A method for detecting and purifying avermectins

Abstract

The application relates to a detection method and a purification method of abamectin, the detection method comprising the following steps: step 1, adding methanol into abamectin fine products to prepare an abamectin methanol solution; step 2, placing the abamectin methanol solution in a volumetric flask and freezing at-18 to-20 DEG C for at least 16 hours; and step 3, placing distilled water in another equal-volume volumetric flask, comparing the frozen abamectin methanol solution with the distilled water under a film reading lamp, and judging the turbidity of the abamectin methanol solution; the purification method comprises the following steps: mixing the abamectin fine products with unqualified impurities determined through turbidity detection with distilled water, heating to boiling, then cooling to room temperature, centrifuging, drying the solid, and then performing turbidity detection again until the turbidity detection is qualified. The application can detect and remove impurities that can cause crop phytotoxicity, improve the quality of abamectin technical material, and make the abamectin technical material capable of being used for preparing emulsifiable concentrate preparations.

Description

Technical Field

[0001] This invention relates to the field of avermectin extraction technology, specifically to a method for detecting and purifying avermectin. Background Technology

[0002] Avermectin is a macrolide antibiotic produced by the fermentation of *Streptomyces avermitilis*. It possesses strong insecticidal activity and is widely used as an agricultural and veterinary insecticide and acaricide. It has a broad insecticidal spectrum, exhibiting strong insecticidal activity against almost all insects, mites, and animal parasites. Furthermore, avermectin is readily degraded in soil and water, and does not accumulate or leave persistent residues in organisms, thus avoiding environmental pollution. Therefore, its development prospects are extremely promising.

[0003] Currently, the extraction process for avermectin typically involves leaching mycelia with methanol, concentrating the extract, desaccharifying with toluene, crystallizing with methanol, and drying to obtain the final avermectin product. However, avermectin prepared by this process often contains a proteinaceous impurity that is difficult to remove completely. When avermectin containing this impurity is formulated into an emulsifiable concentrate and sprayed on crops, it can easily cause phytotoxicity, mainly manifested as the formation of a few spots on crop leaves. However, existing national standards do not include requirements for the detection of this impurity. Therefore, developing a detection and purification method for this impurity in avermectin is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting and purifying abamectin, which can detect and remove impurities that can cause crop phytotoxicity, improve the quality of abamectin technical material, and enable it to be used in the preparation of emulsifiable concentrate formulations.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Technical Solution 1:

[0007] A method for detecting avermectin includes the following steps:

[0008] Step 1: Add methanol to the avermectin concentrate, stir to dissolve, and prepare avermectin methanol solution;

[0009] Step 2: Place the methanol solution of abamectin in a volumetric flask and freeze at -18°C to -20°C for at least 16 hours;

[0010] Step 3: Place distilled water in another volumetric flask of equal volume as a control, and then compare the frozen avermectin methanol solution with distilled water under a film reading lamp to determine the turbidity of the avermectin methanol solution.

[0011] If the turbidity of the frozen avermectin methanol solution is no greater than that of distilled water, the avermectin product is deemed to have acceptable impurities; if the turbidity of the frozen avermectin methanol solution is greater than that of distilled water, the avermectin product is deemed to have unacceptable impurities.

[0012] Furthermore, in step 1, the avermectin concentrate contains: avermectin mass fraction ≥92%, avermectin B1a to avermectin B1b ratio ≥10.0, acetone insoluble matter ≤0.2%, and pH range of 44.5-7.0; all of which meet the relevant requirements of the national standard GB / T 19336-2017.

[0013] Furthermore, in step 1, the mass-to-volume ratio of avermectin to methanol is 1g:80-90mL.

[0014] Furthermore, the volumetric flask is a 100mL volumetric flask.

[0015] Furthermore, the amount of avermectin methanol solution added is equal to the amount of distilled water added;

[0016] The amount of abamectin methanol solution added is 1 / 2 of the volumetric flask volume.

