Preferably, in step 1, 10 mg (accurate to 0.0001 g) each of the solid standard isobutylethoxyquinoline and its metabolite are accurately weighed into a volumetric flask, dissolved in acetonitrile, and diluted to volume to prepare a standard stock solution with a concentration of 1000.0 μg / mL. An appropriate amount of the standard stock solution is then diluted with acetonitrile to prepare a mixed standard intermediate solution with a concentration of 20.0 μg / mL.

[0011] Preferably, in step 2, the sample is any one or more of grapes, cucumbers, pears, Chinese cabbage, tomatoes, goji berries, rice, and barley.

[0012] Preferably, in step 2, weigh an appropriate amount of homogenized sample (10 g for fresh fruits and vegetables and 2.5 g for dried sample, add 7.5 mL of water, soak for 15–25 min, vortex for 1–2 min), place it in a 25–50 mL stoppered centrifuge tube, add 10.0 mL of acetonitrile, vortex for 1–2 min, add 2.0 g of sodium chloride, 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium citrate dihydrate, and 0.5 g of disodium citrate sesquihydrate, shake vigorously for 1–2 min, and centrifuge at 8000 r / min for 3–5 min.

[0013] Preferably, in step 3, 2.0 mL of the supernatant is taken into a stoppered centrifuge tube pre-filled with PAX (25 mg), C18 (25 mg), and XFM64 (10 mg), vortexed to mix, centrifuged for 1-2 min, and the purified supernatant is taken and filtered through a membrane.

[0014] Preferably, in step 4, the mobile phases A (2 mmol / L ammonium acetate, containing 0.1% formic acid) and B (methanol) are used at a flow rate of 0.40 mL / min.

[0015] Preferably, in step 4, the gradient elution program is 0–1.5 min (phase B 10%–65%), 1.5–6.5 min (phase B 65%–90%), 6.5–8.5 min (phase B 90%), and 8.5–10 min (phase B 10%).

[0016] Preferably, in step 4, the ionization mode is as follows: all gases used are high-purity nitrogen; collision gas: 6 kPa; curtain gas: 35 kPa; atomizing gas: 50 kPa; auxiliary gas: 50 kPa; desolventizing temperature: 500 ℃; electrospray voltage: 5.5 KV; collision chamber inlet voltage: 10 V; collision chamber outlet voltage: 10 V.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. Optimization of mass spectrometry conditions: With the instrument set to MassOnly detection mode and using a syringe pump injection method, isobutylethoxyquinoline and its metabolites have better ionization efficiency in ESI+ mode.

[0019] 2. Optimization of chromatographic conditions: When methanol was used as the organic phase, the response values ​​obtained for isobutylethoxyquinoline and its metabolites were significantly higher than those in the acetonitrile system. Under the combined conditions of methanol and 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid, the chromatographic peak half-widths of the two target compounds were narrower, and the peak areas and peak heights were larger, showing good detection sensitivity.

[0020] 3. Optimization of extraction solvent: When acetonitrile is used as the extraction solvent, the recovery rate of the target compound isobutylethoxyquinoline and its metabolites is relatively high, reaching 80% to 120%. In addition, acetonitrile is more compatible with the polarity of most pesticides, and at the same time, it has better selectivity for matrix components such as lipids and proteins during the extraction process, which helps to reduce co-extraction interference and thus improve the accuracy and sensitivity of the analysis.

[0021] 4. Optimization of the extraction environment: In this invention, pH≈5.5 helps both to achieve efficient and stable extraction. The pH conditions have a significant effect on the extraction behavior of the two. In the citrate buffer system, the recovery rates of isobutylethoxyquinoline and its metabolites are all in the range of 80% to 120%.

