Separation and purification process of high-purity ursodeoxycholic acid mother liquor

Abstract

This invention relates to the field of separation technology in biopharmaceuticals and chemicals, and discloses a process for separating and purifying high-purity ursodeoxycholic acid (UDCA) mother liquor. The process includes: adding basic amino acids to the UDCA-containing mother liquor and stirring to dissolve it, obtaining an aqueous phase system; adding a neutral chiral hydrophobic eutectic solvent composed of L-menthol and a long-chain fatty alcohol to the aqueous phase system for mixed extraction; separating and removing the upper oil phase after settling and collecting the lower aqueous phase; finally, adding acid to the aqueous phase to adjust the pH value, followed by ultrasonic-assisted crystallization, washing, and drying to obtain the UDCA product. This invention utilizes the specific hydrophilic anchoring effect of basic amino acids on UDCA, combined with the chiral hydrophobic recognition effect of the eutectic solvent on impurities, to achieve efficient separation of the target product from chenodeoxycholic acid and lithocholic acid. This process is environmentally friendly, the solvent can be recycled, and the obtained product has high purity, making it suitable for industrial applications.

Description

Technical Field

[0001] This invention relates to the field of separation technology in biopharmaceutical and chemical industries, and in particular to a separation and purification process for high-purity ursodeoxycholic acid mother liquor. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid widely used clinically to treat cholesterol gallstones, primary biliary cirrhosis, and other cholestatic liver diseases. In industrial production, UDCA is typically prepared from chenodeoxycholic acid (CDCA) via a chemical or enzymatic epimerization reaction of the C7 hydroxyl group. However, due to reaction equilibrium and side reactions, the mother liquor inevitably contains unreacted chenodeoxycholic acid and the dehydroxylation byproduct lithocholic acid (LCA).

[0003] The separation and purification of ursodeoxycholic acid (UDCA) has always been a technical challenge in the pharmaceutical industry. This is because UDCA and its impurity chenodeoxycholic acid are epimers of each other, differing only in the stereoconfiguration of the C7 hydroxyl group in their steroid skeletons (UDCA is β-configuration, CDCA is α-configuration), while lithocholic acid is a structural analog lacking the C7 hydroxyl group. These three substances have almost identical steroidal core structures, resulting in extremely similar physicochemical properties such as solubility and polarity. This high degree of structural similarity makes them prone to co-crystallization or inclusion phenomena in conventional solvent systems, making complete separation through simple physical crystallization difficult.

[0004] Existing purification processes mainly rely on organic solvent crystallization or column chromatography. Solvent crystallization typically requires repeated recrystallization using volatile organic solvents such as ethyl acetate, chloroform, or acetone to achieve the purity required by the pharmacopoeia. This not only leads to cumbersome operations and extended production cycles but also results in low yields due to mother liquor loss from multiple crystallizations. Furthermore, the large-scale use of organic solvents poses flammable and explosive safety hazards and severe environmental pollution problems, and the unavoidable solvent residues in the product also affect drug safety. While column chromatography offers high separation precision, its consumption of silica gel packing material and eluent is enormous, and its limited throughput leads to high production costs, making it difficult to meet the needs of large-scale industrial production. In addition, some technologies have attempted chemical derivatization, converting bile acids into ester derivatives for separation before hydrolysis, but this increases the number of process steps and the risk of introducing exogenous impurities. Therefore, developing a separation and purification process that can efficiently identify differences in bile acid isomers, is environmentally friendly, and cost-effective is a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The technical problem solved by this invention is that in existing ursodeoxycholic acid (UDCA) separation and purification processes, the target product UDCA and its isomer impurity chenodeoxycholic acid and byproduct lithocholic acid have extremely similar chemical structures, differing only in the hydroxyl configuration or number of hydroxyl groups at the 7-carbon position of the steroid skeleton. This results in small differences in their solubility in conventional solvents and low separation coefficients. Existing technologies often rely on multiple recrystallizations, leading to significant yield losses and a lack of highly selective separation media for impurities with specific stereoconfigurations.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] This invention provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, comprising the following steps:

[0008] Step S1: Constructing the anchored aqueous phase: Add basic amino acids to the mother liquor containing ursodeoxycholic acid, stir to dissolve, and obtain an aqueous phase system;

[0009] Step S2, Chiral Extraction: A neutral chiral hydrophobic eutectic solvent is added to the aqueous system for mixed extraction. The eutectic solvent is formed by complexing the hydrogen bond acceptor L-menthol with the hydrogen bond donor long-chain fatty alcohol.

[0010] Step S3, Phase Separation: Allow the mixture to stand and separate into layers, remove the upper oil phase, and collect the lower aqueous phase;

[0011] Step S4, Crystallization: Add acid to the collected lower aqueous phase to adjust the pH value for crystallization. After solid-liquid separation, wash and dry to obtain ursodeoxycholic acid product.

