A method for determining the residue of amido alcohols and their metabolites in milk

Abstract

This invention relates to the field of food safety testing technology and discloses a method for determining the residual amounts of amide alcohols and their metabolites in milk. The method involves adding an internal standard to a milk sample, followed by the addition of a composite desorption solvation reagent and shaking to induce the desorption of the target analyte from its protein-bound state into the free aqueous phase. Subsequently, magnetic HLB microparticles are introduced and an external magnetic field is applied to anchor the microparticles to the inner wall of the container for spatial isolation. Under these conditions, low-speed horizontal cross-flow is applied, and a phase transition triggering and cross-flow flocculation reagent is added dropwise to induce flocculation of protein impurities at the center of the system, simultaneously driving the free target analyte phase to transfer to the internal pores of the inner wall microparticles. Finally, after draining the central flocculation waste liquid, matrix washing, and targeted elution, quantitative analysis is performed using liquid chromatography-tandem mass spectrometry. This invention, through the combination of in-situ desorption and cross-flow phase transfer, avoids protein flocculants clogging the extraction channels, achieving simultaneous and efficient enrichment of impurities and target analytes.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically a method for determining the residual amounts of amide alcohols and their metabolites in milk. Background Technology

[0002] Amide alcohols, including chloramphenicol, thiamphenicol, florfenicol, and their metabolites, are commonly used as broad-spectrum antibiotics for disease prevention and treatment in dairy cattle and other livestock farming. These drugs and their metabolites have certain toxic side effects. If they remain in milk during lactation, long-term human ingestion can pose potential harm to the hematopoietic system and other systems. Therefore, establishing accurate and reliable methods for detecting amide alcohol residues is fundamental to food safety monitoring. Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the routine analytical method for quantifying these residues; however, the sensitivity and accuracy of the analysis largely depend on sample pretreatment techniques.

[0003] Milk is a complex matrix containing high concentrations of proteins, fats, and large molecular aggregates. Current sample pretreatment methods typically involve adding organic solvents or acidic precipitants to denature and precipitate proteins. However, in practice, high-concentration proteins tend to form large flocs during sedimentation. These dense flocs often adsorb or directly encapsulate free amide alcohol molecules on their surface and internal structure, leading to the loss of the target analyte with the waste liquid or precipitate, resulting in a low overall extraction recovery rate. Furthermore, conventional centrifugation combined with solid-phase extraction (SPE) is cumbersome, requiring multiple independent steps such as centrifugation, supernatant transfer, activation, sample loading, rinsing, and elution. If precipitation separation is incomplete, the tiny suspended protein particles remaining in the liquid phase can easily adhere to the packing surface or directly clog the microporous sieve plate when flowing through the SPE column, causing a sudden drop in flow rate or even column failure.

[0004] To simplify traditional solid-phase extraction (SPE), some technical solutions have introduced dispersive SPE or magnetic SPE. However, when solid extraction particles are directly introduced into a milk matrix system where protein precipitation is occurring, a large amount of precipitated protein flocs physically mix and entangle with the added extraction particles. Macromolecular impurities not only cover the active sites on the surface of the extraction material, hindering the diffusion and mass transfer of the target analyte into the material's internal pores, but also cause the particles to be encapsulated within viscous flocs. This mixed state makes it difficult for an external magnetic field to completely separate the adsorbent material from the protein residue, and the extraction phase easily carries a large amount of matrix impurities. After these impurities are eluted and enter the mass spectrometry system, they can cause severe ion suppression or enhancement phenomena, i.e., produce a significant matrix effect, thus seriously interfering with the stability and accuracy of quantitative results. Existing pretreatment techniques generally face a core contradiction when dealing with high-fat, high-protein emulsion matrices: the in-situ precipitation and flocculation process of matrix macromolecules and the solid-phase enrichment process of the target analyte occur in the same physical space and time dimension. This spatiotemporal overlap inevitably leads to competition between physical entanglement and chemical adsorption for interphase mass transfer. Existing technologies struggle to effectively exclude macromolecular impurities and efficiently recover trace amounts of polar target substances within a single reaction system. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for determining the residual amounts of amide alcohols and their metabolites in milk. This method solves the problems in existing milk sample pretreatment where protein precipitates easily encapsulate the target analytes and become entangled and blocked by extraction particles, resulting in low extraction recovery rates and severe matrix effects.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a method for determining the residual amounts of amide alcohols and their metabolites in milk, employing the following technical solution: A method for determining the residual amounts of amide alcohols and their metabolites in milk includes the following steps: Take the milk sample to be tested and place it in the reaction vessel. Add the isotope internal standard mixture, vortex mix and let stand to obtain the sample mixture. A composite desorption solubilizing reagent is added to the sample mixture and subjected to isothermal oscillation to inhibit macromolecular aggregation and promote the desorption and release of the target analyte into the free aqueous phase; magnetic HLB microparticles are added, and after deceleration and oscillation, a strong magnetic field is applied to the outer wall of the reaction vessel, and the mixture is allowed to stand so that the microparticles are directionally adsorbed onto the inner wall for physical spatial isolation. Maintaining the magnetic field anchoring state, a low-speed horizontal cross-flow is applied to the mixture in the reaction vessel, and a phase change trigger and cross-flow flocculation reagent is added and then aged; micro-phase separation is induced and a solvent-rich wetting layer is formed on the surface of the particles, driving the free target material to undergo thermodynamic phase transfer into the internal pores of the particles, while causing protein impurities to undergo cross-flow flocculation in the center of the reaction vessel; Maintain the outer wall magnetic field and discharge the central protein flocculation waste liquid; remove the magnetic field, add matrix washing solution to wash, apply the magnetic field again and discard the waste liquid; remove the magnetic field, add targeted elution solution, sonicate and apply the magnetic field to collect the supernatant; The collected supernatant liquid nitrogen was blown to near dryness, reconstituted, and injected into an ultra-high performance liquid chromatography-tandem mass spectrometer for quantitative analysis.

[0007] By employing the above technical solution, and through the use of specific reagent combinations combined with physical spatial isolation and cross-flow hydrodynamic control, this invention achieves simultaneous matrix desorption, macromolecular exclusion, and target enrichment. The corresponding microscopic mechanism and separation process are specifically manifested as follows: After adding the complex desorption solvation reagent, the reagent components penetrate into the milk protein molecules, thereby disrupting the hydrogen bonds and hydrophobic interactions that maintain the protein's spatial conformation. This leads to a moderate unfolding of the protein, causing the amide alcohols and their metabolites that were originally embedded or attached to the binding sites to lose their binding force and be released into the free aqueous phase. Combined with isothermal oscillation treatment, this not only accelerates the aforementioned mass transfer and denaturation processes but also inhibits the disordered aggregation of macromolecules within the system, preventing the target substance from being re-encapsulated.

[0008] After the microparticles are added and a magnetic field is applied, the magnetic HLB microparticles, which have a hydrophilic-lipophilic balance, are fixed to the inner wall of the reaction vessel. This operation achieves physical separation of the solid-phase extraction medium from the liquid phase at a macroscopic level, reserving independent space for subsequent matrix precipitation reactions and fundamentally preventing the generated protein flocs from entangled with the extraction microparticles or from clogging the pores.

[0009] When phase-change triggering and cross-flow flocculation reagents are added, the ionic strength and pH of the system change. Due to the salting-out effect and isoelectric point convergence, the hydration layer of free proteins dehydrates, transforming into insoluble flocs. Simultaneously, the fluid shear force generated by the low-velocity horizontal cross-flow causes these flocs to aggregate towards the central axis region of the system. From a chemical thermodynamic perspective, the high-salt environment drives the original organic solvent molecules in the system to migrate towards the surface of the particles anchored on the inner wall, thus forming a locally solvent-rich wetting layer. Driven by the chemical potential difference, free amide alcohol molecules in the aqueous phase spontaneously cross the phase interface, enter this wetting layer, and further diffuse into the internal pores of the particles, where they are fixed. The synergy of these steps effectively decouples impurity sedimentation and target analyte enrichment in both space and time.

[0010] Preferably, the composite desorption solvation reagent is composed of the following components in parts by weight per 100.0 parts of the milk sample to be tested: 10.0-15.0 parts of urea; 0.4-0.8 parts of sodium octyl sulfate; 12.0-18.0 parts of tert-butanol; and 20.0-30.0 parts of deionized water.

