Refining method of dioleoyl phosphatidylcholine

Abstract

The invention provides a refining method of dioleoyl phosphatidylcholine, which comprises the steps of synthesis, adsorption, deep eutectic solvent extraction, recrystallization, drying and the like, by reasonably designing a multi-stage purification process, adsorption treatment, deep eutectic solvent (DES) selective extraction and recrystallization process are organically combined, and the purity of the dioleoyl phosphatidylcholine is greatly improved. According to the present invention, the unreacted fatty acid, the condensation catalyst and the by-product thereof, and other polar impurities are efficiently removed, the purity of the DOPC product refined through the method can stably achieve more than 99.5%, the physical and chemical properties are excellent, the batch-to-batch difference is small, and the consistency and the stability of the product are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of synthetic phospholipids, and more specifically relates to a method for purifying dioleoylphosphatidylcholine. Background Technology

[0002] Synthetic phospholipids are a class of functional phospholipid derivatives prepared through chemical methods, with well-defined structures and compositions, high purity, and high batch-to-batch stability. They are widely used in liposome drug delivery systems, solid lipid nanoparticles, mRNA vaccine carriers, and the construction of artificial cell membrane models. Compared with naturally derived phospholipids, synthetic phospholipids allow for precise control over the carbon number, saturation, and spatial configuration of fatty acid chains, enabling targeted design of membrane fluidity, phase transition temperature, and drug loading performance. This provides them with irreplaceable advantages in the field of high-end injectable formulations.

[0003] Dioleoylphosphatidylcholine (DOPC) is a typical unsaturated synthetic phospholipid containing two cis-double oleic acid chains. It exhibits a low phase transition temperature and good membrane fluidity, making it widely used in liposome formulations, cell membrane mimicry systems, and biophysical research. However, the presence of double bonds in the DOPC molecular structure makes it sensitive to oxygen, heat, and impurities during synthesis and purification, thus placing higher demands on the gentleness and selectivity of the purification process.

[0004] Currently, DOPC is typically prepared by chemical acylation reaction of glycerol phosphatidylcholine (GPC) with fatty acids in the presence of a condensing agent. For example, CN111454289A discloses a method for obtaining DOPC by condensation reaction of GPC and oleic acid in a DCC / DMAP system, followed by solvent slurrying and crystallization. CN117820361A discloses a method for preparing unsaturated phosphatidylcholine using a similar condensation route, achieving product purification through multiple dissolution-crystallization processes. These methods have clear reaction pathways and stable raw material sources, and have become the mainstream technical route for phospholipid synthesis.

[0005] However, such chemical condensation reactions typically require excessive use of fatty acids, condensing agents, and organic base catalysts. After the reaction, unreacted fatty acids, condensation byproducts (such as urea compounds), phase transfer catalysts, and impurities with polarities close to the target product are easily left in the system. These impurities are highly similar to DOPC molecules in terms of polarity and solubility, and it is often difficult to achieve effective separation by simply relying on solvent slurrying or recrystallization. This results in cumbersome purification steps, high solvent consumption, and difficulty in simultaneously ensuring product purity and batch stability.

[0006] To address the issue of catalyst and byproduct residues in synthetic phospholipids, some existing technologies attempt to refine them using methods such as metal salt complexation, adsorption, or multi-stage washing. For example, Chinese invention patent CN120718058A discloses a method for refining synthetic phospholipids by combining metal salt complexation and recrystallization, which can effectively remove residual catalysts and metal ions, improving product purity. This type of method has certain advantages in the refining of saturated fatty acid phospholipids (such as DPPC and DSPC), but for unsaturated phospholipids containing double bonds, metal ions or strong complexing environments may induce double bond oxidation, side reactions, or molecular structure damage, thereby affecting the chemical stability and safety of the product and limiting its application in the refining of unsaturated phospholipids such as DOPC.

