Preparation method of a fish meat veterinary drug residue analysis matrix standard substance

Abstract

The application discloses a preparation method of a fish meat veterinary drug residue analysis matrix standard substance, and belongs to the technical field of standard substance preparation. In view of the defects of the existing skin-meat complex system, such as difficult homogenization, high water residue and blind value setting, the application adopts a medicated bath to enrich the medicine in the fish body with skin, and immediately quenches at-80 DEG C for 24 hours after stopping the medicine to achieve ice crystal embrittlement of collagen fibers; after impurity removal and preparation of skin-mush, based on the measured eutectic point, the phase is locked at-40 DEG C under supercooling, and the limit freeze-drying is carried out under a-80 DEG C cold trap to have a water content of less than or equal to 0.8%; then the high-concentration freeze-dried powder and the blank freeze-dried powder are mixed in proportion in solid phase, and after redissolution and detection, the closed-loop fine adjustment is carried out to the target concentration; finally, the nitrogen-filled vacuum double-sealing and extremely low water activity are carried out for 4KGy 60 Co-gamma in-situ sterilization. The application breaks through the bottleneck of skin-meat homogenization and water-fat micro-phase separation, and the value setting precision is increased by orders of magnitude; the stability of the standard substance is up to 24 months at-20 DEG C, and the standard substance is suitable for quality control of aquatic product veterinary drug residue detection.

Description

Technical Field

[0001] This invention relates to the field of standard substance preparation technology in analytical chemistry and metrology, specifically to a method for developing a matrix standard substance for analyzing veterinary drug residues in fish meat. Background Technology

[0002] In modern analytical chemistry and food quality control systems, matrix reference materials (MRMs) are a core pillar for ensuring the accuracy of trace analysis and achieving international traceability of metrological values. In the field of veterinary drug residue detection in aquatic products, the complex composition of actual biological samples makes them highly susceptible to matrix effects—that is, the inhibition or enhancement of the ionization process of the target analyte by matrix components—leading to serious deviations in test results. Traditional methods of directly adding pure reference materials to a blank matrix cannot realistically simulate the microscopic environment of the intercalation between drugs and biological macromolecules, and are insufficient to effectively compensate for measurement errors caused by matrix effects. Therefore, the development of highly realistic animal-derived matrix reference materials has become an urgent need in the industry.

[0003] However, the development of aquatic animal matrix standard materials is relatively lagging behind. Not only is the variety of matrices limited, but existing development methods also have significant defects and shortcomings in terms of matrix simulation accuracy, tissue homogenization, thermodynamic phase transition control, and long-term stability preservation. These defects are specifically manifested in the following aspects: First, there are issues with the simulation of in vivo metabolism and the absence of legally defined target tissues. Some existing technologies involve directly mixing veterinary drugs into blank fish paste or administering them via intramuscular injection. This completely violates the drug-drinking metabolism pathway in aquaculture, where most drugs are absorbed through water osmosis and gills, resulting in extremely uneven drug distribution gradients in tissues. More critically, there is a bias in the selection of matrix materials. For example, Chinese invention patent CN102288464B discloses a method for preparing a standard sample of sulfamethoxazole residue freeze-dried powder from carp muscle. Although this method uses drug-drinking administration, it explicitly requires skin removal and the collection of muscle during pretreatment. This practice seriously deviates from the legal requirement in current Chinese national food safety standards, such as GB 31650-2019, that the target tissue should be "skin + meat," i.e., fish skin and fish meat. Because the fish skin, rich in polyunsaturated fatty acids and collagen, is removed, the resulting matrix is ​​severely distorted in its biochemical composition and cannot fully cover the matrix effect of routinely sampled specimens.

[0004] Secondly, the complex matrix and freeze-drying process are severely mismatched, resulting in poor microscopic uniformity. When whole fish skin is introduced strictly according to national standards, the matrix changes from a single muscle fiber to a complex "skin-meat complex system." The tough collagen fibers in fish skin are difficult to completely pulverize at normal temperatures or shallow freezing conditions such as -18°C or -20°C. For example, Chinese invention patent CN106813963A discloses a method for preparing a fish meat powder standard material containing chloramphenicol. This method measures the eutectic point of the pure meat matrix to be -15°C and pre-freezes it at -20°C. Simultaneously, after freeze-drying, the material is pulverized and passed through a 35-mesh sieve. However, if this conventional process for pure meat is simply applied to a multiphase matrix containing fish skin, it will face a double dilemma: First, mechanical grinding at room temperature will cause protein denaturation due to the heat generated by strong friction, and due to the lack of the ice crystal expansion and tearing effect brought about by extreme cryogenic treatment, i.e., physical embrittlement, the fish skin cannot be homogeneously pulverized at the micron level, resulting in extremely high inhomogeneity of the standard material; Second, the multiphase system is rich in fat and collagen, and water-lipid microphase separation is very likely to occur during the vacuum stage of conventional freeze-drying, causing local boiling or structural collapse, which seriously hinders the sublimation and escape of internal water, not only greatly reducing freeze-drying efficiency, but also making it difficult to reduce the water content of the final product to the ideal level.

[0005] Third, high residual water activity can induce radiation-induced by-products and lead to insufficient long-term stability. During the repackaging and storage stage, to prevent psychrophilic microorganisms from degrading the target drug, some existing technologies, such as the aforementioned methods in CN106813963A or CN109613148A for preparing AOZ residual standard materials using turbot muscle as a matrix, employ... 60 Co-γ ray irradiation sterilization. However, the moisture content of lyophilized powders prepared using these techniques can typically only be controlled at 3% to 5%. At this moisture content, high-energy γ rays can induce radiolytic hydrolysis of residual water molecules, generating highly destructive hydroxyl radicals (·OH). These radicals attack the chemical structure of target drug molecules, inducing oxidative degradation of organic drug molecules—a side-killing effect of radiation—which is particularly deadly for antibiotics containing specific sensitive groups. Due to the lack of protective mechanisms against drug molecules, the long-term stability of existing aquatic matrix standard materials is extremely difficult to exceed 12 months, severely limiting the practical application value of these standard materials.