[0011] By employing the above technical solution, urea, as a non-ionic denaturant, can penetrate protein clefts to disrupt hydrogen bonds, while sodium octyl sulfate inserts into the hydrophobic core region of proteins via hydrocarbon chains to unfold polypeptide chains; simultaneously, tert-butanol acts as a regulator of dielectric constant and a phase transition precursor in the system. These components work synergistically to promote the conversion of protein-bound analytes into free states without inducing macroscopic matrix precipitation, thereby improving extraction recovery.

[0012] Preferably, the phase change triggering and cross-flow flocculation reagent is composed of the following components in parts by weight per 100.0 parts of the milk sample to be tested: 12.0 to 18.0 parts of ammonium sulfate; 0.3 to 0.8 parts of a buffer salt with a pH of 4.6; wherein the buffer salt is composed of glacial acetic acid and anhydrous sodium acetate; and 80.0 to 100.0 parts of deionized water.

[0013] By employing the above-mentioned technical solution, the sulfate and ammonium ions dissociated from ammonium sulfate can competitively bind to water molecules on the protein surface. Combined with a buffer system composed of glacial acetic acid and anhydrous sodium acetate, the pH of the solution is adjusted to near the isoelectric point of casein. This dual effect promotes the neutralization of surface charges on protein molecules and the rupture of the hydration film, leading to flocculation and sedimentation. Furthermore, the localized high ionic strength also promotes the precipitation of tert-butanol within the system, thereby creating a suitable microenvironment for the extraction of the target analyte on the surface of the magnetic particles.

[0014] Preferably, the conditions for the isothermal oscillation treatment are as follows: the temperature is controlled at 30-40°C, and an oscillation treatment at 1500-2500 rpm is applied for 2-5 minutes; the speed of the deceleration oscillation is controlled at 300-500 rpm, and the treatment time is 1-3 minutes; the apparent magnetic field strength of the applied strong magnetic field is greater than 0.5 Tesla, and the static anchoring time is 45-90 seconds.

[0015] By adopting the above technical solution, setting a temperature of 30-40°C and coordinating with high-speed oscillation increases the frequency of molecular thermal motion, which helps to improve desorption efficiency; subsequent deceleration oscillation promotes uniform dispersion of the added magnetic particles in the liquid phase. Due to the application of an apparent magnetic field greater than 0.5 Tesla, the magnetic particles can overcome the viscous resistance of the liquid within 45 to 90 seconds and stably adhere to the inner wall of the container, thereby establishing a stable solid-phase extraction isolation zone.

[0016] Preferably, the rotation speed of the low-speed horizontal cross-flow is 40-80 rpm; the constant rate of adding the phase change trigger and cross-flow flocculation reagent is controlled at 0.2-0.8 parts by weight per second; and the aging time after the addition is completed is 2-4 minutes.

[0017] By employing the above technical solution, the low-speed cross-flow of 40 to 80 rpm maintains a suitable mass transfer motive force for the liquid phase, promoting the collision and aggregation of suspended protein particles towards the center, thus avoiding the problem of floc fragmentation or dispersal of anchored particles due to excessive shear force. Controlling the droplet acceleration rate within this range can prevent local solution supersaturation and non-specific co-precipitation on the particle surface. The aging process provides the necessary time for thermodynamic phase transfer equilibrium and central flocculation reaction.

[0018] Preferably, the matrix cleaning solution is prepared by mixing methanol and deionized water at a mass ratio of 1:19; the targeted elution solution is prepared by mixing acetonitrile and deionized water at a mass ratio of 4:1; based on 100.0 parts by weight of the milk sample to be tested, the amount of the matrix cleaning solution is 140.0 to 160.0 parts by weight, and the amount of the targeted elution solution is 110.0 to 130.0 parts by weight.

[0019] By employing the above technical solution and using a washing solution with a low methanol ratio, water-soluble impurities and residual free proteins adhering to the surface of microparticles can be washed away without causing leakage of the target analyte. A targeted eluent with a high acetonitrile ratio easily penetrates into the lipophilic pores of the microparticles, disrupting the retention between the analyte and the microparticles, thereby achieving desorption and recovery of the target analyte.

[0020] Preferably, the isotope internal standard mixture is prepared by mixing chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicol-D3 in a mass ratio of 1:1:1:1; the resolution is specifically performed by resolution with an aqueous solution of ammonium formate with a concentration of 10 mmol / L.

[0021] By employing the above technical solution and adding isotopic internal standards corresponding to the structures of the four target analytes, it is helpful to correct for material loss during the pretreatment process and matrix effects during the mass spectrometry ionization stage. Using a 10 mmol / L ammonium formate aqueous solution as the reconstitution medium can better match the chromatographic flow, improving the chromatographic peak shape while enhancing the ionization efficiency of mass spectrometry detection, thus ensuring the accuracy of quantitative analysis.

[0022] A method for preparing magnetic HLB microparticles includes the following steps: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were dissolved in deoxygenated deionized water, stirred and aged with ammonia water dropwise under nitrogen protection, magnetically separated, washed and dried to obtain iron oxide nanoparticles. The iron oxide nanoparticles were dispersed in anhydrous toluene, and a silane coupling agent was added and refluxed. After magnetic separation and washing, modified magnetic microparticles were obtained. The modified magnetic microparticles were dispersed in acetonitrile, and hydrophilic monomers and crosslinking agent monomers were added. After deoxygenation, an initiator was added, and a thermally initiated polymerization reaction was carried out at a constant temperature. Unreacted monomers were extracted and the mixture was freeze-dried to obtain the magnetic HLB microparticles.

[0023] By employing the above technical solution, this invention prepares a core-shell structured magnetically responsive solid-phase extraction medium, combining the macroscopic displacement control properties of an inorganic magnetic core with the microscopic mass transfer and enrichment characteristics of an organic polymer shell. Regarding its specific formation process and mechanism: In an alkaline environment, ferrous and ferric ions in a deoxygenated deionized water system undergo a co-precipitation reaction, forming superparamagnetic magnetite (Fe3O4) crystals. Continuously introducing nitrogen gas to isolate the air prevents the oxidation of ferrous ions and maintains the purity of the crystal form. These magnetite nanoparticles constitute the magnetic core of the microparticles, providing the basis for their subsequent directional migration and spatial anchoring under an applied magnetic field.

[0024] In the subsequent silanization modification, the alkoxy group at the terminal of the silane coupling agent molecule hydrolyzes in anhydrous toluene to generate a silanol group, which then undergoes condensation dehydration with the hydroxyl group on the surface of iron(III) oxide to form a stable silicon-oxygen bond. This bonding method grafts an organic functional group with polymerizable double bonds onto the surface of the inorganic magnetic core. Using anhydrous toluene as the reaction dispersion medium restricts the self-polymerization reaction of the silane coupling agent in the liquid bulk, enabling it to form a relatively dense monomolecular coating layer on the magnetic core surface, thus constructing a chemical bond bridge for subsequent polymer coating.

[0025] In the anaerobic in-situ surface grafting copolymerization stage, the initiator undergoes homolytic cleavage upon heating, generating free radicals that attack the double bonds in the monomers to initiate chain polymerization. Because unsaturated double bonds are pre-grafted onto the magnetic core surface, the added hydrophilic monomer and crosslinking agent monomer segments undergo random copolymerization and crosslinking on this surface, growing to form a three-dimensional network of polymer shells. In this structure, polar hydrophilic monomers provide wettability in the aqueous phase and hydrogen bonding sites, while nonpolar crosslinking agent monomers are responsible for constructing the hydrophobic framework and channels. The combined effect of these two components endows the microparticles with a balanced hydrophilic and lipophilic property, facilitating their wetting in complex phase-transfer systems and effectively trapping amide alcohol target molecules through hydrophobic interactions and electrostatic attraction.

[0026] Preferably, the ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved in deoxygenated deionized water at a molar ratio of 2:1, and under nitrogen protection at 75-85°C, the mixture is stirred at 800-1000 rpm, and ammonia water with a mass fraction of 25% is added dropwise at a uniform rate until the pH of the system reaches 9.5-10.5, and the mixture is allowed to mature for 30-45 minutes.

