A method for detecting bromonitrile dibenzodiamide in ginger

Abstract

This invention discloses a method for detecting bromofenac in ginger, belonging to the field of pesticide residue detection technology. The method includes: homogenizing and extracting ginger samples using an acetonitrile solution containing a hydrophobic deep eutectic solvent; adding a salting-out agent to obtain a supernatant through separation; purifying the supernatant using a boric acid-functionalized magnetic carbon nanotube metal-organic framework composite material as an adsorbent via a temperature-switching adsorption process, first removing phenolic interference through thermal adsorption, then removing lipids through freeze precipitation; and finally detecting the supernatant using liquid chromatography-tandem mass spectrometry. This invention effectively solves the problem of severe matrix interference from phenols and lipids in ginger, and features high recovery rate, low matrix effect, high sensitivity, and simple operation.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection technology, and in particular to a method for detecting bromofenac in ginger. Background Technology

[0002] Brombutamide, a novel meta-diamid insecticide, exhibits excellent control efficacy against lepidopteran and coleopteran pests due to its unique GABA receptor allosteric regulatory mechanism, and is gradually becoming a key pesticide in the cultivation of high-value crops such as ginger. With increasingly stringent food safety standards in various countries, establishing highly sensitive and accurate detection methods for this type of novel pesticide has become an important technical requirement for ensuring the safety of agricultural exports and domestic consumption.

[0003] However, the detection of pesticide residues in ginger, a specific matrix, remains a significant challenge within existing pesticide residue detection technologies. Current general detection standards primarily rely on the QuEChERS method or traditional solid-phase extraction (SPE) techniques, but these have significant limitations when processing ginger samples. Ginger is rich in phenolic substances such as gingerol and shogaol, as well as a large amount of volatile oils and resins; the physicochemical properties of these matrix components are extremely complex. Gingerol and other phenolic substances exhibit severe co-eluent interference with bromuconazole in chromatographic behavior and generate strong charge competition in the mass spectrometry ion source, leading to a severe matrix inhibition effect and significantly reducing detection sensitivity.

[0004] A more challenging aspect is the inherent technical bottleneck of commonly used purification adsorbents in existing technologies, which often suffer from trade-offs. For instance, while the widely used primary and secondary amino acid (PSA) adsorbent can remove organic acids, its adsorption capacity for weakly acidic phenolic impurities is limited. Octadecylsilane (C18) adsorbents primarily rely on hydrophobic interactions to remove lipids, but due to the extremely high lipid solubility of bromonitrile dimethyl ether (LogP > 5), excessive C18 can irreversibly adsorb the target analyte, leading to a significant decrease in recovery rate. Furthermore, to remove dark pigments from ginger extract, graphitized carbon black (GCB) is often introduced. However, GCB exhibits strong non-specific adsorption for molecules with planar conjugated structures, and it will also adsorb bromonitrile dimethyl ether containing benzene rings, resulting in extremely unstable recovery rates that often fail to meet the quantitative requirements for residue analysis. Simultaneously, the large amount of oleoresin in ginger, if not effectively removed, can easily contaminate the chromatographic column and the ion transport system of the mass spectrometer, leading to high equipment maintenance costs. Therefore, there is an urgent need to develop a new pretreatment technology that can specifically identify and remove phenolic and lipid interferences in ginger while ensuring high recovery rates of highly lipid-soluble target compounds. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting bromonitrile dibenzodiamide in ginger. This method not only solves the extraction challenge of the target analyte through an innovative solvent system, but also achieves precise removal of interference from complex matrices through the combination of functional materials and temperature-controlled processes.

[0006] According to a first aspect of the present invention, a method for detecting bromonitrile dibenzodiamide in ginger is provided, comprising the following steps: firstly, an extraction step S1 is performed, in which an extraction solvent is added to a ginger sample for homogenization and extraction, wherein the extraction solvent is an acetonitrile solution containing a hydrophobic deep eutectic solvent (HDES); subsequently, a separation step S2 is performed, in which a salting-out agent is added to the mixture and the mixture is shaken, utilizing the salting-out effect and the generated heat of hydration to separate the organic phase and the aqueous phase, and a supernatant is obtained; next, a purification step S3 is performed, in which the supernatant is mixed with an adsorbent, wherein the adsorbent is a boric acid-functionalized magnetic carbon nanotube metal-organic framework composite material, the purification process including a temperature-switching adsorption step: firstly, thermal adsorption is performed at a first temperature, followed by cryoprecipitation at a second temperature lower than the first temperature, and the purified liquid is obtained after magnetic separation; finally, a detection step S4 is performed, in which the purified liquid is qualitatively and quantitatively analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0007] In a preferred embodiment of the present invention, the hydrophobic deep eutectic solvent (HDES) in the extraction solvent plays a crucial role in solubilization and penetration enhancement. Preferably, the HDES is prepared by mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:0.5 to 1:2, wherein the hydrogen bond acceptor is selected from at least one of menthol, thymol, or quaternary ammonium salts; and the hydrogen bond donor is selected from at least one of C8-C12 organic acids or alcohols. More preferably, it is prepared by mixing DL-menthol and decanoic acid in a molar ratio of 1:1. The reason for choosing this specific component and ratio is that the eutectic system formed by menthol and decanoic acid has extremely low polarity and excellent lipid permeability. When dispersed in acetonitrile as a hydrophobic solubilizer, it can significantly enhance the penetration ability of the extraction solvent into the oily matrix of ginger tissue, promote the mass transfer of lipid-encapsulated bromofennig diamide into the extraction solvent, thereby improving the extraction efficiency; if the molar ratio deviates significantly from 1:1, it is difficult to form a stable supramolecular fluid, resulting in an increase in melting point or a decrease in homogeneity. Furthermore, the volume percentage concentration of the hydrophobic deep eutectic solvent in the extraction solvent is strictly controlled between 0.5% and 5.0%. This concentration range is set because: if the concentration is below 0.5%, the solubilization and penetration enhancement effects are not significant, and the extraction efficiency cannot be significantly improved; if the concentration is above 5.0%, due to the high viscosity of HDES itself, the extract will become too viscous, which will not only easily form a severe emulsion layer in the subsequent salting-out step, hindering stratification, but also increase the injection pressure of the chromatographic system.

[0008] To achieve precise removal of specific interfering substances in ginger, this invention designs an adsorbent with a special microstructure. According to a preferred embodiment of the invention, the adsorbent has a hierarchical loading structure, including carboxylated multi-walled carbon nanotubes (MWCNTs) as a framework layer, magnetic nanoparticles of iron(III) oxide (Fe3O4) loaded on the surface of the framework layer via in-situ co-precipitation, and an outer MOF layer in-situ grown on the framework and modified with boric acid groups. The magnetic nanoparticles are introduced to replace cumbersome centrifugation, achieving rapid magnetic separation; MWCNTs not only act as a nanoframework to prevent MOF aggregation but also utilize their large specific surface area to assist in the adsorption of some hydrophobic impurities. More importantly, the outer MOF layer is preferably an amino-functionalized aluminum-based metal-organic framework MIL-53(Al)-NH2, which uses 2-aminoterephthalic acid as an organic ligand and contains abundant free amino groups (-NH2) in its framework. The surface of this material is grafted with boric acid groups derived from 4-carboxyphenylboronic acid (CPBA) via an amidation reaction. In this invention, the removal of phenolic interfering substances such as gingerol and shogaol by the adsorbent relies on a multiple synergistic mechanism. Gingerol and shogaol molecules contain phenolic hydroxyl groups, and the boric acid group, acting as a Lewis acid, can coordinate with these phenolic hydroxyl groups to form BO bonds. Simultaneously, the free amino groups on the MIL-53(Al)-NH2 skeleton that do not participate in the grafting reaction can form hydrogen bonds with the hydroxyl groups of phenolic compounds. The superposition of these two forces results in the efficient capture of phenolic interfering substances. Furthermore, some phenolic metabolites in ginger (such as caffeic acid derivatives) contain ortho-diphenol structures, which can form more stable cyclic borate esters with the boric acid group, further enhancing the broad-spectrum removal capability of the adsorbent for various phenolic interfering substances. Meanwhile, bromuconazole, lacking a phenolic hydroxyl structure, remains in solution, fundamentally solving the problem of poor selectivity in adsorbents.

