A high-purity spinosad A and D standard and its preparation method

Abstract

The present invention relates to a high-purity spinosad A and D standard product and a preparation method thereof. After the spinosad mother drug is crystallized by methanol, it is prepared by high-performance liquid chromatography to separate and obtain spinosad A and spinosad D with a purity of ≥90%. The present invention can be applied to the quality detection of spinosad in the current production of enterprises, fills the technical problem of the blank of the current spinosad A and D reference products, greatly reduces the quality control cost of spinosad, effectively solves the problem of uneven quality of spinosad, and is conducive to enhancing the sustainable development of biological pesticide enterprises.

Description

Technical Field

[0001] The invention belongs to the technical field of purification of agricultural antibiotics, and specifically relates to a high-purity spinosad A and D standard product and a preparation method thereof, and in particular to high-performance liquid preparative chromatography conditions for preparing the spinosad A and D standard products. Background Art

[0002] Spinosad, also known as spinosad, is a macrolide non-toxic and highly effective biological insecticide extracted from the fermentation broth of Polyspora spinosa. The parent strain of spinosad, soil actinomycete Polyspora spinosa, was originally isolated from an abandoned brewery in the Caribbean. Researchers at Dow AgroSciences (now Dow AgroSciences) in the United States found that the bacteria 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.

[0003] Spinosad has a novel mode of action that can continuously activate the target insect acetylcholine nicotinic receptors, but its binding site is different from nicotine and imidacloprid. Spinosad can also affect GABA receptors, but the mechanism of action is unclear. It is not known whether there is cross-resistance with other types of insecticides. These compounds can cause rapid mortality of target herbivorous insects such as caterpillars, leaf miners, thrips, and leaf-feeding beetles. Although management departments strongly recommend that they be used when resistance has not yet emerged, the moderate residual activity of the compounds reduces the possibility of resistance and group occurrence. When applied at 12-150g / hm, no phytotoxicity was found.

[0004] According to literature search, the separation and purification preparation process of spinosad is mostly recrystallization or column chromatography, but spinosad is a mixture of spinosad A and spinosad D. Even after multiple separation and purification preparations, spinosad A and spinosad D cannot be separated. In our actual quality inspection process, we need to conduct separate quantitative and qualitative analysis of spinosad A and spinosad D, which requires the separation and preparation of separate spinosad A and spinosad D standards for detection. The present invention, based on the high-purity spinosad original drug, further utilizes the recrystallization process to obtain a high-purity original drug product, and further separates and purifies it through high-pressure preparative liquid phase to obtain spinosad A and spinosad D standards with a purity of ≥90%. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a high-purity spinosad A and D standard product and a preparation method thereof. After the spinosad mother drug is crystallized by methanol, it is prepared by high-performance liquid chromatography to separate spinosad A and spinosad D with a purity of ≥90%. The present invention can be applied to the quality detection of spinosad in the current production of enterprises, fills the technical problem of the blank of the current spinosad A and D reference products, greatly reduces the quality control cost of spinosad, effectively solves the problem of uneven quality of spinosad, and is conducive to enhancing the sustainable development of biological pesticide enterprises.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical scheme: a method for preparing high-purity spinosad A and spinosad D standards, wherein the spinosad mother drug is crystallized by methanol, prepared by high performance liquid chromatography, and then concentrated under reduced pressure and freeze-dried to obtain spinosad A and spinosad D with a purity of ≥90%.

[0007] More specifically, the preparation method comprises the following process steps:

[0008] 1) Crystallization: Weigh the spinosad stock drug, dissolve it, let it stand, filter it, and remove the insoluble matter; then let it stand in a low temperature environment until the spinosad crystals are precipitated, centrifuge and filter it, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

[0009] 2) HPLC preparation: HPLC conditions are as follows

[0010] Mobile phase: methanol, ammonia solution;

[0011] Chromatographic column: filled with reverse phase C18 filler;

[0012] Flow rate: 10-15 ml / min; wavelength 245 nm; sample volume 1 ml-5 ml;

[0013] Collecting according to the chromatographic peaks to obtain spinosad A and spinosad D solutions with a purity of ≥90%;

[0014] 3) Concentrating under reduced pressure and freeze-drying: Concentrating the solution in 2) under reduced pressure and freeze-drying to obtain white powders of spinosad A and spinosad D with a purity of ≥90%.

[0015] In a preferred embodiment of the present invention, in step 2), the mobile phase is prepared to be methanol and 0.5% ammonia solution, and the volume ratio of methanol to ammonia solution is 95:5 to 90:10.

[0016] In a preferred embodiment of the present invention, in step 2), the preparation column packing is internal reverse phase C18, 5 μm, 20*250mm~50*250mm packing;

[0017] In a preferred embodiment of the present invention, in step 2), the flow rate is 10 to 12 ml / min.

[0018] In a preferred embodiment of the present invention, in step 2), the sample loading volume is 1 to 3 ml.

[0019] In a preferred embodiment of the present invention, in step 2), the HPLC conditions are as follows:

[0020] Mobile phase: methanol: 0.5% ammonia solution = 95:5;

[0021] Chromatographic column: internal reverse phase C18, 5μm, 20*250mm packing;

[0022] Flow rate: 10 ml / min; wavelength 245 nm; sample volume 1 ml.