[0017] Technical Solution Two:

[0018] A method for purifying avermectin, comprising the following steps:

[0019] Step a: Mix the ivermectin concentrate that was determined to be substandard due to impurities by turbidity testing with distilled water, heat to boiling, then cool to room temperature, centrifuge, dry the solid, and then perform turbidity testing again using the aforementioned testing method.

[0020] Step b: If the turbidity test in step a still determines that the impurities are unqualified, then repeat step a again until the turbidity test in step a determines that the impurities are qualified.

[0021] Furthermore, in step a, the mass ratio of avermectin to distilled water is 1:1; in step a, cooling is performed at a cooling rate of 10℃ / h.

[0022] Furthermore, in step a, the drying temperature is 60℃-80℃, and the drying time is 60-90min.

[0023] Technical Solution 3:

[0024] An avermectin prepared using the purification method described above.

[0025] Technical Solution Four:

[0026] The use of the aforementioned avermectin in the preparation of avermectin emulsifiable concentrate formulations.

[0027] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0028] This invention uses a turbidity detection method to detect impurities in abamectin (which can cause phytotoxicity to crops and cause spots on crop leaves), and removes these impurities by boiling in water, cooling, and centrifuging. This solves the problem that abamectin prepared into emulsifiable concentrates is prone to causing phytotoxicity to crops and causing spots on crop leaves due to these impurities. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments.

[0030] Example 1

[0031] A high-quality avermectin is prepared as follows:

[0032] Step A, Extraction: Add methanol to the dried mycelium of Streptomyces avermectin and extract 3 times. The mass ratio of mycelium to methanol is 1:100 for each extraction and the extraction time is 4 hours. Combine the extracts.

[0033] Step B, Evaporation and Concentration: After filtration, the collected extract is placed in an evaporator for evaporation and desolvation until the temperature inside the evaporator rises to 80℃~82℃ and the vacuum degree reaches -0.08MPa~-0.082MPa, to obtain avermectin paste.

[0034] Step C, Desugaring: Add toluene to the avermectin paste at a mass-volume ratio of 1g:6mL:1mL. Heat until completely dissolved, then add 80℃ hot water and stir continuously for 1 hour. Let stand for 1 hour, then drain the lower water layer and place the paste in an evaporator to evaporate and desolvent. Continue evaporating until the evaporator temperature reaches 80℃ and the vacuum degree reaches -0.08MPa to obtain the desugared avermectin paste.

[0035] Step D: After crystallizing the desugared avermectin paste four times in methanol, the avermectin crystals are washed with methanol and dried to obtain refined avermectin B1 (tested according to the test items specified in the national standard GB / T 19336-2017, and all meet the requirements).

[0036] The specific method for crystallization is as follows: Both the desugared avermectin paste and avermectin crystals are calculated as avermectin. Methanol is added to the desugared avermectin paste or the avermectin crystals obtained in the previous crystallization at a mass-to-volume ratio of avermectin to methanol of 1 g: 15 mL. The mixture is heated to 75°C and stirred for 30-40 minutes. Then, it is cooled and crystallized. When the temperature drops to 30°C, it is kept at that temperature for 2 hours. The mixture is then filtered to obtain avermectin crystals.

[0037] The prepared avermectin B1 refined product was tested using the specified detection method.

[0038] The detection method includes the following steps:

[0039] Step (1): Add 50 mL of methanol to 0.6 g of avermectin concentrate, stir to dissolve, and prepare avermectin methanol solution;

[0040] Step (2): Place the avermectin methanol solution in a 100 mL volumetric flask and freeze at -18℃ to -20℃ for 16 hours;

[0041] Step (3): Place 50 mL of distilled water into another 100 mL volumetric flask as a control. Then, compare the frozen avermectin methanol solution with the distilled water under a film reading lamp to determine the turbidity of the avermectin methanol solution. If the turbidity of the frozen avermectin methanol solution is not greater than that of the distilled water, the avermectin refined product is deemed to be of acceptable quality. If the turbidity of the frozen avermectin methanol solution is greater than that of the distilled water, the avermectin refined product is deemed to be of unacceptable quality.