[0009] Another core innovation of this invention lies in the design of the "temperature-switching adsorption" process. According to a further embodiment of the invention, in the purification step, the first temperature is controlled at 35°C to 45°C using the heat of hydration generated by salting out or external heating, and adsorption is performed at this temperature for 3 to 8 minutes with oscillation. The selection of this higher temperature range is based on both thermodynamic and kinetic considerations: on the one hand, the MIL-53(Al)-NH2 material exhibits a "breathing effect," with its pores in an open macroporous state at around 40°C, which facilitates the entry of large-molecule gingerol into the pores and its binding to active sites; on the other hand, the higher temperature reduces solvent viscosity and accelerates the mass transfer rate. Subsequently, the system is placed in a freezing environment, with the second temperature controlled at -15°C to -25°C, and allowed to stand for 5 to 15 minutes. This rapid cooling step serves two purposes: the low temperature causes the MIL-53(Al)-NH2 framework to shrink into a narrow-pore state, physically "locking in" the adsorbed impurities; simultaneously, utilizing the difference in solubility, HDES carrying some lipids undergoes phase separation with acetonitrile at low temperature and co-precipitates on the surface of the nanomaterial. If the freezing temperature is higher than -15℃, lipid precipitation cannot be effectively triggered, resulting in impurities in the purification solution. If it is lower than -25℃, although acetonitrile itself will not solidify, the viscosity of the system will increase significantly, and the extended low-temperature standing time will reduce operating efficiency. Considering both economic efficiency and ease of operation, the lower limit is set at -25℃.

[0010] Furthermore, the pH environment of the salting-out system was precisely controlled to facilitate the aforementioned chemical reactions. This invention preferably employs a composite salting-out agent comprising anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate, with a ratio that maintains the pH of the aqueous phase after stratification between 5.0 and 5.5. This slightly acidic environment is crucial: on the one hand, this pH effectively inhibits the hydrolysis of the amide bonds in bromobenzamide, ensuring the chemical stability of the target compound; on the other hand, although the classic complexation reaction of free phenylboronic acid with diols typically prefers an alkaline environment (the pKa of phenylboronic acid is approximately 8-9), in this invention, the boric acid groups are linked to the surface of the MIL-53(Al)-NH2 framework via amide bonds. The electron-withdrawing effect of the amide bonds significantly reduces the pKa of the linked phenylboronic acid groups. Simultaneously, the high density of amino groups on the MOF framework provides a locally alkaline microenvironment, resulting in a higher degree of effective dissociation of the boric acid groups near the material surface than the level corresponding to the bulk solution pH. The synergistic effect of the two aforementioned effects enables the functionalized material to retain effective adsorption capacity for phenolic compounds even under weakly acidic conditions (pH 5.0-5.5). Simultaneously, this invention explicitly excludes the use of graphitized carbon black (GCB) and octadecylsilane (C18), avoiding non-specific adsorption of the target analyte and ensuring high recovery rates. In the specific purification process, the amount of adsorbent is preferably added at 30 mg to 80 mg per 1.5 mL of supernatant. For detection, the mobile phase of the liquid chromatography-tandem mass spectrometry preferably includes mobile phase A (an aqueous solution containing 0.05%–0.2% formic acid and 1–5 mM ammonium acetate) and mobile phase B (a methanol solution containing 0.05%–0.2% formic acid and 1–5 mM ammonium acetate); the chromatographic column is preferably a C18 column, and the elution method is gradient elution. Furthermore, the detection method of the present invention may also include the detection of brofentanil metabolites, said metabolites including at least one of DM-8007 and S(PFH-OH)-8007; the ion pairs monitored during detection preferably include: brofentanil m / z 665.0 > 556.0; DM-8007 m / z 648.9 > 242.1; S(PFH-OH)-8007 m / z 660.9 > 454.1.

[0011] Through the implementation of the above technical solution, this invention has achieved significant technical progress in the detection of bromofenac in ginger matrix, and its beneficial effects are mainly reflected in the following aspects:

[0012] First, this invention significantly improves the recovery rate and detection accuracy of the target analyte. By introducing a hydrophobic deep eutectic solvent (HDES) to assist extraction, its excellent permeability and lipophilicity effectively enhance the extraction solvent's ability to penetrate the lipid matrix in ginger tissue, promoting the mass transfer of deeply bound bromofenofibrate to the organic phase. More importantly, this invention abandons the traditional methods that easily produce "dead adsorption" of planar pesticides using graphitized carbon black (GCB) and C18, which easily adsorbs lipophilic pesticides, and instead uses boric acid-functionalized aminated magnetic MOF materials. The Lewis acid coordination of the boric acid groups and the hydrogen bonding of the MOF skeleton amino groups synergistically capture phenolic interfering substances such as gingerol, while having no adsorption effect on bromofenofibrate, which does not contain phenolic hydroxyl structures. This strategy of "targeted capture of interfering substances" rather than "adsorption of target substances" allows the recovery rate of bromofenofibrate to remain stable at over 90% even at extremely low addition levels, solving the problem of low and unstable recovery rates in existing technologies.

[0013] Secondly, this invention significantly eliminates the matrix effect, improves detection sensitivity, and protects analytical instruments. Through a unique "temperature-breathing" purification process, this invention cleverly utilizes the thermosensitive breathing effect and solubility temperature difference principle of the MIL-53(Al)-NH2 material: in the high-temperature stage, the material's pores open to accelerate chemical adsorption; in the low-temperature stage, pore contraction locks in impurities; and low-temperature freezing induces the co-precipitation of HDES and ginger lipids. This dual physical and chemical purification mechanism not only thoroughly removes phenolic substances that cause ion inhibition but also efficiently removes oleoresins and waxes that easily contaminate instruments. Experiments show that this method controls the matrix effect within ±10%, effectively extends the lifespan of the chromatographic column and mass spectrometer, and reduces the method's limit of quantitation (LOQ) to the 0.001 mg / kg level.

[0014] Finally, this invention simplifies the operation process and improves detection efficiency. Utilizing magnetic nanocomposite materials as the adsorption carrier, combined with magnetic separation technology, it replaces the cumbersome column chromatography, rinsing, and elution steps of traditional solid-phase extraction, and also avoids multiple centrifugation operations. The entire pretreatment process can be completed in a single container, achieving "one-step" purification. This reduces sample pretreatment time from over 40 minutes to less than 15 minutes, and significantly reduces organic solvent consumption, resulting in good environmental and economic benefits, making it suitable for rapid screening of large batches of samples. Detailed Implementation

[0015] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments. The following examples and comparative examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise stated, the reagents and instruments used in the following examples are commercially available or prepared according to known methods.