[0007] In summary, while existing purification technologies for synthetic phospholipids can remove impurities to some extent, they still suffer from low purification efficiency, complex processes, and difficulty in controlling solvent residues. Particularly for unsaturated phospholipids, a purification technology that balances high purity and structural stability is lacking. Therefore, developing a purification method that is mild, easy to operate, environmentally friendly and safe, and can effectively remove polar impurities while maintaining the structural integrity of DOPC is of great significance for the industrialization of high-purity injection-grade synthetic phospholipids. Summary of the Invention

[0008] To address the problems in existing technologies regarding the complete removal of impurities with similar polarity, difficulty in controlling solvent residues, easy oxidation of unsaturated structures, and insufficient process repeatability and scale-up stability during the synthesis of DOPC, this invention proposes a purification method for dioleoylphosphatidylcholine. By rationally designing a multi-stage purification process, adsorption treatment, selective extraction with deep eutectic solvent (DES), and recrystallization are organically combined to achieve efficient removal of unreacted fatty acids, condensation catalysts and their byproducts, as well as other polar impurities.

[0009] The technical solution of this invention is implemented as follows: A method for purifying dioleoylphosphatidylcholine includes the following steps: S1. Synthesis: Glycerylphosphatidylcholine (GPC), oleic acid, organic base and phase transfer catalyst are mixed in chloroform. The condensing agent is dissolved in chloroform and added to the mixture to carry out the condensation reaction. After the reaction is completed, acetone is added to concentrate the mixture, filtered, and the solid is collected to obtain crude DOPC. S2. Adsorption: Add crude DOPC to an alcohol solvent, heat and stir until clear under nitrogen protection, add adsorbent and keep warm and stir, then filter while hot, collect the filtrate and concentrate to obtain the first concentrate; S3, DES extraction: Add a haloalkanes organic solvent and a small amount of alcohol solvent as a co-solvent to the first concentrate. Under nitrogen protection, heat and stir until clear. Add a deep eutectic solvent and stir to promote mass transfer. Then stop stirring and let stand to separate the layers. Collect the haloalkanes phase and concentrate to obtain the second concentrate. S4. Recrystallization: Add mixed solvent to the second concentrate, heat until clear under nitrogen protection, cool and crystallize, filter and collect the solid; S5. Drying: The obtained solid is dried to obtain a high-purity DOPC product.

[0010] Preferably, in step S1, the organic base is at least one of 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA), or triethylamine (TEA); the phase transfer catalyst is at least one of tetrabutylammonium bromide (TBAB), benzyltriethylammonium chloride (TEBA), or tetrapropylammonium bromide (TPAB); and the condensing agent is at least one of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0011] Preferably, in step S1, the molar ratio of glycerophosphatidylcholine, oleic acid, organic base, phase transfer catalyst and condensing agent is 1:(2~4):(0.5~1.5):(0.1~0.5):(2~4), the condensing agent is added at a temperature of 5~15℃ and for 50~100 minutes; the reaction temperature is 40~60℃ and the reaction time is 24~36 hours; the mass ratio of acetone added to GPC during the pulping process is (6~10):1, the pulping temperature is 10~30℃, and the pulping time is 120~240 minutes.

[0012] Preferably, in step S2, the alcohol solvent is at least one of methanol, ethanol, or isopropanol; and the adsorbent is at least one of neutral alumina, silica gel, or magnesium silicate. Preferably, in step S2, the mass ratio of crude DOPC to alcohol solvent is 1:(4~8), the heating and dissolution temperature is 35~55℃, the mass ratio of crude DOPC to adsorbent is 1:(1~3), and the stirring time is 60~100 minutes.

[0013] Preferably, in step S3, the halogenated hydrocarbon organic solvent is at least one of chloroform or dichloromethane; the alcohol solvent is at least one of methanol or ethanol; the deep eutectic solvent (DES) is a mixture of a quaternary ammonium salt and a hydrogen bond donor, wherein the quaternary ammonium salt is at least one of choline chloride, methyltriethylammonium chloride, or ethyltrimethylammonium chloride, and the hydrogen bond donor is at least one of glycerol, urea, or ethylene glycol, the molar ratio of the quaternary ammonium salt to the hydrogen bond donor is 1:(1.5~3), and the deep eutectic solvent is a dried product with a water content ≤2%.

[0014] Preferably, in step S3, the mass ratio of the first concentrate to the halogenated hydrocarbon organic solvent is 1:(6~10); the alcohol solvent as a co-solvent accounts for 0.5~2.0% of the mass of the halogenated hydrocarbon phase, and the temperature for heating and stirring to dissolve is 45~55℃; the mass ratio of the first concentrate to the deep eutectic solvent is 1:(0.5~1.0), the stirring temperature is 25~40℃, and the stirring time is 30~100 minutes; the standing and layering time is 60~120 minutes; the concentration is carried out under reduced pressure, the concentration temperature is 40~60℃, the vacuum degree is ≤-0.09MPa, and the concentration time is 5~10 hours.

