Method for detecting impurities of 22-ene-ursodesoxycholic acid and 22-ene-chenodesoxycholic acid in ursodesoxycholic acid

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting impurities of 22-ene-ursodesoxycholic acid and 22-ene-chenodesoxycholic acid in ursodesoxycholic acid. According to the method, a high performance liquid chromatograph is used, a sodium dihydrogen phosphate dihydrate aqueous solution-acetonitrile is used as a mobile phase, isocratic elution is adopted, an octyl silane bonded silica gel column is used as a chromatographic column for separation, and an ultraviolet detector is used for detection. The detection method can effectively separate and identify 22-ene-ursodesoxycholic acid and 22-ene-chenodesoxycholic acid impurities in ursodesoxycholic acid, methodology verifies that all inspection indexes meet regulations, the detection result is accurate and reliable, and the method has the advantages of being simple, convenient, exclusive, accurate and high in sensitivity. And a guarantee is provided for monitoring the quality stability and clinical medication safety of the ursodesoxycholic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid. Background Art

[0002] Ursodeoxycholic acid, CAS: 83-49-8, chemical name is 3α,7β-dihydroxy-5β-cholestane-24-acid, is the main component of bear bile in traditional Chinese medicine, and is in very short supply. This product is used for cholesterol gallstones, primary cholestatic cirrhosis, primary sclerosing cholangitis, bile reflux gastritis and bile deficiency steatorrhea, and can also be used to prevent drug-induced stone formation and treat steatorrhea (after ileal resection).

[0003] In the process of preparing ursodeoxycholic acid, in addition to the target compound, impurities are also produced. The structural formulas of ursodeoxycholic acid and related impurities are shown in the following table:

[0004]

[0005]

[0006]

[0007] Impurities A, B, D, E, F, G, H, I, K, L, M1, N, P, and R of ursodeoxycholic acid are all process impurities that affect the purity of the drug and are related to the effectiveness and safety of the drug.

[0008] Patent application CN 115232853A and patent application CN115404260A mention that there is an impurity 22-ene-chenodeoxycholic acid in chenodeoxycholic acid and an impurity 22-ene-ursodeoxycholic acid in ursodeoxycholic acid. Ursodeoxycholic acid is mainly prepared from chenodeoxycholic acid by chemical synthesis or biosynthesis. Since chenodeoxycholic acid is derived from animal bile, its composition is complex and there are many impurities. The possibility of the presence of 22-ene-chenodeoxycholic acid is very high. The structure of 22-ene-chenodeoxycholic acid is similar to that of chenodeoxycholic acid and is difficult to remove. In the process of preparing ursodeoxycholic acid from chenodeoxycholic acid, 22-ene-chenodeoxycholic acid will also be partially or completely converted into 22-ene-ursodeoxycholic acid. Based on the requirements for impurity control of chemical drugs and relevant guidelines, the limits of impurities 22-ene-chenodeoxycholic acid and 22-ene-ursodeoxycholic acid are controlled below 0.10%, which is of great significance to the quality and safety of raw materials and preparations.

[0009] The prior art all uses high performance liquid chromatography-differential detector to detect related impurities in ursodeoxycholic acid. There is no report on the detection method of 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid. The polarity difference between 22-ene-ursodeoxycholic acid impurities and ursodeoxycholic acid is very small, and the polarity difference between 22-ene-chenodeoxycholic acid impurities and chenodeoxycholic acid (ursodeoxycholic acid impurity A) is also very small. Under the existing related substance detection method, they completely overlap, making it impossible to effectively control the content of 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities. The existing detection methods for ursodeoxycholic acid in the United States Pharmacopoeia, British Pharmacopoeia, and Chinese Pharmacopoeia standards cannot effectively separate the impurities 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid, affecting the quantitative determination of impurities. Therefore, it is of great significance to provide a method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid. Summary of the invention

[0010] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid, which has the characteristics of simplicity, exclusivity, accuracy and high sensitivity, and can better monitor and control the impurity content of ursodeoxycholic acid, ultimately improving the quality of drugs and ensuring the safety of drugs.

