A method for detecting amantadine residue in animal-derived agricultural products based on parallel concentration under reduced pressure

Abstract

The present application relates to the technical field of amantadine detection, and particularly relates to a method for detecting amantadine residues in animal-derived agricultural products based on vacuum parallel concentration, which comprises the following steps: using magnetic molecularly imprinted polymers to specifically adsorb amantadine in the extraction solution of animal-derived agricultural product samples, eluting, and obtaining a test solution; vacuum parallel concentrating the test solution to obtain a concentrated solution; using liquid chromatography-tandem mass spectrometry to detect the concentrated solution to obtain the residual amount of amantadine. In the method, vacuum parallel concentration technology is used to simultaneously and quickly concentrate multiple samples, effectively shortening the detection period. At the same time, by accurately controlling the vacuum conditions and concentration parameters, the loss of samples and the interference of impurities in the concentration process are reduced, and the accuracy and sensitivity of the detection are improved.

Description

Technical Field

[0001] This invention relates to the field of amantadine detection, specifically to a method for detecting amantadine residues in animal-derived agricultural products based on vacuum parallel concentration. Background Technology

[0002] In large-scale farming environments, animals are susceptible to influenza viruses. The use of amantadine has effectively reduced the incidence of animal diseases, safeguarding the economic benefits of the farming industry. For example, in chicken farms, amantadine is often used to prevent chickens from contracting avian influenza, reducing losses caused by large-scale outbreaks. However, the harmful residues of amantadine in animal-derived food products are gradually becoming apparent.

[0003] From a toxicological perspective, amantadine is metabolized slowly in the human body, and long-term consumption of animal-derived foods containing residual amantadine will lead to its continuous accumulation in the body. Therefore, it is imperative to develop strict methods for detecting amantadine residues.

[0004] Currently, the mainstream method for detecting adamantane residues is liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the key lies in sample pretreatment. Existing pretreatment techniques mainly suffer from the following drawbacks:

[0005] Existing standard methods and literature reports mainly employ solid-phase extraction (SPE, represented by MCX mixed cation exchange columns) or the QuEChERS method. SPE methods suffer from drawbacks such as cumbersome operation (requiring multiple steps including activation, sample loading, rinsing, and elution), long processing time (approximately 1.5 hours per sample), and unstable recovery rates (typically between 70% and 85%). Furthermore, for animal-derived matrices high in fat and protein (such as pig liver and egg yolk), the matrix effect is significant, easily leading to ion inhibition or enhancement, affecting quantitative accuracy. While the QuEChERS method is rapid, purification is incomplete, with significant interference from co-extracted phospholipids and proteins, limiting detection sensitivity. Moreover, adamantane is easily lost and has low throughput; adamantane is a thermosensitive compound that easily volatilizes or degrades at high temperatures. Traditional rotary evaporation concentration is difficult to precisely control temperature, resulting in significant target analyte loss (recovery rates can drop below 70%). Nitrogen blowing concentration, while capable of processing multi-channel samples, is time-consuming (30-60 minutes per sample), subjective endpoint judgment, and poor batch-to-batch repeatability. More importantly, existing concentration methods are all single-channel or low-throughput, which cannot meet the needs of rapid detection of large batches of samples. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a method for detecting amantadine residues in animal-derived agricultural products based on vacuum parallel concentration. This method utilizes vacuum parallel concentration technology to achieve rapid simultaneous concentration of multiple samples, effectively shortening the detection cycle. Simultaneously, by precisely controlling the vacuum conditions and concentration parameters, sample loss and impurity interference during the concentration process are reduced, thereby improving the accuracy and sensitivity of the detection.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for detecting amantadine residues in animal-derived agricultural products based on depressurized parallel concentration, the process comprising:

[0008] (1) Adamantane in the extract of animal-derived agricultural products was specifically adsorbed by a magnetic molecularly imprinted polymer. The adamantane was separated and enriched under the action of an external magnetic field. Then, the adamantane was eluted from the magnetic molecularly imprinted polymer with an elution buffer of methanol-acetic acid volume ratio of 8~10:1. The elution volume was 2~5 mL and the elution time was 10~15 min to obtain the test solution.

[0009] (2) The test solution is subjected to reduced pressure parallel concentration for 20-25 min under the conditions of vacuum degree of 40-50 mbar, water bath temperature of 40-50℃ and oscillation rate of 1500-200 rpm to obtain concentrated solution;

[0010] (3) The concentrate was analyzed by liquid chromatography-tandem mass spectrometry to obtain the residual amount of adamantane.

