CN116082425B

CN116082425B - High-purity spinosad A, D standard substance and preparation method thereof

Info

Publication number: CN116082425B
Application number: CN202310075537.3A
Authority: CN
Inventors: 张楠; 潘忠成; 杨宏勃; 李皓瑜; 高波
Original assignee: Shaanxi Microbe Biotechnology Co ltd
Current assignee: Yuanda Crop Science Shaanxi Co ltd
Priority date: 2023-02-07
Filing date: 2023-02-07
Publication date: 2025-01-17
Anticipated expiration: 2043-02-07
Also published as: CN116082425A

Abstract

The invention relates to a high-purity spinosad A, D standard substance and a preparation method thereof, wherein a spinosad mother medicine is prepared by high performance liquid chromatography after methanol crystallization, and spinosad A and spinosad D with purity more than or equal to 90% are obtained by separation. The invention can be applied to the quality detection of spinosad in the current enterprise production, fills the blank technical problem of the current spinosad A, D reference substance, greatly reduces the quality control cost of spinosad, effectively solves the phenomenon of uneven spinosad quality, and is beneficial to enhancing the sustainable development of biological pesticide enterprises.

Description

High-purity spinosad A, D standard substance and preparation method thereof

Technical Field

The invention belongs to the technical field of agricultural antibiotic purification, and particularly relates to a high-purity spinosad A, D standard substance and a preparation method thereof, in particular to a high-performance liquid phase preparation chromatographic condition for preparing the spinosad A, D standard substance.

Background

Spinosad, also known as spinosad, is a macrolide nuisance-free high-efficiency biological pesticide extracted from spinosad sugarcane multi-spore fungus fermentation liquor. The parent spinosyn-producing strain, actinomycetes spinosad, saccharopolyspora spinosa, was originally isolated from one abandoned brewery of the Caribbean. Researchers from the company Tao Shiyi agricultural, now the Dow agricultural science company, have found that this bacterium can produce very high insecticidal compounds, and that the product of practical use is a mixture of Spinosyn A and Spinosyn D, and is therefore known as Spinosad.

The novel mode of action of spinosad allows sustained activation of the target insect acetylcholine nicotinic receptor, but its binding site is different from that of nicotine and imidacloprid. Spinosad can also affect GABA receptors, but the mechanism of action is unclear. It is currently unknown whether there is cross-resistance to other types of pesticides. These compounds can cause rapid death of target phytophagous insects such as caterpillars, leaf miner, thrips, and leaf eating beetles, although regulatory authorities strongly require use when resistance is not present, the moderate residual activity of the compounds reduces the likelihood of resistance and swarming. No phytotoxicity was found when applied at 12-150 g/hm.

According to the document inquiry, the separation and purification preparation process of the spinosad is mostly recrystallization method or column chromatography method, but the spinosad is a mixture of spinosad A and spinosad D, and even though the spinosad A and the spinosad D can not be separated through repeated separation and purification preparation. In the actual quality detection process, the independent quantitative and qualitative analysis of the spinosad A and the spinosad D is needed, and the independent spinosad A and spinosad D standard products are needed to be separated and prepared for detection. On the basis of the high-purity spinosad-based raw material, the recrystallization technology is further utilized to obtain a high-purity raw material product, and then the high-pressure preparation liquid phase is further utilized to separate and purify to obtain spinosad A and spinosad D standard products with the purity of more than or equal to 90%.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a high-purity spinosad A, D standard substance and a preparation method thereof, wherein a spinosad mother drug is prepared by high performance liquid chromatography after methanol crystallization, and spinosad A and spinosad D with purity more than or equal to 90% are obtained by separation. The invention can be applied to the quality detection of spinosad in the current enterprise production, fills the blank technical problem of the current spinosad A, D reference substance, greatly reduces the quality control cost of spinosad, effectively solves the phenomenon of uneven spinosad quality, and is beneficial to enhancing the sustainable development of biological pesticide enterprises.

In order to achieve the aim, the invention adopts the following technical scheme that the preparation method of the high-purity spinosad A, D standard substance is adopted, the spinosad mother medicine is prepared by high performance liquid chromatography after methanol crystallization, and then the spinosad A and spinosad D with the purity of more than or equal to 90% are obtained by reduced pressure concentration and freeze drying.

More specifically, the preparation method comprises the following process steps:

1) The crystallization comprises the steps of weighing the spinosad raw medicine, dissolving, standing, filtering to remove insoluble substances, standing in a low-temperature environment, separating out spinosad crystals, centrifuging, filtering, and removing the solvent to obtain a spinosad A and spinosad D mixed sample;

2) The high performance liquid chromatography is prepared under the following conditions

Mobile phase is methanol, ammonia water solution;

The chromatographic column is internally provided with reversed phase C18 packing;

The flow rate is 10-15 ml/min, the wavelength is 245nm, and the sample loading volume is 1 ml-5 ml;

collecting according to chromatographic peaks to obtain spinosad A and spinosad D solution with purity more than or equal to 90%;

3) Concentrating under reduced pressure, and lyophilizing to obtain white powder of spinosad A and spinosad D with purity not less than 90%.

In a preferred embodiment of the present invention, in step 2), a mobile phase is prepared from methanol and 0.5% aqueous ammonia solution, and the volume ratio of methanol to aqueous ammonia solution is 95:5 to 90:10.

In a preferred embodiment of the present invention, in step 2), the column packing is prepared as a built-in reverse phase C18,5 μm,20 x 250mm to 50 x 250mm packing;

In a preferred embodiment of the present invention, in step 2), the flow rate is 10 to 12ml/min.