[0012] By employing the above technical solution, a two-phase competitive partitioning system was constructed using the specific hydrophilic retention of the aqueous phase and the chiral hydrophobic extraction of the oil phase, significantly improving the separation efficiency of UDCA from impurities. The specific physicochemical mechanism is as follows:

[0013] First, the specific hydrogen bond retention effect in the aqueous phase: In step S1, basic amino acids (such as L-arginine or L-lysine) act as hydrophilic ligands, forming salts with the carboxyl groups of bile acid molecules through acid-base reactions, thus increasing their water solubility. More importantly, the β-oriented hydroxyl group at the C7 position and the α-oriented hydroxyl group at the C3 position of the UDCA molecule tend to form stable intermolecular multiple hydrogen bond complexes with the amino or guanidinium groups of the basic amino acid side chains. In contrast, the C7 position of CDCA is an α-oriented hydroxyl group, which, due to steric hindrance, has a weaker ability to form hydrogen bonds with basic amino acids; while LCA lacks a C7 hydroxyl group, resulting in even fewer hydrogen bond sites. Therefore, basic amino acids preferentially form strongly binding hydrophilic complexes with UDCA, retaining them primarily in the aqueous phase.

[0014] Second, the chiral cavity recognition function of the oil phase: In step S2, L-menthol, as a chiral source, assembles with long-chain fatty alcohols to form a eutectic solvent (DES) with a specific chiral microenvironment. This DES system exhibits strong hydrophobicity and, according to the principle of "like dissolves like," tends to dissolve the more hydrophobic CDCA and LCA. Simultaneously, the cyclic framework structure of L-menthol constructs a chiral cavity within the solvent, which has the ability to recognize the stereoconfiguration of guest molecules. The 7α-hydroxy configuration of CDCA results in a larger overall hydrophobic surface area, leading to a higher matching degree with the chiral hydrophobic cavity of L-menthol, thus making it easier to enter the oil phase; while UDCA, due to its tight binding with amino acids in the aqueous phase and low stereoconfiguration matching degree, is excluded from the oil phase.

[0015] Third, the amplification effect of the partition coefficient at the two-phase interface: In the mixed extraction system, the hydrogen bonding forces of the aqueous phase and the hydrophobic solvation forces of the oil phase are opposite in direction. For UDCA, the aqueous phase complexation forces dominate; for CDCA and LCA, the oil phase solvation forces dominate. This superposition of differentiated two-phase forces results in a relative separation coefficient of impurities for UDCA that is much higher than that of a single solvent system, thus enabling a significant reduction in impurity content with a single extraction.

[0016] Preferably, the long-chain fatty alcohol in step S2 is selected from one of 1-decyl alcohol, 1-dodecyl alcohol, or 1-tetradecyl alcohol; the molar ratio of L-menthol to the long-chain fatty alcohol is 1:1 to 1:2.

[0017] By employing the above technical solution, fatty alcohols with carbon chain lengths of C10-C14 are selected to control the hydrophobicity and viscosity balance of the eutectic solvent. Fatty alcohols within this range can form a stable hydrogen bond network with L-menthol, inhibiting its volatilization while maintaining the solvent's low-viscosity liquid characteristics at room temperature, which is beneficial for interphase mass transfer. The molar ratio is controlled between 1:1 and 1:2 to ensure an appropriate hydrogen bond donor-acceptor ratio, avoiding the precipitation of uncomplexed solids or damage to the solvent structure due to excessive component content.

[0018] Preferably, the neutral chiral hydrophobic eutectic solvent described in step S2 is prepared by the following method: L-menthol is mixed with a long-chain fatty alcohol and stirred and complexed at a temperature of 40-60°C for 30-60 minutes until a homogeneous transparent liquid phase is formed, and then cooled to room temperature.

[0019] By employing the above technical solution, thermally induced molecular thermal motion overcomes lattice energy, promoting the formation of intermolecular hydrogen bonds between components and constructing a homogeneous, thermodynamically stable liquid-phase system. Controlling temperature and time parameters ensures complete complexation while preventing the oxidation or volatilization of L-menthol due to high temperatures.

[0020] Preferably, the basic amino acid in step S1 is selected from L-arginine or L-lysine; the amount of the basic amino acid added is 0.4 to 0.6 times the total molar amount of bile acids in the mother liquor.

[0021] By employing the above technical solution, the guanidino group of L-arginine or the amino group of the side chain of L-lysine is used to provide multi-site hydrogen bond donors. The amount added is strictly controlled at 0.4 to 0.6 times the total molar amount of bile acids (i.e., around half equivalent), and a competitive complexation environment is constructed by limiting the ligand concentration. Since the binding constant of UDCA to basic amino acids is greater than that of CDCA and LCA, in the case of insufficient ligands, basic amino acids preferentially bind to UDCA and remain in the aqueous phase, while CDCA and LCA, with weaker binding forces, are "squeezed out" to the oil phase because they cannot obtain enough ligands. Thus, the separation selectivity is further enhanced by competitive equilibrium.

[0022] Preferably, in step S2, the volume ratio of the neutral chiral hydrophobic eutectic solvent to the aqueous phase system is 1:1 to 2:1; the temperature of the mixed extraction is 25°C, and the stirring time is 30 minutes.

[0023] By adopting the above technical solution, the appropriate ratio provides sufficient organic phase capacity to accommodate the extracted impurities; room temperature operation avoids the destruction of the hydrogen bond structure of the eutectic solvent by high temperature, maintaining its chiral recognition ability; and the 30-minute stirring time ensures that the system reaches the two-phase distribution equilibrium.

[0024] Preferably, the crystallization process described in step S4 is ultrasonic-assisted crystallization, which includes: slowly adding acid solution dropwise to the lower aqueous phase under stirring; when the pH value drops to the range of 6.0 to 6.5, turning on the ultrasonic generator and continuing to add acid solution dropwise until the pH value drops to 4.0 to 4.5; stopping the addition of acid solution dropwise; and performing crystal growth under the condition of maintaining ultrasound.