[0042] Testing revealed that the turbidity of the frozen avermectin methanol solution was greater than that of distilled water, thus indicating that the avermectin refined product prepared above was substandard due to impurities.

[0043] The prepared avermectin product was purified using an avermectin purification method.

[0044] The avermectin purification method specifically includes the following steps:

[0045] The abamectin was mixed with distilled water at a mass ratio of 1:1. The mixture was heated to boiling and then cooled to room temperature at a cooling rate of 10°C / h. After centrifugation, the solid was dried at 60°C for 60 minutes.

[0046] After purification, the turbidity was tested again using the aforementioned detection method. The results showed that the turbidity of the frozen avermectin methanol solution was no greater than that of distilled water. Therefore, it was determined that the impurities in the purified avermectin product were qualified, and it was thus considered a high-quality avermectin product.

[0047] Example 2

[0048] A high-quality avermectin is prepared as follows:

[0049] Step A, Extraction: Add methanol to the dried mycelium of Streptomyces avermectin and extract 3 times. The mass ratio of mycelium to methanol is 1:120 for each extraction and the extraction time is 5 hours. Combine the extracts.

[0050] Step B, Evaporation and Concentration: After filtration, the collected extract is placed in an evaporator for evaporation and desolvation until the temperature inside the evaporator rises to 80℃~82℃ and the vacuum degree reaches -0.08MPa~-0.082MPa, to obtain avermectin paste.

[0051] Step C, Desugaring: Add toluene to the avermectin paste at a mass-volume ratio of 1g:6mL:1mL. Heat until completely dissolved, then add 80℃ hot water and stir continuously for 1 hour. Let stand for 1 hour, then drain the lower water layer and place the paste in an evaporator to evaporate and desolvent. Continue evaporating until the evaporator temperature reaches 80℃ and the vacuum degree reaches -0.08MPa to obtain the desugared avermectin paste.

[0052] Step D: After the desugared avermectin paste is crystallized twice in methanol, the avermectin crystals are washed with methanol and dried to obtain refined avermectin B1 (tested according to the test items specified in the national standard GB / T 19336-2017, and all meet the requirements).

[0053] The specific method for crystallization is as follows: Both the desugared avermectin paste and avermectin crystals are calculated as avermectin. Methanol is added to the desugared avermectin paste or the avermectin crystals obtained in the previous crystallization at a mass-to-volume ratio of avermectin to methanol of 1 g: 15 mL. The mixture is heated to 73°C and stirred for 30-40 minutes. Then, it is cooled and crystallized. When the temperature drops to 25°C, it is kept at that temperature for 2 hours. The mixture is then filtered to obtain avermectin crystals.

[0054] The prepared avermectin B1 refined product was tested using the specified detection method.

[0055] The detection method includes the following steps:

[0056] Step (1): Add 50 mL of methanol to 0.6 g of avermectin concentrate, stir to dissolve, and prepare avermectin methanol solution;

[0057] Step (2): Place the avermectin methanol solution in a 100 mL volumetric flask and freeze at -18℃ to -20℃ for 16 hours;

[0058] Step (3): Place 50 mL of distilled water into another 100 mL volumetric flask as a control. Then, compare the frozen avermectin methanol solution with the distilled water under a film reading lamp to determine the turbidity of the avermectin methanol solution. If the turbidity of the frozen avermectin methanol solution is not greater than that of the distilled water, the avermectin refined product is deemed to be of acceptable quality. If the turbidity of the frozen avermectin methanol solution is greater than that of the distilled water, the avermectin refined product is deemed to be of unacceptable quality.