[0015] Preferably, in step S4, the mixed solvent is a mixture of an alcohol solvent and acetone in a mass ratio of 1:(25~40), and the alcohol solvent is at least one of methanol, ethanol or isopropanol.

[0016] Preferably, in step S4, the mass ratio of the second concentrate to the mixed solvent is 1:(6~10), the temperature at which the system is heated to clarity is 40~60℃, the cooling rate is 1~3℃ / minute, the temperature for holding the crystallization is -10~0℃, and the crystallization time is 50~100 minutes.

[0017] Preferably, the drying in step S5 is vacuum drying, with a drying temperature of 50~70℃, a vacuum degree of ≤-0.09MPa, and a drying time of 24~36 hours.

[0018] The beneficial effects of this invention are as follows: (1) This invention uses a multi-stage refining process, including adsorption, deep eutectic solvent (DES) extraction and recrystallization, to effectively remove residual fatty acids, catalyst byproducts and polar impurities, so that the purity of DOPC can be stably maintained at 99.5% or higher and the yield is ≥85%; (2) This invention does not introduce metal ions or strong acid or strong base conditions, and each step can be carried out under inert atmosphere and relatively mild temperature conditions, which effectively avoids the oxidation or structural damage of unsaturated fatty acid chains. The obtained product has stable physicochemical properties and small batch-to-batch differences, ensuring the quality consistency of injection grade DOPC. (3) The present invention uses environmentally friendly solvent systems such as alcohols, halogenated hydrocarbons and DES, with mild operating conditions and simple process design, which reduces repeated operations and solvent consumption and is conducive to industrial scale-up production. (4) This invention can be widely applied to the high-purity preparation of injectable DOPC, providing reliable technical support for liposome drugs, cell membrane models and fine chemical fields, and can provide feasible solutions for the industrial production of related high-purity phospholipids. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a process flow diagram of the present invention.

[0021] Figure 2 To prepare DOPC according to Example 1 of the present invention 1 H-NMR spectrum.

[0022] Figure 3 This is an HPLC chromatogram for the content detection of DOPC sample prepared according to Example 1 of the present invention.

[0023] Figure 4 The HPLC chromatograms of GPC, TBAB, and DCU standards in DOPC prepared according to Example 1 of the present invention are shown.

[0024] Figure 5 The HPLC chromatograms of GPC, TBAB, and DCU in the DOPC sample prepared according to Example 1 of the present invention are shown.

[0025] Figure 6 The image shows the HPLC chromatogram of the DMAP standard in DOPC prepared according to Example 1 of the present invention.

[0026] Figure 7 The image shows the HPLC chromatogram of DMAP in the DOPC sample prepared according to Example 1 of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] Synthesis: Weigh 50 g of glycerol phosphatidylcholine (GPC), 137.3 g of oleic acid, 23.7 g of 4-dimethylaminopyridine (DMAP), and 12.5 g of tetrabutylammonium bromide (TBAB) into a 3000 mL three-necked flask. Add 1000 mL of chloroform and stir at 10 °C until homogeneous. Maintain the solution temperature at 10 °C. Dissolve 100.3 g of N,N'-dicyclohexylcarbodiimide (DCC) in 500 mL of chloroform and add it dropwise to the reaction system over 60 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 60℃ and the reaction was carried out for 24 hours. After the reaction was completed by TLC, the temperature was lowered to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (40℃, -0.09MPa, 9 hours). 300g of acetone was added to the concentrate, and the mixture was stirred at 15℃ for 150 minutes. The solid was collected by filtration and dried under vacuum (50℃, -0.09MPa, 24 hours) to obtain 175.5g of crude DOPC.

[0030] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 600g of methanol was added, and the mixture was heated and maintained at 45℃ under nitrogen protection, and stirred until the system was clear; 150g of neutral alumina was added, and the mixture was kept at 45℃ and stirred for 70 minutes. The mixture was filtered while hot, the filtrate was collected and concentrated (50℃, -0.09MPa, 6 hours) to obtain 90.2g of the first concentrate.

