A method for purifying eicosapentaenoic acid ethyl ester by single column semi-continuous chromatography

Abstract

The application provides a method for purifying eicosapentaenoic acid ethyl ester by single-column semi-continuous chromatographic separation, which comprises the following steps: 1) dissolving and filtering crude eicosapentaenoic acid ethyl ester to obtain an eicosapentaenoic acid ethyl ester solution; 2) loading the eicosapentaenoic acid ethyl ester solution into a chromatographic column filled with a chromatographic medium to perform chromatography, and eluting by using a mobile phase; 3) collecting the solutions of target peaks after chromatography and elution in sections, and collecting the component liquids meeting the requirements to obtain purified eicosapentaenoic acid ethyl ester; 4) after the main peak of eicosapentaenoic acid ethyl ester is eluted, loading the same amount of eicosapentaenoic acid ethyl ester solution in step 2) into the same chromatographic column in step 2) to perform chromatography and elution; 5) repeating steps 3) and 4) by keeping the interval time of loading, and obtaining purified eicosapentaenoic acid ethyl ester after continuous purification separation and collection.

Description

Technical Field

[0001] This invention belongs to the field of separation and purification technology, and relates to a method for separating and purifying ethyl eicosate, particularly a method for separating and purifying ethyl eicosate by single-column semi-continuous chromatography. Background Technology

[0002] Eicosapentaenoic acid (EPA) is one of the essential omega-3 polyunsaturated fatty acids for the human body, and it has good therapeutic effects in treating and preventing cardiovascular and cerebrovascular diseases, inflammation, inhibiting tumors, and preventing Alzheimer's disease. EPA is mainly derived from fish oil. In addition to EPA, fish oil also contains various fatty acids such as docosahexaenoic acid, docosapentaenoic acid, octadecanoic acid, linoleic acid, oleic acid, stearic acid, and palmitic acid. Due to their similar molecular structures and physicochemical properties, these non-EPA fatty acids can interfere with the therapeutic effects of EPA-EE. EPA mainly exists in the form of ethyl eicosapentaenoate (EPA-EE). Therefore, the demand for high-purity EPA-EE is increasing daily.

[0003] High-purity eicosapentaenoic acid ethyl ester (EPA-EE) can be used as a raw material in the pharmaceutical industry. For example, Vascepa, a drug developed by the Irish biopharmaceutical company Amarin, uses EPA-EE with a purity greater than 96% for the treatment of hypertriglyceridemia and dyslipidemia.

[0004] Currently, the main publicly available methods for preparing EPA-EE include urea adduct crystallization, metal salt precipitation, and molecular distillation. However, most of these methods only remove pigments, cholesterol, and a certain amount of saturated fatty acids from the raw fish oil, making it difficult to achieve a purity of EPA-EE above 80%, which fails to meet high purity requirements. There are few purification and preparation technologies for high-purity EPA-EE (above 97%), and each has its own limitations. For example, Nissin Chemical's technology for purifying EPA-EE using a silver nitrate diatomaceous earth chromatography column yields less than 30%, and the raw material needs to reach a purity of over 90%. In addition, the product carries the risk of heavy metal contamination. CN102391112A discloses an industrial method for producing ethyl eicosapentaenoic acid (EPA-EE), comprising the following steps: (a) using molecular distillation to increase the EPA-EE purity of raw fish oil with a purity of 68%-72% to 79%-83%, with a yield of 50%-70%; (b) using a chemical method of salt precipitation to separate the fish oil with a EPA-EE purity of 79%-83% obtained in step (a) through salt precipitation, thereby increasing the EPA-EE purity to 88%-92%, with a yield of 50%; (c) using industrial preparative chromatography to purify the fish oil with a EPA-EE purity of 88%-92% obtained in step (b) through preparative chromatography, thereby increasing the EPA-EE purity to over 96%. This method uses YMC-PackODS-AQ reversed-phase packing for EPA-EE purification, but the raw materials need to reach a purity of over 90%, and the sample loading amount is also relatively low.

