CN112666294B - Method for separating and determining Shakubatu calcium salt and impurities thereof - Google Patents
Method for separating and determining Shakubatu calcium salt and impurities thereof Download PDFInfo
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- CN112666294B CN112666294B CN202011597815.4A CN202011597815A CN112666294B CN 112666294 B CN112666294 B CN 112666294B CN 202011597815 A CN202011597815 A CN 202011597815A CN 112666294 B CN112666294 B CN 112666294B
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Abstract
A method for separating and measuring Sacubitril calcium salt and its impurities comprises 1) taking Sacubitril calcium salt, adding diluent to dissolve, to obtain sample solution with concentration of 0.1-10 mg/ml; 2) taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution by 50-1000 times to obtain a control solution; 3) and respectively injecting sample solution and control solution with the same volume into a high performance liquid chromatograph, performing gradient elution by using a mobile phase A and a mobile phase B as mobile phases, recording a chromatogram, and completing separation and determination of impurities in the sample solution. The method is simple and convenient to operate, and can effectively realize the separation and determination of the shakubatroxobin calcium salt and the impurities thereof, and the impurities, thereby realizing the purpose of controlling the quality of the shakubatroxobin calcium salt and the product thereof.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for separating and determining a shakubatroxobin salt and impurities thereof.
Background
The calcium salt of Sacubitril is a prodrug of NEP inhibitor developed by Nowa, and is also an experimental drug. The action mechanism can block 2 polypeptides threatening the blood pressure reduction, and is mainly used for developing the medicine for treating hypertension and heart failure. LCZ696 formed by combining the sabotara calcium and the valsartan is the first dual inhibitor of angiotensin receptor type 2 (AT II) and enkephalinase, the clinical curative effect is obvious, the LCZ696 is approved by FDA to be on the market in 2015 7 months, and the sabotara calcium is used as a structural fragment in the molecular structure of the LCZ696 tablet and can also be regarded as a single medicine, so that a control index is established for related substances of the LCZ696 tablet and is a very key control item in quality standards. The chemical name of the Shakuba kojic calcium salt is as follows: the chemical structural formula of the calcium salt of (2R,4S) -5-biphenyl-4-yl-4- (3-carboxyl propionyl amino) -2-methyl-ethyl valerate is shown in the formula (a).
In the process of synthesizing the compound, there are several important intermediates and unknown impurities which may affect the purity and quality of the drug due to incomplete removal, and the known intermediates and unknown impurities and the resulting degradation products are known as related substances (i.e., impurities) in the quality control of the drug. Fourteen known impurities that are primarily controlling for the synthesis of the calcium salt of shakubata are: the structural formula of the impurity-containing composite material is shown as the following formula (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n) and (o).
Therefore, the impurities of the Sacubitril calcium salt are more and similar in structure, which brings difficulty to separation, and in addition, the polarity of each impurity is different, so that the separation between the impurities is also met on the premise of meeting the separation of the Sacubitril calcium salt and each impurity, so that the detection difficulty is increased. Effective separation of the sargasobatrox calcium salt from impurities and impurities from impurities is difficult to achieve by adopting a conventional detection means, which can affect the quality control of the sargasobatrox calcium salt.
In order to accurately control the quality of the calcium salt product of Sacubitril, a method for simply, quickly and accurately separating and detecting related substances of the calcium salt of Sacubitril needs to be researched.
Disclosure of Invention
The invention aims to provide a method for separating and determining the Sacubitril calcium salt and impurities thereof, which is simple and convenient to operate and can effectively realize the separation and determination of the Sacubitril calcium salt and the impurities thereof and the impurities and impurities thereof, thereby realizing the purpose of controlling the quality of the Sacubitril calcium salt and products thereof.
The technical scheme of the invention is as follows: a method for separating and determining a shakubatrox calcium salt and impurities thereof comprises the following steps:
1) preparation of sample solution
Dissolving Sacubitril calcium salt with diluent to obtain sample solution with concentration of 0.1-10 mg/ml;
2) preparation of control solutions
Taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution by 50-1000 times to obtain a control solution;
3) injecting equal volumes of the sample solution of step 1) and the control solution of step 2) into a high performance liquid chromatograph respectively, wherein the injection amount is 5-100 mul,
performing gradient elution by using mobile phase A and mobile phase B as mobile phase with flow rate of 0.8-1.2ml/min, detecting with wavelength of 200 nm-280 nm, recording chromatogram, separating and determining impurities in sample solution,
wherein, the chromatographic column filler of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, the mobile phase A is a buffer solution with the concentration of 0.0001-1.0mol/L, the mobile phase B is methanol, the mobile phase enters the chromatographic column by adopting a gradient elution mode for 0 minute, the volume percentage of the mobile phase A is 35% -45%, and the volume percentage of the mobile phase B is 65% -55%; from 0 minutes to 40 minutes, the volume fraction of mobile phase a decreased linearly to 15% -5%, the volume fraction of mobile phase B increased linearly to 85% -95%; from 40 minutes to 45 minutes, the volume fraction of mobile phase a increases linearly to 35% -45%, the volume fraction of mobile phase B decreases linearly to 65% -55%; from 45 minutes to 50 minutes, the volume fraction of mobile phase A is from 35% to 45%, and the volume fraction of mobile phase B is from 65% to 55%.