[0016] The key raw materials used in the implementation of this invention, their specifications, and sources are as follows: Brombutamide standard (purity ≥98.5%) was purchased from Shanghai Anpu Experimental Technology Co., Ltd., catalog number CDCT-C20184500. Metabolite standards DM-8007 (purity ≥95.0%) and S(PFH-OH)-8007 (purity ≥95.0%) were purchased from Toronto Research Chemicals Co., Ltd., catalog numbers D453002 and S690010, respectively. The standard stock solution was prepared with acetonitrile as solvent, with a concentration of 100 mg / L, and stored at -20℃ in the dark. Blank ginger samples were collected from an organic planting base in Laiwu City, Shandong Province, with no pesticide application records, and were verified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to be free of brombutamide and its metabolite residues. DL-menthol (purity ≥99.0%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number M813338. Decanoic acid (purity ≥99.0%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number D105320. Ferric chloride hexahydrate (FeCl3·6H2O, analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10011928. Ferrous chloride tetrahydrate (FeCl2·4H2O, analytical grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number I116353. Carboxylated multi-walled carbon nanotubes (MWCNT-COOH, outer diameter 10-20 nm, length 10-30 μm, carboxyl content ≥2.56 wt%) were purchased from Chengdu Organic Chemistry Co., Ltd., Chinese Academy of Sciences, catalog number TNCM3. Aluminum chloride hexahydrate (AlCl3·6H2O, analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd. 2-Aminoterephthalic acid (NH2-BDC, purity ≥98.0%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A107312. 4-Carboxyphenylboronic acid (CPBA, purity ≥97.0%) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., catalog number BD24553. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, purity ≥98.0%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number E808856. N-Hydroxysuccinimide (NHS, purity ≥98.0%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H109330. N,N-Dimethylformamide (DMF, chromatographic grade) was purchased from Thermo Fisher Scientific, Inc., catalog number A21440. Anhydrous magnesium sulfate (analytical grade) and sodium chloride (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium citrate dihydrate (analytical grade) and disodium hydrogen citrate 1.5 hydrate (analytical grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Acetonitrile (chromatographic grade) and methanol (chromatographic grade) were purchased from Thermo Fisher Scientific, USA. Formic acid (chromatographic grade) and ammonium acetate (chromatographic grade) were purchased from Merck AG, Germany.Ammonia solution (25%-28%, analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd. The liquid chromatography-tandem mass spectrometry system used an Agilent Technologies 1290 Infinity II ultra-high performance liquid chromatograph coupled with a 6470B triple quadrupole mass spectrometer. The chromatographic column used was an Agilent Poroshell 120 EC-C18 column (2.1 mm × 100 mm, 2.7 μm). The homogenizer was an IKA T25 high-speed shear homogenizer. The vortex mixer was a Thermo Fisher Scientific LP Vortex. The magnetic rack was a Thermo Fisher Scientific DynaMag-2 magnetic separation rack. Low-temperature freezing was performed using a Thermo Fisher Scientific Forma 900 series -25℃ freezer.

[0017] The preparation method of the hydrophobic deep eutectic solvent (HDES) used in this invention is as follows. The HDES is prepared by mixing a hydrogen bond acceptor (at least one selected from menthol, thymol, or a quaternary ammonium salt) and a hydrogen bond donor (at least one selected from C8-C12 organic acids or alcohols) in a molar ratio of 1:0.5 to 1:2. In a specific embodiment, DL-menthol and decanoic acid are weighed in a 50 mL round-bottom flask at a molar ratio of 1:1. Specific amounts for each embodiment are detailed below. The flask is placed in a 50°C water bath and magnetically stirred at 300 rpm until both components are completely dissolved to form a homogeneous and transparent liquid. After heating is stopped, the mixture is allowed to cool naturally to room temperature (approximately 25°C). The resulting HDES is a colorless to pale yellow low-viscosity liquid that does not exhibit crystallization or layering at room temperature. The preparation of this HDES is based on the formation of a stable hydrogen bond network between the hydroxyl group of menthol and the carboxyl group of decanoic acid, which makes the melting point of the mixture much lower than that of any single component (DL-menthol melting point is about 36°C, decanoic acid melting point is about 31.5°C), thus obtaining a stable liquid supramolecular fluid at room temperature. The prepared HDES is added to acetonitrile according to the volume percentage concentration required in each embodiment, and mixed with a vortex mixer for 30 seconds to obtain the extraction solvent.

[0018] The preparation of the boric acid-functionalized magnetic carbon nanotube metal-organic framework composite material (Fe3O4@MWCNT@MIL-53(Al)-NH2-B(OH)2) used in this invention is carried out in three steps. First, the magnetic carbon nanotube carrier is prepared. 1.0 g of carboxylated multi-walled carbon nanotubes are dispersed in 200 mL of deionized water and ultrasonically treated for 30 minutes to ensure uniform dispersion. Then, 4.0 g of FeCl3·6H2O and 2.0 g of FeCl2·4H2O are added, and the mixture is heated to 80°C under nitrogen protection and mechanical stirring (500 rpm). 25% ammonia is added dropwise to adjust the pH to 10, and the reaction is continued for 30 minutes, allowing Fe3O4 nanoparticles to be deposited on the carbon nanotube surface via in-situ co-precipitation. After the reaction, the product is collected using an external magnet, washed three times each with deionized water and anhydrous ethanol, and then vacuum dried at 60°C for 12 hours to obtain a black powdery Fe3O4@MWCNT composite material. In this step, the carboxyl groups on the surface of carbon nanotubes provide active sites for the nucleation and anchoring of Fe3O4 nanoparticles, enabling the magnetic particles to be uniformly distributed on the tube wall surface and avoiding the problem of easy aggregation of pure Fe3O4 nanoparticles.

[0019] Subsequently, in-situ growth of MIL-53(Al)-NH2 on the surface of magnetic carbon nanotubes was performed. 0.5 g of the above Fe3O4@MWCNT material was dispersed in 60 mL of DMF and sonicated for 15 minutes. 1.21 g of AlCl3·6H2O and 0.91 g of 2-aminoterephthalic acid (NH2-BDC) were added, stirred to dissolve, and then transferred to a 100 mL stainless steel reactor lined with polytetrafluoroethylene. The reaction was carried out hydrothermally at 150 °C for 18 hours. The reaction principle is as follows: aluminum ions and 2-aminoterephthalate anions undergo coordination self-assembly under solvothermal conditions, forming a one-dimensional channel structure with aluminum-oxygen octahedral chains as nodes and 2-aminoterephthalic acid as bridging ligands. The amino group of this ligand is located on the side chain of the benzene ring and does not participate in coordination with aluminum ions. Therefore, it remains as a free amino group on the inner wall of the channel and the outer surface of the material, providing chemical reaction sites for subsequent boric acid functionalization grafting. The residual carboxyl groups on the surface of carbon nanotubes serve as heterogeneous nucleation sites, inducing the preferential growth of MIL-53(Al)-NH2 crystals on their surface, forming a hierarchical supported structure. After the reaction was completed, the product was naturally cooled, collected by magnetic separation, and washed three times each with DMF and methanol to remove unreacted monomers and solvent molecules. The product was then vacuum dried at 80°C for 10 hours to obtain a grayish-brown powder, Fe3O4@MWCNT@MIL-53(Al)-NH2.