[0011] Compared with existing technologies, this invention employs magnetic molecularly imprinted polymers (MMIPs) for specific adsorption and extraction of adamantane, combined with destructive parallel concentration under reduced pressure for non-destructive enrichment, and then detects it using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This is because the inventors discovered that existing SPE or QuEChERS methods suffer from poor selectivity and severe matrix interference in complex matrices of animal-derived agricultural products, resulting in the purified extract still containing a large amount of co-extractants. These impurities are highly likely to cause bumping during subsequent concentration. Furthermore, adamantane itself is extremely sensitive to temperature and vacuum. Traditional rotary evaporation or nitrogen blowing concentration methods suffer from uncontrollable temperature and subjective endpoint determination, often resulting in the loss of adamantane along with solvent vapors, with recovery rates generally below 85%, and high-throughput processing is not achievable. Therefore, this invention combines MMIP with vacuum parallel concentration. On the one hand, it utilizes the imprinted cavities on the MMIP surface that precisely match the spatial configuration of adamantane to selectively capture target molecules from high-fat, high-protein matrices, significantly reducing impurities in the eluent and providing a clean sample environment for subsequent concentration. On the other hand, it optimizes vacuum concentration parameters, establishing for the first time a non-destructive concentration window for adamantane (vacuum 40-50 mbar, water bath temperature 40-50℃, oscillation rate 150-200 rpm, time 20-25 min), effectively avoiding boiling over and thermal degradation. This combination overcomes the technical bias of high concentration efficiency at low vacuum and high temperatures, breaking down the technical barrier that a single technology cannot simultaneously address poor selectivity and large concentration losses. Because the MMIP eluent (methanol-acetic acid, volume ratio 8-10:1, 2-5 mL) is highly compatible with the aforementioned vacuum concentration conditions, the appropriate amount of acetic acid in the eluent ensures complete desorption of adamantane without corroding the equipment during concentration. Moreover, MMIP and vacuum parallel concentration have a significant synergistic effect, which truly solves the long-standing technical problems of "incomplete purification, easy loss during concentration, and low throughput" in the detection of adamantane residues.

[0012] Furthermore, the preparation method of the animal-derived agricultural product sample extract is as follows: weigh the homogenized animal-derived agricultural product sample, add D6-adamantaneamine internal standard working solution and acetic acid acetonitrile solution, centrifuge, and take the supernatant as the sample extract.

[0013] This technical solution significantly improves the extraction efficiency and quantitative accuracy of adamantane in complex animal-derived matrices (such as pig liver, chicken meat, and egg yolk) by introducing a D6-adamantane isotope internal standard and synergistic treatment with an acetonitrile-acetic acid extraction solution. The D6-adamantane internal standard and the target analyte, adamantane, have almost identical chemical structures and physicochemical properties, and can simultaneously undergo the same losses and matrix effects throughout the entire process of extraction, purification, concentration, and mass spectrometry detection. This internal standard correction method effectively offsets uncontrollable errors in sample pretreatment and matrix inhibition or enhancement during mass spectrometry ionization, resulting in a qualitative improvement in the precision and accuracy of the quantitative results. Simultaneously, the acetonitrile-acetic acid mixed extraction solution (1% acetonitrile-acetic acid) utilizes the excellent protein precipitation ability of acetonitrile and the ion-pair promoting effect of acetic acid to efficiently disrupt the non-specific binding between proteins and adamantane in animal-derived samples, fully releasing adamantane from its tissue-bound state. A single extraction can achieve an extraction efficiency of over 85%, avoiding the cumbersome multiple extraction operations required in traditional methods. After centrifugation, the supernatant is taken directly, eliminating the need for filtration or degreasing. This not only shortens the pretreatment time but also reduces the non-specific adsorption loss of the target analyte on the filter membrane or solid-phase extraction column. It provides a high-concentration, low-viscosity ideal injection solution for the specific recognition of magnetic molecularly imprinted polymers, thus ensuring the sensitivity and reproducibility of the entire detection method from the source.

[0014] Furthermore, the magnetic molecularly imprinted polymer comprises a template molecule, a functional monomer, and a crosslinking agent in a molar ratio of 1~3:4~5:16~20; the template molecule comprises adamantane; the functional monomer comprises methacrylic acid; and the crosslinking agent comprises ethylene glycol dimethacrylate. In addition, Fe3O4 magnetic nanoparticles serve as a carrier in the polymer.

[0015] By precisely defining the molar ratio of template molecule (adamantane), functional monomer (methacrylic acid), and crosslinking agent (ethylene glycol dimethacrylate), a magnetic molecularly imprinted polymer (MMIP) with antibody-like specific recognition ability for adamantane was successfully prepared, fundamentally solving the common technical problems of poor selectivity and severe matrix interference in traditional solid-phase extraction (SPE) and QuEChERS methods. Within this molar ratio range, the carboxyl groups in the functional monomer methacrylic acid form stable ionic and hydrogen bonds with the amino groups in the adamantane molecule. Simultaneously, the polymerization of excess crosslinking agent forms rigid three-dimensional cavities, ensuring that the shape, size, and chemical functional group arrangement of the imprinted cavities are precisely complementary to those of the adamantane molecule. After the template molecule is eluted, the polymer surface retains numerous specific recognition sites that match the lock-and-key of adamantane, enabling selective capture of adamantane from complex animal-derived sample extracts, while exhibiting almost no non-specific adsorption of structural analogs (such as rimantadine and memantine) and other co-extracted impurities (such as fatty acids, phospholipids, and pigments). Compared with conventional SPE columns, the MMIP of this invention has more than 3 times higher adsorption selectivity, the color of the purified extract changes from dark yellow to colorless and transparent, the matrix effect is significantly reduced, and the ion inhibition rate during LC-MS / MS analysis is significantly reduced, thereby greatly improving the detection sensitivity and the limit of quantitation. At the same time, it reduces the frequency of contamination of the chromatographic column and ion source and extends the service life of the instrument.

[0016] Furthermore, the amount of the magnetic molecularly imprinted polymer used is 30-40 mg of magnetic molecularly imprinted polymer per 2.0 g of animal-derived agricultural product sample; the adsorption time is 10-12 min.

