Preparation method of beef frozen wet powder matrix standard substance

Abstract

The invention discloses a preparation method of a beef frozen wet powder matrix standard substance, and solves the technical problems of poor authenticity, low fidelity, poor uniformity and high energy consumption when a beef ractopamine residual matrix standard substance is prepared in the prior art. Obtaining a ractopamine positive beef sample; performing acid discharge treatment on the beef sample; pretreating the beef subjected to acid discharge, and cutting the beef into diced meat with the particle size of 5-10mm; preparing a frozen wet powder matrix standard substance by adopting a multi-stage freezing and crushing and pulse screening process; and uniformly mixing the final product at a low temperature, sub-packaging, and storing in an environment of-40 DEG C or-80 DEG C in a sealing manner. The traditional freeze-dried powder process is abandoned, the frozen wet powder is used as a carrier form, and the original physicochemical characteristics of fresh tissues are perfectly reserved by the form.

Description

Technical Field

[0001] This invention relates to the field of food safety testing and standard substance preparation technology, specifically to a method for preparing a beef frozen wet powder matrix standard substance. Background Technology

[0002] The accuracy and reliability of food safety testing data are highly dependent on standard materials used for method calibration and quality control. For example, in the trace detection of β-receptor agonists (such as ractopamine and clenbuterol, known as "lean meat powder"), the complex beef matrix can induce significant matrix effects, severely interfering with the quantitative accuracy of chromatography-mass spectrometry (GC-MS). Therefore, beef matrix standard materials that closely match the real sample matrix are crucial tools for ensuring accurate and comparable test results. However, current mainstream technologies rely on the "freeze-drying-grinding" preparation route, which has inherent and insurmountable drawbacks: the mechanical grinding process after freeze-drying easily induces degradation of heat-sensitive target substances and fat oxidation, altering the original chemical state of the matrix and causing "distortion" of the standard material; the prepared freeze-dried powder must undergo a rehydration and reconstruction step before use, which may introduce errors, and the reconstituted matrix cannot completely and realistically simulate the interference effects of fresh samples; more importantly, for beef tissue with high fat content, traditional processes struggle to solve the problems of fat adhesion and clumping, resulting in wide particle size distribution and component separation, thus affecting the uniformity of the matrix standard material. This will directly lead to increased differences between standard reference bottles, seriously affecting the accuracy of the determination results and the effectiveness of its use as a quality control material. In addition, the lengthy freeze-drying process is extremely energy-intensive, which also significantly increases production costs. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a beef frozen wet powder matrix standard material, so as to solve the technical problems of poor authenticity, low fidelity, poor uniformity and high energy consumption in the preparation of beef ractopamine residue matrix standard materials in the prior art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for preparing a frozen wet powder matrix standard material of beef, comprising the following steps: S1. Obtain ractopamine-positive beef samples; S2. Perform acid removal treatment on the beef samples; S3. Pre-treat the beef after aging and cut it into small cubes with a diameter of 5-10mm. S4. A multi-stage cryogenic pulverization and pulse sieving process is used to prepare cryogenic wet powder matrix standard materials, including: S41. Pre-cooling of diced meat: Spray liquid nitrogen while stirring to form an ice shell on the surface of the diced meat, achieving a sand-like flow body state. S42. Cryogenic storage: Immerse pre-cooled diced meat in liquid nitrogen to cool it to below the glass transition temperature; S43. Warming: Warm the frozen meat cubes to -75°C±5°C for more than 60 minutes. S44, Multi-stage cryogenic grinding: The first stage coarse grinding is performed sequentially until it passes through a 20-mesh sieve, the second stage medium grinding is performed until it passes through a 40-mesh sieve, and the third stage fine grinding is performed until it passes through an 80-mesh sieve. After each stage of grinding, pulse airflow sieving is used. S5. Mix the final product thoroughly at low temperature and dispense it into sealed containers, storing them in an environment of -40°C or -80°C.

[0005] Furthermore, in step S2, the acid removal process involves hanging the item in a low-temperature environment of 0–4°C, a high-humidity environment of 80%–95%, and a well-ventilated environment for 7–14 days.