[0027] By adopting the above technical solution, the stoichiometric ratio of ferric iron (Fe3O4) and ferrous iron (Fe2O4) in a molar ratio of 2:1 was well matched, which to a certain extent suppressed the generation of non-magnetic impurity phases. A temperature of 75 to 85°C combined with stirring provided suitable thermodynamic and mass transfer conditions for crystallization. The addition of 25% ammonia water raised the pH of the system to 9.5 to 10.5, promoting the precipitation of free iron ions; coupled with a continuous ripening process of 30 to 45 minutes, it drove the dissolution of small crystals and their redeposition on the surface of large crystals, helping to perfect the crystal structure and thus obtaining a magnetite core with a relatively uniform particle size distribution and a certain saturation magnetization.

[0028] Preferably, the silane coupling agent is 3-(methacryloyloxy)propyltrimethoxysilane; the hydrophilic monomer is N-vinylpyrrolidone; the crosslinking agent monomer is divinylbenzene; and the initiator is azobisisobutyronitrile. The conditions for the thermally initiated polymerization reaction are: temperature 65-75°C, rotation speed 200-300 rpm, and reaction time 16-24 hours.

[0029] By employing the above technical solution, 3-(methacryloyloxy)propyltrimethoxysilane utilizes the condensation of methoxy groups with iron-atom hydroxyl groups, exposing its methacryloyl groups to the liquid phase, providing grafting sites for subsequent polymerization reactions. The amide ring structure of the hydrophilic monomer N-vinylpyrrolidone endows the shell with hydrophilicity, improving the dispersion state of the extraction material in the aqueous phase; the dual vinyl structure of divinylbenzene forms a porous hydrophobic framework after crosslinking, and the two work together to achieve a balance between the hydrophilicity and lipophilicity of the microparticles. Using azobisisobutyronitrile as an initiator, the free radicals are dissociated at 65 to 75 °C, resulting in a relatively stable initiation rate; combined with an appropriate rotation speed and a reaction time of 16 to 24 hours, the polymer monomers can undergo sufficient addition polymerization on the magnetic core surface. This avoids pore blockage caused by excessively rapid crosslinking, or excessive shielding of the core magnetic field response due to an excessively thick shell, thus preparing magnetic microparticles with a regular structure and an affinity for amide alcohols.

[0030] This invention provides a method for determining the residual amounts of amide alcohols and their metabolites in milk. It has the following beneficial effects: 1. This invention utilizes a composite desorption solubilizing reagent to disrupt the hydrogen bonds and hydrophobic interactions of milk proteins, promoting the full release of bound target analytes. Simultaneously, it incorporates phase transition triggering and cross-flow flocculation reagents to induce matrix proteins to aggregate and settle at the center of the system. This mechanism, combining in-situ desorption and cross-flow phase transfer, physically avoids the blockage and entanglement of protein flocs in the solid-phase extraction microparticle channels, fundamentally cutting off the pathway of polar target molecules being lost through co-precipitation with endogenous macromolecules. This effectively reduces the interference of the complex milk matrix on mass spectrometry detection and improves the extraction and recovery rate of amide alcohol drugs and their metabolites.

[0031] 2. This invention uses an external magnetic field to orient magnetic HLB particles onto the inner wall of the reaction vessel, establishing a physical isolation zone between the extraction medium and the bulk liquid phase. Compared to conventional centrifugal precipitation or multi-step column solid-phase extraction, this operation eliminates the cumbersome liquid transfer steps. Relying on microscopic phase separation to drive free target substances across the interface into the inner wall particles, impurity exclusion and target substance enrichment are simultaneously completed within a single container. This significantly shortens the pretreatment cycle for a single sample and, without compromising the target substance extraction rate, greatly reduces the consumption of toxic organic solvents and the generation of solid waste, making it more suitable for rapid screening and high-throughput detection of large batches of liquid milk samples.

[0032] 3. This invention prepares magnetic HLB microparticles with a core-shell structure using surface grafting in-situ copolymerization technology. The polymer shell is formed by copolymerizing hydrophilic monomers and crosslinking agent monomers. The balanced hydrophilic-lipophilic network within these microparticles allows them to maintain good wetting in an aqueous matrix containing some organic solvents, perfectly matching the mesoscopic liquid film microenvironment induced by the high-salt environment of this invention. Relying on the synergy of this specific microstructure and the thermodynamic phase transfer mechanism, these microparticles not only exhibit strong adsorption and retention of amide alcohol molecules, but also, the encapsulated iron oxide nanocrystals provide sufficient magnetic response intensity, ensuring rapid separation and non-destructive collection of the microparticles in multiphase mixed systems, further guaranteeing the repeatability and accuracy of quantitative analysis. Attached Figure Description

[0033] Figure 1 This is a graph showing the results of the macroscopic fluid phase evolution and solid-liquid separation stability test of the present invention; Figure 2 The diagrams are for verifying the thermodynamic phase transfer-mediated mass transfer mechanism of the present invention, wherein (a) is a mass transfer trajectory diagram between the target phases, and (b) is a diagram of the residual liquid phase of the co-solvent. Figure 3 This is a graph showing the absolute recovery rate and precision distribution of the target analyte under different pretreatment processes according to the present invention. Figure 4 This is a quantitative comparison diagram of the matrix effect of different pretreated extracts on the target compound according to the present invention; Figure 5 The diagram shows the comprehensive pretreatment efficiency and green chemistry evaluation of the present invention. (a) is a comparison diagram of single sample processing time and organic solvent consumption, and (b) is a comparison diagram of green analysis evaluation score and solid waste generation. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] To achieve the above objectives, the present invention provides the following technical solution: The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] Chloramphenicol (CAS No.: 56-75-7) with a purity greater than or equal to 98%; Thiamphenicol (CAS No.: 15318-45-3) with a purity greater than or equal to 98%; Florfenicol (CAS No.: 73231-34-2) with a purity greater than or equal to 98%; Florfenicol (CAS No.: 76639-93-5) with a purity greater than or equal to 98%; Chloramphenicol-D5 (CAS No.: 202480-68-0) isotope abundance greater than or equal to 99%; The isotopic abundance of thiamphenicol-D3 (CAS No.: 2211914-19-9) is greater than or equal to 99%; Florfenicol-D3 (CAS No.: 2213400-85-0) isotope abundance greater than or equal to 99%; The isotopic abundance of florfenicol-D3 (CAS No.: 1217625-88-1) is greater than or equal to 99%.

[0037] Sodium octyl sulfate (CAS No.: 142-31-4) with a purity greater than or equal to 95%; tert-Butanol (CAS No.: 75-65-0) with a purity greater than or equal to 99%; 3-(methacryloyloxy)propyltrimethoxysilane (CAS No.: 2530-85-0) with a purity greater than or equal to 97%; N-Vinylpyrrolidone (CAS No.: 88-12-0) with a purity greater than or equal to 99%; Divinylbenzene (CAS No.: 1321-74-0) with a purity greater than or equal to 80%; Azobisisobutyronitrile (CAS No.: 78-67-1) with a purity greater than or equal to 98%.

[0038] Preparation Example 1: This preparation example provides a method for preparing magnetic HLB microparticles, including the following steps: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were weighed and dissolved in deoxygenated deionized water at a molar ratio of 2:1. Under nitrogen protection at 80°C, the mixture was stirred at 900 rpm and ammonia solution with a mass fraction of 25% was added dropwise at a uniform rate until the pH of the system reached 10.0. The mixture was allowed to mature for 40 minutes. The black precipitate was separated by an external magnetic field and washed alternately with water and ethanol until neutral. The precipitate was then dried under vacuum to obtain ferric oxide nanoparticles. 1.0 part by weight of the above iron oxide nanoparticles were ultrasonically dispersed in 45.0 parts by weight of anhydrous toluene, and 1.0 part by weight of 3-(methacryloyloxy)propyltrimethoxysilane was added. The mixture was refluxed at 105°C for 18 hours. After the reaction was completed, the mixture was magnetically separated and washed with ethanol to obtain modified magnetic microparticles with double bonds on the surface. 1.0 parts by weight of modified magnetic microparticles were ultrasonically dispersed in 35.0 parts by weight of acetonitrile, 2.0 parts by weight of N-vinylpyrrolidone and 2.5 parts by weight of divinylbenzene were added, and after purging with nitrogen for 15 minutes to remove oxygen, 0.10 parts by weight of azobisisobutyronitrile were added. The system was heated to 70°C and subjected to a constant-temperature thermally initiated polymerization reaction at 250 rpm for 20 hours. The obtained product was subjected to Soxhlet extraction with methanol and pure water to remove unreacted monomers, and then freeze-dried to obtain the magnetic HLB microparticles.