[0011] To achieve the above object, the present invention is implemented through the following technical solutions:

[0012] In a first aspect, the present invention provides a method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid, using high performance liquid chromatography, the chromatographic conditions are: the chromatographic column uses octylsilane bonded silica gel as a filler, the mobile phase is a mixed solvent of acetonitrile and sodium dihydrogen phosphate dihydrate aqueous solution, isocratic elution is performed, and the detector is an ultraviolet detector.

[0013] The chemical structural formulas of ursodeoxycholic acid, 2,2-ene-ursodeoxycholic acid, and 2,2-ene-chenodeoxycholic acid are shown in formula (1), formula (2), and formula (3), respectively;

[0014]

[0015] Furthermore, the chromatographic column is preferably a chromatographic column using octylsilane bonded silica gel as a filler, 4.6 mm×250 mm, 5 μm, or a chromatographic column with equivalent performance.

[0016] Furthermore, the chromatographic column is Inertsil C8-3, 4.6 mm×250 mm, 5 μm.

[0017] Furthermore, in the mobile phase, the volume ratio of sodium dihydrogen phosphate dihydrate aqueous solution to acetonitrile is 50:50 to 80:20.

[0018] Furthermore, the mass concentration of the sodium dihydrogen phosphate dihydrate aqueous solution in the mobile phase is 2 g / L to 8 g / L.

[0019] Furthermore, the detection wavelength of the ultraviolet detector is 200nm to 240nm, preferably 210nm to 230nm.

[0020] Furthermore, the chromatographic conditions also include the flow rate of the mobile phase, and the flow rate of the mobile phase is 1.0±0.3 ml / min.

[0021] Furthermore, the chromatographic conditions also include a chromatographic column temperature, which is 25°C to 50°C, preferably 30°C to 45°C.

[0022] Furthermore, the chromatographic conditions also include an injection volume, and the injection volume is 2 to 50 μl, preferably 5 to 20 μl.

[0023] Further, the method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid comprises the following steps:

[0024] (1) Preparation of blank solution (diluent): methanol-water (75:25);

[0025] (2) Preparation of test solution: Take an appropriate amount of ursodeoxycholic acid, weigh it accurately, dissolve it in a diluent and quantitatively dilute it to make a solution containing about 15 mg per 1 ml;

[0026] (3) Preparation of reference solution: Take an appropriate amount of 2,2-ene-chenodeoxycholic acid and 2,2-ene-ursodeoxycholic acid reference substances, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing about 15 μg per 1 ml;

[0027] (4) Determine the blank solution, test solution and reference solution in steps (1), (2) and (3) by HPLC, and calculate the content of 2,2-ene-ursodeoxycholic acid and 2,2-ene-chenodeoxycholic acid by external standard method;

[0028] Among them, the specific chromatographic conditions for HPLC determination are: the chromatographic column is Inertsil C8-3, 4.6mm×250mm, 5μm; the mobile phase is 2g / L~8g / L sodium dihydrogen phosphate dihydrate solution-acetonitrile with a volume ratio of 50:50~80:20; the flow rate is 1.0±0.3ml / min; the column temperature is 25℃~50℃; the ultraviolet detection wavelength is 210nm~230nm; and the injection volume is 5~20μl.

[0029] On the other hand, the present invention also provides the application of the above detection method in the detection of related substances in ursodeoxycholic acid raw materials and preparations.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. The high performance liquid phase detection method of the present invention can effectively separate ursodeoxycholic acid from 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid, with a separation degree greater than 1.5.

[0032] 2. The detection method of the present invention has screened out the best chromatographic analysis conditions and solution preparation method through a large number of experiments. After methodological verification, all test indicators meet the requirements, the test results are accurate and reliable, and it has the characteristics of simplicity, exclusivity, accuracy and high sensitivity, which provides a guarantee for monitoring the quality stability and clinical drug safety of ursodeoxycholic acid.