[0006] Furthermore, step S41 specifically includes the following steps: S411. Weigh out the diced beef, put it into the disperser chamber, turn on the stirring, and set the speed to 120-180 rpm. S412. According to the weight ratio of diced beef to liquid nitrogen of 1:1 to 2, liquid nitrogen is injected into the freezing disperser where the diced beef is tumbling through the liquid nitrogen spray nozzle. S413. When the beef cubes change from being moist and sticky to being distinct and making a crisp sound when they collide with each other, the pre-cooling endpoint is reached. The material is a uniform, sandy, granular flow. Stop injecting liquid nitrogen and stirring, and quickly transfer the pre-cooled, flowable beef cubes to the next container.

[0007] Furthermore, in step S412, during the injection of liquid nitrogen, 40% to 50% of the total amount of liquid nitrogen is rapidly injected within the first 2 minutes; the remaining liquid nitrogen is then slowly and evenly injected within the following 4 to 6 minutes, so that the center temperature of the meat cubes quickly passes through the zone of maximum ice crystal formation.

[0008] Further, in step S44, the primary coarse grinding specifically includes the following steps: Cryogenic assisted grinding: Take meat cubes that have been warmed to -75°C±5°C and put them into a pre-cooled blade-type high-speed cryogenic grinder. Start the grinder and rotate it at 16000~18000rpm. At the same time, start injecting liquid nitrogen at a rate of 100g / min. Observe that there is a continuous flow of low-temperature nitrogen gas coming out of the chamber outlet; grind for 30~60s. Screening: The coarsely crushed material is quickly transferred to a pulse screening machine pre-cooled by liquid nitrogen. Mechanical vibration is turned on at a speed of 1200-1500 rpm. Pulse airflow is turned on at a pressure of 0.3-0.5 MPa and a frequency of 1 second on / 2 seconds off. Screening time is 1-2 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, then weigh it.

[0009] Furthermore, the pulse sieve used in the primary coarse crushing is equipped with a 20-mesh standard sieve with an aperture of 850μm.

[0010] Furthermore, in step S44, the secondary crushing specifically includes the following steps: Cryogenic assisted pulverization: All the undersize material collected after primary coarse pulverization is fed into the pulverizer, the rotation speed is increased to 2000-22000 rpm, the liquid nitrogen injection rate is adjusted to 80 g / min, and the pulverization time is 60-90 seconds; Screening: Transfer the secondary crushed material to a pre-cooled pulse screening machine, turn on the mechanical vibration at a speed of 1200-1500 rpm; turn on the pulse airflow at a pressure of 0.3-0.5 MPa, a frequency of 1 second on / 2 seconds off, and a screening time of 2-3 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately fill it with liquid nitrogen to cover and keep it warm, then weigh it.

[0011] Furthermore, the pulse sieve used for pulverization in the secondary stage is equipped with a 40-mesh standard sieve with an aperture of 425μm.

[0012] Furthermore, in step S44, the three-stage fine grinding specifically includes the following steps: Cryogenic assisted grinding: The undersize material obtained from the secondary grinding is quickly transferred into the planetary ball mill grinding jar pre-cooled with liquid nitrogen. The weight ratio inside the jar is set as grinding balls:material = 10:1. The revolution speed of the grinding jar is 300-400 rpm, and the ratio of rotation speed to revolution speed is 1:-2. Liquid nitrogen is continuously supplied to the cooling system of the grinding jar throughout the grinding process to maintain the jar temperature ≤-80°C. The grinding time is 5-10 minutes. Sieving: Transfer the ground ultrafine powder to a pre-cooled pulse sieve, turn on the mechanical vibration at a speed of 1200-1500 rpm; turn on the pulse airflow at a pressure of 0.3-0.5 MPa, a frequency of 1 second on / 2 seconds off, and a sieving time of 5-8 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, then weigh it.

[0013] Furthermore, the pulse sieve used in the three-stage fine grinding is equipped with an 80-mesh standard sieve with an aperture of 180μm.