[0039] Preparation Example 2: This preparation example provides a method for preparing magnetic HLB microparticles, including the following steps: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were weighed and dissolved in deoxygenated deionized water at a molar ratio of 2:1. Under nitrogen protection at 75°C, the mixture was stirred at 800 rpm and ammonia solution with a mass fraction of 25% was added dropwise at a uniform rate until the pH of the system reached 9.5. The mixture was allowed to mature for 30 minutes. The black precipitate was separated by an external magnetic field and washed alternately with water and ethanol until neutral. The precipitate was then dried under vacuum to obtain ferric oxide nanoparticles. 1.0 part by weight of the above iron oxide nanoparticles were ultrasonically dispersed in 40.0 parts by weight of anhydrous toluene, and 0.5 parts by weight of 3-(methacryloyloxy)propyltrimethoxysilane were added. The mixture was refluxed at 100°C for 12 hours. After the reaction was completed, the mixture was magnetically separated and washed with ethanol to obtain modified magnetic microparticles with double bonds on the surface. 1.0 parts by weight of modified magnetic microparticles were ultrasonically dispersed in 30.0 parts by weight of acetonitrile, 1.5 parts by weight of N-vinylpyrrolidone and 2.0 parts by weight of divinylbenzene were added, and nitrogen gas was purged for oxygen removal for 15 minutes. Then, 0.05 parts by weight of azobisisobutyronitrile was added, and the system was heated to 65°C. The thermally initiated polymerization reaction was carried out at a constant temperature of 200 rpm for 16 hours. The product was subjected to Soxhlet extraction with methanol and pure water to remove unreacted monomers, and the magnetic HLB microparticles were obtained after freeze-drying.

[0040] Preparation Example 3: This preparation example provides a method for preparing magnetic HLB microparticles, including the following steps: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were weighed and dissolved in deoxygenated deionized water at a molar ratio of 2:1. Under nitrogen protection at 85°C, the mixture was stirred at 1000 rpm and ammonia water with a mass fraction of 25% was added dropwise at a uniform rate until the pH of the system reached 10.5. The mixture was allowed to mature for 45 minutes. The black precipitate was separated by an external magnetic field and washed alternately with water and ethanol until neutral. The precipitate was then dried under vacuum to obtain ferric oxide nanoparticles. 1.0 part by weight of the above iron oxide nanoparticles were ultrasonically dispersed in 50.0 parts by weight of anhydrous toluene, and 1.5 parts by weight of 3-(methacryloyloxy)propyltrimethoxysilane were added. The mixture was refluxed at 110°C for 24 hours. After the reaction was completed, the mixture was magnetically separated and washed with ethanol to obtain modified magnetic microparticles with double bonds on the surface. 1.0 part by weight of modified magnetic microparticles were ultrasonically dispersed in 40.0 parts by weight of acetonitrile, 2.5 parts by weight of N-vinylpyrrolidone and 3.0 parts by weight of divinylbenzene were added, and after purging with nitrogen for 20 minutes to remove oxygen, 0.15 parts by weight of azobisisobutyronitrile were added. The system was heated to 75°C and subjected to a constant-temperature thermally initiated polymerization reaction at 300 rpm for 24 hours. The product was subjected to Soxhlet extraction with methanol and pure water to remove unreacted monomers, and then freeze-dried to obtain the magnetic HLB microparticles.

[0041] Example 1: This embodiment provides a method for determining the residual amounts of amide alcohols and their metabolites in milk, including the following steps: Take 100.0 parts by weight of homogenized commercial whole milk sample and place it in a reaction vessel. Add 0.003 parts by weight of isotope internal standard mixture, which is composed of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicol-D3 in a mass ratio of 1:1:1:1. Vortex mix at 25°C for 4 minutes and let stand at room temperature for 8 minutes to obtain sample mixture. A composite desorption solubilizing reagent was added to the sample mixture in one go. The reagent was prepared by pre-dissolving 12.5 parts by weight of urea, 0.6 parts by weight of sodium octyl sulfate, and 15.0 parts by weight of tert-butanol in 25.0 parts by weight of deionized water. The reaction vessel was placed in a constant temperature shaker, the temperature was controlled at 35°C, and the shaking was applied at 2000 rpm for 3 minutes. 1.0 part by weight of the pre-wetted magnetic HLB microparticles obtained in Preparation Example 1 were added to the reaction vessel. The temperature was maintained at 35°C, and the oscillation rate was reduced to 400 rpm for 2 minutes. The oscillation was stopped, and a strong magnetic field with an apparent magnetic field strength greater than 0.5 Tesla was immediately applied to the outer wall of one side of the reaction vessel. The vessel was left to stand and anchor for 60 seconds to allow the magnetic HLB microparticles to be directionally adsorbed onto the inner wall of the vessel. While maintaining strong magnetic field anchorage on the outer wall throughout the process, the container was placed on a track shaker and subjected to low-speed horizontal cross-flow at 60 rpm. A micro-fluid pump was used to slowly drip a phase change triggering and cross-flow flocculation reagent into the continuous phase of the mixed liquid in the container at a constant rate of 0.5 parts by weight per second. This reagent was prepared by pre-dissolving 15.0 parts by weight of ammonium sulfate and 0.5 parts by weight of a buffer salt with a pH of 4.6 composed of glacial acetic acid and anhydrous sodium acetate in 90.0 parts by weight of deionized water. After the dripping was completed, the container was aged for 3 minutes while maintaining magnetic attraction and cross-flow. Maintain the magnetic field on the outer wall to completely drain the protein flocculation waste liquid generated in the center of the reaction vessel. Remove the magnetic field and add 150.0 parts by weight of matrix cleaning solution prepared by methanol and deionized water at a mass ratio of 1:19 to the vessel. Vortex wash for 30 seconds, apply the magnetic field again to separate the solid and liquid, and discard the cleaning waste liquid. Remove the magnetic field and add 120.0 parts by weight of targeted eluent prepared by acetonitrile and deionized water at a mass ratio of 4:1 to the vessel. Sonicate at 35°C for 2 minutes, apply the magnetic field to collect the supernatant. Blow the collected eluent to near dryness with nitrogen at 40°C, redissolve it with 1.0 parts by weight of ammonium formate aqueous solution with a concentration of 10 mmol / L, filter the solution through a membrane, and inject it into a liquid chromatography-tandem mass spectrometer for quantitative analysis.

[0042] Example 2: This embodiment provides a method for determining the residual amounts of amide alcohols and their metabolites in milk, including the following steps: Take 100.0 parts by weight of homogenized commercial whole milk sample and place it in a reaction vessel. Add 0.001 parts by weight of isotope internal standard mixture, which is composed of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicol-D3 in a mass ratio of 1:1:1:1. Vortex mix at 20°C for 3 minutes and let stand at room temperature for 5 minutes to obtain sample mixture. A composite desorption solubilizing reagent was added to the sample mixture in one go. The reagent was prepared by pre-dissolving 10.0 parts by weight of urea, 0.4 parts by weight of sodium octyl sulfate, and 12.0 parts by weight of tert-butanol in 20.0 parts by weight of deionized water. The reaction vessel was placed in a constant temperature shaker, the temperature was controlled at 30°C, and the shaking was applied at 1500 rpm for 2 minutes. 0.5 parts by weight of the pre-wetted magnetic HLB particles obtained in Preparation Example 2 were added to the reaction vessel. The temperature was maintained at 30°C, and the oscillation rate was reduced to 300 rpm for 1 minute. The oscillation was stopped, and a strong magnetic field with an apparent magnetic field strength greater than 0.5 Tesla was immediately applied to the outer wall of one side of the reaction vessel. The vessel was left to stand for 45 seconds to allow the magnetic HLB particles to be directionally adsorbed onto the inner wall of the vessel. While maintaining strong magnetic field anchorage on the outer wall throughout the process, the container was placed on a track shaker and subjected to low-speed horizontal cross-flow at 40 rpm. A micro-fluid pump was used to slowly drip a phase change triggering and cross-flow flocculation reagent into the continuous phase of the mixed liquid in the container at a constant rate of 0.2 parts by weight per second. This reagent was prepared by pre-dissolving 12.0 parts by weight of ammonium sulfate and 0.3 parts by weight of a buffer salt with a pH of 4.6 composed of glacial acetic acid and anhydrous sodium acetate in 80.0 parts by weight of deionized water. After the dripping was completed, the container was aged for 2 minutes under magnetic attraction and cross-flow conditions. Maintain the magnetic field on the outer wall, discharge all the protein flocculation waste liquid generated in the center of the reaction vessel, remove the magnetic field, add 140.0 parts by weight of matrix cleaning solution prepared by methanol and deionized water at a mass ratio of 1:19 into the vessel, vortex wash for 20 seconds, apply the magnetic field again to separate the solid and liquid and discard the cleaning waste liquid; remove the magnetic field, add 110.0 parts by weight of targeted elution solution prepared by acetonitrile and deionized water at a mass ratio of 4:1 into the vessel, sonicate at 30°C for 1 minute, apply the magnetic field to collect the supernatant; The collected eluent was purged to near dryness with nitrogen at 35°C, then reconstituted with 1.0 part by weight of 10 mmol / L ammonium formate aqueous solution, filtered through a membrane, and injected into a liquid chromatography-tandem mass spectrometer for quantitative analysis.