[0022] 5. Optimization of Adsorbent Combination and Ratio: The composite adsorbent combination used in this invention ensures a high recovery rate while exhibiting superior overall purification performance. C18, with its high carbon content and strong hydrophobicity, primarily removes impurities such as lipids and sterols. PAX possesses anion exchange capacity and, compared to other adsorbents, has a larger specific surface area and better pH stability, resulting in a recovery rate closer to 100%. XFM64 further clarifies the sample extract, demonstrating superior purification effects. When the ratio of the three adsorbents is 25 mg C18 + 25 mg PAX + 10 mg XFM64, the purified sample extract becomes clearer, demonstrating better impurity removal.

[0023] This invention is simple to operate, has high pretreatment efficiency, and its precision and recovery rate meet the regulatory requirements for residue limit monitoring. It is suitable for the simultaneous determination of isobutylethoxyquinoline and its metabolites in plant-derived foods. Attached Figure Description

[0024] The invention will now be described in more detail by way of example, with reference to the accompanying drawings, in which:

[0025] Figure 1 : Ion scanning mass spectra of isobutylethoxyquinoline and its metabolites in Example 2 of this invention.

[0026] Figure 2 Chromatograms of isobutylethoxyquinoline and its metabolites in Example 2 of this invention.

[0027] Figure 3 Example 4 of this invention describes the extraction efficiency of isobutylethoxyquinoline and its metabolites using different extraction solvents.

[0028] Figure 4 The effect of different extraction environments on the extraction efficiency of isobutylethoxyquinoline and isobutylethoxyquinoline metabolites in Example 5 of this invention.

[0029] Figure 5 The extraction efficiency of isobutylethoxyquinoline and isobutylethoxyquinoline metabolites during purification under different adsorbents and adsorbent combinations in tomato substrate in Example 6 of this invention. Detailed Implementation

[0030] In this invention, for ease of description, the relative positional relationships of each component are described according to the layout of the accompanying drawings. For example, the positional relationships of top, bottom, left, right, etc., are determined according to the layout direction in the accompanying drawings.

[0031] Example 1: The instruments, reagents, and materials used in this implementation are as follows.

[0032] The system includes a Sciex AB-4500 Qtrap high-performance liquid chromatography-tandem mass spectrometer, a multi-point vortex mixer, a Milli-QAdvantage A10 ultrapure water system, and a high-speed refrigerated centrifuge.

[0033] Isobutylethoxyquinoline (CAS: 376645-78-2) and its metabolite (CAS: 376645-76-0) were both from Tanmo Quality Inspection Technology Co., Ltd. Acetonitrile, n-hexane, ethyl acetate, and formic acid (LC grade) were purchased from Anpel. Ammonia, anhydrous magnesium sulfate, sodium citrate, disodium citrate, ammonium acetate, and sodium chloride were also purchased.

[0034] XFM01 (multi-walled nanotubes, 5-15 nm), XFM03 (hydroxylated multi-walled carbon nanotubes, 5-15 nm), XFM62-1 (aminated multi-walled carbon nanotubes), XFM64 (nitrogen-doped multi-walled carbon nanotubes, 30-80 nm), XFM51 (graphitized carboxyl multi-walled carbon nanotubes, >50 nm), GCB (graphitized carbon black), and XFF19 (Al-MCM-41 molecular sieve) are all from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd. PSA (N-propylethylenediamine, 55 μm), PCX (mixed strong cation exchange polymer, 40-50 μm), C18 (octadecylsilane-bonded silica gel, 50 μm), SAX (strong anion exchange silica gel, 55 μm), PAX (strong anion exchange polymer, 55 μm), PEP (polystyrene / divinylbenzene polar reinforced polymer, 55 μm), and SCX (strong cation exchange silica gel, 55 μm) are all from Beijing Hechuanye Technology Co., Ltd. The KNORTH® m-PFC B-1 (light-colored matrix) rapid filtration purification column, the KNORTH® m-PFC C (dark-colored matrix) rapid filtration purification column, and the KNORTH® m-PFC HF-B (cereals, nuts, and oil crops) rapid filtration purification column are all from Beijing KNORTH Technology Co., Ltd.