[0025] By employing the above technical solution, the microjets generated by ultrasonic cavitation and local high pressure are used to induce uniform nucleation in the nucleation-sensitive region of pH 6.0-6.5, controlling the number of primary crystal nuclei. In the subsequent crystal growth and crystal nucleation stages, ultrasonic vibration continuously acts on the solid-liquid interface, effectively preventing microcrystal aggregation and stripping impurities from the mother liquor adsorbed on the crystal surface, reducing lattice defects and mother liquor inclusions, thereby obtaining crystal products with uniform particle size distribution and high purity.

[0026] Preferably, the ultrasonic generator has a power of 300-400W and a frequency of 28-40kHz; the crystal growth time is 20-30 minutes.

[0027] By adopting the above technical solution, the selected power and frequency parameters can generate a cavitation effect of moderate intensity, which is sufficient to disperse crystal aggregates and avoids UDCA crystal transformation or degradation caused by local overheating due to excessive energy.

[0028] Preferably, the acid in step S4 is a hydrochloric acid solution; the cleaning after solid-liquid separation is to wash the filter cake with purified water until it is neutral; the drying is carried out under vacuum at 60°C.

[0029] By adopting the above technical solution, hydrochloric acid can completely convert water-soluble UDCA-amino acid salts into hydrophobic UDCA free acid precipitates; vacuum drying method prevents the product from oxidizing and discoloring in a high-temperature and oxygen-rich environment.

[0030] Preferably, the process further includes a solvent regeneration step, which comprises: collecting the upper oil phase separated and removed in step S3, adding an aqueous sodium hydroxide solution for back-extraction washing, allowing it to stand and separate into layers, discarding the lower alkaline layer, and washing the remaining upper oil phase with water until neutral, then returning it to step S2 as a regenerated eutectic solvent for recycling. The concentration of the aqueous sodium hydroxide solution is 0.5 mol / L, and the volume ratio of the aqueous sodium hydroxide solution to the upper oil phase during the back-extraction washing is 1:1.

[0031] By employing the above technical solution and utilizing the principle of acid-base neutralization, the fat-soluble impurities (CDCA and LCA) enriched in the oil phase are converted into water-soluble sodium salts, which are then transferred from the eutectic solvent phase to the alkaline aqueous phase, thereby achieving the purification and regeneration of the eutectic solvent. This process not only removes impurities but also avoids the use of complex distillation operations, reduces energy consumption and solvent loss, and realizes the recycling of the separation medium.

[0032] In summary, the present invention has at least one of the following beneficial technical effects:

[0033] 1. This invention constructs a two-phase competitive partitioning system consisting of "basic amino acid aqueous phase anchoring" and "chiral eutectic solvent oil phase extraction." Utilizing the slight differences in stereochemistry between ursodeoxycholic acid and impurities, it significantly amplifies the difference in their partition coefficients between the two phases. Specifically, by controlling the addition of basic amino acids at half-equivalent amounts, a competitive complexation mechanism is employed, preferentially retaining the more hydrophilic and tightly bound ursodeoxycholic acid in the aqueous phase, while repelling the less hydrophilic chenodeoxycholic acid and lithocholic acid into the oil phase. This allows for efficient removal of impurities in a single extraction process, achieving a product purity of over 99.5%.

[0034] 2. This invention eliminates the use of volatile organic solvents such as ethyl acetate and chloroform commonly used in traditional processes, instead employing a eutectic solvent composed of naturally sourced L-menthol and long-chain fatty alcohols as the extraction medium. This solvent is characterized by low volatility, non-toxicity, and good biocompatibility, reducing environmental pollution and solvent residue risks at the source. Simultaneously, the accompanying solvent regeneration process requires only simple alkaline washing and water washing to remove accumulated impurities, enabling the recycling of the extractant and reducing production costs and wastewater discharge.

[0035] 3. This invention introduces a segmented ultrasound-assisted crystallization technology. During the critical nucleation stage, the ultrasonic cavitation effect induces uniform nucleation, and during crystal growth, continuous ultrasound inhibits crystal aggregation and mother liquor inclusion. This control method effectively solves the problems of crystal aggregation and impurity encapsulation that easily occur in traditional natural crystallization processes, resulting in a final ursodeoxycholic acid product with intact crystal form, narrow particle size distribution, and high purity, thus improving the pharmaceutical performance of the product. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0037] The L-menthol, 1-dodecyl alcohol, 1-decyl alcohol, 1-tetradecyl alcohol, L-arginine, L-lysine, ursodeoxycholic acid, chenodeoxycholic acid, lithocholic acid, caprylic acid, choline chloride, urea, tetrabutylammonium chloride, decanoic acid, hydrochloric acid, and sodium hydroxide used in the embodiments and comparative examples of this invention are all commercially available analytical grade reagents and can be used directly after purchase. Among them, L-menthol and L-arginine are both levorotatory isomers with an optical purity greater than 98%.

