Refining method and application of acetamido abamectin

Abstract

The invention relates to the field of veterinary drug raw material medicines, and discloses a refining method and application of acetamido abamectin, and the refining method comprises the following steps: carrying out substrate pretreatment on a crude acetamido abamectin raw material through a multifunctional fusion enzyme to enhance the solubility and improve the reaction selectivity; removing macromolecular and micromolecular impurities by adopting membrane separation; performing multi-stage solvent extraction by using an ethyl acetate-normal hexane-water three-phase solvent system, and separating water-soluble and non-polar impurities; in the multi-stage crystallization process, crystal precipitation is controlled through dynamic solvent proportion adjustment and multi-stage cooling, and high-purity crystals are generated; further separating by using a silica gel column system to remove trace impurities; and carrying out secondary crystallization and freeze drying on the purified product to obtain a crystal product with high purity and high stability. The refining method provided by the invention has the characteristics of high efficiency, safety and environmental protection, is suitable for industrial production, and significantly improves the product quality and purity.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary drug raw materials, and in particular to a purification method and application of avermectin. Background Art

[0002] As a broad-spectrum and highly effective anthelmintic, avermectin is mainly used as veterinary anthelmintic APIs, especially for anthelmintic treatment of ruminants such as cattle. Due to its non-withdrawal period, avermectin will not affect the milk production of dairy cows when used during lactation, and can ensure the safety and continuous supply of dairy products. Therefore, it has a wide range of application value in animal husbandry. However, there are some technical bottlenecks in the refining process of avermectin, which makes it difficult to meet the quality standards of high purity, low impurities and low solvent residues. The current refining process generally faces the following shortcomings:

[0003] In the prior art, it is difficult to effectively remove water-soluble and non-polar impurities in avermectin through traditional solvent extraction and single crystallization processes, resulting in low purity of the final product. At the same time, due to the lack of efficient impurity separation and crystal control methods, more impurities often remain in the product, reducing the safety and efficacy of the drug. In addition, the problem of solvent residue is also prominent in traditional processes. Conventional vacuum drying methods are difficult to completely remove solvents. Solvent residues affect the requirement of no drug withdrawal period for veterinary drugs, do not meet the drug safety standards for ruminants such as dairy cows during lactation, and affect the quality of dairy products.

[0004] In summary, the existing technology is difficult to balance the requirements of high purity, low impurities and low solvent residues in the refining process of avermectin, and there are problems of low process efficiency and poor quality stability, which greatly limits the application of avermectin in veterinary anthelmintics without a withdrawal period. An optimized refining method is urgently needed to achieve higher product quality. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a refining method and application of avermectin. By optimizing the refining process, impurities and solvent residues are effectively removed, thereby improving the purity of avermectin, so that it can meet the application requirements of veterinary drugs without a withdrawal period in ruminants such as dairy cows.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: a method for refining avermectin, comprising the following steps:

[0007] S1, pretreating the crude raw material of avermectin by using a multifunctional fusion enzyme;

[0008] S2, performing membrane separation treatment on the product solution after the enzymatic reaction to remove macromolecular impurities and small molecular by-products by nanofiltration and ultrafiltration;

[0009] S3, refining by multi-stage solvent extraction, using ethyl acetate-n-hexane-water three-phase solvent system to separate water-soluble and non-polar impurities;

[0010] S4, adopting a multi-stage crystallization process, through dynamic solvent ratio adjustment and multi-stage cooling crystallization, gradually precipitating high-purity avermectin crystals;

[0011] S5, separating the crystalline product through a silica gel column to further remove trace impurities;

[0012] S6. The purified avermectin is subjected to secondary crystallization and freeze-drying to obtain a high-purity crystalline product.

[0013] Preferably, step S1 comprises the following steps:

[0014] S11, fusing the acylase with the dissolution auxiliary enzyme to prepare a multifunctional fusion enzyme, wherein the dissolution auxiliary enzyme is lysozyme or lipase, and the concentration of the multifunctional fusion enzyme is 0.5-1.5 mg / mL;

[0015] S12, dissolving the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol, wherein the volume ratio of ethyl acetate to ethanol in the mixed solvent is 2:1 to 3:1;

[0016] S13, adding the multifunctional fusion enzyme to the solution, controlling the reaction temperature to 20-30° C., the pH to 7.0-7.5, and reacting for 3-5 hours under stirring conditions;

[0017] S14. Add 0.05-0.2% polyethylene glycol 400 to the reaction system to improve the solubility of the substrate.

[0018] Preferably, the membrane separation step comprises using a nanofiltration membrane with a molecular weight cut-off range of 5-10 kDa and an ultrafiltration membrane with a molecular weight cut-off range of 0.5-1 kDa, an operating pressure range of 0.04-0.12 MPa, and a temperature of 20-25°C.

[0019] Preferably, step S3 comprises the following steps:

[0020] S31, adjusting the pH of the avermectin product solution after membrane separation to 7.5-8.5;

[0021] S32, mixing the product solution with a three-phase solvent system of ethyl acetate, n-hexane and water, wherein the volume ratio of ethyl acetate to n-hexane is 1:1 to 1:2;

[0022] S33, performing a first stage extraction to separate water-soluble impurities, separating and discarding the aqueous phase;

[0023] S34, adding n-hexane to the ethyl acetate phase for a second stage of extraction, separating non-polar impurities, discarding the n-hexane phase and retaining the ethyl acetate phase.

[0024] Preferably, the retained ethyl acetate phase is used for crystallization in the subsequent step S4.

[0025] Preferably, step S4 comprises the following steps:

[0026] S41, preparing a ternary solvent system by mixing the extracted ethyl acetate phase with isopropanol and water, wherein the volume ratio of ethyl acetate, isopropanol and water is 4:3:2 to 5:3:2;

[0027] S42, placing the ternary solvent system in a temperature-controlled crystallization tank, controlling the initial temperature at 35-45° C., maintaining stirring, and performing dissolution equilibrium;

[0028] S43, slowly cooling the solution to 20-25°C and maintaining for 1-3 hours to induce initial crystal precipitation;

[0029] S44, further reducing the temperature to 8-12° C. and maintaining for 2-4 hours to precipitate intermediate crystals;

[0030] S45, the temperature is finally lowered to 2-5°C to complete the crystallization, and the dynamic solvent ratio is adjusted throughout the process, and ethyl acetate is increased or decreased by 3-5% each time to control the precipitation rate and purity of the crystals.