[0005] CN108640840B discloses a chromatographic method and equipment for purifying ethyl eicosapentaenoic acid (EPA-EE). The chromatographic method involves: pulse injection of a sample containing EPA-EE into zone I; removal of precursor impurities in zone II (eluting EPA-EE until it just penetrates zone II); eluting EPA-EE in zone IV until it just completely enters the last column of zone IV; fine separation in zone VI (eluting EPA-EE until it just penetrates zone VI); fractional collection of EPA-EE and EPA-EE containing a small amount of subsequent impurities in zone VII; and removal of subsequent impurities in zones III and V. The above method is achieved through intelligent control such as valve switching, pump start / stop, and / or flow rate regulation. This invention employs a multi-column continuous chromatography method for EPA purification, but this method requires complex equipment and precise instrument control, resulting in a significant equipment investment.

[0006] In addition, multi-column continuous chromatography, which is popular in existing technologies, is more efficient than traditional purification methods, but it is also very risky and requires very strict control. If a step goes wrong, it will cause huge losses. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for the separation and purification of ethyl eicosapride using a single-column semi-continuous chromatography. This method utilizes a single column, requiring only one step of chromatographic purification to meet the purity requirement of >97%, resulting in high and stable purification yields. The single-column semi-continuous chromatography separation and purification involves one equilibration step followed by multiple sample loading and chromatography steps, saving solvent usage in the mobile phase, improving efficiency, and enabling continuous production. Furthermore, the separation method of this invention is simple and convenient, suitable for large-scale production, and significantly reduces production costs.

[0008] One of the objectives of this invention is to provide a method for the separation and purification of ethyl eicosapride by single-column semi-continuous chromatography. To achieve this objective, the invention employs the following technical solution:

[0009] A method for the separation and purification of ethyl eicosapride by single-column semi-continuous chromatography includes the following steps:

[0010] 1) Dissolve and filter crude eicosapentaenoic acid to obtain an eicosapentaenoic acid solution;

[0011] 2) Load the ethyl eicosapride solution obtained in step 1) onto a single column of a chromatography column packed with chromatography medium for chromatography, and use the mobile phase for elution.

[0012] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0013] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0014] 5) Repeat steps 3) and 4) while maintaining the sample loading interval. After continuous purification, separation and summarization, purified eicosapentaenoic acid ethyl ester is obtained.

[0015] The single-column semi-continuous chromatography method for separating and purifying ethyl eicosapride of the present invention uses a single column and requires only one step of chromatographic purification to meet the requirement of purity >97%, with high and stable purification yield. The single-column semi-continuous chromatography separation and purification involves one equilibration step followed by multiple sample loading and chromatography steps, which saves the amount of solvent used in the mobile phase, improves efficiency, and allows for continuous production. At the same time, the separation method of the present invention is simple and convenient, can be used for large-scale production, and greatly reduces production costs.

[0016] In step 2), the chromatography medium is an equivalent medium of C8, C12, C18, or C20, preferably nano-UniSilC18. Specifically, UniSilC18 microspheres have a particle size of 20 μm and a pore size of [missing information]. The polymer microspheres have strictly controlled particle size and pore structure. They are monodisperse microspheres with a porous structure, which makes them highly targeted when used as chromatographic packing materials.

[0017] Preferably, the particle size of the chromatography medium is 8-30 μm, for example, the particle size of the chromatography medium is 8 μm, 10 μm, 15 μm, 20 μm, 30 μm or other non-uniform particle media; preferably 20 μm.

[0018] In step 2), the mobile phase is a mixture of pure water and an organic solvent.

[0019] Preferably, the volume ratio of the pure water to the organic solvent is (1-0.8):(9-9.2), for example, 0.8:9.2, 0.85:9.15, 0.9:9.1, 0.95:9.05 or 1:9, etc.

[0020] Preferably, the organic solvent is one of methanol, ethanol, or acetonitrile, with methanol being the most preferred.

[0021] Preferably, in step 2), the volume of the chromatography medium packed in the chromatography column is considered as one column volume, and the amount of the mobile phase used is 5-8 column volumes, for example, 5 column volumes, 6 column volumes, 7 column volumes or 8 column volumes.

