Method for simultaneously preparing multiple eicosapentaenoic acid ethyl ester trans-isomers

Abstract

The invention relates to the technical field of eicosapentaenoic acid ethyl ester impurity reference substances, in particular to a method for simultaneously preparing multiple eicosapentaenoic acid ethyl ester trans-isomers. Comprising the following steps: (1) reacting EPAee with the purity of more than 95% at 230-300 DEG C under 4.0-5.5 MPa for 3-5 hours to obtain a crude product; (2) separating the obtained crude product by using reversed-phase preparative liquid chromatography, wherein the adopted mobile phase is a silver nitrate aqueous solution-acetonitrile mixed solution; and (3) collecting fractions corresponding to chromatographic peaks of various trans-isomers in the chromatogram, respectively extracting by adopting an organic solvent, and carrying out rotary evaporation on an organic phase to remove the solvent, so as to obtain various EPAee trans-isomers. According to the method provided by the invention, seven EPAee trans-isomers can be prepared, the purities of the seven EPAee trans-isomers are all greater than 90%, the separation and preparation of the high-purity EPAee impurity reference substance are realized, and the purity requirements of drug research and development laws and regulations on the impurity reference substance are met.

Description

Technical Field

[0001] The invention relates to the technical field of ethyl eicosapentaenoic acid impurity reference substances, in particular to a method for simultaneously preparing a plurality of ethyl eicosapentaenoic acid trans isomers. Background Art

[0002] Omega-3 polyunsaturated fatty acids have become a hot topic in the field of natural product separation and purification and medicine due to their unique physiological effects. Eicosapent ethyl (EPAee) is an ω-3 polyunsaturated fatty acid with the structural formula The chemical formula is C 22 H 34 O2, with a molecular weight of 330.51, is a twenty-carbon fatty acid containing five double bonds. It can replace arachidonic acid on the cell phospholipid membrane, thereby changing the physical properties of the cell membrane and stabilizing the cell membrane. Studies have shown that it reduces the incidence of sudden cardiac death by 31% and cardiac arrest by 48%. In addition, it can also inhibit the progression of atherosclerosis by reducing inflammatory mediators, resisting oxidative stress, improving endothelial dysfunction, inhibiting cholesterol crystal formation, and stabilizing plaques.

[0003] EPAee is a high-purity (EPA≥96%) EPA single-molecule prescription drug. It is deesterified in the intestine, and the active product EPA is absorbed in the small intestine and enters the systemic circulation through the thoracic duct lymphatic system, reducing the synthesis or secretion of very low-density lipoprotein triglycerides in the liver and improving the clearance of TG in very low-density lipoprotein particles.

[0004] (Eicosapentaenoic acid ethyl ester capsules, high-purity EPA ethyl ester preparation) is a patented product of Irish pharmaceutical company Amarin. It is the first and only single-molecule prescription drug approved by the FDA containing the highly purified active ingredient eicosapentaenoic acid ethyl ester. It was first launched in the United States in 2013 to reduce triglyceride levels in adult patients with severe (≥500 mg / dL) hypertriglyceridemia. In December 2019, the FDA approved its new indication, which It is the first and only FDA-approved drug for the treatment of high-risk patients with persistent cardiovascular risk after taking statins. Currently, icosapent ethyl is recommended by many guidelines in developed countries in Europe and the United States as a primary and secondary prevention drug for high-risk cardiovascular disease, becoming a breakthrough new treatment for cardiovascular disease and leading cardiovascular risk management into a new era.

[0005] However, EPAee is prone to isomerization and forms a trans configuration, which may theoretically produce 30 isomers. Although the trans structure is more stable, trans fatty acids can increase blood cholesterol levels, increase the risk of cardiovascular disease, and are harmful to human health. Therefore, the quality of EPAee's trans isomers must be studied and controlled, and it is necessary to obtain purified trans isomer reference substances that meet the requirements.

[0006] In addition, in recent years, drug regulatory authorities in various countries have gradually strengthened the supervision of drug applications, especially generic drug applications, and have put forward higher requirements on the quality of generic drugs. Pharmaceutical companies are required to conduct in-depth and detailed research on various impurities that affect the safety and effectiveness of drugs. Only when a certain amount of impurity reference substances are obtained during the research process can various pharmaceutical research be carried out. Therefore, the preparation of impurity reference substances is a very important task.

