Tulobuterol-containing mutton powder matrix standard substance, preparation and detection method

Abstract

The application belongs to the field of analytical chemistry and relates to a tobradex-containing mutton powder matrix standard substance, a preparation method and a detection method. The preparation method comprises the following steps: administering a sheep, killing the sheep to obtain muscle tissue, freezing the muscle tissue, crushing the muscle tissue in a semi-thawed state to obtain crushed muscle tissue, homogenizing the crushed muscle tissue, first freeze-drying, grinding, screening and second freeze-drying to obtain the matrix standard substance. The homogenization conditions comprise the following steps: an ice water bath, a rotation speed of 8000-12000 revolutions per minute, 5s-20s for each homogenization, and an interval of 30s-120s between each homogenization. The grinding conditions comprise the following steps: an ice water bath, 2s-6s for each grinding, and an interval of 10s-60s between each grinding. The method can improve the accuracy of the quantity value of tobradex target analysis components in mutton.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and in particular relates to a mutton powder matrix standard substance containing Tubuterol, and a preparation method and a detection method thereof. Background Art

[0002] Tubuterol is a beta-agonist drug clinically used to treat conditions such as bronchial asthma and obstructive pulmonary disease. It is also used to treat premature labor and miscarriage in animals such as cattle and sheep. It also enhances fat metabolism and protein synthesis in pigs, cattle, and sheep. Long-term consumption of feed containing beta-agonists can improve feed conversion rates, increase animal weight and muscle mass, and thus increase lean meat percentage, thereby redistributing nutrients. However, this drug accumulates in the animal body, and long-term use can have toxic side effects. Consuming meat containing tubuterol residues may cause consumers to experience acute symptoms of poisoning, such as palpitations, elevated blood pressure, and muscle tremors. Long-term consumption can lead to more serious chronic health problems, such as cardiovascular disease, muscle tremors or pain, rapid heart rate, headaches, dizziness, nausea and vomiting, reproductive system damage, and even an increased risk of cancer. In severe cases, it can lead to shock and death, seriously endangering consumers' health.

[0003] To this end, the Ministry of Agriculture and Rural Affairs' Announcement No. 250 explicitly prohibits the use of beta-agonists and their salts and esters in food-producing animals. Furthermore, the "Regulations on the Administration of Feed and Feed Additives," implemented on May 1, 2012, prohibit the addition of any substance, including beta-agonists, to feed, as specified by the State Council's agricultural administrative department. However, recent national agricultural product quality and safety risk monitoring results show that tubuterol is still detected to a certain extent in animal-derived products in my country, demonstrating the persistent problem of the "banned but not stopped" use of beta-agonists in livestock and poultry farming. Therefore, accurate detection and monitoring of beta-agonists, such as tubuterol, is crucial for effectively monitoring the illegal use of these drugs and ensuring food safety in my country. Currently, various analytical methods are available for the analysis and detection of tubuterol in food-producing animals, but a lack of matrix reference materials consistent with actual samples makes it difficult to ensure the accuracy, comparability, and traceability of measurement results.

[0004] It should be noted that this part of the present invention only provides background technology related to the present invention and does not necessarily constitute prior art or public known technology. Summary of the Invention

[0005] The purpose of the present invention is to establish a method for preparing a matrix standard substance that is consistent with actual samples, and to develop a high-accuracy determination method to further develop a matrix standard substance, thereby overcoming the problem of incomparability and non-traceability of existing analytical detection results of tulbuterol in edible animals, and ensuring accurate and reliable measurement results. In response to the defects of complex matrix and poor value accuracy of the target analysis component of tulbuterol in mutton matrix, a mutton powder matrix standard substance containing tulbuterol, as well as preparation and detection methods, is provided. When the mutton powder matrix standard substance containing tulbuterol is used to detect tulbuterol in mutton, it can fully simulate the actual mutton sample to be tested, avoid the influence of matrix effect and matrix mismatch on the accuracy of the value of the target analysis component of tulbuterol during the detection process, and improve the accuracy of the value of the target analysis component of tulbuterol in mutton.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a mutton meal matrix standard substance containing Tubuterol, comprising: administering a drug to a sheep and slaughtering the sheep to obtain muscle tissue, freezing the muscle tissue, crushing the muscle tissue in a semi-thawed state to obtain crushed muscle tissue, homogenizing the crushed muscle tissue, performing a first freeze-drying, grinding, screening, and a second freeze-drying to obtain the matrix standard substance;

[0007] The homogenization conditions include: a rotation speed of 8000-12000 rpm, an ice water bath, each homogenization for 5s-20s, and an interval of 30s-120s between each homogenization. The grinding conditions include: an ice water bath, each grinding for 2s-6s, and an interval of 10s-60s between each grinding.

[0008] In some preferred embodiments, the method further comprises: in the semi-thawed state, before the pulverization, removing the tendon tissue in the muscle tissue and dividing the muscle tissue into pieces with a volume of 0.5 cm 3 ~1cm 3 The pulverizing conditions include: each pulverizing time is less than 30s, ice water bath cooling is performed between each pulverization, and the particle size of the pulverized muscle tissue block is 10 mesh ~ 50 mesh.

[0009] In some preferred embodiments, the homogenization conditions further include: the homogenization time is 0.5h~3h.

[0010] In some preferred embodiments, the method further comprises: pre-freezing the muscle tissue in a freezing device after the homogenization and before the first freeze-drying; the pre-freezing conditions include: a temperature of -90°C to -70°C and a time of 1 hour to 5 hours.

[0011] In some preferred embodiments, the first freeze-drying conditions include: a time of 72 hours to 120 hours, a cold trap temperature of below -70°C, and a sample chamber pressure of below 500 mtorr.

[0012] In some preferred embodiments, the method further comprises: dividing the muscle tissue into pieces with a volume of 0.5 cm after the first freeze-drying and before the grinding. 3 ~1cm 3 of muscle tissue mass.

[0013] In some preferred embodiments, the method further comprises: the mesh size of the sieve for screening is 30-50 meshes, and after screening, mixing is performed in the dark for 1 hour to 3 hours.

[0014] In some preferred embodiments, the method further comprises: after the second freeze-drying, sequentially placing the muscle tissue in a brown sealed glass bottle, vacuum-sealing the brown sealed glass bottle with an aluminum-plastic bag, sterilizing the vacuum-sealed muscle tissue by irradiation, and freezing the muscle tissue;

[0015] The irradiation sterilization conditions include: using gamma rays produced by radioisotope Co60, an irradiation dose of 4 kGy to 6 kGy, and an irradiation time of 2 h to 6 h; and the freezing storage temperature is -80°C to -20°C.

