CN108614038B - Method for determining related substances of obeticholic acid raw material medicine - Google Patents
Method for determining related substances of obeticholic acid raw material medicine Download PDFInfo
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Abstract
The invention establishes a detection method capable of controlling obeticholic acid related substances, and specifically, a proper solvent is selected, a sample is detected through HPLC-ELSD, known impurities adopt an external standard method, and unknown impurities and total impurities adopt a main component reference substance of 1% to control the related substances of a product.
Description
Technical Field
The invention relates to determination of related substances of a raw material medicine, in particular to determination of related substances of an obeticholic acid raw material medicine.
Background
Obeticholic acid (OBDS), a structure modifier of chenodeoxycholic acid, 6-ethyl chenodeoxycholic acid, is a farnesoid X receptor agonist developed by Intercept pharmaceutical companies, can indirectly inhibit the gene expression of cytochrome 7A1(CYP7A1), a rate-limiting enzyme of cholic acid biosynthesis, by activating a farnesoid X receptor, further inhibit cholic acid synthesis, and can be used for treating primary biliary cirrhosis and non-alcoholic fatty liver disease.
Non-alcoholic steatohepatitis is a fatty liver disease, and is expected to become a main factor of liver transplantation by 2020 according to the name of Intercept. No approved therapeutic drug for the disease is available at present, which promotes a market with a potential heavy pound grade, and promotes biopharmaceutical companies to invest huge funds in drug development in the field.
The obeticholic acid has good clinical value and application prospect, and research institutes and pharmaceutical enterprises at home and abroad pay great attention to the research, development and production of the medicine. At present, as the bulk drug is a triterpenoid, no conjugated system exists, the ultraviolet absorption is weak, the control of related substances of the raw materials is difficult, and relevant documents and patents at home and abroad are searched, and no report of a relevant detection method is found.
Disclosure of Invention
The invention aims to establish a detection method capable of controlling obeticholic acid related substances, specifically, a proper solvent is selected, a sample is detected through an HPLC-ELSD (evaporative light detector), known impurities adopt an external standard method, and unknown impurities and total impurities adopt a main component reference substance with the concentration of 1% to control the related substances of a product; because the sensitivity of the evaporation light detector is poor, the concentration and the sample volume of a sample need to be increased by selecting a proper solvent, so that the measurement requirement can be met; however, when the sample concentration and the sample amount are increased, the main peak is overloaded seriously, if the area normalization and the main component self-comparison method are adopted, the large deviation exists, and if the external standard method is adopted, the content of the impurities can be accurately measured, and the quality of the medicine is ensured, the method has the following advantages: 1. the OBDS is dissolved by adopting a proper solvent, so that the solubility can reach 15-30 mg/ml; 2. the related substances can be effectively separated, and quantitative control can be realized; 3. the method has universal applicability and is suitable for being transferred to production companies; 4. the method has sufficient sensitivity, and can effectively control impurities.
The invention provides a detection method capable of controlling obeticholic acid related substances, which comprises the following steps: 1) dissolving the obeticholic acid raw material medicine by using a mixed solvent of acetonitrile and water: 2) detecting the sample by HPLC-ELSD, wherein the chromatographic conditions are as follows: using a C18 acid-resistant chromatographic column, wherein the mobile phase is acetonitrile and 0.1% formic acid; 3) the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
In a specific embodiment of the invention, the volume ratio of the mixed solvent acetonitrile and water is 75:25-85:15, preferably in a volume ratio of 80: 20.
In a particular embodiment of the invention, the sample concentration is between 15 and 30mg/ml, preferably the sample concentration is 20 mg/ml.
In one embodiment of the invention, the ELSD has a drift tube temperature of 35-40 degrees.
In a particular embodiment of the invention the chromatography column is a C18 acid chromatography column, preferably a ZORBAX SB-C18 chromatography column with parameters of 4.6 x 250mm,5um
In a particular embodiment of the invention, the mobile phase is acetonitrile and 0.1% formic acid in a volume ratio of 50:50 to 60:40, preferably in a volume ratio of 55: 45.
In a particular embodiment of the invention, the flow rate of the mobile phase is between 0.2 and 1.2ml/min, preferably the flow rate of the mobile phase is 1.0 ml/min.
Drawings
FIG. 1: in the case that the diluent is acetonitrile: the main peak is flat under the condition of water (80:20, v/v), and the symmetry is good.
FIG. 2: the diluent is acetonitrile: under water (90:10, v/v), the main peak shape front.
FIG. 3: optimum mobile phase conditions are separation of obeticholic acid and impurities under acetonitrile-0.1% formic acid water (55:45, v/v), retention time of each peak is shown as each peak label, and D-1, SM1, D-5, D-6, D-2, OBDS and D-4 are sequentially arranged from left to right.
Detailed Description
The following examples are presented to further understand the present invention but are not intended to limit the invention thereto.
Reference is made to CN104781272A for preparing obeticholic acid bulk drugs and intermediate substances thereof.