[0007] Currently, most of the literature reports are about the separation and purification of EPAee. The main technologies include urea inclusion method, low-temperature crystallization method, molecular distillation method, supercritical extraction method, adsorption separation method and composite method.

[0008] A Chinese patent document with application number 201210305420.1 discloses a method for purifying EPAee by two-stage vacuum distillation. The first-stage distillation tower is used to remove light components with a boiling point lower than that of EPAee, and the removed light components enter the second-stage distillation tower to separate EPAee from DHAee to obtain EPAee with a purity greater than 97%. This method mainly uses the difference in boiling points of different substances to extract by molecular distillation, but it is difficult to separate the trans-isomer with very similar physical and chemical properties.

[0009] Drug Analysis Impurities Chin J Pharm Anal 2023, 43 (10) reported the use of LC-QTOF technology to identify EPAee-related substances. The reverse phase C18 was used for separation and only one isomer was identified. The method was reproduced and found that the trans isomer of EPAee could not be effectively separated. Multiple isomers co-eluted in the chromatographic peaks, and the impurity reference substance with purity that met the requirements could not be separated.

[0010] In addition, the content determination methods of polyenoic acid ethyl esters included in the pharmacopoeias of various countries are all gas phase methods using quartz capillary columns with polyethylene glycol as the stationary liquid. Although this method can detect five isomers of the sample, it cannot be scaled up for separation and preparation because it is a gas phase method.

[0011] In view of this, the present invention is proposed. Summary of the invention

[0012] The object of the present invention is to provide a method for simultaneously preparing a plurality of trans isomers of ethyl eicosapentaenoic acid, wherein the method can prepare a plurality of trans isomers of ethyl eicosapentaenoic acid at one time, and the purity of each trans isomer is greater than 90%, meeting the purity requirements of drug research and development regulations for impurity reference substances.

[0013] The present invention provides a method for simultaneously preparing a plurality of trans isomers of ethyl eicosapentaenoate, comprising the following steps:

[0014] (1) reacting ethyl eicosapentaenoic acid with a purity greater than 95% at a temperature of 230-300° C. and a pressure of 4.0-5.5 MPa for 3-5 hours to obtain a crude product containing the trans isomer of ethyl eicosapentaenoic acid;

[0015] (2) separating the crude product obtained in step (1) by reverse phase preparative liquid chromatography, using a silver nitrate aqueous solution-acetonitrile mixed solution as the mobile phase;

[0016] (3) collecting the fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram, respectively extracting them with organic solvents, and removing the solvent by rotary evaporation of the organic phase to obtain various trans isomers of ethyl eicosapentaenoate.

[0017] Preferably, the temperature in step (1) is 250-280° C., the pressure is 4.5-5.0 MPa, and the reaction time is 3.5-4.5 hours.

[0018] Preferably, the total content of trans isomers of ethyl eicosapentaenoate in the crude product obtained in step (1) is greater than 60%.

[0019] Preferably, the reverse phase chromatographic column used for reverse phase preparative liquid chromatography separation in step (2) is one of a C8 chromatographic column, a C18 chromatographic column, a pentafluorophenyl chromatographic column, a cyano chromatographic column, and an AQ chromatographic column, more preferably a C8 chromatographic column or a C18 chromatographic column.

[0020] Preferably, the reverse phase chromatography column in step (2) is a semi-preparative column or a preparative column with an inner diameter of not less than 0.9 cm, more preferably a semi-preparative column or a preparative column with an inner diameter of 2-3 cm.

[0021] Preferably, the mass fraction of silver nitrate in the silver nitrate aqueous solution used as the mobile phase in step (2) is 1-10%, more preferably 2-8%.

[0022] Preferably, in the step (2), the volume ratio of the silver nitrate aqueous solution to the acetonitrile in the silver nitrate aqueous solution-acetonitrile mixed solution is (35-37):(63-65).

[0023] Preferably, when performing reverse phase preparative liquid chromatography separation in step (2), the flow rate is 18 mL / min, the equilibrium time is 8 minutes, and the running time is 60 minutes.

[0024] Preferably, dichloromethane is used for extraction in step (3).