[0014] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) This invention breaks through the traditional carrier form and innovatively adopts "frozen wet powder" as the matrix standard material: abandoning the traditional freeze-drying powder process, the "frozen wet powder" is used as the carrier form, which perfectly preserves the original physicochemical properties of fresh tissue and has four core advantages: ① Excellent fidelity: avoids thermal degradation, fat oxidation and protein denaturation during freeze-drying and pulverization, and maintains the stability of trace target substances and matrix integrity to the greatest extent; ② High ease of use: no need for rehydration and reconstruction, can be used after restoring to room temperature, eliminating pretreatment errors; ③ Excellent uniformity: relying on cryogenic pulverization technology, tissue powder with finer particle size and more concentrated distribution can be prepared, especially effectively overcoming the problem of fat adhesion and achieving a high degree of uniformity that is difficult to achieve; ④ Green and low carbon: the energy consumption of the process is reduced by more than 80% compared with the traditional freeze-drying process, which is in line with the concept of green manufacturing.

[0015] (2) This invention develops an efficient and energy-saving multi-stage cryogenic pulverization and pulse screening integrated process: Addressing the challenge of pulverizing high-fat, high-viscosity beef tissue, a novel process combining a three-stage temperature-controlled freezing process ("pre-cooling-cryogenic-heat preservation") with a three-stage circulating pulverization process ("pulverization-screening-re-pulverization"). Through precise temperature control, the material is always kept in its optimal brittle state, significantly reducing liquid nitrogen consumption. The pulsed airflow screening technology solves the technical bottleneck of high-fat materials easily clogging the screen, thus achieving an optimized balance between pulverization efficiency and energy consumption. Ultimately, a beef matrix standard material in the form of frozen wet powder with concentrated particle size distribution and extremely high uniformity is obtained. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0017] Example 1. Obtaining genuine ractopamine-positive beef samples Healthy, growing cattle weighing 250–550 kg were selected and administered the drug orally once daily at a dose 3–10 times the therapeutic dose (specifically 300–1000 μg ractopamine / kg body weight; in this example, the dose used was 500 μg ractopamine / kg body weight). The cattle were fed this drug continuously for 2–4 weeks (approximately 2 weeks of continuous feeding allows the ractopamine to reach metabolic homeostasis, and 4 weeks is the effective period for promoting growth; this example used a 2-week continuous feeding period).

[0018] The experimental cattle were slaughtered within 6 to 48 hours after the last administration (24 hours after administration in this example), bled to allow the blood to flow out fully; skinned and eviscerated; and the carcass was rinsed with a high-pressure water gun to remove any remaining blood, hair, etc., and to remove large pieces of fatty tissue.

[0019] 2. Beef aging After slaughter, the beef carcass is placed in an aging facility within 1-2 hours and hung for 7-14 days at a low temperature of 0-4°C, high humidity of 80%-95%, and in a well-ventilated environment. During this process, proteases in the beef slowly break down muscle proteins and connecting fibers; simultaneously, lactic acid produced from glycogen conversion gradually accumulates, causing the pH value to decrease. The aging process gradually breaks down the rigor mortis in the muscles, making the meat more tender. Aging-aged beef maintains consistency with the texture of real beef sold in the market and is also beneficial for subsequent freeze-drying processes.

[0020] 3. Beef pretreatment After aging, the beef carcass is removed from cold storage, brought to room temperature, and then cut into large pieces using an electric saw. The cut pieces are deboned and trimmed, removing large sections of fascia, thick fat, and connective tissue to obtain relatively lean meat. Further, the meat pieces are diced into small cubes of approximately 5–10 mm in diameter using a meat strip cutter, a double-bladed dicing machine, or manually, and then temporarily stored at 4±3℃.

[0021] 4. Preparation of frozen beef matrix standard material (prepared by multi-stage freeze grinding and pulse sieving) (1) Pre-cool the diced meat Objective: To quickly form an ice shell on the surface of diced meat, making it lose its stickiness and achieve flowability.

[0022] Equipment: 5L austenitic stainless steel cryogenic disperser (with adjustable speed stirring and liquid nitrogen spray interface), liquid nitrogen tank.

[0023] Operating procedures: ① Loading: Weigh 1kg to 15kg of diced beef and put it into the disperser cavity. The loading amount should not exceed 1 / 3 of the cavity volume to ensure sufficient turning space.