[0006] Fourth, the reliance on the blind box effect of in vivo metabolism leads to extremely high uncertainty in the determination of values. Current technologies for preparing matrix reference materials typically involve waiting for the target animal's own metabolism to reach a certain concentration before directly performing whole-body meat grinding for determination. Due to the uncontrollable physiological differences and metabolic rates among individual animals, this method exhibits strong blind box characteristics. After drug withdrawal, the actual drug concentration in the tissue often deviates significantly from the target value, and effective intervention and adjustment are impossible during the preparation process. This not only makes it difficult for the reference material to accurately hit the set target concentration, such as the maximum residue limit stipulated by national standards, but also results in uncontrollable concentration fluctuations leading to persistently high standard uncertainty introduced during the determination process, severely restricting the metrological accuracy and traceability of the reference material.

[0007] Fifth, the lack of synergistic process design means that isolated optimization of each step fails to overcome overall performance bottlenecks. Looking at existing technologies, each preparation step—drug administration, freezing, lyophilization, pulverization, mixing, and sterilization—is typically optimized as an independent process, lacking a systematic synergistic design concept. For example, while CN106813963A discloses eutectic point determination and 4KGy irradiation, its combination of pure meat matrix, conventional pre-freezing, and high-moisture-content irradiation fails to generate synergistic gains. Similarly, CN104155162A discloses a method for preparing standard samples of lyophilized drug residues from animal-derived matrices, but it does not address the correlation between precise control of the eutectic point and low-water-activity irradiation protection. It is precisely because of the lack of a deep understanding and systematic process coupling of the inherent physicochemical relationships between "matrix composition, freeze-embrittlement behavior, eutectic point migration, lyophilization limit moisture content, and irradiation damage threshold" that existing technologies have consistently failed to achieve simultaneous breakthroughs in the three core indicators of uniformity, stability, and metrological accuracy.

[0008] In summary, there is an urgent need in this field to develop a novel method for preparing matrix reference materials for veterinary drug residues in fish meat. This method must perfectly meet the regulatory requirements for "skin + meat" multiphase matrices, and systematically solve a series of problems such as homogenization difficulties, high moisture residue, radiation degradation, and uncontrollable concentration through innovative physicochemical process design, thereby obtaining high-quality matrix reference materials with extremely low uncertainty and long-term stability. Summary of the Invention

[0009] The problem this invention aims to solve is the shortcomings of existing aquatic animal matrix standard material development processes, such as difficulties in homogenizing the skin-meat multiphase system, high residual moisture in conventional freeze-drying leading to irradiation degradation of drugs, and uncontrollable concentration and high uncertainty due to reliance on in vivo metabolic determination. To address these issues, this invention provides a method for developing matrix standard materials for veterinary drug residue analysis in fish meat. This method aims to obtain high-quality matrix standard materials with extremely low uncertainty and long-term stability through the synergistic design of innovative physicochemical processes, including in vivo enrichment, cryogenic quenching, eutectic phase locking, closed-loop determination, and non-destructive irradiation.

[0010] To address the aforementioned problems, this invention provides a method for developing a matrix standard for analyzing veterinary drug residues in fish meat, comprising the following synergistic process steps: Step 1, Live Enrichment: The target veterinary drug is added to the water containing the target fish with skin by means of drug bath administration, so that the drug is evenly enriched in the fish through live metabolism; Step 2, Deep Cryogenic Quenching and Embrittling: Immediately after drug withdrawal, the target fish is slaughtered and its internal organs are removed. Then, the target fish body with intact skin and flesh is placed directly at -80°C for at least 24 hours to stop drug metabolism and achieve physical ice crystal embrittlement of the fish skin collagen fibers and muscle tissue. Step 3, preparation of multiphase matrix: The target fish body after deep cryogenic quenching is taken out, thawed and cleaned, the scales, head, tail and bones are removed, and the skin and meat are kept intact. The skin and meat are cut into pieces and ground with a tissue grinder to obtain a homogenized paste. Step 4, Eutectic Lock-in and Limit Freeze-drying: The eutectic point of the skin and meat homogenate was determined, and the pre-cooling temperature was set to -40°C, which is at least 15°C lower than the eutectic point, for supercooling pre-freezing. After pre-freezing, vacuum freeze-drying was carried out for 72 hours under the conditions of cold trap temperature -80°C and vacuum degree 20Pa to reduce the matrix moisture content to ≤0.8%, and fish meat freeze-dried powder containing high concentration of target veterinary drug and blank fish meat freeze-dried powder were obtained respectively. Step 5, Solid-phase closed-loop determination: The pulverized and sieved fish meat freeze-dried powder containing a high concentration of the target veterinary drug is physically dry-mixed with blank fish meat freeze-dried powder in a certain proportion. The mixed powder is reconstituted and the concentration is tested. Based on the test results, the mixing ratio of the high concentration freeze-dried powder and the blank freeze-dried powder is repeatedly adjusted for closed-loop fine-tuning until the drug concentration of the mixed powder reaches the target concentration requirement and passes the initial uniformity test. Step Six: Non-destructive Irradiation Sterilization: The freeze-dried fish powder reaching the target concentration is nitrogen-filled, sealed, and vacuum-packed. Under extremely low water activity conditions (matrix moisture content ≤ 0.8%), a dose of 4 kGy is applied to the fully sealed packaged product. 60 In-situ irradiation sterilization using Co-γ rays.

