FIELD OF INVENTION:
The present invention reports novel purification processes for Chenodeoxycholic Acid (CDCA), 7-Ketolithocholic acid (KLCA as well as Ursodeoxycholic acid (UDCA). Furthermore, the present invention also reports process for the preparation as well as purification of Silyl- Ursodeoxycholic acid (Silyl-UDCA).
BACKGROUND OF THE INVENTION:
Ursodeoxycholic acid (INN, BAN and AAN), also known as Ursodiol (USAN) and the abbreviation UDCA, from the root-word for bear urso, as bear bile contains the substance, is one of the secondary bile acids, which are metabolic byproducts of intestinal bacteria. Ursodiol has been shown to reduce hyperglycemia in type 2 diabetes mellitus.
Primary bile acids are produced by the liver and stored in the gall bladder. When secreted into the intestine, primary bile acids can be metabolized into secondary bile acids by intestinal bacteria. Primary and secondary bile acids help the body digest fats. Ursodeoxycholic acid helps regulate cholesterol by reducing the rate at which the intestine absorbs cholesterol molecules while breaking up micelles containing cholesterol. Because of this property, Ursodeoxycholic acid is used to treat (cholesterol) gallstones non-surgically. It is also used to relieve itching in pregnancy for some women who suffer obstetric cholestasis.
While some bile acids are known to be colon tumor promoters (e.g. deoxycholic acid), others such as Ursodeoxycholic acid are thought to be chemo preventive, perhaps by inducing cellular differentiation and/or cellular senescence in colon epithelial cells. It is believed to inhibit apoptosis.
Ursodiol may be used for biliary stasis, also known as intrahepatic cholestasis of pregnancy, to relieve the symptoms of itching, and to decrease infant mortality rate, which is generally believed to be 10% when Ursodiol is not administered in this fairly rare, and largely undiagnosed pregnancy related disorder. Maternal mortality from hemorrhage is another outcome of the disease, but Urso is not believed to be the preventive cure for this outcome, and to decrease bile absorption.
In children, Ursodeoxycholic acid use is not licensed, as its safety and effectiveness have not been established. Evidence is accumulating that Ursodeoxycholic acid is ineffective and unsafe in neonatal hepatitis and neonatal cholestasis.
In double of the recommended daily dose, Ursodeoxycholic acid reduces elevated liver enzyme levels in those with primary sclerosing cholangitis, but its use was associated with an increased risk of serious adverse events (the development of cirrhosis, varices, death or liver transplantation) in patients who received Ursodeoxycholic acid compared with those who received placebo.
Serious adverse events, were more common in the Ursodeoxycholic acid group than the placebo group. The risk was 2.1 times greater for death, transplantation, or minimal listing criteria in patients on Ursodeoxycholic acid than for those on placebo.
WO2014020024A1, PCT WIPO application describes a process for the synthesis of Ursodeoxycholic acid wherein the purification of the crude Ursodeoxycholic acid (containing approximately 13-15% of Chenodeoxycholic acid impurity) takes place first passing through a salification with imidazole and a subsequent purification via "methyl ester", which allows a finished product with an extremely low content of known "cheno and "litho" impurities to be obtained. The present invention also describes the recovery steps of cholic acid and 3a-hydroxy-7-ketocholanic acid from the mother liquors of process intermediates.
CN101781350B provides a method for purification of a mixed solvent of Ursodeoxycholic acid, a mixed solvent containing formulation, the mixture was washed and dried to give acid Ursodeoxycholic acid three steps. Feature of the present process is the use of 99-20 parts by weight of a mixed solvent composed of small molecules and small molecule alcohol ester 1-80 parts than formulated. The mixed solvent may selectively dissolve Chenodeoxycholic acid mixture and other impurities retained Ursodeoxycholic acid. The mixed solvent of an alcohol only retains the ability to dissolve Chenodeoxycholic acid stronger, and retains the characteristics of esters of Chenodeoxycholic acid selectively dissolves.
CA1134815A relates to a purification process of ursodeoxycholic acid which comprises treating an alkali metal salt of ursodeoxycholic acid to be purified in aqueous solu-tion in the presence of chloroform with an acid, to the product collection-lir crystallized formed and recovering therefrom purified ursodeoxycholic acid. The method of the invention allows to obtain an ursodeoxycholic acid of very high purity.