[0025] Preferably, in step (3), 7 trans isomers of ethyl eicosapentaenoate are obtained, namely, impurity D, impurity E, impurity F, impurity G, RRT1.017 impurity, RRT1.026 impurity, and RRT1.042 impurity, wherein:

[0026] The structural formula of impurity D is The structural formula of impurity E is The structural formula of impurity F is The structural formula of impurity G is The structural formula of RRT1.017 impurity is The structural formula of RRT1.026 impurity is The structural formula of RRT1.042 impurity is

[0027] In summary, compared with the prior art, the present invention has the following advantages:

[0028] The technical solution provided by the present invention uses eicosapentaenoic acid ethyl ester with a purity greater than 95% as a raw material, undergoes a high-temperature and high-pressure reaction, and then separates seven eicosapentaenoic acid ethyl ester trans isomers at one time through reverse phase preparative liquid chromatography, and the purity of the prepared seven eicosapentaenoic acid ethyl ester trans isomers is greater than 90%, thereby realizing the separation and preparation of high-purity eicosapentaenoic acid ethyl ester impurity reference substances and meeting the purity requirements of drug research and development regulations for impurity reference substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 is a high performance liquid chromatogram of the crude product in Example 1 of the present invention;

[0031] Figure 2 is a TIC graph of GC-MS of impurity D separated in Example 1 of the present invention;

[0032] Figure 3 is the H NMR spectrum of impurity D separated in Example 1 of the present invention;

[0033] Figure 4 is the NMR carbon spectrum of impurity D separated in Example 1 of the present invention;

[0034] Figure 5 This is a TIC chart of GC-MS of impurity E separated in Example 1 of the present invention;

[0035] Figure 6 is the H NMR spectrum of impurity E separated in Example 1 of the present invention;

[0036] Figure 7 is the NMR carbon spectrum of impurity E separated in Example 1 of the present invention;

[0037] Figure 8 This is a TIC chart of GC-MS of impurity F separated in Example 1 of the present invention;

[0038] Fig. 9 is the H NMR spectrum of impurity F separated in Example 1 of the present invention;

[0039] Fig.10 is the NMR carbon spectrum of impurity F separated in Example 1 of the present invention;

[0040] Fig.11 This is a TIC chart of GC-MS of impurity G separated in Example 1 of the present invention;

[0041] Fig.12 is the H NMR spectrum of impurity G separated in Example 1 of the present invention;

[0042] Fig.13 is the NMR carbon spectrum of impurity G separated in Example 1 of the present invention;

[0043] Fig.14 is a GC-MS TIC diagram of the RRT1.017 impurity separated in Example 1 of the present invention;

[0044] Fig.15 This is the H NMR spectrum of the RRT1.017 impurity separated in Example 1 of the present invention;

[0045] Fig.16 This is the NMR carbon spectrum of the RRT1.017 impurity separated in Example 1 of the present invention;

[0046] Fig.17 is a GC-MS TIC diagram of the RRT1.026 impurity separated in Example 1 of the present invention;

[0047] Fig.18 is the H NMR spectrum of the RRT1.026 impurity separated in Example 1 of the present invention;

[0048] Fig.19 This is the NMR carbon spectrum of the RRT1.026 impurity separated in Example 1 of the present invention;

[0049] Fig. 20 is a GC-MS TIC diagram of the RRT1.042 impurity separated in Example 1 of the present invention;

[0050] Fig.21 This is the H NMR spectrum of the RRT1.042 impurity separated in Example 1 of the present invention;

[0051] Fig. 22 This is the NMR carbon spectrum of the RRT1.042 impurity separated in Example 1 of the present invention;

[0052] Fig.23 It is the high performance liquid chromatogram in comparative example 1 of the present invention;

[0053] Fig.24 It is the high performance liquid chromatogram in comparative example 2 of the present invention;

[0054] Fig.25 It is the high performance liquid chromatogram in comparative example 3 of the present invention. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0057] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "multiple" means two or more, unless otherwise clearly and specifically defined. In addition, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal connection of two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0058] Example 1

[0059] A method for simultaneously preparing multiple trans isomers of ethyl eicosapentaenoate, the specific process is as follows:

[0060] (1) Synthesis of a crude product rich in trans-isomers of EPAee (eicosapentaenoic acid ethyl ester): 5 g of EPAee raw material with a purity greater than 95% was placed in a stainless steel autoclave, and the heating temperature was set to 280° C. and the pressure was set to 5 MPa. The reaction was stirred for 4 hours to obtain a crude product with a total content of trans-isomers of EPAee greater than 60%. The HPLC chromatogram of the crude product is as follows: Figure 1 shown.