[0011] Preferably, the target veterinary drug mentioned in step one is a drug selected from antibacterial, antiparasitic, or antiviral drugs that has a maximum residue limit (MRL). Limiting the target veterinary drug to commonly used antibacterial, antiparasitic, or antiviral drugs with clearly defined MRL requirements ensures that the prepared matrix standard material can directly serve the legally mandated monitoring items in routine regulatory testing. Existing technologies sometimes use standard materials targeting only a single drug type, failing to provide systematic coverage of drugs with MRL requirements. This invention, by covering three major categories of veterinary drugs with legal monitoring needs, gives the preparation method good universality and promotional value, allowing for flexible adjustments to dosing regimens based on the physicochemical properties of different drugs, and providing a standardized, universal technical platform for standard material development.

[0012] Preferably, the target veterinary drug is a tetracycline. More preferably, the target veterinary drug is a tetracycline.

[0013] Preferably, the tetracycline drug is doxycycline. This invention specifies doxycycline as the target veterinary drug because this class of drugs has a relatively stable polycyclic skeleton structure. Under the special environment of extremely low water activity (≤0.8%) as described in this invention, it can produce a significant synergistic gain in radiation resistance, maximizing resistance to 4KGy. 60 The radiation-side killing effect of Co-γ rays avoids the molecular breakage that may occur with other antibiotics containing fragile groups, ensuring the absolute stability of the product's concentration. Experimental data shows that the doxycycline matrix standard material achieves long-term stability for 24 months at -20℃, filling the gap in the current market for high-precision certified matrix standard materials for doxycycline.

[0014] Preferably, the target fish with skin mentioned in step one is an artificially bred freshwater fish.

[0015] Preferably, the target fish with skin is the sea bass. This invention limits the target animal to artificially raised sea bass, whose skin-flesh multiphase system has a specific ratio of natural collagen to polyunsaturated fatty acids. The cryogenic quenching and eutectic pre-freezing process designed in this invention is optimally suited to this, accurately locking the sea bass's unique water-lipid microphase separation critical point of -18℃ to -25℃, achieving optimal extreme drying effect while avoiding excessive energy consumption. Using sea bass as a specific example, the complete process successfully prepared 500 individually packaged doxycycline-sea bass skin-flesh matrix standard materials, with a measured standard uncertainty (uCRM) as low as 0.098, far superior to the 0.15~15.1 standard uncertainty level of fish meat powder standard materials in the prior art, filling the technological gap in the field of sea bass matrix veterinary drug residue standard materials.

[0016] Preferably, in step four, the measured eutectic point of the homogenized fish skin and meat mixture containing fish skin and meat is -18℃ to -25℃, and the supercooling pre-freezing time at -40℃ is 24 hours. The eutectic point of the "skin + meat" multiphase matrix is ​​determined to be -18℃ to -25℃ through actual measurement, and the pre-freezing time is set to 24 hours. Existing technology measures the eutectic point of pure meat matrix at only -15℃. If this parameter is directly applied to multiphase matrix containing fish skin, water-lipid microphase separation and structural collapse are very likely to occur during the vacuuming stage. The actual measurement shows that the true eutectic point of the skin-meat multiphase matrix is ​​much lower than the -15℃ of pure meat, and the 24-hour pre-freezing ensures the material is completely frozen, guaranteeing unobstructed sublimation channels during the subsequent 72-hour freeze-drying process. This is the key thermodynamic guarantee for ultimately achieving a water content ≤0.8%.

[0017] Preferably, the method for determining the eutectic point of the skin and meat homogenate in step four is resistance method or differential scanning calorimetry.

[0018] Preferably, in step five, the parameters for pulverizing and sieving are as follows: the freeze-dried material is pulverized for 15 seconds using a blade mixer at 2000 r / min, and then passed through a 0.6 mm sieve. This combination of microscopic parameters ensures thorough homogenization of the freeze-dried loose material while cleverly controlling the heat generation threshold of mechanical work, effectively avoiding localized fat melting and protein denaturation caused by high speed or long-term grinding, and providing a dry powder carrier in a perfect physical state for subsequent high-precision solid-phase blending. Compared to existing technologies such as CN106813963A, a method for preparing a fish meat powder standard substance containing chloramphenicol, which uses parameters of "grinding at 3000 rpm for 1-2 minutes and passing through a 35-mesh sieve," this invention achieves a finer pulverization effect with a lower speed and shorter time, 0.6 mm ≈ 30 mesh, finer than 35 mesh, directly reflecting the synergistic effect of cryogenic embrittlement and gentle grinding.

[0019] Preferably, in step five, when reconstituteing the mixed powder, a water-to-powder weight ratio of 4.5 is used. Specifically, 2g of the mixed freeze-dried fish powder is added to 9mL of water for reconstitution. This invention uses a 4.5 water-to-powder weight ratio for reconstitution, which precisely replicates the natural water-to-dry matter ratio (approximately 81%-82%) in real fresh fish tissue. This ensures that when the matrix standard is reconstituted and used in the terminal laboratory, the microosmotic pressure, lipoprotein concentration, and ionization inhibition level in its chromatographic system are completely consistent with those of real fresh samples, achieving 100% equivalent compensation for matrix effects in routine testing. This provides an injection matrix environment identical to that of real samples for subsequent liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection, fundamentally guaranteeing the accuracy of metrological traceability.