[0023] The present invention also protects the spinosad A and spinosad D standard products prepared by the above preparation method, and the purity of the standard products is ≥90%.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) Spinosad separation and purification preparation processes are mostly recrystallization or column chromatography, but spinosad is a mixture of spinosad A and spinosad D. Even after multiple separation and purification preparations, spinosad A and spinosad D cannot be separated. In the actual quality detection process, it is necessary to quantitatively and qualitatively analyze spinosad A and spinosad D separately, which requires the separation and preparation of separate spinosad A and spinosad D standards for detection. The present invention adopts a high-performance liquid chromatography preparation method, and by studying the high-performance liquid chromatography preparation conditions, spinosad A and spinosad D single products with higher purity are separated and purified, which can achieve effective separation of spinosad A and spinosad D. The methanol and a small amount of ammonia water used in the mobile phase can be removed in the subsequent reduced pressure concentration process, and other impurities will not be introduced, ensuring the purity of the sample.

[0026] 2) The method of the present invention can be applied to the quality detection of spinosad in the current production of enterprises, filling the gap of the current spinosad standard, greatly reducing the quality control cost of spinosad, and effectively solving the problem of simultaneous qualitative and quantitative analysis of spinosad A and D, which is conducive to the sustainable development of biopesticide enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following is a further description with reference to the accompanying drawings.

[0028] Figure 1 This is the liquid chromatogram of the spinosad A standard prepared in Example 1.

[0029] Figure 2This is the liquid chromatogram of the spinosad D standard prepared in Example 1. DETAILED DESCRIPTION

[0030] The following is a further detailed description of the present invention in conjunction with the embodiments

[0031] Embodiment 1:

[0032] 1) Crystallization: Weigh 10g of spinosad original drug into a 100ml beaker, dissolve it with 100ml methanol by ultrasonic, let it stand for 1h, filter and remove insoluble matter. Place it in a refrigerator (4°C) and let it stand for 24h. Spinosad crystals will precipitate, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

[0033] 2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 95:5; chromatographic column: reverse phase C18, 5μm, 20*250mm filler; flow rate: 10ml / min; wavelength 245nm; sample volume 1ml. Collect according to the chromatographic peak to obtain spinosad A and spinosad D solutions;

[0034] 3) Concentrating under reduced pressure and freeze-drying: The solution in 1) was concentrated under reduced pressure and freeze-dried to obtain white powders of spinosad A with a purity of 92% and spinosad D with a purity of 93%.

[0035] Embodiment 2:

[0036] 1) Crystallization: Weigh 10g of spinosad original drug into a 100ml beaker, dissolve it with 100ml methanol by ultrasonic, let it stand for 1h, filter and remove insoluble matter. Place it in a refrigerator (4°C) and let it stand for 24h. Spinosad crystals will precipitate, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

[0037] 2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 92:8; chromatographic column: reverse phase C18, 5μm, 20*250mm filler; flow rate: 12ml / min; wavelength 245nm; sample volume 1ml. Collect according to the chromatographic peak to obtain spinosad A and spinosad D solutions;

[0038] 3) Concentrating under reduced pressure and freeze-drying: The solution in 1) was concentrated under reduced pressure and freeze-dried to obtain white powders of spinosad A with a purity of 91% and spinosad D with a purity of 93%.

[0039] Embodiment 3:

[0040] 1) Crystallization: Weigh 10g of spinosad original drug into a 100ml beaker, dissolve it with 100ml methanol by ultrasonic, let it stand for 1h, filter and remove insoluble matter. Place it in a refrigerator (4°C) and let it stand for 24h. Spinosad crystals will precipitate, centrifuge and filter, remove the solvent, and obtain a mixed sample of spinosad A and spinosad D;

[0041] 2) HPLC preparation: mobile phase: methanol: 0.5% ammonia solution = 90:10; chromatographic column: reverse phase C18, 5μm, 20*250mm filler; flow rate: 10ml / min; wavelength 245nm; sample volume 3ml. Collect according to the chromatographic peak to obtain spinosad A and spinosad D solutions;

[0042] 3) Concentrating under reduced pressure and freeze-drying: The solution in 1) was concentrated under reduced pressure and freeze-dried to obtain white powders of spinosad A with a purity of 90% and spinosad D with a purity of 91%.

[0043] The above contents are further descriptions of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Without departing from the concept of the present invention, several substitutions or modifications made to these described embodiments should be deemed to belong to the protection scope of the present invention.

Claims

1. A method for preparing spinosad A and D standard products, characterized in that: The spinosad mother drug is crystallized by methanol, prepared by high performance liquid chromatography, and then concentrated under reduced pressure and freeze-dried to obtain spinosad A with a purity of ≥90% and spinosad D with a purity of ≥90%; The process steps include: 1) Crystallization: Weigh the original drug of spinosad, dissolve it, let it stand, filter it, and remove the insoluble matter; Then, the mixture was allowed to stand at 4°C until spinosad crystals were precipitated, and then centrifuged and filtered to remove the solvent to obtain a mixed sample of spinosad A and spinosad D. 2) HPLC preparation: HPLC conditions are as follows Mobile phase: methanol: 0.5% ammonia solution = 95:5-90:10; Chromatographic column: internally filled with reverse phase C18, 5μm, 20*250mm~50*250mm packing; Flow rate: 10-15 ml / min; wavelength 245 nm; sample volume 1 ml-5 ml; Collecting according to the chromatographic peaks to obtain spinosad A and spinosad D solutions with a purity of ≥90%; 3) Concentrating under reduced pressure and freeze-drying: The solution obtained in step 2) is concentrated under reduced pressure and freeze-dried to obtain white powders of spinosad A with a purity of ≥90% and spinosad D with a purity of ≥90%.

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