[0031] DES Extraction: ① DES Preparation: Weigh 43g of choline chloride and 57g of glycerol, stir at 50℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤2%, cool and store under nitrogen for later use. ② DES Extraction: Place 90.2g of the first concentrate in a 2000mL three-necked flask, add 810g of chloroform and 8g of ethanol, heat to 35℃ under nitrogen protection and stir until dissolved and clear. Slowly add 72g of dried DES to the solution and stir for 60 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 100 minutes to separate the layers. Collect the haloalkanes phase and concentrate (45℃, -0.09MPa, 8 hours) to obtain 80.1g of the second concentrate.

[0032] Recrystallization and drying: 80.1 g of the second concentrate was placed in a 2000 mL three-necked flask, and 624 g of acetone / methanol mixed solution (600 g acetone, 24 g methanol) was added. The mixture was heated to 50 °C under nitrogen protection and stirred until clear. The temperature was then lowered to -5 °C at a rate of 2 °C / min and kept at this temperature for 60 minutes to crystallize. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (50 °C, -0.09 MPa, 30 hours) to obtain 76.3 g of DOPC.

[0033] Example 2

[0034] Synthesis: Weigh 50 g of glycerophosphatidylcholine (GPC), 164.7 g of oleic acid, 22.6 g of N,N-diisopropylethylamine (DIPEA), and 13.3 g of benzyltriethylammonium chloride (TEBA) into a 3000 mL three-necked flask. Add 1000 mL of chloroform and stir at 7°C until homogeneous. Maintaining the solution temperature at 7°C, dissolve 98.1 g of N,N'-diisopropylcarbodiimide (DIC) in 500 mL of chloroform and add it dropwise to the reaction system over 80 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 55℃ and the reaction was carried out for 30 hours. After the reaction was completed by TLC, the temperature was lowered to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (55℃, -0.09MPa, 6 hours). 400g of acetone was added to the concentrate, and the mixture was stirred at 20℃ for 170 minutes. The solid was collected by filtration and dried under vacuum (60℃, -0.09MPa, 29 hours) to obtain 180.3g of crude DOPC.

[0035] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 550g of ethanol was added, and the mixture was heated and maintained at 40℃ under nitrogen protection, and stirred until the system was clear; 200g of silica gel was added, and the mixture was kept at 40℃ and stirred for 80 minutes. The mixture was filtered while hot, the filtrate was collected and concentrated (60℃, -0.09MPa, 7 hours) to obtain 88.7g of the first concentrate.

[0036] DES Extraction: ① DES Preparation: Weigh 55g of methyltriethylammonium chloride and 45g of urea, stir at 55℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤1.5%, cool and store under nitrogen for later use. ② DES Extraction: Place 88.7g of the first concentrate in a 2000mL three-necked flask, add 670g of dichloromethane and 10g of methanol, heat to 30℃ under nitrogen protection and stir until dissolved and clear. Slowly add 80g of dried DES to the solution and stir for 70 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 90 minutes to separate the layers. Collect the haloalkanes phase and concentrate (54℃, -0.09MPa, 6 hours) to obtain 77.8g of the second concentrate.

[0037] Recrystallization and drying: 77.8 g of the second concentrate was placed in a 2000 mL three-necked flask, and 545 g of acetone / ethanol mixed solution (527 g acetone, 18 g ethanol) was added. The mixture was heated to 55 °C under nitrogen protection and stirred until clear. The temperature was then lowered to -7 °C at a rate of 2.5 °C / min and crystallized for 80 minutes. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (68 °C, -0.09 MPa, 27 hours) to obtain 74.7 g of DOPC.

[0038] Example 3

[0039] Synthesis: Weigh 50 g of glycerol phosphatidylcholine (GPC), 164.7 g of oleic acid, 23.6 g of triethylamine (TEA), and 12.9 g of tetrapropylammonium bromide (TPAB) into a 3000 mL three-necked flask, add 1000 mL of chloroform, and stir until homogeneous at 13 °C. Maintaining the solution temperature at 13 °C, dissolve 100 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in 500 mL of chloroform, and add it dropwise to the reaction system over 70 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 48℃ and the reaction was carried out for 29 hours. After the reaction was completed by TLC, the temperature was lowered to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (44℃, -0.09MPa, 5 hours). 415g of acetone was added to the concentrate, and the mixture was stirred at 28℃ for 200 minutes. The solid was collected by filtration and dried under vacuum (63℃, -0.09MPa, 30 hours) to obtain 178.3g of crude DOPC.