[0043] Example 3: This embodiment provides a method for determining the residual amounts of amide alcohols and their metabolites in milk, including the following steps: Take 100.0 parts by weight of homogenized commercial whole milk sample and place it in a reaction vessel. Add 0.005 parts by weight of isotope internal standard mixture, which is composed of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicol-D3 in a mass ratio of 1:1:1:1. Vortex mix at 30°C for 5 minutes and let stand at room temperature for 10 minutes to obtain sample mixture. A composite desorption solubilizing reagent was added to the sample mixture in one go. The reagent was prepared by pre-dissolving 15.0 parts by weight of urea, 0.8 parts by weight of sodium octyl sulfate, and 18.0 parts by weight of tert-butanol in 30.0 parts by weight of deionized water. The reaction vessel was placed in a constant temperature shaker, the temperature was controlled at 40°C, and the shaking was applied at 2500 rpm for 5 minutes. 1.5 parts by weight of the pre-wetted magnetic HLB microparticles obtained in Preparation Example 3 were added to the reaction vessel. The temperature was maintained at 40°C, and the oscillation rate was reduced to 500 rpm for 3 minutes. The oscillation was stopped, and a strong magnetic field with an apparent magnetic field strength greater than 0.5 Tesla was immediately applied to the outer wall of one side of the reaction vessel. The vessel was left to stand for 90 seconds to allow the magnetic HLB microparticles to be directionally adsorbed onto the inner wall of the vessel. While maintaining strong magnetic field anchorage on the outer wall throughout the process, the container was placed on a track shaker and subjected to low-speed horizontal cross-flow at 80 rpm. A micro-fluid pump was used to slowly drip a phase change triggering and cross-flow flocculation reagent into the continuous phase of the mixture in the container at a constant rate of 0.8 parts by weight per second. This reagent was prepared by pre-dissolving 18.0 parts by weight of ammonium sulfate and 0.8 parts by weight of a buffer salt with a pH of 4.6 composed of glacial acetic acid and anhydrous sodium acetate in 100.0 parts by weight of deionized water. After the dripping was completed, the container was aged for 4 minutes while maintaining magnetic attraction and cross-flow. Maintain the magnetic field on the outer wall, discharge all the protein flocculation waste liquid generated in the center of the reaction vessel, remove the magnetic field, add 160.0 parts by weight of matrix cleaning solution prepared by methanol and deionized water at a mass ratio of 1:19 into the vessel, vortex clean for 40 seconds, apply the magnetic field again to separate the solid and liquid and discard the cleaning waste liquid. Remove the magnetic field, add 130.0 parts by weight of targeted eluent prepared by acetonitrile and deionized water at a mass ratio of 4:1 to the container, sonicate at 40°C for 3 minutes, apply a magnetic field and collect the supernatant; blow the collected eluent to near dryness with nitrogen at 45°C, redissolve it with 1.0 parts by weight of ammonium formate aqueous solution with a concentration of 10 mmol / L, filter the solution through a membrane and inject it into a liquid chromatography-tandem mass spectrometer for quantitative analysis.

[0044] Example 4: This embodiment provides a method for determining the residual amounts of amide alcohols and their metabolites in milk, including the following steps: Take 100.0 parts by weight of homogenized high-fat colostrum sample with high fat content and high viscosity and place it in a reaction vessel. Add 0.003 parts by weight of isotope internal standard mixture, which is composed of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicolamine-D3 in a mass ratio of 1:1:1:1. Vortex mix at 25°C for 4 minutes and let stand at room temperature for 8 minutes to obtain sample mixture. A composite desorption solubilizing reagent was added to the sample mixture in one go. The reagent was prepared by pre-dissolving 12.5 parts by weight of urea, 0.6 parts by weight of sodium octyl sulfate, and 15.0 parts by weight of tert-butanol in 25.0 parts by weight of deionized water. The reaction vessel was placed in a constant temperature shaker, the temperature was controlled at 35°C, and the shaking was applied at 2000 rpm for 3 minutes. 1.0 part by weight of the pre-wetted magnetic HLB microparticles obtained in Preparation Example 1 were added to the reaction vessel. The temperature was maintained at 35°C, and the oscillation rate was reduced to 400 rpm for 2 minutes. The oscillation was stopped, and a strong magnetic field with an apparent magnetic field strength greater than 0.5 Tesla was immediately applied to the outer wall of one side of the reaction vessel. The vessel was left to stand and anchor for 60 seconds to allow the magnetic HLB microparticles to be directionally adsorbed onto the inner wall of the vessel. While maintaining strong magnetic field anchorage on the outer wall throughout the process, the container was placed on a track shaker and subjected to low-speed horizontal cross-flow at 60 rpm. A micro-fluid pump was used to slowly drip a phase change triggering and cross-flow flocculation reagent into the continuous phase of the mixture in the container at a constant rate of 0.5 parts by weight per second. This reagent was prepared by pre-dissolving 15.0 parts by weight of ammonium sulfate and 0.5 parts by weight of a buffer salt with a pH of 4.6 composed of glacial acetic acid and anhydrous sodium acetate in 90.0 parts by weight of deionized water. After the dripping was completed, the container was aged for 3 minutes while maintaining magnetic attraction and cross-flow. Maintain the magnetic field on the outer wall, discharge all the coarse flocculated waste liquid generated in the center of the reaction vessel, remove the magnetic field, add 150.0 parts by weight of matrix cleaning solution prepared by methanol and deionized water at a mass ratio of 1:19 into the container, vortex clean for 30 seconds, apply the magnetic field again to separate the solid and liquid and discard the cleaning waste liquid. Remove the magnetic field, add 120.0 parts by weight of targeted eluent prepared by acetonitrile and deionized water at a mass ratio of 4:1 to the container, sonicate at 35°C for 2 minutes, apply a magnetic field and collect the supernatant; blow the collected eluent to near dryness with nitrogen at 40°C, redissolve it with 1.0 parts by weight of ammonium formate aqueous solution with a concentration of 10 mmol / L, filter the solution through a membrane and inject it into a liquid chromatography-tandem mass spectrometer for quantitative analysis.

[0045] Comparative Example 1: Compared with Example 1, the difference is that the composite desorption solvation reagent and phase change triggering and cross-flow flocculation reagent were not used. Instead, an equal volume of a mixed aqueous solution prepared from conventional zinc acetate and potassium ferrocyanide was directly added to the system as a precipitant for hard protein precipitation. Magnetic HLB particles were not introduced or magnetic field anchoring was performed throughout the process. After flocculation, the supernatant was directly centrifuged and subjected to conventional liquid-liquid extraction and nitrogen blowing redissolution. Everything else was the same.

[0046] Comparative Example 2: Compared with Example 1, the difference lies in breaking the spatiotemporal decoupling operation steps. That is, after the magnetic HLB particles are introduced, a strong magnetic field is not applied to the outer wall for physical force field anchoring in advance. Instead, phase change triggering and cross-flow flocculation reagent is directly added while the magnetic HLB particles are in a free suspension state. After macroscopic protein flocculation appears in the central liquid phase region, a magnetic field is then applied for solid-liquid separation. All other aspects are the same.

[0047] Comparative Example 3: Compared with Example 1, the difference is that the composite desorption solvation reagent does not contain the co-solvent tert-butanol, which plays a role in phase transfer mediation and maintaining metastable homogeneous phase. Instead, it is replaced with an equal part by weight of deionized water. All other aspects are the same.