[0059] Testing revealed that the turbidity of the frozen avermectin methanol solution was greater than that of distilled water, thus indicating that the avermectin product prepared above was substandard due to impurities.

[0060] The prepared avermectin was purified using purification methods.

[0061] The purification method specifically includes the following steps:

[0062] The abamectin was mixed with distilled water at a mass ratio of 1:1. The mixture was heated to boiling and then cooled to room temperature at a cooling rate of 10°C / h. After centrifugation, the solid was dried at 60°C for 60 minutes.

[0063] After purification, the turbidity was tested again using the same detection method. The results showed that the turbidity of the frozen avermectin methanol solution was greater than that of distilled water. Therefore, it was still determined that the avermectin refined product after one purification was unqualified due to impurities.

[0064] The purification method described above was used again to purify the avermectin product that had undergone one purification. After purification, the turbidity was tested again using the same detection method. The test result showed that the turbidity of the frozen avermectin methanol solution was no greater than that of distilled water. Therefore, the avermectin product that had undergone two purifications was still deemed to have qualified impurities, and thus it was considered a high-quality avermectin product.

[0065] Example 3

[0066] A high-quality avermectin is prepared as follows:

[0067] Step A, Extraction: Add methanol to the dried mycelium of Streptomyces avermectin and extract 3 times. The mass ratio of mycelium to methanol is 1:100 for each extraction and the extraction time is 3 hours. Combine the extracts.

[0068] Step B, Evaporation and Concentration: After filtration, the collected extract is placed in an evaporator for evaporation and desolvation until the temperature inside the evaporator rises to 80℃~82℃ and the vacuum degree reaches -0.08MPa~-0.082MPa, to obtain avermectin paste.

[0069] Step C, Desugaring: Add toluene to the avermectin paste at a mass-volume ratio of 1g:5mL:1.2mL. Heat until completely dissolved, then add 80℃ hot water and stir continuously for 1 hour. Let stand for 1 hour, then drain the lower water layer and place the paste in an evaporator to evaporate and desolvent. Continue evaporating until the evaporator temperature reaches 80℃ and the vacuum degree reaches -0.08MPa to obtain the desugared avermectin paste.

[0070] Step D: After crystallizing the desugared avermectin paste five times in methanol, the avermectin crystals are washed with methanol and dried to obtain refined avermectin B1 (tested according to the test items specified in the national standard GB / T 19336-2017, and all meet the requirements).

[0071] The specific method for crystallization is as follows: Both the desugared avermectin paste and avermectin crystals are calculated as avermectin. Methanol is added to the desugared avermectin paste or the avermectin crystals obtained in the previous crystallization at a mass-to-volume ratio of avermectin to methanol of 1 g: 15 mL. The temperature is raised to 75°C, stirred for 30-40 minutes, and then cooled for crystallization. When the temperature drops to 25°C, it is kept at this temperature for 2 hours, filtered, and avermectin crystals are obtained.

[0072] The prepared avermectin B1 refined product was tested using the specified detection method.

[0073] The detection method includes the following steps:

[0074] Step (1): Add 50 mL of methanol to 0.6 g of avermectin concentrate, stir to dissolve, and prepare avermectin methanol solution;

[0075] Step (2): Place the avermectin methanol solution in a 100 mL volumetric flask and freeze at -18℃ to -20℃ for 16 hours;

[0076] Step (3): Place 50 mL of distilled water into another 100 mL volumetric flask as a control. Then, compare the frozen avermectin methanol solution with the distilled water under a film reading lamp to determine the turbidity of the avermectin methanol solution. If the turbidity of the frozen avermectin methanol solution is not greater than that of the distilled water, the avermectin refined product is deemed to be of acceptable quality. If the turbidity of the frozen avermectin methanol solution is greater than that of the distilled water, the avermectin refined product is deemed to be of unacceptable quality.