[0035] In this embodiment, a method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods includes the following steps: Step 1: Preparation of standard solution

[0036] Accurately weigh 10 mg (accurate to 0.0001 g) of solid standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a standard stock solution with a concentration of 1000.0 μg / mL. Take an appropriate amount of the standard stock solution and dilute with acetonitrile to prepare a mixed standard intermediate solution with a concentration of 20.0 μg / mL. Prepare a blank matrix solution according to the method in Section 1.3 Sample Pretreatment. Use the blank matrix solution to gradually dilute the mixed standard intermediate solution to prepare a series of matrix-matched standard curve solutions.

[0037] Step 2: Sample pretreatment

[0038] Fresh fruits and vegetables such as grapes, cucumbers, and cabbage should have their edible parts chopped and homogenized using a tissue homogenizer. Goji berry samples should be placed in a -18°C freezer for at least 6 hours to freeze the moisture in the sample into solid ice. The samples should then be quickly removed and ground into powder using a mixing and grinding apparatus. Rice and barley samples should be pulverized into powder using a grinder. Weigh an appropriate amount of homogenized sample (10 g for fresh fruits and vegetables such as grapes, cucumbers, pears, Chinese cabbage, and tomatoes; 2.5 g for dry samples such as goji berries, rice, and barley; add 7.5 mL of water, soak for 20 min, and vortex for 1 min), place it in a 50 mL stoppered centrifuge tube, add 10.0 mL of acetonitrile, vortex for 1 min, add 2.0 g of sodium chloride, 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium citrate dihydrate, and 0.5 g of disodium citrate sesquihydrate, shake vigorously for 1 min, centrifuge at 8000 r / min for 5 min, take 2.0 mL of the supernatant and put it into a 10 mL stoppered centrifuge tube pre-filled with PAX (25 mg), C18 (25 mg), and XFM64 (10 mg), vortex for 30 s, centrifuge at 8000 r / min for 1 min, take the purified supernatant and filter through 0.22 The filtrate was collected in a dedicated HPLC-MS / MS sample vial using a μm nylon membrane for subsequent instrument analysis.

[0039] The instrument conditions in Example 1 were as follows: a JADE-PAK KF-C18 column (100 mm × 2.1 mm, 2.7 µm) was used, with a column temperature of 30 ℃. The mobile phase consisted of solution A (2 mmol / L ammonium acetate, containing 0.1% formic acid) and solution B (methanol), with a flow rate of 0.40 mL / min. The gradient elution program was 0–1.5 min (phase B 10%–65%), 1.5–6.5 min (phase B 65%–90%), 6.5–8.5 min (phase B 90%), and 8.5–10 min (phase B 10%).

[0040] Ionization mode: Electrospray positive ionization (ESI+); Multiple reaction monitoring (MRM); All gases used in the experiment were high-purity nitrogen; Collision gas: 6 kPa; Curtain gas: 35 kPa; Nebulizer gas: 50 kPa; Assist gas: 50 kPa; Desolventization temperature: 500℃; Electrospray voltage: 5.5 kV; Collision chamber inlet voltage: 10 V; Collision chamber outlet voltage: 10 V; Other mass spectrometry parameters are shown in Table 1.

[0041] In this implementation, matrix effect, linear range, limit of detection, and limit of quantitation were considered.