[0048] Comparative Example 4: Compared with Example 1, the difference is that the composite desorption solvation reagent does not contain sodium octyl sulfate, a short-chain anionic surfactant that competes for targeted desorption; otherwise, they are the same.

[0049] Comparative Example 5: Compared with Example 1, the difference is that the phase change triggering and cross-flow flocculation reagent does not contain the strong salting-out agent ammonium sulfate, but is replaced by an equal part by weight of deionized water; all other aspects are the same.

[0050] Test Example 1: Engineering evaluation of macroscopic fluid phase evolution and solid-liquid separation stability The processing systems described in Example 1, Comparative Example 1, and Comparative Example 2 were used as parallel test objects, and three repeated experimental groups were set up for each to obtain the average engineering parameters.

[0051] During the initial pretreatment reaction stage of the experiment, i.e. after the addition of the composite desorption solvation reagent and shaking in Example 1 and Comparative Example 2, and after the addition of the hard precipitate mixed aqueous solution and vortexing in Comparative Example 1, 100 μL of fluid was taken from the central liquid phase region of each reaction vessel using a micropipette, and its apparent absorbance was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer to quantify the dispersion state of the fluid matrix.

[0052] Continue to perform subsequent flocculation, sedimentation and solid-liquid separation operations according to each scheme, and use a high-precision stopwatch to record the time required from the application of the separation force field (external wall magnetic field applied in Example 1 and Comparative Example 2, centrifuge started in Comparative Example 1) to the continuous phase liquid becoming clear and free of obvious suspended matter.

[0053] After the waste discharge and cleaning operation is completed, the magnetic HLB particles recovered from each group are transferred to pre-weighed aluminum foil dishes and placed in a 60℃ vacuum drying oven to be dried at a constant temperature until constant weight. The apparent mass recovery rate of the magnetic solid medium is calculated by the reduction method.

[0054] The experimental data are shown in Table 1: Table 1: Results of fluid phase and separation engineering parameters measured during the pretreatment process.

[0055] in conclusion: According to Table 1 and Figure 1Comparing the initial optical absorption characteristics of Example 1 and Comparative Example 1, the absorbance of the system in Example 1 remained at a low level of 0.312 at 600 nm after the addition of the composite desorption solvation reagent. This result indicates that the synergistic effect of urea and tert-butanol can effectively inhibit the disordered aggregation of casein macromolecules, maintaining the complex emulsion system in a relatively uniform metastable homogeneous state, thus providing the necessary mass transfer time and space for the target drug molecules to desorb from protein binding sites and release into the aqueous phase. In contrast, Comparative Example 1, using a traditional heavy metal salt precipitation process, showed a rapid increase in absorbance to 2.158 after the addition of the reagent, reflecting the instantaneous formation of irreversible polymer flocs. At this point, drug molecules existing within the matrix are easily physically trapped and difficult to transfer to the free phase.

[0056] In the subsequent solid-liquid separation and enrichment stages, the engineering differences resulting from different operating procedures are significant. In Comparative Example 2, due to the absence of a pre-physical anchoring step, the magnetic particles directly encounter protein flocculation triggered by ammonium sulfate in a free-suspension state, and spatial entanglement between the solid support and the polymer gel inevitably occurs within the microscopic flow field.

[0057] This embedding effect makes it difficult for external magnetic fields to effectively overcome the viscous resistance of the gel network, significantly extending the solid-liquid separation time to 615.2 seconds, with an apparent particle recovery rate of only 23.8%. Example 1 utilizes a pre-set external strong magnetic field to directionally anchor magnetic HLB particles to the inner wall of the reaction vessel before the system undergoes a drastic phase transition, spatially isolating them from the protein flocculation process in the central liquid phase region. Test data verified that Example 1 achieved clear solid-liquid separation in just 14.5 seconds, with a particle recovery rate of 96.3%, confirming the effectiveness of the chemical homogeneity maintenance combined with physical spatial isolation mechanism in overcoming interference from high-fat, high-protein matrices, and providing a stable operational basis for subsequent phase-transfer-mediated targeted extraction.

[0058] Test Example 2: Verification of the thermodynamic phase transfer-mediated mass transfer mechanism The treatment systems described in Example 1, Comparative Example 3, and Comparative Example 4 were selected as parallel test objects. A highly polar target compound, florfenicol, with a final concentration of 100.0 ng / mL was added to homogenized commercial whole milk samples in advance, and the samples were vortexed and incubated for 2 hours to simulate the drug residue process under natural conditions.

[0059] After adding the composite desorption solvation reagent and completing the first stage of isothermal oscillation treatment, 50 μL of homogeneous fluid was taken from each group system. After ultracentrifugation to settle the macromolecules, the supernatant was taken for precursor analysis. The concentration of florfenicolamine in the continuous aqueous phase during this stage was determined by liquid chromatography-tandem mass spectrometry to quantify the initial desorption release.

[0060] Continue to add magnetic HLB particles and perform deceleration oscillation and static anchoring operation with strong magnetic field on the outer wall. Then, slowly drip phase change triggering and cross-flow flocculation reagent under low-speed horizontal cross-flow on the track shaker.

[0061] After the addition is complete and the aging process is maintained under magnetic attraction and cross-flow conditions, the protein flocculation waste liquid generated in the center of the system is collected and discharged. The waste liquid sample is filtered through a 0.22-micron microporous membrane, and the concentration of the final residual florfenicol is determined by liquid chromatography-mass spectrometry (LC-MS), and the absolute residual concentration of tert-butanol in the waste liquid is determined by gas chromatography (GC).

[0062] The experimental data are shown in Table 2: Table 2: Liquid phase concentration distribution and migration parameter determination results of key substances in each stage of pretreatment.

[0063] in conclusion: According to Table 2 and Figure 2 The data showed a significant difference in the concentration of free florfenicol in the continuous aqueous phase between Example 1 and Comparative Example 4 in the initial stage. Comparative Example 4, without the addition of sodium octyl sulfate, relied solely on the hydrogen bond disruption effect of urea, resulting in a free target concentration of 31.57 ng / mL in the liquid phase. Conventional milk matrix treatment studies have observed that polar drug molecules readily bind to the hydrophobic regions of casein or phospholipids, and a single reagent often struggles to break this stable binding state to achieve complete desorption. In Example 1, the introduction of sodium octyl sulfate increased the free concentration in the aqueous phase to 91.24 ng / mL. This data indicates that the competitive displacement effect of short-chain anionic surfactants can promote the transfer of the target drug from its polymeric binding site to the free aqueous phase. Establishing this desorption state is a fundamental prerequisite for achieving high recovery rates in complex matrix pretreatment processes.

[0064] Whether the free state of the target drug in the aqueous phase can be converted into effective solid-phase adsorption depends on the mass transfer efficiency of the flocculation separation stage. Although Comparative Example 3 achieved a relatively high free concentration of 88.93 ng / mL initially, the residual amount of the target drug in the discharged waste liquid was still as high as 75.22 ng / mL after ammonium sulfate-induced protein precipitation. In conventional systems, the spontaneous diffusion process from the liquid phase to the solid phase channels is constrained by the decrease in concentration gradient, making it difficult for solid-phase adsorbents to effectively capture low concentrations of polar molecules within a limited operating window. Example 1 changed this process by preparing a composite reagent containing tert-butanol. After the addition of ammonium sulfate, the measured concentration of tert-butanol in the waste liquid was 36.15 mg / mL, lower than its theoretical initial addition concentration. The salting-out effect of high concentrations of salt ions in the system disrupted the hydrogen bond network between water and tert-butanol, leading to microscopic phase separation of some co-solvents, which then spontaneously diffused onto the surface of magnetic HLB particles with similar surface properties. This process forms a locally rich solvent-wetting layer on the particle surface, and the change in chemical potential gradient drives the free drug to accumulate in this mesoscopic liquid film and enter the internal channels. The measurement result of florfenicol concentration in the final waste liquid of Example 1, which decreased to 4.38 nanograms per milliliter, confirms from the perspective of material distribution that the phase transfer mechanism can replace simple passive diffusion and effectively improve the solid-phase extraction efficiency of trace polar molecules in complex matrices.