[0061] (2) Reverse phase preparative liquid chromatography separation, reverse phase preparative liquid chromatography conditions: UniSil C8 (20×250 mm, 7 μm) as the preparative column; silver nitrate aqueous solution-acetonitrile mixed solution as the mobile phase, wherein the volume ratio of silver nitrate aqueous solution to acetonitrile is 35:65, and the silver nitrate content in the silver nitrate aqueous solution is 5%; isocratic elution at a flow rate of 18 mL / min, the equilibrium time is 8 min, and the running time is 60 min; take the crude product in step (1), add ethanol to dissolve it into about 50 mg / mL and pass it through a 0.45 μL filter membrane, and then after the reverse phase preparative system is balanced, inject 2 mL to separate the crude product.

[0062] (3) The fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram were collected separately, and the fractions corresponding to the same chromatographic peaks of trans isomers were combined together, and then extracted with dichloromethane respectively. After extraction, the organic phase was taken and the solvent was removed by rotary evaporation at 30°C, and finally 7 EPAee trans isomers were obtained, namely impurity D, impurity E, impurity F, impurity G, RRT1.017 impurity, RRT1.026 impurity, and RRT1.042 impurity. The 7 trans isomers were tested by GC-MS and NMR (such as Figure 2-22The structure and purity of the product were determined, and the specific information is shown in Table 1.

[0063] Table 1 Structure and purity of the seven EPAee trans isomers prepared

[0064]

[0065] The instrument used in the nuclear magnetic resonance test method is an Agilent 400NMR superconducting pulse Fourier transform nuclear magnetic resonance spectrometer, the solvent is CDCl3, and the reference material is CDCl3( 1 H spectrum, 13 C spectrum); GC-MS test method is shown in Table 2.

[0066] Table 2 GC-MS test method

[0067]

[0068] The specific NMR data of each impurity are as follows:

[0069] Impurity D: 1 HNMR(CDCl3):5.52-5.35(10H,m),4.12(2H,q,J=7.1Hz),2.84-2.77(m,6H),2.76(2H,t,J=5.7Hz),2.30(2H,t,J=7.5 Hz), 2.11 (2H, q, J = 7.5Hz), 2.00 (2H, p, J = 7.5Hz), 1.70 (2H, p, J = 7.4Hz), 1.25 (3H, t, J = 7.1Hz), 0.96 (3H, t, J = 7.5Hz); 13 CNMR(CDCl3):173.78,132.70,129.14,128.93,128.43,128.39,128.34,128.26,128 .16,127.09,60.37,33.85,30.54,26.69,25.75,25.73,25.69,24.93,14.38,13.95.

[0070] Impurity E: 1 HNMR(CDCl3): 5.42-5.35(10H,m),4.12(2H,q,J=7.1Hz),2.80-2.73(8H,m),2.30(2H,t,J=7.5Hz),2.10(2 H,q,J=6.9Hz), 2.03(2H,p,J=7.3Hz), 1.69(2H,p,J=7.4Hz), 1.24(3H,t,J=7.1Hz), 0.95(3H,t,J=7.5Hz); 13CNMR: 173.74,132.34,129.16,129.07,128.94,128.49,128.39,128.29,128.24,128.12 ,126.95,60.33,33.82,30.48,30.37,26.66,25.70,25.66,24.91,20.55,14.40,14.36.

[0071] Impurity F: 1 HNMR: 5.41-5.36(10H,m),4.12(2H,q,J=7.1Hz),2.82-2.76(8H,m),2.28(2H,t,J=7.6H z),2.11-2.01(4H,m),1.72-1.66(2H,m),1.26(3H,t,J=7.1Hz),0.97(3H,t,J=7.5Hz); 13 CNMR: 173.88,132.16,129.74,129.33,128.65,128.48,128.29,128.28,128.10,128.03 ,127.15,60.33,33.81,32.01,30.52,25.74,25.69,25.67,24.79,20.69,14.41,14.38.