Example 1: selection of diluents and solution concentrations
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; quantitative mode: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
Table 1:
test steps and conclusions:
and (4) conclusion: the volume ratio of the diluent is 75:25-85:15, the sample is well dissolved, and the peak pattern is good. The best diluents are acetonitrile: the water content is 80: 20. see figures 1 and 2 for results.
Selection of solution concentration:
and (4) conclusion: the sample concentration range is 15mg/ml-30mg/ml, and the concentration range has better peak shape and can meet the limit requirement of sample determination.
Example 2: selection of evaporative light scattering detector parameters
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; quantitative mode: the known impurities are controlled by external standard method, and the unknown impurities and total impurities are controlled by 1% of main component reference substance
The test steps are as follows:
and (3) adjusting the temperatures of different drift tubes by adopting the chromatographic conditions, and optimizing by taking the noise (6 × SD) and the peak shape of a spectrogram as investigation standards.
Table 2: and (3) test results:
| drift tube temperature (degree) | Noise (6 × SD) and peak shape |
| 30-34 | Does not peak |
| 35-40 | 0.13, good peak shape |
| 41-45 | Difference of peak shape |
And (4) conclusion: the drift tube temperature is in a good peak shape at 35-40 degrees.
Example 3: determination of flow phase ratio
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water, flow rate 1ml/min, sample amount 20 μ l, ELSD drift tube temperature 38 deg.C; quantitative method: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
The test steps are as follows:
system applicability solution: accurately weighing appropriate amount of OBDS and impurities, placing in the same volumetric flask, diluting with diluent to constant volume to scale, and shaking to obtain the final product (OBDS concentration of 20mg/ml, and impurities concentration of 0.1 mg/ml).
And (3) adjusting the proportions of different mobile phases by adopting the chromatographic conditions, and optimizing the proportions of the mobile phases by taking the separation degree of impurities as an investigation index.
Table 3: and (3) test results:
and (4) conclusion: the ratio of mobile phase acetonitrile-0.1% formic acid water is 50:50-60:40, v/v, and each impurity can be completely separated
The optimum mobile phase conditions were acetonitrile-0.1% formic acid water 55:45 (v/v), under which conditions obeticholic acid and the respective impurities are separated as shown in the following table (see figure 3):
table 4:
and (4) conclusion: by adopting the elution condition, the separation degree of each impurity meets the requirement.
Example 4: determination of column type
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, ultraviolet detector model G1315D, and Chemstation processing software, Evaporative Light Scattering Detector (ELSD), acetonitrile and water (80:20, v/v) as solvents, 20mg/ml solution concentration, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; the quantitative method is characterized in that known impurities adopt an external standard method, and unknown impurities and total impurities adopt a main component reference substance with the concentration of 1 percent to control related substances of the product.
The test steps are as follows:
and (3) adjusting different types of chromatographic columns by adopting the chromatographic conditions, and inspecting the types of the chromatographic columns by taking the impurity peak shapes as inspection indexes.
Table 5: and (3) test results:
and (4) conclusion: from the above data, the optimal column should be an acid resistant column such as Agilent zorbaxSB-C18(4.6 x 250mm,5 um).
Example 5: determination of optimal flow rate
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), sample amount 20 μ l, and ELSD drift tube temperature 38 deg.C; quantitative method: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
The test steps are as follows:
system applicability solution: accurately weighing appropriate amount of OBDS and impurities, placing in the same volumetric flask, diluting with diluent to constant volume to scale, and shaking to obtain the final product (OBDS concentration of 20mg/ml, and impurities concentration of 0.1 mg/ml).
And (3) adjusting different flow rates by adopting the chromatographic conditions, and inspecting the flow by taking the retention time and the separation degree of the impurities as inspection indexes.
Table 6: and (3) test results:
and (4) conclusion: from the above data, the flow rate range of this method is 0.8-1.2ml/min, with the optimal flow rate of 1.0 ml/min.
Example 6: method specificity test of the invention
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; quantitative mode: the known impurities are controlled by external standard method, and the unknown impurities and total impurities are controlled by 1% of main component reference substance
The test steps are as follows:
diluent agent: acetonitrile: water (80:20, v/v)
System applicability solution: accurately weighing appropriate amount of OBDS and impurities, placing in the same volumetric flask, diluting with diluent to constant volume to scale, and shaking to obtain the final product (OBDS concentration of 20mg/ml, and impurities concentration of 0.1 mg/ml).
Table 7: and (3) test results:
and (4) conclusion: the diluent does not interfere with the sample measurement, and the method has good specificity.
Example 7: sensitivity testing of the method
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; quantitative method: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
The test steps are as follows:
taking appropriate amount of SM1, D-3, D-5, D-6 and OBDS samples, precisely weighing, placing in different volumetric flasks, diluting with diluent to a certain concentration, and taking the response of each impurity of 10S/N (S/N: signal/noise) or less than 20S/N (S/N: signal/noise) as the appropriate value, namely the quantitative limit concentration of each impurity.
Table 8: and (3) test results:
| code of matter | Limit of quantitation (ug/ml) | Corresponding to the concentration (%) |
| SM1 | 5.12 | 0.02％ |
| D-3 | 16.42 | 0.08％ |
| D-5 | 10.57 | 0.05％ |
| D-6 | 12.48 | 0.06％ |
| OBDS | 15.75 | 0.08％ |
The conclusion is that the impurities with the concentration of the sample below 0.1 percent can be quantitatively measured, and the sensitivity of the method meets the relevant requirements.