[0038] Preparation Example 1:

[0039] This preparation example provides a method for preparing a neutral chiral hydrophobic eutectic solvent for the separation and purification of ursodeoxycholic acid, comprising the following steps:

[0040] Accurately weigh 156.3 g (1.0 mol) of L-menthol and 279.5 g (1.5 mol) of 1-dodecyl alcohol, and mix them in a glass reactor equipped with a heating jacket and a mechanical stirrer. Turn on the heating circulation system and maintain the system temperature at 50°C, while simultaneously turning on the mechanical stirrer at 200 rpm. Complexe the mixture under constant temperature and stirring conditions for 40 minutes until the solid particles completely disappear and the system transforms into a colorless, transparent, and homogeneous oily liquid. Stop heating and stirring, and allow the resulting liquid to cool naturally to room temperature (25°C) to obtain a eutectic solvent of L-menthol / 1-dodecyl alcohol with a molar ratio of 1:1.5. Seal and store for later use.

[0041] Preparation Example 2:

[0042] This preparation example provides a method for preparing a neutral chiral hydrophobic eutectic solvent for the separation and purification of ursodeoxycholic acid, comprising the following steps:

[0043] Accurately weigh 156.3 g (1.0 mol) of L-menthol and 186.3 g (1.0 mol) of 1-dodecyl alcohol, and mix them in a reaction vessel. Maintain the system temperature at 45°C and the stirring speed at 150 rpm for 30 minutes until a clear, transparent single liquid phase is formed. After cooling to room temperature, a eutectic solvent of L-menthol / 1-dodecyl alcohol with a molar ratio of 1:1 is obtained.

[0044] Preparation Example 3:

[0045] This preparation example provides a method for preparing a neutral chiral hydrophobic eutectic solvent for the separation and purification of ursodeoxycholic acid, comprising the following steps:

[0046] Accurately weigh 156.3 g (1.0 mol) of L-menthol and 372.7 g (2.0 mol) of 1-dodecyl alcohol, and mix them in a reaction vessel. Maintain the system temperature at 55°C and the stirring speed at 250 rpm, stirring continuously for 50 minutes until a clear, transparent single liquid phase is formed. After cooling to room temperature, a eutectic solvent of L-menthol / 1-dodecyl alcohol with a molar ratio of 1:2 is obtained.

[0047] Preparation Example 4:

[0048] This preparation example provides a method for preparing a neutral chiral hydrophobic eutectic solvent for the separation and purification of ursodeoxycholic acid, comprising the following steps:

[0049] Accurately weigh 156.3 g (1.0 mol) of L-menthol and 237.4 g (1.5 mol) of 1-decyl alcohol, and mix them in a reaction vessel. Maintain the system temperature at 40 °C and the stirring speed at 200 rpm for 30 minutes until a clear, transparent single liquid phase is formed. After cooling to room temperature, a eutectic solvent of L-menthol / 1-decyl alcohol with a molar ratio of 1:1.5 is obtained.

[0050] Preparation Example 5:

[0051] This preparation example provides a method for preparing a neutral chiral hydrophobic eutectic solvent for the separation and purification of ursodeoxycholic acid, comprising the following steps:

[0052] Accurately weigh 156.3 g (1.0 mol) of L-menthol and 321.6 g (1.5 mol) of 1-tetradecyl alcohol, and mix them in a reaction vessel. Given the slightly higher melting point of 1-tetradecyl alcohol, control the system temperature at 60°C and the stirring speed at 300 rpm, stirring continuously for 60 minutes to ensure complete melting and complexation of the components to form a clear, transparent single liquid phase. After cooling to room temperature, a eutectic solvent of L-menthol / 1-tetradecyl alcohol with a molar ratio of 1:1.5 is obtained.

[0053] In the following examples and comparative examples, to ensure the comparability of experimental data, artificially prepared UDCA stock solution was used as the raw material. The preparation method of this simulated solution is as follows: ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), and lithocholic acid (LCA) are dissolved in a dilute sodium hydroxide aqueous solution, the pH is adjusted to 12.0, and a mixed solution with UDCA concentration of 40 g / L, CDCA concentration of 40 g / L, and LCA concentration of 5 g / L is prepared.

[0054] Example 1:

[0055] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0056] (1) Anchoring complexation: Take 100 mL of the above UDCA mother liquor simulation solution and place it in the extraction vessel. Add 1.9 g of L-arginine (about 0.5 times the total molar amount of bile acids), stir to dissolve, and utilize the guanidinium group of arginine to form a specific hydrophilic anchoring effect with UDCA to construct an aqueous system.

[0057] (2) Chiral extraction: 100 mL of the L-menthol / 1-dodecyl alcohol eutectic solvent (volume ratio O / A=1:1) obtained in Preparation Example 1 was added to the aqueous phase system and mechanically stirred for 30 minutes at 25°C and 300 rpm. The chiral microenvironment of the eutectic solvent was used to preferentially extract impurities such as CDCA and LCA.

[0058] (3) Phase separation: Stop stirring and let stand for 15 minutes to separate the layers. The system quickly separates into an upper oil phase (rich in impurities) and a lower water phase (rich in UDCA). Collect the lower water phase.

[0059] (4) Ultrasonic crystallization: Place the collected lower aqueous phase in an ultrasonic crystallizer, control the temperature at 20℃, and slowly add 1mol / L hydrochloric acid solution while stirring. When the pH drops to 6.5, turn on the ultrasonic generator (300W power, 40kHz frequency), continue adding hydrochloric acid until the pH drops to 4.5, stop adding, and continue ultrasonic crystallization for 20 minutes.