[0031] Preferably, step S5 comprises the following steps:

[0032] S51, dissolving the crystalline product in acetonitrile solvent to prepare a sample solution with a concentration of 8-12 mg / mL;

[0033] S52, loading the sample solution into a column chromatography system, wherein the column chromatography system uses a silica gel filler, and the mobile phase is a mixed system of water, acetonitrile and methanol, wherein the volume ratio of water, acetonitrile and methanol is 60:30:10 to 55:35:10;

[0034] S53, separating the target product from impurities by controlling the solvent flow rate and gradient elution;

[0035] S54, collecting eluted components in stages, and subjecting the collected target product to repeated column chromatography separation 2-3 times to further remove trace impurities;

[0036] S55. Concentrate the purified target product components using a rotary evaporator at 30-40° C. to remove residual solvent.

[0037] Preferably, step S6 comprises the following steps:

[0038] S61, dissolving the purified avermectin in a mixed solvent of ethanol and water, wherein the volume ratio of ethanol to water is 3:1 to 4:1;

[0039] S62, placing the solution in a low-temperature crystallization tank, with the initial temperature controlled at 10-15° C., to induce crystal precipitation;

[0040] S63, gradually cooling to 4° C. and maintaining for 2-4 hours to promote complete precipitation of high-purity crystals;

[0041] S64, collecting the crystallized product through a microporous filter membrane, and placing the crystallized product in a vacuum freeze dryer;

[0042] S65. Control the freeze-drying temperature to -15 to -25°C, the vacuum pressure to 0.01-0.02MPa, and the drying time to 18-24 hours to obtain a high-purity crystalline product.

[0043] The invention also provides a preparation process of avermectin, comprising preparing a crude raw material of avermectin, and purifying it by the refining method to obtain an avermectin product with high purity and low residue.

[0044] The present invention also provides an application of avermectin. The avermectin is obtained through the preparation process and is used as an anthelmintic drug for animals without a withdrawal period.

[0045] The present invention provides a method for refining avermectin and its application, which has the following beneficial effects:

[0046] 1. The present invention significantly improves the purity of avermectin by combining multi-step processes such as multifunctional fusion enzyme pretreatment, multi-stage solvent extraction, multi-stage crystallization process and silica gel column separation. The selective removal of impurities in each step ensures that the purity of the final product can reach more than 99.5%, meeting the requirements of high-quality veterinary drugs.

[0047] 2. The solvent extraction system and multi-stage crystallization process in the present invention effectively control the impurity content by removing water-soluble and non-polar impurities step by step and dynamically adjusting the solvent ratio. Compared with traditional methods, the impurity content is significantly reduced, improving the safety and stability of the product.

[0048] 3. By rationally selecting the solvent system and optimizing the membrane separation technology, the scheme of the present invention can remove impurities while retaining the target product to the maximum extent, so that the recovery rate can reach more than 98%. This not only reduces costs in industrial production, but also reduces resource waste.

[0049] 4. The multifunctional fusion enzyme pretreatment of the present invention reduces the generation of byproducts and accelerates the reaction rate by improving the solubility of the substrate and the reaction selectivity. The total reaction time is significantly shorter than that of the traditional method, providing support for efficient industrial production.

[0050] 5. The present invention adopts multi-stage solvent extraction and freeze-drying process, which greatly reduces the solvent residue. The residual amount of the final product is less than 0.1ppm, which meets the requirements of veterinary drugs without withdrawal period, improves the safety of the product, and ensures its safe application in animal medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the method flow of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Please refer to the attached Figure 1 , the embodiment of the present invention provides a method for refining avermectin, which achieves high purity and low impurity content of avermectin. This method solves the problems of difficulty in removing impurities, controlling residual solvents and improving product purity in the prior art, and is particularly suitable for the production needs of veterinary anthelmintics without a drug withdrawal period. The following is a detailed description of the steps of the refining method of the present invention.

[0054] S1. Substrate pretreatment

[0055] Step S1 is to perform substrate pretreatment on the crude raw material of avermectin using a multifunctional fusion enzyme, and this step comprises the following specific operations:

[0056] 1. The acylase is fused with a dissolution auxiliary enzyme to prepare a multifunctional fusion enzyme, which has both highly selective acylation reaction and substrate dissolution promotion functions, and the dissolution auxiliary enzyme can be lysozyme or lipase. The fusion enzyme can selectively catalyze the acylation group reaction in avermectin, and on the other hand, effectively reduce the reaction time and reduce the risk of by-product generation by improving solubility and substrate dispersibility.

[0057] 2. The multifunctional fusion enzyme is used at a concentration of 0.5-1.5 mg / mL, and the crude avermectin raw material is dissolved in a mixed solvent of ethyl acetate and ethanol, wherein the volume ratio of ethyl acetate to ethanol in the mixed solvent is 2:1 to 3:1. Subsequently, the fusion enzyme is added to the solution, the reaction temperature is controlled at 20-30°C, the pH value is within the range of 7.0-7.5, and the reaction is carried out for 3-5 hours under stirring conditions to ensure that the enzymatic reaction is fully carried out.

[0058] 3. Add 0.05-0.2% polyethylene glycol 400 (PEG 400) as a cosolvent to the reaction system to further improve the solubility of the substrate and make the reaction more thorough and uniform. This step not only improves the solubility and reactivity of the substrate, but also reduces side reactions through the specific action of the enzyme, ensuring the purity of the product.

[0059] S2. Membrane separation

[0060] Step S2 uses nanofiltration and ultrafiltration technology to perform membrane separation on the product solution after the enzymatic reaction. By reasonably selecting the molecular weight cutoff range and operating conditions of the membrane, macromolecular impurities and small molecular by-products are effectively removed, providing a high-purity initial product solution for the subsequent refining step.

[0061] 1. Select nanofiltration membranes with a molecular weight cutoff range of 5-10kDa and ultrafiltration membranes with a molecular weight cutoff range of 0.5-1kDa to separate larger molecular impurities and small molecular by-products, respectively. The operating pressure of the nanofiltration and ultrafiltration processes is controlled at 0.04-0.12MPa, and the temperature range is 20-25℃.

[0062] 2. The purpose of this membrane separation step is to achieve efficient separation by selectively intercepting impurities without introducing other solvents, ensuring that the target compound remains in solution and providing a pure solution for subsequent solvent extraction and crystallization steps.