[0022] In step 2), the specific elution process is as follows: elution is performed on the ethyl eicosapate solution using a mobile phase with a volume ratio of pure water to organic solvent of (1-0.8):(9-9.2), and the elution time is 50-70 min, for example, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, or 70 min, etc.; the flow rate of the mobile phase is 70-80 mL / min, for example, 70 mL / min, 71 mL / min, 72 mL / min, 73 mL / min, 74 mL / min, 75 mL / min, 76 mL / min, 77 mL / min, 78 mL / min, 79 mL / min, or 80 mL / min, etc.

[0023] In step 1), the crude ethyl eicosapentaenoic acid has an EPA-EE purity of 70-75%, for example, 70%, 71%, 72%, 73%, 74%, or 75%.

[0024] Preferably, the concentration of EPA-EE in the ethyl eicosapentaenoic acid solution is 8-25 mg / mL, for example, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, or 25 mg / mL.

[0025] In step 1), the dissolution is performed using an organic solvent.

[0026] Preferably, the organic solvent is methanol.

[0027] Preferably, the filtration is performed using a filter membrane with a pore size of 0.3-0.5 μm, such as a filter membrane with a pore size of 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm or 0.5 μm.

[0028] In step 5), the interval for loading the sample is 40-60 minutes, for example, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

[0029] Step 2) Before loading the sample, the chromatography column also needs to be pre-treated.

[0030] Preferably, the specific process of the pre-column treatment is as follows: the chromatography column is purified by methanol, and then the purified chromatography column is equilibrated by a flow phase with a volume ratio of pure water to methanol of (1-0.8):(9-9.2).

[0031] As a preferred embodiment of the present invention, the method for separating and purifying ethyl eicosapogenate by single-column semi-continuous chromatography includes the following steps:

[0032] 1) Dissolve crude eicosapentaenoic acid ethyl ester with a purity of 70-75% in methanol, filter it through a filter membrane with a pore size of 0.3-0.5μm, and obtain an eicosapentaenoic acid ethyl ester solution with a concentration of 8-25mg / mL of EPA-EE.

[0033] 2) The chromatography column is purified with methanol, and then the purified chromatography column is equilibrated with a mobile phase of pure water to methanol at a volume ratio of (1-0.8):(9-9.2). The ethyl eicosate solution obtained in step 1) is loaded onto a single chromatography column packed with UniSil C18 particles with a particle size of 8-30 μm for chromatography. Elution is performed with the mobile phase for 50-70 min. The mobile phase is a mixture of pure water and organic solvent at a volume ratio of (1-0.8):(9-9.2), the flow rate of the mobile phase is 70-80 mL / min, and the amount of the mobile phase used is 5-8 column volumes.

[0034] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0035] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0036] 5) Repeat steps 3) and 4) with a loading interval of 40-60 min. After continuous purification, separation and summarization, purified eicosapentaenoic acid ethyl ester is obtained.

[0037] A second objective of this invention is to provide an ethyl eicosate prepared by the method described in one objective.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] The single-column semi-continuous chromatographic separation and purification method for ethyl eicosapride (EPA-EE) of this invention utilizes a single column, requiring only one step of chromatographic purification to meet the purity requirement of >97%, with high and stable purification yield. The single-column semi-continuous chromatographic separation and purification involves one equilibration step followed by multiple sample loading and chromatography steps, saving solvent usage in the mobile phase, improving efficiency, and enabling continuous production. Furthermore, the separation method of this invention is simple and convenient, suitable for large-scale production, and significantly reduces production costs. Specifically, the purity of EPA-EE in the eluent obtained by elution with 5-8 column volumes of mobile phase is 98.3-99.2%, with a yield of 93.6-95.7%. Attached Figure Description

[0040] Figure 1 The spectrum is for the single-column semi-continuous chromatography separation and purification of ethyl eicosapride according to the present invention;

[0041] Figure 2 This is a gas chromatogram of EPA-EE before purification in Example 1 of the present invention;

[0042] Figure 3This is a gas chromatogram of purified EPA-EE from Example 1 of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0044] Unless otherwise specified, all raw materials used in this invention are commercially available or can be prepared using conventional methods in the art.