[0040] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 770g of isopropanol was added, and the mixture was heated and maintained at 50℃ under nitrogen protection, and stirred until the system was clear; 230g of magnesium silicate was added, and the mixture was stirred at 50℃ for 90 minutes. The mixture was filtered while hot, the filtrate was collected and concentrated (55℃, -0.09MPa, 6 hours) to obtain 89.3g of the first concentrate.

[0041] DES Extraction: ① DES Preparation: Weigh 40g of ethyltrimethyl chloride and 60g of ethylene glycol, stir at 55℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤2%, cool and store under nitrogen for later use. ② DES Extraction: Place 89.3g of the first concentrate in a 2000mL three-necked flask, add 785g of chloroform and 14g of ethanol, heat to 37℃ under nitrogen protection and stir until dissolved and clear. Slowly add 70g of dried DES to the solution and stir for 90 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 110 minutes to separate the layers. Collect the haloalkanes phase and concentrate (56℃, -0.09MPa, 7.5 hours) to obtain 82.4g of the second concentrate.

[0042] Recrystallization and drying: 82.4 g of the second concentrate was placed in a 2000 mL three-necked flask, and 740 g of acetone / isopropanol mixed solution (720 g acetone, 20 g isopropanol) was added. The mixture was heated to 45 °C under nitrogen protection and stirred until clear. The temperature was lowered to -8 °C at a rate of 1.5 °C / min and crystallized at this temperature for 90 minutes. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (55 °C, -0.09 MPa, 32 hours) to obtain 73.5 g of DOPC.

[0043] Example 4

[0044] Synthesis: Weigh 50 g of glycerol phosphatidylcholine (GPC), 148.2 g of oleic acid, 26.1 g of 4-dimethylaminopyridine (DMAP), and 15.5 g of benzyltriethylammonium chloride (TEBA) into a 3000 mL three-necked flask. Add 1000 mL of chloroform and stir until homogeneous at 6 °C. Maintaining the solution temperature at 6 °C, dissolve 111.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in 500 mL of chloroform and add it dropwise to the reaction system over 89 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 57℃ and the reaction was carried out for 34 hours. After the reaction was completed by TLC, the temperature was lowered to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (50℃, -0.09MPa, 8 hours). 380g of acetone was added to the concentrate, and the mixture was stirred at 13℃ for 138 minutes. The solid was collected by filtration and dried under vacuum (55℃, -0.09MPa, 24 hours) to obtain 183.6g of crude DOPC.

[0045] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 480g of ethanol was added, and the mixture was heated and kept at 35℃ under nitrogen protection, and stirred until the system was clear; 250g of neutral alumina was added, and the mixture was kept at 35℃ and stirred for 65 minutes. The mixture was filtered while hot, the filtrate was collected and concentrated (55℃, -0.09MPa, 6 hours) to obtain 92.9g of the first concentrate.

[0046] DES Extraction: ① DES Preparation: Weigh 50g of choline chloride and 50g of urea, stir at 58℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤2%, cool and store under nitrogen for later use. ② DES Extraction: Place 92.9g of the first concentrate in a 2000mL three-necked flask, add 604g of dichloromethane and 5g of methanol, heat to 28℃ under nitrogen protection and stir until dissolved and clear. Slowly add 84g of dried DES to the solution and stir for 40 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 70 minutes to separate the layers. Collect the haloalkanes phase and concentrate (46℃, -0.09MPa, 9 hours) to obtain 85.6g of the second concentrate.

[0047] Recrystallization and drying: 85.6 g of the second concentrate was placed in a 2000 mL three-necked flask, and 556 g of acetone / isopropanol mixed solution (542 g acetone, 14 g isopropanol) was added. The mixture was heated to 48 °C under nitrogen protection and stirred until clear. The temperature was then lowered to -10 °C at a rate of 1 °C / min and crystallized for 70 minutes. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (55 °C, -0.09 MPa, 30 hours) to obtain 71.7 g of DOPC.