[0016] In some preferred embodiments, the matrix standard substance is tested for uniformity and stability using solid phase extraction-isotope dilution mass spectrometry; the matrix standard substance is quantified using solid phase extraction-isotope dilution mass spectrometry to obtain a nominal value and an uncertainty range; the extractant for the uniformity and stability test and the quantification is an aqueous perchloric acid solution.

[0017] In a second aspect, the present invention provides a mutton meal matrix standard substance containing Tubuterol prepared by the preparation method of the mutton meal matrix standard substance containing Tubuterol described in the first aspect.

[0018] In a third aspect, the present invention provides a method for detecting Tubuterol in mutton muscle tissue, comprising a matrix standard substance prepared by the method for preparing a mutton powder matrix standard substance containing Tubuterol described in the first aspect and / or the mutton powder matrix standard substance containing Tubuterol described in the second aspect.

[0019] The preparation method of the mutton powder matrix standard substance containing Tubuterol of the present invention comprises the following steps: freezing muscle tissue, crushing it in a semi-thawed state, improving the thermal conductivity of the mutton muscle tissue, accelerating the heat dissipation during the muscle tissue crushing process, homogenizing the crushed muscle tissue, rotating at a speed of 8000-12000 rpm, bathing in ice water, homogenizing for 5s-20s each time, and leaving an interval of 30s-120s between each homogenization, freeze-drying to remove moisture from the muscle tissue, grinding the sample after the first freeze-drying, bathing in ice water during the grinding process, grinding for 2s-6s each time, and leaving an interval of 10s-60s between each grinding, and inhibiting the activity of endogenous enzymes (such as proteases, oxidases, etc.) in mutton (mutton The activity of proteins such as proteases and oxidases decreases exponentially with decreasing temperature), thereby inhibiting the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites, inhibiting tubutrol oxidation to ensure the stability of the tubutrol content in mutton; it can inhibit the thermal decomposition of tubutrol to ensure the stability of the tubutrol content in mutton; it can inhibit the denaturation of mutton muscle protein (such as myosin aggregation caused by temperatures above 50°C) to prevent the exposure or dissociation of mutton muscle protein-tubutrol binding sites; it can form a glassy matrix in mutton muscle tissue samples at low temperatures, the mutton protein molecules are fixed in a rigid network, and the tubutrol binding sites remain stable due to the lack of molecular movement, thereby preventing the exposure or dissociation of the tubutrol binding sites.

[0020] In the preparation process of the matrix standard substance of the present invention, the muscle tissue is homogenized in the homogenization process without adding additional water, which can prevent the precipitation of tulbuterol from the mutton muscle tissue. The mutton muscle tissue after homogenization is subjected to a first freeze-drying, which can remove the trace moisture in the muscle tissue itself and the moisture entering the muscle tissue during the crushing and homogenization process, thereby avoiding the precipitation of tulbuterol from the mutton muscle tissue during the subsequent grinding and screening process. The mutton muscle tissue after screening is subjected to a second freeze-drying, which can further remove the trace moisture in the muscle tissue itself and the moisture entering the muscle tissue during the crushing, homogenization, grinding and screening processes, thereby avoiding the precipitation of tulbuterol from the mutton muscle tissue after the matrix standard substance is prepared. The present invention homogenizes the muscle tissue through the homogenization process without adding additional water, performs a first freeze-drying on the homogenized mutton muscle tissue, and performs a second freeze-drying after grinding and screening to avoid the precipitation of tulbuterol from the mutton muscle tissue, thereby affecting the uniformity and tulbuterol content of the mutton powder matrix standard substance.

[0021] The present invention freezes muscle tissue, crushes it in a semi-thawed state, homogenizes it without adding additional water under specific conditions, removes moisture in the muscle tissue by freeze drying, grinds it under specific conditions, performs a first freeze drying on the homogenized mutton muscle tissue, and performs a second freeze drying on the sieved mutton muscle tissue. The present invention can inhibit the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites through synergistic effects, inhibit the oxidation and thermal decomposition of tubutrol, ensure the stability of the tubutrol content in mutton, inhibit the denaturation of mutton muscle protein, prevent the exposure or dissociation of the mutton muscle protein-tubutrol binding sites, and avoid the precipitation of tubutrol from the mutton muscle tissue, thereby fully simulating actual mutton test samples, avoiding the influence of matrix effect and matrix mismatch on the accuracy of the target analysis component value of tubutrol during the detection process, reducing the deviation of the detection result caused by matrix matching, and improving the accuracy of the target analysis component value of tubutrol in mutton. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The present invention is a flowchart of the process steps of the method for preparing a mutton powder matrix standard substance containing Tubuterol in Example 1. DETAILED DESCRIPTION

[0024] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0025] The inventors of the present invention have found that the existing analytical detection methods for Tubuterol in edible animals have limited accuracy in the quantitative values ​​of the target analytical components of Tubuterol.

[0026] In this regard, in a first aspect, the present invention provides a method for preparing a mutton meal matrix standard substance containing Tubuterol, comprising: slaughtering a sheep after administering the drug, obtaining muscle tissue, freezing the muscle tissue, crushing the muscle tissue in a semi-thawed state to obtain crushed muscle tissue, homogenizing the crushed muscle tissue, performing a first freeze-drying, grinding, screening, and a second freeze-drying to obtain the matrix standard substance;

[0027] The homogenization conditions include: a rotation speed of 8000-12000 rpm, an ice water bath, each homogenization for 5s-20s, and an interval of 30s-120s between each homogenization. The grinding conditions include: an ice water bath, each grinding for 2s-6s, and an interval of 10s-60s between each grinding.

[0028] The preparation method of the mutton powder matrix standard substance containing Tubuterol of the present invention comprises the following steps: freezing muscle tissue, crushing it in a semi-thawed state, improving the thermal conductivity of the mutton muscle tissue, accelerating the heat dissipation during the muscle tissue crushing process, homogenizing the crushed muscle tissue, rotating at a speed of 8000-12000 rpm, bathing in ice water, homogenizing for 5s-20s each time, and leaving an interval of 30s-120s between each homogenization, freeze-drying to remove moisture from the muscle tissue, grinding the sample after the first freeze-drying, bathing in ice water during the grinding process, grinding for 2s-6s each time, and leaving an interval of 10s-60s between each grinding, and inhibiting the activity of endogenous enzymes (such as proteases, oxidases, etc.) in mutton (mutton The activity of proteins such as proteases and oxidases decreases exponentially with decreasing temperature), thereby inhibiting the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites, inhibiting tubutrol oxidation to ensure the stability of the tubutrol content in mutton; it can inhibit the thermal decomposition of tubutrol to ensure the stability of the tubutrol content in mutton; it can inhibit the denaturation of mutton muscle protein (such as myosin aggregation caused by temperatures above 50°C) to prevent the exposure or dissociation of mutton muscle protein-tubutrol binding sites; it can form a glassy matrix in mutton muscle tissue samples at low temperatures, the mutton protein molecules are fixed in a rigid network, and the tubutrol binding sites remain stable due to the lack of molecular movement, thereby preventing the exposure or dissociation of the tubutrol binding sites.