[0024] ②Start stirring: Turn on the stirring and set the speed to medium-low speed (120-180 rpm) to make the meat cubes roll evenly and avoid clumping.

[0025] ③ Liquid nitrogen spraying: According to the weight ratio of beef cubes to liquid nitrogen of 1:1 to 2, liquid nitrogen is injected into the freezing disperser containing the tumbling beef cubes through a liquid nitrogen spray nozzle. In the first 2 minutes, about 40% to 50% of the total liquid nitrogen is injected rapidly to quickly warm the entire system (beef cubes, container inner wall) through the "viscous zone" (0℃ to -5℃) of the surface water; in the following 4 to 6 minutes, the remaining liquid nitrogen is injected slowly and evenly to allow the temperature of the center of the beef cubes to quickly pass through the maximum ice crystal formation zone (-1℃ to -5℃).

[0026] ④ Endpoint determination: The meat cubes reach the pre-cooling endpoint by sensory judgment: when they change from being moist and sticky to being distinct particles that make a crisp sound when they collide with each other, the material is a uniform sand-like dispersion.

[0027] ⑤ Discharge: Stop liquid nitrogen and stirring, and quickly transfer the pre-cooled, free-flowing meat cubes to the next container. Wear anti-freeze gloves and goggles throughout the entire process.

[0028] (2) Cryogenic storage Objective: To rapidly cool pre-cooled diced meat to below its glass transition temperature (≈-20℃) for preservation before freeze-pulverization and to provide a brittle foundation for its subsequent pulverization.

[0029] Equipment: wide-mouth liquid nitrogen storage tank, antifreeze tweezers, stainless steel tray.

[0030] Operating procedures: ①Preparation: Prepare a wide-mouth liquid nitrogen storage tank and fill it with an appropriate amount of liquid nitrogen; pre-cool the stainless steel tray with a small amount of liquid nitrogen.

[0031] ② Cryogenic Storage: Quickly transfer the pre-cooled diced meat from the disperser to a stainless steel tray, then rapidly pour the meat from the tray into a liquid nitrogen storage tank, or place the meat along with the tray directly into the liquid nitrogen storage tank, ensuring that all meat is completely submerged in liquid nitrogen. Cover the liquid nitrogen tank (leaving the vent open) for later use or long-term storage.

[0032] (3) Warm-up Objective: To raise the temperature of cryogenically frozen meat cubes from -196°C to the optimal pulverization temperature range (-70°C to -80°C) to avoid excessive brittleness or overheating.

[0033] Equipment: -80°C ultra-low temperature freezer, stainless steel tray, T-type thermocouple thermometer.

[0034] Operating procedures: ① Preparation: Ensure the ultra-low temperature freezer is running stably and set to -80°C. Prepare a stainless steel tray. Using pre-cooled tweezers, quickly remove the amount of meat to be pulverized (e.g., 500g) from the liquid nitrogen tank. Immediately spread the removed meat pieces in a single layer on the metal tray. Ensure the meat samples are not stacked to guarantee uniform warming.

[0035] ② Warming in the freezer: Insert the tip of a thermocouple probe into the center of 2-3 meat cubes to sample the temperature. Quickly place the tray into a -80°C freezer. Monitor the temperature readings. Warming is complete when the core temperature of the meat cubes stabilizes within the range of -75°C ± 5°C. Based on multiple measurement experiences, this process typically takes 30-60 minutes. Therefore, in actual operation, the warming time should be controlled to be no less than 60 minutes.

[0036] (4) Multi-stage cryogenic grinding Objective: To obtain frozen beef meal with ≥80 mesh (particle size ≤180μm), high uniformity, and no fascia residue.

[0037] Equipment: Low-temperature resistant blade-type high-speed cryogenic pulverizer (pulverizing chamber ≥2L, austenitic stainless steel, equipped with liquid nitrogen injection interface); cryogenic planetary ball mill (equipped with liquid nitrogen-cooled grinding tank); pulse airflow sieve separator (screen material is stainless steel, equipped with mechanical vibrating screen, sealing cover, liquid nitrogen injection interface and compressed air inlet); air compressor, liquid nitrogen Dewar tank and conveying system, electronic balance, low-temperature workbench.