[0048] Example 5

[0049] Synthesis: Weigh 50 g of glycerol phosphatidylcholine (GPC), 170.2 g of oleic acid, 20.1 g of N,N-diisopropylethylamine (DIPEA), and 12.5 g of tetrabutylammonium bromide (TBAB) into a 3000 mL three-necked flask. Add 1000 mL of chloroform and stir until homogeneous at 15 °C. Maintaining the solution temperature at 15 °C, dissolve 104.3 g of N,N'-dicyclohexylcarbodiimide (DCC) in 500 mL of chloroform and add it dropwise to the reaction system over 100 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 40℃ and the reaction was carried out for 36 hours. After the reaction was completed by TLC, the temperature was lowered to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (53℃, -0.09MPa, 8 hours). 500g of acetone was added to the concentrate, and the mixture was stirred at 30℃ for 120 minutes. The solid was collected by filtration and dried under vacuum (50℃, -0.09MPa, 24 hours) to obtain crude DOPC of 176.7g.

[0050] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 660g of methanol was added, and the mixture was heated and maintained at 55℃ under nitrogen protection, and stirred until the system was clear; 180g of silica gel was added, and the mixture was stirred at 55℃ for 100 minutes, filtered while hot, and the filtrate was collected and concentrated (58℃, -0.09MPa, 6 hours) to obtain 91.1g of the first concentrate.

[0051] DES Extraction: ① DES Preparation: Weigh 59g of methyltriethylammonium chloride and 41g of ethylene glycol, stir at 56℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤2%, cool and store under nitrogen for later use. ② DES Extraction: Place 91.1g of the first concentrate in a 2000mL three-necked flask, add 640g of chloroform and 10g of ethanol, heat to 25℃ under nitrogen protection and stir until dissolved and clear. Slowly add 64g of dried DES to the solution and stir for 30 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 60 minutes to separate the layers. Collect the haloalkanes phase and concentrate (47℃, -0.09MPa, 8 hours) to obtain 80.3g of the second concentrate.

[0052] Recrystallization and drying: 80.3 g of the second concentrate was placed in a 2000 mL three-necked flask, and 682 g of acetone / isopropanol mixed solution (657 g acetone, 25 g isopropanol) was added. The mixture was heated to 40 °C under nitrogen protection and stirred until clear. The temperature was lowered to 0 °C at a rate of 3 °C / min and crystallized at this temperature for 50 minutes. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (50 °C, -0.09 MPa, 35 hours) to obtain 73.6 g of DOPC.

[0053] Example 6

[0054] Synthesis: 50 g of glycerol phosphatidylcholine (GPC), 197.7 g of oleic acid, 13.8 g of triethylamine (TEA), and 17.7 g of benzyltriethylammonium chloride (TEBA) were weighed into a 3000 mL three-necked flask. 1000 mL of chloroform was added, and the mixture was stirred at 5 °C until homogeneous. While maintaining the solution temperature at 5 °C, 98.1 g of N,N'-diisopropylcarbodiimide (DIC) was dissolved in 500 mL of chloroform and added dropwise to the reaction system over 50 minutes under nitrogen protection. After the addition was complete, the temperature was raised to 51 °C and the reaction was carried out for 26 hours. After the reaction was completed by TLC, the mixture was cooled to room temperature, filtered, and the filtrate was collected and concentrated under vacuum (50 °C, -0.09 MPa, 8 hours). 500 g of acetone was added to the concentrate, and the mixture was stirred at 10 °C for 240 minutes. The solid was collected by filtration and dried under vacuum (50 °C, -0.09 MPa, 27 hours) to obtain crude DOPC of 184.9 g.

[0055] Adsorption: 100g of crude DOPC was placed in a 2000mL three-necked flask, 800g of isopropanol was added, and the mixture was heated and maintained at 39℃ under nitrogen protection, and stirred until the system was clear; 300g of magnesium silicate was added, and the mixture was kept at 39℃ and stirred for 60 minutes. The mixture was filtered while hot, the filtrate was collected and concentrated (60℃, -0.09MPa, 10 hours) to obtain 92.7g of the first concentrate.

[0056] DES Extraction: ① DES Preparation: Weigh 33g of ethyltrimethylammonium chloride and 67g of glycerol, stir at 60℃ to form a homogeneous system, then dehydrate under reduced pressure at 60℃ until the water content is ≤2%, cool and store under nitrogen for later use. ② DES Extraction: Place 92.7g of the first concentrate in a 2000mL three-necked flask, add 927g of dichloromethane and 18g of methanol, heat to 40℃ under nitrogen protection and stir until dissolved and clear. Slowly add 92g of dried DES to the solution and stir for 100 minutes to promote the migration of polar impurities to the DES phase. Then stop stirring and let stand for 120 minutes to separate the layers. Collect the haloalkanes phase and concentrate (45℃, -0.09MPa, 10 hours) to obtain 86.5g of the second concentrate.