[0029] During the preparation of the matrix standard substance of the present invention, the muscle tissue is homogenized in the homogenization process without adding additional water, which can prevent the precipitation of tulbuterol from the mutton muscle tissue. The homogenized mutton muscle tissue is subjected to a first freeze-drying process, which can remove the trace moisture in the muscle tissue itself and the moisture that enters the muscle tissue during the crushing and homogenization process, thereby ensuring the stability of the moisture content of the muscle tissue and preventing the precipitation of tulbuterol from the mutton muscle tissue during the subsequent grinding and screening process. The sieved mutton muscle tissue is subjected to a second freeze-drying process, which can further remove the trace moisture in the muscle tissue itself and the moisture that enters the muscle tissue during the crushing, homogenization, grinding and screening processes, thereby ensuring the stability of the moisture content of the muscle tissue and preventing the precipitation of tulbuterol from the mutton muscle tissue after the matrix standard substance is prepared. The present invention homogenizes muscle tissue without adding water through a homogenization process. The homogenized mutton muscle tissue undergoes a first freeze-drying step, followed by grinding and sieving and a second freeze-drying step. The synergistic effect prevents tulbuterol from precipitating from the mutton muscle tissue, which could affect the homogeneity and tulbuterol content of the mutton powder matrix standard material. The absence of water in the homogenization process also prevents emulsification of the mutton muscle tissue, which could affect the stability of the mutton and tulbuterol. The absence of water in the homogenization process and the double freeze-drying step also inhibit microbial growth and chemical reactions (such as the Maillard reaction).

[0030] The present invention freezes muscle tissue, crushes it in a semi-thawed state, homogenizes it without adding water under specific conditions, removes moisture in the muscle tissue by freeze drying, grinds it under specific conditions, freeze-dries the homogenized mutton muscle tissue for the first time, and freeze-dries the sieved mutton muscle tissue for the second time. Through synergistic effects, the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites can be inhibited, the oxidation and thermal decomposition of tubutrol can be inhibited, the tubutrol content in mutton can be ensured to be stable, the denaturation of mutton muscle protein can be inhibited, and the mutton muscle protein can be prevented from degenerating. -The tulbuterol binding site is exposed or dissociated, preventing the precipitation of tulbuterol from mutton muscle tissue, thereby fully simulating the actual mutton sample to be tested, avoiding the impact of matrix effects and matrix mismatch on the accuracy of the target analytical component of tulbuterol during the detection process, reducing the deviation of the test results caused by matrix matching, and improving the accuracy of the target analytical component of tulbuterol in mutton. It is used for analytical method evaluation, quality control, laboratory capability assessment and quantity comparison of tulbuterol content in mutton. It can also be used for calibrating measuring instruments and evaluating measurement methods. It is simple to operate and easy to promote.

[0031] Existing methods for detecting tulbuterol in animal sources mostly involve external standard quantification or extraction and quantification by adding tulbuterol to a blank sample matrix. Both methods have certain limitations when used to detect tulbuterol in mutton.

[0032] The external standard method relies on free-state tubuterol standards (such as the pure product dissolved in methanol). However, tubuterol in mutton is adsorbed to macromolecules such as proteins through a variety of forces. There are fundamental differences between the two in terms of solvation behavior, matrix interactions, and detection response: the standard exists as a single molecule in methanol, while tubuterol in mutton samples forms complexes with myosin and myoglobin. Despite sample pretreatment, the solvation shell contains polar groups from proteins, which still results in matrix effects and affects the accuracy of the measurement. Furthermore, after mutton sample processing, the protonation efficiency of tubuterol during electrospray ionization (ESI) is reduced by 20%-30% due to the charge shielding effect of proteins in the mutton matrix, while the ionization efficiency of the free-state standard is stable, resulting in inconsistent mass spectrometry signal intensities. The multiple binding interactions (ionic bonds, hydrogen bonds, and hydrophobic interactions) between tulbuterol and mutton protein lead to unstable release efficiency during pretreatment, and the external standard method does not introduce internal standard correction; endogenous substances in the mutton matrix (protein degradation products, phospholipids, free fatty acids) have an inhibitory or enhancing effect on the detection signal, and the external standard method relies on blank matrix correction. The two have a large difference, affecting the accuracy of the test results.

[0033] When tubuterol is added to a blank sample matrix for extraction and quantification, the tubuterol in the blank matrix may be primarily in the free form, differing from the partially bound form in the actual sample. Tubuterol, being weakly basic (pKa ≈ 9.5), can bind to acidic groups (such as aspartic acid and glutamic acid residues) or lipid components of muscle proteins through ionic, hydrogen, or hydrophobic interactions. In the natural matrix, some tubuterol may be embedded within the three-dimensional structure of the protein, forming an irreversible or slowly dissociating bound state. When tubuterol is directly added to the blank mutton matrix, the binding time between tubuterol and the matrix components is short, primarily through physical adsorption or weak interactions. Tubuterol does not undergo the long-term dynamic binding process of tubuterol-mutton matrix in the natural sample. This may result in tubuterol in the blank matrix being more easily dissociated by the solvent during extraction, resulting in higher recoveries. When tubuterol is added to the blank sample matrix for extraction and quantification, the complexity of the mutton matrix and the specific nature of the bound form of tubuterol may distort the estimation of recovery and matrix effects, ultimately affecting the accuracy of the test results. The fundamental reason is that the blank addition method is difficult to simulate the dynamic binding process of tubuterol-lamb matrix in actual samples, and it is easy to ignore the dual interference of matrix components on extraction efficiency and instrument response.

[0034] Matrix standard substances are standard substances that possess the characteristics of actual samples. The present invention, through drug administration and feeding, can produce positive samples consistent with actual mutton samples, ensuring matching with the actual samples and ensuring accurate and reliable measurement results. The present invention prepares a mutton meal-based tulbuterol component analysis standard substance, ensuring consistency between the standard curve and the sample matrix during actual testing, effectively reducing test result deviations caused by matrix matching, and effectively avoiding the impact of matrix effects and matrix mismatches on the accuracy of the target tulbuterol analytical component values ​​during testing.

[0035] The mutton powder matrix standard substance containing Tubuterol of the present invention can obtain a minced meat state consistent with the actual meat sample after being reconstituted with water. In conventional detection, muscle tissue is mainly used, and this standard substance can completely simulate the actual sample to be tested.