[0033] 3. This method is not only applicable to the detection of related substances in ursodeoxycholic acid raw materials, but also to the detection of related substances in solid preparations such as tablets and sustained-release capsules. The detection method is not interfered by common excipients. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the blank solution chromatogram of Example 1.

[0035] Figure 2 It is the chromatogram of the reference substance solution of Example 1.

[0036] Figure 3 This is the chromatogram of the test solution of Example 1.

[0037] Figure 4 This is the chromatogram of the spiked solution of the test sample in Example 1.

[0038] Figure 5 This is the solution chromatogram of the system suitability of Comparative Example 1.

[0039] Figure 6 This is the chromatogram of the test solution of Comparative Example 2.

[0040] Figure 7 It is the chromatogram of the reference substance solution of Comparative Example 2.

[0041] Figure 8 This is the linear relationship diagram of 22-ene-ursodeoxycholic acid in Example 4.

[0042] Fig. 9 This is the linear relationship diagram of 22-ene-chenodeoxycholic acid in Example 4. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail through examples below. The following examples are only used to explain the present invention and do not represent the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0044] The reagents and raw materials used in the present invention are all commercially available.

[0045] Example 1

[0046] 1. Solution preparation

[0047] Blank solution (diluent): methanol-water (75:25).

[0048] Solutions of each impurity reference substance (impurity A, impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M1, impurity N, impurity P, impurity R): take an appropriate amount of impurity (impurity A, impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M1, impurity N, impurity P, impurity R) reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 15 μg per 1 ml.

[0049] Test solution: Take an appropriate amount of ursodeoxycholic acid, weigh it accurately, add diluent to dissolve it and quantitatively dilute it to make a solution containing about 15 mg per 1 ml.

[0050] Reference solution: Take an appropriate amount of 2,2-ene-chenodeoxycholic acid and 2,2-ene-ursodeoxycholic acid reference substances, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing about 15 μg per 1 ml.

[0051] Test sample spiked solution: Take about 300 mg of the product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 2 ml of the impurity reference stock solution, dissolve it with a diluent and dilute it to the scale, shake well, and you have it.

[0052] 2. Chromatographic conditions

[0053] Chromatographic column: Inertsil C8-3 4.6mm×250mm, 5μm or other column with equivalent performance

[0054] Mobile phase: 4.7 g / L sodium dihydrogen phosphate dihydrate solution-acetonitrile (60:40)

[0055] Flow rate: 1.0ml / min Column temperature: 40℃ Detection wavelength: 220nm Injection volume: 10μl

[0056] Running time: 50min

[0057] 3. Determination method

[0058] The blank solution, reference solution, and test solution were accurately measured respectively, and the test solution spiked with the test solution was injected into the liquid chromatograph to record the chromatogram.

[0059] The chromatographic data are shown in the following table:

[0060] Table 1 Specific solution chromatographic data

[0061]

[0062] The results showed that the blank solution and the test solution did not interfere with the detection of 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid. Chenodeoxycholic acid and other impurities had no response under this method. The separation degree of 22-ene-ursodeoxycholic acid, 22-ene-chenodeoxycholic acid and adjacent peaks in the test solution met the requirements. The specificity of this method was good. Figure 1 to Figure 4 .

[0063] Example 2

[0064] Take an appropriate amount of 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid reference substances, accurately weigh them, dissolve them in a diluent and quantitatively dilute them to make a solution of a certain concentration. Accurately pipette 10 μl and inject it into a liquid chromatograph. The chromatographic conditions are the same as those in Example 1. Record the chromatogram. The detection limit is calculated based on a signal-to-noise ratio of about 3:1, and the quantitative limit is calculated based on a signal-to-noise ratio of about 10:1. The results are shown in Tables 2 and 3.