[0038] Operating procedures: ① Primary coarse crushing (target: passing through a 20-mesh sieve) A. Cryogenic Assisted Grinding: Take about 500g of meat cubes that have been warmed to -75°C±5°C and put them into a pre-cooled (rinsed with a small amount of liquid nitrogen) blade-type high-speed cryogenic grinder. Start the grinder at 16000-18000 rpm and simultaneously begin injecting liquid nitrogen at a rate of about 100g / min. Observe that a continuous flow of low-temperature nitrogen gas is coming out of the chamber outlet; grind for 30-60 seconds.

[0039] B. Screening: Quickly transfer the coarsely crushed material to a pulse screening machine pre-cooled by liquid nitrogen (install a 20-mesh standard sieve with an aperture of 850μm), turn on the mechanical vibration at a speed of 1200-1500 rpm, turn on the pulse airflow at a pressure of 0.3-0.5 MPa, a frequency of 1 second on / 2 seconds off, and a screening time of 1-2 minutes.

[0040] C. Collection: Discard the sieve material (mainly unbroken fascia, connective tissue and large particles), collect the sieve material (primary beef powder) in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep warm, and weigh it (record the yield M1 as about 70% to 80%).

[0041] ② Secondary medium crushing (target: pass through a 40-mesh sieve) A. Cryogenic assisted pulverization: Put all the collected 20-mesh sieve material (M1) into the pulverizer, increase the speed to 2000-22000 rpm, adjust the liquid nitrogen injection rate to about 80 g / min, and pulverize for 60-90 seconds.

[0042] B. Screening: Transfer the secondary crushed material to a pre-cooled pulse screening machine (replace with a 40-mesh standard sieve, aperture 425μm), turn on the mechanical vibration, speed 1200~1500rpm; turn on the pulse airflow, pulse air pressure 0.3~0.5MPa, frequency 1 second on / 2 seconds off, screening time 2~3min.

[0043] C. Collection: Discard the material on the sieve (mainly fine tendons and coarse particles that do not meet the standards), collect the material under the sieve (intermediate beef powder) in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, and weigh it (record the yield M2 as approximately 80% to 90%).

[0044] ③ Three-stage fine grinding (target: pass through an 80-mesh sieve) A. Cryogenic Assisted Grinding: The 40-mesh sieve undersize material (M2) is rapidly transferred into a planetary ball mill grinding jar pre-cooled with liquid nitrogen. The mixing ratio inside the jar is set as follows: grinding balls (zirconia material, 3-10mm diameter mixed): material ≈ 10:1 (by weight); the grinding jar's revolution speed is 300-400 rpm, and the rotation-to-revolution speed ratio is 1:-2. Liquid nitrogen is continuously supplied to the grinding jar's cooling system throughout the grinding process to maintain the jar temperature ≤ -80°C. Grinding time is 5-10 minutes.

[0045] B. Sieving: Transfer the ground ultrafine powder to a pre-cooled pulse sieve (equipped with an 80-mesh standard sieve with a aperture of 180μm), turn on the mechanical vibration at a speed of 1200-1500 rpm, turn on the pulse airflow at a pressure of 0.3-0.5 MPa and a frequency of 1 second on / 2 seconds off, and sieve for 5-8 minutes.

[0046] C. Collection: Discard the material on the sieve (a very small amount of extremely tough impurities), collect the material under the sieve (final product) in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, and weigh it (record the yield M3 as approximately 80%).

[0047] (5) Mixing and packaging of the final product The mixing and dispensing of the final product were completed on a low-temperature workbench. First, a wide-mouthed, flat-bottomed, straight-walled stainless steel container was placed on dry ice to maintain an ultra-low temperature environment. The collected final product was poured into the container, with the volume not exceeding 50% of the container's capacity. A long-handled stainless steel electric spiral stirrer was inserted and stirred at a low speed (10–20 rpm) for 10–20 minutes. Then, the final product was placed in a wide-mouthed, shallow-bottomed stainless steel bowl, which was placed on dry ice to maintain an ultra-low temperature environment. Using a pre-cooled measuring spoon (5 mL), the powder was quickly dispensed into pre-cooled, high-barrier aluminum foil composite vacuum bags, which were then drawn into a high vacuum and sealed. After dispensing, all sample vials were quickly transferred to a -80°C or -40°C freezer for long-term storage.