CN106928306A discloses a purification method of Ursodesoxycholic acid. The method comprises the following steps: firstly, preparing a triethylammonium Ursodesoxycholicate; secondly, hydrolyzing the triethylammonium Ursodeoxycholicate; and finally, performing recrystallization and refining. The purification method of Ursodeoxycholic acid disclosed by the invention is simple in step and easy to operate. The used solvent is simple and easily available, and small in energy consumption. The purity of the refined Ursodeoxycholic acid obtained by the purification method reaches up to 99.8%.
CN102702294A provides a purification and preparation method for direct extraction and synthesis of Ursodeoxycholic acid from pig gall paste or scraps thereof. The purification and preparation method comprises: removing non-cholic acid impurities by pretreatment with a low content of 15-35% of the pig gall paste or the scraps thereof, removing the Cholic acid impurities below Chenodeoxycholic acid in thin layer chromatography by oxidation of bromine or chromium, removing the cholic acid impurities above Ursodeoxycholic acid in the thin layer chromatography by conventional reduction, preparing Ursodeoxycholic acid proprietary complex crystals, and hydrolyzing to obtain the Ursodeoxycholic acid of pharmaceutical grade.
The Journal Prep Biochem. 1982;12(4):307-21, discloses the enzymatic and chemical synthesis of ursodeoxycholic and chenodeoxycholic acid from cholic acid. Herein three approaches to the synthesis of ursodeoxycholic acid (UDC) from cholic acid have been investigated: (i) oxidation of cholic acid to 3 alpha, 7 alpha-dihydroxy-12 keto-5 beta-cholanoic acid (12K-CDC) with Clostridium group P 12 alpha-hydroxysteroid dehydrogenase (HSDH), isomerization of 12K-CDC to 3 alpha, 7 beta-dihydroxy-12 keto-5 beta-cholanoic acid (12K-UDC) with Clostridium absonum 7 alpha- and 7 beta-HSDH and reduction of 12K-UDC by Wolff-Kishner to UDC; (ii) isomerization of cholic acid to ursocholic acid (UC) by C. absonum 7 alpha- and 7 beta-HSDH, oxidation of UC to 12K-UDC with Clostridium group P 12 alpha-HSDH and Wolff-Kishner reduction of 12K-UDC to UDC;
(iii) oxidation of cholic acid to 12K-CDC by Clostridium group P 12 alpha-HSDH, Wolff-Kishner reduction of 12K-CDC to chenodeoxycholic acid (CDC) and isomerization of CDC to UDC using whole cell cultures of C. absonum. In the first two approaches (using cell free systems) the yields of desired product were relatively low primarily due to the formation of various side products. The third method proved the most successful giving an overall yield of 37% (UDC) whose structure was verified by mass spectroscopy of the methyl ester.
The Journal “Huang and Cao Bioresources and Bioprocessing (2015) 2:27” shows that Ursodeoxycholic acid can be prepared by electroreduction with high stereoselectivity. The method developed here offers a potential application for large-scale production of ursodeoxycholic acid and an interesting reference to asymmetric electrochemical reduction of the keto group.
The Journal “Acta Chemica Scandinavica 17(1963), 173-186” provides methods of preparation of Chenodeoxycholic acid & other intermediates for preparation of Ursodiol (UDCA). The method comprises the steps of performing selective oxidation on the Chenodeoxycholic acid to obtain 7-O-Lithocholic acid; performing selective reduction on the 7-O-lithocholic acid to obtain the Ursodeoxycholic acid containing a certain amount of the Chenodeoxycholic acid; reacting the obtained Ursodeoxycholic acid with a silicified agent to form Ursodeoxycholic acid silicide which is insoluble in organic solvents, wherein the Chenodeoxycholic acid contained in the Ursodeoxycholic acid reacts with the silicified agent to form Chenodeoxycholic acid silicide which is soluble in organic solvents; and acidizing and hydrolyzing the obtained Ursodeoxycholic acid silicide to obtain a high pure Ursodeoxycholic acid product.
US Patent Application 4,316,848 is directed to the preparation of some new derivatives of UDCA that are useful for the purification of UDCA. Since long time UDCA is used in the human medicine as a colagogue but only very recently it has been discovered that it is endowed with gallstone dissolving activity. UDCA is epimeric with CDCA in respect to the hydroxyl group at C7. UDCA may be prepared from CDCA by steric conversion of the hydroxyl group at C7, after having protected the hydroxyl group at C3 in a selective way.