[0063] S3. Multi-stage solvent extraction

[0064] Step S3 uses multi-stage solvent extraction based on a three-phase solvent system of ethyl acetate, n-hexane and water, and separates water-soluble and non-polar impurities by pH control.

[0065] 1. First, the avermectin product solution after membrane separation is adjusted to a pH range of 7.5-8.5 to ensure that the distribution coefficient of the target product in the ethyl acetate phase is high. Subsequently, the pH-adjusted solution is mixed with a three-phase solvent system of ethyl acetate, n-hexane and water, and the volume ratio of ethyl acetate to n-hexane is controlled at 1:1 to 1:2 to achieve phase separation of the solvent.

[0066] 2. Perform the first stage of extraction, distribute the water-soluble impurities into the aqueous phase, and discard the aqueous phase. This step separates and removes the water-soluble impurities by controlling the pH value, which helps to improve the purity of the product.

[0067] 3. In the second stage of extraction, n-hexane is added to the retained ethyl acetate phase to separate non-polar impurities. This step removes non-polar impurities dissolved in n-hexane, and finally retains the ethyl acetate phase for subsequent crystallization operations to further ensure the purity of the product.

[0068] S4. Multi-stage crystallization

[0069] Step S4 utilizes a multi-stage crystallization process to control the crystallization process of the target compound through dynamic solvent ratio adjustment and multi-stage cooling crystallization to obtain high-purity avermectin crystals.

[0070] 1. First, prepare the ethyl acetate phase with isopropanol and water into a ternary solvent system, wherein the volume ratio of ethyl acetate, isopropanol and water is 4:3:2 to 5:3:2, and place the ternary solvent system in a temperature-controlled crystallization tank, control the initial temperature at 35-45°C and keep stirring to reach a dissolution equilibrium state.

[0071] 2. Under the control of the intelligent temperature control system, slowly cool the solution to 20-25°C and keep it for 1-3 hours to induce the precipitation of preliminary crystals. Then, further reduce the temperature to 8-12°C and keep it for 2-4 hours to continue to precipitate intermediate crystals and avoid impurity coprecipitation.

[0072] 3. Finally, the temperature is lowered to 2-5°C to complete the crystallization process. During the cooling process, the crystallization rate is controlled by dynamically adjusting the content of ethyl acetate, increasing or decreasing by 3-5% each time, so as to obtain uniform, high-purity avermectin crystals.

[0073] S5. Silica gel column separation

[0074] Step S5 is to further remove trace impurities by silica gel column separation to ensure that the final product meets high purity requirements.

[0075] 1. The crystalline product was dissolved in acetonitrile to prepare a sample solution of 8-12 mg / mL, and the solution was loaded into a column chromatography system filled with silica gel. The mobile phase was a mixed system of water, acetonitrile and methanol in a volume ratio of 60:30:10 to 55:35:10.

[0076] 2. Use a silica gel column separation system for purification and separation, collect different elution components in stages, and then use a liquid chromatography or ultraviolet detection system to detect the collected components at a wavelength of 240-250nm to confirm the main peak of avermectin. Accurately collect the main peak components and repeat the silica gel column separation 2-3 times as needed to further remove trace impurities.

[0077] 3. The collected main peak components are concentrated by rotary evaporation at 30-40°C to remove the residual solvent, thereby obtaining a high-purity product for subsequent refining.

[0078] S6. Secondary crystallization and freeze drying

[0079] Step S6: performing secondary crystallization and freeze-drying on the purified avermectin to further improve the purity and stability of the product.

[0080] 1. The purified avermectin was dissolved in a mixed solvent of ethanol and water, with a volume ratio of ethanol to water of 3:1 to 4:1, and placed in a low-temperature crystallization tank for crystallization. The initial temperature was controlled at 10-15°C to induce crystal precipitation.

[0081] 2. Slowly cool the solution to 4°C and maintain for 2-4 hours to promote the complete precipitation of high-purity crystals. Subsequently, the crystallized product is collected through a microporous filter membrane to ensure uniform crystal morphology.

[0082] 3. Place the collected crystals in a vacuum freeze dryer, control the drying temperature at -15 to -25°C, the vacuum pressure at 0.01-0.02MPa, and the drying time at 18-24 hours to obtain a high-purity crystalline product.

[0083] The present invention significantly improves the substrate solubility and reaction selectivity by optimizing the substrate pretreatment of the multifunctional fusion enzyme, thereby avoiding the generation of side reactions. The membrane separation technology is combined with multi-stage solvent extraction, and water-soluble and non-polar impurities are effectively removed through reasonable pH adjustment and solvent ratio. The multi-stage crystallization process ensures the uniformity and high purity of the crystals by dynamically regulating the solvent ratio and cooling rate. Silica gel column separation and secondary crystallization further remove trace impurities and residual solvents, and finally obtain a high-purity, low-residue avermectin product.

[0084] The method solves the problems of difficult purification of avermectin and high solvent residue in the prior art, ensures that the product meets the application standards of veterinary drugs with no withdrawal period, and has good industrial feasibility and environmental friendliness.

[0085] In order to better understand the present invention, the above method is described in detail below in conjunction with specific embodiments.

[0086] Embodiment 1:

[0087] The embodiment of the present invention provides a method for refining avermectin, comprising the following steps:

[0088] Step S1: Dissolve the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol (volume ratio 2:1), use a multifunctional fusion enzyme (acylase and lysozyme fusion) at a concentration of 1 mg / mL, the reaction temperature is 25° C., the pH value is 7.0, and the reaction time is 4 hours. Add 0.1% polyethylene glycol 400.

[0089] Step S2: Membrane separation was performed using a 5 kDa nanofiltration membrane and a 1 kDa ultrafiltration membrane, with an operating pressure of 0.08 MPa and a temperature of 25°C.

[0090] Step S3: The solution after membrane separation is adjusted to pH 8.0 and mixed with ethyl acetate-n-hexane-water (volume ratio 1:1:1). After separating the water-soluble impurities, n-hexane is added to the ethyl acetate phase at a ratio of 1:1 to remove non-polar impurities, and the ethyl acetate phase is retained for crystallization.

[0091] Step S4: Using ethyl acetate-isopropanol-water (volume ratio 4:3:2) as the solvent system, the initial temperature is 40° C., slowly cooling to 20° C. and maintaining for 2 hours, then to 10° C. and maintaining for 3 hours, and finally to 4° C. The proportion of ethyl acetate is dynamically adjusted, increasing by 3% each time.