[0020] Preferably, in step six, the nitrogen-filling sealing and vacuum packaging are performed as follows: the re-inspected and qualified freeze-dried fish powder is packaged in brown plastic or brown glass bottles at a quantity of not less than 2.5g, and then sealed with nitrogen; the outer layer of the bottle is then vacuum-sealed using an aluminum foil bag. This invention employs a dual packaging design: an inner brown bottle filled with nitrogen and an outer aluminum foil vacuum seal. The brown bottle first isolates the photosensitive drug, doxycycline, from the degradation threat of ultraviolet light; the double dense physical barrier formed by nitrogen filling and vacuum aluminum foil completely cuts off the pathway for the ultra-dry powder with a moisture content ≤0.8% to absorb moisture from the environment during a storage period of up to two years, preventing an increase in water activity and the resurgence of psychrophilic bacteria due to secondary moisture absorption, which is a key physical guarantee for maintaining long-term stability.

[0021] Preferably, before step one, a target drug determination step is included: selecting commonly used antibacterial drugs, antiparasitic drugs, or antiviral drugs in fish farming as target drugs, and the target drugs are clearly specified in the national standard maximum residue limit or are required not to be detected.

[0022] Preferably, before step one, a target concentration determination step is included: the target concentration is determined by referring to national standards, the U.S. Food and Drug Administration, or the EU's maximum residue limits for veterinary drugs in fish tissue.

[0023] Compared with the prior art, the present invention achieves the following beneficial technical effects: This invention closely conforms to legally mandated testing standards and accurately compensates for matrix effects. Strictly adhering to national standards, this invention employs live drug administration via bath and retains intact fish skin and flesh as the matrix, maximally replicating the microscopic biochemical environment of real aquatic samples in testing. It contains abundant collagen and polyunsaturated fatty acids, fundamentally avoiding the matrix distortion problems associated with traditional injectable drugs or pure meat matrices. The matrix standard prepared in this way can accurately replicate the matrix effects of actual tested samples, providing an accurate calibration benchmark for subsequent trace analysis of veterinary drug residues. This effectively compensates for the inherent deficiency of traditional pure standard materials or pure meat matrix standard materials in accurately compensating for matrix interference.

[0024] This invention utilizes extreme cryogenic physical embrittlement to overcome the bottleneck of homogenization in multiphase matrices. Addressing the industry challenge of tough fish skin collagen fibers that are difficult to completely pulverize at room temperature, this invention employs a treatment involving immediate -80°C cryogenic quenching for 24 hours after drug withdrawal. The extremely low temperature causes intracellular free water to instantly form fine ice crystals and expand in volume, irreversibly tearing the dense collagen fiber network at the microscopic physical level. This physical embrittlement effect allows the thawed skin-meat matrix to be easily pulverized to the micrometer level during grinding, while avoiding protein denaturation caused by the heat generated by forced grinding. This significantly reduces the matrix inhomogeneity (u_hom) to 0.19, fundamentally solving the technical obstacle of homogenizing the skin-meat multiphase system.

[0025] This invention employs precise eutectic point lock-in to achieve extremely low water activity. Addressing the core defect of the skin-flesh multiphase system, which is prone to water-lipid microphase separation during freeze-drying leading to structural collapse, this invention uses a measured matrix eutectic point of -18°C to -25°C and forcibly sets a pre-freezing temperature of -40°C, at least 15°C below the eutectic point. This cryogenic lock-in mechanism ensures that the matrix remains in a robust porous glassy / crystalline state during subsequent vacuuming, completely avoiding localized boiling and surface hardening. This allows water to escape completely through sublimation channels, ultimately reducing the finished product's water content to ≤0.8%, far exceeding the existing 3%-5% water content levels. This lays a decisive foundation for subsequent non-destructive irradiation and ultra-long-term stability.

[0026] This invention breaks the radiation degradation chain, granting an ultra-long stable lifespan. Traditional matrix standard materials have a high residual moisture content of 3%-5%, which hinders their effectiveness during radiation degradation. 60 During Co-γ ray irradiation sterilization, residual water molecules undergo radiation hydrolysis under the action of high-energy rays, generating a large number of highly reactive hydroxyl radicals (·OH), which attack and destroy the chemical structure of the target drug molecules, leading to a significant decrease in drug content. This invention reduces the water content to ≤0.8% through the aforementioned extreme freeze-drying process, creating an extremely low water activity environment with virtually no anhydrous free radicals. Under these conditions, applying 4 kGy irradiation can completely kill cold-resistant spores and psychrophilic microorganisms to undetectable levels, while also maximally blocking the damage of radiation-induced by-effects to the target drug molecules, transforming irradiation from a double-edged sword into a pure sterilization tool. The final prepared standard material achieves excellent long-term stability of up to 24 months at -20°C, representing a significant leap compared to the existing technology's storage limit of 6 to 12 months.

[0027] This invention breaks through the blind box of biological metabolism, achieving extremely low uncertainty across orders of magnitude. It completely abandons the outdated process of relying solely on animal metabolic enrichment and arbitrary value determination, instead employing a closed-loop feedback mechanism of fine-tuning through the mixing of high and low concentration solid-phase dry powders. This mechanism transforms the uncontrollable metabolic process of living organisms into a dry powder formulation process that can be precisely controlled by a high-precision balance. Combined with the high homogeneity (u_hom=0.19) resulting from cryogenic embrittlement, this invention drastically reduces the uncertainty (u_char) introduced by the value determination process to 0.0604, achieving an excellent overall standard uncertainty (u_CRM) of 0.098. Compared to the standard uncertainty of 0.15-15.1 in existing methods, this invention achieves a leap in metrological accuracy across orders of magnitude, providing the highest-precision certified matrix standard material currently available for the detection of veterinary drug residues in aquatic products. Attached Figure Description

[0028] Figure 1This is a process flow diagram of the method for developing the matrix standard material for the analysis of veterinary drug residues in fish meat according to the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] Example 1: Refer to Figure 1 As shown, this embodiment provides a complete process for preparing doxycycline-based standard materials based on a sea bass skin and meat multiphase system, including the following preliminary preparations and mutually synergistic core process steps: I. Preliminary Preparation Stage (1) Determination of target drug and target tissue: Doxycycline is a commonly used broad-spectrum antibacterial drug in freshwater fish farming. It belongs to the tetracycline class of drugs and has a relatively stable polycyclic skeleton structure. It is currently a mandatory test item in the detection of veterinary drug residues in sea bass. According to the current National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food (GB 31650-2019), the legal target tissue for doxycycline residue detection is "skin + flesh", and the maximum residue limit (MRL) is 100 μg / kg. Therefore, in this embodiment, the fish meat tissue and fish skin (multiphase matrix) of sea bass are selected as the matrix, and 100 μg / kg is set as the target concentration of the matrix standard substance.