[0017] Furthermore, the liquid chromatography mobile phase gradient elution program is as follows: 0~1.5 min, 90% water, 10% methanol; 2~5 min, 10% water, 90% methanol; 6~10 min, 90% water, 10% methanol; wherein the water contains formic acid at a mass percentage of 0.1%;

[0018] The injection volume is 0.8~1.2μL; the column temperature is 38~42℃; and the flow rate is 0.2~0.3ml / min.

[0019] Compared with existing technologies, the water-methanol gradient elution program containing 0.1% formic acid can improve the peak shape of adamantane, enhance resolution, and shorten analysis time. Combined with an injection volume of 0.8~1.2μL, a column temperature of 38~42℃, and a flow rate of 0.2~0.3 mL / min, it achieves an optimal match between chromatographic separation efficiency, response intensity, and operational stability, reduces matrix interference, improves mass spectrometry ionization efficiency, and provides a reliable chromatographic basis for accurate quantification.

[0020] Furthermore, the mass spectrometry conditions are as follows:

[0021] Electrospray ionization (ESI) source; ion source temperature 280~350℃; detection method: multiple reaction monitoring (MRM); scanning method: positive ion scanning.

[0022] Compared with existing technologies, the use of an electrospray ionization source, an ion source temperature of 280~350℃, positive ion scanning and multiple reaction monitoring modes can achieve efficient ionization and high selectivity detection of adamantane, effectively reduce background noise, improve signal-to-noise ratio and detection sensitivity, ensure accurate qualitative and reliable quantitative results, and enable the entire detection system to maintain excellent detection performance even at low residue levels.

[0023] Secondly, the present invention improves the application of the above-described detection method in the detection of amantadine residues in animal-derived agricultural products.

[0024] Compared with existing technologies, this detection method clarifies the direct application of this method in the detection of amantadine residues in animal-derived agricultural products. Based on the aforementioned optimization of the entire process of extraction, purification, concentration, and detection, it can achieve accurate, rapid, and high-throughput detection of various matrices such as livestock and poultry meat, eggs, milk, and aquatic products, providing a directly usable technical means for food safety monitoring.

[0025] Thirdly, the present invention provides the application of the above-described detection method in food safety supervision, third-party testing, enterprise quality control, and import and export inspection and quarantine.

[0026] Fourthly, the present invention provides a pretreatment kit for detecting adamantane residue, comprising:

[0027] The product includes the aforementioned magnetic molecularly imprinted polymer powder, eluent, adamantane amine standard, D6-adamantane amine internal standard solution, sample tubes adapted for vacuum parallel concentration, and instructions for use.

[0028] In summary, the technical effects achieved by this invention are as follows:

[0029] 1) This invention is the first to combine vacuum parallel concentration technology and MMIP technology for the detection of amantadine residues in batches of animal-derived foods, breaking through the bottlenecks of traditional detection methods in sample pretreatment and detection efficiency, and providing a brand-new technical idea and method for the detection of veterinary drug residues in animal-derived foods.

[0030] 2) This invention establishes a novel method system for detecting amantadine residues in animal-derived foods, integrating extraction, purification, vacuum parallel concentration, and high-sensitivity instrumental analysis. This method system not only improves detection efficiency and accuracy but also possesses good versatility and scalability, and can be extended to the detection of other veterinary drug residues.

[0031] 3) The simultaneous processing of multiple samples through the depressurized parallel concentration technology greatly shortens the sample pretreatment time. Combined with optimized instrument analysis conditions, it significantly improves the detection efficiency of amantadine residues in batches of animal-derived foods, meeting the current food safety regulatory requirements for rapid detection.

[0032] 4) Cost-effectiveness advantage: The detection method established in this study reduces reagent consumption and equipment wear and tear while ensuring detection quality, thus lowering detection costs. At the same time, it improves detection efficiency and reduces labor and time costs, demonstrating a significant cost-effectiveness advantage and facilitating its widespread application in practical testing work. Attached Figure Description

[0033] Figure 1 The graph shows the linear relationship between different concentrations of amantadine in pig liver and the peak area.

[0034] Figure 2 The graph shows the linear relationship between different concentrations of adamantane in eggs and the peak area. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0036] In their previous research, the inventors discovered that while gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS / MS) techniques offer high sensitivity and accuracy in detecting amantadine residues, sample pretreatment efficiency becomes a key factor limiting detection speed when dealing with batch sample testing. Traditional concentration methods, such as rotary evaporation, can only process a single sample at a time, failing to meet the demand for rapid detection of large numbers of samples. This results in low detection efficiency and hinders timely monitoring when facing the continuous influx of animal-derived food products requiring inspection.

[0037] To address the aforementioned problems, in a first aspect, embodiments of the present invention provide a method for detecting amantadine residues in animal-derived agricultural products based on depressurized parallel concentration, the process comprising:

[0038] (1) Adamantane in the extract of animal-derived agricultural products was specifically adsorbed by a magnetic molecularly imprinted polymer. The adamantane was separated and enriched under the action of an external magnetic field. Then, the adamantane was eluted from the magnetic molecularly imprinted polymer with an elution buffer of methanol-acetic acid volume ratio of 8~10:1. The elution volume was 2~5 mL and the elution time was 10~15 min to obtain the test solution.

[0039] (2) The test solution is subjected to reduced pressure parallel concentration for 20-25 min under the conditions of vacuum degree of 40-50 mbar, water bath temperature of 40-50℃ and oscillation rate of 1500-200 rpm to obtain concentrated solution;

[0040] (3) The concentrate was analyzed by liquid chromatography-tandem mass spectrometry to obtain the residual amount of adamantane.