[0092] Step S5: The crystalline product was dissolved in acetonitrile (10 mg / mL) and further removed from trace impurities by silica gel column separation. A mixed solvent of ethyl acetate and n-hexane was used as the eluent, and the solvent ratio was adjusted to control the elution process, and the target product components were collected and purified. The separation was repeated twice to improve the purity.

[0093] Step S6: The purified product was dissolved in ethanol-water (volume ratio 3:1) at an initial temperature of 12°C, and then gradually cooled to 4°C and maintained for 3 hours. The product was freeze-dried at -20°C and 0.01 MPa for 24 hours.

[0094] Embodiment 2:

[0095] The embodiment of the present invention provides a method for refining avermectin, comprising the following steps:

[0096] Step S1: Dissolve the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol (volume ratio 3:1), use a multifunctional fusion enzyme (acylase and lipase fusion) at a concentration of 1.2 mg / mL, the reaction temperature is 22° C., the pH value is 7.5, and the reaction time is 3 hours. Add 0.15% polyethylene glycol 400.

[0097] Step S2: Membrane separation was performed using a 7 kDa nanofiltration membrane and a 0.8 kDa ultrafiltration membrane, with an operating pressure of 0.1 MPa and a temperature of 23°C.

[0098] Step S3: The solution was adjusted to pH 7.8 and mixed with ethyl acetate-n-hexane-water (volume ratio 1:2:1). After removing the aqueous phase, n-hexane was added to the ethyl acetate phase at a ratio of 1:2 to remove non-polar impurities and retain the ethyl acetate phase.

[0099] Step S4: using ethyl acetate-isopropanol-water (volume ratio 5:3:2) as the solvent system, with an initial temperature of 35°C, slowly cooling to 25°C and maintaining for 3 hours, then cooling to 8°C and maintaining for 4 hours, and finally cooling to 5°C, dynamically adjusting the proportion of ethyl acetate, reducing it by 4% each time.

[0100] Step S5: The crystal product was dissolved in acetonitrile (8 mg / mL) and separated using a silica gel column with ethyl acetate-n-hexane as the elution solvent. The solvent flow rate was controlled to ensure an appropriate elution rate, and the solvent ratio was adjusted to optimize the separation effect. The separation was repeated 3 times to further improve the purity.

[0101] Step S6: Dissolve in ethanol-water (volume ratio 4:1), with an initial temperature of 15°C, cool to 4°C and keep for 2 hours, and freeze-dry at -15°C and 0.02 MPa for 20 hours.

[0102] Embodiment 3:

[0103] The embodiment of the present invention provides a method for refining avermectin, comprising the following steps:

[0104] Step S1: Dissolve the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol (volume ratio 2:1), use a multifunctional fusion enzyme (acylase and lysozyme fusion) at a concentration of 0.8 mg / mL, the reaction temperature is 28° C., the pH value is 7.2, and the reaction time is 5 hours. Add 0.05% polyethylene glycol 400.

[0105] Step S2: Use a 6 kDa nanofiltration membrane and a 1 kDa ultrafiltration membrane, with an operating pressure of 0.09 MPa and a temperature of 24°C.

[0106] Step S3: The solution was adjusted to pH 8.2, ethyl acetate-n-hexane-water (volume ratio 1:1.5:1) was added, and after separating the aqueous phase, n-hexane (ratio 1:1) was added to the ethyl acetate phase to remove non-polar impurities, and the ethyl acetate phase was retained.

[0107] Step S4: using an ethyl acetate-isopropanol-water (volume ratio 4:3:2) solvent system, the initial temperature was set to 42°C, the temperature was lowered to 22°C and maintained for 1.5 hours, then lowered to 9°C and maintained for 3 hours, and finally lowered to 2°C, and the proportion of ethyl acetate was dynamically adjusted, increasing or decreasing by 5% each time.

[0108] Step S5: The crystalline product was dissolved in acetonitrile (10 mg / mL) and purified twice using a reverse phase C18 column at a flow rate of 1.0 mL / min and a detection wavelength of 250 nm.

[0109] Step S6: Dissolve in ethanol-water (volume ratio 3:1), initially at 10°C, cool to 4°C and keep for 2 hours, and freeze-dry at -25°C and 0.015 MPa for 22 hours.

[0110] Embodiment 4:

[0111] The embodiment of the present invention provides a method for refining avermectin, comprising the following steps:

[0112] Step S1: The crude raw material was dissolved in ethyl acetate and ethanol (volume ratio 2.5:1), a multifunctional fusion enzyme (acylase and lipase fusion) was used at a concentration of 1.3 mg / mL, the reaction temperature was 21°C, the pH value was 7.3, the reaction time was 3.5 hours, and 0.1% polyethylene glycol 400 was added.

[0113] Step S2: Use 8 kDa nanofiltration membrane and 0.7 kDa ultrafiltration membrane, the operating pressure is 0.1 MPa, and the temperature is 20°C.

[0114] Step S3: The solution was adjusted to pH 7.6, mixed with ethyl acetate-n-hexane-water (volume ratio 1:1:1), and after separating the aqueous phase, n-hexane (1:1.5) was added to the ethyl acetate phase to remove non-polar impurities.

[0115] Step S4: using ethyl acetate-isopropanol-water (volume ratio 5:3:2), with an initial temperature of 35°C, cooling to 25°C and maintaining for 2 hours, then cooling to 10°C and maintaining for 2 hours, and finally cooling to 4°C, increasing or decreasing ethyl acetate by 4% each time.

[0116] Step S5: Dissolve in acetonitrile (12 mg / mL), separate using a silica gel column, use ethyl acetate-n-hexane as the elution solvent, control the solvent flow to ensure an appropriate elution rate, and adjust the solvent ratio to optimize the separation effect. Repeat the separation 3 times to further improve the purity.

[0117] Step S6: Dissolve in ethanol-water (volume ratio 4:1), initial temperature 14°C, reduce to 4°C and maintain for 3 hours, freeze-dry at -20°C and 0.02 MPa for 24 hours.

[0118] Embodiment 5:

[0119] The embodiment of the present invention provides a method for refining avermectin, comprising the following steps:

[0120] Step S1: The crude material was dissolved in ethyl acetate and ethanol (volume ratio 3:1), a multifunctional fusion enzyme (acylase and lysozyme fusion) was used at a concentration of 1 mg / mL, the reaction temperature was 26°C, the pH value was 7.1, the reaction time was 4 hours, and 0.15% PEG 400 was added.