[0057] Recrystallization and drying: 86.5 g of the second concentrate was placed in a 2000 mL three-necked flask, and 865 g of acetone / isopropanol mixed solution (843 g acetone, 22 g isopropanol) was added. The mixture was heated to 60 °C under nitrogen protection and stirred until clear. The temperature was lowered to -6 °C at a rate of 1.8 °C / min, and crystallized at this temperature for 100 minutes. The mixture was filtered under low temperature conditions, and the solid was collected. The solid was dried under vacuum (57 °C, -0.09 MPa, 29 hours) to obtain 72.2 g of DOPC.

[0058] Comparative Example 1

[0059] DOPC was prepared according to Example 1 of CN111454289A.

[0060] The DOPC products obtained in Examples 1-6 and Comparative Example 1 were tested. The test items were the content of organic base, glycerol phosphatidylcholine, phase transfer catalyst, condensing agent and DOPC content.

[0061] Determination of organic base content: An octadecyl-bonded silica gel column (25 cm × 4.6 mm, 5 μm) was used. The mobile phases were 0.1% formic acid aqueous solution (A) and 0.1% formic acid methanol solution (B). The mobile phases were maintained at 90% A for 5 min, then switched to 100% B after 0.5 min and maintained for 15.5 min, before returning to the initial ratio for 25 min. The detection wavelength was 254 nm, the column temperature was 25℃, the flow rate was 1.0 ml / min, and the injection volume was 20 μl. A series of control solutions were prepared using organic base standards. A linear regression equation was established based on the logarithm of concentration and the logarithm of peak area, with a linear correlation coefficient ≥ 0.99. After sampling, the test sample was dissolved in toluene, diluted, shaken, and centrifuged. The supernatant was then injected. The content of organic base in the test sample was calculated based on the regression equation.

[0062] Determination of glycerophosphatidylcholine, phase transfer catalyst, and condensing agent byproduct content: An octadecyl-bonded silica gel column (25 cm × 4.6 mm, 5 μm) was used with isocratic elution of water / triethylamine / glacial acetic acid and methanol / triethylamine / glacial acetic acid (95:5) as the mobile phases. The column temperature was 40℃, the flow rate was 1.0 ml / min, and evaporative light scattering detector was used for detection. A series of mixed reference solutions of glycerophosphatidylcholine, phase transfer catalyst, and condensing agent byproduct were prepared. A linear regression equation for the three components was established by comparing the logarithm of concentration with the logarithm of peak area, and the linear correlation coefficients were all not less than 0.99. The test sample was dissolved in toluene, diluted, and centrifuged in layers. The lower layer solution was injected into the sample. The contents of glycerophosphatidylcholine, phase transfer catalyst, and condensing agent byproduct in the test sample were calculated according to the regression equation.

[0063] DOPC content determination: An octylsilane-bonded silica gel column (25 cm × 4.6 mm, 5 μm) was used with methanol / triethylamine / glacial acetic acid as the mobile phase for isocratic elution. The column temperature was 40 °C, the flow rate was 1.0 mL / min, and an evaporative light scattering detector was used for detection. A series of reference solutions were prepared using DOPC standard, and a linear regression equation was established between the logarithm of concentration and the logarithm of peak area, with a linear correlation coefficient ≥ 0.99. The test sample was dissolved in methanol and injected for determination. The DOPC content in the test sample was calculated based on the regression equation.

[0064] The test results are shown in the table below:

[0065] Therefore, it can be seen that the purity of 80.5% and the yield of 74.3% in the comparative example are much lower than those of the product prepared in the embodiment of the present invention. The DOPC prepared by the present invention has a stable purity of over 99.5%, a yield of ≥85%, and excellent physicochemical properties with small batch-to-batch differences, which significantly improves the consistency and stability of the product.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for purifying dioleoylphosphatidylcholine, characterized in that, Includes the following steps: S1. Synthesis: Glycerylphosphatidylcholine, oleic acid, organic base and phase transfer catalyst are mixed in chloroform. The condensing agent is dissolved in chloroform and added to the mixture to carry out the condensation reaction. After the reaction is completed, acetone is added to concentrate the mixture, filtered, and the solid is collected to obtain crude DOPC. S2. Adsorption: Add crude DOPC to an alcohol solvent, heat and stir until clear under nitrogen protection, add adsorbent and keep warm and stir, then filter while hot, collect the filtrate and concentrate to obtain the first concentrate; S3, DES extraction: Add a haloalkanes organic solvent and a small amount of alcohol solvent as a co-solvent to the first concentrate. Under nitrogen protection, heat and stir until clear. Add a deep eutectic solvent and stir to promote mass transfer. Then stop stirring and let stand to separate the layers. Collect the haloalkanes phase and concentrate to obtain the second concentrate. S4. Recrystallization: Add mixed solvent to the second concentrate, heat until clear under nitrogen protection, cool and crystallize, filter and collect the solid; S5. Drying: The obtained solid is dried to obtain a high-purity DOPC product.