For this purpose, the 7-a-hydroxyl group is oxidized to the corresponding 7-keto group and then the 7-keto group is reduced as to obtain the 7-ß-hydroxyl group. Unfortunately, no reducing agent is able to reduce quantitatively the 7-keto group to the 7-ß-hydroxyl group. In practice UDCA thus obtained contains from 10 to 40% of CDCA. This patent describes some new silyl-derivatives of UDCA possess different solubility rates in many organic solvents with respect to the corresponding derivatives of CDCA so that it is possible to separate UDCA from CDCA and to prepare UDCA substantially free from CDCA on industrial scale.
US Patent 4,579,819 relates to a method for production of Ursodeoxycholic acid, and more specifically to a method for production of Ursodeoxycholic acid, the final product, which comprises subjecting Lithocholic acid, the starting material, to the action of one or more specific Ursodeoxycholic acid producing microorganisms.
Chinese Patent CN 1217336 A, relates to a preparation method of Ursodeoxycholic acid by using stereo selectivity synthesis process. Said Ursodeoxycholic acid is an effective component of Chinese valuable medicinal material bear's gall. Said invented preparation method possesses the following advantages: cheap and easily available initial raw material, mild reaction condition and high stereo selectivity and yield, and is favorable for implementing industrial production and its synthesis line has no need of high-temp. and high-pressure reaction condition, and its every step of reaction possesses high yield.
EP2221313 provides a process for preparing the disodium salt of Ursodeoxycholic acid 3,7-disulfate comprising the following steps: a) reaction of Ursodeoxycholic acid with a complex of trimethylamine-sulphuric anhydride, to obtain the Ursodeoxycholic acid 3,7-disulfate di-trimethylammonium reaction intermediate; b) treatment of the di-trimethylammonium salt of Ursodeoxycholic acid 3,7-disulfate with organic or inorganic sodium bases to obtain the tri-sodium salt of Ursodeoxycholic acid 3,7-disulfate; c) treatment with an inorganic acid in presence of isopropanol up to a pH comprised in the range between 3.0 and 4.5, to obtain the disodium salt of Ursodeoxycholic acid 3,7-disulfate in solution; d) removal of the salts present and obtaining the disodium salt of Ursodeoxycholic acid 3,7-disulfate.
US Patent No. 9,206,220 B2, describes a process for the synthesis of Ursodeoxycholic acid wherein the purification of the crude Ursodeoxycholic acid (containing approximately 13-15% of Chenodeoxycholic acid impurity) takes place first passing through a salification with imidazole and a subsequent purification via “methyl ester”, which allows a finished product with an extremely low content of known “Chenodeoxycholic acid and “Lithocholic acid” impurities to be obtained. The present invention also describes the recovery steps of cholic acid and 3a-hydroxy-7-ketocholanic acid from the mother liquors of process intermediates.
The Journal “Process Biochemistry 50 (2015) 598–604”, gives two-step enzymatic synthesis of ursodeoxycholic acid with a new7ß-hydroxysteroid dehydrogenase from Ruminococcus Torquesming.
The Journal “Journal of Biotechnology 191 (2014) 11–21”, gives review of several bacterial regio- and enantio-selective hydroxysteroid dehydrogenases (HSDHs) to carry out thedesired reactions, for example 7-HSDHs from strains of Clostridium, Bacteroides or Xanthomonas, HSDHs from Clostridium, Collinsella, or Ruminococcus, or 12-HSDH from Clostridium or from Eggerthella.However, all these bioconversion reactions need additional steps for the regeneration of the coenzymes.Selected multi-step reaction systems for the synthesis of ursodeoxycholic acid are presented in this review.
Morepen laboratories in collaboration with Cambrex (Germany) decided to pursue with chemoenzymatic method of preparation of Ursodiol or UDCA. It basically involves enzymatic oxidation of CDCA to 7-KLCA as shown in figure below.

Second step involves enzymatic reduction of 7-KLCA to UDCA or Ursodiol or Ursodeoxycholic acid.

SUMMARY OF INVENTION:
The present invention reports novel purification processes for Chenodeoxycholic Acid (CDCA), 7-Ketolithocholic acid (KLCA as well as Ursodeoxycholic acid (UDCA). Furthermore, the present invention also reports process for the preparation as well as purification of Silyl- Ursodeoxycholic acid (Silyl-UDCA).