[0036] The uniformity of the matrix standard material of the present invention meets the requirements of the national "Technical Specifications for First-Class Standard Materials" and can maintain stability for at least 20 months when stored at -20°C, and can maintain stability for 7 days when the transportation temperature is lower than 50°C.

[0037] In some preferred embodiments, acclimatization is performed for one to two weeks before dosing begins. Under this preferred approach, sheep are sensitive to changes in their feeding environment (e.g., housing, temperature, lighting, group structure), feed type, or management personnel, which can trigger a "stress response" and lead to elevated levels of glucocorticoids (e.g., cortisol). Under stress, the activity of tulbuterol-metabolizing enzymes in the sheep's liver may change, leading to abnormalities in tulbuterol's absorption rate, volume of distribution, metabolic pathways, and excretion rate. This preferred approach allows the sheep's gastrointestinal flora and digestive enzyme activity to match the feed composition, ensuring that tulbuterol is absorbed at a stable rate after administration and forms a reproducible binding state with muscle tissue. This avoids variations in tulbuterol absorption or binding due to sudden changes in feed, further improving the accuracy of the target tulbuterol analytical component values ​​in lamb meat.

[0038] In some preferred embodiments, the administration process includes administering the drug to healthy goats at a dose of 0.5-2 mg / kg•BW•d for 5-14 consecutive days. In some preferred embodiments, the goats are slaughtered immediately after administration.

[0039] In some preferred embodiments, the freezing temperature is -90°C to -70°C, which instantly inactivates the enzymes and is more conducive to inhibiting the activity of endogenous enzymes such as proteases and oxidases in mutton, thereby inhibiting the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites, inhibiting tubutrol oxidation and ensuring the stability of the tubutrol content in mutton.

[0040] In some preferred embodiments, the method further comprises: in the semi-thawed state, before the pulverization, removing the tendon tissue in the muscle tissue and dividing the muscle tissue into pieces with a volume of 0.5 cm 3 ~1cm 3 The conditions for the pulverization include: each pulverization time is less than 30 seconds, ice water bath cooling is performed between each pulverization, and the particle size of the pulverized muscle tissue block is 10 mesh to 50 mesh. Under this preferred embodiment, the muscle tissue is divided into 0.5 cm 3 ~1cm 3 The muscle tissue blocks are crushed for less than 30 seconds each time, and ice water bath cooling is performed between each crushing, which can keep the temperature of the mutton muscle tissue sample below 10°C during the crushing process, and is more conducive to avoiding the increase of endogenous enzyme activity in the mutton due to frictional heat generation, the degradation of mutton muscle protein and the destruction of mutton muscle protein-tubutrol binding sites, the influence of tubutrol oxidation on the stability of tubutrol content in mutton, and the influence of tubutrol thermal decomposition on the stability of tubutrol content in mutton (the phenolic hydroxyl group in its structure is easily oxidized), thereby improving the accuracy of the target analytical component value of tubutrol in mutton; crushing can ensure that a uniform mutton powder sample can be obtained during subsequent freeze-drying and grinding and sieving; the particle size of the crushed muscle tissue is not less than 50 mesh It can prevent the complete destruction of the mutton cell structure and release a large amount of endogenous components (such as proteins, nucleic acids, fats, and polysaccharides). These substances may compete with tulbuterol for extraction solvents or adsorption sites, and may also cause tulbuterol to re-bind with exposed mutton protein sites to form a more stable complex, resulting in "secondary adsorption", which leads to a decrease in extraction efficiency and measurement accuracy. It can also prevent tulbuterol from being oxidized by contact with air, thereby improving the measurement accuracy of the target analytical components of tulbuterol in mutton. The particle size of the crushed muscle tissue is no more than 10 meshes, which can ensure that a uniform mutton powder sample can be obtained during subsequent freeze-drying and grinding and sieving; in the semi-thawed state, removing the tendon tissue in the muscle tissue is more conducive to fully removing the tendon tissue.

[0041] In some preferred embodiments, the homogenization conditions further include a homogenization time of 0.5 to 3 hours. Under this preferred embodiment, under the aforementioned specific homogenization conditions, it is more conducive to ensuring that the muscle tissue is crushed and mixed while inhibiting the degradation of mutton muscle protein and the destruction of mutton muscle protein-Tubuterol binding sites, inhibiting the oxidation and thermal decomposition of Tubuterol, ensuring the stability of the Tubuterol content in the mutton, inhibiting the denaturation of mutton muscle protein, preventing the exposure or dissociation of mutton muscle protein-Tubuterol binding sites, and improving the accuracy of the target analytical value of Tubuterol in mutton.

[0042] In some preferred embodiments, the method further comprises pre-freezing the muscle tissue in a freezer after the homogenization and before the first freeze-drying step, wherein the pre-freezing conditions include a temperature of -90°C to -70°C for 1-5 hours. Under this preferred embodiment, the eutectic point of mutton is approximately -25°C to -30°C. Compared to direct freeze-drying, pre-freezing the muscle tissue at a temperature of -90°C to -70°C completely solidifies the muscle tissue, thereby avoiding the "spray bottle" phenomenon caused by boiling of unfrozen liquid during freeze-drying sublimation. The pre-freezing temperature of -90°C to -70°C also allows for rapid freezing of the sample, forming uniform and fine ice crystals, thereby preventing large ice crystals from piercing the cell structure and causing the tulbuterol to fall off the mutton protein binding sites (mechanical damage). By controlling the molecular movement speed, the tulbuterol is prevented from dissociating from the mutton muscle protein due to thermal motion, thereby fixing the binding state of the tulbuterol to the mutton muscle protein.

[0043] In some preferred embodiments, the first freeze-drying process includes a duration of 72-120 hours, a cold trap temperature below -70°C, and a sample chamber pressure below 500 mtorr. This preferred solution effectively removes trace moisture inherent in the muscle tissue, as well as moisture introduced during the pulverization and homogenization process. This prevents precipitation of tulbuterol from the mutton muscle tissue during subsequent grinding and sieving, ensures the sublimation of free (unbound) water in the sample under vacuum, preserves the solid structure of the tulbuterol-mutton muscle protein conjugate, and improves the accuracy of the target tulbuterol component in mutton. A cold trap temperature below -70°C, below the temperature at which the mutton protein matrix begins to soften, prevents softening and collapse of the mutton protein matrix, which could block water vapor channels and thus improve drying efficiency. A sample chamber pressure below 500 mtorr also lowers the boiling point of water, promoting direct sublimation of ice crystals and preventing the formation of liquid water, which could dissolve some tulbuterol and free it.