[0031] (2) Determination of detection method: The current national standard GB 31656.11-2021 "National Food Safety Standard for Determination of Residues of Oxytetracycline, Tetracycline, Chlortetracycline and Doxycycline in Aquatic Products" was selected as the standard method for subsequent concentration retesting and value determination.

[0032] II. Core Process Steps Step 1, Live Enrichment: Fifty healthy sea bass, approximately 1.5 kg in weight, raised in captivity and never treated with antibiotics, were selected. They were divided into a treatment group (40 fish) and a control group (10 fish), with the control group raised alone without medication. The treatment group was administered a bath of 50% doxycycline hydrochloride soluble powder (with a standard approval number) at a concentration of 50 mg / L, once daily for two consecutive days before harvesting. This bath method realistically simulates the natural metabolic pathway of drug absorption through water and gills during aquaculture, allowing the drug to be evenly enriched in the fish's skin and flesh tissue through live metabolism.

[0033] Step Two, Cryogenic Quenching and Embrittlement: Immediately after the drug was discontinued and the fish were caught, the two groups of bass were slaughtered and gutted. The target fish, with intact skin and flesh, were then quickly placed directly into an ultra-low temperature freezer at -80°C for at least 24 hours. This extreme low-temperature thermal shock not only instantly terminated all enzymatic reactions and further drug metabolism within the living tissue, but also caused intracellular free water to form fine ice crystals. The expansion of these ice crystals irreversibly tore apart the dense collagen fiber network in the bass skin at the microscopic level, achieving the crucial physical ice crystal embrittlement.

[0034] Step 3, Multiphase Matrix Preparation: After freezing, the quenched target fish torso was removed, thawed with room temperature water, and thoroughly cleaned. After cleaning, the scales, head, tail, and bones were removed, leaving only the intact skin and flesh. The skin and flesh were then cut into pieces and ground into a paste using a tissue homogenizer, yielding both drug-containing skin-flesh homogenate and blank skin-flesh homogenate. Thanks to the pre-treatment at -80℃, the originally tough fish skin was easily pulverized to the micron level, resulting in a highly homogeneous multiphase paste, effectively avoiding protein denaturation caused by mechanical grinding and frictional heat generation.

[0035] Step 4, Eutectic Lock-in and Extreme Freeze-Drying: Using a vacuum freeze dryer equipped with a eutectic point testing module, preferably the German Christ Alpha1-2 LD plus, the eutectic point of the sea bass "skin + meat" multiphase meat paste was determined. The eutectic point was determined using the resistance method: the resistance measuring electrode was vertically inserted into the skin-meat homogenate sample, and the sample was cooled at a rate of 1-2℃ / min, while continuously recording the resistance value change. When the temperature drops to the eutectic point, all the remaining liquid water in the sample is converted into solid ice, ion migration is completely blocked, and the resistance value jumps sharply. The temperature corresponding to this abrupt change is the eutectic point of the skin-meat homogenate meat paste. The measured eutectic points were distributed between -18℃ and -25℃.

[0036] To prevent water-lipid microphase separation and localized boiling in the high-lipid / high-collagen complex system during vacuuming, the pre-cooling temperature was strictly set to -40°C, at least 15°C below the eutectic point, for a supercooling pre-freezing period of 24 hours. After pre-freezing to ensure complete freezing and phase locking, the material was subjected to deep vacuum freeze-drying for 72 hours under high vacuum conditions of -80°C in the cold trap and 20 Pa. This parameter combination not only did not damage the molecular structure of doxycycline but also ensured unobstructed sublimation channels, ultimately reducing the matrix moisture content to ≤0.8%. This, measured by the Karl Fischer method, yielded high-concentration drug-containing freeze-dried fish powder and blank freeze-dried fish powder, respectively, with no surface hardening and an extremely loose texture.

[0037] Step 5, Solid-phase closed-loop grading: The two lyophilized powders were pulverized separately for 15 seconds using a blade mixer at 2000 rpm. This combination of low speed and short duration ensured fine pulverization while cleverly controlling the heat generation threshold of mechanical work, preventing the critical temperatures for localized fat melting and protein denaturation from being reached. The pulverized samples were then passed through a 0.6 mm sieve (approximately 30 mesh) and collected in a stainless steel container. After mixing for approximately 2 hours, the samples were sealed and stored at -20°C for later use.

[0038] To obtain a precise target concentration of 100 μg / kg, freeze-dried fish powder containing a high concentration of doxycycline and blank freeze-dried fish powder were added to the mixing tank of a mixer in a predetermined ratio for dry phase mixing. 2 g of the mixed dry powder was then reconstituted with 9 mL of pure water. This 4.5 water-to-powder weight ratio accurately restored the natural moisture content of fresh fish meat to 81%-82%, ensuring that the microosmotic pressure, lipoprotein concentration, and ionization inhibition in the reconstituted solution were completely consistent with those of the actual fresh samples. This achieved 100% equivalent compensation for the matrix effect in chromatographic analysis. After reconstitution, the concentration was detected using the GB 31656.11-2021 standard method. If the concentration deviated from 100 μg / kg, closed-loop fine-tuning was performed by adding blank powder or high-concentration powder until the detected concentration accurately approached the target value. Initial inspection confirmed that the batch homogeneity was qualified, thus completing the fixed-value preparation of the dry powder matrix.