[0121] Step S2: Use 7 kDa nanofiltration membrane and 1 kDa ultrafiltration membrane, control the pressure at 0.12 MPa and the temperature at 25°C.

[0122] Step S3: adjust the solution pH to 8.0, add ethyl acetate-n-hexane-water (1:2:1), separate the aqueous phase, add n-hexane (ratio 1:2), and retain the ethyl acetate phase.

[0123] Step S4: using ethyl acetate-isopropanol-water (volume ratio 4:3:2), the initial temperature was 40°C, lowered to 24°C for 3 hours, then lowered to 8°C for 4 hours, and finally lowered to 3°C, dynamically adjusting the ethyl acetate to reduce by 3% each time.

[0124] Step S5: The crystal product was dissolved in acetonitrile (8 mg / mL) and separated using a silica gel column with ethyl acetate-n-hexane as the elution solvent. The solvent flow rate was controlled to ensure an appropriate elution rate, and the solvent ratio was adjusted to optimize the separation effect. The separation was repeated twice to further improve the purity.

[0125] Step S6: Dissolve in ethanol-water (3:1), initial temperature 12°C, lower to 4°C and keep for 3 hours, freeze-dry at -18°C and 0.015 MPa for 18 hours.

[0126] Test experiment:

[0127] The following is a performance test experiment for the avermectin final product obtained from the above five embodiments, and the experimental content covers key performance indicators such as purity, solvent residue, impurity content and stability.

[0128] Experimental objectives:

[0129] 1. Evaluate the purity of the final product in each example;

[0130] 2. Determine the residual solvent content in the product;

[0131] 3. Detect the content of trace impurities;

[0132] 4. Evaluate the storage stability of the product.

[0133] Experimental setup:

[0134] 1. Sample preparation: Samples were taken from the final crystal product of each embodiment and divided into five groups, each group representing a sample of one embodiment.

[0135] 2. Experimental instruments and equipment:

[0136] High performance liquid chromatography (HPLC) was used for purity and impurity content testing;

[0137] Gas chromatograph (GC) was used for residual solvent determination;

[0138] Stability test chamber, used for storage stability testing with temperature and humidity control.

[0139] The experimental steps are as follows:

[0140] 1. Purity test: Use high performance liquid chromatography (HPLC) to detect product purity.

[0141] 1) Dissolve the sample in acetonitrile to prepare a solution with a concentration of 10 mg / mL;

[0142] 2) A reverse phase C18 column was used with a mobile phase of water-acetonitrile-methanol (volume ratio 60:30:10), a flow rate of 0.8 mL / min, and a detection wavelength of 245 nm;

[0143] 3) Record the main peak area of ​​avermectin and calculate the purity based on the ratio of the main peak area to the total peak area.

[0144] 2. Solvent residue test: Use gas chromatography (GC) to detect the residual amount of ethyl acetate and ethanol.

[0145] 1) Place each sample in a 20 mL sealed headspace bottle;

[0146] 2) heating to 80°C, extracting the headspace gas and injecting it into a gas chromatograph;

[0147] 3) Use polar column separation to detect residual solvents in ppm (parts per million).

[0148] 3. Impurity content test: The content of trace impurities was analyzed by high performance liquid chromatography (HPLC).

[0149] 1) Use the same solution configuration and chromatographic conditions as for the purity test;

[0150] 2) Record the peak areas of other impurities except the main peak;

[0151] 3) Calculate the percentage of impurity content in the total peak area.

[0152] 4. Stability test: Evaluate the stability of the product under different temperature and humidity conditions. The test time is 30 days.

[0153] 1) Each sample was divided into three groups and placed under the following three conditions:

[0154] A: 25℃, humidity 60%;

[0155] B: 40℃, humidity 75%;

[0156] C: 60℃, humidity 85%;

[0157] 2) Take samples every 10 days to detect purity changes, taking the initial purity as 100% and record the purity decrease ratio.

[0158] The experimental results are shown in the following table:

[0159]

[0160] Results Analysis

[0161] Purity: The purity of the final products of the five embodiments is all above 99.3%, indicating that different process parameter combinations can obtain high-purity avermectin, and sample 3 has the highest purity, reaching 99.6%.

[0162] Solvent residue: The residual contents of ethyl acetate and ethanol are both less than 0.15ppm, meeting the safety requirements for veterinary anthelmintics without a withdrawal period.

[0163] Impurity content: The impurity content was between 0.18% and 0.25%, showing that sample 3 had the least impurities, further verifying the optimization effect of the process parameters.

[0164] Stability: After being stored under different temperature and humidity conditions for 30 days, all samples performed better under lower temperature and humidity (condition A), with purity remaining above 98%. Under high temperature and high humidity conditions (condition C), the purity decreased, but was still above 95%, indicating that the product has good stability.

[0165] Comparative experiment 1:

[0166] Objective: To verify the role of multifunctional fusion enzyme in improving substrate solubility and selectivity, and to analyze its influence on reaction time, impurity formation and purity.

[0167] The comparison settings of this comparative experiment are as follows:

[0168] Experimental group 1 plan:

[0169] The substrate pretreatment is performed according to step S1 of Example 1, and the specific design is as follows:

[0170] 1. Solution preparation: dissolve the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol, with the volume ratio of the mixed solvent being 2:1.

[0171] 2. Addition of fusion enzyme: Use a multifunctional fusion enzyme fused with acylase and lysozyme, with an enzyme concentration of 1 mg / mL.

[0172] 3. Reaction conditions: The reaction temperature was controlled at 25°C, the pH value was 7.0, the reaction time was 4 hours, and the stirring speed was 150 rpm.

[0173] 4. Addition of cosolvent: Add 0.1% polyethylene glycol 400 to the reaction system to improve the solubility of the substrate.

[0174] The rest of the steps are the same.

[0175] Comparative Example 1:

[0176] The scheme of comparative example 1 is exactly the same as the design of experimental group 1, the only difference is that the multifunctional fusion enzyme is not used, and the crude avermectin raw material is directly dissolved in a mixed solvent of ethyl acetate and ethanol (volume ratio 2:1), and other conditions remain the same, including solution configuration, reaction temperature, pH value and co-solvent addition.

[0177] The experimental steps are as follows:

[0178] 1. Sample configuration: According to the embodiments and comparative examples of the present invention, two groups of experiments were configured, which were recorded as implementation samples and comparison samples.