[0044] In some preferred embodiments, the method further comprises: dividing the muscle tissue into pieces with a volume of 0.5 cm after the first freeze-drying and before the grinding. 3 ~1cm 3 This preferred embodiment, based on the aforementioned specific grinding conditions, is more conducive to inhibiting the degradation of mutton muscle protein and the destruction of mutton muscle protein-Tubuterol binding sites, inhibiting the oxidation and thermal decomposition of Tubuterol, ensuring the stability of the Tubuterol content in mutton, inhibiting the denaturation of mutton muscle protein, preventing the exposure or dissociation of mutton muscle protein-Tubuterol binding sites, and improving the accuracy of the target analytical value of Tubuterol in mutton.

[0045] In some preferred embodiments, the method further comprises: the mesh size of the sieve for sieving is 30 to 50 meshes, and the sieve is mixed in the dark after sieving, and the time for the mixing in the dark is 1-3 hours. Under this preferred embodiment, the mesh size of the sieve is not greater than 50 meshes, which is more conducive to preventing the exposure of the binding site of tulbuterol to mutton muscle protein and reducing the risk of tulbuterol being released. The mesh size of the sieve is not less than 30 meshes, which is more conducive to shortening the internal moisture diffusion path and preventing the second freeze-drying time from being too long. The different water absorption levels of the samples during the grinding and sieving process can easily cause sample unevenness. Mixing in the dark for 1-3 hours after sieving is more conducive to improving the uniformity of the mutton powder matrix standard substance.

[0046] In some preferred embodiments, the second freeze-drying process includes a cold trap temperature below -70°C and a sample chamber pressure below 500 mtorr. This preferred solution further ensures the sublimation of free water (unbound water) in the sample under vacuum, preserving the solid structure of the tulbuterol-lamb muscle protein conjugate and improving the accuracy of the target tulbuterol component in the mutton analysis. It also prevents the mutton protein matrix from softening and collapsing, blocking water vapor channels and thus improving drying efficiency. Furthermore, it prevents the generation of liquid water that could dissolve some of the tulbuterol, freeing it.

[0047] In some preferred embodiments, the method further comprises: after the second freeze-drying, sequentially placing the muscle tissue in a brown sealed glass bottle, vacuum-sealing the brown sealed glass bottle with an aluminum-plastic bag, sterilizing the vacuum-sealed muscle tissue by irradiation, and freezing the muscle tissue;

[0048] The irradiation sterilization conditions include: using gamma rays produced by radioisotope Co60, an irradiation dose of 4 kGy to 6 kGy, and an irradiation time of 2 h to 6 h; and the freezing storage temperature is -80°C to -20°C.

[0049] Under this preferred embodiment, the muscle tissue is placed in a brown sealed glass bottle, and the brown sealed glass bottle is vacuum-sealed with an aluminum-plastic bag, which is more conducive to preventing microorganisms and moisture in the air from affecting the stability of the properties of mutton and the tubuterol in the mutton, and avoiding the oxidation of tubuterol from affecting the stability of the tubuterol content in the mutton, thereby improving the accuracy of the target analysis component value of tubuterol in the mutton; freezing the muscle tissue at a temperature of -80°C to -20°C after irradiation sterilization is more conducive to reducing molecular thermal motion, inhibiting covalent bond rupture, ensuring the stability of the properties of mutton and the tubuterol in the mutton, and improving the accuracy of the target analysis component value of tubuterol in the mutton; the hydrophobic cavity formed by the protein conformation at room temperature may be partially opened due to the thermal motion of the protein, and exposure to the water environment may reduce the hydrophobic effect The results show that the tumbuterol-mutton muscle protein binding stability is weak, which affects the stability of tumbuterol-mutton muscle protein binding. Storage at a low temperature of -80℃~-20℃ with isolation from moisture can quickly fix the natural conformation of the protein, ensure the closure of the hydrophobic cavity and isolate moisture, ensure the stability of tumbuterol-mutton muscle protein binding, and improve the accuracy of the target analysis component value of tumbuterol in mutton; the muscle tissue after vacuum sealing and packaging is irradiated and sterilized using γ rays produced by radioactive isotope Co60 with an irradiation dose of 4kGy~6kGy and an irradiation time of 2h~6h, which is more conducive to preventing the putrefactive microorganisms in the mutton tissue from affecting the mutton and the stability of the tumbuterol properties in the mutton, affecting the stability of the tumbuterol content in the mutton, and improving the accuracy of the target analysis component value of tumbuterol in the mutton.

[0050] The brown sealed glass bottle of the present invention is preferably subjected to cleaning steps such as acid soaking, secondary water rinsing, and high-purity water rinsing, and is dried at 100° C. to 110° C. and cooled to room temperature for use.

[0051] In some preferred embodiments, the matrix standard substance is tested for uniformity and stability using solid phase extraction-isotope dilution mass spectrometry; the matrix standard substance is quantified using solid phase extraction-isotope dilution mass spectrometry to obtain a nominal value and an uncertainty range; the extractant for the uniformity and stability test and the quantification is an aqueous perchloric acid solution.

[0052] Quantitation using the external standard method is susceptible to matrix interference, affecting the accuracy of sample measurement results. For lamb matrices containing components such as protein, fat, and nucleic acids, the target compound may experience fluctuations in recovery due to adsorption or co-precipitation during pretreatment (e.g., protein precipitation or solid-phase extraction), or matrix components may interfere with the mass spectrometry signal (e.g., ion suppression / enhancement). This reference material is quantified using isotope dilution mass spectrometry. The labeled internal standard is chemically identical to the target compound (different only in isotopes). During sample pretreatment, it undergoes protein binding, extraction and release, and matrix interaction simultaneously with the target compound. The mass spectrometry signals of the labeled internal standard and the target compound are subject to the same degree of matrix interference. The differences in their ionization efficiency and chromatographic retention behavior are determined solely by their mass number (a difference of 9 Da). Matrix effects can be offset by isotope peak intensity ratios, effectively eliminating matrix effects and operational errors, achieving absolute quantification and ensuring accurate and reliable measurement results.

[0053] The chemical structure of Tubuterol contains polar groups such as amino group (-NH2) and phenolic hydroxyl group (-OH). On the surface of mutton protein (such as myofibrillar protein and myoplasmic protein in mutton), there are a large number of acidic amino acid residues (such as aspartic acid and glutamic acid, containing carboxyl group - COOH) and basic amino acid residues (such as lysine and arginine, containing amino group - NH2). The two are combined through electrostatic bonding to form ionic bonds and hydrogen bonds. The extraction agent is a strong acidic solution of perchloric acid aqueous solution, which can react with the protein carboxyl group (-COOH) and the protein carboxyl group (-COOH). - ) binds to it, protonating it (-COOH) and losing its negative charge, making it unable to bind to the positively charged amino group (-NH3 + ) forms an electrostatic effect, and at the same time, the perchlorate ion (ClO4 - ) as a strongly hydrophilic ion, can form an ion pair with the positively charged amino group of tulbuterol, replacing its binding to the protein; on the other hand, the perchloric acid aqueous solution has a strong polarity, which can increase the ionic strength of the solution, reduce the hydrophobic interaction between mutton protein and tulbuterol, and further promote the dissolution and elution of tulbuterol; the extractant is selected as a strongly acidic solution of perchloric acid aqueous solution. Through the above two effects, the matrix standard substance can be accurately determined, thereby improving the accuracy of tulbuterol detection in mutton.