[0042] The relative response value method was used to evaluate the matrix effect of isobutyloxyquinoline and its metabolites according to the formula ME = Am / Ac × 100% (where ME is the matrix effect, Am represents the response value of the standard in the blank matrix, and Ac represents the response value of the standard in the pure solvent). When ME is between 80% and 120%, the matrix effect is negligible. When ME is between 50% and 80% and between 120% and 150%, it is necessary to consider how to eliminate the matrix effect. As shown in Table 2, the matrix effect of some samples is significant and cannot be ignored, requiring matrix-matched standard curves to compensate for the influence of the sample matrix on the analytical results. A series of mixed standard solutions with concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μg / L were prepared by diluting the intermediate mixed standard solution with blank matrix solution. Linear regression was performed on peak area (y) against pesticide mass concentration (x). Isobutyloxyquinoline and its metabolites exhibited good linearity within their respective linear ranges (correlation coefficients r² were all greater than 0.9954). Matrix effects, linear ranges, linear equations, and correlation coefficients are shown in Table 2. The limits of detection (LOD) and quantitation (LOQ) were determined based on the signal-to-noise ratio (S / N). The concentration values ​​corresponding to S / N ≥ 3 were defined as LOD, and the concentration values ​​corresponding to S / N ≥ 10 were defined as LOQ. The LOQ and LOD of isobutyloxyquinoline in eight matrices (grape, cucumber, pear, Chinese cabbage, tomato, wolfberry, rice, and barley) are shown in Table 2: the LOQ of isobutyloxyquinoline and its metabolites were all in the range of 0.1–1.0 μg / kg, and the LODs were all in the range of 0.03–0.3 μg / kg.

[0043] The accuracy and precision of the detection using the method of this embodiment

[0044] Grapes, cucumbers, pears, Chinese cabbage, tomatoes, goji berries, rice, and barley were selected as blank samples. Recovery experiments were conducted at spiking levels of 1.0, 2.0, and 10.0 μg / kg. After standing for 30 min to allow sufficient absorption of the target analyte, analysis was performed according to "1.3 Sample Pretreatment" and "1.4 Instrument Conditions," with each level measured six times. Table 3 shows that the recovery rate of isobutylethoxyquinoline was 80.0%–115.0%, with a relative standard deviation of 0.8%–13.4%; the recovery rate of isobutylethoxyquinoline metabolites was 80.3%–108.3%, with a relative standard deviation of 2.6%–11.7%.

[0045] Example 2: Optimization of mass spectrometry conditions

[0046] With the instrument set to MassOnly detection mode, a syringe pump injection method was used to scan the precursor ion of a single standard solution in both electrospray positive ion (ESI+) and negative ion (ESI-) modes. The results showed that isobutylethoxyquinoline and its metabolites exhibited superior ionization efficiency in ESI+ mode. Further optimization of parameters such as daughter ion, fragmentation voltage, and collision energy yielded full-scan mass spectra of isobutylethoxyquinoline and its metabolites (see...). Figure 1 The ion with the strongest signal response was selected as the quantitative ion, and the ion with the highest signal stability was selected as the qualitative ion. Finally, in multiple reaction monitoring (MRM) mode, the system optimized key mass spectrometry parameters such as declustering voltage and collision energy. The final identified characteristic ion pairs of isobutylethoxyquinoline and its metabolites are summarized in Table 1, and the optimized mass spectra are shown below. Figure 2 As shown.

[0047] Example 3, Optimization of chromatographic conditions

[0048] When using positive HPLC-MS / MS in positive ion mode for detection, a suitable amount of formic acid is usually added to the mobile phase to enhance the ionization efficiency of the target compound in [M+H]+ mode, thereby improving its mass spectrometry response intensity and detection sensitivity. Furthermore, introducing ammonium acetate or ammonium formate into the mobile phase can effectively improve the peak shape of basic compounds during chromatographic separation. In this example, the effects of different solvent systems were systematically investigated. Methanol and acetonitrile were used as organic phases, and the effects of pure water, an aqueous solution containing 0.1% formic acid, a 2 mmol / L ammonium acetate aqueous solution, and an inorganic phase containing 2 mmol / L ammonium acetate (containing 0.1% formic acid) were compared. The results showed that when methanol was used as the organic phase, the response values ​​obtained for isobutylethoxyquinoline and its metabolites were significantly higher than those in the acetonitrile system, thus methanol was determined to be the optimal organic mobile phase. Further optimization of the mobile phase ratio was screened by combining methanol with different inorganic phases. The results showed that under the combined conditions of methanol and 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid, the chromatographic peaks of the two target compounds had narrow half-widths, large peak areas, and large peak heights, indicating good detection sensitivity. Based on these results, methanol-2 mmol / L ammonium acetate (containing 0.1% formic acid) was finally selected as the mobile phase system.