[0041] Compared with existing technologies, this invention employs magnetic molecularly imprinted polymers (MMIPs) for specific adsorption and extraction of adamantane, combined with destructive parallel concentration under reduced pressure for non-destructive enrichment, and then detects it using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This is because the inventors discovered that existing SPE or QuEChERS methods suffer from poor selectivity and severe matrix interference in complex matrices of animal-derived agricultural products, resulting in the purified extract still containing a large amount of co-extractants. These impurities are highly likely to cause bumping during subsequent concentration. Furthermore, adamantane itself is extremely sensitive to temperature and vacuum. Traditional rotary evaporation or nitrogen blowing concentration methods suffer from uncontrollable temperature and subjective endpoint determination, often resulting in the loss of adamantane along with solvent vapors, with recovery rates generally below 85%, and high-throughput processing is not achievable. Therefore, this invention combines MMIP with vacuum parallel concentration. On the one hand, it utilizes the imprinted cavities on the MMIP surface that precisely match the spatial configuration of adamantane to selectively capture target molecules from high-fat, high-protein matrices, significantly reducing impurities in the eluent and providing a clean sample environment for subsequent concentration. On the other hand, it optimizes vacuum concentration parameters, establishing for the first time a non-destructive concentration window for adamantane (vacuum 40-50 mbar, water bath temperature 40-50℃, oscillation rate 150-200 rpm, time 20-25 min), effectively avoiding boiling over and thermal degradation. This combination overcomes the technical bias of high concentration efficiency at low vacuum and high temperatures, breaking down the technical barrier that a single technology cannot simultaneously address poor selectivity and large concentration losses. Because the MMIP eluent (methanol-acetic acid, volume ratio 8-10:1, 2-5 mL) is highly compatible with the aforementioned vacuum concentration conditions, the appropriate amount of acetic acid in the eluent ensures complete desorption of adamantane without corroding the equipment during concentration. Comparative data show that when MMIP is used alone with rotary evaporation concentration, the recovery rate is only 69%~75%; when vacuum parallel concentration is used alone with SPE purification, the pretreatment time is as long as 1.5 hours and the operation is cumbersome; however, when the two are combined, more than 30 samples can be processed simultaneously in a single batch, the pretreatment time is shortened to less than 0.5 hours, the amount of organic solvent used is reduced by more than 40%, and MMIP can be reused 3~5 times while the recovery rate remains above 85%. This demonstrates that MMIP and vacuum parallel concentration form a significant synergistic effect, truly solving the long-standing technical challenges of "incomplete purification, easy loss during concentration, and low throughput" in the detection of amantadine residues.

[0042] In some embodiments, the preparation method of the animal-derived agricultural product sample extract is as follows: weigh the homogenized animal-derived agricultural product sample, add D6-adamantaneamine internal standard working solution and acetic acid acetonitrile solution, centrifuge, and take the supernatant as the sample extract.

[0043] This technical solution significantly improves the extraction efficiency and quantitative accuracy of adamantane in complex animal-derived matrices (such as pig liver, chicken meat, and egg yolk) by introducing a D6-adamantane isotope internal standard and synergistic treatment with an acetonitrile-acetic acid extraction solution. The D6-adamantane internal standard and the target analyte, adamantane, have almost identical chemical structures and physicochemical properties, and can simultaneously undergo the same losses and matrix effects throughout the entire process of extraction, purification, concentration, and mass spectrometry detection. This internal standard correction method effectively offsets uncontrollable errors in sample pretreatment and matrix inhibition or enhancement during mass spectrometry ionization, resulting in a qualitative improvement in the precision and accuracy of the quantitative results. Simultaneously, the acetonitrile-acetic acid mixed extraction solution (1% acetonitrile-acetic acid) utilizes the excellent protein precipitation ability of acetonitrile and the ion-pair promoting effect of acetic acid to efficiently disrupt the non-specific binding between proteins and adamantane in animal-derived samples, fully releasing adamantane from its tissue-bound state. A single extraction can achieve an extraction efficiency of over 85%, avoiding the cumbersome multiple extraction operations required in traditional methods. After centrifugation, the supernatant is taken directly, eliminating the need for filtration or degreasing. This not only shortens the pretreatment time but also reduces the non-specific adsorption loss of the target analyte on the filter membrane or solid-phase extraction column. It provides a high-concentration, low-viscosity ideal injection solution for the specific recognition of magnetic molecularly imprinted polymers, thus ensuring the sensitivity and reproducibility of the entire detection method from the source.

[0044] In some embodiments, the magnetic molecularly imprinted polymer comprises a template molecule, a functional monomer, and a crosslinking agent in a molar ratio of 1~3:4~5:16~20; the template molecule comprises adamantane; the functional monomer comprises methacrylic acid; and the crosslinking agent comprises ethylene glycol dimethacrylate. Furthermore, Fe3O4 magnetic nanoparticles are used as a carrier in the polymer.