2. The method as described in claim 1, characterized in that, In step S1, the organic base is at least one of 4-dimethylaminopyridine, N,N-diisopropylethylamine, or triethylamine; the phase transfer catalyst is at least one of tetrabutylammonium bromide, benzyltriethylammonium chloride, or tetrapropylammonium bromide; and the condensing agent is at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

3. The method as described in claim 1, characterized in that, In step S1, the molar ratio of glycerophosphatidylcholine, oleic acid, organic base, phase transfer catalyst and condensing agent is 1:(2~4):(0.5~1.5):(0.1~0.5):(2~4), the condensing agent is added at a temperature of 5~15℃ and for 50~100 minutes; the reaction temperature is 40~60℃ and the reaction time is 24~36 hours; the mass ratio of acetone added to GPC during the pulping process is (6~10):1, the pulping temperature is 10~30℃, and the pulping time is 120~240 minutes.

4. The method as described in claim 1, characterized in that, In step S2, the alcohol solvent is at least one of methanol, ethanol, or isopropanol; the adsorbent is at least one of neutral alumina, silica gel, or magnesium silicate.

5. The method as described in claim 1, characterized in that, In step S2, the mass ratio of crude DOPC to alcohol solvent is 1:(4~8), the heating and dissolution temperature is 35~55℃, the mass ratio of crude DOPC to adsorbent is 1:(1~3), and the stirring time is 60~100 minutes.

6. The method as described in claim 1, characterized in that, In step S3, the halogenated hydrocarbon organic solvent is at least one of chloroform or dichloromethane; the alcohol solvent is at least one of methanol or ethanol; the eutectic solvent is a mixture of a quaternary ammonium salt and a hydrogen bond donor, wherein the quaternary ammonium salt is at least one of choline chloride, methyltriethylammonium chloride or ethyltrimethylammonium chloride, and the hydrogen bond donor is at least one of glycerol, urea or ethylene glycol, the molar ratio of the quaternary ammonium salt to the hydrogen bond donor is 1:(1.5~3), and the eutectic solvent is a dried product with a water content ≤2%.

7. The method as described in claim 1, characterized in that, In step S3, the mass ratio of the first concentrate to the halogenated hydrocarbon organic solvent is 1:(6~10); the alcohol solvent as a co-solvent accounts for 0.5~2.0% of the mass of the halogenated hydrocarbon phase, and the temperature for heating and stirring to dissolve is 45~55℃; the mass ratio of the first concentrate to the deep eutectic solvent is 1:(0.5~1.0), the stirring temperature is 25~40℃, and the stirring time is 30~100 minutes; the standing and layering time is 60~120 minutes; the concentration is carried out under reduced pressure, the concentration temperature is 40~60℃, the vacuum degree is ≤-0.09MPa, and the concentration time is 5~10 hours.

8. The method as described in claim 1, characterized in that, In step S4, the mixed solvent is a mixture of an alcohol solvent and acetone in a mass ratio of 1:(25~40), and the alcohol solvent is at least one of methanol, ethanol or isopropanol.

9. The method as described in claim 1, characterized in that, In step S4, the mass ratio of the second concentrate to the mixed solvent is 1:(6~10), the temperature at which the system is heated to clarity is 40~60℃, the cooling rate is 1~3℃ / minute, the temperature for holding the crystallization is -10~0℃, and the crystallization time is 50~100 minutes.

10. The method as described in claim 1, characterized in that, In step S5, the drying is vacuum drying, with a drying temperature of 50~70℃, a vacuum degree of ≤-0.09MPa, and a drying time of 24~36 hours.

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