[0060] (5) Post-processing: The filtered slurry and filter cake are washed three times with purified water until neutral, and then dried in a vacuum drying oven at 60°C for 6 hours to obtain high-purity ursodeoxycholic acid product.

[0061] Example 2:

[0062] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0063] (1) Anchoring complexation: Take 100 mL of the above UDCA mother liquor simulation solution, add 1.9 g of L-arginine, and stir to dissolve evenly.

[0064] (2) Chiral extraction: 200 mL of the L-menthol / 1-dodecyl alcohol (1:1) eutectic solvent (volume ratio O / A=2:1) ​​obtained in Preparation Example 2 was added to the aqueous phase and stirred for 30 minutes at 25°C and 300 rpm. The removal capacity of LCA, which is more hydrophobic, was enhanced by increasing the amount of solvent.

[0065] (3) Phase separation: Let stand for 15 minutes to separate and collect the lower water phase.

[0066] (4) Ultrasonic crystallization: Place the lower aqueous phase in a crystallizer and add hydrochloric acid at 20°C to adjust the pH. When the pH drops to 6.0, turn on the ultrasound (300W, 40kHz), adjust the final pH to 4.2, and continue ultrasonic crystal growth for 20 minutes.

[0067] (5) Post-processing: filtration, water washing, vacuum drying to obtain ursodeoxycholic acid product.

[0068] Example 3:

[0069] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0070] (1) Anchoring complexation: Take 100 mL of the above UDCA mother liquor simulation solution, add 1.9 g L-arginine, and stir to dissolve.

[0071] (2) Chiral extraction: 100 mL of the L-menthol / 1-decyl alcohol eutectic solvent obtained in Preparation Example 4 was added to the aqueous phase and stirred at 25 °C and 300 rpm for 30 minutes to verify the separation effect of solvent systems constructed with fatty alcohols of different chain lengths.

[0072] (3) Phase separation: Let stand for 15 minutes to separate and collect the lower water phase.

[0073] (4) Ultrasonic crystallization: Same as step (4) in Example 1, with the endpoint pH controlled at 4.5.

[0074] (5) Post-processing: filtration, water washing, drying to obtain ursodeoxycholic acid product.

[0075] Example 4:

[0076] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0077] (1) Anchoring complexation: Take 100 mL of the above UDCA mother liquor simulation solution, add 1.6 g of L-lysine (equal molar replacement of L-arginine), stir to dissolve, and use the side chain amino of lysine to play an auxiliary anchoring role.

[0078] (2) Chiral extraction: Add 100 mL of the eutectic solvent obtained in Preparation Example 1 to the aqueous phase and stir for 30 minutes at 25°C and 300 rpm.

[0079] (3) Phase separation: Let stand for 20 minutes to separate and collect the lower water phase.

[0080] (4) Ultrasonic crystallization: Same as step (4) in Example 1.

[0081] (5) Post-processing: filtration, water washing, drying to obtain ursodeoxycholic acid product.

[0082] Example 5:

[0083] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0084] (1) Anchoring and bonding: Same as step (1) in Example 1.

[0085] (2) Chiral extraction: Same as step (2) in Example 1.

[0086] (3) Phase separation: Same as step (3) in Example 1.

[0087] (4) Ultrasonic crystallization: Change the timing of ultrasonic intervention. When the pH of the lower aqueous phase is adjusted to 5.0 (at which point the system begins to become turbid), turn on the ultrasonic (power 400W, frequency 28kHz) immediately, continue to add hydrochloric acid until pH 4.0, and keep ultrasonic stirring for 30 minutes to regulate the crystal particle size distribution.

[0088] (5) Post-processing: filtration, water washing, drying to obtain ursodeoxycholic acid product.

[0089] Example 6:

[0090] This embodiment provides a process for separating and purifying high-purity ursodeoxycholic acid mother liquor, including the following steps:

[0091] (1) Solvent regeneration: Collect the upper oil phase (containing extracted CDCA and LCA) separated in step (3) of Example 1, add an equal volume of 0.5 mol / L sodium hydroxide solution for back-extraction and washing, stir for 15 minutes and let stand to separate the layers, discard the lower alkaline solution (containing impurity salts), and wash the upper oil phase once with purified water until neutral to obtain the regenerated eutectic solvent.

[0092] (2) Circulating extraction: Take 100 mL of fresh UDCA mother liquor simulation solution, add 1.9 g of L-arginine to dissolve it, add 100 mL of the above-mentioned regenerated eutectic solvent, and extract and separate the phases according to the process conditions of Example 1.

[0093] (3) Subsequent processing: The separated aqueous phase was subjected to ultrasonic crystallization and drying according to steps (4)-(5) of Example 1 to obtain ursodeoxycholic acid product.

[0094] Comparative Example 1:

[0095] Compared with Example 1, the difference is that the extraction solvent used in step (2) is an equal volume of ethyl acetate, while the rest are the same.

[0096] Comparative Example 2:

[0097] Compared with Example 1, the difference is that L-arginine was not added in step (1), and the subsequent extraction operation was carried out directly. All other aspects are the same.

[0098] Comparative Example 3:

[0099] Compared with Example 1, the difference is that DL-menthol (racemate) was used instead of L-menthol when preparing the eutectic solvent, otherwise the same.

[0100] Comparative Example 4:

[0101] Compared with Example 1, the difference is that the extraction solvent used in step (2) is pure 1-dodecyl alcohol (a non-eutectic system), while the rest are the same.