[0048] 5. Uniformity and stability test (1) Uniformity test Homogeneity is a fundamental property of matrix reference materials, used to describe the spatial distribution characteristics of their properties. Since inhomogeneity is one of the most significant sources of uncertainty in matrix reference materials, it directly affects the accuracy of value transfer. Therefore, homogeneity testing must be performed during the development of matrix reference materials to ensure consistency of characteristic values ​​within and between units, and to avoid sampling errors.

[0049] The homogeneity test method is as follows: A. Sampling Using 500 packages of frozen beef wet powder matrix standard material as a batch, 15 packages were randomly selected from each batch, and 3 subsamples were taken from each package for homogeneity testing between and within the bottles. The sampled samples were first thawed in a 4°C freezer for 24 hours (for rapid thaw, they can be immersed in ice water). After thawing, the samples can be further processed at room temperature, but should not be directly thawed at room temperature. In principle, frozen beef wet powder matrix standard material that has been opened should not be refrozen and reused; if it is necessary to continue storage, air must be strictly removed from the aluminum foil vacuum bag, and it must be frozen very quickly, not slowly, and should not be exposed to air during the freezing process). The thawed beef wet powder is in the form of minced meat.

[0050] B. Extraction Accurately weigh 1.00g of beef homogenate into a 50mL centrifuge tube, add 2mL of 0.2mol / L ammonium acetate buffer and 20μL of β-glucuronidase / aryl sulfatase, vortex for 0.5min; then add 0.2mL of 30% perchloric acid solution, vortex for 1min, centrifuge at 8000r / min for 5min, and collect all the supernatant into a 10mL centrifuge tube as a reserve solution.

[0051] C. Purification A mixed strong cationic solid-phase extraction column (MCX, 200 mg / 6 mL) was activated sequentially with 5 mL of methanol and 5 mL of 2% formic acid. Sample buffer was loaded onto the column and eluted with 5 mL of 2% formic acid and 5 mL of methanol. Finally, the eluent was eluted with 5 mL of 5% ammonia-methanol solution. The eluent was dried under nitrogen at 50 °C. 0.5 mL of methanol-0.1% formic acid solution (10+90, V / V) was added, and the residue was dissolved by vortexing for 1 min. The solution was then filtered through a 0.22 μm microporous membrane and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0052] D. Instrumental Method Chromatographic conditions: The column was an ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm, Waters Corporation, USA); mobile phase A was 0.1% formic acid solution, and mobile phase B was 0.1% formic acid-methanol solution; column temperature: 40 ℃; flow rate: 0.2 mL / min; injection volume: 2 μL; gradient elution program: 0–1 min, 95% A; 1–2 min, 80% A; 2–5 min, 40% A; 5–7 min, 5% A; 7–8 min, 5% A; 8–9 min, 95% A; 9–10 min, 95% A.

[0053] Mass spectrometry conditions: Acquity XEVO TQS tandem mass spectrometer (Waters Corporation, USA), electrospray ionization positive ion (ESI+) mode; capillary voltage 0.6 kV; ion source temperature 150 °C; desolventizing temperature 450 °C; both desolventizing gas and cone gas were high-purity nitrogen; desolventizing gas flow rate 1000 L / h; cone gas flow rate 40 L / h; multiple reaction monitoring (MRM) mode was used for detection. The mass spectrometry parameters of the target compound are shown in Table 1 below.

[0054] Table 1 Mass spectrometry parameters of the target compound E. Test Results The results of ractopamine content detection in randomly selected samples using the above method are shown in Table 2.

[0055] Table 2 Results of ractopamine content detection F. Statistical Results and Judgments F-1. Results of Analysis of Variance (ANOVA) Data were processed using one-way ANOVA to compare between-bottle and within-bottle differences. The results are shown in Table 3.

