Separation of ursodeoxycholic acid by silylation crystallization
© Ma and Cao; licensee Springer 2014
Received: 5 May 2014
Accepted: 30 May 2014
Published: 24 July 2014
Ursodeoxycholic acid is an important clinical drug in the treatment of liver disease. In our previous work, ursodeoxycholic acid was prepared by electroreduction of 7-ketolithocholic acid. The separation of ursodeoxycholic acid from the electroreduction product (47% (w/w) ursodeoxycholic acid) by silylation crystallization is described herein.
N,N-dimethylformamide was used as the solvent, whereas hexamethyldisilazane was the reaction agent. The optimal material ratio of electroreduction product/N,N-dimethylformamide/hexamethyldisilazane was found to be 1:10:2 (w/v/v). The reaction proceeded for 2 h at 60°C, and the corresponding silylation derivative was separated by crystallization and pure ursodeoxycholic acid was recovered by 5% acid hydrolysis at 50°C for 0.5 h. The maximum recovery and purity of ursodeoxycholic acid were 99.8% and 99.5%, respectively.
Ursodeoxycholic acid with high purity and high recovery can be prepared directly. The developed method offers a potential application for large-scale production of ursodeoxycholic acid.
KeywordsUrsodeoxycholic acid Silylation Separation
Ursodeoxycholic acid (3α, 7β-2-hydroxy-5β-cholanic acid, UDCA) is an important clinical drug used in the treatment of liver disease, such as gallstones [], alcoholic fatty liver [], nonalcoholic fatty liver [], viral hepatitis [], primary biliary cirrhosis [], primary sclerosing cholangitis [], and cholestatic [].
Researchers have focused on the separation and purification of UDCA. Guillemette et al. [] described the purification of UDCA by reacting an aqueous alkali metal salt solution of UDCA in the presence of chloroform with an acid to recover crystalline UDCA. Bonaldi et al. [] prepared high-purity UDCA, starting from cholic acid by forming the tris-trimethylsilyl derivative acid thereof, reducing the acid by the Wolff-Kishner method into UDCA, and the total impurities were less than 1.3%. Xu et al. [] separated UDCA from its isomeric mixture using a core-shell molecular imprinting polymer, and the separation factor of the molecular imprinting polymer with acrylamide for UDCA was 2.20. Tian et al. [] produced UDCA by catalytic transfer hydrogenation of 7K-LCA with Raney nickel, then UDCA was purified by column chromatography and recrystallized. Ninety-seven percent UDCA was obtained via this last method.
Silylation is a powerful tool to improve the production process and the quality of the product in modern pharmaceutical and organic synthesis, and has been applied in increasing the volatility [], changing the solubility in organic solvents [], and the protection of sensitive functional groups such as hydroxyl and carboxyl moieties []. Silylation of alcohols and phenols with hexamethyldisilazane (HMDS) has been achieved using various types of catalysts [–]. The reaction conditions were nearly neutral, and the corresponding silyl ethers yields were high. Mormann et al. [] reported the silylation of cellulose by HMDS in liquid ammonia and the reaction gave high degrees of silylation.
In this work, UDCA was isolated from the electroreduction product with analogues by silylation crystallization and UDCA. The process afforded UDCA with high purity and high recovery via a direct method.
The standard samples of UDCA, CDCA, and silylating reagents were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). 7K-LCA was prepared according to our previous work []. Acetonitrile and methanol were of high-performance liquid chromatography (HPLC) grade and purchased from Shanghai Xingke Biochemistry Co. Ltd (Shanghai, China). All other reagents were of analytical grade. HPLC (LC-20A, Shimadzu Corporation, Kyoto, Japan) with a UV detector (SPD-20A) using a C18 column (Welchrom-C18, 4.6 × 150 mm, 5 μm, Welch Materials Inc. Shanghai, China) was used for quantification of the reaction products. Electron impact ionization time-of-flight mass spectrometry (EI-TOF-MS) (Micromass GCTTM, Micromass UK Ltd, Lancas, UK) and Fourier transform infrared spectroscopy (FTIR) (Magna-IR 550, Thermo Nicolet Ltd, Wisconsin, USA) were used for the characterization of the product and provided by the Analysis and Test Center, East China University of Science and Technology.