[0045] Example 1

[0046] The method for separating and purifying ethyl eicosapride by single-column semi-continuous chromatography in this embodiment includes the following steps:

[0047] 1) Dissolve crude ethyl eicosapentaenoate (EPA-EE) with a purity of 75% in methanol, filter it through a 4.5 μm pore size filter membrane, and obtain an EPA-EE concentration of 16 mg / mL in ethyl eicosapentaenoate solution.

[0048] 2) The chromatography column was purified with methanol, and then the purified chromatography column was equilibrated with a mobile phase of pure water and methanol at a volume ratio of 1:9. The ethyl eicosate solution obtained in step 1) was loaded onto a single chromatography column packed with UniSil C18 particles with a particle size of 20 μm for chromatography. Elution was performed for 45 min with a mobile phase consisting of a mixture of pure water and organic solvent at a volume ratio of 1:9, a flow rate of 1 mL / min, and a volume of 7 column volumes.

[0049] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0050] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0051] 5) Repeat steps 3) and 4) with a 30-minute interval between sample loading. After continuous purification, separation and summarization, purified ethyl eicosate is obtained.

[0052] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 99.2%, and the yield was 95.6%.

[0053] Figure 1 This is a schematic diagram of the single-column semi-continuous chromatography separation and purification of ethyl eicosapride according to the present invention.

[0054] Figure 2The image shows a gas chromatogram of EPA-EE before purification in Example 1, revealing certain impurities. Specifically, the characteristic peak of EPA-EE is at 21.4 min, while impurity peaks are observed at 17.8 min, 19.9 min, and 26.7 min.

[0055] Figure 3 The image shows the gas chromatogram of the purified EPA-EE from Example 1. As can be seen, there are very few impurities and very small impurity peaks.

[0056] Example 2

[0057] The method for separating and purifying ethyl eicosapride by single-column semi-continuous chromatography in this embodiment includes the following steps:

[0058] 1) Dissolve crude ethyl eicosapentaenoate (EPA-EE) with a purity of 75% in methanol, filter it through a 4.5 μm pore size filter membrane, and obtain an EPA-EE concentration of 10 mg / mL in ethyl eicosapentaenoate solution.

[0059] 2) The chromatography column was purified with methanol, and then equilibrated with a mobile phase of pure water to methanol at a volume ratio of 0.9:9.1. The ethyl eicosapentaenoic acid solution obtained in step 1) was loaded onto a single column packed with UniSil C18 particles with a particle size of 20 μm for chromatography. Elution was performed with the mobile phase for 45 min. The mobile phase was a mixture of pure water and organic solvent at a volume ratio of 0.9:9.1, the flow rate of the mobile phase was 1 mL / min, and the amount of mobile phase used was 6 column volumes.

[0060] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0061] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0062] 5) Repeat steps 3) and 4) with a 30-minute interval between sample loading. After continuous purification, separation and summarization, purified ethyl eicosate is obtained.

[0063] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 98.8%, and the yield was 94.8%.

[0064] Example 3

[0065] The method for separating and purifying ethyl eicosapride by single-column semi-continuous chromatography in this embodiment includes the following steps:

[0066] 1) Dissolve crude ethyl eicosapentaenoate (EPA-EE) with a purity of 75% in methanol, filter it through a 4.5 μm pore size filter membrane, and obtain an EPA-EE concentration of 20 mg / mL in ethyl eicosapentaenoate solution.

[0067] 2) The chromatography column was purified with methanol, and then equilibrated with a mobile phase of pure water to methanol at a volume ratio of 0.8:9.2. The ethyl eicosate solution obtained in step 1) was loaded onto a single column packed with UniSil C18 particles with a particle size of 20 μm for chromatography. Elution was performed for 45 min with a mobile phase consisting of a mixture of pure water and organic solvent at a volume ratio of 0.8:9.2, a flow rate of 1 mL / min, and a volume of 5 column volumes.