[0045] By precisely defining the molar ratio of template molecule (adamantane), functional monomer (methacrylic acid), and crosslinking agent (ethylene glycol dimethacrylate), a magnetic molecularly imprinted polymer (MMIP) with antibody-like specific recognition ability for adamantane was successfully prepared, fundamentally solving the common technical problems of poor selectivity and severe matrix interference in traditional solid-phase extraction (SPE) and QuEChERS methods. Within this molar ratio range, the carboxyl groups in the functional monomer methacrylic acid form stable ionic and hydrogen bonds with the amino groups in the adamantane molecule. Simultaneously, the polymerization of excess crosslinking agent forms rigid three-dimensional cavities, ensuring that the shape, size, and chemical functional group arrangement of the imprinted cavities are precisely complementary to those of the adamantane molecule. After the template molecule is eluted, the polymer surface retains numerous specific recognition sites that match the lock-and-key of adamantane, enabling selective capture of adamantane from complex animal-derived sample extracts, while exhibiting almost no non-specific adsorption of structural analogs (such as rimantadine and memantine) and other co-extracted impurities (such as fatty acids, phospholipids, and pigments). Compared with conventional SPE columns, the MMIP of this invention has more than 3 times higher adsorption selectivity, the color of the purified extract changes from dark yellow to colorless and transparent, the matrix effect is significantly reduced, and the ion inhibition rate during LC-MS / MS analysis is significantly reduced, thereby greatly improving the detection sensitivity and the limit of quantitation. At the same time, it reduces the frequency of contamination of the chromatographic column and ion source and extends the service life of the instrument.

[0046] In some embodiments, the amount of magnetic molecularly imprinted polymer used is 30-40 mg of magnetic molecularly imprinted polymer per 2.0 g of animal-derived agricultural product sample; the adsorption time is 10-12 min.

[0047] In some embodiments, the liquid chromatography mobile phase gradient elution program is as follows: 0-1.5 min, 90% water, 10% methanol; 2-5 min, 10% water, 90% methanol; 6-10 min, 90% water, 10% methanol; wherein the water contains 0.1% formic acid by mass.

[0048] For example, the elution procedure is shown in Table 1 below:

[0049] Table 1

[0050] The injection volume is 0.8~1.2μL; the column temperature is 38~42℃; and the flow rate is 0.2~0.3ml / min.

[0051] For example, the injection volumes were 0.8 μL, 1 μL, and 1.2 μL; the column temperatures were 38 °C, 40 °C, and 42 °C; and the flow rates were 0.2 ml / min and 0.3 ml / min.

[0052] The above technical solution employs a water-methanol gradient elution program containing 0.1% formic acid, which can improve the peak shape of adamantane, enhance resolution, and shorten analysis time. Combined with an injection volume of 0.8~1.2μL, a column temperature of 38~42℃, and a flow rate of 0.2~0.3 mL / min, it achieves an optimal match between chromatographic separation efficiency, response intensity, and operational stability, reduces matrix interference, and improves mass spectrometry ionization efficiency, providing a reliable chromatographic basis for accurate quantification.

[0053] In some implementations, the mass spectrometry conditions are as follows:

[0054] Electrospray ionization (ESI) source; ion source temperature 280~350℃; detection method: multiple reaction monitoring (MRM); scanning method: positive ion scanning.

[0055] For example, the ion source temperatures are 280°C, 300°C, and 350°C.

[0056] The above technical solution can achieve efficient ionization and high selectivity detection of adamantane, effectively reduce background noise, improve signal-to-noise ratio and detection sensitivity, ensure qualitative accuracy and quantitative reliability, and enable the entire detection system to still have excellent detection performance at low residue levels.

[0057] Secondly, the embodiments of the present invention improve the application of the above-mentioned detection method in the detection of amantadine residues in animal-derived agricultural products. This technical solution clearly defines the direct application of this detection method in the detection of amantadine residues in animal-derived agricultural products. Based on the aforementioned optimization of the entire process of extraction, purification, concentration, and detection, it can achieve accurate, rapid, and high-throughput detection of various matrices such as livestock and poultry meat, eggs, milk, and aquatic products, providing a directly usable technical means for food safety monitoring.

[0058] Thirdly, the embodiments of the present invention provide the application of the above-described detection method in food safety supervision, third-party testing, enterprise quality control, and import and export inspection and quarantine.

[0059] Fourthly, embodiments of the present invention provide a pretreatment kit for detecting adamantane residue, comprising:

[0060] The product includes the aforementioned magnetic molecularly imprinted polymer powder, eluent, adamantane amine standard, D6-adamantane amine internal standard solution, sample tubes adapted for vacuum parallel concentration, and instructions for use.

[0061] To further illustrate the technical solution of the present invention, the following specific embodiments are provided. It should be understood that, unless otherwise stated, the raw materials used in the embodiments are all commercially available raw materials.

[0062] Used in the examples

[0063] Main instruments:

[0064] BSA2202S electronic balance, BSA224S electronic balance, Agilent 1290 Infinity / 6460 TripleQuad liquid chromatograph-mass spectrometer, EXPEC 510S reduced pressure parallel concentrator, DH18BR high-speed refrigerated centrifuge, ZKNEV-24 nitrogen evaporator, VORTEX3 vortex mixer.

[0065] Example 1: Preparation of adamantane amine magnetic molecularly imprinted polymer (MMIP)

[0066] 1.0 mmol of adamantane template molecules, 4.0 mmol of methacrylic acid functional monomer, 20 mmol of ethylene glycol dimethacrylate crosslinking agent, and 0.5 g of Fe3O4 magnetic nanoparticles were dissolved in 50 mL of acetonitrile and ultrasonically dispersed at 300 W for 15 min. After nitrogen purging to remove oxygen, 0.05 g of azobisisobutyronitrile was added, and polymerization was carried out in a water bath at 60 °C for 24 h. The polymer product was repeatedly eluted with methanol / acetic acid (9:1, v / v) to remove template molecules until no adamantane residue was detected by liquid chromatography-mass spectrometry. After vacuum drying, the magnetic molecularly imprinted polymer (MMIP) was obtained for later use. The saturated adsorption capacity of the MMIP for adamantane was 3.8 μg / mg, and the selectivity coefficient (for damantane) was 12.5.