[0179] 2. Solubility determination:

[0180] Prepare 0.1 g of crude avermectin raw material and add 15 mL of solvent.

[0181] Under the reaction conditions (25°C, 150 rpm stirring), the dissolution of the substrates treated with the multifunctional fusion enzyme and those without the fusion enzyme was measured respectively.

[0182] The time required for complete dissolution was recorded.

[0183] 3. Determination of impurity content:

[0184] The solution was filtered through a microporous filter membrane, and the filtrate was collected and tested for impurity content using HPLC.

[0185] A reverse phase C18 column was used, the mobile phase was water-acetonitrile-methanol (volume ratio 60:30:10), the flow rate was 0.8 mL / min, and the detection wavelength was 245 nm.

[0186] Record the percentage of impurity content in each sample to the total peak area.

[0187] 4. Purity determination:

[0188] The purity of the target product in the two groups of solutions was measured by HPLC, using a reversed-phase C18 column and keeping the same mobile phase and detection wavelength.

[0189] The purity in each sample was recorded.

[0190] 5. Reaction time determination:

[0191] Start the reaction and record the time required for the complete reaction.

[0192] The experimental results are shown in the following table:

[0193] Test items Implementation samples (experimental group 1) Comparative sample (Comparative Example 1) Dissolution time (minutes) 10 25 Impurity content (%) 0.2 0.35 purity(%) 99.5 98.9 Complete reaction time (hours) 4 6

[0194] The experimental results verify the significant advantages of the multifunctional fusion enzyme in the present invention in the purification process of avermectin. Through the application of the fusion enzyme, it shows superiority in substrate solubility, reaction selectivity, reaction efficiency and impurity control. Specifically:

[0195] 1. The dissolution auxiliary enzyme (such as lysozyme or lipase) in the multifunctional fusion enzyme can physically interact with the crude raw material of avermectin through its unique molecular structure, increase the hydrophilicity and surface area of ​​the substrate, and make the substrate more easily dissolved in the solvent system. In the embodiment of the present invention, the dissolution time is shortened to 10 minutes, while the comparative sample takes 25 minutes, which fully demonstrates the advantage of the multifunctional fusion enzyme in improving the efficiency of substrate dissolution.

[0196] 2. The acylase, through its highly selective catalytic action, reacts only with specific groups in avermectin without affecting other functional groups, thereby reducing side reactions and impurity generation. The results of the comparative experiment showed that the impurity content of the implemented sample was significantly lower than that of the comparative sample (0.20% vs. 0.35%), indicating that the selective reaction mechanism of the fusion enzyme effectively inhibits unnecessary side reactions. This mechanism ensures the cleanliness of the reaction and the high purity of the product, fully demonstrating the innovation of the present invention in terms of impurity control.

[0197] 3. Through the synergistic effect of the fusion enzyme, the present invention effectively shortens the reaction time and significantly improves the reaction efficiency. While the dissolution auxiliary enzyme increases the solubility of the substrate, the specific catalysis of the acylase accelerates the target reaction, shortening the complete reaction time from 6 hours to 4 hours. This innovative combination of dual enzymatic reactions makes the reaction more efficient and energy-saving, and is suitable for industrial production.

[0198] 4. Due to the highly selective catalysis and substrate pretreatment of the multifunctional fusion enzyme, the present invention can significantly improve the purity of the final product. In this experiment, the purity of the implemented sample reached 99.5%, which was significantly higher than 98.9% of the control sample. This purity improvement is attributed to the molecular recognition and selective catalytic properties of the fusion enzyme, which explains the effect of the present invention on improving product purity from a mechanistic perspective, and provides a guarantee for the high standard purity requirements of veterinary drugs without a drug withdrawal period.

[0199] In general, the scheme of the present invention integrates multiple functions of substrate dissolution improvement, reaction selectivity improvement, impurity control and reaction efficiency optimization through the design of multifunctional fusion enzyme, overcoming the bottlenecks of low dissolution efficiency, high impurity generation and long reaction time in traditional refining methods. This unique combination of dual enzyme synergistic reaction not only meets the industrialization requirements of high purity and high efficiency, but also meets environmental protection and safety requirements.

[0200] Comparative experiment 2:

[0201] Objective: To verify the effect of the multistage solvent extraction system (ethyl acetate-n-hexane-water three-phase solvent system) in the present invention in removing water-soluble and non-polar impurities, and to compare the effects of different solvent combinations on impurity removal rate, product purity and target product recovery rate.

[0202] Experimental Group 2 Program:

[0203] According to step S3 of Example 1, ethyl acetate-n-hexane-water three-phase solvent system is used for refining, and the specific design is as follows:

[0204] 1. Solution preparation: Adjust the pH of the avermectin product solution after membrane separation to 8.0.

[0205] 2. First stage extraction: Add a three-phase solvent system of ethyl acetate-n-hexane-water, with a solvent volume ratio of ethyl acetate:n-hexane:water = 1:1:1. After extraction, discard the water phase and retain the ethyl acetate phase.

[0206] 3. Second stage extraction: Add n-hexane to the retained ethyl acetate phase, with the volume ratio of ethyl acetate to n-hexane being 1:1. After separation of non-polar impurities, the n-hexane phase is discarded and the ethyl acetate phase is retained for crystallization.

[0207] Comparative Example 2:

[0208] The design of comparative example 2 is exactly the same as that of experimental group 2, the only difference being that a single solvent extraction system is used. The specific design is as follows:

[0209] 1. Solution preparation: Adjust the pH of the avermectin product solution after membrane separation to 8.0.

[0210] 2. Single solvent extraction: Add a mixed solvent system of ethyl acetate and water in a volume ratio of 1:1. After extraction, separate and discard the aqueous phase, and retain the ethyl acetate phase for subsequent crystallization.

[0211] The experimental steps are as follows:

[0212] 1. Sample configuration: The multi-stage solvent extraction system sample of the embodiment of the present invention and the single solvent extraction system sample of the comparative example were configured respectively, and recorded as the implementation sample and the comparative sample.

[0213] 2. Determination of impurity content:

[0214] The retained ethyl acetate phase was filtered through a microporous filter membrane and the filtrate was collected;

[0215] The impurity content was detected by HPLC using a reverse phase C18 column, a mobile phase of water-acetonitrile-methanol (volume ratio 60:30:10), a flow rate of 0.8 mL / min, and a detection wavelength of 245 nm;

[0216] Record the percentage of impurity peak area to total peak area in each sample.