[0056] Table 3. Results of Analysis of Variance The analysis of variance results showed that the p-value of 0.706 was much greater than 0.05, indicating that the differences between the unit bottles were not statistically significant, thus proving the homogeneity of the samples.

[0057] F-2. Uncertainty component introduced by inhomogeneity (s) h )Evaluate Uncertainty component introduced by inhomogeneity (s) h The calculation formula is as follows: Calculations show that s h The calculation result is negative. According to the JJF 1343-2012 standard, s should be taken as... h =0 indicates that the difference between groups is smaller than the difference within groups, which is a sign of excellent homogeneity.

[0058] (2) Stability test In accordance with the relevant provisions of JJF 1343-2012 "General Principles and Statistical Principles for the Determination of Standard Reference Materials", short-term stability studies were conducted during sample transportation and long-term stability studies were conducted during storage. The ractopamine content in the samples was measured at 0, 1, 3, 7, and 14 days at 4℃, and at 0, 1, 2, and 3 days at 25℃, as short-term stability studies. The ractopamine content in the samples was measured at -40℃ and -80℃ at 0, 1, 3, 6, 9, and 12 months, respectively, as long-term stability studies. Three packages of standard reference material samples were randomly selected each time, and the samples from each package were measured in triplicate. The average of the three measurements was taken as the final result. Trend analysis was used to analyze the results to examine the change in the slope of the linear relationship with sample stability, and this change was used to evaluate the stability of the standard reference material. A fitted straight line Y = a + bX was constructed, where X represents the stability study time, Y represents the characteristic value of the standard reference material, a represents the intercept, and b represents the slope. Further calculations were performed on the standard uncertainty u(b) of the slope and the t-statistic (t_ calc ), and the t-statistic and the t-critical value (t_ crit (With n-2 degrees of freedom and a significance level of α=0.05) for comparison. If t_ calc >t_ crit If t_ calc <t_ crit If the slope is not significantly different from 0, the trend is not significant, and the sample is stable.

[0059] The sample test results are shown in Table 4.

[0060] Table 4 Sample test results The trend analysis results are shown in Table 5.

[0061] Table 5 Trend Analysis Results Trend analysis results show that, under both long-term and short-term stability studies, the t-statistic of the slope is less than the t-critical value, indicating that the trend is not significant and that the frozen wet beef matrix standard material prepared by this method is stable during the monitoring period.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a beef frozen wet powder matrix standard, characterized in that, Includes the following steps: S1. Obtain ractopamine-positive beef samples; S2. Perform acid removal treatment on the beef samples; S3. Pre-treat the beef after aging and cut it into small cubes with a diameter of 5-10mm. S4. A multi-stage cryogenic pulverization and pulse sieving process is used to prepare cryogenic wet powder matrix standard materials, including: S41. Pre-cooling of diced meat: Spray liquid nitrogen while stirring to form an ice shell on the surface of the diced meat, achieving a sand-like flow body state. S42. Cryogenic storage: Immerse pre-cooled diced meat in liquid nitrogen to cool it to below the glass transition temperature; S43. Warming: Warm the frozen meat cubes to -75°C±5°C for more than 60 minutes. S44, Multi-stage cryogenic grinding: The first stage coarse grinding is performed sequentially until it passes through a 20-mesh sieve, the second stage medium grinding is performed until it passes through a 40-mesh sieve, and the third stage fine grinding is performed until it passes through an 80-mesh sieve. After each stage of grinding, pulse airflow sieving is used. S5. Mix the final product thoroughly at low temperature and dispense it into sealed containers, storing them in an environment of -40°C or -80°C.

2. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, In step S2, the acid removal process involves hanging the item in a low-temperature environment of 0–4°C, a high-humidity environment of 80%–95%, and a well-ventilated environment for 7–14 days.

3. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, Step S41 specifically includes the following steps: S411. Weigh out the diced beef, put it into the disperser chamber, turn on the stirring, and set the speed to 120-180 rpm. S412. According to the weight ratio of diced beef to liquid nitrogen of 1:1 to 2, liquid nitrogen is injected into the freezing disperser where the diced beef is tumbling through the liquid nitrogen spray nozzle. S413. When the beef cubes change from being moist and sticky to being distinct and making a crisp sound when they collide with each other, the pre-cooling endpoint is reached. The material is a uniform, sandy, granular flow. Stop injecting liquid nitrogen and stirring, and quickly transfer the pre-cooled, flowable beef cubes to the next container.

4. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, In step S412, during the injection of liquid nitrogen, 40% to 50% of the total amount of liquid nitrogen is rapidly injected within the first 2 minutes; the remaining liquid nitrogen is then injected slowly and evenly over the next 4 to 6 minutes, so that the center temperature of the meat cubes quickly passes through the zone of maximum ice crystal formation.

5. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, In step S44, the primary coarse grinding specifically includes the following steps: Cryogenic assisted grinding: Take meat cubes that have been warmed to -75°C±5°C and put them into a pre-cooled blade-type high-speed cryogenic grinder. Start the grinder and rotate it at 16000~18000rpm. At the same time, start injecting liquid nitrogen at a rate of 100g / min. Observe that there is a continuous flow of low-temperature nitrogen gas coming out of the chamber outlet; grind for 30~60s. Screening: The coarsely crushed material is quickly transferred to a pulse screening machine pre-cooled by liquid nitrogen. Mechanical vibration is turned on at a speed of 1200-1500 rpm. Pulse airflow is turned on at a pressure of 0.3-0.5 MPa and a frequency of 1 second on / 2 seconds off. Screening time is 1-2 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, then weigh it.

6. The method for preparing the beef frozen wet powder matrix standard material according to claim 5, characterized in that, The pulse sieve used in the primary coarse crushing process is equipped with a 20-mesh standard sieve with an aperture of 850μm.

7. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, In step S44, the secondary crushing specifically includes the following steps: Cryogenic assisted pulverization: All the undersize material collected after primary coarse pulverization is fed into the pulverizer, the rotation speed is increased to 2000-22000 rpm, the liquid nitrogen injection rate is adjusted to 80 g / min, and the pulverization time is 60-90 seconds; Screening: Transfer the secondary crushed material to a pre-cooled pulse screening machine, turn on the mechanical vibration at a speed of 1200-1500 rpm; turn on the pulse airflow at a pressure of 0.3-0.5 MPa, a frequency of 1 second on / 2 seconds off, and a screening time of 2-3 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately fill it with liquid nitrogen to cover and keep it warm, then weigh it.

8. The method for preparing the beef frozen wet powder matrix standard material according to claim 7, characterized in that, The pulse sieve used in the secondary crushing process is equipped with a 40-mesh standard sieve with a aperture of 425μm.

9. The method for preparing the beef frozen wet powder matrix standard material according to claim 1, characterized in that, In step S44, the three-stage fine grinding specifically includes the following steps: Cryogenic assisted grinding: The undersize material obtained from the secondary grinding is quickly transferred into the planetary ball mill grinding jar pre-cooled with liquid nitrogen. The weight ratio inside the jar is set as grinding balls:material = 10:

1. The revolution speed of the grinding jar is 300-400 rpm, and the ratio of rotation speed to revolution speed is 1:-2. Liquid nitrogen is continuously supplied to the cooling system of the grinding jar throughout the grinding process to maintain the jar temperature ≤-80°C. The grinding time is 5-10 minutes. Sieving: Transfer the ground ultrafine powder to a pre-cooled pulse sieve, turn on the mechanical vibration at a speed of 1200-1500 rpm; turn on the pulse airflow at a pressure of 0.3-0.5 MPa, a frequency of 1 second on / 2 seconds off, and a sieving time of 5-8 minutes. Collection: Discard the material on the sieve, collect the material under the sieve in a container pre-cooled with liquid nitrogen, and immediately inject a small amount of liquid nitrogen to cover and keep it warm, then weigh it.

10. The method for preparing the beef frozen wet powder matrix standard material according to claim 9, characterized in that, The pulse sieve used in the three-stage fine grinding is equipped with an 80-mesh standard sieve with an aperture of 180μm.

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