[0065] Table 2 Results of detection limit and quantification limit

[0066]

[0067]

[0068] Table 3 Quantitation limit repeated injection results

[0069]

[0070] The results showed that the quantitative limit solution was injected 6 times continuously, the retention time RSD of 22-ene-ursodeoxycholic acid was 0.17%, and the peak area RSD was 4.55%; the retention time RSD of 22-ene-chenodeoxycholic acid was 0.12%, and the peak area RSD was 2.79%, and the quantitative limit met the requirements.

[0071] Example 3

[0072] Take the reference solution and the test solution, inject and test them at intervals, record the chromatogram, and the results are shown in Table 4.

[0073] Table 4 Solution stability results

[0074]

[0075]

[0076] From the results in the above table, it can be seen that the reference solution is stable within 61 hours and the test solution is stable within 41 hours.

[0077] Example 4

[0078] Six concentration points were evenly taken for 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid within the range of the quantitative limit to 200% of the limit concentration, and a linear regression curve was drawn with the sample concentration as the abscissa and the peak area as the ordinate.

[0079] Accurately measure 10 μl of each of the above concentration linear solutions, inject them into the liquid chromatograph, record the spectrum, and the test results are shown in Table 5.

[0080] Table 5 Linearity test results

[0081]

[0082]

[0083] Note: “ / ” indicates not applicable.

[0084] The results showed that the correlation coefficient of the linear equation of 22-ene-ursodeoxycholic acid met the requirements, and the linear relationship was good in the concentration range of 0.5107μg / ml to 30.0388μg / ml; the correlation coefficient of the linear equation of 22-ene-chenodeoxycholic acid met the requirements, and the linear relationship was good in the concentration range of 1.2016μg / ml to 30.0393μg / ml. The linear relationship diagrams of 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid are shown below. Figure 8 , Fig. 9 shown.

[0085] Example 5

[0086] Take 2 portions of reference solution and 6 portions of test solution, inject and test, record the chromatogram, and the results are detailed in Table 6.

[0087] Table 6 Repeatability test results

[0088]

[0089] The results showed that 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid were not detected in the 6 test solutions, and the method had good repeatability.

[0090] Comparative Example 1

[0091] 1. Solution preparation

[0092] System suitability solution: Take appropriate amount of ursodeoxycholic acid reference substance and 22-ene-UDCA reference substance, add diluent to dissolve and dilute to make a solution containing approximately 15 mg of ursodeoxycholic acid and 0.675 μg of 22-ene-UDCA per 1 ml.

[0093] Take 10 μl of each of the above system suitability solutions, inject into the chromatograph, and record the chromatogram.

[0094] 2. Chromatographic conditions

[0095] Chromatographic column: Octadecylsilane bonded silica column (Waters Spherisorb ODS1, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance)

[0096] Mobile phase: 1.2% (w / v) sodium dihydrogen phosphate dihydrate, 0.4% (w / v) disodium hydrogen phosphate dihydrate and 1.5% tetrabutylammonium hydrogen sulfate in water-acetonitrile (60:40)

[0097] Diluent: Weigh 6 g of sodium dihydrogen phosphate dihydrate and 2 g of disodium hydrogen phosphate dihydrate, dissolve them in 535 ml of water, add 65 ml of tetrabutylammonium hydroxide solution (25% aqueous solution), and then add 400 ml of acetonitrile and mix well.

[0098] Flow rate: 1.5 ml / min; column temperature: 50 °C; injection volume: 10 μl; detection wavelength: 210 nm

[0099] Running time: 20 minutes

[0100] The results showed that 22-ene-UDCA and ursodeoxycholic acid in the system suitability solution did not achieve baseline separation, and the resolution was only 1.18, which did not meet the quantitative requirements. Figure 5 .

[0101] Comparative Example 2

[0102] 1. Solution preparation

[0103] Blank solution (diluent): methanol-water (75:25).

[0104] Test solution: Take an appropriate amount of ursodeoxycholic acid, weigh it accurately, add diluent to dissolve it and quantitatively dilute it to make a solution containing about 15 mg per 1 ml.

[0105] Reference solution: Take an appropriate amount of 22-ene-ursodeoxycholic acid reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing about 15 μg per 1 ml.