Further, the diluent in the step 1) or 2) is a mixture of methanol and water, and the volume ratio of the methanol to the water is 65-95: 35-5.
Preferably, the volume ratio of methanol to water is 80: 20.
preferably, the concentration of the mobile phase A in the step 3) is 0.001-0.01 mol/L.
Further, the buffer solution in the step 3) is a phosphoric acid solution and/or a phosphate solution, the phosphate is one or a mixture of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate and diamine hydrogen phosphate, and the pH value of the buffer solution is 2.0-3.5.
Preferably, the mobile phase in the step 3) enters the chromatographic column by adopting a gradient elution mode, and the volume percentage of the mobile phase A is 40 percent and the volume percentage of the mobile phase B is 60 percent within 0 minute; from 0 minutes to 40 minutes, the volume fraction of mobile phase a decreased linearly to 10%, and the volume fraction of mobile phase B increased linearly to 90%; from 40 minutes to 45 minutes, the volume fraction of mobile phase a increased linearly to 40%, the volume fraction of mobile phase B decreased linearly to 60%; from 45 minutes to 50 minutes, the volume fraction of mobile phase a was 40% and that of mobile phase B was 60%.
Preferably, the sample amount in the step 3) is 10 μ l, the detection is carried out by using a wavelength of 255nm, and the column temperature of the chromatographic column is 20-40 ℃.
Preferably, the column temperature of the column is 35 ℃.
Adopt above-mentioned technical scheme to have following beneficial effect:
1. the separation and determination method adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, adopts a gradient elution mode, ensures that the calcium salt of the Sacuqu can be effectively separated from impurities and impurities, and has high separation degree (the separation degree of the calcium salt of the Sacuqu and adjacent nearest impurities is 3.48, and the separation degree of the impurities is more than 1.5). The phosphate buffer solution is matched, so that the retention can be enhanced, the separation degree can be improved, and the good symmetry and the higher column efficiency of chromatographic peaks can be ensured. The mixed solution of methanol and water is selected as a diluent to dissolve a sample, so that the interference of a solvent peak and the solvent effect can be effectively eliminated.
2. The diluent used in the separation and determination method does not interfere with the determination of impurities and has strong specificity; the sensitivity of each impurity meets the requirement. The content of the impurities is calculated according to a self-contrast method added with a correction factor, and the separation degree of the main peak, the adjacent impurities and the adjacent impurities meets the requirement. The minimum detection limit of the product and each impurity is 0.0003%, which shows that more than 0.0003% of the impurities can be detected, and the accuracy is high.
The following further description is made with reference to the accompanying drawings and detailed description.
Drawings
FIG. 1 is a liquid chromatogram of a methanol-water mixture in example 1;
FIG. 2 is a liquid chromatogram of the mixed control solution of example 1;
FIG. 3 is a liquid chromatogram of the sample solution in example 2;
FIG. 4 is a liquid chromatogram of the control solution of example 2.
Detailed Description
Apparatus and conditions
The high performance liquid chromatograph selects Agilent 1260 type liquid chromatograph and chemical workstation, and is set as automatic sample introduction. A ZORBAX Eclipse Plus C18 column (5 μm, 250X 4.6mm) was used as a separation column. Wavelength of ultraviolet detector: 255 nm. Mobile phase: gradient elution was carried out using 0.01mol/L dipotassium hydrogenphosphate solution (taking 2.28g dipotassium hydrogenphosphate trihydrate, dissolving in 1000ml water, adjusting pH to 2.5 with phosphoric acid) as mobile phase A and methanol as mobile phase B: at 0 minute, the volume percent of mobile phase a was 40%, and the volume percent of mobile phase B was 60%; from 0 minutes to 40 minutes, the volume fraction of mobile phase a decreased linearly to 10%, and the volume fraction of mobile phase B increased linearly to 90%; from 40 minutes to 45 minutes, the volume fraction of mobile phase a increased linearly to 40%, the volume fraction of mobile phase B decreased linearly to 60%; from 45 minutes to 50 minutes, the volume fraction of mobile phase a was 40% and that of mobile phase B was 60%. Column temperature 35 ℃, flow rate: 1.0 ml/min. The injection volume was 10. mu.l.
Example 1
Respectively taking 20mg of impurity IPA, impurity AHUE, impurity AHUQ, impurity AHUH, impurity AHUP, impurity AHUD, impurity AHUJ, impurity AHUN, impurity AHUM, impurity AHUG, impurity AHUK, impurity AHUI, impurity AHUL and impurity AHUF (the purity of each impurity is more than 99%), precisely weighing, placing in a 100ml measuring flask, adding methanol-water (the volume ratio is 80: 20) to dissolve and dilute to a scale, shaking uniformly to serve as an impurity stock solution; LCZ696 (about 40 mg) is precisely weighed and placed in a 50ml measuring flask, 1ml of impurity stock solution is precisely added, methanol-water (volume ratio is 80: 20) is added for dissolving and diluting to a scale, and the mixture is shaken up to be used as a mixed control solution (provided by Chongqing Sansheng industry Co., Ltd.).