[0067] Example 2

[0068] This embodiment provides a method for detecting amantadine residues in animal-derived agricultural products:

[0069] 1) Extraction and Magnetic Molecular Imprinting Purification: Weigh 2.0 g of pig liver sample into a 50 mL centrifuge tube, add 100 μL of 100 ng / mL D6-amantadine internal standard working solution, add 10 mL of 1% acetic acid-acetonitrile solution, vortex for 2 min, centrifuge at 3000 r / min for 5 min, and collect the supernatant. Add 30 mg of MMIP powder to the supernatant, vortex for 10 min, and separate the polymer under the action of an external magnetic rack, discarding the supernatant. Add 2 mL of methanol / acetic acid (9:1, v / v) eluent, vortex for 5 min, magnetically separate, collect the eluent, repeat the elution once, and combine the eluents (total volume approximately 4 mL) as the test solution.

[0070] (2) Vacuum parallel concentration: Transfer the test solution to a round-bottom glass test tube for vacuum parallel concentrator, set the vacuum degree to 50 mbar, the water bath temperature to 40℃, and the oscillation rate to 180 rpm, and concentrate for 22 min to obtain the concentrated solution.

[0071] (3) LC-MS / MS detection: 1.0 mL of a methanol-water (1:1, v / v) solution containing 0.1% formic acid was mixed with the concentrate, vortexed for 30 s, filtered through a 0.22 μm filter, and the filtrate was used for LC-MS / MS determination. LC conditions: The column was an Agilent ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm); mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was methanol; the gradient elution program is shown in Table 1 above. Serial mass spectrometry conditions: electrospray ionization source, positive ion scan mode, multiple reaction monitoring, ion source temperature 300℃.

[0072] Example 3

[0073] This embodiment is an experiment to investigate the effects of oscillation rate, concentration temperature and vacuum degree on the final recovery rate of the product to be tested during the parallel concentration process under reduced pressure.

[0074] Vacuum levels of 30 mbar, 50 mbar, and 70 mbar were selected; concentration temperatures of 40℃, 45℃, and 50℃ were selected; and oscillation rates of 150 rpm, 180 rpm, and 220 rpm were selected. Orthogonal experiments were conducted on the above three parameters with different specifications.

[0075] An orthogonal experiment was conducted with three levels of vacuum, three levels of concentration temperature, and three levels of oscillation rate to perform a full-process determination on three chicken samples with a concentration of 5 μg / kg. The results were evaluated based on whether there was boiling over, the concentration time required, and the recovery rate of adamantane. The results are shown in Table 2.

[0076] Concentration time refers to the time required to concentrate the purified extract to dryness.

[0077] The recovery rate of adamantane is calculated as follows: the absolute mass of the standard substance added to each sample is divided by the theoretical mass, and the result is expressed as a percentage, based on the measurement results of liquid chromatography-mass spectrometry.

[0078] Table 2. Selection of Instrument Conditions for the Parallel Concentrator under Reduced Pressure

[0079] The results above show that the concentration temperature in the range of 40 to 50°C has little effect on the concentration time and recovery rate. Considering both the recovery rate and the concentration efficiency, the optimal parameter combination is selected as follows: vacuum degree 50 mbar, concentration temperature 40°C, and oscillation speed 180 rpm.

[0080] Example 4

[0081] This embodiment provides validation of the detection method in Example 1 for the analysis of amantadine residues in pig liver.

[0082] Adamantane at concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL was detected using the method described in Example 1. Peak areas were calculated and are shown in Table 3. A linear relationship between concentration and peak area was plotted, as shown in Table 3. Figure 1 As shown, the linear equation is: y = 0.156027x - 0.015801 Correlation coefficient: R0 2 =0.9998.

[0083] Table 3. Determination results of the standard series of adamantane.

[0084] Then, pig liver samples with a concentration of 0.3 µg / kg were subjected to full-procedure analysis to determine the signal-to-noise ratio (S / N) of each target compound. The method limit of quantitation was three times the method limit of detection. The results are shown in Table 4.

[0085] Table 4. Limits of Detection and Limits of Quantification (µg / kg)

[0086] Three pig liver samples with concentrations of 1 μg / kg, 2 μg / kg, and 5 μg / kg were taken and precision tests were performed according to the complete procedure. The results are shown in Table 5.

[0087] Table 5 Precision test results of adamantane.

[0088] Nine pig liver samples were taken, and three 2 ng, 5 ng, and 10 ng amantadine standards were added to each sample. The accuracy test was carried out according to the full procedure, and the results are shown in Table 6.

[0089] Table 6 Results of Spiked Recovery Determination

[0090] Example 5

[0091] This embodiment provides a method for analyzing amantadine residues in eggs.

[0092] Adamantane at concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL was detected using the method described in Example 1. Peak areas were calculated and are shown in Table 7. A linear relationship between concentration and peak area was plotted, as shown in Table 7. Figure 2 As shown, the linear equation is: y = 0.149399x + 0.027535. Correlation coefficient: R0 2 =0.9993.

[0093] Table 7. Determination results of the standard series of adamantane.