[0054] In a second aspect, the present invention provides a mutton meal matrix standard substance containing Tubuterol prepared by the preparation method of the mutton meal matrix standard substance containing Tubuterol described in the first aspect.

[0055] The matrix standard substance of the present invention can completely simulate the actual mutton sample to be tested, avoid the influence of matrix effect and matrix mismatch on the accuracy of the quantitative value of the target analysis component of tulbuterol during the detection process, reduce the deviation of the detection result caused by matrix matching, and improve the accuracy of the quantitative value of the target analysis component of tulbuterol in mutton.

[0056] In a third aspect, the present invention provides a method for detecting tulbuterol in mutton muscle tissue, comprising a matrix standard material prepared using the method for preparing a mutton powder matrix standard material containing tulbuterol described in the first aspect and / or the mutton powder matrix standard material containing tulbuterol described in the second aspect. The tulbuterol detection method of the present invention can avoid matrix effects and matrix mismatches that can affect the accuracy of the target analyte value of tulbuterol during detection, thereby improving the accuracy of the value.

[0057] The present invention will be further described in detail below with reference to specific embodiments.

[0058] Example 1

[0059] A preparation method of mutton powder matrix standard substance containing Tubuterol, referring to Figure 1 , the steps are as follows:

[0060] Step 1: Select goats weighing 50-55 kg and in good health and condition and administer Tubuterol. The goats should be acclimated for one week before administration. Feed should be stopped 12 hours before administration, but water is not restricted. After that, the goats should be fed freely and water should be replenished regularly every day. Tubuterol should be dissolved in normal saline and administered orally with a syringe after eating in the morning at a dose of 1.2 mg / kg•BW•d. The administration should be continued for 7 consecutive days. After the administration period, the goats should be slaughtered to obtain muscle tissue and frozen at -80°C. The remaining unused tissue should be incinerated for harmless disposal.

[0061] Step 2: Place the muscle tissue from step 1 at room temperature. When it is half-thawed, remove the tendon tissue from the muscle tissue and cut the muscle tissue into 0.8cm pieces. 3 ~1 cm 3 Muscle tissue blocks of different sizes were crushed using a stainless steel meat grinder to obtain crushed muscle tissue, which was placed in a sealed bag and refrigerated at 4°C. The crushing conditions included: each crushing time was 20 seconds, ice-water bath cooling was performed between each crushing, and the particle size of the crushed muscle tissue blocks was 10-50 mesh. The crushed muscle tissue was homogenized using a tissue homogenizer to further crush the muscle tissue. The homogenization conditions included a blade speed of 10,000 rpm, an ice-water bath, each homogenization was 10 seconds, an interval of 60 seconds between each homogenization, and a homogenization time of 1 hour.

[0062] Step 3: The muscle tissue homogenized in step 2 is evenly spread on a clean freeze-drying tray and pre-frozen in a refrigerator. The pre-freezing conditions include: temperature of -80°C and time of 3 hours. After pre-freezing, the muscle tissue is placed in a freeze dryer for a first freeze-drying. The first freeze-drying conditions include: time of 96 hours, cold trap temperature of -80°C, and sample chamber pressure of 400mtorr~500mtorr.

[0063] Step 4: Divide the freeze-dried muscle tissue from step 3 into 0.5 cm 3 ~0.7cm 3 Muscle tissue blocks of different sizes were ground using a grinder. The grinding conditions included an ice water bath, 5 seconds per grinding, and 30 seconds between each grinding. The ground muscle tissue was sieved using a 40-mesh sieve. The sieved mutton muscle tissue powder was collected and poured into a clean plastic bag. The sieved mutton muscle tissue powder was poured into a V-shaped mixer and mixed in a dark environment for 1 hour.

[0064] Step 5: placing the mixed muscle tissue from step 4 into a freeze dryer for a second freeze drying, wherein the muscle tissue has a constant weight after the second freeze drying, and the conditions for the second freeze drying include: a cold trap temperature of -80°C and a sample chamber pressure of 400 mtorr to 500 mtorr;

[0065] Step 6: Place the muscle tissue after the second freeze-drying in a brown glass bottle with a sealed inner lid for sample packaging, and vacuum-seal the brown sealed glass bottle with an aluminum-plastic bag. The brown glass bottles have undergone acid soaking, secondary water rinsing, high-purity water rinsing and other cleaning steps, and are dried at 105°C. The packaging unit is: 4g / bottle, and a total of 160 bottles are packaged. The vacuum-sealed muscle tissue is irradiated and sterilized to obtain the matrix standard substance. The irradiation sterilization is carried out using γ rays generated by the radioactive isotope Co60. The irradiation dose is 5.5 kGy and the irradiation time is 4h. The irradiated samples are stored in a freezer at -20°C.

[0066] Comparative Example 1

[0067] Refer to Example 1, except that, in step 2, homogenization is not performed. Other conditions remain unchanged.

[0068] Comparative Example 2

[0069] Refer to Example 1, except that, in step 2, during the homogenization process, water is added at a ratio of 1:3 between the weight of the pulverized muscle tissue and the weight of water, and all other conditions remain unchanged.

[0070] Comparative Example 3

[0071] Refer to Example 1, except that the second freeze-drying in step 5 is not performed. Other conditions remain unchanged.

[0072] Comparative Example 4

[0073] Refer to Example 1, the difference is that in step 2, the blade speed during the homogenization process is 5000 rpm.

[0074] Experimental example

[0075] Experimental Example 1

[0076] The matrix standard substances of Example 1 and Comparative Examples 1 to 4 were subjected to a uniformity test according to the requirements of the "Technical Specifications for Primary Standard Materials" (JJG1006-1994) and the "Value Determination, Uniformity and Stability Evaluation of Standard Materials" (JJF1343-2022).

[0077] The uniformity inspection process specifically includes:

[0078] The packaged matrix standard materials were uniformly numbered. Eleven packaging units were randomly sampled from the beginning, middle, and end of the entire packaging process. These randomly sampled matrix standard material samples were numbered from 1 to 11. Three parallel subsamples of each randomly sampled matrix standard material were tested, each weighing 0.5 g. Homogeneity was tested using the established solid-phase extraction-isotope dilution high-performance liquid tandem mass spectrometry method. The results were statistically analyzed using analysis of variance. Homogeneity was confirmed at a 95% confidence level by comparing the F test value with the F critical value.