[0039] Step Six, Dispensing and Non-destructive Irradiation Sterilization: Dispense the well-mixed and re-inspected qualified freeze-dried fish powder into 10mL brown glass bottles, preparing a total of 500 portions, each containing no less than 2.5g. After filling with nitrogen, seal the bottles, and then wrap the outer layer of the bottle with an aluminum foil bag for vacuum sealing.

[0040] Without removing the double-sealed packaging, all 500 lyophilized samples were treated with a dose of 4 kGy. 60 In-situ irradiation sterilization was performed using Co-γ rays. Since the matrix moisture content had been compressed to ≤0.8% in the previous steps, there were almost no free water molecules available for radiation hydrolysis in the system. The chain reaction of hydroxyl radicals (·OH) was effectively blocked, and doxycycline molecules were thus protected from the damage caused by radiation by-effects. This successfully achieved a perfect balance between killing cold-resistant microorganisms and protecting the target drug without damage.

[0041] III. Evaluation and Verification Results The 500 samples of doxycycline matrix standard material prepared above were stored at -80℃ in the dark, and transported at ≤-20℃ using ice packs. A systematic metrological evaluation was then performed on them. Homogeneity evaluation: Based on JJF 1343-2022 "Standard Reference Material Value Assignment and Homogeneity and Stability Assessment", one-way ANOVA (F-test) was used for evaluation. Fifteen samples were randomly selected for inter-unit homogeneity testing, with each sample measured three times in parallel under repeatability conditions. The critical value F0.05(14,30) = 2.04 was obtained from the table, and the calculated value was F = 1.61 / 1.29 = 1.24 < 2.04. The results demonstrate that, thanks to the synergistic effect of -80℃ cryogenic embrittlement and low-speed gentle grinding, the homogeneity of this multiphase matrix standard reference material is excellent, with the uncertainty (uhom) introduced by inhomogeneity being only 0.19.

[0042] Stability evaluation: A classical linear model was used to evaluate short-term stability over 15 days at two storage temperatures: 4℃ and 25℃. Samples were taken and measured on days 0, 3, 6, 9, 12, and 15. The calculated values ​​were t0.95,n-2×s(b1)=0.35401>0.03000 and t0.95,n-2×s(b1)=0.402571>0.16620, indicating good short-term stability.

[0043] Long-term stability was evaluated at -20℃ for up to 24 months, with samples taken and measured at months 0, 3, 6, 9, 12, 18, and 24. The calculated values ​​were (t0.95, n-2×s(b1)=0.17030>0.13266). The results show that due to its extremely low water activity (≤0.8%) and non-destructive irradiation protection, this standard material maintains excellent stability after 24 months of storage in a conventional freezer (-20℃), breaking through the lifespan bottleneck of 6-12 months in existing technologies.

[0044] Assigned value and uncertainty assessment: The joint assignment method of 8 authoritative laboratories was adopted, and the final assignment result was 100.32 μg / kg, which is highly consistent with the target concentration of 100 μg / kg, with a relative deviation of only 0.32%.

[0045] Calculations show that the uncertainty introduced by inhomogeneity (uhom) is 0.19; the uncertainty caused by short-term stability (usts) is 0.63021; ​​the uncertainty caused by long-term stability (ults) is 0.56710; and the uncertainty introduced by the settling process brought about by the solid-phase closed-loop blending process (uchar) drops sharply to 0.0604. The overall assessment yields a standard uncertainty (uCRM) of 0.098 and an expanded uncertainty (UCRM, k=2) of 0.196.

[0046] Example 2: Process Parameter Comparison and Verification Experiment. To verify the synergistic effect of the key process parameters of this invention and its superiority over the prior art, the following comparative verification experiment was set up: Comparative Experiment 1: Effect of Cryogenic Temperature on the Fineness of Skin and Flesh Matrix Powdering The bass carcasses from the same batch, after being administered the drug, were divided into three groups: Group A was frozen at -80℃ for 24 hours according to the method of this invention; Group B was frozen at -18℃ for 24 hours, referring to the method in prior art CN102288464B; Group C was treated directly at room temperature without freezing, referring to the method of direct slaughter and mincing in some prior art. All three groups were thawed, impurities removed, and ground under the same conditions, and the particle size distribution of the minced meat after grinding was measured using a laser particle size analyzer.

[0047] The results showed that in Group A (-80℃ group), over 92% of the minced meat contained particles smaller than 100μm, and no intact collagen fiber bundles were observed under a microscope. In Group B (-18℃ group), only 65% ​​of the minced meat contained particles smaller than 100μm, and a large number of incompletely pulverized collagen fiber bundle fragments were visible. In Group C (room temperature group), less than 40% of the minced meat contained particles smaller than 100μm, and the fish skin tissue was basically in fragmented form, making homogenization impossible. This comparative experiment confirms that the ice crystal physical embrittlement effect of -80℃ deep freezing on fish skin collagen fibers is irreplaceable by -18℃ shallow freezing. The former performs irreversible microscopic tearing of the collagen fiber network through ice crystal volume expansion, while the latter only hardens the tissue and cannot effectively destroy the fiber structure.