[0106] Take 10 μl of the test solution and reference solution, inject them into the chromatograph, and record the chromatogram.

[0107] 2. Chromatographic conditions

[0108] Chromatographic column: Octylsilane bonded silica gel column (Inertsil C8-3 4.6mm×250mm, 5μm or chromatographic column with equivalent performance)

[0109] Mobile phase: 14 g / L sodium perchlorate monohydrate solution-acetonitrile (60:40)

[0110] Flow rate: 1.0 ml / min; column temperature: 40°C; detection wavelength: 210 nm; injection volume: 10 μl

[0111] The results showed that the peak times of 22-ene-ursodeoxycholic acid in the reference solution and ursodeoxycholic acid in the test solution partially overlapped, and baseline separation was not achieved, which did not meet the quantitative requirements. Figure 6-7 .

[0112] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application, which should be deemed to fall within the scope of protection of the present application.

Claims

1. A method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid, characterized in that: The chemical structural formulas of ursodeoxycholic acid, 2,2-ene-ursodeoxycholic acid, and 2,2-ene-chenodeoxycholic acid are shown in formula (1), formula (2), and formula (3), respectively; The method adopts high performance liquid chromatography, and the chromatographic conditions are: the chromatographic column uses octylsilane bonded silica gel as a filler, the mobile phase is a mixed solvent of acetonitrile and sodium dihydrogen phosphate dihydrate aqueous solution, isocratic elution is performed, and the detector is an ultraviolet detector.

2. The detection method according to claim 1, characterized in that: The chromatographic column is Inertsil C8-3, 4.6 mm×250 mm, 5 μm.

3. The detection method according to claim 1, characterized in that: In the mobile phase, the volume ratio of sodium dihydrogen phosphate dihydrate aqueous solution to acetonitrile is 50:50 to 80:

20.

4. The detection method according to claim 3, characterized in that: The mass concentration of the sodium dihydrogen phosphate dihydrate aqueous solution in the mobile phase is 2 g / L to 8 g / L.

5. The detection method according to claim 1, characterized in that: The detection wavelength of the ultraviolet detector is 210nm-230nm.

6. The detection method according to claims 1 to 5, characterized in that: The chromatographic conditions of the high performance liquid chromatography method also include one or more of the following I to III: Ⅰ. The flow rate of the mobile phase is 1.0±0.3ml / min; Ⅱ. The column temperature is 25℃~50℃; III. The injection volume is 5 to 20 μl.

7. A method for detecting 22-ene-ursodeoxycholic acid and 22-ene-chenodeoxycholic acid impurities in ursodeoxycholic acid, characterized in that: The following steps are involved: (1) Preparation of blank solution: Blank solvent (diluent): methanol-water (75:25); (2) Preparation of test solution: Take an appropriate amount of ursodeoxycholic acid, weigh it accurately, dissolve it in a diluent and quantitatively dilute it to make a solution containing about 15 mg per 1 ml; (3) Preparation of reference solution: Take an appropriate amount of 2,2-ene-chenodeoxycholic acid and 2,2-ene-ursodeoxycholic acid reference substances, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing about 15 μg per 1 ml; (4) Determine the blank solvent, test solution and reference solution in steps (1), (2) and (3) by HPLC, and calculate the content of 2,2-ene-ursodeoxycholic acid and 2,2-ene-chenodeoxycholic acid by external standard method; Among them, the specific chromatographic conditions for HPLC determination are: the chromatographic column is Inertsil C8-3, 4.6mm×250mm, 5μm; the mobile phase is 2g / L~8g / L sodium dihydrogen phosphate dihydrate solution-acetonitrile with a volume ratio of 50:50~80:20; the flow rate is 1.0±0.3ml / min; the column temperature is 25℃~50℃; the ultraviolet detection wavelength is 210nm~230nm; and the injection volume is 5~20μl.

8. Application of the detection method according to any one of claims 1 to 7 in the detection of related substances in ursodeoxycholic acid raw materials and preparations.

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