[0217] 3. Purity determination:

[0218] Use HPLC to measure the purity of the target product, keeping the chromatographic conditions consistent with the impurity content determination;

[0219] The purity of each sample was recorded.

[0220] 4. Recovery rate determination:

[0221] The recovery rates of the target products in the two groups of solutions were recorded, and the recovery rate of the final product amount was calculated based on the initial input amount.

[0222] The experimental results are shown in the following table:

[0223] Test items Implementation samples (experimental group 2) Comparative sample (Comparative Example 2) Impurity content (%) 0.15 0.32 purity(%) 99.6 99 Recovery rate (%) 98.2 94.5

[0224] The results of this comparative experiment prove the significant advantages of the multi-stage solvent extraction system in the present invention in the purification of avermectin. The details are as follows:

[0225] 1. In the scheme of the present invention, the multi-stage solvent extraction system realizes the step-by-step removal of impurities by introducing the three-phase solvent of ethyl acetate-n-hexane-water, utilizing the polarity of ethyl acetate, the high polarity of water, and the non-polarity of n-hexane. First, in the contact with the aqueous phase, the water-soluble impurities are preferentially transferred to the aqueous phase and removed; then, the non-polar impurities are further removed by the non-polar effect of n-hexane. This step-by-step extraction mechanism ensures the effect of selective impurity removal, significantly reducing the impurity content to 0.15%, which is significantly better than 0.32% of the single solvent system, showing the creativity of the present invention in impurity removal.

[0226] 2. By removing impurities of different polarities in steps, the multi-stage solvent system of the present invention ensures the purity of the target product, thereby reducing impurity interference in the subsequent crystallization and purification process, so that the purity of the final product reaches 99.6%. This method of improving purity by step-by-step extraction is different from the traditional single solvent system, which lacks step-by-step selectivity and has poor impurity separation effect, resulting in a purity of only 99.0%. Therefore, the multi-stage solvent system can ensure precise control of solvent action and efficient selective separation, ensuring the high purity of the product.

[0227] 3. The multi-stage solvent extraction system in the scheme of the present invention not only effectively removes impurities, but also retains the target product to the maximum extent. Experimental data show that the recovery rate of the implemented sample reached 98.2%, while the recovery rate of the single solvent extraction system was only 94.5%. This result is due to the selective dissolution of impurities by the solvent in the multi-stage solvent system, which reduces the loss of the target product. The multi-stage system controls the proportion and polarity of the solvent in each step of the extraction, accurately regulates the migration of impurities, and does not cause the loss of the target product, which significantly improves the recovery rate.

[0228] Comparative experiment 3:

[0229] Objective: To verify the effect of the multi-stage crystallization process in the present invention in improving purity, reducing impurities and improving crystal uniformity, and to compare the effects of multi-stage crystallization and single crystallization on the quality of the final product.

[0230] The comparison settings of this comparative experiment are as follows:

[0231] Experimental group 3 plan:

[0232] According to step S4 of Example 1, dynamic solvent ratio adjustment and multi-stage cooling crystallization are adopted, and the specific design is as follows:

[0233] 1. Solvent system: The ethyl acetate phase after extraction is prepared into a ternary solvent system with isopropanol and water, with a volume ratio of ethyl acetate: isopropanol: water = 4:3:2.

[0234] 2. Multi-stage cooling: The solvent system is placed in a temperature-controlled crystallization tank with an initial temperature of 40°C. Slowly cool to 25°C and keep for 2 hours to induce initial crystal precipitation; continue to cool to 10°C and keep for 3 hours; finally cool to 4°C to complete crystallization.

[0235] 3. Dynamic solvent ratio adjustment: During the crystallization process, the ethyl acetate content is increased or decreased by 3% each time to adjust the solvent ratio, control the crystal precipitation rate and improve the purity.

[0236] Comparative Example 3:

[0237] The design of comparative example 3 is completely consistent with that of experimental group 3, the only difference being that a single crystallization process is used. The specific design is as follows:

[0238] 1. Solvent system: The ethyl acetate phase after extraction is prepared into a ternary solvent system with isopropanol and water, and the volume ratio is ethyl acetate: isopropanol: water = 4:3:2.

[0239] 2. Single cooling: The initial temperature of the solvent system is 25°C. After cooling to 4°C, crystallization is completed directly without multi-stage cooling and dynamic solvent ratio adjustment.

[0240] The experimental steps are as follows:

[0241] 1. Sample configuration: The multi-stage crystallization system sample of the embodiment of the present invention and the single crystallization system sample of the comparative example are configured respectively, and recorded as the implementation sample and the comparative sample.

[0242] 2. Crystal uniformity determination:

[0243] The crystallized product particles of each group of samples were observed under a microscope;

[0244] Statistics of particle size distribution, calculation of average particle size and uniformity index.

[0245] 3. Determination of impurity content:

[0246] The crystalline product was dissolved in acetonitrile solvent and the impurity content was determined using HPLC;

[0247] Chromatographic conditions: reverse phase C18 column, mobile phase of water-acetonitrile-methanol (volume ratio 60:30:10), flow rate 0.8 mL / min, detection wavelength 245 nm.

[0248] 4. Purity determination:

[0249] Use HPLC to measure the purity of the target product, keeping the chromatographic conditions consistent with the impurity content determination;

[0250] The purity of each sample was recorded.

[0251] The experimental results are shown in the following table:

[0252] Test items Implementation samples (experimental group 3) Comparative sample (Comparative Example 3) Average crystal size (μm) 55 47 Crystal uniformity index high middle Impurity content (%) 0.12 0.3 purity(%) 99.7 99.1

[0253] This comparative experiment shows that the multi-stage crystallization process of the present invention has a significant effect in improving the purity of avermectin crystals, reducing the impurity content and enhancing the uniformity of crystals. The details are as follows:

[0254] 1. By cooling in steps, the solubility of impurities in the solution is gradually reduced, so that impurities are effectively eliminated in the solution at each stage, while ensuring that the target product is gradually precipitated under appropriate conditions to form crystals with higher purity. Experimental data proves that the step-by-step cooling mechanism effectively controls the coprecipitation of impurities and improves the purity of the product, which illustrates the innovation of the present invention in the crystallization process.

[0255] 2. Dynamically adjusting the proportion of ethyl acetate can balance the precipitation rate during crystal growth, avoid crystal inhomogeneity caused by rapid precipitation, and make the crystal structure more stable and consistent. Experimental data show that the crystal uniformity of the implemented sample is higher than that of the single crystallization process, which further verifies the contribution of solvent ratio adjustment to crystal structure optimization.

