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CN-116655469-B - Method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semicontinuous chromatography

CN116655469BCN 116655469 BCN116655469 BCN 116655469BCN-116655469-B

Abstract

The invention provides a method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semicontinuous chromatography, which comprises the following steps of 1) dissolving and filtering a eicosapentaenoic acid ethyl ester crude product to obtain an eicosapentaenoic acid ethyl ester solution, 2) loading the eicosapentaenoic acid ethyl ester solution into a single chromatographic column filled with a chromatographic medium for chromatography, eluting by adopting a mobile phase, 3) collecting a solution of a target peak value after chromatography and elution in a sectionalized manner, summarizing component liquid meeting requirements to obtain purified eicosapentaenoic acid ethyl ester, 4) loading the eicosapentaenoic acid ethyl ester solution with the same quantity as the eicosapentaenoic acid ethyl ester solution in the step 2) into the same single chromatographic column in the step 2) again after the main peak of the eicosapentaenoic acid ethyl ester is discharged, 5) repeating the steps 3) and 4) at intervals of loading, and obtaining the purified eicosapentaenoic acid ethyl ester after continuous purification and separation and summarization.

Inventors

  • WANG XI
  • TAO YE
  • YU MINGJUN
  • ZHANG BANGWEI
  • CHEN QIANGQIANG

Assignees

  • 苏州纳微科技股份有限公司

Dates

Publication Date
20260512
Application Date
20220218

Claims (7)

  1. 1. A method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semicontinuous chromatography, which is characterized by comprising the following steps: 1) Dissolving and filtering the coarse eicosapentaenoic acid ethyl ester to obtain an eicosapentaenoic acid ethyl ester solution; 2) Loading the eicosapentaenoic acid ethyl ester solution obtained in the step 1) into a single chromatographic column filled with a chromatographic medium for chromatography, eluting by adopting a mobile phase, wherein the chromatographic medium is nano-micro UniSilC, the mobile phase is a mixture of pure water and methanol with the volume ratio of (1-0.9) to (9-9.1), the volume of the chromatographic medium filled in the chromatographic column is 1 column volume, the dosage of the mobile phase is 5-8 column volumes, the eluting time is 50-70min, and the flow rate of the mobile phase is 70-80mL/min; 3) Collecting the solution of the target peak value after chromatography and elution in step 2) in a sectional way, and summarizing the component liquid meeting the requirements to obtain purified eicosapentaenoic acid ethyl ester; 4) After the main peak of the eicosapentaenoic acid ethyl ester is out, loading the eicosapentaenoic acid ethyl ester solution with the same amount as that in the step 2) into the same single chromatographic column in the step 2) again for chromatography and elution; 5) Repeating the steps 3) and 4) at intervals of sample loading, and obtaining purified eicosapentaenoic acid ethyl ester after continuous purification, separation and summarization; In the step 1), methanol is adopted for dissolution, and a filter membrane with the pore diameter of 0.3-0.5 mu m is adopted for filtration; The step 2) is characterized by further comprising the step of carrying out column pretreatment on the chromatographic column before loading, wherein the specific process of the column pretreatment is to adopt methanol to remove impurities from the chromatographic column, and then adopt the flow of pure water and methanol with the volume ratio of (1-0.8): 9-9.2 to balance the purified chromatographic column.
  2. 2. The method of claim 1, wherein the chromatographic medium has a particle size of 8-30 μm.
  3. 3. The method of claim 2, wherein the chromatographic medium has a particle size of 20 μm.
  4. 4. The process according to claim 1, wherein in step 1) the crude eicosapentaenoic acid ethyl ester has an EPA-EE purity of from 70 to 75%.
  5. 5. The method according to claim 1, wherein in step 1), the concentration of EPA-EE in the eicosapentaenoic acid ethyl ester solution is 8-25 mg/mL.
  6. 6. The method according to claim 1, wherein in step 5), the loading interval is 40-60min.
  7. 7. The method according to claim 1, characterized in that it comprises the steps of: 1) Dissolving the eicosapentaenoic acid ethyl ester crude product with EPA-EE purity of 70-75% with methanol, and filtering with a filter membrane with aperture of 0.3-0.5 μm to obtain eicosapentaenoic acid ethyl ester solution with EPA-EE concentration of 8-25 mg/mL; 2) Removing impurities from a chromatographic column by adopting methanol, balancing the purified water and the methanol by adopting a flow ratio of (1-0.8) (9-9.2) to the chromatographic column after the impurities are removed, loading the eicosapentaenoic acid ethyl ester solution obtained in the step 1) into a single chromatographic column filled with UniSilC with the particle size of 8-30 mu m for chromatography, and eluting by adopting a mobile phase for 50-70min, wherein the mobile phase is a mixture of the purified water and the methanol with the volume ratio of (1-0.9) (9-9.1), the flow rate of the mobile phase is 70-80mL/min, and the use amount of the mobile phase is 5-8 column volumes; 3) Collecting the solution of the target peak value after chromatography and elution in step 2) in a sectional way, and summarizing the component liquid meeting the requirements to obtain purified eicosapentaenoic acid ethyl ester; 4) After the main peak of the eicosapentaenoic acid ethyl ester is out, loading the eicosapentaenoic acid ethyl ester solution with the same amount as that in the step 2) into the same single chromatographic column in the step 2) again for chromatography and elution; 5) And (3) repeating the steps (3) and (4) with the sampling interval time kept between 40 and 60 minutes), and obtaining the purified eicosapentaenoic acid ethyl ester after continuous purification, separation and summarization.