[0072] Impurity G: 1 HNMR: 5.42-5.27(10H,m),4.10(2H,q,J=7.1Hz),2.77-2.75(8H,m),2.28(2H,t,J=7.5H z),2.09-2.03(4H,m),1.71-1.66(2H,m),1.23(3H,t,J=7.1Hz),0.94(3H,t,J=7.4Hz); 13 CNMR: 173.73,131.99,128.96,128.78,128.77,128.66,128.46,127.94,127.71,12 7.16,60.29,33.77,30.42,30.41,26.60,25.58,25.51,24.86,20.60,14.34,14.31.

[0073] RRT1.017 Impurities: 1 HNMR: 5.51-5.32(10H,m),4.12(2H,q,J=7.1Hz),

[0074] 2.78-2.71(8H,m),2.30(2H,t,J=7.5Hz),2.10(2H,q,J=6.9Hz),2.00(2H,p,J (6.8Hz), 1.69(2H,p,J=7.5Hz),1.25(3H,t,J=7.1Hz),0.96(3H,t,J=7.5Hz)? 13 CNMR:173.80,132.65,129.04,129.02,128.92,128.70,128.61,128.51,128.09,128.04 ,127.19,60.37,33.85,30.50,30.45,30.42,26.67,25.69,25.65,24.93,14.39,13.96。

[0075] RRT1.026 New: 1 HNMR:5.42-5.29(10H,m),4.12(2H,q,J=7.1Hz),

[0076] 2.81–2.72(8H,m),2.29(2H,t,J=7.5Hz),2.08–2.05(4H,m),1.68(2H,p,J=7.5Hz),1.25(3H,t,J=7.1Hz),0.75(J=3H),0.75(3H), 13 CNMR:173.81,132.10,129.26,128.87,128.74,128.71,128.56,128.51,128.07,127.98 ,127.16,60.33,33.83,30.48,30.45,26.57,25.64,25.63,24.92,20.66,14.40,14.37。

[0077] RRT1.042 New: 1 HNMR:5.45–5.29(10H,m),4.12(2H,q,J=7.1Hz),

[0078] 2.74-2.71(8H,m),2.28(2H,t,J=7.6Hz),2.10-2.00(4H,m),1.68(2H,p,J=7.4Hz),1.25(3H,t,J=7.1Hz),0.75(J=3H),0.75(3H), 13CNMR: 173.84,132.35,129.26,129.14,128.85,128.84,128.81,128.62,128.36,128 .25,127.02,60.36,33.87,30.47,30.43,30.40,26.59,24.95,20.58,14.42,14.39.

[0079] Example 2

[0080] A method for simultaneously preparing multiple trans isomers of ethyl eicosapentaenoate, the specific process is as follows:

[0081] (1) Synthesis of a crude product rich in EPAee trans-isomers: 5 g of EPAee API with a purity greater than 95% was placed in a stainless steel autoclave, and the heating temperature was set to 250° C., the pressure was set to 5 MPa, and the reaction was stirred for 4.5 hours to obtain a crude product with a total content of EPAee trans-isomers greater than 60%.

[0082] (2) Reverse phase preparative liquid chromatography separation, reverse phase preparative liquid chromatography conditions: UniSil C8 (20×250 mm, 7 μm) as the preparative column; silver nitrate aqueous solution-acetonitrile mixed solution as the mobile phase, wherein the volume ratio of silver nitrate aqueous solution to acetonitrile is 35:65, and the silver nitrate content in the silver nitrate aqueous solution is 5%; isocratic elution at a flow rate of 18 mL / min, the equilibrium time is 8 min, and the running time is 60 min; take the crude product in step (1), add ethanol to dissolve it into about 50 mg / mL and pass it through a 0.45 μL filter membrane, and then after the reverse phase preparative system is balanced, inject 2 mL to separate the crude product.