[0065] Test Example 3: Determination of absolute recovery and precision of target analytes The treatment systems described in Examples 1 to 4 and Comparative Examples 1 to 5 were selected as test subjects, and commercially available whole milk and high-fat colostrum (corresponding only to Example 4) that had been rigorously screened and confirmed to be free of residue were used as matrices.

[0066] Accurately add the mixed standard solution to each blank matrix, setting the spiking concentration of florfenicol and chloramphenicol to 10.0 μg / kg. Disperse with low-frequency ultrasound for 5 minutes and incubate at 4°C in the dark for 12 hours to allow the target analyte to fully penetrate and bind to the casein micelles and lipid bilayer.

[0067] Six replicates were prepared in parallel for each group of spiked samples. Extraction, purification and elution were carried out in strict accordance with the pretreatment steps specified in their respective protocols to obtain the final nitrogen-blown reconstituted test solution.

[0068] Each group of reconstituted analytes was injected into a liquid chromatography-tandem mass spectrometer for quantitative analysis. The absolute recovery rate of each target analyte and the relative standard deviation obtained from six parallel tests were calculated using the matrix-matched standard curve method combined with isotope internal standard correction to evaluate the accuracy and reproducibility of the extraction process.

[0069] The experimental data are shown in Table 3: Table 3: Recovery and Precision of Target Substances for Each Extraction Scheme

[0070] in conclusion: According to Table 3 and Figure 3 The data from the examples and comparative examples show significant differences in the recovery rate and precision of the target substances in complex emulsion matrices. In routine analytical detection, highly polar metabolites with basic groups (such as florfenicol) typically exhibit low extraction efficiency due to their susceptibility to being trapped by the three-dimensional network of large protein molecules in the matrix. Comparative Example 1, using a traditional heavy metal salt precipitation process, achieved a recovery rate of only 34.2% for florfenicol, with a relative standard deviation of 14.7%. This data reflects the extreme instability of the solid-liquid phase mass transfer process in traditional hard precipitation methods when dealing with high-protein systems. Examples 1 to 3 combined homogeneous defolding and cross-flow enrichment operations. Through the intervention of in-situ thermodynamic mechanisms, the recovery rate of polar metabolites was stably increased to the range of 91.8% to 96.1%. Even when processing high-fat colostrum matrices with significantly higher viscosity and lipid content, Example 4 still achieved a recovery rate of 89.5%, indicating that this spatiotemporally decoupled extraction process has excellent anti-interference ability and adaptability to extremely complex biological sample matrices.

[0071] The comparative data lacking specific reagent components further pointed to the specific technical aspects affecting extraction efficiency. In Comparative Example 4, due to the absence of sodium octyl sulfate, the lack of competitive displacement by surfactants resulted in insufficient desorption and release of the target analyte from the protein micelles, leading to a florfenicol recovery rate of 48.7%. Under the condition that the initial desorption stage was established, the data from Comparative Examples 3 (lacking tert-butanol) and 5 (lacking ammonium sulfate) showed that if an effective thermodynamic phase transfer driving force could not be constructed, the target molecules in the free phase could not spontaneously transfer to the solid-phase microparticle channels within the brief protein flocculation window, resulting in recovery rates of less than 60% for both groups. The timing of the extraction operations also directly affected the results. Comparative Example 2 omitted the pre-existing physical force field anchoring step, and the freely suspended magnetic medium was directly encapsulated by the rapidly generated protein gel network, obstructing the mass transfer channels at the solid-liquid interface, causing the recovery rates of both target analytes to drop significantly to around 15%. The test results fully reveal that the present invention fundamentally solves the technical problem of the easy co-precipitation and loss of trace polar target substances in complex high-fat and high-protein matrices through the deep coupling of chemical release, physical isolation and thermodynamic phase transfer.

[0072] Test Example 4: Quantitative evaluation test of matrix effect (ME) The pretreatment methods described in Examples 1, 4, and Comparative Example 1 were selected as evaluation subjects, and pure solvents (prepared according to the initial mobile phase ratio for liquid chromatography) free of the target analyte were used as reference standards. The blank matrices used in the experiments were commercially available whole milk and high-fat colostrum samples that were screened for residues by the instrument.

[0073] According to the operating procedures of each set scheme, the blank sample without the target drug is subjected to extraction, purification and elution process, and the test solution after nitrogen evaporation and reconstitution is collected to obtain blank matrix extract containing potential background impurities.

[0074] Different volumes of target compound mixed standard working solution were accurately added to the pure solvent control group and the above blank matrix extracts to prepare pure solvent standard solutions and matrix-matched standard solutions with concentration gradients of 10.0, 20.0, 50.0 and 100.0 ng / mL, respectively.

[0075] Each set of standard solutions was sequentially injected into a liquid chromatography-tandem mass spectrometer for analysis, and the peak area of ​​the target analyte at different concentration points was recorded. A linear calibration curve was plotted with the absolute concentration of the analyte on the x-axis and the peak area on the y-axis. The quantitative percentage data for evaluating the intensity of the matrix effect was obtained by calculating the ratio of the slope of each matrix-matched standard curve to the slope of the pure solvent standard curve.

[0076] The experimental data are shown in Table 4: Table 4: Results of the determination of the ionization matrix effect of the extracts from each pretreatment process on the target mass spectrometry

[0077] in conclusion: According to Table 4 and Figure 4 The data shows that the test solutions prepared by different pretreatment processes exhibit varying degrees of competitive ionization when entering the mass spectrometry electrospray ionization source. Complex dairy product matrices are rich in endogenous phospholipids and residual protein fragments, which are macromolecular components that easily co-elute with trace target analytes during liquid chromatography separation. During the ionization stage, matrix impurities compete for limited charges on the droplet surface, thus reducing the ionization efficiency of target molecules. Comparative Example 1, using a traditional heavy metal salt precipitation process, failed to effectively remove the aforementioned interfering components; the slope of the standard curve matching the prepared florfenicolamine matrix was only 531.7. Compared to the pure solvent control group, the absolute value of the matrix effect of this method was 42.6%, indicating that more than half of the target analyte signals were attenuated due to matrix suppression. This significant ion suppression not only reduces the instrument's detection sensitivity but also easily causes data deviations in the quantitative analysis of actual samples.

[0078] In comparison, the sample schemes demonstrated better purification effects in controlling endogenous impurities. The matrix effect of florfenicol in the extract of Example 1 was 95.5%, and the slope of the standard curve was basically consistent with that of the pure solvent control group, reflecting the good purity of the extract. This test result verifies the physical role of the thermodynamic cross-flow mechanism in improving extraction selectivity. Under the state of magnetic field anchorage of particles, free target substances are adsorbed onto the solid phase following the chemical potential gradient; simultaneously, polar macromolecular fragments and inorganic salts that easily induce ionization inhibition are retained in the central liquid phase region where cross-flow aggregation occurs, and subsequently discharged from the system with the waste liquid. Physical spatial isolation cuts off the path for matrix impurities to co-enrich into the pores of the adsorption medium. When processing high-lipid colostrum matrices with higher lipid content and viscosity, Example 4 controlled the matrix effects of florfenicol and chloramphenicol at 86.8% and 93.2%, respectively. Comparison of quantitative parameters confirms that the spatiotemporal decoupling process, while ensuring the extraction rate of target substances, reduces competitive ionization caused by co-extracted impurities, providing reliable data for the quantitative analysis of trace residues in complex matrices.

[0079] Test Example 5: Comprehensive pretreatment efficiency and green chemistry index accounting The pretreatment schemes described in Examples 1 and 4, as well as Comparative Examples 1 and 3, were selected as the subjects of comprehensive evaluation. The complex biological matrix used in the experiment consisted of fresh raw milk collected from different batches to cover the natural fluctuations in lipid and protein content in real samples.

[0080] Each pretreatment protocol was designed to process 50 independent samples in parallel, simulating a high-throughput, high-volume testing scenario typically faced by analytical laboratories. A timing device was used to record the cumulative time taken from matrix homogenization to obtaining the final nitrogen-blown reconstituted analyte solution ready for instrument injection. The average processing time per sample was calculated by dividing the total processing time by the sample size.

[0081] Throughout the extraction, purification, and elution process, graduated cylinders and analytical balances were used to quantitatively collect all organic solvent waste, aqueous waste, and solid consumable waste discharged during each sample operation. The total volume of toxic organic solvents such as acetonitrile and methanol, which are listed in the hazardous chemicals catalog, was calculated separately for each operation, and the mass of solid waste such as centrifuge tubes and waste particles generated per operation was weighed.

