CN116082425A - High-purity spinosad A, D standard substance and preparation method thereof - Google Patents

Abstract

The invention relates to a high-purity spinosyn A, D standard product and a preparation method thereof. The spinosad mother drug is crystallized by methanol and prepared by high-performance liquid chromatography, and the spinosyn with a purity ≥ 90% is separated and obtained A. Spinosad D. The present invention can be applied to the quality detection of spinosad in the current enterprise production, fills up the technical problem of the current blank of spinosad A and D reference products, greatly reduces the quality control cost of spinosad, and effectively The phenomenon of uneven quality of spinosad is solved, which is conducive to enhancing the sustainable development of biopesticide enterprises.

Description

A kind of high-purity spinosyn A, D standard substance and preparation method thereof

technical field

The invention belongs to the technical field of purification of agricultural antibiotics, and in particular relates to a high-purity spinosyn A, D standard product and a preparation method thereof, especially relates to the high-efficiency liquid phase preparation of the described spinosyn A, D standard product preparation Chromatographic conditions.

Background technique

Spinosyn, also known as spinosad, is a macrolide pollution-free and high-efficiency biopesticide extracted from the fermentation broth of Polyspora spinosa. The spinosad-producing parent strain, the soil actinomycete Polyspora spinosa, was originally isolated from an abandoned winery in the Caribbean. Researchers from Dow AgroSciences (now Dow Agricultural Sciences) found that the bacterium can produce compounds with very high insecticidal activity. The practical product is a mixture of Spinosyn A and Spinosyn D, so it is called Spinosad.

The novel mode of action of spinosad can continuously activate the target insect acetylcholine nicotinic receptor, but its binding site is different from that of nicotine and imidacloprid. Spinosad also affects GABA receptors, but the mechanism of action is unclear. It is not known whether there is cross-resistance to other types of insecticides. These compounds can cause rapid death of target phytophagous insects such as caterpillars, leafminers, thrips, and leaf-eating beetles, and despite regulatory urges to use them in the absence of resistance, the compounds' moderate residual activity reduces resistance and possibility of group occurrence. When applied at 12-150g/hm, no phytotoxicity was found.

According to the literature search, the separation and purification of spinosad is mostly recrystallization or column chromatography, but spinosad is a mixture of spinosad A and spinosad D, even after multiple separations and purifications. , still unable to separate spinosad A and spinosad D. In our actual quality inspection process, we need separate quantitative and qualitative analysis of spinosad A and spinosad D, which requires separation and preparation of separate spinosad A and spinosad D standards for detection . On the basis of the high-purity spinosad original drug, the present invention further utilizes the recrystallization process to obtain a high-purity original drug product, and then further separates and purifies the liquid phase through high-pressure preparation to obtain spinosad with a purity ≥ 90%. Mycin A, spinosad D standard.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a kind of high-purity spinosyn A, D standard product and its preparation method. Spinosad A and spinosyn D with a purity ≥ 90% were obtained. The present invention can be applied to the quality detection of spinosad in the current enterprise production, fills up the technical problem of the current blank of spinosad A and D reference products, greatly reduces the quality control cost of spinosad, and effectively The phenomenon of uneven quality of spinosad is solved, which is conducive to enhancing the sustainable development of biopesticide enterprises.

In order to achieve the purpose of the invention, the present invention adopts the following technical scheme: a preparation method of high-purity spinosyn A, D standard product, the spinosad parent drug is crystallized by methanol, prepared by high performance liquid chromatography, and then reduced Concentrate under reduced pressure and freeze-dry to obtain spinosad A and spinosad D with a purity ≥ 90%.

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

1) Crystallization: Dissolve the original drug of spinosad, stand still, filter, and remove insoluble matter; then stand in a low temperature environment, at most spinosad crystals are precipitated, centrifugally filter, remove solvent, and obtain spinosad A , Spinosad D mixed sample;

2) HPLC preparation: HPLC conditions are as follows

Mobile phase: methanol, ammonia solution;

Chromatographic column: built-in reversed-phase C18 filler;

Flow rate: 10~15ml/min; wavelength 245nm; sample volume 1ml~5ml;

Collect according to the chromatographic peaks to obtain spinosad A and spinosad D solutions with a purity ≥ 90%;

3) Concentrate under reduced pressure and freeze-dry: Concentrate the solution of 2) under reduced pressure and freeze-dry to obtain spinosad A and spinosad D white powders with a purity ≥ 90%.

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

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

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

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

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

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

Chromatographic column: built-in reversed-phase C18, 5μm, 20*250mm filler;

Flow rate: 10ml/min; wavelength 245nm; sample volume 1ml.

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

Compared with prior art, the beneficial effect of the present invention is:

1) The spinosyn separation and purification preparation process is mostly recrystallization or column chromatography, but spinosyn is a mixture of spinosad A and spinosad D, even after multiple separations and purifications, still Spinosad A and spinosyn D could not be separated. In the actual quality detection process, separate quantitative and qualitative analysis of spinosad A and spinosad D is required, which requires separation and preparation of separate spinosad A and spinosad D standards for detection . The present invention adopts the preparation method of high performance liquid chromatography, and by studying the preparation conditions of high performance liquid chromatography, separates and purifies the single products of spinosad A and spinosad D with higher purity, which can realize spinosad A, spinosad Efficient isolation of cidamicin D. Moreover, the methanol and a small amount of ammonia water used in the mobile phase can be removed in the subsequent process of concentration under reduced pressure, and no other impurities will be introduced to ensure the purity of the sample.