[0020] Finally, graft modification with boric acid groups was performed. 0.4 g of Fe3O4@MWCNT@MIL-53(Al)-NH2 was dispersed in 50 mL of anhydrous DMF, and 0.30 g of 4-carboxyphenylboronic acid (CPBA), 0.35 g of EDC·HCl, and 0.21 g of NHS were added. The reaction mechanism of this step is as follows: EDC first activates the carboxyl group on the CPBA molecule, generating an unstable O-acylisourea intermediate. Subsequently, NHS reacts with this intermediate to form a relatively stable active ester (succinimide ester). This active ester further undergoes a nucleophilic substitution reaction with the free amino group on the 2-aminoterephthalic acid ligand in the MIL-53(Al)-NH2 backbone, covalently bonding the benzene ring containing boric acid groups to the outer surface of the MOF via amide bonds. The above mixture was stirred and reacted at room temperature for 24 hours. After the reaction was complete, the product was magnetically separated, washed three times with DMF to remove unreacted CPBA and coupling agent, washed twice with methanol, and washed twice with deionized water. It was then vacuum dried at 60°C for 8 hours to obtain the final light brown powdered adsorbent Fe3O4@MWCNT@MIL-53(Al)-NH2-B(OH)2. The material was tested by BET and found to have a specific surface area of ​​412 m². 2 / g; Vibrating sample magnetometer (VSM) tests showed a saturation magnetization of 31.2 emu / g, achieving complete magnetic separation within 15 seconds; Inductively coupled plasma optical emission spectroscopy (ICP-OES) determined a boron content of 1.83 wt%, confirming successful grafting of boric acid groups. Fourier transform infrared spectroscopy (FT-IR) analysis showed a boron content of 1.83 wt% at 1650 cm⁻¹. -1 The characteristic absorption peak of amide I appears at 1540 cm⁻¹. -1 The characteristic absorption peak of the amide II band appears at [location], while the original NH2 symmetric stretching vibration peak (3380 cm⁻¹) also appears. -1 The weakening of the intensity further confirms that CPBA was successfully grafted onto the amino site of the MOF backbone via amide bonds.

[0021] The preparation method of the composite salting-out agent used in this invention is as follows: 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium chloride, 1.0 g of sodium citrate dihydrate, and 0.5 g of disodium hydrogen citrate 1.5 hydrate are pre-mixed evenly and dispensed into 15 mL polypropylene centrifuge tubes for later use. The design principle of this combination is as follows: Anhydrous magnesium sulfate is a strong hydrated salt, which releases a large amount of heat of hydration (approximately 91 kJ / mol) upon dissolution, providing an initial heat source for the subsequent thermo-adsorption step; at the same time, its strong water absorption effect can effectively promote the phase separation of the acetonitrile-water system. Sodium chloride further enhances the salting-out effect and improves the recovery rate of the organic phase. Sodium citrate and disodium hydrogen citrate constitute a citrate buffer system, which precisely controls the pH of the aqueous phase after separation to the range of 5.0 to 5.5. This slightly acidic condition is crucial for protecting the chemical stability of the amide bond in the bromfenac bisamide molecule. Too high a pH will promote the hydrolysis of the amide bond, while too low a pH is not conducive to the effective binding of boric acid groups and phenolic compounds on the adsorbent surface.

[0022] The following describes the complete operation flow of the detection method of the present invention. The key parameters of each embodiment and comparative example are adjusted based on this general flow.

[0023] Step S1 (Extraction): Accurately weigh 10.0 g of homogenized ginger sample and place it in a 50 mL polypropylene centrifuge tube. Add precisely 10 mL of extraction solvent (acetonitrile solution containing the specified concentration of HDES), and homogenize using an IKA T25 high-speed shear homogenizer at 13500 rpm for 2 minutes to ensure thorough disruption of the ginger tissue and release of pesticide molecules from the matrix structure. After homogenization, place the centrifuge tube on a vortex mixer and vortex at 2500 rpm for 1 minute to further enhance the extraction effect.

[0024] Step S2 (Separation): Add the pre-prepared composite salting-out agent (6.5 g / tube) all at once to the homogenized solution obtained in Step S1, and immediately shake vigorously by hand for 1 minute. Due to the heat of hydration released by the dissolution of anhydrous magnesium sulfate, the temperature inside the tube will naturally rise within about 30 seconds. Infrared thermometer monitoring shows that the external temperature of the tube wall can reach 38-42℃. Continue vortexing for 1 minute, then centrifuge the tube at 5000 rpm for 5 minutes. At this point, the system will clearly separate into an upper acetonitrile organic phase and a lower saline phase. Take about 6 mL of the supernatant and transfer it to a new 15 mL centrifuge tube for later use.

[0025] Step S3 (Purification): Add the specified mass of Fe3O4@MWCNT@MIL-53(Al)-NH2-B(OH)2 adsorbent (the dosage for each example is detailed below) to a centrifuge tube containing 6 mL of supernatant. Under the residual heat of hydration from the salting-out step or with the assistance of an external water bath, control the system temperature to the specified first temperature. Place the centrifuge tube on a vortex mixer and vortex at 2000 rpm for the specified adsorption time. After vortexing, quickly transfer the centrifuge tube to a low-temperature freezer at the specified second temperature and let it stand for the specified time, allowing HDES and co-dissolved ginger lipids to precipitate as a white flocculent co-precipitate at low temperature. Immediately after removal, place the centrifuge tube on a DynaMag-2 magnetic separator and let it stand for approximately 15 seconds. The adsorbent and co-precipitate will be attracted and aggregated on the tube wall by the magnet. Carefully aspirate approximately 4 mL of the clear supernatant using a disposable pipette and filter it through a 0.22 μm nylon membrane. The filtrate is the purified solution.

[0026] Step S4 (LC-MS / MS detection): Take 1 mL of the purified solution and place it in a 2 mL vial for direct injection analysis. The LC conditions are as follows: Mobile phase A is an aqueous solution containing 0.1% formic acid and 2 mM ammonium acetate; mobile phase B is a methanol solution containing 0.1% formic acid and 2 mM ammonium acetate. The gradient elution program is: 0–1 min, 10% B; 1–3 min, 10%–90% B; 3–6 min, 90% B; 6–6.5 min, 90%–10% B; 6.5–9 min, 10% B (column equilibration). The flow rate is 0.3 mL / min, the column temperature is 40℃, and the injection volume is 5 μL. The mass spectrometry conditions are as follows: electrospray ionization (ESI+) mode, drying gas temperature is 300℃, drying gas flow rate is 10 L / min, nebulizer gas pressure is 35 psi, and capillary voltage is 3500 V. The multiple reaction monitoring (MRM) ion pair parameters are as follows: for bromfenac, the quantitative ion pair m / z 665.0 > 556.0 (collision energy 15 eV) and the qualitative ion pair m / z 665.0 > 409.0 (collision energy 30 eV); for DM-8007, the quantitative ion pair m / z 648.9 > 242.1 (collision energy 22 eV) and the qualitative ion pair m / z 648.9 > 448.0 (collision energy 18 eV); for S(PFH-OH)-8007, the quantitative ion pair m / z 660.9 > 454.1 (collision energy 20 eV) and the qualitative ion pair m / z 660.9 > 228.1 (collision energy 28 eV).