DETAILED DESCRIPTION OF THE INVENTION:
According to the first embodiment of the present invention, a novel process for the purification of Chenodeoxycholic acid is disclosed which comprises (Example 1, 2 & 3):
i. Heating Chenodeoxycholic Acid with stirring in a C1-C3 aliphatic ester. e.g. ethyl acetate, propyl acetate, butyl acetate and/or cyclic or acyclic ether like tetrahydrofuran, 2-Methyl Tetrahydrofuran, cyclopentylmethyl ether, dioxane and/or an aliphatic nitrile such as acetonitrile, propionitrile or a mixture thereof at reflux temperature as to obtain a clear solution.
ii. Allowing the reaction mass to cool to the ambient temperature naturally.
iii. Further cooling the reaction mass to 0-15°C & stirring for 1-5 hours to ensure complete crystallization.
iv. Isolating pure Chenodeoxycholic acid by filtration followed by drying of the wet cake at 50-60°C for 8-12 hours to get pure Chenodeoxycholic acid.
According to another aspect of current embodiment, the temperature in step (i) is solvent dependent.
According to the second embodiment of the present invention, a novel process for the purification of 7-Ketolithocholic acid is disclosed which comprises (Example 4 & 5) :
i. Refluxing of 7-Ketolithocholic acid with stirring in C1-C4 aliphatic alcohol like methanol, ethanol, 1-propanol, 2-propanol, butanol, monoethylene glycol, diethylene glycol and/or straight chain or branched chain ketone such as selected from acetone, ethyl methyl ketone, diethyl ketone, dimethyl ketone, diisopropyl ketone, dibutyl ketone or a mixture thereof for 1-2 hrs
ii. Adding water with stirring under refluxing for 1-2 hours.
iii. Allowing the reaction mass to cool to ambient temperature naturally.
iv. Further cooling the reaction mass to 0-10°C & stirring for 1-2 hours to ensure complete crystallization.
v. Isolating pure 7-Ketolithocholic acid by filtration followed by drying of wet cake at 50-60°C for 8-12 hours to get pure 7-Ketolithocholic acid.
According to the third embodiment of the present invention, a novel process for the preparation & purification of Silyl Ursodeoxycholic acid (Silyl UDCA) is disclosed which comprises (Example 6 & 7):
i. To Ursodeoxycholic acid in an aliphatic nitrile such as acetonitrile, propionitrile or aliphatic amides such as N, N-dimethylformamide, N, N-dimethylacetamide or a mixture thereof.
ii. Adding Hexamethyldisilazane (HMDS) at room temperature.
iii. Refluxing of reaction mass with stirring for 4-6 hours to obtain a clear solution.
iv. Allowing the reaction mass to cool to 20-30°C naturally with stirring for 1-2 hrs to ensure complete crystallization.
v. Isolating Silyl Ursodeoxycholic acid by filtration followed by drying of wet cake at 40-50°C for 8-12 hours to get Silyl Ursodeoxycholic acid.
vi. Furthermore, The material Silyl Ursodeoxycholic acid obtained in step (v) above can be purified by recrystallization in the solvents mentioned in step (i) above.
According to the Fourth embodiment of the present invention, a novel process for the purification of Ursodeoxycholic acid is disclosed which comprises (Example 8 ):
i. Dissolving any polymorphic form of Ursodeoxycholic acid by refluxing in aqueous solution of straight or branched chain aliphatic ketone selected from acetone, ethyl methyl ketone, diethyl ketone, dimethyl ketone, diisopropyl ketone, dibutyl ketone or a mixture thereof under stirring at reflux temperature.
ii. Charcolization and hot filtration of the reaction mass through hyflow-bed
vii. Cooling of the resulting filtrate to room temperature naturally.
viii. Further cooling of the mass to 0-10°C with stirring for 2-4 hours to ensure complete crystallization.
ix. Isolating pure Ursodeoxycholic acid by filtration followed by drying of the wet cake at 50-60°C for 8-12 hours to get Ursodeoxycholic acid.
Another important aspect of present invention is that charcolization and hot filtration of the reaction mass through hyflow-bed in step ii is very effective in removal of additional signal in residual solvent analysis of UDCA due to the use of HMDS in the process.
The above mentioned invention is supported by the following non limiting examples.