[0048] Comparative Experiment 2: Effect of Pre-freezing Temperature on Freeze-drying Efficiency of Skin-Flesh Multiphase Matrix The same batch of sea bass skin and meat homogenate was divided into three groups: Group A was pre-frozen at -40℃ for 24 hours after the eutectic point was measured to be between -18℃ and -25℃ according to the method of this invention; Group B was pre-frozen at -20℃ for 24 hours according to the method of existing technology CN106813963A, based on the pure meat eutectic point of -15℃; Group C was directly vacuum-dried without pre-freezing, following the practice of not measuring the eutectic point in some existing technologies. All three groups were freeze-dried under the same freeze-drying parameters: cold trap -80℃, vacuum degree 20Pa, and duration 72 hours. After freeze-drying, the water content was determined by Karl Fischer method, and the macroscopic morphology of the freeze-dried product was observed.

[0049] The results showed that: Group A (pre-frozen at -40℃) produced a uniformly loose and porous product after freeze-drying, without structural collapse, with a water content of 0.76%; Group B (pre-frozen at -20℃) showed locally hardened areas on the surface of the product after freeze-drying, with some areas of the cross-section being dense, and a water content of 2.8%; Group C (without pre-freezing) exhibited obvious local boiling and bubbling during freeze-drying, with severe structural collapse and a water content as high as 6.5%. This comparative experiment confirms that for the actual eutectic point of the "skin + meat" multiphase matrix (-18℃ to -25℃), the pre-freezing temperature must be lowered to -40℃, at least 15℃ below the eutectic point, to effectively suppress water-lipid microphase separation and structural collapse; directly applying the freeze-drying parameters of -20℃ for pure meat matrix to the skin-meat multiphase matrix will result in a significant decrease in freeze-drying efficiency and a significantly higher water content.

[0050] Comparative Experiment 3: Effect of Irradiation on Doxycycline Content under Different Moisture Contents The freeze-dried sea bass skin and flesh powder prepared in the same batch was divided into three groups, and their moisture content was adjusted accordingly: Group A was freeze-dried powder with a moisture content ≤0.8% prepared by the method of this invention; Group B had its moisture content adjusted to 3.0% by controlling the freeze-drying time, referring to the moisture content level of existing technologies such as CN106813963A; Group C had its moisture content adjusted to 5.0% by controlling the freeze-drying time, referring to the moisture content level of some existing technologies. All three groups of samples were subjected to 4 kGy under the same conditions. 60 The product was packaged in Co-γ, nitrogen-filled and sealed, and vacuum-packed with aluminum foil for irradiation sterilization. The doxycycline content was determined before and after irradiation using the standard method of GB 31656.11-2021, and the content retention rate was calculated.

[0051] The results showed that after irradiation, the doxycycline content retention rate of group A (water content ≤0.8%) was 99.2%, with almost no loss; the doxycycline content retention rate of group B (water content 3.0%) was 91.5%; and the doxycycline content retention rate of group C (water content 5.0%) was only 84.7%. This comparative experiment confirmed that small differences in water content are significantly amplified during irradiation. As the water content increased from ≤0.8% to 5.0%, the irradiation loss rate of doxycycline increased sharply from less than 1% to over 15%. This fully demonstrates the core mechanism of this invention, which reduces the water content to ≤0.8% through extreme freeze-drying, thereby achieving non-destructive irradiation at extremely low water activity: in a near-absolutely dry state, the radiation hydrolysis free radical chain reaction initiated by gamma rays is effectively blocked, and the target drug molecules are protected from oxidative damage.

[0052] Example 3: Applicability of the Target Drug to Other Fish Species. The method of this invention is not limited to the combination of perch and doxycycline. Based on the same process principles and parameter system, it can be applied to other artificially raised freshwater fish such as carp, grass carp, crucian carp, tilapia, etc., as well as other antibacterial drugs with maximum residue limits such as oxytetracycline, chlortetracycline, enrofloxacin, sulfonamides, antiparasitic drugs, or antiviral drugs. When applied to different fish species or different drugs, only the dosage concentration, dosage duration, and withdrawal time need to be adjusted according to the drug's pharmacokinetic curve. Other process parameters, such as -80℃ cryogenic freezing for 24 hours, measured eutectic point and -40℃ pre-freezing, -80℃ cold trap / 20Pa / 72h freeze-drying, 4.5 times water-to-powder ratio reconstitution detection, and 4KGy irradiation sterilization, remain unchanged to obtain a matrix standard material with equally excellent performance.

[0053] The eutectic point of the skin-meat composite matrix for different fish species may vary slightly due to differences in fish species, fat content, and collagen ratio, but all fall within the range of -18℃ to -25℃. In practice, it is only necessary to follow the method described in Example 1 to measure the eutectic point of the skin-meat homogenate of a specific fish species using the resistance method or differential scanning calorimetry, and set the pre-freezing temperature to be at least 15℃ lower than the measured eutectic point. Other freeze-drying parameters, such as cold trap temperature of -80℃, vacuum degree of 20Pa, and freeze-drying time of 72h, do not need to be adjusted. This universality further demonstrates the technical value of the method of the present invention as a standardized preparation platform for matrix standard materials for the analysis of veterinary drug residues in fish meat.

[0054] Through the deep synergy and precise control of the above six process steps of this invention, the following technologies have been successfully developed: drug bath enrichment to achieve uniform drug distribution in skin and flesh tissues; -80℃ deep cryogenic quenching to achieve physical ice crystal embrittlement of fish skin collagen fibers; -40℃ precise phase-locked pre-freezing based on eutectic point measurements to inhibit water-lipid microphase separation; 72h extreme freeze-drying to reduce water content to ≤0.8%; closed-loop mixing of solid-phase dry powder to achieve precise control of target concentration; and 4KGy non-destructive irradiation sterilization at extremely low water activity. These technologies have successfully overcome long-standing technical bottlenecks in the industry, such as the difficulty in processing fish skin-fish flesh multiphase matrix and the radiation degradation of drugs due to high residual moisture. Comparative experimental data further confirmed the irreplaceable nature of each key parameter: the micron-level pulverization effect of -80℃ cryogenic treatment, with a particle size <100μm ratio of 92%, was significantly better than the 65% particle size ratio at -18℃ and less than 40% at room temperature treatment; the moisture content of -40℃ supercooled pre-freezing (0.76%) was much lower than the 2.8% moisture content of -20℃ pre-freezing and the 6.5% moisture content without pre-freezing; the drug retention rate of irradiation with a moisture content ≤0.8% (99.2%) was significantly higher than the 91.5% retention rate of 3.0% moisture content and the 84.7% retention rate of 5.0% moisture content.