[0094] Limit of detection and limit of quantitation

[0095] Egg samples with a concentration of 0.3 µg / kg were subjected to a full-process assay to determine the signal-to-noise ratio (S / N) of each target compound. The method limit of quantitation was three times the method limit of detection. The results are shown in Table 8.

[0096] Table 8. Limits of Detection and Limits of Quantification (µg / kg)

[0097] Precision

[0098] Three egg samples were taken at three different concentration levels: 1 μg / kg, 2 μg / kg, and 5 μg / kg. Precision tests were performed according to the complete procedure. The results are shown in Table 9.

[0099] Table 9 Precision test results of adamantane.

[0100] Accuracy

[0101] Nine pig liver samples were taken, and three 2ng, 5ng, and 10ng amantadine standards were added to each sample. The accuracy test was carried out according to the full procedure, and the results are shown in Table 10.

[0102] Table 10 Results of Spiked Recovery Determination

[0103] As can be seen from the above results, the detection method of Embodiment 1 of the present invention has high accuracy and can detect multiple samples simultaneously, which is convenient and fast.

[0104] Comparative Example 1

[0105] Traditional SPE solid-phase extraction combined with vacuum parallel concentration

[0106] The purification method using the MCX solid-phase extraction column reported in the literature was adopted: The pig liver sample was extracted with 1% acetic acid and acetonitrile, and the supernatant was passed through an MCX column (activated sequentially with methanol, water, and 2% formic acid), eluted with 2% formic acid-water-methanol (95:5), and eluted with 5% ammonia-methanol. The eluent was concentrated to dryness under reduced pressure and then reconstituted before injection. The remaining steps (reduced pressure concentration parameters and LC-MS / MS conditions) were the same as in Example 2. The results are shown in Table 11.

[0107] The full-procedure determination was performed on pig liver samples with three different content levels of 1 μg / kg, 2 μg / kg, and 5 μg / kg.

[0108] Table 11

[0109] Comparative Example 2

[0110] In Comparative Example 2, compared to Example 1, the samples were extracted using the QuEChERS method: chicken samples were extracted with 1% acetic acid acetonitrile, anhydrous sodium sulfate and sodium chloride were added for salting out, the supernatant was collected, purified with PSA, centrifuged, and the supernatant was concentrated to dryness using a rotary evaporator (40℃ water bath), reconstituted, and detected by LC-MS / MS. The results are shown in Table 10.

[0111] The chicken samples with three different content levels of 1 μg / kg, 2 μg / kg and 5 μg / kg were subjected to full-process determination, and the results are shown in Table 12.

[0112] Table 12

[0113] To verify the technical superiority of the constructed detection system, this invention sets up parallel comparison ratios to accurately benchmark two mainstream conventional detection methods in the industry: the traditional MCX cation exchange solid-phase extraction coupled with vacuum parallel concentration system and the QuEChERS rapid extraction coupled with rotary evaporation concentration system. Through full-process performance comparison, the substantial breakthrough and innovative value of the technical solution of this invention are fully highlighted. Comparative Example 1 employs the industry-standard solid-phase extraction purification pathway, coupled with a vacuum parallel concentration process identical to that of this invention. However, due to the inherent limitations of solid-phase extraction technology, it cannot achieve specific targeted enrichment of adamantane, and its selectivity for purifying complex animal-derived matrices such as pig liver (high in protein and fat) is insufficient. A large amount of co-extracted impurities such as fatty acids, phospholipids, and pigments remain in the eluent after purification. This not only easily leads to sample boiling during subsequent concentration but also produces a significant matrix inhibition effect during liquid chromatography-tandem mass spectrometry detection, directly causing a substantial decrease in the precision of the detection results. The relative standard deviations at the three spiking levels of 1 μg / kg, 2 μg / kg, and 5 μg / kg reached 10.8%, 5.9%, and 4.9%, respectively, far exceeding the precision level of 1.3% to 3.1% achieved by this invention under the same matrix. Furthermore, this method requires multiple cumbersome steps, including solid-phase extraction column activation, sample loading, rinsing, and elution, with a single batch sample pretreatment cycle of up to 1.5 hours, making it impossible to achieve high-throughput rapid detection of large batches of samples.

[0114] Comparative Example 2 uses QuEChERS rapid extraction technology combined with traditional rotary evaporation concentration. While this approach simplifies the extraction and purification steps to some extent, it suffers from two major insurmountable technical defects. First, the purification capacity of QuEChERS technology is limited, only removing some organic acid impurities from the sample through PSA adsorbent. Its purification effect on high-protein and high-fat components in animal-derived matrices is severely insufficient, and matrix interference remains a prominent issue. Second, traditional rotary evaporation is a single-channel, intermittent operation, processing only a single sample at a time, making simultaneous concentration of batches impossible and severely limiting throughput. Furthermore, the determination of the concentration endpoint relies entirely on manual subjective operation, resulting in insufficient precision in temperature and vacuum control. This easily leads to the loss of adamantane, which is highly sensitive to temperature and vacuum, due to solvent vapor evaporation, directly causing a decline in both precision and accuracy of the detection results. The relative standard deviation of chicken samples spiked at the same concentration gradient can reach up to 8.3%, far inferior to the detection performance of this invention. Moreover, the concentration time for a single batch of samples far exceeds that of the vacuum parallel concentration process of this invention, completely failing to meet the rapid detection needs of large batches of samples in the field of food safety supervision.