EXAMPLES:
Purification process for Chenodeoxycholic Acid (CDCA)
Example 1: Chenodeoxycholic acid (50g) is dissolved in ethyl acetate (500 ml) at reflux temperature with stirring till get clear solution. The resulting solution is slowly allowed to cool to room temperature with stirring to crystallize the product (CDCA) which is cooled further to 20-30°C with stirring for 1-2 hrs to get complete crystallization. The resulting solid is filtered and wet cake is given running washing with ethyl acetate followed by drying of wet cake at 50-60°C to get 45g of pure CDCA (HPLC purity=99%).
Example 2: Chenodeoxycholic acid (50g) is dissolved in a mixture of ethyl acetate (500 ml) and Tetrahydrofuran (100 ml) at reflux temperature with stirring till get clear solution. The resulting solution is slowly allowed to cool to room temperature with stirring and then cooled further to 0-5°C with stirring for 2-5 hrs to get complete crystallization. The resulting solid is filtered and wet cake is given running washing with ethyl acetate followed by drying of wet cake at 50-60°C to get 42g of pure CDCA.
Example 3: Chenodeoxycholic acid (50g) is stirred in a mixture of Acetonitrile (150 ml) and water (500 ml) for 3-4 hrs at room temperature. The resulting mass is slowly allowed to cool to 0-10°C with stirring for 1-2 hrs to get complete crystallization. The resulting solid is filtered and wet cake is given running washing with water followed by drying of wet cake at 40-50°C to get 40g of pure CDCA (HPLC purity= 98.47%).
Purification process for 7-Ketolithocholic acid (KLCA)
Example 4: 7-Ketolithocholic acid (10g) is taken in Isopropanol (40 ml) and heated at reflux temperature with stirring for 30-60 minutes. Then water (20 ml) was added at reflux temperature and resulting mass was stirred for 1-2 hrs. The resulting mass was slowly cooled to 20-30°C with stirring and then cooled further to 0-5°C followed by stirring for 1-2 hrs to get complete crystallization. The resulting solid was filtered and wet cake is given running washing with IPA followed by drying of wet cake at 50-60°C to get 8g of pure 7-Ketolithocholic acid (HPLC purity = ~98%)
Example 5: 7-Ketolithocholic acid (10g) is taken in Acetone (40 ml) and heated at reflux temperature with stirring for 30-60 minutes. Then water (20 ml) was added at reflux temperature and resulting mass was stirred for 1-2 hrs. The resulting mass was slowly cooled to 20-30°C with stirring and then cooled further to 0-5°C followed by stirring for 1-2 hrs to get complete crystallization. The resulting solid was filtered and wet cake is given running washing with Acetone followed by drying of wet cake at 50-60°C to get 7g of pure 7-Ketolithocholic acid (HPLC purity ~97%).
Purification process for Silyl-Urodeoxycholic Acid
Example 6: Ursodeoxycholic acid (50g) is taken in Acetonitrile (150 ml) and 75 g Hexamethyl disilazane (HMDS) is added to it. The resulting reaction mass is refluxed for 4-6 hrs to get clear solution. The resulting solution is slowly allowed to cool to room temperature with stirring to crystallize the product (Silyl-UDCA) which is cooled further to 20-30°C and stirred for 1-2 hrs to get complete crystallization. The resulting solid is filtered and wet cake is given running washing with Acetonitrile followed by drying of wet cake at 40-50°C for 8-12 hrs to get 60g of pure Silyl-UDCA which is further recrystallized in Acetonitrile and product is isolated by filtration and recommended drying (HPLC purity = 99.77%).
Example 7: Ursodeoxycholic acid (50g) is taken in N,N-Dimethylformamide (400 ml) and 75 g Hexamethyl disilazane (HMDS) is added to it. The resulting reaction mass is refluxed for 4-6 hrs to get clear solution. The resulting solution is slowly allowed to cool to room temperature with stirring to crystallize the product (Silyl-UDCA) which is cooled further to 0-5°C and stirred for 1-2 hrs to get complete crystallization. The resulting solid is filtered and wet cake is dried at 25-35°C for 8-12 hrs to get 45g of pure Silyl-UDCA which is further recrystallized in DMF and product is isolated by filtration and recommended drying (HPLC purity = 99.37%).
Purification process for Ursodeoxycholic Acid (UDCA)
Example 8: Ursodeoxycholic acid (50g) is dissolved in Acetone (250 ml) & water (100ml) mixture at reflux temperature with stirring till get clear solution followed by its charcoalization. The carbon is filtered off and the resulting solution is slowly allowed to cool to room temperature with stirring to crystallize the product (UDCA) which is cooled further to 0-5°C with stirring for 2-4 hrs to get complete crystallization. The resulting solid is filtered and wet cake is given running washing with Acetone followed by drying of wet cake at 50-60°C to get 35 g of pure UDCA (HPLC purity= 100%).