[0079] Extract from the total unit m Units, pairs i carried out n i Measurements ( i = 1,…,a), calculate the within-group sum of squares ( Q 1) and the sum of squares of the between-group variance ( Q 2) The calculation formula is as follows:

[0080]

[0081] F distribution function degrees of freedom ( v 1 , v 2), where the between-group variance is , the within-group variance is , statistics .

[0082] According to the degrees of freedom ( v 1 , v 2) and significance level α, given by FThe critical value table of the distribution is used to find the critical Fα Value, calculated F value ＜Fα There is no significant difference between the data groups, and the matrix standard material samples are uniform.

[0083] like ,in this case, Equivalent to the uncertainty caused by unevenness between bottles .Right now

[0084]

[0085] like hour,

[0086]

[0087] At a confidence level of 95%, variance analysis was used to test the homogeneity of the matrix standard material samples. The experimental data and statistical analysis results of the matrix standard material of Example 1 are shown in Table 1.

[0088] Table 1

[0089]

[0090] The results of the uniformity test of the matrix standard material of Example 1 are shown in Table 2.

[0091] Table 2

[0092]

[0093] From the experimental data and statistical analysis results, it can be seen that the F test value (statistical value) of the uniformity test result of the matrix standard substance in Example 1 is F value) is less than the F critical value (uniformity test critical value) F 0.05 (10,22) : 2.3. The matrix standard substance prepared in Example 1 meets the uniformity requirement.

[0094] Experimental Example 2

[0095] The stability of Example 1 was tested according to the technical specifications for the preparation of standard substances and the principle of first dense and then sparse.

[0096] The stability testing process specifically includes:

[0097] Long-term stability monitoring studies were conducted at 0, 2, 3, 6, 12, and 20 months. Randomly sampled matrix standard material samples were stored at 50°C. Stability monitoring was performed on days 0, 1, 3, 5, and 7. Three aliquots were taken each time, and each unit was measured in triplicate. The measurement method used was the same as the homogeneity test: isotope dilution high-performance liquid chromatography-tandem mass spectrometry. The average of the three aliquots was taken as the long-term stability monitoring result. Trend analysis was used to analyze the results, fitting a straight line between the monitoring time and the results, and statistically analyzing the results. The long-term stability assessment used the classical stability assessment protocol, while the short-term stability assessment used the synchronous stability assessment protocol.

[0098] When the underlying kinetic mechanism is unknown, the basic model for stability assessment is expressed as:

[0099]

[0100] Where, represents the regression coefficient, Y is the characteristic value, X For time.

[0101] Standard deviation The calculation formula is as follows:

[0102]

[0103] based on The standard deviation of The test makes the following judgments:

[0104] like , it indicates that the slope is not significant and no instability is observed.

[0105] The uncertainty introduced by stability, .

[0106] The long-term stability monitoring results of the matrix standard substance of Example 1 are shown in Table 3.

[0107] Table 3

[0108]

[0109] The results of the short-term stability monitoring of the matrix standard substance of Example 1 at 50°C are shown in Table 4.

[0110] Table 4

[0111]

[0112] The results of a 20-month stability study of the tulbuterol concentration of the matrix standard material in Example 1 showed that the characteristic concentration of the matrix standard material was stable over the 20-month period. Under ice pack transportation conditions, the concentration of the matrix standard material remained unchanged for 7 days at an ambient temperature below 50°C.

[0113] Experimental Example 3

[0114] The matrix standard material from Example 1 was quantified using isotope dilution mass spectrometry (IDMS) and a network of qualified laboratories was established for joint quantification. Tulbuterol in the matrix standard material was equilibrated with an internal standard of the same isotopic concentration and extracted with aqueous perchloric acid. The supernatant was centrifuged and cleaned up using MCX solid-phase extraction, followed by quantification using high-performance liquid chromatography-tandem mass spectrometry.

[0115] The valuation process specifically includes:

[0116] Weigh 0.5 g of matrix standard material and place it in a 50 mL centrifuge tube. Add 1.5 g of water for reduction, add 148 μL of tulbuterol-D9 isotope labeling solution (71.4 μg / L), vortex to mix, add 15 mL of 0.1 mol / L perchloric acid aqueous solution, vortex for 10 min, sonicate for 10 min, adjust the pH to 1.0±0.2 with perchloric acid, centrifuge at 12000 r / min for 8 min, and transfer the supernatant to another 50 mL centrifuge tube for later use.

[0117] An MCX solid-phase extraction column was activated sequentially with 3 mL each of methanol, water, and 2% formic acid in water. The entire reserve solution was passed through the column, followed by 3 mL each of 2% formic acid in water and methanol, which was then drained. Finally, the column was eluted with 3 mL of 3% ammonia in methanol. The eluate was blown dry with nitrogen at 40°C. The residue was made up to volume with 1 mL of a 1:9 methanol-water solution, vortexed, and passed through a 0.22 μm Jinteng nylon filter membrane for analysis by liquid chromatography-tandem mass spectrometry.

[0118] HPLC conditions: Column: Waters Xbridge C18 3.5 μm, 2.1×150 mm; aqueous phase: 0.1% formic acid in water (A), organic phase: methanol (B), flow rate: 0.3 mL / min; injection volume: 5.00 μL; column temperature: 40°C.

[0119] The HPLC separation and elution conditions are shown in Table 5.

[0120] Table 5

[0121]

[0122] Mass spectrometry conditions were as follows: electrospray ionization positive multiple reaction monitoring mode, ion source spray voltage of 4500 V, ion source temperature of 550 °C, nebulizer pressure of 55 psi, auxiliary gas pressure of 55 psi, curtain gas pressure of 55 psi, and ion dwell time of 100 ms.

[0123] The mass spectrometry parameters of Tubuterol are shown in Table 6.

[0124] Table 6

[0125]

[0126] Each collaborating laboratory will conduct a valuation study based on the joint valuation plan and the uniformly distributed purity reference materials, deuterated isotope internal standards, and randomly sampled matrix reference materials, and submit a valuation report. The joint valuation data will first undergo a normal distribution test to determine if they conform to a normal distribution. The Dixon criterion will be used to statistically test the valuation data of each laboratory to determine if there are any outliers. The Cochran criterion will then be used to determine if any data groups are suspicious and to perform an equal precision test to determine the valuation data. Finally, the arithmetic mean of the determined data will be used as the valuation result. The joint valuation data is shown in Table 7.