[0082] An analytical ecological scale assessment system was introduced to calculate the green analytical chemistry comprehensive assessment score for each pretreatment process based on the toxicity of reagents used in each operation step, the distribution of equipment energy consumption, the degree of potential occupational exposure risk, and the burden of waste treatment. The full score is 100 points.

[0083] The experimental data are shown in Table 5: Table 5: Results of Measurement of Comprehensive Treatment Time and Green Chemistry Evaluation Indicators for Each Pretreatment Process

[0084] in conclusion: According to Table 5 and Figure 5 The data shows significant differences in sample throughput and environmental assessment indicators among various pretreatment schemes. In routine pesticide and veterinary drug residue screening, the concurrent processing of large batches of samples imposes strict limitations on the preparation time of a single sample. Comparative Example 1 uses a traditional heavy metal salt precipitation and liquid-liquid extraction process, with an average processing time of 52.4 minutes per sample and a single extraction consuming 14.65 ml of toxic organic solvent. Routine laboratory observations indicate that the traditional extraction mode, which relies on large volumes of solvent for phase partitioning, often consumes a long drying time in the subsequent nitrogen blowing concentration stage, resulting in a prolonged overall detection cycle. Example 1 uses homogeneous defolding and cross-flow enrichment operations, reducing the processing time per sample to 17.8 minutes. The magnetic field-mediated solid-liquid separation mode replaces the ultracentrifugation step, and the phase transfer-driven mechanism allows target molecules to be directionally enriched within a locally solvent-wetted membrane, reducing the extraction process's dependence on large volumes of eluent. When processing a high-lipid colostrum matrix with high lipid content and viscosity, the single-sample processing time in Example 4 was 19.3 minutes. The test results show that the kinetic process can still maintain stable separation efficiency in complex and viscous samples.

[0085] The level of greenness in pretreatment operations is directly related to the amount of organic solvent used and the amount of solid waste generated. Comparative Example 1 generated 3.84 grams of solid waste in a single run, achieving a green analytical chemistry evaluation score of 61.7. In Examples 1 and 4, the organic solvent consumption was controlled to within 1.30 ml, and the solid waste sources were mainly trace adsorption media and pipetting consumables, resulting in comprehensive evaluation scores of 88.5 and 86.2, respectively. In the development of residual analysis methods, reducing reagent usage is a common method to improve green evaluation indicators. Comparative Example 3 omitted the co-solvent tert-butanol used to induce microphase separation, achieving an evaluation score of 89.8. Combined with previous recovery test results, the absence of the thermodynamic gradient constructed by tert-butanol prevents the system from driving effective solid-phase transfer of polar target substances. Focusing solely on improving green evaluation indicators without considering extraction recovery is insufficient to meet the accurate quantitative requirements of complex residual analysis in practice. The measured parameters show that the spatiotemporal decoupling and thermodynamic phase transfer mechanism of the present invention maintains the high efficiency of system mass transfer while significantly reducing the use of harmful reagents and the frequency of physical transfer operations, and successfully achieves a deep unity between the analytical method's detection performance and its green and environmentally friendly attributes.

[0086] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for determining the residual amounts of amide alcohols and their metabolites in milk, characterized in that, Includes the following steps: Take the milk sample to be tested and place it in the reaction vessel. Add the isotope internal standard mixture, vortex mix and let stand to obtain the sample mixture. A composite desorption solubilizing reagent is added to the sample mixture and subjected to isothermal oscillation to inhibit macromolecular aggregation and promote the desorption and release of the target analyte into the free aqueous phase; magnetic HLB microparticles are added, and after deceleration and oscillation, a strong magnetic field is applied to the outer wall of the reaction vessel, and the mixture is allowed to stand so that the microparticles are directionally adsorbed onto the inner wall for physical spatial isolation. Maintaining the magnetic field anchoring state, a low-speed horizontal cross-flow is applied to the mixture in the reaction vessel, and a phase change trigger and cross-flow flocculation reagent is added and then aged; micro-phase separation is induced and a solvent-rich wetting layer is formed on the surface of the particles, driving the free target material to undergo thermodynamic phase transfer into the internal pores of the particles, while causing protein impurities to undergo cross-flow flocculation in the center of the reaction vessel; Maintain the magnetic field on the outer wall and discharge the waste liquid from the central protein flocculation; remove the magnetic field, add matrix cleaning solution to clean, and then apply the magnetic field again to discard the waste liquid; Remove the magnetic field, add the targeted elution buffer, sonicate, and apply a magnetic field to collect the supernatant; The collected supernatant liquid nitrogen was blown to near dryness, reconstituted, and injected into an ultra-high performance liquid chromatography-tandem mass spectrometer for quantitative analysis.

2. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The composite desorption solvation reagent, based on 100.0 parts by weight of the milk sample to be tested, is composed of the following components mixed in parts by weight: Urea 10.0–15.0 parts; Sodium octyl sulfate 0.4–0.8 parts; 12.0–18.0 parts of tert-butanol; 20.0 to 30.0 parts of deionized water.

3. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The phase change triggering and cross-flow flocculation reagent is composed of the following components in parts by weight per 100.0 parts of the milk sample to be tested: Ammonium sulfate 12.0–18.0 parts; A buffer salt with a pH of 4.6, consisting of 0.3 to 0.8 parts; wherein the buffer salt is composed of glacial acetic acid and anhydrous sodium acetate; 80.0 to 100.0 parts of deionized water.

4. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The method for preparing the magnetic HLB particles includes the following steps: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were dissolved in deoxygenated deionized water, stirred and aged with ammonia water dropwise under nitrogen protection, magnetically separated, washed and dried to obtain iron oxide nanoparticles. The iron oxide nanoparticles were dispersed in anhydrous toluene, and a silane coupling agent was added and refluxed. After magnetic separation and washing, modified magnetic microparticles were obtained. The modified magnetic microparticles were dispersed in acetonitrile, and hydrophilic monomers and crosslinking agent monomers were added. After deoxygenation, an initiator was added, and a thermally initiated polymerization reaction was carried out at a constant temperature. Unreacted monomers were extracted and the mixture was freeze-dried to obtain the magnetic HLB microparticles.

5. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 4, characterized in that, The ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved in deoxygenated deionized water at a molar ratio of 2:

1. Under nitrogen protection at 75-85°C, the mixture is stirred at 800-1000 rpm, and 25% ammonia solution is added dropwise at a uniform rate until the pH of the system reaches 9.5-10.

5. The mixture is then allowed to mature for 30-45 minutes.

6. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 4, characterized in that, The silane coupling agent is 3-(methacryloyloxy)propyltrimethoxysilane; The hydrophilic monomer is N-vinylpyrrolidone, the crosslinking agent monomer is divinylbenzene, and the initiator is azobisisobutyronitrile; The conditions for thermally initiated polymerization are: temperature 65–75℃, rotation speed 200–300 rpm, and reaction time 16–24 hours.

7. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The conditions for the isothermal oscillation treatment are as follows: Control the temperature at 30-40℃ and apply oscillation at 1500-2500 rpm for 2-5 minutes; The rotation speed of the deceleration oscillation is controlled at 300-500 rpm, and the processing time is 1-3 minutes; The applied strong magnetic field has an apparent magnetic field strength greater than 0.5 Tesla, and the static anchoring time is 45 to 90 seconds.

8. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The rotational speed of the low-speed horizontal crossflow is 40–80 rpm; The constant rate of dripping the phase change trigger and cross-flow flocculation reagent was controlled at 0.2–0.8 parts by weight per second; The aging time after the addition is complete is 2 to 4 minutes.

9. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The matrix cleaning solution is prepared by mixing methanol and deionized water at a mass ratio of 1:

19. The targeted eluent is prepared by mixing acetonitrile and deionized water at a mass ratio of 4:

1. Based on 100.0 parts by weight of the milk sample to be tested, the amount of the matrix washing solution is 140.0 to 160.0 parts by weight, and the amount of the targeted elution solution is 110.0 to 130.0 parts by weight.

10. The method for determining the residual amounts of amide alcohols and their metabolites in milk according to claim 1, characterized in that, The isotope internal standard mixture is composed of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3 and florfenicol-D3 in a mass ratio of 1:1:1:1; the resolution is specifically carried out using an ammonium formate aqueous solution with a concentration of 10 mmol / L.

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