[0027] The performance evaluation of the detection method of this invention was conducted according to the following standards and indicators. Recovery and precision tests were performed in accordance with GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food" and SANTE / 11312 / 2021 "Analytical quality control and method validation procedures for pesticide residues analysis in food and feed". Three concentration levels of bromufenproxil fumarate standard were added to blank ginger samples: low (0.001 mg / kg), medium (0.01 mg / kg), and high (0.1 mg / kg). Each concentration level was tested in parallel six times, and the average recovery rate and relative standard deviation (RSD) were calculated. The acceptable criteria for the method were a recovery rate between 70% and 120%, and an RSD ≤ 20% (at the 0.001 mg / kg level) or RSD ≤ 15% (at levels of 0.01 mg / kg and above). Matrix effect evaluation was conducted by preparing a series of matrix-matched standard solutions and solvent standard solutions, establishing calibration curves within the range of 0.5-100 μg / L. Matrix effect (ME) was calculated using the formula ME(%) = (slope of matrix-matched standard curve / slope of solvent standard curve - 1) × 100%. ME values ​​within ±10% were considered negligible matrix effects, ±10%-±20% were considered moderate matrix effects, and values ​​exceeding ±20% were considered strong matrix effects. The method limit of quantitation (LOQ) was determined based on a signal-to-noise ratio (S / N) ≥10, combined with the minimum spiking concentration required to meet the above acceptable standards, following the guidelines of NY / T 788-2018 "Guidelines for Pesticide Residue Testing in Crops". Pretreatment operation time was recorded in minutes, from weighing the ginger sample to obtaining a purified solution suitable for instrument analysis. The contamination of the chromatographic column and ion source was assessed by continuously injecting 50 spiked samples of purified ginger matrix. The peak area deviation of bromfenac in the 1st and 50th injections was used to characterize the degree of instrument contamination. A deviation within ±15% was considered negligible.

[0028] Example 1

[0029] This embodiment employs the preferred process parameters of the present invention, as follows: The volume percentage concentration of HDES in the extraction solvent is 2.0%, and HDES is prepared from DL-menthol and decanoic acid in a molar ratio of 1:1. The salting-out agent formulation is: 4.0 g anhydrous magnesium sulfate, 1.0 g sodium chloride, 1.0 g sodium citrate dihydrate, and 0.5 g disodium hydrogen citrate 1.5 g. The measured pH of the aqueous phase after separation is 5.3. The adsorbent dosage is 50 mg per 1.5 mL of supernatant (i.e., 200 mg of adsorbent is added to 6 mL of supernatant). The first temperature for temperature-switched adsorption is controlled at 40℃ (utilizing the residual heat of hydration during salting-out combined with a 40℃ constant temperature water bath for auxiliary heat preservation), with an adsorption time of 5 minutes by shaking; the second temperature is controlled at -20℃, with a freezing and settling time of 10 minutes. Recovery tests were conducted at three spiking levels (0.001, 0.01, and 0.1 mg / kg), with each level repeated 6 times. The recoveries of brofentanil were 92.4%, 96.8%, and 98.1%, with RSDs of 4.6%, 3.2%, and 2.5%, respectively. The recoveries of DM-8007 were 89.7%, 94.5%, and 97.2%, with RSDs of 5.8%, 3.9%, and 2.8%, respectively. The recoveries of S(PFH-OH)-8007 were 88.3%, 93.1%, and 96.5%, with RSDs of 6.2%, 4.3%, and 3.1%, respectively. The matrix effect was -3.7%. The limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 14 minutes. After 50 consecutive injections, the peak area response deviation of brofentanil was -4.2%.

[0030] Example 2

[0031] In this embodiment, the HDES concentration was adjusted to near the lower limit of the parameter range of this invention to verify the extraction effect of low-concentration HDES. The HDES volume percentage concentration was 0.5%, and the other conditions were exactly the same as in Example 1, including the HDES composition (DL-menthol to decanoic acid molar ratio 1:1), salting-out agent formulation, adsorbent dosage (50 mg / 1.5 mL), shaking at 40°C for 5 minutes, and freezing at -20°C for 10 minutes. Recovery tests were conducted at three spiking levels. The recoveries of bromufenproxil fumarate were 83.6%, 89.5%, and 94.7%, with RSDs of 7.1%, 4.8%, and 3.4%, respectively. The matrix effect value was -6.2%. The method limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 14 minutes. The response deviation after 50 consecutive injections was -5.8%. Compared with Example 1, although the low concentration of HDES still meets the requirements for quantitative analysis, the recovery rate at the lowest addition level (0.001 mg / kg) is lower, indicating that the permeation enhancement and solubilization of HDES are positively correlated with the concentration. However, 0.5% can still provide higher extraction efficiency than blank acetonitrile.

[0032] Example 3

[0033] In this embodiment, the HDES concentration was adjusted to near the upper limit of the parameter range of this invention. The HDES volume percentage concentration was 5.0%, and the other conditions were the same as in Example 1. Recovery tests were conducted at three spiking levels. The recoveries of brofentanil were 90.1%, 95.3%, and 97.6%, with RSDs of 5.9%, 4.1%, and 2.9%, respectively. The matrix effect was -5.4%. The method limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 16 minutes (centrifugation time was extended to 7 minutes due to slightly slower layering caused by the increased viscosity of 5% HDES). The response deviation after 50 consecutive injections was -6.1%. The recovery rate of high-concentration HDES was comparable to that of Example 1, but the layering efficiency was slightly lower, and there was slightly more residual HDES in the purified solution. In the subsequent freezing step, more HDES was removed by co-precipitation, but a small amount of residue still entered the chromatographic system, resulting in a slightly larger response deviation after 50 injections than in Example 1. This indicates that when the HDES concentration exceeds 2%, the improvement in extraction efficiency is limited, but the pretreatment efficiency and instrument maintenance costs increase slightly.

[0034] Example 4

[0035] This embodiment increases the first temperature to the upper limit of the parameter range of the present invention. The first temperature is controlled at 45°C (using an external 45°C water bath for heating), with an adsorption time of 3 minutes of shaking; the second temperature is controlled at -15°C, with freezing and standing for 15 minutes. The HDES concentration is 2.0%, the adsorbent dosage is 50 mg / 1.5 mL, and the other conditions are the same as in Example 1. Recovery tests were conducted at three spiking levels. The recoveries of bromufenoxam were 91.8%, 95.6%, and 97.3%, with RSDs of 5.2%, 3.6%, and 2.7%, respectively. The matrix effect value was -5.1%. The method limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 15 minutes. The response deviation after 50 consecutive injections was -7.3%. At 45°C, the pores of MIL-53(Al)-NH2 are more fully open, and the mass transfer rate is accelerated, thus effective adsorption can be completed in only 3 minutes. However, at the second temperature of only -15℃, the cryoprecipitation effect was relatively mild, and some lipids failed to precipitate completely, resulting in a slight increase in the matrix effect (-5.1%), and a slightly larger response deviation after 50 consecutive injections. Overall, this parameter combination is still within the acceptable range of the method.