[0102] Comparative Example 5:

[0103] Compared with Example 1, the difference is that the ultrasonic generator is not turned on in step (4), and hydrochloric acid is added dropwise under mechanical stirring at 200 rpm for conventional crystallization. The rest are the same.

[0104] Test Example 1:

[0105] This test case verifies the specific retention ability of amino acid components in the aqueous phase for ursodeoxycholic acid (UDCA), and the chiral recognition ability of the oil phase eutectic solvent for isomers.

[0106] 1. Aqueous-phase specific complexation effect test

[0107] The experimental steps are as follows:

[0108] (1) Accurately weigh UDCA standard, dissolve it in deionized water, add an equimolar amount of L-arginine, prepare an aqueous solution with UDCA concentration of 10 mmol / L, and measure the pH value to be 10.24, which is used as the experimental group.

[0109] (2) Dissolve another UDCA standard in deionized water, add sodium hydroxide solution to adjust the pH to 10.24, so that the UDCA concentration is 10 mmol / L, and use it as a control group.

[0110] (3) Take 20 mL of the experimental group and the control group solution respectively and place them in a separatory funnel, and add 20 mL of n-octanol (as an inert organic phase reference).

[0111] (4) Shake at 200 rpm for 4 hours in a constant temperature shaker at 25℃, let stand and separate the layers, and take the upper organic phase and the lower aqueous phase respectively.

[0112] (5) The equilibrium concentration of UDCA in the two phases was determined by HPLC, and the partition coefficient (the ratio of the concentration of the organic phase to the concentration of the aqueous phase) was calculated.

[0113] The test data is shown in Table 1.

[0114] Table 1. Effect of L-arginine on the partitioning behavior of UDCA in the n-octanol-water system:

[0115]

[0116] 2. Oil phase stereoselectivity recognition test

[0117] The experimental steps are as follows:

[0118] (1) Prepare a mixed aqueous solution containing UDCA and chenodeoxycholic acid (CDCA) at a concentration of 5 mmol / L and adjust the pH to 7.0.

[0119] (2) The L-menthol / 1-dodecyl alcohol eutectic solvent obtained in Preparation Example 1 was used as the extractant for the experimental group; 1-dodecyl alcohol was used as the extractant for the control group.

[0120] (3) Mix the above aqueous solution and extractant at a volume ratio of 1:1 and shake at 25°C for 4 hours.

[0121] (4) After standing and separating the phases, the concentrations of UDCA and CDCA in each phase were determined by HPLC.

[0122] (5) Calculate the partition coefficient of each component and calculate the separation factor by the ratio of the partition coefficient of CDCA to the partition coefficient of UDCA.

[0123] The test data is shown in Table 2.

[0124] Table 2. Comparison of extraction performance between eutectic solvents and conventional solvents:

[0125]

[0126] The data analysis based on Tables 1 and 2 is as follows:

[0127] Regarding the aqueous phase retention mechanism, in the control group under purely alkaline conditions (pH 10.24), the partition coefficient of UDCA was 0.4, indicating that a considerable proportion still allocated to the organic phase. In the experimental group, after the introduction of L-arginine, the partition coefficient significantly decreased to 0.079. The data confirm that the guanidino group of L-arginine forms a strongly hydrophilic complex with UDCA, effectively anchoring the target product to the aqueous phase.

[0128] Regarding the oil phase recognition mechanism, the separation factor was only 1.18 when 1-dodecyl alcohol was used in the control group, indicating that conventional fatty alcohols have no significant selectivity for UDCA and CDCA. In the experimental group, using a eutectic solvent system, the separation factor increased to 3.71. This result demonstrates that the chiral supramolecular network of the eutectic solvent has preferential extraction capability for CDCA, achieving efficient separation of isomers by utilizing steric hindrance differences.

[0129] Test Example 2:

[0130] This test case evaluates the contribution of each process element to the separation efficiency by comparing the key quality attributes and process indicators of the products obtained from Examples 1-6 with those from Comparative Examples 1-5.

[0131] The experimental steps are as follows:

[0132] (1) Determination of product purity and impurity content: High performance liquid chromatography (HPLC) was used for detection. The chromatographic column was a C18 reversed-phase column; the mobile phase was acetonitrile-0.02 mol / L potassium dihydrogen phosphate solution (50:50); the detection wavelength was 210 nm. The dried products of each group were accurately weighed, dissolved and diluted, and then injected. The chromatograms were recorded. The purity of UDCA was calculated by the area normalization method, and the residual percentages of impurities chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) were determined.

[0133] (2) Overall yield determination: Collect the crystallized, filtered and vacuum dried product to constant weight, and weigh it. Calculation formula: Yield (%) = (Weight of dried product / Theoretical UDCA content in raw material mother liquor) × 100%.

[0134] The test data is shown in Table 3.

[0135] Table 3 Summary of purity, impurity content, and yield data for each example and comparative product:

[0136]

[0137] Based on the data in Table 3, the impact analysis of each process element is as follows:

[0138] The purity of the product in Example 1 was 99.62%, while the purities of Comparative Example 1 (ethyl acetate) and Comparative Example 4 (pure 1-dodecyl alcohol) were only 88.45% and 89.67%, respectively, with residual CDCA levels as high as 10.23% and 9.14%, respectively. The data indicate that conventional solvents cannot effectively identify the subtle structural differences between UDCA and CDCA, while the eutectic solvent system constructed in this invention significantly improves the extraction and removal capacity of the impurity CDCA through its specific supramolecular structure.