[0049] Example 4, Optimization of the Extractant

[0050] Using tomato as the analytical matrix, the extraction efficiencies of four solvents—acetonitrile, n-hexane, ethyl acetate, and toluene—on isobutylethoxyquinoline and its metabolites were compared. Results ( Figure 3The results showed that when acetonitrile was used as the extraction solvent, the recoveries of the target compound isobutylethoxyquinoline and its metabolites were all in the range of 80%–120%; however, when using n-hexane and toluene, the recoveries of the metabolites were significantly lower, both below 60%; when ethyl acetate was used as the extraction solvent, the recoveries of both were also below 80%. Further analysis indicated that compared with methanol, acetonitrile is more compatible with the polarity of most pesticides, and also has better selectivity for matrix components such as lipids and proteins during the extraction process, which helps to reduce co-extraction interference and thus improve the accuracy and sensitivity of the analysis. In summary, acetonitrile was selected as the suitable extraction solvent in this embodiment.

[0051] Example 5: Optimization of the extraction environment

[0052] In this embodiment, acetonitrile was used as the extraction solvent. 4.0 g of anhydrous magnesium sulfate was added to absorb moisture and reduce the water content in the acetonitrile phase. Simultaneously, 2.0 g of sodium chloride was introduced to promote the transfer of pesticides to the organic phase through salting-out. This embodiment was conducted under different environmental conditions, and the recovery rate of the target pesticide under each condition was compared. For example, the following four extraction environmental conditions were used: ① pH≈2.1: 0.1 mL formic acid, 2.0 g sodium chloride, and 4.0 g anhydrous magnesium sulfate were added (formic acid environment); ② pH≈4.8: 0.15 mL acetic acid, 2.0 g sodium chloride, 4.0 g anhydrous magnesium sulfate, and 1.5 g sodium acetate were added (acetic acid-ammonium acetate environment); ③ pH≈5.5: 2.0 g sodium chloride, 4.0 g anhydrous magnesium sulfate, 2.0 g sodium citrate dihydrate, and 0.5 g disodium citrate sesquihydrate were added (citrate environment); ④ pH≈10: 0.5 mL ammonia, 2.0 g sodium chloride, and 4.0 g anhydrous magnesium sulfate were added (ammonia environment). The results show (see) Figure 4 Under conditions ① and ④, the recoveries of isobutylethoxyquinoline and its metabolites were both below 70%, presumably due to hydrolysis of the target compound under extreme pH conditions, leading to structural damage and thus a decrease in recovery. In the acetate-ammonium acetate buffer system (condition ②), the recovery rate of isobutylethoxyquinoline was higher (97.2%), but the recovery rate of its metabolites was only 67.2%, showing a significant difference in the effect of pH conditions on the extraction behavior of the two. In the citrate buffer system (condition ③), the recoveries of isobutylethoxyquinoline and its metabolites were both in the range of 80%–120%, indicating that this near-neutral environment facilitates efficient and stable extraction of both. Therefore, considering the recovery performance under various conditions, pH≈5.5 was selected as the optimal extraction environment in this embodiment.