[0036] Example 5

[0037] In this embodiment, the first temperature and the second temperature were set as lower limits. The first temperature was 35°C, and the adsorption time was 8 minutes with shaking. The second temperature was -25°C, and the adsorbent was frozen and allowed to stand for 5 minutes. The HDES concentration was 2.0%, and the adsorbent dosage was 50 mg / 1.5 mL. The other conditions were the same as in Example 1. Recovery tests were conducted at three spiking levels. The recoveries of brofentanil were 90.5%, 94.8%, and 97.0%, with RSDs of 5.5%, 3.8%, and 2.6%, respectively. The matrix effect was -4.1%. The method limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 17 minutes (the shaking time was extended to 8 minutes due to the slow mass transfer at low temperatures). The response deviation was -3.5% after 50 consecutive injections. Although the pores of MIL-53(Al)-NH2 were open at 35°C, they were not as fully open as at 40-45°C, so the adsorption time needed to be extended to compensate for the insufficient kinetics. However, the deep freezing at -25°C resulted in very thorough lipid precipitation, extremely high purity of the purified solution, a matrix effect of only -4.1%, and optimal stability for continuous injection. It is worth noting that although acetonitrile has a freezing point of -45°C and will not solidify at -25°C, its viscosity increases significantly at low temperatures, requiring rapid sampling to ensure accurate pipetting.

[0038] Example 6

[0039] This example investigated the effect of adsorbent dosage on purification efficiency, using two sets of adsorbent dosages for comparison. The low dosage group used 30 mg of adsorbent per 1.5 mL of supernatant (i.e., 120 mg of adsorbent added to 6 mL of supernatant), while the high dosage group used 80 mg of adsorbent per 1.5 mL of supernatant (i.e., 320 mg of adsorbent added to 6 mL of supernatant). All other conditions were the same as in Example 1. Results for the low dosage group: The recoveries of brofenoxam at the three spiking levels were 93.8%, 97.2%, and 98.5%, with RSDs of 5.8%, 4.0%, and 3.0%, respectively. The matrix effect was -8.4%, and the response deviation after 50 consecutive injections was -9.7%. Results for the high dosage group: The recoveries of brofenoxam at the three spiking levels were 91.2%, 95.4%, and 97.0%, with RSDs of 4.9%, 3.4%, and 2.4%, respectively. The matrix effect was -2.8%, and the response deviation after 50 consecutive injections was -3.1%. The above results indicate that the low-dosage group, due to insufficient adsorbent, did not completely capture phenolic interfering substances, resulting in a larger matrix effect (-8.4%) and causing the continuous injection response deviation to approach the acceptable limit. The high-dosage group, however, showed the best purification effect, but at the cost of increased adsorbent. It is noteworthy that although the lowest recovery rate of the high-dosage group (91.2%) was slightly lower than that of the low-dosage group (93.8%), this is because the excess adsorbent surface contains a small number of non-specific physisorption sites. However, since the chemoselectivity of the boric acid-amino synergistic effect is much higher than that of physisorption, this effect is very limited. Considering both economics and analytical performance, the dosage of 50 mg / 1.5 mL in Example 1 represents the optimal balance point.

[0040] Comparative Example 1

[0041] This comparative example used pure acetonitrile as the extraction solvent, without adding any hydrophobic deep eutectic solvent, to verify the necessity of HDES in the extraction process. All other conditions were the same as in Example 1, including the salting-out agent formulation, adsorbent type and dosage (50 mg / 1.5 mL), temperature-dependent adsorption conditions (40℃ / 5 min; -20℃ / 10 min), and LC-MS / MS detection parameters. The recoveries of brofentanil at the three spiking levels were 62.4%, 71.8%, and 82.5%, with RSDs of 11.3%, 7.6%, and 5.1%, respectively. The matrix effect was -4.5%. The method limit of quantitation was 0.005 mg / kg (signal-to-noise ratio less than 10 at the 0.001 mg / kg spiking level). The total pretreatment time was approximately 14 minutes. The response bias after 50 consecutive injections was -5.0%. The results were significantly worse than in Example 1, especially at low spiking levels where the recovery was only 62.4%, far below the lower limit of acceptable standard of 70%. This demonstrates that pure acetonitrile is insufficient for extracting bromufenproxil fumarate encapsulated in a lipid matrix from ginger tissue. The addition of HDES significantly improves the mass transfer and release efficiency of the target analyte by enhancing the solvent's penetration and solubilization of the lipid matrix, making its contribution to improving extraction efficiency irreplaceable. Simultaneously, due to insufficient extraction, the absolute amount of the target analyte entering the purification step is reduced, the signal-to-noise ratio decreases, and the limit of quantitation deteriorates to 0.005 mg / kg, failing to meet the detection requirement at the 0.001 mg / kg level.

[0042] Comparative Example 2

[0043] This comparative example uses a dispersion solid-phase extraction (d-SPE) purification combination commonly used in the traditional QuEChERS method instead of the boric acid-functionalized aminated magnetic MOF of this invention. Specifically, the extraction and separation steps are exactly the same as in Example 1 (acetonitrile extraction with 2.0% HDES, and separation with a composite salting-out agent). 6 mL of the supernatant was added to a 2 mL d-SPE purification tube containing 400 mg anhydrous magnesium sulfate, 100 mg PSA (primary and secondary amino adsorbent), 100 mg C18 (octadecylsilane adsorbent), and 20 mg GCB (graphitized carbon black). After vortexing for 1 minute, the tube was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane. Temperature-dependent adsorption was not performed. The recoveries of brofentanil at the three spiking levels were 38.5%, 52.7%, and 67.3%, with RSDs of 18.4%, 12.6%, and 8.5%, respectively. The matrix effect was -17.2%. The method limit of quantitation was 0.01 mg / kg. The total pretreatment time was approximately 18 minutes. After 50 consecutive injections, the response bias was -22.6%. The results of this comparative study showed a significant deterioration. The substantial decrease in recovery was attributed to the combined effects of two factors: firstly, the strong hydrophobic adsorption of brofentanil dimethyl ether (LogP>5) by the C18 adsorbent resulted in an irreversible loss of approximately 20%-30% of the target analyte; secondly, the strong π-π stacking adsorption of brofentanil dimethyl ether containing a planar conjugated benzene ring by the GCB resulted in an additional loss of 15%-25% of the target analyte. The combined loss of the target analyte by the two adsorbents reduced the recovery to 38.5%. Meanwhile, PSA could only remove organic acid impurities and had extremely limited ability to remove phenolic substances such as gingerol, the main interfering agents causing matrix inhibition, resulting in a moderately strong matrix effect of -17.2%. More seriously, the conventional method failed to effectively remove oleoresin from ginger. After 50 consecutive injections, the chromatographic peak area decreased by more than 22%, indicating that the chromatographic column and ion source were significantly contaminated.