In a preferred embodiment of the present invention, in step 2), the sample volume is 1 to 3ml.

In a preferred embodiment of the present invention, in step 2), the conditions of high performance liquid chromatography are as follows:

Mobile phase methanol 0.5% aqueous ammonia solution = 95:5;

Chromatographic column filled with reversed phase C18,5 μm,20 x 250mm packing;

The flow rate is 10ml/min, the wavelength is 245nm, and the loading volume is 1ml.

The invention also protects the spinosad A and spinosad D standard products prepared by the preparation method, and the purity of the spinosad A and spinosad D standard products is more than or equal to 90 percent.

Compared with the prior art, the invention has the beneficial effects that:

1) The separation and purification preparation process of spinosad is mostly recrystallization method or column chromatography method, but spinosad is a mixture of spinosad A and spinosad D, and even though the spinosad A and spinosad D cannot be separated out after multiple separation and purification preparation. In the actual quality detection process, the spinosad A and spinosad D are required to be separately and quantitatively analyzed qualitatively, and separate spinosad A and spinosad D standard products are required to be prepared for detection. According to the method, the high-performance liquid chromatography preparation conditions are studied, and the high-purity spinosad A and spinosad D single products are obtained through separation and purification, so that the effective separation of spinosad A and spinosad D can be realized. And methanol and a small amount of ammonia water adopted by the mobile phase can be removed in the subsequent decompression concentration process, other impurities cannot be introduced, and the purity of the sample is ensured.

2) The method can be applied to the quality detection of the spinosad in the production of the current enterprises, fills the blank of the current spinosad standard, greatly reduces the quality control cost of the spinosad, effectively solves the difficulty of qualitative and quantitative analysis of the spinosad A, D at the same time, and is beneficial to the sustainable development of biological pesticide enterprises.

Drawings

Further description is provided below with reference to the accompanying drawings.

FIG. 1 is a liquid chromatogram of a spinosad A standard prepared in example 1.

Fig. 2 is a liquid chromatogram of the spinosad standard prepared in example 1.

Detailed Description

The invention will be described in further detail with reference to examples

Example 1:

1) And (3) crystallizing, namely weighing 10g of the spinosad original drug in a 100ml beaker, carrying out ultrasonic dissolution by using 100ml of methanol, standing for 1h, and filtering to remove insoluble substances. Placing in a refrigerator (at 4 ℃) and standing for 24 hours, crystallizing and separating out spinosad, centrifuging and filtering, and removing the solvent to obtain a spinosad A and spinosad D mixed sample;

2) The high performance liquid chromatography preparation comprises the mobile phase of methanol 0.5% ammonia water solution=95:5, the chromatographic column filled with reversed phase C18,5 μm,20×250mm packing, the flow rate of 10ml/min, the wavelength of 245nm, and the loading volume of 1ml. Collecting according to chromatographic peaks to obtain spinosad A and spinosad D solution;

3) Concentrating under reduced pressure, and lyophilizing to obtain white powder of spinosad D with purity of 92% and 93% by concentrating the solution of 1) under reduced pressure and lyophilizing.

Example 2:

2) The high performance liquid chromatography preparation comprises the mobile phase of methanol 0.5% ammonia water solution=92:8, the chromatographic column filled with reversed phase C18,5 μm,20×250mm packing, the flow rate of 12ml/min, the wavelength of 245nm, and the loading volume of 1ml. Collecting according to chromatographic peaks to obtain spinosad A and spinosad D solution;

3) Concentrating under reduced pressure, and lyophilizing to obtain white powder of spinosad D with purity of 91% and 93% by concentrating the solution of 1) under reduced pressure and lyophilizing.

Example 3:

2) The high performance liquid chromatography preparation comprises mobile phase methanol 0.5% ammonia water solution=90:10, chromatographic column filled with reversed phase C18,5 μm,20×250mm packing, flow rate 10ml/min, wavelength 245nm, and loading volume 3ml. Collecting according to chromatographic peaks to obtain spinosad A and spinosad D solution;

3) Concentrating under reduced pressure, and lyophilizing to obtain white powder of spinosad D with purity of 90% and 91% by concentrating the solution of 1) under reduced pressure and lyophilizing.

The foregoing is a further description of the invention in connection with specific/preferred embodiments, and it is not intended that the invention be limited to such description. Several alternatives or modifications to these described embodiments are contemplated as falling within the scope of the present invention without departing from the inventive concept.

Claims

1. A preparation method of a spinosad A, D standard substance is characterized in that a spinosad mother drug is prepared by high performance liquid chromatography after methanol crystallization, and then is subjected to reduced pressure concentration and freeze-drying to obtain spinosad A with the purity of more than or equal to 90% and spinosad D with the purity of more than or equal to 90%, and the preparation method comprises the following process steps:

1) Dissolving spinosad raw medicine, standing, filtering to remove insoluble substances, standing at the temperature of 4 ℃ to separate out spinosad crystals, centrifuging, filtering, and removing solvent to obtain a spinosad A and spinosad D mixed sample;

Mobile phase methanol 0.5% ammonia solution=95:5-90:10;

The chromatographic column is internally provided with reversed phase C18,5 mu m and 20-50 mm 250mm packing;

3) Concentrating under reduced pressure, and lyophilizing to obtain white powder of spinosad with purity not less than 90% and spinosad with purity not less than 90%.