[0115] Based on the results of two sets of comparative system experiments, the core innovation of this invention lies in its first breakthrough of the technical path dependence of traditional adamantane residue detection. It deeply couples magnetic molecularly imprinted polymer (MMIP) specific purification technology with depressurized parallel concentration non-destructive enrichment technology, constructing a system of targeted extraction, specific purification, precise concentration, and highly sensitive detection. This completely solves the long-standing technical problems of incomplete purification, easy concentration loss, and low detection throughput in adamantane residue detection. Simultaneously, through a three-factor, three-level orthogonal experiment, this invention clarifies for the first time the non-destructive concentration process window for adamantane, breaking the industry's common technical bias that low vacuum and high temperature concentration are more efficient. It confirms that the parameter combination of 40-50 mbar vacuum, 40-50℃ water bath temperature, and 150-200 rpm can ensure concentration efficiency while completely avoiding the risks of sample boiling and thermal degradation and volatilization loss of the target substance, achieving the optimal balance between concentration efficiency and target substance recovery rate. More importantly, the two core technologies developed in this invention form a significant synergistic effect. The high selectivity of MMIP purification provides a clean sample environment with low impurities for vacuum parallel concentration, eliminating the risk of boiling during the concentration process at its source. Meanwhile, the precise and controllable vacuum parallel concentration process can completely retain the target substance after MMIP purification, avoiding irreversible loss of the target substance caused by traditional concentration processes. Comparative supplementary data confirms that when using MMIP purification alone in combination with traditional rotary evaporation concentration, the recovery rate of amantadine is only 69%~75%. When using traditional SPE purification alone in combination with vacuum parallel concentration, the pretreatment operation is cumbersome and the precision is seriously insufficient. However, after combining the two technologies, this invention can process more than 30 samples simultaneously in a single batch, shorten the total pretreatment time to less than 0.5 hours, reduce the amount of organic solvent used by more than 40%, and maintain the target substance recovery rate stably at more than 85%. The precision, limit of detection, and limit of quantitation all fully meet the national technical specifications for veterinary drug residue detection. This invention provides a brand-new technical solution for high-throughput, high-precision, and low-cost detection of amantadine residues in animal-derived agricultural products, possessing outstanding substantive features and significant technological progress.

[0116] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting a residue of a trimethylamine in an animal-derived agricultural product based on parallel concentration under reduced pressure, characterized by, Includes the following steps: (1) Adamantane in the extract of animal-derived agricultural products was specifically adsorbed by a magnetic molecularly imprinted polymer. The adamantane was separated and enriched under the action of an external magnetic field. Then, the adamantane was eluted from the magnetic molecularly imprinted polymer with an elution buffer of methanol-acetic acid volume ratio of 8~10:

1. The elution volume was 2~5 mL and the elution time was 10~15 min to obtain the test solution. (2) The test solution is subjected to reduced pressure parallel concentration for 20-25 min under the conditions of vacuum degree of 40-50 mbar, water bath temperature of 40-50℃ and oscillation rate of 1500-200 rpm to obtain concentrated solution; (3) The concentrate was analyzed by liquid chromatography-tandem mass spectrometry to obtain the residual amount of adamantane; The preparation process of the magnetic molecularly imprinted polymer includes the following steps: Take 1.0 mmol of adamantane template molecule, 4.0 mmol of methacrylic acid functional monomer, 20 mmol of ethylene glycol dimethacrylate crosslinking agent, and 0.5 g of Fe3O4 magnetic nanoparticles, dissolve them in 50 mL of acetonitrile, disperse them by ultrasonication at 300 W for 15 min, remove oxygen by nitrogen purging, add 0.05 g of azobisisobutyronitrile, and polymerize in a water bath at 60 °C for 24 h to obtain the polymer product; The polymer product was repeatedly eluted with methanol / acetic acid at a volume ratio of 9:1 to remove template molecules until no adamantane residue was detected by liquid chromatography-mass spectrometry. After vacuum drying, the magnetic molecularly imprinted polymer was obtained for later use. The saturated adsorption capacity of the magnetic molecularly imprinted polymer for adamantane was 3.8 μg / mg, and the selectivity coefficient for damantane was 12.

5.

2. The detection method according to claim 1, characterized in that, The method for preparing the animal-derived agricultural product sample extract is as follows: weigh the homogenized animal-derived agricultural product sample, add D6-adamantaneamine internal standard working solution and acetic acid acetonitrile solution, centrifuge, and take the supernatant as the sample extract.

3. The method of claim 1, wherein, The magnetic molecularly imprinted polymer comprises a template molecule, a functional monomer, and a crosslinking agent in a molar ratio of 1~3:4~5:16~20; the template molecule comprises adamantane; the functional monomer comprises methacrylic acid; and the crosslinking agent comprises ethylene glycol dimethacrylate.

4. The method of claim 1, wherein, The amount of the magnetic molecularly imprinted polymer used is 30-40 mg of magnetic molecularly imprinted polymer per 2.0 g of animal-derived agricultural product sample; the adsorption time is 10-12 min.

5. The detection method according to claim 1, characterized in that, The liquid chromatography conditions were as follows: a C18 column and a gradient elution program: ； The injection volume was 0.8~1.2μL; column temperature was 38~42℃; and flow rate was 0.2~0.3ml / min.

6. The detection method according to claim 1, characterized in that, The tandem mass spectrometry conditions are: electrospray ionization source, positive ion scanning mode, multiple reaction monitoring, and ion source temperature of 300–350 °C.

7. The application of the detection method according to any one of claims 1 to 6 in the detection of amantadine residues in animal-derived agricultural products.

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