[0077] Testing revealed that the turbidity of the frozen avermectin methanol solution was no greater than that of distilled water. Therefore, the avermectin concentrate prepared above was deemed to have met the impurity standards, and thus was considered a high-quality avermectin concentrate.

[0078] Example of effect

[0079] The avermectin refined product without purification process in Example 1, and the avermectin refined product that passed the turbidity test after purification and was determined to be of acceptable quality for impurities in Example 2, as well as the avermectin refined product without purification process, the avermectin refined product after one purification, and the avermectin refined product that passed the turbidity test after two purifications in Example 2, and the avermectin premium product in Comparative Example 3, were all prepared into emulsifiable concentrates. In each group, the formula and process of the emulsifiable concentrate (including the amount of avermectin, the type and amount of excipients, and the preparation process of the emulsifiable concentrate, etc.) were the same. Then, the prepared emulsions were sprayed onto tobacco leaves, and the formation of phytotoxic spots on the leaves was observed after 48 hours. The results are shown in Table 1.

[0080] Evaluation criteria for pesticide damage spots:

[0081] Severe pesticide damage spots: more than 4 pesticide damage spots per square centimeter of leaf;

[0082] The pesticide damage spots are relatively mild: the number of pesticide damage spots on each square centimeter of leaf is 1-1.5;

[0083] No pesticide damage spots: The number of pesticide damage spots on each square centimeter of leaf is less than 0.2;

[0084] Table 1

[0085]

[0086]

[0087] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for purifying avermectin, characterized in that, Includes the following steps: Step a: Mix the avermectin concentrate, which was determined to be substandard due to impurities by turbidity testing, with distilled water, heat to boiling, then cool to room temperature, centrifuge, dry the solid, and perform turbidity testing again. The turbidity testing adopts the avermectin detection method, which includes the following steps: Step 1: Add methanol to the avermectin concentrate, stir to dissolve, and prepare avermectin methanol solution; Step 2: Place the methanol solution of abamectin in a volumetric flask and freeze at -18°C to -20°C for at least 16 hours; Step 3: Place distilled water in another volumetric flask of equal volume as a control, and then compare the frozen avermectin methanol solution with distilled water under a film reading lamp to determine the turbidity of the avermectin methanol solution. If the turbidity of the frozen avermectin methanol solution is no greater than that of distilled water, the avermectin product is deemed to have acceptable impurities; if the turbidity of the frozen avermectin methanol solution is greater than that of distilled water, the avermectin product is deemed to have unacceptable impurities. Step b: If the turbidity test in step a still determines that the impurities are unqualified, then repeat step a again until the turbidity test in step a determines that the impurities are qualified.

2. The purification method for avermectin according to claim 1, characterized in that, In step 1, the avermectin concentrate contains: avermectin mass fraction ≥ 92%, avermectin B1a to avermectin B1b ratio ≥ 10.0, acetone insoluble matter ≤ 0.2%, and pH range of 4.5-7.

0.

3. The purification method for avermectin according to claim 1, characterized in that, In step 1, the mass-to-volume ratio of avermectin to methanol is 1g:80-90mL.

4. The purification method for avermectin according to claim 1, characterized in that, The volumetric flask used is a 100mL volumetric flask.

5. The purification method for avermectin according to claim 1, characterized in that, The amount of avermectin methanol solution added is equal to the amount of distilled water added. The amount of abamectin methanol solution added is 1 / 2 of the volumetric flask volume.

6. The purification method for avermectin according to claim 1, characterized in that, In step a, the mass ratio of abamectin to distilled water is 1:1; in step a, cooling is performed at a cooling rate of 10℃ / h.

7. The purification method for avermectin according to claim 1, characterized in that, In step a, the drying temperature is 60℃-80℃ and the drying time is 60-90min.

8. An avermectin prepared by the purification method according to any one of claims 1-7.

9. The use of avermectin as described in claim 8 in the preparation of avermectin emulsifiable concentrate formulations.

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