HSGC of Residual solvent analysis of UDCA from processes reported in prior art) – Extra Signal marked.

HSGC of Residual solvent analysis of UDCA from processes reported in current invention.

WE CLAIM:
1. A novel process for purification of Chenodeoxycholic acid which comprises:
I. Dissolving Chenodeoxycholic acid in C1-C3 aliphatic ester selected from ethyl acetate, propyl acetate, butyl acetate and/or cyclic or acyclic ether selected from tetrahydrofuran, 2-methyl tetrahydrofuran, cyclopentylmethyl ether, dioxane and/or an aliphatic nitrile selected from acetonitrile, propionitrile or a mixture thereof refluxed to obtain a clear solution at 20-30°C thereafter;
II. Cooling the reaction mass to 0-10°C to recrystallize the material;
III. Stirring for 3-5 hours at 0-10°C to ensure complete crystallization;
IV. Isolating the pure Chenodeoxycholic acid as wet cake by filtration; and
V. Drying of the wet cake at 50-60°C for 8-12 hours to get pure Chenodeoxycholic acid.

2. A novel process for purification of 7-Ketolithocholic acid which comprises:
I. Dissolving 7-Ketolithocholic acid in C1-C4 aliphatic alcohol selected from methanol, ethanol, 1-propanol, 2-propanol, butanol, monoethylene glycol, diethylene glycol and/or straight chain or branched chain ketone selected from acetone, ethyl methyl ketone, diethyl ketone, dimethyl ketone, diisopropyl ketone, dibutyl ketone or a mixture thereof, at reflux temperature with stirring for 1-2 hours;
II. Adding water with stirring for 1-2 hours at reflux;
III. Allowing the reaction mass to attain room temperature naturally;
IV. Cooling the reaction mass to 0-10°C to recrystallize the material;
V. Stirring for 1-2 hours at 0-10°C to ensure complete crystallization;
VI. Isolating pure 7-Ketolithocholic acid as wet cake by filtration; and
VII. Drying of the wet cake at 50-60°C for 8-12 hours to get pure 7-Ketolithocholic acid.

3. A novel process for preparation & purification of Silyl Ursodeoxycholic acid (Silyl UDCA) which comprises:
I. Dissolving Ursodeoxycholic acid in an aliphatic nitrile selected from acetonitrile, propionitrile or aliphatic amides selected from N, N-dimethylformamide, N, N-dimethylacetamide or a mixture thereof at ambient temperature by adding Hexamethyldisilazane (HMDS);
II. Refluxing the reaction mixture for 4-6 hours to obtain a clear solution;
III. Allowing the reaction mass to attain room temperature naturally;
IV. Cooling the reaction mass to 20-30°C to recrystallize the material;
V. Stirring at 20-30°C for 1-2 hours to ensure complete crystallization;
VI. Isolating the pure Silyl Ursodeoxycholic acid as wet cake by filtration; and
VII. Drying of the wet cake at 40-50°C for 8-12 hours to get pure Silyl Ursodeoxycholic acid.

4. An improved process for the preparation & purification of Ursodeoxycholic acid (UDCA) which comprises:
I. Dissolving any polymorphic form of Ursodeoxycholic acid in aqueous solution of straight or branched chain aliphatic ketone selected from acetone, ethyl methyl ketone, diethyl ketone, dimethyl ketone, diisopropyl ketone, dibutyl ketone or a mixture thereof, at reflux temperature under stirring for 1-2 hours;
II. Charcolizing the reaction mass & finely filtering through hyflow-bed;
III. Cooling the reaction mass to 0-10°C to recrystallize the material;
IV. Stirring for 2-4 hours at 0-10°C to ensure complete crystallization;
V. Isolating the pure Ursodeoxycholic acid (UDCA) as wet cake by filtration; and
VI. Drying the wet cake at 50-60° C for 8-12 hours to get pure Ursodeoxycholic acid (UDCA).

5. The charcolization and hot filtering of the reaction mass through hyflo-bed as claimed in claim 4 (step II) which removes additional signal in HSGC of Residual Solvent analysis of UDCA due to the use of Hexamethyldisilazane (HMDS) in the process.