HPLC was used to analyze the product. The mobile phase was a mixture of phosphate acid buffer (pH 3.0) and acetonitrile (50:50, v/v) at a flow rate of 1.0 ml/min at 25°C. Detection was performed using a UV detector at 208 nm. UDCA was quantified by an external standard.
EI-TOF-MS was used to identify the molecular weight of the derivatives.
FTIR spectra with KBr pellets were used to analyze the structure of the product and the standard UDCA.
Preparation of UDCA
The synthesis procedure followed Yuan's method []. UDCA was synthesized in a divided electrolytic cell by direct electroreduction of 7K-LCA. A titanium ruthenium mesh electrode was used as the anode, and a high-purity lead plate was used as the cathode. Under the optimized process conditions, the content of UDCA was 47%.
Silylation crystallization experiments were carried out as below. One gram of the electroreduction product (47% (w/w) of UDCA) was added into a 100-ml conical flask containing 10 ml N,N-dimethylformamide (DMF) at 30°C, and following the dissolution of the material under magnetic stirring at 150 rpm, 1 ml of HMDS was added into the solution. The conical flask was sealed with thread seal tape. The reaction was carried out for 2 h at 30°C at a speed of 150 rpm. After the reaction was complete, the flask was moved to glacial water and maintained at 0°C for 24 h. The crystalline material was collected by filtration and washed with the same silylating reagent and dried in a vacuum oven.
Results and discussion
Selection of silylating reagents
There are many kinds of silylation reagents. In this study, hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS), and 1,3-bis (trimethylsilyl) urea (BSU) were chosen as the silylating reagents.
Since the boiling point of TMCS is 57.7°C, the reaction took place intensively after TMCS was added to the system under the experimental temperature (30°C), and TMCS evaporated. Consequently, the contact between reactants was reduced, and this made the final product yield lower. BSU is a silylation reagent that can increase the solubility of the derivative of UDCA in DMF, so it is difficult to form a precipitant. HMDS, the commercially available reagent for trimethylsilylation of reactive hydrogen, is stable and yields ammonia as the only by-product, which is simple to remove from the reaction medium. Therefore, HMDS was chosen for the following study.
Difference between the silylation derivatives
Since a silylation reagent can replace the hydroxyl groups in UDCA, CDCA, and 7K-LCA, the difference between their corresponding silylation derivatives was the key to the subsequent success of the separation step. As such, silylation was conducted using standard samples (UDCA, CDCA, 7K-LCA, and their mixtures at different proportions) as the silylation reaction substrates and HMDS as the silylation reagent.
Difference between the silylation derivatives
Influence of temperature
The silylation reaction is a strongly exothermic reaction. Thus, temperature could affect the reaction to some extent. Thus, silylation was conducted over the temperature range of 20°C to 70°C.
Influence of the material ratio
Influence of different material ratios
Electroreduction product (g)
When the material ratio of electroreduction product/DMF/HMDS was 1:10:2 (w/v/v), the recovery and purity of UDCA were 99.8% and 99.5%, respectively. With 1 ml HMDS present, the reaction did not complete; however, when 3 ml of HMDS was used, the silylation derivative of UDCA did not readily form a crystal because of the increase of HMDS in the solution. A comparison of group 2 with groups 4, 5, and 6 showed that excessive crude UDCA made the reaction system too viscous to react.
Characterization of the product
After being hydrolyzed and dried, the product was determined by HPLC, EI-TOF-MS, and FTIR.
Characterization by HPLC
Characterization by EI-TOF-MS
Formula evaluated by multiple mass analysis
Double bond equivalents
Characterization by FTIR
Ursodeoxycholic acid was purified by silylation crystallization in this study. By optimizing process conditions, recovery and purity of ursodeoxycholic acid was up to 99.8% and 99.5%, respectively. HPLC, EI-TOF-MS, and FTIR analysis showed that silylation is a highly efficient purification method of ursodeoxycholic acid. Compared with previous methods, the UDCA preparation methods presented herein (i) gave higher purity and recovery, (ii) avoided cumbersome procedures, (iii) was more cost efficient, and (iv) did not require harsh reaction conditions. The silylation crystallization approach was easy to operate, economic, and timesaving, and the by-product (i.e., ammonia) was simple to remove from the reaction medium.
electron impact ionization time-of-flight mass spectrometry
Fourier transform infrared spectroscopy
high-performance liquid chromatography
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