[0139] Compared with Comparative Example 1 and Comparative Example 3 (DL-menthol), when the chiral component in the solvent was replaced by the racemate, the product purity decreased to 91.23% and the residual CDCA increased to 7.85%. This confirms that the chiral microenvironment constructed by L-menthol in the solvent is the key to achieving isomer separation. The racemate, lacking a uniform chiral cavity, loses its specific adsorption capacity for CDCA.

[0140] Compared with Comparative Example 1 and Comparative Example 2 (without L-arginine), the absence of L-arginine caused the total yield to plummet from 92.45% to 68.34%, and the residual CDCA content in the product increased (4.82%). This indicates that L-arginine not only significantly reduced the loss of UDCA to the organic phase through the anchoring effect, but also indirectly assisted the selective extraction of impurities by the oil phase (extractant) by enhancing the stability of UDCA in the aqueous phase. Example 4, using L-lysine, also achieved good results (purity 99.35%, yield 90.62%), proving that this anchoring mechanism is universal in basic amino acid compounds.

[0141] Comparing Example 1 and Comparative Example 5 (without ultrasound), the product purity without ultrasound assistance was 98.12%, with slightly higher residual impurities. Example 5 further improved the purity to 99.78% by optimizing the timing of ultrasound intervention. The cavitation effect of ultrasound effectively suppressed crystal aggregation and mother liquor encapsulation, improving the purity of the final product.

[0142] Data from Example 6 shows that after five cycles of solvent recycling, the product purity remained at 99.25% and the yield remained above 90%, indicating that the eutectic solvent system is structurally stable, the regeneration process is effective, and it has the potential for industrial application.

[0143] Test Example 3:

[0144] This test case aims to verify the effect of ultrasound-assisted crystallization process on improving the microstructure and packing properties of the product, and to evaluate the chemical stability and separation efficiency of the eutectic solvent system during multiple cycles.

[0145] The experimental steps are as follows:

[0146] (1) Crystal grain size distribution test:

[0147] Dry UDCA powder samples prepared in Example 1 (ultrasonic crystallization process) and Comparative Example 5 (conventional mechanical stirring crystallization process) were used. Particle size distribution was determined using a laser particle size analyzer. The samples were dispersed in pure water (with a trace amount of Tween-80 added as a dispersant), and ultrasonic dispersion was performed for 1 minute to eliminate pseudo-agglomeration. Measurements were taken within a suitable range of light shading, and the particle size distribution curves and D10, D50, and D90 values ​​were recorded. The span was calculated using the formula: Span = (D90 − D10) / D50.

[0148] (2) Bulk density and flowability test:

[0149] Take 10g of each of the two samples mentioned above and slowly pour them into graduated cylinders. Record the initial volume and calculate the loose density. Then fix the graduated cylinders on a tapped density meter, set the vibration frequency to 250 times / min, and vibrate until the volume no longer changes. Record the final volume and calculate the tapped density.

[0150] (3) Solvent cycling performance verification (5 cycles of pressure test):

[0151] To verify the long-term stability of the solvent, a continuous cycle experiment was designed, referring to the solvent regeneration method described in Example 6:

[0152] 1) First cycle (i.e. Example 1): Extraction was performed using freshly prepared eutectic solvent, the upper oil phase was separated, and product data was recorded.

[0153] 2) Second cycle (i.e., Example 6): Collect the upper oil phase after the first cycle and regenerate it by back-extraction with NaOH solution according to step (1) of Example 6. Mix the regenerated solvent with fresh UDCA mother liquor for extraction and record the data.

[0154] 3) Cycles 3-5: Repeat the above steps of "collecting the oil phase - washing and regenerating with alkali - adding to the next extraction" until the 5th cycle is completed.

[0155] 4) Sampling points: Focus on monitoring the product purity, yield, and solvent phase separation after the 1st (fresh), 3rd, and 5th cycles.

[0156] The test data are shown in Tables 4 and 5.

[0157] Table 4. Test data on crystal grain size distribution and packing density:

[0158]

[0159] Table 5. Separation performance monitoring during solvent recycling:

[0160]

[0161] According to the data in Table 4, the particle size range of the product in Example 1 was 1.27, significantly lower than that of Comparative Example 5 (2.77). In Comparative Example 5, the D90 was as high as 168.45 μm, while the D10 was only 5.14 μm, indicating that under conventional mechanical stirring, the crystal growth process was uncontrollable, resulting in severe crystal agglomeration and fine-grained inclusions. In Example 1, the introduction of an ultrasonic field effectively broke up the primary agglomerates through the localized high-pressure microjets generated by cavitation, promoting secondary nucleation and resulting in a more uniform crystal size distribution. Regarding bulk density, the loose packing density (0.48 g / mL) and tap density (0.62 g / mL) of Example 1 were significantly higher than those of Comparative Example 5, indicating that the product obtained by ultrasonic crystallization has a more regular and dense crystal form, better flowability, and is beneficial for subsequent formulation processes such as tableting or capsule filling.