The diluent (methanol: water: 80: 20) and the mixed control solution were taken, respectively, subjected to liquid chromatography analysis under the above-mentioned chromatographic conditions, and chromatograms were recorded, and the results are shown in fig. 1 and fig. 2.
Figure 1 shows that the methanol-water mixture and the chromatographic system do not interfere with the assay.
In fig. 2, the sequential order of peaks is IPA as impurity, AHUE as impurity, AHUQ as impurity, AHUH as impurity, AHUP as impurity, acarbostrium salt as impurity, AHUD as impurity, AHUJ as impurity, AHUN as impurity, AHUM as impurity, AHUG as impurity, AHUK as impurity, AHUI as impurity, AHUL as impurity and AHUF as impurity.
Fig. 2 shows that the separation and determination method of the invention can effectively separate the impurities with unknown structures and the impurities with known structures which may exist in the calcium salt of shakubatu, and the detection sensitivity of each impurity can meet the requirement, and the separation degree between the main peak and the adjacent impurities and among the impurities can meet the requirement, namely the method can be used for determining the impurities of the calcium salt of shakubatu and the preparation thereof.
Example 2 determination of Sacubitril calcium salt drug substance (supplied by Chongqing Sansheng industries, Ltd.)
Taking 40mg of a shakuba kojic calcium salt raw material medicine, precisely weighing, placing in a 50ml measuring flask, adding methanol-water (volume ratio is 80: 20) for ultrasonic treatment, dissolving and diluting to a scale, and shaking up to obtain a sample solution; precisely measuring 0.5ml of sample solution, placing in a 100ml measuring flask, diluting with methanol-water (volume ratio of 80: 20) to scale, and shaking to obtain control solution; liquid chromatography was carried out under the chromatographic conditions of example 1 and the chromatogram was recorded. If there is impurity peak (except solvent peak) in chromatogram of sample solution, calculating impurity content according to self-contrast method of adding correction factor. The results are shown in FIGS. 3 and 4. The detection results are shown in table 1:
TABLE 1
Claims (7)
1. A method for separating and determining a shakubatu calcium salt and impurities thereof is characterized by comprising the following steps:
step 1) preparation of sample solution
Dissolving Sacubitril calcium salt in diluent to obtain sample solution with concentration of 0.1-10mg/ml, wherein the Sacubitril calcium salt contains impurities of the following structural formula:
step 2) preparation of control solution
Taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution by 50-1000 times to obtain a control solution;
step 3) injecting equal volumes of the sample solution in the step 1) and the control solution in the step 2) into a high performance liquid chromatograph respectively, wherein the injection amount is 5-100 mul,
performing gradient elution by using mobile phase A and mobile phase B as mobile phase with flow rate of 0.8-1.2ml/min, detecting with wavelength of 200 nm-280 nm, recording chromatogram, separating and determining impurities in sample solution,
wherein, the chromatographic column filler of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, the mobile phase A is a buffer solution with the concentration of 0.0001-1.0mol/L, the mobile phase B is methanol, the mobile phase enters the chromatographic column by adopting a gradient elution mode for 0 minute, the volume percentage of the mobile phase A is 40%, and the volume percentage of the mobile phase B is 60%; from 0 minutes to 40 minutes, the volume fraction of mobile phase a decreased linearly to 10%, and the volume fraction of mobile phase B increased linearly to 90%; from 40 minutes to 45 minutes, the volume fraction of mobile phase a increased linearly to 40%, the volume fraction of mobile phase B decreased linearly to 60%; from 45 minutes to 50 minutes, the volume fraction of mobile phase a is 40% and that of mobile phase B is 60%.
2. The method according to claim 1, wherein the diluent of step 1) or 2) is a mixture of methanol and water, and the volume ratio of methanol to water is 65-95: 35-5.
3. The method according to claim 2, wherein the volume ratio of methanol to water is 80: 20.
4. the method according to claim 1, wherein the concentration of the mobile phase A in the step 3) is 0.001-0.01 mol/L.
5. The method of claim 1, wherein the buffer solution in step 3) is a phosphoric acid solution and/or a phosphate solution, the phosphate is any one or a mixture of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate and diamine hydrogen phosphate, and the pH value of the buffer solution is 2.0-3.5.
6. The method according to claim 1, wherein the sample volume in step 3) is 10 μ l, the detection is carried out at a wavelength of 255nm, and the column temperature of the chromatographic column is 20-40 ℃.
7. The method of claim 6, wherein the column temperature of the chromatography column is 35 ℃.
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| CN114409558A (en) * | 2022-02-15 | 2022-04-29 | 重庆市碚圣医药科技股份有限公司 | Sacubitril intermediate impurity and synthetic method thereof |
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| CN110646519A (en) * | 2018-06-26 | 2020-01-03 | 广东东阳光药业有限公司 | Method for measuring LCZ 696-related substances |
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| CN110133150B (en) * | 2019-05-31 | 2022-04-12 | 重庆三圣实业股份有限公司 | Method for separating and measuring LCZ696 isomer impurities |
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