[0083] (3) The fractions corresponding to the various trans-isomer chromatographic peaks in the chromatogram were collected separately, and the fractions corresponding to the same trans-isomer chromatographic peaks were combined together, and then extracted with dichloromethane respectively. After extraction, the organic phase was taken and the solvent was removed by rotary evaporation at 30° C., and 7 EPAee trans-isomer impurities with a purity greater than 95% were prepared by separation. The purity of each impurity is as follows: impurity D is 97.3%, impurity E is 95.2%, impurity F is 95.9%, impurity G is 99.2%, RRT1.017 impurity is 97.3%, RRT1.026 impurity is 95.4%, and RRT1.042 impurity is 97.5%.

[0084] Example 3

[0085] A method for simultaneously preparing multiple trans isomers of ethyl eicosapentaenoate, the specific process is as follows:

[0086] (1) Synthesis of a crude product rich in EPAee trans-isomers: 5 g of EPAee API with a purity greater than 95% was placed in a stainless steel autoclave, and the heating temperature was set to 280° C., the pressure was set to 5 MPa, and the reaction was stirred for 4 hours to obtain a crude product with a total content of EPAee trans-isomers greater than 60%.

[0087] (2) Reverse phase preparative liquid chromatography separation, reverse phase preparative liquid chromatography conditions: UniSil C8 (20×250 mm, 7 μm) as the preparative column; silver nitrate aqueous solution-acetonitrile mixed solution as the mobile phase, wherein the volume ratio of silver nitrate aqueous solution to acetonitrile is 37:63, and the silver nitrate content in the silver nitrate aqueous solution is 3%; isocratic elution at a flow rate of 18 mL / min, the equilibrium time is 8 min, and the running time is 60 min; take the crude product in step (1), add ethanol to dissolve it into about 50 mg / mL and pass it through a 0.45 μL filter membrane, and then after the reverse phase preparative system is balanced, inject 2 mL to separate the crude product.

[0088] (3) The fractions corresponding to the various trans-isomer chromatographic peaks in the chromatogram were collected separately, and the fractions corresponding to the same trans-isomer chromatographic peaks were combined together, and then extracted with dichloromethane respectively. After extraction, the organic phase was taken and the solvent was removed by rotary evaporation at 30° C., and 7 EPAee trans-isomer impurities with a purity of more than 95% were prepared by separation. The purity of each impurity is as follows: impurity D is 98.5%, impurity E is 95.4%, impurity F is 97.2%, impurity G is 98.1%, RRT1.017 impurity is 96.1%, RRT1.026 impurity is 95.1%, and RRT1.042 impurity is 98.2%.

[0089] Comparative Example 1

[0090] A method for synthesizing a crude trans-isomer of EPAee: 5 g of EPAee API was placed in a stainless steel autoclave, and the heating temperature was set to 150° C., the pressure was set to 2.5 MPa, and the reaction was stirred for 10 hours. Fig.23 As shown, the crude product with a total content of EPAee trans-isomers of less than 5% was obtained, which was not suitable for separation and preparation.

[0091] Comparative Example 2

[0092] A method for preparing a trans isomer of ethyl eicosapentaenoate, the specific process is as follows:

[0093] (1) Synthesis of a crude product rich in EPAee trans-isomers: 5 g of EPAee API was placed in a stainless steel autoclave, and the heating temperature was set to 280° C. and the pressure was set to 5 MPa. The reaction was stirred for 4 hours to obtain a crude product with a total EPAee trans-isomer content of more than 60%.

[0094] (2) Reverse phase preparative liquid chromatography separation: A UniSil C8 (20×250 mm, 7 μm) preparative column was used as the preparative column; a water-acetonitrile mixed solution was used as the mobile phase, wherein the volume ratio of water to acetonitrile was 35:65; isocratic elution was performed at a flow rate of 18 mL / min, the equilibrium time was 8 min, and the running time was 30 min. Samples were injected under the above conditions, and the typical spectrum obtained was as follows Fig.24 As shown, when silver nitrate is not added to the mobile phase, the isomers are difficult to separate effectively and it is impossible to obtain an impurity reference substance of qualified purity.

[0095] Comparative Example 3

[0096] A method for preparing a trans isomer of ethyl eicosapentaenoate, the specific process is as follows:

[0097] (1) Synthesis of a crude product rich in EPAee trans-isomers: 5 g of EPAee API was placed in a stainless steel autoclave, and the heating temperature was set to 280° C. and the pressure was set to 5 MPa. The reaction was stirred for 4 hours to obtain a crude product with a total EPAee trans-isomer content of more than 60%.