[0068] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0069] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0070] 5) Repeat steps 3) and 4) with a 30-minute interval between sample loading. After continuous purification, separation and summarization, purified ethyl eicosate is obtained.

[0071] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 98.6%, and the yield was 94.2%.

[0072] Example 4

[0073] The method for separating and purifying ethyl eicosapride by single-column semi-continuous chromatography in this embodiment includes the following steps:

[0074] 1) Dissolve crude ethyl eicosapentaenoate (EPA-EE) with a purity of 75% in methanol, filter it through a 4.5 μm pore size filter membrane, and obtain an EPA-EE concentration of 20 mg / mL in ethyl eicosapentaenoate solution.

[0075] 2) The chromatography column was purified with methanol, and then the purified chromatography column was equilibrated with a mobile phase of pure water and methanol in a volume ratio of 1:9. The ethyl eicosate solution obtained in step 1) was loaded onto a single chromatography column packed with UniSil C18 particles with a particle size of 20 μm for chromatography. Elution was performed for 45 min with a mobile phase consisting of a mixture of pure water and organic solvent in a volume ratio of 1:9, a flow rate of 1 mL / min, and a volume of 7 column volumes.

[0076] 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified ethyl eicosapride.

[0077] 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution.

[0078] 5) Repeat steps 3) and 4) with a 30-minute interval between sample loading. After continuous purification, separation and summarization, purified ethyl eicosate is obtained.

[0079] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 99.1%, and the yield was 95.7%.

[0080] Example 5

[0081] The difference between this embodiment and Embodiment 1 is that in step 2), the chromatography medium is C8 with a particle size of 20 μm, while the rest are the same as in Embodiment 1.

[0082] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 98.3%, and the yield was 93.6%.

[0083] Example 6

[0084] The difference between this embodiment and Embodiment 1 is that in step 2), the mobile phase is a mixture of pure water and methanol with a volume ratio of 8.5:91.5, while the rest is the same as in Embodiment 1.

[0085] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 98.6%, and the yield was 93.4%.

[0086] Example 7

[0087] The difference between this embodiment and Embodiment 1 is that in step 2), the chromatography medium is 20μm UniSil PS, while the rest are the same as in Embodiment 1.

[0088] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 96.8%, and the yield was 90.5%.

[0089] Example 8

[0090] The difference between this embodiment and Embodiment 1 is that in step 2), the mobile phase is a mixture of water and isopropanol with a volume ratio of 1:9, while the rest is the same as in Embodiment 1.

[0091] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 97.2%, and the yield was 88.6%.

[0092] Comparative Example 1

[0093] The difference between this comparative example and Example 1 is that step 2 is a multi-column continuous chromatography method. Specifically, the sample containing EPA-EE is pulse-injected in zone I; precursor impurities are removed in zone II (EPA-EE is eluted until it just penetrates zone II); EPA-EE is eluted in zone IV until it just completely enters the last column of zone IV; fine separation is performed in zone VI (EPA-EE is eluted until it just penetrates zone VI); EPA-EE and EPA-EE containing a small amount of subsequent impurities are collected in fractions in zone VII; and subsequent impurities are removed in zones III and V. The above method is achieved through intelligent control such as valve switching, pump start / stop, and / or flow rate regulation. Zones I, II, III, IV, V, and VI all require the same chromatography columns packed with chromatographic media to be used simultaneously, i.e., six chromatography columns are needed.

[0094] The solutions containing the target peak were collected in segments, and the components that met the requirements were summarized. Gas chromatography analysis showed that the purity of EPA-EE in the eluent was 98.9%, and the yield was 95.2%.

[0095] Comparative Example 2

[0096] The difference between this comparative example and Example 1 is that it does not include steps 4) and 5), the purity is 98.8%, and the yield is 94.6%.

[0097] Using this comparative method, the average mobile phase required per needle during elution is 10 CV, and the required time is 1 hour.