2) The method of the present invention can be applied to the quality detection of spinosad in the current enterprise production, fills up the blank of the current spinosad standard product, greatly reduces the quality control cost of spinosad, and effectively solves the problem of spinosad Solved the problem of simultaneous qualitative and quantitative analysis of spinosyn A and D, which is conducive to the sustainable development of biopesticide enterprises.

Description of drawings

Further description will be made below in conjunction with the accompanying drawings.

Fig. 1 is the liquid chromatogram of spinosyn A standard product prepared in Example 1.

Fig. 2 is the liquid chromatogram of spinosad D standard product prepared in Example 1.

Detailed ways

Below in conjunction with embodiment, be used for illustrating the present invention and do further detailed description

Example 1:

1) Crystallization: Weigh 10 g of the original spinosad into a 100 ml beaker, dissolve it ultrasonically with 100 ml of methanol, let it stand for 1 hour, and filter to remove the insoluble matter. Put it in the refrigerator (4°C), let it stand for 24 hours, spinosad crystallizes out, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 95:5; chromatographic column: built-in reverse phase C18, 5 μm, 20*250mm filler; flow rate: 10ml/min; wavelength 245nm; sample volume 1ml. Collect according to the chromatographic peaks to obtain spinosad A and spinosad D solutions;

3) Concentrate under reduced pressure and freeze-dry: Concentrate the solution of 1) under reduced pressure and freeze-dry to obtain a white powder with a purity of 92% spinosyn A and a purity of 93% spinosyn D.

Example 2:

1) Crystallization: Weigh 10 g of the original spinosad into a 100 ml beaker, dissolve it ultrasonically with 100 ml of methanol, let it stand for 1 hour, and filter to remove the insoluble matter. Put it in the refrigerator (4°C), let it stand for 24 hours, spinosad crystallizes out, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 92:8; chromatographic column: built-in reversed-phase C18, 5 μm, 20*250mm filler; flow rate: 12ml/min; wavelength 245nm; loading volume 1ml. Collect according to the chromatographic peaks to obtain spinosad A and spinosad D solutions;

3) Concentrate under reduced pressure and freeze-dry: Concentrate the solution of 1) under reduced pressure and freeze-dry to obtain a white powder with a purity of 91% spinosyn A and a purity of 93% spinosyn D.

Example 3:

1) Crystallization: Weigh 10 g of the original spinosad into a 100 ml beaker, dissolve it ultrasonically with 100 ml of methanol, let it stand for 1 hour, and filter to remove the insoluble matter. Put it in the refrigerator (4°C), let it stand for 24 hours, spinosad crystallizes out, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 90:10; chromatographic column: built-in reversed-phase C18, 5 μm, 20*250mm filler; flow rate: 10ml/min; wavelength 245nm; sample volume 3ml. Collect according to the chromatographic peaks to obtain spinosad A and spinosad D solutions;

3) Concentrate under reduced pressure and freeze-dry: Concentrate the solution of 1) under reduced pressure and freeze-dry to obtain a white powder with a purity of 90% spinosyn A and a purity of 91% spinosyn D.

The above content is a further description of the present invention in conjunction with specific/preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. On the premise of not departing from the concept of the present invention, several substitutions or modifications made to these described embodiments should be considered as belonging to the protection scope of the present invention.

Claims (8)

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

2. The preparation method according to claim 1, characterized by comprising the following process steps:

1) And (3) crystallization: weighing spinosad raw medicine, dissolving, standing, filtering and removing insoluble substances; standing in a low-temperature environment, crystallizing and separating out spinosad at most, centrifuging and filtering, and removing the solvent to obtain a spinosad A and spinosad D mixed sample;

2) High performance liquid chromatography preparation: the conditions of the high performance liquid chromatography are as follows

Mobile phase: methanol, aqueous ammonia solution;

chromatographic column: a reversed phase C18 filler is filled in;

flow rate: 10-15 ml/min; wavelength 245nm; the 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: concentrating the solution obtained in the step 2) under reduced pressure, and freeze-drying to obtain white spinosad A and spinosad D powder with the purity of more than or equal to 90%.

3. The method according to claim 2, wherein in step 2), the mobile phase is methanol and 0.5% aqueous ammonia solution, and the volume ratio of methanol to aqueous ammonia solution is 95:5 to 90:10.

4. The method according to claim 2, wherein in step 2), the column packing is a built-in reverse phase C18,5 μm,20 x 250mm to 50 x 250mm packing.

5. The method according to claim 2, wherein in step 2), the flow rate is 10 to 12ml/min.

6. The method according to claim 2, wherein in step 2), the loading volume is 1 to 3ml.

7. The method according to claim 2, wherein 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: reverse phase C18,5 μm,20 x 250mm filler is filled;

flow rate: 10ml/min; wavelength 245nm; the loading volume was 1ml.

8. The spinosad A and spinosad D standard prepared by the preparation method according to any one of claims 1 to 7, wherein the purity is not less than 90%.

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