Example 8: linear testing of the method
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent ZorbaxSB-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample volume of 20 μ l, drift tube temperature of ELSD of 38 deg.C; quantitative method: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
The test steps are as follows:
taking a proper amount of each impurity, precisely weighing, uniformly preparing 5 concentration points from the quantitative limit concentration to 10 times of the quantitative limit concentration, and performing linear measurement.
Table 9: and (3) test results:
| foreign matter code | Linear Range (μ g/ml) | Coefficient of correlation (R)2) |
| SM1 | 5.12-51.2 | 0.998 |
| D-3 | 16.42-164.2 | 0.999 |
| D-5 | 10.57-105.7 | 0.999 |
| D-6 | 12.48-124.8 | 0.999 |
| OBDS | 15.75-157.5 | 0.999 |
And (4) conclusion: considering that the original product is not on the market and the impurity limit is unknown, the concentration is increased by 10 times in the quantitative limit concentration, 5 points are taken to perform a linear experiment, and the linear relation is good.
Example 9: accuracy testing of the method
Instruments and conditions:
agilent1260 liquid chromatograph, pump model G1311A, uv detector model G1315D, and Chemstation process software, column: agilent zorbax sb-C18(250 x 4.6mm, 5 μm), Evaporative Light Scattering Detector (ELSD), solvents acetonitrile and water (80:20, v/v), solution concentration 20mg/ml, mobile phase: acetonitrile-0.1% formic acid water (55:45, v/v), flow rate of 1ml/min, sample size of 20 μ l, and temperature of drift tube of ELSD of 38 deg.C; quantitative method: the known impurities adopt an external standard method, and the unknown impurities and the total impurities adopt a main component reference substance of 1 percent to control related substances of the product.
The test steps are as follows:
control solution: taking a proper amount of SM1 reference substances, precisely weighing, placing in a volumetric flask, diluting with diluent to a constant volume to scale, and shaking up to obtain the final product (the concentration is 0.1 mg/ml).
Test solution: and (3) precisely weighing a proper amount of OBDS sample, placing the OBDS sample in a volumetric flask, diluting the OBDS sample with a diluent to a constant volume to a scale, and shaking up to obtain the OBDS sample (the concentration is 20 mg/ml).
Recovery test solution: taking SM1 reference substance, precisely weighing, and adding into the test solution, wherein the SM1 concentration is 50%, 100%, and 150% of the limit concentration respectively.
Table 10: and (3) test results:
and (4) conclusion: and (3) carrying out recovery rate measurement on the residual impurities in the step of the finished product of the obeticholic acid, and according to the data, the accuracy of the method meets the requirement for the impurities.
Claims (9)
1. A method for detecting obeticholic acid related substances comprises the following steps: 1) dissolving obeticholic acid by using a mixed solvent of acetonitrile and water to obtain a sample, wherein the concentration of the sample is 15-30 mg/ml; 2) the samples were tested by HPLC-ELSD under the following chromatographic conditions: using a C18 acid-resistant chromatographic column, wherein the mobile phase is acetonitrile and 0.1% formic acid; 3) known impurities in the sample adopt an external standard method, unknown impurities and total impurities adopt 1% of main components as reference substances, and related substances of obeticholic acid are controlled; wherein the related substances include 3 α -hydroxy-7-oxo-5 β -cholanic acid, chenodeoxycholic acid, 3 α -hydroxy-6-ethylene-7-oxo-5 β -cholane-24-oic acid methyl ester, 3 α -hydroxy-6 α -ethyl-7-oxo-5 β -cholane-24-oic acid methyl ester, and 3 α -hydroxy-7-oxo-5 β -cholane-24-oic acid methyl ester; wherein the volume ratio of acetonitrile to 0.1% formic acid in the mobile phase is 50:50-60: 40.
2. The detection method according to claim 1, wherein the volume ratio of acetonitrile to water in the mixed solvent is 75:25-85: 15.
3. The assay of claim 1 or 2 wherein the sample is at a concentration of 20 mg/ml.
4. The detection method of claim 3, wherein the ELSD has a drift tube temperature of 35-40 degrees.
5. The detection method according to claim 3, wherein the column is a ZORBAX SB-C18 column.
6. The assay of claim 3 wherein the volume ratio of acetonitrile to 0.1% formic acid in the mobile phase is 55: 45.
7. The detection method according to claim 3, wherein the flow rate of the mobile phase is 0.2 to 1.2 ml/min.
8. The assay of claim 7 wherein the mobile phase has a flow rate of 1.0 ml/min.
9. The detection method according to claim 3, wherein the concentration of the sample in the mixed solvent is 20mg/ml, the drift tube temperature of the ELSD is 35-40 degrees, the chromatographic column is a ZORBAX SB-C18 chromatographic column, the volume ratio of acetonitrile to 0.1% formic acid in the mobile phase is 55:45, and the flow rate of the mobile phase is 1.0 ml/min.
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