[0098] Comparative Example 3

[0099] This comparative example uses a conventional EPA-EE purification method, which directly employs molecular distillation. Specifically, the sample is directly subjected to molecular distillation, and the fraction collected at 250°C is obtained. The purity is measured to be 80%, and the yield is 85%.

[0100] This invention illustrates the detailed process equipment and process flow through the above embodiments. However, this invention is not limited to the detailed process equipment and process flow described above, meaning that this invention does not necessarily depend on the detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, all fall within the protection scope and disclosure scope of this invention.

[0101] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0102] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0103] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for the separation and purification of ethyl eicosapride by single-column semi-continuous chromatography, characterized in that, The method includes the following steps: 1) Dissolve and filter crude eicosapentaenoic acid to obtain an eicosapentaenoic acid solution; 2) The ethyl eicosate solution obtained in step 1) is loaded onto a single column of chromatography packed with chromatography medium for chromatography, and elution is performed using a mobile phase; the chromatography medium is Nano-Micro UniSil C18; the mobile phase is a mixture of pure water and methanol in a volume ratio of (1-0.9):(9-9.1); the volume of chromatography medium packed in the column is considered as one column volume, the amount of mobile phase used is 5-8 column volumes, the elution time is 50-70 min, and the flow rate of the mobile phase is 70-80 mL / min; 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified eicosapentaenoic acid ethyl ester; 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution. 5) Repeat steps 3) and 4) while maintaining the sample loading interval. After continuous purification, separation and summarization, purified ethyl eicosate is obtained. In step 1), the dissolution is performed using methanol, and the filtration is performed using a filter membrane with a pore size of 0.3-0.5 μm. Step 2) Before loading the sample, the chromatography column is also pre-column treated. The specific process of the pre-column treatment is as follows: the chromatography column is purified with methanol, and then the purified chromatography column is equilibrated with a flow ratio of pure water to methanol of (1-0.8):(9-9.2).

2. The method according to claim 1, characterized in that, The particle size of the chromatography medium is 8-30 μm.

3. The method according to claim 2, characterized in that, The particle size of the chromatography medium is 20 μm.

4. The method according to claim 1, characterized in that, In step 1), the crude ethyl eicosapentaenoic acid has an EPA-EE purity of 70-75%.

5. The method according to claim 1, characterized in that, In step 1), the concentration of EPA-EE in the ethyl eicosapentaenoic acid solution is 8-25 mg / mL.

6. The method according to claim 1, characterized in that, In step 5), the interval between sample loading is 40-60 minutes.

7. The method according to claim 1, characterized in that, The method includes the following steps: 1) Dissolve crude eicosapentaenoic acid ethyl ester with a purity of 70-75% in methanol, filter it through a filter membrane with a pore size of 0.3-0.5μm, and obtain an eicosapentaenoic acid ethyl ester solution with a concentration of 8-25 mg / mL of EPA-EE. 2) The chromatography column is purified with methanol, and then the purified chromatography column is equilibrated with a mobile phase of pure water to methanol at a volume ratio of (1-0.8):(9-9.2). The ethyl eicosapentaenoic acid solution obtained in step 1) is loaded onto a single chromatography column packed with UniSil C18 particles with a particle size of 8-30 μm for chromatography. Elution is performed with a mobile phase of pure water and methanol at a volume ratio of (1-0.9):(9-9.1) for 50-70 min, the flow rate of the mobile phase is 70-80 mL / min, and the amount of mobile phase used is 5-8 column volumes. 3) Collect the solution of the target peak after chromatography and elution in step 2) in segments, and summarize the component solutions that meet the requirements to obtain purified eicosapentaenoic acid ethyl ester. 4) After the main peak of ethyl eicosapentaenoate has eluted, load the same amount of ethyl eicosapentaenoate solution as in step 2) onto the same single column of the chromatography column used in step 2) for chromatography and elution. 5) Repeat steps 3) and 4) with a loading interval of 40-60 min. After continuous purification, separation and summarization, purified eicosapentaenoic acid ethyl ester is obtained.

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