[0053] Example 6, QuEChERS purification

[0054] In the QuEChERS pretreatment process, specific adsorbents can be used to achieve efficient purification of different types of interfering substances in the sample. For example, graphitized carbon black (GCB) has a significant adsorption capacity for pigments (such as lutein, chlorophyll, and carotenoids); C18 has the characteristics of high carbon content and strong hydrophobicity, and mainly removes impurities such as lipids and sterols; the main function of PSA is to adsorb and purify acidic compounds in the sample, such as organic acids, fatty acids, and certain pigments. In addition, it can also adsorb moisture in the sample to reduce the influence of moisture on the analytical results. Tomatoes rich in pigment were used as the analytical sample. A mixed standard solution was added to a concentration of 5 μg / kg of the analyte. The purification effects of 14 adsorbents, including GCB, PSA, PCX, PEP, SCX, SAX, PAX, C18, and novel adsorbents XFM01, XFM03, XFM62-1, XFM64, XFM51, and XFF19, were compared. The dosage of non-carbon-based materials (such as PSA, PCX, PEP, SCX, SAX, PAX, and C18) was 25 mg, while the dosage of carbon-based materials (such as GCB, XFM01, XFM03, XFM62-1, XFM64, XFM51, and XFF19) was 5 mg. The results showed (see...) Figure 4 Among non-carbon-based adsorbents, PCX, PEP, and SCX showed recoveries of less than 70% for both target compounds, indicating poor purification performance. PSA showed a recovery rate of less than 60% for isobutylethoxyquinoline metabolites, suggesting potentially strong adsorption for compounds with specific structures. In contrast, C18, PAX, and SAX maintained recoveries between 70% and 120%, meeting routine analytical requirements. Although both PAX and SAX possess anion exchange capacity, PAX, with its larger specific surface area and superior pH stability, achieved a recovery rate closer to 100%, outperforming SAX. Therefore, C18 and PAX were ultimately selected as non-carbon-based adsorbents for subsequent experiments. Among carbon-based adsorbents, XFM03 and XFF19 showed recoveries of less than 50% for isobutylethoxyquinoline metabolites, and XFM62-1 also showed a recovery rate of less than 65%. XFM01 and XFM51 showed recoveries of less than 70% for both target compounds, indicating that their adsorption selectivity may not be suitable for this type of compound. In comparison, the recovery rates of XFM64 and GCB are both within a reasonable range of 70% to 120%, and the sample extract purified by XFM64 is clearer, indicating that it has a better purification effect. Therefore, the examples use C18, PAX and XFM64 as a composite purification adsorbent combination.

[0055] Further optimization of the adsorbent ratio was conducted, resulting in four combination schemes: ① C18 25 mg + PAX 25 mg + XFM64 5 mg; ② C18 50 mg + PAX 50 mg + XFM64 5 mg; ③ C18 25 mg + PAX 25 mg + XFM64 10 mg; ④ C18 50 mg + PAX 50 mg + XFM64 10 mg. The purification effect is shown in [the figure]. Figure 5 The recoveries of isobutylethoxyquinoline and its metabolites in combinations ② and ④ were both below 60%, which could not meet the relevant requirements of the laboratory quality control specifications for food physicochemical testing; while the recoveries of combinations ① and ③ were between 60% and 120%, which were acceptable. Among them, the sample extract after purification by combination ③ was clearer, indicating that its impurity removal effect was better.

[0056] Example 7, Rapid Filtration Purification Column Purification

[0057] The purification effects of three rapid filtration purification columns—KNORTH® m-PFC B-1, KNORTH® m-PFC C, and KNORTH® m-PFC HF-B—were compared. Tomato was used as the analytical sample. A mixed standard solution was added to a concentration of 5 μg / kg of the analyte. The analyte was extracted, and 1 mL of supernatant was transferred to each of the three rapid filtration purification columns: KNORTH® m-PFC B-1, KNORTH® m-PFC C, and KNORTH® m-PFC HF-B. A 0.22 µm filter was connected to the bottom of each rapid filtration column for filtration. The filtrate was collected in a dedicated HPLC-MS / MS sample vial for subsequent instrumental analysis. The results showed that the recoveries of isobutylethoxyquinoline and its metabolites were between 60% and 80% when processing the sample using the three types of rapid filtration purification columns. Although such rapid filtration columns perform better in reducing matrix effects compared to the traditional QuEChERS purification method, their recovery rate is still lower than that of the composite adsorbent purification scheme composed of C18 (25 mg), PAX (25 mg), and XFM64 (nitrogen-doped multi-walled carbon nanotubes, 10 mg). This composite adsorbent combination exhibits superior overall purification performance while ensuring a high recovery rate. In addition, rapid filtration purification columns are more expensive. Therefore, this application adopts the combination of C18, PAX, and XFM64 as the preferred purification method.