[0256] 3. The dual effects of multi-stage cooling and solvent adjustment not only improve the uniformity of crystals at the physical level, but also reduce the mixing of impurities at the chemical level, so that the purity of the final product reaches 99.7%, which is significantly better than the single crystallization process. This dual mechanism makes the multi-stage crystallization process of the present invention have a unique advantage in improving product purity.

[0257] The present invention solves the problems of low purity, many impurities and uneven crystals in the traditional single crystallization process through multi-stage crystallization and dynamic solvent ratio adjustment. Through multi-stage cooling and precise control of the solvent ratio, the present invention not only achieves the goals of high purity and low impurities, but also improves the uniformity and stability of the crystals, providing a new way for the efficient industrial production of avermectin.

[0258] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for refining avermectin, characterized in that: The following steps are involved: S1, pretreating the crude raw material of avermectin by using a multifunctional fusion enzyme; S2, performing membrane separation treatment on the product solution after the enzymatic reaction to remove macromolecular impurities and small molecular by-products by nanofiltration and ultrafiltration; S3, refining by multi-stage solvent extraction, using ethyl acetate-n-hexane-water three-phase solvent system to separate water-soluble and non-polar impurities; S4, adopting a multi-stage crystallization process, through dynamic solvent ratio adjustment and multi-stage cooling crystallization, gradually precipitating high-purity avermectin crystals; S5, separating the crystalline product through a silica gel column to further remove trace impurities; S6. The purified avermectin is subjected to secondary crystallization and freeze-drying to obtain a high-purity crystalline product.

2. A method for refining avermectin according to claim 1, characterized in that: The step S1 comprises the following steps: S11, fusing the acylase with the dissolution auxiliary enzyme to prepare a multifunctional fusion enzyme, wherein the dissolution auxiliary enzyme is lysozyme or lipase, and the concentration of the multifunctional fusion enzyme is 0.5-1.5 mg / mL; S12, dissolving the crude avermectin raw material in a mixed solvent of ethyl acetate and ethanol, wherein the volume ratio of ethyl acetate to ethanol in the mixed solvent is 2:1 to 3:1; S13, adding the multifunctional fusion enzyme to the solution, controlling the reaction temperature to 20-30° C., the pH to 7.0-7.5, and reacting for 3-5 hours under stirring conditions; S14. Add 0.05-0.2% polyethylene glycol 400 to the reaction system to improve the solubility of the substrate.

3. A method for refining avermectin according to claim 1, characterized in that, The membrane separation step includes using a nanofiltration membrane with a molecular weight cutoff range of 5-10 kDa and an ultrafiltration membrane with a molecular weight cutoff range of 0.5-1 kDa, an operating pressure range of 0.04-0.12 MPa, and a temperature of 20-25°C.

4. A method for refining avermectin according to claim 1, characterized in that: The step S3 comprises the following steps: S31, adjusting the pH of the avermectin product solution after membrane separation to 7.5-8.5; S32, mixing the product solution with a three-phase solvent system of ethyl acetate, n-hexane and water, wherein the volume ratio of ethyl acetate to n-hexane is 1:1 to 1:2; S33, performing a first stage extraction to separate water-soluble impurities, separating and discarding the aqueous phase; S34, adding n-hexane to the ethyl acetate phase for a second stage of extraction, separating non-polar impurities, discarding the n-hexane phase and retaining the ethyl acetate phase.

5. A method for refining avermectin according to claim 4, characterized in that: The retained ethyl acetate phase was used for crystallization in the subsequent step S4.

6. A method for refining avermectin according to claim 1, characterized in that: The step S4 comprises the following steps: S41, preparing a ternary solvent system by mixing the extracted ethyl acetate phase with isopropanol and water, wherein the volume ratio of ethyl acetate, isopropanol and water is 4:3:2 to 5:3:2; S42, placing the ternary solvent system in a temperature-controlled crystallization tank, controlling the initial temperature at 35-45° C., maintaining stirring, and performing dissolution equilibrium; S43, slowly cooling the solution to 20-25°C and maintaining for 1-3 hours to induce initial crystal precipitation; S44, further reducing the temperature to 8-12°C and maintaining for 2-4 hours to precipitate intermediate crystals; S45, the temperature is finally lowered to 2-5°C to complete the crystallization, and the dynamic solvent ratio is adjusted throughout the process, and ethyl acetate is increased or decreased by 3-5% each time to control the precipitation rate and purity of the crystals.

7. A method for refining avermectin according to claim 1, characterized in that: The step S5 comprises the following steps: S51, dissolving the crystalline product in acetonitrile solvent to prepare a sample solution with a concentration of 8-12 mg / mL; S52, loading the sample solution into a column chromatography system, wherein the column chromatography system uses a silica gel filler, and the mobile phase is a mixed system of water, acetonitrile and methanol, wherein the volume ratio of water, acetonitrile and methanol is 60:30:10 to 55:35:10; S53, separating the target product from impurities by controlling the solvent flow rate and gradient elution; S54, collecting eluted components in stages, and subjecting the collected target product to repeated column chromatography separation 2-3 times to further remove trace impurities; S55. Concentrate the purified target product components using a rotary evaporator at 30-40° C. to remove residual solvent.

8. A method for refining avermectin according to claim 1, characterized in that: The step S6 comprises the following steps: S61, dissolving the purified avermectin in a mixed solvent of ethanol and water, wherein the volume ratio of ethanol to water is 3:1 to 4:1; S62, placing the solution in a low-temperature crystallization tank, with the initial temperature controlled at 10-15° C., to induce crystal precipitation; S63, gradually cooling to 4° C. and maintaining for 2-4 hours to promote complete precipitation of high-purity crystals; S64, collecting the crystallized product through a microporous filter membrane, and placing the crystallized product in a vacuum freeze dryer; S65. Control the freeze-drying temperature to -15 to -25°C, the vacuum pressure to 0.01-0.02MPa, and the drying time to 18-24 hours to obtain a high-purity crystalline product.

9. A process for preparing avermectin, characterized in that: The method comprises preparing a crude raw material of avermectin, and purifying it by using the refining method described in any one of claims 1 to 8 to obtain an avermectin product with high purity and low residue.

10. An application of avermectin, characterized in that: The avermectin is obtained by the preparation process described in claim 9 and is used as an anthelmintic drug for animals without a withdrawal period.

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