Description

Method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semicontinuous chromatography Technical Field The invention belongs to the technical field of separation and purification, relates to a separation and purification method of eicosapentaenoic acid ethyl ester, and particularly relates to a method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semi-continuous chromatography. Background Eicosapentaenoic acid (EPA) is one of several omega-3 polyunsaturated fatty acids essential to human body, and has good curative effects in treating and preventing cardiovascular and cerebrovascular diseases, inflammation, inhibiting tumor, preventing senile dementia, etc. EPA mainly originates from fish oil, and besides EPA, the fish oil also contains various fatty acids such as docosahexaenoic acid, docosapentaenoic acid, stearidonic acid, linoleic acid, oleic acid, stearic acid, palmitic acid and the like, and due to similar molecular structures and physical and chemical properties, the non-EPA fatty acids interfere with the treatment effect of EPA-EE, and EPA mainly exists in the form of eicosapentaenoic acid ethyl ester (EPA-EE). Thus, the demand for high purity EPA-EE is increasing. The high-purity eicosapentaenoic acid ethyl ester (EPA-EE) can be used as a raw material medicine and applied to the pharmaceutical field. For example, drug Vascepa developed by Ireland Bio-pharmaceutical company Amarin is an eicosapentaenoic acid ethyl ester (EPA-EE) having a purity of greater than 96% used for the treatment of hypertriglyceridemia and dyslipidemia. The main methods for preparing EPA-EE disclosed at present are urea adduct crystallization method, metal salt precipitation method and molecular distillation method, but most of the methods are remained in the simple method for removing pigment, cholesterol and a certain amount of saturated fatty acid in raw material fish oil, so that the purity of the prepared EPA-EE is difficult to reach more than 80%, and the requirement of high purity cannot be met. There are few purification techniques for preparing EPA-EE (97% or more) of high purity, and there are drawbacks. For example, the technology of purifying EPA-EE by using silver nitrate diatomite chromatographic column is adopted by Niqing company, the yield is less than 30%, the purity of raw material is required to be more than 90%, in addition, the heavy metal pollution risk exists in the product, CN102391112A discloses a method for industrially producing eicosapentaenoic acid ethyl ester, which comprises the following steps of (a) adopting a molecular distillation method to improve the EPA-EE purity in raw material fish oil with the EPA-EE purity of 68% -72% to 79% -83%, the yield is 50% -70%, and (b) adopting a chemical method of salt precipitation to separate fish oil with the EPA-EE purity of 79% -83% obtained in the step (a) by adopting a salt precipitation method, so that the EPA-EE purity is improved to 88% -92%, and the yield is 50%, and (c) adopting an industrial preparation chromatographic method to purify fish oil with the EPA-EE purity of 88% -92% obtained in the step (b) by adopting the preparation chromatographic method, so that the EPA-EE purity is improved to be more than 96%. The method adopts YMC-PackODS-AQ reverse filler to refine EPA-EE, but the raw material of the EPA-EE reverse filler needs to reach more than 90% purity, and the sample loading amount is low. CN108640840B discloses a chromatographic method and equipment for purifying eicosapentaenoic acid ethyl ester, the chromatographic method is that a sample containing EPA-EE is pulsed and injected in a zone I, impurities are removed in a zone II (EPA-EE is eluted to just penetrate the zone II), EPA-EE is eluted to just enter a final column in a zone IV, fine separation is carried out in a zone VI (EPA-EE is eluted to just penetrate the zone VI), EPA-EE and EPA-EE containing a small amount of post impurities are collected by sections eluted in a zone VII, and the post impurities are removed in a zone III and a zone V. The method is realized through intelligent control such as valve switching, start-stop of a pump or/and flow speed regulation and control. The invention adopts a multi-column continuous chromatography method for EPA purification, but the method requires complex equipment and precise instrument control, and has large equipment investment. In addition, the multi-column continuous chromatography popular in the prior art is high in efficiency compared with the traditional purification method, but the risk is also high, the control requirement is very strict, and once the steps are out of order, huge losses are caused. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a method for separating and purifying eicosapentaenoic acid ethyl ester by single-column semicontinuous chromatography, which adopts a chromatographic column single colum