[0098] (2) Reverse phase preparative liquid chromatography separation, reverse phase preparative liquid chromatography conditions: UniSil C8 (20×250 mm, 7 μm) preparative column; silver nitrate aqueous solution-methanol mixed solution as mobile phase, wherein the volume ratio of silver nitrate aqueous solution to methanol is 20:80, and the silver nitrate content in the silver nitrate aqueous solution is 5%; isocratic elution at a flow rate of 18 mL / min, equilibrium time of 8 min, and running time of 40 min; injection under the above conditions, the obtained typical spectrum is as follows Fig.25 As shown in the figure, when the mobile phase is changed from acetonitrile to methanol, the isomers are difficult to be effectively separated and it is impossible to obtain an impurity reference substance of qualified purity.

[0099] Compared with the prior art, the preparation method of the trans isomer of ethyl eicosapentaenoate provided by the present invention uses high pressure and high temperature to destroy EPAee, which can effectively obtain a mixture with multiple types and large content, and is conducive to the simultaneous separation of multiple isomer impurity reference substances from the same sample. In addition, a method of adding silver nitrate to the mobile phase of reversed-phase preparative liquid chromatography is used to separate and prepare 7 types of EPAee trans isomer impurity reference substances with purity that meets the requirements on reversed-phase chromatography at one time for the first time, which meets the purity requirements of drug research and development regulations for impurity reference substances, and greatly promotes the application process of EPAee drugs.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously preparing a plurality of trans isomers of ethyl eicosapentaenoate, characterized in that: The following steps are involved: (1) reacting ethyl eicosapentaenoic acid with a purity greater than 95% at a temperature of 230-300° C. and a pressure of 4.0-5.5 MPa for 3-5 hours to obtain a crude product containing the trans isomer of ethyl eicosapentaenoic acid; (2) separating the crude product obtained in step (1) by reverse phase preparative liquid chromatography, using a silver nitrate aqueous solution-acetonitrile mixed solution as the mobile phase; (3) collecting the fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram, respectively extracting them with organic solvents, and removing the solvent by rotary evaporation of the organic phase to obtain various trans isomers of ethyl eicosapentaenoate.

2. The method according to claim 1, characterized in that: The temperature in step (1) is 250-280° C., the pressure is 4.5-5.0 MPa, and the reaction time is 3.5-4.5 hours.

3. The method according to claim 1, characterized in that The total content of trans isomers of ethyl eicosapentaenoate in the crude product obtained in step (1) is greater than 60%.

4. The method according to claim 1, characterized in that: The reverse phase chromatographic column used in the reverse phase preparative liquid chromatography separation in step (2) is one of a C8 chromatographic column, a C18 chromatographic column, a pentafluorophenyl chromatographic column, a cyano chromatographic column, and an AQ chromatographic column.

5. The method according to claim 4, characterized in that In the step (2), the reverse phase chromatography column is a semi-preparative column or a preparative column with an inner diameter of not less than 0.9 cm.

6. The method according to claim 1, characterized in that The mass fraction of silver nitrate in the silver nitrate aqueous solution used as the mobile phase in step (2) is 1-10%.

7. The method according to claim 6, characterized in that In the step (2), the volume ratio of the silver nitrate aqueous solution to the acetonitrile in the silver nitrate aqueous solution-acetonitrile mixed solution is (35-37):(63-65).

8. The method according to claim 1, characterized in that: When performing reverse phase preparative liquid chromatography separation in step (2), the flow rate is 18 mL / min, the equilibrium time is 8 minutes, and the running time is 60 minutes.

9. The method according to claim 1, characterized in that: In the step (3), dichloromethane is used for extraction.

10. The method according to claim 1, characterized in that In the step (3), 7 trans isomers of ethyl eicosapentaenoate are obtained, namely, impurity D, impurity E, impurity F, impurity G, RRT1.017 impurity, RRT1.026 impurity, and RRT1.042 impurity, wherein: The structural formula of impurity D is The structural formula of impurity E is The structural formula of impurity F is The structural formula of impurity G is The structural formula of RRT1.017 impurity The structural formula of RRT1.026 impurity is The structural formula of RRT1.042 impurity is

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