[0044] Comparative Example 3

[0045] This comparative example used the exact same extraction solvent (2.0% HDES acetonitrile solution), salting-out agent, and adsorbent (Fe3O4@MWCNT@MIL-53(Al)-NH2-B(OH)2, 50 mg / 1.5 mL) as Example 1, but the temperature-switched adsorption operation was omitted. Instead, adsorption was performed by shaking at room temperature (25°C) for 5 minutes, followed by direct magnetic separation and liquid extraction, without the cryoprecipitation step. The recoveries of brofentanil at the three spiking levels were 85.2%, 91.4%, and 95.8%, with RSDs of 6.8%, 4.5%, and 3.3%, respectively. The matrix effect was -14.8%. The method limit of quantitation was 0.001 mg / kg. The total pretreatment time was approximately 10 minutes. The response deviation after 50 consecutive injections was -16.5%. Although the recovery rate was still basically acceptable (lowest level 85.2%), the matrix effect significantly deteriorated to -14.8%, which is within the range of moderate matrix effects. This result is due to two factors: First, at 25°C, MIL-53(Al)-NH2 is in a relatively contracted, narrow-pore state (this material is predominantly narrow-pore below 30°C), making it difficult for the large-molecule gingerol to penetrate deep into the pores and fully contact the boric acid and amino active sites, resulting in a reduction in chemisorption efficiency of approximately 30%-40%. Second, the absence of a freezing step means that HDES and its carried lipids remain entirely in the purification solution. These high-boiling-point lipids severely inhibit the ionization efficiency of the target analyte during electrospray ionization. Continuous injection stability data (-16.5%) also confirms the continuous contamination of the instrument by a large amount of lipids. This comparative example clearly demonstrates that both the warming stage (promoting pore opening and chemisorption of MIL-53(Al)-NH2) and the freezing stage (inducing lipid co-precipitation) in the temperature-dependent adsorption process are indispensable.

[0046] Comparative Example 4

[0047] This comparative example uses the Fe3O4@MWCNT@MIL-53(Al)-NH2 intermediate obtained in the second step of the adsorbent preparation process, namely a magnetic carbon nanotube MOF composite material containing free amino groups but without boric acid functionalization modification, to verify the necessity of synergistic recognition and enhanced removal of phenolic interfering substances by boric acid groups. The adsorbent dosage remained at 50 mg / 1.5 mL, and the temperature-switching adsorption conditions were 40℃ / 5 min and -20℃ / 10 min, with all other conditions identical to those in Example 1. The recoveries of brofentanil at the three spiking levels were 93.5%, 96.7%, and 98.4%, with RSDs of 4.8%, 3.5%, and 2.2%, respectively. The matrix effect was -12.6%. The method limit of quantitation was 0.002 mg / kg (due to the increased matrix effect leading to a decrease in signal-to-noise ratio at low concentrations). The total pretreatment time was approximately 14 minutes. The response deviation after 50 consecutive injections was -12.8%. The recovery rate of this comparative example was slightly higher than that of Example 1. This is because the MIL-53(Al)-NH2 surface without modified boric acid groups relies solely on hydrogen bonding of the amino groups and physical adsorption through MOF channels. Its ability to capture phenolic interfering substances is significantly insufficient compared to the synergistic effect of boric acid and amino groups, and naturally, there is no additional adsorption of the target analyte. However, due to the lack of Lewis acid coordination provided by the boric acid groups, a large amount of phenolic interfering substances remain in the purification solution, causing the matrix effect to worsen from -3.7% in Example 1 to -12.6%. Although the freezing step of temperature-controlled adsorption effectively removed some lipids (thus the matrix effect was better than -14.8% in Comparative Example 3), the charge competition and signal suppression problems caused by phenolic substances in the electrospray ionization source were not resolved. After 50 consecutive injections, the response deviation reached -12.8%, indicating that the unremoved phenolic substances gradually accumulated in the ion source region, leading to a gradual decrease in ionization efficiency.

[0048] Table 1 summarizes the performance test results of each embodiment and comparative example.

[0049]

[0050] The following distinct technical patterns can be extracted from the above experimental data.

[0051] Comparing Example 1 (HDES 2.0%, 92.4% recovery at 0.001 mg / kg) with Comparative Example 1 (no HDES, 62.4% recovery at 0.001 mg / kg), the introduction of HDES improved the low-level recovery by approximately 30 percentage points. This significant difference stems from the high lipophilicity of bromuconazole (LogP>5), which causes it to be encapsulated and bound by the lipid matrix in ginger tissue. While pure acetonitrile is a moderately polar organic solvent, its penetration and solubilization capabilities in the lipid matrix of ginger tissue are insufficient to completely release deep-seated drug molecules. The HDES formed by DL-menthol and decanoic acid possesses extremely low polarity and excellent surface activity. As a hydrophobic solubilizer, it significantly enhances the penetration of the extraction solvent into the lipid matrix, promoting mass transfer of encapsulated pesticide molecules into the organic phase. The gradient data from Examples 2 (0.5%, recovery rate 83.6%) and 3 (5.0%, recovery rate 90.1%) show that the HDES concentration has the most significant impact on extraction efficiency in the range of 0.5% to 2.0%, with diminishing marginal returns beyond 2.0%. This aligns with the general principle of surfactants acting on interfacial processes; when the HDES concentration exceeds the critical aggregation concentration, excessive HDES no longer effectively enhances interfacial penetration, but instead causes inconvenience to subsequent operations due to increased viscosity (e.g., the pretreatment time in Example 3 increased from 14 minutes to 16 minutes).

[0052] The comparison between Comparative Example 4 (MIL-53(Al)-NH2 without boric acid modification, containing only amino groups, matrix effect -12.6%) and Example 1 (boric acid-functionalized MIL-53(Al)-NH2, matrix effect -3.7%) reveals the decisive role of the synergistic effect of boric acid groups and amino groups in the purification effect. Although the adsorbent without boric acid modification can remove some impurities through the pore-based physical adsorption of MIL-53(Al)-NH2, amino hydrogen bonding, and the hydrophobic effect of carbon nanotubes, it lacks the Lewis acid (BO) coordination force provided by the boric acid group, resulting in a significantly insufficient overall capture capacity for phenolic interfering substances. When the boric acid group is introduced, it acts as a Lewis acid and can form BO coordination bonds with the phenolic hydroxyl groups in phenolic compounds such as gingerol. Simultaneously, the remaining free amino groups on the MIL-53(Al)-NH2 skeleton further stabilize this bond through hydrogen bonding. This dual synergistic effect of boric acid coordination and amino hydrogen bonding endows the adsorbent with a highly efficient capture capacity for phenolic compounds. Furthermore, some phenolic secondary metabolites in the ginger matrix contain ortho- and tho-diphenol structures (such as caffeic acid and its derivatives), which can form more stable cyclic borate esters with boric acid groups, further enhancing the removal of such components. This multi-synergistic reaction exhibits high structural specificity: only molecules containing phenolic hydroxyl structures can be effectively captured, while bromoxynil dimethyl ether, due to the absence of such functional groups in its molecular structure, remains unaffected by the chemical action of the adsorbent throughout the purification process, thus maintaining a high recovery rate. Notably, the recovery rate of Comparative Example 4 (93.5%) is slightly higher than that of Example 1 (92.4%). This seemingly contradictory phenomenon precisely verifies the selective capture of phenols by boric acid groups and the chemical inertness of the target analyte: a very small amount of non-specific boric acid-amide hydrogen bond interactions may exist on the surface of the boric acid-modified adsorbent, leading to a trace loss of the target analyte (approximately 1%). However, this loss results in a significant purification gain, reducing the matrix effect from -12.6% to -3.7%, which is crucial for improving the accuracy of ultra-trace quantitative analysis.