[0055] The final doxycycline-based standard material exhibits an exceptionally long stability of 24 months at -20°C, far exceeding the 6-12 month storage limit of existing technologies. Its standard uncertainty (uCRM) is as low as 0.098, significantly better than the 0.15-15.1 levels of existing technologies. The relative deviation between the assigned concentration and the target concentration is only 0.32%. Furthermore, its "skin + flesh" multiphase matrix can achieve 100% equivalent compensation for the matrix effect of the actual test sample during final reconstitution. These interconnected and progressively superior technical indicators fully demonstrate the significant synergistic effect among the various process features of this invention, achieving remarkable technical results that were unpredictable to those skilled in the art.

[0056] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a matrix standard for analyzing veterinary drug residues in fish meat, characterized in that, This includes the following mutually synergistic process steps: Step 1, Live Enrichment: The target veterinary drug is added to the water containing the target fish with skin by means of drug bath administration, so that the drug is evenly enriched in the fish through live metabolism; Step 2, Deep Cryogenic Quenching and Embrittling: Immediately after drug withdrawal, the target fish is slaughtered and its internal organs are removed. Then, the target fish body with intact skin and flesh is placed directly at -80°C for at least 24 hours to stop drug metabolism and achieve physical ice crystal embrittlement of the fish skin collagen fibers and muscle tissue. Step 3, preparation of multiphase matrix: The target fish body after deep cryogenic quenching is taken out, thawed and cleaned, the scales, head, tail and bones are removed, and the skin and meat are kept intact. The skin and meat are cut into pieces and ground with a tissue grinder to obtain a homogenized paste. Step 4, Eutectic Lock-in and Limit Freeze-drying: The eutectic point of the skin and meat homogenate was determined, and the pre-cooling temperature was set to -40°C, which is at least 15°C lower than the eutectic point, for supercooling pre-freezing. After pre-freezing, vacuum freeze-drying was carried out for 72 hours under the conditions of cold trap temperature -80°C and vacuum degree 20Pa to reduce the matrix moisture content to ≤0.8%, and fish meat freeze-dried powder containing high concentration of target veterinary drug and blank fish meat freeze-dried powder were obtained respectively. Step 5, Solid-phase closed-loop determination: The pulverized and sieved fish meat freeze-dried powder containing a high concentration of the target veterinary drug is physically dry-mixed with blank fish meat freeze-dried powder in a certain proportion. The mixed powder is reconstituted and the concentration is tested. Based on the test results, the mixing ratio of the high concentration freeze-dried powder and the blank freeze-dried powder is repeatedly adjusted for closed-loop fine-tuning until the drug concentration of the mixed powder reaches the target concentration requirement and passes the initial uniformity test. Step Six: Non-destructive Irradiation Sterilization: The freeze-dried fish powder reaching the target concentration is nitrogen-filled, sealed, and vacuum-packed. Under extremely low water activity conditions (matrix moisture content ≤ 0.8%), a dose of 4 kGy is applied to the fully sealed packaged product. 60 In-situ irradiation sterilization using Co-γ rays.

2. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, The target veterinary drug mentioned in step one is a drug selected from antibacterial drugs, antiparasitic drugs, or antiviral drugs that has the highest residue limit requirement.

3. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, The target fish with skin mentioned in step one are artificially bred freshwater fish.

4. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, In step four, the measured eutectic point of the homogenized meat paste containing fish skin and fish meat is -18℃ to -25℃, and the supercooling pre-freezing time at -40℃ is 24h.

5. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, The method for determining the eutectic point of the skin and meat homogenate in step four is either resistance method or differential scanning calorimetry.

6. The method for preparing the standard reference material for analyzing veterinary drug residues in fish meat according to claim 1, characterized in that, In step five, the parameters for crushing and sieving are as follows: the freeze-dried material is crushed for 15 seconds using a blade mixer at a speed of 2000 r / min, and then passed through a sieve with a pore size of 0.6 mm.

7. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1 or 6, characterized in that, In step five, when reconstituted the mixed powder, water is added at a weight ratio of 4.

5. Specifically, 2g of the mixed freeze-dried fish powder is added to 9mL of water for reconstitution.

8. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, In step six, the specific method of nitrogen filling and vacuum packaging is as follows: the re-inspected and qualified freeze-dried fish powder is packaged in brown plastic bottles or brown glass bottles in a quantity of not less than 2.5g, and then sealed with nitrogen; the outer layer of the bottle is then vacuum-sealed with an aluminum foil bag.

9. The method for preparing the standard reference material for analyzing veterinary drug residues in fish meat according to claim 1, characterized in that, Before step one, there is also a target drug determination step: select commonly used antibacterial drugs, antiparasitic drugs or antiviral drugs in fish farming as target drugs, and the target drugs are clearly specified in the national standard maximum residue limit or are required not to be detected.

10. The method for preparing the standard reference material for veterinary drug residue analysis in fish meat according to claim 1, characterized in that, Before step one, there is also a target concentration determination step: the target concentration is determined by referring to national standards, the U.S. Food and Drug Administration, or the EU's maximum residue limits for veterinary drugs in fish tissue.

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