[0058] Table 1 Retention times and mass spectrometry parameters of the two target compounds

[0059]

[0060] Table 2. Linear range, matrix effect, and linear relationship of isobutylethoxyquinoline and its metabolites.

[0061]

[0062] Table 3. Spike recoveries and relative standard deviations of isobutylethoxyquinoline and its metabolites in eight matrices (n=6)

[0063]

[0064]

[0065]

[0066] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting residues of isobutylethoxyquinoline and its metabolites in plant-derived foods, characterized in that, Includes the following steps: Step 1, Preparation of standard solutions: Dissolve the solid standard isobutylethoxyquinoline and its metabolite in acetonitrile to prepare a standard stock solution. Dilute the standard stock solution with acetonitrile to prepare a mixed standard intermediate solution. Step 2, Sample pretreatment: Add acetonitrile to the homogenized sample for extraction. The optimal extraction environment is pH≈5.

5. Step 3: The supernatant after step 2 is purified by PAX, C18 and XFM64 adsorbents and filtered through a membrane. The mass ratio of the adsorbent combination is: C18 20-25 mg + PAX 20-25 mg + XFM64 8-10 mg. Step 4: HPLC-MS / MS detection. The filtrate after membrane filtration in step 3 is collected in a dedicated HPLC-MS / MS sample vial and separated by chromatography using a C18 column. The mobile phase is methanol-2 mmol / L ammonium acetate (containing 0.1% formic acid), and the elution is isocratic at a flow rate of 0.40 mL / min. Electrospray positive ionization (ESI+) is performed, and the detection is performed in multiple reaction monitoring mode (MRM).

2. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 1, characterized in that: In step 1, accurately weigh 10 mg (accurate to 0.0001 g) each of the solid standard isobutylethoxyquinoline and isobutylethoxyquinoline metabolite into a volumetric flask, dissolve and dilute with acetonitrile to prepare a standard stock solution with a concentration of 1000.0 μg / mL. Take an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare a mixed standard intermediate solution with a concentration of 20.0 μg / mL.

3. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 2, characterized in that: In step 2, weigh an appropriate amount of homogenized sample (10 g for fresh fruits and vegetables and 2.5 g for dry samples, add 7.5 mL of water, soak for 15–25 min, vortex for 1–2 min), place it in a 25–50 mL stoppered centrifuge tube, add 10.0 mL of acetonitrile, vortex for 1–2 min, add 2.0 g of sodium chloride, 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium citrate dihydrate, and 0.5 g of disodium citrate sesquihydrate, shake vigorously for 1–2 min, and centrifuge at 8000 r / min for 3–5 min.

4. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 1, characterized in that: In step 3, take 2.0 mL of the supernatant into a stoppered centrifuge tube pre-filled with PAX (25 mg), C18 (25 mg), and XFM64 (10 mg), vortex to mix, centrifuge for 1-2 min, take the purified supernatant and filter it through a membrane.

5. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 1, characterized in that: In step 4, the mobile phases A (2 mmol / L ammonium acetate, containing 0.1% formic acid) and B (methanol) were used at a flow rate of 0.40 mL / min.

6. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 5, characterized in that: In step 4, the gradient elution program is 0–1.5 min (phase B 10%–65%), 1.5–6.5 min (phase B 65%–90%), 6.5–8.5 min (phase B 90%), and 8.5–10 min (phase B 10%).

7. The method for detecting the residues of isobutylethoxyquinoline and its metabolites in plant-derived foods as described in claim 6, characterized in that: In step 4, the ionization mode is as follows: all gases used are high-purity nitrogen; collision gas: 6 kPa; curtain gas: 35 kPa; atomizing gas: 50 kPa; auxiliary gas: 50 kPa; solvent removal temperature: 500 ℃; electrospray voltage: 5.5 KV; collision chamber inlet voltage: 10 V; collision chamber outlet voltage: 10 V.

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