[0053] The significant difference between Comparative Example 3 (purification at room temperature and 25°C, matrix effect -14.8%) and Example 1 (warming + freezing at 40°C, matrix effect -3.7%) reveals the dual purification mechanism of the temperature-controlled process. MIL-53 type MOF materials are recognized in the literature as metal-organic frameworks with flexible breathing behavior. Their one-dimensional rhombic channels can reversibly switch between narrow pore (np) and macropore (lp) configurations, with the switching temperature depending on the guest molecules and solvent environment. In the acetonitrile system of this invention, experimental observations show that when the temperature rises to 35-45°C, the MIL-53(Al)-NH2 channels significantly expand to a macroporous state, allowing the relatively large molecular weight gingerol (approximately 294 g / mol) to enter the pores and contact the active sites. A comparison of Examples 4 and 5 shows that effective adsorption can be completed in only 3 minutes at 45°C, while it takes 8 minutes at 35°C. This is because the larger pore size at higher temperatures accelerates the diffusion-controlled mass transfer process. When the system temperature drops sharply from 35-45℃ to -15 to -25℃, HDES (the solubility of the menthol-decanoic acid system decreases significantly at low temperatures), originally dissolved in acetonitrile, rapidly precipitates into fine droplets or solid particles. Similarly, the oils and waxes dissolved in the ginger matrix also experience a sharp decrease in solubility at low temperatures, are captured by the HDES droplets, and co-precipitate. Simultaneously, the MIL-53(Al)-NH2 channels, which have already adsorbed phenolic impurities, shrink into narrow pores at low temperatures, physically preventing the desorption and release of the captured impurities. The presence of magnetic nanoparticles allows the adsorbent containing impurities and the HDES-lipid co-precipitate to be rapidly removed together under the influence of a magnetic field, achieving a synergistic effect of physical and chemical purification. In Example 5, although the freezing time at -25℃ was only 5 minutes, the matrix effect was as low as -4.1%, and the response deviation for 50 needles was only -3.5%, confirming the superior effect of lower temperatures on lipid removal.

[0054] Comparative Example 2 (PSA + C18 + GCB conventional purification, 0.001 mg / kg recovery 38.5%, matrix effect -17.2%) showed significantly worse performance than all embodiments of the present invention, due to a combination of factors. The C18 adsorbent relies on van der Waals forces between its C18 alkyl chain and highly lipophilic molecules for hydrophobic retention. This mechanism exhibits strong irreversible adsorption of brofenoxuronide with LogP>5, requiring extremely strong elution conditions for desorption of the target analyte on the C18 surface. Selective desorption is not achievable in the simple mix-separation mode of d-SPE. The graphitized planar structure of GCB can undergo π-π stacking interactions with any molecule containing aromatic rings or conjugated double bonds. Brofenoxuronide contains multiple benzene rings and amide conjugated structures, thus inevitably leading to adsorption by GCB. The combined loss of the target analyte by the two adsorbents resulted in a recovery rate of only 38.5%. Even more problematic is that PSA primarily removes organic acids and fatty acids through acid-base interactions between amino and carboxylic acids. Its adsorption capacity for weakly acidic phenolic substances like gingerol is extremely low, resulting in the failure to remove major matrix interferences and a worsening matrix effect to -17.2%. Furthermore, this conventional approach completely ignores lipid removal mechanisms. Large amounts of oleoresins and waxes from ginger directly enter the detection system through the purification process, leading to a response deviation as high as -22.6% after 50 consecutive injections. In practical applications, this means that instrument maintenance and cleaning are required approximately every 20-30 injections, severely impacting throughput.

[0055] In summary, this invention achieves high recovery rate (over 90%), low matrix effect (within ±10%), and ultra-low limit of quantitation (0.001 mg / kg) of bromonitrile dimethyl ether (BDE) in the challenging detection scenario of ginger matrix through the organic synergy of HDES-assisted extraction, boric acid-amino synergistic functionalized magnetic MOF specific purification, and temperature-switching adsorption process. All indicators are superior to existing technical solutions, and the pretreatment process is efficient and simple, making it practically valuable for large-scale application.

[0056] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting bromothiallcyohydrazide in ginger, characterized by, Includes the following steps: Step S1, Extraction: Add an extraction solvent to the ginger sample for homogenization and extraction. The extraction solvent is an acetonitrile solution containing a hydrophobic deep eutectic solvent. The hydrophobic deep eutectic solvent is prepared by mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:0.5 to 1:

2. The hydrogen bond acceptor is selected from at least one of menthol, thymol, or quaternary ammonium salts, and the hydrogen bond donor is selected from at least one of C8-C12 organic acids or alcohols. Step S2, Separation: Add salting-out agent to the mixture obtained in step S1 and shake. Utilize the salting-out effect and the heat of hydration generated to separate the organic phase and the aqueous phase, and obtain the supernatant. The salting-out agent includes anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate. The ratio of the salting-out agent is such that the pH value of the aqueous phase after separation is maintained between 5.0 and 5.

5. Step S3, Purification: The supernatant is mixed with an adsorbent, which is a boric acid-functionalized magnetic carbon nanotube metal-organic framework composite material. The adsorbent has a hierarchical loading structure, including: a carbon nanotube framework layer, which is a carboxylated multi-walled carbon nanotube; magnetic nanoparticles, which are nano-ferric oxide, loaded on the surface of the carbon nanotube framework layer by in-situ co-precipitation; and an outer metal-organic framework, which is an amino-functionalized aluminum-based metal-organic framework, with 2-aminoterephthalic acid as its organic ligand. The adsorbent is then purified by... The amide reaction is used to graft 4-carboxyphenylboronic acid onto the amino sites of the amino-functionalized aluminum-based metal-organic framework to obtain the product; the purification process includes a temperature-switching adsorption step: using the heat of hydration generated by salting out in step S2 or external heating, the first temperature is controlled at 35°C to 45°C, and the adsorption is carried out by shaking at this temperature for 3 to 8 minutes. Then the system is placed in a freezing environment, and the second temperature is controlled at -15°C to -25°C. The system is allowed to stand for 5 to 15 minutes to allow the hydrophobic deep eutectic solvent to co-precipitate with the lipids in the ginger matrix. The purified liquid is then taken after magnetic separation. Step S4, Detection: Qualitative and quantitative analysis of the purified solution is performed using liquid chromatography-tandem mass spectrometry.

2. The detection method according to claim 1, characterized in that, The hydrophobic deep eutectic solvent is prepared by DL-menthol and decanoic acid in a molar ratio of 1:1; In the extraction solvent, the volume percentage concentration of the hydrophobic deep eutectic solvent is 0.5% to 5.0%.

3. The method of claim 1, wherein, In step S3, the amount of adsorbent used is 30 mg to 80 mg per 1.5 mL of supernatant; The purification process does not use graphitized carbon black or octadecylsilane as adsorbents.

4. The method of claim 1, wherein, In step S4, the mobile phase of the liquid chromatography-tandem mass spectrometry method includes mobile phase A and mobile phase B; The mobile phase A is an aqueous solution containing 0.05%~0.2% formic acid and 1~5mM ammonium acetate; The mobile phase B is a methanol solution containing 0.05%~0.2% formic acid and 1~5mM ammonium acetate; A C18 column was selected for the chromatographic column, and gradient elution was used.

5. The detection method according to claim 1, characterized in that, The method for detecting bromoxynil diamide also includes the detection of its metabolites; The metabolite includes at least one of DM-8007 and S(PFH-OH)-8007; The ion pairs monitored during the detection included: bromfenacin diamide m / z 665.0 > 556.0; DM-8007 m / z 648.9 > 242.1; S(PFH-OH)-8007 m / z 660.9 > 454.1.

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