[0127] Table 7

[0128]

[0129] After statistical inspection, all data were found to be of equal precision and without outliers, meeting the requirements of the fixed value. The characteristic quantity of the matrix standard substance in Example 1 was the average value of the joint fixed value results of multiple laboratories, 21.32 μg / kg.

[0130] The uncertainty of the matrix standard substance in Example 1 was evaluated. The uncertainty sources introduced in the preparation process of the matrix standard substance mainly consist of three parts: the uncertainty introduced by the standard substance value u char , the uncertainty introduced by the homogeneity of the standard substance u bb , uncertainty introduced by the stability of standard substances u lts and u sts , combining the uncertainty components into the total uncertainty.

[0131] The Class A uncertainty introduced by the joint determination of multiple laboratories is estimated using variance analysis. .

[0132] The type B uncertainty introduced by the experimental operation and result calculation formula is: .

[0133] The uncertainty introduced by the fixed value is .

[0134] Uncertainty introduced by uniformity u bb Including the unevenness of the processing process of the matrix standard material sample, the unevenness of the packaging and the deviation between parallel experimental analyses, .

[0135] The uncertainty introduced by long-term stability is .

[0136] The uncertainty introduced by short-term stability is .

[0137] The uncertainty of the determination result of matrix reference material consists of three parts: the determination value, the uncertainty introduced by the inhomogeneity and instability of the reference material. The overall uncertainty calculation formula of matrix reference material is as follows:

[0138]

[0139] The expanded uncertainty at is:

[0140] The expanded uncertainty of the matrix standard substance of Example 1 is:

[0141]

[0142] The mass value and uncertainty of the matrix standard substance in Example 1 are 21.3±2.1μg / kg ( k =2).

[0143] Experimental Example 4

[0144] The mutton meal matrix standard substances of Example 1 and Comparative Examples 1 to 4 were subjected to three parallel tests of the tulbuterol content in the mutton meal samples using solid phase extraction-isotope dilution mass spectrometry, and the RSD (relative standard deviation) was calculated. The test and calculation results are shown in Table 8.

[0145] Table 8

[0146]

[0147] Through Table 8, comparing Example 1 and Comparative Example 1, it can be seen that homogenizing the crushed muscle tissue can improve the uniformity of the mutton meal matrix standard substance and improve the consistency of the measurement value. Comparing Example 1 and Comparative Example 2, it can be seen that homogenizing the crushed muscle tissue without adding water can improve the uniformity of the mutton meal matrix standard substance and improve the consistency of the measurement value. Comparing Example 1 and Comparative Example 3, grinding and screening are followed by a second freeze-drying, which can improve the uniformity of the mutton meal matrix standard substance and improve the consistency of the measurement value. Comparing Example 1 and Comparative Example 4, the blade speed during the homogenization process is 8000-12000 rpm, which can improve the uniformity of the mutton meal matrix standard substance and improve the consistency of the measurement value.

[0148] The mutton meal matrix standard material containing Tubuterol has undergone homogeneity and stability testing and has been jointly determined by multiple laboratories using established, high-accuracy determination methods. It can be used in instrument calibration, method validation, quality control, and other measurements. The matrix standard material of the present invention effectively ensures accurate, comparable, and traceable measurement results for Tubuterol residues in mutton, providing significant social and economic benefits for food safety supervision and testing.

[0149] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for preparing a mutton powder matrix standard substance containing Tubuterol, characterized in that: include: After administering the drug to the sheep, the sheep is slaughtered to obtain muscle tissue, the muscle tissue is frozen, and in a semi-thawed state, the muscle tissue is crushed to obtain crushed muscle tissue, and the crushed muscle tissue is homogenized, freeze-dried, ground, sieved, and freeze-dried to obtain the matrix standard substance; The homogenization conditions include: a rotation speed of 8000-12000 rpm, an ice water bath, each homogenization for 5s-20s, and an interval of 30s-120s between each homogenization. The grinding conditions include: an ice water bath, each grinding for 2s-6s, and an interval of 10s-60s between each grinding.

2. The preparation method according to claim 1, characterized in that Also includes: In the semi-thawed state, before the pulverization, the tendon tissue in the muscle tissue was removed and the muscle tissue was divided into pieces with a volume of 0.5 cm 3 ~1cm 3 The pulverizing conditions include: each pulverizing time is less than 30s, ice water bath cooling is performed between each pulverization, and the particle size of the pulverized muscle tissue block is 10 mesh ~ 50 mesh.

3. The preparation method according to claim 1, characterized in that The homogenization conditions also include: the homogenization time is 0.5h~3h.

4. The preparation method according to claim 1, characterized in that Also includes: After the homogenization and before the first freeze-drying, pre-freezing the muscle tissue in a freezing device; The pre-freezing conditions include: a temperature of -90°C to -70°C and a time of 1 hour to 5 hours.

5. The preparation method according to claim 1, characterized in that The conditions for the first freeze-drying include: a time of 72 hours to 120 hours, a cold trap temperature of below -70°C, and a sample chamber pressure of below 500 mtorr.

6. The preparation method according to claim 1, characterized in that Also includes: After the first freeze-drying and before the grinding, the muscle tissue was divided into pieces with a volume of 0.5 cm 3 ~1cm 3 of muscle tissue mass.

7. The preparation method according to claim 1, characterized in that Also includes: The mesh number of the sieve for screening is 30-50 mesh, and after screening, the mixture is mixed in the dark for 1 hour to 3 hours.

8. The preparation method according to claim 1, characterized in that Also includes: After the second freeze-drying, the muscle tissue is placed in a brown sealed glass bottle, the brown sealed glass bottle is vacuum-sealed with an aluminum-plastic bag, the vacuum-sealed muscle tissue is sterilized by irradiation, and the muscle tissue is frozen for preservation; The irradiation sterilization conditions include: using gamma rays produced by radioisotope Co60, an irradiation dose of 4kGy to 6kGy, and an irradiation time of 2h to 6h; The freezing storage temperature is -80°C to -20°C.

9. The preparation method according to claim 1, characterized in that Solid phase extraction-isotope dilution mass spectrometry is used to test the homogeneity and stability of the matrix standard substance; solid phase extraction-isotope dilution mass spectrometry is used to determine the value of the matrix standard substance to obtain a nominal value and an uncertainty range; the extraction agent for the uniformity and stability test and the determination is an aqueous solution of perchloric acid.

10. A mutton powder matrix standard substance containing Tubuterol prepared by the method for preparing a mutton powder matrix standard substance containing Tubuterol according to any one of claims 1 to 9.

11. A method for detecting Tubuterol in mutton muscle tissue, characterized in that: The invention comprises a matrix standard substance prepared by the preparation method of the mutton powder matrix standard substance containing Tubuterol according to any one of claims 1 to 9 and / or the mutton powder matrix standard substance containing Tubuterol according to claim 10.

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