[0162] According to the data in Table 5, in the solvent cycling experiment, as the number of cycles increased, the solvent appearance gradually changed from colorless to light yellow, and the phase separation time slightly increased from 14.5 minutes to 15.8 minutes, presumably due to the accumulation of trace pigments or impurities in the organic phase. However, from the perspective of key indicators, the UDCA purity of the product obtained in the 5th cycle remained at 99.25%, and the total yield was 90.33%, which was not significantly different from the data of the 1st cycle (purity 99.62%, yield 92.45%). This result confirms that impurities such as CDCA and LCA enriched in eutectic solvents can be effectively removed by a simple alkaline washing and back-extraction process. The chiral recognition structure of the solvent remains stable during multiple acid-base environment switching, demonstrating good potential for industrial recycling.

[0163] Based on the data from Test Examples 1-3, the oil-water interface competitive recognition system constructed in this invention achieves efficient separation of UDCA. Test Example 1 data shows that L-arginine reduces the partition coefficient of UDCA in the aqueous phase to 0.079, retaining it in the aqueous phase; while the L-menthol-based eutectic solvent achieves a separation factor of 3.71 for CDCA, superior to conventional fatty alcohols. Test Example 2 results show that if racemic menthol is used, the residual CDCA content in the product increases to 7.85%; if L-arginine is missing, the total yield decreases to 68.34%. This confirms that the stereorecognition effect of chiral solvents and the retention effect of aqueous phase anchoring agents are key to improving purity and yield.

[0164] Regarding crystal properties, Test Example 3 showed that ultrasonic-assisted crystallization reduced the product particle size range from 2.77 with conventional stirring to 1.27, and increased the tap density to 0.62 g / mL, improving crystal agglomeration and increasing bulk density. Furthermore, solvent recycling experiments showed that after five regeneration cycles, the product purity remained at 99.25%, and the yield remained above 90%, indicating that the eutectic solvent system maintained structural stability during multiple acid-base back-extraction operations and could meet the requirements for recycling.

[0165] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for separating and purifying high-purity ursodeoxycholic acid mother liquor, characterized in that, Includes the following steps: S1. Constructing the anchored aqueous phase: Add basic amino acids to the mother liquor containing ursodeoxycholic acid, stir to dissolve, and obtain an aqueous phase system; S2. Chiral extraction: A neutral chiral hydrophobic eutectic solvent is added to the aqueous system for mixed extraction. The eutectic solvent is formed by complexing a hydrogen bond acceptor, L-menthol, with a hydrogen bond donor, a long-chain fatty alcohol. The long-chain fatty alcohol is selected from one of 1-decyl alcohol, 1-dodecyl alcohol, or 1-tetradecyl alcohol. S3. Phase separation: Allow the mixture to stand and separate into layers, remove the upper oil phase and collect the lower aqueous phase; S4. Crystallization: Add acid to the collected lower aqueous phase to adjust the pH value for crystallization. After solid-liquid separation, wash and dry to obtain ursodeoxycholic acid product.

2. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, The molar ratio of L-menthol to the long-chain fatty alcohol in step S2 is 1:1 to 1:

2.

3. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, The neutral chiral hydrophobic eutectic solvent described in step S2 is prepared by the following method: Mix L-menthol with long-chain fatty alcohols and stir to complex at 40–60°C for 30–60 minutes until a homogeneous and transparent liquid phase is formed. Cool to room temperature.

4. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, The basic amino acid mentioned in step S1 is selected from L-arginine or L-lysine; The amount of basic amino acids added is 0.4 to 0.6 times the total molar amount of bile acids in the mother liquor.

5. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, In step S2, the volume ratio of the neutral chiral hydrophobic eutectic solvent to the aqueous phase system is 1:1 to 2:1; the temperature of the mixed extraction is 25°C, and the stirring time is 30 minutes.

6. The separation and purification process for high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, The crystallization process described in step S4 is ultrasound-assisted crystallization, which includes: While stirring, acid solution is slowly added dropwise to the lower aqueous phase. When the pH value drops to the range of 6.0 to 6.5, the ultrasonic generator is turned on, and acid solution is added dropwise until the pH value drops to 4.0 to 4.

5. Then, the addition of acid solution is stopped, and crystal growth is carried out while maintaining the ultrasonic condition.

7. The separation and purification process for high-purity ursodeoxycholic acid mother liquor according to claim 6, characterized in that, The ultrasonic generator has a power of 300-400W and a frequency of 28-40kHz; the crystal growth time is 20-30 minutes.

8. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 1, characterized in that, The acid mentioned in step S4 is a hydrochloric acid solution; the cleaning after solid-liquid separation is to wash the filter cake with purified water until it is neutral; the drying is carried out under vacuum at 60°C.

9. The separation and purification process for high-purity ursodeoxycholic acid mother liquor according to any one of claims 1-8, characterized in that, It also includes a solvent regeneration step, which includes: collecting the upper oil phase separated and removed in step S3, adding sodium hydroxide aqueous solution for back-extraction washing, allowing it to stand and separate into layers, discarding the lower alkaline solution, and then washing the remaining upper oil phase with water until neutral, and returning it to step S2 for recycling as a regenerated eutectic solvent.

10. The separation and purification process of high-purity ursodeoxycholic acid mother liquor according to claim 9, characterized in that, The concentration of the sodium hydroxide aqueous solution is 0.5 mol / L, and the volume ratio of the sodium hydroxide aqueous solution to the upper oil phase during the back-extraction washing is 1:1.

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